Demonstration of 2,3,7,8-tetrachlorodibenzo- p-dioxin attenuation of P450 steroidogenic enzyme mRNAs in rat granulosa cell in vitro by competitive reverse transcriptase-polymerase chain reaction assay

Abstract

We investigated the effects of 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD), in prepubertal (PP) and adult (A) rat granulosa cells (GC) in vitro by examining the changes in estrogen secretion, aromatase enzyme activity and mRNAs for steroidogenic enzymes P450scc, 3β-HSDI, P450arom; and for components of the AHR signaling pathway-CYP1A1, aromatic hydrocarbon receptor (AHR), and the AHR nuclear translocator protein (ARNT). In PP and A rat GC, TCDD (3.1 nM) reduced estrogen secretion at 48 h without altering aromatase enzyme activity. Addition of FSH (50 ng/ml) increased aromatase activity in GC with or without TCDD. FSH-induced aromatase activity was significantly reduced by TCDD (3.1 nM) at 48 h. Semi-quantitative RT-PCR showed a significant increase in CYP1A1 mRNA both at 24 and 48 h with TCAP, while a significant reduction in P450scc and P450arom mRNA was observed with competitive RT-PCR. All steroidogenic enzyme mRNAs were significantly lower in adults than in PP GC. We conclude that in rat GC, TCDD modulates the level of cytochrome P450 enzymes involved in the steroid biosynthetic cascade. This effect may be attributable to AHR interaction with dioxin-responsive elements present in the genes encoding these enzymes.