Effects of Heavy Metals on the in vitro Follicular Steroidogenesis in Amphibians

Heavy metals are well known as important environmental pollutants and also considered as endocrine disrupters. This study was performed to evaluate the direct effects of heavy metals such as cadmium (Cd), zinc (Zn), mercury (Hg), lead (Pb), cobalt (Co), and arsenic (As) on the various steroidogenic enzymes in frog ovarian follicles. Ovarian follicles from Rana catesbeiana were isolated and cultured for 18 hours in the presence of frog pituitary homogenate (FPH, 0.05 gland/ml) or various steroid precursors with or without heavy metals (0.01-100 ${\mu}M$), and steroid levels in the follicle or culture medium were measured by radioimmunoassay (RIA). Thus, the steroidogenic enzyme activities were indirectly evaluated by measuring the converted steroid levels from the added precursor steroid. Among heavy metals, Hg, Cd and Zn significantly inhibited FPH-induced pregnenolone ($P_5$) production by the follicles ($EC_{50},\;4.0{\mu}M,\;25.6{\mu}M\;and\;5.7{\mu}M$, respectively ), and also suppressed the conversion of testosterone (T) to estradiol $17{beta}\;(E_2)\;(EC_{50},\;4.2{\mu}M,\;7.5{\mu}M\;and\;80.0{\mu}M) while Pb, Co and As are not or less effective in the inhibition. Other enzymes such as $C_{17-20}$ lyase and $17{\beta}$-hydroxysteroid dehydrogenase ($17{\beta}$-HSD) were suppressed only in the high concentration of Hg, Cd and Zn. Taken together, these data demonstrate that cytochrome P450 side chain cleavage (P450scc) and aromatase are much more sensitive to heavy metals than other steroidogenic enzymes and Hg, Cd and Zn show stronger toxicity to follicles than other heavy metals examined.