Exogenous steroid substrate modifies the effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin on estradiol production of human luteinized granulosa cells in vitro

The in vitro effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on steroid metabolism in human luteinized granulosa cells (hLGC) have been summarized as a decreased estradiol (E(2)) production without altering either E(2) metabolism or cytochrome P450 aromatase activity. In the present study, hLGC were used to analyze the fate of different substrates for cytochrome P450 17alpha-hydroxylase/17,20-lyase (P450(c17)) in the presence or absence of TCDD. Human LGCs were plated directly on plastic culture dishes in medium supplemented with 2 IU/ml of hCG. TCDD (10 nM) or its solvent was added directly to the cells at the time of medium change, every 48 h for 8 days. The objective of the experiment was to test the hypothesis that exogenous steroid, substrate for P450(c17), would reduce the TCDD effects on E(2) synthesis. With dehydroepiandrosterone (DHEA) (a P450(c17) product), a dose-related increase in E(2) production was observed and the effect of TCDD on lowering E(2) production disappeared. In contrast, with increasing doses, up to 10 micro M, of pregnenolone (P(5)), no change in E(2) production was observed. However, 17alpha-hydroxypregnenolone (17P(5)) at 10 micro M produced a modest but significant increase in the E(2) production. Treatments with P(5) and 17P(5) did not alter the effect of TCDD on E(2) production. Radiolabeled substrate utilization by hLGC suggests that the principal metabolic pathway for Delta5 substrates is the conversion to a Delta4 product probably by a very active 3beta-hydroxysteroid dehydrogenase. We conclude that estrogen production by hLGC is limited at the level of lyase activity. Thus, these data suggest that the most likely target for the TCDD-induced inhibition of estrogen synthesis by hLGC is the 17,20-lyase activity of the P450(c17) enzyme complex.