DIOXIN PERTURBS, IN A DOSE-DEPENDENT AND TIME-DEPENDENT FASHION, STEROID-SECRETION, AND INDUCES APOPTOSIS OF HUMAN LUTEINIZED GRANULOSA-CELLS

Dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDD) is the most toxic c ongener of a large class of environmental pollutants. Several studies have shown that TCDD exposure reduced fecundity and ovulatory rate in rats and increased the incidence of endometriosis in monkeys. Recent w ork suggests that TCDD's endocrine-disrupting effects are, at least in part, caused by a direct action at the ovary. Although the factors in volved in TCDD-induced toxicity are still under investigation, several studies have shown that TCDD induces programmed cell death, or apopto sis, in various tissues and may act in a similar fashion in the ovary. In the present study, we set out to evaluate the in, vitro effects of TCDD on steroid secretion, specifically estradiol-17 beta (E-2) and p rogesterone, by human luteinized granulosa cells (LGC), and to further determine whether TCDD is capable of inducing apoptosis in this cell type. Human LGC were obtained from women participating in an in vitro fertilization program. Medium, with or without three different concent rations of TCDD and substrates [androstenedione (A(4)) or pregnenolone ], was added to each culture. The media were collected at 4, 8, 12, 24 , 36, and 48 h and were assayed by RIA. At 24 and 48 h, the LGC were f ixed for assessment of DNA fragmentation via an in situ immunofluoresc ence technique. Transmission electron microscopy was also performed on LGC after 24 and 48 h with TCDD. TCDD, at all concentrations tested ( 3.1 pM, 3.1 nM, and 3.1 mu M), significantly reduced E-2 accumulation in the media at 8, 12, and 24 h, compared with controls. At 36 and 48 h, TCDD treatment (at 3.1 mu M) caused a significant increase in E-2, compared with controls. The effect of TCDD on E-2 was abolished with t he addition of A(4). TCDD treatment did not alter progesterone accumul ation. Apoptosis increased at 24 h with 3.1 mu M TCDD, with no apparen t effect at 3.1 nM. By 48 h, however, TCDD increased apoptosis in a do se-dependent manner. Transmission electron microscopy showed ultrastru ctural differences in LGC with 3.1 mu M TCDD at 24 and 48 h. Collectiv ely, the results of the present study suggest that TCDD perturbs E-2 s ecretion by depletion of A(4) precursor and increases apoptotic cell d eath of human LGC in a dose- and time-dependent fashion.