Tobacco Documents Online

D R A F T TOBACCO ANDHEALT'.Hi RESE:ARCIii SOME PROPOSED STUDIES', I. CANCERS A. Human Studies 1. Select populations of high, low risk, a. Test for proteins and antigen levels, blood grou-ps, b. Test for known genetic markers. (1) HLA tytings (show relationship to lung, sto:nach, bladder, leukemia and other cancers) in susceptibles and non- susceptibles, (2) Chromosomal anomalies. (3) Test biopsy cells for transformation, e.gi., oncogenic expression~ (antigen), (4) Fusion experiments to simulate genetic crossing experii::enus. C. Repeat in vitro experiments on differentiiatedicells. 2. New prospective epidemilological study like that of A.C.S. but without its shortcomings. 3. Predisposingifactors in human lung cancer. E xhaustive multifactorial studies of constitutional, environ:rental, rsyc?ological, biochemicali, hormonal, physiological, morphologieal, imr.•unoLogical, virogenetiic,, or other characteristics. 4. Depth study of special popul'ations such as that of Graz, Austria where lung cancer is high and smoking r.roddrate. A1so regiional difference studies like those of Eleanor Macdonald. 5. Compare total and percentage lung cancer prevalence by auto:;sy versus clinical or death certificate reoorts with _r:okinc reaores collected! from kin. If disparity exists, search for erreneous dilagncses. 6. Campaign to make cancer a reporta!Dle discase as a means of evaluating diagnostic criteria. 7. Study classification of lung cancers bv ne.,°er and davelc.;ing criteria. 8. Study time trends in the relative inaiidencc of lung t=,or tyces.

B'. Animal Studies 1. Genetic definiti~on of animal strains. a. Blood gzoupings. b, Specific protein (antigen) expression. c. Crossing experiments to locate gene transformation sites. d. Other genetically defined determinants. (1) Enzyme indinction: define genes responsible. (12) Relate to inborn errors (e.g.,, trisomy 21). (3) Immunologic determinants. 2. Metlhodi i~mprovement. a. Define tobaccos more precisely. b. Literature and ITC review to determine tobacco components contributing to smoke. c. Standardize lung exposure methods. (1) Exposure equipment development and tPsting: standard dosage and delivery. (2) Animal exposure methods.. (a)i Nose plugged. (b): Nose breathing. (c) Mouthrtracheal intubation. (d) Tracheostomv. (e) Lung exposure "fistula". (f) Beeswax im.pLantation, thread or metal i^.plantation. (3) Animal conditi~oning: base rate determinations. ('a) Breathing rates, plethvs:o5raohy. (ib). Lung~~ ciearance.(ic) stress analysis after machine ext)osure simulation. (d) Lung physiology of animals in highlCO:environment: additive smoke effects. (49: Lung disage determinations (vsing various methods of exposure). (a) Particulate chemiical tracers inclcdina flucrescent materials (e.g., dotricontrane, chlorilnated hydrecsrbons) (b) Gas ehase tracers ('C^-hemoglobin, C::' bir.di:„ _-,ossi5lv C ~.~ '~.. C14'aldehydebinding). (c) Simultaneous corcconent r•easurer.rent in aninals to att~r=t of a^.o'.....t cf oxc rs. ~

C -3 C (d) With continuous exnosure in a chamber attempt to develop an equation for gas absorption, relate to intermittent gas exposure. (e) Lung uptake and clearance of dofined particles in the presence of gas phase, whole smoke, condensate, other air pollutants (e.g., dusts). 3. Experiments using animals of knownigenotype, predictable tumor incidence and viral expression andifor which antigen and other assay reagents are available, using chemically and virally primed and unprimed animals: a. Exposure to particulate, gas phase, whole smoke in alanned experiments. Use different tobaccos (e.g., high and low nitrate, sugars ) ,. Smoke, smoke constituent and dust synergisms (asbestos and metal dusts). c. Individual smoke component treatment of susceptible or primed anilma ls . d. Saffioti iron oxide, carcinogen. e. Andervont thread with carcincgens. f. Stanton beeswax pellet impregnated with carcinogen, possibly coupled with noxi~ous gas or particles. g. Palladium wire or other radioisotope implantation method. h. Fistula experiments counlied with exoosure method to show natural recovery after gas or other exposure: transformation stages in susceptible animal. i. Endocrine involvement: progesterone ilnibeeswax effect on sroked aniinalior carcinogen treated animal lung (protective). j. Assays for specific antigen, tumor ty:e and antigen, tunor transplantibility in immunologically tolerant host (e.g., fetali tissue, ALS treated'). k. Apply new,electron microscoce technioues and behavilor in culture media and to transplant, to_define better "_nrecancerous lesions"' and~distinguish,them from dysolasias. 1. Develbp other r.oc~itive models, in,animals, for the controlLed production of squamous carcinoma of the lung, by use of: (1) Asbestos as ai primer with polynucleas hydrocarlions or saoke condensates. (2) Nickel or nic;el carbonvl. (3) Niltirosar.uines ornitrosarr.inenrec_urso_rs fed, inhaled, or injected in various corbinationc, I

m. Rodent skin assay: P• (1) Effects of solvents used. (2) Transplantation of papil'lomas to other sites. (3) AcroLein 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). Study smoke and chemical effects on immunity (RES) system. (1) Cel'1-mediated immunity, effects (thymocytes). (2) Humoral antibody synthesis effects ('B,lymcnocytes) (3) Efflects;on predigestibn of anticen by maarophage. (4) Message from thymocvte to B lymphocyte: R:1A implication. (5) Specific smoke related questions: (a) Effects of smoke on reolicase levels, specific protein production, intracellular message transport. (b) Smoke effects on phagocy•tosis, energy system of macrophace. (e) Net influence cn P,ES (additive effect). C. Tissue Culture Studies 1. Elucidate mechanisms of transformation, especially biochemical factors. 2. Develop and qpantify assay methods for com_oaring weak and strong carcinogens. 3. Develop system~using human tissues to measure species and strain differences. 4. Measure oncogenic activity of tc:,acco and smog residues (suk9iractions of inethanol' extracts). 5. Carcinogen, gas phase, particulate, whole, reconstituted (minus nicotine) smoke fractions exposure. a. b. c. d. e. Specific protein production (iantigens). Pathology, time and type of transformation, transplantability. Studies with temoerature sensitive transfornaticn strains (transform at 37°' and revert at 41°') Studies with differentiated cell, types, eDitheli~al, endbthelial from different tissues. Effects o= tobacco vnrieties (c.g., hiclanri lo:: nitrate, sugar). C ~ ~ ~ ~ ~ N

I 6. Cell fusion experiments. a. Define transformingigene locus. b. Attempt to design chemical carcinogen,experinents to show effects at gene level, DNA, RNA replication effects. c. Effects of fusion on enzyme induction. 7. Basic molecular biology. a. Define cell polarity,, immunologic sites;, relate to contact inhibition, transformation. b. Define period! iini divilsion when transEormation by chemicals is possible. c. Show effects of hemagglutinin induced cell division and net cells at risk, attempt to relate to rate of in vitro transformation events. d. Differentiation of cells, involvement in transformation? Chlorinated hydrocarbons cause reversion to fibroblasts but affect susceotibililty to transfornatiion, tyce of transforired cell, type of resultant tumor? e. Experiments to define repressor protein expression, controll'ing gene or operon er.oression. Then dl:sicn exaeri_ments to show s^,cke,, chemical effects on a molecularly defined system. II. CARDIOVASCULAR D15:AcES A. Human Studies. 1. Absorption of ni~cotine by human smokers. a. Kinetic patterns of absorption and:dispositiond b. Rapid, specific, accurate nicotine assay usina small blood sam:cies. C. Application to smokers undersCandgrcize3'conditions, then.tolargen populations. d. Observation of variations in the mechanics of smoking,re blood nicotine levels. 2. Study cardiovascular diseases in id'entical '_tLins with discarate r.,e'<ina habits to end-ooints of clinicsl disease and'death, witi autopsy confir:. aticn.

3. Differences between cigarette smokers, cigar smokers, nipe smokers and non-smokers. a. Basic genetic or constitutional differences. b. Life style and habits, patterns and'practices. 4'. Smoking effect on blood sugar levels re hypoglycemia in cardiovascular disease. 5. Acute episodes: related to past history or immediate effect of smoking?' 6. Effects of smoking on blood platelets. 7. Effects of smoking on blood level's of diphosghoglycerate. 8. Thyroid'acti'vity as related to cardiac pathology, stress andismcking effects. B. Animal Studies. 1. Develop better animal model of coronary artery atherosclerosis. a. Study effects of nicotine. b. Cornpare effects of whole cigarette smoke, gas vapor phase, carbon monoxide, carbon disulifide, smoke from "cigarette'" ::ithout nicotine. 2. Search for mechani.s^s of infarction and atherosclerosis, e.g,., effects of stress on the electron transport m,.echanism. 3. Study origins, mechanisms and conseciuences of cardiac arrhytlYSmias in animal models, considering hornor,es, drugs, toxins, caffein, nicotine_, carbon monoxide, fibrillation threshold. 4. In atheroselierosis, study competition of nicotine, cholesterol',with, NAD, NADH. 5. Using,defined animal strains characterize response to nicotine and metabolites at various aaes. a. Absorption. h. Enzyme induction. a. Lipid and cholesterol oroduction. d. Catecholamine and~ cvcl'.ic (and other rece_etor moiecule)' level in cellis and systemically. e. Calcification. f. Modified animal studies. (1)Yharmaco]locicall bl'rckadeand en?iance7,n st~o ies. (2) Surgical noc:i`_ication (e.g., adr .aic,ct _ ) G_=

C 6. In defined animal strains, study biochemical and physiological responses to stress. a. Catecholamine responses, cyclic AMP, et al. release sequellae. b. Intermediary metabol'ism resconse (muscle oxygen debt, fibriln, formation,,,lipid patterns, bLocks in pathways). c. Cytochrome changes (including SH reversal of oxidation by smoke). d. Specific ribasomal orotein 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 steroidinterrelationshi'p in system with defined stress resoonse. 7. Study thrombosis in dafined animal strains. a. Biochemical mechanisms of fibrin formation clotting and lysi~s. b. External factors affecti~ng above. c. Genetic studies leading to model system. d'. Effect of age on enzymatically defined ..hrombus syatc;;. e. Pliatelet turnover rate after smoke exposure, fibrinolysiini studies. 8, Study hemodynamics and cardiac function. a. Mechanisns of increased blood flow in coronary and other vascular beds; overall! andinutritional flow. b. Effects of nicotine and'smoke on systemic and tissue catecholiamines, energy state of muscle, including glycogen mobilization, acrobic and anaerobic response, and cytochrome resnonFes. C. TilssueCulture Studies. 1. Characterize nicotilne resoonse at cellluliar lcvel; tests as for in vivo. 2. Nicotine effects on cells of defined strains, of various tissues to show dif.`erentiation influence on tested excressions. 'rvr,othala:-us, sympathetic ganclion„ medu17ai 5 HT release from throrwoc_tes. Cholinergic resnonse.

r- 3. Stress responses in vitro. a. Catecholiamine responses, cyclic AMP,, et al. release sequellae. b. Intermediary metabolism response. c. Cytochrome changes (including SH reversal of oxidation by smoke). ISI. CHRONIC LUNG DISEASES A. Human Studies. 1. Longitudinal clinical study to define clinical entities;and i:nprove diagnosis,, including smokers and non-smokers, with exhaustive historiies,, examinations, and tests. 2. Post mortem study of the lungs of the same subjects, using the best modern methods,,including electron microscopic examination, and correliation of degree of emphysema with records of lung function in vivo. 3. Long-terrn canvass study on a geographical basis, of rando^ samples of lungs post mortem, to measure anatomic efuhysema, sr:okers and non- smokers, in various locations. Could~be confined to relatively healthy victims of accidents or homicide. 4. Comparison of _moki- and non-=*^okina ho*.^:oz•_,-gotes (.al~hal-antitr:zsi_n deficiency) and normals re ace of incidence and severity of emphysema. 5. Repetitive lung functioni studies of healthy subjects, s:-okers and non*-smokers, and antitrypsin assays. 6. Comparison of emphysema recorted on death certificates with that found at autopsy, to determine incorrect cl'inicalidi.agnosis. 7. Critical review of existing epidemiological studies that have associated emphysema with smoking. 8. Study, in healthy neo_;le, the reliation bet-ween smokimg and respiratory infections, including acute and;chronic brorchitis. 9. Further InF estigation of the role of autlo-i;.r;une reactions in hn.^~~.an chronic respiratory disease, backed by ani.:al inhalation studies. 10s Natural enphysema incidence. Anonalous cases (e.g,., six in one fa7-~iliv) 11. Specific industrial or other ex.nosures: def;ne a pepulation. 12. Specific air pollution exnosures: define po.ulation hackground incidence. L=

r C B. Animal Studies - 9 - C 1. Lung function changes in animals inhaling gas-vapor phase of reference cigarette smoke. If the gas-varor phase nroduces the same effects known to be produced by whole smoke, further fractionation shouldd identify the responsible agents. 2. Improvement of animal models for human emnhysena by inbreeding to produce a strain resembling the human homozygptes for anti'trypsin deficiency. Artificial emphysema by iinjection of proteolytic enzymes ilnto animals needs study to see whether the model is analogous to the hur,an disease, and to estatbl:ish the right doses of enzyme. 3. Long-term whole-smoke and a3s-vapor phase inhalation studies on several strains of rodents, dogs, and prirnates to~establishistrain and scecies comparisons, and to seek changes predictive of eventual disease. Iin ruminants,,study analogy of f•,:ngal exposure, etc. Might be developed in susceptible mouse model. 5. Lung physiology and biochemistry in genetically defined ar.im,als. a. C57 or other specified low tumor strain of kno~m cenetic constitution: define natural emohysema incidence in old age. b. Dust, chromate, etc., exi:)osure of same strains mav be need'ed to express lesi=. c. Breed7ng exoeriments to,concentsate ex,Dressien, show proxi:nity other gene expressi!ons (e.g. , histocompatibililty genes). d. With genetically defined model,, test for (1) Alpha -antitry.osi~n, ilnhibitor. (2) Antielas.tasei~n}iiibitor.. (3) Collagen structural differences. (4) Autoimmune nesoonse to denatured collacen. (5) Aerobic-anaerobic pathways (LDd isoenzymes, TCA enzymes, etc.)i. e. Animal (defined natural incidence) exaosure to indiice change. (1) Whole smoke. (2) Gas uhase. (3) Parti'cuLate. (4) Sscoke co:zpor:ents (e.g. , aldehydes) , measure bindinc to collacen. (5) Smoke and dusts. (6) ,,_,.z;e from differiing tobacco types (e.g. , high and low nitrate, suga,r)'. 4 G-=

IN'. SMOKE CHEMISTRY A. Sulfhydryll compounds in smokes from various t'obaccos. (Because of effects of such compounds in recoupliing site II of electron transport systeca in mitochondria). BS Grosser gas-phase component's of cigar and pipe tobacco smokes versus those of cigarettes.. C'. Nickel carbonyl in smoke of ordinary reference cigarettes and cigarettes primed with metallic nickel or nickel compounds. D. Cadmium inicigarettes, mainstream smoke, and smoke conddnsate. V. UNDEREXPLOITED.STUDYSYSTEMS A. The g,ibbon ape, which could be exposed'to,smoke inhalation and is, phylogenetically close to nani. B. Inbred mice with genetically determined blood pressure levels. C. American I~ndi~an malles;, relationship of smoking habits to lbw,cancer death rates. VI. NICOTINEABSORYTIOJd!.,. ME'PAB(JLISS:,. e'iNDi FFFECT9 A. Effects on metabolism of common dYugs (e.g.,, aspirin,, tranquilizers),. B. Effects of common drugs on the efficiency of nicotine metabolism. C. Effects of chronic smoking on efficiency of the metabolism of administered ni~cotine. D. Congenital differences in the ability to metabolize nicotine. E'. Nicotine absorption from various types of smoking. F. Effects of nicotine on blood'pressure. G. CNS effects of nicotine; arousal. W G_= VII. BENEFTCIAL RFFiiCTS OF SMOKING A. Hunran measurenents of effects of nicotine on,lsarning, and otirer behavi.or, reportedl from animal studies. B. Seek further phar:nacolo:?ical! and biochemical e:echanis^s of trunquil!izinq, and arousal effects of nicotir.e. Lr 459= ~..~ C'"_ ar-