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Ettr ir~u c tlr Per.rpec•tires Vol. 29, pp. 63-69. /979 April ~ W uo/ Biological Activity of Tobacco Smoke and Tobacco Smoke-Related Chemicals by Richard E. Kouri,* Thomas H. Rude,* Rodger D. Curren,'` Karen R. Brandt, ~- Ronald G. Sosnowski,* Leonard M. Schechtman,* William F. Benedict, i and Carol J. Henry >` Exposure to whole cigarette smoke from reference cigarettes results in the prompt (peak activity is 6 hrs). but fairly weak (- 2 fold), induction of murine pulmonary microsomal monooxygenase activity.. This activity can be detected by using as substrates either benzo(a)pyrene or ethoxyresoruftn, and can be inhibited by treatment with cycloheximide or actinomycin D. Unlike the induction of pulmonary monooxygenases following intratracheal administration of 3-methylcholanthrene, these cigarette smoke-induced increases were not unequivocally linked to the Ah locus. Whole smoke condensate and fractions derived from these condensates can; a) induce pulmonary monooxygenase activity, b) inhibit benzo(a)pyrene metabolism in citro, cl be metabolized to forms mutagenic to Salmonella typhitnurium tester strains TAI538 or TA98, d) transform C3H 10T'h cells in ritro, and e) enhance the carcinogenicity of benzo(a)pyrene in murine pulmonary tissue. A potentially important observation is that whereas hepatic tissue is capable of activating whole cigarette smoke condensate to mutagenic forms in c•itro, murine pulmonary tissue does not seem capable of such activa- tion. Although these pulmonary-derived tissue homogenates have significant AHH activity and can metabolize Aflatoxin B. 2-aminofluorene and 7;8-dihydro-7,8-dihydroxybenzo(a)pyrene to mutagenic forms, these homogenates fail to activate both cigarette smoke condensate and the pro-mutagen, 6- aminochrysene. These results are discussed with reference to the concept that whole cigarette smoke may be both a potential "initiator" and "promotor" of lung cancer in mice, and that this latter property may be the most important in determining cancer risk. Introduction Cancer is not randomly distributed among the human population (/ ), rather, both environmental (2) and genetic (3-5) factors strongly influence its occurrence. Lung cancer is a specific example of a neoplasia whose occurrence is nonrandomly distributed. Its incidence is often associated with cigarette smoking (6), but the actual role that to- bacco smoke or smoke-related chemicals play in this association is unclear. A potential role of tobacco smoke involves in- teraction with those microsomally-bound mono- ' Microbiological Associates. Department of Biochemical On- cology. 5221 River Road. Bethesda. Maryland 20016. ' Children's Hospital of Los Angeles. Los Angeles. California 90027. oxygenases that seem important in determining sus- ceptibility to chemically-induced cancer in model animal systems (7. 8). These enzymes are important because not only do they metabolize many en- vironmental chemicals, but also naturally occurring variations in their steady-state levels are genetically linked with susceptibility to toxicity. mutagenesis, and carcinogenesis induced by many of the same chemicals (7. 9-1 / ). Three basic questions can be addressed con- cerning the role of tobacco smoke in cancer suscep- tibility in man: (a) Does tobacco smoke contain chemicals that interact with the microsomal monooxygenases? (b) If they do interact, what are some of the in t•iru and in ritru effects? (c) Can certain individuals or tissues within an individual be at greater risk than others from the effects of tobacco-related chemicals'? April 1979 ASCA June 15, 1979 63 g~K,'•r.r~~'~ 1Z __.*_t,'~

Does Tobacco Smoke Contain Chemicals Capable of Interacting •ith the Monooxygenase 6ystems? Kinetics of increase of pulmonary AHH levels in C57BL`6 (B6): DBA;?J (D2). and C3H/fCum (C3) strains uf mice exposed to I A I cigarettes are shown in Figure 1. Results ,ug ,gested that there were rapid increases in pulmonary AHH activity that peaked by 6 hr post-exposure. and that these induced en- zymes remained at this level for at least 24 hr. The D2 strain w•us virtu,tlly nonresponsive to this smoke expouu•e compared with B6 and C 3 strains: this re- sult paralleled that found when a pulycyclic aro- matic h_vdrucarbun (PAH). 3-methvlcholanthrene (MC), was given intratracheally to the same strains (see Fig. 1). However. the kinetics of induction of pulmonary AHH by smoke are different from those obtained when MC was used as an inducer. The peak of activity in the. smoke induced lungs oc- curred at 6 hr (vs. 24 hr for MC). Also the half-life of the smoke-induced enzyme was much longer (-24 hr vs.. -4 hr): Finally, these smoke exposure condi- tions did not induce the AHH activity in liver. colun. and kidney (data not shown) whereas MC- treatment did: moreover. filtered smoke (gas phase) did not induce enzyme activity in lung. liver, or 1.0 ~ 0.751- 66- CIGARETTE SMOKE ~ 02 C3H ~ 050 ~ 3 0 25 ~ ,"~--°-~+.-.o ~3 1 ~-I MCA 1200~Lug~IT1 I, T ~ 66 p 02 ~ C3H I _ 20 f a r r toi ~ os~ s~~ ,-0 3 6 9 12 24 3 6 9 12 24 HOURS 3 6 9 12 24 FicuttE I. Pulmonary response ofC3HifCum. C57BL6Cum. and DBA•2J inbred strains of mice to MC and whole cigarette smoke. Mice were either treated with 200 µg MCA/0?rir gelatin-saline solution intratracheally (IT) as described previ= owsly (12) and sacrificed at the time periods specified or ex- posed to four cigarettes from a Walton smoking machine employing a 2-sec puff. 10rh smoke. 30-sec exposure, and 30-sec purge (/.l). Mice were given a 2-min rest period before exposure to the next cigarette and were sacrificed at varying times after the fourth cigarette. Lungs were excised and fro- zen at -70'C until assayed. AHH units are expressed as nmole 3-OH-BP formed:mitng wet weight tissue (12). Aver- age activity of three animals is given. kidney tissue (data not shown). Smoke-induced AHH in the lung was similar to MC-induced AHH. in that both tue -capable of (1-deethylation uf ethoxyresorulin (Table 1): see Burke and Mayer (15) for data on MC as an inducer. Data with ethoxyresiirufin were particularly interesting be- cause studie, have suggested that this chemical is a specific substrate fur the P-448 (or P,-450) cyto- c:hrome (/6 ). This is the cytochrome that is inti- mately associated with metabolizing hen- zo[alpyrene (BP) and MC to cvtotoxic (17-19). mutagenic (2). DNA-binding (?/. 22). and car- cinogenic (y. !(/) forms. Similar to MC-induced AHH ievels. the smoke-induced enzyme levels ttre dependent upon in t•iro protein and RNA synthesis (see "I <tble 1). Although tobacco smoke induced AHH activity in the B6 strain preferentially to the D2 titrain. the genetic basis for this difference may not be the same ati that for the difference in AHH activity induced by MC. Figure 2 showti that, whereas MC given intratracheally to (B6D2) FI x D2 progeny induced pulmonary AHH activity in approximately 501-,~ (13 29) of the mice (12. 13). no clearcut discrimina- tion was observed in the smoke-exposed animals. How•ever, it may be that the inducing capacity of cigarette smoke for pulmonary tissue is just too low to easily evaluate its genetic regulation. Whole.tobacco smoke can be collected bv con- densation with the use of Dry Ice, and this cigarette smoke condensate (CSC) or fractions derived therefrom (23) can be evaluated for potential biological effects. The fractionation scheme and Table t. Effect of cycioheximide and actinomycin D on smoke- induced BC3F, lung AHH and ethox.•resorufin O-deethylase activitv.' reatment AHH activity, nmole;min-g tissue'• O-Deethylase activity nmoleimin-g tissue!" Machine controls 0.35 <0.04 Smoke - 0.70 0.61 Smoke + saline 0.67 0.83 Smoke - propylene glycol 0.53 0.83 Smoke + cycloheximide, 500 µg/g 0.32 0.14 Smoke + actinomycin D. I µg~g 0.42 0.15 ".Cycloheximide and actinomycin D were injected in- traperitoneally (tP) in saline or in propylene glycol respective(y, immediately before exposure to one 2A1 cigarette on a Walton smoking machine. Methods given in legend to Fig. I. Mice were sacrificed 6 hr after smoke exposure. Procedures were as de- scribed by Van Cantfort and Gielen (14). AHH units are expressed as nmole 3-OH-BP formed.min.g wet weight of tissue. Average activity of three animals. ()-Deethylase units are expressed as nmoles resorufin furmed.• mia g wet weight of tissue according tu the procedures of Burke and Mayer (15). 64 Environmental Health Perspectives

n a ~ Z a NON-RESPONSIVE RESPONSIVE ~ i m.23 2 o.1m m 95 -0 301 i . ' o aa o o ~ L~ o0o ao 0o e~ o 0 i 0 z 9 Z 0 o~oa,o~ooooa .i 0.1 0.5 1.0 1.5 AHH ACTIVITY IUNITS g TISSUE'MINI SAIt•, actually inhibiting AHH levels in the lungs. The fractions that induced AHH activity (e.g. B,a or B,b) contained chemicals that could metabolically compete with BP in an in ritrv assay (see Table 2). This implies that CSC contains not only chemicals that induce pulmonary AHH. but also chemicals that are potential substrates for these enzymes. What Are Some in Vitro and in . Vil'ro Effects of Tobacco-Related Chemicals? Ftct:RE'_. Pulmonary response of B6D2F, x D2 progeny to (top) MC and (bottom) whole cigarette smoke. Animals were gen- erated and housed as described before (/2). Inoculation of MC and smoke exposure was given as described in legend to Figure 1. AHH activity was determined 24 hr after IT instilla- tion of MC and 6 hr after exposure to cigarette smoke. Mean and standard deviation is given for the MC-treated popula- tions that are representative of the nonresponsive (0.23 - 0.10) and responsive (0.95 = 0.30) populations. nomenclature were as described by Swain et al. (26). Table 2 summarizes some of these effects. In- tratracheal administration of the whole CSC and re- constituted fractions induced a small. but signifi- cant, increase in pulmonary AHH activity. The CSC fractions varied in their capacity to induce AHH activity with the fractions B,a, B,b, N.tr.•r~rr• and N.,.1f the best inducers, fractions BF• and WAr weaker inducers. and fractions B,, SAr, SAF• and In Vitro Assays The preceding section showed that tobacco smoke contains chemicals which interact with the monooxygenase system. This interaction can be beneficial if it produces nonbiologically active: polar metabolites which can be excreted from the body. or it can be detrimental if it results in inter- mediates with exceptional biological potency. Table 2 shows that 1 A 1 CSC fractions are metabolized bv enzyme fractions from rat liver to forms mutagenic to tester strains TA1538 (25. 27) of Scrlmonellu tYp/rinurrium. and also by endogenous cellular en- zymes to forms capable of transforming the C3H 10Ttiz cells in culture. This table also depicts the levels of nicotine, phenols, and BP in fractions B,,•. WA,.., and N.,•.tr-the fractions which contain virtu- Table 2. Effects of fractions of tAI. cigarette smoke condensate (CSC) in various,model systems. Fraction" Content, mgicig AHH inducibility° (X)lBP to give 50r/c inhibition'' Mutagenesis'' Transformation'' Whole CSC 23.50 1.7 5.0 +++ + Reconstituted CSC 23.00 1.8 - 5.2 +++ + B,a 0.81 3.6 0.8 + + - B,b 0.29 2.5 0.5 + + + BF• 0.95 1.5 3.0 + - B,,. 0.36 0.5 > 10.0 - - WA, 2.27 1.6 5.0 + + + WAF. 1.98 1.1 2.0 - SA, 0.39 0.5 >10.0 _ SAF. 0.78 0.3 > 10.0 SAR. 8.69 0.4 > 10.0 N.,,,,oN 1.19 2.5 3.0 _ N,,, 4.58 1.2 ND N,s, 0.70 3.2 1.0 ° Whole CSC has 21.0 mg nicotine/g, 5.70 mg phenols/g, and 0.98 µg BP/g. Reconstituted CSC has 22.0 mg nicotine/g. 5.5 mg phenols/g, and 0.90 µg BP/g. BE has 31.0 mg nicotine/g. WAt• has 41 mg phenolslg, and N,.,,, has 31.1 µg BP/g. Details given by Patel et al. (23). '' AHH inducibility = effect of fractions of 1 A I CSC fractions on pulmonary AHH activity of C57BL6 Cum mice relative to a corn oil control (12). ' BP inhibition = competitive in vitro effect of CSC fractions of BP metabolism by hepatic microsomes from 3-methylcholanthrene- treated C57BL6 mice (/3). " Mutagenesis = mutagenic activity of lAl CSC fractions in Sa(nonella t.•phinrurism TA 1538 in the presence of liver microsomal S-9 mix (24). ` Transformation = malignant transformation frequency in C3H 10T1%2 Cl. 8 cells treated with CSC fractions (25). April 1979 - 65

ally all of these.three chemicals. The most biologi- cally active fractions are not those that contain the BP or phenols. The Bla and Blb fractions have not '-Aen analyzed for their chemical content. However, cause of the nature of the fractionation scheme (B,a = bases insoluble after ether, B,b = bases in- soluble before ether), these fractions could contain some aromatic amine-like chemicals. A similar distribution of mutagenic activity was found with CSC fractions from the 2A1 reference cigarette (Table 3), and such activity was observed to be stable even if the fractions were stored frozen for one year. Thus, for at least two different types of cigarettes., certain fractions of their condensates can be metabolized to forms that are active in an in vitro mutagenesis test system. However, subsequent studies (Table 4) revealed a potentially important hindrance to the extrapolation of the foregoing studies to an in vivo situation. Mouse pulmonary tissue preparations, under con- ditions where BP is significantly metabolized to 3-OH-BP (see AHH levels), failed to significantly activate either 2A1 condensate or 6-aminochrysene (6-AC) to forms mutagenic to the TA98 tester strain. Table 3. Mutagenesis of TA 98 with 2A1 cigarette smoke condensate (CSC) fractions." ontent, Mutants per plate per 250 Ecg of sample Calculated mutants per plate per cigarette Activity. % Fraction mg/cig Expt I Expt 2 Expt I Expt 2 Expt I Expt 2 Whole CSC 40.00 133 122 21.280 19,520 Reconstituted CSC 39.50 159 148 25.122 23,284 BI° 0.60 987 1.149 2.369 2.758 11 10 BI° 0.24 3.510 5.266 3.370 5.055 16 18 Be 1.04 1.258 2,065 5.233 8.590 25 30 Bµ. 0.52 102 151 212 314 1 I WAt 3.63 289 394 4.196 5.721 20 20 WAE 2.66 141 206 1.500 2,192 7 8 SA, 1.44 53 43 305 248 2 1 SA£ 0.88 8 14 28 49 0 0 SA„• 15.60 8 20 499 1.248 2 4 NM,o„ 2.16 65 93 562 804 3 3 NcH 9.54 57 24 2.175 916 10 3 N,..,, 1.24 80 139 397 689 2 2 ° The 2A1 (low nicotine) CSC fractions were generated by Meloy Laboratories according to the methods of Patel et al. (23) and the ,)ur plate incorporation mutagenesis assay was performed according to the methods of Kier et al. (24). Table 4. Capacity of TCDD-induced mouse hepatic and pulmonary S-9, and Aroclor 1254-induced rat hepatic S-9 to activate whole cigarette smoke condensate to form(s) mutagenic to S. typhimurium TA 98P Compound AHH Survival Mutants/ late Source of S-9 Type /cg/plate Protein. mg . pmole/tube° . ''/er p (minus background) C57BL/6 hepatic Pour plate 2A 1 650 3.14 4929 115 AFB, 1 3.14 4929 170 Suspension 2A1 260 3.14 4929 109 61 6-AC 5 3.14 4929 102 115 C57BL/6 pulmonary Pour plate 2A1 1300 11.5 8800 8 AFB, 25 11.5 8800 250 Suspension 2A1 1300 1.44d 1071 105 5 6-AC 125 6.90 3208 83 3 Rat hepatic Pour plate 2A1 1300 4.43 9625 185 AFB, 1 4.43 9625 760 Suspension 2A 1 650 2.95 7371 106 83 6-AC 0.5 0.03 190 82 315 " Pour plate incorporation mutagenesis assays were performed according to the method of Kier et al. (24). Suspension assays consisted of a 35 min incubation of a mixture of bacteria. S-9, and test chemicals in a buffer composed of 3.6 mM NADPH. 4.2 mM NADH. 3 mM MgClz, 8.1 mM NazHPO4. 1.5 mM KH2PO,. 2.7 mM KCI and 137 mM NaCl. ° As pmole 3-OH-BP formed per assay tube: total time was 35 min: 25 µg BP/ml was substrate. * Percentage of bacteria surviving 35 min suspension relative to TA 98 alone. d Subsequent assays with 11.5 mg protein. 8800 units AHH, and 1300 tc.g 2A 1 still showed no induced mutants. 66 Environmental Health Perspectives

Under similar conditions both rat and mouse he- patic tissue could activate both 6-AC and the 2A1 CSC to mutagenic intermediates. However, the pulmonary S-9 preparations were capable of ac- tivating aflatoxin B, (AFB,) (see Table 4), 7,8- dihydro-7,8-dihydroxybenzo(a)-8-dio[ pyrene-7, and 2-aminofluorene (2-AF) to mutagenic forms (Table 5). Thus, the lung preparations were metabolically active by some measures, but did not activate 6-AC or CSC. That the tissue hypotheti- cally at risk to tobacco-associated carcinogenesis seems unable to activate the tobacco smoke con- densate allows many interesting interpretations which will be discussed in the last section of this report. In Vit;o Studies Tobacco smoke condensate fractions have been shown to "promote" carcinogen-initiated skin tumors in mice (28, 29). Table 6 demonstrates that at least some fractions from the' 2A I CSC are capa- ble of acting synergistically with intratracheally in- stilled BP (with or without FeLOa), resulting in a much higher incidence of lung cancers in BC3Fl mice; see Saffiotti (30) for discussion on use of Fe..O„ in pulmonary cancer model systems. BP alone (given every other week for 6 weeks) resulted in only one malignant tumor by 39 weeks after treat- ment out of a total of 82 treated animals (combining animals,given 0.6 mg and 1.2 mg BP per treatment). Table 5. Activation of 7,8-dihydro-7,8-dihydroxybenzo[alpyrene (7,8-diol) and 2-aminofluorene (2-AF) by pulmonary S-9:' Pulmonary S-9, mg protein 2-AF. µg/tube 7.8-diol. µg/tube AHH. pmole/tube° Survival, ~7e'' Mutants/ plate 6.57 25 1904 48 536 10 1925 81 668 10 1904 61 521 3.28 25 1452 52 458 10 1452 52 540 5 1680 101 627 0 25 - 82 39 10 - 84 52 6.57 0.5 1928 57 544 3.28 0.5 1680 18 338 0 1.0 - 88 76 0 0.1 - 99 84 TA 98 alone - - 100 20 " Suspension assays performed according to procedures given in Table 4. ° AHH = pmoles 3-OH-BP formed per 35 min incubation in separate tubes containing 25 fcg BP/ml as substrate. Percentage of viable bacteria after 35 minute assay relative to TA 98 alone. Table 6. Evaluation of cocarcinogenic effects of 2A1 CSC and selected fractions of the condensates on BP-initiated lung cancers in BC3F, mice.a Gel CSC fraction° Treatment group saline° B,a B,b WA, N.,,o„ RFT SM° No other treatment 0/118 0/37 1/47 0/28 1/40 0/68 0/50 0.6 mg Fe._O, 0/48 1/47 0/71 0/48 0/46 2/65 0/51 0.6 mg BP 1/43 10/64 5/74 14/59 19/82 4/60 13/58 1.2 mg BP 0/39 9/62 3/35 12/46 34/79 4/79 10/58 9/66 0.6 mg BP + mg FeZ0, 3/60 7/64 4/40 2/40 3/50 3/42 1/56 1.2 mg BP + 0.6 mg FeZO, 7/69 9/71 13/57 6/55 10/74 5/47 i l/50 " Groups of BC3F,/Cum female mice (8-14 weeks old) were intratracheally (IT) inoculated once every 2 weeks for a total of 6 weeks with 0.02 ml gelatin-saline solutions of BP, FelO3, BP:FeZO, alone or in combinations with the cigarette smoke condensate (CSC) fractions (10 µg each). At the end of the "car.cinogen treatment" period. IT treatment with the appropriate CSC fractions was continued once every 2 weeks until 4 weeks prior to sacrifice. Data presented represents tumor incidence for mice randomly sacrificed after 26 and 39 weeks on test. The tumor types diagnosed histopathologically include alveologenic adenocarcinomas, adenosquamous carcinomas, squamous cell carcinomas, and squamous neoplasms. A more complete description of these lung cancers is given elsewhere (33). b Data given in terms of the number of mice with lung carcinomas per the total number of animals at risk. RF = reconstituted fractions. ° SM = starting material. April 1979 67

Addition of ferric oxide (Fe,O,) with BP resulted in 8rl~ malignant tumors (30). However, treatment with selected 2A I condensate fractions every other week for the duration of the study (39 weeks), resulted in significantly increased malignant lung lesions in animals treated with both BP alone or with BP plus Fe..O;,. The fractions alone, or with Fe.:O,,, resulted in few malignant tumors (5 out of a total of 764 treated animals). Thus, in an in riro lung tumor model system, the fractions seemed to have mainly "promotor-like" activity. Can Individuals or Tissues within an Individual Differ in Risk to the Effects of Tobacco-Related Chemicals? The answers to this question are at best complex. The results of studies discussed in the previous sec- tions can be somewhat difficult to reconcile. On the one hand, tobacco smoke contains chemicals that interact with hepatic mixed-function oxidases such as AHH, and this metabolism generates inter- mediates that are mutagenic and transforming in in i•itro test systems. On the other hand. CSC is not activated to mutagenic forms by mouse-derived pulmonary tissues in t•itrv. Also, CSC material does not seem to efficiently cause lung tumors in t•iru. Many interpretations are feasible. Perhaps the lack of activation is characteristic of mouse pulmonary tissue alone and cannot be generalized to other species. In point of fact, rat pulmonary tissue has been reported to activate certain smoke conden- sates to mutagenic forms (24). However, both rat and human lung tissues have also failed to activate CSC material (24. 31). and so the generality of the activation (or lack thereof) is still not understood. Second, the fact that hepatic tissue can activate these condensates suggests that tobacco material could be activated by this organ and, following that, the tissues at risk (e.g., lung) would be exposed to these active forms. Or third, the lung is at risk only for the effects of those chemicals in cigarette smoke capable of "promoting" the carcinogenic event that may be initiated by a myriad of environmental pol- lutants. The first interpretation is difficult to assess be- cause no lung cancer model animal system directly applicable to man is currently available. However, the kind of pulmonary lesions observed in mice fol- lowing exposure to known chemical carcinogens, that is, bronchogenic squamous cell carcinomas, al- veolar adenocarcinomas, and adenosquamous car- cinomas. resemble lesions found in man (32. 33). Some data suggest that the liver plays a major role in the eventual susceptibility of other organs to chemically-induced cancers (34). However, no evi- dence is available to suggest that lung tissue is at risk from liver-metabolized chemicals. Evaluation of this alternative in t•it•u will be difficult because of the close liver/lung relationship. The third interpretation is particularly intriguing and suggests that "promotion" represents the real risk of cigarette smoke to lung tissue. This idea is consistent with the following facts: (a) tobacco smoke condensate is capable of "promoting" mouse skin carcinogenesis: (b) tobacco-smoke con- densate can "promote" lung carcinogenesis in model animal systems (see Table 6): (c) the condi- tions of cigarette-smoke exposure that result in the highest risk of human lung cancer are quite similar to those that are most promotive in animal test sys- tems: that is, frequent and relatively prolonged treatment: (d) the chemicals in cigarette smoke (particulate phase) that are known initiators of car- cinogenesis may be too low in concentration [total = 400 ng/cigarette (35)] to- initiate significant trans- formation in vivo. The evaluation of whole cigarette smoke as a potential initiator and promoter of lung cancer in the inbred strains of mice is now being studied in our laboratory. NOTE ADDED IN PROOF: We have recently found that a pulmonary S-9 preparation from Aroclor 1254-induced mice is capable of weakly activating 6-AC to a bacterial mutagen with the use of a pour plate assay (2-3-fold over background). We have still not observed an increase in bacterial mutations using this S-9 preparation and 2A 1 cigarette smoke con- densate. This study was supported in part through contracts from The Council for Tobacco Research Inc., U.S.A.. New York. N. Y. The authors thank Ms. A. Ventura and Ms. C. Dwyer for their assistance in typing this manuscript. REFERENCES I. Lynch, H. T. Cancer Genetics. Charles C Thomas. Springfield. III., 1978. 2. Mason, T. J. Atlas of Cancer Mortality for U. S. Counties: 1950-1969. U. S. DHEW. Public Health Service NIH. Bethesda. Md., 1975. 3. Lynch, H. T.. Guirgis. H. A.. and Albert. S. Familial associ- ation of carcinoma of the breast and ovary. Surg. Gynecol. Obstet. 138: 717 (1975). 4. Knudson. A. G. The genetics of childhood cancer. Cancer 35: 1022 (1975). 5. Petrakis. N. L.. and King, M. C. Genetic markers and cancer epidemiology. Cancer 39: 1861 (1977). 68. Environmental Health Perspectives

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