Chlorinated polyfluorinated ether sulfonates (Cl-PFAESs) are the alternative products of perfluorooctanesulfonate (PFOS) in the metal plating industry in China. The similarity in chemical structures between Cl-PFAESs and PFOS makes it reasonable to assume they possess similar biological activities. In the present study, we investigated whether Cl-PFAESs could induce cellular effects through peroxisome proliferator-activated receptors (PPARs) signaling pathways like PFOS. By using fluorescence competitive binding assay, we found two dominant Cl-PFAESs (6:2 Cl-PFAES and 8:2 Cl-PFAES) bound to PPARs with affinity higher than PFOS. Based on the luciferase reporter gene transcription assay, the two Cl-PFAESs also showed agonistic activity toward PPARs signaling pathways with potency similar to (6:2 Cl-PFAES) or higher than (8:2 Cl-PFAES) PFOS. Molecular docking simulation showed the two Cl-PFAESs fitted into the ligand binding pockets of PPARs with very similar binding mode as PFOS. The cell function results showed Cl-PFAESs promoted the process of adipogenesis in 3T3-L1 cells with potency higher than PFOS. Taken together, we found for the first time that Cl-PFAESs have the ability to interfere with PPARs signaling pathways, and current exposure level of 6:2 Cl-PFAES in occupational workers has exceeded the margin of safety. Our study highlights the potential health risks of Cl-PFAESs as PFOS alternatives.

A B S T R A C TPerfluoroalkyl substances (PFASs) have been shown to cause abnormal levels of thyroid hormones (THs) in experimental animals, but the molecular mechanism is poorly understood. Here, a fluorescence displacement assay was used to determine the binding affinities of 16 PFASs with two major TH transport proteins, transthyretin (TTR) and thyroxine-binding globulin (TBG). Most of the tested PFASs bound TTR with relative potency (RP) values of 3 Â 10 À4 to 0.24 when compared with that of the natural ligand thyroxine, whereas fluorotelomer alcohols did not bind. Only perfluorotridecanoic acid and perfluorotetradecanoic acid bound TBG, with RP values of 2 Â 10 À4 when compared with that of thyroxine. Based on these results, it was estimated that displacement of T4 from TTR by perfluorooctane sulfonate and perfluorooctanoic acids would be significant for the occupationally exposed workers but not the general population. Structure-binding analysis revealed that PFASs with a medium chain length and a sulfonate acid group are optimal for TTR binding, and PFASs with lengths longer than 12 carbons are optimal for TBG binding. Three mutant proteins were prepared to examine crucial residues involved in the binding of PFASs to TH transport proteins. TTR with a K15G mutation and TBG with either a R378G or R381G mutation showed decreased binding affinity to PFASs, indicating that these residues play key roles in the interaction with the compounds. Molecular docking showed that the PFASs bind to TTR with their acid group forming a hydrogen bond with K15 and the hydrophobic chain towards the interior. PFASs were modeled to bind TBG with their acid group forming a hydrogen bond with R381 and the hydrophobic chain extending towards R378. The findings aid our understanding of the behavior and toxicity of PFASs on the thyroid hormone system.

Numerous studies have indicated estrogenic disruption effects of bisphenol A (BPA) analogues. Previous mechanistic studies were mainly focused on their genomic activities on nuclear estrogen receptor pathway. However, their nongenomic effects through G protein-coupled estrogen receptor (GPER) pathway remain poorly understood. Here, using a SKBR3 cell-based fluorescence competitive binding assay, we found six BPA analogues bound to GPER directly, with bisphenol AF (BPAF) and bisphenol B (BPB) displaying much higher (∼9-fold) binding affinity than BPA. Molecular docking also demonstrated the binding of these BPA analogues to GPER. By measuring calcium mobilization and cAMP production in SKBR3 cells, we found the binding of these BPA analogues to GPER lead to the activation of subsequent signaling pathways. Consistent with the binding results, BPAF and BPB presented higher agonistic activity than BPA with the lowest effective concentration (LOEC) of 10 nM. Moreover, based on the results of Boyden chamber and wound-healing assays, BPAF and BPB displayed higher activity in promoting GPER mediated SKBR3 cell migration than BPA with the LOEC of 100 nM. Overall, we found two BPA analogues BPAF and BPB could exert higher estrogenic effects than BPA via GPER pathway at nanomolar concentrations.

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In addition to PPARα and PPARγ, PPARβ/δ mediated pathway might also be a potential adverse outcome pathway for PFASs.

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Organophosphate esters (OPEs) have been reported to induce endocrine disruption effects, and several well-known nuclear receptors have been investigated as cellular targets of OPEs in their mode of action. Here, we demonstrated for the first time that an orphan nuclear receptor estrogen-related receptor γ (ERRγ) is another possible target of OPEs. Using the fluorescence competitive binding assays that we established, we measured the binding affinity of nine OPEs with different substitution groups, including aromatic rings, chlorinated alkyl chains, and alkyl chains. Seven of the OPEs were found to bind to ERRγ, with tri-m-cresyl phosphate (TCrP) showing the highest binding affinity (K d, 0.34 μM). By using an ERRγ-mediated luciferase reporter gene assay, we found seven OPEs showed inhibitory effects toward ERRγ. Both the binding affinity and the inhibitory effect of the OPEs correlate positively with the hydrophobicity of their substitution groups in the following rank order: aromatic rings > chlorinated alkyl chains > alkyl chains. On the basis of molecular docking, the mechanism of the inhibitory effect of OPEs was proposed to be ligand-triggered displacement of the activation function-2 helix from the active position in the receptor. The ERRγ pathway may provide a new mechanism for the endocrine disruption effects of OPEs.

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