Basic investigation on influence of environmental chemicals on Sex-steroid hormone secretion

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The in vitro assay system to examine the influence of the environmental chemicals on the androgen production was constructed by using the primary culture of pig Leydig cell. We found that organotin compounds, such as tributyltin chloride (TBT), dibutyltin dichloride and triphenyltin chloride, strongly suppressed the testosterone production level at a concentration without cytotoxicity by using this assay system. It was clarified as action mechanism that TBT exposure suppressed the rise of intracellular cAMP level and decreased the CYP17 mRNA level up-regulated by human chorionic gonadotropin or forskolin (FSK). Furthermore, TBT inhibited 17β-HSD activity in vivo as well as in vitro, which enzyme was essential for testosterone production. The estrogen biosyntheses enzyme, aromatase is a rate-limiting enzyme of the estrogen biosyntheses, and an indispensable enzyme to the estrogen production. We clarified that aromatase was expressed in human adrenocortical carcinoma cell line, H295R and promoter II and 1.3 were selected for its expression, and the transcription was activated by EGF and PGE_2 stimulation. Then, the in vitro assay system to estimate aromatase activity or the transcriptional activity was constructed by using this H295R cell, and the influence of the environmental chemicals on the estrogen production was examined. Mono-(2-ethylhexyl) phthalate (MEHP), a main metabolite of di-(2-ethylhexyl) phthalate (DEHP) which is widely used as plasticizers for polyvinylchloride plastics suppressed aromatase activity in a dose-dependent manner and its transcription level, which are up-regulated with FSK. It was suggested as action mechanism that the suppression of aromatase activity and the transcription level by exposure of NCI-H295R cells to MEHP was regulated through the rapid and transient expression of orphan nuclear receptor Nur77 gene.