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Strain Rsfterences in the Response of Inbred Syrian Hamsters to Cigarette Smoke Inhaiation " P. Bernfeld,' F. Homburger,` and A. B. Russfield' SUMMARY-•Mah hamsters, 102 from each of 2 inbred hamster liiss, were exposed to cigarette smoke twice a day. S daysJw•2ek for up to 100 weeks, in a mod ified Walton revents+r,uking machine. Sixty sham-smoked and 60 cage-held controls wero used for each strain. Smoke eposure for up to 100 weeks had no eHect on mortality in either saairr. hut -educed body weight. Carbo:yhemo- globin levels i>:creased ntarkedly immWlately after each smokk exposure but rtaturned to baseline levels in less than 24 hours. Sorum triglyqride levels and virus profiles of smokteiposeA animals were unchanged. Chronic smoke exposure incrro.-ed relative weight of the lungs and heart in both stroin?, but to different deyrees. Over 99 of t e smokauxpor.d animals of both strains s owed hv>Nr• plastie~: pTaiHe cha arynx. However, mieromvas3ve cancer was nom imes more fr.g.uent iq 1 s~LraTn 4han ehi other. In the inbred~line more suseep- tible to laryngeal hyperplasis, 2 animals developed naso- pharyngeal tumors, one of which was malignant. Smoke exposure induced rare benign squamous papillomas in the air passages of botlt stralns. The strain less susceptible to laryngeal hyperplasla exhibited more pulmonary adenoma- tosls, but its incidence was not significantly affected by sntoke exposure. Clumping of pulmonary macrophages was proof that smoke had reached the lungs; 1 strain was more •usceptible to this phe"emenon than the other. In neither rain did snsoke exposure affect the incidence of tumors .rising outside the respiratory tract or the degenerative changes characteristlc of aging hatnsters.-J Natl Cancer Inst 53: 1141-1157, 1974. A`IODEL SYSTEM for studying the effects of chronic inhalation of cigarette smoke was developed by Dontenwill and co-workers using randornbred Syrian golden hamsters (1). This paper reports the effects of genetic differences between 2 lines of inbred Syrian hamsters in a related model system. The most appropriate methods for studying bio- logic effects of tobacco smoke in experimental animals are necessarily those based on chronic inhalation simulating conditions in the human smoker. One ^iain principle of toxicologic experimentation in animals is the use of dose levels many times higher than those safely tolerated by human subjects. With tobacco smoke, it is difficult to do this, since smoke conta,ns not only substances of possible chronic toxicity but also compounds of known acute toxicity, such as nicotine, carbon monoxide, and others which sharply reduce the tolerated dose levels (2). Hartr sters are desirable test subjects for tobacco-smoke inhalation studies, since they have a much greates resistance to toxic effects of nicotine than do rats or mice (3). We used inbred hamsters in order to im- prove reproducibility of results and to detect possible strain-related differences in response to tobacco smoke. MATERIALS AND METHODS .ac:.Talr.-BIO harrsters %.ere purchased from Trenton Experimental Laboratory animal Co., Bar Harbor, Maine. Five strains, the inbred BIO 2.4, BIO 87 20, BIO 82.62, and BIO 13. 16 lines, and the RB (randombred) line, %.ere used in prelirninaryy acute toxicity studies (to be reported elsew•here). F:om these studies, the BIO 87.20 and BIO 15.16 strains were selected for chronic toxicity erperi- merits. All animals weighed 108t4 g at the beginning of the c^ronic toxicity studies. -• Onl•v males were used, since atale hamsters are :ess belligerent than fe:nales and can therefore be housed se%eral per cage. In the present study, ham- sters were housed in groups of 6 in 12X 14X6.5-inch ?otvpropylene boxes covered with wel!-fitting, wire- screen ! ds. San-I-CeI, Deodor grade (Paxton Proe- emng Inc.. Paxtor,, I!I ), was used as bedding material. It L.as changed once a week, at which time the cages %.ere washed in 180° F%.ater and detergent in a com:r.ere:al cage-washing machine. The animal room %.as centinuously ventilated (32.2 exchanges;hr). Temperature was kept between /2° and 16° F. Fluorescent bulbs, the only light source, were auto- T1atlcallV controlled to-eperate between 7.00 a.:n. and 7 00 p.m. No other animals were kept in the room housing the namsters, and no smoke was generated within this room. The animals received Wayne -Mouse Breeder Blox and fresh tap water ad libitum. Twice a day, 5 days,week, hamsters were transferred to a nearby laboratory for exposure to smoke. Crnnatron of cig,acttr rmok..-`fodified Walton reverse-smoking machines were used (3) and are described and illustrated elsewhere (4). In these machines, air is pushed through lighted cigarettes by application of positive pressure at the burning end. This contrasts with the conventional smoking machine or the human smoker, since both suck air through the cigarette by application of negative pressure at the mouth end. All animals wore well-fitting, permanently attached, felt rings around their necks (y,s of an inch thick. 2 inches outside diameter, and '{-Y of an inch inside I Reeci.ed .1ipril 24. 1974; accepted July 8, 1974. ' Supported by a cootraet fmn the Couuc+l for Tobacco Re- uarch-l1 S A. The view, expreued are rhoee ot the autLors and do not netessarsly re3ect the opinions of the Councul for Tobacco Researeb. i Bio-Resnarcb Conaultann, Ine., 9 Cornmercial Ave.. Cambridge, Ntaas. 02141. ~ Bio-Researcb Coesultants, Ine.; ResearcE Professor of Oncology, Di.vion of Basie Scicnces, School o! Graduate Dentistry, Boston Univenity; Research Pro(essor of PatAologY Boston C'oiveruty School of Medicine, Boston. MQ. ' Pathology Depuoneat, St. Vincmt Horpital, Worcnt Q.Stass.0l6t0. JURYAL O! TtiZ NAT7ONAL CA-VCY{t NsTrTSTrt, VOL. 57, vO. 4, OCTOEER 1974 1tJ. 4 4 1 J • t.• a 1141 c"~"I-~ ) la ~ 04,2253

I I -...r.~. . 1142 HER.VFELD, HO)18CRGER, A.YD RCSiF:ELD diameter, depending on t.`.e s:ze of -he t;arrster). The felt rings were reinforced on both s:des by tcin, : mg- shaped alumtnum plates of slightly smaller dimen- sions (l'i inches outside diameter and 's;, of an inch inside diameter). The heads of the animals tvcre in- serted into the smoking machine by means of these collars, so that only the heads were in contact with the smoke. Because :he bodies remained outside the machine and were firmly restrained by the collars, the need to confine the animals in tubes, with attend- ant excessive sweating and stress, was eliminated. Six animals were exposed simultaneously to smoke inhalation. Since the felt collars prevented the ani:nals from grooming their eyes, these t•ere washed with penicillin solution on a cotton swab daily 5 davs w-!ek. - Smoke generated from 4 I q 1 Kentucky reference cigarettes s burning simultaneously ~s is administered to the animals in 60-secor.d purt c•.c'•es Each cycle consisted of 2 seconds of smoke ;er.eratton followed by 15 seconds of addir,onal s-..)ke -t--osure and 43 seconds of exposure to fresa a,r :3 •-..-.tn%:ze -6e toxic effects of carbon monoxide. During the 2-second >:n.ie r---r1::-M• 35 :nl air was pushed through -,_i i:-e ~ c.;are(ces, yielding a total volume of :=1) ntroduced into the 72.i-ml exposure :r.agnette stirrer provided instant :r.:r.rq t`•e s~ :ke daution faetor was 725: 140, or 5 2. T-.ts::•.uke corcen1[yuon_ inhaled by the hjQy~tt;n ~,•as •..ere:orimatelv ITfZi',at'tFiat~heavtn the mou:h -n ~o::.ec~t ~a~retta. mparuon,_ t e a~:er33~_z2ncentrar:on oT smok`e iti- tlte"Iungf or a human smoker a aoout (aVePage puff volume, 3~ r~"^;uman TunciioriaT residual capacity, 3.3 liters; :^•u:nan ::dal %olume, 0.7 liters). The smoke, seen through the t: araparent'chamber, took less than I second to tra%,erx the 6 inches between the mouth end of the c:garettes and the harnsten' heads, which assured that the hamster inhaled smoke no less fresh than that reaching the human smoker. C/nortrt irthelofron ttudy.-A prelimtnsrv study was performed to determine a dose ur.edule at which mortality would be negltgtble during :ne :ntttal months of the experiment. An exposure of approxi- mately 5% of the median !ethal dose, ;.re%:ously determined under identical exposure condtttons, constituted the upper safe limit. Ths .ar,ed with the strain of harruter. For obvtous :eisocs• exposure time suitable for the most senstuve strain -as used for all aniznals in the long-term study Accordingly, each hamster was e.pr,sed :Mice a day, 5 days/week, to 8 consecutive purf ;ticles from the IRI Kentucky reference cigarettes P,,fs 1-8 from the cigarettes were used; the resjit.nq 5•itt ength was slightly longer than 30 mm. T`,e per:od :f exposure was 45-100 weeks. Two simultaneous control groups ~cre Taintained for each inbred strain. One group cons,sted of cage- held animals of comparable age and initial weight that were maintained stmultaneo~..jlv n •hc same animal room and handled like all otner -arrsten with - --......+... .--- -- reg3rd to weekly ielt-al-trtunum collars. etc exposed to the smoking machines. The group consisted of comparable harrsten exoo~ -sham smoking" on the machines; i e , the ma- were operated under ;:andard condit:ors, -.c-_ no cigarettes were inxr:ed :n:o t=em. For each 2 lines, 102 hamsten were esposed to smoke, 61) exposed to sham smok:::g, ir.d 60 were cag- controls. Since it was impossible to obtain ail 222 harr required for each inbred iir:e at the ;-ime animals of the proper ir.:t al age ard ..e:g'^t introduced into the erperi^ient :^ :ots or o, over a period of 37 weeiu. Th.:s procedure :s cc on!y when homozcgo -s J'Jc ]n ,, ~rec are used, and provided that all f..ct^,rs s•:c- is .. sex, and age are uniform. .ti iar a-s 6 of each of the cont:ols vas ;tar:ed at L~.: sar.:e as 2 lots (12 antma:s) of srroKe!CDosed ::.lmst-" 0btrro2twnl J.rrT; weights were recorded •.^--K:y ~h-o,:g~oct periment. CarSor~he:r.oi!eb n .-:e!t . ... ured bv a mtcrom.ec::od i, i:er ~: i::ocs :..- smokee<posure. Blood waSobta:ned by':eart ?-r. •inder 'ight \embutal a-.-ahesia. T~ a•:oid ,` deleterious effects'of h.ear•t Dur.c:-_,r: an n:-;te:s chronic inhalation st.:dv, :ubov1.7-e:~,V",,o1^ were determined in 6 male harr.crer+ •'.tk`-!_ body weight) of each inbred scr,:a tt to smoke for 1-3 weeks but chronic inhalation ;tttdy Car~r;. . . z. : '.. were also determined in i:=•,V .. ~i . haauters from the chroni of exposure. Tnmrrtawn of rt.sv.-.•1ni-sjs were ::I!o!d .. they appeared mortbund, 3s •--dzed by ,::,n;i. weight loss or the appearance of edema. tn accit. some hamsten of all groups •..ere kllled at 45, 75, a-td 9C weeks. Survival time calculations w corrected for the hartuten killed when not moribu Complete autopsies ~%ere pe-^or;ned. The e respiratory tract and sny o.:^.er :;gans or tis grossly appearing abnormal •.ere ;c•:d:cd histo: cally. Ungs were indated and Fred ~-i Teiivesnicz• fluid in an apparatus devised by Dr. Sadarn-u I kawa (personal eottununication/ r'td+ .•:.. ec ibration between internal aod :.'',:.:r prtssure. The larynx was remo%cc: ,ac '... _~ ,:"o" connection of the trachea :o r.::. r.'!a tc:a the fixation fluid into the I..ng ..:c • 40 mrn Hg. At the completion of •:xs:.or, cr; .rs was equaLized at 10 mm Hg. Organ weights of some ~%aT.Ste7 ^'er•7 •7e Serum triglycerides [by :^.e +.arometr:c (7)) and virus profiles t,5y co.:: tcsv -)f Ur ~:xrt r I R I Kentucky refaence vq,re••ro jr•- :)^x!.ced b1. 1 were obttine7 lrom, the L'n:"n.w -i K-o:_ck. Tobxco Health Rex.reh Insutute, Kent•<.r 2ex.vch Fo.a, stt• LeainSton, Ky. Cnmpowtuoa of •ne r•+rr.-•ce c:,3ret e f R' 12 : moivure .vaa as tollowl , S' ., :e i ,r-d fluetured +terrt, 14 2 :; Burter :.:-- l. r rk ,n . leafl, II 6^.; \faryiand lun,na, sucrou, 5.3;. - ....,,.~,~ C T R H N 0 "'!'a:..22 5 F "r

C:CA.2ETTE i%IOKE !NHkLkTION :N ;\aREO HaMiTTRS !!43 I~ytebner, \at:onal [r.sti;utes of_Fiealth~ %.ere s:udied . n scme antmaTs ot eac group. ~ - e - _ - ;< Mortality Morwlity was .cry low in all groups until ibout :!:e ti0th i%cck of thc c.perir.unt (text-fi;s. l, 2), at..hich time the animals' chronoloqic agcs were 3p- proximately 73 %veeks. Thereafter, mortality increased rapidly in all groups, reaching 100 r, at about :00 weeks. Ncither smoke exposure nor sham smoking had a significant effect on +r,ortality. There was ^o difference in survival between the 2 strains. Changes in Body W.igpt (Tabl. 1; Text•Fiys. 3, 4) In both hamster strains, individual body weights were affected by smoke inhalation. The mere erper,- mental manipulations, such as sham•smoking con- ctitions and'or vress, also changed the body weiQhL Initial weight in both groups a%,eraged 108 q. Pe BIO 87.20 cage-held controls attained a final a.era¢- weight of 144.3 q, whereas the BIO 15.16 caqe-ae.d controls attained only 123.0 q. „ -3 > > ~ ~ I j \ L \'~ \y- ~ ' 1 7 i .i\NCf < I' 1 >>0 1 i ? Y u: ~ 5 i n - - ~ ~ o .o w i0 '2 +o to » or +cts ~ a Text•nct:a t.-Moctality of B[O 15.16 bartuten eipo.ed to tmoie from IRI cyarettes and of uneapo.ed cortroL. i ~ Y - ..:.~f \\\\ ^? N •., er o ~f ~\\\ xrf`~a ,y,naOP ~~nn O- w 3 ,V-,'...t` _.~ r y pn+~- .o- cr,rf ~ - -f..L.. ~ ~a=o _- Z Ts+cT-necss 2.-Ntorrality of BIO 87 20 hartuters arpo.ca to smoke from I RI ci`arecces and of une:po.ed coetmla i<n i 3 3 s T T ~ j ~ IC ==i: 3 : ~ M1 U \ \ \ \ 5 771 I 3 -O;! i A 399~i BnaS /\ O-,On . ae. .u11- M 7 a C T R taI N 0 4'~~1.5 ; 'ES

I t 1144 BERVFELD, HOMBt:RCER, AND RUSSFILLD Exposure to cigarette smoke reduced body weight of both inbred strains by about 10 q during the first 2 weeks of treatment. Subsequently, weights increased again, but somewhat more slowly in BIO 15.16 than BIO 87.20 animals. Because of the low final weight of cage-held BIO 15.16 hamsters, the weight difference between the controls and smoke-exposed hamsters was much leu in the BIO 15.16 line than in the BIO 87.20 line. Sham smoking produced body weights intermediate between those of the cage-held controls and the smoke-exposed animals in line B1O 15.16; it had no effect on body weight in line 810 87.20. It thus appears that the long-term response to smoke inhalation, i.e., the failure of the smoke- exposed animals to gain was due largely to direct effects of smoke lnhaiation rather than to nonipeciftc stress. Carson Monoxid* Content ot Blood Baseline levels of carboxvhec,oglohin rar.ged from about 0.1 to 0 5c'r. fmmediacelv afcer erposure of hamsters not previously inhalir.g smoke to a sequence of 20-puff cycles, carbor%her•.ogiobin lesels rose 20--40"'t. Within 4 hours after erposure, these high levets fell to 4-10c';. A second rx:~csure on :-e same da,v again raised the :e~el :o 30-30~':. After nvo w.- _, r 2 - ~ ~.,,. ~.•a.,.. ; --'r---- '"' . ...,... s.:.r • ~ ~ ~. .,...r. ~. .r!•, Tvcr-rrecat ).-Changea in a.erage bodv we ehq o( 9tO 1 S 15 hamsten during exposure o c:¢are•.ce smoie i-.d hose o! convols. ! .lC ar ID ..rf~eF ....~. ~. ..r.s Tr.r-r:CL0te 4.-Changey in a~erace oody w•-ghu or RIO a7':"1 ham.ten durins eepo.ure to c,gare<<e s~oke ind :hose of conVols. 20-cycle exposures to cigarette smoke wit'tin s- hours on the same day, and :hen 18-19 ^.ours rest during the night, the carboxyhemoglobin - turned to near pretreatment levels in all insta.n.ces. From these results on animats not included in t- chronie inhalation study, exposure of hamsters cigarette smoke for much shorter time periods, i e to 8-puff cycles twice a day as in the chronic inhalatio: experiment, may thus be assumed to cause no lasttn6 accumulation of carbon monoc:de in the blood. Thi was confirmed in 22 hamsters after 13-45 weeks in ;hr chronic inhalation study. Blood carboxyhemoglobi-n measured on a Monday morning before the tirs: exposure to smoke inhalation of the week ranged f:cr: 0.2 to 2.0hr, with a mean of 0.03 ;. Chan9H in Ssruro Triglyeeride Levets Serum triglyceride levels, determincd in :est ir,4 control animals at week 45 of the experi^nenr, rarz,-a from 145 to 222 mg; 100 ml, w,th sts:.dard deviat:ons from 28 to 97 mg; 100 ml. Because of these lar;e :a- dividual standard deviations, the d:fferences were fac from significant. Thus neither s^^.oke erposc:re -cr sham smoking produced changes in serum triglycer.des. Ylrus Profites The virus profiles were deter.^-::ned at 45 weeks :n 6 animals from each experimental group and in i young, untreated animals of eacn strain that ^orre- sponded in weight and age to the -es: ^a::.sters at t.-.e beginning of the experiment. The da-a c:esrly s^cwed that the'vtrus profile was not changed by smcke ex- posure, sham smoking, or prolonged cagL-ig under !he conditions of the experiment. InftuMel of Sntoke taposurl on Orqan Wetqhts Because of strain differences in body stze (table 1) and the weight changes produced by t.he e.xperi rnental procedures, absolute organ weights in these anlma:s were diff'icult to interpret. T4erefore. only relative organ weights were analyzed (tabl-s ':-4). %%'e:gnu were tabulated only for the animals caled, not for those dying spontaneously. This wu r!one because changes in the respiratory tract were )f •he greatest interest relative to smoke exposure. ind inctu.s on ~)i the lung weights of spontaneously :ead hamsten would have introduced a variable rnd unknown amount of agonal pulmonary congescon and p1l- monary edema into the averages. Relative weights of organs (liver, spleen, kidneys, adrenals, heart, or lungs) did not dtder ugnificanely between animals of the 2 inbred strair.s at any of :`:e ages studied, as found by comparison with data ror cage-held controls. Relative organ w,!tghts tended to increase with advancing age in e.xrr-^ental a:,d control groups. They also became more .arsable, as evidenced by higher standard deviattoru n c`e older animals. The age-related increase in relative w••q'+t Nu particularly marked in the kidneyy of ~tn ner-d cn J strains and was caused by amytoid deorn.t -~n R amyloidosia was, in fau, the leading ca;.x of .- - .C7w „K • T` . .f, -+. • . { _ TR 1f f 7 0 "-i .6~ ~ s.,J~ .+

'I'Art.c 2. -Hc)atwc .plun tacrphts of Aumaerc l.rllcd Jur Aulolupic .tudtca 0 - - - - -- - ; Lenglh of time on Inbred tar hu t l ha d r t(clalUvn aplccn wi• of banulun (n4g) Jtrlwltc.l nigndic.uce ut dlHercncea bctweeu rncanr u( 2 gruupa of sumc inhrcd 1ur0 ( l i . meo a m .ape oondrliooa ($10) tlmukc-eapuaed group tihrm-antokcJ control ---- --- --- r-v. uca) C.gc-hrld cmStrul group -- - (wk) (captl. group) gruup (cuutrul 1) (cuutrul 2) 1•:aptl. group E:ptl. group Cuutrul I vs. va cunlrul I va. cuntrul 2 control 2 40-60 IS. IB 78. 2(11t 56/0) 6U. 8(3. 49/S) 81 1( 1 t. 97/U) (2.04) A 61-76 170.0(9tl. 14/6) (--/2) S /0) n 76-90 . 124. 2(31. 51/24) 111i i31/17) 0(71 1lti li(45 LN/11) > >90 .. . . 141.6 (((5S. 78/9S) 135.0 (48. 24/IS) . . 117. 8(41. 23/12) 40- 60 57 . 20 64.0(16.43/6) 62. u(2a. 30/7) ri3. 7(IU UO/6) 2. 99 (1.80) ~ 61-75 83. 4 14.50/9) 92. B(32.28/13) 185. 1(107.91/11) - 2.79 2.94 76-90 j 88 1 19. 62/22) ti9 5(31 61/6) 63/17) - 108 5(25 2.82 i >90 .. . . 10a0 4ll 13/30) 11I4.9(36.85/12) . . 110.9(38.26/7) 0 A dl.ldd 4r ladlvlau.l L.dr u.yuts 4.l{). a.WUYY.d by Iu0 Y..n r.km.a Iu jawaW..r.:.utN.u W Wu.J. •ACwArt. v.Ir4b , .ua.ar.. uw dawtwur uamaw.t. z wlwu uu a..W. b1A•ea .. Y N t%a.aaty.r Lwl (PSm/) a&t 1 u t.o1ns F. Yr L.a t u t rmYcw u lO oJ 1 . / /r' 1:a w) _ - n da . . n.u. . ram . . u a .u" o . I --UMAc o.. au a w.. . . r w > llarmu.ot aat. r > y T.ut.tc 3- /1eGulve Aeurt u.eryAts of Aunutrrs till..d fur Aralulupic dudua 0 c ~ tuna uu lubrcd ItelrUvc hr.rl wt• of h.uralur. (i i JoBctcucra bcl wccu 6%) wcr~u u( 2 gruupn .d tawc tnLrrd luM ~ capenmental h.nuler hne (1-v.lur.)( 0 (810) dit n 6ruukcra(.recd gruup Jhruu-a~nukud cuntrul u-hrW 4-uutrul U. roup -- ` " x tu a cun (w`) (eaptl gruup) group (euntrul 1) g g (cuntrul 2) liayll. gruuy Eaptl. gruup (:uutrul 1 va. s va. Cuulrul I va. cuntrul 1 wntru 1'L S - --- --- --- ---- -' -- ----- - -- - - ~ 40-60 15.16 407. tl(2tl. tl7/U) - ---- - ----- 34tf. 0116 l16/S) - --- - 339. tl(31i. -J1/li) 4 07 3 S5 P 61-75 " 494.3 (67.89/7) -( /2) -( /0) 76-90 " 448 6( 12ti. S/24) 41K g(74. A4/12f) 417. U(7J. 86/10) >90 . 674. 0(132. 5/25) 439. 5(MY. 05/)tBI 442. tl( S.i. 83/ 12) 'L. 69 3 27 40-60 117 20 407.8 (57. 95/6) 330. 9(21. 65/7) 'J'lfi. S(2ti. 36/6) 4 67 .1. 1.1 61-75 . ^ 437.7 (26. 10/9) 415A(tl6. 43/14) :177.7(3K ti6/11) - 3. 'Js 76-90 " 477.4 (63.67/22) 41i8.2(74.44/6) 422 4(84.87/I7) - >00 .. 4617 (62. b5/30) 441. 7(b2. 0//12) 461. 7(2'l. Oy/6) •A WoWb W.t{ey 410" by lott/eldwl 4ads ~.Ia01rr Uo U. muW tallal b 1~. W.n .YU.~ ta pv. W..rw.u/NU .LU/u.N. lYlaWYf~14W Wor Y Y l~.MOOC.s 1.NI lPSOAq. a.Mr 1-takr bset 1s v.r.W..u t. Wtrat. aaWau.rw a 616 wWMYUw 4..1 oWr (Y_n Ir) wt~ ts r1•.u, .LCa.ou uul a.UauWly uawTot.nt da~ V.

I 1146 ~ > e -~ ~V ~ C - e_ O y U 1~ N v0 t: =v b ~ ^ yi - ~ lV S~ I , ~ s a J 1 ap ~ ~ F E N ~ NI n Y . 1 ] I i 2 ~ N ~-.. ~ ~-V a ti_ . ^I q -! ^ T y ~ 7 -- Q ! 1 ~ YI ~1J * I ~ . .e ~ ' " av y -.~.;. - '~ o~-+o i I =1a '° n > _ r P =I : ~ .. a . ( I I r I ~ ~ D \\ ?pOX `ia0in a f9hC '•1 av ~ a Ph--! '-o A 0 S na°o :un~\ BER.VFELD, HOS18t:RGER, A;(p RttiSSPtELD 1~ Yf T 3 ,+1-iy-' y - h . 3 3 -3 a O z ~ .,i 0 ~:.. ° 1 a i in the hamsters of this experiment. In the adrerlas, age-related weight increase was due variably to amyloidosis, hyperplasia of capsular cells, and formation of adrenal tumon. The prevalence of these changes precluded use of adrenal weight as a measure of stress. No weight changes in liver, spleen, kidneys, or adrenals could be attributed to smoking. In the BIO 87.20 hamsters during weeks 40-90 of the experiment, both smoke exposure and sham smoking reduced relative spleen weight significantly (table 2). This difference, possibly repttisenting a d'u- solution of lymphocytes due to stress, d'uappeared in very old hamst:rs when intrinsic diseeses of the spleen began to appear. No such efffect was demon- strated in the BIO 15.16 line. " Relative heart weights of hamsters exposed to smoke for 40-60 weeks were markedly and significanJy greater in both strains than those of cage-held or sham•smoked animals (table 3). In BIO 87,20 ham- sters, the hearts of smoke-exposed animals were sigoiflcantly heavier than those of cage-held controls after 60-75 weeiu, but the difference disappeared as the experiment progressed. In contra~t, there were many extremely large hears in hamsters of the BIO 15.16 line after 90 weeks of smoke exposure, but none in the control groups. This observation may represent an important strain-limited effect of smoke exposure. Lung Weights showed no systematic di.frerences be- tween experimental and control animals of the B[O 15.16 line until after 90 weeks, when the lungs of the smoke-exposed animals became signiflcantly heavier than those of the controls (table 4). In the BIO 87.20 hanuten, lung weights of smoke•exposed animals were consistently higher than those of the controls and by 90 weeks exceeded the weights of the cage-held con- trob by about 50?',. Because of the large variability of individual weights during the later part of the ex- periment, the statistical significance of these diEfer• ences is somewhat reduced. Strain-related differeocea in lung histology will be described below. Histopafholqte nndlnss 4rynr By far the most important findinp in this experi- ment occurred in the •larynges, approximately two- thirds of which were studied hucologically. The larynges of the last 49 surviving animals, including 34 BIO87.20's and IS BIO 15.16's, were transplanted into hamster cheek pouches for an experiment which will be reported separately. Among the smoke- exposed hasasten, only 2 of 48 larynges in the BIO 15.16 line (4%) and 3 of 45 from the BIO 87.20 line (7%) were regarded as histologically normal, as op- posed to 68-90%, of the larynges from the correspond- ing controls (tables 5, 6). Patholo ie chan es in th ke•ex were con to e s uar~ taluaiaar-}utscswn oP'T1s~~+ a cor an were c aa--(Ouow++ Ff}•p`rip arra was e e~^hyperplasuc thickening of the squamous epithelium with acanthosa and mild CTR HN ~~~'~,2-~~

CIOARET'rE SMOKE r?1HALATt^V L`1 tV17RED HAMSTERS 1147, nuclear dysplasia. The basal laver remained intact d fairly straight (figs. 1-3). This change was seen 40-, of the larynges of smoke-exposed BIO 15.16 nauuters and in 53?~ nf the BIO 87.20 line, but in none of the control animals (tables 5, 6). Basal «ll h~peractlotty was defined as r ra~t~ion of the squamous epithelium in .v 'lls Qrew down into th~dermts, o ten in a retecular Nuclesr dysplasi n nutotte activity tended to be more intense than in simple hvperplasia (fig. 4). This type of change was more fre uent in BIO 15.16~y) than in the BIO 87.20 ~ 13e1, anim U and it was absent in the controls. many cases of this tvpe of change, the basement membrane of epidermu appeared to be intact (figs. 5, 6). It sometimes became very irregular, with apparently isolated cell clumps in the dermis, a few suggesting lyrnphatic invasion (figs. 7-10). In advanced staqesl basal_cell hy ep ractivity,w ~aiffscuTt io ~if~erenaate fromsnrl)t i vas~tv~e c.a,,~t1pQm3 ~weve.r.,N,n Istan tCtastass~ wee found i_n apy ~timal. The a vanced.-,•I1dSS]L_ c asT`iifi"e3-as 'severe basaTcelT-Tiv peractivit ' In_ ia7iiei 5 and-6, ICoften t~d'jitinguuFiaTle~mml~r~ invasive cancer, occurred in 1950 of tht;scntike~.xaDscd aaliiia ~ _tn_ e IO.1S Ib`~itl'e, duq In only ~4t'o of L):cse of~t'Fie ._.BIO_.8_7.20.,.1it)e:__they._were abseat in the controls. Small squamous paprllomar similar to those occur- ring in the trachea and bronchi were found in the :ant)geal epithelium of 25 a of smok_-e_xposed harn- s:ers ar•,d in 13o of t.he BIO 13.16 animals. Occa- s:onailv, :here was downgrowLh of ceils at the base of papillomas kfigs. 11, 12). A few larynges of smoke-erposed• hamsters also showed chronic inflamrnation andr'or squamous meta- ?l,uia of mucous glands (fig. 13). Chronic inflamrna- tion in a few controls was associated with slight eptrheLal th)ckenin¢ but aever with true~h~er ~las,i~, _ pseudoe tt eFi-liomatous charge or ~a~i lol ma forma- t:on- smo ~ cage~uld' controls 3td-not dtder siqrilflcantly in this respect. L.nqs H:s:otogic examination of 90°0 of the lungs from B(O 13 16 hamsters and i11 of those from the BIO i: :0 strain revealed clear-cut strain differences and i:zr.tticant er{ecta of smoke exposure (tables 7, 8). r.::~,p;~q .~f macrophoqef: Pulmonary rnaerophages ut 'x;tn s;rs:ns of hamsters formed small clumps within •ne :itieolL This tendency was much greater in the BIO 87 :0 than in the BIO 15.16 strain, as seen from i:o:-par,son of the cage-held control groups (W7, . t ):. I t was accentuated by smoke exposure in ••- ar~ rs k924'', vs. 53%, :egpectively). In neither s•ri.n as :ne incidence of rna:roph:ge clumptng ,:{ec!ed ov sham smoking. :':ere •ere qualitative as weU as quantitative :.-Terences in macrophage clumptng among the sari- T...Le S.-Hiatotopie )Sndinqi in tA. !o.-i-)u iJ 810 15.18 hanteftrs• Number of aoim.l>t w(tD Leogtb of Treatment group time on e:peri- - mental condi- tiona (ak) Smoha-e:posed----- 40-60.......... ............ 61-75---------- .•-------.---. 78-90.......... .............. > 90----------- " -- Subtota)-..... Sham-smoked-..... 40-60.......... .............. 61-75.....---.. .............. 76-90.......... .............. > 90.....------- "---------.-- Subtotal-__... Cage-held......--- 40-60.......... ............ 61-75.......... .............. 76-90..-....... .............. > 90........... ••------------ SubtotaL..... Total per straln .............. Vum- Num- ber of ber of animals larqnges suto studied sler (iii) (iv) 7 17 31 29 S 7 16 20 $4 6 S 21 10 44 8 4 17 13 40 166 48(S7) 6 4 le 8 36(861 3 4 11 7 2S(831 l09(66) \ PDt Hyper- Toul pluu BCHt Micro- inva- • uvs eancer Pap1- lott• Chronic InHam- mauon Squ.- mous meta- laala •v) v,) (vu) ivtii) ;1a) (:) p :1) 4 1 3 1 4 9 1 3 7 8 l 8 l 2 2 4 1 7 3 3 2l4) :9(40) 19(40) 9(19) 7(15) 7(IS) 8(17) S 1 2 2 as 3 S 3 27;75) `r 0 ) 0(0) 0(0) 0(0) • 9(25) 0(0) 3 7 2 9 9 3 4 17cU) )(0) 0(0) 0(0) 0(0) 8(32) 0(0) 16(4Z) t9(17) 19(17) 9(8) 7(6) Z4(27) 3(7) •lforr 1s MwYY nPrN onmOt d vwr RodlM ElrtokdeaU7 ie pgramot d=me.n or .n/m&11 sutopfW: 100 U.I/llU): 69mr La Nrr"Aiw n0' ramt oumtan d AlMMad~ ~4~e..IJeor ~ s D~t d eamb1n d erpn rraCi.A . 100 i. Wougd O/f U.). tnmr ol ONrWIn D&MeL9..a ma7 6s-.A t00 o.esas tGn mer Oan tar mon tMs l eb.s..uos psr areaa. ~ Ve pad-ied4 N.aWA6 tD...l a.Y Rrp.r..owttf. C `°R ~#`N 0~' ~~ ~'-

I 1148 BER-VFEI-D, FOHHGRCER, AND Ri:SlFLEI.D T.at.e 8.-Kirlaloqic,i1diaq.f m fAe larjnqst of 810 87,20 Ao+vtesf• .....~.. -.. Number of animala wttb Len;th of Treatment group t,me on experi- mental condi- tion3 (wh) (i) u) 1um• ber of sntmsla autop- e~ed ;i1i) ?lum- ber of 'aryngea studied (iv) NPD v) Hyper. plaaia (vi) Total BCH (vii) lticro- inva- sive cancer (vti)) Papd- loma (iu) Chronic Iodsm- mation 'x) 8( m m. p(r '~ S moke-esposed..... 40-60....••-... "._.._._..._. 61-75........ . 75-40.......... .............. > 90........... 8 23 25 31 7 18 13 9 - 2 1 - 7 9 5 3 - 2 3 1 - - 1 1 - 3 4 4 - - 1 _ - 3 4 .............. Subtotal.... . , y7 45(S2( 3(7) 24(53) 8(13) 2(4) l1(°.4) .~2} , Sbam-imoked...... 40-60..... .....r 81-75.... ...... .............. 76-90..., ...-••-•-..... 5 90........ ) i :2 5 17 3 S 5 18 3 3 - - - - - -- - - - - - - - - - -- - I -- 2 - 1 ".........•.. 5ubt.ta' ... 14 30(88) 27(90) 0(0) 0(0) 0(0) 0(0) 3(10) Cagrheld......... 40-50.-.. . 51-7.i. . ...._.... ............ 15 y0.. ............. > 90..... •' ) ) i 5 :3 :4 . 5 11 !0 5 - - -- - - - - - -- - - - - - - - - 2 4 2 - - - - .............. 44 ;)'31) 31t79) 0(0) 0(0) 0(0) 0(0) 8(21) 9.) Tota) peretrun........... ') 114(54) 61(•rt) 24(21) 6(5) 2(2) 11(l0) 12(ll) 3(7) . •J." e9ataotwterqbl./. _- T.at.t 7--Kutoloqu Jindinqs in tAe (unqs of Bf0 16.(6 Amns4n• • Len th of tne \umber ' tumber Number of snimab ntb , g Treatment group on ecpenmental of of lung Clumped 8ron• In- Meta.- condit.ons .-k) anmma)a samples ?lPD maero- chiolar Ectopic dam. static autop- studied phaae" meta- bonee roation tumor tied plasu (i) u) w) riv) (v) (vi) (vu) %I u) tl Smoke-etpoeee......... 40-eo........ . 7 7 s 4 - 2 - - , . 81-73 .............. ................. 76-90.............. „ .......... > 9r)___........ .. 17 31 :9 17 28 24 a S 1 - 6 IB 5 13 2 2 - 4 2 9 4 3 1 2 ................. Subtotal... .... 34 76(90/ 21(:8) 40(53) 3(4) 17(22) 8(8) 6(3) Sbam.moked.......... 40-80 ......... .... B 6 4 - 1 - - - " ..-...... 6l••75.......... S S 1 - - 1 t ! . ............ 7E-90........ ................. > 90.._...... . . 21 10 20 9 lS - 1 3 -- - 4 - 3 1 - 2 ................. Subtotal... ... 42 40(95) 23(58) 0(0) 2(S) 8(20) 2(5) 3(9) Cage-held.....•.....-. 40-80....... ,...•............ 81-75......... "............... 76-90......... ................. > 90.......... . . i 4 17 13 6 5 - - 4 2 -- - 18 3 - - 8 S 2 - 1 - I - 2 1 2 - - I - - ".....,.....•..• Subtotal....... .. 40 34(85) 1S(N) s(e) 0(0) 8(1g) 1(3) 1(3) Tota) peratrsin ................. . :66 :50(30j 54(39) 42(28) 5(3) 31(2l) 9(8) 10(7) • se bamouw • aea t. wr a I . ,-r+w." ~.+' t f""a { CTR y i~ 4..A'.°.!' ~ d"~ ~ ti..~'F

GICARE2TE SHORE cNHALAt'OV LV t~aAED HANSTERS TAaca 8.-Riatologie )Sndi"qi i'L tAe (.'!1s iJ 3(0 97.:0 Aanuter.• Length of time Treatment group on e:penmental conditiona (wk) Nunber of inimaa tiith Number `umber of •~f : inq C1-3mped Bron- (n- \leta- snimals samples `PD mscro- ch olar c,:opic :'.am- s*Atuc autop- studied phages meta- boce rnstion tumor sied Plasia - (i) (ii) tiii) it•) V l sn) sv) '%'w) ix) ' a) Smoke-ezposed.--..-... a0-60 .............. 3 3 ............... 61-75.............- 23 23 ................. 76-90..-•----••---. 25 25 ............... >90............... 31 3l - 3 2 3 19 l - 1 23 18 6 - 30 22 4 2 " -. SubtotaL.-.-.-... 37 37(1001 4(3) 30(92) 46(33) l0(11) 2(2) Sham-smoked..-.--.... 40-60 .............. 7 7 ................. 61-75.............. 19 19 ...........:... 76-90.............. 6 6 ................. >90...... ........ 12 12 5 6 l - - l l 2(2) 7 4 4 2 - 3 3 2 - I 10 5 5 l 2 " ... Subtotal-.....-..- 44 44(100( 12(27) 20(45) 13(30) 11(23) 3(7) Cage-held ............. 40-60.............. 7 1 ................. 61-75--•----------- 13 13 ................. 78-90 .............. 20 20 .. ---...- >90 ............... 8 3 3 3 - 1 1 2 7 6 9 - 4 6 11 7 2 l 5 5 3 1 3(7.) 1 3 ................. Subtotal.......... +9 t41100110(21) 2l(44) 22(48) 20(42) 4(8) 4(9) Total per strain ...................... 179 :79(IJ01 :8(15) 121(68) 31(45) 41(23) 9(S) ). S, •S..wosaoua•Imt.uora. ous groups. In hematoxylin and eosin-stained slides of BIO 15.16 hamster lung from all groups and BIO 87.20 hamster lung from the control groups, the clumps consisted of comparatively few, sraall, loosely packed macrophages containing dark-brown to black -pigatent (fig. 14). In lungs of smoke-exposed BIO 87.20 hamsters, the dumps were larger, more abun- dant, andcomposed of very large cells containing pale gold pigment. These cells were frequently mtxed with polymorphonuclear leukocytes (fig. lS). Rare smoke- exposed BIO 87.20 hamsters not having such dumped macrophages in their lungs were usually those found dead of some interturrent diaease. - Macrophages of both strains, both isolated cells and those occurring in clumps, charaeterisdcally gave a positive Prussian-blue reaction for iron. Thia was intense in the small macrophages of all BIO 15.16 animals and B1O 87.20 controls. It was weak in the large macrophages of smoke-exposed BIO 87.20 ham. sters; these cells also appeared to contain yellowish iron-negative pigment. Oceaaional granules of black, iron-negative pigment consiatent with carbon were found in a few macrophages. BroncAiolar rnemplaria was defined as metaplasia of A at alveolar epithelium into columnar epithelium of the bronchiolar type (fig. 16). The strain difference was highly significant: Metaplasia occurred in nearly half of all BIO 87.20 hatnsters, but in less than 5% of the BIO 15.16 strain. In both strains, the appearance of broncbiolar meuplasia was age related but was not significantly affected by smoke expaaure. Ectoptc bon. Jonnalion: Pulmonary parenchytru in both straina contained small foci ot ectopie bone not associated with inAammation or other obvious disease processes. These foci occurred in 18',''0 of the cage- held BIO 15.16 and 420 of the B1O 87.20 animals. Incidence was not affected by experimental proce- dures in the BIO 15.16 hamsters. It was reduced by both smoking and sham smoking in the 87.20's. A few lungs in both strains showed acute or chronic pneumonitis or contained metastatic tumors of some sort, usually adrenal carcinoma, lymphotna, or :eu- kemic infiltrate. No differences in these parameters could be ascribed to strain or to smoke exposure. rracnta ana eroncAl Abnormalities of the air passages were rare in all groups. A few small patches of squamous metaplasia were seen in 5-10% of BIO 15.16 hamsters, in which the incidence was not significandy affected by smoke exposure. In the cage-held BIO 87.20 animals, squamous metaplasia was not seen in the trachea and in only i%, of the bronchus specimens. In sr^oae- exposed BIO 87.20 animals, these figures increased 'o 26 and 1l%a, respectively (figs. 17, 18). `o tumors of the air passa ea weie found in conaol animals of either stratn. ~hree benign squamous papolomas in the trachea were found in smoice- exposed BIO 15.16 hamsten. Four benign squamous paptilomas were found in smoke-exposed BIO 87 :0 hamsters, 2 occurring in the trachea and 2 n trse main bronchus (figs. 19, 20). Vasopha rynx Cross-sections were taken of the nasopharynx i'.d adjacent stsucttues of the head in all groups A L.,r f f"~'i f f f"f 0422261

I 1150 SEANFLLD, HOSltitrAGER, MyD At:SS9tLLD hamsters. Approximately half contained only normal adrsnats tissues. The remainder showed various pathologic The most frequent change obsetvr.,, in ci:: 3dr processes including gingivitis, dental caries, ocular of both strains was proliferatiort cf capsuiar inflammation with phthtsis bulbi, and thromboses of orbital veins. None of these phenomena could be This was reported previously in hamotu-• ::l i related to smoke exposure. mice, where it was called "A-8 ce)1 hy}-. „~asa' This proliferation, not affected by smokt e.pc ~On! 2_ ~rumon were found in the nasopharynx, was xen in { 1'~'e of BIO 15.16 and in '100s, ot bot oci eurr~in.g y~$I~~26~sitttais xnRosed to- 87•20 hamsters. Of the BIO 15.16 hau.~sters, i4~ SertoicE•6U-~5 weeks. One was an adenoid cystie tumo ` adrenal adenomas and 1;, carcinomas, as comE be ieve to h`ave o~riginaied?rom mucous glands with 135'a with adenomas and 5% with carcinom (fig`Zl~. Ttfi other tvas "a"fib~d32~com3_thaE2i,zd pro' the BIO 87.20 animals. Rarcly carcinomas t: duced nuastT6ui'"itaall puTiiionary metastases (figs, tasized to the lungs. The occurrence of adrenal ru 22, 23)--••--- was not related to smoke exposure. Of the anim: both strains, 5-10 ; exhibited adrenal amy!oidcs Heart The heart itself was not sectioned routinely in this DISCUSSION I However, it was included with the lungs The model system used 'by us differs frotn of 20-25%, of the animals. Of the cage-held control reported by Dontenwtll et al. l!) and Donten.nf I B1.O 15.16 hearts, 25 ; showed myocardial degenera- in several important respects in addition to our : tion as compared with 1'r, of the BIO 87.20 heartt. inbred hamsters. The composition of the 1R1 b This consisted of myolvsu with (in the 8IO 15.16 tucky reference cigarettes was different than tha animals only) an abundant intiltratron of lymphocytes. Dontenwill's cigarettes. i,4..pQnun;ziUl..iL%.d_v_.- Smoke exposure appeared to increase myocardial de- sters' bodies were enclosed in a tuoe and ::us se~- ¢eneration ;n both strains, especially in the BIO ---s`ff&Ted-. IN-6un e ere-nor.-emokeexpo.uM - 15.16 animals. However, the number of animals epti tnuous tn o~tS 7i11's't t3t`ttttt; "i~terrn: =^' examined waa too small to permit definitive conclu- ours. Concentration of inhaled smoke was about 11 s:ons (data not stacisncally slgnificant)• in the German model and 20cl, in ours, and scr - Mural thrombi were seen in approximately one- was administered by different dosage schedu!es. ftfth of the hearts of both strains. Incidence was not Nonetheless, both model systems permit ,he sa affected by the experimental procedures. major condusioas: Chronic exposure o cigare smoke produces severe hyperplasac and neopla: t.iver changes in the squamous epithelium of :he hams The most frequent abnormality in the livers of larynx and is associated with decreased body weig these hamsters was cystic hyperplasia of the bile ducts, In neither system did smoke esposurL increase pi in rare instances verging on cholangiocarcinoma. This liferative. changes outside the respiratory tract change occurred after the 60th week of the experiment increase the non-neoplastie degenerative chang and was more frcquent in the 8IO 87 20 (60%) than characteristic of aging hamsters. (Our observations ( in the 810 15.16 (I7?r'a) animals. Other abnormalities possible smoke-related changes in the heart 3re pre noted were not strain related and consisted of sporadic ently equivoeal; further investigation is desirable.) intiltration with amyloid and leukemic material, fatty Chronic smoke exposure signi6cantly reduced su metamorphosis, focal inflammation, and focal necrosis. wa1 time in the Dontenwill model; no such erTr One primary hemangiosarcoma of the liver was found was seen in ours. This difference may be attnbute in a csge-held BIO 15.16 control. None of these to the twofold higher carbon monoxide concentratic changes could be related to smoke exposure. inhaled by Dontenwill's haas:ers than by ours. I fact, previous observations of Dontenwill (10) showet Spie1n a sustained increase in blood carboxyhemoglobin in b.: A irnatel three-quarters the s model, whereas ours produced only transient increisa PProx Y -quatten o Pleeru ex- Both systems produced no change in blood triglycer amined in both strains were histologically normal• ides. In addition, we showed no change in vtru: Lesions in the remainder included hemangiomas, profile. lymphotnataus or leuketnic infiltrates, and amyloid. The po int of greatest practical importance to None of these conditions was related to strain or to emerge from otu work is the stnktng differencea arnong smoke exposure. various Lines of hamaters with respect to suxepubilitv to acute toxie effects of smoke and to hyperplasac Kidneys response of the larynx to smoke (3). Animals of the Some renal amyloidosis was seen in 86q', of the inbred BIO 15.16 line have both the higheet resistance BIO 15.16 and 69%, of the BIO dJ 10 hamsters. In to smoke or nicotine toxicity and :he qreatest laryngeal susceptibiliw-qualities greatly :.^.crea.sing the scnsi- r.etther strain was it affected by smoke inhalation. tivity of the model. Further studies w,th larger num- The only other renal pathology consisted of rare, bers of animals will be necessarv to iscertain :he minute foci of calcification in a few hamsten of significance of the laryngeal char:ges in different each strain. strains. .... _.r..- - C T R ~~~~ 0 42~~~

CIGARLTrL 3NOK6 NHALAttOV IN (NHRED HAseSTE0.S ! Ijl No tumon of the lung parenehv;tta aiter___ j SRoke A. Weirihouse s, eda.), vol 8. Sew York, Acade_n~: e`ct)OSure w t'T ontenwill_ or by Presf Inc., 15ti4, Pp 2i9-253 ~ ~~~ 11 BeR~RIO P, HOMRCRC[R F: High n cocine toleraace of uf.~` 'e did not co_ntti tl'lt~h K~, nt.ulcre35e_ __ Svnan golden hamsters. Toxtcol .ippl Pharmacol in a enom-a toad_lesions.obsetved_hv-Dontenw{It. In 22:324. 1972 bo__tl~-ir.odel systems, so-called "smoke cells" increased in the al.eoli after smoke exposure. The observation that %ellow iron•negative and also black particlee occur in the macrophages suggests that the particulate phase of smoke reached the alveolar wall to be taken up by the macrophages. Use of the 2 inbred lines in our experiment permitted the conclusion that these celJs are not directly related to hyperplastic changes in the respiratory tract. They were much more abun- dant in the BlO 87.20 hamsters, which had the lower ncidence of hyperplastic lesions. The tracheal and bronchial papillomas and the nasooharn•ngeal tumors (one with clear•cut metas- •ises) observed in our smoke-exposed hamsters resem- hle tumors induced by others with nitrosamines (11) :nd suggest the presence of this class of substances in ;.-e smoke from our reference cigarettes. The only -itrosamine so far detected in cigarette smoke con• :ensates, however, is dimethytnitrosamine (1?). =EFERENCES 1) Do.rza~itt tt', Cacvaaa H•J, HAARS H•P, et al fn- es::¢a:.ons on 'he et[eeu of chronie uqare::e•er oce n• h.vat:on in Syrnan ;olden huruten J Nad Cancer (^st it Iv73 `1 1V1vocR Ett Horrwwrv D: Expenmental tobacco car- anogenesu. /a .%dvances in Cancer Rexarch (Haddow HorrxA-4>r D, WyxotR EL: Chamber development and aerosol dispersion. In Inhalation Carcinogenes t Hanna \tG Jr, Nettesheim P, Gilbert JR, eds.). .iEC Symp Series No. 18, 1970, pp 173-189 AntLMso. WO: Production of sample c garettes for tobacco and health rexareh. Tobacco and Health Conf, Rept Yo. Z, 1970, 28 Cowstt~s BT, LawtxlR PP A tensitive method for the determination of carboxyhaemoglobin in a fu,ger prick sample of blood. Br J Ind %ted 22:139-13 3, 1965 KsssteR G, LeoeatR H: Fluoromevie measurement of -iglycendes. !+ Automation in Analvtical Chcmacry, Techntcon Symposia 1963 (Skegss LT, ed. ). New t'oric, \tediad, 1965, pp 341-345 HowavsectR F, Rvssrato A8: An inbred line of Svr an hamaterf with frequent spontaneow adrenal tumon. Caocer Res 30 305-308, 1970 Weot.LCr GW: Tumors of the adrenal cortex. !n C ba Foundation on Endrocrlnology. 12. Hormone Produc- tion in Endocrine Tumoun (tvolstenholme GE. O'Connor ' f, edt.l. Boston, Little Brown & Co., 1958, Pp 122-136 DoNrevwstt W: Experimental invesdsations on the erfeet of c;garette smoke inhalation on err.lil '.s5ora*ory aainalt /a Inhalatton Curenogenesu , Har.na MG Jr. Nettesham P, Gilbert JR, eds.l. .>EC Symp ienes \o. 18, 1970, pp 389-411 Hea.cto K~f: [nducuon of olfactorv -euroeo the!ui tumors in Svnan hamatess by diethylaisysa:rune. Cancer Il 11i-121, 1964 MeCORMIei A, Nteecotsov MJ, Bartes NfA, et al. Nitro- samtna in c garctte smoke condensate. Nature ;\ew Biol) 24i:237-238, 1973 's) ,6) (7) l B) '9) W 12) i .~- ~ 11 t Y i..N I R 1 1 f ? 042ef L..9 ..,.7t

tg 0 C.. • N . , ~ -_. . & F _ . _. /'. . ~_~--- . Ftotfas I.-Latyns of male 810 I S 16 harrato exposed to cigarette tmoke for 71 week.. Epttbelium thows m+aamal tluckeaa .You promr,aeot, regular layer of baaal cetla. Henucoeylin and eoua (H & E). X 800 Ftotns 2.-Hyyerplaaia in laryns of male BIO 97 ;0 !harroter exposed to smoks Wr 60 wee4a. Yar# moderitely thickened epiderr rtuai+aaUy aeypieal nuelei, and nuet baW iaver H & E. x E00 Ftotns 7.-Foe:al hyperplaaia in larvn: of Tale 810 15 16 bannter exposed to .moke for 73 weeks. .You .:.arkrdJy atyp cal auc and iatacn basal layv. H & E. x 900 FteCns C-Earlybasal cell hyperacav,rv n'arvne :f male 810 IS 16 haanter exposed to+moke foe ! 70 -'u .Y,u th:cY cptchel . with nanovw aone of wpernual keratLn,rauon Downqrowtlt of basal cella ~nth matouc acu"cy and wrr.e oudear arvpca; hu..e, normal oeteotauaf of cellb fauly weJl tr+unta~ned H & E. X 400 Ftouws 5.-Early basal cell hvDeracu•irv -n 'arvne of male BfO 97 20 hanuter exposed to imoke for 51 .eeki B.ual laver fo!ne- ^ ureBular but raL iotact. .You atvpical n~c.o ano aonorrrul keeauowuon tn deep ceU ae n fAC. H & E. x 300 Fiet.is 6.-`tarked ba.aW cell hyperacn-rv n'arvnt of male 810 13.16 huratrr exposed to smoke fcr 't2 weeta 6e.ere hvp~ keratoaif and aeanthaaa wuh nuclear aevpia. pan cularly tn the baul agim. .You re6ulal polanaat o0 of b.ual celh .n ae cdl oeata. H & E. X 250 1152 BERNIELD, HOMBL'4C?R, •ND RtaiF[T_C L' TR 1-If 7 04^'.-. 2 64

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Ftovas 10.-L,aryns of male 810 15 16 hamrter e:poted to tmoke for 91 weelo. (Juwra ofatypieal ep,:DeLal cub leep m ttro:= +uggest.lymphatic invauoo. H & E. X 400 Fzctru 11.-Lsr;e paptiloma nearly occlud,nS larvrnu of maJe BIO 87 20 0 hamryer e:po.ed to.moke for n -eeLS H t E. x 50 Ftccits 1?.-PspJloma of larynx from maJt 810 87 20 hamrtQ exposed to emote for 63 weela. Xwe a.e.,doep,anclwm,tous SrpQ- plaaia of covenag eptthdium. H & E. x;00 Ftctas I7.-Early tquamoui metaplat,a nmucui•secreung epit6elium of Larynx io male BIO 87 20 ~-a-wer eapo.eC ;o +rr.cac lor 86 weeka. H & E. x 800 1154 BLRIHRLD, HONalllc.tR, LND RCSStfE-:) • • - . - - . - •~ Vb- V Y\, .r. • • -VIwww_• - - -* .... CTR I IN V. 4i,., i! 66

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. FicU.a I8.-Pocts af Iquamoas meuplaos ia broocbial epithdium of male 810 15.16 bsmsrer expoaed to tmoke foe 92 weelu Neis ili8bt arocisujd cbrooic La6ammauoe aad dJated lympbauc. H Et E. X 400 Fictzs I9.-Papilloau ariains u rracbeal bJurcaum Ln male BIO 87.20 hamaer e:poaed to unoke for 78 wetlo. Geaioo parauli ob.uvcujd tracheal lumeu. H i E. x 50 Ficcu 20.-Lsrte papdlloma of broocMal onM in auk BIO 87 20 buoate e:po.ed to tmoke fa 82 weeh H S Ir X IS 1156 sRRNRLD, AOHIVAC><lt, AYD RUSSPSL.7 \..a' T1'`tr ! 1! 7 042268

M -)-) ~ ~. " 73. 2 F,ctxt :I.-.tdenoid c.+tac tumas of eawphar7ms in ma]e H1O I S:6 h.rdter eapa.ed to amoke far 70 weeis. No areae of icYauoa or dutant rutaatatn wae fouad. H & E. X 50 : ct tt 2?.-Su.or..atow tumor of naaopYaryn: in mak B1O I S i5 hurwtu arpo.ed to tmo~ke for 73 weeb. .~eu aliated rop rator~ ep~tCelium abo%e tu:-.or. H& E. TOOX -:ctat :7.-Pulr:onar% r-..etauaru from nuopharynReol tumor 0o.+o a rsura 22. H & L x 800 1157 OER.\fZLD, HONDL'RCER, A%D RUSSl1ZLD 4 I r 1~ t T1'` F t 1 0422 LJt F