Triclocarban and triclosan promote breast cancer progression in vitro and in vivo via activating G protein-coupled estrogen receptor signaling pathways.

Triclocarban (TCC) and triclosan (TCS) have been detected ubiquitously in human body and evoked increasing concerns. This study aimed to reveal the induction risks of TCC and TCS on triple negative breast cancer through non-genomic GPER-mediated signaling pathways. Molecular simulation indicated that TCC exhibited higher GPER binding affinity than TCS theoretically. Calcium mobilization assay displayed that TCC/TCS activated GPER signaling pathway with the lowest observed effective concentrations (LOEC) of 10 nM/100 nM. TCC and TCS also upregulated MMP-2/9, EGFR, MAPK3 but downregulated MAPK8 via GPER-mediated signaling pathway. Proliferation assay showed that TCC/TCS induced 4 T1 breast cancer cells proliferation with the LOEC of 100 nM/1000 nM. Wound-healing and transwell assays showed that TCC/TCS promoted 4 T1 cells migration in a concentration-dependent manner with the LOEC of 10 nM. The effects of TCC on breast cancer cells proliferation and migration were stronger than TCS and both were regulated by GPER. TCC/TCS induced migratory effects were more significantly than proliferative effect. Mechanism study showed that TCC/TCS downregulated the expression of epithelial marker (E-cadherin) but upregulated mesenchymal markers (snail and N-cadherin), which was reversed by GPER inhibitor G15. These biomarkers results indicated that TCC/TCS-induced 4 T1 cells migration was a classic epithelial to mesenchymal transition mechanism regulated by GPER signaling pathway. Orthotopic tumor model verified that TCC promoted breast cancer in-situ tumor growth and distal tissue metastasis via GPER-mediated signaling pathway at human-exposure level of 10 mg/kg/d. TCC-induced tissue metastasis of breast cancer was more significantly than in-situ tumor growth. Overall, we demonstrated for the first time that TCC/TCS could activate the GPER signaling pathways to induce breast cancer progression.

Triclocarban exhibits higher adipogenic activity than triclosan through peroxisome proliferator-activated receptors pathways.

Previous epidemiological and animal studies have showed the lipid metabolic disruption of antimicrobial triclocarban (TCC) and triclosan (TCS). However, the present in vivo researches were mainly devoted to the hepatic lipid metabolism, while the evidence about the impacts of TCC/TCS on the adipose tissue is very limited and the potential mechanism is unclear, especially the molecular initiation events. Moreover, little is known about the toxic difference between TCC and TCS. This study aimed to demonstrate the differential adipogenic activity of TCC/TCS as well as the potential molecular mechanism via peroxisome proliferator-activated receptors (PPARα/ß/γ). The in vitro experiment based on 3T3-L1 cells showed that TCC/TCS promoted the differentiation of preadipocytes into mature adipocytes at nanomolar to micromolar concentrations, which was approach to their human exposure levels. We revealed for the first time by reporter gene assay that TCC could activate three PPARs signaling pathways in a concentration-dependent manner, while TCS only activate PPARß. The molecular docking strategy was applied to simulate the interactions of TCC/TCS with PPARs, which explained well the different PPARs activities between TCC and TCS. TCC up-regulated the mRNA expression of three PPARs, but TCS only up-regulated PPARß and PPARγ significantly. Meanwhile, TCC/TCS also promoted the expression of adipogenic genes targeted by PPARs to different extent. The cellular and simulating studies demonstrated that TCC exerted higher adipogenic effects and PPARs activities than TCS. Our mice in vivo experiment showed that TCC could lead to adipocyte size increase, adipocyte lipid accumulation growing, fat weight and body weight gain at human-related exposure levels, and high fat diet exacerbated these effects. Moreover, male mice tended to be more susceptible to TCC induced obesogenic effect than female mice. This work highlights the potential obesogenic risks of TCC/TCS via PPARs signaling pathways, and TCC deserves more concerns for its higher activity.

Triclocarban and triclosan exacerbate high-fat diet-induced hepatic lipid accumulation at environmental related levels: The potential roles of estrogen-related receptors pathways.

Triclosan (TCS) and triclocarban (TCC) have become ubiquitous pollutants detected in human body with concentrations up to hundreds of nanomolar levels. Previous studies about the hepatic lipid accumulation induced by TCS and TCC were focused on pollutant itself, which showed weak or no effects. High-fat diet (HFD), as a known environmental factor contributing to lipid metabolism-related disorders, its synergistic action with environmental pollutants deserves concern. The present study aimed to demonstrate the combined effects and potential molecular mechanisms of TCS and TCC with HFD at cellular and animal levels. The in vitro studies showed that TCC and TCS alone had negligible impact on lipid accumulation in HepG2 cells but induced lipid deposition at nanomolar levels when co-exposure with fatty acid. TCC exhibited much higher induction effects than TCS, which was related to their differential regulatory roles in adipogenic-related genes expression. The in vivo studies showed that TCC had little influence on hepatic lipid accumulation in mice fed with normal diet (ND) but could exacerbate the lipid accumulation in mice fed with HFD. Meanwhile, TCC-induced dyslipidemia in mice fed with HFD was more significant than that fed with ND. Therefore, we speculated that TCC might increase the risk of nonalcoholic fatty liver disease (NAFLD) and atherosclerosis in HFD humans. Molecular mechanism studies showed that TCC and TCS could bind to and activate estrogen-related receptor α (ERRα) and ERRγ as well as regulate their expression. TCC had higher activity on ERRα and ERRγ than TCS, which explained partly the differential regulatory roles of two receptors in the lipid accumulation induced by TCC and TCS. This work revealed synergistic effects and molecular mechanisms of TCC and TCS with excessive fatty acid on the hepatic lipid metabolism, which provided a novel insight into the toxic mechanism of pollutants from the perspective of dietary habits.

Foot and Ankle Motion after Tibiotalocalcaneal Arthrodesis: Comparison with Tibiotalar Arthrodesis Using a Multi-Segment Foot Model.

Background: Tibiotalocalcaneal arthrodesis is an established surgical procedure for treating patients with end-stage ankle joint arthritis and subtalar joint arthritis. Although it greatly relives pain, a major drawback is loss of range of motion. Although it is known to restrict an additional subtalar joint compared to tibiotalar arthrodesis, there is a lack of gait analysis studies comparing the two methods. This study aimed to evaluate the differences in kinematics of the foot and ankle joints between tibiotalar and tibiotalocalcaneal arthrodesis. We also compared preoperative and postoperative statuses for each surgical method. Methods: The study included 12 and 9 patients who underwent tibiotalar and tibiotalocalcaneal arthrodesis, respectively, and 40 healthy participants were included in the control group. The DuPont foot model was used to analyze intersegmental foot and ankle kinematics during gait. Results: Compared to controls, both tibiotalar and tibiotalocalcaneal arthrodesis resulted in slow gait speed with reduced stride length, increased step width, and decreased range of sagittal plane motion. Both fusion methods showed similar range of motion in all segments and planes following surgery. Coronal positions showed more supination of the forefoot and pronation of the hindfoot segment after each operation, particularly tibiotalocalcaneal arthrodesis. Gait after tibiotalocalcaneal arthrodesis did not significantly differ from that after tibiotalar arthrodesis, but there was a tendency of more pronation in the hindfoot segment. Conclusions: Both fusion methods limited foot and ankle motion in similar ways. Comparing tibiotalar and tibiotalocalcaneal arthrodesis suggests that additionally fusing the subtalar joint does not cause greater movement restriction in patients. Objectively comparing tibiotalar and tibiotalocalcaneal arthrodesis will facilitate further understanding of the effect of tibiotalocalcaneal arthrodesis on movement and the value of subtalar joint motion for improved preoperative counselling.

Neonicotinoid insecticides promote breast cancer progression via G protein-coupled estrogen receptor: In vivo, in vitro and in silico studies.

Neonicotinoid insecticides (NIs) have been widely detected in environmental media and human body with concentrations reaching hundreds of nanomolar to micromolar levels. However, the information about their human health toxicology and mechanism is deficient. Previous studies have implied that NIs might exert estrogenic disruption and promote breast cancer progression, but the molecular mechanism is unclear, especially the molecular initiating event. G protein-coupled estrogen receptor (GPER), as a candidate therapeutic target, plays vital roles in the development of breast cancer. This work aimed to reveal the potential mechanism through GPER pathway. Firstly, we screened the activities of seven most common NIs on GPER signal pathway by calcium mobilization assay. Clothianidin, acetamiprid (ACE), and dinotefuran activated GPER most potently and ACE displayed the highest agonistic activity with the lowest observed effective concentration (LOEC) of 1 µM. The molecular docking and dynamics simulation showed favored interaction trend between the NIs and GPER. The three NIs with GPER activity induced 4T1 breast cancer cells migration and ACE showed the highest potency with LOEC of 100 nM. ACE also induced 4T1 cells proliferation at high concentration of 50 µM and up-regulated GPER expression in a dose-dependent manner. We speculated that both the induction effects of ACE on 4T1 cells proliferation and migration might be owing to the activation and up-regulation of GPER. By using 4T1-Luc cells injected orthotopic tumor model, we found that ACE also promoted in-situ breast cancer growth and lung metastasis in normal mouse dependent on GPER. However, ACE only promoted in-situ breast cancer growth through GPER but not lung metastasis in ovariectomized mice, implying that the ACE-induced lung metastasis should be related to endogenous estrogen from ovary. Overall, we demonstrated that NIs promoted breast cancer progression via GPER pathway at human related exposure levels and their female health risks need urgent concerns.

Benzotriazole Ultraviolet Stabilizers Promote Breast Cancer Cell Proliferation via Activating Estrogen-Related Receptors α and γ at Human-Relevant Levels.

Benzotriazole ultraviolet stabilizers (BUVSs) are ubiquitous emerging pollutants that have been reported to show estrogenic disruption effects through interaction with the classic estrogen receptors (ERs) in the fashion of low activity. The present study aims at revealing the potential disruption mechanism via estrogen-related receptors α and γ (ERRα and ERRγ) pathways. By the competitive binding assay, we first found that BUVSs bond to ERRγ ligand binding domain (ERRγ-LBD) with Kd ranging from 0.66 to 19.27 µM. According to the results of reporter gene assays, the transcriptional activities of ERRα and ERRγ were promoted by most tested BUVSs with the lowest observed effective concentrations (LOEC) from 10 to 100 nM, which are in the range of human exposure levels. At 1 µM, most tested BUVSs showed higher agonistic activity toward ERRγ than ERRα. The most effective two BUVSs promoted the MCF-7 proliferation dependent on ERRα and ERRγ with a LOEC of 100 nM. The molecular dynamics simulation showed that most studied BUVSs had lower binding free energy with ERRγ than with ERRα. The structure-activity relationship analysis revealed that molecular polarizability, electron-donating ability, ionization potential, and softness were the main structural factors impacting the binding of BUVSs with ERRγ. Overall, our results provide novel insights into the estrogenic disruption effects of BUVSs.

The potential endocrine disruption mechanism of anthelmintic drug niclosamide by activating estrogen receptors and estrogen-related receptors.

Niclosamide (NIC), a helminthic drug used widely for controlling schistosomiasis, can reportedly disrupt the endocrine system. However, its underlying mechanisms are still unclear. In this study, we revealed the potential endocrine disruption mechanism of NIC by activating estrogen receptors (ERs) and estrogen-related receptors (ERRs). The binding potency of NIC with ERα, ERß and ERRγ were determined by fluorescence competitive binding assays, which shows an IC50 (the concentration of NIC needed to displace 50 % of the probe from the receptor) of 90 ± 4.1, 10 ± 1.7 nM and 0.59 ± 0.07 nM respectively. The IC50 for ERRγ is the lowest one among the three detected receptors, which is three orders of magnitude lower than the known agonist GSK4716.The transcriptional activities of NIC on ERs and ERRs were detected by MVLN cells (stably transfected with ERs reporter gene) and HeLa cells (transiently transfected with ERRs reporter gene)-based luciferase reporter gene assay. The lowest observable effective concentration (LOEC) ranked as follows: ERRγ (0.5 nM) < ERRα (10 nM) < ERs (100 nM). The maximum observed induction rate for ERRγ (294 %) was higher than that for ERRα (191 %). The maximum observed induction rate of NIC for ERs was 30 % relative to 17ß-estradiol. In addition, we simulated the interactions of NIC with ERs and ERRs by molecular docking. NIC could dock into the ligand binding pockets of ERs and ERRs and form hydrogen bonds with different amino acids. The binding energy ranked as follows: ERRγ (-8.90 kcal/mol) < ERß (-7.57 kcal/mol) < ERRα (-7.15 kcal/mol) < ERα (-6.53 kcal/mol), which implied that NIC bound to ERRγ with higher binding affinity than the other receptors. Overall, we clarify that ERRγ might be the dominant target for NIC in cells rather than ERRα and ERs. We reveal potential novel mechanisms for the endocrine disruption effects of NIC by activating both ERRs and ERs at environmentally-related nanomolar levels.

Bioaccumulation and human health risk assessment of DDT and its metabolites (DDTs) in yellowfin tuna (Thunnus albacares) and their prey from the South China Sea.

DDTs were detected in yellowfin tuna (Thunnus albacares, 92.1-221.8 ng‧g-1 lipid weight) and their prey (54.9-93.5 ng‧g-1 lipid weight) from the South China Sea (SCS). DDT levels reported in this study were lower than those of the previous studies indicated the recent mitigation of DDT contamination in the SCS. Higher DDT levels were observed in fat abdominal muscle than lean dorsal muscle in adult yellowfin tuna. Meanwhile, DDT levels in adult yellowfin tuna were higher than the young ones. The composition profiles of DDT and its metabolites suggested DDTs in fish in the SCS were mainly derived from the historical use of technical DDTs. DDTs were biomagnified through food chains with the trophic magnification factor of 2.5. Risk assessment results indicated that dietary exposure to DDTs through lifetime fish consumption from the SCS would pose little cancer and noncarcinogenic risk to coastal residents.

Simultaneous removal of iron and manganese from acid mine drainage by acclimated bacteria.

A bacterial consortium for efficient decontamination of high-concentration Fe-Mn acid mine drainage (AMD) was successfully isolated. The removal efficiencies of Fe and Mn were effective, reaching 99.8 % and 98.6 %, respectively. High-throughput sequencing of the 16S rRNA genes demonstrated that the microbial community had changed substantially during the treatment. The Fe-Mn oxidizing bacteria Flavobacterium, Brevundimonas, Stenotrophomonas and Thermomonas became dominant genera, suggesting that they might play vital roles in Fe and Mn removal. Moreover, the pH of culture increased obviously after incubation, which was benefit for depositing Fe and Mn from AMD. The specific surface area of the biogenic Fe-Mn oxides was 108-121 m2/g, and the surface contained reactive oxygen functional groups (-OH and -COOH), which also improved Fe and Mn removal efficiency. Thus, this study provides an alternative method to treat AMD containing high concentrations of Fe and Mn.

Perfluoroalkyl Substances Stimulate Insulin Secretion by Islet ß Cells via G Protein-Coupled Receptor 40.

The potential causal relationship between exposure to environmental contaminants and diabetes is troubling. Exposure of perfluoroalkyl substances (PFASs) is found to be associated with hyperinsulinemia and the enhancement of insulin secretion by islet ß cells in humans, but the underlying mechanism is still unclear. Here, by combining in vivo studies with both wild type and gene knockout mice and in vitro studies with mouse islet ß cells (ß-TC-6), we demonstrated clearly that 1 h exposure of perfluorooctanesulfonate (PFOS) stimulated insulin secretion and intracellular calcium level by activating G protein-coupled receptor 40 (GPR40), a vital free fatty acid regulated membrane receptor on islet ß cells. We further showed that the observed effects of PFASs on the mouse model may also exist in humans by investigating the molecular binding interaction of PFASs with human GPR40. We thus provided evidence for a novel mechanism for how insulin-secretion is disrupted by PFASs in humans.

Experimental and theoretical insights into kinetics and mechanisms of hydroxyl and sulfate radicals-mediated degradation of sulfamethoxazole: Similarities and differences.

Hydroxyl radical (•OH)- and sulfate radical ()-based advanced oxidation technologies (AOTs) have been proven an effective method to remove antibiotics in wastewater treatment plants (WWTPs). This study aims to gain insights into kinetics and mechanisms of neutral sulfamethoxazole (SMX) degradation, a representative antibiotic, by •OH and using an experimental and theoretical approach. First, the second-order rate constants (k) of SMX with •OH and were determined to be (7.27 ± 0.43) × 109 and (2.98 ± 0.32) × 109 M-1 s-1 in UV/H2O2 and UV/persulfate (UV/PS) systems, respectively. The following theoretical calculations at the M06-2X level of theory revealed that addition of radicals to the benzene ring is the most favorable first-step reaction for both •OH and , but that exhibits higher energy barriers and selectivity than •OH due to steric hindrance. We further analyzed subsequent reactions and, interestingly, our findings closely corroborated HOMO/LUMO distributions of SMX to the oxidation pathways. Finally, the estimation of energy consumption for UV alone, •OH-, and -mediated oxidation processes was compared. These comparative results, for the first time, provide insights into the similarities and differences of degradation of SMX by •OH/ at the molecular level and can help improve antibiotics removal using radical based AOTs in WWTPs.

Estrogen-related receptor γ is a novel target for Lower-Chlorinated Polychlorinated Biphenyls and their hydroxylated and sulfated metabolites.

Airborne lower-chlorinated PCBs are vulnerable to metabolization to PCB sulfates through further sulfation of the hydroxylated metabolites (OH-PCBs). However, studies on the toxic effects and mechanisms of PCB sulfates are still very limited. Here, we investigated for the first time the potential endocrine disruption effects of PCB sulfates through estrogen-related receptor γ (ERRγ) in comparison with their OH-PCBs precursors and PCB parent compounds. The binding affinity of thirteen PCBs/OH-PCBs/PCB sulfates was measured by using fluorescence competitive binding assays based on fluorescence polarization (FP). All of the tested chemicals could bind to ERRγ with the Kd (dissociation constant) values ranging from not available (NA) to 3.2 µM 4'-OH-PCB 12 showed the highest binding affinity with Kd value of 3.2 µM, which was comparable to that of a synthetic ERRγ agonist GSK4716. The effects of the thirteen chemicals on the ERRγ transcriptional activity were determined by using the luciferase reporter gene assay. We found the PCBs/OH-PCBs/PCB sulfates acted as agonists for ERRγ, with the lowest observed effective concentration reaching 3 µM. The binding affinity and agonistic activity of PCBs towards ERRγ were both enhanced after hydroxylation, while further sulfation of OH-PCBs decreased the activity instead. Molecular docking simulation showed that OH-PCBs had lower binding energy than the corresponding PCBs and PCB sulfates, indicating that OH-PCBs had higher binding affinity theoretically. In addition, OH-PCBs could form hydrogen bonds with amino acids Glu316 and Arg247 while PCBs and PCB sulfates could not, which might be the main factor impacting the binding affinity and agonistic activity. Overall, ERRγ is a novel target for lower-chlorinated PCBs and their metabolites.

Investigation of binding and activity of perfluoroalkyl substances to the human peroxisome proliferator-activated receptor ß/δ.

Previously, perfluoroalkyl substances (PFASs) have been found to be associated with many adverse effects mediated by the peroxisome proliferator-activated receptor α (PPARα) and PPARγ. Here, we found another subtype of the peroxisome proliferator-activated receptors (PPARs); the PPARß/δ mediated pathway might also be a potential adverse outcome pathway for PFASs. We investigated the direct binding and transcriptional activity of PFASs toward human PPARß/δ, and further revealed the structure-binding and structure-activity relationship between PFASs and PPARß/δ. The receptor binding experiment showed that their binding potency was dependent on the carbon chain length and the terminal functional group. For twelve perfluoroalkyl carboxylic acids (PFCAs), an inverted U-shaped relationship existed between the PPARß/δ binding potency and the carbon chain length, with perfluorododecanoc acid (C12) showing the highest binding potency. The three perfluoroalkane sulfonic acids (PFSAs) exhibited a stronger binding potency than their PFCA counterparts. The two fluorotelomer alcohols (FTOHs) showed no binding potency. In receptor transcriptional activity assays, they enhanced the PPARß/δ transcriptional activity. Their transcriptional activity was also related to the carbon chain length and the terminal functional group. Molecular docking analysis showed the PFASs fitted into the ligand binding pocket of PPARß/δ with a binding geometry similar to a fatty acid.

Structure-Dependent Activity of Polybrominated Diphenyl Ethers and Their Hydroxylated Metabolites on Estrogen Related Receptor γ: in Vitro and in Silico Study.

Estrogen-related receptor γ (ERRγ) is an orphan nuclear receptor having functional cross-talk with classical estrogen receptors. Here, we investigated whether ERRγ is a potential target of polybrominated diphenyl ethers (PBDEs) and their hydroxylated metabolites (OH-PBDEs). By using a fluorescence competitive binding method established in our laboratory, the binding potencies of 30 PBDEs/OH-PBDEs with ERRγ were determined for the first time. All of the tested OH-PBDEs and some PBDEs bound to ERRγ with Kd values ranging from 0.13-13.61 µM. The OH-PBDEs showed much higher binding potency than their parent PBDEs. A quantitative structure-activity relationship (QSAR) model was developed to analyze the chemical binding potencies in relation to their structural and chemical characteristics. The QSAR model indicated that the molecular size, relative ratios of aromatic atoms, and hydrogen bond donors and acceptors were crucial factors for PBDEs/OH-PBDEs binding. By using a reporter gene assay, we found that most of the low-brominated PBDEs/OH-PBDEs exerted agonistic activity toward ERRγ, while high-brominated PBDEs/OH-PBDEs had no effect on the basal ERRγ activity. The docking results showed that the low-brominated PBDEs/OH-PBDEs tended to take an agonistic binding mode while the high-brominated ones tended to take an antagonistic binding mode. Overall, our results suggest ERRγ to be a potential novel target for PBDEs/OH-PBDEs.

Erratum: "Hydroxylated Polybrominated Biphenyl Ethers Exert Estrogenic Effects via Nongenomic G Protein-Coupled Estrogen Receptor Mediated Pathways".

[This corrects the article DOI: https://doi.org/10.1289/EHP2387.].

Hydroxylated Polybrominated Diphenyl Ethers Exert Estrogenic Effects via Non-Genomic G Protein-Coupled Estrogen Receptor Mediated Pathways.

BACKGROUND: Numerous studies have indicated the estrogenic effects of polybrominated diphenyl ethers (PBDEs) and hydroxylated PBDEs (OH-PBDEs). However, the previous mechanistic studies focused on their estrogenic effects through genomic transcriptional activation of estrogen receptors. OBJECTIVE: The present study aimed to investigate the estrogenic effects of PBDEs and OH-PBDEs via nongenomic G protein-coupled estrogen receptor (GPER) pathways. METHODS: The binding affinities of 12 PBDEs and 18 OH-PBDEs with GPER were determined by a fluorescence competitive binding assay in a human breast cancer cell line (SKBR3). Molecular docking was performed to simulate the interactions. Their activities on GPER pathways were investigated by detecting calcium mobilization and cyclic adenosine monophosphate (cAMP) accumulation in SKBR3 cells. The effects on SKBR3 cell migration were investigated using Boyden chamber and wound-healing assays. RESULTS: Our results showed that 11 of the OH-PBDEs but none of the PBDEs bound to GPER directly. Relative binding affinities ranged from 1.3% to 20.0% compared to 17ß-estradiol. Docking results suggested that the hydroxyl group played an essential role in the binding of OH-PBDEs to GPER by forming hydrogen bond interactions. Most of the OH-PBDEs activated subsequent GPER signaling pathways. Among them, 4'-OH-BDE-049, 5'-OH-BDE-099, and 3'-OH-BDE-154 displayed the highest activity with lowest effective concentrations (LOECs) of 10-100 nM. These three OH-PBDEs also promoted SKBR3 cell migration via GPER pathways with LOECs of 0.1-1 µM. CONCLUSION: OH-PBDEs could bind to GPER, activate the subsequent signaling pathways, and promote SKBR3 cell migration via GPER pathways. OH-PBDEs might exert estrogenic effects by a novel nongenomic mechanism involving the activation of GPER at nanomolar concentrations. https://doi.org/10.1289/EHP2387.

Chlorinated Polyfluorinated Ether Sulfonates Exhibit Higher Activity toward Peroxisome Proliferator-Activated Receptors Signaling Pathways than Perfluorooctanesulfonate.

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.

Bisphenol AF and Bisphenol B Exert Higher Estrogenic Effects than Bisphenol A via G Protein-Coupled Estrogen Receptor Pathway.

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.

Binding interactions of perfluoroalkyl substances with thyroid hormone transport proteins and potential toxicological implications.

Perfluoroalkyl 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 3×10(-4) 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 2×10(-4) 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.

In vitro assessment of thyroid hormone receptor activity of four organophosphate esters.

Previous animal experiments have implied that organophosphate esters (OPEs) have a disruption effect on the thyroid endocrine system. However, knowledge of the toxicological mechanism remains limited. In this study, the activities of four OPEs have been characterized against the thyroid hormone (TH) nuclear receptor (TR) using two in vitro models, with the aim of evaluating their toxicity mechanisms towards the TR. The results of a TH-dependent cell proliferation assay showed that tris(2-chloro-1-(chloromethyl)ethyl)phosphate (TDCPP) could induce cell growth, while the other three OPEs had no effect. The results of a luciferase reporter gene assay revealed that all four of the OPEs tested in the current study showed agonistic activity towards TRß, with TDCPP being the most potent one. Moreover, molecular docking revealed that all the tested OPEs could fit into the ligand binding pocket of TRß, with TDCPP binding more effectively than the other three OPEs. Taken together, these data suggest that OPEs might disrupt the thyroid endocrine system via a mechanism involving the activation of TR.