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SF0250162 II. COROLLARY STUDIES A. C'"R 119. Deposition and Distribution of 2Rl and 3al C: carette Smoke Using the Corti.^.uous Exposure Rec_^en . The results from comparative smoke toxicity studies zave shown that the continuous exposure regimen (C';R 101B) resulted in :fai:ly low toxicity to BC3F1/Cum male and female mice (Progress Report, 1980). A dosimetry experiment using 14C-dotriacor.tane (DTC) labeled 2R1 cigarette smoke was therefore required to deter:nine the actual TPM deposition and distribution under the exposure conditions. The tissues for this dosimetry experi^en= were analyzed for radioactive content at MA using the Packard 306 TRI-CARB Tissue Oxidizer. Previous dosimetry experiments ::s:nc the Walton Horizontal Smoking Machine (WHSM) and the tissue oxi- dizer had demonstrated extremely low and uni_orm background values °or all tissues analyzed (Progress Report, 1980). This was in marked contrast to results previously obtained using a:=o- :^.clic-cotassium hydroxide solutions to solub:lize the tissues. Thus the level of sensitivity and detection tor these exper::::er.:s should be greatly improved. As will be discussed below, the higher background levels due to tissue solubilization solution in terac tions obtained previously may have con tribu:.ed to higher :'P!rM deposi=ioc values than actually occurred when the background :evels a:e low. A brief description of the dosimetry experiment fol:.cws. BC3Fl/Cum female mice, 12-14 weeks old, were exposed to the c;r.- t:nuous exposure regimen for 4 weeks prior to the dosimetry experiment. On the day of the experiment, 64 of these mice we:e exposed to radiolabeled 14C-DTC-2R1 cigarette smoke, along w:::: arn addi _ional 10 mice which had never beern exposed to cicare =_e smoke ("uzadapted"). The experiment was designed so that 3-_re wculd be removed from smoke exposure after each r•.:.^ and the =_ss::es analyzed for radioactivity. The deposition and dis=.:ibu- _ior, of 14C-DTC disinteqrations per minute (dpm) is nresen_er :n '^a!:,le 35 _°or 3C3F1/Cum female mice exposed to 14C-DTC-2R1 ciga- -et :.e smoke under the continuous r egi:nen. Deposi =:on in the l.:::g .::creases linearly with increasing smoke exposure time ( corre:.a- .ion coefficient (r)s0.97), with a slope of -260 dpm/120 second xposure time or "run". An average of 74% (cv - 0.04) of the 4C-OTC dpm was found in the lung for all eight "runs". Coef=i- :ents of variation of deposition in the lung ranged from 0.21 to 39 for the individual "runs". A substantially higher cv of 50 was observed in "unadapted" mice. The average 14C-DTC dpm the lung was also substantially -higher in these animals, sug- sting that alterations in respiratory rates occurred with :ger periods of exposure. This is in contrast to previous sults using a different exposure regimen (CTR 100, 30 seconds :ke alternating with 30 seconds of air for 10•minutes), where :e exposed for different periods of time (3 weeks, 3 months, 6 months) were found to have similar TPM deposition and tribution. 8 Q J .Ki~,`i'Ob10iOgRU 1 :U60CiAteS -~ ~ 11N 044484 C f

Sr026; :n order to convert the 14C-DTC-dpm to micrograms of TP"!, the specific activity of the TPM :aust be determined. A brief review of the mechanics of the procedures will be given here. The specific activity of the TPM is the :4C-D^_'C d=:.. pe_ ,:c TPM. In the WHSM, this is de _er:nined by burning cigare __es before and after mice are exposed, collecting a:+ of --'-.e par=:c;:- lates on a"condi=ioned" Cambridge filter, weighing the filter, eluting the particulates and counting an aliquot to determine =;:e amount of dpm on the filter. These procedures have bee.^n deta:led previously and data presented (Progress Report, 1980). The amounts of TPM collected on the filter is 30-50 mg and is a resonable amount to weigh quantitatively. . Because the SEM is a dynamic smoke exposure machine and t::e smoke is generated in a flowing stream slightly di:fere.^.: oro- cedures must be employed in order to determline the speciric activity. The optical sensor is calibrated to detect ::ze tota_ ar.iour.t of TPM generated from the cigarettes. A samplinc pump w::hdraws (at a known rate ) smoke ...^.rouch 3"cord= :_c.^.ed" C3m- bridce filter. The amount of particulates i.^. _..is filter '_s generally not sufficient to accurately and reproducib'_y we:g;: ;<5 :ng ), however, sufficient radioacti -jity can be detected a: _er e:.._ ::g the particulates and analyzing by scint:liatiorn counting. vicqtine can also be analyzed from this sample. Thus the amount c~ TPM is detbr:ained from the opt:cal sensor data and the pump sampling rate. The amount of radioactivi _y is determined after el;a:.^.g from the filter and counting. These measurements and the calculated specific activity are presen:ed in Table 36 for t::is dosimetry experiment. Using the specific activity calculated in Table 36 to cor.- ver= 14C-DTC-dpm to microgram TPM results in lung depcsi=ior. ..: 4, 9, 11, 19, 24, 29, 31, and 34 ug TPM for 120, 240, 360, 480, 600, 720, 840, and 960 seconds total smoke expos::re time, :esoectively. These values are far lower than expected and :u=_::er evaluation is rea_uired. The specific activity „_ ='.:e labeled cigarettes will be determined aga_n. If these dosimetry data are confirmed, a dosi:netry experi- ment L:sir.g the exposure regimen used for CTR 100 and C^R lOla will be per=ormed before June, 1981. It is most important to verify the previous results with the SEM where the background valses for the tissues were 3-5 times higher than those irn the present studies, suggesting that the deposition values deter- mined previously may have been influenced by the background. Should this experiment be necessary, SEM II time can be scheduled in April. B. CTR 109. Alteration of the I:nmune Resnonse After Exposure to Cigarette Smoke (Dr. H. Herscowitz). Collaborative studies with Dr. H. Herscowitz (Georgetown University, Washington, D.C.) have investigated the 9 ~~ .KiC10biOlO~CRI ASSOcialPS _wjmn CTR HN 044-48"IE-5

SF025C15^ immunosuppresive effects of cigarette smoke in BC3F1/Cum female mice. The following criteria have beern used to determine the immune reponse: 1) antibody production at the cellular level as reflected by the spleen plaque assay (?FC assay), 2) antibody oroduction in vivo as determined by circulating hemagglut'_:.a- ting antibody 711A7,.and 3) mitogen induced proliferation of spleen cells. Previously Dr. Herscowitz has demonstrated that all 3 responses were significantly suppressed in BALB/c mice after exposure to 1R1 cigarette smoke but not to lAl cigarette smoke using the WHSM (Progress Report, 1979). Exposure of BC3F1/ Cum mice to 2R1 cigarette smoke did not result in a consistent depression of the antibody production (?FC assay) when compared to sham-exoosed controls. :n order to deter:nine whether the lack of deoress;orn of the ?FC respor.se in BC3F1/Cum mice was due to differences :n strains, experiments were designed to expose both 3C3F1/C;:m and 3AL9/c C::- ~nice under the same conditions and at the same time. The 3,a.3;"c :nice would serve as positive controls of _nhib:=iorn and _::e resul :.s f?om the PFC assay for the two str ai.^.s could be .._: ec::1 comDared. As reported previously (Progress Report, 1980), neither ac::te r.or 'czronic e•xposure to 2R1 cigarette smoke caused significant i.-unur.osuoflression in either BALB/Cum or BC3F1,/Cum mice under the conditions employed in these experiments. Further analYsis of these data by Dr. Herscowitz has raised the following issues (CTR 3rant #1045B, Progress Report $2, 1980 Dr. H. Herscowi:z). -f the number of PFC observed are presented per spleen, rat^er than per million spleen cells, a reduction in the number of PFC,' spleen is observed in the smoke-exposed compared to the shar••- exposed animals. The number of cells recovered per spleern decreased 30-50% for smoke-exposed, compared to sha.•n-exposed animals, and the numbers of cells recovered seemed :cw compa:ea to oublished results. Further analysis and in:erpreta::on of these data are in progress. C. C'"R 105. Deoosition, Distribution and Clearance cr 3H- Catechol (CAT in BC3F1 Cum Mice a::er to 3H-CAT-2R1 Cigarette Smoke. As reported previously (Progress Report, 1980), the pharma- cokinetics of inhaled 3H-CAT have been Ieter.^•iined in 3C3F1/Cum mice after exposure to 3H-CA': aerosolized in 2R1 cigarette smoke. CAT, 1,2-dihydroxybenzene, is of interest in that it represents the diphenols in cigarette smoke and is present in moderately high concentrations (220-550 mg/cigarette), and has been reported to be a weak carcinogen (Van Duuren, B.C., Katz, C., and Gold- schmidt, J., J. Natl. Cancer Inst. 51, 703, 1973). More recently, its weak carcinogenicity has been confirmed. It has also been reported to be a potent cocarcinogen (aecht, S.S., Car.nella, S., Mori, H., and Hoffmann, D., J. Natl. Cancer Inst., 66, 163, 1981). 10 ~s .~~~~ .~SSOCiateS fl^~Il .r..-. .....' TR PIN ~'..~. 44• I•....r.x.J'

~.~ 2 .~ ~ C r U ;~ 3H-CAT in cigarette smoke was rapidly cleared- from the respiratory tract, with rapid redistribution occurring dur:.^.c the 10 minute exposure period (Table 37). The half-life (tli2) of 3H-CAT in the lung was less than 4 minutes. 3H-CA': was absorbed in the blood and rapidly removed. Signi::cant levels of :ad:;- ac:ivity were found in the liver and kidney im.mediately after exposure (Table 37); however, these decreased rapidly. Two hours after exposure, -908 of 3H-label' was recovered in the urine. Further analyses indicated that CAT was metabolized and excreted to a conjugated metabolite, since >90% of the 3H-label was released as authentic CAT after enzymatic digestion with B-glucu- ronidase and arylsulfatase. These studies, along with previous work with 14C-DTC, 14C-Nicotine, and 14C-BaP in cigarette smcke, point out the complex nature of cigarette smokd and the dive=s::v of the oossible biological responses to cigarette smoke expos:::e. D. CTR 128. Evaluation of Chromosomal Damace or Al_era=_on After Exnosure to Whole Cicare:te Smoke ';s:nc a Sister Chromatid Exchance SCE) assav (Dr. W. 3enedict a. introduction The sister chromatid exchange (SCE) assay measures the number of ti:;ies the genetic material of the chromosome is exchanged or recombined per metaphase chromosome. It has beer. suggested that human smokers have an increased number of SCS's ia-their blood lymphocytes compared to non-smokers (:opki^, :.u. and Evans, H.J., Nature 283, 388, 1980). Thus it was of in :.er- est to determine whether mice exposed to various doses of who:e cigaret _e smoke would also display arn inc: ease in SCE' s:n ei _::e= lymphocytes or bone marrow cells. In collaboration with :,r. W. 3enedic: (Children's Hospital of Los Angeles, Los AnceLes, CA), bone .;,a=row cells from :nice exposed to cigarette smoke are ::nde= evaluation for SCE. b. Results to date• Data reported previously (Progress Report, 198C) suc- ges _ed that the SCE rate was two fold higher in BC3Fli C:m Mice exposed to 3A1 cigarette smoke than sham exposed animals. These results have been confirmed, as shown in Table 38. BC3F1/C::m female mice were exposed to 10% 3A1 cigarette smoke using the continuous regimen for a period of 4 weeks. Mice were then shipped to Dr. Benedict and the SCE assay performed. The 3UdR pellets (required for differential staining of the chromosomes) were implanted -24 hours after the last smoke exposure. Experi- ments are in progress to determine whether exposure to 2R1 c::a- rette smoke results in a'similar SCE rate, and the mini:num expo- sure period required to observe this effect. /M( MCrObiOlOgiCal A&SOCiateS ---W~:. CTR MN 04448f` `