MECHANISM OF TOXIC ACTION OF 2,3,7,8-TETRACHLORODIBENZO-P-DIOXIN (TCDD) IN CULTURED HUMAN LUTEINIZED GRANULOSA-CELLS

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) caused a significant decrea se in estradiol (E(2)) production when it was administered to human lu teinized granulosa cells (hLGCs) in culture. We investigated the invol vement of the epidermal growth factor receptor (EGFR) and protein tyro sine kinase (PTK) in this TCDD-induced toxicity, Upregulation in I-125 -EGF binding to EGFR was measured after 24 h of TCDD treatment, while downregulation in EGFR binding was measured after 72 h of TCDD treatme nt. Upregulation of EGFR binding was associated with a significant dec rease in postnuclear (7000 x g supernatant) PTK activity, but this act ivity was stimulated after 72 h of TCDD treatment. TCDD altered the le vel of tyrosine phosphorylation in proteins with molecular weights 35, 40, 43, 45, 60, and > 205 kDa. TCDD caused a significant increase in postnuclear cAMP-dependent protein kinase (PKA) after 24 h of treatmen t. The actions of TCDD on protein kinases were partially blocked by th e protein synthesis inhibitor, cycloheximide. On the other hand, TCDD increased nuclear PTK and decreased nuclear PKA activity. E(2) inhibit ed the postnuclear and nuclear activity of both PTK and PKA in control samples, but did not affect TCDD actions on either postnuclear or nuc lear PTK activity. However, E(2) abolished the stimulatory effect of T CDD on PKA activity in postnuclear protein. In the presence of insulin , TCDD did not induce any additional changes in postnuclear or nuclear PTK. Forskolin (PK) alone inhibited postnuclear PTK activity and stim ulated its nuclear activity. The addition of TCDD 20 min after FK resu lted in an increase in postnuclear PTK, but there was little change in nuclear PTK as compared to the effect of FK alone. The stimulatory ef fect of TCDD on postnuclear PKA activity was enhanced by insulin and T CDD reversed the negative effect of FK, but there was no effect of eit her insulin or FK on the inhibition by TCDD of nuclear PKA activity. T CDD decreased the activity of MAP2 kinase and reduced the binding acti vity of AP-1 DNA when given alone, and also blocked the E(2) stimulati on of MAP2K. These findings suggest that TCDD may interrupt the endocr ine function of hLGCs through the blockage of the mitotic signal direc tly or indirectly through the interaction of PTK/MAP2K and PKA signali ng. (C) 1996 Elsevier Science Inc.