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H T®01 2005•} MIA 5221 RIVER ROAD BEYHESDA, MARYLAND 20016 ;

llGtR~~` y~c~~ HT80120035 A CAPABILITIES STATEMENT FOR INHALATION TOXICOLOGY STUDIES OF WHOLE CIGARETTE SMOKE AND/OR AEROSOLIZED CHEMICALS USING AN ANIMAL MODEL AND NOSE-ONLY INHALATION SYSTEM. Prepared by Carol J. Henry, Ph.D. Ken K. Kanagalingam, Ph.D. Richard E. Kouri, Ph.D. Microbiological Associates 5221 River Road Bethesda, Maryland 20016 (301) 654-3400

( 1. INTRODUCTION HT10120036 Lung cancer among all malignant diseases is the major cause of death among humans. A strong correlation has emerged based on epidemio- logical and other evidence linking cigarette Emoking to this disease (14, 20). Cancer of the oral cavity, esophagus, pancreas and bladder (15, 16, 17, 19) have also been traced to cigarette smoke. The mech- anism by which cigarette smoke, either alone or with other factors, may exert.its wide ranging toxicological effects is not known. However an excess of 2,00C chemical constituents have been Identified in cigarette smoke suggesting that a complexity of interactions may be involved. The consituents of cigarecte smoke include known initiators of cancer, such as polynuclear aromatic hydrocarbons, aromatic amines and nitro- samines and also known promocers of cancer, such as pyrggallol, catechol, etc. (18). Thus the study of cigarette smoke induced alterations in target tissues has become an important focus of attention in many laboratories. In order to properly understand and analyze the various molecular, biochemical, metabolic, immunologic, developmental, physiologic, and pathologic events associated with the toxicology of pulmonary exposures to cigarette smoke, it is Important that a suitable and convenient animal model systan be available for such diverse studies. Such a model system must incorporate the following capabilities: • Adequately simulate human smoking conditions. • Facilitate smoke exposure via inhalation, while at the same time avoiding whole body exposure. • Readily allow exposure of both small and large numbers of animals. • Allow for quantitative monitoring of the "dosage" of snoke reaching the animals. • Have the flexibility to allow changes in the length of exposure and number of exposures presented to the animals. ® Include built-in technology to characterize the smoke as to size of particles reaching the targets, estimation of the depo- sition in the respiratory tract of the total particulata matter, monitoring of sel:cted ;,onstitueits in cigarette smoke (e.g., CO and nicotine), and the documentation of these parameters. ® Have well defined histopathologic criteria. e Have ready access to automated data processing. A model system for conducting inhalation toxicology studies of the effects of whole cigarette smoke, which encompasses all of these features was evolved in our laboratories at Microbiological Associates. The uniqueness of the system arises from its capacity to facilitate exposure of small rodents which are obligate nose-breathers by inhala- tion of whole cigarette smoke via only the nasal orifice.. -1-

N T1C 120C'37 II. SMOKE-GENERATION SYSTEM A. Small CaPacity, `,Static System The Walton Horizontal Smoke Exposure Machine Is shown in Figures la and lb. This machine is designed to expose a small number of experimental animals (12-20 mice) to cigarette smoke under the condi- tions of a 2-sec,puff,35-m1 volume,once every minute. The features of this machine have beeq described elsewhere (3, 7). The Walton provides exposure to standing or static smoke from a single puff that is uniformly mixed by a rotating fan. The animals breathe the smoke for a preselected time ranging from 5 to 45 sec. The particle size of the smoke aerosol has a geometric mean diameter of 0.40 to 0.63 pm during a 30 sec exposure period (9). The chamber is flushed with fresh air between puffs. The smoke concentration can be varied by changing either the chamber volume (between 384 and 1150 ml) or the number of cigarettes (1, 2 or 3) simultaneously smoked. B. Large Capacity, Oynamic Svstem The Smoke Exposure Machine (SEN II) is a dynamic exposure system which has the capacity to expose up to 480 mice to smoke at any one time. The SEM II shown in Figure 2, operates In a manner simulating human smoking and automatically and sequentially loads, lights, puffs, e,lects, and extinguishes a series of 30 cigarettes. The operating principles of this machine are presented elsewhere (12). The standard smoking conditions employed are: puff frequency (one/minute), puff duration (2 seconds), average puff volume (35 ml) and cigarette butt length (23 mm average). These conditions are maintained independently of the animal containment system. Variable amounts of dilution air can be introduced at the butt end of the cigarette allowing a range of smoke concentrations from 5-100% (v/v) to be offered to the test animals. A diagram of the smoke generation system and animal contain- ment unit is presented in Figure 3. Smoke can be alternated with breathing air for various intervals within a one minute cycle. Air is provided to all channels in the absence of smoke. Cigarette smoke is delivered to the test animals within 2 seconds after generation at the cigarette. Previous studies have shown that smoke particle size was log normally distributed with e geometric mean diameter of 0.34 microns and geometric standard deviation of 1.35 microns (9). 111. AER050L•-GENERATION SYmMS Aerosol generation systems utilizing a nebulizer for chemicals such as catechol and the ettanol-soluble tetradeconyl phorbol acetate (1), as well as a dust-generating-system for non-soluble solids, such as asbestos, have also been developed In our laboratories. They can readily be adapted to studies of aerosols of chemicals and mixtures, fumes, pre- and post-cambustion products, as we11 as gases. The Nose-Only-Inhalation system as developed in our laboratories has the further advantage that the same aerosol characterization system and the same animal holding system (see below) used for cigarette-smoke- inhalation studies can be extended for use 1vitn other substances that can be aerosolized. -2-

( IV. SMOKE AND AEROSOL MONITORING SYSTEM HTE0120038 A monitoring system was developed for continuous measurement of particulate aerosols (see Figure 3). The aerosols are monitored with an optical-type scattering detector (8). Each exposure is documented on a strip chart recorder, which provides a permanent record of aerosol concentration and exposure period. In the case of cigarette smoke, this monitor provides both a puff by puff profile of smoke eposure and a cumulative value for the total particulate matter from each individual exposure. The monitoring system also allows for continuous measurement of carbon monoxide and carbon dioxide concentration in cigarette sinoke or other aerosols. V. ANIMAL HOLDING SYSTEFI The animal containment system for smoke exposure is shown in Figure 4. Groups of five animals are placed in a "stock-like" holder using a combination of a neck slot and a restraining spring. A chin rest insures that the nose of the animal is aligned with the cone shaped opening on the exposure modules (Figure 3). The nose of each animal passes through a dental rubber dam diaphragm forming a seal that prevents exposure of the animal body to the smoke or particulate aerosol. Daily restraint for up to three hours (twice per day) results in no mortality or obvious ill effects to the animals. VI. RELEVANT BACKGROUND STUDIES A. Animals_and End-P_o_ints_in Chemicall Induced Lung Cancer We selected the mouse for our cigarette smoke inhalation experi- ments because it offers several advantages over other species. These advantages are: ® Availability of defined genetic stocks. o Availability of many strains. ® Availability of colcriPs free nf two important adventitious agents for the lung. Sendai and pneumonia virus of mice. ® Small size for ease in manipulation and relAtive ease and economy in housing. o Deposition and distribution of smoke particulates in these strains has been documented by us (5, 6, 71. a Inbred strains of mice have the capacity to express those types of lung r.anr.er normally associated with cigarette smoktng in humans (e.g., squamous cell carcinomas, alveolar adenocar- cinomas, adenosquamous carcinomas and poorly differentiated r.arcinomas). s As has been suggested in humans, susceptibility to lung cancer in m'.-ce may be genetically regulated in that certain strains of mice have a higher capacity to metabolize chemical carcino- gens (11). -3-

( B. Deposition and Distribution 0 Ci9arette Smoke Particulates Hr0o1zoo35 Using standard smoke exposure conditions, the deposition and internal distribution of the total particulate matter from cigarette smoke has been determined in various strains of mice (5, 6, 7). Results show: a) smoke exposure conditions can be varied so that depo- sition from 30 pg to 200 4,g total particulate matter/lung can be obtained, b) 80-90t of the deposition of the total particulate matter is found in the respiratory tissues, c) the mouse-to-mouse variation for particulate deposition in pulmonary tissue is ti20i;, d) similar. deposition and distribution of particulates are observed in both male and female mice, and e) deposition and distribution of particulates are not altered in mice exposed to smoke on a daily basis over 6 months. Deposition, distribution and clearance of a specific con- stituent of smoke is dependent upon the metabolic fate and chemical class of constituent used. The pharmoj(okinetics of four particulate C phase constituents of cigarette smoke have been studied in our labora- tories (6). Measurements of carboxyhemoglobin levels reflect the up- take of the gas phase smoke and are highly correlated with pulmonary deposition of total particulate matter. C.. Toxicology of Whole Cigarette Smoke Several short-term markers have been determined for the evaluation of the biological effects of whole cigarette smoke. These include: a) induction of mic,)omal monoxygenases in the lung and liver (10), b) induction of pulmonary ornithine decarboxylase, an enzyme which is rate limiting in polyamine biosynthesis and thought to be a marker for tumor promotion (4), c) determination of levels of DNA repair capacity of lung tissue (13), d) determination of the extent of DNA damage in pulmonary tissue, e) determination of rates of pulmonary macromolecular synthesis (i.e., DNA, RNA, protein and collagen) f) separation and identification of cigarette smeke specific meta~olites using high perforimance 'iquid chromatography, and g) physiologi response in the lung such as the formation and persistence of adhert~it cell types as measured by both light and electron microscopy. The long-term end- points of toxicology and lung cancer can also be determined. VII. FUTURE CONSIDERATIONS We feel that the combination of this animal model and nose-only inhalation system affords the capabilities necessary to address the following difficult issues pertaining to the toxicological effect and biological activity of cigarette smoke and/or aerosolized chemicals: • What are the long term (life time) toxicological effects of whole cigarette smoke? • Are these effects related to the composition of the smoke (e.g., high tar vs. low tar cigarettes, high nicotine vs. ,,low nicotine cigarettes)? • What are the toxicological effects of additives to tobacco? • Are the effects of smoke modified by exposure to respiratory viruses? -4-

i HT®0120040 s Can the effects of smoke be modified by exposure to environ- mental or occupational agents such as asbestos, diesel fuel exhaust, smog, pesticides, etc.? ® Does cigarette smoke act as an "initiator" or a"promoter", or both, in lung carcinogenesis? e What are the conditions under which the effects of cigarette smoke may be altered by anti-carcinogens? VIII. FACILITIES Smoke inhalation experiments are carried out in our inhalation facility, which occupies 8000 square feet and employs 25 individuals. The facility has restricted access to ensure the health of the animals and only known animal breeders are used as sources of mice. These sources are screened by our Animal Disease Diagnostic Testing Labora- tory to ensure and document that the animals are disease free. All animals are vaccinated with Sendai vaccine, which was developed and tested within this program. We feel that the exclusion of Sendai virus and pneumonia virus of mice is extremely important to any pulmonary toxicology or carcinogenesis studies. IX. PERSONNEL C k Directly involved in these studies are seven personnel at the Ph.D. level who have a wide range of technical expertise in the bio- logical and chemical sciences. Their combined experience has resulted in significant contributions to the fields of toxicology, carcinogenesis, and biochemistry, with over 100 publications resulting from these studies. These endeavors are aided by a well qualified and trained technical staff, (M.S., B.S., and AALAS certified). The scientific staff at Microbiological Associates who are involved in these projects have ongoing collaborative efforts with colleagues at such ins*:`6tions as: NCI; NICH; NIAMDD; NIADR; NIAID; NINCm; UaK Kidge National Labora-• tory; University of California, Irvine;, University of California, San Francisco; Children's Hospital, Los Angel.s; Baylor College of Medicine; Georgetown University; University of South Carolina; and University of Maryland. In addition to this research team, expertise in the areas of veterinary care, veterinary pathology, histology, and data processing are integral components of the program. Microbiological Associates ,r has on its staff a vekterinarian, board certified in laboratory animal science and a veVterinarian, board certified In animal pathology. The v histology department has 4 of its 7 personnel certified in histochemical technology. The Data Processing department is comprised of 6 profes- sionals with experience ranging from computer science to automated statistical applications. This varied background has stood in good stead over 25 years at MA in achieving renown and proficiency in meeting the research and toxicology testing needs of the Federal Government and industr.ial clients around the world. -5-

NT00120041 1. l 2. 3. 4. 5. ~ 6. 7. 8. 9. l REFERENCES Dinowitz, M., Nims, R., Bhooshan, B., Kouri, R. E., and Henry, C. J. Induction of Ornithine Decarboxylase (ODC) by 12-0-Tetradecanoyl- phorobo1-13-acetatP (TPA) in Pulmonary Tissue: A Model System for Tumor Promotion in Mouse Lungs. Proc. Am. Assoc. Cancer Res. 21: 31 (1980). Green, C. R. Recent Adv. Tobacco Sci. 3: 94 (1977). Guerin, M. R., Stokely, J. R., Higgins, C. E., Moneyhun, J. H.; and Holmberg, R. W. Inhalation Bioassay Chemistry -- Walton Horizontal Smoking Machine ior Inhalation Exposure of Rodents to Ciaarette Smoke. J. Natl. Cancer Inst. 63: 441 (1979). Henry, C. J., Billups, L. H., Dinowitz, M., Rasmussen, R. E., Avery, M. D., Dansie, D. R., Lopez, A., Breth, L. A., Mullinax. H. D., and Kouri, R. E. The Effect of Exposure to Whole Cigarette Smoke on Pul- monary Mixed Function Oxidase, Ornithine Decarboxylase, DNA Repair Capacity and on 3-Methylcholanthrene (MCA) Induced Lung Tumors in BC3F1/Cum Mice. In: Symposium on Cocarcinogenesis and Biological Effects of Tumor Promoters. October 13-16, 1980. Henry, C. J., Caton. J. E., Stokely, J. R., Guerin, M.R., Lopez, A., Avery, M. 0., Dansie, D. R., Henderson, G. M., Gayle, T., Whitmire, C. E., and Kouri, R. E. Deposition and Distribution of the Total Particulate Matter of Cigarette Smoke in Mice Using a Large Capa- city Smoke Exposure System. Toxicol. Appl. Pharmaco'o. (1981). Henry, C. J., Lopez, A., Dansie, D. R., Avery, M. D., Whitmire, C. E., Caton, J. E., Stokely, J. R„ Guerin, M. R., Curren, R. D., and Kouri, R. E. D4stribution and Clearance of Three Cigarette Smoke Constituents, Dotriacont3ne (DTC), Nicotine (NIC), and Benzo- (a)pyrene (BP), after Exposure of Mica to Whole Cigarette Smoke. Proc. Soc. of Tox. (1981). Henry, C. J., Whitmire, C. E., Lopez, A., Dansie, D. R., Avery, M. D., Caton, J. E., Stokely, J. R., Hcimberg, R. W., Guerin, M. R., anti Kouri, R. E. The Dosimetry and Distribution of Whole Cigarette Smoke Particulates in Inbred Strains of Mice: Comparison rf a Large Smoke-Exposure Machine (SEM) with a Small-Capacity Smoke-ExpGaure Machine (Walton), In: Pulmonary Toxicology of Respirable. C. L. Sanders, F. T. Cross, G. E. Dagle and J. A. Mahafl ey, Eds., Tech- nical Information Center, U.S. Department of Encerg, NT1S, 1980. Higgins, C. E., Gayle, T. M., and Stokely, J. R. Sensor for Detec tion of Tobacco Smoke Particulates in Inhalation Exposure System. Beitr. Tabakforsch. 9: 185 (1978). Holmberg, R. W. Determination of Particle Size in Tobacco Smoke Inhalation Devices Using Methylcyanoacrylate Fixation and Scannig Microscopy. In: Tobacco Smoke Inhalation Bioassay Chemistry. H. R. Guerin, J. R. Stokely, and C. E. Higgins, Eds., DOE Report ORNL-5422, Oak Ridge National Laboratory, NTIS, 1979. -6-

REFERENCES HT®0120042 10. Kouri, R. E., Rude T. H„ Curren, R. D., Brandt K. R. Sosnowski ~ , , , , R. G„ Schechtman, L. M., Benedict, W. F., and Henry, C. J. Bio- 11. logical Activity of Tobacco Smoke and Tobacco Smoke-Related Chemi- cals. Environm. Hlth. Perspect. 29: 63 (1979). Kouri, R. E., Schechtman, L. M., and Nebert, D. W. Genetic Control of Carcinogen Metabolism. In: Genetic Differences in Chemical Car- cinogenesis. R. E. Kouri, Ed., CRC Press, Boca Raton, Fla., 21-66, 1980. 12. Moneyhun, J. H., Stokely, J_ R, and Florant, L. Process and Instru= 3. ments Corporation Automatic Smoke Exposure Machine -- SEM II. In: Tobacco Smoke Inhalation Bioassay Chemistry. M. R. Guerin, J. R. Stokely, and C. E. Higgins, Eds., DOE Report ORNL-5422, Oak Ridge National Laboratory, NTIS, 1979. Rasmussen, R. E., Boyd, C. H., Dansie, 0. R., Kouri, R. E., and 14. Henry, C. J. DNA Replication and Unscheduled DNA Synthesis in Lun s of Mice Exposed to Cigarette Smoke. Cancer Res. (Submitted) (1980qj. U.S. Dept. of Health, Education and Welfare: "Smoking and "Health", 5. U.S. Publ. Hlth. Serv. Mo., 1103, 1964 "The Health Consequences of Smoking", DHEW Publ. No. (HSM) 71, 7513, 1971 "The Health Con- sequences of Smoking", DHEW Pubt. No. (CDC) 76-8704, 1975. Wynder, E. L., Bross, I. J. and Feldman, R. M. A Study of the l 16. Etiological Factors in Cancer of the Mouth. Cancer. 10: 1300 (1957). Wynder, E. L. and Bross, I. J. A Study of Etiological Factors in 17. Cancer of the Esophagus. Cancer. 14: 389 (1961). Wynder, E. L. and Goldsmith, R. The Epidemiology of Bladder Cancer, 18. A Second Look. Cancer. 40: 1246 (1977). Wynder, E. L. and Hoffinann, D, Tobacco and Tobacco Smoke. Acedmic 19. Press, New York, 1967. Wynder, E. L., Mabuchi, K., Maruchi, N., and Fortner, J. G. Epi- demiology of Cancer of the Pancreas. J. Natl. Cancer Inst. 50: 645 (1973). 20. Wynder, E. L. and Stellman, S. D. Comparative Eaidemiology of Tobacco-related Cancers. Cancer Res. 37: 46c)8 (1977). l -7-

HT®0120043 Fig. 1(e) Welton Nozisontal Smoidng 111aahine. Mice are senwu teetta5nr.d in the cylied.-tcaf Cubea. -8-

HTE0120044 t ( Fig. 1(b) Weltoa ezposute chamber. One ride plate has been temoved to ahos' the inafds 'a[ e:posute chamber (CH) with the cou:ca4ehoped esposure holes (H) visible. Duuing an espoaute the ciprette dome (D'. movee forward and automatically fgnitee the cigarette (C). and 35 ml of puff air is [oreed through the cigetatte into tbo chembar. A continuously rotating [en (F) ie tequirtd to ®tume rapid and uniform mixing of the smoke. At the end of an e:poaur® Intervall. emok+l Is aenred through ohe exlmuat port (E). -9-

H T®0120045 Figure 2. The SEM II with front and side panels open to show electronic components. See text for full operational details. Cigarettes (C) are loaded from a hopper (HO) into a rotating drum holder (DH). The lighter (L) automatically ignites the cigarette and puff air is forced through each cigarette in turn. Flow is caused by a constantly held differential pressure provided by the positive pressure in the dome (D0) between the ignited end and the butt end of the cigarettes. Dilution air (DA) is introduced at the butt end of the ~,iga- rette. Side stream smoke is removed through the vent (V). Cigarettes are removed by the automatic ejector (E) and discarded in the butt shutte (S) -10-

Figure 2 HtC0120U4b -11-

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HT§0120048 ( Figure 4. SEM II animal containment system. One side of the unit isshown,with the capacity for exposing 240 mice per side (480 mice total). Rubber stoppers are used in the exposure holes when less than the full capacity of mice are to be exposed. -13-

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H1Mp120050 X. BIBLIOGRAPHY The appendix attached to this document contains selected references that highlight some of the scientific contributions of Microbiological Associates in the areas of toxicology and carcinogenesis research. Reprints of papers most salient to application of the lung cancer model system and nose-only Inhalation ara also included. -15-

Y H T O01 20051 ( APPENDIX I k

HTE0120052 l l PUBLICATIONS Kouri, R.E., Salerno, R.A., and Whitmire, C.E. - Relationships between Aryl Hydrocarbon Hydroxylase Inducibility and Sensitivity to Chemically-Induced Subcutaneous Sarcomas in Various Strains of Mic.e. J. Natl. Cancer Inst., 50: 363-368, 1973. Kouri, R.E., Ratrie, H., and Whitmire, C.E. - Evidence for Genetic Relationship Between Susceptibility to 3-Methylcholanthrene Induced Subcutaneous Tumors and Inducibility of Aryl Hyd:ocarbon Hydroxylase. J. Natl. Cancer Inst., 51: 197-200, 1973. Lubet, R.A., Brown, o.Q., and Kouri, R.E. - The Role of 3-OH-Benzo(a)pyrene in Mediating Benzo(a)pyrene Induced Cytotoxicity and Transformation in Cell Culture. Res. Comm. in Chem. Path. and Pharm., 6: 929-942, 1973. Oemoise, C.F., Kouri, R.E., and Whitmire, C.E. - Cell-Mediated Immunity After Intratracheal Exposure to 3-Methyicholanthrene and its Relationship to Tumor Transplant Growth in C3H/f Mai Mice. In: E. Karbe and J.F. Park (Eds.) Experimental Lung Cancer. Springer- Verlag, New York, p. 72, 1974. Kouri, R.E., Ratrie, H., and Whitmire, C.E. - Genetic Control of Susceptibility to 3-Methytcholanthrene-Induced Subcutaneous Sarcomas. Int. J. Cancer, 13: 714-720, 1974. Nebert, D.W., Benedict, W.F., and Kouri, R.E. - Aromatic Hydrocarbon Produced Tumorigenesis and the Genetic Differences in Aryl Hydrocarbon Hydroxylase Induction. In: P. Ts'o and J. DiPuolo (Eds.) Chemical Car0nogenesis, Part A., Marcel Dekker, New York, pp. 271-288, 1974. Benedict, W.F., Rucker, N., Mark, C., and Kouri, R.E. - Correlation Between the Balance of Specific Chromosomes and the Expression of Malignancy in Hamster Cells. J. Nat1. Cancer Inst., 54: 157-162, 1975. Benedict, W.F., Rucker, N., Faust, J., and Kouri, R.E. - Malignant Trans- formation of Mouse Cells by Cigarette by Cigarette Smoke Condpnsate. Cancer Res., 35: 857-860, 1975. Kouri, R.E., Whitmire, C.E., and Benedict, W.F. - In Yivo and In Vitro Effects of Cigarette Smoke Condensate Fractions. Proc. M-Assoc. Cancer Res., 16: 173, 1975. Kouri, R.E., Rude, T., Thomas, P.E., and Nhitmire, C.E. - Studies on Pulmonary Aryl Hydrocarbon Hydroxylase Activity in Inbred Strains of Mice. Chem. 8io1. Interactions, 13: 317-331, 1976. Kouri, R.E. - Relationship Between Levels of Aryl H,vdrocarbon Hydroxylase Activity and Susceptibility to 3-Methylcholanthrene and Benzo(a)pyrene Induced Cancers in Inbred Strains of Mice. In; Polynuclear Aromatic Hydrocarbcns: Chemistry, Metabolism and Carcinogenesis. Freudenthal & Jones, (Eds.) Raven P-ess, New York, p. 139-151, 1976. i

HTE0120053 ( Kouri, R.E., Imblum, R.L., and Prough, R.A. - Measurement of Aryl Hydro- Carbon Hydroxylase and NADH-Dependent Cytochrome c Reductase Activities in Mitogen-Activated Human Lymphocytes. Proc. 3rd Intl. Symp. on Detection and Prevention of Cancer, H.E. Nieburgs (Ed.) Marcel Dekker, Nerr York p. 510, 1977. Kouri, R.E., and Nebert, D.W. - Genetic Regulation of Susceptibility to Polycyclic Hydrocarbon-Induced Tumors in the Mouse. In: Origins of Human Cancer, H.H. Hiatt, J.D., Watson, and J.A. Winsten, (Eds.) Cold Spring Harbor Press, p. 811-835, 1977. Nebert, O.W., Boobis, A.R., Yagi, H., Jerina, D.M., and Kouri, R.E. - Genetic Differences in Benzo(a)pyrene Carcinogenic Index In Vivo and in Mouse Cytochrome P 450-Mediated Benzo(a)pyrene Metahol~te Binding to DNA In Vitro. In: Biological Reactive Intermediates, D.J. Jallow, J. J. Kocsis, R. Snyder, and H. Vainio, (Eds.) Plenum Press, New York, 1977. Schechtnian, L.M., and Kouri, R.E. - Control of Benzo(a)pyrene-Induced Mammalian Cell Cytotoxicity, Mutagenesis, and Transformation by Exogenous Enzyme Fractions. In: Progress in Genetic Toxicology D. Schoot, B.A. Bridges, F.H., Sobels, (Eds.) Elsevier/North Holland Press, New York, Vol. 2, p. 307, 1977. Billups. L.M., Henry, C.J., Whitmire, C.E., and Kouri, R.E. - Pathogenesis of Chemically Induced Lung Lesions in Mice. Proc.Am. Assoc. Cancer Res., 19: 142, 1978. Nebert, D.W., Atlas, S., Guenther, T., Kouri, R.E. - The Ah Locus: Genetic Regulation of the Enzymes wnich Metabolize Polycyc)ic Hydrocarbons and the Risk of Cancer. Polycyclic Hydrocarbons and Cancer: Chemistry Molecular Biology and Eniironment, P.O.P. Ts'o and H.V. Gelboin, (Eds.) Academic Press, New York, p. 345-390, 1978. Radtke, H.E., Rude, T., and Kouri, R.E. - Evidence of Heterogeneity of Hepatic and Pulmonary Epoxide Hydratase (EH) Activity in Inbred Strains of Mice. Proc. Am. Assoc. Cancer Res.,19: 142, 1978. Henry, C.J., Billups, L.H., Avery, M.D., Dansie, D.R. Lopez, A., Breth, L.A., Rude, T.R., Kouri, R.E. - Lung Cancer Model Systems in Inbred Strains of Mice. Proc. Am Assoc. Cancer Res., 20: 242, 1979. Henry, C.J., Lopez, A., Whitmire, C.E., Caton, J., Henderson, C.M., Kouri, R.E. Deposition and Distribution of Total Particulate Matter (TPM) in BC3F1 Mice After Exposure to Cigarette Smoke. Toxicology & Applied Pharmacology,48: A5, 1979. Kouri, R.E., Kurtz, S., Vernon, M.L., Schechtman, L.M., Nicholson, M., Klement, V., and Benedict, W.F. - C-Type RNA Virus from Hamster Cells Transformed In Vitro by 1-B-D-Arabinofuranosylcytosine. Arch. of Vi.ology,62: NT--252, 19'9. ii

r HTC012005~ f l Kouri, R.E., Brandt, K., Sosnowski, R., Benedict, W.F „ and Schechtman, L.M. - In Vitro Activation of Cigarette Smoke Condensate Materials to their Mutagenic Forms. In: Application of Short-Term Bioassays in the Fractionation and Analysis of Complex Environmental Mixtures. M. Waters, S. Nesnow, J.I. Huisingh, S.S. Sandhu, L. Claxton, (Eds.) Environanental Protection Agency, Research Triangle Park, N.P. pp. 495-512, 1979. Kouri, R.E., Rude, T.H., Curren, R.D., Brandt, K.R., Sosnowski, R:G., Schechtman, L.M., Benedict, W.F., Henry, C.J. - Biological'Activity of Tobacco Smoke and Tobacco Smoke-Related Chemicals. J. Environ. Health Perspectives, 29: 63-69, 1979. Rasmussen, R.E., Boyd, C.H., Henry, C.J., Kouri, E.F.. - DNA Repair is Reduced In Lungs of Mice Exposed to Cigarette Smoke. Proc. Am. Assoc. Cancer Res. 20: 242, 1979. Dinowtiz, M., Nims, R., Bhooshan, D., Kourl, R.E., and Henry, C.J. - Induction of Ornithine Decarboxylase (0DC) by 12-0-Tetradecanoyl-phorbo1-13- acetate (TPA) in pulmonary Tissue: A Model System for Tumor Promotion in Mouse Lungs. Proc. Am. Assoc. Cancer Res., 21: 31, 1980. Henry, C.J., Whitmire, C.E., Lopez, A., Dansie, D.R., Avery, M.D., Caton, J.E., Stokely, J.R., Holmberg, R.W., Guerin, M.R., Kouri, R.E. - The Dosimetry and Distribution of Whole Cigarette Smoke Particulates in Inbred Strains of Mice: Comparison of a Large Smoke-Exposure Machine (SEM) with a Small-Capacity Smoke-Exposure (Walton). In: Pulmonary Toxicology of Respirable Particles, C.L. Sanders, F.T. Cross, G.E. Dagle, and J.A. Mahafley (Eds.) Technical Infor- mation Center, U.S. Department of Energy, NTIS, pp 177-192, 1980. Henry, C.J., Avei-y, M.D., Dansie, D.R., Lope%, A., Breth, L.A.. Billups, L.H., Kouri, R.E. - The Effect of Exposure to Whole Cigarette Smoke on 3-Methylcholanthrene (MCA) Induced Lung Tumors in BC3F1/Cum Mice. Proc. Pm. Assoc. Cancer Res., 21: 126, 1980. Henry, C.E., Billups, L.H., Dinowitz, M., Rasmussen, R.E., Avery, M.D., Dansie, D.P.., Lopez, A., Breth, L.A., Mullinax, H.D., Kouri, k.E. - The Effect of Exposure to Whole Cigarette Smoke on Pulmonary Mixed Function Oxidase, Ornithine Decarboxylase, DNA Repair Capacity and on 3-Methylcholanthrene (MCA) Induced Lung Tumors In BC3F1/Cum Mice Symposium on Co-Carcinogenesis and Biological Effects of Tumor Promoters, Castle of Elmau, D-8101 Klais/Baveria, Federal Republic of Germany, Book of Abstracts, p. 56, 1980. Kouri, R.E., Rude, T., Whitmire, C.E., Henry, C.J., Sass, 8., Billups, L.H., Correlation of Inducibility of Aryl Hydrocarbon Hydroxylase with Susceptibility to 3-Methylcholanthrene-Induced Lung Cancer. Cancer Letters, 9: 277-284, 1980. Kouri, R.E., Schechtman, L.M., and Nebert, D.W. - Genetic Control of Car- cinogen Metabolism. In: Genetic Differences in Chemical Carcino- genesis. R.E. Kouri (Ed.) CRC Press, Boca Raton, Fla., pp. 21-66, 1980. iii

O --- -~ : w= ( H T10120055 Rasmussen, R.E., Boyd, C.H., Dansie D.R. Kouri, R.E., Henry, C.J. - DNA Replication and Unscheduled DNA Synthesis in Lungs of Mice Exposed to Cigarette Smoke (Submitted) 1980. Schechtman, L..M., Henry, C.J., and Kouri, R.F.. - Exposure, Uptake and Distribution of Chemical Carcinogens. In: Genetic Differences in Chemical Carcinogenesis. R.E. Kouri (Ed.) CRC Press, Boca Raton, Fla., pp. 1-20, 1980. Henry, C.J., Caton, J.E., Stokely. J.R., Guerin, M.R., Lopez, A., Henderson, C.M., Gayle, T., Moneyhun, J., Whitmire, C.E., Kouri, R.E.- Deposition and Distribution of the Total Particulate Matter of Ciga- Rette Smoke in Mice Generated by a High Capacity Smoke Exposure System. Toxicology & Applied Pharmacology (In press) 1981. Henry, C.J., Geihard, J.C., Gosnell, S., Knipscher, R.C., Lopez, A., Dansie, D.R., Billups, L.H., Kouri, R.E. - Pulmonary Localization of Cigarette Smoke Particulates After Exposure of Mice to Cigarette Smoke. (Submitted) 1981. Henry, C.J., Breth, L.A., Gerhart, J.M., Whitmire, C.E., Kouri, R.E. - Carboxyhemoglogin Levels as a Measure of Cigarette Smoke Exposure in Mice (Submitted) 1981. Henry, C.J., Lopez. A., Dansie, D.R., Avery, M.D., Whitmire, C.E., Caton, J.E., Stokely, J.R., Guerin, M.R., Curren, R.O., Kouri, R.E. - Distribution and Clearance of Three Cigarette Smoke Constituents Dotriacontane (DTC), Nicotine (NIC) and Benzo(a)pyrene (BP), after Exposure to Mice to Whole Cigarette Smoke (In press) Proc. Society of Toxico!ogy 1981. Hwang, K.K., Sonko, 0., Dansie, O.R., Kouri, R.E., Henry, C.J. - Studies on the Deposition and Distribution of the Catechol of Whole CigaretLe Smoke in,8C3F1/Cum Female Mice. (Submitted) 1981. iv