In vivo biotransformation of testosterone by phase I and II detoxication enzymes and their modulation by 20-hydroxyecdysone in Daphnia magna

Abstract

Phase I and phase II chemical detoxication processes were elucidated in Daphnia magna using in vivo techniques and [ 14C]testosterone as a substrate. Testosterone was used because this compound undergoes multiple biotransformations and its metabolites are well characterized in other species. In addition, regulation of these processes by the endogenous steroid hormone, 20-hydroxyecdysone, was investigated. Daphnids produced at least ten polar phase I metabolites and four nonpolar phase I metabolites of testosterone. Six of the ten polar metabolites have been identified as monohydroxy-products of testosterone. The polar metabolites were preferentially excreted while the nonpolar metabolites were preferentially retained by the daphnids. In addition, testosterone and all phase I metabolites were also excreted as glucose conjugates. A polar metabolite designated ‘C’ was preferentially conjugated with glucose over the other metabolites. Testosterone and its polar phase I metabolites were also excreted as sulfate conjugates with 2α-hydroxytestosterone being the predominant sulfate-conjugated metabolite. In contrast to glucose conjugation, no nonpolar phase I metabolites of testosterone were sulfate conjugated. Twenty-four hour pre-exposure of daphnids to 4.2 μM 20-hydroxyecdysone did not affect phase I metabolism of testosterone, but differentially modulated phase II conjugation in a manner suggesting the presence of at least two glucosyltransferases and two sulfotransferases. Treatment with 20-hydroxyecdysone significantly increased the elimination of sulfate conjugates due largely to increased sulfate conjugation of unmetabolized testosterone. These results demonstrate that daphnids can convert polycyclic compounds to multiple polar and nonpolar metabolites resulting from both phase I and phase II biotransformations, and that some phase II activities are under the regulatory control of 20-hydroxyecdysone.