The metabolism of testosterone by the periwinkle ( Littorina littorea) in vitro and in vivo: Effects of tributyl tin

Abstract

Exposure to tributyl tin (TBT) at concentrations as low as 0.5 ng/liter has been associated with disrupted sexual physiology (imposex) in 40–50 species of marine gastropod. Imposex is a state of pseudohemaphrodism in which females exhibit nonfunctional secondary male characteristics. It has been suggested that the mechanism underlying imposex involves disrupted metabolism of endogenous sex hormones and in particular inhibition of cytochrome P450-dependent aromatization of androgens to estrogens. In the current study, the effects of TBT on the ability of the Periwinkle ( Littorina littorea) to metabolize the androgenic sex steroid testosterone was examined in vitro and in vivo. Microsomes were prepared from ‘digestive gland’ (visceral complex including the gonads) and combined kidney and gill fractions. CO-ligated reduced difference spectra contained peaks at 459 nm and calculated P450 contents of 0.3 and 0.05 nmol/mg. Digestive gland microsomes were found to be capable of oxidative metabolism of testosterone in either the presence or absence of NADPH. In the absence of NADPH, testosterone was metabolized to androstenedione, 6β-, 6α- and 15β-hydroxytestosterone, 17β-estradiol and an unidentified metabolite possibly 3α-androstene-17β-diol. In the presence of NADPH the same products were produced at a higher rate but the major products formed were the products of the NADPH-dependent steroid 5α-reductase-3α(β)hydroxysteroid dehydrogenase pathway; dihydrotestosterone (DHT) and 3α(β)androstane-17β-diols (DHT diols). TBT, even at high concentrations up to 100 μM, had only modest effect on P450-dependent testosterone metabolism in vitro, producing increases in androstenedione formation and only 30–40% inhibition of aromatase. In vivo, [ 14C] testosterone was injected directly into the cephalopedal sinus of adult snails and animals maintained in sea water at 15 °C for 42 h in the presence of 0, 0.5 and 5 mM TBT. Testosterone was almost completely metabolized during this time, predominantly to water-soluble sulfur conjugates of testosterone, the 5α-reduced products and 6α-hydroxytestosterone. However, with increasing concentrations of TBT, more radioactivity was retained within the animal and was increasingly associated with organic extractable unmetabolized testosterone and its phase I products androstenedione, dihydroandrostenedione (DHA), DHT and DHT-diols. Thus it appears that, in vivo, TBT inhibits sulfur conjugation of testosterone and its phase I metabolites and their excretion resulting in a build-up of pharmacologically active androgens in the tissues. This data are consistent with the hypothesis that TBT-induced imposex in sensitive gastropods, such as stenoglossans, may arise from peturbations in sex steroid metabolism. However, the major biochemical targets of TBT appear to be steroid conjugation and excretory transport mechanisms rather than P450-dependent oxidative pathways such as aromatase.