Tobacco Documents Online

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m Ln ~- m a m J co W TIIE COIINCII. FOR TOBACCO RFSEARCH - U.S.A.

MA ~deatibe lldvboiy aoud, e.~otlr ao.aittie+)< of teD .cientists and ; ~ i:. dt3tton who tssalntaln tbdt rcapoctlve Inatltstlioosl tt~'iliatlons, has full t^••. 1'' reapoesibility for rerearch polky and propamMIGff; Aa. Hoard it does not ~:' dimcOr COpa I* reacarch tor The C,ouocll, .5C 70q~ Ibe Council itself ~~,+Mw jaz~~~ ~~;~ j.-~--~ ~ research ,h.~~..j.,,•, . i :Orants-laaid for research are made b~.• . 'ZL/hu wbo are asaured eomplete scienti8e fr • z- .•~` r~atc~. Grnetcey alooe ue responsibte for reporting or publisbin` their ~ t±'!L ~tp In the accepted acientific manner - through medical and scientific ~ ~ • tu-A -4Ct1. e1 VM ~ • " .:Ibrout3s June 1970 research projectt have been approved by the 41 i h i '~a O inat ,- ties and researc atd to 249 kayestipfoA Ia 189 boapitab univers iB ~... 12}aut/oa4. These tiwarda MhOed =17 262 000. ` : , , , : pro}ecb ouPPated bv ' Ibe Council is Included in litt of ruearth %A ~~'.'a . ~.U>J•'Repott'Alw induded ate abatraets ~d 93 research papen, accnowl- ,, 1t ,~ ~t'drlnt Coupol support, that have appeared In ndeati8c }ournab since the '~pryvlotr jteporl and through June 1970. A total ci 821 such paprn has been publisbed by grant recipients. W. T. Flort Exrcutl.e 1)Ircetor t TIIF. CnIIN(7L FOR 'i'nBA(:C(1 RESF.AItCII-11.S.A. 110 F.ast S9tb Street, New York, N.Y. 10022 .o..' . . ApW to independent i 1rj cooductin3 their I ANNUAL REPORT o/ the SCIENTIF IC I)IRECTOR CI.AItEiNCE COOK L1TT[.E, Se.U.

S(:IENTIFIC ADVISORY IiOARI) to The Council for Tobacco Research - U.S.A. SI IELDON C. SOMMERS. M.D.. Chairman Research Director. The Council for Tobacco Research - U.S.A. Director of Laborotories, Lenox Hill Hospital Professor of Pathology Columbia University College of Physicians and Surgeons New York. New York HOWARD B. ANDERVONT. Sc.D. Scientific Editor (rrrbed), The Journal of the National Cancer Institute Bethesda. Maryland RICHARD M. BING, M.D. Director of Cardiology and Intramura/ Medicine Huntington Memorial Hospital. Pasadena. California Professor of Medicine Univenity of Southern California School of Medicine Loa An6eks, California McKEEN CATTELh Pw.D., M.D. Professor Emeritus o/ P/rarrnarolory Cornell University Medical College. New York, New York ROBERT J. HUEBNER. M.D. Chief. Viral Carcinosenesis Branch National Cancer Inuitute Bethesda. Maryland LEON O. JACOBSON. M.D. Dean of the Division of Biological Sciences Regenstein Pro/rssor of Biological Sciences University of Chicago. Illinois CLARENCE COOK LITTLE, Sc.D., LL.D., Ltrr.D. Scientific Director. The Council tor Tobacco Research - U.S.A. Director Emeritus. Roscoe B. Jackson Memorial Laboratory Bar Harbor, Maine CLAYTON G. LOOSLI, Pn.D., M.D. Hastings Professor of Medicine and Pathology University of Southern California School ot Medicine Los Angeles. California KENNETH MERRILL LYNCH. M.D.. Sc.D.. LL.D. Chancellor and Professor Emeritus of Pathology Medical College of South Carolina. Charleston. South Carolina WIl.I.IAM F. RIENl1OFF, Ju., M.D. Pro/tssor Emeritus of Surgery Johns llopkins l)nivrrsity School of Medicine, Baltimore. Maryland I ROBERT C. H(X.'KETT. PM.D. Assor(att Scientific Dirertor J. MORRISON BRADY. M.D. JOHN FI. KREISHf?R. PH 1). I Associate Scientific Director Associate Scientific I)irecror VINCENT F. LISANTI, D.M.D. ! Scientific Associatt

CONTENTS fntroduction . 5 Status of Current Researth . , - , - . , , , , 6 /The Cancer Program . . . . . . . . . . . . . 6 x'ardiovascular Res+carch . 9 Chronic Respiratory Diseases . . , . . . . . . . . 12 .1leuropharmacobgy and Psychology . . . . . . . . 13 Other Studies . . . 14 Abstracts of Reports . . . . . . . . . . . . . . . . 16 1 Psycho-PhysioloRical Studies . . . . . . . . . . . 16 Farcinogenesis Studies . . . . . . . . . . . . . 18 Cardiovascular System . . . . . . . . . . . . . 25 Respiratory System . . . . . . . . . . . . . . 32 / NeuroQhysrobgy . . . . . . . . . . . . . . . 45 J'issue and Organ Culture . . . . . . . . . . . . 50 J Pharmaoology . . . . . . . . . . . . . . . 52 l Metabolic Studies . . . . . . . . . . . . . . 63 ~Cbemistry and Biochemistry . . , . . . . , . . . 65 JiRe views . . . . . . . . . . . . . . . . . 67 Recipients of Active Projeet.s . . . . . . . . . . . . . 69 Recipients of Compkted Projeets . . . . . . . . . . , . 79 i I Iiitroduction The research program of The Council for Tobacco Research - U.S.A. continued to move ahead in 1969-70 with additional support for studics into smoking and health. The Council's eRort to help develop mote scien- tific data was further expanded during the year with the inauguration of several conlracls for research with institutions and laboratories geared to petform the required work in the shortest possible time. 'Me Council, by augmenting its regular gant-in-aid program with research contracts, is re-emphasizing its desire to speed up the search for the answers to lung cancer, heart diseases, chronic respiratory ailments, and other diseases. The process is naturally stow and painstaking because these diseases are immensely complex and have been afflicting mankind for hundreds, even tlxwsands, of years. While bits and pieces of information about them are rcported by scientists almost every day, the time when the numerous parts of the full puzzle will be assembled into a cohesive picture cannot be predicted. (.'laims Ihat the puzzle has been solved are totally unrealistic by scientific standards and misleading to the public. There are far too many questions that remain unanswered at this time. It is noteworthy that in 1955, one year after the Scientific Advisory floard to The Council began its research program, a progress report was issued that said in part: "The history of cancer research is a record of slow but steady progress. There is every reason to expect marked advances in the prevention, treatment and cure of eaneer. This also is true nf the other constitutional diseases, such as heart ailments, which are our grcatest present and future health challenges. "But this will take time, and we cannot count on shrxtcuts. Neither the generating of unnecessarr fears, nor the arousing of unfounded hopes, witt hasaen the coming of significant discuveries. "1'roFress in the battle against these great health prubtems has dependert and •will depend on solidly planned and wcll- cxecuted scientific research. 'I hcsc words are still true today. 1 hc Council for Tobacco Research - U.S.A. will continue its rescafch program in the belief that valid scientific conclusions can conic only from unbiaced and complete research. • 5

Status of Current Research Since 1954, when The Council for Tobacco Research - U.S.A. began to support studies by independent investigators on tobacco use and heaith, reports of its activities have been made annually to the scientific and lay publics. Tlris is such a report for 1969-70. Through the yran, the staff of The Council and the membcrr of the Scientific Advisory Board have seen the research program devciop and mature along lines and Into fields which give an opportunity for much more objective results. It remains clear that the complexity of the origin of different cancen, cardiovascular diseases and chronic respiratory diseases is very great and still littlr understocxi Ifence, as heretofore, The Councii's emphasis is still upon the etiolofy or pathogenesis of these diseases, since any possibte effects of tobacco use can he interpreted only in this context. A summary and overview of the current status of The Council's pro- gram are briefly prescnted in the following sections. rI'he Cancer Prograrn Virruea and Cancer Several present studies are directly related to the recent theory that cancer is one of several possible expressions of a latent C-type RNA viral genome, or genetic potential, found in all mammalian cells. The frenome, according to the theory, is normally repressed and inactive, and there is a complex interplay of internal and external factors that determines whether, when and how the rcprcssiim control mechanism is somehow broken and active expressicxt occurs in the form of cancer or uncontrolled growth. One study in this rea is aimed at developing nKxe realistic, as well as more sensitive, test systems to illuminate the interactions between exter- nal agents and internal Influences affecting the susceptibility of the biological substrate as determined by the state of the viral genome. Hiological test systcnn for this purlxne are expected to be of three kinds: ( 1) in vhro studies of cells that are controlled and standardized with respect both to source and state, (2) skins of intact animals in vivn from standardized, controlled and state-defined strains, and (3) lungs of intact animals in vivo from similarly defined and standardized sources exposed to monitored inhalation of external agents. One imnrcdiatc objective is to develop rapid yet realistic test systems that may "magnify" the effects of external agents so that the systems can be used to guide fractionation of complex mixtures and isolation of the ingredients most likely to be active in a suseeptibk host. Such assay systems should also provide new information on the components of host suscepti- bilrty through study ot step-by-step modificatiorn in the substrate system. An ultimate hope, of course, is to contribute toward discovery of methods for inhibiting or dclaying dcrepressian of the latent cancer genome as a means of preventing or deferring cancer in the human population. Other Ccwncil-sponsored studies are aimed at filling gaps and round- ing out needed basic knowkdge about the nature and applications of Ihe virus-cancer hypothesis. These include studies of epithelial cell transforma- tion and carcinoma induction by C-type RNA viruses; the behavior of avian tumor viruses in mammalian hosts; and oncogenesis in the rabbit in terms of genetic susceptibility, vertical transmission of virus, and environ- mental influences. The Irnrnunological System The concept that cancer may result from virus activity has renewed interest in studying the body i complex immunological machinery to see whether stimulating it will help prevent the induction of cancer. Research supported in this field includes studies of: the action of interferon as one type of virus-repressing substance; the function of the reticulo-endothelial system as it may be related to tumor induction and growth; the possible effect of tobacco smoke inhalation on the immunolotjcal system in animals and man; and how carcinogenic hydrocarbons may possibiy suppress cellu- lar inrnrunologicat mechanisms. 1'olxrcco Smoke Studies Sn.okinR Machinea . After an extensive period of experiments'and tests, progress was made in the last year toward development of inechanica! devices for exposing experimental animals to thc monitored inhalation rif wholc, fresh, normal 6 7

cigarette smoke under conditions simulating actual human exposure as closely as possible. Previous Annual Reports have described the numerous and complex criteria necessary for reaiistic smoke inhalation studies. The aim in the design of such machines is to generate the smoke ur der defincd conditions and to permit delivery of the whole smoke to the res- piratory tracts of the animal in the same physical and chemical condiiion as that which reaches the respiratory tract of a human. Mechanical and biological evaluations have been made of several such devices developed through the years by various organizations. Also, ana,yti- cal data were obtained for the gasean and particulate phases of cigarette snroke generated by each device. Some of the machines are now being used in preliminary studici by various Council granteea for inhalatkors tests with animals. Full descriptons of these devices will be published with operational details as soon as the progress of these preliminary tests and trials justifies. Exposure oJ Anim.l@ to Smoke In all cancer-cxiented projects invoiving direct exposure of waole animals to smoke, including viruslancer studies, some of which involve work with tobacco srrxike, it is now Council policy to require defining, standardizing and controlling the virus content of all animals being used. In one investipation aimed at elucidating the possible significance of freshness and physical state of smoke, a concentrated stream of whole, fresh, normal cigarefte smoke is blown directly on the skins of mice of the sitne strain that had been used previously in a conventional skin-painting experi- ment with tobacco-smoke condensate. All components of the smoke inrpinge on the skin and many of the constituents condense upon the surface. The treatment is being carried out with and without acetone, which has been gencraily used as a solvent for "tars" in skin-painting work, and in one group of animals a"promotin6' agent is also being used to intensify any possible "carcinogenic" effects that may exist. This experiment has been under way for a year. While the study still involvcs the "wrong tissuc of the wrong animal," it docs use natural smoke in the condition encountered in normal human smoking, and it should pro- vidc further insight into the widely prevaicnt mouse skin-painting with stale, artificially prepared "tars" in a sdvent. No Squamous (:elf Tumora Found In a long-term chronic smoke inhalation experimcnt, two strains of mice are being expcxed to smoke with the use of improved equipment. Mice thus exposed to inhalation of whole smoke, or of the gas phase alone, have developed only the types of lung tumors - adenomas and adenocarcino- mas - that develop in the same mice without any exposure. No squamous cell tumors of the type considered by some to be associated with smoking have been found in any of the hundreds of mice that have so far been exposed and then subjected to pathological examination. To study the incidence and appearance of lung tumors and pneumo- nitis in mice, a Council-supported researcher exposed three inbred strains of mice to prolonged inhalatjon of ambient and filtered Los Angeles air. Mice in the ambient air group showed no lung pathology whik lung adcno- mas were found in one strain exposed to filtered air. Autopsy examination of the animals' lungs showed that kngthy exposure to the ambient atmos- phere seemed to be associated with an increased susceptibility to pulmo- nary infection but not to an increased incidence of pulmonary neoplasia. This particular project is relevant both to cancer and to chronic respiratory diseases. In studying the effects on the lungs of mice of chronic inhalation of several pure gases that are present in polluted urban ir, and certain mix- ~ tures of these, the same researcher has developed a remarkably elegant technique for infialing. fixing and diRcrentialfy staining the lung tissues. Among the gases studied were oxides of nitrogen, ozone, carbon monoxide, and sulfur dioxide at various dosages and over various periods of time. Other Cancer Studies Other studies related to cancer include one dealing with host facton, especially abnormalities of the endocrine system, aa they may be related to lung cancer in humans. Another researcher is eompkting a study of bladder cancer in man. This particular project has produced findings to the effect that, contrary to some reporta, there is no paralklism between reported disturbances in tryptophan metabolism In bladder cancer patients and reputed effects of nicotine on the same metabolic function. Support is also being continued for research on the mechanisms by which urethan affects cells in the alveoli of certain mice. Cardiovascular Research There are now two key problems considered to be of first priority in regard to cardiovascular ailments and any possibk relationship to cigarette smoking: 8 9

1. Since arteriosclerosis, including atherosclerosis, is widcly considered to be thc hasic factor underlying most of the cardiovascular diseaus that are the leading causes of death in the United States, the ques- tion of preveming, delaying or reversing the process is paramount. As regards tobacco, a prime question is whether smoking and/or nicotine have any effect in speeding the development of artcrio- sckrosis, especially of the coronary arteries. Tlwugh there ar-- many studies that question whether smoking can affect the process, there is a need for mcxe research. Still lacking is a really satisfactory animal model for reproducing the human eoronary disease under reasonably physiological conditions and such a model is being sought. 2. Once artericxclerosis or atherosclerosis has been established, par- ticularly at an advanced age, another questiort is what factors or influences may possibly trigger an acute event such as a heart attack or a stroke. f lere too a prime question Is whether smoking or nico- tinc can be involved. Especially important are the basic differences between the kinds of people who like or need to smnke and those who do not. The issue is confused, particularly in epidemiological investigations, because people predisposed to cardiovascular disease may also be of the type cx types who feel a need for tobacco and, consequentrf, sepa- ratic.ns of populations on the basis of smoking behavior may produce statistically non-equivalent groups. Various twin studies have sup- ported the concept that epidemiological studies have generally pro- duced and attempted to compare non-equivalent groups. Method for Meaaurirsg Blood Flow The Council has helped support research for several years from which a method has been developed for measuring blood flow in the arte ies that feed the heart muscle without inserting tubes or resorting to surlery. By this new approach, it has been shown that nicotine does not reduce blood flow in normal arteries that supply the myocardium, but generally increases it after the manner of mild exercise. In addition to an increase in "nxchani- cal" coronary blood flow, there is an increase in "nutritional" flow as well. This method, repcated and confirmed in the past year with tests on animals and humans, has important implications in the entire field crf heart disease. The same research project, with continued support from The Council, is now approaching the athero.ckrosis problem by studying human arteries in tissue culture by perfusion methods. Tbe purpose is to determine whether nicotine affccts drposition of fatty materials in the artery wall from serum or plasma. The conditions are made to resemble those in life, with control and variations in pressure on the vessel wall and in the concentration of alhumin, cholesterol, free fatty acids, ete., in the serum. Blood flow in the microcirculation is also being studied to see whether it is affected by nicotine. Among other studies that have been contributing knowledge in various phases of the complex cardiovascular problem are: one suggesting a nega- tive effect of nicotine on thrombus formation; the relationship of blood- sugar level to nicotine effects; measurement of carbon monoxide in the biood; the interrelationship between carbon monoxide and oxygen clution from hemoglobin; the degree of fibrous thickening of blood vessels in smokers and nonsmoken; and possible prediction of early heart disease by longitudinal study of precursor conditions. 7hese arKl other interesting findings of The Council's grant-in-aid program are presented in more technical form in the various summaries ol research included later in this Report. Predietora o/ F,arly He,.rt Diaeaae Being continued is a long-term study of medical students to leam what characteristics, measured at an early age, will turn out to be predictors of early cardiovascular disease. Part of this project includes determining the patterns of measurable characteristics that correlate with a family his- tory of cardiovascular disease. During the year, the investigator conducting this study reported that 16 of the mature men originally studied as students had developed clinical hypertension by 1968 while 89 others had exhibited transitory hypertension. In 62.5 percent of the subjects with clinical hypertension, both parents evidenced hypertension and/or coronary disease as compared with 21.9 percent of the parents of the control group. Another development reported from this study was that there have bec 1 31 premature deaths amon& the subjeets, 14 of them from suicide. The other 17 deaths were due to accidents, coronary heart disease, neo- piasfn, chronic alcoholism, nephritb with hypettcniion, acute ulcerative colitis, and subacute bacterial endoarditis. Statistical evidence was reported that certain precursors of suicide, accident, fatal heart attack, and fatal stroke are already present and can be identifkd In youth. Uther research is being supported to karn whether changes in eakium ion mobility, induced by nicotine, play a role in addition to that of catecho- Iamines in bringing about the short-lerm cardiovascular changes that follow smoking; and to measure the short-term effects of smoking upon regional blood flow in the brain. , 10 II

Chronic Respiratory Diseases A hasic problem involved in the study of chronic respiratory diseases, particularly in determining their cause or causes, is the lack of generally accepted clinical distinctions among the various respiratory difficulties. These include emphysema, bronchitis, asthma, and even certain heart ailments. Inadequate definitions and the lack of uniform clinical distinction of these conditions have confused attempts at epidemiological studies of causation or aggravation. Further, It appears that diagnostic uncertainties and doubts, as well as changes in the popularity of such terms as "emphy- sema," may have resulted in a fictitious increase in reported prevalence rates. Nence, The Council ia supporting a long-term study in a pufmonary disease clinic where comprehensive and repetitive observations can be car- ried out on many chronic patients over a{ong period of time. T[x hope is that numerous bioehemical, pathological and radiological observations can eventually be correlated with the clinical course to produce better definition as well as imp.oved diagnosh and trcatment of chronic lung disease entities and also provide new etiological insights. Metaplaais in Nonamolrera A Council grantee examined the bronchial epithelia of 500 apparently "healthy" adults who died suddenly and unexpectedly, after living and working in an urban area noted for its air pollution, but who had had no known disease history. Metapfasia was found in the lungs of both smokers and nonsmokers. (x great importance, however, was the observation that various degrees of roetaplasia were present in a surprisingly high proportion (50 percent) of "healthy" adult men who had never smoked ciMrettes, cigars or pipes. There were no cases of carcinoma In situ In either the smokers or non- smokers. Thus. In the study of lung tissue t autopsy, it is important to distinguish between patients who die suddenly and those who die following wastingor chronic ailments in a hospital after being treated with drugs and other therapies. The Council has supported several studies through the years that have contributed evidence showing that a deficiency of alpha,-antitrypsin appears to predispose to emphysema. Other rrseamh includn: the eHecta of hypoxia on function of the respiratory system; the bioebemkal activities of sputum cells from patients with various types of chronic lung disease; and how lung celis and Mher products recovered by lavage from normal smokers and normal nonsmok- ers may differ. One investigator is studying the cyclic changes in the structure of lung cells of young women, recovered by lavage, that correlate with the men- strual cycle. An investigator is conducting a long series of studies on the effects of cigarette smokc inhalation on lung function, especially pulmonary resist- ance, in several species of animals. He has found short-term effects that differ among species and that a female sex hormorse, progesterone, reduces or blocks them. No permanent changes of the kind that occur in emphy- sema have been found in these chronic smoke inhalation tests. Additional experimental studies include: isolation of functioning ribo- somes from the lungs of animals and relating the speed of their formation to certain ksions produced in animals by high-oxygen exposure known to cause such lesions in humans; the effects of smoke exposure on the pul- monary macrophages; clarification of the relationship between lung lym- phocytes and pulmonary macrophages; and the lymphatics of the lung, their structure and role in fluid transport and in ekarance of airborne par- ticulate matter from the lung. Neuropharmacology and Psychology Most of the pharmacological studies currently being supported by The Council are concerned with the effects of nicotine and/or smoking on the central nervous system (the brain) with the object of learning more about why people like, want or need to smoke. These should help reveal the basic differences between smokers and nonsmokers, which previous work of other character has shown to exist. In a Council•supported study of high school and junior high schcrol students, a psychologist has reported results quite similar to those found earlier with adults, namety, that there are personality differences between smokers and nonsmokers. Brain Waoe Patterna An ongoing study deals with the effects o( nkotine or smoking on the brain waves of human subjecta. By use of computer methods to analyze these electroencephalograms, the meanings of the wave patterns In tenns 13 12

of "deep slecp," "arousal." "awakc but in rrpose," etc., have become known. One siKnificant observation ftom the study so fat is the irnpl-cation that heavy smokers may have a different basic prevailing brain wa.e pat- tern from that of nonsmokers. The investigator bclieves that thic (,attern may be congenital and that it antedates and influences the adoption of tobacco use. ConJerente on Nirofine A conference on the effects of nicotine and/or smoking on the central nervous system was held June 1, 1970, at The Council's oftice. In addition to staff and menttlcrs of the Scientific Advisory Board, the following scien- tists participated: Budhdev Iihagat, hh.D., Professor of Physiology, St. I.cwis Ilnivcrsity School of Mcdicine, St. Louis. Barbara B. Brown, Ph.D.. Chief. Esperimental Psychiatry. Veterans Ad- ministration f lospitaf, Sepulveda, Cal. Edward F. Dcxnino, M.D., Professor of Pharmacology. University of Michigan, Ann Arbor. Walter B. Essman, Ph.D.. Professor of Psychology, Queens College of the City of New York, Flushing. Leonide Gofds,cin, D.Sc., Research Scientist, Bureau of Research in Neurology and Psychiatry, New Jersey Neuropsychiatric Institute, Princeton. Henry B. Murplrree, M.D., Associate Professor of Psychiatry, Center of Alcohol Srudies, Rutgers Univenity, New Brunswick. N. 1. S. N. Pradhan, M.D., Ph.D., Professor of Pharmacology. Ifoward Uni- versity Colkge of Medicine, Washington, D. C. Ulrich N. Sehaeppi, M.D., Director of Neuropharmacology, Mason Re- search Institute, Worcester, Mass. '['homas C. WcsNall, Ph.D., Assistant Professor of Pharmacology. Uni- versity o( Virginia School of Medicine, Charlottesville. Other Studies The Council is contributing to a projeet that is supported mainly by the Veterans Administration. Its aim is to make numerous periodic exami- nations of a large group of male veterans, with a high initial level of good health, in order to describe the changes Ihat corne during normal apng. Support is being provided for a study of certain biochemical effects of chronic smoke inhalation by guinea pigs. Using a smoking machine, the investigator has it:c>aated the mitochondria from lungs of eRpcned animals and is studying the electron transport system in these cell components. The Co'incil is continuing sponsorship of a project that has been of great benefit to researchers and others interested in smoking nd health: the collection, abstracting and andysis of the world literature on tobacrn, including experimental and clinical studies. A second supplement to the monograph on this subject, titkd "Tobacco - Experimental and Clinical Studics," first published in 1961, is expected to be published soon. 14 15