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1 MICROBIOLOGICAL ?,SSOC:?.'_"ES PROGRESS REPORT FOR C':R-oo3o---SMOK:. I_NFik:.A':ZO:r _N `".ZCE Prepared by ^.arol J. Her.ry, Ker. K. Kanagalinaam, ?'.-:. ~ . Kin-Kai :iwanc, ?h . O . Marshall Oinowitz, Sc.:. Ronald A. Lubet, ?h.--. R:chard E. Kouri, ?'.:.:. March :5, 1981 ,`)L 1 AT. 3~r 0010 1141. &mvgff$T / \ cKkTOtTiOiO&w -uSoCi2iiQ5 „ok~ : L r^ v ~ .. .,. .. v . CTR PIN 044474

~:f0250?53 . TABLE OF CONTENTS Pace _ Abbreviations Chronic Inhalation S tuCies A. CTR 101A. Chronic Exposure of BC3F1/Cum Mice to 2R1 Cigarette Smoke. I 3. CTR 1013. Chronic Exposure of BC3F1/Curn Mice to 2R1 and 3A1 Cigarette Smoke. 2 := Corollary Studies A. 3. :.. ... CTR 119. Deposi:iorn and Distribution of 2R! and 3A1 Ci.arette Smoke lisinc =:^e Continuous Smoke Exposure Regime.^.. r':R 109. Alteration of the Izunu:.e Response After Exposure to Cigarette Smoke (Dr. a. I?erscowitz). 3 C':R 105. Deposition, Distribution and Clearance of 3H-catechol (CAT) in 3C3F1/ C:un Mice After Exo_ osure to 3Fi-CA^-2R1 Cigarette Smoke. 3 ~:'R 128. Evaluation of Chromosomal Damac_e or Alteration After Exoosure to Whole Cigarette Smoke using a Sister Chromatid Exchance (SCE) Assay (Dr. W. Benedict). C':R 96. Inhibition of Pulmonary D~1A Reaai= :.apaci_y After Exposure to Cigarette Smoke (Dr. R. RasmLsser.) .'T`:ese studies have been izcorporated under CTR 1013. _. C':R 127. Alkaline Elution Assay (C'"R Grant #1241). These studies have been incorporated under CTR 101B. Tables )C'.N3C!'ObiO~ .U50Ci3teS _ W,41~ ~~~ ~~~ 0~~~~4t~"`~

ABBREVIATIONS AAC alveolar adenocarcizoma s,FIH ar-yl hydrocarbor. :^.yd= oxv: ase BaP, BP benzo(a)pyrene BaP-7,8-diol (+)trans-7,8-dihydroxv-7,8-dihydrobenzo- (a)pyrene • CAT catechol COHb carboxyhemoglobin cv coefficient of variation DTC. :4:.-DTC dotriacontane DPIyS disintegrations per T:::ute 3H-HvP '3H-hydroxyproli.^.e HPLC high performance li=id chroma='og:ap'r.v 3H-TdR 3H-thymidine ~ intratracheal :? intraperitoneal labeling index MCA 3-methylcholant::rene MFC mixed function oxidase MMS methylmethanesul:or.a :e nicotine ODC ornithine decarboxylase PA. polycyclic aromatic hydrocarbons ?A.N.A pigmented aveolar macrophage accumulation accumulation PDC poorly differentiated carcinoma ?FC plaque forming ce_1 3'r:-?=o 3H-oroline ?&= Process and Instr::ments _ correlation coe=f:cier.t R.^S replicative DNA syr.t::esis SCC scuamous cell carc4noma S.V sister chromatid exchange SEM Smoke Exposure Machine SR3C sheep red blood cells T?A tetradecanoyl phorbol acetate "PM total particulate matter UDS unscheduled DNA syr:thesis wi•iSy Walton Horizontal Smoking Machine 1 :7!!r M.'MsCi~ObiOiOgiCa1 .A.S60CiateS - vess E..,. TR I"IN 0~-~ 4'4f=`~'~

~~025C~5~ I. C::RONIC INHALATION STUDIES A. C^R 101A. Chronic Exaosure o_° 3C3F1/Cum M:ce to 2R! C:carette Smoke. This study was initiated in September, 1978, with a total of 2053 mice exposed to 2R1 cigarette smoke alone, a total of 10-4 mice sham-exposed, and a total of 449 mice for shelf•controls. An additional 710 mice were utilized to evaluate the cocari:.o-. genic effects of 2R1 cigarette smoke and benzo(a)pyrene. .M:ce were exposed to 2R1 cigarette smoke for 110 weeks. This time was based of the number of animals alive at that time and which would be available for actuarial analysis. •Smoke and sham exposures were stopped after 110 weeks and the ar.imals have been hela and allowed to die "naturally". Because of the larc_e n,:mber of animals in this study, mice were grouped and star:ec or. test at three different dates. Thus, October 1980 was 1:0 weeks for mice on test since September 1978, November 1980 :or mice cr. test since October 1978, and March 1981 for mice or. test since Februa:y 1979. The numbers of animals alive in each series when smoke or sham exposure was stopped and those presently alive are g:ven in Table 1. Thus, 308 mice in the smoke exposed groups and 224 animals in the sham-exposed groups will be used for probabi- lity analysis of the effects of chronic exposure to 2R1 cigarette smoke. With these numbers of animals, it will be possible to distinguish differences in tumor probability between test and control groups of, for example, 0.10 and 0.18, or 0.50 and 0.65. ':'.:e number of animals required per group to discri:ni::a _e betweer. t::mor probabilities is presented in Table 2. The disposition of all animals for CTR 1Cla is given in '.'able 3. A total of 1647 mice have been zistopathoioc:cally examined and another 463 are being processed. Every ar.i:nal has been accounted for. 1. Exposure to 2R1 cigarette smoke alone. A total of 601 mice have been diagnosed in the smoke a:one groups, a total of 382 in the sham exposed group, and a total of 179 in the shelf controls. The only smoke-associated lesion observed to date has been the accumulation of pigmented alveolar macrophages (PAMA, "11$, Table 4). Congestion has been observed in both smoke, sham and shelf control animals.. Lympho- cytic leukemias, lymphosarcomas, fibrosarcomas, and sarcomas have been observed in smoke-exposed, sham-exposed and shelf control groups. Eight (1.4%) alveolar adenocarcinomas have been observed in the smoke exposed group, compared to 2(0.58) and 6(3.88) for the sham-exposed and shelf control groups, respectively. The d'_stribution of the lung lesions at various time intervals for these groups are given in Tables 5, 6, and 7. .HiCrObi0~ A3SOci8te5 CTR 1-11-4 0444'i"-"1P'

sF025oi 2. Effects of exposure to 2R1 smoke on BaP-induced lung tumors. :.ung lesions observed fol.:owing :ntra:racheal inoc;:la:i:- nn of 3 doses of 1.2 mg BaP, with and without smoke exposure, are sum- marized in Table 4. Several lung carcinomas were induced by 3aP, including AAC, squamous cell carcinomas (SCC), and poorly d:_°'er- entiated carcinomas (PDC). The total incidence of lung carcino- mas was higher in the BaP + sham exposed compared to the BaP + smoke exposed animals (84/188 vs. 71/198, respectively). The only lesion which could be attributed to smoke exposure was PAMA. The incidence of these lesions at 4 weeks intervals is presented in Tables 8, 9, and 10 for the Ba? + smoke, BaP + sham, and Ba? + shelf groups, respectively. Preliminary analysis of the data from those ani:aal's wh:c:: died during the experiment and which are known to have died w? ;.;. a random distribution, (139 BaP + smoke exposed and 117 Ba? + sham exposed animals ) has been performed usi ng the Mantel ciae.^.szel statistical procedure (Tables 11, 12, and 13). The results suggest that there are no differences between the BaP + smoke and BaP + sham exposed groups up to 80 weeks on test. After that time, there are significantly more malignant =;:ng canoers (AAC,,SCC, PDC) in the BaP + sham-exposed groups, com- pa=ed to the BaP + smoke-exposed groups (p=O.C23 at 92 weeks on test, Table 11). There is no difference in the time denender.t 4ncidence of premalignant lesions (p-0.293 Table 12). '"he :i:ne dependent incidence of premalignant and malignant lesions beca.me significar.tly different after 80 weeks on test (p-0.036 at 92 weeks or, test, Table 13). Preliminary analysis of data from those animals which were found at death to have lung tumors and in which it is reascr.ab:v assumed that the tumors caused the death of the animal, indicated no difference in lung tumor probability between the 3aP • smoke- exposed (46/60) and BaP + sham-exposed (57/77) groups (p=0.5, :'ab_e 14). 3. CTR 1013 Chronic Exposure of BC3F1/Cum Mice to 2F/ and 3A1 Cicarette Smoke. The animals in CTR 101B will be used to evaluate the effects of 2R1 and 3A1 cigarette smoke in 6 short term markers at 3, 6, 9, and 12 months of smoke exposure. The short term markers to be analyzed in male and female mice are: a) inhibition of pulmonary DNA repair (collaboration with Dr. R. Rasmussen), b) stimulation of pulmonary DNA synthesis, c) induction of pulmonary AHH activity, d) induction of pulmonary ODC activity, e) augmentation of the DNA damaging effects of particular lung carcinogens, and ~ 2 ~' .%Cl+obio~.UsO(.`i3te5 _. r t '~° -~ +~ ~ ~ f i.~ ~f~ I ~ "f

SrGZ5U15% ~) alteration of certain physical characteristics such as lung weight and incidence of pigmented alveolar macrophages. The smoke exposure regiffien for this study is sigr.ificant:: different from other exposure regimens used previously: a) there are no rest periods between successive cigarettes or "runs", b) the exposure period for smoke is 15 seconds per minute, c) each "run" results in exposure to 120-150 seconds of smoke from 2Rl or 3A1 cigarettes (- pu_'f/minute, 15 seconds smoke/minute 8 ouffs/cigarette), and d) smoke exposures are repeated "cor.:inuously", resul=i::c _ "runs" over 140 minutes. T!:is experiment is composed of two par_s. C-118, 3C3F1!C::- female mice only and C-131, SC3F1/Cum age matched male and re:r,a:e mice. The schedule for C-118 and C-131 is given in Table 15, with the cisposition of the animals given in Table 16 =.,_ and Table 17 for C-131. Results to date are as follows: 1. Inhibition of pulmonary DNA repair (collaboration wi_h Dr. R. Rasmussen). No marked effect on unscheduled DNA sy.^.t:^.es'_s ("D::?A repair") has been detected to date for mice exposed to the cor:- ..inuous regi:aen with either 3A1 or 2R1 cica=et_es for •c to 9 months (Tables 18 and 19). This is in contrast to results obtained with the exposure regimen used in ;,'^R 96 wit:: 2R1 a:.d 3a1 cigarettes, where a marked effect was observed after `3 mcn=:s exaosure. 2. Stimulation of DNA synthesis. a. Biochemical Analysis (collaboration with Dr. R. Rasmussen). DNA replication is increased -2 fold in mice exposed to 2R1 or 3A1 cigarette smoke for up to 9 months, compared to sham exposed mice (Tables 19 and 19). These results are similar to those obtained with the exposure regimen used in CTR 96 for both 3A1 and 2R1 cigarettes. b. Autoradiographic Analysis: Labelling Index (LI). BC3F1/Cum female mice were exposed to cigarette smcoke using the continuous regimen for 3 months. Immediately following the last exposure each mouse was administered i.p., 100 uCi - tiated _hyrnidine. The animals were sacrificed one hour later, 3 ~! .`'tiCi'ObiOk3g1Ca1 kSsOCiateS C~N~~ ~'~~"",~ 0,-~•'::~,'~ t'"~~S_~

SF02501158 and their lungs, trachea, liver, spleen, kidney, bladder, sma!: and large intestines were fixed i:n neutral :ormalin and processed for autoradiography. Priori.t:• was given to analysis of the labe:linc index in the lung.• 'or each l:::c 3 sections were counted over 5-10 different areas per section and the trac:icrn of total counted cells labelled per lung is presented in Table 20. A ma-;ority of the labelled ce:ls from smoke-exposed mice had a grain number per nucleus of 20-50 and these cells were generally in isolated areas of the lung. The sham-exposed and shelf con- trol mice showed far fewer labelled cells and those had heavierr grain accumulation. These observations suggest the possibility of detecting normal DNA replication and unscheduled DNA sy:,tzesis :nd::ced by exposure to cigarette smoke. Whereas there was very iit~le labelling in the shelf-control and sham exposed m'-ce, :hose animals exposed to 3A1 and 2R1 cigarette smoke ind:ca:ed sig::*=_:an:ly higher labelling indices. Prel::ninary data are presented for the labelling index the trachea (Table 21) and urinary bladder (Table 22). There anpeared to be li tt? e increase in the :: achea or urizasy t1adder LI :or animals exposed to 3a_' smoke, wh4le a significant increase was observed in the LI's for mice exposed to 2R1 cigarette smoke 3. Induction of pulmonary AiY''H activity. Pulmonary AHH activi _y is stimulated '3 fold within 3 hours after exposure to 2R1 or 3A1 cigarette smoke for 3 mcn :::s (Table 23). AiiH levels remain elevated at this level for 9::oL:s or longer after exposure. T'.:ese resul=s are similar to those ;,bta:::ed for other exposure reci:aer.s. 4. Induction of pulmonary DDC activity. Pulmonary ODC activ_ _y is stimulated '2 fold wi:--.. _ zour s after exposure to 2R1 or 3A1 ciga: e:te smoke =or -mcr. _::s ;'"ab=e 24). ODC levels, were elevated for 3 and 6 hou: s a= _.e: exposure, but by 9 hours had returned to sham-exposed and she:=- cont=ol levels. There results are sim,i:ar to those obtained =or other exposure regimens. 5. Augmentation of DNA damaging effects of lung carcinogens. :'he alkaline elution assay measures the rate of elution of DNA from a polyvinylchloride filter in the presence of an alka- line buffer system. The rate at which DNA elutes from such a filter is dependent upon the molecular weight of the DNA. Dti'a from many chemically or physically damaged cells elute from the filter at a faster rate than DNA from untreated control cells. 4 ~.ni ~ .~1iCT~Ob1o~OgiCa1 .~SSOCi3t.eS ~ Cf R f-If °I 041"f"48S...a

SFC2~~.~~ The results to date employing the alkaline elutior. assay :c measure the effects of smoke exposure have been mixed. Repro- ducibiYity of the assay has been a problem, particularly :n :::e amount of DNA recovered when -4sing dlaminobenzoic acid to deter- mine the DNA spectrofluorometrically. The extent of damage induced in the positive controls has also beer. variable and, in general, lower than expected. For these reasons, experiments are in progress to radiolabel DNA in intact animals and then measure by scintillation counting, the extent of damage induced by smoke exposure alone and the combined effects of smoke exposure and chemical treatment. Results for C-118 after 3 and 6 months exposure are : r e- sented in Table 25 and 26. Bxposure to 3rk: or 2R1 cigarette smoke alone resulted in minimal effects or. pulmor.arv DNA re:a::ve• to that found in the sham-exposed aninals (Table 25). . These results support those found previously in C':R 96 after exposure to 2A1 cigarette smoke. The effects of smoke exposure combined with in:racraczea: trea:.ent with BaP or BaP-7, 8-diol are presented in ': able 26. For this exposure regimen, no significant increase was obse:ved in the amount DNA eluted for BaP + smoke or BaP-7,8-diol + s:~cke treatment, compared to the sham-exposed groups. Previous resul_s had shown an enhanced elution of lung DNA from animals exposed :o 2A1 cigarette smoke ('1 mg TPM/day) for 40 weeks and then treated with 3aP-7,8-diol (Progress Report, 1980). Preliminary results for C-131 are presented in Tables 27 and ~_ 29. No enhanced DNA damage was observed after short periods sr.icke exposure and no differences were observed between male and fema'_e mice. 6. Alteration of certain physical characteristics of _::e lung. a) Lung Weight The data are presented in Tables 29 and 30 for the effects of the continuous exposure regi-men on body weight, lung wet weight, and lung dry weight. The continuous exposure regimern resulted in an increase in the wet weights of lungs from mice exposed for 3 or 6:nonths to both cigarette types. The dry weights of the lung were also increased, with the dry weight con- sistently representing 22-24% of the wet weight of the lung. The wet and dry weight lung to body weight ratios are given in Table 31. Thus the increase in weight of the lung occurs within 3 months using this exposur.e-regimen. b) DNA/Protein ratios It has been demonstrated that smoke exposure results i.n increased cellular proliferation and enzyme activity - either d::e 5 ~+ XiClUbi0iog1Ca1 A.SSOCiateS 6r.1' TR / I ! / 04448./.

to new cells being formed to replace cells rendered necrotic by smoke-exposure, or due to smoke-induced hyperplasia - which i.n turn causes an increase in size and weight of lung. Ar. approach to test this postulate was to determine the DNA and protein co: - tent in lung tissue following'various'treatment conditions (':a!::e 32). There appeared to be a measurable increase in the amour.: of total DNA per lung in 3A1 exposed (1.65 mg) and 2R1 exposed (1.42 mg) mice, relative to sham-exposed (1.04 mg) or shelf control (1.06 mg) mice (Table 32). A comparison of ug DNA per mg dry weight of lung also showed that 3A1 exposure (36.31 ug) and 2R1 exposure (37.04 ug) gave higher values than sham-exposed (31.14 ug) or unexposed shelf (29.25 ug) mice. Total protein content per lung was higher in the lungs of mice exposed to 3A1 (26.31 mg ) or 2R1 Z 25 .11 rdg ) cigarettes, compared to sham-exposed (19.13 mg) or shel_°-control (12.61 mg) mice. A comparison of the. ug protein per mg dry lung weight showed a slight increase :o: the smoke-exposed mice, compared to the sham-exposed and s::el- con=roi (Table 32). The DNA to protein ratios were app: ox'_ mate? y the same for all conditions. c) Collagen synthesis in lung One of,the most consistent morphologic observations of the conseauence of exposure of mice to whole cigarette smoke has been the finding that smoke-exposed lungs were bigger with a larger surface area and heavier (wet weigh=) than lungs from shamexposed or unexposed mice. aistopathologic examination showed that the alveolar walls were somewhat thicker in the smoked exposed lung, but no evidence of edema or accumula_iorn of fluid was found. A comparison of the wet and dry weights of .::r.c suggested that lungs from smoke-exposed animals had larger we= weigh= a.^d dry weight. Both smoke- and sham-exposed ?.:nc; :os = 74-764 of fluid when lyophilized. Zt is known that ama4or con- sequence of inhalation of many aerosolized chemicals is _::e al_eraticr= in collagen synthesis in the lung. We have a:=emp:.ed to s:.udy this phenomenon by 3 different approaches. ::: all 3. me:::ods the basic assay•is the deter;ai nation of hydroxyproline (=vp) in collagen. This is because collagen is unique in that it alone among proteins has a high concentration of HyP i.n its amino acid composition. The 3 approaches are: 1) Use minced lung from smoke-exposed and non-exposed mice in a short term organ culture to follow the uptake of 3H-proline into collagen in vitro. A modification of the procedure described by Greenberg et al (J. Lab. Clin. Med. 92: 1333- 1042, 1978) will be used (Table 33). 3H-HyP will be determined by the method of Juva and Prockop (Anal. Biochem. 15: 77-83, 1966) (Table 33). 2) Homogenates of lung will be prepared, acid hydrolyzed and the levels of HyP determined by a spectrophotometric assay adapted from Woessner, Arch. Biochem. 9iophys. 93: 440-447, 1961. Thus levels of collagen in smoked-exposed and control lt;ncs may 6 ~~ .HKTObi0~2Ca1 .~SSOCiSteS CTR HN 044~--~' 62

s~o2Jol6; be compared (Table 34). 3) Treat mice wi =h 3H-Pro and est::^a =e 3H-HyP in Iunc ^omo- ce::ates as to obtain a:,ieasure of the.co-'laaen synthesis rate v:vo. 7 ~'.1~Ki~OblO~OgiC31 ISSOCiBteS ~%~:~ CTI 1 I IN •.J'' I+44 ) 83'