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~~.-. ~-.. .. Be:nfeld, Ho::zburger, Russfield --_ Bio.Research Consultants - C?garette Smoke Inhalation Srndies... I`inety 'percent of the lungs from BIO 15. 16 hamsters and_ : ' - - all of those from the BIO 87.20 strain were examined histologically. This revealed both clear-cut strain differences and signiLcant effects of smoke exposure. Pulmonary macrophages in both strains of hamsters can form small clurnps 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 cor::-aarison of the cage-held control groups (=417e vs. 6 e). I: is accen:uated by smoke e:-oosure in both. st:ai:•s (°'_','j vs. 53',':, respccti~ely). In neither strain is the incidence of nacrc:`_Zge cl•s:nping affected by sham smokizg. There were qualitative as well as c,.:ar.t_tat:ve difi_: ences ir: :nacrophage c1u.-i?ing a_-nong the varicus grcu?s. In her-accxylir.- and eosin-sta:ne= slides o! BIC 1:.-16 harr.ste- lung of aI: g:oups, and in 310 87. 20 ha:nster lung fterrn the control groups, ta clu.-nps consisted of comparatic-ely few, small, loosely packed ^z=: op~ages con:aining dark brov.a to black p?em.ent. In i_-.gs o- srno:<e-e•eposeZ B:O 87. 20 hamsters, the clumos were larger, :nore a:.d they were composed of very lar.ge ceUs cor.taini ;; pale golden pigment. These cells were frequently mixed •xith polyr•.torphonucleaz leukocytes. Rare smoke-exposed BIO 87.20 hamsters .%khich did r.ot have such clumped macrophages in their lu:.gs %vere usuill~ those found dead of -8. cTR ca~(TRacTS oa~%'~o 11225471 CTIR HN 04,2239

- - _ - - '~--_- _ - - -_, Bernleld, Homburger, Rnes2ield_.- ~.Cigarette Smoke In'r.alation Stndies... - _ - -• - --'_ :'-.~ r Bio Research Consultants - `samte intercurzent disease. - ~ . ...~~ .... - - Macrophages of both atraias, both the isolated cells and those occurring in clumps, characteristically gave a positive Prussian blue reactioa for iron. This was intense in the small macrophages of all BIO 15. 16 animals and of the BIO 87. 20 contrcls. It was weak in the large macrophages of smoke-e:cposed BIO 87. 20 hamsters, which also appeared to contain y-eliow•ish iron-negative pigment. Occasional granules of black, iron-negative pigment consistent ec-ith care:ao::Za we: e fu,_-ci in a few r--lacrophages. Fvl--nonary pare nc3y-rna in both strains contained sma'.1 foci of ectopic bone not associated wit : ir.flammation or other o:~:o s disease ^rocesses. These occnrred i: lS-, of c: e_cage-he:_ SiJ 15. :6 ani-als, 4270 of the B:O =7. 2'J's. Spc:a3:c 1-.-gs .= b:,:h s:-a_-s-sho-•:'c- c.-..:e or chron^c :neurr.onicis or centa4-ned metastic t~-^ors of so-ae scr:, usually adre-z'_ carcino:r.a, 1.-:-ip:c;na, or leL.-emic infilt:ate, There were r.o dif-`ererces i:: these pa-a:-ie'ers which could be ascribed to s:rz.^. or smo:•._ e.,,osure. Abnormalities of the trac.`.ea and t.ronc'-:i %~ere com7arat:Vely- rare irn all gro•.:?s. A few small pa:ches of sc,uar:-icus metaplasia were see : in 5- 1 i~*e of the B10 15. 1i :-.ar.-isters, in -which tbe ~c:dence uas not s:;r.ificantly affected by snzoke exposure. In the cage-: e'.d BIO 87. 20 animals, squ3.-no,_3 mctaplasia r.M seen in t:e tracnea ar.d in only -:-,) of the bronchi. in sr-..cF:e-enp )sed 3:0 37.'_0 animals. -q- CTR CaNTRRCTS 405(31 11225472 c"TR HN 0422`240

BerrSeld, Homburger; Rusafield~ _ Bio-Reseaich Consultanta C' - t I S igae e tu_3es.. e mo e ation these figures increased to. 26 and 21 ,e, respeccively. ^ --- - _ -_--_ -_ = ~-- ., „ - • .- _ - \'o tumors of the air passages were found_in control animals of either strain. Three benign squamous papillomas weie fonnd in smoke- exposed BIO 15. 16 hamsters, a11 of which occurr~ed'in the trachea. Four benign squamous papillomas were found ir• smoke- exposed BIO 87. Z0 har.^.sters, 2 occurring in the trachea and 2 in the main bronchus. Sections were taken through the nasophary-nx and adjacent structures of t=e head in all groL?s of hamsters. Approx;tr:a:ely half contained o-1y normal tissues. ': ie re:-i:inder showed a vari ety of pathological processes, inc'_udinq gingi•.-itis, dental caries, ocular irria.:r..z:ion p::hisis bu?:i, azd thromboses of orbital veins. None of these phenomena could be rela:ed :o sr.;oKe~e_X-posure. Only two turncrs were found in the nasop:har}•nx, bott ac;urr'.:g i.n 2::r I:. 16 arimals which had beern exoose= to s-ne:e c0-75 •&ee=:s. One -aas ar. adenoid cysti, t~i-nor be1:°:e? _, ^zve or?ginz!e= :zo t: racccus glands. The other was a fibcosarcor..a «hEch had or xucec nurr.ercus s:r.all : ulraonary metastasec. The heart, as such, was not se::'oned reucinel.• in t='s exper:- ment. Ho,.%-ever, it was included aith t^e lua%-,s in 20-35'0 of :he ani:nals. Tu•enty-five percent of the cage-held cuntrol S:O 15. 16 hearts showed myocardial degeneration, as compared witi: l-.', of the liiG 67.20 hearts. This consisted of myolysis uiti~ ~in t: e BlO 15. 16 a•-i:mals onlyl an abundant infiltration of lyn-npFc,c.::es. Smoke exposure appear e= - 10- CTR COHTRACTS 005732 11225473 f~ TR HN 04.'''`41

= --_-_-Bernfel_d, Fiomburger, Rus-field Cigarette Smoke Inhalation Studies... ~_to increase myocard-ial degeneration in both strains, especially', in the BIO 15. 16 animals. However, the number of ?r+**+als e.~camined was too amall to permit definitive conclusions. Mural thrombi were eee.a in approximately one-fifth of the hearts of both strains. Incidence was not affected by the experimental procedures. Discussion Chronic e-Kposure to cigarette sm.o':e produces severe hyper- plastic changes in the -qaamous epiti:e'_iu.:• of the bamster lary--Lx and is associated with decreased body weight. S:nolce exposure cid not increase prolifera.ive changes outside of the respiratory tract or the non-neoplastic degenerative cl:z_g_s c;aratteristic of ag~.ng ::a:::s:ers. (Ou: c:servations on p:oss:b'e smoke-rela:ed c'cinges in the :ez:t are presently ecuivc,zL r_::he: inves:igation •x-cc_ be highly cesirable. ) Chr:)--'c smoke ex-oosure signi:'ca-..1%• reduced sur%-i%-z1 tirr.e in t=.e prev:ously pubL's :ed Donter..•.?i: :-:ode1: r.~ such effec: was see= in ours. This difference may be attri'o_:ed to the t~o-fold'cig?te: carbon :noao-cide concentration inhaled by Dor•tenwill's harr:sters tca_- by ours. in fact, previous observatit-,s oi Docten.%.:11 (14iU, 1973) sshowed sustained increase in blood carbo.rylte-r,cglcbin i n his model, while ours produced only transient iacreases. CTR Ct~(~TC~ACTS 04~~'~~ 11225474 I C~`~ ~' ~°°~ `~ ~.~.~ ~'~ ~ '''~~ ~

Berafeld, Homburger, Russfield .- _-Cigarette Smoke Inhalation Studies... __The.p(?int of greatest practical importance to emerge-from .... our work is the demonstration`of.striking strain differences among various lines of hamstere with respect to snsceptibility to acute to)dc effects of smoke and to hyperplastic response of the larynx to smoke (Bernfeld and Homburger, 1972). Animals of the inbred B1O 15. 16 line have both the highest resistance to smoke or nicotine to-,ccity and the greatest laryngeal susceptibility--qialities greatly increasing the sensitivity of the model. Further studies with large numbers of animals will be necessary to ascertain the sign:':cance of the laryngeal strain differences. No turnors of the lung.parenchyma follocvir-g sznoke r.rosure were observed either by Doptenwill or by us. Theslig :tly significant increase in adenomatoid Zesions observed by Dozten•.vili was n,,. cen.firmed by us. In both model systar:s there K-as an increase in so- called "smoke ce/ls`irn alveoli fo11o.ving smoice ezoposure. i^e observa- tion that yellew iron-negative a:.d also black particles occur in the macrophages suggests that the particulate phase of smoke did reach the alveolar wall to be taken up by the macrophages. Use of t:e two inbred lines in osr cxperiment permitted the conclusion that these cells are not directly related to hyperplastic changes in the respiratory tract. They were much more abundant in the SIO 87.20 hamsters which had the lower incidence of hyperplastic lesions. - 12- ~ m 2 ~ X I-M cTR cONTRacTS 005734 'f" 11225475 ~" T"R ~'~'~~ 0~-~~''~~~'

! ~ =$ernfeld~ Hor~burger, Rasefield R _ _ Bio-Researeh Consultants -t.,igarecte -,moxe lnnatation Jcnaies... -Table I L_4RYNGEAL PATF:OLOGY Strain Procedure No. of Autopsies (Larvn-x) Nor.^ al Patholozical Eoithelium+ 21,fost Secer e C_a=-¢e={ BIO 15.16 Smoke 84(57) 2 =-+ 9 Sha:n smote 42(36) 27 . 0 Cage-held 40(25) 17 ~ 0 B?O 87. 20 Smoke 87(45) 3 ' 2 Sni... sr.:c:<e .=r3~j 27 ~ 0 Cag~- ;elc 4 8/3i) 31 0 x' c rom 1eu}:oolakia to s eve-e pse,--ccep::helio:r:acouS =. ,-?lasia a=.c mic: oiavasive c. ^.cer ! \u_:.ber of lary:ees h:scolo;ical:.- exa:nine3. i'.:oee exa^:i=e_ were t-ansplar.ted. CTR cONTRRCTS o0,5%'35 11225476 CT R H N 0 4222 4 4

< -- ---- _ -. -; .-~- - -Bernfeld, Homburger, Russfield -_ ^ Cigarette Smoke In•halation Studies... Figure 1. -- Severe pseudoepitheliomatous hyperplasia in lary:x of male BIO 87. 20 hameter exposed to pmoke for 7S weeks. Although some degree of basal ceL orientation is retained in the upper portion of this lesion, it is disappearing in the deep portion. Note nuclear variatior.. He:-iatox}•lin and eos'_-. X 1:0 Figure 2. -- Severe pseudoepitheliomatocs hy-perplz•sia im- lary=x of male B1O 15. 16 hamster exposed to smoke for 84 weeis. Note retic•.1ar pattern of grov.:i and complete loss of norm..a`_ orier-:a- tion of cells in the deepest porti:.^n of the lasior.. Her:atoxylin a--%d eosia. X 25C Figure 3. -- Marked psecdoepit.^•e:1DCT1atoLLs h7perplasia in laryr-~: of male B?G 15. 16 hatns:er exoosed :o srnoke for ~?'_ ••a•ee%:s. -^ere is s~: er:ic:a! hyper:<e-atosis and massi%'e do•«gro~~h c: epi:hel:•.:^: into the st: orna. Note r egular polarization o: basal ce:1s in deep cell nes:s. Hen:ato.n•lin =d eosi::. X 230 Figure 4. -- Large papillorna nearly occludir.g the lar;rnv of a male BIO 87. 20 hamster which had been e:izposed to srloke for 92 weeks. Hematoxylin and eosin. X 50 CTE~ CCIt~{TRRCTS Qd~~-'3j6 11225477 CTR t-IN 0"••"i•` ,d",.. 2-"'i• EE-5

- j::1.,:`~" •--_---- : .~ernfeld, Homburger, Ruesfield BicLResearch'Coin sultan.` •s _-_-;;Cigarette Smoke In.halation Stndies... - -- --- 77 ...--..~~~-... _ Fignre 5. -- Cluster of small macrophages irn lung' 6£ a.Lale BIO - ---• - -. _ _..: . . _- - --- --- - --~~ ~ Hen~atoryliz~ . - ___.15. 16 hxmster exposed to smoke for 45 weeks. and eosin. X 50 Figure 6. -- Multiple large clueters of :aacrop`:ages in lunc of rnale BiO 87. 20 hamster exoosed to smoke for 45 Geeks. Hematoxylin and eosin. X 50 CTf2 CONTRRCTS 0057737 11225478 4 ~ C TR HN 0422.11

Bernfeld, Fo:nburger, Russt'ield_.: .;= . Bio-o-Researeh Consultants Cigarette Smoke Inhalation Studies... T Footnotes 1 BIO"I'hamsters from TELACO, Bar 1-Iarbor,'Maine. ~ IR1 Kentucky Reference cigarettes are produced by, and were obtained from, the University of Kentucky, Tobacco and Health Research Institute, Kentucky Research Foundation. ' ComposiNons of the Reference cigarette (1R1) at 12ro moisture were as follo•x•s (Atldnson, 1970): flue-cured larnina, 40. 1~0: :lue-c•.:rec s:ern, 14. 2ro; Burley la:nina, 24. 9 0: Turkish ( vhole leaf), 11. 5":: tfaryla^.d 1arlina, 1. 1;o; glycerine, 2. 8~:,: i.•:e-: sucrose, 5. 3-,. ~ m S r ~ ~ CTR CClf~T{~RCTS 00~~~~~ ~ 11225479 CFR I-IN ~'~422-47`

~~ -~ _ - M ~-T i Beirifeld, Hombusger, .Raspfi Bio:.Reseaieb Coniultante ` ~ ~,Clgar e'Siuoke~Inhalation Studies.. ,.,...._~._•-:.FER -__=Litsrahue References ~ Atkineon W. O._: Pioduction of sample cigaiettes foi tobacco`drid' -- .~~, .'--' _-`_~'r_ - "'- ~...~•- research. Tobicco and Healt Conference,4 Report No, 2, _ a~`t~-w -'~~•-.-'=~f'~ -P ~28 (1970). - ' r Bernfeld,YP., Homburger F. Higb nicotzne toler.aaee of Syiiaa•.1 ~~.•. ~... " olde.ri harnsters. Toucol Appl Pharmacol -22, 324 (1972): -- •~- _~-~ _~ ... _ . - - - - - - -- . • R.+-_ :Dontenw'ill, W.: Ex, erimeatal investigations on the effect of ciga: et e smoke inbalation on small laboratory aaimals. In: Inhalat:on Carcinogenesis, AEC Symposium-Series No: 18 (eds. L1. G. Hanna, P. Nettesheim, J. R. Gilbert) pp.389-~11, 1970. Donten:vill W. , Chevalier H-J. , Harke, i- °. , Lafrenz, i;. Rechzeh, 0. , Schneider, B. : Investigations on the effects of ci:rosic cigarette- smoke ir-:ala_ion in Syrian golden hamsters. J Natl Ca-cer Inst 51, 1781-1807 (1973). F:errold K. M. : Induction of olfactory neuroepitnelial :u-m-ors in hamsters by diethylna.trosamine. Cancer 17, 114-121 i:9o4). Hoffrn ann D. ,Wyr.der, E. L. : Chamber development and aer osal dispersior.. In: Inhalation Carcinogenesis, AEC Syn:posiur:: Series No. 18 (M. G. Hanna, P. Nettesheirn, J. R. Gilbert, e_s. )pp. 173- 189 (1970). Ishikawa, S.: Personal Coraznunication (1972). McCormick, A. , Nicholson, M. J. , M. A. Baylis, J. G. Underwood: Nitrosamines in cigarette smoke conde.Zsate, Nature (New Biology) 244, 237-238 (1973). ~ ~ CTR caNTRRCTS Oa5739 CS 11225480 L.x TR f f f"i 0 r"'Y' 2 2 1"Y` ~