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

以原代培养的猪睾丸间质细胞为材料，建立了检测环境化学物质对雄激素分泌影响的体外实验体系。我们发现，有机锡化合物，如三丁基氯化锡（TBT），二丁基二氯化锡和三苯基氯化锡，强烈抑制睾酮的生产水平，在没有细胞毒性的浓度，通过使用该测定系统。TBT暴露抑制细胞内cAMP水平的升高和降低由人绒毛膜促性腺激素或毛喉素（FSK）上调的CYP 17 mRNA水平，阐明了其作用机制。此外，三丁基锡化合物在体内和体外都能抑制17β-HSD的活性，而这种酶是睾酮产生所必需的。雌激素生物合成酶芳香化酶是雌激素生物合成的限速酶，是雌激素产生不可缺少的酶。本实验证实人肾上腺皮质癌细胞系中存在芳香化酶的表达，选用H295 R及其启动子II和1.3进行表达，EGF和PGE_2可激活芳香化酶的转录。然后，通过使用该H295 R细胞构建评估芳香化酶活性或转录活性的体外测定系统，并检查环境化学物质对雌激素产生的影响。邻苯二甲酸二（2-乙基己基）酯（DEHP）的主要代谢产物邻苯二甲酸单（2-乙基己基）酯（MEHP）被广泛用作聚氯乙烯塑料的增塑剂，其以剂量依赖性方式抑制芳香酶活性及其转录水平，这与FSK上调。MEHP对NCI-H295 R细胞芳香化酶活性和转录水平的抑制作用可能是通过孤儿核受体Nur 77基因的快速瞬时表达来调节的。

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.