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I ( ( V ( ( , 7 ( ( '1'1n: COUxc11. T'c,:e 'I'e11sAcco I2>tal:Auci1-U.ti..1.. Irc. 11U F:Ai:T 51si1t s9TRF'.M.T \F:N' YORK. \. 1'. 10022 (1., 121 4 t1.rx%. M:. Applicotion for Research Grant (Use extra pages as needed) 1. Prinrpal Investiqotor (give title and degrees): Carol J. Henry, Ph.D. and Richard E. Kour:, Ph.D. Director, Department of Director, Department of Experimental Oncology ' Biochemical Oncology 2. Insriwtion & address: Microbiological Associates 5221 River Road Bethesda, Maryland 20016 3. Oeportmenr(s) where reseerch will be done or collaboration provided: Department of Experimental Oncology Department of Biochemical Oncology SSP i g 1978 G- ~ - 6'.,1 4. Short ude o( s+udy: Evaluation and Characterization of an Alkaline Elution Assay as a Measure of Pulmonary DNA Damage Induced by Chemical Carcinogens or the Chemicals in Cigarette Smoke. S. Proposed storrinp dote: November 1, 1978 6. Estimored time ro complete: Three years 7. 8rief description of specific research oims: Exposure to many carcinogens in vivo has been shown to result in damage to DNA which can be detecte3 as single strand breaks using an alkaline elution (AE) assay. We propose to characterize the AE assay simultaneously using two specific approaches: 1) as a tool in predicting lung tumorigenic capacity of chemical carcinogens, and 2) as a tool in predicting the capacity of the chemicals In cigarette smoke to interact with lung DNA in vivo. Using our model systems for lung carcinogenesis and cigarette smoke inhalation, we intend to address the foliowing questions: 1. Can damage to lung DNA be detected after intratracheal (IT) treatment with chemicals under conditions where these chemicals inducellung carcinomas? 2. For a particular-class of chemical carcinogens, Is there a correlation between conditions that yield high inci- dences of lung carcinomas and amount of DNA damage? 3. Is the amount of lung DNA damage related to the capacity of lung tissue to metabolize these chemicals? -1- ( 50137808 Dote: 9/ 11/78 EXHIBR NO.-I-L S. NELSON I C`'~' ~`~~~ ~~4;~ ;~"`~~

A Continuation of Brief Description of Specific Research Atms: 4. Does exposure to whole cigarette smoke cause damage to DNA as detected by the AE assay? 5. Does exposure to the chemicals in smoke as repre- sented by the cigarette smoke condensate and con- densate fractions cause damage to DNA as detected by the AE assay? . 6. Does exposure to whole cigarette smoke alter the DNA damage lnduced by other chemicals? ABBREVIATIONS ) • AE alkaline elution AHH aryl hydrocarbon hydroxylase ASG alkaline=sucrose gradients BaP benzo(a)pyrene C3 C3H/Anf mouse strain CSC cigarette smoke condensate CSCF cigarette smoke condensate fractions D2 DBA/2 mouse strain DEN diethylnitrosamine DMH 1,2-dimethylhydrazine DMN dimethylnitrosamine DMNA N-nitrosodimethylamtne EMS ethyl methylsulfonate ENU N-ethyl-N-nitrosourea MFO mixed function oxidase MMS methyl methanesulfonate MNNG N-methyl-N'-nitro-N-nitrosograndine MNU N-methyl-N-nitrosourea 4-NQO 4-nitroquinoline oxide PAH polycyclic aromatic h ydrocarbons RCM reconstituted material RNA ribonucleic acid TCDD 2,3,7,8-tetrachlordibenzo(p)dioxln TPM total particulate matter -2- 50137809 ~~~ ~ ~~ CTR HN

L. ( _ 8. Brief statement of 'working hypolhcsis: Our working hypothesis Is that chemical carcinogens or the metabo- lites of the carcinogens have the potential to Interact with DNA and that this interaction or DNA damage is one of the early steps In the carcinogenic process. This DNA damage can be quantitated in vivo by a simple, sensitive, short-term, and inexpenstve techni uqe w~ ch measures the rate at which this damaged DNA elutes from a filter under alkaline conditions. In this way, a method that has the capacity to quantitate one of the earliest presumed steps In the initiatlon of carcinogenesis can be s ecificall used to address the question of defining those classes o c em ca s and those in vivo conditions responsible for determining the susceptibility _oT 'iung tissue to chemical carcinogenesis. . ( ( 9. Deio11s of experimenrol design and procedures (append extra pages as necessary) I. Background Many chemicals tho.ght to be toxins, mutagens, or carcinogens are believed to exert their effects through interactions with DNA. This interaction can be a covalent attachment of the chemital to a s peci- fic nucleotide, an Intercalation between bases which resultsiin helix distortion, a cross-linking between nucleotides on opposing DNA strands - or phosphotrlester formation (see reviews 1, 2, 3). Such damage can ' cause localized perturbations In the DNA structure and either directly lead to single-stranded breaks, or indirectly result In single stranded breaks formed because of the action of endonucleases (part of the normal repair processes). The amount of damage can be quan- titated by measuring the size of the DNA strands after exposure to alkaline conditions (>pH 12.0). Exposure to alkali results in the unwinding of the double strands of DNA and the size of these strands can be directly quantitated by either alkaline-sucrose gradient (ASG) centrifugation (4) or alkaline elutlon (AE) (5, 6, 7). The ASG method is tedious, time-consuming, limited in the number of individual assays that can be performed, and sometimes suffers from poor recovery 8f labeled DNA. Moreover, single strand sizes of up to only 2-5 x 10 daltons can be measured (8). Above this size, no generally applicable method has been available. The AE assay,on the other hand, provides size measurements of extremely long DNA single strands (7). This assay has been used to study DNA replica- tion (9), DNA repair followin X-ray exposure (10), DNA scission In cells treated with bleomycin ?il), and DNA cross-linking in cells treated with nitrogen mustard or with chloroethylnitrosoureas (12). The AE method is based on the very simp le Idea of re~ainin cells in which the DNA has been prelabeled with 3H-Thymidine ( H-TdR~ on poly (vinylchloride) filters, lysing the cells on the filters, and eluting the DNA from the filters in an alkaline buffer (pH 12.2). • The elution rate seems to be governed by the properties of the DNA itself because:(7) -31 50137810 ~' ~~' ~'"I~>~ 04,23724

it ge l, Continutation of Details of Experimental Design and Procedures: a) almost all non-DNA material 1s removed by the lysis solution Including protein, lipids and memb ranes, b) removal of protein with proteinase-K and RNA by ribonuclease does not alter the elutlon kinetics, therefore,proteln and RNA play no role In the DNA elution, c) the rate of DNA elution is independent of the number of cell~ on the filter in the range of 0.1 - 2.0 x 10 cells; therefore, retention on the filter does not depend on the aggregation or trapping of DNA strands in a large-scale d) conditions that cause DNA shearing result In an immediate and striking Increase in DNA elution, 0 e) retention of DNA Is independent of the filter material, also essentlally independent of pore size from 1-5 µm, f) DNA under alkaline conditions is susceptible to' strand scission by visible light (13), and the elution of DNA is markedly sensitive to exposure to room light. The limit of sensitivity of the AE assay has been estimated following exposure to low-level X-rays. Th int ajeil lar NA siieet~)are at least 1.3 x 107 nucieotides ~4 x~0 da~tonsg in Current speculation is that the rate-limiting process Involves the search for a molecular conformation that will allow a long DNA single strand to be pulled through a single filter pore. Since the strand dimensions are much larger than the filter pores, portions of a single strand may be pulled through many pores at the same time. Elution must require that the strand segment In one pore acquire enough dominance to pull the entlre strand th rough . The AE assay can be streamlined Into a rap id and inexpensive method to test the capacity of unknown chemicals to Induce damage to DNA (5,6). Moreover, the AE assay can be used In the Presence of exogenous metabolic activation systems (6). Assay of 46 differ- ent chemicals(with and without metabolic activation) Indicate that every known carcinogen causes DNA damage (ncluding such weakly active chemicals as safrole, DDT, dieldrin and cyclophosphamide, while all non-carcinogens yield no DNA damage (5). Recent studies In our • laboratory suggest that the amount of damage to DNA Imparted by benzo(a)pyrene (BaP) is directly related to the aryl hydrocarbon hydroxylase (AHH) levels in tissue hemogenates from lung or liver, -4- 50137811 CTR MN 043`2`2215

c r r r Continuation of Details of Experimental Design and Procedures: ~ and not the level of epoxide hydrase (14). Thus, at least In 1n vitro systems, the amount of BaP metabolized to forms that cause YNA aamage, parallels quite nicely the amount of BaP metabolized to 3-OH BaP (i.e., AHH activity). The AE assay can also be used to quantitate the level of DNA damage following In vivo exposure of chemical carcinogens (5,15). This DNA ls prelabel'ey 3H-TdR and•the DNA detected by radio- metric means, or directly measured by a fluorometric method (15). DNA damage can be assessed in liver, lung, thymus, brain, kidney, stomach, duodenum, colon, bone marrow and mammary gland (5,15). Most direct acting carcinogens (e.g. N-methyl-N'-nitro-N-nitroso- guanidine (MNNG), N-methyl-N-nitrosourea, N-ethyl-N-nitrosourea, methyl methanesulfonate, and ethyl methanesulfonate) caused sig- nificant DNA damage in target and non-target organs, but ten other known chemical carcinogens induced the greatest DNA damage In the target organs for carcinogeniclty (5). In fact, dimethylhydrazine, a potent colon carcinogen, causes dose-dependent DNA damage In colonic tissue, and strains of mice that are susceptible to di- methylhydrazine-induced colonic cancers are more sensitive to DNA damage in this tissue, than are strains that are resistant to the tumorigenic effect of this chemical (15)'. Since there Is adequate evidence that methods for evaluating ' the capacity of a compound to damage DNA and to Initiate DNA repair may be utilized as a rapid prescreening test for assessing potential carcinogenic activity (5,15), this evidence coupled with the relative ease and sensitivity of the AE assay suggests a rather straight forward, sensitive, short-term bioassay that should be able to quantitatively evaluate the kind of chemicals and the In vivo conditions necessary for optimal induction of lung cancer.-T~F e- animal model developed In our laboratories for the defined chemical induction of lung cancer ts described below. The use of this model in cigarette smoke lnhalation studies is also described. Both the chemical induction of lung tumors and bioevaluation of cigarette smoke require long-term studies (one year or longer) before an effect can be observed or the lack of an effect can be determined to besignificant. Hence, the desirability of the AE assay as a sensl- tive, short-term test which would show direction In the chronic, long-term In vivo studies. Doses of 250 µg MCA given intratracheally one, two, four and six times to C3H/Anf Cum mice yielded approximately 35, 45, 76 and 77% lung carcinomas, respectively, within one year after treatment (Table i). Doses of 1.2 mg BaP given Intratracheally five, ten and fifteen times to C3H/Anf mice yielded approximately 19, 15 and 24% tumor Incidences within one year after treatment. The derivative, 7,8-dlhydro dth ydroxybenzo(a)pyrene (BaP-7,8 diol), is much more tumort genic to lung tissue than is the parent chemical, BaP. Data in Table 1 show that IT administration of 250 µg BaP-7,8-dlol given • five times at biweekly intervals results In approximately 40-50% lung carcinomas after about one year after treatment. The tumors -5- I 50137812 ~ r TCTR ' MN 0437,26-)

I Continuatlon of Details of Experimental Design and Procedures: observed In these experiments Include bronchogenic squamous cell c arcinoma, squamous neoplasm, alveolar adenocarcinoma and alveolar adeno-squamous carcinoma. These malignant lesions have been defined and characterized In the mouse and are representative of the major classes of the lung cancers found In man. The characterization of these lesions In our model animal systems has included a descrip- tion of the pathogenesis of these lesions; that is, a documentation of the steps Involved In the progress'ton or formation of the tumors from specific preneopl-asttc lesions (Table 2). This model system has also been characterized for levels of aryl hydrocarbon hydrox- ylase (AHH) In the pulmonary tissues of inbred strains of mice after IT inoculation of chemical carcinogens (16), and the susceptibil- ity of lung tissue to cancers induced by these chemicals is geneti- c ally linked to the capacity of lung tissue to metabolize these chemicals (see Appendlx A). Thus, we have an In vivo model system for lung cancer that is sensitive, well-characterTzed, analogous to the human situation, and specifically dependent on the capacity of the lung tissue itself for the bioactivation of these chemicals to forms carcinogenic to•the lung. It is interesttng to note that an Inhibitor of MFO activlty dl- sulfiram, has been shown to alter the amount of hepatic DNA damage induced In rats by diethylnitrosamine (DEN) (23). In addition, multi- ple feeding of disulfiram has also been shown to alter and delay the onset of hepatocarclnogenesls induced by DEN (24). Thus a known ln- • hlbttor of MFO activity Is capable of altering the amount of DNA damage Induced by a chemical requiring these enzymes for bioactlvations. This murine lung model s ystem has been utilized In our labora- tories for evaluating the biological activity of whole cigarette smoke In vivo. Cigarette smoke has been characterized as a weak carcinogen orto-carclnogen and hypothetically can exert Its effects at various stages during the process of tumor formation and ex- pression (see discussion, 25). Cigarette smoke contains between 5,000 and 10,000 chemicals (17), however, only a few have been characterized with regard to concentration In smoke and potential biological activity. Experiments evaluating the carcinogenic potential of such a complex chemical mixture as cl garette smoke In animal models must satisfy several specific criteria matnly because cigarette smoke possesses, at best, very weak biological activity. Thus, conditions must be found that mimic those that exist for man; thatis, exposure for long periods of time (in humans 30-40 years) at fairiy hl gh levels (3-4 packs/day), and then oniy 0.5-2% of the subjects will be observed to have smoke-associated lung cancers. The smoke exposure conditions presently in use In our labora- tory for the inbred strains of mice have been developed to assure maximal de posltion of total particulate matter (TPM) In the lung. Fresh, whole cigarette smoke ls generated using the Smoke Exposure Machine (SEM 11, Process and Instruments, Brooklyn, N1(.), delivered • as a smoke aerosol (0.3-0.5 µm mass median diameter) to the animal containment unit (Figure I). The mice are restrained In stock- type head restraint trays (5 mlce/tray) which are placed on the -6- 50137813 I I C T R 111N 0 4 3 1-"2 -7

: 1 > Continuation of Details of Experimental Design and Procedures: ~ supports of the polycarbonate module In the animal containment unit for smoke exposure. Smoke Is delivered through a channel In the polycarbonate module,into which the noses of the mice protrude. A soft rubber dam material provides a seal around the nose to prevent smoke leakage. Mice are obligate nose breathers and this exposure system, therefore, forces the mice to breathe the air or smoke provided In the channel (see Figure 1). The deposition and distribution of TPM In SC3F1lCum mice after exposure to cigarettes In the SEM 11 system has been characterized and representative data are shown In Table 3 and Figure 2. Such dosimetry experiments utilize a radioactive tracer 1n the cigarette smoke to quantitate the TPM distributed through- out the body of the mouse after smoke exposure. The SEM 11 allows the smoke concentration and exposure time to be varied and as the results Indicate, a dose response is obtained for TPM In the lung with Increasing smoke concentration or Increasing smoke exposure time. Elghty-90% of the TPM was found In the total respiratory tract of the mouse (head, larynx and lung) and 60-80% of the TPM was found 1n the lung itself. The deposition In the lung represents 80-90 µg TPM per cigarette at the lon ger exposure times (200-300 seconds total). Mice can tolerate this level of exposure fairly well and doses of 10 cigarettes per day (Kentucky reference 2A1 cigarettes) have routinely been used for periods of longer than ~ one year. This dose of 10 ctgarettes per day results In almost I mg TPM/day mouse lung or 50 mg TPM/kg body weight, which Is equivaient to a human smoker exposed to the smoke from 3-4 packs of cigarettes per day (18). The capacity of clgarette smoke to induce pulmonary DNA d amage requires several approaches In order to be bhoroughly evaluated. The question addressed Is whether there are chemicals in cigarette smoke which Interact with DNA and whether this lnter- action can be detected as damage by the AE assay. The most direct approach Is to expose mice to known quantlties of smoke and monitor the DNA damage at various times after the exposure. The use of whole smoke alone, however, precludes ascribing ~ the effect (i.e., DNA damage) to specific chemicals or even a class of chemicals. Condensates of whole cigarette smoke (20) and frac- tionation of those condensates (21) represent one of the only logical methods for breaking down the complex chemical mixtures found in smoke into some workable number of subcomplexes. The procedures for making clgare.tte smoke condensates (CSC) and the resulting fractions (CSCFs) have been previously discussed (20, ~ 21,22) and are only briefly described here. Cigarettes are smoked i on an analytical smoking machine using a two-second puff of 35 ml volume, once a minute to a butt iength of not less than 20 mm. The smoke is condensed In traps cooled in a dry-ice acetone mixture and the condensate removed from the traps with acetone. The • solvent is removed In vacuo at a low temperature and the conden- sate stored under nlcrogen at -20°C or lower until fractionation. - 7- ( 50137814 I CTR HN 043 '.1,26

~ Continuation of Details of Experimental Design and Procedures: The fractionation scheme is given In Table 6. Twelve fractions are obtained. A reconstituted sample was prepared by pooling one-third portions of each fraction. This reconstituted material (RCM) is needed to control for possiblE. alterations in chemical composition during the fractionation procedure. The need for standardization of procedures and establishment of quality stand- ards in this area has been recognized (20,22). The twelve con- densate fractions and reconstituted material from several research cigarettes are available to us for evaluation as potential Inducers of pulmonary DNA damage (Table 8). As discussed previously, compounds which can alter MF0 activ- ity can alter lung cancer susceptibility and DNA damage caused by other chemicals as measured by the AE assay (23). Cigarette smoke and CSC contain chemicals which lnduce pulmonary AHH and theoret- ically could alter the lung cancer susceptibility and amount of DNA damage induced by other chemical carcinogens. The effect of whole cigarette smoke In altering the amount of DNA damage caused by other chemicals may require long-term studies in ll yht of recent information from our laboratories. In collaboration with Dr. R. Rasmussen, we have observed that whole cigarette smoke is capable of lnhibiting DNA repair capacity in an in vitro assay (Table 7), however, the effect Is not observed unttl-after 2-3 months of con- tinuous exposure (Table 7). Even though DNA repair is inhlbited , in this assay, the role of this lnhibition on the DNA damage or ' persistence of this damage Induced by other chemicals is diffi- culttio predict. There could be an enhancement of the effect, tnhtbition or no effect on DNA damage.lnduced ]M vivo. Special Considerations The Departments of Experimental and Biochemical Oncology possess special expertise required for: 1. Short-term and long-term animal handling under conditions where such factors as adventitious agents, parasites, and housing conditions that may alter the carcinogen metabolizing capacity of the Inbred strains of mice are well controlled. 2. On-hand and in-depth experience in the model systems for inducing lung cancers in the Inbred strains of mice. 3. On-hand and In-depth experience In exposing the in- bred strains of mice to varying doses of whole cigarette smoke under a variety of exposure conditions. 4. More than a year.of experience in analyzing the amount of DNA damage produced In mammalian cells using the alkaline elution assay. This experience includes on-slte visits by the departmental staff to the laboratories of Drs. J. Swenberg and G. Petzold of the Upjohn Corporation to gain first-hand experience at the nuances of this assay • precedure. -8- 50137815 CT1 ! I .• I 04' 1.W I~ E-1^

Continuation of Details of Experimental Design and Procedures: II. Experimental Approach ( ( i 0 A. Evaluation of AE assay as a tool In predicting lung tumorigenic capaclty of chemical carcinogens. 1. Can damage to lung DNA be detected after intratracheal (IT) treatment with chemicals under conditions where these chemicals lnduce carcinomas? Intraperitoneal trea'tment with such chemicals as dimethylnitrosamine, 4-nltroqulnoline-N-oxide (4-NQO), 1,12-dimethylbenz(a)anthracene, ethylnitrosourea, methyl methanesulfonate (5) and subcutaneous or oral treatment with dimethylhydrazine (15) all cause damage to lung tissue DNA as measured by the AE assay. The major question 1s, can the AE assay detect DNA damage In lung tissue under conditions where a significant incidence-of malignant lung carcinomas will eventuate? The question will be addressed by measuring the amount of DNA damage in lung tissue using the AE assay at 0.5, 1.0, 2.0, 4.0, 8.0, 24 and 48 hours after IT instillat-ton of MCA, BaP, and BaP-7,8 diol to female SC3F1 mice. The procedure for the AE assay ls given in Table 4. The negative control will be the 0.2% gelatin- saline vehicle and the positive control will be 2.0 mg 4-NQO given IT to these mice. Recent studies In our labo- ratory have indicated that an In vitro positive control _ should also be Incorporated Into tFiTs test to insure that ' the buffers, filters, lysing solution etc. are functional. This control will be MNNG-treated V-79 cells (treated In culture) that are stored frozen at -120°C until assayed. The total DNA eluted from the filter will be determtned simultaneously method (the animals will be IP treated as neonates with a total of 150 pCI 3H-TdR over a 3 week period of time and a fluorometric method (15,19). This latter technique, although a little less sensitive, has the advantage of not requiring prelabelling of the DNA. This is especially Important when analyzing for DNA damage after multiple treatments with these carcinogens, because the In vivo labeling Index of lung tissue has a halflife of abou`=-3 weeks and thus by 6-9 weeks the ability to detect DNA damage may be severely impaired because of an Inability to detect the DNA which may elute f rom the filters. 2. For a particular class of chemical carcinogens, is there a correlation between conditions that yield high Incidence of lung carcinomas and amount of DNA damage? The major factors that Influence lung cancer susceptibility In BC3F1 mice are type of chemical carcinogen and dose of carcinogen. The data from section 1 should determine those conditions whereby MCA, BaP, or BaP-7,8 dlol _ Induce lung DNA damage. The second approach will be to use _s various doses of MCA (10-500 jig) and BaP (600-1800 µg) and various times of treatment (1-6 times) in order to observe -9- 50137816 ~ CTR PIN 043730

, 0 L• Continuation of Details of Experimental Design and Procedures: if the extent of DNA damage parallels the ultimate suscepti- bility of that organ to cFiemical carcinogenesis after a specific dose and treatment. Thus, we propose to evaluate the extent of DNA damage at only 2-3 times after multiple IT treatments with these chemlcals (e.g. 2, 4 and 6 hours, the exact time will be determined by the results of thE study in section 1). 3. Is the amount of lung DNA damage related to the capacity of lung tissue to metabolize these chemicals? The answer to this question also provides support for the question addressed in section ii. That is, strains of mice are variable in their susceptibility to lung cancer induced by MCA and this variability at least partly results from the relative levels of those enzymes capable of metabo- lizing this chemical carcinogen (see Appendex A). Therefore, by measuring the amount of DNA damage in various strains of mice with known AHH levels following IT treatment with MCA, we can determine not only if the levels of AHH are playing an'important role, but also if this damage parallels the ultimate susceptibility of these strains to MCA-induced lung cancers. We propose to answer this question by measuring the amount of DNA damage in lung tissue of B6D2F1 x D2 mice following IT treatment with MCA. These progeny animals represent an ideal model system, because the capacity to recognize MCA and metabolize this chemical to a variety of biologically active forms segregates as a sin e autos.omal dominant gene in this cross (see Appendix B for discus3~ on). Thus 50% of the progeny animals from this cross will be capable of responding to the IT-instilled MCA by Increased )evels of pulmonary AHH, and 50% will not. The genetic linkage that may exist In lung tissue between AHH responsiveness, cancer susceptibility, and degree of DNA damage can, therefore, be unequivocably obtained. Approximately only 20 B6D2F1 x D2 animals would have to be analyzed in order to arrive at this conclusion. A simple method of confirming the relationship between AHH responsiveness and amount of pulmonary DNA damage ls based on the observation that there exists a chemical that can specifically alter the AHH capacity of the genetically non-responsive strains of mice. That is, 2,3,7,8-tetrachlorodibenzo(p)dioxin (TCDD) treatment of DBA/2 mice will result in increased AHH levsls. Thus, TCDD-treated D2 mice have AHH capacities very similar to that of the responsive B6 strain. TCDD, under conditions where it induces AHH in D2 mice, selectively enhances the tumor susceptibil ity of this strain (see Appendix ). The most direct test for establishing a relationship between AHH levels and amount of DNA damage in pulmonary tissue is to compare the level and kinetics of DNA damage in control MCA-pretreated, and TCDD-treated D2 strains of mice. -10- 50137817 G `"~~' HN 4~.0 4 3 ,~~'_'4p .11, i

L• ( I c 0 • Continuation of Details of Experimental Design and Procedures: If the AHH enzyme complex plays a major role in determining the amount of pulmonary DNA damage induced by MCA, BaP and/or BaP-7,8 diol, then TCOD should specifically enhance the sus- ceptibility of the D2 mice to this DNA damage. The effect of co-treatment and pre-treatment with TCDD will be carried out In a manner similar to that described in manuscript form in Appendix C. B. Characterization of the AE assay as a tool In predicting the capacity of the chemicals in cigarette smoke to interact with lung DNA In vivo. 1. Does whole cigarette smoke contain chemicals capable of causing damage to lung DNA as detected by the AE assay? The general approach will be to expose mice to a known quantity of smoke and monitor damage to pulmonary DNA at various times after the exposure. Several variables will be examined: 1) the exposure conditions Including the cigarette type (hi h vs low nicotine), the smoke concentration, the total dose; 23 exposure of different strains of mice to determine whether a difference exists in susceptibility to smoke-induced damage, and 3) exposure for various lengths of time to determine long-term effects of smoke exposure. The smoking conditions currently In use In our laboratory utilize the SEM II, the Kentucky reference 2A1 or 2R1 cigarettes at a smoke concentration of 10%, total exposure time of 300 or 200 seconds Per ctgarette, respectlvely. Shorter exposure time is used for the 2R1 cigarette because of the high nicotine content and resulting toxicity. A description of the reference cigarettes is given In Table 5. BC3FI/Cum mice (7-8 weeks of age) will be adapted to smoke from the 2A1 or 2R1 cigarettes under adaptation procedures requiring 3 weeks to reach an exposure level of 2 cigarettes per day. The AE assay will be performed on adapted mice exposed to: 1) one 2A1 cigarette, 10% smoke concentration, 300 seconds exposure time, and 2) one 2R1 cigarette, 10% smoke, 200 seconds exposure time. Mice of the same age but unadapted to smoke will be exposed once on the day the AE assay Is performed. Mice used as machine controls will serve as ne ative controls and mice treated IT with 4-NQO (2 mg per mouse) will serve as positive controls. The AE assay will be performed at 0.5, 1, 4, 6, 8, 24 and 48 Fiours post exposure. I f any DNA damage Is observed under the conditions of 300 or 200 seconds total exposure time for 2A1 or 2R1 cigarettes, respectively, then a dose response curve will be determined for Increasing smoke concentration and varying total exposure times. If no DNA damage is observed after one 2A1 or one 2R1 cigarette, the number of cigarettes per day will be lncreased to 5 and then 10 and the AE assay performed at the times indicated after the last cigarette offered. 50137818 ~ CTR I-IN 0437 `32

i O a • Continuation of Details of Experimental Design and Procedures: If an effect is observed with one cigarette type but not the other, the cigarettes listed in Table 5 will be evaluated under the same conditions In which DNA damage was observed. The optimal conditions for Inducing DNA damage by cigarette smoke will be determined using BC3F1/Cum mice. These conditions will be used to evaluate the effects of whole cigarette smoke in two other strains, the C3H/Anf (C3) and DBA/2 (D2). The C3 and 02 represent extremes In sensi- tivity to PAH induced carcinogenesis, the C3 being most sensitive, while the D2 Is resistant. The BC3F1 strain Is presently being evaluated and the C3 and D2 strains are scheduled for evaluation as model strains for the determi- natlon of carcinogenic and/or cocarcinogenic capacity of whole cigarette smoke during lifetime exposure. Using the optimal times for detecting pulmonary DNA damage determined in the above, BC3F1/Cum mice chronically exposed to cigarette smoke will be monitored by the AE assay at monthly Intervals for one year for DNA damage. 2. Are tliQre chemlcals In smoke as represented by cigarette smoke condensate and condensate fractions whic h have the capacity to cause damage to DNA as detected by the AE assay? As discussed in the Background (section 1), a logical extension of the studies evaluating the potential damaging effects of whole cigarette smoke on lung DNA is the use of cigarette smoke condensates and fractions derived from the condensates. This approach would not only confirm the result of using whole smoke, but also, should give indications of the type or class of chemicals that may be responsible for these effects. Cigarette smoke condensates from different cl garettes have been made avail- able to us and are currently being stored at -120°C. The CSC material will be lnitlally dissolved in acetone, vi gorously added to 0.2% gelatln-saline solution, the acetone will then be removed, leaving a working suspension of CSC in gelatin-saline. CSC from 3A1 and 2R1 ci garettes will be evaluated in BC3F /Cum mice after IT administration for the capacity to induce DNA damage using the AE assay. The 3A1 and 2R1 cigarettes represent low and high nicotine containing cigarettes (Table 5). If differences are observed between the 3A1 and 2R1 condensates, then 2A1 and 1R3 con- densate material (representing low and intermediate nicotine containing cigarettes,'Table 5) will be evaluated under conditions where the effect is scen. The twelve cigarette smoke condensate fractions (Table 6) and the reconstituted material from the 3A1 and 2111 cigarettes will be evaluated after intratracheal inocu- lation into BC3F1/Cum mice by the AE assay. The strong acid fractions will be neutralized with IN NaOH so as to a void acid-associated toxicity. -12- 50137819 CTR H~t~ ~ ~ ~ ~ ~~ '3 -

----- ~ --------••-•------------------- --• ( 0 • Continuation of Details of Experimental Design and Procedures: The dose response will be determined for each CSCF and an LDSO dose determined. This dose and two dilutions of this dose will be evaluated for the capacity to damage DNA. 3. Does exposure to whole cigarette smoke alter the DNA damage induced by other chemicals? Cigarette smoke contains chemicals which alter MFO activity and hypothetically,•such chemicals may have the potential to al.ter DNA damage induced by other chemicals (see Background, section 1). An experiment designed to test this hypothesis Is outlined 1n the accompanying table (Table 9). The general approach will be to expose mice to cigarette smoke for one year. At several times during the year, mice will be treated IT with a chemical known to induce DNA damage (see section II.A.1). The AE assay will be performed at monthly intervals for one year. Results from the chemically-treated smoke exposed mice will be compared to chemically-treated machine control mice. Variations on this approach will include evaluation of different ciga- rette types (Table 5), different chemicals, different mouse strains, and different treatment schedules. -13- 50137820 1 I (.r f i"C f f f "f 0"f ,'~ 73"'f'

/. . III. Schedule for Experiments 1979 1980 1981 J F M A M J J A S 0 N D J F M A M J J A S 0 N D J F M A M J J A S 0 N D Lung Carcinogens Evaluated (Sect. II.A.I) Dose Response of Carcino ens for Damage ~II.A.2) Damage Correlated to Metabolism Whole Smoke Evaluated (II.B.2) BC3F1 C3 02 CSC and CSCF Evaluated (11.6.3) Whole Smoke and Carcinogens Evaluated (II.B.3)- d - ... I I

• Table I CUMULATIVE TUMOR INCIDENCE AND TIME OF OCCURRENCE IN C3H/Anf MICE AFTER INTRATRACHEAL INOCULATION OF MCA, BaP OR BaP-7,8-DIOL Treatmenta Tumor Incidence(%) b Number of Animals with "Lung Tumors" Per Number of Survivors Moriths on Tes t 4 5 6 7 8 9 10 11 12 13 14 MCA IX300 µg 12/35(35) 2/35. 4/27 1/17 5/12 2X300 µg 18/40(45) 3/40 2/32 4/31 2/19 7/15 4X300 µg 31/41(75) 1/41 2/36 2/30 3/29 10/21 13/15 6X300 Pg 19/22(77) 2/22 2/17 4/14 2/9 5/8 4/5 u+ 0 w ~ BaP v ~ 5X1.2 mg 4/28(19) 1/20 1/22 1/17 1/9 5/13 OD N IOXI.2 mg 5/33(15) 1/33 1/29 1/18 1/15 1/12 N I5X1.2 mg 4/17(24) 1/17 1/12 2/10 , BaP-7,8-Diol 5X250 µg 25/56(45) 1/56 1/55 3/50 4/42 3/33 4/25 10/15 BaP 5X250 µg 0/90(0) 0/90 1 a. Treatment with chemicals was at the designated doses and given biweekly for the designated numbers of times. b. "Lung tumors" are defined to be either of the type alveolar adenocarcinoma, squamous cell carcinoma, adeno-squamous carcinoma, or squamous neoplasms.

• r tw Table 2 POSTULATED PROGRESSION BRONCHOGENIC NEOPLASIA I OF SQUAMOUS, A N MOUSE LUNG T LVEOLOGENIC AND REATED WITH MCA . HYPERPLAStA , METAPLASIA BRONCHIOLAR-ALVEOLAR I SQUAMOUS METAPLASIA ~ LINING CELL HYPERPLASIA BRONCHIOLAR ALVEOLAR REGION ~ I cn o , J w cy% cw N ALVEOLAR LINING MUCINO CELL HYPERPLASIA HYPER t ` MINIMAL NODU 1 US CELL PLASIA AR (?) SQUAMOUS NON-KER y MINI METAPLASIA ATINIZING AL SQUAMOUS KERATI KERATIN CYST MINI METAPLAS/A '~ NIZING 1 y MAL w MODERATE BRONCHOGENIC MUCINOUS t ADENOCARCINOMA NODULAR. NON•COMPRESSING T NODULAR. COMPRESSING OOE I NOD NON-COM ATE LAR, PRESSING. MODE I NOD NON-COM ATE LAR, PRESSING L t PAPILLARY AND PLEOMORPHIC CHANGES t ALVEOLARADENOCARCINOMA •'•"""">" 1 • 1 _~ NODULAR. COMPRESSING SQUAMOUS NEOPLASM f- NODULAR. COMPRESSING t ADENOSQUAMOUS __< ....................... SQUAMOUS CELL CARCINOMA CARCINOMA

` Table 3 DEPOSITION AND DISTRIBUTION OF TOTAL PARTICULATE MATTER IN BC3FI MICE AFTER EXPOSURE TO CIGARETTE SMOKE ON THE SEM 11 Exposure Conditions Total Particulate Matter (µeS.D .) Smoke Concen. (°6) Exposure Interval Smoke/Alr (Secs.) Total Smoke Exposure (Secs.) Mean Body Wei ht (Gg} ex ead arynx ung tomach Total Body 10 5/55 50 26.6 F 2±1 1±5 9±2 3±1 20±5 10 10/50 100 22.3 F 2t2 3*1 15t4 4t1 23t6 10 10/50 100 26.1 M 3t1 4*5 16t4 3*1 26*6 10 20/40 200 25.1 F 5*1 4tl 57*7 8*2 85t13 10 20/40 200 25.8 M 5}1 6t4 54t12 6*2 77f14 10 30/30 300 22.9 F 8t2 4t3 85t24 9t3 109t26 10 30/30 300 26.7 M 8*2 3t1 93t37 13*3 121t30 20 5/55 50 22.8 F 2t l 2f 1 10t4 - 3t 1 19*5 L 20 10/50 100 23.0 F 5t1 ltl 42f1~ 5*2 55*14 20 15/35 150 23.2 F 7*2 3*2 75*23 6t2 93t24 * 20 20/40 200 23.3 F 8*2 4f2 92t19 6t3 114 20

Table 4. Methodology for Alkaline DNA Elutlon Assay of Tissues from Treated Mice. i 1. 2. 3. 4. 5. , 6. 7. • A. Cell Preparation Intratracheally Inoculate chemical or exposed mice to cigarette smoke. Sacrifice by cervical dislocation. Remove lungs rapidly and place in cold 0.02 M EDTA - 0.9% NaCI solution. Mince lungs with scissors and homogenize gently in a Teflon- glass homogenizer at 0•C. Centrifuge homogenate at 100 x g for 2-3 minutes to remove large fragments. Remove supernatant and apply allquots to polyvinyl chloride filters for elution of DNA. B. DNA Elution Procedure Add the aliquot to 2.0 ml lysing solution (2 M NaCI - 0.02 M EDTA - 0.2% Trtton X-100, pH 8.2) in the filter holder reservoir, draw the solution through the filter add additlonal lysing solution (5.0 ml), wash with 0.001 M EDTA, pN 10.0 (2.0 mt/min.). -18- 50137825 CTR NN 04~~ ~ 39-

L. > i c I 8. Elute single stranded DNA from the filter with 0.02 M tetraethylammonium hydroxide - 0.02 M EDTA, pH 12.2 (0.05 ml/min., 75 ml total). 9. Collect the eluate, determine the amount of radioactivity In two 1.5 ml aliquots or assay--for DNA content (19). Determine the amount of radioactivity remaining (n the filter or assay for the amount of DNA on the filter. -19- ( ( 50137826 CTR HN 04,3"""40

L.. ' TA B LE 5 Analyses of Kentucky Reference Cigarettes Cigarette Type TPM m/ci . Nicotine mg/cig.., Water mg/cig. Puffs 2A1 40 0.51 3.1 10 3A1 43 0.35 6.1 12 iR2 35 1.70 4.2 10 IR3 26 1.25 3.8 8 2R1 50 2.81 6.6 13 -20- Cf R Hf'i 043741

Table 6 I ( 40 CIGARETTE SMOKE CONDENSATE FRACTIONATION SCHEME (21) CCi:::E"S:.TE IcHC.! _Er_d; l::) I •i .: ll j1 ..J • `f.... ----;;.....1110 !U;! '.::• v:,lt, ~ _.l__....' '.11•tte S~~IS I I l ILC E1o lS:.l! lSRF- ,. :.v~:pro1L, IOC••OI ule ttON ! I FiitfC:t ~0!IS (SAw/ C~:IO~tiOne • `.~eON-H=U r---r-- '1 C.;Ic:.ti:nt t:lirc1nerSOn; (11cN1 (I~Rm I -21- ( 50137828 , l1 A A : ( ' i DH I J, ~ I ~ i C T R N N 0 43 ' .1 I Ar2

4# Table 7 DNA REPAIR AND REPLICATION IN LUNG TISSUE FROM BC3F1/CUM gICE EXPOSED TO 2A1 CIGARETTE SMOKE FOR THE INDICATED PERIODS. Cumulative Exposure (Weeks) Smoke DNA Repair 3H-DPM/µg DNA Machine S/M DNA Replication 3H-DPM/µg DNA Smoke Machine S/M 1 254f68 189±17 1.34 1838±615 622t208 2.95 4 88f12 101±20 0.88 927t160 369±102 2.51 6 123t28 138t15 0.89 963±127 556± 97 1.73 8 94t45 125f30 0.75 1240t 13 581t116 2.13 11 101t22 176t32 0.57 1300t172 737±202 1.76 15 89±15 131t32 0.68 1231t 92 1082t226 1.14 17 119±23 254t19 0.47 1532± 46 699t365 2.19 24b 79t29 155±27 0.51 1951t275 840t170 2.32 n aR. Rasmussen and C. J. Henry, personal communtcation. bMMS used to damage DNA; 4NQ0 used in all others. • -22- I 50137829 ' W 7 R I 7 7 / 043743

J Table 8 YIELDS OF CIGARETTE SMOKE CONDENSATE FRACTIONS FOR 1A1, 2A1, 3A1 and 2R1 CIGARETTES. Fractions 1A1 Mg/Cig. 2A1 Mg/Cig. 3A1 Mg/CIg. 2R1 Mg/Ctg. i Whole CSC 23.50 40.00 43.0 50.0 Reconstltuted 23.00 39.50 42.9 49.8 Bla 0.81 0.60 0.58 1.04 B1b 0.29 0.24 0.21 0.32 BE 0.95 1.04 1.30 3.77 eW 0.36 0.52 0.83 0.63 WAI 2.27- 3.63 3.66 3.57 WAE 1.98 2.66 4.45 3.77 SAI 0.39 1.44 1.26 2.62 SAE 0.78 0.88 1.36 1.60 ~ SAW 8.69 15.60 16.9 19.59 NMeOH 1.19 2.16 2.17 2.10 NCH 4.58 9.54 9.29 9.40 NNM 0.70 1.24 0.98 1.54 ( ( • -23- ( 50137830 CTR NN 043744

. . i Table 9 PROPOSED OUTLINE FOR ANALYSIS OF THE CAPACITY OF CIGARETTE SMOKE TO ALTER DNA DAMAGE INDUCED BY KNOWN CHEMICAL CARCINOGENS Treatment Schedule Months after Chemical Treatment 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 C X 1 1. Smoke E xpos u reb X a. The chemical used for IT inoculation will be determined as outlined.in Section II.A.1 ' under treatment conditionswhere maximal DNA damage is Induced. b. The cigarette type to be used for smoke exposure will be determined in Section 11.8.1 under the conditions where maximal DNA damage is induced. Smoke exposure will be continuous (5 days/week) for one year. I LJ

• i Figure 1 Animal Containment and Smoke Flow System 1 1 n,,T~~N ~i~s~nt i ~ t~ AIR ~ ~lutomatic ignition, pulf, ejection Ismoke I -z j oPTICAL DISTRIBUTION orair SENSOR VALVE C C r RECORDER I~J Sampling - c•l..~ ANIMAL CONTAINMEN T Vent Manifold nr-nrrc ~ ven r, L1 Cross Section of Polycarbonate Module vent smoke Close-up View of Animal Restraint in Relation to Polycarbonate Module uten RF¢TReINT NOSE SEAL CHIN SUPPORT t

Figure 2 TOTAL PARTICULATE DEPOSITION IN MICE AFTER EXPOSURE TO SMOKE GENERATED ON THE SEM II "A 0 LUNG 100 µ g TPM 50 100 TOTAL RESPIRATORY TRACT 100 µ g TPM TOTAL BODY µg TPM 50 100 50 -A. 100 200 -.6 200 I 300 300 -4-•- 300 20';6 Smoke Concentration 10% Smoke Concentration CUMULATIVE EXPOSURE TIME (Seconds) -26- t ~~~ ~N 043747

L• REFERENCES ' 1. Irving, C. C., Methods Cancer Res. 7:189-244 (1973). 2. Kleihues, P., Lantos, P. L. and Magee, P. N. Intern. Rev. Expt1. Pathol. 15: 153-230,(1976). 3. Sarma, D. S. R., Rajalakchmi, S., and Farber, E. In Cancer: A Comprehensive rreatlse (F. F. Becker, ed.) TTew York: Plenum Press (1975), Vol. 1, pp 235-287. 4. Damjanov, I., Cox, R., Sarma, D. S. R. and Farber, E. Cancer Res. 33:2122-2128 (1973). 5. Petzold, G. L. and Swenberg, J. A., Cancer Res. 38:1589- 1594 (1978). 6. Swenberg, J. A., Petzold, G. L. and Harbach, P. R. Blochem. Biophys. Res. Comm. 72:732-738 (1976). 7. Kohn, K. W., Erickson, L. C., Ewi 9,R.A.G., and Friedman, C. A., Blochem. 15:4629-4637 (1976). 8. Lett, J. T., Klucis, E. S. and Sun, C., Blophys. J. 10:277-292 (1970). , 9. Kohn, K. W., Friedman, C. A., Ewig, R. A. G. and iqbal, F. M. Blochem. 13:4134-4139 (1974). 10. Kann, H. E., Jr., Kohn, K. W. and Lyles, J. M., Cancer Res. 34:398-402 (1974). 11. Kohn, K. W. and Ewig, R. A. G., Cancer Res. 33:1849 (1973) 12. Ewiy R. A. G. and Kohn, K. W., Proc. Am. Assoc. Can. Res. 17:147 (1976). 13. Elkind, M. M. Blophys. J. 11:502-520 (1971). I 14. Shechtman, L. M. and Kourl, R. E., In Progress in Genetic Toxicology, D. Scott, B. A. Bridges and F. H. Sobels,(eds.) Amsterdam, The Netherlands: Elsevier/North-Holland Biorned. Press (1977), pp 307-316. 15. Brambilla, G., Cavanna, M., Parodi, S., Sciaba, L., Pino, A. and Robbiano, L. Int. J. Cancer 22:174-180 (1978). ( • l -27- ( I 50137834 r , CTR H~~ 043748

L. A 16. Kouri, R. E., Rude, T. H., Thomas, P. E. and Whitmire, C. E., Chem.-Bio. Interactions 13:317-331 (1976). 17. Wakeham, H. In Chemistry of Tobacco and Tobacco Smoke (I. Schmeltz, ed.) New York: Plenum Press 1972, pp 1-20. 18. Binns, R. Toxicol. 7:189-195. (1977). 19. Kissane, J. M.- and Robins, E., J. Blol. Chem. 233:184-188 (1958). 20. Stedman, R. C. Chem. Rev. 68:153-207 (1968). 21. Swain, ARCooper, J. E. and Stedman, R. L.,Cancer Res. 29:579-5630969). 22. Benner, J. F. and Keene, C. K., in Proc. of the Univ. of Kentucky Tobacco and Health Research Inst., pp 185-190 (1972). 23. HadJlolov, D., Frank, N. and Schm'Ahi, D. Z. Krebforsch.90: 107-109 (1977). 24. Schmahl, D., Kruger, F. W., Habs, M., Diehl, B. a Z. Krebsforsch.85:271-276 (1976) 25. Van Duuren, B. L., Cancer Res.28:2357-2362 (1968). • -28- 50137835 C 7"R iI N 0 4,3"1`4 q-

L. ( ( i ( ( r ) 10. Space and facilities ovoilob(e (when elsewhere than item 2 indicales, slole location): All necessary space and equipment will be provided for these studies by the Departments of Biochemical and Experimental Oncology. The model systems for chemical Induction of lung cancer and for cigarette-smoke Inhalation have been developed and are current investigations of these two departments. The following major pieces of equipment are available for use In this work in conjunction with CTR Contract 0030. Beckman scintillation counter, Aminco Bowman s pectrophotofluorometer, Gilson peristaltic pumps, fraction.collectors and all necessary centri fuges. Animals required for these studies will be housed at 4807- 4809 Bethesda Avenue where adequate quarantine, isolation and holding facilities are available. Mice will be exposed to smoke using the SEM II (see Section I and Figure 1 for description) as a service of the smoke inhalation facility provided under CTR Contract 0030. 11. Additional fociliries required: None 12. Biographical s4elches of invesligolor(s) and other professional personnel (oppend): See Append 1 x D • 13. Publico:ions: (Fve mosl recent and pertinent of inveuigoror(s); append GN, and provide reprints if ovoiloble). See Appendices A, B. C and E -29 - 50137836 C f R f ~N t-'.~4:3 e°'`~-0

L. 4. I 14. first yoor budpet: A. Salaries (give names or stote "to be rccruited") % time Profestionol (give % time of investiqotor(s) even if no salary requostcd) Amount C. Henry, Ph.D. 10% 2,698 R. Kouri, Ph. D. 10% .__ R. Curren, Ph.D. 10% ___ Technical R. Sosnowski 9 922 Merit Increases @ 7% 1V, M 883 Fringe Benefits @ 32% 4,321 a. Consumable supplies (by major coteyoriis) Mice 2,500 Chemicals and Solvents 1,000 Labor Supplies 2,000 Radioactive Sub.Total for A -._17.,824._:.._ Materials 1,000 Sub.Total for 8 .. 6,500. " C. Other eapenses (itemize) Equipment Repair 100 Publication Costs 500 ~ ~ Travel Freight Laundry 500 100 144 J Centra.l & Office Sub•Totol for C • 2;568 ' ' Services 1,224 ~ Running Totol of A•I a-1 C 26,892. 0. Permanent equipment (itemize) • E. Indirect costs ~. Fi>e~i Fee 1S. ttimoteo ulure requirements: Sub.Totol for D .~ _0 . .. . ... E _.._.-7.,443_._ F. Total Request 38,150 ( Solories Consumoble Suppl. Other E.penses Permonent Equip. Indirect CousFee Total Year 2 19,071 7150 3,3 ___ 8,074 4180 41,800 Year 3 20,406 7865 R_a3T e9e0 a5,40n 50137837 CTR I`IN 04•31-1:x 1

L. 1 I . CAROL JANICE MORGAN HENRY ADDRESS: 4807 Bethesda Avenue, Bethesda, Maryland 20016 DATE AND PLACE OF BIRTH: June 24, 1944 - Tampa, Florida SOCIAL SECURITY NUMBER: 474-46-8073 Education Ph.D. - 1972 - University of Pittsburgh, Pittsburgh, Pennsylvania (Microbiology) B.A. - 1966 - University of Minnesota, Minneapolis, Minnesota • (Chemistry) Employment ~ ~ Departmental Director and Project Director - Microbiological Associates - 1977-present Assistant Project Director - Microbiological Associates - 1976-1 97/ Research Associate - Sloan-Kettering Institute - 1975-1976 Research Assistant - Sloan-Kettering Institute - 1974-1975 Postdoctoral Fellow - Princeton University, Princeton, New Jersey - 1973 Postdoctoral Fellow - Friedrich Miescher Labor, Max Plack Institute, Tubingen, W. Germany - 1972-1973 Graduate Student - University of Pittsburgh, Pittsburgh, Pennsylvania - 1968-1971 Research Assistant - Mellon Institute, Pittsburgh, Pennsylvania - 1968-1968 Junior Scientist - University of Minnesota, Minneapolis, Minnesota - 1966-1967 Undergraduate Research Assistant - University of Minnesota, Minneapolis, Minnesota - 1964-1966 ( Awards, Distinctions and Lectureships Sigma Xi, Scientific Honorary Iota Sigma Pi, Chemistry Honorary ( Chimes, Junior Women's Honorary Postdoctoral Fellow - National Institutes of Health, Sloan-Kettering Institute - 1975 European Molecular Biology Fellow - Biozentrum der Universitat Basel, Basel, Switzerland - 1973 Predoctoral Fellow - National Institutes of Health, University of ~ Pittsburgh Department of Biophysics Pittsburgh Pennsylvania ( , , , and Microbiology - 1970-1971 Andrew Mellon Fellow - University of Pittsburgh, Pittsburgh, Pennsylvania, Department of Biophysics and Microbiology - 1968-1969 C 50137838 ~ C T R H N 0 4 3 7- " 552

L. a C. J. M. Henry Kouri, R. E., Rude, T. H., Whitmire, C. E., Henry, C. J., Sass, B. and Billups, L. H. Correlation of Inducibility of Aryl Hydrocarbon Hydroxylase with Susceptibility to 3-Methylcholanthrene-Induced Lung Cancer. J. Natl. Cancer Inst. (Submitted, 1978) Kouri, R. E., Rude, T. H., Brandt, K. R., Sosnowski, R. G., Schechtman, L. M., Benedict, W. F., Henry, C. J. - Biological Activity of Tobacco Smoke Condensate and Condensate Fractions. In: Proc. lst Intl. Symp. on Pollution and High Risk Groups. (in press) 1978. 0 50137840 CTR MN ~".~4..3f"`53

CTR MH 043754

Txr. Covxclr. FoH r!'oBAcco REa)Wi;czt- 130 EA6T aOTts tsTREFT \EN' YORK. T. Y. 30022 (212).23•69e5 Application For Renewal of Research Grant (Use extra pages as needed) First Renewal ® Second Renewal 0 Dat.: 8/15/79 ( ( ( ( ( ( 1. Principal Investigator (give title and degrees), Carol J. Henry, Ph.D. Richard E. Kouri, Ph.D. Rodger D. Curren, Ph.D. Director, Department of Director, Department of ProJect Director, Experimental Oncology Biochemical Oncology Department of Biochemical 2. Institution & address: Oncology jp Microbiological Associates 5221 River Road Bethesda, MD 20D16 3. Department(s) where research will be done or collaboration provided: Department of Experimental Oncology Department of Biochemical Oncology 4. Short title of study: Evaluation and Characterization of an Alkaline Elution Assay as a Measure of Pulmondry DNA Damage Induced by Chemical Carcinogens or Cigarette Smoke. 3. Proposed renewal dote: November 11979 6. How results to dote have changed earlier specific research aims: No changes have been made in our specific research aims. We have characterized the alkaline elution assay using both a radiometric and fluorometric assay for the quantitation of DNA. For reasons given in the Progress Report, we have opted to use the fluorometric assay in the proposed studies. 7. How results to dote hove changed earlier working hypothesiu No changes have been made in our working hypothesis. Our hypothesis is that chemical carcinogens or the metabolites of the carcinogens have the potential to interact with DNA and that this interaction or DNA damage is one of the early ( steps in the carcinogenic process. This DNA damage can be quantitated by the alkaline elution assay. 501377R2

L. 2. 8. Any additional facilities now required? Describe brieflyr No. 9. Any changes in personnel? Append biographical sketches of new key professional personnel: No. ft 1 10. Append outline of experimental protocol for ensuing year. See proposed s tudies . 11. List publications or papers in press resulting from this or closely related work. (append reprints or manuscripts not previoutly sent). None at this time. do 12. Summary progress report (append in standard form as separate document, unless recently submitted). 50137783 ~.r f ~~.'~' f f t 'f ~.,r~ `"f' .~ f' t ~~ ~

( ( ( Technical R. Sosnowski 100',6 10,200.00 Merit increases ® 7% 945.00 Fringe Benefits ® 32% 4,623.00 Sub-Total for A 19, 071. 00/ ( I i 3. 13. Budget for the coming year: • A. Salories (pive nomes or state "to be recruited") Professional (give % time of investigator(s) ~ even if no salary requested) % time Amount C. J. Henry, Ph.D. 10% $ 3,303.00 R. E. Kourl, Ph.D. NC R. D. Curren, Ph.D. NC r B. Consumable supplies (by mojor.catepories) Mice 2,500 Chemicals and Solvents 2,300 Lab Supplies 2,350 C. Other expenses (itemize) Equipment repair 500 Publication cost 500 Travel 600 Freight 150 Laundry 144 Central Services 1431 Sub-Total for B Sub-Total for C > 7,150.00 / 3,325.00 Running Total of A + B+ C29, 546.00 D. Permanent equipment (itemize) -0- E. Indirect costs F. Fixed Fee 50137784 Sub-Total for D 8,074.00 4,180760 Total request !f 1,8.0.0rQ0 i CTR NN 0437"57

L. 4. we 14. Other sources of financial support: List financial support from all sources, including own institution, for this and reioted research projects. CURRENTLY ACTIVE Title of Project Smoke Inhalation Studies in Mice Title of Project • Source Inclusive (give grant numbers) The Council for ' Tobacco Research I, Inc, U.S.A. PENDING OR PLANNED It is understood that the investigator and institutional officers in applying for a grant have read and accept the Council's "Statement of Policy Containing Conditions and Terms Under Which Project Grants Are Mode." . . re , our . :, . . , T rf~ Name sianat ur. at. 8/ 16 / 7 9 Source inclusive (pive grant numbers) r v- Checks payable to crobiological Associates Mailing address for checks 5221 River Road .,~thesda. MD 20016 _ Amount 1,170,000 Amount Dotes September 1, 1978 December 31, 1979 Dates Principoi investipator C. J. Henry, Ph.D. Ph.D. R D Cur n Ph D R E K i Telephone.~~1) 654-34Q0 _ 303• 241, 284 AreeC~e Nv~Mr lHqrJ.e Responsible officer of institution Typed Name Mary Ann Montgomery r,l, Director Contracts and Marketin Admin. Signature r1L ~ • }~1~ (301) 54-3400 241 Telephone AINCrrdrr NVmbr •' aarl~ri~~ 50137785 CTR NN 043f-56

we Section 10. Proposed Studies For Second (Renewal) Year. ( i ( ( ( r The studies described in the Progress Report (section 12) demonstrate that the alkaline elution assay may be used to quantitate the amount of chemical ly-i nduced damage to pulmonary DNA. A series of standardized pro- tocols have been developed in order to make this assay efficient, sensi- tive and reproducible. A major question that was answered during this first year of work is that a non-radiometric assay can be used to quanti- tate the DNA. A fluorometric assay was developed and standardized so that DNA levels in the order of 0.1-0.3 ug/ml can be routinely detected. A. Two studies are presently on test and will be completed during the next year. 1. Evaluation of ability of MCA, B P, 7,8-dihydro-7,8-dihydroxy -BP (BP-7,8-diol) to induce damage to pulmonary DNA. These chemicals have previously been shown to be carcinogenic to lung tissue by our laboratory. Under these conditions of inducing lung carcinomas, the chemicals will be given intratracheally (IT) to BC3F1 female mice and the rate of DNA elutlon at 0.5, 1.0, 2.0, 4.0, 8.0, 24 and 48 hours will be determined. The positive control will b e 4-riitroquinoline-N-oxide (4-NQO) and 0.2% gelatin-saline will be the negative control. 2. Measurement of the damage induced by certain lung carcinogens (e.g. MCA and BP-7,8-diol) following multiple treatments. The carcinogenicity of both MCA and BP-7,8-diol is enhanced by multi- treatments given IT at biweekly intervals. We will evaluate the amount of DNA damage induced by 1,3 and 6 treatments of those two chemicals. The exact times at which those assays will be done will be determined by the data generated in the previous study. 50137786 I CTR HN 043f 5..,

r OR B. Studies to be initiated during this second. year. 1. Evaluation of the capacity of whole cigarette smoke to damage pulmonary DNA. The general approach will be to expose mice to known quantities of whole smoke and monitor damage to pulmonary DNA at various times after the exposure. Several variables will be examined: a) the immediate exposure conditions, including the cigarette type (high vs low nicotine), the smoke concentration, and the total dose, b) ex- posure of different strains of mice to determine whether a difference exists in susceptibility to smoke-induced damage, and c) exposure for various lengths of time to determine long-term effects of smoke exposure. These studies have not been initiated because of the higher priority assigned particular supplemental studies to our smoke in-• halation program also funded by The Council for Tobacco Research. These studies have utilized most of the available space on our Smoke Exposure Machines (SEM) and thus long term exposure for this proposed study will not begin until February, 1980. In addition, the initiation of this study has been delayed by the observation by our collaborator, Dr. R. Rasmussen, that unlike the smoke exposure regimen used for 2A1 cigarettes, the smoke ex- posure regimen for 2R1 cigarettes has not caused any DNA repair in- hibition in pulmonary tissue. We presently are scheduling long- term exposure of BC3F1 mice to 2A1, 3A1, and 2R1 cigarettes for the simultaneous measurement of: a) DNA repair capacity, b) ability of pulmonary tissue to decarboxylate ornithine (ornithine decarboxy- lase is the proposed rate limiting step for promotion of the carcino- genicis process), and c) mixed-function oxidase activity. 2 50137787 ! 1 I CTR HN 043-760

:. I < ( I r I This study should show direction as to the type of smoke expo- sure (and length of time of exposure) necessary to cause some bio- chemical alteration in pulmonary tissue. 2. Evaluation of the metabolic capacity of pulmonary tissue as a prime determinant of the amount of damage induced by MCA. This approach-is a corollary to the studies proposed in section A. That is, strains of mice differ in their susceptibility to lung cancer induced by MCA. Such variability at least in part results from the relative levels of the enzymes capable of metabolizing chem- ical carcinogens. Therefore, by measuring the amount of DNA damage in various strains of mice following IT treatment with MCA, we can determine if the levels of AHH are playing an important role in the extent of damage, and if this damage parallels the ultimate sensi- tivity of these strains to MCA-induced lung cancers. We propose to answer this question by measuring the amount of DNA damage in lung tissue of B6D2F1 x 02 mice following IT treat- ment with MCA. These progeny animals represent an ideal model sys- tem, because the capacity to recognize MCA and metabolize this chemical to a variety of biologically active forms segregates as a single autosomal dominant gene in this cross. Thus 50% of the pro- geny animals from this cross will be capable of responding to the IT-instilled MCA by increased levels of pulmonary AHH, and 50% will not. The genetic linkage that may exist in lung tissue between AHH responsiveness, cancer susceptibility, and degree of DNA damage can, therefore, be unequivocably obtained. Approximately 20 B6D2F1 x 02 animals would have to be analyzed in order to perform these studies. 3 50137788 I ir f R f i f'i 04,,:s 761

. 40 r W. A simple method of confirming the relationship between AHH res- ponsiveness and the amount of pulmonary DNA damage is based on the observation that there exists a chemical that can specifically alter the AHH capacity of the genetically non-responsive strains of mice. That is, 2,3,7,8-tetrachlorodibenzo(p)dioxin (TCDD) treatment of D2 mice will result in increased AHH levels. Thus. TCDD-treated D2 mice have AHH capacities very similar to that of the responsive 86 strain. TCDD, under conditions where it induces AHH in D2 mice, selectively•eahances the tumor susceptibility of this strain (Kouri, ~t,a.1,Can. Res. 38, 2777, 1978). The most direct test for estab- lishing a relationship between AHH levels and the amount of DNA damage in pulmonary tissue is to compare the rates of elution of DNA in the alkaline elution assay in control, MCA-pretreated, and TCDD-treated D2 strains of mice. If the AHH enzyme complex plays a major role in determining the amount of pulmonary DNA damage induced by MCA, BP and/or B P- 7,8-diol, then TCDD should specifically enhance the susceptibility of the D2 mice to this DNA damage. The effect of co-treatment and pre-treatment with TCDD will be carried out in a manner similar to that described (Kouri gl A-1, Can Res 38: 2777, 1978). 4 50137789 C~'~`R I-I~~ 0~'.~ ~`~ ~ i !

Cf R i f f`i 043763

a f ( i ( C .e /24i. ?'Z 0 Tim Covxcu. ron TOBACCO Rja1:A;::c;fl-U.S.A.. INc. O 130 FABT 50TH 6TREF:T NEH• YORK. N. Y. 2 0022 (232) 421.9090 Application For Renewal of Research Grant (Ute extra pages as needed) Firu Renewal Q Second Renewal ® Date: 7/7/80 !* ~'•,~ 3UL 10 1980 1. Principal Inve,tiQator (give title and depree,): Carol J. Henry, Ph.D. Richard E. Kouri, Ph.D. Ronald A. Lubet, Ph.D. Director, Department of Director, Department of Co-Project Director Experimental Oncology Biochemical Oncology Dept. of Biochemical 2. Institution & address: Oncology Microbiological Associates 5221 River Road Bethesda, MD 20016 Evaluation and Characterization of an Alkaline Elution Assay as a Measure of Pulmonary DNA Damage Induced by Chemical Carcinogens or Cigarette Smoke. 5. Proposed renewal date: December 1, 1980 6. How results to date hove changed earlier specific research oimt: No Changes have been made in our specific research aims. We have characterized the alkaline elution assay using a fluorometric assay for the quantitation of DNA. 3. Department(s) where research will be done or collaborotion provided: Department of Experimental Oncology • Department of Biochemical Oncology 4. Short title of ttudy: 7. How results to dote hove changed earlier working hypothesis: No changes have been made in our working hypothesis. Our hypothesis is that chemical carcinogens or the metabolites of the carcinogens have the potential to interact with DNA and that this interaction or DNA -_ damage is one of the early steps in the carcinogenic process. This f~ DNA damage can be quantitated by the alkaline elution assay. 50137760 I t. r f f°6 i f f'f 043764

2. 8. Any additional focilities now required? Describe brieAys 0 No. 9. Any changes in personnel? Append biographical sketches of new key professional personneli R. A. Lubet, Ph.D. (C.V. Attached) 10. Append ouiline of experimentol protocol for ensuing year. (See Proposed Studi es ) 11. List publications or papers in press resulting from this or closely related work. (append reprints or manuscripts not previously sent). None at this time. 12. Summary progress report (append in standard form os separate document, unless recently submitted). 50137761 I i CTR HN 04,3765

( X Time Amount C. Henry, Ph.D. 10% $'3,237 R. Kouri, Ph.D. NC 13. Budget for.the coming year: A. Salaries (give names or state "to be recruited") Profeassional (give X time of investigator(s) • even if no salary requested) R. Lubet, Ph.D. NC ' Technical C. McKinney 90% 13,109 Merit Increases 8% 1,308 Fringe Benefits 36% 6,355 Sub-Total for A $24,009 B. Consumable supplies (by major categories) ( • Mice Chemicals and Solvents Labor Supplies 2,500 1,280 2,400 ub-Total for B 6,180 V C. Other expenses (itemize) Equipment Repair Publication Coats 500 500 ~ 0 Travel 600 Freight 100 Laundry 144 \ Central & Office Services 1,052 / Sub-Total for C $ 2,896 L,- Running Total of A + B + C $33,085 'Y D. Permanent equipment (itemize) ( Sub-Total for D -0- E. Indirect Costs E 7,775 F. Fixed Fee F 4,540 / Total Request $4 5~400 50137762 ~" w / ~ I yI I I .r' I rY Iryt 1l1/~ 6

4. 14. Other sources of finoncial supporti List financial support from all sources, including own institution, for this and related research projects. • CURRENTLY ACTIVE Source Inclusive rtie of Project Smoke Inhalation Studies in Mice (give grant numbers) The Council for Tobacco Research, Inc., U.S.A. Amount 1,301,700 Dates January 1, 1980 - Dece.mber 31, 1980 PENDING OR PLANNED Tirle of Project • It is understood that the investigator and institutional officers in opplyinp for a grant have read ond accept Ihe Council's "Stotement of Policy Containing Conditions and Terms Under Which Project Grants Are Made." Source Inclusive (9ive grant numbers) Principal investigator Amount Dotes C. Henry, Ph.D. R. Kouri, Ph.D.. 8• Lubet, ; Ph.D. R,A 40 !~Cl~.:.(~ ote 7/08/80 r Si , pna ure Checks payable to - %1lrrnhlnlnglrnl Aecnrietae Mailing address for check: 5221 River Road Bethesda, Marvland 20016 T.lephone (30T).654-3400 x303. 284 Ar.a Cod• Hvwf..r 4b".w Responsible officer of institution • Typed Name' James H. Ludwick Title, SiQnoture Director of " Con r L A Date i/n Teleahon 50137763 (301), 654-3400 x328 /Ar! CTR ININ 0437G*i'-"

CTR Grant A' 1241 Proposed Studies 7/1/80 - 11/31/81 10. Proposed studies for the period July, 1980 - 4ovember, 1981. ( ( • • The specific aims of this grant are to: o Characterize the capacity of whole cigarette smoke to cause damage to the DNA from pulmonary tissue as detected by the alkaline elution (AE) assay. o Characterization of the capacity of whole cigarette smoke to alter the DNA damage Induced by other chemicals. o Characterization and detection of damage to lung DyA after Intratracheal treatment with chemical carcinogens under varying conditions (i.e. carcinogen dose, carcinogen metabolizing abilities). 50137764 Cf R HN 043'-f"3i~J E3

CTR Grant # 1241 • Proposed Studies 7/1/80 - 12/31/80 0 • • I. Studies for the period July, 1980 to December 1980. A. Smoke-induced DNA damage. Our Initial results (Table 10) showed that chronic exposure to 2A1 cigarette smoke for a period of 40 weeks did not result In an in- creased elution of DNA. We will continue to Investigate this phenomenon in more detail using both chronically and acutely exposed animals. Our first set of studies will involve the effects of the 2R1 ciga- rette smoke. Male and female BC3F1 mice have been placed on chronic exposure to high levels (• 600 L'g TPM/day) 2R1 cigarette smoke starting in January 1980 (CTR 117). Thus, in the next six months we will test for the existence of DNA damage In animals exposed for periods of 6, 9, or 12 months. At the same time we will test for the existence of DNA damage in animals acutely exposed to the 2R1 cigarette smoke. These studies should tell us: posure, 2) when is cumulative. Ch ron i c 1) If direct DNA damage this damage and initlated June 1980 sition 1s estimated mice will be tested can be detected from smoke ex- can first be detected, and 3) If this damage acute exposure to 3A1 and 2R1 cigarette smoke were and are scheduled for lifetime exposure. Smoke depo- to be - 600 1ig TPM per day. At 6, 9, and 12 months, for the existence of DNA damage. Mice will also be tested for AHH activity, ODC activity, DNA repair capacity, and evidence of pigmented alveolar macrophage accumulation. Acute studies following the two week exposures to smoke from both cigarette types will also be scheduled. The third study will be the analysis of the lung DNA In animals chronically exposed to low levels of 2R1 cigarette smoke (.. 200 yg/day). -1- 50137765 I CTR HN 043i;"69

I L• CTR Grant # 1241 • Proposed Studies 7/1/80 - 12/31/80 ( • The source of these animals will be CTR 101 which will have animals at 24, 30, and 36 months of smoke exposure by September 1980, March 1981 and September 1981, respectively. B. Cigarette smoke plus chemicals as Inducers of DNA damage. Our preliminary results studying the lnteraction of smoke exposure in conjunction with intratracheal BaP or BaP-7,8-diol administration showed that smoke exposure augmented the DNA damaging effects of these compounds. (Table 11). The exact mechanism for this augmentation. i.e. effects on carcinogen metabolism, effects on DNA repair, etc., is not ( w e • carc nogens w e pursue pr, mar y as support ata n ans er ng i i questions outlined in parts A and B. known. Duririg the next six months we will Initiate studies designed to more fully detail the specifics of this interactlon. We will employ both long termsmoke exposed animals (up to 12 months) as well as animals exposed acutely (up to two weeks) to 3A1 or 2R1 cigarette smoke. These studies should help answer a number of questions with respect to our finding of smoke induced augmentation of BaP-7,8-diol damage, including: 1) how long an exposure to cigarette smoke ls necessary to see this lnter- actton? 2) is there any specificity in terms of which cigarette type can induce the interaction? Experiments lntended to determine mechanistic questions with respect to this Interaction will be addressed during the last year of this grant (December 1980 - November 1981) and will be discussed in section II. C. Chemical carcingoen-induced lung DNA damage. Further studies on the DNA damaging capacity of certain lung i th I 111 b d l d 1 -2- 50137766 0 C TR HN 0,43 . 7`,70

CTR Grant N 1241 Proposed Studies 7/1/80 - 12/31/80 0 First, attempts to optimize the use of inethyl methane sulfonate with respect to dose, and time post administration to yield reproducible and substantial DNA damage will be performed. These experiments will be aimed primarily to optimize the MMS treatment conditions which will be employed as a positive control In the in vivo lung experiments. Second, the optimal conditions for DNA damage with BaP-7,8-dlol or BaP will be determined so that further studies anent the Interaction of cigarette smoke with this carcinogen can be performed. By October 1980 we will have optimized the use of this agent with respectto dose, time post treatment,etc. Having optimized these procedures, we can then readily screen various groups of smoked animals for the Interaction be- tween smoke and carcinogen exposure. • . -3- 50137767 C T R 11"I N 0 4°t '7'- "' I

( 0 ( I • l • CTR Grant 1 1241 Proposed Studies 7/1/80 - 12/31/80 II. Proposed studies for the period December 1, 1980 - November 31, 1981. The exact nature of the studies to be performed in the last year of this study will be significantly determined by the results obtained in the preceding six months. The following series of experiments are scheduled. A. Cigarette smoke exposure and pulmonary DNA damage. By the early part of 1981 we will have animals which have been chronically exposed to 3A1 or 2R1 cigarette smoke at a high dose daily of TPM for up to nine months and animals exposed to 2R1 cigarette smoke for - 30 months at a low dose of TPM. Thus, these results together with the chronic studies previously performed (I.e. Table 11) and those to be performed between July 1980 - December 1980 with the 3A1 and 2R1 ciga- rettes should provide information as to: 1) whether there is any measur- able increase in DNA damage following chronic cigarette smoke exposure, 2) does the presence or the amount of damage depend on the particular type of cigarette employed, and 3) how long of an exposure to cigarette smoke will be necessary to detect measurable DNA damage employing the alkaline elution assay? At the same time that the chronically exposed animals are being studiedjBC3F1 female and male mice will be studied following acute (2 week) exposure to 3A1 or 2R1 cigarette smoke. Should there prove to be signifi- cant DNA damage following acute exposure, we will attempt to determine the role of the Ah locus in this effect. Thus, we would expose C57BL/6 and DBA/2-derived congenic animals which differ only at the Ah locus, being either AHH inducible or AHH non-inducible, and look for differences in DNA damage in the two groups of mice. -4- 50137768 ~ CTR ~`~N 043r ` ~-~`~

CTR Grant ,f 1241 Proposed Studies 7/1/80 - 12/31/80 I t • The congenic line which would initially be studied would be the 86 --> 02 line where the Ahb locus from the B6 mouse has been genetically inserted onto a D2 backgound. Thus, the study will be to compare the DNA damaging effects of whole cigarette smoke In this congenic line compared to the D2 strain itseif. This approach Is more efficient than the previously proposed use of B6o2F1 x D2 progeny because the congenic lines would not have to be phenotyped for their AHH responsiveness prior to smoke exposure, and fewer animals would have to be evaluated In order to generate significant results. The rationale for such an approach Is that cigarette smoke contains many chemicals that have the capacity to (nduce AHH activity and these chemicals are more readily activated to mutagenic moieties by AHH induced microsomes (Kouri, et al, Environ. Health Perspec. 29: 63, 1979). Together,' these findings suggest the real possibility that genetic differences In AHH function may regulate the lnteraction of the organism with whole cigarette smoke. B. Cigarette smoke exposure plus chemical carcinogens as inducers of pulmonary DNA damage. Mice from ongoing smoke exposure studies will be used to evaluate the synergistic effects of BaP-7,8-d1o1 and exposure to whole cigarette smoke in Inducing pulmonary DNA damage. The ongoing studies CTR 101, CTR 117 and CTR 118 (from Contract 0030) wlll provide female and male BC3F1 animals which have been exposed to smoke from 2R1 and 3A1 reference ciga- rettes for periods of time ranging from 6 months to 30 months. At -. 6 month intervals, mice will be treated with BaP-7,8-diol intratracheally using the conditions which are presently being standardized (see Section A- • Proposed Studies). -5- 50137769 i 1 C ~`R N~>~ 0~•:~ s~` ~`~

( ( • • • CTR Grant 1 1241 Proposed Studies 7/1/80 - 12/31/80 These studies will be a continuation of those presented In Section A. A second approach will be to study the actual mechanism of this BaP-7,8-diol-whole cigarette smoke interaction. The genetic system out- lined in the previous section is proposed. That Is, mice from the congenic ~ line 86 -> D2 will be compared to the D2 strain in their susceptibility to pulmonary DNA damage induced by BrP-7,8-diol plus cigarette smoke. These genetic studies will be a continuation of those proposed In Section A. Sufficient congenic animals should be available for smoke exposure by November 1980. Studies will continue for 6, 9, and 12 months of smoke exposure and will end by October 1981. C. Chemical carcinogen-induced pulmonary DNA damage. Analysis of BaP-7,8-diol pulmonary DNA damage will be done simul- taneously with the proposed studies described In the previous section. Both shelf controls and sham controlanimals will serve as source of animals for determining conditions for optimal pulmonary DNA damage by this chemical. Ongoing studies and thosescheduled for completion during the next 3-6 months will utilize Intratracheal treatment with this diol. We presently have the capabilities to generate aerosols of several chemicals and these will be tested for their interactions with pulmonary DNA. The aerosols will be yenerated using the conditions which have been used to generate particulate aerosols of phorbol esters (Dinowitz et al. Proc. Amer. Assoc. Cancer Res. 21: 31, 1980). A collision nebulizer has been modified to generate an aerosol of respirable particles in ethanol or water with an estimated mass median diameter of 0.4 micron. This aerosol generator will be used for exposures of catechol, nicotine and tetradeconyl- phorbol acetate(TPA) from 10-200 ug/day and for daily exposures for 1, 2, 4, 8, and 16 days. BaP and BaP-7,8-dlol have also been generated from a -6- 50137770 ( I CTR HN 043774

L. CTR Grant A' 1241 Proposed Studies 7/1/80 - 12/31/80 DMSO/ethanol solution, and this approach is under active investigation so that aerosols of these chemicals could be examined for their inter- .action with pulmonary DNA. At the appropriate times after exposure, BC3F1 mice will be sacrificed by cervical dislocation and analyzed for pulmonary DNA damage. The more uniform distribution of this chemical throughout the lung tissue of mice should put more cells at risk to these chemicals and lung DNA damage should be more easily and repro- ducibly detected. The role of the Ahb locus (AHH responsiveness) in determining the amount of DNA damage can easily be determined. Mice from the 86 - D2 congenic line (AHH responsive) and D2 mice (AHH non-responsive) are proposed to be compared for their susceptibility to aerosol generated chemical-induced lung DNA damage. • -7- 5UT377Tl ~ ~ C T R H N 0 43 7

RONALD A. LUBET t i r ( a ADDRESS: 5221 River Road, Bethesda, Maryland 20016 DATE AND PLAC E OF BIRTH: July 7, 1946, New York, New York SOCIAL SECURITY NUMBER: 085-36-4880 Education Ph.D. - 1977 - University of Texas, Dallas (Radiation Biology) Dissertation Title: Studies on the Therapy of and Development of Tumor Immunity Against the Murine Plasmacytoma MOPC 104E. M.S. - 1973 - University of Tennessee, Knoxville (Radiation Biology) Thesis title: The Induction of Aryl HyArocarbon Hydro- xytase in Individual Hamster Cells in Culture B.S. - 1969 - University of Tennessee, Knoxville (Zoology) Employment Assistant Project Director - Microbiological Associates, 1979 - Present Postdoctoral Fellow - Laboratory of Immunodiagnosis, National Cancer Institute - 1978-1979 Postdoctoral Fellow - Department of Biochemistry, University of Texas HSCD - 1977-1978 Research Interests Mechanisms of chemical carclnogenesis. Mutagenicity of chemical carcinogens and chemotherapeutic compounds. Metabolism of a variety of xenobiotics including carcinogens and chemo- therapeutic agents. Mechanism ot action of chemotherapeutic agents. Effects of chemotherapeutic agents on the murine response. Induction of tumor immunity in vivo. Methods for studying tumor immun~Ly in vitro.. Professional Memberships ( • Sigma X Publications Kouri, R. E., Lubet, R. A., Brown, D. Q. In vitro cellular uptake of benzo(a)pyrene by a microfluorimetric technique. Proc. Soc. Expt. Biol. Med. 136: 1038-1044, 1971. 50137772 t I GfR ffN 043776

L• a 4h • R.A. Lubet Kouri, R. E., Lubet, R. A., Brown, 0. Q. Quantitation of AHH activity in individual fetal Hamster embryo cells in vitro. J. Natl. Cancer Inst. 49: 993-1005, 1972. Lubet, R. A., Brown, 0. Q., Kouri, R. E. The role of 3-OH B(a)P in mediating B(a)P induced toxicity and transformation in cell culture. Res. Comm. Chem. Path. Pharmacol. 6: 929-941, 1973. Lubet, R. A., Turner-Lubet, H., Whitson, G. L. The relationship of cellular metabolism of B(a)P to the induction of cytotoxicity in cell transfor- mation in fetal hamster cells in vitro. Eur. J. Cancer 11: 139-145, 1975. Carison, D. E:, Lubet, R. A. Effects of sublethal gamma radiation on T and B cell activity in the antibody response of mice. Rad. Res. 65: 111-119, 1976. Grunow, J. E., Lubet, R. A., Furgeson, M. J., Gaulden, M. E. Preferential decrease in thymus dependent lymphocytes during storage at 4oC in anti- coagulant. Transfusion 16: 610-615, 1976. Lubet, R. A., Carlson, 0. E. Tumor immunity against MOPC 104E. Effect of various therapeutic regimens. Cancer Immunol. Immunotherap. 2: 267-271, 1977. Lubet, R. A., Carlson, D. E. Tumor immunity against MOPC 104E: Effect of tumor size and time post theraphy on in vivo immunity. J. Natl. Cancer Inst. 60: 1007-1011, 1978. Lubet, R. A., Carlson, 0. E. Therapy of the Turine plasmacytoma'l1OPC 104E;-Role of the immune response. J. Natl. ~ancer Inst. 61: 897-905, 1978. Prough, R. A., Capdevilla, J., Lubet, R. A. The metabolic activation of benzo(a)pyrene and 9-OH benzo(a)pyrene by tissue fractions from rat liver and lung. Biochem. Soc. Transact. 7: 44-47, 1978. Lubet, R. A., Capdevilla, J., Prough, R.A. The•Aicroso:nal Activation of Benzo(a)•py.reft and• its •9• PhenoT to..Mutagenic and'alkyt'ating moieties. Intl. J. Cancer 23: 223=228, 1979. Dunn, D., Lubet, R. A., Prough, R. A. The Oxidative Metabolism of Procarbazine. Cancer Research 39: 4555-62. Lubet, R. A., Carlson, D. E. Tumor immunity against MOPC 104E: Leukocyte adherence assay. Eur. J. Cancer, 1•7s663=692, 1980. Lubet, R.A., McCoy, J. L., Herberman, R.H. Cellular Immunity in BALB/c Mice Against the Syngeneic Madison 109 Lung Tumor. Submitted to J. NCI. 50137773 i I CTR NN 043f~`7r`

R.A. Lubet 0 ( 1 ( • Capdevilla, J. Lubet, R.A., Prough, R.A. Metabolism of 3 and 9-Oh Ben- zo(a)pyrene by Microsomal Rat Liver Fractions in Microsomes, Drug Oxidations and Chenical Carcinogenesis. Editor: N.J. Cone (In Press, 1980). 50137774 I CTR HN 04~~ 7,76