Mono-(2-ethylhexyl) phthalate suppresses aromatase transcript levels and estradiol production in cultured rat granulosa cells

The female reproductive toxicity of di-(2-ethylhexyl) phthalate and its act ive metabolite mono-(2-ethylhexyl) phthalate (MEHP) is attributed to suppre ssion of ovarian granulosa cell estradiol production. In these studies, sev eral structurally related phthalates (0-200 muM) and Wy-14,643 (0-100 muM) were compared to MEHP for their effects on granulosa cell estradiol product ion and transcript levels of cytochrome P450 enzyme CYP 19, also known as a romatase (P450arom), the rate-limiting enzyme in the conversion of androgen s to estrogens, Granulosa cells were obtained from 28-day-old Fisher 344 ra ts and were cultured for 48 h, Test chemical or DMSO was added at the time of culture, along with testosterone as a substrate for aromatase. 17 beta - Estradiol production was measured by standard radioimmunoassay, mRNA was me asured by fluorescent RT-PCR, and protein was measured by Western blot anal ysis. MEHP was unique among the phthalates in its ability to decrease estra diol production, while Wy-14,643 had effects similar to MEHP at 100 muM MEH P and Wy-14,643 also significantly decreased aromatase mRNA levels. The dec rease in mRNA was concentration dependent and was paralleled by a decrease in aromatase protein. MEHP did not alter levels of CYP 11A1, the cholestero l side-chain cleavage enzyme (P450scc). Treatment with a cAMP analogue incr eased expression of P450scc in the presence of MEHP (100 to 200 muM) while the decrease in aromatase remained. Thus, these studies suggest that MEHP i s distinct from several structurally related phthalates but similar to the peroxisome proliferator Wy-14,643 in its action on granulosa cell estradiol production. Moreover, the suppression of estradiol by MEHP is likely media ted through its action on aromatase transcript levels independent of cAMP-s timulated regulation. (C) 2001 Academic Press.