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91 a Subchronic Cigarette Smoke Inhalation Studies in Inbred Syrian i I t Golden Hamsters That Develop Laryngeal Carcinoma Upon Chronic Exposure 1.2 Peter Bemfeld,3 F. Homburger,3.` and E. Sotos.s ABSTRACT-Inbred 8100 15.16 Syrian golden hamsters were exposed for 6-20 weeks to smoke from three types of expenmentar cigarettes. The tnadence and seventy of laryngeal hyperplasia increased in these hamsters. a few (2) laryngeal papillomas appeared, alveolar macrophages became more frequent and aggregated, and hyperplasia of terminal brortchiolar epithelium occurred. This sutxhrortic response of hamsters to smoke markedly differed for the three types of cigarettes. Statistical evaluation of the data by log linear models proved these differences to be significant. At equal doses of srvoke. the most severe resporse was caused by an au-tobacco cigarette. The weakest subcnronic effects. next to those seen in the negative control group, were elicited by smoke 'rom a cenulose-denved tobacco supplement. The effects of smoke from a 1:1 blend of the two smoking materials were intermediate. The seventy of the suochrornc response of the respiratory tract paralleled the extent of malignant transformattons of the larynx previously observed in the same animal mocel with the same three types of cigarettes in ^hrontc mnaiation studtes.-1NCI 1983: 7t:6i9-623. The St rian golden hamster is the onlv laborator.' rodent that responds to tobacco smoke inhalation with the formation of malignant tumors of the respiraton' trsct in substantial aad statistically significant numbers of animals at risk (1-4). The end point in this test svstem is carcinoma of the lannx. t.hich starts to manifest itself after 59 t•eehs of smoke inhalation and t.hich, under the :no>t se% ere experimental conditions. ma% affect as much as 4'`% of the hamsters after 73 t.eeks of exposure to. tobacco ;moke (4). The use of different t%pes of ciga- rettes and of different concentrations of smoke in rhronrc inhalation studies (4I demonstrated a dose-response re- lationship for cigarette smoke and permitted us to distin- guish the biologic responses to various t~pes of cigarettes according to the carcinogenic potencv of the smoke. Thepresenr %%ork deals t%ah subchromr cigarette smoke inhalation studies. ranging in duration of exposure from 6 to 20 %eeks. in t.hich %.e used three of the same types of cigarettes. the same inbred hamster strain, and the same experimental conditions of smoke exposure as in our, . l,r.,mr studies (4). These subchronic studies did not and %%ere not expected to elicit a tumorigenic response, because of the short time of exposure to smoke. \ever- theless, the histologic changes observed in the respira• tor, tracts of the animals after short-term inhalation may not. be related to the response of the hamsters after chronic smoke exposure. because of the identical exper- imental conditions in both the subchronic and chronic e.periments (The chronic experiments eventualh re- sulted in int•asive larnngeal carcinomas.) Such a compar- ison has now shot<n that the inclusion in cigarettes of a cellulo~.e-deri.ed tobacco supplement reduced the extent of both the subchronic histopathologic changes and the chronic carcinogenic response in the respiratory tract. Although we do not imply that the short-term-response to cigarette smoke is of precancerous nature, we believe ,that a similar relationship may well be true also for the comparison of other tobacco blends or smoking mate- rials. The results of subchronic inhalation in S.rian hamsters may thus predict the effects of chronic inhala- tion. particularly the potential turnorigenic potency of different t%pes of cigarettes. On the basis of the results presented herein, it t:ould become possible. therefore, to evaluate and screen different types of cigarettes in relati.•eh' inexpensive, short-term smoke inhalation tests and to assess the carcinogenic potential of their smoke for later verification by actual chronic studies of selected t..pes. MATERIALS AND METHODS Attrinrrls.-Male BIOj' 13.16 inbred S\rian golden hamsters bred in our own laboratorv %%ere used as de- scribed pretiouslv (4). Crgarrtres.-The three ttpes of experimental cigarettes %%ere the came as those pre%iouslt used in chronic inha- lation studies. Thet %%ere 1) t%pe .a. a flue-cured. all- tobacco cigarette. as found ori the market in the l nited Kingdom around 1975: 2) ttpe B. a cigarette made of l0Q'c Cttrel.. a cellulose-derned tobacco supplement: and 3) a cigarette that consisted of a 1:1 blend of the t%pe Aand tcpe B smol.inq materials. AII cigarettes t•ere tilter-tipped and had a length of 72 mm. Thet \%ere described in more detail pte,6 tt)usi% t-t. Srrrokrn~ toudruorr;.-Smol.e t.as generated and the an- .\leREVtATION L'SED' df=rieRrees of freedom. ' Recei.ed Januara 28. 1983: accepted .apnl 19. I!+aJ 'Animals vere maintained in facilities fulh accredttet: bs the kmer- ican ii,,.ocuuon for Accreditation of htwrators Animal Care ind upcr-ated in accordance wtth the Guide for the Use and Care of tiboratorc Animals and the Animal Welfare Act of 1980 'Bio-Rcse+rch Institute. 9 Commercial ise.. Cambridge. Mass. 02141. . 'tkpartrnem of Patholog.. Boston L'ni%ersuv. Boston. Mass. Q':'-1 5 ' Department of Patholog.. St. \' ncent Hospital. Ss'orcester. Mass. 01604 'We thanl Robert A. Parler. D. Sc.. for cunductmq the +tat stinl essluauons. 'A tndemarl of the Celanese Fibers Co.. a ,ub.idiars of the Ce- lanese Corp.. Ch.trlotte. \.C. 619 JNCI. VOL. 71. NO. 3. SEPTE>1BER 19d3 ~ ;~~~~

620 Bemfeld, Homburger, and Soto imals were exposed to it in the modified reverse Walton smoking machine (4). Smoke exposure was intermittent: 27 seconds of a 42c cigarette smoke concentration was alternated in the exposure chamber with 33 seconds of fresh air. Fr• ch hamster was exposed to 12 minutes of this regimeii, twice a da.• (with at least 4 hr between exposures). 7 davs each week. DtrrrNio,r nJ.auukv r.%posurr.-Twelve hamsters were ex- posed to smoke from each of the three types of cigarettes. as well as to sham smoking conditions, for total durations of 6. 9. 12. and 15 weeks, and 18 hamsters were exposed for 20 weeks. A total of 264 hamsters were used in the experiments. All 6- and 9-week exposures were started simultaneousls. the 12-.+eek exposure was started 4 ..eeks later, the I 5-week exposure Qweel:s later. and the 20-week exposure 14 weeks after the start of the 6- and U-%,ee{k exposures. H,uopnib,pln,,ir ;nrdir~.-The animals were killed at the end of the smoke exposure period. and the histologic sections of their lungs. tracheas. larvnges, and heads were prepared as previoush described (4). The histopathologic findings %%ere evaluated as pre% i- ousls reported (4), except that for the alveolar macro- phages and Iar%ngeal hyperplasia, a three-point svstem of grading the se%erit% and.,or the frequenc% of occur- rence of these phenomena in individual animals %.as substituted for the pre%iouslh used t%%o-stage s%stem of presence or absence of either increased frequenr% of al% eolar macrophages or of se.ere h% perplasia. on„o .,pu,r,•,.-These s%ere graded bs three step• uf se%erit%,!i rare: None to occasional single mac- roDha_e• %•ere seen in the tntra-akeolar spaces. nl 1-: NInstls >in,le macrooha,e~ %%ere seen. and seseral fields h,rci to be scanned tor their detection: macrophage ag- gre,ates utcurred onk occa,~iunall\. ) '?': Macrophages \.ere fiound in nto~t field~ and often occurred in small intraak eolar a„re,ates or clusters. This three-point s%stem replaced the pre\iousl% used none-or-all arrangement. and the present °' stage Nas equi%aient to the pre%iou5 state of 'increased frequenc\ of nr,,crophages ' t-t). -Four degrees of se\eritc were disttnzuished, but the first t\•o (normal and 1') Nere treated as a sinQie categor\ because thev occurred ..ith about equal frequenc\ in the control animals. The de- grees of se\eritv %%ere (i) normal: The lanm was lined b% columnar ciliated epithelium. except for stratified squamous epithelium on the %ocal cords. b) 1': The anterior lat\ngeal uall sho%%ed epithelium similar to the .ocal cords. probably a normal variation). r) 2': The entire Iarnngeal ~all Mas covered by stratified squamous epithelium on a smooth basal laver, and keratinization Mas present. d) 3': This degree was the same as 2' h.perplasia but 'A ith do% ngro\<th of epithelium into the submucosa. Sr(.na,r, -Statistical significance of the differences in response to the three types of smoke or to no smoke (i.e.. sham smoking conditions) was assessed by means of log linear models t;). The model evaluated the effects of the three (%pes of smoke or of the Lontrol conditions JNCI. VOL. 71. NO. S, SEPTEMBER 1983 on the following four tvpes of response: 1) presence or absence of hyperplasia of the terminal bronchiolar epi- thelium. 2) frequency and degree of aggregation of al\eolar macrophages. 3) presence'or absence of squa- mous metaplasia of the trachea, and 4) incidence and severits• of laryngeal hyperplasia. Allowance Was made for the five different durations of exposure to smoke. Models for each tcpe of response H•ere constructed. and the influence of the response to smoke was assessed bs use of the likelihood ratio statistics (G2). All but one model provided a satisfacton• fit (P_.3): 'the fit of the exception was marginal. The method used provided an overall test for differences among all three tvpes of smoke and the control conditions. Tests for differences between the effects of certain combinations of opes of smoke (e.g., between responses in control animals and responses in all smoke-exposed animals. regardless of tvpe of smo{.e).%ere performed in a fe%% instances. Tests were done with the use of a standard x"- anaksu t%e here the duration of exposure could be ignored) or t%ith the use of a log linear model with duration effect. The occurrence of all other tvpes of response uas too sparse to require statistical anah sis. RESULTS Lungs and Bronchi Increased frequencs of alveolar macrophaze~ and macrophage accumulation was observed in hamsters e.- posed to smoke from tvpe A cigarettes. was ;ess often obsen ed in those inhaling smoke from the 1: I blend. and onl\ rarelc %.as obser\ed in hamsters tre.ued mn^ smoke from t\ pe B cigarettes or in control anrnral: ;ab;e 1 r. The histopathologt of this phenomenon %,.t5 de- scribed earlier for chronic smoke inhalation studies ;:- figs. 14 and 15 in (?)J. In the present stud\. the increa>ed frequencs occurred as earls as 6 s%eel.s after the start of smoke exposure and did not intensifv thereafter When %%e ignored the duration x t\pe-of-smol.e x re<oonse interaction, the best model pro\ ided onl% a mar;;nal 'n of the data (P=.049). This model in.ol.ed all the t\.o• wa% interactions: i.e.. grade-of-response x duratton. grade-of-response x tvpe-of-smol.e. and duration X ope• of-smol.e. The grade-of-response x t\pe-of-substartce in- teraction, which assesses the importance of the t\pe of smoke on the occurrence or on the frequenc\- of al\ eolar macrophages. was found to be essential to the model (G==233 on 14 df. p<.00001). This means that the ope of smoke markedlv influences the response. and the differences in alveolar macrophages among the 4 groups. as shoNn in table 1. are highl\ significant b\ statistical criteria. H% perplasia of the terminal bronchiolar epithe!ium. a benign change that was rarels found in older smoke- exoosed or control hamsters in our chronic studies. %%as seen in 16 of 263 hamsters in the present short-term experiment (table I). A similar lesion in aged hamsters after tobacco smoke exposure was described bv Wehner et al. (6), who termed it "bronchiolization of aheolar c - rR HN 0427984

1 a Subchronic Cigarette Smoke Inhalation 621 I TABLE i.-Histopathology of lungs and trachea in hamsters after subchronic exposure to smoJu from three types of cigarettes Histopathologic findings,' No. of animals with: Smoke- Cigarette exposure No. of animals Hyper- Alveolar plasia of macrophages t.pe duration, studied Squamous terminal ' wk bronchio- Rare metaplasia 1" Z- ` f h lar epithe- lium ea o trac A 6 12 3 8 4 3(25) 9 12 2 8 4 3(25) 12 12 8 4 4(33) 15 12 9 3 5(42) 20 18' 2 10 8 11 (65) 1:1 blend' 6 111 10 1 0(0) 9 12 10 2 5(42) 12 12 2 12 6(SO) 15 12 - 11 1 4 133) 20 18 1 8 10 8(44) B 6 12 7 5 4(33) 9 12 2 12 5 (12) 12 12 3 & 1 3 c25) 15 12 2 12 4(33) 20 18 2 15 3 3(17) None' 6 12 10 2 2(17) 9 12 12 4 (33) 12 12 10 2 2(17) 15 12 12 2(17) 20 15 18 0(0) 'Data were excluded for t animai with edema and congestion of the :ur.gs and :or four :nstances of tracheal hyyerplasia in 4 different groups -Asex:a:ed w:t!: Fyperxeratosis. Values :n parentheses are percentages. A few animals showed ^•.;erpiasia; all others had normal tracheas. :' 3n:mals were examtned for the trachea tno tracheal histologic exa°:;::atnon for : antmall. :: cter.d o: c:¢arette types A and B. : ar.t^ai as lost for !::swio¢u esamin3tion of lungs and trachea. `An::r,ais aere exposed to snat^ s:noKtng conditions. eptt!irlium ''•., fig. 5 in (6)]. In the present subchronic stud%. the changes Here restricted to small areas of the ternt,ral hronchiolrs. and the% occurred %•ith approxi- matek equal frequenc% in animals that inhaled smoke from tspe .-k and t.pr B cigarettes. Changes occurred somr~t hat more rarek in those animals that inhaled smoke of the blend, and changes were absern 'rr, t+tc control animals. Statistical evaluation showed that the duration of smoke exposure t.as not associated with the prr%alence of h.perplasia of terminal bronchioles iG==1.1 7 on 4 df, p>.8). and the exposure duration. therr'ore. could pr ignored. Prevalence of h%•perplasia Of,e-nunal bronchto!es t.as then associated with the tspe of sn,ol,r tG'=1 1.-1 on 3 df. p<.01). Clearls, the result is ,igntfcant onlk because the control group differs from each of the 3 smoke-exposed groups. This contrast gi%es Y'=-+ 37 (df = 1, with Yates' correction), p<.05. Since hsperplasia of terminal bronchioles was absent in ham- sters in our previous smoke inhalation studies. even after onk 33-4U t.eeks of rxposure, this phenomenon must be assumed to be an early response that disappears in older hamsters of this inbred strain.° Lung congestion and edema were limited to one ani- mal that was exposed to smoke from type B cigarettes for 20 weeks. Bronchial hyperplasia %as not observed at all in the present experiments. Trachea A frequent abnormalits''qf the trachea was squamous metaplasia, often a focal replacem(;rit of the ciliated respiratory epithelium associated with h} perkeratosis [see fig. 1 7 in (2)]. As the duration of exposure progressed the incidence of squamous metaplasia increased some- t. hat in hamsters that inhaled smoke from type A ciga- rettes (table 1). The incidence did not change, hok-ever, with duration of exposure in any of the other smoke- exposed animals. Statistical evaluation of the data showed no association between the presence or absence of tracheal squamous metaplasia and the duration of exposure. but a marked association was observed be- tween the type of smoke and the occurrence of squamous metaplasia in the trachea (G"=11.7 on 3 df. p=.008). .again, as in the case of hyperplasia of terminal bron- chioles, the result is only significant because the control group differed from each of the 3 experimental groups. When we thus ignored the duration. the contrast of the combined 3 smoke-exposed groups %,ith the control group gase x"=6.69 on df=1 (t.ith 1'atrs' correction) and p<.03. but there was no statistical significance to the differences among the 3 e.perimrntal groups: e.g.. X'=1.36 on df = IO.ith Yates' correction) and ' D>.'?. Hence the t%pe of smoke does not hase an%- influence on tracheal metap!asia. Trucheui h%perplasia was seen rarel%, i.e.. in on!% 4 of the hamsters examined. Three of them had been r.- posed to smoke from tspe B cigarettes .tftrr different exposure durations, and I had been esposed to >moi.r from tspe A cigarettes. The occurrence of tracheal hs- perplasia t.as ttot deemed to be re!ated to smoke inha- lation. Mucus %%as frequentlt present in the trachea and did not appear to be related to e\posurr of the animals to smoke. Larynx L'nlike the situation in pre%ious chronic inhalation studies. in which a rather high incidence of '?' h\perpla- sia occurred in the control animals tr%en at intermediate killing of the animals tfter onis 35- to 40-wk exposuret. °?' hsperplasia of the lar.•nx aas.er. rare in the control hamsters of the present short-term inhalation stud\ Ho%.e.er, 2' h%perplasia occurred rather frequentl\ in 'What Ne now call - h%perplasia of the termtnal bronchiolar eptthe- hum- is relerred to in a lorthcommg boo(, as -.tdenom.ttos s- in S%rtwn himaters The .tuthors sute that -conudennq the incidence of .iden- umatosis in nonareated .tntmals. Me belte%e that the interpretation ot adenomatosts as rn expenmenuih tiused lesion should be made -th caution - t5chmidt RE. Eason RL. Hubbrrd (.B 1'our.q JT. Euen- bnndt DL. Patholoqt of.tqtnq Strun hamsten &xa R.tton. Fla CRC Press. 1983. In press.) JNCI. VOL. ll, `O. 3. SEPTEMBER 1985 4 2•i 85

I i 622 Bemfeld, Homburger, and Soto subchronicalh' smoke-exposed animals. This degree of h%'perplasia was differentiated, therefore, from both the 3' and normal (including 1') stages in the present sub- chronic studt. in contrast to our procedure in the earlier chronic inhalation studv. Various degrees of laryngeal hyperplasia in smoke- exposed hamsters were previously described [see figs. 3. 4, and 6 in (4)]. In the present short-term experiments. laryngeal hyperplasia was clearlv more frequent and more severe in hamsters exposed to smoke from any of the three types of ci-arettes than in the control animals (table 2). Obviously, the laryngeal response was most severe in hamsters that inhaled smoke from type A cigarettes. Hamsters that inhaled smoke from type B cigarettes had a considerably lower laryngeal response than did animals exposed to smoke from tvpe A ciga- rettes. Inhalation of smoke from a 1:1 blend of the tt%o smoking materials elicited an intermediate response. In contrast to smoke from type A cigarettes, which caused the lan'ngeal response to increase as duration of expo- sure progresseo. the effect on the lar%•nx of the other two types of cigarette smoke had alreadv leseled off after 6 weeks of exposure. For statistical evaluation. the best-fitting model re- quired both the grade-of-response X duration interaction and the grade-of-response X tt'pe-of-smoke interaction. The grade-of-response x duration interaction Has statis- ticallt significant (G'=13.4 on 8 df. p<.0`?5), and the grade-of-response X t%pe-of-smol.e interaction t.as eten more significant on 22 df, p<.00001 j. Because the lar%ngeal response in the control group t%as consid- TABLE 2 -C.e^.nQPa. ^.S:o_Ja:h.O{oh'.. !'n hATSters al:fr sufKNronic ff?Ji4rP to ~^ICSP,'•^^I tnree :~F1Ps o/ CI(dorf:tes igarette ~moke• exposure Noof Hutopathoiog:c frndrngs. No of anrt::als with: tVTe durauon. an.^a:_ studied Hy~perplasra Pa illo a Rk p m A 6 11 1 9 12 11 1 12 12 6 6 1 15 12 9 3 20 1"' 8 9 1 1:1 blend' 6 11' 10 1 9 11' 11 12 12 ' 5 15 12 12 20 1-' 14 3 B 6 11' 1 10 9 12 1 11 12 12 12 15 11' 11 20 1"' 1 16 None' 6 12 10 2 9 12 12 12 11' 11 15 12 12 20 1 - 1 7 'No histotogy of the larynx for I animal. ' 1:1 blend of cigarette types A and B. ' 1 animal was lost for histotoggic examination. 'Exposed to sham smoking conditions. JNCI. VOL. 71. NO. S. SEPTEMBER 1983 erabh' ~:eaker than that in ant of the 3 experimental groups. the data were anahzed for the 3 groups of hamsters exposed to smoke without inclusion of the data for the control group. Again, the best-fitting model required both the grade-of-response x duration inter- actton and the grade-of-response x- type-of-sntoke inter- action. The grade=of-response x type-of-smoke interac- tion was still statistically significant. with G'=36.5 (df=1 °) and p<.0005. Hence there is no doubt that the type of smoke influences the severity and pretalence of laryngeal hyperplasia and that the response-increases significantlt as the tobacco content increases in the cig- arettes. Laryngeal papillomas were seen in the present short• term studt only in 2 hamsters, both exposed to smoke from type A cigarettes (table 2). The histopathologt of such lesions has pre%iousl% been described (- fig. 3 in (a))• DISCUSSION The observation of lar%ngeal ht'perplasia. lar%ngeal papillontas, or changes in al%eolar macropha,e popula• trons iti experinaental animals that had inhalyd cigarette smoke is not net~. Nonmalignant lesions t.ere reported after tobacco smoke exposure in rats (; -1!rt and in mice (11-1 3). Malignant lung tumors were described in beagle dogs (1 a-1 7) after cigarette smoke exposure for 29 months. The present results can be distinguished on t\,o count, from all pretiouslt reported effects of tobacco smoke inhalation in laborator% rodents: 1) Our firtdw,> %.ere obtained under ;i,n,inor,n' experimental conditwr> ir t~,r samr test anirnals in \.hich oter -tOt`c are l.n,%\n tt, de\elup carcinomas of the respirator, tract Jarxn\ up"m chronic smoke inhalation. ''t The se\erin and ini:Crncr of the nonneoplastic changes in the respiratoi \ :!.ir: ,-,: the hamsters tn the .,,%, ;: .. stud\ and the tnc,cirnt e w malignant lart ngeal lesions of the animais in thc ' - - - TABLE 3.-Tne most rmpor:ant char.ges in !rc :-.:: - hams:ers exposed to cigarette smoke ,n soocr:ronic er.;z chronic rnhalatron su.dws Type of ciga• Alveotar mac• Laryngeal hy- rophages perplasia °c of anima.s r: risk A Highest Highest 4"4 1:1 blend' titedwm 1Nedium 21 7 B Low Low 0 None' Lowest Lowest 0 'After 6- to ?0-wk exposure. 'The differences among animal groups were found to be statrsucatl\ significant (see text). 'After t59•wk exposure Idata from (41). ' 1:1 blend of cigarette types A and B. 'Sham smoking conditrons, negative controls. Hrstopathologtc findings in respirator. hamsters Subchronic study." severity Chronic stuc\ rette and :ncidence o!: lan ngeai car:. noma inncer.cr CTR MN 042786

- ..~...-....,, a Subchronic Cigarette Smoke Inhalation 623 study each permitted differentiation among exactlv tbe same three types of cigarettes. The salient points that summarize the response of the respiratory tract of the hamsters to the present sub- chronic and the previous chronic cigarette smoke expo- sure are shown in table 3 for comparison. Smoke from the most carcinogenic of the three types of cigarettes upon chronic exposure (type A) also induced the most severe response in the subchronic study. The least car- cinogenic smoke of the chronic study (type B cigarettes) elicited also the lowest subchronic response, and smoke from a 1:l blend caused intermer:zte effects in both chronic and subchronic studies. Although there is no evidence to assume that the short- term phenomena observed were of precancerous nature, the obvious parallelism between the subchronic and chronic response for the three types of cigarettes strongly suggests that the short-term results may be of predictive nature for what will happen with a given type of cigarette upon chronic smoke exposure of hamsters. REFERENCES i1) DONTENk'ILL W. CHEYALIER H -J. HARKE H-P, LAFRENZ U. RECK- zEH G. SCHVEIDER B. Insestigations on ttie effects of chronic cigarette-smoke inhalation in Ssnan golden hamsters. J\atI Cancer Inst 1973: 5 I:178 1-1832. 12) BERYFELD P. HOMBL'RGER F. RCSSFIELD AB. Strain differences in the response of inbred S%rtan hamsters to cigarette smoke inhalation J\"atl Cancer Inst 19-4: 33:1 I41-I 137. ,i1 DOVTE%N'tLL W. CHEVALIER H.J. HaRKE H-P. et al. Untersu- chungen uber den Effect der chronischen Zigarettenrnuchin- halation beim ssrischen Goldhamster und uber die Bedeutung des Viumin A auf die be Berauchung gefundenen Orgart.er- anderunQen. Z f:rebsforsch 1977. ?9:133-130. =i BERWELD P. HO%,IBLRGER F. SOTO E. Pat f:J Cigarette cmoke inhaiation studies ~-i :nbred.isnan Zolden hamsters. J\CI 1979: 0)6"5-6 59. ts. BtSHOP 1M. FIEI~BERG SE. HOLLIN;D P%%*. Discrete multi%ariJte analysis: Theory and Practice. Cambridge. 3tau.: MIT Fress. 1975. (6) WEHVER AP. BL'SCH RH. OL50N RJ. Effect of chronic exposure to cigarette smoke on tumor incidence in the Syrian golden hamster. In: Karbe E, Park JF. eds. Experimental lung cancer, carcinogenesis and bioau»s. Berlin. Heidelberg, and New York: Springer-Verlag. '1,9: i:360-368. (7) KENDRICK J. NETTESHEIM P. GCERIN M, et al. Tobacco smoke inhalation studies in rats. Toxicol Appl Pharmacol 1976: 3 7:SS7-369. (8) SMtTH G, WILTON LV, BtvNs R. Sequential changes in the struc- (9) ture of the rat respiratory system during and after exposure to cigarette smoke. Toxicol Appi Pharmacol I978r96:379-391. DALBEY WE. NETTESHEIM P, GRIESEMER R. C'ATON.JE. GCERIN MR. Chronic inhalation of cigarette smoke by F344 rus. J\CI 1980: 64:383-390. 1/O) WEHNER AP, DAGLE GE. `1ILLIMAN EM. et al. Inhalation bioassay ofcigarette smoke in rats. Toxicol Appi Pharmacol 1981: 61:1- 17. (!!) EasENeERG J%t. Cigarette smoke and the incidence of prtman neoplasms in the lung of the albino mouse. Science 1952; 1116:361-562. (/2) LECCHTENBEROER C. LECCHTENBERGER R. The role of influenzl virus in the development of malignant transformauon in .itto and in the respiratory tract of mice. with and s.ithout exposure to cigarette smoke. In: Severi L. ed. Lung tumors in animals. Proceedings of the third quadrennial conference on cancer. L'nisersitv of Perugia. Perugia, Itah: L'nt. Perugia. 1966 445- 463. 0 i) H.RRts RJ. `EOROVt G. Production of lung carcinomas in C37BL mice exposed to a cigarette smoke and air mirture. Br \led J 1967; 4:63'-6a 1. (l4) AL'ERBACH O. HANMOND EC. KIRMAN D. G4,RFI\KEL L. STOLT AP. Histologic changes in bronchial tubes of cigarette-smokmg dogs. Cancer 1967. "0:4033-'20ti6 t 1`) The effect of direct cigarette smoke inhalation on the respirator. tree of dogs. \atl Cancer Inst StonogT 19,58. 28 03- ,7. + l5t ALERB.CH O. HAaMoND EC. Effects of cigarette smoke on do¢s. 171 I1. Pulmonan neoplasms. Arch En.tron Health 19-0: 21 '68. Zt.ICKER GNL FtuPN RE. P.RK JF. LosccTOrF SM. R.c.. HA. STE.•Ea DL. C1in cal and pathological etfects of c„z,rette smoke erpv3ure in beagle dogs. Arch Pathoi Lab Med :9"i. 10_:o':1-ti_8 JNCI, VOL. 71. NO. 3. SEPTEMBER 1983 CT ~ ~"'~~'`~ 0427987 -