Assessment of cytochrome P450 fluorometric substrates with rainbow trout and killifish exposed to dexamethasone, pregnenolone-16alpha-carbonitrile, rifampicin, and beta-naphthoflavone

Abstract

Cytochrome P450s (CYPs) are important xenobiotic metabolizing proteins. While their functions are well understood in mammals, CYP function in non-mammalian vertebrate systems is much less defined, with function often inferred from mammalian data, assuming similar function across vertebrate species. In this study, we investigate whether in vivo treatment with known mammalian CYP inducers can alter the in vitro catalytic activity of fish microsomes using eleven fluorescent CYP-mediated substrates. We investigate the basal metabolism and induction potential for hepatic CYPs in two fish species, rainbow trout (Oncorhynchus mykiss) and killifish (Fundulus heteroclitus). Species differences were found in the baseline metabolism of these substrates. Killifish have significantly higher metabolic rates for all tested substrates except 7-benzyloxyquinoline and 7-benzyloxy-4-trifluoromethylcoumarin (both mammalian CYP3A substrates); significant differences were also seen between male and female killifish. Treatment with dexamethasone, pregnenolone-16alpha-carbonitrile, and rifampicin did not cause broad, measurable CYP induction in either fish species. In trout, dexamethasone (100 mg kg(-1)) significantly induced 3-cyano-7-ethoxycoumarin metabolism and rifampicin (100 mg kg(-1)) induced the dealkylation of 7-methoxyresorufin, although both were highly variable. Female killifish exposed to pregnenolone-16alpha-carbonitrile (100 mg kg(-1)) showed significantly higher metabolism of 7-pentoxyresorufin. Overall, dexamethasone, pregnenolone-16alpha-carbonitrile and rifampicin did not appear to consistently increase CYP activity in fish. Trout treated with 10 or 50 mg kg(-1) beta-naphthoflavone (BNF), a CYP1A inducer, showed significantly induced activity across almost all substrates tested, exceptions being 7-benzyloxyquinoline, 7-benzyloxy-4-trifluoromethylcoumarin and dibenzylfluorescein. 7-Methoxy-4-(aminomethyl)coumarin, a typical CYP2D substrate in mammals, was not metabolized by untreated fish liver microsomes; however, treatment with BNF significantly induced the metabolism of this substrate in trout. Induced substrate metabolism in BNF-treated microsomes was only correlated across selective substrates, suggesting that BNF induces multiple CYPs in fish liver. These include the known BNF inducible CYP1s plus a number of as yet unidentified fish CYPs. Overall, many of these catalytic assays could be valuable tools for identification of the function of specific CYP subfamilies and individual isoforms in fish.