Zhang Yun-Hui, Lin Ling, Liu Zhi-Wei, Jiang Xue-Zhi, Chen Bing-Heng
Department of Environmental Health, School of Public Health, Fudan University, Shanghai 200032, People's Republic of China.
Environ Toxicol. 2008 Jun;23(3):302-8. doi: 10.1002/tox.20343.
Phthalates are suspect environmental endocrine disruptors that may affect male reproduction and development by disturbing androgen synthesis and cell-cell interactions in the seminiferous epithelium. The in vivo metabolites, monophthalates, are thought to be the active agents, and toxicant effects including testicular damage and decreased sperm motility have been described previously. In this study, the aim was to investigate the effect of monophthalates on Sertoli cells using a two-compartment cell culture model, asking whether tight junction protein structures are affected, compromising the blood-testis barrier and contributing to male-mediated toxicity. Sertoli cells were isolated from Sprague Dawley rat testes and seeded onto the filters of two-compartment wells. A Sertoli cell monolayer was allowed to form, whereupon the cultures were treated with 0, 10, 30, 150, and 600 micromol/L monobutyl phthalate (MBP) or mono-2-ethylhexyl phthalate (MEHP) for 24 h. Effects on the tight junctions between adjacent Sertoli cells were studied by light and transmission electron microscopy, the transepithelial electrical resistance (TEER) assay, and immunofluorescence localization. Results showed that exposures to monophthalates destroyed tight junctional structure in Sertoli cell monolayers in a dose-depended manner, as evidenced by a loss of single-cell layer organization in the cultures, decline of TEER value, and decreased expression of proteins associated with tight junctions such as zonula occludens-1 (ZO-1), F-actin, and Occludin. The changes were observed at doses of 150 and 600 micromol/L, which is 10-100 times higher relative to estimated human exposures from the environment. These results are consistent with monophthalate-induced damage to tight junctions between adjacent Sertoli cells, suggesting that damage to Sertoli cell tight junctions induced by monophthalates may be an underlying mechanism of their male-mediated reproductive toxicity.
邻苯二甲酸盐被怀疑是环境内分泌干扰物，可能通过干扰雄激素合成以及生精上皮中的细胞间相互作用来影响男性生殖和发育。其体内代谢产物单邻苯二甲酸盐被认为是活性物质，此前已有包括睾丸损伤和精子活力下降在内的毒性作用的相关描述。在本研究中，目的是使用两室细胞培养模型研究单邻苯二甲酸盐对支持细胞的影响，探究紧密连接蛋白结构是否受到影响，从而损害血睾屏障并导致雄性介导的毒性。从斯普拉格-道利大鼠睾丸中分离出支持细胞，并接种到两室孔的滤膜上。待形成支持细胞单层后，用0、10、30、150和600微摩尔/升的邻苯二甲酸单丁酯（MBP）或邻苯二甲酸单（2-乙基己基）酯（MEHP）处理培养物24小时。通过光学显微镜和透射电子显微镜、跨上皮电阻（TEER）测定以及免疫荧光定位研究对相邻支持细胞之间紧密连接的影响。结果表明，暴露于单邻苯二甲酸盐会以剂量依赖的方式破坏支持细胞单层中的紧密连接结构，培养物中单细胞层结构丧失、TEER值下降以及紧密连接相关蛋白如闭合蛋白-1（ZO-1）、F-肌动蛋白和闭合蛋白表达降低均证明了这一点。在150和600微摩尔/升的剂量下观察到了这些变化，这比估计的人类环境暴露量高10至100倍。这些结果与单邻苯二甲酸盐诱导的相邻支持细胞之间紧密连接的损伤一致，表明单邻苯二甲酸盐诱导的支持细胞紧密连接损伤可能是其雄性介导的生殖毒性的潜在机制。
