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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