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SCIENTIFIC ADVISORY BOARD to Thc Council for Tobacco Rcscarch-U.S.A., Inc. as of December 31, 1975 SHELDON C. SOMMERS, M.D., Chairman Director of Laboratories. Lenoz Hill Hospital Clinical ProJessor of Pathology College of Phyciciana & Surgeons of Columbia University New York, New York RICHARD M. BING, M.D. Director of Cardiology and Intramural Medicine Huntington Memorial Hospital, Paaadena, California Professor of Medicine University of Southern California School of Mcdicine Los Angeles, California JOSEPH D. FELDMAN, M.D. Head, Department of Immunopathology Scripps Clinie and Research Foundation La Jolla, California WILUAM U. GARDNER, PH.D. ScientiJ'ic Direcmr, The Council for Tobacco Research-U.S.A., Inc. E. K. Hunt ProJessor of Anatomy (emeritus) Yale Univera+ty School of Modicine New Haven, Connecticut ROBERT J. HUEBNER, M.D. Chie/, Laboratory of RNA Tumor Viruses National Cancer Institute Betheada, Maryland LEON O. JACOBSON, MD. Director, The Franklin McLean Memorial Research Institute RegeRrtein Pro%aroi of Biological Sciences University ol Chkayo Chiu8o, ]llinoia AVERILL A. LIEBOW, M.D. ProJesaor and ChaLman, Department of Pathology University of California School of Medicine San Diego. Catifocnia FUNRY T. LYNCH, M.D. ProJesaor and Chdrmarr Deparitment of Preventive Modkine and Public Health Cie3 ton Unlverslty School of Medicine Om.~a„ Nebcaaka iIANS MEIER, D.V.M., Dr. Mcd. Vct., M.R.SJi. Senior .StafJ Scientist The Jackson Iatxxatory Bar I larbor, Maine LEE W. WA7TENBERG, M.D. Professor of Pathology Department of Laboratory Medicine and Pathology University of Minnesota Medical School Minneapolis, Minnesota JOHN P. WYATT, M.D. Director Tobacco and Health Research Institute Univcrsity of Kentucky Lexington, Kentucky Scientific Staff of The Couneil WILLIAM U. GARDNER, Prt.D. Scientific Director ROBERT C. HOCKETT, Ptt.D. Research Director lfr 00. 1,- JOHN H. KREISHER, Px.D. DAVID STONE, Ptt.D. Associate Research Director .4ssociate Research Director VINCENT F. LISANTI, D.M.D. Research Associate

C I CONTENTS Introduction . • . - • • • • • • ' 5 Studics Related to Respiratory Function and Chronic Pulmonary Diseases . . • . 7 Abstracts of Reports . . . • . . . . . . . . . . . . 14 Canccr-Rclatcd Studies . . • • - - • • • • • • 14 The Respiratory System . . . • • • • • • • • • 24 Heart and CSrculation . . . . . . . . . . . . . 35 Ncuropharmacology and Psychophysiology . . • • • • 47 Pharmacology . . . . . . . . . . . . . . . 53 Immunology and Adaptive Mcchanisms . . . . . . . 55 Epidemiology . . . . . . . . . . . . . . . 62 Active Projects . . . . . . . . . . . . . . . . . 66 Completed Projects . . . . . . . . . . . . . . . . 73 Indcx of Principal Investigators . . . . . . . • . . . 83 Index of Senior Authors . . . . . . . . . . . . 84 Introduction The Annual Report of 1973 had one of its seven sections devoted to the presentation of some research on lung (unction nd chronic respiratory dis- cases. During the past two years, studies on respiratory functions of man and lahtuatory animals have advanced greatly. Sophisticated biochemical methods have been used to study thc dcfcnsc mechanisms of the lungs, their response to the internal environment as determined by the genetic background of the = subject and their response to external stimuli. The abstracts of the investiptions that have been supported during 1975 in this and in other research areas are [ included in the third section of this report. F. The previous Annual Report emp hasized the researches that were relatcd ( to cancer and to the considerable effort that has been made to develop methods for the delivery to, and retention of smoke or smoke components in, different parts of the respiratory tracts of mice, the animals that had been selected for the smoke exposure experiments. This year's Annual Report emphasizes investigations that have been under- taken on the respiratory system and on the epidemiology of respiratory dis- eases. In some ways the classification is somewhat arbitrary. For example, em- physema might be considered under the respiratory system, but because many studies on this disease involve hereditary and environmental components, certain studies might be classified under epidemiology. The rapid development in these areas during the last few yean led The Council to convene a two-day informal conference during 1975 at which a number of inveatitators concerned with the lung's protein-dcstroying enzymes and their inhibitors had an opportunity to communicate and compare their most recent investigations. The subjects dis- cussed in detail are presented in the following section of this report. Identical twins, like laboratory animals of highly inbred strains, provide the best means of determining the impact of the environment on the senotype. When environmental factors are discordant the expression of the genotype may be modified. The New Swedish Twin Registry - twins born in Sweden from 1926 to 1959 - has been completed with some 32,000 entries of like-sexed twins. A Finnish Twin Registry has been compiled and much information is being obtained through responses to questionnaires. The contrasting incidences of pulmonary cancer and coronary heart disease in Sweden and in Finland should add significance to the data that may be derived from studies of these registries. The studies will involve not only the identical and the noo-ideoticat, like-sexed twins, but also the twins as individuals making up a erou section of the populations. Pulmonary function of populations at different ages and different areas of residcnce and diRcrent occupations hu been determined and the dfeets of familial or hereditary inftucnces have been deseribed and diacussed. New observations have also been made on lung structure; neuroepithelial bodies have been found in the human lungs and described in detail. Their (unction is not known with certainty. They probabty contribute to the normal function of the lung. Even at this time, new structures and potential regulatory mechanisms of lungs, which provide that narrow interface between environ- ment and blood, can still be discovered• The following review of the stud'w related to respiratory functios and chronic pulmonary diseases reveals the interests of The Couacil in these areas 5

of research. Bccaux o( concern with human well-being. studics arc being danc with human subjccts when possible. Epidemiological studies cspccially must relate to human populations. In other studies, animal models must hc u.ed to reveal information to augment our knowledge of Ihe etiology and pathogencsis of those aging-associated diseascs of the pulmonary and cardiovascular systems. WILLIAM U. GARDNLR, PII.D. Scientific Director ` '- ~ J i._.. Studies Related to Respiratory Function and Chronic Pulmonary Diseases ® . The Council's 1973 Report presented general concepts and plans underly~ ing the rescarch program in respiratory function and disease. Along these linet- a doren projects in a number of research institutes were maintained during &4,) year just ended, with collateral support by contract studies for equipment de- velopment and methodology. i1 nimal Studies Involving /nhalation Exposures In four studies animals were exposed chronically, by inhalation, to agents widely present in human environments. One investigator, who had developed methods for measuring pulmonary functions in mice, subjected animals of two strains to cigarette snake inhalation daily for five to ten weeks. High- and low-niootine reference cigarettes were used to compare the effects of different nicotine kveis in smokes otherwise very similar. Effects of whole, fresh smoke inhalation were similarly compared with those of the gas-vapor phase alone. The latter, being substantially free from particulates, contains only traces of nicotine. Measurements of functional residual capacity of the lungs showed that emphysema was not produced. The strains differed in susceptibility to increaae in pulmonary resistance (bronchospasm), which was elicited by whole amoke and to some extent also by the =arvapor phaae, but both strains developed almost compiete tolerance within ten weeks. A decrease in pulmonary eompii- ance was reported to be associated with Avo- to teo-week inhafatioo of smoka from the high-nicotine reference cigarette, but this was not elicited by the sas• vapor phase alone. Effects of acetaldehyde, acrolein and skatole, which ara low- level, but in other contexts known to be chemically aetive, components of the gas-vapor phase, were measured in separate but parallel experiments. Meanwhile, because of suyscstions in the literature that cumulative traces of inhaled cadmium might be a factor in the etiology of emphysema, a study similar to that just described was begun with eijarettes "prinsed' with three levcls of added cadmium compounds. Because fly ash and nitrogen dioxide are found In the atmospheres of many cities where chronic pulmonary ailments are prevalent, anorher invcstiptor tx- posed hamsters for prolonged periods to alrtsospherea containing these eom- pounds. He too had an impressive series of physiological and morphological test methods applicable to the hamster, an animal regarded from other studies as being relatively susceptible to lung damage. The animals were exposed chroni- cally for about 14 months either to dust alooe, to dust with nitrogen dioxide or to ambient air. Some of the animals were rr-exposed to ambient air aloos for two weeks following the other exposurts. After sacriAce, mcuuremeots were made of static cornpliaoee of the lun=.

'S L static clastic recoil pressurc, total pulmonary rc.istancc and flow rates, forced dcllatiun, and morphumetry (or mean lirxar intcrccpts and internal surfacC arca, a% wcll as general patholugical and hi.tological evaluations. In summary, no significant abnurmahtics wcre observed in the animals exposed to dust with or without nitrogen dioxide. This study drd not produce an animal model for emphysema. In another study by a difierent investigator, hamsters were exposed to long-term chronic cigarette snwke inhalation but gross emphysematous changes were not sccn. been measurcd as well as the mobilization following such aspirations, of ir creased numbers of recoverable macrophages. The clkcts of tobacco smoke it halation upon rates of disappearance of viable organisms from the lung in sit have been reported, and the total engulfment and killing activity of macrophag populations recovered from smoke-exposed animals arc now being determine in vitro. Studiea of Ozone Ozone, a powerful oxidant and a very prevalent and active constituent of atmospheric smog, can severely damage the lung as can exposure to abnormally high levels of oxygen. A scientist sponsored by The Council undertook to study the effects of these agents in rats and monkeys in order to discover what changes occur in the lung metabolic and enzymc systems and how they might be prc- vented or reverscd. When the study is complcted, the information obtuincd should he useful as background for analogous itudies of tobacco smoke inhala- tion arid provide a basis for observations on possible interactions between to- bacco smokc and smog. Two distinct aspects of ozone effects were found. Short-term, high-lcvcl exposures (2 to 4 p.p.m.) for two to eight hours destroyed large proportions of the sulghydryl compounds present in different portions of the lung cells, with profound lowering in the levels of enzyme activitics involved in cell division and repair. In contrast, exposures at the level of 0.2 and 0.8 p.p.m. for two to seven days produced no destruction of sullhydryl compounds or depression of these enzyme systems; instead, the activity of the systems was increased. These effects of low levels of ozone may explain in part the tolerance or adap- tation to such exposures that this scientist and others have noted in animals and in man. Morphological studies have paralleled these biochemical obscrvations. Exposures to relatively high concentrations of oxygen are being madc (or comparison, and studies of tobacco smoke inhalation by rats with or without previous ozone exposures are now under way. Preliminary experiments with mice, employing realistic dosages of cigarette smoke daily for three weeks, showed no histological evidence of major patho- logical change. The lungs, however, had higher levcls of specific activitics of the antioxidant cnzymcs, glucosc-6-phospharc dchydrogcnasc, glutathione rc- ductase " glutathione peroxidase, which may re/lcct adaptive response to oxi- dants present in the smoke. Other lung biochemical assays have been added to the protocols; (or example, the effects of oxidants on collagen synthesis. Pulmo- nary fibrosis is o(ten a sequel to long-term chronic respiratory damage by such agcnts. Defense Mechanisms of the Lung A previous Annual Report (1973) mentiooed studies on the defense me- chanisnu of the respiratory tract against inhaled particulates, both inanimate "dusts" and microorganisms, especially by mobilization and (unction of pulmo- nary macropbatea. Rata of clearanee for aspirated Srayhylococcu.r aureus have !n/luensa Infections Influenza infections, still prevalent, result in significant persistent lun damage in man. A veteran investigator of this problem has published a detailc description of the sequential effects of influenza PR8-A infections in mia With the advantages provided by the opportunity to sacrifice animals scriall at 211 stages of the process, these observations generally have confSrmed and ei tended those available from human posr-morrcns examinations. A separate rt view applied these findings to interpretation of potentially serious consequenct of human influenza and suggested preventive measures that should be applie to prevent such infections. A method for producing more consistently a persistent vitamin A deficicnc in mice made it possible to study the consequences of such deficiency upon th scquclae of infection. Squamous metaplasia and keratinization of the bronchia membranes were significantly greater in the animals with little or no hepat~ vitamin A and the post-in(ection lesions showed more extensive epithelia nodule formation. These and observations from other quarters have renewa interest in relationships between this vitamin and cancer susceptibility or rc SlstanCc. Clinical Surueya of Lung Function "Captive" populations in the Boston area and on the West Coast are pro viding the opportunity to describe relations of age, ux, race, light smoking heavy smoking, industrial exposures, and other facton to pulmonary functiona changes over an extended period of time, through repetitive reexaminations. Respiratory Distress Syndrome of Newborn !n fanta Hyalinc membrane disease in newborn infants, associated with prematurity maternal diabetes and complications of pregnancy and delivery, produces hitl mortality. It is due to failure of the inlant lung to expand properly after de livery and is usually accompanied by formation of a"hyaline membrane." Thi structure, observed in the alveolar spaces, ducta and bronchioles, consists of de generated lung surface cells and blood elemenu, and contains large utsounts o fibrin, an clastic, thread-like, insoluble protein material. The atekctasis (non expansion) has been attributed to a defSciency in surfactant and the flbrin sc cumulation to a defective balance between the systems normally responsiblL for its formation and its removal. 3 9

Only very recently it was discovered that normal hum;,n cmbryonic lung fibroblast cells (and those of many other mammals) rcgularly produce high levels of plasminogen activator. This is a protein that converts plasminogen, ,,n inaetive substance present in the serum, into plasmin, an enzyme that dcstroys fibrin. The serum from infants with hyaline membrane disease is dcficicnt in plasntinogen though it is not clear whether this is due to tlie continued produc tion of the activator or to excessive inhibition of the plasmin produced from it. In a Council-supported project, plasminogcn activator is being prepared in quantity from cell cultures; it will be concentratcd, purificd, characterized and used to produce labeled antibodies for studying its presence and concentration at specific sites, and its role in normal cells as well as in disease states. Relevance of this work extends beyond the problem of the respiratory dis- tress syndrome. It has recently been discovered that normal cells, other than the exceptional ones of embryonic 1ung, though they quite generally produce very little pts.smirtogen activator, will nearly always increase this production at least 50-fold when transformed into the malignant state. Indeed, this property may be a useful indicator of malignant cell transformation. Such transformed cells show a number of changes in behavior. They will grow on soft agar (unlike most normal eells), they show Increased migration, they are agglutinated more readily, they display an altered morphology, and they produce tumors if trans- planted into mice with low or impaired immune responsivenesa. While normal embryonic lung cells show a similar ability to grow on soft agar, they do not show the altered morphology or produoe tumors when so implanted. Ingenious systems have been designed and applied for studying the bio- chemical conditions within the cell that govern the production of plasminogen activator and its accumulation or dispoul in normal lung cells as compared to canoer cells. Proteaaeaatad Proteaae Inthibitors in Relation to Emphysema 'TLere are substantial indications that human pulmonary emphysema may result from "digestioa" of the lunj s elastic structural material by enzymes (proteaacs) that attack the proteins of which it is composed. One irxllcation is that paprun, a plant protease mixture that docs not occur naturally in animals, will neverthekas produce destructive ksioos similar to emphyacma when in- uillod directly into the lungs of animals of several species. Another, rcported only a few years ago, is that pcrsons having an ab- nornully low blood level of a protease inhibitor, originally designated as alpha,- antitrypsin•, are particuLrly likely to develop emphysema at an early age. This observation rcinforeed the idea that the disease might be caused by proteases from sorne aource, either internal or exkrnal, if or when their action is not property controlled or "turned-oft" by an antiprotease. A third, still more reeent indication is that certain human or dog blood kukocytes, particularly polymorplsoouckar kukocytes and macrophages from the periton,eum or lungs, when is+olated, concentrated, broken down, suspended in a medium, and instilled into dog lunp, produce emphysema. The extent of the lesions produced is dependent upon the protesae activity of the preparations, •$iaws tbis Lobibilnt is not apocitie for trypsla but binds a number of. proecaaes, ,ipba,- aatiproteaae is a betur aamo. c- and thu.c (rom polyrnorphonuclcar lcukocytcs arc more active than those from~ macruphagc.. (Ioth typcs of cclls concentrate in thc lung in dcfcnu against ,n 1 halcd particulate matter, including infectious organisms. They contain power(ul protcolytic enzymes packaged in small vesiclcs inside the cell walls and had not hccn con.idcrcd likcly to harm the lung structure under normal conditions. The new expcrimcnts, thcrcforc, raised the questions whether cnzyme leakage from the Icukucyte cells or disintegration of these cells within the lung might bring the proteascs into direct contact with the lung tissues and thus contribute to pathogencsis of emphysema under real life conditions, if not properly countered by protcase inhibitors available in adequate concentrations in the critical areas® An overproduction of leukocyte protcases or an abnormal access to critical~ zones might contribute to the process. I- U A Delicate Balance A delicate balance between protcolytic and antiproteolytic activities in the hmg is necessary for defense against infections, but damage to the lungs them- sclvcs might occur if the balance is disturbed either by overproduction or exces- sive rclease of protcases, or by underproduction or inadequate access of in- hibitors. Extensive studies by many investigators, including several sponsored by The Council, have shown that alpha,-antitrypsin deficiency follows definite hereditary patterns in human families. These studies support the concept of an inherited susceptibility to emphysema and provide one explanation of the em- pirical observation that the disease lends to "run in families." The picture is complicated, however, by the existence of several variant forms of this anti- protease, which differ in theirefficieneiesof antiprolease activity. Atkut twenty differcnt phenotypes have been described that represent the various possible combinations of the genes controlling the alphar-antiprotease types and produe- tion. Moreover, there appear to be familial aggregations showing high em- physcma predisposition that is unrelated to this antiprotease, suggesting that other factora not yet described may be involved. There is no reason to suppose, lur examplc, that antiproteases other than the serum Ipha -xntiprotease and its variants may not exist in other organs and play a rok in the total picturr. Ad- ditionally, the role of lung surfactant and its effect, if any, upon protcases and antiprotcases is not fully understood. Several grants by The Council have fostered studies of proteases and anti- protcases in rclation to emphysema. Much eRort has been devoted to purifks- tion of serum alpha,-antiprotease and its variants so that their structures and molecular weights could be determirxd, the sequential arrangement of the con- stituent amino acids worked out and the biochemical modes of action in the inhibition of protcase activity described. Rsdiolabeled antibodies have been used to show where these inhibitors bind to tissues. In these studies, compari- sons with other protease inhibitors from different sources arc helping to define and describe their likenesses and differertces and especially their specific actions. The isolation and description of proleases elaborated from lung macro- phages and granulocytes have also been undertaken to find out which ones arc most active in attacking lung structural proteins and how they react with in- hibiton. This has entailed exploration of methods for oollecting, separating and culturing these several types of cells. 10 11

Detecting the Enrphyserrra-Prone Indiuiduaf Avowcd aims of these investigators are to develop bettcr methods for de- tecting high entphysema predisposition in individuals, ma(criats for early diagno- sis of the discasc and, it is hopcd, agents that might bolster defensive mcchan- isms in the body and arrest progression of thc discasc. A related but compatible aim ia to determine whcthcr, how, to what cx- tent, under what circumstances, and in whom tobacco srnoke exposure could contribute to the etiology or pathogcnesis, with a view to countering any such cfiect that may be determined. This aim obviously rcquires studics to elucidate the uages and mechanisms of pathogcncsis. In this context a study was supported by The Council to measure levels of lung kukocyte proteases in groups of persons characterized respectively by normal, intermediate and low levels of alpha,-antitrypsin. The purpose was to determine whether there were differences in protcase levels between persons with and without certain pulmonary functional abnormalities (considered in- dicative of emphysema) within each of the groups or between those within these groups who smoked and those who did not. The protcascs assayed were granulocyte elastase, granulocyte eathepsin and monocyte cathepsin, all of which are capable of damaging lung structure and all of which are known to he in- hibited by alphar-antiprotease. No significant differences in the leukocytc pro- tease levels were found between persons with or without the lung functional ab- normalities or between those who smoked or did not smoke. Thus, no explana- tion of the reported associations between smoking and such functional ab- notmalities emerged from this study. Modified experimental designs may be dc- vdoped for exploring this question further. Another research Ieam, supported by The Council, has been concentrating attention especially upon the proteascs that can be extracted from the poly- morphonuckar kukocytes and pulmonary macrophagcs. These extracts produce emphysema-like lesions when instilled into an isolated dog lung maintained in vitro. Results obtained by this method were shown to be comparablc to those found in living dop when similarly treated. The introduction of protcascs throupls the air passages in the living dog was more effective in producing cm- physema than ia)oction via the blood vessels. A new antrprotcinaso has been recovcred in washings from dog lungs. It differs from the alphar-antiproteinau in the blood and from other known pro- tease inhibitorn, and may function as a regulator of lung protcolysis. In the model syatem, this antiproteinaae prevented the production of emphysematous lesions by clastaae (an effoctive ditcatant of the structural lung substance, elastin), if mixed with an elastare before instillation of the latter into the lung. Similar antiproleasea have beea rocovered in washings from monkey an.l human lungs and are being studied similarly. New Studies of Proteases and Protease Inhibitors Tltree new studies Ia thia area have boen inaujurated. One of these, at the biochemical kvrol, involves study ot two proteaaea isolated from human leuko- cytes, an elauale and a ehyasowrypaia-like proteaae, to characterize their spe- cific aaivities, their fahibitioo or iaactivatioa by the various naturally occurring antiprotcascs and by synthetic ones, and their abilities to break down trachco- hrunchial cartilagc. 'l wo others arc more clinically oriented. In widely separatcd geographical arcas, I:ugc, unsclcctcd populations of newborn infants of various ethnic origins will be phcnutypcd with respcct to the alpha, -antiprotease tnentioncd above. The plra is to conduct rnultifactorial studies of these children and their relatives prospcctivcly over a period of years in the hope of elucidating the interaction ul gcnctic and environmental factors. Conclusion I~resh clucs and concepts have stimulated a strong wave of new interest and activity in this long obscure and baffling field of chronic lung discases and pro- ductrvc rescarch appcars to be reaching an unprecedented level. As The Council participates in this progress, we will be approaching the objective set forth in our 1973 Annual Report: to contribute toward meeting the challcngc posed by the need to establish the etiology and pathogenesis of the chronic obstructive and other pulmonary diseases. RoaeaT C. Hocxerr, Prt.D. Rcscarch Director L 12 13

Abstracts of Reports Following arc abstracts, approved by the authors, of reports on new re- search acknowledging support from The Council that have appeared in scicntific journals since pubiication of the 1974 Report. 7he name of the recipient is in italics. The abstracts are grouped under these headings: 1. Cancer-Related Studies, 11. The Respiratory System, 111. Heart and Circulation, IV. Ncuropharmacology and Psychophysiology, V. Pharmacolo;;y, VI. Immunology and Adaptive Mc- ehanisms, VI1. Epidemiology. I. Cancer,Related Studies CORRELATION BETWEEN BALANCE OF SPECIFIC CHROMOSOMES AND EXPRESSION OF MALIGNANCY IN HAMSTER CELLS In cell culture, abnormal chromosome patterns occur rapidly in hamster fetal (HF) cells after treatment by various polycyclic hydrocarbons. The close association between this ancuploidy and malignant transformation has been dis- euascd, and it has been suggested recently that malignant cell transformation and its reversion are determined by the balance between specific chromosomes containing information for either the "expression" or "suppres,sion" of malig- nancy. In this study. HF cells transformed by 1-p-D-arabinofuratsosylcytosine (ara-C) and dimethylnitroaamine (DMN) were used to see if a specific chromo- some imbalance could be correlated with the expression of malignancy in trans- formed cells. Four ara-C- and one DMN-transformcd Syrian hamster ccll lines were established. All produced tumors when inoculated into newborn hamsters. Speciffe chromosonse changes were observed in these lines which were con- aistent with changes desaibed by other investigators. Clones that had either high or low malignant potential were derived from two fibrosarcomas produced by one of the ara-C-transformed cell lines. The expression of malignancy in these clortes was auoeiatod with an exceu 5, chromosomes over 7, chromo- ,omes. It is importaat to determine it the same relationship between the balance of spocillc chromosomea and the expression of malignancy can be extended to other mammalian species, particularly the murine and human systems. Some of this work is now in progress. Beaedicr, W. F. er al. Journal of rlra Natfonal Cancer lnsrlrrus 34(1) :137-162, 1975. Ot3sar aupportr National Caacer Imtitute. From the Divisioo of Hematology-Oncology, Departrnent of Pediatrics, Chil- dren's Hospita) of Los Angeles; the Pediatric Oncology Program, University of Southern California School of Medieine, Los Angeles; and the Department of Viral-Chemical Oneoiogy, 1llcrobiological Aasociates, Inc., Bethesda, Md. STRAIN DIFFERENCES IN THE RESPONSE OF INI3RED SYRIAN HAMSTERS TO CIGARETTE SMOKE INHALATION Striking diRcrcnccs occur among diflcrent lines of hamstcr: with respect to the susccptihility to acute toxic ef1'ects of smoke and to the hypcrplastic response of the larynx to smoke. In this study, male hamsters, 102 from each of two inbred lines, were exposed to cigarette smoke twice a day, five days/ week (or up to 100 weeks, in a modified Walton reverse-smoking machine. Sixty sham-smoked and 60 cage-held controls were used for each strain. Smoke exposure for up to 100 weeks had no effect on mortality in either strain, but reduced body weight. Carboxyhemoglobin kvels increased markedly imnxdi- atcly after each smoke exposure but returned to baseline levels in less than 24 hours. Serum triglyceride kvels and virus profiles of srnoke-exposed animals were unchanged. Chronic smoke exposure increased relative weight of the lungs and heart in both strains, but to different degrees. Over 90% of the smoke-exposed animals of both strains showed hyperplastic or steoplastic changes in the larynx. However, microinvasive cancer was nearly five times more frequent in one strain than in the other. In the inbred line more stu- ccptible to laryngeal hyperplasia, two animals developed nasopharyngeal tumors, one of which was malignant. Smoke exposure induced rare benign squamous papillomas in the air passages of both strains. The strain less susceptible to laryngeal hyperplasia exhibited more pulmonary adctsomatosis, but its incidusce was not significantly affected by smoke exposure. Bernfeld, P., Homburger, F. and Russfkad, A. B. (Bio-ReJaarch Conrrdranu, Inc.) Journal o/ the National Canccr lnuiru/e 53 (4) :1 141-1157, 1974. From Bio-Rcsearch Consultants, Inc., Cambridge, Masa. MODF_S OF GROWTII AND SPREAD OF A TRANSPLANTABLE, VIRUS-PRODUCING MURINE (MOLONEY) SARCOMA: KARYOTYPIC ANALYSES According to karyotypic analyses, cell clones from a virus-prnducing, uans- plantabk murine (Molortcy) sarcoma line (MSC) contained a aet of stabk, structurally rearranged "marker" chromosomes. These identifying markers were not present in cells newly transformed by murine sarcoma virus (MSV). Tltus, after MSC injection, it was possible to determine if there was any causal na lationship between MSV produced in vivo and the subsequent development of primary and secondary neoplasms. Each adult and neonatal mouse given 104 MSC developed a progressing primary sarroma. Many had secondary pul- monary neoplasms as well, and some neonates developed secondary apken and periosteal tumon. All the 879 metaphase spreads prepared from primary sar- comas and secondary pulmonary neoplasms contained MSC marker chramo- somes. In contnst, cells cxplanted from a perlaateal neonatal mousc turnor uni- formly lacked such markers, even tboujh the primary sarcoma of the same mouse consisted of MSC cells exclusively. Sirtillarly, tsooe of the 180 meuphase spreads from sarcoma virus-indueod primary tuawt>e contained MSC marker chromosomes. Primary sarcomas and seeoodary pulmonary tseopaasnts, the 14 13

lesions most frequently encountered in this system, developed by replication and metastasis of MSC eells, rapectively. Virus recruitment of new tumor cells appears to have only a ntinor role in the spread of sarcomas in neonatal mice. Russell, S. W., Franckc, U., Bucttncr, L. and Cochrane, C. C. lournal of the National Cancer lntriture 53 ( 3):801-806, 1974. Other support: National Institute of Allergy and Infectious Diseases, Nation- al Institute of Child Health and Human Development, National Foundation - March of Dimes, and American Heart Association. From the Department of Experimental Pathology, Scripps Clinic and Research Foundation, and the Department of Pedistria. University of California at San Diego School of Medicine. La lolla. THE ONCORNAVIRUS GLYCOPROTEIN gp69/7l: A CONSTITUENT OF THE SURFACE OF NORMAL AND MALIGNANT THYMOCYTES The gp69/71 glycoprotein, which has group, type and interspecies anti- genic determinants, is one of the important antigens involved in the neutraliza- tion of virus by antibody. In this report, the authors extended their previous studies of oncornavirus-related protcins and showed that gp69/71 is present on the surface of virus-induced lymphoma cells, as well as on virus particles, and the surface of normal thymus cells of some mice. In gp69/71- mice, conver- sion to the gp69/71• phenotype accompanied leukcmogcnesis. An interesting difference in apparent mokcular size of virus-related antigens of the 70.000 dalton size clasa was detected in lymphoma cells present in involved spleens as compared to those found in involved thymuses. Also, it was shown that mice infected as neonates with Scripps leukemia virus make antibody to gp69/71 and some make antibodies to molecules associated with the surface of their own tumors. Results of this study show that ntibody to gp69/71 can react with the surface of three independent structures with rcplicativc potential (vints, rsonaal thymocytes and tumor cells). Such antibody may have different con- aoquencu for oocogeneaia, depending on the localization of the antigen. Del Villano, B. C.. Navt, B., Croker, B. P., Lerner, R. A. and Dixon, F. 1. The lo.wssaf o/ EapsrbnentaJ iiledkine 141 (1) :172-187, 1975. Other support: National Foundation - March of Dimcs, U. S. Public Health Service assd the National Cancer (mtitute. From the Department of Imraunopathology, Scripps Clinic and Research Foun- datioo, La Jolla, CaL CELL-MEDIATED IMMUNITY AFTER INTRATRACHEAL EXPOSURE TO 3-METHYLCHOLANTHRENE, AND ITS IIELATIONSHIP TO TUMOR TRANSPLANT GROWTH IN C3H/f MAI MICE Immstnologfc deficiencioa an often noted In assoctation with cancer, but the eaact nauure of this relatiombip has oot boea fully characterizod. The rela- 16 i tivc irnmunocompelence of an individual, however, definitely plays a major role in the uldmate susceptibility or resistance to cancer. Numerous studies support the concept that it is the cell-mediatcd immunity (CMI) which is largely re- sponsible for thc body's defcnsc against cancer. Here, C31i/f Mai mice were instilled intratracheally with MCA (four 500 yg doses of 3-methykholanthrene in corn oil, at weekly intervals) as part of an investigation aiming to determine the levels of chemicals at which tumorigenesis occurs in various strains and whether the animals' immunocompetcnce is affected. Effects on CMI were as- scssed three days after each treatment by mcuuring DNA synthesis rates withez sli-thymidine in allogencic and spleen lymphocyte cultures. Spleen, thymus and lung were weighed and peripheral blood leukocytes counted. Syngeruic and ~., allogeneic tumor cells were inoculated into control and test mice to determineo whether CMI data are biologically relevant to tumor growth. The CMI and tissue responses were again evaluated 7, 14 and 28 days later. Preliminary data based on spleen lymphocyte responsa to phytohemagglutinin, pokeweed mito- gen and allogencic antigen indicate that MCA suppresses CMI. This effect was most pronounced in response to pokeweed mitogen, however, indicating that the B-lymphocytcs were most afCected. Although only syngeneic transplants were succcssful, lymphocyte cultures from all tumor-inoculated mice demon- stratcd enhancement of T-cell activity and thus CMI, regardless of MCA ex- posure. It will be of intcresl to follow the kinetics of this effect in the hou and compare it to the rate of tumor transplant growth. This study suggests that pul- monary exposure to polycyclic hydrocarbons in mice provides a useful model for characterizing the underlying mechanism of respiratory carcinogenesis and host immunocompetence. Demoise, C. F., Kouri, R. E. and Whitmire, C. E. (Microbiological Associates) In: Karbe. E. and Park, 1. F. (eds.): E.rprrirrknral Lung Cancer. Carcino- gfncsis and Diourrays, New York: Springcr-Vcrlag, 1974, pp. 72-80. From the Department of Experimental Oncology, Viral-Chemical Carcino- genesis Section, Microbiological Aasociates, Inc., Bcthesda, Md. ARYL HYDROCARBON HYDROXYLASE INDUCTION IN HUMAN LYMPHOCYTE CULTURES BY 2,3,7,8-TETRACHLORODIBENZO-p- DIOXIN Recent studies have shown that 2,3,7,8-tetrachlorodibenzo-p-dioxia (TCDD), a toxic contaminant formed during commercial synthesis of the herbicide 2,4,5-trichlorophcnoxyacetic acid, is about 30,000 times more potent than )-methylcholanthrcrx (MC) as an inducer of aryl hydrocarbon hydroryl- ase (AFIH) activity in the rat liver. Furthermore, In contrast to MC or A- naphahoflavone, it fully induces the hydroxylase activity in the liver, kidney, bowel, lung and skin of so-called "nonrcsponsive" mice. The biological half-life of TCDD in the rat is about 17 days, with the induced hydroxy)ase activity and associated cytochrome P,450 remaining elevated for more than 35 days. Thus. TCDD may become a scrious environmental contaminant foe man. Evidence for the appearance of this toxic agent In the food chain has already been re- ported. This report indicates that individuals having genetically lower basal and MC-inducibk hydroxylase activities also have lower TCDD-inducibk bydroxyl- 17