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ANDREWS OFr1CE PRODUCTS CAPITOL HEIGHTS, MD (K) I

1975 REPORT oJ TIIE COUNCIQ. FOR TOBACCO RESEARCII-U.S.A., Inc. TIIE (:OIIN(:Il. FOR TOItA(YY/ Rtat:AR/'11-11.ti.A., loe. 110 t:..l 59t6 11.er1, New Yo.k, N.Y. 1 1N122

SCIENTIFIC ADVISORY BOARD to The Council for Tobacco Re.earch-U.S.A., Inc. aa ot Decembe: 31, 1975 SHELDON C. SOMMERS, M.D., Chalrnmn Director of l.a6oratories, (.eeo: Hill Ho.pitd Clinlcal Professor of PatAolory Co1k~e of Physiciuu ! SurBoo.s of Columbia University New York, New York RICHARD M. BINO, M.D. Diredor of Ca.Jiology aid Intr+rnv,d Medicine Iluetington Memorial Hwpltal, Paa.deaa, California Pro/essor of Med(dne Ueivenity of Southern California ScSool of Medicine Los Ans ela. California JOSEPH D. FELDMAN. M.D. Hed,Depa rtment of lano.nop.tho{op Scripps dioic and Research Fotrdatio. La Jolla. California VVii1.lAM U. GARDNER. Pu.D. Scientific Director, The Council tot Tobacco Reatarch-U.S.A., Inc. E. K. Hunt Professor of Anatow"y (emer(h.s) Yak Univcr.it7 School of Medicine New Havea, C,onoecticvt ROBERT 1. IiUEBNeR, M.D. Chief. Laboratory of RNA Tumor Viru.ea National Canoer Institute Betbesda, Maryland LEON O. JACOBSON. M.D. Dbecto., The Fnnklia McLta. Meawrial Research Institute Regeiuteln Professoe of A/oloslca/ Sclencw Univenitr of Chiup Cbicaso, luisoir AVERIIL A. LIEBOW. M D. Pro/essw a.d CAad.mmn. Departmeat of Pathology University of California School of Medkme San Diego. California HENRY T. LYNCH. M.D. Profes.or and C.hebw.on Ocp.rt..c.t of Phevc.tive Medicine and Public Health (lrcighk,. Urvctrt7 School of Medicine O.aha, Ntbf..ka HANS MEIER, D.V.M., Dr. Med. Vet., M.R.S 11. Senior Staf/ Scientist The Jackson I.abotalory Bar Harbor, Maine LEE W. WATTENBERG, M.D. Professor of Pathology DePartment of Laboratory Medicine and Pathology lJniversity of Minnesota Medical School Minncapolis, Minnesota JOHN P. WYATT, M.D. Director Tobacco and Health Research Institute llniveraity of Kentucky Leaie6toa, Kentucky Seleetiie S1aR.t The C.o..ndl WILLIAM U. GARDNER, PH.D. Scientific Director ROBERT C. HOCKETT, P>t.D. Research Director JO11N 11. KREISIIER, PH.D. DAVID STONE, Pw.D. Associate Research Director . Associate Researcb Director VINCENT F. i.1SANT1, D.M.D. Research Assoclate I

i CONTENTS Introduction . . . . . . . . . . . . . . . . ' . 5 Studies Related to Respiratory Function and Chronic Pulmonary Diseases . . - . 7 Abstsscts of Reporb . . . . . . . . . . . . . . . . 14 Cancer-Related Studies . . . . . . . . . . 14 7 he Respiratory System . . . . . . . . . . 24 1lcart and Qrculatlon . . . . . . - . - . . . 33 NeuropharmaooiM and PsydwQbysioloty . . . . . 47 pbarm.co{op . . . . . 53 Itnatutw{op and Adaptive Mochadsms . . . . . . . 55 Epidemlobpr . . . . . . . . . . . . . . . 62 Active Projects . - . . . 66 Compkted Projccta - - - - - - - - - - - - - - 73 Indc: of Principal Invcstipton . - - - . . . . 83 lndei of Senior Authors . . . . . . . . . . ' . . 84 Introduction The Annual Report of 1973 had one of ib seven aoctiona dcvolod 1o the presentation of some research on hmg functioo and chro.ic respiratory dirr eues. During the past two years. studies on respiratory functioru of nun and I laboratory aniat.ds bave advanced greatly. Sophisticated biochemical rnethods i have been used 1o study Ihe defense mechanisms of the lunp, their response to Ihe internal environment as determined by the genetie background of the i I like-seaed twim, but also the Iwins as Individuals making up a crore section o4 the populations. Puheonary function of populations at different ages and different arere of residence and diAerent oecuPalions hae been detetmincd and the eAects of familial or hereditary In/luencaa have been described and discussed. New observalions have also been made on lung slructurc; neuroepMhellal bodies have been found is the human lungs and described in detail. ihcir function Is not known with certainty. They probably contribute to the normal function of 1he lung. Even at this tine, new Mtucturee and potential regulatory mechanisms of lunp, which provide that narrow interfact between envitoa ment and blood, can atdl he discovcrcd. i The following review of the studies related to reapitalory function and chronic pulmonary diseases reveals the interests of The ('ouncJ in tbeae anaa sub/ect and theur response b eaternd rrnwh. The aburacu of the tnvesliptioem that have bten supported during 1973 in this and in other research areas .re included in the third section of this report. lhe previou. Annual Report emphasized the researches that were related to cancer and to the considerable effort that has been made so develop mnhods for the delivery lo, aed rekntion of smoke or .moke components in, different Parts of the respiratory tracle of mioe, the animab that had beea aelected fot the snwke eaposure eaperiments. Thir year i Annual Report emphasitea Inve.tiptiosr that have been u.dsr- taken on the respiratory systern and on the epidemiology of respiratory di.- easee. In some ways the classilkatiow ie eomewhat arbitrary. For eaunpk, em- pbysenu might be eoraidered under the respiratory ayslern, but becatw masy studies on this d'aease involve hereditary and environmental componeds, otrtals studies might be clnsiAed wader epiderniolop. The rapid developnKN 1s theae arer during the but few years led The Council 1o convene a twoday idornal conference during 1975 at which a number of investigators coneerned with the luafe proaeia-destrqring en:ynw .nd their inhibitors had an opportu.Yy to communicate and compare their moel reeeat investigations. Tlr aubjocto di.- cus.ed in detail are presented in the following aection of Ihim reporl. Identical twin., like laboratory animals of highly Inbred Nrdr. Provide the best meaae of dclamining the imPact of the environment on the genotype. When enviroemental factors are direordawl the eapression of the oenotypc nray be modilled. TM New Swedish Twin Registry - twins born in Swedcn from 1926 1o 1959 - hae been completed with some 32,000 eMrica of like-eeaed twina. A FisriaA Twin Registry has beew eospibd and anuch ksforsnatlon i being obtained through responses to aueetionnaites. The contrasting Mcidcaoes of pulmonary cancer and coronary heart disease in Sweden a.d 1. Finland should add ddniAeanoe so the dala that may be derived rrom Mudia of thne registries. The Nudia wiN involve not only the identical and the .o.-idedical. t J 3

static clastic recoil pressure, total pulmonary resistance and flow rates, forced deflatKrn, and nsorphunsctry for mean linear rntercepts and intcrnal surface area, as well as general pathological and histological evaluations. In summary, no sitnilicant ahnurmalnies were observed in the animals exposed to dust with or without nitrogen droaides lhis study dNd not produce an animal model (or emphysems. In another study by a different investitator, hamsten were exposed to long-term chronic cigarette smoke inhalation but gross emphysematous changes were not seen. Studies o/ Osone Orone, a powerful oxidant and a very prevalent and active constituent of atmospheric smot, can severely damage the lung as can exposure to abnormally high levels of oxygen. A scientist sponsored by I he Council undei took to study the cflects of these agents in rats and rworskcys in order to discover what changes occur in the lung meubdrc and enzynse syslersu and how they might be pre- vented or reversed. When the study is compkted, dse informalion obtained shouW be useful as background for analogous studies of tobacco unoke inhala- tion and provide a basis for observationa on possible interactions between to- bacco smoke and smog. 1 wo drstinct aspects of ozone effects were found. Short•te: m, high-level c.psnures 12 to 4 p p m I for two to tight hours destroyed large Ixoportans of the suilhydryl compounds present in different portans of the lung cells, with profound lowering in Ihe levels of enzyme activities involved iu cell division and repair. In contrast, exposures at the level of 0 2 and 0.8 p p m. (or two to seven days produced no deuructwn of sullhydryl compounds or depression of these enzyme syslcros, urstead. the activity u/ the systems was increased. thcse etlects of low Icveh of ozonc may ecplam in part the tolcrance or adap- Iation to such eapusures that thrs scientist and oncers have noted in animals and in man Morphr,tosiscal studies have parallelcd these hsaxhemrcal uhscrvations. laposures to relatively high concentrations of oxygen are being made for comparison, and studies of tobacco snsokc inhalation by rats with or without previousozorse espvsures are now under way. Preliminary eapcriments with mke, employing realistic dosages of cigarette smoke daily fur three wceks, showed no histological evidence of maior patho- logical change. lhe lunp, however, had higher levels of specific activities of the antiosidant enzymes, glucme-6-phosphate dehydrogenase. Slutathione re- ductase and gluuthione peroaidase, which may refket adaptive response to oai- dants present in the smoke. Other lung biochemical asoys have been added to the protocols; for esampk, the effects of o.idants on collagen synthesis. Pulma nary fibrosis is olten a sequel lo long-term chronic respiratory damage by such agents. De/en.e Mec/h.ni.n.s o/ tbe Lung A previous Annual Report (1977) mentioned studies on the defense me- chanisms of the respiratory aract against rnhakd partrculales, both inanimate 'du.ts*' and mKroortannms, especially by mobilization and functron of pulmo- n.ry roacri-phatcs Rates of clearanCe for aspirated SwOhyfoCocCW aureYJ hare 0 a been measured as well as the mobilization following such aspiralions, of creased numbers of recoverable macrophages. 1 he effects of tobacco smoke halation upon rates of disappearance of viable orpniams from Ihe lung /a , have been reported, and Ihe total engulfinent and killing activity of macrophr populations recovered from amokes.posed animals are now being dctcrmrr in vitro. In jluensa In jeetlona Influenza idections, atili prevaknt, resuM in significant persfsknl Ir damage in man. A veteran investiptor of this problem has published a detu description of the sequential effects of influenza PRII-A infections In mi Wi1h the advantt#es provided by the opportunity to sacrifice anirnds seri. at all stages of thz process, these observations generally have conflrmed and tended those avaiJabk frons human por-nso.krn e.aminatiorsa. A scparuc view applied Iheat findinp 1o interpretatioe of potentially serious ovesequen of human hsthrcrsa and suggested preventive meaaures that should be appl to prevent such iafectioos. A tnethod for producing rrsore consistently a persistent vitami. A defkie, in mice made it possible to study the consequences of such deficiency upon aequelae of infection. Squamous metaplasia and keratinization of the broncl membranes were significantly greater in the animals with little or so heps vitamin A and the pou-infection lesions showed more e.teesive epthr noduk formation. These and observations from othcr quarten have renev interest in relationships betweea this vitamin and cancer susceptibility ot sistaoce. Clinical Surtreys oJ Lung Frnetion "Y'aptive" populations in the ftoaoa area and on she West Coaat are 1 viding the opportunity to describe relations of age, sea, raos. Ii$M unok, heavy sewking, industrial exposures, aad other factors to pulnso.ary functr changes over an extended period of time, through repetitive resaami.aioaa Respiretory Distress Syndrome o/ Newborn In/enta Ilyalrne membrane disease in newborn infants, associated witb prcrwatu, maternal diabetes and complications of pregnancy and deiivery, produces I mortality. It is due to IaMure of the infanl lung to expand propesly dta livery and ia usually accompanied by formation of a"hyaliee rnembrane" I structure, observed in the alveolar spaeea, ducts and bronchioles, coasisu of generated lung surface cells and blood ekmenls, and contains large .mount fibrin, an elastic, thread lrke, insoluble protein material. The ate{ectasia (r expansion) has been ataributed to a deficiency in wr/actant and the flbtin cumulation to a detective balance between the systems normally rapoor for its formation and its removal. 9 I

Abstracts of Reports Following are aburacts, approved by the authors, of reports on new re- search acknowledging support from The Council that have appeared in scientifk 'ournals since publication of the 1974 Report. The name of the recipient is tn italics. The ab.tracts are grouped under Ihese headinp: 1. Cancer-Related Studies. 11. The Respiratory System. Ill. Heart and Circulalion, IV. Neurophwrmacolopr and Psychophyuob6y, V. Pturta.co/oRy, VI. Immunology and AcJaptive Me- chaniams, VI1. Epidemiology. 1. (',.wc.r.R.l.t.d Stadiea CORRELATION BETWEEN BALANCE OF SPECIFIC CHROMOSOMES AND EXPRESSION OF MALIGNANCY IN HAMSTER CELLS In cell cullure, abnormal chromosome patterns occur rapidly in hamster fetal (HF) cells after Irea/mesd by various polycyclic hydrocarbons. The close association between thia aneuploidy and malignant transformation has been dis- cussed, and it has been suynled recently that malignant cell Iranslormation and tts revenion are determined by the balance between specdic chromosomes containing information for either the "espression" or "suppression° of malig- nancy. In this uudy. HF cells transformed by 1-/l-D-arabinoluranosykptosine (ara<;) and dimelhylmrro.amine (DMN) were used to aee if a specifk chromo- some imbalance could be correlated with the expression of malignancy in trans- fonned cells. Four ara1;- and one DMN-transformed Syrian hamster cell lines were established. AN produced tumors when inoculated into newborn hamsters. SpeciDc chromosome changes were observed in these lines which were cort- sntent with changes described by other investigaton. ('lones that had either high or low malignant potential were derived from two Bbrosarcomas produced by one of the ara-C-transformed cell lines. The expression of malignancy in these clones was associated with an excess S, chromosomes over 7. chromo- sornes. It ia important lo determine il the same relationship between the balance of speciAc chromosomes and the expression of malignancy can be extended to other mammalian species, particularly the murine aod human systems. Some of this work ia now in progress. Ornrdkr, W. F. rr d. lournal o/ the N.Non.l C.nrrr lnrtlrrrt 34( I):157-162, 1975. OtAoe srrrortr National Cancer Irlilute. From the Division of Hematology Oncolopy. Deparlment of Pcdialrics, Chil- dien's Ilo.ptal of Ins Anjcks; the Pediatric Oncology Pro4ram, University of S4wthern ('a6fornia School of Medicine. l.os Angeks; and the [kpartment of Vual Chenucal Uncoloty, Mucrobwbgical Associales, Inc.. Belhesda, Md. I I I STRAIN DIFFERENCES IN THE RESPONSE OF INBReD SYRIAN HAMSTERS TO CIGARETTF. SMOKE INHALATION Striking diAerences occur among different lines of hamslen with respect to the susceptibility to acute lo.k effects of smoke and to the hyperplauk response of the laryna to smoke. In this qudy, male hamsten, 102 frore each of two inbred lines, were exposed to cigarette smoke twice a day, fiva darsl week for up lo 100 weeks, in a modified Walton revcrse-unokinR machine. Sixty sham-unoked and 60 cage-held controls were used /or each urai.. Smoke exposure for up so IC11 weeks had no effect on mortality in either draiw, but reduced body weight. Carboxyhemoglobin levels increased markedly innrodi- ately after each smoka expowre but returned 1o baseline levels in ler than 24 houn. Serum Iriplyceride levels and virus profiles of smoke-eaposed a.imak+ were unchanged. Chronic smoke exposure increaaed relative wc*M of the lunp and heart in both uraies, but /o different degrees. Over 9" of the smoke-esposed animds of both strains slwwed hyperplaatic or .eoplartic chanRes in Ihe larytu. However, microinvasive eancer was nearly Ave ti.w more fretJuenl In one strain than in the other. In the inbred li.e tnae su&- cepttbk to laryngeal hrperplasis, two animals developed naaopluryngesl tutwot., one of which wr mdisnant. Smoke exposure induced rare beni6n squawwtr papilbmas in tha air paaagen of both uraios. The alraia less suaceptible lo laryngeal hrperpluia cshibiled more pulmonary .denomatosis, but Na Lcide.w was not si6nilkastly affected by suake exposure. Bernfeld, P., Hornburyer, F. and Ruulkld, A. B. (elo-Rei..rcA Cowrrbaar., /nc.) lournn/ oJ rhe Narlon+t C.ncrr lnuirrre 33(4):1141-1137, 1974. From Bio-Research Corodtanu, Inc., Cambrid6e, Mau. MODES OF OROWi'H AND SPREAD OF A TRANSPLANTASLA VIRUS-PRODUCING MURINE (MOLONEY) SARCOMA: KARYOTYPIC ANALYSES According 1o karyotypie ..alYses, cell elones from a visua-producia& tra..- plantabk nsurins (Moloney) sarcoma Nr (MSC) eontaised a aet of d.kla, structurally rearranged "nurker° chronwaorees.'TAese identifying m.rken were not present in cells newly transroneed by reurine sarcoma virus ( MSV ). Tlur, after MSC injection, il was possible to delermine it there was any causal tw lationship between MSV produced in viro and the sub.eyuent development of primary and aecondary neoplasms. Each adult and neonatal mouse {ivea IM MSC devck.ped a proprenin{ primary sarcoma. Many had aoco.dary put- monary ncopla.ms as well, and soate neonates developed secau.dary, ap{eee snd periosteal tumors. AH the •79 metaphase spreads prepared from primary s..- comas a.d secondary puhnonary neoplasms contained MSC masker chronw- sornes. In con/rast, cells explanted (rom a perbsteal neonatal mouw (unsor uni- formly lacked such markers, even thoirXh the primary sarcoma of the s.... mouse consisted of MSC cells exclusively. Sirnilarly, none of the 140 metaphaw spreads from sarcoma virus-induced primary lumora contaised MSC murkcr chromosomes. Primary sucomas and secoodary pulmonary ooopiaads, the IS 14

i INHIBITION OF DNA REPLICATIVE SYNTIIESIS AND DNA REPAIR SYNTHESIS IN HUMAN AND MOUSE CELtS IN CULTURE BY CI(;ARETTE SMOKE CONDENSATE FRACTIONS As part of an investiptioo Into the mode of actioa of lumor promoten, a study was made of the eRens of cigarette smoke eondeosata (CSC) frac- tions on replicative DNA synthesis and DNA eacision repair syolhesis. A total of 12 CSC fractions were tested for their abilily to inhibil these functiooa is cultures of human Abroblasts and Srrir twoare embryo cells. None of the /rac- tions showed say specificity toe Iha WMtion of DNA repair aed, in Aeneral, repair synthesis was less acaeitlvs to iohMioe tlan was rsplicative synthesis. Tbere was aowu: correlation between Ib WibMory, actioa of tbs variow /rae- tioas and their activity in bioassays perforrned in other laboratories, including in vitro cetl traroformation and baclerial arutapeoicity. 1s most instanues. DNA synthesis was more sensitive to i.hibitioo Iw tha huwun ce8s than is the mour cells. lTr specific compounds re.po..ibM for ,he activity of the condensate Iractiom hsv..o1 been ideetitled. Ranuusen. R. E. L!/e Sciences 17(S):767-77), 1973. From Ibe Cancer Research IosUtwa, University of California. San Francisco. MFASUREMeNT OF NITROOEN OXIDES 1N TOBACCO SM1)KE BY MEANS OF THP. CHI?MILUMINE9CENC13 M13THOD Chemical analysis of fresh lobaeco raoke to determine the ronteot of nilrosamines that may be prescN under the conditions of actual hunun smok- inA has been ddficutl with traditional methods because of the tirtre reQuired to carry them out. Formation of precursors neceswry for oitro.amine produc- tion in the presence of aminea occurs only by tinte depeoden( resctioos, so that artelactual formation duriaj the oourtr of smoke analysis by older methods has appeared very probable. lo order to obtain reli.ble bask data, it was ooe- sidered necessary Arst to rehvestillNe 1ho kinetics of the precurso. reactions under the conditions actually present is smoke. T3e actual nitrosating a" for twnioa Ia believed to be dinitrogeo trl- oaide (N,O,). an unstable compound which Y oot present in tresl.ly formed tobacco ssaoke but formed only when .quiraolecular proportioa of aNric oaide (NO) aod of nitroAco dioaide (NO,) are present aimult.aawdy. The authas underbok, r a Ars1 atep, b rtatwn the concentratiorn of tba /wo latter osides of nitrogen in sreoke at a brief bul carefully deAned Interval fol- lowing its formaticM+. Earlier work by diffkuk methods had Indicated that NO is by /ar the predominating oaide of oitrogeo In fresh smoke (Norman and Keith) and is only slowly osidizod to NO, under actual smoking conditions. TAe present authors undertook to repeat such determinations by use of the recently developed chemilumineseence method which can determine NO auao- trtativrly- and almo+l in+lantaneoudy, in the presence of NO, It should. tfirrrl..re. he m.w h k.+ LlorMws, rnwe accurate and allow better control of the tmv 1aMVN 1hu nrw trthntq..e depeod+ upon the selective oxrdauon of Nt1 h. ..r..nr U. I...m rk•tt..o..allt •.uord NO,..hrch rcacuon is tmmediate- ly followed by the unique and measurabk emission ol light in the 0 6-3 pru wavelength region. Thirty-uven diRerent tobacco products were thus tested to determine the NO content of their freshly produced smoke. Thn ranred from a minimum value of 190 ag/mt of fresh smoke for Aue-cured tobacco cigarettes to a mawl- mum of 1,300 nA/ml of smoke from Spanish black tobacco cigarettes. In Aen- eral, high values were obtained from cigaretten made from air-cured (burky, black, and cigar) tobaccos and low values front 1ho,e containing mainly Aue- cured or oriental types. Following this type of specific NO determinatiow in the total smoke mis- ture, which may also contain smaR amounls of NOf at the defined time of the assay. the mi.ture can be passed through a catalyst 1o reconvert all the naro- lien oaides back to NO. By immediate reappliestion of ehe chemiluminescence procedure the total o.ides of nitrogen can then be estimated; the differeoce be- tween the two assays serving as an assay for the original small content of NO, present at the time of the initial assay. Systematic eatension of the present studies to this and other aspects of the total problem is contemplated. Nerra6. G. e. and DOoger, M. In: Bo6ovski, P. and Wa:ker, E. A. feds.): N-Ninoio Compounds !n the Es- vironmenr. rroceediwp of the Wor4inp Con/erence Held ar the Inrern.tbnd Apency for Research on Cancer. Lyon. France 17-IO Ocrol.rr, 1973. Lyoo: International Agency for Research on Cancer, 1973, pp. 177-179. From the Microanalytical Laboratory, Hamburg, Federal Republic of Germaey. Q-NAPIITHOFI.AVONE ACTIVATION OF 6-HYDROXYMETHYI. BENZO(o)PYRENE SYNIHETASE In their series of studies on the properties of aryl hydrocarbon hydroay- methyl symhesase (IfMS). Sloane er .t. have demonstrated that model ao- maic compounds could be metabolized to the aryl hydrosymethyl derivative by either the direct hydroaymethylatioo of the benzene ring or by osidalioo of the aryl side<haio methyl compound. These studies showed that the methyl &roup hydrosylatioa was mediated via eytoehrome P-4SO, whereas the 11MS reaction was independent of this pathway. Of interest here is the fact that 6- hydrosymelhylben:o( s)pyrene, a metabol'Ne of hen:o( a )pyrene, has been shown to be carcinogenic by several sels of investigators Now the preseat ,uthor re- ports that a-napthoAavone activates the aryl IIMS ol both the microrowral membrane-bound and soluble enzymes of ra/ liver and rat lung. The enzyme catalyzes the hydrosymethytation of benru(s)pyrene to the 6 hydroaymcthyl derivative. , Sloane, N. H. Cancer RereorcA )S:)7)1-)7)1. 1975. Other srpportr American Cancer Society. From the Department of Biochcmisuy. Univcusuty ol ienr.esuc Cenkr (or Heahh Sciences, Memphis. .21 20 I

Only very recently it was discovered that normal human embryonic lung fibrohlast celis (and those of many other mammah) regularly produce high levels of plasminogen aclivator. This is a protein that converts plasminogen, an inactive substance present in the serum, Into plasmin, an enzyme that destroys fibrin. The aerum from infants with hydine membrane disease is deficient in plasminogen though it is not cksr whether this is due to the continued produc- lion of the activator or to eacessive inhibition of the plasmin produced from it. In a Council-supported project, piasminogen activator is being prepared in quantity from cell cuitures; it will be concsntrated, purificd, characterized and used so produce labeled antibodies for studying its presence and conccntration at specific sites, and its roie in normal eells as well as in disease states. Relevance of this work estends beyond the problem of the respiratory dis- tress syndrome. It ha. reteswly bee. discovered that normal cetls, other than the esceptional ones of embryonic ha.jL though they quite generally produce very little plasrnirwlles aclivator, will aearly always increase this production at least 3Pfold when transformed into the malignaM staN. Indeed, this property may be a useful indicator of malignant cell lransforwution. Such transformed cells show a number of chanees in behavior. Tlrey will grow oa soft apr (unlike mosl normal alh), they show incre.sed mipation. they are agglutinated more readdy, they display an altered morphoiopr, sd they produce tumors if trans- planted into mice with low or irnpaired 'weamtee nspomiveness. While normal embryonic bng cells show a similar ability to grow on soft a=ar, they do eot show the altered morphology or produa tumors when so implanted. Ingenious systenr have been designed and applied for studying the bio- chemical c.orditiom within the cell that govern the production of plasminogen activator and iu accumulation or drsposal is normal lung cells as compared to cancer eelb. Protesea end Protea.e /nhi6itors in Reletion to En.phy.em• TAere are subuantial iodications that human pulmonary emphysema may result (rom "di{estion' of the lunlfs elastic structural material by enzymes /fxrxe..n) that attack the proteim of which it ia composed. One indication is that papaisi, a plant protease miature that does not occur naturally in animda, wip nevertheless produce destructive lesions similar to emphysema when in- stilled directly into the luop of animals of several species. Another, reported only a few years allo, is that persons having an ab- normally low blood level of a protease inhibilor, originally designated as alpha,- antitrypsie•, ue particularly likely to develop emphysema at an early age. This observation reinforced the idea that the disease might be caused by p.oleases from some sourse, either internal or eaternal, if or when their action is not properly controlled or "wrned otT" by an antiprotease. A third, still more recent indicatioa is that certain human or dog blood kukocy/es, particularly polymorphonuckar kukocytes and macrophaloes from the peritoneum or lungs, when isolated, eoncentrated, broken down, susperded in a medium, and imtilkd inlo d" lunp, produce emphysema. The eatent of the lesions produced is dependenl upon the prolease activity of du preparalions, •SGnic i tug onh.h.oow is n..4 .trcAk fow to ypwln but bon,ls n numhcr of p.otta.r., alpba.- •MOIN.+t.s n a Mw. wu.-t I I I I and those from polymorphonuckar kukocytes are more active than those from macrophages. Both types of cells concentrate in the lung in defense against in- hakd particulate matter, including infectious organisms. 1 hey contain powerful proteolytic enzymes packaged in small vesicks inside the cell walls and had not been considered likely to harm the lung structure under normal conditions. Thc new e.perimenls, therefore, raised the questions whether enzyme leakage from the kukocyte cells or disintegration of these cells within the lung might bring the prodeases into direct contact with the lung tissues and thus contribute to pathogenesis of emphysema under real life conditions, if not properly countered by protease inhibiton available in adequate concentrations in the critical areas. An overproduction of leukocyte proteases or an abnormal access to critical zones might contribute so the process. A Delie.te Bel.nee A delic.ne balance between proteofytic and antiproteolylic activitia i. Ihe lung is necessary (or defense against infections, but damage to the lungs t6em- aelves might occur if the balance is disturbed either by overproduction or eaa+- sive release of prole..es, or by underproduction or inadequate access of in- hibilon. Ealensive studies by many investidators, including several sponsored by The Council, have shown that alpha; antitrypsin deficiency follows deNnite hereditary patterns in human families. T1rcse studies support the concept of an inherited susceptibility lo emphysema and provide one erplanation of the em- pirical observation that the disease tends to "run in families." The picture is complicated, however, by the e.istence of several variant forms of this a.ti- protease, which differ is their efficiencies of antiprotease activity. At least twenty different phenotypes have been described that represent the various possible combinations of the genes controlang Me alpha; antiproteaae types and produc- tion. Moreover, there appear to be familial aggregations showing high em- physema predisposition that is unrelated to this antiprotease, suggesting that other factors awt yet described may be invdved. lhere is no reason to suppose, for eaample, that aruiproteases other than the serum alpha -antiprotease aad its variants may not eaist in other organs and play a role in t6 total picture. Ad- ditionally, the role of lung surfactant .nd its effect, it any, upon proleaas and antiproteases is snot fully understood. ' Several grants by The Council have fostered studies of protessea aurd anti• proteases in relation to emphysema. Much effort has been devoted to puriAca- tion of serum alpha, -anliprolease and its variants so that their Nructurd aaid molecular weighls could be delennined, the sequential arrangement of the com- atiwent amino acids worked out and the biochemical nw.des of action ir the inhibition of protease activity described. R.diolabeled antibodies have bees used to show where these inhibitors bind to tissues. In these swdies, wmp.ri- sons with other protcase inhibuors from different sources are helping to define and describe their likenesses and differences and especially their specific actiw.s The isolation and description of proleases elaborated from lung macto- phages and granulocytes have also been undertaken to find out which onee ara musl active in attacking lung structural proteins and how they react with is• hibrtors Ihis has entailed eaplwation of mdhoda fur collectrng, scp.rating and culturing Ihese several lypesof cells. II

I Detecting the Em phy.ema-Prone Individual Avowed aims of these investigators are to develop hetter methods for de- tecting high emphysema prcdisposuron in individuals, materials for early du`no- sis of the disease and, it is hoped, agents that might bolster defensive mechan- isms in the body and arrest progression of the disease. A related but compatible aim is to determine whether, how, to what es- tent, under what circumstances, and in whom tobacco smoke eapusure could contribute to the etiology or patholie.esi% with a view to countering any such effect that may be determined. Tlris aim obviously reQuires studies to ehscidate tlse uages.nd mcchanams of pathoK.esis. In this contest a study was supported by The Council to measure levels of hurg kukocyle proleases in groups of persons characterized respectively by normal, intermediate and low levels of alph.,-antilrypain. The purpose was to delerwrine whether there were differences irt protease kvels between persons with and without certain pulmonary functional abnormalities (considered in- dicative of emphysema) within each of the groups or betweew those within these groups who uooked and those who did ssw. The proteases asuyed were granulocylt elastase, granutocyte calhepsim and arooocyte eathepsin, all of which are capable of damaging lung structure and aN of which ate known to be in- hibited by alpha,-anlprotease. No signiAca.t differences in the kukocyte pro- tease levels were found between perwns wilh or without the lung functional ab- aorrsulities or between those who senoied or did not smoke. Thus, no esplana- tion of the reported associations between nssoking and such functional ab- normalitrn emertled from this study. Modieed eaperirnental desipu may be de- veloped for espbring this question further. Another research team, supported by TLe Council, has been concentrating aqention especially upon the proteaaes Ih.t can be eatracted fronm the poly- rnwphonuckar kukocytes and pulmonary macrophages. Theae esuacts produce emphysema-like lesions when instilled into as isolated dog lung maintained /n vbro. Results obtained by Ihis method were shown to be comparable to those fowsd iw living dogs wben similarly treated. Tho introduction of proteases through the air pasuges (s the living dog was tnors effective in producing ao- physema than isjection via the bbod vessels. A new sntrproteiwass has been racovered In washinp from dog lunp. It differs from the alpha,-antiproleinase iN the blood and from other known pro- teasn inhibitors. and may function as a regulator of lung proteolysis. In the model system, this antiproteinase prevented thr: production of emphysematous kaioas by ela.taar: (aa effective digestant of Ihe structural lung substanu, elastirt). it rnised with an tlatase before instillation of the latter into the lung. Similar aMiproteasea have been recovered is washusp from monkey and human lungs a.d are being studied similarly. antiproteases and by synthetic ones, and their abilities to break down trachoo- bronchial cartilage. Two others are more clinically oriented. In widely separated roliraphical areas, large, unselected populations of newborn intants of various ethnic origins will be phenotyped with respect to the alpha,-antiprotease mentioned above.l he plan is to conduct mulufactorial studies of these children nd their relatives prospectively over a period of years in the hope of elucidating the interaction of genetic and environmental factors. I (:onclu.ion Fresh clues and concepts have stimulated a strong wave of new interest and activity in this bng obscure and bdlling field of chronic lung drseases and pro- ductrve research appears to be reaching an unprecedented level. As The Council participates in dws propess, we will be approaching the objective set forth is our 1973 Annual Report: to contribute toward meeting the chalknge posed by the need to establish the etiology and pathogerrcsis of the chrooic obdructive and other pulmonary diseases. I I New Studies o/ Protewa.a .nd Proteaw /nhibitor. ibree ncw swd'ws in this area have been Inaugurated. One of ttse,e, at the biochemical kvel, r.volves study of two Vrote..es iwlated from human kuko- cytes. an elwaas aad a cAysorrypre hke protease, to characterum their spe- cdfc .crrvqres, tAcw rwhdaaa+ or lnaetivalan by the various nalursfly oceurrisg Ro.e.r C. lloc[aTr, hs.U. Research Diroesor 12 ' 13

lesions most frequently encountered in this system, developed by replication and metastasis of MSC cells, respectively. Virus recruitment of new tumor cells appears to have only a minor role in the spread of sarcomas in neonatal mice. Russell, S. W., Francke, U., [luenner, L. and Cochranc, C. G. lorrwd o/ the Nurionaf Cr.ccr lnnlhwtt 3)()):f101-d06, 1974. Other arrrorf: National Institute of ANerp and Infectious Disea!es, Natior- ai Institute of Child Iledth and Hw..n Development. National Foundation - March of Dimes, and American Heart Asaociation. From the Dep.rtment of Eaperimen/al Path~. Scripps Clink ard Research Foundation, aed the Department of Pediatrica. University of Cslifcrnia at Saa L)rego School of Medicine. La ldla. 711E ONCORNAVIRl1S GLYCOPROTEIN gp69/71: A CONSTITUENT OF 7HE SURFACE OF NORMAL AND MAUGNANT TIIYA(OCYTES The gp69/71 Slycoprotein, which has group, type and inkrrpeeies anli- kenic determinanta, is one of the importaM asMigeas involved in tir: sicutralira- tion of virus by antibody. In this report, the authon extended th:ir previous audies of oacornavirus-reialed proteins ard showed that gp69/71 ii present on the surface of virus-induced IympMma eeds, as well as on virus puticks, and the surface of normal thymus cells of noene mice. In gp69/71- rnice, conver- siors 1o the gp69/71' phenotype accompanied kukemogenesis. An interesting difference in apparent molecular size of vinu-related antigens of the 70,000 dalton site class was detected in lymphoma cells present in involved apkens as compared to those found in involved thymuses. Also, it was shown that mice infected as neonates with Scripps leukemia virus make antibody to Sp69/71 and some make antibodies to mokcules aasociated with the surface of their own tumors. Results of this study show that antibody to gp69/71 can react with the surface of three independent structurea with replicative potential (virus, eoreral thymocytee and tumor ce1M). Such uMibody may have diAercnt coa- seque.cee for oocornesw, depending on aho locdialioo of the antillen. Del Villaeo, /. C., Nave. 11., Crokee, 1111. P.. Lsrner, R. A. and Diaon. F. 1. TAe lorrssd o/ Erperbwenu./ k/elkine 141(1) :172-1 87, 1975. Other.r'rortr National Foundatioe - March of Dimea. U. S. Public Healtb Service a.d Ilse National Cancer IrtitulY. Prom the DepartmeM of /mmuoopatholopr. Scripps Clinic aad Research Fou.- dalio., La )dla. Cal. ('P-I.L-MEDIA7F.D IMMUNITY AFTER INTRATRACNEAL. EXPOSURE TO I-MEil1YLCHOLANTNRENE, AND CTS RELAl1ONS11IP TO TUMOR TRANSPI.ANf GROWTH IN C)11// MAI MIC@ ImmunoM(ic deficiencies are often noted In association with eanur, but the esact nature of this relationship has not beea fully cbaracterired. lbe eela- 16 i live immunocompetence of an individual, however, definitcly plays a mapr role in the ultimate susceptibility or resiuance lo cancer. Numerous studies support the concept that it is the cell-mediated immunity (CMI) which is largely re- sponsible for Ihe body's defense against cancer. Ilere, ('l11/1 Mai miu wen inslilkd inlralracheally with MCA (four 500 rg doses of l-me/hyklsolanlhrene in corn oJ, at weekly intervals) u part of aa investigation aiming to deteraune the levels of chemicals at which tumorigenesis occurs in various saraies and whether the animals' immunocornpeleace is affected. Effects on CMI wen r- sessed three days after each treatment by measuring DNA synthesis raks with sH-thymidine in allogerseie and splecn lymphocyte euNures. Spleen. Ihymus aod hmit were weighed arw) peripheral hlood kukocytea counted. Syngeneic and allogeneic tumor cells were inoculated into control and test mice to determine whether CMI data are biologically relevant so tumor gtowth. Tlre CMI and tissue responses were aeain evaluated 7, 14 and 2!i days later. Preliminary data based on spleen lymphocyte responses to phytohemagglutinin, pokeweed mito gen and albgeneie anli,len indicate that MCA suppresses CMI. -Lhis effect was naw pronounced in response to pokeweed miloden, however, indicating that the e-lymplsocyla were most affected. Although only syndeneic transplants were successful, lymphocyle cultures from all lumor-inoculated mice demon- Mrated enhancement oC T-cell activity and thus CMI, regardkss of MCA es- po.ure. It will be of iscerest to follow the kinelics of this effect in the bost ud compare N to the rale of tumor transplanl growth. Tlsis study suggests ahal pul- tswaary exposure to pdycyclic hydrocarbons in mice provides a useful wtodd for characterizing the undalyinR mechanism of respiratory carcinogeeetis and hoel immunocompeleace. Demoise, C. F., Kouri, R. E. and Whitmire, C. E. (Microbloloil,k.f Aaaoclwa) In: Karbe. E. and Park, 1. F. (eds. ): E+rrri+nrntal lynt C.ncer. CercMo- ptnesL awd sioars.yr, New York: SprinlIer-Veri•g, 1974, pp. 72-d0. From the Departmen/ of Experimental Oncology. Viral-Chcmicd Carcieo- getsesis Section, Microbiological Associales, lac., Belhesda, Md. ARYI. IiYDROCARBON HYDROXYLASE INDUCTION IN HUMAN L.YMPIIOCYTE CULTURES BY 2,1,7,t-TETRACHLOROD19ENl(lr DIOXIN Recent studies have shown that 2,),7,11-lelrachlorodibenro-rdbai. (TCDD), a toxic contaminant formed during commercial aynlhesis of tAe herbicide 2,4,5-trklslotophenosyacelie acid, is aboul 10,000 1imes sran po1eN than 7-methykholanthrene (MC) as an inducer of aryl hydrocarbon hydrosyl- ase (Alll/) activity in the ra1 liver. Furlhermon, In contrast so MC or a naphlho/lavone, it fully inducqs the hydrosylase aclivity in the livsr, kidney, bowel, luns and skin of so-called "nonresponsive" mice. l he biological hal/ life of TCDD in the ral is about 17 days, with Ihe induced hydrosylase aclivily aod associated cylochrome P,4S0 remaining elevated for more Ihan )S days. lbus. TCDD may become a serious environmental conlamsnant for m.e. Evidence for the appearance of this losk agent in the food chain has already been n- porled. lhis report indreales that individuals having genetically Mwer ba.al and MC-inducible hydroaylase actwitres also have lower ICDIDrnducibk hydiosyl 17 0