Monooxygenase catalysts play key roles in activation and detoxication of organic xenobiotics including carcinogens, drugs, and pollutants. Cytochrome P-450 is dominant in oxidative metabolism and activation of foreign compounds, and isozymes of cytochrome P-450 could determine the effects of chemicals in different organs, individuals or species. The overall objective of this research is to establish the nature of biotransformation enzymes, especially cytochrome P-450 isozymes, in non- mammalian and aquatic species. Currently it is difficult to critically compare cytochrome P-450 isozymes of non-mammalian species with each other, because definitive knowledge of these isozymes in these species is scant. Basic aspects concerning cytochrome P-450 isozymes and their regulation must be known in order to understand the similarities and differences of their functions among different species, and to evaluate and compare species responses to xenobiotics. Multiple forms of cytochrome P-450 will be purified from liver of the marine fish Stenotomus chrysops (scup). Efforts will be directed toward cytochromes P- 450A to P-450E, which compose the majority of scup liver microsomal P-450. Isozymes will be characterized for catalytic and physico-chemical properties. NADPH-cytochrome P-450 reductase and cytochrome bs will also be purified. Polyclonal antibodies to cytochrome P-450 forms A-E and cytochrome b5, and monoclonal antibodies to cytochrome P-450A, will be prepared. Antibodies will be used in immunoblotting and ELISA to study regulation of cytochrome P-450 forms in fish liver by selected inducers, assessing the turnover of cytochrome P-450, and the regulation of major forms of cytochrome P-450 in extrahepatic organs of scup. Antibodies will also be used to evaluate the contribution of different isozymes to xenobiotic and steroid metabolism in non-mammalian microsomal systems, and to evaluate the similarities of cytochromes P-450 in selected non- mammalian and mammalian species. In addition to the above aims, we will determine the feasibility of using molluscan species to study flavoprotein monooxygenase function, evaluating the hypothesis that his enzyme is involved in activating carcinogenic amines in these invertebrates. Results could provide information and probes for evaluating various non-mammalian species or even cell lines for mechanisms of xenobiotic metabolism.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Research Project (R01)
Project #
5R01ES004220-02
Application #
3252246
Study Section
(SRC)
Project Start
1987-01-01
Project End
1991-12-31
Budget Start
1988-01-01
Budget End
1988-12-31
Support Year
2
Fiscal Year
1988
Total Cost
Indirect Cost
Name
Woods Hole Oceanographic Institution
Department
Type
Graduate Schools
DUNS #
001766682
City
Woods Hole
State
MA
Country
United States
Zip Code
02543
Stegeman, J J; Woodin, B R; Singh, H et al. (1997) Cytochromes P450 (CYP) in tropical fishes: catalytic activities, expression of multiple CYP proteins and high levels of microsomal P450 in liver of fishes from Bermuda. Comp Biochem Physiol C Pharmacol Toxicol Endocrinol 116:61-75
Morrison, H G; Oleksiak, M F; Cornell, N W et al. (1995) Identification of cytochrome P-450 1A (CYP1A) genes from two teleost fish, toadfish (Opsanus tau) and scup (Stenotomus chrysops), and phylogenetic analysis of CYP1A genes. Biochem J 308 ( Pt 1):97-104
White, R D; Hahn, M E; Lockhart, W L et al. (1994) Catalytic and immunochemical characterization of hepatic microsomal cytochromes P450 in beluga whale (Delphinapterus leucas). Toxicol Appl Pharmacol 126:45-57
Hahn, M E; Stegeman, J J (1994) Regulation of cytochrome P4501A1 in teleosts: sustained induction of CYP1A1 mRNA, protein, and catalytic activity by 2,3,7,8-tetrachlorodibenzofuran in the marine fish Stenotomus chrysops. Toxicol Appl Pharmacol 127:187-98
Kloepper-Sams, P J; Stegeman, J J (1994) Turnover of hepatic microsomal cytochrome P4501A protein and heme in beta-naphthoflavone-induced Fundulus heteroclitus. Mol Mar Biol Biotechnol 3:171-83
Hahn, M E; Poland, A; Glover, E et al. (1994) Photoaffinity labeling of the Ah receptor: phylogenetic survey of diverse vertebrate and invertebrate species. Arch Biochem Biophys 310:218-28
Lester, S M; Braunbeck, T A; Teh, S J et al. (1993) Hepatic cellular distribution of cytochrome P-450 IA1 in rainbow trout (Oncorhynchus mykiss): an immunohisto- and cytochemical study. Cancer Res 53:3700-6
Kloepper-Sams, P J; Stegeman, J J (1992) Effects of temperature acclimation on the expression of hepatic cytochrome P4501A mRNA and protein in the fish Fundulus heteroclitus. Arch Biochem Biophys 299:38-46
Smolowitz, R M; Schultz, M E; Stegeman, J J (1992) Cytochrome P4501A induction in tissues, including olfactory epithelium, of topminnows (Poeciliopsis spp.) by waterborne benzo[a]pyrene. Carcinogenesis 13:2395-402
Gray, E S; Woodin, B R; Stegeman, J J (1991) Sex differences in hepatic monooxygenases in winter flounder (Pseudopleuronectes americanus) and scup (Stenotomus chrysops) and regulation of P450 forms by estradiol. J Exp Zool 259:330-42

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