Tobacco Documents Online

i D R A f T TOBACCO AND HEALTH RESEARCH SOKE PROPOSED STUDIES I. CANCERS A. Human Studies 1. Select populations of high, low risk. a. Test for proteins and antigen levels, blood groups. b. Test for known genetic markers. (1) HIJ1 typings (show relationship to lang, stomach, bladder, leukemia and other cancers) in susceptibles and non- suseeptibles. (2) Chros+osomal anomalies. • i (3) Test biopsy cells for transformation, e.g., oncogenic expression (antigen). (4) tusion experiments to simulate genetic crossing experiments. c. Repeat in vitro experiments on differentiated cells. 2. New prospective epidemiological study like that of A.C.S. but without its shortcominqs. 3. Predisposing factors in human lung cancer. Exhaustive multifactorial studies of constitutional, environmental, psychological, bioch:mical. hormonal, physiological, morphological, immmological, virogenetic, or other-characteristics. 4. Depth study of special populations such as that of Graz, Austria where lung cancer is high and smoking moderate. also regional difference studies like those of Eleanor Macdonald. S. Compare total and percentage lung cancer preralence by autopsy versus clinical or death certificate reports with ssioking records,collected from kin. If disparity exists, search for erroneous diagnoses. 6. Campaign to make cancer a reportable disease as a means of avaluating diagnostic criteria. 7. Study classification of lung cancers by newer and developing criteria. 8. Study time trends in the relative incidence of lung tumor types. THIS DOCUMENT SUBJECT TO CONFIOEl1TiAUTr A(,Ktt.MIEMT. !!11 N 14 0 : CTR HN 014980

2 B. 1lnimal Studies THIS DOCUMENT SUBIECT Tp CONfIDENiiaEilv ar,REEMENT. 1. Genetic definition of animal strains. a. Blood groupings. b. Specific protein.(antiqen) expression. e. Crossing experiments to locate qene transformation sites. d. Other genetically defined determinants. (1) Enzyme induction: define genes responsible. (2) R.late to inborn errors (e.g., trisosry 21). (3) Isenunologic determinants. 2. Method improvement. a. Define tobaccos more precisely. b. Literature and ITC review to determine tobacco coeiponerSts contributing to smoke. c. Standardize lung exposure methods. / (1) Exposure equipment development and testinq: standard dosage and delivery. (2) Anisul exposure methods. (a) Nose plugged. (b) Nose breathing. (e) Mouth-traeheal intubation. (d) Tracheostoiey. (e) Lung exposure "fistula". (f) Beeswax implantation, thread or metal implantation. (3) Animal conditioning: base rate determinations. (a) Breathing rates, plethysmoqraphy. (b) Lung clearance. (c) Stress analysis after machine exposure simulation. (d) Lung physiology of animals in high CO environment: additive smoke effects. (4) Lung disage determinations (using various methods of exposur (a) particulate chemical tracers including fluorescent materials (e.q., dotricontrane, chlorinated hydrocarbon (b) Gas phase tracers (Ca-hemoqlobin, CN binding, possibly C14 aldehyde binding). (c) Simultaneous conoonent measuren+ent in animals to atteer "profilc" cf amount of cxposuro. CTR HN 014981

- 3 - THIS DOCUMENT SUBJECT TO CONFIDENTIALITY AGREEMENT. (d) With continuous exposure in a chamber attempt to develop an equation for gas absorption, relate to intermittent gas exposure. (a) Lung uptake and clearance of defined particles in the presence of gas phase, whole smoke, condensate, other air pollutants (e.g., dusts). 3. Experiments using animals of known genotype, predictable tumor incidence and viral expression and for whioh antigen and other assay reagents are available, using chestically and virally primed and unprimed animals: / a. b. c. d. e. f. q. h. i. j. k. Exposure to particulate, gas phase, whole smoke in planned experiments. Use different tobaccos (e.g., high and low nitrate, sugars). Smoke, smoke constituent and dust synerqisms (asbestos and metal dwts) - Individual smoke component treatment of susceptible or primed anima ls . ' Saffioti iron oxide, carcinogen. AnderJont thread with carcinoqeru. Stanton beeswax pellet impregnated with carcinogen, possibly coupled with noxious gas or particles. Palladium wire or other radioisotope implantation method. Tistula experiments coupled with exposure method to show natural recovery after gas or other exposure: transformation stages in susceptible animal. Endocrine involvem.nt, proqesterone in beeswax effect on smoked animal or carcinogen treated animal lung (protective). Assays for specific antigen, tumor type and antiqen, tumor transplantibility in imsunoloqically tolerant host (e.q.o fetal tissue, ALi treated). Apply new electron microscope techniques and behavior in culture media and to transplant, to detine better "precancerous lesions" and distinguish them from dysplasias. 1. Develop other positive models, in animals, for the controlled production of squamow carcinoma of the lung, by use of: (1) Asbestos as a primer with polynuclear hydrocarbons or smoke condensates. (2) Nickel or nickel carbonyl. (3) Nitrosamines or nitrosamine precursors fed, inhaled, or injected in various combinations. !IN 14 0 -,~ ' CTR HAN 0149-82-

- 4 - THIS DOCUMENT SUBJECT TO m. Rodent skin assay: CONFIDENTIALITY AGREEMENT. (1) Effects of solvents used. (2) Transplantation of papillomas to other sites. (3) hcrolein as a co-carcinogen. n. Study relative effects of carcinogens on mice having chronic 20% carboxyhemoglobin. o. Study apparent differences in susceptibility among tissues directly exposed to cigarette smoke (mouth, nose, pharynx, larynx, trachea, lung). / P• Study smoke and chemical effects on isssunity (1tES) system. (1) Cell-s+ediated ima+unity effects (thymocytes). (2) Hunoral antibody synthesis effects (d lymphocy tes). (3) Effects on predigestion of antigen by macrophage. (4) Message from thymoryte to s lymphoryte: 1Qik implication. (5) Specific smoke related questions: (a) Effects of smoke on replicase levels, specific protein production, intracellular message transport. (b) Smoke effects on phagocytosis, enWrgy systasi of Macrophag- (c) Net influence on RES (additive effect). C. Tissue Culture Studies 1. Elucidate mechanisms of transformation, especially biochemical factors. 2. Develop and quantify assay methods for comparing weak and strong carcinogens•. 3. Develop system using human tissues to mtasure species and strain differences. 4. Measure oncogenic activity of tobacco and ss+oq residues (subfractions of methanol extracts). S. Carcinogen, gas phase, particulate, whole, reconstituted (minus nicotine) smoke fractions exposure. a. Specific protein production (antigens). b. Pathology, time and type of transforssation, transplantability. c. Studies with temperature sensitive trassformation strains (transform at 37• and revert at 41•). d. Studies with differentiated coll types, epithelial, endothelial from different tissues. e. Effects of tobacco varieties (e.g., high and low nitrate, sugar). kZN 1403 CTR HN 014983

S 6. Cell fusion experiments. a. Define transforming gene locus. ThiS DOCUMENT SUBJECT T) CONFIDENTIALITY AGREEMEJVT. b. Attempt to design chemical carcinogen experiments to show effects at gene level, DNA, RNA replication effects. c. Effects of fusion on enzyme induction. 7. 8asic molecular biology. a. Define cell polarity, isseunoloIIic sites; relate to contact inhibition, transformation. i b. Define period in division when transformation by chemicals is possible. c. Show effects of hemaqqlutinin induced cell division and net cells at risk, attempt to relate to rate of in vitro transformation events. d. Differentiation of cslls, involveieent in transformation? Chlorinated hydrocarbons cause reversion to fibroblasts but affect susceptibility to transformation, type of transformed cell, type of resultant tumor? e. Experiments to define repressor protein expression, controlling qene or operon expression. Then design experiments to show sr..oke. chemical effects on a molecularly defined system. II. CAFDIOVASCULJIR DISEASES A. Human Studies. 1. Absorption of nicotine by human smokers. a. Kinetic patterns of absorption and disposition. b. Rapid, specific, accurate nicotine assay using small blood samples c. Application to smokers under standardized conditions, then to largo populations. d. Observation of variations in the mechanics of smoking re blood nicotine levels. 2. Study cardiovascular diseases in identical twins with disparate snokin habits to end-points of clinical disease and death, with autopsy confirmation. I/1N 1403S CT\ `•. H,4' 014.e/TSsr \

rLI; CCC6%'E'!T : Ci:"; J CONfIDENTIAUTY AGREEMENT. 3. Differences between cigarette smokers, cigar smokers, pipe smokers and non-smokers. a. dasie genetic or constitutional differences. b. Life style and habits, patterns and practices. 4. Smoking effect on blood sugar levels re hypoglycemia in cardiovascular disease. ~ S. Acute episodes: related to past history or immediate effect of so+oking? 6. Effects of smoking on blood platelets. 7. Effects of smoking on blood levels of diphosphoQlycerate. 8. Thyroid activity as related to cardiac pathology, stress and smoking effects. B. Animal Studies. 1. Develop better animal model of coronary artery atherosclerosis. a. Study effects of nicotine. b. Compare effects of whole cigarette smoke; gas vapor phase, carbon monoxide, carbon disulfide, smoke from "ciqarette" without nicotine. i 2. Search for mechanisms of infarction and atherosclerosis, e.g., effects of stress on the electron transport mechanism. 3. Study origins, mechanisms and consequences of cardiac arthythmias in animal models, considering hormones, druqs, toxins, caffein, nicotine, carbon monoxide, fibrillation threshold. 4. in atherosclerosis, study competition of nicotine, cholesterol with NAD, NADH. S. IJsinq defined animal strains charaeterise response to nicotine and s-etabolites at various ages. a. Absorption. b. Enzyme induction. C. Lipid and cholesterol production. d. Catecholae+ine and cyclic 1W (and other receptor molecule) level in cells and systemically. e. Calcification. f. Modified animal studies. (1) Pharmaeolonical bloekade and enhancement studics. (2) Surgical modification (e.g.. adrenaleeto:++y). CTR ~~ 0 1 49BE-1

V - 7 _ iMIS DOCuhtENf ~udj~~,i iu GONFIDENIlAL11Y AGREFtdENj. 6. 2n defined animal strains, stuay biochemical and physioloqical responses to stress. a. Catecholamine responses, cyclic AMP, et al. release sequellae. b. Intersnediary metabolism response (muscle oxygen debt, fibrin formation, lipid patterns, blocks in pathways). c. Cytochrome changes (including SN reversal of oxidation by smoke). 4. Specific ribosomal protein synthesis in cerebral tissue and its inhibition. e. Genetic differences with different strains at different ages in (1) Cathecholamine response, recovery rate. (2) Age and steroid interrelationship in system with defined stress response. 7. Study thrombosis in defined animal strains. a. siochemical aechanissss of fibrin formation clotting and lysis. b. External factors affecting above. c. Genetic studies leading to model systsz. 4. Effect of age on .nsymatically defined throraus systam. e. Platelet turnover rate after smoke exposure, fibrinolysin studies. 6. Study hesiodynamics and cardiac function. a. Mschanissas of increased blood flow in ccronary and other vascular b.dss overall and nutritional flow. b. Effects of nicotine and smoke on systeRic and tissue catecholasnin energy state of muscle, including 9lyeoqen mobili:ation, aerobic and•anaerobic response, and cytochrome responses. C. T!.ssue Culture Studies. 1. Characterise nicotine response at cellular level; tests as for in vi* 2-. Nicotine effects on cells of defined straiss, of various tissues to show differentiation influence on tested espressions. Hypothalanus. sympathetic qanqlion, medulla 5 HT release from thrombocytes. Cholinorqic response. , CTR HN 014986

a THIS DOCUMENT SUBJECT TO 3. Stress responses in n vitro. CONfIDENTIALITY AGREEMENT. a. Catecholamine responses, cyclic Ate, et al. release sequellae. b. intermediary metabolism response. c. Cytochrome chanqes (including SN reversal of oxidation by amoke). III. CHRCNIC LUNG DISEASES A. Ituman Studies. 1. 2. 3. ~ 4. S. Lonqitudinal clinical study to define clinical entities and improve diagnosis, including smokers and non-smokers, with exhaustive histories, examinations, and teats. Post mortem study of the lungs of the same subjects, using the best modern methods, including electron microscopic examination, and correlation of deqree of emphysema with records of lung function in vivo. Lonq-t6rm canvass study on a geographical basis, of random 'sar+ples of lungs post mortem, to measure anatomic eiphysema, smokers and non- smokers, in various locations. Could be confined to relatively healthy victims of accidents or homicide. Comparison of smoking and non•smokinq homrosvqotes (alphal-antitr,vosin deficiency) and normals re age of incidence and severity of emphysema. non-smokers, and antitrypsin assays. Repetitive lung function studies of healthy subjects, smokers and 6. Comparison of emphyseria reported on death certificates with that fount at autopsy, to determine incorrect clinical diagnosis. 7. Critical review of existing epidemioloqical studies that have associated emphysema with smoking. 6. Study, in healthy people, the relation between smoking and respirator 4nfections, including acute and chronic bronchitis. 9. rurther investigation of the role of auto-issiun* reactions in human chronic respiratory disease, backed by animal inhalation studies. 10. Natural emphysema incidence. Anomalous casss (e.g., six in one famil 11. Specific industrial or other exposures: define a population. 12. Specific air pollution exposures: define population background incid• CTR HN 01496'

8. Mimal Studies 9 THIS DOCUGIENT SUBJECT rC CONFIDENTIALITY AGREEh1ENTJ 1. Lung function changes in animals inhaling qas-vapor phase of reference cigarette smoke. If the qas-vapor phase produces the same effects known to be produced by whole smoke, further fractionation should identify the responsible agents. ' 2. Improvement of animal models for human emphysema by inbreeding to ' produce a strain resembling the hunan homosyqotes for antitrypsin deficiency. Artificial emphysema by injection of proteolytic enzymes into animals needs study to'see whether the model is analogous to the human disease, and to establish the right doses of ensym.. 3. Long-term whole-smoke and gas-vapor phase inhalation studies on several strains of rodents, dogs, and primates to establish strain and species comparisons, and to seek changes predictive of eventual disease. 4. in ruminants, study analogy of fungal exposure, etc. Might be developed in susceptible mouse s+odel. 5. Lung physiology and biochamistry in genetically dtfinad ani"ls. i a. b. C57 or other specified lorn tumor strain of known genetic constitution: define natural emphysema incidence in old age. Dust, chromate, etc., exposure of same strains may be needed to c. d. express lesion. Breeding experiments to concentrate expression, show proximity to other gene expressions (e.q., histocompatibility qanes). With genetically defined model, test'for (1) Alpha -antitrypsin inhibitor. (2) Antielastase inhibitor. (3) Collagen structural differences. (4) Autoiswine response to denatured collaqen. (S) •Aerobio-anaerobic pathways (LDH isoenzymes, TCA ensymes, t. Anis+al (defined natural incidence) exposure to induce change. etc.) (1) Whole smoke. (2) Gas phase. (3) Particulate. (4) Smoke components (a.q., aldehydes) measure binding to collaqer. (5) Smoke and dusts. (6) Smoke from differing tobacco types (e.q., high and low nitratE sugar). I;N 14 o-. CTf' HN 01 49eS

- 10 - IV. SMOIQ CHEMISTRY THIS DOCUMENT SUBJECT TO CONFIDENTIAUTY AGREEMENT. A. Sulfhydryl compounds in smokes from various tobaecos. (Because of effects of such compounds in recoupling site II of electron transport system in mitochondria). B. Grosser gas-phase components of cigar and pipe tobacco smokes versus of cigarettes. those C. Nickel carbonyl in smoke of ordinary reference cigarettes and cigarettes primed with metallic nickel or nickel compounds. D. Cadmiuw in cigarettes, mainstream smoke, and smoke condensate. V. UNDLRtXPLflITLD STUDY SYSTBMS• A. The gibbon ape, which could be exposed to smoke inhalation and is phyloqenetically close to man. B. Inbred siiee with genetically determined blood pressure levels. C. American Indian nales; relationship of smoking habits to low cancer death rates. / VI. NICOTINE ABSORPTION, METABOLISM, AND EFFECTS A. LffeCts on metabolism of cononon drugs (e.g., aspirin, tranquili:ers). B. Effects of coasAon drugs on the efficiency of nicotine metabolism. C. Effects of chronic smoking on efficiency of the metabolism of administered nicotine. D. Congenital differences in tfie ability to metabolise nicotine. E. Nicotine absorption from various types of smoking. F. Effects of nicotine on blood pressure. . CNS effects of nicotines arousal. VII. BENElICIAL LffSCTS 0? SMOKING A. Human measurements of effects of nicotine on learninq, and other behavior, reported from animal studies. B. Seek further pharmacological and biochemical saschanisme of tranquilisinc and arousal effects of nicotine. 140 CTR_ HN 014.989

/ TNIS DOCUMENT SUBIECT TO CONFIDEMTIALJTY AGREEMENT. C. Repeat observations suggesting that congenital brain-vave patterns are predictive of smoking habits developed later. Could EEG's be predictive of psychosomatic diseases? D. Study relative increase in physical titness of smokers and non-smokers on standardized training regimens. i I 1404 IIN C T R_ It"'I 1-4 0 14 SI 9 0