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HT10123005 MICROBIOLOGICAL 4SSOClATES PROGRESS REPORT CONTRACT CTR-0030 FOR THE PERIOD September 1, 1978 - Apri 1 1, 1979 C. J. Prepared By Henry, Ph.D. R. E. Kouri, Ph.D. L. M. Schechtman, Ph.D. R. 0. Curren, Ph.D. M. Dinowltz, Sc.D B. Bhooshan, Ph.D. April 1 1, 1979 ~%~ F(. •61 • ~rv+ i ~_ . ames M. u w c Director of Contract Administration . ~ . icar our, . . Director of Research

1 Contract CTR--0030 HT®0123006 TABLE OF CONTENTS Page S ynops i s i I. Introduction I II. Update on Ongoing Studies on the Bioevaluation of Risk to Low Nicotine-High Tar Cigarettes: CTR-100 (1-100) Cocarcinogenesis and Ch ronic Inhalation of 2A1 Cigarette Smoke 2 Tables and Figures for I-100 9 III. Update on On going Studies in the Bioevaluation of Risk to High Nicotine-High Tar Cigarettes. A. CTR-101 (C-101A). Chronic exposure of BC3F1/Cum mice to cigarette smoke. 37 B. CTR-102. De position, dosimetry and ch ronic smoke exposu re using 2R1 cigarettes compared to 2A1 cigarettes In BC3FI/Cum mice. 40 C. CTR-103. Evaluation p4 SEM II animal contain- ment capacity using C-DTC-zR1 cigarettes 42 D. CTR-104. Deposition and dosimetry of14C-DTC- 3A1 cigarette smoke In BC3F1/Cum mice. 42 E. CTR-105. Comparative evaluation of deposition and dosimetry after exposure to aenaphthylamine or catechol In a smoke aerosol. 43 F. CTR-106. Autoradiographic analysis of lung tissue after exposure of mice to 2R1 cigarette smoke. 44 G. CTR-107 3H- Thymidine labeling index (LI) in lung, liver, bladder, or kidney after exposure to smoke or t reatment with carcinogen. 45 (Dr. R. Rasmussen). H. CTR-108. The effect of cigarette smoke from different types of cigarettes on pulmonary riixed function oxidases (MFO) levels in three strains of mice. 46 I. CTR-96. Inhibition of pulmonary DNA repair capacity afte r ex osure~ to cigarette smoke. (Dr. R. Rasmussen~. 47

Contract CTR-0030 HIT®0123007 Page J. CTR-82. Characterization of pulmonary cyto- chromes involved in smoke associated MFO induction (Dr. i. Wang). 49 K. CTR-109. Alterations In immune response after exposure to cigarette smoke. (Dr. H. Herscowi tz) 50 L. CTR-110. Evaluation of adaptation and stress i n the i nbred s t ra i ns of m i ce expos ed to cigarette smoke. (Dr. W. Essman) 51 M. CTR-111. Comparison of smoke-induced ODC in three strains of mice and discrimination between ODC induction and AHH induction by cigarette smoke. 52 N. CTR-112. Evaluation of feasibility of a focus enhancement assay. 56 0. CTR-113. Acute toxicity of aerosolized TPA In the inbred strains of mice. 58 P. CTR-114. Deposition and retention of aeros olized TPA. 60 Q. CTR-115. Feasibility study of biochemical response to aerosolized chemicals. 64 Tables and Figures 65 IV. Appendix A 104 V. Budget 111

a Contract CTR-0030 HT®0123008 SYNOPSIS OF PROGRESS: September 1, 1978 - Apri 1 1, 1979 A. Evaluation of risk due to low nicotine - hi h tar ci arettes; c ron c exposure o_ C F Cum m ce to c garette smoce. This experiment was originally designed to continue through S eptember, 1978, but has been extended due to the survival of the animals. it is anticipated that the experiment will be completed by June, 1979. 1. As of April 1, 1979 (approximately 19 months of exposure), 20 animals remain in smoke exposed groups. 2. No neoplastic lesions have been observed in the smoke- a 1 one ex'posed groups. 3. Such lesions as pulmonary alveolar macrophage accumu- lation, squamous metaplasia of trachea, rhinitis, otitis media, and otitis externa have been observed only in the smoke exposed groups of animals. (One machine control animal was observed to have pulmonary alveolar macrophage accumulation). 4. Pathology on all organs has been comr~leted up to 60 weeks on test and all lung specimens should be completed by approximately May 15, 1979. 5. Both smoke exposed and machine control animals fail to gain weight in contrast to the shelf control animals. 6. Exposure to whole cigarette smoke did not influence the in cidence or severity of the lung cancers induced by IT instilled 3-methylcholarthrene (MCA). Data has been analyzed by probability analysis. B. Evaluation of risk due to hi h nicotine-hi h tar ci arettes; c ron c exposure o BG F um m ce to eRl c garette_smo_e. 1. As of April 1, 1979 (approximately 6 months of exposure), 1278, 898 and 449 animals remain in the smoke exposed, machine control, and shelf controls, respectively. 2. Current death rates indicate that this study is proceeding almost exactly as scheduled, so that approximately 300 animals will be at risk per group after 24-30 months on tes t. 3. Analysis of the circumstances surrounding the deaths so far observed suggest that approximately 50% die on the module itself (either smoke overdose or improper air flow) and approxi- mately 25% die because the animal's head moves too freely In the holders so that non-alignment with the module results. Three ongoing supplemental studies should resolve which technical modifications can be used to decrease this death rate. Data should be availabl6 by June 14, 1979. -i-

r Contract CTR-0030 ,yi®0123009 4. Pathological examination of some of these dead animals yielded no identifiable reason for the death. Routine patholo- logical examination of the smoke exposed animals will begin after one year exposure (approximately September, 1979). 5. Ability of 2R1 cigarette smoke to alter BaP-induced lung cancer is on test with no results to date. C. Deposition and distribution of whole smoke in model animal s ys tems . 1. The 2A1 and 2R1 cigaret tes contain similar levels of tar and deposition studies reflect similar levels of total partic- ulate matter (TPM) in mice after exposure to either 2A1 or 2R1 cigarette smoke. 2. Approximately 90% of the TPM taken up by mice is depos- ited in the respiratory tract. 3. Approximately 100 µg TPM is deposited per lung per cigarette. 4. Exposure for 3 weeks, 3 months or 6 months to cigarette smoke failed to alter this deposition, either in quantity or distribution. 5. Deposition is dependent on time of exposure, and con- centration of smoke, but indepe ndent of strain of mouse. 6. TPM is distributed alon g major airways, with lictle deposition in the te nninal alveoli. This is the same distri- bution pattern that has been in ferred for man. U. Update on the standard lung carcinoma model system. 1. MCA alone produced a va riety of pulmonary lesions, with at least 54 different combinations of lesions observed In animals randomly sacrificed over the ,fi rst 40 weeks following MCA treatment. 2. Data analysis procedures for these serially sacrificed BC3F1/Cum mice have been developed and data suggest that: a. There is a progression of alveologenic lesions from preneoplasia to frank neoplasia through at least four well-defined intermediate steps. b. No real progression of s quamous lesions could be documented. These lesions seem to grow rapidly and quickly result in the death of the animal. c. More multiple tumors are observed as the time after MCA treatment increases. , 3. The alveologenic compre.s sing nodule, the alveologenic -i i-

Contract CTR-0030 ''T®01?301P adenocarcinoma, and the squamous cell carcinomareadily transplant into newborn BC3F1/Cum mice. 4. At equivalent doses of MCA, the BC3F1/Cum and C3H/Anf strains are similarly susceptible to MCA-induced tumors. 5. There is a dose response to MCA-induced tumors for doses of MCA of 750, 1500 and 2250 µg MCA. 6. The assay of different chemicals for their ability tu induce carcinomas in BC3F1/Cum shows MCA > 7,B-dihydr:),-7,8- dihydroxybenzo(a)pyrene > benzo;a)pyrene. E. U date on romotion of carcinogenesis - in vivo and in vitro stu ies. 1. Standard procedures for che assay of pulmonary ornithine decarboxylase (ODC) activity have been developed. 2. ODC increases 20-40 fold following exposure to IT or ae rosol administered TPA. 3. This increase Is observed In BC3F1/Cum, C57BL/6, C3H/Anf or DBA/2 strains of mice. 4. Both 2111 cigarette smoke exposed and machine control animals were observed to have Increased ODC activity relative to shelf control animals. 5. The in vitro enhancement of the transformed phenotype Is a promotion assay that is influenced by the following parameters: a. Ratios of normal to transformed cells. b. Preferential toxicity of test chemical to normal or transformed cells. F. Aerosol studies 1. Aerosols of TPA, nicotine, BaP, ~-naphthylamine, and catechol have been generated. 2. LD50 for aerosoiized TPA is approximately 470 ng and is similar f9r both BC3FI/Cum and C57BL/6 Cum strains. 3. LD50 for aersolized nicotine is approximately 4-5 µg and is similar for both BC3FI/Cum and C3H/Anf strains. 4. Intermitten exposure to aerosolized nicotine yields an LDSOof approximately 8 µg. I -ili-

Contract CTR-0030 N TG0123011 5. Dosimetry of aerosolized TPA shows: a. Approximately 50% deposited in lungs. b. Approximately 75% deposited in total respiratory tract. c. in vivo half life of approximately 20 minutes fo`r tT`lungs, and approximately 10 hours fcr the total body. d. A very even distribution throughout the lung -- in contrast to the central hylar distribution of IT-instiiled TPA. G. Collaborative proiects. 1. DNA repair (with Dr. R. Rasmussen). a. 2A1 cigarette smoke (approximately 1.0 mg TPM/day for 15-20 weeks) inhibits DNA repair in BC3FI/Cum mice -- gas phase filtered smoke does not. b. 2R1 cigarette smoke (approximately 0.4 m TP14/day for 15-20 weeks) has not inhibited DNA repair In C3H~Anf mice to date. 2. Pulmonary cytochromes and smoke exposure (with Dr. i. Wang). a. Hepat;c cytochromes are separated and quantitated easily on polyacrylamide gel electrophoresis (PAGE). b. Pulmonary cytochromes c:an only be separated on long gels (approximately 30 cm). c. Frozen and fresh pulmonary tissues can now be assayed on these long gels. 3. immune response following smoke exposure (with Dr. H. Herscowitz). a. Mice can be treated at MA and sent to Georgetown University with no loss In the sensitivity of a spleen plaque assay, circulating hetnagglutinating antibody, or mitogen proliferation. -iv,-

B Contract CTR-0030 H-T®0123012 b. Exposure to 2111 cigarette smoke for two weeks de- presses the number of plaque forming cells in BC3F1/Cum mice by approximately 40y,. Machine controls and shelf controls were unaltered. -v-

I Contract CTR-0C3C 8 tE012301? I. INTRODUCTION The inhalation facility at Microbiological Associates (MA) is a state-of-the-art facility designed to define the potential biological effects of whole cigarette smoke in an animal model. Large-scale chronic cigarette smoke inhalation studies using the inbred strains of mice have been in progress since June 1977. During this time, over 7,000 mice have been exposed to smoke from over 500,000 cigarettes. The studies performed during this time have required continuous (5 days/week, 11 hours/day) use of the smoke inhalation equipment, which has included the SEM II B and C, animal containment units and animal re- straints. Smoke exposure related manipulations include indi- vidual animal identification (by ear tag), individual vaccination against Sendai virus, Individual loading and unloading of animals for smoke exposure, monthly weighing of each animal, and approximately 5-10 data entry points for each animal within any given experiment. Additional daily effort in technical support services is required to autoclave food and bedding, wash cages, and water bo:tles, dismantle and clean smoke- exposure equipment each day (16 animal containment modules, 2,000 animal restraints, the smoke exposed surfaces of the SEM II B or C, the flow thermistors and the optical sensors). Finally, another level of effort in professional support services Is required for complete necropsy, histology, patho- logy, data evaluation, reexamination and final analyses of each experiment. The level of effort required to perform these studies amounts to roughly 11,000 animai related mani- pulations per day. -1-

I Contract CTR-0030 Hr9u123u14 I:. UPDATE OF ONGOING STUDIES ON THE BIOEVAL UATION OF RISK TO LOW NICOTINE-HIGH TAR CIGARETTES CTR 100 1-100 . Cocarcinogenesls and Chronic Inhalation of 21 7'igarette moke. In June 1977, the first of a series of s tudies was initiated that we re designed to assess the biological risk of whole cigarette smoke in model animal systems. This first effort was designed to a) evaluate the large capacity of the SEM's and animal containment units, b) determine the expected mortality rates associated with the use of these machines, c) dete rmine the effect of whole 2A 1 cigarette smoke in BC3F1/C um mice for a 12-15 month exposure period, and d) determine the effects of whole 2A1 ci arette smoke as a cocarcinogen with 3-methylcholanthrene ~MCA). The following groups of mice were plac~d on test with the fol- lowing treatments: Group No. of Mice Treatment Smoke Exposure 1 220 Uni nocu 1 ated None 2 220 9X gelatin saline None 3 680 9X 250 µg MCA None 4 220 Uninoculated Mack,inp Control 5 220 9X gelatin saline Machine Control 6 280 9X 250 µg MCA Machine Control 7 325 Uninoculated 2A 1 Smoke 8 320 9X gelat!n saline 2A 1 Smoke 9 430 9x 250 )ig MCA 2A1 5moke Total 3155 MCA was given in 0.02 ml of gelatin saline once very two weeks. Smoke exposed and machine control groJps were started three 'days after chemical or vehicle treatment and continued during the 18 wee k-treatment period. Mice were adapted to increasing amounts of 10% smoke from 2A1 cig.arettes, 30 seconds exposure time, 10 puffs/cigarette, eventually reaching 10 ciga- rettes/day, Smoke exposure was carried out 5 days/week. Mice were allowed to die or were killed when moribund. How- ever, included in this study in Groups 1-3, were shelf controls, vehicle controls and MCA treated mice scheduled for serial sacrifice at timed intervals. Results from these mice were to confirm previous preliminary results which suggested that there existed specific steps of progression f rom pr-eneoplastic to frank carcinomas of the lung. Samples of lung tumors from these mice were also scheduled for transplantation Into newborn animals t o define the Talignant potential of the in- duced tumors. -2-