Abstract

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 combinat