Tobacco Documents Online

MICROBIOLOGICAL ASSOCIATES MAry Ann Montg ' September 1, 1978 - August 31, 1979 Prepared By C. J. Henry, Ph.D. R. E. Kourl, Ph.D. L. M. Schechtman, Ph.D. R. D. Curren, Ph.D. M. Dinowitz, Sc.D. B. Bhooshan, Ph.D. August 24, 1978 Director of Co Administration , ' Proposed Studies For CTR 0030 - Smoke Inhalation Carcinogenesis Studtes in Mice 6 (o4&1 ery ,S Richard E. Kourl, Ph.D. tract v Director of Research CTR CaNTRRCTS 027833 11247583 f CTR VIN 0442..

PROPOSED STUDIES Contract CTR-0030 TABLE OF CONTENTS ABBREViATiONS 1. CHRONIC INHALATION STUDIES A. •Introduction B. CTR-101A. Chronic exposure of BC3F1/Cum mice to cigarette smoke. CTR-1018. Chronic exposure of C3H/Anf-Cum mice to cigarette smoke. CTR-101C. Chronic exposure of DBA/2J mice to 1 cigarette smoke. i ii. SUPPORT SERVICES A. Dosimetry 1. CTR-102. 2. CTR-103. 3. CTR-104. 4. CTR-105. 10 aerosol. 11 B. Distribution of Cigarette Smoke Constituents 1. CTR-106. Deposition, dosimetry and chronic smoke exposure using 2R1 cigarettes compared to 2Al cigarettes in BC3F1/Cum mice. 8 Evaluation of SEM 11 animal containment capacity using 14C-DTC-2R1 cigarettes.• 10 Deposition and dosimetry of 14C-DTC-3A1 cigarette smoke in BC3F1/Cum mice. Comparative evaluation of deposition and dosimetrv after exposure to • B-naphthylamine or catechol in a smoke Autoradiographic analysis of lung tissue after exposure of mice to 2111 cigarette smoke. 12 2. CTR-107. 3H-Thymidine labeling index (LI) in lung,-liver, bladder, or kidney after exposure to smoke, or treatment with carcinogen. (Dr. R. Rasmussen). 14 3. CTR-108. The effect of cigarette smoke from different types of ci yarettes on pulmonary mixed function oxidases (MFO) levels in three strains of mice. 15 C. Short-Term 4ssays Relating to Possible Initiation Events 1. CTR-96. Inhibition of pulmonary DNA repair capacity after exposure to cigarette smoke. (Or. R. Rasmussen). 18 CTR coNTRacTs a2-7a34 11247584 CTR 1111N 04•42'2'9B

PROPOSED STUDIES Contract CTR-0030 2. CTR-8?A. •3. CTR-109. 4. CTR-110. Characterization of pulmonary cytochromes involved in s.-.,oke associated MFO induction. (Dr. 1. Wang). 19 Alterations in the immune response ' after exposure to cigarette smoke. (Dr. H. Herscowitz). 20 Evaluation of adaptation and stress in the inbred•strains of mice exposed to cigarette smoke. (Dr. W. Essman). 22 D. Short-Term Feasibility Assays Relating to Possible Promoting•Events 1.' CTR-111. Comparison of smoke induced ODC in three strains of mice and discrimination between ODC i•nduction and.AHH induction by cigarette smoke. 24 2. CTR-112. Evaluation of feasibility of a focus enhancement assay. 27 E. Aerosol Studies 1. Introduction 2. CTR-113. Acute toxicity of aerosolized TPA in the Inbred strains of mice. 3. CTR-114. Deposition and retention of aerosolized TPA. 29 30 30 4. CTR-115. Feasibility study of biochemical response to aerosolized chemicals. 31 F. Budget 32 CTR CQNTRRCTS 027835 11247585 CTR MN 04429013*

PROPOSED STUDIES Contract CTR-0030 > A(3BREVIATIONS AHH aryl h drocarbon hydroxylase BA benz(a~anthracene BaP, BP benzo(a)pyrene COHb carboxyhemoglobin CSC cigarette smoke condensate ConA concanavalin A DMBA 1,12-dimethylbenz(a)anthracene DTC, 14C-DTC doctriacontane EH epoxide hydrase ETR ethoxyresorufin FA flucinolone acetonide HA hemagglutinating Hb hemogTobin 3H-TdR 3H-thymidine IT intratracheal IgG immune gammaglobulin LI labeling index LPS lipopolysaccharide MCA 3-me th.y 1 cho 1 an th rene MFO mixed function oxidase ODC o*rnithine decarboxylase ORNL Oak Ridge Nattonal Laboratory PAG polyacrylamide gel PFC plaque forming cells PHA phytohemagglutinin SEM Smoke Exposure Machine SRBC sheep red blood cells TPA tetradecanbylphorbol acetate TPM total particulate matter WHSM Walton Horizontal Smoking Machine CTR CQNTRRCT6 027836 11247586 ~ ~~ ~-I~>~ 044,300

PROPOSED STUDIES Con t ract CTR-0030 I. CHRONIC INHALATION STUDIES A. Introduction. These studies proposed will describe those conditions necessary for maintenanc,e, utilization and implementation of a state-of-the-art inhalation facility for the determination of the potential biological effects of whole cigarette smoke in the inbred strains of mice. In order to carry out successful chronic cigarette smoke inhalation studies, it is required that: 1) the facility must be capable of generating and characterizing large quantities of cigarette smoke; 2) the facility must be capable not only of documenting and quantitatina the level of smoke generated, but also documenting the smoke deposition patterns in the test animal; 3) the facility must be capable of utilizing large numbers of animals in chronic studies; and 4) the facility must be capable of evaluating and characterizing the potential biological response of the test animal to these exposure conditions. The inhalation facility at M.A. meets these rather stringent conditions. The following proposed studies should ensure a continuation of the state-of-the-art inhalation facility. B. CTR-101A. Chronic exposure of BC3F1/Cum mice to cigarette smo e. CTR-101B. Chronic exposure of C3H/Anf-Cum mice to ci arette smo e. CTR-101C. Chronic exoosure of OBA/2J mice to - cigarette smoke. The chronic inhalation studies are designed to evaluate chronic exposure of rJiole cigarette smoke as a potential com- plete carcinogen in.three strains of mice (BC3F1/Cum, C3H/Anf-Cum, and DBA/2J).Data to date suggest that cigarette smoke at best, possesses weak biological potency to man. Because of this low biological potential and because of the conditions under which man is exposed to cigarette smoke, it is a necessary•requirement that any smoke exposure study use conditions whereby: a) the smoke deposition and distribution parallels that of human smokers b) the length of time of exposure parallels that of human smokers, and c) the animal system employed is capable of - expressing those types of biological lesions associated with cigarette smoking in man. 1. Rationale.. Several parameters must be considered for this experiment including: 1) estimated lifetime of the animals under these test conditions; 2) numbers of mice required initially in order to insure a significant number of animals during the time when lung lesions may be expected; 3) an assumption as to the incidence and latency of lung lesions -1- CTR CONTRRCTS 027837 11247587 CTR I-IN 0'44301

PROPOSED STUDIES Contract CTR-0030 4) the type of cigarette employed; 5) dose of cigarette employed and 6) equipment capacity for smoke exposure. The experiment will be scheduled so that mice are exposed to smoke for their "lifetime". In an attempt to deter- mine what the lifetime is for each strain, the following data were assembled. Table 1 Mean Life Span Strain (LD50) References i3C3F1/Cum 30 Months Peter, et al (Mech. A ing Develop. +T~c'251, 1975? C3H/Anf-Cum 28-30 Months Peterson (Fersonal Com- munication) DBA/2J 22 Months Myers (Birth Defects 14:41, 1978) Realistic estimates for the lifetimes of the mice are difficult to predict because of variations in laboratory con- ditions and because of the treatment conditions employed. Data from our laboratory'(CTR-i100) suggest that BC3F1/Cum mice exposed to 2A1 cigarettes at a dose level of 10 per day yield an LD5o (50% of the animals are dead) of about 44 weeks. The machine control animals also yield an LD50 of about 44 weeks. However, less than 1% of the uninoculated control , animals have died during this observation period. The factors causing this high death rate include, human and equipment associated accidents, stress, toxicity of treatment and a combination of these factors. The accidental deaths have decreased dramatically during the past four months mainly' for two•reasons: 1) the technical staff has become experienced in the day-to-day handling of the animals, and 2) equipment malfunctions have been an increasingly rare occurrence. The death rates in the machine control groups have decreased significantly as the result of the installation of flow meters at the end of the smoke line in the animal containment units. These flow meters insure adequate flow of air and/or cigarette smoke and have significantly decreased the numbers of animals dying on the smoke exposure machines. Because.of these latter changes, we now assume'that the LD50 for BC3F1/Cum mice for uninoculated controls, machine controls, and smoke exposed animals will be approximately 30 months, 15 months, and 12 months, respectively. Although not directly comparable, results from experiments CO-91:and CO-94 tend to corroborate these assumptions.that is,handling animals every 4-5 days throughout their lifetime has resulted in at least 20% survival at 118 weeks of age. Therefore,• it would seem for the BC3F1/Cum, C3H/Anf-Cum, and DBA/2J strains that the initial numbers of mice should be 1600, 800, and 400 for smoke exposed, machine controls, and uninoculated controls, respectively. A tentative schedule for the bioassay of the effects of whole cigarette -2- CTR CONTRRCTS 027838 11247588 CTR NN 0443022

I ~E 1 . 1 It BC3F1 9/78 C311/f 7/79 0BA/2J. 3/80 TABLE 2 SCHEDULE FOR CHRONIC INHALATION STUDIES -•CTR-0030 8/21/78 ESTIMATED NUMBERS OF SURVIVORS 1978 I • 1979 I 1980 1981 Strain Group S 0 N D J F M A M J J A S 0 N D J F M A.M J J A S 0 N D 1 J F M A M J J A S ~ k 1 1 1600 :1200 800 400 200 ^-0 I . Smo e BC3F 2. Mach. ~00 • 550 380 • 200 NO j. Uninoc. 00 400 350 200 nv0 4. BPs .~ Smoke 200 150 100 ...0 5. BP t i Mach. 200 1S0 . 100 ti 0 ~ 6. 8P 100 100 100 ~ 0 1 TOTAL SI:OKE 1800 1350 9oo 600 400 200 C3N/f Cum I. Smoke 1600 1200 ' 800 600 400 2. Mach. 800 550 380 3. Uninoc. 400' 400 350 ~ 4: BP + Smoke 200 150 100 '~0 N 5. BP • I 100 L• 0 ~ Mach. 200 i50 v (7 , 6. BP 100 100 100 A,0 0Cn0 t0 TOTAL SMOKE ~ 1800 . 1350 9oo 600 4 # (~ OBA/2J 1. Smoke 1600 1200 600 350 2. Mach. 800 550 300 250 , 3. Untreat. 400 400 275 250 4. BP + Smoke 200 150 100 .v O 5. BP• ~ Mach. 200 150 ' 100 100 / 0 O rw. 6. BP 100 1UO 0. TOTAL SMOKE ~ 1800. 1350 700 .~ ~ OVERALL SMOKE Ln IOTAL SPACE 0 Pres.. Equlp. 1800 2000 1350 2000 2700 2000 . 3750 2000 2650 2000 1500 2000 750 ~ OVERALL 'AVAIL. NJ SPACE 200 650 -700 -1750 -650 +500 ~ CD TOTAL SPACES. a 4 4000 ~ 051. Pres. Equip. 4000 000 W AVAIL. SPACES 1300 250 1350 the equipment capacity will be doubled at this time. aIt Is proposed that

PROPOSED STUDIES Contract CTR-0030 smoke from the 2R1 cigarette on BC3F1/Cum, C3H/Anf-Cum, and DBA/2J strains is given in Table 2. The schedule was made with the assumption that approxi- mately 200 animals would be alive during the time at which the LD50 Qccurred to the time at which the L090 occurred.. This is the minimal number of animals which must be evaluated in order to insure that an assumed 2% squamous cell carcinoma incidence In the smoke exposed group and a zero incidence in the control groups will be statistically significant. A 2% .inc.i.dence was based on the extrapolation of the data of Hammond, et al (Origins of Human Cancer, =d. Hiatt, Watson, Winsten, Co1U-Spring Harbor Laboratory, 101-1t.2, 1977). on the inci.dence of lung cancers in humans exposed to high levels of cigarette-smoke (approximately 2 packs/day) for extended periods of time (>40 years). These proposed studies will use the 2R1 reference cigarette. A comparison of its total particulate matter (TPM), nicotine, and puff characterisitics relative to the 2A1, 3A1, and 1R3 cigarettes are given in table 3. Cigarette Type Table 3 TPM m /Ci .) Nicotine m /cl .) Puffs (#1 Cig. ) 2A1 40 0.51 10 3A1 43 0.35 11 2111 50 2.81 12 1R3 26 1.25 8 Recent studies in our lab have shown that this relatively "high" nicotine cigarette can be used If given for 20 second exposure times, eleven puffs/cigarette, and a total of 5 cigarettes per day. it is assumed that this exposure regimen will result _ (n the deposition of about 25 mg TPM/kg/day. (A dosimetry experiment with the 2R1 cigarette has been proposed (CTR-102) to quantitate this deposition.) Such a dose is equivalent to a human smoking approximately 3.5 packs of cigarettes per day. Also presented in Table 2 Is the schedule of an experiment to study the potential co-carcinogenicity of ciga- rette smoke in BaP Induced lung cancers. The rationale for this study is as follows: 1) previous studies have shown that IT instillation of cigarette smoke condensate materials enhanced BaP induced lung tumorigenesis (see progress report, 1978), and b) this enhanced tumorigenicity was observed within 1.0 year afte.r BaP treatment. This pa-rt of the chronic inhalation study would be expected to end by 12-18 months. ' The schedule in Table 2 shoais that the BC3F1/Cum strain will be put on test September, 1978 and in order to keep to the scheduled initiation dates of Sept.1979 (for C3H/Anf-Cum) -4- CTR CONTRACTS Q27B4'Q 11247590 CTR MN 044304

PROPOSED STUDIES Contract CTR-0030 and March, 1980 (for ')EA/?J), the capacity for animal exposure must be doubled. We propose to evaluate two alter- native arrangements of animal containment units that should double :he number of anim?ls that could be exposed per day. The alternatives are a tandem (one behind another) arrangement o" animal containment units, or the doubling the number of smoke •lines coming from the SEM. These alternatives will be evaluated by testing for total flow rate, smoke particulate deposition and levels of CO/CO~, in line. These details are presented in proposed study CTft-103. Table 4 Schedule for Mice Exposed to 2R1 or 3A1 Cigarettes (12 puffs, 12 min) Time- Smoke Exposed Dos # Ci e Machine as. Exposure Dose # Ci E gs. auiv. Run # # Mice . Run # = Mice 6 a.m. (~ 7 8 (S-1) 480 ~ 5a (M-1) 480 5 9 10 (S-2) 480 5 '(M-2) 480 5 11 ,~ Clean Machine 12 QS-3) 480 5 (M-3) 480b 5 2 1-4) 48ob 5 Dismantled & Clea n 3 4 Set up for next day 5 p.m. a. Each 2R1 or 3A1 cigarette requires 12 min to burn to the 23 mrn butt. A rest period of 12 min then follows. Thus, approximately 120 min are required for a dose level of• 5 cigarettes. Loading and unloading 480 mice requires an additional 20-30 min. b. Total Capacity: a) 19?0 mice ex posed to 5 cigarettes, or 960 mice exp osed to 10 cigarettes b) 1440 mice ex posed to 5 . cigarettes equivalent (machine controls). -5- CTR CONTRRCTS 027641 11247591 CTR t-IN 044,~OE;

PROPOSED STUDIES Contract CTR-0030 2. Procedure. The following groups of BC3FI/Cum, C3H/Anf-Cum, and DBA/?J mice will be used at the scheduled times. (see Table 2) No. of G rouo pS i ce T rea tmen t 1 1600 Smoke exposure 2 800 Machine control 3 400 Uninoculated 4 200 BaP and smoke exposure 5 200 BaP - machine control 6 100 BaP control The procedures followed will be those used and developed under 1-100 and the daily schedule is presented In Table 4. a. Mice will be 8-12 weeks old when ear-tagged and put on test. Each mouse will be individually marked. b. Observation records•will be kept on Individual mice. Weekly observations will be recorded in all groups and will become part of the permanent record for the experiment. c. All mice will be weighed monthly, at the same time of day, two days after cages have been changed. For the smoke exposed and machine control mice, weights will be obtained at least two hours after exposure. d. Smoke and machine exposure will be initiated gradually until exposure reaches 5 cigarettes/day and will continue 5 day/week for the lifetime of the animal. e. Carcinogen treatment of the designated groups will be given at weekly intervals and be completed before the first smoke exposure. The dose will be 3 x 1.2.mg BaP/0.02 ml gel-saline. f. Urine will be routinely collected from each group of animals and assayed for mutagenic components according to procedures described. g. Carboxyhemoglobin, hemoglobin and methemoglobin determinations will be made for mice from each group at monthly intervals. h. Several assays will be used to monitor this long-term study: OOC, AHH, and pulmonary DNA repair capacity. These will be performed periodically on 3-4 mice and compared to known controls. i. Mice will be taken off test when moribund and the following tissues examined histopathologically: lung, trachea, larynx, head, and any other abnormal tissue. -6- CTR coNTRaCTS 027842 11247592 CTR HN 044,306

PROPOSED STUDIES Contract CTR-0030 j. The follo+qing data will be determined: (1) Toxicity due to carcinogen treatment, toxicity due to carc:nogen-smoke treatment, toxicity due to smoke alone. (2) The probability of a lung tumor developing at certain times after carcinogen treatment alone, after car- cinogen-smoke treatment, or after smoke treatment alone. (3) The latency for each tumor type. (4) A comoarison of the tumor probability after carcinogen treatment in this experiment using BaP with experiment 1-100 using MCA. -7 - CTR CONTRRCTS 027843 11247593 4..r' TI `! NI I 0-441.M` •rf• !

DATA TYPE ~u EXP'-t ® GROUP 1 0 0 o a a ANIMAL w TREATMENT c]- # REASON FOR ~ ' WEIGHT SACRIFICE 19ramsl 33 1 2 3 4 5 G 7 8 9 10 MORPHOLOGY 11 12 11 14 16 ~ [] Q PATHOLOGY# a- l 20 21 22 23 24 25 26 ® NUMBER OF LUNG LESIONS DATA TYPE ~ ~ 1 TISSUE TOP.COOES Hist. GROSS FINDINGS MICROSCOPIC FINDINGS Salivary Gland 5500 C.rrical Nod. 0820 Nesr+t.ne Node 0851 Inquind Node 08811 Nod. ISpaclN) Mammary Tisw• Ot00/0404 Pancnac 5900 Spleen 0700 Llv.r 6600 Kidney 7100 Adnn.l 9300 L8. Int. 6700 $rr. InL 6400 . Stem.ah 9300 uterua $200 OraryfTasc 7800/8700 V.qin.-C.rv; 8100/8300 Bl.ddar-urin.ry 7400 Ha.rt 3:200 Luny 2800 N BA PI PC', Thrmuc 9800 Murcl. ISpeci(Y) S.O. Tisu. 0300 8rain-Mny.. X200-X100 BoM ISp.oihr) Skin 0100 HardeNan GI. XX90 Jolnu (SpecilYl BOM Mar.ow 0600 Thyroid 9600 Veswls (Spccrtvl Oth.r ISp.cify) -a l I I I 16 I 17 18 19 DATE OF DEATH ® 34 35 ® 38 19 40 1 m [= f ] 27 28 29 30 31 FIXATIVE OF PATH SPECI6IEN 37 PATHOLOGIST Q 41 K.ypunchd OuPlical. Cols 2-19 Above ~ 1 2 3 4 5 6 7 6 9 10 11 12 13 14 IS 16 17 18 19 EXTERNAL PATHOLOGY NO. TOPOGRAPHY 1G l I I ] 2M 1 I 20 21 22 21 a 44 45 4G 47 Report prepared by I I I J 24 25 26 27 ® 46 49 60 51 2G 3M TOPOGRAPHY ( I 1 I I 28 29 30 31 I I I 52 53 64 55 Autopsy by 1 I -7a- MORPHOLOGY ~ 32 33 34 35 ~ 66 67 68 69 110 TOPOGRAPHY ~ 4M 36 37 36 39 ~ 60 61 62 63 MORPHOLOGY ~ I I I 1 40 41 42 43 11247594 CTR CONTRRCTS 027844 64 66 66 67 (,ilaB-G01-OIilUIiS/ I I I AUTOPSY DATA FORM C TR VIN 0 -4, 43 0 a

PROPOSED STUDIES Contract CTR-003t. 11. SUPPORT SERVICES A. Dosimetry. 1. CTR-102. Oeoosition. dosirr.etr and chronic smoke ex osure using 2Rcig_arectes comparea Co Z ciqarettes ~n B Cum mice. a. Rationale. After completion of preliminary experiments to determine the tolerance of BC3FI/Cum mice to smoke from 2R1 cigarettes, a dosimetry experiment using 14C-DTC and 14C- nicotlne-labeled 2R1 cigarettes is proposed. Comparison of the 2R1 with the 2A1 cigarette with regard to particulate deposi- tion Is important because these cigarettes differ in: 1) toxicity of smoke, 2) procedures for adaptation to smoke, and 3) the resulting level of carboxyhemoglobin. The 2R1 and 2A1 cigarettes have slightly different TPM (50 and 40 mg/clgarettes, res pectively), and are known to differ In the amount of nicotine (2:81 and 0.5 mg/cigarette, respectively), the amount of water delivered under analytical smoking conditions (6.6 and 3.1 mg/ cigarette, respectively) and the number of puffs per cigarette (12 and 10 puffs/cigarette, respectively) (see Table 3). We thus propose to evaluate whether the particulate deposition and internal distribution can account for the differences ob- served in a biological effect using 14C-DTC and 14C-nicotine labeled 2R1 cigarettes. b. Procedure. . (1) 14C-DTC - 2R1 cigarettes BC3FI/Cum female mice (7-8 weeks old) will be adapted to smoke on the SEM II according to the schedule which results in the lowest toxicity. Exposure to smoke will continue for one month before the dosimetry study with 14C-OTC. Labeled 2R1 cigarettes will be prepared using the cigarette labeled injection machine described previously. The smoke exposure conditions used for the experiment will depend upon which conditions are best tolerated by BC3FI/Cum female mice (10% smoke concentration, 20 or 30.seconds ex- posure time). Immediately after smoke exposure, retroorbital blood samples will be taken for carboxyhemoglobin analysis. One group of mice (15 mice) will then be sacrificed by C02 asphyxiation to determine the Immediate deposition of smoke particulates and another group (15 mice) sacrificed 24 hours post exposure to determine the level of retention. The following tissues will be analyzed to determine the amount of particulate deposition: head, larynx and upper trachea, lung and lower trachea, stomach, liver, bladder, kidneys, remains and hide. The data will be presented in micrograms mi crogramss~P~1~/mg vre t wei ghte ti ss~i ei1 1 be presented i n -8- CTR CQNTRRCTS 027645 11247595 C) R 1 Il 7 0-l-4tir..n 0..n~

PROPOSED STUDIES Contract CTR-003L (2) l4C-Nicotine - 2R1 Cigarettes The smoke exposure conditions will be as described for 14C-DTC-2R1 cigarettes, with the exceptlon that one group of BC3F1/Cum mice will be unadapted to smoke, while the other group will be adapted and exposed to smoke for one month prior to the dosimetry experiment. Ten mice per time point will be sacrificed after smoke exposure at zero, 0.25, 0.50, 1.0, 2.0, 4.0 hours. Tissues and data will be analyzed as described for 14C-DTC-2R1 cigarettes. -9- CTR coNTRacTS 027846 11247596 CTR NN 0'4`- ti,.,°~ 10

PROPOSED STUDIES Contract CTR-003G 2. CTR-103. Evaluation of SEM il Animal Contain- ment Caoacity Usinq 14C- -c Cicarette Dosimetrv Studv. a. Rationale. The feasibility of increasing the inhalation capac•ity through use of additional•animal containment units will be evaluated. The amount of smoke generated by the SEM II , is ln excess of the amount required for one animal containment unit, so that the possibility of attaching a second unit in series or in parallel seems to be a likely way to increase capacity of the SEM 11 machine. Preliminary determinations of flow rate, CO1C02 changes along the extended smoke line, and total particulate matter along the smoke line are presently under- way. In the event these results indicate that two animal containment units can be successfully utilized with one SEM 11, a dosimetry experiment to verify the deposition and dosimetry in the BC3F1/Cum mice will be performed. b, Procedure. The procedures will be as described for CTR-102 using 14C-DTC-2R1 cigarettes. Fifteen mice per module (60 mice total) will be sacrificed after smoke exposure, with the position on the smoke line carefully noted. Tissues and data will be as described as for CTR-102, with the additional information of the TPM deposition given as a function of the distance from the SEM 11. 3. CTR-104. De osition and dosimetr of 14C-DTC-3A1 cigarette smo eC3F um mice. a. Rationale. Dosimetry results presently available have all been obtained using the 2A1 cigarette. It is proposed that a low-alkaloid cigarette may be used in the chronic inhalation studies. The 2A1 cigarette is not available in sufficient quantities for these studies and therefore, the 3A1 cigarette would have to be used. The particulate deposi- tion and distribution pattern of this cigarette have not been determined, and as indicated in Table 3, the TPM, nicotine and number of puffs pe r cigarette are different compared to both the 2A1 and 2R1 cigarette. It is anticipated that deposi- tion and dosimetry from a 3A1 cigarette would most resemble results from a 2A1 cigarette. b. Procedure. The experimental procedure will be as described for CTR-102 using 14C-DTC-3A1 cigarettes. CTR CONTRRCTS 027847 11247597 CTR 11N (:~4' 431 1

PROPOSED STUDIES Contract CTR-003G 4. CTR-205. Comnarative.evaluation of deoosition and_ dosimetry arter_exposure to <-naont v amine or catec o in a smo e aeroso . _, a. Rationale. ' Previous studies (CTR-0022 Progress Report) have examined the physiologic response in the mouse to nanogram quantities of BaP, MCA, nicotine and DTC when aerosolized In cigarette smoke. All are constituents of smoke with the exception of MCA. These specific chemicals represent, respectively, the polycyclic aromatic hydro- carbons, the tobacco alkaloids and an inert particulate con- stituent of tobacco smoke. BaP, MCA and nicotine were shcxqn in these studies to be rapidly cleared (t12<2 hours) from the lung, while DTC was retained at significant levels up to a week post exposure. There are many more classes of chemicals in cigarette smoke capable of exerting a biological effect, for example, catechol (220-550 µg/cigarette) or e'-naphthylamine (22 ng/cigarette) Weisburger, J. L., et al.,(Origins of Human Cancer Ed. Hiatt, Watson, Winsten, CoTd-9'pring Harbor Laboratory, 573-574, 1977). The chemicals examined In our studies to date may not reflect the types of chemicals responsible for the biological effects In the lung. The chemicals responsible may be the cocarcinogens or "promoters", a few of which are known to be specifically present In tobacco smoke. Dosimetry studies using radloactively labeled f'-naphthylamine and catechol will be carried out so as to determine the conditions of retention and clearance from the lung and whole body. _ b. Procedure. BC3F1/Cum female mice (8-12 weeks old) will be adapted to smoke on the Walton Smoking Machine using standard procedures (10% smoke concentration, 30 seconds smoke exposure alternatin with 30 seconds purge air per puff for a total of 10 minutes}q. Exposure to smoke will continue for one month before the dosimetry study. Labeled 2A1 cigarettes will be prepared using the cigarette label, injector machine described previously. Standard dosimetry procedures'will be used that Is: (exposure to one unlabeled 2A1 cigarette, followed by exposure to one radio-labeled cigarette, and 4 mice per time-point) and a total of 6 time-points. Mice will be sacrificed at the following times after exposure: 0, 1, 2, 4, 12 and 24 hours. The following tissues will be analyzed for radioactive content: head, larynx and upper trachea, lung and lower trachea, stomach, liver, bladder, kidneys, remains and hides. The data will be p'resented as micrograms or nanograms chemical per tissue per time-point, the data for the lung normalized to mg wet weight/tissue. The time for one-half of the original dose to clear the lung (t 1/2) will be estimated from this data. cTR CaNTRacTS 027848 11247598 CTR IuIN 044312

PROPOSED STUDIES Contract CTR-0030 B. Distribution of Cigarette Smoke Constituents. 1. CTR-106, Autoradio raohic analysis of luna tissue after ex6osure o mice to 2-ciQarette smo e. (1 C-DTC. ?4C-BaP), • a. Rationale. Autoradiography of lung tissue after appro- priate exposure to biologically relevant chemicals allows deposition and retention patterns at the cellular level to be established. The'distribution pattern within the lung after exposure of mice to 14C-DTC-2R1 cigarette smoke will be determined using the Walton Horizontal Smoking Machine (WHSM) with the techniques and procedures developed under CTR-Contract 0022 last year: The WHSM utilizes fewer animals and cigarettes than the SEM 11 and comparative dosimetry studies have demon- strated equivalent TPM deposition between the two machines. By their nature, dosimetry studies cannot provide information except in an approximate fashion, regarding the specific dis- tribution of chemicals within an organ. Thus, autoradiography Is proposed to evaluate the imnediate deposition and distribu- tion patterns within the lung for 14C-DTC and 14C-BaP aero- solized In 2R1 cfgarette smoke. In addition, an evaluation of the methods of administration of chemicals to the lung can be made; that Is whether a different localization pattern would be found for )4-BaP delivered in a smoke aerosol com- pared to 14C-BaP delivered by intratracheal )noculation. b. Procedure. BC3F)/Cum mice (8-12 weeks old) will be ex- posed to smoke on the WHSM using 10% or 20~ smoke concentra- tion, 20 or 30 seconds exposure time. 14C-DTC-2R1 and 14C-BaP-2R1 ci garettes will be prepared as described using the cigarette label injector machine (10-100 µCi/cigarette). Four mice will be sacrificed by C02 asphyxiation at 5 minutes, 6 hours and 24 hours post exposure. Lungs will be rapidly removed,, quickly frozen in dry ice snow and stored at -70° C. Frozen lungs will be embedded in paraplast for sectioning at 6 hM in the International Cryostat. Two sections per glass slide are collected and allowed to dehydrate slightly in the cryostat. The sections then adheres well to the slide, which is then treated with methanol for I minute to complete fixation and dehydration. The dried slides are dipped in a 2% solution of NTB-2 gelatin solution and allowed to harden briefly at room temperature in the dark. Lead shielded slide boxes are used for storing the slides at -20° C for the 4-8 week exposure period. The exposed slides are developed in Kodak D-19 developer, fixed and stained with nuclear fast red (O.lY). The developed, stained sections are then examined microscopically for the distribution of silver grains. CTR C0NTRACTS 027849 11247599 vwJ I m R ! I I 1 0,44313 •

PROPOSED STUDIES Contract CTR-0030 For the comparison between smoke aerosol delivery and intratracheal inoculation, five BC3F1/Cum. mice will be inoculated -oith I uCi-14C-BaP in gelatin saline and sacrificed immediately. Two mice will serve as controls, inocu,lated with gel-saline only. The procedures will be as described for the smoke exposr.d mice. The cellular localiza- tion of BaP inoculated intratracheally will be determined on the basis of the distribution of the silver grains, and com- pared to the results from BaP aerosolized in smoke. -13- CTR CaNTRRCTS 027850 11247600 C TR VIN 0 44;~ 14

PROPOSED STUDIES Contract CTR-0030 2. CTR-107. 3H-Thvmidine labelina index LI in lun 1 iver. a a er or •iancv arter exoosure to smo e. or Lreacmenc arIc 1 carcinooen. a. Rationale. Carcinogenesis appears to be a focal reaction • of tissues, that is, the eventual manifestations of neo- plastic growth do not involve the whole tissue but appear as discrete and separate Islands of cellular aberration. It has been suggested that damage to cellular DNA may represent an early essential step in carcinogenesis. For some carcinogens, such damage has been shown to result in an inhibition of DNA synthesis, followed by an increase in the incorporation of 3H- TdR into DNA (Shimkin et al., Cancer Res. 29, 994 (1969)). These changes in 3H-TdR incorporatlon can be specifically de- tected after autoradiographic treatment of sections from fixed tissue. The fraction of labeled cells can be determined and Is expressed as the 3H-TdR labeling index (LI). In collabora- tion with Dr. R. Rasmussen (University of California at Irvine, Irvine, Ca.), we have used this technique to evaluate tissues from mice exposed to cigarette smoke. Preliminary results suggest that one of the earliest effects of exposure to 2A1 or 2R1 smoke In the lungs of BC3F1/Cum mice is an in- crease in the L1. Thus, the LI may be a sensitive monitor of organ or cellular response and may be used to detect the earliest effects of smoke or carcinogen treatments. The analysis of cell type and histopathology of the smoke associated lesions will be performed at MA with Dr. L.H. Billups. We propose to continue these studies In collaboration with Dr. Rasmussen to evaluate tissue specific or cellular specific responses to whole cigarette smoke. Particular attention will be given to comparisons between the methods of delivery, intratracheal Inoculation, and aero- solization in solvent vs. aerosolization in smoke. b. Procedure. As discussed previously, Initial studies are in progress with regard to the effects of 2A1 and 2R1 cigarette smoke on the LI of lung, liver, bladder and kidney. Mice have been exposed to smoke on the SEM II (10% smoke concentrate, 30 seconds exposure time) for varying periods of time, then shipped to Dr. Rasmussen for evaluation of DNA repair capacity. Two mice from each group sent to Dr. Rasmussen have been used at MA to evaluate the LI immedi- ately after exposure to smoke. Dr. Rasmussen has determined the LI at subsequent times. Mice have been injected after smoke exposure with 100 wCi-3H-TdR for 30 minutes, sacri- ficed by pentobarbital overdose, tissues removed and fixed in formalin. Lungs and bladders have been embedded in -14- CTR COhtTRRCTS 027651 11247601 ~ T~"~ t-IN 044,31tIE"i

PROPOSED STUDIES Contract CTR-003u parafin, l iver and kidney embedded in paraplast, and 6}cM sections cut. The sections have been dipped in NTB-3 emulsion and developed for a minimum of 4 weeks. The following schedule has been followed for the 3C3F1/Cum strain and will be followed for the C3H/Anf Cum and DBA/2J. Strain Treatment X o o ~ # Mice s u e 1. BC3F1/Cum 2A1, 10% Smoke I week 2 4 weeks 2 12 weeks 2 16 weeks 2 Machine Controls I week 2 4 weeks 2 12 weeks 2 16 weeks 2 2R1, 10% Smoke (As above) 2. C3H/Anf Cum (As above) 3. DBA/2J (As above) CTR-108. The effect of ci arette smoke from different types o cigarettes onpu_monary mix_~e 'function oxiaases 0 evels in t ree strains or mice. Alterations In pulmonary MFO activity a. Rationale. are among the earliest reponses following exposure to whole cigarette smoke. It is not known whether different cigarette types engender different biochemical responses among the in- bred strains of mice. The proposed use of the three different cigarette types, 2A1, 3A1, and 2R1 and the use of the three different strains, BC3FI/Cum, C3H/Anf Cum and DBA/2J In our chronic inhalation studies (CTR-101), make it necessary to evaluate the lung MFO response under these different conditions. The use of ethoxyresorufin (ETR), propoxyresorufin and methoxyresorufin as substrates for MFO will be particularly important because the use of these different substrates may differentiate between the type of cytochromes (e.g. P-450, P-448) present in specific tissue preparations. Moreover, ETR Is the most sensitive substrate 'for smoke-induced MFO activity because non-induced pulmonary tissue cannot metabolize this substrate at all, but induced pulmonary tissues, resulting from exposure to only one puff of whole cigarette smoke, exhibits significant levels of c• deethylase activity 6 hours post exposure. -15- CTR CONTRRCTS 027852 11247602 CTR HN 0''°431 G

PROPOSED STUDIES Contract CTR-003U The use of ETR-as a substrate for MFO will permit evaluation of the following: a. The effect of different types of cigarettes (2A1, 3A1, and 2R1). b. The use of different strains of mice in order to observe if enzymatic response to whole smoke is genetically linked to AHH responsiveness (BC3F1/Cum, C3H/Anf Cum, DBA/2J). c. Correlation of smoke particulate deposition with Induced levels of pulmonary MFO activity using the SEM to generate and deliver whole smoke, and d. Comparison of Induced pulmonary enzyme levels following treatment with IT administered chemicals or exposure to aerosol generated chemicals. This latter study must await the implementation of the aerosol generator to be constructed and developed by ORNL for this work In this con- tract. The main thrust of all of these studies will be the characterization of the differences between smoke chemicals-induced and chemically-induced MFo levels in the lung. • . b. Procedures. The SEM Ii will be used in these studies to generate smoke from either 2A1, 3A1 or 2R1 ci ar- ettes. Initial studies will use standard conditions of ~0% smoke concentration, 20 or 30 seconds exposure time. BC3F1/Cum, C3H/Anf Cum, DBA/2J mice, 8-12 weeks old, will be exposed to 2A1, 3A1 or 2R1 smoke once on the day of the assay. Mice will be sacrificed 6 hours post exposure, lungs removed and assayed for MFO levels using methoxy-, ethoxy-, and propoxyresorufin as substrates. Six mice per substrate- will be used, in addition to control groups. The assay for ETR 0-deethylase activity is performed on lung homogenate in the presence of 0.01 mM ethoxyresorufin (dissolved in 0.1 M Tris-Hcl, pH 8.5) in a total volume 0.4 ml. NADPH (0.125 mM) is added to initiate the reaction and the f.luorescence of the suspension is measured at an emission waveleng th of 586 nanomoles with an excitation wavelength of 520 nm using an Aminco-Bowman spectrophotofluorometer. A known amount of resorufin is added after the reaction has proceeded sufficiently in order to standardize the fluorometer. Data is given in terms of nanamoles resorufin formed per minute per wet weight tissue. As part of CTR-102, particulate deposition and distribution in BC3F1/Cum female mice will be determined after exposure to smoke generated on the SEM using ]4C-DTC-2R1 cigarettes. Twelve mice from this exposure will be sacrificed 6 hours post exposure to determine the induced pulmonary MFO levels using ethoxyresorufin as substrate. -16- CTR cONTRaCTS a2?'®53 11247603 CTR PIN 04431f `

PROPOSED STUDIES ^ Contract CTR-0030 The data will be presented as nmoles resorufin formed per minute per gram wet weight tissue. This MFO activity will be correlated directly to micrograms TPM in the lunc after the remaining mice have been necropsied according tL standard dosinjetry procedures and analyzed in collaboration with ORNL. When an aerosol generator for BaP becomes available, ETR will be used as substrate to discrim- inate between smoke-induced and chemically-induced pulmonary MFO. Three groups of 12 mice each, BC3F1/Cum mice, 8-12 weeks old, will be used. Group 1: Exposed to 2A1 cigarette smoke. Group 2: Exposed to 10 µg BaP aerosolized in solvent. Group 3: inoculated intratracheally with 10 µg BaP/0.02 ml gal-saline. Mice will be sacrificed by cervical'dislocation at 0, 6, 24 and 48 hours post treatment. Data will be presented for each group in terms of: 1) maximum induced MFO levels; 2) time of maximum Induction; and 3) half-life of MFO activity. cTR coNTRaCTS 027654 11247604 ~ CTR I I I I 044,318

PROPOSED STUDIES Contract CTR-0030 C. Short-Term Assays Relating to Possible Initiation Events. 1. CTR-96. Inhibition of pulmonary DNA repair capacity after exDosure to cioarette smoke. a. Rationale. Collaborative studies with Dr. R. Rasmussen (University of California at Irivne, Irvine, CA) are In progress. These studies are designed to characterize the effects of whole cigarette smoke on DNA repair capacity of pulmonary tissue in vivo. As reported in the CTR-0022 Progress Report, inhibition o7-iSNA repair was observed after exposure to whole 2A1 cigarette smoke, but not 2R1 filtered smoke. Exposure to smoke for four weeks was required for the effect to be first observed. The inhibitory effects of chronic smoke exposure on DNA repair capacity and the apparent persistence of this effect will be investigated during the next contract year using 2R1 cigarettes and mice of three strains (BC3F1/Cum, C3H/Anf-Cum and DBA/2J). Mice•will be exposed to smoke at M.A. then sent to Dr. Rasmussen for evaluation of In vitro DNA repair capacity. b. Procedure. • The SEM li will be used to generate smoke from 2A1, 3A1, or 2111 cigarettes. Mice will be rapidly adapted to smoke, using 10% smoke concentration and 20 or 30 seconds exposure time depending upon the cigarette type. The C3H/Anf- Cum will be evaluated first followin~ the methods developed for BC3F1/Cum mice. Approximately 100 m ce will be exposed to smoke; 100 mice used as machine controls, and 50 mice used as shelf controls. Shipment Schedule for C3H/Anf-Cum Mice Weeks on Test 2R1 Cigarette/Day No. of Mice Smoke Mach. Shelf 4 5 3.0 10 5 8 5 10 10 5 12 5......-- --. .. 20 10 5 16 5 20 20 5 20 5 20 20 5 The same procedure will be followed with DBA/2J mice aftPr completion of the C3H/Anf-Cum strain or when space becomes available'on the machine (Table 2). CTR CONTRRCTS 027855 11247605 CTR HN 044,.»"~ 1H

PROPOSED STUDIES Contract CTR-0030 2. CTR-82A. Characterization of ulmonarv cvto- chro.iies invol-yea in smot;e assoctateo M in uction Dr. I. Wano`,. a. Rationale. This effort will be a continuation of the ongoing collaborative studies with Dr. I. Wang (Univ. of So. Carolina, Charleston, S.C.). BC3F1/Cum, C3H/Anf-Cum, DBA/2J mice will be exposed to cigarette smoke (2A1 and 2R1 ciga- rettes) in our laboratory and either the mice or the pulmonary tissues will then be sent to Dr. Wang for isolation and identi- fication of the pulmonary cytochrome P-450's by polyacrylamide gel electrophoresis. MFO activity will be determined at M.A. on mice from the same groups as sent to Dr. Wang. The experi- mental protocols for these studies have been discussed at length in the proposed studies prepared by Dr. Wang and will not be presented here. b. Procedure. Mice will be exposed to smoke generated on the SEM II from 2A1 or 2R1 cigarettes. BC3F1/Cum mice will be evaluated first under several conditions: 10% and 20% smoke concentration and 20 or 30 seconds exposure time. Six hours post exposure, 3 mice from the treated and 3 mice from the inachine controls will be sacrificed, lungs removed and the MFO activity will be determined by ETR or AHH assays. The remaining treated and control mice will be utilized in Dr. Wang's assay. C3H/Anf-Cum and DBA/2J mice will be evaluated under the procedures developed for BC3F1/Cum mice. An example of the protocol to be used is given belcw. Number of Cigarettes/Day (2A1, 2R1, or 3A1) Hours Post Exposure 0 6 24 0.1 5a 5 5 0.5 5 5 5 1.0 5 5 5 5.0 5 5 5 10.0 5 5 5 aNumber of animals per time point; two to be utilized at M.A. for MFO levels and three sent to Or. Wang for analysis of lung cytochromes. -19- CTR CQhtTRRCTS 027856 11247606 C T R N ~~~ 0 :1, - 4. ~~~:~~

PROPOSED STUDIES Contract CTR-0030 3. CTR-109. Alterations in the i-rrnune res onse after exoosure to cia-' arette smo•e Dr. H. Hersco-:iitz.. a. Rationale. The immune response has been shown to be suppressed by MCA inoculated intratracheaily into the inbred strains of mice (Levy, aS ,a_l,. Can. Res. 37, 3892, 1977). The suppression could be correlated with the genetic suscepti- bility of the various mouse strains to chemically induced lung tumors and also to the inducibility of AHH. Herscowitz has recently demonstrated (In Pulmonary Disease: Defense Mechanisms and Populations at Risk, Tobacco Health Research Institute, Lexington, Ky., 1977) the immunosuppressive effects of cigarette smoke in BALB/C mice, which are inducible for AHH and susceptible to MCA induced lung tumors (Kouri, et al. J. Natl. Can. Inst. 51, 197, 1973). The criteria used by Herscowitz to determin~e the immune responses were: 1) antibody production at the cellular levelas reflected by the spleen plaque assay; 2) antibody production In vivo as determined by circulating hemagglutinating antibody ZHA'j, and 3) mitogen induced prolifer- ation of spleen cells. All three criteria were.shown to be suppressed by 1R1 cigarette smoke but not by 1A1 cigarette smoke. Similar results have been reported in mice exposed to "high tar" cigarettes (16 mg tar, 1.1 m9 nicotine) (Holt, et al., Arch. Environ. Health 31, 258, 1976). These same autios had previously demonstrated that 1f murine sarcoma virus was introduced intothe lungs of BALB/c mice, only those mice chronically exposed to cigarette smoke were susceptible (Thomas, et al., Br., J. Can. 10, 459, 1974). No such effect was observed-for mice exposed to smoke for short periods of time. Thus, several questions can be posed with regard to immune - suopression and_ the mouse model forr lung carcinogenesis. 1) What effect does chronic exposure to cigarette smoke have on the immune response of BC3F1/Cum female mice? 2) Can any effect observed be quantitatively related to the immunosuppressive effects of chemicals used to induce lung tumors? 3) What Is the effect of various levels of nicotine and tar in this system? We propose to address these specific questions in collaboration with Dr. Herscowitz as part of our ongoing chronic inhalation studies. Briefly, as shown by Herscowitz, the immuno- suppressive effects of cigarette smoke in BALB/c mice may be summarized as follows: BALB/c mice exposed to smoke for 1 week der~onstrated a decrease In the plaque-forming cells (PFC) per 10 cells from 2671 ± 487 for machine controls to 21 t 6 for smoke exposed mice. For BALB/c mice exposed to 1R1 cigarette smoke for approximately the reqimen proposed for the BC3F1/Cum mice, the hemagglutinating antibody (HA) titer log2 decreased from 8 for the machine controls to 4 for the smoke exposed mice. Chronic exposure to. cigarette smoke in BALB/c mice resulted in approximately 40% reduction in responsiveness to phyto- hemaggluttnin .(PHA) and concanavalin A(con A), but not to E. coli lipopolysaccharide (LPS) of splenic lymphocytes. -20- CTR CONTRRCTS 027857 11247607 CTR ~~~~ Ct~~ 4~~2 ~

PROPOSED STUDIES Contract CTR-0030 No effect was observed in BALB/C mice exposed to 1R1 cigarette smoke fcr 15 days. Our initial studies in collaboration with Dr. Herscowitz (Georgetcwn University, Washington, D.C.) will measur.e the immunosuppressive effects of 2R1 and 2A1 cigarette smoke in BC3F1/Cum mice and the results will be compared directly to the results for BALB/c mice. Using the smoke exposure regimen developed in previous studies with the SEM ii, mice will be adapted to increasing amounts of cigarette smoke up to 2 ciga- rettes/day (a period requiring at least 3 weeks). Mice used as machine controls will be similarly treated, placed in holders, etc. but without exposure to smoke. Approximately 20 mice will be used per group to assure statistical signifi- cance for the results. . After the initial evaluation In BC3F1/Cum mice of the immunosuppressive effects of smoke, the combined effects of-MCA and smoke exposure will be evaluated using the above assays. This would confirm the previous findings with regard to MCA and indicate whether smoke caused an increased immunosuppressive effect. MCA (250 ug/0.02 m1 gel-saline) will be given intratracheally concurrently with the first smoke exposure (day 1). Mice will be immunized with sheep red blood cells (SRBC) on day 11 of the smoke exposure. Smoke exposure will continue for the 3-week period, with the PFC and HA•assays performed on day 15 of smoke exposu re. Mitogen stimulation of spleen cells will not be performed. b. Procedure. All mice will be exposed to smoke at M.A. using either 2A1 or 2R1 cigarette smoke generated on the SEM il. BC3F1/Cum female mice, 7-8 weeks old at the time of the first exposure, will be adapted gradually to the machine or smoke exposure up to the equivalent of 2 cigarettes per day. For the 2A1 cigarette; 2 complete cigarettes of 10 puffs each can be offered for the 2R1 cigarette recent results suggest that the equivalentof 2 complete cigarettes can be given of 5 puffs from 4 cigarettes are given with rest periods between cigarettes. Mice or tissues will be transported directly to Dr. Herscowitz's laboratory for assay. In the PFC or HA assays, mice will be immunized I.p. at M.A. with 0.1 ml of 12.5% suspension of SRBC washed at least three times in phosphate buffered saline, pH 7.2. Mice will be immunized with SRBC 5 days before sacrifice (day 11 or smoke exposure). The mice will be exposed to the equivalent of 2 cigarettes per day. The PFC and HA assays will be performed by Herscowitz as described. Mice assayed for mitogen stimu.lation of spleen cells will be exposed to smoke on the 3-week regimen as described, (not immunized with SRBC), transported to Herscowitz's laboratory where the spleens will be removed, cultured, and mitogen activated as descri.bed. -21- CTR CONTRRCTS Q2?'E358 11247608 CTR HN 044,32.2

PROPOSED STUDIES Contract CTR-0030 r After analysis of the first PFC and HA assays, if BC3F1/Cum mice respond to 2A1 and 2111 cigarette smoke exposure similarly to BALE/c mice. then HA assays. which require only serum, will be used to monitor chronic smoke exposure studies. Serum will be prepared at M.A. and transported to Herscowitz's l aboratory for ana i ys i s. 4. CT_ R-y0_. Evaluation of adaotation and stress in the inbred strains or mice exposea to ci arette smo e. Or. ssman . . a. Rationale. Previous studies with Dr. Essman have evaluated the effects of restraint and exposure to cigarette smoke in BC3F1/Cum female mice using several assays as stress Indicators (plasma corticosterone (PC) levels, cardiac norepinephrine (NE) and digoxin uptake, and in vitro adrenal corticosterold systnesis). These studies employed tF-ie WAM,- the 2A1 cigarette, and either the cone or neck restraint. There was no apparent difference between results obtained when the mice were restrained using the cones compared to the neck type. The effect of the machine or cigarette has not been evaluated and will be the aim of the present' study. While an exact protocol must await some further results from the previous studies with Dr. Essman, the following general protocol will be employed. b. Procedure. All'mice will be exposed to smoke at M.A. using 2111 cigarette smoke generated on the SEM 11. In the first study, BC3F1/Cum female mice, 7-8 weeks old at the time of the first exposure, will be adapted gradually to the machine or smoke exposure up to 2 cigarettes per day or the equivalent machine time. The following groups of mice will be treated, placed in cages for 10 minutes, then sacrificed by pontobarbital overdose. Five mice per group will be sacrificed after 1 day, 7 days, 14 days, 28 days and 56 days smoke exposure (125 mice total). Group 1 smoke, daily 2 smoke, one time on the day of sacrifice 3 air, daily (machine control) 4 air, one time on the day of sacrifice 5 shelf control Groups 2 and 4 are age controls to determine whether age, rather than treatment, dictates the degree of response for PC levels and cardiac NE uptake. -22- CTR coNTRacT S Q2?'859 11247609 CTR HN 044,322,3

PROPOSED STUDIES Contract CTR-0030 - Mouse weights will be determined, then blood samples obtained from the abdominal aorta using heparinized syringes. Heart, lung and adrenal tissues will be trimmed, blotted, and weighed. Pulmonary AHH levels will be evaluated at M.A. using the lung tissue, while the hearts, adrenals, and blood will be frozen and shipped to Or. Essman for analysis of plasma,corticosterone levels, cardiac NE uptake and, perhaps, selected In vitro corticosteroid synthesis. When space on the machine permits, C3H/Anf- Cum and DBA/2J mice will be similarly evaluated. -23- CTR Cah4TRACTS Q2?'660i 11247610 CTR ! NI I id' , I`/ 13I"rm.e -11

PROPOSED STUDIES - Contract CTR-0030 0. Short-Term Feasibility Assays Relating to Possible Promoting Events. 1.. CTR-111. Comoarison of smoke induced OOC in three strains of mice and discrimination between ODC induct on ano • Frin uction by c garette smoke Am. a. Rationale. OOC induction has been shown to be an obligate step in the promotability of at least certain chemicals. As reported previously, mouse pulmonary ODC activity was induced after 1) exposure to whole cigarette smoke and 2) exposure to TPA inoculated intratracheally. Thus, at least in principle, there are chemicals in cigarette smoke both capable of inducing ODC activity and promoting chemically induced cancers. In addition, the observation that ODC induction is also an obligate step in the induction of MFO acitivty In mice makes it necessary to discriminate between ODC induction'as the result of Increases in AHH activity and that observed specifically In response to a promoter. b. Procedure. Mice will be exposed to cigarette smoke us i ng the SEM 11 and us i ng 2A1, 3A1, or 2R1 c igarettes, 1004 or 20% smoke concentration, and 20 or 30 seconds exposure time.. The levels of pulmonary induced ODC, the time of maximun iriduction and the half-life of the enzyme will be determined for several exposure conditions and in 3 strains of mice. Exposure Schedule for Evaluation ODC Induction in Mice S train Cigarette Smoke Ex osure Sacrifice Times # Mice onditions (hrs. post exp.) BC3F1/Cum 2A1 10% smoke 0, 2, 4, 6, 8, 12, 24 21 or C3H/Anf-Cum or 2A1 30 secs. 20% smoke 0, 2, 4, 6, 8, 12, 24 21 DBA/2J St.rains , 30 secs. 2111 10% smoke 0, 2, 4, 6, 8, 12, 24 21 3A1 20 secs. 10% smoke 0, 2, 4, 6, 8, 12, 24 21 30 secs. After the optimal conditions for induction and the kinetics of induction have been determined, those conditions will be selected for use with the recombinant inbred lines (C57BL/6 x OBA/2J, available from Jackson Lab, Bar Harbor, ME) to discriminate ODC induction from AHH induction. Ten lines will be used initially, five AHH responsive and five AHH non- responsive. -24- CTR CaNTRRCT6 027861 11247611 G T R V I N 0 4 4 '3,~.Er

PROPOSED STUDIES Contract CTR-0030 RI Line Treatment Sacrificp Time (hrs. post exp.) ;; Mice Lines 1-10 Cigarette 0,6,8 9 • Smoke Control for 0,8 6 ODC -T PA Control for 6,24 6 AHH-BaP The conclusions to be reached will be 1) whether the same mice that are positive for ODC induction by cigarette smoke are positive for AHH induction, 2) whether the same mice that are positive for OOC induction by cigarette smoke are positive for ETR 0- deethylase induction by BaP. The assay protocol for ETR was presented in CTR-108. The protocol for ODC in lung tissue is as follows: 1. Pulmonary hepatic tissue is placed In a pestle and lass homogenizer containing 2 ml of homogenization buffer at 4Y C. -Homogenization Buffer (PH 7.8 for lung, pH 7.5 for liver). Ill 100 mM Hepes 2 1 mM EDTA 2.5 mM dithlothreitol ml of Buffer ~) 100 pM pyridoxal phosphate -Total protein content of this mixture is determined by the standard fluorescamine'assay previously described by our laboratory (see Progress Report). 2. The homogenate is spun at 2,000 rpm for 20 min at 4°C. The supernatant is saved and the pellet is discarded. 3. At this point, the supernatant can be f rozen or recentrifuged. The supernatant is spun in an ultracentrifuge at 100,000 xg for 30 min at 4°C. This super- natant contains only the soluble form of the enzyme. 4. The supernatant from the 100,000 xg spin 'is saved and the pellet is discarded.(at this point the super- natant material can be assayed directly for OOC activity or forzen and assayed later. The 100 µM pyridoxal phosphate has been found to stabilize to enzyme.) 5. 0.5 ml.of the 100,000 xg supernata t is added to a 6 ml narrow mouth vial, and then 25 ~ci (1-C1~) ornithine (0.025 µCI/3.5 nmoles/fcl) is added, and immediately sealed with a rubber stopper fitted with a plastic wail con- taining a paper wick and 0.2 ml ethanolamines:2-methoxyethanol (2:1) to trap 14C02. Reaction is run in a slow shaking water bath at 37°C. -25 - CTR caNTC2ACT5 -a27s62 11247612 I I C TR VIN 04439'2>16

PROPOSED STUDIES Contract CTR-0030 6. The reaction is stopped after 20 minutes by the addition of 0.5 nil 3Q:.', perchloric acid and the still sealed vial is allyrd to stand for 1 hour for complete absorption of the C02. 7. The plastic well (including the paper wick) is then removed or cut into a vial containing 10 ml liquifl or-toluene cocktail with 2 ml ethanol and counted for total 14C. Zero time controls are run by stopping with perchloric acid prior to the addition of substrate. 8. Activity is given as pmoles 14 C02/mg protein/20 minutes, 10 cpm's = 1 pmole of activity. -26- CTR cohtTRaCTS 027863 11247613 `~ CT~"~' HN 04434 r

PROPOSED STUDIES Contract CTR-0030 2. CTR-112. Evaluation of feasibility of a focus enhancement assay. a. Rationale. Agents which have the ability to increase the yield of tumors in tissues previously exposed to initiators of carcinogenesis are cortmonly called promoters. Presently, one of the basic needs is to establish assays which would permit the rapid screening of compounds to which man is exposed (e.g. cigarette smoke, environmental pollutants) which may be potential promoters of carcinogenesis. The need to be able to detect promoter properties of chemical agents is obvious, since such properties could account in part for the biohazardouse nature of certain compounds. We feel that if , a test compound fails to cause neoplastic transformation in culture or neoplasia In vivo, this may simply reflect its inability to initiate the transformation process, but does not rule out the possi6iTity that such a compound could promote a transformation event previously initiated by another chemical. We have shown that promoters such as TPA can enhance expression of the transformed phenotypes in vitro (see Progress Report, 2/78), which demonstrated the uiTlity'- of the BALB/3T3 focus enhancement assay. This asssay, developed by Sivak (Cancer Letters, 2: 285, 1977), is based upon the fact that chemicals which possess promotion pro- perties enhance the development of phenotypically transformed foci In a mixed population of transformed and non-transformed cells. We have also reported that whole cigarette smoke condensatels capable of enhancing expression of phenotypic transformation in BALB/3T3 celis (see Progress Report, 2/78). Thus, the focus enhancement assay appears to be capable of detecting potential promotion properties associated with known model promoting agents (e.g. TPA) as well as complex biological mixtures. The added advantage of being a rapid, relatively inexpensive technique make the focus enhancement assay a promising short-term in vitro method for screening potential promoters. - . Prior to implementation of this,bioassay as a screen, it will first be necessary to validate the accuracy, repcoducibility and precision of the test. The specificity of the focus enhancement assay also needs to be established, In order to show that enhancement of focus expression is not a non-specific event, inducible.by non-promoters as well. b. Procedure. 1. 50-400 .4hemically transformed 3T3 cells are co-incubated with 2 x 10..untransformed 3T3 cells In 60 mm diameter petri dishes at 37°C In a humidified atmosphere of 57. C02 plus ai r. 2. Cultures are then treated # test chemical at various concentrations and further incubated for 24 hours. -27 - cYR caNTRac-rs 027864 11247614 CTR VIN 0'44328

PROPOSED STUDIES Contract CTR-0030 3. The cultures are then washed free of ths test agent and .nre incubated with scheduled medium changes for approximately two weeks. 4. After 10 to 14 days, cultures are fixed with atisolute methanol, stained with 10% aqueous Geimsa stain, and scored for multi-layered foci on a background monolayer of contact Inhibited cells. 5. The increase in the.number of foci treated cultures over untreated controls Is Indicative of promotion properties associated with the test chemical. 6. Protocol Chemicals Concentration Comment 1. TPA 0. 1, 0. ,.... 0.0 µg os~it ve control 2. Phorbol 0. 1. 0:3, 1.0,3.0. 10.0••µg Negative control 3. DMSO Solvent control 4. Acetone Solvent control -5. Acetic acid ~ 5 doses (w/v) Non-specific toxin 6. Formaldehyde ranging from Non-speci fi c toxi n 7. 2A1 Whole Condensate LD90 to LD10 Unknown. 8. 3A1 Whole Condensate Unknown 9. 1R2 Whole Condensate UnknovNn -28- CTR CONTRRCTS 027865 11247615 CTR M-1 0'44,.~.~ 9

PROPOSED.STUDIES Contract CTR-0030 E. Aerosol Studies 1. Introduction. - The animal model for lung carcinogenesis developed in our laboratories has relied upon'intratracheal inoculation for the precise and reproducible delivery of carcinogens or tobacco-related material to the lung. The intratracheal technique in carcinogenesis has elucidated the pulmonary response with respect to: 1) chemical, 2) strain suscepti- bility, 3) dose, 4) biochemical responses, and 5) the types of histologic lesions that can be induced in a mouse lung. How- ever, now that some of the histopathologic and biochemical end points have been investigated in this animal system, a refinement of the delivery method of the chemical to the lung is desired In order to pose more biologically relevant questions concerning lung carcinogenesis. Aerosolization of these chemicals may be a more biologically relevant method of delivery for the following reasons: 1) aerosolization of chemicals should result In tissue deposition and distribution more similar to that observed during normal respiration; 2) the effect of particle size on cellular distribution within tissues can be efficiently studied, and 3) the biochemical response of such pulmonary enzymes as AHH, ODC, and EH to certain chemicals may change after aerosol delivery compared to intratracheal delivery. In addition, aerosolization of single chemicals or groups of chemicals would allow direct comparison with the effects of ovhole cigarette smoke on the physiological and biochemical responses of the lung. •As described In the progress report from Dr. M. Guerin and Dr. R. Holmberg at ORNL, an aerosol generator has been developed which will allow aerosolization of TPA. This equipment will hypothetically permit the aerosolization of any chemical soluble in ethanot TPA has been fully characterized in this generator with regard to concentration and particle size. The following experiments will utilize TPA aerosolized with this generator, but the conditions and procedures. developed willbe applicable to any aerosolized chemical. -?9- CTR CQNTRRQTS 027866 11247616 CTR MN 044330

PROPOSED STUDIES Contract CTR-0030 2. CTR-1_3. Acute toxicity of aerosolized TPA in the inbre3' strains o7 mice. a. Rationale. Recently we studiej the acute toxicity of aerosolized TPA in BC3F1/Cum female mice. Three groups of animals, 15 animals in each group were exposed to aerosolized TPA for 5, 10, and 15 ninutes. Our results indicated that LD5o for total TPA aerosolized was 2340 jig and that the estimated LD50 lung dose is 2.20 yg/mouse, which may be appreciably higher than the LD50 dose of 0.25 µg/mouse observed earlier for intra- tracheal inoculation. The LD50 doses will also be determined for the C3H/Anf-Cum, C57BL/6, and DBA/2J mice. b. Procedure. A 0.2% solution of TPA in ethanol is used in the aerosol generator, which is run for 5, 10, and 15 minutes. Each exposure must be cal-ibrated at the time of exposure in order to quantitate the dose. C3H/Anf-Cum and DBA/2J mice will be evaluated and data presented for the LD50, for the total TPA aerosolized, and the•LD50 for estimateddose to the lungs. TPA. 3. CTR-114:• Deposition and retention of aerosolized a. Rationale. Quantitatton of the amount of aerosolized TPA reaching the lung can only be determined by dosimetry experiments. Acute toxicity experiments.(CTR-113) have suggested that the estimated tolerable doses of TPA are higher when given in aerosol form than when given intra- tracheally. Use of 3H-TPA for aerosolization will document and quantitate the deposition, distribution, retention and c;earance of this chemical after aerosol exposure of mice. The amount of TPA found in the lung will also be compared to the Induction of pulmonary ODC (see CTR-111). b. Procedure. BC3F1/Cum mice, 8-12 weeks old, will be exposed to aerosolized TPA or ethanol at approximately one half the LD50 dose. Five mice will be sacrificed by cervical dislocation after exposure at the following times: zero, 0.25, 050, 1, 4, 8, and 24 hours. The control group will be sacrificed after 8 hours. The following tissues will be removed, stored, frozen and the shipped to ORNL for radio- active analysis; head and law, upper trachea and larynx, lower trachea and lungs, 1 ver, stomach, and esophagus, fur and remains. -30- CTR CONTRACTS 027867 11247617 C Tk;' kif'I 0+4331

PROPOSED STUDIES Contract CTR-0030 i 3. CTR-115. Feasibility study of biochemical response to aerosolized chemicTs. a. Rationale. The aerosol generator and aerosolization as a more biologically relevant technique will be evaluated with regard to a b.iological response in the whole animal. The end points to be used for this evaluation are the induction of ODC and MFO by the aerosolized chemicals in the Inbred strains of mice. At this time. TPA will be the chemical evaluated and when a generator is available for BaP, the same techniques_and assays will be applied. b. Procedure. Strain BC3F1/Cum or C3H/Anf-Cum or DBA/2J Strains Treatment TPA-aerosol-LD50 Assay and Sacrifice Time MFO-4,6,24 hrs. ODC-6,8,10,24 hrs. TPA-aerosol-1/2LD50 as above EtOH controls as above The MFO assay will use ETR as substrate and is as described in CTR-108. The ODC assay is as described in CTR-111.. Data will be presented for each strain In terms of: 1) N nomoles resorufln/minute/gram wet weight tissue and 2] 1~C02 released from ornithine per minute per gram wet weight tissue. -31- CTR CCNTRRCTS Q27666 11247618 CI R H1 I 04 l 4sd"3dun

PROPOSED STUDTES Contract CTi:-0030 BUDGET September 1, 1978 - August 31, 1979 A. Total Labor Cost (see attached support schedule) $357,349 . Other Direct Costs: Materials and Supplies $100,000 Services 8,000 Equipment Rental And Repair 4,000 Publication Costs 3,000 Travel 3,000 Consultant Fees .29,600 Overtime Premium 1,000 Recruitment Expense 500 Freight 5,000 Laundry 7,800 Classsnshing 2,000 Data Processing 49,392 Central and Office Services 27,701 Sistology . 28,000 B. Total Other Direct Costs 268.993 C. Total Direct.Costs 626,342 D. Overhead * 226,290 E. Total'Direct Costs and Overhead I 852,632 I F. General and Adm.taistrative (23.5x of Line E) 200,368 C. Total Cost • 1,053,000 H. Fixed Fee 117,000 I. Total Price $1,170,000 * Fixed Overhead will be billed in twelve equal installments during the contract year -32- CTR CONTRRCTS 027869 11247619 C T R I - I N ~'0 4' 4 3 311 •

PROrOSGD STUDIES Contract CTR-0030 SCHEDULE IN SUPPORT OF TOTAL LABOR COST September 1, 1978 - August 31, 1979 Time on Contract , Hourly Labor Name Function x Hours* R . ate Dollars• C. ltenry, Ph. D. Project 90 1684 13.23 22,279 Dircctor R. 3:ouri, Ph.D. Projcct 30 561. 16.35 9,172 Director L. Schechtman, Ph.D. Co-Project Director 20 374 12.28 4,593 R. Curren, Ph.D. Co-Project Director 25 469 13.23 6,205 " L. Serrano, D.V.M. Veterinarian 5 .94 19.72 1,854 2i. Dinowdtz, Sc.D. Immunologist 20 374 13.23 4,948 B. Bhooshan, Ph.D. Senior IavestiAator 100 1872 9.14 17,110 R. Kaipscher, M.S. Laboratory 50 936 7.52 .7 039 Suvervisor , E. Kiss let 4 months only Tech. III 100 624 6.35 3,962 D. Avery Tech.:'III 100 . 1872 5.68 10,632 A. Lopez Tech. IIT 100 1872 6.19 11,588 D. Dansia Tech. II 100 1872 5.26 9,847 T. Rude Tech. II 100 1872 6.05 11,325 R. Sosnowaki ` lst 4 months only Tech. II 100 624 5.30 3,307 S. Beard Tech. -I 100 1872 5. 08 9,510 -33- CTR COh4TRACTS 027870 11247620 CTR MN cf4'-4"-3"-34

PROPOSID STUDIES Contract CTR-0030 September 1, 1978 - August 31, 1979 .SCtIEDULE IN SUPPORT OF TOTAL LABOR COST (Continued) • Time on Contract Hourly Labor Name Function X Hours Rate Dolln L. Broth Tech I 100 1872 4.60 rs 8,611 P. Gradwell Data Tech. 85 1591 4.92 7,828 R. Osburn Lab Aide 100 1872 4.50 8,424 J. Celhard Lab Aide 100 1872 4.30 8,050 G. 2fartinez Lab Aide 100 1872 3.44 6,440 R.•1Yitch Lab Aide 100 1872 3.94 7,376 M. Ricketta Lab Aide 100 1872 • 4.30 8,050 _.D.-Sinclair Lab Aide 100 1872 4•130 8,050 L. Espin Lab Aide 100 .1872 3.85 7_ 207 -~ R. Pena-Parra Animal Caretaker 50 936 3.60 3,370 A. 2una Animal Caretaker 100 1872 3. 85 7,207 A. Victory Animal Carataker 100 1872 3.75 7,020 P. ?iarroquin Animal Caretaker 100 1872 3.30 6,178 P. Ilarbin Contracts Assist, I 100 1872 4.57 8.555 J. Williams Secretary 100 1872 3.98 7,451 -34- CTR CaNTRRCTczj 027871 11247621 CTR NN 044335

1 r- , r PROPOSED STUDIES September 1, 1978 - Contract CTR-0030 August 31, 1979 SCHEDULE IN SUPPORT OF TOTAL UBOR COST (Continued): Time on Contrect Name Functlon X_ _ Hours E. Williinan Secretary 75 1404 Hourly Labor Rate Dollars 5.57 7,820 Overtime - straight time portion average only 2,000 * MA operates on a 2,080 hour wrork year. From experience, we have determined that 10% of an employee's time is for vacation, holidays, sick leave. Our policy is to include these costs in fringe benefits. Total Base Labor 253,008 Merit Increases @ 7Z 17,711 Total Direct Labor 270,719 Fringe Benefits Q 32Z 86,630 TOTAL LAB08 COST $357,349 ,,;. :1.5• -35- CTR CClNTE2RCT5 027872 11247622 CTR HN 044,3"3G-