Typical environmental endocrine-disrupting chemicals (EDCs) such as estradiol valerate (EV), diethylstilbestrol (DES), di-2-ethylhexyl phthalate (DEHP), mono-2-ethylhexyl phthalate (MEHP), and bisphenol A (BPA) have a strong reproductive and developmental toxicity at low concentrations. However, information on their joint toxicity is scarce. In this study, we evaluated the combined effects of EV and other four EDCs (DES, DEHP, MEHP, and BPA) on the human breast MCF-7 cells by detecting the cell proliferation, intracellular reactive oxygen species (ROS) levels, and estrogen receptor alpha (ERα) protein expression using equal concentration ratio method. The results showed that, after exposure for 24, 48, and 72 h, single EV, DES, and BPA can promote the proliferation of MCF-7 human breast cancer cells, and EV has the strongest effect in inducing cell proliferation. DEHP and MEHP cannot induce MCF-7 cell proliferation for all exposure time, while cell proliferation induced by EV was significantly attenuated by DES, BPA, DEHP, and MEHP when they mixed with EV. For intracellular ROS, single EV, BPA, DES, DEHP, and MEHP elevated intracellular ROS levels for different exposure time. Similar to the cell proliferation, DES and BPA decreased intracellular ROS levels induced by EV when they mixed with EV for 24 h. EV, DES, and BPA exposed alone or combined with EV upregulated the ERα protein expression. However, DEHP and MEHP exposed alone or combined with EV had no effect on ERα protein expression, indicating that DEHP or MEHP could attenuate ERα protein expression upregulated by EV. These results showed that the joint toxicity of binary mixtures of EV and other EDCs do not interact in a synergistic fashion in inducing cell proliferation, intracellular ROS levels, and ERα protein expression. These findings have important implications in the human risk assessments of EV mixed with other EDCs in the environment.

中文翻译：

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使用体外生物测定法进行内分泌干扰化学物质二元混合物的相互作用试验

典型的环境内分泌干扰化学品 (EDC)，如戊酸雌二醇 (EV)、己烯雌酚 (DES)、邻苯二甲酸二-2-乙基己酯 (DEHP)、邻苯二甲酸单-2-乙基己酯 (MEHP) 和双酚 A (BPA)在低浓度下有很强的生殖和发育毒性。然而，关于它们的关节毒性的信息很少。在本研究中，我们通过检测细胞增殖、细胞内活性氧 (ROS) 水平和雌激素来评估 EV 和其他四种 EDC（DES、DEHP、MEHP 和 BPA）对人乳腺 MCF-7 细胞的联合作用受体α (ERα) 蛋白表达使用等浓度比法。结果表明，暴露24、48、72 h后，单一EV、DES和BPA均能促进MCF-7人乳腺癌细胞增殖，EV在诱导细胞增殖方面的作用最强。DEHP 和 MEHP 不能在所有暴露时间内诱导 MCF-7 细胞增殖，而当它们与 EV 混合时，由 EV 诱导的细胞增殖被 DES、BPA、DEHP 和 MEHP 显着减弱。对于细胞内 ROS，单个 EV、BPA、DES、DEHP 和 MEHP 会在不同的暴露时间提高细胞内 ROS 水平。与细胞增殖类似，DES 和 BPA 与 EV 混合 24 小时后，可降低 EV 诱导的细胞内 ROS 水平。EV、DES 和 BPA 单独暴露或与 EV 联合暴露上调 ERα 蛋白表达。然而，DEHP 和 MEHP 单独暴露或与 EV 结合对 ERα 蛋白表达没有影响，表明 DEHP 或 MEHP 可以减弱 EV 上调的 ERα 蛋白表达。这些结果表明，EV 和其他 EDC 的二元混合物的联合毒性在诱导细胞增殖、细胞内 ROS 水平和 ERα 蛋白表达方面不会以协同方式相互作用。这些发现对 EV 与环境中其他 EDC 混合的人类风险评估具有重要意义。