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the Toxicologist. An Official Publication of the Society of Toxicology. Abstracts of the 27th Annual Meeting.

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e Preface This issue of the Toxicologist is devoted to the abstracts of the presentations for the platform, poster/discussion, and poster sessions of the 27th Annual Meeting of the Society of Toxicology, held at the Loews Anatole Hotel, Dallas, Texas, February 15-19, 1988. The issue also contains a Keyword Index (by subject or chemical) to the titles of all the presentations, beginning on page 271. An alphabetical Author Index, cross-referencing the corresponding abstract number(s), begins on page 317. The abstracts are reproduced as accepted by the Program Committee of the Society of Toxicology, and appear in numerical sequence. Copies of this issue are available at $20 each plus $3.00 postage and handling (U.S. funds) from: Society of Toxicology 1133 Fifteenth Street, N.W., Suite 1000 Washington, D. C. 20005 © 1988, Society of Toxicology
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`e , . I ~~ ~~XI~;~~~~~ST An Official Publication of the Society of Toxicology Abstracts of the 2? th Annual Meeting Vol. 8, No. 1, February 1988
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21 SELECTIVE TOXICITY OF 3-TRIFLUOROMETHYLPYRIDINE (3-FMP) TO RAT OLFACTORY EPITHELIUM. E A Lock and P M Hext. ICI PLC, Central Toxicology Laboratory + Macclesfield, Cheshire, UK. - . 3-FMP is a semi-volatile by-product from the synthesis of a process intermediate. .Toxicological studies showed that via inhalation 3-FMP produced necrosis to the olfactory epithelium (OE) and liver (L) of rats. Whole body autoradiograptiystudies with [14C]-3-FMP showed selective retention of radioactivity in the OE and L. We have examined the distribution of radioactivity from [14C]-3-FMP in rat OE and L following in vivo and in vitro administration. Rats were,treated (po) with 125mg/kg 3-FMP and killed 0.5 to 24hr later and total and protein bound radioactivity determined in the two tissues. Both tissues showed marked accumulation of radioactivity with time. Studies in vitro with isolated OE and L slices showed that these tissues accumulated radioactivity from [14C]-3-FMP. This accumulation of radioactivity was abolished by inhibitors of cytochrome P-450 (eg metyrapone). Prior treatment of rats with metyrapone (150mg/kg, ip) followed by [14C]-3- FMP prevented the retention of radioactivity in the OE but not L and the onset of the lesion in the OE. These studies show that radioactivity from 3-FMP is selectively retained in the rat OE and L by a cytochrome P-450 dependant process and that inhibitors of cytochrome P-450 can prevent the toxicity to the OE. 22 KINETICS OF NASAL MUCOSAL CARBOXYLESTERASE- MEDIATED HYDROLYSIS OF DIBASIC ESTERS. C A Patterson, C R Kee, and M S Bogdanffy. E I du Pont de Nemours & Co, Inc, Haskell Laboratory for Toxicology and Industrial Medicine, Newark, DE. Ninety-day,inhalatioa exposure of rats to dibasic esters (DBEs), a mixture of dimethyl -succinate, -glutarate and -adipate, causes mild lesions of the nasal olfactory mucosa (OLF) of rats at concentrations that do not affect the nasal respiratory mucosa (RESP). A possible mechanism of toxicity may be metabolism of these compounds in nasal tissue to toxic acids. The purpose of this study was to determine the kinetic constants for carboxylesterase-mediated hydrolysis of DBEs and correlate these findings with lesion formation. Formation of mono- and diacid metabolites of DBEs in nasal mucosal homogenates was monitored by HPLC. However, none of the diacid metabolites were detected. Vmax values for the formation of monomethyl succinate (MMS), monomethyl glutarate (MMG), and monomethyl adipate (MMA) were approximately 8 to 10 times larger in OLF than RESP. V/K values for the formation of MMG and MMA were approximately 9 and 10 times larger in OLF than RESP. For the formation of MMS, V/K was approximately 2 times larger in RESP than OLF. Thus, hydrolysis of these esters occurs more readily in OLF than RESP and the efficiency of hydrolysis at low substrate concentrations is related to carbon chain length and mucosal type. 6 23 SUBCHRONIC INHALATION STUDY IN RATS WITH DIBASIC_ ESTERS (DBE): RECOVERY OF NASAL LESIONS. C M Keenan, M S Bogdanffy, and D P Kelly. E I du Pont de Nemours & Co, Inc, Haskell Laboratory for Toxicology and Industrial Medicine, Newark, DE. .! DBE is a solvent mixture of dimethyl -succiWte, -glutarate and -adipate used in the paint and coating industry. A suV@qrronic inhalation•study with a recovery period was initiated to e aliuate nasal lesions induced by DBE vapors. Male ~~ female rats were exposed to DBEs at ? concentrations of 20, 80, or 400 mg/m3 for 45 or 90 days. An additional group was allowed a recovery period of 45 days, After 45 days of exposure, slight degeneraV'bn af- the olfactory epithelium was observed in both male and female rats at 80 and 400 mg/m3. After 90 days, degeneration of the olfactory epithelium was present at all DBE concentrations in female rats but only at the mid and high concentrations in male rats. The severity and incidence of the lesions were dose-related for both sexes with female rats being more sensitive than males. Following the recovery period, histological changes compatible with repair in the olfactory mucosa included an absence of degeneration, focal disorganization of the olfactory epithelium, and respiratory metaplasia. Inhalation studies of other esters demonstrate similar pathology in the olfactory epithelium. Since olfactory mucosa is high in carboxylesterase activity, acids may be the toxic metabolites of these compounds. 24 DIFFERENTIAL METABOLISM AND MUTAGENESIS OF 2-ACETYLAMINOFLUORENE BY HUMAN AND RAT HEPATOCYTES, S9 AND MICROSOMES. K Rudo',Z, W Dauterman2, and R Langenbach' 'CGTB, NIER , RTP, Nff_.zToxicology Program, NCSU, Raleigh, NC. Human and rat hepatocytes, liver S9 and microso- mal fractions were compared by measuring the metabolism and mutagenicity of 2-acetylamino- fluorene (AAF). Human tissue mediated mutagene- sis with Salmonella was highest in hepatocytes followed by S9 and microsomes, respectively, with variation existing from individual to indi- vidual. Mutagenesis with rat S9 and hepatocytes were roughly equivalent with rat microsomes teing inactive. Human hepatocyte and microsomal activation was higher than rat hepatocytes and microsomes, with human and rat S9 mutagenesis appearing to be similar. Organic-soluble AAF metabolites produced by hepatocytes, S9 and microsomes were qualitatively similar but exhi- bited quantitative species variation. Ring hydroxylated products, N-hydroxy-AAF and AF were the major metabolites. Human and rat hepatocy- tes produced only trace levels of the mutageni- cally active N-hydroxy-AAF in contrast to the high levels produced by subcellular fractions. Human hepatocyte levels of water soluble metabo- lites were higher than human S9, althouth human S9 exhibited a capacity to conjugate AAF. Human microsomes, rat S9 and microsomes only produced water soluble products at very low levels. This study illustrates the differences between tissue fractions and that species differences can vary with the tissue preparation utilzed. 50875 8131,
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EFFECT OF ZINC ON THE DISTRIBUTION AND TOXICITY OF CADMIUM IN ISOLATED INTE$$TITIAL CELLS OF THE RAT TESTES. T Koizumi and M P Waalkes. Labor- atory of Com'parative Carcinogenesis, National Cancer Institute-FCRF, Frederick, MD It is known that pretreatment with zinc prevents the eventual formation of interstitial cell (IC) tumors by cadmium in the rodent testes. However, the mechanism by which zinc redurel the toxicity of cadmium in these target cells of cadmium car- cinogenesis is not known. Therefore, in this study we assessed the effects of in vivo pre- treatment with zinc on the cytotoxicity and sub- cellular distribution of cadmium in vitro in ICs isolated by collagenase dispersion from Wistar rats. Such zinc pretreatment resulted in marked reduction of cadmium-induced cytotoxicity, as reflected by a reduced loss of cellular K and glutamic-oxaloacetic transaminase (GOT). Although the overall cadmium uptake was modestly increased the subcellular distribution of cadmium showed a marked alteration in zinc-pretreated cells, including a major shift of cadmium away from the nuclei to other components. Cadmium content in isolated nuclei was also decreased by zinc pretreatment. The uptake of cadmium into nuclei isolated from zinc-pretreated cells also was sub- stantially reduced. Thus, it is speculated. that a major preventive effect of zinc-pretreatment against cadmium induced testicular tumor forma- tion is due to the ability of zinc to decrease cadmium uptake into the nuclei, the presumed target site for the genetic toxicity of cadmium. 50 CADMIUM INDUCES TWO SMALL PHOSPHOPROTEINS IN SERTOLI CELL CULTURES. S R Clough, M J Welsh and M J Brabec. Dept. Chemistry, Eastern Michigan University, Ypsilanti, MI The hypothesis that cadmium (Cd) alters protein phosphorylation as a mechanism of toxicity was examined in primary cultures of rat Sertoli cells. Sertoli cell cultures were exposed to sub-lethal concentrations of Cd, and the changes in 32P-labelled phosphoproteins was examined utilizing autoradiography and 2- dimensional gel electrophoresis. The phosphorylation of two acidic proteins of 26 kD molecular weight increased in a time- and dose- dependent manner. The phosphorylation of one protein, GC1, is calcium and germ-cell dependent. The phosphorylation of the other, CdSP, was stimulated specifically by Cd, and not by lead, zinc or mercury. Cycloheximide treatment reduced the increased phosphoryla- tion of both proteins to that of control, while actinomycin D caused only a slight reduction in intensity. The alteration in phosphoproteins occurs at concentrations of Cd similar to that which cause an increase in lactate secretion and germ cell release in Sertoli-germ cell co- cultures. Our tentative conclusion is that the apparent induction of the two phosphoproteins represents a primary biochemical event of Cd toxicity, and may be important in understanding how Cd disrupts testicular function. NIEHS 04141. 13 ., ~-~ ..,,~, 51 CYTOTOXICITY OF SIX METALS TO TESTICULAR CELLS IN VITRO. C D_Brown, Q Li'and M J Brabec. Dept. Chemistry, Eastern Michigan University, Ypsilanti, MI. .t The testes is'a target organ for some toxic metal cations. This study compared the ° relative cytotoxicity of metal cations in ~ primary cultures from •rat ole•hes. Primary -. cultures of testicular cells isolated from.-~ r• rats were exposed to one of six divalent metal cations: Cd, Pb, Zn, Cu, Mg or Ca. r Two cutures systems were used: Sertoli cells from 21 day old rats, and co-cultures of Sertoli-germ cells from 27-30 day old rats. Cytotoxicity was assayed aft .0 a 24 h incubation with the metal cationtby neutral red incorporation, reduction of a tetrazolium dye (MTT) and lactate secretion. Germ cell detachment was assayed in co-cultures. Results from the four assays showed strong correlation between the relative cytotoxicities of the 6 metals. Lactate secretion was the most sensitive (p<0.5 with 1 µM Cd) assay. All four assays ranked the cytotoxicities in the order Cd>Pb>Zn>Cu>Ca,Mg. The assays used are relatively simple, rapid, and use few animals. Quantitative in vitro assays such as these may be useful for initial screening of suspected testicular toxins. NIEHS R01- 04141. 52 THE EFFECTS OF SALIGENIN CYCLIC-o-TOLYL PHOSPHATE (SCOTP) ON PRIMARY aERTOLI CELL CULTURES. R E Chapin, S G Somkuti*, J L Phelps, J J Heindel, D M Lapadula*, M Othman*, and M B Abou-Donia*. Developmental and Reproductive Toxicology, NTP, NIEHS, Research Triangle ParkYand *Department of Pharmacology, Duke University Medical Center, Durham, NC Since in vivo studies identified Sertoli and Leydig cells as targets for tri-o-cresyl phosphate (TOCP) in testis, we investigated the effects of TOCP and its active metabolite, SCOTP, on cultured Sertoli cells. Daily repeat exposure for 5 days to up to 100 u M of TOCP produced no changes in lactate or pyruvate secretion or morphology. But SCOTP produced morphologic effects at 20 u M, and depressed non-specific esterase (NSE) activity at and above 0.1 u M by up to 402. A 24-hr exposure to SCOTP produced equivocal changes in media lactate, pyruvate, ABP, and cellular ATP, protein synthesis, and FSH-stimulated cAMP. However, 5 daily exposures to SCOTP increased media lactate and pyruvate, and further depressed NSE. The NSE decrease did not recover for 5 days after 24 hr. exposure to SCOP. These studies define some effects of a neurotoxic organophosphate metabolite on testicular Sertoli cells~and imply that Sertoli cells lack the metabolic ability to transform TOCP to SCOTP.
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DECREASED TESTICULAR GLUTATHIONE (GSH) LEVELS DO" NOT EXACERBATE THE REPRODUCTIVE TOXICITY OF 1,3- DINITROBENZENE (m-DNB) IN MALERATS. V Slott, R Linder, L Strader, S Perreault. USEPA, HERL, Re- productive Tox.!%r., RTP, NC. Sponsor: R Chadwick - The intracellular thiol GSH is known to protect cells against many toxic chemicals. To investi- gate a protective role for GSH in adult rat tes- tes, GSH was decreased to 30-50% of control lev- els in one testis of each rat by injratesticular injections of a mixture of diethyl maleate. and buthionine sulfoximine prior to oral gavage with the testicular toxicant m-DNB (0, 16 or 24mg/kg). At 14 days posttreatment the reproductive effects on the GSH-depleted and contralateral control sides were compared to determine if reduced lev- els of GSH enhance the effects of m-DNB. GSH de- pletion alone did not affect testis or epididymal weight, sperm motility, testicular spermatid counts, or cauda sperm reserves compared to vehicle-injected controls, but an increase in histologically abnormal tubules localized to the injection site occurred in some GSH-depleted testes. Among the m-DNB-treated rats, 24 mg/kg significantly reduced all of the above parameters while 16 mg/kg reduced all but sperm motility and testicular spermatid counts. Both 16 and 24 mg/kg increased the number of abnormal tubules, however GSH depletion did not enhance any of the effects of either dose of m-DNB. Thus, since depletion of, testicular GSH to 30% of control did not exacerbate the reproductive toxicity of m-DNB, GSH may not protect against this toxicant. 58 TESTICULAR TOXICITY OF THE CHLORONITROBENZENES. K L Mohr and P K Working. CIIT, Research Triangle Park, NC. Many nitroaromatic compounds, including nitrobenzene and 1,3-dinitrobenzene (DNB), are testicular toxicants in rodents. We have assessed the testicular toxicity of the chloronitrobenzenes (CNB) in Fischer 344 rats. Males (n=5 or, 6) received a single oral dose of one-half the LD50 of 2-, 3- or 4-CNB (150, 200 and 250 mg/kg, respectively) and were sacrificed I d or 25 d later. Neither 2- nor 4-CNB had any effect on testicular histopathology (at 1 d) or testes weight and daily sperm production (DSP, at 25 d). In contrast, 1 d after exposure to 3- CNB, signs of early degenerative change were observed in the testes including condensed nuclei and cytoplasmic vacuolation in pachytene spermatocytes of stages VII-IX, loosening and disorganization of spermatid layers and Sertoli cell cytoplasmic vacuolation in stages X-XIV and chromatid margination in steps 1 and 2 spermatids. By 25 d after treatment, testis weights and DSP were both significantly lower in 3-CNB exposed males than in controls (1.9 + 0.2 g and 6.8 + 2.2 million/g testis per d, respectively, vs. 2.6 + 0.1 g and 17.8 + 1.5 million/g testis per d in controls). Thus, 3- CNB but not 2- or 4-CNB is a testicular toxicant in rats under these exposure conditions. Similar isomeric specificity is seen in both CNB and DNB, i.e., only the meta isomer of each is a testicular toxicant after a single dose. 59 INHALATION TOXICITY OF.COBALT SULFATE. J-R Bucher,.National Toxicology Program, Research ~ Triangle Park, NC, and B J Chou, R A Renne, J R Decker, H A Ragan, Battelle Pacific Northwest Laboratories., Richland, WA. Male and female B6C3F1 mice and F344/N rats (ten_ per sex and group) were exposed to 0, 0.3, 1.0, ~ 3.0, 10.0 or 30.0 mg/m3 of aerosolized cobalt ~ sulfate by whole body inhalat.&n-exposure, 5 - J4iays/week for 13 weeks. Two high dose male mir&_. ' died during the exposures and high dose rats arid' mice gained less weight than controls. Increases' = in lung weights (up to 70%) were dose related in both species. Testis weight was decreased by 60% in high dose male mice, where testicular atrophy, mineralization and abr~ rmq-sperm were frequently observed. Sperm mdtility was de- creased in the top 3 dose groups of mice. Hema- totogic changes were prominent in rats and included polycythemia and decreased platelets. Urinalyses showed dose-related cobalt content, and in male rats there was a dose-related increase in the number of sloughed epithelial cells in urine., Histopathologic findings included inflammatory and regenerative changes throughout the respiratory tract with the larynx appearing most severely affected. Polypoid- shaped nodules of connective tissue covered by stratified squamous epithelium were observed at the base of the epiglottis in 28/40 rats exposed to 10 or 30 mg/m3. Squamous metaplasia of respiratory epithelium at the base of the epiglottis occurred at all chamber concen- trations in both species. 60 CHARACTERIZATION OF NICKEL CHLORIDE-RESISTANT BALB/C-3T3 MOUSE FIBROBLAST CELLS. X W Wang, R J Imbra and M Costa, Institute of Environmental Medicine, New York University Medical Center, Tuxedo, NY- Nickel is a toxic and carcinogenic agent that is a contaminant in the environment. We have begun to study the metabolic fate of nickel in mamma- lian cells by selecting mutant cells that con- tinue to grow and divide in the presence of con- centrations of nickel chloride that are toxic to the parent cell line. The phenotype of these resistant cells is being examined by several methods, including cell survival studies, chro- mosome analysis by Giemsa or C-banding, 63NiC12 uptake, and one- and two-dimensional electro- phoresis. Preliminary results indicate that the nickel-resistant phenotype is associated with changes in chromosome structure and number and in protein expression. Specifically, increased nickel resistance appears to correlate with an increased number of centric fusions, increased rearrangement of heterochromatic DNA, decreased chromosome number, and increased accumulation of a 44 Kd (p2 ti7„5) protein. The nickel- resistant phenotype is relatively stable when these cells are grown in the absence of nickel, but the LC50 decreases by one-half after approxi- mately 40 cell generations. These nickel- resistant cells appear to be a useful model for investigating the metabolic response of cells to nickel in the environment. (Supported by Grant •No. R813140 from the U.S. EPA) - - 50875 8140 15
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65 CADMIUM AND POSTMENOPAUSAL BONE LOSS. *M H Bhattacharyya, tP H Stern, and *C Kuhn. *Argonne National Laboratory, Argonne, IL and tNorthwestern University, ChXcago, IL. To determine U cadmium (Cd) exposure causes increased bone loss in ovariectomized mice, we labeled bones of female CF1 mice with '+5Ca. 4 w after labeling, mice were either ovariectomized (OV) to simulate human menopause or sham- operated (SO). 2 w after OV, half the OV and half the SO mice were given dietaaq,Cd at 50 ppm; remaining mice received no Cd. 45Ca excretion in urine and feces (a measure of 45Ca release from bone) increasedy immediately after Cd, and the increase in OV mice (26t4X) was clearly greater than in SO mice (6±3%). To determine if added Cd increases resorption in bones in culture, we exposed 't5Ca-prelabeled fetal rat limb bones (FRLB) to 0.01 or 1 UM Cd. 0.01 pM Cd (estimated level in serum of 50 ppm Cd mice) produced a 68f6% release of '+5Ca from the FRLBs as compared to a 27f2% release in control bones. In contrast, Cd at 1 pM had no significant effect alone and inhibited para- thyroid hormone-induced bone resorption. In smokers, blood Cd is 0.01-0.03 pM, in the range of levels in both our studies; blood Cd in nonsmokers is 10-fold less (0.002 pM). Our results suggest that Cd in cigarette smoke may be one cause of the increased incidence of post- menopausal osteoporosis in women who smoke and that Cd may enhance bone mineral loss by a dir- ect action on bone. Work supported by U.S. DOE under contract No. W-31-109-ENG-38. 66 EFFECT OF CADMIUM ON EPITHELIAL TRANSPORT SYSTEMS IN WINTER FLOUNDER: STUDIES WITH BRUSH BORDER MEMBRANE VESICLES. C Bevan, R K H Kinne, E. Kinne-Saffran, and E C Foulkes, U. Cincinnati, Col. Med., Dept. Environ. Health, Cincinnati, OH; Max-Planck-Institut fur Systemphysiologie, Dortmund, FRG; and MDIBL, Salsbury Cove, ME. Chronic exposure to Cd can result.in impairment of amino acid reabsorption in the proximal tubule of the kidney. Using isolated brush border membrane vesicles from the kidney of the winter flounder, we have studied the effect of Cd on L- alanine transport. Pretreatment of vesicles with 0.1 mM Cd resulted in time-dependent inhibition of L-alanine transport in the presence of a NaCI (but not KCl) gradient. Inhibition was due to a direct interaction with the transport system and not a change in driving force for alanine trans- port, since Cd did not affect Na-D-glucose co- transport. Cd uptake itself is time- and temperature-dependent, and results in consider- able binding to internal sites. Inhibition of alanine transport could not be correlated to binding of Cd at either side of the membrane. Furthermore, inhibition continued to increase even at time points when Cd uptake was at equilibrium. When EDTA was added to Cd-exposed vesicles, inhibition of alanine transport was not reversed. These results suggest that Cd inhibits Na-L-alanine cotransport by binding to the alanine transport system at buried sites in the membrane. (Supported by DFG grant Ki 333/1-1 and NIEHS grant 1 P30 ES03828) 67 EFFECT OF CADMIUM TREATMENT IN VIVO ON GLUCOSE PRODUCTION FROM HEPA'fOCYTES. R R Bell, J L' Early, V K NOnvinakere, and Z Mallory. Florida A&M University, College of Pharmacy, Tallahassee, FL 32307. Sponsor: R C Schnell Cadmium (Cd) is,an environmental contaminant which produces toxic effects following long and short-term exposure. The objective of this stuny g~.Cd on glucose pre- was to determine the effect, n duction from hepatocytes after Cd treatment _,n vivo. Male Spraque Dawley derived rats (180-360g ) received sodium acetate (1.23 mg/kg) or Cd (". 4 mg/kg) as cadmium acetate intraperitoneally at` 30, 60, 90, 120, 150, and 180 min. prior to iso- lation of hepatocytes. Hepatocytes were isolat- ed using a revised method of~err~,and Friend (1966). The average viabili of isolated hepat- ocytes determined by trypan blue Eexclussion test was 80 %. Glucose production from hepatocytes incubated with or without sodium lactate (10 mM) was measured usir;y a glucose analyzer. Cd,pre- treatment 180 min. prior to isolation of hepat- ocytes signficantly increased glucose output from hepatocytes (p < 0.05) incubated with sodium lactate. Results from this study tend to indi- cate that Cd promotes gluconeogenesis at 180 min. post treatment. (Supported by NIH GRR08110/RCMI G12 RR03020-02). 68 THE EFFECT OF SODIUM ON THE ACCUMULATION OF CADMIUM BY SINUSOIDAL HEPATIC PLASMA MEMBRANE V_ESICLES. H B Eastman and J M Frazier. Johns Hopkins University, Baltimore, MD. The effect of Na+ on the transport of Cd2+ across hepatic plasma membranes was investigated using plasma membrane vesicles. Sinusoidal hepatic plasma membrane vesicles were isolated from Male Wistar rats (175-200g) according to the method developed by Bartles et al. (Journal Cell Biolo~y 10:1126-1138, 1985). Accumulation of Cd + by the vesicles was measured using a rapid filtration method. For the uptake experiments, the sinusoidal plasma membrane vesicles were incubated in media containing 0.035uM Cd109 (1.25mCi/ug), 0.25M sucrose, 100mM KC1, 0.5mM MgC12, 5mM Tris-HC1(pH7.4) for 20 min at 37°C. To determine the effect of Na'r" on Cd2+ accumulation by the vesicles, KC1 in the incubation media was replaced by NaCl in a stepwise manner. It was found that as Na"F' replaced K+ in the incubation media, the total accumulation of Cd2+ by the vesicles increased. When K+ was completely replaced by Na+ Cd2+ accumulation increased 2.2 fold. Both nonspecific and specific uptake of Cd2+ by the vesicles were increased in the presence of Na{'. 17
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THE DISTRIBUTION OFLEAD IN Y1LK AND THE FATE OF MILK LEAD IN THE aASTROINTESTINAL TRACT OF SUCKLING RATS. J R Beach and S J Hennina. Biology Department, University of Houston, Housto"ii,' TX. i Milk can be a significant source of Pb for young mammals, including humans. Certain essential trace elements have previously been shown to be associated with particular milk components, thus increasing bioavailability. Our first goal was to determine the distribution on Pb in cream, casein and whey ftactions using 203Pb as a tracer. In rat milk almost 90% of the Pb was associated with the casein micelles, regardless of: a) whether the milk was labeled in vivo or in vitro; b) whether the milk was fresh or frozen; and c) the added concentration of Pb (up to 75 µg/mI). A virtually identical pattern of Pb distribution was observed with bovine milk. Our second goal was to determine whether Pb remained associated with casein as it traversed the gastrointestinal tract of infant rats. For this purpose, rat pups were gavaged with 203Pb-labeled rat milk and sacrificed 2 h later. Differential centrifugation of the homogenized lumenal contents showed that in the stomach the Pb was associated primarily with the casein curd. By the time chyme reached the distal small intestine, Pb was predominantly in a fraction that was not precipitable by high-speed centrifugation (thus, not intact casein micelles), but was nondialyzable. We conclude that Pb in milk is protein bound and remains this way as it traverses the stomach and proximal small intestine of the infant rat. 99 ASSESSMENT OF LEAD EXPOSURE OF TIAN WORKERS IN DIFFERENT LEAD-RELATED OCCUPATIONS. A M Soffar.,•S El-Melegy, A Abd-El-Hakeim, and S A Soliman*. Department of Biochemistry, Faculty of Medicine, Tanta University, Tanta, and *Laboratory of Environmental Chemistry and Toxicology, Faculty of Agriculture, Alexandria University, Alexandria, Egypt. -f Blood lead level and d-4winolevu- linic acid dehydratase activity were measured in 60 subjects•of different occupations. The data obtained were analyzed to evaluate occupation, age and length of occupational exposure. Results demonstrated that blood lead level of lead smelters was 49.68 ±8.56og/~il and that of the battery manufacture Workers was 73.04 ±15.84 ug/dl. The blood lead levels of workers at gasoline stations and the gasoline-mixing company were 27.74 ±4.57 and 17.17 ±3.27 ug/dl, res- pectively. Blood lead level of what was considered to be the control group was 30.51 ±10.49 Ng/dl. Generally, the ob- tained data demonstrated that 8-amino- levulinic acid dehydratase could be used as a measure of lead occupational expo- sure. Data also indicated that increas- ing age and length of occupational expo- sure lead to high blood lead levels.- L-% RAY FLUORESCENCE (XRF): A RAPID ASSESSMENT 100 OF CORTICAL BONE LEAD (Pb) IN Pb-TOXIC CHILDREN. J F Rosen, M E Markowitz, S T Jenks, D N Slatkin, and L Wielopolski. Dept. Ped., Albert Einstein Coll. Med., Montefiore Med. Ctr., Bronx, NY; Med. Dept., Brookhaven National Lab., Upton, NY. The current method to determine the need for chelation in Pb-toxic children (blood Pb: 25-55 ug/dl; erythrocyte protoporphyrin >35 ug/dl) is based on the result of the CaNa2EDTA provocative test (PbP). The PbP requires an injection and a quantitative 8-hour urine collection - difficult to achieve in young children. In this study, a low energy x-ray generator with a silver anode was used to measure Pb La XRF from the tibias of 41 untreated Pb-toxic children. With the leg immobilized, partially polarized photons were directed at the anteromedial skin surface of the °mid-tibia. Based on spectra from a leg phantom and from seven surgically amputated adult legs, the nominal detection limit was N 2 ug Pb/gm of bone, when the skin sµrface dose reached 1.0 rad. Sixteen minute XRF measurements were compared with PbP results performed one week later. When the XRF was <2 ug Pb/gm of bone, 21of 25 Pb-toxic children had a negative PbP. When the nominal XRF measurement was >2 ug Pb/gm of bone, this was predictive of the need for chelation in 14 of the 16 %RF-positive Pb-toxic children. These results indicate that L-XRF measurements of cortical bone Pb will be useful to rapidly discover Pb toxicity in large populations of children. 25 RISK ASSESSMENT FOR -LEAD IN FIRST-DRAW RATER. M J Miller and A J Grey. Bureau of Toxic Su s tance Assessment, New York State Department of Health, Albany, NY. Elevated concentrations of lead (Pb) in drinking water are associated with the use of Pb pipes and Pb solder for interior plumbing and with soft water supplies. The greatest exposure to Pb in drinking water occurs during consumption of "first -draw" tap water that has been in contact with Pb plumbing for more than 6 hr. The health consequ- ence of chronic exposure to Pb in first-draw water, as reflected by increased blood Pb levels (PbB), is estimated for high-risk populations (i.e., children and pregnant women). Assuming background exposure to Pb from air (1 ug/m3), food (19-25 ug/day), water (10 ug/1) and dust (1000 ppm), estimates of background PbB levels in children and adult females living in urban areas are 13 and 8.5 ug/dl, respectively. Consumption of two 4 oz. glasses of first-draw water per day at concentrations of 50, 100, 200 and 500 ug/1 is estimated to increase background PbB levels"in children by 10, 25, 50 and 140%, corresponding to PbB levels of 14, 16, 20 and 31 ug/dl, respect- ively. Similar consumption of two 8 oz. glasses by adult females is estimated to increase PbB levels by 5, 10, 20 and 50%, corresponding to PbB levels of 9, 10, 11 and 16 ug/dl, respectively. The current US EPA (1985) PbB threshold level of concern for Pb exposure by high-risk populations is 15 ug/dl. Thus, exposure to Pb in first-draw water may significantly elevate PbB levels. .....j
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45 PSiEPARATION OF HAFYEN-CONJIiGATE A_NPIGIISS 47 ELICITATION OF IMMUNE RESPONSES IN GUINEA PIGS EFFECTIVE IN DEZI:CPION OF A_NTIDODIES IN FERSONS EXPOFED TO DIPHENYL,MEPHANR -4,41-DIISOC.'YANATE (hIDI). R Jin and M H Karol Jilin Province, FOLLOWING INHALATION-EXPOSURES TO RAT URINARY ` PROTEINS. J C Stadler. E I duPont de Nemours and Co, Haskell Laboratory for Toxicology and Indus- People's Republic of China and Dept. Ind. Env. trial Medicine, Newark, DE. Sponsor: L S Mullin. Hlth. Sci., ihkiv. Pittsburgh, Pittsburgh, PA. - . Considerable controversy exists regarding the Animal handlers frequently develop dermal and respiratory symptoms of allergy on exposure to-- pathogenesis of isocyanate induced respiratory disease. Much of the difficulty resides in lack of a,dequate attention to preparation and characterization of hapten-protein conjugates employed in diagnostic assays. (•Se prepared a series of MDI-human serum albtmin (MDI-HSA) conjugates by varying the starting proportions of hID7 and protein, and the reaction time. The reaction products were isolated and analyzed using ultraviolet absorbance, migration in SDS gradient gels and reactivity with trinitroben- zene sulfonic acid. Extensive intra- and intermolecular cross linking of protein was observed. Using Western blot, antigen frac- tions were compared for ability to react with antibodies in sera from patients with MDI respiratory disease. The most effective antigens were those extensively cross-linked and having high haptenic substitution. Less substituted antigens often failed completely to identify the presence of antibody. These results emphasize the importance of standar- dized procedures for preparing antigens to detect isocyanate-specific antibodies and the need for careful characterization of the antigens. Supported by NIEHS ES01532. laboratory animals. Urine, pelt extracts, and ;. serum have been identified as allergen sources,= but little is known about t1ft`etiology of the e_ ' f allergies. A guinea pig model of pulmonary `s i.- tivity was used to.study sensitization from in!''-' halation exposure to rat urinary protei.ns. Total"' protein fraction was extracted from the urine of male rats to use for immunization and challenge. Guinea pigs were exposed to 18~ m,g/m,3 or 3 mg/m3 protein aerosols 10 min/day, days/week or to 30 mg/m3 or 5 mg/m3 10 minutes ea6h on day 1 and day 15. Respiratory rates were monitored continu- ously before, during, and one hour following ex- posures. Some reactions were noted on day 11, but the majority of positive sensitivity responses among repeatedly exposed animals were first seen on day 15. Guinea pigs exposed to 18 mg/m3 had more responders, longer duration of response, and more severe reactions (gasping or anaphylaxis) than those exposed to 3 mg/m3. Single exposures to 30 mg/m3 elicited positive responses at day 15 challenges, but single exposure to 5 mg/m3 did not. Guinea pigs with pulmonary responses also displayed dermal sensitivity and antibodies. This animal model has potential for use in further study of laboratory animal allergies. 46 ELICITATION OF A CELL MEDIATED IMMUNE RESPONSE TO A REACTIVE INTERMEDIATE' OF HALOTHANE. AK Hubbard, TP Roth and AJ Gandolfi. Dept Anesthe- siology, University o Ari1~z-ona Tucson, AZ Halothane exposures of rabbits and humans elicit a humoral immune (antibody) response with specificity for the reactive intermediate of halothane, trifluoroacetyl halide moiety (TFA), conjugated to a carrier protein, rabbit serum albumin (RSA). Since T cells could also potentially mediate liver damage, experiments were conducted to determine if a cell mediated immune response for TFA was elicited in halothane exposed rabbits. Male New Zealand white rabbits were exposed 4 times to 1% halothane (80% 0) at 2 wk intervals. Splenic leukocytes were ?solated and- cultured for 723hr in the presence of TFA-RSA, RSA, or media. H- thymidine was used to measure the 24 hr pro- liferation of T.lymphocy3tes. Data are expressed as stimulation indices ( H-thymidine cpm antigen /media). TFA-RSA (5,1,0.1 mg/ml) stimulated leukocytes from halothane exposed rabbits (3.5,3.7,2.2, respectively) and only mildly stimulated leukocytes from• unexposed. rabbits. RSA (0.1-5 mg/ml) did not stimulate either source of leukocytes. These studies suggest that multiple halothane exposures induce a cell mediated immune response with reactivity toward a reactive intermediate of halothane (GM 34788). 48 ACTIVE REGULATION OF THE AFFERENT PHASE OF CONTACT ALLERGY FOLLOWING CONVENTIONAL SENSITISATION. "Ian Kimber. Julie Mitchell and Avril Kinnaird, Immunology Group,.Central Toxicology Laboratory, ICI Plc, Alderley Park, Cheshire, UK Sponsor: I F H Purchase Epicutaneous exposure of mice to 4- ethoxymethylene-2-phenyloxazol-5-one(oxazolone) results in both the induction of specific contact sensitisation and the systemic depression of subsequent proliferative responses, which at least initially, is antigen-non-specific in nature. Inhibition of T lymphocyte proliferation can be transferred to naive syngeneic animals with draining auricular lymph node cells (LNC) isolated from oxazolone exposed mice. We have demonstrated that the efficiency of contact sensitisation to the antigenically- , non-cross-reactive chemical picryl chloride is significantly impaired in mice previously exposed to oxazolone at a distant site. In addition the adoptive transfer of draining LNC from ctazolone-treated mice which non- specifically inhibit proliferative responses similarly depresses contact sensitisation to picryl chloride. These data suggest the existence of an important lymphocyte-mediated regulatory mecha-:ism which serves to control cutaneous immune responses following topical exposure to chemicals. 50875 8137 12
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50875 8123
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41 ASSESSMENT OF EFFECTS OF DIETARY EXPOSURE TO TOXIC COMPOUNDS ON LOCAL IMMUNITY IN THE RAT. J G Vos, and H Van Loveren. National Institute of Public Health and Enviionmental Hygiene, Bilthoven, The-*Netherlands. _ In toxicology the most commonly used experimental animal is the rat. Hence, also in the overlapping field with immunology i.e. immunotoxicology, rats are often chosen. We are interested in the study of effects of potential immunotoxicants on local immtmity in the intestines, since the alimentary traut is one of the major routes of exposure to toxicants. We have developed a mouse monoclonal ant body, that specifically recognizes rat IgA. This reagent proved valuable in an enzyme-linked immttnosorbent assay (ELISA) for the titration of specific IgA in the serum of rats that were imminized via the Peyer's patches with ovalbumin, or that were orally infected with muscle larvae of the parasite Trichinella spiralis. Using this assay we have determined the effect of dietary exposure during 6 weeks to upto 80 mg bis(tri-n- butyltin)oxide (TBTO) per kg food on IgA responses. Whereas no significant effect on IgA responses to ovalbumin was observed, exposure to TBTO enhanced in a dose-related fashion the IgA response to T.spiralis. As a possible explanation may serve the fact that TBTO, know to suppress immiine responses, inhibits expulsion of T.--+iralis worms from the gut, thus prolonging the stimulus to produce IgA. Thus in this system imn,-.iosuppression by TBTO can paradoxically lead to mmune stimulation. Another explanation may be thc TBTO corrupts immune regulatory mechanisms. 42 EVALUATIOtI OF SU2FACTAPIT TA FOR SYST ~.'*iIC ANAPHYL&CIS IN GUINr;A PIGS. C L Yang, S Tetceli, and ll x Patterson. Aboott Laboratories, Abbott Park, IL. SurYactant TA (STA), a pulmonary surf actant extracted Yrom bovine lung, is intended for use ].n hyahne memoranedisease which occurs in premature infants. Intratracheal (IT) auministration is an intended clinical route. The potential of STA to produce anapnylaxis was assessed in male Hartley guinea pigs weighing 3MU-45U g. Animals received three IP injections of s1A in saline at a dose of 20 mg/animal/injection for inductions. Tnree injections were given on alternate days. Egg albumin at doses of 0.2 and U.5 mg/animal/injection was used as a positive control. Three weeks after the last 1P injection, animals were challenged with respective test material by an IT instill- ation. Doses or U.5 and 1.5 mg/animal were used for egg albumin. A dose of SU mg/animal was used tor STA. None of the' animals that received STA or saline showed signs of anaphylaxis or death. Convulsions followed by death occurred shortly after the IT challenge in animals receiving egg albumin. Gross and histologic examinations of the lungs from the animals that died showed various degrees of pulmonary changes which were compatible with the anaptrylatic responses of guinea pigs. 11 43 ON THE POPLITEAL LYMPH NODE (PLN) ASSAY FOR THE DETECTION OF AUTOIMMUNOGENS IN NICE. X Joseph, ~ J P Uetrecht,-and T Balazs. FDA, Nashington, DC and Univ. of Toronto, Toronto, Ontario, Canada. The PLN reaction, a locally induced graft- versus-host reaction, has been used recently as a predictive test system for chemically induced.r immune disregulation (Kammuller et al., Abstr. ;y 6th Int. Congr. Immunol., 64$~~ 1986). We _ ~d examined the reliability of ~fiis assay for identifying the autoimmunity-inducing potentia9r of various chemicals, viz., procainamide (PRO)=---1~-:: and its metabolites [nitro (N) and hydroxyl- ~ amine (HY) derivatives], hydralazine, methyl- dopa, and propranolol in inbred mice. Phenytoin (PT) was used as a positive cWtroj.. Groups (8 each) of 6- to 8-week-old maW CB6F1/J mice were given a single sc injection 6f 0.05 ml (2.5 mg) of each drug or its vehicle to one of the hind footpads. Certain irritants (1% acetic acid or 50% ethanol) were also given to other groups of mice. PLNs were removed 7 days after injection and PLN indices (PI) were calculated as the ratio of lymph node weights of the injected over the untreated side. The N derivative of PRO (PI 4-5) and PT (PI 5-10) gave strong positive reactions, the HY derivative was intermediate (PI 1.5-3), and.all other drugs were negative. However, acetic acid and ethanol were positive (PI 4-8), indicating that the PLN reaction is not specific for autoimmunogens. 44 LOCALIZATION OF HALOTHANE-INDUCED ANTIGEN IN SITU BY SPECIFIC ANTI HALOTHANE METABOLITE ANTIBODIES. TP Roth, AK Hubbard, AJ Gandolfi, Department of Anesthesiology, University of Arizona, Tucson, AZ Multiple halothane exposures of rabbits induce an antibody cross reactive with trifluoro- acetylated (TFA) proteins. The anatomical location of the halothane-induced antigen that induces this immune response was investigated in livers from halothane exposed rabbits. Rabbits, immunized with TFA-rabbit serum albumin were exposed multiple times to halothane anesthesia. Antibodies from these animals will detect TFA-conjugated proteins induced by halothane exposure. Using an immuno-staining technique, binding by the antibody, to the antigen, was detected in liver tissue from all 6 halothanee exposed rabbits. Antigen could be detected only in the centrilobular area around the central vein where staining intensity was concentrated in an area 7-9 cells deep. Approximately 60-75% of the central veins within each tissue section were positive for antigen detection and staining around one vein became contiguous with staining around another central vein. No necrosis, however, was observed in this animal model. These data suggest that halothane exposure by inhalation generates an antigen in the centrilobular area of the liver. which is antigenically similar to the reactive intermediate, TFA halide. (NIH GM 34788) 50875 8136
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37 IN VITRO BIOACTIVATION OF 2,4-DIAMII9DIOLUhNE BY 39 ARQCHIAR-I254-iDIDLICED RAT LIVER S9. M L Cunningham, L T Burka, and-Ii B Matthews, NIEHS, Research Triangle Park, NC. -_ 2,4-Diaminotolu®ge (2,4-DAT) is used as an in- termediate in the synthesis of various dyes and for the production of toluene diisocyanate8for the manufacture of polyurethane. 1.6 x 10 pounds of 2,4-DAT, a potent bacterial mutagen and rodent carcinogen were produced in 1982. In order to characterize the mutagenic (and by inference, carcinogenic) metabolite(s)~ of 2,4- DAT, an Arochlor 1254-induced rat liver S9 mix was used as an activation system, and the metabolite(s) separated and purified by HPLC and characterized by mass spectroscopy. 2,4-DAT metabolism exhibited Michaelis-Menton kinetics with a K and V of 5.29mM and 18.1 Nmoles/mg protein,~min., re'spectively. 2,4-DAT was rapidly and quantitatively metabolized to one major metabolite in this S9 activation system. It had a molecular weight of 256 daltons and mass fragmentation pattern consistent with a structure of dimethyl, diamino, dihydrohydroxy- phenazine ( CH3 NHZ C6 HZ NOHNHC6 H NHz CH, ). This metabolite was not produced when 2,4-DAT was incubated with boiled S9. The major metabolites of 2,4-DAT found after in vivo exposure, N-acetyl conjugates or cenzoic acid derivi- tives, were not found in this in vitro S9 activation system. The noncarcinogem.c isomer, 2,6-DAT, did not appear to form a similiar dimeric metabolite in this activation system. This dimeric metabolite of 2,4-DAT is planer and may be a mutagenic intercalating agent. 38 -QONTACT,EYPERSENSITIVITY RESPONSE TO GLUTARAL- DEHYDE IN GUINEA PIGS AND MICE. M L Stern, J A McCay, R D Brown and M P Holsao_Dle. Dept. of Pharmacology and Toxicology. Medical College of Virginia/VCU, Richmond, VA. Glutaraldehyde (GL) has a wide spectrum of uses which can result in dermal contact with the agent. The low number of reports of hypersensitive reactions to GL indicates a low incidence of sensitization. Female albino Hartley strain guinea pigs and female B6C3F1 mice were sensitized with 0.3, 1.0 and 3.0% GL and challenged with 10% GL. Doses of GL were selected by assays for primary irritancy. Guinea pigs received 100 µl by direct dermal application for 14 consecutive days, and mice received 20 µl by direct dermal application for 5 or 14 consecutive days, to sites prepared by shaving and dermabrading. Rest periods were 7 or 14 days for guinea pigs and 4 or 7 days for mice. Measurement of the contact hypersensitivity response in guinea pigs was by both visual evaluation (scoring) at 24 and 48 hours following challenge and radioisotopic assay at 48 hours, and in mice by radioisotopic assay 48 hours after chal- lenge. Both guinea pigs and mice demonstrated dose dependent contact hypersensitivity responses to GL The radioisotopic assay appeared to be more sensitive than visual evaluation in detecting contact allergic hypersenstivity to GL. (Supported by NIH contract ES 55094). SiTRUCTURE-ACTIVITY STUDIES OF CYCLIC' &NHYDRIDES WHICH CAUSE PULMONARY SENSITIZATION. C L Leach, N S Hatoum, C R Zeiss, and P J Garvin. IIT Research Institute, Veterans Admin., and Amoco Corporation, Chicago, IL. Trimellitic anhydride (TMA) is known to cause.: pulmonary sensitization and lung pathology; however, s there are many structurally similar cyclic anhydrides and = carboxylic acid precursors wR*li'' have not beeL~ - ~{investigated for their potential to cause sensitization: ' Inhalation studies were coniiucted on (I) trimellitic acid~: (TMAC), a precursor to TMA, and (II) pyromellitic dian- " hydride (PMDA), a chemical used similarly to TMA. (I) Rats were exposed for 2 weeks to 300 ug/m3 of TMAC, rested 2 weeks, and challenged with TIvtAC prior to termination. There were no lungAesions or TMAC antibody at any time point. In vitro studies showed that the TMAC reactivity to protein, a necessary prerequisite for immunogenicity, was minimal compared to TMA. (II) Rats exposed once to 2.2 mg/1 of PMDA, rested 2 weeks, and challenged with 500 ug/m3 showed hemorrhagic lung foci, and increased lung weights, lun§ volumes, and IgG antibody. Rats exposed to 500 ug/m of PMDA for 5 days, rested 2 weeks, and challenged also showed lung foci, and increased lung weights and IgG antibody. The effects of PMDA inhalation were similar to those caused by TMA, however, there were no effects caused by TMAC. hi vitro studies of antibody reactivity to TMA and PMDA coupled to albumin showed substantial cross-reactivity between the two anhydrides. Thus, there appeared to be comparable activity between TMA and PMDA suggesting that other similar anhydrides may also be sensitizers. 40 A TWO WEEK INTRAVENOUS SAFETY STUDY OF T-CELL MODULA TORY PEPTIDE IN RATS. J E Atkinson, Bio/dynamics, Inc, E Millstone, NJ, J L McCoy, ImmuQuest Laboratories, Inc, Rockville, MD, and S P Richieri, G S Hahn and J M Plummer, Immunetech Pharmaceuticals, San Diego, CA. The safety and immunomodulatory activity of a synthetic T-Cell Modulatory Peptide (TCMP-80) derived from the human IgG Fc region was assessed in CD rats (12/sex/group) given daily i.v. injections of vehicle or 1, 2, 10 or 100 mg/kg TCMP-80 for 14 days. Routine safety evaluations (body weight, clinical chemistry, hematology, gross postmortem exam- ination) revealed no toxic effects. Spleno- cytes from 8 rats per group were collected for lymphocyte subset and lectin mitogenesis analyses. Lymphocyte subsets were not affected in females but T helper cells in males tended to decrease at all dose + levels after 14 days. The percentage of Ia cells increased independent of dose. PHA proliferation was inhibited at the 1 and 2 mg/kg doses, while 100 mg/kg enhanced PHA proliferation by 133% in males and by 193% in females. B lymphocyte proliferation (LPS) was enhanced (178-635%) in both sexes at the high dose of TCMP-80. The 1 and 2 mg/kg doses were inhibitory and stimulatory for B cell mitogen proliferation (PWM, LPS). Thus, TCMP-80 did not produce any short term toxicity but did demonstrate strong dose dependent T and B lymphocyte modulation. 10 50875 8135
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53 MONO-2-ETHYLHEXYL PHTHALATE (MEHP) STIMULATES PROTEIN AND RNA SYNTHESIS IN RAT SERTOLI CELL CULTURES. A C Savage, D M Creasy, and T J B Gray. BIBRA, Carshalton, Surrey, England. Phthalate esters which cause testicular toxicity act primarily on the Sertoli cells. To - investigate early biochemical effects of these toxicants;-protein and RNA synthesis was evaluated in primary rat Sertoli cell-enriched cultures (appox. 85% Sertoli cells), using a 60 min pulse with 3H--leucine and 3H-uridine respectively. In cultures treated for 24 hr with MEHP, 3H-leucine incorporaxion into Sertoli cell proteins was significantly increased by 15% at 1 pM, up to 50% at 200 pM. At 200 yM this effect was first evident after a 3 hr treatment and was maximal after 18-24 hr. An increase in RNA synthesis was evident after 1 hr, maximal after 3 hr (125% control) and absent by 6 hr. Blocking RNA synthesis with actinomycin D abolished the stimulation of protein synthesis by MEHP. Exposure to MEHP for only 30 min produced a significant stimulation of 3H-leucine incorporation assessed 24 hr later. None of the above effects were observed with phthalate monoesters which do not affect the testis in vivo. Thus MEHP, at concentrations relevant to tissue levels in vivo, rapidly initiates increased transcription and translation in cultured Sertoli cells. The signals involved and their role in the toxicity of MEHP remain to be established. (Supported by the UK Ministry of Agriculture, Fisheries and Food) 54 STAGE-SPECIFIC UNSCHEDULED DNA SYNTHESIS (UDS) IN RAT SPERMATOGENIC CELLS K S Bentley and P K Working, Chemical Industry Institute of Toxicology, Research Triangle Park, NC DNA repair in spermatogenic cells at various stages of maturity was determined by UDS. F-344 rats were exposed to 35 mg/kg (i.p.) methyl methanesulfonate (MMS) or methyl nitrosourea (MNU). One hour later segments of seminiferous tubules were cut corresponding to stages II, IV, VI, VII, VIII, X, XII, and XIV of spermatogenesis using the unique transillumination pattern of the tubules as a guide. Intact tubule s5gments were cultured 24 hours in the presence of H-thymidine and UDS was measured by autoradiography as net grains per nucleus (NG). In MNS-exposed animals NG increased as primary spermatocytes (SC) matured from leptotene SC (3.5 NG) up to stage VIII and X pachytene SC (22 NG), after which NG in stage XII pachytene SC and in diplotene SC decreased (16 NG and 8 NG, respectively). A similar stage-specific UDS pattern was observed in primary SC isolated from MNU-exposed animals. Round spermatids of steps 2 to 8 all exhibited approximately the same UDS response (8 NG).- Spermatids as mature as step 14 exhibited UDS responses after exposure to MMS or MNU in vivo, but step 15 and later step spermatids apparently lacked the ability to repair DNA. Whether the variation in UDS response observed in primary SC was due to a difference in DNA repair capabili*_y or in the degree of DNA alkylation cannot oe determined from these data. 14 55 { DI-N-PENTYL PHTHALATB°'~DPP) INDUCED INFERTILITY: CORRELATION WITH SERUM ANDROGEN BINDING PROTEIN (sABP). P Lindstrom, M Harris, M Ross, J C Lamb, R C~ha pi_n_. DART, STB, NIEHS/NTP, Researc T Triangle Park, NC. We showed that changes in sABP correlate with changes in morphological and biochemical repro- ductive endpoints in DPP-treated F344 rats:- sABP may be a good marker in the rat for geami- nal epithelial damage. However, it should &1so indicate changes in tot*&°reproductive f2V- ' tion. One objective was to study effects o~ DPP on fertility, •then to correlate the resu3:t's with changes in sABP. Histopathology from tfi€ earlier study suggested a very slow recovery; the second objective was to evaluate recovery up to 30 weeks postexposure. Eighty male rats were dosed once via gavage*vith:'.DPP in corn oil (0, 0.25, 1.0, and 2.0 g/k'g BW) then mated to " untreated females at 3, 6, and 10 weeks postex- posure. Controls and high-dose rats were killed at 14, 18, and 30 weeks after dosing, and the germinal epithelium was evaluated his- tologically. Two g/kg DPP caused reduction in pregnancies and live pups and increase in pre- implantation loss. One g/kg DPP did not change these endpoints. sABP had more than doubled during the first 2 weeks postexposure to 2 g/kg DPP, while after 1 g/kg DPP sABP showed 50% in- crease during the first week postexposure. No high-dose animal showed signs of recovery. In summary, 115% initial increase in sABP corre- lated with subsequent infertility, while 48% increase did not. The epithelium did not recover within 30 weeks. 56 CIRCADIAN FLUCTUATION OF GLUTATHIONE (GSH) LEVELS TN THE REPRODUCTIVE TRACr OF THE MALE RAT. H K Bates, R D Harbison, J Gandy, Pathology Associ- ates, Inc. NC R, Jefferson, AR and University of Arkansas for Medical Sciences, Little Rock, AR. Studies in our laboratory have shown that chemical depletion of reproductive tract GSH increases susceptibility to germ cell mutations. Hepatic glutathione in the rat has been demon- strated to fluctuate on a circadian cycle linked to food intake. Extrahepatic pools of GSH have not been adequately investigated. Fluctuations in GSH levels were examined in the male reproductive tract as a possible mechanism for varying indi- vidual susceptibility to reproductive toxicants. Male rats 12 weeks old were maintained on a 12:12 hour light:dark cycle starting at 6:00am with ad libitum access to food. Total GSH was measured every four hours in the liver, kidney, testis, caput and cauda epididymis, and the seminal vesicles. Liver, testis, and caput epididymis exhibited significant (p > 0.05) circadian dif- ferences in GSH content with maximal levels of 2490 + 156 (SE), 935 + 57, and 1604 + 85 ug/g wet tissue at 1400, 060-0, and 0600 hours,respec- tively. No significant alterations in GSH content occurred in the remaining tissues. Fluctuation of the high GSH content in the male rat reproductive tract indicates a potential for variable suscep- tibility to chemically induced toxicity. (Sup- ported in part by PHS grant 0H02258 and the National Foundation of the March of Dimes.) 50875 8139
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13 HALOTHANE-INDUCED INHIBITION OF SUPEROXIDE RADICAL PRODUCTION AND MOBILIZATION OF INTRACELLULAR C1ALCIUM. J E Ryer-Powder, H J Forman, and R J Dorio. Chi"ldrens Hospital of Los Angeles anj University of Southern California, Los Angeles,°Ca. Halothane is a routinely used general anesthetic. .The incidence of pneumonia, a common post- surgical complication, is directly related to time under anesthesia. We examined the hypothesis that halothane interferes with antibacterial defenses, such as superoxide (02T) production by alveolar macrophages (AM). 02: production can be stimulated by agonists, which either mobilize Ca2+ or activate protein kinase C, such as phorbol esters. In vitro exposure of rat AM to 1 mM halothane induced a 50Z inhibition of phorbol ester-stimulated 02T production as measured by cytochrome c reduction. Halothane also caused a 50 nM increase in intracellular calcium as measured by fura-2 fluorescence. AM were labeled with myo-[3H] inositol. Labelled products were separated by Dowex 1-%8 anion exchange chromatography. Halothane (10 mM) induced a 40% increase in the level of inositol trisphosphate 60 seconds after halothane addition. Inhibition of phorbol ester stimulated 02T production has been found with other agents, which elevate intracellular Ca2+, such as A23187. Thus, the halothane induced inhibition of phorbol ester stimulated 0~= production may be related to its effect on Ca + mobilization. 14 QUALITATIVE CHANGES IN CYTOCHROME P-450-LINKED MONOOXYGENASE ACTIVITY IN LUNG MICROSOMES AND ISOLATED CLARA CELLS DERIVED FROM 0_ZONE-EXPOSED RATS. L van Bree, I M C M Rietjens, J A M A Dormans and P J A Rombout. National Institute for Public Health and Environmental Hygiene, Bilthoven, The Netherlands. Sponsor: R Kroes. This study was designed to determine the effect of prolonged ozone exposure (1.6 mg/ms; 7 days; 24 h/day) on cytochrome P-450 (P-450) dependent xenobiotic metabolism in whole rat lung micro- somes as well as in isolated bronchiolar Clara cell preparations. It was demonstrated that the increases in components of the pulmonary micro- somal P-450 electron transport system were not accompanied by a concomitant increase in speci- fic model substrate conversions. 0-dealkylations of ethoxycoumarin and ethoxyresorufin were de- creased, whereas, in contrast, the 0-dealkyla- tion of pentoxyresorufin was increased. Clara cell populations isolated from ozone-exposed rats showed a comparable qualitative shift in P-450-linked monooxygenase activities. In vitro experiments revealed that ozone is able to inac- tivate the various 0-dealkylation activities but the results could not expl,ain the in vivo data. Lung morphometrics and Clara cell isolation data supported the view that the in vivo effects of ozone should rather be ascribed to proliferation and/or renewal of P-450 containing cell popula- tions and to intrinsic cellular biochemical changes. 4 15 EFFECT OF OZONE EXPOSURE ON DEFENSE TO RESPIRATORY INFECTION IN THE RAT. H Van Loveren, Sj :c Wagenaar, P J A Rombout, and J G Vos. National Institute of Public Health and Environmental Hygiene, Bilthoven, The Netherlands. We have investigated the effect of ozone expqsure on defense to a respiratory infection 'SWith l,is+eria monocytogenes the rat. We "iaill { prerent data that indicate that cout3Auous expc-sure of rats to ozone during 7 days iWirs phaf;ocytic and lytic activity of alvear mac:•ophages. Moreover, also development ' of cellular immune responses to Listeria is supiressed. As a consequence control of the res-iratory Listeria infection ~n exposed rats is inac'equate. Pathological Aesions induced by pul:ionary Listeria infections are characterized by multifocal inflammation of the lung par•:nchyma. These foci comprise infiltration of his'iocytic and lymphoid cells. The cellularity of interstitial tissue is increased, and many al-.•..olar macrophages are observed. Since ozone inf:.uences both these types of inflammatory cells, we have investigated the influence of ozone on the pathological lesions associated with pulmonary infection with Listeria. After ozone expcsure the severity of the lesions is more prcitounced, and the duration prolonged. Moreover, the tendency of pulmonary Listeria infection to indnce granulomatous alterations is enhanced in ozoue exposed rats; in exposed and infected rats sev.:re granulomas can be observed. Consequently ozoi,e exposure adds to the loss of lung functions aftcr respiratory infection. 16 RAPID INCAPACITATING AND LETHAL EFFECT OF HCL IN Gt1INEA PIGS DURING E}D1I3CISE. D E Malek, M F Stock and Y Alarie. Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA. The guinea pig ergometer (Toxicol. 7, 123, 1987 permits continuous measurement of tidal volume, respiratory frequency, 02 uptake (V02) and Ws output and is featured with a clear incapecita- tion endpoint (collapse) was used for this study. Guinea pigs were exercised on the ergometer at 25-30% of their V02 max according t.o a defined 55 min protocol. Four groups of exercising guinea pigs were exposed to 107, 142, 162 or 586 ppm HC1. All animals collapsed at the- 3 higher concentrations after 20, 2 and 1 min of exposure respectively. At 586 pp® they died within 4 min. Collapsed animals were coughing, gasping for air and the dark color of their eyes and skin of the ears and paws indicated asphyxiation. No collapse or death occurred in sedentary animals exposed to 1,300 ppm. In humans exposed up to 100 ppm HC1 Matt (Dissertation, University of Wurzburg 1889) concluded that work would be impossible in the range of 50-100 ppn. Thus the guinea pig is less sensitive than man to HC1. These experi- ments were conducted to investigate escape capabilities in fires where HC1 is released from polyvinylchloride. The direct action of HC1 on the respiratory tract was greatly enhanced by an increase in minute ventilation. Supported by NBS 60NANB4D001. 50875 8129
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29 GLUTATHIONE (GSH) DEPLETION AND INHIBITION OF HEPATIC MIXED FUNCTION OXIDASE (_MFO) BY A SERIES OF ALPHA,BETA-UNSATURATED ALDEHYDES (ABUA). K 0 Cooper, G Witz and C Witmer. The Joint Graduate Program ifi Toxicology, Rutgers University/UMDNJ, Piscataway, NJ. ' . ABUA are putative mediators of hepatotoxicity and inhibitors of hepatic MFO activity despite high concentrations of GSH(10 mM) in the hepatic cytosol. Male rats were given a single dose of muconaldehyde (MUC)(36umole/kg), acro- lein (ACR) (89umole/kg), croton'aldehyde (CR0) (450umole/kg) or the saturated aldehyde, pro- pionaldehyde(PRO) (89umole/kg), and sacrificed 30 min, 4 hr or 24 hr later. Hepatic cytochrome P-450 concentration (P-450), NADPH-cytochrome c reductase activity (CCR), ethylmorphine N-de- methylation (EMD) and glucose-6-phosphatase activity (G-6-Pase) were unchanged compared to controls at 30 min and 4 hr after dosing with any aldehydes. At 24 hr the ABUAs, ACR, MUC and CR0 decreased P-450 (60-70% of control) EMD (30-60% of control) and G-6-Pase (75-85% of control) while CCR was unchanged. In livers from animals treated with ACR or MUC and sac- rificed 4 hr later GSH was decreased to 51 and 75% of control, respectively, and had returned to control levels at 24 hr. PRO had no effect on MFO activity or GSH concentration. The number of SH groups in.microsomes from animals treated with any aldehyde remained unchanged compared to controls. Thus, cytosolic GSH does not protect the MFO from the action of ABUA. 30 I-NDUCTION OF CYTOCHROME P-450 BY PHENOBARBITAL AND 3-METHYLCHOLANTHRENE: DIFFERENCES BETWEEN. RATS AND HAMSTERS. D R Dutton, S K McMillen and A Parkinson . Kansas University Medical Center, Kansas City, KS. Previous studies have shown that treatment of rats with phenobarbital (PB) causes a marked induction (>30 fold) of P-450b, which is associated with a comparable increase in the rate of liver microsomal testosterone 16,8-hydroxylation and . pentoxyresorufin O-dealkylation. In the present study, , we show , that treatment of hamsters with PB caused a marked incr- ease (>25 fold) in the intensity of a liver micro-l somal protein immunochemically related to P-450b (as determined by Western blot). However, treatment of hamsters with PB caused less than a 5-fold increase in testosterone 16,8-hydroxylase and pentoxyresorufin O-dealkylase activity. Similarly, previous studies have shown that treatment of rats with 3-methylcholanthr- ene (3-MC) causes a marked induction (>30 fold) of P-450c, which is associated with a comparable incr- ease in the rate of liver microsomal benzo[a]pyrene 3- hydroxylation and ethoxyresorufin O-dealkylation: Treatment of hamsters with 3-MC caused a marked increase (>25 fold) in the intensity of a liver micro- somal protein immunochemically related to P-450c, and caused a comparable increase in ethoxyresorufin 0- dealkylase activity. However, treatment of hamsters with 3-MC caused a slight decrease in benzo[ajpyrene 3-hydroxylase activity. These results suggest that rat P-450b and P-450c differ catalytically from the corre- sponding hamster proteins. Supported by NIH grants ES-03765, ES-00166 and ES-07079. 8 31 .f IDENTIFICATION OF A T_CDD-INDUCIBLE HAMSTER LIVER MICROSOMAL PROTEIN IMMUNOCHEMICALLY RELATED TO RAT _Q'YTOCHROME P-450a BUT WITH- OUT _TESTOSTERONE 7oc-HYDROXYLASE ACTIVITY. M P Arlotto, S K McMillen and A Parkinson. Kansas University Medical Center, Kansas City, KS. Polyclonal antibodies against purified rat " liver microsomal cytochrome P-450a were raised in rffibbits, and rendered monospecif~,by absorption chroinato- graphy. In Western immunoblots, the r,aatitiody recognized a singlg protein (Mr 48,000) in, Jiver microsomes from rats of different age and s~eor from rats treated with phenobarbital, 3-methyl- cholanthrene (3-MC) or dexamethasone. The antibody also completely inhibited (>98%) the 7a-hydroxylation of testosterone catalyzed ~b.y ~~these same liver microsomal preparations. Im~unoblots of liver micro- somes from hamsters revealed dhe presence of two proteins (Mr 48,500 and 50,000) immunochemically related to rat P-450a. Treatment of hamsters with TCDD or 3-MC caused a marked intensification (>25 fold) of the larger protein recognized by antibody to rat P-450a (Mr 50,000), but slightly decreased the intensity of the smaller protein (Mr 48,500). The rate of testosterone 7oc-hydroxylation catalyzed by liver microsomes was slightly decreased after treatment of hamsters with TCDD or 3-MC. These results indicate that hamster liver microsomes contain two proteins immunochemically related to rat cytochrome P-450a, the larger of which is highly inducible by TCDD and 3-MC, but is apparently devoid of testosterone 7cc- hydroxylase activity. Supported by NIH grants ES- 03765, ES-00166 and ES-07079. 32 S_PECIES DIFFERENCES IN THE OXIDATIVE B IOTRANSFORMATION AND TOXICITY OF DIGITOXIN M R Halvorson and A Parkinson. Kansas University Medical Center, Kansas City, KS. The cytochrome P-450-dependent biotransformation of . digitoxin (dt3) catalyzed by liver microsomes from various laboratory animals was studied by HPLC. When liver microsomes from male rats were incubated with dt3 and NADPH, the predominant metabolite formed was digitoxigenin bisdigitoxoside (dt2), which formed by oxidative cleavage of the terminal sugar of dt3. When hamster liver microsomes were incubated with dt3 and NADPH, very little dt2 was formed. However, hamster liver microsomes converted dt3 to 17«- hydroxy-dts, a metabolite not formed by rat liver microsomes. Based on the profile of dt3 metabolites formed - by liver miciosomes, mice resembled rats, whereas guinea pigs resembled hamsters. Liver microsomes from cynomolgus monkeys and rabbits also converted dt3 to 17«-hydroxy-dts, but the major pathway catalyzed by monkey liver microsomes was 12,6-hydroxylation of dts to digoxin. Treatment of rats with dexamethasone caused a 4-5-fold increase in the rate of conversion of dt3 to dt2, whereas treatment of hamsters with dexamethasone caused a 4-5-fold increase in the conversion of dts to 17«-hydroxy=dt3. Treatment with 10 mg/kg digitoxin was lethal to 100% of male rats, whereas male hamsters completely tolerated dosages of 100 mg/kg digitoxin. We are currently investigating whether differences in the pithways of digitoxin biotransformation are the reason for the •marked species difference in digitoxin toxicity. Supported by NIH grants ES-03765 and ES-00166. 50875 8133
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61 N_ICKEL-MAGNESIUM IN'1'ERACTION IN SPECIFIC PROTEIN BINDING TO MOUSE SATELLITE DNA. D M Latta, R J Imbra and M Costa. Institute of Environmental Medicine, New York University Medical Center, Tuxedo, NY. Treatment ofaanouse or Chinese hamster ovary cells with various nickel compounds results in decon- densation of heterochromatin (HC); this effect can be reversed by increasing the extracellular level of magnesium (Mg) ions.. We have used the band shift assay to study the effects of Ni and Mg on specific protein binding to19NA. Protein extracts from cells were incubated with a mouse satellite DNA (SD) probe, a sequence that com- prises the HC of mouse chromosomes, in the . absence or presence of metal ions. Complexes of DNA and protein are detected by their migration in non-denaturing polyacrylamide gels. Protein binding to the human metallothionein-IIA (MT) gene promoter was examined for comparison. Ni or Mg alone inhibit protein binding to the SD and the MT probes. When Mg and Ni are added together to the MT probe, the inhibitory effects are additive; in contrast, Mg reverses the Ni- induced induced inhibition of protein binding to the SD probe. Preliminary results indicate that effects of other metal ions (Pb, Cd, Zn, and Hg), which also reduce protein binding to both the SD and MT probes, are not reversed by the addition of Mg to the reaction. (Supported by.Grant No. CA-43070 from NCI and by NRSA 1-F32-ES-05423) 62 NICKEL GENOTOXICITY IN TYPE II ALVEOLAR EPITHELIAL CELLS. AM Burke, CR Shoaf and DB Menzel. Duke U. Med. Ctr., Depts. Pharm. and Med., Compre. Cancer Ctr., Durham, NC. Genotoxic effects in human (A549) and rat (L2) type II alveolar epithelial cells exposed to NiClZ were measured by quantitating single strand breaks, DNA-protein crosslinks and DNA- DNA crosslinks utilizing the alkaline elution technique. When both cell lines were exposed to 0, 0.13, 0.68, and 1.3 mM NiClZ for 24 hr, a dose dependent production of single strand breaks occurred. Based on intracellular nickel, A549 cells were more sensitive than L2 cells. Wher_ converted to rad equivalents, a nickel intracellular concentration of 50 fcM resulted in single strand break frequencies of 85 and 120 rad-equivalents in L2 and A549 cells, respec- tively. When both cell lines were treated with proteinase K to break any DNA-protein cross- links, no difference in the elution rate occur- red. Thus, nickel does not produce DNA-protein crosslinks as part of its genotoxic mechanism. Alkaline elution assays for DNA-DNA interstrand crosslinks were carried out by further treating nickel exposed and control cells with a high dose of radiation and proteinase K to eliminate the effects of single strand breaks and DNA- protein crosslinks. Comparison of the slow eluting portions of the control and nickel exposed curves showed no change in slope between the two. Thus, no DNA-DNA interstrand cross- links result from nickel exposure. (Supported by :1IH Grants RR01693, ES07031 and CA14236.) 16 63 RISK FACTORS OF LUNG CANC R MONG CADMIUM SMELTER ./ EMPLOYEES. S Lamm; M Anderson, S Tirey, W Taylor. Consultants in Epidemiology and Occupgtional Health, Inc., Washington, DC. The increased lung cancer risk of cadmium smelter workers might reflect cadmium exposure, arsenic exposure, or cigarette smoking. Cohort mortality analysis of 597 white males emplayed over six months in cadmium smelter production area durk 1940-69 and a within cohort case-cohort study of lung cancer mortality prior to 1984 was conduft3a assess the contr%ti.on' of these factors. Cumulative cadmium exposure estimates..had been developad by NIOSH fdr each employee at their warks*_ based on work-category, time-period exposure estimates: Smoking histories were obtained by company personnel from interviews and company records for 45% of the cohort Plant process history indicated three peri~s_ ~f potential arsenic exposure based on arsenic concentrati~#'of the feedstock-prior to 1926, very high: 1926-39, high: 1940-69, moderate to low. Based on 25 cases and 75 matched controls, the relative casa-to- control cadmium exposure (mg-yrs/m3) was 1.0 (range 0.9- 1.1). The relative cigarette smoking exposure (pack- year/employee with known smoking history) was 2.5 (range 1.6-8.3). SMRs for lung cancer by period of hire were 492 (hired prior to 12926), 283 (hired 1926-39), and 88 (hired 1940-69). The lung cancer risk appears to reflect arsenic exposure rather than cadmium exposure and to be confounded by cigarette smoking. 64 A MODULATING ROLE FOR THE TESTES IN THE RESPONSE OF THE ADULT MALE RAT TO CADMIUM. M Mautino and J_ U Be11. Department of Physiological Sciences, University of Florida, Gainesville, FL. Testicular toxicity has been reported to exist in rodents following exposure to cadmium. This study was designed to determine if there is a modulating role for the testes in the hepatic and renal response to this heavy metal. Adult male rats were castrated, then treated with testosterone (1 mg/day) or vehicle. Twenty-five days after surgery, a single iv dose of cadmium chloride (0, 0.5 or 1.0 mg Cd/kg body weight) was administered and the animals were killed 72 hr later. A sham-surgery control group was also included which did not receive testosterone but was treated with cadmium. In liver and kidney, castration had no effect on the concentrations ' of either cadmium or metallothionein. In serum, castration led to an exaggerated increase in both copper.concentration and ceruloplasmin activity and a decrease in zinc concentration following the high cadmium dose. These responses were attenuated by testosterone. In a similar fashion, the high dose of cadmium caused a fall in serum aliesterase activity in castrated rats, which was not observed in the intact animal. It too was reversed by testosterone. Cholinesterase activity was decreased in serum by castration, yet appeared to be unaffected by cadmium. The decrease was partially reversed by testosterone. Thus, the testes do appear to play a modulating role in the response of the male rat to cadmium. 50875 8141
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EFFECT OF METHYLMERCURY ON LYMPHOCYTE MICROTU- BULES AND MITOGENIC RESPONSIVENESS. KR Reuhl and DL Brown, Neurotoxicology Lab., 1Tept. Pharmaco- logy and Toxicology, Rutgers University,Piscata- way, NJ and Dept. $iology, University of Ottawa, Ottawa, Ontario, Canada. Sponsor: H L Lowndes. Lymphocytes exposed to methylmercury (MM) display many deleterious effects,including chromosomal breakage,abnormal mitosis,and changes ;Ln mitogen- ic responsiveness. Studies with cultueeS cells have shown that MM induces microtubule (MT) de- polymerization, and many MM effects may be medi- ated through MT damage. We examined by immunoflu- orescence microscopy the 14T system of nonstimu- lated and stimulated lymphocytes following expo- sures to MM. For in vitro studies,splenocytes from adult BALB/c mice were exposed to MM (1-10 µa) during incubation with and without the mito- gen conconavalin A(con A). Lymphocytes from mice treated in vivo with 10 mg MM/kg were examined for MT injury and in vitro mitogen responsiveness. MM caused MT disassembly in vitro and in vivo. Response to con A was inhibited in a concentra- tion dependent fashion following both in vivo and in vitro exposure to MM. Inhibition correlated well with the degree of MT disassembly. MM treat- ment causing extensive MT damage suppressed con A response and blocked cells early in the stimula- tion sequence. MM exposure late in mitogenesis caused rapid,concentration-dependent inhibition of 3H-thymidine uptake. These data suggest that MT damage may underlie MM effects on lymphocyte function and may serve as an index of MM toxicity. BRAIN HALFLIFE OF rIETHYLMERCURY IN THE MONKEY IS LONGER THAN BLOOD HALFLIFE. D C Rice. Health Protection Branch, Ottawa, Ontario, CANADA. Estimated halflives of mercury following methyl- mercury exposure in humans are about 75 days for whole body and about 60 (40-160) days for blood. In its most recent review , the World Health organization (1980) concluded that there was no evidence to suggest that brain halflife differed from whole body halflife. In the present study, female monkeys (MM fascicularis) were dosed for at least 1.5 years with 25 or 50 µg/kg/day of mercury as methylmercuric chloride. Dosing was discontinued, and blood halflife was determined to be approximately 15 days. Approximately 230 days after cessation of dosing, monkeys were sacrif iced and regional brain total mercury levels determined. One monkey that died while still being dosed had brain mercury levels three times higher than levels in blood. Theoretical calculations were performed assuming steady state brain:blood ratios of 3, 5 or 10. Brain mercury levels were three orders of magnitude higher than those predicted assuming the halflife in brain to be the same as that in blood. Estimated halflives in brain were between 58 (brain:blood ratio of 3) and 40 (brain:blood ratio of 10) days. In addition, there was a dose-dependent difference in halflives for some brain regions. These data clearly indicate that brain halflife is considerably longer than blood halflife in the monkey under conditions of chronic dosing. 83 ROLE OF HEPATIC GSH AND RENAL 1'-GTP IN RENAL . UPTAKE OF METHYLMERCURY AND INORGANIC _MERCURY IN MOUSE. A Naganuma, T Tanaka and N Imura, Dept. of Public Health, Sch. of Pharmaceutical Sciences, Kitasato University, Minato-ku Tokyo, Japan The role of hepatic glutathione (GSH) and . renal Y -glutamyltranspeptidase (Y-GTP) in renal ' uytake of mercurials by uITng a specific depletor for hepatic GSH, 1,2-dichrolo-4- nitrobenzene (DCNB) , and 1' -GTP inhibitor, *wti acivicin. DCNB pretreatment reduced the renal uptake of both methyl and inorganic mercuric Hg and prevented the renal toxicity of inorganic Hg. Furthermore, renal mercury uptake was increased by intravenous coadAnistration of the mercurials with GSH. These results suggest that the hepatic GSH or GSH released from the liver into the plasma plays a key role in the renal accumulation of mercury compounds. Inhibition of renal Y-GTP by acivicin pretreatment reduced renal Hg uptake and increased urinaly excretion of Hg and GSH, consequently ameliorating the renal and lethal toxicity of inorganic Hg. These facts indicate that methyl and inorganic mercury are transported to the kidney as Hg-GSH complex and Hg in the complex is incorporated into the kidney by a t-GTP dependent system. 84 THE EFFECTS OF KETAMINE:XYLAZINE ANESTHESIA ON HEPATIC -aULFHYDRYL (SH) DISPOSITION AND BILIARY EXCRETION OF CH3Hg. C A White and C D Klaassen. Univ. of Kansas Med. Ctr, Kansas City, KS. Many metals, including CHsHg, are excreted into bile as SH (glutathione and its degradation products) complexes. Thus, compounds which affect. SH disposition may also alter the biliary clearance of metals. We have shown previously that ketamine:xylazine (KX, 100:10 mg/kg) anesthesia increases biliary excretion of SHs 2-4 fold when compared to urethane-anesthetized (1.5 g/kg ip) rats. The purpose of the present study was to determine the influence of KX-induced anesthesia on the biliary excretion of CH3Hg and total hepatic SH disposition by quantitating SH composition in the blood entering the liver, venous blood exiting the liver as well as in liver and bile. SHs were separated by reverse-phase HPLC and quantitated using electrochemical detection, while 20SHg- CHSHg (10 µmol/kg, iv) was measured using gamma scintillation spectrometry. KX increased biliary SH excretion 2.1-fold compared to urethane-anesthetized rats. Although the biliary concentrations of CH3Hg were similar after administration of either anesthetic, KX increased both bile flow (78%) and the cumulative amount (112%) of CHgHg excreted into bile. While the biliary excretion of SHs was higher in KX-treated rats, the concentration of SHs in liver and the blood entering and leaving the liver were equivalent between the two groups. In conclusion, KX increases the efflux of SHs into bile but not from liver into blood. It appears that the increase in biliary excretion of CH3Hg produced by KX is largely due to the increase in biliary SH excretion. (Supported by USPHS Grants ES-01142, ES-03192 and ES-07079) ' 21
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THE ROLE OF _PHARMACOKINETICS AND METABOLISM IN AGE-DEPENDENT A_CETAMINOPHEN NEPHROTOXICITY IN MALE SPRAGUE-DAWLEY RATS. J B Tarloff, RS Goldstein, R S So°t3o and J B Hook. Dept. of Investigative Toxicology, Smith Kline & French Laboratories, King of Prussia, PA. Increased susceptibility to acetaminophen (APAP)-induced nephrotoxicity in 12-mo old male Sprague-Dawley (SD) rats is due, in pa~rt, to an age-dependent reduction in APAP volume of distribution. To define more clearly the role of pharmacokinetics, APAP nephrotoxicity was evaluated under conditions in which blood APAP concentrations were equivalent in 3- and 12-mo old male SD rats. APAP was administered iv at 750 mg/kg in 3-mo old and 500 mg/kg in 12-mo old rats. Despite equivalent blood APAP concentra- tions, only 12-mo old rats manifested nephrotox- icity 24 h following APAP administration. To assess the role of age-dependent differences in APAP metabolism, APAP metabolism via deacetyla- tion or glucuronidation was determined J-n vitro in 3- and 12-mo old rats. Deacetylation of APAP to p-aminophenol by renal cortical cytosol in vitro did not differ with age. In vi r glucur- onidation of APAP by 3- and 12-mo old liver and kidney microsomes was not different. These data indicate that differences in nephrotoxicity persist under conditions in which blood APAP .concentrations are equivalent in 3- and 12-mo old rats. However, differences in APAP de- acetylation or glucuronidation cannot account for enhanced nephrotoxicity in 12-mo old rats. 14 DIFFERENTIAL ACETAMINOPHEN TOXICITY AS A FUNCTION OF GENOTYPE IN MICE. D W Roberts, R W Benson, N R?umford, D W Potter, K L Rowland, J A Hinson, and G L Wolff. National Center for ToxicT oTogTE_al Researc , e ferson, AR. Congeneic yellow Avy/A and agouti A/a (C3H x VY) F-1 hybrid mice; known to have -differential responsiveness to hepatic tumorige~, were used to test the effects of the obese A/A phenotype on susceptibility to acetaminophen-induced hepatotoxicity. Since acetaminophen hepato- toxicity is known to correlate with the covalent binding of acetaminophen to protein as 3-(cys- tein-S-yl)-acetaminophen, an immunochemical technique was used to quantitate this adduct as a function of dose and genotype. Serum alanine and aspartate aminotransferase (ALT and AST) levels were measured as indices of hepatotoxicity. Mice fasted overnight were dosed by intraperitoneal injection of 0, 50, 150, 250, 350, or 450 mg/kg acetaminophen. Four hours after dosing, yellow mice receiving toxic doses of acetaminophen had elevated ALT and AST levels and elevated serum acetaminophen adduct levels as compared to similarly treated agouti mice. In non-fasted mice, resistance to acetaminophen toxicity was increased regaraless of genotype, but agouti mice were more susceptible as indicated by greater elevation of serum ALT and AST. These data show differential susceptibility to acetamino8~en toxicity as a function of the obese A/A phenotype and nutritional status. 115 ACETAMINOPHEN HEPATOTOXICITY IN OBESE ZUCKER RATS: MECHANISM OF RESISTANCE. I Chaudhary, P J McNamara, R A Blouin. Graduate Center for Toxi- cology, University of Kentucky, Lexington, KY. Sponsor: L Robertson. The hepatic cytochrome P-450 system in obese Zucker rats is not inducible b3;,,&enobarbital (PB) and these rats are more resistant to acet- aminophen overdose after PB-treatment. The pur- pose of this study was*to assess the influence of PB on glutathione transferase (GST), Y-glut- amyl cysteine synthetase (Y-GCS) and hepatic - glutathione (GSH). PB was administered orally to lean (34.7 mg/Kg/12 hr) and ob~ae (20.4 mg/ Kg/12 hr) rats for 5 days. The lfversI of 9 male control and induced lean and obese rats were homogenized and the final supernatant after 105,000 g was assayed for GST and Y-GCS acti- vities. PB treatment caused statistically significant increase in liver protein (mg/g protein) in lean control vs treated (74.12 ± 5.54 vs 80.58+4.82) rats, but no change was observed in obese control vs treated (80.39 + 5.13 vs.83.42+7.54) animals. PB induced GST activity (pmole/mg protein) in lean treated vs control (1.77+.07 vs 0.98 + 0.8) and obese tre- ated vs control (1.31 ±.07 vs 0.81+.07). There was no increase in the Y-GCS and GSH level in PB treated lean and obese animals compared to controls. Results from this study showed that resistance of obese Zucker rats to acetamino- phen could in part be due to the preferential induction of GST over cytochrome P-450. 116 In Vitro Metabolism and the Age-dependency of Acetaminophen (APAP)-induced Hepatotoxi.city in CD-1 Mice. J T Brady, W P Bei-erschmitt, D S Wyand , E A Khairallah and S D Cohen. Univ. of Connecticut, Toxicology Program, Storrs, CT Two and 3 month (M) old fasted (18 hr), male, CD-1 mice were given APAP (600 mg/kg, po) or 50% propylene glycol vehicle. Plasma sorbitol dehy- drogenase (SDH) activity was measured and liver histopathology assessed 12 hr after dosing. Three M old, APAP-treated mice had significantly greater (7 fold) plasma SDH activities compared to 2 M old, APAP-treated mice. Verification of damage was supported by liver histopathologic evaluations. These data suggest that 3 M old mice were more susceptible than 2 M old mice to APAP hepatotoxicity. In vitro, hepatic cytochrome p-450 levels, an_iline hydroxylase, p-nitroanisole 0-demethylase, glutathione (GSH)-S-transferase (GST) activities and APAP-GSH adduct formation were measured in fasted, untreated 2 and 3 M old mice and no differences were detected. These data suggest that factors other than age-related differences in APAP bioactivation or GST-mediated detoxification of the APAP reactive metabolite were responsible for the difference in suscept- ibility of 2 and 3 M old mice to APAP-induced liver damage. (Supported in part by NIH Grant # Qy31460 and FS07163 and a Stauffer Chemical Company Fellowship in Toxicology to JTB.) 29 .~.-
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77 ARSINE (AsH3) AND GALLIUM ARSENIDE (GaAs)-INDUCED ALTERATIONS IN tiEME METABOLISM. W E Bakewell, P L Goering, M P Moorman, and B A Fowler, NIEHS, Research Triangle Park, NC Arsine gas (AsH3) is a pof'ent hemolytic agent which is widely used in the manufacture of GaAs semiconduct®rs. Prolonged exposure to AsH3 results in hemolysis with development of a regenerative anemia. GaAs has also been shown to produce alterations in heme biosynthesis and development of porphyrinuria. These studies examined AsH and GaAs-induced alterations in reduced ce113 6-aminolevulinic acid dehydratase (ALAD) in relation to urinary excretion of ALA and 6 porphyrin isomers. Acute intratracheal instillation of GaAs (50-200mg/kg) produced a marked inhibition of ALAD activity with an associated increase in urinary ALA. Increased urinary excretion of the 7 & 8 COOH uroporphyrins and lesser amounts of the 4-COOH coproporphyrin was also observed. In contrast, exposure of rats to AsH3 (0.5-5.0) ppm for 4 weeks produced a dose-related increase in the activity of ALAD and a modest depression of urinary ALA. A marked increase in the urinary excretion of the 7 & 8 COOH uroporphyrins but not the 4-COOH coproporphyrin was observed. These data suggest that AsH3 and GaAs produce highly specific alterations in the heme biosynthetic pathway which may lead to the development of biochemical "fingerprints" for early detection of ongoing biological activity associated with exposure to these agents either alone or in mixtures. 78 EFFECT OF ARSENIC ON CARBOHYDRATE METABO- LISM AFTERrSINGLE OR REPEATED INJECTION IN GUINEA PIGS. F X Reichl, L Szinicz, H Kreppel, B Fichtl, and W Forth. Walther-Straub-Institute for Pharmacology and Toxicology, MUnchen, FRG. Sponsor: D A Cory-Slechta Further experiments were performed to in- vestigate whether the metabolic effects of arsenic after repeated administration may differ from those in acute poisoning (cf. Arch.Pharmacol. 335: R17, 1987). Male guinea pigs were sacrificed lh after a single dose of,As 0(10 mg/kg s.c.) or after repeated trea~ment(2.5 mg/kg b.i.d. for 5 days). The liver was removed using a freeze stop technique and the content of glycogen and several glycolysis inter- mediates was measured. As compared with untreated controls, both dosing regimens resulted in a decrease of total carbohydrate content which was due mainly to a depletion of glycogen (single dose -24%; repeated -81%). There were, however, distinct differences in the pat- tern of metabolic changes,e.g. an increa- se of pyruvate after the single dose but a decrease after repeated dosing of As203 It.is concluded that the metabolic conse- quences of arsenic poisoning are depen- dent on the duration of exposure sugges- ting a possible different mechanism of toxicity in acute and chronic poisoning. 20 79 DEVELOPMENT OF AN IN'1/ITRO SCREEN FOR ARSENIC ANTIDOTES USING PYRUUATE DEHYDR06ENASE COMPLEX ENZYME ACTIVITY. DW Hobson, TH Snider, MJ Chang, and RL Joiner. Battelle Columbus Division, Columbus, OH. Sponsor: CT Olson.. An in vitro assay to screen candidate antidotes to systemic arsenic poisoning was developed_- using a commercially-available preparation of pyruvate dehydrogenase complex (PDHC). Inhibition of PDHC activity was deta"rined for three As(+3) ~ compounds: sodium arsenite, Lewisite (Lj';="-' and chlorovinylarsenous acid (CVAA). L and''CaIAA were, respectively, 25 to 50 times more poten~ inhibitors of PDHC activity than was sodium arsenite. The effectiveness of three candidate antidotal compounds, 2,3-dimercaptopropranol (BAL), 2,3-dimercapto-l-pry#an'e sulfonic acid (DMPS), and meso-2,3-dimercaptosuccinic acid (DNSA), was tested against each PDHC inhibitor. The millimolar ratios (mM reactivator/mM inhibitor) of BAL, DMPS, or DMSA required for complete reactivation of PDHC activity from 90 percent inhibition by sodium arsenite were 1.82, 2.00, and 2.78 respectively. The respec- tive millimolar ratios of BAL, DNPS, or DMSA required for complete reactivation of PDHC from ~_,a percent inhibition by L were 1.99, 2.13, and 3.20, and 2.42, 3.18, and 7.25 from 90 percent inhibition by CVAA. The relative ranking of PDHC reactivator effectiveness against the three inhibitors tested using the in vitro assay was BAL > DMPS > DMSA. 80 COMPARATIVE TOXICITY AND TISSUE DISTRIBUTION OF ANTIMONY POTASSIUM TARTRATE IN RATS AND MICE DOSED BY DRINKING WATER (DW) OR INTRAPERITONEAL INJECTION (IP). M.P. Dieter, C.V. Jameson (NIH, NIEHS, National Toxicology Program, RTP, NC); J.W. Lodge (Research Triangle Institute, RTP, NC); M. Hejtmancik, S.L. Grumbein, A.C. Peters (Battel e Columbus Division, Columbus, OH) Antimony potassium tartrate (APT) is given i.v. to treat schistosomiasis, and was tested by NTP because of its possible association with bladder tumors. Both sexes of F344 rats and B6C3F1 mice were given APT by two different routes. DV doses in rats were 0, 16, 28, 59, 94 and 168 mg/kg and in mice were 0, 59, 98, 174, 273 and 407 mg/kg; IP doses in rats were 0, 1.5, 3, 6, 11 and 22 mg/kg and in mice were 0, 6, 13, 25, 50 and 100 mg/kg. APT was much more toxic by the IP route, causing mortality and histopathological lesions at doses about one order of magnitude below those used by the DW route. In DW studies only one animal died; in•IP studies 3 rats and 29 mice died. In DW studies there were no toxic responses in rats; there were body and organ weight decreases, liver and forestomach lesions in mice. APT given IP in rats and mice caused liver, kidney, and mesenteric lesions. Antimony accumulated in blood, liver, spleen, heart, and kidney of rats given APT in DW (94 mg/kg) or by IP (11 mg/kg), at equivalent levels of 15, 6, 4, 3, and 2 pg/gm. In mice there was no antimony in blood, heart, and kidney; there was 24 pg/gm in liver after 273 mg/kg (DV) or 50 mg/kg (IP), and 5 ug/gm in spleen of mice given the IP dose. 50875 8145
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EFFECTS OF VITAMIN B6 STATUS ON LB.AD TOXICITY IN THE RAT. C McGowan, V Wiley and L Matthews. Food Science and Human Nutrition' Department, University of Florida, Gainesville, FL_ The influence ofi vitamin B6 status on lead (Pb) intoxication •was investigated in a 2x2 factorial experiment. The role-of B6 as a cofactor for several enzymes of the trans- sulfuration (TS) pathway is important in the conversion of dietary methionine to cysteine. Since cysteine is rate-limiting in glutathione (GSH) formation, Pb-induced changes~.in GSH metabolism may be affected by B6 status. Male weanling Spraque-Dawley rats were maintained for 5 weeks on either a B6-deficient or-adequate diet (AIN-76A purified rat diet, Research Diets, New Brunswick, NJ) supplemented with 0 or 2000 ppm Pb as Pb acetate •3H20. Both Pb and B6 deficiency caused significant (p<_O5) reductions in body weight gain, but no Pb x B6 interaction was evident. Hepatic cysteine con- centration and renal y-glutamyl transpeptidase activity were not significantly affected by either Pb or B6 deficiency. In the liver, Pb had a significant effect on GSH content and glutathione reductase (GSSG-R) activity showed significant influences due to both Pb and B6 deficiency with a significant Pb x B6 inter- action. The data indicate that Pb-induced changes in certain aspects of GSH metabolism may be influenced by B6 status but do nott clearly show it is due entirely to the.role of; B6 in cysteine metabolism. =90 LEAD EXPOSURE AND SKELETAL.DEVELOPMENT. J D : Aamilton and E J 011ahert . Department of nvironmental Hea th, University of Cincinnati, Cincinnati, OH. Reduced neonatal body weight associated with maternal lead exposure has- been reported. in humans. Skeletal ossification site counts and other morphometric indices of skeletal develop- ment are correlated with body weight .in young experimental animals and neonatal humans. It was hypothesized that lead exposure will result in retarded skeletal development and reduced body weight in rats. Female Sprague-Dawley weanling rats received 0, 50, 250, 500 or 1000 ppm lead in drinking water for 56 days. Food consumption, water intake, tail length and body weight were measured every 2 to 3 days. The results indicated that lead exposure is asso- ciated with decreased tail length growth rate in the 1000 ppm lead treatment group (p=0.0009), and reduced body weight in the 500 and 1000 ppm lead treatment groups (p=0.0436 and p=0.0001, respectively). Food intake rates in the lead treatment groups were not reduced when compared to the food intake rate in the control group. Water consumption was reduced in the 250, 500 and 1000 ppm lead treatment groups relative to the control group water consumption. Studies are underway to examine the effect of maternal lead exposure on fetal and neonatal offspring skeletal ossification. (Project supported by NIOSH 1R01-0H02376-01). 23 91 EFFECT OF LEAD ON INTRACELLUTaAR,;~Ca'2+ IN ROS 17/2.8 OSTEOBLASTIC BONE CELLS DETERMINED BY 19F-NMR. F A X Schanne, T L Dowd,•R K Gupta, and J F Rosen. , Depts. of'Pediatrics, Pathology, Physiology and Biophysics, Albert Einstein College, Bronx, NY. Lead (Pb) has been shown to interfere with cellu- lar calcium homeostasis, altering sizes and flux rates of cellular pools of exchangeable calcium and perturbing calcium-mediated cell processes. The present study examined directly the effect of Pblon the intracellular free calA.°ihm ion concen- tration, tration, [Ca2i"]i, in rat ROS 17/2.8 cells. ROS-d- 17/2.8 bone cells were.attached to collagen- coated microcarriers (Cytodex 3) and loaded with the fluorinated calcium ion indicator, 5,5'-di- fluoro-l,2-bis(o-aminophenoxy) ethane N,N,N',N'- tetra acetic acid (5F-BAPTA). The 19F NMR was observed using a Varian VXR-500 pM-spectrometer operating at a magnetic field of 11.1 Tesla. ROS 17/2.8 cells were maintained in a 10 mm NMR tube at 30oC by superfusion at 2 ml/min with.F-12 medium saturated with 95% 02-5% C02. Treatments were added to the superfusion medium. The basal [Ca2"E']i was estimated to be 175±17 nM. Treatment with parathyroid hormone (400 ng/mi) produced a 30:% increase in [Ca2+]i. Treatment with Pb (5-50 uPi) produced a 25 to 100% increase in [Ca2+]i as well as observable intracellular Pb2+. These observations are the first direct demonstration of intracellular Pb2+ and Pb-induced increases in intracellular free Ca2+. 92 LEAD METABOLISM IN CULTURED OSTEO BLAS- TIC BONE (OB) CELLS. G,J Long. University of Ark. for Med. Sciences, Little Rock, AR. J F Rosen:: Albert Einstein Coll. Med., Bronx, NY. J G Pounds. ~ Brook- haven National Laboratory, Upton, NY. Characterization of lead metabolism in bone is impor- tant to establish:the contribution of skeletal lead_to its toxicity in soft tissues. Experiments were conducted to establish a steady-state kinetic model for 210Pb in cul- tured OB cells. Cultures were obtained from mouse cal- varia by a sequential collagenase digestion, labeled with 2iopb , and kinetic parameters were derived-from. 210Pb washout curves. Three kinetic compartments:were ade- quate to describe the intracellular metabolism of 210Pb_ The relative sizes of the three compartments were de- termined to be Si - 12%, S2 - 15%, and S3 - ?3%. - Total cell lead was 1.1 nmol/mg cell protein. There was a concentration dependent increase in all three kinetic compartments in cells exposed from 5wM to 10014M.Pb. There was a large increase in S3 and relatively smaller in- crements in Sl and Sa. In this concentration range, none of the kinetic compartments displayed saturation kinet- ics. Treatment with EGTA had no effect on any of the kinetic compartments, indicating that all three compart- ments were intracellular. All three kinetic compartments were increased by phosphate with small increases in Sl and S2 and a large (lOx) increase in S3 at 8.OmM phos- phate. This suggests that S3 includes mitochondrial lead. (Supported by NIH grants ES01060 and ES04040) 50875 8148
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ay INDUCTION OF DIFFERENT DRUG METABOLIZING ENZYMES BY ELOTRIMAZOLE IS NOT INITIATED BY THE SAME PARAMETERS OF DRUG EXPOSURE. Wr Hopson, and M R r nklin. Depariment of Pharmacology and Toxicology,- University of Utah`, Salt Lake City, UT. Clotrimazole, when administered daily for 3 days at 75 mg/kg is a high magnitude (3-4 fold)cytochrome P-450 inducer. The induction exhibits dose-differentiated isozyme induction. Erythromyci.n dem9thylase activity, unlike p-nitroanisole demethylase and pento esorufiin dealkylase activities, is not induced by a lower25 mg/kg x 3 days) dose. Since overall dose has changed m the two treatments, (225 mg/kg vs 75 mg/kg) the inductive effect of a single 75 mg/kg dose was investigated. The effects, except for the extent of cytochrome P-450 induction (2- 2.5 fold) paralleled those of the 75x3 dose, not the 25x3 dose. Changes in Phase II drug metabolizing enzymes were also compared. Microsomal UDP- glucuronosyltransferase (p-nitrophenol) was induced to the same extent (1.5 fold) by all three treatments. Cytosolic GSH transferase (1 chloro-2,4-dinitrobenzene) was induced 3 fold by the 225 mg/kg total dose and only 1.5 fold by the 75 mg/kg total dose given either as a single or divided dose. Depression of cytosolic sulfotransferase (p-nitrophenol) was only seen with the 225 mg/kg dose. Thus, differential cytochrome P450 isozyme induction appears to be triggered by a bolus concentration, not total dose. UDP- glucuronosyltransferase induction is independent of either bolus concentration or total dose and cytosolic GSH transferase induction and phenolsulfotransferase depression appear proportional to total dose. 26 ISOZYME-SELECTIVE INHIBITION OF THE PULMONARY CYTOCHROME P-450-MEDIATED BIOACTIVATION OF 3-METT-IYLINIDOLE. _Q ,S `: ost, I C Huijzer, J D Adams, Jr., and J Y Jaw. Department of Pharmacology and Toxicology, University of Utah, Salt Lake City, UT. The bioactivation of the highly selective pulmonary toxin, 3-methylindole (3MI), by isozymes of, cytochrome P-450 was studied through the synthesis and use of isozyme-selective aminobenzotriazole suicide inhibitors of P-450. ~~th 3MI metabolic turnover and covalent binding of C-labeled 3MI were inhibited to an equal extent (50% of controls) by 1-aminobenzotriazole at a concentration of 0.01 mM in goat lung microsomes. Concentrations of the inhibitor greater than 0.1 mM caused a complete loss of 3MI metabolism and covalent binding in a time-dependent manner. The isozyme-selective inhibitor, alpha-methylbenzylamino- benzotriazole (AMB), was synthesized and used to assess the involvement of P-450 forms selective for benzphetamine metabolism as contrasted to ethoxyresorufin metabolism. A concentration of AMB of 0.01 mM inhibited approximately 80% of 3MI metabolism or benzphetamme demethylation but only 28% of ethoxyresorufin O-deethylase activity. These results show that cytochrome P-450 is responsible for the bioactivation of 3MI to an alkylating mtermediate, and that the "phenobarbital-inducible" isozymes are grimarily responsible for the activation process. pported by USPHS Grant HL13645. GSY is a USPHS Research Career Development Awardee (HL02119) ~ 27 IN VITRO EFFECTS OF ENVIRONHENTAL ALKANES ON RAT PULMONARY CYTOCHROME P450-DEPENDENT MONOO%YGENASE ACTIVITIES. J Rabovsky and DJ Judy. NIOSH/DRDS. Morgantown, WV. Sponsor: V Castranova. Alkanes are environmental contaminants that are metabolized through cytochrome P450-dependent enzyme activities(P450). The reactivities-between alkanes and two rat lung microsomal P450 activi-r ties,constitutive benzyloxypkSnoxazone dealkyl- = +) ase(BzOPh'ase) and B-naphthoflavone(BNF)-indi.tsed_' ethoxyphenoxazone dealkylase(EtOPh'ase),were studied by measuring the inhibition of resorufin,1-, formation. The data are expressed as the con- centration of alkane required for 50% inhibition (I50)• I50/BzOPh for n-hexane through n- undecane(nC6-nCll) ranged fromj2-1®pM. Some branch-chain octanes(C8) eXhif>ited6decreased 150/ BzOPh;i.e.I5O=7uM-C8,6pM-2methylheptane,lpM-4- methylheptane,0.9uM-2,5dimethylhexane. No effect on EtOPH'ase activity was observed for straight- chain alkanes or branch-chain C8s at concentra- tions up to 1-1omM. Hydroxyl and carbonyl C8s exhibited increased I50/BzOPh'ase (I50=54UM-3- octano1,85pM-3octanone) and measurable I50/ EtOPh'ase(I50=0.5mH-3octano1,1.2mM-3octanone). The data show in rat lung microsomes,the alkanes were more reactive towards constitutive BzOPh'ase than towards BNF-inducible EtOPh'ase and the re- activities depended on structure and functional groups. BzOPh'ase and EtOPh'ase activities are useful tools for the study of specific interac- tions between environmental pollutants and pulmonary P450-mediated reactions. 28 HEPATIC MONOOXYGENASE ACTIVITIES IN THE ADULT RHESUS MONKEY: COMPARISON WITH THE RAT. J E A teakey, J Bazare, Jr, H C Cunny, P J Webb, W Slikker, Jr and J R Bailey. Division of Repro- ductive an Developmental Toxicology, National Center for Toxicological Research, Jefferson, AR. Hepatic cytochrome P-450(s) (P-450) and their resultant monooxygenase activities exhibit sexual dimorphism in adult rats. For instance, hepatic testoterone 6B and 16a/2a hydroxylase activities are relatively high in males due to the presence of the P-450g and h isozymes respectively, but are low or absent in females, where testosterone is predominantly reduced at the 5a postion. Furthermore, 7-ethoxy- resorufin-O deethylase activity (EROD) is also present at low activities in (uninduced) adult rat liver due to the low amounts of P-450c. Monooxygenase activities were investigated in liver biopsy samples from adult male and female rhesus monkeys. No significant sex differences were seen in the hepatic metabolism of testos- terone which was hydroxylated predominantly at the 6B position in both sexes and was not reduced at the 5a position. EROD activity was 18-fold greater in both male and female monkey liver than in rat liver but these high acti- vities did not correlate with an increased capacity to form benzo[a]pyrene epoxides. These findings suggest that large differences occur between the rat and rhesus monkey in both quan- tity and substrate specificity of their analogous P-450 isozymes. 50875 8132
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93 DO LEAD TOXICITY AND DIETARY FAT INTERACT WITH LEUKOTRIENE PRODUCTION? S Knowles and W E Donaldson. N.C. State Univ., Raleigh, NC. Previous work has demonstrated an effect of dietary Pb on several precursors of leukotriene (LT) produssion including increased hepatic glutathione and increased tissue arachidonic acid (AA 20:4) / linoleate (18:2) ratio. We investigated the possible role of LT's as mediators of Pb toxicity. Chicks were reared with diets supplying varying levels of linoleic acid (ti55% of total fatty acids. in cottonseed oil). In two 3-week trials, adequate fat (2%) vs. 4 or fi$ fat and 0 vs. 2000 ppm Pb as Pb acetate were fed in a 3 x 2. factorial arrangement. The decreased growth rate associated with 2000 ppm Pb was not moderated by fat intake. Serum and liver were analyzed for LTC4 by radio-immuno assay. Average concentration of all treatments was 2.42 ng/ml in serum and 1.58 ng/g in liver. Neither Pb nor fat affected tissue LT concentration due to high variation within treatments. Pb significantly increased hepatic glutathione concentration and 20:4/18:2 ratio in hepatic fatty acids. Despite these increased precursor concentrations, Pb did not show an effect on LT concentration. Although the results do not support a Pb effect on LT synthesis, such an effect could be masked by increased LT concentrations in other tissues or by increased LT turnover, 94 INTERACTION OF TOXIC DIETARY LEVELS OF LEAD AND SELENIUM. W E Donaldson and C McGowan. North Carolina State Univ., Raleigh, NC. Two experiments were conducted in which varying levelss of lead (up to 2000 ppm as Pb acetate 3H20) and selenium (up to 40 ppm as Na2Se03)) were fed, either alone or in combination, to chicks from 0 through 18 or 20 days of age. Pb additions depressed growth in a dose- dependent manner without affecting mortality. Se addition at 20 ppm was severely growth inhibitory, also without affecting mortality. The growth inhibition by 20 ppm Se was partially alleviated by feeding it in combination with 2000 ppm Pb, however mortality was increased significantly by the combination. In contrast, 40 ppm Se resulted in almost complete cessation of growth and !i5$ morta l i ty, wh i le the comb i na- tion with 2000 ppm Pb partially overcame the growth inhibition and eliminated the excess mortality. When Pb or Se were fed alone, hepatic levels of the fed element were elevated. There were further significant elevations when both elements were fed in combination at identical dietary concentrations as the single element additions. The results suggest that Pb and Se are antagonistic. The nature of the antagonism is such that while Pb par- tially overcomes growth inhibition by Se, the reverse is not observed. 24 95 INDUCTION AND ACTIVATr~N RAT RENAL EPOXIDE HYDROLASE BY LEAD. E Graichen, B Conway, K Phi.pps, T Leonard, Smith Kline & French Laboratories, Swedeland, PA ' '! Treatment of rats with salts of metals such as Hg. Ni, and Pb causes dose-related increases in renal microsomal epoxide hydrolase (EH) : activity; however, the increases observed are> 2 to 8-fold greater in Sprague-Dawley (SD) rats than in Fischer 344 tPT"rats. To furt er examine the basis of the differential EH "~ ` response to metals, the enzyme kinetics were `~' studied in kidney microsomes from control (C) " rats, or rats treated with 3 daily doses of 300 pmol/kg lead acetate (Pb). Using benzo(a)pyrene-4,5-oxide substr_aje, sigmoidal velocity curves were observgf. When fit to the Hill equation, v/Vmax=[S]n/(sK'+[S]n), Vmax was found to be 0.88, 2.5, 1.0, and 20.5 nmol/mg pro/min; K' was 0.23, 0.36, 0.87, and 9.0 uM; and the apparent n was 2.3, 2.2, 2.1 and 1.5, in F C, F Pb, SD C, and SD Pb groups respectively. The increases in Vmax in microsomes from Pb-treated animals suggests that EH is induced in both strains; however, the differences in K' and n between C and Pb SD groups suggests that in SD rats renal EH is also altered by Pb treatment. Purified renal EH preparations had specific activities of 24 (SD C) and 96 (SD Pb) nmol/mg pro/min, consis- tent with activation of the enzyme. These data suggest that renal EH is both induced and activated by Pb treatment of SD rats. 96 EFFECT OF LEAD TOXICITY ON INTRACELLU- LAR CALCIUM HOMEOSTASIS.J G Pounds. Brook- haven National Laboratory, Upton, NY. Cellular Ca++ -mediated cell functions are implicated as important targets of lead toxicity in a wide variety of cells, tissues, and organs. Alteration of the temporal and spatial regulation of cytoplasmic free Ca++ levels by lead is the critical event in Pb++ -Ca++ interactions. Al- though many studies report alteration of cellular calcium homeostasis, and thus implicate perturbation of [Ca++]i , this effect has not been verified by direct measurement of [Ca++]i. Experiments were conducted using ion-selective electrode techniques to evaluate the effect of lead on the "set point" for [Ca++]; homeostasis with mitochondria, microsomes, and permeablized cells. Hepatocytes were isolated from Sprague-Dawley rats, placed in to primary culture, and treated with 0,10,50, or 100 EcM lead ac- etate,and mitochondria, microsomes, and permeablized cells prepared after 20 hr exposure to lead. Previous studies have shown that at 20 hr, the cells are in steady state with respect to Pb++ and perturbations in cellular 4sCa are observed. The rate of Cat+ dearance, and the steady state level was altered in the mitochondrial and permeablized cell preparations. These studies indicate that lead toxicity alters the dynamic regulation of Ca++ rather than altering the steady state levels, thus explain- ing in part, the sensitivity of Ca++ -mediated process to lead toxicity. (Supported by NIH ES04040) . 50875 8149
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69 CALMODULIN MODULATION OF CD-INDUCED INHIBITION OF MICRO'SSSBULE (MT) ASSEMBLY. B A Perrino and I N_Chou, Dept. of Microbiology, Boston Univ. Sch. of Medicine. Boston, MA Sponsor: C T Walsh We have shown that CdCl cau'ses MT disassembly in cultured ~e11s. Inhibition of MT assembly ri vitro by Ca+ ols enhanced by calmodulia (CaM), when microt~~ule-associated proteins (MAPs) are present. Cd has been shown to inhibit MT assem- bly in vitro by ~jnding to sulfhydryls ~f tubul- in. However. Cd also binds to th~ZCa -binding domains of CaM as effectively 1~ Ca apparently because the ionic rflius of Cd (0:097nm) is close to that of Ca (0.099nm). To determ~e whether the inhibition of MT assembly by Cd is simi%ly en%ced by CaM, we examined the effect of Ca or Cd on the assembly of bovine brain MT protein, containing tubulin and MAPs, in the presence and absence of CaM. MT assembly was monitored by measuring the increase in turbidity at 350nm following the addition of GTP. MT assem- b1T2was i%bited by micromolar concentrations of Ca or Cd alone. As expV2ted, CaM enhanced the inhibitory effect 9f Ca . Similarly, the in- hibitory effect of Cd+ was enhanced by CaM. Com- pound 48\80, a potent+gaM inhibitor, reversed the CaM enhancement of Cd -induced inhibition of MT assembly. These resVi ts demonstrate that when CaM is available. Cd can inhibit MT assembly by binding to and activating CaM, and suggest that tubul~2 sulfhydryls may not be a primary target of Cd binding in vivo. 70 CADMIUM-INDUCED ALTERATIONS IN PULMON- ARY ANTIOXIDANT ENZYMES AND METAL LEVELS. P K Bennett and I Jamall. Toxicology Program, St. John's University, NY. Weanling, Sprague-Dawley rats were fed a diet containing 05 ppm selenium (Se) with either 0, 10 ppm or 50pp m copper (Cu) for 7 weeks. There were two sets of Cd-treated rats. One set (Cd-OP) received 5 mg Cd via osmotic minipumps (Alzet 2002). The second set (Cd-D) received 50 Ppm Cd admixed)with their feed. Catalase (CAT) activity was increased by 23% and 32% in the rats treated with dietary Cd and fed the diets supplemented with 10 ppm and 50 ppm Cu, respectively. Cd-OP treated rats exhibited a 27% reduction in CAT only in the 10 p m Cu-supplemented group. Superoxide dismutase /~SOD) activity was mcreased by increasing dietary Cu but was unaffected by either Cd treatment. Glutathione peroxidase (GSH- Px) was also unaffected by the Cd treatments. Tissue peroxidation, measured by thiobarbituric acid reactive substances, was also unaffected by Cd. Increasing dietary Cu resulted in marked reductions in Cd levels in Cd-OP treated rats but increased Cd accumulation in rats treated with dietary Cd. Dietary Cd treatment resulted in up to 48% reductions in lung Se compared to untreated controls. In contrast, Cd-OP rats exhibited increased Se levels in their Iungs. This effect was most pronounced (up to 47%) m rats fed the diet sup lemented with 0 Cu. Thus, the effects of dietary andp parenteral Cd treatments elicit very different responses in the rat lung. .' ~~`-'' 71 DEPRESSION OF SUPEROXIDE?-PitODUCTION IN LAVAGED LUNG CELLS FOLLOWING CADMIUM CHLORIDE EXPOSURE. S H Gavett, N M Corson and G Oberdbrster. Env. Health Sci. Ctr., Univ. of Rochester, Roch., NY. Oxygen radical generation by activated alveolar macrophages (AM) and other infiammatory cells may be a mechanism of damage to the lung foilow- ing cadmium chloride exposure. To investigate'_ this possibility, Fisher 344 rats were intra- " tracheaily instilled with lQ" Cd as CdC12. Numbers of lavaged neutrophils peaked 1 to.2--- days later, while numbers of lavaged macro- .~ phages and mononuclear cells peaked after 3 to 4=` days. Despite the increased potential for super- oxide (SO) production in neutrophils as com- pared to macrophages, SO production in both resting and phorbol ester (PM~-stimulated lavaged cells of Cd exposed r~a s alas signifi- cantly depressed. This depression was noted as early as 1 hr after Cd exposure and was most pronounced 1 to 2 days later. SO production did not reach control levels even after 5 days. Since Cd may have induced a quick burst of SO production, AMs from control uninstilled rats were exposed to 0, 1, 10, or 100 uM Cd with the SO detection system. PMA-stimulated SO pro- duction was unaffected, but resting production showed a dose-dependent depression to 38% of control levels with 100 pM Cd. The results indicate that SO generation is not a mechanism of damage to the lung following acute CdC1Z exposure. Instead, the results further show the inhibition of oxidative metabolism by ionic Cd. 72 DMPA INCREASES 109-CADMIUM EXCRETIC{4. 9 M Aposhian, W zheng, P Tobias, K Brendl, R M MaiorinQ and H V Aposhian. Univ. Arizona, Tucson,"AZ There is a continuing search for better chelat- ing agents for the mobilization and excretion of cadmium. N-(2,3-dimercaptopropyl)phthalamidic acid (DMPA), DMPS and DMSA are water soluble analogs of BAL and are antidotes for As, Hg or Pb poisoning. The 10 day urinary excretion of 109-cadmium is increased four fold and the fecal excretion two fold when a twice daily sc injec- tion of 0.20mmo1 DMPA/kg is given to young male rats for 10 days beginning three days after the administration of 109-cadmium (1 mg/kg). The biliary excretion of Cd increases 20 fold when rats receive 0.02mmo1 DMPA iv three days after the administration of cadmium-109 (1mg/kg). DMPS, DMSA, EDTA, DDfOQr GSH did not increase biliary excretion of yCd. DMPA also increases the biliary excretion of Cd when given iv 10 min after the iv administration of 109-Cd. The increase in biliary Cd is not due to an increase in the rate of bile flow since the latter in- creases only 15% while the Cd increases 1900%. Since DMPA increases biliary GSH, it would appear that DMPA has more than one mode of action as far as its antidotal properties and differs in this respect from DMSA. (Supported in part by ES03356 and oH02185) 50875 8143 18
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-1 101 LEAD EXP~)SURE IN AN OUT~)OOR FIRING RANGFy. RK Tripathi i PC Sherertz~1, GC Llewallyn31, CW Ar,strong , AS1Phillips , and SL RzmsPy _ ~A Dept. of Health ,.VA Dept. of Llor and Industry, and VA State Police Academy , Richmond, VA. Health hazaf'ds from lead (Pb) exposures in poorly ventilated indoor firing ranges are well document- ed. However; possible concern about over-exposure to Pb in an outdoor firing range initiated an industrial hygiene and medical survey. Substan- tial over-exposures to airbor7 ~ Pb were found among personnel during firing of no:p-jaciceted bul- lets. The mean lead levels (ug/m , 8-hr TWA) in personal breathing zone samples and area samples were 122, and 68, respectively. Twelve (57%) of 21 personal breathing zone samples and 8 (44%) of 18 ar3ea samples exceeded the OSHA standard of 50 ug/m . Blood lead levels (PbBs) were found to increase substantially in all individuals after firing non-jacketed bullets, but none of the val- ues exceeded the OSHA standard of 40 ug/dl. Test of a totally copper-jacketed bullet resulted in low leveis of Pb in both personal3 breathing zone (6 ug/m ) and area samples (9 ug/m ). Analy- sis of airborne copper revealgd levels in both personal ~reathing zone (3 ug/m ) and area samples (0.83ug/m ) well below the OSHA standard of 100 ug/m . No substantial changes in PbBs were found in individuals after firing totally copper-jacket- ed bullets. We conclude that a potential health hazard due to Pb exposure existed at the range, and that the use of a totally copper-jacketed bullet reduced this exposure. 26 103 G.UPATHIONE CONTENT OF THE BILE IS INCREASED By 'I4iE ADMINISTRP.'1`ION OF 2HE MMAL CHEIATING AGENTr DMPA. W. Zheng and H V_~asken A oshian. Dept Pharm &Toxic and Dept Mol & CeTlHiol, 6niv of Arizona, Tucson, Az. 102 BIOTRANSFORMATION OF THE METAL CHELATING AGENT MESO-2,3-DIP9ERCAPinSUCCINIC ACID ZDMSA). R M Maiorino, D C Bruce, and H V Aposhian. Dept. Molecular & Cellular Bio ogy, University of Arizona, Tucson, AZ. DMSA has been used in the treatment of acute and chronic exposure to lead, mercury and arsenic compounds. Virtually nothing is known about its biotransformation. Urine from fasted normal young men (N=6) given,0.055mmo1 DMSA/Kg po was collected at various times over a 14 hr period. Urine samples, before and after electrolytic reduction treatment, were analyzed for DMSA and its biotransformants by bromobimane derivatiza- tion, HPLC separation and fluorescence detection. Metabolites were isolated by anion-cation ex- change extraction and TLC. By 14 hr after admin- istration, 18.1%±2.1 SE of the administered DMSA was excreted in the urine as altered DMSA (88% of the total DMSA found in the urine) and only 2.53% ±0.40 SE was excreted as unaltered DMSA (12%of the total DMSA found). The urinary excretion of altered cysteine versus altered DMSA at 1,2,4,6,9 and 14 hr after administration of DMSA had a correlation coefficient of 0.952 and p<0.003. Approximately 90$ of the altered DMSA is a mixed disulfide with cysteine (2 cysteines to 1 DMSA). DMSA disulfide is a minor metabolite. The DMSA- CYS mixed disulfide indicates a thiol-disulfide interchange between DMSA and cystine. The forma- tion of a water soluble DMSA-CYS mixed disulfide suggests the use of DMSA as a potential treatment for cystinuria. (NIEHS RO1ES03356). If N-(2,3-dimercaptopropyl)phthalamidic acid (DMPA), DMPS and DMSA are antidotes for poison- ing by As, Hg or Pb. After iv injection of mRle rats with 0.20 mmol DM%&, the concentration of GSH in bile is increased 100% within 1Q.m3ns. This is not due tQ an increase in bile ~low since bile volume increases only 27% during this time period. The amounts of GSH, GSSG, unaltered and altered DMPA in the bile have been determined by the bromobimane assay for dithiols. The increases o,f -D.OPA and GSH in the bile coincide. DMSA~rinjection, however, does not increase biliary GSH or GSSG and DMSA is not detected in the bile. When rats are challenged with HgC1 or CdC12 and treated with DMPA, the increase o?mercury and cadmium excre- tion in the bile may be due not only to the chelating action of DMPA but also to its activity in increasing the GSH content of the bile. (Supported in part by ES03356 and OHO2185) 104 PHYSICO-CHEMICAL PROPERTIES OF THE CHELATING AGENT DMSA AND ITS DIMETHYL ESTER. 1 Rivera, H V Aposhian, and Q Fernando. Dept Chem and Dept Mol & Cell Biol, Univ. Ariz. Tucson, Az. DMSA is becoming an important chelating agent for therapeutic use in humans. Information about its physico-chemical properties has been very limited even though such knowledge could be helpful in fully understanding its pharmacolog- ical properties and potential. The acid dis- sociation constants, complex formation constants and metal complexing properties of the ligands, meso-dimercaptosuccinic acid (DMSA) and the dimethyl ester of DMSA, CH3OOC-CH(SH)-CH(SH)- COOCH , have t~een investigated. DMSA reacts with Pb2+ or Cd + to form chelates consisting of five membered rings that have oxygen and sulfur as the donor atoms. In contrast the fiy2e membered rings formed with DMSA and Hg`2+ or Ni + have two sulfur atoms as the donors. The acid dissociation constants of the dimethyl ester are greater than thc~se of DMSA. The formation con- stants of the Ni + chelate of the dimethyl ester are comparable wit~ the values that have been obtained for the Ni + chelate of DMSA. (Support- ed in part by ES03356 and OH02185). o, m 00 J o, 00 N Ln N
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5 CYTOTOXICITY OF BENZYL 1,2,3,4,4-PENTACHLOROBUTA- 1,3-DIENYL SULFIDE (I) AND BENZYL 2-CHLORO-1,1,2- TRIFLUOROETHYL SULFIDE (II) IN ISOLATED HEPATO- CYTES. J C Veltmanl, W Dekantl, F P Guengerich2, and M W Andersl, 1) Dept. o£.Fharmacology, Univ. of Rochester, Rochester, NY, 2) Center for Molec_ ular Toxicolo*y, Vanderbilt Univ., Nashville, TN. The toxicity of the cysteine S-conjugates S- (pentachiorobutadienyl)-L-cysteine and S-(2- chloro-1,1,2-trifluoroethyl)-L-cysteine is asso- ciated with their metabolism to unstable thiols. The bioactivation and cytotoxicity.nf benzyl sul- fides I and II were studied to test the hypoth- esis that both would undergo cyt. P-450-dependent benzylic hydroxylation and that the intermediate hemimercaptals would eliminate the cytotoxic thiols. Benzyl sulfides I and II were cytotoxic in isolated rat hepatocytes; I was more cytotoxic than II. The corresponding tert-butyl sulfides were not cytotoxic. The cytotoxicity of I was increased after phenobarbital treatment and was greater in cells from male versus female rats. Benzyl sulfide I cytotoxicity was inhibited by CO and by SKF 525-A. Benzyl sulfides I and II were metabolized to benzaldehyde by hepatic microsomal fractions and by purified reconstituted cyt. P-450pB_B systems. These results support the hypothesis that the benzyl sulfides and the cor- responding cysteine S-conjugates yield unstable thiols that give rise to acylating agents or to stable, but toxic, terminal products that cause cytotoxicity. (Supported by grants ES03127, HL07475, and AFOSR 86-NL-007.) 6 'INSIGHTS INTO THE RELATIONSHIP BETWEEN _CARCINO- GEN-DNA ADDUCT FORMATION AND TUMOR LOCATION IN THE RESPIRATORY TRACT FOLLOWING EXPOSURE TO DIE- SEL EXHAUST. J A Bond, J R Harkema, J L Mauderly, R 0 McClellan, and R K Wolff. Love- lace Inhalation Toxicology Research Institute, Albuquerque, NM. r` N r-i 00 Rats exposed chronically to high concentrations of diesel exhaust (DE) have increased levels of whole lung DNA adducts and develop tumors in the lung parenchyma. We examined the relation- ship between tumor location and the distribu- tion of DNA adducts in the respiratory tract (RT) following exposure to DE. DNA adducts were measured in rat RT following exposure to a car- cinogenic concentration of DE (10 mg soot/m3) for 7 hr/day, 5 days/wk for 12 weeks. Controls were exposed to air only. The nasal tissue, tra- chea; mainstem bronchi, intrapulmonary axial airway, and parenchyma were removed. DNA was analyzed for DNA adducts using the 32P-postla- beling method. Levels of DNA adducts in tissues from rats exposed to DE were greater than those in controls. Quantities of DNA adducts ranged from -1 adduct in 109 bases (mainstem bronchi) to 2,000 adducts in 109 bases (parenchyma). Thus, a good relationship exists between tumor location (parenchyma) and level of adduct forma- tion (highest in parenchyma) in rats exposed to DE. These data suggest that DNA adduct levels in discrete locations of the RT may be good measures of "effective dose" of carcinogenic compounds. (Research sponsored by the U.S. DOE/OHER under Contract No. DE-AC04-76EV01013.) K,iLj4 7 SUSCEPTIBILITY TO 1,3-BIT'f1JIENE-INDUCED LEUKEMO- 't GENESIS CORRELATES WITH ENDOGENOUS ECOTROPIC RETROVIRAL BACKGROUND IN THE MOUSE. R D Irons, H P Cathro, W S Stillman, W H Steinhagen, and R S Shah, Chemical Industry Institute of Toxicolo- gy, Res Triangle Pk, NC, and M W Cloyd, Univ Texas Med Br, Galveston, TX. Chronic exposure of B6C3F1 mice to 1,3-butadiene (BD) results in a high incidence of thymic lyi- phoma/leukemia (TL). Endog.e,gaus ecotropic retro= virus (eMuLV) is frequently associated..,aziLh murine TL. Therefore., the influence of ret;o !- viral background on BD leukemogenesis was exa` ~= ined by comparing ' the incidence of TL between B6C3F1 and NIH Swiss mice. Proviral sequences for eMuLV are truncated' in the NIH Swiss, and the virus is not expressed. E os~re to BD (1250 ppm) for up' to one yr result'~~in marked differ- ences in the incidence of TL between B6C3F1 (65%) and NIH Swiss (14%) mice. This has previ- ously been shown to be preceded by anemia and bone marrow cytogenetic abnormalities which are indistinguishable between the two strains. In contrast, a marked increase in eMuLV replication was observed only in B6C3F1 mice. These find- ings suggest that retroviral background influences susceptibility to BD leukemogenesis. Selective eMuLV replication may correlate with TL, whereas hematologic and cytogenetic param- eters are not adequate predictors'of leukemo- genicity. The respective roles of eMuLV and BD in this leukemia model system are currently under study. DIFFERENTIAL POTENTIAL FOR EXPRESSION OF THE HARVEY-ras ONCGGENE (Ha-ras) IN B6C3F1, C3h/He, AND C57BL/6 MICE. R L Vorce and J I Goodman. Dept. Pharmacology & Toxicology, Cent. Env. Toxicol., stlichigan State Univ., E. Lansing, MI. The male hybrid B6C3F1 mouse (C57BL/6 female X C3h/He male) has a 30% incidence of spon- taneous hepatomas, whereas the male C3h/He has a 60% incidence and the female C57BL/6 has a negligible incidence. The Harvey-ras oncogene has been implicated in a wide variety of solid tumors, including hepatomas in the B6C3F1 mouse. The objective of this study was to examine a possible point of transcrip- tional control of this gene in the liver of all three mouse strains. Msp I/Hpa II re- striction enzyme analysis was used to determine the methylation state of the Ha-ras oncogene. There is a negative correlation between methyl- ation and expression of a gene. It was found that Ha-ras is hypermethylated in the female C57BL/6 mouse (5/5). By contrast, Ha-ras usually was found to be relatively hypomethylated in both male B6C3F1 (3/4) and C3h/He (3/5) mice. These results indicate that Ha-ras has a lower potential for transcriptional activity in the C57BL/6 mouse as compared to B6C3F1 and C3h/He mice.. The Ha-ras oncogene may therefore be "primed" for aberrant expression by virtue of its hypomethylated state in B6C3F1 and C3h/He mice, and this condition may facilitate hepatoma development in these animals. 2
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85 MICROSCOPIC DISTRIBUTION OF HG IN HGCL2-EXPOSED MOUSE KIDNEY. P M Rodier and B Kates, Department of Obstetrics and Gynecology, University of Rochester, Rochester, NY. Sponsor: T W Clarkson. When mice are exposed to a single dose of 203~g as methylmeitury, grain counts of autoradiographs reveal an uneven pattern of distribution in the kidney, but all regions contain significant levels of Hg. In contrast, the inorganic Hg formed over time after MeHg exposure and visual- ized by silver staining is foundj exclusively in the cells of the proximal tubules (Rodier and Kates, 1987; Rodier, Kates, and Simons, 1987). The distribution of Hg after a single dose of HgC12 was investigated by the same methods in the present study. Silver stain again demonstrated Hg only in the apical portions of the cells of proximal tubules and on the luminal surface of the brush border. As in MeHg-exposed kidney, there was evidence of active movement of Hg into kidney cells by pinocytosis and into the lumen by sloughing of cytoplasm. Although the resolution of autoradiography was not adequate to describe the intracellular placement of Hg, it was possle to confirm the exclusive localization of 28 Hg in the proximal tubules. Thus, the kidney seems to respond similarly to inorganic Hg whether its source is exogenous or endogenous. (ES 01247, 01248 and 04428). 86 HEIGHTENED VULNERABILITY TO LEAD DURING ADVANCED AGE. D A Cory-Slechta. Environmental Health' Yciences Center, Dept. of Biophysics, Univ. of Rochester School of Medicine, Rochester, NY.- While the degenerative processes comprising aging have been extensively described, little research has been directed towards understanding whether such processes may be accelerated by: exposure to toxicants like lead (Pb), an environmental contaminant to which exposure occurs over the lifespan. Using a combined cross-sectional/longitudinal study design, groups of young (21 day old), adult (8 mo) and- old (16 mo) male Fischer-344 rats were exposed to 0, 250 or 500 ppm Pb acetate in drinking water for 7 mo. Blood Pb (PbB), hematocrit, ZPP and urinary D-ALA measurements were made after 3 & 7 months of exposure; tissue Pb concentrations, organ weights and urinary Pb (PbU), Ca, Fe, Zn and Cu excretion after 7 mo. Results suggested that aged animals were more vulnerable to Pb, and that Pb exposure may accelerate aging: elevations of D-ALA and ZPP were greater, morbidity and mortality were highest, and liver weights and hematocrit declined more rapidly in old-Pb rats. Complex changes in tissue distribution with age were seen. Most notably, a marked reduction in bone and brain Pb levels occurred while other soft tissue Pb levels increased. Interactions of age by dose of Pb were noted in both Pb6 and PbU. Patterns of lead toxicity may reflect stage cf life cycle. 22 87 C01iF3INED EFFECTS OF LEAD AND AGING ON KIDNEY FUNCTION. C. Cox, G-i. Diamond and 0 A Cory-Slechta. Environmen a ealth ciences Center, r; On`iversity of Rochester School of Medicine, Rochester, NY. Degenerative changes in kidney,function over-the life span have been well documented. The kidney is also a well-known target organ for lead, an environmental toxicant to*wha-ch exposure occurs- r over the life span. The extent to which aft='- related renal impairments may be exacerbated -bat,: chronic lead exposure was the subject of this =' study. Using a combined cross-sectional/ longitudinal study design, groups of young (21 days), adult (8 mo) and old (16 mo) male rats were exposed to 0, 250 or 5A ppm.Pb acetate in drinking water for 7 months and;maintained at a body weight of about 230 gm. Kidney function was evaluated after 3 and 7 months of exposure. Characteristic age-related changes in kidney function were observed, including a decline in glomerular filtration rate and urine concen- trating ability, albuminuria and enzymuria. Pb modified the progression of age-related changes in certain parameters of kidney function, although no evidence of overt Pb nephropathy was found. Specifically, fractional excretion of alpha-amino nitrogen tincreased, and that of glucose decreased in old Pb-treated animals. These changes could not be explained by alter- ations in filtered load, suggesting combined effects of lead and aging on renal handling of glucose and amino acids. . "88 MODELING THE EFFECT OF EXPOSURE DURATION ON BLOOD LEAD LEVELS. M W Himmelstein and E J 0'Flaherty. Department.of Environmental Health, University of Cincinnati, Cincinnati, OH.. Few controlled animai studies have been perform- ed to investigate how body burden of lead influ- ences blood lead levels (Pb-B) upon removal from chronic exposure. The specific aim of this pro- ject was to assess the effect of exposure dura- tion and animal age during exposure on,post- exposure Pb-B. Two groups of weanling male Sprague Dawley rats were exposed to 1000 mg Pb/l drinking water for 31 and 60 days. Post-exposure Pb-Bs were compared to those in a third and a fourth group of male Long Evans rats exposed to 5500 mg Pb/1 drinking water for 56 days beginning at 406 days of age and 440 days beginning at weaning. Post-exposure Pb-B curves were fit to two- and three-compartment mammillary models. The three-compartment model was required to fit data for rats beginning exposure at weaning. The two-compartment model adequately fit data for rats beginning exposure in adult life. Estimates of terminal half-lives from the sum-of-exponen- tial compartmental analysis for rats beginning exposure at weaning were unsatisfactory because no linear decay constant was identifiable for the terminal compartment. A power function was nec- essary to fit the terminal Pb-B data from 1000 mg Pb/1 exposure groups. The power function has important implications-for developing a physio- logically-based pharmacokinetic model for lead. 50875 8147
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13 DIFFERENTIAL EFFECTS OF CADMIUM AND METHYL- MERCURY ON Na -K -ATPase INHIBITION BY 5,5'- DITHIOBIS-2 NITROBENZOIC ACID) AND N-ETHYL- t9ALBIMIDE. K I Ahammadsahbi and D Desaiah. Dept. of Neurology, Univ. Med. Ctr., Jackson, MS .` Previous studies from this laboratory have indicated that while CdCl affects Na+ -pump by binding to ligands 1hat are protected ATP, CH3HgC1 binds to some non-specific allosteric sites. In the present study the relative accessibility of CdCl jand CH HgC1 to the vital SH groups which 2 are sens~.tive to NEM and DTNB were determined. The reaction of DTNB with Na+ -K+ -ATPase was prevented by previous blocking of SH groups with CdC17 DTT failed to reverse NEM inhibition ofCdC12 inhibited enzyme. Although DTT can protect the enzyme against CH 3 HgC1 as well as DTNB inhibition, the protection by ATP was evident only against DTNB but not against CH 3 HgC1 suggesting that CH3HgCl reacts with an additional vital SH group than DTNB. Further, protection by DTT was evident only against CH 3HgC1 but not against NEM inhibited enzyme. However, ATP can partially protect against NEM but not against Ch 3HgC1 inhibited enzyme. All effects of CH3HgC1 and CdC12 on DTNB and NEM inhibited enzyme suggest that while CdC1~ binds to vital SH groups reacting with botFi DTNB and NEM, CH 3HgC1 appear to bind to some additional vital SH groups apart from those sensitive to DTNB and NEM. 74, Eri'ECTS OF SUBCHRONIC EXPOSURE TO A-RSINE ON IMMUNE FUNCTION AND HOST RESISIANCE. G J Rosenthal, M M Fort, D R Germolec, M F Ackermann, P Blair, K R Lamm, and M J. u i r. NIEHS, NIH, Research Triangle Park, NC. Arsine is a potent hemolytic agent primarily used in the semiconductor industry. We examined the effects of arsine inhalation on immunological parameters in female B6C3F 1 mice. Animals exposed for 14 days to 0.5, 2.5 or 5.0 ppm arsine demonstrated a dose dependent decrease in natural killer cell and cytotoxic T lymphocyte function. Macrophage function was not affected at any concentration as measured by tumor cell cytostasis nor were numbers of peritoneal exudate cells recovered following arsine inhalation. Arsine induced marked changes in splenic cellular populations. Lympha.yte percentages fell significantly in all arsine exposed groups from 83.4% in air controls to 45.6Z, 54.3%, and 73.8% in 5, 2.5, and 0.5 ppm arsine, respectively. Expansion of the splenic rubricyte population indicated that arsine induced splenic erythropoiesis. Splenic T cell percentages were significantly depressed at all cuncentrations of arsine while B cells were depressed only at the highest concentration. On a per spleen cell basis, arsine resulted in an impaired ability to respond to sub optimal concentrations of Con A while B cell proliferation in response to LPS remained unaltered. However, on a per lymphoycte basis, the lymphoproliferative data is strongly suggestive of lymphocyte hyperreactivity. Host resistance to challenge with infiuenza, PYB6 tumor cells, and B16F10 melanoma was unaffected by arsine. However, increased susceptibility to L. morrocytogmes and P. ycelii was observed. Taken together, these data show that arsine inhalation can have marked effects on the murine immune system and these effects implicate the T cell as a sensitive target. 19 75 ARSINE: TOXICITY DATA FfitOM-'SHORT-TERH INHALATION EXPOSURES. M P Moorman , R A Sloanel, B Alkins~, ~t V O'Connor2, S L Eustisl, and B A Fovler . National Institute'of Environmental Health Sciences; ZNorthrop Environmental Sciences, Research Triangle Park, NC 27709 The effects of exposure to arsine have been 'investigated in a series of inhalation studies.: Male and female Fischer 344 rats and B6C3F1 mic e. have been exposed 6 hours/day for periods ranging from 14 days to 13 weeks. AftYne concentrations . ! included levels causing no observable effectal,1-10 ppb to 250 ppb; levels•causing significant but~~: tolerated effects, .5 ppm to 5 ppm; and levels ~ which were lethal within 4 days or less, 10 ppm to 50 ppm. Chamber concentrations were monitored by a gas chromatograph and maintained within 10X of nominal levels. Body weig~s-wbich were taken weekly showed no treatment r ateq. differences. Organ weights and hematocrits, taken at necropsy, showed a substantial exposure dependent increase in spleen weights, an exposure dependent decrease in packed cell volume, and a slight increase in liver weights in the highest dose groups. Analysis of the arsenic concentration in livers taken at necropsy showed a marked exposure dependent increase. Small differences were observed in the magnitude of the effects of exposure between sexes and species. Results of these studies indicate that rats and mice respond in a similar fashion to arsine exposure under the conditions used in these studies and that inhalation exposure to this agent results in dose related tissue uptake. 76 EVIDENCE FOR OXIDATIVE DAMAGE TO ERYTHROCYTES IN RATS AND MICE INDUCED BY ARSINE GAS. P C Blair, M Bechtold, M B Thompson, C R Moorman, M P Moorman and B A Fowler, NIERS, Research Triangle Park, NC Male and female B6C3F1 mice and F344 rats, 6 weeks of age, were exposed to arsine gas (6 hours/day, 5 days/week) at concentrations of 0.000, 0.025, 0.500, and 2.500 ppm for 90 days. During the study, whole blood samples were collected at 5, 15, and 90 days. Complete hemtological profiles (which included methemo- globin concentrations only in mice at 90 days) were performed at each time point. Decreased erythrocyte counts, hematocrits and hemoglobin concentrations and increased mean corpuscular volumes, erythrocyte indices and reticulocyte counts occurred in animals exposed to 2.500 ppm at all time points and, additionally, in those exposed to 0.500 ppm after 90 days of exposure. Increased concentrations of inethemoglobin occurred in male and female mice exposed to 2.500 ppm for 90 days. Examination of blood films revealed marked polychromasia, poikilocytosis and anisocytosis accompanied by a presence of Heinz bodies. Treatment with arsine gas produced a regenerative anemia secondary to hemolysis. Glutathione levels in rat and mouse erythrocytes were reduced markedly after in vitro exposure to arsine gas for 1 hour. Data from in vivo and in vitro studies are consistent with a mechanism of oxidation of the heme iron accompanied by a denaturation of hemoglobin. 50875 8144
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157 EFFECTS OF gUINOLONES ON PROTEOGLYCANS (PGs) IN ADULT AND JUVENILE CANINE CARTILAGE. M J Palmoski, P D Williams, D A Laska, J S Bean. Bristol-Myers, -Eyracuse, NY Quinolones induce lesions in cartilage from joints of juvenile dogs. Since PGs are neces- sary for cartilage integrity and function, the effects of quinolones on cartilage PG metabo- lism were studied. Dose dependent inhibition of PG synthesis was observed in aflult canine knee cartilage s133r5es cultured for 48 h in media containing S and either ciprofloxa- cin, norfloxacin, oxolinic acid or nalidixic acid with IC50 values of 80, 85, 230 an A 290 ug/ml, respectively. PG degradation, S-PG release into the culture medium, was increased only with norfloxacin and ciprofloxacin (200% of controls at 200 ug/ml). Degradation of total PGs, measured by dimethylene blue, was not increased. Samples from juvenile dogs (18 wks) responded similarly to adult cartilage after norfloxacin exposure with respect to inhibition of PG synthesis. PG degradation was not increased. Adult and juvenile dogs were dosed orally with norfloxacin (100 mg/kg x 7 days) and after35sacrifice cartilage slices were .cultured with S for 48 h. PG synthesis was reduced to a similar extent in adult and juvenile cartilage (30% of controls), while degradation of total PGs was increased only in juvenile cartilage (287% of controls). The results suggest that quinolone effects on PG metabolism may be important in the pathogenesiss of articular lesions. 158 Bacillus thuringiensis israelensis CYTOLYTIC TOXIN: INTERACTION WITH CELL MEMBRANE. E Chow, L Shi, ill. Division of Toxicology and Physiology, University of California, Riverside, CA. Bacillus thuringiensis israelensis (BTI) is an effec-* tive agent for the control of disease vectors. This bacterium produces parasporal crystalline inclu- sions, containing 28, 65, and 130kDa protein toxins, which are selectively toxic to mosquito and biackfiy larvae. Upon alkaline solublization the 28kDa toxin is hydolyzed to a 25kDa toxin by proteases. The interaction of the 25kDa toxin of BTI with cell membrane lipids was investigated utilizing radiola- beled toxin, monoclonal antibodies and sucrose density gradient centrifugation. Once bound, it is not possible to remove the toxin even after exten- sive washing. The membrane bound toxin is, however, recognized by most of the twenty six monoclonal antibodies (9 IgG,17 IgM) tested. All of these monoclonal antibodies can decrease the cytotoxicity of the 25kDa toxin. None of the mono- clonal antibodies, however, significantly inhibits the binding of the radiolabeled toxin. Analysis of the cell membrane bound toxin by sucrose density gradient reveals aggregation of the toxin at the cell surface with an approximate molecular weight of several hundred thousand kDa. These and addi- tional data indicate the probable existence of three different domains in the toxin molecule for binding, cell lysis, and toxin aggregation. y 161 159 PURIFICATION AND CHARACTERIZATION OF A I EPOXIDE HYDROLASE FROM THE'"MITOCHON- DRIAL/PEROXISOMAL FRACTION OF MOUSE LIVER. ill and C. Chang, Division of Toxico- logy and Physiology, University of California, Riverside, CA Epoxide hydrolase activity has been reported to ~ occur in most subcellular fractifts• of mouse liver.. `The epoxide hydrolases in the microsomal an&--=~ cytosolic fractions have been purified and character--~_-zf,~: ized, however, the nature of the epoxide hydrolase(s) - in the crude mftochondrial/peroxisomal fraction is not known. Therefore the epoxide hydrolase from this fraction was purified. Crude 12,0009 pellet from mouse liver, free from cytosolic qintarFSmation, was sonicated to obtain a 105,000g solubie fraction containing 80% of the original activity. Epoxide hydrolase in this fraction was purified, using trans- stilbene oxide (TSO) as substrate, by a combination of affinity and hydroxyapatite chromatography. The purifed mitochondrial/peroxisomal epoxide hydro- lase had a native molecular weight of 56kDA, a molecular weight of 59kDa by SDS-PAGE, and a pi of 5.5. The purified enzyme was observed to be immunologically similar to the cytosolic epoxide hydrolase. The kinetics of hydrolysis of TSO were also similar. The purifed mitochondrial/peroxisomal enzyme thus appears to be very similar, if not identi- cal, to the cytosolic epoxide hydrolase. The basis for the subcellular localization of this epoxide hydrolase in the mitochondrial/peroxisomal fraction is not known and is currently under investigation. 160 40 ANATOXIN-A(S) EFFECTS ON ISOLATED MUSCLE. E G Hyde and W W Carmichael. Wright State University Dayton, OH. Sponsor: R Koerker. Anatoxin-a(s) [antx-a(s)], produced by the fresh- water cyanobacterium (blue-green alga) Anabaena flos-aquae NRC-525-17, has been shown to be an anticholinesterase with chemical/functional characteristics similar to the organophosphate anticholinesterases (ki: DFP 0.03 uM-1 min-l. antx-a(s) 1.36 uFS 1 min 1). The symptoms of' antx-a(s) intoxication include salivation, lacrimation, respiratory distress and muscle fasciculations which suggest direct nicotinic and muscarinic receptor activity along with its anticholinesterase activity. Isolated frog rectus abdominis muscle and the isolated, de- nervated guinea pig ileum were used to test for nicotinic and muscarinic activity, respectively. Acetylcholine (ACh, 0.01 to 100 uM) was used to generate a standard curve. Antx-a(s) (4 uM) was tested and showed a slowly rising spontaneous contraction in both the frog rectus abdominis and the guinea pig ileum (DFP at 1 uM has been shown to produce a similar response in the guinea pig ileum). Extensive washing brought the muscles back to resting levels and addition of 1 uM ACh showed an increase in twitch height above that elicted by ACh alone.
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33 PHENOBARBITAL (PB) PROTECTION AGAINST THE LETHAL EFFECT OF MONOCHLOROACETIC ACID (MCA) IN RATS. J G Mitroka, K R Cooner and R Snyder, Joint Graduate Program in Toxicology, Rutgers University, Phscataway, NJ. - We have previously reported on the toxicology of MCA in mice (Toxicologist 6,199,1986). In Sprague-Dawley rats injected with MCA (80 mg/kg, iv) followed 15 minutes later by either saline, phenytoin (80 mg/kg, ip), or PB (40 mg/kg, ip), the overall 24 hour survival rate for the three treatment groups was 25%, 38%, and 100%, respectively. Relative to the saline treatment group, the survival rate was significantly higher in the PB but not the phenytoin treatment group. When the order of treatment was reversed, i.e., PB or saline was administered to rats 15 min prior to MCA, the 24 hour survival rate was significantly higher in the PB (87%) group than the saline (53%) group. PB had no effect on plasma or cerebrospinal fluid levels of 14C-MCA or its radiolabeled metabolites. PB had no effect on 14C-MCA equivalents covalently bound to brain proteins. These results suggest that (1) PB is an effective antidote to MCA intoxication in rats and (2) this effect does not result from an alteration in the metabolic disposition of MCA. 34 pUR1F1CATION OF RAT LIVER MICROSOMAL ESTERASES. A Howell, D Greenway and A Parkinson. Kansas University Medical Center, Kansas City, KS. Rat liver microsomes were subjected to non-denaturing electrophoresis in 7.5% acrylamide gels containing 4M urea. Esterases were located by their ability to hydrolyze 1-naphthylacetate to 1-naphthol (which was detected by formation of an insoluble product with Fast Blue RR or freshly diazotized - pararosaniline). Liver microsomes from mature male rats contained 3 esterases, designated hydrolase A, B and C, based arbitrarily on their electrophoretic mobility (C>B>A). Liver microsomes from phenobarbital-treated rats contained a fourth esterase, designated hydrolase D, that migrated even further than hydrolase C. Treatment of liver microsomes from mature male rats (5 mg protein/ml) with 0.2-0.25% sodium cholate solubilized >80% esterase activity (measured with para- nitrophenylacetate), but solubilized <30% of cytochrome P-450 and b5. The solubilized hydrolases were further purified by fractionation with 9-25% polyethylene glycol or 50-65% ammonium sulfate. Following these procedures, hydrolases A and B were purified to homogeneity by a combination of anion- exchange chromatography (on Whatman DE-52 and DE- 53 DEAE cellulose), adsorption chromatography (on hydroxyapatite), and chromatofocussing on Pharmacia PBE 96. The highly purified hydrolases could be resolved by SDS-PAGE, and could be distinguished by differences in their substrate specificity toward carboxylic acid esters and amides. We conclude that hydrolase A and B are distinct proteins. Supported by DAMD17-86-G-6038 and NIH grant ES-00166. ---~-:- 35 DEVELOPMENTAL, TISSUE-SPECIFIC, AND INDUCER- MEDIATED EXPRESSION OF RAT CYTOCFHtOME P-450s. C J Omiecinski and C M Giachelli. Depts of Envir Hlth and Pharmacology, Univ of Wash, Seattle, WA. The cytochrome P-450s (P-450s) catalyze pivotal detoxication and bioactivation reactions in mam- malian tissues, transforming a large variety of endogenous and xenobiotic substances. To exami&e P-450 gene regulation durigg-rievelopment, RNA - ~{ hybridization analyses were performed with _WA - oligomer probes specific for the highly inducible rat P-450s: b, c,'d, and e. Whereas P-450b and___ P-450e were inducible in fetal livers by trans-' placental administration of phenobarbital (PB), P-450c and P-450d mRNAs were not detectable in fetal hepatic tissues, regard,lesar,of pretreatment. Fetal expression of P-450b ~`r P-450e was not stim- ulated by dexamethasone, nor inAbited by metyra- pone administration in newborn animals, suggesting that glucocorticoids alone are not critical for P-450b or P-450e gene expression. In untreated 1 week-old neonates, low levels of hepatic P-450d and P-450e mRNAs were apparent and, along with P-450b and P-450c, were markedly responsive to either PB (P-450b,e), or 3-methylcholanthrene (P-450c,d). The levels of these constitutive forms, as well as the response of the mRNAs to inducer pretreatment, increased with age and peaked in three week-old animals. The,expression patterns in lung, kidney or testis were highly distinctive for each isozyme, indicating the in- fluence of several modes of genetic regulation. Supported by NIH Grants GM-32281 and EH-07032. 36 ADULT BIOTRANSFORMATION OF XENOBIOTICS FOLLOWING NEONATAL EXPOSURE TO DIETH-YLSTILBESTROL (DES). C A Lamartiniere and G A Pardo. Environmental Hea t ciences, University of Alabama at Birmingham, Birmingham, AL. We have investigated the effects of neonatal exposure to DES (3 x 1.45 ti,moles) on hepatic activation/detoxicai:ion enzyme levels (males>females) in Sprague Dawley CD rats. Adult males treated neonatally with DES (DES males)- have lower endogenous levels of UDP-glucuronyltransferase than control males. Adult females treated neonatally with DES (DES females) have higher endogenous epoxide hydrolase and glutathione transferase activity levels than control females. Adult 3-methyl- cholanthrene treatment results in higher benzo(a)pyrene hydroxylase and lower UDP- glucuronyltransferase activity levels in DES males and lower benzo(a)pyrene hydroxylase and epoxide hydrolase activity levels in DES females as compared to respective controls. Adult phenobarbital treatment causes lower cytochrome P-450 and glutathione transferase activity levels in DES males and lower activity levels of all enzymes in DES females as compared with respective controls. Neonatal DES exposure therefore alters the endogenous levels of specific biotransformation enzymes (altered imprinting) and changes the metabolic response to a carcinogen and drug. 50815 8134
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117 SELECYLVE PROrTEIN ARYLATION AND THE AGE DEPEND- ED1CY OF ACETAMINOPHEN (APAP) FIEPATOTOXICITY. W P Beierschmitt, J T Brady, J B Bartolone, E A Khairallah, an Sd D Clohen. Toxicology Program, Univ. of Connecticut, Storrs, CT. _ -4 Three montfi-(M) but not 2 M old male CD-i mice are susceptible to APAP (600 mg/kg, po) hepato- toxicity. APAP metabolism and covalent binding were studied in vivo to determine if such changes correlated with the toxicity. Fasted 2 and 3 M old mice were given APAP (600 ugjkg, po) and killed at selected times for assessment of APAP metabolism. APAP concentrations in both plasma and liver, as well as hepatic glutathione depletion were similar through 4 hours after dosing for the 2 and 3 M old mice. Similar amounts of APAP and it's glucuronide, sulfate, cysteine and mercapturate conjugates were excreted by 2 and 3 M old mice 24 hours post dosing. Covalent binding of APAP reactive .r metabolite to whole homogenate was simil.a between ages at 4 hours. Im[nunochemical analysis revealed that at 4 hours after APAP, there were age-related differences in the specific proteins bound by APAP, in liver cytosolic and microsomal fractions. The age dependency of APAP hepatotoxicity in CD-1 mice is not related to metabolism differences but may result from differences in the specificity and outcome of selected protein arylation. (Supported in part by NIH grants CM-31460 and ES 07163 and a Stauffer Chemical Company Fellow- ship in Toxicology to JTB.) 118 COMPARISON OF INKRMCHEMICALLY DETECTED PROTEINS BOUND BY ACELMIENOPfEN (APAP) IN LIVER, IKIDNEY AND TA3NG -S D Cohen, W P BeierscYmitt, J B Bartolone, and E A Khairallah. Toxicology Program, t7niv. of Connecticut, Storrs, C'T. Administration of APAP (600 mg/kg, po) to fasted male CD-1 mice results in damage to the liver, kidney and lung. An affinity purified antibody for covalently bound APAP was used to identify APAP-protein adducts among electrophoretically resolved proteins from these target organs. Microsomal and cytosolic fractions were prepared from liver, kidney and lung 4 hours after APAP (600 mg/kg, po) and were resolved on 10% SDS PAGE , transblotted to nitrocellulose and analyzed inmunochemically. In the microsomal fraction, APAP was predominantly bound to proteins corresponding to approximately 44 kD in liver, and 58 kD in liver and lung. No binding was detected in kidney microsomes. APAP-bound proteins corresponding to 33,*44, and 58 kD were detected in cytosol from both liver and kidney, and only 58 kD in lung cytosol. 71m, in all,, three tissues, APAP binding to proteins was not random but highly selective. As we have suggest- ed for liver, this selective binding may also be mechanistically involved in APAP-induced kidney and lung toxicity. Distinct differences in protein arylation among the three tissues may provide mechanistic insight into APAP target organ toxicity. (Supported in part by NIH grants # (M 31460 and ES 07163.) 30 119 ANALYSIS OF COVALENT BINDING AND CHARACTERIZATION OF THE MAJOR ACETAMINOPHLN (APAP) PROTEIN ADDUCTS. J B•Bartolone, R B Birge, S D Cohen and E A Khairallah. Univ. of CT, Storrs, CT. The covalent binding of APAP to proteins has been implicated in eliciting hepatotoxicity~ We have produced an affinity purified antibody which detects electropheAtetically resolved. :t APAP-protein adducts (Biochem. Pharm. 36t'4••t93, 1987). Using this immunoassay, we recentiK ,:~-: reported that proteins of 44 and 58 kD are the major targets in livers of mice treated with 600 mg/kg of APAP. By 2-D PAGE / Western blotting, the 44 and 58 kD proteins contain clusters of 3 and 4 moietJps'wfth isoelectrio points of 7.0 to 7.1 indS"4 to 6.6, respec- tively. Although by H-NEM fluorography both clusters exhibited high protein sulfhydryl content (PSH), other proteins which contained a high PSH content did not bind APAP. Analy- sis of liver extracts derivatized directly with NAPQI resulted in a binding profile dif- ferent,from that observed in vivo with decreased relative binding to the major adducts. These data indicate that the selec- tivity and extent of APAP arylation in vivo may depend upon the intact cell structure, the protein thiol affinity, and the cellular localization of the proteins. (NIH GM-31460). 120 IMMUNOCHEMICAL DETECTION OF 2,6-DIMETHYL ACETAMINOPHEN (2,6-DMA) PROTEIN ADDUCTS. R B Birge, J B Bartolone, S D, Cohen, and E A Khairallah. Univ. of CT, Storrs, CT To evaluate the mechanistic importance of cova- lent binding in acetaminophen (APAP) induced hepatotoxicity, we have compared the effects of 2,6-DMA to those observed with APAP. In cul- tured mouse hepatocytes 10 mM APAP is cytotoxic and, by immunochemical analysis, can be shown to bind to a few specific proteins. By contrast, at equimolar concentrations 2,6-DMA is not cyto- toxic yet radiometrically binds 30% as much as does APAP. The differences in toxicity between these compounds may reflect the differences in the specificity of the targetted proteins. Because the anti-APAP antibody exhibited no anti-2,6-DMA activity, an antibody specific for 2,6-DMA was constructed by covalently linking 2,6-DMA via a p-amino benzoio acid linker to keyhole limpet hemocyanin. Following immuniza- tion with that antigen, antibodies specific for :2,6-DMA were purified using a 2,6-DMA affinity Sepharose 6B column. Using 2,6-DMA covalently bound to BSA, we have demonstrated that the anti-2,6-DMA antibody can detect protein-bound 2,6-DMA by solid phase ELISA and Western blot- ting. This new antibody will enable the compar- ison of the binding specificity of 2,6-DMA to that of APAP and may help elucidate the "criti- cal" versus "non-critical" nature of the protein targets. (NIH GM-31460). 50875 8155
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NEPHROTOXICITY OF ACETAMINOPHE ~~HE4 RAT - 131 POTENTIAL INVOLVEMENT OF A REVERSIBLE INHIBITION EFFECTS OF ANTIDOTES. W Molle~ rtmann, and C-P OF M_ITOCHONDRIAL RESPIRATION IN Sie ers, Institute of Toxicology, Medicaf- ACETAMINOPHEN-INDUCED METABOLIC ACIDOSIS AND m versity.of LUbeck, Llibeck, FRG. COMA. R Esterline and S Ji. Rutgers University, Piscataway, NJ Metabolic acidosis:gnd coma have recently been associated with severe overdoses of acetaminophen (AA) in humans in the absence of overt liver damage. AA (2-25mM) and its nonhepatotoxic isomer, 3-hydroxyacetanilide (3HAA), have been shown to inhibit NAD-linked respiration in rat liver mitochondria arid cause an increase in glucose and lactate output from the isolated perfused rat liver. Both isomers of AA cause profound alterations in serum glucose and lactate levels in vivo. Following administration of either isomer (1 g/kg ip in saline), serum lactate increased from 33.0 + 2.8 mg/dl (mean ± SEM; n=6) to 84.0 ± 5.4 mg/dl one hour after AA and to 78.8 + 4.5 mg/dl one hour after 3HAA. Serum glucose increased from 189.2 ± 4.1 mg/dl to 460.1 ± 35.1 mg/dl one hour after AA and to 392.6 ± 9.0 mg/dl one hour after 3HAA. Glycogen levels in the livers of AA and 3HAA treated rats one hour post-dosing were also diminished. Serum AA and 3HAA concentrations ' one hour after treatment were 5.4 ± 0.4 mM and 5.9 ± 0.5 mM, respectively. Similar concentrations of acetaminophen have been reported following overdoses in humans. These studies suggest that AA and 3HAA cause toxicity by inhibiting NAD-linked mitochondrial respiration. Supported by AA5848. 130 CYTOCHROME P-450-INDEPENDENT A_CETAMINOPHEN HEPATOTOXICITY IN ACUTELY ALCHOHOL TREATED RATS. S Ray, R Esterline and S Ji Joint Graduate Program in Toxicology, Rutgers University, Piscataway, NJ When acetaminophen (AA) (2-25 mM) is infused into isolated perfused livers from rats acutely treated with ethanol (E) (4g/Kg for 1-4 hrs), the rate of hepatic 02 uptake was inhibited in two steps - the rapid inhibition complete within 2-3 minutes (19 ± 1.39'o basal respiration inhibited) (mean ± SEM, n=5) and the slow inhibition proceeding at a rate of 79±119'o basal respiration inhibited per hr (n=8). The slow inhibition was (1) negligible in livers from control fed rats, (2) unaffected by metyrapone (.5 mM), (3) accompanied by lactate dehydrogenase leakage, (4) blocked by 10 +TM mannitol, (5) dependent on extracellular Ca , and (6) abolished by hypophysectomy and thyroidectomy. Since (1) the granulocyte content of the liver increased by 3 fold upon pretreating rats with E for 4 hrs, (2) an incubation of human neutrophils with H-acetaminophen caused covalent binding of AA metabolite (likely N-acetyl-p-benzoquinoneimine (NAPQI)), and (3) synthesized NAPQI irreversibly inhibited 0 uptake by isolated mitochondria, we postulate tRat the AA-induced slow inhibition of hepatic respiration is in part due to NAPQI formed in granulocytes which then enter hepatocytes to covalently inhibit mitochondrial enzymes. Supported by AA5848. Antidotes known to prevent acetaminophen-induced hepatotoxicity were tested for their ability to protect against acetaminophen-induced renal failure. Male Wistar-rats were treated with acetaminophen (0.5-1.5 g/kg p.o.), liver and kidney damage were monitored by measuring plasma > aminotransferase and sorbitol,,dehydrogenase, = ure$, creatinine as well as urinary enzyme ~_ excretion of N-glutamyltranspeptidase and N-ace- ` tyl-B-glucosaminidase over 4 days. Methionine (0.5 g/kg p.o.) partially reduced acetaminophen hepatotoxicity, but had no effect on the nephrotoxicity; a lower dose of methionine (0.15 g/kg p.o.) even enhanced the -nqphrotoxic response to acetaminophen. With N~cet~Tcysteine (1.0 g/kg p.o.) a safe hepatoprotection was seen, kidney damage, however, was only partially reduced. Only diethyldithiocarbamate (200 mg/kg p.o.)- was capable of totally preventing acetaminophen-induced hepato- and nephrotoxi- city. It is concluded, that antidotes known as precursors of GSH-synthesis (methionine, N-acetylcysteine) are ineffective in protecting against acetaminophen nephrotoxicity. Inhibition of microsomal bioactivation of acetaminophen by dithiocarb resulted in a complete protection against hepato- and nephrotoxicity. 132 ASCORBIC ACID ESTERS PROTECT AGAINST ACETAMINO- PHEN (APAP) HEPATOTOXICITY IN MICE: POSSIBLE ROLE IN GLUTATHIONE (GSH) REGENERATION. A K.Mitra and V C Ravikumar. Division of Pharmacology ~ Toxicology, School of Pharmacy, Northeast Loui- siana University, Monroe, LA. 33 APAP is primarily excreted in the urine as sulf- ate and glucuronide conjugates. Following over- doses, a reactive, electrophilic metabolite of APAP causes liver GSH depletion with subsequent centrilobular necrosis. Continued availability of hepatic GSH is, therefore, considered a key factor in attenuating hepatotoxicity. .Groups of adult, male SW mice,were gavaged with (i) APAP alone, (ii) APAP + Ascorbyl Palmitate (AP), or (iii) APAP + Ascorbyl stearate (AS), at a dose of 600 mg/kg of each drug. Hepatic GSH levels (15 min - 2 hr) and sulfobromophthalein (BSP) excretion were monitored for all groups. GSH levels were significantly decreased in all groups compared to vehicle controls. However, starting 4 hrs post-dosing, the levels in AP and. AS treated groups recovered and reached control values in 12 hours. At 30 min post-dosing, APAP alone caused significant BSP retention; plasma BSP levels in the vehicle, AP and AS treated animals were undetectable. Since BSP is known to be excreted as a GSH con- jugate, we suggest that ascorbyl palmitate and ascorbyl stearate provide protection against APAP-hepatotoxicity by increasing GSH turnover. ;y'
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189 MODULATION OF )Lf,PP+ ~TEUROTOXICITY iN VITRO. S J Simmons and M F D-Notter. University of Rochester, Rochester, NY Sponsor. V G Laties - ~s A mouse adrenergic neural tumor cell line (N2 AB-1) was used as an in vitro model to investigate the toxicity of _ 1- methyl-4-phenylpyridinium iodide (MPP+), the active metabolite of 1-methyl-4-phenyl-1,2,3,6 tetrahydropyri- dine(MPTP). Cells were differentiated by P(~El/dbcAMP to induce a neuronal phenotype. Both mitotic anddifferentiated cells were exposed to a moderate concentration of MPP+ (34,uM) for 24 to 48 hours. Differentiated cells were less sensitive to the toxic effects of MPP+ than mitotic cells, as determined by morphologic observation. MPP+ toxicity manifested itself morphologically by increased cytoplasmic inclusions(or vesicles), cytoplasmic blebbing and cell death, noted as an increased number of floating cells and debris. Gangliosides(GA), a neurite promoting factor in the CNS, have been shown to protect neurons in vivo from MPP+ toxicity. Cells were treated with 200u g/ml mixed GA. GA induced neurite formation in both mitotic and previously differentiated cells. Mitotic cells appeared to be significantly protected from MPP+ toxicity with 24 hour GA pretreatment; while differentiated cells showed slight morphologic protection during the same time period. Tubulin, a structural protein in neurites, was observed for disturbances by MPPf; none were seen until the cells were near death. Tetanus toxin immunofluorescence confirmed GA interaction with the cell surface. Neurotransmitter storage and uptake in N2AB-1 cells was confirmed by glyoxylic acid induced histofluorescence. 190 SPECIES DIFFERENCES IN THE SUBSTRATE AND I_NHIBITOR SPECIFICITY OF BRAIN ACETYL- CHOLINESTERASE. J R Kemp and K B Wallace. Dept. of Pharmacol., Univ. of Minnesota, Duluth, MN. Rodent brain acetyicholinesterase (AChE) is more sensitive to ' vi r inhibition by diethyl-p-nitrophenyl phosphate than are fish preparations. This investigation assessed differences in the relative size of the catalytic site in rat and rainbow trout detergent- solubilized brain.AChE by examining the kinetics of hydrolysis for thiocholine substrates (acetyl-, proprionyl-, and butyryl-) and the inhibitor potencies for a series of dialkyl-p-nitrophenyl phosphates (dimethyl-, diethyl-, dipropyl-). The Michaelis-Menten constants (Km) for rats were determined to be: acetyl- (78.9 ± 4.7 µM), proprionyl-(83.3 ± 4.2 µM) and butyryl-(337.8 ± 40.5 µM). The corresponding values for trout were: 202.0 ± 54.1 µM, 2078.5 ± 203.2 p.M and not detectable for the butyryl-analog. The inhibitor data revealed similar trends and species. differences: the IC50 for dimethyl- and diethyl- were comparable for rats whereas in trout the ICSo increased progressively with increasing methylene substitutions. These data are consistent with the exclusion of large substrates and inhibitors by the relatively small catalytic site of fish AChE and may have important implications regarding the comparative toxicity of anticholinesterase agents of varying molecular size. (Supported in part by U.S. EPA CR-810963 and by a grant-in-aid from the Univ. of Minn. Graduate School.) 48 191 NEUROCHEMICAL PITUITARY-HYPOTHAS.AMUS-PINEAL , MEASUREMENTS IN STEERS GRAZED ON ENDOPHYTE' INFECTED FESCUE. J K Porterl L B Lipham2, ,T A Stuedemann3 and F N Thompson1. 1R. B. Russell Agricultural Research Center, USDA/ARS, Athens ; ,_ GA., 2University of Georgia, College of O, Veterinary Medicine, Athens, GA and 3Southeru Piedmont Conservation Researcbweenter, USDA/ARS::! . 'D7atkinsville, GA. Sponsor: W P. Norred. , ~^__ Endocrine mechanisms associated with fescue summer toxicity in steers (n-6, each treatment) grazed on endophyte infected vs. endophyte free fescue have been studied. Reduced prolactin (9.28 vs. 30.49 ng/ml) and av#a`ge°-`daily gains (-0.15 vs. +0.57 lbs) have'been correlated (P<0.05, t-15 wks) with elevated anterior pituitary dihydroxyphenylacetic (108 vs. 59 ng/g) and 5-hydroxyindoleacetic acids (265 vs. 148 ng/g) (major metabolites of dopamine, DA, and serotonin, 5HT, respectively). In addition diurnal pineal 5-hydroxytryptophan (5HTP, precursor to 5HT) was elevated (502 vs. 280 ng/g, n=4) while the 5HT/5HTP ratio was depressed (0.589 vs. 0.879). No significant differences were observed in the neurochemicals measured in the hypothalamus. The results suggest a possible pituitary-pineal (indoles) involvement in the seasonal fescue summer toxicity observed in steers grazed on endophyte infected fescue. 192 EFFECT OF C:YANIDE ON BRAIN A_NTIOXIDANT ENZYMES AND LIPID P_EROXIDATION. G E Isom, J L Borowitz _ and B K Ardelt. Dept. of Pharmacology & Toxicology, School of Pharmacy and Pharmacal Sciences, Purdue University, West Lafayette, IN. Accompanying inhibition of cytochrome oxidase,. CN initiates a series of intracellular events resulting in cellular injury. CN alters neuronal Ca2+ homeostasis resulting in accumulation of cytosolic Ca2+ and subsequent peroxidation of lipids via a Ca2+-activated xanthine oxidase mediated process. In the present study, the status of the brain antioxidant enzymes in mice intoxicated with KCN was correlated with lipid peroxidation. Thirty min after the administration of KCN (7 mg/kg, sc), activity of brain superoxide dismutase (SOD), catalase (CA), glutathione peroxidase (GP) and glutathione reductase (GR) were significantly reduced (percent inhibition: SOD 16%, CA 44%, GP 23%, GR 38%) . Reduction of enzyme activity was accompanied by lipid peroxidation of brain membranes as measured by the conjugated diene method. These intracellular responses may partially account for the neurotoxicity of CN. (Supported in part by PHS grant ESO4140). m m 00 J Ln co N ~ w P a
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THE EFFECTS OF 5-THIO-D-GLUCOSE, N-2- MERCAPTO-GLYCINE, AND OTHER SULFHYDRYLS ON THE BINDING OF MERCURY I1P'BRAIN AND OTHER TISSUES OdF MICF. K Amoako-Ahabio.. Department of Pharniocodynamics, University of Oklahoma College of Pharmacy, Oklahoma City, OK. Sponsor: J A Rieger. The affinity of methyl mercury r4dicals for sulfhydryl compounds is often`great- er than that for proteins. To examine this effect, several sulfhydryl com- pounds, including N-2-m.ercapto-glycine and 5-thio-D-glucose, were injected into mice that had been given methyl mercury chloride per os 72 hours earlier. Blood, brain, liver and kidney samples of ho- mogenates were analyzed for concentra- tion of free methyl mercury by gas chromatography with an electron capture detector. N-2-mercapto-glycine and 5- thio-D-glucose were much more effective in releasing methyl mercury from binding sites in mouse brain and liver than were any of the other six compounds. The levels of total methyl mercury chloride were lowest in tissue samples of mice injected with 2,3-dimercapto-succinic acid. The results of the study indicate there is considerable variation in the ability of sulfhydryl compounds to release methyl mercury from the bound state. 06 pH AND CITRATE EFFECTS ON BIOAVAILABILITY OF AL- UMINUM (Al) FROM DRINKING WATER (DW). B Fulton, S Jaw, and E Jeffery. U. of Illinois, Urbana, IL. Present in our DW as hydroxide or sulfate, Al is limited by solubility to 2.5 mg/L at pH 7.0. Al given at 100 mg/kg rat/day only accumulates in bone or brain if citrate (C) is co-administered (Slanina et al. Fd.Chem.Toxic. 22:391,1984). We determined if Al, in doses found in DW, accumu- lates in rats, when C at neutral or acid pH is co-administered, or at acid pH in the absence of C. Al, as (OH) or C13, was given ad lib. in DW to male S-D ra2s at 0, 0.1, 2.0, or 100 mg/L, in 4 mM acetate pH 3.2(A), 4 mM C pH 2.6, 4 mM C pH 7.0(C7) or distilled water pH 7.0 (W). Growth rates did not differ between groups. Rats given 100 mg Al/L drank significantly less. Thus the DW dose range was approx. 0, 0.01, 0.2, or 5.5 mg Al/kg rat/day. After 10 weeks, rats were killed and tissues wet-ashed in nitric acid for determination of Al by graphite furnace AA. Al in tibia, brain, and liver of W rats were 8.57 ±.94, 5.25 ±.88, and 5.57 ±1.01 {ig/gww, respec- tively. In rats receiving 100 mg Al/L tibia Al levels were 9.83 ±1.76, 10.87 ±2.38, and 11.84 ±1.08 pg/gww in W, A, and C rats respectively. Thus, mean tibia Al was increased in all 100 mg Al/L rats, but the increase was not significant. No consistent increases were observed in other tissues. Results suggest that Al, up to 40x that found in DW, does not accumulate significantly in tissue, regardless of acidity or citrate. Supported by Water Resource Center, USGS. 27 107 METAL INHIBITION OF CALMODULIN ACTIVITY TN MONKEY BRAIN. R Nath, P J S.Vig and D Desaiah, Dept. of Biochem. PGIMER, Chandigarh, India and Dept. Neurol.Univ. Miss. Med. Ctr. Jackson, MS. Metals have been shown to alter calmodulin ~ (CaM) activity in various tissues. The pre- ~ sent studies were conducted*mto investigate - ther effect of aluminum (Al) cadmium (C d).a...., lead (Pb), manganese • (Mn), mercgy (Hg) and vanadium (V) on CaM regulated Ca ATPase ~ activity in Rhesus Monkey Brain (RMB).Cerebral cortex of RMB was dissected and homogenized in 10% sucrop buffer and synaptosomes were prepared. Ca ATPase activity Was+4determined by inorganic phosphate method. 'Different concen- trations of metal ions were incubated with RMB fractions containing endogenous CaM and without CaM. The results show that no metal ion inhibited the basel enzyme b2$ showed a profound inhibition on total Ca ATPase containing CaM. The order of potency of metals was Hg > Cd > Pb> Mn> Al > V. Exogenous addition of CaM restored the metal inhibited enzyme activity to near normal level. The basal enzyme when reconstituted with CaM showed sensitivity to metal ions. These data indicate that the metal ions alter CaM activity in RMB. (D. D. was supported by UNDP/TOKTEN Program). 108 M.ULTIELEMENTAL ANALYSIS OF T H E HEPATOPANCREAS OF $ELENIUM-EXPOSED 5-UNFISH. E M B Sorensen. T L Bauerl, and M G Krausel. College of Pharmacy, Department of Pharmacology and Toxicology, 1Nuclear Engineering Teaching Laboratory, Department of Mechanical Engineering, University of Texas, Austin, TX. The concentrations of zinc (Zn), iron (Fe), mercury (Hg), and selenium (Se) were determined in the livers (i.e. hepatopancreas) of redear sunfish (Lepomis microlophus) from Martin Lake (Se-contaminated) and a reference Lake (Lake Tyler). Selenium concentrations increased four-fold (p<0.001) and Zn levels increased 1.3 times (p<0.001). The level of Fe decreased from 450 ppm in livers of Lake Tyler fish to 240 ppm in Martin Lake fish (p<0.001). Martin Lake fish form a distinct population of fish compared with the Lake Tyler fish when compared on a multielemental basis. The average Se-contaminated sunfish from Martin Lake accumulated 8 ppm Se, 30 ppm Zn, and 240 ppm Fe. Whereas, the reference average sunfish from Lake Tyler accumulated 2 ppm Se, 20 ppm Zn, and 450 ppm Fe. Therefore, despite the passage of nine years since the initial discharges of Se-contaminated particulate wastes (i.e. fly ash, scrubber sludge, and bottom ash) into Martin Lake and despite the reduction in the discharge of selenium, hepatic levels of a number of elements were altered compared to that of reference sunfish. U1 m 00 a L7 OD Y Csi N
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109 jUSTOPATHOLOGICAL ALTERATIONS IN TELEOSTS FOLLOWINGaELENATEEXPOSLTRE. C S Boecker and E M B Sorensen. College of Pharmacy, Division of Pharmacology and Toxicology, iFffiVersity of Texas, Austin, TX. a - . To simulate environmental exposures of Lepomis microlophus (redear sunfish) to selenium at cooling reservoirs at electric generating stations, a laboratory exposure of this same species was conducted. Young adults were exposed for 7 or 14 days to sodium selenate at 100, 45, 10, 1, or 0 ppm selenium. Liver (i.i. ,hepatopancreas) was prepared for optical and electron microscopy. The mesonephros (i.e. kidney), heart, gills, ovaries, testes, and spleen were prepared for optical microscopy. Livers showed focal necrosis, vacuolation, fatty infiltration, and architectural disorganization. Kidneys showed proliferative glomerulonephritis, perigiomernlar fibrosis, and degenera- tion of proximal convoluted tubule cells. The severity of these alterations increased with exposure to higher concentrations of selenium. Other organs showed minimal histopathological alterations. These changes were consistent with those observed following environmental exposures of this or congeneric species to selenium in Martin Lake and Belews Lake. 110 THE EFFECTIVENESS OF VARIOUS oC-RETOCARBO%YhIC. ACIDS IN PREVENTING SULFIDE-INDUCED LETHALITY. A S Hume and M D Dulaney, Jr. Department of Pharmacology and Toxicology, University of Mississippi Medical Center, Jackson, MS The effectiveness of several a-ketocarboxylic acids in reducing the lethality of sodium sul- fide (37.5 mg/kg, i.p.) was examined in male CD-1 mice (Charles River). This dose of sul- fide resulted in 100% lethality in these mice. Equimolar (0.45 M) concentrations of pyruvic acid (PYR), a-ketobutyric acid (M), a-keto- valeric acid (AKV), a-ketoglutaric acid (ARG), and oxaloacetic acid (OXA) were administered (i.p.) 15 min prior to the sulfide. PYR re- duced the lethality from 100% to 15% while ARB reduced the lethality to 40%. OXA reduced the lethality to 20%, however, OXA itself proved to be toxic. ARV only reduced the mortality to 70% and ARG had no effect on mortality. PYR provided the best protection of all the a-ketocarboxylic acids tested with no visible signs or symptoms of toxicity. This data suggests that PYR may be an effective measure against sulfide-induced lethality and that further studies should be undertaken to deter- mine its value as a therapeutic agent. (Supported by 5M01 RR02303.) 28 111 PYRUVIC ACID PROTECTION AGAINST SULFIDE LETHA- LITY,. M D Dulaney, Jr. and A S Hume, Dept. of Pharmacology and Toxicology, University of MS Medical Center, Jackson, MS Sulfide is a deadly industrial toxin to which many workers are constantly at risk. At pre- sent, treatment is symptomatic using sodiumik nitrite and oxygen but tlAo,,is controversial= '! and there is no generally accepted treatmeag., ' The protective actiom of pyruvic acid against sulfide lethality was examined in male CD-1-~_:- mice (Charles River) of two different weights (22-30 g and 33-45 g). The lethality curve of sodium sulfide (i.p.) was significantly differ- ent between the two groups Qf- mice with the larger more resistant to •'ls"ulfide. Pyruvic acid (1 g/kg, i.p.), 15 min prior to sulfide, shifted the lethality curves for both groups of mice to the right with a protection factor of 1.8 for each group. This protection factor is the same as that for nitrites. Pyruvic acid also increases the effectiveness of the current therapy for sulfide poisoning, sodium nitrite. The time course of pyruvic acid protection was also examined. Pyruvic acid (1 g/kg, i.p.) reduced the lethality of sul- fide (37.5 mg/kg, i.p.) from 100% to 5R at 15 min, 40% at 20min, and 50% at 30 min. This data suggests'that pyruvic acid may be a safe and effective new antidote against sulfide poisoning, both prophylactically and anti- dotally.(Supported by 5M01 RR02303.) 112 ABSORPTION AND ELIMINATION OF I2 AND I IN THE RAT. K D Stout and R J Bu17. Pharmacology/Toxicology ro~ gram, College of Pharmacy, Washington State University, Pullman, WA. Use of iodine as a drinking water disinfectant for extended space flight raises questions about its chronic toxicology. Whether the chemical form of iodine modifies the uptake of 125-I was investigated. The uptake of 125-I into blood was measured over a 72 h interval following a gavage dose in fasted (F) and non-fasted (NF) animals. Excretion of 125-I was followed in urine and feces. At 72 h, thyroids were removed and counted. Blood I- and I values peaked at 2 h in both F and NF an4als. Urinary excretion paralleled blood concentration. The uptake of I- into the thyroid gland at 72 h was greater than the uptake of 12 = 0.03 in NF). In separate experiments, the rate of uptake of 125-I was measured in.the thyroid. The rate of uptake of I- was greater than the rate of I (P = 0.02 in F). The difference in thyroid &ptake can be related to higher amounts of I retained in the stomach contents and stomaC2h wall. This indicates that I is less available for uptake in the th§roid apparently due to decreased absorption. (Supported by NASA Grant No. NAG 9-226). 50875 8153
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PYRETHROID INSECTICIDES ALTER' MEMBRANE POTENTIAL IN FISH AND RAT BRAIN BYNAPTOSOMES. J T Eells and P A Bandettini. Medical College of Wisconsin, Milwaukee, WI. Sponsor: M~ V ini. , a. Pyrethroid insecticides modify the ionic permeability of nerve membranes producing distinctive poisoning syndromes in insects, fish and mammals. Fish are more sensitive this than mammals to pyrethroids. The aim o'fs investigation was to compare the actions of the pyrethroid, deltamethrin (DM), on membrane potential in trout and rat brain synaptosomes. Accumulation of 3H-tetraphenylphosphonium (TPP) was used to estimate membrane potential. In trout synaptosomes, DM decreased TPP accum- ulation in contrast to its actions in rat synaptosomes where an increase in TPP accum- ulation was observed. These data indicate that DM depolarized fish synaptosomes and hyperpolarized rat synaptosomes. The hyperpolarizing effect of DM in rat synapto- somes was reversed by the addition of the sodium channel activator, veratridine (Vtd), .resulting in a decrease in TPP accumulation greater than that observed with Vtd alone. Fish •brain synaptosomes were more sensitive to the depolarizing actions of DM and aconitine than rat brain synaptosomes suggesting a species difference in sodium channel function. The mechanism of the unusual hyperpolarizing action of DM on rat synaptosomes is under , investigation. (ES 01985, NSF-OCE 8713027) '94 EFFECT OF TRIORTHOCRESYL PHOSPHATE (TOCP) ON MEMBRANE BOUND ATPases IN HEN BRAIN AND SPINAL CORD. J A Wisler, H R Besch,Jr., and R_B Forney.Sr., Indiana University School of Medicine, Indianapolis, IN. The mechanism for organophosphorus-induced delayed neuropathy (OPIDN) is unknown; but inhibition of neuropathy target esterase (NTE) has been correlated with the development of OPIDN. Previously we reported that NTE activity was positively associated with Ca2+/K+-ATPase (CRA) in canine cardiac membrane vesicles (MV) and subfractions. Therefore we undertook to evaluate NTE, _CRA, and Na+/K+-ATPase (NKA) activities in MV from nervous tissue, taken from brain and spinal cord of adult white leghorn hens dosed orally with TOCP (750mg/kg) or corn oil and sacrificed at 24 and 72 hours later. NTE activity was significantly decreased in brain and spinal cord at 24 and 72 hours (p<0.01). NKA remained unchanged from control values at both times and in both tissue preparations. CKA was found to be significantly increased (p<0.05) in brain preparations 24 hours after dosing, but had returned to control values by 72 hours. Spinal cord CRA activities were unchanged from control at both time points. Although statistically significant, the small change in CKA activity may not be of pathologic significance. These data suggest that alterations of NAK and CAR may not be involved in the initial development of OPIDN. 195 ALTERED PHOSPHORYLATION OF PHOSPHOLIPIDS IN HEN SCIATIC NERVE BY TRI-o-CRESYL PHOSPHATE: POS- SIBLE ROLE IN ORGANOPHOSPHORUS-INDUCED` DELAYED NEUROPATHY (OPIDN). C N Pope* and S Padilla. Neurotox. Div., EPA, RTP, NC. Some organophosphorus compounds can induce a delayed progressive degeneration of certain long tracts in the central and peripheral nervous systems (OPIDN). We examiued the ef- fect~ of TOCP (590mg/kg, p.o.) on in vitro labeling of hen sciatic nerve phospholipids ei- ther 2 or 7 days after,administration. Nerve segments were incubated for 30 or 60 min in oxygenated, physiological buffer containing 20- uCi [32P]phosphoric acid and lipids extracted. Extracts were separated by TLC, lip ds .)~dentif- ied by autoradiography and counte by `liquid scintillation. There was a tendency1for all lipids to be phosphorylated less in the TOCP-treated nerve with a significant reduction (p<.025) at 7 d post-TOCP after 60 min incuba- tion. When individual lipids were examined at either 2 or 7 days post-TOCP, phosphatidlyinositol (PI) labeling appeared to be reduced at 30 min but similar to controls at 60 min; apparently, the rate of labeling of PI was increased in the TOCP-treated nerve: Because no change was seen in total phospholipid concentration in the nerves after TOCP treatment, changes in phosphorylation may indicate altered turnover of phospholipids. The observed changes in lipid phosphorylation following OP exposure are early events and may be involved in the pathogenesis of OPIDN. (*Supported by NRC Research Associateship) 196 EFFECT OF DIISOPROPYL PHOSPHOROFLUORIDATE (DFP) ON AXONAL TRANSPORT IN THE CAT. C D Carrington, D M Lapadula, and M B Abou-Donia. Duke University Medical Center, Durham,.NC. Experiments were performed to assess the role of axonal transport in organophosphorus compound- induced delayed neurotoxicity (OPIDN). Cats were dosed with 5.0 mg/kg DFP, s.c. which resulted in over 80% inhibition of neurotoxic esterase in brain and produced a mild to severe hindlimb ataxia. Proteins were labeled by injecting 35S- methionine into both L7 dorsal root ganglia. In one experiment, the sciatic nerve was doubly ligated 24 hours after injection. After an addi- tional 24 hours, accumulation was measured both proximal and distal to the ligated area. An increase of about 100% was observed on both sides. No significant difference was observed between control and treated animals. The retro- grade accumulation was too variable to be indica- tive. In a second experiment, the radioactivity per mg tissue was measured in 9 mm segments along the entire sciatic nerve and the tibial and pero- neal branches (19-25 cm). In control animals and animals treated 4 days prior with DFP, the labeling decreased along the sciatic nerve and then increased in the branches. Animals treated 7 (n=6) and 14 (n=7) days prior exhibited a simi- lar pattern except that there was little or no increase at the distal end. The dpm/mg in the distal nerve was significantly less (about 50%) in the 7 and 14 day animals when compared to either the proximal or middle portions of the nerve. Supported by NIOSH grant OHO 2003. 49
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181 fOLCHICINE-INDUCED ALTERATIONS IN THE STIMULATED TURNOVER OF INOSITOLPHOSPHATES IN THE RAT H_IPPO- CAMPUS. Pushpa Tandon, J.G. Harry, and H.A. Tilson. NIEHS, Research Triang:~e Park, NC. . Administratior o1'.colchicine into the hippocamp- us has been reported to produce a reduction in 3H-Quinuclidinyl B.enzilate [QNB] binding and an increase in choline acetyltransferase (CHAT) activity. The muscarinic cholinergic receptors are known to be linked to the inositolphosphate system inside the cell. To study theli;ime- dependent effect of colchicine on agonist- stimulated turnover of inositolphosphates, col- chicine was injected into the dentate gyrus of the hippocampus. The rats were sacrificed 1 wk, 3 wk, or 12 wk post treatment. The hippocampi were removed and sliced. Carbachol, a choliner- gic receptor agonist, was used to study the stimulated turnover of inositolphosphates. Col- chicine was found to alter the normal pattern of inositolphosphate turnover as.early as 1 week after administration. The degree of carbachol- induced stimulation of inositolphosphates was also found to be lower in colchicine treated rats at the same time periods. Thus, colchicine appears to alter the signal transduction process in the rat hippocampus. These changes may be associated with the compensatory changes associ- ated with the damaged hippocampus. 182 EFFECTS OF ALUMINIUM ON CHOLINERGIC,NEURONES IN RAT BRAIN REAGGREGATE CULTURES. P iN Collins & C K Atterwill. Sponsor: G Leslie. Smith Kline & French Research Ltd., The Frythe, Welwyn, Herts, U.K. The neurotoxic effects of aluminium and its association with dementia is a subject of great interest. Aluminium plays a role in dialysis encephalepathy and is found in the senile plaques of Alzheimers Disease. Its effects may be mediated through the cholinergic system, this .being confirmed in rat brain synaptosomes where high affinity choline uptake (ChU) and glucose metabolism were inhibited. We have now exposed foetal rat brain reaggregate cultures from 9 DIV for 96 h to 0.1 mM and 0.01 mM A1C1 and measured choline acetyltransferase ictivity (ChAT) at different time-points. Exposure for 48h produced no effect on ChAT but after 72 and 96h exposure persistent 30-40% losses of ChAT activi't;y (P<0.01) were found. (Control ChAT activities ranged between 40-50 pmol/min/mg protein). These data are consistent with chronic AlCl exposure producing a cholinergic functional dificit or neuronal loss. The IC50 for.inhibition of synaptosomal ChU is 0.5mM although 0.1mM A1C13 as used here depresses glucose metabolism, and carbachol-stimulated inositol phosphate release. The degree of selectivity of this lesion and extent of neuronal loss will be delineated, e.g. by measuring neurofilament protein. 46 ~~. 183 STUDIES ON ECMA-INDUCED._HOLINERGIC LESIONS AND NEUROTROPHIC FACTORS IN RAT BRAIN REAGGREGATE CULTURES. C K. Atterwilr, P Coilins', A Pillar and A Prince Sponsor: G Leslie. - Dept Toxicology, Smith Kline & Frenc Fi eT~search Ltd., Welwyn, U.K. and Dept. Pharmacology, Kings College, London. We have recently shown that at low ~ concentrations in vitro (12 5 -%1Q ECMA roduces ` . .. p , dholinergic lesions in rat brain reaggregate .~ cultures in a serum-supplemented (S+) medium. Injured or denervated'brain secretes -_ neurotrophic factors (NTF's) in an age-dependent manner and NTF's such as TRH and NGF stimulate cholinergic regeneration. We have now tested ECMA in serum-free (S-) reaggregait es~tp examine the production and action of NTF's. #,Foetal rat brain reaggregates were prepared in S+ or S- medium. ECMA was added to the cultures at 9 DIV (12.5 pM). ECMA (12.5 pM) caused a rapid, HC3 sensitive loss of ChAT activity in S+ reaggregates (38%, +2h). In contrast, S- reaggregates showed no immediate loss of ChAT activity but a 40-50% loss appeared by 48h. Although this was also less than that in S+ reaggregates it may correspond to the HC insensitive phase representing cholinerg?c neuronal loss. In both reaggregate types NGF produced increased ChAT activity with more marked effects in S+ (45%) than in S- medium (20-25% increase). TRH had no effect. Despite a residual ChAT pool in the treated cultures, neither NTF reversed the neurone loss. 184 IN VITRO METHODS FOR ASSESSING NEUROTOXICITY. G C Siek & J K Mar uis. Dept. of Pharmacology & Experimenta Therapeutics, Boston University School of Medicine, Boston, MA. In vitro cell culture represents a new approach for screening and mechanistic studies of sus- pected neurotoxicants. The effects of several drugs and toxicants have been observed in neural cell cultures at concentrations similar to those found in the brain after exposure of animals to doses which cause effects in vivo. Four types of neuronal cell cultures can be used in neuro- toxicity studies: organotypic cultures, disperse primary cultures, reaggregated cultures, and cell lines. These are compared and evaluated for routine screening assays and test protocols. Our work on PC12 and NG108-15 cell lines, as well as on primary cultures of rat dorsal root ganglion, is used to provide examples for methods development. Although the specific mechanisms of neurotoxicity are not thoroughly defined, endpoints which can be detected in cell cultures must be identified in order to develop standardized study protocols. Proposed neural-specific endpoints include: neurite outgrowth, neurotoxic esterase activity, morphological counts/scores, immunological markers, electrophysiological parameters, and neurotransmitter-related endpoints. Non-neural endpoints, su.ch as protein synthesis, lipid . peroxide levels, and cytotoxicity, should be evaluated in parallel. 50875 8171
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DOSE-DEPENDENT DISPOSITION OF d-LIMONENE: RELA- TIONSHIP TO MALE RAT-SPECIFIC NEPHROTO%ICITY. L D Lehman-McKeeman and D Caudill. Miami Valley Laboratories, Procter & Gamble, Cincinnati, OH. it, Exposure to d-limonene exacerbates hyaline drop- let accumulation in male rat kidneys. Studies were conducted to evaluate the relationship between the disposition of d-limonene and its nephrotoxicity. Dose-response studies indicated that d-limonene exacerbated hyalipe droplets over control at dosages of 0.3 mmol/kg and high- er. Disposition studies with [14C] d-limonene indicated that, at 0.1 mmol/kg, renal concen- trations of d-limonene equivalents in male and female rats were similar. However,_ at I mmol/ kg, renal concentrations of d-limonene equiva- lents were significantly higher in males than in females. To determine whether retention of d-limonene in male rat kidney resulted from an interaction between d-limonene and or2u-globulin (ar2u), rats were dosed orally with [14C] d- limonene and 24 hr later, the distribution of radioactivity among renal proteins was deter- mined by reverse-phase HPLC. The results indicated that in male rat kidney, radioactivity co-eluted with the ar2u peak, whereas, no radio- activity was seen in the corresponding fraction of female rat kidney proteins. These results indicate that d-limonene is retained in male rat kidney at dosages that exacerbate hyaline drop- lets which may result from an interaction between d-limonene or its metabolites with a2u. HOW DOES CADMIUM CROSS CELL MEMBRANES? E C Foulkes, Depts. Environ: HeaTth & Physio-i.; Univ. Cincinnati Med. Ctr., Cincinnati, OH. Details of how Cd enters cells remain to be clarified. Although saturable and sensitive to inhibition, cellular Cd uptake in.rat jejunum appears to involve neither specific carriers nor competition with other metals (Toxicol. 37:117, 1985). It can be conveniently explained by A) non-specific bi.nding of Cd to anionic membrane sites; binding is reversed by EDTA. Reaction A) is followed by B) temperature-dependent internalization (Am. J. Physiol. 253:G134, 1987); internalized Cd is not accessible to extracellular EDTA except after sonication of the tissue. In similar experiments, Cd taken up by rat erythrocytes was extracted by EDTA only after osmotic hemolysis of the cells. When everted sacs of jejunum were suspended in 20 }iM CdCI 2- saline for 5 s at 37°C and rinsed, 60 s further incubation in fresh saline at 37°C sufficed for internal Cd to reach a plateau. The plateau does not reflect saturation of the internal compartment, as this was 3 times higher after an original exposure of 40 s. Although maximum internalization had occurred, the mean of means from 8 expts. showed 45+13% (SD) of tissue Cd still accessible to EDTX. It follows that not all binding sites on the membrane filled during process A) participate in the further process B). The sites involved in step B) have not yet been identified. (Supported by NIH grant ES-02416) 3 EFFECT OF TRANSPORT AND RIOTRANSFORMATION ON THE ! RENAL• SITE-SPECIFIC TOXICITY OF DICHLOROVINYL t=YSTEINF (DCUC). GHI Wolfgang, AJ Gandolfi, K Brendel, RB Nagle. Arizona Hea th Sciences e~ nter, University of Arizona, Tucson, AZ. DCVC, a model cysteine conjugate, is known to produce an S3 lesion in vivo and in rabbit renat corti cal sl ices ( R(.S) : fhe RCS system was" utilized to understand ther~fte-specificity of ., this lesion. RCS from male NZW rabbits were i ncubated o( serum-free DME/F12 medi u%, 9~A~5~ 0/C02, 25 C) for up to 12 hr with 10- -10 M~ DFVC. Transport studies (steady state and kinetic) show that DCVC did not use the organic anionic transporter (PAH) nor was its toxicity inhibited by co-incubationAith~ the anionic transport inhibitor probenecid. There, however, does appear to be a role for neutral amino acid transport. When beta-lyase, which is maintained in control slices, is inhibited by aminooxy- acetic acid, the toxicity of DCVC is inhibited. Thus emphasizing the critical role of beta-lyase in activating DCVC. DCVC, which has been shown to be a mitochondrial toxin, only causes a 30% decrease in slice 0 consumption. However, ATP content decreases by2more than 90%, indicating a disruption of the energy status. EM studies were used to confirm the site-specific ultrastruc- tural changes in mitochondria. The mechanism of the site-specific injury of DCVC in RCS appears to be linked to its uptake, bioactivation, and subcellular targets. (J.H. CAAT/GM 38290/NIEHS- ES-(170-91) POLYCHLORINATED BIPHENYL CONGENER INDUCTION AND INACTIVATION OF MONOOXYGENASE ACTIVITY IN THE FISH SCUP (STENOTOMUS CHRYSOPS). J W Gooch, A A Elskus, P J Rloepper.-Sams and J J Stegeman. Woods Hole Oceanographic Institution, Woods Hole, MA. Sponsor: M 0 James Studiess have shown that chlorobiphenyl (CB) effects on mammalian monooxygenase activity are structurally determined. We examined effects of environmentally common PCB congeners that are MC- or mixed-type inducers, on hepatic monooxy- genase systems in fish. Scup were given single ip injections and after 5 days were analyzed for levels of microsomal cytochromes P450 and b5, ethoxyresorufin-O-deethylase (EROD) and amino- pyrine-N-demethylase (APD) activities, immuno- detectable P450E (the major BNF-inducible scup P450) and translatable P450E mRNA. 3,3',4,4'- tetra-CB produced a parabolic dose-response for EROD activity, with strong induction at 1 mg/kg and lower activity at higher doses; APD activity was unchanged. By contrast, immunodetectable P450E and mRNA levels were strongly elevated at all doses. 2,3,3',4,4'-penta-CB, 2,3,4,4',5'- penta-CB and 2,2',3,4,4',5'-hexa-CB had only limited effects on the parameters. measured. The response of fish to pure MC-type PCB inducers thus involves transcription and translation, but there can be strong inhibition or inactivation of the P450 induced, by as yet undefined mech- anisms. The results also show a relative insen- sitivity sitivity of P450E to mixed-type CB inducers. •(Supported by USPHS ES-4220 and USEPA CX-813567.) 50875 8126
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153 THE EFFECT OF BIS-(BETA-CHLOROETHYL) SULFIDE (BCES) ON DNA SYNTHESIS OF A STRATIFIED KERATINOCYTE -GULTURE SYSTEM. S. Zaman, F L Vaughan and I A Bernstein., +.Toxicology Program, Dept. Env. Ind. Health, Univ. of Michigan, Ann Arbor, MI. Previous studies in our laboratory demonstrated that after a topical exposure of a stratified kdzatinocyte culture system to BCES, the DNA synthesis of the culture was inhibited in the conce9tration range of 0.01 to 500 nmoles/cm . This investigation was initiated to determine if this resulted from the inhibition of semi-conservative DNA synthesis. A method that distinguishes between semi-conservative DNA synthesis and repair synthesis was used which involved labeling the cells with 5-bromodeoxyuridine and separating the newly synthesized heavy DNA from the total DNA in a CsC1 density gradient. From these studies it has been determined that semi-conservative DNA synthesis was indeed inhibited in thg concentration range of 1-10 nmoles/cm up to 6 h after a 30 min exposure to BCES. These results indicate that sulfur mustard prevents cells from undergoing division which could seriously affect tissue integrity. 154 STUDIES ON THE EFFECT OF NONGENOTOXIC DRINKING WATER CONTAMINANTS ON LIVER DNA SYNTHESIS AND ON THE ACTIVATION OF ONCOGENES IN DIFFERENT MOUSE STRAINS. T V Reddy, A B DeAngelo, M A Pereiral, F B Daniel, J C Kandala2 and R V Guntaka2. U.S. EPA, HERL, Cincinnati, OH, EHRT1, Cincinnati, OH and University of Missouri2, Columbia, MO Strains of mice with different susceptibilities for spontaneous tumors (C3H > B6C3F1 > C57B1C/6) were implanted (S.C) with osmotic mini pumps filled with [H3] thymidine and then intubated daily for 5 days with nongenotoxic drinking water contaminants (perchloroethylene 7 mmole/kg, tri- chloroethylene 18 mmole/kg, trichloroacetic acid 2 mmole/kg, and chloroform 1.6 mole/kg) and phenobarbital as positive control (20 mole/kg). On day seven the mice were sacrificed and liver UNA specific activity and mitotic index were determined. All test chemicals and positive con- trol exerted a stimulatory effect on DNA synthe- sis as compared to corn oil control. However, the differences between the strains were not significant. Hence no positive correlation was obtained between DNA synthesis and spontaneous tumor susseptibility in mouse strains. We are currently investigating the levels of expression of m-RNA for pro-tooncogenes C-Ha-ras, C-Ki-ras, C-myc, C-fos, raf, and Src in liver and their response to chemical treatment in an attempt to implicate any differences in the pattern of onco- genes to the differences in susceptibility of mouse strains to spontaneous tumors (Abstract does not necessarily reflect EPA policy). 155 THE EFFECT OF THE PYRROLIZIDINE ALKALOID SENE- CIONINE AND THE ALKENALS TRANS-4-OH-2-HEXENAL AND TRANS-2-HEXENAL ON INTRACELLULAR CALCIUM COMPART- MENTATION IN ISOLATED HEPATOCYTES. D S Griffin and H J. Sega11.VM/Pharmacology and Toxicology, University of California, Davis, CA The pyrrolizidine alkaloid seneconine produced- an increase in cytosolic.free Ca + concentration_' .p isolated hepatocytes whicAftbrrelated with an . increase in cellular toxicity. The cytotoxict~ was greater in the absence of extracellular Ca ~-, than in it's presence, su~~esting that alter- >- ations in intracellular Ca dis2tribution, and not an influx of extracellular Ca +, was respon- sible for the senecionine-induced hepatotoxicity. Th2effect of senecionine on t~ sequestration of Ca + in mitochondrial and extramitochondrial com- partments was examined in isolated hepatocytes; as well as the effects of trans-4-OH-2-hexenal, a microsomal metabolite of senecionine, and a re- lated alkenal, trans-2-hexenal. Each of the test compounds elicited a decrease in the available extramitochondrial Ca2+ stores which was inhibit- ed by pretreatment with dithiothreitol. Senecio- nine and t-4HH decreased the level of Ca2+ seque- stered in the mitochondrial compartment of hepa- tocytes. The presence of a pyridine nucleotide reducing agent, B-hydroxybutryrate, inhibited this 'reduction. These results suggest that sene- cignine and t-4HH inhibit the sequestration of Ca + in extramitochondrial and mitochondrial com- partments possibly by inactivating free sulfhy- dryl groups and oxidizing pyridine nucleotides. 156 CELLULAR AND MOLECULAR EFFECTS OF DI-N-OCTYLTIN DICfILORIDE (DOTC) ON THE RAT THYMUS. S.G. Volsen, N. Barrass, M.P. Scott and Mi11er,.R.. BIBRA, Carshalton, England. Sponsor: S D Gangolli. - , The oral administration of DOTC to young rats induces marked thymic atrophy. Previous experiments conducted at BIBRA have demonstrated that this characteristic lesion is associated at an early stage with both a suppression in spontaneous in vitro thymocyte proliferation and concomitant loss of MRC OX 18 positive, mature medullary thymocytes. Since recent evidence suggests that interleukin-2 (IL-2) plays a central role in the proliferation seen in the active thymus, studies were conducted to evaluate whether DOTC exerts its anti- proliferative effects by compromising IL-2` production. Using an mRNA cytoplasmic dot blot technique, the examination of thymocyte lysates from treated and control animals revealed that DOTC markedly down-regulates, and at high doses abolishes, the normal expression of the IL-2 gene. The housekeeper gene CY-actin is however unaffected by its action thus demonstrating the selective effect of DOTC. (Supported by the UR Ministry of Agriculture, Fisheries and Food). 39
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,37 MOLECULAR BASIS OF INDUCIBILITY OF CYTOCHROME P-450 IN OBESE RODENT MODEL. P Jones, I Chaudhary, L Robertson, R A Blouin. Department of Biochemistry atad Graduate Center for Toxi- cology, University of Kentucky, Lexington, KY. Biochemical characterization of hepatic cyto- chrome P-450 system in obese Zucker rat model has shown that phenobarbital (PB) induced cyto- chrome P-450 is present at the basal level.but is not inducible. In order to further c~haracter- ize the above phenotype at a molecular level, hepatic mRNA levels expressed in both obese and lean Zucker rats, treated (experimental) with PB and untreated (control) were studied. Induction of cytochrome P-450 was carried out by orally administering PB to both lean (34.7 mg/Kg/12 hr) and obese (20.4 mg/Kg/12 hr) animals. Hepatic total cellular RNA was isolated, and examined by Northern analysis using radiolabelled B and E c-DNA probes. B and E c-DNA probes produced a faint signal in.control rats which shows that basal level m-RNA for the B and E form are present. Both probes produced a stronger signals in PB-induced animals. However, the difference in induction signal was 5-7 fold greater in the lean animals. The results indicate that the genetic defects in the obese Zucker rats results in the decrease in the responsiveness of cytochrome P-450 response to PB- treatment. 139 DNA BINDING FORMS OF RAT Ah RECEPTOR COMPLEX.- R R Hannah, Washington State University, Pullman, WA. Sponsor: R J Bull. The Ah receptor is a cytosolic protein and is involved in gene expression of cytochrome : P-450 formation with ligand and nuclear Janslocation. The ability of the Ah receptor to activate in vitro as been used i:0 examine the nuclear bing orm of this receptor. In this study, DNA-cellulose column chromatographywas used to detect formation of activated Ah receptor complex. A linear NaCI gradient generated two forms of the receptor with differential binding affinities for DNA. Form I hadA. low, binding affinity - 0.12M NaCl and form haa-a higher binding affinity - 0.33M NaCV, for DNA. Form II was comparable to the salt extractable nuclear receptor from C57BL/6 and DBA/2 mice, however form I was not observed in these extracts. The addition of ATP converts form I to form II in vitro. The significance of these result's wi be discussed in terms of receptor phosphorylation/dephosphorylation during- activation of the complex. 138 SYNERGISTIC TOXIC INTERACTIONS OF HEXACHLORO- 140 BENZENE (HCB) AND 2,3,7,8 TETRACHLARODIBENZO-p- DIOXIN (TCDD) IN THE RAT. M A Li, M A Dencnme, R 4bwner, B Leece and S Safe, Departinent of Veterinary Physiology and Pharmacology, College of Veterinary Medicine, Texas A&M University, College Station, TX. and Dapartment of Chanistry and Biochemistry, University of Guelph, Guelph, Ontario, Canada. 2,3,7,8-TCDD and HCB elicit several camion biologic and toxic effects including the induction of hepatic microsomal aryl hydrocarbon hydroxylase (AHH). Cotreatment of rats with 2,3,7,8-TCDD, HCB and 2,3,7,8-TCDD plus FLB resulted in some minor non-additive changes in the induction of hepatic and extrahepatic AHH which could not be linked to HCB-mediated changes in organ Ah receptor levels. However, the most dramatic interactive effects of HCB (400 umol/kg) and 2, 3, 7, 8-TCDD (10 or 30 ug/kg) were observed with two Ah receptor-mediated toxic effects, namely body weight loss and thymic atrophy. HCB synergistically exacerbated both body weight loss and thymic atrophy caused by 2,3,7,8=PCDD whereas HCB administered alone at doses as high as 3000 umol/kg did not cause any significant body weight loss or thymic atrophy in the rats. (Supported by the Natural Sciences and Engineering Research Council of Canada.) 35 METAL OXYANION INHIBITION OF THE Ah RECEPTOR. M E Jazwin and R R Hannah, Washington State University, Pullman, WA. Sponsor: R J Bull. The Ah locus, important in the induction of the multi-faceted cytochrome P-450 systems, is also of focal interest in receptor studies. Exposure of the rat cytosolic Ah receptor in vitro with the ligand TCDD produces a DAA-6inding form. This methodology is conducive for studies of the conformational and mechanistic elements involved in the activation and inhibition of this receptor complex. Metal oxyanions such as molybdate, tungstate, and vanadate seem to prevent significant activation of the DNA binding form of the rat hepatic receptor in the in vitro system. Incubation of rat liver cytosolic preparations with 10 or 20 mM sodium molybdate (V) for one hour at 4° C followed by addition of the ligand, heat treatment and subsequent DNA-cellulose chromatography demonstrated about a two-fold decrease in the DNA binding form. Activation of the receptor by heat treatment prior to addition of the sodium molybdate did not significantly inhibit receptor binding to DNA. This suggests that presence of molybdate may somehow modify receptor sites that are pivotal during activation to the DNA-binding form. ;~.
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A VIBRATING ELECTRODE STUDY Uh' EXTRACELL- 227 ili,AR MEMBRAIIE CURRENTS FROM ACETABULARIA EXPOSED TO IRIBUTYLTIN METHOXIDE._S B Baumann, Northrop Services, Inc, Research Triangle Park, NC.. ftonsor: K T Kitchin - Tributyltin (TBT) is used as an anti- fouling agent in.marine paints, and it appears in sea water samples from many harbors. The large-celled, marine algae, Acetgbularia, has been studied previo}usly with a vibrating electrode system that measures slowly varying ionic currents near the cell exterior (Bowles and Allen, in Ionic Currents in Development, 1986). In this study stable, baseline measure- ments were taken for one hour at 10 points around the rhizoid and stem regions. Within 5 minutes of treatment with 6 nanomolar (2ppb) TBT, currents decreased from an average of 22 uA/cm2 to 4 uA/cm2. Although current densities recovered to about 1/3 of original levels after 5 hours of TBT exposure, both current magnitude and polarity showed great disturbance from the normal pattern. Since TBT is one of many toxic compounds that affects cell membrane permeability, this study demonstrates that a vibrating electrode system can be used as a sensitive bioassay for membrane toxicity. WATER QUALITY C_RITERIA FOR H_EXACHLOROETHANE. P S Hovatter, K A Davidson, and R H Ross, Oak Ridge National Laboratory*, Oak Ridge, TN. Sponsor: P. Y Lu. Hexachloroethane is a USEPA designated priority pollutant, which has been detected in drinking water, groundwater, surface water, and waste- water effluents. It is primarily used by the military as a constituent of white smoke grenades and markers. Based on the existing data concerning the environmental fate, aquatic toxicity, bioaccumulation, and mammalian toxicity of hexachloroethane, water quality criteria for the protection of aquatic life and its uses and of human health are calculated using USEPA guidelines. The aquatic criteria consists of two values, a criterion maximum concentration based on acute toxicity data and a criterion continuous concentration based on chronic toxicity and bioaccumulation data. The human health criterion is derived from car- cinogenicity data based on concentraifions 9f the che?ical related to risks of 10- , 10- , and 10- or on chronic toxicity data based on acceptable daily intake. *Operated by Martin Marietta Energy Systems, Inc., under contract No. DE-AC05-840R21400 with the U.S. Department of Energy. ORGANOSILANES: HEALTH AND ENVIRONMENTAL EFFECTS. M W Daugherty, R H'Ross, Oak Ridge National Laboratory*. Oak Ridge, TN. P Wagner, J S Leitzke. U.S. Environmental Protection Agency (USEPA), Washington, D.C. Sponsor: P:Y Lu' The health and environmental effects of eight structural categories of organosilanes have been reviewed under the Chemical Hazard Informat;fon Profile program of tft"`USEPA. Organosilanes are bifunctional (able to react with both organic and inorganic°substrates) and many are used in industry as coupling agents or surface modifiers. Structure-related trends in toxicity were not observed for any of the categories reviewed. Most organosilanes are generally of low to moderate toxicity X h'uffans and animals, but some have elicited serious chemical-specific effects in experimental systems. For example, methacryloxypropyl- trimethoxysilane, administered acutely or subchronically as an aerosol, caused an unusual form of granulomatous laryngitis in rats; trimethoxy[2-(7-oxabicyclo[4.1.0]hept-3- yl)ethyl]silane (TMOHS) was carcinogenic in C3H/HeJ male mice; and others were positive for genotoxicity. Limited data indicate that these organosilanes are of low acute toxicity in aquatic species. *Operated by Martin Marietta Energy Systems, Inc., under contract No. DE-AC05-840R21400 with the U.S. Department of Energy. 228 TSCA INTERAGENCY TESTING COMMITTEE (ITC). E K Weisburqer. National Cancer Institute, Bethesda, MD. The ITC recommends chemicals to the EPA for priority testing consideration. All chemical substances regulable under the Toxic Substances Control Act (TSCA) are subject to ITC review. The ITC consists of statutory members from 8 agencies specified in TSCA and representatives from seven agencies invited to participate. The ITC has developed a process to screen large numbers of chemicals to select a few.for detailed review. The process involves evalua- tion of exposure and biological effects potentials and current consumption values to score and rank chemicals. Since 1977, more than 25,000 chemicals have been screened, approximately 7,000 have been scored for exposure and/or biological effects and over 1,000 have been selected.for detailed review. The ITC has issued 21 Reports to the EPA Administrator recommending over 400 chemicals for priority testing considerations. The status of all chemicals scored, reviewed and recommended by the ITC is maintained on a computerized tracking system. On-line database searching capabilities, which are available to all interested parties, will be demonstrated during the session. Interested parties are also encouraged to nominate chemicals to the ITC for its consideration. 57 50875 8182
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173 THE DECREASE IN AXONAL TRANSPORT OF PROTEINS IN 175 ISOLATED RAT RETINAL MITOCHONDRIAL~RESPIRATION: THE RAT OPTIC SYSTEM PRODUCED BY"XYLENE INHALA- IN VITRO AND IN VIVO LEAD STUDIES. D A Fox, C J TION IS REVERSED BY ETHANOL CONSUMPTION. S Padilla, C N. -Pope*, ann D P Lyerly**, U.S. Medrano Houston, and S D Rubinstein. College of Optometry, University Houston, of TX. EPA, **Northrop Services, RTP, NC. Because of the possible metabolic interaction between ethanol and xylene, coupled with the knowledge that human exposure to solvents are often complicated by ethanol inges4ion, we investigated the effect of ethanol on the p-xylene-induced decrease in axonal transport in the rat optic system previously reported by our laboratory. Long-Evans, hooded, male, rats were divided randomly into 4 groups (n=8/group): no ethanol and no xylene (Group A); ethanol (10% in drinking water 6 days prior to and during inhalation exposure) but no xylene (Group B); no ethanol, but xylene (inhalation,1600 ppm, 6 hr/d, 5 d/week, for 8 exposure days)(Group C); or both ethanol and xylene exposure (Group D). Ethanol alone produced no decrease in the axonal transport of [35S]methionine-labeled proteins, i.e., there were no significant differences in radiolabeling of the retinal ganglion cell projections in groups A and B. There was, however, >40% decrease in axonal transport of proteins in the xylene-exposed animals (Group C), while the axonal transport in the xylene-exposed animals receiving ethanol (Group D) equaled that measured in the ethanol controls (Group B). These data suggest that the substan- tial reduction in protein transport seen in xylene-treated animals was reversed by sub-chronic ethanol consumption. (*Supported by NRC Research Associateship.) 1'74 EFFECTS OF LEAD AND POTASSIUM'ON RETINAL ATPases. I: S D Rubinstein and D A Fox. University of , Houston, College of Optometry, Houston, TX. In vitro and in vivo lead exposure produce an ichibition of ncn-stimulated respiration (NSR) and an enhancement of 50 mH potassium-stimulated respiration (KSR) in isolated whole rat retina (SOT, 1987). Our recent experiments show that: (i) ouabain produces an inhibition of NSR and blocks the enhanced KSR produced by Pb and (ii) 30% of retinal metabolism is due to Na-K ATPase activity. These results suggest that Pb may alter retinal metabolism via changes in Na-K ATPase. These studies examined the effects of Pb (10 500 uM) and 50 mM KC1 on total, ouabain insensitive (basic) and ouabain sensitive (Na-K) ATPases in rat retinal homogenates using Winkler and Riley°s procedures (1977). In adult hooded rat retina, basic and Na K ATPase comprise 53% and 47X of the total ATPase activity. A two min pre-incubation of the homogenate with Pb produces dose-response inhibition of total and Na-K ATPase, with Na-K exhibiting a greater inhibition at all Pb concentrations. Ion-substitution studies reveal that KC1 decreases Na-K activity (-8%). In contrast, pre-incubation with Pb (100 or 250 uM) prior to KC1 increases the activity of total ATPase (10%) and Na-K ATPase (40-70%). These results are similar to those seen in the isolated whole retina and suggest that Pb alters retinal metabolism via a yet to be determined interaction with Na-K ATPase. Supported by ES 03183, EY 07088, Sigma Xi and Beta Sigma Kappa. 44 Developmental Pb exposure produces long-term rod „ selective degeneration. Although Pb has multiple _ s~tes and proposed mechanisms o'Paction, a direct iAibitory effect of Pb on mitochondrial respira=`" tion is suggested to be a fundamental and common underlying mechanism accounting for neuronal necrosis. The inhibition of energy metabolism by Pb in isolated whole rat retina (SOT, 1987) supports this idea. However, to determine if Pb inhibits retinal mitochondrialAesp~ration we developed an isolation methodology since, to our knowledge, there were none. Based on the brain preparation of Clark and Nicklas (JBC, 1970) we isolated retinal mitochondria with good respira- tory control ratios (mean RCRs: 6.0), State 3 and 4 respiratory rates (mean S3: 31.5 and mean S4: 5.2 nM 02/mg/ min) and ADP/O ratios (mean: 3.1) using the NAD-linked substrates glutamate and malate. Further characterization will use enzymatic assays, substrates and EM. In vitro Pb (10-300 uM: incubated for 5 min) produces large. dose-dependent decreases in RCR, S3 and ADP/0. S4 is increased at low Pb and decreased at high Pb. In vivo Pb produces large decreases in RCR, S3, S4, ADP/O and more labile mitochondria. These results demonstrate a direct inhibitory effect of Pb on retinal mitochondria and suggest that this effect may contribute to the Pb-induced retinal degeneration. Supported by ES 03183 and EY 07088. 176 INCREASES IN FREE INTRACELLULAR Ca++ ACCOMPANY EXPOSURE OF NEUROHYBRIDOMA CELLS TO THE ZNSECTICIDE, JLINDANE. R M Joy, V W Burns and L G Stark. Depts. of Pharmacology/Toxicology and Physiological Sciences, School of Veterinary Medicine and Dept. of Pharmacology, School of Medicine, University of California, Davis, CA. Exposure to the gamma-isomer of hexachloro- cyclohexane (lindane) produces enhanced neuronal excitability and convulsions. A role for increased intracellular Ca++ in these effects has .been proposed. We report here that cultured neurohybridoma cells exhibit dose-de endent elevations in free intracellular Ca upon exposure to lindane. Free intracellular Ca++ was measured using the INDO-1 method. Ca++ levels were determined after exposure of cells to various concentrations of lindane for periods ranging from 5-45 minutes. Control Ca++ levels were 234 ± 4.8 nanomolaC (X ± S~, N = 48). Exposures to lindane (10-b - 4x10' M) resulted in a dose-dependent increase in free Ca++ that ranged from 15 220 nanomolar. A time-response study using 10-4 M lindane indicated that the maximal effect had occurred by 5 minutes, the shortest time period tested. Free Ca++ remained elevated above basal levels throughout the remaining 40 minutes examined. 01 m 00 v Ln 0o N ~
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', ptJBLIC INFORMATION SLIDE TAPE PROGRAMS. S ~upanger and A L Craigmill, Environmental =ouicology Extension, University of California ` at Davis, Davis, CA. , i. public perception about the risks of exposure to chemicals in the environment, and recent legislation have prompted the need for educa- tional programs to address public concern on these issues. Two slide tape programs were a~ developed to present background information and practical management tools for target audiences in rural and urban areas of tenth to twelfth grade education levels. One objective of the project is to promote a wider perspective on the role of chemicals in our daily lives, and just what risk they present to us. A second objective is to present infor- mation in an easily digestible format to assist county staff in answering questions about environmental risk and hazardous wastes. THE TOXICOLOGY RESOURCE INFORMATION SERVICE. J S Aoods, Battetle Seattle Research Center, Seatt e, WA and A L Craigmill, University of California, Davis, CA The SOT COmnittee on Public Communications has recognized a growing need for a comprehensive computerized registry of educational and infor- mational resources in toxicology and related to- pics which could be utilized by members of the SOT and other interested professionals to assist in teaching and public communications efforts. To meet this need, the Committee has established the Toxicolo Resource Information Service (TRIS , containing information about audiovisuals (films, slides, videotapes); publications (books, monographs, brichures); computer software (c(xm- puter-assisted teaching, analysis and utility programs); arid teaching materials (syllabi, course/curricula outlines) which have been ac- quired or developed by members of the SOT and other professional groups. The TRIS currently holds over 300 specific entries. A prototype version of the TRIS will be displayed during the special session on Coamunicating Basic Concepts in Toxicology at the 1988 annual meeting of the SOT. All items submitted to the TRIS up to that time will be included in the display. It is anticipated that, when brought to fruition, the TRIS will constitute a resource of major import- ance to members of the SOT in enhancing their individual teaching and public communications efforts in toxicology and related topics. 235 POISON CONTROL. CENTERS (PCCS): A UNIQUE OCCUPATIONAL/ENVIRONMENTAL HEALTH RESOURCE,FOR THE PUBLIC. C S Clark, V H Sublet,L T Sigell, J F Bonfiglio, Drug and Poison Informa tion Center (DPIC) and Dept. Envir. Hlth., Univ. of Cincinnati, Cinti, Oh. Sponsor: C S Baxter. At the present time, occupational/environ- mental health requests for iormation constituti up to 10% of the calls received by PCCs. Health professionals are„responsible for the greatest number of - these inquiries, currently. The volume of requests to PCCs regarding occupational/environmental health issues is expected to escalate due to the recent right to know laws. At present, most PCCg.~.anggwer these inquiries using a background 4n actite toxicology. The objective of the present research has been to assess resource utilization and staff training needs of PCCs on a national scale. Ninety-eight PCCs participated in this survey. The results determined that most PCCs underutilize government agencies primarily using the Poisindex system to answer occupa- tional/environmental requests. Education about right to know laws, hazardous spills, reproductive and cancer risks were regarded,as as very important needs by PCCs. Most PCCs desired 40 hours/year of occupational/environ- mental health training. Future research will demonstrate how the development of efficient resource materials and training programs can aid PCCs to implement a unique occupational/ environmental health program for the public. 236 THE INQUIRY-RESPONSE SYSTEM AS A MEANS OF PUBLIC EDUCATION. M A Kamrin. Center for Environmental Toxicology, Michigan State University,.East Lansing, MI. The Center for Environmental Toxicology has operated an inquiry-response system for over five years. Center staff have responded to about one thousand inquiries involving a variety of environmental toxicology issues. Inquiries have been received from a diverse audience including public agencies, Cooperative Extension Service agents, industry, the media, and private citizens. Each inquiry has been utilized as a means to public education since time has been taken to explore the situation fully with each individual and to impart information appropriate to the issue involved in the inquiry. In addi- tion, the distribution and nature of inquiries have proven very useful in identifying issues which require more general education. Yearly summaries. of the data reveal a number of trends including an increased inte•rest in groundwater contamination, pesticide toxicity, and testing of environmental samples and a decreased interest in chlorinated hydrocarbons. A currently emerg- ing issue is indoor air. Recognition of these trends has led to the development of publications and presentations to address issues of impor- tance. Examples of these materials will be part of the poster presentation. 50875 8184 59
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45 GENE EXPRESSION EFFECTS OF TOXIC AGENTS: USE OF 2-D ELECTROPHORESIS TO MONITOR CHANGES IN THE EXPRESSION OF HUNDREDS OF LIVER PROTEINS FOLLOWING EXPOSURE OF MICE TO T-VARIETY OF XENOBIOTICS. N~ Anderson*, F A Giere**, and N G Anderson*. Large Scale Biology Corp.(*), Roekville, MD and Lake Forest College(**), Lake Forest, IL. Recently developed techniques based on computer analysis of two-dimensional electrophoretic protein patterns allow accurate measurbment of the abundances of hundreds of individual proteins in tissue samples. Many toxic agents cause characteristic changes in the amounts of specific, proteins as determined by this technique, offering a relatively comprehensive picture of intoxication effects at the molecular level. Drawing on appropriate computer databases, an analysis of these changes can provide insight into mechanisms and sites of action, as well as allowing classification of chemical effects. Compounds investigated in this study include carbon tetrachloride, cycloheximide, Aroclor 1254, phenobarbital, ibuprofen, ethanol, indomethacin, ethylene glycol, aspartame, clofibrate, and alloxan. Extension of such studies to a range of organs and the construction of a larger database of reference compound effects may allow a systematic and quantitative general approach to gene expression toxicology in rodents. 146 ETHANOL POTENTIATION OF THE HEPATOTOXIC RESPONSE TO ACUTE !rOCAINE ADMINISTRATION IN MICE. C S Bover and D R Petersen. Hepatobiliary Research Center, Molecular and Environmental Toxicology Program, and School of Pharmacy, University of Colorado Health Sciences Center, Denver, CO - Cocaine has been docu mented to be a potent hepatotoxin in mice. The theorized toxic metabolite of cocaine is thought to be produced by a multistep metabolic pathway beginning with N-demethylation by P-450. Ethanol, because of its P-450 induction capabilities with chronic ingestion and its probable concomitant social use is an obvious choice for interaction . studies. Male and female C3H mice were fed an ethanol-containing liquid diet for 6 days. On day .5 of the treatment, mice were injected ip with 50 mg/Kg cocaine and serum glutamic-pyruvic transaminase (SGPT) levels were determined 24 hours later. Of the mice receiving both the ethanol pretreatment and acute cocaine, males showed a five-fold increase in SGPT levels over naive animals receiving the same dose and females showed a twenty-fold increase over naive animals receiving the same dose. In both sexes, cocaine N-demethylase activity was increased 1.8 fold over control animals, thus indicating an inductive effect of chronic ethanol. This evidence indicates that in mice, chronic alcohol ingestion can potentiate the hepatotoxic response to an acute dose of cocaine. (Grant Support: NIH AM34914) 147 $YNERGISTIC EFFECT OF j;,THANOL AND AMITRIPTYLINE (AMI) ON lIA,K-ATPASE ACTIVITY OF SYNAPTIC PLASMA' MEMBRANES. M A Carfagnal and B B Muboberac2, Department of Pharmacology/Toxicology, Indiana University School of Medicinel and Department of Chemistry2, Indiana University-Purdue University at Indianapolis, IN Sponsor: R B Fornev. Sr.l The effects of simultaneous exposure of synaptic a plasma membranes (SPMs) to,&t:hanol and other = 6fIS-active drugs may be important in undsc_K ' standing the apparent po.tentiation of respiratory depression and psychomotor impairment induced by k;'e combinations of such drugs. In this study Na,R- '' ATPase (NKA) and Mg-ATPase activities of rat SPMs were measured after acute, in vitro exposure to ethanol (100 & 200 mM) and AM6.Sa5 & 9.01 uM) both separately and in combi on. Three of the four ethanol/AMI combinations stuAied showed a statistically significant (p<0.01) difference between the mathematical sum of ethanol- and AMI- induced NKA inhibition measured separately and the experimentally measured effect of the two drugs in combination. For example, ethanol (100 mM) inhibited activity 6.42% ± 0.53 and AMI (9.01 uM), 41.46% ± 1.28 which gives a mathematical sum of 47.88% ± 1.80, whereas the experimentally measured combined effect was 60.77% ± 0.72 inhibition. In contrast, an additive effect was observed with Mg-ATPase at the same concentra- tions of ethanol and AMI. These data show an acute, in vitro synergistic inhibition of NKA by ethanol and AMI which may be a basis for enhanced CNS impairment. (Supported by NIAAA AA06935) 148 a TOCOPHERYL SUCCINATE AS A UNIQUE AND POTENT CY70PROTECTIVE AGENT. M.W Fariss. Environ- mental Toxicology, Department of Pathology, Medical College of Virginia/ Virginia Common- wealth University, Richmond, VA We previously de~~nstrated, using 1 mM free extracellular Ca (physiological), that a- tocopheryl succinate (a-TS) protects hepato- cytes from the toxic effects of chemicals. To determine if the cytoprotective ability of a-TS is related to the release of a-tocopherol (a-T) we compared the protective capacity of a-T and a-TS in rat hepatocytes exposed to a variety of toxic insults. With each insult investigated (ethyl methanesulfonate, ionophore A23187, cad- mium and 95% 0 ) a-TS was found to be a more potent protecti2ve agent than a-T. For example, complete protection against ethyl methanesulfo- nate-induced toxicity was observed with 2 to 250 uM a-TS whereas 250 uM a-T offered 1 i ttl e protection (30%). Cellular a-TS was found only in protected cells (2 to 250 pM a-TS) at cong centrations ranging from 0.1 to 7.6 nmoles/10 cells. The concentration of a-T in protected hepatocytes (a-TS, 2 uM) however was 200 times lower than that observed in unprotected hepato- cytes (a-T, 250 uM). It is concluded that cyto- protection observed after a-TS administration does not result from the cellular accumulation of a-T but rather from the presence of the in- tact a-TS molecule. Thus the a-TS molecule itself appears to be the protective agent. 37
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I ,g COMPARISON OF UPTAKE AND DISTRIBUTION OF DIETHYLNITROSAMINE (DENA) IN ORYZIAS CATIPES AND PIMEPHALES PROMELA.1j, TL Holliday, ME Davis, *DE Hinton. West VlrgirPia University Medical Center, Morgantown, WV *School of Vet. Medicine, University of California=Davis. 0. lati es, Japanese medaka, and P. promelas, fathead minnow, are small laboratory fishes of similar body size and culture requiremeAs'. Exposure to DENA caused tumor formation in 0. latipes but not P. promelas. Uptake and distribution of 14C-DENA was compared in these two species. Two week old fry of both species were exposed to 10, 100 or 200 ppm 14C-DENA for 3, 6, 12, 24 or 48 hr. Following exposure, fish were rinsed and radioactivity determined in whole fish or visceral mass (liver, kidney, intestine and gonads). A dose dependent uptake of radioactivity into whole fish and visceral mass of both species was seen. The time course of incorporation and disappearance of radio- activity in the visceral mass differed in the two species. The peak uptake into the visceral mass occurred at 12 hr in P. promelas and 24 hr in 0. latipes. Disappearance, presumably by elimination occurred earlier in P. rop melas. 0. latipes also had higher tissue concentration of radioactivity than P. promelas. Therefore, it appears that differences in tumor production by these two species may be related to avail- ability of the carcinogen. (Supported by USGS Grant No. 14080001G1052). 219 A MODEL SYSTEM FOR STUDYING THE INTESTINAL ABSORP- TION OF A HEPATOTOXIN FROM BLUE-GREEN ALGAE. A M Dahlem 1 A S Hassan,l S P Swanson 1 W A Carmi- chael,~ and V R Beasley.' Dpatment of Veteri- nary Biosciences, Urbana, IL, and Department of Biological Sciences, Wright State University, Dayton, OH. Rats nre used to evaluate a model .S~s'tiem for studying the hepatotoxicity caused by micro- cystin-A, a cyclic peptide.tox'in produced by the cyanobacterium, Microcystis aeruginosa, and for evaluating the therapeutic potential of cholesty- ramine resin (CTR). Female rats were assigned to one of two groups and treated with either'toxin (5 mg/kg) or an equivalent volume ofAline-.vehi- cle instilled into the lumen of an in situ isolated ileal loop. Male rats were dosed with toxin as described above and then were dosed with either CTR (50,mg/rat) or an equivalent volume of vehicle. The surviving animals in both studies were killed six hours postdosing and heptatotoxi- city was assessed by change in liver weight as a percent of whole body weight. In all groups given toxin alone, there was a significant (p < 0.05) increase in liver weight. Liver weights of the toxin plus CTR treated rats were similar to those in vehicle-treated rats. When the toxin was administered into a similarly isolated jejunal loop, liver weight was significantly (p < 0.05) less than that found when an equivalent dose was administered into the ileal loop. These results indicate that a site specificity exists for intestinal absorption, and that CTR produces a reduction in harmful effects. _METABOLISM AND LIPID PEROXIDATION IN THE TROUT 220 IN VITRO GLUCOSE AND SULFATE CONJUGATION OF AND RAT. Y Singh, S McEuen, D Warren, D Hinton and M Miller. Dept of Env Tox and Vet Med, Univ 4-METHYL UMELLIFERONE (4-MeU) BY THE SPINY LOBSTER (PANULIRUS ARGUS). J D Schell and M.O. of California, Davis, CA. Sponsor: L Shull. Examination of similarities and differences be- tween chemically-induced toxicity in teleost and mammalian model systems could lead to better un-g derstanding of the mechanisms underlying toxic action. The present studies examined metabolism using the rainbow trout and the Sprague-Dawley rat as models. The trout's susceptibility to car- bon tetrachloride (CC14)-induced lipid peroxida- tion (LP) was also investigated. In metabolism studies utilizing liver microsomal and cytosolic preparations, kinetic constants for glucuronyl transferase and sulfotransferase were obtained with 1-naphthol as substrate. The metabolic capa- city (Vmax) and affinity (ICm) of the trout conju- gative enzymes were markedly lower than those of the rat. For example, the Vmax for the trout and the rat glucuronyl transferases were 0.77 and 32 nmol/min/mg protein, respectively. Similarly, the teleost Vmax for 1-naphthol sulfation was approx. Is that for the rat. Further studies investigated CC14-induced LP in fish and rat liver microsomes. LP was measured by HPLC analysis of the thiobarb- ituric acid adduct of malondialdehyde (MDA). CC14 induced a concentration dependent increase in the amount of MDA formed in both species. Further studies are underway to examine'LP and metabolism in freshly isolated trout and rat hepatocytes, and to evaluate the in vivo sensitivity of the trout to CC14 hepatotoxicity. James. ifiitney Labora ory and Dep. ~icinal ~ Chemistry, Univ. of Florida, St. Augustine, FL. The ability of various tissue fractions of the spiny lobster to catalyze conjugation reac- tions was studied using the fluorescent mole- .cule, 4-MeU as the acceptor substrate. The hepatopancreas (HP) exhibited UDP-glucosyl- transferase (UDPGT) activity, and used UDP- g}ucose as a co-factor. The activity was localized in the microsomal fraction. HP microsomes had no UDP-glucuronyl tranferase activity with UDP-glucuronic acid as a co- factor. Under assay conditions of 25 uM 4-MeU, 2.6 mM UDP-glucose, pH 7.6 and 30oC, UDPGT activity was 268 + 100 (mean + SD, n=17) pmol/min/mg protein. HF cytosol haa no sulfo- transferase activity and completely inhibited the sulfotransferase activity of fish liver cytosol. The cytosolic fraction of the green gland (GG) contained sulfotransferase activity. This enzyme had a rate of 63.4 + 49.3 (n=4) pmol/min/mg protein, at the pH optimum of 7.0. GG contained no detectable UDPGT activity with 4-MeU in any subcellular fraction. The role of these enzymes as in vivo detoxification path- ways in xenobiotic meabolism is currently being investigated. Supported by USPHS CA44297. 55 50875 8180
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9 USE OF RAT HEPATOCYTES CULTURED ON EXZBACELLULAR MATRIX AS AN IMPROVED SYSTEM TO STUDY LIVER GENE EXPRESSION IN VITRO. P S. Guzelian, and E G Scheutz. Medical College of-Virginia, Richmond, VA. a W Traditional monolayer cultures of adult rat hep- atocytes on type I Collagen (VIT) lose many liver functions including the cytochromes P-450- (CP450) induced by phenobarbital (PB), P450b/e, or by isosafrole (ISF), P450d. when we incub- ated freshly isolated rat hepatocyCjeiz in serum- free medium on matrigel (MGel), a reconstituted basement membrane, the cells remained rounded as viable clusters for over 30 days in contrast to the flattened, confluent monolayers on VIT. Analyses of RNA by Northern blots and of pro- teins by immunoblots revealed that MGel cultures exposed to PB contained amounts of P-450b/e mRNAs and proteins comparable to PB treated rat liver whereas no responses were detected in PB treated VIT cultures. Moreover, treatment of VIT cultures with ISF, 3-methylcholanthrene or PCBs induced the mRNA and protein only for P450c. The same treatment of MGel cultures re- sulted in co-induction of P450c and P450d mRNAs and proteins provided the medium was supple- mented with hormones including epidermal growth factor. We conclude that MGel permits cultured hepatocytes to retain many liver-specific func- tions including inducible expression of saane CP450 not previously reported in vitro and thus provides a new way to rigorously investigate mechanisms by which xenobiotics affect liver cell physiology and toxicity. ANTIFIBROTIC EFFECT OF POLYINOSINIC-POLYCYTIDYLIC ACID IN BLEOMYCIN MODEL OF LUNG FIBROSIS. S N Giri, and D. M Hyde. Depts. of Vet. Pharmacol. Toxicol. and Anat., Univ. of Calif., Davis, CA. Interferon Y was found to ameliorate the bleo- mycin (BM)-induced lung fibrosis in mice. The effects of polyinosinic-polycytidylic acid (Poly IC), an inducer of interferon, on BM-induced lung fibrosis was studied in hamsters. Poly IC (10 mg/kg IP) was administered for two days and im- mediately prior to IT instillation of BM (7.5 U/kg) and thereafter, daily for 13 days. The lung hydroxyproline in Poly IC, BM and Poly IC + BM groups were 84%, 1499'o and 97% of the control (791. µg/lung), respectively. Prolyl hydroxylase act- ivities in the corresponding groups were 83%,183% and 142% of the control (1x10 dpm/lung). Protein in bronchoalveolar lavage (BAL) supernatant in Poly IC, BM and Poly IC + BM groups were 72%, 286%, and 206% of the control (1157 pg/lung), respectively. There was no difference in total leukocyte counts between Poly IC + BM and BM groups but the differential cell counts were changed. The numbers of PMN, Mono, Lymph and Eosin were 50%, 849'0, 919'o and 10% of the BM group, respectively. The hamsters in Poly IC + BM gr3up had significantly less volume of lesion (1.Ocm ), which was restricted to t~ie interstitium as com- pared to BM group (1.6 cm ) where there was also alveolar fibrosis. In addition, lesions in BM group were multifocal and primarily proximal aci- nar in location, had fewer extracellular fibers, PMN and Mono. (NHLBI Grant # 5R01 HL 27354-07) 3 11 PRETREATMENT WITH.QYCLOPHOSPHAMIDE DOES NOT PROTECT AGAINST THE LUNG DAMAGE AND BMROSIS OF A SECOND DOSE. R D Smith and J P Kehrer. - Division of Pharmacology and Toxicology, College of Pharmacy, The University of Texas at Austin, Austin, TX. Administration of a single intraperitoneal (i.p.) dose of cyclophospha mide (CP) produces lung damage and pulmonary fibrosis in mice. CFis metabolically activated by the mixed-funcdon oxidase (Ivff+O) system agd can reportedly inhibit this system. Othe vestigators have reported that s a 50 mg/kg dose of CP, 7-14 days prior250 mg/kg, protectedwinst• lung damage in male mice. The objective of the present study was to determine whether this protective effect was evident using a higfiec~ preliminary dose and more sensitive indices of lung damage. Maf~ BALB/c mice were injected with 100 mg/kg CP followed by an additional 100 mg/kg on days 3, 7 or 14. Control animals received the same volumes of saline. Incorporation of r¢dioWlled thymidine into pulmonary DNA has been shown to be an ~rect.~rect ineasure of the extent of lung damage. Mice in each treatment group were injected i.p. on various days with 0.5 {tCi of 14C-thymidine and sacrificed after 90 min. Tlryniidine incorporation was maximal on Day 7 in mice receiving only a single dose of CP, and returned to control levels by Day 21. Tbis peak was unaffected by a subsequent CP dose on Day 3. Those mice receiving a second dose of CP on days 3, 7 or 14 experienced a second surge of thymidine incorporation on Days 21-28. Measurements of total lung hydroxyproline (HOP), an amino acid found primarily in collagen, accurately reflects the degree of fibrosis. All groups given a second dose of CP had levels of HOP on Day 35 significantly greater than saline or CP controls. The increases with two 100 mg/kg doses of CP were more than twice those seen with a single 100 mg/kg dose, but less than those with a single 200 mg/kg dose. These results indicate that a preliminary dose of CP delays the lung cell proliferation induced by a subesequent dose, but does not prevent additional lung damage and librosis. (JPK is the recipient of Research Career Development Award HL 01435.) 12 GLUTATHIONE IN HYDROPEROXIDE TOXICITY IN RAT ALVEOLAR MACROPHAGES. H J Forman, G A Loeb, and D C Skelton. Childrens Hospital and University of Southern California, Los Angeles, CA. Glutathione is an essential component of defense against oxidant injury. Release of oxidized glutathione (GSSG) has been u"sed as a- semi- quantitative measure of oxidant stress in a number of model systems. In the rat alveolar macrophage, the transport system responsible for the release of GSSG appears to be absent. Therefore, we focused upon another indicator of altered glutathione metabolism in these cells, glutathione-protein conjugate (PSSG) formation. PSSG is formed by the interaction of GSSG with the protein cysteinyl moiety in a reaction catalyzed by glutathione S-transferase. When 10 mM t-butyl hydroperoxide (tBOOH) was incubated with alveolar macrophages there was no release of GSSG above control until the appearance of concomitant lactate dehydrogenase (LDH) release, which began after 30 min. LDH release indicates gross membrane damage. Prior to LDH release however, total reduced glutathione (GSH) and GSSG decreased from 8.6 ± 1.6 to 3.0 ± 0.6 nmol/)tg protein with the major fraction converted to PSSG. Under less severe oxidant stress (10 pM tBOOH), GSSG was maintained at control levels of < 1% of the GSH + GSSG pool and PSSG did not increase. These results suggest that formation of PSSG can be a sensitive indicator of hydroperoxide stress and a better quantitative measure than is GSSG release. 50875 8128
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229 HAZARD COMMUNICATION: THE CASE FOR CATEGORY 4 "CANCER INFORMATION". J E Betso, R J Kociba. The Dow Chemical Company, Midland, MI. Today's demarylis to provide scientific infor- - mation to'a bAoad spectrum of the public suggest changes in the way chemical labeling is conducted. It'is now appropriate to provide supplemental information beyond a simple warning--that is, enumeration of a broader range of possible adverse effects that may be associated with overexposure. Wiiti regard to cancer hazard warnings, it is fairly easy to identify known human carcinogens and even confirmed animal carcinogens--ones without dispute among professionals. The new ANSI (American National Standards Institute, Inc.) guidelines have recommended that cancer warnings be limited to known human and con- firmed animal carcinogens. It seems appropri- ate, however, to extend cancer label statements to include information about certain animal tests which were indicative of some degree of carcinogenic activity, but which are not believed to be relevant to human risk when consideration is given to all of the scientific data. Information concerning carcinogenicity test results and their relevance to humans can be added to label text but need not be in the form of a warning. Examples are given of chemicals for which certain positive cancer test data exist, but which do not deserve full precautionary labeling. 231 THE ENVIRONMENTAL AND OCCUPATIONAL HEALTH INppR_ MATION PROGRAM (EOHIP): A BROAD-BASED APpROACg TO COMMUNICATING RISK TO THE PUBLIC. A Gotsch, R Kashdan, C Rovins. UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ Sponsor: M Gallo. Responding to the public's need for accurate apd reliable information about environmental and oc- , cupational health risks, UMDNJ-Robert Wood Johnson Medical School has developed EOHIP $g_g- model program to provide information and ser-x~_ vices to the general public, lay and profession*-- al employees, small industry, schools, and physicians. The program produces a variety of educational materials on specific risk issues. An Advisory Committee guides&vtlie"'p.rogram with.. representation from the diverse sectors involved in these environmental and occupational health issues, including labor, industry, government, media, public interest organizations, and academia. This presentation will review the goals of the program and discuss materials and services available to date. The "Healthy Environment- Healthy Me" school curriculum on environmental and occupational health and a videotape series on "Teaching Our Children About Hazardous Sub- stances" will be highlighted. EOHIP is designed to be a model program based in New Jersey that : can be replicated in other states. 230 AN INTERACTIVE ROLE FOR TOXICOLOGISTS IN 232 gESTICIDE INFORMATION PROFILES. A M Beale.and' COMMUNITY RISK MANAGEMENT. J S Heath and J A L Craiqmill. Environmental Toxicology Ex- Fessenden-Raden, Cornell University, Ithaca, NY. tension, University of California, Davis, CA. People misunderstand toxicological information, and hence risk management options, not because they are incapable of reasoned thinking and sound judgement, but rather because adequate, relevant toxicological risk information has not been provided. Case studies, part of a multi- disciplinary research project on environmental chemicals and community risk management, illus- trate how lack of involvement of toxicologists either directly or through an integrated risk management team resulted in misunderstanding and mismanagement. Understanding of toxicologic risk information is prerequisite to constructive participation in state, federal and corporate risk management efforts. In the absence of toxicologists, this information is provided by engineers and others not necessarily qualified to interpret or even convey information pertain- ing to the basic concepts of toxicology and tox- icologic risk. We will focus on the many ways that toxicologists,could participate meaning- fully in a community's effort to understand and manage environmental risk situations: inter- preting analytical results, epidemiologic data and potential health effects; explaining routes of entry, biotransformation, etc.; identifying relevant laws and regulations. Toxicology, as a profession, has failed to participate meaning- fully in risk communication at the local level. We propose a more active role. A series of 20 informational pamphlets were developed as part of a project in.conjunction with Cornell University, Oregon State and Michigan State for the EPA. These pamphlets were created to address the needs of pro- fessional/agricultural or home applicators of a variety of commonly used herbicides and pesticides. Information regarding the effect of the chemical on humans as well as target and nontarget species of wildlife and plants; Environmental issues such as accumulation/ magnification and ground water contamination; physical/chemical properties of the chemical and standard accepted uses was garnered from a variety of sources. This information was then distilled into an easy to read format, standardized between the participating insti- tutions, and readied for future distribution on an as requested basis. 58
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149 1NTRACELLULAR a-TOCOPHEROL CONTENT AND CHEMICAL TOXICITY IN HBPATOCYTES. M S Sandy, D Di Monte and M T Smith. School of Peblic Health, Univer- . sity of California, Berkeley, CA. _ a. The a-tocopherol (a-T) content of isolated rat hepatocytes exposed to several types of chemical injury was measured by HPLC-EC. Cellular a-T was depleted following exposure of BCNU-treated hepatocytes to cytotoxic doses of the redox cy- cling compound diquat. This loss 1of a-T could be prevented by addition of the antioxidant N,N'- diphenyl-p-phenylenediamine, although diquat- induced loss of soluble and protein thiols still occurred, as did cell death. Addition of dithio- threitol not only prevented this loss of a-T, but also protected against diquat-induced solu- ble and protein thiol loss, and cytotoxicity. In contrast, exposure of hepatocytes to cyto- toxic doses of either menadione, MPTP, or MPP+ did not deplete cellular a-T. Although peroxi- dation of cellular lipids occurs following ex- posure to diquat, none was observed in ~ells ex- posed to either menadione, MPTP, or MPP . On the other hand, aWoxidizing, but non-cytotoxic dose of ADP-Fe rapidly decreased cellular a-T levels. These data suggest that cellular ac-T loss is neither a prerequisite for, nor a neces- sary consequence of toxicity. Furthermore, al- though a-T protects against the oxidation of cellular lipids, the maintenance of hepatocyte a-T content does not prevent the oxidation of soluble and protein thiols. 150 LATEAIUEHP~ ANDIUITN-UCT1~L(NHTHALATEXYp~,)PHTAA- B DeAngelo, J Cicmanec, L P McMillan, and P-T Wernsing. U.S. Environmental Protection Agency Health Effects Research Laboratory, Cincinnati, OH Toxic responses of male F344 rats to DEHP and DOP were measured to gain insight into DOP's ability and DEHP's inability to promote the outgrowth of liver enzyme-altered foci. Groups of animals received DEHP (U.1, 0.5 and 2.0%) or DOP (0.5 and 1.0%) in the diets for 11 weeks. DEHP, but not DOP, increased liver weight and palmitoyl CoA oxidase activity. DOP did pro- duce marked hepatic changes such as hepatocell- ular cytomegaly, increased mitotic activity, vacuolization, chronic inflammation, and necro- sis. DEHP was only slightly effective at the high dose. The other pathological change seen was severe testicular tubular degeneration in the 2.0% DEHP group. Serum enzyme alterations reflected the liver pathology observed in the- UUP treated animals. These studies suggest that DOP might increase the outgrowth of altered liver cells through a regenerative response. (Abstract does not necessarily reflect EPA policy). 38 151 EFFECT OF FATTY ACIDS AND PEROXIDES ON ENDO- 4'HELIAL CELL 1024BRAINE STRUCTURE" AND FUNCTION. J M Patel,E R Block, and M K Raizada. College of Medicine, University of Florida and VAMC, Gainesville, FL. We investigated the effects of linoleic acid (18:2) and linoleic acid hydroperoxide (18:2 ' OOH) on membrane structu3t-and function of - vascular endothelial cells (EC). Conf1QftC' porc3ne pulmonary artery (PA), coronary artei,~-: (CA), and aortic (60) EC were treated with 15µM -=- each 18:2 or 18:2-OOH. After 2-4 hr 3ncubation at 37° C. membrane fluidity was monitored by measuring fluorescence anisotropy,(rs) of two membrane probes, DPH and PH. Insulin receptor binding was measured using 125 Insulin. Exposure of CA and PA EC to 18:2 for hr caused significant reductions in r for DPH (p <0.001) and for TMA-DPH (p < 0.02).sHowever, for AO EC it required a longer (4 hr) exposure to cause identical changes in r for both DPH and TMA-DPH. Exposure to 18:2-001f for 2 hr also caused significant reductions in r for DPH in CA and PA (p < 0.001) EC and in ASO (p < 0.02) EC. With more prolonged exposure (4 hr), the decreases in r were proportionately greater in all cell ~ypes. In addition, specific insulin binding was increased (p < 0.05) in all cell types exposed to 18:2. These results demonstrate that fatty acids and their perox- ides alter membrane physi.cal state and receptor -ligand binding of EC from a various vascular beds.(supported in part by AHA-FL). 152 COMPARATIVE EFFECTS OF BIS-(BETA- CHLOROETHYL) SULFDE (BCES) ON THE DNA OF BASAL AND DIFFERENTIATED KERATINOCYTES R Scaverelli, F L Vaughan and I A Bernstein. Toxicology Program. Dept. Env. Ind. Health, Univ. of Michigan, Ann Arbor, MI. Isolated rat epidermis and stratified rat keratinocyte cultures were exposed topically to BCES for 0.5 h with concentra~ ions ranging from 0.01 to 50 nmoles/cm . After exposure, the cells from each source were harvested and separated into basal and. differentiated cell populations. The formation of DNA single strand breaks was measured in both populations by the nucleoid velocity sedimentation assay. The level of DNA alkylation in the two cell types was also measured by first exposing rat geidermis and keratinocyte cultures to C-BCES for 30 ' min. ConcenTrations ranged from 5 to 50 nmoles/cm . DNA from the cell populations obtained from each source was then isolated using a CsCl density gradient. These studies demonstrated that the basal cells are more adversely affected by BCES than differentiated cells as indicated by more single strand breaks and higher DNA alkylation levels resulting in the former after similar exposure. 50875 8163
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221 TISSUE DISTRIBUTION, METABOLISM AND ELIMINATION OF PENTACHLORODIPHENYL ETHER IN THE RAT. E Komsta, I Chu, D C Villeneuve, F Benoit aia II- Wrdoch. Env ronmen a a th Directorate, Health Protectten Branch, Ottawa, Ontario. Canada. Chlorinated diphenyl ethers are demonstrated environmental contaminants in the Great Lakes region. One of the most predominant congeners found in Lake Ontario fish is 2,2',4,4'5-penta- chlorodiphenyl ether (PCDE) but little information is available on its toxicity or metabolism. The present study was carried out as part of an ongoing program on chlorodiphenyl ethers and provides information on the tissue distribution and metabolism of this isomer in the rat. PCDE was distributed in all tissues examined with the highest concentrations being found in fat followed by skin, liver, kidney and muscle. Most of the radioactivity found in the tissues was due to the unchanged PCDE. The decay profile of PCDE in the blood was fitted to a four compartmental pharmacokinetic model (4 exponential terms) and the last exponential term had a half-life of 5.8 days. A total of 55% and 1.3% of the administered dose was excreted in feces and urine respectively in seven days. More than 64% of the fecal radioactivity was due to the unchanged PCDE, while the hydroKylated PCDE accounted for 23% of the excreted radio- activity. :222 A COMPARATIVE STUDY OF MACROPHAGE: DEVELOPMENT OF A FISH MODEL FOR TOXICOLOGICAL STUDIES. JT Zelikoff, RB Schlesinger, KS Squibb, JM O'Conner. New York University Med. Cntr., Inst. of Env. Med., Tuxedo, NY. The immune defense mechanisms of fish are close- ly related and similarly competent to that of mammals. Because of this, interest in the im- mune response of fish as a model for higher ver- tebrates in toxicological studies, is increasing. Investigations to establish normal baseline cri- teria for different components of the immune re- sponse of fish are needed prior to toxicological studies. Macrophages are a pivotal cell in the immune response and host defense of fish and mammals. In this study we have examined the morphology and in vitro phagocytic activity of unexposed rainbow trout peritoneal macrophages and compared these results with those of rabbit alveolar cells (RAM). Results showed that trout and rabbit.macrophages were similar in size, shape and nuclear configuration and both'were esterase positive. To determine the phagocytic index (PI) and capacity (PC), cells were incu- bated with latex beads for 30, 60 and 90 minutes using culture conditions appropriate for the species. Though cells from both species take up particles in a similar manner (i.e., by membrane engulfment) the PI and PC for trout cells were low compared to RAM. These results provide com- parative information needed to evaluate the use- fulness of fish macrophages to serve as a model for higher vertebrates in immunotoxicological studies. Supported by NIEHS Ctr. Grant ES00260. 223 EFFECT OF METHOD AND'DURATION.OF EXPOSURE 0F. ~ PIPERONYL BUTOXIDE ON THE HEPATIC MONO;:;;,-O XYGENASE. OXYGENASE ACTIVITY OF RAINBOW TROUT. D A:' Erickson, M L Haasch, and L h. Departmenti of Pharmacology and Toxicology, Medical.`' College of Wisconsin, Milwaukee, WI. The induction of hepatic monooxygenase (MO)~6 activity in fish by ag%t1. other than~ ,Iyolycycl i c aromati c hydrocarybns i s not weL,.:,,.Y ' documented. This study characterizes the-,., ~_ induction of hepatic MO in rainbow trout using piperonyl butoxide (PB0). When trout were- exposed by bath to PB0 over 21 days, hepatic~:- MO activity increased to maximal levels in 1b days. Following . transfer to,.- clRean water, trout (induced by PBO by bath ~5r 14 days) had, hepatic MO activity that was half the fu11y= induced level after 9 days. When trout were exposed to PBO by i.p. injection, hepatic MO., activity increased to relatively low maximal levels and rapidly returned to control - levels. When levels of P1450 mRNA were measured over time following i.p. injection of . either a-naphthoflavone (BNF) (100mg/kg) or PB0 (450 mg/kg), the P1450 mRNA reached_ maximal levels at 18 hrs. and 48 hrs.,,° respectively. These data indicate that.°.: induction of hepatic MO activity in trout by PBO is affected by the type and/or duration of . exposure and that the genetic regulation of ..`` induction of hepatic MO activity by PB0 and BNF in trout may be mechanistically .'. different. (Supported by NIEHS grant ES 01080) 224 COMPARATIVE INDUCTION OF HEPATIC C_YTOCHROME P450 mRNA AND CATALYTIC ACTIVITY IN VARIOUS SPECIES: '. STUDIES USING A COMPLEMENTARY DNA PROBE. M L Haasch, P Wejksnora, J J Lech. Medical College of Wisconsin and University of Wisconsin- Milwaukee, Milwaukee, WI. Comparative induction in rainbow trout ( aS lmo gairdneri), brook trout (Salvelinus f n inali ), painted turtle (Chrysomvices Dicta) and rat was examined by determination of levels ofP)450 mRNA and corresponding catalytic activities. Levels of P1450 mRNA were determined utilizing - pfP1450-3', a 3'-specific, 1.5 kb comple- mentary DNA (cDNA) clone derived from 3-methyl- cholanthrene-induced P1450 mRNA of rainbow trout (gift of D W Nebert, Bethesda). Animals were treated with beta-naphthoflavone,(13-NF, 100 mg/Kg, i.p., 24 hrs.) Rainbow trout, brook trout and rat were significantly induced (25-, 76-, and 16-fold, respectively) with respect to ethoxyresorufin-0-deethylation (EROD). In addition rat was significantly induced (4.4- and 1.4-fold) with respect to ethoxycoumarin-0- deethylation (ECOD) and total cytochrome P450. All of the species examined possessed mRNAs which were recognized by pfPl 450-3' with rainbow trout and brook trout exhibiting an increase in an mRNA of about 2.5 kb (8.3- and 6.8-fold, respectively). These data support previous findings and provide evidence for the usefulness of pfP1450-3' for examining induc- tion and P450 gene regulation in many different aquatic species. (Supported by ES 01080). 56 50875 8181
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17 N1EAS(k2EMENT OF TIDAL VOLUME, RFSPIRATORY k'NSQl1ENt;Y, 02 UPTAIiE ANI) COz OUTPUI' IN EXERCIS- ING (.U1NEA _PIGS. Y Alarie and D Malek. Graduate School of Public Aealth, tiniversit.,y- of Pittsburgh, fi.ttsburgh, PA. - . We have built a guinea pig ergometer within an enclosed ventilated chamber which served as a whole body plethysmograph permitting continuous measurement of tidal volune (VT), (measured indirectly from pressure change,(P) created with each breath, respiratory frequenc,y, (f), 02 uptake (V02) and C0z output (VCbz). Guinea pigs were trained to run with no shocking device. The above measurements were made at rest and during 30 minutes of running at twice or three times their basal V0z. With these increases in V02 there was an increase in minute volume (W) but this increase was mainly due to an increase in f rather than an increase in VT. This is opposite what we have previous- Ly found in guinea pigs challenged with 10% Cbz in inspired air which increased VT much more than f. Adding 10% Cbz during exercise resulted in a large increase in VT with the increase in f still maintained. This combina- tion of exercise and 10% Cbz resulted in a final increase in MV of 9 to '12 times above baseline. The large increase in MV was sustained with no evidence of stress on the animal. Thus this protocol could be used in toxicological studies of airborne contaminants. Supported by NIEHS 1R01-FS02747 and NBS 60NAN134D001. 18 DOSE-DEPENDENT CHANGES IN AIRWAY CONDUCTANCE AND EDEMA IN GUINEA PIGS EXPOSED TO I_NHALED fNDOTOXIN. rdon J Balmes, J Fine, and D Sheppard. UCSF, CA There is evidence that some of the acute airway symptoms associated with exposure to inhaled cotton and grain dusts are correlated with the level of endotoxin (ENDO) in these dusts. In the present study, changes in specific airway conductance (SGaw) and airway edema were examined In guinea.pigs exposed to aerosols of ENDO (1 and 10 ug/ml in saline) or saline for 3 hrs. SGaw began to drop by 90 min and decreased by 22% and 33% by 3 hrs in animais exposed to i and 10 ug/ml ENDO, respectively. Exposure to saline alone did not alter SGaw. Exposure to ENDO produced a dose-dependentincrease in vascular permeability (iv Evans blue dye, 50 mg/kg, was used as a marker of protein extravasation) In the trachea and mainstem and hilar bronchi. The dye content of tracheas from animals exposed to 10 ug/ml ENDO (49.0t 6.4), but not tug/ml ENDO (23.2±2.4), was significantly greater than that of tracheas from animals exposed to saline (20.7±3.5). Dye leakage was more pronounced in the mainstem (50.5±8.1 and 101.5±12.7) and hilar bronchi (66.1t 11.0 and 102.3±14.8) in 1 and 10 ug/ml ENDO animals (0.05 < p(0.01), respectively. In saline animals, the dye content In the mainstem bronchi was 24.5±3.4 and in the hilar bronchi was 29.5±3.2. These results suggest that inhalation of aerosols of ENDO (approximately 10 to 100 ug/m3) in concentrations that approach those found In the workplace can produce airway edema as well as airway obstruction. Airway edema may contribute to symptoms experienced by workers during acute exposures to cotton and grain dusts. 5 19 BRONCHI AL REACT I V I TY %TO..i Hs" •1~?8TNu1 I NE : TEST i N3 C,LJ I NEA PIGS IN BODY PLETHYSM31APHS. P S Thorne and M H Karol. Dept. of Industrtal Envlronmental Health Sciences, Univ. of Pittsburgh, Pittsburgh, PA. :f Bronchial hyperreactivity has been associated with late-onset allergic reactions. The aim of this work was to incorporate a method for assQss- ing bronchial reactivity in guinea pigs intoP'our animal model for lmnediatfts and late-onset pu'Imo- nary sensitivity without interruption +af -1ong term pulmonary monitoring. A multiple dose.,(Np) protocol was developed In which guinea i4gs received 15 min exposure to 1.5-fold (ncreasing histamine doses. This protocol was tested with and without breathing augnented by exposure to, 10% C;02. Plethysmgraphjpressure changes (pP) associated with each brOgth , were monitored. Histamine exposures with C02 revealed a dose- dependent decrease In a P fran the C02-induced increase. The concentration required to reach a 33% decl(ne frcm the 0 P increase, PC,33(C02), averaged 0.58±0.17 mg/m3 (±SD,N=38). Exposures to histamine in air produced an increase (n LP due to bronchoconstriction with dose-dependent onset. The dose which doubied dP, PC;2 a p, was 2.1±0.9 mg/m3 (N=20). Parallel tests using this protocol and a head-on i y system shaved that PC:2. 6 p corre- lated with a decrease in VT. Exposures on 3 con- secutlve days demonstrated repeatability with no significant difference in PC2.QP or in P033(C02). The Intersubject variability with these protocols was half that reported for PC2OFEV1 (n hunans. Supported by NIEHS ES01532. 20 HISTOCHEMICAL LOCALIZATION OF F ORMALDEHYDE DEHYDROGENASE FDH AC IV TY I E _ R D ( ) N T I TH AT. A Keller, H d'A Heck, H W Randall, and K T Morgan. Chemical Research Industry Institute Triangle Park, NC. of Toxicology, Formaldehyde (HCHO) is a nasal carcinogen in the rat and is metabolized by FDH (EC to non-genotoxic formate. FDH activity is known,to exist in the olfactory and respiratory mucosae of the rat. The cellular localization of FDH activ- ity has not previously been investigated. Cold processed glycol methacrylate (GMA) embedded tissues were used to localize FDH activity in the rat respiratory tract and kidney. Ten )tm GMA sections were incubated at 37oC with a reaction mixture containing HCHO, glutathione, NAD, pyr- azole, and nitroblue tetrazolium. Disulfiram was used to inhibit nonspecific aldehyde dehydroge- nases. FDH activity yielded a blue formazan precipitate. The kidney was stained intensely at the brush border and in the cytoplasm of the epithelial cells of the proximal tubule. FDH staining was weaker in the respiratory tract, with the exception of the Clara cells. However, FDH activity was detected in the nasal passages, with diffuse cytoplasmic staining of both respi- ratory and olfactory epithelial cells, Bowman's gland, and seromucus glands. Identical prepara- tions lacking glutathione, a required substrate for FDH, had little or no activity. These results indicate that FDH activity is present in the rat respiratory tract and kidney in specific cell types. 50875 8130
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177 BLOCK OF 45Ca UPTAKE BY METHYLMERCURY (MeHg) INTO NERVE TERMINALS IS Na-DEPENDENT AND PARTIALLY REVERSIBLE: T J Shafer and N D Atchison:` Dept. Pharmacol./Tox., Michigan- State Univ., E. Lansing, 14i1. Experiments. were performed to investigate the ability of increasing extracellular [Cal ([Cale) to relieve block of Ca uptake by MeHg into rat fore- brain synaptosomes. Ca uptake was assessed by measuring 45Ca content following 1.' 1"fast phase") or 10 ("total") sec of depolarization induced by elevated K+ (77.5 rn(l1), or after 10 sec of depolarization in synaptosomes that had been predepolarized for 10 sec in solutions free from Ca and h1eHg ("slow phase"). CJhen compared to control, MeHg (100 NM) reduced "total" uptake of 45Ca by >70% at all [Cale tested (.019 to 2.3 mM). Over the same range of [Cale, 100 NA1 A9eHg reduced "fast" uptake by 20-60% when compared to control. For [Cale of .019 to .29 mM, 100 Nft•1 P,1eHg reduced "slow" uptake by 75-90%. However, for [Cale >0.58 mM, Ca uptake returned to control levels. Thus, block of this • component of uptake by A4eHg was reversed by increasing [Cale. A portion of the "slow" phase of uptake is dependent on [Na]e. To test for dependence of block of this phase by P.lelag on [Nale, 45Ca uptake was compared in synaptosomes incubated in normal or Na-free (choline substituted) buffers. MeHg (100 pM) completely blocked 45Ca uptake into both groups, whereas 50 pM, MeHg reduced uptake by 86.5±2.5 and 49.5t14.5 0 in N a- and choline-synaptosomes, respectively. These results indicate that the blocking action of MeHg on the "slow" phase of Ca uptake in synaptosomes is Na-dependent. (Supported by NIH grant ES03299.) 178 NO CORRELATION BETWEEN METHYLMERCURY-INDUCED TRANSMITTER RELEASE AND ZrALCIUM EFFLUX FROM SYNAPTOSOMES. D Minnema, Dept. Environ. Hith., U. Cinti., Cinti., OH. Sponsor: P Hammond. Methylmercury (MeHg) in vitro has been shown to increase spontaneous acetylcholine (ACh) release from presynaptic motor terminals. This effect has been attributed to an increase in the intra- neuronal ionized calcium [Ca]i that apparently results from a MeHg-induced increase in mitochondrial Ca efflux (Atchison, 1987). In the present study, MeHg was shown to produce a concentration-dependent (0.5-5uM) increase in the spontaneous release of several CNS transmitter substances (ACh, dopamine, GABA) from superfused rat brain synaptosomes. However, MeHg (under similar exposure conditions) did not produce a corresponding increase in 45Ca efflux from superfused synap- tosomes preloaded with 45Ca. An increase in synaptosomal 45Ca effl ux would be expected if MeHg was increasing intraneuronal [Ca]i. These results suggest that if the MeHg-induced increase in spontaneous transmitter release results from an increase in intraneuronal [Ca]i, then this [Ca]i is not being extruded from the nerve terminal, due to either increased intra- neuronal buffering of the [Ca]i, or the simulta- neous inhibition by MeHg of [Ca]i extrusion mechanisms (e.g. Ca-Mg-ATPase). Alternatively, MeHg may be increasing spontaneous transmitter release by mechanisms not involving an increase in intraneuronal [Ca]i. (Supported by ES-03992). 179 EFFECTS OF MERCURIC CHLORIDE (Hg) ON,SPONTANEOUS TRANSMITTER RELEASE AND Na+,K+-ATPase (NKA) IN SYNAPTOSOMES. M F Hare and D Minnema. Dept. Environ. Hith., U. Cinti., Cinti., OH. Sponsor: E J O'Flaherty. Electrophysiological studies employing amphibian' neuromuscular preparations have shown that Hg in~ vitro increases both spo~ n~~eous and evoked:. 'itransmitter release. These H"g effects are simi- - lar to those produced by the NKA inhibMF ouabain. A number of reports have shown tha"t~:_ brain NKA is sensitive to inhibition by Hg. The -` present study examines the role of NKA in mediating the effects of Hg on neurotransmitter release. Purified rat str~tal.Y syHap do somes were either: (a) preloaded with 3 pamine (DA), superfused, and release examined, or (b) lysed, and membranes exposed to Hg and NKA acti- vity measured. Hg (3 uM) causes an increase in spontaneous DA release from intact synaptosomes that is insensitive to changes in extracellular Ca2+ levels. Lowering the Na+ concentration (replaced by choline chloride or sucrose) in the superfusing buffer attenuates the Hg effect. Addition of ouabain to Ca2+-free superfusing buffer causes an increase in spontaneous DA release but does not inhibit the Hg effect. Specific activity of NKA in lysed synaptosomal membranes is sensitive to Hg inhibition (IC50=160nM). These data suggest that Hg inhi- bition of NKA activity may, in part mediate Hg- induced increases in DA release. (Supported by ES-03992). 180 MANGANESE IRANSPORT ACROSS THE $LOOD- BRAIN BARRIER IN THE RAT. L E Kerper, M Aschner. J D Obourn, and T W Clarkson. Environmental Health Sciences Center, School of Medicine and Dentistry, University of Rochester, Rochester, NY. The mechanism of Mn transport across the rat blood-brain barrier was investigated in the rat. Radiolabelled carrier free 54Mn was injected into the rat common carotid artery in a Ringer's buffer solution at pH 7.55. Fifteen seconds after injection rats were decapitated and brains were removed and counted by means of gamma scintillation spectrometry. Injection with [U-14C]-sucrose, an inert polar substance which does not penetrate the blood-brain barrier, was used to determine the residual radioactivity in the cerebrovascular space. The transport of 54Mn across the blood-brain barrier appears to be a rate-limited process. [U-14C]-sucrose and 3H-methionine uptake does not change with increased concentrations of injected 54Mn up to 0.55 x 10-6 M, suggesting that the blood brain barrier remains functionally intact at these concentrations. Calcium and magnesium do not appear to inhibit 54Mn transport. 54Mn transport does appear to be inhibitable by iron-dextran venous infusion in a dose related manner. Supported by NIH grants: ES-01247, ES-01248 and ES- 07026. 45
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iAECI3At1ISN1S OF IN VITRO ACELAMIIQOPM II0IBITION OF RFSPIP.ATION IN HEPATIC MITOGHONDRIA. L L Nleyers, W P Beierscbmitt, E A Khairallah, and S D Cohen. Toxicology Program, University of Connecticut, Storrs, CT. + Inhibition of both glulamate- and succinate- supported mitochondrial respiration is associated with acetaminophen (APAP) hepatotoxicity in vivo. AP.W, in vitro, rapidly inhibits mitochondeial respiration with glutamate but not succinate as substrate, suggesting a direct effect at or prior to Comolex I of the respiratory chain. T8 test this, the effect of APAP on respiration of mitochondria isolated from control male CD-1 mice Taas studied polarographically with alpha-keto- glutarate and beta-hydroxybutyrate as additional Complex I substrates. As with glutamate, state 3 respiration and respiratory control were decreased frora 10 to 90% in a dose dependent manner by 2.5 to 20 mM APAP. In contrast to in vivo findings, no covalent binding was detected in mitochondria exposed to APAP in vitro. Washing APAP from inhibited mitochondria resulted in complete return to control respiratory rates. This indicates that APAP's in vitro action differs from that which predomi.nates in vivo and may result from a direct and reversible inter- ference with electron transfer at Complex I. 123 CYTOCHROME P-450-MEDIATED CATALYSIS WITH CUMENE HYDROPEROXIDE OF ACETAMINOPHEN TO N-ACETYL-_E- BENZOQUINONE IMINE AND R-ACETYL-P- BENZOSEMIQUINONE IMINE. D W Potter, and J A Hinson. National Center for Toxicological Research, Jefferson, AR. The oxidation of acetaminophen (APAP) by P-450 results in the formation of reactive intermediates which are involrved in APAP toxicity. daiftent data indicates that the 2-electron oxidation product, N-acetyl-Q- benzoquinone imine (NAPQI), both binds to and oxidizes cellular sulfhydryl groups. To better understand the mechanisms of APAP oxidation b P-450, reaction mixtures containing ro°somal P-450, cumene hydroperoxide and APAP were evaluated for APAP oxidation - products. It was shown that P-450 catalyzed APAP to NAPQI which reacted with GSH to form 3-(glutathion-S- yl)APAP and to N-acetyl-g-benzosemi- quinone imine to give APAP polymeriza- tion products. However, in reaction mixtures containing NADPH, the APAP polymerization products were not (Supported in part by NIH grant GM 31460' and ES 07163) EFFECT OF P_REGNENOLONE-16a-CARBONITRILE 124 (PCN) ON ACETAMINOPHEN-INDUCED HEPATO- TOXICITY IN HAMSTERS. C Madhu and C D Klaassen. Univ. of Kansas Med. Ctr, Kansas City, KS. Excessive dosages of acetaminophen (AA) are known to produce acute liver toxicity in both humans and laboratory animals. Hamsters are especially sensitive to the hepatotoxic effects of AA. In the present study, hamsters pretreated with PCN (75 mg/kg, ip, daily for 4 days) were given a single dose of AA (300-1200 mg/kg, ip) and liver function determined at 24 hr. Serum activities of alanine aminotransferase (ALT) and sorbitol dehydrogenase (SDH) as well as histopathology were used as indices of hepatotoxicity. PCN pretreatment decreased AA-induced mortality. PCN dramatically decreased ALT (93-97%) and SDH (63-98%) activities relative to control values from hamsters treated with AA alone and remarkably decreased hepatic centrilobular necrosis produced by AA. To investigate the mechanism of this protective effect, the biliary and urinary excretion of AA metabolites were measured for 1 hr after administration of AA (150 mg/kg, ip) in bile duct-cannulated hamsters anesthetizedd with pentobarbital. PCN pretreatment resulted in increased urinary and biliary excretion of AA-glucuronide and decreased biliary excretion of AA-glutathione. Microsomes from PCN-pretreated hamsters produced less reactive benzoquinoneimine intermediate than controls, as determined by the formation of AA-GSH. In conclusion, PCN has a dramatic protective effect against AA-induced hepatotoxicity. The mechanism of this protection appears to be due to decreased formation of the reactive metabolite by the cytochrome P-450 pathway, and an increased detoxication by enhanced glucuronidation of AA. (Supported by USPHS Grant ES-03192) 31 detected. This was possibly due to rapid reduction of APAP free radicals by NADPH. These results indicate that P-450 catalyzes both the 1-and 2- electron oxidation of APAP. EVIDENCE FOR THE IN VIVO FORMATION OF p-BENZO- QUINONE AS A REACTIVE INTERMEDIATE IN ACT- AMINOPHEN METABOLISM. G A Pascoe, C J.Calleman, and T A Baillie. Dept. of Medicinal Chemistry, University of Washington, Seattle, WA. Sponser: S D Nelson. Acetaminophen (APAP) undergoes metabolic activa- tion by mixed function oxidases to the highly reactive N-acetyl-p-benzoquinone imine (NAPQI). NAPQI readily forms thioether conjugates with both hepatic glutathione and cysteinyl residues of liver proteins, and has been speculated to undergo hydrolysis to -benzoquinone. In mice treated with 14C-APAP p(200 mg/kg, i.p.), we have identified S-(2,5-dihydroxyphenyl)-cysteine and S-(2,5-dihydroxyphenyl)-N-acetyl-cysteine [the cysteine and mercapturate adducts, respectively, of hydroquinone (HQ)] as urinary excretory products. Adducts were isolated from 24h urine by HPLC fractionation and were converted to a common derivative, O,O,S-tris-acetyl-3-thio-HQ, which was characterized by GC-MS. Quantification of urinary HQ-thiol adducts by HPLC indicated that they accounted for 6.3% of urinary APAP- thiol conjugates. In hemoglobin hydrolysates from 14C-APAP-treated mice, HQ-cysteine was similarly identified as a minor component of the covalently bound 14C-residues. These findings provide strong indirect evidence that p-benzo- quinone is indeed formed in vivo as an addition- al reactive metabolite ofi-APKP_. Supported by NIH grant DK 30699. 50875 8156
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141 MOLFICULAR PROPERTIES OF THE Ah RECEPTOR COMPLEX USING DIFFERENfP pOLYCHLORINATED AROMATIC RADIOLIGANDSS. J PiskorskaaPliszczynska and S Safe, Department of Veterinary bfiysiology and _ Pharmacolog_,aS. College of 4eterinary Medicine, Texas A&M Chiversity, College Station, TX. The mole,^ular properties of the rat hepatic Ah receptor complexed with [3H]-2,3,7,8-tetra- chlorodibenzo-p-dioxin (TCDD), [3H]-2,3,7,8- tetrachlorodibenzofuran (TCDF) aDk7,other radiolabeled polychlorinated dibenzofurans were evaluated by velocity sedimentation on sucrose gradients. In conditions of low (no salt), moderate (0.1 M KC1) and high (0.4 M SC1) ionic strength, there were significant differences in the sedimentation constants for the Ah receptor- radioligand canplexes. For [3H]-2,3,7,8-2CDD, the sedimentation constants were 8.7, 9.6 and 6.8 S under conditions of low, moderate and high salt. In contrast using [3H]-2,3,7.8-TCDF, both the high and low molecular weight binding subunits wzre observed in low salt conditions (8.9 and 5.6 S respectively), whereas in high salt only the peak sedimenting at 5.0 S was observed. Incubation in moderate salt condi- tions (0.1 M IC1) gave a broad peak which sedimented between 8.9 and 5.2 S. It was evident that the molecular properties of the bound receptor complex depended not only on the incubation conditions but on the structure of the radioligand. (Supported by the National Institutes of Health.) 142 TCDD EFFECTS ON MOUSE UTERINE CYTOSOLIC PROTEINS. S A MacKenzie, D S Brandewene, P Scala, T H Umbreit, and M A Gallo. Jt.Grad.Prog.in Toxicology, UMDNJ-R.GI.Johnson Medical School/Rutgers Univ., Piscataway, NJ. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) has been shown to antagonize certain estrogenic effects, such as estrogen-induced increase in uterine weight and tumor growth in estrogen- responsive tissues. TCDD down-regulates the estrogen receptor.. Recent studies indicate marked changes• in specific mouse liver cytosolic proteins. 30 day old C57B/6 female mice were given 0, 20, or 40 ng estradiol s.c. in corn oil, daily for 14 days. On days 7, 9, 11, and 13 they also received 0, 5 or 10 ug/kg TCDD in corn oil, by gavage. After sacrifice on day 15, cytosol was prepared from uteri and livers pooled from each group. Cytosolic proteins were separated on one-dimensional SDS- 10% polyacrylamide gels and stained with Comassie blue. Multiple bands of 20-200 kd MW were found. A prominent band of Mr 65 kd was found in uterine cytosol from all groups. This Mtd is near that of the 68 kd estrogen receptor. This band appeared reduced in intensity in mice receiving TCDD but not estrogen. The dioxin effect was less apparent in estrogen-treated mice. Further studies are in progress to identify this protein. Dioxin increased liver/body weight ratios, which were not altered by estradiol, and uterus/body weight ratios were increased by estrogen exposure. 36 143 A CANTRARIDIN BINDING SITE IN MOUSE LIYER CYTOSOL: CORRELATION OF BINDING AFFINITY AND ACUTE TOXICITY. M J Graziano, A L Waterhouse, and J E Casida. Pesticide Chemistry and Toxicology Laboratory, Dept. of Entomological Sciences, U of California, Berkeley, CA. Cantharidin is a potent natural vesicaA isolated from blister beeAAes. To investigate. s its mechanism of mammalian toxicity, [3H}ean- tharidin (14 Ci/mmol; >98X radiochemio4._, purity) was synthesized and assayed as radioligand using mouse liver as the model target organ. [3H]Cantharidin was shown to interact in a specific and saturable manner with a binding site in the.AytoWolic fraction with apparent Kd and Bmax value~s of 30 nM and 1.8 pmol/mg protein, respectively. There was no appreciable binding to any of the membrane fractions. The potency of cantharidic acid (the dicarboxylate form of the anhydride) and 20 other structurally related compounds, including the widely used herbicide endothal, as inhibitors of [3H]cantharidin binding in vitro correlated extremely well with their acute toxicity to mice (r=0.962). Furthermore, [3H]cantharidin binding to the cytosol of mice pretreated with unlabeled cantharidin was inhibited in a dose-related manner. The results of this study strongly suggest that the mechanism of cantharidins' toxicity involves interaction with a cytosolic binding site. (Supported in part by NIEHS Grant P01 ES00049). 144 MEASUREMENT OF SERUM ALBUMIN GENE EXPRESSION AS A FUNCTIONAL INDICATOR OF HEPATOXICITY. J S Ray, R L Vorce, R K Jensen and J I Goodman, Dept. Pharm./Tox., Cent. Env. Tox., Michigan State Univ., E. Lansing, MI and (RKJ) Path. Tox. Res., The Upjohn.Co., Kalamazoo, MI. Our objective is to test the hypothesis that alterations in gene expression might serve as an indicator of chemically-induced hepato- toxicity. Decreased serum albumin gene ex- pression was used as an indicator of liver dysfunction because it is expressed in normal hepatocytes. Actin gene expression was chosen as an internal control; actin is produced at a constant rate in all cells. Bromobenzene (BrB) was administered to phenobarbital-induced male Sprague-Dawley rats at doses of 1.0, 2.5 or 5.0 mmoles/kg; they were sacrificed 24 or 48 hr after BrB. Histopathology and serum alanine aminotransferase (ALT) levels were assessed, and RNA was isolated from hepatic cells. Levels of albumin and actin mRNA were measured by slot-blot analysis using a 32P-labelled cDNA probe for each gene. The higher doses of BrB resulted in hepatic necrosis and increased ALT with proportional decreases in albumin mRNA. The low dose of BrB did not result in necrosis or changes in ALT; there did appear to be decreases in albumin mRNA. These results indicate that monitoring albumin gene expression might -serve as a useful adjunct to traditional methods of assessing hepatotoxicity. 50875 8161
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p-XYLENE POTENTIATION OF CARBON TETRACHLORIDE HE1sATOTOXICITY. 3 E Simmons, E C Grose, B L Robinson and J W Al_Tis Health ffet„ks Research Laboratory, U.S.E.P.A., Research Triangle Park, NC . ?. Because inhalation of 1600,ppm p-xylene for 3 days appeared to enhance the hepatotoxicity of orally administered carbon tetrachloride (CC14) in rats, we conducted experiments to determine if a single exposure to p-xylene could potentA; ate CC14 hepatotoxicity. Male F344 rats were exposed to 0 or to 1600 ppm p-xylene by inhala- tion for 6 hours, gavaged 48 hours later with 0 or with 0.075 ml CC14/kg, and killed 24 hours later for assessment of hepatotoxicity. Exposure to CC14 only, but not p-xylene only, increased serum alanine aminotransferase and aspartate aminotransferase activity, increased the liver- to-body-weight ratio, and caused mild centri- lobular vacuolar degeneration as well as minimal centrilobular necrosis. Combined exposure (p-xylene plus CC14) did not result in increased hepatotoxicity relative to CC14 only. These results indicated that a single 6 hour exposure to 1600 ppm p-xylene was not sufficient to potentiate the hepatotoxicity of CC14. The induction of hepatic cytochrome P450 at the time of CC14 dosing, observed after 3 exposures to pxylene, suggests a possible mechanism of the p-xylene mediated potentiation. (This is an abstract of a proposed presentation and does not necessarily reflect EPA policy.) EFFECT OF 24-HOUR INFUSION OF SK&F 93944 ON HEPATIC FUNCTION IN RAT, DOG, AND MONKEY. A Poole, W Hewitt, and G Betton. Smith Kline & French Research Ltd., Welwyn, UK and Philadelphia, PA. Sponsor: J B Hook SK&F 93944, a competitive histamine Hi-receptor antagonist, produced appreciable, but intermittent elevations in the plasma activity of alanine aminotransferase (ALT) and glutamate dehydrogenase (GLDH) when administered chronically to dogs but not rats. The objective of this study was to assess the potential of SK&F 93944 to produce acute hepatocellular dysfunction in 3 species: rats, dog and monkey. Male Wistar rats, beagle dogs, and cynomolgus monkeys received iv infusions of SK&F 93944 at rates ranging from 5 to 35 mg/kg/hr for 24 hrs. Hepatocellular dysfunction was assessed by monitoring serum concentration of bi.lirubin (BILI), and the activity of ALT, GLDH, and alkaline phosphatase (ALK) at 0 (control), 12, 24, and 48 hour from the start of the infusion. The results, at large dosages, indicated that: (1) SK&F 93944 could produce hepatocellular dysfunction in the dog: and (2) the dog was more susceptible to SK&F 93944-induced hepatic dysfunction than the monkey: the rat was the least susceptible species. . a- 266 RESPONSES OF THE LUNG TO INHALED CARBON BLACK AND INHALED DILUTED DIESEL EXHAUST. RF Henderson, R K Wolff, J L Mauderly and R McClellan. Lovelace Inhalation Toxicology 267 67 Research Institute, Albuquerque, NM Inhalation of diesel exhaust by rats causes an inflammatory response in the lung at lung bur- dens of soot H.5 mg/g lung. We hypothesized that this response was due to the par icle load in the latig and not to the gases or a r ed or- ganic compounds associated witD the soot. To test this hypotheses, F344 rats were exposed 7 hr/day, 5 days/wk for 12 wks to diesel exhaust or carbon black at 3.5 or 10 mg particles/m3. The inflammatory response of the lung was meas- ured at the end of the exposure by analys_itof bronchoalveolar lavage fluid (BALF) fpF macro- phages, neutrophils (PMN), lactate dehydroge- nase, a-glucuronidase, acid proteases and pro- tein. Lung burdens achieved by the low and high level exposures were (x t SD) 0.94 = 0.37 and 2.8 ± 0.6 mg/g lung for carbon black and 0.99 = 0.40 and 2.5 = 0.5 mg/g for diesel soot. A simi- lar response was caused by both types of parti- cles. There was no indication of pulmonary inflammation at the lower dose of either parti'- cle. At the higher dose, there was an increase of PMN's and acid protease activity in BALF, in- dicating a mild inflammatory response to both particles. The data indicate that the inflamma- tory response to diesel exhaust is due mainly to the accumulation of carbonaceous particles in the lung. (Research supported by U.S. DOE/OH- ER under Contract No. DE-AC04-76EV01013.) PULMONARY EFFECTS OF COMBINED INHALATION EXPOSURES OF RATS TO OIL SHALE DUST AND _DIESEL EXHAUST. J A Pickrell, E B Barr, A F Eidson, R F Henderson, J R Harkema, and J L Mauderly. Lovelace Inhalation Toxicology Research Institute, Albuquerque, NM Because diesel powered vehicles may be used to mine oil shale, workers may be exposed to com- bined aerosols of oil shale dusts (S) and die- sel emissions (D). We studied potential inter- actions of D (carbonaceous particles) and S (mineral particles) in rats exposed for 7 hr/day, 5 days/wk for up to 30 mo to raw or re- torted S at 5 mg/m3, to D at 3.5 mg soot/m3, to combinations of S and D, or to air as con- trols. Histopathology demonstrated more chron- ic inflammation, parenchymal epithelial prolif- eration, and focal fibrosis in D-than in S-ex- posed groups. Bronchoalveolar lavage (BAL) measurements suggested progressive mild inflam= mation, cytotoxicity, and active phagocytosis. BAL collagenous peptides were increased between 18 and 30 months of exposure, indicating per- sistent remodelling. Increased lung collagen accompanied histologic evidence of fibrosis. There was a mild restrictive impairment of res- piratory function. Effects were more severe in D- than in S-exposed rats, and most severe in those exposed to both. S and D interact in an approximately additive (independent) fashion to produce lung disease. (Research supported by the U.S. DOE Office of Health and Environmental Research under Contract No. DE-AC04-76EV01013.)
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125 EFFECTS OF SULFUR-DEFICIENT DIET ON ACETAMINOPHEN METABOLISM AND TOXICITY IN RATS. VF Price & DJ Jollow. Dept. Pharmacol. Med.U. of SC, Chas.,SC Cysteine is required for ths-.synthesis of cosub- strates for two pathways of acetaminophen (ACET)_ metabolism: PAgS for sulfation and GSH for detoxification of the reactive metabolite (RM). Decreased cysteine may reduce PAPS and/or GSH and thereby reduce clearance by sulfation and/or detoxification leading to potentiation of liver injury. Conversely, limitation of sulfur amino acids may result in depression of -or.otein synthe- sis and hepatic cytochrome P450, and hence in decrease in RM formation and in liver injury. To determine whether the potentiating effects exceed the protective effects, rats were fed isocaloric AIN-76 liquid diets for three weeks containing various levels of inethionine (1.2, 2.4 and 7.2 mmol/kg/day) as the sole source of . sulfur in the diet. Sulfur deficiency (SD) was assessed by urinary inorganic sulfate levels. SD retarded growth and suppressed hepatic GSH levels. SD decreased ACET sulfation capacity, whereas ACET RM formation was increased. The predicted potentiation of ACET liver injury in these animals was confirmed by histologic studies which showed increase in both incidence and severity of ACET hepatic necrosis in:SD . animals. These observations raise the possibil- ity that nutritional inadequacy of sulfur amino acids in man may similarly enhance suscepti- bility to ACET liver injury. (Supported by GM 36687). 126 IMMUNOCHEMICAL QUANTITATION OF ACETAMINOPHEN- IFROTEIN ADDUCTS IN MICE. N R-Pumford, J A Hinson, IT W Potter, K L Rowland, and D-V Roberts. Natl. Ctr. Tox. Res., Jefferson, AR and Univ. Arkansas Medical Sciences, Little Rock, AR. The correlation between the formation of acetaminophen-protein adducts and the development of hepatotoxicity has previously been shown using a radiolabeled assay. An immunochemical assay was used to quantitate acetaminophen bound to cysteinyl groups on protein. Serum alanine and aspartate aminotransferase (ALT and AST) levels were measured as indicators of hepatotoxicity. ALT and AST levels did not increase following doses of 50, 100, and 200 mg/kg; however, at doses of 300, 400, and 500 mg/kg, levels were significantly increased. Binding to liver proteins was only detected following doses of 300, 400, and 500 mg/kg. Protein adducts in serum were detected and showed a dose response similar to serum aminotransferases. A time course following a dose of 400 mg/kg indicated that liver protein adducts peaked at two hours after dosing, whereas the appearance of serum adducts was delayed concomitant with the decline of liver adducts and the increase in ALT and AST levels. Serum adducts reached sustained peak levels at six to twelve hours after dosing. Collectively, these data suggest that adducts were of hepatic origin or that binding to hepatic and serum proteins occurs simultaneously. It is postulated that serum adducts may be used as a biomarker to study acetaminophen toxicity in humans. ~~~~ ~: 127 ACTIVATION OF LIVER^~?MACROPHAGES (MP) FOR KILLING OF HEPATOCYTES (HC) FOLLOWING ACETAMINOPHEN (AA) TREATMENT OF RATS. D L Laskin and A M Pilaro, Toxicology, Rutgers University, Piscataway, NJ. Treatment of rats with AA results in accumulation of MP in centrilobular regions of the liver. To determine 'If these MP were nonspecifically activate$, we examined their cibtc.toxic activify. 'f towards normal and transformed HCs---MP were isolated from livers of contr,p.I (RKC) or AA treated rats (1.2 g/kg, 2~ hr) by coliagenase/pronase perfusion. Using a 3H-TdR release assay, both RKC and AA-MP were found to be cytotoxic towards N1S1 hepatoz va" mells in an effector(E):target(T) tependent manner, but only after 48-72 hr coincubation. AA- MP were 2-3 times more cytotoxic than were RKC. AA-MP also displayed cyto- toxicity towards normal HC as determined by trypan blue dye exclusion. After 24 hr incubation with AA-MP at a 1:10 E:T ratio, HC viability decreased by 15-20%. Cytotoxicity towards HC was stimulated (20%) by factors released from HC (H-MAF) treated with 100 uM AA. RKC were not cytotoxic towards HC unless they were coincubated with AA and H-MAF. These data support our model that AA causes activation of MP in the liver and that these cells may contribute to AA hepatotoxicity. Supported by NIH GM34310. 128 ON THE PREDICTION OF CHEMICAL TOXICITY IN VITRO: BIOTRANSFORMATION OF ACETAMINOPHEN (APAP) AND 7- OH-ACETYLANIINOFLUORENE (7-OH-AAF). C Harris, K L Stark and M.R. Juchau. Department of Pharmacology, University of Washington, Seattle, WA 7-OH-AAF, a putative detoxication product of the carcinogen 2- acetylaminofluorene, was previously shown to be embryotoxic to rat embryos grown in vitro without an exogenous bioactivation system. Investigations into the role of bioactivation of embryotoxic agents led us to propose that exposure to a chemically similar N-acetyl- aminophenol, APAP, could also result in the same form of embryotoxicity. Our comparisons showed, for the first time, that 7-OH- AAF and APAP elicited virtually identical forms of dysmorphogenesis in cultured rat embryos. Doses of APAP and 7-OH-AAF at concentrations of 0.50 and 0.25 mM, respectively, resulted in a 50% incidence of open neural tubes (ONT) on gestational day 11. Addition of an exogenous bioactivation system resulted in increased toxicity for both compounds as indicated by severe hypoplasia and necrosis in the caudal and c.ephalic regions, but no clear changes in the incidence of ONT could be detected. A similar form of dysmorphogenesis was observed when conceptuses were cultured with lower concentrations of p-aminophenol and 7-OH-AF, indicating that deacetylase activity may modify the toxic response. Preinduction of embryos in utero with the P450-IAl/L inducer, 3-methylcholanthrene, produced no significant changes in the incidence of ONT with either APAP or 7-OH-AAF. Conceptuses cultured in a CO atmosphere also resulted in no changes in ONT incidence or severity. This showed a possible lack of P450 involvement in the bioactivation step. Bioactivation via the cyclooxygenase pathway was also considered unlikely due to the lack of protection following incubation with inhibitory concentrations of indomethacin. Evidence now suggests that peroxidative metabolism may be involved in the conversion of 7-OH-AAF and APAP to reactive chemical intermediates which are capable of eliciting dysmorphogenesis in rat embryos in vitro. Supported by NIH grants ES-04041 and ES- 04342.
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~ 165 OXIME REACTIVATION OF OP-TREATED ACHE IN CULTURED MUSCLE. M J Hooper, P S Nieberg and B W Wilson. University of Califernia, Davis, CA. Acetylcholinest%rase (AChE) activity in living cells rapidly recovers from inhibition by organophosphorus,esters (OPS) by synthesis of new enzyme. The fate of OP-inhibited AChE and its effect on apparent AChE synthesis was examined using 2-pralidoxime (2-PAM) to reactivate OP-inhibited AChE in q6il embryo skeletal muscle cultures. Ten day embryo muscle cells were grown for 10 days in 10% horse serum, 2% embryo extract and 88% Eagles MEM. Cultures were treated with 0.1 uM paraoxon for 10 minutes and reappearance of AChE activity in cells and medium was studied after various times, treatments and 10mM 2-PAM. The increase in newly synthesized AChE and the decrease in reactivatable OP-inhibited AChE over time were such that the total AChE of the cell remained relatively constant for more than 72 hours. Release of newly synthesized and reactivatable AChE into the medium was consistent with the idea that AChE moves in an orderly sequence from the inside to the outside of the cell. Supported by NIH ES-00202. 166 HOMOGENIZING MEDIUM MAKES A BIG DIFFEI2ENCE IN THE MEASUREMENT CF THE EFFECT CF 2=4 ON SUB- CELLULAR SC,ALCIUM TRANSPORT. V Prakash and A Agarwal. Toxicology Research and Training Center. John Jay College of CIINY. New York. NY. Sponsor: H M Mehendale Five different homogenizing media -(1) Su- crose (2) Sucrose. Hepes. Mannitol (3) Tris. MgCl . RCl, ATP, EDTA (4) Sucrose. Tris, EDTA and (5) Sucrose. EGTA. HEPES were used to homogenize rat liver tissues for isolation of mitochorxlria and microsomes by differential centrifugation. Mitochondria or microsomes were suspended in4the same media but without EDTA or EGPA and Ca uptake in the Organelies was quickly measured. Male Sprague-Dawley rats were also administered 0.5 ml CCl4/kg ip in corn oil vehicle. 7be animals were sacri- ficed at 244kr and liver mitochondrial and microsomal Ca uptake was measured using five different homogenizing media. It was observed that medium III containing Tris. MgC12. RCl. AZT and FD'!'A exhibited a significant in~~ease in both mitochondrial and microsomal Ca uptake as compared to the other four media. A 60% decrease in microsomal and 20% decrease in . mitochondrial calcium uptake was observed using sucrose medium in CC14 treated rats where as with medium IIIr only 30$ decrease in microsomal and 25% decrease in mitochonclrial calcium uptake by CC14 was observed. Homo- genizing medium might play a big role in the viability of microsomes. 42 167 _PRINCIPLE OF "EMERGENT TOXICOLOGICAL ENDPOINTS" SUGGESTED BY ACETAMINOPHEN ZOXICIT`1 DATA. S Ji and R Esterline. Joint Graduate Program in Toxicology, Rutgers University, Piscataway, NJ The toxicological endpoints of acetaminophen.= (AA) can be grouped into 5 categories: (I) Fn-i, z mes ((a) Covalent binding of AA metabolite to :- roteins catalyzed by horsed`ish peroxidasg,~b) Covalent binding catalyzed by prostaglandifi~ synthetase), (II) Subcellular oraanelles ((a)'"k;: Covalent binding catalyzed by microsomes; (b) ' Inhibition of oxidative phosphorylation), (III) Cells ((a) Enzyme leakage from hepatocytes; (b) Covalent binding catalyzed b phobol ester-activated neutrophils; (G)I' Activation of superoxide anion production in Kupffer cells), (IV) Organs((a) Lactate production in the liver; (b) Coma in the brain), and (V) Organisms (Potentiating effect of acute alcohol on acetaminophen hepatotoxicity mediated by the pituitary and thyroid glands). Experimental evidence to support Categories IIb, IIIb, IIIc, and V have been obtained recently in our laboratories. Although certain toxicological endpoints observed at higher levels of organization can be linked to those manifested at lower levels (IIa and IIIa), some toxic effects of AA are not directly related to toxic manifestations at lower levels (IIIc and V); certain toxicological endpoints emerge as a result of the increased complexity of the test system, reminescent of "emergent properties" in physics and chemistry. Supported by AA5848. 168 ETHANOL-INDUCED MICROENCEPHALY AND INHIBITION OF PHOSPHOINOSITIDE METABOLISM. Lucio G. Costa and Walter Balduini, Dept. of Environmental Health, Univ. of Washington, Seattle, WA 98195 The pattern of muscarinic receptor (MR)-stimula- ted phosphoinositide (PI) metabolism during post- natal development has a striking resemblance with the curve of rat brain growth spurt (JPET,241: 421,1987). Therefore, the enhanced hydrolysis of membrane PIs by cholinergic agonists during this period may have a relevant role in cell prolifer- ation and differentiation. We have investigated whether exposure of rat pups to ethanol (EtOH) during the brain growth spurt would alter MR-sti- mulated PI metabolism in cerebral cortex. Female Long-Evans rats were administered 4 g/kg EtOH by gastric intubation from postnatal day 4 to day 10. This treatment caused microencephaly but did not have any effect on the pups' body weight as compared to an equally-handled, sucrose-fed group of animals. MR-stimulated PI metabolism was mea- sured in cerebral cortex slices of control and EtOH-treated rats at days 7,12,20, and 45 of age and was significantly reduced in EtOH-exposed an- imals only on day 7. A similar treatment in adult rats did not cause alteration in any of the biochemical parameters measured, despite similar blood EtOH concentrations. These results confirm that the develooing brain is particularly sensi- tive to the effects of EtOH, and suggest that the PI system coupled to MR could represent a likely biochemical target for its toxicity. (Sup . by ADAI, U. of Wash, and the Dana Foundation.p) 50875 8167
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213 071DENCE FOR REACTIVE CPIIARIOALDEHYDE INTERI)IB/DIATES IN THE P4ETABOLISM OF 1-C HIARO•- I-MER4iYLPROPENE (DMVC). P Sriasivas and L T Burka. NIEHS, Research Triangle Park, NC. Sponsor: H,B Mbtthews. Cysteine and N-acetylcysteine (NAC) conjugates of 3-chloro-2-methylpropenoic acid with trans (E) stereochemistry were recently identified as the two major urinary metabolites of DMVC. Conjugates with z stereochemistry yle?e not detected. The apparent stereospecificity in the formation of these metabolites has been investigated. Cytochrome P-450 catalysed hydroxylation of DMVC was found to be only stereoselective. In vitro conjugation reactions of sulfur nucleophiles with both E- and Z-3-chloro-2- methylpropenals were rapid; with NAC the E adduct was the sole product from either aldehyde. In contrast, Michael reaction of the corresponding acids with NAC was sluggish. Also, while the E acid yielded exclusively E adduct, the Z isomer afforded both Z and E adducts in 3:4 ratio. Glutathione S-transferases were ineffective catalysts for reaction of either of these acids with glutathione (GSH) and formation of only an E adduct was observed. However, direct Michael reaction of GSH with the E acid gave both E and Z adducts in 95:5 ratio. Based on the above observations, a metabolic pathway involving reactive aldehydic species as the metabolic intermediates has been proposed. The electro- philic haloenoic aldehydes appear to be the likely candidates responsible for the toxicity and carcinogenicity of DMVC. 214 RESPONSE OF HOUSE BRAIN TO SL's3CUTAI:EOUS ADi•IINISTRATIOY OF LUTYL 2-CIILOROET;IYL SULFIDL' N i•i ilsayed, S• T Omaye, G J .: i:lain, J L Inase, E T Dahlberg, and D Korte. Letterman ar.my Institute of Research. San Francisco, CA. F7e examined the eCfects of butyl 2-chloroethyl sulfide (BCci), a potent vesicant analog of bis(2-chloroethyl) sulfide (sulfur mustard). '.Ie injected 3 groups of athymic nude mice (n=u), weighing 30-35 g, subcutaneously with 5 ul of neat i:C:;. After 1, 24 and :J" lirs, we sacrificed the r.iice along witiian untreated control -rou?, and analyzed the brains for bioc;iemical markers of oxidative injury. Compared to controls, the activity of olutathione (GS::) peroxidase increased 76:>, P<O.t)5 at 24 hrs, and GS:I S-transferases 27;i;, P<0.05 at 48 hrs. GSH content was nrar::edly lower (25 and •20;0) after 1 and "=+ firs. Concomitant with decreased GS!I, lipid peroxidation increased almost 3-fold. iie conclude that BCS administered subcutaneously affects mouse brain via an oxidative mechanism, possibly reflecting the intitial injury phase by vesicants. These observations may contribute to the understanding of the molecular mechanisia of vesicant toxicity and could offer a new approach for treatrrent and protection. 54 215 LETHAL AND SUBLETHAL POTENCY OF VARIOUS DIOXIN CONGENERS TO THE JAPANESE _EDAKA EMBRYO (0r Zias latiges). J D Wisk and K R Cooper. Joint Graduate Program in Toxicology, Rutgers University, Piscataway, NJ. In most animal species, toxicity to dibenzo-p- dioxin is observed when 3 of the 4 latera~' positions are chlorine subs~ektuted and at least . ! one other position remains non-chlorinated. -fte purpose of our studies were to determine if similar structure activity relationship (SAR) -" would be observed in a developing fish embryo. Individual embryos were exposed immediately post-fertilization to nominal concentrations of dioxin congeners under s*tid== conditions. Embryos were staged daily and• checked for visible lesions and death. The 2,3,7,8- tetrachloro congener was the most toxic to the developing medaka with 100% lethality and lesions occuring at the 1 ng/L level. The major lesions were vascular hemorrhage and pericardial edema, resulting in the collapse of the yolk sphere. No visible lesions were observed prior to the formation of the embryonic liver rudiment. The 1,2,3,4,7,8-hexachloro congener caused a similar sequence of lesions with an LC50 of 230 ng/L (std. error 107 ng/L) and EC50 for lesions of 35 ng/L (std. error 19 ng/L). At 10 ug/L, the non-chlorinated, 2,3-dichloro, and octachloro congeners were not toxic. Based on these findings, a similar SAR is observed in the medaka embryo to that found in mammals and birds. (USGS 28322) 216. DIETHYLNITROSAMINE INDUCED JiEPATIC CARCINOGENE- SIS IN THE BROWN BULLHEAD (Ictalurus nebulosus) CATFISH. J A Hampton, P J 5o1"d6i-aff anU-S"E-- K~l~au~_n_i~g. Department of Pathology, Medica•I- t~ege of Ohio, Toledo, OH. Nine months following a 28 d aqueous exposure of diethylnitrosamine (DENA-100 PPM) to I yr old male and female brown bullhead catfish (N=68) resulted in hepatic lesions similar to those observed in mammals. Differences in the incidence of hepatic lesions between males and females were not seen. Earliest lesions obser- ved were focal areas of cellular alteration (foci < 0.5 mm in diameter) which exhibited tinctorial staining differences (increased baso- philia, acidophilia, clear cell) from surround- ing normal tissue. Acidophilic and basophilic lesions greater than 0.5 mm in diameter exhibit- ed expansile growth and frequently produced raised protrusions of the hepatic capsule. While little is known about the biology of hepatic neoplasia in the bullhead, we felt the term "adenoma" was appropriate in labeling indeterminant hepatic lesions that exhibited expansile growth. Trabecular hepatocellular carcinoma was characterized by broad discontin- uous laminae (2-7 cells thick) of hepatocytes which alternated with sinusoids. Within trabe- cular lesions, bile ducts or duct-like struct- ures and sparse supporting connective tissue were seen. Cholangiomas identical to those observed in feral bullheads from polluted waters were also seen in DENA treated fish. Ducts were separated trom surrounding connect- ive tissue by a basal lamina. Laboratory results indicate that the brown bullhead may serve as a sentinel organism for the detection of biohazardous material in the environment. 50875 8179
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4 COMPARATIVE ASPECTS OF OXIDATIVE CELL INJURY. C E Thomas and D J Reed, Dept. of Biochemistry & Biophysics, Oregon State Univ., CorOailis, OR a Previous studies have Idemonstrated that incuba- tion of isolated rat hepatocytes in Ca2+ free media results in malondialdehyde (MDA) produc- tion and cell injury (LDH and K+ leakage). We have further investigated this phenomenon and compared it to oxidative cell injury induced by paraquat and 1,3-bis(2-chloroethyl)-1-ni Cr®so- urea (PQ/BCNU) treatment. Vitamin E(vE) and desferrioxamine (Desf) prevented MDA formation and LDH leakage in both systems. Ruthenium.red (RR) and La3+, which block Ca2+ translocation through the mitochondrial uniport, prevented MDA formation, GSH and protein-SH loss, VE loss, and LDH leakage induced by Ca2+ omission, but had no effect on cell damage resulting from PQ/BCNU treatment. Ca2+ omission promoted a marked loss of mitochondrial transmembrane potential (A*) which was prevented by RR, EGTA, VE and Desf. in contrast, PQ/BCNU exposure had little effect on A*. Treatment with the protonophore CCCP resulted in no LDH leakage nor MDA formation, but caused a complete loss of A* which was not ` prevented by VE or RR. These studies indicate that in the absence of extracellular Ca2+ mitochondrial Ca2+ cycling contributes to the observed oxidative stress and resultant loss of cell viability. (Supported by USPHS ES05422 and ES01978) .162 PARAQUAT RESISTANCE DUE TO ALTERATIONS IN CLUTATHIONE ENZYMES AND NOT INCREASED SUPEROXIDE DISHUTASE OR CATALASE. MJ Kelner and R Bagnell, University of California-San Diego, CA. Paraquat (PQ) normally induces superoxide dismutase (SOD) and catalase in cells. This demonstrates that superoxide & hydrogen peroxide are involved in PQ toxicity. The ultimate target in cell death has not been conclusively identified although lipid/DNA damage, and alterations in dNTP pools have all been suggested. We attempted to identify the cellular target in paraquat toxicity by breeding a PQ-resistant cell line (PQR) (resistant to >8OuM/3 days) containing low SOD and catalase activity as compared to the parent line, and studying how such a cell defends itself. The resistance is not due to alterations in PQ uptake. Increases in , glutathione peroaidase, transferases, and reductase were found suggesting that peroxidative damage is important in cell death from PQ. The major PQ reducing enzyme NADPH- cytochrome C reductase was unaltered.. The alterations in anti-oxidant enzymes were not due to mutations as activities reverted to •normal upon removal from PQ. We are currently studying isoenzyme alterations in GSH- transferase and other important enzymes. We are also altering the SOD concentration of the PQR l.ine by using a pSVNeo-pSV(Cu/Zn-SOD)cDNA vector to see if increased SOD activity will either decrease or further increase PQ resistance. 163 THE EFFECTS OF A MIXTURE OF 25 GROUNDWATER CONTAMINANTS.ON SOME BIOCHEMICAL INDICES IN F344 RATS. H Kermani, B Ferguson, S Gangjee, A Greenwell, F Harrington, W Jenkins, R Melnick, K Tomaszewski, ~R~ Yan~ , NIEHS/NTP, Research Triangle Park, NC - A mixture of 25 chemicals, frequently found in groundwater, including some heavy metals, aro- matic,hydrocarbons and halogenated solvents was formiflated in water for toxicologi"tudies (see Yang et al., this issue). Male F344 rats were ,.,6.: treated with the mixture•at two levels, both near environmentally detected concentrations, for 14 days via drinking water. Body weight and rela- tive liver weights were decreased in the high dose group concommitant with decreased water intake of about 50% compared to cogsFrolt:. The ADP-stimulated succinate oxidation rata-in iso- lated hepatic mitochondria was inhibited by 33% in the high dose group and 19% in the low dose group. Liver microsomal cytochrome P-450 and aryl hydrocarbon hydroxylase (AHH) showed positive trends of induction. AHH activity was significantly higher (60%) in the high dose group than the control group. Clinical chemis- try showed little change. The results of this study show that a chemical mixture, at very low concentrations of its component chemicals, could inhibit ADP phosphorylation in hepatic mitochon- dria and induce microsomal AHH. Comparative In vivo (gavage) and in vitro studies on this mix- ture are being performed. 164 ERYT1iROCYTE GLUTATHIONE S-TRANSFERASE: A POSSIBLE IlARKER OF CHEMICAL EXPOSURE. S V Singh, G A S Ansari and Y C Awasthi, University of Texas 2fedi- cal Branch, Galveston, TX. Glutatnione S-transferases(GST; EC play an important role in the detoxification of xeno- biotics by several different mechanisms. Many co- mpounds of industrial interest are toxic and can be conjugated to GSH through GST. On the other , hand, some of the recent studies indicate that a number of compounds present in the environment innibit tnis enzyme system. Therefore, this study was designed to explore the possibility of using GST activity of erythrocytes as a marker of che- mical exposure by studying the inhibitory effect of industrial compounds such as acrolein, styrene oxide, propylene oxide, ethylene dichloride, and ethylene dibromide on the activity of human eryt- nrocyte GST in vitro and in situ. In vitro stud- ies witn purified GST indicated that all these compounds inhibited this enzyme to varying degree. Among these compounds, acrolein was found to bZ most innibitory with an I_ value of 5.7 x 10 M. A dose-dependent inhibition of erythrocyte GST activity was observed by all these compounds when incubated with intact erythrocytes and 150 values were in tne same range as those observed 3.n vitro. These results indicate that the extent of inhibi- tion of GST activity of human erythrocytes may possibly be used,as a marker of chemical exposure. (Supported in part by NIH grant GM 32304 and NIOSH grant OH 02149). 41 50g75 8166
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ASSOCIATION OF SPERM, REPRODUCTIVE ORGAN WEIGHT AND VAGINAL CYTOLOGY (SMVCE) DATA WITH FERTILITY OF SWISS (CD-I) MICE. R E Morri~ssey, J'C Lamb IV*, B A Schwetz, J L TfagueT, and R W Moiris NTP/NIEHS, Research Tria4flgle Park, NC; *US EPA, Washington, DC; and +ASA,•Research Triangle Park, NC. To evaluate the effectiveness of the SMVCE as a screen, changes in SMVCE endpoints were compared with results of 25 continuous breeding reprol-, duction studies in which crossover mating trials were conducted to determine the affected sex. Historical control data were used to calculate statistical power of each endpoint. For male reproductive toxicants, sperm motility was the endpoint most frequently decreased (90%) fol- lowed by epididymis and testis weights. Among studies with no detectable change in male breed- ing performance, no change in epididymal weight was most frequently observed (87%), followed by lack of an effect on testis weight. Endpoints providing the best overall association with breeding data and the greatest statistical power were epididymis and testis weights, sperm motility, and cauda weight; sperm concentration and percentage abnormal sperm were not as well associated with breeding data. A change in fe- male cycle length was associated with an adverse effect on female breeding performance. Results with this group of 25 chemicals suggest that testis and epididymis weights and sperm motility have a high association with changes in fertility. THE EFFECTS OF A SYMPATHOLYTIC,HYPOTENSIVE AGENT (LOSULAZINE) ON THE ACCESSORY SEX GLANDS OF THE MALE RAT ' G M Mesfin, A E Buht, T A Marks, M 1 Higgins, and M V Williams T~pjohn Co., Kalamazoo, Ml Losulazine hydrochloride, a qumoienyt compound, . is an effective hypotensive agent in expenmentatl animals and . man Reprvductive and developmental studies in rats, revealed interrupted estrous cycles in females, decrea-sed fertility in . males, and delayed development in offspring of treated dams. This presentation will discuss the pathologic changes in the accessory sex glands of rats treated with losulazine, and associated ejaculatory disturbances Rat were treated with 2 to 32 mg/kg/day of losulazine for up to 1 year for pathologic evaluatio- of the reproductive organs; reversibility was determined in rats treated with losulazine for 24 weeks followed by a 20-week' drug withdrawal' period Ejaculatory performance was evaluated by mating male rats (treated with 4 or 16 mg/kg/day for 4 weeks followed by a recovery period of up to 11 weeks) to untreated females Losulazine at 4 or more mg/kgtday resulted in reversible exudative prostatitis, resolving ampullary gland sperm granu~omas, and nonreversible seminal impaction There were reversible decreases in ejaculatory cc+erm count and seminal plug weights in treated rats The pathologic lesions in the accessory sex glands of rats treated with losulazine were consistent with pharmacologically-mediated ejaculatory stasis 63 250 THE SEMINAL VESICLE AS A TARGET ORGAN OF TOXICITY. R_ E. Bac7don, C J Molloy and J.D Laskin, Joint Graduate Program in Toxicology, UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ The seminal vesicle is sensitive to hormonal (androgens, estrogens) and chemical (gossypol, hexachlorobenzene, pesticddes) stimuli. It has''"9'`tubular mucosal structure and is lined with a discontinuous layer of .basal cells and a layer of columnar epithelial cells containing secretory granules. We have examined alterations in the seminal vesicles of mice following vitamin A deficiency. Male CF-1 mice, maiined on a vitamin A deficient diet from birth, developed abnormalities including body wasting and xeropthalmia within 16 weeks. Severe testicular degeneration was evident and morphological examination revealed that the seminal vesicles were thickened, enlarged and fibrous. Numerous keratin containing cysts were apparent. .Micrascopic examination revealed that the normal luminal epithelium was replaced by a squamous metaplasia with histological_ features resembling epidermis. Our data suggests that vitamin A is required for normal seminal vesicle development. Furthermore, alterations in retinoid metabolism by seminal vesicle toxins may in part underlie organ specific toxicity in this tissue. 251 TOXIC SIDE EFFECTS OF CHEMOTHERAPEUTIC AGENTS ON THE RAT TESTIS: A STUDY OF THE SHORT-TERI1 PtORPHOL06ICAL PATTERNS OF RESPONSE. J A Pickford, and L D Russell. Dept. of Physiology,- Southern Illinois University, Carbondale, IL. Sponsor: D P Waller. . As cancer survival rates increase, there is an - escalating concern about.the adverse effects of chemotherapeutic agents on male fertility. Agents affect proliferation, metabolism and synthetic processes, however, there is only sparse information as to initial insult to the testis. The present study using plastic embed- ment, employed four chemotherapeutic agents (actinomycin-D, A-D; doxorubicin, DX; cis- platinum, C-P; 5-fluorouracil, 5-F) to deter- mine the initial site affected. Morphological patterns of response were found that were highly characteristic for each agent and in- variably stage-specific. For example, certain spermatogonia (A-D, C-P, DX), preleptotene (A-D. DX) and pachytene spermatocytes (A-D) were targeted in a stage-specific manner; the chromatoid body was often abnormal (A-D, C-P); an unusual asynchronization'of the spermato- genic cycle was accompanied by meiotic meta- phase arrest (5-F); spermatid and Sertoli nucleolar changes (A-D), nuclear vacuolation (C-P) and failed release of sperm (DX) were noted. Although agents show some shared m responses, many sites of injury differ, sugges- m ting that strategies may be developed to m minimize testicular damage. -.4 cn ~ ~ 00 co
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; 268 SUBCHRONIC INHALATION TOXICITY OF NICKEL OXIDE TO RATS AND MICE. C H Hobbs-, J M Benson, D G Burt, Y S Checlg," J K Dunnick*, A F Eidson, P J_ Haley, and• J!t Pickrell. Lovelace Inhalation Toxicology Research Institute, Albuquerque, NM; *NIEHS/Research-Triangle Park, NC Occupational exposure to nickel oxide may occur during nickel refining. This study evaluated the subchronic inhalation toxicity 1of nickel ox- ide (calcined at 1350°C) to F344/N rats and B6C3F1 mice exposed to 0, 0.6, 1.2, 2.5, 5 and 10 mg NiO/m3 6 hr/day, 5 day/wk for 13 wks. There were no significant exposure concentra- tion related effects on clinical signs or mor- tality. Weight gain was depressed in mice but not rats exposed to 10 mg/m3. Lesions in rat lung included inflammation in rats exposed to 2.5 mg/m3 and greater and alveolar macrophage hyperplasia and perivascular lymphoc3ytic infil- trates in rats exposed to 1.2 mg/m or great- er. Similar lung lesions occurred in mice with infl~immation occurring in mice exposed to 10 mg/m and alveolar macrophage hyperplasia and perivascular lymphocytic infiltrates occurring in mice exposed to 2.5 mg/m3 or greater. Bio- chemical endpoints in bronchoalveolar lavage fluid indicated an inflammatory response in lungs of both species, with rats being affected to a greater extent than mice. Results suggest that exposure of rodents to NiO at concentra- tions near the current TLV produces minor le- sions in the respiratory tract. (Research con- ducted under IAA Y01-ES-30108 between U.S. DOE Contract No. DE-AC04-76EV01013 and NIEHS/NTP.) 269 SUBCHRONIC INHALATION TOXICITY OF NICKEL SULFATE TO RATS AND MICE. J M Benson, D G Burt, Y S Cheng, J K Dunnick*, A F Eidson, F F Hahn, C H Hobbs, and J A Pickrell. Lovelace Inhalation Toxicology Research Institute, Albuquerque, NM; *NIEHS/NTP, RTP, NC Occupational exposure to nickel sulfate (NiSO4) may occur during nickel smelting.and electro- plating. This study evaluated the subchronic inhalation toxicity of NiSO4 to F344/N rats and B6C3F1 mice exposed to 0, 0.12, 0.25, 0.50, 1.0 and 2.0 mg Ni/m3 as NiSO4•6H2O6 hr/day, 5 day/wk for 13 wks. Lesions in rat lung includ- ed necrotizing pneumonia and degeneration of the bronchiolar epithelium. Lesions in mouse lung included focal chronic inflammation with focal fibrosis, chronic active pneumonia infil- trates in the interstition and alveolar macro- phage hyperplasia. Atrophy of the olfactory ep- ithelium of the nose occurred in rats and mice. Biochemical and cytologic endpoints eva1= uated in bronchoalveolar lavage fluid indicated the presence of inflammation in lungs of both species. The incidence and severity of histopa- thological changes and the magnitude of changes in biochemical parameters were related to expo- sure concentration in both species but were greater in rats than mice. Results suggest that inhalation exposure of rodents to NiSO4 at concentrations near the TLV produces substan- tial lesions in the respiratory tract. (Re- search conducted under IAA Y01-ES-30108 between U.S. DOE Contract No. DE-AC04-76EV01013 and NIEHS/NTP.) 68 270 A SUBCHRONIC INFIALATION STUDY OF A-SPECIAL TEST• TONER IN RATS. R Kilpperi, U Mohr2, S Takenak,2 0 Creutzenberg2, R Mermelsteini, and H Muhle2," ° 1 Corporate Env. Health & Safety, Xerox Corp;'` Rochester, NY; 2 Fraunhofer Inst. for Toxicology; Hannover, FRG; University of Rochester, Rochester, NY .; ~ 't A subchronic inhalation study,~using SPF, F-3t}j~,__ rats, was conducted for, 6 hr/da, 5 da/wk for 13 r_ weeks. The special test toner was enriched about 10-fold, respirably (ACGIH), relative to product. Nominal exposure levels were 0, 1.0, 4.0, 16.0 and 64 mg/m3 (0, 0.35, 1.4, 5.6 and 22.4 mg/m3, respirable). ,~ The exposures were well-tolerated, with no unsched- uled deaths. Significant findings were restricted to the high exposure groups. The MTD (pulmonary) was determined relative to a defined Maximum Functionally Tolerated Dose (MFTD). At the lower exposures, clearance measurements of toner and a spike of iron oxide (Fe-59) were essentially unchanged from controls. At 16 mg/m3, slight effects were observed. At 64 mg/m3, no appreciable toner clearance occurred after 60 da exposure. The MFTD was deemed to be exceeded at 64 mg/m3 and 16 mg/m3 was chosen for. the chronic study high concentration. 271 LONG-TERM INHALATION STUDY OF TEST TONER IN RATS. R Mermelsteinl, U Mohr2 W Koch2, C Dasenbrock2, R Kilpperl, J MacKenziel, P Morrow3, and H Muhle2 1 Corporate Env. Health & Safety, Xerox Corp., Rochester, NY; 2 Fraunhofer Inst. Toxicology, Hannover, FRG; 3 University of Rochester, Rochester, NY SPF, F-344 rats were exposed 6 hr/da 5 da/wk for up to 24-mo to a special test toner at 0, 1, 4 and 16 mg : m-3, Ti02 at 5 mg m-3, Si02 at 1 mg m-3 by the inhalation route. The test toner material was enriched in fine particles, such that the respirable ,. aerosol concentrations (ACGIH) corresponded to 0, 0.35, 1.4 and 5.6 mg m-3. The design of the investigation, which included periodic measurement of lung retention, collagen determination, broncho- alveolar lavage, alveolar clearance and histopatho- logical examinations will be discussed in detail. Body weight development, food consumption, and clinical chemistry as well as animal health and " survival were satisfactory. Mortality rates and causes of death were independent of treatment and in accordance with published values. The relative lung weights of both the toner high and Si02 ex- posed groups increased during the study, such that compared to concurrent controls, they reached 117% and 173% after 15 months and 144% and 235% after 24 months of exposure respectively. Both the Maximum Functionally Tolerated Dose (MFTD) and Maximum Tolerated Dose (MTD) were exceeded at the toner high exposure level within the study. 50875 8193
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PULMONARY DEPOSITION, CLEARANCE AND RETENTION OF TEST TONER, T102 AND QUARTZ DURING A LONG TERM INHALATION STUDY IN RATS. H Muh1e1.,.B Bell- mannl, 0 Creutzenbergl [,I~ Stoberl, R ~Ki~l p.e_r2, J MacKenzie2, P Morrow5 , and R Mermels 1 Fraunhofer Inst. for Toxicology, Hannover, FRG; 2 Corporate Env. Health & Safety, Xerox Corp, Rochester, NY; 3 University of Rochester, Rochester, NY Male and female F-344 rats were exposed 6 hrs/dV. 5 days/week for up to 24-months to a special test toner at 0, 1, 4 and 16 mg m-3, Ti02 at 5 mg m-3, Si02 at 1 mg m-3 by the inhalation route under SPF conditions. The dynamics of alveolar clearance and pulmonary retention were measured after 3, 9, 15, 21 and 24 months exposure. The quantity of test toner retained in the lungs increased with exposure level and duration. At the toner high exposure level, the retained quantity off material in the lungs was significantly higher than the results projected from either low or middle exposure levels. Iron oxide (Fe-59) and polystyrene latex (Sr-85) periodically inhaled by the nose-only route were used to measure alveolar clearance. Clearance of both tracers was substantially retarded in the toner high and Si02 exposure groups, while only polystyrene clearance was retarded in the mid- dle toner group. The excessive quantity of retained toner and retarded clearance in the toner high exposure groups are indicative of lung overloading. BRONCHOALVEOLAR LAVAGE FLUID (BALF) ANALYSIS FOLLOWING ALUMINUM OXIDE (A1203) AND TITANIUM DIOXIDE (Ti0z) ADMINISTRATION. M A Perkins, R C Lindenschmidt, K E Driscoll, J K Maurer, and J M Higgins. Procter & Gamble, Miami Valley Laboratories, Cincinnati, OH. This study characterized the time and dose= related changes in selected biochemical and cellular endpoints following intratracheal instillation of the nonfibrogenic nuisance-type dusts, A1203 and Ti02. F344 rats were intra- tracheally instilled with 0, 1.0, or 5.0 mg/100 gm body weight of the test materials. Groups of 5 animals/treatment were sacrificed 1, 7, 14, 28, and 63 days post instillation, and the changes in biochemical and cellular components of BALF determined. Both dusts elicited similar responses. At the initial time point the dose- d related increases in BALF levels of lactate dehydrogenase, beta-glucuronidase, n-acetyl- glucosaminidase, and numbers of neutrophils and lymphocytes represented the most sensitive indicators of response. Importantly, however, the changes in these endpoints were transient. At the low dose most parameters returned to baseline levels by day 14, while changes at the higher dose level were decreasing, but persisted beyond day 28. The magnitude and temporal nature of this response to these nuisance-type dusts is in marked contrast to the persistent alterations seen in these same parameters following administration of silica, a known fibrogenic agent. 274 BRONCHOALVEOLAR LAVAGE FLUID (BALF)-ANALYSIS . FOLLOWING SILICA ADMINISTRATION. R C Lindenschmidt, K E Driscoll, J K Maurer, M A Perkins, and J M Higgins. Procter & Gamble,' - Miami Valley Laboratories, Cincinnati, OH. ,. The purpose of this study was to characterize the dose and temporal response of several biochemical and cellular endpoints following intratracheal instillation of the fibrogenic agent, sAica. F344 rats were instilllff-with either 0, 0.2, 1.0, or 5:0 mg/100 gm body weight of silica on day 0. Animals.(n="5/group) were sacrificed on days +1, +7, +14, +28, and +63 following instillation, and the pulmonary damage chararterized by acellular and cellular assays on the BALF. Histopathological changes were determined at the final sacrifice timewAnt:`'i'. Lactate dehydrogenase, beta-glucuronidase, t n-acetylglucosaminidase, and total protein demonstrated a significant dose response effect that increasQd in magnitude with time. Total ' cell numbers, neutrophil, and lymphocyte numbers were the most sensitive cellular indicators of the pulmonary response, and also demonstrated a dose response which increased in magnitude with time. There was a good correlation between the biochemical and cellular elevations and the resulting lung pathology (e.g., macrophage and neutrophil influx, type II cell hyperplasia, fibrosis). This persistent and increasing pattern is markedly different from the trans- ient pattern seen with nuisance-type dusts. 275 DIFFERENTIAL RESPONSES IN RATS FOLLOWING ACUTE INHALATION OF NUISANCE DUSTS. MA Hartsky and DB Warheit. Du Pont-Haskell Lab., Newark, DE. ^ Nuisance dusts are generally defined as part- iculates which when inhaled produce few adverse toxic effects. We sought to compare the lung res- ponses to 2 nuisance dusts, zinc oxide (ZnO) and titanium dioxide (Ti02) with a known fibrogenic dust, i.e., alpha quartz silica dust (Si). CD rats were exposed to aerosols of either Si, ZnO, or Ti.02 for 6 hrs at 100 mg/m3. Time course studies were carried out on cells and lavage fluid (BAL) from groups of sham and dust-exposed rats. LDH, alkaline phosphatase (AP), and protein were measured in concentrated BAL fluid. Our re- sults showed that inhalation of Si or Zn0 result- ed in a PMN inflammatory response. ZnO-induced inflammation was resolved within 8 days, while Si changes persisted. In contrast, no increases in PMN or cell numbers were observed at any time after Ti02 exposure. In addition, acute and perm- anent lung cytotoxic and permeability changes, respectively, were manife"sted,.by-.increases 3:n . LDH, AP, and protein (P :-0; 053..` iri-.ZnO and.'Si-- exposed rats. No significant differences were seen in these parameters following exposures to Ti02. Our results suggest that all nuisance dusts are not alike; brief exposures to ZnO pro- duced acute pulmonary changes while Ti02 pro- duced no adverse effects. 69 I Y
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ANTICHOLINESTERASE EFFECTS OF TRITOLYL PHOSPHATE TTTP) IN THE RAT. A J Krueger, J J Yang, T A Roy. Mobil Environmental Health _&nd Science Laboratory, Princeton, NJ. Sponsor: C Kommineni An experimental lubricant formulation was found to significantly reduce serum and erythrocyte cholinesterase (ChE) activities in the Sprague- Dawley rat in a 13-week dermal study. The mag- nitude and onset of such effects were unexpected since the only known ChE inhibitor in the lubri- cant formulation was a reported tri-orth~olyl phosphate (TOTP) contaminant (<0.03% w/w) in the commercial technical grade tritolyl phosphate (TTP) component (3% w/w). In follow-up studies it was shown that technical grade TTP inhibited ChE activities in the serum, erythrocyte and brain of the rat-and that the TTP component was the sole cause of the observed anti-ChE activit- ies in the lubricant. Although the technical grade TTP was not as potent as TOTP, the anti-ChE activity of TTP could not be explained based on the reported TOTP content alone. Chemical ana- lysis of the TTP by gas chromatography/mass spectrometry shoved that TTP was composed of at least 20 organophosphorus compounds in addition to TOTP (0.4% w/w) and its meta (20%) and para (2.4%) positional isomers. Two other mixed-TTP isomers accounted for 35%. Triaryl phosphate homologs of molecular weight 382 and 396 account- ed for additional 35% and 7%, respectively. Inspection of the mass spectra of these compounds indicates the presence of four ortho-substituted triaryl phosphates with a molecular weight of 382 (e.g. ditolylxylyl phosphates) representing 3-5% of the technical grade TTP. AMINO ACID ADDUCTS FROM LIVER PROTEINS OF BROMOBENZENE TREATED RATS. P E Weller and R P Hanzlik. Department of Medicinal Chemistry, University of Kansas, Lawrence, KS. Bromobenzene hepatotoxicity has been associated with the covalent binding of reactive intermediates (presumably epoxides and/or quinones) to protein. To elucidate this chemistry, we have pursued isolation of modified amino acids from liver protein of phenobarbital pretreated (i.p., 50 mg/kg, 3 d) and fasted (16 h) rats treated with [14C]-bromobenzene (i.p., 2.5 mmol/kg). S-(4-Bromophenyl)-L-cysteine,. from acid hydrolysis of urinary 4-bromo- phenylmercapturic acid, was used as a prototype hydrophobic amino acid reference compound. Hydrolysis (6 N HCI, 140 oC for 16 h under N2) of trichloroacetic acid-precipitated and solvent washed liver homogenate protein, followed by chro- matography on XAD-2, strong cation exchange chromatography, and reverse phase HPLC demonstrated two peaks with behavior similar but not identical to S-(4-bromophenyl)-L-cysteine. Identifica- tion of these materials is being pursued (GM-21784). SS'•'M 203 iNVESTIGATIONS INTO THE ROLE~OF~`BIOTRANS- FORMATION IN THE COVALENT BINDING OF 1,2,3- TRICHLOROPROPANE (TCP) TO HEPATIC 'PROTEIN AND DNA. GL Weber and ~IG Si es. Dept. of Pharmacology and Toxicology, College of Pharmacy, Univ. of Arizona, Tucson, AZ. Trichloropropane (TCP) is used as an intermediate in the manufacturing of pesticides and polysulfide rubbers. TCP requires rat liver S-9 to be mutagenic. In a National Toxicology Program ch[ ic bioassay, TCP pr6duced gastrointestinal tract anTliver tumors in the rat and mouse. We-have undertaken preliminary investigations into the role of biotransformation in TCP induced tumor formation. Male Fischer 344 rats (n = 6) were administered 30 mg/kg 14C-TCP (100 gCi/kg) intraperitoneally and killed 4 hours later. The extent of coval ~_nt.~jnding to hepatic protein and DNA was 418 ±.~I'9 pmol/mg and 244 ± 29 pmol/mg, respectively (mearF ± SE) Incubations with hepatic cytosolic and microsomai fractions were performed to determine their respective roles in the covalent binding of TCP to macromolecules. Glutathione decreased protein binding and increased the formation of aqueouss soluble 14C-TCP equivalents in cytosolic incubations. However, in vitro cytosolic and microsomal incubations of TCP with DNA failed to show any significant covalent binding under the conditions used. At this point the in vivo binding suggests that TCP is genotoxic, -'Tiowever the role of biotransformation in TCP induced genotoxicity is still unclear. (Supported by NIEHS N01-ES-3-5031.) 204 COVALENT INTERACTION OF A REDUCTIVELY ACTIVATED 5-NITROIMIDAZOLE WITH DNA. C'rL Kedderis, LS Argenbright, and GT Miwa. Merck Sharp & Dohme Research Laboratories, Rahway, NJ. The interaction of reductively activated 5-nitro- imidazole drugs with DNA is believed to be the mechanism of the antiparasitic properties of these agents. However, the nature of the interaction of the reactive intermediate with DNA is controversial, and nitroimidazole-DNA adducts have not been character- ized. Therefore, we have investigated the interaction of reductively activated ronidazole [(1-methyl-5-nitro- imidazole-2-yi)-methylcarbamate] with DNA in vitro. After anaerobic reduction of radiolabelled ronidazole (50 uM) with dithionite in the presence of calf thymus.DNA (1 mg/ml), the DNA was recovered by ethanol precipitation, extensively washed, and the DNA concentration and radioactivity were deter- mined spectrally. The anaerobic covalent binding of [14 C]ronidazole to DNA was maximal at 2 moles of dithionite per mole of drug, indicating that 4 electron reduction of the nitroimidazole to a hydroxylamine is required for covalent binding. Characterization of the ronidazole-DNA adduct with variously radio- labelled drug molecules demonstrated the presence of the 1-[''tC]methyl, 4,5-[14C], and 2-[14C]methylene labels in the adduct while the 4-[sH] and 2- [14C]carbamoyl labels were not present. This labelling pattern is the same as for the enzyme-catalyzed covalent binding of ronidazole to protein sulfhydryl groups. These results demonstrate the formation of a ring-intact ronidazole-DNA adduct after anaerobic reduction of the nitro group to a hydroxylamine. 51
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I G THE INTEGRATED RISK INFORNATION SYSTEM (IRIS) OF 242 THE U S ENVIRONMENTAL PROTECTION AGENCY (U S EPA). 3 C Swartout, J Patterson, R Picardi, P Preuss, U S Environmental Protection Agency, Office of Health andAEnvironmental Assessment, Cincinnati, Ohio'and'Washington, DC. Sponsor: ip Fenner-Crisp. " The U S EPA is developing a centralized health risk information system in response to the need for easy access to risk assessment and risk management data by U S EPA personnel and Ae general public. The U S EPA's IRIS is an electronic database of the U S EPA's risk assessment and regulatory information on chemical substances. Each chemical file contains up to five sections: reference doses (RfDs) for systemic toxicity (non-carcinogenic effects); risk estimates for carcinogenicity; drinking water health advisories; regulatory actions; and supplementary information. Each health hazard assessment contained in the first two sections is a consensus of one of two intra-agency review groups of EPA scientists: the Reference Dose Workgroup or the Carcinogen Assessment Review Workgroup. These sections each provide a description of the basis for the hazard assessment and a discussion of the uncertainties in that assessment in a sumanary format (2-5 pages) IRIS will be made available to the public in early 1988. Future plans include the development of an interactive mainframe and/or PC version of the database. NEONATAL IMPRINTING OF RAT HEPATIC MICROSCMAL 243 BENZO [a] PYRENE HYDRO7fltLASFS BY A_ROCLAR 1254. . J M Haake and S Safe, Reprod. Develop. Toxicol., N3t1. Ctr. for Toxicol. les., Jefferson, AR and Department of veterinary Physiology and Pharmacology, College of Veterinary Medicine, Texas A&M University, College Station, TX 77843. Male and female rats were neonatally exposed to Aroclor 1254 (100 or 300 umol/kg) and the effects of this KB mixture on neonatal imprinting of benzo[a]pyrene hydroxylases were determined in 12PJ-day-old animals. Aroclor 1254 inducibility with respect to the rate of formation of the 9,10-, 4,5- and 7,8-dihydro- diol, quinone and 9-hydroxybenzo[a]pyrene metabolites was significantly decreased in both male and fe,nale rats exposed neonatally to Aroclor 1254. Only neonatally imprinted male rats showed any differences in constitutive benzo[a]pyrene hydroxylases and these were decreased formation of the 4,5-dihydrodiol metabolite and increased 3 hydroxylase activity. The most significant effects of Aroclor 1254 on the neonatal imprinting process were observed at a dose of 300 umol/kg, which is considerably higher than the potential environmental exposure to these canpounds via lactation. (Supported by the Texas Fgricultural Experiment Station.) 61 MITOCHONDRIAL INHIBITION BY CATIONIC RHODAMINES AS A POSSIBLE TERATOGENICITY MECHANISM. S Ranganathan and R D Hood. Biology Dep'artment, The University of Alabama, Tuscaloosa, AL. Teratogenic cationic rhodamine (Rh) dyes, Rh 123 and 6G, accumulate in mitochondria, interfering with energy metabolism. We investigated the possibility that rhodamines affect embryonic mitoghondria. On gestation day (g~:1 12 (plug = g.d-: 1), mitochondria were isolated from embryos of mice treated on g:d. 7-10 (with 15 mg/kg/day Rh 123 or 0.5 mg/kg/day Rh 6G) or left untreated. Effects were assessed by polaro- graphic measurement of oxygen use. Control mitochondrial 02 consumption increased from 51 to 180 ngatom/mg/min after additionA 1a8.nM ADP, with succinate as substrate. Values for mitochondria from Rh 123-treated embryos were 62 and 135 ngatom. Similar values were obtained following in vitro Rh 123 exposure of mito- chondria from untreated embryos (70 and 130 ngatom/mg/min with 5.ug Rh 123/mg protein). Much lower amounts of Rh 6G caused similar results in vivo and in vitro. Association of rhodamines with embryonic mitochondria was assessed by spectrophotometric measurement. Mitochondrial rhodamine content was 4-5 x greater under energized than nonenergized conditions. These results support the possibility that interference with oxidative phosphorylation is a mechanism for the developmental toxicity of cationic rhodamines. Supported by PHS Grant BRSG 1751-08. pKa VALUE DETERMINES RETINOID EMBRYOTOXICITY IN VITRO. C E Steele, R Marlow, J Turton and - R M Hicks. SK&F Research•Ltd., Welwyn, U.K. and Middlesex Hospital Medical School, London, U.K. Sponsor: G B Leslie. To determine what factor(s) controls intrinsic retinoid embryotoxicity (i.e. independent of the effects of maternal pharmacokinetics) we have cultured rat embryos for 48h in serum containing one of ten retinoids at concentrations ranging from 0.5-400 ug/ml. Trans-retinoic acid, 13-cis-retinoic acid and etretin caused abnormal development in the concentration range 0.5-1 ug/ml. Six retinamides (NER, 13cisNER, OHNER, 4HPR, NBR and TZR) were . less toxic and adverse developmental effects were only evident at concentrations of 50 or 100 ug/ml. Etretinate, the parent compound of etretin, was without'effect at 10u ug/ml. In this culture system, TRA and 13-CRA caused abnormal development at very low concentrations but in contrast, ETR was non-toxic at 100 ug/ml. Therefore these findings indicate that in vivo, maternal pharmacokinetics, and bioactivat~on in particular, play a major role in inducing abnormal development. Cis/trans isomerization was not a major determinant of toxicity. However, there appeared to be a relationship between abnormal development and the actual or estimated pKa values of the 10 retinoids tested. --._---._'
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284 EFFECTS OF BENZENE METABOLITES IN COMBINATION ON FE UPTAKE INTO ERYTHROCYTES IN MICE. E Dimitriadis, R L Guy, P Hu, K R Cooper and R Snyder. Joint Graduate Program ~ Toxicology, Rutgers Universit3j., Piscataway, NJ. . . Eastmond et al.' (Toxicologist 7_,4,1987) suggested a toxicological interaction among benzene (B) metabolites. Using the ferrokinetic method of Lee et al. (Env. Hlth. Persp. 39,29,1981) we have shown that the redpction in 59Fe uptake produced by B is a mAsure of B-induced experimental anemia. A similar procedure was used to evaluate the effects of mixtures of B metabolites on red cell production in mice. Phenol (P), catechol (C) and hydroquinone (H) were administered ip at doses between 25 and 100 mg/kg 3 times over a 36 hr period. 59Fe was administered iv 36 hrs later after which 24 hr uptake was measured. Using this protocol P produced no decreases in 59Fe uptake; a dose related decrease was observed with H. Mixtures of P+H or P+C produced decreases which were greater than predicted by the effects of the compounds given alone. No decreases were observed when the mixtures were given at the same time as the 59Fe indicating there was no effect on hemoglobin synthesis. The results suggest that B-induced decreases in red cell production may be the result of a cooperative effect of B metabolites. (Supported by ES02931) 285 ACTIVATION OF BONE MARROW MACROPHAGES (MP) AND PMN FOLLOWING BENZENE TREATMENT OF MICE. L. MacEachern, R Snyder, and D Laskin. Rutgers Univ., Piscataway, NJ. Flow cytometry/cell sorting was used to study the mechanism underlying benzene induced bone marrow (BM) toxicity. Analysis of BM cells from male Balb/c mice revealed two distinct subpopulations differing in size and density. Differen-. tial staining of sorted cells showed that the larger population was PMN and the smaller, mononuclear cells consisting of MP and immature blast cells. Treatment of mice with benzene (lml/kg s.c., 3 d), resulted in a 45-50% decrease in the total number of cells recovered from the BM. However, the proportion of MP and PMN was not altered. Using dichlorofluores- cein, we analyzed 'the production of H202 by BM MP and PMN. We found that phago- cytes from benzene treated mice produced an average of 25% more H202 following phorbol myristate acetate stimulation than did cells from control animals. In addition, using the calcium probe, Indo- 1, benzene treatment was found to induce the appearance of a unique subpopulation of phagocytes in the BM that displayed elevated levels of intracellular calcium. These results suggest that MP and PMN in the BM are cellular targets for benzene toxicity. Supported by NIH Grants ES02931 and GM34310. 286 HYDROQUINONE INHIBITS MACROPHAGE REGULATION. UF STROMAL CELL DEPENDENT B-LYMPHOPOIESIS. A Kin , K Landreth, and D Wierda. Depts. of; Pharmacology/Toxicology an Microbiology and Immunology, West Virginia University Medical Center, Morgantown, WV. Previous investigations have shown that the ben- - zene metabolite, hydroquinone (HQ), inhibits B'° > cell production by preventing, maturation of - pft-B cells. Our data indicate i'fiat HQ altereil.__ ' the production of Interle,pkin-4 (IL-4) by stro= mal cells necessary for 8-lymphopoiesis. The '. purpose of this study was to identify the stro= mal cell population affected following HQ expo- sure. A cloned bone marrow stromal cell line ' (SCL-173) was developed which propuce0 IL-4 activity. HQ exposure of SCL-17,Sw-celTs did not... alter IL-4 production at any dose tefted. Addition of untreated macrophages to HQ treated marrow cells restored pre-B cell maturation to IgM expression. However, addition of HQ treated . macrophages was without effect. Further analy- sis suggests that HQ inhibits macrophage produc- tion of IL-1 which, in turn, induced production of IL-4 by stromal cells. In summary, HQ affects the ability of the bone marrow macropha- ges to regulate stromal cell production of IL-4 which is apparently required to sustain B cell, formation. Supported by grants from the EPA R813986, and NIH AI-23950,ES-04808, and an SOT fellowship sponsored by Hoffman La Roche. 287 BONE MARROW STROMAL MACROPHAGE PRODUCTION OF INTERLEUKIN-1 ACTIVITY IS ALTERED BY BENZENE METABOLITES. D J Thomas, D Wierda. Dept. of Pharmacology an Toxico ogy,best Virginia University Medical Center, Morgantown, WV. Previous investigations have shown that ben- . zene metabolites alter bone marrow stromal cell function. To determine which stromal cell population was most sensitive to benzene metabolites, we assayed the effect of hydro- quinone (HQ); benzoquinone (BQ), phenol (PH), and catechol (CT) on independent populations _ of bone-marrow-derived stromal macrophages and' fibroblasts. Fibroblast cell function was expressed as the capacity of fibroblast con- ditioned media to stimulate the formation of granulocyte/macrophage colonies in agar. The level of lipopolysaccharide inducible interleukin-1 (IL-1) secreted by macrphage was used as an index of macrophage function. IL-1 regulates fibroblast dependent myelopoiesis. Macrophages or fibroblasts were cult red with different concentrations (10-4 - 10-~ M) of metabolite for 24 or 48 hrs, and conditioned media assayed for activity. Metabolites did not effect fibrob~ast function except at con- centraions of 10- M. In contrast, macrophage production of IL-1 activity was markedly inhi- bited by HQ and BQ and stimulated by CT and PH. These results indicate that benzene can exert its myelotoxic action by selectively influencing stromal macrophage production of IL-1. (iJIH ES04808)
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260 TEMPORAL SEPARATION OF K+ AND Ca+2 MOVEMENTS IN ;,1iLTURED RAT LIVER SUICES TREATED WITH MODEL HEPATOTOXINS. M S Connors, M V Bell, A J Gandolfi, and K BrendeT. Departments of Pharmaco gy ind Anest e~4i s oTogy, Health Sciences- Center, Urriversity of Arizona, Tucson, AZ. Heal thy cel 1 s° mai ntai n 1 ow intracel l ul ar Ca+2 and high K+ against large electrochevical'. gradients. Maintenence of intracellular K is a ustlul index of viability. Inward movement of. Ca has often been proposed as ttie'final common pathway in toxic liver cell death. Precision- cut liver slices were incubated with for example al ly1 ai chohol (0.5 mM) and K+ and Ca+ val ues determi ned by f 1 ame photometry. After 1~ir of incubation, > 90% of the intracellular K wf i lost, whereas the intracellular Ca concentrations had only risen 10%. After 2 hrs of incubation ~~ere was a 56% increase in intracellular Ca which progressed to 120% by 6 hrs. Low temperature incubation or variouI ionophores anVhannel blockers also separate K efflux and Ca influx. The same is true for 02 limitation or uncoupling of oxidative phosphorylation. Selected hepatotoxins are incubated in liver slices and the temporal relationship between ion fluxes compared with agents or treatment of known mechanisms such as verapamil, A23187, valinomycin, dinitrophenol, detergents, low temperature and low 02. It might then be possible to exclude certain .mechanisms as relevant to the toxicity of an unknown hepatotoxin. (NIH GM 38290). 261 MECHANISM-BASED TOXICITY OF HMG-CoA REDUCTASE INHIBITORS (HRI's) IN RABBITS. D Kornbrust, C P Peter and J S MacDonald. Merck Sharp & Dohme Research Laboratories, West Point, PA HRI's have been shown to be highly effective in lowering serum cholesterol in animals and man, and thus represent a promising approach to the treatment and prevention of cardiovascular dis- ease. During preclinical safety evaluations of several HRI's, oral doses which were tolerated in chronic studies by dogs, rats and mice were found to be lethal to rabbits in subacute studies. Postmortem findings in rabbits consist- ed of centrilobular hepatic necrosis frequently accompanied by renal tubular necrosis. These lesions did not occur in rats, mice, dogs or monkeys which received comparable or higher doses for much longer periods. Qualitatively similar effects in rabbits were produced by (5) structurally diverse HRI's, including ?%-^8^3 (lovastatin), MK-733 and CS-514, but not by a pharmacologically inactive isomer. The liver and kidney damage were preceded by marked anorexia '° and body weight loss. Forced-feeding greatly reduced the toxicity (of I&C-733), thus demon- strating the potentiating role of decreased food intake. The liver and kidney damage induced by MK-733 and MK-0803 were completely prevented by coadministration of mevalonic acid, the product of the inhibited HMG-CoA reductase enzyme. These findings indicate that the unique susceptibility of rabbits to HRI's is due to extreme sensitivity to the pharmacologic action of these compounds. 262 .! 263 HEPATIC ENERGY STATUS DURING CCL TOXICITY 1~ RATS• PRETREATED WITH CHLORDECOR, MIRE$ AND PHENOBARBITAL. K S Prasada Rao; U M Joshi and H M Mehendale. Department of Pharmacology and Toxicology, University of Mississippi Medical. Center, Jackson, MS. Previous studies indicated perturbation of Ca2+;' homeostasis and decreased hepatocyte divisioa:." in chlordecone (CD) + -L'*f4 hepatotoxiciv~ Since these phenomena indicate perturbed energy status of liver, studies were designed to tesf>~ this possibility. Hepatic ATP levels were'' determined after CC14 (0.1 ml/kg) administra- tion to rats on diets with or without CD (10 ppm) for 15 days. Rats pretreated with pheno- barbital (PB, 225 ppm) and'4ikreX-(M, 10 ppa) were selected as positive and znegative con- trols. There were na+significant differences in hepatic ATP or Mg -ATPase in CD, M or PB treated -rats. CC14 alone did not alter hepatic ATP in control rats. CC14 administration to CD pretreated rats significantly decreased the hepatic ATP as early as 1-hr and 2~his decrease was progressive with time. Mg -ATPase was significantly decreased starting at 6 hr after CC14 administration. This was not observed in M or PB pretreated rats. These results indi- cate that.severely compromised energy status of liver after CC14 administration to CD, but not PB or M, pr1e+treated rats leads to the distur- bance of Ca -homeostasis and of cell division. (Supported by EPA R-811072 and CR814053010.) SUBACUTE 14-DAY "INHALATION TOXICITY OF`_ 'F-METHYLFURAN IN -THE SPRAGUE-DAWLEY RAT.. S-Laham and S Anderson, Environmental Health ~rTS' ectorate, Health and Welfare Canada, Ottawa;,; and N. Hamelin, Bio-Research Laboratories Ltd._,- . Senneville, Canada. 2-Methylfuran (2-MF) was administered by inhala-.: tion (6 hours/day for 14 consecutive days) to randomized Sprague-Dawley rats (wt. range: 180-201 g for females; 270-299 g for males) divided into low-level (80 ppm), high-level (160 ppm) and control groups (15 rats/sex/group). Twenty hours after the last exposure, 10_ rats/sex/group were necropsied and the remaining 5 rats/sex/group were kept for a 12-week recovery.study. Hypertrophy of hepatocytes was observed in both high-level females (10/10) and males (10/10). Hyperplasia of the splenic white pulp (1/10 female and 7/10 males) and thymuS atrophy (1/10 female and 3/10 males) were also , observed in the same group. In addition to hypertrophy of hepatocytes observed in low-level females (2/10) and males (1/10), proliferation of bile duct was observed in low-level males (9/10). After a 12-week rest period, morpho- logical changes in the bile duct were still observed in both high-level females (2/5) and males (2/5). (Sponsored in part by Canadian Panel on Energy Research and Development). 50875 8191 66
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252 DEVELOPMENTAL TOXICITY OF BROPIRIMINE. T A 253 for day 6 and days 12-17 of gestation, such treatment led to significant decreases in the survival of the offspring within 72 hours of exposure. The results of these studies have provided us with clues about the mechanism(s) involved in the developmental toxicity of bropirimine. Hopefully future studies will enable us to better understand why in utero exposure to bropirimine can be lethal whereas the maternal animal is able to survive the effects of this drug. Marks, D L Black, S M Poppe and R D Terry, the Up n Company, Kalamzoo, Mt- Bropirimin•e & an immunomodulator and interferon inducer with antiviral and antitumor activities. During routine Segment II Teratology Studies, oral administration of bropirimine to pregnant rats resulted in ronounced develo mental toxicity at doses ~200 and 400 mg/kg) which also were mater4l ally toxic (Teratology 35:30A, 1987). In subsequent rat studies, maternal toxicity always was observed at the doses which caused developmental toxicity, even when bropirimine was given only once. Bropirimine administration to pregnant rats, resulted in a 4-8% decrease in weight within 24 hours of a single dosage (200 or 400 mg/kg), while control rats given vehicle alone gained weight. Bropirimine did not cause maternal lethality at the doses employed. In order to determine the periods of in utero development which were most susceptible to its effects, bropirimine was given once between days 3 and 19 of gestation. Except TQXICITY STUDIES OF 2,3,4,6 - TETRACHLGI2OPHENOL A T Bathija, B R Sonawane, C DeRAsa and R Rubenstein. US EPA, Washington, DC. Sponsor: P A Fenner-Crisp. 2,3,4,6 - tetrachlorcphenol (TCP) was evaluated for subchronic toxicity and teratogenicity in Sprague-Dawley rats. Groups of rats (30/sex/ dose) were administered 0,25,100 or 200 mgAg/ day TCP by gavage for 90 days. At 200 mgAg/ day there was a significant decrease in the male body weight; liver and kidney weights were significantly higher than controls in males and females at sacrifice. Also, cen- trilobular hypertrophy was observed in 15/ 30 males and 6/30 females and a number of clinical pathology parameters were significant- ly changed at this dose. At the 100 mg/kg/day dose liver and relative liver weights were significantly elevated in both males and fe- males. In females, both absolute kidney and relative kidney weights were elevated; centri- lobular hypertrophy was seen in 12 males and one female. At 25 mg/kg/day there were no significant adverse effects. In the teratology study, rats administered 0, 25, 100 or 200 mgAg/ day TCP by gavage daily on day 6-15 of gestation showed a statistically significant reduced maternal weight gain at the highest dose; no significant maternal effects were seen at the tvx> lower doses. No adverse develop- mental effects were observed at any of the doses tested. 64 254 COMPARISON OF THE DEVELOPMENTAL TOXICITY OF OCTABROMODIPHENYLOXIDE AND PENTABROMODIPHENYL- OXIDE IN Cr1: CDB(SD)BR RATS. A M Hoberman, E A Lochry, M N Pinkerton and M S Christian. Argus Research Labs., Inc., Horsham, PA; Ethyl Corporation, Baton Rouge, LA Octabromodiphenyloside and Pentabromodiphenyl- .psi.de are chemically similar'Sterials used inIhe ' manufacturing of flameretardants. Pregnant rats~ (25/group) were given (gavage) Octabromodiphenyl-'-`_c:: oside at dosages of 0(corn oil), 2.5, 10 or 25 mg/kg/day or Pentabromodiphenyloaide at dosages of 0(corn oil), 10, 100 or 200 mg/kg/day on days 6 to 15 of gestation. The dams were.sacrificed on day 20 of gestation, and thAfetuses were ex- amined for esternal, visceral and s-keletal alter- ations. Octabromodiphenyloxide produced develop- mental toxicity (resorption and decreased fetal weight) at dosages of 10 and 25 mg/kg/day, re- sulting in a developmental NOEL of 2.5 mg/kg/day. Maternal toxicity (decreased maternal body weight) occurred only in a pilot study at dosages of 100 mg/kg/day or greater. Pentabromodiphenyloxide at 200 mg/kg/day produced a slight decrease in the average fetal body weight and maternal toxicity (decreased maternal body weight gain). Maternal toxicity (decreased body weight gain) was also. produced at 100 mg/kg/day. Based on these data Octabromodip'henyloaide was a unique hazard to the conceptus with an A/D ratio (adult/developmental ratio) of 10, while Pentabromodiphenyloxide was shown not to be a unique hazard (A/D ratio of 0.5). 255 CYTOTOXICITY MEASUREMENTS WITH PRIMARY CULTURES OF CRYOPRESERVED (CP) RAT HEPATOCYTES. K,S Santone, D C Melder and G Powis. Mayo Clinic, Department of Pharmacology, Rochester, MN. We have previously reported a technique for bulk CP of rat hepatocytes that maintains functional viability. Presently we have performed experi- ments to assess the usefulness of primary cul- tures of these CP hepatocytes for cytotoxicity measurements. 2 x 106 viable fresh and CP hepa- tocytes were cultured for 24 hr before exposure for 2 hr to various concentrations of either chlorpromazine (CPZ), cadmium chloride (Cd) or menadione (MND). Lactate dehydrogenase (LDH) leakage and intracellular reduced glutathione (GSH) concentration were chosen as indices of hepatocyte damage. The results listed below are the drug concentrations (pM) to produce a 50% release of total LDH or a 50% decrease in GSH (mean + S.E., n = 3 separate preparations. The following LDH results were obtained, fresh vs. CP: CPZ 215 ± 30 vs. 235 ± 20, Cd 272 ± 23 vs. 200 ± 5 and MND 44 ± 8 vs. 24 ± 7. GSH results fresh vs. CP were: CPZ 235 ± 8 vs. 200 ± 8, Cd 213 ± 7 vs. 242 ± 19 and MND 21 ± 3 vs. 22 ± 2. The above results show that CPZ, Cd and MND toxicity in CP hepatocytes correlates well with the toxicity seen in fresh hepatocytes. These results indicate that CP hepatocytes may be a useful model in determining xenobiotic toxicity. Supported by NIEHS Contract ES55110 and The John Hopkins Center for Alternatives to Animal Testing. 50875 8189'
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ACTIVATION OF PHENOL AND HYDROQUINONE TO COVA- 290 LENTLY BINDING METABOLITES BY MOUSE MACROPHAGE LYSATES. M J Schlosser and G F Kalf. Depart- ment of Biochemistry and o ecu ar_Bioloey, Jefferson Medical College, Thomas Jefferson University, Philadelphia s PA. - Benzene(B) -induced bone marrow (BM) hypoplasia is due in part to its metabolites phenol (P) and hydroquinone (HQ) which are metabolized in BM to reactive intermediates via peroxidases. A prostaglandin H synthase(PHS)-peroxidaseJ ,is likely since B-induced myelotoxicity is prevent- ed by inhibitors of PHS activity. Macrophages, a major BM cell-type, contain relatively large amounts of PHS-peroxidase and are kffwn to Tatabolize P. Incubations of either C-P or C-HQ with lysates of macrophages collected from mouse peritonium resulted in a H 0, dose, and time-dependent metabolic activ3t?on, as indicated by covalent binding (CVB) of labeled metabolites to albumin. Production of metabo- lites from P (0.5mM) was inhibited by aminotria- zole, ascorbic acid and 8ti,M HQ (52%), whereas binding from HQ (0.5mM) was increased by 5mM P (17%). Glutathione (GSH), a nucleophile and PHS-peroxidase substrate, inhibited CVB from bo4 P and HQ in a dose-dependent manner. CVB of H-GSH (0.5mM) was increased by 5mM P(36%); however, GSH binding was decreased by 5mM HQ (11%), suggesting the presence of a HQ-GSH conjugate. These results suggest that P and HQ are metabolized by a macrophage peroxidase to CVB intermediates. (NIEHS grant ES03724). EARLY VERSUS DELAYED BEHAVIORAL EFFECTS OF ACUTE TRIETHYLTIN EXPOSURE. Y L T Ting, S B Fountain, H L Andre, and T J Teyler. Department of Neurobiology, NE Ohio Univs Coll of Med, Rootstown, OH. Sponsor: Z Annau. The timecourse of triethyltin (TET) induced effects on hypothalamic brain-stimulation reward (BSR) was examined. Male hooded rats were trained in a threshold procedure that daily determined the shortest stimulus duration supporting discrete-trial leverpress responding for BSR. After a stable baseline threshold duration was obtained, 3 mg/kg TET-Br was administered by i.p. injection. Threshold duration was elevated 1 hr postexposure but returned to baseline by 24 hr postexposure. A second increase in threshold occurred approximately 5 days postexposure, returning to baseline by approximately 4 weeks postexposure. The later increase probably reflects the myelinotoxic effects of TET, whereas the early increase in threshold may reflect a rapid suppression of synaptic transmission like that observed following acute TET exposure in the in vitro hippocampal slice (Soc for.Neurosci Abstr 13: 695, 1987). To evaluate the latter hypothesis, neurochemical analysis of the timecourse of TET effects will be presented that may help explain the frequently observed dissociation in timecourse of behavioral and anatomical consequences of TET exposure. (Supported by EPA coop. agreement #813394, NIH DA03755, and ONR 86K0664.) NEONATAL EXPOSURE TO TRIMETH ~~~ Y~~ ~ (TMT) DISRUPTS THE ONTOGENY OF LONG-TERH OLFACTORY MEMORY IN THE RAT. M E Stanton. U. S. Environmental Protection Agency, Research Triangle Park, NC Sponsor: P J Bushnell "' TMT is a potent limbic-system neurotoxin with known effects on cognitive function in adult animals. The present study examined the effect of neonatal TMT administration on cognitive development. Long-Evans rat pups received an i.p. injection of either TMT (6 mg41W,,:in 10 ul/g saline) or saline vehicle on postnatal day ten (PND 10). These pups were .then trained and tested for retention on an' aversive olfactory discrimination task (Kucharski and Spear, Develo Ps chobiol, 1984, 17, 465-479). Train ng took p ace on either PND`12 or PND 18 and "testing took place either immedi tel~;or after 24 hrs or 7 days. Saline p~treated animals showed the typical, age related emergence of long-term retention at the 7-day retention interval: ie., 18-day-olds showed good retention of the discrimination after 7 days whereas 12-day-olds did not. TMT pretreatment prevented this age-related increase in retention capacity. A second experiment revealed a similar pattern of results when TMT injection followed original training. These findings in icate that neonatal exposure to organotin compounds can impair cognitive development, and illustrate the value of olfactory conditioning as an animal model for assessing neurotoxic effects on on the ontogeny of learning and memory. 291 THE ACUTE EFFECTS OF BIS(TRI-N-BUTYLTIN)OXIDE ON MOTOR ACTIVITY: ORAL VS. INHALATION EXPOSURE. K M Crofton, M E Hiteshew, L P Sheets, V M Boncek, K F Dean, and L[J Re~ter, Neurotoxicology Division, US EPA, ATPt NC and Northrop Services, Inc, Environmental Services, RTP, NC Bis(tri-n-butyltin)oxide (TBTO) is an organotin compound used as a biocide in marine paint. The purpose of the present study was to deter- mine and compare the effects of oral and inhalation exposure to TBTO on neurobehavioral function using motor activity (MA). Dosage-. effect functions were determined in male Long Evans hooded rats (n=8/group). Acute oral (0-150 mg/kg TBTO) or nose-only inhalation exposure (0-11 ppm TBTO as vapor for 2 hr) was followed (1 hr or 1/2 hr, respectively) by activity measurement for 1 hr in figure-eight mazes. Oral exposure produced a dosage- dependent decrease in MA (ED50 "20 mg/kg). Inhalation exposure reduced MA in a concentra- tion-dependent manner (ED50 -10 ppm). The relationship between a behaviorally effective dosage and lethality differed with route of exposure. Inhalation exposure produced respiratory distress (at > 9 ppm) and mortality (LD50 -11 ppm). Oral exposure to TBTO decreased activity at -1/10 the oral LD50, whereas inhalation exposure decreased activity only at dosages that produced respiratory distress or lethality. These findings suggest differences in the toxicity of TBTO following oral and inhalation exposure. 50875 8198 73
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NDUCTION OF I4ICRONUCLEI BY _BEPfZENE AND ITS ETABOLITES. H Shirada, T Sato and S Takavasa. esearch Institute of Daiichi Seiyaku Co.,Ltd., okyo, Japan. ~ . he relationships betveeri DNA single-strand reakes (SSBs) and 'icronuclei formation nduced by benzene and its netabolites were nvestigated. The ricronucleated polychroiatic rythrocytes in bone 'arroa cells of nale nice ere aarkedly increased at 48 h after a single ral ad'inistration of benzene. SSBs detect& by alkaline elution assay in bone narrow cells were aarkedly increased at 36 h after the administration of benzene. In addition, the ti.e-dependent response of SSBs was siailar to that of the aicronuclei foraation. In the micronucleus test of the benzene tetabolites, phenol and hydroquinone induced ®icronuclei but catechol, hydroxyhydroquinone, 1,4-benzo- quinone and 4,4'-biphenol did not. In vitro studies using the Chinese ha.ster cells, hydroxyhydroquinone induced SSBs in the alkaline elution assay, and hydroquinone, catechol and nuconic acid induced aetaphase arrest in the aetaphase/anaphase analysis. Moreover, hydroquinone, catechol, hydroxyhydro- quinone and 4,4'-biphenol exhibited clasto- genicity in the cultured Chinese hamster cells. These results suggest that the induction of iclonuclei by benzene follows after SSBs, and the several ietabolites play an i.portant role to induce nicronuclei. PROTECTION AGAINST BENZENE-INDUCED MYELO-AND GENOTOXICITY IN MICE BY NON-STEROIDAL ANTI- INFLAMMATORY AGENTS. S J Pirozzi, J R Renz, M J Schlosser, and G F Kalf. Dept. of Biochemistry and Molecular ill'o7ogy; Jefferson Medical Col- lege, Thomas Jefferson Univ., Phila. PA. Benzene (B) affects hemopoietic progenitor cells leading to bone marrow depression (BMD) and genotoxic effects such as micronucleus forma- tion. Progenitor cell survival and differentia- tion is inhibited by prostaglandins produced by macrophages. Administration of 800 mg/kg B ip to DBA/2 mice caused BMD and a significant increase in marrow PGE level (radioimmunoassay) that was inhibited by indomethacin (I). Phenol, the major metabolite of B is converted in macro- phages by the endoperoxidase activity of prosta- glandin synthase (PS) to species that covalently bind to protein and DNA. This, suggests a role for PS in BMD and genotoxicity. The ability of PS inhibitors to prevent BMD and genotoxicity was tested. Administration of B (200-1000 mg/kg) ip to DBA/2 or C57B1/6 mice caused a dose-dependent decrease in nucleated cells which was prevented by I(2 mg/kg), aspirin (50 mg/kg) or meclofenamate (4 mg/kg). B (400 mg/kg) caused a 50% decrease in cellularity and a 3 to 5-fold increase in micronucleus formation in peripheral blood polychromatic erythrocytes both of which were prevented by the coadministration of I. PS maybe i nvol ved i n causi ng the myel o- and genotoxic effects from B. Supported by NIEHS grant ES 03724. 282 IN VITRO AND IN VIVO STUDIES OF`-~C-ENZENE METABOLITE REACTIONS WITH DNA, NUCLEOSIDES AND NUCLEOTIDES. H Bauer, E Dimitriadis, K R Cooper, and R Snyder, Joint Graduate Program iii _ Toxicology, Rutgers University, Piscataway, NJ. We have previously reported (Arch. Toxicol. 60,61,1987) on the structure of an adduct formed between hydroquinone (HQ) or benzoquinone (BQ) and 2'deoxyguanosine. Here we report furtier studi es on. the reacti ons,,bp;~_ween HQ or BQ with 2'-deoxyguanosine, 2'-deoxyadeno- sine, 2'-deoxyguanosine-3'-phosphate, 2'-deoxy- guanosine-5'-phosphate and 2'-deoxyguanosine- 3'5'-phosphate. The reaction products were analyzed using both thin layer chromatography on PEI cellulose in 0.1M phosphate buffer, pH 6.8, and HPLC using a C-18 column r+d-i a water:methanol gradient between !C-100~• methanol. In each case at least one new pea~c, separable from the starting materials was found. DNA was purified from the livers of rabbits treated on 4 consecutive days with benzene (586 mg/kg/day, sc). DNA adducts were detected using the P-post labeling technique (Reddy and Randerath, Carcinogenesis 7_,1543,1986). (Supported by ES 2931) 283 HPLC ANALYSIS OF [32P1-POST LABELED DNA ADDUCTS FROM BENZENE TREATED RATS. R L Guy, and R Snyder. Joint Graduate Program in Toxicology, Rutgers University, Piscataway, NJ. The formation of DNA adducts in liver and bone marrow of male Sprague-Dawley rats treated with either benzene (1 ml/kg, sc, 1 dose/day, 4 days) or a combination of phenol (P) + hydroquinone (HQ)(50 mg/kg/day of each, ip, 1 dose/day, 4 days) was studied. DNA from bone marrow and liver _was isolated by solvent extraction and enzymatic digestion to 3'deoxynucleoside phosphates and analyzed using a modification of the technique of Haseltine (Env. Hlth. Persp. 48,29,1983). We have detected DNA adducts following both benzene and P+HQ treatments. In in vitro studies, fluorescence spectra of fractions separable by DE-52 chromatography, suggest the formation of adducts from the reaction of benzoquinone and 3'dGMP. The fractions were 32P labeled and analyzed in the HPLC system. The adduct eluted at a position corresponding to one of the peaks seen with marrow DNA. It is concluded that benzene gives rise to several DNA adducts; benzoquinone, as a metabolite of benzene, may be responsible for at least one adduct, and other metabolites of benzene may also form adducts. It is suggested that DNA adduct formation may play a role in the mechanism of benzene toxicity. (Supported by ES02931). 50875 8196 71
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197 BRAi13 REGIOI3AL SPECIFICITY OF GUANOSINE 3',5'- hFONOPHOSPHATE (cGMP) RESPONSF..TO DIISOPROPYLr- FLUOROPHOSPHATE (DFP) ADMINISTRATION. _ L Davenport, G,Gianutsos, and SD Cohen. Toxicology Program, University of Connecticut, Storrs, CT. Organophosphate cholinesterase (ChE) inhibitors, such as DFP, induce toxicity through an increase in synaptic concentrations of acety7.choline subsequent to inhibition of ChE. c2'D.' has been implicated as a second messenger following muscarinic receptor stimulation. The role of cGMP in DFP toxicity was investigated in male SD rats administered 2 mg/kg DFP sc. At selected times after DFP animals were sacrificed for determination of cGMP and ChE levels in the brainstem (BS), cerebellum (CE), and cortex (CX). ChE was inhibited by approximately 60% by 15 minutes post dose in all brain regions and further declined to greater than 90% by 1 hour, the time when overt signs of poisoning were first observed. c(NIIP levels exhibited selective regional sensitivity to DFP. BS c(NP levels were elevated three fold thirty minutes after DFP and remained elevated at 2 hours. No effect was observed in the CX or CE through 2 hours. . These results suggest that the cGMP accumulation following ChE inhibition is brain region specif- ic. (Supported in part by the Center for Bio- ' chemical Toxicology and the UCONN Research Foun- dation.) 198 NEUROPATHY TARGET ESTERASE (ME) IN CHICKENS AFTER TREATMENT WITH ISOPROPYL METHYLPHOSPHO- NOFLUORIDATE (SARIN-TYPE I & II). J A Crowell, R M Parker, T J Bucci, and *J~C" Dacre. Pathology Associates Inc., NCTR, Jefferson, AR and *US Army Biomedical R&D Labora*_ory, Fort Detrick, Fredrick.MD. To estimate the potential of small doses of the Sarin-I and Sarin-Il to cause delayed neuropathy, 400, 200, 61, & 0 ug.>Kg (Sarir,-I).: and 281, 141, 70, & 0 ug/Kg.(Sarin-II) by gavage were compared with 510 mg/Kg tri-o-cresyl phosphate (TOCP), in 14-16 month-old SPF white Leghorn hens (4!dose), protected with atropine (100 mg/Kg). The NTE activity 24 h after dosing was determined inn brain,..spinal cord, and lymphocytes and in plasma and brain for cholinesterase (ChF-) and carboxylesterase. TOCP inhibited the 3 tissue NTE's 60%. Sarin-I showed no NTE effect statistically. Sarin-II showed an effect only in lymphocytes. Sarin-1 inhibitec` ChE and carboxylesterase in plasma (69Y% and 47%, respectively) and in brain (46% and 16%). Sarin-II inhibited ChE and carboxylesterase in plasma (86'/% and 56%) and in brain (65`/. and 23%). Using 60'/% inhibition of brain NTE to predict delayed neuropathy, Sarin-I & 11 are biochemically predicted not to cause delayed neuropathy at nonlethal doses. (Supported by US Army Medical Research & Development Commmand, contract *85PP5868). 199 ENZYME INHIBITION IN CHICKS INJECTED WITH Dp, .! Organophosphorus esters (OP's) inhibit acetyii_; cholinesterase (AChE), causing acute neuroto;.~ icity. Certain OP's are also associated witb" inois, Urbana IL. I tSKVMVLL'r1VrriV5 Al LWV rP,K1VL5 LuK1NG INC13BA71~-_ M Farage-Elawar and B M Francis. University- op ll organophosphate induced delayed neuropathy o~:_ ;:r rather than a structural, teratogen. conclude that DBL teratogenesis is a functional._ at hatching and even 6 days after hatching. ,k1e-,- ;- stand and had curled toes. Some were paralyzed - not seen, but treated chicks were unable to beak and other overt teratogenic effects were.: mortality in the chicks; the weight of surviving chicks was not decreased. Wry neck, crossed : ing. Both treatment regimens caused around 50A.~- hibition >70%, at least until day 2 after hatch-';" day, NTE inhibition remained >80%, and AChE in-, ,. and 15 of incubation. After treatment on eithe= AChE inhibition when DBL was injected on days 3--. of DBL on chick development, we compared NTE aud nate between teratogenic and neurotoxic effects until day 4, and affects the gait of the chick:M,; for at least 6 weeks posthatching. To discriqd_:-:,- NTE inhibition until day 10, AChE:inhibition '.„.•. (DBL), injected on day 15 of r*cubation, cauASs ' We previously showed that desbromoleptophos to chick embryos early in incubation. also cause teratogenic effects if administered " o neuropathic target esterase (NTE). Some Op!s ~ OPIDN, which is correlated with the inhibitian°>1 f 200 DELAYED NEUROPATHY OF METHYL-CYANOFENPHOS, 0- METHYL-O-(4-CYANOPHENYL) PHENYLPHOSPHONO- THIOATE, IN CHICKEN. S A Soliman, K A Osman,, N S Ahmed, K S El-Gendy, and I E E1-Shennawy*_ Laboratory of Environmental Chemistry and Toxicology, Faculties of Agriculture and Medicine*, Alexandria University, Alexandria, Egypt- compound either once, or with subsequent chicken. Hens were orally treated by the phosphonothioate, was studied in the phos, O-methyl-0-(4-cyanophenyl)phenyl - ganophosphorus ester, methyl-cyanofen-.... The delayed neurotoxic effect of the or- daily doses. Some of the control and 50 hens. This compound was also able to produce such effect in chickens given 25 mg/Kg/day for 13 days. The effect was demonstrated by the standard clinical and histological changes and manifested by inhibition of NTE activity. However, methyl-cyanofenphos did not produce neurotoxic effect in chickens treated daily at 1 mg/Kg for 180 days. was able to produce delayed neuropathy inn pathy target esterase (NTE) was meas- ured. The remaining hens were used for clinical and histological examinations. Results demonstrated that methyl-cyano- fenphos at a single dose of 200 mg/Kg the last treatment and their brain neuro-.: treated hens were sacrificed 24 hrs after:_;
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PHARMACOKINETIC EVALUATION OF PYRIDOSTIGMINE IN 187 THE RHESUS A Staubus*, MONKEY. G Wient3es, J Chinn, T Hayes, R Joiner, and-W Kluwe. Battelle Columbus Division and *Ohio State University, Columbus-ti A ' Pyridostigmine.(PYR), a.reversible inhibitor of acetylcholinesterase (AChE) is used to prevent intoxication with irreversible AChE inhibitors. To evaluate the monkey as a model for human re- sponse to PYR, the pharmacokinetics of orall PYR (0.29, 0.57, 1.14 mg/kg) and its relationship to the time course of AChE inhibition (in red blood cells, RBC) were determined and compared with human data. The gastrointestinal absorption of PYR was erratic, resulting in multiple concen- tration maxima. The half-life of PYR in plasma was 2-3 hr (means per dose group). Maximal plasma concentrations of 14 to 45 ng/mL occurred ~1 hr after drug administration and increased proportionally with dose. Maximum inhibition of RBC AChE was 30 to 55% and occurred -16 min after peak plasma concentrations. Pharmaco- kinetic-pharmacodynamic modeling showed that this delay is consistent with the slow reversi- bility of AChE Inhibition, or with a slow dif- fusion of PYR to the enzyme site. Comparison with existing human data showed that the absorp- tion and disposition of PYR are similar in- humans and Rhesus monkeys, and that 50% AChE inhibition can be achieved at lower plasma con- centrations then used clinically in humans. (Supported by USAMRDC, Contract No. DAMD-17- 83-C-3129) PERSISTENT REDUCTION OF BRAIN SEROTONIN (5-HT) BY MDMA IN THE RHESUS MONKEY. W Slikker, Jr., E Wood, S F Ali, A C ScaTlet, G D Newport, J R Bailey and M G Kolta. NCTR, Jefferson, AR. MDMA, an amphetamine derivative, has previously been reported to reduce brain 5-HT concentra- tions and produce serotonergic nerve terminal pathology up to 4 months after 8 successive doses (40 mg/kg po, BID) in the rat. To deter= mine the sensitivity of the nonhuman primate to MDMA, a total of 9 rhesus monkeys were dosed with either vehicle, 5 or 10 mg/kg MDMA (n=3) by gastric intubation twice per day for 4 days. Home cage spontaneous behavior assessed with an activity check-list was not significantly altered by MDMA dosing. Neither body weights nor follow-up spontaneous behavior assessments 3 days after the last dose were altered by treat- ment. One month after the last MDMA dose, the monkeys were overdosed with pentobarbital and their brains removed, quickly dissected into frontal cortex (FC), caudate nucleus (CN), hippocampus (H) and brain stem (BS) and frozen on dry ice. HPLC/EC analysis of 5-HT and 5-HIAA revealed a significant, dose-related % reduction from control for these monoamines: 5-HT (FC) 82, 91%; (CN) 17, 30%; (H) 74, 83%; (BS) 22. 25% and 5-HIAA (FC) 39, 92%; (CN) 23, 45%; (H) 63. 80%; (BS) 20, 30% for 5 and 10 mg/kg po, respec- tively. These results indicate that the monkey may be more sensitive than the rat to the per- sistent serotonergic neurotoxicity of MDMA. DISPARATE CONSEQUENCES OF TWO DISTINCT 6- HYDROXYDOPAMINE (6-OHDA) BRAIN LESIONS IN RATS. B E Mileson, R B Mailman. University of North Carolina Curriculum in Toxicology and Biological Sciences Research Center, Chapel Hill, NC. Behavioral and biochemical endpoints of .two different models of dopaminergic "denervation super.sensitivity" were compared. These were intracisternal (lC) administration of 6-OHDA (20 Ng), and bilatetal (BIL) infusion of G-OHDA into the satbsUtntia nigra (SN) (8 Ng /side). Both lesions resulted in 85% or greater decrease in dopamine (DA) concentrations in lhe striatum, the major terminal area of the SN. Pharmacological challenge with a low dose of the nonspecific DA agonist, apomorphine (03 mg/kg) elicited a greater increase in locomotor activity in IC Iesioncd rats than control. BIL lesioned rats responded to apomorphine by engaging in self-injurious behavior, including paw and tail bi6n ~This«j;5 the first systematic evidence that 6-OHDA lesions r~'sult in~ seff. injurious behavior in adult rats lesioned after developement. Apomorphine treated control rats exhibited mild stereotypies including sniffing and licking. It has been accepted based on studies with unilateral nigral lesions that increase in D receptor density is the primary mechanism responsible for tchavioral "supersensitivity". Receptor binding studies performed on striatall tissue of IC lesioned rats revcaled no change in the maximum number of binding sites, nor the dissociation constant (K ) of either D r or D= receptors. Data from initial binding experiments using BIL lesioned rats support the same conclusions. Preliminary data indicate there is a small, but significant, increase in the affinity of striatal adenylate cyclase for dopamine in IC lesioned rats. These data suggest alterations in recognition characteristics of populations of receptors in gross regions of the brain are not sufficient to predict changes in drug sensitivity resulting from toxicant perturbation. 188 TRIADIMEFON INDUCES STEREOTYPED BEHAVIOR AND ALTERS BIOGENIC AMINE ACTIVITY IN RATS. 0.13 Walker, M.H Lewis, K.C Crofton, and R B Mailman. University of North Carolina, Curriculum in Toxicology and Biological Sciences Research Center, Chapel Hil1, NC, and Environmental Protection Agency, Research Triangle Park, NC. Triadimefon, [1-(4-chl(irophenoxy)-3,3-dimethyl-1-(1 H-1,2,d- triazol-l-yl)-2-butanonel, is a lriazole fungicide licensed for use on fruits and grains. Because initial behavioral investigations showed that triadimefon induces hyperactivity in mice and rats, the present study examined its behavioral effects using a computer-supported observational method designed to quantify changes in multiple behavioral topographies. Additionally, neurochemical effects were assessed by quantifying monoamine concentrations in terminal fields of nigrostriatal and mesolimbic dopamine pathways. Triadimefon was administered i.p. (0, 50, 100, or 200 mg/kg) in corn oil (2 mI/kg) four hours prior to behavioral assessment to female Sprague/Dawley rats (n = 10). Each animal was observed for a one minute period every five minutes for one hour. Triadimefon administration resulted in a highly significant dose- dependent increase in stereotyped behavior. At the highest dose, triadimefon induced intense gnawing, head weaving, backward locomotion, and circling. At lower doses, less intense stereotyped behaviors including gnawing, licking, rearing, and locomotion were observed. Immediately after behavioral testing, rats were sacrificed, and striata and olfactory tubercles removed. Concentrations of dopamine (DA), and its acidic metabolites, 3,4-dihydroxyphenyl- acetic acid (DOPAC) and homovanillic acid (HVA), as well as serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA), were determined by HPLC with electrochemical detection. Significant dose-related increases in HVA and 5-HIAA were observed in the absence of changes in DOPAC in both brain regions. No significant changes in 5-HT, and an decrease in DA at only the highest dose, were detected. 47
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MANGANESE ADMINISTRATION INDUCES YERSISTIIIG 298 DDT AND PICROTOXIN EFFECTS ON THE ACOUSTIC EFFECTS ON EFFORTFUL BEHAVIORAL RESPONSES. B Weiss and M C Newland, Environmental Health STARTLE RESPONSE (ASR) IN PREWEANLINGcRATr RESEMBLE THE EFFECTS OF*TYPE I AND TYPE II Sciences Center, Univ of Rochester School of PYRETHROIDS. L P Sheets , K M Crofton and Medicine and,Dentistry, Rochester NY. 'y- L V Reiter. Neurotox. Div., EPA, RTP, NC. i High doses of manganhse ('Mn) produce signs of basal ganglia dysfunction-in primates. To characterize the effects-of lower doses, three Cebus monkeys were trained to execute a rowing- type motion through a 10 cm displacement against a 4 kg force. Performance was maintained by a multiple fixed ratio-fixed interval (mult 73 fI) schedule of reinforcement. Two monkeys received saline, followed by two doses of 5 mg/kg Mn, iv, at intervals of at least 4 weeks. The third monkey received a total of 30 mg/kg in four doses at least one week apart. The hi,gh-dose monkey exhibited a persistent movement tremor, but no other sign of toxicity. In all monkeys, Mn increased the number of incomplete responses (those that did not extend to the full 10 cm) by 10- to 100-fold. This effect appeared a few days after the last dose, persisting up to,several weeks,and undulating in cycles of several weeks duration. Schedule patterning remained intact in the two low-dose animals, but vigorous FR-like responding began to appear in the FI performance of the high-dose monkey several months after the last dose. (Supported by ES-01247. ES-01248. and AA-01588) CHOLINESTERASE INHIBITION AND NEUROBEHAVIORAL EFFECTS IN PARAOXON-TREATED LONG-EVANS RATS. W 0 Cook, S S Singh, V R Beasley, and J A Dellinger. Dept. of Veterinary Biosciences, University of Illinois, Urbana. To determine the effects of the organophosphate paraoxon on neurobehavior using a battery of neurobehavioral tests, 24 male 400-600 g Sprague-Dawley Long-Evans rats were by intraperitoneal injection administered in groups of eight 200 or 400 ug of paraoxon/kg BW or a control solution. Brain, plasma, and erythrocytes were assayed for cholinesterase (ChE) activity at the end of the experiment. Brain ChE was inhibited 57% and 19% with 400 1ig/kg and 200 ug/kg paraoxon, respectively. Neurobehavioral endpoints included; Figure 8 (F8) maze acitvity (counts/5 min), Accelerating Rotarod (AR) (sec on rod), Grip Strength (GS) (kg), and Startle Reflex (SR) (amplitude; voltages, and latency in seconds). No significant differences were detected in GS or AR performance between toxicant and control groups of rats. Audio and tactile SR amplitudes were increased with 200 ug/kg of paraoxon and decreased with 400 ug/kg of paraoxon. initial exploratory activity and activity during the remainder of the 60 min F8 session were decreased in paraoxon treated rats. Overall, paraoxon affected the sensory and exploratory behavior paradigms, but did not affect neuromuscular or balancing performance at these treatment levels. (Funded in part - US Army.) 299 75 Previous work from this lab showed that Type I pyrethroids (T-I) increase and Type II pyrethroids (T-II) decrease ASR amplitude in adult rats. We have recently found age-related differences in their effects: T-I do not alter and T-II decrease ASR amplitude in preweanling, rats. These differences could reflec4oRqparate mechanisms; a DDT-like effect of T-I on axonal sodium channels and a picrotoxi.n (PTX)-like effect of T-II on the GABA receptor complex. The present study examined the effects of DDT and PTX on the ASR during development to compare with the effects of T-I and T-II. Long Evans preweanling (ages 17 and 21 days)_and adult (70 day old) rats (n=12/group) ye~re V. tested 3 hr after DDT (po in corn oil) or k immediately after PTX (ip in saline). The results confirm reports that DDT (25 mg/kg) increases and PTX (1-2 mg/kg) decreases the ASR in adult rats. In preweanling rats, DDT did not alter ASR amplitude even at much higher dosages (125 mg/kg), whereas, PTX decreased the ASR at the same dosages used in adults. Age-related similarities between DDT and T-I support a sodium channel mechanism for T-I to increase the ASR and indicate that critical development occurs after postnatal day 21. The results are consistent with a PTX-like mechanism for the effects of T-II on the ASR. ( Supported by NRC Research Associateship.) ASSESSMENT OF MEMORY DEFICITS FOLLOWING REPEATED ORGANOPHOSPHATE EXPOSURE. K Raffaele, D Olton, and Z Annau. The Johns Hopkins University, Baltimore, MD. To assess changes in memory and development of tolerance following repeated exposure to organo- phosphate anticholinesterases, and to investigate a possible mechanism for the development of tol- erance, rats were trained in a serial reversal procedure. Each day, a rat was required 'to re- member the correct response learned the day be- fore, and then learn a new correct response. _ After rats achieved stable performance they were divided into two groups. One group received di- isopropylfluorophosphate (DFP), injected i.p. i mg/kg on the first day and 0.5 mg/kg every 3rd day thereafter. The other group received saline injections. To determine whether DFP-tolerant rats would be more sensitive than normal rats to stresses on the cholinergic system, rats were also injected with scopolamine, a muscarinic re- ceptor blocker, 20 min.•prior to testing on some days. A given dose of scopolamine produced , greater impairment in DFP-tolerant rats than in control rats, even if the DFP-tolerant rats were performing normally when no scopolamine was present. This demonstrates that, although there may be no obvious impairment following repeated exposure to organophosphate anticholinesterases, a tolerant system is not the same as a normal system and may not be able to react normally when challenged. (Supported by NIEHS 5T32 ES 07149). 50875 8200
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205 MECHANISMS OF 1,2-DIBROMO-3-CHLOROPROPANE (QBCP) INDUCED T3NA-DAMAGE, BACTERIAL MUTAGENICITY, AND CYTOTOXICITY IN ISOLATED RAT LIVER CELLS. J A Holme*, E J Soderlund*, G"' Brunborg*, J Omi- chinski*, S.Nelson**, and E Dvbing*. Natl. Inst. Publ. •Hlth., Oslo, Norway* and Univ. Washington, Dept. Med. Chem., Seattle, WA**. DBCP was found to induce DNA damage in liver cells at low concentrations (1-10 pM), as measured by alkaline elution. At higher con5entrations (0.5- 2.5 mM), DBCP was metabolized to' products that were mutagenic to Salmonella typhimurium TA100 co- incubated with the liver cells. At these concen- trations a marked depletion of cellular GSH was seen and at 2.5 mM DBCP was cytotoxic. Preincuba- tion of the liver dells with diethylmaleate (DEM), reduced DBCP-induced DNA damage.-In contrast, DEM lead to increases in DBCP-induced bacterial muta- genicity and cellular toxicity. Perdeuterated (D )-DBCP induced less DNA damage in the liver ceIls than DBCP. A greater reduction than this was seen on mutagenicity with D-DBCP compared to DBCP, whereas the two compoundsswere equally cyto- toxic. The cytotoxic effect was inhibited by ascorbate. Apparently different mechanisms are in- volved in DNA damage, bacterial mutagenicity and cytotoxicity induced by DBCP in vitro. We suggest that: 1) Both oxidation and formation of an epi- sulfonium ion are involved in cellular DNA damage, 2) oxidation leads to formation of products muta- . genic to the bacteria, whereas 3) the cytotoxicity induced by DBCP in the liver cells is caused by oxidative damage following GSH depletion.. 206 SPIN TRAPPING OF FREE RADICALS IN VIVO: A NEW APPROACH TO TOXICOLOGY. P B McCay, L A Reinke, E K Lai, C M DuBose. Sponsor: R A Floyd. Okla. Medical Research Foundation, Okla. City~OK. Spin trapping has made the detection of highly reactive free radicals in biological systems possible by converting non-persistent radicals to persistent ones. We have used this technique to trap free radicals which are generated in specific organs of intact animals which had been subjected to toxic substances. The method can determine in which subcellular organelle(s) the free radicals were formed. Both the intensity and duration of free radical formation can be assesed by this procedure. It has been demonstrated that highly reactive free radicals (•CC13) are produced in the endoplasmic reticulum of animals exposed to CC14 vapors. Other studies have demonstrated that chronic consumption of alcohol results in the production of carbon-centered lipid radicals in the liver and heart of rats. The production of these radicals continues for at least 4 days after administration of ethanol is stopped, suggesting that ethanol initiates a chain reaction (probably lipid peroxidation) which persists for a significant period of time. In vivo spin trapping has also been used in our laboratory to demonstrate that exposure to paraquat and other related pyridinyl diradical compounds also produces reactive radicals in the lung, liver, heart and spleen of intact animals. Supported by NIH Grant GM 36512. 52 ;~- 207 CALCULATIONS ON THE REACTIVITY OF ACRYLATE ANION WITH BIOLOGIQAL NUCLEOPHILES. C H Reynolds and C B Frederick Rohm and Haas Co., Spring House, PA. A recent paper has concluded that the ionized form of acrylic acid, acrylate anion, may form Michael adducts with ! biological nucleophiles basec3'on a 40 day in vitrQ reaction at pH 7.0 (Segal et al., Chem: i~c I. Int r .1, 189-197, 1987). Since the reaction of a negatively-charged species (acrylate anion) with a nucleophile appeared contrary to conventional reaction thegry, the pathway for the addition of I& representative nucleophiles (methylamine and imidazole) was expldred with the semi- empirical quantum model, AM1. The results indicate that there is no viable reaction pathway for the addition of acrylate anion to the nucleophiles. An alternative route for the formation of the Michael products in vitro via the non-ionized form of acrylic acid was explored and found to be theoretically possible. The afternative route is plausible, but is considered to be insignificant in vivo based upon the rapid metabolism and excretion of acrylic acid (excretion half-life of 1-8 hrs after oral dosing). 208 PROTECTIVE EFFECT OF DILTIAZEM AGAINST COCAINE INDUCED HEPATOTOXICITY AND HEPATIC LIPID PEROXIDATION. K A Suarez and S Bhonsle. Dept. of - Pharmacology, Chicago College of Osteopathic Medicine, Chicago, IL. Intracellular accumulation of calcium (Ca) has been postulated to be important in hepatotoxin induced injury. We decided to determine whether influx of Ca was a feature off cocaine (C) hepatic injury and whether damage could be altered by diltiazem (D). C was administered (70 mg/kg) to phenobar- bital induced, female ICR mice. D (20 mg/kg) or saline was administered 1 hr prior to and 6 hrs after C. Mice were sacrificed at various time intervals af ter C for determination of hepatic glutathione (GSH), malondialdehyde (MDA) and Ca.; levels or serum ALT, AST and ICDH. C produced a dramatic increase,compared to controls, in MDA (> 5x), ALT (>100x), AST (>15x), and ICDH (>50X) measuresl 24 hrs after C. The elevation in hepatic Ca (2x) was far lower than the rise (6x) reported for CHC13 and CC1 in rats. D pretreatment produceQ virtually complete protec- tion against C induced hepatic injury, the decline in,hepatio GSH (at 4 and 6 hrs) and the elevation in total Ca content (at 10 and 24 hrs). Because the C induced rise in hepatic Ca was far less than reported for other hepatotoxins, it may be a consequence rather than a cause of cocaine induced hepatoxoxicity. Supported by a grant from CCOM. 50875 8177
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EFFECTS OF ACUTE ADMINISTRATION OF O,O,S-TRI- IMMUNOTOXICITY OF TRIBUTYLTIN OXIDE IN jATS 318 METHYL PHOSPHOROTHIOATE ON THE RESPIRATORY BURST EXPOSED AS ADULTS OR PRE-WEANLINGS.t R J AND PHAGOCYTIC ACTIVITY OF SPLENIC AND PERITO- Smialowicz, M M Riddle, R.R. Rogers, R.W NEAL LEUKOCYTES.g RE Rodgers, and DD Ellefson. Luebke, C B Copeland and A.C Adams. U.S. EPA, School of Medicine, University of Southern Cali- Research Triangle Park, NC fornia, Los Angeles, CA. An impurity in malathion, O,O,S-trimethyl phos- phorothioate (OOS-TMP), was previously shown to be immunosuppressive. The immune cell type which induced immune suppression caused by OOS- TMP at 24 hrs after administration was found to be splenic macrophages. Further characteriza- tion of macrophages from OOS-TMP treated. mice indicated that OOS-TMP led to macrophage differ- entiation. In this study, these initial studies were continued and extended to examine the effects of OOS-TMP on splenic and peritoneal macrophages at various times following exposure. Administration of OOS-TMP increased the size heterogeneity of cell volume, phagocytic capa- bility and respiratory burst activity of splenic and peritoneal macrophages. However, by day 7 splenic and peritoneal macrophages from treated animals had size frequency histograms, phago- cytic capability and respiratory burst activity similar to control. These data would suggest that macrophages not previously exposed to inflammatory stimuli migrated to the spleen and peritoneum of treated animals. This migration may allow the restoration of the ability of splenocytes from treated animals to generate an immune response. Supported by PHS Grant ES04437. HOST RESISTANCE TO MURINE MALARIA IN ADENOSINE DEAMINASE-DEFICIENT MICE. R W Luebke, A C Adams, C B Copeland, M M Riddle, R R Rogers and R J Smialowicz. U.S. EPA, Research Triangle .Park, NC The purpose of this study was to profile host resistance to a clone of murine malaria derived from the nonlethal strain of Plasmodium oy elli. 2'-Deoxycoformycin (2dCF), a potent adenosine deaminase inhibitor, was administered before initiation of infection at a dose of 0.8 ug/g/d for 5 days. Previous studies in our lab have shown that 2dCF has immunomodulating properties, enhancing some functions and suppressing others. Patent infections were demonstrated in both 2dCF- and saline-pretreated mice on day 3; however, the peak of parasitemia occurred about 2 days later in 2dCF-treated mice than in controls. In addition, treated mice eliminated the parasitemia more gradually than in controls. Erythrocyte counts decreased to 50% of uninfected values where parasitemia reached approximately 20% in both treated and control mice and returned to normal within 2 days of parasitemia clearance. These results indicate that 2dCF alters the course of nonlethal P. oy elli in the treated host. This is an abstract of a proposed presentation and does not necessarily reflect EPA policy. 80 Previous studies from this laboratory demonstrat-' _ ed that exposure of pre-weanling rats to dioctyl- ~' tin dichloride (DOTC) resultedAuia.a more severe ` Ind persistent immunosuppression than exposure-a- of adults. In order to ;determine if this also occurs with other organotins, rats were exposed_ '; to tributyltin oxide (TBTO). Male Fischer rats were dosed by oral gavage with TBTO as adults (9 weeks old) at 5, 10, or 20 mg/kg/dose or as pre-weanlings (3 to 24 days old1gat 4.5 or 5 mg/ kg/dose three times per week for a total of ten doses. At various times after dosing rats were examined for changes in body and lymphoid organ weights and for mitogen, mixed lymphocyte (MLR), natural killer (NK) and cytotoxic T lymphocyte (CTL) responses. A dose of 5 mg/kg or more to adults or pre-weanlings resulted in thymic invol- ution. Reductions in mitogen responses were ob- served in adults at 10 and 20 mg/kg and at 5 mg/ kg in pre-weanling rats. The MLR response was suppressed only in the adult rats at 20 mg/kg. NK and CTL responses were not affected in either age group. Within 3 weeks all parameters return- ed to normal in rats dosed as pre-weanlings. These data suggest that exposure of young rats to TBTO results in immune alterations, but that unlike DOTC these changes are not persistent. 319 FLOW CYTOMETRIC ANALYSIS OF LYMPHOCYTE SUBPOP- ULATIONS IN MICE EXPOSED TO 2,3,7,8-TCDD: CON- STITUTIVE AND FACULTATIVE EXPRESSION. J A ` Brauner and N I Kerkvliet. College of Veterinary Medicine, Oregon State University, Corvallis, OR. Mouse spleen and thymus cells were studied for surface marker expression by multiparameter flow cytometry to characterize the subpopulations present in vehicle- and TCDD-treated mice and to determine relative changes in subpopulations following sheep red blood cell (SRBC) challenge. Cells were analyzed for the expression of the major murine T and B cell differentiation antigens including Ig, Thy 1.2, L3T4, Lytl, and Lyt2 and for the dual expression of Lytl and Lyt2 or L3T4 and Lyt2. Spleen cells from C57B1/6 mice exposed orally to 2 ug/kg TCDD two days prior to sacrifice showed.no significant alterations in the percent of cells expressing these surface markers whereas the percent of thymus cells expressing L3T4 was reduced (p <_ 0.05). Spleen cells from mice exposed to 2 or 5 ug/kg TCDD were analyzed . daily for four days following SRBC challenge for . possible effects of TCDD on antigen-induced cell surface marker expression. The percent of Lyt2+ cells was modestly yet significantly reduced (p < 0.05) in both 2 and 5 ug/kg treatment groups on day 3 after SRBC challenge. No other changes were observed. The relationship between this transient alteration in the number of Lyt2+ cells and suppression of the antiSRBC antibody response by'TCDD remains to be determined. Supported by NIH grant RO1-ES03966. 50875 8205
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92 ANALYSIS OF THE COGNITIVE IMPAIRMENT INDUCED IN RATS BY TRIMETHYLTIN: AUTOMAINTAINED REVERSAL LEARNING_ D New*, P J Bushnell, and D D Dunn*. Neurotoxicology Division-` , US EPA, RTP, NC and *Northrop Serviceszt Inc. - Environmental Sciences, RTP, NC.' . . Trimethyltin (TMT) is a potent neurotoxicant known to disrupt learning and memory. It thus provides a positive control treatment for characterizing cognitive dysfunction in test animals. Eight male hooded Long-Evans rats, maintained at 350±10 g body weight, were presented two retractable bars for 15" (CSs) per trial on identical but independent random (ITI=45") schedules. A food pellet (UCS) was delivered upon retraction of one of the bars (hot CS), but not the other (cold CS). Presses on both bars (CRs) were counted but had no programmed consequences; thus one pellet was delivered per trial regardless of the rat's behavior. At asymptote, hot CRs exceeded 10/trial, while cold CRs approached 0/trial. When the positions of the hot and cold CSs were reversed (R), CRs shifted to the new hot bar. Successive reversals (2/week) occurred at faster rates: by R12, reversal was complete within one 100-trial session. Injection of TMT (7 mg/kg, iv) to half the animals after R44 reduced hot CR rates for 1 week, after which rates were normal. Thereafter, ratios of hot CRs to total CRs were normal when no delay separated the CS and UCS; with delays of 2 to 4 sec, treated animals reversed more slowly than controls. These results indicate that TMT increases rats' sensitivity to temporal factors. '293 ANALYSIS OF THE COGNITIVE IMPAIRMENT INDUCED IN RATS BY TRIMETHYLTIN: EPISODIC MEMORY AND VISUAL DISCRIMINATION. P J Bushnell, D D Dunn*, and D New*. Neurotoxicology'Diisfon, US EPA, RTP, NC and *Northrop Services, Inc. - Environmental Sciences, RTP, NC. 'Trimethyltin (TMT) is a potent neurotoxicant known to disrupt learning and memory. It thus provides a positive control treatment for characterizing cognitive dysfunction in test animals. Eight male hooded Long-Evans rats, maintained at 350±10 g body weight (bwt), were trained to press one of two retractable bars for food reward in the choice phase of a trial if that bar had been extended in the prior sample phase of the same trial. On half the 200 trials per session, the correct response was cued by a light above one of the bars, not by the position of the preceding sample stimulus. After training, half the rats received 7 mg/kg TMT hydroxide iv, and the other half saline. Behavior and bwt were assessed daily for 12 weeks. Treated rats lost 1-16 g bwt 1 vk post TMT and recovered baseline bwts in 2-5 wks. TNT caused a significant and irreversible reduction in uncued choice accuracy at long delays in 3 of the treated rats, with degree of impairment loosely related to degree of previous bwt loss. In contrast, accuracy on cued trials was affected more strongly at short than at long delays. ' When the light cue was made redundant with sample position, cued accuracy returned to control levels. These results indicate that the cognitive effects of TMT involve impairment of both episodic memory and cue discrimination. 294 IMPAIRMENT OF CONDITIONED AVOIDANCE RESPONSE r:.;, jCAR) INDUCED BY ALUMINUM IN MICE. Yen-Koo Balazs, T and Baer, H. Food and Drug Administration, Washington, DC Aluminum (Al) is used in medicinal products and some parenteral products (vaccine) contain A1, _ In a study of the neurotoxicity of Al in mice, four groups of CD1 mice (5 M and 5 F per dose lev4) were treated as follows: Mup 1 drank:- 1.0% Al (as A1C1g) during the weaning period _. from day 1 to 8 weeks of.age; group 2 drank : A1C13 from 1 month to 4 months of age; group 3 mice (1 month old) were injected i.p. with 10, 30 and 100 mg Al (as A1C13)/kg/day for 2 days; . group 4 mice (one month old) were injected s.c. -- with 3, 10 and 30 mg Al (as A1C13)/,1~g/day for 2 days; controls received vehicle. All mice were' tested for CAR 5 times at about 2 months of age. The CAR of mice that ingested A1C13 during the weaning period to 8 weeks of age was lowered by 30% or more compared to the control group, which achieved 50% of CAR after 5 training sessions. ' Also, retention of CAR was reduced to 55%, whereas that of the control group remained at the same level after 1 month. CAR values of group 2 did not differ from those of its control. CAR of group 3 (at 30 mg/kg) was 87% ' lower than controls; CAR retention was reduced 60%. CAR of s.c. groups (10 to 30 mg) was lowered to 11-37% of the control; CAR retention _ was reduced to 16-27%. Oral ingestion of Al . induced toxicity in mice only at an early age, - but injection caused neurotoxicity at any age. 295 CHELATORS ATTENUATE THE BEHAVIORAL CONSE- QUENCES OF CADMIUM ADMINISTRATION. DB Peele, JD Farmer, and RC MacPhail. Northrop Environmental Sc en ces, F(TP, NC, and U.S. EPA, RTP, NC. The conditioned flavor-aversion paradigm was used to determine the behavioral toxicity of acutely administered cadmium and the interac- tion of cadmium with the heavy-metal chela- tors dimercaptosuccinic acid (DMSA) and dimercaprol (BAL). Rats received cadmium (ip) either alone or in combination with BAL or DMSA (sc) 20 min after consuming saccha- rin. Seventy-two hrs later they were given concurrent access to saccharin and water, and saccharin preferences were recorded. Rats receiving cadmium displayed significant reductions (compared to controls) in saccha- rin preference (i.e., conditioned flavor aversions). BAL and DMSA were also capable of producing conditioned flavor aversions when given alone. Rats receiving cadmium in combination with either BAL or DMSA displayed significant attenuations of conditioned flavor aversions when compared to the flavor aversions of rats receiving cadmium alone. Chelator-induced attenuation of cadmium-induced flavor-aversion conditioning was not obtained when BAL or DMSA administra- tion was delayed by 4 hrs. These results ex- tend earlier findings of an attenuation of lead- but not thallium-induced flavor-aversion conditioning by these heavy-metal complexing agents. 74 MA EF B Sc ME 297
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,244 POLYCHLORINATED BIPHENYL (pCB) CONGENERS WHICH ANTAGONIZE THE TERATOGENIC EEFECTS OF 2,3,7,8-' TETRACHLORODIBENZO-p-DIOXIN (2CDD) IN C57BL/6 MICE. L Biegedl, J M Haake, S Safe, K Mayura and T D Ph411ie, Department of Veterinary Physio ogy an Pharmacology and Department of Veterinary Public Health, College of Veterinary Medicine, Texas A&M University, College Station, TXo Previous work in our laboratorp has demonstrated that the cottmercial PCB mixture, Aroclor 1254 (750 umol/kg), protects C57BL/6 mice against 2,3,7,8 1CDD-induced cleft palate. Several R'B compounds present in Aroclor 1254 exhibited low but measurable affinity for the Ah receptor protein but did not elicit 2,3,7,8- TCDD-ttediated responses. Two of these con- geners, namely 2,2',4,4',5,5'-hexa- and 2,2',5,5'-tetrachlorobiphenyl, at dose levels of 750 umol/kg also antagonized the teratogenic effects of 2,3,7,8 -TCDD. Administration of [3H]-2,3,7,8-TCDD to C57BL/6 mice showed that there was a rapid uptake of radiolabel into the fetal palate. However, cotreatment with [3H]- 2,3,7,8 3CDD plus 2,2',4,4',5,5'-hexachlorobi- phenyl resulted in decreased uptake of radio- label into the fetal palate tissue. The sig- . nificance of these interactive effects on the mechanism of PCB-ntediate8 antagonisn will be discussed. (Supported by the National Institutes of Health.) . 245 THE ROLE OF ALTERATION IN THE DISTRIBUTION OF SECALONIC ACID D IN THE ANTITERATOGENIC EFFECT . OF DMSO. M.M R ELDEIB AND"C S. REDDY. Depart- ment of Veterinary Biomedical Sciences,"Univer= : sity of Missouri-Columbia, MO. DMSO is known to antagonize teratogenic effects . of secalonic acid D (SAD) in mice. To establish optimum protective dose of DMSO, pregnant CD-1, mice were treated,.ip, with 30 mg/kg of SAD.in: NaHCO with 0, 10; 20 or 30% DMSO on day 12 of gestahon. Data indicate that 10 and 20Z DMSt) afford a dose-related protection against SAD- induced cleft palate whereas 30% DMSO enhanced SAD-induced fetal resorptions with no reduction in the incidence ofl&ft palate. Distribution and elimination of C-SAD was studied in fetal and maternal tissues from pregnant mice qt 24 and 48 hr after exposure.to 30 mg/kg of C-SAD, ip, in NaHC03 (control) or with 20% DMSO. ..-. Maternal exposure to DMSO: (A) significantly reduced (42 to 75%) radioactivity in fetal heads and bodies; in placenta; and in maternal tissues including GI tract, uterus, salivary gland, bone and tongue; (B) significantly increased (2222) the radioactivity in maternal liver; and (C) significantly reduced (44 to 58%) fecal and urinary elimination of radioactivity; compared to control. These results suggest that the antiteratogenic effect of DMSO against SAD may be mediated by increased SAD (or its metabo- lites) retention by maternal liver leading to both reduced SAD uptake by the fetus and reduced elimination in urine and feces. Supported by NIH grant 11DE07107. 62 246 SECRETION OF HIGH CONCENTRATIONS OF CIMETIDINE INTO RAT MILK DURING LACTATION. L:A. Dostal, ; R W Weaver, and B A Schwetz, National Toxicology Program, NIEHS, P.O. Box 12233, Research Triangle Park, NC 27709 High milk/plasma ratios (4-12) for cimetidine (pK 7.1) have been reported in humans after in- ~ gestion.. To investigate thewaaceretion of cimet.• _ {idine into milk and its effect on lactating r&8- and their pups, rats were given daily oral doses~;;- of cimetidine on Days 13-16 of lactation. Ma_ :. ternal exposure to cimetidine (18 or 180 mg/kg/ day) had no effect on milk protein, lipid, or . water content, nor on total mammary DNA or RNA content. However, a small inc0ase?-in milk lac- tose was found at 180 mg/kg/day, and the RNA/DN& ratio was increased at both doses. There was no effect on growth, testis weight, anogenital dis- tance, or hepatic aminopyrene N-demethylase ac- tivity in the pups. Four hrs after the last dose of 18 mg/kg/day, plasma and milk cimetidine levels were 0.77 + 0.16 and 16.7 + 1.8 yg/ml (Mean +SE, na6), respectively; at 180 mg/kg/day,. cimetidine levels were 3.6 + 0.4 and 1733 + 9 y.g/ml in plasma and milk. In vitro, [ H]cimeti- dine is mainly distributed in whey (69%) and casein (28%) with only 18% protein binding in whey. Protein binding was 60% in skim milk and - 45% in plasma. Thus, milk/plasma ratios of 22-. 31 in rats cannot be explained by ionic equilib- - rium or protein binding in t~e milk. The possi- bility of active uptake of [ H]cimetidine into' the mammary gland is under investigation. 247 2,5-HEXANEDIONE-INDUCED TESTICULAR INJURY - AND MICROTUBULE ALTERATION. K Boekelheide:',-: Brown University, Providence, RI. We have proposed alterations in Sertoli cell microtubule as=. sembly as the basic "mechanism of 2,5-hexanedione-induced., =. testicular injury, an hypothesis supported by.the diffeienCial " " testicular toxicity and microtubule effects of two y-diketone congeners (TAP 88, 370-382 and 383-396) and the early on- set of altered assembly kinetics during 2,5-hexanedione in- _ toxication (TAP, two articles in press). Two new experimental" approaches further strengthen this association. Charles Kiver CD rats (220 g) were intoxicated with 131 ± 2 mmol/kg 2,5-hexanedione delivered in the drinking water at dose rates of 6.1, 3.8 and 1.9 mmol/kg/day over 21, 35 and 69 days, respectively. The extent of testicular injury, as determined by - testis weights and histopathology, correlated with the dose rate and not total dose. The extent of biochemical abnormality in ' testis pyrrole content and microtubule assembly kinetics, determined in rats intoxicated at the various dose rates for 3 weeks, was also a function of dose rate. In a morphometric study, the closest inter-microtubule distance was measured for cytoplasmic Sertoli cell microtubules in stage VII seminiferous tubules. When compared with controls, rats intoxicated for 3 weeks with 1% 2,5-hexanedione had significantly decreased numbers of microtubules with intermediate inter-microtubule distances (25 - 50 nm), with the majority of microtubules closer than 25 nm. This morphometric result is consistent with the in vivo induction by 2,5-hexanedione of an increased number of shorter microtubules, a known in vitro morphologic effect of 2,5-hexanedione tubulin treatment. 50875 8187
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TOXICITY OF ELSAMICIN, A POTENTIAL ANTICANCER AGENT. T J Davidson, C L Bregman, R A. Buroker, R,S Birth, and H Madissoo, Department of Pathology and Toxir-ology, Bristol-Myers Company, S~racuse, NY. Toxicity evaluationsyof.the succinate salt were conducted IV in mice, ratg and dogs. The LD 0 in CD-1 mice was 16.3 mg/kg (M) and 26.0 mg/k5g (F). Most deaths occurred within 2-4 days after treatment. When the drug was admini- stered for 5 consecutive days to mice, the IJ)was between 9 and 12 mg/kg/day (M and F). In rats, single doses of 5.5 mg/kg or more resulted in transient bone marrow and lympho- cytotoxicity. Systemic toxicity and mortality were noted within 3-4 days after a dose of 16 mg/kg. Following 5 consecutive daily doses of 4 or 8 mg/kg, hematologic and lymphocytotoxicity were apparent within 3-4 days. In the x1 and x5 studies, neutrophilic leukocytosis was evident shortly after treatment and surviving males showed testicular atrophy at 30 days. Elsamicin was significantly more toxic in the beagle dog. Single doses of 0.16 mg/kg were lethal within 1=3 days and 5 daily doses of 0.07 mg/kg were lethal 24 hours after the last dose. Hemoconcentration and neutrophilic leukocytosis with a left shift were detected within 24 hours after treatment and a shock or shock-like state preceded deaths in both studies. The single toxic dose low in the dog is 0.008 mg/kg based on a transient neutrophilic leukocytosis. RELATIVE BIOAVAILABILITY OF TISSUE RESIDUES OF PQLYETHER IONOPHORE ANTIBIOTIC (P-2546) IN RATS. C IC Parekh, S M Ghiasuddin, J W Fernandes, and T M Sullivan. Research & Development, Pitman-. Moore, Inc., Northbrook, IL. P-2546 is being developed as a feed additive to increase the feed efficiency and rate of weight gain in beef cattle. To determine bioavailability of tissue residues of P-2546 present in beef liver, modified Gallo-Torres (1977) procedure was used. In the modified procedure rats intubated for bile infusion and collection were free moving with free access to food, water and survived upto 7 to 10 days. The surgically prepared rats were gavaggi with a suspension of beef liver spiked with C-P2R6 or liver from beef cattle orally dosed with C-P2546. Distribution of radioacti- vity in all the tissues was monitored. Bioavail- ability is expressed as the percentage of an oral dose absorbed from the gastrointestinal trach Rats orally fed with beef liver spiked with C- P2546, only 44.8% of P-2546 was bioavailable while P-2546 residues present in liver were less than half or only 19.2% were bioavailable. The modified procedure in free moving rat model, is useful for determining bioavailability of resi- dues or free drug. 338 LIPID AND VITAMIN LEVELS IN 30 uAY«A1D`4RATS AD- AfTNISTERED SODIUM SACCHARIN SINCE CUNCEPTION. E M Garland, P Kraft*, R Shapirot and S M Cohen, Univ. Nebraska Med. Ctr., 0maha; AEZ * epsiCo, Valhalla, NY, and tNutr; Intl. Inc., ' New Brunswick, NJ. Lipid and vitamin levels in liver, bladder, and serum were evaluated in•Sprague-Dawley rats ex- posed to 7.5% sodium saccharin (NaS) in the diet from conCeption until sacrifice at ~Q~a,~as post- birth (d~b). Litter sizes and body weights (BW) at birth were similar in the NaS and control groups. BW of.NaS pups were depressed after 4 dpb and were 60% of control at 30 dpb. Food consumption was not affected by NaS treatment in F0 rats, but it was reduced in weaned NaS- fed F1 rats. NaS-treated F1 rats exhib~'.ted,~, several changes in lipid and vitamin lt~iels.^ Serum cholesterol, triglycerides, and vitathin (vit.) E increased by approximately 45%, 1000% and 100%, respectively. Serum high-density lipoprotein, vit. A, and folate decreased by 45-60%. Hepatic concentration of vit. A palmi- tate increased in males only, vit. E increased in females only, and folate decreased in males and females. Total amounts of folate, vit. A, vit. A palmitate, and vit. E in the liver de- creased with NaS treatment. Cholesterol in bladder epithelium was unchanged. In summary, high doses of NaS from conception through 30 dpb produced profound biochemical changes in the weanling rat. Supported by a grant from the Intl. Life Sciences Institute. 339 85 EFFECTS OF ASPARTAME ON PENTYLENE- TETRAZOL(PTZ)-INDUCED CONVULSIONS IN CD- I MICE. P C Jobe, A F Bettendorf, S_ M Lasley. and J W'Dailey. The University of Illinois College of Medicine at Peoria, Peoria, Illinois 61656. Large doses of aspartame were administered orally to CD-1 mice. For each dose of aspartame, a convulsive dose fifty (CD50) was determined for PTZ at one hour following gavage with aspartame or a 0.5% methylcellulose vehicle. Each animal employed received only one dose of PTZ. The convulsive endpoint was a generalized clonus with loss of righting reflex. Milder forms -of response such as facial and forelinrl3 clonus with or without rearing were not assessed. First, CD50s were determined in fa ted male mice weighing 13 to 28 grams each and housed in rou s before and after the injection of PTZ. Under these conditions, the CD s for tests of 3 different doses of aspartame . mafcOhed with the appropriate vehicle controls were: (1) 1500 mg/kg aspartame CD50 3 63.9 mg/kg (confidence interval - 57.9 - 70.6) versus vehicle CD50 = 54.5 (45.5 - 65.3); (2) 2000 mg/kg aspartame CD5 = 67.1 mg/kg (61.3 - 73.3) versus vehicle CD50 = 56% mg/kg (49.3 - 64.9); (3) 2500 mg/kg aspartame CD = 59.5 mg/kg (50.1 - 70.7) versus vehicle CD50 = 56.~ mg/kg (49.4 - 64.5). In a second study grou ped mice were fed, rather than fasted, and the individual body weights ranged from 9 to 13 grams. Aspartame was administered in a dose of 1500 mg/kg. The CD50 for this dose was 69.2 mg/kg (61.6 - 77.8), whereas the CD50 for vehicle was 65.5 mg/kg (59.0 - 72.8). According to these data, aspartame does not facilitate PTZ seizures in grouped male CD-1 mice. Neither does aspartame produce a significant anticonvulsant effect. (Supported in part by a grant from the NutraSweet Company). 50875 8210 ,i I
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169 ETHYLENE DIBROMIDE (EDB): MULTIGENERATIONAL NEU~OTOXIC EFFECTS OF PATERNAL EXPOSURE. L.L. Hsu , M S Legator2, and P.M Adams , Depts. of _ HBC&G1 and.PMkH2, UTMB, Ga veston, TX, Dept. of Psychiatry, UTHSC, Dallas, TX Sponsor: G A S Ansari. In this study, we assessed the male mediated EDB effects on the brain chemistry of F2 progeny. Young male"Fisher 344 rats were tteated (i.p.) daily with corn oil or 1.25 mg or 5 mg of EDB/kg for 5 days. After 7 days, the EDB and corn oil treated males were crossed with untreated virgin females. Young adult male F progeny were crossed with their respective litter mates. Brain regions of F progeny at 90 days old, including cerebel- lum 2(CB), corpus striatum (CS), frontal cortex (FC), hippocampus (HIPP) and hypothalamus (HY) were assayed for ChAT, AChE and.GAD. Results in- dicated: (1) paternal exposure to 1.25 mg EDB in- creased ChAT in CB and FC of both male and fe- " male, it increased ChAT in only male CS and had no effects on ChAT in HY or HIPP; it decreased AChE in all male (but not female) brain regions, and increased GAD in only the male FC. (2) pat- ernal exposure to 5 mg of EDB increased ChAT in the female CB but decreased ChAT in the female CS and HIPP, and did not change ChAT in the male brain; it markedly increased (114%) the AChE in the female CB but decreased (40%) AChE in female HIPP and did not affect the male brain AChE, and it increased the GAD in both the male (82%) and female (65%) CB but had no effect on GAD in other brain areas. (Supported by NIOSH Grant No.OH01245) 170 EFFECPS OF NEOROFIhANIENTOUS AXONOPATfD[ PRODUCING NEUROTOXICAN'PS UPON FAST ANTEROGF2ADE TRANSPORT. D W Sickles. Medical College of Georgia, Augusta, GA. We hypothesize that neurofilamentous axonopathy- producing toxicants which also produce nerve degeneration adversely affect fast anterograde transport. The effects of single neurotoxic doses upon the rate and/or capacity of transport of radiolabelled protein in rat sciatic nerve after injection of 3H-leucine into the DRG were determined. Acrylamide (A(Tt; 50-100mg/kg) produced a 9.2-20.8% decrease in rate and 42.7- 51.4% decrease in quantity of protein transport (Sickles and Pearson, Toxicologist 7:132,1987). 2,5-Hexanedione (2,5-HD; 4-8 mmoles/kg) and 3,4- dimethyl-2,5-HD (0.25-0.5 mmoles/kg) caused a 16.8-22.9$ and 18.5-36% decrease in rate, respectively; the total protein transport was decreased 49.7-61.6% and 46.8-69.6%. The incorporation of 3H-leucine was not decreased by ACR, 2,5-HD or DNgID. IDPN (0.1%), which causes a neurofilamentous axonopathy without nerve degeneration, and methylene-bis-ACR (108.5 mg/kg) had no significant effect upon rate or quantity of protein transport. This is the first report of a correlation between nerve degeneration and primary decreases in quantity of protein transport caused by single doses of these neurotoxicants. Supported by National Institute of Occupational Safety and Health, #QH 02020. 43 171 2,5-J3EXANEDIONE-INDUCED ALTERATIONS IN AXONAL PROTEIN AND PHOSPHOLIPID PHOSPHORYLATION. K.L. Horan, R M LoPachin, D Caprette and J Eichberg. Dept. Pharmacology and Dept. Biochemistry and Biophysical Sciences, U. Houston, Houston, TX 2,5-Hexanedione (HD) produces a central-peripheral distal axonopathy which is characterized by mufti-focal swellings oCL distal axons. The present study examined the possibility thar alterations in the phosphorylatiogof" axonal proteins and= 'r phospholipids are involved in the neurotoxic mechanism of HD. - Male Sprague-Dawley rats (275-300 gm) were divided'fiStii groups (n=6) based on the daily treatment to be received: HDt_ (400 mg/kg), 1,6-Hexanediol (414 mg/kg) or saline (3 mUkg).".' Groups of animals were injected with chemicals or saline according to one of the following treatment schedules: 7, 15 or 24 days. In a separate experiment rats were treated with HD, hexanediol or saline for 24 days at which time treatments were discontinued for 17 days. At the appr,*riate°time animals were sacr'rficed by decapitation and both sciatic nerves removed. Nerves were desheathed and incubated for 2 hrs in oxygenated Krebs buffer containing 32P orthophosphate. Nerves were cut into proximal and distal halves and incorporation of radiolabel was assessed by liquid scintillation spectrometry. Morphotogical analysis (axonal diameter) was conducted to correlate biochemical changes with nerve damage. Results showed that in proximal nerve segments, HD-treated animals exhibited reversible, temporally-related selective increases in isotope incorporation into the 180K (unidentified) and 55K (tubulin) protein bands, while in distal segments changes were not evident. Radiolabel incorporation into myeiin proteins P1, P2 and Po as well as into phospholipids revealed inconsistent changes. Morphological analysis showed HD intoxicated animals (24 day treatment) to have significant increases in fiber diameter both proximally "and distally (68% and 70%, respectively) when compared to controls. These results suggest that alterations in phosphorylation of proteins might play a role in HD-induced nerve damage. (Supported by NIH grants ESO 3830 and DK 30577). 172 MICROSOMAL ATPASE ACTIVITY FOLT,Oin1ING LONG-TERM EXPOSt7RE TO 2, 5-BEXANEDIONE. J K Pearson and D W Sickles. Medical College of Georgia, Augusta, GA 2,5-Hexanedione (HD), and 3,4-Dinethyl-2,5- Hexanedione (m-ID) are neurotoxicants known to produce a neurofilamentous axonopathy form of neuropathy by an unknown mechanism. It has been proposed that inadequate supply and transport of vital macromolecules to the distal axon is responsible for the nerve degeneration. We have , hypothesized that ganana-diketones produce these changes through interference with the microtubules. It has been demonstrated that HD and DNEID affect both the rate and the quantity of radiolabeled protein transported in rat sciatic nerve (Sickles, Toxicologist 8, 1988). The current study tests the possibility that HD conproaises the energy utilizing protein (ATPase) associated with axonal.transport, which has been demonstrated to be a Ca/Mg-deperxiQnt ATPase associated with the microtubules found in the microsomal fraction. This ATPase activity of the mi.crosanal. fraction of rat sciatic nerve, following 3 and 4 week exposure to HD (4 nm/kg daily), showed no significant differences between control and experimental nerves. We conclude that HD does not affect transport via inhibition of the transport ATPase. Supported in part by National Institute of Occupational Safety and Health #OH-02020. Ln m 00 J ~ co ~. 01 00
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'340 INDUCTION OF CLEAR CYTOPLASMIC VACUOLES (CCV) IN 342 CULTURED CELLS BY SC-35311. S DLAnderson, Z Ruben, H T Gaud, and R B Johnson and V Gupta. R & D, G. D. 5eax%e & Co., Skokie, IL. SC-35311 was found as a minor impurity in a syn- thetic process of the cardiac antiarrhythmic SC-40230. SC-35311 is a bis-piperidine ring (pKa1=8.2, pKa2=9.6) analogue of disobutamide. Disobutamide induces CCV in vivo and 3n,vitro, which are signs of intracellular drug storage and not of overt toxicity (Human Pathol. Dec. 1987). The three compounds are structurally similar cationic amphiphilic bis-tertiary amines, but differ in basicity of the amine functions. To test whether SC-35311 is vacuologenic, we incu- bated it with rat urinary bladder carcinoma (RBT CC-8) and rabbit aorta muscle cells at 0, 1, 2, 4, 6, 8 & 10 x 10-4M for 24 hr. We examined cells in situ by light microscopy and the medium for lactic dehydrogenase (LDH). Disobutamide and SC-40230 served as positive and negative controls, respectively, for CC7 induction. SC-35311 in- duced CCV at 4 x 10- M or higher. There was no morphologic evidence of cell death nor signifi- cant release of LDH. We concluded that, similar to disobutamide Proc. Soc.184:165-171,1987), the relative high basicity of the bis-tertiary amine functions of SC-35311 determined induction of CCV, and that the induced vacuolation is not accompan- ied with overt toxicity. " 347 THE EFFICACY OF SUPERACTIVATED fHAR- COAL IN TREATING RATS EXPOSED TO A LETHAL ORAL DOSE OF POTASSIUM.QYANIDE. R J Lambert, B L Kindler, and D J Schaeffer. Illinois Animal Poison Information Center, Dept. of Veterinary Biosciences, University of Illinois, Urbana, IL. Due to the relatively low binding capacity of regular acti- vated charcoal (AC) for potassium cyanide (KCN) in vitro, the use of oral activated charcoal therapy for oral exposure to cyanide compounds is controversial. In this study rats were given a lethal oral dose ofground granular KCN (35 or40mg/ kg) in a gelatin capsule followed immediately by either 4 g/ kg of superactivated charcoal (SAC) in a 20% suspension or a similar volume of deionized water. Signs of cyanide toxi- cosis occurred rapidly, with a mean time to signs of 3.3 and 2.7 min incontrol animals receiving 35 or40 mg/kgKCN, re- spectively. All 26 of the control rats showed signs, and all but 1 in the 35 mg/kg group died within 19 minutes. Only 12 of ' 26 rats treated with SAC showed signs of KCN toxicosis and 8 of those animals died. A regression model for the 33 animals dying indicated that time to onset of signs was correlated with the time to death (time to death = 5.21 + 2.16signs, R = 0.73). Oral exposure of rats to lethal doses of KCN can be effectively treated by immediate administration of superactivated charcoal. SUBCHRONIC ORAL TOXICITY STUDY OF CYCLOPIAZONIC ACID (CPA) IN MALE SPRAGIIE-DAWLEY-RATS. K A' Voss, W P Norred, R J Cole and J W Dorner. Richard B. Russell Research Center, ARS, USDA, Athens, GA and the National Peanut Laboratory, ARS, USDA, Dawson, GA. CPA is a mycotoxin produced by Aspergillus and Pe,nicillium species. It has b?i'i"isolated from . meats, cheese, corn and peanuts. Effects oi`~`"-~' •'• subchronic CPA exposure Were studied. Groups of'~', ° 12 male Sprague-Dawley rats were orally dosed with 0.2, 0.6, 2.0 or 4.0 mg/kg/day CPA for 13 weeks. Twelve controls were dosed with vehicle (1.0 N Sodium Bicarbonate) only. Animals were observed daily. Body weight aA f od consump- tion were measured weekly. Selectedxhematology, serum chemistry and urinalysis determinations were done after 7 and 13 weeks. All animals were subjected to necropsy and organ weight mea- surements. Detailed histopathological examina- tion of control and high dose animals was done. Gross lesions and stomachs of remaining animals were microscopically examined. CPA failed to produce overt systemic toxicity at any dose level. Weight gain and food consumption of all groups were similar. Clinical laboratory stud- ies, necropsy and organ weight determinations did not reveal definite treatment-related ef- fects. Slight acute inflammation of the gastric submucosa, considered a local effect of CPA ad- ministration, was noted at doses >0.6 mg/kg. 343 KINETICS AND METABOLISM OF THE RADIOPROTECTIVE AGENT, S-2-(3-METHYLAMINOPROPYLAMINO)ETHYLPHOS- PHOROTHIOIC ACID (WR 3689), IN RHESUS MACAQUES. A Buckpitt, H Chung, D Baggot, S Hietala, D Jo nson an M GoTdman. Institute for Environ-. mental Health Research, UC Davis, Davis, CA and Walter Reed Army Medical Center, Washington, DC. WR 3689 is one of several aminothiols which have protective actions against gamma irradiation. These agents also enhance the therapeutic index of several antineoplastic agents. The present studies have examined the kinetics of 14C-WR 3689 in Rhesus Macaques after IV and PO ad- ministration of 150 mg/kg. WR 3689 is cleared rapidly from plasma with a t1J2 of 15 min and a Vd of 1600 ml/kg after IV admtnistration. Parent drug could not be detected after P0 administra- tion. Radioactivity in the plasma and whole blood after IV 14C-WR 3689 had elimination tl/2's of 10 and 26 hours, respectively. The long tl/2 of radioactive parent and metabolite in comparison with the short tl/2 of parent drug in plasma suggests that metabolites are sequestered in the blood. Urinary and fecal excretion were 70 and 10% of the administered label after IV and 37 and 52% after oral administration. Based on whole blood radioac- tivity, the oral bioavailability was 30%. Four metabolites and the parent compound were isolated from urine; no qualitative differences were noted between the metabolites excreted after P0 vs IV administration. Supported by DAMD 17-86-C-6177. 14 :i°: 50875 8211 - 86
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,~ 324 IMMUNE MODULATION PRODUCED BY INTRATRACHEAL INSTILLATION OF.Q4LLIUM ARSENIDE. J A McCay, E E Sikorski, K L White. Jr, and A E Munson. Department of Pharmacology and-,Toxicoiogy. Medical College of Vir- ginia/ VCU, Ritthmo}id, VA Gallium arsenide (GaAs), used in the semiconductor industry, was administered to female B6C3F1 mice by intratracheal instillation at doses between 50 and 200 mg/kg in a 100 µi volume. The AFC respogse to SRBC was reduced by day 6 and remained suppressed through- out the 25 day period. Dose response studies on immune functions were conducted on day 15 post i.t. instiilation. The spleen IgM and 1gG AFC response to SRBC was suppressed dose dependently with i.v. immunization, but the IgM AFC response on the lung associated lymph nodes increased with i.t. immunization. The IgM AFC response of spleen cells to the T-independent antigen, DNP-Ficoll, ewas not altered. In vitro exposure of spleen cells to GaAs produced a suppression of the AFC response to the T- dependent antigen, SRBC, which was reversed with Interleukin 1. No modulation occurred using the T- independent antigens, DNP-Ficoll and LPS. The DHR re- sponse to KLH of mice exposed to GaAs was suppressed dose dependently, to a level of 20% of control. The MLR was suppressed dose dependently, to a level of 46% of control; however, the response to T- and B-cell mitogens was not suppressed. Fc-mediated phagocytosis of peri- toneal cells was increased, but phagocytosis of latex beads was decreased. These studies show that GaAs administered by the i.t. route modulates the immune response. (Supported by NIEHS Contract ES 55094). 325 DIFFERENTIAL EFFECTS OF CO- ADMINISTRATION OF AMINOACETONITRILE ON D-IMETHYLNITROSAMINE -- INDUCED jMMNUNOSUPPRESSION AND HEPATOTOX- ICIT~.' PRODUCED IN VIVO. H.G. Haggerty, L.H. Boise, S.D. Jordan, and M.P. _ Holsannle. Dept. of Pharmacology & Toxicology, Medical College of Virginia/VCU, Richmond, VA Aminoacetonitrile (AAN) is an inhibitor of the high affinity form of dimethylnitrosamine (DMN) demethylase. The reversal of the immunosuppression and hepatotoxicity induced by the in vivo exposure to DMN, by AAN was investigated. Female B6C3F1 mice were exposed to either 3 or 6 mg/kg of DMN (in saline) I.P. for 7 consecutive days. The animals were also treated (IP) 1 hour prior and 6 hours after the DMN exposure with either 10, 30, 100 or 300 mg/kg AAN or saline. Mice were sensitized with sRBC LV. on day 8. On day 12 body and organ weights were determined, serum chemistry and histopathology were evaluated, and day 4 IgM antibody response was measured (antibody forming cells were enumerated). Hepatotoxicity caused by DMN, as reflected by an increase in body weight ;n rributed to the production of ascites and a 266.7% increase in the SGPT levels, was reversed by doses of AAN as low as 10 mg/kg. Conversely, doses of AAN as high as 300 mg/kg were unable to reverse the suppression of the AFC response to sRBC produced by DMN. Currently, alterations in the distribution of reactive species to known target organs (liver) and immune organs (spleen) after subchronic exposure of (methyl-14C)DMN and AAN are being evaluated. (Supported by NIH grant ES03564 and Training grant ES07087 ). 326 ADHERENT AND NON-ADHERENT FRACTIONS OF SPLENO. CYTES ARE TARGETS OF BENZO(a)PYRENE (B(a)P] AND 7,12-pIMETHYLBENZANTHRACENE (DMBA) INDUCED SUPPRESSION OF THE HUMORALIMMUNE RESPONSE.K-L White. Jr , M C Parrrott, , and T T Kawabata. Depts. of Pharmacology and Toxicology, and Biostatistics. Medical ` College of VAIVCU, Richmond, VA. s; .1~(a)P or DMBA exposure lp.vivo _Mftvitro suppress,gs,_ ' the T-dependent antibody forming cell (AFC) response "to- : sRBC. Since T- and B-IympFtocytes and macrophages ar6 '_-- required in the generation of AFCs to sRBC, studies were undertaken to determine which cell type(s) was affected by these polycyclic aromatic hydrocarbons (PAH). Female B6C3F1 mice were administered B(a)P_ (200 mg/kg) or DMBA (10 mg/kg) s.c. daily for 7 de`'~s, and splenocytes were separated into adherent (macrophages; AD) and non- adherent (Sephadex G-10 purified; non-AD) fractions. Combinations of fractions from vehicle (VH; corn oil) or PAH dose groups were exposed to sRBC and evaluated after 5 days of culture. B(a)P and DMBA treatments resulted in a decreased AFC response in unfractionated splenocytes by 80-95%. This suppression was completely attenuated with 2-mercaptoethanol (25 µM) in the cultures. Cultures con- taining AD cells from PAH treated mice and non-AD cells from VH treated mice or AD cells from VH-treated mice and non-AD cells from PAH exposed mice had significantly decreased responses, as compared to cultures containing AD and non-AD splenocytes from VH treated mice. The greatest- suppression was observed when both splenocyte fractions were obtained from PAH treated mice. These results suggest that B(a)P and DMBA suppress the AFC response by affecting cell types present in both non-AD and AD frac- tions. (Supported by NIH grant ES03434). 327 82 ASSESSMENT OF FEMOGIABIN .1SOVARIENTS AS A FACTOR OF CHEMICAL-INDUCED FOLYCYTHEMIA IN WISTAR RATS. G M Henningsen, T D Eurelll, and L D Kol er2. NIOSH, DBBS, ABPB, BMRS, Cincinnati, OH; Dept. Vet. Bioscience, Univ. Illinois, Urbana, IL; and 2Coll. Vet. Med., Oregon St. Univ., Corvalis, OR. Erythrocytosis is the central feature of polycy- themia, a blood disease with diverse etiologies. Inbred Wistar,rats developed a 40% incidence of polycythemia.:following transplacental exposure to methylmercury and ethylnitrosourea. Mean onset was 8 weeks of age, hematocrits exceeded 60%, and death occurred within months. An etiologic algo- rithm was used to determine the probable underly- ing cause of the polycythemia. Physiologic or myeloprolific mechanisms were ruled out, whereas secondary polycythemia was diagnosed based on . elevations in red cell mass and serum erythro- poietin. A 10mm Hg mean left shift of the 02- RBC dissociation curve, with normal pH and levels of 2,3-diphosphoglycerate, indicated that RBCs or hemoglobin had an abnormally high affinity for 02. To determine if a high-affinity mutant hemoglobin existed, isoelectric focusing in an agarose gel matrix was performed on purified hem- oglobins. None of the 5 major hemoglobin bands significantly differed between affected rats and age/sex matched controls. High 02 affinity of the RBCs could still result from a mutated hemo- globin that is electrophoretically silent, or is insufficiently present in the borderline (hemato- crits - 56-59%) polycythemic rats tested. Further studies will assess severely polycythemic rats and alternate mechanisms for high 02 affinity.
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~ 308 THE DIRECT APPLICATION OF NMDA TO THE HIPPOCAMPUS PRODUCES MEMORY DEFICITS IN RATS. B.C Rogers, W R. Mundy, P Pediaditakis, and H A Tilson. Toxicology Curriculum, Univet*ity o or Carolina, Chapel- Hill, NC 27514 and NIEHS, _ Research Triadgle Park, NC 27709. Reductions in cortical and hippocampal N- methyl-D-aspartate (n-MDA) receptors have been reported in patients with senile dementia of the Alzheimer's type (SDAT) raising the possibility that glutaminergic hyperactivity ig -these areas may contribute to the progression of this di- sease. We report that direct application of n-MDA (1,2, or 4 mg) to the frontal cortex has no effect on overall motor activity, water maze acquisition, or cholineacetyltransferase (CHAT) activity despite producing reductions in corti- cal width at the site of application. The ef- fects of n-MDA on the hippocampus were studied by injecting rats bilaterally with either arti- ficial CSF, 2.5, 5.0, 10.0, or 20 ug/site n-MDA. Intrahippocampal n-MDA damaged pyramidal and to a lesser extent granule cells. n-MDA produced . a dose-related increase in motor activity each week of testing. In addition, n-MDA produced selective dose-related elevations in hippocampal CHAT activity. Animals receiving 10 ug/site injections were also significantly impaired in water maze acquisition. These results suggest that n-MDA may be useful in studying hippocampal glutaminergic dysfunction which may be associ- ated with SDAT. (B.C.R. was funded by ES07126). :!309 BEHAVIORALLY CONDITIONED SUPPRESSION OF MURINE T-DEPENDENT ANTIBODY RESPONSES. G E Schulze, R W Benson, M G Paule, D W Roberts, NCTR, Jefferson, AR. Sponsor: W Slikker, JR. The aversive and immunosuppressive effects of cyclophosphamide (CY), an unconditioned stimulus (UCS), were paired with a novel saccharine drinking solution (SACC), a conditioned stimulus (CS), in Balb/c mice. The objective was to determine the temporal relationship between re- exposure to the CS and immunization with sheep red blood cells (SRBCs), a T-dependent antigen, and Type III pneumococcal polysaccharide (S3), a T-independent antigen, on subsequent antibody responses. Reexposure to the CS or UCS occurred on days -4, -2, 0, +2, or +4 relative to immun- ization. Antibody responses were measured six days after immunization. A strong association between the CS and UCS developed producing flavor aversions evidenced by decreased SACC consumption on the second presentation relative to the first, or to unpaired controls. CY consistently suppressed both types of antibody responses. CS• presentation (i.e., SACC) had no effect on anti-S3 antibody response. However, the anti-SRBC response was significantly depressed following CS exposure. Exposure to the CS only on days -4, or +2 relative to immunization resulted in anti-SRBC suppression. These results support the premis that conditioned immune suppression is specific for T-dependent antigens and CS presentation relative to immunization is an important factor in eliciting this response. 78 310 RELATIONSHIP OF ffYPOTHALAMO-PITUITARY-ADRENAL 'r (HPA) ACTIVITY TO IMMUNE FUNCTION IN MICE EXPOSED TO B_ENZENE AND TOLUENE. G C Hsieh, R P Sharma. and R D R Parker. Toxicology Program, Utah State University, Logan, UT. Benzene and toluene via oral ingestion possess neurotoxic and immunotoxic effects. Male CD 7J mice were continuously fed drinking water- containing 0, 31, 166 and-4MJ{i' mg/L benzene and - 0, 17, 80 and 405 mg/L toluene, respectiv€Flyi. The hypothalamic norepinephrine (NE) and .•Ig-S-_-: metabolite vanillymandelic acid (VMA), plasma- adrenocorticotropic hormone (ACTH), and interleukin-2 (IL-2) were evaluated after 28 days of exposure. Serum corticosterone was measured before and at 2, A-1$= and 28 days after exposure. Concentrations of NE, VMA, and ACTH increased following both chemical treatments. Corticosterone levels were significantly higher in mice after 7 days (166 and 790 mg/L) and 28 days (790 mg/L) of benzene exposure. Toluene elevated corticosterone levels at 14 and 28 days at the highest dose. IL-2 production by ConA-stimulated mouse T- lymphocytes was suppressed in the two higher benzene dosed groups, while toluene decreased IL-2 at the 405 mg/L dose. Results suggest that both benzene and toluene ingestion activate HPA activity; elevation of corticosterone has been reported to inhibit IL-2 production and impair immunocompetence. (supported in part by U.S. Geological Survey Grant G-1255) 311 PERINATAL IMMUNOTOXICITY OF BENZENE. D Wierda, R W Leubke and R J Smialowicz. Dept. Pharmacology and Toxicology, West Virginia University Medical Center, Morgantown, WV and U S EPA, Research Triangle Park, NC., Development of the immune system involves a precise sequence of steps which begins early in. gestation. In mammals, the fetal liver is a major site of lymphopoiesis during embryonic develop- ment. We initiated'studies to examine whether benzene exposure of mouse fetuses in utero com- promises the development of fetal B lymphopoiesis. Pregnant Balb/c dams were injected i.p. with 200 mg/kg benzene twice a day beginning on day 12.5 of gestation. Fetal livers were obtained on day 1S.5Vof gestation and a dispersed suspension of hemopoietic cells was plated into liquid culture. Maturation of B lymphocyte precursors into pre-B cells and B cells was analysed pheno- typically over a two day culture period by immuno- fluorescence. Fetal liver cultures from benzene exposed dams contained 30-50% fewer pre-B and B cells than did control cultures. Separate cul- tures of adherent,fetal liver, stromal cells established from corresponding fetuses also con- tained 50% fewer cells. These results indicate that benzene does not impair fetal B cell matura- tion at discrete stages of B cell development but instead causes a nonselective reduction in all fetal cells involved in B cell development. (Supported by EPA Coop Agreement CR813583;•and does not represent EPA policy) 50875 8203 t
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276 PARTIC[E4MaCffPGE RE[ATICidSEIIPS D1ktING RfIE CLEAR- ANCE OF PARTICrM FfMJM ZHE ALVDOM Hg1CROPHAGE GCMARMYEW. B E I,ehnert,--R E 4bevs, Y E Vaide2, and R J SebringrLos.amos National Laboratory, Los Alamos, 'N,1 Little experimental attention has been given to examining the relationship(s) between particles retained in the alveolar space arntaartment and the lung's populaticn of alveolar macrophages (AM) during alveolar phase clearance. ~'In this study, we quantitatively characterized the distributicns of particles among lavageable AM over a 30 day period after the acute intratracheal instillation of - 3 mg of 1.9 in dia polystyrene microspheres into the lungs of rats. Information obtained for particles retained in the lavageable AM caapartment and particle-M distribution data were collectively examined using a simple, first order kinetic andel for AM removal from the lung. The results of our analyses suggest that a volume load of pprticles in a macrophage up to at least - 450 pm does not inhibit the nobilization of macrophages from the alveolar coanDartment. Additionally, the kinetic analyses of the particle-macrophage distributions suggest that macrophages that replenish those AM that are translocated fran the lung on a continual basis during alveolar, clearance are not and/or do not remain particle-free in the alveoli. 9:hi.s latter observation can be explained by: 1) thr influx of particle-bearing macrophages into the alveoli, or 2) the in situ proliferation of particle bearing AM, or 3) tf~e release of particles by AM and the subsequent phagocytosis of the particles by newly arrived cells, or 4) a canbi.nation of these possibilities. 277 SHORT-TERM INHALATION EXPOSURE TO BENZENE PRODUCES MYELODYSPLASTIC SYNDROME AND LEUKEMIA IN C57BL/6 MICE. H P Cathro, W S Stillman, W H Steinha en and R D Irons, CIIT, Research Triangle Park, NC. Benzene (BZ) is an established human leukemogen with chronic exposure known to result in myelodysplastic syndrome (MDS), myelogenous leukemia and lymphoma. However, little is known about its mechanism of action. Historically, studies on the mechanism of BZ toxicity have been hampered by the inability to reliably reproduce MDS in experimental animals following benzene exposure. The present study was undertaken to characterize the regimen-dose dependence of BZ myelotoxicity in male C57BL/6 mice and was not designed to quantitate tumor incidence. Nevertheless, NIDS was encountered in 12 of 20 mice exposed 100 or 300 ppm (3-6h/d, 5d/wk) for 12 weeks and held post-exposure for an additional 6 mo. Lesions constituted a continuous spectrum ranging from inversion of the lymphoid/myeloid cell ratio in peripheral blood and moderate myeloid hyperplasia in spleen to marked myelodysplasia, preleukemia and leukemia. Neoplastic lesions included T cell lymphoma and early myeloid leukemias as defined by histopathology, surface marker analysis and growth requirements in culture. These findings suggest that, for BZ, regimen as well as dose may be important considerations for modeling carcinogenesis. 70 t ts 278 LIVER CYTOSOLIC METABOLISM OF IRANS,TRANS-MUCON- "' ALDEHYDE TO TRANS,TRANS-MUCONIC ACID. T A Kirley, B D Goldstein, and G Witz. Joint Graduate Program in Toxicology, UMDNJ-Robert Wood Johnson Medical School/Rutgers University, Piscataway, NJ. .r Trans,trans-muconaldehyde (MUC) has been show'fF-to be a hematotoxic metabotil„gf benzene in mice.. In the present study we examined the in vilro- me- tabolism of MUC by piice and rat liver cytosa .:to trans,trans-muconic acid (MA), a urinary metafia= lite in mice and rats treated with benzene. MUC incubated with liver cytosol from male DBA/2 mice and Sprague-Dawley rats in the presence of NAD+ and pyrazole, an_atcohol debydro,genase inhibitor, resulted in.NADH productiod`as monitored at 340 nm, suggesting the formation oeMA. In mouse li- ver cytosol metabolism of MUC gave a biphasic Lineweaver-Burke plot, with Km's of 0.07 and 0.01 mM and corresponding Vmax values of 13 and 10 nmol min-lmg-1. A Km of 3.3 mM and Vmax of 80 nmol min-lmg-1 was obtained for metabolism of MUC by rat cytosol. HPLC analysis of ether extracts of the cytosolic metabolism mixtures indicated the formation of MA by showing a peak at the re- tention time of authentic MA. Omission of MUC, NAD+, or use of boiled_cytosol resulted in no peak on the HPLC chromatogram corresponding to MA. Preliminary data indicate that intraperito- neal injection of 2 mg/kg MUC into mice results in the formation of MA as detected by HPLC analy- sis of urine extracts. The data suggest that MUC is metabolized to MA by cytosolic aldehyde dehy- drogenases. Supported by NIH grant # ES02558. 279 METABOLISM OF 3H-BENZENE IN F344/N RATS AND B6C3F1 MICE: SPECIES A~,ND DOSE EFfECT. P~ Sabourin, L S Birnbaum, G Lucier and R F Henderson. Lovelace Inhalation Toxicology Research Institute, Albuquerque, NM; *NIEHS, Research Triangle Park, NC. Benzene causes aplastic anemia and leukemia in humans. Studies by the NTP. indicate that B6C3F1 mice were more susceptible than F344 rats to benzene toxicity. We have initiated studies to determine the effect of dose on the metabolism of benzene in the two species. Wa- ter-soTuble benzene metabolites were measured .in blood, urine, liver, lung and bone marrow during and following a 6 hr exposure to 5, 50 and 600 ppm 3H-benzene. The area under the curve (AUC) of metabolite concentration vs. time was determined. In both rats and mice, the AUC of hydroquinone glucuronide (HQG) and muconic acid (MU) in blood, lung and liver was not linear with vapor concentrations above 50 ppm. Between 50 ppm and 600 ppm metabolism shifted to formation of phenylglucuronide and pre-phenylmercapturic acid. In all tissues ana- lyzed, mice had a higher concentration of HQG and MU, markers for pathways of metabolism lead- ing to the toxic benzene metabolites, benzoqui- none and muconaldehyde. In rats, HQG was bare- ly or not detectable. These results may, in part, explain why mice are more susceptible to the toxic effects of benzene than rats. (Re- search supported by NIEHS through IAA ES-20092 under U.S. DOE Contract No. DE-AC04-76EV01013.) 50875 8195
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1300 EFFECT OF THE ANTICHOLINESTERASE PARAOXON ON SHUTTLE AVOIDANCE BEHAVIOR AND BRAIN MUSCARINIC RECEPTORS. J E Chambers and H W Chambers. Depts. of Biol. Sci. and Entomology, Miss. State Univ., Miss. Stqte, MS 39762. The effect . of acute sub-lethal and antidoted/lethal doses of paraoxon were studied on the retention of avoidance behavior and concurrently on 3H-quinuclidinyl benzilate (QNB) binding in the corpus striatum. Aduat~male rats were trained to avoid a shock in response to a buzzer in a shuttle box. Following stabilization, rats were injected i.p. with the toxicant and/or antidote (atropine). Their performance was mointored at 1 and 2 days after treatment. On day 2, membranes from the corpus striatum were obtained for 3H-QNB binding studies. About 90% inhibition of brain acetylcholinesterase was observed on the day of injection with both doses of paraoxon, with still about 50% inhibition remaining on day 2. There were some decreases in avoidance percentages and increases in escape percentages on day 1, with recovery by day 2. Avoidance latencies were unaffected and inconsistent effects on escape latencies were seen on day 1. There were no statistical differences in affinities or densities of muscarinic receptors. Despite the extensive and persistent inhibition caused by an acute exposure to a high dose .of paraoxon, relatively minor effects were observed in performance and no effects on receptors. (Supported by EPA R-811295). ;30; . REVERSIBILITY AND TOLERANCE TO TRICRESYL PHOS- PHATE-INDUCED NEUROTOXIC EFFECTS IN F344 RATS. G B Freeman, R Irwin * R Trejo, M HeJtmancik, M-- Ryan, and A C Peters. Battelle Columbus Div- : ision, Columbus, OH andl4IEHS, Research Triangle Park, NC. After 13 weeks of dosing with tricresyl phosphate (CAS No. 1330-78-5), in feed, hindlimb grip strength decreased "in male rats (600 and 300 ppm), but not in female rats. Serum cholinester- ase was reduced significantly"relative to control in the 600 (-27%, -49%) and 300.(-18%, -35%) ppm male and female dose groups, respectively. Animals in the 600 ppm dose groups were fed tricresyl phosphate-dosed feed through week 23, after which they were maintained on blank (un- dosed) feed. At the 39-week interim termination, all clinical pathology, organ weight and hindlimb grip strength data for the 600 ppm male and -female dose groups were statistically similar to those of controls. Therefore, all previously seen compound-related effects were apparently reversed when 23 weeks of exposure to'tricresyl phosphate in feed at a concentration of 600 ppm was followed by 16 weeks of undosed feed. Hind- limb grip strength scores of male rats in the 300 ppm group that remained on the tricresyl phosphate were no longer different from control animals after 39 weeks, although serum cholin- esterase was still reduced significantly relative to control (-19%). This suggests a development of tolerance to the neurobehavioral effects of tricresyl phosphate in the diet as well. (Sup- ported by Contract No. N01-ES-45050 from NTP). ~ 302 THE >2UMMCK OF cA1zeox rXMXmE wrM ETHANOL, CHECRPRafZINE, PE3fI'OBARBITAL OR d- AMAMTAMINE CK FIXED-RATIO PERFOFMANCE IN 7iiE NX7IJSE. J S Rnisely, D C Rees, R L Balster, and L J Thomas. Dept of pharmacology and Zhxicology, Medical College of Virginia, Ricrmrond, VA. Zhe behavioral effects of variws dosage .~=binations of CO with '"''LEFIdnol (EtCai), rhlorprcnazine (CPz), pentobarbital (PB) or d amphetamine (d-ANVHr'were investigated in mice trained to lever press nxier a fixed- ratio 32 schedule of water reinforoement. Following a dose-response curve determination for each drug, animals were then"administered intraperitoneal Cn (3.75, 7„~, ±5 and 30 ml/kg) alone and in c~nbi~tion with the ED50 of EtOH (1.1 g/kg), CPZ (2.2 mg/kg), PB (23 mg/kg) or d-ANPf3 (1.4 mg/kg). Only the highest dose of OD (30 ml/kg), when given alone, produced significant decreases in rates of responding in some animals. Haaever, ' when 00 was given in conbinatioai with EtOH, a significant interaction was observed at doses of 00 as low as 7.5 ml/kg (associated oOHb levels of <20%). Also, response-rate suppression following 30 mljkg CD was enhanced when administered with CPZ and d-AMPfi. Frhen PB was given with Cb, no significant interaction was obtained. (Supported by grants ES-03809 and ES-07087) 303 PROCONVULSIVE ACTIVITY OF gUINOLONE ANTIBIOTICS 30~ IN AN ANIMAL MODEL. P D Williams and D R Helton, " Lilly Research Laboratories, Toxicology Division,'.`" Greenfield, IN. The side,effect profile of quinolone antibiotics =. in man includes CNS disturbances such as dizzi=- ess, vertigo, and convulsions. Although the°" convulsive liability - has been suggested.to in-.. :_ volve interaction with GABA receptors in the CNS, ' ` no animal model has been described to evaluate"or<< confirm the mechanism of this effect. The pro-;.- convulsive behavior of the quinolones, naladixic,' (NAL) and oxolinic (OXO) acid, were examined in- male CF1 mice utilizing pentylenetetrazol, picrotoxin, strychnine and electroshock as con- vulsants. Following single oral doses of 10, 30, : 100 mg/kg NAL or OXO, the threshold for penty-lenetetrazol, picrotoxin, and strychnine-induced convulsions was not altered; however, electro- • shock-induced seizures were potentiated in a:'~` dose-dependent fashion. OXO and NAL (100 mg/kg) produced a 60 and 50% incidence of convulsions, respectively, at an ED10 dose of electroshock (6.9 mA). These resul-ts suggest that 1) the mechanism of convulsive liability of quinolones may not involve GABA antagonism (as tested by picrotoxin) but may involve pathways related to electroshock seizure activity and 2) the electroshock model may provide a useful tool " for evaluating the convulsive liability of new quinolone antibiotics. -76 50875 8201 A
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STUDIES ON DAPSONE-N-HYDROXYLAMINE (DDS-NOH) IN- DUCED MORPHOLOGICAL CHANGES IN RAT ERYTHROCYTES. RA Budinsky, JV Simson, V Price, a~d DJ Jollow, Depts Pharmacol & Anatomy, Med U SC, Chas., SC. Previous studies have indicated that the hemoly- tic activity of dapsone in rats results from the action of its N-hydroxymetabolites (DDS-NOH and monoacetyl DDS-NOH) on the red cells. Incubation of rat red cells in vitro with hemotoxic concen- trations of DDS-NOH have been observed t9.Anduce striking transformation in cell morphology DDS- NOH treated red cells developed echinocyte mor- phology (types I,II,and III) with significant numbers of severely deformed cells designated as "Jack-shaped". The changes were observed under light microscopy, SEM and TEM. Few, if any, Heinz bodies were seen under TEM. Phenylhydra- zine used as a positive control induced a spher- oechinocytic morphology with numerous Heinz bodies. Quantitation of the morphological trans- formation using a computer/microscope (IBAS) indicated an EC50 of about 120uM, which is simi- lar to those of GSH (depletion and commitment) to splenic sequestration 51Cr-T50, 180O. Time studies indicated linearity with a maximal re- sponse occurring by 20 min. This response was significantly faster than was commitment of the cells to sequestration (51Cr-T50). Cysteamine prevented but did not reverse the shape changes. It is suggested that the morphological transfor- mations reflects intracellular changes important in the premature sequestration of the cell by the spleen. (Supported by HL30038). 210 BUTYLATED-HYDROXYTOLUENE (BHT) INDUCED INCREASES IN NAD(P)H-QUINONE-REDUCTASE(QR) ACTIVITY IN MOUSE LUNG AND LUNG CELLS. D Siegel,A Malkinson and D Ross. Molecular and Environmental Toxicol- ogy Program, School of Pharmacy, University of Colorado, Boulder, C0. BHT, a widely used food additive, can modulate the toxic activity of several xenobiotics and the pulmonary toxicity of BHT itself is reduced after repeated BHT administration. BHT forms a quinone during metabolism and its protective actions may be related to increased activity of QR - a protective enzyme against quinone toxicity. More quinone is formed from BHT in lung microsomes isolated from A/J& than from C57&mice so QR activity in these two strains was examined. Lung QR activity was increased .in both strains by treatment of mice with BHT and was observed as early as 6h*post-BHT (300mg /Kg ip) in C57 mice. Dose-response data indicated that QR activity in A/J mice could be increased by BHT (100mg/Kg ip) whereas higher doses were needed in C57 mice. Clara cells and lavaged macrophages isolated from mouse lung and a Clara cell line had little basal or BHT-induced QR activity whereas alveolar type II cell lines exhibited high QR activity. These data show that QR activity may be localized to certain cell types and increased activity may play a role in BHT -mediated protection against lung toxicity. Supported by UROP, Univ. Colorado and the American Institute for Cancer Research. 53 211 OXIDATION OF CATECHOL BY HORSERADISH PEROXIDASE AND HUMAN LEUKOCYTE PEROXIDASE. REACTIONS OF o- BENZOSEMIQUINONE (BSQ) AND o-BENZOQUINONE (BQ). V V Subrahmanyam, A Sadler and D Ross. Molecular and Environmental Toxicology Program, School of Phamacy, University of Colorado, Boulder, CO. Hydrogen peroxide dependent oxidation of catgchol by horseradish peroxidavr-and peroxi- dases present in human leukocytes resulted in BQ production, which was characterized as its bro- mothiophenol adduct. BQ-glutathione conjugates were formed during peroxidatic oxidation of catechol in the presence of glutathione (GSH). As much as 80% of catechol removed during pero- xidatic oxidation could be recov$*'ed Vs mono- and di-GSH conj ugates of BQ. GSH had ng inhibi- tory effect on the removal of catechol during peroxidatic oxidation. In the presence of M Fg or Zn++, which slow the rate of BSQ disproport- ionation, GSH was found to inhibit catechol removal. This suggests that in the absence of stabilizing metal, reduction of BSQ by GSH can- not compete with other rapid reactions of the radical such as disproportionation. No inter- action of BSQ with oxygen could be detected even in the presence of stabilizing metals or super- oxide dismutase : hich inhibits the reverse reaction of the SQ + 02 ~ Q + OZ equilibrium. These data show that generation of thiol conju- gates of BQ can be used as probes of peroxidatic oxidation of catechol. Supported by ES04112. 212 EVALUATION OF 3-METHYL-2-BEIQZOTHIAZOLIHONE HYDRAZONE HYDROCHLORIDE (MBTH) FOR ACUTE T_OXICITY, PRIMARY IRRITANCY, AND BOTAGENICITY. R C Myers, R S Slesinski, and B Ballantvne, Bushy Run Research Center, Union Carbide Corporation, Export, PA MBTH, used extensively in analytical laborator- ies, was evaluated for potential handling hazards. It'had moderately high acute oral . toxicity in both the rabbit (LD50s of 177 and 268 mg/kg for males and females, respectively) and the rat (LD50s of 308 and 149 mg/kg). By 24-hr occluded cutaneous contact, MBTH had low acute toxicity in the rat (LD50 > 16 g/kg), but was more toxic in the rabbit (16 g/kg killed 1/5 males; LD50=12.3 g/kg in females). Cutaneous doses Z 4 g/kg and oral doses Z 125 mg/kg elic- ited convulsions in the rabbit. No cutaneous inflammation was evident from 4 hr of occluded contact with 0.5 g,of moistened MBTH; the 24-hr test produced erythema, edema, and instances of necrosis. MBTH caused mild to moderate eye irritation depending on the amount dosed. No adverse effects were apparent among male or female rats enclosed for 6 hr in an inhalation chamber containing solid MBTH (equilibrated overnight). MBTH was mutagenic in the Ames assay, particularly in the absence of metabolic activation. These studies demonstrate a need for skin and eye protection, as well as avoidance off ingestion, during handling of MBTH. Copyright O 1987 Union Carbide Corporation 50875 8178 1 i
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380 A STUDY OF THE JOINT HEPATOTOXIC ACTION OF CARBON TETRACHLORIDE (CC1 ) AND CHLOROFORM (CHC1 ) FOL- LOWING SIMULTANEOU~ GAVAGE ADMINISTRATION 3IN THE RAT. T M O'Hara, R H Granger;`L W Condie* and J F Borzelleca. a, Depts. of Pharmacology/Toxi- cology and Pathdlogy, Medical College of VA, Richmond, VA and-*U.S.E.P.A., Cincinnati, OH. The joint action of CC14 and CHC13 following simultaneous oral administration has been inves- tigated in the chronically arteria}-cannulated male CD rat. CCL , CHC1 or their mikture were administered at dAes of ~, 100, 250'and 400 mg/ kg in a 5% Emulphor vehicle, completing a 4x4 grid design. Hepatotoxicity was evaluated as a measure of AST, ALT and SDH plasma enzyme activ- ity at 0, 3, 6, 12, 24, 36, 48 and 72 hours post gavage. Additional blood samples were taken between 0 and 6 hours post gavage for CC14 and CHC13 pharmacokinetic analysis. Response data were analyzed for possible interactions using response surface methods (RSM). Hepatotoxicity increased in a dose-dependent manner for both CC1 and CHC1 when administered alone. Inter- actions betweh CCl and CHC1 appeared biphasic with an initial "Af ra-additive" response at 6 hours followed by a"supra-additive" response at 24, 36 and 48 hours. Data indicate alterations in the pharmacokinetics of CC14 when co-admin- istered with CHC13. (Supported by EPA Cooperative Agreement 812558.) ' 381 THE INFLUENCE OF STRUCTURAL ANALOGUES OF CARBON _ TETRACHLORIDE (CC14) ON HEPATOCYTE FUNCTIONS IN VITRO. J B Coleman, L W. Condie*, J F Borzelleca and R G Lamb. Department of Pharma- cology/Toxicology, Medical College of VA, Richmond, VA and *USEPA, Cincinnati, OH Isolated and cultured hepatocytes were incubated (15 min to 18 h) with various concentrations (0.1 to 1.0 mM) of bromotrichloromethane (CBrC13), CC1 , flurotrichloromethane (CFC1 ) and chloro- forg (CHC1 ). Phospholipase C (PL2) was rapidly rapidly Ativated (translocation) by membrane _(phospholipid)-associated chemical metabolites and preceeded chemical-dependent alterations in endoplasmic reticulum (ER), mitochondria and plasma membrane functions. The observed rank order of PLC activation and alterations in cellu- lar functions: CBrC13 > CC1 > CHC1 > CFC1 , corresponds to the reportW hepatoto3Xicity o? these agents in vivo. Therefore, the rapidly activated, PLCrtiediated degradation of membrane ' phospholipids may: 1) represent the event that injures membranes, such as the ER; 2) explain in ' part the alterations in membrane functions asso- ciated with reactive metabolite-mediated hepato- cyte injury. (Supported by NIH AM 31115 and EPA Cooperative Agreement 812558.) 382 383 96 METABOLISM AND DISPOSITION OF LOW DOSE CCL~~IN PARTIALLY HEPATECTOMIZED, CHLORDECONE PRETREAT- ED RATS. R A Young, F Siddiqui,< and H M MEHENDALE Univ. Miss. Med. Ctr., Jackson, MS Previous work in our lab has indicated sup- pressed hepatocellular regeneration to be a potential mechanism for the potentiation of CC14 hepatotoxicity by chlordecone (CD). Addi- tional studies demonstrated thei"vers induced :64to active regeneration by partial hepatectomy.aa»- (PH) provided protectior~,.against this amplified ~ toxicity. Since bioactivation of CC14 is ne- cessary for expression of toxic effects, it was imperative to determine if CC14 metabolism and disposition was altered in PH rats. Sham ope- rated (SH) and PH (male SpragM t Dawley, 150- 175g) were given dietary CD ('f0 ppm) for 15 days. On day 15 the rats were surgically mani- pulated (SH or PH) and on day 16 challenged with a singR dose of CC14 (100 ul/kg), i.p.) containing CC14 20uCi). Over a 6 hour pe- riod, there was no difference betweeA the SH and PH rats iY4 recovery of exhaled CC14 or formation olf4 CO2 . As f~ticipated, hepatic content of CC14 derived C(per g t#asue) was greater in PH rats14 Values for free CC14 and covalently bound C were similar for livers from SH and PH rats. The data suggest that metabolism and disposition of CC14 are not altered by PH, thereby serving as a valid model for studying the CD-CC14 interaction in an actively regenerating liver. (Supported by EPA, R-811072 and CR814053010.) TIME-COURSE OF LIVER INJURY AND 3H-THYMIDINE INCORPORATION IN CHLORDECONE-POTENTIATED CHC1? HEPATOTOXICITY_ K R Purushotham and H M Mehendale. Department of Pharmacology and Toxi- cology, University of Mississippi Medical Cen- ter, Jackson, MS. We have shown that chlordecone (CD)-potentiated CHC13 hepatotoxicity leads to increased lethal- ity in male S.W. mice. We intended to study tJhe time-coursje of such hepatotoxicity and H-thymidine 'A H-T) incorporation into,nuclear DNA in mice fed CD (10 ppm), mirex (M, 10 ppm) or phenobarbital (PB, 225 ppm) diets for 15 days. The mice received 0.1 ml CHC13/kg in corn oil. Liver damage was assessed by plasma ALT, AST and histology at 4, 12, 24, 36, 48, 72 and 96 hr`after CHC13. Elevation in plasma enzymes and the hepatocellular damage.were seen only in CD pretreated mice. The enzyme eleva- tions were maximum at 24 hr. The centrilobular and midzonal necrosis was seen from 12 hr on- wards. The pretreatments did not show any chan_*e in hepatic nuclear DNA levels but alter- ed H-T incorporation. Nqne of the dietary treatments alone altered H-T inc:?rporation. Among the combination treatments, H-T incor- poration was maximal and biphasic (36 and 72 hr) with CD. M showed a single peak at 72 hr and PB showed a progressive but least increase. These results indicate the impact of dietary CD + CHC13 on the hfpatocellular damage and the incorporation of H-T into nuclear DNA. (Sup- ported by EPA R-811072.) 50815 8221
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: 237 S[RlMARY OF LITERATURE REVIEW ON ME_TAT c IN HIItSAN URINE AS A BIOLOGICAL INDICATOR OF E%POSURE. P Y Lu, J Stengel*, S M Hubner, B C Pal, and R A Faust, qgk Ridge National Laboratory**, - Oak Ridge: TN ' Potential worker exposure to metals in fumes or dusts by ingestion, skin contact, or inhalation can take place during industrial processes using metals and'alloys. One useful approach to monitor such exposure is through the assay of the metals in the urine of the exposed workers. However, exposure data to obtain average baseline urine levels of nonexposed (background) and exposed (occupationall and medical treatment) populations are scattered or scarcely available. A comprehen- sive literature search of 20 metals excreted in human urine was perfoimed on TOXLINE and HEDLINE. From the limited information available we were able to compile the following data for,(1) non- exposed population: uranium, thorium, iridium, tantalum, and zirconium, <_0.1 pg/L; platinum, bismuth, < 1 pg/L; lead and mercury, < 10 µg/L; (2) occupationally exposed population: thallium, platinum, and zirconium, :5 5 pg/L; mercury, < 50 pg/L; lead, uranium, and selenium, .5 100 pg/L; titanium, -5 3000 pg/L; and (3) population under medical treatment: gold and bismuth, <_ 20 µgjL; mercury, < 100 pg/L; and platinum, < 20,000 µg/L. *Lawrence Livermore National Iaboratory, Livermore, CA. **Operated by Martin Marietta Energy Systems, Inc., for the U.S. Department of,Energy under Contract No. DE-AC05-840R21400. ; 238 . HEALTH EFFECTS ASSOCIATED WITH B_ROMINE AND BROMINE COMPOUNDS. F M Martin, Oak Ridge National Laboratory*, Oak Ridge, TN. Sponsor: P Y Lu. The purpose of the overview is to determine whether or not evidence exists which suggests that bromine and its compounds exert effects on human health at concentrations commonly encountered by the general public under ambient air exposure conditions. Acute and chronic health effects are addr9ssed, including systemic toxicity, genotox_iti`ity, carcinogeni- city, and reproductive and developmental effects. Other topics discussed are chemical and physical properties, uses, production, sources, environmental fate, ambient levels, pharmacokinetics, and regulations and stan- dards. Bromine is known to be highly toxic via oral and inhalation routes. Chronic exposure leads to fatigue, tremors, delirium, and schizophrenia in man. Several compounds, such as methyl bromide, ethylene dibromide, and developing countries such as Mozambique where liver cancer is more common, infection with di bromochl oromethane, have given positive results for genotoxicity, and some like dibromochioropropane, a banned pesticide, have shown carcinogenic effects and/or adverse effects on reproduction. The ambient con- centrations reported for bromine have been below the O§HA maximum time-weighted average of hepatitis B virus is a compounding factor in individuals also exposed to aflatoxin. Health hazards associated with fungicides, which are limited in their effectiveness on Aspergillus are evaluated. Many of these compounds are still in the testing stage. 0.1 ppm. Operated by Martin Marietta Energy Systems; Inc., for the U.S. Department of Energy under Contract No. DE-AC05- 84021400. m ~ ~ 60 Ln co N ov cn 239 IMMUNOTOXICITY AND RISK ASSESSMENT OF CONTAMa.~ NANTS IN DRINKING WATER. S Sriharan, Selma ,.:,::: University, Se1ma,AL and E V Ohanian, OfficeLLaf Drinking Water, EPA, Washington, . . '! The process of risk assessment is being recognized as essential to every organized hu~q'`•~ society. The Safe Drinking"Vater Act Amend%&~t' ° of 1986 require the U.S. EPA to promulgate ~.-. national drinking-water regulations which spec risk of adverse human health effects from exposure to environmental contaminants. Quanti- tative risk assessment usually involves perforQ• ing two separate evaluations.,~ (14.a hazard assessment to determine poterYtial'toxicity, and (2) an exposure assessment. These separate assessments are combined to derive an estimate of risk to the exposed human population. This paper evaluates the incorporation of immunotoxicity data into EPA's health research aimed at improving risk assessment methodologiea. The EPA's Office of Drinking Water (ODW) has developed Health Advisories (HAS) that describe the concentrations of contaminants in drinking water at which adverse effects would not be anticipated to occur following 1-day, 10-day, longer term or lifetime exposure. Studies carried on tetracholoro-p-dioxin (TCDD), a potent immunosuppressant are discussed here for risk assessment and deriving ten-day Health Advisory number. 240 HAZARD EVALUATION OF AFLATOXIN IN FOOD. P E Berteau and A M Fan. Hazard Evaluation Section, Calif Dept Health Services, Berkeley, CA. This study evaluates the basis of the action parative health hazards associated with aflatoxinn level for aflatoxin B1 in peanuts and the com- ingestion and the fungicides used to control duces liver cancer in many species of experimen- tal animals, but epidemiological data are dif- ficult to interpret. Liver cancer is relatively.; molds, such as Aspergillus flavus, which produce aflatoxin. The action level of 20 ppb was es- . tablished in 1969, based on the analytical detection limit, which, combined with toxico- logical data and the decision to permit peanuts and peanut products to remain on the market, serves as the basis for the Food and Drug Admin- istration's regulatory action. Aflatoxin in- rare in the United States particularly in the rural Southeast where peanuts are grown. In
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r;T 384 STATUS OF SOME ENZYMES INVOLVED IN POLYAMINE METABOLISM DURING CHLORDECONE (CD) -INDUCED P0- TENTIATION OF CC14 HEPATOTOXICITSF:- S B Rao, R A Young and H M Mehendale, University of Missis sippi Medical Center, Department of Pharmacology and Toxicology, Jackson, MS. Ornithine decarboxylase (ODC), S-adenosyl meth- ionine decarboxylase (SAMD), and spermidine acetyl transferase (SAT) are altered during CC1 -induced liver injury. We inve~bigated polyamine-related enzymes associated with CD+ CC1 hepatotoxicity. Rats fed CD (10ppm, 15 days) were injected with 0.1 ml CC1 /kg (low dose) and compared with high dose (2.5k ml/kg) of CC14 without CD pre-tf~atment. Liver ODC and SAMD were measured as CO2 released from re14 spective substrates and SAT was measured as C- acetylation of spermidine. While ODC but not SAMD showed a steady rise from 6 to 24-hr post- treatment with CD+CC14 (low dose), CC14 (high dose) showed greater increase in both enzymes. SAT showed elevation in 6-hr and decreased in 24-hr for both treatments but CC14 (high dose) showed significant increase in 6-hr., CC14 (low dose) alone showed insignificant change of ODC and SAND and minor elevation for SAT at 6 and 24-hr. The enzyme status indicates that rats treated with CD + CC14 show lesser induction of enzyme synthesis compared to high dose of CC14 alone. Substantial changes in.polyamines could be a factor for liver regeneration, a determ- inant in animal recovery or death. (Supported by EPA R-811072 and CR 814053010) 386 MONOCHLOROACETATE (MCA) INCREASES CHLOROFORM (CHCL3) AND VINYLIDENE CHLORIDE (VDC) SOXICITIES. M E Davis an3 W 0 Berndt. West Virginia Univ e ica enter, Morgantown, WV and Univ of Nebraska Medical Center, Omaha, NE Mono-, di- and trichloroacetate (DCA and TCA) are Z all by-products of water chlorination and DCA and ;~ TCA have been shown to increase, icity of : CHGd3. MCA has effects that are~ferent from ~ either DCA or TCA. The interactions between MCA_. ;. and either CHC13 or VDC; a contaminant of ground water, were studied. MCA was administered by oral gavage of a neutralized solution for 3 doses. CHC13 (0.75 ml/kg) or VDC (200 mg/kg) was injected ip 1 hr after the last MCQ•do$~e. CHC13 given to saline controls had no ef~ects, in either male or female rats, on either "GPT or BUN. MCA (188 mg/kg) increased GPT in male rats (954 + 275 vs 21 + 6 SF u/ml) but did not affect BUN (20.2 + 2.7 vs 15.4 + 0.7 mg/dl). In female rats, NX;A (94 mg/kg)'increased both GPT (253+120 vs 19+1) and BUN (50+12 vs 25+1). HepatotoxTcity was seen 24 hrs after VDC in T/5 control males and 4/5 MCA males. GPT was elevated in none of the controls and 3/6 of MCA treated females. MCA decreases hepatic glutathione by 80% in males and this likely accounts for the increases of CHC13 and VDC hepatotoxicity. (Supported by Air Force Contract # F49620-86-C-0096. 385 BIOCHEMICAL EFFECTS OF THREE CARCINOGENIC CHLORINATED METHANES IN RAT LIVER. K T Kitchin end J L Brown. Health Effects Research Labora- tory, US EPA, Research Triangle Park, NC The mechanism (initiation or promotion) by which three chlorinated methanes cause rodent liver tumors was investigated. Varying oral doses of either carbon tetrachloride, chloroform or methylene chloride were given to adult female rats both 21 and 4 hours before sacrifice. Then DNA damage, ornithine decarboxylase activity (ODC), cytochrome P-450 and glutathione content were assayed in liver. DNA damage and ODC induction are considered markers for carcino- genic initiation and promotion, respectively. With or without increased serum alanine amino- transferase (SGPT) activity, carbon tetrachlo- ride increased rat hepatic ODC and decreased cytochrome P-450 content. Chloroform increased hepatic ODC with minimal or no elevation in SGPT activity. Methylene chloride (1275 mg/kg) caused a small, but significant amount of hepatic DNA damage. When these three compounds are compared on either an equimolar or equitoxic (1/5 LD50) basis, the order of potency of induc- ing hepatic ODC or increasing serum alanine aminotransferase activity was carbon tetrachlo- ride > chloroform > methylene chloride. As the degree of chlorination increases in this series, the compound is more likely to promote, rather than initiate, the carcinogenic process. This is an abstract of a proposed presentation and does not necessarily reflect EPA policy. 387 97 DIFFERENTIAL POTENCY OF POLYCHLORINATED BIPHENYL CONGENERS ON CYTOCHROMES P-450 AND ACCUMULATION OF U_ROPORPHYRIN IN CHICK EMBRYO HEPATOCYTES. S I Shedlofsky, L E Rodman, L W Robertson, and A T Swim. VA Hosp. & Grad. Ctr. for Toxicology, University of Kentucky, Lexington KY. To assess effects of PCB structure on induction of hepatic cytochromes P-450 and accumulation of uroporphyrin(URO), cultured chick embryo hepato- cytes were tre4ted for 18h with pure synthetic PCB congeners as shown below (0.001 - 10pM). Cyt P-450 concentrations, benzphetamine demeth- ylase (BPDM), ethoxyresorufin deethylase (EROD), and porphyrins (after 150uIi aminolevulinate for the final 3h) were assayed. For each PCB except 245,2'4'5', Cyt P-450 and EROD peaked and then declined and BPDM was increased only minimally (<45X). 245,2'4'5' increased Cyt P-450 and BPDM 3 to 4-fold with plateau 2z50pM, but.EROD peaked and fell. For each PCB congener, the DOSE (uM) at which MAXIMAL INDUCTION occurred for CYT P450 and for EROD ACTIVITY and the DOSE AT WHICH URO ACCUMULATED WITH NO PROTOPORPHYRIN ver_ as follows, respectively: 34.3'4' (0.34, 0.034, & 0. 34 ); 345,3141 ( 0. 01, 0. 0034, & 0. 0034 ); 345.2'3' (1.0, 1.0, & 1.0); 345.2'3'4' (0.1, 0.034, & 0. 1); 4. 2' 3' 4' 5' ( 0. 34, 0. 34, & 0. 34 ); 245. 2' 4' 5' ( 250. 0, 1. 0, & 3.4). We conclude that the PCB congener 345,3'4'vas the .ost potent of this series with 345,2'3'4'>34,3'4'=4,2'3'4'5'> 345,2'3'. 245,2'4'S'mas a°.ixed" inducer, but also caused URO accumulation at higher doses. N N
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400 THE INTERACTION OF HALOACETUNITRILES (HAN) WITH GLUTATHIONE AND GLUTATHIUNE-S-TRANSFERASE (GSH- T). E L C Lin and C W Guion. U.S. EPA, HERL, Cincinnati, OH. Sponsor: T V Reddy _ . 1~s GSH was incubated with HAN including chloroace- tonitrile (P1CAN), dichloroacetonitrile (DCAN), trichloroacetonitrile (TCAN), dibromoacetonitrile (DBAN) and bromochloroacetonitrile (BCAN) under various conditions. HAN reacted with GSH in the absence of GSH-t and the reactiwities were OBAN > BCAN >>TCAN > MCAN. DCAN failed to react with GSH either in the presence or absence of GSH-t. In the presence of cytosol the depletion of GSH was increased by MCAN and decreased slightly by TCAN and DBAN. Addition of microsomes to the incubation mixture increased the depletion of GSH by MCAN and DCAN and decreased that by TCAN and OBAN, suggesting metabolism of HAN to metabolites which reacted either more or less readily with GSH than the HAN itself. In the presence of BSA, TCAN depleted less GSH while UBAN showed no dif- ference. All five HAN inhibited GSH-t in vitro with the activities of TCAN >.DBAN > BCAN > MCAN > DCAN. GSH concentration and GSH-t activity were measured 1, 3 and 18 hr following orally administered HAN to rats. GSH concentration was depleted by MCAN at 1 and 3 hr, and by dihalogen- ated acetonitriles (DHAN) at 1 hr. GSH was ele- vated by DHAN at 18 hr and by TCAN at 3 and 18 hr. Only DBAN inhibited GSH-t significantly. The tox- icity of HAN may result from their direct inter- action with cellular macromolecules. (Abstract does not necessarily reflect EPA policy). 401 DETERAlINATION OF THE SUBCHRONIC ORAL TOXICITY OF HALOCARBON 27-S OIL. E R Kinkeadll, B T Culpepperl, S S Henryl, R S Kutzman , J F Wyman2, R H Bruner2. 1Northrop Services, Inc., Dayton, OH, 2NMRI/TD, Wright-Patterson Air Force Base, OH. Halocarbon 27-S (H 27-5), a polymer of chlorotrifluoroethylene (CTFE), is used as a lubricating oil for pumps in hyperbaric chambers. Although monomeric CTFE has been shown to produce renal lesions in rats the toxicity of CTFE polymers have not been investigated. To assess the toxicity of subchronic exposure to H 27-S, three groups (N=6/group) of male and female Fischer-344 rats were repeatedly dosed with 2.5 g H 27-S/kg for 7 or 21 days. Groups were sacrificed at 7, 21, and 35 days (14 days after the 21-day dosing). Corresponding control groups (N=6) were dosed with deionized water. Decreased water consumption and urine output were apparent in all test groups. Statistically significant increases in fluoride excretion were noted in 24-hour urine samples assessed periodically during the study. Neurotoxic signs were observed in female rats but not in male rats. Significant increases in liver and kidney weights were seen in all rats, regardless of number of dosing days. (Supported by US Navy through Air Force Contract # F33615-85-C-0532.) 402 LOCUS AND SPECIES SPECIFICITY IN MUTAGENESIS: W Caspary1, R Langenbach', D MGGregorz, B^Myhr', A Mitchell', B Penman5, C Crespis. 1NIEHS, Research Triangle Park, NC, zIRI, Musselburgh, Scotland, 'LBI, Kensington, MD, 'SRI, Menlo Park, CA, and SGentest, Woburn, MA. Sponsor:,. J R Bucher. ~ ~ We exami ned the mutageni.c*f~"ponses of the tk"- . . 'V and hgprt loci in mouse lymphoma (MOLY) c_e3Ss-0 of the tk locus in;,kuman lymphoblasts (HULY.' and of the hgprt'locus in CHO cells to determi,e the relative sensitivity of these targets. Initially, twelve compounds that were positive at the MOLY tk locus were examined at the HULY tk locus. In the absence o~;S9Vthere was, agreement for all the chemi~cals, In the pre- sence of S9, there was substantial disagreement. These same compounds were then tested at the CH0 hgprt locus. Only three compounds were found to be positive. We then examined eleven compounds that were strongly positive at MOLY tk and were mutagenic at the Salmonella histidine locus or chromosomal aberration assay. These compounds were negative at CHO hgprt. Five of these ele- ven were also tested at MOLY hgprt and found to be negative. Thus, the tk locus appears to be the most sensitive of the loci, the Salmonella histidine locus of intermediate sensitivity and hgprt the least sensitive of the loci. Species differences do not appear to account for the differences observed. 403 101 THE PREDICTIVE CAPABILITY OF THE IN VITRO UDS AND TN VIVO RODENT HEPATOCYTE UDS AND SDS ASSAYS FOR RODENT HEPATOCARCINOGENS. J W 3-paldingl, 0 A Cascianoz, and J C Mirsalis'. 'NIEHS, Research Triangle Park, NC, zNCTR, Jefferson, AR, and 3SRI International, Menlo Park, CA. Sponsor: J K Dunnick. More than 50% of all carcinogens identified by the NTP/NCI carcinogenesis assay increase the incidence,f of liver tumors in at least one rodent sdi</ species. The liver may be the only site or one of several sites of tumor induc- tion. It was expected that both the in vitro and in vivo DNA damage/repair (UDS) assays which were performed in male F344 rat hepato- cytes would readily detect liver carcinogens. This proved not to be the case. Only 7 of 22 hepatocarcinogens were detected in the in vitro UDS assay. Of 18 hepatocarcinogens tested, only 4 were detected in the in vivo UDS assay; however, 10 of these did induce scheduled DNA synthesis (SDS), a response indicative of either mitogenic or hepatotoxic activity. All of the hepatocarcinogens detected in both UDS assays were active in one or more other genetic toxicity assays; however, both genotoxic and nongenotoxic liver carcinogens induced in vivo SDS in rodent liver:- These results indicate that while some liver tumors are induced by genotoxic mechanisms, in other instances chemicals may induce tumors by nongenotoxic mechanisms such as by chronic induction of hepatic proliferation.
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ZACOpRIDE: A PROMISING RADIATION ANTIEMETIC. *V Bogo, *C Boward, N Fiala and A Dubois *Behavioral Sciences Dept., AFRRI; and Dept. of Medicine, USUHS; Bethesda, MD Zacopride (p.o., 0.3 mg/kg) was tested in mon- keys (mean weight 3.5 JCg)4#or its ability to block radiation-induced emesis and to assess be- havioral toxicity. In the emesis study, monkeys were tested after placebo- or zacopride-only, and after placebo- or zacopride-radiation (N=6/group). Zacopride or placebo were given 15 min before 8 Gy whole-body, gamma radiation. Np~ emetic effects were noted after placebo- or zacopride-only. After radiation, 70 emetic epi- sodes occurred in 5 of 6 monkeys in the placebo/ radiation condition compared to 1 episode after zacopride/radiation (p < 0.01). Also, 353 retch- ing episodes occurred in the 6 radiation-only monkeys compared to 173 episodes in 4 of 6 zacopride/radiation monkeys (p <0.01). In the toxicity study, monkeys performed a visual dis- crimination task (VDT). Maximum response time was 0.7 sec to discriminate between a circle and' square (correct), randomly presented every 10 sec. Monkeys (N=6) were tested for a 3 hr in control-, placebo-, and zacopride-only condi- tions. Monkeys maintained performance above 95% correct in all behavioral conditions. These re- sults suggest that zacopride.significantly in-s hibits radiation-induced retching and vomiting and that it is not behaviorally toxic, which may have important operational and clinical implications. BEHAVIORAL ASSESSMENT OF X_YLENE AND ETHYLBENZENE USING THE STARTLE REFLEX OF THE RAT. J M Russo, National Institute for ' Occupational Safety and Health, Cincinnati, OH, and K Junnila, Finnish Institute of Occupational Health, Helsinki, Finland. Sponsor: W K Anger. Rats were exposed via inhalation to xylene (XY), ethylbenzene (EB), or a mixture of xylene'and ethylbenzene (XY+EB), and examined : for behavioral•impairment using tests of motor activity-and the startle reflex. -In Experiment I, daily exposures up to 4 days to XY (300 ppm), EB (230 ppm),- or XY+EB (220 •!-._--•` 80 ppm) failed to produce reliable behavioral changes. In Experiment II, similar exposures to XY+EB (150 + 150 ppm) and XY+EB (300 + 300, ppm) significantly elevated motor activity and depressed the startle reflex after 4 days. In Experiment III, 16 weeks of daily exposure to XY (300 ppm) impaired the usual inhibitory impact of a leading acoustic prepulse on the startle reflex, and an . equally long exposure to EB (230 ppm) reduced the startle reflex itself. No effect of similar exposures to combined XY+EB (220 + 80 ppm) was noted. The results revealed that there are some unique behavioral effects of separate exposures to XY and EB which differ from the more compelling behavioral effects of combined exposures to XY and EB. 77 306 COGNITIVE EFFECTS OF LONG-TERM CHRO~,TIE,sr$ NEUROLEPTIC ADMINISTRATION. ED.yevin and PE Johansson.Psychiatric Research Center, UllerAker Hospital, Uppsala, SWEDEN. Sponsor: DE Woollev. Cognitive deficits in humans accompany the motor dysfunction, tardive. dyskinesia (TD), which results from chronic neuroleptic administration. It is not clear if the cognitive deficits result from neuroleptic exposure or if preexisting neurological damage'Vfflch causes cognitive deficits predisposes patients to TD. We injected"male and female albino rats monthly with haloperidol or fluphenazine decanoate in doses equivalent to (0.5, 1, 2 and 4 mg/kg/day). After 16 months the rats were tested for exploration in an,,*-a~ radial maze. Both haloperidol (p('.025e) and fluphenazine (p<.001) caused decreases in the distribution of choices as measured by entries to repeat. Response speed was slowed by haloperidol (p<.005), but fluphenazine only,had marginal effect (p<.10). The decrease in choice distribution demonstrates that long term neuroleptic administration does affect cognitive function. In humans chronic neuroleptic exposure may induce not only motor disorders but' cognitive dysfunction as well. (Supported by Swedish MRC grant # 4546). 307 BEHAVIORAL IMPAIRMENT IN THE RAT AFTER COLCHI- LINE LESIONS OF THE NUCLEUS BASALIS. W R Mundy and H A Tilson. NIEHS, Res. Tri. Park, NC. Neuronal loss in the nucleus basalis of Meynert .(NBM) has been consistently associated with mem- ory impairment. In the present study rats were . given bilateral injections of coichicine (2.0 ug/site) into the.NBM and examined for changes in three models of learning and memory. Reten- tion of a step-through.passive avoidance.task was examined 28 days after surgery. Rats with NBM lesions had decreased step-through latencies compared to controtli-. Training began in a Morris water maze on the next day. NBM lesions had no effect on escape latencies during water maze acquisition. After water maze training, rats were food-deprived to 85% original body wt. and trained on an autoshape task, in which they learned to associate lever presentation with food pellet delivery. NBM-lesioned rats initi- ally made a greater number of responses (lever touch) compared to controls, but both groups reached crfterion in the same number of trials. NBM-lesioned rats also made a greater number of responses during the intertrial interval. At the completion of autoshape training rats were given free access to food and tested for reten- tion of the water maze task. There was no dif- ference in retention between lesioned and con- trol rats. These data indicate that NBM lesions do not affect acquisition but selectively impair retention of a nonspatial reference memory task. 50875 8202 I
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133 OLTIPRAZ-INDUCED PROTECTION IN ACETAMINOPHEN FISPATo=Cr'1'Y IN MALE HAMSTERS. II. ACETAMIARr Pl)13N MEI'ABCLISM. M H Davies* and R C Sclaiel l, S C Johnson and Son, Inc *,-12acine, WI and North Dakota State jJniversity, Fargo, ND. - . s Oltipraz (OPP: 5-(2-pyrazinyl)-4-methyl-1, 2-di- thiol-3-thime, 2.0 nmole/kg, po), 48 hr prior to acetamincphest (AAP: 2.6 nmole/kg, ip), decreases AAP hepatotcaicity. These studies were performed to determine whether CJI'P altered AAP metebolism. Urinary recoveries of AAP+AAP-~6tebolites were similar (80%). However, more AAP was found as the glucurcnide (GLUC) and less AAP was recovered as glutathione (GSH)-derived conjugates in OTP- treated hamsters. The calculated UDPGS synthetic rate, liver UDPGA content and UDP-glucurenyl transferase activity, as well as, the foanation rate constant (kf) for GLUC were markedly increased by OTP. OTP treatment decreased in vivo binding of AAP to liver protein, concani.tant with decreases in kf for GSH-derived metabolites and AAP reactive fraction (kf GSH/K). Markedly increased rate and extent of AAP glucurcnidation, which shunts AAP metabolism, may be a primary mechanism of OTP hepateprotecticn. (SuppOrted by a Sandoz Research Institute Fellowship (MEID) and a Burraughs-9Tellcane Toxicology Award (RCS). 134 OLTIPRAZ-INDUCED PROTECTION IN ACETAMINOPHEN HEPATClPCp{ICI'I'Y IN MALE HAMSMRS.. I. ACETAMDXI- PHESJ 'tCICIOCFCIl=CS. R C ScYuiel l. L J Lutz and . M H Davies*, S.C. Johnson and Son, Inc.*, Racine, WI and North Dakota State University, Fazgo, rD. Our previous stuciies indicated that hepatic glu- tathione was not central to the.protection provided by oltipraz (OTP: 5-(2-pyrazinyl)-4- . methyl-1, 2-li.thiol-3-thiene) in acetaminophen (AAP)-induced hepatotoxicity. These studies were designed to determine if AAP (2.6 nmol/kg, ip) disposition was altered by prior (48 hr) dl'P (2.0 mmol/kg, po) treatment. OTP did not influence AAP absorption, as shown by similar PCNONLIN- generated ka (0.2 min-1), Tmax (16 min) and Cmax (2200 uM) estimates. AAP was eliminated more rapidly by OTP-treated hamsters. Decreased plasma Tl/2 (49%) and AUC (46%) were associated with correspond3ng increases in the elimination rate constant (87%) and systematic clearance (70$) in hamsters receiving OTP. During AAP elimination, plasma OTP concentraticats were low (10 uM); thus a direct interaction is unlikely. Theae data indicate that OIP single treatme nt may protect against AAP hepatotoxicity by enhancing overall AAP elimination. (Supported by a Sandoz Research Institute Fellowship (MHD) and a Burrcughs-wellcane Tmcicology Awaxd (RCS)). 135 STUDIES ON THE MECHANISM OF COUMARIN-INDUCED HEPATOTOXICITY IN THE RAT. B G Lake, T J B Gray, J G Evans, J A Beamand, and K L Hue BIBRA, Carshalton, Surrey, England. .{ Coumarin (1,2-benzopyrone) is known to produce liver damage in the rat but controversy exists. as to whether this is due to coumarin r se or to a coumarin metabolite(s). We have compared"'- the toxicity of coumarin ar,"ihydrocoumarin ' (DHC), which lacks the 3,4-double bond, both4n vivo and in primary hepatocyte cultures from , male Sprague-Dawley rats. The administration o~-- coumarin (125 mg/kg, i.p.), but not DHC (127 and 254 mg/kg), markedly reduced hepatic reduced glutathione (GSH) levels after 2 hr and produced centrilobular hepatic necrosi#- and-elevated serum enzyme activites aftet'~24 hr. In hepatocyte cultures coumarin, but not DHC, produced a dose dependent inhibition of protein synthesis and depletion of GSH levels. GSH depletion was not due to GSSG formation or to leakage from the cells. Coumarin-induced toxicity was reduced by the cytochrome P-450 inhibitors metyrapone and ellipticine but enhanced in GSH depleted (diethyl maleate treated) hepatocytes. These studies demonstrate a good correlation between the in vivo and in vitro effects of coumarin and DHC and are consistent with the hypothesis that a coumarin 3,4-epoxide intermediate is responsible for coumarin-induced hepatotoxicity in the rat. (Supported by the UK Ministry of Agriculture, Fisheries and Food) 136 ALLYLAMINE AND ACROLEIN TOXICITY IN CULTURED FIBROBLASTS AND-MYOCYTES FROM NEONATAL RAT HEART. M Toraason, M E Luken, M J Breitenstein, J A Krueger, and R E Biagini. CDC, NIOSH,* Experimental Toxicology Branch, Robert A. Taft Laboratories, Cincinnati, OH Allylamine is toxic to the cardiovascular system causing aortic, valvular and myocardial lesions: Acute toxcity is believed to involve metabolism of allylamine to highly reactive acrolein. Comparative toxicity of allylamine and acrolein was evaluated in cardiac fibroblasts and myocytes, which were obtained from neonatal rat hearts by a differential plating technique. Allylamine and acrolein were added directly to serum supplemented culture media (M199). Toxicity was assessed by measuring lactate dehydrogenase (LDH) release, ATP levels, and spontaneous beating activity. Beating activity was arrested by 0.05 mM acrolein and 0.1 mM allylamine. Acrolein at a concentration of 0.05 mM was equally toxic to myocytes and fibroblasts reducing ATP levels and causing marked leakage of LDH 4 hrs after treatment. In contrast, 10 mM allylamine was required to reduce ATP levels and cause leakage of LDH in fibroblasts, whereas a comparable response was produced in myocytes by 0.1 mM allylamine. Semicarbizide,.a benzylamine oxidase inhibitor, protected myocytes from allylamine toxicity, whereas clorgyline, a monoamine oxidase inhibitor, was ineffective. Semicarbizide was ineffective against acrolein toxicity. The findings support the hypothesis that the toxicity of allylamine in cultured myocytes is dependent on its metabolism to acrolein. 50875 8159 34
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OPTIMIZATION OF rRYOPRESERVATION PROCEDURES FOR RAT AND HUMAN HEPATOCYTES. L J Loretz, A P Li, M W Flye and A G E Wilson. Monsanto Environmental Health Laboratory and Washington Univ Med.Sclftol, St. Louis, MO. Rat hepatocytes were cryopreserved using a number of procedures ahd the viability, attachment, and metabolic activity of the cryopreserved cells were compared. Several cryopreservation agents (DMSO,glycerol,PVP, dextrans) and combinations of these.agentsi . were tested. Other variables tested included the freezing rate and the concentration of serum in the freezing medium. The greatest recovery of viable attached cells was obtained using DMSO at concentrations of 10% or higher, and a freezing rate of ~1°C/minute. Varying serum concentration in the freezing medium did not affect cryopreservation results. Using this procedure, the recovery of viable hepatocytes was 70%. Metabolic endpoints used to evaluate cryopreserved cells included activation of pro-mutagens in the CHO/HGPRT gene mutation assay, ethoxycoumarin-0-deethylase activity, p-chloromethylaniline demethylase activity, and urea synthesis. Each of these endpoints remained unchanged following cryopreservation. In addition, peroxisome proliferation at a level similar to that found in freshly isolated hepatocytes was observed in cryopreserved hepatocytes following treatment with Wyeth 14,643. Similar results were obtained with hepatocytes isolated from two human livers. ROLE OF THE 4S BINDING PROTEIN IN THE INDUCTION OF ARYL HYDRO.:ARBON HYDROXYLASE IN THE RAT. M Harris, C Kamps and S Safe, Departments of Veterinary Ehysiology and Pharmacology and Biochemistry and Biophysics, Texas A&M University, College Station, TX A survey of several rat strains demon- strated that the levels of the hepatic cytosolic 4S binding protein (using [3H]-benzo(alpyrene as the radioligand) were highly variable. The ' concentrations of this binding protein in Sprague Dawley (Harlan, -4S), Sprague Dawley (Sasco, +4S), Fischer 344, Wistar, and Lewis rat hepatic cytosol were 0, 208 + 57, 0, 244 + 21, and 216 + 40 fmol/mg cytosolic protein, respec- tively. Dose-response induction of hepatic microsomal aryl hydrocarbon hydroxylase by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 3-methylcholanthrene (MC) in Sprague Dawley (+4S) and Sprague Dawley (-4S) rat strains gave comparable ED5g values for AHH induction. Stu3ies with these inducers and other poly- nuclear aromatic hydrocarbons with affinity for the 4S binding protein suggest that this protein plays a minimal or antagonist role in the regulation of AHH induction in the rat. (Supported by the Texas Agricultural Experiment Station and the N3tional Institutes of Health.) 65 258 259 2,3,7,8 T_ETRACHLARODIBENZO-p-DIOXIN (TCDD) ANTACI)NISTS PROTECT AGAINST TCDD-MEDIATED pORPHYRIA. C Yao and S Safe, Department of Veterinary Physiology and Pharmacology, College of Veterinary Medicine and Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX Administration of 2,3,7,8-ICDD (75 ug/kg) to C57BL# mice causes significant hepWdc accumulation of octa and hepta carboxy-sub- stituted porphyrins within 3 weeks after treatment. In contrast, treatment of the animals with either Aroclor 1254 (250 umol/kg) or 6-methyl-1,3,8-trichlorodibenzofuran (MCDF, 750 umol/kg) did not cause hepatic porphyria. Cotreatment of the mice with 2, 3, 7, 8-T^~Z fi'k~. ug/kg) plus either Aroclor 1254 (250, '7~ and 25 unol/kg) or MCDF (750, 250 and 75 umol/kg) ~ completely inhibited the accumulation of hepatic hepta and octa carboxy-substituted porphyrins. The inhibition of hepatic microsomal aryl hydrocarbon hydroxylase by MCDF or Aroclor 1254 was not observed. Using a staggered treatment protocol for administration of the antagonist, significant protection from the porphyrino- genicity of 2,3,7,8=ICDD was observed when the antagonist, was administered 2 weeks after the toxin. The mechanistic implications of these and other observations will be discussed. (Supported by the National Institutes of Health.) EFFECTS OF NITROUS OXIDE AND BODY WEIGHT ON THE GUINEA PIG MODEL OF HALOTHANE HEPATOTOXICITY. RC Lind and AJ GandoTfi, Department of Anes- thesiology, Univers'ity _oT Arizona, Tucson, AZ. Halothane (H), is often administered con- currently with N 0. Since N 0 can exacerbate liver injury, th2ts combinatiX of anesthetics was evaluated in a guinea pig model of halothane hepatotoxicity. Male and female strain 13 guinea pigs (300-1000 g) were exposed to 1% H, 40% 02 for 4 hr with or without 60% N20. The addition of N 0 affected neither plasma concentration of 2H metabolites nor the degree of hepatic injury (ALT and histopathology). Animal weight was a factor with larger (572-970 g)- animals of both sexes demonstrating signifi- cantly greater elevations in 48 hr ALT and incidences of centrilobular necrosis vs smaller (318-565 g) animals (ALT=134 + 74 units/ml vs 27 + 7; necrosis=17/24 vs OT8, respectively). There were no significant differences between large and small animals in levels of plasma metabolites of H. Following 0.1 ml/kg CC1 (ip), larger guinea pigs also developeA significantly greater elevations in ALT over those in smaller animals. Further studies will be required to elucidate factors involved in this variation in hepatotoxic response in guinea pigs of different sizes. (NIH AM16715). 50875 8190
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332 VITAMIlN K DEPENDENT TOXICITY OF LY163443 SODIUM, A NEW LTD4 ANTAGONIST, IN LABORATO- RY RATS. R B L v~n Lir, J P McGrath, and L D Cherry, Toxicology Division, l.illy Research Laboratories, Green- field, IN. LY163443 sodium, 1-[2-hydroxy-3-propyl-4-[[4-(1H-tetra- zol-5-ylmethyl)phenoxy]methyl]phenyl]ethanone, sodium salt (LY) is a new LTD4 antagonist being developed for the treatment of asthma. Hemorrhage and prolohged clotting times were the principal signs of toxicity observed in rats and rabbits during toxicological testing. These signs were not observed in mice, dogs, or monkeys. Analysis of plasma from affected rats with specific coagulation factor free plas- ma indicated that at least factors II and VII were reduced suggesting that vitamin K absorption or utilization was af- fected. The effect of vitamin K on LY toxicity was studied in F344 rats (5 males/group) given diets containing 0, 0.25, 0.3 or 0.4% LY in Purina Certified Rodent Chow (#5002) or in Teklad 4% diet which contained 9 ppm added menadione. The mortality in animals maintained on non-menadione ford- fied diet was 0, 20, 100, and 100%, respectively, with a mean time to death of 5 days. No mortality was observed with LY in fortified diets. Activated partial thromboplastin times and prothrombin times were both elevated in all surviving ani- mals receiving the non-fortified feed. Coagulation parame- ters were completely unaffected in animals maintained on fortified feed at these doses. Mean APTT and PT values for animals receiving LY in fortified diets were below those for animals receiving LY in non-fortified diet suggesting that some commercially available rodent chows may contain in- sufficient quantities of vitamin K to sustain normal clotting function in rats during periods of stress. These data demon- strate that LY toxicity can be modulated by dietary vitamin K. 333 TOXICITY OF A NOVEL ANPICONVULSF1Nr, 2-AXa4O-N-(2- MEZi3Ylr1,2-DIPFIH3QYLEIHYL) ACE.AND?)E (PR 934-423A). CF Luke, DW Moore, GC Palmer, ML Coan, JC Strand, and CF Morris. Pennwalt Pharmaceutical Division Rochester, NY. PR 934-423A (PR) is effective orally to control grand mal seizures in animal models [ED50 for maximal electroshock seizures (mouse, 23 mg/kg, ip, 33 mg/kg, po; rat, 20 mg/kg, po); T050 for neural impairment (mouse-inverted scr~een test, 85 mg/:cg, ip, 581 mg/kg, po; rat-plank walk test, 690 mg/kg, po)t therapeutic indices in mouse (ip= 3.8, po= 17.6) and rat (po=34)]. In anesthetized dogs, 10 and 30 mg/kg, iv of PR depressed myocar- dial contractility, cardiac output and heart rate. In hypertensive rats, 100 mg/kg, po did not alter arterial blood pressure. PR exhibited a large volume of distribution [Vd=3.3 L/kg (rats) and 3.8 L/kg (dogs)] and followed a two canpartment model with rapid elimination [Tl/2=0.36 hour (rats) and 1.12 hour (dogs)]. LD50 values were deter- mined for: rat (897 mg/kg, po: 142 mg/kg, ip) - and mouse (781 mg/kg, po; 51 mg/kg, iv). Single oral doses of >400 mg/kg produced focal necrosis and hemorrhage of the duodenal epithelium in mice. Daily oral doses of 400 mg/kg for 30 days were lethal in 8 of 20 rats and produced lymphoid atrophy of the thymus, spleen and lymph nodes. Daily oral doses of 60 mg/kg for 90 days were lethal in 2 of. 10 beagle dogs. The desirable efficacy/safety ratios and the lack of signifi- cant toxicological effects in animals define PR as a promising anticonvulsant now in clinical trials. 334 ACUTE AND SUBACUTE TOXICITY OF THE ANTI- CONVULSANT CI-953. R Walker, M Seefeld, and G Wolfe, Warner-Lambert/Parke-Davis Res. Inst., Mississauga, ON, and Ann Arbor, MI; and Hazleton Laboratories America, Inc., Vienna, VA CI-953 (N-(2-chloro-6-methylphenyl)-N'-4-pyri- _ ° dinylurea, monohydrochloride) has potential ~ efficacy for the treatment of•gQuaralized tonic- ` clonic and partial seizures in human beings atmtr-.~ doses of 5-10 mg/kg.:• Acute oral toxicity- studies in fasted mice and rats revealed 14-day = median lethal doses of about 350 mg/kg. Subacute oral toxicity studies were conducted in Beagle dogs for 2 weeks (escalating regimen of 10-220 mg/kg and repeated doses~ t' ~0, 40, 80, and 120 mg/kg) and rats for 3Q dayp (30, 100, 300, and 600 mg/kg by gavage). Mortality in the rat study occurred at 600 mg/kg. Reduced food consumption, body weight loss/gain suppression, and CNS depression occurred at high dose levels in both species. Biochemical analyses and pathologic examination revealed the liver, kidney, and stomach to be target organs at high dose levels. Hepatic changes were mild in both species and were probably related to induction of mixed function oxidase. Renal effects occurred only in dogs and included pyelitis, associated necrosis, and transitional cell erosion. Gastric erosin/ulceration occurred in the nonglandular mucosa of rats, and in the glandular mucosa of dogs. (Supported in part by NINCDS Contract N01-NS-3-2358) 335 EXCRETION AND TISSUE DISTRIBUTION OF METHYLPHENIDATE-HCI (MPH) IN RATS AND MICE AFTER A SINGLE ORAL DOSE. CR Duerson, [2.ESartgr, IG Sipes, Department of Pharmacology and Toxicology, University of Arizona, Tucson, AZ. MPH is a central stimulant widely prescribed for treatment of attention deficit disorder in children., Recently, MPH has been shown to cause liver tumors in male mice but not in rats. Our experiments are designed to determine if the toxicity finding can be explained by differences in MPH disposition. Excretion and tissue distribution of total radioactivity were examined in male Fischer F344 rats and male B6C3F1 mice after a single oral dose of 14C-MPH. Rats were treated with 7, 35, or 70 mg/kg, while mice were treated with 2.1 or 19 mg/kg. The treated animals were placed in plastic metabolic chambers and feces and urine were collected separately at 4, 8, 12, and 24 - hours. At termination, necropsies were performed, tissues were sampled and analyzed for total radioactivity after oxidation to C02. Less than 1% of the radioactivity remained in the tissues at 24 hours. Liver showed the highest tissue level of radioactivity. Total radioactivity in feces of rats at 24 hours after 7, 35, or 70 mg/kg MPH was 24.2, 18.9, and 22.7 percent of the dose, respectively. Total radioactivity in rat urine was 83.4, 86.2, and 78.4 percent of the dose, respectively. Total radioactivity in feces of mice at 24 hours after 2.1 or 19 mg/kg MPH was 15.7 and 19.7 percent of the dose, respectively. Urinary recovery was 80.5 and 75.0 percent of the dose, respectively. Excretion and tissue distribution of MPH were fairly similar in the F344 rat and B6C3F1 mice. Supported by NIEHS NO1-ES-3-5031. n At B, D B T C i I 337
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388 CHIRAL EFFECTS IN THE INDUCTION OF DRUG- METABOLIZING ENZYMES USING SYNTHETIC ATROPISOMERS OF POLYCHLORINATED BIPHENYLS. M Puttmann, A Mannschreck, and -°' L W Robertson. kaduate Center for Toxicology, University of Kentucky, Lexington, KY and Department'of Organic Chemistry, University of RegeP:sburg, Regensburg FRG. Atropisomers of the polychlorinated bi- phenyls 2,2',3,4,4',6-hexachlorobiphegyl (II) and 2,2',3,3',4,4',6,6'-octachlorobipFieliyl (III), stable to racemization under physiologi- cal conditions, were administered to immature male Sprague-Dawley rats. The racemic hexa- chlorobiphenyl (II) was found to be a potent (phenobarbital-type) inducer, whereas (+)-II and (-)-II, administered at 100 umol/kg, showed clearly differing potencies as inducers with (+)-II enhancing aminopyrine N-demethylase, aldrin epoxidase and cytochrome P-450 content more potently than (-)-II. In contrast, the racemic octachlorobiphenyl (III) and its indi- vidual enantiomers were only weak phenobarbi- tal-type inducers of cytochrome P-450 and the enantiomers of III were equally (weakly) po- tent. Separate studies conducted to investi- gate the differential potency of the enantio- mers of II showed that (-)-II was apparently more rapidly metabolized than its antipode. Therefore, enantioselectivity in disposition rather than in recognition is responsible for the differential potency seen. (Supported by A. von Humboldt Foundation) 389 A UNIQUE APPROACH TO THE SYNTHESIS OF 2,3,4,5-SUBSTITUTED POLYBROMINATED BIPHENYLS (PBBs): QUANTITATION IN FIREMASTER FF-1 AND FIREMASTER BP-6. G Kubiczak, F Oesch, and L W Robertson. Graduate Center for Toxicology, University of Kentucky, Lexington, KY and Institute of Toxicology, University of Mainz, Mainz FRG. Unraveling the toxicity of a complex chemical mixture necessitates the isolation or synthe- sis of the individual components. From a single starting material, 2,6-dibromo-4-nitro- aniline, a synthetic scheme is presented which provides in good yields 4 anilines (3,5-di-, 3,4,5-tri-, 2,3,4,5-tetra- and 2,3,4,5,6- pentabromoanilines), as well as the 1,2,3,4- tetrabromobenzene, all useful precursors for the synthesis of polybrominated biphenyls (PBBs). The aryl-aryl coupling of these and other bromoanilines with 1,2,3,4-tetrabromo- benzene provides a versatile approach to the synthesis of 2,3,4,5-substituted PBBs. The synthesis and characterization of nine such PBBs will be reported. Aside from the desired coupling product, the 2,2',3,3',4,4',5,5'- octabromobiphenyl was a by-product of each coupling reaction, ranging from less than 2 to 63% of the polybrominated biphenyl products. Capillary gas chromatographic quantitation of the nine synthetic PBBs in fireMaster FF-1 and fireMaster BP-6 will be presented. 390 TERATOGENICITY OF 2,3,4,7,8-PENTACHLORODIBENZO- F'URAN(4-PeCDF) IN F344 RATS.z L A Couture, M W Harris, and L S Birnbaum. NIEHS, RTP,,.NC and UNC, Chapel H-iI3., NC ' PCDFs are ubiquitous environmental contaminants that have also been detected in human tissues. 4-PeCDF, similar in chemical structure and toxicity to 2,3,7,8-tetrachlorodibenzodioxin, is one of the most toxic members in the class 4 halogenated aromatics. To-vooluate the teratogenicity of 4-PeCDF in F344 rats, dams were dosed po on gestatisn day 12 with doses of 0,10,30,100, or 300 ug/kg. All animals were sacrificed on gestation day 20 and maternal and fetal toxicity was assessed. Determination of fetal toxicity involved both soft tissue and skeletal examinations. Maternaltieiot gain decreased in a dose-related fashton, while the liver:body weight ratio increased an$ the ratio of thymus:body weight decreased. A steep dose- response curve was observed for fetal mortality reaching 81% at a dose of 300 ug/kg. There was a 100% incidence of cleft palate in all surviving fetuses in the 300 ug/kg group. Soft tissue anomalies observed in the high dose group were a=eduction in the size of the lungs and thymus or an absence of the thymus altogether. In conclusion, the spectrum of teratogenic and maternally toxic effects are indicative of a pattern similar to those observed following exposure of murine species to polyhalogenated aromatic compounds. Further experiments are currently on-going to verify such a pattern. 391 IMMUNOTOXICITY OF FOLYCHIARINATED DIBENZOFURANS: STRUCTURE-ACTIVITY RELATIONSHIPS AND INTERACTIVE EFFECTS. D Davis and S Safe, Departinent of `7eterinary Physiology and Pharmacology, College of 4eterinary Medicine, Texas A&M University, College Station, TX. The dose-response effects of several polychlorinated dibenzofuran congeners on the splenic plaque-forming cell (PFC) response to sheep red blood cells (SRBC) were determined in C57BL/6 mic(k The doses required for a 50% reduction in PFC's/106 viable cells (ED50) for 2, 3, 7,8-tetrachlorodibenzo-p-dioxin ('ICDD), 2,3,4,7,8-pentachlorodibenzofuran (PeCDF), 2,3,7,8-tetrachlorodibenzofuran (ZCDF), 1,2,3,7,9-PeCDF and 1,3,6,8-'ICDF were 2.4, 3.0, 14.0, 710 and 35,700 nmol/kg respectively. The relative potencies and structure-activity relationships (SARs) for the BCDFs were com- parable to those observed for other Ah receptor- mediated responses. The interaction of 1,3,6,8- ZCDF and 2,3,7,8-TCDD demonstrated that sub- toxic dose levels of 1,3,6,8 TCDF, (6.6 - 49.3 umol/kg) a weak agonist, significantly an- tagonized the immunotoxicity of the more potent agonist, 2,3,7,8-TCDD (3.7 nmol/kg). The effectiveness of the partial antagonist was dependent on several factors including the dose and the time of adninistration relative to both the immunotoxin and the SRBC antigen. (Sup- ported by the National Institutes of Health and the United States Environmental Protection Agency•) 98 50875 8223
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ONTANEOUS AND INDUCED ACCUMULATION OF ~?u OBULIN IN THE KIDNEY CORTEX OF RATS AND MICE. M Ridder, E C Von Bargen, R D Parker, and L A7den. Procter & Gamble, Cincinnati, A~kl. onsor: L Lehman-McKeeman~ ' show that several strains of male rat that oduce a2u globulin (Fischer ~44, Sprague Dawley, ffalo, and Norwegian Brown) accumulate this otein in the kidney cortex as hyaline droplets. osure to decalin increases the accumulation of nly otLu globulin and creates larger hyalinea roplets that are believed to be nephrotoxic. We lso show that the NCI-Black-Reiter male rat which oes not synthesize this protein, forms no hyaline roplets, and does not accumulate any protein in ts kidney either spontaneously or when exposed to ecalin. We expect that this strain will emonstrate no chronic toxicity. Mice produce an a2u globulin called MUP. Its sequence differs slightly from rat o2u globulin yet it does not accumulate in the kidney of either sex of mice either spontaneously or after decalin exposure. To determine whether this difference between rats and mice is due to specific characteristics of rat a2u globulin, both sexes of mice were injected with male rat o2u globulin at a level consistent with male rat output. Histology shows only very small droplets in the kidneys of the injected mice. Dosing with decalin had no effect on either the histology or the protein content of the tissues. Other factors such as metabolism or renal function must be required to explain the differences between mice and rats. QUANTITATION OF ETHOXYQUIN IN MOUSE TISSUES: H L Kim, veterinary Physiology and Pharmacology, Texas A&M University, College Station, TX. Sponsor: S Safe Dietary administration of ethoxyquin (EQ); an antioxidant, reduces the carcinogenic effects of a variety of chemicals in rodents. Rapid and nearly complete excretion of orally administered 14C-EQ is reported. However, traces of radioactivity are reportedly remaining in tissues for up to 4 weeks and it has been asscaned that the persistent radioactivity is due to the presence of EQ metabloites, though not identified. 4he uptake and metabolism of EQ in mice was determined using an HPLC-fluorometric detection method. Mice were given powdered feed containing EQ-HC1, 0.125% or 0.5%, and the EQ content of tissues including liver, kidney, lung and brain was quantified 1-7 days after the feeding started, and 1-7 days after the feeding terminated. The highest EQ residues were found in the liver; 0.74 and 7.72 ug/g tissue, respectively, 24 hours after consuming feed containing 0. 125 and 0. 5% EQ-HC1. EQ residues were detected in all tissues 7 days after the feeding terminated; the level ranged from 0.08 ug/g brain to 0.7 ug/g liver. These results suggest that the persistent redioactivity is, in part, due to the presence of EQ. (Supported by the Texas Agricultural Experiment Station.) 89 354 CONTINUOUS INTRAVENOUS INFUSION STUDIES;bWITC' 2`,3'-DIDEOXYADENOSINE (adA) IN BEAIiCE UOGS. J G Page, M E Placke, K A Colling, L E Mezza, 6 M Wientjes, C K Grieshaber*, and J E Tomaszewski*. Battelle Columbus .Division,,;. - Columbus, OH and *National Cancer Institute, Bethesda, MD. ddA inhibits the cytopathic effects of human immunodeficiency virus (HIV) in vitro at a minimum inhibitory concentrationZMIC o 10 pM (Mitsuya an(# 'Broder, PNAS, 83, 1911.t,~98 )6 Dogs rapidly deaminate c~dA to Z,3'-dideoxy~no- sine (ddI). ddA was infused in•dogs at 3.13, 31.3 and 93.9 mg ddA/kglhr. Administration of 31.3 mg/kg/hr for 240 hrs produced a mean plasma level (Cpss) of 30.5 jAg/mL (129pM) and few clinical signs of toxicity. Severe GI tox- icity occurred in dogs receiving 93.9 mg4g/hr, starting on day 6 of the infusion. I+'th~ group, C ss was 142 µg/mL (males) and 205 µg/fiL (females). Infusion was terminated at 215 hrs in this group and 3 of 4 dogs were judged moribund and necropsied over the next two days. Moderate to severe necrotizing inflammation and focal mucosal hyperplasia in the GI tract, bone marrow hypocellularity and marked thymic lymphocyte depletion were detected in most high dose dogs. Dose-related tachycardia and hypo- tension were also observed. (This work supported by NCI contract No. N01-CM-67869) 355 SINGLE-DOSE PHARMACOKINETICS AND BIOAVAILABI- LITY STUDY QF 2',3'-DIDEOXYADENOSINE (ddA) IN BEAGLE DOGS AND RATS. M E Placke, J ~G Page, K A Colling, G M WientJes, C K Griesha erb -*, and J E Tomaszewski*. Battelle Columbus Division, Columbus, OH and *National Cancer Institute, Bethesda, MD. ddA is currently being considered for treatment of AIDS. ddA inhibits DNA synthesis and shows antiviral activity in vitro against human im- munodeficiency virus-(HlV at concentrations of 10-200 pM without., evidence of cytotoxicity. These pre-clinical'~ studies were conducted to determine the pharmacokinetics and bioavaila- •biiity of ddA in dogs following IV and oral administration and in rats following IV, SC, and IP administration. ddA was rapidly deamin- ated to dideoxyinosine (ddI), with suggested dose-dependent kinetics. The half-life in dogs was approximately 61 min following an IV dose 500 mg/kg of and 24 min after 100 mg/kg. The half-life in rats was 17 min after 175 mg/kg of ddA, IV. Plasma concentrations and clearance rates of ddI were disproportionaty related to ddA doses, suggesting there was a, concentration-limited metabolism of ddl. Oral doses of ddA were poorly tolerated in dogs and only partially retained, complicating the de- termination of bioavailabiiity. When doses were retained, the oral bioavailability in dogs ranged from 28-50%. The bioavailability in rats was calculated to be 48% after SC doses and was greater than 100% after IP doses. (Supported by NCI contract No. N01-CM-67869) f; z
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E USE OF CULTURED HUMAN PERIPHERAL BLOOD HOCYTES (PBL) FOR IMMUNOTOXICOLOGY EVAL- TION. JB Cornacoff, AN Tucker and J ean. IT, Research Triangle Park, NC. . .. large volume of information has been accumu- ted in rodents on the effects of various xeno- otics on the immune system. This has provided, luable information regarding dose, kinetics, d target cell populations affected. To gain nsight into the relevancy of these observationT o man, a study is underway to determine the nfluence of 5 immunotoxicants of rodents on uman lymphocyte function. Lymphocyte function s being assessed by 2 assays: mixed lymphocyte esponse (MLR) and quantitation of IgM and IgG y an ELISA following stimulation of B lympho- ytes with pokeweed mitogen. In each assay, PBL re preincubated with xenobiotics' at 4-5 con- entrations and then cultured in vitro. The hemicals under study include: TCDD, PCDF, 0 MBA, BaP, and 2-AAF. Preliminary data for the values of rovided approximate ED R have ~ p 10 $M (TCDD), 10-8M (PCDF),1.4 }1I10(BaP), and 2 ;tM (DMBA). 2-AAF did not alter the MLR prior to or following metabolic activation with a liver microsome preparation (S9) at any of the con- centrations studied (0.2-100 }iM). Additional functional assays are currently being evaluated. These studies should improve risk assessment for human populations regarding relevant dose and better define pertinent biomarkers for immune dysfunction following xenobiotic exposure. THE DISRUPTION OF LYMPHOCYTE TRANSMEMBRANE SIGNALLING BY XENOBIOTICS. LM Thurmond, RV House, JH Dean. CIIT, RTP, NC. Early events in lymphocyte activation include the interaction of mitogen or antigen with p~embrane receptors, followed by release of Ca + from endoplasmic reticulum, activation of protein kinase C, and turnover of membrane phospholipids. Exogenous induction of calcium flux using Ionomycin and kinase activation using phorbol myristate acetate (PMA) bypasses normal signalling events. Experiments have been designed to identify the stages of lymphocyte activation affected by exposure to the model PAH immunosuppressant 7,12- dimethylbenz[a]anthracene (DMBA). Proliferation of DNBA-exposed lymphocytes to concanavalin A and cellular alloantigens was suppressed in the presence of PMA and Ionomycin, as was the production of IL 2. IL 2 receptor function was }T3aired by a two-fold decrease in affinity f2+ I-IL 2. Measurements of intracellular Ca using the fluorescent probe'Quin 2 show that DMBA- nosed lymphocytes have three-fold higher basal Ca + concentrations, (~ut are less responsive to Ionomycin-induced Ca t flux. Estimations of membrane phosphoinositol lipid turnover show altered accumulation of 3H-inositol phosphates in DMBA-exposed cells. These data suggest that normal signal transduction across lymphocyte plasma membranes is impaired by DMBA. Thus, the immune system may be vulnerable to adverse effects of hydrophobic xenobiotics due to the dependence of the immunoregulatory network on membrane-mediated activation events. 79 314 MODULATION OF PHA-INDIICED T-C87,~ CAhCIIIM MOBILIZATION BY DMBA T A Thompson, R H Fincher, and S W Burchiel,•University of New Mexico College of -Pharmacp, - Albuquerque, NM 87131 The purpose of these studies was to evaluate the effect of 7,12-dimethylbenz- (a)anthracene (DMBA) on calcium mobiliza- tion in murine and human T-cell lines exposed to phytohemagglutinin (Psk)-. We have previously shown that DMBA inhibits T-cell proliferation induced-by PHA. DMBA may inhibit PHA-induced calcium mobiliza- tion that is necessary for T-cell activa- tion. In these studies, Indo-1 was used. to analyze calcium mobilization in a murine (WEHI-7 ) and a human (Jur~#t)-I2- cell line. A FACS Analyzer was used, to determine the fluorescence ratio of Izido- 1 in its calcium-bound and calcium-free states. The kinetic changes that occurred in the fluorescence ratio (violet to blue) was monitored for up to 10 minutes following the addition of 50 ug/ml PHA to WEHI-7 or Jurkat. Exposure of WEHI-7 and Jurkat to 10 uM DMBA for 5 hrs resulted in a decrease in calcium mobilization induced by PHA. A 20 hr exposure of Jurkat to DMBA demonstrated a dose- dependent decrease in calcium mobiliza- tion at 1, 4 and 10 uM. These studies suggest that one effect" of DMBA is to modulate activation of T-cells by altering calcium mobilization. 315 EFFECTS OF 2-ACETYLAMINOFLUORENE ON IN VITRO IMMUNE RESPONSES IN MURINE SPLENOCYTES. K H Yang, D H Kim, T Kawabata*, and M P Holsapple*. Korea Advanced Institute of Science and Technology, Seoul, Korea.and *Medical College of Virginia, Richmond, VA. The immunosuppressive effects of 2-acetylamino- fluorene fluorene (AAF) were investigated in female BALB/c mouse splenocytes. Direct addition of AAF into spleen cell suspensions resulted in a dose- related suppressionV:,of the in vitro antibody responses to LPS and SRBC. AAF also produced a dose-related suppression of the proliferative responses to LPS and Con A. Time course study showed that AAF produced the maximum suppression of the lymphoproliferative responses when was added to the culture along with mitogen at the initiation of cultures. To determine the role of metabolic activation of AAF for the immunosuppressive effects, mouse splenocytes were cocultured with rat hepatocytes for 4 hr along with AAF. AAF did not produced the suppression of the in vitro antibody response to LPS in this coculture system. Meanwhile, AAF induced a dose- related unscheduled DNA synthesis in spleen cells only if activated by hepatocytes in the splenocytes-hepatocyte coculture system. These results suggested that the reactive metabolites of AAF which mediates its mutagenicity are not essential for its immunosuppressive activity. (Supported by the Korea Science and Engineering Foundation Research Grant). 50875 8204
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~ TOPOIETIC EFFECTS IN FEMALE B6C3F1 MICE EX- POSED TO ARSINE GAS. H L Hong, B A Fowler and G A Boorman. National Institute of Environment- al Health Sciences (NIEHS), Research Triliftgle Park, NC. Sponsor: G A$p an Arsine gas is a potent hemolytic agent. Concern about semiconductor workers prompted an in-depth study of arsine at NIEHS to determine the hemato- poietic effects of prolonged exposure to the tolerated doses of this gas. Female B6C3F1 mice were exposed via inhalation to 0, 0.5, 2.5, ak-5 ppm arsine 6 hr/day for 14 days. Body weights in exposed mice were comparable to controls, but a marked, dose-related splenomegaly was observed. Arsine exposure produced significant dose-related decreases in red blood cells, hematocrit and he- moglobin, with increases in white blood cell counts and mean corpuscular volume of red blood cells. Furthermore, erythropoiesis as measured by quantitation of erythroid precursors in cul- ture revealed a significant reduction of CFU-E/ femur cells for all treated groups on day 3 post- exposure and only at the 5 ppm dose group on 24 days postexposure. There was no alteration in bone marrow cellularity and a less significant effect on granulocyte-macrophage progenitors. A 12-week study of arsine at 0, 0.025, 0.5 and 2.5 ppm (6 hr/day) via inhalation showed similar but less effects on hematopoiesis in mice. These ef- fects were seen 3 weeks postexposure at 2.5 ppm . group. In conclusion, arsine exposure at well- tolerated doses produces a dose-related stress on the hematopoietic system characterized by a hemo- lytic anemia. THIRTEEN-WEEK DOSED FEED TOXICIT`l STUDIES OF m-NITROBENZOIC ACID IN F34 RATS AND B6C3F1 MICE. K M Abdoi, M E1we111, B S Levine2, L T Mulligan2, R Kovatch3. 1NIEHS, NTP, RTP, NC; 2Department of Pharmacolog~, Microbiological Associates, Bethesda, MD; PAI, Ijamsville, MD. Ten F344 rats of each sex received 0, 0.063, 0.125, 0.25, 0.5 or 1.0% m-nitrobenzoic acid in their diet for 13 weeks. Similarly, groups of 10 B6C3F1 mice of each sex received 0, 0.125, 0.25, 0.5, 1.0 or 2.0% of the compound in their diet. No rats or mice died during the study. Final body weights of rats at 1% and mice at z0.5% were lower(p<0.05) than those of the controls. Male rats at all dose levels had significantly more Heinz bodies . than controls. Methemoglobin concentration in males at 1% dose level was significantly increased. Females at 0.5 and 1% dose levels had lower hematocrit values, hemoglobin con- centration and red blood cell count. Methemo- globin concentration was increased (p<0.05) in females at Z0.25% dose levels. There was a significant decrease in total and free plasma levels of T-4 in males at 0.5 and 1% dose levels. Hematology and clinical chemistry were not done on mice. The only compound- related histopathological change was testicu- lar atrophy in rats at 1% dose level. No compound-related histopathological changes were observed in mice. _ ~~ ~~ 330 ACUTE, SUBCHRONIC, AND CHRONIC TOBI,C,~IT,3~'~` STUDIES OF THE CARDIOTONIC ISOMAZOLE (LY175326) IN RATS AND DOGS. J R Means, G E Sandusky, and D B Meyers. Lilly Research Laboratories, Toxicology Division, Greenfield, IN 331 83 Isomazole (LY175326, IMZ) was evaluated for tox- icity in a series of acute, subchronic, and chronic studies. Minimum lethal oral doses were 125 and 400 mg/kg in rats and mice, respectively. Dogs and monkeys survived single oral doses of 50 and 100 mg/kg, respectively; however,--*ue,of two female dogs died at 100 mg/kg of IMZ. Rats were given IMZ in the diet for three months at an average daily intake of 0, 20, 65, or 198 mg/kg. Prominent effects were increased relative liver weights and centrilobular fat deposition in the liver in the 198 mg/kg group and periarteritis of mesenteric vessels in both the 65 and~,98-ing/kg groups. Similar findings were seen ia they one- year study (0, 10, 25.5, and 68 mg/kg) wit1~ the hepatic effects occurring at doses as low as 25.5 mg/kg. Beagle dogs given oral doses of 0, 2, 6, or 16 mg/kg/day for one year did not exhibit any overt signs of toxicity. Slight increases in heart rate were observed at two hours post-dose in the 6.mg/kg females and moderate to marked increases occurred in the 16 mg/kg animals of both sexes. Basal heart rate was slightly de- creased relative to control in the 16 mg/kg group after one month of treatment. The heart was the target organ with increased relative heart weight (577) in the 16 mg/kg group and multi-focal myo- cardial fibrosis in the 6 (1/4 females) and the 16 mg/kg (2/4 males, 3/4 females) groups. ACUTE AND SUBACUTE TOXICOLOGIC EVALUATION OF THE ANGIOTENSIN-CONVERTING-ENZYME INHIBITOR SQ 29,852. R A Soltys. C A Johnson. M M Miller. R J Shuren. D L Tuomari. Y H Yoon. A DePaoli. and P L Sibley. The Squibb Institute for Medical Research. New Brunswick. NJ. Sponsor: J S Kulesza. SQ 29,852 (SQ) is the first of a new class of ACE inhibitors containing a phosphonic ester function. It is being developed as an orally active antihypertensive agent. Acute studies of SQ;in mice and rats revealed that its minimum lethal dose -was greater than 8000 mg/kg po and 4000 mg/kg or more iv. for both species. In an iv study. dogs given SQ at daily doses of 2 to 50 m g/kg for 2 weeks showed no significant drug-related effects. Mice given doses of 4 to 100 mg/kg iv for 2 weeks showed no changes, other than a slight decrease in total leukocyte counts at 100 mg/kg. In dogs. SQ given po at 30 mg/kg for 3 months induced no drug-related changes. At doses of 150 and 750 mg/kg, slight increases in serum glucose and magnesium were observed, but no serious adverse effects were noted in dogs at these dose levels. Rats given SQ po at 25 to 3000 mg/kg for 3 months did not show any major adverse effects. Cecal distention was noted grossly in high-dose rats. Histologically, a reduced incidence of renal tubular casts was noted at doses of 120 mg/kg or greater, and mild juxtaglomerular-cell hyperplasia was seen at 600 and 3000 mg/kg. These renal changes were probably associated with the pharmacologic properties of the drug. SQ was not genotoxic in a battery of mutagenicity assays, with and without metabolic activation. The results of these studies demonstrate that SQ is one of the least toxic ACE inhibitors reported to date. 50875 8208
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GENOTOXICITY STUDIES OF TETRANDRINE. T Ong, J D Stewart, C H Lu, H-% Jian, and W-Z Whong. Division of Respiratory Disease Studies, NIOSH, Morgantown, WV. Sponsor: V Castranova Tetrandrine, a compound isolated from Stephania tetranda, has been found to inhibit silicosis in laboratory animals. Results from clinical studies have shown that treatment of patients with tetrandrine significantly reduced pathological symptoms associated with silicosis. Since this compound may be used for the treatment of silicosis, the potential genotoxic and carcinogenic hazards of tetrandrine to the treated patients need to be investigated. The genotoxicity of tetrandrine and the effect of tetrandrine on the genotoxicity of chemicals and complex mixtures have been studied in our labora- tory with the Ames Salmonella and the SOS/umu assay systems. Results show that tetrandrine was weakly mutagenic to Salmonella typhimurium TA98 and did not induce SOS response. However, tetrandrine was found to be a potent mutagenesis enhancer. It highly increased (over 100%) the sutagenic activity of benzo(a)pyrene, trinitro- fluorenone, 2-aminoanthracene, diesel emission, airborne particles, fried beef, and cigarette smoke. The mechanism for the mutagenesis enhancement is not known. Data from this study seem to indicate that tetrandrine may enhance error prone DNA repair. Further studies with in vivo assay systems need to be performed to evaluate the potential health hazards of tetrandrine. 104 412 A STUDY ON THE IiIITAGENICI-TY OF LOW TAR CIGAgETTE SMOKE. - 0 T Chortyk, J L Baker, and W J Chamberlail. Tobacco Quality and Safety Research Unit, USDA, Athens, GA. Sponsor: A P Norred. A series of 10 low tar cigarettes, yielding from 1 to 10 mg tar, were smoked on an automatic cigarette smoking maAine that collected both mainstream (HS, inhaled) and sidestream (SS, between puffs) smoke. The collected MS and SS cigarette smoke condensates were evaluated for mutagenicity by the Ames test and compared to MS and SS condensates from a high tar cigarette. Both B(S and SS condensates were dissolved in DMSO and tested, at concentrations of 50-200 µg/plate, with the bacterial strain TA 1538, previously shown to be highly responsive too cigarette smoke condensate. The results for two experiments were averaged. Except for two of the cigarettes, the MS mutagenicities of the low tar cigarette smoke condensates were significantly reduced (5-507.), when compared to the high tar (23 mg) cigarette. Just the opposite result was found for the sidestream smoke condensates.. Most of the SS condensates produced more revertants/plate than the SS of the control (high tar) cigarette, indicating greater concentrations of mutagenic compounds (10-30%) in their SS smoke. The results will be discussed in terms of MS:SS distribution of known cigarette smoke mutagens. 414 MUTAGENICITY OF AZIDOALANINE: POSSIBLE ROLE OF TRANSAMINATION. J M LaVelle, J B Mangold, M R Mischke,Toxicology Progra.m, Section of Pharmacology and Toxicology and Section of Medicinal Chemistry, School of Pharmacy, University of Connecticut,'Storrs, CT. ~ 4eidoalanine (AZAL), a metabo-ilTe of azide is probably metabolized to produce an ultimate mutagenic species. TraIIsamination is a common ~; 415 means of metabolizing aminoacids; such reactions could lead to activation of AZAL. To test this, three azido- compounds were prepared and tested for mutagenicity in S. t himuriumastrain TA1530. Alpha-methyl substitution, whic _ nhibits trans- aminase action, virtually eliminate(Ymutagenesis, a finding consistent with postulated activation of AZAL via transamination. Further, azido- acetate (AA) was only weakly mutagenic. Since the expected transamination product, azido- pyruvate, should form AA upon further metabolism, the ultimate mutagen may form either before this point, or by an alternate reaction. Thus, AA and metabolites (such as TCA cycle inter- mediates) seem not to be involved in the produc- tion of the ultimate mutagen. Finally, the homo- logue proved several times more potent than AZAL . itself. Clearly, mutagenic potential is not limited to AZAL; other azidoaminoacids share this property, perhaps due to activation via trans- amination. Current findingsare thus consistent with the suggestion that transamination plays a critical role in activation of AZAL and, hence, in the mutagenic action of.azide. QUANTITATION OF UNSCHEDULED DNA SYNTHESIS (UDS) IN HEPATOCYTES FROM RATS TREATED WITH THE PER- OXISOME PROLIFERATOR WY-14,643 (WY). RC Catt- ley, T Smith-Oliver, BE Butterworth, and JA Popp, CIIT, RTP, NC. Sponsor: E Gross-Bermudez The _ peroxisome proliferator hepatocarcinogens lack genotoxic activity in in vitro assays. The effect ok-in vivo treatment with the potent carcinogen WY on DNA repair was quantitated in P344 rat hepatocytes. Peroxisomal palmitoyl CoA oxidase in isolated cells was elevated 9- fold by day 5 of WY gavage (50 mg/kg) and 13- fold by day 27 of WY feeding (0.1j). Cultures of these cells were incubated with H-thymidine and repair as UDS was measured in autoradio- graphs as net nuclear grains (NG). Neither gavage treatment for 1 to 5 days (NG = -3.8 WY 5 days vs. -3.8 ctrl) nor feeding for 27 days (NG = -4.6 WY vs. -4.5 ctrl) induced DNA repair. WY treatment partly blocked the in vitro repair response to 2-AAF (NG reduced , 30%). Cells from WY gavaged rats (0-5 day) were treated with H,02 (0.8 mM 3x) to evaluate the ability of UDS Eo detect repair which might be induced by peroxisomal metabolism. H202 did not induce repair or block 2-AAF-3nduced repair. Higher doses of H 0 (1.9-3.6 mM 3x) to cells from a naive rat iesulted in -20% of cells in repair. In summary, the peroxisome proliferator hepatocarcinogen WY lacked geno- toxic activity detectable as UDS in hepatocytes despite peroxisome proliferation due to in vivo treatment. _
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436 THE INFLUENCE OF HUMIC ACID ON TRACE METAL COMPLEXATION AND CHROMIUM SPECIATION. R A Stackhouse and W H Benson, Coll~r of Pharmacy and Health Sciences, Northeast Louisiana ~, LA. University, Monro-. . Dissolved organic"-cbmpounds (DOC), such as humic acid (HA), can affect the speciation of trace metals in natural waters. The physico-chemical form of metals may, in turn, affect their bio- availability and toxicity. Although 5dequate information is available concerning the influence on HA on the speciation of several trace metals (e..&., Cd, Cu), there is a paucity of information available on chromium. Dialysis and diffusion cell studies were used to determine the influence of HA (0, 0.5, 5 and 50 mg/L) on the bioavail- ability of selected chromium species (Cr III, Cr VI and chrome lignosulphonate). Daphnia pulex was used as an animall model to resolve HA's influence on toxicity. The dialysis studies revealed that increasing concentrations of HA decreased the bioavailability of Cr III while having no significant effect on Cr VI and chrome lignosulphonate. These results were reflected by a significant decrease in toxicity of Cr III in the presence of the 5 and 50 mg/L HA concentra- tions and the absence of any effect of HA on the toxicity of Cr VI. However, the 50 mg/L concen- tration of HA did significantly decrease the toxicity of chrome lignosulphonate. These results indicate that the influence of HA is dependent on HA concentration as well as metal speciation. ;0: 437 SEQUESTRATION OF ENVIRONMENTAL CADMIUM IN GILL AND LIVER CYTOSOLIC PROTEINS OF THE FRESHWATER TELEOST, Lepomis macrochirus. C F Watson, K N Baer, and W H Benson, College of Pharmacy and Health Sciences, Northeast Louisiana University, Monroe, I,A. Industrial processes, agricultural practices, and fossel fuel combustions provide a multiplicity of point and diffuse sources of cadmium contamination. Differential sensitivity to environmental cadmium has been observed in freshwater teleost species. There is, however, a paucity of information concerning the biochemical basis for such differential susceptibilities in aquatic species. It has been suggested that metallothionein (MT) and other metal-binding proteins function in the metabolic detoxification and storage of cadmium. It is further postulated that "spill-over" from the MT-like proteins to low-molecular weight (LMW) proteins may correlate with the onset of pathological.damage. Recent investigations in our laboratory have indicated that acute exposure of bluegill sunfish (Lepomis macrochirus) to 1, " 10, and 100 ug Cd/L produced a characteristic profile of cadmium distribution in the cytosolic fraction of the gill and the liver. Of the three classes of cadmium-binding proteins identified, the MT-like proteins consistently sequestered the highest percentage of metal. The cadmium-, copper-, and zinc- cytosolic protein interactions reported may further delineate the cellular aspects of cadmium toxicity in freshwater teleosts. ~ :.. 438 THE DISPOSITION OF URL-14C-DICOFOL IN THE RING DOVE: THE QUESTION OF DDE AND EGGSHELL THINNING. B A Narloch, S E Schwarzbach and L RtShull. Departments of Environmental Toxicology and of Avian Science, University of California, Davis, CA. The disposition of URL-14C-dicofol in the ring dove was determined following a single oral dose ir of pure compound. Ring doves_z %ji&tained on com- - m~rcial feed and a 16-hour light/day reproductivtz= ,' cycle were exposed to a.single dose of 50.0 mg , (4.5 uCi)/kg b.w. URL=14C=dicofol by gastric intubation. Fecal samples were collected at 24- hour intervals and analyzed for parent compound, degradation products and metabolites. Eggs layed post-exposure were collected and~s hel thickness was measured. Groups of birds ere sacrificed at 24, 48 and 72 hours, necropsied and organs and tissues were analyzed for radioactivity. By 72 hours post-exposure, 35.7% of the dose was eliminated in feces. The organs/tissues with the highest amounts of radioactivity present after 72 hours were liver (1.0% of dose), gastrointe- stinal tract (4.6%), fat pad (5.2%), and carcass (55.4%). The oviduct retained 0.2% of the dose after 72 hours, with the highest amounts of radioactivity present in the shell gland and the developing follicle. Metabolic profiles were obtained for liver, fat, oviduct, eggs and feces, with emphasis on the presence or absence of DDE. 439 EFFECT OF REFERENCE HEPATOTOXINS UPON HEPATIC INTEGRITY AND P-450 ISOZYMES IN THE WINTER FLOUNDE~t (P Fl? (1jt N AMERIS~ffiIS) . K M Kleinow ~ B F Droy , D R Buhler and D1E Williams . Louisiana State te ~Jniversity Baton Rouge, LA., West Virginia Universit~Z Morgantown, WV., Oregon State University , Corvalis, OR. Sponsor: J J Lech. Induction of hepatic P-450 in fish exposed to environmental xenobiotics has received attention as a biological monitoring tool for the screen- ing of aquaG4c environments impacted by pollu- tants. Oftein hepatotoxins are present in con- junction with inducers in polluted waters and therefore reference information is required on, this possible interaction. The inducer beta-naphthoflavone (BNF)(100 mg/kg IP) and ref- erence hepatotoxins jcarbon tetrachlo- ride(CCL4)(1 ml/kg) or allyl formate (AF)(75ug/kg)(via gavage)] were administered alone and in combination as single administra- tions to (160-450g) flounder. The BNF, CCL4, and AF were administered respectively 5, 1 and 2 days prior to sacrifice. Polyclonal antibodies against BNF inducible trout LMA and constitu- tive LM2 isozymes were utilizedT for immunolog- ical staining of western blots. CCL~, exposure resulted in moderate declines in LI~f4, content and nearly comp lete loss of LM2 when compared to controls. BNF resulted in an induction of LM~ and appeareda to be artially, protective agAinst CCL4 mediated reductions in LM4 and LM2. No significant differences were noted for AF or, AF and BNF treatments. Serum trans- aminase levels and histology reflected the iso- zyme data. These data suggest that at the dos- ages utilized, inducer-hepatotoxin interactions may occur for select agents in the flounder. Support to KMK, By NIEHS Center for Membrane Toxicity Studies and, Markey Foundation - Mount Desert Island Biological Laboratory; Medical College of Wisconsin BSRG. 110 50875 8235
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404 HUMAN CARCINOGENS: DETECTION OF GENETIC TOXICITY. M.O.Shelby, NIEHS, Research Triangle Park, NC. Sponsor: J H Mennear. Twenty-three chemacals and chemical combinations have been dgsiftated by the International Agency for Research on Cancer as causally associated with cancer in humans. The literature was searched for reports of their activity in the Salmonella mutagenicity assay and for evidence of their ability to induce chromosomal damage in the bone marrow of rodents. The purlpose of this study was to determine the extent to which human carcinogens exhibit genetic toxicity in vitro and in vivo and to what extent they can be detected using these two widely employed short- term tests. Nineteen of the 23 carcinogens have been found active in one or both short-term tests. Two others, for which short-term test results are not available, are predicted to be active based on their structures. Asbestos and conjugated estrogens are not mutagenic to Salmonella; asbestos is not likely to induce cytogenetic effects in the bone marrow and the potential activity of conjugated estrogens in the bone marrow is difficult to anticipate. These findings indicate that genetic toxicity is characteristic of the majority of human car- cinogens. If the 23 IARC human carcinogens are representative of potential human carcinogens in general, then two short-term tests may serve as an effective rp imary screen for chemicals that present a carcinogenic hazard. 405 EXCRETION OF MUTAGENS BY GREENHOUSE WORKERS FOLLOWING EXPOSURE TO P_ESTICIDES B S Shane, J M Scarlett Krantz, W S Reid and D J Lisk, Louisiana State University, Baton Rouge, LA and Cornell University, Ithaca, NY. 9r~ Sponsor: C R Sh Deleterious effects on health following exposure of greenhouse workers to pesticides has become a concern in recent years. Attempts to assess exposure by measuring the concentration of the compound on clothing or by air monitoring has been inconclusive. Monitoring the excretory pattern of workers is mone informative with regard to exposure. In this study, the urine of twelve greenhouse workers, exposed to different spraying regimens was monitored for mutagens, eight hours after exposure to pesticide. These workers served as their own controls following assay of urine samples 72 hours after exposure. Urine was evaluated by both Salmonella tvohimurium TA 98 and TA 100 for frameshift and base pair mutagens respectively. A 9000 x g (S9) preparation from Aroclor- pretreated rats was used as the activating system. Five workers demonstrated urinary mutagens following exposure to benlate, orthene, kelthane and sumithrin. Benlate provides an equivocal response in the Ames mutagenicity assay whereas orthene has been shown to be mutagenic in short term tests. An inverse relation was observed between the use of protective clothing and the absence of mutagens. As excretion of mutagens in urine is indicative of exposure to deleterious genotoxic agents, use of a monitoring system as described in this study is useful in assessing the health effects of pesticides. 406 STRUCTURE-ACTIVITY RELATIONSHIPS OF PYRROLI2IDINE ALKALOID INDUCED G_ENOTOXICITY. J.R_ Hincks, H Y Kim, H.J. Segall and R.A Coulombe, Jr. Graduate Programs in Toxicology and Molecular Biology and Biochemistry, Departments of Veterinary Sciences, Utah State University, Logan, UT, and Pharmacology and Toxicology, School of Veterinary Medicine, University of California, Davis, CA Pyrrolizidine alkaloids (PQQ,,4,.-~ve been shown t8 ~yinteract with cellular DNA in a number ofa. vitro assay systems. Further characterization qf,_, the mechanism by which: PAs interact with cellular'_:-= DNA may be useful in understanding the carcinogenic and antineoplastic capabilities of these compounds. In this study, we investigated the potential DNA-interstran -(DD) and DNA- protein (DP) cross-linking acvi~' of seven PAs in cultured Madin-Darby.bovine kidney epithelial cells (MDBK) using gravity-flow alkaline elution. Cells were cultured two hr with the PA (100, 300 and 500 uM) and rat liver S9 containing a NADPH- generating system. The alkaloids which produced either DD or DP cross-links (ranked from highest to lowest) were: seneciphylline (DP > DD), riddelline (DP > DD), retrorsine; (DP > DD), senecionine (DP > DD), monocrotaline (DD > DP), heliosupine (DP only), retronecine (neither DP nor DD). None produced DNA single-strand breaks. Since the tested PAs differ only in the diester . substituent of the pyrrole ring, the characteristics of these groups may be an important determinant in the genotoxicity of pyrrolizidine alkaloids. (Supported by ES 03591). 407 LACK OF CORRELATION BETWEEN THE DEBRISOQUINE POLYMORPHISM AND AFLATOXIN B1 (AFB1) GENOTOX- ICITY. C A McQueen, B M Way and G M Williams. American Health Foundation, Vallaha, NY Hydroxylation of debrisoquine is under polymor- phic genetic control. Two phenotypes, poor me- tabolizers (PM) and extensive metabolizers (EM) have been identified. Studies in humans and rats have suggested that the debrisoquine polymorphism plays a role in susceptibility to AFB1 toxicit*.. The present study was undertaken to compare AFB1 genotoxicity in hepatocytes iso- lated from two rat strains of the EM phenotype, Lewis and F344, and from DA rats, of the PM phenotype. DNA repair, determined by autoradiography, was used as a measure of DNA damage. AFB1 induced DNA repair in hepatocytes from all strains, but quantitative and qualita- tive differences were observed. At 10-4M, AFB1 was toxic to hepatocytes from Lewis rats (EM) while maximum repair was observed at 10-5 to 10-6M. Maximum repair was observed at 10-4M with hepatocytes from both F344 (EM) and DA rats (PM). Thus, no correlation was found between phenotype and AFB1 genotoxicity. Nevertheless, the results suggest that Lewis rats should be more susceptible to AFB1 toxicity than DA or F344 rats. 50875 8227 102
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I i 432 RESIDENCE TIME AND TUMOR-INITIATING ACTIVITY OF BENZO[A]PYRENE AND A COMPLEX MIXTURE. D D Mahlum. Battelle, Pacific Northwest Laboratory, Richland, WA " _ It is of bqth bpsic and pragmatic interest to know how long a carcinogen needs to be in con- tact with the skin in order to be effective in initiating tumo`rs. We studied the effect of residence time on initiating activity of BaP and a complex organic mixture on mouse skin. BaP (25 ug) or a coal-derived heavy distillate (HD; 25 mg) was applied to the skin of female CD-1 mice. At times varying from 10 minutes to 24 hours, the skin was washed with soap and water. Control mice were initiated but not washed. After two weeks all animals were promoted twice weekly with 5 ug of TPA for 24 weeks. Tumor incidence and tumors/mouse were monitored biweekly. BaP controls had a 93% incidence with a mean of 5.3 tumors/mouse; HD controls had an 83% incidence with a mean tumor yield of 2.1 per mouse. Washing BaP mice 10 min after administration reduced the incidence and the tumor yield; washing after 30 or 60 minutes reduced the tumor yield, but not the incidence. Washing HD mice up to 4 hr after application reduced both the incidence and the tumor yield significantly. These data suggest that initiation occurs relatively quickly when BaP is used in ug amounts. How- ever, washing is effective for longer periods when a mixture of carcinogens is applied in mg amounts. U.S. Dept. of Energy Contract DE- AC06-76RL0 1830. 433 FAT, VITAMIN A DEFICIENCY AND CALORIC INTAKE COLON CARCINOGENESIS. PM Newberne, D BUECHE, and TF Schrager. Mallory Institute of Pathology. Boston, MA Epidemiological studies have correlated high fat intake with increased risk of colon cancer. Experimental studies have indicated that high intake of unsaturated fat after carcinogen exposure can significantly increase colon tumor incidence suggesting a promotional effect. In this laboratory no enhancing effect of high fat diets on colon carcinogenesis was demonstrated, after using both direct and activated carcinogens and different types of fat at different stages of the carcinogenic process. However, in rats on a high fat diet (24% corn oil) and made vitamin A deficient, tumor incidence was.significantly increased from 52% to 75%, P< 0.01. High levels of vitamin A were not protective. This suggests a more complex interaction; in some studies vitamin A levels may not have been controlled. In a separate set of experiments, the effect of postnatal alteration of caloric intake on colon cancer was examined by reducing liter size at birth from 8 to 4. This allowed greater access to milk (calories). Rats at weaning ate ad libitum and then were dosed with DMH. Increased access to food (calories) only from birth to weaning significantly increased colon tumor incidence (85% vs 48%), even when rats were pair fed to control rats. This indicates the complexity of dietary factors on colon carcino- genesis. 434 435 109 EFFECT OF HEATING OR THE 4MtSENCE OF VEGETABLES AND FRUIT IN HUMAN DIETSl ON THE SPONJANEOUS TUMOP, RATE IN RATS. T M Alink , H A Kuiper , R B Beems , and J H Koeman. iDepartment of To~i.cology, Agricul- tural University, Wageningen, Institute for Quality Contr3ol of Agricultural Products (RIKILT), Wageningen, TNO-CIVO Toxicology and Nutrition Institute, Zeist, The Netherlands. Sponsor: V J Feron. - 0. To study the influence of di4"actors like total ~and' ~f composition, thermal processing and vegetabl' fruit on tumor rate a;,.chronic experiment was L; designed. Five diet groups were tested in 100 ~' Wistar rats each. Diet A was a semisynthetic ~ animal diet, diet B an animal diet to which vegetables and fruit were added, diet C a human diet with raw products, diet ~-human diet with fried and baken products and Siet,E a complete ., human meal consisting of heated products, vegeta- bles and fruit prepared according to mean consump- tion figures in the Netherlands. The animal diets consisted of 25 E % (E=energy) protein, 14.5 E % fat, 60.5 E % carbohydrate and 3.5 % (w/w) fibre. For the human diets these figures were 13, 40, 47 and 5 % respectively. Animals were treated for 142 wk and fed ad libitum. In male rats but not in female rats human diets gave a higher tumor inci- . dence than animal diets (p < 0.01). Frying and baking, and vegetables and fruit induced minor differences in tumor rate, although these differences were, not statistically significant (p < 0.1) further research is needed to get more conclusive results. LONG TERM EFFT;CTS OF CLOFIBRATF TBEATMENT IN THE PRIMATE (CALLITHRIX JACCHUS ) M J Tucker, ,I-f,- TOUheID. A M Marsden'ICI PlC Pharmaceuticals Div. Macclesfield U K Lipid lowering agents in the rat produce liver enlargement due to proliferation of peroxisomes and smooth endoplasmic reticulum; long term administration of these compounds to rodents produces liver cell tumors. Clofibrate is a hypolipidaemic agent which has been used in man for more than twenty years; it has been shown to prolifera#2 peroxisomes and produce liver cell tumours in the rat; in the non-human primate, the marmoset Callithrix jacchus the effect of administration of clofibrate at.levels of 94, 157, 218 and 263mgs/kg for 343 weeks was studied There was no evidence of toxicity from daily observation, body weight records, veterinary examination, ophthalmology or pathological examination.No carcinogenic effect was seen in the liver or any other organ.
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APPLICATION OF PREIMPLANTATION RODENT EMBRYO CULTURE SYSTEMS TO MONITOR THE ABORTICATIVE TOXICITY OF AFLATOXIN B1. P C Mertes and T R Irvin. Lab of Toxicology, Veterinary Anatomy Department, Texas A&M, Col 1 egL-Stati on, Texas Sponsor: A C Ray ' e -s We have characterized the in vitro developmental taxicity of aflatoxin B via preimplantation mouse embryo culture to compare in vitro the embryotoxicity of these compounds with prenatal toxic effects observed in vivo. Whole mouse sembryos, explanted 48 hours after conception, were cultured in serum-supplemented Eagle's basal media for 96 hours with each test compound and ultrastructurally evaluated for toxic indices. Over a 10 fold dose range, AFB1 alone demonstrated minimal embryotoxicity; after culture supplemention with rodent hepatic S-9 fractions isolated from animals preadministered Aroclor 1254, embryolethality . as well as embryotoxicity was observed Ultrastructural effects of AFB1 in culture included decreased embryo hatching, attachment, and inner cell mass. These findings parallel reports in vivo of the prenatal toxic effects associated with AFB1 and strongly supports application of this test system for testing environmental agents suspect as prenatal and aborticative toxicants. 457 COMPARISON OF DYSMORPHOLOGY INDUCED IN VITRO BY TWO YOLK SAC ACTIVE TERATOGENS IN RAT. F Elder- kin, R Marlow and S J Freeman. SK&F Research Ltd., Welwyn,, Herts, UK. Sponsor: J B Hook The placental function of the visceral yolk sac (YS) of the early post-implantation rat conceptus is well established as a target for certain teratogens. Especially sensitive to insult are the processes of pinocytic uptake and degradation of protein by YS. Interference with either of these processes is considered to produce a similar nutritional deficit and consequent embryonic malformation: this latter point however has not previously been demonstrated. Using 48h rat whole embryo cultures, we have compared the dysmorphogenic action of 2 agents that are teratogenic through an effect on YS: leupeptin (proteolysis inhibitor) and anti-YS antiserum (pinocytosis inhibitor). At the lowest dysmorphogenic doses (i~ag/ml leupeptin; 10fd/ml anti-YS) the pattern of abnormalities was similar: retardation or absence of optic vesicles and some growth retardation but no apparent YS pathology. At higher doses (2,ag/ml leupeptin; 15,u1/ml anti-TS) gross brain deformities and severe growth retardation were evident but still YS appeared normal. These data demonstrate that, as anticipated, disturbance of YS placental function in either of 2 ways produces similar embryonic dysmorphology and, furthermore, occurs in the absence of gross effects on YS appearance. 115 458 INHIBITION OF DNA SYNT$ES3'gIN WHOLE EMBRYO CULTURE BY _METHOXYACETIC ACID (MAA) AND ATTEN- UATION OF THE EFFECTS BY ACETATE AND FORMATE. F` Welsch, and DB Stedman, C.I.I.T., Research Triangle Park, NC. ` 2-Methoxyethanol (ME) is oxidized to MAA which is the proximate toxin. ME is ineffective in whole embryo culture (WEC), while MAA is em-: bryotoxic. We have shown that the teratogenici, effects of ME in vivo are a tenuated by simple:. ;iphysiological compounds, among them acetate (A.) • and formate (F), which provide carbon-._f4i` puri~~ and pyrimidine ' bases in DNA. Label from'*~__ 1,2- C-ME was present in all macromolecular !;i- fractiqR s of embryos and dams, and dams ex- haled CO2, suggesting that MAA was further metabolized and that this mip3ht..k~ relevant to embryotoxicity. The presentll WEC~"`experiments were designed to distinguish maternal from em- bryonal effects of MAA. Embryos on gestation day 11 (40-44 somites; maximally sensitive to ME in vivo) were maintained in WEC for 6 hr in serum free medium. The WECs were exposed to JAA, A, F or J~AA+A and MAA+F for 5 hr before H-thymidine ( H-T; 5 µCi/culture bottle) in- corporation into DNA was measursd during the last hour. MAA (5 mM) reduced H-T in DNA by 50%. Neither A (5 mM) nor F(1 mM) affected DNA synthesis. However, when added to WEC together with 5 mM MAA, both A and F completely abol- ished MAA's effects. These findings suggest that A and F block the entry of MAA into react- ions that inhibit DNA synthesis in embryos. 459 ATTENUATION OF THE INCIDENCE OF 2- METHOXYETHANOL-INDUCED DIGIT MALFORMATIONS BY SARCOSINE. C Mebus and F Welsch. Chemical Industry Institute of Toxicology, Research Triangle Park, NC The ethylene glycol ether 2-methoxyethanol (ME) is selectively embryotoxic and teratogenic in several species. Results from other studies led to the conclusion that methoxyacetic acid (MAA), the oxidation product of ME, is the proximate teratogen. However, our data reveale14 the incorporat~n of label derived from 1,2- C-ME into macromolecular fractions of the embryo suggesting that MAA enters into further biochemical reactions. Sarcosine (S) is an intermediate in the biosynthesis of glycine and a structural analogue 'of MAA; MAA inhibits S oxidation to glycine. The entire backbone of glycine is incorporated into the purine ring. In the present experiments mice received concurrent oral doses of 3.3 mmol ME/kg and 43 mmol S/kg on gestation day 11. Animals dosed with ME alone exhibited digit malformations (any digit) in 47% of the fetuses or 83% of litters. S significantly reduced the digit malformation incidence (p < 0.05) to 8% of the fetuses or 42% of litters. This outcome suggests that MAA might inhibit purine biosynthesis by depleting glycine pools in the embryo. Another mechanism leading to disruption of purine and pyrimidine supply, supported by earlier results, indicates that a product of further MAA metabolism might interfere with one-carbon moiety availability. 50875 8240
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HE EFFECT OF 2,3,7,8-TETRACHLOROOIBENZO-p-DIOXIN TCDD) ON HEPATIC GLUCURONIDATION OF RETINOIC CID. P A BANK, K L SALYERS, AND M H ZILE, ept. of Food Science and Hum,an Nutrition; ichigan State Universi,ty,aJ. Lansing, MI. ponsor: S Sleight fCOD and related compounds dause a decrease in iver vitamin A reserves and an increase in uri- ary and fecal excretion of vitamin A metabo- lites. No correlation has been established for CDD increased UDP-glucuronosyltransferase (UD?_ GT) activity toward the substrate p-nitrophenol and decreased hepatic vitamin A concentration. We have investigated the effect of TCDD on hepatic vitamin A metabolism in male Sprague- Dawiey rats receiving a single oral, dose of 10 ug TCDD/kg. Ten day post-exposure to TCDD there was a 30% reduction in hepatic vitamin A esters as compared to pair-fed controls. The hepatic microsomal UOP-GT activity toward the substrate 3H-all-trans-retinoic acid (RA) was five-fold greater for TCDD-treated rats compared to pair fed contro~s. Verification of the in vitro for- mation of H-retinoyl glucuronide (RG) wds by cochromatography of authenic R§ ori reverse phase HPLC and by identification of H-RA as the hydro- lysis product after B-glucuronidase treatment. Increased retinoic acid ylucuronidation may be a contributing factor to the hepatic depletion of vitamin A following TCBD exposure. Supported by NIH grant ES 05347-03 and by USDA grant 87-CRCR- 1-2449. FACTORS INFLUENCING THE INDUCTION OF DNA SINGLE STRAND BREAKS IN RATS BY 2,3,7,8-TETRACHLORO- DIBENZO-P-DIOXIN. Z Z Wahba, S J Stohs, T A Lawson, W J Murray. University of Nebraska " Medical Center, Omaha, NE. TCDD induces hepatic DNA single strand breaks (SSB) in conjunction with lipid peroxidation in. rats. The mechanism involved in DNA damage by TCDD is not known. The oral administration of 100 µg TCDD/kg resulted in 1.3-, 4.3-, 7.2- and 7.8-fold increases in DNA elution constants 3, 7, 10 and 14 days post-treatment, respectively. Similar changes occurred in hepatic nuclear lipid peroxidation as determined by the content of thiobarbituric acid reactive substances (TBARS). Treatment of rats with the dithiol- thione antioxidant oltipraz (30 mg/kg/day) inhibited the formation of DNA SSB by TCDD. Incubation of hepatic nuclei from control animals with microsomes, mitochondria and cytosol from rats 3, 7, 10 and 14 days after treatment with TCDD resulted in increased DNA elution constants of 1.2-, 1.9-, 2.0- and 2.3- fold, respectively. Mitochondria and microsomes but not cytosol from TCDD treated rats enhanced DNA damage in nuclei from control rats. The addition of the iron chelator desferrioxamine inhibited in vitro DNA damage by mi.crosomes and roitochondr+~ from T9D treated rats. The addi- tion of Fe and Fe to nuclei from control animals enhanced DNA damage. Thus, TCDD.induced DNA SSB involves iron and the formation of reactive oxygen species anO/or free radicals. 91 362 2,3,7,8-TETRACHLORODIBENZO-P-DIOXIN INDUCED LIPID PEROXIDATION IN RESPONSIVE AND NON-RESPONSIVE MICE. S J Stohs and H Mohammadpour. University of Nebraske Medicei ,.. _ Center, Omaha, NE. TCDD induces lipid peroxidation (LP) in hepatic and extrahepatic tissues of rats. Induction of LP in rats by congeners of TCDD correlates with induction of aryl hydrocarbon hydroxylase (AHH) activity. .,IThe effect of TCDD administra*bw on LP in various strains of mice was determined. The hepatic content of thiobarbituric acid reactive substances (TBARS) was used as an index of LP. Six days after a single oral dose of 0.5 pg TCDD/kg to congenic male C57BL/6J rice, which were either homozygous (bb) or heterozy- gous (bd) with respect to the gene for the_-.Ah.V locus, significant increases in hepatic )SP were induced. Hepatic LP was induced in homozygous non-responsive (2.d) mice by a dose of 25 pg TCDD/kg but not 0.5 pg/kg. In kidney, heart andd testis, lower doses of TCDD produced LP in bb and bd mice as compared to dd mice. A dose of 2500 µg TCDD/kg produced the same level of LP in DBA/2J mice that was observed with 180 pg TCDD/kg in C57BL/6J bb and B6D2F1/J mice. LP was not induced in kidney, heart or testis by TCDD in DBA/2J mice. A strain difference exists in the induction of LP by TCDD. In the liver, the ability to induce LP by TCDD is controlled in part by the Ah "b" allele, although other loci may play a major role. 363 THE EFFECT OF 2,3,7,8-TETRACHLORODIBEN- ZO-p-DIOXIN (TCDD),2,3 DICHLORODIBENZO- p-DIOXIN (DCDD),AND 2,3,7 TRICHLORODI- BENZO-p-DIOXIN (TrCDD)ON aYTOCHROME P450 GENERATED REACTIVE OXYGEN. D S Brandwene, P C Kahn. Rutgers Uni- versity, Joint Graduate Program in `. Toxicology, Piscataway, NJ. Sponsor: G Witz. Studies have implicated reactive-oxygen as a possible mechanism of toxicity for TCDD.. We compared the effects of TCDD, DCDD, and TrCDD on hydrogen peroxide production from TCDD induced rat liver microsomes. Male Sprague-Dawley rats were given a single oral dose of TCDD (25 ug/kg) and sacrificed 3 days later. Microsomes were assayed for hydrogen peroxide production in the presence of TCDD,DCDD, or TrCDD as substrate. Con- centrations of 10-6M-10-9M were used for all three congeners; hydrogen peroxide was measured at 3,7,11, and 15 minutes. There was no significant difference between TCDD, DCDD, and TrCDD treated microsomes in hydrogen peroxide pro- duced. TCDD at a dose of 40 ug/kg in- hibited glutathione peroxidase activity 39% compared to corn oil controls. These results suggest that a decrease in the metabolism of active oxygen species may play a role in TCDD toxicity. 50875 8216
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348 PATHOLOGY OF SK&F L-94901, A POTENT THYROMIMETIC . IN THE RAT. S J Kennedy, C K Atterw311 & A Poole; Smith Kline & French Research Ltd, The Frythe, Welwyn, Herts, U.K. Sponsor: J Hook. a A potent thyra'nimetic, SK&F L-94901, was designed for the treatment. of hyperlipidemia and had been demonstrated to be'devoid of the adverse cardiac side effects which limit the use of conventional non-selective thyromimetics. However, oral administration of this compound to rat,s,revealed several proliferative lesions. SK&F L-94901 was given to rats over a period of 30 days at dose levels between 0 and 100mg/kg/day. A positive control group of lmg/kg/day of L-triiodothyronine (L-T ) was included in an attempt to separate a toxi2 from a thyromimetic effect. SK&F L-94901 produced treatment-related lesions in three main sites: bone, serosal surfaces and kidney. The bone changes were a focal deposition of new trabecular bone with associated spindle cell proliferation in the medullary cavity. In the kidney there were multi-nucleated epithelial cells in the distal tubules, and in the abdominal cavity there was focal capsular fibrosis primarily on the serosal surfaces of the liver and spleen. The rats which received L-T3 had a low survival due to extensive myocardial damage, and there was a low incidence of lesions similar to those mentioned above. The proliferative lesions were therefore attributed to a thyro- mimetic effect, and the possible association with a local over-production or ampli'cation of growth factors will be considered. ` 349 HIPPOCAMPAL ~IISTOPATHOLOGY IN -ETHANOL- EXPOSED RATS. R M Sioco, N D T Nguyen, E J Root, S W Leslie, and E M B Sorensen. Division of Pharmacology and Toxicology, College of Pharmacy, University of Texas, Austin, TX. Following eight-week exposures of Sprague-Dawley rats to ethanol (37% caloric substitution), brains were preserved for optical and electron microscopy or impregnated using the Golgi-Cox staining procedure. Dense bodies were observed in processes and excess lipofusion was observed at the electron microscopy level of ethanol-fed (EF), but not pair- fed (PF) or chow-fed (CF) controls. Morphometric analyses indicated that surface density of the processes of EF rats , were significantly different than those of CF rats. With ' respect to hippocampal areas, PF rats also were statistically different from CF rats. Areal measurements of whole brain, -hippocampai space, and ventrical space showed no statistical differences between EF and CF rats. However, differences . were evident in the surface density of the processes, whole brain areas and hippocampai areas of PF and EF rats. 88 350 351 EFFECTS OF DOXYLAMINE SUCCINATE ADMINISTERED.TO FISCHER 344 RATS FOR 65 WEEKS. C D Jackson,-G 21 Cronin, and B N Blackwell,. National-Center for Toxicological Research and Pathology Associates Inc., Jefferson, AR. Sponsor: G W Wolff. Doxylamine succinate, a commonly used antihista- mine, was administered in the feed to male and °' female Fischer 344 rats at dose levels of 0, ~ 500, 1000, and 2000 ppm. Food,4momwmption, body `, we4ghts, and signs of clinical toxicity were-~, determined weekly. Gross• and microscopic path- a~..;:.., ology examinations were performed. Weight gain was depressed greater than 1oX in the 2000 ppm dose group of females only. Liver weights were .elevated in the two highest dose levels for males but no effect was observ ilh', females. Serum enzyme levels of ALAT andSDS~ were ele- vated in the 2000 ppm dose level in males. In males, reticulocyte counts were elevated in the 1000 ppm and 2000 ppm dose groups and hemoglobin levels were decreased in the highest dose group. Both sexes exhibited a dose-response effect for cytoplasmic alterations of the acinar cells of the parotid salivary gland. Dose-related fatty change occurred in all treated groups of males and the highest dose group of females. Centri- lobular hypertrophy of the liver was observed in the 1000 ppm and 2000 ppm dose groups of females but none was observed in males. These results indicate the need for longer term studies of . doxylamine for possible carcinogenicity activity in the rat. LOW DIETARY BIOAVAILABILITY OF OXALIC ACID PRESENT IN REFINED SUGAR BEET PULP COMPARED To SPINACH AND SODIUM OXALATE. C F Hanson, Okla. .. State Univ., Stillwater, OK, V H Frankos, ENVIRON Corp., Washington, DC, W 0 Thompson, Med. Coll. GA., Augusta, GA. Sponsor: R G Tardiff Bioavailability of oxalic acid from common foods varies widely. Oxalate availability from a high fiber food- Sugar Beet Pulp (SBP)- compared to spinach and so$ium oxalate, was tested in nine women using a triplicated 3 by 3 latin square arrangement with a 2 day control period and 3 test periods consisting of a test day followed by a control day. Test material which provided 120 mg oxalate was consumed at breakfast on test days. Subjects consumed the same diet every day. Intake of test materials was 40 g beet pulp, 25 g spinach, and 182 mg sodium oxalate. Total 24 hr urines were collected and analyzed daily for oxalate. Oxalate excretion was similar follow- ing spinach or sodium oxalate ingestion (23.6 • and 25.7 mg/day), however, it was higher (P<.05) following sodium oxalate intake than fcllowing beet (19.0 mg/day) or control (18.2 :--g/day) diets. Oxalate excretion did not differ among control days and was not significantly increased with SBP consumption. Low bioavailability of oxalate from SBP may be attributable to its high ratio of calcium and magnesium to oxalate, its high fiber content or the loss of soluble oxalate during processing. 50875 8213 I 53
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I1IIiIBITION OF HUMAN SERUM COMPLEMENT (C') ACTIVITY BY DIISOPROPYLFLUOROPHOSPHATE (DFP) AND SELECTED A_NTICHOLINESTERASE INSECTICIDES. S Bavari, J J Connolly, and 5 P Casale. _1Tniv. Of Nebraska Medical Center, GQllege of Pharmacy, Qnaha. NE. Activation of the human C'~systen, a major line of defense against infections, requires the participation of serine esterases. The insecticides carbaryl (Ca), carbofuran (Cf), j ~ diclzl.orvos (DDVP), and paraoxon (Ps) inhibit serine esterases including CHE.. The present study evaluated potencies of the latter to inhibit C'-mediated lysis of sheep red cells (SRC), by a panel of 6 normal, human sera. Test chenicals were added to diluted sera, just prior to incorporation into C' reaction mixtures. Potencies to inhibit C' and serum cholinesterase (CHE) were compared to potencies of DFP, a potent serine esterase inhibitor. At 0.5 to 3.0 mM, Ca, Cf. DDVP, and DFP produced a dose-dependent inhibition of lysis, while Ps was not inhibitory. On a molar basis. Ca was 3 times more potent than DFP, and inhibited lysis 15-25% and 26-45% at 1.0 and 3.0 mM, respectively. Cf. DDVP and DEP were equipotent. Mean IC50s for inhibition of CHE by DEP, Px~ DDVP, Cf, and Ca werg 1.Ox10 8M, 2.Os10"M, 1.Qa10 7M, 3.3s10 °M, and 1.8s10-5M, respectively. Potencies of insecticides to inhibit CHE did not predict absolute or relative potencies to inhibit human, serum C' activity. Supported by NAPIAP, ES/USDA. THE IMMUNOMODULATORY ROLE OF D-[ALA2] METHIONINE ENKEPHALINAMIDE IN INHIBITION THE TUMOR EXPRESSION OF PYB6 TREATED MICE. B Srisuchart, L E Sikorski, A E Munson. S E Loveless. Dept. of Pharmacology and Toxicology, Medical College of V3rginiaNCU, Richmond, VA, and E I duPont de Nemours & Co, inc, Glenolden, PA. Tumor growth has been shown to be influenced by the endogenous opioid system. The objective of the present study was to investigate the immunomodulatory role of the synthetic peptide, D-[Ala2jmethionine enkephalin- amide (AS), in inhibiting tumor growth of the PYB6 fiibrosarcoma. Daily intraperitoneal injections of AS (between 0.5 and 1 mg/kg) for 7 days decreased tumor size. The magnitude of the antitumor response by AS was a function of the number of tumor cells transplanted. Pretreatment with the opioid receptor antagonist, naloxone (0.05-4.00 mg/kg), reversed the suppressive effects of AS on tumor growth, suggesting that the pharmacological action of AS may be mediated via the opioid receptor. AS did not directly affect the viability of PYB6 tumor cells, as evaluated by the measurement of cellular ATP levels in AS treated PYB6 cells. Therefore, the antitumor activity of AS acts via an indirect mechan- ism. Immunological studies indicated that AS selectively enhanced the lymphoproliferative response to the T-cell mitogen, concanavaiin A, but not to the B-cell mitogen, iipopolysaccharide. Natural killer cell activity and the number of IgM plaque forming cells in response to the T- dependent antigen, sheep red blood cells, were unaffected after both in vivo and in vitro exposure to AS. (Supported by E I duPont de Nemours & Co. Inc.) 81 322 SUPPRESSION OF }j,UMORAL 1MMUNITY~8~~6ONO- NITROTOLUENES (A STRUCTURAL ACTIVITY STUDY). H H Lysy, J A McCay, K L White. Jr, and A E Munson. Depts. of Pharmacology and Toxicology,. and- Biostatistiss. - Mec(ical College of VANCU,•Richmond, VA. Mononitrotoluenes are intermediates in the manufacture of a variety of products, from explosives to pharmaceu- ticals. It has been estimated that over 8000 occupational exposures occur annually. The effects of toluene and three montanitrotoiuene isomers on humorai+wirrt[nunity were evaluated. Female B6C3F1 mice were exposed by oral gavage to toluene, 2-nitrotoksene (2-NT), 3- nitrotoluene (3-NT), or 4-nitrotoluene (4-NT) for 14 days: Doses for 3-NT and 4-NT were 200, 400, and 600 mg/kg/day. Toluene and 2-NT were administered at 600 mg/kg/day. The IgM antibody forming cell response to the T-dependent antigen, sheep erythrocyjps; v..as suppressed in a dose dependent manner. Th4 greatest suppression observed was 38% and 61% for 3-NT`and 4-NT, respectively. Toluene and 2-NT did not alter the IgM response. The IgG antibody forming cell response to sheep erythrocytes was not suppressed by any of the chemicals. A slight trend toward a decreased lymphocyte proliferation response was observed only with 4-NT. Host resistance assays to bacterial challenges revealed that susceptibility to Streptocococcus pneumoniae was not altered, while susceptibility to Listeria monocyto- genes was enhanced when animals were exposed to the nitrated toluenes. These results suggest that the immunosuppression observed with the nitrotoluene compounds is related to both the presence and position of the nitrate moiety. (Supported by NIEHS contract ES 55094). 323 IMMUNO'!'OXICOLOGY IN THE RAT: _ AN II+PRUVED MODEL. J L Bussiere, J H Exon and G G Mattier. Dept. Veterinary Science, University of Idaho, Moscow, ID. Research in this laboratory during the past sev- eral years has been directed to developing a ccxYr- prehensive panel of assays to assess imnunotoxi cologic activity of chemicals and drugs. Partic- ular attention has been given to assays that are sensitive, reproducible, economical, sinple to ., perform and representative of the major aazms of the i.miame systen: " Furthezmore, the rat has been utilized as the animal ntodel due to the general familiarity of many toxicologists with this species and because a preponderance of general toxicologic testing is currently perfouned in the rat. We have previously reported an economical, multiple assay approach to innnziotoxicohogic testing in the rat (Exon et al., 1986). Herein we report sign.ificant improvements and expansion in that model that increase verstility and better facilitate application to a variety of toxicity testing protocols. These improvements include. 1) eliminatiQn of EYeumd's adjuvant fran the ant- igen treatmeat schedule, 2) use of a single anti gen to assess cell-madiated and htmroral immune resparises, 3) coordination of timing for testing DTH and antibody responses and 4) inclusion of several additional optional assays for diffRrent types of inarauie responses. 'Ihe improvermnts in this rat model should increase its attractiveness as an economical and versatile paradim to accani date tier 1 and 2 inanunotoxicologic assessment. 50875 8206
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372 - REDUCED GLUCONEOGENESIS IN TCDD-TREATED RATS. LWD Weber, JR Gorski and K Rozman, University of Kansas Medical Centef, Kansas City, KS U.S.A. and Institiit 'fur Toxikologie, GSF Munchen, Neuherberg, F.R.G. ' The effect of - 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 125 µg/kg) was studied on the conversion of 1gC-alanine into 14C-glucose in male Sprague-Dawley rats by established procedures (determination of alanine and glucose by enzymatic assays -6d isolation of 14C-alanine and 14C-glucose from whole blood by column chromatography). TCDD-treated rats converted only 1/2 as much 14C-alanine into 14C-glucose as did their pair-fed or ad libitum-fed counterparts indicating reduced gluconeogenesis in TCDD-treated rats. This finding suggested that reduced gluconeogenesis in TCDD-treated rats may contribute to the profound hypoglycemia observed in intoxicated animals. Corticosterone, a key hormone in gluconeogenesis, provides partial protection from TCDD-induced toxicity in hypophysectomized (HXD) rats. Therefore, the conversion of 19C-alanine into 14C-glucose was also determined in HXD rats. dosed with TCDD (125 µg/kg) and given corticosterone (25 µg/ml in drinking water). These rats also converted only 1/2 as much 19C-alanine into 14C-glucose as did their pair-fed counterparts. However, in contrast to non-HXD rats, these rats maintained normoglycemia is even at 64 days after dosing with TCDD. It s concluded that TCDD reduces gluconeogenesis in rats but the protective effect of corticosterone in HXD rats is unrelated to gluconeogenesis. (Supported by NIH ES-07079) 374 373 PHARMACOKINETICS (PK) OF VOLATILE HALOCARBONS: 375 COMPARISON OF SINGLE ORAL BOLUS VERSUS GASTRIC . INFUSION OF 1,1,1-TRICHLOROETHANE (TRI). S Muralidhara, R Ramanathan,. J M Gallo*, C E Dallas, and J V Bruckner. Depts. of Pharmacol.. & - h rm~cs*, College of Pharmacy, Toxicol. and P a University of Georgia, Athens, GA. The objective of this study was to assess the influence of different patterns of ingestion on the PK of TRI, a halocarbon-contaminant of water- supplies. TRI was given to unanesthetized male S-D rats as an Emulphor emulsion, either orally as a single bolus or infused through a surgi- cally implanted gastric cannula over 2 hr at a, dose of 48 mg/kg. Blood samples were collected from an indwelling carotid arterial cannula from 0 to 540 min during and post infusion. The blood samples were analyzed for TRI using a GC-ECD headspace technique. Cmax, AUC, elimination half-life and other PK parameters were determined and compared for these two oral dosage regimens. Reductions in the AUC and - Cmax and increased terminal elimination half-life were observed when TRI was given by continuous gastric infusion. Similar experiments were also performed using a low dose of 6 mg/kg of TRI. These results indicate that the pattern of consumption of contaminated water can significantly influence the PK of TRI, and thereby likely affect the toxic potential of the chemical. (Supported by EPA Cooperative A reement #CR812267 and U.S. Air Force AFOSR 870248? TRICHLOROETHYLENE (TCE) AND THE AREA UNDER CURVES (AUC) FOR ITS METABOLITES IN BLOOD. J L Larson and R J BuTl. Pharmacology/ Toxicology Program, of Pharmacy, Washington State University, Pullman, WA. Mice have been shown to be more sensitive than rats to the hepatocarcinogenic effect of ~ TCE. Several studies have dem~s,trated that ~ t#ie metabolism of TCE is saturated at doses y above 1 g/kg in the rat.. It has been suggested that this saturation is responsible for the lower sensitivity in rats. The present study more carefully analyzes the blood concentration versus time AUC for TCE and metabolites at doses of 3.0Vkg-~and 0.6 g/kg TCE. Blood samples wefe collected at 1, 2, 4, 8, 12, 24, 48 and 72 hours following oral dosage of TCE in a 1% Tween 80 vehicle. The AUC for TCE increased by 6.3, and the AUC for the metabolites dichloro- acetic acid, trichloroacetic acid and trichloroethanol were increased 1.9, 2.3 and 3.0, respectively. Therefore, the saturating dose of TCE resulted in a substantial prolongation of the period of time that appreciable amounts of TCE and metabolites were present in blood. This must be . considered when assessing the impact of saturating, doses of TCE on carcinogenic response. (This work was funded by U.S. Air Force.Grant #AFOSR-86- 0284) PRECISION AND A_CCURACY OF PHYSIOLOGICALLY-BASED -` PHARMACOKINETIC MODELS IN REGULATORY RISK ASSESSMENTS. -F Y_Bois,'L Zeise and T N Tozer. Department of Pharmacy, University of California San Francisco and California Public Health Foun-.., dation, Berkeley, CA Sponsor: C C Willhite. Physiologically-based pharmacokinetic (PBPK) models have been applied to correct for inter- ' species and interdose differences when estimat- ing human ca~cer risk from animal data. Differ- ences in uptake, absorption, metabolism, excre- tion and tissue partitioning may explicitly be taken into account through these models. Model input parameters are derived from various in vitro and in vivo experiments, usually performed on relatively few specimens under conditions different from those of human exposure or the ` cancer bioassays. Inaccuracies in the input parameters influence the overall accuracy of the model predictions. Monte Carlo simulation is ' used to evaluate the precision of model predic- - tions resulting from the parameter uncertainties quantified from the published data. PBPK models developed for tetrachloroethylene and vinyl chloride are used as examples. Under the speci- fied model assumptions, these analyses provide a means of quantitatively assessing the relia- bility of using PBPK models to scale between species and high to low dose, as well as the sensitivity of model predictions to particular parameters. Supported by the State of Calif- ornia, Department of Health Services, Contract 85-87088. 311 94 50875 8219
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396 SPECIES DIFFERENCES IN RENAL NECROSIS AND pNA DAMAGE, DISTRIBUTION AND METAB4LISM OF 1,2- DIBROMO-3-CHLOROPROPANE (_BCP). E J Soder- lund*, M L$g*,I.J G Omichinski*, J A Holme*, G Brunborg*, S 0 Nelson**, and E , b.in *. Natl. Inst. Publ. H1th., ` Oslo, Norway* and Univ. Washington, Dept. Med. Chem., Seattle, WA**. The ability of DBCP to cause acute retpal nec- rosis was determined 48 hr after single i.p. doses (170-680 .umol/kg) in male rats, mice, hamsters and guinea pigs. DBCP showed appreciable species differences with respect to induction of proximal tubular necrosis. Substantially less necrosis was found in mice compared to rats, whereas no necrosis was observed in hamsters. To investigate the reason for these differences in nephrotoxic potential, renal distribution of DBCP was studied 1, 3 and 8 hr after a dose of 85 pmol/kg. Furthermore, in both time- and dose- dependent experiments, the ability of DBCP to induce DNA damage in kidney nuclei isolated after single i.p. doses of DBCP was compared in the four species. In order to study the involvement of GSH-mediated metabolism in the renal toxicity of DBCP in rats, mice, ham- sters and guinea pigs, bromide release from DBCP with kidney cytosols fortified with 1 nM GSH was performed and correlated with DBCP- induced renal necrosis and DNA damage. 398 MORPHOMETRIC ANALYSIS AND STRENGTH DETERMINATION OF OSTEOSCLEROTIC BONE RESULTING FROM•HEXACHLO- ROBENZENE (HCB) EXPOSURE. J E AndrewsT and W E Donaldson2. 1U.S.E.P.A., HERL, RTP, NC; 2NCSU; Toxicology Program, Raleigh, NC We have shown previously that hexachlorobenzene (HCB) exposure induces hyperparathyroidism and osteosclerosis in rats. Theref4o,,.we investi- - gafed the effects of HCB on femur morphometry as- _. weii as breaKing strength. Flscher 344 rats were dosed 5 days/wk for 15 weeks with 0, 0.1, 1.0, 10.0 or 25 mg HCB/kg body weight. Femur weight was significantly increased in the rats receiving 0.1, 1.0 and 25.0 mg HCB/kg body weight whereas density was increa d- signifi- cantly at 1.0, 10 and 25 mg HCB/Ir~dose"levels. Bone strength was also significantly Increased at the three higher dose levels. Cross sec- tional area of the midpoint of the femur was used to determine effects on cortical and medul- lary parameters. Cortical area as well as the proportion of the total area of the bone which the cortex occupied were significantly increased at the three higher dose levels. Medullary cav-: ity area was significantly decreased at the two higher dose levels of HCB. The right femur was significantly predominant to the left femur in weight, volume and density through all dosing regimens. Data from this study indicate that HCB does affect bone development and morphometry as well as strength. (This is an abstract of a proposed presentation and does not necessarily reflect EPA policy.), 397 SUBACUTE AND SUBCHRONIC TOXICOLOGICAL RESPONSES 399 OF RATS AFTER ORAL EXPOSURE TO CHLOROPICRIN (CP). L W CONDIE, M ROBINSON, AND B A MERRICK. USEPA, Health Effects Research Laboratory, Cincinnati, OH The toxicology of CP, a chlorination disinfec- tion byproduct, was evaluated by oral gavage in malP and female Sprague Dawley rats. Subacute treatment (SA-Tx) of rats was for 10 consecutive days at dose levels of 10, 20, 40 and 80 mg/kg/ day. The subchronic 90 day treatment (SC-Tx) exposure levels were 2, 8 and 32 mg/kg/ day. Controls were treated with corn oil. SA-Tx with CP showed significant decreases in final body and thymus weights in both sexes. Thymic lymphoid depletion was noted in the male high dose treatment group while increased spleen, weight was detected in the high dose female treatment group. Examination of gastric mucosa revealed inflammation-associated necrosis with increasing dose. Gastric epithelial hyper- plasia was noted at all doses but ulceration occurred only at higher doses of both sexes. SC-Tx with 32 mg/kg of CP produced 60% and 80% lethality in males and females, respectively. Histopathology of survivors showed that the primary affected organ was the stomach. Gastric mucosa was rough, pitted and thickened. While these studies indicate that the stomach was the major target organ of CP toxicity, other obser- vations suggest that the immune system may be adversely altered. (Abstract does not neces- sarily reflect EPA policy). 1,3-DICHLOROPROPANONE REDUCES CARDIAC OUTPUT. R D Laurie and T K Wessendarp. USEPA, HERL, Cincinnati, OH. Sponsor: L W Condie It has been shown that 1,3-dichloro-2-propanone (DCP) (also known as 1,3-dichloroacetone), when given 2 hours before carbon tetrachloride (CC14), inhibits the hepatotoxic effects of CC14. Pharmacokinetic studies in our laboratory have shown that the amount of CC14 exhaled is decreased when preceded by DCP (19 mg/kg). We have shown that histopathological analysis of the stomach and intestines revealed that there did not appear to be enough necrosis to account for the decrease in CC14 absorption after animals had been dosed with DCP. The current studies were completed using a radiolabeled microsphere technique. The basis of the proce- dure involved intra-arterial (right carotid) injection of 0.1 to 0.2 ml of microspheres averaging 15 pn in diameter and labeled with cerium 141. Starting five seconds prior to this injection, a one ml blood sample was taken from the left femoral artery over a period of one minute. The resulting data clearly indicate that cardiac output and the consequent blood flow to individual organs is reduced by DCP. (Abstract does not necessarily reflect EPA pol- icy). 50875 $225 100
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476 TESTICULAR TOXICITY OF 2-METHOXYETHANOL APPLIED DERMALLY TO OCCLUDED AND NONOCCLUDED SITES IN MALE RATS. M H Feuston, K R Bodnar, M J Belcak, S L Kerstetter, and C. P. Grink. Mobil Oil Corporation, Princeton, NJ. 2-Methoxyethanpl'(2-ME) was applied to the backs - of Sprague-Dawiey male rats, at dose levels of 0, 625, 1250, and 2500 mg/kg on occluded sites, and 0, 1250, 2500, 5000 mg/kg on nonoccluded sites for 7 consecutive days. Changes in testicular histology were evaluated on weeks 4, 7, 10, and 15 of the study. In an attempt to examine the time needed to induce 4sticular toxicity, the testes of animals exposed to 1250 mg/kg (occluded) and 2500 mg/kg (nonoccluded) were examined on days 2 and 5 and weeks 1 and 2 of the study (Day 1 = 1st day of dosing). After fixation in 10% formalin, the testes were embed- ded in paraffin and in glycol methacrylate (GMA). The paraffin sections were stained with hema- toxylin and eosin (H&E) and the GMA sections stained with H&E or Periodic Acid Schiff. Tox- icity to spermatocytes was apparent in Occluded and Nonoccluded groups after one day of dosing. Recovery (presence of spermatocytes in a major- ity of the seminiferous tubules on week 15) from 2-ME toxicity was apparent in 50% (2/4) of the Occluded group animals exposed to 2500 mg/kg and in 100% of the Nonoccluded group animals exposed to 5000 mg/kg. Histological evaluation of the testes of the mid- and low-dose Occluded groups is underway in an attempt to establish the dosage for which recovery is apparent. 478 477 ACRYLAMIDE (ACR)-INDUCED FERTILIZATION FAILURE 479 IN RATS. V Subletl, M K Smith,2 and H Zenick,2 Dept. of Environ. Hith. Univ. of Cin- ci nati, Cincinnati, OHi, USEPA, Cincinnati, OH2, and USEPA, Washington, DC3 Dominant lethal studies indicated that the exposure of male rats to ACR produced signifi- cant pre-implantation loss at Weeks 1-4 after treatment (spermatozoal and spermatid stages of spermatogenesis). The present study was designed to determine if ACR-induced fertilization fail- ure might contribute to a reduced rate of im- plantation. Adult male Long Evans rats received 0, 15, or 45 mg/kg ACR (p.o.) for 5 days and were then.mated to proestrous fenales on Weeks 1-4 after ACR. Ova were flushed from the oviducts 12 hours after mating. The presence of a sperm head and tail or two pronuclei in the oocyte provided evidence of fertilization. Females mated to males at Week 1 after exposure showed 84%, 41% and 29% fertilization for the 0, 15, and 45 mg/kg ACR, respectiveiy. At Week 3, fertilization rates were 65%, 73%, and 12% for 0, 15, and 45 mg/kg ACR, respectively. These results represent signi'- ficant reductions in fertilization at both ACR exposures at Week 1 and at the 45 mg/kg dose of ACR at Week 3. During Weeks 2 and 4, fertiliza- tion was not affected. These data suggest that fertilization failure may, in part, account for the subsequent reduction in implantation associ- ated with ACR exposure. (This abstract does not reflect U.S. EPA policy or opinion). 120 .~.., DIACETOXYSCIRPENOL-INDUCED~~TESTI~CULAR INJURY IN F344 RATS. M W Connez~;''B H Conner, L Wagonner-Pogue, A E Rogers. Boston Univ. School of Medicine, Boston, MA. Diacetoxyscirpenol (DAS, anguidine) administered ip at 75% of the LD50 causes testicular degeneration in CD-1 mice and Lewis rats. The purpose of this study was to characterize the testicular damage in a " strain of rats in which testicular structure 'F and function have been moremOtAly " :~ characterized. Mature (12 wk) male F344 rats-,-- were given DAS in 10%-aqueous DMSO by ip injection at 50x (0.8 mg/kg) or 75% (1.2 mg/kg) of the LD50 and studied 7, 45 or 90 days later. DAS caused dose-dependent decreases in testicular and epididymal weights, sperm production andiepiflidymal sperm reserves. These decreases were significant at all times studied after exposure to 1.3 mg/kg but only at 45 and 90 days after exposure to 0.8 mg/kg. Morphologic changes were characterized by dose- and time- dependent decreases in diameter of seminiferous tubules and increases in the percent of hypocellular tubules. These results are consistent with damage to proliferating cells early in the maturation sequence and to mature spermatids. (Supported by contract DAMD 17-82-C-2235 from_the U.S. Army Medical Research and Development Command.) EVALUATION OF THE EFFECTS OF NIACIN AND ZINC SUPPLEMENTATION ON METHYLXANTHINE-INDUCED TESTICULAR ATROPHY IN RATS. C A Shively, J L Apar, N J Worley, and S M Tarka, Jr. Hershey Foods Corporation Technical Center, Hershey, PA. Previous studies have shown that methylxanthines (caffeine and theobromine) exert testicular and thymic toxicity in rats when fed at high dietary levels (0.6-0.8%). This study determined the effects of niacin and/or zinc supplementation on theobromine (TBR)-induced thymic and testicular atrophy in.weanling and adult male Sprague- Dawley ratAfed control, 0.6% TBR, 0.6% TBR + 0.12% zinc, 0.6% TBR + 0.2% niacin, or 0.6% TBR + 0.12% zinc + 0.2% niacin in a purified diet. After 28 days of TBR exposure, body weights were reduced in all TBR treatment groups although effects were more severe in the weanlings. Rela- tive thymus and testes weights in weanlings treated with TBR were 30% and 70% of controls, respectively. Adult animals exhibited no testic- ular atrophy from TBR, but relative thymus weights were 40% of control. Analytical results following TBR exposure indicated: 1) reduction in sperm motility, 2) decreased plasma/testes testosterone, 3) increased testes malate de- hydrogenase, and 4) decreased testes sorbitol dehydrogenase. Plasma/testes alkaline phospha- tase and zinc levels were similar among all animals. Supplementation with niacin and/or zinc had no significant positive effects on TBR-induced organ or body weight changes. 50875 8245
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468 THE METABOLISM OF TRI-S,I-CRESYL PHOSPHATE (TOCP) BY RAT TESTIS. S G Somkuti, R E Chapin+, D M Lapadula,_1J,A Othman, and M.B. Abou-Donia.,Dept. of Pharmacology, Duke Univ. Med, (~ter, Durham, NC, and +Dev. and ReproductivQ,Toxicology, NTP, NIEHS, NC. The metabolism after 10 daily oral doses of 50 mg/kg (6.8uCi/kg) of [C14] TOCP was investi- gated in adult Fischer 344 male rats. Groups of 3 animals were killed at interval,s (24,48,72 and 96 hrs) following the last dose. The highest concentrations of radioactivity were found in urine and feces and g.i. con- tents. Metabolites (as determined by HPLC) were found in all organs, including the testes. Sufficient amounts of the active meta- bolite saligenin cyclic-o-tolyl phosphate (SCOTP) were detected at all time points in testes to suggest local activation. Thus, [C14]TOCP was injected directly into the left testis (0.55uCi/testis). After two hours, testes were removed for determination of local metabolism of TOCP. Metabolites of TOCP, including SCOTP, were present in testis, liver and blood. No radioactivity was detected in the contralateral (non-injected) testis indi- cating that the metabolites were not origi- nating from the liver and being sequestered in the testis. The cellular site of activation is being investigated. 470 5 469 CATIONIC MODULATION OF ADENYLATE CYCLASE (AC) ACTIVITY 471 IN A HETEROGENOUS POPULATION OF MALE GER.*f CELLS (MGC). L Beebe, K Pendino, R 0 Warwick Jr., and D A Barsotti. Philadelphia College of Pharmacy & Science, Phila., PA and A.T.S.D.R., Atlanta, GA. Germ cell specific AC is located in the cytosol of haploid germ cells, where it demonstrates an obligatory requirement for manganese, and insensitivity to hormones or magnesium. Investigations in our laboratory have demonstrated AC activity in a membrane enriched fraction isolated from a heterogenous population of MGC. These studies have focused on the cationic requirements for activation of this membrane associated enzyme, and its responsiveness to activators of the Gs protein. MGC membranes are incubated in the presence of magnesium (10 mM), magnesium and manganese (5 mM each), or manganese (10 mM) alone, in an assay buffer containing HEPES (50 mM, pH=7.5), EDTA (1 mM) and IBMX (1 mM). The conversion of 32P-ATP to 32P-cAMP is quantitated utilizing successive chromatography over Dowex and alumina columns. Our results indicate that membrane associated AC is stimulated by both magnesium and ' manganese, although the magnitude of this stimulation is greater in the presence of manganese. Responsive- ness to GTP (10-100 uM) is demonstrated only in the presence of magnesium, and is abolished upon the addition of manganese to'the incubation buffer. These data suggest that the membrane bound AC in MGC has a divalent cation requirement which may be fulfilled by either magnesium or manganese, however expression of G protein activation is magnesium dependent and attenuated by the presence of maganese ions. 118 LDH-C4: A SPECIFIC MARKER OF ACUTE-PHASE TESTICULAR DAMAGE. S C J Reader, C Shingles and M D Stonard. ICI plc, Centr9I Toxicology Laboratory, Macclesfield, Cheshire, U.K. Sponsor: E A Lock 1,3 dinitrobenzene (DNB) and ethylene glycol monomethyl ether (EGME) markedly effect rat f testis Sertoli and germ-cells respectively. We. have monitored biochemicar-i"onses to such damage including the testis specific isozym2'-.°'` LDH-C4. Rats (6/grohp) were dosed P.O. with DN&•-, (0-30mg/kg) or EGME (0-500mg/kg), after 48h plasma/tissue samples were analysed. Further studies used a single oral dose of DNB (25mg/kg) or EGME (500mg/kg), samples were taken at 6,16, 24,48,96h and 14 days. LDH 1pozymes were profiled using electrophoresis, 04 was assayed using a kinetic NADH-linked reaction with a ketocaproate as substrate. Testicular C4 activity was reduced after.DNB (25-30mg/kg) from control 242t14 IU/mg protein to test 149±36 IU/mg protein; P<0.02, with a concomitant increase in plasma C4 activity at 10mg/kg DNB from control 3±2 IU/L to test 31±11 IU/L- P<0.05. C4 was increased in plasma and decreased in testis after 6h (P<0.02) remaining so for upto 14 days (P<0.05). Similar results were found with EGME upto 96h. Testicular lesions were seen at 16h with 20mg/kg and 300mg/kg for DNB and EGME respectively. In conclusion, these results suggest disruption of the blood-testis barrier allowing C4 to be measured in blood as an early diagnostic marker of acute-phase damage before apparent pathological lesions. f AUTOMATED SPERM ANALYSIS: EPICHLOROHYDRIN (ECH) A MODEL COMPOUND. G P Tothl, J A Stoberl, E J Read2, H Zenick3, M K Smithl. 1HERL, USEPA, Cincinnati, OH, 2Computer Sciences Corporation, Cincinnati, OH, 3USEPA, Washington, D.C. .The Cellsoft computer-assisted sperm motion analysis system (CRYO Resources) has been used for the evaluation of motion characteristics of sperm. Using previously published system set- tings in our analyses, we identified the poten- tial for error in evaluating dose effects in rats exposed to ECH, a compound previously shown to reduce fertility in experimental animals. We then examined the effect of changes in Cell- soft system settings on motion endpoints. In controls, sperm tracking was significantly shortened at a maximum velocity setting of 300 pm/sec, resulting in artificially lower mean velocities. Mean linear velocities appear- ed to level off when maximum velocity was set to 600 um/sec. Using a paired t-test, Cell- soft values for the percentage of motile sperm were not significantly different from manually determined values when the minimum sampling mo- tile parameter was set at 10 frames rather than 1. ECH dose-response relationships were then evaluated at these settings and compared to those determined at the previously published settings. Use of the new settings reduced system biases and expanded the range of measure- ments to allow a greater sensitivity in measur- ing toxic dose effects. (Abstract does not necessarily reflect EPA policy). 50875 8243
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392 TOxICiTY OF PERFLt30RODECANOIC ACID (PFDA) IS UN- LIKE THAT OF TCDD. D W Brewster, M W Harris and L S Birnbaum. NIEHS, Researd;-Tri. Pk., NC. PFDA is an industr$al surfactant that has been reported to produce similiar signs of toxicity as does TCDD upon administration to rats. To deter- mine whether PFDA toxicity is mediated by the Ah locus and to characterize the toxicity of PFDA in the mouse, congenic female C57B1/6J mice differ- ing only at the Ah locus, (Ah b/b, Ah bfd, and Ah d/d) were administered a single oral dose of PFDA. The wild type (b/b) strain was killed 2, 7, 14, or 30 days after administration of 0, 40, 80, 100, 120, or 160 mg PFDA/kg. The 2 substrains were killed 30 days after dosing with 0, 40, 80, or 160 mg/kg. In all 3 strains the LD50 was esti- mated to be "120mg/kg. PFDA produced a 30% loss of body weight, a 2.5 fold increase in liver weight, a decrease in thymus and spleen weights, only a 20% increase in hepatic palmitoyl Co-A oxidase activity, a 70% decrease in hepatic ethoxyresorufin-o-deethylase (EROD) activity and an increase in serum T4 concentrations. Total hepatic lipids were increased at an early time point and at the lowest dose. At later time periods and/or higher doses lipid concentration was decreased "70% from that of controls. There was little difference in any of these parameters between the'3 strains and the effects were dose and time dependent. These results suggest that the Ah allele has little effect in regulating the toxicity of PFDA, that the biochemical response to PFDA is markedly different from that of TCDD, and that the biochemical response to PFDA in the mouse is different from that in the rat. 393 TERATOLOGIC EVALUATION OF PERFLUORODECANOIC ACID .(PFDA) IN C57BL/6N MICE. M W Harris and L S Birnbaum, Systemic Toxicology Branch, NIEHS, RTP, NC PFDA is a representative of the perfluorinated carboxylic acids used as commercial wetting agents and flame retardants. Signs of PFDA toxicity have been reported to resemble those seen after exposure.to TCDD. To determine if PFDA is teratogenic, time mated C57BL/6N mice received PFDA by gavage in corn oil (10 ml/kg) on gd 6-15 or gd 10-13 at levels ranging from 0 to 12.8 mg/kg/day or 0 to 32.0 mg/kg/day respectively. Dams were sacrificed on gd 18 and maternal and fetal toxicity assessed. Fetuses were examined for external malformations, cleft palate as well as other soft tissue or skeletal anomalies. Maternal body weight gain was significantly reduced as a result of PFDA - treatment at 6.4 and 12.8 mg/kg/day (gd 6-15) and 16.0 and 32.0 mg/kg/day (gd 10-13). Fetal viability was decreased only in those groups showing reduced maternal weight gains. Fetal body weights were significantly reduced at levels as low as 0.1 mg/kg/day (gd 6-15) and 0.5 mg/kg/day (gd 10-13). No hydronephrosis, cleft palate or edema were observed nor were any other soft tissue or skeletal anomalies detected. Thus, unlike TCDD, PFDA is not teratogenic in C57BL/6N even at doses which are maternally toxic. 394 DIFFERENTIAL EFFECTS OF DIETARY SELENIUM ON GLUTATHIONE-DEPENDENT ENZYME ACTIVITIES IN RATS TREATED WITH PEROXISOME-PROLIFERATORS. L C Chen, T Borges, L W Robertson, H P Glauert and C K Chow. Graduate Center for Toxicology and Department of Nutrition & Food Science, University of Kentucky, Lexington, KY. One-month-old male Sprague-Dawle*wzts, fed a diet containing either 0.04 or 1.0 ppm Se for 14 days, were treated witFi.either ciprofi.brate (CIP, 0.025 ppm in the diet) or perfluorodeca- noic acid (PFDA, 35 mg/kg, single IP) 2 weeks prior to sacrifice. Control animals were - pair-fed. Hepatic GSH levels and GSH peroxi- dase (GSH Px) activity in S9 and cjjtoscx,r were significantly increased in PFDA-tfreate# rats fed the low Se diet, whereas the activity of GSH transferase (GST) was decreased. In contrast, a significant decrease in GSH Px was found in PFDA-treated rats fed the high Se diet. Likewise, CIP caused a significant decrease in GSH Px activity in the high Se group, whereas GST activity was diminished in both groups. CIP had no significant effect on GSH levels. Serum GSH Px activity was significantly decreased by CIP, but not by PFDA, in both dietary groups. The results suggest that dietary Se modulates GSH levels and related enzymes in rats treated with peroxisome proliferators. The mechanism of these differential effects remains to be elucidated. (Supported by AICR.86B66 and NIH CA43719) 395 EFFECTS OF CIPROFIBRATE AND PERFLUORODECANOIC ACID ON LIPID METABOLISM ANC GROWTH OF MALE SPRAGUE-DAWLEY RATS FED 2 LEVELS OF SELENIUM. T Borges, L C Chen, L W Robertson, C K Chow, and H P Glauert. Graduate Center for Toxicology and Department of Nutrition & Food Science, University of Kentucky, Lexington, KY Rats fed diets containing either 0.04 ppm or 1.0 ppm selenium were treated with the hypolipidemic iirug ciprofibrate (CIP, 0.025% in the diet) or the industrial chemical perfluorodecanoic acid (PFDA, 35 mg/kg in corn oil IP). Rats administered either peroxisome proliferator gained less weight than their pair-fed controls. Toxicity of PFDA, particularly apparent in rats fed the low selenium diet, was reflected by lowered feed intake, feed efficiency, weight gain and thymus weights as compared to pair-fed controls. These effects were less evident in rats fed the high selenium diet. Peroxisomal beta-oxidation was increased in all CIP-fed rats but was decreased in PFDA-treated animals. Plasma triglycerides and cholesterol were decreased in CIP-fed rats consistent with increased lipid metabolism. Plasma trigly- cerides but not cholesterol was lowered in PFDA-treated rats. The results suggest that selenium influences PFDA-induced toxicity whereas no effect of selenium was seen in CIP-fed animals. (Supported by AICR 86B66 and NIH •CA43719) 99
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408 PARAMETERS OF HYDROXYL FREE RADICAL MEDIATED DNA DAMAGE. J E Schneider, M M Browning, J J Watson, and R A Floyd, Oklahoma Medical Research Foundation, Oklahoma City, OK. a as Ascorbic acid.and its oxidation products have been implicated,as both causative agents and as protective agents in disease processes involving oxidative stress. We are developing in vitro model systems to explore the potential roles for ascorbate mediated •OH formation it~causing DNA damge.. Two hydroxyl free radical (•OH) generating systems were shown to cause single- stranded nicks in supercoiled pBR322 plasmid DNA. In one system (0.03% hydrogen peroxide plus U.V. light), the amount of single-stranded DNA nicking activity correlated with the accumu- lation of an adduct of DNA, 8-hydroxydeoxyguano- sine (8-OHdG), which is caused by •OH attack on guanosine. In another system, ascorbate and iron(III) caused DNA nicking which was inhibited by known hydroxyl radical scavengers. DNA nicking was temperature dependent according to' Arrhenius theory. An initial lag in the rate DNA of nicking was not due to a corresponding lag in •OH production as assessed by measurement of salicylate hydroxylation products in this system. The •OH dependent formation of salicylate hydroxylation products was quantitated by HPLC electrochemical detection. Measurement of 8-OHdG levels, and its correlation with loss of ascorbate that occurs in this system, are in progress. Supported in part by NIH Grant No. CA42854 409 HYDROXYL FREE RADICAL MEDIATED FORMATION OF 8- HYDROXYGUANINE IN ISOLATED DNA. R A Floydl, M S Westl, K L Eneffl, W E Hogsett2, and D T Tingey2, Oklahoma Medical Research Foundationl, Oklahoma City, OK. and Environmental Research Laboratory, U.S.E.P.A., Corvallis, OR. Formation of 8-hydroxyguanine within calf thymus DNA has been studied after exposure to UV-H202 as a hydroxyl free radical generating system. Using high pressure liquid chromatography with electrochemical detection, we measured the amount of 8-hydroxy-2-deoxyguanosine (8-OHdG) in the enzymatically digested DNA. The 8-OHdG content of UV-exposed DNA increased with increasing H202 levels up to 0.03%, but then increased only slightly at higher levels. All hydroxyl free radical scavengers studied (mannitol, ethanol, thiourea and salicylate)) caused a decrease in the amount of 8-OHdG formed in DNA exposed to the UV-H2O2 system. Thiourea reacted with 8-OHdG as an integral part of DNA but not with the nucleoside free in solution, to cause a decrease in the amount of the hydroxyl free radical modified guanine present. In contrast to thiourea, however, reduced glutathione did not react with 8-OHdG either as an integral part of DNA or free in solution. Ozone, which has been shown to react with DNA to cause damage, did not initiate formation of 8- OHdG within DNA solubilized in a buffer system. Supported by EPA Proj. No. CR-812710-01-0, and NIH Grants CA42854, NS23307, and ES04296. Glycerol is a widely distributed const- ituent of the food supply and is also a common additive in manufactured cigar- '`" ettes. Although generally regarded as"'; safe (GRAS) by the U.S. Food and Drug Administration, it has not been evalu-.°: ated for its potential to cause DNA damage in a modern battery of short-term genotoxicity assays. In the present study glycerol was evaluated for geno- toxic potential in the Ames bacterial , mutagenesis assay (strains TA 98, 100, 1535, 1537 and 1538), the rat hepatocyte: unscheduled DNA synthesis assay, the CHO'O sister chromatid exchange assay, the CHO`'O chromosomal aberration assay and the CHO:: mammalian mutagenesis assay. All assays_ (except the rat hepatocyte unscheduled DNA synthesis) were conducted both with and without the addition of Aroclor- induced rat liver S9. The maximum concentration of glycerol evaluated was 1 mg per plate for bacterial mutagenesis assays and 1 mg per ml for all mammalian assays. The results of all tests were negative, showing that neither glycerol nor its metabolites have apparent intrinsic genotoxic activity, as meas- ured by the battery of tests used. 50875 8228 410 THE GENOTOXIC POTENTIAL OF CONDENSATE FROM.A CIGARETTE WHICH DOES NOT BURN TOBACCO. D J Doolittle, G_ ~T~~B_~u~~r e~r~~, A W Ha es and C K Lee. R.J. ReynolcTs Tobacco Co, Winston-Salem, NC. The genotoxic activity of cigarette smoke condensate (CSC) from a cigarette- which does not burn tobacco (test) was _ compared to that of CSC from the 1R4F !0cigarette (reference):-ABSE was collecteti by standard techniques, which involvga,,._, ' collecting smoke;particulate matter ori„. Cambridge filter pads, under FTC condi- ta__ ions. The pads were extracted with DMSO; the CSC obtained (10mg/ml) was subjected to an in vitro test battery. The reference CSC was positive in Ames bacterial strains TA 98And 100 with S9 activation (dose range 25-140 ug/plate), while test CSC was negative in both strains, with and without metabolic activation, at doses up to 1400 ug per plate. The reference CSC was positive in the CHO chromosome aberration assay (50-250 ug/ml), the CHO-sister chromatid exchange assay (10-150 ug/ml) and in the rat hepatocyte UDS assay (25-125 ug/ml). Test CSC was negative in all three of these assays under similar conditions. Test and reference CSCs were negative in the V79-HGPRT and the CHO-HGPRT mammal- ian mutation assays, with and without metabolic activation. These results indicate that test CSC has less geno- toxic potential than reference CSC, as measured by the battery of tests used. 411 THE GENOTOXIC ACTIVITY OF GLYCEROL IN AN IN VITRO TEST BATTERY. C K Lee, G T Bur, er, A W Hayes and,D J DoolittTe-. R.J. Reynolds Tobacco Co, Wlnston-Salem, NC. 103
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A 13-WEEK S_KIN IRRITATION STUDY WITH ACRYLIC ACID IN3MSTRCarneOF ~ICE•ThomasTeSeRisMurphY, Balmer. F J E, McLaughlin, J.L. Sevmour;. Tegeris Laboratories, Inc,., Laurel, MD and Basic Acrylic Monomgr Manufacturers Association. Washington, b.C:' The degree and =•course of irritation from dermallY applied acrylic acid (AA) at o, .1a and 4% in acetone were assessed in 3 strains of mice. For each level, groups of 30 female ICR, 30 male C3H and 30 female B6C3F1 a mice were treated 3 times weekly for 13 weeks, with skin irritation graded daily. Five mice per group were necropsied after weeks 1, 2, 4 and 8; the remaining mice after week 13. Four percent (4%) AA in acetone caused significant skin irritation in ICR, C3H and B6C3F1 mice. Desquamation, fissuring, and eschar were observed with 4% AA after 1 or 2 weeks (depending on strain), and continued through week 13, peaking between weeks 3 and 5. Little or no gross irritation occurred in any strain dosed with 0% (acetone only) or 1% AA. Microscopic findings of proliferative, degenerative, and inflammatory changes in the epidermis and dermis were seen in all strains dosed 4% AA, beginning at week 1 and continuing through week 13 with little change in severity. A low incidence of minimal proliferative changes were noted at 1% AA, but not in control animals. Skin irritation and histological changes caused by AA do not differ significantly among three different strains of mice. COMPARATIVE EFFECTS OF TRIETHANOLAMINE (TEA) AND DIETHANOLAMINE (DEAT IN SHORT-TERM DERMAL STUDIES. R Melnick*, M Hejtmancik, L Mezza, M Ryan, R Persing, and A Peters. Battelle Columbus Division, Columbus, OH and *NIEHS, Research Triangle' Park, NC. TEA and DEA are aminoalcohols frequently included in cosmetic preparations as emulsifiers and thickeners. Twelve doses of TEA (undiluted) or DEA (in ethanol) were administered to the inter- scapular region of F344 rats or B6C3F1 mice during a 16-day period. Dosages ranged from about 0.1 to 2.0 g/kg body weight in rats and 0.2 to 3.0 g/kg in mice. Administration of TEA did not cause any deaths. Necrotizing inflamma- tion of the epidermis and dermal fibrosis were observed at the site of application in rats of both sexes (but not mice) at doses of 0.6 g/kg and above. DEA caused mortality in the highest dose groups of rats and mice. Chronic active inflammation, ulceration, and acanthosis of the epidermis were observed at the site of application in rats (0.5 g/kg and higher) and in mice (at 1.25 mg/kg and higher). Minimal acanthosis was, also present in mice in the lower dose groups. Renal tubular necrosis and necrosis of the semini- ferous tubules were observed in high dose rats.. These dataindicate that rats are more susceptible to ethanolamines than mice, and that systemic toxicit can result from dermal DEA administra- tion. (Supported by Contract No. N01-ES-45068 and N01-ES-65148 from NTP). 506 SHORT-TERM TOXICOLOGY STUDTd0 THE MONOMER OF 1,2-DIHYDR0=2,2,4-TRIMETHYLQUINOLINE IN F344/N RATS AND B6C3F1 MICE (SKIN PAINT). J E, French; ' *A G Manus; *J Heath; *R Thompson; J J Collins; NIEHS, RTP, NC and *SoRI, Birmingham, AL Sponsor: J Bucher. Thirteen week studies were conducted by _ applying 1,2-dihydro-2,2,4-trimethylquinoline ~ (monomer) in acetone to rats (0, 20, 50, 100, ~ and 200 mg/kg) or mice (0, 2,&r-5, 10, 20, and ~ b0 mg/kg) by skin-paint (ix/da, 5 da/wk). A].]~~ ' animals survived to th%-end of the study. Male.' rats treated with 200 mg/kg had reduced body weights (7%) and increased liver/body weight (BW) ratios. Thymus to BW ratio in male rats was increased at the 20 mg/kg and greater dose levels. Female rats had dose-~lated increased liver/BW and kidney/BW ratiosr ~ H'0topathology revealed treatment related lesions of the skin in rats at all doses. Hepatocellular vacuoli- zation was observed in 200 mg/kg treated male rats. For mice, there were no significant decreases in survival, body weights, or organ weights due to treatment. Body weights of female mice were increased in the 2.5 mg/kg and greater dose levels. Treatment related skin lesions at the dose site were observed micro- scopically in all dose groups of mice. Based on these results, long term toxicology and car- cinogenesis studies in F344 rats and B6C3F1 mice and study designs to determine the ini- tiation and promotion potential of TMQ are pro- posed in male F344 rats and female SENCAR mice. 507 EVALUATION OF MODIFIED METHODS FOR DETERMINING SKIN IRRITATION IN ANIMALS AND HUMANS. G A Nixon, E A Bannan, T W Gaynor, D H Johnston and J F Griffith. The Procter & Gamble Co, Miami Valley Laboratories, Cincinnati, OH Animal skin irritation testing is normally con- ducted following governmental regulatory agency procedures. Often more than one test must be conducted in order to meet the various require- ments. Tests in rabbits were compared with identical human tests to evaluate potential alternative`~~' to the current regulatory proced- ures. Variables evaluated in the study were 19 mm Hill Top chambers vs. standard gauze patches (DOT procedure), intact vs. perforated cham- bers, and time of exposure (1 and 4 hours). Variability in the irritation responses pro- duced in both species was attributed to subject susceptibility, and not procedural factors. Most rabbit responses were conservative estim- ates of human test responses. The use of Hill Top chambers or equivalent offers the potential to 1) reduce the number of animals used for skin irritation screening (smaller group size and up to eight test substances/concentrations per animal), 2) eliminate the need for con- ducting multiple tests to satisfy governmental requirements and 3) reduce animal stress by reducing exposure times. It appears that these benefits could be achieved without compromising the value of the irritancy patch test for making human dermal safety judgments. 127 50875 8252
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EVALUATING PERCUTANEOUS ABSORPTION PROPERTIES OF A LIGHT PETROLEUM MIDDLE DISTILLATE IN MICE. J J Yang, T A Roy, V Neil, A J Krueger. Mobil Environmental and Health Science Laboratory, Princeton, NJ. STonsor: C Ko'mmineni .6 Chronic deftal `treatment of C3H/HeN mice with a mildly hydrotreated light petroleum middle dis- tillate (MD) resulted in pronounced skin irrita- tion and an atypical tumor response with many animals developing malignant tumors late in the study (mean onset: 79 wk). Results from subse- quent subchronic studies indicatedAhat Sencar BR mice are more resistant to MD-induced skin irri- tation than C3H mice. In order to better under- stand the effects of MD on skin, in vivo percu- taneous absorption studies with the MD were performed in C3H and Sencar mice. Absorption of the MD was measured over 96 hr following a single topical dose containing [3H]n-dodecane and [14C]- 2-methyl naphthalene as surrogates representing the aliphatic and aromatic ' components, respec- tively, of the MD. The results indicate that there is no significant difference in the perme- ability of C3H and Sencar mouse skin to the MD. This similarity suggests that the different MD-induced skin irritations observed in C3H and Sencar mice are a result of biological and/or immunological variations between the two strains. 489 PERCUTANEOUS PENETRATION OF NICOTINE IN,YOUNG.' AND ADULT BATS.. .L L Hall, H L Fisher;.==.: M R Sumler'll and P V Shah*.. U.S. EPA, *Northrop Services,.Inc., Research - Triangle Park, NC A e dependence in percutaneous absorption of - 1RC-nicotine was assessed in 33 and 82 day old Fischer 344 rats. Nicotine was applied in acetone to the previously clipped back' skin and the radioactivity in the treated . skin, tissues, urine.and feces was determined at 24, 48, and 72 hours. In vitro dermal absorption was also measuren Tn tFe same aged.: rats by static and flow through methods. -In vivo dermal penetration at 48 hrs. was 81% in young and 79% in adult rats. Adults excreted about 67% in urine and 5% in feces in 48 hours while excretion in the young,.was 73% urinary and 5.5% fecal. Age dependence .. in tissue content was found in kidney, blood, and carcass. Age dependence in tissue con- centration was seen also in kidney, blood, , and carcass. Dermal absorption was " slightly higher in young rats while reten- tion was greater in the adult. Skin absorption by the in vitro methods was somewhat less than the in vivo absorption and was greater in young than adults. This is an abstract of a proposed presentation and does not necessarily reflect EPA policy. ~_. ,~. 490 Dermal absorption of diso~d"ium`an dum`and mono- sodium methylarsenates (DSMA and MSMA) in young and adult rats. S_P Shrivastava, H L Fisher*, M R Sumler, P.V. Shah, and L L Hall.* Northrop Services, Inc., * USEPA, Research Triangle Park, NC 491 123 Dermal absorption of DSMA and MSMA was determined in 33 and 82-day old Fischer .,.344 rats. Water solution ofochemicals r - was applied to previously clipped skin - - and the radioactivity:.fn the treated skin, tissues, urine and feces was deter- ~ mined at 6-120 hours. A comparison of the in vitro dermal absorption via flow-through and static systems was also made. The dermal absorpti on of DSMA i n young a2k adt#l.t was 2.6-7.5 and 4.5-12.4% respectively,. The values for MSMA were 3-11 and 5.3-17%. The variability in penetration between rats and at different times was quite large. At 72 hours in vitro dermal absorp- tion of DSMA by continuous flow system was 36% for both age groups and 7.4 and 22% for young and adults by static system. For MSMA the absorption by the continuous system was 30% in young and 46% in adult, and by the static system the absorption was 11% in young and 4.1% in adults. This is an abstract of a proposed presentation and does not necessarily reflect EPA policy. DERMAL AND TRANSDERMAL ZOXICITY OF THE. CALCIUM IONOPHORE, A23187. R W Wannemacher, Jr., D L Bunner, and R E Dinterman.. U.S.. Army Medical Research Institute of Infectious Diseases,_ Fort Detrick, Frederick, MD. A23187 is an antibiotic which has properties as a Ca{'+ionophore. 'Its dermal toxicity and transdermal lethality was assessed in a guinea pig model. For the dermal toxicity studies, a 2-ul sample of varying concentrations of A23187 in DMSO or.,ethyl,acetate was applied to a shaven area of th' ' skin. At 1, 2, 3,.7,.and 14 days,` the resultant lesions were scored for erythema, edema, or necrosis. Six hours after dermal exposure to A23187, we observed a severe erythema and edema, with a minimal effective dose of 20 ng. A severe necrosis developed by day 3, and the lesion was still detectable by day 14. Various concentrations of A23187,in DM.SO (0.1 ml) were applied to the shaven area of, skin to assess transdermal lethality. We used a protective barrier to prevent oral ingestion. We did not observe transdermal lethality with doses of A23187 up to 13 mg/kg in IIN.SO. In mice, the minimum lethal dose by the i.p. route was approximately 7 mg/kg, while doses up to 25 mg/kg s.c. were not lethal. Thus, we conclude that A23187 is a severe dermal irritant but has a low transdermal toxicity. 50815 8248
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430 CHARACTERIZATION OF AN IN VITRO MODEL TO ASSESS MALE GERM CELL (MGC) TORLCITY. K Pendino, L Beebe, R O Warwick Jr., and D A Barsotti. Philadelphia College of Pharmacy & Science, Phila., PA, and A.T.S.D.R., Atlanta, GA. We have previously developed a model to investigate the toxicityvof ftgmotional agents on a heterogenous population of MGC.. Exposure regimens in vitro have necessitated the qnantitation of cell viability as detetmined by trypan blue exclusion dye and lactate dehydrogenase (LD{i) leakage from these cells. ATP production has also been quantita ted utilizing a purified luciferin/luciferase enzyme asday. MGC have been exposed to the promotional agents tetradecanoylphorbol-l3-acetate (TPA) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) over time (1.7 nM, 30-180 min) and concentration (lnM-luM, 60 min) ranges. DMSO (0.5% v/v) and PBSG were incubated as vehicle and non-vehicle controls, respectively. Viability was statistically reduced at 150 and 180 minutes for the DMSO treatment. Neither TPA nor TCDD decreased viability at any time interval or concentration investigated, as compared to vehicle controls. ATP production was statistically decreasedd by all treatments at 60 minutes, with recovery by 90 and 120 minutes. Incubations exceeding 120 minutes reduced ATP levels as compared to the 30 minute value. Neither TPA nor TCDD inhibited ATP production at any time interval or concentration investigated. These data suggest that TPA and TCDD are not directly cytotoxic under our exposure conditions. Additionally, incubations should not exceed 120 minutes utilizing this model. 481 GOSSYPOL INDUCED CHANGES IN ZINC CONTENT OF HAMSTER EPIDIDYMAL SPERMATOZOA. Y Wang and D P Waller. University of Illinois at Chicago, Chicago, IL. Gossypol is known to cause infertility in several species of animals and man. A decrease in sperm motility usually precedes or accompanies a decrease in sperm production. Zinc is an essential element incorporated into a spermatozoa during spermatogenesis and is required for motility. We have investigated the zinc content of epididymal spermatozoa in hamsters treated with gossypol. Mature male hamsters were treated orally with vehicle, 20 mg/kg or 40 mg/kg of gossypol for 13 days, and euthanized on day 14. The testes and epididymides were removed and weighed. Spermatozoa were then obtained from the cauda epididymides and counted. Zinc content of the spermatozoa was determined utilizing atomic absorption spectroscopy. The body weight of both treated groups decreased compared to controls. Testes weight, epididymal weight and caudal epididymal sperm counts were dec~eased in the 40 mg/kg group only. Zinc content/10 spermatozoa was increased in the, 40 mg/kg group. These data demonstrate the alteration of zinc levels in epididymal spermatozoa by gossypol and may be related to the male antifertility effects of gossypol. 482 EFFECT OF LINURON ON 1HE BRAINx= PITUITARY = TESTICULAR REPRODUCTIVE AXES£~-I' THE RAT. G L Rehnberg, J M Goldman, R L'Cooper, J F Hein, W K McElroy, K C Booth, and L E Gray, Jr. USEPA, HERI. DCTD, RTP, NC. Sponsor: R W Chadwick _ The pesticide Linuron (LRN) has been reported to cause testicular damage in rats. Consequently, the present study was designed to explore the effects of LRN on the brain-pituitary-testicular (H-P-T) axis after short term exposure. Adul~ male rats were gavaged with LRN (50, 100, or 2 jj0 --,mg/kg) in corn oil for 4 day_,A*!0'_"Controls received . ~ corn oil only. Rats were killed 1, 3 or 7-Ityii post-dosing and serum°was collected for analys~; of pituitary, thyroid and gonadal hormones. Pi-~ tuitaries and hypothalami were assayed for tissue hormone concentrations. Testicular and epididy- mal (epi) measures were recorded and testosterone and androgen binding proteinABP~"^^were assayed in individual testicular compartmefits. Data anal- ysis showed a reduction in caput epi weight and interstitial fluid volume on day 1 at the highest doses. A dose-dependent reduction in the thyroid hormone, T4, occurred on day 1. No other hormon- al alterations were seen. The data indicate that LRN under these conditions has minimal effects on the H-P-T axis. Additional research is needed on the influence of more prolonged exposure to LRN. This is an abstract of a proposed presentation and does, not necessarily reflect EPA policy. 483 SKIN ABSORPTION AS A ROUTE OF EXPOSURE FOR fUNGAL TOXINS. B W Kemppainen, R T Riley, and J G Pace. Department of Pharmacal Sciences, School of Pharmacy, Auburn University, AL; USDA-ARS, Athens, GA; USAMRIID, Frederick, MD. Airborne occurrence of mycotoxins in agricultural and residential environments has generated concern that these toxins may be absorbed via the skin. and respiratory tract. This paper summarizes information available on the in vivo and in vitro cutaneous permeability of several myjotoxins (aflatoxin, T-2 toxin [T-2], diacetoxyscirpenol, and verrucarin A). Published data is used to calculate the dose absorbed during cutaneous exposure to fungal toxins in hypothetical agricultural and research laboratory environments. These estimated doses of T-2 (0.004 and 0.041 ug/kg, respectively) were 25,000 and 2,439 times less than a reported oral dose (100 ug T-2/kg/day) which caused immunosuppression , in monkeys. In general, exposure of human skin to mycotoxins results in slow absorption. The risk of systemic toxicity resulting from dermal exposure increases in the presence of high toxin concentrations, occlusion, and vehicles which enhance penetration. un m , OD -1 121 tst 00 N iP In
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KINETICS AA)D METABOhISM OF SULFADIMETHO%INE IN C13At3*~F.L CATFISIH. CMF Michel, KS Squibb, JT Zeli- koff and JM O'Connor. NYU Med. Ctr., Inst. Env. Med., Tuxedo, NY Sulfadimethoxine (SD1A) 0,a sulfonamide drug used to control bacterial infections in the aquacul- ture industry. Studies of_the pharmacokinetics and metabolism of SDM have been undertaken to aid in predicting the uptake and tissue disposition of this drug in catfish and to estimate the accu- mulation of drug residues in e #ble flesh. Can- nel catfish were administered 'C-or 35S-SDM by intravenous (IV) injection or per os to determine vascular clearance, absorption from the gut, tis- sue disposition, metabolism and elimination. Vas- cular clearance of SDM administered IV was rapid (t1/2o,30.06;t1/2F=12.8hr) with a volume of distri- bution (Vd) of 662 mi/kg. Plasma protein binding of SDM was low (18%) and dose-independent. Na± SDM administered in food had a bioavailability of 40.7% and distributed rapidly to muscle tissue. Within 3 hr after dosing, 13% of the dose was pre- sent in muscle as parent compound. However, elim- ination of SDM from muscle occurred quickly with a t1/2 of 14 hr. SDM metabolism in channel cat- fish was also rapid. Six hr after IV dosing, about one-half the SDM in urine was present as N-acetyl-SDM and greater than 90% of the SDM that accumulated in bile was present as the N-acetyl metabolite. Thus, N-acetylation appears to be the most important transformation pathway leading to elimination of SDM in the channel catfish. Sup- ported by FDA-CVM-000150 and NIEHS Ctr. Grant ES-00260. SUBCHRONIC TOXICITY OF ORALLY-ADMINISTERED THEOPHYLLINE (GAVAGE AND DOSED-FEED) IN F344 RATS RND B6C3F1 MICE. J J Collinsl, J C Lamb IV2, A G Manus3, J. Heath3, and T Makovec37 iNTP NIEHS, RTP, NC; 2EPA, Washington, DC; 3Southern Research Inst., Birmingham, AL. Theophylline (TPL) is a methylxanthine pharmaceu- tical agent widely used for the treatment of var- ious respiratory disorders and certain acute car- diovascular conditions. 13-week subchronic toxi- city studies of TPL were conducted by gavage and dosed-feed in F344 rats and B6C3F1 mice (10 ani- mals/group). TPL in the feed (0,0.1,0.2,0.4%) re- sulted in no mortality or body weight effects in F344 rats, but did induce polyarteritis (nodosa) of the pancreas (males and females) and an in- creased severity of chronic nephropathy (males). Dietary TPL resulted in no mortality in B6C3F1 mice but terminal body weights were significantly decreased in all dosed groups and there was a dose-related increased incidence of hepatocellu- lar glycogen depletion in males and females. Ad- ministration of TPL by gavage in corn oil to F344 rats (0,37.5,75,150 mg/kg') resulted in slight mortality and significant body weight effects at the high dose. There was a dose-related increased incidence and severity of mesenteric perivascular infiammation in male and female rats. Gavage ad- ministration of TPL to B6C3F1 mice (0,75 150,300 mg/kg) resulted in the early death of ali high- dose females and 3/10 high-dose males and de- creased terminal body weights in high- and mid- dose males and low-dose females. As in the dosed- feed study, hepatocellular glycogen depletion was observed, although only in females. 346 THE EFFECT OF SUCROSE POLYESTER (SPE) ABSORPTION (ABS) OF SELECTED DRUGS EN OBAGUE- DAWLEY (SD) RATS. K Y Johnson, M W Perkins, and F E Wood, Jr., Procter & Gamble, Cincinnati, OH. Sponsor: R C LINDENSCHMIDT. „ w. SPE is a nonabsorbable, noncaloric lipid-like material which has the potential to be used as a fat replacement in many foods. Studies were de- signed to evaluate the effect of SPE on the ABS of 3 drugs with a range of partition coeffi- cients (PCs): diazepam (DM), propranolAJ_QL), and aspirin (AN). Cholesterol (CH) was used as a positive control since previoos studies showed that SPE decreases the ABS of this highly lipo- philic (high-PC) material. Male SD rats were orally administered a single dose of radio- labeled compound followed by 1 ml of either H20, corn oil, or SPE. ABS profiles wer~~e 4~- evaluated by monitoring total radioacti't~ity in plasma, urine, and feces over time, and in 3~e- lected tissues taken at necropsy. Results indi- cate that SPE had no effect, relative to H20, on the ABS profiles of DM, PL, or AN. Corn oil reduced the rate, but not the amount of ABS of all 3 drugs. SPE decreased the ABS of CH as reflected by increased fecal excretion of radio- label. These results indicate that the poten- tial effect of SPE on the ABS of a material can be predicted on the basis of its PC. A compari- son of the relative PCs of the 4 materials test- ed suggests that SPE will not affect the ABS of commonly-prescribed oral pharmaceuticals•. 347 PRECHRONIC TOXICITY OF SCOPOLAMINE HYDROBROMIDE IN RODENTS. D D Dietz*, E J Rauckman*, J D Prejean+, A G Ma, J E Hea and 0 R Farnell+, *NIEHS/NTP, Research Triangle Park, NC, and +Southern Research Institute, Birmingham, AL. Groups (0, 15, 45, 135, 400, and 1200 mg/kg) of ten F344 rats or B6C3F1 mice/sex received 5 daily doses of Scopolamine Hydrobromide per week for 13 weeks. The vehicle was deionized water. Body weights, food consumptions, and clinical signs were recorded, hematology anal- yses performed, Ad pathology evaluations were conducted. Significant mortality was noted in male (6/10, 400 mg/kg; 8/10, 1200 mg/kg) and female (7/10, 1200 mg/kg) rats. Food impac- tion of the pharynx appeared to be a primary cause of death. Body weights were depressed in all treatment groups and food consumption was increased in treated rats (400 mg/kg female; 1200 mg/kg male and female). Primary clinical signs involved the eyes and included dilated pupils (all treated animals), reddened eyes (13/40 rats, 400 and 1200 mg/kg) and lacrimation (15/40 rats, 400 and 1200 mg/kg). Hypoactivity was noted in 8/20 rats and 9/20 mice (1200 mg/kg). The WBC differential shifted toward increased segmented neutrophils/ lymphocytes' in all treated male rats and female mice and the three highest dose male mice groups. No histopathologic changes were associated with drug treatment. 5gg7$ 8212 87
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460 IN VIVO AND IN VITRO TOXICITY OF FOUR HALOGEN- ATED DIPHENYL ETHERS B M Francis, S A Engl, and M Farage-Elawar. University_,Cf Illinois, Urbana IL. . ~ As part of`ong,oing studies of the teratogenicity of diphenyl ethers, four compounds that can be unambiguously ranked with respect to embryotox- icity in vivo were also evaluated in an in vitro assay for cytotoxicity. The 4 compounds and the dose required to cause statistical3.y, significant perinatal mortality in CD-1 mice are [in ascend- ing order of effective dose]: 2,4,5-trichloro- phenyl 4-nitrophenyl ether [TCN], 4 mg/kg/day; 2,4-dichlorophenyl 4'-nitrophenyl ether [nitro- fen], 50 mg/kg per day; 2,4,6-trichlorophenyl 4'-nitrophenyl ether [CNPI, 250 mg/kg/day; phen- yl 4-bromophenyl ether [4BR], > 1000 mg/kg per day. The same order pertains for teratogenic potency, but decreased Harderian gland size is seen at lower doses than is perinatal mortality. In short-term culture, 100 ug toxicant per ml culture medium, the toxicity of these compounds to human lymphocytes was: 4BR > CNP > nitrofen > TCN, both in the absence and in the presence of rat microsomes. Cytotoxicity of 4BR decreased, and that of the remaining diphenyl ethers in- creased, with addition of microsomes to the sys- tem. At cell mortalities in excess of 502, dis- crimination between compounds was unsatisfactory because total cell counts were too low. ` 461 PHARMACOKINETIC MODELS FOR FETAL EXPOSURE TO DEVELOPMENTAL TOXICANTS. A H Marcus, P Feder, D Hobson. Battelle Columbus Division, Research Triangle Park, NC and Columbus, OH. Time-dependent compartmental models with physiological parameters are used to study the effects of pregnancy and lactation on the delivery of toxic substances and their metabolites to the fetus and neonate. Examples are shown for lead, hexachloro- benzene, and ethanol. The model shows lead accumulation, in growing fetal organs. The model also illustrates the effects of differing acetaldehyde metabolism of ethanol in mother and fetus, and may be useful in studying effects of time-varying exposures e.g. "binge drinking". Parameters used in modeling rat data are adjusted statistically to provide good-fitting models. 462 PLACENTAL TRANSFER OF FLUOXETINE IN THE RAT. R C Pohland, T K Byrd, M Hamilton, and J R Koons. Lilly Research Laboratonies, Greenfield, IN. Results from reproductive studies in Wistar rats at maximum daily 12.5 mg/kg oral doses of fluoxetine from two weeks prior to mating through lactation indicated that fluoxetine produced no compound-related adverse effects on fertility and no teratogenic effects. In order ~i to support these find s, the placental- transfer of 24C-fluoxetine in pregnant Wftt*&r rats was determined 1,- 4, 8, and 24 hours aft~- a single 12.5 mg/kg oral dose. Quantitation of* radioactivity within placenta, whole fetus, and amniotic fluid was determined during organo- genesis (day 12) and post-organog~eltesis (day 18) and compared with radio.al~bon`-- content in maternal tissues. Radioactivity -~`readily distri- buted to maternal and fetal tissues on both gestation days. Fetal tissue levels of radio- activity were low compared to maternal tissues. Fetal concentrations were similar to maternal plasma concentrations on gestation day 12; fetal tissue/maternal plasma ratios rose to 1.81 at 4 hours before declining to 0.92 at 24 hours. However, on gestation day 18, fetal concentra- tions were consistently higher than maternal plasma concentrations; fetal tissue/maternal plasma ratios rose to 3.54 at 4 hours before declining to 2.77 at 24 hours. This study demonstrated that 1``C-fluoxetine and/or its metabolites traverse the placenta and distribute within the fetus during the periods of organo- genesis and post-organogenesis. 463 MULTIPLE P450 ISOZYMES IN TBE CONCEPTUS DURING EARLY ORGANOGENESIS. H.L Yang, M J. Namkung and M R Juchau. Department of Pharmacology, University of Washington, Seattle, WA Highly sensitive probe-substrate analyses revealed that three separate tissues of day 11 rat conceptuses contained xenobiotic-oxidizing P450 isozymes. The embryo proper contained constitutive P450(s) ttat catalyzed readily measurable O-depentylation and 0- debenzylation but no measurable CI-demethylation and barely detectable O-deethylation of phenoxazone ethers. Higher activities for O-depentylation and O-debenzylation were measured in the yolk sac which also contained constitutive P450(s) for readily detectable 0-deethylation. O-deethylation could not be detected in the yolk sac. Only O-debenzylation was detected in the ectoplacental cone. Treatment with 3-methylcholanthrene (MC) significantly incresed 0- deethylation in yolk sac and embryo'but not ectoplacental cone. Demethylation was not detectable in the same preparations. Phenobarbital, pregnenolone 16 a-carbonitrile or isosafrole produced no effect on any reaction. CO markedly inhibited all reactions and inhibition was reversed by 02. Deethylation and debenzylation were inhibited by anti-P4501A1 IgG after MC induction but were not affected by the same IgG fraction in untreated conceptuses. Depentylation was not inhibited by anti-P450IA1 or anti-P450I1B 1/2 under any conditions utilized. Deethylation was strongly inhibited by 1.0 pM 7,8-benzoflavone in MC-treated but not untreated conceptuses. Metyrapone (1.0 µM) failed to inhibit depentylation reactions. The results indicated four (or more) functional P450 isozymes in the conceptus during organogenesis: a constitutive depentylase(s) in yolk sac and embryo, a constitutive deethylase(s) in yolk sac, an MC-inducible deethylase(s) in embryo and yolk sac and constitutive debenzylase(s) in all three tissues. No O-demethylation was detectable in any tissues, even after exposure to inducers. Supported by NIH grants ES-04041 and ES-04342. 116 50875 8241
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THE EFFECT OF EXPOSURE TO 14C-2,4,5,2',4',5'- L-IEXACHLOROBIPHENYL (6-CB) ON PLASMA _TESTOSTERONE (T) IN hjEONATAL MALE RATS. P D Calvert,,B 4 Ring and M J Vodicnik. Medical -- College of Wisconsin, Milwaukee, WI. Treatment of neonatal male rats with phenobarbital (PB)-Iike inducers results in irreversible effects on reproductive function and hepatic monooxygenase activities. The present study was ohrried out to determine whether maternal exposure to 6-CB influenced T levels in male perinatal rats, thereby modulating CNS "imprinting" of these activities. 6-CB .(100 mg/kg) or corn oil (CO) was administered ip to d14 pregnant rats. On d19 of gestation (dl9g) and d4 and 15 postpartum, plasma was collected from individual male offspring. Hepatic microsomes were prepared from individual litters and assessed for ethoxycoumarin-Q-deethylation (ECOD), benzphet- amine-h: demethyiation (BND) and P-450 content. No differences were seen on d19g correlating to the minimal placental transfer of 6-CB. Activities were elevated in nursing offspring of 6-CB treated mothers (d4-ECOD:19-fold; BND:25-fold; P-450:4-fold; d15- ECOD:6-foid; BND:6-fold; P-450:2.5-fold). T levels (pg/ml) as determined by RIA were not different following 6-CB exposure (d19g:CO=1038±222, 6-CB =1073±225; d4:CO=752±159, 6-CB=852±172; d15: CO=556-t96, 6-CB=474±114). This suggests that the neonatal endocrine system can compensate for elevated hepatic PB-like monooxygenase activities. EFFECTS OF NEONATAL EXPOSURE TO A_RCCLAR 1254 ON ADULT RAT HEPATIC MICROSCMAL TESTOSTERONE METABOLISM. M Kelley, J M Aaake and S Safe, Department of 4Teterinary Physiology and Pharmacology, College of 4eterinary Medicine, Texas A&M University, College Station, TX and Reprod. Develop. Toxicol., Natl. Ctr. for Toxicol. Res., Jefferson, AR. The effect of neonatal exposure to Aroclor 1254 (100 or 300 unol/kg) on hepatic microsanal testosterone hydroxylases was determined in 60-, 90- and 120-day-old rats. Most alterations in testosterone metabolism were observed in 90-day- old male and 60-day-old fenale rats. Males at 90 days exhibited decreased basal 7a-hydroxyla- tion and increased basal 16a-, 2a-, 20- and 15s- hydroxylations and androstenedione formation. 60-Day-old females treated neonatally with Aroclor 1254 exhibited decreased basal 16a-, 2a-, 6a-, 15s- and 16S-hydroxylase activities and androstene3ione formation. Testosterone hydroxylase inducibilities by Aroclor 1254 were. also modulated by neonatal exposure to this co:miercial mixture. In many cases, the lower dose of Aroclor 1254 (100 umol/kg) x,as more effective than the higher dose (300 anol/kg) in imprinting hepatic testosterone hydroxylases and further research on the neonatal effects of lower "environmettal" doses of fCBs is required. (Supported by the Texas Agricultural Ecperiment Station.) 466 ETHYLENE GLYCOL MONOMETHYL ETHER ~ (EGME)' EFFECTS ON TESTIS LACTATE DEHYDROGENASE (LDH) AND °' SORBITOL DEHYDROGENASE (S_DH) IN THE MOUSE. A R Nikurs, D P Waller, and L J D Zaneveld, University of Illinois at Chicago, and Rush-Presbyterian-St. Luke's Medical Centre, Chicago, IL. . Glycol ethers (GEs) are known male reproductive " .joxins in a number of species, idefttfng man. Groups of male CD-1 mice received p.o. eithei -. 467 117 distilled water or EGM1f at one of two dose levels•~,,~ (100 or 500 mg/kg per day) for one or two weeks. ~ The males were euthanized, and the testes, epididymides, seminal vesicles, liver, kidneys," and spleen were removed and weighed. The testes were homogenized, pelleted, and resuspgf~de&--dn a Triton X-100 solution. After 12 hours of incubation at 4oC, supernatants were filtered, and aliquots analyzed for LDH or SDH activity. For LDH assays, sodium pyruvate substrate and NADH in buffer were added; LDH activity was measured as a change in optical density at 340 nm. For SDH, fructose substrate was added, and the measurements made at 366 nm. Testes weights decreased with increasing EGME doses after both one and two weeks of exposure. No differences were observed in other organ weights between any of the groups. Decreases in LDH and SDH activity were observed in groups exposed to 500 mg/kg EGME for one and two weeks. ,. These results confirm previous observations that EGME exhibits toxic effects on the male mouse reproductive system at the testis level. TESTICULAR XENOBIOTIC, _METABOLISM IN HUMANS. K W DiBiasio, M H Silva, B D Hammock, and L R Shull. Depts. of Toxicology and Entomology, University of California, Davis, CA. Reproductive toxicology studies are routinely conducted using laboratory animals, or more rarely using non-human primates, which are thought to be better models for humans. Testicu- lar xenobiotic metabolism was compared in humans, monkeys, r4s, and mice to determine the validity of using ttiese animals as models for humans in reproductive toxicology. Human tissue samples were obtained from adult male organ donors. Rhesus monkey tissues were obtained at necropsy from adult males. Rodents were sexually mature male Fisher 344 rats and CD-1 mice. The activ- ities of epoxide hydrolase (EH) in microsomes (m) and cytosol (c) and cytosolic glutathione S- transferase (cGST) were measured using a radiometric technique. Testicular mEH activity in humans was comparable to mice, as opposed to 3.5-fold greater than rats, and 15-fold greater than monkeys. Testicular cEH activity in humans was comparable to rats and monkeys, but 2-fold greater than mice. Testicular cGST activity in humans was comparable to monkeys, however it was 60-fold lower than rats and 35-fold lower than mice. These results indicate that none of the animals studied here are an accurate model for testicular xenobiotic metabolism in man. 50875 8242
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484 IN VIVO PERCUTANEOUS ABSORPTION OF 4 pESTICIDES, AS AFFECTED BY ANATOMTC REGIONS OF THS RAT. L L Tromp, C Brownie, and F E Guthrie. Toxicology Program, N.C. State University, Raleigh, NC. A caiparative study was ca ,,rried eut in 7 week male Sprague Dawley rats (150 + 5 grams) with -1''C-labelqd peStiCides. The conparative - variablb wasg anatomic site (back, stomach, feet, ear and tail)'with dose (33 ug/sq cm), application site (+ 4.5 cm2), solvent (50 pL acetone), and methodology being constant. Quantitative measurement was based on excreta (urine and feces) and carcass r6CQveiy. The penetration of radioactive malathion, carbofuran, lindane, and fervalerate was determined in the rat after separate applications. After 48 hr the percent of malathion absorbed was greater than absorption of other pesticides for all anatomic sites except the ear where lindane was the highest penetrant. There was no consistent pattern of penetration among the 4 insecticides studied although malathion absorption was greatest after 48 hr. Absorption at 48 hr was generally greatest by the feet and lowest by the tail. 485 liERMAL ABSORf+T10N KINETICS OF NEAT HND AQUEOUS VOLATILE OR6ANIC CHEMICALS. DL Morgan, JR Tus- chall, RS Kutzman, Northrop Services, Inc, RTP, NC, and DR Mattie, AAMRL/TH, WPAF'8, OH - The objective of this study was to determine the dermal absorption kinetics of volatile orqanic chemicals detected in contaminated water. Male Fischer 344 rats were dermally exposed to neat, saturated, 2/3 saturated, and 1/3 sat«rated. aqueous solutions of trichioroethylene ilt;E), perchloroethylene (PCE), and toluene for 24 h. Blood samples (100 ul) were withdrawn prior to exposure and after exposure for 0.5, 1, 2, 4, 8, 12, and 24 h. Samples were analyzed by head- space gas chromatography. The volumes and con- centrations of the chemical solutions remaining in the exposure cells were determined after ex- posure for 24,h. Dermal exposure to neat PCE produced higher peak blood levels than exposure to neat TCE or neat toluene. Blood levels of each chemical reached a peak value rapidly and remained elevated throughout the 24 h exposure. Exposure to saturated aqueous chemicais resulted in rapid absorption, however bluod Levels did not remain elevated but decreased back to con- trol levels by 24 h. Exposure to ii3 and 2/3 saturated solutions of TCE, pCE, and toluene resulted in very low, but potentially sig- nificant levels of these chemicals in the blood. These data will be used in the development of a refined model of dermal absorption. (Supported by AAMRL/TH contract No. F:33615-a:5-4-Q532). 122 486 : 487 $LKANE INDUCED ZDEMA AND,,~,kARRIER DYS- FUNCTION. S J Molosaey`~and J J Teal. Avon Products, Suffern NY. Sponsor 3 Chang. Certain mineral oils and hydrocarbons require repeated topical applications to cause irritation. A structure activity relationship of pure n-alkanes in a mouse ear edema model was undertaken to investigate the mechanism of cumulative irritancy. Alkanes were applied twice > daily over a 96 hr ppr. Dodecane was non-irritating. Tridecane (C13) eliGaitec3 a response only.~t 96 hr. Tetradecank (C14) was the •s£r-ongest irritant with~.:--- significant increases at 48 hr. Hexa- decane, octadecane and eicosane showed progresively decreasing activity. Perm- eability of the ears to~•,contisol was monitored ~ vitro dur"r"ng C13 and C14 induced irritation. Significant increases in permeability were observed 24 hr before edema formation. Larger increases were observed with C14 than C13. Loss of barrier function resulted in increased cutaneous availabilty of the alkanes. Increased permeabilty prior to edema fQrmation indicates that induction of barrier dysfunction may be a factor in the mechanism of alkane induced irritation. Appreciation of such factors is important in new method development such as j,a vitro alternatives. UTILIZATION OF LANTHANUM TO DETECT CHANGES IN THE PER.*SEABILITY BARRIER OF RAT SKIN AFTER EXPOSURE TO SIX ORGANIC SOLVENTS. C J Hixson, J N McDougal*, M R Chase, and D R Mattie. AAMRL/THT, Wright-Patterson AFB, OH, *EOARD, FPO, NY. Sponsor: M E Andersen Rat skin was exposed in vivo to 6 organic sol- vents to correlate ultrastructural changes with the location of an electron dense tracer, lan- thanum (La), which is normally excluded by the permeability barrier in the stratum corneum. Male F-344 rats were exposed for 24 h to sterile saline, qichloroethylene, perchloroethylene, toluene, xylene,.hexane or benzene using dermal cells developed in this laboratory (surface area of 2.54 mm ). The cells were filled with 2 mL of saline or neat chemical. After exposure, the cells were rinsed with saline and filled with 1% La in saline for 1 h. After exposure to La, rats were sacrificed, cells removed and the exposed area of skin prepared for light and electron microscopy. Rat skin exposed to saline for 24 h was normal. La was found only on the surface of the stratum corneum indicating the permeability barrier was intact. In rat skin exposed to an organic solvent for 24 h, La was found throughout the epidermis and upper dermis indicating that these six solvents altered the barrier properties of the stratum corneum. 50875 8247
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364 IMPLICATIONS OF.ZCDD-ESTROGEN INTERACTIONS. T H Umbreit and M A Gallo. Dept. Environmental and Community Medicine, UMDNJ/Robt. W. Johnson Medical School, Piscataway, N.J,s a TCDD has been'showa to downregulate several receptors for estiogen, prolactin, androgens, corticosteroids, epidermal growth factor, low density lipids, and thyroxine. Many of the known effects of TCDD can be attributed to the downregulation of estrogen receptors .3nd the compensatory responses. We suggest that down- regulation of the estrogen receptor results in an increased synthesis of the natural ligand via feedback regulation. Therefore, both hypo- and hyper-hormonal responses will occur. Species that cannot excrete estrogens well are able to elevate estrogen levels sufficiently to overcome TCDD's toxic effects and will survive. Species that excrete estrogens well are unable to build up sufficient hormone levels and will die from toxic effects of over-synthesis of estrogens and the ensueing hormonal imbalance. Because hormonal systems are closely interregulated, other hormones are probably involved. (Supported by USEPA CR# 812114-01-1). 365 CCMPARATIVE EFFF7CTS•OF 2,3,7,8 TETRACHIARODI- BENZO-P-DIOXIN AND PROGESTERONE AS ANPIESTROGENS - IN THE FEMALE RAT VTERUS. M Romkes and S Safe, Department of Veterinary Physiology and Pharmacology, College of Veterinary Medicine, Texas A&M University, College Station, TX. The canparative antiestrogenic activities_ of 2,3,7,8-tetrachlorodibenzo-p-dioxin (ZCDD). and progesterone on nuclear and cytosolic -' progesterone (PRn and PRc) and estrogen (ERn and ERc) receptor levels in the rat uterus were - determined. Treatment of the rats with estra-- diol (5 ug/kg) and estradiol plus either progesterone (1 mg/animal) or 2,3,7,8-= (8PJ0 ug/kg) showed that both progesterone and 2,3,7,8TCDD significantly antagonized the estradiol-mediated increases in uterine ERc, ERn, PRc and PRn; moreover for 2,3,7,8-TCDD, the decreased receptor levels persisted for up to 7 days. In an in vitro rat uterine strip assay systen, both 2,3,7,8-ZCDD and progesterone act as antiestrogens. In contrast, the antiestro- genic activity of progesterone was inhibited by both protein and RNA synthesis inhibitors whereas the antiestrogenicity of 2,3,7,8 -TCDD was inhibited only by the RNA polymerase inhibitor, actinanycin D. These results suggest that the antiestrogenic effects elicited by 2,3,7,8 TCDD and progesterone are expressed through different mechanisms. (Supported by the Texas Agricultural Experiment Station.) .w~ _. ~.~. 366 ANTIATROPHY EFFECT OF 2,3,7,8-TETRACHLORODIBENZO- p-DIOXIN. (TCDD) ON RAT GASTRTC MUCOSA AND ITS POSSIBLE RELATIONSHIP TO HYPERGASTRINEMIA. H M Theobald, T A Mably, G B Ingall and R E Peterson. Sch. Pharmacy, Univ. Wisc., Madison;-WT. Atrophy of the gastrointestinal (GI) mucosa that • occurs in pair-fed control rats is not observed - in TCDD-treated rats (Christian et al., 1986). ~ Q~r objective was to determine~t~ie~I trophic hormone, gastrin, is involved in the antiatrophyA'~ ' effect of TCDD. TCDD-treated rats (100 pg/kg) were markedly hypergastrinemic 14 days posttreat- ment whereas pair-fed rats were normogastrinemic. Feed-restriction-induced atrophy of both fundic and antral mucosa was observed in pair-fed rats but not in TCDD-treated animals.,Aince hypergas- trinemia stimulates cellular p'roliferation and growth of fundic but not antral mucosa, the antiatrophy effect of TCDD on fundic mucosa could in part be due to hypergastrinemia. However its antiatrophy action on the antral mucosa must occur by a different mechanism. Reductions in the antral content and concentration of both gastrin and somatostatin were observed 7 days after treatment in TCDD-treated rats but not in pair-fed controls. The dose of TCDD required .to reduce antral levels of gastrin and somato- statin was significantly less than that needed to produce hypergastrinemia. In addition the TCDD-induced reduction in antral gastrin was not due to a decrease in the number of gastrin-contain- ing cells in the antral mucosa. (Supported - by NIH ES01332). 367 DECREASED GASTRIC ACID SECRETION AS A POSSIBLE: MECHANISM UF HYPERGASTRINEMIA IN 2,3,7,8-TETRA-., CHLORODIBENZO-p-DIOXIN (TCDD)-TREATED RATS-. 1 7- Mably and R E Peterson. School of_Pharmacy, University of lsc~l nsin; Madison, WI. - Hypergastrinemia occurs during the later stagess of the wasting syndrome. in. TCDD-treated rats (40-100 ug/kg).. Our objective was to determine the mechanism. Serum disappearance of gastrin-17 was similar i~ normogastrinemic control and hyper-, gastrinemic `ITCDD-treated rats suggesting that , reduced serum clearance of endogenous gastrins is not involved. Since reduc:•d acidity of stomach contents normally leads to elevated serum gas- trins, a TCDD-induced decrease in parietal cell acid secretion was postulated. In support of this hypothesis, TCDD-treated rats that were hy- pergastrinemic had decreases in gastric secretory volume, gastric acidity and total gastric acid output. Additionally serum gastrin concentrations of these animals were negatively correlated with total gastric acid outputs. The decreased gastric acid secretion was not due to a reduction in fundic mucosa parietal cell mass but appeared due to TCDD treatment inhibiting the ability of endogenous gastrins or exogenously administered pentagastrin from stimulating parietal cell acid secretion. 'Neither hypergastrinemia nor reduced gastric acid secretion was observed in pair-fed control rats. Thus hypergastrinemia in TCDD- treated rats appears due to a decrease in gastric acid secretion resulting from parietal cell dys- function. (Supported by NIH ESO1332). 50815 821.'1 92 i8
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IN VITRO PENETRATION OF PESTICIDES THROUGH HUMAN NE1dBORN FORESKIN. -li Shehata-Karam, N A Monteiro-Riviere and F C-6-uTfirfe.Tozico ogy rogram, Nort Carolina State University, *North Carolina State University School of Veterinary Medicine, Raleigh,-RC The in vitr& der4hal penetration of 1'+C-labeled paratiion, fenvalerate, carbofuran, and lindane through fresh fu1-1-thickness human newborn foreskin was determined at 1, 6, 24, and 48 hours. The pesticides were applied to a constant dosing area (0.031cm2), and a fixed Yose (1.18 µg) for each of the compounds studied. Ninety percent, or greater, of the labeled pesticides were recovered in all cases. Carbofuran showed the greatest mean penetration of 82% followed by parathion and lindane with mean penetrations of 79% and 66%, respectively. Fenvalerate exhibited a mean penetration of 9% which is significantly lower than that of the other three compounds. No difference was noted in the penetration of pesticides through human skin from blacks and whites. MAINTENANCE OF SKIN VIABILITY. DURING IN VITRO PERCUTANEOUS ABSORPTION/_METABOLISM STUDIES. S W Collier, N M Sheikh, A Sakr, J L Lichtin, R F Stewart,. R L Bronaugh, Division of Toxicology, FDA, Washington DC and University of Cincinnati, College of Pharmacy, Cincinnati, OH Evaluation off first pass xenobiotic metabolism in in vitro.percutaneous absorption studies requires the use of a receptor fluid which maintains skin viability. The fluid should allow extraction of parent compound and metabolites. Organ culture media (minimal.' essential media (MEM) t 10%'fetal bovine serum (FBS)), physiological"'.buffers ;and balanced salt solutions (BSS) were evaluated for these properties. Skin viability,was determined through aerobic C14 glucose utilization, maintenance of estradiol and testosterone metabolism for 24 hr and histological appearance of skin. Results show HEPES-buffered Hank's BSS with 0.1% glucose was equivalent to MEM in. maintaining metabolic activity for 24 hr in the diffusion cell at a flow rate of 1.5 mi/hr. Phosphate baffered saline was unable to maintain skin viability as exhibited by declining glucosee utilization and steroid metabolism. The exclusion of FBS and other organic compounds from perfusion fluids simplified extraction of parent compound and metabolites and precluded masking of metabolism due to protein binding of steroids. Recovery of the metabolite, estrone, was enhanced 5 fold by exclusion of FBS. Thus a HEPES-buffered BSS is adequate for metabolic maintenance of dermatomed skin (200 um) in flow-through cel absorption/metabolism experiments. 498 SORPTION, DISTRIBUTION, AND~-ELIMINATION OF 4C-BENZETHONIUM CHLORID"TC) IN FISCHER 344 RATS AFTER IV ADMINISTRATION OR DERMAL APPLICA-, TION. J D JOHNSON, J W CHINN, N HEJTMANCIK, W M KLUWE, A C PETERS, AND W C EASTIN*. BatteMe Co umbus Division, Columbus, OH and *NIEHS, Research Triangle Park, NC. The potential for toxicity to result from dermal ~ absorption of benzethonium chloride (BTC) exists- because it is widely used in cosmetics, disin- ~ fectants, herbicides, and top;LG&Lanti i nfecti ves. ~ lThe objectives of this study were to determiae.., ' the amount and rate of.percutaneous absorption,,;^ tissue distribution, and elimination of BTC •~= after a single IV administration (0.15 mg/kg) and a single (0.15 or 1.5 mg/kg) or 10-day re- peated (1.5 mg/kg) dermal application. Serial blood samples collected for 24.ihrs.kfollowing IV administration of 14C-BTC werd"usepto estimate bioavailability and determine pharmacokinetic parameters of BTC. Blood samples collected following the single or repeated dermal applica- tion of 14C-BTC were generally undetectable (low dose) or slightly above (high dose) detection limits (5.6 ng/mL). Low levels of 14C-BTC equiva- lents were measured in selected tissues from animals sacrificed at 6, 24, 48, 96 or 168 hrs after dosing. Urine and fecal samples were collected at 24 hr intervals after dosing. The urine contained detectable levels (>2.29 ng/mL), and the feces low levels of 14C-BTC equivalents. These results suggest that BTC penetrates the skin after dermal application. (Supported by Contract No. N01-ES-75177 from NTP). 499 SIGNIFICANT FIRST-PASS BIOACTIVATION OF PARATHION (P) DURING PERCUTANEOUS ABSORPTION IN THE ISOLATED PERFUSED PORCINE SKIN FLAP (IPPSF). M. P Carver, :. - P E Levi, and J.E. Riviere. Toxicology Prog. " & School of Vet. Med., NCSU, Raleigh, NC. Organophosphate insecticides have demonstrated unusual percutaneous absorption kinetics- and systemic toxicity patterns in vivo. - . The . metabolic profile of P was therefore studied -. in the IPPSF, an in vitro model for examining absorption mechanisms in living skin. Radio- labelled [1OC]-P was applied in ethanol _(40 pg/sq. cm) . Cumulative absorption was biphasic; - with an average penetration rate (APR) = 70 ng/sq.cm/hr for the first 5 hr, and peak flux = 320 + 50 ng/sq. cm/hr (mean + SE, n=3) at 3.5 hr post-dosing. The APR dropped to <10 ng/sq. cm/hr in the remaining 5 hr, producing a plateau at about 1% total absorption. TLC separation of - terminal perfusate samples revealed that almost 70% of the radiolabel penetrated as paraoxon, 6% as p-nitrophenol, and 24% as parent (P). P injected .into perfusate (no IPPSF) did not degrade significantly (<15%), and few metabolites (<10%) were found from a flap perfused 18 hr post-in situ dosing (APR = 8 ng/ sq. cm/ hr) . No P penetration occurred in a• non-viable IPPSF. Thus, biotransformation may play a crucial role in the disposition of topically dosed 'P. [supported by USAMRDC # DAMD17-84C-4103] 125 50875 8250 ~
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RATS. J.M. Andress, C.P. Chengelis, S. C. Gad, C.D. Port, and M,S. Tegtmeyer. G.D. Searle & Co., Skokie, IL The nephrotoxicity of D- and L-arginine was investigated. Each enantiomer was infused at 6 g/kg over 75 min into the caudal vein of eight male rats. Kidney function was evaluated by measuring several constituents of serum and urine. Structural changes were assess- ed by light and electron microscopy. Both D- and L-arginine caused changes in kidney function. Urinary protein and glucose were increased and urinary pH was decreased. Glomerular filtration rate was reduced as evidenced by reduced creatinine clearance, and increases in serum urea and creatinine. In addition, half of the rats treated with D-arginine but none treated with L-arginine had marked increases in urinary malate dehydrogenase activity, probably indicative of injury to epi= thelial cells of the proximal tubules. D-arginine caused obvious swelling and vacuolation of the epithelial cells of the proximal tubules, but L-arginine caused less obvious changes. In conclusion, D-arginine was moderately nephrotoxic, based on tests of kidney function and structure, , while L-arginine caused less serious changes. 133 §2' ACUTE, SUBACUTE, AND CHRONIC ORAL TOXICITY STUD- IES WITH BENAZEPRIL, A NOVEL 6NGIOTENSIN CON- VERTING ENZYME (ACE) INHIBITOR. J Hazelette, H Han Hsu, J Green, and V Traina. Res. Dept., Pharma. Div., CIBA-GEIGY Corp „ Summit, NJ. Benazepril `is a`structurally novel nonsulfhydryl ~ inhibitor of ACE: Toxicology studies demon- strated that Bes'.azepril is well tolerated in mice, rats, and dogs at doses well in excess of those needed for effective ACE inhibition. Acute toxicity was low in all three spec~es. Clinical signs appeared in the rat at 3000 mg7kg and in the dog at 250 mg/kg. In toxicity studies of 2-3 weeks duration, doses as high as 100 and 90 mg/kg were considered to be nontoxic in the rat and dog, respectively. Thirteen week rat and dog toxicity studies revealed no drug-related toxicity at high multiples (up to 75x) of the intended human dose. Chronic administration for 6-12 months to rats and dogs produced no unex- pected findings or adverse target organ changes. Minimal and mostly reversible compound-related effects were observed in rats and/or dogs after repeat exposure, and included: reductions in body weight, erythrocytic parameters, and heart weight; increases in serum BUN; and microscopic evidence of hyperplasia and/or hypertrophy of renal juxtaglomerular cells, and hypertrophy of afferent arteriolar and interlobular arterial walls: The majority of these findings were pharmacologic in nature and have been previous- ly reported for other ACE inhibitors. 328 NEPHROTOXICITY OF D- AND L-ARGININE IN 529 i2! THE TOXICITY OF 2-METHYL-L""1,4-NAPHTHOQUINONE CM) AND TWO THIOETHER CONJUGATES. P C Brown and T 6C Jones. Department of Pathology, University of Maryland School of Medicine, Baltimore, MD. _ The cytotoxicity of M has been associated with depletion of celiular thiols due to direct covalent interaction and oxidation as a result, of redox cycling. Excretable thioether conju= gates represent important quinone detoxicatiorp' ~ products. However, since-*Ahe- quinone nucleus" % remains intact, these conjugates may pose_AF-, threat of oxidative damage to tissues involve~; in their elimination. Zn the present study, the -;f,- toxicity of M and its glutathione (MG) and N-acetyl-L-cysteine (MNAC) conjugates was stud- ied using subcellular fractions and freshly isolated renal epithelial ce EC). Unlike M, MG and MNAC did not reac~wi~th (~#~t ' soluble or protein thiols in a cell-free system indicating a lack of alkylating potential. However, all three compounds were capable of redox cycling in the presence of renal microsomes (M=MNAC>MG) or mitochondria (M>MNAC>MG). All three compounds were cytotoxic to IREC (M>MNAC>MG). In all cases, cell death was associated with a deple- tion of soluble and protein thiols. M and MNAC resulted in a similar degree of cell death at 2 hr, but M caused more rapid initial cell killing and was associated with a larger reduction of thiols. These results indicate that the thio- ether conjugates of M are toxic to IREC by a mechanism involving oxidative stress. 530 STUDIES OF _METHYLCYCLOHEXANE INDUCED NEPHRO- TOXICITY AND METABOLISM IN MALE FISCHER 344 RATS. M J Parnell, G M Henningsen, K 0 Yu, M P Serve, and G M McDonald. Harry G. Armstrong Aerospace Medical Research Laboratory, Toxic Hazards Division, Wright-Patterson AFB, OH. Methylcyclohexane (MCH), a common solvent and a component of many fuels, was examined for its ability to produce a type of nephropathy characteristic of branched chain and cyclic hydrocarbons. Additionally, in a continuing effort to 4nderstand what effects specific hydrocarbon metabolism may play in the pathogenesis of this nephrotoxic syndrome, MCH urinary metabolites were isolated and identified. Fischer 344 male rats were dosed with MCH over a 14 day period. Histopathologic,examination of the rat kidneys revealed only a minimal amount of renal damage. Forty-eight hour rat urine metabolites of MCH identified were cyclohexylmethanol, t-3-methylcyclohexanol, t-4-methylcyclohexanol, 2c-hydroxy-4t- methylcyclohexanol, 2t-hydroxy-4c--methylcyclo- hexanol and 2c-hydroxy-4c-methylcyclohexanol."
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DIFFERENTIAL HISTOPATHOLOGY IN TCDD- TREATED AND PAIR-FED RATS. MJ Iatronoulos, JR Gorski, D Perera, G Muzi, RJ Arceo, LWD Weber and K Rozman. University of Kansas MedicaT Center, Kansas City, KS U.S.A, atatL Institut ffir Toxikologie, GSF MOnchen, Neuherberg, . F.R.G. and Medical Research Division, American Cyanamid, Pearl River, NY, U.S.A. ~ ( Histopathology of a usually nonlethal (25 µg/kg) and of a usually lethal (125 µg/kg) single dose of 2,3,7 8- tetrachlorodibenzo-p-dioxin (TCDD, ip in 95:5 ,ofn oiL•acetone) was studied in male Sprague-Dawley rats and compared to pair-fed as well as to ad libitum-fed controls at 1,2,4,8,16 and 32 (28 for lethal dose) days after dosing. Pair-feeding itself had numerous effects on the morphology of tissues as compared to ad libitum-fed controls. However, TCDD had dose- dependent differential effects as compared to pair-fed controls on the following tissues and organs: pituitary, pancreas, adrenal, thyroid, testes, thymus, lymphoid tissue of the gut, liver, brown adipose tissue and non- lymhoid tissue of the gut. Tissue-specific histopathology is discussed in the context of known endocrine, immunological and metabolic effects of TCDD. In addition, this study demonstrates the importance of appropriate viz, pair-fed controls also for histopathological studies, when body weight loss is a prominent feature of toxicity as is the case in many instances. The "normalization" of morphology using pair-fed controls aids in avoiding potential misdiagnosis of toxic effects, which in fact are attributable to reduced feed intake rather than to the toxic agent. (Supported by NIH ES-07079) TISSUE-SPECIFIC ALTERATIONS OF - DE. NOVO F_AT7Y ACID SYNTHESIS IN TCDD-TREATED RATS. JR Gorski,- LWD Weber and K Rozman. University. of Kansas Medical Center, Kansas City, KS . U.S.A. and Inst. fur Toxikol., GSF Munchen, Neuherberg, F.R.G. De novo fatty acid synthesis (FAS) was determined by the 3H20 method in 2,3,7,8-tetrachlorodibenzo-p- dioxin (TCDD)-treated (125 µg/kg), pair-fed and ad libitum-fed rats to examine if this energy-inefficient - process plays a role in the wasting away caused by TCDD.. Of the 12 tissues and organs examined, liver- showed an increased and interscapular brown adipose tissue (IBAT) a decreased FAS in TCDD-treated vs. pair-fed or ad libitum-fed controls. De novo FAS was unaffected in other organs • and tissues examined. Increased FAS in liver coincided with increased plasma triiodothyronine (T3) whereas decreased FA synthesis in IBAT parallelled decreased plasma thyroxine (T4) 1eVels. Thyroidectomy decreased FAS in both liver and IBAT. TCDD elicited no response in either of these organs in thyroidectomized (TXD) rats. These findings suggest that changes observed in non- TXD rats are probably secondary effects. Known tissue-specific effects of T3 on liver, and of T4 on IBAT provide a likely explanation for the. altered FAS in these organs. It is suggested that this increased FAS in the livers of TCDD-treated rats is responsible for the additional wasting away observable in TCDD- treated vs. pair-fed rats. The partial protection from TCDD toxicity in TXD rats may now be explained as a result of a reduction of an energy-inefficient metabolic pathway, viz. de novo fatty acid synthesis. (Supported by NIH ES-07079) 370 fORTICOSTERONE MODULATES ACU4E``TOXICITY OF 'I'CDD IN RATS. K Rozman, JR Gorski, T Rozman and H Greim. University of Kansas Medical Center, Kansas City, KS U.S.A. and Institut fnr Toxikologie, GSF Munchen, Neuherberg, F.R.G. Bilateral adrenalectomy or adrenal demedullation was performed in male Sprague-Dawley rats by established surgical techniques. Subsequently, the dose-response (mortality and mean time to death) to 2,3,7,8- tetrachlorot}ibenzo-p-dioxin (TCDD) was delii0ined in both adrenalectomized (10, 20, 40 µg/kg TCDD ip in 95:5 corn oil:acetone) and demedujlated (15, 30, 60 µg/kg) rats. Adrenalectomy resulted 'in a dramatically increased mortality and a much shorter mean time to death. The estimated LD50 was about 5 times lower than in non-adrenalectomized rats. Conversely, adrenal demedullation had no effect on mqEtalit,b~ or mean time to death. It was concluded thAT' factoi(s) modulating the acute toxicity of TCDD reside in the adrenal cortex and not in the medulla. Administration of corticosterone (25 µg/ml in drinking water) to adrenalectomized rats restored toxicity of TCDD to "normal" suggesting that this hormone is another key factor (in addition to the thyroid hormones) in the acute toxicity of TCDD. Corticosterone supplementation (25, .50, 100 µg/ml in drinking water) to non-adrenalectomized rats, or to thyroidectomized and adrenalectomized rats, resulted in no additional beneficial effect indicating that factor(s) other than thyroid hormones and corticosterone are also involved in the acute toxicity of TCDD. (Supported by NIH ES-. 07079) 371 CORTICOSTERONE DECREASES TOXICITY OF 3:CDD IN HYPOPHYSECTOMIZED RATS. M HBfler, JR Gorski and K Rozman. University of Kansas Medical Center, Kansas City, KS U.S.A. and Institut ffir Toxikologie, GSF MOnchen, Neuherberg, F.R.G. (HXD) by an established surgical technique. Similar to adrenalectomy, hypophysectomy aggravated the toxicity (mortality and mean time to death) of 2,3,7,8- Male Sprague-Dawley rats were hypophysectomized 93 tetrachlorodibenzo-p-dioxin (TCDD; 125 -µg/kg ip in _ 95:5 corn. oiL•acetony' ) when compared to sham-HXD rats (100% mortalit''with 9 ± 1 days mean time to . death versus .90°Po mortality with 32 ± 6 days mean time to death, respectively). However, administration of corticosterone (25 µg/ml in drinking water) to HXD rats resulted in an attenuation of the dose-response to a range of TCDD doses (125, 250, 500 µg/kg) much higher than the LD50 in non-HXD : rats (about 60 µg/kg TCDD). , Based on the data and those obtained in adrenalectomized rats it is concluded that one or more key factors which are capable of modulating the toxicity of TCDD, reside in the pituitary. (Supported by NIH ES-07079) s ~ t y`
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GUINEA PIG SKIN SENSITIZATION TESTING OF FIVE CHEMICALS USING THE MAXIMIZATION AND CLOSED PATCH TESTS. G W Trimmer and J J Freeman. Exxon Biomedical Sciences, Inc., Easr Millstone, NJ The skin sengitizIng potential of five chemicals was assessed in guinea pigs using both the Maximization Test*(MT, Magnusson and Kligman, J. Invest. Dermatol. 52:268,1969) and the Closed Patch Test (CPT, Buehler, Arch. Dermatol. 91:171, 1965). The test materials included two unknowns ("A", magnesium alkylphenol sulfide dnd "B", 3,4, 5-trichloropyridazine), two sensitizers ("C", formalin and "D", mercaptobenzothiazol) and a primary irritant ("E", sodium lauryl sulfate). Female Hartley albino guinea pigs (15/group, 310- 470 g) were used for both procedures. Dermal responses were graded 24 and 48 hr after chal- lenge (and after rechallenge) according to the method of Draize. The average rate of positive responses (Test values-concurrent irritation control values) in the MT and CPT, respectively, were: "A":0%, 9%; "B":93%, 51%; "C":71%, 78%; "D": 97%, 29~; "E":0%, 0%. Stronger responses were observed for "A" (24%) and "D" (53%) after re-challenge in the CPT. Thus no evidence of sensitization was observed for "E" in either assay or for "A" in the MT, "A" appeared to have sensitizing potential in the CPT (but has tested negative in a human patch test) and "B", "C" and "D" exhibited sensitizing potential in both assays. In addition, the sensitizing potentials of "B" and "D" were more readily detected with the MT. 513 HAIKLESS GUINEA PIGS: USE IN PHOTUTUXICITY TESTING. F L Fort and R V Kotz. Abbott Laboratories, Abbott Park, IL. Euthymic hairless, Cr1:IAF(HA)BR, and Hartley albino guinea pigs were compared to determine the suitability of hairless guinea pigs as a model for investigation of phototoxicity. 8- Methoxypsoralen'(8-MOP) given intraperi- toneally was used to induce phototoxicity. Hartley guinea pigs were shaved and treated with a depilatory 24 hrs. prior to treat- ment. Groups composed of 2 animals per sex each of hairless or hartley guinea pigs were given a single treatment: no treatment, UV (ultraviolet) irradiation only, 5U mg/kg 8-MOP plus UV, or 10 mg/kg 8-MOP plus UV. UV irradiation was a 3U min. exposure to a GE L4 Sunlamp positioned 15 inches above the animals beginning 1 hr. after injection of b-MOP. The skin of the back and ears was evaluated for phototoxicity (erythema) at 1, 2, 3 and 5 days after irradiation. Hairless and Hartley guinea pigs proved to be equally sensitive to phototoxicity under the conditions used. Hairless guinea pigs offer the advantages of 1) eliminating the need for hair removal prior to irradiation or evaluation of the erythema response, thus avoiding the potential for interference with the erythema response and 2) when an erythematous response was observed on the back, a definite line of demarcation separating the exposed and unexposed skin facilitated grading the response. 129 514 A COMPARATIVE METHOD OF PRESENTING DRAIZE EYE IRRITATION SCORES. G A Smith, C E Dick. S C Johnson & Son, Racine, WI. The FHSA test for eye irritation, using the Draize technique, has been used for many years• as the industrial standard for assessing poten- tial irritancy of test materials. The assay uses ,~ a dose of 100 ju1. The scoring results in a number. of time dependent eye scores which creates com- " -V9ex data relationships. The-ff"nt study inte-. _. grates the time dependent data by using a simpW,- time-weighted average otier 7 days by applying.` Simpson's rule. This method produces a single 'nF- value which improves comparisons and enables rankings between different products and different studies on similar products. It is-o4r experience that a reduced dose of 30 y~l ca~'improve discrim- ination between the potential eye irritancy of new formulas. Thus, 30 ,ul of each of 15 marketed shampoos was instilled into rabbit eyes without washing and standard Draize readings were per- formed. The shampoos were ranked according to this new integration method. Irritation compari- sons were made by observing the shape of time dependent plots and time-weighted average. Scores ranged from 0.4, a non-irritant, to 35.0, a severe irritant. In all cases, the time- weighted averages provided simplified, single- figure comparisons of eye irritation. This new method enables a graphic presentation of complex eye irritation scores that can be easily under- stood by non-specialists. 515 A MODIFIED CHORIOLLANTOIC MEMBRANE (CAM) ASSAY: A PRACTICAL ALTERNATIVE TO THE DRAIZE EYE.TEST. D M Bagley, P Y Rizvi, B M Kong, S J De Salva, Colgate-Palmolive Co., Piscataway, NJ . Concern for the use of animals in safety testing must be balanced with the need to ensure the safety of new products and compliance with reg- latory requirements. To meet these needs an alternative to the in vivo Draize eye irritation test was investigated which uses the chorioall- antoic mem, Tane (CAM) of a fertile chicken egg. Various metTiods have been published using this membrane but have shown limited correlation with in vivo. Only one report addressed the applica- tion of their method to predicting regulatory labelling requirements for eye irritation (Rong, B.M. et al in In Vitro Toxicology - Approaches to Validation. Vo. 5, A. M. Goldberg (Ed.) M. A. Liebert Inc., NY (1987)). However, a shortcoming of the CAM assay was the occurrence of false-positive reactions. Investigating the time course of the CAM response to irritants suggested that the initial event of hemorrhage could be used as an endpoint. The time of onset and the incidence of hemorrhaging were related to the ocular irritancy of the test material. Determining the incidence of CAM hemorrhaging at 30 min. after treatment resulted in a better separation of irritants from nonirritants. This modified CAM assay also offers the advantages of a shorter assay completion time, a more easily fleegfgined endpoint and the use of a less developed 50875 8254
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492 DERMAL P,BSORPTION OF POLYCHLORINATED DIBENZOFURANS (PCDFs) AND TCDD. Y B Banks-Case, D W Brewster, and L S Birnbaum. NIEHS, Research Triangle Park, NC 493 PCDFS andpplychlorinated dibenzodioxins are -. toxic envir7)nmental contaminants which accumulate in-human tissues. In order to examine the potential..for systemic exposure following dermal exposure, the absorption, distribution and elimination of 1,2,3,7,8- (1-PeCDF) and 2,3,4,7,8-pentachlorodibenzofuran (4-PeCDF), 2,3,7,8-tetrachlorodibenzofuranl(TCDF), and 2,3,7,8-tetrachlorodibenzodioxin (TCDD) were evaluated in male F344 rats. The compounds were applied to the back at doses of 0.1, 0.5, and 1.0 pmo1/kg in -60 pl acetone and covered with a perforated cap; rats were held in individual metabolism cages for 3 days. The percentage of dose absorbed decreased between 0.1 and 1.0 pmol/kg. Absorption of 1-PeCDF, 4-PeCDF and TCDD was similar: 22% of the dose at 0.1; 11% at 0.5; 10% at 1.0 ,umol/kg. Absorption of TCDF at 0.1 ,umol/kg was 44% which was significantly greater than that of the other compounds. Absorption at the higher doses resembled the other compounds. Major tissue depots for all 4 compounds were liver, fat, skin and muscle. Very little of the absorbed dose of 1-PeCDF, 4-PeCDF and TCDD was eliminated, however at the low dose of TCDF,_56% of what was absorbed was excreted as metabolite(s) in 3 days: Results indicate that all the compounds are poorly absorbed after dermal exposure and that metabolism is required for elimination. EVALUATING THE PERCUTANEOUS ABSORPTION OF POLY- NUCLEAR AROMATICS USING IN VITRO TECHNIQUES AND . STRUCTURE ACTIVITY RELATIONSHIPS. T A Roy, V Neil, J J Yang, A M Starrett, A J Krueger. Hobil Environmental Health and Science Laboratory, Princeton, NJ. Sponsor: M A Hehlman In vitro percutaneous absorption studies have een--u-nZe-rtaken to investigate the development of appropriate surrogate compounds to represent the major classes of compounds found in refinery" streams. The dermal penetration of a series of three through five fused-ring PNA has been deter- mined using a modified in vitro procedure using anthracene as a model for 3-fused-ring aromatics and benzo[a]pyrene (BaP) as a model for 4- and 5-fused-ring aromatics. The extent of absorption of nearly all the compounds in both classes fell within a factor of two of the chosen standards. In order to evaluate the "operational window" of anthracene and BaP as surrogates for other PNA, a mathematical model was developed based on struc- ture-activity relationships and multiple linear regression. Molecular descriptors (independent variables) were generated for each of the com- pounds whose dermal penetration (dependent-vari- - able) had been experimentally determined. A model was developed which correlated well with the experimental absorption data. The descrip- tors that best described the variance in the PNA study set seem to support the suggestion that percutaneous absorption can be assessed in terms of molecular shape and size and other common physicochemical properties. 494 IN VIVO AND IN VITRO SKIN ABSORPTION OF PCBs. R C Wester, H.I Maibach, D A W Bucks, J McMaster, M Mobayen, *R Sarason and *A Moore. Department of Dermatology, University of California at San Francisco, and *the California Primate Research Center at Davis, CA. PCBs are a major contaminant of the environment. Knowledge of their entry through the skin into the body and subsequent disposition is necessetiy for estimating human healli"azard. [14C]-42%-- : PCBs and [14C]-54% PCBs were separately ac~ ni= stered intravenousl-y_ and topically to rhesus monkeys. Following 5.v administration, 30 da Y`' Y`~== excretion was 39.4+5.9% urine and 16.1+0.8% feces (total 55.5+5.1%) for 42% PCBs and 7.0+2.2$ urine and 19.7+5.8% feces (tcta.L 26.7+7.5$) for 54% PCBs. Skin absorption o 42%:PCBs was 20.4+8.5% formulated in mirai., oil and 18.0+3.8% iil trichlorobenzene. Absorption of 54% PCBs was 20.8+ 8_3% in mineral oil and 14.6+3.6$ in trichiorobenzene. Mineral oil and trichloroben- zene are common PCB vehicles in transformers. Therefore, in vivo skin absorption of PCBs is considered high and disposition from the body is considered low. in vitro skin absorption in human cadaver skin was not predictive of the in vivo data in the rhesus due to lack of PCB pa,_tition frotu sk:n into water reservoir fluid. Eren cri_tit w0d:i.F.icn of 6% Oleth 20 solubilize::. 495 DESIGN OF CHEMICALS ZO TEST PERCUTANEOUS ABSORP-. - TION (PA)"PARAMFTF'ttS. D H Gould. US Environ- :: mental Protection Agency, Washington, DC. It is known that the rate of PA (flux) is rel-. ".'le ated toa the octanol-water coefficient (logP) of test chemicals; and also to the molecular weight (MW). Thus, for the.first few members of a homologous series, the logP and MW increase with: each additional -CH2- and so does the flux. How- ever, the increasing size (MW) operates to decrease the PA after a:naximisn is reached. In a serie~':of phenols and nicotinates, the maxi- mum flux is reached at logr of 2-2.5. Clearly at some maximum MW and/or lcg°, the flux will become negligible. To determine the maximun MW which can show PA, a series of polyesters was designed with a monomer core, with a calculated logP incr,ment of -0.15 and a MW increment of 144, malonylpropylenyl." This was capped as nicotinate and benzyl esters giving a monomer of MW 357 and ClogP 252. This series leads to the pentamer of MW 933 and ClogP 1.92. Thus, the log P is held relatively constant while the MW al- _, most triples. This should allow determination of MW maximum for PA independent of change of logP. 124
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< CHLORINATED DRINKING WATER DECREASES SERUM HIGH DENSITY LIPOPROTEIN IN TWO' MONKEY SPECIES. J p Berczl, L Jongsl, T Mills~; J Stoberl, J Cicmanecl, a4d Condiel. lU.S. EPA, Health Effects Research Laboratory, Cincinnati, OH, 2Computer Sciencea'Corporation, Cincinnati, OH. Chlorinated (0.87 mM NaOCI) drinking water de- creased HDL cholesterol and increased low density forms of lipoproteins in thg serum of two species of non-human primates maintained on an atherogenic diet consisting of a normal high protein monkey chow containing 15% top lard and 1% free cholesterol. Food and water was admini- stered ad libitum, and weekly fasting serum samples were collected for determination of cholesterol,.triglycerides, HDLc and lipoprotein electrophoresis. The effect on lipoproteins apeared within four weeks after start of expo- sure and persisted after cessation of exposure to hypochlorite. Similar effects however could not be elicited by comparable doses of Na0C1 when the monkeys received a non-atherogenic, low lipid and high protein diet (Purina Monkey Chow). This observation raises suspicion about the possible role of residual chlorine in drinking water as a co-atherogen which may exacerbate the atherogenic risk of dys-lipopro- teinemia in susceptible or diet-stressed human populations. (Abstract does not necessari- ly reflect EPA policy). 552 INCREASED LEVELS OF LUNG DNA QDDUCTS IN RATS EXPOSED TO PARTICLE-ASSOCIATED BENZO(A)PYRENE (BP) COMPARED TO PURE BP. R K Wol ff, J A Bond, J D Sun, R F Henderson, and J L Mauderly. Lovelace Inhalation Toxicology Research Institute, Albuquerque, NM Exposure of rodents to benzo(a)pyrene (BP) asso- ciated with particulate matter results in en- hanced lung tumorigenicity and retention of BP and metabolites in lungs compared to BP alone. We examined whether DNA lung adducts differed following inhalation of pure BP or particle-as- sociated BP. Groups of rats were exposed nose-only to air, BP (2 mg/m3) or BaP adsorbed on carbon black (BP/CB) for 4 hr/day, I day/wk, for 12 weeks. Inhalation of BP/CB resulted in higher lung burdens of BP and metabolites com- pared to pure BP. DNA isolated from lungs was analyzed for DNA adducts using the 32P-postla- beling assay. Lung DNA adducts in rats exposed to BP alone ranged from <1-15 adducts in 109 bases (medians3) and in rats exposed to BP/CB from 7-32 adducts in 109 bases (median-16); con- trol rats had (1 lung DNA adduct in 109 bases. One of the DNA adducts was tentatively identi- fied as the BP diol-epoxide deoxyguanosine ad- duct. The data suggest that there may be a re- lationship between the higher levels of lung DNA adducts and enhanced tumorigenicity of BP when it is associated with particles. (Re- search sponsored by the U.S. DOE/OHER under Contract No. DE-AC04-76EV01013.) 553 COMPARATIVE PHARMACOKINETICS OF INHALED AND INGESTED 1,1-DICHLOROETHYLENE (DICE) JN RATS. C E Dallas, R Ramanathan, S Mu`ralidhara, J M Gallo*"'° and J V Bruckner. Depts. of Pharmacol. & Toxicol. and arm~Fi aceut cs*, College of Pharmacy, University of Georgia, Athens, GA. _ 554 139 In order to assess the feasibility of route ~, to route extrapolation of phar c._okinetic data ~E'or volatile organics, the upta't'ce~''and elimination*-. of DCE were contrasted.i,p male S.-D. rats -""`:"'- subjected to inhalatfon`-and equivalent oral DCE'~&.~ exposures. Unanesthetized rats of 325-375 g with'~ an indwelling arterial cannula inhaled 100 or 300 ppm DCE for 2 hr through a 1-way valve. Repetitive samples of the separa,t e.,i0haled and exhaled breath streams, as wel^. s arterial blood, were collected concurrently 6nd analyzed for DCE by GC. Based on cumulative uptake at the end of the 100 and 300 ppm inhalation exposures, an equivalent oral dose of 10 or 30 mg/kg, respectively, was given in an aqueous emulsion as a single oral bolus to rats and serial blood samples taken from an arterial cannula. Peak blood levels of DCE were achieved within 4 min of the oral bolus, but levels declined` rapidly thereafter. Rats inhaling DCE attained near steady-state blood levels during the 2-hr exposure that were 3 to 4 times lower in magni- tude than peak levels achieved after an equiva- lent oral bolus dose. Nevertheless, AUC values were higher for each inhalation group than for the corresponding ingestion group. (Supported by U.S. EPA CR 812267 and U.S. Air Force AFOSR 870248) A COMPARISON OF THE TOXICITY OF ASBESTOS, NON- ASBESTOS FIBERS, AND SEMI-METALLIC BRAKE RESIDUE IN A MACROPHAGE-LIKE CELL LINE. C S Wheeler and C D Garner. Biomedical Science Dept., GM Research Labs, Warren, MI. Sponsor: E S Wright A variety of materials have been developed as replacements for asbestos but the potential health impact of these materials is largely unknown. The purpose of this study was to evaluate 4nd compare the cytotoxicity of crocidolite, amosite, and chrysotile asbestos, with two asbestos TAiber substitutes, calciuW sodium metaphosphate (Monsanto) and Fiberfrax (Sohio), as well as semi-metallic disk and drum brake lining residues generated using a dynamometer under simulated driving conditions in a macrophage-like cell line, J77uA.1. Following 24-hour incubations of cells with the test materials, changes in cell morphology, number and viability were assessed. A viability index was calculated to reflect changes in both cell number and viability and used to establish the relative cytotoxicity of the various materials under study. At equivalent concentrations (µg/mL), the 'eytotoxicity of the test materials was amosite, crocidolite, chrysotile > calcium sodium metaphosphate fibers >> semi-metallic drum brake residue > semi- metallic disk brake residue, Fiberfrax fibers > semi-metallic disk and drum brake lining filings. 50875 8264
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401 547 SUBCHRONIC CARDIOVASCULAR EFFECTS OF SOMAN INTOXI- CATION. R Moutvic, C R Hassler, *R L Hamlin. Bat- telle Columbus Div., Columbus, OH; *Ohio State University, Columbus, OHk.. Sponsor: G L Fisher This study fnvestigated the effects of a single, non-ldthal`dose of Soman on the mechanical func- tion, electrical properties, and nervous control of the canine heart. Chronically instrumented animal models were prepared to examine irritabil- ity, nervous control, hemodynamic properties and arrhythmic activity. 81j animals were observed for one month post-exposure. The elec- trophysiology data suggest a temporary decrement in response to direct .6-sympathomimetic.stimula- tion and direct sympatholytic stimulation hypoten- sion. Chemical denervation experiments suggested a direct effect of Soman upon cardiac activity. Repetitive ventricular response data indicated an increase in irritability. Cardiovascular data indicated a mild decrease in cardiac perform- ance; however, none of these decrements were life threatening and indicated the cardiovascular system was capable of providing sufficient blood supply. Holter arrhythmia analysis indicated animal-specific increases in ectopic events post-Soman exposure. Overall, a prolonged state of autonomic imbalance was characterized primarily by parasympathomimetic activity. The autonomic imbalance was further suggested by the appearance of potential life-threatening arrh thmic events in Soman-intoxicated animals. (Supported by U.S. Army Contract No. DAMD17-85-C-5038). 948. EFFECT OF PRENATAL EXPOSURE TO SODIUM SALICYLATE . (NaS), ASPIRIN (ASA), OR GENTAMICIN (GT ON BLOOD_. PRESSURE IN RATS. G L Johnson, F R Alleva`and.., T Balazs. FDA, Washington, DC. Prenatal exposure to NaS or G has been reported :`'too result in elevated blood -pressure - (BP) in.. female rats. To confirm these findings,.preg- . nant Sprague-Dawley rats.were given either NaS. (125 or 175 mg/kg, p.o., q.d), ASA •(1.75 mg/kg,. : p.o., q.d) or G (25 or 75 mg/kg, i.p., b.i.d.).-) on days 9-11, .9-il, and 14-18 °of gestation, respectively. BP was measured by the tail-cuff method (indirect) in: 1) lightly anesthetized (pentobarbital, 30 mg/kg, s.c.) 3-month-old male and 5-month-old female pups from the NaS and ASA groups and 1-year-old female pups in the G group; and 2) conscious 3-month-old male pups from the high-dose NaS group and 5-month- old females from the NaS and ASA groups. BP was also measured by cannulation of a carotid artery (direct) in more deeply anesthetized 5-month-old females from the NaS and ASA groups. Heart rate (HR) was determined during all BP measurements by counting pulses. In no instance was either the BP or HR of rats ex- posed prenatally to NaS, ASA or G significantly different from that of respective controls. Also, there was no drug-related histopathologic change in kidneys from 5-month-old pups exposed prenatally to NaS or ASA. Treatment with G resulted in renal histopathologic changes in dams at 21 days of gestation but not in 21- day-old fetuses or 1-year-old offspring. 549 Si,ARDIOMEGALY IN NEONATAL RATS EXPOSED TO 500 PPM _QARBON JJONOXIDE. F J Clubb, Jr, D G Penney, and S P Bishop. Dept Path, UTHSCD; Dallas, TX, Dept Physiol, WSU, Detroit, MI, Dept Path, UAB, Birmingham, AL. Sponsor: Z Ruben Carbon monoxide (CO) hemodynamic workload Kas induced in growing neonatal rats to study ~ changes in myocyte (MC) structure and number and to determine if CO proftftott volume-overload - f cardiomegaly. Newborn rats were exposed to:`300 ppm CO for up to:.32 days of age (d), at wK4c.b-: time the remaining CO exposed rats and ambie»t air controls continued development in room air until 200d. In the CO group, ventricular weight: body weight ratio was 26% greater than controls at 6d, more than 100% at,*d and 477. greater at 28d. Although absolute MC volumes were not different between the two groups at any time period, the CO group did have greater MC:body weight ratio at 6, 15 and 28d. Binucleated MCs of 15 and 28d CO rats were both longer and had increased length:width ratios than controls. By 200d, MCs from LV+S of CO exposed rats were significantly shorter. CO exposed rats at 200d had more total MCs (36X106 versus 32X106 for controls, p<0.05). Cardiomegaly induced by 500 ppm CO from birth to 32d was primarily due to MC hypertrophy, with MCs having increased length: width ratios (i.e., changes' consistent with a volume-overload model). Regression of cardio- megaly occurred after removal from CO, with smaller MC length and volume and greater MC number.- 550 REVERSAL OF PROPRANOLOL TOXICITY WITH AMINOPHYLLINE, AMRINONE OR FORSKOLIN. Vick, J, Whitehurst, V, Joseph, X and Balazs, T Food and Drug Administration Washington, DC. Hypotension and bradycardia leading to irreversible shock and death can be the result of overdoses of propranol-ol, a widely used beta blocking agent. We attem~'~pted to reverse the effects of lethal overdos~s"of propranolol with one of three agents, aminophylline, amrinone or forskolin. Anesthetized beagle dogs were given a ten minute infusion of propranolol at a dose of 1 mg/kg/min. The five control dogs exhibited profound hypotension and severe.bradycardia which lead to cardiogenic shock and death at 15-30.min.. Treatement with each compound was initiated within five minutes following the end of propranolol infusion. Aminophylline (20 mg/kg iv) increased heart rate and blood pressure to near normal levels in 7 treated dogs within 30 to 60 seconds. Both amrinone (2-3 mg/kg, iv) in 5 dogs and forskolin (1-2 mg/kg, iv) in 4 dogs were also effective; however, recovery was slower than with amino- phylline. All treated dogs survived with no apparent residual damage. Results of this study show that each of these drugs is capable of reversing the otherwise lethal effects of propanolol overdose. 138 50875 8263
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531 THE INTERACTION OF SODIUM IHIOSULFATE (Na S 0) AND 1,4-(N,N-DIMETHYL) sETRASULFIDE WITH ORA PIG LIVER.BHODANESE. S I Bdskin and S D Kirby. US Army Med. Res. Inst.em. 6efense, APG, MD. Availability,_of Na S 0 for mitochondrial rhodanese is "the rati 3iliting factor for the detoxification of cyanide to thiocyanate (SCN). WR146108, (1,4-N,N-dimethyl) tetrasulfide) was examined for its capacity to act as a sulfane-sulfur donor. Guinea prg,rhodanese was isolated following the method of Westley (Adv. Enz. 39, 1973). Rhodanese activity was measured by following the formation of the ferric-SCN complex at 460 nm as an in vitro model of cyanide detoxification. Using Nb (50 mM) as a substrate, rhodanese can m~dx~mIlly form 0.45 umol SCN/mg/min, while WR146108 (32 mM) acting as a substrate can form 1.5 umol SCN/mg/min. WR146108 (2 or 8 mM) inhibits the ability of Na2S 0 (> 5 mM) to form SCN. Conversely, i";Ueasing the WR146108 concentration (> 8 mM) potentiates rhodanese maximum response to 7.4 umol SCN/mg/min in the presence of 50 mM Na2S 0 Comparing the half-maximal response of eicR*substrate, Na2S 0 appears to be 4-6 times more potent tQ WR146108 although it displays less intrinsic activity. Our data suggests that Na S 0 and WR146108 act synergistically to 2St?iAlate rhodanese activity. We propose that under certain conditions, a polysulfide combined with Na S 0 may provide improved antidotal benefit agd2i~isi cyanide toxicity. 532 STUDIES OF IN VIVO NEPHROTOXIC POTENTIAL OF CYS- TEINE CONJUGATES AND MERCAPTURATES OF STYRENE AND BROMOBENZENE. S Chakrabarti and A Malick. Med.trav.hyg.mil., Faculte de medecine, Universi te de Montreal, Montreal, Quebec, Canada. Our previous studies reported the in vitro nephrotoxicity of cysteine-and N-acetylcysteine conjugates of bromobenzene (BB) and styrene (S) using isolated renal tubule suspensions (Clin. Res. 35: 544A, 1987). The present study was designed to determine the in vivo nephrotoxic potential of cysteine conjugates and mercaptura- tes of BB and S. Groups of male Fischer-344 rats (150-160 g b.w.) were treated i.v. with 0 and 0.025 mmole per rat of S-(l-phenyl-2-hydro- xyethyl) cysteine (PEC), N-acetyl-S-(l-phenyl-2- hydroxyethyl) cysteine (NAPEC), S-(0-bromophenyl) -L-cysteine (0-BPC) and N-acetyl-S-(0-bromo- phenyl)-L-cysteine (0-NABPC) in phosphate buffer, pH 7.4 Urines were collected for 8, 24 and 48 h after the administration and the animals were finally sacrificed for blood withdrawal and tis- sue removal. A significant increase in urinary glucose and N-acetyl-S-D-glucosaminidase (lysoso- mal) due to NAPEC, O-BPC and O-NABPC as well as that of urinary LDH (cytosolic) due to PEC, NAPEC and 0-NABPC was observed at 48 h. A significant increase of urinary y-GT (brush border) due to NAPEC at 24 h and an increase close to signifi- cant due to NAPEC and O-BPC at 48 h were noticed. An increase of urinary GDH (mitochondrial) close to significant due to NAPEC at 24 h and O-NABPC at 48 h was also seen (MRC grant # MA-9705). 533 INABILITY OF PHENOBARBITAL TO MODIFY.EENTAMICIN- INDUCED NEPHROTOXICITY IN RAT. S Kacew. Dept. of Pharmacology, Univ. of Ottawa; Ottawa,. Ontario. 'f . by the Medical Research Council of Canada). inability to increase GEN excretion. (Supported elevated tissue aminoglycoside levels as . compared to GEN alone. Data show that PB appears to be an ineffective inhibitor of GEN- induced kidney damage as reflected by an nephrotoxicity was associated with signif icantly days, PB failed to prevent aminoglycoside- induced phospholipidosis, morphologic changes and decrease in alkaline phosphatase. The inability of PB to inhibit GEN-induced given GEN and PB (30 mg/kg/day) (ip) daily for 4 alkaline phosphatase. In rats concurrently activities of phospholipase C, Na -K ATPase and category of agents, the objective of this study was to determine whether the effects of GEN on rat kidney metabolism and morphology could also be blocked by PB. GEN (10~*gJiFg/day) administered (sc) daily fot 4 dgys to male Sprague-Dawley rats increased the renal concentration of phospholipid and+de~reased the phospholipidosis and GEN belongs to this The renal toxic manifestations associated with gentamicin (GEN) include phospholipidosis, inhibition of phospholipase C, hist~pa~hological alterations as well as decreased Na -K ATPasd' and alkaline phosphatase-attt![vities. Since ` phenobarbital (PB) was previously found to•r-_ ~ prevent cationic am}thiphilic drug-induced 534 INFLUENCE OF DECREASED RENAL MASS ON G-ENTAMICIN (G) NEPHROTOXICITY: ULTRASTRUCTURAL MORPHOMETRIC AND FUNCTIONAL STUDIES IN DOGS. D L. Frazier, B. Fowler and J.E Riviere. Univ. Tn. College Vet Med., Knoxvil e-1 Tn; NIEHS, Research Triangle Park, NC; North Carolina State Univ. Sch. Vet. Med.; Raleigh, NC. Subtotally nephrectomized dogs were used as a model to study morphological alterations in sub- clinical renal disease that may predispose to nephrotkicity from gentamicin. After 12 days of gentamicin treatment, serum chemistries did not differ significantly between groups. Light micro- scopy showed differences in proximal tubule cell (PT) necrosis and regeneration between nephrecto- mized control and treated dogs but not between intact control and treated dogs. Ultrastructural morphometric studies showed volume densities of mitochondria and lysosomes were increased in nephrectomized dogs. Lysosomal volume increased in G-treated groups relative to their control groups. Lysosomal diameter and number per unit volume increased in treated intact dogs whereas only lysosomal diameter increased in nephrectomi- zed treated dogs. Surface density of outer and inner mitochondrial membranes increased in neph- rectomized dogs. Rough endoplasmic reticulum increased in intact G-treated dogs. These data suggest partial nephrectomy results in altered morphology of remnant PT cells and these alter- ations may result in a predisposition for nephro- toxicity at clinically useful levels of G. 134 50875 8259
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416 SYNTHESES OF N-OXIDIZED DERIVATIVES OF 4,4'-.Ut ETHYLENEBIS(2-CFILORB'ANILINE) Ul•1l3OCA) AND THEIR -DIRECT -MUTAGENICITIES TOWARD. ~ TYPHIP:tURNP.1 TA90 AND TA100. B I Kuslikis, T H Chen and VJ •E Braselton. Dept. of Pharmacology and Toxicology,.P,lichigan State Univ., East Lansing, AAI. (Sponsor: W D Atchison) AABOCA, a commercially important crosslinking agent and an environmental contaminant 1is-an arylamine, a class of compounds thought to be activated to proxi- mate carcinogens by metabolic oxidation to the corre- sponding hydroxylamine. Consistent with this hypothe- sis, f;1BOCA requires rat liver S9 activation to become a mutagen in the 1. typhimurium mutagenicity assay. This study tested the hypothesis that AABOCA is acti- vated through N-oxidation. We synthesized N-oxidized derivatives of PABOCA by oxidation with m-chloroper- benzoic acid. The N-hydroxy and nitroso metabolites formed by liver microsomal enzyme systems were identical to the chemically synthesized derivatives. The N-hydroxy metabolite showed strong frame shift mutagenicity towards 1. typhimurium TA98 as well as strong base pair mutagenicity towards TA100. The nitroso derivative showed a slight positive effect on TA100. The dinitroso compound was not active. A third major metabolite of microsomal oxidations, .4llb.Q:-hydroxy 141BOCA, was also not mutagenic at any of the concentrations tested. These studies show that the N-hydroxy derivative of MBOCA is highly mvtagenic and may account for the majority of the mutagenicity of MBOCA seen in the S9-activated bacterial test system. (Supported by the RAich. Dept. of Public Health.) 417 SISTER CHROMATID EXCHANGE IN EPILEPTIC PATIENTS ON ANTICONVULSANT THERAPY. V B Winge, B Schaumann*, V F Garry. University of Minnesota, Environmenta:_ Pathology Laboratory, Minneapolis, MN. * Veterans Administration Medical Center, Neurology Service, Minneapolis, MN Sister chromatid exchange (SCE) techniques were used to test the mutagenic potential of anti- convulsant drugs in epileptic patients. The following drugs were studied: valproate (n=13), phenytoin (n=17), phenytoin and phenobarbital in combination (n=9). Each patient was matched with a control (n=30) by sex, age and smoking habits. All individuals with exposure to known environmental mutagens or taking other drugs were excluded. No statistically significant differences in SCE level were found between the patient and control groups, indicating a lack of mutagenic potential of the tested anti- convulsant chronically administered (six months or more) within the therapeutic dose range. Supported by the Veterans Administra- tion. 105 418 A FILTER BINDING ASSAY TO DETECT E.HROMIUM- INDUCED DNA-PROTEIN CROSSLINKS IN ISOLATED ' NUCLEI, T P Coogan and M Costa. Institute of Environmental Medicine, New York University Medical Center, Tuxedo, NY. Exposure to chromium results in DNA-protein i, crosslinks (DPC) that are pe~sistent and stable;.- 'fi Since this type of lesion may playan import~tt role in chromium carc ,nogenicity, the mechanisia~ of chromium-induced DPC was investigated using'_4_ a filter binding assay. Prior to isolation of CHO cell nuclei, DNA and proteins were labelled using 3-H thymidine and 35-S methionine, re- spectively. Nuclei were suspendecj;,.in 10 mM HEPES buffer (pH 6.8), and e;o'osed'to either UV irradiation, CrC13, or K2CrO4. Following expo- sure, nuclei were incubated at 370C for 20 min in a high salt solution; aliquots were loaded onto nitrocellulose filters'and washed with a low salt solution, DNA (3-H) retained on each filter was normalized based on the protein re- tained (35-S). Exposure of isolated nuclei to UV irradiation resulted in a dose dependent in- crease in DPC as indicated by the increasing 3-H/35-S ratio. CrC13 induced DPC in a biphasic manner over the concentration range,tested (0- 200 uM), whereas K2CrO4 only induced DPC at the higher conc. (>100uM)o Incubation of K2CrO4 with sodium bisulfite prior to nuclei exposure resulted in DPC similar to that observed with CrC13 alone. (Supported by Grant No. CA-43070 from the NCI) 419 INDUCTION OF ANEUPLOIDY.BY Ni(II) AND Cr(VI) IN A HUMAN/MOUSE HYBRID CELL SYSTEM, K Conway, 1R S Athwal- and M Costa, Inst. of Environmental Medicine, NYU Medical Ctr., Tuxedo, NY and 1Dept. of Microbiology, NJ Medical School, Newark, NJ. Increasing evidence indicates that aneuploidy may play an important role in the multistage process of carcinogenesis. In the present study, the abiliV of the carcinogenic metals, Ni(II) and Cr(VI), to induce aneuploidy was assessed in a cell culture system using a human/mouse somatic cell hybrid containing a single copy of human chromosome 2. The human chromosome present in the mouse cells served as a cytogenetic marker to quantitate abnormal chromosome segregation and could be easily identified by the G-11 staining technique. The frequency of cells con- taining 0 or 2 human chromosomes in the progeny of metal-treated hybrid cells provided a direct measure of aneuploidy. Preliminary results in- dicate that R-crystalline NiS, NiC12, and K2Cr04 induced aneuploidy in this system in a dose- dependent manner and the highest frequency of aneuploidy observed for all three metal compounds was in the range of 2 to 3-fold above background. Each of these compounds also produced an increase in the number of polyploid cells. MgC12, a non- carcinogenic metal compound, did not induce aneuploidy or polyploidy in this system, SimilaY results were obtained when aneuploidy was assayed with primary Chinese hamster embryo cells. (Supported by Grant No. R813140 from the U.S, EPA) 50876 8230
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524 EFFECT OF O_XYGEN TENSION AND ANTIOXIDANTS ON 525A SUSPENSIONS OF ISOLATED RAT RENAL PROXIMAL TUBULES (RPT). J E Dabbs, C E Green, K L Allen, C A Tyson, and *E J Rauckman. SRI Interna- tional, Menlo Park, CA and *?iational Toxicology Program, NIEHS,•Research Triangle Park, NC. ~ -t. A decline in.the viability and functional capabilities of.isolated RPT limits their •utility for many in vitro studies. Tubules were isolated from adult male F-344 rats by a one- step collagenase perfusion method and were incubated in Waymouth's 752/1 + 2$ ZSA + 5 mM lactate in gas-tight flasks with an atmosphere of 95%, 19%, or 10% 02:5% C02:balance NZ. Tubule viability was determined by measuring lactate dehydrogenase release (LDH-R). LDH-R at 4 hr increased progressively with higher 02 tensions. Butylated hydroxytoluene (0.1 mM) in the medium of RPT incubated with 95% 02 decreased LDH-R significantly from 24.6 ± 2.6% to 11.7 ± 1.3%. Deferoxamine (DEF; 0.1 mM) decreased 4-hr LDH-R (27.0 ± 3.2% to 22.4 ± 1.8%) and ascorbic acid (1 mM) was ineffective. Modification of the isolation method to include DEF in the perfusion buffer significantly improved tubule quality. LDH-R at 4 hr was 10.6 ± 1.7% vs 21.0 ± 4.7% for the previous best isolation method. The basal rate of 02 consumption decreased about 10% in 4 hr commen- surate with the change in viability. Nystatin stimulation of 02 uptake was maintained for 2.hr and decreased 15-20% between 2 and 4 hr. (Supported by NIEHS contract ES-65145). 525 A RAT KIDNEY PROXIMAL TUBULE CELL (RPTC) CULTURE MODEL FOR CHRONIC RENAL TOXICITY STUDIES. P B Hatzinger and J L Stevens. W Alton Jones Cell Science Center, Lake Placid, NY. Sponsor: T W Jones Primary cultures of RPTC are important for investigating mechanisms of chronic toxicity since RPTC in suspension are only viable for short periods of time. Previous attempts to culture RPTC have met with mixed success, therefore, we have developed an improved method for the rapid isolation and culture of pure (93 ± 3%) RPTC. Plating efficiency is 80 ± 10% at 24 hr. Epithelial cells grow from tubular fragments which stain for g-glutamyltransferase (GT) and alkaline phosphatase. RPTC grow to confluence at 4-5 days and can be maintained for 2-3 weeks. There is a time-dependent change in cellular morphology. At 7-14 days the mono- layers form linear aggregates which stain more, intensely for GT than the monolayer cells. Ini- tially, the cells have high fructose-1,6-biphos- phatate (FBP) and low hexokinase (HK) activity, but HK increases with time while FBP decreases. These changes may represent adaption to culture. GT activity is maintained at 50% of the tubule value at 7 days. The cells respond to para- thyroid hormone (PTH), but not vasopressin, with an increase in cAMP, consistent with proximal tubule origin. Nephrotoxic cysteine conjugates kill the cells with a structure-activity re- lationship similar to that.determined in other models. This model will have broad application in studies of chronic nephrotoxicity. STRUCTURE ACTIVITY RElATIflNSMP^~OF CYSTEINE .. CONJUGATE TOXICITY IN T_iAT KIDNEY MITOCHDNDRIA. J L Stevens and P J Hayden. W Alton Jones Cell Science Center, Lake Placid, NY,,Sponsor: TW Jones ` Some-cysteine conjugates are potent nephrotoxins. Metabolism by a renal S-lyase is thought to mediate the toxicity of some of these conju- gates. There is evidence to suggest that mitochondria are the ultimate site of toxiciti-,.. -` rate/ malate (site I) and succinate (site,Il)' We have studied the inhib,i;i6i.un of a-ketogluta- ~ dria by pentachlorobutadienyl- (PCBDC), df~ stimulated respirati®n in rat kidney mitochgnr . mechanism for the mitochondrial toxicity of cysteine conjugates. Metabolism by S-lyase will be discussed with regard to inhibition of mito- chondrial respiration. PCBDC, DCVC, TFEC, CTFC and HFPC respectively. For site II the ICS, values are 45 ± 8 pM, 236 ± 40 pM, 216 ± 81 pM, 236 ± 72 pM and 779 ± 142 pM. (Aminooxy)acetic acid, a 0-lyase inhibitor, blocks the toxicity. Only PCBDC was shown to uncouple oxidative phosphorylation. DCVC shows a significant difference in inhibition of site I vs. II stimulated respiration. The other conjugates inhibit both sites to about the same extent. The data may suggest more than one chlorotrifluoroethyl- (CTFC), and hexafluoro- propyl-L-cysteine (HFPC). The ICs, (concen- tration which gives 50% inh,~bit4op) values for .site I respiration are.65 ~'11 yM, 76 ± 26 pM, 191 ± 80 uM, 260 ± 67 pM and 979 ± 203 pM for. chlorovinyl- (DCVC), tetrafluoroethyl- (TFEC);, 526 OCHRATOXIN A (OTA) TRANSPORT IN RENAL PLASMA MEMBRANE VESICLES. P P Sokol, P D Holohan, C R Ross, SIINY-Health Sci. Ctr., Syracuse, NY. Sponsor: P D Williams. The effect of the fungal 3metabolite, OTA, on the transport of p-[ H]aminohip- purate (PAH), a prototypic organic anion was examined in canine renal brush border (BBMV) and basolateral membrane vesicles (BLMV). OTA was as effective an inhibitor of PAH transport in both membranes as was probenec:Ld, a competitive blocker. The IC~O values for cis inhibition by OTA in BRi4 and BLMV were 20vM and 32uM, respect- ively. The effect was specific lince the transport of the organic cation N-methyl- nicotinamide was not affected. The pheno- mena of counterflow was studied to evaluate OTA translocatability. OTA produced trans stimulation of PAR transport in both BBMV and BLMV. The observation .that OTA produced trans stimulation of PAH transport is conclusive evidence that it is translocated across both of these membranes. The data suggest that the reason OTA accumulates in the kidney is because it is a substrate for the renal organic anion transport system. (Supported by NIH#02835). 50875 8257 132
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PHARMACOKINETICS (PK) OF VOLATILE HALOCARBONS: COMPARISON OF SINGLE ORAL BOLUS VERSUS INFUSION OF TRICHLOROETHYLENE (TCE). R Ramana-AA-an, S Muralidhara, J M Gallo*,,C E Dallas, and J V Bruckner. Depts. ofi Phlrmacol. & Toxicol. and *Pfiarmaceutics, College of Pharmacy, University of Georgia, Athens, GA.. The objective of this study Was to gain a better understanding of the PK of TCE, a common halocarbon contaminant of drinking water. 1FCE was given to unanesthetized male S-D rats as an aqueous Emulphor emulsion, either orally as a single bolus or infused through a surgically implanted gastric cannula over 2 hr at a dose of 76 mg/kg. Blood samples were collected from an indwelling carotid arterial cannula during and post infusion from 0 to 540 min. The blood samples were analyzed for TCE using a GC-ECD head space technique. Cmax, AUC, elimination half-life and other PK parameters for the two patterns of ingestion were determined and contrasted. Significant reductions in the AUC and Cmax and increased terminal elimination half-life were observed when TCE was infused intragastrically. Similar experiments were done using a low dose of 8 mg/kg of TCE in order to evaluate the linearity of the PK of the chemical. Our findings indicate that the PK of TCE can be significantly affected by both the route of. administration and regimen of dosing employed. (Supported by U.S. EPA Cooperative Agreement CR812267 and U.S. Air Force AFOSR 870248) DIFFERING TOXICITY AFTER SUBACUTE TRICHLORO- ETHYLENE (TCE) EXPOSURE IN AQUEOUS AND CORN OIL GAVAGE VEHICLES IN MICE. B A Merrick, M Robin- son and L W Condie. USEPA, HERL, Cincinnati, OH Subacute toxicity of TCE was evaluated in male and female B6C3F1 mice using a corn oil or an aqueous (20% Emulphor emulsion) gavage vehicle. Mice received oral doses of TCE 5 times a week for 4 weeks at 600, 1200 and 2400 mg/kg/day for males and 450, 900 and 1800 mg/kg/day for females. Control mice were dosed with either corn oil or Emulphor. A dose-related increase in lethality occurred in male and female mice receiving TCE in Emulphor but not in corn oil during the first week of treatment. Lethality was consistent with CNS depressant effects of TCE. At sacrifice, body weights were not al- tered by TCE treatment but liver/body weight ratios were uniformly increased by TCE admin- istered in either vehicle in both sexes. Only male mice treated with TCE in corn oil, however, sustained elevations in serum enzyme levels accompanied by liver histopathology. TCE in corn oil produced inflammation-associated focal necrosis in one-third of the male mice with in- creasing severity from low to high dose. Lipid accumulation by Oil-Red-O staining was most prevalent in male mice treated with TCE in corn oil. This study indicates that the type of oral gavage vehicle is an important factor in determining the nature of TCE toxicity. (Ab- stract does not necessarily reflect EPA policy). 95 378 A STUDY' OF THE JOINT ACTION OF CARBON TETRA- CHLORIDE (CCL ) AND TRICHLOROETHYLENE (C HC1 ) FOLLOWING SIMATANEOUS GAVAGE A_DMINISTRAZ120N 13N THE RAT. R H Granger, T M 0'Hara, L W Condie*, and J F Borzelleca. Depts. of Pat~o o-i~gy and Pharmaco ogy ozi oTogy, Medical College of VA, Richmond, VA and *U.S.E.P.A., Cincinnati, OH. The joint action of (CC14) and (C,HC1 ) following simultaneous oral adminiStration h a;~en inves- tigated in the male CD rat. This study, prompted by previous reports of enhancgd CC14 hepatotoxic- ity following C HC1 pretreatment, is one of a series designed2 to3 identify and characterize interactions occurring among common drinking water contaminants. Rats with indwelling arte- rial cannulas were gavaged with mixtytes #f CC1 and C HC1 at doses of 0, 100, 250 affid 400 mg/k8 in a 44 g+id design. Hepatotoxicity was evalu- ated as a measure of AST, ALT and SDH plasma en- zyme activity at 0, 3, 6, 12, 24, 36, 48 and 72 hours post gavage. Additional blood samples were taken between 0 and 6 hours post gavage for CC1 and C9HC1 pharmacokinetic analysis. Time coursa respoP~se gata from individual rats were calculat- ed as area-under-the response/time-curve (AUC). Response data were analyzed for possible inter- actions using response surface methods (RSM). Statistically significant "greater than additive" interactions between CC14 and C2HC1 were ob- served under these conditions and may b3e related to the pharmacokinetics of the combination. (Sponsored by EPA Cooperative Agreement 812558.) 379 THE SYNERGISTIC HEPATOTOXICITY OF CARBON TETRACHLORIDE AND TRICHLOROETHYLENE IN MALE F-344 RATS. DA McMillan, M Tokars, C Eskelson and IG Siges. Dept. of Pharmacology and Toxicology, Univ. of Arizona, Tucson, AZ The interactive hepatic effects of carbon tetrachloride (CCI4) and trichloroethylene (TCE), two common drinking water pollutants, were studied using male F-344 rats. The chemicals were administered orally in an aqueous (10% Emulphor) vehicle. Dose-response studies with CCI4 and TCE indicated that a non-toxic dose of TCE (0.2-5 or 0.50 mI/kg) (as indicated by plasma alanine aminotransferase, ALT, and sorbitol dehydrogenase, SDH, activities) administered simultaneously with a minimally hepatotoxic dose of CCI4 (0.05 mI/kg) produced potentiation of the halogenated hydrocarbon-induced hepatotoxicity. A time-course study using CCI4 (0.05 mI/kg) + TCE (0.5 ml/kg) showed that the binary combination maximally elevated ALT and SDH activities and depressed hepatic reduced glutathione content at 24 hrs after administration. Ethane exhalation, as a measure of lipid peroxidation, was found to be similar for rats receiving CCI4 (0.05 mI/kg), or TCE (0.5 mI/kg) or both chemicals simultaneously. Rats treated with either 14CCI4 or 14CCI4 + TCE did not differ in the amount of 14C-equivalents expired either as 14CO2 or as exhaled organic compounds. Gas chromatographic analysis of the exhaled organic samples showed that the 14CC14 + TCE group expired more chloroform than the 14CCI4 alone group. These data indicate that the synergistic hepatotoxicity of CCI4 + TCE may involve changes in CCI4 metabolism. (Supported by EPA No. CR-812557.) 50875 8220
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440 INFLUENCE OF ENVIRONMENTAL FACTORS ON THE RADIATION EFFECTS IN GRANARY WEEVIL: CHANGES IN EPICUTICULAR HYDROCARBONS. S Sriharanl, T P Sriharan2, S Sellers3, W Bertseh4 and R S Saini5. 1,2Selma University, Selma, AL, 394The University_ of Alabama,-UnNersity, AL and5Tuskegee Univer- sity, Tuskegee,.AL, all in the USA. Sponsor: E V Ohanian. , One of the non-chemical methods of protecting stored-product commodities is disinfestation by irradiation. An understanding of Ae environ- mental factors like temperature and humidity with reference to insect mortality due to irra- diation is essential for effective pest control. The mechanism of radiation damage to adult in- sects is poorly understood. As such, studies were undertaken by exposing granary weevil to different dose rates (0.05, 0.15 and 0.30 kGy) of gamma radiation, maintaining at different temperatures, humidity and analyzing the epicu- ticular hydrocarbons by gas chromatography-mass spectrometry (GC/MS). The results indicate that gamma radiation in- duced greater water loss leading to desiccation and early death of weevils. Low humidity en- vironment (17% R.H) accelerates lethal effects. GC/MS analysis showed significant variations in the n-Alkanes (C23 to C35) of epicuticular hydrocarbon mixture between the irradiated and control weevils. Quantitative changes among the treated groups were observed. (Department of Energy Grant No. DE-FG03.86CH10288.AOOI). 441 BIOASSAY AND ANALYSIS OF PINE NEEDLES FOR POLYCHLORINATED DIBENZO-p-DIOXINS (PCDDS) AND DIBENZOFtkiANS (pCDFs): A NOVEL MONITORING SYSTEM FOR AIR POLLUPANPS. T Zacharewski, S Safe, A Reischl, M Reissinger, H'Ihoma ancT-0 Hutzinger, Department of Veterinary Fnysiology and Pharmacology, College of Veterinary Medicine, Texas A&M University, College Station, TX, Ecological Chemistry and Geochemistry, University of Bayreuth, Bayreuth, FRG. The lipophilic waxy surfaces of pine needles fran several urban, non-urban and industrialized areas contained canplex mixtures of fCDD and pCDF congeners and the highest concentrations of these toxins were detected in extracts from industrialized areas. Cas- chromatographic-mass spectrometric (GC-MS) analysis showed that PCDD/pCDF levels in dry pine needles were as high as 660 pg/g (parts per trillion). Utilizing the induction of aryl hydrocarbon hydroxylase (AHH) in rat hepatoma H- 4-II E cells as a bioassay, the "2,3,7,8- tetrachlorodibenzo-p-dioxin (TCDD) toxic equivalents" of the pine needle extracts were determined and showed an excellent correlation with the (1:-MS data. The results clearly illustrate the utility of the bioassay for assessing toxic equivalents and also suggest that plant surfaces may be useful monitoring systens for lipophilic atmospheric pollutant (Supported by the Environmental Protection Agency.) 111 442 HEPATOTOXICITY OF ALLYL FORMATE AND EFFECT ON TROUT LIVER GLUTATHIONE. BF Droy, ME Davis, and *DE Hinton. Dept. of Pharmacoiogy an~Toxi'cology, WVU SchooT of Medicine,'Morgantown, WV. *School of Vet. Medicine, Univeristy of Cal.-Davis. In mammals, glutathione (GSH) serves a protective role against allyl formate-induced hepatotoxicity by combining with reactive metabolites and ren-& dering them inert. Therefore the role of GSH irL- 'r the protection against all^oirmate-induced - hepatotoxicity in trout was investigated. Rainbow trout received -10,30, or 100 ul/kg gavage. Forty eight hrs later, SGPT, tissue Na+ ~ and K+, and histopathology revealed greatest change in the 100 ul/kg group. Near massive necrosis and severe hemorrhage,chqActerized . parenchymal space. Significaft change in tissue electrolytes were seen with the 30 ul/kg group (Na+ 202%, K+ 71% of control), however no eleva- tion in SGPT was noted. Normal parenchyma was the most common finding histologically, with some sections revealing necrosis and hemorrhage around venular profiles. No indication of toxicity was seen with the 10 ul/kg group. A dose-dependent decrease in liver GSH was seen at 3,6, and 24 hrs following 10,30, and 100 ul/kg alyl formate (51,40, and 29% of control respectively) with maximal depression seen at 6 hr. All groups showed partial recovery in GSH by 24 hrs. These results indicate that greater than 50% decreases in liver GSH are required before"toxicity is expressed at 48 hrs by allyl formate in trout. 443 CYTOTOXICITY OF FUSARIUM MONILIFORME CONTAMI- NATED CORN. W P Norred, C W Bacon, J K Porter and R D Plattner, Richard B. Russell Agricultu- ral Research Center, ARS/USDA, Athens GA and Northern Regional Research Center, ARS/USDA, Peoria, IL Corn believed to cause equine leukoencephaloma- lacia (ELEM) was found to be heavily infected with F. moniliforme. Rat primary hepatocytes were used to screen fractions of chloroform:me- thanol ext.racts of the corn for cytotoxicity (release (;k lactic dehydrogenase), mutagenicity (unscheduled DNA synthesis, UDS), and effects on protein synthesis (incorporation of [3H]valine). Extracts had low cytotoxicity and caused no UDS. However, the neutral fraction inhibited protein synthesis when cells were exposed to as little as 0.125 gram-equivalents of the toxic corn per 106 cells. Neither the acidic fraction nor similarly prepared extracts of fungal-free corn had an effect on protein synthesis. Isolates of F. moniliforme obtained from the toxic corn were capable of producing inhibitors of protein syn- thesis in culture. The inhibitory components do not appear to be trichothecenes (potent inhibi- tors of protein synthesis), since the neutral fraction did not produce irritation of rabbit skin nor was there evidence of trichothecene- like compounds when the fraction was analyzed,by tandem mass spectrometry. Whether or not the components of the corn responsible for the inhi- bition of protein synthesis also cause ELEM remains to be determined. 50875 8236
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EFFECT OF CHLORPROMAZINE ON PARAQUAT AND NADPH- DEPENDENT LIPID PEROXIDATION IN LUNG MICROSOMES. P0 Ogunbiyi and HP Misra. VA-MII'Regional College of VeterinaryMeckic~.ne, VirginiaTech, Blacksburg, VA. * 'O' Chlorpromazine (CPZ) has been shown to ameliorate or exacerbate paraquat (PQ) toxicity in experimental animals. The involvement of lipid peroxidation in PQ toxicity is controversial and the effect of CPZ on the process is inconclusiv;. In this study, the role of PQ and the effect of CPZ on lipid peroxide formation by lung microsomes were investigated using thiobarbituric acid reactive substance (TBA-RS) assay. NADPH stimulated lipid peroxidation with or without exogenous Fe3+. PQ (10-6 to 10-3 M) inhibited microsomal TBA-RS production under aerobic condition irrespective of the presence of Fe3+. In the absence of PQ, CPZ (10-7 to 10-2 M) significantly inhibited microsomal lipid peroxidation. In PQ-treated microsomes, CPZ at 10-7 to 10-5 M, significantly inhibited,while at 2.5x10-5 to 10-2 it enhanced micros5mal lipid3peroxidation. Addition of Fe3+ (5x10- to 5x10- M) potentiated these responses. These results indicate that PQ-induced lung injury in guinea pig does not involve lipid peroxidation. The reported beneficial effect of CPZ in PQ toxicity probably involves mechanisms other than inhibition of peroxidation' of lung lipids. (Support: HIH HL 36366, HL 35656). SENSORY IRRITATION STRUCTURE ACTIVITY RELATION= SHIPS OF SOME BENZYLCHLORIDE CONGENERS. B R Dudek, M V Roloff, R D Short, M A Council. Monsanto Company, St. Louis, MO.. . Inhalation sensory irritation causes stimulation of trigeminal nerve endings in the nasal mucosa.. One of the reflex reactions in mice resulting from such stimulation is a decrease in respira- tory frequency. The concentration of chemical that causes a 50Z reduction in respiratory rate is referred to as its RD50. The RD50 values (ppm) for benzylchloride and some of its congeners were determined in mice by whole body plethysmography to be: benzylchloride (27), benzylbromide (5.2), benzyliodide (4.3), ortho- (5.7), meta- (27), and para- (14) chlorobenzylchloride, and a,a-di- chlorotoluene (20). The structure activity rela- tionships can be rationalized in terms of the reaction of the irritant chemical with-a receptor protein in a lipid layer. A good correlation (.92) between the RD50's and the bond strength of the benzyl-halogen bond was found for the • benzylhalides. For the ortho-, meta-, and para- substituted congeners, the RD50's appear to be related, to the electron withdrawing ability of the ring halogen during a nucleophilic substi- tution reaction at the alpha carbon by a nucleo- philic group such as a sulfhydryl group asso- ciated with the receptor protein. 145 577 PERTURBATION OF LUNG SUBCELLULAR •~ CALCIUM TRANSPORT BY $ARAQUAT. J W Coleman and A K Agarwal. To'xicology Research and Training Center, John Jay College of CUNY, New York, NY. Sponsor: H M Mehendale Pervious studies from this laboratory ~ indicated a perturbation of lung mito- -phondrial and micros'a%i3P7I calcium ~. transport by paraquat. In the present'-: study, male SprageYe=-Dawley rats were = ¢ given paraquat ip at doses from 10 to 578 30 mg/kg. The animals were sacrificed at 72 hr following paraquat. Lungs were removed and mitoc.l~o,~}p7ria,oro microsomes were isolated,~9y biffegn- ' tial centrifugation. In vitro Ca uptake and ATPase were measured. There was a severe inhibition of mitochondrial and microsomal calcium uptake but no dose response relation- ship was.observed. The rate of calcium uptake in lung mitochondria and microsomes was almost negligible at higher doses of paraquat. A dose dependent decrease was observed in ATPase. The results suggest that perturbation in subcellular calcium transport might be associated with lung damage. INDUCTION OF 0_XIDATIVE-STRESS IN UPPER RESPIRATORY TRACT (URT) TISSUES. D G Cavanagh and J B Morris. Toxicology Program, School of Pharmacy, University of Connecticut, Storrs, CT. Oxidant gases deposit efficiently in the URT, but it is not known if this site is sensitive to these gases, or to oxidants in general. To provide preliminary data on the sensitivity of URT tissuet.to oxidative stress, the URT of the anesthetized F344 rat was exposed to the known oxidant tert-butyl hydroperoxide, via continuous lavage. This system allowed for. precise control of dosage, and for assessment of lactate dehydrogenase (LDH) leakage over the course of the exposure. The URT was lavaged att a flow rate of 2 mi/min for 60 min with 0.9% NaCl-1% dextran containing 0.0, 0.2, 1, or 5 mM TBHP. Immediately post-exposure URT tissues were isolated for biochemical analysis. At the highest level TBHP was cytotoxic as evidenced by increased lavage LDH content (p< 0.01). - Enzyme leakage became apparent 20-30 min after initiation of exposure. Tissue nonprotein sulfhydryls were decreased in a dose-dependent manner by TBHP (p<0.01). TBHP also produced dose-dependent elevations in tissue malondialdehyde levels, but the changes were not statistically significant. These results indicate URT tissues are sensitive to TBHP- induced oxidative stress. (Supported by NIH grants ES 03676 and ES07163, and a Sandoz Institute Fellowship in Toxicology to DGC.) 50875 8270
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428 CHRC[dIC ORAL TQXICITY/CARCINOGE3JICITY STUDY OF FORNALDEHYDE IN RATS. J J Cla , H P Til, R A Woutersen, V M H Ho rs, and V J Feron. Hoechst CeJ.anese Corporation, Somerville, NJ. TND CIVO Toxicology and Nutrition institute. Zeist. The Netherlands. I Wistar Rats (4 groups of 70 males and 70 females) were given formaldehyde in their drinking water at doses of 0, 5, 25 or 125 mg/kg b.w./day for a period of 2 years. Significant effects were only seen in the high-dose group and comprised markedly decreased liquid intake, lower body weights and food intake, decreases in urinary PH, plasma protein and cholesterol level, and increased potassium concentration in blood plasma. Pathological changes in the stomach (raised limiting ridge, ulcers and epithelial hyperplasia in the forestomach, and gastritis and glandular hyperplasia in the glandular stomach) and kidneys (necrosis of the papilla) were also observed. Some of these changes might partially or entirely be due to the strongly reduced liquid intake. No compound-related tumours were found. The no adverse effect level was judged to be 25 mg/kg b.w./day. Chronic oral administration of formaldehyde to rats at the NTD caused severe damage to the gastric mucosa but did not result in gastric tumours or tumaurs at other sites. 429 NASAL TUMOURS AND DAMAGE TO THE OLFACTORY EPI-- THELIUM IN FORMALDEHYDE-EXPOSED RATS WITH A SE- VERELY INJURED NASAL MUCOSA. V J Feron, R A Wou- u tersen, A van Garderen-Hoetmer, J B Bruijntjes . and A Zwart. TNO-CIVO Toxicology and Nutrition Institute, Zeist, The Netherlands To study the significance of damage to the nasal mucosa of rats for the induction of nasal tumours by formaldehyde (FA), a study was conducted in male rats with an injured nose (bilateral intra- nasal electro-coagulation) or with an intact nose exposed to 0, 0.1,-1.0 or 10 ppm FA for 6 h/day, 5 days/week during 28 months or during 3 months followed by a non-exposuie period of 25 months. The damaged nose was more susceptible to 10 ppm FA than the undamaged nose as appeared from in- creasestin incidence of rhinitis, hyper- and metaplasia of the respiratory epithelium, and de- generation and hyper- and metaplasia of the ol- . factory epithelium in rats with a damaged nose. In addition, exposure to 10 ppm FA for 28 months resulted in a much higher incidence of nasal squamous cell carcinomas in rats with a damaged nose (17/60) than in rats with an intact nose (1/29). No significant differences in tumour in- cidence between rats with a damaged and undamaged nose were found at the 0.1 or 1.0 ppm level or after exposure to 10 ppm for only 3 months. In conclusion: severe damage to the nasal mucosa may be an important factor for the induction of nasal tumours by FA. 430 EARLY.CELL PROLIFERATIVE AND CYTOTOXIC EFFECTS OF ORAL PHENACETIN ON RAT NASAL MUCOSA. M S Bogdanffy, T J Mazaika, and W J Fasano. E I du Pont de Nemours & Co, Inc, Haskell Laboratory for Toxicology and Industrial Medicine, Newark, DE. Phenacetin binds covalently to rat nasal olfactory mucosa (OLF) and causes nasal tumors in rats when administered in the diet for 18 months. The, purpose of this study was tovoeeamine changes in tell proliferation during 1 or 2 weeks of phenacetin dosing. Rats Were gavaged with phenacetin at 100, 625, or 1250 mg/kg for 7 (group A) or 14 (group B) days and were implanted with osmotic minipumps containing [3H]-thymidine during days 1-7 (group A) or 8-14 (group B). Noses were then processed for histgJautdradio- graphy. No lesions were seen at i00 mg/kg in groups A or B, but there was a slight increase in cell proliferation in OLF. In both groups, 625 or 1250-mg/kg caused extensive necrosis of Bowman's glands, a loss of PAS positive material in the subepithelium of OLF, and a dose and time related increase in cell proliferation in OLF. In Group B at 1250 mg/kg, the labeling index was increased approximately 10 fold. In the neuronal cell layer, this was accompanied by disorganization and the appearance of clusters of PAS positive cells. Phenacetin had no effect on cell proliferation in respiratory mucosa. These results demonstrate that phenacetin causes early changes in cell proliferation in the nasal olfactory mucosa consistent with covalent binding and carcinoma formation. 431 TISSUE HYDROXYLATION OF METHYL-n-AMYLNITROSAMINE (MNAN) TN NEONATAL TO ADULT RATS AND HAMSTERS. S S Mirvish, C Ji, and S Rosinsky..Eppley Inst Res Cancer, Omaha, NE. In studies to help explain.carcinogen organo- tropy, fresh adult rat tissues converted the esophageal carcinogen MNAN into 2- to 5-hydroxy- MNAN and some 3- and 4-oxo-MNAN. In assays, g0- 250 mg tissue slices were incubated (3 h, 37 ) with 23 pM MNAN/5 ml Eagle's medium. CH C1 extracts were 1nalyzed by g.l.c.-thermaT egergy analysis. We measured sum of hydroxy- and oxo- MNANs, expressed as % yield/100 leg tissue, that were produced by MRC-Wistar rat and Syrian _ hamster tissues from animals 1 day prenatal to age 6-8 weeks. In rats, metabolism peaked at 9.7% in 6-day esophagus, 1.4% in 3-day forestom- ach, and 14.9% in 9-day liver (adult levels 3.3, 0.02, 4.2%, respectively). In hamsters, yield peaked at 10.0% in neonatal esophagus, 4.4% in 3-day forestomach, 5.1% in 6-day trachea, and 3.7% in 1-day-prenatal lung (adult levels 0.5, 0.3, 1.9, 1.4%). Metabolite ratios varied with conditions. Incubation for 3 h with varied [MNAN] showed apparent E~ of 150 pM for esopha- gus and 300 uM for liver of adult rats. Differ- ences between adult and 6-day esophagus, and between adult and 9-day liver of rats dis- appeared when incubation was with 300 µM (esophagus) or 600 pM (liver) MNAN. Hence metabolism in young tissues was increased only with 23 µM MNAN. Support: NIH grants R01-CA- 35628 and CA-36727. 108 50875 8233
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S_ILICONE IMPLANTS IN THE RAT VAS DEFERENS. D P Waller. A Martin, M Szarley, A R Nikurs, N A Nuzzo, and L J D t.isneveld, University of Illinois at Chitago, and Rush Presbyterian-St. Lukes Medical dster, Chicago, IL. A silicone plug for.the vas deferens (SHUG) is being developed as a new reversible male contraceptive device. Previous studies have demonstrated its ability to block the flow of spermatozoa when placed in the monkey vas deferens. Our laboratory 4veluated the effect of chronic exposure of the rat vas deferens to the presence of the silicone material used to make the SHUG. Four hundred rats were divided into two equal groups. One group had sham operations and the other, group had silicone noodles implanted in the vas deferens and secured by a suture. Animals were euthanized, and the vas deferens removed, fixed, and histologically evaluated after six, twelve, or 22 months of implantation. Changes in the vas deferens attributed to the presence of the silicone were minimal-to-mild epithelial hyperplasia in the proximal, surgical, and distal segments. Their incidence and severity were directly related to the length of exposure to the silicone. An increased incidence of spermatoceles, aspermatic granulomas, and chronic inflammatory changes within the inner walls of the vas deferens were also observed in all implanted groups. These vas deferens changes represent normal tissue responses to the presence of a foreign body. Supported by the program for Applied Research on Fertility Regulation (Agency for International Development, PARFR 339). 473 RESTRICTING bATING TRIALS ENHANCES THE DETECTION OF,LTHOXYETHANOL (EE)-INDUCED FERTILITY IMPAIR- t4ENT IN RATS. E D Cl egg and H Zeni ck , U. S. Environmental Protection Agency, Washington, DC In fertility testing, male and female rodents are allowed to mate overnight. That practice allows numerous copulations that usually result in deposition of an adequate number of sperm to insure fertility even in males that have been severely compromised. To test the hypothesis that restricting the number of copulations would increase the sensitivity of fertility testing, male rats were exposed to EE (0 or 450 mg/kg/ day) by gavage for 7 weeks at a dose level that produced severe depressions in sperm counts. Each control and EE-treated male was initially mated, in a counterbalanced design, to a female in proestrus, with either a single copulation or a minimum of 3 copulations allowed. Three days later, each male was mated using the alternate condition. Despite the extreme reduction in , sperm counts in EE males, no differences in fer- tility rate were observed relative to controls witn multiple copulations (72% vs. 80%, respec- tively). However, a marked decrease in fertility was seen in the EE group when only a single mat- ing was allowed (22% vs. 60% in control males). These results suggest that the sensitivity of breeding protocols may be enhanced by limiting the number of copulations. Such a model is also more analogous to the copulatory pattern of humans. 474 THE EVALUATION OF 3 MOUSE STRAINS IN THE CONTIN- UOUS BREEDING DESIGN (RACB) USING ETHYLENE GLYCOL MONOMETHYL ETHER (EGME).-D K Gulati*, E Hope*, L Barnes*, R Mounce*, R Morrissey+, and R E Chapin+. *Environmental Health Research & 'resting, Inc., Lexington,-KY, and +National To::icology Program. (NTP), NIEHS, Research Triangle Park, NC. - ~ The rodent strains generally used for testing '~ „ xequire significant reproduef6ar-organ damage ` rbefore fertility is impaired. It has been pxs*~,~-, posed that using animals of marginal fertility would better mimic human fertility potential; thus, we tested the CD1 (the strain used in ~ RACB studies), C57BL/6, and C3H strains in_the RACB protocol using EGME as the toxicant at 0.0, 0.03, 0.1, and 0.3% w/v i the._drinking water. The percent fertile ~trs khaving > 1 litter) was equal for all strains.t However, while 23 of 30 pairs of control CD1 mice had fifth litters, only 3/30 pairs of control C3H and C57BL/6 strain had fifth litters. This decline was most dramatic after the third litter. The C3H strain had the greatest EGME-induced change in number of litters per pair, and in number and percent live pups, while CD1 showed the least change. In the second generation with continuous treatment, fertility was reduced in all strains at the 0.1% level. However, the inability of the C3H and C57BL/6 strains to produce more than 3 litters resulted in insuf- ficient pairs in the F1 generation to adequately test fertility, and suggests that the C3H and C57, while more sensitive to EGME than are the CD1 mice, are unsuitable for a 5-litter design. 475 REPRODUCTIVE TOXICITY IN MALE RATS FOLLOWING ORAL ADMINISTRATION OF CGS 15863. G Batastini, R H Spaet, R N Infurna, E T Yau, and V M Traina. Research Dept., Pharmaceuticals Div., CIBA-GEIGY Corp., Summit, NJ CGS 15863, a thromboxane synthetase inhibitor, was evaluated for effects on fertility and repro- ductive performance in rats. The compound was administered orally via gavage to groups of male and female,~D rats at daily doses of 0, 25, 75 or 225 mg/kg."Females were dosed for 14 days prior to mating and throughout gestation and lactation. Males were dosed for 63 days prior to mating and received a total of either 98 or 105 daily doses. Following the dosing period, selected males from the control and 225 mg/kg groups were placed on recovery (untreated) for 70 days and then mated with a separate group of untreated females. Com- pound-related effects were not evident at daily doses of 25 or 75 mg/kg. In contrast, rats from the 225 mg/kg group exhibited compound-related reproductive toxicity, which included: a) re- duced fertility, decreased numbers of implants and viable.neonates, and no litters at the end of the recovery/remating period; b) reduced testes weights; c) gross testicular atrophy; and d) mi- croscopic evidence of testicular vacuolar degen- eration, progressing to tubular atrophy. These effects were increased in incidence arid severity in recovery animals, indicating non-reversibil- ity. The etiology of these degenerative testic- ular changes in response to CGS 15863 has not been established. 11J 50875 8244
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424 L, Liver E,ndonuclease Activity Stimulated by Elevated Cytosolic Qa++ Induced by Halogenat- ed jj,ydrocarbons? II. In V..itro Studies. R M Long, D R. Schoenberg, and L. Moore, Dept, of Pharmacology, L3$UHS, Bethesda, MD. We have previously reported (Pharmacologist 29:122, 1987) that rising cytosolic Ca•+ concentrations do not activate endonucleases in liver exposed in vivo to carbon tetrachloride (CCh) and 1,1-dichloroethylene (DCE). We have now examined endonuclease activatipR following in vitro exposure of liver cells to halogenated hydrocarbons. CCl4 (1.5 mM) and DCE (6 mM) were added to primary cultures of rat hepatocytes. DNA was prepared and sepa- rated on agarose gels. No generalized DNA fragmentation was observed until very late times (2 hr.) when plasma membrane integrity was disrupted as measured by enzyme release. Endonuclease activity was further examined by specifically monitoring hypersensitive sites in serum albumin gene. This gene is very actively transcribed in liver and thus should be extremely sensitive to nucleolytic attack. DNA was digested with restriction enzymes Eco R1 or Hind III, electrophoresed on agarose gels, and blotted onto nitrocellulose. Albumin sequences were detected by hybridization to a32P-labelled 1400 bp geno- mic clone of rat albumin. No cleavage at hypersensitive sites was observed at early times (15 to 45 min.) when Ca** homeostasis was first disrupted. Thus as had been found in vivo, there is no evidence to suggest that activation of endonucleases by Ca*• is responsible for mediating the hepatotoxicity accompanying halocarbon exposure. (Supported by ES03437 and GM30270 from NIH.) 425 FORMATION OF CARBON DIOXIDE FREE RADICAL BY - LIVER MITOCHONDRIA FROM KREB CYCLE INTERMEDIATES WHEN HYDRALAZINE IS PRESENT. P K Wong, J L Poyer, C M DuBose, and R A Floyd Oklahoma . Medical Research Foundation, Oklahoma City, OK. Isolated rat liver mitochondria incubated with - malate-glutamate in the presence of hydralazine and spin-traps yielded free radical spin adducts. Studies with mitochondrial inhibitors and the use of succinate as substrate showed that free radical formation was dependent upon" mitochondrial electron transport in the NADH-: _ dehydrogenase region. The spin adducts showed that the free radicals trapped were carbon centered. The coupling constants suggested the trapped radical was Cq. This was proven to be the case by synthesizing the C0,:~ trapped radical in a chemical system and isolating the authentic compound and the one produced by mitochondria - using HPLC and conducting EPR measurements on the isolated fractions. Studies with mitochondria substrates demonstrated pyruvate produced much more of the COZ radical, than malate/glutamate. Studies with 1-13C-pyruvate demonstrated that the hydralazine dependent COZ production arose from the carboxyl carbon of pyruvate. 426 FORMATION OF FREE RADICAL PRODUCTS FROM HYDRALAZINE BY RE.D BLOOD CELLS AND OXY- HEMOGLOBIN. J L Poyer,-C M DuBose, and R A Floyd, Oklahoma Medical Research Foundation, Oklahoma City, OK. Human red blood cells treated with hydralazine-: (3-phthalazyl hydrazine) produced free radical§= which could be spin-trapped, and observed usine electron paramagnetic resonaaoe (EPR) ` techniques. The free radical trapping agent,+_-;-, DMPO (3,3-dimethyl-l-.pyrroline-N-oxide) trappec~,z,:, a nitrogen-centered free radical whereas the %;-- free radical trapping agent PBN (cx- phenyl-tert- N-butylnitrone) trapped a carbon-centered radical as well as the hydrogen free radical. Free radical formation was de.~endent upon the presence of oxygen, indicating the involvement of oxy-hemoglobin. One of the spin-trapped free radicals which could be identified was the 3- phthalazyl radical. This radical could be formed chemically by the reaction of potassium ferricyanide with hydralazine and trapped with PBN. Bovine oxy-hemoglobin plus hydralazine yielded an EPR spectrum similar to that for red blood cells indicating that the free radicals were similar. Carbon monoxide inhibited free radical formation in both the red blood cell and bovine hemoglobin systems. This work was supported in part by NIH Grant No. 2-RO1- . ES03067-04. 427 - 6-ME'PHYL-1,3,8 TRICHLORODIBENZCFURAN (MCDF) AND RELATED ANALOGS AS 2,3,7,8-TETRACHLORODIBENZO-p- DIOXIN (TCDD) A_NTA(3ONISTS: STRUCTURE-ACTIVITY RELATIONSHIPS. B Astroff and S Safe, Dzpartment of Veterinary PhysioT357 -apd Pharmacology, College of Veterinary Medicine, Texas A&M University, College Station, ft. 1,3,6,8-Substituted dibenzofurans, inclu- ding MCDF, antagonize a broad spectrum of 2,3,7,8-TCDD-mediated biologic and toxic responsesAn mice and rats; however, MCDF is the only cccnpound which specifically inhibits the induction of rat hepatic microsomal aryl hydrocarbon hydroxylase (AHH) and the associated cytochrome P-450 isozymes. The requirement of the 1,3,8-substituted Cl and the 6-CH3 sub- stituents for this inhibitory activity has been investigated using a series of analogs in which a single substituent (Cl or CH3) has been replaced with a H. The most active antagonist of AHH induction was MCDF; however, replacement of the 1- or 8-Cl group with H did not eliminate the antagonist activity. In contrast, replace- ment of the 3-Cl or 6-CH3 group with H or the 6- CH3 group with C1 gave congeners with no inhibitory activity. These results correlated in part with the Ah receptor binding activities of these hcmologs and suggest that the an- tagonists interact stereoselectively with the receptor. (Supported by the National Institutes of Health.) 107 50875 8232
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TOXICOKINETIC EVALUATIONS OF _BEAGLE DOGS THAT jNHALED BERYLLIUM OXIDE. G L Finch, J A Mewhinney, M D Hoover, P J Ha1-~.Ty, A F Eidson, D E Bice, and A C, F(armsen. Lovelace Inhalation Toxicology Rtseartch Institute, Albuquerque, NM Sponsor: R 0 McElellan .,~., The effects of inhaled BeO were studied in Bea- gle dogs to evaluate the suitability of a dog model for human chronic berylliuln disease. Groups of dogs received single acut2',inhalation exposures to respirable radiolabeled aerosols of 7BeO calcined at either 500 or 1000°C result- ing in lung burdens of 18 ug Be/kg and 6 µg Be/kg. Serial sacrifices through one year af- ter exposure were conducted to permit quantita- tion of tissue Be content and histological exam- ination. BeO calcined at 500°C was cleared more rapidly from lung, and Be was translocated more rapidly to blood, liver, and skeleton, com- pared with BeO prepared at 1000°C. Pulmonary histological abnormalities demonstrated marked individual variation between dogs and were most severe at 64 days after exposure. Animals scheduled for sacrifice at 2 years after expo- sure underwent pulmonary lavage for cytological evaluation at intervals up to 18 months after exposure. Pulmonary lymphocyte stimulation re- sponses peaked at 7 months after exposure then declined. This study, still in progress, indi- cates that the Beagle dog is a useful model for examining the induction of chronic beryllium disease. (Research sponsored by the U.S. DOE Office of Health and Environmental Research un- der Contract No. DE-AC04-76EV01013.) 350 SEX AND SPECIES DIFFERENCES IN THE INHALATION TOXI- CITY OF THIOPHENE. R Irwin,* M He tmancik, M Ryan, D Craig, and A Peters. Batte e o u us Divi- sion, Columbus, OH and *NIEHS, Research Triangle Park, NC. Thiophene (CAS No. 110-02-1) is widely used as an industrial solvent and pharmaceutical synthetic intermediate. A two-week inhalation study (12 six-hour exposures) was conducted in F344 rats and B6C3F1 mice at concentrations of 0, 500, 1000, 2000, 4000 and 8000 ppm." Thiophene was vaporized by nebulization into a common plenum distribution system, and chamber concentrations were measured using a Miran-80® infrared analyzer. The first exposure to thiophene produced 100% mortality in male mice at all exposure concentra- tions and in female mice at the two highest levels. Clinical signs of toxicity included hypoactivity, prostration, and dyspnea. A single exposure to thiophene also produced complete mortality in the high dose male and female rats. Mortality also occurred in male rats at the 2000 and 4000 ppm dose levels. Rats of both sexes at the 4000, ppm dose level showed an abnormal gait immediately following exposure, possibly indicative of cere- bellar dysfunction. Thiophene exposure produced reduced weight gain, gross liver abnormalities, and an increased liver-to-brain weight ratio in survivors of both species. These repeated dose studies suggest that mice are more sensitive than rats to similar thiophene concentrations by the inhalation route, and that males of both species are more sensitive than females. (Sup- ported by Contract No. N01-ES-65163 from NTP). 561 a-TOCOPHEROL AND ASCORBATE OXIDATION IN , LIPOSOMES EXPOSED TO N_Oz. CR Shoaf and D Menzel. Duke U. Med. Gtr., Depts°4-•Pharm. and - Med., Compre. Cancer Ctr., Durham, NC. Oxidation of lung membrane lipids is thought to be _he primary mechanism of NO toxicity by in- i halation. The rate constant for lipid peroxida-..C tion by this free radical in the presence of en- dogenous dogenous antioxidants, such ~~tocopherol and ~ a~scorbate, are required before lung models can, predict effects from buman exposure to NO~`K=~' Liposomes composed of diiinoleoyl phosphatidy~=':; choline (18:2) and dilinolenoyl phosphatidyl- '74choline (18:3) had second order rate constants of 1.5X10-3 and 3.lX10-3 Pt1s 1, respectively. These rate constants are consis~eith a mech- anism of allylic hydrogen aby+fYa.,;tion. Lipo- somes composed of 18:2 and dipaltiitoyl phos- phatidylcholine (16:0) and containing 2 mole % a-tocopherol had second order rate constants of 4.9X1O-z and 8.9X10-2 M-ls-1, respectively, for a- tocopherol oxidation. a-Tocopherol is preferen- tially oxidized by NOZ in the presence of these memPrane systems. The oxidation product of a- tocopherol was a-tocopherol quinone. When 18:2 and 16:0 liposomes were exposed to NOz in the presence of 50 µM ascorbate, there was no dif- ference between the rate of ascorbate oxidation at 0 ppm NOZ (air) and 0.5, 1, or 2 ppm NOz. The reaction rate of NOZ with ascorbate in the presence of lipid is not consistent with a sec- ond order model, and ascorbate is not preferen- tially oxidized. (Supported by EPA Coop. Agree. CR809715 and NIH Grants RRO1693 and CA14236.) 562 Eti'ALUATION OF CARBON DIOXIDE RESPONSE CURVES IN GUINEA PIGS. M Schaper, K Detwiler and Y Alarie, University of Pittsburgh, Pittsburgh,PA it is well-recognized that humans increase their ventilation during exposure to a mixture containing COz• Furthermore, the level of ventilatory response is proportionall to the concentration of COz in inspired air. This is the premise of the test in which the COz re- sponse c is generated. From this curve, the slope and intercept are then obtained. We have simulated this test in unanesthetized, unre- strained guinea pigs. Animals were placed in whole-body plethysmographs, to which sensitive pressure transducers were attached. These animals were first exposed to room air and then to mixtures containing increasing levels of COz (1.5, 2.8, 4.4, 7.0, 10.0 or 15.0% in COz in 20% Oz, balance Nz). Tidal volume (A P) and respiratory frequency (f) were continuously monitored and all data were collected by an IBM-AT PC. The level of change in AP and f was assessed at each concentration of ODz and plots of COz concentration vs. response level for pP and f were obtained. For each plot, the slope and intercept were determined. Our results indicate low variation in the responses to COz in normal. guinea pigs. This approach may be usefull in assessing lung function or changes in central chemoreceptor sensitivity to COz in an- imals acutely or chronically exposed to toxi- .cants. Supported by NIEHS grant RO1-ES02747. 141 50875 8266
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4' 444 A CALIFORNIA PROGRAM FOR EVALUATION OF CHEMICAL CONTAMINATION OF FISH. A M Faa, G A Pollock, and R J Jackson. Hazard Evaluation Section, Calif. Dept Health.SeAr,ices (CDHS), Berkeley, CA. 5 t 4 445 We describe our procedures for assessing risks from chemical contaminants in fish and issuing health advisories (consumption guidelines) for public health protection. Requests for evalua- tions come from both within and wit8qut the Department as a result of accidental contamina- tion situations, routine monitoring programs, , and mandated CDHS studies. Review considera- tions include chemical concentrations in fish, consumption patterns, toxicological data, safety factors, acceptable daily intake, sources of exposure, and sensitive subpopulations. The chemicals involved have included methylmercury, selenium, PCBs, and DDT. Methylmercury affects the nervous system and the fetus. Selenium can cause gastrointestinal disturbances, loss of hair and nails, nervous system effects, skin lesions, and, based on animal data, may cause adverse reproductive and developmental effects. Certain PCBs and DDT are animal carcinogens. Maternal ingestion of PCB contaminated fish is reported to be associated with low birth weight and small head circumferences in newborn infants. Health advisories published in the State Sport Fishing Regulations have been issued for persons consuming fish taken from 13 sites in California. News releases and posting may be done with local health departments or the Department of Fish and Game. AGASS PHASE TECHNIQUE FOR DETERMINING THE KINETIC CONSTANTS OF CHEMICAL METABOLISM IN THE RAT. M L Gargas and M E Andersen„ AAMRL/TH, Wright-Patterson AFB, OH. The kinetic constants of chemical metabolism are needed to develop physiologically-based pharmacokinetic (PB-PK) models which predict the time course distribution of volatile chemicals in a matma3lian system Gas uptake proved useful in determining kinetic constants for a variety of volatile chemicals, but low vapor pressure compounds could not be examined and an alternative gas phase method was developed. Rats were first exposed by constant concentration inhalation for six hours and then placed in a 2.5L chamber with a fresh air flow (120m1/min). The chamber effluent ass serially analyzed for test chemical. The resulting elimination behavior is extremely sensitive to metabolism and kinetic constants can be estimated by simulation with a PB-PK model containing equations describing the experimental conditions. Optimized constants were obtained for 1,1,2-trichloroethane by allowing the model to vary only the kinetic constants until a best least squares fit vas achieved between predicted and experimental results (Vmax = 51 umDles/hr/ kg; Km = 3.0 uM). This technique has also been applied to several other chlorinated ethanes with still lower vapor pressures (the twro tetrachloroethane isomers, and penta-and hexachloroethane). 446 PHARMACOKINETIC DISTRIBUTION OF !NTRATRACHEALLY ADMINISTERED MICROCRYSTAL•LINE AND GRAIN PARTICLE- ADSORBED AFIATOXIN B1 IN THE RAT. J M Huie, R A Coulombe. Jr. and R P Sharma Graduate Programs in Toxicology and Molecular Biology and Biochemistry, Department of Veterinary Sciences„ Utah State University, Logan, UT. ~ .,fHigh concentrations of the•-javcinogen aflatoxit~ ' B1 (AFB1) are commonly found in respir.ab~e-, airborne grain dusts. Since the pulmona epithelium metabolically activates AFB1, theQ= characteristics of AFB1 exposure via the respiratory tract is of interest , in the determination of occupational risk. The pharmacokinetic disposition ,4 itftratracheally- administered (i.t.) AFB1 , either in microcrystalline form (MC) or adsorbed onto grain dust particles (GD) was studied. Blood and tissues were sampled for three weeks at selected intervals following (i.t.) administration of a single dose of MC or GD [3H]-AFB1 (6 uCi; 300 ug/kg) in male Sprague-Dawley rats. The blood concentration data from both groups approximated a two-compartment open model with first-order absorption. The time-to-peak for the blood concentration of label was significantly greater in the animals given GD than those receiving MC AFB1 (12 hr vs 2 hr), although the first-order elimination rate constants for both groups were nearly identical (0.00928 and 0.00921 hr'1 respectively). Tissue concentrations of label closely followed those in the blood. in part by PHS grant ES 03591). (Supported 447 WATER-SOLUBLE METABOLITES OF 2-AMINO-3-METHYLIMI- DAZO-[4,5-FJQUINOLINE (I0) AND -2-AMINO-3,4-DIME- THYLIMIDAZO[4,5-F)QUINOLINE (MeIQ). J Alexander, J A Holme,G Becher, and H,E. Wallin, Dept. of Toxicology, National Institute of Public Health, Oslo, Norway. Sponsor: E. Ovbing IQ, MeIQ and related compounds, a new class of chemical carcinogens, are formed during thermal processing of protein rich food. Due to widespread human expos,ure, studies on metabolism and mode of action ar8''~.} important. Metabolism was studied in rats, isolated rat hepatocytes and rat a.iver microsomes. PCB pretreated hepatocytes (1•10 /ml) metabolized IQ and MeIQ to three 'main groups of metfPolites. After 3 hr of incubation with 100 uM of C-labelled substrate, 616 pmol IQ/mg prot. and 885 pmol MeIQ/mg prot. was found covalently bound, more than 961 of IQ/MeIQ had been metaboli- zed into water soluble products,while only 2-4y could stxll be extracted into ethyl acetate. For both compounds 3 major polar conjugates were found in the aqueous phase: one glucuronide, one sulfa- mate and most likely one sulfate. The sulfamates were acid labile and the parent substances were recovered after hydrolysis. The glucuronide (S- glucuronidase treatment) and sulfate (acid hydrol- ysis) conjugates yielded different kinds of ethyl- acetate extractable hydrolysis products, both more polar than the parent compounds. The main water- soluble conjugates were also found in urine and bile of IQ or MeIQ exposed rats. The glucuronides were also formed from IQ and MeIQ in vitro with microsomes in the presence of NADPH and UDPGA. .Among the nonpolar compounds (ethylacetate extrac- table) the N-acetylderivatatives were found as well as several other metabolites. Some of these seem to correspond to the hydrolysis products of the conjugates. Further work on the chemical iden- tification of the metabolites are in progress. 112 50875 8237
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619 A BIOLOGICALLY-BASED PHARMACOKINETIC MODEL FOR DERMAL ABSORPTION. C B Frederick and I M Chang-Mateu. Rohm and Haas Co., Spring House.r. PA. ; A biolqgiftlly-based computer model has been developed to examine chemical absorption.~into skin. The model includes the metabolic characteristics of the stratum corneum, viable epidermis, dermis, and either the circulating blood in the catp~llaries in vivo or the bathing solution of a Franz cell in vitro. The partitioning of a compound between skin compartments, the rapid initial absorption of compounds through hair follicles, and the evaporation of volatile compounds are included. This model is developed in a simulation language that is simple to use and easy to modify so that biochemical pathways specific to a compound may be included. The model has been applied to several sets of data illustrating various metabolic and reactivity profiles. This approach may be useful for risk assessment and the simulation of exposure scenarios involving dermal exposure to xenobiotics. 620 INSIGHT INTO THE INTERSPECIES DIFFERENCES IN BENZENE TOXICITY PROVIDED BY A PHYSIOLOGICAL MODEL. M A Medinsky, P J Sabourin, R F Henderson, G Lucier*, L S Birnbaum*, Lovelace ITRI, Albuquerque, NM and *NIEHS, RTP, NC Studies on the chronic toxicity of benzene indicated that B6C3F1 mice are a more sensitive species ,than are F344 rats. A physiological model was developed to describe the uptake and metabolism of benzene in rats and mice and to determine if differences`in toxic effects are consistent with differences in pathways for metabolism or by differences in total metabo- lism. Compartments incorporated into the model included lung, blood, liver, fat, a group of poorly perfused tissues such as skin and muscle, and a group of richly perfused tissues such as kidney, bone marrow, and heart. Simulations of 6-hr inhalation exposures to benzene indicated that up to 1000 ppm, mice metabolized 2-3 times more benzene than rats per kg body wt. Simula- tions of oral exposure to benzene up to 150 mg/ kg body weight resulted in similar amounts of benzene metabolized in both species per kg7body wt. However, patterns of metabolites formed were different. Rats formed primarily the detoxifi- cation metabolite, phenyl sulfate. Mice formed more hydroquinone glucuronide and muconic acid which are associated with formation of putative toxic metabolites of benzene. (Research sup- ported by the NIEHS through Interagency Agree- ment ES20092 with U.S. DOE Contract No. DE-AC04- 76EV01013.) 156 621 IN VITRO STUDIES OF MEfHYLENE CHLORIDE (MEC) METABOLISM IN HUMAN AND ANIMAL TISSUES: USE IN PHYSIOLOGICALLY-BASED PHARMACOKINETIC (PB-PK) MODELS. R H- Reitz, A"`"L Mendrala, and F P Guen er c. ow hemical Co., Midlan , MI, an anderbilt Univer., Nashville, TN. ~ Andersen et al. (Tox. Appl. Pharm., 87, 185, "' 1987) devefoped a PB-PK model for MET-risk 3'zt assessment. To confi rm tfi'L4frnnan metabol ic . rate constants used in this model, cytosoli&_'.~;._ ,. and microsomal enzyMes were prepared from and liver of F344 rat, B6C3F1 mouse, hamsters,~ and humans. Radiochemical assays (36CL-MEC) . of glutathione S-transferase (GST) and mixed function oxidase (MFO) gave kinetic parameters for these enzymes. Michaelj*'constants (liver . enzymes) were 1-2 mM (MFO)'and >50 mM (GST). MFO activities (nM/min/mg at 5 mM MEC) for mouse, rat, hamster, and human were 11, 4, 14, and 5 nM/min/mg (liver) and 5, 0.2, 1, and <0.1 (lung). GST activities (at 40 mM MEC) were 26, 7, 1, and 2 nM/min/mg (liver) and 7, 1, <0.2 and <0.4 (lung) respectively. These data provide support for the human constants used in the PB-PK model of Andersen et al. and suggest that the lung and liver tumors - observed in mice exposed to MEC vapor may result from the high exposure concentrations (which saturate MFO) and the high levels of GST enzymes found in mice but not hamsters or, humans. 622 PHYSIOLOGICAL PHARMACOKINETIC MODEL FOR HEXACHLOROBENZENE (HCB) IN THE SPRAGUE - DAWLEY RAT AND RHESUS MONKEY. A G E Wilson, K K Rozman, J D Wilson, and R A Freeman. Monsanto Company, St. Louis, MO and University of Kansas Medical Center, Kansas City, KS Numerous studies have been performed.on the absorption, distribution and elimi- nation of HCB in the rat. In addition, studievq~have also been conducted in the rhesuss`monkev. However, no unifvine model appears to have been developed which would permit examination of HCB pharmacokinetics in different species, including man: In this paper we des- cribe the development of a physiologi-d cally-based pharmacokinetic model (PB-PK) for HCB in the rat. The PB-PK - model simulations for HCB in the rat were shown to be in excellent agreement with the published data. The use of PB-PK in species extrapolation was demonstrated by scaling the rat model to the rhesus monkey. Model simulations for the pharmacokinetics of HCB in the rhesus monkey were found to be in good agreement with the experimentally determined data. tn B ou v cn 00 N co Y
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d 508 EFFECT OF VITAMIN A ACETATE ON IRRITANT INFLAM- MATORY RESPONSES IN MTCE. E.Patrick,*S Volsen and *K.Mi.L1er, Dermatology Department, University of California, San Francisco, CA and *Immunology Department, Briti,gh Industrial Biological Research Association, Carshalton, Surrey, Grpata§ritain. Sponsor: H I Maibach Suppiementing tiie diet with Vitamin A acetate (VAA) enhances the ability of laboratory mice to develop delayed contact hypersensitivity. We evaluated the effect of VAA on irritant inflam- ation in unsensitized mice. One QK of groups of five to six female Balb/c mice, maintained on normal diet or diet suppiemented with 0.477gm/kg VAA, were dosed with 25 ul of the irritants in a 1:1 acetone:corn oil mixture. Six concentrations of 1 chloro-2,4 dinitrobenze (DNCB), 1 chloro-2, 4,6 trinitrobenze (TNCB), and 4-etnoxymethylene- 2 phenyloxazolone (oxazolone) were tested. Infiamation was quantified as change in ear thickness measured with a micrometer at 1,2,4,6 aizd 24 hours after application of the irritants. Ear thickness changes in animals maintained on VAA diet were significantly greater than controls dosed with DNCB or TNCB. Early thick- ness changes produced by oxazolone were not different but 24 hour responses were signifi- cantly greater in animals fed VAA. Histology of responses and expression of immune response antigens in ear tissue of animals fed each diet were compared to investigate the mechanism of differences in irritant responses to DNCB, TNCB and oxazotone. 509 MODIFICATION OF THE NON-IMMUNOLOGIC QPNTACT URTICARIA PREDICTIVE ASSAY IN GUINEA PIGS H I Maibach and E.Patrick, Department of Dermatology, University of California, San Francisco, CA. The predictive assay for non-immunologic contact urticaria (NICU) in the guinea pig used change in ear thickness to evaluate the inflammatory response. A significant increase in ear thick- ness was evidence for a positive response. We performed assays of 27 consumer products and ingredients, not previously shown to produce urticaria. Evaluation by change in ear thickness measured with a micrometer and visuai grading for the presence of discrete lesions and vaso- dilation were compared. Ears of ten 300-400 gm Hartley strain guinea pigs were dosed with 0.1 ml of the test material; the opposite ear was dosed with a suitable control. Thickness mea- suremencs and visual examinations were performed 15, 30, 45, and 60 minutes and 24 hours after application of the test materials. No signifi- cant change in ear thickness was produced by' any of the test materiais. Seven of the 27 materials produced visible lesions which were reproducible on the same animals when retested at 48-96hours. Human testing or use tests have confirmed that three of the seven materials, visually positive in the NICU, produce urticaria in man. This comparison demonstrates that incorporating visual evaluation by a trained observer will increase the sensitivity of the NICU assay. . 510 CORRELATION OF AN IN V T -_KERATINOCYTE SYSTEM WITH THE RABBIT PRIMARY'DERMAL IRRITATION MODEL K C Norbury, W J Powers, and P Tischio Ortho Pharmaceutical Corp. Research Laboratories of Raritan.~ NJ. Studies were conducted to determine the feasibility of using an in vi r system as a predictor of dermal irritation potential. Commercially obtained normal human epidermal keratinocytes were grown in the presence cYf various concentrations of-*err-chemicals. These; chemicals were selected for their known e€4ect in vivo, ranging:.:- _from non-irritating severely irritating on intact rabbit skin. - vitro toxicity was determined by the inhibi- tion of DNA or protein synthesis as measured by either the incorporation of 3H-thymidine or 3H-leucine, respectively. kPaftllel studies with the same chemicals were Fonducted using the standard in vivo rabbit primary dermal irritation test. The test compounds were ranked in terms of severity. The preliminary results indicated a good correlation between the in vitro and in vivo tests. We are encouraged by the possi- bility that this in vi r system offers an alternative to the animal assay for identifying potential dermal irritants. 511 CONTACT SENSITIZATION FOLLOWING APPLICATION OF K PYRIDOSTIGMINE BROMIDE TRANSDERMAL DRUG DEIVERY SYSTEM. G L Harris, J L Allen, and . H I Maibach. Pathology/Toxico o6epartment, •ixer a s 3M Co. St. Paul, MN and University of California, San Francisco, CA. Pyridostigmine bromide (PB), is a useful pre- treatment for organophosphate poisoning. PB transdermal formulations were developed since this route provides more consistent blood levels than oral administration. The formulations con- 128 tained 30;or 50% PB in a gel. matrix and some also. contained~~a surfactant (eg, 0.2% sodium lauryl sulfate) as a penetration enhancer. Guinea pig sensitization studies (Split Adjuvant Method) were conducted as part of this formulation's safety evaluation. Eleven groups of 10 animals were induced and challenged with combinations of PB and excipients such that the sensitizer could be identified. Four of nine animals induced with 50% PB alone had positive responses to a PB challenge dose. Most animals induced with PB-surfactant had positive responses to PB and PB-surfactant formulations. Surfactants appeared to potentiate the response since the incidence of sensitization was greater. PB is structurally similar to quaternary ammonium compounds which are known sensitizers. Thus, PB may represent an example of contact sensitization from a cationic tertiary ammonium compound. This work supported by the U.S. Army, but the findings and conclusions are not necessarily the official position or policy of the U.S. Army. 50875 8253
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356 A95ESSMEiV'P OF RHE Cfi%kTlC QRAL TOXICITY OF d-L'IIMNIIQE IN DOGS. D R WeY~b, D K Hyse11 and C L Alden. Rne Procter & Gamble Co., Cincinnati, Cgi. A vrlde variety o* hydrocarbons, incltxling _d-13asone.ne (DL) ,. have been slxxai to iixhzce a specific triad of.nephzot:oadc events which to date have been unique to the mature male rat. Me present study was designed to further evaluate the unigieness of this lesi by determining the ~Sic toxicity of ~ in a non-rod,2nt species. Five male and five fema.7.e adult Beagle dogs were gavaged twice daily over a 6-month period with tap water (control) or M at 0.12 or 1.2 ml/kg body weight/day (100-1, 000 ng/Jag BkT/day). Feed consumpticn and body weight were unaffected by treatment. 'Ilve highest dose of DL resulted in slight increases in seYum alkaline phoJphatase and cholesterol values in both sexes, and slight decreases in urine specific gravity and Pi in females. Linear regression analyses indicated a positive dose-related trend for absolute and relative male and female liver weights, and absolute and relative fema].e kidney weights. There was no microscopic evidence of histopathologic c2sange in the liver and kidneys which would correspond to the organ weight chatxses. Most importantly, this incl.udes an absence of light-7niaroscapical7.y evident hyaline droplets in renal tubules which is recognized to occur in the kidneys of male rats treated with DL. 357 TOXICITY STUDIES WITH QUININE HYDRO- CHLORIDE. J C Colley, J A Edwards, R Heywood and D Purser. Huntingdon Research Centre, Cambs., England. Quinine is the main alkaloid and active principle of cinchona bark, a febrifuge used for 350 years. 40%.of the world market is now taken up by the food industry, where it is used as a bittering agent in carbonated drinks. In 1980 the scientific committee of the EEC asked UNESDA to supply safety data, because few formal toxicity studies had been done. This poster presents the results of 3 month studies in the rat, a rat embryo-toxicity study and a specific study to investigate ototoxicity. The result of these studies led to the setting of an acceptable daily intake of 40mg quinine hydro- chloride for an adult. There were no indications of teratogenic effects and sophisticated audio- metric techniques showed no indication of inter- ' ference with auditory function in rats receiving up to 200mg/kg. 358 TO EFFECT OF 'hCM CN RAT FIEPATIC VITWQti A LMIEES,. AND RETIZ10YIrAM P NI'IIiOPFIIMIrUI3? GINCORONO6YL RRANSFERFLSE (GT) ALR:'IVITIFS. R H Powiers, L C Gilbert and S D Aust. Department of Bioc;hmistYy, Michigan State University, F.ast L3nsing, ML. A single po dose of RCDD caused a dose dependent depression of Yepatic retinyi ester levels in male Sprague Dawleysmts by 12 days '£ollowing treatment. Los.s of hepatic retinyl. esters in rats treated ;vith > 10 naol/}og was significantly greater than in untreated rats fed a vitamin A deficient diet. A dose dependent increase in both p-nitrophenol- and retirwyl-UDPGT activity was observed in the liver micirosomes from ZCDD-treated rats. p- Nitrcphenol-UDPGT activity in R&D treated rats was elevated to a maximam of about 7x that of untreated controls, and was unaffected by feeding rats a vitamin A deficient diet for 12 days. Retinayl-UDPGT activity in TCDD- treated rats was elevated to a maximm of about 4x that of untreated oontrols, and was significantly depressed in untreated rats fed a vitamin A deficient diet, when ccanpar+ed to rats fed a ccnplete diet. Micrrosomall retinol- UDPGT activity was not detected. We suggest that elevated rates of formation of retinoyl- fl-glucurronide in the livers of TCM-treated rats contributes to the depletion of vitamin A reserves. (Supported by NIIi Grant FS3585.) 359 90 SELECTIVE IIJIiANCEMENT OF TERATOGENICITY IN MICE BY TCDD AND VITAMIN A(RA). L S Birnbaum, M W Harris, and R E Morrissey. NIEHS, Research.... Triangle Park, NC. Many of the signs of TCDD toxicity resemble those seen during RA deficiency. Both TCDD and RA are also well known teratogens. In mice, TCDD causes cleft palate and hydronephrosis at doses where there is no overt fetal or maternal toxicity. A similar situation exists for RA in the induction of cleft palqte and skeletal abnormalities. In order to dete'=mine if excess RA could overcome the teratogenic effects of TCDD, or vice versa, C578L/6N mice were treated po on gestation day (gd)10 with 10 ml corn oil/kg containing TCDD (0-18Ng/kg), all trans retinoic acid (RA)(0-100 mg/kg) or combinations of the two chemicals. Dams were killed on gd 18, and developmental toxicity assessed. Coadministration of TCDD and RA had no effect on maternal or fetal toxicity beyond what would be expected by either compound alone. The incidence of cleft palate was dramatically en- hanced by administration of the two compounds together. No increase in the incidence or severity of hydronephrosis was seen in the com- bination treatments over that expected by TCDD alone, nor was there any increase in the inci- dence or pattern of limb bud anomalies in the combination groups over that caused by RA alone. No other soft tissue or skeletal abnormalities were detected. Thus, coadministration of TCDD and RA selectively increases the incidence of cleft palate, a conmon target tissue for these chemicals, without interacting to affect devel- opment in target organs unique to each compound. 61
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34 500 INTERSTRAIN AND SPECIES.DIFFERENCES IN XENOBIOTIC METABOLIZING CAPACITY IN SKIN: EVIDENCE OF ENHANCED ACTIVITY IN SENCAR MICE. J E Storm, R F Stewart, and R L Bronaugh._Div, of Toxicology, FDA, Washingtot3, DC. a + SENCAR m~ce aie especially susceptible to epidermal tumorigenesis. The basis for this is unknown, but"perhaps greater metabolic activation of initiators (Phase I metabolism) and/or decreased detoxication of reactive intermediates (Phase II metabolism) in skin ar(3 involved. Thus, metabolic capacity of SENCAR mouse*skin was compared with another mouse strain and two rat strains. Benzo(a)pyrene (BP) was applied to skin in flow-through diffusion cells and was measured with metabolites in receptor fluid. Skin of SENCAR mice metabolized substantially more BP to ' ethyl acetate- and water- soluble metabolites than skin of BALB C mice, Osborne Mendel rats or Fuzzy rats; e.g., in SENCAR mice 73% of BP penetrating the skin was present as ethyl acetate- extractable metabolites while in Fuzzy rats the value was only 18%. In enzyme kinetic studies, SENCAR mouse skin cytosol had substantially more glutathione transferase activity than Fuzzy rat skin cytosol. Apparent Vmax for the.conjugation of dinitrochlorobenzene in SENCAR mice was nearly 2.5X that in Fuzzy rats. These results together are evidence that skin of SENCAR mice has substantially more metabolizing capacity than another mouse strain and species; and, that both Phase I and Phase II metabolism is involved. ~ 501 EFFECTS OF UVA IRRADIATION ON ACTIVE OXYGEN SCAVENGING ENZYMES IN DOG AND MOUSE BLOOD. D G Robertson, D L Bailey, and R A Martin. Dept. Path. & Exp. Tox., Parke-Davis Pharm. Res. Div., Warner-Lambert Co., Ann Arbor, MI. In exploratory studies, using a photohemolysis model, substantial species differences were noted in susceptibility of red blood cells to phototoxicants and to UVA light alone. We examined the effects of UVA light (1300 uW/cm2) on catalase (CAT), superoxide dis-mutase (SOD) and glutathione peroxidase (GPX) in dog and mouse washed red blood cells. UVA induced hemolysis was more rapid in the mouse than in dog. Control levels of CAT and GPX were approximately 10 and 3 times greater in the mouse. SOD levels were higher in the dog (2x), however, individual variation was high. Mouse CAT and GPX activity declined rapidly. Enzyme degradation was more pronounced in UVA irradiated samples. Dog CAT was stable for 5 hours but activity was significantly decreased after 3 hours of UVA irradiation. SOD levels were unaffected by UVA irradiation in both mouse and dog. These results suggest that species differences in susceptibility to hemolysis by phototoxicants may be due to differences in oxygen scavenging enzyme activities and their inactivation by UVA light. Therefore, in vitro phototoxicity screens from differing species should be interpreted with caution. 502 NINE-DAY REPEATED DOSS15UTANEOUS TOXICITY OF DIETHYLENE GLYCOL MONOHEXYL ETHER (DGHE) IN • ALBINO RABBITS. S W Frantz, M W Gill, J P Van Miller, P E Losco, C MTroup, and B Ballantyne. Bushy Run Research Center, Export, PA and Union Carbide Corporation, Danbury, CT. «f The toxicity of DGHE from repeated cutaneous =_ on hematology, clinical chemistry measurements, or organ weights were observed. Based on this study, DGHE is considered to be a mild skin irritant at 100 mg/kg/day and a severe skin irritant at 1000 mg/kg/day. However, short- term cutaneous exposure to DGHE did not cause systemic toxicity in this species. (1000 mg/kg). Microscopic -hiriges in the skin _ included acanthosis, hyperkeratosis (100-1000 mg /kg), suppurative epidermitis (300-1000 mg/kg), hemorrhage (females, 300-1000 mg/kg) and dermal fibrosis (300-1000 mg/kg). Transient depressions in body weight and food consumption, (1000 mg /kg) possibly related to the stress caused by the skin lesions, were observed during the first week of the study. No treatment-related effecte Local clinical signs of toxicity from DGHE included edema, erythema, fissure formation (100-1000mg/kg), encrustation, and ecchymoses 300, or 1000 mg/kg;-body weight/day for a totak W- of 9 applications (6 hours/day) over 11 days.'47 exposure was assessed. New Zealand White '~~" rabbits (5/sex/group) weflWexposed to DGHE by ~: occluded cutaneous exposure at doses of 0,--"100j 503 DERMAL TOXICITY OF A HIGH BOILING (BP 250-450°C) COAL LIQUEFACTION PRODUCT IN THE RAT. I Chul, D C Villeneuvel , M_ C6te2, V. Secoursl , R tso an Valli3. lEnvironmental Health Directorate, Ottawa, Ontario, 2 Department of Pharmacology, University of Montreal, Montreal, Quebec and 3Biopath Analysts Ltd, Guelph, Ontario. Coal liquefaction products (CLP) have been con- sidered as an alternate source of energy to replace ,~ onventional crude oil. The present study was designed to investigate the dermal toxicity of a heavy fraction of CLP (bp 250- 450°C) in the rat. Groups of 10 male and 10 female Sprague-Dawley rats (180-200 g) were painted dermally with the CLP at dose levels of 0, 100, 200, 400 or 800 mg/kg b.w./day for 6 weeks. Growth suppression was observed in all CLP treated groups of males and in the 2 highest dose groups of females. Increased liver and kidney weights were observed in the diesel fuel (400 mg/kg) as well as CLP treated groups of females. Decreased red cell count, hemoglobin and packed cell volume and mild bone marrow hyperplasia occurred in some CLP and diesel fuel groups of both sexes. Mild histological changes were observed in the thyroid, liver, bone marrow and skin of rats of both sexes treated with CLP and diesel fuel. Based on the data presented, CLP produced systemic toxicity at a dose level of 100 mg/kg b.w., and both CLP and diesel fuel possess toxic effects of a similar nature and magnitude. 126
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0 EFFECTS OF gIvIITRIPTYI.INE ON CALCIUM IjpTAKE AND HIGH ENERGY PHOSPHATES•IN PRIMARY MYOCARDIAL CELL CULTURES. $Ar'&U, Y Park, *J Bradlaw, and AA Welder. University of,Texas College of Pharmacy, Austin, TX and *Food and Drug Administration, Washington, D.C. Tricyclic antidepressants (TCAs) are used in the treatment of endogenous mental depression. There are an estimated 5,000 to 10,000 cases of overdosing or poisoning each year with TCAs. Of the TCAs, amitriptyline is the most cardiotoxic, with evidbqe of arrhythmias and contractile disturbances. In a previous study (J. Toxicol. Envir. HIth. ,14_:137, 1984), we demonstrated that TCAs were directly cytotoxic and arrhythmogenic to cultured myocardial cells. The purpose of this investigation was to determine the direct effects of amitriptyline on 45Ca''-'' uptake and high energy phosphate stores in primary cultures of rat myocardial cells. Cultures obtained from hearts of 3-5 day old Sprague-Dawley rats were exposed to various concentrations of amitriptyline (1 x 10-4 and 1 x 10-5 M) for 4 to 24 hr. Glucose utilization, 45Ca++ uptake, adenosine triphosphate (ATP) and creatine phosphate (CP) levels were evaluated after amitriptyline treatment. Glucose utilization was not affected by either concentration of amitriptyline after 24 hr of treatment 45Ca++ uptake was depressed after the 4 hr exposure to the highest concentration of amitriptyline tested. ATP levels were also depressed after 4 hr and depleted after 8 and 24 hr exposure to I x 10-`i M amitriptyline. In contrast, CP levels were not affected by the drug. These data suggest that aberrant contractile activity of myocardial cells after exposure to amitriptyline may be associated with depletion of high energy phosphates and ionic disturbances. (supported by FDA contract #223-86-2109) 421 STUDIES ON THE MECHANISM OF PSORALEN INDUCED PHOTOTOXICITY USING HUMAN EPIDERMAL A431 CELLS. F_ Mermeistein, M.A Gallo, and J D Laskin, UMDNJ-Robert` . W. Johnson Medical School, Piscataway, NJ The psoralens are furocoumarins that, when combined with ultraviolet light.. (UVA), cause skin phototoxicity.- We have previously shown that psoralens and UVA light are potent'inhibitors of epidermal . growth factor (EGF) binding in several different cell types. This inhibition appears to be modulated by a high affinity psoralen receptor. The human epidermal cell line, A431, expresses over 106 EGF receptors/cell. The EGF receptor in these cells has been characterized as a 160-170 kd transmembrane protein with an intrinsic tyrosine kinase (TK) cytoplasmic domain. Binding of EGF to A431 cells stimulates TK activity. We found that the psoralen analog 4,5',8- trimethylpsoralen in combination with UVA inhibits EGF binding to A431 cells by 20- 25%. Using an antiphosphotyrosine mono- clonal antibody, we purified a population of tyrosine phosphorylated EGF receptors from control and psoralen/UVA treated cells. Treated cells were found to have decreased levels of EGF stimulated tyrosine phosphorylated EGF receptors. Our data provide evidence that psoralens modulate EGF receptor binding and function. Supported by NIEHS ES 03647. 422 THE PSORALEN RECEPTOR AS A MEDIATOR OF CHEMICAL PHOTOTOXICITY- J D, Laskin, E J Yurkow and M A Gallo, UMDNJ-Robert W. Johnson Medical School, Piscataway, NJ Psoralens, or furocoumarins, are potent skin photosensitizing agents. Epidermal cells are known to contain specific high afffsiity binding sites for -"lft- psoralens and we have hypothesized that these.-A,, . binding sites medi.ate_ the biological effects of these compounds in the skin. We have characterized the psoralen binding sites from PAM 212 mouse epidermal cells and HeLa cells. We have found that they can be alkyla,*d"by 3H-8- methoxypsoralen following 'ultraviolet light (UVA) exposure. Covalent binding of the label was inhibited by an excess of unlabeled psoralen indicating that covalent psoralen binding was saturable. Fractionation studies revealed that the binding sites were present in membrane and cytoplasmic fractions of the cells and were sensitive to protease but not nuclease treatment. Using SDS-polyacryl- amide gel electrophoresis, the labeled psoralen binding sites were found to have a molecular mass of 22,000 daltons. The identification of a specific cellular protein.which binds psoralen supports our model that the biological effects of these compounds are receptor mediated.' Supported by NIEHS 03647. 423 ALLYLAMINE (AAM)-INDUCED ALTERATIONS IN THE PHOSPHOINOSITIDE/IN0SIT0L PHOSPHATE PROFILE OF CULTURED AORTIC SMOOTH MUSCLE CELLS. L R Cox, S K Murphy, and K Ramos*. Philadelphia College of Pharmacy & Science, Phila, PA and *Texas Tech University Health Sciences Center, Lubbock, TX. Previous studies in our laboratory have shown that smooth muscle cells (SMC) cultured from . AAM-treated rats exhibit morphologic features and synthetic capabilities characteristic of A proliferative phenotype. As an increased turn- over of membrane phosphoinositides has been implicated in cell proliferation, the present studies were undertaken to examine the phospho- inositide/inositol phosphate levels in SMC cultured from control and AAM-treated animals. Adult male Sprague-Dawley rats were dosed daily with AAM-HC1 (70 mg/kg) or tap water for 21 days. The levels of phosphatidylinositol 4-phosphate, phosphatidylinositol 4,5- bisphosphate and phosphatidic acid in SMC from AAM-treated rats were lower than those of control SMC by 31, 35 and 22%, respectively (n=10). Inositol phosphate levels were similar in cells cultured from control and treated animals (n=7-9). Our results show that the cellular toxicity of AAM is associated with changes in the phosphoinositide/inositol phosphate profile of aortic SMC. AAM-induced alterations are affected by manipulation of cyclic AMP levels. The phenotypic expression of vascular SMC upon AAM exposure may be modulated by changes in phosphatidylinositol turnover. 106 50875 8231
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S20 THE TOXICOLOGY AND PATHOLOGY OF 5-AMINOSALICYLIC ACID KERATOCONJUNCTIVITIS SICCA IN THE BEAGLE DOG. E C Joseph, G R Betton, K C Barnett and J M Faccini. SK&F Research Ltd. Welwyn, Herts, UK; The Animal"Realth Trust, Kennett, Ne wrnar~et, Suffolk, UK; Les Ouldes, Blere, France. ~ponsor: J B Hook. Keratoconjunctivitis Sicca (KCS) is an inflamma- tory eye condition affecting the cornea and conjunctiva, caused by the deficiency in the aqueous fraction of tears. A number of sulph- onamides including salicylazosulphapiyridine (SA) have been associated with KCS in the dog. KCS has been noted in a 1-year dog toxicity study with the nonsulphonamide 5-aminosalicylic acid (5-ASA), with females being more affected than males (Incidence: Males 12%; Females 71%). There was a close correlation between KCS and reduced lacrimation. Primary atrophic changes were observed in the lacrimal and nictitans glands and parotid salivary gland of treated dogs. These changes were associated with lymphoid cell infiltration suggestive of a cell mediated immune reaction. Lesions were also present in the cornea, eyelids and nictitating membranes. In contrast to the effects noted in dogs, there have been no reports of KCS in man following SA administration (the prodrug of 5-ASA) during four decades of clinical use. Therefore, it is highly probable that the dog is not a predictive model for man for this effect. 521 IN VITRO ALTERATION OF EPOXIDE HYDROLASE (EH) ACTIVITY BY METABOLITES TF THE RENAL CYSTOGEN 2-AMINO-4,5-DIPHENYLTHIAZOLE (DPT). T T Hjelle, T Guenthner, R Whalen, G R Flouret, and F A Carone. University of Illinois College of Medicine at Peoria and Chicago, IL and North- western University Medical School, Chicago, IL Sponsor: S M Lasley The mechanism by which subchronic feeding of DPT causes renal cysts and the observed changes in renal enzyme activities is unknown. Because DPT is metabolized in vivo, we examined the ability of DPT and its para-hydroxylated metabolites to alter various enzyme activities in vitro. Using enriched prep- arations of mouse liver microsomes and cytosol, the microsomal and cytosolic forms of EH were assayed using the specific substrates styrene oxide and transtilbene oxide, respectively. cEH was inhibited by the 4-hydroxy and 4,5 dihydroxy metabolites, but not the 5-hydroxy or parent compound. In contrast, only the 4-hydroxy form inhibited mEH; 5-hydroxy DPT actually stimulated mEH activity. When the known renal cystogens nordihydroquaiaretic acid and diphenylamine were tested, only inhibition of cEH was observed. The hydroxylated metabolites of DPT also inhibit catalase activity in a hydrogen peroxide- facilitated manner. Thus, metabolites of DPT share with two known cystogens the capability of directly altering the activity of cEH in a manner consistent with pro-oxidative mechanisms of cellular stress. (Supported by PHS grants AM33003 and CA34455.) ~~1-'+K522 EFFECT OF.DEFEROXAMINE ON:NOXIA-INDUCED INJURY IN RAT RENAL SLICES. W Hewitt and A Silver. Smith Kline & French Labs, King of Prussia, PA." 4~- Deferoxamine (DEF) has been shown to reduce the renal dysfunction in rats produced by ischemia/ reperfusion. It is unclear if DEF acts directly on renal tubular cells or on extrarenal cells ` (e.g., neutrophils). The objective of this study was to determine if DEF prevented proximal> tubular dysfunction produced„W.An s vi r ~ "'anoxic insult. Longitudinal renal slices pre-._:~ ~ pared from male CD rat.kidneys were incubated dt37°C. One-half of the' slices were subjected t~: a 45-min anoxic (100% NZ) period followed by - 8 hr of reoxygenation (100% O2); control . slices were incubated continuously under 100% 02- Proximal tubular function asaestimated hourly during reoxygenation by~liee organic ion (PAH, TEA) accumulation; viabilityrwas assessed by LDH leakage. Malondialdehyde (MDA) genera- tion was used as an index of lipid peroxida- tion. PAH accumulation by slices subjected to anoxia was reduced 40% and 60% after 1 and 8 hr of reoxygenation, respectively; TEA uptake was reduced approximately 30% after 8 hr reoxygena- tion. LDH leakage and MDA generation increased in a time-related fashion and were greater in slices exposed to 100% N2. DEF (1 or 20 mM) did not ameliorate the anoxia-induced altera- tions in slice organic ion accumulation, LDH leakage or MDA generation. Thus, DEF may not exert a direct protective effect on renal cells damaged by anoxia/reoxygenation. 523 RENAL CYSTEINE CONJUGATE S-LYASE (S-LYASE)- MEDIATED TOXICITY STUDIED WITH PRIMARY CULTURES OF HUMAN PROXIMAL TUBULAR CELLS (HPTC). J C Chen, J L Stevens, and T W Jones. Univ. of MD School of Medicine, Balto., MD and W. Alton Jones Cell Science Center, Lake Placid, NY. B-lyase-mediated, S-cysteine conjugate-induced nephrotoxicity has been described in a variety of in vitro and in vivo animal models. In this study, we have extended these observations to include human by investigating the toxicity of S-(1,2-dicki].orovinyl)-glutathione (DCVG) and S=(1,2-dichlorovinyl)-L-cysteine (DCVC) to HPTC. Primary HPTC were treated overnight with either. DCVG or DCVC at concentrations ranging from 100 uM to 1.0 mM. In each case, a dose-dependent toxicity was seen with both DCVG and DCVC. Despite a wide degree of variation in sensitivity observed between cases, DCVC was consistently found to be more toxic than DCVG. However, inclusion of glycylglycine (10 mM) or Y-glutamyltranspeptidase (0.5 U/mi) increased the toxicity of DCVG to that of an equimolar dose of DCVC, indicating that metabolism to the cysteine conjugate is an important rate-limiting step. Aminooxyacetic acid (250 uM), an inhibitor of pyridoxal phosphate;Adependent enzymes such as g-lyase, provided complete protection, suggesting a critical role for g=lyase in the HPTC toxicity of DCVG and DCVC. (Supported by ACS/BC°570.) .131 50875 8256
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452 CHARACTERIZATION OF _QLUTATHIONE S-TRANS- FERASE ACTIVITY TOWARD VApMUS SUBSTRATES IN F(IJMAN ON(*JqUCLEAR LEUKOCYTES. J A - Richards, D, S K Homung, A G Motulski and B D Hammock, University of Washington, Seattle, WA and University of California-Davis, Davis, CA. Glutathione S-transferases (GST) are involved in the detoxi- fication of many xenobiotics. A genetic tri-modal distribution of GST activity in human mononuclear leukocytes has been found toward trans-stilbene oxide (TSO). It has been sug- gested that leukocyte GST activity toward TSO may be used as a marker for genetic susceptibility to cancer. The aim of this study was to determine if leukocyte GST activity ex- pressed toward TSO is characteristic of activity toward environmentally relevant carcinogenic substrates. HPLC as- says were developed to measure GST activity in leukocytes towards aflatoxin-8,9-oxide (AFBO), benzo(a)pyrene-4,5- oxide (BAPO), and p-nitrostyrene-7,8-oxide (PNSO). Ac- tivity toward CDNB was also assayed. These assays were conducted on leukocyte preparations from 31 apparently normal individuals, with the exception of the aflatoxin assay, in which no measurable GST activity was found toward AFBO in a subset of these samples. Comparisons of GST activity toward TSO yielded statistically significant correla ' tions between GST-TSO activity and GST BAPO activity (r=0.74); and GST-TSO activity and GST-CDNB activity (r=0.48). GST-PNSO activity was not significantly corre- lated with GST-TSO. No apparent modality in GST activity toward these substrates was observed. These results suggest that it may be misleading to make inferences about GST ac- tivity towards environmentally relevant compounds from the use of surrogate substrates. (Supported by The Dana Foun- dation). s 453 ANALYSIS AND SCREENING OF XENOBIOTIC MERCAPTURIC ACID CONJUGATES USING NEGATIVE IONIZATION AND TANDEM MASS 5PECTROMETRY. C K Winter, A D Jones*, and H T Seeall, Department of Veterinary Pharmacology and Toxicology and *Facility for Advanced Instrumentation, University of California, Davis, CA. The mass spectra and fragmentation pathways of the mercapturic acid conjugates of 1,3-dichloropropene, styrene oxide, trans-4-hydroxy-2-hexenal, trans-4- hydroxy-2-nonenal, naphthalene oxide, and benzyl chloride were investigated using chemical ionization and fast atom bombardment mass spectrometry in conjunction with linked scan and mass-analyzed ion kinetic energy spectrometric techniques. Fragmentation patterns of the pseudomolecular ions of these mercapturic acids were simple and consistent, with the dominant mode of decomposition of [M-H]-involving scission of C-S bonds with loss of a neutral fragment of mass 129 u. _ Screening for mercapturic acids in urine samples was performed by introducing samples via desorption chemical ionization probe, and neutral loss scanning was performed to obtain selective detection of inercapturates. Limits of detection were determined to be at low nanogram levels. 454 CHICK EMBRYO RETINA CELL CULTURE: TERATOGEN SCREEN AND MECHANISTIC PROBE. G P'°' Daston, J E Yonker, D Baines and J I Poynter, Miami Valley Laboratories, Procter & Gamble, Cincinnati, OH. We have determined that a suspension culture of _ aggregated neural retina cells derived from ' incubation day 6 chick embryos is a useful ~ -Vcreen for teratogens, and m"-provide informa- ~ tion on the mechanisms of action of teratogena. -, In culture, the embryoAic cells undergo a number.," of fundamental processes of development, inclu- ding cell-cell interactions, pattern formation, histogenesis, growth and division, and selective expression and suppression of genes to produce a differentiated phenotype. We.#ave$c'._established objective, measurable endpoints for, these devel- opmental events. Perturbation of any endpoint by a chemical is indicative of potential for developmental toxicity. To date, we have tested 17 known teratogens and non-teratogens, and the screen has correctly classified all but one (a false negative). We have successfully used an exogenous metabolizing system (rat hepatic S-9) to activate an indirect teratogen, cyclophos- phamide. Developmental toxicants alter differ- ent sets of endpoints depending on teratogenic mechanism. For example, inhibitors of gene expression alter endpoints of differentiation; enzyme inhibitors or mitoclasts interfere with growth; inhibitors of cell-cell interaction disturb pattern formation. Thus, this assay is a predictive teratogen screen and may be useful in identifying putative mechanisms of action.. 455 THE HYDRA ASSAY AS A PRESCREEN FOR T-ERATOGENIC MYCOTOXINS. E E Smith, E A Maull, M A Taylor, B A Clement and T D Phillips. Veterinary Public Health, Texas A&M University, College Station, TX. More than 200 mycotoxins have been discovered and structurally characterized. A number of these chemicals have been reported to be terato- genic, bu%.for most, the developmental hazard is unknown. TFiis study was designed to evaluate the ability of the hydra assay to accurately detect and predictively rank teratogenic mycotoxins and/or homologs, according to their develop- mental hazard index (A/D ratio). Hydra attenuata were maintained in culture. Mycotoxins, (i.e., T-2 toxin, diacetoxyscirpenol, aflatoxins B1 and Gl, ochratoxin A, and a racemic mixture of ochratoxin alpha) were diluted in an appropriate vehicle and tested according to established procedures to determine the minimal affective concentrations of each for the adult hydra (A) and the developing hydra "embryo" (D). The calculated A/D ratios ranged from 1.0 (for afla- toxin B1, suggesting maternal toxicity at con- centrations shown to affect the embryo) to 5.5 (for T-2 toxin, suggesting more of an effect on the embryo and less maternal toxicity). These findings are in general agreement with previous studies in vivo, supporting the conclusion that the hydra assay may be useful as a prescreen for other important mycotoxins (Supported by AH 6830, USDA Project 84CRSR-2-2434 and USAID CRSP 02-50305-2). 114 50875 8239
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579 METABOLIC CHARACTERIZATION OF ISOLATED,. ENRICHED RAT LUNG CELL FRACTIONS. S Lacy, J Mangum, and J Everitt. Sponsor: J Gibson. CIIT, RTP, NC. Isolated, enriched fractions of alveolar type II cells, non- ciliated bronchJoiar ep(theiial (Ciara) cells, endothellal cells, ar~d p%nonary macrophages were examined to determine reiative xenoblotic metabolism parameters for rat lung cell xypes. Male F-344 rats were given a- naphthoflavone (50 mg/kg) ip 48 hours prior to sacrifice.. Perfused lungs underwent protease-di6gest yielding a heterogeneous cell suspension (102 xt1„0 /rat) containing 33.8% type II cells and 3.6% Clara cells. Enriched fractions of type II cells (73.8%) and Clara cells (21.6%) were prepared by centrifugal elutriation and discontinuous gradient centrifugation. The final Clara cell fraction (97% viable) possessed higher levels (per 106 cells) than the type II cell and endothelial cell fractions, respectively, for cytochrome P-450 [P-450] (33% and 14%), glutathione [GSH] (33% and 51 %), ethoxyresorufin-0-deethylase [EROD] (511% and 104%), and GSH-S-transferase (69% and 277%). Macrophages contained non-detectable P- 450, EROD, and GSH-S-transferase, but possessed 50% greater cellular GSH content than the Clara cell fraction. The biochemical characterization of these metabolically- active isolated, enriched pneumocyte fractions will help elucidate cell specificity of pulmonary toxicants in vitro. ~ ' 580 ACCUMULATION OF CYSTAMINE BY RAT LUNG SLICES. C P L Lewis, *G M Cohen and L L Smith. Imperial Chemical Industries PLC, Central Toxicology Laboratory, Macclesfield, Cheshire, UK, and *School of Pharmacy, University of London, UK. Sponsor: E A Lock. The objective of these studies was to determine the accumulation and fate of the disulphide, cystamine, by rat lung slices. Cystamine was accumulated by two uptake systems that obeyed saturation kinetics with apparent Km values of 12 and 503µM, and maximal rates of 530 and 5900nmol/g wet weight/hr respectively. The high affinity system was competitively inhibited by the diamine, putrescine, and the herbicide, paraquat, which are themselves accumulated. Thus, this pulmonary uptake process appears to be identical for all three compounds. The low affinity process was not inhibited by putrescine and results from the diffusion of cystamine into the cell and its subsequent metabolism. Cystamine was metabolised predominantly to the sulphonic acid, taurine, with 10-20X of the intracellular cystamine covalently binding to protein. Conversion to taurine was unaffected by amine oxidase inhibitors, but was decreased after GSH depletion, suggesting that pulmonary metabolism is GSH-dependent, and is not mediated by diamine oxidase. We have concluded that cystamine is taken up by the lung, whereupon it may protect against oxidative stress, since both cystamine and taurine have been implicated as, antioxidants. 146 581 COMPARATIVE PULMONARY T0%ICITY=`OF TWO ORGANOMETALLIC COMPOU,,VIDS;''"CYCLOPENTADIENYL MANGANESE TRICARBONYL (CMT) AND METTHYL- CYCLOPENTADIENYL MANGANESE TRICARBONYL (MMT)'. R J Cl2 and J B Morris Toxicology Program, Sch- oo Pharmacy, University of Connecticut, Storrs, CT. MMT is a gasoline antiknock additive which is selectively toxic to the lungs. In the current study the importance of the methyl side chai&,of MMT as it relates to the selective toxicity was ~ assessed by comparing thxic responses~to Mifir to those of its analog CMT. MMT and CMT' were administered Sprague-Dawley rats at~'-; : doses containing 1 or 2.5 mg/Kg Mn. Rats wer~d sacrificed 24 hr post injection, aortic blood was removed, lungs were lavaged with 0.9% saline and then removed and homog~z. e,~i.. in 1.15% KC1. Lavage lactate dehydrogen , total protein and albumin were increased in a dose dependant manner by both CMT and MMT with CMT producing a. greater response than MMT at both dose levels. For example, at the low dose CMT produced a 12 fold increase in lavage albumin whereas MMT produced a 4 fold increase. Mn content was increased 16 fold over•control following the low dose of CMT and 9 fold following MMT. Thus, CMT appears to be the more potent agent with respect to pulmonary toxicity and pulmonary Mn accumulation. These results suggest that the methyl side chain is not necessary for the pulmonary toxicity and in fact may allow for detoxication. 582 INTRATRACHEAL INSTILLATION (ITI) OF AROMATIC- RICH HYDROCARBONS (ARH) AND 3-METHYLCHOLANTHRENE (MC) SUSPENDED IN A LECITHIN EMULSION (L). J P Hinz, J J Freeman and F T Whitman. Exxon Biomedical Sciences, Inc., East Millstone, NJ ITI has been proposed as a possible route of ex- posure in assays to detect pulmonary carcinogens. For a preliminary toxicity assessment, suspen- sions of ARH (4%, w/v), which is rich in poly- cyclic aromatic hydrocarbons, and MC (2.5%, w/v)' were prepared in L (5% w/v in phosphate buffered saline) by sonication. Once/wk for 5 wks male F-344 r~ts (285-310g, 5/ group) were anesthetized with enflurane and administered 0.2 ml of ARH, MC or L using•a small animal laryngoscope. ARH- and L-treated animals were sacrificed 1, 4 and 8 wks later. The MC group was sacrificed at 8 wks. Transient rales was frequently observed after dosing. Mortality was spread across treatment groups and appeared to be related to the static anesthetic procedure. (Mortality was altogether eliminated in a subsequent study using a dynamic anesthetic exposure system). Histopathological evaluations of the lungs and tracheas revealed no adverse effects in L treated rats. A single focus of squamous metaplasia was noted in the lungs of a MC-treated rat. ARH elicited accumu- lations of foamy macrophages in the alveoli, multifocal interstitial pneumonitis and terminal bronchiolitis. Similar inflammatory changes have been reported to occur following inhalation studies with mineral oils and might be expected to occur in chronic ITI studies. 50875 8271
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516 REDUCTION IN THE NUMBER OF RABBITS USED TO ASSIGN EYE IRRITATION CLASSIFICATIONS WITH CORNEAL PACHYMETRY. R L Morgan, S S Sorenson and T R Castles. Stauffer Chemical Co. Toxicology epartment, Richmond, CA.'- Reduction o1-the number of rabbits used for an eye irritat-ton test is important for animal wel- fare considerations and cost effectiveness. Cor- neal pachymetry has been shown to be an accurate method for assigning EPA and EEC eye irritation classifications. Due to the pr cision inherent to pachymetry it was reasonableo assume that a smaller number of rabbits per test could be used to reliably assign eye irritation classifica- tions. To test this hypothesis, a variety of materials were tested for eye irritation. Each material was tested in a group of seven to eight rabbits and in a group of three rabbits. Both left (treated) and right (control) eyes were evaluated for irritation and corneal thickness each day for three days. EPA and EEC classifica- tions were assigned using the corneal thickness ratios (left corneal thickness/right corneal thickness). Approximately 89% of the irritation classifications obtained with 3 rabbits were in agreement with or 1 grade higher than those obtained with a minimum of 7 rabbits. The mean coefficient of variations for the ratios were 19% and 10% for the tests using three rabbits and 7 to 8 rabbits, respectively. From these results corneal pachymetry has been shown to accurately assess eye irritation with approximately a 50% reduction in the number of rabbits required. 517 EFFECTS OF BETA-ADRENOCEPTOR AGONISTS ON THE _RETINA OF RATS AND HAMSTERS. G R Lankas •and H L Allen. Merck Sharp & Dohme Research - Laboratories, West Point, PA. Sponsor: R T Robertson Cimaterol and L-644,469 are potent, non-selec- tive beta-adrenoceptor agonists with signifi- cant growth-promoting activity. Both com- pounds produced a focal retinopathy in hamsters, but not in pigmented or albino rats after 4 weeks of treatment at dosage levels Z 0.5 mg/kg/day. These lesions were charac- terized as a focal loss of rods and thinning of the retinal outer nuclear layer. Subse- quent studies showed that these lesions could be inhibited by coadministration of propranol- ol, a beta-adrenergic blocking agent, implica- ting beta-receptors in the induction of the retinopathy. To determine if the hamster was unique in the accumulation of drug in the retina compared to rats, 14C-L-644,969 was given IP to both rat strains and hamsters. Autoradiographic images made 3 hours after dosing showed that despite marked accumulation of drug in the pigmented ocular tissue of both rats and hamsters, retinopathy was found only in hamsters. No drug accumulation or lesions were found in albino rats. These studies in- dicate that the retinopathy induced in ham- sters by beta-agonists involves more than just accumulation of drug in the retinal pigmented layer. 130 518 ZNLTISPECIES COMPARISOIw-0~ CORIYEAL LESIONS PRODUCED DURING A 2-WEEK VAPOR EXPOSURE TO gROPYLEIQE GLYCOL MOHOPROPYL ETHER (PGPE). D R Klonne, D& Dodd, B Ballantvne, and P E Losco. Busby Rua Research Center/Union Carbide Corp., Export, PA. A previous 2-wk study in male and female F-344 rats indicated the coraea to be the primary;. target organ from exposure to PGPE vapor. Tkg, present study evaluated the potential ocular.__ ;i toxicity in male Spraguai3ley rats (SDR), F-344 rats (FR), Hartley guinea pigs (HGPI"f and New Zealand white-iabbits (PZR). Eaposures'irexe 6 hr/d, for 9 days during a 2-wk period to meaa concentrations of 0, 105, 486, or 1824 ppm PGPE. At 1824 ppm ocular irritation, ataxia, and narcosis occurred in all animals except HGP; 3 NZR died; FR had oasriitfes, necrosis, and vascularization of the cornea, with stromal mineralization, splitting, and fibrosis; SDR had corneal opacities and keratitis; 16ZR had opacities (transient), conjunctivitis, keratitis, and corneal degeneration; HGP had no lesions. Similar ocular lesions were observed in FR, SDR, and NZR at the lower PGPE concentrations, but at a lesser incidence. Only lesions (mineralization, vascularization, stromal splitting and fibrosis) found in the FR were considered to be nonreversible. The results of this study indicate that the FR is the most sensitive species with.regard to eye lesions produced by exposure to PGPE. 519 OCULAR TOXICITY OF SYSTEMIC EXPOSURE TO BUTYL 2- CHLULtU6THYL SULFIDE (BS). G J Klain, S T Schuschereba, L M McKinney, and S T Omaye, Letterman Army Inst. Res., San Francisco, CA. We have recently reported that a portion of butyl 2-chloroethyl sulfide (BS), a potent vesicant and a monofunctional analog of bis-2-chloroethyl sui- fide (sulfur mustard) which comes in contact with skin remains free to disperse through various tissues and organs, including the retina and lens of the eye. Because of the potential for occupa- tional~xposure and our concern to establish an effective antidote, we studied metabolic and his- tologic changes in the eyes of rats systemically exposed to BS. Young rats were injected (sc) with 20 ul of neat BS. At various times (24 and 48 hrs) after injection, rats were killed, eyes were'excised, dissected, and prepared for his- tologic and biochemical evaluation. Treated rats had multiple foci of dark, angulated, and vesicu- lated cells in the ganglion cell layer of the retina.. Photoreceptors had an increased number of autophagosomey and disarrayed outer segments. Endothelial cells of. the optic nerve and choroi- dal vessels were elevated from the intima and aggregates of activated platelets were present in the lumens. There was an increase in lipid peroxidation during the first hr which returned to values for untreated rats at 4 hr after injection. The results of this and our previous study show-that systemic administration and perhaps cutaneous penetration of BS, produce metabolic and ultrastructural changes in the eye. 50875 8255
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448 BLOOD CLEARANCE OF CYCLOPIAZONIC ACID IN MALE BROILER CHICKENS. M E Wilson, W M Hagler, Jr., J M Cullen, and R J Cole. Department of Poultry Science, North Carolina State University', Raleigh, NC. sponsor: W A Donaldson. Cyclopiazoni-c• acid (CPA), an indole tetramic acid;- is produced by several Aspergillus and Penicillium species and co-occurs with aflatoxin B1 in feeds and foods. The toxicity of CPA 3aay be due to its chelating activity; the' compound is found unchanged in muscle tissue. Two 28 wk old male broiler chickens were anesthetized and a cannula was placed in the hepatic portal vein. Cyclopiazonic acid, 167 ug/ml (2.5 mg/kg BW), was infused at a rate of 333 ul/min for 90 min. Blood was taken at time 0 and every 15 min for 2 hr. Serum was extracted and CPA quantified by HPLC. After the first 15 min of infusion, an average of 96.1% of the CPA had been distributed to organs, tissues, bile, and urine. During the 90 min infusion period, the average concentration of CPA in serum was 589 ng/ml; more than 75% of the residual CPA was removed from plasma 30 min after termination of infusion. The rapid accumulation of CPA in tissues, particularly muscle, may play a role in the mechanism of toxicity. 449 SPECIES-SPECIFIC OPTIC NEUROPATHY BY M_E'PHYLTHIO- ACETATE IN RABBITS. D W Rosenberg, C M Cisson, Z A Wong. Chevron Environmental Health Center, Inc., Richmond, CA. Methylthioacetate (MTA) has been found to produce optic neuropathy in rabbits but not in rats or monkeys after acute exposure. This effect may be due to species differences in the meta- bolism of MTA. An in vitro metabolism study was conducted to determine the relative esteroly- tic activity of liver, kidney and plasma on MTA in the rat and rabbit. In both species, liver microsomes had higher levels of MTA thioesterase activity than in any other tissue tested. At an MTA concentration of 15 mM, thioesterase activity in rabbit liver was 3 to 4 times greater than in the rat. Maximum thioesterase activity was observed with MTA concentrations between 5 and 10 mM in the rat, whereas in the rabbit, activity still increased at the highest concentration of MTA examined (25 mM). The Km for rat liver microsomes was 2.25 mM, with a corresponding Vmax of 74 umoles product/min/mg protein. In contrast, the rabbit had a Km of 13.4 mM and a Vmax of 343.6 umoles product/min/mg protein. The higher MTA thioesterase activity in rabbit tissues, especially in the liver, may be a significant factor in the toxicity of MTA in the optic nerve of this species. 113 450 CHANGES IN MORPHINE'-~'I~ARMACOHINETICS APTER ETHANOL IlVDUCTION OF TJDR GLUCURONYLTRANSFERASES. S Narayan, D T Kuntz, and Yos. Department of Pharmacology and Toxicology, University of Utah, Salt Lake City, UT. Chronic ethanol treatment (10% in drinking water for 2 weeks) has been shown to increase hepatic microsomal morphine glucuronidation rates. To correlate in vitrol work with in vivo disposition of morphine, male NevJ` .,,Zealand white rabbits were dcrftd-wrth 15 mg/kg ip , morphine sulfate, treated for 2 weeks with ethanol,4t4 - dosed again with 15 mg/kg ip morphine sulfate„t.. Arterial blood samples were collected from the rabbrt' ears from 5 min to 6 hr after dosing. HPLC analyses of plasma samples demonstrated a reduction in the area under the plasma concentration curve (AUC) for morphine and a concomitant ingiase$"an the AUC of m0rphine 3-glucuronide after eth8.no1 treatment. These results demonstrate that ethanol induction of hepatic UDP-glucuronyltransferases may be responsible for changes in xenobiotic disposition, which may result in altered pharmacoldnetics. Supported by USPHS Grant AA06555. GSY is a USPHS Research Career Development Awardee (HL02119) 451 induced isozyme compared to the control iso activitv was two-fold. The large increase in cat SUBSTRATE SELECTIVITY Or PURIFIED i~DP-GLUCURONYL- ETHANOL-INDUCED TRANSFERASE FROM RABBIT HEPATIC MICROSOMES. . R M Hutabaravand _Q JS Yost. Department of Pharmacology and Toxicology, Umversity of Utah, Salt Lake City, UT. We have previously shown that chronic ethanol consumption results - m induction of microsomal UDP- Oucuronyltransferase (UDP-GT) activities. We have isolated and purified an ethanol-induced UDP-GT isozyme t~ homogeneity as determined by electrophor~sis: The ethanol-induced isozyme tryptic digest HPLC profile was compared to the tryptic digest HPLC profile from a similar isozyme from untreated animals and they were found to be different. The etnanol-i_. nuced ee e form does not appear to have high substr-i:: spe city The highestsp ecific activity for this isczyme was found with the GT1 substrate naphthol ,5'~8.92 nM/min mg protein witH a Km of 42.6_ ukl). The specific activity for the GT~ substrate morphiu:•~ -aas 10.26 nM/min mg protein with a Km o: 108.73 uN i. The steroid substrates, estrone and testoster-one had specific activities of 3.22 nM/min mg protein and 0.46 nM/mm mg protein, respectively. The increase in Vmax/Km (naphthol) for the 'ethanol- efficieacy of the ethanol-induced isozyme for naphthol, coupled with only small increases in turnover numbers for other substrates, demonstrates a partial selectivity of this enzyme form for GT -type substrates. Supported by USPHS Grant AP.0~555. GSY is a USPHS Research Career Development Awardee (HL.02119) 50875 8238
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INHALATION TOXICITY OF 3-TRIFLUOROMETHYL PYRIDINE (3-FMP). P M Hext, B A Gaskell and G H Pigott, ICI Central Tox. Lab. Macclesfield, UK. Sponsor: E A Lock~ 3-FMP is a sewi-v%latile by-product from the synthesis of a pr_ocess intermediate. Single or multiple exposure,Q,f rats to 3-FMP vapour resulted in lesions in the nasal olfactory epithelium and liver. The pathogenesis and progression of both lesions were followed in a series of experiments ranging in duratl,on from a single 15 min exposure to 13 weeks exposure for 6 hours/day. The olfactory lesion, a focal necrosis/desquamation of the epithelium, was evident within 24 hours of a single 6hr exposure to 3-FMP at lppm or higher. The liver lesion, typically a hydropic degeneration of hepatocytes, was seen one day after a similar exposure to 50ppm. Recovery of both lesions was evident even with continuedexposure. Additional studies investigated the toxicity of 3-FMP in the mouse, rabbit and monkey. There was considerable interspecies variation in the severity of the lesions seen, particularly with respect to the nasal lesion. The cynomolgus monkey was the least affected with no nasal lesion evident following exposure to 50ppm 3-FMP for 10 days. Effects on bodyweight gain, food consumption, clinical signs and blood biochemistry occurred in one or more species but are considered to be secondary to those in the target organs. THE INFLUENCE OF HYPOXIA ON THE ACUTE TOXICITY OF HA7#ON 1211 FIRE RETARDANT. S Ugwu, J Thilsted and B R Smith. The College of Pharmacy, IIniversity of New Pfexico, Albuq- uerque, NM. *Veterinary Diagnostic Services, New Mexico Dept. of Agric. Albuquerque, NA€. To determine the influence of tissue hypoxia on the acute toxicity of Halon 1211, groups of ten Fisher 344 rats were subjected for 4 hours to one of the following exposure conditionss 1) 15% Halon and 21% O D 2) 15% Halon and 12% 02 3) 12% O2 w~o Halon. Unexposed rats were used as colony controls. Aspartyl aminotransferase (AST), alkaline phosphatase(ALP),blood urea nitrogen (BUN) and serum c=eatinine (Crt) were measured on all rats. AST and ALP levels in group 2 (15% Halon and 12% 02) were increased significantly when compared to control groups. The BUN and Crt levels remained unchanged in treatment and control groups. Histopathological examination showed moderate to marked centrilobular vacuolar degeneration and necrosis in the livers of all group 2 rats (15% Halon 1211 + 12% O2) This lesion was not observed in rats in the other three groups. In another experiment, groups of six Fisher 344 rats were exposed for 4 hours to the same exposure conditions as described above and immediately after the exposure the livers were examined for the formation of thiobarbituric acid (TBA) reactive product. TBA reactive product formation in group 1 or 2 was not significantly different from controls. Our data suggest that hypoxia enh- ces the toxicity of Halon 1211 but the mecha- nism does not involve lipid peroxidation. 609 610 THE GENERATION AND CONTROL OF-VfNYLIDENE FLUORIDE, A FL-AMMABLE GAS, FOR INHALATION TOXICOLOGY. 13 K Craigl, and W C EASTM, Jr., Litton Bionetics, Inc., Rockville, MD and NIEHS., Research Triangle Park, NC. Vinylidene fluoride (VF2) is a flammable gas, nominated for carcinogenicity testing because of its large production volume, possible occupa- -w tional exposure, and chemical relationship to ,~. known carcinogens. The high-4p -gsure level of 6.W,000 ppm for rodent inhalation studies was ~. very close to the lower flammable limit (LFL~"`'~- for VF2 of 55,000 ppm.' 1')Serefore, the inhalation- _'_1~__ exposure room was modified to comply with the ~ National Electrical Code for Class I, Divis.ion I, Group D atmospheres, all metal parts of the inhalation system were grounded tg.pceuent static, discharge, the chambers were mod~l`fied~to include explosion relief panels, and all possible ignition sources were removed from VF2 areas. A specially designed safety control system that closely monitored VF2 pressures.and -flows and chamber air flows was incorporated in the Inhalation dosing system. The VF2 delivery system auto- matically activated audible and visible alarms, and shut off the VF2 supply if pressure or flow increased, or air flow decreased, increasing VF2 concentration toward the LFL. Monitoring pressures, flows and VF2 concentrations was done remotely. This VF2 gas delivery system operated safely without incident and all con- centration parameters were well within acceptable limits. 'Now at Battelle Columbus, Columbus, OH. INTERACTION OF A_MPHIPHILIC DRUGS WITH PHOSPHO- LIPID VESICLES. U M Joshi, K S Prasada Rao, B Coudert, T M Dwyer and H M Mehendale. Depart- ment of Pharmacology and Toxicology, University of Mississippi Medical Center, Jackson, MS. Binding characteristics of several amphiphilic drugs to L-a-dipalmitoyl phosphatidylcholine (DPPC) was studied using fluorescence probes, 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-ani- lino-8-naphthalene sulfonate (ANS) for hydro- phobic and_ hydrophilic interactions, respec- tively. Di%g binding to DPPC was quantitated using Scatchard equations. The drugs used, showed varied binding capacities to DPPC with respect to the probes. The order of binding capacity using DPH was promazine(PZ)>chloram- phenicol(CRP)>amiodarone(AM)>trimipramine(TIP)> chlorpromazine(CPZ)>imipramine(IM)>propranolol (PP). Two binding affinities were calculated for some of the drugs. The order of the drug binding capacity using ANS was chlorphenter- mine(CP)>CRP>TIP>PMZ>PP. IM and CPZ showed intense fluorescence with ANS in the absence of DPPC. CP did not bind at the hydrophobic site of DPPC and AM did not bind to the hydrophilic site of DPPC. CRQ, did not bind to DPPC. These results suggest that induction of pulmo- nary phospholipidosis could be by either bind- ing of the drug to phospholipids or by inhibit- ing enzymatic break-down of phospholipids, or both, depending on the molecular charge of the drug. (Supported by HL-20622.) 153
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e or, S EFFECTS OF ERYTHROSIN B(EB) ON MYOCARDIAL MYOCYTE REAGGREGATE CULTURES (MMR) L K Earl, K Kesingland, C Holland & C K Atterwill, Smith Kline & French Research The Frythe, Welwyn, UK. Spoqsor: J B Hoo-k: i Erythrosin°B is an inhibitor of high affinity ouabain bindin'T to guinea pig cardiac membrane preparAtions and Na+/K+ATPase activity. The present studies set out to investigate the actions of EB on spontaneously beating MMR and comptre them with those of ouabain. Chick MMR we~e prepared from .10 day old embryos and superfused with salt solutions containing test compounds whilst being filmed under the microscope. Superfusion with ouabain (0.25µM-5uM) increased contraction amplitude. Ouabain also inhibited 86Rb+ accumulation (IC50 2.5µM). EB similarly increased contraction amplitude (O.IUM-10µM). and inhibited 86RB+ accumulation(IC50 approx 4uM). In contrast, EB (>lµM) caused unusual contraction patterns in MMR leading to eventual cessation of contraction which were not observed with ouabain toxicity. These results indicate that EB, like ouabain, is a positive inotrope and can inhibit Na+/K+ATPase in MMR. However the unusual contraction patterns observed.in EB toxicity suggest that Ouabain and EB may have different mechanisms of toxicity. $44 ELECTROCARDIOGRAPHIC EFFECTS OF INODILATOR FHOSPHODIESTERASE INHIBITORS IN BEAGLE DOGS. R J Evis, E C Joseph and T F Walker. SK&F Research Ltd., Welwyn, Herts, U.K. Sponsor: J B Hook. The effects on the electrocardiogram of administration of high doses of inodilator phosphodiesterase type III inhibitors, SK&F 94120 [5-(4-acetamidophenyl)pyrazin-2(iH)-one] and related compounds SK&F 95654, SK&F 94836 and SK&F 94418 were assessed in toxicity studies. In the do , i.v. administration of SK&F 94120 (1.5mg/kg~, SK&F 95654 (2mg/kg) and SK&F 94418 (2mg/kg) gave i'se to a transient increase in heart rate (HR) (maximum 290beats/min) and T wave amplitude, also a slight reduction in P-R interval (0.01- 0.03secs). SK&F 94836 did not cause these changes when given p.o. to dogs (up to 800mg/kg). T waves were observed infrequently followiAg exposure to SK&F 94418 (i.v.), SK&F 95654 (i.v.) and SK&F 94836 (p.o.). Ventricular extrasystoles were observed afterr administration of SK&F 94120 at 120mg/kg and SK&F 34418 at 4.,mg/kg. I.V. infusion of SK&F- 941?0 (15mg/kg) gave rise occasionally to atr.io.~entricular dissociation with accrochage. Papillary muscle necrosis was observed in dogs given SK&F 9412~, SK&F 95654 and SK&F 94418, but not SK&F 94836. These changes correlated with the incidence of tachycardia for these compounds and suggested that tachycardia may be a contributory factor in the pathogenesis of this lesion. <' rr 545 r1Y0CARDIAL TOXICITY OF CHL~O1INATED HYDROCARBONS IN DRINKING WATER. B F Nagy, J P Bercz and L W Condie. U.S. EPA, Health Effects Re- ' search Laboratory, Cincinnati, OH, f Sixteen organics in 0.2% glucose-Ringer solu- tion at 10-7 to 10-3 M concentrations were tested for acute toxicity on isolated frog, hearts (R. catesbiana). The heart was perfus -_' ed with glucose-Ringer solution at 25°C. The,;. force of the heartbeat wa.-measured as full;- isotonic contractions from aorta to apex by - longitudinal pull wit.ty a force transducer (Gra~~ FT03) and an externally applied 3g load. contractions were recorded with a Grass 7D _=i` polygraph. The maximum tension was compared in each titration for 5 or 10 minutes with con- trols. Acute toxicity was fo,~nd,~at up to 10~+ M with 1,2,3-trichloropropatt~, 'abetonitrile,: 2-chlorophenol, 2,4-dimethylphenok, 1,2,3-tri- chlorobenzene and 1,2-dichloroethylene causing reversible decrease in heartbeat tensions or heart stoppage. Some organics increased the force of contraction. Other benzene isomers had no effect up to 10-3 M. The compounds were also tested for interference with isolated sarcoplasmic reticular Ca2i' ATPase. No__effect was seen up to a 10-3 M. The ease of reversi- bility of effects indicated no interactions with either with the glycolytic or the oxidative ATP generating systems. (Abstract does not neces- sarily reflect EPA policy). 546 HYPOTENSIVE RESPONSE FROM ACUTE AND S_UBACUTE EXPOSURE TO DIPHENYLI000NIUM HEXAFLUOROARSENATE (PIFA). S L Yurasevecz, E A Emmett, I S Farrukh, T P Kennedy, R J Rubin and L W Smith. General Electric Company, Pittsfield, MA and Johns Hopkins University, Baltimore, MD. 137 PIFA has a high order of acute toxicity. The oral LD50 is 49 mg/kg in rats and < 25 mg/kg in dogs. Rapid and extensive gastric absorption is shown by comparison to the rat iv LD50 of 20 mg/kgA-The dermal LD50 is between 200 and 2000 mg/kg in rabbits and the inhalation LC50 is 1.75 mg/1 in rats. PIFA applied to the eye at 50 mg/kg causes lethality in rabbits. Structure-activity studies show that the diphenyliodonium cation is responsible for the toxic effects. These are expressed by an all-or-nothing reponse and exhibit a steep dose-response curve. Death is associated with respiratory failure and generalized engorgement of blood in the organs. Single iv doses in anesthetized dogs cause hypotension. Repeated oral doses in unanesthetized dogs cause fatty degeneration of cardiac muscle. Subacute oral studies in rats and hairless mice and dermal studies in rabbits and hairless mice confirm the high degree of toxicity. Effects include lethality, gastrointestinal inflammation, myocardial hypertrophy and renal tubule degeneration.
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ALTERATION OF F344 INFLUENZA-SPECIFIC CTL ACTIVITY FOLLOWIJG ACUTE _PHOSGEN5 I13HALATION. 7 P Ehrlich and G R Burleson. TNYU Med1~2Center. Institute of Environmental Medicine, Northrop Services, Ise.; Environmental Sciences, RTP, NC. ~ ~ phosgene (COC12)-ds;a highly toxic gas used in the synthesis of isocyanates. Its target organ of toxicity is the lung. The effects of phosgene on the in vivo generation and kinetics of the influenza virus- specific cytotoxic T lymphocyte (CTL) response were investigated. Male Fischer-344 rats wei~e. infected with influenza/Port Chalmers/l/73 (H3N2) virus via intranasal administration 24 hr following acute exposure to 1.0 ppm phosgene or clean filtered air for 4 hr. Single cell suspensions of lung cells were prepared by collagenase digestion of finely minced lung tissue. The lung cell suspensions were assayed for inf}uenza-virus specific CTL activity using an 18 hr Cr release assay: The pulmonary CTL response was detected 5 days post-infection and reached a peak response 10 days post-infection. Acute phosgene inhalation resulted . in the enhancement of the CTL response on day 7 post- infection and was followed by a suppression in activity by day 10 post-infection. The CTL response in phosgene exposed animals did not differ from those of air exposed infected animals at days 15 and 20 post-infection. This is an abstract of a proposed presentation and does not necessarily reflect EPA policy. P_HOSGENE-SUPPRESSED PULMONARY NATURAL KILLER ACTIVITY: STUDIES ON THE MECHANISM OF DbIMUNOSUPPRESSION. G R Burlesonl, L L Keyesl, M C Madden2 and M Friedman2• 1Northrop Services, Inc., Environmental Sciences, RTP, NC. 2University of North Carolina, Chapel Hill, NC. Natural killer (NK) activity is an important component of the nonspecific innate immunity that is important in defense against both neoplastic and viral diseases. Cells exerting NK activity also have important immunoregulatory responsibilities. Suppression of NK activity will result in an enhanced susceptibility to viral and neoplastic disease. Phosgene is a hazardous air toxic and a.potential occupational health hazard. Exposure to phosgene resulted in local pulmonary and systemic immune dysfunction manifested as suppressed NK activity.. Lavage fluid from phosgene-exposed animals had decreased levels of arachidonic acid metabolites. ' Removal of adherent cells restored the' suppressed NK activity. Pulmonary effector cells from phosgene- and air-` exposed animals were incubated for 18 hr with immunomodulators before assay for NK activity.' Poly (I):poly (C), gamma interferon, and interleukin-2 restored the phosgene-suppressed pulmonary NK activity. Therefore, suppressed pulmonary NK activity can be enhanced by potent immunomodulators. This is an abstract of a proposed presentation and does not necessarily reflect EPA policy. 151 601 EFFECTS OF INHALATION OF PHOSGENE bk BACTERIAL, VIRAL AND NEOPLASTIC DISEASE SUSCEPTIBILITY MODELS IN MICE. M J K Selarade, JW Illing, D M ~ Starnes, `and M J Daniels. U.S. Environmental Protection Agency, Research.-Triangle-Park, N.C., Inhaled compounds first contact the immune sys- tem in the lung (also a common site for infec- tious or neoplastic diseases). Usual methods for assessing immunotoxicity, using blood or spleen ° cells, cannot predict increased risk of these +~ pu,lmonary diseases due to exposwreto compounds like phosgene, which does not have significant.•s,,;.., . extrapulmonary circulat5on. Host resistance-_-~ -models may provide a rapid screen for assessing' this risk. Exposure of •ice to as little as 0.025 ppm phosgene for 4 hrs prior to aerosol exposure to a group C Streptococcus resulted in a 5-fold increase in % mortali!*"dttd:`,decreased" survival time compared to air cbntrq,ls. Results were not due to decreased macrophage phagocytic activity. Effects on bactericidal activity have not been ruled out. Also an,increase in lung tumors was observed in- mice injected with 816 melanoma tumors and exposed 4hr/day for 5 days, to phosgene at levels as low as 0.05 ppm. In contrast, 5 exposures to 0.5 ppm phosgene did not affect mortality or lung weight following influenza infection, or mortality or natural killer cell activity due to murine cytomegalo-s virus infection. Hence, phosgene enhanced bac- terial and neoplastic, but not viral diseases tested here. (This is an abstract of a proposed presentation and does not reflect EPA policy.) 602 Sli3CFiFi(JN I C EXPOSURE OF GU I IJEA P I GS TO A N74- I RR I TAT I Id3 OaVCENTRAT I OIV OF COTTON DUST . I Vyas, A Ogundiran, C Gatty, K Spear and M Karol. Dept. Ind. Env. Health ScI., Univ. Pgh.,. Pfttsburgh, PA. The guinea pig has acute and chronic responses to inhalation of cotton dust(c.d.) resenbling those reported for cotton workers. Effects of sub-chronic exposure to a non-irritating concentration of c.d.(1.6mg/m3) were examined by exposing 30 guinea pigs 6 hrs/day, 5 days/wk for 12 wks. Respirable particles were generated froro bulk C:'2i'. The sham-exposed group contained 20 animals. Daily measurenents included: weight, respiratory rate and breathing volune pre- and post-exposure In air and in 10% C02. Blood was collected monthly for blood chenistry, and antibody measurenent. After 12 wks groups were pre-treated with atropine (.015mg), anesthetized with tJenbutal and the lungs perfused with glutaraldehyde. Heart, liver, and kidneys were renoved for histology. The rate of.welght gain of the exposed group was decreased c:cmpared to controls. Five exposed animals had produced antlbodies. N!o differences were found In respiratory rate or volume. These results lndicate chronic changes due to exposure to a low c.d. concentration and may have Implications for establishing a safe workplace exposure level which Is currently based on acute changes In pulmonary function. Supported under IISDA Cooperative Agreenent 58-434K-0050. 50875 8276
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COMPARISON OF DOSE BY MASS AND NUMBER OF MINERAL TIgERS IN THE INDUCTION OF MESOTHELIOMA IN RATS. 5L C ffanii L D Palekar,2 P M Cook,3 and tea 1U S Environmental-Protection Agency, RTP,NC., 2No$throp Services, Inc., gTp,NC and 3li5S 6vironmental Protection Agency, Duluth, MN:=. Human studies suggest that mineral fibers differ in their potency to induce mesotheliomas. Erionite, crocidolite, chrysotile and attapul- gite were injected into the pleural cAtties of male Fischer 344 rats. The rats were necropsied upon their death. The survival time and the incidence of tumors was determined for each group and compared. The administered dose was calculated according to both mass and number of minerals of various categories of length and widths. The results indicated; 1) there was an inverse correlation between the survival time and tumor incidence; 2) the average survival time of erionite treated animals was lower than that of asbestos treated animals; 3) the average tumor incidence was higher in the erionite treated animals than in those treated with asbestos. The tumorigenic potencies per mass dose ranked in the decreasing order for erionite, chrysotile, crocidolite and attapul- gite. When the dose was computed according to the number of fibers, the ranking was erionite, crocidolite, chrysotile and attapulgite. This iatter ranking correlates with that noted in human exposure for erionite and asbestos. A 90-DAY STUDY OF PETROLEUM MIDDLE DISTILLATE- INDUCED DERMAL IRRITATION. J J Freeman, R D Phillips, R H McKee, R T Plutnick and R A Scala, Exxon Biomedical Sciences,-East Millstone, NJ Some petroleum middle distillates (PMDs) elicit skin tumors in mouse skin painting studies. - This tumorigenic activity is not always explained,by , polycyclic aromatic hydrocarbon (PAH) content: . many PMDs contain low concentrations of PAHs.,-_,, . However, the dermal irritation elicited by PMDs` may play a role in tumor formation. This,study was conducted to study the patterns of dermal- irritation elicited by PMDs of varying aromatic content: a steam cracked gas oil (SCGO), a* mineral seal oil (MSO) and a Jet E~Zel (JF), (aro- matic contents: 95%, 11%, 21%). Male C3H mice (25/group) were treated topically (37.5 ul 2x/ week for 13 weeks) with 10%, 50% or 100% (undi- luted) concentrations of each PMD. The vehicle was a 90 SUS mineral oil. Dermal,changes were evaluated by gross observations and light micros- copy. The vehicle and the 10% PMDs were essen- tially nonirritating. The 50% PMDs elicited slight to moderate proliferative and inflammatory changes. 100% SCGO caused evidence of necrosis on Days 1-7, but not later in the study. 100% MS0 did not cause necrosis but with time induced inflammatory and marked proliferative changes. The effects of 100% JF were similar to 100% MS0 but less severe. Thus the SCGO elicited a differ- ent pattern of dermal effects than MSO or JF. The possible relationships of these dermal changes to epidermal carcinogenesis are under study. 641 ...~,_ . ,~-~~=" COMPARATIVE EFFECTS OF TWO"~MOUSE SKIN TUMOR INITIATORS IN.THREE MOUSE STRAINS. W C Eastin Jr., M R, He'tmancik, G E Wilkinson, and A C Peters. NI HS, Research-.Triangle fark, NC,• Battelle Columbus Division, Columbus, OH. Two-stage mouse skin initiation/promotion stu- dies are routinely used to identify chemicals with initiating and/or promoting properties. ~ For these tests, it is important to select a ;~ mouse strain to minimize totay udy time and ~~ tor maximize the probability identifying a_A promoter. In the curren.t studies, the sensi= ~ tivities of both sexes &-Sencar, Swiss (CD-1)-__'~~~-.: and B6C3F1 mice strains to different combina- ~ tions of known initiators (DMBA and MNNG) and promoters (TPA and benzoyl peroxide) were com- pared using the two-stage mouse Ikin,,-protocol. One week after initiation with DRBA 0:25, 2.5, 25, or 50 }ig) or MNNG (100, 500 o~ 1000 jig), promotion was begun with TPA (5 or 1}ig) or benzoyl peroxide (20 mg) and continued for up to 52 weeks. Gross observations of tumor appearance and number were recorded weekly. Results indicate that males respond similar to females of the same strain, that all three strains respond to a low dose of initiator given repeatedly, that there is an initiator dose response; and that there is a difference between strains in mean time to first tumor appearance. These data suggest that the sen- sitivity of the three strains in the two-stage mouse skin protocol is: Sencar > Swiss (CD-1) > B6C3F1. 642 DERMAL INITIATION/PROMOTION STUDY OF o_-BENZYL-p- CHLOROPHENOL (BCP). IN SWISS CD-1 MICE. M He t- mancik, M Ryarr, A C Peters, *W C Eastin, an L S B9rnbaum. Battelle Columbus Division, Columbus, OH and *NIEHS, Research Triangle Park, NC.. o-Benzyl-p-chlorophenol (CAS No. 120-32-1) is a biocide which is used as.a phenolic disinfectant with a high potential for human exposure. The chemical was tested for activity as an initiator (with TPA promotion), a promoter (after DMBA initiation) : and as a complete carcinogen using a mouse skiri~Wo-stage protocol.'Positive control groups that received DMBA only or DMBA/TPA and negative control groups were included for com- parison with test groups.' All doses were applied to the back in acetone in a volume of 100 micro- liters. Initiator doses were administered once during the first week and promoter doses were applied three times weekly for 51 weeks there- after. BCP was administered once at a dose of 10 mg (for initiation) or three times weekly at doses of 0.1, 1, or 3 mg. The location and di- mensions of tumors were recorded weekly. The survival rate of mice was depressed in the posi- tive control groups that developed tumors. BCP showed little activity as an.initiator. A dose- related increase occurred when BCP was adminis- tered as a promoter, and the time required for the first appearance of a tumor was inversely related to dose. The incidence of tumors produced by BCP was similar in male and female mice. Thus, BCP appears to be a promoter of mouse skin tumorigenesis. (Supported by Contract No. NO1-ES-45042 from NTP). 161 50875 8286
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635 ALTERED IN VITRO GROWTH CHARACTERISTICS OF 637 CANINE TRACHEAL EPITHELIAL CELLS FOLLOWING EXPOSURE TO N_-METHYL-N'-NITRO-N'-NITROSO- GUANIDINE (MNNG). - AF Hubba, FF Hahn, and DG Thomassen. Lovilace Inhalation Toxicology Research Iassti'6ate,Albuquerque, NM and Colorado State University:, Fort Collins, CO. Sponsor: J M. Benson -.;.:_ were switched to a serum-containing medium. In this medium, most cells ceased proliferation and underwent squamous differentiation. A fraction of carcinogen-exposed cultures con- To investigate the stepwise development of neoplasia in a higher mammalian species, canine tracheal epithelial cells have bee4.cultured in serum-free medium and exposed to the direct- acting carcinogen, MNNG. Up to 6% of the normal canine tracheal cells plated in serum- free medium formed colonies of 30 or more cells. Following exposure to MNNG, cultures tained colonies of proliferative cells which approached confluence 3 weeks after exposure. Cells isolated from these colonies were capable of repeated passage, in vitro, showed a decreased tendency to differentiate in culture, and were shown by electron microscopy to be composed of epithelial cells. These results indicate that altered growth patterns can be identified in vitro in canine epithelial cells following exposure to MNNG. (Research sup- ported by the U.S. Department of Energy's Office of Health and Environmental Research, under Contract No. DE-AC04-76EV010013.) 636. EVALUATION OF UNSCHEDULED DNA SYNTHESIS (UDS) AND S-PHASE SYNTHESIS (SPS) FOL- " LOWING TREATMENT WITH p-DICHLOROBENZENE KL Steinmetz, JP Bakke, GM Hamilton', -, KC Pardo, M Ramsey, and JC Mirsalis.. SRI International, Menlo Park, CA Sponsor: CE Green .. p-Dichlorobenzene (PDCB) was found. to be carcinogenic.in mouse Iiver•and male rat_.. kidney in_two-year gavage studies con- ' ducted by the NTP.,PDCB (> 97% pure.y Nias administered in corn-.oil via.gavage to ; B6C3F1 mice 16 and 48 hr prior,to sacri= fice and to Fischer-344 rats 16 and 96 hrr prior to sacrifice. UDS and SPS were - quantitated by autoradiography in mouse`L hepatocytes and rat kidney cells . All dose levels of PDCB and the negative control yielded <0 net grains/nucleus (NG). Positive control groups yielded >15 NG (liver) and > 4 NG (kidney). All con- trol groups yielded < 0.52 percent of cells in S-phase (%S). Treatment of mice with 300, 600, or 1000 mg/kg PDCB yielded 0.46, 1.90, and 1.52 %S (males) and 2.61, . 1.18, and 4.45%S (females) in hepatocytes. The same doses yielded 0.87, 0.67, and' 1.01 %S (males) and 0.48, 0.43, 0.32 %S (females) in rat kidney.. These results indicate that PDCB is not genotoxic in the mouse liver or rat kidney following single administration of doses.comparable to the daily doses given in the NTP bioassay. Increases in SPS may indicate a possible mechanism of tumor induction. Sponsored by the Chlorobenzene Program Panel, Chemical Manufacturers Association, Washington, DC 20037 638 160 COMPARISON OF ETHYLENE OXIDE AND 4-AMINO- BIPHENYL HEMOGLOBIN ADDUCTS IN CIGARETTE SMOKERS. L Latriano, R Perera, D Brenner, A M Jeffrey, Columbia Univ., Inst.' Cancer Res./ Env. Sci. Div., New York, NY. The hemoglobin-(Hb) adducts of ethylene oxide (EO) or 4-aminobiphenyl (ABP) have been used as - markers of cigarette smoking. In the present - a~- studies the level of EO-Hb adducts in smokers . fand non-smokers was determi.i~"~'n blood samples ~• which had been previously analyzed for ABP."*7'` Tannenbaum et al. .(Csn. Res. 47:602, 1987).~-~-; Determination of EO-Hb adducts utilized the ~ modified Edman degradation procedure in which the N-terminal valine of Hb is derivitized with pentafluoroisothiocyanate. The resulting N-2 hydroxyethylvaline derivativffli.s"Nanalyzed by gas chromatography/mass spectrometry. For both compounds, Hb adduct levels for smokers were significantly higher than for nonsmokers; there was an approximate 5 and 10 fold increase for -ABP-HB and EO-Hb adducts respeci:ively. Although neither E0 or ABP Hb adduct levers correlated well with the number . of cigarettes smoked per day, there was a good correlation (R=0.86) between the Hb adduct levels of these two compounds. Studies to determine whether either of these two adducts can be correlated with an increased risk of lung cancer are underway. Supported by NCI Grant No. 5U01CA43013 and NIEHS 5P01ES03881. CHRONIC TOXICITY AND ONCOGENICITY STUDY OF 2,6-DIETHYLANILINE. T G Pullin., R W Naismith, J F Hardisty, E B Whorton Jr, G L Ter Haar. Ethyl Corporation, Baton Rouge, LA. Diethylaniline (DEA) is used in the production of chloroacetanilides which are important herbicides used for corn, soybeans, and other crops. 'It is a mammalian metabolite.for the chloroacetanilide herbicide, alachlor: This . study was conducted to investigate the potential chronic tiM;tcity/oncogenicity of DEA. The test " material was administered in the diet ad libitum at 0.02, 0.16 or 0.32% to groups: of 80 Sprague Dawley rats of each sex for 104 weeks. The vehicle control group, consisting of 100 rats per sex, received diet alone. DEA had no effect on the survival of the rats. There were no consistently significant alterations in food consumption, hematologic or blood chemistry values or organ weight data. Body weights, male and female, were consistently reduced over each 6 month interval. Histopathological examination. of tissues showed no increased incidence of neoplasms. An increase in the incidence and relative degree of severity of eosinophilic foci in the liver was present in the male rats receiving 0.16% and 0.32% DEA. Eosinophilic foci in livers of males receiving 0.02% DEA were similar to controls. No treatment related changes were observed in the female rats. This study demonstrated no evidence of a chronic toxicity or oncogenic effect of DEA in rats. 50875 8285
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603 HIS"LCkKlRPHOME:PRIC fiXAMINATION OF GUINEA PIGS E}0?OBED SUBC:HIdONICALLY TO A NON-IRRITATING CONCIIJTRATION OF COrPPON DUSP. B Cockrell, C Rehfeld, C Gatty, and M Karol. EPL, Inc. Herndon, VA and Dept. --Rbd. Env. Hlth. Sci., Univ. Pittsburgh, Pittsburgh, PA. ..,_ ft ~d The effect,* _of subchronic exposure to an acutely non -4rritating concentration of cotton dust (c.d.) were examined using histopatholo- gic, electron microscopic and morphometric techniques. Examination of the lungs, trachea, major bronchi and small brorrchioles, nasal cavity, and peribronchial lymph nodes from guinea pigs exposed by inhalation to c.d. at 1.6 mg/m3 for 12 weeks failed to reveal sig- nificant morphologic changes when evaluated with H & E stained slides. However, when tissues from the deep lungs were, examined by transmission electron microscopy, differences in the lungs of CD-exposed guinea pigs when compared to controls were 1) increased numbers of alveolar macrophages, many with phagocytized particles; 2) increased numbers of type II cells, particularly around the end airways; 3) recruitment of leukocytes; 4) focal thickening of the alveolar wall caused by deposition of collagen fibers and infiltration of leukocytes. Morphometric analysis revealed slight thicken- ing of the bronchiolar smooth muscle layer. Numbers of alveolar macrophages were increased 40% and type II cells increased 10% over the controls. Supported by USDA Coop. Agrmt. # 43YK-5-0050. 1 604 TIME COURSE OF TOXIC PULMONARY EFFECTS FOLLOWING INHALATION OF TEFLON PYROLYSIS PRODUCTS. DB War- heit, WC Seidel, JR Bamberger, and MA Hartsky. Du Pont-Haskell Lab., Newark, DE. Thermal decomposition products of certain per- fluorinated polymers are toxic to experimental animals in small-scale combustion toxicity tests. Our studies were designed to investigate the time course of pulmonary effects in rats after a 30 min exposure to Teflon (FEP-100) decomp. products. FEP was combusted in a quartz beaker at 560°C (0.03 mg/1). Five groups of rats were exposed un- der varying conditions. Groups A & B were exposed to FEP smoke during 1-15 and 15-30 min..intervals, respectively. Group C was exposed toolFE for the entire 30 min period, while group Dd~~iaexposed to a filtered (particle-free) atmosphere of FEP for 30 min. Group E was sham-exposed to room air. Lung wet wts. were recorded and cells and fluids recovered by lavage were assessed for toxicity. Our results showed that lung wts., inflamma- tion, hemorrhage, alk. phosphatase, B-gluc, LDH, protein, and angiotensin convert. enzyme levels were significantly elevated in groups B & C com- pared to group D (filtered) and group E (sham). Group A rats were intermediate for all parameters. The data suggest that the lung injury observed in groups B & C occurs during the later phase of ex- posure (i.e., 15-30 min segment of exposure), and that animals exposed to a.filtered atmosphere were protected. Secondary decomposition products following pyrolysis of FEP may be responsible for the observed lung toxicity. 152 605 INHALATION TOXICITY Olk:'FLUOROPOLYMER/WOOD SMOKES IN A FULL-SCALE FIRE. R Valentine, B B Baker, J K Bonesteel, D J Kasprzak; F B Clarkel; and C B Herpol and M Jannssep,s2. E.I.adu Pont de, Nemours & Co., Wilmington, DE; Benjamin/Clarke Associates Kensington, MD1; and State University of Khent, Belgium2. Full-scale assessment of the smoke toxicity re- f 606 sulting from the combustion of Teflon® FEP iaF-a wood fire was studied. _?,Vu were started iii-a 40 m3 burn room; the smoke was extracted gum ' the end of a 13 m.c,orridor and conducted"to"the animal exposure chambers. Groups of 10 Cr1eCD~+ rats were exposed nose-only for 30 minutes to ~ the smokes formed from burning 110 kg of wood with 20-30 kg of Teflon® FEP cable. Smoke sam- ples were monitored for hyd~ oiy~able fluoride (FH), CO, C02, and fluoroc'aYbon gases. Blood was obtained from 4 rats immedtately after ex- posure for carboxyhemoglobin (COHb) analysis. Tetrafluoroethylene, hexafluoropropylene, per- fluorocyclobutane or perfluoroisobutylene were not present in lethal concentrations. Time integrated CO concentrations ranged from 70,000 to 110,000 ppm-min; corresponding COHb concen- trations ranged from 54-64%; some animal deaths were attributed to CO exposure. FH concentra- tions ranged from 7000 to 35,000 ppm-min and were,better correlated with animal deaths. The . data suggested that the toxic components present in the smoke include CO and fluorochemical(s) with a toxicity similar to carbonyl fluoride; the presence of unusually toxic substances was not experimentally confirmed. D_IMETHYLETHAYtOLAMIINE (DMEA) : ACUTE, 2-WEEK, AHD 13-WEEK,I_NHALATION TOXICITY STUDIES IN RATS. E H Fowler, D R Klonne, D E Dodd, I M Pritts, C M Troup, D J Nachreiner, and B Ballantyne. Bushy Run Res Ctr/Union Carbide Corp, Export, PA The acute and subchronic inhalation tosicity of DMEA vapor was evaluated. The 4-hr LC50 value (95% confidence limits) for Wistar rats was 1641 (862-3125) ppm with signs of nasal and ocular irritation, respiratory distress, and decreased body weight. F-344 rats exposed to 98, 288j~'A'or 586 ppm DMEA for 9 days (6 hr/d during an 11-day period) had signs of respira- tory and ocular irritation (except at 98 ppm) and body weight loss, with 100X (586 ppm) and 16% (288 ppm) mortality. Microscopic lesions (only 98 and 288 ppm groups evaluated) occurred .in ocular and nasal tissues. Subsequently, F-344 rats exposed to 0, 8, 24, or 76 ppm DMEA for 6 h/d, 5 d/wk for 13-wk, with a portion of each group held for a 5-wk recovery period, had transient ocular opacity (24 and 76 ppm), decreased body weight gain (76 ppm), lesions of .the respiratory and olfactory epithelium within the anterior nasal cavity (76 ppm), and.lesions of the eye (76 ppm females). Recovery rats had nasal tissue lesions which were decreased in incidence and severity. Thus, DMEA is an ocular and upper respiratory tract irritant and toxicant in rats at vapor concentrations of 76 ppm, while 24 ppm or less produced no biologically significant toxicity. 50875 8277
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675 FORMATION AND PERSISTENCE OF D_NA ADDUCTS IN RAT HEPATIC TISSUE FOLLOWING PRETREATMENT WITH 3,3'-DICHLOROBENZIDINE. C S Nessel and MM Iba, Joint Graduate Program in Toxicology, Rutgers University,- Piscataw_a_y, NJ. 3,3'-Dichlorobenzidine (DCB) is hepatocarcino- genic genic in Averal species, including mice, rats, and dogs. In"oeder to assess the potential of DCB to form DKA'' adducts i n vi vo, rats were administered 7.8 umol ~14C-DCB (specific activity = 0.32 mCi/mmol) ip and sacrificed 3 or 14 days following treatment. „~FHepatic DNA was isolated by precipitation and solvent extraction and subjected to enzymatic hydrolysis. Modified nucleosides in the hydrolysate were separated from unbound nucleosides by Sephadex LH-20 chromatography. Chromatographic, radiometric, and spectrophoto- metric analysis indicated. that several persistent 14C-DCB adducts are formed in hepatic DNA following administration of this compound. The binding (molecules of DCB : nucleosides) was 1:19000 and 1:70000 at 3 and 14 days, respectively. Two of the adducts formed in vivo co-chromatographed with deoxyguanosine (dG) adducts formed in NADPH-supplemented rat hepatic microsomal incubations containing 14C-DCB and dG. These results suggest that DCB is bioactivated to reactive intermediates capable of forming persistent adducts with DNA, two of which may be deoxyguanosine-derived. (Supported by US EPA R-812459) ! 676 PREPARATION OF DNA ADDUCTS FOR CHEMICAL CHARACTERIZATION STUDIES, USING ISOLATED RAT HEPATOCYTES. D A Dankovic, D L S rin er, D.B. Mann, B L Thomas, and R M Bean. Pac ic Northwest Laboratory, Dept. of Biology and. Chemistry, Richland, WA 99352. Preparation of carcinogen adducts to DNA in quantities sufficient for chemical character- ization (ie, Ag) is frequently difficult. Bio- synthetic methods are often employed, using rat liver microsomes plus calf-thymus DNA (CT-DNA). However, it has previously been shown that the HPLC profile of benzo[a]pyrene (BAP) adducts ob- tained in this way differs substantially from that observed in vivo. In contrast, the adduct profile obtained rom isolated rat hepatocytes is very similar to that seen in vivo. It has also been previously shown tf~at BAP adducts are formed with CT-DNA added to the hepatocyte in- cubation medium. We now report that the HPLC profiles of BAP-DNA adducts prepared using iso- lated rat hepatocytes plus exogenous CT-DNA are essentially identical to the profiles of mouse skin adducts; in both cases the predominant peak is the BAP-anti-diol-epoxide adduct of deoxy- ° guanosine. An experiment using 60 ml of cell suspension and 180 µM BAP, incubated for two hours with 54 mg of CT-DNA, yielded 1.58 µg of BAP adducted to 28 mg of DNA. The binding level was 217 pmol BAP bound/mg DNA. For comparison, a typical result using mouse skin in vivo is approx. 20 pmol BAP bound per mg DN-A, yielding 2 - 5 ng of adduct/mouse. Supported.by U.S. Department of Energy Contract DE-AC06-76RL0 1830. 677 CHARACTERIZATION OF NON fLASSICAL BaP ADDUCTS TO DNA. D L Springer, B'L Thomas, D A Dan&ovic, D B Mann, E K Chess and R M Bean. Battelle, Pacific Northwest Laboratory, Richland, WA ~ f 678 170 Tumor initiation by BaP and other PAH corre- lates with covalent binding of the diol- epoxide to the exocylic nitrogen group of deoxyguanosine. Classically, these adducts have been studied by isolation of DNA from mouse skin about one-day aTt"reatment with • Using this procedure, about 50% of the radio- activity associated with the DNA elutes as classical adducts, which are derived from reactions with BaP-diol-epoxj6s•._14'he other 50% is more polar and elutes frowthe LH-20 column near the solvent front (non-classical fraction). When treated with acid, the polar material releases radiolabe.led compounds hav- ing reverse phase HPLC retention times co- incident with BaP-quinones. Similar treatment of the classical fraction produces BaP-tetrols characteristic of adducts formed from BaP- diol-epoxides. Hydrolysis of intact adducted DNA yields both quinones and tetrols. The presence of quinone precursors in the non- classical fraction suggests a radical cation mechanism for their formation and represents a previously uncharacterized class of DNA adducts. Supported by U.S. Department of Energy Contract DE-AC06-76RL0 1830. normal nucleosides by LH-20 chromatography. ~ nucleosides and sepaiAtion of adducted from radiolabeled BaP, enzymatic hydrolysis to IN VIVO INDUCTION OF DNA-PROTEIN CROSSLINKS IN RAT TRACHEAL IMPLANTS EXPOSED TO FORMALDEHYDE (HCHO) AND BENZO(A)PYRENE (BAP), G N Cosma, A S Wilhite, and A C Marchok, Biology Division, , Oak Ridge National Laboratory, Oak Ridge, TN. Sponsor: M Costa. HCHO and BAP are ubiquitous atmospheric pollu- tants with known or suspect carcinogenicity in ._ animals and humans. To complement our study of their initiation of carcinogenesis in rat tra- cheal implants, the single and combined effects of HCHO ar~BAP on DNA damage were measured by alkaline e tion of DNA coupled with a fluoro- metric assay in order to detect DNA-protein crosslinks (DPC) in the tracheal epithelium. A maximal response of 85% total DNA retention was obtained from single or multiple exposure (2X/ wk) to a=0.2% HCHO solution, while 43% DNA re- tention resulted from a 0.01% HCHO exposure. The effect was reversed by proteinase treatment of DNA prior to.elution which revealed the pro- tein nature of the crosslink. Removal of HCHO- induced DPC was complete 3d after both single and multiple exposures. Tracheas exposed to BAP exhibited no DPC, regardless of exposure time (24-48h) or concentration (20-40 ltg). Pre- exposure to 20 ug BAP followed by 0.05$ HCHO diminished HCHO-induced DPC, but the combined effect was lost at higher HCHO concentrations.' (USPHS Grant CF, 43857 and OHER U.S. DOE under contract DE-AC05-84OR21400 with Martin Marietta Energy Systems, Inc.) 50875 8295 s
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651 AGING DECREASES HEPATIC METABOLISM OF AZOXYMETH- INE (AZO) BUT NOT COLONIC METABOLISM OF METHYL- AZOXYMETHANOL (MAM). T MeMahon, J Peggins and M Weiner. Universitx.of Maryland School of Pharm- acy, Baltimore,rMD. Sponsori C U Eccles. ~ A Microsomaf° hep`atic hydroxylation of the colon carcinogen AZO =to MAM and colonic metabolism of HAM by cytosoifo alcohol dehydrogenase (ADH) was assessed in young (2-4 mo) and aged (22-24 mo) male Fischer 344 rats. AZO hydroxylase (AZO-H) was quantitated by reversed-phase,~HPLC, and me- tabolism of MAM was measured by •?eduction of NAD+ at 340 nm. Activity of AZO-H declined by 33% in aged rats on a per mg protein basis, and by 15% on a per nmol cytochrome P-450 basis. The latter decline in activity is in agreement with the 21% decrease in hepatic P-450 content found in aged rats. In young rats, phenobarbital (Pb), 80 mg/kg x 5 days, caused a 2.7-fold induc- tion of P-450 and a 1.8-fold induction of AZO-H. Thus, activity on a per nmol P-450 basis in Pb- induced rats was decreased 42% relative to con- trol. This evidence, coupled with the decline in AZO-H seen in aged rats and data that 3-meth- yZcholanthrene, 30 mg/kg x 5 days, showed no inductive effect, implies indirectly that the decline in AZO metabolism is due to a decreased content of a specific isozyme(s) of P-450 respon- sible for metabolism of AZO. Colonic metabolism of HAM by ADH, in contrast to AZO-H, showed no age-related decline in activity. These results suggest a greater role for extrahepatic metabol- ism of AZO in aged rats. 652 CARDIAC GLUTATHIONE AND CARDIOTOXICITY IN HIGH- DOSE CANCER CHEMOTHERAPY. DP Rodeheaver, DB Menze , TM Bashore, RJ Herfkens and WP Peters, Duke U. Med. Ctr., Depts. Pharm. and Med., Compre. Cancer Ctr.,Durham, NC. Cardiotoxicity is a major cause of morbidity and mortality following high dose cancer chemother- apy. The antineoplastic drugs, bis-chloroethyl nitrosourea (BCNU) and melphalan, are alkylating agents known to reduce intracellular glutathione (GSH) in some cell types. A reasonable hypo- thesis is that the decreased GSH increases the cardiac cell's sensitivity to cytotoxic effects of the drugs or decreases its ability to detox- ify peroxides or free radicals formed during drug metabolism. To establish a relationship between GSH and cardiotoxicity, we report here human GSH levels from cardiac biopsies. Cardiac biopsies from cancer patients were analyzed for GSH content before and immediately after high dose cyclophosphamide, cisplatin and either BCNU or melphalan. Cardiac GSH levels in biopsies of less than 1 mg size were determined by the'HPLC method of Keller and Menzel (Anal. Biochem. 151, 418, 1985). A 60-80% reduction in cardiac GSH following chemotherapy was observed in four of five patients examined. Although initial GSH levels varied 100-fold between individuals, a similar degree of reduction occurred. Initial GSH_ _eenLenh and Irercene --de-erease after chemotherapy appear correlated with cardiotox- icity. (Supported by NIH Grants CA14236 and RRO1693 and NCI Grant 5-PO1-CA32672.) 653 MODIFICATION OF N-METH'tr'=~N'-NITRO-N-NITROSO, GUANIDINE-INDUCED STOMACH CARCINOGENESIS ygv ^ , PHENOLIC ANTIOXIDANTS IN RATS. S Tamano,~p Hirose, S Fukushima.. Y. Kurata and N.,It _ lst. Dept. Pathol., Nagoya City Univ. Med,~ ~~-k~~~ Nagoya, Japan. Modifying effects of five phenolic antioxidants on N-methyl-N -nitro-N-nitrosoguanidine (MNNG):~ induced stomach carcinogenesis were investigat;r_l 11, in F344 male rats. Groups-DeaQ rats were givgn^ an intragastric dose of 150mg/kg OW MA91aC%;,; . -:..., Starti ng one week ;,,}ater they received di-' containing 0.8% catec"hol(CC), 1% 2-t-butyT-4::~:`: methylphenol(TBMP), 1.5% p-t-butylphenol(PTBP);`== 1.5% methylhydroquino.ne, 1.5% 4-methoxyphenol-" (4MP) or basal di et al one for 51 weeks,- ; Histological examination revq~ti'led~,that CC, TBMp , and PTBP strongly enhanced~'~he Oevelopment of : squamous cell carcinoma of the forestomach, Y whereas 4MP inhibited induction of carcinoma in l situ. In the pyloric region of the glandular ';_.. stomach, CC strongly enhanced the development of ` adenomatous hyperplasia and adenocarcinoma. Inn addition, CC alone induced adenomatous hyperplasia and adenocarcinoma in 100% and 20$ _. of rats, respectively. These results clearly show that CC, TBMP and PTBP are strong promoters.;., for forestomach carcinogenesis, and that CC, which is known to be widely present in our `. environment as a natural or industrial product, _. is a new glandular stomach carcinogen with`:: strong promoting activity for the forestomach, 654 EFFECTS OF ANTI-INFLAMMATORY AGENTS, A GLUTATHI-- ONE DEPLETING AGENT AND OTHER ANTIOXIDANTS ON THE DEVELOPMENT OF BHA-INDUCED RAT FORESTOMACH HYPERPLASIA. S Yamaguchi, A Masuda, S Fukushima, M Hirose and N Ito. lst Dept. Pathol., Nagoya City Univ. Med. Sch. Nagoya,.: Japan. The effects of various chemicals on BHA-induced. rat forestomach hyperplasia was evaluated in 2 experiments. In the first, groups of male F344 rats were given dik containing 1% BHA together with 50 ppm indomethacin (IM), 1.5 ppm dexamethasone (DEX), 0.15% 6-aminocaproic acid, 0.15% FOY 305, 0.25% diethylmaleate (DEM) or 0.015% retinyl. acetate for 52 wks. Histological examination revealed that the development of forestomach epithelial hyperplasia was completely inhibited by DEM, and significantly inhibited by IM or DEX. In experiment 2, groups of F344 male rats were given diet containing 1% BHA together with 0.7% BHT, 1% sodium ascorbate (SA), 1% propyl gallate (PG), 1% a-tocopherol or 0.2% ethoxyquin for 52 wks. SA enhanced the induction of hyperplasia in the prefundic region, whereas PG enhanced it in the mid-region. These results indicate that glutathione may be i_r-volved_ in the inducti_on of forestomach hyperplasia by BHA, and that some antioxidants (e.g. SA, PG) enhance the induction of BHA- induced hyperplasia at different sites of the forestomach epithelium. 164 50875 8289
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gi VIVO DNA BINDING POSE-RESPONSE STUDIES WITH AFB1 AND THE A_NTI-CARCINOGEN I_NDOLE-3-CARBINOL (I3C). R Dashwood, DArbogast, A Fong, J Hend- ricks and G Bai~ley-6.Oregon State University, Corvallis, OR. Sponsor: D Selivonchick .n - Several recent repoJrts describe inhibitor- mediated reductions in the covalent binding of various carcinogens to DNA in vivo. Most show inhibitory effects after testing at on5,inhib- itor and one carcinogen dose-level only ,°Thus, the detailed relationships between carcinogen dose, inhibitor dose, and iZ vivo inhibitory potency have not been clearly delineated in any species. Therefore, inhibitory potencies were assessed in rainbow trout (Salmo gaird- neri) given a range of carcinogen (AFB1) and inhibitor (I3C) doses by concomitant dietary exposure. In vivo binding of AFB1 to liver DNA was used as an end-point; linear increases oc- curred with increasing AFB1 dose and with time of I3C/AFB1 co-treatment, at each 13C dose- level. Successive increases in I3C dose gave dose-related decreases in DNA binding, yield- ing a series of curves of decreasing slope, with 95% inhibition at the top dose of 4000ppm 13C. Linear inhibitory responses observed at low 13C doses indicate the possible absence of any significant threshold for 13C protection against AFB1-DNA binding. Thus, even low lev- ls of I3C may offer some protection against chemically-induced neoplasia. (Studies suppor- ted in part by grants CA34732,ES03850,ES00210). EXAMINATION OF EVIDENCE FgR THE INTRACELLULAR FORMATION OF AN ADDUCT, N-HYDROXYMETHYLDEOXY- ADENOSINE (hm6dA), BY FORMALDEHYDE. M Casanova and H d'A Heck. CIIT, Res. Triangle Park, NC Formaldehyde (FA)-induced DNA-protein cross- links (DPX) are unstable to enzymatic hydrolysis of the DNA and dissociate, releasing free.HCHO. The released FA may react with deoxyribonucleo- sides (dNs) in the hydrolyzate to form hydroxy- methyl adducts (hmAs). To test whether this mechanism caa explain the detection of hm6dA in hydrolyzates of DNA from mammalian cells incu- bated with FA (Beland et al., 1984), a mliture of the four major dNs was incubated with [ CIFA (1I.tM) in the 3° amine buffer, bis-Tris. All of the dNs formed hmAs in this buffer, which were detectable by HPLC. The labeled compounds were identified from the elution profile and spectra of products obtained by reacting the dNs with FA (t4 and 82 mM). No hmAs were formed when [ C]FA was incubated with the dNs in the 1° amine buffer, Tris, due to preferential reaction° of FA with1t4he amino group of the buffer. The yield of [ C]hm6dA obtained in bis-Tris buffer, adjusted for differences in reactant concentra- tjJons, was almost identical to the yield of [ H]hm6dA reportedly founi in DNA hydrolyzates (2.7 vs 2.3 hm6dA per 10 dA). These results strongly suggest that the hm6dA was not an in- tracellular product, but was3 formed in the hy- _drolyzates by release of3H] [ H]FA from DPX and reaction of the released [ FA with dA. 673 FORMATION 'OF CROSS-L"INKED ADDUCTS ON a REACTION OF AMINO ACIDS, WITH FORMALDEHYDE AND DEOXYRIBONUCLEOSIDES - OR DNA. T R Fennell, F H Deal, and J A Swenher . Chemica n ustry Institute of Toxicology, Researc Triangle Park, NC. Formaldehyde (HCHO) is a nasal carcinogen which is known to form DNA protein cross-links both in vitro anVin vivo, but little is known abheir cheiW'ical nature. Previous studies have identified unstable^"`:.^ adducts formed by reactioff- of HCHO with deoxy- guanosine (dG) and lysine. -We have investigated the cross-linking reaction of HCHO with nucleosides and other potentially reactive amino acids (gly, thr, and N-acetyl derivatives of arg, as'p~ ~cys. glu, his, phe, ser, trp and tyr). After incur~tion of HCHO (100 mM): with nucleosades (10 rnM) rand amino acids (10 mM) at 37 and pH 7.4 for 12 h, products were analysed by reverse phase HPLC. Glycine reacted with dG and HCHO to form two unstable products. On reaction of N-acetylcysteine with HCHO and dG (but not dA, dC, or dT), a cross-linked adduct was found which was stable in aqueous solution. It as characterized by proton NMR spectroscopy as N -methylene-(Na-acetylcystein- S-yl)deoxyguanosine. Reaction of N-acetylcysteine with HCHO and calf thymus DNA under similar conditions in vitro resulted in the formation of the adduct, resolved on HPLC of a nucleoside digest. The formation of a similar cross-linked adduct produced on reaction of DNA with cysteine from proteins in nuclei is under investigation. 674 DETECTION OF N2,3-ETHENOGUANINE ' AND 7-(2-OXO- ETHYL)GUANINE IN DNA FROM RATS CHRONICALLY EXPOSED TO ACRYLONITRILE. S A M Koch, V E Walker and J A Swenberg. CIIT, Research Triangle Park, NC. Acrylonitrile (AN) has been shown to be car- cinogenic in rats. DNA from target and non- target organs of rats chronically exposed to AN was analyzed for the presenae of two postulated promutagenicfA 'adducts, N , 3-ethenoguariine (ethenoG) and 7-(2-oxoethyl)guanine (7-OEG). Tissues were removed at termination of a 2-year study in which rats had been exposed to 500 ppm of AN in drinking water. DNA was extracted from brain, stomach and Zymbal's gland' (target tissues), as well as liver and spleen (non-target tissues):;. The DNA was either acid-depurinated to release ethenoG or reduced with NaBH4 and subjected to neutral thermal lysis to release 7- OEG. The adducts were measured using HPLC with fluorescence detection. Adduct concentrations were highest in the brain (1.6 nmol ethenoG and 1.2 nmol 7-OEG/umol guanine) followed by Zymbal's gland. Only 7-OEG was detected in stomach DNA. Much lower concentrations of both adducts were found in liver and neither adduct was 'detected in spleen._„ Both adducts were formed in calf thymus DNA incubated (37°C, 18 h) with 2-cyanoethylene oxide (ANO), a metabolite of AN. These data suggest that ANO is a,proximate carcinogenic metabolite and that one or both adducts are .relevant to AN carcinogenesis. (Supported in part by NIH grant NS 20023) 169 50875 8294
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699 A COMPARATIVE EVALUATION OF THE LETHALITY OF FENVALERATE AND THE FENVALERATE FORMULATION PYDRIN®, E G Williamson, M.J Kallman, M C Wilson, Department of Pharmacology,-Cchool of Pharmacy, University of Mississippi, University, MS. ~ m, f. A 72 hr LDSp -study involving the fenvalerate formulation," Fydrin® 2.4 EC, was performed using male Swiss mice in order to compare the relative toxicities of Pydrin® to that of technical grade fenvalerate i}n corn oil. Fenvalerate was more toxic when7 administered as the formulated product, than when administered as technical grade material. The LD50 value for Pydrin® was calculated to be 72 mg/kg and 62 mg/kg following P0 and IP administration. Administration of 128 mg/kg and 89 mg/kg of technical grade fenvalerate, which corresponded to the amount of fenvalerate contained in the LD99 of Pydrin® for the P0 and IP routes respectively, resulted in no deaths. These data suggest a substantial effect of the Pydrin® formulation vehicle on the toxicity of technical fenvalerate. The formulation vehicle, did not result in substantial lethality as compared to no-treatment controls. An alternative interpretation of these data is that the fenvalerate was less bioavailable when administered in corn 'oil. The comparative bioavailability of the technical grade material and Pydrin® in corn oil, is currently being investigated. (Work supported in part by the Research Institute of Pharmaceutical Sciences). ~ 700 THE EFFECTS OF A BENZODIAZEPINE RECEPTOR ANTAGO- NIST AND PICROTOXIN ON FENVALERATE TOXICITY. KM Tolson and WN Bourn, School of Pharmacy, North- east Louisiana University, Monroe, LA. Sponsor: PJ Medon. Pyrethroid insecticides of the Type II class have been linked to the GABAergic system in mammals. A variety of evidence indicates that fenvalerate (FNV) and other Type II agents interact with the picrotoxinin domain on the GABA receptor complex. In the present study FNV toxicity was manipulated with a benzodiazepine.(BZ) receptor blocker, CGS- 8216, and picrotoxin (PTX). The time course of FNV toxicity was previously determined for female rats in an electronic ac- tivity monitoring system.-Onset of the syndrome, as shown by an increase in activity, occurred . within one hour following an oral dose of 100 mg/ kg FNV. A subconvulsive dose of PTX (2 mg/kg ip) 30 minutes prior to FNV exposure precluded any signs of FNV toxicity until the fourth hour of testing. Pretreatment with CGS-8216, an established BZ, receptor antagonist, failed to alter the above measured behavioral aspects of the FNV toxicity syndrome. Similarly, pretreatment with the subconvulsive dose of picrotoxin reduced the frequency of elec- troencephalographic spiking induced by continuous intravenous infusion of FNV. In a parallel ex- periment, CGS-8216 did not alter EEG spiking. 701 exposure and susceptibility of aged rats to the immunosuppressive potency of TBTO. 1 Vos et al. Tox.Appl.Pharm. 75 (1984), 387. 2 Krajnc et al. Tox.Appl.Pharm. 75 (1984), 363. 3 Vos et al. The Toxicologist.5 Z'f985), 5. tumor incidences were observed in some endocrine organs which is assumed to be related to the ob- served hormonal disbalance. Ongoing studies focus,; on reproduction, immune effects after perinatal A parallel experiment disclosed functional immune _• alterations at a dose level of 5 mg/kg3. Increased = ting, TBTO suppressed the cellular and the non- specific immunity'. Endocrinologically, effects comprised interference with the pituitary-thyroid axis (diminished TSH productj1n"aVd release associated with decreased ttiyroid activity) and the pituitary-gonad axis (increased luteinizing hormone production and release)2. In a long-term rat toxicity and carcinogenicity study effects were found at the high dose (50 mg/kg feed) indi- cating the same targets as.in short-term tests. immune and endocrine system. In functional tes= for TBTO toxicity ha;i!'e been identified, i.e. studies were sponsored by-1.'~"e"WHO Parasitic Di-`":. seases Program. In short-term tests, two tari-et$ may be performed. Part of the rat oral toxicity._` ; of toxicity data on which human safety assessm%t t t in Schistosomiasis con ro require e pro uctlLon : ,ji.. . ~t e BIS(TRI-N-BUTYLTIN)OXIDE (TBTO) TOXICITY TESTING IN THE RAT: PRESENT STATUS. E I Krajnc, PW Wester, J G Vos and C A van der Heijden. Naational Institute of Public Health and En`vironmental Hygiene, Bilthoven, The Netherlands. Sponsor: J G Vos. Intended large-scale use of TBTO as molluscicide 1 d h d 702 CHRONIC TOXICITY/0_NCOGENICITY FEEDING STUDIES IN SPRAGUE-DAWLEY RATS AND CF1 MICE WITH ETHION., J D McCarty, L D Morrow, J R DeProspo,. L B Kedderis, and M J Fletcher. FMC Corp., Princeton, NJ and American Biogenics Corp., Decatur, IL. The toxicity of the organophosphate pesticide ethion, was evaluated in lifespan feeding studies to assess the chronic toxicity and oncogenic,potential. Groups of 80/sex/dose CF1 mice and#,prague-Dawley rats were administered ethion tec`hnical in the diet for 24 months., The dose levels were 0,.0.75; 1.5, and 8.0 ppm for'mice and 0, 2, 4, and 40 ppm for rats. Interim sacrifices were conducted at 6, 12, and. 18 months (10/sex/group) and a final sacrifice was performed at 24 months (50/sex/group). There were no remarkable differences in body weight, food consumption,'clinical obser- vations, hematology, or clinical chemistry . between control and treated animals except for treatment-related depression of plasma cholin- esteras.e.levels. In mice, plasma cholines- terase depression of 25-44% was observed in the. 8.0 ppm dose group at 6, 12, and 18 months in females and at 12 and 18 months in males. In the rats, cholinesterase depression was 37-578 in the 40 ppm dose group at 12 and 18 months for the males, and at all intervals for the females. There were no treatment-related neoplastic or non-neoplastic histopathological findings. The No Observable Effect Level (NOEL) is 4 ppm in rats and 1.5 ppm in mice. 176 50875 8301
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VE SUBCHRONIC EFFECTS OF ETHYLENE DIBROMIDE ON cYTOCHROME P-450 LEVELS AND GLUTATHIONE-S- TgANSFERASES IN RAT LIVER AND KIDNEY. J W Sauswirth, Center for Veter.,inary Medicine, Food and Drug Administrati~on,~`eltsville, MD. sponsor: T M Farber. y- to determine the sub- this study was undertaken,. chronic effects of ethylene dibromide (EDB) on liver and kidney cytochrome P-450 (P-450) levels and glutathione-S-transferases (GST's). Dosage levels used were 0, 10, 40 and 80 mg/kg givea~toy gavage to male Sprague-Dawley rats for a peridd of 90 days. Six animals were killed on days 7, 14, 30, 60 and 90 and liver and kidney tissues vere removed for analyses. P-450 levels were induced in the kidney by EDB at all dosage levels at 30 days. However, by 60 days, the percentage induction in kidney decreased. By 90 days at the 80 mg/kg dose, the levels were only 80% of the controls. Hepatic levels of P-450 were unaffected at 7 and 14 days, but by 30 days the levels were decreased by treatment. The reduction.reached a maximum at 90 days in the 80 mg/kg group when P-450 levels reached 55% of the control group. GST's were consistently induced in the liver at all dosage levels and in a dose-related manner. In the kidney at 40 and 80 mg/kg, GST's were also induced but not to the same extent and the effect plateaued earlier. At 10 mg/kg in the kidney, there was an initial increase'followed,by a de- crease to or just below control levels. These results indicate a mixed effect of EDB on P-450 levels in liver and kidney whereas, a consistent inductive effect was seen on GST activities. COMPARATIVE TOXICITY OF 4 ALKYL THIOCARBAMATES TN DOGS AND iTATS FOLLOWING REPEATED ORAL ADMIN- ISTRATION. M W Sauerhoff, D R Saunders, and G L Sprague. Environmental Health Center, Stauffer Chemical Company, Farmington, CT. The toxicity of 4, closely related alkyl thio- carbamates was compared in Sprague-Dawley rats (diet) and Beagle dogs (capsule) using repeated, daily administration. The highest dose tolerated, without producing lethality, was the end-point used for comparison. All compounds exhibited rel- atively low toxicity. They were s-ethyl diisopro- pyl thiocarbamate (I), s-ethyl di-isbutyl thio- carbamate (II), s-propyl dipropyl thiocarbamate (II) and s-propyl butylethyl thiocarbamate (IV). The order of potency in dogs was I>IV>III>II (corresponding doses were 60, 100, 125 and 300 mg/kg/day, respectively). The order of potency in rats, (I=III>IV>II; corresponding doses were 25, 25, 60 and 400 mg/kg/day, respectively) was the same as for dogs except for III. For dose levels producing lethality, deaths occurred after 1-2 weeks in both species for II.and III and for I and IV in rats. In dogs, I and IV produced death within 1 week. For I, III and IV rats were slightly more sensitive than dogs when doses were expressed as mg/kg/day (2-4x). For II, rats and dogs were approximately, equally sensitive. When doses were expressed as mg/m2/day rats were 2-15x more sensitive than dogs. In conclusion, rats were more sensitive than dogs for 3 of the 4 compounds. - :...e-- 71$ TOXICITY OF MIXTURES OF HERBICIDES, FOUND IN GROUNDWATER, IN MICE. A K Chaturvedi, L M Dix, W L Liu, I E Berg, and G Padmanabhan, Departments of Civil Engineering, Pharmaceutichl Sciences% Toxicology, and Veterinary Science, North Dakota State University, Fargo, ND. Alachlor(AL), atrazine(AT), and/or picloram(PI) have been reported to be present ( 1 ppb) in groundwater of certain agricultural areas. Though tU toxicity of each of these _W~'cides (HEs) is individually studied, the,.toxicity of their mixtures has not been evaivated. Therefore, AL+AT, AT+PI, PI+AL, and AL+AT+PI•mixtures (10 ppm of each of HEs) and individual HE in drinking water were given to ICR male mice (21-24 g) for 30 days. The exposure to HEs or their mixtu~gs did not produce significant change in fAd 3.r[-~ take, water consumption, body weight gain, d'r liver or kidney to body weight ratio. However, the spleen to body weight ratios in AT and AL+AT groups were higher by 51 and 41%, respectively, than the control (ns10). Histopatholic examina- tion of the above tissues revealed that the enlarged spleens were moderately congested. The pentobarbital(60 mg/kg, ip)-induced sleep time in AL+PI group was lower (24%, p<0.05) than the control. These results suggest that HEs and their mixtures have potential to produce unde- sirable effects.on spleen and to alter liver function. More data, however, from the exposure to HE mixtures for a longer period are crucial in elucidating their toxicity. (Supported by USDIGS Grant #14-08-0001-G1441-NDWRIP). 714 COMPARISON OF CAPILLARY SUPERCRITICAL FLUID CHROMATOGRAPHY (SFC) AND HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) FOR THE ANALYSIS OF PESTICIDES IN BI0L0GICAL SYSTEMS. E R Campbelll, D W Lateri, D N Dankovic2, R C Zangar2 and D L Springer2. 'Lee Scientific, Inc., Salt Lake City, UT and 2Battelle, Pacific Northwest Laboratories, Richland, WA Currently, chromatographic methods for the analysis of pesticides and their metabolites in biological systems ~s tedious, time-consuming, and often lacking in sensitivity. Pesticide compounds were evaluated by capillary SFC and HPLC to determine the feasibility of detecting them in potentially complex cellular matrices. Actual metabolism studies, using radiolabelled pesticides such as aldicarb and carbaryl, were performed with freshly isolated rat hepato- cytes. Metabolic products were analyzed by capillary SFC and flame ionization detection and the results compared to HPLC separation and liquid scintillation counting. Results from analysis with these techniques indicated that approximately 40% of the parent aldicarb was converted to metabolite after 30 min of incuba- tion. It was also shown that aldicarb sulf- oxide and sulfone metabolites could be separat- ed by using a selective cyanopropyl stationary phase for SFC and that these separations provided an enhancement in analytical data 'compared to reverse phase HPLC. It was observed that the data obtained by capillary SFC agree favorably with that of HPLC. Work supported by NCI Contract N44-CP-71086. 179 50875 8304
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ANALYSIS OF 2,4,4-TRIMETHYL-2-PENTANOL (TMP-OH) BINDING TO MALE RAT KIDNEY a2u-GLOBULIN (a2u) AND OTHER PROTEINS. 8J Borgfifif, J Strasser, Jr., M Charb~ onn eau.~t~ d JA Swenbera. CIIT, Research -- Triangle Park, NC. The nephrotoxicit)i of 2,2,4-trimethylpentane (TMP) in male rats is charactenzed by an increase in protein droplet formation and renal concentration of a2u. These changes correlate with an increase In cell protiferat pn noted in maie rat kidney after TMP exposure. TMP OH has been identified as the metabolite of TMP reversibly bound to a protein fraction containing a2u isolated from TMP-treated male rat kidney cytosol. To investigate whether TMP-OH is bound specifically to a2u and not other low molecular weight proteins, a2u was purified from [3H]-TMP-treated male rat kidneys using gel filtration and ion exchang e chromatography. Purified a2u, Identified using SDS-PAGE (18,000 Da) along with Western blot analysis (anti-a2u), co- eluted with TMP-derived radiolabel when injected onto an HPLC anion exchange column. To evaluate whether TMP- OH could.,patentially bind to other low molecular weight proteins, mlitro binding studies were carried out. [14C]- TMP-OH was ni cubated with either purified a2u (untreated male rats), Q-lactoglobulin lac), al-acid-glycoprotein (a1- glyco), 2-microglobulin P2) or lysozyme. The level of [14C1-TMP-OH bound to each protein was assessed using HPLC anion exchange chromatography. In vitro, [14C]- TMP-OH was found to bind to a2u as well as lac arld ai-glyco which are members of the- same family o proteias as a2u. There was no detectable binding of [14C]-TMP-OH to unrelated proteins such as p2 or lysozyme. Although TMP-OH binds to proteins in the same family as a2u in vitro, the only protein found to be associated with radiolabe[ cTenved from [3H]-TMP-treated male rat kidney cytosol was a2u. COMPARISON OF ALPHA-2U GLOBULIN ISOLATED FROM THE URINE OF ALBINO AND NON-ALBINO MALE RATS. T E Eurell, M J Parnell*, and G M Henningsen**. Dept. of Vet. Biosci., Univ. of IL., *AAMRL, Wright-Patterson AFB,OH., and**NIOSH- DBBS, Cincinnati, OH. Alpha-2U globulin has been associated with the hydrocarbon-induced proximal tubular cell degeneration reported in the male rat kidney The Fischer 344 strain has been used most often in these studies, and may be particularly susceptible to the toxic effect. Alpha-2U globulin was isolated from the urine of albino (Fischer 344 and Sprague Dawley) and pigmented (Long-Evans and Fawn-Hooded) male rats for protein characterization by isoelectric focusing techniques.An alpha-2U globulin iso- electric variant profile distinguishing albino from non-albino male rats was not apparent, however, strain differences were revealed. Fischer 344 male rats appear to have higher levels of the (Pi)=5.4 and 5.5 isoelectric variants than the other strains studied. These findings suggest that if a strain susceptibility to the hydrocarbon- induced nephrotoxic lesion exists, it may be associated with the alpha-2U globulin isoelectric variant profile. (Supported by AFOSR grant # 86-0313.) ~~~ 4.-& 537 IN VITRO HYDROLYSIS OF [14C]-a2u-GLOBULFN (a2u) ISOLATED FROM MALE RAT RIDNEY. M Charbonneau, J Strassei:, SJ Bor h'~~f and JA Swenberg. IIT. Research naT - ngle Park. NC. 2.2,4-Trimethylpentane (TMP) causes an increase in the renal concentration of a2u, a male rat-specific In1iv molecular weight protein. A reversible associatiqp between a metabolite of T and a2u has been f observed in kidney cytosol of~-treated male ats. - This study assesses protease hydrolysis of [14C;2uh with or without the bound TMP metabolite. Mali!~~ 344 rats were treated (po) with TMP 500 mg/kg) o~ corn oil at 0, 3. 6 and 9 hr. and [14C -labeled amino acids (ip) at 2.5. 5.0 and 7.5 hr. 14C]-a2u was isolated from kidney cytosol usi -ge flltration : and ion exchange chromatographj'. 1-fydrolysis was performed using a mixture of purifie& proteases (PP), proteinase K (PK) or kidney (untreated male rats) lysosomal enzymes (LE) at their resgective optimum pH. [14C]-a2u was incubated at 37 C for 2 hr with or without the PP. PK or LE. The reaction was stopped by addition of perchloric acid, and protein hydrolysis assessed by measuring the radioactivity in the supernatant. [14C]-a2u from both control and TMP-treated rats was poorly hydrolyzed by PP or LE, and moderately hydrolyzed by PK. However, both [14C]-high and -low molecular weight hepatic proteins -were hydrolyzed by PP, PK and LE. These results show that a2u is resistant to protease hydrolysis. Under these in vitro conditions, a2u hydrolysis was not altered by tffe-7b"inding of the TMP metabolite. 538 LOCALIZATION OF a2u-GLOBIILIN i)ITHIN RENAL PROTEIN DROPLETS OF H®LE RATS EXPOSED TO 2,2,4- TRIHETHYLPENTANE (TMP). V L Burnett, B G Short, J A Swenberg, Chemical Industry Institute of Toxicology, Research Triangle Park, NC. A novel immunohistochemical technique was developed to assess the relationship between protein droplet formation and a2u-globulin (a2u) accumulation induced by light chain hydrocarbons in the maW rat kidney. This procedure utilized a mouse monoclonal antibody to localize a2u within the kidney. Seventy two hours after oral dosing of male rats with 50 mg/kg TMP, kidneys were perfusion-fixed, cold processed into glycol methacrylate, and cut into 211 sections for staining. Results showed that localization of a2u stained by immunohistochemistry corresponded to the localization of protein droplets seen in serial sections stained with Lee's methylene blue-basic fuchsin. Quantitative morphometry by image analysis showed the area of staining of both renal a2u and protein droplets of the treated animals increased 1.5- to 2-fold over respective controls, which was comparable to the increases seen using biochemical and morphological evaluations. Thus, by using this immunohistochemical technique, . a strong correlation between protein droplet formation and a2u accumulation has been observed in control rats and rats treated with TMP. 135 50875 8260
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404 571 ASSESSMENT OF OLFACTORY FUNCTION AFTER INHALATION EXPOSURE OF RATS TO _METHYL BROMIDE. M Hurtt, D A Thomas, K T Morgan, and P K Working. CIIT, Research ~,,riangle Park, NC. ~ Olfactory -*function was evaluated in methyl bromide' (MeBr)-exposed adult male F-344 rats using the 5iuiied food pellet test. Animals (n=6 per group) were trained to locate and retrieve a food pellet buried under 2-4" bedding in a 2x4 ft. open field. The average retrieval time (RT) + SEM for 4 replicates in u)tt.Feated rats was 32.2 + 4.4 sec. Animals were retested 24 hr after a single 6 hr inhalation exposure to either 90 or 200 ppm MeBr. The animals' ability to locate the buried pellet was unaffected by exposure to 90 ppm MeBr (RT = 31.0 + 2.9 sec), consistent with the absence of morphologic damage to the olfactory epithelium seen after this exposure. The exposure to 200 ppm MeBr rendered the animals temporarily incapable of locating the hidden pellet, consistent with the severe destruction of the olfactory epithelium observed immediately after such an exposure. Four to 6 days after treatment, the rats recovered sufficient olfactory function to find pellets (RT at day 5= 39.8 + 11.9 sec), even though histological examination of the epithelium revealed that morphological damage was still extensive at that time. These data indicate that functional recovery precedes complete morphological recovery and, thus, may not be a good indicator of the morphological state of the olfactory epithelium. )5 ~ 572 A COMPARATIVE STUDY OF TEST METHODS USED TO DETERMINE THE TOXIC POTENCY OF PVC SMOKE. W G Switzer, and H L Kaplan Southwest Research Institute, San Antonio, TX, and M M Hirschler, BFGoodrich, Avon Lake, OH Toxic potency of smokes produced by combustion of two standard (SI, SJ) and two experimental (EX B, EX C) polyvinyl chloride (PVC) wire coating materials were evaluated by the National Bureau of Standards (NBS), Radiant Heater and University of Pittsburgh (UPitt) test methods. Analyses of CO and HC1 in the smoke were correlated with lethality data in order to assess the role of these toxicants for each method. In the NBS method, EX B and C produced atmospheres containing 78 to 95% less HC1 than equivalent quantities of SI, resulting in significantly higher LC50 values (p<0.05) for the exper- imental materials. The Radiant Heater method marked- ly decreased HC1 content of smoke from all materials, possibly due to HCI decay at high humidity. Thus, leth- ality was mostly caused by CO, but the method still differentiated between materials. With the UPitt method, HCI content of smoke from both EX B and C was reduced in comparison to the standard materials, but differences between LC50 values were statistieally significant only for EX C compared to SI and SJ. The lack of differentiation in LC50 values between EX B and the other three materials was determined to be due to an extreme sensitivity of mice to even low concentra- tions of HCI. This work indicates that toxic potencies determined by the UPitt method may not accurately reflect the relative toxic hazard of smoke from some materials to humans. (Sponsored by the BFGoodrich Geon Vinyl Division) 573 TOXIC EFFECIS OF VARIWS N_II1MVIINES ON N%s AL TISSUES OF RAZS.' S D Sleic3~t and C RanggEL_ Tahbu. Department of Pathology, Midiigan State Univ., East Iansing,`Pff. 11 574 144 ability of varioas nitrosamines to target specific cells in the nasall cavity of the rat. We postulate that these differences may be jn~prtant in determinim What types of tumors are caused by exposure to these chezoicals. that there are striking differerx:es in the - Rhe acute necrogenic effects of N-Nittrosodiethy_ lamine (DFM , N-Jitrosopyi.2~olidine (NPYR) ,` N.. Nitroc.~orpholine (1~) , N-Nitrosodi.ethanolaati,ne (NDELA) , and N' -Nitrosa-rarnicotine (Nta3) tiere compared in 80 g fema]rague-Dawley rats. SirxJl.e equiiaolar doses o each nitirosamir~, weie given ip. After 24 hr, rats were killed and tissues were colleated. Sections of iia- cavity and liver were evaluated histologically. inhibition of glycoprotein synthesis in cells of Bowman's glands in the olfactory region as evidenced by loss of Alciatvb],ve-periodic acid- Schiff material was seen .A`i.th a11 nitrosamines except NDII.A. DEN and IvNN cabsed changes at a lower dose than NPYR and NMP. Necrosis of Bowman's glands was most extensive with DEtJ and NPYR, was much less severe with NNN and NW and absent with NDELA. Olfactory epithelial cells were severely damaged with NPZ2 and NMP, less so with DEN and were unaffected by raIIi or NDELA. Hepatic centrilobular fatty change was seen only with DEN, NPYR, and 2aIN. 7hese results indicate LIMITATIONS OF THE UPITT METHOD FOR THE SCREENING OF MATERIALS FQR THE TOXI~ POTENCY OF SMO~E. H L Ka lan,l~ M M Hirschle `/r and W G Switzer 1 Southwest Research Institute, San Antonio, TX; 2) BFGoodrich, Avon Lake, OH New York State's requirement for submission of LC50 values of materials, using the University of Pittsburgh (UPitt) combustion toxicity screening method, may lead to the selection of materials based exclusively on these values. In a comprehensive evaluation .of the UPitt method, ~C50 values were obtained for four PVC and one nylon materials. The relative toxic hazards to humans of the corresponding smoke atmospheres were estimated from measurements of the concentrations of the major toxicants in the smoke and compared with UPitt LCoO values. UPitt LC50 values underestimated the relative toxic hazard of smoke from nylon and overestimated that of smoke from two PVC materials. Determinations of LC50 values for CO, HCN, HC1 and -CO + HC1 for mice proved the excessive sensitivity of this animal to HCI. Deficiencies in the UPitt method identified by this study include: the rapid evolution of gases resulting in a brief exposure of animals to very high levels, non-linearity between sample weight and gas evolution with some materials and, particularly, the extreme sensitivity of mice to HCi. The results show that a ranking based on LC50 values obtained by the UPitt method may lead to wrong identifications of those materials that produce smoke most toxicologically hazardous to humans. (Sponsored by the BFGoodrich Geon Vinyl Division) 50875 8269
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~ pIJLMONAI:iY TOXICITY OF ETHOXYLATES GIVEN ENDO- TRACHEALLY TO RATS. T R Tyler2, R C Myers2, S M Christopher2, & E.H Fowler , Union Carbide Corp., Danbury, CT1, Bushy Run"ftesearch Center, Export, PA2 + ~` Studies were condncted on a number of ethoxy- lated alcohols (EAL), nonylphenols (NP) and poly- ethyleneglycols (PEG) to investigate their poten- tial to cause lung injury by aspiration. Male Sprague-Dawley rats received single gndotracheal doses of test chemicals to determing minimal lethal dosages (MLD). The doses were adminis- tered in constant volume (0.5 ml/rat) by diluting the substances with saline. The MLD ranged from 20 ul to 640 ul/kg for the surfactants (EAL and NP), while MLDs for PEGs were greater than 1 g/kg. In a subsequent study, groups of 10 rats of each sex received a single sub-lethal dose of each material. Two animals from each group were sacrificed at 1, 2 and 3 days post-dose, and the remaining 4 at 14 days post-dose. For EALs and NPs, the most commonly occurring gross observa- tion on lungs was color change associated with inflammatory infiltrates, edema, and atelectasis at dosages of 10 to 160 ul/kg. Microscopic findings included fibrinopurulent pneumonia early in the course of the reaction, fibrosis later, and subsequently atelectasis accompanied by bronchoaleveolar mucus accumulation. Pulmonary injury was seen with PEGs only at much higher dosages (1 g/kg). Significant aspiration hazards exist for ethoxylated surfactants but not for PEGs. $84 CHRONIC PULMONARY CHANGES INDUCED BY O,O,S-TRIMETHYL PHOSPHOROTHIOATE (OOS-TMP) IN RATS M J.J Gijbels* and S K Durham, *TNO-IVEG, Rijswijk, The Netherlands, and Hoffmann-La Roche, Nutley, NJ Sponsor: T Imamura The long-term pulmonary effects induced by OOS-TMP, an impurity in organophosphorus insecticides, was examined by morphological and biochemical means. Weanling, female WAG/Rij rats received either corn oil or 40 mg OOS-TMP dissolved in corn oil/kg body weight by gavage and were studied at the following time intervals: 10 d, 30 d, 90 d, 6 mos and 1 yr. No animal died spontaneously. The 14 d LO in pilot studies was. 80 mg/kg body we g~t. OOS-TMP treatment resulted in the fol- lowing morphological changes: hypertrophy of type II alveolar epithelial cells which contained abundant and enlarged osmiophilic lamellar bodies, and increased interstitial fibrosis accompanied by basement membrane alterations. There was also a significant increase in pul-• monary hydroxyproline content in OOS-TMP treated animals as compared to controls at all times examined. Collagen deposition was assoc- iated with the interalveolar septa rather than being oriented around airways. The results of' this study indicates that a single, sublethal dose of OOS-TMP induces long-term pulmonary structural alterations in rats. 147 585 PULMONARY INJURY INDUCED BY TRIMETHYL 15HOSPHOROTHIOATE (OOS-TMP) If`TMICE. . S K Durham* and T Imamura, *Hoffmann- La Roche, Nutley, NJ an`c -Ty-ma Pharmaceutical;-'° Nyon, Switzerland The morphogenesis of pulmonary injury induced by a contaminant in commercially important or- _y ganophosphate insecticides, OOS-TMP, was ex- amined by combined light and electron micro- " ~copy. Weanling, female C~117Ka mice re- ceived OOS-TMP dissolved in corn oil by -11*,.- injection and were sts7fc7ied at intervals from `6 `~, to 168 hrs after treatment. Initial alterations ' were observed in Clara cells at 6 hrs and in- cluded dilated endoplasmic reticulum and loss of secretory granules. Severe in,ju~y of Clara cells occurred from 12 to 77r~'hrs'~> Injury of ciliated cells also occurred during this time. Sequestration of neutrophils was initially ob- served at 12 hrs and accompanied by interstit- ial edema, and mild injury to the endothelium. Endothelial cell injury was most pronounced at 24 hrs and accompanied by a significant in- crease in percent water content. Platelet ag- gregation was occasionally observed in capil- laries and small vessels at 24 hrs. Alterations in type I and 11 alveolar epithelial cells oc- curred from 24 to 72 hours after treatment. The results of this study indicates that the Clara cell was the initial and most severely injured pulmonary cell population in the mouse, which is different from that described in rats. 586 ATROPINE PRETREATMENT DOES NOT ABRO- 'GATE O,O,S-TRIMETHYLPHOSPHOROTHIOATE- (OOS-TMP) INDUCED BRONCHIOLAR INJURY IN MICE. T Imamura and S.K. Durham*, Zyma Co., Nyon, witzerland, and_ *Hoffmann-La Roche, Nutley, NJ OOS-TMP is a contaminant present in organo- phosphorus insecticides. Previous investigators suggested that morphological changes observed in Clara cells following the- administration of these compounds probably constitutes part of the cholin'tergic response to the xenobiotic. Fe- male, weanling C57BL/Ka received 30 mg OOS- TMP per Kg BW by IP injection and were stud- ied at intervals from 30 min to 12 hrs after OOS-TMP. Another group of animals were given 10 mg atropine sulfate per Kg BW by SQ inject- ion 30 'min prior to receiving OOS-TMP. The loss of secretory granules from Clara cells were observed at 30 min in animals that had received OOS-TMP and not atropine, whereas no loss of granules were observed in mice given atropine. Changes indicative of Clara cell injury, includ- ing dilated endoplasmic reticulum and loss of the apical cytoplasmic bulge without loss of sec- retory granules were observed in mice receiving atropine at 6 hours. Clara cell necrosis occur- red in both groups at 12 hrs. The results of this study in dicates that atropine does not a bro- gate the development of OOS-TMP-induced bron- chiolar injury and further suggests that the only cholinergic response from Clara cells in mice to this compound is the release of secre- tory granules. 50875 8272
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MODEL OF INHALED METHANOL: A SPECIES COMPARISON. V L Horton and D E Rickert. CIIT, Research Triangle Park, NC, and Curriculum in Toxicology; Univ. N. Carolina, Chapel Hill, NC. There are minimal data on the disposition of methanol (MeOH) after inhalation. MeOH PB-PK models were developed for F-344 rats, rhesus monkeys, and humans using ACSL software and compared to in vivo results. In rats, a double pathway for MeOH metabolism, using catalase Km and Vmax for one enzyme and high affinity, low capacity values for the second enzyme, simulated blood concentrations for 6h exposures to 200, 1200, or 2000 ppm MeOH. Simulation of 100 mg MeOH/kg intravenously required only the catalase values, suggesting the second pathway was induced during inhalation exposure. In monkeys, modeling of blood MeOH levels after 6h exposures to 200, 1200, or 2000 ppm required catalase parameters; alcohol dehydrogenase values employed as a second enzyme did not greatly influence the simulation, suggesting an interspecies metabolic similarit'y' at low doses. The human urine excretion model used monkey metabolism parameters, an extraction coefficient of 0.007, and a urine production rate of 0.4m1/hr/kg to simulate published data acquired for 8h exposures to 78, 158, or 231 ppm. These studies suggest that there is a high probability of accurately predicting MeOH blood and urine concentrations under varied exposure conditions in various species using PB-PK models. 625 PHYSIOLOGICALLY BASED P_HARMACOKINETICS OF GOBALT REMOVAL FROM THE LUNG AND ITS DEPOSITION AND ` ELIMINATION FROM THE BODY. JR Boger III, DB Menzel, RJ Francovitch, RL Wolpert, MI Tayyeb; and CR Shoaf. Depts.. Pharm. and Med., Duke U. Compre. Cancer Ctr., Durham, NC. Cobalt is a toxic and carcinogenic metal emitted by the combustion of fossil fuels and by indus- ° trial processes. Sprague-Dawley rats were ex- po~ ed to various CoC12 aeros'A~eoncentrations ~ fdr two hours to estimate human lung anii body--,. burdens. Co aerosols wik-te deposited in the NPR •= ? and lung with efficiencies of 2.3 and 5.4%.' Kinetics of NPR Co removal were first-order with estimated clearance rate of 0.16 hr 1.' Consis- tent with a two-compartment model, lung Co removal decreased over time. fo"'dptake into- compartment 1 was 425 ng/hr at'656gug/m3 expo- sure concentration. Elimination from com- partment 2 was 0.075 hr 1 Intercompartmental transitions were 0.022 hr 1(kl,) and 0.018 hr 1 (k21) derived from characteristic relaxation times of 10.3 hr and 59.9 hr, respectively. The relaxation time for the NPR was 6.37 hr. Since these clearance rates are faster than those for insoluble particles removed by mucocilliary clearance, the two compartments probably repre- sent the lung epithelium and capillary endo- thelium, respectively. These data and simu- lations have been incorporated into a model of Co body deposition which predicts organ and body burdens following several exposure scenarios. (Supported by NIH grants ES07031, CA14236 and RRO1693 and by,the ILSI Risk Institute.) 626 CLONAL INSULIN-PRODUCING CELL LINES AS MODELS OF CYPROHEPTADINE-INDUCED PANCREATIC BETA-CELL TOXICITY. C P Miller and L.J. Fischer. Dept. of Pharm./Tox. and Ctr. Env. Tox., Mich. State Univ., E. Lansing, MI. The insulin-secreting cell lines RINm5F and HIT-T15 have been used to investigate mechanisms of insulin,synthesis and secretion. C yproheptadine (C PH) is a pancreatotoxic chemical that reversibly, inhibits insulin secretion and depletes pancreatic insulin in the,&t. Studies were undertaken to deter- mine if the RIN and HIT cell lines respond to CPH in a similar rnanner to rat pancreatic g-cells. CPH produced similar alterations in both cell lines. After a 48 hr. culture period in the presence of 0, U.1, 1.0, or 1U.0 NM CPH, cellular imrnun- oreactive insulin (IRI) stores and media IRI levels were decreased in a concentration-dependent manner. At 10 pM CPH, RIN and HIT cell insulin declined by 77 and 0°6, respectively. C ellular IRI returned to control levels 48 hours after removal of C PH. In experiments designed to assess a direct inhibitory effect on stimulated IRI secretion, 1-10 Wvl CPH was found to inhibit glucose-stimulated release from HIT cells, and K+, alanine and glyceraldehyde- stimulated release from RIN cells. The results show that these cell lines exhibit sensitivity to the IRI-depleting and anti-secretory effects of C PH, and will serve as useful models in which to conduct mechanistic studies into the diabetogenic actions of this compound. (Supported by the Juvenile Diabetes Foundation, Grant #185208.) 157 50875 8282 pHYSIOLUGICALLY-BASED CONIP'lJTER SIMULATION OF a-ILMpENTAETIJCROBENZEIIE (CPF'B) 9FlA1dlA00KINEPICS j~qD ITS QUANTITATION 3H E}PIRED ffixEAZii: A NC3J- If1VASIVE 'hOOL FOR EYAIITATIf1G E06SCIRE HISiOR*Y. A Vinegar, D W Wimsett, R B Conolly, and M E pndersen. Nor&mp` Services, Inc., Dayton OH and _ Wright7"Patterson AFB, OH. --,cn A physiologically-based cmquter siaaal.ation (PB- pg) model has been developed to describe the relationship between CPFB concentrat~ons in ex- pired breath and body tissues. In this 3nodel ven- tilation and cardiac output were i-i~~al1y both set at 14 I!/min/kg X (body weight) (Eq. I). For model validation rats were exposed to CPFB vapor (300, 600, 1200 ppm for 1 hr) and expired CPFB tracked for 1 hr post-exposure. Ventilation was measured with a combination restrainer plethysmograph. It showed significant minute-to- minute variation and was higher than prediet,ed by Eq. I. PB-PK model simulation of expired CPFB levels improved dramatically when the real ven- tilation data were substituted for Eq. I and when cardiac output was set at one half the ventila- tion rate. This study shows that PB-PK modeling, used in conjunction with quantitation of toxicant in expired breath, can function as a non- invasive, retrospective monitor of CPFB exposure. 2he technique should be generally applicable to other.volatile materials and for those with a low blood-air partition coefficient it could also be utilized as a probe of cardiac output. ;2; PHYSIOLOGICALLY-BASED PHARMACOKINETIC (PB-PK)
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715 METABOLISM AND ELIMINATION OF A FLUORINATED 717 SULFONAMIDE INSECTICIDE IN THE RAT. R 0 Manning, K Muralidhara, J V Bruckner, M E Mispagel* and J M Bowen*. Dept.o`-Pharmacol. & Toxicol., College of Pharmacy,.and *Dept. of Physiol. & Pharmacol., College of Veterinary Medicine, University of (se~o,rgia, Athens, GA. - The absorp`tian, distribution and elimination of a fluorinated`sulfonamide being evaluated as a fire ant poison were examined in male and female S-D rats weighing 170-240 g. Rats were housed in glass Roth-type metabolism cages (oye per cage) for the separate collection of urine; feces and expired air. Each rat received a single oral bolus of 50 mg/kg bw of N-ethylperfluorooctane sulfonamideY4which contained approximately 10 uCi of N-ethyl( C)perfluorooctane sulfonamide. Feces, urine and expired air samples were col- lected periodically for 72 hrs after dosing, and radiolabel measurements were conducted. Greater than 70% of the radiolabel was eliminated from the14at within 72 hrs, with the largest quantity of C recovered in the expired air. Cumulative elimina- tion plots and amount remaining to be excreted plots of radioactivity were used to calculate approximate elimination half-lives (ti). W all three routes of elimination, the ti's of C elimination were between 9 and 13 hours. The rapid appearance of substantial quantities of radiolabel in expired air indicated that the N-ethyl group was readily removed from the mole- cule after dosing. (Supported by Univ. of Georgia Veterinary Med. Exper. Station 88-104 ET) ;; 716 DOSE-DEPENDENT PHARMACOKINETICS AND MAXIMUM TOLERATED DOSE OF OXADIXYL IN MICE. Y H Atallah, and C C Yu. Environmental• Sciences, Sandoz n~Tcoz Crop Protection Corporation, Des Plaines, IL. This experiment is designed to determine the dose-dependent pharmacokinetics and maximum tolerated dose (MTD) of oxadixyl fungicide in mice (Naval Medical Research Institute strain). Groups of males and females were dosed with 0, 10, 40, 100 and 400 ppm of 14C-oxadixyl equiv- alent in diet. 14C in the blood was monitored at specific time intervals. Excreta were collected and analyzed. Tissue samples were collected from animals killed at 24 and 96 hr after 14C dosing. Oxadixyl was rapidly absorbed and then rapidly eliminated by mice. The major route of elimina- tion was via urine (95% in 4 days). Fecal elimination accounted for about 5%. The body weight gains in treated male mice was signifi- cantly reduced at day 28. The liver weights as percentage of body weight in both males and females were significantly increased. The half-lives of blood radiocarbon in the 100 and 400 ppm treatment groups were about twice or more than that of the 10 and 40 ppm groups in both sexes. The proportion of unchanged oxadixyl was higher in urine of mice at 400 ppm diet level. These results showed that saturation kinetics of oxadixyl in mice had occurred at or about 100 ppm dietary levels. These effects were more definite at the 400 ppm level. Thus, the pharmacokinetic data in mice demonstrate that the MTD for this species is between 100 and 400 ppm in the diet. THE. META80LISM AND NEPHIiCrltlXICITY, TEfrRALIIV IN FISCHER 344 _ RNTS~ ' M P Serve', C T Olson, B M Llewellyn, R H Bruner, K 0 Yu, and D"W Hcbson. Wright State Universi- ty, Dayton, OH and Arnistrong Aerospace Medi- cal Research Iaboratory;`WPAFB, OH:`•. The C10 cyclic hydrocarbon tetralin, when administered by gavage to Fischer 344 male rats over a 14 day period produced moderate hyaline droplet formation and foci of degeneration in kidney proximal tubules. JAnalysis of 48 hour urine saVtft frcm the tetralin-dosed rats resulted in the identification of the'fcillowing metabolites: 1-tetralol, 2-tetralol, •2-hydroxy-l-tetra- ~ lone, 4-hydroxy-l-tetralone, 1,4-tetralin- diol and 1,2-tetralin3iol. Research sponsored by U.S. Air Forqe Grant No. AFOSR-87-0108. - ~ 718 THE ROLE OF INTESTINAL MICROFLORA ON DEEPOXIDA- TION OF TRICHOTHECENE MYCOTOXINS. S P Swanson, C Helasek, W B Buck and H D Rood. Dept. of Vet. Biosciences, Univ. of Illinois, Urbana, IL. Deepoxidation is an important pathway in the ani- mal metabolism of trichothecene mycotoxins. In this study, the role of intestinal microflora on metabolism of trichothecenes was examined. Micro- flora obtained from feces of six species, or intestinal contents from swine and rats were in- cubated anaerobically with the trichothecenes diacetoxyscirpenol (DAS) or T-2 toxin. Metabo- lites we4extracted with reverse phase cart- ridges and identified by capillary gas chromato- graphy and mass spectrometry. Incubation of DAS with fecal microflora from rat, cow and swine, yielded both deepoxidation and deacylation pro- ducts. By contrast, fecal microflora isolated from horse, chicken or dog failed to reduce the epoxide group in DAS and yielded only the deacy- lated products monoacetoxyscirpenol (MAS) and scirpentriol (SCP). Intestinal microflora ob- tained from rats and swine biotransformed T-2 toxin to a variety of deepoxy and deacylated pro- ducts including: deepoxy HT-2, deepoxy T-2 triol, HT-2 and T-2 triol. Rat intestinal micro- flora also biotransformed the polar trichothe- cenes T-2 tetraol and SCP to their deepoxy ana- logs. These results suggest intestinal micro- flora could play a significant role in the metabolism and toxicity of trichothecenes. 180 50875 8305
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40, 05 539 FUEL RYDROCARBON-INDUCED HYALINE DROPLET (HD) NEPHROPATHY IN MALE RATS DURING AGING. CVR Murtyl, MJ Olson2, BD Garg2, and AK Roy'. 'Dept. of Biol. Sci., Oakland U., Rochester, MI, ZBiomed. Sci. Dept., GM "ttes. Labs., Warren, MI. Sponsor: E W Lee s lyw Young male :rats treated with certain hydro- carbons, inpluding gasoline, develop nephropathy characterized by accumulation of HD in cells of the renal proximal tubules (PCT). However, no information is presently ava~lable on possible age-dependent susceptibility t HD accumulation. PCT of young (3 mo.), untreated, male Fischer rats had numerous HD in the P1 and P2 segments. By 12 no. of age, the number of droplets had declined markedly; at 24-26 no., no HD were visible. Electron microscopy of PCT of aged male rats showed that phagolysosomes, equivalent to HD, were reduced in number. We have also shown that a major constituent of many HD is the male rat urinary protein, a2 -globulin, for which the rate of hepatic synthesYs declines during aging. Levels of both hepatic and renal a-globulin in 26 no. old male rats were abou?u 1.5% of the young adult. Unleaded gasoline (0.4 mL/kg/d, 5 d) caused a 3-fold increase in the renal content of a2 -globulin in young males whereas the same trea~ment did not increase renal a - globulin in old rats. We conclude that age isua major determinant in the development of hydrocarbon-induced nephropathy in male rats and only rats which produce large amounts of a2u globulin are susceptible. 540 POSSIBLE INHIBITION OF RENAL PHAGOLYSOSOMAL (PL) PROTEOLYSIS BY GASOLINE IN MALE RAT: EVIDENCE FROM IMMUNOELECTRON MICROSCOPIC LOCALIZATION OF a-GLOBULIN. MJ Olson', MA Mancinl2, BD Garg', an AK Roy2. 'Biomed. Sci. Dept., GM Res. Labs., Warren, MI, 2Dept. of Bio1. Sci., Oakland U., Rochester, MI. Sponsor: E W Lee a2 -Globulin (aG) is a protein thought to be inFegral to the development of male rat-specific nephrotoxicity caused by unleaded gasoline. A gold-labelled second antibody was used to determine the distribution of aG within cells of the renal proximal convoluted tubules (PCT). aG was localized almost exclusively in PL of saline- or gasoline-treated (2.0. mL/kg/d, 9 d) male rat kidney; no aG was apparent at sites other than PL, microvilli or endocytotic vacuoles. Further, treatment of male rats with leupeptin, to inhibit cathepsin B, caused accumulation of PL identical to those observed following gasoline administration and rapidly increased the renal content of aG. aG in PL of control rats was distributed uniformly. However, the enlarged, angular PL eharacteristic- of gasoline and leupeptin intoxication contained deposits of anti-aG located preferentially over crystalline PL inclusions. In addition, numerous PL, unreactive with anti-aG, were observed in PCT of gasoline- or leupeptin-treated rats. Thus, it is concluded that a defect of lysosomal proteolysis is induced by gasoline and results in sequestration of large amounts of protein, including aG, in PL of PCT. 541 f 542 136 RENAL PROTEIN DR PLET FORMATION IN MALE FISCHER 344 RATS AFTER ISOPHORONE (IPH) TREATMENT. J Strasser, Jr., M Charbonneau, S J Borahoff, M J Turner an`d J A SwenberA. CIIT, Research Triangle Park, North Carolina. Several nongenotoxic carcinogens produce increased protein droplets and cell proliferation in the mate rat kidney, the target organ. These protein droplets co~htain a2u-globulln (a2u), a male,a tnSpecific low moleCular weight protein that Is absorbed by proximal tubula®ceUs. In a two-year bioassay,.IPH has been shown to cause~aJow Incidence of renal tumors In male, but not female rai.g.-or either sex of mice. Our objective was to test whether IPH would induce protein droplet formation and to determine whether IPH or the metaboiites, isophorol and dihydroisophorone, were asso iated with a2u. Male rats treated with 0.5 or 1.0 g/kg exhibited a significant increase in protein droplets. Gel fittra`tion chromatography was used to isolate a2u from other proteins in male rat kidney cytosol 24 hr after treatment with IPH, isophorol or dihydroisophorone. Samples were extracted with ethyl acetate. GC/MS of the organic phase positively identified IPH (parent compound) and dihydrolsophorone in the a2u samples from kidney cytosol of the IPH and dihydroisophorone treated animals, respectively. IPH was identified in samples from animals treated with Isophorol. This may suggest that isophorol is metabolized to IPH, via the alcohol dehydrogenase system, prior to binding to a2u. Biochemical alterations induced by IPH resembled those of 2,2,4-trimethylpentane and 1,4-dichiorobenzene, and thus suggest a similar mechanism of nephrotoxicity and carcinogenicity in male rats. A POSSIBLE THRESHOLD IN THE TOXICITY OF THE PYRROLIZIDINE ALKALOID, NONOCROTALINE PJ Shubat and RJ Huxtable, Dept. of Pharmacology Univ. of AZ, Tucson, AZ. Sponsor: AJ Gandolfi Rat lung and right heart weights Increase following inJection of 40 - 100 mg/kg monocrotaline or following continuous ingestion of 20 mg/L monocrotaline in drinking water. We have investiated the relationship between dose and toxicity by giving male rats (60 or 100 g) monocrotaline in drinking water for 1,2,4,6,10 or 20 days4, Organ weights and pulmonary arterial contractility were measured 20 days after initiating treatment. Rats given 20 mg/L monocrotaline for 4 or more days developed the same degree of right ventricular hypertrophy as rats given 40 mg/L monocrotaiine. Likewise, rats given 20 or 40 mg/L monocrotaline for 6 or more days developed similar and significant increases In lung weights. Pulmonary arteries were removed to assess norepinephrine-induced contractility In tissue baths. Maximum force of contraction was significantly decreased by monocrotaline treatment, but only If rats were exposed for 4'or more days. There was no difference In D50 between t t t o Th thr hold ex os re whicU e es rea men r u p. g p produced changes in pulmonary arteries, lung and of t ht tI f 20 /L h i i we s was on o or more ear nges mg g nonocrotallne for more than 48 hours (a total-dose of less than 15 mg/kg). If damage occurred during the first 48 hr of treatment, it was reversed by day 20. Monocrotaline-induced damage to rat lung and heart appeared to be quantal rather than grade.d Supported by USPHS HL25258 and NIEHS ES-070-91. 50875 8261
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FACTORS INFLUENCING THE ESTIMATION OF HAZARD FROM AN ACCIDENTAL ARSINE RELEASE. G V ,ie~e£f_, California Department of. Health ~ $ervices, Berkeley, CA z Arsine is an- extremely hazardous substance that requires evaluation for the potential consequences of=_.an accidental release. geported rat LC50s indicate that arsine is,of similar toxic potency as methyl isocyanate. Although over 470 human cases of arsine poisoning have been reported i=n, the literature, quantitative dose-response data are lacking. Thus, available data reported for laboratory animals were evaluated to calculate concentrations that could produce fatal or severe toxic effects (hemolysis) in humans. Reported data for mice indicate that the toxic response to arsine varies as a function of concentration2 x time. Calculations based on the administered concentrations indicate that the mouse (10- min LC50 = 85 ppm, 10-min LOEL = 12 ppm) is more sensitive than the rabbit (10-min LC50 = 250 ppm, 10-min LOEL = 16 ppm). However, calculations based on the estimated quantity of arsine absorbed per RBC, indicate that the rabbit may be 5 to 10 times more sensitive to the effects of arsine than the mouse. Based on evaluation of pharmacokinetic parameters, children would receive approximately twice the arsine dose per RBC compared to adults breathing the same concentration. V6 A BIOLOGICALLY-BASED COMPUTER SIMULATION MODEL FOR HEPATOCYTOTOXICITX. J M Gearhartl, L J Good- pasterl, M E AndersenZ and R B Conollyl. 1 Northrop Services Inc., Dayton, H, 2 AAMRL/TH WPAFB, OH. A number of cytotoxicants (C) are carcinogenic in rodent bioassays. Their carcinogenicity may be due to continual regenerative hyperplasia (RH) following repeated toxicity. Evaluation of this hypothesis requires, in part, quantitation of necrosis and RH after C exposure. We have de- veloped a simulation model describing C pharma- cokinetics and a biochemical mechanism leading to cell death. In this model a metabolite of C attacks a target macromolecule (T) and cells die when T falls below a threshold concentra- tion. Soluble enzymes are released by viable, leaky cells while membrane-bound enzymes are only released when cells die. Computer simula- tions of hepatotoxicant inhalation and resultant cytotoxicity are,presented. One aspect of model validation is the quantitation of hepatic en- zyme activity in situ and of its clearance from- blood. SGPT, a so'fu b7e hepatic enzyme, had in situ activity of 19,450 ± 3,700 SF units/g Tiver in male Osborne-Mendel rats and was cleared from the blood by a biphasic process with half-lives of 3.4 and 35 hr, respectively. Full validation of the model may enable activities of membrane- bound hepatic enzymes in blood to be used as quantitative indices of hepatocyte death. 155 617 618 BIOLOGICALLY-BASED COMPUTER SNU~ZATION OF DOSE- RESPONSE (D-R) CURVES FOR CYTOTOXIC CHEMICAL CARCINOGENS. R B Conollyl, H J Clewell, 1112, R H Reitz3, and M E Andersen2.. I Northrop Services; Inc., Dayton, OH; 2 AAMRL/TH. WPAFB, OH; 3 Dow Chemical Co., Midland, MI Predicting the shape of carcinogen D-R curves at low doses is a long-standing problem which can " be addressed by ccuprter simulation. We have 'F dev,eloped a sitnulation niodel fosraa-cytotoacicant ' (CJ, including its phazmacokinetic behavior, biochemical mccha„is„ of.:toxicity, and for a .. linkage between twcicity arid twtar formation. ^' ~= Target tissue ce11s have basal birth and death ^ rates. Mutations occur during replication and cause transition of normal cells to intermediate and then malignant genotypes (0, k-ai&°2 muta- : tions, respectively). For cytotokicity, a metabolite of C wrecks a target macromolecule (T) and cells die when T falls below a threshold concentration. Individual cell;thnesholds are normally distributed. Ce1ll death is followed by regenerative hyperplasia which increases the transition rates. Six hr/day, 5 day/wk for 2 yr inhalation exposures were siaulatecl. The threshold for cytotoxicity was varied and D-R curves obtained frcam 1 ppb through 100 ppm. As expected, shapes of carcinogen D-R curves were a fiuiction of cytotoxic threshold. This study illustrates the use of caViter simu_l.ation to integrate infoYmation on carcinogen exposure, gharmacokinetics, me-hani s,++ of action, and tumor formation. A PHYSIOLOGICAL PHARMACOKINETIC DESCRIPTION OF THE TISSUE DISTRIBUTION AND ENZYME INDUCTION OF 2,3,7,8-TETRACHLORODIBENZO-P-DIOXIN IN THE RAT. H W Leung, *M E Andersen, R H Ku, and D 3 Paustenbach. Syntex (USA) Inc., Palo Alto, CA, and *Consultant, Dayton, OH. The disposition of TCDD in the mouse is primar- ily determined by high affinity hepatic binding to a cytosolic receptor and a microsomal bind- ing domain. Distribution studies provided est- imates of t~i binding constant for the latter, but not for`~he former. We developed and vali- dated a physiological pharmacokinetic model for the mouse which included the 2 hepatic.binding sites. We then modified the model to include enzyme induction, which was assumed to be re- lated to the fractional occupancy of the cyto- solic receptor. This model was scaled up for the rat to evaluate literature data for enzyme induction by TCDD. The cytosolic receptor binding affinity in vivo was estimated by simu- lation to be about 10 pM. This rat model also accurately predicted the tissue distribution following repeated dosing as described by Rose et al. (Toxicol. Appl. Pharmacol. 36 (1976) 209). In both instances, the behavior was ex- tremely sensitive to binding affinities, but much less sensitive to binding capacities in the dose range studied. This physiological model for TCDD which accounts for hepatic bind- ing and enzyme induction is useful for cancer risk assessments when it is coupled with biologically-based models for tumor promoters. 50875 8280
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L. 192 ROLE OF ADRENAL CORTICOSTERONE (CS) IN SUPPRES- 593 SION OF CYTOTOXIC T LYMPHOCYTE (CTL) RESPONSE FOLLOWING EXPOSURE TO 3,4,5,3',4',5'-H_EXACHLORO- BIPHENYL (HxCB). N I Rerkvliet;--B B Smith and L B Steppan. Col,~e e of Veterinary Medicine, ~ Oregon State Nniversity, Corvallis, OR. The CTL response. of"C57B1/6 mice to alloantigen is sensitive to suppression by HxCB, a toxic Ah- receptor binding PCB isomer. However, suppres- sion of the CTL response occurs only at doses of HxCB that also produce thymic atrophyj Since thymic atrophy can result from elevated`lev,els of CS and PCB exposure has been shown to elevate serum CS levels, the possibility that suppression of the CTL response was an indirect effect of HxCB on CS production was examined. Serum CS levels were elevated 3-fold(p <0.01) in mice treated orally with a single dose of 10 mg/kg Hx- CB. Alloantigen challenge alone did not influence serum CS levels nor alter the elevation of CS in- duced by HxCB. Plasma ACTH levels were not alter- ed by HxCB suggesting an effect at the level of the adrenal gland rather than the pituitary. Adrenalectomized (Adx) mice treated with 10 mg/kg HxCB exhibited a high incidence of mortality when challenged with allogeneic tumor cells. Survivors showed suppressed CTL responses. Since Adx did not prevent the suppression of CTL activity by HxCB, a cause-effect relationship between HxCB- induced elevation of CS and suppressed CTL is doubtful. Studies with 2,4,5,2',4',5' - and 2,3, 4,3',4',5' -HxCB suggested that enhanced.CS pro- duction and CTL suppression are coexpressed con- sequences of Ah-receptor activation. INHIBITION OF ANTI-HAPTEN ANTIBODY RESPONSE IN 594 ADOPTIVE HOST RECONSTITUTED WITH T CELLS FROM 2,3,7,8-TETRACHLORODIBENZO-P-DIO%IN (TCDD) EXPOSED MICE. R A Tomar and N I Kerkvliet. College of Veterinary Medicine, Oregon State University, Corvallis, OR. Antibody production to T-dependent antigens is highly sensitive to suppression by polychlori- nated dibenzo-p-dioxins. Previous studies from this laboratory have shown that the cellular mechanisms of suppression is related to effects on T cells. The present study provides evidence for a defect in T helper (Ts) cells in TCDD- exposed mice. Because spleen cells from non-, immune TCDD-exposed mice did not show suppressed antibody response in the adoptive host, we used a hapten-carrier (TNP-SRBC) system with cell . separation/reconstitution techniques to determine the effects of TCDD on carrier specific Ta cells. Lethally irradiated syngenic recipients, reconstituted with virgin B cells (nonimmune) and T cells primed to sheep red blood cells, were immunized with TNP-SRBC. Mice •reconstituted with carrier primed T cells from TCDD-exposed mice produced fewer anti TNP-PFC as compared to controls. In vitro assay of T® cells produced similar results. The findings suggest reduced Te cell activity in mice exposed to TCDD. The inability to show suppression upon transfer of unprimed spleen cells suggests that resting T cells are not sensitive to TCDD. Supported by NIH Grant ES00040. 149 ALTERATION IN PGE2 PRODUCTIO - N FOLLOWING IN VIVO , EXPOSURE TO DIMETHYLNITROSAMINE (DMN). M J Myers, J F Lockwood, and L B Schook, Lab- a oratory of Molecular Immunnlogy, Dept`: of Animal.... Sciences, University of Illinois, Urbana, IL. Previous results from this laboratory have shown DMN depressed T cell responses through changes in macrophage (MPH) functions. As PGE2 is an important MPH derived mediator affecting both a~ and T cell functions, it ws&~Q.€ interest to T~ ertain if DMN affected PGE2 production. Peritoneal exudate MPH eJ:icited with either thioglycollate (TG) or Con A (CA) were cultured- in vitro with medium, LPS, or IFN-y. Both TG and CA MPH obtained from DMN exposed animals showed a 3-fold increase in PGE2 levels following ei- ther LPS or IFN-Y as compared to~,~eh3tle control responses. The production of PGE2 ipduced by LPS and IFN-y in both vehicle and DMN treated animals were completely inhibited by the addi- tion of indomethicin. In contrast, bone marrow derived MPH cultured with either medium, LPS or IFN-y for 24 h prior to examination showed no differences in PGE2 production between vehicle and DMN treated animals at either 3,5,7, or 9 d of culture. These results suggest the DMN- induced decrease in MPH dependent T cell re- sponses may be due to increased PGE2 production by MPH. (Supported by NIH grant ES-04348). DIMETHYLNITROSAMINE(DMN)-INDUCED CHANGES IN TNF-a -EXPRESSION AS DETECTED BY NORTHERN BLOT ANALYSIS._ J.F Lockwood, M J Myers & L B Schook. University o.f Illinois, Urbana, IL Previous results from this laboratory have . shown exposure to DMN in vivo resulted in increased tumoricidal activity of thioglycol- - late elicited macrophage(MPH) following acti- vation in vitro and cultured bone marrow derived macrophage(BMDM). As TNF-a(TNF) is the predominant..MPH effector cytolytic molecule, it was neccess~•ry to determine if DMN exposure was altering the regulation of-this cytokine. There -fore TG-MPH and BMDM were- examined for TNF - RNA by Northern Blot analysis. Examination of TG-MPH from vehicle and DMN exposed animals revealed similar TNF levels. However, following LPS stimulation in vitro, T6-B4PH from DMN animals demonstrated enhanced transcriptional activity of TNF. Additionally, the kinetics of TNF expression by BMDM also was affected by DMN exposure in vivo. BMDM from.control animals demonstrated highest accumulation of mRNA at day 7 following LPS treatment whereas BMDM from DMN exposed animals demonstrated greatest mRNA accumulation at day 5 of differentiation. These results suggest that the previously observed increases in MPH tumoricidal activity are due in part to enhanced levels of TNF mRNA. (Supported by NIH grant ES-04348)
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659 7-(2,3-EPOXYPROPOXY)ACTINOMYCIN D(EPA), A LESS TOXIC AND MORE POTENT ANALOGUE OF ACTINOMYCIN D (AMD). D P Rosenbaum, and S Z-Sengupta, Depart- ment of Pharmaco3togy, Boston University School of Medicine, J~ppst+hn, MA. Sponsor: J K Marquis '` AMD is useful ii}_the treatment of Wilm's tumor in children, choriocarcinoma in pregnant women and Kaposi's sarcoma. AMD has not been applied more widely because it is highly toxic and has a low TI. Its high toxicity is presumed„-tq derive from its lack of metabolism in vivo and rts poor excr- etion from the host system. We designed EPA so that it could be capable of metabolic deactiva- tion in the host and act in tumors by a different mechanism of action than AMD. In enzymatic and in vivo experiments using rodent hepatic enzymes and rat liver, we demonstrated that EPA is metab- olized to inactive conjugates thereby reducing host toxicity. In the presence of epoxide hydro- lase and glutathione-S-transferase, EPA is meta- bolized to DHPA (a dihydroxy derivative of EPA), and the glutathione-S-conjugates, and mercapturic acid conjugates of DHPA. AIID remains untrans- formed. The rate of conjugation with glutathione -S-transferase is 3 to 4 fold faster than with epoxide hydrolase. These transformations are con- sistent with the reduced toxicity of EPA in host compared to AMD. EPA acts in tumors by both in- tercalation (like AMD and DHPA) and adduct forma- tion. The result is that EPA is active in tumors that are resistant to AMD. (Support: NCI CA 26281-06) ~ 660 PERINATAL CARCINOGENESIS INDUCED BY INHALED VINYL CHLORIDE. M J Radike, J Warkanya, K Stemmer, E Bingham. University of Cincinnati College of Medicine, aInstitute for Develop- mental Research, Childrens' Hospital, Cincinnati, OH. Sponsor: D Warshawsky In order to investigate the effects of vinyl chloride (VC) on perinatal carcinogenesis, three groups of pregnant Sprague-Dawley rats were exposed by inhalation to 600 ppm.VC 4 hrs/day, from the 9th to 21st day of gestation. One VC group received 5% ethanol (EtOH) in water, and. one group, with neonates, was additionally exposed to VC through weaning of pups. Off- spring, including those of EtOH and filtered air control groups, were observed for life. The development of angiosarcoma (liver, lung, muscle) in VC-treated groups indicates the transplacental potential of VC to initiate cancer in utero. Ingestion of 5% EtOH by VC- treated dams did not enhance the incidence of treatment-related malignancies. Post-natal ~ exposure of pups to 600 ppm VC increased the incidence of liver tumors, 10 of 72 offspring with angiosarcoma and 48 of 72 with carcinoma. In comparison, exposure to VC in utero alone induced liver angiosarcoma in 1 of 71 rats and hepatocellular carcinoma in 11 of 71. Treat- ment-related malignant tumors included angiosar- coma in the liver, lung and muscle, and carci- noma in the liver, bile duct and mammary tissue. Supported by USEPA 68-03-2402 661 INFLUENCE OF VIRAL INFECTIONS ON INCIDENCES, BODY WEIGHT AND SURVIVAL OF FI ~`::. 344 RATS G N Rao J Edmo ds a ~%` ,_o n on, . nd Haseman. Nat orial Txicology Program, j~t ,.. Institute of Environmental Health Scie~ Research Trian le Park NC g , . Sialodacryoadenitis virus (SDAV), Sendai vi,;-.;t f (SV), and pneumonia virus of mice (PVM) are ii;;f-~. common viral infections o€-imab,s. Influence`;pt, these viral infections on the incidence of.~. _ common tumors (>20%);such as leukemia, pitui i tumors and adrenal'pheochromocytomas in the rat and pituitary tumors, mammary tumors aW" l k ia i th f l t l i eu em n e ema e ra s a ong w th the b~y weights and survival in 29 diet control group3 at 5 different laboratories watr*ld without viral infections were evaluaf`ed. p;The PVM and 91 but not the SDAV infections significantly (P<0.05) decreased the body weights. The rats - with PVM infection had , significantly lowea : incidence of leukemia and this effect can (g;'' explained by laboratory to laboratory '' variability. Male rats with SDAV infection had ~_ significantly higher (P<0.01) incidence o.f, pituitary tumors and this difference can be explained on the basis of time-related trends and.laboratory to laboratory variability. A11 other tumor incidences evaluated in this study' and the survival were not affected by the viral'" infections. 662 TRANSPLACENTAL (TP) TUMORIGENESIS BY N-NITROSO- ETHYLUREA (NEU), N-NITROSODIETHYLAMINE (NDEA)', AND N-NITROSODIMETHYLAMINE (NDMA) IN MICE. L M Anderson, J M Rice, and A Hagiwara, National Cancer Institute, Frederick, MD Childhood cancers including brain tumors are pos- tulated to be associated with exposure to nitros=- amine-contai"ning materials. Mice develop capac- ity for metabolic activation of nitrosamines by day (g.d.) 16, and those of the C3H: gestatioq. ~ strain s4pbw neurogenic tumors after tp NEU. To- '.: test this strain as a model for tp causation of.?:'.: neurogenic tumors by nitrosamines pregnant C3H/ ~: HeNCr mice were treated ip on g.d. 16 or 19 with- '` ' NDEA, NDMA, or NEU (0.5, 0.1, 0.4 mmole/kg, resp.). In the offspring, NEU caused multiple alveologenic lung tumors, increased hepatocellu- lar tumors, and schwannomas in 12% (g.d. 16) or 35% (g.d. 19). Unusual cranial tumors were also ~ ' noted, including 4 glioblastomas. NDEA was more effective after g.d. 19 than 16, causing a sig- nificant increase in both liver and lung tumors compared with controls. NDMA, being tested for the first time as a tp carcinogen in mice, re- in both sexes after g.d. 19 exposure, but had ; sulted in an increased incidence of liver tumors no effect on lung tumors. Five histiocytic 166 occurred. These results confirm that nitrosa- mines can be effective tp carcinogens in rodents once fetal activating enzymes have developed, but do not have a pronounced neurotropic effect analogous to that of the nitrosoureas. sarcomas and a schwannoma-like neoplasm also ; 50875,8291
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691 DEMETHYLATION OF METRYL QRGANOPHOS- PHATES BY RAT HEPATIC -QLUTATHIONE S- TRANSFERASE. J P Rank and D L Eaton, Department of Environmental Health, University of Washington, Seat- tle, WA. Glutathione-S trpnsferases (GST) are a family of dimeric enzymes responsible for the detozification of a variety of '` toxic chenl~'cals,including,,including the widely used insecticide methyl parathi6ii (W). A mechanism of detoxification of MP proceeds via iiemethylation and requires glutathione as a cofactor. This study sought to determine the relative ability of GST to demethylate MP in the presence of other methyl and ethyl organophosphate analogf. A reverse- phase ion-pairing high performance liquid ¢bromatography method was developed to separate all the metabolites of MP. The limit of quantitation of desmethyl parathion (DMP) was 100 pmol. Incubations of rat hepatic cytosol with 0.50 mM MF resulted in a GST-mediated rate of demethylation of 359 pmol/mg /min. Demethylation of MP displayed saturation kinetics with an apparent Vmax of 1.13 nmol/mg/min and Km of 0.56 mM. Methyl paraoxon demonstrated a similar activity towards GST, but did not exhibit saturation up to an incubation concentration of 4 mM. A wide variety of methyl organophosphates inhibited the GST-mediated formation of DMP, indicating that many methyl organo- phosphates may serve as substrates for GST. Ethyl organophosphates also inhibited the demethylation of MP by GST, however, deethylation was not detected, suggesting an affinity for the active site in . GST without serving as a substrate. (Supported by the Dana Foundation). ~ 1692 THE ROLE OF GLUTATHIONE IN THE DETOXIFICATION OF _METHYL PARATHION IN VIVO IN THE MOUSE L G Sultato_s and L Woods, Dept. Pharmacol. Univ. Med. Dent. of New Jersey; Newark, NJ. The dimethyl-substituted organothiosphosphate insecticide methyl parathion is thought to un- dergo glutathione-mediated detoxification in mam- mals. In the present study, depletion of hepatic glutathione in the mouse by pretreatment with diethyl maleate (DEM) potentiated the acute toxi- city of inethyl parathion, whereas depletion of hepatic glutathione by pretreatment with buthi- onine sulfoximine (BSO) did not. Furthermore, incubation of 50 uM methyl parathion with mouse hepatic microsomes for 5 min in the presence of 1 mM DEM led to significantly greater (p < 0.05) production of methyl paraoxon, compared to incu- batiotis in the absence of DEM (2.1 t 0.3 nmol methyl paraoxon/100 mg liver with DEM versus 0.6 t 0.1 nmol methyl paraoxon/100 mg liver without DEM). These results suggest that normal levels of hepatic glutathione are not required for de- toxification of methyl parathion. Moreover, the potentiation of the acute toxicity of methyl parathion following DEM pretreatment could result from enhanced production of methyl paraoxon and not from depletion of hepatic glutathione. There- fore these data raise doubts about the parti- cipation of glutathione in the detoxification of methyl parathion in vivo in the mouse. (Supported by Grant ES04335 from NIEHS). 693 694 174 ACUTE ORAL TOXICITY STUDY IN~CYW0MOLGUS MONKEYS WITH ALDICARB RESIDUE hD}--SANANAS AND WATERMELON. J A Trutter, F E Reno, R H Cox, R L Barona, and J M Charlesa. HazTeton Laboratories America,'. Inc.', Vienna, VA, aRhone-Poulenc,~Ag Company, . Research Triangle Park; NC. The study was performed to determine the acute toxicity and effects on acetylcholinesterase (ChE) activities in cynomolgus monkeys after -=-. intake of bananas or watermelon treated with ar-r„ exaggerated rate of TEMIK-,]QG~„to assure deliver--_ ance of fruit containing h'i`g"h aldicarb resike., ' levels. Twelve monkeys (3/sex/group) were fed., test fruits in amoutfts providing a residue in `ta?I"g of 0.005 mg/kg of body "weight, a value equal to-- the ADI established by the WHO. Equal numbers of controls (0.000 mg of residue/kg) received a similar rate of fruit (20g/kq~ ...,~eriodic evalu- ations of clinical signs anci~hE activities were made through 24 hours post-feeding. There were no clinical signs of toxicity or inhibition of RBC ChE activity. Plasma ChE activity was inhibited through 2 hours''(watermelon) or 4 hours (bananas), with peak inhibition (32-37%) occur- ring at 1- and 2-hours, respectively. These data ~: fit on the same dose response curve as that which ', ; was obtained from human volunteers given an acute aqueous solution of aldicarb corresponding to doses of 0.025, 0.050 and 0.10 mg/kg of body weight. BIUASSAYS FOR ALDICARB IN WATERMELON. B W Wilson, T.E Archer, J N Seiber, M E Stelljes; J D Henderson and J B Knaak. University of California, Davis and California Department of Health Services. The more than 1200 illnesses attributed to aldicarb-contaminated watermelons in the summer of 1985 in California and Oregon sparked this project. to determine whether acetylcholinesterase (AChE) inhibitions and honeybee mortalities would provide rapid, sensitive&ssays for carbamate contamination of melons. Aqueous extracts of melons were concentrated with a rotary evaporator; extracts and concentrates were spiked with aldicarb, aldicarb sulfoxide and aldicarb sulfone. Inhibition of electric eel AChE was measured with an automated colorimetric assay and•an optical plate reader. Honeybees were fed extracts in syrup and the LD50s for the carbamates determined. Aldicarb sulfoxide was the most potent inhibitor. Detection limits were within the range expected for contaminated melons. The lower limit for AChE was 30 ppb, competitive with gas chromatography, and that for honeybees was approximately 1 ppm. Supported by contracts from California Department of Health Services. 50875 8299
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Sd EFFECT OF AGING ON PROSTATE CARCINOGENESIS INDUCED BY 3,2'-DIMETHYL.4-AMINOBIPHENYL(DMAB) IN F344 RATS. A Naka'nura, T ShiraR, S Fukushima and N Ito. lst Department of Pathology, Nagoya City Universitf Merical School, Nagoya, Japan. DMAB, a multipot'ential carcinogen, induces prostate carcinoma in F344 rats. The effect of aging on DMAB prostate carcinogenesis was examined in male F344 rats. Rats 5, )5 and 65 weeks of age were given DMAB s.c. at a"dose of 200 or 150' mg/kg once a week for 4 weeks. Initially, the experiment was started with 200mg DMAB. Since this dose was too toxic for 65-week- old rats, additional groups of 65-week-old and 5-week-old rats were treated with 150mg/kg DMAB for comparison. All animals in the 5- or 35- week-old groups were killed 60 weeks after the beginning of the experiment,'but rats in the 65- week-old group were killed after 46 weeks. Carcinomas of the prostate in each group were found in incidences of 8 to 19%. The incidences of atypical hyperplasia in the prostate were 33% to 75%. An age-related decrease was observed in induction of small intestine tumors. The incidences of tumors of the preputial and mammary gland were the highest in the 35-week- old group. There were no clear differences in the frequencies of tumors of the large intestine, Zymbal gland, subcutis and urinary bladder between groups. Thus, under the present experimental conditions, injection of DMAB in rats at different ages had no effect on prostate carcinogenesis. 657 MARKED ENHANCING POTENTIAL OF PRIOR N-METHYL-N- ~ NITROSOUREA (MNU) TREATMENT ON RAT TUMORIGENESIS IN VARIOUS ORGANS INDUCED BY 6IfIFFERENT. CARCINOGENS. S Uwagawa, K Imaida, H Tsuda, T Masui and N Ito. lst Dept. Pathol., Nagoya City Univ. Med. Sch., Nagoya. Japan. The enhancing effects of 6 different carcinogens on two stage carcinogenesis initiated with MNU in'fats were investigated. Ml1`'e;"6-week-old CELL KINETICS OF PEPSINOGEN DECREASED PYLORIC 658 GLAND CELLS, A PUTATIVE PRENEOPLASTIC LESION, IN RATS TREATED WITH MNNG. M Mutai, M Tatematsu, K Imaida, and N ITO. lst Dept. Pathol., Nagoya City Univ. Med. Sch., Nagoya, Japan. Pepsinogen 1 (Pgl) decreased pyloric glands (PDPG) represent the earliest histopathological- ly detectable preneoplastic change during MNNG induced rat gastric carcinogenesis. In this study, we evaluated the cell kinetics of the PDPG compared with normal pyloric glands (NPG). Forty male WKY rats, aged 7 wks, were divided into two groups. One was given 100 mg/l MNNG in the drinking water for 10 wks and sacrificed at the end of 12 wks and the other was given tap water as a control. Bromodeoxyuridine (BrdU) was applied to both groups as a single injection or by continuous labeling using osmotic mini-pumps for 10, 7 or 4 days before sacrifice. The stom- achs were fixed and routinely processed. The sections were double-stained immunohistochemi- cally for BrdU incorporation and Pgl. The-- labeling indices after a single injection were 0.12% in NPG and 1.7% in PDPG. More than 90% of PDPG cells incorporated BrdU by 7 days of labeling, but NPG.cells incorporated only 70- 80% by 10 days. These results suggest that NPG cells have little cell proliferating activity and are replaced by newly formed cells in about 14 days. In contrast, PDPG cells have increased proliferating activity and are replaced in 7-10 days. PDPG show an independent proliferation pattern as an early preneoplastic event. each. Rats were i n jectLil- wi th MNU (20mg/kgr': i.p.) twice a week for 4 weeks, -then treated with DMAB (3,2'-dimethyl-4-aminobiphenyl, 50mg/kg, s.c., 1/week), DBN (N-nitroso-di-n- butyl ami ne, 0.05%, i n water), DHP,N ~N-bi s-(2- hydroxypropyl) nitrosamine, 0.1%„~h water), DES - (diethylstilbestrol, 2.5ppm, in diet), S-OPP (sodium o-phenylphenate, "2%, in diet), Captafol (0.15%, in diet) or no test chemical. After 20 weeks, rats were killed and.sall organs were carefully examined for preneoplastic and neo- plastic lesions. All six carcinogens enhanced the incidence of preneoplastic and neoplastic lesions in their respective target organs: pancreas, small intestine and urinary bladder with DMAB; liver, esophagus, forestomach, and urinary bladder with DBN; thyroid, lung, liver, esophagus and urinary bladder with DHPN; liver with DES; and kidney and urinary bladder with S- OPP. The results suggest that MNU enhances carcinogenesis by as early as 20 weeks, and that it shows organotropy characteristic of the respective carcinogen. ,. F344 rats were divided into.7 groups of 25 rats-`<;w VINYLACETATE: INHALATION TOXICITY AND CARCINOG- ENICITY STUDY I RATS AND MICE. PE Owen and CA Thompson Hazleton UK, Harrogate, England; JJ Clary, RW Rickard, TR Tyler and MB Vinegar A two year inhalation study was conducted in Sprague-Dawley rats and CD-1 mice with vinyl acetate (VA). Groups of 90 male and 90 female rats and mice inhaled VA at 0, 50, 200 or 600 ppm for 6 hrs/day, 5 days/week. 10 animals/sex/ pecies were designated for interim clinical and~athological investigations at weeks 53 and 83. Similar groups were removed from treatment at week 70 and examined 16 weeks later. No effect on rat survival was noted. A few early deaths in mice were possibly associated with inhalation of 600 ppm of VA. Body weight gain was lower in both species at higher concentrations. Treatment-related pathology in both species was limited to the respiratoryy tract (hyperplasia, atrophy) at 200 and 600 ppm. Generally these changes were evident at 53.weeks and showed little progression. Following recovery, hyperplasia reversed but not atrophy. 11 nasal tumours and 1 larynx squamous carcinoma were noted in rats at 600 ppm. A nasal papilloma occurred in 1 rat at 200 ppm. One squamous carcinoma and 1 squamous nodule were noted in lungs of mice at 600 ppm. There was no evidence that exposure to VA caused adverse systemic effects and 50 ppm was a clear no ' observable effect level. , 165 50875 8290
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-E GAMINOCENIC EOTIICIAL OF TER'=-OON'1ROL CIDES. D V Singh, N P Page,_ and V J• xlliano. U.S. ~vixornt~ental Protection P~ency, ashington, DC, and Dynanr~c~Cdrp., Rockville, NID. .~ . hiordane, heptachlor, aldring and dieldrin are 0 main teani.ticides used~in the U.S. Due to eir hepatocarcinogenicity in mice, their pplications have been restricted to methods that 0 ami.ze human exposure. The relevance of liver 0 rs in mice to human risk, however, has been+k+ ighly debated. ' er the last decade, many new chronic and geno- 0 icity studies have been conducted. HIILi.le iver tumor induction in mice has been observed both sexes of several strains of mice, vidence for carcinogenicity in rats has been conclusive. Results from mutagenicity tests ve been uniformly negative. However, there is ~ . CE evidence for unscheduled DNA synthesis, sccnal aberrations, and interference with 0 nlar cauxmmication. While the mechanissn for inogenicity *emainG unclear, the data are m istent with a promational mechanism. tes of the carcinogenic potency of these sticides indicate that their use poses pe P° tentially high cancer risks. The uncertainties n f cross-species and low-dose extrapolation, however, can affect these risk estimates to a large, but unknown, extent. (This paper does not reflect EPA policy.) DIETARY CHRONIC TOXICITY AND ONCOGENICITY STUDIES aF TRICLUPYR IN RATS AND MICE. D L Eisenbrandt, T D-l.andr , H M Firchau, S Tsuda, and J F Quast.. , e Dow Chemical Company, Midland, MI and IET, Tokyo, Japan. The chronic toxicity and oncogenic potential of triclopyr (3,5,6-trichloro-2-pyridinyloxyacetic acid) herbicide were evaluated in rats for 24 months and mice for 22 months. Rats were fed diets that contained 0, 3, 12 or 36 mg/kg body weight/day and mice were fed at levels of 0, 50, 250 or 1,250 ppm (approximately 0, 5, 28 or 139 mg/kg/day). Standard parameters were examined. Male rats fed 12 or 36 mg/kg/day had increased kidney weights at 6, 12 and 24 months and minimal microscopic degeneration of proximal renal tubules was discernible at 6 and 12 months. Female rats fed 12 or 36 mg/kg/day had a,minimal increase in normal, age-related pigmentation of the renal proximal tubules at 6, 12 and 24 months while females fed 3 mg/kg/day had increased pigment only at 24 months. Body weight gain was decreased in male and female mice fed 1,250 ppm triclopyr. Water consumption was somewhat increased in male and female mice fed 250 or 1,250 ppm; urinary specific gravity was decreased at 6, 12 and 22 months for males fed 1,250 ppm. There were no treatment-related microscopic changes in mice. Administration of triclopyr was not associated with a tumorigenic response in either rats or mice. No adverse effects were observed in rats treated with 3 mg/tg/day or in mice fed at a level of 50 ppm. 705 CHRONIC TOXICITY AND ONCOGENICITY OF INHALED TECHNICAL GRADE 1,3-DICHLOROPROPENE (DCP) IN RATS AND MICE. WT Stott, KA Johnson, LG Lomax and LL- Y Calhoun, e ow emical Co., Midland, MI 48674 The effects of chronic DCP exposure in rodents were examined via inhalation, an appropriate route of potential human exposure. Male and female Fisc-ljer 344 rats and B6C3F1 mice-were- exposed to 0, 5, 20 or 60 ppm DCP 6 hours/ week, 5 days/week, for 2 years.. Boc~y weights of both sexes of rats and mice exposed.to 60 ppm vapors were depressed. A slight degeneration of the olfactory epithelium of the nasal_mucosa, occassionally accompanied by submucosal fi- brosis, also occurred in both sexes of ravr'-V., exposed to 60 ppm DCP. In mice, nonneoplastico changes included: hyperplasia of the trans- itional epithelium of the urinary bladder; slight degeneration of the olfactory eptthelium of the nasal mucosa; and minimal hyperplasia and hypertrophy of the nasal respiratory epithelium of both sexes exposed to 60 ppm vapors. Bladder and nasal effects were also observed in mice exposed to 20 ppm DCP. A minimal degree of hyperplasia also occurred in the nonglandular stomach mucosa of high-dose group male mice. The only tumorigen