Tobacco Documents Online

Of THE COUNCIL FOR TOBACCO RESEARCI I -U.S.A., Inc. 1974 '. : 4t.. •iI

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SC1EtiTIFIC ADViSORY BOARD to The Council for Tobacco Rcscarch-U.S.A., Inc. as of Deecmbcr 31, 1974 SHELDON C. SOMMERS, M.D., Chairman Directorof Laboratories, Lenox Hill Hospital Clinical Pro/cssur of l'athology Collcgc of Physicians & Surgeons of Columbia llnivcrsity Ncw Yurk, Ncw York RICHARD M. BING, M.D. Director of Cardiology and Intramural Medicine Huntington Mcmorial Hospital, Pasadena, Cali(urnia PruJessor of Medicine University of Southern California School of Mcc7icinc Los Angclcs, California JOSEPH D. FELDMAN, M.D. Nead, Department of Immunopathology Scripps Clinic and Rcsearch Foundation La Jolla, California WlLLIAM U. GARDNER, PH.D. ScicntiJic Director, The Council for Tobacco Rcscarch-U.S.A., I nc. E. K. Nunt Professor of Anatomy (enuritus) Yale University School of Medicine New Havcn, Connccticut ROBERT J. HUEBNER, M.D. Chief, Viral Carcinogenesis Program National Cancer Institute Bethesda, Maryland LEON O. JACOBSON. M.D. Dean of rhe Division of Biological Sciences Regenstein Professor of Biological Sciences University of Chicago Chicago, Illinois AVERILL A. LIEBOW, M.D. Professor and Chairman. Department of Pathology University of California School of Medicine San Dicgo, California HENRY T. LYNCH. M.D. ProJessar and Chairman Department of Preventive Medicine and Public Iicalth Creighton University School of Mcdicinc Omaha, Nebraska i HANS M~II:R. D.V.M., Dr. Mcd. Vet.. M.R.S.I1. .Senmr .Sta/J .Scien ti.u Thc Jack.on Latxiratory Rar Harbor, Maine JOHN P. WYA'iT, M.D. I)irector Tubaccn and I Icaith Rcscarch Institutc University of Kcntucky Lcxinl;tun, Kcntucky Scientific St.ff of The Council WILLIAM U. GARDNER, Pri.D. Scientific Director ROBERT C. HOCKETT, Ptt.D. Research Director JOfIN !f. KREaS1iER, Ptt.D. FREDERIC W. NORDSIEK, PH.D. AsslKYare Research I)Irector DA V I D STON E, Ptt. D. Associare Rrsearch Director Associate Research Director VINCENT F. LISANTI. D.M.D. Research Associate

Introduction CONTENTS Introduction . . • • • • • • • • • • • • ' ' 5 Canccr-Rclatcd Studics . . . . • • • • • • • • • ' ' 7 Abstracts of Reports . . . . • • • • • • • • • • • ' 13 Canccr-Rclated Studics . . . . • • • • • • • • • 13 Thc Rcspiratory System . . . . • • • • • • • • 20 Hcart and Circulation . . . . • . . . . . . . . 33 Ncuropharmacology and Psychophysiology . . . . . . 43 Pharmacology . . . . . . . . . . . . . 47 Immunology and Adaptive Mechanisms . . . • • • • 52 Epidemiology . . . . . . . . . . . . . . . . 58 Misccllarxous . . . . • • • • . . . . . 69 Active Projects . . . . • . • • • • . • . . . . . 72 Completed Projects . . . • • • • • • • • • • • • . 79 Index of Senior Authors . . . . • • • • • . . . . . . 88 Indu of Principal Authors . . . . • • • • • • • • • • 89 The invcstigations supportcd by The Council for Tobacco Research - U.S.A.. Inc. that were published during 1974 arc summarized in this Annual Report under their specific titlcs. These investigations relate largcly to studies of cigarctte smoking and smoke derivatives and problems of health. They have ~ bccn grouped into categories that are oricnted toward either specific diseases or organs, or that arc discipline-associated, i.e., epidemiological. immunological. or psycho• or ncuropharmacological. The health-rclated disturbancec associated with cigarctxc smoking are also age-associated manifestations that often require prolonged periods of observation in intact animals; therefore. in vitro cellular studies and even studic% with microbial nwdcls have been undertaken. Man is the only animal that smokes (or his own satisfaction. Hence, stud- ics are continuing on why man smokes and those diseases to which he is sub- ject and which may be associated with smoking behavior. Such observations supply inferences or associations but fail to demonstrate causes or mechanisms. Animal models must be used for such experimentation. Animal models for the study of the problems relating to tobacco smoking and health arc not easy to devise. Smoke exposure is stressful for animals and presumably prolonged periods of smoke exposure will be required. Extensive studics have bccn undertaken during 1974 to determine the most satisfactory animal modcl (or smoke inhalation studies. Mice have been chosen because they have been inbred and selected for differences in response to stress, sus- ceptibilities to different diseases, and differences in histocompatability and antigcnic characteristics. Mice of different inbred strains differ in the capacities of their liver microsomes to increase the levels of enzymes that metabolize a carcinogenic hydrocarbon, 3-mcthykholanthrene, following the injection of inducing agents. These enzymes, aryl hydrocarbon hydroxylases (AHHs), arc inducible in mice of some strains so that cancer may be expressed while not expressed in mice of other strains. These enzymes are under genetic control. The availability of mice of inbred strains provides much genetic control for the animal model. The interaction of environmental variables on essentially identical genotypes also can be studied. The human counterparts to mice of inbred strains are provided by identi- cal twins. The Swedish Twin Registry, which has provided data on the smoking histories and the incidence of pulmonary and vascular disease symptoms in monozygotic and like-sexed twins born in Swedcn between 1886 artd 1925, has been expanded to include similar data on twins born between 1925 and 1959. This will provide additional data on a population that has a long life expect- ancy and a very low lung cancer incidence. Another twin registry has been started in Finland where the rcported incidence of lung canccr is about five timcs greater than that in Sweden. This should augment srcady the numbers of idcntical and non-identical like•sexed twins that arc discordant (or smoking experience and other cnvironmental exposures. Furthermore, the Iwins among those receiving mulliphasic health checkups in the Northern California Kaiser Permanentc Medical Care Program arc being surveyed as a potential source of identical and like-scxed non-idcntical twins that have rathcr extensive medi- cal and health histories and arc from different racial groups. Significant dilfer- ences have already been reported in the pulmonary function tests of rcprc- ~ I@-~- ~ 5

scntativc• of the different racial gmups. Not only is informatiun being ohtainckl on sm.rl.ing cspcricnce but on a wide range of cnvirunmcntal cxposwcs and occupatrunal activiUCs in rclation to morbidity and mortality. 'I he twin rcgr.- trics should providc a valuable source for many other .IUdic% thal will intludc the cRccts of mulrip{c environmental variables on individuals of identical geno- types and during thc process of aging. Studies on familial predisposiuon to disease arc also being done. Thc tenderscy for cancers to occur in some familics is grcatcr than in others. 'fhis tcndency may be restricted to cancers of certain sites or to two or rnore sitc.. The predisposition of persons with known gcnctic constitutrons to acquire emphysema also has been demonstratcd and may be caused by dcfrcrcncy of a substance or substances that inhibit protcolytic cnzymes. lndividuals with this genetic deficicncy, fonunately only a small percent of the population. arc par- ticularly vulnerable in "smog" environments. Emphysema also occurs in inJi- viluals without this specific genetic deficicncy' a special risk indicatur rs pto- vided by the family history, an indication that other unidcntilicd genetic deter- minants of predisposition may exist. The cpidcmiological projects of interest to The Council involve mostly prospective studies of sclcctcd populations that pro- vide special contrasts for genetic or environmental similaritics or diHcrenccs. Basic research is also being funded on the chemical identrlicatron of proteases and their inhibitors and how they interact. The levels of protca5e5 in different organs and in normal, embryonic and cancerous u.sucs arc being studied. Furthermore, cxtensive efforts arc being made to improve the nllti assay, using human tissues, to promote its application to human cpidemiologi- cal studies. Without basic investigation, the improvement and perfcction of analytical techniques and the development of new or modified concepts of discase prevention or control will be limited- WILLIAM U. GARDNER, Ptt.D. Scicntific Director Canccr-Rclated Studies AmcmE the di.ca..cs that have been a.sociatcd uatiaic:rlly with cigarette .mo/,inb in I-lopul:,uon studics. carcinoma of the hmg has apparently received thc grcatest attcntion. Ncvcrthclcss, this disca+c occur% only in a small minor- ity even ol hcavy smokers. This cmphasizes that research on its pathogencsis must include con.idcration of possible substantial differences among individuals both in their genetic characteristics and in the numerous conditioning in0u- cnccs to which thcy havc been subjected. Many anomalous and contradictory aspects of the epidcmiological findings havc been dcscribcd, and the great difliculty of conducting human .tudics with controls adequate for resolution of these anomalies has been recognized. The rca.un% for this arc both ethical and practical. Ilence, the crucial qucstions "whcthcr, how, lu what cxtcnt, under what conditions, and in wlorn" smoking could contribule to pathogcncsis of the disease remain unanswered. An alternative approach to the problem of describing the interactions of intrinsic and extrinsic influences in the process of carcinogcncsis lies in the design of aiitahlc model systems involving animals, animal lissucs or cells or, in .umc instances, human tissues or eclls. Superficial experiments with inade- quatcly defrncd species or strains, whether whole animals or their tissues or ccll.. will not -solvc thcsc problems. There is strong basis for doubting that mou+c skin painting with stored condensates of the particulate phase of smoke, altered in physical slate and in chemical composition and lacking most eom- poncnts of the gas-vapor phase of "wholc, fresh normal smoke," can cstablish, dcfinc or quantitatc the "carcinogenic hazard" of smoke inhalation by man under lifc conditions. Thc design of such model systems is a most exacting enterprise, since the qucstion of the applicability of the results from thcse models to man in his adual environmcnts niust be asked at every stage and evcntually answered. lhc Council has, ncvcrtheless, undertaken to develop a series of model sys- tcnts for the purposes mcntioncd and to explore strategies for relating the results to man. Carr'inogenesis as a 31u11i-Stage Process Though the term "carcinogen" is often applied to individual chemical compounds or nrixtures as if "carcinogcnicity" were a specific or unitary prop- crty of matter, like a molccular weight, an absorption spectrum or a dipole moment, it is wcll-known that Ihis is not the case. Induced carcinogenesis is rather a procrss presumed to require a series of sequential changes in the bio- logical systems of the host. Thcse, in turn- are presumed to depend both upon the initial, genetically influenced characteristics of these aystems arsd also upon a scrics of separate actions by the extcrnaiinciting agent or agents that require quite specific physical or chemical properties and conditions. Such agcnts have habitually been grouped together as "carcinogens" or "potential earcinogens." Ncvcrlhclcss, it is well-known that a"carcinogen" for one spccies, strain or tissuc under particular circumstances, may not be a"carcinogen" for another spccies, strain or tissuc. or undcr other circumstances. Metabolic modification may be necessary to convert the cxternal "poten- ~ Z. 6 7

lial carcinogcn" into a form that can directly attack sotuc particular anrcturc in the ceil. Cclls may be more. or cvcn cxclusivcly. vulnerahlc lo such attack during sumc particular stage of division. Hencc. influences that stimtdatc ccll division or arrest thc process at a particular stagc may play a significant role in thc proccss. Factors that alter the normal DNA repair mcchanism% may be implicatcd in allowing abnormal eombinations to survive. ucreprc.sion of a viral gcnonx or an orrcogene may be implicatcd. Whcn a normal ccll has bccn transformed into a malignant one, the activitics of thc immrnc system in its many manifestationc may determine whether the transformed ecll can survive, clone and proliferate. Undoubtally Ihesc arc only a few illustrativic examples of the kinds of factors involved and many others undoubtedly await discovery and description. A strategic goal in the research program is to identify those animal strains in which such individual factors arc combined in a way to produce maximal cancer susceptibility. Another goal is to create such combinations of traits by genetic manipulation. As a corollary, the discovcry or production of strarns with other combinations of such traits should help assess the relative roles of such putative contributing facton to thc overall process of carcinogcncsis. Systems for Chronic Smoke Exposure by the Inhalation Route For a long time the lung has been the organ of primary intcrest in The Council's cancer research program. Thcrefore, its program has bcen premised upon the concept that appropriate whole animal test systems should bc based upon the chronic inhalation of fresh, whole cigarette smoke generated under dc6ned conditions that simulate as nearly as possible those experienced by human smoken, from cigarettes of defined and constant composition, and administered in quantitated dosages to carefully selected and defined animal species and strains. Development of mechanical devices for accomplishing such exposures of animals has bcen an exacting, time{oncuming task even after the estahlishmcnt of criteria for acccptahle conditions. Despite the seventcen-year history of The Council's smoke inhalation experience, recognition and definition of these cri- teria have developed only with time. These have been detailed elsewhcre. Two devices that meet many or most of the criteria reasonably well for mice and other small animals have now been developed and a number of units have been in trsc on a trial basis in Council projects. This experience has led to rnodifxations arrd improvements on both. With both types of device, current emphasis is being placed upon the quantitation of dosage to the respiratory tract by use of separate tracer ingrcdi- cnts for srnoke particulates and for gas-vapor phase. Though humans smoke voluntarily, often probably for the relie/ of stress, the conditions of smoke inhalation by experimental animals arc involuntary and stressful so that the contributions of tcnsions to the overall experimental results must be consirfcrcd carefully. Animal containers for usc in 'snwkc inhalation devices have had to be designcd to minimize these cffccta. Mcthods for rnit.gat- ing them, the role of habituation in reducing them and the use of uncaposed "machine control" animals for estimating' their influence have all had to be studied extensively to avoid complicating observations on smoke cffccts par u. "I hout;h .mokr inhalation aurdics with animal% have been .ponsorcd almcxt continuously by 'l he ('uuncil since its organization, the progressive improvc- mcnts in controllcd and monitored systcros are expected to justify beginning a new and more extensive series of experiments at a higher level of sophistication within the coming year. Lsscnually the same inhalation systems can obviou%ly he used in studies hcaring on cancer, on cardiovascular diseases, on chronic pulmonary diseases, on pharmacology and on other questions. Selection of Animal Sub jects for Cancer Studies Principles underlying the selection of animal species and strains for carci- nogencsis studies have been described and these are being used as a guide to selection of animals for the forthcoming new smoke inhalation experiments. There appear to be more cogent reasons at this time for use of the mouse than any other spccies, and this is our choice for present major purposes. Aside from the obvious advantages of low cost, ease of handling, modest space re- quircments, short life span, extensive existing information on their viral and bacterial flora and methods of controlling these, there are others of even greater importance. Many inbred mouse strains exist which show greatly contrasting degrees of susceptibility or resistance to carcinogenesis as observed empirically over the years. These diffcrcnces arc now being related progressively to genetic factors and interpreted to a significant degree in terms of biochemical mechan- isms. In such mouse strains, moreover, the implications of competitive con- cepts of the viral etiology of cancer have been explored much more extensively than in any other species. Their immunological responses to carcinogenic events have also been investigated quite intensively, though they are still in- complctcly understood. • Despite the disadvantages of small lung size, meager volume of blood for tests, differences from humans in lung structure, and the fact that mice are nose hreathers, the use of mice from strains that have sharply contrasting susceptibilities to the process of carcinogenesis presents a unique experimental modcl. If a particular regimen produces carcinoma of the lung in one such strain but not in another, and if these strains have been described with respect to genetically influcnccd biochemical dificrences, the results may provide valu- able inferences regarding the roles of such biochemical facton in the process of carcinogenesis. Such inferences should furnish leads toward the search for analogous biochemical bases for cancer susceptibility or resistance in man. In a long series of preparatory studies, a number of biological and bio- chemical characteristics deemed likely to be related to cancer susceptibility have been studied in many inbred srrains of mice. A number of papers have been published describing these studies and others arc in preparation. Among these preliminary studies was an empirical survey to compare and quantitac the relative (and contrasting) overall susceptibilities of a number of mouse strains, both inhrcd and rarxlom•bred, to marginal, subcutaneous doses ol several polycycGc hydrocarbons known from extensive experience to be "carcinogenic° to rnice in the conventional sense. These hydrocarbons in- cludcd 3-methylcholanthrcne (MCA) and bcnzo(a)pyrerx. The suhcuurseous route was used because doses could be administered with precision, and the 8 9

treatmcnt zone kcpt undcr close scrutiny. On the basis of a carcfully dcvr.cd scoring system, very striking differcnccs in tumor responscs were found anwng these strains. Strains sclcctcd from the spectrum of varying subcutancous .usccptihihtics wcre then subjected to dircct instillation of the same polynuclcar hydrucarhons into thc lungs, in various media and by scvcral tcchniqucs. Squamous ccll Iung carcinomas have arisen at ditTerent rates among these animals, showing that sume strains at least arc susceptible to this disease. Continuing experiments arc expcctcd to reveal more fully the magnitude of these strain JilTcrcnccs. With- out the assurance that the animals to be used in smokc inhalation expcrimcnts can devclop the discase under investigation, the intcrpretation of negative rc- sults from such experiments would be impossible. The Metabolic Activation of Polycyclic Aromalic IlyrlrocarLons (PdIL) Some PAHs, at least, must be metabolized to active forms before Ihcy can transform cells, induce tumors or produce mutations. This acuvalion is accomplished by enzymes of the family termeil "mixed function oxidases" found in the microsonxs of the cells of many tissues. The cnzynxs Ihat pro- duce the change arc designated as aryl hydrocarbon hydroxylaxs (A(ills). These enzymes occur in cells of many tissues of both man and animals at moderate levels and arc probably in some way necessary to life. In some ani- mals, however, exposure to certain polycyclic aromatic hydrocarbons stimulates production of an increased level of the enzymes, while in others it does not. Those that respond to such exposure by production of more (and perhaps different types of ) AHH arc said to be "AHH induciblc." fn the expcriments citcd. AHH inducibility ranged widely in mice of different strains and was correlated with the susceptibility of the strain to MCA- induced subeutancous carcinogenesis. This may well be one clue to the bio- chemical difference between more susceptibk and less susceptible animals with respect to induced cancer as contrasted with spontaneous cancer. Many other substances can stimulate AIIH production in "inducible" animals. 7bese include internal agents such as corticosteroid hormones and bilirubin• external ones including barbiturates; chlorinated hydrocarbons such as DDT~and certain defoliating agents; and compounds that occur in several vegetables. Exposure to sueh compounds in thc environment may, thcrcfore, inercsse some aspects of cancer susceptibility in both animals and men who arc AHH inducible. Inducibility of the AHH system by MCA has been reported to be gencti- cally based in mice. Susceptibility to induction by mcthylcholanthrcnc was reported to segregate as a single autosomal dominant gene in crosses between inducible and rwn-inducibk strains. Published reports of these mouse studies from The Council program stim- ulated other investigaton (not Council sponsored) to study AHH levels and inducibility in man, to teat the concept that this function might be under geactic control iu man as well as in mice. Fo[ several tYasorta ta+ch determinations in man have becn much more dif8ettlt than in mioe. 1Le Council tutdertook to assist the research of the invcstil;ator% aliuJ.d to. Al.o. looking to Ihc practicalitics of cxrcnding such titudic% to large hunian gruups fur elucidation of the modc of inhcritancc, l hc C'puncil inaugur:itcJ aJJiliunal cfiorts to improve the AHIi asay systcros with respcct tu scn.iUvity, accuncy, rcpGcahility and speed. Populations of lung c:rnccr patient% as wcll as human family aggrcgrations (kindreds) charactcr- i.rcd respectively by unusually high and very low familial levels of cancer inci- dcncc. alrcady under study for other purposes, arc available for survey as mcthodolugical dcvclopmcnts permit. Other studics rclating to AHH induction include more dctaileJ investiga- lion ol thc metabolic routcs (or bcnzo(a)pyrene, of the intermediates formcd. the activilics of the.c in cell systems, the enzymes that produce and destroy thcm, and of agents that may block their formation. Inur.urtororrrpr7tence of Alorrse Strains Mice of all the contrasting strains that arc eandidates fur uw in chronic cigateue smoke inhalatiuu studies are being given an cxtcmive battery of pre- linrinary tests to dctcct any large differcnces in the ba%ic competence of their imrnunc systems. "l hc tcsts are then repeated during experimental regimens to detect any consistent changcs in the hope of determining whcthcr immunologi- cal impairment may be a factor contributing to the end results of long term cxperiments. A/utagenPSis arrd RPpair bler•haniarns Metabolic activation of potential carcinogens and alterations in immuno- compctcncc arc two of Ihc scqucntial changes that may be involved in the multi-stage process of carcinogenesis in many cases. It appears that both in- cludc genetic bascs for susceptibility as well as for the intervention of external agents in an interplay. There is a widespread belief that the conversion of a normal cell into a potentially ntalignant one is closely related to the process of mutation. Sensi- tive model systems for appraisal of "mutagenic potential" of environmental agents suspected of a role in carcinogencsis' have been devcbpcd by several investigators. In The Council program, some of these arc being assessed for their applicability to tobacco smoke and its major fractions as an adjunct to the inhalation experiments. When ccllular components, especially those in the nucleus that are be- licved to rcgulatc the activities of the cell, including division, have been changcJ, perhaps by mutagenesis, into a potentially malignant stute, ir is widely believed that nornul repair mechanisms may reverse these changes by climinat- ing the modificd ccll componcnts. A deBciency or depression of such repair pwcntial may constuute another step in the overall process of careinogertesis. Model .ystcnrs proposcd for assessing the efficieney of repair mechanisms are under study in the progrant as another step in the investigation of contrasting auscephbilitics in mouse strains. 10 11

tiruuk.• 1rrlrulrNiorr in Re/afiun to the I:nrcinr.Rerreaia Proi'eaa The principlcs, conccpts and developmental studies that have been dc- scritxd hriclly arc bcing incorporated into the design o( the new scrics of long- term chronic cigarette smoke inhalation experimcm.. In these cxpcrimcnts. micc will be used that havc the combination of factors must conducivc to cancer srrsccptibiliry. on the basis of prescnt knowledge. For the rca+uns our- lincd, mice of other suains with contrasting su.ccptihiliucs, on the basis of available biochemical information, will be employed for comparison. I: un- cquivocal squarnous-ccll carcinomas of the lung should develop at a suhst:rnlial kvcl in some strains and not in others, useful leads into the study of human susccptibility ntight be suggested. By "unequivocal" lung carcinoma is meant a cancer analoFou% to the human disease not only in morphology but in bchavior, being aggres.ivcly in- vasive, metastatic and rapidly fatal. Diagnoses arc to be made independently by several experienced pathologists. At the present stage, "prc-canccrous" lesions. though they are to be described and recorded, arc not regarded as acceptable end-points. In one Council-sponsorcd project of several years' duration, a Sendai virus infection invaded a colony of mice that were tinder chronic smoke inhalation exposure. Lung lesions were found that could easily have been mis- diagnosed morphologically as early lung carcinoma. but which were ultimatcly shown to be attributable to the virus, to regress in many cases and to occur in controls not exposed to smoke. This experience recommcnds grcat vigilancc with respect to prevention and dclection of infections in srnokc-inhalation stud- ics, as well as caution in diagnosis. Obviously, results from such mouse experiments could not be extrapolated direcrly to humans, since the highly susceptihle mice repre+cnt conthinatwns of characteristics that may occur never or rarely in man. Development of the dis- ease in all strains would provide less guidance of the kind mentioned. Its oc- eurrence in none of the strains would suggest that the treatment is incapable of bringing about the necessary sequential changes of the carcinogenic process. In any case, however, the model system should have other important u.ccs. Humans do not live under the rigidly controlled "hot-housc" cnvironmcnts pro- vided for experimental mice. They are generally cxpo.ed to considerable amounts of "potential carcinogenic agents" in the contemporary environment. The model can be uud to explore conditioning effects ol small chronic doses of these agents. The human situation can be simulated to some degree in mice by administering "priming" doses of such "careinogens," too small to produce cancer alone, and rhen subjecting these mice to chronic smoke inhalation to determine whether the latter shows somc type of "promotcr" activity. Sintilarly, the possible synergistic roles of controlled bacterial and viral infcctions, ex- po%ure to agents that produce inflammation, mechanical damage, ircat trauma. presence of lung in(arcts, hormone and vitamin ddieicncics or exces.es. deposi- tions of asbestos, immunosuppression or stimulation. liver dysfunction, and many other factors common to human experience can be studicd cmpirrcally or on a rational basis. Ruecrcr C. Ilocrcr•.rr, Pit.D. Rcscarch Director Abstracts of Reports f:oll4,wing are ahstracts, approved by the authurc, of reporta on new re- scarch acknowlc.lbing support front Thc Council that have appeared in .cicn- riGc journals since publication of the 1973 Report. 'I he name of the recipient iti in italics. 'Ihc ah.tracts arc grouped tinder thc%c headings: 1.. Canccr-Rclatcd Srudics,® 11. 'l hc Respiratory Systcm, 111. (Icart and Circulation. IV. Ncuropharmacology, anrl I'sYchnPhYaolobY• V. Pharmacology, V1. Immunology and Adaptive Mech-Ix i V an srns, 11. Epidcmiology, VIII. Miscellancous. 1. Cancer-Related Studiea TRACHEOIIRONCHIAL EPITHELIAL MULTINUCLF.AT(ON IN MAI.IGNANT DISEASE During a continuing study (2,9R3 cases thus far) of trachcohronchial ex- foliated cytology in surgical patients subjected to endotracheal anetithesia. the authors notcd that individuals with known malignancies sccmcd to have un- usually numerous multinuclcated ciliated cells. Further examination of smears taken front 112 patients known to be suffering (rorri, a widc variety of malig- nant tumors confirmed this observation. Muhinuckation was 2.08 times more frequent in this population than in a supposedly malignancy-free control group matchcd by scx, age in dccadcs, and smoking habit. It is hoped that rccog- nition of this phcnomcnon may Icad to the development of a new test for the diagnosis of occult canccr, and may open new pathwayc (or investigation of canccr-host rclationships. The authors are currently tackling the first oh- jcctivc by looking for occult cancer in controls who have high percentages of trachcobronchial epithclial multinucleation. Clralon, J. ci a!. Science IS3:525-52G, 1974. O1her.rrpporr: National Cancer Institute. From the Department of Ancsthesiology, Albert Einstein College of Medicine of Yeshiva University. f3ronx, N. Y. THE CF(.LUI-AR I~.VF.NTS ASSOCIATED WITH REGRESSION AND PROGRESSION OF MURINE (MOLONEY) SARCOMAS Previous histopatholovic classifications of Moloncy sarcomas have diltercd wirlcly, ranging from a bcnign, reparative inflammatory reaction to mescn- chymal sarcoma. Furthermore, a complete sequential analysis of the cellular inflammatory cvcnls accompanying both tunwr proFrosion and rellre.sion is still Ircking. lhe authors, thcrefure, ehinc to induce Moloncy sareorna in neo- natal and Adull mice jn ordcr to characterize both its histologic nature and v ` 12 13

the coursc of events that dcvclops following the injcction of its tran.plantahlc cells. In ncomatcs. intramuscular injections of either 10' or II)° cclls Iruor a cultured murinc (Muloncy) sarcoma line inkluccd ncoplasms Ihal pn+)uc..cJ, In adults. only the larger dose pfoduccd this clicct; adult micc receiving 10' cells usually dcvclopcd tumors that regressed. The growths wcrc cxaminc.l hy light microscopy at 2-3 day inmcrvals throughout the course uf their dcvclup- ment and subscqucnt regression or progression. Initially, all tumors wcre in- filtralcd with polymorphonuclcar Icukocytcs - mainly ncutrophils - and cdcm:r was extensivc. fly thc end of the second weck aftcr inoculation, this acute in- flammatory infiltrate had been replaced in adult mice by one consi.ting of mononuclcar cells; neonatal mice never devcloped significant numbers of these inflammatory cclls in their tumors. Of particular significancc, sincc munonucicar inflammatory cells were associated intimatcly with tumors during the proccss of rcgression, was thc disappearance of these cclls 12-14 days aftcr inocula- tion frorn tumors datined to progress in adult micc. Hyperplastic changcs wcrc found in the corticcs and medullae of regional lymph nodcs draining both progressing and regressing sarcomas. Thc devclopment of secondary neoplasms was common, and the distribution of these lesions was rclatcd to the ages of mice at the time of inoculation. Russell, S. W. and Cochrane, C. G. lnternationalJournalof Cancer 13(1):54-63, 1974. Other support: U. S. Public Health Service. From the Department of Experimental Pathology, Scripps Clinic and Kcsearch Foundatioo, La Jolla, Cal. HEREDITARY LYMPHOSARCOMA IN WH RABBITS AND HEREDITARY HEMOLYTIC ANEMIA ASSOCIATED W1Tli THYMOMA IN S1 RAIN X RABBITS Among their "partially inbred" rabbit strains, the authors searched for those genes which produce susceptibility and resistance to certain types of tumors. They found such a situation in the wirchair (WH) strain: lymphosar- coma occurs at a mean age of eight months; susceptibility is conferred by a singk autosomal recessive gerx designated Is. There is tcntativc cvidencc which points to the presence of a C-type RNA gcnomc coding for the interspccics dc- terminant of the group-speciflc antigen. Highly positive reactions were obtained with potent antisera produced in rau to the p-AGs of murinc C-type virus. Complete (infectious) C-type particles have not bcen found by electron micro- scopy; thus, ccll-free transmission has been negative so far, and assays for "hdpcr" activity have been inconclusive. However, cellular transmission is successful upon inoculation of lymphosarcoma tissue into (ctuses. Presumahly, the !i gene confers susceptrbiGty to virus-induced tumorigcncsis. The invcsti- gaton have also observed thymomas associated with hereditary hcmulytic anemia in strain X rabbits. In these rabbits, which are genetically related to strain WH, anemia occurs at about five months; susceptibility is contcrred by a siogk autosomal recessive gene, symbolized ha. Thc data for these two dis- eaaes are compatible with both conoepts of genetic susceptibility and the vcrtical tnnsmisaion or inheritance of a viral jenome. in Alrirr, 11. and Fox. K. R. !}~ Jtilrliurbrca l/urrnarr+lugir'u (No. 39: Unifying Conccpt. of I.cukcmia):72-93, ~ 1973. Otlrrr auplturr: National Institulcs of Health and the National Cancer In- slitutc. From Thc Jackson Laboratory- Bar Harbor, Mc. INFI.UE?NCE OF PRi:INFfiCT1ON OF C57BL/6 MICE WITH (;RAFFI l.1{UKF.MIA VIRUS ON 3-METIiYLC}iOLANTHRIiNE-INDUCF.D SUBCUTANEOUS SARCOMA W Do oncogenic agents which induce different types of ncoplasms have an effect upon each other? This study on the combined cfkctc of the Grafli Icukcmia vinu and 3-mcthylcholanthrcnc (MCA) ancmptc to answcr this qucsuon. It investigates the mutual influences of virus-induced leukemia and chernically-induced sarcoma in C57B1./6 mice and examinec furthcr the latency pcritxl as a dctcrmining factor in the co-oecurrence of induced neoplasms dilTcring in ccll type. Rcsultc from these cxpcriments showed that when C57A1./6 mice were given )oth Graffi Ieukcmia virus and M(-A the devclop- mcnt of either Icukcmia and/or sarcoma was dependent on the dene of each carcinogen given. A high dosc of virus reduced sarcoma inductiim hccause. due tu the high incidence of Icukcmia. the survival timc of thc mice was Icss than the averagc latency period requircd for tumor development. A high dose of MCA (3(X)rg) increased the incidence of lcukemia induclion by a low dose of virus without affecting the incidcncc of sarcoma. 7his occurred since the latency pcrio.l for sarcoma and Icukensia coincided and 'SS'o of the mice developed both leukemia and sarcoma. The combination of a low drxe of virus and a low dose of MCA did not alter the incidence of Ieukcmia or sarcoma; however, with this combination of virue and chemical c.rrcinogcns. the average latency period (or thc development of leukemia was delayed and the average latency period for sarcoma induction was acceleraled. Crafli virus failed to increase the incidence of MCA-induccd sarcoma under the conditions studied. Whitmire, C. F. and Salerno, R. A. (Microbiological Arsociates) ProcrrdinRs of the Society /or Esperimtntal Biology and Mrdicint 144(2):674- 678, 1973. Other sapport: National Institutes of Health. From the Department of Viral-Chcmical Oncology, Microbiological Ascociates, Bethesda, Md. HYDROCARIION-MF.TABOLI7.ING ACTIVITY OF VARIOUS MAMMALIAN CE(.LS IN CULTURE Recent in viro studies strongly suggest a relationship betwcen levels of hydrocarbon-metabolizing enzyme activity and sensitivity to polycyclic aro- matic hydrocarbon-induced chemical careinogerxsis. In the limited number of in virro systems available. the levels of these enzymes may strongly in(luence the sensitivity of cclls to transformation induced by certain polycyclic aro- 14 15

matic hydrocarbons. Thus. before starting any large scale in rirrn Iransforma- tion program, potential human cell lines should hc scrccncd so a, to idcntify thosc with high enzyme Icvcls. This approach has hccn panicularly uscful in cluciJating the relationships between hydrocarhon-mctaholi/ing cnxyrnc activity and sensitivity to hydrncarbon-induced cytotoxicity. Hcrc. the awhors comparc two procedures (or measuring the enzymcs. The one designed tn mcasurc bcnzolrlanthraccnc-irxluced aryl hydrocarbon hydroxyla.c activity couW dctcct and quantify enzynx activities in low passagc rodent cclls, hut could not rcproJucibly dctcct IcvcJs in intermediate or high passage muu.c, rat, or human cells. The method designed to rneasure the ability of a cell to convert hcnr.o(a)- pyrcne from an organic-soluhlc to an aqueous acctonc-soluhle form. proved more reproducible. When nsodified, this particular technique proved to be an effective screening test for the detection of those lines with highcr lcvcls of hydrocarbon-me(abolizing enzynxs. Kouri, R. E., Kiefer, R. and Zimmerman. E. M. (Microbiological d csocinres) In Vitro 10(1 & 2) :18 ?5, 1974. Other sr.pport: National Institutes of Health. From the Department of Vinl-Chcmical Oncology, Microbiological Associates, Bcthcsda, Md. GENETIC CONTROL OF SUSCEPTIBILITY TO 3-METIIYI.- CIIOLANTIfRENE-INDUCED SUBCUTANEOUS SARCOMAS The authors report on their use of a mouse gcnctic systcm to cxtcnd their observations on the relationship between aryl hydrocarbon hydroxylase (AHH) inducibility and sensitivity to 3-mcthylcholanthrcnc (MCA)-induced lumorigencsis. Observations reveal that AHH-inducible mice are approximately 12 times more sensitive to MCA-induced tumorigenesis than thcir noninduciblc littermates. The type of parental cross (maternal influence) plays no role in this sensitivity. Host-regulated expression of the group-spccihc (gs) antigen of type-C RNA viruses, although also segregating in this genetic system. does not seem to play a major role in this enhanced susceptibility to MCA carcino- genesis. Results are discussed with the view that the enhanced scnsitivity of the AHH-inducihle animals probably results from rapid and efficient mctab- olism of the chemical to its ultimate carcinogenic form. Recent information suggests that these observations may be analogous to what occurs in human populations wherein the degree of AHH inducibility is genetically controlled (probably by a single locus), and inheritance seems to be codominant. Thus, thers may be a correlation between the presence of (or susceptibility to) lung carcinomas and the alleks regulating expression of high or intermediate in- ducibility. lf so. the genetic system presented here may provide a valuable model for studying Ibis problcm in human populations. Kouri, R. E., Ratric, H., III, and Whitmire, C. E. (Micral,ioloy;icaL.tssociatcs) Inrernarlond Journal o/ Cancer 13 (5 ):714-720, 1974. Other support: National Institutes of Health. Fnom the Dcpartnxnt of Viral-Chemical Oncology, Microbiological Associates, Bethesda, Md. IiN('AI'Slil.A l ION OF LYMI't1(X:Y'1 [i DNA BY VI:SICUI.AR SfUMAl7fIS VIRUS Scvcr.kl : nimal viru.cs arc known to incorporatc hoat DNA during their rcplication : nd ncuuration, but this proccss was generally thought to be char- actcristic uf oncogcnic DNA viruses with a nuclear phasc of replication. Hcrc, howcvcr, the awhur. demonstrate that an RNA virus which is rwt considcred oncogcnic- has no known nuclear phax in its rcplication, has its own RNA- dcpcndcnt RNA polymcrasc, and shuts ofl host DNA synthesis can contain up to 30"/0 of its t;cnctic rnais as DNA. In addition to viral RNA, vesicular stnmatitis virus (VSV), grown on thc WILs-3A line of continuously growing human lymphocytcs, contains DNA which is found in both the B and T virus particles and is resistant to Jcoxyribonuclcasc. This DNA is intimately assa ciatcJ with the virus and appears to be incorporated into the viral ribonucleo- protcin structure produced by treating VSV with Nonidct P40 followed by ('sCl isopycnic banding. In conlrast. virus grown on BHK 21-4713 fibroblasts has no Jctcctahlc DNA. The virus-associated DNA has an isopycnic density of 1.699 g X cm 3 in CsCl, identical to that of human DNA. 11s average molecular weight is 9.0 x 10"•, as determined by velocity sedimentation in sucrose density gradicnts, and studies of its contour length in the electron microscopc. The DNA'DNA rcas.ociation kinctics of this particular nucleic acid demonstratc that it is of host origin and rule out the possibility that it originatcs from coNaminating microorganisms or mitoclwndria. lhe analytical coniplexity of the virus-as.ociateJ DNA shows that 50% of its sequences are humologous to thc rcpcatcd DNA scqucnccs of human DNA and that thc rcmaining 50% arc homologous to human unique sequcnces. On the average, there is one molecule of DNA for each four virion particles. Whether or not viruscs such as VSV arc capable of oncogenic information "transduction" is unknown, but it is a possibility which can he tested. Kingsbury, D. T. and Lcrnrr. R. A. Prrxrcdint,•s n/ rhr Nutional .IroJnny of Scitncr.s of the United Statrs ol i(mrrica, 71(5):1753-1757, 1974. Othrr support: National Cancer institWe, National Foundation - March of Dimcs, and U. S. Public }lealth Service. Front the Department of Medical Microhiology, Univcrsity of California Col- Icgc of Medicine. Irvine. and the Department of Experimental Pathology, Scripps Clinic and Research Foundation, La ]olla. Cal. DIFFERENTIAI. RL'SPONSF. OF SNF.LL:S AND C57 BLACK MI('F•. 10 ('HRONI(' INffA1.ATlON OF CiGARETTE SMOKIi. PULMONARY CAR('IN(XJIiNF.SIS AND VASCULAR ALTERATIONS IN LUNG AND HEART Do genetic diffcrcnccs hetNccn mouse atrains influcncc their re.ponse to chronic inhaletion of cigarette smoke? The question is expiorcd in this study 16 17