Cytochrome P450-mediated 17β-estradiol metabolism in zebrafish (Danio rerio)

Abstract

Cytochrome P4501 (CYP1) and CYP3A proteins are primarily responsible for the metabolism of 17b-estradiol (E 2 ) in mammals. We have cloned and heterologously expressed CYP1A, CYP1B1, CYP1C1, CYP1C2, CYP1D1, and CYP3A65 from zebrafish (Danio rerio) to determine the CYP-mediated metabolism of E 2 in a non-mammalian species. Constructs of each CYP cDNA were created using a leader sequence from the bacterial ompA gene to allow appropriate expression in Escherichia coli without 5 0 modification of the gene. Membrane vesicles were purified, and functional CYP protein was verified using carbon monoxide difference spectra and fluorescent catalytic assays with the substrates 7-ethoxyresorufin and 7-benzyloxy-4-(trifluoromethyl)-coumarin. Rates of in vitro E 2 metabolism into 4-hydroxyE 2 (4-OHE 2 ), 2-hydroxyE 2 (2-OHE 2 ), and 16a-hydroxyE 1 (16a-OHE 1 ) metabolites were determined by gas chromatography/mass spectrometry. The 2-OHE 2 metabolite was produced by all CYPs tested, while 4-OHE 2 was only detected following incubation with CYP1A, CYP1B1, CYP1C1, and CYP1C2. The 16a-OHE 1 metabolite was only produced by CYP1A. The highest rates of E 2 metabolism were from CYP1A and CYP1C1, followed by CYP1C2. CYP1B1, CYP1D1, and CYP3A65 had low rates of E 2 metabolism. E 2 metabolism by zebrafish CYP1A, CYP1C1, and CYP1C2 produced similar ratios of 4-OHE 2 to 2-OHE 2 as previous studies with mammalian CYP1As. CYP1B1 formed the highest ratio of 4-OHE 2 to 2-OHE 2 metabolites. Contrary to mammals, these results suggest that fish CYP1A and CYP1C proteins are primarily responsible for E 2 metabolism, with only minor contributions from CYP3A65 and CYP1B1. Similar to mammals, 2-OHE 2 is the predominant metabolite from CYP-mediated E 2 metabolism in fish, suggesting that all vertebrate species produce the same major E 2 metabolite.

Probing the xeno-oestrogenic impact of sediment cores contaminated by the pulp and paper industry: induction of aromatase cyp19a1b in late larval zebrafish

“…By definition, estrogenicity per se is not an adverse effect, as focused, e.g., in females, although disruptions along related pathways often amount to altered homeostasis and regulation of the endocrine system. Compared to the roles of CYP1A and CYP3A in mammalian species, the metabolism of E2 in zebrafish is mainly catalysed by CYP1A and CYP1C enzymes with the formation of 2-hydroxyestradiol (2-OH E2) being the dominant metabolite (Scornaienchi et al 2010). Depending on ligand-activation, both ER and AhR are known to play inhibiting and co-regulating roles in signalling of either pathway (Hasselberg et al 2005, Matthews and Gustaffson 2006, Gräns et al 2010, Ohtake et al 2011, Bugel et al 2013.…”

Specificity Determinants of CYP1B1 Estradiol Hydroxylation

“…Mouse CYP1B1 was reported to not bind or oxidize 17b-estradiol. The zebrafish enzyme was shown to oxidize estradiol, but, in contrast to the human enzyme that exhibits a 4-hydroxyestradiol (4OH E2)/2-hydroxyestradiol (2OH E2) ratio of approximately 3.44 (Lee et al, 2003), the zebrafish enzyme favored 2-hydroxylation and gave a 4OH E2/2OH E2 ratio of 0.32 (Scornaienchi et al, 2010).…”

“…The CYP1B1 orthologs from mouse (Savas et al, 1997) and zebrafish (Scornaienchi et al, 2010) have been cloned and heterologously expressed. Mouse CYP1B1 was reported to not bind or oxidize 17b-estradiol.…”

“…Cytochrome b 5 has been reported to have no effect on substrate oxidation by human CYP1B1 (Shimada et al, 1997;Yamazaki et al, 2002). In contrast, cytochrome b 5 was found to enhance 7-ethoxycoumarin O-dealkylation by zebrafish CYP1B1, whereas the oxidation of 17b-estradiol by this same enzyme was inhibited by cytochrome b 5 (Scornaienchi et al, 2010).…”

Natural Allelic Variations of Xenobiotic Enzymes Pleiotropically Affect Sexual Dimorphism inOryzias latipes

“…Our preliminary screening studies showed high degrees of polymorphism among medaka CYPs and suggested that differing activities of CYP1A and CYP1B1 alleles contribute to differential regulation of sex steroid metabolism between medaka populations from various locations. Indeed, CYP1A and CYP1B1 were highly polymorphic and differentiated across medaka populations, and were also involved in estrogen metabolism in fish [22] ( Figure S1 and Supplemental Experimental Procedures). Therefore, we investigated the role of CYP polymorphisms and the ensuing molecular mechanisms that lead to geographical variations in medaka anal fin morphology, and elucidated the evolution of sexual dimorphisms under CYP genes.…”