Tobacco Documents Online

.v\1A-ERF COtiiMITTEE FOR RF.SEARCH ON TOH ir :CO AND HEALTH \F,E,ninted lunt• 1964 Richard J. B,ing.M. D. 1964-1977 Paul S. Larson, Ph.D. 1964-1977 Professor of Medicine L'niversity of Southern California Director of Cardiology and I ntramural Medicine Huntington Memorial Hospital Haag Professor of Pharmacology Chairman Emeritus, Department of Pharmacology Medical College of Virginia Stuart Bondurant, ?bf. D. 1970-1977 President and Dean Albany Medical College of Union University Charles LeMaistre, M. D. 1964-1966 Chancellor University of Texas Southwestern Medical School, Dallas, Texas Earl A. Evans, Jr., Ph.D. 1969-1977 Richard D. Remington, Ph.D. 1969-1977 Professor and Chairman Emeritus Department of Biochemistry University of Chicago Dean, School of Public Health University of Michigan Robert J. Hasterlik, M. D. 1966-1977 Maurice H. Seevers, M.D., Ph.D.** La Jolla, California CHAIRMAN 1964-1977 ohn B. Hickam, M. D.* 1964-1970 Professor Emeritus Department of Pharmacology University of Michigan Chester M. Southam, M. D. 965-1966 Chairman. Department of Internal Medicine C'niversitv of Indiana Paul Kotin, M. D. 1966-1977 t'ice President, Health, Safety and Environment Johns-Manville Chairman, Dept. of Oncology Jefferson Medical College of Thomas Jefferson University AMA Staff Secretaries John C. Ballin, Ph.D. 964-1965 Greenwood Plaza Marvin Kuschner, M. D. 1969-1977 American Medical Association Ira Singer, Ph.D. 1966-1977 Dean, School of Medicine Health ; ciznce Center State University of New York at Stony Brook American Medical Association Deceased February 9, 1970 * Deceased April 29, 1977 T200235 TIMN 0115744 xi

Sect 27. Effect of Nicotine Treatment on the Metabolism of Nicotine in the %iouse Liver in Vitro ...... 14 28. AIteration of Enzt-me Activitv in Rat Liver Following the Acute and Chronic Administration of Nicotine .................................................................. 15 29. Effects of Increased Liver Metabolism of Nicotine on its Uptake. Elimination and Toxicitv in `lice ..................................................................... 15 30. Stimulation of Nuclear Protein Svnthesis in Rat Liver After Phenobarbital Administration ..... 15 31. Comparison on Effects of Phenobarbital and Nicotine on Nuclear Protein Svnthesis in Rat Liver ..................................................................... 16 32. Potent Inhibitory Action of Pilocarpine on Hepatic Drug Metabolism ..................... 16 33. Copper-Binding Activity of Tobacco Smoke Condensate .............................. 16 34. Isolation of Metal-Binding Fractions from Tobacco Smoke Condensate .................... 17 35. Effects of %tetal-Binding Fractions of Tobacco Smoke on in Vitro Activity of Enzymes ........ 17 36. Potent in Vitro Inhibitors of Tyrosinase: Vtetal-Binding Fractions of Tobacco Smoke Con- densate ................................................................... 17 37. Isolation of Metal-Binding Agents from Lettuce Cigarette Smoke and their Effect on 0,-Uptake of Liver Slices ................................................................ 18 38. Effect of Tobacco Smoke Extracts on Mitochondrial Respiration and Anion Transport ........ 18 39. X%-litol Metabolism in Perfused Rat Liver: Interactions with Gluconeogenesis and Ketogenesis .. 18 40. Molecular Structures and Catalytic Activity. I. Catalytic Polarography of Thiamine. Oxythiamine and Thiamine Phosphates ..................................................... 19 D. Excretion ..................................................................... 19 41. Molecular Structures and Catalytic Activity. II. Proton Reactivity and Catalytic Polarography of Histidine and Related Imidazole Derivatives ....................................... 19 42. Excretion of Nicotine and its Metabolites in Dog and Monkey Saliva ..................... 20 43. GastricExcretionofC"-I`'icotine ................................................ 20 E. Toxic:olog}..................................................................... 21 44. Comparison of Pharmacological Responses to Nicotine and Release of Catecholamines from the -\drenals in Dogs and Monkeys ................................................. 21 45. Comparison of the Effects of Nicotine on Catecholamine Release from Isolated Adrenal Glands of Dogs and Monkeys .......................................................... 21 -t6. Comparison of the Cardiostimulatory Effects of Nicotine in Dogs and Monkeys ............. 21 47. Comparatix e Studies of Hepatic Nicotine Metabolizing Enzyme Activities in Monkeys and Dogs 22 18. Lack of Effect of Chronic Nicotine Administration on Fatty Acid Distribution in the Liver, Testis, and Adipose Tissue of Male Fisher-344 Rats ....................................... 22 -I9. Effects of Cb-ronic Nicotine Adn.inistration arrd Age in Male Fischer-344 Rats .............. 22 50. Hepatic Function after Acute or Subchronic Nicotine Administration in Untreated Mice and Mice Treated with Hepatotoxic Chemicals ............................................. 23 51. Cadmium and Nickel-Common Characteristics of Lettuce Leaf and Tobacco Cigarette Smoke .. 23 52. Biological Disposition of Nicotine: Mechanisms of Protection Against the Acute Toxicity of Nicotine .................................................................. 23 Abs( Excr Sum I n tro( absor t iun i chem 5ecti( r,cloe hePn . contri T tru m c timuki inhali Incon ent eft lipid n marizF tall\'sl tornar\ the cus of toba quantit rettes t hese tc %vithou i n mea: 'i l l not Fo presun stituen ( 2.5.6): carbon isoprer propioi B. Dist Dv tine fo subject leaves ~ ~•arying notablv tributio a.ailabl monkev having toleranc 2 TIMN 0115748 T200239

Section I Absorption. Distribution, Metabolism, Excretion. Toxi(cPlogy Surnnlarx, Abstracts (52) A. Absorption ......................................................... ... ; 1. Effect of Cigarette. Cigar. and Pipe Smoking on Nicotine Excretion. The Influence of fnhalin, t; 2. Distribution of Nicotine in the Rat ......................................... . ...... r; 3. Tissue Distribution of ('H) Nicotine in Dogs and Rhesus ,vtonkeys ..................... . t; 4. Distribution of Nicotine in the Central Nervous Svstem ...................... . ...... - ;. Age Dependent Changes in Nicotine Distribution in the Brain of the Mouse . ......... . . . . . . - 6. Lethal Brain Concentrations of Nicotine in Mice of Different Ages ............... . ........ 8 ;.Accumulation of Nicotine in Pancreatic Islets and Calcitonin-Producing Cells in Mice and Chicks Demonstrated by Micro- and Whole-Body Autoradiography ......................... . . . . 8 8. Catechol- and Indolamines in some Endocrine Cell Systems. An Autoradiographical. His- tochemical and Radioimmunological Study ......................... . . . . . . . . . . . . . . . . t~ 9. Dependence of Nicotine-C" Distribution and Movements Upon pH in Frog Sartorius Muscle .... q 10. Uptake and Distribution of Nicotine-"C in Frog Rectus Abdominis Muscle ................. q 11. Placental Transfer and Distribution of Nicotine in the Pregnant Rhesus Monkey ............. q B. Distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ....................... . . 10 12. Protein Conformation in Biomembranes: Optical Rotation and Absorption of Membrane Sus- pensions ................................................................... 10 13. Circular Dichroism and Absorption Studies on Biomembranes .......................... 10 14. Corrections for Optical Rotation Data on Biomembranes ............................... 10 C. , tetabolism ................................................................... 10 15. Urinarv Excretion of Conjugate Forms of 1-(3-Pyridyl) Ethanol After Administration of 3- Acetylpyridine .............................................................. 10 16. Studies on the Separation of Acidic Metabolites of Nicotine by Gas Chromatography ......... 11 17. Metabolism of (±)-Cotinine-2-"C in the Rat ........................................ 11 18. The Fate and Distribution of 1-(3-Pyridyl) Ethanol Methiodide in Relation to the Toxicih• of 1-(3- P%'ridyl) Ethanol and 3-Acetylpyridine ............................................. 11 19. Studies on the Excretion of 5-(3-Pyridyl)-Tetrahydrofuranone-2 and its Intermediary Role in the Lietabolism of Nicotine ....................................................... 12 20. Additional Routes in the Metabolism of Nicotine to 3-Pvridvlacetate. The Metabolism of Dihvdrometanicotine ......................................................... 12 21. Pvridvl Acids from the Mammalian Metabolism of Trans-Metanicotine ................... 13 22. Synthesis of Trans-4-(3-Pyridyl)-3-Butenoic Acid, A Metanicotine Metabolite .............. 13 23. Structure o[ Dibromoticonine, A Bromination Product of Nicotine ....................... i 3 24. N-3-Pyridylacetylglycine as a Nicotine Metabolite ................................... 13 25. The Metabolism of Nicotine and Cotinine bv a Mouse Liver Preparation .................. 14 26. Metabolism in Vitro of "C-Nicotine in Livers of Foetal, Newborn and Young Mice .......... 1-1 TIMN 0115747 T200238 1

Absorption. Distribution. Metabolism. Excretion. Toxicologv keys in the third trimester. Nicotine was exchanged rapidlv across the placenta between mother and fetus when it was administered into the maternal circulation in a dose of 1 mg per kg of body weight. The concentration of nicotine in the fetal circulation surpassed the maternal level rapidly, reaching maxi- mum in 16 min and remaining at a higher con- centration for over two hours following the in- jection. Disappearance of nicotine from the fetal circulation was slower than that from the maternal circulation. The concentration of nicotine in adrenal glands, heart, kidneys, stomach wall, and spleen of fetuses was high; that in placenta and skeletal muscles was low. The amount of nicotine contained in a fetus, however, was onlv a small fraction of the total dose administered to the mother. Other support: U. S. Public Health Service. B. Distribution 12. Protein conformation in biomembranes: Optical rotation and absorption of membrane suspensions Dan W. Urry Division of Molecular Biophysics, Laboratory of Molecular Biology. t'niversity of Alabama Medical Center. Birmingham, Alabama Biochimica et Biphysica .4cta 265:115-168, 1972 An analytical review of the above subject is presented. Other support: Mental Health Board of Alabama. 13. Circular dichroism and absorption studies on biomembranes Dan W. Urry, and M. M. Long Laboratorv of Molecular Biophysics. University of Alabama .1.fedical Center, Birmingham, Alabama Methods in Membrane Biology, Volume 1, edited by Edward D. Korn. Plenum Press, pp 105-141, 1974 A discussion of the techniques and results of the measurement of circular dichroism (the difference in absorbance measurement between left and right circular polarized light) and the optical absorption of a number of synthetic macromolecules and bio- membranes is presented. Most of the illustrations are derived from studies by the senior author and his colleagues. Other support: Mental Health Board of Alabama. and the U. S. Public Health Service. 14. Corrections for optical rotation data on biomembranes Dan W. Urry Laboratory of Molecular Biophysics, University of Alabama Medical Center, Birmingham, Alabama Methods in Enzymology: Biomembranes 32:220-233, 1974. Edited by Colowick and Kaplan, Academic Press, Inc. Corrections to be applied to measurements of the circular dichroism patterns of biomembranes so as to ensure their greater accuracy are presented. Other support: Mental Health Board of Alabama. C. Metabolism 15. Urinary excretion of conjugate forms of 1-(3- pyridyl) ethanol after administration of 3- acetylpyridine Herbert McKennis, Jr., Lennox B. Turnbull, Edward R. Bowman, and C. Norman Lukhard Department of Pharmacology, Medical College of Virginia. Richmond, Virginia Journal of Biological Chemistry 241:1878-1881, 1966 Oral administration of 3-acetylpyridine (a me- tabolite of nicotine) to the dog led to the excretion of 1-(3-pyridyl)ethanol in two acidic conjugate forms which were fractionated with the aid of ion ex- change resins. The more acidic of the conjugate forms had the properties of a sulfate ester in comparison with synthetic material. Acidic hydrolysis of the sulfate ester fraction led to the release of (-)-1-(3-pyri- dyl)ethanol. Acidic hydrolysis of the other conjugate form, which had the properties of a glucuronide, led to the release of 1-3(3-pyridyl)ethanol which after chromatographic purification was approximately 95% pure on the basis of extinction coefficient and which was approximately 60% in the levorotatory form on the basis of optical rotation. The pyridyle- thanol separated after the hydrolysis of the glu- curonide contained, in addition, two other Koenig- positive materials. By the chromatographic behavior TIMN 0115756 T200247 of tat giL cy e. [)t: Co An 16. me Her %to DeF Virs t'ir_ gas met met y-(3 acic vest a C, Aqu met p roc colu com of c in ( and sent The butx affoi the i OthE and 17. Paolc and I Depa ['irgi. Jo u rn rneta 10

Absorption. Distribution. Metabolism. Excretion. Toxicolog%- and its metabolites into mice foetuses and placentae. abs 15. In addition. also see three papers comprising basic studies on the circular dichroism and optical absorption of synthetic polypeotides on a variety of biomembranes, since these mav ultimatelv become pertinent in understanding distribution of both en- dogenous and exogenous substances (12. 13, 14). C. Metabolism The complex metabolism of nicotine has been extensivelv and precisely studied by the isolation and characterization of intermediate compounds. etc. Such knowledge is not only important to an understanding of the pathways of biotransformation of nicotine, but also makes possible the further study of any biological activities that are inherent in the transformation products. Studies contributing di- rectlv or indirectlv to knowledge concerning the metabolism of nicotine appearing in this subsec- tion include the following: Urinary excretion of conjugate forms of 1-(3-pridyl)ethanol after adminis- tration of 3-acetvlpyridine (15); Studies on the sepa- ration of acidic metabolites of nicotine by gas chro- matographv (16): Metabolism of (±)-cotinine-2-14C in the rat (17): The fate and distribution of 1-(3- pvridvl)ethanol methiodide in relation to the toxic- itv of 1-(3-pvridy1)ethanol and 3-acetylpyridine (18); Studies on the excretion of 5-(3-pyridyl)-tetrahydro- furanone-2 and its intermediarv role in the me- tabolism of nicotine (19): Additional routes in fhe metabolism of nicotine to 3-pvridylacetate: The metabolism of dihvdrometanicotine (20): Pyridvl acids from the mammalian metabolism of trans- metanicotine (21): Svnthesis of trans-4-(3)-pyridvl)- 3-butenoic acid. a metanicotine metabolite (22); Structure of dibromoticonine, a bromination product of nicotine (23): :\'-3-Pvridvlacetvlglycine as a nico- tine metabolite (24): The metabolism of nicotine is carried out most notablv bv the liver. an activity that is shared in lesser degree bv kidnev and lung, but apparently not b%- brain. Of two studies, summaries of which have been placed in this subsection, one deals with the metabolism of nicotine and cotinine by a mouse liver preparation (?5), the other with metabolfsrn in vitro of "C-nicotine in livers of foetal, newborn and young mice (26). A number of compounds are known to cause enz~Itne induction which may accelerate their own metabolism andlor that of other drugs. In mice. treatment with nicotine for up to 17 days in moder- ate dosage does not appear to significantly affect its metabolism by liver, and in toxic dosage depresses it (27). However, pretreatment with nicotine causes an elevation in the metabolism of ethylmorphine. nor- codeine and aniline by liver microsomes, possibly bv inducing enzyme svnthesis (28). Phenobarbital is a well-documented inducer of manv microsomal enzymes including those causing enhancement of liver metabolism of nicotine both in vitro and in vivo (29). Stimulation of nuclear protein synthesis in rat liver after phenobarbital administration has been the subject of another studv (30), followed by a compari- son on effects of phenobarbital and nicotine on nuclear protein synthesis in rat liver (31). In contrast to the stimulating effect of phenobarbital, pilocar- pine. DPEA. SKF 525-A and imipramine. all have been shown to have inhibitorv effects on the me- tabolism of nicotine bv microsomes obtained from the livers of rabbits (32). For studies on induction of aryl hydrocarbon hvdroxvlase by cigarette smoke see Section II, abs 9. 10. Along a different vein, the possibilitv that tobacco (cigarette) smoke mav contain constituents capable of binding with trace metals in the body, in particular metalloenzyznes thereby inhibiting their metabolic functions has been explored. Tobacco smoke condensate was found to have binding activ- ity for copper, zinc, cadmium, iron and lead (33. 34). Subfractions of tobacco smoke condensate were prepared, certain of which showed inhibitory activ- ity in vitro on galactose oxidase and catalase (me- talloenzymes) but not on glucose oxidase (a gly- coprotein containing flavin-adenine dinucleotide and no metals) (35); certain fractions were also found to inhibit tvrosinase (36). It is suggested that the presence of potent inhibitors of tyrosinase (and other metalloenzymes) in tobacco smoke, if absorbed by an organism, could affect the essential trace metal metabolism, thus impairing vital metal-requiring cellular processes, such as oxidative phosphorvla- tion. lipid metabolism, connective tissue synthesis and repair, and microsomal mixed function oxy- aertases. A smaller amount of cepper-c^~.^.-~p!exina f material was also found in the smoke from lettuce- leaf cigarettes (37). This material inhibited the oxygen uptake of rat liver slices, presumably by TIMN 0115750 T200241 intc~ sm(- aver ope; smo lke, Am l una the tam. rem, carh tion i nca is a perfi and of tF (40) (41). D. E 1 and u nde lism nific cotir (42). terer mu0 and( heim- E. T ( fects are tc port gathe C findii resist tine t 4

4 , t; f, f; 7 8 8 8 9 9 t t > 3 3 3 - .r .-i- a -.-_ s. Section I Absorption, Distribution, Metabolism, gxcretion, Toxicolo9y tiunlnlarV fntrr,cjuction-Clasicall\, knowledge concerning ,,l,:urption. tissue distribution. metabolism, excre- ti„ii and tdxicolog} has become basic to the studv of hemicals to which man becomes exposed. In this tir, tion, some of the contributions to such knowl- ,.r{;e achieved by grantees under this Project have hr•r n assembled: cross-reference is made to related ()ntributions appearing in other Sections. \. .-lbsorption The lesser pharmacodynamic effects resulting trnnl cigar and pipe smoking compared to cigarette ;mnking have been attributed to differences in inhallna practices between these modes of smoking. In connec:tion with studies on quantitativelv differ- rnt effects of cigar, pipe and cigarette smoking on lipid metabolism and catecholamine excretion, sum- marized in Section III (Abs 184). it was experimen- tall% shown (1) that on smoking cigarettes in the cus- tomary manner of inhaling, and cigars and pipes in the customary manner of not inhaling (equal weights nf tobacco being consumed). about three times the quantit\ of nicotine was excreted (urine) with ciga- rettes than with cigars or pipes. However, when t hese tobacco forms were smoked, with inhaling and k% ithout inhaling, there was no significant difference i ii mean nicotine excretion when all were inhaled or All not inhaled. For studies on respiratory tract retention (and t)rr",cnned absorption) of other tobacco smoke con- .tituNnts, see Section VII: ammonia (8); acetaldehyde ( 2.;.fi): acetone (2.8): acetonitrile (2): acrolein (7); i,rrhnn monoxide (2): formaldehyde (7); furan (10); :.nprene (2.9): methanol (9); particulate matter (2); ;)n~pionaldehvde (7): toluene (2.11); benzene (11). li Distribution Dvnamicc changes in tissue distribution of nico- tine following its administration have been the suhject of numerous investigations. Nicotine rapidly 'aves the blood to become more concentrated to ~ arving degrees in the body organs and tissues, most notablv in brain, adrenals, liver and kidneys. Dis- tribution data in relation to time have been made ,rvailable for the rat (2) and the dog and rhesus nlnnkey (3'„ the comparative data in the lattcr study having additional interest in view of the higher tolerance of the monkey for nicotine. More detailed studies have been conducted on the distribution of administered "C-nic:otine in sev- eral tissues. In the central nervous system of mice. the highest level of activity was observed in the arev matter (lower in the white matter): the nuclei of the diencephalon and the medulla obiongata and the cellular lavers of the hippocampus also concentrated activity (4). Study of change with age in mice of nicotine distribution in the brain (3 vs. 12 vs. 26 dav old animals) showed shifting patterns (5). This studv (5) followed on an earlier one (6) in which sig- nificant age dependent differences between lethal brain and blood concentrations of nicotine and latent period before death occurred were found. For additional studies on brain area nicotine levels in relation to behavioral effects see Section IV. abs 54. 55: also. for studies on nicotine distribution and movements in brain slices from monkev_ s and rats see the same section. abs 69. 70. There also appears to be a specific accumulation of administered nicotine in the pancreatic islets and in the parafollicular cells of the thyroid in mice. as well as in the ultimobranchial glands of chicks (7). Since biogenic amines are operative in these en- docrine organs (the ultimobranchial glands produce calcitonin), it is suggested that nicotine can share common transport and/or storage mechanisms with biogenic amines in the cells, and that an effect of nicotine on hormone storage and/or release may take place via an interference with aminergic mech- anisms in the cells. Attention may be called here to a autoradiographical. histochemical and radioimmu- nological study of catechol- and indolamines in some endocrine cell svstems (8). In frog sartorius muscle (9) and frog rectus abdominis muscle (10), uptake and efflux of nicotine has been shown to be dependent upon the cellular pH gradient. Placental transfer of nicotine in the rhesus monkey occurs rapidly, the concentration in the fetal circulation surpassing that in the maternal circula- tion (11). The concentration of nicotine in adrenal glands, heart, kidneys, stomach wall, and spleen of fetuses was high: that in placenta and skeletal muscles was low. The amount contained in a fetus. however, was only a small fraction of the total dose administered to the mother. Also see Section VI for an additional stuiiv on the passage of 1a1--nicCtir:c TIMN 0115749 T200240 3

Absorption. Distribution, Metabolism, Excretion. Toxicology medulla spinalis and medulla oblongata, while on the other hand. radioactivity was very low in the forebrain structures. High radioactive concentra- tions were also seen in the hippocampal formation, lobus olfactorius and cerebellum. Again in the 12- day old mouse the highest levels of radioactivity were seen in the medulla and lobus olfactorius, but in this case the radioactivitv in the forebrain struc- tures was comparatively higher than in the 3-day old mouse. A higher radioactivity was also seen in the hippocampal formation. However. in the CNS of the 26-day old mouse a quite different pattern of dis- tribution was seen. Here the forebrain structures had the highest level of radioactivity and the medulla the lowest. At this age too, the radioactivity was high in the hippocampal formation. Other support: The Swedish Tobacco Company. and the Knut and Alice Wallenbergs Stiftelse (for general equipment for isotope work). 6. Lethal brain concentrations of nicotine in mice of different ages Torbjorn Stalhandske, and Premysl Slanina (Carl G. Schmiterlow) Department of Pharmacology, Royal Veterinary College. Stockholm. Sweden Acta Pharmacologica et Toxicologica 28:233-240. 1970 Albino mice aged 3 days, 12 days and 35 days, were injected intraperitoneally with lethal doses of "C-nicotine; mice aged 3 and 35 days were given 23 mg/kg and ones aged 12 days 5.4 mg/kg, the doses corresponding to 2-fold LD5o dose for each age. At death the concentrations of nicotine in the brain and blood were measured. Significant age dependent differences between lethal brain and blood con- centrations and latent period before death occurred were found. The brains of mice aged 12 days were found to have the lowest (2.10 ± 0.15 µg/g wet weight) and the brains of mice aged 35 days the highest (8.35 ± 1.02 µg/g) nicotine concentration at death; in mice aged 3 days the concentration was 4.87 - 0.66 µg/g. The lethal concentration of nic- otine in the blood was highest in mice aged 3 days (8.75 ± 1.11 µglg wet weight) and lowest in mice age 12 days (2.11 ± 0.33 µg/g); in mice aged 35 days the concentration was 4.47 ± 0.47 µg/g. Brain/blood ratios increased with age. The latent period before death occurred was shortest in mice aged 12 days (98 ± 8 sec) and longest in mice age 3 days (375 ± 25 sec); in mice aged 35 days in was 157 - 17 sec. The significance of these conditions for age dependent differences in tolerance to nicotine is discussed. Other support: Swedish Tobacco Company. and the Knut and Alice Wallenbergs Stiftelse (for general equipment for isotope research work). 7. Accumulation of nicotine in pancreatic islets and calcitonin-producing cells in mice and chicks demonstrated by micro- and whole-body autoradiography Premvsl Slanina, and Hans Tjalve (Carl G. Schmiterlow) Department of Pharmacology. Royal Veterinar} College. Stockholm, Sweden Journal of Endocrinology 58:21-30. 1973 By autoradiographic methods, nicotine was shown to be specifically accumulated in the pan- creatic islets in mice. The results also indicated a high accumulation of nicotine in the parafollicular cells of the thyroid in mice and an accumulation was also shown in the ultimobranchial glands in chicks. Like the parafollicular cells of the thyroid in mam- mals, the ultimobranchial glands of birds are known to produce calcitonin. Metabolic studies with nic- otine in vitro and autoradiographic studies with the main nicotine-metabolite cotinine, indicated an ac- cumulation of unchanged nicotine (not metabolites) in the cells. The results are discussed in view of the fact that biogenic amines have been shown to be operative in these endocrine organs. It is suggested that nicotine can share common transport and/or storage mech- anisms with biogenic amines in the cells. An effect of nicotine on hormone storage andlor release may take place via an interference with aminergic mech- anisms in the cells. Other support: Swedish Tobacco Company, and 'Ragnar and Torsten Soderbergs Stiftelse'. 8. Catechol- and indolamines in some endocrine cell systems. An autoradiographical, histochemical and radioimmunological study Hans Tjalve (Carl G. Schmiterlow) Department of Toxicology, University of Uppsala, and the Department of Pharmacology, Royal Veterinary CnIlege .Stnr.kholm. Sweden Acta Physiologica Scandinavica. Supplementum 360:1-122, 1971 A study is reported focusing on extraneuronal localizations and functions of catechol- and in- 9. m Gi Dt 1'1 Jo 16 fin 26 TIMN 0115754 T200245 8

,s Y ;s \V rt re- of ;A, 21. Pyridyl acids from the mammalian -metabolism of trans-metanicotine Roger H., .1,4eacham. Jr.. C. T. Sprouse. Edward R. Bou•man. and Herbert McKennis. Jr. .~fedical College of Virginia. Richmond, Virginia Federation Proceedings 32:511, 1973 The natural occurrence of metanicotine (3-(4- methN'lamino-l-butenyl)pyridine) and dihvdrometa- nicotine has given each of these alkaloids a possible role in the metabolic degradation of the pyrroli- dine ring of nicotine. For current studies on the metabolism of inetanicotine, trans-benzoylmetanico- tine, from the Pinner-Etard reaction of benzoyl chloride and nicotine, was freed of the cis isomer by frational crystallization. Trans-metanicotine from acidic hvdrolysis of the benzoyl compound was administered (15 mg/kg) orally to male dogs. The pyridyl acids in the urine were concentrated with the aid of Dowex 50 (H') and Dowex 21K (OH-), and then converted to methyl esters. Gas chromatogra- phy (30% SE-30, 205°) provided two major com- ponents (retention time 10 min and 25 min). The 25 min component formed a picrate, m.p. 127-128°. Data (NMR, UV, IR and mass spectral) on the free base was consistent with the methyl ester of trans-4- (3-pyridyl)-3-butenoic acid. Similar evidence led to identification of the other major components as 2- pvridylacetate, previously identified as a mamma- lian metabolite of nicotine and dihydrometanico- tine. Other support: Council for Tobacco Research- L'.S.A.. and The American Tobacco Company. 22. Synthesis of trans-4-(3-pyridyl}3-butenoic acid, a metanicotine metabolite C. T. Sprouse. and Herbert McKennis, Jr. Department of Pharmacology, Medical College of l"irginia. Richmond, Virginia 1'irginia Journal of Science 24:220, 1973 in the metabolism of cis and trans isomers of metanicotine in the dog, a number of pyridyl car- boxylic acids are formed. Physical and chemical data suggested that one of the metabolic acids was trans- 4-(3-pyridyl)-3-butenoic acid, which is subsequently metabolized to 3-pyridylacetic acid. To facilitate fLrtl:er studies, and confirmation of structure, a svnthesis of trans-4-(3-pvridyl)-3-butenoic acid was sought. 1-Methoxy-2-(3-pyridyl) ethylene, prepared from pyridine-3-carboxyaldehvde and methox}-me- thylenetriphenyl phosphorane, was reacted w' ith carbomethoxymethylenetriphenvl-phosphorane to form methyl trans-4-(3-pvridvl)-2)butenoate. Upon treatment with aqueous base, for hydrolvsis and isomerization, this ester afforded trans--t-(3-pyrid%•1)- 3-butenoic acid in good yield. The synthetic product was identical in all respects to the metabolite pre- viously isolated from dog urine. Other support: Council for Tobacco Research- U.S.A., and the American Tobacco Compan\. 23. Structure of dibromoticonine, a bromination product of nicotine Herbert McKennis, Jr., Edward R. Bowman, Louis D. Quin. and Ronald C. Dennev Department of Pharmacology, Virginia Commonwealth University, Richmond, Virginia, and the Gross Chemical Laboratory, Duke University, Durham. North Carolina Journal of the Chemical Society; Perkin Transactions I: Organic and Bio-organic Chemistry 1:2046-2049. 1973 The fate of the pyrrolidine ring of nicotine on its oxidative bromination to dibromoticonine has been re-examined. From chemical and spectral (including "C n.m.r.) properties dibromoticonine is now es- tablished as 3,4-dibromo-5-hydroxy-l-methvl-5-( 3- pyridyl)-0'-pyrrolin-2-one. The bromine atoms can be selectively replaced by hydrogen under proper reductive conditions to give the isomeric monobro- moticonines. Other support: American Tobacco Company, and the Council for Tobacco Research-U.S.A. 24. N-3-pyridylacetylglycine as a nicotine metabolite Edward R. Bowman. Ravmond S. L. Chang, C. T. Sprouse, and Herbert McKennis, Jr. Department of Pharmacology, Medical College of Virginia, Richmond, Virginia Abstract, 27th Tobacco Chemists' Research Conference, Winston-Salem, North Carolina, October 3-5. 1973, p32 Occurrence of metanicotine as a possible bac- terial metabolite of nicotine (Wenusch) and as a tobacco smoke constituent led to a study of mamma- lian metabolism of metanicotine, in which 3-pvr- idylacetate was identified as a metabolite. Although TIMN 0115759 T200250 13

Absorption. Distribution. Metabolism. Excretion. Toxicology ternan- ammonium compounds, showed no great tendency to pass the blood-brain barrier. In rats, after i.p. administration. and mice, after i.v. administra- tion, the radioactivity of (+,-)-N-methyl-''`C-3-(1- hvdroxvethvl) pvridinium iodide was eliminated largely (78-90%) by way of the urine within 24 hr. This excreted radioactivity was predominantly in the form of the administered cation. The signs of acute toxicity from large doses of (+.-)-1-methyl-3-(1-hvdroxyethvl) pvridinium io- dide differed from the previouslv cvell-established similarity of those arising from 3-acetylpyridine and (+,-)-1-(3-pyridyl)ethanol; and the levorotatory form of 1-(3-pyridyl)ethanol appeared to produce gross toxic effects similar to those from the racemic alcohol. Other support: U. S. Public Health Service, the Council for Tobacco Research-U.S.A., the Ameri- can Tobacco Company, and the Swedish Tobacco Company. 19. Studies on the excretion of 5-(3-pyridyl)- tetrahydrofuranone-2 and its intermediary role in the metabolism of nicotine Edward R. Bowman (Herbert McKennis, Jr.) Department of Pharmacology, Medical College of Virginia. Richmond. Virginia Virginia Journal of Science 19:115-121, 1968 After the administration of 5-(3-pyridyl)tetrahy- drofuranone-2 to the rat, the urine contains five or more Koenig-positive compounds. Two of these, metabolites of the administered compound, were isolated by means of gas chromatography and identified (in derivative form) as y-(3-pyridyl)--~- hydroxvbutyric acid and 3-pyridylacetic acid. The pattern of excretion of the Koenig-positive com- pounds resembles in part that obtained from the administration of nicotine or cotinine and provides additional evidence for the intermediary role of -y(3- pyridyl)--y-hydroxybutyric acid, or the correspond- ing lactone 5-(3-pyridyl)tetrahydrofuranone-2, in the formation of 3-pyridylacetic acid from nicotine. In the rat 5-(3-pyridyl)tetrahydrofuranone-2 was vir- tuallv devoid of the CNS depressant properties of y- butyrolactone. In common with cotinine and other metabolites of nicotine, the acute toxicity of 5-(3- pyridvl)tetrahydrofuranone-2 was considerably less than that of the parent nicotine. This studv was the subject in part of a preliminary report appearing in Federation Proceedings 26(2):616, 1967. Other support: Council for Tobacco Research-USA, and the American Tobacco Company. 20. Additional routes in the metabolism of nicotine to 3-pyridylacetate. The metabolism of dihydrometanicotine Roger H. vieacham. Jr., Edward R. Bowman, and Herbert McKennis, Jr. Department of Pharmacology, Medical College of Virginia. Richmond, Virginia Journal of Biological Chemistry 247:902-908, 1972 The metabolism of dihydrometanicotine was investigated in the rat and the dog. In preliminary studies, dihydrometanicotine difumarate was ad- ministered to rats. The Rf values and retention times of methyl esters prepared from acidic Koenig-posi- tive metabolites in the urine suggested the presence of 3-pyridylacetate and 4-(3-pyridvl)butyrate. Fol- lowing administration of dihydrometanicotine di- fumarate to dogs. the pattern of excretion of acidic metabolites in the urine was similar to that found in the rat. Chemical conversion of the acidic metabo- lites to their methyl esters for separation by pre- parative gas chromatography afforded methyl 3- pyridylacetate and methyl 4-(3-pyridyl)butyrate. Methyl 3-pyridylacetate was identified by melting point, elemental analysis, and the infrared spectrum of the methyl ester picrate. Methyl 4-(3-pyridyl) butyrate was identified by mass spectroscopy and by the melting point, elemental analysis, and the in- frared spectrum of the methyl ester picrate. Previous studies have provided evidence for the formation of 3-pyridylacetate from (-)nicotine via either (-)co- tinine or (-)demethylcotinine. In view of the report (de Clercq, and Truhaut, Bull Soc Chim Bio144:227, 1962) that dihydrometanicotine is a metabolite of nicotine in the rat, the present findings in the rat and dog suggest a third alternative route (via dihydro- metanicotine) to 3-pyridylacetate. This study was the subject in part of a pre- liminary report appearing in the Virginia Journal of Science 22:133, 1971. Other support: Council for Tobacco Research-USA, and the American Tobacco Company. TIMN 0115758 T200249 21. meta Roge Bo«•' Medr Feder meth nicot role dine meta tine. chlor fratio acidi, admi pvrid the ai then phy I ponei min ~ Data base x ( 3-pvi identi pyrid- lian i tine. Other U.S.A 22. S~ acid, . C. T. S Depart Virgini Virgini In metan boxyli sugge< -}-(3-p, metab, f urthet 12

Absorption. Distribution. `ietabolism. Excretion, Toxicologv prior studies provided no firm evidence for con- jugation of 3-pvridylacetate. analagous substances are well known for their ability to form conjugates in both plants and animals. Administration of synthetic N-3-pvridt•lacetvlglvcine to dogs led to urinary ex- cretion of 3-pvridvlacetate. while administration of 3-pvridvlacetate led to urinary excretion of V-3- pvridvlacetylglvcine. In light of the previous iso- lation of 3-pyridvlacetate as a nicotine metabolite, N- 3-pyridylacetylglycine itself is now implicated in the metabolism of nicotine. Amounts of the con- jugate formed and excreted are theoretically de- pendent upon competing biological factors. 25. The metabolism of nicotine and cotinine by a mouse liver preparation Torbjorn Sta' lhandske (Carl G. Schmiterlow) Department of Pharmacology, Royal Veterinary College. Stockholm, Sweden Acta Physiologica Scandinavica 78:236-248. 1970 The metabolism of nicotine and cotinine by a 10.000 x g supernatant fraction of mouse liver homogenate has been studied by using "C-labelled compounds. The metabolism of nicotine was found to be TPNH and 0, dependent and chromatographi- cal evidence for the formation of cotinine, y-(3- pvridvl)-y-oxo-N-methylbutyramide and hydroxyco- Linine are presented. Three unidentified metabolites and delayed formation of small amounts of 14COZ were also observed. The observed metabolites were also excreted in urine after intraperitoneal adminis- tration of nicotine. Chromatographical evidences revealed that cotinine is metabolized to hydroxy- cotinine, y-(3-pvridvl)-y-oxo-N-methylbutyramide and/or demethvlcotinine and one unidentified me- tabolite. Other support: the Swedish Tobacco Company. and the Knut and Alice Wallenbergs Stiftelse (for general equipment for isotope research work). 26. Metabolism in vitro of 14C-nicotine in livers of foetal, newborn and young mice T. Stalhandske. P. Slanina, H. Tjalve, E. Hansson, and C. G. Schmiterlow Department of Pharmacology. Royal Veterinary College, Stocl:ho!m. Stredcn Acta Pharmacologica et Toxicologica 27:363-380, 1969 The metabolism of nicotine in the livers of fetal, young and adult mice was studied in vitro. The metabolism of nicotine in the fetal liver with the formation of cotinine was seen at the end of fetal life. Cotinine was found to be the major metabolite at all of the ages investigated. After birth the ability of the liver to metabolize nicotine increased and ap- proached the adult level at four weeks of age. The LD,o value for intraperitoneally injected nicotine increased with age in two to eight weeks old mice. A relation between the rate of metabolism in vitro and the lethal toxicity of nicotine in young mice was observed. However, despite a very low metabolism of nicotine in the livers of three day old mice, the LD5o at this age was almost of the same order as for adult mice. In three day old mice, nicotine did not induce the convulsive pattern seen in adult mice. Other support: Swedish Tobacco Company, and the Knut and Alice Wallenbergs Stiftelse (for general equipment for isotope research work). 27. Effect of nicotine treatment on the metabolism of nicotine in the mouse liver in vitro Torbjorn Stalhandske. and Premvsl Slanina (Carl G. Schmiterlow) Department of Pharmacology. Royal Veterinary College. Stockholm, Sweden Acta Pharmacologica et Toxicologica 28:75-80, 1970 'aC-nicotine was incubated with a 10,000 x g supernatant fraction of liver homogenate from mice pretreated with nicotine for 3, 10 and 17 days, respectively. The rate of metabolism of nicotine was measured by the determination of the oxidative nicotine metabolite, cotinine. After intraperitoneal injections of 5 mg/kg of nicotine three times daily for three days, a 50% significant decrease in the me- tabolism of nicotine was observed. A 71% sig- nificant decrease in hepatic glycogen was also seen. Giving nicotine 27.8 and 24.5 mg/kg/24 hours in drinking water for 10 and 17 days respectively, did not significantly change the metabolism of nicotine or the hepatic glycogen levels. Other support: Swedish Tobacco Company, and the Knut and Alice Wallenbergs Stiftelse (for general equipment for isotope research work). TIMN 0115760 T200251 28. foil niu R. IDep .% fec. Tox 19%( mg; pvrc adrr reac retu trati 3 cc mor choi c hro in t elev, code incrE we6 How dav. activ amin An i syntl treat: indic meta acute conv suggt drug that i ncrei possil T repor 28(2): 14