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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