Cytochrome P450-mediated 17β-estradiol metabolism in zebrafish (Danio rerio)
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abstract
Cytochrome P4501 (CYP1) and CYP3A proteins are primarily responsible for the metabolism of 17β-estradiol (E2) 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 E2 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′ 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 E2 metabolism into 4-hydroxyE2 (4-OHE2), 2-hydroxyE2 (2-OHE2), and 16α-hydroxyE1 (16α-OHE1) metabolites were determined by gas chromatography/mass spectrometry. The 2-OHE2 metabolite was produced by all CYPs tested, while 4-OHE2 was only detected following incubation with CYP1A, CYP1B1, CYP1C1, and CYP1C2. The 16α-OHE1 metabolite was only produced by CYP1A. The highest rates of E2 metabolism were from CYP1A and CYP1C1, followed by CYP1C2. CYP1B1, CYP1D1, and CYP3A65 had low rates of E2 metabolism. E2 metabolism by zebrafish CYP1A, CYP1C1, and CYP1C2 produced similar ratios of 4-OHE2 to 2-OHE2 as previous studies with mammalian CYP1As. CYP1B1 formed the highest ratio of 4-OHE2 to 2-OHE2 metabolites. Contrary to mammals, these results suggest that fish CYP1A and CYP1C proteins are primarily responsible for E2 metabolism, with only minor contributions from CYP3A65 and CYP1B1. Similar to mammals, 2-OHE2 is the predominant metabolite from CYP-mediated E2 metabolism in fish, suggesting that all vertebrate species produce the same major E2 metabolite.
