THE METABOLISM OF TESTOSTERONE BY THE PERIWINKLE (LITTORINA-LITTOREA)IN-VITRO AND IN-VIVO - EFFECTS OF TRIBUTYL TIN

Exposure to tributyl tin (TBT) at concentrations as low as 0.5 ng/lite r has been associated with disrupted sexual physiology (imposex) in 40 -50 species of marine gastropod. Imposex is a state of pseudohemaphrod ism in which females exhibit nonfunctional secondary male characterist ics. It has been suggested that the mechanism underlying imposex invol ves 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 s teroid testosterone was examined in vitro and in vivo. Microsomes were prepared from 'digestive gland' (visceral complex including the gonad s) and combined kidney and gill fractions. CO-ligated reduced differen ce 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 capab le of oxidative metabolism of testosterone in either the presence or a bsence of NADPH. In the absence of NADPH, testosterone was metabolized to androstenedione, 6 beta-, 6 alpha- and 15 beta-hydroxytestosterone , 17 beta-estradiol and an unidentified metabolite possibly 3 alpha-an drostene-17 beta-diol. In the presence of NADPH the same products were produced at a higher rate but the major products formed were the prod ucts of the NADPH-dependent steroid 5 alpha-reductase-3 alpha (beta) h ydroxysteroid dehydrogenase pathway; dihydrotestosterone (DHT) and 3 a lpha (beta) androstane-17 beta-diols (DHT diols). TBT, even at high co ncentrations up to 100 mu M, had only, modest effect on P450-dependent testosterone metabolism in vitro, producing increases in androstenedi one formation and only 30-40% inhibition of aromatase. In vivo, [C-14] testosterone was injected directly into the cephalopedal sinus of adu lt snails and animals maintained in sea water at 15 degrees C for 42 h in the presence of 0, 0.5 and 5 mM TBT. Testosterone was almost compl etely metabolized during this time, predominantly to water-soluble sul fur conjugates of testosterone, the 5 alpha-reduced products and 6 alp ha-hydroxytestosterone. However, with increasing concentrations of TBT , more radioactivity was retained within the animal and was increasing ly associated with organic extractable unmetabolized testosterone and its phase I products androstenedione, dihydroandrostenedione (DHA), DH T and DHT-diols. Thus it appears that, in vivo, TBT inhibits sulfur co njugation of testosterone and its phase I metabolites and their excret ion resulting in a build-up of pharmacologically active androgens in t he tissues. This data are consistent with the hypothesis that TBT-indu ced imposex in sensitive gastropods, such as stenoglossans, may arise from perturbations in sex steroid metabolism. However, the major bioch emical targets of TBT appear to be steroid conjugation and excretory t ransport mechanisms rather than P450-dependent oxidative pathways fuel as aromatase. Copyright (C) 1996 Elsevier Science Ltd