Exposure to many kinds of bisphenols (BPs) is common, and the effects of BP mixtures may differ from those of individual BPs. Therefore, evaluating combined exposure effects is necessary. Our study evaluated the individual and combined exposure effects of bisphenol A (BPA), bisphenol S (BPS) and bisphenol AF (BPAF) on embryonic development using an embryonic stem cell test (EST) and a concentration additive (CA) model at relatively high doses to uncover the interaction model of the three BPs. Environmentally relevant concentrations were then used to evaluate the possible effects of the individual and combined BPs at actual human exposure levels. Exposure to relatively high-dose BPA, BPS and BPAF inhibited embryonic stem cell differentiation into cardiomyocytes and exhibited weak embryo toxicity. Individually, BPA, BPS and BPAF inhibited endoderm, mesoderm and ectoderm marker expression but enhanced pluripotency marker expression. Combined exposure to BPs had an additive effect on cardiomyocyte differentiation and embryonic stem cell proliferation based on the CA model. Environmentally relevant individual or combined BP doses (10 ng/ml individual BPA, BPS and BPAF doses or 1 ng/ml and 10 ng/ml BP mixture doses) failed to cause oxidative stress, DNA damage or apoptosis changes in stem cell differentiation. The cardiomyocyte differentiation ratio also did not change significantly. Individual and combined exposure to environmentally relevant BP doses led to a significant increase in collagen expression. BPAF and the combination of BPs increased the type 1 collagen level, while the combination also increased the type 3 collagen level, which may be related to p38 pathway activation. The p38 pathway inhibitor SB203580 inhibited the increase in collagen during cardiomyocyte differentiation caused by low-dose BPs. These results suggest that relatively high-dose BPs in combination have an additive effect on cardiomyocyte differentiation. Low-dose BPs individually and in combination may affect cardiomyocyte collagen through the p38 pathway.

暴露于多种双酚 (BP) 是常见的，并且 BP 混合物的影响可能与单个 BP 的影响不同。因此，评估组合曝光效果是必要的。我们的研究使用胚胎干细胞测试 (EST) 和浓度添加剂 (CA) 模型在相对较高的条件下评估了双酚 A (BPA)、双酚 S (BPS) 和双酚 AF (BPAF) 对胚胎发育的个体和联合暴露影响。剂量以揭示三个 BP 的相互作用模型。然后使用与环境相关的浓度来评估个体和组合 BP 在实际人类暴露水平下的可能影响。暴露于相对高剂量的 BPA、BPS 和 BPAF 会抑制胚胎干细胞分化为心肌细胞，并表现出较弱的胚胎毒性。BPA、BPS 和 BPAF 分别抑制内胚层，中胚层和外胚层标记表达，但增强多能性标记表达。基于 CA 模型，联合暴露于 BPs 对心肌细胞分化和胚胎干细胞增殖具有累加作用。环境相关的单独或联合 BP 剂量（10 ng/ml 单独 BPA、BPS 和 BPAF 剂量或 1 ng/ml 和 10 ng/ml BP 混合物剂量）未能在干细胞分化中引起氧化应激、DNA 损伤或细胞凋亡变化。心肌细胞分化比率也没有显着变化。单独和联合暴露于与环境相关的 BP 剂量导致胶原蛋白表达显着增加。BPAF和BPs的组合增加了1型胶原蛋白的水平，而组合也增加了3型胶原蛋白的水平，这可能与p38途径的激活有关。p38 通路抑制剂 SB203580 抑制低剂量 BPs 引起的心肌细胞分化过程中胶原蛋白的增加。这些结果表明，相对高剂量的 BP 组合对心肌细胞分化具有累加作用。单独和组合使用低剂量 BP 可能通过 p38 途径影响心肌细胞胶原蛋白。