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A k Strain Differences in the Response of Inbred Syrian Hamsters to Cigarette Smoke Inhaiation t' I I P. Bernfeld,' F. Homburger,4 and A. B. Russfield + SUMMARY-Mate hamsters, 102 from each of 2 inbred hamster lines. were exposed to cigarette smoke twice a day. S days/week for up to 100 weeks, in a modified Walton reversrsnwking machine. Sixty shartt-sntoked and 60 ca4e-heid controls were used for each strain. Smoke exposure for up to 100 we.ks had no eMect on mortality in either strain, but reduced body wiOht Carbo:yhetrw- globin levels increased markedly imntediataly after each smoke exposure but returned to baseline levels in less than 24 hours. Serum trialyceride levels and virus profiles of unoke-e.posed animals were unchanged. Chronic smoke exposure increased relative we.i9ht of the Iun's and heart in both strains, but to differsnt degrees. Over 90% of the smoke-exposed animals of both strains showed hyper- plastic or eeoplastic changes in the larynx. However, nricroinvasive cancer was nearly S times more frequent in I strain than in the other. In the inbred line more suscep- tible to laryngeal hyperplasia, 2 animals developed naso- pharyn9eai tumors, one of which was malignant. Smoke exposure induced rare benign squamous papillontas in;he air passages of both strains. The strain less susceptible te laryngeal hyperpiasla exhibited more pulmonary adenoma- tesls, but its incidence was not significantly affected by smoke exposure. Clumping of pulmonary macrophages was proof that smoke had reached the lungs; 1 strain was more •osceptible to this phenomenon than the other. In neither rain did smoke exposure affect the incidenee of tumors driaino outside the respiratory tract or the degenerative changes characleristlc of aging hamsters.-J Natl Cancer Inst 63: 1141-1157, 1974. A`IODEL SYSTEM for studying the effects of chronic inhalation of cigarette srnoke was developed by Dontenwill and co-workers using randombred 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 sasoker. One main pnnciple 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 contains not only substances of posaibie chronic toxicity but also compounds of known acute toxicity, such as nicotine, carbon monoxide, and others which sharply reduce the tolerated dose IeveL (2). Ham- sters are desirable test subjects for tobaoro-smoke inhalation studies, since they have a much greater resistance to toxic effects of nicotine than do rats or mice (3). We used inbred hamsters in order to im- prove reproducibiliry of resulu and to detect poasible strain-related diAetences in response to tobacco smoke. MATLRIALS AND METHODS ./nimolr.-BIO hamsters were purchased from Trenton E!cperimental Laboratory Animal Co., Bar Harbor, \faine. Five strains, the inbred BIO 2.4, BIO 87.20, BIO 82.62, and BIO 15.16 lines, and the RB (randombred) line, were used in preliminary acute toxicity studies (to be reported elsewhere). From these studies, the BIO 87.20 and BIO 15.16 strains were selected for chronic toxicity experi- ments. All animals weighed 108*4 g at the beginning of the chronic toxicity studies. Only males were used, since male hamsters are less belligerent than females and can therefore be housed several per cage. In the present study, ham- sters were housed in groups of 6 in 12X 14X6.S-inch polypropylene boxes covered with well-fitting, wire- screen lids. San-l-Cel, Deodor grade (Paxton Proc- essing Inc., Paxtou, Ill.), was used as bedding material. ' It was changed once a week, at which time the cages were washed in 180° F water and detergent in d commercial cage-washing machine. The animal room was continuously ventilated (32.2 exchanges/hr). Temperature was kept between 72° and 76° F. Fluorescent bulbs, the only light source, were auto- matically controlled to operate between 7:00 a.m. and 7:00 p.m. No other animals were kept in the room housing the hamsters, and no smoke was generated within this room. The animals received Wayne Mouse Breeder Blox and fresh tap water ad libitum. Twice a day, S days/week, hamsters were transferred to a nearby laboratory for exposure to smoke. ' Cennotron of «lmrttr nnoks-Modified Walton reverse-smoking etaeltina wen used (3) aad are described and illustrated elsewhere (4). In these machines, air is pushed through lighted cigarettes by application of positive prtssure 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-5tting, permanently attached, felt rings around their necks (yis of an inch thick, 2 inches outside diameter, and yC-y, of an inch inside I Reeei.ed April 24, 1974; aeeepted July !. 1974. s Supported by a rmtract trom tbe Couneil Iar Tobaeoo Re- searcN-U S. A. 'Ihe via.rs acpre..°d are tboe of the Couoril and do not necasarnly rebaca the opiaioos tor Tobaceo Rrsearcb. t Bio-Raearcb Consultano, Ine., 9 Coeamercial Ave., Cambridge, )•tas. 02141. 4 dio-Rexarcb t;.onsuitants, Inc.; Researeb Profsot of D~entssv~ry, bo~~on Uni~veSsi~R a~rcb~Procfs~or o~f latbol~ote ~11d Doaton Uoivertity School of >ttediane, Sa+to4 ' 1 Patholo~y Deparvneot, St. Vincmt Hoepiul, hiaa 01610. 3URNAL OT TtlZ NATIONAL CA.rCZt DifTlTlrtt, VOL. 33, NO. 4, OCTOfLi 197* »i.ur o • 1. . ,• 1141 , CTR ~'°'~~~ ~~~~~.r';~"

I I -.~r.~... 1142 - - . .._...•---_. DERNFELO, HO)tat:RCER, AND RVSSFCELD diameter, depending on the size of the hamster). The felt rings were reinforced on both sides by thin, ring- shaped aluminum plates of slightly smaller dimen- sions (1 % inches outside diameter and '!;, of an inch inside diameter). The heads of the animals were in- serted into the smoking machine by means of these collars, so that only the heads were in contact with the smoke. Because the 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 animals from grooming their eyes, these were washed with penicillin solution on a cotton swab daily 5 days.iweek. Smoke F enerated from 4 1 R 1 Kentucky reference cigarettes burning simultaneously was administered to the animals in 60-second puff cycles. Each cycle consisted of 2 seconds of smoke generation followed by 15 seconds of additional smoke exposure and 43 seconds of exposure to fresh air to minimize the toxic effects of carbon monoxide. During the 2-second smoke generation, 35 ml air was pushed through each of the 4 cigarettp, yielding a total volume of 140 mi smoke introduced into the 725-ml exposure chamber. A magnetic stirrer provided instant mixing. The smoke dilution factor was 725:140, or 5.2. The smoke concentration inhaled by the hamsters was therefore apprommately 19.2% of that leaving the mouth end of the cigarettes. In comparison, the average concentration of smoke in the lungs of a human smoker is about 0.8% (average puff volume, 35 ml; human functional residual capacity, 3.5 liters; human tidal volume, 0.7 liters). The smoke, seen through the transparent chamber, took less than I second to traverse the 6 inches between the mouth end of the cigarettes and the hamstera' heads, which assured that the hamster inhaled smoke no less fresh than that reaching the human smoker. Ckronec tnhdeeioa trudy-A preliminary study was performed to determine a dose schedule at which mortality would be negligible during the initial months of the experiment. An exposure of approxi- mately 5% of the median lethal dose, previously determined under identiul e?cpoture conditions, constituted the upper safe limit. This varied with the strain of hamster. For obvious reasons, exposure time suitable for the most sensitive strain was used for all animals in the long-term study. Accordingly, each hatnster was exposed twice a day, 5 days/week, to 8 consecutive puff cycles from the IRI Kentucky reference cigarettes. Puffs 1-8 from the cigarettes were used; the resulting butt length was slightly longer than 30 mm. The period of exposure was 45-100 weeks. Two simultaneous control groups were maintained for each inbred strain. One group consisted of cage- held animals of comparable age and initial weight that were maintained simultaneously in the same animal room and handled like all other hamsters with _. .,-_,,.-. .. regard to weekly weighing, permanent wearina felt-aluminum collars, etc.; however, they were erposed to the smoking machines. The second con group consisted of comparable hamsters exposec "sham smoking" on the machines; i.e., the machh were operated under standard conditions, except : no cigarettes were inserted into them. For each of 2 lines, 102 hamsters were exposed to smoke, 60 %% exposed to sham smoking, and 60 were cage-h. controls. , Since it was impossible to obtain all 222 haras• re4uired for each tnbred line at the same ti: animals of the proper initial age .and weight o. introduced into the experiment in Lots of 6, stagge over a period of 37 weeks. This procedure is poss: only when homozygous animals of an inbred 1 are used, and provided that all facton such as weig sex, and age are uniform. As far as possible, I loc 6 of each of the controls was started at the sarne ti: as 2 lots (12 animals) of smoke-exposed hamsters. ObteroationJ dnrins inho/ation rtrrdy.-Indi%idual bo weights were recorded weekly throughout the r pertiment. Carboxyhemoglobin levels were me: ured by a cticromethod (6) after various periods smoke exposure. Blood was obtitined by heart punctL under light Nembutal anesthesia. To avoid possii deleterious effects of heart puncture on hamsters in t chronic inhalation study, carbo~cyhemoglobin leve were determined in 6 male hamsters (100-120 body weight) of each inbred strain that were expos• to smoke for 1-3 weeks but were not included in t: chronic inhalation study. Carboryhemoglobin leve were also determined in a few randomly selecte hamsters from the chronic study after 13-45 wee of exposure. Taminodron of ttudr.-Anitnals were killed whc they appeared moribund; as judged by consiste: weight loss or the appearance of edema. In additio: some hamstrrs of all groups were killed at 45, 6• 75, and 90 weeks. Survival time calculations we• corrected for the hamsters killed when not moribur. Complete autopsies were performed. The enti tYSpiratory tract and any other organs or tissu grossly appearing abnormal were studied histolov cally. Lungs were inflated and itxed in Tellyesniczk. fluid in an apparatus devised by Dr. Sadamu Ist: kawa (personal communication) that permits equ: ibration between internal and external pulmona prasure. The larynx was removed and &xed prior connection of the trachea to the canoula feedir the fixation fluid into the lung under a presaure 40 mm Hg. At the completion of fixation, the pressu. was equalized at 10 mm Hg. Organ weights of some hatnsten wcre determinec Serum triglycerides [by the fluorometric techniqu (7)] and virus profiles (by courtesy of Dr. Robert _ I IRI lCentueky reference cigarenes are produced by, ac were obtained trom, the Univenity of Kentucky Tobacco r' Health Resrareh 1nstitute, Kenrucl•y Resc.vch Fouridauo' Luington, Ky. Compoutioo of the rrference cigarctte I R I• 12% moisture was as follo.n (5): flue-cured Iamina. 401' Rue-cured ritem, 14.2%; Burley lamina, 24 9~,; Turki,h (..no teaf), 11 6%. ; Maryland lamina, 1.1 :, glqcerw. 2 9^,; in`c wtrac, 5.3%. , .. _ f.,~. CTR MN 04*22 558

.....a /1 I I CICARETtL S0lOY.E INHALATiON IN iNaRtD HA%ISTERS Huebner, National Institutes of Health) were studicd 'n some animals o(each group. .ESULTS Mortality 'Mortality was .•cry low in all groups until about the 60th %.•cck of the cxperiment (text-figs. I, 2), at which time the animals' chronologic ages were ap- proximately 73 weeks. Thereafter, mortality increased rapidly in all groups, reaching 100% at about 100 weeks. Neither emolce exposure nor sham smoking had a significant effect on mortality. There was no difference in survival between the 2 strains. Chan4.s in Body Weight (Tabla 1; Text-Figs. 3, 4) In both hamster strains, individual body weights were affected by smoke inhalation. The mere experi- mental manipulations, such as sham-smoking con- ditions and;or s!res,s, also changed the body weight. Initial weight in both groups a%•eraged 108 g. The BIO 87.20 cage-held controls attained a final average weight of 144.3 g, whereas the BIO 15.16 cage-held controls attained only 125.0 g. Tizt-nacsi 1.-Mortaliry of BIO 13.16 barrnmrs e:po.ed to tmoke bom IRI etiuetua and of unespoaed eooteo4. Tsxr-nceas 2.-`tortalitT of BIO 87.20 hamstes e:po.ed to smoke from 1 RI eipretto and d unecpoeed cootrols. - 1143 i+ I 2 c ~3 ' I~ 3 ' ~ I I ll T- i 6. i =i_; :.. I ~ ~ I '11 IT I a V m~.hlV CpN.-~a >6aoa ogo-• v ese•r.- ga"g t~-!Y W ~=-- S,~.db Of~Od .of, .or" , (._. i f•.. MN 0"°!'255-..e

I I /1 -.-ddliftb.r: 1144 ZERYEELD, KOltaURCER, Exposure to cigarette smoke reduced body weight of both inbred strains by about 10 g 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 ofcage-held BIO 15.16 hamsters, the weight difference between the controls and smoke-exposed hamsters was much less in the BIO 15.16 line than in the BIO 87.20 line. Sham smoking produced body weight, intermediate between those of the cage-held controls and the smoke-exposed animals in line BIO 15.16; it had no effect on body weight in line BIO 87.20. It thus appears that the long-term response to smoke inhalation, i.e., the failure of the smoke- exposed animals to gain weight, was due largely to direct effects of smoke inhalation rather than to nonspecific stress. Carbon Monoxide Content of Blood Baseline levels of carboxyhemoglobin ranged from about 0.1 to 0.5%. Immediately after exposure of hamsters not previously inhaling smoke to a sequence of 20-puff cycles, carbon•herr.oglobin levels rose 20-40%. Within 4 hours after exposure, these high levels fell to 4-10%'0. A second exposure on the same ddy again raised the level to 30-36%. After two : , . ; :.. c.s..rR..a.~ . ,_'. •4 A i4 .rffea I ~ f ~ f 10 IS f~ f7 .O •f s0 q TcxT.rnecai 3.-Chsnges in average body weirho of B(O .13 16 harruters during exposure to cisarette smoke and those of contro4. «.a.......e.. , : . . .r.etr a .rt•r 'ej Texr.rret*e 4.-Changes in a.eraee body weighn of BIO bT.2~1 hamsten during expoaure to crsareue smoke and rho+e of controts. AND RUSSFCEtA 20-cycle exposures to cigarette smoke within 4-5 hours on the same day, and then 18-19 hours of rest during the night, the carboxyhemoglobin re- turned to near pretreatment levels in a1J instances. From these results on animals not included in the chronic inhalation study, exposure of hamsters to cigarette smoke for much shorter time periods, i.e., to 8-puff cycles twice a day as in the chronic inhalation experiment, may thus be assumed to cause no lasting accumulation of carbon monoxide in the blood. This was confirmed in 22 hamsters after 13-45 weeks in the chronic inhalation study. Blood carboxyhemoglobin measured on a Monday morning before the 6rst exposure to smoke inhalation of the weeli ranged from 0.2to2.0%,,withatneanof0.6370. Chanaes in Serum Triatyeortd. Levels Serum triglyceride levels, determined in test and control animals at week 45 of the experiment, ranged from 145 to 222 mg/100 ml, with standard deviations from 28 to 97 mg/100 ml. Because of these large in- dividual standard deviations, the differences were far from significant. Thus neither smoke exposure nor sham smoking produced changes in serum triglycerides. Virus Profiles The virus profiles were determined at 45 weeks in 6 animals from each experimental group and in 6 young, untreated animals of each strain that corre- sponded in weight and age to the test hamsters at the beginning of the experiment. The data clearly showed that the virus profile was not changed by smoke ex- posure, sham smoking, or prolonged caging under the conditions of the experiment. Influence ef Smeke Espesu.e en Organ Wet9hts Because of strain differences in body size (table 1) and the weight changes produced by the experimental procedures, abaoluu organ weights in these animals were difficult to interpret. Therefore, only relative organ weights were analyzed (tables 2-4). Weights were tabulated only for the anianals killed, not for those dying spontaneously. This was done because changes in the respiratory tract were of the greatest interest relative to smoke exposure, and inclusion of . the lung weights of spontaneously dead hamsters would have introduced a variable and unknown amount of agonal pulmonary congestion and pul- monary cdeasa into the averaga. Relative weights of organs (tiver, spleen, kidneys, adrenab, heart, or lungs) did not differ significantly between anisnals of the 2 inbred strains at any of the ages studied, as found by comparison with data for cage-held controls. Relative organ weights tended to increase with advancing age in experimental and control groups. They also became more variable, as evidenced by higher standard deviations in the older animab. The age-related increase in relative weight was particularly marked in the kidneys of both inbred strains and was caused by amyloid deposition. Renal amyloidoais was, in fact, the leading cause of death I

0 Ta.Le 2.- Helatw spleen ruyAts of Aam.tars k if4d Jor AaUotooic studiu 0 LenBth of time on t i ) Inbred t Il h • I(elative spleen wl of hanutcr. (mg) Statistical siKniBcancc of diQercnces bctweea means of 2 8roups of same inbred line men •sptr a oonditloo. er au» oe (H10) 8mok"spo.ed gro up 6hamamokrd control Cage-held control group (t-valucs) t (wk) (eapU. group) group (cootrut 1) (control 2) )'sspU. Sroup vs. control I EspU. group vs. control 2 Control I vs. control 2 40-60 !S !b 78.2(/8. 50/6) 69.8(3. 49/5) 81.2(11.97/6) (2 04) e1-76 u 170. 0 98. 14/6) /0) . I 0 70-0 0 N 2 124 51/24 31 110 0(71 AI/t7) I I6 9A/l I) 64s .y ~ >90 . )41.6 . SS. 78/25) . . 135.0(48. 24/8) . . 117.8(41.23/12) > 40-60 87.20 64.0(16.43/6) . 62.0(24.:10/7) 8;f.7(18.00/6) 2.99 . (1. 80) ^ 61-75 . 83. 4S14. 50/9) 92.8 (32. 28/13) 185. 1(107. 94/11) 2.79 2 94 76-9 0 .. 88 1(10. 62/22) 80. 5(31. 61 /6) 1 J8. 5(25. 63/ 17) 2 82 . y ~ >90 . N 108. 0(((44. 13/30) 118. 9(36. 85/12) 110. 9(38. 26/7) . 0 9 •tae.Nrtb..latw. 4a.t41s by ".w..1 s.U wblit• (u.1...n+vWi sr tm. tA..n ..iu.a I e r....w..a.oM...r ww6. /wrwwtsrt+aarr r ts.rt+w.rwr (rS.w. wrrt•wh.rra ta w•r.r... ut.dK." .t:re...e.a.x...e..... r..~ wr (r5e.oy. w... rt ..w.dr..n.eis.n.e...t rtw.uunr uarw.t Itr.rtw aw- > ~ 0 Taa.e 3.-Retati.. Aeart uKiyAts of Aasuttra lrllsd for Aiato(ooie etudi.r z ~ Length of Statiulical .iRnificance of diQerencey betwcen ~  time on Inbred RdaUvo heart wt• of hamstcr. (m8) tncans of 2 8roups of same inbred line (t i O espe nmental hnauter line k d e-held control rou oked control Ca Sh -s -va ues) f eondiUoo. (awk) e-e: (HIO) Bmo (eap po.c grou tl. group) g p g am m p group (control 1) (control 2) EspU• Sroup Esptl. group ('.wUrul I vs. x > v.. control I v.. control 2 contrul 2 Z NJ ~ 40-60 16 407.8 ( 1b 28. 87/6) J48. 6(16. 00/S) 339.8(36. 91/6) 4.07 .L JJ ~ 61-75 . .. 494.3 ( 67.89/7) -(--/1) -(-/0) M O .. 448 6( 1 5/24 ) 28 418. 8( 74. 84/ 18 ) 417. 0(79. 85/ t0) k 76-90 >90 . .. 674. 0( . 132. 5/25) 439. 5( 82. 05/8) 442.8(55.83/12) 2.69 3.27 w 60 40 20 407. 8 ( 97 57. 95/6) 330. 9(21. 65/7) 326. 5(26. 'S6/6 ) 4.67 3.13 - . .. 437 7 10/9) 26 415.4(80.43/14) 377. 7(3A. 86/ 11) - 3.95 61-75 . .. 477 4 . 67/21) 63 44/6) 422. 4 (84. 87/ 17) 2( 74 468 .wr3. z 76-90 >90 . . 46J.7 . 62.55/30) . . 441.7(82.04/12) 46i. 7(22. 09/6) •wtratrlW rrWt., r./W by Ld1.N.J 1dr ~V tl. a). salYp" by ta0. Yra av.a t. p...qrwc roM...t W ak IttuUWSldptffW.rr.t 17r w4A~ tswl (rSlM)...kr b"'.lw I. set to pr..Y..a to t.dkNw d[•IAera N M% ...OOrer 1...I.Yr (r<_..04. wl.w a, t-tYw Yg1•..s dda.ecs mt M.nfUe.iy dsrtae.a. p..rttbt.a dN.

/1 1146  ~ i • I o.. s e~ 0 ~ E~ .C L (Q i E Y lLRNILLD, HOlt1UROIR, AND RVSSTZLLD 2 I I I I I IN N N III~ ~~ N Vi ~i"i Viri PI v ":III .~ ~ ~....~.- i...N0 CN-p ~~V C~rn :.. .en o ~ e`vo oa;:~e4 fr ~ N C1 ~1 p N - -~ P--- O N ^-.. rz--- Sn9e d_,oi\ o:o/\ .on .on I I I i s a I ' I in the hamsters of this experiment. In the adreaals, age-related weight increase was due variably to arnyloidosis, hyperplasia of capsular cells, and formation of adrenal tumors. 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 hamuen during weeb 40-90 of the experiment, both smoke exposure and kham smoking reduced relative spleen weight signi£caady (table 2). This difference, possibly representing a dis- rolution of lymphocytes due to stress, disappeared in very old hatnstera when intriruic diseases of the spleen began to appear. No such effect was demon- strated in the BIO 15.16 line. Relative heart weights of hamsters exposed to smoke for 40-60 weeks were markedly and significantly 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 aaimals were significantly heavier than those of cage-held controls after 60-75 weeks, but the difference disappeared as the experiment progressed. In contmst, there were many extremely large hearts 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-liraited effect of smoke exposure. Lung weights showed no systemadc di.qerences be- tween experimental and control animal+ of the BIO 15.16 line until after 90 weeks, when the lungs of the smoke-exposed animals became significantly heavier than those of the controls (table 4). In the BIO 87.20 hamsters, lung weights of smoke-exposed animals were consistently higher than those of dte controls and by 90 weeks exceeded the weights of the cage-held con- trols by about S0%,. Because of the large variability of individual weights during the later part of the ex- periment, the statistical significance of these differ- ences is somewhat reduced. Strain-related diBerences in lung histology will be described below. Mlstepathrielia r1edingn LaryrW By far the most important findings in this experi- ment occurred in the larynges, approximately two- thirds of which were studied histologically. The larynges of the last 49 surviving animals, including 34 BIO 87.20's and 15 BIO 15.16's, were transplanted into hattater cheek pouches for an experiment which will be reported separately. Among the smoke- ezpaed hamstess, 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). Pathologie changes in the smoke-exposed hatasten were all conPtned to the squamous{olurnaar juncuon of the vocal cords and were dassified as follows: Xype+p/aria was defuted as hyperplastic thickening of the squamous epithelium with ac.anthosi+ and ould , _. CTR HN 042562

nuclear dysplasia. The basal layer remained intact d fairly straight (figs. 1-3). This change was seen -i0cyc of the larynges of smoke-exposed BIO 15.16 namsters and in 53% of the BIO 87.20 line, but in none of the control animals (tables 5, 6). Baial cell hjpoactioiry was defined as proliferation of the squamous epithelium in which cells grew down into the dermis, often in a reticular pattern. Nuclear dysplasia and mitotic activiry tended to be more intense than in simple hyperplasia (fig. 4). This type of change was more f uent in BIO 15.16 (40%) than in the BIO 87.2013%,) animals, and it was absent in the controls. In many cases of this type of change, the basement membrane of epidermis appeared to be intact (figs. 5, 6). It sometimes became very irregular, with apparently isolated cell clumps in the dermis, a few suggesting lymphatic invasion (figs. 7-10). In advanced stages, basal cell hyperactivity was difficult to differentiate from early invasive carcinoma; however, no distant metastases were found in any animal. The advanced stages classified as "severe basal cell hyperactivity" in tables 5 and 6, and often indistinguishable from micro- invasive cancer, occurred in 19% of the smoke-exposed animals in the BIO 15.161ine, but in only 4% of those of the BIO 87.20 line; they were absent in the controls. Small squamous popillomar similar to those occur- ring in the trachea and bronchi were found in the CIGARLTTL SNOICL INHAiATION IV INDRLD HAYSTLRS I - 1147 Lengtb of IVum- Vum. Treatment group time on esperi- ber of ber of NPDt Hyper- Total Micro- Papil- Chronic SQua- mentsl coodi- aaimaL larynses plau. BCH1 icva- loma iaDam- mous tions (wk) autop- atudied tive mation metr ued caacer plaaie (i) (ii) (iii) (iv) (v) (vi) (vii) (viii) (i:) (1) (si) Tsats S.-Ai.tolopie J4ndineo in tM lorynpu of B10 18.18 Aumu(e+i• Number of animals wit.b Smok~-espoaed..... 40-60.......... 7 S - 4 1 - - - - -........... d1-7S.......... 17 7 1 3 3 - 1 2 2 "............ 78-90.......... 31 16 1 4 7 1 2 4 3 .............. >90....-..-... 29 20 - 8 8 8 4 1 3 .............. Subtotal...... 84 48(S7I 2(4) 19(40) 19(40) 9(19) 7(13) 7(1S) 8(17) I Sbamimoked_..... 40-60.......... 6 6 5 .............. a1-7S...----... s 4 1 " 76-90.......... 21 18 lb .............. >90............ 10 8 8 ............ Sabtot;L.... 42 36(88) 27(75) 0(0) 0(0) 0(0) 0(0) 9(2S) 0(0) Ca;e-beld......... 40-60 ......... 6 .............. 81-75: --•----- 4 ,.----------.. 76-90.------... 17 .............. >90 ........... 13 " .... 8ubtotaL..... Tota1 pn atrain .............. a 40 166 3 4 11 7 laryngeal epithelium of 25 ; of smoke-emposed ham- sten and in 13% of the BIO 15.16 animals. Occa- sionally, there was downgrowth of cells at the base of papillomas (figs. 11, 12). A few larynges of smoke-exposed hamsters also showed chronic inflammation and/or squamous meta- plasia of mucous glands (fig. 13). Chronic in9amma- tion in a fet•• controls was associated with slight epithelial thickening but never with true hyperplasia, pseudoepitheliomatous change, or papilloma fotraa- tion. Sham-smoked and cage-held controls did not difier significantly in this respect. Cwps Histologic exatniaation of 90% of the lungs from BIO 13.16 hamsters and all of those from the BIO 87.20 strain revealed clear-cut strain differences and significant effects of smoke exposure (tables 7, 8). Chrmpins oJmanophalu: Pulmonary tnacrophages in both strains of hamsters formed small clumps within the alveoli. This tendency was much greater in the BIO 87.20 than in the BIO 15.16 strain, as seen from a comparison of the cage-held control groups (44% vs. 67,). It was accentuated by smoke exposure in both strains (92% vs. 53ao, respectively). In neither strain was the incidence of macrophage dumping affected by sham smoking. There were qualitative as well as quantitative differences in macrophage clumping among the vari- 3 2 9 3 /1 1 2 3 a 2 9 4 23163) 17(48) 0(0) 0(0) 0(0) 0(0) 8(32) 0(0) 104(66J 46(42) 19(17) 19(17) 9(8) 7(6) 24(22) 8(7) •14ssr.r b MsAm nR•+wY iumlv d ofiri ttoCLd platoiepdQr t. p~n1 d eombn d oalmoL t•otopli.d: ICO C.Unu): 1111111 r ta PIwtlor nP n..nt eumt.in d l4weiape ea.r.aUeoo ta preat d anmbef d arpm teodYd: loo (T t.6tvaaL D)lUT): tomr N ORti u pafinV>~rt at60 Unwd 1m banam u»rm may Gon ts..e stln thaa 1 obm .~tMe pw wSAs, tYO PLLbw+W mKda 1a&N1 tulJ lrpftepap. 1-.+' TR !NI) i 042563

j --~-. ... . 1148 lttlNR1.D, NOMICRCM AND RVSStZLLD TAxct 0-Riatolopic.findiepe ir t11e (eaynPa of 810 67.10 Aonutere• Number of animals with Lenitb of Num- vum- ~~ Treatment group time on e:pen- ber of ber of NPD Hyper- Total -Micro- PapiJ- Chronie• Sque mental condi- animals larynjes pWi& BCH (nva- loma in8am- mot: tioas (wk) autop- studied live matlon meta sied cancer pLL3j: ti) (ii) (iii) (iv) (v) (v!) (vii) (viii) (iX) (:) (xi) Smoke-esposed..... 40-60.......... 8 7 7 " .....-.--- ' 61-73.......... 23 16 2 9 s 3 •~_. _......... 76-90.......... 23 13 1 5 3 1 4 3 ............. >90........... 31 9 3 1 1 4 4 .............. Subtotal-..... 87 45(S41 3(7) 24(53) 6(l3) 4(4) 11(24) 1(2) 7(16 Sbam-emoked...... 40-60.......... 7 S S ............ .. .............. 61-75.......-.. 76-90.......... 19 8 13 13 1 ............ > 90........... 12 S 3 s 1 .............. Subtotal...... 44 30(68J 27(90) 0(0) 0(0) 0(0) 0(0) 3(10) 1(3) Cale-heid......... 40-60.......... 7 3 S ..-......-...- 61-75.......... 13 1 11 ? .. ............ 76-90.......... 20 14 10 4 .. ............ > 90........... 8 7 2 ............... Subtottl...... 48 39(81( r 31(79) .~..~ 0(0) r 0(0) 0(0) 0(0) 8(2l) 0(0) Total per straia .............. 179 114(641 61(34) 24(2!) 6( 5) 2(Z) 11(10) 12(11) 8(7) •Bw taeioow lor IaOL ~. T.u at T-Rtiuolep+e jindiapi in tM lwya of BIO 16.16 Amrut.*i` Length of time Number Number Treatment group on experimental of of lung conditiona (Mk) anitnals eatnplM auto~''' etudiod eilO (il (i!) (iii) (1V) Number of anlmals wfth ; Clumped Bron- Io- yleta- NPD macro- chiolar Ectopic Eam- etatic pbatn met.- boue mation tumor plasi. (v) (vi) (vii) (viii) (is) (:) -• - Sm oBe-erpo.ed_..-..... 40-60...,.......... 7 7 2 4 - 2 " ............... 81-7b.............. 17 17 8 5 1 2 - 3 •.......... .- 76-90.............. 31 29 6 18 4 2 1 ................. > 90............... 29 24 S 13 2 9 4 2 '.... 9ubtotal......... 84 76(90j 41(28) 40(53) 3(4) 17(22) 6(8) 6(8) Sbam4m ok.d....._.... 40-d0 .............. 6 6 4 - 1 -- - -- ".....-.-. 61-TS .............. 5 5 1 - 1 1 1 .............. 76-90.............. 21 20 16 - 1 4 - - ................. > 90............... 10 9 3 - - 3 1 2 ................. Subtotal......... 42 40(9SJ 23(S8) 0(0) 2(5) 8(20) 2(S) 3(8) Ca=e-held ............. 40-60......... .--.. 6 6 5 - - ! - - ••............... 61-75 .............. 4 4 2 - - 1 - 1 .. - ............. 76-90.............. 17 16 a - - 1 .. ::............. >90............... 13 8 a 2 - 2 - - "............... SubtotaL.......... 40 34(85) 15(44) 2(8) 0(0) 6(18) 1(3) l(3) Total per etrsin ...................... 166 1S0(90j S9(39) 44(18) S(3) 31(21) 9(6) 10(7) •Sw betma' erd t. hs1.1. . .......~...-- CTR 11N 0 42 ~~6D 4

a 8 19 23 30 LengtD ot time Number Nuntber \umber of animals nith Treatment group on esperimental of of lung Clumped Bron- In. Nfeta- conditioas (R•k) aaimaL samples \ PD macro- cbiolsr Ectopic Aam- static .uto studied phages meta- bone mation tumor sit~ plasie (i) (li) (iii) (iv) (v) (vi) (vii) (.iu) (ix) (t) I 7 19 6 12 CtOARETTi SHORL VIHALATfOY LV LINDRED HAlISTi1tS TADLt 8-Aittofopie )lndinpi in (As lunps of B!0 97.90 Aarn+ters• Sm oketsposed___...... 40-60 .............. ................. 61-75.............. " .. 76-90 .............. ..:---------_•-_- >90............... ................. SubtotaL......... Shamem oked...-...... 40-60 .............. -------------- 61-75.............. .. -__•___••_... 76-90.............. ................. > 90...... _....... 8 8 - 23 23 3 23 23 1 31 31 - 87 87(100) 4(5) 7 19 6 12 6 6 " -__ Subtotal.......... 44 44(1001 12(27) Cage-beld------------- 40-60.............. ................. 61-75 .............. ................. 76-90 .............. ................. 1 90.._......-_---- 7 7 13 13 20 20 8 8 '• ___ SubtotaL......... 48 /1 2 4 22 6 4 2 80(92) 46(53) 10(11) 2(2) 7 3 10 1 4 3 S 4 2 5 2 1 20(45) 13(30) 11(25) 3(7) 1149 1 1 2(2) 1 2 3(7) 3 3 1 1 - 2 7 6 9 - 1 4 6 11 7 2 - 1 3 S 3 1 3 48(100110(21) 21(") 22(46) 20(42) 4(8) 4(8) - Total perstrain ...................... 179 179(1001 26(15) 121(68) 81(45) 41(23) 9(5) 9(5) •Sn IsomsW • Md t. rbY aL 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, small, loosely packed taaaophages containing dark-brown to black pigment (fig. 14). In lungs of smoke-exposed BIO 87.20 hatruters, the clumps were larger, more abun- danc, and composed of very Large cells containing pale gold pigment. These cells were frequeatly mixed with polymorphonuciear leukocytes (fig. 15). Rare smoke- exposed BIO 87.20 haauters not having such dumped macrophages in their lungs were usually those found dead of some intercurrent diaeasa - Lfacrophages of both strains, both isolated cells • and those otturring in durops, characteristically gave a posiuve Prussian•blue reaction for iron. This was intense in the small macrophages of all BIO 15-16 animaL and B1O 87.20 controls. It was weak in the large macrophages of stnoetxDo.od BIO 87.20 ham- sters; theae u1L also ap ared to contain yellowish iron-negative pigment. Oociaional granules of black, iron-negauve pigment consisteat with earbon were found in a few macrophages. Broncluolm nutcp/ana was defined as taetaplasia of fla t alveolar epithelium into cdumnar epithelium of the bronchiolar type (fig. 16). The si:ain differeace was highly significant: Metaplasia occurred in nearly half of all BIO 87.20 haeute::, but in lea than 5% of the BIO 15.16 strain. In both strains, the appearance of broncbiolar metaplasia was age related but was not significantly affected by smoke expo.ure. Fitoptc bonf fonnetron: Pulmonary parenehytna in both strains contained small foci of ectopie bone not associated with inRammation or other obvious disease processes. These foci occurred in 18% of the cage- , held BIO 15.16 and 42% of the BIO 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, lymphoma, or leu- kemic infiltrate. No differences in these parameters could be ascribed to strain or to smoke exposure. TracMa and ba+eAl Abnormalities of the air passages were rare in all groups. A few smalJ patches of squamous metuplasia were seen in 5-10% of BIO 15.16 hamsters, in which the incidence was not signifiuntly affected by smoke exposure. In the cage-held BIO 87.20 anima)s, squamous metaplasia was not seen in the trachea and in only 4% of the bronchus specimens. In smoke- exposed BIO 87.20 animals, these figures increased to 26 and 21 %,, respectively (figs. 17, 18). No tumors of the air pasugea were found in control animals of either strun. Three benign squamous papillomas in the trachea were found in smoke- exposed BIO 1 S.16 hamsters. Four benign squamous papi)lomas were found in smoke-exposed BIO 87.20 hatnsters, 2 oecurrin6 ia the trachea and 2 in the main bronchus (6gs. 19, 20). NasoO(+arM+t Cross-sections were taken of the naaopharyax and adjacent structures of the head in all groups of L-.r f f"S f f { "'{ 0422 .a.f 65.5

/1 1150 ILRNItt.D, HOflatrAOtll, AND RVSSIttt.D hamstert. Approximately half contained only notatal tissues. The remainder showed various pathologte procaoes including gingivitis, dental earies, ocular tnflatnmation with phthisis bulbi, and thromboses of orbital veins. None of these phenomena could be related to smoke exposure. Only 2 tumors were found in the nasopharynx, both occurring in BIO 15.16 anitnals exposed to smoke 60-75 weeks. One was an adenoid cystic tumor believed to have originated from mucous glands (fig. 21). The other was a fibrosarcoma that had ro- duced numerous small pulmonary metastases (figs. 22,23). Heart The heart itself was not sectioned routinely in this experiment. However, it was included with the lungs of 20-25% of the animals. Of the cage-held control BIO 15.16 hearts, 25% showed myocardial degenera- tion as compared with 17% of the BIO 87.20 hearts. This consisted of myolysis with (in the BIO 15.16 animals only) an abundant infiltration of lymphocytes. Smoke exposure appeared to increase myocardial de- generation in both strains, especially in the BIO 15.16 animals. However, the number of animals examined was too small to. permit definitive eonclu- sions (data not statistically significant). Mural thrombi were seen in approximately one- ftfth of the hearts of both strains. Incidence was not affected by the experimental procedures. Liver The most frequent abnormality in the liven of these hamstcn was cystic hyperplasis of the bile ducts, in rare instances verging on cholangiocateinortu. This change occurred after the 60th week of the experiment and was more fre uent in the B10 87.20 (60%) than in the BIO 15.16 ?17%,) aninlals. Other abnormalities noted were not strain related and consisted of sporadic infiltration with amyloid and leukemic material, fatty metamorphosis, focal inflammation, and focal necrosis. One primary hemattgiosarcoma of the liver was found in a cage-held BIO 15.16 control. None of these changes could be related to smoke exposure. Spl..n Approximately three-quarters of the spleens ex- amined in both strains were histologically normal. Lesions in the remainder included hetuangiomas, lymphomatow or leukemic in6ltratey, and atnyloid. None of these conditions was related to strain or to smoke exposure. Kidntys Some renal amyioidosis was seen in 86%, of the BIO 15.16 and 69%, of the BIO 87.20 hamsters. In neither strain was it affected by smoke inhalation. The only other renal pathology consisted of rare, minute foci of calcification in a few hatnsters of each strain. Adr.na/s The most frequent change observed in the adre: of both strains was proliferation of capsular c- This waQ reported previously in hamsters (8) anc mice, where it wat called "A-B cell hyperplasia" 1 This proliferation, not affected by smoke expost. was seen in 41,70 of BIO 15.16 and in 20% of B 87.20 hamsters. Of the BIO 15.16 hamsters, 14%, l• adrenal adenomas and 1%,, carcinomas, as compa: with 13% with adenomas and S% with carcinomas the BIO 87.20 animals. Rarely carcinomas met tasized to the lungs. The occurrence of adrenal turac was not related to smoke exposure. Of the animals both straias, 5-10% exhibited adrenal amyloidosis. DISCUSSION The model system used by us- differs from tl: reported by Dontenwill et al. (1) and Dontenwill (1 in several important respects in addition to our use inbred hamsters. The composition of the 1 R 1 Ke tucky reference cigarettes was different than that Dontenwiil's cigarettes. In Dontenwill's study, hac sters' bodies were enclosed in a tube and thus severe. stressed; in ours they were notr Smoke exposure w: continuous in Dontenwill's machine, intermittent : ours, Concentration of inhaled smoke was about 6.71 in the German model and 20% in ours, and smok was administered by different dosage schedules. Nonetheless, both model systems permit the sarr. major conelusions: Chronic exposure to cigarett smoke produces severe hyperplastic and neoplasti changes in the squamous epithelium of the hatastex larytu and is associated with decreased body weight In neither system did smoke exposure increase pro liferative changes outside the respiratory tract or increase the non-neoplastjc dcgenerative change characteristic of aging hatnsten. (Our observations on possible smoke-related changes in the heart are pres- endy equivocal; further investigation is desirable.) Chronic smoke exposure significantly reduced sur- vival tirae in the Dontenwill model; no such effect was seen in ours. This difference may be attributed to the twofold higher carbon monoxide concentration inhaled by Dontenwill's hamsters than by ours. In faet, previous observadons of Donteawill (10) showed a sustained increase in blood carboxyhemoglobin in his model, whereas ours produced only transient increases. Both systems produced no change in blood triglycer- ides. In addition, we showed no change in virus profile. The po int of greatest practical importance to emerge from our work is the striking differences among various lines of hamsters with respect to susceptibility to acute toxic eSecti of smoke and to hyperplastic response of the larynx to smoke (3). Animals of the inbred BIO IS.16 line have both the highest resistance to smoke or nieotine toxicity and the greatest laryngeal susceptibility-qualides greatly increasing the sensi- tivity of the model. Further studies with larger num- bers of animals will be necessary to ascertain the signi5caace of the laryngeal changes in diSereat straina. ~ _ . . .......... - CTR 'FAIN 042=566

A h taOAUtTi SStoKL LYNALATTO4 LY t%'l{0.LD KAKS?LRS No tumors of the lung parenchytru after smoke exposure were observed either by Dontenwill or by us. We did not confirm the slightly significant increase in adenomatoid lesions observed by Dontenwill. In both model s)•stemi, so-called "smoke cells" increased in the alveoli after smoke exposure. The observation that yellow iron-negative and alao black particles 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 cells are not directly related to hyperplastic changes in the respiratory tract. They werr. much more abun- dant in the 8IO 87.20 hamsters, which had the lower !ncidence of hyperplastic lesions. The tracheal and bronchial papillomas and the nasopharyngeal tumors (one with clear-cut metas- tises) obsen•ed in our smoke-exposed hamsters resem- ale tumors induced by others with nitrosamines (11) :nd suggest the presence of this clan of substances in ::he smoke from our reference cigarettes. The only r.itrosamine so far detected in cigarette smoke con- :ertsates, however, is dimethy)nitrosamine (1?). xEFERENCES /) Do.TS.~.nl W, CKCVALaR H•J,.HAttus H•P. et a): Ins tes:iQation. on the efttc of chronic ugarette-smoke tn- halanon in S)rian golden bamsters. J`atl Cancer fnst 51 1'81-183?, 1973 ;t %Vtvou EL, HOrrwAVr D- Experimental tobacco car- ctnoger.esu A .>,dvanca in Cancer Research (Haddow (3) A. Weinhoure S, vol 8. New York, Academie Preas Inc., 1964, pp 249-253 S[altRLo P, HOMti7ROtR F: Hish nicotine tolerance of S rian `olden hatsuten. Toxicol .+,ppl Pharmacol 21:324, 1972 (f) HorrNANN D, WvNOSt EL: Chamber developmeot and aerosol dispersion. /e Inhalation Carcinogenesu tHartna MG Jr. Neetesheitn P. Gilbert JR, eds.). AEC Symp Seria No. 18, 1970, pp 173-189 ($) ATk1~c70N WO: Production of sample ci`aretta for (6) tobacco and health researcb. Tobaceo and Health Conf, Rept No. 2, 1970, 28 Cotnavs BT. LAwrre[st PJ: A sensitive method for the determination of tarboxyhae lobin in a finSer pricit sample of blood. Br J tnd Sted:l39-143, 1965 (7) Kassua G, Laoesr H: Fluorometr{c measurement of tsiglycerida. ln Automation in Analyticaa Chemucryl Technicon Symposia 1965 (Skens LT, ed,). New York. Mcdiad, 1965, pp 341-345 (6) HowswtasR F, Russrat.o A8: An inbred line of Syrian hamsters with frequent spontaneous adrenal tumors. Cancer Res 30:305-308, 1970 (9) Wooltsv GW: Tumors of the adrenaJ cortex. !A Ciba (10) Foundation on Endrocrioology. 12. Hormone Produe- tion in Endocrine Turnours (Wobtenhoime GE, O'Connor M, eda.). bosmn, Little 8rown & Co., 1938, pp 122-136 DomreN.,tll W: Etrptrimental investigations on the effect of cigarette smoke inhalation on small laborator7' animals. lA Inhaladon Careinogenais (Hanna \tC Jr, Nettesheim P, Gilbert JR, eds.). AEC Symp Senea No. 18, 1970, p 389-411 (Il) H[.Aot.o KM: Pnduetion of olfactory neuroepithelia) tumors in Syrian harruten by diethylaitrosarrune. Cancer 17:11+-121, 1964 ( 12) M6CORMIc:R A, NteHOCSON MJ, BArnts MA, et a). Nitro• sarrunes in ci6arette smoke condensate. Nature ;1ew Etolj 214:237-238, 1973 , I CTR i ' i 7 0425567

/ Il ... i i Fictrne I.-Laryts: of malc 210 15.16 bam.ter ezpn.ed to ci6arette smoke foT 71 wee:ks. Epitbelium shovn miaimal thickeain: X.u praniaeat, rquhr laycr of baW ceW. Hertutosylio and eoata (H & E). X 600 Ftcuss 2-H79erpls.is ia laryna of male 210 87.20 hanroter expc.ed to uaols feir 60 weel.. Xar moderately tbichmed epiderrr;. rtuaimsUy stypiul audei, and iatact basal layer. H k F- X 800 Frcvsa 3-FouJ 6yperplaaia in Iaryne of male 210 15 16 6 bamuer, eexposed to aatake for 73 vre¢ka. X.u nurkedly atypical outlc and intact basal isytt. H t E. X 600 Ftce-Rn 4-Fariy basal cell hypnactiv{ty in laryne of male B1O IS.l6 harrstar exposed to.mok<fot• 100 weeka. J'Xw thick epitAeliur wtth narrv.r tcoe oi superficial keratinuaooe. Downpowth of basal cells witb mitotic activiq and .ome oucicar atypea; bo-eve: normal orieotatiao of celb fauly well maiatained. H 8; E. X 400 Ftousts S.-Early basal cell hypetactivity in larynx of male Sf O 87.20 bamuer exposed to.moke for 61 weeks. Basal layer tomewha irrcrular bvt still intact. .yea stypical nucJe and abnorrnal keradaiaauon in deep «U at ngki. H & E. X 800 Fict'st 6-Marked bataJ tell hyperactivity in larynx of male 8TO 15.16 harrstn exposed to smoke for 92 weekS Severe hypu kersto.it and acantbow with nuclear atypu, particulsrly in the basal re8im. N.u re8ular polarisatioo of baul celli in dee: cNl oau. H k E. X 250 1152 fERNILLD, HOXDVRCER, A-.D All7SFQLS .. ..~. ~..~.. .. . CTR 1 11 1 012-Si.+I B

. . . i • . ~ -~ ... : . . ~ ~ j. . ,.. .. x % el I ,AWl r ~ '. / c r fa_ ~ t O ~r.., : p~ O M ~ t ~ ;.. p ft. ,i~',~..~, • .__ 11_ , % ~ ~ ~'OR O ' ~. .~ ~t~ , ' 2 ~ , e.A • ".. ~, ! ~p I :. ~ - . .• ) , •.~ -•..: 1 Oq i .- ' . - •~ ~, t~ l ~ , 4 . !: ~ .'t', ~,-~. 0 .Y,;~.~ Oy',' i~ 111111 1.,• tit. o~ iu ii ou u cr 1:, I

/b ., i • _ -'''" - i ,1 1 . . c% . ~ .. a7'~ -.• - •. . ai _ISE-4LZ4bIr d: I ~Ma 1 ; I` I . Fiotnt 10.-t,yryn; ar male 310 13. 16 hartutcr etpotcd to ~ "'ffldd lymphatic wvuiao. H& E. X p~ amka for 91 wft . Cjwta ofat ; rP cal cpithelial t:db deep to ttroma F,cuRt ! 1.-I,aTe papiUoena txarly Wl udir4 1wM; of maJe E10 87.20 darotter e:po.ed to imoke for 92 w.c},i Ha: E. X SO Ftcvaa 1?.-papiUoma of luyws from malt DEO 87.20 hamuer ptaLa of covertijs epitheliurn. H & E. x 100 ~pt"°d ee to+oke far 63 weeka X,u Fiocts 13.-F~ F~doepitheliocaaou~ 6yper• for 86 weeka. HY&qE X 80p ~PI~ io muny.tectetieg eptbeliuro of larr~ io male 810 87.pp 411111 c:pc.ed to ~moke 1154 RtRNFELD, dD1l1l7RCER, A,YD RVSS1ff LD I • _ _- ..w~.. ..,~-.... CTR IIIIN 04~'~".~al-° 700

. . - 0

a /1 ------ • - --•-.... . ---.~.~ VAA Ficuaa 18.-Foiv of iquamw metaplaau ie brmcbial cpithdiurn o( male 310 15.16 Lamrtsr esparod a smeke (or 92 weeka. N.u .ii8hc awoaated cMocic ic8ammaae aod d;lawd lymphatic H & 8. X 400 Fic~~c l4.-PapiUomaarwn4 at aacbaJ bifurcedon in mak E10 $7.20 bamne esptied te aroolr !or 78 weeb. Ledpn parually obuucged tracbeal luroen. H d E. X 50 Fiouu 20.-Lute papilloau ol brmcbial ori8in la auk 310 $7.20 pamner expasd m amoke tar 82 reeta H 3 E. X 15 1156 s1RNFtCD, AOIAITAOS>tti AND Avs3FMLD . . ....... _ ._..., >~~~ CTR HN 04'- 55

- A -..... -- - 0 ~ N. /h Ftcotc : I.-.\denoad catic tuaur d aawphns in ttale H1O 13.16 Bamuer atpr,d to.mokt foe 70 ~.ecb. :!o t~team d(avaeioo or du~ant netu~asrs «ere found H Q E.1750 1 ict, ai 2?.-Sarcorutoui tumot• ot turop6arynu in rtuk 310 15.16 huatm ettpa.af to taaoke for 73 ..aeb, X.u ciiiarod cepiraosy cpitEel~um sbo%e tu-or. H & E. 200X ",ct'Xe; 27.-Pulmonuy metawta.u froro naropharynaet+l tumar .howa in 6rue 22. H & L X 800 BIR7.f1LD, HOUtiL'1lGLA, AND AV33TQLD 1157 ~ ~T R M N 0 42 ED"" 2