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

• DOI: 10.1677/joe-10-0075
• Publication Date: 2010-06-04T02:16:27Z
• Authors: Marcus L Scornaie...; Cammi Thornton; Kristine L Willett; Joanna Y Wilson
• Keywords: CYP3A; Danio; CYP1B1

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