Patterns and regulation of estradiol metabolism by hepatic microsomes from two species of marine teleosts.

Estradiol (E2) metabolites formed in vitro by microsomes from the marine teleosts winter flounder (Pseudopleuronectes americanus) and scup (Stenotomus chrysops) included at least seven products detected by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). The least polar metabolite was shown to be estrone by chromatographic and mass spectrometric identity with authentic estrone. Chromatographic analyses coupled with dual-label experiments also indicated formation of the catecholestrogen 2-hydroxyestradiol (2-OH-E2), which was the most prominent metabolite determined by TLC. Analysis of microsomal E2 2-hydroxylase activity by measuring the specific release of 3H2O from [2-3H]E2 indicated that it is mediated by cytochrome P-450. E2 2-hydroxylase activity normalized to microsomal protein was lower in females than in males for microsomes from both mature scup and winter flounder. Activity normalized to liver weight or body weight in female winter flounder was also lower than that in males. However, activity normalized to cytochrome P-450 content did not show sex differences in either species. E2 2-hydroxylase activity per nanomole cytochrome P-450 was reduced in scup treated with beta-naphthoflavone, which induces the hydrocarbon hydroxylase cytochrome P-450E. Studies employing reconstituted P-450E and microsomes preincubated with polyclonal antibodies against P-450E confirmed that this isozyme does not catalyze E2 2-hydroxylase activity in microsomes. However, preliminary work with scup cytochrome P-450A suggests that it may be an E2 2-hydroxylase. The studies establish that catecholestrogen formation is prominent in fish liver and that it is sexually differentiated, but further investigation is required to define the catalysts as well as the significance and regulation of this function.

Cytochrome P-450 isozymes from the marine teleost Stenotomus chrysops: their roles in steroid hydroxylation and the influence of cytochrome b5.

Two new cytochrome P-450 forms were purified from liver microsomes of the marine fish Stenotomus chrysops (scup). Cytochrome P-450A (Mr = 52.5K) had a CO-ligated, reduced difference spectrum lambda max at 447.5 nm, and reconstituted modest benzo[a]pyrene hydroxylase activity (0.16 nmol/min/nmol P-450) and ethoxycoumarin O-deethylase activity (0.42 nmol/min/nmol P-450). Cytochrome P-450A reconstituted under optimal conditions catalyzed hydroxylation of testosterone almost exclusively at the 6 beta position (0.8 nmol/min/nmol P-450) and also catalyzed 2-hydroxylation of estradiol. Cytochrome P-450A is active toward steroid substrates and we propose that it is a major contributor to microsomal testosterone 6 beta-hydroxylase activity. Cytochrome P-450A had a requirement for conspecific (scup) NADPH-cytochrome P-450 reductase and all reconstituted activities examined were stimulated by the addition of purified scup cytochrome b5. Cytochrome P-450B (Mr = 45.9K) had a CO-ligated, reduced difference spectrum lambda max at 449.5 nm and displayed low rates of reconstituted catalytic activities. However, cytochrome P-450B oxidized testosterone at several different sites including the 15 alpha position (0.07 nmol/min/nmol P-450). Both cytochromes P-450A and P-450B were distinct from the major benzo[a]pyrene hydroxylating form, cytochrome P-450E, by the criteria of spectroscopic properties, substrate profiles, minimum molecular weights on NaDodSO4-polyacrylamide gels, peptide mapping and lack of cross-reaction with antibody raised against cytochrome P-450E. Cytochrome P-450E shares epitopes with rat cytochrome P-450c indicating it is the equivalent enzyme, but possible homology between scup cytochromes P-450A or P-450B and known P-450 isozymes in other vertebrate groups is uncertain, although functional analogs exist.