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

Date: Feb 1988
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List of Scientific Research Articles. List of Scientific Research Abstracts. The Genotoxic Potential of Condensate From A Cigarette Which Does Not Burn Tobacco, by Doolittle Dj, Burger Gt, Hayes Aw, Lee Ck, the Toxicologist. An Official Publication of the
Smoke Chemistry, by James Ra, Avalos Jt, Mosberg at, Coggins Cre, Ayres Ph, the Toxicoligist. An Official Publication of the Society of Toxicology, 880200. Ninety-Day Inhalation Study in Rats, Comparing Smoke From Cigarettes Which Burned or Only Heated To
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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 tumorigenic response observed was an increased incidence of benign lung tumors (bronchioloalveolar adenomas) in male mice ex- posed to 60 ppm DCP (22/50 vs 9/50 in con- trols). No treatment-related effects were observed in rats and mice exposed to 20 ppm and 5 ppm DCP, respectively. 706 LIVER EFFECTS OF LACTOFEN (COBRA HERBICIDE) IN RATS AND C_HIMPANZEES. P Leber, C Fisher#, R Couch, M Erickson*, E But efi r; H Maruyama and G Williams+. #PPG Ind., Barberton,OH, *Primate Research Inst., Holloman AFB, NM, and +Naylor Dana Inst., Valhalla, NY. Lactofen (L), registered in 1987 for use as a soybean herbicide, was found to induce liver tum- mors in rodents following long-term dietary ad- ministration. Lacking genotoxic activity, an al- ternate mechanism wa~~ sought as a basis for the tumorigenic action of-"~this compound. This project assessed the potential of L. to induce liver changes in the reactive rat and in the higher primate comparable to the peroxisomal enhancement seen in the mouse. Rats (M & F) -were fed diets containing 0 or 2000 ppm L for a period of 8 weeks and then sac'd. Six male chimpanzees re- ceived oral doses of 5 or 75 mg/kg.d of L for 3 months. Serial liver biopsies were taken on Days -12, 92 and 239. Both species were subjected to blood chemistries, liver microscopic and bio- chemical study for evidence of hepatic effects. Rats exhibited hepatic peroxisome proliferation as evidenced by 2.5 to 3.5 fold increases in pal- mitoyl CoA oxidase in M and F's respectively, and by microscopic observations. Chimps displayed no changes in these peroxisomal parameters from pre- tr.eatment through 92 days of dosing. These obser- vations indicate that this species is refractory to L induction of peroxisome changes often asso- ciated with hepatic tumorigenesis in rodents. 177 50875 8302 . 44
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643 THE DERMAL CARCINOGENIC POTENTIAL OF LUBRICANT BASE OILS AND CUTTING FLUIDS. R H McKee, R A Scala, and C Chauzy. Exxon Biomedical Sciences, Inc., East Millstone, NJ and Centre Henri Becquerel, Rouen, France.. Epidermal canter in humans has been associated_ with ir}gust4ial exposure to products derived from unrefined Ae_troleum distillates. Because of this association,,rmany of these products are now normally formulated from highly refined (e.g., solvent-extracted) lubricant base oils which are normally inactive in dermal carcinogenesis bioassays. The current studies.J'aesessed the carcinogenic potential of solvent- extracted lubricant base oils and several products .including fresh and used cutting fluids which were prepared from these oils. All materials were tested for tumorigenic potential in mouse skin, and selected samples were analyzed for polycyclic aromatic hydrocarbon content. None of the solvent-extracted base oils induced skin tumors, and, similarly, the cutting fluids prepared from these oils were not carcinogenic. Additionally, there was no evidence that industrial usage influenced either.the carcinogenic potential or the PAH levels in these fluids. 645 CHRONIC TOXICITY AND CARC_FN06IIJESIS STUDIES OF 'i SULFAMETHAZINE IN eFSCHER 344 RATS. N-A Littlefield, D W Gaylor,R R Allen, and W,G.. Sheldon. National Center for lbxicological Research, Jefferson; AR. Sponsgr: G.L Wolff A lifespan dosing study using Sul.famethazine in the diet of Fischer 344 rats at dose levels of 10, 40, 600, 1200, and 2400 ppm in the diet for up to 24 months was conducted to determine-its toxicity and carcinogenicity. Sacrifices Vkre conducted at 12, 18, and.24 months. A total:--of 810 males and 810 females were allocated to „tlie study. Food conspnption was equal among the dose groups and-tt5e-control. A dose effect`:p~ noted at all dose l:evels for body weight gaia. An inverse dose effect was noted for. mortali- ty,i.e., the high dose groups survived longer than the controls. Mortal}'ty„for the controls, 10 and 40 ppn dose groups;n~s about 36 to 40% at 24 months, while the mortality~'in the 600, 1200, and 2400 ppu groups was only about 20 to 26%. Low level statistically significant neoplastic responses consisting of* follicular cell adeno- carcinoma (male and female) and adencmas (male only) of the thyroid gland were noted only in the 24 month sacrifice group. While over-all trends were noted, the effect was positive only in the high dose groups for adenocarcinomas in males and the 1200 pgn group in fenales. Non- neoplastic dose-related responses were noted as thyroid gland hyperplasia and focal cellular changes, atrophy of the retina and of the pancreas, and dilatation of the uterus lumen. f 644 EFFECT OF DURATION OF DERMAL EXPOSURE.TO BEMZ0- a-PYRENE ON THE CARCINOGENIC RESPONSE IN MICE. G. Cruzan, S. R. Carter, and G. E. Cox. Mobil Oil Corporation, Princeton, NJ. The risk of skin cancer from lifetime exposure to Benzo-a-pyrene (BaP) is well established in mice. The present study was conducted to assess the risk of tumors from shorter periods of exposure. Twice weekly applications of 0.05% BaP in acetone were performed in groups of 50 male C3H mice for 6, 13, 26, or 41 weeks. Tumor incidencesafter 80 weeks from the ini- tiation of exposure were 6, 74, 100, and 94%, respectively. In addition, twice weekly applications of 0.01% BaP in acetone were per- formed in groups of 50 male C3H mice for 26, 52, or 80 weeks. Tumor incidencesafter 80 weeks from the initiation of exposure were 84, 96, and 96%, respectively. For all groups, tumors have continued to appear long after treatment stopped. The in cidence was not proportional to the total dose applied, e.g.; 0.01% for 26 weeks = 260 ug total, 84% tumors; 0.05% for 6 weeks = 300 µg, 6% tumors; 0.01% for 52 weeks = 520 ug, 96% tumors; 0.05% for 13 weeks = _ 650 pg, 74% tumors. Thus the duration of exposure is a more important factor than the exposure level in the risk of tumor development from dermal exposure to BaP. 646 CHRONIC ORAL TOXICITY AND CARCINOGENICITY STUDY OF VINYL ACETATE ADMINISTERED IN DRINKING WATER DC Shaw, and AJ Zubaidy Hazleton UK, Harrogate, England; JJ Clary, RW Rickard, TR Tyler, MB Vinegar and F Carpanini This study was designed to evaluate the chronic toxicity and carcinogenic potential of vinyl acetate (VA). The uninhibited commercial VA monomer was administered in drinking water, prepared fresh daily, at nominal concentrations of 0 (control), 200, 1000 or 5000 ppm (v/v). Sixty male and 60 female Sprague Dawley rats at each doV level were used for the 2 year study following in utero exposure. An additional 30 animals of each sex and group were similarly treated and used for interim clinical pathology and post-mortem investigations at 52 and 78 weeks. Chronic oral exposure to VA caused a statistically significant reduction of water and food intake and reduced body weight gain at the highest dose level tested. There were no effects of treatment with VA on mortality, morbidity, clinical signs, haematology, clinical chemistry, urinary constituents or pathology. The only treatment-related organ effect was an increase in relative kidney weight in the high-dose males which was not accompanied by toxicologically tAgnificant pathological changes. There r.as no evidence of treatment-relatFj systemic toxicity or of an oncogenic respr.:nse. 162 50875 8287
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404 05 563 SDlULTANHOUS MEASURP4EIS OF WHOLE BODY PLETIlYSMOGRAPHIC_ PRESSURE AND TRANSPULMONARY PRFSSLk2E DURING E1IR HREATHING AND COz CHAGT.pNGE M F Stock, lr• Alari.a., and M Schaper. University qf Pittsburgh, Pittsburgh, PA. ,~ When using_ a whol.e body plethysmograph, the minima and maxima of the pressure changes (P) due to respiration are taken to indicate the beginning and end of each breath and the amplitude is taken to represent tidal volume (VT). We reported that thiJ3, is correct in norma7l guinea pigs but not during intense bronchoconstriction. To verify this, guinea pigs were anesthetized and a catheter tip pressure transducer was secured into the thoracic cavity to measure transpulmonary pressure (TPP). Two days later P and TPP were measured during air breathing, 002 challenge, and during challenge with histamine (H) or carbaa~lcholine (C). During air breathing or iX:iZ challenge P and TPP were in phase for the beginning and end of each breath. With H or C, the P minima were shifted in time, occurring much before the end of expiration as indicated by TPP. The minima shifted in time and also were much lower due to gas compression. Thus, if the amplitude from minima to maxima is taken to represent 4'I`, overestimation of up to 100% can occur. However, this can be used to indicate bronchoconstriction during challenge with a constrictor without measuring TPP. Supported under NIEHS 1R01-ES02747. ~ 564 C_OMPUTBEIZATIOBt OF ZIULMONA&Y FUNCTION STUDIES IN LABORATORY ANIMALS. H Burleigh-Flayera, M Schaper, R Thompson, and Y Alarie. Bushy Run Research Center/Union Carbide Corporation, Export, PAa and Dept. Ind. Env. Hlth. Sci., University of Pittsburgh, PA. A series of computer programs were developed to permit real time display and storage of pulmonary function data. Changes in pressure due to the respiration of animals can be detected by differential pressure transducers or microphones and displayed on a recorder. These respiratory signals are digitized, allowing the calculation of the following parameters: respiratory rate, amplitude of the respiratory wave (i.e., tidal volume), time of inspiration, and time of expiration. The data are stored on disk of an IBM personal computer AT®. Respira- tory frequency and mean amplitude of the signal every 15 seconds can be seen on the video display and a graph of these parameters can be generated on a printer while the experiment is In progress. A second computer program splits the data file into individual animal files to allow for processing of the data. The third computer program smoothes the data over pressure signals due to animal movement. The final computer program in the series calculates mean values and standard deviations. This software can be used in many different pulmonary function applications including sensory irritation and pulmonary hypersensitivity testing. Supported in part by YQIIBHS 1 RO1-BS02747. 142 ~.~ ~~-4 565 DETERMINING THE.LC50 FOR RATS EXPOSED BY INHAG ATION TO DIMETHYTPH0SPH0ROCHLORIDOTHIOATE. S M MacAskill,.K.. L Pavkow-and G L S ra ue; Environmental Hea ti Center, Stauf er C emica Company, Farmington, CT. This inhalation study with Dimethylphosphoco- chloridothioate (DMPCT) was conducted to deter- mine the 1-hour LC50 using whole body expos',flres. DMPCT is an intermedia#eAu the production of. j pesticides. Weil's adaptation of the mowing average method (~rug and Chemical Toxicolo 6(6); pp. 595-603,"1983) was used which alloiaed the calculation of an LC50 using small group sizes and concentrations producing 0-or 100% • mortality. Five male and 5 female Sprague-Dawle rats per dose group were Aicpn"jd to mean concen- trations of DMPCT of 0. 6-43 r 0.90. 1.06 and 3.n mg/i. Mortality was- 100% for both sexes at 3.00 mg/1 and 40% for males at 1.06 mg/1. Transient body weight loss was observed in all groups exposed to DMPCT excepf at 3.00 mg/1 where 100% mortality occurred by day 2. Clinical and gross pathologic signs of toxicity suggested that pulmonary insufficiency and/or systemic toxicity may have contributed to mortality. This method calculated LC50 values for both sexes to be less than 2 mg/1. It was an acceptable one for calculating an inhalation LC50 with as few as 5 rats per group. 566 INFLUENCE OF THE _LUNG TOXIN _ARAQUAT ON THE FREQUENCY AND FORM OF SPONTANEOUSLY GENERATED AUGMENTED BREATHS IN UNANESTHETIZED RATS. D J Murphy and D A Culp. Dept. of Investigative Toxicology, Smith Kline & French Laboratories, King of Prussia, PA. Sponsor: G F Rush. Changes in the frequency and form of spontan- eously generated augmented breaths following the administration of a single ip dose of paraquat were measured io Sprague-Dawley rats. The ven- tilatAt parameters were measured in unanesthe- tized rats using a head-exposed plethysmograph chamber. Paraquat treatment caused dose-depen- dent decreases in volume and increases in the expiratory time of augmented breaths. Signifi- cant changes were first noted at doses of 16 and 20 mg/kg for volume and expiratory time, respec- tively. The frequency of augmented breaths was least affected by paraquat, with a slight de- crease noted only at the highest dose of 24 mg/kg. Changes in tidal volume and respira- tory rate (tachypnea) were first detected at 16 mg/kg and were highly correlated with the de- creases in the volume of augmented breaths (r = ± 0.7). The changes in frequency and expiratory time of augmented breaths, however, were poorly correlated with the changes in respiratory rate and tidal volume (r < ± 0.5). Thus, changes in the volume of augmented breaths are as sensitive as the changes in respiratory rate and tidal volume in assessing the effects of paraquat on the lung, and all of these changes appear to be influenced by a common factor(s). 50875 8267
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41 587 PULMONARY RESPONSE TO AMIODARONE (AD) IN RATS. M.J. Reasor, C L Ogle, E R Walker, R C Lantz, West Virginia Univ. Medical Center, Morgantown, WV and S Kacew,"Universit~ of Ottawa, Ottawa, Ontario, Canada p ~. Humans treated chronically with the antiarrhyth- mic drug AD,may develop pulmonary damage. To characterize the pulmonary response to AD, male Fischer 344 rats were treated with AD, 150 mg/kg, p.o., for varying periods of time. Throughout 13 wks of treatment, AD and its pri=ncipalmetabolite, desethylAD, accumulated in lung tissue to approx- imately equal levels. Bis-desethylAD was also present, but was minimal by comparison. Two other unidentified polar metabolites were also detect- ed. By 3 wks of treatment, total pulmonary phos- pholipid was increased 2-3 fold and was not fur- ther increased through 13 wks of AD. The molar ratios of AD to phospholipid and desethylAD to phospholipid were both approximately 0.05. All classes of phospholipids measured were elevated. The activity of pulmonary Na+/K* ATPase was in- hibited in a time-dependent manner by AD treat- ment with about 35% of the activity remaining after 9 wks of AD. Alkaline phosphatase levels in the lungs were not changed by AD treatment. Additionally, lysosomal hydrolase activities were not markedly affected by AD treatment. The results indicate that selective biochemical changes occur in rat lung following AD treatment. (Supported by a grant from the American Heart Assoc.) 05 i ~ 588 PULMONARY CHANGES FOLLOWING INTRATRACHEAL INSTILLATION OF EALLIUM ARSENIDE AND ARSENIC AND GALLIUM OXIDES IN HAMSTERS AND RATS. M H Rosner and D E Carter. College of Pharmacy, University of Arizona, Tucson, AZ. Male Syrian Golden hamsters and Fischer-344 rats (n=3) were dosed to assess the effects of gaLlium arsenide (GaAs), sodium arsenite (As (III)) and gallium trioxide (Ga (III)) on several Lung antioxidants. The specific activity of the enzymes, superoxide dismutase (SOD), catalase (CAT], and gLutathione peroxidase (GSH-PX] plus sulfhydryL and ascorbic acid Levels were measured following intratracheal instiLlation of GaAs (100 and 10 mg/kg), As[III)(5 mg/kg) and Ga(III] (50 mg/kg) at 1-96 hours postexposure. In both species the lung/body weight ratio and total Lung protein increased compared to the control .vaLues for aLL test groups. GaAs (after I day) and As(III) (up to 3 hours) treated animals showed Lower CAT activity. GSH-PX activity was Lower than control Levels at times before I day with the GaAs and As(III] only. SOD activity was below control Levels for As(III) and GaAs after 3 hours and up to 2 days. SuLfhydryL and ascorbate LeveLs were different from control levels at the earlier time points for alL test compounds. Lung response to GaAs exposure involves changes in enzymes and biochemicaLs important in oxidant metabolism. 148 589 f 590 THE ROLE OF METABOLISM IN aARBON T_ETRACHLORIDE-MEDIATED I_MMUNOSUPPRES- SION. N E Kaminski, M Y Chapman, S D Jordan, and A_ P Holsapple. Dept. oi:'Pharmacology and Toxicology, Medical College of VA/VCU, Richmond, VA. The role of metabolic bioactivation for carbon tetrachloride- mediated suppression of humoral responses -,was investigated in B6C3F1 mice. Comparisons of imniune responses were performed b m' systems add i8 vitro systems with and wit~onMt metabolic ge_Watiui capability. Treatmettt of mice i.p. with 500, 1000 orY1500 mg/kg of carbon tetraohloride (CC14) for 7 consecutive"d~_sY" s followed by in vivo sensitization with sRBC resulted m a dose dependent suppression of the T-dependent antibody response (36, 48 and 53%, respectively). The in viv,g T- independent antibody respon .tc~-DNP-Ficoll was suppressed only at 1500 mg~g (16%). Antibody responses by splenocytes from mice treated with 1500 mg/kg and sensitized in vitro were suppressed by 65% to sRBC and 25% to DNP-Ficoll. The polyclonal response to LPS was unaffected by 1500 mg/kg CC14. CC14 added directly to culture at a 3.0 µM concentration resulted in decreased antibody responses to sRBC, DNP-Ficoll and LPS, which were directly attributable to cytotoxicity. Direct addition of CC14 to naive splenocytes co-incubated with a S9 metabolic generating system did not enhance the suppression of humoral responses. Direct addition of CC14 to naive splenocytes co-cultured with metabolically active hepatocytes also did not enhance the suppression of humoral responses. Studies utilizing the cytochrome p-450 inhibitor methoxsalen are in progress. (Supported by NIH ES03564 and NRSA ES05415). HPLC SEPARATION OF BENZO(a)PYRENE [B(a)P] METABOLITES GENERATED BY$PLENIC MICROSOMES OF UNTREATED MICE. T T Kawabata and K L White: Jr. Depts. of Pharmacology and Toxicology, and Biostatistics. Medical College of VirginiaNCU, Richmond, VA. Immunosuppression produced by B(a)P is thought to be mediated by its reactive metabolites rather than the parent compound, as demonstrated for its carcinogenic effects. Thus, metabolism of B(a)P by splenocytes to reactive .),ntermediates would be a requisite of B(a)P- induced `~uppression of the splenic immune response. Although murine splenic microsomes were demonstrated to be capable of metabolizing B(a)P to water soluble compounds (Cancer Res. 1:2317, 1987), the types of metabolites generated are not known. To identify the B(a)P metabolites produced by splenic microsomes, 3H- B(a)P was incubated with microsomal preparations and the metabolites were separated by reverse-phase HPLC. The metabolites were identified by comparison to the elution time of standards. The 9,10- and 7,8-dihydrodiol B(a)P were the primary diol metabolites formed, while only low levels of the 4,5-diol were detected. The addi- tion of the epoxide hydrolase inhibitor, trichloropropy- lene oxide (0.1 mM), was found to abrogate the formation of the diol metabolites. Splenic microsomes were also found to mediate the generation of the 9-, 7-, and 3- hydroxy and 3,6- and 6,12-dione metabolites. The cytochrome P-450 inhibitor, alpha-naphthoflavone (0.1 and 1.0 µM), inhibited the generation of all B(a)P metabolites. These results suggest that the spleen is capable of generating reactive B(a)P metabolites that may alter the immune response. (Supported by NIH grant ES03434). . 50875 8273
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611 Organic-Free R_adicals in Freshly Fractured Coal Dust and its Effect on Cytotoxicity V Vallyathan, B Jafari and N S Dalal Div. Respir. Dis. Studies, NIOSH, and West Virginia Univ., gorgantown. WV.. The relatiInAip between the development of ccaal workers^ pcidumoconiosis (CWP) and the geologic variables in 'coal has been a subject of debate and study. -The differences in the prevalence of CWP between geographic regions and mines can only be partly explained by the rank of coal, mineral composition, or its cytotoxicity potential. Increased levels of organic-free radicals were shown to be present in the freshly fractured coal (Artemo and Resnik, 1980; Dalal et al., 1986). These studies have postulated a potential role for organic-free radicals in the pathogenesis of CWP. This present study was initiated to test this hypothesis with the aid of cytotoxicity bio- assay to determine whether organic-free radicals generated are associated with an increased poten- tial for cell injury. To accomplish this task, we ground coal and measured the concentrations of organic-free radicals using electron spin reson- ance. Aliquots of same coal dust used in bio- assays showed that freshly ground coal with greater concentrations of organic-free radicals induced increased cytotoxic effect. The organic- free radicals generated by grinding decayed at a slow rate over time with a corresponding loss in toxicity. The half life of reactivity patterns of free radicals detected by ESR correlated with cytotoxicity of coal in bioassays. ~ 612 UPTAKE IN BLOOD OF 14C DURING AND FOLLOWING EkPOSURE TO METHYL ISOCYANATE (1,CH3NCO). J. Ferguson, Y Alarie, M F. Stock, A L Kennedy and W E Brown. University of Pittsburgh and Carnegie Mellon University, Pittsburgh, PA. Guinea pigs were exposed to i{CH,NCO for 1 to 6 hrs at 0.5 to 15 ppm. A carotid artery cannula was implanted two days prior to exposure. Each animal was exposed in a glass whole body plethysmograph. Blood samples taken during exposure revealed imsnediate and rapid uptake of 3dC. This uptake continued following exposure, followed by gradual clearance over a period of 3 days. The uptake was linear during exposure and, when expressed in cpm/min/ppm exposure concentration was 10 times lower at 15 ppm than at 0.5 ppm. We conclude that nasal secretions which increase with exposure concentrations of this irritant are responsible for this phenome- non. Animals fitted with a tracheal cannula and exposed while under anesthesia showed a much reduced arterial blood IAC uptake, too low to be due to the reduced minute ventilation-due to anesthesia. The reduced uptake is due primarily to bypassing the upper respiratory tract which we conclude to be the major site from which laC was absorbed in normal animals exposed to 14CHsNCO. The results show that it is impossible to extrapolate from the con- centration range used to lower or higher exposure concentrations. Supported under NIEHS 1R01-ES02747. .~-~• 613 A HISTOLOGICAL AND BIOCHEMICAL ANALYSIS OF THE IN VIVO TARGETS OF INHALED RADIOACTIVE METHYL ISOCYANATE. A L Kennedy, Y Alarie, and W E Brown, Carnegie Mellon Univ. and Univ. of Pittsburgh, Pittsburgh, PA. ' 'i 614 MIC is a highly reactive gas with documented physiological effects. Analysis of the distri- bution, cellular damage and fate of MIC.will add to our understanding of its mechanism~ of injury and physiological response. To examine the effects of MIC Vat3Pe subcellular level, guinea pigs were exposed, through inha1ttton, to a range of :;"~ublethal concentration"s•:.GOf radioactive MIC from 0.5 to 15 ppm. Me relative organ distribution of radioactivity, which was concentration independent, showed high levels in trachea, lung and blood, and lower but significant lAelein brain, liver, kidney and spleen. In all cases, clearance of radioactivity was rapid. Histological exam- ination of trachea and lung sections of exposed animals showed minimal ;epithelial damage except at 15 ppm. Inflamatory cell migration was also studied. Autoradiography of these sections showed that the label penetrated to the substrata beneath the respiratory epithelium and rapidly cleared in vivo. Biochemical analysis of respiratory tissues revealed that 90% of trachea and 70% of lung associated radioactivity was saline extractable. This indicated a significant noncovalent interaction of MIC or covalent reaction with a soluble respiratory factor. This work was supported by .grant OH-02214 to WEB.. APPLICATION OF SHORT-TERM LUNG BIOASSAYS TO RISK ASSESSMENT FOR METALS B D Beck) & J D Brain2, Gradient Corporation, Cambridge, MA) & Harvard Schl. Pub. Hith., Boston, MA2 Short-term pulmonary bioassays of metals were used to evaluate dose-response relationships and to assess complex mixtures. Published data were obtained on in vitro macrophage tests and on mouse infectivity models for the following metals:- As, Pb, Mn, Ni, Zn, V, Cd, Fe and Cu. Dose extrapolation from in vitro studies to maximum concentrations in human lungs was per- formed 4suming the volume of I gm of tissue was I ml and metals were evenly distributed in the lungs. Comparison of estimated in vivo concen- trations to in vitro cytoxic concentrations showed in vivo concentrations to be much less than those required to reduce in vitro macro- phage'viability by 50%. Extrapolation from infectivity models based on deposited dose showed that Cd, Cu and Zn (assuming metals were •present as salts) were present in human lungs at levels > those required to increase mortal- ity during infection by 20% in mice. Mixtures were evaluated by comparing effects of mixtures with those of individual metals. For oil fly ash, V could account for in vitro toxicity and V and Ni for infectivity, For Al smelter dust, Cu and As were greater than necessary to en- hance infectivity. In contrast, levels of metals in steel mill furnace particulates and in coal combustion fly ash could not account for tox- icity, suggesting a role for other constituents. 50875 8279 154

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