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Report of the Council for Tobacco Research-U.S.A., Inc. [St]

Date: 1981
Length: 63 pages
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~~ REPORT of .THE COUNCIL FOR ' TOBACCO RESEARCH-IJ.S.A., Inc. k
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r 1 9 ti 1 It 1:1' O lt '1' of TIIE COUNCIL FOR TOitACCO RGSGARCII-U.S.A., Inc. 7711i (a)lINf:II. I'llI!'1'/IIL~/'lY/ Itl:~hVt(:If-1~.~,.1., Inr. 1 I// l:na 5'/t1h ti1rf•ct, Ne.r York, N.Y. 1111/"•!
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~ v ~ r SCIENTIFIC ADVISORY IIOARD to The Council for Tobacco Research-U.S.A., Inc. as of December 31, 1981 LEON O. JACOBSON. M.D., Chairman Joseph Regenstein Professor of Biulogical Scirnces (emeritus) l'ro/rssw of the I)rpartnrent of Medicine (emeritus) University of Chicago Chicago, Illinois RICf fARD J. BING, M.D. Dirrr7or of E.cprrimental Cardiology and Scientific Drvrlopmcnt Huntington Medical Research Institute, Pasadena, California l'ro/%%arr of Medicine (emeritus) University of Southern California School of Medicine Los Angeles. California ROSWELL K. BOUTWELL, Ptt.D. Professor of Oncology McArdle Laboratory for Cancer Research University of Wisconsin Madison. Wisconsin DRUMMOND 11. BOWDEN, M.D. Professor and Nrad Department of Pathology University of Manitoba Health Sciences Center Winnipeg. Canada MICHAEL J. BRENNAN. M.D. President and Mrdical Director Michigan Cancer Foundation Detroit, Michigan JOSEPff D. FELDMAN, M.D. Member. Research Institute of Scripps Clinic Scripps Clinic and Research Foundation La lolla, California WILLIAM U. GARDNER. Ptt.D. F. K. Hunt Professor of Anatomy (emeritus) Yale Universtty School of Medicine New Haven, Connecticut ROBERT J. HUEBNER, M.D. Laboratory of Cellular and Molecular Biology National Cancer Institute Bethesda, Maryland HENRY T. LYNCH, M.D. Professor and Chairman Department of Preventive Medicine and Public Health Cteighton University School of Medicine Omaha, Nebraska HANS MEIER, D.V.M., Dr. Med. Vet., M.R.S.H. Senior Staff Scientist The Jackson Laboratory Bar Harbor, Maine GORDON H. SATO, Ptt.D. 1'roJrssor o/ Biology University of California, San Diego La Jolla, California SIIE:f.DON C. SOMMERS .Scirnrihc Director, The Council for Tobacco Research-U.S.A., Inc. Clinical Professor of Pathology College of Physicians & Surgeons of Columbia University New York, New York Scientific Staff of The Council SHELDON C. SOMMERS, M.D. Scientific Director ROBERT C. HOCKETT, Ptt.D. Research Director DONALD H. FORD, Ptt.D. VINCENT F. LISANTI, D.M.D. Associatr Research Dirrclor Associate Research Director DAVID STONE, Pn.D. Associate Research Director
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r CONTENTS Introduction . . . . . . . . . . • • • • • . • • 7 Abstracts of Reports . . . . . . . . . . . . . . . . 9 Cancer-Related Studies . . . . . . . . . . . . • 9 The Respiratory System . . . . . . . . . . . . 25 Heart and Circulation . . . . . . . . . . . . . 49 Neuropharmacology and Physiology . . . . . . . . . 63 Pharmacology and Biochemistry . . . . . . . . . . 71 immunolory and Adaptive Mechanisms . . . . . . . 88 Epidemiology . . . . . . . . . . . . . . . . 9 5 Active Projects . . . . . . . . . . . . . . • • • IGO Completed Pro jects . . . . . . . . . . . . . . . . 109 Index of Principal Investigators . . . . . . . . . . . . 122 Index of Senior Authors . . . . . . . . . . • • • • 123 HANS MEIER 1929-19a 1 Ilans M.cicr, a memhcr of the Scientific Advisory Iloard for ten ycar.. died May 14. Hc had hccn a tienior Statf Scientist at The lack- .un f.:daratr.ry in It:rr Ilartxwr. Maine, which he joined in 1962, and a ron.ullanl tu the Natirm•rl Cancer Institute since 1970. Hans was a di.tinKui.h.d cancer rrsxarcher with a special interest in viroloEy, a lictd in which hc attaitkd yn international reputation. Qu:et and soft• sIxd.rn, hc had great warrr.th nd empathy and an appreciative sense ul humor. Ile cuntnhracd wiscly and unsel6shty to the efforts of the Srientific Advi.ory ttuard and, in his own Lboralory, worked long and Ir.ud lur the rewlutiim of one of mankind's major killer diseases. He will he m6.ed as a person and a xe:entisr. a
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v' Introduction ....~ ~ a The rrsearch prnXram of the Council for Tobacco Rescarch•U S.A. moved ahead steaJily in 19tt t with thc emphasis continuing to he directed toward gaining adJition•rl knowledge of cancer, cardiovascular diseases and chronic pulmonary ailmcntc. '1 he Scientific Advisory Board to the Council closely reviewed +cores ol applicalions from independent invesligaton for research snprmut, and by yenr's end the number of original grams approved by the Council had risen to 7X9. Council lunding for the research program since its inccption m+w stands at more than $69.000,000. Grant recipients puhli.hed 144 documents in 19R1 that acknow•ledged Councd suppkat; the total of such publications is now 2,026. Abstracts of the t9M1 puhlications are includarl in this report. ticveral ch•rnges nccurrcJ in thc composition of the Advisory Board dur- ing the year. Ilam I.. Meier. D.V.M., died in May. William U. Gardner. resigned •rs ticicnti/ic Director of the Council but remains a member of the Ito•rrd. He w:r. succceded as Scientific Director by Sheldon C. Sommers. M.1)., a tung-timc nxmhcr who had been Chairman of the Advisory Board. %uccecJing Dr. tiumrncrs as Chairman was Leon 0. Jacobson. M.D., a Board member since 1954. Robert 1. Huebner, M.D.. became a memher emeritus at the end ot I9HI. '1 wu more JistinguisheJ scientists joined the Board. They were Drvm- mrnid tl. Ilowden, M.1)., Professor and }lead of the Department of Pathology at ,he University of Manitoha Ilealth Sciences Center in Winnipeg. Canada, and Michael 1. Brennan. M.D.. PresiJent and Medical Director of the Mich- igan C'ancer f'oundation in Detroit. 0 0 F+~ ~ 0 CX: Gly ~_} e~ 7 ~ `a,.~'
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C Abstracts of Reports FOllowing are ab.tncts, approved by the authon, of reports on new research acknowledging support from The Council that have appeared in scien- tific journals since publication of the 1980 Report. The name of the recipient is in italics. The abstracts are grouped under these headinas: 1. Cancer-Related Studies. II. The Rcspiratory Systcm, III..Heart and Circulation, IV. Neuropharmacology and 1'hysiology, V. Pharmacology and Biochemistry, VI. Immunology and Adaptive Mechanisms, V11. Epidemiology. 1. Canccr-Related Studitr FAMILInI.CAN('I:R IN AN ONCOLOOY CLINIC Knowledge of hereditary cancer syndrome identiRcaiion, often based upon tmnor profi)cs, age of onset and tumor distribution within the kindred, aid significantly in the diagnosis of cancer. As reported here, all cancer pa- ticnts (565) treated at the Creighton University Oncology Clinics between June 1978 and April 1980 were entered into a prospective family history a.ccrtainnunt prouwol. '/bis consisted of an initial evaluation by a registered nursc utiliring both questionnaire and personal interview. The initial assess- nient yielded 199 (35.Sryo ) families with two more family members with cancer (all sites) within an informative nuclear component, which constituted parcnts, grandparcn,s, aunls/uncles, sib)ings, and children. One or more of the opcrational criteria for cancer (arniliality, namely vertical transmission of canccr, bilatcrality, and/or multiple primaries, early age of onset, and three or more site specific cancen, were found on physician review in 171 (30.5%) of the familics. This group was referred for comprehensive cancer genetic evaluation concisting of pedigree extension and tumor verification through all second degree and, when possible, third degree relatives. It was determined that approximately 470 of the total clinic population demonstrated findings compatible with hereditary cancer syndromes. The universal extension of these survci))ance mcthods in clinical practice is highly recommended here. Albano, W. A.. f.ynrb, If. T., Recabaren, 1. A., Organ, C. N., Mailliard, J. A.. Illack, 1.. 1:., I'ollett, K. L., and Lynch, 1. Cancrr 47(9):211J-2118,1981. Other ar.pport: Fraternal Order of [agles. from the Inaitute for Familial Cancer Management and Control. Inc., and the Ikpartment of Sorgcry and Preventive Medicine. Crcighton University School of Medicine, Om•rha, Neb. 9
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!'AM11.IA1. HR):AST CANCER AND I'TS RECOGNITION IN AN ONCOI.OGY CLINIC This paper focvses attention upon in-depth clinicopathologic studies of breast caneer-prane families wherein cancers of all anatomic sites were cri- tieally assessed in conlest with genealugy. For this study. family history of cancer was es'aluated for 79 breast cancer probands from among a series of curnatiynively ascertained cancer patients undergoing treatment in the ('reigh- ton-St. )oseph's Oncology Clinic. Cancer prevalence for each family was quan- tificd by u.ing a statistic that accotmts for variable size and age structure among families. To test the null hypothesis that cancer risk is independent of family membenhip, the distribution of this statistic for families in their original configuration was compared with the distributions observed when relatives were randomly assigncd to families in 99 random permutations of family membership. The results indicated significant heterogeneity for cancer risk among relatives of breast cancer probands, which sugbksts that the isola- tion of high risk families can provide a meaningful resource for in-depth studies in breast cancer geneties. The atudy of breast cancer-prone families harbors a powerful potential for better comprehension of nechanisms of car- cinogenesis and the development of novel pursuits toward improved cancer control. l.ynrh, H. T. rr a1. Cancer 47(1 I ):27J0-27)9, 199 1. OOther support: Fraternal Order of Eagles. From the Creighton University School of Medicine. Omaha. Neb. ROUTINE CYTODIAGNOSIS OF PULMONARY MALIGNANCII-S Material routinely suctioned from the tracheal tube of patients under general endotracheal anesthesia for surgery is normally discarded. In this study, however, the suctioned secretions from 10,621 such patients were pre- served, and later examined microscopically. Among these specimens. 1I cy- tologically abnormal smears were obtained from subjects with unsuspectcd pulmonary involvement, an incidence slightly more than 1:1.000. The accu- racy of this method was assessed by calculation of the percent of abnormal smears obtained from patients with prediagnosed hronchogenie carcinomas: 409's when suctioned material was immediately spread on slides, and 6790 when cellular concentration was achieved by mucolysis followed by filtration or centrifugation. In addition, by using this technique, a wide variety of cyto- morphologic and cytochemical changes in tracheobronchial cytology of both intrinsic and extrinsic origin were discovered, changes which otherwise would have been missed. Although routine cytologic screening of 311 patients ttndcr- going general endorraeheal anesthesia is not advocated here, it does seem possible that this method could prove beneficial for the early diagnocis of malignant neoptasms in a population at risk. ('hadnn, !A et ol. Arrhivrr o/ Parholuxy d Lohorarory Mrrlirinr 105:11-14, 1991. OOther ar.pport: U. S. Public Health Service. From the (kpartment of Anesthesiology, New York University Medical Cen- ter, New York. ARE TUMOR MARKERS USEFUL IN DIAGNOSING LUNG CANCER? Hecanse it has been noted that many lung tumors have a long tatency period and a slow growth rate, the idea has been advanced that biochemical markers-hormones and other metabolic products of tumors-might reveal the presence of malignant disease at a very early stage and thus improve the sur- vival ratio. The paper presented here describes some of these marker systems and evaluates them. In one case, armed with a precise and simple assay sys- tem, investigators tested the hypothesis that elevations in peptide hormone concentrations in bloixl might he a sensitive indieator of the presence of a tumor. These and other studies have revealed many interesting endocrinologic intcractinns, hut unfartunately have not provided a suAiciently reliable and sensitive early marker of tumor presence. There are several other substanees which have been found to be present in abnormally high amounts in the blood or urine of paticnts with bronchial carcinoma. Some are produets of the neo- plaslic cclls themselves and thus represent true tumor marken. Carcinoem- bryonic actigen is the best studied and most interesting of these substances. Aryl hydrtwarhnn hydroxylase and free sccretory component of immunoglohu- lin A studies are also touched upon hcre. From a different facet of early de- tection, ffuorescent bronchoscopy shows promise as a means of very early anatomic identification of lung tumon when the tumors are curable surgically but not detectable by traditional methods. Merrill, W. W. and Rrynoldf, 11. Y. Tlrrlonrnaf of Respiratory ntrraars 2:44-SJ, 198 1. OOther nrpport: Charles F. C'ulpepper Foundation. From the Department of Medicine and Internal Medicine, Yale University School of Medicine, New Haven, Conn. REASSF.SSMENT OF TME RF.l.AT1ONSHIP DETWEEN ARYI. IiYDRO('ARiSON HYDROXYLASE AND LUNG CANCER In this study of the relatiunship between aryl hydrocarbon hydroxylase (AIfII) and lung cancer. AHII levels were evaluated in mitogen-activated lymphocytes cullured with 'and without the inducer benzanthracene in tM medium, and in pulmonary alveolar macrophages (PAMs) that were freshl) IavaRed from 114 cigarette smokers, 6) with and 31 without lung cancer. Considered separately, neither lymphocyte AHFI nor PAM AHH )eveh were distinctly differcnt in either nuncancer or lung cancer patients. However, when 10 11
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r 7 v ~ ~ J ~ )O )O consvlereil simoltanernnly, lymphocyte anJ macrophage AIIH Iavcls were quite di/ferent in twncancer and lung cancer patients. The lung cancer paticm group was seen to contain a significantly higher percentage of persons witl. high kvels of AIIH than did an age-matched grcNtp of noncanccr patients. Frorn another vicw, results of this study showed a 1:1 correlation of PAM and lymphoryte AHH levels in noncancer patients and an abscnce of correlation of thc trssue AHN levels in lung cancer patients. Overalt, it scems, on the basis of the work presented here, that single-tissue AHH an•rlyses are not ade- quate for evaluation of AHH activity in lung cancer populations. McLemore. T. L., Martin, R. R., Wray, N. P., Cantrcll, E. T., and Buabee, D. L. C'anrrr 411(6) :14311-144), 1981. Ozber aapport: National Institutes of Health, American Cancer Society and the Veterans Administration Hospital. Houston. From the Department of Medicine, Daytor Collcge of Medicinc, Ilouston• Veterans Administration Hosptal, Nouston; Department of Phannacology, Texas College of Osteopathic Medicine, Fort Worth; and Genetics Center and lkpartnknt of Biological Sciences, North Texas State University, Denton. A MFTHOD FOR DETECTING ARYL HYDROCARBON HYDROXYLASE ACTIVITIES IN CRYOPRESERVED HUMAN LYMPHOCYTES A standard procedure for the fre.zing, thawing and culluring of cryod preserved human lymphocytcs is presented here. Using this methixl, three parameters-aryl hydrocarbon hydroxylase (AHH) activity, NADH-dependcnt cytochrome c reductase (cyt c) activity, and 1.1111 thymidine ('If=1'dR) in- corporation-were monitored in human lymphocytes cryoprescrved for periods up to one year. The kinetics for expression of benz(nlanthr.iccne-(BA)-in- duced AHH activity, cyt c adivity and 'N-TdR incorporation were similar in freshly cultured and cryopreserved cells. For this study, lymphocyte samples from 10 individuals were collected once a month during a three-month period and cells were either cultured at time of donation or cryopreserved for later assay. Results indicated that the cryopreserved lymphocytes efficiently re- sporKkd to mitogen activation. The intn-individual variation in A11H activ. ities was reduced in the cryopreserved lymphocytes compared to the freshly cultured cells, and the relative ranking of these individuals in terms of their AHH activities remained constant for both fresh and cryopreserved samples. It seems, therefore, that cryopreservation offers significant advantages over the freshly cultured lymphocytes because it allows for lymphocyte samples to be collected in diverse geographical locations and over extended periods of time and yet permits for the culture and assay of alI the cell samples at exactly the same time. Kouri, R. E. n d. (Mkroblologlcal Asrodores) Cantvr Leners 14:2940, 1981. From the Division of Toxicology and Oncology. Microbiological Associates, Bethesda, Md. 12 S1C:NI1'IC'AN T VARIA I ION IN MOUSf::SKIN ARYI. HYnROCARDON I1Y1)KOXYI.AJh INDUCIBILITY AS A FUNCTION OF THE 11AIR GRUNr111 CYCLE Skin, which is a good biological model for the study of polycyclic hydro- cartxm mctabolisrn, cxhibits three mujor morphological phases in the hair- growth cycle. In earlier works, it had already been demonstrated that skin possesses activating and dctoxifying enzynses, notably aryl hydrocarbon hy- droxylase (AHH) and cpuxide hydrolase. For the present study, an attempt was made to determine whether AHH activity and its inducibility varied dur- ing the hair growth cycle and could possibly explain certain variations in skio sensitivity of carcinogens. An easy, iapid and Improved technique for homo- gcnizing whole skin was devised for this work. Using the new homogenizing methcHl, it was shown that skin AHFf activity in C57BL/61 and CJFI/Ico mice could be induced by i.p. injected or topically applied methylcholanthrene during a defined period of the hair growth cycle. I.r., between the Rth and 14111 days after depilation (Stage 6 of the anagen phase). In each experi- mental moJcl, there was an optimal mc,hylcholanthrene concentration which yielded a maximum induction. Topical methylcholanthrene was atso respon- ciblc for a sntaller Altll induction when the chemical was applied the same day that the club hairs were plucked. On the other band, skin AHH activity was never induced by niethylcholanthrene in DBA/21 mice, a genetically non- responsive strain. Frum a toxicological point of view, the fact that skin AHH activity could only he induced by polycyclic hydrocarbons at a certain time during the hair growth cycle of a genetically responsive strain might be of great importance. Manil, 1.., Van Cantfort, 1., Lapiere, C. M., and Cirlrn, l. E. Ilritirlt lournul of Cunctr 4):210-221, 1981. From the Lahoratoire de Dermatologie and Laboratoire de Chimie MEdicale, Institut de 1'athologie. UniversitE de Li2ge, Litge, Belgium. CYTCX'IIROMI: P-450 MONOOXYGENASE ACTIVITIES IN HUMAN AND RAT 1-IVER MICROSOMES Only limited data are available at the present time on the biochemical propcrties ol' the human enzymes. However, recent studies have shown that suitable liver samples could be obtained from kidney transplant donon, and the micruuimes used in this study were prepared from human livers acquired from renal donors of various ages and both sexes. Their drug-metabolizing capacity was measured and compared to that of rat liver microsomes. The following parameters were investigated: cytochrome P-450, cytochrome bS. NAUPHrytochromc r reductase, epoxide hydrolase, aryl hydrocarbon hy- droxylase, henzphetamine N-demethylase, p-nitroanisole-O-demethylase, ethoxyl- coumarindl-decthylasc, steruid-t6e-hydroxylase. In addition, the metabolism of hcnzo(n)Pyrene, progesterone, pregnenolone, testosterone, dehydroepian- drosterone, and estradiol was studied in detail in vitro. Results showed that the cytochrome P-450 content in the livers of the kidney transplant donors was 10.2 - 7.9 nmol/g of tissue. It did not differ with the sex of the donor. Comparison of the data obtained with human and rat microsomes demonstrated qualitative and quantitative differences which varied with the parameters 13
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41 studied. Overall, results of these studies indicate that the drug-metaholizing enzyme activities (ethoxyeoumarine deethylase, Ixnzphctamine demethytase, ryl hydrocarbon hydroxy)ase) vary as a function of the cytochrome P-450 content. The steroid hydroxylase activities do not fluctuate in a similar man- ner. Speeifieally, this study shows that the use of (iven from kidney tnns- plant donors is a promising too( for testing the metabolic pathway of new drugs in man or for screening potential toxic or carcinogenic properties of new chemicals. Kremers, P., Beaune, P.. Crestei(, T., De Gneve, J., Columelli, S., Leroux, J. P., and Ciefre, !. E. Erropten lowrnd of eioeAemixrr I 1 g:599-606, 198 1. Other wpporrr Fonds de la Recherche Scientifique MEdicate (Belgium). Frmm the Laboratoire de Chirnie Midicale. Imtitut de Pathotogil. Universitf de Litge, Li2ge, Bel=ium, and Facultf de MEdicine, Itopilal Necker-Enfants Malades, Patis, France. ONTOGENIC DEVELOPMENT OF STEROID 16 n-IIYDROXYI.ASE AS A TOOL FOR THE STUDY OF THE MULTIPLICITY OF CYTOCHROME P-450 This study followed the evolution of steroid 16 s-hydroxylase in the rat liver from birth to adulthood. To do Ihis, the quantitative and qualitative properties of the steroid-htumone-metabolizing system during the period of ontogenic development of the animal were investigated and compared to those of Ihe adult monooxygenase system. In the beginning, activities of progesterone, testosterone, pregnenolone, and dehydroepiandrostcrone 16 ,r-hy- droxylaae were undetectable in the fetal rat liver. During the neonatal period, however, the four enzymic activities increased in parallel to the concentration of eytoehronx P-450. Until puberty, they developed similarly in male and female rat livers. From the 40th to the 55th day, the four steroid 16 n-hy- droxylase activities increased rapidly in lhe male rat liver, but not in the female. The sexual diRerentiation of the steroid 16 e-hydroxylation observed here took place around the 55th day. When the adult rat liver was studicd, ' steroid 16 s-hydroxylase was supported by two forms of cytochrome P-450 ~ (forms I and )1) which diRered in their relative affinities for the various v steroid substrates and by their relative proportions in male and female rat ~ livers. The transition from the immature to the adult rrpartition of the two forms occurred during puberty and was correlated with the sexual diReren- ~ tiation of the steroid 16 w-hydroxylase activities. In another phase of this .~i ~ .'~ study, the in vitro interactions between benzo(n)pyrcne and steroids were eompared during the critical phases of the rat ontogenic development. Puleau, F.. Kolodzici, C., Krcmer, P., and Glrlrn, l. E. European lorrnd of elochrmUrry 120:213-220, 1981. Other support: Fonds de Ia Reeherehe Seientiflque Mfdicale (Belgium). From the Laboratoire de Chemie Midicale. Institut de Pathologie. UniversilE de Litge, Litge, Belgium. ('OMI'1'T1fION AKIW1iliN AIiN7_O(a)PYRENEAND VARIOUS ti I't:RUtl)S VOR ('Y'fO('HROME P-450-DEPENDENT RAT 1-IVF:R MONUOXYGIiNASFS Many previouc reports have described the existence of varian types of in virro competition between munooxygenase substrates. For the present paper, the interaction in vitro of steroids and benzo(a)pyrene (BP) was studied at the level of two rat liver monooxygenases, steroid 16 rhydroxylase and aryl hydrocarhon hydroxylase (AHH). The results obtained in these tests suggest the following conclusions: (I) Steroid-16 a-hydroxy)ase is partially supported hy a specific cytochrome P-450 form which is not inhibited in vitro by exo- genous substracs. titcroid-16 ,rhydroxytase is completely independent from cylochromc P,-450 (or P-44g), as it is insensitive, In virro, to ,.-naphtho- flavune; (2) AHH is supported by two cytochrome P-450 forms: a specific form which is inducible by mNhylcholanthrcne and inhibited ln vitro by e-naphthoflavone, hut is insensitive to metyrapone and steroWs; and another les specific form which is inhibited by metyrapone and steroids In vitro. It .ccros reasimable on these grounds to sssume the existence of specific eyto- chronte t'-450 furms that are respon.ibk for endogenora eompound melabol- icm, or at least for hormonal steroid 16 o-hydroxylation. This observation may pussibly he of physiological importance, as it could preserve endogenous melaboli.m from competition due to environmental xenobiotics. Paslcau, F.. Kremers, I'. and Girlrn, !. F.. Chrnnlro-Rinluglrul Inrrrnrtions 34 :279-2g6, 19K 1, Other suplN.rt: 1'ondc National de la Recherche Scientifique Midicale. From the Lalwratnirc dc C'himie Midicale. Institut de Pathologie. UnivenitE de t.i2gc, l.iege, Belgium. METABOLIC INACTIVATION OF MUTAGENIC BENZO(a)PYRENE METABOLITE.S: SIGNIFICANCE TO CARCINOGENICITY AND IMPLICATIONS FOR IN VITRO TESTS In this very well-reasoned paper, the mutagenieity, carcinogenicity and in vitro testing of benzo(e)pyrene (BP) are coosidered from several different aspects. It is known that BP has a complex metabolism involving many en- zymes and, because of this, complete metabolizing systems for the activation of BP and its premutagenic metabolitcs were used in the bacterial muta- genicity tests presented here. The activating systems used were freshly iso- lated intact hepatocytes or homogenates of their cells supplemented with var- ious cofactor systems. The compounds tested. were the carcinogens 8P and (.-: )nans-7,tt-dihydroxy-7,g-aihydro-[lP (7,g-diol) and the very weak ear- cinogens or tumor initiators 3-hydroxy-BP (3-Off-BP), 9-hydroxy-BP (9-Oli- BP) and ( r )trons-9,10•dihydroxy-9,10-dihydro-BP (9,I0diol). (All are strong mutagens in the Ames test.) Results of these tests showed that BP was not mutagenic when tested directly or in the presence of hepatocyte homog- enate. However, it became strongly mutagenie when an NADPH-generating system was added to the hornogenate or when whole hepalocytes were used. 7 he dose-response relatiunship differed markedly under the two situations. 3-O1i-B1', 9-OH-SP and 9-10-diol were not mutagenic in the presence of 14 15
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! homogenate. However, when an NADPH-generating system was added with the cell homogenate, mutagenic effects were observed with all three. On the other hand, 7.8-diol was activated by both homogenized or intact hepatocytes to a vrry potent mutagen. Overall, the results of this study show that it is possible to activate BP and BP-metabolites to bacterial mutagens with intact hepatocytes. The mutagenicity often is weaker and the relative potencies of varian compounds greatly different from results obtained with NADPH-for- tiAed cell homogenate. Aho, it was shown here that addition of cofacton of further enzymes considerably altered mutagenicity. The alterations differed with different test compounds and the results became more similar to results obtained with Intact celh. When carcinogenicity was taken into account, it was seen that use of intact hepatocytes instead of NADPll-fortifkd homog- enate considerably improved the correlation of mulagenacity with whole ani- mal carcinogenicity. Now, while intact hepatocytes may well not be the optimal metabolizing system In short-term tests for carctnogenicity and mutagenicity, this study shows that the metabolizing system can very strongly affect the result of a test. Thercforc, if inactivation occurs !n vivo it is reasonable to take the inactivating systems Into account also in in vitro tests. Clatt, H. R., Ptatt, K. L, Vogel, M., Bucker, M., Biflings, R., and Oexch, F. In: Holmstedt, B., Lauwer)n, R., Mercier, M.. and Roberfroid, M. (eds.): Mechanirnu of Torfciry and Hazard Evduarion, Elsevier: North Holland Biomedical Press, 1980. pp. 181-186. From the Phartnakologisches Institut der Universitit Mainz, Federal Republic of Germany. RAT LIVER CYTOPLASMIC DIHYDRODIOL DEHYDROCENASE: PURIFICATION TO APPARENT HOMOGENEITY AND PROPERTIES In this biochemical paper, a method is described for purifying a dihy- drodiol dehydrogenase to apparent homogeneity from the cytoplasmic frac- tion of rat liver homogenate. Some properties of the purified preparation, and the effect of this enzyme upon the mutagenkity of benzo(s)pyrene are also considered here. SpeciBcally, the purification involved (NH,)-SO, fractiona- tion, DEAEcellulose chromatography, interfacial salting-in and gel filtration through Sephadex (3-100 superfine. The end product, which was purified over S00-fold with a yield of about 14% when compared to rat liver 100.000 x g supetnatant, was judged to be homogeneous by several criteria. Physical stud- ies suggested that the protein was a monomer with a molecular weight of 35,000 assd one NADPH binding site per molecule. Amino acid analysis showed that the enzyme had a relatively high content of acidic and neutral amino acids in agreement with its isoelectric point. Apparent K_ values for benzene dihydrodiol and NADP+ were found to be 2.2 mM and 7.7 pM, respectively. Substrate specifkity studies showed that, in addition to henzene dihydrod-al, the dehydrivgenase could oxidize acenapthenol and the 3 o-hydroxy group of steroida. No activity was observable with a large number of other hydroxylated steroids possaning hydroxy groups at positions 3ft. 110. 17e, 17a, 20s, 20A 21 and 22 of the steroid skeleton. Furthermore, only steroids which contained a 3-keto group and no double bond at the A4 position were reduced. The, and the fact that a range of nonsteroidal vicinal diols did not scrve as suhstrates. indicates a relatively narrow substrate specificity. The role of the enzyme in the metabolism of carcinogenic polycyclic hydrocarbons is discussed. Vogel, K.. Bentley, P., Platt, K-L, and Oerch, F. The Journal o/ Bioloriral Chemistry 2SS(20):9621-9625, 1980. From the Pharmakologisches institut der Universitiit Mainz, Federal Republic of Germany. f?NZYMIC IIYDROXYI.ATfON OP REN7.O(rv)PYRENL• AT THE 6-POSITION RY VITAMIN K,-IIYDROPEROXIDE AND RAT 1.UN0 IIYDROPEROXIDASE In this biochemical paper, the role of vitamin K, in the hydroxytation of benz.o(,.)pyrene (nP) at the 6-position is correlated with the oxidative formation of vitamin K,-hydroperoxide and its subsequent reaction with BP in the presence of soluble rat lung hydroperoxidase. Furthermore the chem- ical synthesis of vitamin K,-hydroperoxide and the duat inhibitory action of thiodione (menadione-glutathione adduct) both as an aryl hydrocarbon hy- droxylase inhibitor and hydroperoxidase inhibitor is presented here. The spea tral characteristics of vitamin K,-hydroperoxide and the 3,6-BP dione are also shown. It is generally known that the identification of a proximate car- cinogen that results from the metabolism of carcinogenic polycyclic hydro- carbons is a necessary prerequisite in order to study the mechanism of chem- ical carcinogenesis. Studies are considered in this paper that deal with the role of dihydrodiol epoxides of BP, cytochrome P-450-44R-independent reae- tions, and hydroperoxidase reactions in the hydroxylation of BP at the 6-posi- tion in chemical carcinogenesis. Spccifically, the studies reported fully here demonstrate that the mammalian lung hydroperoxidase is capable of reacting with potentially naturally occurring hydroperoxides, e.g., vitamin K,-hydro- pcroxide,to form the carcinogen 6-hydroxy BP. Sloane. N. Xrnobiorica 11(4) :267-274, 1981. From the Department of Diochemistry. University of Tennessee College of Medicine, Memphis. DIALKYLNITROSAMINE DIOACTIVATION AND CARCINOGENESIS This mini-review presents a synopsis of studies on the mechanisms of mnatolic activation and carcinogenesis of dialkylnitrosamines. Since the sim- plcst and most common dialkylnitrosamine is dimethylnitrosamine (DMN), it was studied first. The considerable number of studies that were later car- ried out on DMN are reviewed in the first section of this paper; sections two nd three are devoted to the actions and reactions of diethylnitrosamitses (DEN) and methylethylnitrosamine (MEN) and higher nitrosamines. An overall consideration of these many studies led to the conclusions that: (1) Prior metabolic activation of nitrosamines by various oxidases to alkylating 16 17

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