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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