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MICROBIOLOGICAL ASSOCIATES PROGRESS REPORT FOR CTR-0030 - SMOKE INHALATION STUDIES IN MICE SEPTEMBER 1, 1979 - AUGUST 31, 1980 . l. L ~-- a--.). EXHIBIT S. NELSOH CTR coNTRacTS 028259 11248009 a CTR HN 044. 337`

PROGRESS REPORT Contract CTR-0030 9/1/79 - 8/31/80 TA3LE 0P CONTENTS Page Abbreviations i Synopsis of the Progress Report ii I. Chronic Inhalation Studies A. Introduction 1 B. CTR-101A. Chronic exposure of BC3F1/Cum mice to 2R1 cigarette smoke. 1 C. CTR-101B. Chronic exposure of BC3F1/Cum mice to 2R1 and 3A1 cigarette smoke. 4 0. CTR-116. Comparative toxicity of 2R1 and 3A1 cigarette smoke. 5 F. CTR-117. Comparative toxicity of 2R] and 3A1 cigarette smoke when the rest period between exposures is eliminated. 5 11. Support Services A. Dosimetry 1. CTR-119. Deposition and distribution of 2R1 and'3A1 cigarette smoke using the continuous smoke exposure regimen. 6 2. CTR-105. Deposition, distribution and clearance of 3H-catechol (CAT) in BC3F1/Cum mice after exposure to 3H-CAT-2R1 cigarette smoke. 8 B. Distribution of cigarette smoke constituents 1. CTR-106. Autoradiographic analysis of lung tissues. 10 2. CTR-107. 3H-thymidine labeling (LI) index in lung, trachea, liver, bladder, spleen or kidney after exposure to smoke. 11 3. CTR-108. Effect of cigarette smoke on pulmonary mixed function oxidases. 11 C. Short-term assays relating to possible initiating events. 1. CTR-96. Inhibition of pulmonary DNA repair capacity after exposure to cigarette smoke (Dr. R. Rasmussen). 12 2. CTR-82A. Characterization of pulmonary cytochromes involved in smoke-associated MFO-induction (Or. I. uang). 15 3. CTR-109. Alteration in the immune response after exposure to cigarette smoke (Or. H. Herscowitz). 16 4. CTR-126.-Oocyte toxicity and atresia studies (Dr. 0. Mattison). ' 17 CTR caNTRRCTS 026260 11248010 C/ ! p /M! N 044'1.Yw• •WMM^C3

PROGRESS REPORT Contract CTR-0030 9/1/79 - 3/31/80 Page 5. CTR-127. Alkaline elution assay (CTR Grant !1?41). 19 6. CTR-128. Evaluation of chromosomal damaae or alteration after ex?osure to whole ciqarette smoke using a sister chromatid exchange (SCE) assay (Dr. W. Benedict). 21 0. Feasibility of short tesrrn assays relating to possible tumor-promoting events. CTR-111. Ornithine decarboxylase (ODC) induction as a marker of promotion in pulmonary tissues. 22 E. Aerosol studies 1. Introduction. 26 2. CTR-114. Deposition and retention of aerosolized TPA. 26. 3. CTR-115 and 123. Feasibility studies for aerosolization of selected chemicals and solvent vehicles. 26 4.' CTR.121. Aerosol dosimetry studies with 3H-catechol. 27 5. CTR-122. Studies with nicotine sulfate aerosols. 27 Tables 29 Figures 78 III. Appendices Publications 118 CTR CaNTRRCTS 028261 11248011 C/ / ! H/ f W' 1 I• 1r~•3.d" •

PROGRESS REPORT Contract CTR-0030 9/1/79 - 8/31/80 A83REVIATIOt:S L AHH BA BaP, BP BaP-7,8-diol CAT COHb CSC Co nA DMBA OTC, 14C-DTC EH ETR FA HA Hb HPLC 3H-TdR IT Igu IP LI LPS MCA MFO MMS NIC ODC ORNL PAG, PAGE PAH PFC PHA P&I RDS SCE SEM SRBC TCDO TPA TPM UDS UV WHSM aryl hydrocarbon hydroxylase benz(a)anthracene benzo(a)pyrene (s)trans 7,8-dihydroxy-l,8-dihydrobenzo(a)pyrene catecnoi carboxyhemoglobin cigarette smoke condensate concanavalinA 7,12-dimethylbenz(a)anthracene doctriacontane epoxide hydrase ethoxyresorufin flucir.olone acetonide hemagglutinating hemoglobin high performance liquid chromatography 3H-thymidine intratracheal irtmunoglobulin G intraperitoneal labeling index lipopolysaccharide 3-methylcholanthrene mixed function oxidase methylmethanesulfonate nicotine ornithine aecarboxylase Oak Ridge National Laboratory polyacrylamide gel electrophoresis polycyclic aromatic hydrocarbons plaque forming cells phytohemaggiutinin Process and Instruments replicative DNA synthesis sister chrocoatid exchange Smoke Exposure Machine sheep red blood cells tetrachlorodibenzodioxin tetradecanoyl phorbol acetate total particulate matter unscheduled ONA synthesis ultraviolet Walton Horizontal Smoking Machine i CTR caNTRRCTS 028262 11248012 VwM' l I w / MI I / lrr' /4' 4rrN' 4, .yr~'

PROGRESS REPORT Contract CTR-0030 9/1/79 - 8/31/80 SYNPOSIS OF PROGRESS .I. CiiRONIC INHALATION STUDIES .A. Eauioment. 1. A sham exposure machine was designed, developed, fabricated, delivered and tested. 2. The smoke exposure systems were upgraded by the addition of an additional animal containment unit, an improved air flow monitor- ing system with.temperature ccmpensated thermistors and direct reading air flow gauges. .3. Animal holding trays were evaluated, in terms of reducing neck abrasions., Inserts to cushion the neck edge and allow size changes were designed and developed. Fabrication was not completed due to the phase out of these inhalation studies in 1981. :4. An operation and service manual was drafted and 4 of 7 sections are complete. A fifth section is in final draft stage. B. Chronic Exposure of BC3F1/Cum Mice to 2R1 Cigarette Smoke. .1. As of October 24, 1980 (approximately 25 months of exposure), 389, 276 and 178 animals remain in the smoke-exposed, sham-exposed and shelf control, groups, respectively. :2. Smoke and sham exposures will continue for ti120 weeks (ti27 months). Exposures will be stopped at this time and remaining animals be allowed to die "naturally'. January 1981, February 1981 and June 1981 will be 020 weeks for mice on test since September 1978, October 1978, and February 1979. respectively. .3. All animals in CTR 101A will be removed from smoke and sham exposure by June 1981. 4. Histopathology of randomly selected groups of animals has sug- gested that the only smoke-associated lesion observed to date has been PAHA. Six lung carcinomas were observed in both the smoke and sham- exposed groups. ;5. The capacity of 2R1 cigarette smoke to alter BaP induced lung cancer is also on test. uata to date Indicate a higher incidence of lung carcinomas in the sham exposed animals compared to the smoke exposed animals. The only lesion which could be attributed to smoke exposure was the PAMA. ii CTR CaNTRRCTS 028263 11248013 CTR I I l 1 044341

PROGRESS REPORT Contract CTR-0030 9/1/79 - 8/31/80 ) C. Chronic Exoosure of BC3F1/Cum Mice to 2R1 and 3A1 Cigarette Smoke. l. A new exposure regimen was developed which resulted in higher TPM deposition and low associated toxicity for both 2R1 and 3A1 cigarette smoke. .2. A long term study employing this regimen was proposed, approved and initiated during this contract year, however, in July 1980 it was 'decided this study riould not be completed as proposed. Rather, smoke exposure using these newly defined conditions would be limited to the length of time during which animals in CTR 101A are to be exposed. Thus, all smoke exposure studies at MICROBIOLOGICAL ASSOCIATES will cease in June 1981. .3. The animals on test In this experiment will be used to evaluate the effects of 2R1 and 3A1 cigarette smoke on 6 short term markers at 3, 6, 9 and 12 months of smoke exposure. Both male and female mice will be evaluated. II. SUPPORT SERVICES A. Oecosition and Distribution of Whole Smoke in Model Animal Systems. .1. Analytical techniques h ve been utilized to monitor and quantitate the levels of TPM, NIC, ~4C-DTC and 3H-CAT in cigarette smoke. The procedures have been performed at MA. .2. Methods for reproducibly measuri.ng the radioactive content of tissues after exposure to radiolabeled cigarette smoke (or aerosols) have been determined using the Packard TRI-CARB Tissue Sample Oxidizer. .3. The deposition, distribution and clearance of 3H-CAT has been determined in mice after exposure to 3H-CAT-2R1 cigarette smoke. The half-life for 3H-CAT In the lung is <4 minutes. Over 90% of the radiolabel is found in the urine, conjugated as a glucuronide or sul- fate, within two hours after exposure. 4. Urine from smoke-exposed mice can be shown to contain several UV absorbing species compared to sham-exposed or shelf control mice. .5. Four smoke particulate constituents, DTC, NIC, 8aP and CAT, were shown to have widely different deposition and distribution patterns in mice exposed to whole cigarette smoke. B. Short-Term Effects of Exoosure to Cigarette Smoke. .1. AHH Pulmonary AHH,levelsare 7-8..fQ.ld higher in smoke exposed mice compared to controls after exposure to 2A1, 3A1 or 2R1 cigarette smoke. . iti CTR caNTRacTS 028264 11248014 L.I' 7 R l 1 N 'i./' 44ti..p 41~v+

PROGRESS REPORT Contract CTR-0030 9/1/79 - 3/31/80 2. DNA repair (Dr. R. Rasmussen) Unscheduled DNA synthesis was inhibited in pulmonary tissues from mice exposed to 2A1 ano 3A1 cigarette smoke, but not 2R1 cigarette smoke. Replicative DNA synthesis was stimulated after exposure to all three types of cigarette smoke. 3. Labeling Index The LI for pulmonary tissue was 2-3 fold higher in mice after exposure to 2A1 and 3A1 cigarette smoke, compared to pulmonary tissue from sham-exposed or 2Q1 cigarette smoke exposed mice. 4. ODC Exposure to smoke from 2A1, 3A1 or 2R1 cigarettes resulted in a 2-3 fold induction of ODC 6-9 hours after the final exposure. 5. Immune competence (Dr. H. Herscowitz) Acute or chronic exposure of BC3F1/Cum or BALB/c mice to 2R1 cigarette smoke failed to suppress the splenic PFC response. .6. Oocyte depletion (Dr. 0. Mattison) The rate of oocyte depletion was not influenced in ovaries of mice exposed to either 2A1 or 2R1 cigarette smoke compared to sham- exposed or shelf control animals. .7. DNA damage (Grant 11241) Exposure to 2A1 cigarette smoke resulted in minimal effects on pulmonary DNA compared to sham exposed animals. Enhanced damage to pulmonary DNA was observed in smoke exposed mice treated with BaP and BaP-7,8-diol compared to sham-exposed BaP treated mice. 8. Sister chromatid exchange (Dr. W. Benedict) Preliminary results indicate an. :SCE• rate which is 2 fold higher in mice exposed to 3A1 cigarette smoke, compared to sham-exposed mice. ,9. Lung weights _ Exposure to 2A1 and 3A1 cigarette smoke resulted in an increased lung iveight, compared to sham-exposed of 2R1 cigarette smoke exposed mice. iv CTR CONTRRCTS Q26265 11248015 CTR I-IN 044~~ 43

PROGRESS REPORT Contract CTR-0030 9/1/79 - 8/31/80 C. Aerosol Studies 1. Heasurements of aerosol concentration as a function of solution concentration have been completed for two chemicals. 2. Particle size distribution have been initiated using a 7 stage cascade impactor. 3. Preliminary calculations of a"1 ng deposition factor" have been made for 34-CAT after exposure to ~H-CAT-2R1 cigarette smoke. v CTR caNTRacTS 028266 11248016 CTR HN 044,`344

PROGRESS R:?ORT Contract CTR-0030 9/1/79 - 8/31/80 I. CHRONIC i`HALATION STUDIES A. Introduction The chronic cigarette smoke exposure studies were divided into three areas: a) chronic exposure of BC3F1/Cum female mice to 2R1 ciga- rette smoke (CTR-101A), b) comparison of effects of exposure to 2R1 or 3A1 cigarette smoke in BC3F1/Cum female mice (CTR-101B), and c) de- velopment of a new smoke exposure regimen* which eliminates the rest period (CTR-117). These studies were initiated as scheduled and are on- going at this time. Other studies were also completed during this funding year which were /n preparation for the studies comparing the effects of 2R1 and 3A1 cigarettes. :n March, 1980, a sham exposure machine was delivered to MA from Process and Instruments (P&I. Brooklyn, NY). The sham exposure machine duplicates the performance of the SEli II smoke exposure machines without generating whole cigarette smoke. Control animals can thus be treated exactly as the smoke exposed but without exposure to smoke ("sham- exposed°). The availability of this machine allowed the SEM II B and C machines to be scheduled full time for smo.ke generation, with the control animals sham-exposed on the sham machine. In this way, the capacity for Inhalation studies was greatly increased and the biological effects of both cigarette types could be analyzed simultaneously and with sham- exposed controls. The smoke exposure systems were upgraded by the addition of an additional animal containment unit .(a total of four are now available), improved air flow monitoring with temperature compensated thermistors, and direct reading air flow gauges. The servicing of the exposure systems were also upgraded by the development of the operation and service manual. The production of operation and service manual has been coordinated by Mr. Tom Gayle at ORNL, with assistance provided by Mr. Doyle Mullinax at MA and Mr. Leroy Florant at P&I. The manual provides schematic wiring diagrams and specifications for all components, detailed instructions for operation, ir.cluding trouble shooting sections. Four of the seven sections of the manual are complete (Table 1). :opi'es of the final drafts have recently been distributed to CTR. PSI, and MA. Section IV, Animal Handling Procedures and Techniques is in the final draft stage. The last two sections to be prepared are scheduled for completion in 1981. The tables of contents for Sections I-I'/ are given in Tables 2-5. The manual represents.the culmination of the ' development of not only the sophisticated smoke generation monitoring and exposure system, but the procedures and techniques for conducting long- term inhalation experiments with animals. B. CTR-:01A. Chronic exDosure of BC3F1/Cum mice to 2R1 cioarette smoke. This study was initiated in September, 1978, with a total of 2053 mice exposed to 2R1 smoke alone, a total of 1014 mice sham-exposed, and a total of 449 mice for shelf controls. -1- C1"R caHTRRCTS 028267 11248017 Md' /-R IMII / V• / I 3! Vu.'

PROGRESS REPORT Contract CTR-0030 9/1/79 - 6/31/80 An additional 710 mice were utilized to evaluate the cocarcinogenic effects of 2R] cigarette smoke and benzo(a)pyrene (BaP). As of October 24, 1980, a total of 389, 276, and 178 mice remain on test in the smoke, sham, and shelf control groups, respectively. Of the 389 smoke- exposed mice, 80 have been on test since September, 1978, 147 since October, 1978, and 122 since February, 1979. At monthly intervals, animals were weighed to ascertain their gen- eral health status. The average weights of smoke-exposea, sham-exposed and shelf control'animals at four weekly intervals over the first 80 weeks of the study are presented in Figure 1. There was a significant suppression of weight gain in both the sham and smoke-exposed'animals compared to shelf controls. 7here was no difference in weight gain between the sham and smoke exposed animals. As observed earlier in CTR-100, daily exposure and restraint on either the SEM II or sham exposure machines results in significantly slower weight gain. After 20-30 weeks of exposure, certain animals were observed to have reddened skin and worn away hair around the neck area which fits Into our "stock-type° holders. These- lesions progressed to open sores by 40-60 weeks of daily restraint. Approximately 90% of the animals express some level of neck abrasions by 80 weeks of exposure and 30-40% of these lesions are quite severe. Polycarbonate inserts to cushion the neck slot and of varying sizes were analyzed and found to be quite effective in limiting these neck-cuts, however, the scheduled fabrication and use of these inserts was not carried out because of the decision to discontinue any further life-time smoke-exposure studies. It is our opinion that these inserts must be used for any long term studies which may utilize animals of different ages and/or sizes. At monthly intervals, 3-5 mice per group were tested for carboxy- hemoglobin (COHb) levels immediately after smoke or sham exposure. The percent COHb at 4 weekly intervals is presented in Table 6. Every mouse ' which was exposed to 2RI cigarette smoke expressed high•levels of COHb with the average being %.18X COHb over the ti2 year observation period. No animal in the snam or in the shelf control groups expressed significant levels of COHb. These results indicate that theanimals were unable to avoid smoke exoo- sure. The level of smoke exposure has been documented throughout the experiment. Other studiei in our laboratory have shown that there is a high correlation between COFb levels in an animal and the level of total particulate matter (TPM) deposited in the respiratory•tract of the smoke exposed animals (r-.8, p<.01). Thus, these data confirm that this smoke exposure system generates smoke in a manner which results in the reproducible deposition of both particulate phase and gas phase constituents in the respiratory tract of the exposed animal. The disposition of all animals for CTR-101A is given in Table 7. A total of 1177 mice have been histopathologically examined and another 485 are being processed. Every individual animal has been accounted for. -2- CTR CONTRACTS 028268 11248018 CT~"~ PIN 04#4346