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TIII: COUNCIL FOR TOBACCO t?RSliARCH-j,'.S. a., INC. CTR-SUPPORTED RESEARCF3ES: 1977 CANCER-RF.LATED STIDDIES I. Smoke Exposure Studies Relating to Cancer. 1. Applied Aspects. (a) Contract Research at Microbiological Associates. (MA 22Y* During the summer of 1977, mice that have had a cancer-inducinq chemical, methy,cholanthrene, placed into their tracheas are receiving smoke inhalation from the 2A1 Kentucky reference cigarette generated through the SEM-II-B smoke delivery machine. This experiment is designed to determine whether or not cigarette smoke may have a cancer-promoting, a cocarcinoqenic or a cancer inhibitory effect. 240 mice can be exposed in the SEM at one time. (b) Experiments on dosimetry have been complieted wi!th both the Walton and SEM'machines and with smoke deliivery at several different concentrations. The deposition and retention in mice of particulate matter, of nicotine,, benzo-a-pyrene and methy,lchol!anthrene with radioactive labels has been determined using the Walton machine. Both Microbiological Associates and Oak Ridtr_e National' Laboratories have been doing this work, the former doing the exposures of the mice and the latter the radioactive counting,of the respiratory tract andlother organs. These are all short-term experiments. (c) Mice in smoking machines are subjected to considerable stress but seem to adapt after a few weeks. Stress and adaptation of mice exposed to smoke iin the machine, placed in the machine without smoke but with confinement, and others with confinement alone have been evaluated by MA-22 and Essmanr836. 2. Basic Aspects. (a) Mice of some strains are much more sensitive to carcinogenic chemicals than mice of other strains. The sensitive strains have an inducible enzyme system, arylhydrocarbon hydroxylase (AHH). Mice of inducible strains have elevated ABB in their 1'ungs when exposed to cigarette smoke; 1'~iver AHH i~s,not elevated however (MA-14, Giel!enrli072)',. (b) Some substances act as promoters of carcinogenilc chemicals. A, common quality of these chemicals is their capacity to induce another enzyme, ornithi:ne decarboxylase. Studies are underway to determine the extent of i~nduction by lung exposures, strain differences, the duration of induction ar.-- %o!iether or not cigarette smoke and the smoke condensates may be active. (MA-14) *'-The bracketed,letters and names refer to the contractee or grantee and CTR contract or grant number.

(c) Smoke-exposed mice are being,tested for the rates of deoxyribonuc3eic acid (DNA) replication and repair (MA-14,, Rasmussen-973). Repair of DNA is important because„ if rapid, it may restore ce1Ls, that have been transformed, to normal. (d)' Severallenzymes acting in the transformation of the carcinogenic hydrocarbons are being purified. Some of these enzymes inactivated the polycyclic aromatic hydrocarbons, such as benzo-a-pyrene, by modifications of their structure. Some of these enzymes have now been sufficiently puri~fiedi to have their chemical structures analyzed and to be used to produce antibodies so that some of the steps in~PAH metabolism may be controlled (Oesch-1097). (e) In addition to exposure ofi lung tissue to known carcinogens, with or wi,thout cigarette smoke„ cells grown in.cuSture have been excosed to:ciaarette smoke concentrates and fractions of the concentrates. Some of the fractions of concentrates and fractions of the concentrates. Some of the fractions of concentrates will transform normal~ ce11s into~cancer ce1Ls if properly conditioned (Benedi,ct-1027). Also some concentrate fractions will induce mutations in specialistrains of bacteria (hmes test). At the same time, information is accumulating om.how to prevent transformation and mutagenesis (Oesch-1097, Wattenberg-1031). 3. Arylhydrocarbon Hydroxylase. Some chemicals are carcinogenic when given to animals of one species but not in animals of another species. Benzo-a-oyrene, tons of which are dumped into the atmosphere dai~ly,, is inactive as a carcinogen until its cemnosi'tiion is altered by enzymes which may or may not be present or mobilized in some animalis or indi~viduals. In mice of some strains the enzymes are mobilized ('iinduced)', in others not, and it is known that the inducible strains are more suscentible to cancer, at least of certain types. Inducibility is determined in some strains by a single gene, in others by 2 or 3'. The enzymes of the AHH system,are most concentrated in the liver but also occur in lung, kidney, skin and most other tissues. The animalistudies indicated that AHH inducibilitv can determine individuals at high and at low risk for the development of cancer. Larce amounts of carcilnogen are active in all strains nossibly because of the censtituitive AaH1. Both basic chemical and biological studies on,AHH have been referred to in the section on smoke exposure (Oeschri!097, Gielen-1042, Wanc-1005, Meier-951,. Benedict-1027). After AHH ha&been reported to be inducible and under genetic control in man, the CTR extended its support of researches in this area. A major difficulty was a source of tissues or cells that might contain the enzymes in adecuate and consistent amountls.. The first CTR--suo_oorted research.with human tissue. and AHY.:was done with foreskins (acapnas-802)'. Blood is a most readily ava'_lable source of viable human celils but blood cells contain little endoplasmic reti :=um unless activated. Considerable support is being givenioni-,rojects that r_.--te to assay methods for improvement of sensitivity and reproducibil'ity (t~fA-25, :nirgis-1132, Martin-1094, Rasco-1i102, Cantrell-11i20). I.n.addition to using activated lympnocytes,, pulmonary macrophages and blood monocytes have been used. At this time,,it appears that the determination of AHH may have some value as a risk =edictor even, though such things as diet,, drugs, health status, etc., may modify the levels of AHH' induction or inhibit it. The present levels of refinement of the test now permit quite consistent valiues when the same specimens are tested in~different 1!aboratories.

- 3 - The binding of the PAHs to DNA can also be prevented by certain chemicals and this is another way of reducing cancer incidence (Wattlenberg-1031, Rasmussen-973):. 4. Other Cancer Studies. Small amounts of nitroso compounds are found in tobacco and~possibly also in smoke. Neurath (981J' has been determining nitrosamines in tobacco smoke and some aspects of their formation andltransfer. Extensive studies on the enzymes acting on nitrosamines andihow they may modify AHH responses of PAHS have been undertaken (Arcos-992). Other studies are also being undertaken on tumor induction by nitroso compounds in.rabbits and mice (Meier-951'). These compoundS are active transplacental carcinogens. Dilfferences in the amounts of different metabolites of nicotine have beemnoted'in~persons with bliadder cancer (Gorrod-986). Additional metabolit-s are now being isolated and tested for the induction of nutations and carcinocenesis. Uri~nary glucuronidase levels are inheri~ted in mice and nan (Paigen-1I080)'. Glucuronidase, one of the lysosomal enzymes, is being determined in bladder cancer patients and their sibs andispouses. P4ice are also bei'ng aiven;bladder-cancer- inducing chemicals and their urine being tested by the Ames method. THE R°SPIRATORY SYSTEM - PULMONARYS.TUDIES Polluted environments have been associated with respiratory diisease, chronic obstructive pulmonary disease (COPD) or emonysema for some time. About ten~ years ago an inhibitor of an enzyme or enzymes that digest proteins was found to be deficient in persons unusually predisposed to COPD. At the present time, CTRisR supporting the work of a number of investigators who are working with orotease inhibitors, proteolytic enzymes of different types, their structure and mechanism or action (Cohen-954, Travis-962,, weinbaur.r940, Feinstei'.n'-11033, Geokas-1i068, Crawfordrl'105, Turino,l084). Some of these are basic research activitie~; most, at least in part, are concerned with human enzymes or enzyme inhibitors.J Most of the outstanding~investigators in this research area are included. A second workshop meeting is planned for 1978 to bring together those working on the proteases an,li their inhibitors. Animal models for spontaneous CCPD do not exist. The pathological picture of emphysema can he induced~by instillilng purified enzymes into the lung. Radioimmunoassays can now be done on twolnroteases produced in the pancreas that enable quantitation at levels eaual to 1/80th of the norr.al bliood serumi llevelis (Geokas-1©88). A group of agina mice are being studied to dete:-c^.ine age changes in lung~structure and functioni(Kleinerman-1143)1. The lungs of the mice under chronic smoke exposure are alisobeina studied (MA-22).. The cellular changes in the bronchi and smaller ainaavs of rats er_nosed to smoke and! smoke and~ some drugs are beilna studied in detail for -_,diticns considered to lead to chronic bronchitis (Reid-1082). 1. Other Pulrnonary Studies. One of the oolypentides (Angiotensin II) that reg,ulates,blood'pressu_re is formed in the liungs in the cellis that line the bloodivessels - endothelial cells - from Angiotensini I. The enzyme removes two amino acids from anaiotensi.n I(Ryan-814).

- 4 - Dr. Ryan is also studying the formation, at cellular levels, of prostaglandins and thromboxanes, recently discovered substances that have important roles in blood clotting and circulatory and respiratory'function. Dr. Rifkin (928) has studied the activation of a precursor - plasminogen - by an enzyme found in cultured lung tissue and in cancer cells. A detailed study of the lymphatics of the lungs has been completed and reported in a"State of the Art" publication (Lauweryns-1041). He is now studying the neuroepithelial bodies in the lungs (NEB) of laboratory animals and man. The NEBs produce biologically active materials, have apparent neurosensory functions that are activated by low oxygen levelis in the inspired air but not by low blood oxygen levels. The instillation of nicotine also modified these celils. The function of the neurosecretory cells in the lungs is also being studie d by another investigator (Will-1036). 2. Miscellaneous Pulmonary Studies. Tracheal washings from persons undergoing inhalation anesthesia have shown changes in the respiratory cell's during the menstrual cycle (Chalon-725) and abnormall multinucleated cel'ls, the latter from persons with cancer most frecruently and with cancers of many different organs. HEART AND CrRCULATION~ Studies have been done on the effects of ozone, ozone olus cigarette smoke and cigarette smoke alone on a number of enzymes found i~n the lungs of rats (Cross-937). Few measurements have been made on intracardiac pressures, coronary flow rates, respiration and peripheral blood flow rates and volumes in unanesthetized animals. Dogs so prepared have been given nicotine intra-arterialily. Both sensory and motor aspects have been noted. Both the injection of nicotine and pulmonary distension increased blood flow in the coronary arteries. The studies are being continued wi,th smaller and more prolonged injections of nicotine and'cigarette smoke inhaliation (vatner-979). The uptake of choLesteroli in the presence of CC and some cholesterol analogues has been studied in bothiim,vivo and in vitro creparations (3ing-301). Endothelial cells are being grown in tissue culture (Boo_yse-11133, Mason,939)1. Their reaction to platelets, to enineohrine and hormones as revealed by their growth characteristics and secretory activities in normal crow•thimedia and when exposed to nicotine and smoke are beina studied. Endothelial cell cultures are also being used to provide enzymes for the formation of specific tiromboxanes and some prostagl'andi~ns fromiarachadonic acid (Rvan-814). The effect of smoking andismoke abstinence on the resnor.-_ : of rats and persons to stress as revealed through blood cressure and heart rate changes are being studiedi(Hutchinson-1107).

W_ -5- , NEUROPHARMACOLOGY AND PHYSIOLOGY A number of the researches described under other headings might well be included here as they involve the basic biological aspects. Although much i~s known about the pharmacological action of nicotine on the peripheral nervous system little is known of its action on the central nervous system. Increased emphasis is now being placed'on new approached (Abood-9H3, schmidt-1058, Hall-1128,,and McClearn-1076). The binding site of nicotine on celil organels is being sought. The "right-handed" nicotine is inactive but will preventt the action of the levo- nicotine. Piperidine and other nicotine-like substances are also nicotine competitors. Genetic selection of fruit flies is being used to find nicotine- sensitive individuals (Hall-1126), and McClearn (1076) and his group are working with nicotine-sensitive and more tolerant strains of mice. Dr. McKennis (5054) is providing nicotine-related chemicals for some of these studies. The development of additional radioimmunoassay reagents may also permit another method for nicotine localization. A new study reLates to the effects of nicotine and carbon monoxide oniamino acid trans_nort into brain and brain protein metabolism (Lajtha-11411);. Rats have been trained to discriminate between nicotine and saline as demonstrated by behavioral tests, and substances are sought that the rats cannot discriminate from nicotine (Rosecrans-909). The competition between chemicals for binding sites is in some instances supplemented by augmented rates of drug, metabolism (;7etsko-1i079)~, il.e., cigarette smokers eliminate theophylin twice as fast as nonsmokers. 1. Effects of Smoking on Pregnancy. Much has been written on this subject but most investigations have been complioaried by uncontroliled variables that made evaluation difficult. One study using a Swedish group is following not only birth weicht, paranatal mortalilty,, body liengthy,headidiameter, respiratory distress svndYome, pre-eciamosia, but is also,foll'owing,bloodilevels of hormones, placental proteases and protease inhibitors (Kullander-1039)!. Smoking mothers were 5 kg smaller and'their babies 100 gm smaller. In another study, pregnant monkeys wilil be exposed to cigarette smoke and to CO (Mannimg-1139). Fetal respiratory and cardiac functions are being followedland blbod levels of CO and nicotine ascertained. The effects of nicotine on pregnant rats and their offspring are being studied (Mitchelil-1112, Yoshi~naga-1042),. Large doses of nicotine delay im..pliantationi of eggs and subseguent develoo^entalistages will also be delayed. The amount of nicotine is many times that possible from smoke inhalation. Arrangements are being made to repeat these experiscents coiving, smaller doses and over longer oeri~ods. The ddvelopment of the immune system in fetuses and new-born mice exposed in utero to cigarette smoke is beinc followed (Herscowitz-1045). Rats exoosed i~nlutero to nicotine or cicrarette smoke and aliso after birth are being studied for lung function andirate of develc---.ent (Hamosh-11'30). 0~ GW=

- 6 - 4 I"1MUNOLOGY ANDADAPTATIOiN A specific antigemhas been found in two types of lung cancer iin man and not in other types of Lung cancer or other cancers (Soachim-945). This antigen has been purified and antibodies have been prepared'. These will be used for diagnostic and possible therapeutic purposes. A number of hormones andlenzymes or enzyme deviants are being followed in human lung cancers (Fishman-925). The secretory component of IgA is,being follbwed in lung,lavages of persons with lung cancer and other persons needing lavages (Reynolds-1127). Other inmunoglbbulins, transferin and lysosomal!enzytmes will also be studied. Some very basic research is being supported. The fluid in which macrophages grow contains (1) a ceLl division-indhcing substance, (2)' a thymus-cell differentiating material, ('3)' a specific ce11-activating substance (Unanue-1030), these substances are being purified and cher.ically identified. The function and activationlof a suppressor celli (T-cell) is being intensely investigated (Pierce-L040Y. Basie infliammatory pathways, especially in lung diseases, are being studied (Cochrane-764i). A basic study of macroohage structure andifunction is also being supported (Pierce-11i16). EPIDEMIOLOGY The culture of macrophages with lymphocytes produced a supernatant that suppresses the activation of and proliferation of T 1Vmp1-ocvt'es (Talmage-1086). Smallier number of macrophages, however, activated T-cells and~ augmentedi their cytotoxicity. 1. Lung Studies. High school students in the Washington, D.C. azea are !,)eing given a minimum of three pulmonary function tests at yearly intervals. Some will start smoking during,this time (Hamosh-11i08). These tests may give some idea of the early effects of cigarette smoking. Another grantee starting with an.ihdex group of infants that have the hereditary low activity of orotease inhibitors is following these infants carefully for evidences of modified lung function„ and at the same time is following the lung function of their parents and'sibs (Buist-1044). A somewhat similar study is being,done in New York City. Alil babies born in one hospital are being Pi gene typed and are being followed for at least tne followina 1 1/2'years for the incidence of lung diseases or lung associated illnesses. The nature of their home environment is also being observed (Evans-1043). 2. Twin Studies. The Council has contributed toward the development of' two national twin registries - Sweden and Finland -(Friberq-892'& 1136, Ranta==.=D-953), two countries with contrastina i~ncidences of lune cancer and cardiac c_,ease. The USA registry faile&because of the small number of twins available over 35 years of age (Friedrntanr985). within the next two or three years data should be available on the relative significance of smoking and envisonmental'factors cn the development of disease patterns. The study of the sibs of twins and their children old enough to have started smoking is on schedlale. The auestionnaires have been prepared and tested and'the index twins are being'selected (Cru~moacker-1066). We should emphasize that the twin studies include epidemiological, genetic, cardiac, pulhnonary, dietary, psychological, and many environmental factors.

_ 7 - ~ 3. Additional Epidemiological Studies. The Multiphasi:c Examination groups at Kailser-Permanente in the Oakland area are being studied at this time for the characteristics of smokers, of smokers who quit and started again,who cuit permanently, and of never smokers (Friedman- 787). The Normative Aging group in Boston (Bosse-455, Costa-1085) continues to be a source of a stable population that provides information on several aspects of smoking and health, primarily changes associ~ated to aging. The incidence of cancer of certain types tends to concentrate in families. Lynch (941) is following such families in Nebraska. Twenty-three families have ha&two or more first degree relatives with two or more lung cancers. As of October 1, 1977.