Tricresyl phosphates (TCPs), as representative aromatic organophosphate flame retardants (OPFRs), have received much attention due to their potential neurotoxicity and endocrine-disrupting effects. However, the role of estrogen receptor α (ERα) and G protein-coupled estrogen receptor (GPER) in their estrogen disrupting effects remains poorly understood. Therefore, in this study, three TCP isomers, tri-o-cresyl phosphate (ToCP), tri-m-cresyl phosphate (TmCP) and tri-p-cresyl phosphate (TpCP), were examined for their activities on ERα by using two-hybrid yeast assay, and action on GPER by using Boyden chamber assay, cAMP production assay, calcium mobilization assay and molecular docking analysis. The results showed that three TCP isomers were found to act as ERα antagonists. Conversely, they had agonistic activity on GPER to promote GPER-mediated cell migration of MCF7 cells and SKBR3 cells. Both ToCP and TpCP activated GPER-mediated cAMP production and calcium mobilization, whereas TmCP had different mode of action, it only triggered GPER-mediated calcium mobilization, as evidenced by using the specific GPER inhibitor (G15) and GPER overexpressing experiments. Molecular docking further revealed that the way of interaction of TmCP and TpCP with GPER was different from that of ToCP with GPER, and higher activity of ToCP in activating GPER-mediated pathways might be associated with the alkyl substitution at the ortho position of the aromatic ring. Our results, for the first time, found a new target, GPER, for TCPs exerting their estrogen-disrupting effects, and demonstrated complex estrogen-disrupting effects of three TCP isomers involved their opposite activities toward ERα and GPER.

磷酸三甲苯酯（TCPs）作为代表性的芳族有机磷酸酯阻燃剂（OPFRs），由于其潜在的神经毒性和破坏内分泌的作用而备受关注。然而，雌激素受体α（ERα）和G蛋白偶联雌激素受体（GPER）在其破坏雌激素作用中的作用仍知之甚少。因此，在这项研究中，通过使用两种方法检测了三个TCP异构体：磷酸三邻甲酚酯（ToCP），磷酸三间甲酚酯（TmCP）和磷酸三对甲酚酯（TpCP）。 -杂交酵母测定，并通过使用博登室测定，cAMP产生测定，钙动员测定和分子对接分析对GPER起作用。结果表明，发现三种TCP异构体充当ERα拮抗剂。反过来，它们对GPER具有激动活性，以促进GPER介导的MCF7细胞和SKBR3细胞的细胞迁移。ToCP和TpCP均激活GPER介导的cAMP产生和钙动员，而TmCP具有不同的作用方式，它仅触发GPER介导的钙动员，这通过使用特定的GPER抑制剂（G15）和GPER过表达的实验证明。分子对接进一步揭示了TmCP和TpCP与GPER的相互作用方式不同于ToCP与GPER的相互作用方式，ToCP在激活GPER介导的途径中的更高活性可能与芳环邻位的烷基取代有关。 。我们的结果首次为TCPs发挥了破坏雌激素的作用，设立了一个新的目标GPER，