WP9 Endocrine Disruption: Fish Estrogenicity
Room 12A/B, Level 4
2:10 PM - 5:30 PM, Wednesday, 12 November 2003
Chair: Janz, David ,
Co-chair: Foran, Christy ,
(479) Role of CYP2K- and CYP2M-like isozymes in the regio-selective hydroxylation of nonylphenol in Atlantic salmon.
Arukwe, A.1, Thibaut, R.2, 1 Great Lakes Institute for Environmental Research (GLIER), University of Windsor, Windsor, Ontario, Canada2 Laboratoire des Xe'nobiotiques, INRA, Toulouse, Cedex 9, France
ABSTRACT- We have previously demonstrated that nonylphenol (NP) can increase the activity of steroid metabolizing enzymes at low concentrations that does not induce vitellogenin (Vtg) and zona radiata proteins and decrease the activity of these enzymes at higher concentrations. To more precisely understand the estrogenic effects of NP in fish and a possible interference with steroid hormone metabolism, we investigated in the Atlantic salmon the identity of the cytochrome P450 enzymes involved in NP hydroxylation. Up to 9 metabolites were separated by radio-HPLC when [R-14C]-4n-NP was incubated with hepatic microsomes from juvenile salmon. In control fish the major metabolites were identified as monohydroxylated products at 
-, (-1)- and (-2)-positions of the alkyl chain of 4n-NP. Salmon hepatic microsomes formed -, (-1)- and (-2)-lauric acid (LA) hydroxylation products. The potency of a-naphthoflavone, ketoconazole and ethynylestradiol (non-specific, but strong inhibitors of CYP1A, 2K and 3A, respectively) on LA and NP hydroxylations were assessed. Ketoconazole inhibited -, (-1)- and (-2)-hydroxylations of LA and 4n-NP and was the only inhibitor of -hydroxylation of both substrates. Ethynylestradiol specifically inhibited (-1)- and (-2)-hydroxylations of LA as well as 4n-NP. Interestingly, the lowest NP dose (1 mg/kg) was the most potent inducer of NP-metabolites formation. These results suggest the involvement of CYP2M- and 2K-like enzymes in terminal and sub-terminal hydroxylations of 4n-NP respectively, and were confirmed by the competitive inhibition between LA and 4n-NP. The presence of one unidentified 4n-NP metabolite was not affected by any of the chemicals used, suggesting a possible ring hydroxylation that may involve another cytochrome P450 isoform. Our data reveal a novel aspect of CYP isozymes involvement in NP metabolism that may complicate the assessment of its endocrine effects. Hence, the regio-selective hydroxylation of endocrine disruptors, such as NP, by CYP isozymes may be a possible new marker of estrogenicity.
Key words: Metabolism, Nonylphenol, Regio-selectively, Endocrine Disruption