Bisphenol A/Dibutyl Phthalate/Perfluorooctanoic Acid Ternary Mixture Exerted Additive Estrogenic Effects through ERα Activation

Environmental estrogens are commonly encountered as mixtures, yet the mechanism by which structurally diverse estrogenic chemicals produce combined estrogenic effects remains insufficiently understood. In this study, a ternary mixture of bisphenol A (BPA), dibutyl phthalate (DBP), and perfluorooctanoic acid (PFOA) at a biomonitoring-informed mass ratio was used as an environmental estrogen mixture model to investigate the combined estrogenic effect and underlying molecular mechanism. BPA, DBP, and PFOA individually exhibited estrogenic effects by promoting MCF-7 cell proliferation with distinct potencies, and the mixture induced a concentration-dependent response with an EC50 of 4.6 μM. The dose–response behavior of the mixture fell within the acceptable additivity range for both the concentration addition (CA) and response addition (RA) models, with a slight trend toward the CA model, indicating an additive estrogenic effect of the mixture. In the ER transcriptional activity assay, the three compounds and the mixture all induced dose-dependent responses, with EC50 values of 1.3, 8.3, 15.8, and 3.6 μM, respectively. Additionally, in the cell-free biosensor measurement, the three compounds and the mixture all demonstrated ERα agonistic activity, with EC50 values of 0.01, 0.15, 0.24, and 0.08 μM, respectively. The results of the transcriptional activity assay and biosensor measurement were both fitted better with the CA model, indicating the central role of ERα activation in inducing the additive effect of the mixture. This study provides mechanistic support for understanding the combined estrogenic effects of environmental estrogen mixtures and highlights the importance of receptor activation in mixture toxicity assessment.