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Council for Tobacco Research

Report of the Council for Tobacco Research-U.S.A., Inc. [St]

Date: 1973
Length: 49 pages
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.~ . EY.TPA COPY REPORT of THE COUNCIL FOR TOBACCO RESEARCI-i-U.S.A., Inc. 1973
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Organization and Policy The Cowcil for Tobacco Researzh-U.S.A., Inc. was fortunate this year in having a world-tenowned cancer investiaator, Dr. William U. Gardner, join the ataff as Scientific Director. This post had been vacant since the death of Dr. Clarence Cook I3tt1e in December 1971. Dr. Gardner recently retired a E. K. Hunt Profeaor of Anatomy at Yale University School of Medicine w.~ere he had been Professor and Chairnun of the Department of Anatomy from 1943-1 %7. Dr. Robert C. Hodtett, who has been with The Councll lince 1954, has been appointed Research DtrMor. He had been serving as Actin= Scientiflc Director for the last two yavtl. Two distinguished scientists joined the Sdentifk Advisory Bo.rd during 1973, adding further strengtb and expest3.e to the Board. They are Dr. Averilt A. I3ebo.r, Professor and Cluirm.n of the Department of Patholo=y at the University of CalifornL School of Medicine In San Die=o, and Dr. Henry T. Lynch. Professor and Chairman of the Department of Preventive Medicine and Public Health at Crei=hron Uni.enity School of MedicSne in Omaha. Nebra,%ka. During Ihe ya.r, thrne lon=••t{mm rnembers left the IIoard. Dn. MeKeen Gtten and Kenneth Merritl Lynch had been members since the Doard was esaelid+ed in 1954. wbile Dr. CLyton Loo.ii had }oined .orne yean later. The wir catnset and wt sntpetienoe of theae senior nxn of science and medicine did much to .dvaeoe The Cotmdl't tesearch proyram. The Council i+ the spornar{at agency of a promm of research into quntloet of tobacco u.e and 6ealth. It Is the outarowth of an orpnintion formed early In 1954 by rtpresentatlv" of tobacco manufacturas, growers ; and warehonsernm. Research nrpport hr been mainly through a program of gnnb-in-ald aupQlaeented by oootr.cts for research with institutions and ~~ fabontoriey. The Council does eot itself operate any research facility. ~ The SchntE6e Adrbory Board to The Council meeta regularly to evaluate ~: applicatioos for granb-in-aid and for oontr'.cts., )tadlint them solely on the •. basis of tcientibe merk and relevana. ~ The Com+cil awards research srtnts to kfdependrnt scientists who are ararsd oomptebt scieotlfle freedom in conducting their atudk.. Granteea .lone are re+porrible for reportlej or publishing their Ifndina. In the accepted .t+ekatitk tsunnv - tbrottjb medical and acientlfk }ournals and societies. Thteallh December 1973, t•aeare5 pro*ta have been approved for 29s ~ I ' 'I ~ , 1973 R E P O R T i of ~ ~ THE COUNCIL FOR TOBACCO RESEARCH -U.S.A., Inc. 1J ~ ~ ( ? ln.nti=.tora in 219 medfod .choola, ho.pitah and idearch imtitvtloa.. Tbese + aw.rd- totaled more than f26.000,000 I. This report indoAes a brief snmraray of The Covoctl'a present protram as wetl r li.b of the cotea and pre.iorat reaearch projecta .upported by 'Ilu CoereA. Alae l -1 IF arf abkraeta of l Is naearch papers, acknowledging CarcY wp*ort, tiut wat" pabUgW fe scJeedAc }oareala durin= 1973. A total ,, 'f of 1.170 weD p.pets !u Nepo6tlrA.d by rn+j.cx ndpieets. T1IF. lY)L'N(:IL Ff1R Tl1R.kCC(1 RFSEAftC]i-U.4.A., Inc. ~ IL H. IUsot ~ +l* (]tafrman .d Pre.Went 110 F,,~et .9th titrrrt, New York, N.Y. 10022 , ~ : r~ ..~.•..~ .~. . • ' ` . . •
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n ---1 ~ ® 5(:rF,NTtrtC ADVISORY f3OART) to The Couneil for Tobacco Rewarch-U.S.A., Inc. a,, nf Dcccmbcr 11, 1973 SIIELDON C. SOMMERS,M.D., Chairman Dirrr rnr of Lnhnratnrirs, Lcnox Ifill F1oapital (7inirall'rnfrisnr nf 1'athnlngr Cotumhia University College of Physicianc and Surgconc Ncw York. New York IlOWARD B. ANDERVOW, Sc.D. Srirntifir Editor (rrrirrd), Thr Iornnal n,f thr Nnrionn! ('nncrr Inctirnrr llcthcscL•i, Marylantl RICIIARD M. BING, M.D. 1)irrctnr of CardinloRv and fmramurnf Mrdicinr f funtington Memorial fiospital, Pasadcna. California 1'rnfrriur nf MtAicinr University uf Southem California School of Mcdicine Los Angclcs, California W1LLIAM U. GARDNER, Ptt.D. Scientific Dirrcror, The Council for Tobacco Research-U.S.A., Inc. li. K. flnnt Profts.ror of .inotomy (retired) Yale University School of Medicine New Havcn, Connecticut ROBERT J. f{UEBNER, M.D. Chief, Viral Carcinogenesis Program National Cancer Institute f3cthesda, Maryland LEON O. JACOBSON. M.D. Dran of the Division of Rialogical Scirncr.r Rrgrnrtrin Profts.mr of llioingical.Ccirncr.c University of Chicago Chicago. fllinois AVERILL A. LIEBOW. M.D. Prnfrssnr and Chairman. Department of Pathology University of California School of Medicine San Diego. California f1ENRY T. LYNCH, M.D. Prnfrs.ror and Chairman Department of Preventive Medicine and Public f1calth Creighton University School of Medicine Omaha. Nebraska fIANS MFfF.R, D.V.M.. Dr. Mcd. Vct., M.R.S.H. SrninrStaff Scirnriit I he lackaon Lahoratory flar Ifarbor, Maine 1OTtN F . WYATT. M.D. 1'rnfr.csor anrl llrad, Department of Pathology University of Manitoba Faculty of Medicine Winnipeg, C7nada Sekntifie StafT of the Couneil WILLIAM U. GARDNER, Pn.D. Scientifie Director ROBERT C. HOCKE7T, Ptt.D. Research Director JOHN H. KREiSHER, Ptr.D. FREDERIC W. NORDSTEK, Ptt.D. .1 s.rnciate Rrsrarch Director Assoeiate Researeh Director VINCENT F. LISANTI, D.M.D. Research .t ssociate
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Introduction CONTENTS Tntroduction . . . . . . . . . . . . . . . . . . 5 Current Status of the Research Program .. . 7 Cancer Studies . . . . . . . . . . . . . . . . 7 Chronic Respiratory DiseasCs . . . . . . . . . . . . . 9 Cardiovascular Diseases . . . . . 13 Pharmacology and Psychopharmacology . . . . . . . . . 13 Epidem iology . . . . . . . . . . . . . . . . . . 14 Abstracts of Reports . . . . . . . . . . . . . . . . 15 Studies Related to Cancer . . . . . . . . . . . . 15 Heart and Circulation . . . . . . . . . . . . . 28 The Respiratory System . . . . . . . . . . . . 34 Pharmacology and Psychopharmacology . . . . . . . 51 Immunology . . . . . . . . . . . . . . . . 61 Epidemiology . . . . . . . . . . . . . . . . 65 The Normative Aging Study . . . . . . . . . . . 67 Miscellaneous . . . . . . . . . . . . . . . 75 0 Active Projects . . . . . . . . . . . . . . . . . 80 .~ '~Compteted Projects . . . . . . . . . . . . . . . . 86 Index of Senior Authors . . . . . . . . . . . . . . . 94 Index of Principal Authort . . . . . . . . . . . . . . 95 The interaction of the genetic or constitutional factors and the environ- mental conditions to which an individual is exposed over the course of many years probably contribute to the expression and nature of the aging-assoeiated diseases. Those diseases that have been statistically associated with smoking and health are predominately age-associated diseases. In some instances,.an indi- vidual may have an inherited tendency for a disease, for example emphysema, and the asaociated and presumahly contributory inherited atpha-I-antitrypsin deficieney. During the past few years mueh has been learned of viral expression and cancer in laboratory animats and of chemicals which, when modified by enzymes that may be present, can transform normal body eetls into cancer eells, possibly through initiating viral expression. Chemicals that may themselves be quite in- active in inducing cancer in laboratory animals may make small and ineffective amounts of known cancer-eausing chemicals capable of transforming normal cells into cancer cells. It is now possible to monitor the environment of inbred lahoratory mice so that the virai, chemicai, immunoioginl, and genetic qualities can he knowingly controlled and varied. The environment of laboratory animals can be eontrotied more specifically than can man's environment. Even when genetically controlled and predisposed laboratory animals are used, exposure to known or presumed ancer-invoking chemicak muv extend over prolonged periods. Only monozygotie (identical) twins afford a genetic uniformity In man comparable to that of inbred laboratory animals. If they are discordant in de- terminable environmental exposures, these twins could provide data that may lead to the determination of the relative Importance of genetic and environ- mental factors. Such studies are being undertaken in countries in which the mortality from lung cancer reportedly diRers more than fourfold. The transformation of cells in tissue culture has been studied In hopes of ohtaining a rapid and reliable method of detecting small amounts of eancer- causing chemicals in materials to he tested. The occasional "spontaneous" trans- formation of cultured cells, the presence of latent viral antigens and otha qwti- ties have not permitted exclusive use of cultured eefls for detection or qnsntita- tion or cancer-causing substances. Up to this time, the results afflrm. In general, those of experiments with intact animals. T1x In vltro techniques provide the methods of investigating mechanisms of cell transformation at a subeeifular or molecular level and of search for inhibiton of trarnformation. Revession of transformed to normal cells and selective Inhibition of growth of trinsformed celts can Iso be detected In virro. The discovery that the levels of Inhibitors of proteolytk enzymes in the hkrod of persons highly susceptibte to emphysema are tow, has revealed at least one objective hereditary predictor quality for this pulmonary disease. Further- . morr. information on the sources of proteases, the meehanisms of their action on f.ndy tissues and the characteristics of their inhibitors continue to be sovght. The age-associated atheroxlerotie .lfcutar dbeafes, particularly thotm in- votving the coronary arteries, continne to be invdtipted from their jeoetic and environmental interrelatiomhips. An enzyme that makes cholesterol ttsore soluble, lecithin-cholesteroi-acyt-tnnsferase, may provide Information for titnifl- eant advances in atherosclerosis. 5
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Nicotine anJ its metat.olites, untler aonfe circumctnncct. h: vc Jircct or in. direct cfTccl< ur- nt'rve ct'tl.. Thc Jevelof+mcnt (d raJi~immuni a..ay mcthoJ% for the tlcteclion of nicotine and metaho!ilcv i[ f!cilifafinr work on mt'1.,lqilic %fudics anJ maV reveal specific nerve cella uron which they may act or may have hinding .ilc.. Significant advancc% in the p.vchoph:vmacatnFical :npects of %moking may he expected as methoJ% develop to permit exploration of a direct nicotine binding at cellular or suhcellular Icvet. Some of ,h,! researches supported by The Council are at the cet!uLv and suhccltular levels. It is important that advances continuc to hc made at such methotlntogical and conceptual levclc to aeaurc cnntinueJ progress in other asptets. The support of haeie research in immunokogv or virotogy, for examrfc, may eontrihvte %ignifieantly to an under.tanding tN tmokinf and heaith. "1 hc more than 1(N1 papers reporting investications supported hv The Counci! that were puhtishcJ during Ihi% past year reveal the hreacdth of the ovcrall rc~tarch intcrezt. Wn.t tnM U. CnRnNrrt, rrr.T). Scientific f)ircctor Current Status of the Research Program The tesearch program of The Council for Tobaeco Research-US.A., 1nc. expanded further in several directiorn during 1973, with studies relating to cancer still receiving the greatest- emphasis. Studies of chronie pulmonary diua"s and epidemiological investigations have also been expanded. Among the tatter, the study of identical twin poputations as a means of separating genetic from environmental facton in the genesis of diseaxs has been extended. Researches on cardiovascular diseases and on pharmaeoiogy and psyeho- pharmacofogy were continued at arprt.ximately previous levels. As herctofore, the emphasis was mainly upon the etiology or pathogenesis of these aging.associated constitutional diseases with t special interest in the ttiscovery of faclors predictive of susceptibility or resistance to them. Since the published resuits of Council-sponsored studies are abstracted elsewhere in this Report, our intention here is to give perspeetives on the nature and extent of the program as a whole, the distribution of emphasia among its parts and on the purposes and plans of apptoach in such a way as to aid in fitting individual research teports into a context. 1t will be recognized that many studies acknowledging Council aid represent smatl methodological steps toward ultimate goais and that some are by-products of the main effort. Cancer Studies During the year just ended, The Council concentnted major attention upon an extencive, systematic, muttifactorial and step-wise atudy of eartinosetxeis in a number of contrazting mouse strains. The objeR of the ongoing study is to define and measure several genetically mediated biochemical eharoeteristies that are thought to determine and predict the responses of these animals to external "carcinogenic inftuence.s," especially to chemical substancea introduced into the lung with or without promoters or speeial conditioning. This undes• taking it to discover and deflne eondittons under which truly mafi=nant, aggressively invasive and fatal squamous carcinoma of the lune, with metasuses, analogous to the human disease, can be obtained repetitively and predictably in such animals. The importance of this toat is emphasized by experkttce showing the un- certainties in diagnosis of microscopic, incipient lesions by purely rrarphofogical criteria and in the use of such ksiom as end-poinh in experimental researth. If this aim is attained, in the context deseribed, ft should provide infor- mation in biochemical, genetic, viral or immunototic tetrrn how the animals that develop this disease differ from those that prove to be reslstant or immune. This is the kind of information from animal experiments deemed most likely to provide promising clues for application to human studies. 6 1 7
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lnedeqreocv of MouaeSkirr PQintFng Thc C-ouncil's often repeated dissatisfaction with the cnnventinnat mouse- skin painting with smoke condensates as an indicator of "tobacco carcinop,eni- city" stems in good part from the welt-known and very great diRerences among animal strsins and species in their responses to treatments of this kind with known and potent chemical agents. Without information atrout the biotngical bases for such differences, any assumption that the results of such mouse eaperiments can he extrapolated directly to other species, esf.eciatly man, is highly p.esumptuous. A Council-sponsored study completed during the year employed mouse-skin exposures to whole fresh smoke in its aerosol form in comparison with skin painting with smoke condensate from the same reference cigarettes. The rrsults appeared to show that the condensates produced highly artifactual results. If oonfirmed, this study seems likely further to reduce confidence in the skin-painting technique. Similar uncenainties penain to the significance of nearly all smoke in- hatation studies heretofore carried out with animals because of inadequate definition of the animis and poorly controlled or unquantitated exposure conditions. For the current multifactorial project, the mouse has been the species selected because it oRen the maximum potential in terms of definable genetic and biochemical suscepti!rility or resistance factors and thus may hopefully provide the shortest discernible route toward human relevance. It should be undentood that the highly inbred mouse strains used in the study are essentially laboratory creations and never existed as such in the undisturbed state of nature. They must therefore be regarded as "bundles of traits," not as direct models of man, so that experimental results will be relevant to man (or other animals) only insofar as the same traits are present and similar in import. "Feeder" Studies Carried Out The prennt project became possible only after a Iarge number of pre- limu+ary or preparatory biological projects had been carried out as "feeders" to provide basic information essential to its design. Previous Annual Reports have descn'bed or listed many such investiaations. The program is still passing through systematie, step-wise developmental phases including subcutaneous injeeticm and lung implantations of known "earcinogens." When sufficient information about susceptibility and resistance to such carcinogens has been aceumatatcvt, long-term, chronic exposures of contrasting "defined" animals to inhalation of whole, fresh tobacco smoke or its =as-vapor phase re con- templated. These will require the production of smoke under controlled con- ditions, from tobacco products of known composition and eharacteristiesr delivered quantitatively and monitored by tis+ue dosage measurements. A battery of tests and observations is being developed and refined for application to the treated animals. Se.eral service contracts for urryin: out the necessary chemical, physico- ehemipl, analytic, meehanial, and calibration work continue in operation at a high level to support the design of the experiments. fs such carefully controlled long-term smoke inhalation experiments, using highly rr.oeptible mice of laboratory design and other strains with contrasting comhinations of putative susceptibility characteristics, either the appearance or non-appearance of squamous lung carcinoma (or other carxer), should provide, not immediate conclusions, but scientific information on a new plane of interpretability and potential relevance to man. ITiochemical and Cenetic Factori in Cancer Among the biochemical or genetic facton relevant to cancer susceptibtlity or resistance in mice, three may be mentioned here: (I) the Eenetically- mediated presence of aryl hydrocarbon hydroxylase (AHH) inducibility, which determines the ability of the animal to convert various inactive but potential carcinogenic substances into an active form capable of attacking the cellular nuclear materiat, (2) the genetically determined expressions of indigenous "C-type viral genome," and (3) competence of the immune system and its susceptibility to depression by "carcinogenic" influences or agents. During the past year. Council-sponsored stud'ees reported evidence of a genetic relationship between susceptibility of mioe from several strains to 3-methylcholanthrene (MCA)-induced subcutaneous tumors and the inducibility in these mice of aryl hydrocarbon hydroxylase. Inducibility was controlled by a single autosomal dominant gene and reflected in flve-to-ten times greater MCA susceptibility in "inducibte° animals. These reports stimulated other investigaton to study AHH levels and inducibility in man, with results suggesting that in man also, inducibility is controlled by a single (incompletely) dominant gene. This results in a distribu- tion of inducihility in a random population into three groupings - low, inter- mediate, and high - essentially in accord with theoretical predidion. A preliminary study of AHH levels in human lung cancer patients indi- cated that nearly all the victims of this disease in the sample had either inter- mediate or high levels of AHH indtxibiliry. The Council has undertaken to support eRorts at simplifying and expediting the test procedurrc for use in clinical studies to evaluate their potential in assessing cancer susceptibitity or resistance in human populations. Other cancer investigations, collateral to the project described, inc(uded: !n vitro studies of malignant transformation of lung epithelial ee(ls and of possible induction of squamous nxtaplasia in cells already malignant; efforts to identify the agent or agents that produce sheep-funt adenomattxis and to transfer these to mice; immunological atudies of tvmor-anociated antisens in human cancers of ovary and lung; research into plasma-rnembrane associated DNA; genetic studies of oncogenesis and vertical virvs transrnissions in the rabbit: mechanisms of suppression of cellular Immunity by carcinogenic hydro- carbons; work on the role of cyclic AMP in arrino=enesi.; exploration of methods for enhancing the immune responses to neoplastie tissves; and evalua- tion of chromosome changes as indicaton of malignant transformation. Chronic Respiratory Distases The lung is a unique orpn. It is a double tdtnetvre with two very aimifar halves in practically constant motion, expanding and contracting in a unison so perfectly balanced that it would appear to defy the laws of physics. We 9 E3
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know that the balance is possihle only becaute of an internal coating by a material, the "•urfactant,° that equalites differcnces in tcnsirn that would othcrwisc result rrorn different dcgrees of distension of the air s:res. In addition, the lung has an enormous internal surfacc of thin memhrane, eqrnivalent in an adult man to about half the area of a tennis cotitt. The entire hkrid supply of the body must he transported at frequent intcrvals to one side of this mcmhrane surfaet, spread over its extent and then carricd awav, whilc air is moved in and out from the opposite side for ahsorption of oxygcn and removal of carbon dioxide and other substances. Thu% the lung is trcmendousty involved in tranaport of materials. Its vertical position in m.in, Mntrasting with the horhontal positnon in most Mher marnmals, alters the effccts of gravity and creates ccrtain pmhlems. Dcsides these mechanical operat'rons. the lung and hronchial surfaccs :trc exposed to a great variety of hacteria, viruaes, dusts, gases, smokes and other extraneous materials that must he killed, removed, dctoxified, inactivated or otherwise disposcd of. The lung has remarkable capacities for these function.. Chronic diseases of the lung such as "bronchitis" and "emphysema" are disorden that generally develop gradually and progress over a long period, suggeuing that they have their basis in a gradual loss of c(Ticicncy of one or more of the mechanisms mentioned. Their association with the aging process and a tendency to "run in famiiies" also suggests that some congenital de- ficieney in one or more of the functions described may predispose to relatively early development of these disorders. The pathogenesis of these diseases, that is, the successive stcps in breakdown of normal mechanisms which finally bring them to recognition at the clinical level, has been very obscure until recently and still presents a great challenge to science. They are not by any means new diseases but they have become more prevalent as causes of illncss, incapacitation and death, as the acute fatal infectious diseases of the past have been eliminated, allowing potential vietims to reach a more advanced age. Neither bronchitis nor emphysema occurs naturally in animals in a form that could hitherto he regarded with assurance as duplicating the human disorder~ and thus serve as a reliable model for experimentation. Although "chronic bronchitis" and "emphysema" are separate conditions, each occurring in several distinguishable types, they are often associated in the same individual, which complicates the problems of etiology and pathogenesis. Chronie bronchitis is necessarily defined in clinical rather than in path- ological terms, chiefly because uncomplicated bronchitis is seldom seen by pathologists in the normal course of events. The disease, or syndrome, is characterized by recurrent acute infeetions of the lowcr respiratory tract, especially in winter, by mixed flor of viral and bacterial agents, and is generally accompanied by chronic cough and abundant production of mucus. It is thus unlike the acute infectious diseases caused by specific organivns acting in a specific manner and running the familiar type of course to death or recovery. Biopsies have shown over-growth and activity of mucus-secreting cells in bronchitis. Nevertheless, the nature of the basic defect has not really been estahlished, whether exttss mucus, a defect in the germicidal systems, an autoimmune manifestation, or some other factor. Where the disease has become established, inhaled environmental irritants may well aggravate the symptoms. While there Is an impression of long standing that familial pre- dispositions may eaist, the various clinical entities grouped under the term "hronchitis" have not so far been clearly enough distinguished to permit clear scparation of genetic from environmental inftuences. Tobocc.r Smokr lnhaletion Studietr As one approach to the bronchitis problem, The Couneil ha_s maintained long-term studies of the cffeets of many factors, ineluding tobacco srnoke inhalation, on the dispcxal of living bacteria implanted into the lungs of miee. When bacteria are inhaled, a mobilization of white scavenger cells (macro- phages) into the lung is stimulated. These while cells engulf and destroy many tyf,cs of organisms and forei,r,n particles- The studies with mice have shown thnt inhalation of tobacco smoke produces a similar mobilir.ation of macro- phages. Though some of these defense cells are Inactivated by contact with the smoke, the available numben are so gt'eatly inereased that the overali capacity for bacterial destruction is not diminished- Similar studies of macrophages recovered from the lungs of human suhjects after smoking have shown that they maint>"ia a high bactericidal capacity despite some changes in appeannce and in oxygen requirements. These observations do not support the hypothesis that smoking oontributes to development of bronchitis by deprrssion of the macrophage defense against infection. Emphysema is more definable than bronehltis In physiological or path- ological terms. Basically, it is an increase in the size of the ,it sacs (alveoli) beyond the terminal bronchioles (air ducts) due to lots of elasticity or actual destruction of the alveolar wa11s. Obstruction of the terminal bronchioles may contribute to these processes. Emphysema can either be diffuse throughout the lung or located in "islandi" distributed through normal tissue. It tends to be progressive, with increasing hvperintiation, airway obstnxtion, and residual lung volume, defective mixing of gases, and eventual breathlessness and oxygen starvation. The tissue destruction, once it has oecurred, cannot be repaired. Emphysema can be diagnosed anatomically, with measurement of the type and extcnt of destruction, by postmortem examination of lungs suitably inflated and fixed. Clinical diagnosis has been more diffieult so that death ceriificates in the past have made poor distinction between bronchitis and emphysema and alsn have provided rather generally unreliable classYicat'ans of either disorder as to type, Creatty improved methods of ineuurint the mechanical functions of the lung and more sophisticated radiological techniques tire now being employed increasingly. F,nsyme•a That Deatroy LunR Tittane Because damage or destruction of the elastic strudtaai substances of the lung is a basic feature of emphysema, it has long been surmised that enzymes capable of breaking down these substances, psrtieularty elastin and eollagen, might be involved. This concept has been supported by experiments In which the injection of various "forrign" proteolytic enzymes into antmals was fol- lowed by lung destruction more or less resembling human etnphyxm.. At present a great deal of study is being concentrated npon the aeatth for enzymes capable, of destroying lung tissue that might be present in the lunt under real life eonditions. The lung itselt produces proteases, particularly 10 11
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during its embryonic stages, and the nature and functions of these are being invettigated under a new Council grant. Moreover, the macrophaRes and tmall lymphocytes, which play a major role in defente of the lung aFainst infectious organitms, produce and utilize proteotytic enzymes in the prncets. Proteaset, released by disintegration of these defensive white cells or leakage of enzymes from them, can themselves damage the lung. Normally th'rs is prevented by enzyme inhibitors such as the a/pha-I-antitrypsin that circulates in the blood. The emerging picture is one of a delicate balance hetween proteolytic and anti-proaeolytic activities in the lung, necessary for its defente against infection but capable of damaging its structure if the balance is disturtxd either by over-production of proteases or under-production of inhibitors. A number of the studies currently being sponsored by The Council, as will be evident from the listing of current projects, are concerned with lung proteaus and their inhibitors as possibly contributory to emphysema. There has been considerable publicity over the discovery that one such enzyme inhibitor, alpha-l-antitrypsin, is congenitally deficient in some persons and that such persoos may be especially susceptible to emphysema. Though the situation has been complicated by the discovery of several different forms of antitrypsin with differences in activity, it nevcrtheless appears to he firmly esublished that a hereditary predisposition does exist. One Council-sponsored project is studying the possible relations of the several genotypes, as determined by blood assay, to responses to environmental stresses in a large human population. This study has also produced evidence of familial predispositions other than those attributable to antitrypsin deficiency. Another hypothesis, not necessarily in conflict with the proteaue-anti- prolcase concept, views lung tissue destruction as due to changes in its anti- genicity that invite ttack by the body's own "foreign tissue" rejection mechanisms. Such changes conceivably could be produced by relatively slight chemical alterations of the lung structural substsnces by inhaled pollutants. Cotmcil-sponsored studies based on this hypothesis continue to produce sug- gestive results. As implied by our comments on the function of lung surfactant, a de- ficiency or maldistribution of this agent would be expected to produce significant malfunct'ans of the lung. A Council-supported investigator suceceded in adapting certain physiological lung function measurements for application to small animals, including living mice. This promises to be useful In combination with postmortem lung ex- aminations tor studying sgarelated changes in mice of different strains, in the search for a better emphysema model. Such function messuremenh may also become a useful tool In determining whether effects of various inhaled agents, including tobacco snake, are tempotary nd reversible or persist and progress to the stage of postmortem observation. Besides the numerow fumctions mentioned above, the lung also has metabolic activities such as trnwvint from the blood stream a number of hormones that are active In the mediation of inftammation, in the regulation of blood pressure and in mobilizatioo of white blood cells. Two Council- spoosorrd studies are exploring these sub}ects. Seventeen projects in the field o( chronic pulmonary disease were active in 1973. Two of these terminatcd during the year nd four new ones were approved for activation earty in 1974. Cardiovascular Discascs Notabie protrets was made during the year, under Council spo~hip and independently by other investigaton, in the development of immuno- chemical anays for nicotine and its major metabolites. These show promise of providing a npid, precise and highly speciflc method for a_ssay of nicotine and related compounds in body fluids, especially blood plasma. Such methods, when perfected, should make possible the studies of nicotine pharmscodynamics that have long been needed for solution of many important problems. Among these are the kinetics of nicotine absorption from the various tobacco products under normal conditions of human use (cigarettes, pipes, cigan, snuR, chewing tobacco), the peak levels to which nicotine rises In btood, rates of inetabolism and excretion, etc. The effects on these kinetics of genetic differences, of habituation, of taking common household medicines, of tempenture, exercise, and many other factors should then become measurable. Application of such measurements to human populations could add new dimensions to the interpretation of epidemiological atudies of cardiovascuiar disease in relation to various forms of tobacco use by showing whether or not nicotine can be involved. Conferences with scientists from a number of countries are further ex- ploring the feasibility of international twin studies to test more npidly and decisively the implications of Swedish researches on identical twins with dis- cordant smoking habits. These suggest that genetic factors are predominsnt over environmental ones in the genesis of cardiovascular diseases. Assistance to the Swedish study continued meanwhile. Aside from epidemiological studies bearing upon cardiovascular diseax, thirteen experimental or clinical studies were supported during the year. These proceeded a)ong the lines described in the 1972 Report, with a yield of many publications that aro abstracted elsewhere. Two of these studies have now terminated. A new project was approved for study of possible effects of tobacco smoke inhalation and of nicotine upon development of collateral coronary blood flow following acute or chronic occlusion of a coronary artery in dogs. Pharmacology and Psychopharmacology This phase of The Couneil'a program continued along the 11nes described in last year's Report. Current emphasis is being placed upon aherations in nicotine pharmacology that result from chronic exposures through habituation, adaptation or enzyme induction. Other pharmacological problems await further development of the new analytical techniqtxs to provide precise infortnation on the concentrations of nicotine and of Its various metabolites present at particular times in speciflc tissues and at specific sRes, Studies of nicotine effects on animal behavior ate presently receiving little emphasis, not because there is any dearth of academically interestint probfcros that could be approached by existing techniques, but rather becatrse the new analytical methods mentioned as being in reasotnbte prospeet should Increase the sophistication of any future studies in this area. In addition, there aze the 12 13
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iml+ortam prohlcros of whether and to what extcnt the impiicnt;ons of rat- IearninR and behavioral studics can be translerred to man and thcn o( evaluatinl, thc r practical eignificance in human life. One new animal behavior study of unique character is, hnwever, heinFy inaugurated currently. The investigator has shown that a rat behavior Icarned under the in0ucnce of a particular drug X can he made comingent upon presence of the drug state. Such beMvinral patterns can then he usrd as teats to determine whether the rats (ub!ecthvcly perceive nther drups as X, as non-X, or as rcinforccrs <x antagonists of X. 'ihe eRects of nicotine and some of its analogs and mctaholitea will be studied -in this contcxt. A human behavioral study, rceenttv hegun, will explore the effects (i( smokinR deprivation on group problem solving proceases in terms of speed and accuracy. Epidemiology Including con(erence% on potentials for international twin studies, six epidemiological investigations were active during 1973. These on-going projeets, described in previous Rcports, are producing a large number of significant and relevant findings on the constitutional and behavioral differences at many different levels among persons who have placed themselves by se!f-setection into the several categories of smoking behavior. As the numerous reports from these etudies reach publication they are abstracted in the Annual Reports. Several, as well as many by-product papers, are included in this issue. RonrRr C. Ff(x'tcrrT, Ptt.i). Rcsearch I)irector Abstracts of Reports Folltewing are ;ehatrncts, apprc.vcd by the atrthon, of rcport% on new re- %carch ocknr.wic Iging atr,lxert frorn The Couneil that have appeared in seien- ti/ic jrnrrnali, %incc ruhtication of the 1972 Report- The name of the recipient is in itatic%, 'I hc ah.tr:ects :erc rroupcd uncler thete headingt: 1. Studirs Related tn ('anccr, It, Ite:ert :rnd ('ircularinn, 111. The Respiratory SyNem, 1V, Pharma- cningv and 1'.yrheqrh:trmacnlogy, V. Imnttrnotogy, Vt, Epidcminlop,y, VI1. The Normative Aging titudy, and V111. Miaecllaneous. 1. ,Strr(lies Refaterl to Canrer Mf'TA)lOL)(' (-ONVf'RS1ON OF BF.N7-O(y)PYRENE (tY SYRIAN HAMSTf.R LIVF.R MI(-ROSOMFS AND IIINDING OF METABOLITES TO DEOXYRIf1ONUCLEIC- ACID f)inding studies reported here show that epoxides, phenolt, and other mct:tho!itcc td hcnzolilpyrenc (BP) formed in hamster liver microu+me systems may not just hc end products, but may he further metaholized to bind to DNA. Specifica!)y, analysis of inetahotites of generally tritiated benzo[:)pyrene ('){.Rp) produced by a Syrian hamster liver mierosomal system has revealed the presence of a number of dihydrodihydroxy detivatives of DP including 4,5-dihydroxy-4,5-dihydrobenzo(a)pyrene. Detection of this metabolite is taken as evidence that the K-region 4,5-double bond is acted upon by a mierosomal hydroxyfase to form the 4,5-epoxide of IiP which is subsequently converted vio epoxide hydrase to the dihydrodio)- Incubation of severa! `H-F1P metabolites with DNA alone gave !ittle evidence for spontaneotn covalent binding. How- ever, when hamster liver microsomes were present, a metabolite recently identified as 7,R-dihydroxy-7,R-dihydrobenzo(e)pyrene was found to covatent!y hind to DNA to a tenfold greater extent than ©P itself, suggesting that this compound may hc an intermediate in the pathway leading to binding of BP to DNA in vivo. Borgen, A., Darvey, H., Castagnoli. N., Crneker, T. T., Rasmussen, R. E. and Wang, 1. Y. lnnrnol of Mrdicinol Chrndsrry 16(5):502-506, 1973. Otlrer arrptxrrr: U. S. Puhlic Health Serviee, and the Cancer Research Funds of the University of California. From the Cancer Research Institute and Department of Pharmaceutical Chem- istry, University of ('alifornia, San Francisco. INDUCTION OF ARYL HYDROCARBON HYDROXYf.ASE IN f1UMAN SKIN The aryl hydrocarbon hydroxytau which hydroxylates henrofTlpyrene (fIP) has been shown to he markedly induced in animala expoeed to polycyelie 14 15
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hvdronrbons such as 1.mcthytcholanthrene or BP. In the prccent study, tiaue culture wis utrd to determine whether the carcinop,cn-merahotirinR enzyme. RP hydroxylace, is present in neonatal human forckin, and whether it is inducib!c in this tissue. The resultant data show that human ckin has detectahte fevels of DP hydroxylase, and that this enzyme is inducihie; moreover, sub.tantiat diRer- eneet in basal and inducible levels of the enzyme were dcmnnctrated. This difference may he genetically determined. No correlation wa% found between the inducibility of hydroxylase activity in the neonatal foreskin and the race or age of the mother, or the medication given to the mother prior to delivery. The enzyme system that hydroxylates BP requires nicotinamide adenine dinueleotide phosphate and molecular oxygen. Expoture of the skin homogenates to nitrogen or CO resulted in little or no enzymatic activities. It was also shown that induci- bility of hydroxylase activity was greater in human skin than in neonatal rodent skin. The presence of hydroxylase in human skin and its ability to he induced may he important protective mechanisms against, earcinngenesis in man, since human skin is often exposed to polycyclic hydrocarbons in the environment. However, the exact role of BP hydroxytz,se in carcinogenesis is still unclear. Alvares, A. P., Leigh, S., Koppa.r, A.. Levin, W. and Conney, A. II. Drug Mr•taboli.tm ond DGporfrfon 1(1) :3R6-390, 1973. Other aupport: U. S. Public Health Service. From Rockefeller University. New York, and the Department of Biochemistry and Drug Metabolism, Noffmann-La Roche Inc., Nutley, N. l. INDUCIBiLITY OF BENZO[aJPYRENE HYDROXYLASE IN ifUMAN SKIN BY POLYCYCLIC HYDROCARBONS Benzo(aJpyrene (BP) hydroxylase activity is inducible by polycyclic hydro- arbom in cultures of neonatal human foreskin. This study shows that the enzyme system which hydroxylates BP requires nicotinamide adenine dinucleo- tide phosphate (NADPH) and molecular oxy=en, and that its optimum pH is 7.4. Exposure to carbon monoxide completely inhibits hydroxyiase activity. When skins were Incubated in a growth medium containing txnr(a/anthracene (DA) two- to flve-fotd inereasea in the arnosnt of BP hydroxyLse were obxrved. Maximum enzyme levels were found when the skin was incubated in the presence of BA for 16 to 24 houn. Tnducibility of hydroxylase activity was greater in htmun skin than in neonatal rodent skin. Comiderabk variability in the buai levets and In the inducibility of SP hydroxylax was observed in foreskins obtained from 13 subjects. These studies indicate that human skin can metabolize polyeyclie hydrocarbons and that the levels of the hydroxylase, is we11 as the inducibility of the enzyme In skin, may provide a uceful means for evaluating individual diffetrnces in the capacities of humans to metabolize environmental earcinotens. Aivares, A. P., KoDpas, A., Levin, W. and Conney, A. H. Clinlcel Pharmacofogy .nd Thervpexrkr 14(1) :30-40, 1973. Other arpport: U. S. Public Health Service. From Rockefeller Univenity, New York, and the Department of Bioe)xmistry and Drug Metabolism. Hoffmann-La Roche Inc., Nutley, N. 7. POLYC-HLORINAT17-D fl/PFiJrVYLC:A NEW TYPE OF INDUCER OF C-YTO('HROME P-44R IN THE LIVER Thic ctudy ~hows that Aroclor 1254, one of the polychtorinated bipheny(s mixtures manufactured in the United States, is a potent inducer of the micro- tomal hemnprofein, cytnchrome P-44R, and of henzo(a)pyrene hydroxylase and eth,vlmorphine N-demethytase. The CO-difference spectrum of microsomes from rats treated with Aroclor 1254 has an absorption maximum at 44R nm. With erhytisocyanide as the IiRand for reduced microsomes, Aroelor 1254 treatment causes a shilt in the 455-nm peak to 453 nm and increases the ratio of absorb- ance of 455 nm to that at 430 nm from 0.53, obtained with untreated rats, to 1.24. These findings are similar to those seen in nts treated with the polycyclic hydrocarbon, 1-methylcholanthrene, hut differ from those that characteriu cytochrome P-450 in control or phenobarbital-treated rats. Aroclor 1254 trtat- ment results in a tripling of cytochrome P-44R content and a 10-lold inerease in benzo(aJpyrene hydroxylatitm. However, it causes a signifiant enhancement of ethylmorphine N-demethylase. These data suggest that Arocior 1254-induced cytochrome P-44R may be catalyticatly different from the 3-methylchoianthrene- induced P-44R or that the hemoprotein(s) induced by Aroclor 1254 may be a mixture of cytochromes P-44R and P-450 exhibiting catalytic properties of both cytochromes. Alvares, A. P., Bickers, D. R. and Kappa.t, A. Prnrrcdrnps of rAr National Academy of Sclrnces of the United States of .fmnica 70(5):1321-1325. 1973. h Other aapport: Hoffman-La Roche Tnc., U. S. Public Health Ser.iee, Na- tional Institutes of Heatth, and Scaife Family Charitable Trusts. From The Rockefeller Univen'rty, and the Department of Medicine, Cornell University Medical College. New York. EVIDENCE OF A C[•.NETiC RELATIONSIIIP DET1'VEEN SUSCETTIDiL- ITY TO 3-METIIYLCFiOi-ANTfIRENE-INDUCED SUBCUTANEOUS TUMORS AND tNDUCIBILITY OF ARYL HYDROCARDON I IYDROXYLASE The presence (or inducibility) of aryl hydrocarbon hydroxylase (AHH) is known to be associated with the cytotoxic action of polycyclic aromatic hydrocarbons. In this study, the authors report a close correlation between susceptibility to 3-methylcholanthrene (3MC)•induced subcutaneous tumors and the genetically mediated presence of AHH Induction. In a genetic system where AHH inducibility segregated as a single autosomal dominant gene, mice carrying the Ah" (or inducible) allele were 5-IO times more sensitive to MCA tumorigenesis than were their noninducible (Ahd/Ahf) littermates. In baekcross animals ((C57DL/6XDSA/2)XDBA/2). 10 of 12 (R3%) mice with tumon were AHH inducible. This was statistially signiflant (P <0.025) (the expected rrsponse was 50°k ) and clearly indicated a relationship between this enzyme system and MCA earcinogenesis. Kouri, R. E., Ratrie, H. and Whitmire, C. E. (M(croblofodical A.rsocfatrt) 16 17

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