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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.

A new chemical toxicity sensor was developed based on the electrochemiluminescence (ECL) quantification of apurinic/apyrimidinic sites (AP sites) in a DNA monolayer with a covalent aldehyde reactive probe (ARP). In the sensor, a uracil-containing DNA duplex was first immobilized on a gold electrode by self-assembly. The duplex was then reacted with uracil-DNA glycosylase (UDG) to convert uracils into AP sites. ARP was employed to tag the AP site with a biotin. After reacting with a ruthenium complex labeled streptavidin, ECL was measured for quantitative analysis. The DNA monolayer was characterized by cyclic voltammetry, electrochemical impedance spectroscopy and chronocoulometry, and its density was measured to be 2.89 × 10−12 mol/cm2. Characterization of the reaction product between ARP and DNA AP sites in solution by nondenaturing polyacrylamide gel electrophoresis and mass spectrometry confirmed successful biotinylation. ECL intensity of the labeled DNA monolayer on the electrode was found to correlate with the number of AP sites, and the detection limit was estimated to be about 1 lesion in 512 DNA bases, which meant that 8.5 fmol AP bases on the electrode were detected. ECL response of the DNA monolayers containing either 8-oxodGuo or methylated bases was very low, indicating that ARP-based AP sites detection method was highly selective. The sensor successfully detected the AP sites in normal DNA induced by methylmethane sulfonate, a carcinogenic chemical. The novel combination of covalent probe and ECL measurement in a sensor configuration therefore provides unique advantages in selectivity and sensitivity, and can be potentially employed in the screening of chemicals for their genetic toxicity.

Ecotoxicity of engineered nanoparticles (NPs) has become the focus of considerable attention because of their wide applications. Reactive oxygen species (ROS) play important roles in the toxicity mechanisms of engineered metal oxide NPs. This work aimed to understand quantitatively the contribution of photogenerated ROS on metal oxide NPs to their toxicity. The dynamic generation of O2•–, •OH, and H2O2 in aqueous suspensions of photoilluminated metal oxide nano- and bulk particles (TiO2, ZnO, V2O5, CeO2, Fe2O3, and Al2O3) was measured by a continuous-flow chemiluminescence (CFCL) detection system. Superoxides were generated on all six nanoparticles as well as bulk TiO2 and ZnO, with nano TiO2 producing the highest concentration (180 nM). Hydroxyl radicals were detected on both nano- and bulk TiO2 and ZnO, whereas H2O2 was detected only on TiO2 and ZnO nanoparticles. The generation of ROS can in general be interpreted by the electronic structures and surface defects of the NPs and the ROS redox potentials....

// Tianyi Dong 1, 2, * , Yanping Peng 1, * , Ning Zhong 3 , Fengyan Liu 3 , Hanyu Zhang 1 , Mengchen Xu 4 , Rutao Liu 4 , Mingyong Han 5 , Xingsong Tian 2 , Jihui Jia 1 , Lap Kam Chang 1 , Liang-Hong Guo 6 and Shili Liu 1 1 School of Medicine, Shandong University, Jinan, Shandong, 250012, China 2 Department of Breast Thyroid Surgery, Shandong Provincial Hospital, Shandong University, Jinan, Shandong, 250021, China 3 Department of Gastroenterology, Qilu Hospital of Shandong University, Jinan, Shandong, 250012, China 4 School of Environmental Science and Engineering, Shandong University, Jinan, Shandong, 250100, China 5 Cancer Therapy and Research Center, Shandong Provincial Hospital, Shandong university, Jinan, Shandong 250021, China 6 State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China * These authors contributed equally to this work Correspondence to: Liang-Hong Guo, email: lhguo@rcees.ac.cn Shili Liu, email: liushili@sdu.edu.cn Keywords: PFDA, proliferation, sPLA2-IIA, TCF4 Received: September 06, 2016 Accepted: April 05, 2017 Published: April 20, 2017 ABSTRACT The association of perfluorodecanoicacid (PFDA) with tumor promotion and associated effects is not clear. Given that PDFA is mostly consumed with food and drinking water, we evaluated the effects of PFDA on a gastric cell line. When added to cell cultures, PFDA significantly increased growth rate and colony forming ability compared with control treatment. We found that suppression of cell senescence, but not apoptosis or autophagy was associated with PFDA-induced promotion of cell amount. To determine the molecular mechanism that was involved, DNA microarray assays was used to analyze changes in gene expression in response to PFDA treatment. Data analysis demonstrated that the vascular endothelial growth factor signaling pathway had the lowest p -value, with sPLA2-IIA ( pla2g2a ) exhibits the most altered expression pattern within the pathway. Moreover, sPLA2-IIA and its transcription factor TCF4, known as a direct target and a binding partner of Wnt/β-catenin signaling in gastric cells respectively, were the third and second most varied genes globally. Cells transfected with expression plasmids pENTER- tcf4 and pENTER- pla2g2a show reduced cell proliferation by more than 60% and 30% respectively. Knockdown with sPLA2-IIA siRNA provided additional evidence that sPLA2-IIA was a mediator of PFDA-induced cell senescence suppression. The results suggest for the first time that PFDA induced suppression of cell senescence through inhibition of sPLA2-IIA protein expression and might increased the proliferative capacity of an existing tumor.

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 β 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 β cells (β-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 β 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.

Chlorinated polyfluoroalkylether sulfonic acids (Cl-PFESAs) have been shown to have potential thyroid hormone (TH) disruption effects. Here, we further investigated their estrogenic effects and underlying mechanisms. In vivo results revealed that exposure of zebrafish to Cl-PFESAs induced disorder of sex hormones during the early embryonic stages and caused histopathological lesions in the gonads of adult zebrafish relative to control groups. To find out whether the estrogen receptor is the molecular target of Cl-PFESAs, the binding interaction between Cl-PFESAs and ERs was investigated using a series of in vitro assays. We found that all tested chemicals could bind directly to ERs and exhibit relatively weak agonistic activity toward ERs, suggesting that the ER-mediated signaling pathway is directly involved in the estrogenic effects of Cl-PFESAs. The internal dose of 8:2 Cl-PFESA was significantly higher than the others, which explained why it obviously displayed an ER agonistic effect despite its weak ER binding affinity. Taken together, these results uncover that, in addition to the TH disruption effect, Cl-PFESAs might also cause estrogenic effects by activating ER pathways.

Microplastic (MP) accumulation in the environment has become an issue of human and environmental importance. Great efforts were made recently to identify the sources of MP exposure to humans and their release into the environment. Here, we employed spectroscopic techniques to identify and characterize MP in consumer plastic food containers that are, in huge quantity, used for food delivery and disposable plastic cups for daily drinking. We determined the average weight of isolated MP per pack to be 12 ± 5.12 mg, 38 ± 5.29 mg, and 3 ± 1.13 mg for the round-shaped, rectangular-shaped plastic container and disposable plastic cups, respectively, with various morphological features including cubic, spherical, rod-like as well as irregular shapes, which may either be consumed by humans or released into the environment. This study demonstrates that new plastic containers can be an important source of direct human and environmental exposure to microplastics. Most importantly, our results indicated that necessary attention must be given to morphological features of realistic MPs when evaluating their risks to humans and the environment.

Carbon nanomaterials (CNMs) are widely used in industrial and medical sectors. The increasing exposure of CNMs necessitates the studies of their potential environmental and health effects. High-mobility group box-1 (HMGB1) is a nuclear DNA-binding protein, but when released from cells, may cause sustained inflammatory response and promote cell migration and invasion. In this work, we found that 7-day exposure of 2.5 mg/kg/day CNMs, including C60, single-walled carbon nanotubes, and graphene oxides significantly elevated the level of HMGB1 in blood and lung lavage fluids in C57BL/6 mice. Subsequently, cellular effects and underlying mechanism were explored by using Raw264.7. The results showed that noncytotoxic CNMs enhanced HMGB1 intracellular translocation and release via activating P2X7 receptor. Released HMGB1 further activated receptor for advanced glycation endproducts (RAGE) and downstream signaling pathway by upregulating RAGE and Rac1 expression. Simultaneously, CNMs prepared the cells for migration and invasion by modulating MMP2 and TIMP2 gene expression as well as cytoskeleton reorganization. Intriguingly, released HMGB1 from macrophages promoted the migration of nearby lung cancer cell, which can be efficiently inhibited by neutralizing antibodies against HMGB1 and RAGE. Taken together, our work demonstrated that CNMs stimulated HMGB1 release and cell migration/invasion through P2X7R-HMGB1-RAGE pathway. The revealed mechanisms might facilitate a better understanding on the inflammatory property and subsequent cell functional alteration of CNMs.

A formation model of O

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 PPARβ/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 PPARβ/δ, and further revealed the structure-binding and structure–activity relationship between PFASs and PPARβ/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 PPARβ/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 PPARβ/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 PPARβ/δ with a binding geometry similar to a fatty acid.

Polybrominated diphenyl ethers (PBDEs) can be metabolized to hydroxylated PBDEs (OH-PBDEs), which play important roles in their disruption effects on the thyroid hormone (TH) system. Recently, multiple in vitro studies suggested that OH-PBDEs might be further metabolically transformed to PBDE sulfates. However, information about the bioactivity of PBDE sulfate metabolites is limited. In the present study, we explored the possible disruption effects of PBDE sulfates to the TH system by studying their binding and activity towards TH transport proteins and nuclear receptors. We found PBDE sulfates could bind to two major TH transport proteins (thyroxine-binding globulin and transthyretin). Besides, PBDE sulfates could also bind to two subtypes of TH nuclear receptors (TRα and TRβ) and showed agonistic activity towards the subsequent signaling pathway. Moreover, the PBDE sulfates showed higher binding potency to TH transport proteins and TRs compared with their corresponding OH-PBDE precursors. Molecular docking results showed that replacement of hydroxyl groups with sulfate groups might lead to more hydrogen bond interactions with these proteins. Overall, our study suggested that PBDE sulfates might disturb the TH system by binding to TH transport proteins or TRs. Our finding indicated a possible mechanism for the TH system disruption effects of PBDEs through their sulfate metabolites.

Reactive oxygen species (ROS) play an essential role in TiO

Bisphenol analogues including bisphenol A and its derivatives are ubiquitous environmental contaminants and have been linked to adverse neurodevelopment effects on animals and humans. Most toxicological research focused on estrogen receptor mediated pathways and did not comprehensively clarify the observed toxicity. O-GlcNAcase (OGA), the highest level in brain, plays a critical role in controlling neuronal functions at multi-levels from molecule to animal behaviors. In this work, we intend to investigate the underlying molecular mechanisms for the neurotoxicity of bisphenol analogues by identifying their cellular targets and the resultant effects. The inhibitory actions of seven bisphenol analogues on the OGA activity at molecular level were investigated by our developed electrochemical biosensor. We found that their potency varied with substituent groups, in which tetrabromo bisphenol A (TBBPA) was the strongest. The seven bisphenol analogues (0–100 μM exposure) significantly inhibited OGA activity and up-regulated protein O-GlcNAcylation level in PC12 cells. Inhibition of OGA by bisphenol analogues further induced intracellular calcium, ROS, inflammation, repressed proliferation, interfered with cell cycle, induced apoptosis. And especially, 10 μM tetrabromo bisphenol A (TBBPA) exposure could impair the growth and development of neurite in human neural stem cells (hNSCs). Molecular docking for OGA/bisphenol analogue complexes revealed the hydrophobicity-dominated inhibition potency. OGA, as a new cellular target of bisphenol analogues, would illuminate the molecular mechanism of bisphenol analogues neurotoxicity.

Coexistence of nanomaterials and environmental pollutants requires in-depth understanding of combined toxicity and underlying mechanism. In this work, we found that coexposure to the mixture of noncytotoxic level of single-walled carbon nanotubes (SWCNTs) (10 μg/mL) and Ni2+ (20 μM) induced significant cytotoxicity in macrophages. However, almost equal amount of intracellular Ni2+ was detected after Ni2+/SWCNT coexposure or Ni2+ single exposure, indicating no enhanced cellular uptake of Ni2+ occurred. SDS-PAGE analysis revealed 50% more SWCNTs retained in Ni2+/SWCNT exposed cells than that with SWCNT exposure alone, regardless of the exposure sequence (coexposure, Ni2+ pre- or post-treatment), suggesting inhibited SWCNT exocytosis by Ni2+. The increased cellular dose of SWCNTs could quantitatively account for the elevated toxicity of Ni2+/SWCNT mixture to cells. It was then found that agonist (ATP) and antagonist (o-ATP) of P2X7R could regulate intracellular SWCNT amount and the cytotoxicity accordingly. ...

Exocytosis of single-walled carbon nanotubes (SWCNTs) determines therapeutic efficiency and toxicity of nanoproducts but its underlying mechanism remains elusive. In this study, it is found that the exocytosis mechanism of SWCNTs is mediated mainly through the activation of P2X7 receptor (P2X7 R), an ATP-gated membrane receptor highly expressed in macrophages. Inhibition of P2X7 R signaling by either a specific inhibitor (oxidized ATP) or small interfering RNA targeting P2X7 R largely prevents the exocytosis of SWCNTs from Raw264.7 cells, resulting in significant accumulation of SWCNTs within cells. In contrast, activation of P2X7 R with ATP promotes exocytosis of SWCNTs. Specifically, it is elucidated that internalized SWCNTs are accumulated in lysosomes and induce transitional release of ATP into extracellular space, which further activates P2X7 R, leading to the influx of calcium ions, phosphorylation of protein kinase C, ERK1/2, p38, and JNK, as well as alkalization of lysosomes. SWCNTs exposure also induces microtubules reorganization that facilitates the secretion of SWCNTs-containing lysosomes. It is also found that P2X7 R simultaneously mediates secretion of IL-1β from Raw264.7 cells during the process of SWCNTs exocytosis. The combined data reveals that P2X7 R-mediated pathway is the predominant molecular mechanism for exocytosis of SWCNTs in Raw264.7 cells. Moreover, SWCNT-induced inflammation is closely coupled with the exocytosis of SWCNTs through P2X7 R.

The environmental behavior of engineered nanomaterials is complex due to their photochemical transformation and interactions with the surrounding aquatic environment. In this report, the aggregation of UV-irradiated TiO2 nanoparticles under different aquatic conditions (ionic strength, ionic composition, and humic acids) was investigated. With increased UV irradiation time and elevated ionic strength, TiO2 aggregation was remarkably accelerated due to the suppression of the electrostatic repulsion between the nanoparticles. Humic acids normally stabilize nanoparticle suspensions in salt solutions by the steric hindrance effect. But the change of TiO2 surface chemistry induced by UV irradiation reduced their adsorption capacity toward humic acids and thus weakened their stabilizing effect. In a simulated aquatic solution with 450 mM NaCl and 10 mg L− 1 SRHA, aggregation rate a TiO2 nanoparticle suspension increased from 5.8 nm min− 1 before UV irradiation to 26.7 nm min− 1 after 40 h irradiation. These results demonstrate the critical role of light irradiation in mediating the environmental behaviors of nanomaterials in aquatic environments.

Pentachlorophenol (PCP), a highly toxic halogenated aromatic compound, and its direct photolysis or TiO2 photocatalysis may generate toxic intermediates and induce secondary pollution in the environment. It is urgently needed to design a strategy to inhibit the toxic intermediates in the photodegradation of PCP. To achieve this, polydopamine (PDA), a non-toxic substance, modified TiO2 (P25/PDA) nanoparticles were synthesized and used to improve the PCP photodegradation process. The dynamic tracking of toxic intermediates tetrachloro-1,4-benzoquinone (TCBQ) and trichlorohydroxy-1,4-benzoquinone (OH-TrCBQ) produced in the PCP photodegradation process were obtained by continuous flow chemiluminescence. Combined with reactive oxygen species (ROS) measurements, P25/PDA could approximatively depress 70 % TCBQ and 40 % OH-TrCBQ generation through the regulation of ROS especially the generation of a fairly large amount of H2O2 (about 30 μM) and O2- (about 20 μM) on the surface of the P25/PDA. The toxicity evaluation showed that the photodegradation of PCP by P25/PDA was a safer and green approach. Therefore, it was instructive to inhibit the formation of highly toxic intermediates in the photodegradation of environmental contaminants by regulating the ROS generated on the surface of the photocatalysts.

The design and synthesis of antibacterial nanomaterials (NM) with improved efficiency, stability, fouling resistance, biocompatibility and recyclability is a fast-developing field. Here we review the recent research directions in the antibacterial NM development for potential applications in the environmental and consumer products, with the focus on the antibacterial mechanisms and novel material synthesis approaches. We identify the major application areas for antibacterial NMs and the types of NMs under research and already in use in the consumer products. The main synthetic approaches for preparation of antibacterial NMs range from surfaces coated with vertically aligned 2D NMs, photocatalytic membranes with extended photoactivation range and composite materials for solar-to-thermal energy conversion to layered nanocomposites and metal-organic frameworks for controlled release of antibacterial compounds. The strategies for increasing antibacterial efficiency of NMs include contact-mediated damage via the edges of 2D NMs, efficient generation of reactive oxygen species (ROS) by photocatalytic composite NMs with or without concurrent mechanical entrapment of bacteria, photothermal damage, and synergistic action of photocatalysts and metal ion release. For the research in antibacterial NM synthesis to successfully progress into the product development phase it is crucial to incorporate NM safety assessment into all steps of the research and development process. Based on the synthesis approaches and antibacterial mechanisms of NMs reviewed herein we discuss considerations for safe-by-design strategies for antibacterial NMs, crucial for the sustainable development of nano-enabled products in the environmental and consumer applications.

As a class of widely used ultraviolet filters, hydroxylated benzophenones (BPs) have been detected in various environmental and human samples, raising concerns about their ecological and health effects. BPs were found to impair reproductive functions, and their disruption effects on estrogen receptor, G protein-coupled estrogen receptor, and androgen receptor were investigated as possible mechanisms of action. In the present study, we investigated the binding and activation of estrogen related receptor γ (ERRγ) as an alternative mechanism of BPs reproductive toxicity. In competitive fluorescence binding assays, all of the 8 tested BPs were found to bind to the ligand-binding domain of ERRγ but with varying binding affinity. Molecular dock analysis indicated that binding of BPs to ERRγ in the agonistic conformation is preferred. In luciferase reporter gene assays in HepG2 cells, all the 8 BPs activated the transcription of ERRγ in a concentration-dependent manner in the range of 3–100 μM,consistent with the docking result. Hazard quotient calculation revealed a high risk on the BP-type personal care products users. The combined in vitro and in silico results suggest that binding and activation of ERRγ might be the molecular initiating events of an alternative mechanism of BPs endocrine disruption toxicity.

Hexafluoropropylene oxide trimer acid (HFPO-TA) and hexafluoropropylene oxide dimer acid (HFPO-DA) have been used as perfluorooctanoic acid (PFOA) alternatives in the fluoropolymer industry for years. Their widespread environmental distribution, high bioaccumulation capability, and human exposure have caused great concern. Nevertheless, their potential toxicity and health risk remain largely unknown. In the present study, we compared potential disruption effects of HFPO-TA, HFPO-DA, and PFOA on peroxisome proliferator-activated receptor γ (PPARγ) via the investigation of receptor binding, receptor activity, and cell adipogenesis effects. The receptor binding experiment showed HFPO-TA exhibited 4.8-7.5 folds higher binding affinity with PPARγ than PFOA, whereas HFPO-DA exhibited weaker binding affinity than PFOA. They also showed agonistic activity toward PPARγ signaling pathway in HEK 293 cells in the order of HFPO-TA > PFOA > HFPO-DA. Molecular docking simulation indicated HFPO-TA formed more hydrogen bonds than PFOA, whereas HFPO-DA formed fewer hydrogen bonds than PFOA. HFPO-TA promoted adipogenic differentiation and lipid accumulation in both mouse and human preadipocytes with potency higher than PFOA. Adipogenesis in human preadipocytes is a more sensitive end point than mouse preadipocytes. Collectively, HFPO-TA exerts higher binding affinity, agonistic activity, and adipogenesis activity than PFOA. The potential health risk of HFPO-TA should be of concern.

Pentachlorophenol (PCP) caused water quality problems owe to its past widespread application and stability, harmful to human health. Photocatalysis, which was mainly involved in the reactive oxygen species (ROS) reaction, has large potential as water treatment process. However, the roles of ROS on the degradation process of PCP are not yet clearly defined. The main objectives of this work were to investigate the roles of ROS involved in the whole degradation of PCP and main toxic intermediates and elucidate the degradation mechanisms. Tetrachloro-1,4-benzo/hydroquinone (TCBQ/TCHQ), trichlorohydroxy-1,4-benzoquinone (OH-TrCBQ) and 2,5-dichloro-3,6-dihydroxy-1,4-benzoquinone (OH-DCBQ) were identified as main intermediates. The roles of generated ROS including OH, O

Heterogeneous catalytic reactions usually proceed at the surfaces of materials, where many intermediates, such as free radicals, usually were believed to be short-lived. Herein, surface long-lived superoxide radicals (O2˙-) were identified in UV-irradiated aqueous suspensions of TiO2 and other metal oxide nanoparticles using an online chemiluminescence system. From the decay dynamics process of O2˙-, a long-lived O2˙- radical was observed on anatase TiO2 at pH = 12. After separation of the photo-excited suspension via filtration, CL was detected from the particles but not the filtrate, thus confirming O2˙- surface adsorption. The unusual stability of O2˙- was also verified using density functional theory (DFT) calculations. The lifetimes of the radicals were estimated on the different kinds of semiconductor surface according to the decay dynamics curves, and followed the order: TiO2ZnOSnO2CeO2Fe2O3. Furthermore, the function of surface long-lived O2˙- in TiO2 suspensions with regards to photochemical conversion was investigated using NBT as a chemical model; it was found that half of the molecules were reduced by the surface-adsorbed O2˙-. The finding of surface-stabilized, long-lived superoxide radicals may have important implications in relation to the chemistry, biology and toxicology of these radicals.

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 ...

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 17β-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, 4ʹ-OH-BDE-049, 5ʹ-OH-BDE-099, and 3ʹ-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 0.1–1μM. 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

Biotransformation of 8:2 fluorotelomer alcohol (8:2 FTOH) can form potentially more toxic metabolites. However, the responsible cytochrome P450 (CYP) isoform(s) and phase II metabolism have not been studied in humans. Here, we characterized the in vitro metabolism of 8:2 FTOH by recombinant human CYPs, human liver microsomes, and human liver cytosol. The results showed that among the 11 isoforms investigated, CYP2C19 was the only enzyme capable of catalyzing 8:2 FTOH with Km and Vmax values of 18.8 μM and 8.52 pmol min−1 pmol−1 P450, respectively. The phase I metabolite was identified as 8:2 fluorotelomer aldehyde (8:2 FTAL). HLMs also catalyzed 8:2 FTOH transformation, with the Vmax and intrinsic clearance (CLint) values similar to those of CYP2C19 after the protein content is taken into account. Molecular docking showed that the hydroxyl group of 8:2 FTOH accesses the heme iron-oxo of CYP2C19 in an energetically favored orientation. 8:2 FTOH was also transformed by phase II enzymes to form O-glucuronide and O-sulfate conjugates. The CLint values follow the order of sulfation > oxidation > glucuronidation, suggesting that conjugation is the major metabolic pathway, which explains the low yield of perfluoroalkyl acids (PFCAs). These results provide new insight into fluorotelomer alcohol biotransformation and indirect human exposure to PFCAs.

Environment-friendly metal-free photocatalysts represent a promising alternative to conventional metal-based semiconductors. In this report, a carbon dots (CDs) decorated graphitic carbon nitride (g-C3N4) photocatalyst was synthesized via a facile impregnation-thermal method. Under visible light irradiation, a very low CDs content of 0.5 web in the g-C3N4/CDs composite resulted in a 3.7 times faster reaction rate for phenol photodegradation than pristine g-C3N4. Spectroscopic and photoelectrochemical characterizations revealed that impregnation of CDs into g-C3N4 not only enhanced the production of photogenerated electron-hole pairs by extending the visible light absorption region due to the upconverted photoluminescence character of CDs, but also facilitated electron-hole separation by band alignment in the g-C3N4/CDs junction, thus yielded more holes, O-center dot(2)- and (OH)-O-center dot radicals to promote phenol degradation. These results highlight the potential application of sustainable metal-free photocatalysts in water purification. (C) 2015 Elsevier B.V. All rights reserved.

Perfluorooctane sulfonate (PFOS) is a persistent organic pollutant that is used worldwide and is continuously being detected in biota and the environment, thus presenting potential threats to the ecosystem and human health. Although PFOS is highly immunotoxic, its underlying molecular mechanisms remain largely unknown. The present study examined PFOS-induced immunotoxicity in the mouse spleen and explored its underlying mechanisms by gene expression profiling. Oral exposure of male BALB/c mice for three weeks followed by one-week recovery showed that a 10 mg/kg/day PFOS exposure damaged the splenic architecture, inhibited T-cell proliferation in response to mitogen, and increased the percentages of T helper (CD3(+)CD4(+)) and cytotoxic T (CD3(+)CD8(+)) cells, despite the decrease in the absolute number of these cells. A delayed type of PFOS immunotoxicity was observed, which mainly occurred during the recovery period. Global gene expression profiling of mouse spleens and QRT-PCR analyses suggest that PFOS inhibited the expression of genes involved in cell cycle regulation and NRF2-mediated oxidative stress response, and upregulated those in TCR signaling, calcium signaling, and p38/MAPK signaling pathways. Western blot analysis confirmed that the expressions of CAMK4, THEMIS, and CD3G, which were involved in the upregulated pathways, were induced upon PFOS exposure. Acute PFOS exposure modulated calcium homoeostasis in splenocytes. These results indicate that PFOS exposure can activate TCR signaling and calcium ion influx, which provides a clue for the potential mechanism of PFOS immunotoxicity. The altered signaling pathways by PFOS treatment as revealed in the present study might facilitate in better understanding PFOS immunotoxicity and explain the association between immune disease and PFOS exposure.

Photocatalytic degradation is a powerful technique for the decomposition of pollutants. However, toxic intermediates might be generated which have become a great concern recently. In the present work, a continuous flow chemiluminescence (CFCL) method was developed for dynamic monitoring of toxic intermediates generated in the photocatalytic degradation of pentachlorophenol (PCP). Among the main intermediates, tetrachloro-1,4-benzoquinone (TCBQ) and trichlorohydroxy-1,4-benzoquinone (OH-TrCBQ) showed higher or similar toxicity to PCP. As both TCBQ and OH-TrCBQ can produce chemiluminescence (CL) in the presence of H2O2, a CFCL system was established for the dynamic tracking of the two toxic intermediates. A PCP/TiO2 suspension was irradiated in a photoreactor, pumped continuously into a detection cell, and mixed with H2O2 to produce CL. The time-dependent CL response displayed two distinctive peaks at pH 7, which were attributed to the generation of OH-TrCBQ and TCBQ, respectively, by comparing with their c...

Polybrominated diphenyl ethers (PBDEs) are widely used as flame retardants and are often detected in the environment, wildlife, and humans, presenting potential threats to ecosystem and human health. PBDEs can cause neurotoxicity, hepatotoxicity, and endocrine disruption. However, data on PBDE immunotoxicity are limited, and the toxicity mechanisms remain largely unknown. Both immune cell death and dysfunction can modulate the responses of the immune system. This study examined the toxic effects of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) and decabromodiphenyl ether (BDE-209) on the immune system by using peritoneal macrophages as the model. The macrophages were exposed to PBDEs, and cell death was determined through flow cytometry and immunochemical blot. The results showed that after 24h of exposure, BDE-47 (>5 μM) and BDE-209 (>20 μM) induced cell apoptosis, increased intracellular reactive oxygen species (ROS) formation and depleted glutathione. BDE-47 was more potent than BDE-209; the cytotoxic concentrations for BDE-47 and BDE-209 were determined to be 5 μM and 20 μM, respectively, during 24h of exposure. However, pretreatment with n-acetyl-l-cysteine (ROS scavenger) partially reversed the cytotoxic effects. Further gene expression analyses on Caspase-3,-8,-9, TNFR1, and Bax revealed that both intrinsic and extrinsic apoptotic pathways were activated. More importantly, non-cytotoxic concentrations BDE-47 (

Transformation of nanomaterials in aqueous environment has significant impact on their behavior in engineered application and natural system. In this paper, UV irradiation induced transformation of TiO2 nanoparticles in aqueous solutions was demonstrated, and its effect on the aggregation and photocatalytic reactivity of TiO2 was investigated. UV irradiation of a TiO2 nanoparticle suspension accelerated nanoparticle aggregation that was dependent on the irradiation duration. The aggregation rate increased from

Perfluorinated compounds (PFCs) have been shown to disrupt lipid metabolism and even induce cancer in rodents through activation of peroxisome proliferator-activated receptors (PPARs). Lines of evidence showed that PPARα was activated by PFCs. However, the information on the binding interactions between PPARγ and PFCs and subsequent alteration of PPARγ activity is still limited and sometimes inconsistent. In the present study, in vitro binding of 16 PFCs to human PPARγ ligand binding domain (hPPARγ-LBD) and their activity on the receptor in cells were investigated. The results showed that the binding affinity was strongly dependent on their carbon number and functional group. For the eleven perfluorinated carboxylic acids (PFCAs), the binding affinity increased with their carbon number from 4 to 11, and then decreased slightly. The binding affinity of the three perfluorinated sulfonic acids (PFSAs) was stronger than their PFCA counterparts. No binding was detected for the two fluorotelomer alcohols (FTOHs). Circular dichroim spectroscopy showed that PFC binding induced distinctive structural change of the receptor. In dual luciferase reporter assays using transiently transfected Hep G2 cells, PFCs acted as hPPARγ agonists, and their potency correlated with their binding affinity with hPPARγ-LBD. Molecular docking showed that PFCs with different chain length bind with the receptor in different geometry, which may contribute to their differences in binding affinity and transcriptional activity.

Perfluoroalkyl compounds (PFCs) have been shown to disrupt thyroid functions through thyroid hormone receptor (TR)-mediated pathways, but direct binding of PFCs with TR has not been demonstrated. We investigated the binding interactions of 16 structurally diverse PFCs with human TR, their activities on TR in cells, and the activity of perfluorooctane sulfonate (PFOS) in vivo. In fluorescence competitive binding assays, most of the 16 PFCs were found to bind to TR with relative binding potency in the range of 0.0003-0.05 compared with triiodothyronine (T3). A structure-binding relationship for PFCs was observed, where fluorinated alkyl chain length longer than ten, and an acid end group were optimal for TR binding. In thyroid hormone (TH)-responsive cell proliferation assays, PFOS, perfluorohexadecanoic acid, and perfluorooctadecanoic acid exhibited agonistic activity by promoting cell growth. Furthermore, similar to T3, PFOS exposure promoted expression of three TH upregulated genes and inhibited three TH downregulated genes in amphibians. Molecular docking analysis revealed that most of the tested PFCs efficiently fit into the T3-binding pocket in TR and formed a hydrogen bond with arginine 228 in a manner similar to T3. The combined in vitro, in vivo, and computational data strongly suggest that some PFCs disrupt the normal activity of TR pathways by directly binding to TR.

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.

Sensitive and selective detection of Pb(2+) is of great importance to both human health and environmental protection. Here we propose a novel fluorescence anisotropy (FA) approach for sensing Pb(2+) in homogeneous solution by a G-rich thrombin binding aptamer (TBA). The TBA labeled with 6-carboxytetramethylrhodamine (TMR) at the seventh thymine nucleotide was used as a fluorescent probe for signaling Pb(2+). It was found that the aptamer probe had a high FA in the absence of Pb(2+). This is because the rotation of TMR is restricted by intramolecular interaction with the adjacent guanine bases, which results in photoinduced electron transfer (PET). When the aptamer probe binds to Pb(2+) to form G-quadruplex, the intramolecular interaction should be eliminated, resulting in faster rotation of the fluorophore TMR in solution. Therefore, FA of aptamer probe is expected to decrease significantly upon binding to Pb(2+). Indeed, we observed a decrease in FA of aptamer probe upon Pb(2+) binding. Circular dichroism, fluorescence spectra, and fluorescence lifetime measurement were used to verify the reliability and reasonability of the sensing mechanism. By monitoring the FA change of the aptamer probe, we were able to real-time detect binding between the TBA probe and Pb(2+). Moreover, the aptamer probe was exploited as a recognition element for quantification of Pb(2+) in homogeneous solution. The change in FA showed a linear response to Pb(2+) from 10 nM to 2.0 μM, with 1.0 nM limit of detection. In addition, this sensing system exhibited good selectivity for Pb(2+) over other metal ions. The method is simple, quick and inherits the advantages of aptamer and FA.

We firstly studied the chemiluminescence behavior of branched poly(ethylenimine)-functionalized carbon dots (BPEI-CDs). The results demonstrated that BPEI-CDs can be used as a novel chemiluminescence probe in alkaline solution for rapid detection of iron(III) ions with high sensitivity and selectivity. A possible CL mechanism was studied by UV-Vis, fluorescence, CL, FTIR, XPS and EPR spectroscopy. Iron(III) could be selectively captured by the surface functional groups of BPEI and injected holes into the carbon dots which resulted in a great improvement of the BPEI-CDs' CL signal in alkaline solution. The work sheds new light on the characteristics and further application of functionalized carbon dots.

The noncovalent interactions of 24 diversely structured hydroxylated polybrominated diphenyl ethers (OH-PBDEs), ranging from hydroxylated monobromodiphenyl ether (OH-monoBDE) to hydroxylated octabromodiphenyl ether (OH-octaBDE), with bovine serum albumin (BSA) have been examined by employing an electrospray ionization source fitted on a quadrupole time-of-flight hybrid mass spectrometer equipped with a traveling wave ion mobility cell. The mass spectrometric parameters were finely optimized to favor the observation of noncovalent complexes. The experimental data confirm that due to the close structural resemblance with thyroid hormone thyroxine, some OH-PBDEs have been shown to conjugate with BSA. It is found that BSA preferentially interacts with one OH-octaBDE (4′-OH-BDE-201) and two hydroxylated heptabromodiphenyl ether (OH-heptaBDE) isomers (4-OH-BDE-187 and 6-OH-BDE-180) with 1:1 and 2:1 binding stoichiometries. The state of bound OH-PBDEs is inferred to be as both neutral and anionic species. The dissociation constants of corresponding noncovalent complexes are calculated by titration curve data fitting. Investigation into various OH-PBDE analogs demonstrates that both the degree of bromination and the positions of hydroxylation and bromine moieties may exert influence on the binding of OH-PBDEs to BSA. The possible conformational changes in BSA induced upon the binding of OH-PBDEs were explored by use of ion mobility-mass spectrometry (IM-MS) to understand the details of the interactions in structural aspects.

Quantitative detection of small molecules by a photoelectrochemical system was demonstrated in a competitive assay for biotin. The system for the determination of Biotin was employed by using ruthenium tris(bipyridine) (Ru-bpy) as the label, oxalate as the sacrificial electron donor to recycle the label, and tin oxide nanoparticle as the semiconductor electrode. Upon irradiation with 470 nm light, electrons of Ru-bpy were promoted to the excited state, and then injected into the conduction band of the tin oxide semiconductor, thus producing photocurrent signal. The oxidized Ru-bpy was reduced back to its original state by oxalate in solution, and was used again in the next cycle of signal generation. In the competitive assay of biotin, avidin was adsorbed passively on the tin oxide surface as the recognition element. Adsorbed protein was found to be stable under assay conditions, and reached maximum surface coverage when the concentration of avidin solution for adsorption was 0.5 g L −1 or higher. A mixed solution of 1 μM tracer and various concentrations of biotins were reacted with surface-immobilized avidin. As biotin concentration was increased, less tracer molecules bound to avidin, leading to a reduction in its photocurrent signal. A detection limit of 8 μg L −1 biotin was obtained in the competitive assay. The method can be easily extended to the detection of competitive immunoassays for organic chemicals.

Perfluorinated compounds (PFCs) are known to accumulate in liver and induce hepatotoxicity on experimental animals. Liver fatty acid binding protein (L-FABP) is expressed highly in hepatocytes and binds fatty acids. PFCs may bind with FABP and change their ADME and toxicity profile. In the present study, the binding interaction of 17 structurally diverse PFCs with human L-FABP was investigated to assess their potential disruption effect on fatty acid binding. The binding affinity of twelve perfluorinated carboxylic acids (PFCAs), as determined by fluorescence displacement assay, increased significantly with their carbon number from 4 to 11, and decreased slightly when the number was over 11. The three perfluorinated sulfonic acids (PFSAs) displayed comparable affinity, but no binding was detected for the two fluorotelomer alcohols. Circular dichroism results showed that PFC binding induced distinctive structural changes of the protein. Molecular docking revealed that the driving forces for the binding of PFCs with FABP were predominantly hydrophobic and hydrogen-bonding interactions, and the binding geometry was dependent on both the size and rigidity of the PFCs. Based on the binding constant obtained in this work, the possibility of in vivo competitive displacement of fatty acids from FABP by PFCs was estimated.

The wide application of carbon nanomaterials in various fields urges in-depth understanding of the toxic effects and underlying mechanisms of these materials on biological systems. Cell autophagy was recently recognized as an important lysosome-based pathway of cell death, and autophagosome accumulation has been found to be associated with the exposure of various nanoparticles, but the underlying mechanisms are still uncertain due to the fact that autophagosome accumulation can result from autophagy induction and/or autophagy blockade. In this study, we first evaluated the toxicity of acid-functionalized single-walled carbon nanotubes and graphene oxides, and found that both carbon nanomaterials induced adverse effects in murine peritoneal macrophages, and GOs were more potent than AF-SWCNTs. Both carbon nanomaterials induced autophagosome accumulation and the conversion of LC3-I to LC3-II. However, degradation of the autophagic substrate p62 protein was also inhibited by both nanomaterials. Further analyses on lysosomes revealed that both carbon nanomaterials accumulated in macrophage lysosomes, leading to lysosome membrane destabilization, which indicates reduced autophagic degradation. The effects of AF-SWCNTs and GOs on cell autophagy revealed by this study may shed light on the potential toxic mechanism and suggest caution on their utilization.

A bead-based competitive fluorescence immunoassay for the detection of triazophos has been developed. In this method, a conjugate of hapten and ovalbumin covalently bound to carboxylated fluorescent bead was used as a coating antigen and goat anti-mouse IgG/R-phycoerythrin conjugate was used as the fluorescent molecular probe. For standards (in 5% methanol in phosphate-buffered saline), the detection range was 0.02–50 ng/ml and the average coefficient of variation (CV) was 8.9%. This bead-assay was highly specific for triazophos, and none of other the structurally similar compounds (diazinon, malathion, parathion and fenitrothion) were recognised (

O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) plays a critical role in modulating protein function in many cellular processes and human diseases such as Alzheimer's disease and type II diabetes, and has emerged as a promising new target. Specific inhibitors of OGT could be valuable tools to probe the biological functions of O-GlcNAcylation, but a lack of robust nonradiometric assay strategies to detect glycosylation, has impeded efforts to identify such compounds. Here we have developed a novel label-free electrochemical biosensor for the detection of peptide O-GlcNAcylation using protease-protection strategy and electrocatalytic oxidation of tyrosine mediated by osmium bipyridine as a signal reporter. There is a large difference in the abilities of proteolysis of the glycosylated and the unglycosylated peptides by protease, thus providing a sensing mechanism for OGT activity. When the O-GlcNAcylation is achieved, the glycosylated peptides cannot be cleaved by proteinase K and result in a high current response on indium tin oxide (ITO) electrode. However, when the O-GlcNAcylation is successfully inhibited using a small molecule, the unglycosylated peptides can be cleaved easily and lead to low current signal. Peptide O-GlcNAcylation reaction was performed in the presence of a well-defined small-molecule OGT inhibitor. The results indicated that the biosensor could be used to screen the OGT inhibitors effectively. Our label-free electrochemical method is a promising candidate for protein glycosylation pathway research in screening small-molecule inhibitors of OGT.

This chapter introduces the safety of carbon nanotubes (CNTs), a type of nanomaterial widely used in industry. We start by presenting potential human exposure routes to CNTs during the life cycle of this material based on a few reports and the principles of nanoparticle biodistribution in living systems. Protocols and guidance on preventive and protective measurements are given for potential occupational and environmental CNT exposure. We then discuss the dynamic changes and mechanisms for the interactions between CNTs and organs. The toxic effects of CNTs on potential target organs are reviewed and the influencing factors are explored. At the end of the chapter, a discussion on the challenges currently existing in the risk assessment of CNTs is provided along with an outlook for future development.

Coexistence of nanomaterials and environmental pollutants requires in-depth understanding of combined toxicity and underlying mechanism. In this work, we found that coexposure to the mixture of noncytotoxic level of single-walled carbon nanotubes (SWCNTs) (10 μg/mL) and Ni

Exocytosis of single-walled carbon nanotubes (SWCNTs) determines therapeutic efficiency and toxicity of nanoproducts but its underlying mechanism remains elusive. In this study, it is found that the exocytosis mechanism of SWCNTs is mediated mainly through the activation of P2X

Mercury compounds are well-known toxic environmental pollutants and potently induce severe neurotoxicological effects in human and experimental animals. Previous studies showed that one of the mechanisms of mercury compounds neurotoxicity arose from the over-activation of the N-methyl d-aspartate (NMDA)-type glutamate receptor induced by increased glutamate release. In this work, we aimed to investigate the molecular mechanisms of Hg compounds neurotoxicities by identifying their biological targets in cells. Firstly, the inhibitory effects of four Hg compounds, including three organic (methyl-, ethyl- and phenyl-mercury) and one inorganic (Hg2+) Hg compounds, on the activity of arginine decarboxylase (ADC), a key enzyme in the central agmatinergic system, were evaluated. They were found to inhibit the ADC activity significantly with methylmercury (MeHg) being the strongest (IC50=7.96nM). Furthermore, they showed remarkable inhibitory effects on ADC activity in PC12 cells (MeHg>EtHg>PhHg>HgCl2), and led to a marked loss in the level of agmatine, an endogenous neuromodulatory and neuroprotective agent that selectively blocks the activation of NMDA receptors. MeHg was detected in the immunoprecipitated ADC from the cells, providing unequivocal evidence for the direct binding of MeHg with ADC in the cell. Molecular dynamics simulation revealed that Hg compounds could form the coordination bond not only with cofactor PLP of ADC, but also with substrate arginine. Our finding indicated that MeHg could attenuate the neuroprotective effects of agmatine by the inhibition of ADC, a new cellular target of MeHg, which might be implicated in molecular mechanism of MeHg neurotoxicity.

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.

Perfluoroalkyl acids (PFAAs) are widespread environmental contaminants which have been detected in humans and linked to adverse health effects. Previous toxicological studies mostly focused on nuclear receptor-mediated pathways and did not support the observed toxic effects. In this study, we aimed to investigate the molecular mechanisms of PFAA toxicities by identifying their biological targets in cells. Using a novel electrochemical biosensor, 16 PFAAs were evaluated for inhibition of protein tyrosine phosphatase SHP-2 activity. Their potency increased with PFAA chain length, with perfluorooctadecanoic acid (PFODA) showing the strongest inhibition. Three selected PFAAs, 25 μM perfluorooctanoic acid (PFOA), perfluorooctane sulfonic acid, and PFODA, also inhibited SHP-2 activity in HepG2 cells and increased paxillin phosphorylation level. PFOA was detected in the immunoprecipitated SHP-2 from the cells exposed to 250 μM PFOA, providing unequivocal evidence for the direct binding of PFOA with SHP-2 in the cell. Molecular docking rationalized the formation of PFAA/SHP-2 complex and chain length-dependent inhibition potency. Our results have established SHP-2 as a new cellular target of PFAAs.

Human G protein-coupled receptor 40 (hGPR40), with medium- and long-chain free fatty acids (FFAs) as its natural ligands, plays an important role in the enhancement of glucose-dependent insulin secretion. To date, information about the direct binding of FFAs to hGPR40 is very limited, and how carbon-chain length affects the activities of FFAs on hGPR40 is not yet understood. In this study, a fluorescein-fasiglifam analogue (F-TAK-875A) conjugate was designed and synthesized as a site-specific fluorescence probe to study the interaction of FFAs with hGPR40. hGPR40 was expressed in human embryonic kidney 293 cells and labeled with F-TAK-875A. By using flow cytometry, competitive binding of FFA and F-TAK-875A to hGPR40-expressed cells was measured. Binding affinities of 18 saturated FFAs, with carbon-chain lengths ranging from C6 to C23, were analyzed. The results showed that the binding potencies of FFAs to hGPR40 were dependent on carbon length. There was a positive correlation between length and binding potency for seven FFAs (C9-C15), with myristic acid (C15) showing the highest potency, 0.2% relative to TAK-875. For FFAs with a length of fewer than C9 or more than C15, they had very weak or no binding. Molecular docking results showed that the binding pocket of TAK-875 in hGPR40 could enclose FFAs with lengths of C15 or fewer. However, for FFAs with lengths longer than C15, part of the alkyl chain extended out of the binding pocket. This study provided insights into the structural dependence of FFAs binding to and activation of hGPR40.