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PROGRESS REPORT Contract CTR-0030 9/1/79 - 8/31/80 Analysis of the reasons for death in this study are given in Table 8. Virtually 80: of animals were,taken off test by the impact of the•exposure system itself. These animal deaths were randomly distributed over the 104 week exposure period and the incidence of any histopathologic lesion for any 4 week period should reflect the incidence of that lesion. in the population of animals on test at that time. The abbreviations used for the lung lesions observed are given in Table 9. A selected sumnary is given in Table 10 of all pulmonary histopathology for all six groups of CTR-101A animals. 1. Exposure to 2R1 cigarette smoke alone. A total of 415 animals have been diagnosed in the smoke alone groups, a total of 295 in the sham exposed group, and a total of 66 in the shelf controls. The only smoke-associated lesion observed to date has been the accumulation.of pigmented alveolar machrophages (PAMA). Six lung carcinomas have been observed and they were found.in both smoke- and sham-exposed animals. Leukemia, lymphosarcoma, osteogenic sarcoma and reticulum cell sarcoma have also been observed, and these tumors also seem to be evenly distributed over both smoke- and sham-exposed groups. The actual time of occurrence of these various lesions for the smoke, sham or shelf groups is presented in Tables 11 through 16, respectively. 2. Exposure to 2R3 smoke after intratracheal inoculation of BaP. Lung lesions observed following intratracheal treatment with 1.2 mg BaP given with and without smoke exposure are summarized In Table 10. Several lung carcinomas were induced by BaP, including AAC, SCC. and POC. The total incidence of lung carcinomas was higher in the sham exposed compared to the smoke-exposed animals (67/162 vs 43/159). The only lesion which could be attributed to smoke exposure was PAMA. The incidence of these lesions at 4 week intervals is presented in Tables 17-22 for the smoke + BaP, Sham + 9aP, and shelf + BaP groups. Preliminary analysis of the data from those animals which died during the experiment and which are known to have died with a random dis- tribution (125 smoke exposed and 106 sham exposed animals) has been performed- using the Mantel-Haenszel statistical procedure. The results suggest that there are significantly more malignant lung cancers (AAC, SCC and POC) in the sham-exposed compared to s.oke-exposed animals (p-.0386). There is no difference in the time-dependent incidence in premalignant lesions (SN, ANCN, ACN) (p-.5535). Incidence of premalignant and malignant lesions was also not different between the two groups (pa.8145). Since many animals remain to be analyzed it is premature to draw firm conclusions. However, it seems that as we observed in the C'R-100 study, whole cigarette smoke has very little activity as a cocarcirogen with BaP at the dose used in this study. -3 - CTR CONTRRCT6 029269 11248019 CTR `4 M/ / 04/' 34•! t

PROGRESS REPORT Contract CTR-0030 9/1/79 - 8/31/80 C. CTR-101B. Chronic exoosure of BC3F1/Cum mice to 2R1'and 3A1 Ciaarette smoke. ' 1. Background During the last year, an exposure regimen was developed which was estimated to result in high TPH deposition (ti1 mg TPM/day) and low associated toxicity for'both 2R1 and 3A1 cigarette smoke (see discussion CTR-117). These exposure conditions allowed the scheduling of a long term study whereby the biological effects of smoke from both cigarette types could be simultaneously evaluated. Such a long term study employing this new smoke exposure regimen was proposed, approved and initiated during the 1980 contract year (this study was referred to as CTR-118 in the Proposed Studies for 1980, which i's now termed CTR-101B). In July, 1980, however, it was decided that this study should not be complPted as proposed.and that smoke exposure under these newly defined conditions would be limited to the length of time during which animals in CTR-101A are to be exposed to smoke. As a result, all smoke exposure studies at Microbiological Associates will•c.ease in June, 1981. The animals in CTR-lO1B 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. The short term markers to be analyzed in male and female mice are: a) inhibition of pulmonary DNA repair, b) stimulation of pulmonary DNA synthesis, c) induction of pulmonary AHH activity, d) ip- duction of pulmonary ODC activity, e) augmentation of the DNA damaging effects of particular lung carcinogens, and f),alteration of.certain physical characteristics such as lung weight and incidence of PAMA. The smoke exposure'regimen for this study is significantly different from other exposure regimen carried out in this laboratory. • There are no rest periods between successive cigarettes. • The exposure period•for smoke exposure is :5 seconds per minute. • Smoke expasures are repeated "continuously" over 140 minutes. • Each "run" results in exposure to the ecuivalent of C 17 2R1 or 3A1 cigarettes (8 puffs/cigarette). The disposition of animals in this study are presented in Table 23. A total of 417 mice are presently be,ing exposed to 3A1 smoke, 322 mice are being exposed to 2R1 smoke, 401 mice are being sham exposed, and 156 mice are shelf controls. These animals will be removed from test at 3, 6, 9, and 12 months of smoke exposure and analyzed for the 6 markers previously described. -4- CTR C0NTFZRCTS5 026270 11248020 CTR NN 044-7;'3~• A-1

PROGRESS REPORT Contract CTR-0030 9/1/79 - 3131/80 The precent COHb in these mice irmediately following this exposure regimen is presented in Table 24. These values for the 2R1 smoke exposea animals are approximately twice that for the mice in CTR-101A. The COHb values for the 3A1 smoke-exposed animals are similar to that observed in CTR-100. These data suggest that. much more smoke is being taken up by these animals during this new smoke exposure regimen. A more complete discussion of the biological effects of this smoke exposure regimen is discussed in CTR-117. 0. CTR-116. Comoara:'.ve toxicity of 2R1 and 3AI cicarette smoke. This study was to be implemented if the animals could not survive the continuous exposure regimen. Data presented in CTR-117 show that this continuous exposure regimen worked very well and CTR-116 was, therefore, not required. A comparison of the toxicity of 2R1 and 3A1 cigarette smoke is given in the discussion of CTR-96. E. CTR-117. Comoarative toxicit of 2R1 and 3A1 ci arette smoke when the rest oeriod between exoosures is e iminated. As discussed in CTR-101B, a new exposure regimen. has been utilized where the rest periods between successive cigarettes has been eliminated. TPM deposition per mouse has been estimated to be twice that deposition observed in CTR-101A while requiring the same amount of machine time. The effect of this smoke exposure regimen on BC3FI/Cum female mice after exposure to 2P.1 cigarette smoke Is given In Table 25. The toxicity was 0.9% per week. The toxicity observed for the exposure regimen used in CTR-lOlA was ti1.6S per week, while TPM deposition and percent COHb were approximately one-half that in this regimen. Thus, this exposure regimen seems to result in a higher dose of smoke taken up by the animals with less toxicity. This exposure regimen was also used to evaluate the toxic effects of smoke exposure to male BC3F1/Cum mice. Table 26 shows the toxic effects of 2R1 cigarette smoke in male 8C3F1/Cum mice. Tnxicity was ti1.8S per weex, which is higher than that observed in female BC3FI/Cum mice suggesting that male mice are slightly more sensitive to the toxic effects of 2R1 cigarette smoke (see Figure 2). The percent of COHb was similar in both female and male mice. Histopathology on female BC3F1/Cum mice over the first 20 weeks of smoke exposure is presented in Table 27. Twenty-three of 24 animals which were examined•after 1? weeks of smoke exposure were observed to have PAMA. This is in direct contrast to CTR-10IA, where high levels of PAMA have still not been observed after approximately 2 years of daily smoke exposure. These data indicate that this exposure regimen results in a higher dose of smoke to the mice, with a higher T?M deposition In the respiratory tissue. -5- CTR COHTRRCTS 028271 11248021 . CTR HN 044,349

PROGRESS REPORT Contract CTR-0030 9/1/79 - 8/31/80 II. SUPPORT SERVICES A. Dosimetry 1. CTR-119. Deoosition and distribution of 2R1 and 3A1 cigarette smoke usina the continuous smoke exoosure regimen. The results frcm'CTR-117 have shown that the new exposure regimen results in fairly low toxicity to BC3F1/Cum fe.male and male mice. The actual distribution and dosimetry of TP?4 from radiolabeled smoke from the 2R1 and 3A1 cigarettes under these exposure conditions are scheduled for CTR-119. This study has not been completed due to scheduling difficulties on the SEM II B and C, which have not allowed shut down of the machines for the necessary 3-4 day period in order to generate the radiolabeled smoke. It is anticipated that machine time will be available in early December for this experiment. As a part of the monitoring of th smoke generated for the dosimetry experiments, T?M, nicotine and 14C-DTC content of radio- labeled cigarette smoke was determined in our laboratory at MA and the data compared and verified with previous experiments at ORHL.(see below). The tissues for this dosimetry experiment will be analyzed for radioactive content using the new Packard 306 TRI-CARB tissue oxidizer. In order to insure that techniques and procedures have been adequately implemented with this instrument, dosimetry studies using the small Walton Horizontal Smoking Machine (WHSM) were scheduled. Data could then be compared with that of previous dosimetry experiments analyzed by tissue solubilization procedures. Results from both the smoke monitoring and the WHSM dosimetry studies are presented in the following sections. a. Analysis of smoke constituents. Nicotine (NIC) is the principal alkaloid present in cigarette smoke. It is associated with the particulate phase of smoke and is found with the total particulate matter (T?M) collected on a Cambridge filter pad. NIC was quantitated by extractions with various organic solvents to compare the extraction efficiency of nicotine from Cambridge filter pads. Table 28 shows that hot chloroform extraction from Cambridge filter pads gives 100% recovery, whereas 95%, 89% and 33% of recovery was found by using pyridine, ethanol, and p-dioxane, respectively. NIC in these solvent extracts was quantitated by utilized gas liquid chromatography (GLC,PERKIN ELMER, Model 3920 B). The extracts of Cambridge filter pads were dissolved in chloroform, and 1 ul and 2 ul were infected into the GLC equipped with a flame ionization detector. The column was a silanized 3 ft x 4 irm ID coiled glass tube packed with ~ 3% OV-17 on gas chrome Q (80-100 mesh, Supelco). The column and in,iec- tion port temperature were 1400C and 2000C, respectively, and detector oven temperature was 3000C. -6- CTR CCNTRRCTS Q28272 11248022 C T R I ~~N 0 - -1• 4 3 1: V' 0

PROGRESS REPORT Contract CTR-0030 9/1/79 - 8/31/80 L.. Helium gas was used as a carrier at a flow rate of approximately 40 ml/min and attenuator setting of 8 x 10. The NIC elution profiles shown in Figures 3 and 4 were compared with NIC standards (Eastman, Rochester, N.Y.) dissolved in chloroform. Figure 5 demonstrates the linearity response of a flame ionization detector against the various nicotine concentrations using an external standard method. Figure 6 indicates that it is feasible to use 3-methylindole as an internal standard for GLC analysis to quantitate the amount of NIC on the pads as well as in biological samples. NIC samples generated from reference cigarettes on a Phipps and Bird-Smoking Machine were analyzed both at ORNL and MA, to compare the analytical methods used at both laboratories. The results from both laboratories are shown In Table 29. Chloroform extraction and the 0V-17 chromatography column were highly efficient in determining the amount of NIC in these samples. The 20% recovery difference observed resulted from differences in inf ection techniques. In the laboratory at MA, the dead space volume of the injection syringe is accounted for, yielding higher values than if it is not accounted for. A correlation coefficient of 0.99 was obtained for the relationship between NIC and TPM for NIC determined at MA, while a correlation coefficient of 0.97 was obtained for the data obtained by ORNL. These analytical techniques have therefore been utilized to reproducibly quantitate levels of NIC generated in the smoke exposure• stud es at MA. Oata are presented in Table 30 for analysis of TPM, NIC and 4C-DTC levels generated from 14C-DTC-2R1 cigarettes on the WHSM. A11 of these experiments and subsequent analyses were performed at MA. These data demonstrate the direct relationship between increase In TPM, NIC and 14C-OTC-dpm with increasing smoke exposure (puffs per cigarette). The correlation between these three constituents is also demonstrated, indicating that any two measurements of TPH, NIC, or 14C-DTC-dpm can be used to monitor the levels of the third parameter. These data also point out the difference in NIC yield when cigarettes are burned on the WHSM, compared to cigarettes burned on the Phipps and Bird analytical sroking machine. On the WHSM cigarette butts are "free" to the atmosphere between puffs, while in the Phipps and Bird, the cigarette butts are "restricted" between puffs and are not exposed to the atmosphere. NIC yields are significantly higher from the WHSM, ccrpared to the yield from the Phipps and Bird (Stokely, J.R., Woneyhum, J.H., Guerin, M.R., Florant, G., and Greenspan, J. In Tobacco Smoke Inhalation Bioassay Chemistry, edited by M.R. Guerin, 3:R. Stokely and C.E. Higgins, ORNL-5424, 1979, pp 1-17). These data point to the need for moni.toring of the smoke generated under actual experimental conditions rather than extrapolating from the analytical data. It should be noted that the NIC levels in the smoke generated on the wHSH from 2R1 cigarettes are. approximately 1.5-2.0 times higher than when the smoke is generated from the Phipps and Bird. Since the cigarette butts are also "free" in the SEH II smoking machine, the levels of NIC were determined for smoke generated on the SEM U. -7- CTR CaHTRRCTS5 026273 11248023 C T R VIN 0 4 4,3 =5 I

PROGRESS REPORT Contract CTR-0030 9/1/79 - 8/31/80 The monitoring system for the SEM II has the capacity to instanteously and simultaneously determine TPM, COZ and CO levels in smoke. For determinations of nicotine or radioactivity present in smoke, the same Cambridge filter is analyzed which is used to filter the smoke for analysis of the gas phase constituents. The data in Table 31 summarize the measurements of TPM, C02, CO and nicotine in 2R1 cigarette smoke generated on the SEM II. The NIC/TPM ratio from 2R1 cigarette smoke is lower than that observed on the WHSM. The effect of the "free" vs. "restricted" configuration for the cigarette butt does not appear to be as great in the SEM II as in the WHSM, but again points to the necessity of monitoring the smoke actually generated for inhalation_experiments. b. 14C-DTC dosimetry on the WHSM. The previous section showed that techniques re now available for the rapid quantitation of TPM, nicotine and t4C-DTC in cigarette smoke. No preliminary 14C-DTC-dosimetry experiments were performed using the WHSM so as to become familiar with the tissue sample oxidizer and to be able to compare the data in terms of previous experi- ments andlyzed by tissue. solubilization. Data are presented in Tables 32 & 33 for TPM deposition and distribution in BC3F1/Cum female mice after exposure to 10S-2R1 cigarette smoke•using the 15/45 second (smoke/ air) exposure cycle. Mice were sacrificed by C02 asphyxiation immed- iately after smoke exposure. Tissues (lungs, trachea, larynx and turbinates) were removed and placed directly into combusto cones In scintillation vials, stored at -60oC until analyzed with TRI-CARB Sample Oxidizer (Packard, Downers Grove, Iil.). The procedures for sample combustion were those recommended by Packard Instruction Manual 2130/1. Combusted samples were analyzed for radioactivity using a Beckman LS-330 Liquid Scintillation System. All samples were corrected for background and quenching as determined by the External Standard Method. When mice were used which had not been previously exposed to smoke before the dosimetry experiment, a large coefficient of variation (CV) for lung deposition was observed (Table 32). However, when smoke "adapted" mice were used, the CY was reduced (Table 33) and a linear dose response for TPM deposition in the lung was observed as a function of total smoke exposure time (Table 33). The fifteen second smoke exposure interval on the WHSM results in a lower deposition than that estimated after exposure on the S-7M II. Techniques and procedures have now been established at MA to perform the large dosimetry experiment using the SEM fI. This experiment will quantitate.the TPM deposition and distribution in BC3F1/Cum mice after exposure to the "continuous" regimen discussed previously. This experiment will be completed by December, 1980. 2. CTR-105. Ceoosition, distribution and clearance of 3H- catechol (CAT) in BC3F1/Cum mice after exposure to 3H-CAT-2R1 cigar- ette smoke. Previous studies have examined the pharmacokinetics of quantities of BaP, MCA, UIC and DTC in mice after exposure to these aerosolized chemicals in cigarette smoke. -8- CTR CoNTRRCTS 028274 11248024 C T R N V 1 0 4 4'-~~.', -a

PROGRESS REPORT Contract CTR-0030 9/1/79 - 3/31/80 BaP, HIC and OTC are constituents of smoke found in the particulate phase and represent, respectively, the polycyclic aromatic hydrocaroons, the tobacco alkaloids and a straight chain hydrocarbons. BaP, MCA, and NIC were shown in previous studies to be rapidly cleared (th<2 hours) from the mouse lung, while OTC was retained at significant levels up to a week post exposure. CAT, 1,2-dihydroxybenzene, is of interest for these studies iri that it represents a fourth class of chemicals (diphenols), is present in moderately high concentration in cigaret:e smoke 220-550µg/cigarette, (Mold, J.D., Pepton, M.P., Means, R.E., and Walker, T.B., Analyst 91, 189, 1966), and has been reported to be a weak carcinogen. (van Ourren, B.C., Katz, C., and Goldschmidt, J. Naa . Cancer Inst. 51, 703, 1973). We report h re the pharmacokinetics of inhaled 3H-CAT in mice after exposure to ~H-CAT aerosolized in cigar- ette smoke. BC3F1/Cum female mice were exposed to 10% (v/v) smoke from 3H-CAT-2R1 cigarettes under standard conditions on the WHSM for a total of 300 seconds. Mice were sacrificed by C02 asphyxiation at the following times after exposure: 1:25, 2, 4, 4:25, 5:25, 6:30, 7, 7:60, 8:30, 9:25, 10, and 10:9 minutes. Levels of radioactivity in Individual tissues was determined by tissue oxidation (using a Packard TRI-CARB Tissue Oxidizer). 3H-CAT was rapidly cleared from the respiratory tract as shown in Figure 7. The half-life (t4) of H-CAT In the lung was less than 4 minutes. 3H-CAT was rapidly absorbed in the blood and rapidly removed (tk<4 minutes, Figure 8). Significant levels of radioactivity were found in the liver and kidney immediately after exposure (Figures 9 and 11). These levels decreased rapidly (th-. 4 minutes, Figures 9 and 11). Small amounts of radioactivity were observed In the spleen and pancreas (Figure 9) and brain and heart (Figure 10). Two hours after exposure, approximately 90% of the 3H label was excreted in the urine (Table 34). Less then 10% of the labeled compound remaine~ in the lung, turbinates, liver, or kidney (Table 34). Over 90% of the H label in the urine was not extracted by chloroforn- methanol (3:1 , v/v, Figure 12) or an ethyl acetate (Figure 13), suggesting that CAT was metabolized to another form before excretion. High performance liquid chromatoqraphy (HPLC) was used to analyze both the ultraviolet (UV) absorbing and radioTabeled metabolites in u-rine. As shown in Figure 13, free CAT Is not detected in urine from smoke-exposed mice. Following g-glucuronidase and arylsulfatase treatment of the urine, both UV and radiolabeled CAT were detected (Figure 14) in HPLC profiles. These data indicate that CAT is detoxified through conjugation as a glucuronide and/or a sulfate before excretion ine the urine. Enzymatic digestion of the urine from smoke exposed mice resulted in additional UV absorbing peaks in the HPLC elution profiles (compare Figures 138 and 148). Urine from mice on test in CTR 1018 was analyzed ` to determine whether these additional peaks were smoke-associated. :n Figures 15, 16 and 17 are presented thg. HPLC elution profiles of. enzyme treated urine from shelf controls, sham-exposed and smoke-exposed mice from CTR 101B. -9- CTR caKTRacTS a2-8275 11248025 rM I R ! I N •-/' , I43M5V/."

PROGRESS REPORT Contract CTR-0030 9/1/79 - 8/31/80 > The HPLC profiles of urine from shelf controls and sham-exposed mice are almost identical (Figures 15 and 16). The HPLC profile of urine from smoke exposed mice reveals many additional peaks, suggesting that many constituents inhaled in smoke are excreted in the urine in con- jugated form. Longer exposure of BC3F1/Cum mice to smoke aerosols containing 3H-CAT resulted in'deposition and distribution of label similar to that seen after shorter exposures. Data in Table 35 indicate that TPM deposition does not accumulate in the respiratory tract after 1200 seconds of exposure. TPH deposition as represented by 3H- CAT is observed in the liver and kidney immediately after this 1200 second exposure (Table 35). Data are summarized in Table 36 for the percent distribution of the four smoke constituents, DTC, NIC, BaP and CAT, in the internal tissues of the mouse after exposure to whole cigarette smoke. Deposition in the lung of the four particulate constituent ranges from 2.5 to 69%. TPM can be represented by all four of these chemicals but different results are observed for the deposition and'distribution of each, thus indicating the complex nature of cigarette smoke and the potential difficulties in ascribing biological activity to cigarette smoke. B. Distribution of Cigarette Smoke Constituents 1. CTR-T06. Autoradiograohic analysis of lung tissues. Autoradiography of lung tissue after appropriate exposure to radioactively labeled chemicals allows determination of the deposition and distribution pattern within the lung. After exposure of the mice to 14C-DTC aerosolized in 2A1 or 2R1 cigarette smoke, we demonstrated that the tracer could be seen to have reached all portions of the lung and was somewhat concentrated along the bronchi and bronchioles (Progress Report, 1979). Since the technique of using frozen cut sections without organic solvents has been reproducible and routine in our laboratory, we wished to extend these studies to determine the irrmediate localiza- tion of either 3H-tetradeconyl phorbol acetate (TPA) generated as a chemical aerosol or 3H-CAT, generated both in a smoke aerosol and as a single chemical aerosol. Data from CTR-105 demonstrated that the half-life of 3H-CAT in the lung was less than 4 minutes. Due to rapid clearance from the lung wnich occurs with 3H-CAT and low quantities of radioactivity found in pulmonary tissue, autoradiographic analysis was not attempted. Should hi gher quantities of radioactivity be observed after aerosolization of 3H-TPA or 3H-CAT from ethanol, lungs from the aerosol exposed mice will be quick-frozen and autoradlograpnic analysis performed. L -10- CTR CoNTRRCTS o2e276 11248026 CTT%'> I-IN 0443 -4

PROGRESS REPORT Contract CTR-0030 9/1/79 - 3/31/80 2. CTR-107. 3H-Thymidine labelina index (LI) in luno, trachea, liver, biadder, saleen or kidney after exoosure to srroKe. Results obtained in collaboration with Dr. R. Ramussen (University of California at Irvine, Irvine, CA) suggested that one of the effects of exposure of mice to 2A1 cigarette smoke is an increase In the fraction of cells in the lung in which 3H-Thymidine (3H-TdR) is incorporated (Progress Report, 1979). These data are presented as a labeling index (LI), defined as the number of cells labeled with 3H-TdR divided by the total number of cells examined x100Y. The LI is deter- mined from autoradiographic analysis of sections of fixed tissue. Since in most eukaryotic cells the 3H-TdR-LI is proportional growth rate, measurement of LI was undertaken to further investigate the effect which cigarette smoke might have on cellular turnover rate in the lung and other tissues. Experiments performed in collaboration with Dr. Rasmussen and reported previously have indicated that pulmonary replicative DNA synthesis (RDS) was stimulated after exposure of mice to smoke from 2A1 cigarettes but not 2R1 cigarettes (Progress Report, 1979). To Investigate these observations, the LI was determined in BC3F1/Cuar female mica after exposure to three different cigarette types, 2A1, 3A1 and 2R1, as part of a rather large comprehensive experiment with Dr. Rasmussen. Results and details of this experiment, Including the LI data, are discussed under CTR-96, however, the data suggest that the LI is 2-3 fold higher in lung tissue from mice exposed to 2A1 or 3A1 cigarette smoke, compared to lung tissue from mice exposed to 2R1 cigarette smoke or sham exposed. These effects were observed after 9 and 13 weeks of exposure. Tissues from mice exposed for 24 and 41 weeks are under investigation. 3. CTR-108. Effect of cigarette smoke on pulmonary mixed function oxidases. Measurement of MFO activity in lung tissue is a rapid, sensitive and reproducible method to biochemically assess the effect of exposure of various chemicals to pulmonary tissue. The substrates used for these assays are either BaP or ethoxyresorufin. An advantage to the latter chemical is that unexposed or control tissues have little capa- city to metabolize this chemical and, therefore, low levels of induced MFO can be detected. MFO levels have been determined in pulmonary tissues of BC3F1/Cum mice after exposure to smoke from 2A1, 3A1 and 2R1 cigarettes. Results and details of this experiment, including the Induction of MFO, ,• are discussed under CTR-96, however the data suggest that the AHH levels in the smoke exposed lung tissues are 7-8 fold higher than the lung tissue from sham-exposed mice. Further, the level of induction was constant over 24 hours after exposure to smoke from all three cigarette ~ types. CTE2 CONTRRCTS 0282 77 11248027 CTR NN 0443' r,."~ 5

PROGRESS REPORT Contract CTR-0030 9/1/79 - 8/31/80 As discussed under CTR 101B, the levels of pulmonary and hepatic MFO will be determined in BC3F1/Cum female mice after exposure to 3A1-and 2R1 cigarette smoke for 3, 6, 9 and 12 months using the "continuous" exposure regimen. Data will thus be available concerning whether differences exist in HFO-induction kinetics or levels following exposure to different cigarettes using different smoke exposure regimens. C. Short-term Assays Relating to Possible Initiating Events. 1. CTR-96. Inhibitton of oulmonar DNA reoair caoacity after exposure to cicarette smoke (collaboration with Dr. R. Rasmussen). a. 'Introduction Collaborative studies with Dr. R. Rasmussen (University of California at Irvine, Irvine. CA) continue to further characterize the effects of whole cigarette smoke on the capacity of pulmonary tissue to repair DNA damaged in an in vitro assay. Previous results indicated that chronic exposure of BC3F1/Cum mice to 2A1 cigarette smoke resulted in an inhibition of DNA repair as determined by unscheduled DNA synthesis (UDS) and that this inhibition persisted for at least 6 months after exposure was terminated (Progress Report, 1979 and Appendix A, Rasmussen, R.E., Boyd, C.H., Dansie, D.R., Kourt, R.E. and Henry, C.J., DNA Repli- cation and Unscheduled DNA Synthesis in Lungs of Mice Exposed to Cigarette Smoke, submitted to Cancer Research, 1980). When these studies were extended to include exposures using 2R1 cigarettes and C3H/Anf Cum mice, in addition to BC3F1/Cum mice, no inhibition of UDS was observed up to 27 weeks exposure (Progress Report, 1979). To investigate these observations a rather large comprehensive study was proposed last year and the results are presented here. b. Experimental Protocol The SEM II 8 and C were used to generate smoke from 2A1, 3A1 and 2R1 cigarettes. Groups of BC3F1/Cum mice, 8-10 weeks old, were adapted to smoke from all three cigarette types. In the following table the exposure conditions are summarized for each cigarette, all utilizing 10% (v/v) smoke concentration and standard exposure ccnditions of 2 second puff duration, 35 ml average puff volume: Cigarette Type Exposure Cycle Smoke/Air (sec) Total Smoke Exposure/ Day (sec) Number of Cigarettes 2A1 30/30 2400 10 3A1 30/30 2400 10 2Rl 20/40 1600 10 Sham 0 0 0 Shelf 0 0 0 -12- CTR CaNTRRCTS 02E3278 11248028 C.; T R N t 4 0 4, 4, °~G