Tobacco Documents Online

ANDREWS OFFICE PRODUCTS CAPITOL HEIGHTS, MD (K)

1979 REPORT o/ THE COUNCO. FO7t TOBACCO RF.SEARCH-U.9.A., I.e. TEE COUN(71. FOR T()BA(:CO RE3EAR(71-11.9.A., lat. l 10 F...1 59t6 3eree1, New Y.ric, N.Y. 10022

SCIFIV'17FIC ADVISORY BOARD to The Council for Tobacco Re.aucb-U.S.A., Inc. as ot December 31. 1979 SHFII)ON C. SOMMERS, M.D., Cho"sr Dlrenor of Laboratorle.r, Ltaos Hill Hospital Cpnkd Pro/estor of Pathology Colkge of Ph)sician. t Surteoas of Columbia University New Yort, New York RICHARD J. BtNO, M.D. Dlrertor of Crdlolosy and Introrwao/ Afedklne Huntington Memorial Ho.pital, Paadeea, Catitorda Professor of Medklne Unlrenity of Soutl+er. CalYornia Scbool of Medicine [.o. AngeJea, California JOSFPII D. FELDMAN. M.D. Head, Department of leununopatbolop Seripp C1mie and Research Foundation La 1 _olla, Cati(ornia iVQ.UAM U. GARDNER. Pn.D. Sdent!/ic Dirertor, The Council for Tobacco Research-U.S.A., Inc. E.1C. Hrnt ProJcsavr of Anaton+y (en.rritut) Yab Unireniq School of Medicine New Haren, Conoecticut ROBERT J. HUEBNER, M.D. Chfe/, laboratory of RNA Tumor Vinues National Cancerr Institute Betbe.d., Maryland LFrON O. JACOBSON, M.D. loseph Retentteln Professor of Biological Scknres icago Cbkambiooi. HENRY T. LYNCH, M.D. Professor and ClwJrnwn Department of Preventive Medicine and Public Heatth Crel~too Uoivenity School of Medicine Om a, Nebraaka HANS MEIER, D.V.M.. Dr. Med. Vet., M.R.S.H. Senior StaO Scknttrt Tbe Jackson IAboratory Bar Harbor, Maloe JOHN P. WYATT, MD. Tlrector obaoco and Health Research Institute University of Kentucky Les)nSton, KeatucJ:y 9eleetlfe Staa./ T`. Cw.dl WILLJAM U. OARDNP.R, Pa.D. Scientific Dbrelw ROBERT C. HOCKE7T, Pe.D. Research Director DONALD H. FORD, Ptt.D. VINCF.NT F. USANT4. D.MD. Associate Research Director Assodate Reserc h Director DAVID SfONB, Pa.D. As.ociate ResrrdY DLMctor

CONTENTS Introduction . . . . . . . . . . . . . . . . . . 7 Abatracta of Reporta . . . . . . . . . . . . . . . . 9 Caocer-Related Studies . . . . . . . . . . . . . 9 The Respiratory System . . . . . . . . . . . . 22 1leart and Circulation . . . . . . . . . . . . . 32 Neuropharmaoolop and Phrtiobp . . . . . . . . . 39 Pharmacology .ed BiothemLtry . . . . . . . . . . 49 Immunology and Adaptive Mechanisms . . . . . . . 67 Epidemioio8,y . . . . . . . . . . . . . . . . 78 Active Projects . . . . . . . . . . . . . . . . . . 83 Completed Projects . . . . . . . . . . . . . . . . 92 Itsde: of Priscip.l Investigators . . . . . . . . . . . . !03 Index of Scnior Authors . . . . . . . . . . . . . . 104 i I / JOHN P. WYA7T n 916.1980 lohn P. Wyatt, a member of the SckntiAc Advisory Board for aeven years. died January 22. A distinguished pathologist and specialist in pul- monary disorders, he had heew, sinoe 1974. Director of the Tobacco and Health Research Institute at the Univasity of Leaington. Kentucky. Before that he was Professor and Head of the Department of Pathology .t the University of Manitoba Faculty of Medicine, Winnipeg, Canada. lack Wyatt was a modern version of the Renaissance man, aervin8.ocieq well as a physician and researcher at the same lime that he explored (or truths in literature and the arts. He frequently enlivened Advisory Board meetinp with quotationf from literary {re.u that struck directly to the core of the matter under discussion. Articulate, witty, gregarious. ).ct Wyatt dwars sought to Improve the Imperfect world of scknoe. He took panicui.rly to bean his responsibiliy as a member of tha Scknti6e Ad- visory Board and arove in thN body, in his owo institution and el.ewhert, to advance acieati/k knowledge and better the human conditloa !1e wiY be deeply missed.

Introduction The Council /or Tobacco Research continued ouriaQ 1979 to pravide L oancial support (or independent scientists who are aeeking to add to knowledr of canur, cardiovascular diseases and chronic pulmoaary ai4nema. The tact is that the etiology of ttrese map' ~ailmenb r~emaias a mystery despite the vast and evertxpaadics researcb cAort (or . solution. Coo.ideries their compleaNy, there should be no doubt that the aoswer, or amwer., wiY be slow in comind asd will be b uod on discoveries by researchers is masy diecr- tot disciplines. The (acton o[ genetic makeup aad aging are important eoosiderasior is the scientific effort to determine the puhoRenesis of these ailments. Aiso ies- portaot are the recognition and examination of the impact m.de by the toul environment of our iodustrialized society. The areases and strairr of livinL exacerbated by worsening economic tww- ditions and international crises, are being studied more closely for their porr sibk role in body processes and d'ae..e accurretsce. The overall situatioe, /rom a scicatiik viewpoint, is immensely ooesplen, and one cannot expect a simple or easy solution. The Couocil, however, r.- mains optimistic that answers wiN be found eventually; so too ar, other &raM- ing agencies as well as aientists invdved in the research. The Council believes that new ideas and fresh approaches to old but still good ideas will continue to come from the world of scienee. l hat this is true ir aece in the number of requests (or research aupport that The ('ouncjl receivea rc11u- lariy. The ('ouncil remains dedicated to helping support independent isvesd- gauas in their search (or definitive answers to eancer, heart disease and chroaic pulmonary ailments. 7 he following section contains abstracts of research reports published durins 1979 by rccipientc of Couqeil support. 7

Abstracts of Reports Following are aburacts, approved by the autlwri, of reports on oenr research acknowledging support /rom The Council that have appeared ie .cka lifk journala since publication of the 1978 Report. The oame of tho recipket ia in Nalics. The abstracts are grouped under these headinp: 1. Cancer-Related Studie., 11. The Respiratory System. 111. Ileart and Circulation. IV. Neuropharmacolory and Physiob`y. V. phatmacolo6y and Biochemistry, V1. Immunology aod Adaptive Mechanisms, VII. Epidemiology. I. (;ancer-Re/ated S'tudIe. BIOLOGICAL ACTIVf3Y OP TOBACCO SMOKE AND TOBACCO SMOKE-RELATED CIIEMICAIS There are three basic questions concerning the role of tobacco amoke IN human cancer susceptibility: (1) Does tobacco amoke contain aubataooa that interact with the microeomal munooayjenaset (2) I/ ao, what are some of Uw in vivo and in vitro eflectsT (3) Are such eQecb more hmtdoua for urtais individuals or tissues than for others? As ahows here, eaposure to w6ole cle- ueue smoke Irom relerenee cigarettes promptly, but rather weakly, ioducea murine pulmonary tnkro.amal matoosy6enase activity (peak activity at 6 hours; about 2-told). This can be detected with Ihe uae of benzo(a)pyrem or ethoayresorulln as suburates, and is inhibited by cyclohesimide or actiaomycis D. 7 hese smoke-induced incrcnes. Aowever, are not umequivocally linked to the Ah locus as they are when caused by the Intratrache.l admini.uatioa of 3-methykholanthrene. Both whole amoke condenaa/e and its /ractiom ca.: (1) induce pulmonary monoosygenase activity; (2) Mhibit btnto(a)pyrene metab- olism in ritro; (3) be metabolized to subMances muta6enie to SdtnoweBa typAl.nrrirm (TA133t or TA9e); (4) transform C311 IOT% oelb in vltro; and (5) inlensi(y the carcinogenicity of benzo(a)pyrene ht murina lwsue. ro- leatially important ia the observalion that while hepatic tis.ue has the capacity to activale whole cigarette smoke condensate to mutagenic lorma !n v/t% murine pulmonary Iiuue docs nut seem lo have Ihis capacity. Although murina hutg homottenales posse.s slgnilkanl Allil activity and can metaboliae AAa- toain 8,, 2•aminolluorene and 7,tdihydro•7,ldihydroaybenro(a)pyrena to mu• tagenic aubstances. they show only weak activity with the ptommutcn, 6- aminochrysene and no activity with 2A1 cigarette smoke eoodeoaale. Macus- sioa of these results centers on the concept that whole cigarette .moke may be both a potential "iniliator" and it "ptomotor' of lung cancer in mice, and on the notion that the lalter may be the most important determinant of cancer risk. Kouti, R. E. et d. (Alicro6iofoticd Auociatti) and flenrdict, W. F. Environmentd Ilealth lertpertiver 29:63•69, 1979. From the neparament of Biochemical Onedo6Y, Microbiological Associatn, Inc, Bethesda. Md., and Childtcn's 1lospital of Los AnIleks. 9

INHALATION BIOASSAY CHEMISTRY-WALTON HORIZONTAL SMOKING MACHINE FOR INHAl.Al1ON EXPOSURE OF RODEN fS TO CIOARETTE SMOKE Until now, esperimenlal studies pertaining to tobacco smoke carcino- genesis have been hampered by the lack of a convenient, readily vailabk and wcll-deRned system capable of producing smoke for inhalation by (est animals under standardized conditions. TM Wallon Horizontal Smoking Machine ( WIISM ), a commercially available derioa designed to expose as rnany as 20 mice to the smoke of a single citurtle, way provide the answer. Sfudies indi- cate that this machine produces a uniform smoke aeroaol of predictable con- centration with both high and low nicounctontent cigarettes. The aerosol's composition was appropriate to both types of cigarettes: those with a high nicotine conteot each delivered 40 mg total particulate ma/ler, 2 4 mE nico- Iine, aml 17 cc carbon monoxide; thosa with a low nicotine cnnt:nl yielded 30 mg particulate matter, 0.3 mg nicoUna, and 17 cc carbon munoxide. For this atudy, two diRerent qrains of mice. C378L and DBA/2Bd, were exposed in the WNSM. In spite of limitations inherent to a system exposing animals to standing smoke, the WHSM oQers the animal a uniform quaotity of smoke whose concentration and composition (all within those predicted ky standard analytical smoking. The period of exposure is characterized by a~kcrease in the concenlrations of particulate matter aod asost gas phase constiwents, and by an increase in particle size and carbon dioxide concentration in the exposure chamber. Guerin, M. ft. er al (Union Carbide) Journal of the Nariona! Cancer lntrirWe 67(2) :411-41E, 1979. OtAer.upport: U. S. Department of Energy. From the Analytical Chemistry Division, Oak Ridge National Laboratory, Oak Ridge, Tena_ ARYL HYDROCARBON NYDROXYLASE INDUCTION IN MITOGEN- STIMULATED LYMPHOCYTES BY BEN2ANTIIRACENE OR CIGARETTE TARS ADSORBED TO ASBESTOS FIBERS Soma chemical and physical properties of amosile asbestos (AS) fibers were examined here using Ihe human lymphocyte aryl hydrocarbon hydroi- ylase (AHt1) enzyme system as an inveqigative tool. When cultures of mito- gen-stimulaled lymphocytes were Incubated with AS, an increase in Atilt activity was noted. A much grealer increase in enzyme aclivrty followed addi- lion of the inducen benzanthracene (BA) or cigarette lars (CI ) to cell cut- tures. Significant enzyme induction also occurred when AS fibers were 8rst preincubaled with CT or BA, washed with acetone, then added to lymphocyte cultures. lhis increase in Atilt activity was not as great, however, as the in- durliun uMervcd when BA or CT was added to cell cultures. Overall, the cnrynsc .clr.rly in cells rncubated with (T preuealeJ AS par/rcks was not as Rrs.r .% she aar...ry in ('1 rnJrrceJ IyrnpMrcyres ur nr lymphrnyla 14- whrah t 1•u.l \\ ,.crr .u..ulr.nc.ruslr aJJeJ Ihese rc.ulls mJrC.le 1h./ puly:yclie a ru.ru hsc6- %.sh .. NA .nJ cumirrnrnl% ui ( 1, cau Ire ad.urhed i to AS parlicka, which then function as carriers for the transport of these com- pounds into cells. McLemore, T. L., Jenkina, W. T., Arnorr, M. S., and Wray. N. P. Canrir Lerrerr 7(2,3):171-177, 1979. Other lupporer Veterans Administration Hospital. From the Veterans Administration Hospital; Departmcnt of Biology. "ibe Uai- versity of Texas System Cancer Center; M.D. Anderson Hospital and Tunar Ins1i1u1e, and the Dep.rtment of Medicine, Baylor College of Medicina, Ilouston. INDUCTION OF ARYL HYDROCARBON HYDROXYI.ASE IN HUMAN PERIPIIERAI. BLOOD LYMPH(X:Y7E3 BY CIIRYSENE Many polycyclic aromalk hydrourbons (PAIIa) Induce aryl hydrocarbon hydroxylase (AIIH) in human lisasres. This report describes the rok of chrys- ene-an environmental contaminant formed aa a result of the pyrocondeaaa- tioa of various cornpounds, including tobacco products-as an inducer of AHH in human lymphocytes in vitro. Lymphocytes obtained (rom healthy human subjects were cultured for •tl hours, then incubated with chrysene i. rcetone for an additional 24 hours. The chrysene-induced cella showed a aill- nifkant increase in cnzyme induction when compared to controls incubated with acetone alone. Preliminary observations suggest that the time course (or chrysene induction of Atilt is similar to that of benzanthracene (BA) and 7-nxlhykholanthrene (3-MC) induclion, the maximum being reached linearly after 24 hours of incubation. Other s/udin with pregnant rats suggest that chrysene also increases AHH activity !w r,iro. Tirc present study, moreo.er, indicates inlerindividuat variation in ANN inductibility with ehryaene, but this is commensurate with that obtained with SA or 3-MC. While there Is a theoretical relationship between Atilt induction and chemical earciaoRenesis, the connection between carcinogen metabolism and cancer susceptibility is man has not been established. Consequently, reports wch as these might help to determine the role of enzyme induction in Iha susceptibilily to diaease caused by exogenous agents. Although Ihe meubolilea of chrysene produced by At1Fl are only weak carcinogns. the abundance of this hydrocarbon ia cigarette smoke might have an idirect but significant rok in the amplifkaliow of the Atilt system in the lungs of cigarette unokers. Snodgrass. D. R., Mcl.emore, T. 1.., Marshall, M. V., Wray. N. P. Cantrell. E. T., Busbee, (). 1.., and Arnar, M. S. (MarNw, R. R.) Cancer [.erters 7 : )1)-)1 e, 1979. Other .o'portr lhe Welch Foundation, Nalional Institutes of Health, Amer- ican Cancer Society, and the Veterans Administration Hospital, HouMon. From the Department of Biological Sciences, North Texas Slate University. Denton; [kpanment of Pharmacology, Teaas College of Oaleopathic Medicine, For/ Worth; /kpartmenl of Environmental Biology. l)nivenity of Tesas, M D. Anderson Hosprul and Tumor Institute; (kpanment of Medicine, Baylor C'olkge of Medicine; and Veterans Administration Ilospilal, Houston. 11 10

INDUCf1ON, INIIIBITION, AND SOME ENZYMOLOGICAL PROPI:RTIFS OF ARYI. IIYDKO('ARBON HYDROXYLASE IN FRL'SII MITOGEN-AClIVAT1:D HUMAN LYMPHOCYTES This report describes the induction and Inhibition speciflcities and some other enrymologic properties of lymphocytic aryl hydrocarbon hydroaylase (AHHI• Basal and/or polycyclic hydrocarbon (PAII)-induced AlitJ in mito- ten-activated cultured lymphocytes from healthy donors were used to study these properties. Several compousds with an Inducibility ralio > 2.0 were found among the 24 chemicab lested: 2,I,7,S-telracMorodibenzo-o-dioain (TCDD), dibenz(a,li)anlhracene (D<!A), benz(e)anthracene, 7-methykhot- anthrene, P -naphthoflavone, chokcakiltrol, and tx.-isoproterenol. The Rrs1 four, Iisled in descending order of potency, were Ihe mwt vigorous inducen. lhcse appear to activate the common penelitally determined factors involved in Alllt induction, suggesting that they could be used Inlerchanlieably. EAects ranging from inhibition to mild inducliorr were obtained with other substances: 1-bromoffavone, .-naphthofiavont, chrysene, p.,*-1,1,1-Irichbro-2,2-bis ( p<hloro- phenyl)elhane, 7,12dimelhylbewnthrateee, pyrene, o,z-norepinephrine, bcnzo(r)pyrene, lindane, n octylamine, Ie.toueroee, 2.3diphenybsar.ok, 170estradiol, mclyrapone, and phenobarbital. The inhibilon were ranked In descending order of potency as follows: mr-napMhoflavone, p-naphlloflavone, 2dielhylanainoe/hyl-2,2-diphenylvakrak, nsetyrapone, 1,1,1-Iricholoropropene oaide, and cyclohesene ozide. Depending on their concentratioe, the Iaa1 four produced moderate inhibition to moderate stimulation. However, the inhibitioe pattern for the basal and the PAH-induced AIIH was Indistirguishabk. Both the half-life of the enzyme during cell culture and the K. values of A/IH in uninduceJ and PAII-induced cells were similar as well. Based on this and other evidence, it can be concluded that: (1) 'ICDD. DBA, benz(.)anlhracene, and .l-methykholanlhrene have a common mechanism of action whereby only the degree of AHH induction produced dt8ers; and (2) basal and induced AHII are qualitatively similar and only quantitatively different in comparabk unin- duced and PAH-induced cells. Gurtoo. H. L. ct ol. Cancrr RrsrarcA )9(11) :1620-1629, 1979. Other aupp.rtr National Cancer Institute. From the Departments of Eaperimental Therapeutics. Molecular BirJoty. and Immunobpr, Roswell Park Memorial Institute. New York Slale Department of I/ealth, suflalo. ARY1. HYDR(X'ARBON HYDROXYLASL' IN A STABLE HUMAN B I.YMPII()CYl F. ('EI.I. LINN, RPMI-17H0, CUL7URED IN TIIE ABSENCE OF MI t(X3ENS K/'M1 170e is an imorunoRlnhulin synlhesiring B-lymphncyle cell line that has shown hish basa and indrKrbk aryl hydrocarbon hydroaylasc (/1/11t) at tivily in the ab.rne c rtl nutuRcmc preslimulation Durin6 the course of the c.l,~nruts ir/NUi.J hrec. .ri,aut p.ramcters influcncmg the enzyme activity i and inducibilily were studied and optimized. This allowed altlainmeett of enzyme levels and inducibility ratios comparable to those previously reported for fresh milosen-aclivaled human lymphocytes. However. Ihio cell line haa several advantages over fresh lymphocytes: (a) high AIIlI aclivNy in the absence of milogen preslimulation; (b) availability in unlimited quantities le the absence of donors; and (c) the consistency gained by using a ain6le huma. cell line free from seasonal variation of donors. It'seems, therefore, that this system could provide a good esperimental tool for studies on human mised- tunction osygenases. In addition, using cells cultured under oplimal,conditions and induced optimally with dibeoz(.,h)anahraccne. the melabolisrn of bee- zo(a)-pyrene was analyzed by high-pressure liquid chromatography. The melabolile profik produced by these cells had severd similarities with Ihosa produced by mitogen-uimulated, abort-term cultured human lymphocytes de- rived from fresh blood. Freedman, 11. 1., Gwroo, Il. L.. Mieowada, l., PalSee. S., and Vaught, l. 0. Cwscrr RerearcA )9(11) :1605-1611, 1979. ' Other.rpportr National Cancer Institute. Front the Departments of Eaperimenlal Therapeulics, Molecular Sioloq aed Immunology, Roswell Park Mernorial Instilute, New York State Department of Health, Buffalo. INDUCTION, INIIISITION, AND eIOLOOICAL PROPERTIES OP ARYL HYDROCARBON HYDROXYLASE IN A STABLE HUMAN B-LYMPHOCYTE CELL LINE. RPMI-178t The present paper reports on the ieductbn ud Inhibition by .atious chemicals of aryl hydrocarbon hydrosylaae (AHH) iw a slabk, imnwteolog. kally de6ned (ISM-yiek/ing) S-lymphocyle line (RPMI•17llU), as well aa ae the kinetic and biological properties of the ewzyme In these cells. This pr- ticular line had previously beee selected as a biochemical ruodel for AHII .ludies mainly because of Its hi6h enzyme activity ie the absence of mitopeos. In the dose ranges tested and on a molar baia, the inducers, in deaoeodie6 order of polency, were: 2,),7,!-telrachlorodibenzo-p-diotin, dibenz(.,A)aa- thracene (DBA), l-methykholanthrene, benzo(.)pyrene, and 1,2-beezanthra- cene. Included among the potemial inducers that paradoaically lowered basal AIIlI, were 7,12-dimethylbenzan(hracene, 2,Shcphenylosazok, and chryacee. Under optimal culture curdilions, induc/ion promoted maaimum enzyme activ- itics three- to four-fotd that of batal AIIH. lYm characterhllcs of the 1)sA-In- duced and basal enzymes were virtually idenikal. lhe pat curves were similar for brMh (R.23 optimum), as was inhibilor specificity. Induced and basal enzymes displayed similar half-lives, apparent activation encrgies, optimal tem- peratures. denaturation temperature range, end apparent Kr„ with benzo(.) pyrene. lhe small differences observed in Ihe latter were related to the enzyme concentratarn during incubaion rather )han to its yuai/y. Freedman. 11. 1., Parker, N. 8,, Marinello. A. 1., (irrroo, 11. l.. and Mino- wada, 1. 1) 12

Cancer Rerrarch )9:4612-4619, 1979. Ottier.upportt National Cancer Itatitute. Prom the Departments of Experimental Thenpeutia and Immunology. Roswell Park Memorial Institute, New York State Department of 1(ealth, Buf(ab. CELL SOR i'ER ANALYSIS OF CARCINOGEN METABOLITES IN HUMAN 71SSUES The biochemkal parameters of a betesv~eoaw culture of human (ympho- cytes were eaamined with a f)sortsoeeoe-activated ceN sorler (FACS-11). During culture, the cells were incubaled i. the presence of benz(a)an:hracene (BA) to induce the enzyme system that metabolizes carcinogens. Direct assay of carcinogen metabolism was achieved by measuring the phenolic meubolites of cells e.posed to benso(a)pyrene (BP). According to single cell measure- ments. BP metabolism was grealer in tbe larser cells than in Ihe smaller ones. In the cells exposed to BA during cultura, the entyme aclivity also was greater (or a given cell size. In some of the studiea, dabk celb were Ont sorted out sccordind to size nd the wbpopulations were tusayed (or the o-deethylation of eihosyresorufln, a compound that measures eylochrome P-44E1 activ:ty more specifically. In corroboration with the direct metuuremenu, the larger cells demonstrated higher en:yme activity levels thas 1he smaller ones. The technique of sorting before enzyme assay. as described here, can be applied to other tiswes, as well as to other cell types asd enzyme systems that cannot otherwise be measured directly. Tyrer, lf. W., Adams, L. A.. Tdlany, S. M., O'Connell. J. P., and Cantrei(, E. T. The /ownd of !llytocheminry and Cytochemirtry 27(1) • 30N-S 11, 1979. From the Becton, Dickinson Research Center. Research Triangle Park. North Carolina; Tesas College of Osleopathic Medicine. Fort Worth; and Division of Cytopslhdop, 1)epartment of Pathobp, The Johns Hopkins University School of Medicine, Baltimore. ACTIVITIES OF POLYCYCLIC HYDROCARBON ACTIVATING AND INACTIVATINO ENZYMES IN HUMAN LUNGS OF SMOKF.RS, NON-SMOKERS, LUNGCANCER AND NON~CANCER PATIENiS Polycydk hydrocarbons (PAH), which are present everywhere, eaert lheir adverse effects only afler metabolic activation. The mkrowmnl nwno- o,ygtnase system (MO) catalyses tM osidaliw biotrans(ormation of asreat number of endo6enous and exogenous compounds and ia also rtsponstbk for the formation of eiectrophilically reactive eposides from PAII. Concentrations within the ceUs of Ihese reactive epo.idet, which are suspected ultimate car- cinoRens, may be conerolkd no1 only by the MO aclivitits, but alur by the eposrde•metabuli:ing epoaide bydratase (PH) and tlutalhione-Suarrs(erase ((iF) ectivilies. Since such dats did not seem to eaisl, the epoaide (orming and metaboli:ind enzyme activities were meuured in 47 ssmpks from lunp of smukers, nonsri>,.\ers, lung cancer and noneaneer patients and correlated to the rMcurnence rd e.ncer When uand.rd assays were used, no Ml) activity .uulJ f.r dcte.ted m hun..n lunp, alth.>,t6h the very setadrve assays with ben:o(a)pyrene, 7<thoaycoumariq 7-etboayreaoru(in and biphenyl as wtb. strates were used. However, these activities in rat lung mkrosornes used as positive controls were always clearly meawrabk. By ooincub.tios of ral lueg microsomes with human hms microsomes, It could be demorulrated 16at 7- etho.ycoumarin-odedhyl..e activity wu inhsbiled by the presence of hurn.n lung microsomes after preiacubation for 13 minutes. 'Ibes. Bndinp wyeM the presence of  diffusible inhibitor is human hmR microoaees. 1. 16e enzyme activities of human lung aampks, considerable i.lerindi.idwl va,riiatioas were observed. However, no sisa(kant diRereuces (P>0.1) were found wbes enzyme activities in IunRs from notscsaeer patients awd .oa.rnoken ww compared with thow from lung cancer p.lie.ts, suggesting Wy other (acfors may affect differences is lung tumor wuceplibility. Lorens, 1., Schm.asmann, H.. Ohnhaw, E. and OercA, F. Archlver of Toairofoq. Suppl. 2(Meehanitrn of Toxic Action on Soene Tarpt Orpns) :4l1-4l9, 1979. From the Section on Biochemical Plurm.eobQr, Institute of Phann.oo{ogy. University of Main:, Main:, Wes1 Germawy; a.d Inaelspitd Bers, Switzerland. A NEW ASSAY FOR GLUTATHIONE S-TBANSFERASE USINO (•H)- BENZO(A)PYRENE 4,S-OXID6 AS SUBSTRATE. INDUCIBI(.ITY BY VARIOUS CHEMICALS IN DIFFERENT RAT TISSUES COMPARED TO THAT OF ARYL HYDROCARBON HYDROXYLASE AND EPOXIDE HYDRATASE A new assay for glutathione S-tratuferass that uaa (s11)-benrp(a)pysw. 4.5-oxide as a substrate Is described. Compared to previously propo.eA net6ob. this assay offers several adoantapes: (1) it ia easily .od rapidly per(orsnea, requiring only one extraction step and eliminating the usual chromatorphic sep.ratioa of substrate and products; (2) it tNilires as a substrate am s ids metabolically produced from a ea(einodetsie polycyelie aromatic hydrocarbon; and (3) it is performed under optimum conditions for the pll and eo.oes- trations of the two substrates (ghNalhione and epoaide). This assay wr used 1o study the effect on Shnathione S-transferase of compourds kt.ows w induce drug metabolizing enzymes. The activity of gIWathions Stransfetase was oaly slightly aflected by varioua inducers. In this respe.ct, it ia very diRerent from other enzymes inswlved in polycyclic hydrocarbon metabolism: liver epoalde hydralase was very sianifkanty enhanced by phenobarbilal and 16„tyaoo- pregnerwrlon. Iwatmenl, and aryl hydrocarbon hydrosylasa appeared to be very easily arnd strongly induced (n many wgans by several chemkals. 'Ihese results further support the eoMention that in contrast to other en:yaw, glutathione S-transferase itduetion probably has only a minor role in the regulation of polycyclic arumalic hydrocarbons elwninalion. Van ('anlfort, I.. ManU, 1.., (ikkn, I. E.. (Ball, 11. R., aad UcrcA, F. Biochemical Pharmacology 2(1(4 ):4S S•460, 1979. From the I ahoratoire dc ('himk MEdicak, Imtitul de Palholugic, San Tdmaa par I itae, Nelsium; and the Section on BNrchemKal Pharmacolo6y, Institute of Pharnsacology. Universtty of Mainr, Mainr, Wcst ('.ermany. 14 I'S

i i { RI:Dl1C1lUN OF t11:NZO(a)PYRENE MUTAGt:NICITY BY DIIIYDRODIOL UEHYDRO(iENASti Since epoaide hydratase cannot inactivate the dihydrodiol epoaides pro- duced by the environmental carcira8en, benzo(a)pyrerse, the abiLty of a dihydrodiol dehydro8enase to sequester the precursor dihydrodioh an,' thereby to aflord protection from the later, more reaclive, dihydrodiol ept..i•Ics was investigated. To carry out this study, a purified form of the enzyme had to be prepared. The effect of pure dihydrodiol dchydro8enase on llse mula- Senicity of bento(n)pyrene was then studied in the microsomc/Salnwnclla lest. Here two metabolic pathways mainly contribule to the muts8enicily of benro(a)py'ene: (1) activation to bento(a)p)rene-,,s-oaide and (2) drhydro- d'wl epoaides. ihe Salnwnrlla rypAlnrrrlvrw arains TA 98 or TA 1110 are highly susceptible to bulh Sroup" of muta8enie wrelabuliles, whcrea Iisc strain lA 15)7 is much mote susceptible Io the 1,l-oside than to the drhyQrodnA epoaidts. Results showed that when benzo(a)pyreno was activated by liver microsomes from 3 nselhylcholanthrene-trealed mice, addition of pure dihy- drodiul dehydrogenase ekarly reduced the reula8enicily. The effect increased with the duse up to 0.5 units per plate; (urther addition of dihydnAiul de- hydro8enase had no si8nifkant effect. The percentage of the bcn:ol.)pyrene mutarcnicrty which could be removed by dihydrodiol dehydro8enase depended oo the bacterisl strain. With 1A 98, it was 30-609i, and with TA IS37, as eapecled from the lower sensitivity of this strain (ot benzo(e)pyrerse drhydro- dio) eposides, it was smaller. lhus, it was concluded that dihydrodiol de- hydru8enase can protect atainsl muu8enie effects ol benzo(a)pyrene, but that not all the mula8cnic mcuhuhtcs ul bttuo(a)pyrene esn be rensuved by lhis enzyme. Howevcr, the mutagemctty mediated by the drhydrodiu6epuaide pathway opens an intriguing perspecttve for the entire field of polycyclic hydroearbontvoted muls8enicity, carano8enicity and cyloloaicity. (11a11, II. R., Vogel. K., Bentley. P., and Orsch, F. Naturr 277:319-320, 1979. From the Section on Biochemical Pharmacology. Institute of Pharmacolop. University of Mainz, Maioz, West Getmany. MUTAGENICI TY TFSiING OF NITROGENOUS COMPOUNDS AND 1H1iIR N-OXIDIZt:D PRODU('iS USING TRP+ IN E. COU The N-oaidalion of certain aromatk amirses, aromatic ansiJcs and carbamates can produce mula8enic and carcinogenic compounds. Consequenlly, the development of short-term tests designed lo evaluate potential nsYs front new compounds is highly desirable. lhis report describes a spot test ut:lizin8 tryM"phan ( Trp t) reversion In E. coli to detect substances that cause Aase-pair substitWion, a measure of mula8enicity. A variety of nitro8entontainin8 compoundi and their chemically synthesized N-oxidation products was es- amined 1 be N oarnlalion of certain aromatic amines and earb+malcs was assuaiatcd with mutagenic aclivrly, bul that of aliphalie amincs, tertiary sro- matrc annnes. helrnMyclic numrtic amines, and aromalic aosides did nu1 yicld pnMlutts srl;nificandy nwre nnwagcnic than the parent cumprwnds. 16 I i t Pai, V., Bloomfkkl, S. F., lonca, l. and Gorro/, !. W. In: Gorrod, l. W. (ed.): siiolosird Otidatlow o/ Nlnotew. New Yortt: Elsevier/North-Holland Biomedical Press, 1978, pp. 375-382. From the Department of Pharmacy, Chelsea College. Univenity of l.ondon, Londoo. TIIE IN-VIVO N-0XIDATION OF 3-SUBSTITUTED PYRIDINES FOt.1.OWING 111E1R INTRAPERITONE'AL ADMINISTRATION Previous Mudin have shown that several 3-subslitutcd pyridina ara oaidired /n rlno al the pyridyl nilro8e.. ihe in rlro meubulism of pyridiae and of its l•chkxo and 3-methyl derivatives was therefore resaamined Is order to determine the role of N-oaidation In the metabolism of these com- pounds. Following the intraperitoneal admi.istralion of thc parent pyridina, pyridine-Noaide, )-methylpyriditse-N-oaide and 3<hloropyridine-N-oside were detected in the urine of mice, hamslen, rNS. Suinea pip, rabbils, and fcrrcu. N-oaidalion proved to be a quantitatively Important metabolic pathway /or pyridine. The urinary eacrelion of the N-oaide ranged from about 10% o/ the dose adnsinistered in ra/s to about 40% In mice and Suinea pip. With 3chbropyridine and 3-melhylpyridine, the N-oaides accounted for kss Ihan seven % of the administered dose. Mice prelrealed with phersobarbitnne showed increased urinary levels of pyridinc-N-oaide, but 3-nrcthykholanthrene pro- treatment had no noticeable eRect. Damani, 1.. A., Disky, L. 0. and Gorrod, J. W. In: Gorrod, l. W. (ed): eidorkal Oa/datlon o/ Nluotew; New YorT: t:lsevier/North-Ilolland Biornedical Press, 1978, pp. 157-162. From Ihe Deparlment of Pharmacy, Chelsea CoqeBe, University of l.oedo., Londas. SOME FACTORS INVOLVED IN THE N-OXIDATION OF 3. SIIBSTfI UTt:D PYRIDINES BY MICROSOMAL PREPARKiIONS IN Vf7'RO While the uaidalion of nkrNinamide to the N osWe has been demon- slratcd ln r/tro. the N-osidaliun of the pyridyl nitrogen in various drup and c.Hnpounds with this type of amine function has not yet been studied ea- tensively, either in rirro or its riro. There/ore, in order to establish the occurrence of pyridine-N-usidatiun and to establish the en:ymolo8y of Ihe various reactions invulved, a systematic study of hetero-arornatie N-osidation was begun with the use of a series of 3•substituted pyridines as ns.Klet com- pounds. It has prevronsly been shown that oaidation at the pyridyl nitrogen appears to be a Ecncral melalxslie In virro routc /ur several 3-substiluted pyrwlines. lhc elkcts of various factors on the C- and N•oaidation of 3-melhyl- 17