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CiaR NN 042214

inhalation carcinogenesis Proceedings of a Biology Division, Oak Rid;e Nationsl Laborator+, conference :e!d in Gatlinburg. Teanessee, October 9-11, 1"69 Sponsored b}• Vational Cancer Institute and U. S. Atomic Ener;: Commissic- Editors M. G. Hanna, Jr. P. Nettesheim J. R. Gilbert April 1970 .U. S. ATOMIC ENERGY COMMISSION Division of Technical Information _ _• ~..r.-~. . . ~ L, f R H N 0 ~'`-2 ,-2 1

EXPERIMENTAL INVESTIGATIONS ON TI IF l_1=F1=(;TC/I- CIGARET'fE SMOKE INt-IAI_A'1'1ON ON SMI\b_I_ I_l).Itt Ils P, I(11sY ANIMALS f. I lV. IXIN"I'IiN1VII.L IllstiUllC UI Scientific IlCSC:IrCII, AsiUci:ItlUll U! 111C l;rlu,.iil t'1t;.,I.:l1G t,I:1l)U- [acturing Industry, IlamburE, Gcnn:luy ARSTIiACT Fapclimcnla wrlt pcllnnncd .m ham<L:u, luirc, .,n.l -I. i.. ..n rll,ul I., Iind a ph'... 1,1.: Inh.d.,llon In,~Jrl Lu Imnl~ Icun c~poam" I,• i„I~.,.r,. :/u„I.c. 'I'llcst i~IK,wlcflla Wa11c d.:al~nc,l lu IirC:d .dl IiJrllu,n ul Iln. ..,d.a.J, .lu~lu~t, I..-fn•a alllul:C cvp.,aulc w Ihal lllc cIlc..ls .,I I,uv.lvc ,lu.,l.inl; .,n .mw,.1l, ,..~J.I L; ...uq'.u-l Iv1111 Il,t clf:LI. ..I.,cllvc aiuul:aq; un lu.,n. -Ihc lu'.ICII)' ul -,c...1 ....,J.: pu..,ula, aucll .u (I .nl.l nli„liuc, :IUJ ll,e iulnl,Ad.illly a.I .Illlc,,:ul . .I.,...u. lcele ulvc~Ui;.ilr.l; inllucllaea nl i~,l,h.ll.ny lI:,lu.:u.y ,..n~.,. ..i r. lU'l...)Ir.an.l IcuL~.i)•Ka, ai~I1,uU,l.llluu IJ1C, hu.1) H.CII;1,1, .u,,l Lanl \vCIC al.,, w-,wlyd. fJu,':uvtl, lvc li.,v: lu..l r~p:ninenLJl, ..L~ull II~, .I_aull:..ncc,d Ihc v111'L,nCCi lu IIiC lu. .r ... ,.II ... ,14: ,,,cIJLUllmll ul al.iy I,c,l.,d I,I lalClnul(CIIIc ea,elu..leuie sul,al:M.:ca. Illal..lut•IC:d .li.ultrt:. III II.C n..l.,,.~i,~ ) u.~,.I ,.,Il.,lunt; Iung-lrnd ItJ. It)n,,~.~o...,.. p.u/1.lca, v:c li.,ac Illt.l 1.. c~pl.du II,c .d II... ..I, ....~.~~, .1. ~, .. I..u,'J. IAKII;-ICItII CXpCIi1llCllls willl syll:lll gul,liu I/:nli,l,',:., ..,,ull-lcl1:1t Ill 0111 hhuIn• IIHic) Ill I'1h1,1 'I ,IClnuniU:IlCII J IcIJGUl.:IllIc ..II•,In.,llul,~ Ill Illc IC)Illl:lluly ILICI I.,IIU)vill(; 71111i11i:411i~,u ulIlilluLlllli/,c,. 1,I,~/lun:nll v.•CIC IIICICliqd uallcrl:ll.cll tu invcslil;:llc Illc suilallilily ul III,:.; Il,nl,..J.a; Ila Icsling ulllcl iJtluilieJ ur susl)cclell l::ucinuEcni.: iul,sluncc!:. 1Vc bcp:/n (iur Inll:ll:lluull cxpeluncnh. :Illuul 11) ycals :Ilrn.a Syrian h:unsters wClc cxlwxll i/) /;ilt,:nell: snn,l.e Ily lu::.n. ,I .1 musd.illl; Clrulll,el Ilcll \vai rrl:llivrly I:ul. ' c :lull ul Iln I}'llc ,Ic.'lll„rll Ly 1•... ,Il,.:,.1'' (I•11;. 1). s,lun, lluwevel, wC IC:IIilell 111.71 Illls IllullCl by~.IClll I,,, ll~l,.il.,u.,li a1'I,IICs ICSUlls Ill a vdlY Iu4V CuI1l:CI111JIliIH1 UI, YI11„Lc ill Ilil; ':IGIIlII1Cl, I.,i,lb-Ul.lil~ .II IIIC IICIt11111111l; l11 IIIC cXl/rlilncnl. I11 allllil/ull In Illc I,I- I,ulcllt p:I1liClllale5 in Ihc sn1ul:inl; clI:unhcl, Illrlc :u.: scvcl.ll „111,:1 Ln:l.,l~ lu becunsiJtrell, sucll :u Ihe I,I/s,if)Ilily ul nus:ll'Illll:lliom :lu,l IIIe I,,,d,.nl;oJ „I :Illele/l hass:lgc u/ inll:llcll sInul.c IIIuuugh Illc n:ual i:rv11y.

X 4 r.Ale wr UUNTENtvILL PII;. I 1 u,/, w.,rLinc wilh a largc vacuuln chambcr designeJ for 10 Itanutcrs /1'lulll l'f,C111,1•16's1 I Ig _ ~ Sulod.intt ul•L lu... • Uh c ulum.ltte .uloAe/:/ir venlil:l/ion, dcalenr.l I„r , 1,~1~1 Icnu.l.•n it) pr I,..,,1. ,. ,II ", I„i.J sluuking syslcul"1. (II.,u, I),ulcuwlll er'I'I'(TIS t)P c1(inltEt rt srelul.l IIIII. l. lll I1 Iu ulu ulliniun Illcrc tvelc Iwu Ilruldellts Ilcll II•IJ 1,'~ La .,dkrJ: 1 I 1 JI'sy;uiult a swukiul; dlalullcl device Ihal Inudln:cs .Intl ull~ 1:. Ilic :uuul.ll, I,q-l, r„n.'I nu,l liu(ls lJl' snlUkle witlllllU entl:ulga(illg Illclll UIJIIUI r.%I,i11iU:111.11 ,_I.ulllu,.lu; ('t) puisuning :ultl niculinc inluxic:l(iun wclc csl,c..l.lllN ll, I,1 (2) pruviug, ill expelilnenl:ll slullics, Illc :lssinlil:llil,u „I ,In,.Lr u, III l:,llu.llw~ org:uls autl Ihc ellccls uf slnuhc culnpuuculs Im Illc I,...pilulul, i)wing llle 'llliltl (1uaJrellnial ('wllclcllic uu (•uu.••I, Ilcl,l lu Pclugi:l in 1 965, wC6 reporletl tesulls ul c.\IlCsinlcnls with u Iwv. l) I„ 1.1 wl.,l.ull, nl:l.:hinc that was.hasctl tln Illc clusell syslclu (lypc I).,nlen,vlll) I I. uull (I'ig. 2) tv:ls su snlall Ihal lllc Il:lnlstcls welc eal,l,,.:ll l., .1 Inl•1113' ..,u,cnlr:llcll aUuuspllcrc of snwke and :lit. 'I•Ilc ln:lt:hinc I,11„•IJ.•J CyClc with condiliuns slantlculliied :ICculllinl; lu Illnu:ln sulul•nll: I<r ults ul Illcsc cxpclinleuls Icve:dcd suluc Ilallilluly prulilclaliuu., „I IIl, In,cII,:;J c1/ilUcliuul :Is wcll as Iuclalllaslic ch:ingcs, intluJin); :llypic:ll nurl:l .u.,l '•rVrlC I.t.dllcraliun ul the ullper P:ul uf Ille Iradlc:r, Illcsc Ili~lu(~alllul,,, n.Jl, .lu. ll. sc:nlrll tu bc cotup:lr,lhle willl precautceruus ill:ull;es. In IV(,S we Ileg:nl exllclilucnl:ll sln/lics7 un 11. .' I.i. L.c ,lull nn:l:lhllllilu ul ('O (Fir. 3) in Ilillctcnl slnul.iul; syslclns, wllil:L ~.., ,.JI•,I I11,: ':uIJ Ilk: ulxu•' syslcnls; Illrst stutlies ,lulwcll 111,11 :ul cunl:lin% lilllc lisk lur CU illtuxu::lli.w. AItcl Ill0nllulrlll ,u .'.nl.,nnl 111c loulu•lUuu I.I ('(1Jlelnul;lullin :nul Illc :usullilaUun ul" Ili.:uliln i,u•. Ulr I1u,,•, .lu,l utllcl Ilig,nli I D0 •ul IU l, '/ - /;. / / I lIJ au IU u U II • .. . I I :IZ, 1 1f.11 , . ~.• 111t. ) In.,..u•: ,I 1/ lln•,.n.+•I•~Ln~ ( u, Il,c lunl:, ,I I,.wnlrl.

OON7ENWILI 392 EFFECTS OF CIGARETTE SMOKE INIfAI.ATION • . 393 60 so 10 0 T StoNf'f t) S6ttln t NG 1 2 1 itt. 4- ItcJu,tiun of ('O hcrnoglot)in in the blood of smoke•expoced hamsters: in thc ptc.cu : (d Ilc.it air I-•-) or after addition of Oz to Ihe otfcrcd air voluinc /-/. ll touu I)inAcumdl rr vL~1 30 0 120 /- (7 ll z 10 x x StOPPEO SMOKINti r A x x x x I.ig. S- Itcduction of ot, ~,line in Ihe hamster lung after smoke exposure. x= mcan front lttrce anintats; •- tlieall frnlll six a/11111aIs. (I'rt1n1 I)nnlcnwitt er n/.~) in different inhalation systems (Figs. 4 and 5), we developed a ncw smoking machinc, type [lamburg. This machine has sig,tilic:ntt ativantages, especially for lung-tenn inhalalion studicsa (Figs. 6 and 7): I. It is possible lu expose animals to citiarelle sinuke liver a lung period of lime. Al present, we Itavc ltad nwre than 2 ycals of cstmicnce wittl 1Lis type of Iuaclline. 2. "I'Ite maclline offers freslt smoke in umt:eutrat'suns and quality that rctnain constanl during Ihc cuurse of exhcriment, and it guarantecs a uniform rhytltnt of sulukc flow. Alorcuver, the distance bctwecn cigarette and animal is kept as small as possible. Fig. 6 - Snwking rnachinc lype ILmthurg ("open swoking systenl"), edaptable to either 10 Itamsters, IO rats, ur 11) utice. The concentration of !he stnoke/air adtoixture is adptstahtc, and Ihe puff vulonle is Lnlonlatlcally controtterl. (Frorll Itcckich rr af. "0 1

394 DONTENWILL Fig. 7 Animal-holding wbe of the smoking machine type Ilamborg. Within tlre lubc arc iuuu •I d/z.luun equipment and an opening into the smoking chambcr for Iht nuec dud ,u•,uIh uf Ihc hamster. 3. I'ilut sluJi.. Ilave determined tthe smoking cycles Ihat assure tlle most el7eclive Josc :II Irllvest toxicity (Table 1). 11 has becn possible to dclnunslrate cxpcrilnentally Iitr luinb concentration uf ('C) (ref. 9), which is a Iimiting I:Iclor in lung-tcnn inlt:dafiull tests. Nicotine, on the olher 1)anJ, is tuleraleJ even in high doses, but the Ilu,r tulcratell varics according to tlle species tcstcll. 1'A IS I.I: I Snwking ('r, fr. I)urarioa ojEx/wsure to Smoke, Nmnber ojCrgorettcs Sn)okcd, m,J /./)StI of lla)nsters Snroking Reference ('igarettes irt the 5rnoking Alaclune 7'ype llaprburg• Jmuklnl; : yric (rifaretues/day) 3 X ((1 3X3u 1 x 60 I X `/11 2 x 111 I/ullc.,lcrl cunltuls •I•rom Ilcckzch et al. " I I:ivc days a week. Ll)su Iwlc (w.cks) U.,y%l uicxp•,mulc lu .ulukc/ uuwbtr of iil,arcllc% sutukcd 1.5 7/135u 34.1) 17n/15,3n(1 39.0 1') 5/ 1 1,7(1(1 )Il.ll 150113,500 >34-(1 > 170/ 10.200 n2 >S? 1/ i EFFECTS OF CIGARET'rE SMOKE INHALATION . 3f 4. According to inhalation experirnents carried out with aerosols and investigations on the intake of nicotine when the nose is opened or closed, it w dcmonstrated that one cduld count on thigl) smoke assin)iL•Itiun by selecting : inhalation apparatus that is technically appropriate. When experimental cigarette smoke inhalation in animals is to be cornpar( with active smoking in man, many factors )nusl be carefully controlled al considered to assure that cigarettes "smoked" fay auimals copy actual hlnu; smoking pallerns as closely as possible. In studying the tespiratury frequency of hamsters in the "closed" and in tl "open" smoking systems, we' o found lhat respiratory frequency as well 1 respiratory volume is increasingly dirninishcll during long periods of inhalatiu and (Ilat this effect is especially pronounced iu "clusell" smuking systems (Fil 8). Accordingly, Ihe assimilation of smoke decreases. When animals are expose to cigarette smoke in "open" smoking syslenls (Fig. 9), respiratory frequency not affected to so great an extent. According lu uur experience there are tw lin)iting facturs fur inhalation cxperiments: C'O-heuwglubin concentration an respiratory function uf (Ite test aniulals. Fig. 8 Ilcspirulury 1'reyuency of hamsters aflcr slnukc c\pusurc in a"closcd system•. (lyhe I)tlllttll\VIII). Itl'nel:lory f1Ul) of respira- ,tiou is ,cen allcr beginnirlg of smoke ex- pusule. (t-tuln Ituckcr aud Uunlcntvill10) t

JJU I'~ IY6~'IYbY~ ~'~ r11~l4tilh'~ 1-t5 9 35 c'1 101 35 /... t 11 30 DONTENWILL 4 5 70 106 1-ig. 9 Ite,ln,.l,,,, r,.,locncy of two Ilamsters after smoke exposure In the ..ul,cu au„ll,.g ,, -1.,., ' Itcduction Of rcNpiratory frrc(Iueucy aflcr conlimlouf ,.,,. I. -i'. I)uration of amuking, 17(1 ur I(111 uun. Numben uu,J g,.el.ll IuJI,.,, ,_.LUAuly Ircyueucy per u,inutc; Il,ose on Ihe oulside Iud,c.Jc 11411116,1 ,., 1.1 ... , In 0garetlcs c.,ch). (I:ruul Itucket aud Uontcnwilllo) 1YIICn rc,puns,~ ,./ ,,I,,:linlen(al anim:lls to l't7 are lu be interpreted for'.. j ..lupauNun wttll Iluulal, „,lu,nscs, it must be stressed that CO-11b valuef ;T I,uing relatlvcly rlul, 1.1, lu animals llllrlllb passive SmUl:illb' are never reacllell " t,l 111.111 after a,:uv%: slu.,l.,... : :\s we have recently reportetl,' 1 the number of ': ',}•11ur,cyles is signitic:l,lll, u,crcased after hantsters are exrosed to cigarette ,1„1.4.c; this ioclcasc I,I.,I...I,ly cau be explained by a rcduction uf lho ,I 11 1');ul-Iranshurtilll; I,clw,l L.I,uI. I luwever, no rclnliunsltip betwecu smoking ;? ,lu.l ,Illcl:,liun in wllitc ,nult cutlld be tlclnunslraled (Fig. It))• 'fho ~ bclllnlent:,tiuu rate in Ilaul.l.. , is uut accelerated (htring exposure to cigarette ' auu,l.e. I/rvestigaliuns ul Il.,lil l,•,:il;llt dcvclupntenl" 1 distinctly intlicaled that '' ,Ilun,,lls cxl,usell lu cig:uctl,. e nuke reacherl, and maintained Ihroughout the c;I,~nlnl:l,1:1J peliud, nNiLnill .-Ily luwer body weight Ihal was:us,>LiateJ wilh :' Jcclca~.c.t I.,ult cc,nsun,y,li,.., I.i Iuol, detailed inspection uf growth curves ull/lc:,ics 111•It ttays willl,/nt :.1-.1 lut; :Ile associated witll a slightly increased body lvcilall I I'1,: I I ). 'I-h"c cxll, 1,.. „u :Il,ain slluw Ihe a),rclitc-inllillilinl; cllect uf EFFECTS OF CIGAHE•fTE SMOKE INIiA1.ATIOrI 7 I CONTROL hi < m {:, lV^ 1 I; 5 t; .: ` ~. t:- 4F04 SIAl7T 10 ;.Q I,,./ I I' ,d.: Juyo Flg. I11 I(ffccl of clvalellc 5u1.,Lc ..u c11'dn,,, Ic ...,... 1 .., I...., ..,. 11 „w, Nc,kicll :,u.l 11,ulcnwllll' I ------ C.(BlTkOL ANIAtt.l J lU ~.I1,Akf I Il ):G.)I;1 . 1. ,' I,. I..... --'-' Li.rut,uHl- I,. ..L,•.ul I II I , . , L..-, , .1 l --. I.---- -t----..I -.I ... I I I I I -I..__._.I-..-. I-.---~- (/Uy! IJ " l) •1:1 la) /', '.hl I., . 1,'1/ 1:', 1:.11 Il.l) 1/ll fill fill tm Imt mn mm mm mit nm tmt tlm fill mn 14 Iml mll n10 fill fill Iln tm 11.. 1....... 1:1'. .I.:tclupnlcld ,t buJp

3`.ltf DONTENWILL TAU1.1; 2 .Sttrdk•s u%.l dnptive Z prrtu,rertcsis itt Str)oke-1:•x posed Ra ts • liuw ul iu.~....,Lu„incl I',ctrcatmcnt luu 12 lixposures tus,nukcuf I+,u 3 X)tl cig.trctles daily luu Inicctiunof •li) tug/kg rigaretlc smoke cundensate i.p. IUU )t1 mg/kg 100 1 bcntpyrene i.p. 160 1'arlial rescclion 1611 ul Ihc Iivcr -Time of p:unlysist (uliu) C'unlruls( Difference ('f) 146(414) 177 (4(4) IX 234(19/2U) 335(IX/211) 31) 232 (18120) 388 (27/3U) 40 79(13/15) 140(11/15) 44 44 (15/30) 143 (2(1/30) 69 23 (6/lU) 87 (8/10) 74 262 (9/9) 192(819) 36 700 (X/9) 29X (9/9)Q 1 SS •('totn I lunlcna•rll Ci at. 1 T I, I u wb c nt+r+. ~to1 C. I 2-aln l nu-5 , 3Nund,rr, ui p.nrull,c.c> indi.:atc animals willt paraly5lS (1Cr total CX(1CfII1LL'rltJl 8r111119ls. 4S(wu+ upct.,ti..n upcning of the pctitoncal cavity. -I'AItL)•: 3 Strr.h. I, .•( I.laplirc• Zyrnugc•rresis in Snwke•Expuscd 1/arnstrrs• I lu+c ul to.~atul~+nln~I r+rlrralmcnt Uupf (.);1 I1111 I.' I~t,usutcs I(,l/ +,• .,,+ut u( + +n,q:,rcllcs 1l1(1 I„p, tt•.u ul- •1uuuJ1K . Ir.u, n: satokC t:un.l. ,,,•,tc 1.(t- funuvl, brnil. I imc uI p:+ralysis) (a+in) ('u,dtulsl 11i11iacncC ('J ) 49 (16/2/)) 67 (1 X/20) 27 I2X(15/2(1) 16i(IX/2(l) 22 dX ( 15/2() 57 (13/a1) 16 49(ta/21) 57(13J2/1) 14 51(X/1(1) 73('1/111) 311 51 (29/30) (,1 (28130) 16 •I+um I)untcnwdla•ta/ `' ( ! EFFECTS OF CIGAHETfE Sh1t)KE INIIAI A f Ivt•) . ' 3( i Slutlit:s uf :u)aplivc zy'nturtalcsis lulluwiltl; .null.c cxlt++surclt Irltrvatl 1h: II1e futnlaliuu u)' hytlruxyLlsc is iulcnsi(tctl a(l.:t wtul.c c\l,umuc ('l:dtlc. .'2 au .51, ant) tllalt ttlc hytlnrxyhtsc is :tlllc tu cluuin:rir a1+,ulaU+' 1+V,Ituc:ubuns. Ou uhsetv:Uiuns Ilave :dteatly llcctl cunliuttcll I/}• 11'clclt rr +d. +' nl lct iuveslig:tlitw in ul:ul. As I:ulin t.'t ul" t't have (ruiu/ctl ultt, Il+k Inu,:cs, uf cllr+litrl(iuu, whita ubviuusly funt;liuns vt;ly wt:H in Iruttsltas, nl,,) IIl.; tlcci;lvc (+n Illa cifcct u t::rtt:iuoltrnit: sullst:lllccs. In uttlt:r Itr keep l+lxicily as luw as 1wsNil,lr Jlnlnl. inlr.Jaliun cxOclilncrtls scvcl:fl Ics( t:ig:lrc(lci a'clc usctf uu tlilll;tcul :,nuuc+I .I+ecic, lu (IctCrntinc llh' acutc :ultl chruuit: loxit;ily ul' ccrtain sul„hr ,-,ulpunents." 7'hc reslllts w, t,bt:linc,l have a prtm:uy bt::lling unly un I11e ;,iunr+l cxl,cuttlcnl bcc:nlsc, a Plt:viuusly statctl, a('U-tcslturlx; uct:ultinl; tlululg I,a.bivc Sluul:istg iu auiut;lls i: ncvcr rtt:It:heJ tluriny active snluking in ucln. Idcccrllclc;s, we trust tllat surtti rt:sulls fruln (lie cxltctimen( may be applictl 1„ Ilt;w. 'f luac toxicity sUnlit:: tlculuns(ralc Illt: fulluwillg ('I'ablt;s •1 aud 5): 'I'AIS1 1` •1 C'untparative Studic•s urr Tusic•it)' u/ 1t./, r, nr,•.in.! 11uJi/ird ('i-~arrttrs (Srnu!-tng.11ac•ltirre l)yrc llarnLrirg/, tritlr l'.u rrrular 1(. ,:.n J to Srvvirdl (irnr an+/ ( U unt( .ti'iru(inr ( •unr"nt I ,+1 N,c ( •iyur ctrc>' - 1 u,,, ul c\Swituc lu amuke until dcath Vu1. Ni, Miuc l Nn,nbc+ l hnuN Typc uf cigarrtlc ( Yl eunl.n u A(ousc l`') uf S+uuS.t pu11o pc! Il +msicr Itat 1i/c ((SAl (uttJcig.) . pl.+rrl (Ill) . )as) 1.GIauJ~rJ) ` 4.6 2A5 lu It+-I IL 76 15 f 1( hutnidtty < (1; / 1. S >.t,U I++ I I I S! 4 4 It• I F 1csltdrlcJ will+ 5,1 U.LX Iu I 1L 6•1 3X 7lP„. cthanull 1(il ((i - rcconstilut.,l 7.7 (/,X2 a 15 X H 12 Inb.ti i n) lt:t (f.(; / t NaNt)~) 7m u--17 I [I M /. 14 l'~ (h t,d,ara:a .t.•ut.l 1. S u, tt, v / I !-1 -1 1 1') I n Iwcuu~litutcJ 11.1 u.6•I lu !6 I I I I 13 lubrccu ttwu I.s ) K(1'.+ L:harcual lillcr) 5.1 1.1,8 IU 1 14 17 25 19 A(I'.+ ]ICId(C tilt+:r) 4.2 1.11 SlU IhU 27 .Sll 411 I. +('amtm,lgc lille, 4,6 I/ lu 1•17 158 1~ 1O2 11,64 10 49 22 17 1 1 0 Ui 2311) Iu 20 1` •1 It k h l v % r : ru/n c. tc rta . A t., .. . ,. n+ u, .... ,.

DUNTL-NWILL •1'yp` uf/ ly,Illlu 'S,:c I I.r I 1 TAI3LC 5 Comparative Studles on Toxfclty of (Ygnrlllos" Nicotine Number conlent of ot'sluoke puf('sper (mg/cig•) plate "t'ime of cxposnre to smoke until rlelithl Itmnstcr Rat 12.3 2.9 20 53 9 '_.9 20 100 26 I_',l (1 20 63 15 1?It 2.5 20 67 24 (ntin) " htOllse litOutO c (I('1) (DAL9/cJix In 12 19 21 16 Illvestit;;llittns of Iile gaseotls arul Ihe particttlate phases.y~ inJiL,llo 111.11 u,. /( 1 Icsltttuse is equal in uniln:lls such :Is hantslers, mice, and .:;~ 1,115. !. ILlnul.,, .,. 1,,,1 at uIl as susceptible tu nicotine as are rats and certain . 51t71ills t11 Ini,Q. /: i. It;3I 11,: willl cl very high ('O level are unsuitahle for chronic lull:d:lliuu cxIl,allll...,l, 4. 1 I1c1,: S,..;iu•. 1,. 1•.; •1 suluur:ltiun effect of CO and nicotine. These findings, .is wcll as :dl ltl. .••I1. , 1.::,ulls nlenliunctl, en:lblerl us to detertnine all oplimal .I,la•Igc Iol ulu inll,d.lll..,l expeuluenls. Irt lllis connecliun, wc wO1rld like to I„unt uu( II1:11 Illc ,t.. •..ly.lliltiun ul nicnline, fur exanlple, in llle h:unster liver, ia signilicanlly •I,:cclcl..1.,1 :11 ctrmp:uetl with tlle tlecolnprrsiliun uf nicnline in • ulc rat livc( iu r•itro. r' Actually, Illc Il.u.nl. , w,ls fuuntl to be the most suit:lltle test anintal for il,ll.lle(iun exltcunlcuIS, l.. I•.1,I hec:ruse of ils resisl:lncc It) Iwllntmary infectiuns I,IJ its :Ibilily Itl tlc,:,ll,.l..•,.: niculirte, and especially because uf llle type and L,:.Itr.m of tllc cxllcll..l. ,.I.Illy inJuccd Iunl; tutuors. In this cunneclion, wr,:tugaliurts ul J.11(tlllrl ..., lo Iiellultl anll 1)unll:lnl,t7,`n :uul Kux:hnerlv lu.ly llc cttctl. 1lcl~lre: Ihr: effccl of I•••., L:rnl exposure cm the respiratury tract of hantsters is rcl,t/rletl, a I•ew ex:lnll/l. ..II„ultf lie menliuncJ lo denlunslr:lle the primary tl.::.lt:rt of cxpcrilneuts. 11'. .•., vcry grale(ul to lhe nwnogr:lph of Wyntler and II.Illncnln,lu wllicll l/lacc. ..• I,i/lclallle emphasis on (lie rlevelopment uf a"less Il,n/lllut CiZ:uettc" ul "•1 h•11. , Ilr:uelle" mtll raollecls su nt:lny cunlribuliuns of nllllulll:llive Jisctls;luns I.•. .,c shecied :Ire:u. Our own cuJe:rvurs, Ittu. have llc.a/ I/unl,u lly :unlcll :11 tlcl..,,,,l.onll; Iwsslblc risks uf tub:lccu slnuking, as well as Iln,llnl; :, w.ry of cllnliuaullt ,., .:Jucing Ilannllll substances. lVllen ttbservalions of a rcduCcls Ilnnttril,cnic acll. . 1I1 slxlnlnt nilratc :uttl ill'ccrlain tolrtcco shet:ts wclc lalllll,llctl ', 11 \V.li h111. 11; .ulvisctl that Ihis :ultlilive slluulll ntH lie useJ LI-I LC IS ul M,+ f. . • . . . -"-' _ `••'' ---- . n-....1._ ... .....1 l/ ..............." ....1 d:.n, o,. .,. ......... .. 1.... ..,. .. .. .. _ . _ . . . .. Others have repe:Uedly puilttetl uut Illnt tlle arklitiOn 411 s1lJlulu I/lu•11: lhe causr ul :w increascd li/rntaliun :unl uplake of nilntsaluincs. iinswer the tluesliun ot' whether tllc atllliliuu ul suJiuln nil/al.: t•. II•I1;1.,:,( possibly inhiltils lllc Inutoligcnic activity unly in Ille :uc;l of lll,; ,Liu, tn t•:lu,(Ilcl the activily ul Illis :ultlilivc is enll;lncetl in IIIe Ilull;s. I'llc,,: rl ll,ut,:l.lll'.Ilz S'provided a valn:lblc inlpetus tuw:ntl' dcsiEning :m inlllul lwll Iu1,J,:l sy..l, ul Illr , ;enimal inll:llaliun sludies un lot:d sluol:e. In our total progr:nu uf expelilnental inllalation slutlic,, llw plill,:il,:ll slr.:,; was given lu Ilte :usutnptiun 111:1t tullacw sluuke Inust Ic•IJ lu a Ileiiuile IC:I, Liun at syute place un Ille lesltir:llury Iract wllCn :Itlluiuislclctl iu :.Ir1li,icully lut!ll tluanlilies lo Illc aninlals. 'flle tesliug of lllis wull;ing I1~'I•.111 ~:.1, a,:culcJ I., us urEenlly rtccess:lry. Irt llle c:ue'thal sut/uL u.ual liltlauuu I•::1 .:,•uSl,l,a:,lllc qualitative ;nnl lpl:ntlil:llivc Ietluc(H/u ul Ille SIl1ULC a+~+1u111.i1,:J, 111e I,::I:Uuu V ;ir ntusl altpc;tr iu Illd arca of rtiu d Irusagcs; ul, in Illo L:.c.c tl-l ,ul ll. i:nl I/;ll llclcs , have p:nsetl llle nas'll regittu, Ille Icacu,m Inusl :Il)l,.;,u al .1 /~~„Ir Ill:~t.lnl I•Ia~e in lhe resllil:uury llucl, i.c.,:ll :1 Illacc %•:Ilclc Illlyslt::dly 11 , I l•I •l ,14I,..Illl,luun is lo lie exl,et:lctl. In lcnus u1- llle su•calletl slu,a.c-Irus•It! , e Ic.nliut; I1"t1, 111, n•.Ilil 1., 111e Iung peripllcly, wc Il:rvc lusltll,ltically luvcbulsllC,l ,cll:d „• II•1-: ,•1 Ilu.; I.,.luun uf 16e leSllnallny lrtct. IIiSIolugi.:'ll ullsclv'lliuui lu Illr 1.." I•1.•In:lli, :InJ alveuli Ieiuulilnled r.aulls wlu, ll I1:1,1 .IL::IJy l,:.vl L; u, ullf u(Iltr InvcsUt,:Iluls. 'I'llc n:u:d c:rvity t FIt;. I'/ :IUJ lllc ..I1Illll,,•I n,ll;ullnl:lltay nruCua:d t.leulVcs :IS vvcll as cl1i111cl1:11 luel.llllusi.l, ul .unl~l.lls v:llicll 11.141 been cXlrustll lu slnu6e fur a luug Ilnlc SIU.JI lyl.l• .ll I. Inll.n l.l:. a'.:I,: (,luutl in Ille n:u:d ccrvlly ul Iwu aulnl•Ils cxlnlx,I 1„ ,luol.c I- Irlt• Illln:. ,\Iveuli (Fil;. 13) slnlwetl :I ctlncenll:lliun ul uelcl,ll,ll:ll;cs wllll I11 •. ne.ll , Illlll+ llal inclllsi,nts, which welc tlclinetl Ity OIlul2 :Is "snlukc-cclIs. . IC•:! .Ilwll:lls I.Il.lt werc ' explrscd ttl snwkc f,tr a luut; lintc sln.wctl SIn.lll ".1,1_rtlllll;ll.nd Icsiuui" in Ittc Iung.l" tliltcul ex:wunali,m ul linlllut:S ul Illc u:c..l ,.nll) . Il.u llca, culd Iuu}; is ttnwrrranlctl in Ihe cunlcxt lil 111.; lul.ll Icsull,. I I/, SIlall ntll lliscuss Ihcsc liulliul:,s in tlelail hul will lurn, illslc:ul, 1.. 11.. I.u;u;, .vllidl Il:l,l,:li I:u :u we have wcn, the nluNl Iclu:rll.allle allcl:lluln•:. I•ul cllrunic snl(tke expusutc, I•Itl Il:nuslcls ".,.1c ~elc.llal. As shuwn ill Fig. 14, 93 :ruilu:ds sulvivcd snwke exl>usulc lul lu+•IC Ill,ln Itl luunlhs. 'I'llc ligtue alltrws nul unly an in1e11ncl:llilm of Stnriv.ll .I-u.,-:• I1l 111: cuulse of luug-tenn cxpuNule Ilul alsu :I Icullr./lal nltcllnclullun 1•I ..,,rll.ll/lrtl Ilislulugical cll:ingt:s. ' In In J.a lu Il:rvc Ilcttcl Cunll ul 111 II1..• In,hl. •.I we tlilleren(ialetl live stat;cs ,11 cllilllrllal cll:ull•. : nl I11.: I:nyu\ I I.II~L u l, u,u lr Illc .lNui I;f Ywnl,r /i!(l1/-l/r1•t' lll 111e A1111C11 l',11,:CY I11,111111,:"1 I~.ill~..l.ll')'~ .li Ital'llall~. As rll Inlluau Ilalllulul!) , a Jlllclcnliala• ••1 ++.Ilr,l I,;eutlue1/i111.liunt:tluns ch:nlgcs :ultl lIlc Iluscl ,/1 .:Il.:inulu:n 111 Illc I.w, 1.; 1S t•cly llllIicult. Alrylme willl lunl, cal,Clirnce In Illn Ilclll .lull wlll. IIW .11„IVC l.l,:nlltlncll :Ill;ls n olller 111i I.llulC .Ill IIIi\ 1I11)jCI I Ill Ilnn/I lll.1l Ili,: t•/ll. lIun (UI In:Ilil-llLlnCy ill 111e 1

I •11111:1 JI11 It• n•IlcCnatlll Ilue uc111rlnas?IJ (rul~ilifl :,rll r'1 rllr,l.u •Irlll' UI• •1 li I ainal.( :~Inu •clrrllll.ll r.ln(nn?n•.•.',Ir~'.~nl tllr.lln'ca~nllc'I~n,n(1/~1'~CI alnYlt.l (h r~( •s91;1) sa3els a/:+nas .(I.'n 1•' c:•I/lnil", al,uK tJMClllc ?nrll ,M 'saqurlp Ir.l(11j1!cla Ilaulr.IJnnsr. lo tJnlss.-l(IUtI nP ;:'nll•• "I .1.11m, nl •c,~irlc crl(IIa.lUr•-aJd /(.Illlrlil(qrl air. 59111111111 lialllscr.l, ,,vl „II I" 1•1`+.,c tr'lll Inn IIrJ11cl 111 a'f!I I'Int,nt In(I '111i.c UI rtulwlanra•, tu1a1 -lill pw,cr .clIcli,•I I1I,InI ?r11'ulr(dXJ Ill Ilnall.l~l) /(1?n cI xllr(IP.I 4 / r Ir) rr 1!/ IrJ n ; ~rrirldn•n,l-- , r I anolrmnqarplJ+.•rnx,l •s npl'In.rul .h q r an~l.ru..lp•lql •~ ~ r r r1~rpM+n,1 sm..nv>n •( 1. 1 r . 1 rl~rld.nn, 1~_ irlcrlJuJCrI rli,•In•1,1 ------------ Ir..q ,rp )^ n• • ua<rl (nrn,rir•,. rn_•,' '•11 /rr~ll,.qlnm snnl]nn +•rl •r' nnnrqc vl mn• nsolll~ '•1 a+fi~r'I• nr•qunn, aArIS Ilnll^•111"r"~ (•?Ir,l?nl) ,n.l.rn•rll r~..rJ.l/71r1.'J.~r).cJSnl.~l'JLrJ.cl7.r"lJJLq .,I/J f011(H1n.rllIrSq.J 1 :1 IfIV.I. •,ntp, llllc?Ir/nl.l'. ~', r~'r••i"• L"•. •rrr:r•rl~:+ oyt•ulc ~n ur.llctnl~!/unllcllucplr ln1'! I•.~arI'.) °' ~n.I •ril rn .P:IItP11J IIIIH111AC laut IIIIM cIr1111111' Il:KP1IYP pyn111c 111 1?rlrnnP cln:.nqr yl• .i nn1lr•II'11111 J111) Ir, (Ilncpl JIII J11 M:,Id?2I hI ''II:I •li:llv/qrlll••1 °rl'qq 1-P gqlnfl lyYJlryy~v ltlLYlqtl 31J0-- •'llo0ul ' 3MncOdr. 7 aN01'JS rl' •. Ir• ul n1 t'1 71 r11~ 14 9 1- . ~ ~ - -. __. ...... . .... ._ . .... . .. I - . . . _ 7 r ~ I - s < i1'1aa-a-1 I / nl It r 11 -_, L 01 q Sf I I/ II I V lV I ItJ! 3:It)4'JS -l l 1~IIHE)I )•1(l S LJ7 J 13 bf /' s I 'IUauluu.ul plr cnrqcnl .n, i-1 .r~IPI~ q,tnln,rnrll 1/.1 l.~iculry p..coJ~a.ayouls e•lu aunl my1 rrl c~arqdr.l .. ..' p. umlrlnwni:,y I 1 -0I:1 -Il l+'.,rl l I rlnrl

UUNIENWILI. I pIIhcli,,1 , haul-c~ m Ihc Lupnx. Scc Table 6 lim clas%i(iealinn ul' Ihe I' 1', cAp,,.w, lunc. 1:l1 I'.,dipJr,nu,i ul Ilic Irrynh ll:T = 2.1 wcckxl; ,,IdL,I,1.i „I Ihe I,uyn~ 1U I' - 18 :,nd 5b tvccAa, rrspcclivclyl: (,1) . I, ,,.upL,l,., nl II,i I:uyu% IPI' = 71 %cccka; /c,I/ p+ipill.,m.iluus L.,.,•I.,I ,., „I Ii,c I.,n'u. 11 1- S.1 ,,ud 72 wrrl.s, ir>prrlncly!; lti q/ ku:upL,l.ia /hypeipL,.i:,1 ul Ihc I.uynx Ilil tu, g- SS ~.,. 1., l.., h, 51 ; lur I. Gj; lur j:wd k, l.l; lur 1, lu, :~nd u, '1'1; lur u, 55; :mJ fur p u.l y. 12 rcccl.o; (1) early I11V:1)IVC C8ICIIWIIW (I.I ° 72 K'CC{:SJ. : t , -t •~ • '~ nlw~Y:a3 ~ ' , ' . •. r ' , ' .,: .. tnl i..• f~' t,r _ ~~~~' ~~., rrl~~~ ! ; =. . 4~Pk ;'1 ~• ' r

t)UN7ENWILL I-tt;ruc 1•1 clearly shows that (1) only a few animals are surviving after a s/rlrlcl.:ully long period of exposure; (2) all animals exposed to smoke for more :.''y~ tll.ul 11) luurulu exhibited changes in thc area of the larynx; (3) Ihe degree of,' ~,v~lity I,I file changes corresponds to the duration, concentration, and Ilctftlcrhly ul exOoYllrt:; and (4) llle smoking cycle is, therefore, of lltnlost tl„Ih„ rl, Illt1 ulJtcome of the exrerinlent. 1!'Irat rrr.rl• Irc ctmcluded from file results of ttlese experiments? I. L,r,rl; I,:I,u ex(lusure to high but wcll-toleratcd concentrations of cigarette sul,rl,c Il c, 1., „Irtiulal (loses) induced epithelial changes in the larynx which are cunsidclc,l I., Lc Inecancerous.• Results of our experiments give evidence that CalwNure (u NuuoLc, /nlder the conditions and metllods just described, is Irl(Illcing IIIIIIUIS 111 Ilre IJt Vrl\. !. Ihnwy Il,,,~ (rrcatlling in smoke-exposed animals, (hc fast-moving smoke stre:wl is sltnt•c•I J.rwn considerahly as it passes tlle narrow nasal orifice and culels file L•Ilgc u:ls•d cavity (Fig. 16). Particles are deposited by impact on file innicate tullliua systcln of tlle hamster and the smoke flow is slowed down consitlerably (I:IIr. I7). Since deposition takes place on :I large surface, there is ubviously uu liu»ilnllly lilr the particles to settle and to concentrate at definite parts uf any surl:r,t: .nca. It is not Iikely, therefore, tllat tlle am(lunt of smoke acrusuls re:rcung I.cr ,urfnte unil of file n:lsal area is high enough to intluce lulnun. The Ilieli r„lnwlury fretluency of the obligatory nose-breathing auiul:Js •I'iua1 JL%,ifi.rn,.n ..i rLe Iiiidings in the area of the larynx after Iong•tcnn exposure to s„toke, aI wdl .u Iil,.J ,. ...I.. ,.1 rhu eaperirocnt, will I)e re(lortell ClSewtlcre. 16 kuugh wndcl rN16. .. ...il antl oral cavilics uf file hrwslcr :rtr'ording lu lu.rnl..r,i..d utr.d Icrrr,.niul I..~.~~ ..L„ 6c.iJ rcl:,.•ua. LFI•LCIS(1F- CIGAHL-1 IL Sh1UKt INIIIU Al I(N) Ir// I u:. 17 (luss SCiti.nl 11uou1•Ir Il.:ad „/ h:w,a.A .L.- lu,; uJU,.d., luildn.ll curn.hlcls Ilre snn)ke sllc:lnl in less Illan Ic,ll a ac, ,.,..I I„ I:uerl 0r1;.lu uf ilupail, file Iarynx, I>Io,lucilil!, u t:rqlslJel~rl,IC .I,. • n„1 II ,: ;luul.c Iluw u1d leadinl; lu file JcIxylLluu „( IIIt; I+,ienlc%t :unuuul r.l I•.,I II, nL.,tCS uu a iln:rll :uea. IlcacIiuns ultservctl Itl I11t: I:uyllx in:ry lie czlrl:un.:,l 1,, Iliu I, .,rlc 1.1 acliuu. •I•lus liyluwlllclic:d cxldauaLlull is aulllwrlcd I.1 .I tulr:d:rlitrn eXplri- nlenls willl ''t('-I.il,elcJ CIl:,ncllc slu.rl,t .inJ II. I'. It:rrke, wt1ntlrlislictl ul>servILiuuS)A (u Conyp.unlg Ilic i.l.,lr„n ,I JcI).,slletl la('•Lilrcled pal lit:lci in tlreilnukc I)ass:ll•c (lnuullr r,i u.i.:d uaclle:l, :md lung), it iu111tI lie tlcluirtlSllal~rl Ilr.ll file :rlu„ntil ,.I Jcl,.,:,ur,I Ii:IIiICItY rlue5 Ilul t:urrespunrl witll file sull: cc :uta „l file Imull"d.u ~tael,uo In Ilie pasiage ( I'able 71. Tllc vnuunt ul p:nll,:ul:1lta Jcp,r,ilcJ lu 11..: 1., A:Ilitht::ullly n ctt:uetl, Inl nuolc Ilsul wuul,I Irt: .xl.ccl,:rl I n U,,iul.1,', :/,up.i,a :uul rlcluisllwll uf I,:uLlrlcN :rlc Culihi,lcl:d,l.: licic. Ilic .uu.,uiir .I I,.rilirl•,, ,Irlr.lsilctl in llic I:uyllx, Irer surl:)ce unil, ni:rlx, file urlcli;il)• .,1 Ili,; I,i.~„I..ill~,.l,,,•I ,J IinJiut;i nl Illis arc:r iwdr:lslanrlald~. WII:It alt: file pluipeils (-I Ilrr: ic;ult'. „1 I. I lus is plulral~l~ _Ilie /li;l uu ': I1-1 II,.: clt.. I I lutel sulrrLe on :rninclls r,)nltl lir ICSICJ uutlil L „r,li,-IL:J, :,l ,..I.Jlliulu :lurl Inu.•c(llucs; arl urtlrluvc.l luu,hl %).I••u I,u r.ui„I r i, i, .1 .i-Jir. li;l% Iliis slUtly I,,,,,il,l,: r

...•.,"....,_,. 7•A Il I-I: 7 I)ry,urtliull o/' 14C-l.abeleJ Smoke Parricles irl Parlicltlar Regions uj tlre Rerpira(ory l}ac•(• 4 '. ...u..,. h.lly t)r~an tirllmaeJ t'Jiuc/iv11 y11y neporilion ul paftKlc/ Proporhiunal are/ u( Ih6 t u 'fraculJcpmlliue i/1lclaliun In lli „~ n (rJ'4 (,~/ rc.p a ury pru1wr11unN Irul area S.S 37.4 p_L1- pal.le fu.,~... ,r 1 I Or.I t:avlly 1.6 10.9 m luld 0.1-0.3 XS6I-187 7.6 - IraceJ I 51.7 0.6 x62.3 10110 X I lur.l I1.1: 14.71 100 hlracllu LI,,I,J ,..d. I'1'. I+nh..aJeun; J.u repreunl mun values frum 10 .nnnab, cal.ulneJ (ruw aur(..c ,II,II ILul.,,u ),. 11.- 1...1. 1 I 1,. vluc uf 1-1./ ,,-,U.SB nanu:uncr ar erlunaleJ from quanlily of deposition in Ihe nonlnceJ otcl ?. 1Vu :ue :1111.: I,. 1.:st lvllule smoke of rnodified cibarelles, which previously r:ullid Ilc rxanllu,:J „uly as slurllcc cundrnsate ull skin ur suhculancuus lissuc. Only Ille iuvcstle;lu.n, .,I' whule slnuke un the respiratory tract gives us ;1 clear ;I1lYwl:r IU IIIC IC;d (1.Im 1,)'. ItEFERENCES I W. I)onlcnw111 :luJ 11 r.Ld.l , I IIICr 'I rJCI1eJl- Illlll ItrnnilllJl•cJrcllllllllc bci Coldllanulcrn 11.601 ItchanJlnng rull I.1.,.1.) Inlll.n.uuin. A7irl. IYur•llsrlrr.. 39 ( 1961 )•7'11. '. W. I)unlcuwdl .,u.l II : 1.•1,,, l',uL ln,rlue Jcs Itcal,lr.llwns-Iraclus nach 11¢bandlung vorl r:ulJh,uual.aul.ul,•,,.nuul.'l.. A•rcbr/urscA..6d (1'161) {IIS-J1?. I. U'. Uuu/cnlv/ll, 11. t L.lu, .,,.1 AI. L.Ibcl, I1Gcr .IIJ unlcraihicdhcllc I.unUcn-carrlnngcnc l1'u6wlg .Ie. Ihalh)I„Ir,.. .,,,lu bet Ilalns/cr uud Ita1/J. Z. A-rehsJirrsrd., 6•I (19h!) -1 •/'1 ll) 2, 1 1:• Uuul.nwill ,In.l 11 I{%(krimcnlcllc llnlcrsocllungcn zlnn 1'rublcm Jcr w}• uauunlculalchuut; lul Ic. ,...,uunara/ l. II. Illc lVul.unb vun'fal):Ilrauchkuudcrnal.n n..l Ylg.ucll~ru.nl.'I. .IIJ d.. 1./..~: Jca liul.lh luufcra. 'l.. l~re/ afura'rA..nS (IY6I)e! 68. ~ •~r• ~ i I I I a:cubcrg, t 1, cu1J Illt inclJtucc ol prilnary ucupl.urn ul Ilic Inng m r, .. .JLIU., lnulrsc..1'.vrr/,., I i_, I'1%1) 561 -56L. I n,ulcuwlll .In.l I/ 1S'1. ) 1,, f/,ri6c:,I and pulmqnaty allcr,lllulu fulluwiu); Ihc 0 L,L..L,II.,u ui .Ig,u;ll, au)•,I . _ Ihc guldcn harnsler. In, l.unV '%unuqrrs irr:lnimrlr, pp. i 1/ ~ o, I.. .lclru Ic.l.l I). . „,.1 l'~Jnrcr ltcx:nih, 11, ul' I'enlgla, I I:dy, 196e. ~ s, N. Ii,.W:rllvlll, ti. I1e.1.r.J, .,..1 1., 11,,dIC1, Iu11Al.llrullx•\(,crlruculC nlll (lt}Irtllcu• /.,Bdd/u,r.A., , r/ ~./, 1 ~137 •t-t7. II 11'. I,,.nlcu.vill, li. Itc..Atct,, .~,,.1 1 Slydl.r, IlCrJ11cI111116'sJ)II7JrJtllr Iilr I.abot:UOriunu- t,, ~ uc,c. h. tlr. /:rGuk%ura.A., •1 11'•. ', IS J'I. ~J •1. t. It~,I,tch, K. Ku,Let, II I 11•,rl,c, wnd 1V. Uunlenwill, llntersuchuugcn zur ~ IIC,In,uu.... y dcl aLl)Icu uuJ 'foxitilal vun ('11;.IrCllcnr•ul.al hk:i pav,tvcr ~; IJ, 1.,1„ I...... ; v.ul Vcl,u.l..n.l... , .,, n,ultrll.u),/rum;. ?/1'/n'1) 2 I7 !.11. br 1.1 1 I.I:IS U1* I:Il,nl(l I 11 :;t•1u1.1: II'JIIl11 ,111tIrJ 10. K. Itii.•Lcr, anJ \V.1)unlenwill,'I'icrcxpcriwenlcllc Irulenu.llungcu IJ..1 lic II•;1„/ln:• sung Jcr .almmig w:ihr.uU Jcr Inh:ll.lliun vun (ig:neUcnl:ul.ll. la:r„ (:d.,rl./,,,.,ir, 1 (196'/) •tll •1•!. II. G. Rccktch and \V. I)unlcnwill, FIfcrh uf cig:ucttc anwkc un,ham,m,. I\I-Inu.•ul•J sludica un akin Iclupcr,llorc. buJy wcir.lll, Ieul,ucylc :lud c1y11,t,,.y 1. l.rL J:irrirurr. llrul(ll, 21/ (19711) 7-I i. 12. 1V. 1luulcnwill, I1,•I', llalkt:, (1. I.aticat, and G. Rtckzrll, I)ic \VI/A)uq: v.m Ilcl.rpyl.u, Ligarcllcnrallchkundcn.)11 und passlvel lIcl.wchoug aul Jic Ilddun,; .Icl lu~../..l..uu). IlyJruxyluac.l:'rprrirnria, 24 (I96ti) 71•1 715, 13. It. AI. \VcL-b, 1'. Iti. Il,lrnwn, It, \V. Guwwi, I'. J. 1'uppcl;, M. I In,Icr, ..uJ A. II. ('unucy, Slirnulalury et(ccl ulc'ig,uclle slnohing .,u Ihc ,i 3,•1-bcutpyrcnc anJ Ihc N-Jcmclhyluliuu )l 3-Inr(llyl-I ulunu u.cIL) L.lul)n...tul , necu Ily cutyu,cs ill Iluluan pla/:cula. (7rnl. I'!lurrr,dru(. '171rrup., I1111•4, II Iul/ Illv. 14. I'. F:ulln, I. .\. Alllchell, cuwd 11. L. F,dl., '1'Lc rule ul In)l1u1cJ .d, .1,1.1 laclllla In IIIC I)JIIIUgC11Cal] UIICAI,II:Il.u)' l/.ICl i,NICCr. In, A'r.'/,a h.~l,lllrt'Nl..(,.,rl INI./ J7ulisfiR rnu/igrlrr 7irrll.rr4n, pp. I~YI 181, (7. W:Igucl (cd.). I. 'Jr,LIE, 5111116:111, 1')/,tS. 15. \1'. 1)unlculeill, II• Fllncuhurst, C. ttca.trh, II.-1'. I1:)rLe,:,tlJ I. rtl.,,ll;l. I~pclila~nl ll~ Ilulcnuchullgcn liber Aulu,,hmc, :1btr.,nspurl uuJ .11.L..1. ,.. ,..la r uc) G,Jlku- w,,» cralullc ml IlcrciLh Jcs I(t.plr.rlluiulr,)I;Ira. l.. l rrha/..r,, l. , I I/ I'h.t4/ -' 25 243. 16. O. 5,dliwli, F. (btia, anJ 1.. II. F:ull., A Incllwd (u/ tt,, .I,.: Ia1c1.1.J Iwl.),nun ol' I)rUI1C1111E~111.: l"JR:I/IUIIIJ. ~arfl't'r I(tU'., 28 ( I'I611 ) 101 - I-'.1. 17. K. AI. I IcrruLl auJ 1.. 1. I)unhau/, Indu.aiun ol' Lun ...nl p..pill,)ma of Ihe Iracllcub,unr•InLI mucusa ill Il.c Syliau II-,wtslc/ b)~ uil..,u.,,l..•J unrlll.,liuui u( bentululpylcuc.l. Nurl. Caplcrr(nsl., _'1i (IHn?) •Iri7 -1'll 18. I:. AI. IlcrrulJ aud I.. J. Ilnuh,)m, luductiuu ul Iwuul% u, 11,e SYli:.ll Il,m,>ICr willl IlclhylniUua:uuint (N-niuuau,Ilclhyl:nuiucl. ('rn,-;r l'rs I'11. 1/ // I 171. 17. III. I:oa.llucr, llrc J. Ilullu AnJ)claun I cclluc. I hC ,..u.. ..,1 I.u.g c1.:1n1. 1te'r. J(es7nra(. l)is<'ases, !113 ( I9611) S74 SYI). 20. F. I.. 1VyuQcr and I/. Ilunw.lnu, I'ul,,rrr„ nnJ Tubur.,. .1'furlir; in ii.ip.•rinrrnldl ('arrinu4.~lrsis. Atadrlulc 1'Ic.., Ncw Yull,, I'11.7. 21. IL Ilnn l,lcy nnJ It. Ihc1l.,/u.,nn, Yur I flll.,...dulue:nu Ilclapicl Jt.l',di,ILrau,Ila.A'unuuncrni.h,l/l.vr, -I'/ /l'/t,?) I'IN 1.v 2?. 11. (luu, I•~l)c11u1cnIrI1C Ill,ldl>nlllllllACrI ,111 (11.,11.1. ,ul I,J•M,.1 1i1;:IfYtll'nl.ul:ll- Lr,duu/ul;. l•ianAJlu-l.•I i. ISrNn,(., 'I 1 11'16 11 Ill t,. ?l. 1. I.:\,h, f.I. It. Ilc.l,,:w.l I I/. 11'r11.C u,, I uuluu..t Ih, .Lp.. .: .I,n..l„ly Ir,n:l anJ c•Ir1 hl,.l rl•1) u/ lilt,lrn /'a/ln,l1.4,', Sc... I\', 1•,,-•.- I! I I I I' I1. I'h, I f)IS(:USS1t7n1 T. Clur••kcr: I cnu vcly iulclcsitll iu Ilic ,II11• Irlll/.ll, luclahr)Ilc I:I,Ie.:Ii 111 Itil!,IIcIIC \IIJI)LC CXIIUsr)IC run ILlIs, II,IIU11.:1], ll1i,l ).d, i, ill ••:I11~11 6'IC:lICt ]IlJVivAl v:.n Jc1u1,I1sUaItJ ill rwc sIn:clts Illan ill ..,u IJ:Ite Ihc luugef lilc ul $ul)IC :n1i/1J:115 IU InC1:111Ullk: CUIIJIICI.:IJ,..: 1..1 II1,~I..IIU, II„1. .,I IJm:uliUC. Is 111(:(d :1 C.,Ilclalnmu I)cl\\'Ccl/ 111e :)IIIIII)' I.) ]IIlvlvti, I....n1 I,/,.I.II,I,IIC III:ICllvallull .)1 ,uculinc, 1,11 caaulplc, au.l Illc tlcl-l.:,: .I /d Il:lcllcubl.mchiul cpilllclla7 Ilucs an :Illuu:il 111.11 I...,liuc ul ili Inclab(lt.; IIl1,lCl:11Vd InCChallialrlN, Cxllil)i1 (1,IC.ild1 .u L.. .:Inlll,.ll:d :Ihnulln:JiliJ~ in II1~: .)il w,l p? I

.11r) DONTENWILL W. Dontenwill: Thcre is not unly a metabolic difference wilh respect to nicutinc: we also measured Ihe diflerence in hydruxylase activity anJ futurd Ihat suhcuurncuus treatntent wilh benzolnlpyrene is able to induce zuxazulamin- hydroxyl:ue (2-aniino-5•clllorubenzoxazolc hydroxylasel. Furlhcnnure, we fuunJ a rliffercnt arlaptive enzyme pruduction in rliffcrcnt :tnirnal species: yt cuiyrnc :rctivily in Irrmsters is an order higher, whereas file enzyme system rats is c:qrablc of higher inducibility. Crocker: 1Vas tltis dilTerence in enzyrue activity assrwi:rtetl wilh greatcr or Irss Jel;rc.:, .rl clritlrclial :dbuuuncility? Dontcnwill: 1Vell, I Ilrink one of file diffcrenl reactions in :miin;ds, or in htnnans, willr respect tu rlilferenl carcinubens, comes frurn llre diffcrenl enzyiuc aclivily. We fuund in Itaruslers, for cxainple, a very tlitierenl enzynic activity. flie ranrc tiI tlie dilicrcucc is very high in one colony. Y. Alarie: Ii:•owIJ like to ask butli you and I)r. Aucrbach, wlral do yuu scc iu file Itiunclri.d lyiupli nudes ul your ;uuinals exposed tu cigarette snruke? 1'uu have presen[rJ .1 hiI ul Inslulogical slides on utlier areas, and I Iliink I)r. Aucrbaclr Irrr.rurrJ .i few un tlie lympli nutle. What kintJ of specific change do you see Iheic" Dontenwill: I ilunk file shecific change is a brownish pibrnent in file lymph nude, t,r a piulilcr.1ti"m uf file sinus ehitlreliiun. 13row)tislt pibntent, :urd black pignrent, tliat is all I I cre are not more reactiuns. N. Nelson: I)iJ N ..u w.inl lu add lu that, t)scar? 0. Auerbach: 1Vc ~~ hruwn pignrenla/iun within llte lymph nodes. We also see Ial witliin Ilicnl. x. %•.ell as reticulum anrl lyrnphocylic hypcrplasia. I would s:ry Ihal hecausr u1 Uir,r cli:rngcs file lyntph nudcs irr Ihe siuuking dogs are alruut fuur to tive tuur, i. I:nge as thusa: uf file nunsnwking dogs. A. Wehner: Iluw Ju you keel) Ilie animals in these restraininhcages,:rt :uiy given srnukin6 peo.,J, :rnil huw inany tirnes a Jay can you safely do Ihis urtliuul impu:int; an uii,lu. stress? t7ontenwill: In uuo Ittuccrlure wc expose aninr:ds tu sinukc Ilucc Uiues a J:ry liu I1/ rniu t:.. I. Rcliore calcul:rtint, Ilie arrtuunl uf suruke inlr:dcd hy tlir aninrals, it is nccess:rI> i.r calculale llle respiratory rate as well as Ilie number ol iuipiraliuiu by tlie Ica .niiiuals at a given liute. In lianisters the rcslm•,t--iy rate is ahpruxiinately 100 inspirationsprr ntinule, ,u wlicii we expose tlieni i•, smoke Ilrrec lintes a day for 10 nrin caclt. Iliis would arnuunt lu 30(10 inhaLatimu tiI smoke/air mixture for eacli Itatnster. There is nu pussrhdity during thit tini: lor file animals to inhale stale snroke, because file siuukmg chanrber is filled vcry ullier secuntl with fresh smoke, so that llie li.uiutcrs ale exlwsrd iuntulu,wsl)' to Ireslt smokc tlurinb tire inlr:daliun periud. 11 is mrnnrt:nrl Itr Ilre srnut:in); cycles and lirucedures uf ullrer I EFFECTS OF CIGARETTE SMOKE INHALATION 411 smuked for 10 min. I don't know, however, huw long tite srnuke uf one rnff stays in (he srnuking charuber. In case it stays lin only 2 sec, eaclr h:unster will make appruxiniately 330 inlr:datiuns wllen srnukinb IU cigarettes a day. As we have seen in carlier experirncnls in which inlraLrliun niaclrines of utlter cunstruction were nsetl, auinrals are Iiable lu stul) brcalhing for 2 sec or nrore as suon as smoke euters the ch:unber, and then begin breathing nurnrally as soun ats file srrloke is replaced by fresh air. This means tlr;il :r slay period uf suluke in the i ch:nnber of' only 2 sec does uul necessarily inake Ilir :rnim;Js inlrale tlre ufferrctl sinukc. Un file ullrer h:uul, wlicn snwke slays ill IIw rlcinrlrcr fut I nrin befure llre next pu17 is rlrawn, slale and aged snruke is inliulrd by file :rninrals ne:rrly :dl (lie linre. M. Kuschner: 1Ve have becn plagued tiu a Imnt, tiinc I)y file inabilily lu pruduce pulnwnary changes with cig:ueltc prudurl;. I)r. Nelsun rtferred earlier in llre conference to II)e hazards uf puur experirucnl:d Jesigu, rn Ilre face uf very slruiit; cl>iderniulogic evidence. I firsl saw Dr. I)unlenwill's resulls earlier IIsi; yccn in Lusanne with a bruuh of people who were tlrere I',r a UIl'C Cuurnrillrc Mccting uu inhalaliun cancer. l.uuking at Iris changes in file larynx, I was cunvinccJ Iluat tliese were, to tlale, file most impressive ciranges pruduced in llie reslriratury clrrllrelium by exposure to cigarette smoke products. As a rnatter uf I:rct, I Ihuuglrt it wuuldn'1 take rnucli rnure to convince file lllat these changes 1t•erc aIready invasive carcinunla. I think 11)at I)r. Donlenwill Iras been very mouhst in his cl:riin as to lhe nature of these changes. All of us would feel a little h:rppiei, uf cuurse, if the clranges extendcd heyond the larynx and everuually rnct:ut:aizerl. Iluwevcr, I think we all feel tlrey would if the animals were kepl alive Ioug tnuuglr. I think these results dernunslr:de tlre facl Ilcll wlrat we have been sufferinb fruin, up to now, Iras1 bccn (I) :r dosage rlcficicncy in uur experimental lechniquts, and (2) a failurc to use whole siuul:e, a very Cunrplcx inatcrial, which perrnils llie suntnlaliun of file nunl6er u(efle'ls. Nelson: Befure we end /he rliscussion I helievc Iliat I)r. Ftust has several conunents lie wuuld like to rnake iibuut file (uAurs tu bc cunsirlererl in tobacco sniuke inhal:rliun studies. A. Furst: For file pasl 15 yc:us tlie ('~ruiicil fur 'ful)accu Rescarclr -- U.S.A., becausc uf its interesl in file prublcn) uf liuni:rn snruking and Irealtli, has bcen cuncerucd wilh file proper biu:us:ry ruulliuds fur lubaccu stnuke inlialatiun research. '1•Ire scienlilic stal7' recumnreuds tli:,l tlre fulluwirtg facturs be cunsiJered wlren usinb:ur aninral ruudel. First and foremost, IllealnngfnI dala can only be obtained if file whole smuke delivered to file variuus parts of' file respiratory tract of lhe anirnal is in file s:une physical anJ clrcuric:d slate as that which reaches similar :utatontical :nc:u uf, tfile liuru:ur rliuinl; nunual cig:ucllc srrtuking. Iu urJcr to :rccunqilislr tlris, it is ueccss:uy lu have a nlecllanical device -a

412 DONTENWILL device should be able ttr dcliver pulsetl amounts of sntoke in a quantitative and tcltruducible manner.'t'Ite machine should be so dcsigned and constructed Iltat it will be possible tu measure accurately the time between two successive pyffs, file tiine of file puff; file volume of the puff, the extent of the dilution with air, and file number uf puffs per cigarette. Care must be taken so that the temperature of tlte burning tip dues nul exceed tlcN reacbed when a human snwkes-a cibarelte, mtr should the cigarette be "smoked" beyond a minimum butt length. 'llte macltinc shuuld have a port to pcnuil sampling of lhc smoke slremn for ,Itcntic:d ur gas cltnmtalugralthic att:dysis, if dcsired. I'or fracltutlallrtg (llc whole ,inol.e :uul tit permil /lte bioasscry of llte gaseexts plt•ase, lhe snutking nctchinc ,It~~ul~l Itc tlesiguetl so 11et1 a CantbriQge filter c:ut he attachctl, l~ur cumparisuns hetwcen lahuralurics, it is desitabla that a refcrenco-typc cigarette be used, such as Iltc urne nuw being prutlut:ed at the Univcrsity ol' Kentucky. 'Ilic mmtrnal tcceivinb file strntkc should be placed so lltat only its lrreatlting altpatmus is iu Ilte nt,tinslrcant uf file smoke and no fartlter lltan lltc Qisl:utcc ul file ltuur,tu nuse tu the ntain brauching of Ilte airways in Itumwt tungs. The auiuctt tnrrl slwultl bc futly described as to age, genetie backgruund, tveigltt, nultifiun.d st;ttc, physical eondition, virus ce>tnponents, extent uC surgical lrautna, ,nill tlte degree of stress tu which it is subjected. Apprupriate numbers of hutlt sh:+uranuketl and tage•Itetd animals should serve as conlruls. llle cml Itomts suugltt include more than morphological alterations and file y,irscticc •,, d,cncc of ttunors. They also should include :qppropriate bio- il1ru1i;.1l iur.1,utcutcnls and their analysis. A1uflivariale analysis should Iheu he al>IrIicJ 1" tlw ,I.1t;t nhtained frunt the systematic uhscrvations ctf' tlte c:unlrotleJ tmu,nneIet~. 1. Liuvc fu file sntukinb tuacltine, the animal model, and file I+i.+tut;tcat ;wd lui.rltentical uteasurcntcnts. lly these means, il may bc possible to inletlncl sum. ..I tltc Itiulubital elfects resulling frunt Cxpusure uf animals tu cig:nette snwL.• P,ra exlreriulents, wltete llte sule ubjective was the attetnpt lu inQnce ltunut,, mnt hc c:uetitlly and cautiously intetpretetl in lit;ltl td tud:ry's kttuwledge.