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I Microbiological Associates 5221 RIVER RCAD • BETHE::,x, MARYLAhD 29016 TELEPHCNE: I::1/ E5a.3tDo rzy 24, :Sia Cr. ililliazi U. Gardner The Courcil for Tobacco Research 11i0 East :Sth Street New York, New York 1C:22 Cear Bill, I Just a little r.ote to affirm that your concept a•^.d cur ar.ce;t cf the events of last wetk are cor.yatible. Here is our versicr.: a) the proposed stadies are sound and scientifically cc,-peta-:; b) the existing mechanism and funding will continue for a 4-6 nccth period of t:re at a level similar to that proposed; c) during :;is 4-6 month f:;nding period, alterr.ative methods of funding will be st.a ed, evalu- ated and hopefully, implenented; d) based on these contir.;eicies, a ";rar.t-like' proposal will be submitted for contracts 14, 25, and 2B, for evaluation at the fall CTR-SA8 meeting; e) contract 22 will continue in Its present form. Please advise if you are not in agree:x nt with these views. Ncpe to hear froo you in the near future. Sincerely, ~ Richard E Kouri, 'h.0. Director•of Researci ( R:Y:av -.. Nenry L. Schechtman P.S. Please keep in aird that while the meetings wiii :e tzting place '.n four x nths it wiil take time to ieple:x nt whatever c`anees are agreed u:on, ttierefore, these prc;ects should be f:,nced for a-:^:~%m of six -cnt"s at this tir,e. A OfVISION OF ~`~hittakar CTR COF(TRACTS 020285 ( I 11240033 ovrr EXHIBIT NO./6 S. NELSON ... t..+ i i'`e f 7 i 7 04377q

I . ! TABLE CF ceNTEVTS Pac,e l. Brief Surrniary of Progress 1 It. Progress of Smoke Inhalation Studies ; A. Introduction 4 B. C. Equi,:mcnt StatuS f'hysiologic Effzcts of Smoke or Carcinogens In thc Lung i; 10 0. Biochertiical Respcnse of Pulmonary Tissue t to Carcinoyens and Tobacco Related Materials 21 I t E.. Inltiation and Promotion of Cancer in the . t ~ Lung 27 I 1 ~ F. Ref:rences 34 G. Tables 36 ~ H. Figures 62 1: 'Appcndices CTR'-CONTRACTS 020266 11240034 GTR H~~ 04371130

?RCC'._:S ..=PC;,T I. _•ier su--ary of cr -zress. .,. Intrc ~cticn .qui,-ent s:at.:s re;.crt 1. S-cke Ir.^a:2:icri a. Sct1 I, :, SII C b. S-c:c_ r_.i:ors - ,;ar.icula:e serscr_ c. Ini-a1 c:rit2in-ent c. Flc'•I safety syste'7s / 2. AereSo' cenerazor 3. Ci:arette R.7D selector 4. CC-Dxirneter 5. Cryostat and measurer..ents Physioiceic ef'ec:s of srrolce in the lung 1. De^csitier., distributio^, retention a-+d clearance of __ z'.nece^s and to~acc~ related r..aterial. a. './altc^ Scrizcr.:al S-okino 113chine (:~~5!!). u.ar.us^ a ..escr.,I , the firs_ cc....ietec ser __ ,,- thes= ex: .•.:e-.;s are a:pended. 1) _xceri-e,:s 20: Effect cf ;articu- late __ os : c-. :s ac; nu-.:er of c:;are--:es or le^ - _revicusl, expcsed ._ s-oke. 'atais ;.resentisd i .. . s re;c: .. „ 2) Ex~-eri-ents 6, „ 1.4, 17: Depcsi:icn and clear_-•ce of SaP, or u.CA aeresolized in s-cke. Data is presen:e: i;, tiis report. 3) Exrerir,sent 18: Chrcnic exposure to j4C-MCa aeres:.'.ized in srcke. Tnis study o-,as not initiated as describec. 1-(:2-, :21, C_;, s ~piants. Data is presented in t`is repcr:. 1 C CTR CONTRACT~ 02026? CTR HN 043->~ 81

, L ) :x:erir..ent 19 _ffect c~ E9~? in*-a:rac.- eai ns..ile= c^ :l:e ._:ention and cleerc-„_ ,. 1_ oa? aeresalizec i:; s-c:e. This study is ce,olete c-t a-a1',=_is is in prog-2ss. 51 x;,eri-ents 1;, 22. P,eter.:i=- zn- •-8nce CT o'ldd aft°_r i tratracheal '.oC..ia These s' _=s are .:.- cte crid preli^inary res:.:s are ;re- ser.__ i.. t s re=.,... b. S-,c:e ...csure Itc:chine (SZ1•1 I I). 1) Cx;.erirenis 1, _, L: Ef=ec. ez.,,__._ tir,.e ar sc- _=r r_-atica cn par.icalate ca^-s: icn an- cc. c- res_i `.s v+i tin those e_:ei n3o us ir the 1•ia i_-. These s:~-,dies are c~~.z.iete c:1d data i: r'ese.^..__ i^ ...is reDJ'.. 2) l:cute Toxicity Erperinants. 'D ata is prese-ted i,+ tl,is re::cr.. 2. Lung structure and morpticlogy. a. -xzeri-ent A-001: Autoradiecra:y of rrouse lur_ a.`ter expos_ to 14C-DTC labeled cicare:e s^oke. This study is cr.:cinc, however, prelininary res--:i:s are .re- ser.:ec in this re:crt: b. Ex.erine.^.t 1-022, 1-023: Recress ion cf sn.^.:e- ir,d;:^.d lunc lesicns in 3CSF1/Cur fe-ale -ice a-:er '`.c..i_ s-cice exocsure. ;,rei..-inary '-ese st~.;dies are still en are presented .- this re:. _. hcwever, c. =x:=-i-er I-040: 'cxicity r -= aeresc..= i. src<e ar ..is.olocic evalua: - c- .-e ex.ese= a^-2:s. T'isis cC^r1e:_ a'd resu;:s c'- ..isC..:sc_ in ,ris t'epCr:. ~ Jioc'e'• CBi resC ^se of p',:ic.orary ,1s5:.. ..c car...r.:,ce-_ and .:.taceo .'el'e:e= -a:erialS l. ~x.~, :,era 1-C;O, E-CC?, E-00, Seie:-t.•: assays or t`e s ress -esPonse in ,,..ce as a f:._ cn cr a:e and exoesure :e --e racnine or smicice. These s:ucles are ccr,.pie:c and pre .-inary results are preseate--~ ..n this reDcrt. 2 V CTR CON : RAC"5 I ()L.-C5 1 C~"~' ~'IN 0~'~~ r`~:>

2 . Ex-~e-...•ent CT?.-32ACete.-m naticn c- -ha r,ature cf c•.;__-..,,-es involved in smoL:e-•nduce_~~"r4 levels. ( ( 3. _ ;er.reat C7R-8Z5rE ffects of i„ ibi tcrs cr F.;j:, and pro;ein synt-esis on s-oke-induced ~.H:~ ac:ivity. L . Meas-.--ent of orni,-ine decarbor.ylase (CC~, in pulr•„-,ary tistt:=5. Use _- ..-`er substrates to charec;eri:e s-:o:•:e- inducc~ or.icase activity. 6. Cc-• _-, . cn cf smol;e- i nduced and f'.C,- i nduce= 1 7. Carccx;-e-cclobin Assays. 8. Sco,<o- tlated inhibition of pul-onary DN:+ repair in BC,-=1 femaic -,ce. E. Initiatic., and prcmocion of cancer in the lung. 1. Er.peri•:.ent 1-100: Chronic and co-carcinooenic smoke inhalaticn s:_dies. 2 Exoerire• I 1C1. Relative suscert:...iity of C3H/::r, f/:r,f C:.., mice _.",CA-induced lung turaors. COF4 ~ CTR CTR HN 043-~ 83

C, I L• A II. Procress • `-'cre Inhaia;icn St..dies A. I n t r c :..~n . The s-..._ ,..-alaticn unit (L2~07 ^oe:^esda r•as '.;aen ,'.l` _-"a:icnal Since June 1977 ..;a.^. t'e .., carci-ocenes.s a-.: cnic s-:o:ce inhalation s. ies .ere ini a:ec I-,..ae i?i), all animals ware tra.^.sferr__ ._: cf :_::ni t. -. _-... re uni t::as then c;eaned, cec ..__- na:c__ cnc a,es:. _:ed access policy initiatecl fcr ., -.icaard -_ as to prevent the occur•ence of cinfec_icns ir r.- -?ce. In particular SenCai .:r.sl•.cs tee^ _-;.ie _ from tie inhaia:i^ . !.n coi a~crati • .:i. Drs :ack Parker e,d biike~Collins cC ^Cal vir•s .::cine has been tested and found to es :rc;ec- .. -i^e that received an i^., rcnasal ch al1er^_>. Y~ice the inhalation unit areroutineiy vac cinate: and or cccplete seroloSY tes;ing. ne•.a r..ic= bro;:c: t in _.-e unit are vaccinated, su5jected to isolatien fcr -r__: weeks, and tested serologically c:`rir tiis ;ime. Cniy ....e.~ the serological results are ne;a:ive for t;;e cos: ...r-_n ,r.urine viruses are the mice moved ou: of. isolaticn a,C :-, on test. Duri-= :'-e past year, collaborative studies have ~een carrie-~ - -- sc,oke inhalation end related areas ::i:- .,. J. Catcn,~ .. . -'.olrberc, Dr. J Stokely and Dr. 11. C.er..j at C'tl:l; Mr. _er- iorant aid Dr. Joseph Greenspan of ?r„cess and Instrur-en: .Ne,-e York; the evaluacien of stress as a=ac:cr in mice excose= . s-•cke with Dr. W. Essman at Queens Cci:e;e; :e s-oke rela:e: -ni--ition of C?lA repair `.,/iCh Dr. R. =asr,:ssen a; ;-.__ ..:vers i,y of Cal i forni a a; I rvi ne; . nc__- ti , cr l;,r_ cy s^~ofc_ ~:ith Dr. i. wanl at .e _ ..:ers y „- ___.- Carol ina. `Nith CRN'iL an„ , 1. $-J:°_ ...`aia:icn a. _::~ 1 1 3, S_M II C. -=e S~_K II 3 has heen in use since Se:.,;e^.:er 1976. Upon e:•a'__. :n and saccessful ccrple:icn of prel..-.rar, experiments, :ne -=sicn anc ooeratien of the SEM II ° was a-Proved, thus ai a secor,d machine, tne S:M 11 C, Ie I;:ilt and deliver:: .,, r!A. In lonc-teras studies, these ^s:,- -.•:s are subiec:e_ ,., heavy use so that hav:. _ a cec:<_, ^as :ee.. -osclutei•,y r.-essary to assure continuity in tne caily ex=osures. in acz::cr., several exzeriments required -`:e use c- a sec_.... -a_..i-.. ....i le .-.e ; irst was in use. ':x-.eri-en:s H (a~o~~o c?~~ ~~~-c CTR HN 04-~.3f~`84

(. ( ( ( r• 'di `-h the r,_dification in the shate of the hol e,'a ole, t=e ..yiein the rubber cam material r:as raiscc 1/1c , ".;s a--ance-_ :l,,.:eo a better fi t for ,he no•ses of the mice. T-.e ..._.:les ar,c c=- are •roshed daily and then cnce .,•eekly ;ake^. a:)ar; :nc .`--.. etely cleaned, dried and reasser..t. led. Da-s' are re'.lac_da: !ncrlChiy intervals, so as to avoid the atc:-:,latic^. of c:care:te tar %.hich wi l l causa ;he ca-.s t,:r Cracl;- c. =iC':! safety systems. i-ere are two flo;+ safety systess in c:crc:'sc-n a-= _r evaluation on the SE!•t I I. i=.e fi rs: s:s i_ lc=a:__ ,n rcom ari th the S~:1 I I and --•.. :crs ,~e '!c, cf <_-.c< . ai r) after the SE11 1 I but be,'„rca i, cac`.es t:••a __-;airenc unit. If a lacic of smoke cr ai ecc::rs .-- machine, a pressure crop is lost and c^ s::i , is t' '_= so that auxi i iLry ai r -wi i 1 bZ divP. :e: .,, -'•.e a,i irals. 'nis system is also interconne_ted to the par;ic''.a;e sensors and '::hen a predecermined unacce:;a51c level of parcic:la:es is detected, an alarm signals, the smoke is diver.ec --,... ;ha animals and replaced by e.^areency ai.; lf the auY.iii2i~ cir supply is filulty and no flc'.J Is dete'.sd, the __ -o-acnets holding the modules toget'her are reieased, allc•.+incl:=.a mice to breathe rcom air. The flow systems described a:,ove '.ri 11 Pro- tec: the animals r.cm :aulc, exposure equipment, electrical mai=unction, anc _-.ac:e=table levels of cigerette smoke. :1 saf ty system o:as -ee=ec, however, that rrould absolutely prc;a-- ~.gains; a ~.c: smoke (or air) flow after it passed c.`.e -ice. Suc. _.:_. =f flow in the animal containment system _. be causEC s-.o<a or air leatcace in the modules either acause ef ...ci_s -.., saal ina proceri.i or fau'.,y ing fic.r safety device coavoi ;eee c-cc _,s a+as :ir:=- = Process and Ins;rur,e^;s Esc is ...es=-.ly te. - ___ i~.is d:vice c:.._is;s of a..eat s:nsi..ve = s-- .-. ci resoends to fluc .:a:icns ^air '-a -=s:onse of the :her-~isccr is eiec- -.n cally cc,.re--=- :he safety systs- so tna; i_-e -.- (smoke cr a ._ :s beiow a certain preset vaiue, auxi iiary ai w i11 -e civerc_d to the animals. If acxiiia-y a:- is caul v,,' or connections prevent any flow of ai r eie- .-ac-e a to the animal:,a-:=r~a en second celay, the are released cn ,_ mc__:es, chus.ailowing t`e animals to breathe rcc-, ai r. 6 ( I CTR CQNTRP.C~~ 020292 CTR MN 0~-~~ ~ ~~

L. -his , iow -.er-..stor was tested `cr t•::o %•:eel,.s e^ a syste- in ,.hich rachir.e control ^ia;als r:3re ex=osed to air cnly in t`~e anir;al containr.a-: uni; The air !ic•a values a: t^e end of the modules %:ere 550 `,r (fzet ;._r nir :e) for auxiliary air and purse air, arid 450 fr-. for nin chine air. The safe:y system was set to trigccr %;~en ;e air cec _ase^ to iess than 3C0 fpm. The cevice %•:or ed c•ui._ sa:is4ac:eril except for the inconvenience ef loac n5 r•i i c_ ~ri . .-e -oc_ . °5 ^0:1. 2. :,erescl .,,rator. In C,_-=r..ce- 1977, CRt:L delivered e~ ee-csci ca• tcr c__a':le celivering 0.2-0.; p pcr:iclca :. a^.-•a s co, ;zi .ec in a system identical to t`.a- usec c- -~.^ Sc.. I 1 The ..cse cr, aerosol ized chemical car, be con:rci ied by -e ie. :, ire c= exposure or using a' zprcoria:e co,.cen- ,ra-i,,^s ..; -`•e -e-.icais of intcrest. Ex=csures will be ronitorec usir t=e sa:e particula[e sensor used on the S=:•1 II. The precise tec::nic:l detai Is concerning the aerosol cer.erator can be `cc:nd; in the CRti! Progress Repert. Initial ex:erinents utilizine the aerosol yen- era:or will be cescribe: in a later section. 3. Cieere:._ =TO selector. Duri ; z'%:ast year, the sr:ekiny characteristics of ..:c_are:-es of ci,=:r_.., types has been investigated by -easuring -_ .esi=-=-_ to draw, (RTD) with a F,ltrona Resis- tance to Cr~:! >tac::ne. It has been observed that variations i.• avera: " c:., ?."~ ~ ccc . tet•.,een cigarettes rrcm differert car ns c- . s=.-e -e ci ,-e-te type, althcuch :he lar es va- a;:cns c: _rre- cn n i. the _al ciaare::es (-abie Ij .:S _ ..'•Z =..-:-.^.c -. a '.t:een NractlCai Cr1 _-Ine, iarce v ..,, .. '_re-.,rat!;e CiCaret:e .' selecter sc= e e- ec _ ^ _ _ . mac•. .^~(~ „ -SSc.1Gi-:~ Tells) \':as dE\':,: '°C.. _11e c. w d .J:' . ~ s ar. _:a;.:a: _.. c- ..•_ a-_o.atic seiector. The c^c:cr_. cauaes on the RT: selector macnine were set -cr ,~e =oTlawir.g RTD's 1/ery icw (:! ) < 65 rr~ r20 :ow (L) 65/75 -^+ H-0 r•edi u.. (M) 75/85 •.. H-~0 'r.ich N) 85/95 ~r1 •r.2C 'Je~y Hici (HH) > 95 rr:^•• •`•2C 7 CTR CG~`!.T cAC_T~ 0 ~:~'1:.r CTR HN 043~ 86o

L. T`e -... , r.: ..=s I•r Icac^d wi th a:aroxir~at21 v i cicar: ^s The ..,- !n: sclec:ed ciaarettes a: a~rz.:c :- ato.c 1t ci^zret ^s/-ir.;,:e or 1100 cigrrct-2s/- .:s a resu!t cf this sellec:ic', obt2ined the fcz_,.cx'r..a:e r,;.-._- cf ci =r....es i.n each RTD vzlue. LL 13 (< tharn 1%) L lc'[ (< '~~r 14io) ti La (< t`an 22A) k 7E'~ (< than 60%) F - 13 (< th2a . Lcs:. .-.. I y -ac.•ne :~ (< t;an '2Y) 5=M ! I was a1 ibrated for three c' =ere^., ?.' 's anc ,_ p_.r ress_._ eccaireo frc- chese ca:•..-Z :ion: .,,re. RTD P::f` Pressura b3/7: 9.7 mn 1420 78/82 10.3 mm H20 90/95 11.5 mm H20 Afce- t.ie cs!i:re~:`cn oi the machine, cigare::~s~~s with ci =er- enc R'0's ~~ere s-crec at a fixed puff pressura(inter.-edia:= valua of 10.3 r- ~;:C) z--~ also at the puff pressure c:taine= ~y ac;rV_riate r.a: bra; on of the differer: "'s. The res:1 :sere sho: n i•, Ta='•e 2 from these res; :s, and cc sider,-o ,.•:e varia: cr _,Y70 enonc di =erer- _.._-es cr C: ca' .:=s r e_ i., i i, r.e ean '_Orel - e,~,. 'hut •- y~:._r to ~~ ~~anC .._ ver a cJ:a. n a Cc.^.s:c'. _...' •^!'~eof Tt C-.^.s.~.. smckC:...ce".ir_:ICn to tht ariraa!s, :. =are :'es •_- t0 :._ selu cte = C•. _^7 e ___is C' RTi arG a: .'.e E=rrC,.rlate'_- pres:_res. 8 r~C/ Q. ~ ~ Q, , 1,.RC ' ~ ~ (~ ~ ~.G~ CT;~ ~Fl C T R H N 0 437 E 37" I

, 4. CC-0xi-e:_ . I n Oc;^te. 1977, an I l ,--2 C0-Cxir.: t=r :as dei... r=_ ':, to be •sed primarily fcr czrboxyher._nc,,.n d_-_ - .._. - in rice ar;er cigarecte ax^osure. Theex__ri men;t! 'es:.it5 Ce:.°•r"^.'O:! c''er vc.'icus cigarette e>••csar_ leve:_ b2 rcaorte: n a later secticn In addi: -, to CC'-:b e.^.elysis, however, -'-.e CC-=•. -._ autc7wtica::y, and on the :e-ie blood sa-:ie ), c>:y'C'.:clo~i~t (r-zrrenc) Lnd vGiu-° Ox•.cen 'E6 c' ' LrQ~hc_..-:tric r..2ihcd ( ) •:i.9 2^d 1 to 15 s2.^••ples !:3' Gay. 1.• cC_. :c-,, a-es ysis c- ^e va-i_ ic is c~,ser veo i- •.eas ri- CC•- toch r..et^ _, the :0-^•:i.._ter r_ •ires se~•:ar . has iCr.,.. ,•_..,Ticte J', vari.::icn. c'C: \•ery fe1•1 ee'~:•les I;r+ les; c=r:atureti - The cor.c'.-^erttion of hecerin used to - .:!c ;,-Ieoc coi1:_..ng c~_bes,usec with the CC-Cxire:er; w•as~ nc.__.-sed to preve^, ..ouse bicad frcm clottl-; zr. then t!): ir.;tr„nc-nt could be us2d •.rith no iurt`.•.cr rr.odi`.caticns. 5. Cryoste:. An IEC 1•:o::l CTD Harris Cryostet~~•:as purchcsed in :.cc st 1;77 to ens~le'le us to carry out c r 1•C-OTC auco- racicarao~v ex:)erir-ants. lur.c sec'ions`cr 2Utoradic^.r:;Orv pre.zret -=ec, forraiic ,ixed i!ssce are cc-plecely unsatis- fac:_-y if :e suos:=-__ r.= !nter?sc dce. not s^aci`ically 3nd -.vaie!v bind tc allclar ccm.or.ents cr if the su bs :nce is +^_ Gurina t`: prepara:ior. pror;.is. ?rel ir,inary resu::s are s`:c-..ir. in sec:ion D.C.2., ca^cnscratina the I~. d i s 'r i =j :icn and i r.:+ts.=te depcsi:ic^ wic=.n t^.e ju^•g of C- uT .Z e:• :sL i_=: i r, sfn c::e. s C=. RR CQNT~RCT~ t]202°~ 1 CTR VIN 043'7`86

L• r i ( C. Phys;ol^;ic er-e:ts ef s-,oke or carcino;>-.s in ,-_ lunc Dosiretry Studies The e: °ct r sr.,cke ccncertrcticn and e>:;es~._ :ic_ on th_ ;.artic_ia:e depositicn in h.e m.cuse has been --e- vic,:s!y recc:. -r.e c^nclusions will be stateo here "°li•j G2,-•- Slt ic' is llnear wlt1 excosure tim::; cc'^<_.-.,,~. -;c:.s a polyncnizi func:ien as sc!oke ccacentra;icn is c.:a-r_ed; over 5.:=. c- :re _:rticula:e de,:ositien in the -cuse is .~c in cr 1;-- ••an_scripts have been drt;:ec, a-_ __i-_ revi5e,d and .:i il =_-S_.-,i tted. The e-~ _: of sex anc strain cn particulate ositicn has als- en =-evicusly reported. No sicr ica-. C.fErEnces i-i ce:'s :ic, .:ere founc ber.-iaen male anc fe-aie r..ic,,. Di-,' fere^ces n c=Dosition w•ere ebserved amona several mouse strains: ?C;FI/Cum > Cz,731/5 Cu-n > D6A/2J > Cun > Scr "1. ('•lanuscripts dascribinc these studies `cve~ be2n c rat ._d, are 'leiag revised and wi 1 i be sub'ii ttec. The use of 14. C-dotr:aco-tar.e (OTC) as a tracer fer tt5p particulate c=ase of smoke has been described in prcvious reports. It is relatively inert and as shcwn in Fie-:re 1, is ^.c: r_pidly cleared from the mouse lung. The results shownin Fi::ure I are from three experiments usinc t`:e Walton kcrixcr.:_i Sr..cic:ng Plachine (14H.SM) under the sa.:•e ,.,,..citions: smoke __-cen:ration of 10°;, 30 secends smeke ex^. :ure a l;crne;:-c .:it` ;0 seconds purca air for a tozl c• ve~ minittes. !.. _x;eri-ants 5 and 2C, C"H/Anf Cum and - -!!•Cur,, mice, -. =ec, were exposea to s.moke fre, .,,._ -:C,1 a-,d sacri ; ice-i a; the times .ndica:__. - Cxzfri, ' ;, ;i--- __m mice :.e._ ex?csec to smoke ..,,- . • ve ' :C--i.. . _o re ::=s . Is in 'ieure 1, .:e :eccsi;icn of par i_- ._:_' ..12t:er is a--c%.C ra_e'?old hiehe' .' :X;eriment 9 rat`_' -ar -.e 'ive-alc -e- -ec from _xp_ -._,.. 5 Depos i ti ., n mice chroricail ;> -ronths, :xper. en: 20) excosec to srcke :nay be ;ess .-an .,..' mice exoec_z ,.o lcw levels of s-oi;_ for shorter :ariccsor time (< -o-ths, Experiment ~) T`e relative s:ancarc: ceviations are richer for the circn-` ically exposec mica, suaces;ing more vari=_ciiity in the response of c?%e mice to s-olke. i0 C?"R CO!~TRFC~~ 020296 I C T R M N 0 4 11769

The re:=^zion aad clearance for particulates in the lunc seerr~s -a:>:alDly simiiar in ail three e.*.ceri-,ents. The hal, - liie ( l/=) o• cice chrc^ically e:c?esed ;o s-,ok: prior tot`.: cosi-.e-ry er.periment -::here cne I `C-DiC cicaret:e cs cf=_ :c' is about the saFe as for riice exposed to snoke frc m'ive i=C-DTC cigarettes (24 hours <;) /~ < 120 hours). Thus, clearance of smoke par;iculates see-ee sc-..at l.•-~ e::ance. c4 -,e initial dose, ~r.d no er stimule:ien o; _zrance could be dotec•ed after c ex;c.L:re to hic1'-_r ,_.eis or cigarette s-oke (;-I0 cicar„_:as per day). - Cic_. ' _ S'tolCe centtin5 "anY cc:-,pc'.nt5 reac. t z i. 2" -. i":a:ation, not all c' h1ich C:r_ as iner; asCTC.r although part of tne par: _:late phasu, is ._ re=ioiy clear frc.-,% the 'espira;ory .ra: after inhaiation. a •~is~-ed to verify this cbservaticr. usir•g the WHSM +•:i:i, o_r -.-sa ...oce1 and compare nicotine dos o:i th t;:o c;=_- c.`.e,` cz:s of interest wi ch recerd to' lung carcinoc-^r.=sis, :' =nc ?aP. Thus, the foi:owi^g experir..er:s %,ere t~cr;c,-e:. Tra er cuantities of i4C-nicotine, 1`'C-Bap ar•d 14C-1•fC:. were in;ec::c into 2A1 cigarettes as previously described. The s-cce `:Gm these cigarettes were then offered und • r s ta-::a-d cz:nc i: i cr.s to m i ce on the 1.'uSM (smokc conc=n- trati.an of 10%, :0 sec=ncs smoke exposure.followed by 30 seconds purc_e air 'cr a .otal of 10 minutes). In addition, i n the case er and BaP, a one-p:.=f ( che f i f th) exposure was given rr.c~-!%e mice sacrificed ir.mediacely. The resua for pulmonary deocs i;ion wi th r.ico:ir.e o:ay ~-e ,- - =igure 2. with Ba? in Ficure „ and PtCA in Ficure ~:Ze:ic :nitial clearance was observed for r,1I three _.,_:^icais, -- tl/2 <<0 ^inucec. Little differ- ence in t!l,, rateS ==2nce was --ser\'P.c after eXpOsl'.^e ._ C^ or „,_- , 01-- -- - -er nicc; 'a or zaa labeled ci ca.'_-:.es In c:_. _ -.,l i ctle C"2re' ^e •vas otserve^ in "(: rat.~-5 c- clee.'a':e• - t'1e lunc CG'Cd'P.C t0 t:•e %•:itole tGC.v. - II V cTR CONTRRCTS 0201Q% I CTR HN 04379WO

1 ( 7he i•[ernal distrituticn ot la'-eiec ni v -,: and _-D <.fter ene-__.~ exposurc, is tabuiated in 7aolel. The relatively la-ce s:ancard deviatic•r.s .- the re';ains after to T`:-zcp indicates extensive varia. n il v in this c:ecsu-=..ent ::ie:eau value fcr all tissues seers to have cccur td try i5 mi es. hior2 nicotire is de:c: _ iri the heac t•I -er „-'e -an in the lunc, tut acai.. ..~_ __ levels I•:tve teen cy 15 rinutes. :it`~ suc` ra:.C„learar ratcs, ..'^ara-.ce is occ,..rin-, .I r.c the '_ rec te c-:er .-•_ er.,ire ciczret:e The ore-puf, :c=2. -2^_ %iZs :!1 F: ..Z. .. C.L'antitLt th1 ini ial CarCs _,.. ' . nicotil•. Ln„ ^? 'he _:;a for nicotine in the iur _i- suc;;.3 : t;:at t`•e = .- .. esent at zero ..r..e arte is :ss than ;. _ciicte! frcm the cne-p_.r ca:e. For t,-_ %:=.c: c:.~, -.._•.er, the a-c-int present Zt zero _ after tC'r F._ •_i: eXactly ;a!: c.'cunt predictec --o.- tie cne-p.if; cate. For 3e?, the lung c::a for one pu~` t-d ten puff• ac:'c.=s :ite y.eil for all points 15 minutes after exposure. 'f•.c tl!= va;ue based on the one-puff data ~~ouid be less than 1; c.ir:_-es; a much differenc val,.e wrould have beer, c~er:tr.e= f-c- :he te-1-puff data (t: > 60 minutes). In contrasz to nicotir.e, 5a? apparently may not be cleared as rEp'dly c;_ri,c -e ex.csure since the xmcur.t for ten puffs i s abou t 10 fo'd d.. i-e r,-an for cne pu f f Ho•, eve r, 4uan _ i- tative diffcrences ca-.rct be determined frorn this data. t The lunc clearance of (=icure ':) is similar to r.icc;i-e er.: _a?, with tl/2 about 15 minutes. The ir.tcrr.al of MCA (Table 2) shows an increas=d retention --`.e~.eac and stcmach et zero and 15 r..inutes after ex:os:.re. ' For -i - -e, .a9, and MCA, t.`•e clearance fr - .. _ tocy ;ara11 'e- .-e =er_-ce frcm the i;.- indicating no •._. na I i.- o : s e=_e .; n: r tie -n-+i cz 1. exa=- _te cr cye_ra-ce is ..c. a-c is Yresentiy uncer i wc=:i ca: ic~. ;he - -•_ - oei Ceveic.e- • :`e i, _, 'on c- .,:r:g C2:Cincce.'.ess s..es.s -•c- :_t.'1 =ai. y v:.+.ll c.-arac:e.^i-eC.- ;.:'evi^,:s C-'R-C, r.sc-e= _ at r::, :itr re_ar" to: i) tne i ra- ;racheal ccse •ac_. re= _ ir.cuce ^ai ic-j='-cy; 2) the aPprcxi-a:e .i^e course of __-a =.•z les io-s; ~) tne ceneti s.:sce- tibility of --.e :ari^_=_ -.:se strains to carcir.oser.esis; -) the dose a.^.dtyce osr -.e-ical that is carcinogenic; ar.e =! _`e induction o: aryi nycrcc=---.cn hycrcxylase (.`.i4:•i) activity+ as it correla:as :o t`_ ce ne: c s;:sce?ti-ility to neop;asian CT~ CGNTRAC : S C ~. O~ ~G: S ;a~ I CTR tMIN 043791

I• e':a lvacirc the •.:rir.e .....del wi t^ rc =a -o to .,;3 sslcle ' :ie c carecte sr..cic= may hz _ in lunc _a.r.o- ger.esis, several .•csicrs or a bicc!~emEcal nature have: ,.een raised. I) ~-.c'., l:ne is :he chemical in the !L;r.e after .. :ra- trac'.•.eal inccu !a:ic::? 2) 1fhat is the dis.r..._ ion or --.e chemical? 3) ',tr.y are several larc: doses reeuired fcr -1_ i n c ior o a r ..i -a^ lunc lesicns? 4) Are ,:he bieic= -I variatior:s in t -.cr incicence related to the variaticns n inctui ,,.'cn ^.':.c:- .; z i varia:iCns? 5) is it pcssit! to de:er-ic_a nc. ^ic cically rtlevzn: ,,,,;e after e:<2 . ^ ,.. a:., cer:an.- . lur- 6) C:•es s^c>;e exposure hc;e an e'i;c- cn. ~~ a rc ^__ r:te of a c'^e nica' intra:rccr.e=_I~y ins:illed' ') -. _rc2 - cells in the ,:re d i-=era -n fcr ch:...•cais ii. _rsucirtrat:'ach2ai y i~isci l lec. -e E) -e tha -c_s celis~ci -ent for iner- versus --^iize d A c!nemicals? 9 -y a-e t^e i;inetics of ~Hh~ nduct:en cifr;;r- ent in mouse pu'.ry nary tissue %:`+en c`e^icais are iven IT (24' • hc •s) c=-oar_^ _ -_ aeresolized (6 hours;? 103 Does the carticl: sizs ^ r_c^smical play a role in target cclls, enzym= ind::c:ien,-e:abc) ism or mechanical ciear3nce? !nitial studios have ad.^:ressec these questions and results to date will bc descri::c. 2. Intratrecheal dosimetry studies. The clearance of j4C-MCA from the lung s;hen civen by intra:rac:-esi inoculaticn was deter-iined ar.d the results are presen:e= in Fioure 5. Four different doses ^- N.C:+ were used in --is experi-,enc to specifically answer the question of •:aet`=r the clearance rate :•:as ti function of the size of the cose •:.s shown in Figure 5, :he semilcaarithnic curves for the _;-: and 1C L:o initial dose are reTari:ably sir,ilar a,ith :he clea 5C`t cf t!,e .ial dose e' u:ou: I - _r. ~ne . ..rents cc varia::cn (C.v.) for -nese es:cerir._ -s are - even when ,:si. "v rice per exneri- ^e!.:ai ^oin: i!:e ,-ascn . this variab • :y is mcs- 1.<ely c'_ i^CC_.a:.' a :`--!lire su°.ensi.^.. cver whiCn :.ne a-..,,al has no cc..- -.. 'n r.^ala:ion dcs.-et-y exper.-er.ts `a'.e much ic'::er -:'s because when .^e animais are s!milarlv res.-air.ec an: a'ic':ied to trea_-e, similar amou..:s are taken uz. The cur'.es cr =e :O ILg arc 10 }:c dcse azpear biphasic (or rossi:ly t=asic) in that - the first 6 i-curs e-+earance is rapid ..^ile relatively after inocuiation, '- little is clearec c...- to Z= hears Mic- _cram quantities are detectabie ,:•:o weeks cf:er =ese ini::ar doses were ci'.er. Similar results cn :`e nature cf :earance has :.een reported in humans as a,,,r.c:io^ of partic:e size (2). 13 ~ CT~2 „ „ (_f< <.: o ~'CC~^C''~ t ~ C T R N N 0 43s `9- 2

L. / i J The 1„ . _oses, C. ICC L:c -.C C.010 ,: s: -cs, a....^er co- i-,eus ..eara,ce 'o^ the I; _ (Thes cses a-e z;jrci:.c.aal•• ..na: .:I.. 'e c°.l•ivered by a I-C-mC:: ciccrc: °) The varia-,ili[y Ir t^ESe .r.Cses is su5stan:ial, bL't, . u1C app=ar'thcc t1/z ;or C•.IC0 ~g is about 4-~ hours, fc•r`_. ._ ' Lc, over 6 hours b:., less ,r.an 24 hours. The a--zunt cf ._r..ei..- Ing a: 1E' hours afterv.iL'~/ :9 Inocl:l<;;Ic^ is s lc.'l l•.~icz.^ and 'ctcvCv~u;, i`~e a'CJnt re-ainSz z at 24 hcur5 c?:c. _~_ :round. is -ot a~.,cve The er.__ cf sr:^k: on the c'ear:nc_ ef exer,.ir,ed :si, :;F.c .p V -i^ r initial Tcses (Oo:tc: ti -.=s, Fic.:re ~) ,ice :.ere.., us•~ z~ z, as 4 anes _..ic, al ici c_ to re^..ver a^ ;,soc ,. ' -„_ S::11 I`or o1: -.,l ci ca `._. I t r.as :hz fi rs t ancJcni y s,rm oi;:• e r. : csur(-. the a:'ii-;alex:erienced. Ti:ere may be a s 1 i c . retzrcaticn uo to 2L ho_rs due to scoice a: 6- e ;0 r; ese,^ however. after 1 we=c r- levels of MC:. remaining ir the i;;n; werc the same. t;o e;`ec; due to sTokc _s coserved a; the 10 jug dose. The ccef,`icients of vari: ^ were larcer for the s-clce er^c=::d and r..eciine control• ;e i.ot she..r.) , end this is probably ,,e to t`a ir.iti ._;ess of the res;ra!nt and associated iiandlinc. Thus, `or !".CA i t has beer, d tabl i shed frcra th i s exoeriment thac th.e clearance rate is L :ur.ctien of cose. The kinetics es clear;:nce are different fcr initial nicroc_ran quantities ccr;cred to r.ancoren quantities. It has been assu-.ed that t!e czr;icie size of the N•C_' for the four ccses was sinilzr since the _nia . zeled and labeicc c'CA was precarec . i n the same c,ar.ner. Pretiminar results recardi -.-.e -nec-.anism c= ...,;arance or •:C.1 ... -e _c . y indicatc .._ ac _r,:la:ic, in a-y ci .^e _ c_a-s znaivze . the -c-ain.s. 'ranszort •cn ,^e -g intc ;ne not irdica:a_ - ta 6 hours - . an•v c" chc cc•es ';-ic:s._ blcod ar.d '---rsys _-s -cv - r~sc.rsicie ~-. c!eara-__ s^_esci -ha: ; t`e~:zr; s zx^c= ,`e MCA %:as s-_1`. :o be'reTc•:ec =v ^ .•.,,co-ciiiarv ciearance r..?c^anisr.,s; "; - MCA was aepar_..lvre-evec frcr not cnly cha lunc b;,; ._ cv in less than _ hcurs !1 or the craan se-.,ies have ...- :een analyzec as yet a-1 .. firm ,, ,, .• clusions about bcdy c!etrance must wait L-:91 then. The cieara-ce _. 1~3aP after -tratrac=eal inocu- lation was also deter.^irec ar.= :hr results for both BaP and MCA are presented ir Table I- Cne dose (i7 mc)of °aP, with er wi t^out 0.6 .-a_ Fe-0. •:.asir.tr•atrac:.oei iv insci i led into either BC3FI/Cum or CSA/2J mice. These t.:o strains were selected on the'basis e-1 -eir cenetic s::sce:;ibili;y to t•1CA• incuc lunc cancer, „zinr sus: =tible s;-ain anc :JDA/G„ beic a resis;an; s:rain. mice are :.hN ^.cuci'Zie, •i'e are nor-i .C.e. 4o -!earar,ce c, c=is .,cse „ aa? ~.as^c_servec 'ir. e~:tt:er s;rain fcr :wo 14 c7K_^o~~-~~c-~ 010~00 I C TR- ~N 0~'~~~'~~

L• -1 y _ r the 1 I. i.l j~:is 'ath° r s tC,.!.:'?_ res'.:i.is npre5eltly C_irc i:lveSticAted .:i:n ccnsiCerZ:c'1 beinc~ civen to: 1} the io:al cose that r.zy paralyze 1_, cle'c:=^ce r.!ech.:nisr.s; s) the internal Cis`ribu:ion .:i .i- the lung :r such a UCSe; 3) Y.het'_r s-aller _Oses of 6? .:i.~ te clCereC aL ratCs -..-;.ura~ie to P':CSc; 4) 'e innate C.'E-...t pro;-,.,ie: os Ca?~ccm;:arac I'CA (s;._r es crystals,.re and 5)ti/2 fcr such a ccse. 3. S_,1 :I ,._-si-_.-y Studies. Our r.-.•ir^- r.c~el for its bSS.^c on cilu .cais -'2:i' .. -SIy inc- i'-~. l- sC•. ..._-'C i ccy f'.as EGVa^ ~, i: `.cs ...:~ ccSe pCss'Ie to Co5` c.--c rre„7es GiesiiOn in an ex:ef.-.,,1 of the .-:.,el; t1F. is, cces chrr.-,ic inhalac ..., of toeac smc;e havc ar. effect in r. ..., r,cdel for lung ._.:%oc_nesis^ In order to dc`ine ar._ -ar._ terize the role chronic in^aia:ion of tccacco smoke may have- in a murine -cdt fcr 1; carcinoeenesis, an instru-enc wns nePC :d ca;c51e of rel iac;% aenerction and delivery of smoice to l;:rc_ nwrb:rs of anir..als_ The Smoke :xposu.•e Macnir.e (S_;t) w,;s dc'.clra.. fcr esc in chrcr,ic, lar e-scale sircke inhaiaticn studies. The S:M auccmacicaily f oads, icni:es, pur`s on cicer=.t:es, ;tlivers and reni:„rs a precise emcun: of freshly c'iluted s.-c:<: for nose crity ex~osure of 480 -ice at cn,~ time. In .^is sectlcn, cosir,etrv experirnents usir.c the SEM II will b2 desc:ib c. in a latcr section, the preiinninary results of a chrcnic ir.=elaticz experiment will be descri'--e:. exc^r Ir entcl A cor,_. , dose rssocns;,.-ias cbse-ved for a varie:y of o-s and 8C-:= of c-a smoke taken was 'cuna int7e lunes, .'i ^ .-.ir,c. :uanti,ies .:nd in otier :zr:s .. of ,-a :•cdv. Ir. ;acie .r rtsults three desic ^. :.x,^v°1' '.en:S are ~en!ri.l)v iejs :t1ar lCi '.~ ::Es r,,,;c in eny of ;e ...-_ r orca-s, inaica:.^c that t~e in s-.c!:= .:as c_' iv_re= r.- to t;e -.c. The ex.csu-e conci _„ns of iCi~ s-o<e or -se :ics ;arterntti-g ti, sC sec..r.cs purce air .r_r a :ai10 mir ts) result_., in a..._. :ne sa:ce ce;cs i ticnas or =0 seccr.cs. Severe: ex=er --ents were carrie,^', cut to ce:=r mine whether the ce:cs.-?cr: :,cuid cnance with smoke concen;ra- :r3ticn or time of ex:cs_re. An almost iinear response was observcd for smoke cance^:re:7.n r•,f IC; and 20% between i0 tr.d 30 seconds (Figure 6). The V linear pc-.ion of the line in =i gure b~.~as the IC•`; src:ce. s..econcs excesure time and :rob- ably represents a minime.i - ~sio'c ievei for detection. :ata presentec in Ficure 6 i.._i.:ces ,ar_.- ate cepcstic.. in :otri male and `er:ale rnice. No oi"erer.ce in depcsition was cbserved as a fur.c:icn :.° sex ur.der several conditions ; Tab i 3 7). 1't i c3 e:<2CSld s-ciCe rCr 'e f i rs t time may bave slic"tl,v low2r (20`,i) Ce,^•csi:ic:1 than-nada-Ptecn ij C"+,~R CQN! R~'-tC'~ 0220301 ) I CTR 11N 043;7`9 4

azi-•a'.s, as the data .•• FicLre present. The ceaninc and ei 2nc the ceneralized stress r>s=zns= :o restrai^ and sco<_ exposure wiil be discussed ir, a lacer sec:i~n.(11.0.1 ). l ( The p__'c'_-1ate ce^.ositionin the total re= r;:cry -a_- . (~ ic;,._ 7) r:-. _, t=•e ceposi ior in th e Iunc a ne. 'hus ••.c increase in ce-osi ien occurred in the head or la_r -x t`,a: .:as not refiec:== iri lung with ei•her increasec s-..~_ _::nc_. tra:ic,•or e•=_-_,_ .•-.e. Ln ev.' of the nur.erical methods cf es,.. ..n_ dcsi-_•.•y dat= -= =e°.. -cce. Lungs %•-ere car lly ._ _-__ a- , ^.,, ,•,eo= sac-.- -e anc the cart iculate depos i ,, .:as _..en orrelated wi:- .`•e lu-c .eicht (:•.et tissue). ::s ,•`.e results in Ficure E c_-~cnstrate, a linecr response is acain observed `cr bot.`. -•_.e _cncen:rations and between 10 a~c ;:, seconcs ex:esure t'.-e The res:.its presented in Ficures 6 anc 5 are Suitz similar, in :hat increasino the exposure tice ro. 1~ to 3 0 seccn ds r =s_.tec in a T?:t or 7PM/.1a lun _ increase of 7-8 `oid. These 7nea=__rem,ents are more precise than those relating TFi•i/c bcc -:.eicht, par.icularly when deposition is the sam° in animais havi.- cifferen: body weights. For c.--=risen. data fron other sroke inha,a:io~ laboratories is pre:e-::c in Table 8. Animal exposures usira ne S:M Ii, the h-Sli ar.c :he Maddcx/ORNL (3) all result in c0-9;'6 of the pa rt i- 1ates i n s-noke to be depos i ted i r, the lungs. No substan: a: deposition is observed in the head, iarynx =r ste-,ach. In cz-:rzst, ex:osures usinc the Mason (L) mac`•ine and the conciticns c, ton:ir,ueus smoke resulted not only in -~%ree deaths bit . nic_n part.,,,:late cexsition in the ne:_. 'r: e particle size - smckeaerosoi mus be carefully ccr. .._ lec anc cuan:...=_ if •. is not, the target crcar. c=y -.,. be ;ne lune c: --a • hic;-.er in the .es:iratCry .^ac as by Ccr -..... S._cics acc-essinc the c.ues.• ..- a :arcet ^. car and ._ ;_- _iis are in progress usine aut..rac:c:- --z:i;ict__-nic__- _... .•;.1 -e ..;s:.ussec .a:er in this se,.. on. The -- Z^+sn'r -e• een ;ar ., •late depcsi.i„. _•- _ car!:exyhemclc= leveis in mice after smoke ex:cs-re_re s.shown in Ficure - T-e s:e- :notome: ;c ..•„thod was used :o evaluate CCr:S • a_:~e:s in `- S=!" lI dosiretry exoerir,e^:s and the levels are -=-e•.-a:ic%•er than t^ose cbtained routinely :ith CO-Oximeter, r.=::=•:er, :er i late cepcsition could ,.be r,,, (r=. =_ ). These measurementsreflect correlated to percent ^ :he upcake o` CO .cn ••`•eca ..,ase and indicate that CC'Hb can be used as a reni: ..- s:nc,cs ex_csurt cr exposure ..~es creater -an _-0 sec ncs (= __ wcre detaiied results concerr,ing =~!b measured usir- a C:-Oxirt:e: will be discussed in another section 16 cn~!TRRc-s o2a3a2 c~~ C I CTR VIN 043795

4. Acu:c toxicity. :.ll cf ;e s-e'r.e exposures reported far .:sed ,-._ 2 :1 cic_,are:ce (hieh tar, !er, nicotine). In cr`- . _ : . , co-:are~the e _ r -:s cf s -,oke cn pzrcicutate ce -cs i;ie.; „_. leveis, anci varicus biochemical, and histoiecic resoer;es, other cieare:te ;y;es were evaluated under stancard ex , .. . .;ors cr. the :!1 1 i . l. oC; l fe-.ale mice that had no; teen c-z icusi! ex.cs= s:,cce ..ere csec to Ceterc•ir,e the ie:nti ccse c. `our c` ;~e .ollcwinc ciczre;te types: i 'e -a.' N 1 f! Averac' 2i.1 L"v.90 2.81 13.2 1R2 2C91 1.70 10.3 1R3 2C.L2 1.2,= E.0 2A1 36.13 C.S1 10.2 The mice were aeaoted to the stock-type neck holder for 3 days :~rior to smoke exposure cn the S:M II a under the follov,ino conditions: 10„ s-cke, ;0 seccnds exposure time. On the rrrst cay of smoke cxpcsure, the mice were first given 3 puffs fr one cigarette, allc::ed a 10-minute rest, then 3 puffs fro-n --_ second cicore:te. The nu-ber of ouffs was increased to L . each cicaret:e c'1 :he second day, to 6;.uffs on the third Ca'/, E Fuf`s cn c::e .ourt, day and 10 puffs on the fifth day. T`e experiment was tercinacec ar;-r five days. The data has been tabulated as a functic- of :he intecraced CO and par ticulate levels reac hed durinc t=e s-~oke ex~osur=_ (Ta:!e o; The data from Tabl -lo:tee in i' cures .1 ar.d 12. The fcllo%•iir._ :ee•, c:te.-ine_ , c- these -a:-s - e 9 has 5eer: LD_ val~es r:av_ - L~ ~.... ..c;,:= ExPes ~ in -e .. Thre- : c_are:._ Types In:eeratee Part _..:la:e ^ Intecraced Cicare: Value CO yalu_ :;RI 1'~ 13 1R2 14 , 15 1R3 13 17 The inte^r_:z:artic::late and CO values are remarica6.-ly simi:ar. Sfr.oKC ..••, ;hs -':.1 cicare:te reac-ed ric°er leveis cs ..::c C.,and yet did not cacse ceath at these iGveis. :uc`. results suggest nicotine tcxicit•( as the cause =- Cea:- for c-_ ccher three cicare:;e types. =.ecent resul ts s=:__ __... f.' ica=:a: i c.n., t':at i s, i anct ..-_ CTR CC~~'~RC'~ (~~0~0~ I CTR MN 043->79-GEo

L. ( I ( to i^crease t`= of puffs/c:carettc, results in -ea• sc,-vival. ,i arc presently beinc adapted to sra, ~.=cn c3l cicarc::es usirc this slower adaptation regimen. The foreeoing discussion and resuit; h:.ve emph:sized -hc ^hysiolocic effects of smoke in the lL:nc rcgcrc tc the whole l!e now discuss experir. -s di rect: tc•.rarc lacc! ., ng the effects of smoke wi th^,i t.`.e lung usi ; au:crcoicc-c;.hic and histclo.ic tecl.niques 5. .%.'Jtcradiooraphy. j1'C-Do--iacontan_ (14 C-DTC) has be''r usec ._ char.x ,L:.za t` cis:ribution of par iculate matter in -he varicus orc;ns ef the rouse after s-oke erposure. :TC is a naturally cccurrir.e cc-pcnent of sr.,aice end remains ~sscci=. ed %•:i th the l;,ne far at least 24 hours after smoke excosure (Figure 1). The autorzdiographic studies described here are the prelir.+inary attempts to determine the intercellular distribution of the DTC in the lunc. The following is a description of the techniques involved. BC3F1 feTale mice were exposed to smoke or. a WHSIt usi-e cne 2A1 cicarette loaded with 100 uCi 1kC-DTC. They were-exposec under standard con=itions (10~ smoke, 30 seconds expcs::re tice). Irrmediatcly following sr,.ok: exrc- sure, all mice were sacrificed by cervical dislocation, the lungs infiatec with approximztely I cc of air,excised and quick-frozen in cry ice snow. Specimens were mounted on a Minot custcm rotary -icrctcre ,tithin an IEC Model CTO Harris - Cryostat at a chzr.ce+ set:inc of - 15' C wit` the knife blade ;.revicus- ly cocie ..o a te~pera; below tha: of the specima^ short r•lasts c' Freon. Loncitudinai •:.hcle-lung sections (iC ~.:). re .._: anc -:eunted or, cela::n-co~:ted micresc -e sl ides -,a : ar.m si ice ,iethod. Th:s'= .e re placee in tne be: cm e` „ c~ycstat cha-oer to re'reeze and desicca:e Iz•y cublir,a:ic- for several hours and were subsequently fixec in r.etharoi a: rcci: te^:erature for i minute. AutoraCiccraphy was -erformed cn these slices utilicine the cir:ine technique (16) with Kodak NTo-2 ruciezr track e^_isicn. Slides with sections were dipped once in the emulsion a: Lj` C and allowed to air dry at room temperature for I hour at %•:hich time they were piaced in slide boxes and placed in a light tight container over CaSOq desiccant over- nicht. !n .^e morning, slide boxes .ere closed, maintained in the dark a: -~' C for exoosure resultir,g frcm nuciear decay cf Ca-zcr. 14. Aut--rad i cc rams kere developed in Kodak D-19 deveicoer,• ixe= in Kcdak fixer and subsequently s:ained with n;,,.ltar `ast red (K3rnecntro:). 18 %.. cTR ["O'orl-; RAc-S. 020304 i I CTR NN 04'-R79..'7

;.. ihe raata• diffi ,;l.v encount_red .:as =z- taiainc suitable ticns frc- whole, frozen 1••nc d.e o the larce size o` :=e sazcir n (approxi-:a:ely 1.5 c- s_.`ace area).'anc the e•r.: _-e friability of fresh frozen 1a_,issue. The ecco^panying photeeraph (Figure 1;) ce-.c-- strc es tne resuits ca: In oeneral reduced si ve. -_ a.r.s are d i s ,-i;.icr .`e large, air %:eys, the brcnc`i -.c br„-.,.-ie'es, •:i,r s...ailer a-cunLs ct)scr•.e.d in the a;.ecl~. i:r.,:e-i- •,:s are in pr --ess to impr . -_ rescluicn e` ',_ s=_ ,.. In addithe dis: t:'.°_ Crains B: 1^-' .:ir•-es after smoke exposure ':ri i iy.,,1 y~a'-in._ to ca:e nr•ine i: :ransioca:icn occ;:rs. =rem previc_s s:u=:es (Fic::re 1), ;ittle iearanca of CTC fro:•i the lung eccurs befcre 2L hcurs. T,)e-efcre, it is of irterest to cetern-,na whether the celi types invclved with s-,oke particulates change wit`i time. Such informatier, r..av explain the diffe-e-cc in the kinetics of s-oice-incuced I:HH (~ hcur peak) ccmparec to che:nicaily-i:,iuced A'r;H (24 hours). 6. nis:c:athotcgy: t•:hiie experiments are in progress tc iden;ify and charac:erizs s ecific lung tarce: eils `or s-cke par::__ lates histci -ic data :ras cbtained recardino the biolccic results in ,rie 1L:nc os chronic smoke ex:osure. Af:cr i-: months' expcaure to 5-10 2;l cigarettes, BC3F1/Cum mice exhibi:ed no ror-ei !;:ncs and a high :L_rcentage of ir,fla~a- tory lesicns (Table 10). Adenomatesisand pulmonary aiveoir,r macrochace ac__r..uia:icn (?~~lA) were asscciated with s-...:a ex:esure, exci:aiveiv causa: tv smoke expos;:re si-ce Cc^.'^l ^..iCe ::ere casiC-.aily cC°_e•':!: to have these les._,s aisc. 'o ca:e, no -c -crs have :::-, o-servec in ~nice ex:csed - y to s-cka. 1 a^ zt:e-. ..Z determine the ._:ers ibiiity cr .ne cbservad, s-cka :xcas,:._ :s s-:': enc mice t-ave sacrif;ce.c a: r.cnt. intervals. ''- e inr.a-...=_=rv lesicns -ave persisted c_ :e ieng a=:er t~e last sccke exposure (cver - months). '7,-.e mest severe l;c lesicn obscrved in any o= :ie mice ctserved was an alveeier non-ccm.pressina -ocule. Preiiminary studies were performed to de:er'ir.e the effect cr. -,`e i,rng of tetradecancyi phorbal ace:a:e (-=A) aerosolized ins-oke. In collaboration with Dr. Peter ?crc=ert, itt was dr-ermine: `.a: T?A could be aeresolized in cicaret:e smc:e anc~ =a: ~•elevant cese would :e available :`+e ani-ais. ::.3F1,C:.- . .-aie mice beca.:.e cuite sick i--e._~ia:ely after exces_re- to s.-o:ce containinc TPa a: two concentra:ions. The es:im.a•ac dose ._ :`e lung aras 0.6 _ or 0.06 La T'... ~c -:ic eiec inc _-e _we-week coservaticr pericc, ,c%•:eve', ! -- Ose ^'OuDs aCDear_C s ick th.'C'•C.`CU: tnis .-e. s.=. ;: e:ai a;ior, cf _.ssues fror.. ,ne anir..ais 19 CTR CQN?'RAC~~ 020305 I CTR HN 043796

I eur.: t=e - :ericd did not she-:, an~• a-nc. -zl -,_z;,,res. t:o c~? iula r~prci i rcratien or cei lulzr s;eci iici ty wzsc'=sel.•ec cue :o TF-S. in t`e luny. ( 20 c-: R %OoN:-RRc7"S 02o3"aE CTR MH 043"1'`9'5.11,

D• icc ^ical Response of Pulno-ary Tissue to Carc;.-.ocens and Tobacco-~.elated Ptater;als. l, Selected assays for the stress response ir, micc as a functien of ace and expcsure to the ractiin.- or Smo"'2. In cel latora-cion wi th Dr. Y+, Ess,-an at Queens Colle- in York, s:udies have been initiated ;o r•.eas;! stres^ .r. ...e exposed to cicare::e smoke. Several ti est,,:s have =n accressed: How car stress be r.aasured in a mouse, and ..ri taria should be used? How can aa2~tatic' to stress be r'aasi.ed in a:noC:se? .'haC effeC dC2S ,.- ace • strei :, er sex of the mouse have cr, any criteria -Fat are usec? What efrec;s co c.hronic restraint and chronic smoke e::,:esure have cn these? Several criteria have been invest;gated as possible measures o; stress and adaptation in the mouse; body and organ weiaht, plasma ccrticosterone (PC) levels, cardiac norepire- phrir•e u;.take in vitro, adrenal corticcsteron,; synthesis ir• vitro, and otas=a cotechol ariine levels. ~ As a cross r.•easure of stress, body and organ we i ghts were daecrmir.ec after BC3F1/Cum mice had been er.posed to smoke on ei ther the 1•r-St•1 or the Sia•1 1 I under various recimens (Tab1E 11), The aata fcr appropriate groups presented in Table 11 was statistically treated (t-tes:) to deternine sieni- ficar.t differences (Table 12) for organ weights and organ-bcdy %•reicht ratics. The heart, liver and body weights were sicni- ficantly dif=E,rent for all grcuos cor„pared except the r,.ale shelf and -achine .....,trols on the SEN, and the female shelf and smoke e:::cs;= "crossovers" (alternately exposed to smol•e on the t•lHSAt cr •=is is of ir•.teres: in the case of the cross- overs, since :-e male crossovers ~e^:cns:rated a siani=icar.t difr„-ence r:,r =e i;2art. liver anc body :,cich:s bets•:een s~%ei4 and s-„ka e: r-:e_ a-ir,.a1,, :ale ar: ,'e-+ale crosscvers wert the sarr:e ace2r•c star;eds,T.Cke exoosure at the same ,ime, L_~c_ weich: t•:as-a- indicator of differences bet:reen crouos in two cases: °,ales, shelf a-c smoke exzo.ec for 1L weeks oq the Sc'•1 (P - 0.001, crouzs I and 11); fe.-nales, shelf and s;noke ex;.csed for 21 wee;cs on the klal;cn (P < 0.0001, groups IV and V). Since the males were given 2 cigarettes per day and the fe:nales 5-10 cigare;:es per day, it may be that dese (cigarettes per day) does not have as creat an effect as the iencth of time eposed, In s~~port o~ this, the male and female crossovers (4 weeks' smcke exDosure) did not demonstrate statistical differences in i::ne weigh: when ccmpared to the proper shelf ..Cnt,,.•. 21 CTR CQNTRAC } 5 020307 j CTR MN 043800

( ( ihre: cr5an t•.eicl:t ratios ape:ared to be sig- nificant -arkers o.-differences bet:•:een shelf controi and sco~c-ex;.osed :nice: h:art/lung, lung/liver, and lun:/body. When eil test ;tr•d control animals in their respective creu:s were ex=_mir.ec, crotabilities of P< 0,01 were calcuiated. Res:lts cm a preliminary experirant to deter- r:r r_ e'tec; c` ace cr a single exposure to s-cke on PC levels a-e res:^:e' in Figure lh. Mice of 4 different aces v:ere c>:zs=_d :o one 2:1 ciserette c.n the 1•1HSt•!, expcse- to t! c=„ _. s-.ck= or %:cre untreated. A, cce- i -- th.e sh:i^_cc- -.ols, PC-le'.els change r•aturally in EC3"1/C::nfe- male r••*ce, -!` the maxit.^.urm CCCurri nC at about 8 waek5 of age. Very li;:le difference in PC levels was observed be- tween e-e-:~.. ?c!^.ire exoosed or one-time smoke exposed mice. -=us,-:-e ^.. ?c !^.ire added effect of scoke on the mouse did not resuit in any hicher PC levels. PercFnt carboxyheT.oalnbin (CCt;b) %ia^ determined as a fu- ;ion cf, adaptation and incre-sing exposure to smoke or r.:achine c^ the 4rHS"t. using either the stock-type r.eck restTaint or t::e cone holders. Mice exposed to smoke daily were observed to have hicher COHb levels (6-7% COHb, Fic_ure 15), after reachinc 8 puffs plus 8 puffs/day (the third o:ee4: cn test). Mice exposed to smoke one time on the day of the essay •..-ere shown to have levels of COHb comparable to control mice until two entire cicarettes were offered, sug5esting that :he first exoosure :•:as so stressful that either r.,, mice co nct breathe for the amount of time re- cuirec ~~offer o cuf,°s or the clearance rate is accelerated during t=.Es L:-,ic::e `i-st exposure. Continued analysis of this is in .rc_rass with reeard to 1='C-DiC de- posi - i_::is. carciac norec:'fe^ .rine uptnke and acreaal 2. ^eterr`r.a:icn of the .e re of pul-ona-y cyt....n•,.-es i^:e'.vec in s-oke-inducec :.HH levels (C'.,-s+2A)_._ In coila~-cra:ion with Dr. I. Wang (University of South Carciina) s; cies using :olyacrlyanide gel-elec;ro- phoresis to ce:ara;e tne various P-'-50 cytochrc,,,es of hepat:c micreseres have :een initiated. Dr. Ir.ang has succeeded in separttine and ider,tifying four different cytechromes. The studies ti•:ith pul-or•ary tissue have been initiated and al- thouch sc.:.e seoaration of cytochre.:es can be seen, identi- fication of ;~e nat::re of cytochre:es has as yet not been pcssit7r, :•:ar,c now has pulmonary tissue from C5i°L/6 (86), ..,,:./2,! (^2), I-3H/Ar,f (C3) nice induced by MC:., TCDD 22 C v C"IR ~0 f~ ; ~R C i ~ (12G :0C C I CTR VIN 043801

and control :•ehicle. I_entifict.:icn of the cytochrc-2s in thes= pre:,=ratiors is presently beinc done. S-oKe ex- posed ;.ulr„cnary tissve has not been anajyzed to date. 3. Effects of inhibitors of R.`:AA and pro:ei- synthesis on s-oke-ircuc_d A;-:H activity (CTR-E2o). Cycloh=::i,-ic_-, an inhibitor of protein synt~%esis, ond Actine- c.r D, ar i,~ititor of Fi~lA synthesis a,ere •s_r _ in crcer to ci,s2rva if t::einduction of pulmonary Lr-: ing exposure to ..-.ci: cicarette s-oke is dependent cr;. zrc/ or protein s;nthesis. The results of the effects c` c~c -_...- nide (5 and ;i'.D c!^__ bc:y -:,eicht) and Act..norycir. D(5 _c/c+, body a:eig' ht) on 2;i cigarette s^oke-inc::ced AHH is i^ Table 13. ls cbserv:c previously, whole smoke elevated ..' levels are ::ccut ~- :ios~old. Use cf the substrate et'-o:cyre:cru'in (ETR) insteac c` iaP ectually magnifies tais induc:ion. Usins either substra;e (:Tr shows it better), cycloher.inide (;OC :: )g and A•ctinomycir, ;: inhibits this smoke-induced nulr..onary mixec- function oxid-=st. Cycle;:eximide given a: a dose of 5 c:c/c body ti•:eight dicd r.ot fect the levels of these sr.,ok:-..^cuced enzy:r.es. Actii~cmycin D civen 2 hours prior to smoke ex- posure•seemed t~ cause a super-induction of pulmonary AK'j activity. 5;het!ier this super-induction is the result of an inhibition of breakdo~•:n of those RUA species responsible for AHH activi ty has not been determined at t~is time. The data shc-:;s that 2A1 ciaarette smoke-induced pulmonary A;;;; leveis are dependent on both RNA cr,d ;,rctein synthesis. Thus, this 6 hour induction.seems to be fvnc:ie,i- ally similZr .c =a '_--hour induction observed folle:•+ir ex- acSure to po::•C'!clicaro-atic hydrocarbons; however, - rate Of induction 81: l:Va: C' e.'1~•.9nes induCB= ~9re still ^..:i'e di fferer,t. t•:he:`.e- sucl,factors as 2erasoi vs. cornci . vehicle, internal is:. .:tiCn and l:otake, or physicai s- :e of the indJez. C--ui--4 Z;;p ain this d.ffere.^.c;: reTains to _e elucidated. 4. MeasL;re^en: of ornithine dxca-r.,oxylase (:~C) in pulmor,ary tissues. Quantities o` the CCC levels in pulmonary tis- sues following exocsure~to whole ciaaret:e sr..oke ano CSC= is described at lencth in the progress rt:ert for CTR-GCi4. 23 cT~ cof~ ; KP c-~ (~~o~a9 S I t CTR HN 043802

C 5. Use cf other substra;es to charecterize -ixed-f..rction oxi2asE aetivi ty. As stated ;reviously(II•D.2.),i:TR is an ei~fectI ve subs:r.~te fe'r s.-oke-inducec -ixed-,'unctior •..ase ~ acti+•ity• This subs:rete seems speCit'ic or the P-+= c,toc„ „-•e 7=e-_'ore, th2se observa:ions suecest •ha: oxiceses -ha: -,rire.ari ly u:i I:_ .- F-'-;8 cytoci)rc-e^as the site of electron transfer fro-% re:_ culer c::voen to ne su*,-strate. Ide are attempting to = _ so.r nfcr^a'icr, co^ce•nin; the r.=_t -e of the c;:oc-• involve= i.n t-,is cxication by meas_rina the ebilit11 c-he s-,c::e in_ c:c enzy-es to ceta5olize a i'a-i1y of resorufin derivativ;s (in coiiatoration with :r, i. i•Sayer, Uni•:ersity of Taxas, Collcce Station, Texas). The profile wiil then be ccr,.p, red to :::e prcfi le observed +•,=en enzy:r.: prepara- tions cre induced by inducers of kr.ewn specificity. T!-•e use of six substr=tes of resorufin (the methor•y-, et.":cy-, prcooxy••, isoprcpcxy, butoxy-, and isobutoxy- derivatives) and he;_tic ,-icroscre preparations 'ron MCA-. TCOD-. 5-6-benzoflavor.e, and control treated ZC3F1 mice is sho.•:n in fig:,re 16. Differences in the rate of for-ation of resorufin using the:e.vario:,s substrates were observed, but final interpretation -.:st await da;a using phenobarhital-induce^ preparations. Results to date succest that the Vmax ratio using ret!:oxyresorufin and propoxyresorufin as substrates is about 3 for ccntrcl preparation (?-i~50) and 0.5 - 0.9 for N.CA-, 5, •-berzoflavone, and TCCO-induced preparations (P-44u). This would suggest that the type of cytochrc-e cresent .•, the enzyme pre?arations can ir=luence the abil- ity o` these _•,:~res to metabolize this family of rescrufin ceriva:?ve:, 6. Ca-_ar• sc . of sr..oke-.. __ee= and 1•'.C:`.-i nd~;cec BC3F1 ...ice ::era exposed ,_ s-•oka from 2A1 eigar e' ss ~:.., F.ac -een , tified wi -•C.C . MCA (in =enze-e) and to co~tral 2:.1 cicarettes• T'-e :+H:X ac;ivity at vari- cus times after exocsure to these :aret:es are shown in Table 14. The 2al cigarette causec a two-fold increase in pulmonary AHH tia: reached a peak b•! 6 hours after the ex- posure and atayed at this level for at least 48 hours. The MCA-treated cicarette caused a slightly higher level of pulmonary AHH at all times examined, but the kinetics of induction were quite similar to that observed with the 2A1 cicarette alone. Co-treatment with 2-5 MCA-for;i fied cigarettes did r,ot enhance the AHH level oCserved at 6 hours post-exposure. There seems to be a saturation level of 24 cTR coW-: RRcTs 010,Z 10 I CTR HN 04,3803

L• r• AHH irduction ._cardless of the level of exccsure tc cigarett: smoke or to 1;=A aerosolized in _-o.l•e. NC%A aiore (given IT) at 10 * u5 also induced aulr..or.ary GH.y to a~cuc tf~e level observed ..ith either the 2:,1, or the MCA-fcrtified 241 cicarette. t'Ca at this conce^tration plus expcs:,-e• :o the 2A1 cicarette did yield hioh2r:.HH levels; ho'::eve ., a,he:her this is a true additive effect a:ill require E:di- ticnal stu:ies •::ith r..ore anirr.als. It seems that tt;e - fortifie= cicare:tes have little, if any, ircreasec e'=`ec: on „lt-orary ::her cc-n^ared to t=.e 2A1 cicare• a'„-e. SC7N animals that %:.;re previously exposed to sm,c'<e .,r 10 cir,arettes/ccy fer cne month %•:ere not further ird~ce: by exposure i:o i-:CA-,`ortified cicare:tes. Thus, these -ice were alrecc•; ir ced at zero tire and the level rcmai-td uncharced for the next 24 hours recardless of the nur.:e.- of h' =ortifiec c;gare:tes (1-- ) :nich t•.ere offered to the mice (data ^ot lcno'.:n). This lack of a striking effect in either of these studies could result frem the fact that the MCA %,,as given in an aerosol, or that the use of EeP as a substra:e fcr Ai;H is just Coo insensitive to detect the differences that exist. This study o-:ii1 be repeated using ETR rather than BaP a_ the substrate for the pul- monary enzy-es. Moreover, MCA given in an aerosol, given in smoke, or civen iT must be corpared for its ability to induce pulmarary AHH levels as r:eas red %:i;h ETR as the substrate. 7. Car_-cxyhernoclobin Assays. Cicar_;:e smaking has bee- reoorted as the most importEnt source of carbon monoxide ex:esure to al- r,•os- er,e-^a1= of the adult American epulation (5). Car=cx•;i:er„oclczi- (CCN;) levels in n_r..an sc.o:cers may !:e a r..easure of -e -:tn cr i^-ala; -, he::2-, levels :an ree::• 2-85~ (l,;,c) CC hasbeen i-„iicat:c as a cause c- ar;eriescleres:s in animai studies _sina_ ra:--its and s:,::i,rei r•.on:ceys Usinc .hs animal model for ciroric cigarette smo::e inhaTa;Ecr, st_cies, we have ce`ir,ed t=.e CO,ib resccis:e of the SC3F'1 fe-aie mouse to various doses of cigarette smoke. A ccr,iparison of two rre-t^ccs cf analysis for COhb is,given in Ta!:ie 15. The values c:tained with the CO- Oxirr•eter are almost two-fold higher than those obtair,e- using the spectropnotc-eter (1). The intra-assay varia- tion as reflected by the coefficient of variation for the individual assays is lower for the L3-0xir,eter in all duplicate assays performed. The ir;erassay variation is 25 CTR caNTRacTS 02431 t 1 CTR NN 043804

L• also lc.:er. Tie Cc:a sugcests that the a.mount of CO teken up micht vary frcm one day to another a-d .-ay be a function of a particular group of cicarettes (1G). Tabie.•16 sho,:is data `rcn individual mice and also ~e-.-,-•- strates one o' the biceest drawbacks to using the s;e-'ro- photc-ater. T%•:o values could not be determined beca::se no difference spectr~:-m was obtained, thus no calcula:::ns couid be r-cde. Cthers have ex:erienced this proble-• _si- the -etho~] (11) and althouci- the reason for the lost sa--~es is r:ot kro::. it m,,y be associated with sample prere-e;:cn. In Table 17, the cc.-plete blocd ar.alysis is given as dcterr:r.ed by the CO-Oxiceter for mice er.to s,-oke 3weel:s, 3 con;h;, and 6 months. After exoosure-to two cic8rettes there is no sicnifican*t difference in levels of COH~ as deter-ir:ej with the CC-Cximeter. The mice =rom whici ctta was obtained for Figure 17 ware exposed to cic- arette snoke Elone ;or 6 rr.onths (5-10 cigarettes per cay). The mice from %•:hich data was obtained for Figure 18 were treated with 9 instillations of 250 µg MC:, cor.cc:rrer.tly with ciaarette s-,oke (5-10 cigarettes per day) for 6 months. T;;e dose respcr.se -:ras almost identical for both groups. After one cic=-ettr.. 20-25'A' COHb was detected, incre:sing to beV:reen 5,5-60`,; after 5 cigarettes. CC3F1 fenrale mice seem to b: able to sustain a chronic dose of 5 ciearettes t,::ice a day :•iithout r.:ortality, thus suggesting that this strain is not :articularly susceptible to CO toxicity. The reten:ion end clearance of COHb is plotted in Fioure 19 for 3C3i'1 female mice after exposure to two-2ti1 cicarettes. The three different curves re:- resent three cirrera-it _rouos of mice, all of a!~out the same ace but ex=ose= to s-•oke for ci ffere-+t lcncths o` tir.e- 3 weel:s, -onths, and 6 mcrchs. The ,ice to clear SC:6 or the l_ve1 (20--=-) is bet••:een 30 ar.d L0 c;i nutes for a i l -re: c-oups. One nour• a.°ter er.posure, ..._ level o; C:.=t is still atcve c:ntrol values. ( ( Additional -aasureTe-:s will be r.eeded to determine whether the ^ercent oxv^e-+oglc~-in and volume percent oxygen -cnti.,,:e to decrease wit^ i,.,,reasir.c ...,,,..r.s of exposure to smoke. 8. Smoke-related inhibition of pulmonary D";:, repair in BC3FI female mice. In colla6~oration with Dr. R. Rasmussen at the University o.' California at Irvine, we have investigated the smoke-related inhibition of pulmonary DNA repair in 26 C CTF2 CONTRRCT_ 010351 1"2) I CTR HN 0438055

r 5C3fi fe-0le ..:ice.A complete report of the find;-cs of the initiai studies may be reund in Dr. Rasmussen's prccress repcrt for 1977. Sur-aarized briefly, however, the results from'these initial st--dies de^enstrated that mice chrcnically exposed to cigarettes smoke WHSM, 10% smoke, 30 second ex- posure time, 2A1 cigarettes, 5-10 cigarettes/day, L-5 r..c^ths) appeer to remain repair deficient for up to 2 months after the lcst smoke exposure. neplicative Ci:A synthesi: was cb- serva_ to increase at ~`e sarrz time Cf•tA repai r sync~-esi s aras i n h i'; i:eo The lat±linc index (LI) usir.g triti:.;ec thy- midi',a (sH-icR) t-jas sl;c%:ri to in:.reese in the lung, liver and ble.'._~er Cel:.:lar proliferation hes been in-,':erred,'rc~ s:ch ob-tervctions. Yiith these preliminary observa*.io^s and results, an e::^eriment h3s been initiated to answer the folio•.:inc question:: 1) l•lhat is the earliest time after minimal (two 2A 1 cicarettes/doy) smoke exposure that DtlA repair is deficient in mouse lungs? 2) Is the defiriency in re- pair reversibie in non-chronically exposcd nice and how much time is reeuired to return to r.ormr.l? 3) What are the earliest tirres after ninimal smoke exposure that the LI is increased? L) Is there a difference in the LI when determined i.r.-ediately after smoke exposure compared to several days after? Preliminary results from this experi- ment, which is still in pro~Cress, suggest that 2 weeks' cxpesure to t•::o 2Al or t~•ro ._R1 cioarettes/day i s not a sufficient dose to cause the inhibition in repair, ai- thou0h the dose did result in a very slig!•,t cellular pro- liferation in the lung for both cigarette types during this time. i:. Initiaticn ar,6 ;.remcticn of cancer in the lung It has been s_ccested thrt carcinc-cenesis is at leas: a two stace process. The first staoe, called initia- tion, results in a irreversi.ie event which alone cannot result • in r-alicnancy. The initiation step has beer, os:..- latedto be scre form of damace to D1.A or to the replica- tion of DNA. One prerec.uisite for studying initiation is that the event can be delineated in time fre--i subsec_ent events leading to neoplasia. The second stace. called promotion, when it occurs after an initiating event results in malignancy. Alone, the promoting event has little effect. The most well docur,ented "tumor prcmcting" agent is TPA for skin tumors. 27 • CTR CONTRAC"5 O20?? ~ I CTFe MN 04,3606,

The results we have presented in Cort-c_ C^1=.I!.C., Tablcs 2 end 3, suecest a tuncr prc,-oting or co-carcir.oce,ic effec: for '-= cic_erette smoke cendensate end the C::,-'s in the l::no. A biological expcrirrr-nt was initie:ed to test the -theory tha: exposure to chronic levels of cigar<.:te sr-.et:e had a co-ca-r,.inccnnic or a pro,^otine e`fect .cr lu-- t:,- -s in mice aftCr i,.itiating 'with a known luna cPrCin.cz^ (,`?C ) The follo%iin; consideretio-s were nade v:hen this (I-i00) wa; . , on test. ( The i;C.;-sro!ce co-carcir.ocenesis study in fe- male ^ice ..as cesicned to deternine t-:hether .re ir:c.__. ty,r cr ,ir.: cc rse of develcp^ent of carcir.c-? of i„-_ observ:dir• pre:~io_s intratracheel experir:en:s ~:culc be chencev; t~e mice 4:ere also exposed to c:caret:_ smc.e (5-10 cigarettes/day). The chronic inhalatic= stuc• r,as air..=u at cEterrining the chronoleoical progression o' sc~;ce indLc=d lunc lesions. The criteria by which :`:e mice are taken off test i n e i ther experiment ti•ri l 1 deter•..i ne what types of conclusions can be drawn at the end cf the exoeri- ment as di:c:.issed extensively in CTR 0014. If moribundity and death ~.re used as criteria, then the follc:+ing can be determine6: --leath rate due to treatment (both IT inoculated and smoke ex;osed), the death rate due to the effects of the dose of carcir.ocen, the latency of lesions and the probability of tumor formation as calc.ulated 1--v Saffiot:i (15). If nice are sacrificed rendonly at specified ti..es (serial sacrifice), then the tumor incidence at those times and the pro?ression of lesions can be determi-ed. Table 13 surtrsarizes selected data fram orevious ir.tratrach.;;a': er.oerir,ents where hich dosas or `tCA were used. The n-=:er c= OC?F1 .-ics t-at rievelo.e= t;.-:ors (Si•! :::+C. SCC, ASc) seems to be a~)o::t -ree -aic~ lo-,ier than C3•r:/:.n` and about the saane as CSi:_/c nica a_- ec::ivslent doses: =ck-:ever, :tie length of tirx tes- ::as not the sace :zc::t 2`; cf, iiC3F1 remale mice c:ed a-- 6 inoculations c` :SC;: given every other week. The -'sFi nice did not seem to die or beccme noribund early i-- experiment (ccmoare the D-v,/N for similar doses in Ta:ie 18) while the C3;/::nf nice a:oarently did. T=is mav :a a reflection of the numoer of nalianar.: t:.-ors `ounc the C3ii/:nf L:p to L0 weeks on test. Concurrer.: sr.c~:e ex• posure may hasten this tumor development or c=ance -t frequency or have no effect at all. In order to maximize the effect due to sa:ol:e, the amount of particulate deposition in the lc-c sheulcd be considered. 28 ~oN-RAc?-~ 0a~ ~ ~ cT~ CTR HN 04380f7

Ptousc Strc,in Nicroo_~a- ?articuiate/ gm Eody 1•ie i ght C3H/-'n4. 9 5.61 3.97 C57°L/b . 7.77 SC3F1 9 5.58 E.17 EC3F1 .`c-aie rice `ad the highes: deposition in c-e ex:;ri mer.t and %:As a:oc.' `.`.e s_c,e in another. It has tuc,l ce-y-r, sL'rateG :he' -., 1'emai_ mice can be exposed to ten G.,l cicaret:=s/c=y withcut ar.y acutely toxic effects •..-:n cb- served up to e ive mcnths. It would ac^ear therefore that 6C3F1 °c-:ale r..ice are able to :olerater.'airly laree doses of NCA by ir.tra- tracheal instillation and also tolerate fairly larce doses cf smoke cn a daily b=sis and do not die premat::re;y in the er,;.arir.;3nts of ncr.--elignant causes. The amour.t of information from the two inhalation studies has ~--etn creatly enhanced since the cocarcino- genesis a,c chronic inhalation studies were cer..'--ined and both moribur:'ty and serial sacrifice criteria rrre used for taking .iee off test Thus, the probability of luny lesions can =a co-pared for carcir,ooer.-smoke, cercinocen, or smoke tre:tac ani,rsls. A serial sacrifice is noc con- templated at this tir^e for the s-noke exposed crc_ps. The arrance.n_nt an--~ n_-ber of animals used has taken into account -a itoints to be ans~•:ered in -he ex--er.- ments ~•:hi ie -a.<ic most er-Ficient use of contr^.:os. Fewer ani-ais have -j__.t usec overall after the seriai sacrir.ce-tra-solar, :xz •i,r.ent prcpcsed on C', i-., .:es also cc...bir-_.:i n,-s inhalation studies. These animals then servec:s the ..cn-s^oice anc non-machine cen:rcls and have been the source V- tissues for transDlar.,a:icn. In additior., in orcer :ococs:rent that more tumors (S`i, SCC, AAC, ASC) are found in the C3ii/Anf. -ice, a parallel `!C,; intra;rac^eal stucy in this strain was put on test at the same tire (I-i01). These mice have been taken c'= test when dead or roribunc only, a us allowing direct cc-.carison of the tumor probabiiities for SC3F1 females and C:H/Anf females. The exDericental data to be obtained from the coroined ex;.eri- men:s includes the following: 29 CT!2 ^QNTRnCT5 020315 I 4r f f`s. i i f`f 043808

_j Mcriound - death criteria ( 1. Death rate due to carcinenen dose ir. both cC3=' an-d C3X/An=; death rate due to carcinoc_en-ssai;e int=racticn, teath rate due to smoke alone. 2. The probability of a lung tumor deveic:i-_ Z: a certain :irr afZer carcinoCen treatment alor.e, aft=r car- cinoc=n-s:r,;,:ce together and a`ter smoke alone. 3. The le:ency for each tumor type. L. :+ coo:.parison of the tumor probabi i i ty a`:er c; rcir.,~;e^ ,.eat-.ent in EC3F1 and C3'r./Anf feraie r.•i,,e. 5. total tur„or incidence ror both EC,~'FI ai:d C31!/l.n.' ;;ic=_ after equivalent times on test. G. AA comparison of the types of tumcrs founci and ~:I;sti~er ti:=se cause ecual numbers of deaths '.n the t•.:o strains, e':en if the tir.e course of developr.ent is different. 7. The severity index can be tested as to its p,±- dictive vcl_e in both moribund-death and the serial sacrifice stuaies and the results compared. Serial sacrifice criteria: 1. Histcaatholcgical progression of all lesions ef the lung ...:: tir,.e, due to carcinogen alone. 2. Oocu-entation of the relationship of prenialicr.ant lesic^s to reiicnant. 3. Tumor incieence a; s.ecified times due to car- r.c .n carc: .oc=n-s-c,<a .;etF,er, and due to _r.1o;ce a lo;:_. 4T-ansclan:cbility of rren.alicnant and raiicnan: lesions in „~5F1 `e^aies only. A comparison bets.reen the tur„or types a::earinc at specified tirss in the scheduied sacrifice arc t=e r.atural death rate 11 indicate the lethality of specific iesicns. Preiiminary results frem the serial sacrifice date from the P!CA' treated croups are presen.ted in Table ig. After 22 weeks cn test and 9 insti l tations of 250 }cg MCA, 3C3i=1 30 ( CTR CONTRRC T 5_ 0.20316 1' c I CTR MN 0431-809.

fen:ale r••~ice cc-ons:rat~d 21"; St! a~d 2155 SCC. I;o alveclc -ic carcinc-.as have b~en e~served after 22 o-:eeks, l:orrev~r, t!~e follo~.~inc !•;ype•a lastic lesions ~:+ere found: 64y mininei hy:er- plasia, 2„ non-ccmpres:,ir,g nodular hyperplasia, ar~ ; cc~- pressing nodular hyperplasia. Tie celatin-sali:e sacri.;ce? controls ~:r~re e~serv~d to have completely norral luncs. For rice trected ti~+ith :CA alone (~Y.) a-d ::~,._•. _~_ or ~.•ere ki-i lec.' ~:her m.o, ibund, -4e t_-or probabi i i ty a--2= ~:ee%s was catc:.~latec to be 0.C2b for SCC, 0.002 .,,. ~':, ~r,~ 0.C2 for sti e-.ous r'a:a;,lasia (Tab'.e 20). For mic„ ~~a:_„ ~~+iCh I;C:: (;`;) and placed cn the SC`I as r.*.acisir,e c= ~-rc[s, t~.e tu-or pre~e~ii ~y a~ 22 •:eek: :•+as celculated to ~__ C.C:_ "or SCC, 0.032 fcr S?~, a~d 0.1~ for s;,uarr.ous m~ta?l~sia (;a~le 21). For mice trea~Ld :•+ith f1CA (~X) and then ex~ose~ to cironic hici•~ levels o` cicare::e s-olce (5-10 cic:rettes/ day), t5e t_r.•or probabili;y at 22 weeks was calc:late= :o be 0.033 for SCC, 0.030 for S,1 and 0.121 for sa,ucr..c::s r,~taplasia (Table 22). ~(o alveoiooenic carcinomas have bee^ observed in an,v of these groups uo to 22 weeks on test. The prc~- abi 1 i ty resul ts for sc;:a~ous cel 1 carcinora a-d sc,;;a•^ous metaplasia are c,rophically presented in Fiaure 20. 0 nly inflar,mator~~ lesions i±ave been observed in the cro_ps ex- posed only .o s~oke after 22 weeks on test. No I~istoiccic resuits ~re available as yet for the C3H/Ani` mice put on test in a parallel study of t~C: induced lunc tumors (1-101). 'r.owever, the toxicity of 9 intratracheal insti l lations of 2:0 c:g 11CA in i3C3F i and C3H/Anf mice has been found to be Q.5X and 1i::, ~escec- tively (Table 23). Bcdv :eicht r,as ~eer. •Se: as a rat-er •;;n-s~ec. •°ic parzr-_-er o -~ac- -c effec:s- s~:eral in~ala:i s cies. Con;e .v+i ll ( i2) and S•lel.ner, e' a_ 13) r°portec si -i' ca^:~y dec'ecse~ ~o~'f ::ei :s - -2-5:Er5 ~?x~:3~~ :G C y': ette smoke. '*ehner, e,_t ai ;l-) aiso rc~ortec cec-easec body r+eichts in Syrian ccicen ha-sters exrosed :o cEcare::e sloke in cC~~inatiCn '++i:h cocal; c.YiCe comptred cr _~s receiving shan sroke e~:pesure and cobalt oxide. ~Scoke er.- posed animals had t=,e io.~est bocy ::ei5it under any conc'i- tion in these studies. Body weights :.re dete•rmir.ed at approximately monthly intervals in t!:e co-carcir.ocenic and chronic in- halation studies in orcer to dccu.:.ent the relaticnship between body ~•:eiaht anc treat-ent. Comoarison c.' :=e a:prc- priate BC3F1/Cu~ fe-ale mice (I-100) with C3H/An.' Cum fe- male mice (1-1~1) will also determine the relative strain susceptibility to decreased body weight (or lack of in- crease) durin; carcir:oaenesis studies. 31 CTR CONTRFCTS 0~Q~ ~ ~~ ~~~~~ ~~~ ~~~ ~ ~

01 ( T;- .: results of body weieht measurements for BC3F1/Cu-1 are sho:•+n in Ficures 21A, throuch 21J. The unir•oculated shelf controls (Figure 21A) cained between 15-20 grams after 25-30 weeks on test. Thc crouo whic'i received 0.C2 ml cel-saline every two ~,eeks ror 9 a,e=ks increased as r..uch as the uninoculated. More surprisino is the c5servation that c,roups which received 250 1:g H.CA in 0.C2 nl cei-saline every two weeks for 9 weeks cair.ed orly slic '•"y less .:eieht than the uninoculated rice (Ficures 21C. C). This same group wos shown to have a sSuanous c=ii carcino-,a incidence of 21„ in a rande.^ sacri- fice after 22 a,eeics on test. Thus, the implication tha: tumors pres±n: in this croup between 2L-32 weeks had little eifect c:: t~e tody weig~t. An entirely different set cf circurasta-c>s exists in the crouc:s which received either sham ciearette ex- posure lmac=tr.e controls) or actual cigarette smoke exposure. The nachir.e controls (Figure 21E) gained weight ct a much slower rate and aftei 25-30 weel:s on test, weiched almost 10 grams less than the uninoculated, gGl-saline or 1•lCA treated oro::ps. All of the groups termed machine co ntrol (unindculatec, Figure 21E; gel-saline, Figure 21F; MCA, Figure. 21G) responc;ed very similarly in general caining less than ; arams over 32 weeks on test. The effect of sr+oke exposure on body weight was not much greater than the effect of the manipulations required for smoke exposure. The BC3FI/Cum arouas exposed to smoke (uninocula:c-, Ficure 21ii; gel-:eline, Figure 211; MCA, Figure 2iJ) :aincc between 2-3 gracu aftcr 32 weeks on test. T~-e :nc-ease in body weic-t is shown in Ficure 22 for C3H/=,-i'Ccm -^ate nice (I-iCT.) a.`ter 9 ir.oc::la:ions of eithcr C.." -i ^ei-saiine cr 250 ;'CA inO.02 ml c_el-salina. The rat .;' i•^•crease i s a?Tos~ t=.e same for C?rl -_.:.-n as for oC3-1/C- =enaie raice after i., weeks on test^(cor:.are Ficure 21C,. anc 22). Conclusions: a) Hic_ii ir,tra:racheal doses ef carcinogen (2;0 _g MCA), repeated 9 times over IS weeks do not appear to iffect body weight increases in BC3F1/Cun female mice. b) The manipulations associ.ated with cioare::e s.-oke exposure in this system resulted in almost 10 crams differ- ence in body weight between machine and shelf controls after 32 weeks o~ test. Smoke e=-csure resulted in an 32 cTR coNTRRC-s o2o.3iE I CI 0.R HN 043811

(I ( additional 2-3 cra- ci;.-erence. . c ) A 1 t ' r , o u c ^ a 21% scuamous cel l tur.,or i nci eer,cr was observed from a sacri=ice of MCA treated "eC'sFl/Cu- mice at 22 weeks, no cene-ai weight decline %•+,,s observed for this croup. i`~ese results have implicaticns with -ec=.. to the co-c=_rcir.acenic studies with MCA and cieare::;- s-oke ! !-es ;.Een -- that cecreasec bocy V.>_i nt asso iate" .:i", exros:.re to cicarette smo!<e resultec a celay in a?pcerance of cvloidosis in Syrian :i_en h2r.,sters (1=) ~ Suc` es_lts cust be kept in mind ~:^en evaluatir ~e resul:s from e>:;.aricents presen:ly cn :es: (I-1CC ene 1-101). I 33 u CTR CQNTRACTS 020221'~ CTR HN 043612

F.EFER :!:C:S 1. Cc.--ins, B.T. and Lawther, P.J., 1965. A Sensiti•.•e 1':ethoc for the De:errir.ation of Carboryhaemoclohir. .n a Fincer Prick Sa-•Dle of Blooc. Br. J. of Indust. ..__~. 22; 13°-1L3. 2. --an-, :•1., . lber: A: Yeates, D.B., Bercer, J.`'•., / Fcs:er 'r'.M., Boh,i- D.E., 1977. Factors A fec:i-- Trac.`•eC 'y .^hial ~:ucc-Cilia'y Transoort. In Part.cltslV., eci;ec oh. Walton, Pergar.non Fr_ss, hew ','ork. 3. i<endric:c, J., ?letteshein, P., Guerin, M., Catc-s. J., Dalbey, 'd., Gries-.er, y„ ?;;bin, R., and Madco>:, W., 1976. Tobacco Smoke Inhalation Studies in Rats. 4. Toxiccl. Appl. Pharcacol. 37, 557-569. Binns, S., Bcveri, J.L., Wiltcn, L.V., and Lugton, 1976. Inhalatio- Toxicity Studies on Cigarette Sr--;;e II. Tobacco S,r.cke Inhalaticn Dosimetry Studies c; Small Laboratory Animais. Toxicol. 6, 197-206. 5. Golcsr-ith, J.R., 1970. Contribution of motor vehicle l exhaust, industry and cigarette smoking to ce,.,:uni:v carbon r;oncxidc zxzosures. Ann. N.Y. Acad. Sci. i7=:, 122-124. 6. Russell, M.A..H., Wilson, C., Patel, V.A., et al., i5',. Cer..:ariscn of effect on tctecco consumotion and car:cn ncr.cxic_ a~scrction of chancir to high and ic. -i,,,,::ne ( cicar_.-:s. Br. ~'ac. J. ' . 7. Ast _:, .., I:ieldcc-, K., ilens=u:). J., 1967. _rhar- r_ in`l,;er.ce of carbon -...nexice on the develo:^ , o- at^er-c'.tosis in c:loiestercl fec rabbits. J. .,t`er'oscle.. Res. %, ?', S. Astrup, ?, 1972. Scre physiological and pat~• - al effects of Tocera;e carbon ronoxice exposure. ?'e: J. 4, 447-=152. 9.' Webster, W..S., Dendincer, J., Rokaw, S.N., 1971. r- rate and carbon mor.cxide level after smokine hic_h-'• - and r.on-nicotine cicarettes. Ann. Intern. Mec. 7=. 697-7C2. c-R ^a4T-KAr.--c CTR 11N 043813

A f 10. Rob-inscn, J.C, ar.d Forbes, ',J.F., 1975. The Role of Czr'-nn t•1or.exidc in Cigarette Scro:cir,q. Arch Environ. F:ea1 th 31, 4_5-434. 11. Caton, J.C., personal corsrunicaticn. 12. Cont:-•.,i 11, 1969. Inhalation Carcinocenesis, L'.S AEC S•,^-;osiuri Series No. 18, April 1970. Procec. c: 6ioiocy Div., Cek Ridc+ !;atl. Lab Ccnf., Oct. S- ~ 389-~ie. • 13. 1%ehr.er, t. ?., Busch, R.K., and Clson, R.J., 197"Effec of C~-rcnic Er•cosure to Cigarette Sccsce an '-or Inciee^c3 in r,e tyrien Colden Harster, In Exre:•c.entai l.::ng C:ncer Carcirccene5is and Bioassays.-TE. Karbe and J.F. Par::, Eus. ), 350, Springer-Verlag, t.zw York. 1•'+. 1'!e!;'2r, t•.i°., Busch, R.H., Olson, R.J., and Craia, D.K., 1977. Chronic lnhalation of Cobalt Oxide and Cicarette S'liot;e =y I?arsters. Am. Ind. Hyg. Assoc. J., Vol. 38, 33~-?4b. 15. Saffiot:i, 'J., ~Sontesero, R., Sellakunar, A.R., Cef.s, F. and Kaufmar, O.G., 1972. Respiraiorv Trzcc Carcinocenesis in Hamsters induced by Oifferent Numbers of Adnil:istraticns of 8enzo(a)pyrene and Ferric Oxide. Cancer Research 32, 1073-IC81. 15. Ro,ers, 1973. Liquid C.^uision Techniques for Grain Density Autorad?o- cra~hs. ^e:ziled Cescria:ion of TechniGues using Eascc-an hcda~c ti-5-- ar.,. N' - 3 u i s i ons t ~ i ec~ n I o,ues c' ~u c0- racic; =.,y, cp 3C°-,i;, _:sevier ~Lcientific Pubiis!sing Cc~?a: ;:, .._:, 7ork CTR .CC1N! RACrS& 02u311 I CTR HN 043814

Table I w(IC~i7 a*:7 Rl;ISTSnCt 1: On;a: Gua !C•'. :r.RICCi L(S!ntCUt Ctf•A"(tPa ft^:S Mv-ec• Ot Rt0 - R70 I/ei,4t C:•,a.ette+ _ St! Cev ~•l t roc 7e. e' 7 (^^ .,7U1 !al (S) ;q) iRl I ICJ 74 -S 7S 7: . 1.1720.0'. I.t; 2 100 , 6~~t2 7C.S S4 . 1.12:0.06 t.li 3 1:0 E:=1t 12.5 S4 1.I7-0.C6 1.12 C.72 4 1C0 9}:~6 SS 137 1.I1 1.71 0.V, S I:0 75-17 . 73.5 e6 I.Ia1C•C? 1,17 C.ct to:aieo +.e=it,: Ca. 31=;? )3 tSt 1.16]~.Oe t.S6 O.SS totil•:e s.c=.e-, 61=..5 I.t6:0.02 1R1 I ICO 71Z6 )1 42 1.161.03 1 .1 7 17 7 ICO 63:6 63 77 1.II=.01. ..tl • 0. 19 3 1 CO 56-. 59 . 25 1 .I;=.01 1 .1) 0.111 4 100 6E': 11 64 47 1.16=.06 1.15 0.27 S ICO 6t._S 63 • 23 1.1)l.03 I.)C 0.15 Totaiee Ave-St! Jev 65 0 6) 52 1.11.:,05 I.). 0.20 Totatc! tvc=.c" 65=2.? I.I4=.01 11.) I Ico 6S=•6 65 2e I.n,-.04 I..!3 0.20 2 IQO . 62J, 62 24 I.12=.0) 1.12 0.20 3 93 6}0 70 38 1 .1 t.=.0; 1 .IS 0.15 4 100 6So 68 32 1.14=.0) 1.14 0.18 S t00 59=7 60 )2 1.12 0.20 Toeolea ..•e-ftd Cev 65=7 6S )a I.I)=M 1.13 0.25 Totaled Ave=sc+, 65=1.9 ~ !.1):p.t706 'stl I 100 79 30 t.70=.03 1.13 0.15 I' . 2 99 atz; e2 ~ 37 1.13-.03 1 .13 C.16 I 3 99 e=-9 E4 44 ' I.I)-.o4 1. u 0.17 b teo JS-7 75 36 I.It_.0! I•It 0.16 ~ t 5 IC0 -11 `) 64 I.OS .06 I.10 0.30 i ( G co -t) 6. 60 I.1.:.CS I.12 0.:9 ; 'e:atc•+ nve-•Ste ::v ;;=t0 79 71 0.32 ' Tn!ate! nve=se•+ 1-4 .: t.t7-.C `2 {{{ !:1 I iC0 S1=S S: 33 1.iS=.03 I.IS 0.20 F 2 ICo 57=5 S6 29 1.16=.11 1.17 1.17 3 I00 O5~ 64 33 1.7t=.73 I.7 1 :.11 I 4 IC0 60LS 60 3) t.:fl=.0t+ I.:a 0.20 ~ S 100 60 ='. 60 21 t.te=.03 .IS 0.14 Terate! nve=Std Oev 6 014 60 4) 1.1a•C.U6 1.18 1.22 Totaled we=see• 6'r:.7.) I.IO•=.:C) CTR. rnNt&PrT-c;. n~n =~~ CTR HN 04•3615

: +.J Table 2 TIIC EFFCCT OF RID ON TPM AND CO DELIVERY FROM TIIE 2R1 CIGARETTE WHEN SMOKED ON THE SEM I I ItfD Fixed 1'uff Pressure Intcyrat•cd Valucs Corresponding Puff Pressure Integrated Values TPM CO TI'M CO I 65/73 20.3 65.2 58.5 9.7 65.4 57.9 i 78/82 10.3 56.1 49.6 10.3 56.1 90/95 7.0.3 50.4 +,2.4 11.5 57.C LS'2.G' -J Note: 1he number of puffs was const•ant for thesc cigarette, (1) however, only those cigarettes with the RID range 65/73 burneJ to the 23 nm butt under these conditions. Cigarettes in the other RTD ranges resulted In butt lengths greater than 23 n.n. RID Fixed Puff Pressure Integrated Values Correspondiny Puff Pressure Integrated V:1 Iucs TPM CO _ Tf 11 CO 65/73 30.3 - - 9.7 69.8 61.9 78/82 10.3 62.5 59.7 10.3 62.5 59.7 90/95 10.3 G4.7 57.1 11.5 65.4 5ll.G Note: (r C-% (r C ~ (- I i I I I The numl,er of puffs in this case was 12 except for ciqarettes wl th RID's 65/73 at 10.3 nm 1120; at 9.7 "N» 1120 the cigarette butt length for 6;/73 was slightly shorter than 23 nrn. 37

c I i , Table 3 I)LPOSITION A/d0 CI.EARANCE 0F 1"C-DaP AND 1I'C-NICOTINE AF 1 LR LXI'OSURE TO ONE PUfF 01" RA010LA!iCl_f.b CIGARETTE SMOKE (EXP XIV) I OTAI. I'/,RT ICULATE MATTER (ji9 ) Timc After Smokc Exposurc (Fllnutes) Tissuc 0 5 15 30 60 111 C-DaP Lung 7. 7! 1.8 3.2-1- 0.6 2.6-1 0.9 2.3 1' 0.5 2.61 0.3 Larynx 1.6± 0.9 1.5± 0.2 0.8} 0.3 0.7!' 0.1 0.6± 0.2 11car1 1. 5!0.2 1.010.2 1.2T 0.3 0.8 1 0.1 0.7! 0.1 ~ 5 tcxn::c:h 1.8! 0.8 5. 1± 2.2 4. 31 2.0 2. 91 3.5 3 1.' 0.11 ~ ReRa ins 36.044. 5 13.5±13.0 It.5± 0.2 5.2± 0.4 2.1* 0.1 ~ . Total 60.0t57.0 23.0±13.0 12.5± 5.2• 11.0± 3.5 8.5± 3.0 D ~ C-:1lcotlne Lung 2.01 0.1 1.21• 0.4 0.8i 0.1 1.21 0.6 0.8± 0.2 .~ Larynx 0.8± 0.1 0.5± 0.2 0.3± 0.2 0.3± 0.2 0.31 0.2 ~ Ilead 3.2 t 0.5 2. 0± 0.2 0. 9 F 0.2 0. 7!• 0.2 0. Ii± 0.2 ~ S tomach 1.. 01 0.1 1. )t~~ 0.2 1. 2t 0.2 1.2± 0.2 1. ]t 0.2 ~~ Remains 10.51• 1.5 9.OF 0.5 5.0~• 0.2 h.01• 0./1 2.4F 1.5 Tota1 13 .0!' 3.2 14.0!' 0.5 8.5± 1.9 7.5!' 1 8 52!- 2.6 ~ a) ..~ 38 ~ r',

'a=le 4 DE?C: IT I i.N 0.t D Q= .A: ;CE OF "C-i1CA Ai"Ti:R EXaOSU::_ TO R:.OI::LA°EL_D CIGARETTE SMOKE (EX°Erlh::l:T XVII) Total Fart:c:..ate f•tatter (rg) Tir,_ Ater Sr~oke _xposure (!?curs; I Tiss::^_ 0 4 _6 2'-: Lu^_ 50=_L 22- Larynx 7= 1 7= _ 4= 3 5= 2 7= 3 Yead 219 2 4- 1 3= 1 3= _ Storoach 22- 8 22= 3 11' 4 5= 1 3= _ Rer^a i ns 10= 7 12==5 - - - f Total 11604 7200 72i10 1S= 4 2_ 4 r'P i nN'KQr.Tc; nM~q9c; CTR 11N 043"816

-1 l TADLE 5 RETENTIO[I AND CIEARAIICC OF IItC-MCA AND 14C-8aP AfTER INTRnTRACIIEAL INOCU(ATION IN TttO STRAINS 0F MICE.a (Cxp. 13 and 22) b Micru,jr.tm. (! S.D.) Rexwlnlnj In Lunj T1mc atter Inoatlatlon (Ilours) Itls or Irc.it.wsnt 0 1 2 4 6 24 168 316 9C;FI/Cun 2i0p9MCA 4[tU ~ !:O 140 ! IU' 29 ! 20 10 ! 6 4 : 2 • 1.• 0 Inpgll(4 L / ~. fO t [t 2/ 1 : t 1 0- 0 0 s O . 1 1 t/ '. -j MC ` .14n ! •0•16 .0/4 ! .060 IU6 ~.116' .032 ! .0?0 .002 ! .003 .001 '-.001 .0n) t.o13 .014 .021 .006 * .006 .018 t .041 0 ! 0 .7 ."~ ~ 9.8 ! )10 646'- 187 931 + 2)9 757F205 9Zb = 231 650 * 169 1038 * 83 928+186 .7 ~ l aer.Ir Z . 1200 1.9 o>v 8)9 '- 176 1029 t 115 003 ! 230 752 ' 120 1200 pg AsP • 0.6 my fe O ; 1R)8 1 )49 1007 ! 211 900 ! 202 377 ! 83 n j ) 7 u. Mlce were anesthetlteJ wl tL octulan.:. Intrntrachcally Inoculated wIth the eppropriote Jose of 14C-MCA or 1tt C-GaP -{ In 0.02 ml 9cl-sullne. 10-13 mice .+ere snerlfleed per time point. Results ar.: presenteJ only (or 141,11. n b. Standard devlatlon. v ~ r ro

r• t TAOLE 6 ~ I ~ C-DOTRtACONTANi LA6ElLE0 SMOKE IN 0C3F1/Ct1M I:ICE AFTER SHOYE E:(70;ti1:E ON THE SEM 11 DEPOSITION OF TOTAL PARTICULATE M4TTiA ~ b c d t D t d k T S.:•o.e Sex liead larynx Lung li~•er Storoch Ren~lns o a c y Ccnccntratlon Pg v9 Pg F7 1, 7 I'g 1~9 e - 1. I C::. 30 F F 7.5 1.9 -- 7.7 ' 3.0 05.2 2b.2 2.6 t 0.7 7.! :.0 16.9 s 20.0 Ie).4 c 2:.7 '0 ft 9 7.f1 T. . t !.! 11.11 93.0 ~ 36.6 1.5 * 0.4 13.4 ~ 3.2 9.9 ~ 2.3 120.6 = 3C.3 f 7.9 2.1 4.0 1.8 82.9 '- 16.8 0.4 ~ 0.2 1.4 ~ 0.7~ 0.0 ~ 0.0 10?.4 -` 16.1 a. /!e:.t :n:i..es la+er jaw, excludes skin. . Larynx and upncr trachea were analyzed tog_ther. c. Ltmg snd It-.ar traehes were nnalyzed togother. d• Excludes Sldes (see text)• --4 "~ e. :11ce .r_re ex;osed to IOX smckc for 30 sccs. followed by 30 secs. ef air, for a to:al o: 10 minutes. ~ D f. Thirty mice were used, mean body wcl,ht was 22.9 ~ 1.2 g. I7) g. Thlrty mice were used, menn body .+ctght was 26.7 * 1.0 g. 1 h. Mice were exposad to 20% smoke for 20 secs. followed by 40 secs. of air for a total of 10 mlr`otcs. I. Thirty mice ..•cre uscd, meen body .n:l,lit was 23.3 * 0.5 g. J. The stomach tias separated fron the conten,sand the TPM determined to be 4.3 t 2.4 g. ~ v k. The mean values for total body were determined from the total body for each animal and nay not hc cractly the s,x..e as ~, tne jig total body determined (ru.e he n:cans of the organs. (1t ~ V

Tab1e 7 PARTICUL'TE _; _ ITICt: (µg TPH) 0F EC3=1 ?!ICE AS A FU!-lCTI:Pi ltl S=:( AttO Ap:PTAT10;! (1rJ`Y, S1h!OK_) ( ( Exposure Tir,e Tiss:e Females u.=_1 es u9 T" } o T rm :0 Sec.,.._: __ 13.6 = 3.4 7 Fea: 1.7 ± 1.7 3.0 - 3.0 Lar2.4 t 0.9 3.3 = 4.7 20 Seco..--~s Lu: 56.9 = 7.3 54.4 - 11.7 Hea= 5.4 5.3 = 0.9 Lary-x 4.0 = 1.2 5.7 = 3.5 30 Seccr.cs Lur._ 85.2 t 24.2 93.0 = 36.6 Xea= 7.5 ~ 1.9 7.8 j 2.1 Larv-x ' 3.9 = 3.0 = 3.3 0.8 r.-;~ r.nNT*ar7= n-?n -?~ c I C TR HN 04382 1

I I Tablc 8 COMPARISON 01: DATA fROM OTIIER SMOKE INIIALATION LA;,ORATORIES tlachInc E xiu>sure -Con<I I t lons. Nunib.:r of AnimiIs Lung (1~<I fPlt/nt~ lung) j<g TPM ± S.D. (% I Larynx Respiratory Tract) I Ilcad S L ontaCh ` ' S I4 I I 10y Smol;c 30 110.6 ''- <2.6(82) 14.6 ~'12.2 (1) ) - 2.0(7) 9.6 ± 2.7 9.9 . 'C-nlC . 3')"/30'a (0.51 ± .11) 3.9 i 3.0 (11) 7.5 C 1.9(8) 9.3 t 2.8 "1 ml ns. b 80.2 : 27.6(88) (.36 F .1h) WAI TttN I tt% Smoke 1.: 135.3 ' 16.4(93) 8.3 =. 3.1(G) 1.3 ~~ 0.5(1) ,-':!. 7 .± 3.0 . Ih ';-ItTr 30''/'sn,< Ll lo "Ins. d 2 ± ._.~ Mas-on 7~ Sir.oke 5 (58) 233.7 ~ 87. 14.8 ± 10..6(3.8) 1b.3 144.6 = 24 1,0.0 :1S DClSI: l ! Continuous 10 mins. (1.8'-0.5) f (36.8) n MAODIix/ or.Nl_ 10% Smoke 10 "5 3''- 21. 7( 95 ) 2 t 0(1) 10 'F 3(q) • ~ 1 t'c-DrC ~ , 30"/30, ( I . 8 !* 0. ?_ ) ~ l 10 mins. 7.) ~ ---t . ~ a. Exposurc- condltluu::: 30 smoke, follow<:d by 30 sccs. air. ..~ b. The altr.rnatin9 cycle was carrled out for 10 min:.. `T f) c. Total cap:tcity uf thu SiM I I for any one cxposur.: Is 240. .~..7. d. Three mice died durinq the exposure. e. Data for luvad and lower jaw were contbined to allow canparition. ~ . f. Data for Iun9s, bronchi, and trachea were combined to allow comparison ._l . 1) J .0 113 ~ ~ 8`sd

( Ta51c 9 "==- ' OF AC'JTE EXPOSURE TO FOUR C_IG ~~7i~ TYPES IN BC3r'1/CUi•1 P1ICEa' ( Cicaret._ Tyoe Percant S::rvivcrs Inteorated CO Value Intec-a;ed Par,i..,'a;_ ~a?.= 2:._ .9/:~ = 93.. 5 5 Scl:: = 95.0 ?6 15 93.3 21 19 5"'/50 = 90.0 24 24 2n1 57/60 = 95.0 2 3 5-6/60 = 93.3 6 7 43/C0 = 71.6 13 17 30/60 = 50.0 13 14 0/60 = 0.0 22 25 1R2 5;/60 = 98.3 10 9 1.5/6J = 25.0 18 17 '_~3 57/60 = 5;. 7 5 5_/60 = c5.o '_S ;0 P/o.. = j.. 20 17 Mac., i ne cC/--' 0 =_00.., 0 0 'a. . -. - were ex:os•= - ^creas ino r.cmoers y ;uT`s . rcn +o ,.i care:-:s c- -e I I, i0;',-s-+o~;e, 30sr eco^cs -oke exccs::re zolic::ec b y accr.cs Durc_ sir ^ ., 10 minu:es. Mice ::ere aile•::ec a ter -i-u:e rest betweencicaret:es. The CO anc car:ic:,late values were observed ourine tne exPcsure anc represenc :r.e amount °or one ci7aretce. -4 I CTR HN 041-:38213

l I . . 1'ahlc 10 SKJXE INOIICI:O turtG LESICNS (OC3I'I MICE) (I-c22. 1-02)) ~ 11,aks ~ i..l.used to ~ Swo::_ lfecks Off. S rank e 2z-;6 . 0 22 (? cI9s.) Controls 0 Ilorma 1 r A da,nma - tusIs PAMA Alv. Ilyperpl. Othe0tht•rc Luag Tissue ALnnrr..al. AbnOrmal. o/i2 41/6,(66r) 42/ET.(67Y) 0/s2 7/~~'(11z) 0/cZ 0/4 3/4 (75iC) l;/+(100;:) ' 0/4 0/4 0/t. 3n/49(61'L) 14/49(291,;) I/!.9 (2%) 0/1-9 Ic/4)(20") 0/49 0/41 2u/41(63:.) 3)/41(80Y.) CaxscI uls - 7/J (no;.) a/n 0/8 -~ 38 13 0/I4 9/!4(643:) 14/14(100%) •Ccn:rols - 4/9 (4h%) 2/9 (22;) 1/9 (1I;:) 1/41(2r) 0/9 3 1/I4 0/9 I4/4I(31•Y.) 0/1.1 I/C (12;O 0/0 7/14(50y,) 1/14 (7%) 4/9 (44r) 0/9 27-38 19 0/)t 1]/;2 (5:!C) 26/:2 (al%) 0/32 -'/32 (63:) 2/32 (6%) 25 . 19 9/10 (90'. ) 1/10 (10X). 0/IO 0/10 0/10 0/10 Controls - . i 1/e ( 12z) 3/8 (37::) 6/8 (759.) 0/8 0/0 0/e . 1. Includes: Chronic bronchioliti:; GranuIo,3otous bronchlolltls; Pcribronchtolitls, acutr., m.iItifncal; PerlvesculltIs; perlbronchiolsr tymphold Inflltraclon; Interstltlal pncurr.onla acute, dlffusc; Lipid granuloea; Eoslnophlllc inclusion (bronchlola:, crystallina). 2. Includa : Urinary blad.'er - cystic, chronic, diffuse; kidney - hydronephrosis; Llver - fatty met:.•norphosls. 3. Alveolar non-conpre:sing nodule. 115 \ Ll

I::Y ((10' 11oo' p(uo• 9p(0' ,611' Sc:7' l0]0' (S00• SS10'_p011' 0(00' S(10' 1(S/' ,040• 9fGO' pSlo' tSll' f(:0' pppl' ::CO' (S00' 9010' t/l1• 91.0- LSSI' ?IS7' t000' [SOO' 1Y10' 9oU ' Ci:l' 6;~Y (=o' CS7o' (11o• [(01' •:-/:oi •t.!FOQ /a+t] 911 U (S:O00' q100• 6SOO' 99001 !((0' ioo0' 6(00' 0[uo' SOGo' 4Cio' pOff' f6p:'9 l(C9'91 CI . tl 60.'000_p000• C,W' l(00• ,9(0' l :000' 9(00'_SroO'9h(0' _ Y%:(C'_(6i9_11Y0'2_66:5'll 9 11. `. O(1000• 0100' 0(00' p/00 10:0' 1000 0100' 1/00 0990' YO• 9)[f' 6p9:'YI GIi-'(( 01 /LS G :11000• [OOO' Stoo_0800' _ (Ilo_ 1000' p(uo' :900' GP(O' 6)'10' S)p9'IY .p'YI 06l('f( 9 1.'. 0l[000' 9100' 1900' Sf r00' 9t(O' - 1000 6(u0' 0600' lifo' _ 6f10' 1Y[1' 90.'l'i( 0= (~ h:000• 60C0' 0500' 9700' 1 N0' (_000' 1(00' iS00' (9(O' ffSo' 61(9 • 91:0'S 9aT'S1 0: ~1 E+ S91000' 6o0J' S(00' ZfW' 1(.0' (000' 11u0 :GOO' fb60' 1950' _ Ee(f' 0016't: 6: /11 2tlo00' 9000• 9:00' 1900• p1Z0' (OM' YIOO' :t0o' /1C0' (oCo' S1(l' 6!(9'(1 (e5,'i( C 11 .!. C[1000' SooO' S.00' (900' ,Y;.o' (000' YY70' 9100' p6pT 9=•:[9' Sl!i' S6(YE OJ(S'S( f 1 C '1n/0o1 /!J] •22 Ar.01 L j•u+.lrv ;+. +u a s l w:11 'O.t i/•+/1 '0't 11•+.1 .I•y.P . ena+f ()JIM 1J(JY) MtMSi) 'YO ///IN IOnIS S[)NIS Y0/ S.100107 AY f1071)Y N.7W) /3 f0.(tY 10 /:J11.M(7l •J (.,•^~ (I) 6 a61 ....6 0l .Ia%_^u /1• Yp(i 9((1' O.:YG' [010' If91' (l00' SS/N1' 96(w' 61p0' 6(S:'I oCOf'1: 61 ta I/"'IS /•^^a•o•) (0(~ 91Y:' 60l<'1 Gllo• 00,1, 0077' ,:u0' (0.)0' :(6C' !S60'( C?:C'9' 9 11'• _ .,o•+i ~+nosfo;7 ipld (S96' SO.i.1 s(io* -iJli'-_21oo--%(oo* ico0'~Oo6O- olyi'~ aP.-: 6i 11.•. ll^vS 10[e •19,1' 4Y.Y'1 S(10' ((91' 0100' S(00' 190G' [11/' 1A o„^-S Op[w ,Spo' .:040'1 Zu.O' _ 4561 /IJ0~1!'00 9[uo' S16o' ,if0'I c.b(': .(: /• Jla'/S '•••1•N 6(/t 1(S/' [0:7*1_p(10'_II(I' 01C0' 0(0J' ((00' 1/01' ((Y)'( Ci::':( O: AI _ aW.r'S p[(i 9111' 96h1~1 pa0' 9(G1 IIUO ^S130' SSOO' 0111' OIS1*1 O:JO /11 l(lt 0111' S(09'I 1910' Y911' 0100' O(co' 1900' 9S(1' 6l1S'1 G7:C'If 1 11 /la•IS M)S 911.1 ' .`(.2' (SYS'1 Y.OI' I/G:1' 6M10' p(Ib' t(r10' p((1' !IS_-( OO:S'I( 1 wlldl.:i..0 / •Y '0'S la^II •o.S 6un7 O.S .(-iP7 '0'S S/eall .'S /l•oi .1...1.;• . Cry+: I ' ()JIM IJ(Jp) I/rNSS1 'YO IIIIM A001S SS)pIS )111 110J IC.0b7 .9 1117::.. Jt7Y0 i4 K01:•SMfI I I r Il .,lq(?l I

1;" I i Table 12 Strilltl.al :Irynlfl..mr. I... tl.e Olf/ercnc.. eul...cn t/e.n. of Or .n Ntl..t. a..J O.g.n Vel)•.l R+1/e/ fnr sveC swcy vlu. Dr. f....n rt/9/nl/ . Iu4.llalon of P V4lu.s 7 Grd.y. Ohcrlpt/on Ile+.l I/vvr Oody Rdr.:..l luny /lrt/l.un 11rt/ Ilrt/ lon/llv lun/OoJy llv/OOJy Ilrt/LJr •dr/tun •dr/ll.. .dr/e o.y J C.^C:!Y/ _ ._ Iwr Ilody 1 a I. 11 StM. d..lf L ..cAlne d .)64 .s.:J .)ut 9,11 .06) .031 .164 .9)9 Jt) .00) .1o9 .877 .4_2 .7J1 .951 I. 111 StM. .ne1f a 6 .-4. d .006 .00) .005 .001 L:On07 .900 .074- LZI001 L;aJ07 .057 .Ole .L02 (_CT17 .G01. 1. 111 StM. ..ocAln. L trul. r U00' L,..__ .ll.".ti/ AGO] .1J9 .0;6 L;G6Q/ .0)2 .0)6 L~!~Z ~Z+AD7 .00) . .012 .6J0 [ 0l7. .c07 IV. V walton, .n.lf a .' L ..w. r L'nnA7 Lt.nu/L;rt-7 .175 /ZA1II LAnn7 ~II _Ai17EMKoo7 (_R ! [.0--7 .276 .419 .00± .016 .007 V1, V11 •'tro.fov.rf" .hell L J : ;OJ/. ~DO~ ;Dpl .tlot .2)4 .001 ^:1 .100 fTr /-Ai L1TF77 os6 .le) .71.) .21s )72 J ...k. _ . V111. 11 °cro:.ov.rt' 7 .L.If L Y .wM. On/ .u19 .014 .OOJ .042 ;no .oo; .00) L,~hm7 L;?A/7 o)t )o0 1ss .o1s .061. j J a. C.alculatctl statistical significancc > 0.0001; computer designated 0.0 as/lmor " slgniflcantl' than 0.000. t 1/7

( TA3LE 13 Effect o` ryclchexic•ide and A~-inc.^yci- 0 cn S-cke Induced SC3=1 L::nc ~-H and ETn~O-Deethylase Activity / Tre?t-e^'a AHH Activitv5 0-Ceet-:;ase c 1. 2. Mac=•ir:e controlsd Smoke 0.35 0.70 <0.^- C.--' l 3. Sroke T sa 1 i r,e 0.67 0.83 4. Smo!<e = propylene clycol 0.53 0.83 5. Smoke - cyclehexi-ice, I_g/g 0.92 0.65 6. Srr•ol:c + cyclohex'imide, 500 hg/g 0.32 NDe 7. Smoke = actire-nycin 0, 1}i9/9 0.42 0.15 8. Cyclchexir.iide, cn0 c/c 2 hr. prior to sr-.oice 0.39 NO ~. Actinomycin 0, 2 hr. ;rior to smoke 1.51 ND a Cycle~-3xiride and ac; ^cr..vcin 0 were injected ir -a=eri.,,ne=;i• in 0.~°•a sa11,e i"ec l2:e y'eceeclne or 2 hr. ~e-ore ex:cs: - to or•e 7_..1 ciceret:s • a e~in; ^3chir•e (10; ar.- 30 sec.exposure) Mice -,.ere sacrificec 6 hr. after scoice. t :HH units are •roles ^H-3? `orm.ed/:^in/g wet lunc_ weicht. Averace activity of 3~ar,i-als. c 0-Deetl;ylase units are •.-oies e:hoxyresorufin dsethylz;e=/ri-/ g wet lung weight. averace activity of 3 animals. d The 3 rachir•e-sham controls: 1) machine alone, 2) machine with cyrlohaxi^ide, ar,d 3) machine with actinomycin were crouped tccether since they did not vary significantly in 1,imi}i or ETR 0-::~eethylase activi ty. e Not done. 48 0 2a'T .rd' C t.r f R f f f'f 043627

r- T:.°LE 14 Compariscn. cs` p;;lr..o-ary „-„ levels in cigarette s-oke er.;osed .• andlor ;',C:-ex;osed "cC3Fl!Cun mice.a'S Time after expcsure (hrs.) T-ea :- ^t 0 6 24 a 1, None 0.26 0.22 - - 2. 1-2.;1 cic=ret:e 0.50 C.5- 0 C.54 3. 1 - 2 A ; cigaret._ 0.66 0.61 0.56 plus ';C:. 4. 2-21,1 cicarettesc 0.84 - - plu: t!C:: 5. 3-2:1 ciaarettes 0.75 - - plus MICr 6. 4-2A1 ciaarettes 0.71 - - plus !•tCA 7. 5-2a1 cicaret:es 0.71 - - plus MCA 8. MCA giver, iT 0.50 0.76 - 9. MCA f,1C_c) cl_s - 0.8= - carette e 1C. PSC' "C M1as - 1.02 - l ~ ) 1-2ai cicaretce a Mice were exresed to :~11 cigarettes in a tilalton smoking machine; 10ro snoke, 2.0 sec. ;,ui` and 30 sec. holding period. 5 Ai-iH data iven in terms o' nmoles 3-OH-9P formed per min. per g wet ..~t. :issue. c One mc I-tCA was "adced cer cicarette and - l0ug should be delivered. 49 I CTR HN 043-626

, r ( Table 15 A Cc.-rarison _. :arbo,:yheroc)cbin Levels in Grc;:ps cf Mice Usin: '.:o Different ?rocedures 1 Percent Ca:~--oxyhe-oclc'Din (- S.D.) Assay Nucber :;;-oxir„eter Date of Sac.ples Spectrophotc-e:er 3 A 6 --.3 ± 5.0(.11) 26 9 ± ~ 9 S . . ~ 5.0(•i3) 21.2 " 8.5(• =) C 8 ...1 ~ 5.9(.17) 20.5 t 4.2 0 9 _.1 * 6.S(.17) 25.2 t 10.3 (.;1) E S 1 -.•3 - 3.5(•15) 14.8 t 7.3 (.50) + Mean 33 . 5 8.1(.15) 21.7 t 4.7 (.37) 1. BC3F1 .`e-+ale mice %.ere exposed to snoke from two succes<_ive 2A1 cigare:tes on -e S_.M II B. 2. Stancard deviation. 3. Coefficient of var:a:ien. 50 ( rTR s"a1NT;Pr-r-C- s t~ c ~ CTR H~~ ~~~~ 82-9

Tat lt: 16 -, Co-:zriso^ c` Car!:cxyi,eneclctin levels in Indivic ~l !-;ice Using 7%•:o Differen; Proceduresl Percen. Carboxyhe-loglctin (: S.D)2 Mouse Number CC-oxi-ater Spectro,ho;cm,eter 7081 27.1 21.5 7051 2 L..9 15.7 7CE? 23.0 - 3 70EL 18.5 1.1 7085 21.0 - 3 + 4 Mean 23.3 S 19 ) 14.8 = 7.3(.50 ) 1. 14ice werc exposed to smoke from two successive 2::1 cicarettes on the S_i•1 I I ii. 2. Standard Caviation. _ 3. Sample was lost due to lack of a dirferenca in spectrum• 4. Coe-Hicient of variation. 51 - r.-t~ ^n~4 ; rm r , - = n~, r~ ..~ . ..~, I CTR V11-~11 04,38,30

t I I I ~ Table 17 Carboxyhemocllobin, Ilemoglobin, Methemoglobin and Oxyhc:moglobin Measurements In Mice Exposecl to Smoke 1'or Varying Lengths of Time. r, . ~ t Time Exposed 1 C0111Z lib Met. Mb 0; !Ib Vol. 02 to Smoke M (g/ 100 nil ) M (%) (~ ) Shelf Control(10)1.9 ~ 0.4 1(,.6 L.10 0.1:0 ~' 0.15 30.6 1 14.6 10.0 '~ 1.6 Machine Control 0.6 .2 15.5 ~' 0.5 0.20 'F 0.10 25.1 t 8.8 5. 8 ~ 1.9 .7 3 o,ccks' (t1) 38.9 t 3.5 15.6 ! 1.7 0.73 r 0.10 30.11 1 5.6 6.5 f 1.0 u 3 months (3) 3Z.0 F 6.2 16.3 ~~ 0.5 1.00•± 0.20 31.6 ± 4.1 7.1 ± 1.1 6 mon1ihs (3) 35.h ! I I.u 13.t1 'F 0.9 0.60 '-' 0.26 16.5 ± 6. 1 3.0 i' 1. 1 ~ (1 .7 ~ 11 ~) a) ~ aa ~CD W I. 1lally (5 clcrys/wc.a:) ~.a~~.!:u cxl:a;urc. 5-10 clcarettes/day. 3. Iflunel ..nnlcle takcn utl.er exposure to two 2A1 clgarettes. Number of mlce. '-4

r i Tab1c 18 SE U ClcO DATA rRoM MCA INIRAIMCIrtAt [ltrERItKRTS IN Ci11/Anf, C57ct/6 nnd eC)rl rEMAIE MICE Strele I.p. Gcoup lotal Oose Inoc. Do.e p Iroc. I/tlce I M1ce Ihat Oled f~ efOr ) Er Meeks / . 1 / (M9 MCA) (.g MCA) R.. S!hed. Gr-p Rw 0 K Q , On T.-ored / lc.lons ,r. (f) Te.t e/lee SI/ A.C SSC ASC CSM/Anf COSS o1C2A 77S 62.3 G I/•w Sn 1 4r.6 0)Sr S/SO 10/SO 79/10 SG 6• 0 S 0 t 0lC2A 7S0 L.:.S 12 1/..k 47 0 2r.S 0 ~Tr 0/47 7/W 1-447 St I2 0 II 0 I 010A 750 12S 6 1/..k 50 3 4p.5 0 t.Op )/50 //So W./SO S6 19 S e /I 2 02D/A 1500 125 I2 I/..k 1.5 f/ 7p.10 0 2op 0!1.5 17/4S 2)/4S ' 4D 19 • 5 e 11 2 • o1I2A 1500 250 6 I/.•k 50 10 9r.9 0 22r 10/So 10/SO JI/50 $0 21 6 6 12 I oa 2A 2250 ' 2;0 9 1/.•. 51 • 20 7p.IZ 0 11P.1 2c/SI 19/51 Ie/SI )S I/ 10 2 11 6 • C067 o6e)A 1200 300 4 1/2 wks 44 1 orc22 0 15% 0 7/44 22/44 42 e 0 6 0 0 oSe4A 6oo 1S0 6 1/2 ..k. 50 2 op.29 0 19p.o 2/S 29/50 17/S0 41 6 0 6 0 D OSe)A 600 I50 A. 1/2 wws So I Ir.I2 0 S6p.o 1/S0 1)/SO N/So 4S 11 0 11 0 0 CS761/6 Co/) D4AtA 1500 250 6 1/.•k 100 29 ~9r.21 0 41r.o 29/100 30/100 50/100 47 17 2 10 7 2 tKtI2A 1500 zs0 6 1/2 ..ks 100 22 11r.2) 0 ur.o 22/100 !4/100 SS/100 47 11 5 5 1 1 V eC1f) CO7) OIA2A 1500 25O 6 1/.1k S0 lt - Sp.I0 1 2)p 11/SO 16/So 20/SO '10 7 1 6 0 0 J 02314 iS00 250 6 I/2 wws So I ep.12 0 29p 1/50 20/S0 37/50 40 9 6 5 2 I -.~ Coel 07ctA 1100 210 6 1/2 ..rs 100 9 4 0 elp.6 9/100 4/100 e//700 2e 1 I C 0 0 OSCZ• 1300 z50 6 11 ..ks too IS 3 0 77p.7 17/100 )/t00 77/100 2e 0 0 0 0 0 7 0)C2C 1500 2S0 6 1/: •ks 100 Is 5 0 lTpe7 15/100 5/100 71/100 2e 0 0 0 0 C ~ 0)tz0 1500 250 6 1/2 ..ks 100 e e 0 76'se (l/100 6/100 76/100 28 2 0 0 2 0 1 1 z -4 O7C2E o)C2! IS00 IS~ 2S0 2S0 6 6 1/2 w4s 1/2 .a.. 100 100 13 14 , 2 2 0 0 6Sp•20 6)r.21 1)/100 14/1C0 . 2/100 6S/100 2/100 63/100 2C ze 2 0 I 0 0 0 0 0 1 0 i n 1- r...ber of .Ice per yrmq. Rw -.Ice that dled during Ire.tioent perlod 0 - wlce lhet were foond dead. 0p Indicates pathology K-.1ee tha wece killed because worlbond Q-.Ice that wece kllled beca.ne of 'test secrlflce' or scbeduled sacrifice or ter.lnal sacrlflc /- vudrer of w,lce +ent to hlstoloqr ' 53

C7?, I-!CC. Table lg !ncicence cf Lung Lesiens in 6C3F1 Female Mice :fter !ntratracr.eai Inoculation of 3 (Serial Sacrifice) reatr..e^: C:aSncsis 12 wks. .16 wkS. 2 2 w{s. (6x-) 2 (8x)2 (9x)2 z 4 ~ninoc_.a:__ ~c.-z: l 10/10'(ICO) 10/10(1C0) 8ii: (c') > ( I n f la-.-. at o",r C.her6 1/10 (1C) C:el'-Sal ir.e tior-al 10/10(100) 9/9000) 10/lC(100) 250 µg 1!CA Nor.na1 5/29 (17) 0/29 (0) 2/28 (7) lX/2 wee:cs Infiarr-iatory 11/20, (37) 5/29(17) 2/28 (7) Hyperplasia7 Non-Ccmpress.7 16/29 (55) 20/29(68) 18/28(64) Nocule . Cor^oress inc_~ 0/29 (0) 2/29 (6) 7/2°(25) Nocule Sc. Metapla- 0/29 (0) 0/29 (0) 1/28 (3) sia 8/29 (27) 19/29(6:) 8/28r28) ( SqNeopias- Sc. Cel : 0/_9 (0) 0/29 (0) Carcinc.-a 1/29 (3) 0/29 (IV) 6/2~icli Other 1. Weeks on test. 2. At 12, 16 and 22 weeks on test, 6, 8 and 9 inoculations r' rIC:, 3.. had been civen. Nunber of nice wit.i lesions per total number of mice sacr•ific.nc:. 4. Percentac_e. 5. Inciuces PAIt!A; :.denc-atosis, Perilymph., Brenc^. (ac L suhaz), Cor.c_esticn, ?neunenias,Peribrorc~., Fibrosis, ?erivasc., Brenc`.., anc i:deTa, Sronchici. polyp. 6. MeCiai nvoer :hy arteriole. '. Ail are eivec ~enic iesions. 54 r.T2 r.rNri2r^r.TS n9ri -1;an c CI I! I I I 1 0'/36..R t.+^ 4M.,`

l.• l TA3LE 20 CTR I-? C^., I;.~ i ~EYCE ..' LUNG LES I01dS Ik! 2C3F 1 F;:-'L:LE K I CE AFTE? It77«::TRACHEAL IkOCULI.TIC:I C= 3 NC:, (Spc-;aneous Deaths) Group: 2;: _g MCA I (03C, Shelf Cc^:rc1s) Vleeks „- Tes: - 8 12 16 20 22 riu-ber c: :i,s!:` ,-, 518 516 513 511 SC.- (liarros; S flor,-e1 2/i 1/2 Inflarrr•ztorY - - - - 1/7 - H•yperplasia - - - - 1/7 - Plon-Co-,D. re s s . Nodule Compress.NoC;:le - - - - - - Alv. Carcinoma - - - - - - Sq. Met. 1/7 - 1/3 2/2 2/7 4/12 Sq. P:ecplas- - - - - - 1/12 SCC - - - 1/1 2/7 9/12 Cther Ciacr,oses ~• _ 1/7 2/12" No Diac-csis at present - - - - - - No Itecr psy 1/2 2/3 - 2/7 - Prc!;ab i i:y c' Sq. Me_aplasia Ccs- CC2 .C04 OC6 .~1 .02 Probabi 1 i:y c° S;j C 0 0 0 0 .0:12 Probability cf SCC 0 0 CIC2 CG4 ."C° .^126 Total Tv;,.ors/Tota1 1;;;3 Deaths = 39, 4;<_ » CTR CaNTRACTS 0103~~ I I CTR I-IN 04~~8:~~~

( Note: .c, 7. ab le 2 0 c^ 1` r^ rols 1 250 }:; :1CA i n Ge l Sa l i r,e/2 Weeks, 9X 2 Mice :'-:at died d;;e to accidents caused by har,dlina, have `cen e>:clud_d from these cai:_iations 3 ttuci. ...el l Hy;er. lasie 4 Ly-, eserc:.-a enc in far,.t etc. 5 Tu-c-s incl;;de A::C, ASC, SN and SCC A tumored animal is counte_ only once sor calc:;laticrn o; the C.I. ( 5 o /7 /7 /7 .7 ~ - CTR CC~TRACTS 020342 C T R M N ~'0 4 ~~8 ~:3 =E;

( Notes for Tablc 2 l 0c~•., 1:achine 1 2;0 ~: P1C:, in Cel Saline/2 Weeks, 9X. 2 Mice that died d'ue to accidents ceused by handlir,c have been er.cluded fre- these calcula:icns. ~ ~ AtSCess C tc. L ~ .:.~~ ;_ SN and S: tc-,orec anir-', is cc_-ted .::', once ,, calculeticn ef .-: C.I. I i 58 V CT~2 CQHTRRCTS 0220344 CTR / I I 1 0438Sr+' iu.i'

C 22 T.:oL; _ ( ( CTR I-100 1hCI0ENCE Gi" LUi;G LESIONS In EC3=1 FEMr.LE i•.IC_ I+FT_ . It.inbTni+Cn:AL IP;OCU:".i!C!d OF 3 11CA (S.pontaneous Deaths) Grou:): 2:3 r: MCA1 (C: c-:<e1 ~, , '.1__.s on ies; - 8 12 i6 20 22 Ru,, t er at Ri sk ` 25^ 2E2 267 _:. 2.6 231 Diac^csis tto^,a1 5/16 4/15 In=1ar.Fnatory 8/15 7/15 I.yperplasia - 3/15 t;on-Cc.-press. - - Nodule. Co-press. Nodule - - Alv. Carcincs,a - - 1/12 6/12 2/12 1/12 - - 3/^- 1/= - - - - 1/15 1/15 - - - - 2/13 2/13 - 1/13 1/13 • - Sq. I•let. 2/16 4/15 4/12 10/15 10/13 Sq. t;eoplat~ - - - 1/9 3/1: 3/13 SCC - - - 1/9 4/15 3/13 Ot ti, Diagnoses 2/1' - - 1/°; - - No D'ac-osis at Presen: - - - - - - 40 Necro:sy Cor.e 2/16 1/15 ProbaJiii,y of c" Metaolasia c C' .C21 ;i._ ._;6 .080 .i21 Probability of S.14 0 0 . := .017 .030 ?rooability of SC: 0 0 0 .C20 .0:3 Total Tur:ors/Total 12/80 Deaths ~ 15.0°41 59 CTR CQ~!'R nC! S rJ203 45- C I C T R M N 0 4:'-a 3 7

l:otcs for 'a:1e 22 t3S, 5-oko 1 250,}; 111:A in Gei Saline/2 Weeks, ;X 2 idice t',a: died d::e t: a=cidents caused by h=-diinr, etc. have bee n ezctude: frem these calculations. 3 ~rcnc-:c~a'r ~yre-:'as a 4 Abscess S T:-,+ors :nclu:e :•i.:. :SC, SN and SCC. A t6-more- a-ir..a1 is countcd cnly or.a sar cclcuiation of the C.1. f 60 CTR CC1NTk~... ; 5 020346 CTR HN 04:~838

TABLE 23 TOXICI TY OF It~T=.:.Tn~C-EAL INSTILLATION C= IN EC3F1/Cu- C;H/Anf FEMALE MICE.a No. Laaci S.rain C3,'/A nf Ge!-:eiine, 9X 1/SC -) EC3=1/C:.r.l Gei :aline, 9X 7/lc^ ~y) C3H/Anf 25: -_ MCA, 3Y. 2/1G~ (2) 250 ;;g MCA, 6x 13/l0C ;13) 250 :=g MCA, 9X 25/ 150 ( 17) 6C3F..1 /Cun 250 r:; MCA, 9r. 3/650 (o. 5) a. Mice were trea:ed with 250 hg MCA/O.C2 -l cel-saline or cel-sal ine aicne once every :wo weeks fcr appropriate nc-zer of weeks. Mice that aiec c_rina Creatmient or 2= hours post anesthetic were ca:rted-as "trea:-ent ce::-s." ( 61 CTR CCW+'kr~CTS U203Q %' C TR M.N. 04 3639,

I Fig. 1 CL p C= 14 C-DTC fRGt1 THE LUt:G CfT_n ?C:UnE TO R:.DIOL:'E=L.D SMCiCE I". C;~j%=CU:I 0R tC;FI/Cu.; MICE 1 ~ ......................... ~ 1 ..............~ "cxDS T T .1 I Y. ExP 20 12 16 20 ~L iF J; 120 TI4E (haurc)• 6:. I ,1 CTR CCF•;?"RRCTS 020348 L• 50 1 i0 [ i 1 I CTR HN 04-3840

C. Fic_. 2 Clearance of 14 C-Hicotine after ex;.osure to radiolaoaled cioaret;c s-oke. ( 300 1C . SG 20 c, ; 11 1 ten puffs ~ on ff e pu 1 ~ L ~ . J ten puffs i 1 \ G,- n one puff .S !- O luna *rhole bocv M, cONTRACTS 020349 CTR ILIN 04-384.1

I F i c. 3 CIE~R `:CE OF 14C-3a° AFTER EX= TO n.lD10L .,.L"cD CIG:.;ETTE S;;D;E 2C El 5 2 1 Le.n. purfs one puffs ten puffs one puff ~ TiN: (-i, ... e lunc ~ wncie body v CC~; ;'RRCTc 4243~G I CTR IwIN 043842

I L. F i c. 4 OF 1 .• C-MCA AFTER EX?C•SUn_ TO F-. i.-:11:-C lE, C I Gi+IZC T T E SiiCKE T ~~ .. \\ 10 .1 . s t U n s I C ~ : 8 16 24 T '•E (-:;l rs? CTR CoNTRACTS 020351 rwI' I R I lI I 043843

~ Ficure 5. Fete-:icn e'^d I~ Clearance of '"C-PSC-, aFter ~ In:r"racheal Inocu!a;ion ~ of Fcv, Di fferer.; D~ses . . in 5c's:l/CUM nice (Exp 22) I 66 CTR CCNTRACTS 020352 CTR HN 043-844

I i L. FIGUn: E. SFM i1 DOSIME T RY: PARTICULATE DEPOSiTION IN LULNIG AS.A FUNCTION OF EXPOSURE TIME AND SMOKE CONCENTRATION (aC3F1 MICE) ( VLI l 1 I 1 t 5' 10. . i5~ 20 25' ;:XP05i.!:.~' ?Iiv',E (SECONDS) ( CTR CONTRACTS 020353 I CTR MN 04384S

, r fIGJR ; ). : j~i:.r~ ~ ~: f~ n......n q L A, . t ++ f ivd c`i I E:t! Iv%I ~ i E; i,~ -+-- r-~` ?•~...~ J ~<<0 ~-;?0 L~ .._. ._ F-- < u '00 t- =--- - I f I I r. M I~0._ EX?GS::: i'ii;iE ~SEC'O \'~c) = . .. 6E CTR CONTRRCTS 0203.5141 UO r- U I CTR IIN 04384.6

( ( • I Gt;;: 9 . r- a 4, ( I 0 a I I ~0 20 30 "~cE u- - =.E 0 x~ ~ E~,~OeLOR!~ --' ^.~,~. ... .I en~r- - i : i 4r..:~ : ; .~l f ,.~...:/'~ ..- k ::`v C.Ci/~7.1 . .~. ~ - :1'r +--i I~snP/.. 120 r, 0 ~ ( .. C'R CONTRRCTS 02035b ( I CTR HN 043647

L. FIG. 10 Perce,; C:::S in CC3F1 b,ice as a Func;ion of _x;cscre 7ire. 20 18 7 1 c-rR cONtRAC Ts o2C135 -7 I G TR HN 043848

I ; FIGURE 11 ACUTE TOXICITY PERCENT SU=.VIVORS `!S. RELATIVE P:.?,T;.._...TE CCRC:Nin.`.TICR ( I NTEC~: ":0 PARTI CC::.,TE VALU: 72 CTR CCNTRRCTS Q2a3.503 c I G TR HN 04,384.9

r ( L. FIGURE 12 ACUTE TC7.ICITY PEnCEWT SURVIVC'?S V. P.ELATIVE CO CC"CENTRATICNS Il2TEGrZ:.T:D CO Vi.LUE 73 CTR CQNTRACTS 020s~5 I CTR HN 04'3850

( ( ( ( FIGURE 13 ::UTCRAOlOGRA`"• OF C=7CSTAT CUT LUNG, AFTE?, EXPOSURE OF THE NOUSE 70 1`C-CTC LABELED LIGaRETTE SMOKE 3C3F_ fe-ale mice were exDPsed to smoke frc,-.i one 2A; cicare;te loaced with 1CG :Ci - C-DTC. Mice were sacrificed ano t^e lur,cs frczen. Ten micron sections of lung were obtained thrcuch the frontal plane :o include major air ways. The sec:icr.s were fixed in met`anol for one minute, Kodak NT3-2 nuclear track eTulsien was used and emulsion ccated sections were exposed for fcur wee;cs. The above photcgraph was taken by transmitted licht at a-aanification of 63X. The .-ajor?ty of the reduced silver crain can be seen to line the bronchi (3) and bronchioles (b), with Scattered activity present in the alveoli (A). 73a -1 c CTR CCNZRRC i 5 02103360 I CTR HN 043851

I 0.8 0.41. F IGU.:E 14 PLASM! CC;TICCSTERONE L:VELS IN EC3F1•FErAL£ MICE OF CIFFE;:uT :.CES (C0LLA6vRATIC` WITH DR. W. ESSMA`) 12 AGE HEEK: SMCi;: EXoCs:: 11 '+--7~Mica (6) we't exoosed to sc.oke one time from two - 2aI-Cigars:ces us::+g neck hoicers on the walton Horizontal S-okine Mac-fne. The m:ce were then returned to to cage for 10 .r.inu:ts, removed or,e at a time, sacrificed quickly by cervical dtslocation. Blood samples were obtained from t!se•abocrinal aorta. MACHINE EX?CSEO: Mice (6) were :rested as above without the cic,arettes. S~E L. C~NTn::S : . ..._ ~ _ . abovo. Mice (6) were placed in cages and sacrificed as 74 GG CTR CONTRACTS 020361 CTR NN 043852

r S O u . c e 6 ~ u L. ~ 0 a ~n {,..I 4 _ ,'.z :7u c7 2 _ Figure 15 Percent C0lIb in I9C3F1 Female Mice After Increasii;g Exposure To Cigarette Smoke (E003) I 2 3 . r i s (G1 G) (O-1-ll) (1af10) (10r10) Puffs/CI(larcttc I t 6 (10110) smoke dally smoke 1X ~ machine ~ (I0i-10) controls ,r L 75

L. F I GURE 16' Substrate spcciiicity cf ?C3I'1 Hcpatic Microsomes Using D1!rQrC7a t.^.CUce-s 0.6 0. 5 0. 4 E E 0. 3 r, I _ 0.2 ~ I x ~ 0.1 ~ ~ 0 t _4Lrl~ =A-- ISO3 C 4 IS04 1 ~ 2 3 NumScr of carbons inroaoru:4n side chain at 2 position 76 CTR CCNTRACTS 020363 I GTR I-IN 043854

C ( Ficure 16. T^._ effect of known ~*H inducers on the 0-deet!~•,.•lase V -ex profiles of BC3F1 h•epatic ricrosorr.es tested %•:ith a n r•olo;c:s series of rescrufin substrates. The ~~cax for each substrate was calculated frea, a Lineweaver-Surk plot of the initial velocities obtained at four differFnt con- centraticas (0.5, C.2;, 0.1, and 0.05 HM). Ti,e six subs.rates tested •.•:ere: r..et-:cxy, et5or,y, procoxy, isopr•opoxy, butcxy, and ..ssays co, tained: 2 rl 0.05 M TRIS, p~4 5 hl iC ;:1 ;0 ,a N:,O?P2. Reactions were run a; roo tcr, _ra: re and ,roduct forc.ation was deterrnir.ed by fluc,•ic•=_;ry at an excitation waveienc,•;h of 520 n^ and an er:^rissior waveier_-h o` 5c6 nm. Inducers were administered IT in 10 Hl corn oil at these doses: 1) 5,6BF (100;:c/g), 2) TCCO (60 ny/:), 3) NCA (180 pg/g). 77 CTR CONTRRCTS Q2Q'36a c I CTR HN 043855=5'

FIG. 17 70 6 C 50 :o CCt-,b in EC3F1 Female Mice After Exposure to Multiplc 2A1 Cigorettes on the SEM II B. (1-100, Group 07S1A) ! t1k 30 10 0 1 . 2 3 4 .Number of Successive Cioarettes Offered 78 r CTR CONTRRCTS 020365; CTR HN 043856-

FIG. 15 r i0 50 ( 30 20 10 0 ro CG-b .. ---_,=1 Fe.^ale h!ice After Expos.ure 2A1 Ci;ar..:es cn tne SE:•t II B. (I-iOG, Grou.p CqSIA) to kultiple 1 2 3 4 5 f~um--er „r S_,.~ess:ve C::aret`.es C"ered cTR cGN?"RNC t S a2o3C'C: c I CTR HN 043857

Figurs l? Reten:icr. c' COHo in EC?F. Female I•tice A`ter E>:pesure tc 2-+1 Cicerettts cn the SE.N II =. 100. C 10.0 1.0 Bleedinc 71r..e (!"in) :fter T•.+o Cigaretre: e0 .t C?"R CCN?'RACTS Q:2G. = I CTR IIN 043"BSO

L. ( FIC! - :=: 20 CTR I-ICO, HCA A!:D S~,;:OF, LOCtiRCINOGEI•!ESIS, P?Go:.3ILITY OF SQUAMDUS HETAPLAS1: t.t:D SQL..r,-~US CEL! C..RCII:v.',A I;7 6C~r i/ Ut9 FENALE MICE 1+FTER VARIOUS TncwTp;EN;S ~ ilITH MCA (C50 }:9 X 9) ............. .._'r P;ASii. SCC c .050 r,. 2A I S' OF:_ e K ~ .?A, SM.r :~:' . . ..~. ( 4rJcZ.L-, S On iBSt l.! R i.0 NT f:R C! Sw .• `-'u36S C I CTR HN 043859,

L. 0 0 FIGURE 21A 1100 XNLIPi- ::Clr uR:e,•;v.att0 01:10 uNlwG::.'lR7co OitIC U-1Ir.,:;,'LR7C0 n1;10 Usti4:JtRtCO 19.1^70 i 0 0 C......~~1_ V . V O WC' KS ON I[.. 1 i .. . . ~ ~i J . ~ V 02 CTR CQNTRACT: 4241-:6 9 CTR Nt,,-l 04'~860

L• C C 0 0 0 Y) m l ! % FIGURE 216 1100 JRIU0.jT 19,197s 02C1L Cr--:ruMc - ~~n r oz:1e Ot.IC C:L-au.c - :nRr Ctt-:nLInc - :-nr c-o ~-e ot:i: CCl-On:rFC - 9nclr +--« r.- -1 c 0•00 16.00 WCCK$ wy 40.1.i0 E3 L C CTR coNYRt,-, . S 02a~r o E I CTR HN 043861

C I ro ~ ~ 0 •..~ .7_ ;u t ~ ...r. : .~ --~ ~ ; Ln ~ r1Ef1N ANIMAL WEIGFiT. GRAMS alo.oo l5. 01) 20.Oo 25.00 So.O. F/5.0o 130.00 ----~-- . ~ I . m U Q M 1• {) I] u D , ~~ ~

( FIGU?._ 2i D i 100 JF%U?1T 19.1:10 ( l ( ( 0 0 n 0 0 oa__ -b.00 ..... 23:JC hCr~ ...... Zs:J. IncR ...., zs:uc nca - :nnr :nnr :nar 6.Ou" 16.00 24.00 WEEKS ON TEST 32.CC .4 C.Oo CTR CCNTRACTS 02031."2 „ 0 CTR MN 04-3863

FIGURE 21E j i U o ~aso-n t:.1s7e CJMSN Cvrltl CUt1C Otlv..^.SP'.;7 - !il1CM!~:C C+1NOCL1P1C0 - Y.ecM1C[ Cx1KG:J197C0 - RRCKIl:: E---a i -o ~-.-+ cvu10 C'R1SC CC.1r.;;.;tFiCr- p:dUn: 'Ol1>rSCVLniCO - F.,CN1:iC <-~ O O - i ~.oo . 0.00 16.00 ^~ '^.u"0 V0.0c n=~KS G'ti ;_~' 86 CTR CaNTRRCTS Q2Q--<3 CTR HN 04-13864

C C FIGURE 21F I 1 G 0 JAwuAAT t:.1~7J CG-.?L-IMC - MCtJ6t 0-0 0i~t: CSMSC CSatO :SMSC CC:-7FL1NC - dAM1vt Ci;-:F:1KC - MnCM1NC C :-SCUat - MAC;t1wC - 1irlCH1NC t.---~ •-F %--K 0---•O O O -b.00 fl'.0ot 16.OG ^C 3i.00 40.00 ~lEcKS ON 87 ( C CTR CONTRRCTS Q20-3-74 I CTR I-IN 04,38635

( L• -A FIGURE 21G 1 100 JAxUrnT u.lsie O C7 O, p I l7 Z'.:J: n;n - r-ACnrY.C o---o C:nl: 2S:Jc R.A - It.CNINC C:r::C 25CJC ACA = KACnl:;C ~y CCn10 2SCV0 RCA - !!FCn1KC X--K OtnlC 25C'JO RCA - InACM1nC a--o ! M 2 _ 0 0 ~b.00 0.00 !t.00 211.00 WE='r.G Ct; T C.`:T . 32.OT0 4b.00 £Q lU ~ r 5 CTR CaNTRRCTS 020 V i R i 7 f i S-A -."T 3866

C, FIGURE 21H JP~IL'f~T 1~.1570 1100 n G O O 1f 17~ n I 7 f 9 (/7 C o 1 vo. 1 i 1- Z LD o ! C .-. 0 0 +- 0 ~.00 C7:1ft 07:1E l'N1Y.C:~L~TCO - Qn1 Uwl~:O:LlF1CJ - 2A1 6-4 0771C UwIwCCULPICO - 2F1 :?tOKC ~.._. 07:10 UNIwOCULPTCO - 2al 07:IC Uw)neCLtPTCC - 2,31 !^:aC ~q 8.00 r:b.1:C _21:.00 32.C0 4 0. 00 1::.~ l ~S I CTR coNTRac i S 0203376 I CTR HN 043867

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L• FIGURE 22 1101 iiAi.1tT 1°.1C7a f^C2p C^.-!.tliML. tII c--o 1sC2:, 2j0pg MCA, 9X ~ 0 0 o, 0 0 m- I i6.Q0 24.C0 32.00 40.00 ~N I ES't CTR CCNTRRCT5 020-31; il CTR HN 043870

L• ( ( I ~ .v:$ I lt.%('1:: >tlJS"r 11.1' ItLC01\`L'u u'1' NU~ICNlil):It H. 1977 h.< ..r.t..,..1 r,r.J wr. ,•) .n., nn.1K.\r'r 1•n . u. rur r 1 u.t\I r. r..•. , ..... n-,a•iv . ..J .r..~•....n :. r~.rn...,a... • <..p.. V./n ..,rPr..re.... aa Ih.)eln..l•.I:n..i..NV1+J. ~ e . nJ .nrr..J.,. KI C[.\HCn fJ,...r.ai llua<: Id<Monr.:1S/ l ALL CUESTIONS Illil.Oll' UUST DE ANS'W[RL:D DfrORL l'llf AUSTR AC7 fAN UF. COSS1.7t:RCD U1' THE r oOGRA'.( CUMM1•fTLE: St•.•nn rn .nt~ r•a^rr n~t.r ne .<1<JvMJ Itr,et'. n,• n~./ L k~.ci,.•.nn.n C]ct•na•al In.< ~•;.r~,•rw -(J-+ L ei-ar ehcrwu.: rarl 01 TT_M1.:..pe O 1•.rni,.tr I h<.<nqlun prtt<?<.ee (eh«I nnt enty)p rn..er It the pr ~<mari•.n enrkrrn<t a, ind.oreJ eai. nel !Y )vnir:J P, r1r r.ngran CCnrrnnne. x the SPV.VIOR an.;vt Iht sbenu.ne J.<nion. O rhe S?(:t.SO4 M•f nrw at<t1r the all<rwaat drw..n a<.J Ye. nn .M rn Ir nn rht rnyrarn. O n,t !rY)~SON .nA. x• .nr\n.v u~ aN.ra<x Irrvw ru. n•r.n re r~ • p4OCCCDINGS. It r•< a1..va<. n n.w rn R rr<v:mN.~ :Fsrrt.: SrONSO4 •..ill.n/ ru M.r fr pvrvaJ.r. rkoceecl>GS. `; q.•: SrO'5U4 ...r.•..r ..reJra.•.r l.ar. r.. .n rht rxocz'_-,I.Ncs. Absrra-' SPCNSORED Oe D: Csr- _ ^~~-- - . `,.AO is -'M:reGucinj" this ahstract. the ou:r:nrs~:p o(~`icn is enlirtly nonmemDer. ' Teie;hone numrir nf firtt aulhor: " !'N:.ncs of nrcm~cr aulhor(s) uther than SPONSOIL• ° t eh.-a ' ~nr• r 2! •lhc is :n :cthor of t.••1s obslrx;. CR 1973 ~• AACR aasra•aC- _... _ FOn.a.1 PATFiOCEt:ESIS'0? ClG•`fICI.LLY INDUCED LBNC LESIO;:S Ia 1i:CE.r'• L: •11: Bi:lups, C. J. Henry, C. _. -hicr.,lre,;•. R. E.\Kouri::r- Y.ScrcS!ologicsl 4ssociates, bethesde, ~c 20015. Criteria have been cscablished for the p:og:ess!ve bistolo;ic ehan;os induced by intratracbeal iroculac:o^s of 3-mathylcholanch:cne (/iCa) in the lunss of va.ious in~:ed strains of eico. Trected niee (250 )je bi~aeay for 12 •:ks.) stere cerially sac:ificed at eopropriete c!=e !_:c -ais i1 order to decucent and characterize p:oli:_:a:!•:e c::~r,aes :n the lung srit4 regard to cell type and d!s::_ sL: l„ suggest tuat at lx:st three early•mot,=.o:=;. t•:,c+ -.y y:o- 6:ess to caligr.;at ntopla-ia. Y.,3:vho:ce_ec:!:e:!a a:or.: are noc Euff!ciEnt: to elaasi.'y cr p:edic: che __..:_.. b:o- logic b.lavior of lesioas. These scudies scx;ea:,Ch.: the p:olifcraticn of alveologcnie c¢lls prog:estes °:en a s:age te::•,ed hyperplas!a, throug4 a stage 4aich is ncculn: and co:presaes the surrounding parer.chyrna, finali7 :cac: ir.g a stage vhe:e papillary ar.d plcooorphie cta-ecs cceu:, i.e., alveolar adenocarcir,omr (AAC). Progressive s:ages of early aqueaous lesior.s (squ=ous cetaplasia) are leas clearly de- fioe4, appear to be,treatmenc related and may :e;:ess 4tlcn treacment ends. Squaaoua eall-earcinona (SCC) appears to arise froa the bronchioles or alveoli ad;acea: to the terai- na1 bror.chioles. The difference ia pachogeaesis betFeen alveolo;enic and squamoa:s lesions sugge:cs a di:fe:ent :ode of progression for DJIC and SCC. The lesions prcc--nd have at least one type that is mo:pnologically the sax as that four.d in huaans, i.e.a•SCC. In tcodel systeas •.there pul:o- aary aryl hydrocarbon hyd:oxylaae (AHH) isvels a:e hos:-ser.c regulated (C3H/fCum and flUA/2J; C57C1/SC:.-m and Lr:/2J), sus- ceptibility to these IdC'r induced pulmona:y ea:ci._=as is genetically linked.co these AF.}i levels. (Suppcr:ed by The Couacil for Tobacco Rrsearci, USA, Inc.). vu n'urrucn 9[L ___...a e... (t.7uC netnt o/SPONSOR) (urrn toAt• tdeyAon: mru;r: Namcs of nnnmemLyer au/hnr(s): Dr. Carol J. Heary. Dr. Leonnrd H. Billuos .I 1 i S.gmm~ren/~l'r1r~.lUK h<U^n- et rrnrimsrr.l Ornleev t U. (nnnnrnr aard brwnuann 5221 River Rord I AJJeraa err?.n<A, ~^1 7rltl 1 R ~ ~ L'ir,r and Starr (Cbwury) 2.p ('.r. I i CTR CCNTRRCTS 020361 ~ rf ~ CTR HN 043