Estrogenic Activity of Structurally Distinct PFASs Through Estrogen Receptor Pathway

Perfluorooctanoic acid (PFOA), perfluorooctane sulfonate (PFOS), and perfluorohexane sulfonic acid (PFHxS) have been listed under the Stockholm Convention and are being progressively phased out worldwide; environmental prevalence and human exposure to other poly- and perfluoroalkyl substances (PFASs) with different structures are on the rise. However, the toxic effects and mechanisms of other PFASs remain unclear. This study explored and compared the estrogenic effects and underlying mechanisms of eight typical PFASs. PFOA, PFOS, PFHxS, and perfluorooctane sulfonamide (PFOSA) promoted the proliferation of MCF-7 cells, with PFOSA exhibiting the strongest estrogenic effect among the eight targeted PFASs. Specifically, for the PFASs with the same number of carbons and different functional groups, the estrogenic effect is in the decreasing order of PFOSA (sulfonamide group) > PFOS (sulfonic acid group) > PFOA (carboxylic acid group). The observed enhancement was likely caused by estrogen receptor (ER) activation, as an ER antagonist significantly inhibited the pro-proliferation effect. Luciferase reporter gene assays, fluorescence competitive binding assays, and molecular docking simulation were used to furnish supplementary evidence for the direct action of PFASs on the ER. PFOA, PFHxS, PFOS, and PFOSA can activate ER transcriptional activity and bind to the ER, demonstrating that ER is a potential target of PFASs. For PFOA, PFOS, and PFOSA, the rank order of binding affinities with ER in fluorescence competitive binding assays and the calculated binding energy in molecular docking simulation were consistent with the results of the cell proliferation experiment and luciferase reporter gene assays, suggesting that receptor binding affinity also plays a critical role in the activation effect of the ER pathway. Overall, our results indicate the estrogenic effects of other PFASs through ER-mediated pathways and elucidate the molecular mechanism for the different activity of eight PFASs, as well as suggest that PFOS precursors require more attention when determining the environmental risk of PFASs.