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! Preprint ~T®012005E December 198 Accepted in Toxicology and Applied Pharmacology C Depositiot and Distributioa oY the Total Particulate tSstaer of Cigarette Saaice in Hice Osing a Large Capacity Smdse Esposare Systeml Rusoi,ag Title: Cigarette Smoka Dosimatry ia Mice C. J. Bearyao5, J. ::. Caton3, J. $. Stokaly3,. 3. R. CaeriXL3, e. LapesZ, •L'. D. &very2, D. 8. Dansie2, G. 11. Sea3ersoa3, T. vayle3, C. E. Wb,itmire2•4, and R. E. b,ouri2 - f 1. f'reseated ia part at the 18th Aaanal aeetiag of the Society of Tosicology, :TSw Orleans, Louisiana, aarch 11.-15, 1979. 2. Department of Fxperimeatal Oncology and Department of 8iochemical Oncology, aicrobialogical Aesociates, 5221 River Road, Hethesda, 1larylaod 20016. 3. The Aaalyticat Chemistry Division, Oak Ridge Nettonal Laboratory, Oak RidBe, Tennessee 37830. ~ 4. Preseat Address: ;Iationsl Cancer Iaetituta/fttiooal Tosacology Program, Sethesda, c;arylaad 20205. 5. To wham correspondenta sbould be aeat. l

HTe0120057 "sTSacT Deposition and Distribution of the Total Particulate Matter of Cigarette Smoke in Mice tJsing a Larga Capacity Smoka ' Eaposure Systea Henry, C. J., Caton, J. E., Stokol.y, J. R., Guerin, M. a., i.4pea, A., Avery, M. D., Dansie, D. R., Benderson, G. M., Gayle, T., Whicmire, C. 8., aad Rouri, 8. E. (1981) Tosicol. Appl. Pbarmaaol. 00, 00-00. I A nevly developed autoaatic Smoke Eaposare Machine (SEM II) ees ased to generate 14C-datriacontane labeled tJniversity of EemtuCky reference 2AL or ZR1 cigarette smoke. The SEM II is a large capacity (480 mice) dyaamic smoke eaposdre systen -ia which smoka is routed throngh the anjagL contaiament system as a contimioualy flowing stream. Mice ure restrained about the neck in stocklike b,olders for °nose-oaly" exposure. Using standard amo[ce .ocposure conditions, the deposit3ou and iatesaal deposition of thm total particulate matter (TPZi) frcm cigarette smoke vas determinad in SC3F1lCum male and fematm mice. Results show: a) smoke exposure conditions can be varied so that deposition from 30 ug to 200 µg TIW]amg caa be obtained, b) 80-90X of the TP!i deposition was found in the re9piiatory tissues, c) the monse-to-mouse variation for TFM depogitioa in pulmoti;ary tisssue was ti20x, d) similar deposition and distribution of T8PH was observed in sale and f®ale mice, and e) depoeition and distribution iF TPM was not altered in mica exposed to smoke oa a daily basis over a 6 month period of time.

a- - HT®0120058 1 UrTAODUCTION There are two major objectives when studying the effects of cigarette mmoke in esperimental animal systems: a) the s+aoking machine ssast stimu- late bs-- making conditions and these conditions must be easily ra- yroduced on a daily basis, and b) the particulate phas® of the seoke aerosol generated must reach the Langs of the an3mald. Every lamma amoicer has aa almost unique puffing pattern, within which there may be consider- able Eluctuat.ioa (Wynder and goffman, 1967). Although no s®oUeing device can duplicate h-a smolci.ng habits, machines can approsimate human making by usiag standard smoking coaditions which are the average values obtained from a large ottatber of saohsrs. Four aariables which influence the yield of total tobacco s®oke, total partiauiate matter (TP:!), and iadividael smoke constituents, have besa staadardized for machine smoiciag: puff voLrme, puff frequency, puff duration, and butt length (Wynder and Hoffman, 1967). ® standardized making regamen'tsas'been established where the cigarette is puffed onee per minute generating a 35 ml puff during a two second period. ihe cigarette is saoked to a 23 = butt length. In the present studies, SC3F1/Cuu (C57B1/Cue x C3S/Aaf CuR) =ls and fe'ma7.m mice eere eaposed to whole cigarette smoke %sing this standard smoking regtaAen wxth a nev Smoke Esposure Machine (SFId II).'The S}2t II is an automatic smoking system, designed to generate and deliver a con- tirnsoue strum of fresh snoke for large scale inbalation studies. Ic is a dyaemic systan in that the aai.4als do not rebreath the s®oke, In contrast to a static systen where animals breaths into a chamber. The SSti II uses revarse making (positive puff) and automatieally loads, lights, puffs, and distributes the smoke from as maay as 30 cigarettes to special holders where "nose on.ly'r exposure is effected. A total of 480 miee can be eapoaed to smake uVing various ®aposnre regimens with the SIIK II anSmal contaicaent system. The distribution r,aa dosimetry of whole cigarette smoke was deter- m3aed using this SIIi II system and the results are presented here. LiSTHODS Smoke Generation The,SF2f 11 (Process and Inetruments Corporation, Brooklyn, .i) shoM.n in Figure i, operates in a maaner simulating bmaa making and automatically and sequentially loeds, lights, puffs, ejects, and eatinguishes a series of -1-

NT®0120059 ( 30 cigarettes. The operating principles of this machine are presented else- where (Moaeybua,,a~t al, 1979). The staadard smoking conditions employad ars: puff fsraqueacy (oaeaminuta), puff duration (2 seconds), average puff volume (35 ml) and cigarette butt Length (23 amt average). These conditions are maintained completely independent of the aaimel contadment systea. Thirty cigarettes are losrled from a hopper into holders fitted oa the srsr- face of a rotating drum. The drum advances one holdes position every Z• seconds resulting in all 30 cigarettes being sampled every 60 secoads. Puff air is forced through the cigarstte being sampled by a constantly held d3fferential pressure betueea r.ha lit end and the butt end of the cigarette. 7ariable amcuats of dilatioa air can be introduced at the butt and of the cigarette. This a]loros a range of smoke concentrations from 5-100Z (v/v) which can be offared to the test aaima2s. The diluted snake flows at a predetermined rate through a programeabla distribution valve which a+eqnen- tially directs the smoke, at precise time iatervals, into oae of four .hsanals leading to the aaimal contaioment.onits, or to an esbiust system. A diagran of the smoke generation system and aaLm,l containment unit is presented in Figure 2. Smoke can be alteraated with.breathing air for various intervals within a one minute cptle. dir is provided to all a!an*+pla ia the absence of s9aoke. Fach chanaeal hsa the capacity to provide smoke or, air to L20 mice at one time. Cigarette smoke is delivered to the test animals within 2 seconds after generation at the cigarette. Preyious studies have shosra that smoke particle siae was (as determiaed by methylcyaaoacrylate fixation) log normally distributed with a geometric mean diameter of 0.34 microns and geometric standard deviation of 1.35 microns (Holmbarg. 1979). In the studies presented here, two of the four chaemels were used. Smoka was provided to aae clauaa®l for a set amoumt of tima (e.g., 30 secomds) while air aas ptovided to the sacosd•chamael. At these predetermined t3me intervals, the distributiov va.lve was aucomatieaLl.y activated so tbac the first channel received air whila the second chaanel received smoke. ftoks exposure is presented in terma of total smoke eaposure tlme, e.g., 30 setoads smoke eapoeare per minute, alte=aated with 30 seconds of air par miauta, for 10 consecutive minutes resulted in 300 seconds of total smoke ( euposura.

H r®o120060 I ( Smoke moIIitoriIIg A smoke monitoring systam was developed for the SFM iI. Tha monitor provided• coatiauous measurement of smoka particulate phase ('rpri) and smoke gas phase carbon moaozjde (CO) i;mmadiatel.p up stresm from the aa3male (see Figure :). Smoke particulates were measured with an optical-type scattering detector (Siggias st a1., 1978), while a.noa-dispersive infrared aoalyzar. (Beci®an Model 864-Source Infrared Aealyzer, Fullerton, CA) was eatployed for CO determiaaticns (Si,ggias at al., 1978, Gayle ae al., 1979). Each exposure was doewaeated on a•Speedamaag M 2lark II Recorder (Leeds and Northrap, ttorth hiales, FA) which provided a permaaent recor.d of the puff by puff profile and fita]l ictegrsted values for both 1'F!i and CO. The detector signals vere e processed to interface with 'approprixte saf ety appara- tus to protect the aaima.ts from accidental over exposure. - The integrated sigaa.t was coaverted to mg TPM by using a correlation factor derived by simultaaeously. cpmparing this sigual to lmoan weights of ~ '=PM which were col.lected oa Cambry.dge filters (Caabridge Filter Corporation, Syracuse, NY, see Gayle at al., 1979, for details). For smoke concentrations between 5% and 20g, the sensor readout oas linear with respect to TPM. The concentration of a 10Z smoba' aerosol was approximatelp 9-10 {ig I3Wml (v/v). Male and female BC:3F1/Cum mice (C5781 x C3H/Aaf - Cumber].and Vi®w Farma, Clinton, TiQ) were purchased at 4-6 weak9 of ag€, housed S mice per cage (str.ialass steel cages equipped with plastic fronts and filter bonnets) oa Bed-0-Cob coYa cob bedding (Chesapeake Feed Company, Beltsvil7.e, !ID) and allowed free access to Pnscina Lab Chow and water from an autamatic watering systea. 8aclc9 containing the animal cages v®re kept in a room at 70-740r with a light cycle of 12 • hours darimess and 12 lsours light from fluoreseent lights. dica were initially esposed •to 80 seconds of 10Z 281 or 120 seconds of l0x 2A1. Xwtwcky reference cigarette smoke, twice per day with a taa mimata rest between exposures (see Doaimatry). Eaposnre was inr.zeased rnily by 20 or 30 seconds of amnke•per exposure until a total of 360 or 600 seconds per day was achieved for ZR1 ar 7A]I smoke, respectively. Nice were exposed on a daily basis (5 days/yeek) to this Level of emoke natil dosiaecry studies were performed. .

H T®0120061 ( i ansmal Hoiders The an.ima1 conta3ament system for smoise esposure Ss showa in Figure 3. Groups of f ive aaimals are held Ln a "stock like" holder using a co®biaation of a neck slot and a restra~A~ spring. The chin rest insures that the nose of the anima3l is aligaed with the cone shaped openin8 on the eaposure modules (Fig,use 2). The mesa of each saimal passes through a deatal rubber dam d3aphragm (Process and Instrsmtents Corporation, Brooklyn, NY), Foraang a seal that preroats eaposure of the body to the smoke aerosoL. Bubber dsm diaphragms must be changed frequently (at least monthly) to ma+n*9tn the seal around the nosa. Daily restraint for up to three hours (twice par day) resulted in mortality and mo obvious ill effects to the anIma].s. Cigarettes Kentucky refereoca 2A1 or 281 cigarettes (University of Santneky, Leu3agton, SY) wore used in these studies. These cigarettes have beea characterized at Oak Ridge N,atioaal.Laboratory using fhe Phipps and Bird Analy- tical Smotcing Machine. tTnder these analytical conditions, 2A1 cigarettes delivered 38.9 t 1.9 mWTI+M, 0.54 t 0.04 mg nicotia, 19.0 - 0.6 mI. C0, (yoneytum et al., 1979) and 2I4 cigarettes delivered 45.+ = 1.9 mg T!!i, 2.10 - 0.13 mg nicotine and 21.6 s 2.0 ml Co. Cigarettes were preselected on the basis of weight (1. 7.Z t 0.10 g) and resistance to drav (80 = 5= IL20), measured with a Filtroua Pressure-i?rop Tester (CSgarette Components L.imitmd, ttembley, England). Gigarattes were labeled with 0.Z5 - 0.5 uCi 14C-dozria- coatane (14C-DTC, 3merican 8adiochemical Corporation, Teaford, FL) as described ,previonsly (Cacon, 1979). Dosimetry 8ine esperimental measurements 4ra required for each eigarette smake doshoatr3+ study. These are siaaoarized in Table 1. The smoks TPti aae - datecm3aed using the opt3cal seasor (see Smoke Monitoring) place directly in the stooke Line lmmediataiy prsceeding the +`aioals, iass than 0420 csn fram the eaposure module. Total radioactivity was determined.by withdrawing tmofan volumes of smoice from the sampa.ing port (see Figure 2), col.'.ecting this sample oan Camyridge filters, elatir.g these,seoke particn]ates with pytidiae, and caunt3ag the pyridine solution by liquid scintillation speacro+s.etry simo.l.taneously with interna]l standards to correct for quenching and counting effienGy. The specif ic acxivity for the 14C-DTC radiolabeled

snoke was 15-25 disiasegrations per minute (dpm) /ug TPM. Ia addit+i0dq %!!e~ 00 62 smoke delivered to che aalmals was further moo.itored by withdrawiag samples of smoke from the first and Iast an3mal, eaposnre port in the aniesl costaia- mear, unit. These mpnsuremeat9 were asad to qua].itatively check the r.adio- labeled snoice bafore and after esposure•of the anima]s. Differences of lose than 5% of L4C-DTC content saera observed between those two points. Tha amouat of TP".i res,oved by the animels in Chese eaposuras was less than C 4X of the total generated. Mice were exposed co smoke for various tiae periods ranging from 3 weeks to 6 months to allow for adaptioa to esposure to cigarette smoke. For the dosimatry studies the fo]lowing protocol was used. Mice were espoaed to either 180 seconds of lOT 2IY1 onlabeled cigaretta smoke using an eaposara cycle which alteraated 20 seconds of smoke with 40 seconds of air for l0 consecutive minutes, or 300 seconds of 1OZ 2A1 unlabeled cigaritte smoise using an exposure cycle which alteraated 30 seconds of smoke with 30 seconds of air for 10 consecutive miautes.' After a 10 minute.rest, mice were then exposed to selected esposure conditions utilizing 14C--DTC:-= of 14C-=-2Al cigarette smoke. Imeediately after ssak,e exposure, mica were killed by C02 asphyaiation and selected tissues were removed, triaed, blotted to remova excess blood and fluids and stored at -60oC until aaalyred for total radioac,ivity. The following tissues were selected: head, larynx and upper trachea, lungs and lover trachea, esophagus and stomach, liver, hides and a eomaosite sample of all remaining tissues. Lung and lower trachea ci.!sue samples were weighed before storage. All tissue samples were coded at Microbiological Associates, packed in dry ice and shipped to Oak Atdge Nat.ional Laboratory for detera.iaatioa . of radioactivitq. Tissues rere qutcklT thawed and solubiliaed in an alcoholic potassiom hydroside solutioa as described previously (Ceton, 1979). Tissue samples fro® mice exposed. to noa-radiolabeled smoke were used to determine background values. Background values varied among the different t.Asaues and averaged 165 dpa for lung and lower trachea, 155 dpe larynx end upper rrachee, 82 dpm for head, 13.2 dpm for stomach and esophagus, 60 dpa for hides, 55 dpm for lAver, and. 88 dpm for the remain.tag tissues. Because of background variabi].ity, deposition was not considered sigaifi- cant imless test tissucs ware found to have values three times above 'i3ack- ground. The only tissue sample which met these criterala were the head, 3aryoa and upper trachea, lung and lower trachea, and stomach and esophagus. -5-

H T10120()63 . l All radioactive samples were corrected for background, quenching, and counting efficiency, and final data are preaented in terms of ug TPM deposited per total tissue. The affect of total s®oke exposure time oa the deposition of TPl9 in BCiFl/aam mice is shown in Tables 2 and 3: Mice were exposed to 10Z (q/v) 2R1 cigarette smoka for a. totaL of 180, 360, or 540 seconds• (Table 2) or to 20Z (v/v) 2A1 cigarette smoke for a to.tal of 50, 100, 150 or 200 seconds (Table 3). Depositioa increased in a].L tissues arLh increasing; exposure times for both smoke concentrations. L tjme dependent linear increase is evident from Tablee 2 and 3 for deposition ia the long and iower trachea, the total respiratory tract and the total body. Correla- tion coefficients (r) of 0.99 were calculated for the deposition in thaa Lung and lower trachea as a function of total smoke exposure time for both 281 and 2A1 cigarette smoke. the amount'o! radioactivity detected is the lung sample was always 5 to 10 times above background. No other tissue samples reflected this amovat of radioactivity, however, the head, laryna and upper trachea, and esophagus and stomach were generally 2 to 3 times above thetr respective backgrounds. The liver and composite remains semples were not above background levels and eere not considered in furtber eaperiments. TPM deposition on the hides of the animals was found to be a functiaa of the seal around the nose provided by the rubber dam diaphragms. Changing these diapbrages routinely resulted in TPM deposition of less tbatt 25X of the total internal tissue deposition (data not shown). The intesaaimal variation in TPM deposition in the lung and looser trachea vss found to be dependent upon exposure time. The coeff icieat of veriation (cv) for deposition in the lung and iover trachea from 13 nice per group decreased from 0.30 to 0.14 for 10Z smoka when exposure time vas increased froq 180 to 540 seconds. For 20S amoke, the cv's for '0 mice par group decreased from 0.67 tc, 0.21 when uposure '.ime vas in- creased from 50 to 200. The deposition in eatrapulmanary tissues was more variable and these variations were not dependent upon exposure tjae. Oeposi.tion in the laryns and upper trachea was the most variable, as reflected by cv's ranging from 0.16 to 0.80 (see Tables 2 amd 3). At a 10$ smolse concentration, equivalent deposition of ZPX from 2Aa1 or 2Rl cigarette moke was observed. For a range of exposure tiaea, -6-

I ( HTQ0120064 the rate of deposition of TPH in lung and Lower trachea was 0.45 ug/sec for ZLL cigarette srake and 0.43 ug TFlVsec from 2RL cigarette smotca. As can be dsearmined fram data in Table 3, the rate of depositim for 20% U1 smoke, i.e., 0.9 u6 TP"_s/ssc, is double that for I0X 2Al smoke. Compsrisoe of 10% and 20% 231 cigarette amolce could not be made because of eoaieity to the anioals of 20X 2R1 saoke. The SEi tI is a dyoamic smoke exposure machiae which generates smolse in a flowing stresm to the animals. The snoke flow rate to the aaimels is 5.0 Iiters/miautPs far = saoks concentration and 2.5 Litprslminute for 20X smoLce concentration. Dosimetry studies were p®rgormed to determine whether TPH deposition was altered by a change In flow rate. 8C3F1/(am fema7.e mica mace esposed to 10Z 2A.L cigarette smols.a for 300 seconds total eaposnrs time, with smoke flow rates of 1.0, 2.5 or 5.0 liters/minuts. The smoks scream was mechsnically altered to provide the 1.0 and 2.5 liter/ aiiaate flow rates. Palmouary TPH deposition was 123, 142 and 123 ug TPH for smoke flow rates of 1.0, 2.5 x* 5.0 liters/minutd, rsspectively. No change in deposition was observed when the flow rates were changed. The distribation of TPH within the,mouse was analyzed in all o: these esperiments. As prqsented in Table 2 far 10X 2R1 cigarette smake, the percent of total deposition of TPH in lung tissue was over 802 for the three exposure timea evaluated, with an average of 882 found in the total respiratory tract. The esophagus and stomach contained the rema-detectable radioactivity. Data from Table 3 for 20X TAL cigarette staake, demonstrated that the percent TPTI foand Sn lung tissue was 60, 78, 80 and 81Z for 50, 100, 150 and 200 seconds aaposure tiee, respectively. For thess same erposare times, the percent TPH found in the total respiratory tract was 83, 89, 81 and 944, respectively. As seen in dal:a from Table 2,' the rema ins radioactivity was in the stomach and esopbagtss. The effect of previous ®aposure to smoke on '!PM deposition and dis- tribution was determined in three groups of SC3F1/Cum female mics and compsred to one group of BC31!'l/Ciaa male mice. One group of female mice and the male group were esposed to tan-T0 minute ~asposnr®s per day (5 days/ week) for 6 manths. A second group of fe9ma].e mice was exposed to ten-10 minute suposusrea per day for 3 moncbs, and the third group of female mice ums espoacd to ewo-10'miasite exposures per day for 3 veeks. The female mica from these three groups were exposed simultaaeously during % doaSmetry eaperiment utilizing 102 ?A]l cigarette smoke for 300 seconds total eaposare

i / time. The ®a].e nice were exposed to che same eaposur.e conditions "MS~' 2 a u 6 5 a different day. As shown in Table 4, TPM depo9itioa vas observed tobUe equivalent for ell four gsoups, with no sigaifi.caat differences observed between any of the groups for deposiLSoa in th® luag and lover trachea. Thus the average daposit ian in luog and Lower trachea f or 40 fema].a mico was 145 pg TPH (average cv • 0.15), 167 pg TpK for the total respiratory tract, and 183 Ng for the total body, compared to deposition ia 30 male mics of 1&6 ug TPM (a+v ° 0.26) in lung and lower traehea, 170 {ag VN in the total respiratory tract, and 198 µg TPM for the total body. No sigaS.ficant differewe in TPM distributiaa was found between m®le and female asice. The percent distritwtioet vas detsrstiaed from aI]. TO nice. Bn average of 78% of the TPM was found in the luog and loseer trachea and 93Z was fosmd in the total respiratory tract. These distributioes vere qaita reproducible with cv's of 0.05 and 0.03 for lung and low®r trachea and tota]l respiratory tract, respectively.