Identification of multiple steroid hydroxylases in Daphnia magna and their modulation by xenobiotics

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Abstract
Steroid hydroxylase activities were characterized in Daphnia magna and evaluated for potential use as biomarkers of xenobiotic exposure. Microsomes prepared from Daphma magna generated a single NADPH‐dependent metabolite of [14C]testosterone. However, intact daphnids excreted at least 10 polar metabolites of [l4C]testosterone into the test medium. Six of these metabolites were identified as 2α‐, 16β‐, 6β‐, 6α‐, 7α‐, and 15α‐[14C]hydroxytestosterone. The unidentified metabolites are also presumed to be hydroxylated products of testosterone, based on their relative migrations during TLC. The inefficient metabolism of [14C]testosterone during the in vitro microsomal incubations may have been due to the release of P450 inhibitors during microsome preparation. Exposure of daphnids to the P450 modulators phenobarbital, β‐naphthoflavone, piperonyl butoxide, and malathion differentially inhibited the steroid hydroxylase activities. Results from this study indicate that Daphma magna expresses several P450 enzymes and that these enzymes are differentially modulated by xenobiotic exposure. Steroid hydroxylase activities may serve not only as a biomarker of toxicant exposure, but also as a predictor of toxicant effects involving perturbations of steroid hormone homeostasis.