Document k6kaXJj5rQZz7rVn9VgopdNNy

BiO-hemital PMrrrucolojiy, Vo> 21 pp 5*9 596 >I> Ptrsjmon Prtss LtJ, 1919, Pnrucri in Great Britain 0006 295! 79,0301 0589 S0100.0 METABOLISM OF [ 14C ]- and [36CU~LABLED VINYL CHLORIDE IN VIVO. AND IN VITRO * F. Peter Guengericii Department of Biochemistry and Center in Environmental Toxicology. Vanderbilt University School of Medicine, Nashville, TN 37232, U.S.A. Philip G. Watanabe Toxicology Research Laboratory. Health and Environmental Research, The Dow Chemical Co., Midland, MI 48640, U.S.A. (Received 5 May 1978; accepted 24 July 1978) Abstract--Label from [ |4C Ivinyl chloride was covalently bound to protein and nucleic acids in riro and in vitro in the presence of rat liver microsomal fractions or highly purified cytochrome P-450 and NADPHcytochrome P-450 reductase preparations- The ratio of bound to total non-volatile metabolites increased in going from the in vivo to the microsomal to the purified system. ["ClIvinyl chloride was metabolized by microsomes and highly purified systems; no label was bound and most could be accounted for as chloride ion. Phenobarbital pretreatment or rats did not induce total metabolism of vinyl chloride in vivo at either 10 or 250 ppm exposure levels; however, binding to protein and RNA was enhanced at the 10 ppm but not the 250 ppm level. Phenobarbital pretreatment increased the in vitro microsomal conversion of vinyl chloride to both total and bound metabolites. A sizeable fraction of the label of [ IJC Ivinyl chloride metabolized in vivo was recovered in the microsomal fraction of the liver, but sodium dodecyl sulfate polyacrylamide gel electrophoresis of in vitro incubations indicated that the metabolites were distributed among many microsomal proteins and not localized to cytochrome P-450. Evidence was obtained for the metabolism of the suspected vinyl chloride metabolite chloroethylene oxide by microsomal epoxide hydratase. However, the epoxide hydratase inhibitor 3.3,3-trichloropropylene oxide, which blocks the microsomal degradation of chloroethylene oxide, did not enhance the level of vinyl chloride bound to either protein or adenosine. Vinyl chloride is an industrial chemical that has been demonstrated to be carcinogenic in laboratory ani mals [ 11 and humans [2, 31. The concept that a metabolite(s) of vinyl chloride is responsible for the observed carcinogenicity [4, 51 is supported by the findings that NADPH-dependent microsomal activation is neces sary for vinyl chloride-mediated mutagenesis in Salmo nella typhimurtum tester strains [6, 7l and for binding of vinyl chloride to protein and nucleic acids [8-101. Th,e vinyl chloride metabolite responsible for these activities has been postulated to be the potent electro phile chloroethylene oxide [111. Liver microsomal cytochrome P-450 has been shown to activate vinyl chloride to a species that de stroys the heme of that cytochrome [12]. Cytochrome P-450 has also been suggested, on the basis of COinhibition experiments 191, as the enzyme responsible for activating vinyl chloride to metabolites bound to tissue nucleophiles. Spectral evidence [ 131 for a rote of cytochrome P-450 in vinyl chloride metabolism is rather weak when the limitations of such data are considered [ 141. Previous workers had reported that induction with phenobarbital increases the ability of the liver to convert vinyl chloride to a P-450 destructive species in vivo [ 15] and in vitro 1121, observed in vivo vinyl chloride hepatotoxicity f 161, and the capacity to * This work was supported by National Institute of Envi ronmental Health Sciences Grants ES 01 590 and ES 00267 and Biomedical Research-Support Gram RR-05424. convert vinyl chloride to mutagenic products In vitro [61; however, induction with phenobarbital had no effect on the levels of vinyl chloride metabolism by rats in vivo and in vitro [ 81. Recently, Watanabe et al. [ 17] reported that, in rats exposed in vivo to 100 ppm vinyl chloride, phenobarbital increased binding approxi mately 2-fold but had no effect on the level oftotal vinyl chloride metabolism. The studies described in this report were initiated in order to establish the roles of liver microsomal cyto chrome P-450 and epoxide hydratase in the biotrans formation of vinyl chloride to metabolites, particularly those bound to protein and nucleic acids. MATERIALS AND METHODS Materials. 1, A"-ethenoadenosine was purchased from P-L Biochemicals (Milwaukee. WI). calf thymus DNA and Escherichia coli soluble RNA from Calbiochem (La Jolla, CA), research grade vinyl chloride from Matheson (E. Rutherford, NJ), and 3,3,3-trichloropropykne oxide. (TCPO)from Aldrich (Milwaukee, WI), Chloroethylene oxide was prepared, as described previously [12. 181, on the day of use; identity was confirmed by N.M-R- and mass spectra and by reaction with 4-(pnttrobenzyl)-pyridine [ II |. [ 1.2-Ivinyl chloride was prepared from [ l,2-uCIdichloroethane [191 and was diluted with carrier vinyl chloride: the purity was as described previously (17, 191. [ ,AC| |vin> I chloride (0.10 mCi/m-mole) was synthesized from H34C1 (New England Nuclear) and acetylene (201; the 589 SL 101947 Environmental Teratogens Their Significance and Epidemiologic Methods of Detection M.A. KUngberg and C.M. Papier Department of Epidemiology, Israel Institute for Biological Research, Nesj-Ziona. and Department of Preventive and Social Medicine, Tel-Aviv University Sackler School of Medicine, Tel-Aviv Although the past few years have shown a great increase in fundamental research in the field of congenital malformations, particularly the development of new techniques in genetics, biology and biochemistry, progress in the epi demiology of birth defects has been less remarkable. Following Gregg's discovery of the teratogenic effects of rubella in 1941 (44) It was widely but over-optimistically presumed that the discovery of other environmental agents responsible for malformations would be just around the corner. In the ensuing two decades the number of environmental factors dis covered to be crucial determinants of congenital malformations can be counted on the fingers of one hand. After the 1960-61 epidemic of congenital mal formations caused by thalidomide there was even more hope regarding the discovery of new environmental teratogens and for establishing new methods of detection and evaluation of teratogens in the human environment. More than 15 years have elapsed since this widely-publicized observation of thalidomide embryopathy in Europe arid elsewhere, but even today only about 10% of malformations can be ascribed to environmental factors: those which can' be attributed to genetic transmission and chromosomal aberrations account for approximately 25% of the detects. The causes of the great majority remain uncertain (127). What then is the etiology of the estimated 60 -70% of malformations that cannot be pin-pointed to arty known causes? Many investigators believe that most presently unaccounted for malformations and syndromes result from the poteutiatue effect of two or more factors(127). But the thalidomide disaster remains a sivid reminder of the need for developing new methodologies of uncovering the etmlogie hetois responsible for the majority of birth defects. Epidemiologists must wotk in coii]uiictnm and cooperation with many disciplines 'in striving tocether towards the goal ot prevention of congenital malformations. SL 101948