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19 76 RI:11 0 RT oJ 'I'H1: COUN(:IL FOR TOBACCO RESEARCtI-U.S.A., Inc. Ti1"r. COUNCIL F'OH TOIiAf:(:O IitaF:ARCaI-U.S.A., Inc. 110 Enat 59111 Strret, Neh York, N.Y. 10022

'IT ^~' / y Y SCIENTIFIC ADVISORY ItOAKi) fIANS MEIER, D.V.M., Dr. Mcd. Vct., M.R.S.H. 00 ti..J Senior Staff Scientist The Jackson Laboratory ~ to Thc Council for Tobacco Rcscarch-U.S.A., Inc. as of Dcccmbcr 31, 1976 SHELDON C. SOMMERS, M.D., Chairman Bar Harbor, Mainc LEE W. WAT'I'ENI3ERG, M.D. ProJrssou of l'uthology i... .. .1i Director of Laboratories Lenox Hill Hospital Dcpartmcnt of L. boratory Medicine and Pathology .~... , Clinical Professor of Pathology University of Minnesota Medical School ~ Colicgc of Physicians & Surgeons of Columbia University Minneapolis, Minnesota .~ Ncw York, New York JOIIN P. WYA'IT, M.D. ~ RICHARD M. BING, M.D. Director of Cardiology and Intramural Mcdicint Huntington Mcoiorial Hospital, Pasadcna, Cafifornia Professor of Medicine University of Southern California School of Medicine Director Tobacco and Health Research Institute University of Kentucky Lexington, Kentucky F-- U U Los Angcles, California JOSEPH D. FELDMAN, M.D. Kead, Department of Immunopathology Scri ps Clinic and Rcxarch Foundation La Polla, California WILLIAM U. GARDNER, N.D. Scientific Director, The Council for Tobacco Research-U.S.A., Inc. E. K. Hunt Professor of Anatomy (emeritus) Yale University School of Medicine New Havcn, Connecticut ROBERT J. HUEBNER, M.D. Chief, Laboratory of RNA Tumor Viruses National Cancer Institute Bcthcsda, Maryland LEON O. JACOBSON, M.D. Director, The Franklin McLean Mcmorial Research Institutc Regen.stein Professor of Biological Sciences Univcrsity of Chicago Chicago, Iliinoia AVERILL A. LIEBOW, M.D. Professor of Pathology (emeritus) University of California School of Medicine San Dicgo, California HENRY T. LYNCH, M.D. Pro/essor and Cltairman Department of Preventive Medicine and Public Health Creighton Univeriity School of Medicine Omaha, Ncbraska Scienti5c Staff of The C.ouncit WILLIAM U. GARDNER, N.D. Scientific Director ROBERT C. HOCKETT, N.D. Research Director JOHN I:. KREISIIER, N.D. VINCENT F. LISANTI, D.M.D. Associate Research Director Associate Research Director DAVID STONE, N.D. Associate Research Director I _~

CON"Y'F.hi :'S Studics Rclatcd to Cardiovascular Uiscascs a;id Functiun .... 5 Abstracts of Reports . . . . . . . . . . . . . . ... 14 Canccr-Rclatcd Studics . . . . . . . . . . . . t •1 Tiic Rcspiratory System . . . . . . . . . . . . 28 i{cart and Circulation . . . . . ... . . . . . . 41 Ncuropha macology and Physiology . . . . . . . . 54 Immunology and Adaptive Mechanisms . . . . . . . 61 Epidemiology . . . . . . . . . . . . . . . 64 Activc Pro;ccts . . . . . . . . . . . . • . . . . 67 Completcd Projects . . . . . . ... . . . . . . . . 75 lndex of Principai Invcstigators . . . . . . . . . . . . 85 Index of Scnior Authors . . . . . . . . . . . . . . 86 I Studies Related to Cardiovascular Diseases and Function cr 252 F-- Prcvious annual reports havc preseMed the general plan and rationale of Council-sponsored studies of carcinogenesis and the etiology of chronic pul- monary Jisorcicrs. Their purpose was to provide a framework in which the rclevancc of individual contributions would he more easily apparent. in the prescnt issue, we describe similarly some of our approaches to study of cardiovascular diseases and function. Cardiovascular Diseases Among thc many disorders of the cardiovascular system, those derivin; from progressive atherosclerosis rank first as causes of disability and death in the United States. These include heart attacks (myocardial infaretion), angina, stroke, arterial blockages in the limbs, and some cases of congestive heart failure. Hypcrtcnsion is believed not only to accekrate atherosclerosis, but also to precipitate acute events in damaged circulatory systems. Atherosclerosis is, therefore, a prirxipal focus of the present discussion. Epidemiology of A therosclerosia-Rela(ecl Diseases Numerous epidemiological ttudies during the last 20 years have sought possible "causci' (primary or contributory) of Ihe atherosclerosis-rclated dis- eases. These studies often summarized lheir Andings in terms of "Yisk facton." The -risk factor" is essentially a statistical concept based upon mathematical relationships without necessarily any known or established mechanism by which it might contribute to etiology. Among the many such -risk factors" frequently reported are hypertension, elevated scrum cholcsterol, diabetes or a prcdiabetie diathesis. cigarette smoking, personality type, inadequate physical activity, and emotional stress. Genetic predisposition (e.g., hyperlipopro(einemia, hornocysteinemia) is another such factor, both in its own right and as a contributor to rtwst or possibly all of the others. Biochemists. physiologista, pharmacologlsts, psychologists, and other scientists have thus been challenged to All the gaps and learn whether, how, to what cxtent and in what kinds of persons each factor might actually be operative. The stimulus to conjecture, speculation and hypothesis has been strong, and many scientists have apparently aeizcd the occasion to teat their personal hunches in the hope of a lucky strike. This was entirely legitimate, but has tended to produce a welter of (ragmentary, confusing and inconclusive observations subjected to speculative projections. ClarifScation may come as coherent, integrative theories emerge that can accommodate all the flndints that prove valid. 5

S CiFarclle Smoking as a "Risk Faclor" The identification of a "risk factor" can be no morc rcliahle than the statistics on which it is based. Smokers select themselves from among thc general population on a basis or bascs that arc litrJc undcrstcxxl. Is it Icl;itim:rtc to compare thcm with nonsmoker cuntrols as aninial experimenters comparc their test animals with gcnctically identical lincr-matcs? Tobacco use has many variations, both qualitative and quantitative. To what extent does voluntary adoption of any particular behavioral pattern select out a sub-population with innate charactcriNtics or life-stylcs that arc linked to cardiovascular disease susceptibility? There arc many evidences from studies of man that genetic prcdisposition plays an important part in determining which individuals arc potential victims of atherosclerosis-related diseases. In several types of rather extreme suscepti- bihty, the patterns of inheritance have been well establishcd. Revicw of the "risk (actori" listed above indicates that most of them are already known to be influcnced, it not determined. by hcrcdity. Studies of spontaooous alcohol eonsumption by inbred mice have shown substantial strain differences in appetite, in behavioral responscs and in meta- bolism. Analogous mouse studies of spontaneous nicotine and tobacco smoke intake have now been inaugurated by a Council grant to learn whether similar genetic variations occur. Ttoin Studies Identical twins provide the human counterpart of animals of inbred strain. Studies of all like-sexed twins in Sweden and Finland arc being assisted by The Council in an effort to determine broadly the relative influences of heredity versus environment upon the incidence of atheroscicrosis-bascd cardiovascular diseases. A key strategy is to comparc the incidence of such disordcrs in smoking and nonsmoking identical twins, including symptomatology during life and, eventually, age at dea/h, the primary cause of decease and post-mortcm cvalua- (ion of vascular pathology. Non-identical, li'r:c-sexed twin siblings serve as contrrols. While the incidence of discordance in smoking practices is low among idcntiul twina, an observation that in itself evidenccs a atrong role of hcrcdi- tary influences (even among twins reared apart) in determining initiation and maintenance of amoking, the numbers of discordants are great enough to accrue to increasingly significant levels in substantial twin populations followed over a sufficient period of time. Theae studies have now been extended beyond identical twins to other rclatives of various defined degrees of consanguinity, such as half-sibs and the offspring of twins. Theae tinsetonsuming 'investiptions will require a considerable induction period before comprehensive new reporta emcrge. They may eventually provide more direct evidence whcther dprette smoking per rr is a truly significant "risk factor" in theae and perhaps aorrse o<her diseases. They cannot, however, be expected to add greatly to elucidation of pathogenic proccsses or to provide mcthMl% uscful in reducing risks due lo biochemical abcrrations impartcd by thr gcnes from anccsuors. For this, other types of rescarch are requircd. Clroleslerol 31 r•laLolism Anuthcr prevalcnt "rhk factor" in cpidcmiological studics has been "cle- vated scrum cholesterol." Contradictory findings over the years suggest thal this conccpt was an ovcrsimplification, especially since many victims of heart attacks had no hictory of high cholcstcrol levels. That cholesterol is implicateG in athcrosclcrosis has been assumed because mature arterial plaques eontair the steroid both free and in combinations. Nevertheless, long-tcrm adminiss tration of drugs that lower serum cholesterol has rtot been as generally eflective in arresting or reversing the process in man as had been hoped. Accumulating information about the siates of combination of cholesterol in blood is directing attention to the several eholestcrol-eontaining combina- tions, classified as chylomicrons. very low density lipoprotcins (VLDL), low density lipoprotcins (LDL) and high density lipoproleins (HDL). The LDLs are the major carriers of blood cholesterol and there arc a number of txperi- mcntai as well as epidemiological findings that an elevated serum kvel of VLDL is correlated with progression of atherosclerosis, whereas elevated HDL is a favorable indication. Thus certain "lipoprotein profiles" are now considered by many to be bct(cr indicators of susceptibility to atherosclerosis than elevated total chol- estcrol. Certain of these profiles, associated with very high susceptibility to ' discasc, have been shown to be genetically determined. Barring some unforeseeable empirical discovery (which does occur in medical research and is a perennial hope), the rational route toward etTective control is through systematic study of the pathogenesis of atherosclerosis, at biochemical and physiological levels. This obviously must include a better understanding of thc regulation of synthcsis, transport, function, and climin- atinn of cholesterol and of the aberrations in disease in the hope of altering thesc by targeted treatment. The Council is presently reviewing the most promising eorscepts and leads that now exist in this complex tkld with the intent of increasing its support of basic study of athercroscicrosis. The intent is not only to assist these im- portant developments, but also to seek more directly relevant assay systems for assessing the posaible effects of cigarette snsoke inhalation. The task is additionally difficult because the athcrosckrrolic disorders appear to he peculiarly human ones with few counterparts among animals under natural conditions. Highly contrived manipulations to produce animal "counterparti' tend to diminish probability of relevance to human experience. Human studies have been impeded by the lack of non-interventioo tech- niqrxs for assessinj initiation and progression of the atherotenesis processes in the vasculature of man. New experitnrntal techniques for viwaGrLs= the condition of the arterial walls, including scintillation photography, appear to show promise forthe future. 6 7

In Vitro Studies of Arteries and Veins Meanwhile, in virro techniques providc one method of studying synthcais and uptake of lipids and cholesterol by human arteries and vcins, txxh "normal" and atherosclerotic, in direct comparison with those of animals. Though in 'virro conditions cannot be made to duplicate thc in vir•o situation pcrfectly, they may provide useful guidcs to in vivo research. Such studies have already been made with CCTR support as describcd in the Council's Annual Reports for 1972-1975. It has been reported (and is reitcrated in a current review) that human atherosclcrotic and normal coronary arteries as well as saphenous veins, pcrfuscd under pulsatile artcrial pressures with human plasma containing labelcd cholestcrol and acctalc, do not synthe- size cholesterol from acctate and produce only small amounts of cholcstcrol estcrs. Cholesterol is taken up in identical amounts by normal and discasui vessels and this uptake is incrcased at higher pulsc pressures, which secros conaistant with the reputed efiects of hypertension. l.ahclcd acctate is incor- poratcd into various lipids. IIoth normal and discascd coronary artcrics and saphenous veins synthesize free fatty acids, triglycerides and phospholipids. Addition of nicotine to the pcrfusion fluid did not influcncc cholesterol uptake. Analogous experiments with dog arteries appeared to shown an in- flucnce of nicotine upon cholesterol uptake. If this is confirmed, it suggests caution in extrapolations from this species to man (1972 report). Carbon monoxide in the perfused plasma was reported to enhance chol- esterol uptake by all arteries in this In vlrro system. Several new studies report a dramatic inhibition of cholesterol uptake by both human and animal arteries, under these in vitro conditions, by 7-keto- etwlesteroi. Uving rabbits also showed an inhibition of cholesterol uptake, hut to a much smaller extent. The low solubility of the 7-keto compound imposed technical problems. Improved techniques may increase the cfficicncy of the effect. Meanwhile, the disparity between the in vitro and whole animal rc- sponses is anothcr reminder that extrapolations must be cautious. Other current studies deal with elucidation of possible mechanisms of the inhibition and with the metabolic fale of the injected 7-keto compound in rabbits. Role of Smooth irfuacle Cells in Atheroaclerotic Plaques The heretofore prevalent theory of atlerosclerotic plaque formation postu- lated that the indltration of fatty substances from the bloodstream into the arterial wall giva rise to cholesterol deposits that act as an irritant, causing inftammatioa and proliferation of cells by processes akin to ordinary healing. This "irnudation" ooneept was eonsistent with the associationy of atherosclcr- osis with elevated blood cholesterol, high blood pressure and high fat diets. Animals led Iargo amoutfts of saturated fats and chokstcrol, sometimes sup- pienrcnted with hoctnorsal or other ttcatments, developedi lesions superficially resembling those observed in man at autopsy. These lesions often regressed in animals when the dieta were ailercd, an observation that stimulated human dietary uudia invoiving restriction of cholesterol and saturated (at consump- 8 tion. Complicatcd by di(Gcultics of control, the results in man have been equivocal. Rcccnt clcctron microwopic studics of human arterial plaques have re- vcalcd that the major component of authentic plaqucs i% proliferating smooth muscle cells like thusc normally compoting Ihc center (mcdial) layer of arter- ial walls rathcr than fihrobiast ccll. such as proliferate to heal a wound. Early hurnan artcrial Icsions (strcaks) contain littlc lipid, suggesting that insudation ie not the prime factor in typical atherosclerosis. Cholesicrol deposits and cellular debris appear latcr.-Present debate centers on what incitcs normal smooth muscle celis to migrate from the medial layer and what causes them to prolifcrate. Presumahly, damage to the inner surface cell layer of the vessels is implicated and a number of theories as to how thi% endothclial layer may bccomc damagcd have txen advanced. Many of 1he plaques generated in animal arteries by non-physiological manipulations arc reported to he radically different in composition from those o( genuine human athcnosclcrosis and their relevancc to the human diseases is therefore questionahle. Recognition of the diRercnce has, however, led to reports that ccrtain animals can develop human-type atherosclerosis under othcr more appropriate conditions. Smooth muscle cella from primate arteries arc now being maintained successfully in culture media. Low-dcnsity Iipoprotcins added to the media stimulate them to multiply. Further, it has been reported that thc ever-present blood platclcts, normally involved in the clotting process in response to injury and in other functions, also secrcte a substance that strongly stimulates pro- liferation of thcw smooth muscle cells. Damage to endahclial and intimal ccll layers of an artery, which ordinarily separate the blood from contact with the smooth muscle cells of the medial layer, can bring these cells into contact with platclets and presumably incite multiplication in the artery wall. Another concept holds that the smooth muscle cells multiply as the consequence of a mutation akin to that which transforms other normal cells into malignant ones. This "monoclonal" theory, which is supported by striking evidence but is ncvcrthcless in some dispute. would suggest quite different mechanisms of action by external agents than those describcd heretofore. Endothelial Cells and Blood Platelets A single layer of endothelial cells composes the thin membrane lining the inner surfaces of arteries, performing an important function in retaining the red corpuscles while allowing water and some other substances to pass through. As mentioned, damage to the endothellum is believed to contribute to all three of the mechanisms of atherosclerosis described. Many possible causes of cndothelial damage have been suggested and are being studied. a is possible tSat several may be Involved. The damaged endothclium can repair itself, rather slowly, but it damage is sustained or repeated too often, repair may not be achieved. Clots (thrombi) forming in an artery are thought to injure the endo- thclium by pressure and by impeding the access of oxygen. The blood platelets have long bcen• known to play a role in the complex events that produce 9 .y ~.

!?0 thrombosis in both arteries and veins, and may thus contribute indirectly to cndothelial injury. At the same time, it has been thought likely that the plate- Icts may contribute in some way to maintaining integrity of the endothclium. Recently developcd techniques for growing human endothclial cclls in culture media, after harvest from umbilicrl cords, have madc it possible to study (acton that influence their replication. A Council-sponsored investigator has reported that platelcts added to the medium, as wcll as some individual substances secreted by the platclcts, will stimulate their growth. It is, thcreforc, suggested (hat in vivo, platelets may expcditc endothclial repair. 11c has also isolated from normal blood substances that inhibit platelet adhesion AMI thus impede the processes of thrombus formation. The implication is that a dClicatr balance among opposing functions maintains integrity undcr normal conditions. Y:xtcnsion of such studics should help dclincatc thc mechanisms of throm- bosis and atherosclerosis. Another investigator has used in virrrr techniques to culture rat heart muscle and epithelioid cells to determine the effects of cholestcrol, cholestcrol- esten and fl-lipoprotein in producing cellular lipid inclusions and in lahiliting lysosomes or mitochondria. Lecithin: Cholesterol.lcyl Trans/eraae (LCiiT) Lecithin: cholesterol acyl transferase is an enzyme thought to be respon- sible (or the esterification of lipoprotein cholesterol in plasma. It is postulated by several investigators to be important in the mechanisms that remove chol- esterol from the arterial wall. A number of previous papers, abstracted in these Annual Reports- have reported clinical studies of LCAT activity in the plasma of animals and man under different circumstanccs to elucidate its clinical significance. Instability of the enzyme complicated these studies. Experimentation to delineate the enzyme's modes of action required its isolation, purification and stabilization so that enzyme concentrations could be controlled and substrate compositions better deflned. A recent publication reports that a concentrated etfort has achieved the isolation of LCAT in substantial quantity in a virtually pure slate with greatly improved stability. DeveSopment of a radioimmunoassay is reported to be under way. Two publications on applied studies of LCAT have also appeared recently. Availability of the puriBed enzyme and of its radioimmunosssay should stimulate and facilitate studies of cholesterol metabolism and the rolcs of the several lipoproteins and contribute to understanding of atherosclerosis. MetabolicActivities oj Pulraonary Endothelial Cells Five years of research assistod by The Council have produced numerous reports on this subject, Including aevert.l published during 1976. It has been shown that the vasoactive polypeptldes bradykinin and angiotensin I arc meta- bolically altered during a single passage through the vast pulmonary vascular bed. Bradykinin, a substance that tends to lower blood pressure, is completely C C inactivated whilc angiotcnsin I is convcrtcd to angiotcnsin It, which is a potent G - ~ hypertensive agent. f3radykinin, under suitable conditions, can inhibit this angiotcnsin conversion. Thcse relationships suggest a role of the pulmonary circulation in blood pressure regulation. ti..J ~ These mctabolic changes have been traced to peptide hydrolase enzymes of the lung. which are not present in the bloodstream but arc attached to the luminal surfaces of cndothelial cells, especially ahcne of capillaries and vcnulcs. '17ie same cncynu inactivates bradykinin and converls angiotcnsin 1. Stcps in thc hydrolytic process have been dcscrihcd in considerable dctail; antibodies ~ 'r.. J... to thc cncynu wcre prepared and Ialxlcd to provide tools for research. ~ New tunctions uf bradykinin are now being discovered, including an efTect l i ~ on prusteg and n synthclasc which remains to be explored in depth. It also ~ remains for future studies to inquire how dys(unctions of these systems may be related to vascular or pulmonary diseases. ~ S...J Chronic Smoke Inhalation by Dogs A technique for chronic exposure of beagle dogs to cigarette smoke via trachcostomy was employed by a Councii-supponed scientist to look for poui- blc u3ects on clotting mechanisms and on cardiovascular function. Despite recognized limitations of this technique as a model of human practice, rather extensive experience with its use for other purposes suggested that extension of observations to the cardiovascular system might,, provide some preliminary data and guidelines for future studies. No clinically evident disease was found after 18 months' exposure under different dietary regimes. Some indications of possible enhancement of eoagu- lation mechanisms and of relatively minor altcrations in (unction were re- ported. The author described these minutely with cautions against transfer to man in view of experimental limitations, specks differences, and the greater complcxity of human environments. Studies Involving Nicotine A number of past Council-sponsored studies, mainly using animals and with a varicty of objectives, have involved the administrat'an of nicotine. Experience through the years suggests that comments on some of the problems involved in selection of dosages are pertinent for evaluation of past results and for designing new and better experiments. Smokers receive nicotine by way of the oral cavity and the lung, in small successive doses over a period of several minutes and repeated at variable intervals. The entire dosagc range they experience is very bw in contrast to the levels often used by pharmacologiats in exploratory studies of nicotinei pharmacological potentials. The view has been espressed that ordinary smok- ing rarely, it ever, produccs high enough levels to act upon the sympathetic gangtia, but stimulates only the several special aenaory receptors to iocite rapid but brief systemic responses of reflex origin. Thae responses may be quite contradictory and paradoxical in nature, depending on the conditions that predominate at any moment. 10 I1

Yet. within the low range of dosages that can he achieved from smoking, thcre is still very considerable variation. Rate of absorption via oral cavity or Iung is highly dcpcndent upon the acid-hasc balance in the smoke. Maintenance of blood level is strongly aGccted by the hydrogen-ion conccntration of the hladder contents. The degree of acidity influences nicotine resorption and is altered by dict and nervous state. Nicotinc metabolism is rapid and metabolic rates not only vary markedly among normal humans but are probably in- flucnced by duration of smoking expericncc, a recognized but little undcrstood phenomenon. In addition arc the variations due to choice of tobacco product used and individual dif[crences in pufi volumc, puff frequency, depth of inhalatiai, duration of smoke retention, etc. Design of animal experiments intended to mimic human smoking cx- pcrience, long or short, must include consideration of all the factors cnumcr- atcd and species differences as well. Experimental control or standardization of all the variables mentioned in any large-scale or long-continued human study appears to be virtually im- possible. However. an expansion of information on the actual ranges or dura- tions of plasma nicotine kvcls attained by human smokers (and uscrs of other forms of tobacco) under actual conditions of life should be attainable. Recent studies by laborious methods have provided such data for small numbers of human subjects. These are a most valuable interim guide to the design of animal experiments. However, mass data on largc representative populations are ncrdcd to define, on a sound statistical basis, the ranges, peaks antl mcdiane of plasma nicotine on a time scale, if important refinements are to be achieved in epidemiological studies of smoking. Radioimmunological Assay of Nicotine Sensitive, specific and rapid assays for plasma nicotine and its ma}or metabolitcs have long been needed. They should be able to he carried out with very small samples of blood, with simple. inexpensive and mobile cquipment, and by competent technicians without extraordinary special traininb, have long been needed for such purposes. Radioimmunoassays would appear to have promise of meeting these re- quirements and The Council is continuing to support studies in this arca. Antibodies sensitive to nicotine have been obtained and reported (1975). Continuing efforts are now directed toward improving scnsitivity. tcstin,t for specificity and simplifying procedures for broad application. Nicotine E,(Jrcts on Coronary Circulation in Conscious Dogs To avoid the effects of anesthetics on the pharmacological responses to nicotine. a method was pcrfected for intracarotid administration of the alka- loid to conacious dop in doscs described by the investigator as "realistic." A striking increax in coronary blood flow was observed which was traced to two separate (acton. The major component was found to be due indirectly to 12 an increase in thc depth of respiration produced by nicotine. the minor one to a chcmorcflcx via a diffcrcnt pathway. EPII)EAf1OLOGIC STUDY OF SMOKING CESSATION A gcncral difl'iculty in making and interpreting studies of smoking eessa- tion is that tho,c who discontinue smoking are not chosen at random but by their own dccision, so that the influence of selection on comparability of the group% has hccn unknown. Nor has it generally been possible to assess the infiucnces of concomitant or "compensatory" changs in life-style, such as aitcrations in dict, exercise, use of alcohol or drugs, etc. A Council-sponwred compari%on of continuing smoken, with smokers ( who have discontinued, and with smokers who have stopped for a period and then resumed, sometimes repeatedly, is under way. In the population under study, a large body of data had been collected by uniform methods when all were smokcrs, before any had stopped. Hence, some bases may be found for assessing selection biases that may have occurred in the subsequent groupings into the categories mentioned. 13 . ii

Abstracts of Reports Following arc abstracts, approved by the authors, of reports on ncw rc- search aeknowlcdging support from The Council that have appeared in scicntific journals since publication of thc 1975 Rcport. The namc of thc recipient is in italics. The abstracts arc grouped under these hcadings: I. Canccr-Rclated Studies. 11. Thc Rcspiratory System. 111. Heart and Circulation. IV. Ncuropharmacology and Physiology, V. Immunology and Adaptivc Mcchanisms, V 1. Fpidcmiology. I. Cance.r-Relateri Studiea HYDROCARIION-NITROSAMINE SYNERGISM AS A POSSIE3l.L• AMPLIFYING FACTOR IN LUNG TUMORIGENtS1S BY TOBACCO SMOKE A number of studies have shown that inducers and repressors of microsomal mixed-function oxidascs can powerfully influence thc effects of chemical car- cinogcns. A significant incidence of pulmonary tumors has been reported in both rats and micc as a result of the simultaneous administration of 1-mcrhyl- cholanthrcne (MC) and dimcthylnitrosamine (DMN) at Icvels at which neither compound is carcinogenic in the lung. Nitrosarnincs, including DMN, as well as 3.4-bcnzopyrcne and 3.4-benzoAuoranthenc whose carcinogenic properties arc very similar to those of MC, arc present in tobacco smoke. "I hcrcfore, it is pro- posed that a substantial proportion of the lung lumor incidence of smokers is due to synergism between the carcinogenic hydrocarbons and nitrosamincs in the smoke, rather than to hydrocarbons alone. Two alternative mechanisms which may account for this synergism arc considered. Preliminary results, how- ever, support the concept that DMN increases the hydrocarbon epoxide pool by lowering the activity of microsomal cpoxidc hydrascs in the lung while substan- lially increasing the arylhydrocarbon hydroxy)asc activity. The demonstration of a DMN-hydrocarbon synergism in human lung carcinogcnesis could en- courage unexplored approaches for the development of partial means of pro- tection for smokers, such as the usc of DMN-demethylase repressors and in- hibiton to block the DMN-hydroearbon synergism. 11 is also suggcstcd that dietary nitrosamines or prcnitrosamine components, such as nitrites and secon- dary amincs, may be found to have some role in the etiology of lung cancer. Argus, M. F. and Arcos, J. C. Journal of Throrrricd Biology 36:491-498, 1976. Ot)rer supporlr National Caruoer Inullute. From the Seamen's Memorial Research Laboratory, U. S. Public Health Service Hospital, and the Department of Medicine, Tulane University Medical Ccntcr, New Oricans. SrRUCIURAI. I.IMrrS OF SPECIFICITY OF Cy M1:1'tiYLC'f1Of.ANfliKliNE-REPRLi.SSIE3LE NITROSAMINI: . a N-DL•:AL.KY1-Atik;,S. INlfll)ITION BY ANALOG SUSSTRA'1't:S In order lu gain an insight into the structural specificity of dimcthytnitro.a- minc (DMN)-dcmcthylase, a systematic investigation was carried wrt on the cumparativc dcalkylation of DMN, higher dialkylnitrosamincs and DMN-ana- logs, as well as on the inhibition of DMN-dcmethylatiun by thc DMN-analogs. Other experiments in this framework explored the effcct of pretrcatmcnt by )- mcthyl-cholanthrcnc (MC-), a potent repressor of DMN-demcthylase, on these © diffcrcnt dcalkyla~cs. Resulas showed that the dealkylation of dimcthyl-, diethyl- and .lipropyinitnnaminc by hepatic microsomes of Sprague-Dawky rats was 4 rcprc.vcd by prc(rcatmcot of the animals with MC. '1 his. rcpression progres. n sivcly dccrcascd with thc increase of alkyl chain kngth. In contrast Io its cQcct F on tlac dcnuthylation of DMN, in vivo pherwbarbital induced rather than rc-~- presscd the dccthytation of dicthylnitrosamine (DEN). The rates of demethyla- tion of the DMN analog substrates, although low as compared to DMN, in- creased with the acyl chain length. These analogs were potent in virro inhibitors of DMN dcmcthylation when uscd in combination with DMN as substrates, and the inhibition dccrcascd with the length of the acyl chain. Although the rate of dcmcthylation of mcthylphcnylnitrosaminc was not influenced by MC- prclrcatment, the compound was, however, a potent inhibitor of dertxthylation when used as substrate in combination with DMN. Morcovcr, beyond the ap- parent distinctness of DMN-demcthylase and DEN-decthylax there is now in- dication that more than one enzyme, having the same substrate specificity but different kinetic and regulatory characteristics, underlie DMN-dcmethylase ac- tivity. .ircor, l. C. et al. Tiiuchri/r /iir Krcht/orrchung und Kflnlichc Onkologie 86:171-183, 1976. Other aupport: National Cancer Institute. From the Seamcn's Memorial Research Laboratory, U. S. Public Health Service liospital, and the Dcpartmcnt of Medicirx, Tulane University Medical Center, New Orlcans. DIMI:THYLNITROSAMINE-DEMETHYLASE: ABSENCE OF INCREASED ENZYME CATABOLISM AND MUI.TIPLICITY OF EFFECTOR SITL:S IN REPRESSION. HEMOPROTEIN INVOLVEMENT Evidcnce is presented here that the rate of decay of dimethylnitrossmine (DMN)-demcthylaso' following pretreatment with 3-mcthykholanlhrcne (MC) is no greater than that to be expected from normal enzyme catabolism. These results also show that thc ob.xrved decrease of DMN.krnethylase V_ follow- ing MC administration is not due to increased rate of breakdown but to de- crcased dr novo synthesis. Other experiments in this study indicate that differ- cnt receptor sites arc involved in the repression of DMN-dcmethylase by hydro- carbons and by phcnobarbital (PB), and that a P-450 type microsomal cyto- chrome is involved in the demethylation of DMN. The totality of these ex- pcrimental obscrvatiotu prescnta an apparent paradox which may be sttmmar- 14 15 ><r'

izcd as follows: (1) MC and PB arc polent repressors of the DM N-dcmcthyl- ase: (2) MC and PB arc potent inducen of a number of othcr mixcd-function oxidases as wcll as of the synthesis of the essential components, cytochrumes P-4y8 and P-450, and (3) the MC- and PB-rcpressihlc enzyme. DMN-dc- methylase, is inhibited by carbon monoxide, just as are MC- and 1'U-induciblc mixed-function oxidases. The implications of these findings arc considered. Argus, M. F.. .IrcoJ, J. C., Paator, K. M., Wu. B. C., and Vcnkatcsan, N. Chamico-Bioloyical Inreracrions 13:127-140, 1976. Other aupporl: National Cancer Institute and Hoffmann-La Roche Inc. From the Scamcn's Memorial Research Laboratory, U. S. Public Health Service )Iospital, and the Department of Medicine. Tulanc University Mcdical Ccntcr, New Orleans. MALIGNANT DISEASE AND TRACHEOBRONCH1AL EPITHELIAL MULTINUCLEATION Trachcobronehial washings of patients with a wide variety of cxtrathoracic malignancies have been shown earlier to contain significantly more multinucleatcd ciiiated cells than those of a rnatchcd control group without prcdiagnoscd cancer. This study, whilc confirming the original findings, also determined some (actors which influence multinucleation. One half of the total group of 824 pa- tients had malignant disease and the othcr half, matched by xx, age (decades) and smoking habit but without prediagrwsed malignancies, served as controls. Multinuc)cation was found to be 2.03 times more frequent in the cancer pa- tients than in the controls. In patients with invasive tumors without known mctastases, multinuckation was seen four times more frequently than in the controls. Site of origin, stage of tutrwr and excessive smoking habit in control (emaia influencod statistical aigniP,cance, but smoking habit in males did not. A prospective study is now being planned in which incidence and degree of tracheobronchial epithelial multinuclcation will be uscd in conjunction with biochemical tests for the diagnosis of occult cancer. Chalon, J. et al. Cancer 37(4) :I874-188i, 1976. Other aupport: U. S. Public Health Service. From the Departments of Anesthesiology and Pathology, New York University School of Medicine, New York. ECTOPIC ISOENZYMES: EXPRESSION OF EMBRYONIC GENES IN NEOPLASIA Awareneas of the phenonxnon of ectopic polypeptide hormone production by tumors haa coincided with the roooytition of ectopic isocnzymcs in expcri- menW rodent tumors and in tbo serum and tumor tisstxs of human cancer ~ patients. Many ncwlY-reco8nizcd embryonic protein phenotypes are receivin ~ E wide attcntion as "markeri' of malignancy. A number of other propcrties both ~ cnzymic and noncnzymic arc shared by embryonic and ncoplastic cells. The ever-inereasing accumulation of findingc of embryonic gene products in neo- plmia gathered from rescarch on isoenzymcs, hormones, and protein antigens is causing reevaluation of the current viewpoint regarding the nature of cancer. This disciission cxamines the authors' recent experiences with the carcinopla- cental antigen, Rcgan isocnzyme, as a model system for studying the regulation of emhryonic gcnc expression in cancer cells, the relevance of the cell cycle, and the nature of the gene product in membranes. Their hope was to construct ~ a perspective on the naturc of cancer from the point of view of ectopic iso- ~ enzymcs. The cvidcncc is then examined from the standpoint of a single central ~ qucstion: "Is Ihc selective activation of embryonic gencs a necessary step in ~ ncoplastic tnnsformation.T' Several areas arc scrutinizcd in an attempt to answcr this qucstion. These inciude the relationship between embryology and oncology, the possible embryonic origin of viral transforming genes, normal host gcncs, and oncogcnic mechanisms. It is concluded that: (1) ectopie iso- enzymes, present in tumor tissues but not in the tissue of tumor origin, are predominantly embryonic in type; (2) the Regan- and non-Regan-isoenzyme- producing IfcLa cells provide a suitable model system for the study of a variety of factors which arc involved in the expression of ectopic isoenzymes of the carcinocmbryonic category; and (3) information on cell cycle and hornxxul regulation of embryonic gene expression in cancer cells may contribute to our understanding of the role of this phenomenon in the process of ncpplastic transformation which may reflect a disorder of gene regulation with the re- appearancc of trophoblastic propertics. Such traits evidenced by transforming cclls may hc a conscquence of whatever oncogcnic agent (ctxmiuls, radiation, viruses) produces a specific loss of regulatory control of embryonic genes. F(thman, W. 11. and Singer, R. M. In: Bccker, F. F. (ed.): Cancer: Biology o/ Tumors: Crflwlar Biology and Growth, New York, Plenum Publishing Corporation, 1975, vol. 3, chapt. 3. pp. 57-80. Other aupport: National Cancer Institute. From Tufts Canccr Research Center and the Department of Pathology, Tufts Univcruty School of Medicine, Boston. A SIMPI.I: RAD~OIMMUNOAS.SAY OF HUMAN PLACENTAL ALKALINE PHOS)'iiATASE (REGAN ISOENZYME) USING SPGCIFIC ANTIBODY POLYMERS Ectopic placcntal alkaline phosphatase (Regan isoenzyme) has been found in the sera of cancer patients, reported in variant forms, and considered to strongly reinforce the contemporary view of the bioiogical importance of em- hryonic genc activation during ncoplattic transformation. However, the ex- trcmcl7( minute amount of Regan lsocnzyrne In .eta has made its de(ection, pri- marily by cnzymological quantitation, diffkult. Now, this paper presents a eotn- pctitivc-protein-binding assay of Regan isocnzyme using a specific polymeriaed 16 17