Exogenous Steroid Substrate Modifies the Effect of 2,3,7,8-Tetrachlorodibenzo-p-Dioxin on Estradiol Production of Human Luteinized Granulosa Cells In Vitro1

Abstract

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.

TCDD Increases Inhibin A Production by Human Luteinized Granulosa Cells In Vitro

“…An enzyme located earlier in the steroidogenic pathway is therefore implicated. When dehydroepiandrosterone (DHEA), a CYP17/ 17,20-lyase product was added along with TCDD, TCDD's action on estradiol s ecretion w as attenuated [7]. However, as TCDD also caused a decrease in progesterone production [7, 23, our preliminary results], TCDD may act on more than one enzyme in the ovarian steroidogenic pathway.…”

“…2,3,7, is the most toxic of the halogenated dioxins and one of the most poisonous substances known to man. The major toxic effects of TCDD on reproduction are decreased fertility and diminished ability to maintain a pregnancy.…”

“…Therefore, TCDD during relatively brief exposures likely affects steroidogenesis at or before the conversion of androstenedione to estrone or testosterone. The addition of dehydroepiandrosterone (DHEA), a CYP17/17,20-lyase product, also prevented TCDD's decrease in estrogen secretion [7]. When either 17 α hydroxypregnenolone or 17 α hydroxyprogesterone, precursors of androgens, was added to the hGLC culture, TCDD was not hindered in its ability to decrease estrogen secretion [7].…”

“…The addition of dehydroepiandrosterone (DHEA), a CYP17/17,20-lyase product, also prevented TCDD's decrease in estrogen secretion [7]. When either 17 α hydroxypregnenolone or 17 α hydroxyprogesterone, precursors of androgens, was added to the hGLC culture, TCDD was not hindered in its ability to decrease estrogen secretion [7]. This suggests that aromatase activity remains intact during TCDD treatment.…”

2,3,7,8-Tetrachlorodibenzo-p-dioxin alters steroid secretion but does not affect cell viability and the incidence of apoptosis in porcine luteinised granulosa cells

“…TCDD is considered to be a potent disrupter of ovarian steroidogenesis in humans (Enan et al, 1996a;Heimler et al, 1998a;Morán et al, 2000Morán et al, , 2003 and rats (Son et al, 1999;Franczak et al, 2006;Shi et al, 2007;Jablonska et al, 2010). In the pig, the toxic effects of TCDD on steroidogenesis have also been reported (Piekło et al, 2000;Gregoraszczuk et al, 2001;Grochowalski et al, 2001).…”