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a /1 323 Fig. 1 100 ~ 60 20 l I . ~ i0` 00 Fig. 2 l I 1 I so 60 70 eo NUMBER OF wEEKS L 90 10 2. Changes in Bod wei ht (Figs 3 and 4): In both lines of hamsters, ir3'ividual o y weignts were affected by srr•cke inhalation and, in addition, by mere experimental manipulaticns, s.;ch as sham-smoking conditions and/or stress produced by the latter. Initial veignt in both groups averaged 108 g. The BI0 87.20 ca;e-r.eld controls attained a final weight of 144.3 g, whereas the BIO 15.16 animals attained only 125.0 g. I ~ I L.r f R MN 04,22584

a /It 1 324 150 I u ¢t00 > a 90 0 Fig. 3 Fig. 4 C4GE•NELD 010 15.16 MaYSTERS .0 •5 23 25 30 35 NuM9ER Oir wEEKS ~ _ ... „_ SNaM•SMORING I CIGaRETTE SMOKE - I I 40 45 SO r--~-- . l 90 Exposure to c:;3r--::e s-oRe reduced body weig`: of tc:n :r.ered scrai^s Cy atcjt 10 •; c:r.-i .r.a first 2 weeks of tree:ment. S_csc_uen::;. weights increased a;aln. out se-+e.+hat mcre slo•.ly in :ne 610 15.16 than in the 810 jr:rals. Because of t.•.e low :inal .e:q~: of t-.e cage-held 010 15.:e -aws:e,;s, the weight difference betdeen tne cc,- trols and smo<e--!•:cfed hnmsters was much less in the BIO 15.16 1:^e than :n the B:0 9'.:~ :1,ne. Sham-smok>.ng produced bcdy -e>,qnts :n:er- mediate between :~,zse zf the cage-held controls and t~ie a-+oKe-exFesed animals in line B:0 15.16; it had no effect on body veiq-t in line 810 87.20. ' . CTR . PIN 0,42258=5

a J . . t . • • " . , ~. /1 _.`_ . ~ ' • . .. • . 1 . 323 - i I It thus appears that the long-tera+ response te smoke lnhalation,•, i.e., the failure of the sr..oke-exposed animals ta gair. wei,tit; •.+as' due, at least in large part, to ,3irect e:fects uf smcke :n::alac3qn rather than to nonspecific stress. 3. Larynqeal Lesiois: By far the most important findings in this ex- periment occurred n the larynges (Table 1), ap;:roxicotely two-thir,:s of which were studied histologically (Figs (Tne rvr.,ainder :+~ra transplanted into hamster cheek pouches for ar. experiT.er.t wr.ich wil: be reported separately.) Among the s-o<e-expcse(: hamsters, only 2..,t Cf 48 larynges in the 910 15.16 lir.e +4t) ar.d ? o-t o.` 45 from tne 8I0 d7.20 line (7i) were regarded as h:stol.:,i:ally nor-.a1, as c;.pcsed to 68 to 90% of the larynges from the c4;rresp•:,r.::Lr.q cclcrol a-iTa:s. Tr.e only change seen in the ccntrol _ni7j:s i-.d sr.J:r•-snc..vd anir..als •.as chronic ir.f la-.!~ation. I Table 1. Laryr.geal pathology ~ I --- .'::vrs of animais No of ' 1•r• i. . autopsies i t.p:.r.vlial .lr in:•as:ve .,r..ail Strain Procedure (larlr.x) ~ :crr..sl !•.j n s ca:cer r.illsr.a a - •----~---- - -- -------- , - BI0 15.16 S-iOke 84 (49) 1 2 ja 9 7 S6a--smoke 42 1 36) ~ 2 7 0 0 0 Cace-::eld 40 (25) ~ 17 :. 0 0 Bi0 87.20 Smoke 87 (45) 3 30 2 11 Sham-smoke 44 (30) ~ 27 0 0 0 Cage-held 4d (39) j 31 0 : J () Number of larynges histologically examined. T'.ose not examined •.rere transp:anted. 'Tnese numbers are ir.cludca ...0se animals wi:h epi:he.ial changes. Pathological changes in the smoke-exposed :.jr.st.rs mere classified as follcws: hyperplasia was defined as r pvr:,:ast:c t-.icke:•.tnq cf the squamous epithelium with acanthosis an.: -ilJ -.:cloar dysplasia. Tr.e basal layer remained intact and fairil ;tra:;-.t. :his change was seen in 401 of the larynges of smcke-exwased hl'7 :;.16 nansrers and in 531 of the BI0 87.20 line, but in ncr.o .: : re :_..l rc 1ani-a1 s. Epithelial hyperplasia also occurred with ;,roli!,•raticn of the squarois epithelium in which there was a downgrowtn I I:s i,co tne conr•vc- tive tissue, often in a reticular pattcrn. •..~.~.~r »•;s;,1as,.a and mitotic activity tended to be more intcnse :-.jr. .- :•1yporplasia. This type of change was more frequent in bIJ 1;.10 ir.imals (:Ut) thon in the BIO 87.20 line (13%) and it was atsent in t-e L•cntrols. In many cases of this type of change, the basal layvr .ytJVr"lis was clearly intact. In some, it became very irregular jnJ j;•;.jrvntly isolated cell clumps were seen in the connective tissue, s fr. ;u(qvstinq lymphatic invasion. In advanced stages, this type of r.yFerPl,,sia is excecdinyly difficult to differentiate from early invasive raresncma. This occurrad in 18.81 of the BIO 15.16 animals and in 4% of tnc BIO 87.20 hamsters. It should be emphasized that no distant retastasvs .vre found in any animal. None of these tissue cnanges occurred in :ne :entrols. Small squamous papillomas, similar to those ucc•,.rrinq in sorn of the tracheas and bronchi, were found in the larynqejl vpitnelium in 241 of the 8:0 87.20 and in 151 of the BIO 15.:6 ,ininals erpcseJ to smckc. Occasionally, cnere was pseudoep>.theliomatous ,i...^yrJ•m:h of ce11s .it t.`.e base of pap>.lloma. I I L.r f f"G Hf'i 0422586

/1 Legends see op;.osiee page I i.1 f R HN 0422587

/\ 327 A few larynges of smoke-exposed hamsters also showed chronic inflamma- tion and/or squamous metaplasia of mucus glands. Chronic inflammation also occurred in a few control animals and was associated with sllght epithelial thickening, but never with frank n,vperplasia, pseudoepitne- liomatous change, or papillora formation. There was no significant difference between sham-smoked and cage-he19 controls in this respect. 4. Lun Lesions: 901 of the lungs from BI0 15.16 hamsters and all of t~Tose rom the 8I0 87.20 strain were examined histologically. This revealed both clear-cut strain differences and significant effects of smoke exposure.l Pulmonary macrophages in both strains of hamsters can form small clumps within the alveoli. This tendency is much more pronounced in the BIO 87.20 strain than in the BIO 15.16 strain, as seen from a comparison of the cage-held control groups (44% versus 6i1. It is accentuated by smoke exposure in both strains (929 vers,:s 531, respectively). In neither strain is the incidence of aacrcrr•age cl_rping affected by sham-smoking. There were qualitative as well as quantitative differences in macro- phages clumping among the various groups. Irn nem•atox;lin- and eosin- stained slides of 610 15.16 nax.ster 1.:-g of all crc:ps, and in 810 87.20 hamster lung from t`e ccr.trcl grc,;ps, t`e clumps consisted of comparatively few, s^al;, lc+osely-pacKed macrephayes containing dark brown to black pigment. In lungs of s:^eke-cxpcsej 9I0 67.20 hamsters, the clumps were Iarger, more atundant, and tr.e;. were cc:,posed of very large cells ccntair.ing pale golden picmer•t. :-ese cells were frequent- ly mixed witn polyr••orphonuciear leukoc;tes. Rare smorte-exposed BIO 87.20 hamsters which d:d not have s,~ch cl_:-iped macraphages in their lungs were usually tr.ose found dead of sc:re i::terc:rrant disease. 1The authors are grateful to Drs. STANLEY ROBBINS of Boston University and WALTER BAUER of Washington University. St. Louis..for having re- viewed the histological slides of the most important laryngeal lesions. Fig. 5. Severe epithelial hyperplasia in larynx of male BI0 87.20 hamster exposed to smoke for 78 weeks. Althc::qn some degree of basal cell orientation is retained in the upper pcrcion of this lesion, it is disappearing in the deep portion. ::ote nuclear variation. Hema- toxylin and eosin. X 300 Fig. 6. Severe epithelial hyperplasia in larynx of male 810 15.16 hamster exposed to smoke for 84 weeks. t:ote reticular pattern of growth and complete loss of normal orientation of cells in the deepest portion of the lesion. Hematoxylin and eosin. X 150 Fig. 7. Larynx of male 810 15.16 hamster exposed to smoke for 96 weeks. There is complete loss of normal polarity: nuclei show marked pleomorphisr: 3 mitotic figures are seen. This lesion is difficult to differentiate from early invasive squamous carcinoma. Hematoxylin and eosin. X 190 F~ig 8. Large papilloma nearly occluding the larynx of a male 910 87.20 hamster which had been exposed to smoke for 92 weeks. Hema- toxylin and eosin. X 38 Fig. 9. Cluster of small macrophages in lung of male 810 15.16 hamster exposed to smcice for 45 weeks. Hematoxylin and eosin. X 38 Fig. 10. Multiple large clusters of macrophagos in lung of male 810 87.20 hamster exposed to smoke for 45 weeks. Hematoxylin and eosin. X 38 L..' w/ R  f) i 04f 51...T 8

329 Macrophages of both strains, both the isolated cells ar.d t!:cse occur- ring in clumps, characteristically gave a pos.tive Prussian blue reac- tion for iron. This was intense in the small macropna?es of all 3I0 15.16 animals and of the BIO 87.20 controls. It ~.ras :+eak in the large macrcphages of smoke-exposed 810 87.20 hamsters, which also aipeared to contain yellowish iron-negative pigment. Occasional granules of black, iron-negative pigment consistent with carbon were found in a few..maCro;L~O.Q"•._-....-....~. • ... . Pulmonary parenchyma in both strains contained small foci of ectopic bone not associated with inflammation or other obvious disease pro- cesses. These occurred in 18% of the cage-hald 8i0 15.16 animals, and in 421 of the 810 87.20's. ,Sporadic lungs in both strains showed acute or chronic pneumonitis or contained metastic tumors of some sort, usually adrenal carc:ncma, lymphoma, or leukemic infiltrate. There were no differences in these parameters which could be ascribed to strain or smoke exposure.. Atnormalities of the trachea and bronchi: They were comparatively rare in all groups. A few small patches of squamous metaplasia were seen in 5 to 151 of the BI0 15.16 hamsters, in which the ir.cidence was not significantly affected by smoke exposure. In the cage-held BIO 87.20 animals, squamcus metaplasia was not seen in the trachea and in only 4% of the bronchi. in smoke-exposed BIO 87.20 animals, these figures increased to 261 and 211, respectively. !:o tumors of the air passages were found in control a.^.:r+als of either strain. 3 tenian sqja.-oss papillomas were foun,+j in sroke-exposed BIO 15.16 hamsters, all of which occurred in the trachea. 4 benign squamous papillomas were found in smoke-exposed BI0 87.20 hansters, 2 occurring in the trachea and 2:n the mrin bronchus. 5. Lesions in Xascpharvnx: Sections were taY.en throogh the nasorharynx an3-eclacent struct~:es of the head in all groups of hamsters. Approx- imately half contained only normal tissues. The remainder sh.:+ed a variety of pathological processes, including gingivitis, dental caries, occular inffaaTaticn with p':thisis bulbi, and thromboses cf crt:tal veins. None of these phenomena could be related to smoke exposure. Only 2 tumors were found in the nasophar7.^.x, both occurrir•g :n 3:^- 15.16 anirals •.rrich had been exposed to smoke fcr 60 to 75 weeks. 1 was an adenoid cystic tumor, believed to have originated f:cm -.jc~s glands. The other was a fibrosarcoma that had produced numerous small pulmonary metastases. 6. Heart Lesions: The heart, as such, was not sectioned routinely :n this experiment. However, it was included with the lungs :n 20 to 25% o'f the animals. 251 of the cage-held control eI0 15.16 hearts shc4ed myocardial degeneration, as compared with 171 cf the °IO 8'.20 hearts. This consisted of :^yolysis wlth (in the 8I0 15.16 animals onlyl an abundant infiltration of lynphocytes. Smoke-exposure appeared to in- crease myocardial degeneration in both strains, especially in the 810 15.16 animals. However. the number of animals examined was too small to permit definitive conclusions. Mural thrombi were seen in approximately ).01 of the hearts of both strains. Incidence was not affected by the experimental procedures. CTR NN 042-589

a /1 ® 329 Discussion Chronic exposure to cigarette smoke produces severe hyperplastic changes in the squamous epithelium of the hamster.larynx and is associated with decreased body weight. Smoke-exposure did not in- crease proliferative changes outside of the respiratory tract or the non-neoplastic degenerative c!:ar.ges characteristic of aging hamsters. (Our observations on possible smoke-reiateC changes in the heart are presently equivocal. Further investiga-ion ~iould be highly desirable.) Chronic smoke-exposure significantly reduced survtval :ime in the previously-published DONTENWILL model; no sucn e'!e:t was seen in ours. This difference nay be attributed to t.:e t•:-fold higher carbonmonoxide concentration inhaled by DOt:TENWILL's !%aT.sters than by ours. In fact, previous observations of GG::TE'.W1LL (1970. 1973), showed sustained increase in clood carbex;ne-oq::a::: in his model, while ours produced only transient inc:tases. The point of greatest practical importance to eae:,e from our work is the demonstration of striking stra u: d:f:erca.-ei a-.ong various lines of hamsters with respect to susceptibility to acute toxic effects of smoke, and to hyperplastic resgonse of :ne 1a-ynx to smoke (BERNFELD and HOMBL'RGER, 1972). Animals of tne :r.trec 810 15.16 line have both the highest resistance to smoke or nicot.ne tcxicity and the greatest laryngeal susceptibility - qualities ;reatly increas:ng the sensitivity of the model. Further studies with large n::nbers of animals will be necessary to ascertain the signif:can:e of the laryn- geal strain differences. No tumors of the lung parenchyma following smcke exposure were ob- served either by DONTENWILL or by us. The slightly s:gnificant in- crease in adenomatoid lesions observed by DO'::E':wILL .as not confirmed by us. In both model systems, there was an increase in so-called "smoke cells" in alveoli following smoke exposurt. T`t observation that yellow iron-negative and also black particles ::cur in the macro- phaoes suggests that the particulate pnase of smoke did reach the alveolar wa11 to be taken up by the macrophages. ~:se of the 2 inbred lines in our experiment permitted the conclusion t`at t~ese cells are not directly related to hyperplastic changes in the respiratory tract. They were much more abundant in the BIO 87.20 hansters which had the lower incidence of hyperplastic lesions. References ATKINSON, W.O.: Production of sample cigarettes for tot~acco and health research. Tobacco and Health Conference. 2. 28 (19'Z). BERNFELD, P., HOMBURGER, F.: High nicotine tolerance of Syrian golden ha:nsters. Toxicol Appl. Pharmacol. 22, 324 (1972/. DONTENWILL, W.: Experimental investigations on thL effect of cigarette smoke inhalation on small laboratory animals. In: Inhalat:on Carcino- genesis (M.G. Hanna, Jr., P. Nettesheim, J.R. G:lbert, eds). AEC Symposium Series ho. 18, pp. 389-411. Oak Ridge. Tennessee: U.S. Atomic Energy Commission, Division of Technical Inf. 1970. DDONTENWILL, W., CHEVALIER, H.-J., HARKE, H.-P., LAFRENZ. U., RECKZEH, G.. SCHNEIDER, B.: Investigations on the effects of c`ronic cigarette smoke inhalation in Syrian golden hamsters. J. nat. Ca^cer Inst. 31, 1781-1807 (1973). Ci R. i i N 0 4 ,.!.. v.. .~+~0

/1 330 HERROLD, N.M.: Ind:,ction of olfactcry ncv:oepithelial :-,crs in Syriarr hamsters by diethylnitrosamine. Cancer 17, 11i-121 (195:). HOFFMANN, D., hYNDER, E.L.: Chamber dcvelcrre~: ar.d acresal dispers;or. In: Inhalation Carcinogcnesis (M.G. Hanna, Jr., P.•Ne:tesnei:a, I.R. Gilbert, eds). ACC Symposiam Series no. 18, np. 173-189. oik R:dge, Tennessee: U.S. Atomic Energy Commiss3on, Divlsion of Technical Inf. 1970. ISHIKAWA, S.: Persor.al Cormunlcation 1971. MCCORMICK. A., NICttOLSON, M.J.. 9~%YLIS, M.A., :'tiOEPt:00D, J.G.: Nitro- samines in cigarette smoke condensate. ':ature (New Baol.) 244, 237- 238 (1973). (..r rf f"b iMiN 0'"T' 25..s 1