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 label-free biosensor for the assay of OGT inhibition has been developed.•Protease-protection strategy and electrocatalytic signal of tyrosine were used.•The biosensor could be used to screen the OGT inhibitors effectively.•We provide a complementary method for antibody and labeling techniques.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.

•Full-lengths of HSP60 and HSP70 were first obtained from bivalve Anodonta woodiana.•AwHSP60 and AwHSP70 have a widely tissues distribution.•Expression of AwHSP60 is significantly induced by PBDE-47 in during whole experiment.•Expression of AwHSP70 is significant up-regulated by PBDE-47 in experiment observed.Heat shock proteins (HSPs) play an important role in adaption of environmental stress by protein folding, membrane translocation, degradation of misfolded proteins and other regulatory processes. Our previous study showed oxidative stress generated from polybrominated diphenyl ether-47 (PBDE-47) could cause an acute toxicity on freshwater bivalve Anodonta Woodiana, but the effect of chronic toxicity need to be elucidated. In order to further investigate the chronic effect of PBDE-47, clams A. Woodiana were randomly divided into the PBDE-47 treated group administrated with PBDE-47 at a concentration 3.36 μg/L and control group treated with a similar volume dimethyl sulfoxide. Two complete HSP sequences were isolated from A. Woodianaa and respectively named AwHSP60 and AwHSP70. They were widely distributed in foot, gill, hepatopancreas, adductor muscle, heart, hemocytes and mantle. Administration of PBDE-47 could result in a significant up-regulation of AwHSP60 and AwHSP70 expressions in the hepatopancreas, gill and hemocytes. In the hepatopancreas, compared with that of control group, mRNA level of AwHSP60 increased more than 89.9% (P < 0.05) from day 1–15, AwHSP70 increased more 2.79 times (P < 0.01). In the gill, during experiment observed, expression of AwHSP60 increased more 2.09 times (P < 0.01) in contrasted with that of control group. Significant up-regulation of AwHSP70 expression showed a reversed U shape. In the hemocytes, AwHSP60 and AwHSP70 expressions of PBDE-47 treated group respectively increased more 2.09 times (P < 0.05) and 1.81 times (P < 0.05) compared with that of control group. These results indicated that up-regulations of AwHSP60 and AwHSP70 expression are contribute to enhancing adaption of bivalve A. Woodiana exposed to PBDE-47 treatment.

•ECL-based toxicity sensor was developed for AP site detection in damaged DNA.•Aldehyde reactive probe was employed for specific labeling of AP sites.•A low detection limit of 1 AP site in 512 normal bases was achieved.•The sensor can be used for the screening of genetic toxicity of chemicals.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.Download high-res image (118KB)Download full-size image

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.

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. In addition, inhibition of P2X7R by P2X7-targeting siRNA diminished the inhibitory effect of Ni2+. It was therefore concluded that Ni2+ impeded SWCNT exocytosis by inhibiting P2X7R, leading to higher intracellular retention of SWCNTs and elevated cytotoxicity. Our work identified exocytosis inhibition as an important mechanism for SWCNT/Ni2+ toxicity, and revealed the crucial role of P2X7R in mediating such inhibitory effect.

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.

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.

An efficient photocatalytic process involves two closely related steps: charge separation and the subsequent surface redox reaction. Herein, a ternary hybrid photocatalytic system was designed and fabricated by anchoring Cu(II) clusters onto a TiO2/reduced graphene oxide (RGO) composite. Microscopic and spectroscopic characterization revealed that both TiO2 nanoparticles and Cu(II) clusters were highly dispersed on a graphene sheet with intimate interfacial contact. Compared with pristine TiO2, the TiO2/RGO/Cu(II) composite yielded an almost 3-fold enhancement in the photodegradation rate toward phenol degradation under UV irradiation. Electron spin resonance (ESR) spectra and electrochemical measurements demonstrated that the improved photocatalytic activity of this ternary system benefitted from the synergetic effect between RGO and Cu(II), which facilitates the interfacial charge transfer and simultaneously achieves in situ generation of H2O2 via two-electron reduction of O2. These results highlight the importance to harmonize the charge separation and surface reaction process in achieving high photocatalytic efficiency for practical application.Keywords: oxygen reduction; phenol degradation; photocatalysis; TiO2

The selectivity of molecular oxygen activation on the exfoliated graphitic carbon nitride (g-C3N4) and its influence on the photocatalytic phenol degradation process were demonstrated. Compared with bulk g-C3N4, the exfoliated nanosheet yielded a 3-fold enhancement in photocatalytic phenol degradation. ROS trapping experiments demonstrated that although the direct hole oxidation was mainly responsible for phenol photodegradation on both g-C3N4 catalysts, molecular oxygen activation processes on their surface greatly influenced the whole phenol degradation efficiency. Reactive oxygen species and Raman spectroscopy measurements revealed that oxygen was preferentially reduced to ·O2– by one-electron transfer on bulk g-C3N4, while on g-C3N4 nanosheet the production of H2O2 via a two-electron transfer process was favored due to the rapid formation of surface-stabilized 1,4-endoperoxide. The latter process not only promotes the separation of photogenerated electron–hole pairs but also greatly facilitates reactive oxygen species formation and subsequently enhances phenol degradation.

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.

•An electrochemiluminescence (ECL) sensor was developed for the detection of 8-oxodGuo lesion repair by FPG enzyme.•A novel bifunctional chemical probe was designed as an 8-oxodGuo ECL label.•The sensor was employed to assess the inhibition of FPG activity by heavy metals.•Methyl Hg(II) showed very potent inhibition effect on FPG.A new electrochemiluminescence (ECL) sensor was developed for 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) quantification and Escherichia coli formamidopyrimidine-DNA glycosylase (FPG) activity assay. The sensor employed a novel spermine conjugated ruthenium tris-(bipyridine) derivative (spermine-Ru) which binds specifically with 8-oxodGuo through a one-step reaction and also acts as an ECL signal reporter. In the sensor, an 8-oxodGuo-containing ds-DNA film was first immobilized on a gold electrode by self-assembly. The DNA film was then incubated with spermine-Ru under oxidative condition for 8-oxodGuo labeling. The ECL intensity was found to correlate with the amount of 8-oxodGuo on the surface and the detection limit was estimated to be about 1 lesion in 500 DNA bases. Addition of FPG resulted in some loss of the signal due to the excision of 8-oxodGuo by the enzyme. An inverse relationship between ECL intensity and FPG concentration was observed in a range from 0 to 4.0 U/µL, demonstrating that this sensor could be used for FPG activity assay. A number of metal ions were screened by the sensor for their inhibition effect on FPG activity. Among them, Hg2+ and methyl Hg(II) shown very potent inhibition, with IC50 values of 4.04 µM and 4.34 nM respectively. The result may suggest that interference on the DNA repair system could be another mechanism for the high toxicity of MeHg.

Reactive oxygen species (ROS) play very important roles in the photocatalytic reactions of semiconductors. Using a continuous flow chemiluminescence (CFCL) system, we developed three methods for the selective, sensitive, and online detection of O2• –, •OH, and H2O2 generated during ultraviolet (UV) irradiation of nano-TiO2 suspensions. TiO2 nanoparticles were irradiated in a photoreactor and pumped continuously into a detection cell. To detect O2• –, luminol was mixed with TiO2 before it entered the detection cell. For the detection of short-lived •OH, phthalhydrazide was added into the photoreactor to capture •OH, and then mixed with H2O2/K5Cu(HIO6)2 to produce chemiluminescence (CL). To detect H2O2, an irradiated TiO2 suspension was kept in darkness for 30 min, and then mixed with luminol/K3Fe(CN)6 to produce CL. The selectivity of each method for a particular ROS was verified by using specific ROS scavengers. For a given ROS, a comparison between CL and conventional method showed good agreement for a series of TiO2 concentrations. The sensitivity of CL method was approximately 3-, 1200-, and 5-fold higher than the conventional method for O2• –, •OH, and H2O2, respectively. To demonstrate the utility of the methods, ROS in three different types of TiO2 suspensions was detected by CFCL. It was found that photodegradation efficiency of Rhodamine B correlated the best (R2 > 0.95) with the amount of photogenerated •OH, implying that •OH was the major oxidant in Rhodamine B photodegradation reaction. CFCL may provide a convenient tool for the studies on the reaction kinetics of ROS-participated decomposition of environmental contaminants.

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 <0.001 nm/s before irradiation to 0.027 nm/s after 50 h irradiation, resulting in aggregates with a hydrodynamic diameter of 623 nm. The isoelectric point of the suspension was lowered from 7.0 to 6.4 after irradiation, indicating less positive charges on the surface. ATR-FTIR spectra displayed successive growth of surface hydroxyl groups with UV irradiation which might be responsible for the change of surface charge and aggregation rate. UV irradiation also changed the photocatalytic degradation rate of Rhodamine B by TiO2, which initially increased with irradiation time, then decreased. Based on the photoluminescence decay and photocurrent collection data, the change was attributed to the variation in interparticle charge transfer kinetics. These results highlight the importance of light irradiation on the transformation and reactivity of TiO2 nanomaterials.

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.

•Noncovalent interactions of 24 diverse OH-PBDEs with BSA are examined by ESI-MS.•BSA preferentially interacts with one OH-octaBDE and two OH-heptaBDE isomers.•OH-PBDEs are inferred to be bound as both neutral and anionic species.•Possible conformational changes in BSA induced on binding are explored by IM-MS.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.

We report the surprising chemiluminescence (CL) behavior of fluorescent carbon dots in the presence of a strong alkaline solution, such as NaOH or KOH. The CL intensity was dependent on the concentration of the base and carbon dots in a certain range. A possible CL mechanism was studied by UV-Vis, fluorescence, CL, FTIR, XPS and EPR spectroscopy. Radiative recombination of the injected electrons by “chemical reduction” of carbon dots with thermally excited generated holes was proposed, which sheds new light on the characteristics of carbon dots.

A graphene-based two-dimensional (2D) nanoplatform provides new opportunities for fabricating 2D heterojunction interfaces to fortify charge transfer in semiconductor assemblies. In this report, TiO2 nanosheet/graphene composite based 2D–2D heterojunctions were fabricated by a solvothermal process. Microscopic and spectroscopic characterization revealed a homogeneous sheetlike morphology with intimate interfacial contact between the TiO2 nanosheet and graphene due to chemical interactions. Compared with 0D–2D Degussa P25 (TiO2)/graphene and 1D–2D TiO2 nanotube/graphene composites, the 2D–2D TiO2 nanosheet/graphene hybrid demonstrated higher photocatalytic activity toward the degradation of rhodamine B and 2,4-dichlorophenol under UV irradiation. Radical trapping and ESR experiments revealed the enhanced generation of ·OH and O2•– in the 2D–2D heterojunction system. By analyzing TiO2 excited state deactivation lifetime, the interfacial electron transfer rates determined for 0D–2D, 1D–2D, and 2D–2D TiO2/graphene composites were 1.15 × 108 s–1, 3.47 × 108 s–1, and 1.06 × 109 s–1, respectively. It was therefore proposed that the fast charge separation in the TiO2 nanosheet/graphene photocatalyst promoted the generation of reactive oxygen species and enhanced the photodegradation reactions. The results underscore the key role of nanomaterial dimensionality in interfacial charge transfer processes.Keywords: 2D−2D heterojunction; graphene; interfacial charge transfer; photocatalysis; TiO2 nanosheet;

Exogenous chemicals may produce DNA methylation that is potentially toxic to living systems. Methylated DNA bases are difficult to detect with biosensors because the methyl group is small and chemically inert. In this report, a label-free photoelectrochemical sensor was developed for the selective detection of chemically methylated bases in DNA films. The sensor employed two DNA repair enzymes, human alkyladenine DNA glycosylase and human apurinic/apyrimidinic endonuclease, to convert DNA methylation sites in DNA films on indium tin oxide electrodes into strand breaks. A DNA intercalator, Ru(bpy)2(dppz)2+ (bpy=2,2′-bipyridine, dppz = dipyrido[3,2-a:2′,3′-c]phenazine) was then used as the photoelectrochemical signal indicator to detect the DNA strand breaks. Its photocurrent signal was found to correlate inversely with the amount of 3-methyladenines (metAde) produced with a methylating agent, methylmethane sulfonate (MMS). The sensor detected the methylated bases produced with as low as 1 mM MMS, at which concentration the amount of metAde on the sensor surface was estimated to be 0.5 pg, or 1 metAde in 1.6 × 105 normal bases. Other DNA base modification products, such as 5-methylcytosine and DNA adducts with ethyl and styrene groups did not attenuate the photocurrent, demonstrating good selectivity of the sensor. This strategy can be utilized to develop sensors for the detection of other modified DNA bases with specific DNA repair enzymes.

Perfluoroalkyl acids (PFAAs) have been reported to interfere with the endocrine system in vivo by mimicking endogenous hormone activities and causing adverse effects. Some exoestrogens bind to estrogen receptor (ER) and subsequently induce an ER-mediated response. The transcriptional activity of ER is regulated by its distinct conformational states that are the results of ligand binding. In this work, a biosensor based on surface plasmon resonance (SPR) technique was developed which can discriminate between agonist and antagonist of human ERα (hERα) by monitoring the conformation state of the protein induced by ligand binding. The biosensor utilized the specific interaction between hERα and conformation-selective peptides. Six PFAAs with different chain lengths and acid groups were tested by the biosensor, and perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) were found to be ER agonists. Kinetic analyses of direct interaction between PFAAs and hERα by SPR revealed that PFOS and PFOA were both weak binders of ER with KD values of 2.19 and 107 μM respectively, whereas the other four PFAAs did not bind with ER. To understand the differences in ER binding affinity and estrogenic activity among the six PFAAs, molecular docking based on the crystal structure of hERα ligand binding domain was performed. PFOS and PFOA were efficiently docked with hERα and formed hydrogen bonds with Arg394 in a manner similar to estradiol. Overall, the two 8-carbon PFAAs were assessed as weak agonists of hERα and are of potential concern.

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.

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.Quantitative detection of small molecules by a photoelectrochemical system was demonstrated in a competitive assay for biotin. In this experiment avidin was immobilized on a SnO2 electrode to capture the biotin and tracer in solution, and the amount of biotin was quantified by measuring the photocurrent of the tracer upon irradiation with 470 nm light. A detection limit of 8 μg L−1 biotin was obtained in this competitive assay.

Polybrominated diphenyl ethers (PBDEs) are used in large quantities as flame retardant additives in commercial products. Bio-monitoring data show that PBDE concentrations have increased rapidly in the bodies of wildlife and human over the last few decades. Based on the studies on experimental animals, the toxicological endpoints of exposure to PBDEs are likely to be thyroid homeostasis disruption, neuro-developmental deficits, reproductive ineffectiveness and even cancer. Unfortunately, the available molecular toxicological evidence for these endpoints is still very limited. This review focuses on the recent studies on the molecular mechanisms of PBDE toxicities carried out through the hormone receptor pathways, including thyroid hormone receptor, estrogen receptor, androgen receptor, progesterone receptor and aryl hydrocarbon receptor pathways. The general approach in the mechanistic investigation is to examine the in vitro direct binding of a PBDE with a receptor, the in vitro recruitment of a co-activator or co-repressor by the ligand-bound receptor, and the participation of the ligand in the receptor-mediated transcription pathways in cells. It is hoped that further studies in this area would provide more insights into the potential risks of PBDEs to human health.

Exposure of DNA to oxidative stress conditions results in the generation of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo). 8-OxodGuo is genotoxic if left unrepaired. We quantified 8-oxodGuo lesions in double-stranded DNA films by using a photoelectrochemical DNA sensor in conjunction with a specific covalent labeling method. A lesion-containing DNA film was assembled on a SnO2 nanoparticle modified indium tin oxide electrode through layer-by-layer electrostatic adsorption. The lesions were covalently labeled with a biotin conjugated spermine derivative, and ruthenium tris(bipyridine) labeled streptavidin was introduced as the signal reporter molecule. Photocurrent increased with the number of lesions in the strand and decreased as the film was diluted with intact DNA. Quantification of 8-oxodGuo was achieved with an estimated detection limit of ∼1 lesion in 650 bases or 1.6 fmol of 8-oxodGuo on the electrode. Incubation of the film with a DNA base excision repair enzyme, E. coli formamidopyrimidine–DNA glycosylase (Fpg), resulted in complete loss of the signal, indicating efficient excision of the isolated lesions in the nucleotide. Oxidatively generated DNA damage to a double-stranded calf thymus DNA film by the Fenton reaction was then assessed. One 8-oxodGuo lesion in 520 bases was detected in DNA exposed to 50 μM Fe2+/200 μM H2O2. Treatment with Fpg reduced the photocurrent by 50%, indicating only partial excision of 8-oxodGuo. This suggests that tandem lesions, which are resistant to Fpg excision, are generated by the Fenton reaction. Unlike repair enzyme dependent methods, the sensor recognizes 8-oxodGuo in tandem lesions and can avoid underestimating DNA damage.

Polybrominated diphenyl ethers (PBDEs) have been shown to disrupt thyroid hormone (TH) functions on experimental animals, and one of the proposed disruption mechanisms is the competitive binding of PBDE metabolites to TH transport proteins. In this report, a nonradioactive, site-specific fluorescein–thyroxine (F–T4) conjugate was designed and synthesized as a fluorescence probe to study the binding interaction of hydroxylated PBDEs to thyroxine-binding globulin (TBG) and transthyretin (TTR), two major TH transport proteins in human plasma. Compared with free F–T4, the fluorescence intensity of TTR-bound conjugate was enhanced by as much as 2-fold, and the fluorescence polarization value of TBG-bound conjugate increased by more than 20-fold. These changes provide signal modulation mechanisms for F–T4 as a fluorescence probe. Based on fluorescence quantum yield and lifetime measurements, the fluorescence intensity enhancement was likely due to the elimination of intramolecular fluorescence quenching of fluorescein by T4 after F–T4 was bound to TTR. In circular dichroism and intrinsic tryptophan fluorescence measurements, F–T4 induced similar spectroscopic changes of the proteins as T4 did, suggesting that F–T4 bound to the proteins at the T4 binding site. By using F–T4 as the fluorescence probe in competitive binding assays, 11 OH–PBDEs with different levels of bromination and different hydroxylation positions were assessed for their binding affinity with TBG and TTR, respectively. The results indicate that the binding affinity generally increased with bromine number and OH position also played an important role. 3-OH–BDE-47 and 3′-OH–BDE-154 bound to TTR and TBG even stronger, respectively, than T4. With rising environmental level and high bioaccumulation capability, PBDEs have the potential to disrupt thyroid homeostasis by competitive binding with TH transport proteins.

Metal nanoparticles and carbon nanotubes have been shown to possess high electrocatalytic activity. Indium tin oxide (ITO) is a popular electrode material, but the electro-catalytic properties of its nano-materials have not been reported. We demonstrate here for the first time facile electrocatalytic oxidation of ascorbic acid on ITO nanoparticle-modified electrodes. Compared to the conventional ITO thin film electrode, the voltammetric peak potential for ascorbic acid oxidation was lowered by 800 mV on ITO nanoparticle-modified electrodes to a potential similar to metal electrodes. The ITO nanoparticle was composed of 90% In2O3 and 10% SnO2. Since the electrocatalytic activity was also found on In2O3 nanoparticle electrodes but not on SnO2 nanoparticle electrodes, the In2O3 composition in ITO nanoparticle is mainly responsible for the high activity. In photoluminescence measurement, two intense emission peaks at 415 nm and 438 nm associated with surface oxygen vacancies were observed on the semiconductor electrodes. It was hypothesized that the oxygen vacancies could be the active sites for electrocatalytic reactions. A linear relationship between the oxidation current and ascorbic acid concentration was found in the range of 10 μM to 5 mM, with a lower detection limit of 5 μM and 7.9% RSD (n = 11). The high electro-catalytic activity and transmittance of In2O3 and ITO nanoparticle electrodes make them potentially very useful in opto-electronic devices and chemical/bio-sensors.Highlights► Indium tin oxide (ITO) nanoparticle modified electrode exhibited high electrocatalytic activity for ascorbic acid oxidation. ► Voltammetric peak potential on the nanoparticle electrode was 800 mV lower than conventional ITO. ► Electrocatalytic activity was correlated with the defects on electrode surface.

The binding interactions between ligands and receptors play a vital role in their biological functions. In this work, a label-free indirect competitive binding assay based on surface plasmon resonance (SPR) detection was developed, using fatty acid–human serum albumin (HSA) as a model system. In the assay, a fatty acid analog was immobilized on the surface of an SPR gold chip by self-assembly, and was allowed to bind with HSA in solution. Addition of a ligand under investigation into the solution set up a competition with the analog for the protein, causing a reduction in the SPR signal. Due to the asymmetric structure of the ligand binding channel, HSA exhibited remarkable selectivity for the orientation of the fatty acid analog on a gold surface. When the carboxylic acid group of the analog was exposed on the sensor surface, the amount of bound HSA was very low. However, it increased by more than 7 fold when the alkyl chain was exposed. Dilution of the surface coverage of the fatty acid analog also enhanced HSA binding by reducing steric hindrance on the surface. Using a 1:4 mercaptoundecanoic acid–mercaptohexanol modified surface derivatized with octylamine, competitive binding assays for octanoic acid and perfluorooctane sulfonate with HSA were conducted. Using a derived kinetic equilibrium model, the equilibrium association constant was determined to be 4.57 × 105 M−1 and 2.11 × 105 M−1, respectively, which are consistent with the values measured by other established methods. The engineered sensor chip surface can be used to study the binding interactions of other chemicals with this important serum transport protein.

A comprehensive two-dimensional system coupling hydrophilic interaction chromatography (HILIC) and ion mobility-mass spectrometry (IM-MS) has been developed for the separation and analysis of alkylphenol ethoxylates (APEOs). The first-dimensional HILIC was performed on porous silica stationary phase using acetonitrile–water gradient elution, which was readily compatible with electrospray ionization (ESI), and enabled good chromatographic separation of APEO oligomers on account of their differences in ethoxy chain length. Maintaining the fidelity of pre-ionization resolution in the first dimension, the second-dimensional IM-MS employed a hybrid quadrupole ion mobility time-of-flight mass spectrometer and added an orthogonal post-ionization separation for APEOs based on their size, shape and electric charge during a very short period of 13.0 ms. By virtue of the combination of HILIC and IM-MS, comprehensive resolution according to both hydrophobicity difference and mobility disparity has been achieved for APEO ethoxy homologues. The orthogonality of the developed two-dimensional system was evaluated with the correlation coefficient and peak spreading angle of 0.2191 and 77.34° for octylphenol ethoxylates (OPEOs), and 0.1490 and 81.43° for nonylphenol ethoxylates (NPEOs). A significant enhancement in peak capacity was achieved for the comprehensive two-dimensional plane with the actual peak capacity calculated to be approximately 7 and 122 times higher than that of the two dimensions used alone, respectively. The attractive potential for removing the effects of isobaric interference of APEOs by the rapid and solvent-free ion mobility approach was also highlighted.Graphical abstractHighlights► A novel two-dimensional system combing HILLIC and IM-MS was constructed for the separation and analysis of APEOs. ► The developed plane exhibited unique selectivity and increased peak capacity. ► Ion mobility approach demonstrated attractive potential to remove isobaric interference.

Indirect competitive immunoassays were developed on protein microarrays for the sensitive and simultaneous detection of multiple environmental chemicals in one sample. In this assay, a DNA/SYTOX Orange conjugate was employed as an antibody label to increase the fluorescence signal and sensitivity of the immunoassays. Epoxy-modified glass slides were selected as the substrate for the production of 4 × 4 coating antigen microarrays. With this signal-enhancing system, competition curves for 17β-estradiol (E2), benzo[a]pyrene (BaP) and 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47) were obtained individually on the protein microarray. The IC50 and calculated limit of detection (LOD) are 0.32 μg L−1 and 0.022 μg L−1 for E2, 37.2 μg L−1 and 24.5 μg L−1 for BaP, and 31.6 μg L−1 and 2.8 μg L−1 for BDE-47, respectively. LOD of E2 is 14-fold lower than the value reported in a previous study using Cy3 labeled antibody (Du et al., Clin. Chem, 2005, 51, 368–375). The results of the microarray immunoassay were within 15% of chromatographic analysis for all three pollutants in spiked river water samples, thus verifying the immunoassay. Simultaneous detection of E2, BaP and BDE-47 in one sample was demonstrated. There was no cross-reaction in the immunoassay between these three environmental chemicals. These results suggest that microarray-based immunoassays with DNA/dye conjugate labels are useful tools for the rapid, sensitive, and high throughput screening of multiple environmental contaminants.

Many chemicals have been found to induce DNA damages which may lead to gene mutation and tumor generation. In this report, a microplate-based photoelectrochemical DNA sensor array was developed for the rapid and high throughput screening of DNA damaging chemicals. A 96-well plate with built-in electrodes was fabricated on a plastic substrate by the standard electronics industry processes. The working electrode in each well was deposited with SnO2 nanoparticles, and the resulting film was sintered at low temperatures tolerable for the plastic substrate. The film was characterized by scanning electron microscopy, X-ray diffraction and X-ray photoelectron spectroscopy. On the plates sintered at 150 °C, a significant amount of photocurrent was obtained in a Ru(bpy)32+ (bpy = 2,2′-bipyridine) solution. To construct a DNA sensor, poly-(diallydimethyl ammonium chloride) and double-stranded DNA were sequentially assembled on the SnO2 electrode by electrostatic interaction, and a DNA intercalator, Ru(bpy)2(dppz)2+ (dppz = dipyrido[3,2-a:2′,3′-c]phenazine) was used as the photoelectrochemical signal indicator. After the DNA film was exposed to tetrafluoro-1,4-benzoquinone (TFBQ) or TFBQ/H2O2, the photocurrent dropped by 38% and 73% respectively. The photocurrent reduction can be attributed to less binding of Ru(bpy)2(dppz)2+ to the electrode after DNA damage. Photocurrent measurement of the entire 96-well plate was completed within 22 min automatically.

A “turn-on” photoelectrochemical sensor for Hg2+ detection based on thymine-Hg2+-thymine interaction is presented by using a thymine-rich oligonucleotide film and a double-strand DNA intercalator, Ru(bpy)2(dppz)2+ (bpy=2,2′-bipyridine, dppz=dipyrido[3,2-a:2′,3′-c]phenazine) as the photocurrent signal reporter. The presence of Hg2+ induces the formation of a double helical DNA structure which provides binding sites for Ru(bpy)2(dppz)2+. The double helical structure was confirmed by circular dichroism and fluorescence measurements. Under the optimized conditions, a linear relationship between photocurrent and Hg2+ concentration was obtained over the range of 0.1 nM to 10 nM Hg2+, with a detection limit of 20 pM. Interference by 10 other metal ions was negligible. Analytical results of Hg2+ spiked into tap water and lake water by the sensor were in good agreement with mass spectrometry data. With the advantages of high sensitivity and selectivity, simple sensor construction, low instrument cost and low sample volume, this method is potentially suitable for the on-site monitoring of Hg2+ contamination.Highlights► Photoelectrochemical DNA sensor was developed for Hg2+ detection. ► Linear range was 0.1–10 nM, detection limit 20 pM. ► Interference from 10 common metal ions was negligible. ► The sensor was validated with Hg2+ spiked tap and lake water samples.

Label-free electrochemical (EC) protein biosensors that derive electrical signal from redox-active amino acid (AA) residues can avoid disruption of delicate protein structures, and thus provide a great opportunity to reveal valid information about protein functions. However, the challenge is that such a signal is usually very limited due to the sluggish EC reaction of free AAs on most common electrodes and slow electron-transfer rates from the deeply-buried AA residues in a protein to the electrode. Signal enhancement therefore becomes crucial. We first survey recent progress in this area.We present a signal-enhancing system that relies on the electrocatalytic oxidation of tyrosine mediated by osmium bipyridine or phenoxazine complexes. We describe several applications of label-free protein EC biosensors based on this detection principle for the analysis of protein functions, including the monitoring of protein-conformation change, study of ligand/protein binding, and detection of protein oxidative damage and protein phosphorylation.We describe related works on protein-function analysis using other signal-enhancing methods. The results suggest that label-free EC protein biosensors are suitable for the rapid survey of protein functions due to their fast response, ease of integration, cost effectiveness and convenience. Proof-of-concept work on the application of our system is paving the way for bio-analytical detections and protein-function analysis in future work.Highlights► Work on electrochemical (EC) biosensors for protein function analysis is reviewed. ► Focus is on label-free EC sensors based on electrocatalytic oxidation of tyrosines. ► Studies on protein conformation change and ligand binding are described. ► Detection of protein oxidative damage and protein phosphorylation is described.

Electrochemiluminescence (ECL) systems using Ru(bpy)32+ (bpy = 2,2′-bipyridyl) and tripropylamine (TPA) on metal and carbon electrodes are widely used in immunoassay and DNA hybridization detections. The large overpotential of TPA oxidation on indium tin oxide (ITO) electrodes has limited their ECL application. In this work, significant enhancement of ECL signal from the Ru(bpy)32+/TPA system was achieved on ITO nanoparticle-modified electrode. Due to the electrocatalytic activity of ITO nanoparticle electrodes, TPA oxidation potential was found to shift negatively by 600 mV, leading to 24-fold increases in ECL peak intensity over the conventional ITO electrode, and 2-fold increases in the integrated signal over the conventional gold electrode. As a result, the detection limit for Ru(bpy)32+ in solution was 20-fold lower than the value on the conventional ITO electrode. Because the transmittance of ITO at 610 nm (the wavelength of Ru(bpy)32+ emission maximum) was reduced only slightly after nanoparticle modification, it may offer a better alternative than metal and carbon electrodes in ECL measurements.

A novel label-free electrochemical method for measuring the activity of protein tyrosine kinases (PTK) has been developed. Epidermal growth factor receptor (EGFR), a typical PTK associated with a large percentage of all solid tumors, was used as the model kinase. Poly(glu, tyr) (4:1) peptide, as a substrate of EGFR, was covalently immobilized on the surface of indium tin oxide (ITO) electrode by silane chemistry. The tyrosine (Tyr) residue in the polypeptide served as an electrochemical signal reporter. Its voltammetric current was catalyzed by a dissolved electron mediator Os(bpy)32+ (bpy = 2,2′-bipyridine) for increased sensitivity. Phosphorylation of the Tyr led to a loss of its electrochemical current, thus providing a sensing mechanism for PTK activity. Experimental conditions for the silanization of ITO surface and immobilization of polypeptide were investigated in details to facilitate the generation of Tyr electrochemical signal. The proposed biosensor exhibited high sensitivity and excellent stability. The limit of detection for EGFR was 1 U mL−1. Furthermore, this biosensor can also be used for quantitative analysis of kinase inhibition. On the basis of the inhibitor concentration dependent electrochemical signal, the half-maximal inhibition value IC50 of three EGFR inhibitors, PD-153035, OSI-774 and ZD-1839, and their corresponding inhibition constants Ki were estimated, which were in agreement with those obtained from the conventional kinase assay. This electrochemical biosensor can be implemented in an array format for the high throughput assay of in vitro PTK activity and PTK inhibitors screening for practical diagnostic application and drug discovery.

Nanomaterials have been used increasingly in a wide variety of applications, and some of them have shown toxic effects on experimental animals and cells. In this study, a previously established photoelectrochemical DNA sensor was employed to rapidly detect DNA damage induced by polystyrene nanosphere (PSNS) suspensions. In the sensor, a double-stranded DNA film was assembled on a semiconductor electrode, and a DNA intercalator, Ru(bpy)2(dppz)2+ (bpy = 2,2′-bipyridine, dppz = dipyrido[3,2-a:2′,3′-c]phenazine) was used as the photoelectrochemical signal indicator. After the DNA-modified electrode was exposed to 2.0 mg/mL PSNS suspension, photocurrent of DNA-bound Ru(bpy)2(dppz)2+ decreased by about 20%. The decrease is attributed to the chemical damage of DNA and consequently less binding of Ru(bpy)2(dppz)2+ molecules to the electrode. Gel electrophoresis of DNA samples incubated with PSNS suspension confirmed DNA damage after the chemical exposure. However, in both photoelectrochemical and gel electrophoresis experiments, extensively washed PSNS did not induce any DNA damage, and the supernatant of PSNS suspension exhibited comparable DNA damage as the unwashed PSNS suspension. Furthermore, UV-visible absorption spectrum of the supernatant displayed a pattern very similar to that of styrene oxide (SO), a compound which has been shown to induce DNA damage by forming covalent DNA adducts. It is therefore suggested that styrene oxide and other residual chemicals in the PSNS may be responsible for the observed DNA damage. The results highlight the importance of full characterization of nanomaterials before their toxicity study, and demonstrate the utility of photoelectrochemical DNA sensors in the rapid assessment of DNA damage induced by chemicals and nanomaterials.

A comprehensive two-dimensional system coupling ultra-performance liquid chromatography (UPLC) and ion mobility-mass spectrometry (IM-MS) has been applied for the separation and analysis of hydroxylated polybrominated diphenyl ethers (OH-PBDEs). A complex mixture containing 23 OH-PBDE congeners ranging from hydroxylated monobromodiphenyl ether (OH-monoBDE) to hydroxylated octabromodiphenyl ether (OH-octaBDE) was satisfactorily separated within 16 min of analysis time. The first-dimensional reversed-phase UPLC was performed on a sub-2 μm BEH C18 chromatographic column using acetonitrile-water gradient elution program with a flow rate ramp. It enabled excellent chromatographic separation for both between-class and within-class OH-PBDEs based on their differences in hydrophobicity. Following the pre-ionization resolution in the first dimension, the second-dimensional IM-MS employed a hybrid electrospray quadrupole ion mobility time-of-flight mass spectrometer and added an extra post-ionization separation for between-class OH-PBDE congeners on account of their relative mobility disparity during a very short period of 8.80 ms. The orthogonality of the developed two-dimensional system was evaluated with the correlation coefficient of 0.9665 and peak spreading angle of 14.87°. The peak capacity of the system was calculated to be approximately 2 and 15 times higher than that of the two dimensions used alone, respectively. The two-dimensional separation plane also contributed to the removal of background interference ions and the enhanced confidence in the characterization of OH-PBDEs of interest.

A simple and sensitive electrochemical biosensor was used to detect tyrosine oxidation induced by hydroxyl radicals generated by Fenton reaction (Fe2+/H2O2). Poly(glu, tyr) (4:1) peptides were immobilized on indium tin oxide (ITO) electrode surface via layer-by-layer assembly technique, and Os(bpy)32+-mediated tyrosine oxidation current was employed as the signal reporter of the biosensor. It was found that the electrochemical signal of the peptide decreased markedly after incubation with Fenton reagents. Interestingly, l-dopa, the oxidation product of tyrosine, was likely to form complexes with Fe(III), which could suppress the electro-oxidation of l-dopa and resulted in decrease of current response. Our results indicate that the peptide damage involved two steps and was a second-order reaction. X-ray photoelectron spectroscopy was used to quantitatively determine nitrogen elemental percentage on peptide-coated electrode surface, which eliminated the possibility that signal decrease was caused by peptide backbone cleavage. Moreover, the lowest concentration of Fenton reagents that could be detected was 10 μM Fe2+ or H2O2, similar to the level in vivo. We suggest that the biosensor can be used to detect protein damage induced by Fenton reaction.

Strategies for electrochemical sensing of DNA can be classified into label-free and label-based approaches, categories of which include enzyme-, nanomaterial- and redox labels that are attached to DNA either by covalent or non-covalent means. Metallointercalators represent one group of small molecule redox labels that non-covalently enter the groove of a DNA. The metallointercalator plays a dual-role in acting as a structure indicator (for hybridization) and a signal generator. Labeling is not needed, and electrochemical measurements can be carried out in a label-free solution of an electrolyte. However, such metallointercalators lack the option of catalytic signal generation as in the case of enzyme- and nanomaterial-based labels. Therefore, signal amplification becomes crucial. We first survey here recent progress in this area. A signal-amplifying system is presented that relies on the electroatalytic oxidation of a metallointercalator ruthenium(II)bipyridine/phenoxazine complex in the presence of electron donor species such as oxalate, DNA bases, or tripropylamine. Recent work on such DNA sensors is discussed. Results suggest that such metallointercalator-based DNA sensors represent a viable platform for developing high-throughput and automated PCR/lab-on-a-chip devices as well as visualized multifunctional DNA sensors.

Non-covalent binding interactions of small molecules with DNA play important roles in regulating gene expression and gene function. In this work, a highly sensitive electrochemiluminescence (ECL) displacement method has been developed to investigate such interactions, particularly for weak DNA binders. This ECL method relies on a double-stranded DNA film deposited on an indium tin oxide electrode (ITO) surface by layer-by-layer self-assembly. A DNA intercalator, [Ru(bpy)2(dppz)]2+ (bpy = 2,2′-bipyridine, dppz = dipyrido[3,2-a:2′3′-c]phenazine), is employed as the ECL signal indicator. If a test compound competes with the indicator for the same binding sites in DNA, it would displace the indicator from the film and reduce ECL signal. The new method was validated by measuring five well-known DNA-binding organic molecules including quinacrine, H33258, thiazole orange, ethidium bromide and 4,6-diamidine-2-phenylindole dihydrochloride. Due to high ECL sensitivity, only 0.4 μmol L−1 [Ru(bpy)2(dppz)]2+ was needed in the ECL displacement measurement, which is about 75-fold less than the concentration in the voltammetric measurement. The lowered concentration permitted direct measurement of IC50 values of eight hydroxylated polycyclic aromatic hydrocarbons in their ECL displacement curves and subsequent calculation of their binding constants with DNA. The ECL displacement method is particularly useful for investigating weak DNA binders with limited aqueous solubility.

Facile electrical communication between redox-active labeling molecules and electrode is essential in the electrochemical detection of bio-affinity reactions. In this report, nanometer-sized indium tin oxide (ITO) particles were employed in the fabrication of porous thick film electrodes to enhance the otherwise impeded electrochemical activity of redox labels in multi-layered protein films, and to enable quantitative detection of avidin/biotin binding interaction. To carry out the affinity reaction, avidin immobilized on an ITO electrode was reacted with mouse IgG labeled with both biotin and ruthenium Tris-(2,2′-bipyridine) (Ru–bipy). The binding reaction between avidin and biotin was detected by the catalytic voltammetry of Ru–bipy in an oxalate-containing electrolyte. On sputtered ITO thin film electrode, although a single layer of Ru–bipy labeled avidin exhibited substantial anodic current, attaching the label to the outer IgG layer of the avidin/biotin–IgG binding pair resulted in almost complete loss of the signal. However, electrochemical current was recovered on ITO film electrodes prepared from nanometer-sized particles. The surface of the nanoparticle structured electrode was found by scanning electron microscopy to be very porous, and had twice as much surface binding capacity for avidin as the sputtered electrode. The results were rationalized by the assumption of different packing density of avidin inner layer on the two surfaces, and consequently different electron transfer distance between the electrode and Ru–bipy on the IgG outer layer. A linear relationship between electrochemical current and IgG concentration was obtained in the range of 40–4000 nmol L−1 on the nanoparticle-based electrode. The approach can be employed in the electrochemical detection of immunoassays using non-enzymatic redox labels.

Silica nanoparticles are most commonly modified with amino-silanes, followed by post-modification activation for protein immobilization. In this work, epoxy-functionalized silica nanoparticles were prepared by modification with glycidyloxypropyl trimethoxysilane (GPTMS) for direct protein immobilization. Silica nanoparticles possessed an average size of 46 nm, but increased to 63 nm after GPTMS modification. Reaction time, reaction temperature and GPTMS content had no significant effect on particle size. Zeta potential of SiO2 changed from −26mV to +38mV after modification. Fourier-transformed infrared spectroscopy revealed alkyl C-H bending and stretching bands at 2944 cm−1, 1343 cm−1 and 1465 cm−1, respectively, for the modified nanoparticles. Fluorescein cadaverine was found to bind to GPTMS-modified SiO2, but not to bare SiO2, indicating the chemical reactivity of epoxy groups on the modified nanoparticle with amines. Finally, fluorescently labeled bovine serum albumin (BSA) was used as a model protein to investigate the capacity of epoxy-SiO2 nanoparticles for protein immobilization. The results showed that more proteins were immobilized on the particle with longer reaction time, higher NaCl concentration, lower pH, and less GPTMS content. More importantly, proteins bound to epoxy-SiO2 nanoparticle were highly stable. Under optimized reaction conditions, as much as 25 mg BSA/g nanoparticle was covalently attached to the nanoparticle. The epoxy silane modification of silica nanoparticles offers a reactive surface for one-step and high-density protein immobilization.

A redox-labeled direct competitive electrochemical immunoassay for polycyclic aromatic hydrocarbons (PAHs) was developed. A ruthenium tris(bipyridine)-pyrenebutyric acid conjugate was synthesized as the redox-labeled tracer. Its recognition by an anti-PAH monoclonal antibody was confirmed by surface plasmon resonance. In the immunoassay, the antibody was immobilized on (3-glycidoxypropyl)-trimethoxysilane (GPTMS)-modified indium tin oxide (ITO) electrodes. The assay was quantified by measuring the electro-catalytic current of the redox label in an oxalate-containing electrolyte which served as a sacrificial electron donor to amplify the current signal. Formation of GPTMS film on ITO and subsequent antibody immobilization were characterized by X-ray photoelectron spectroscopy (XPS) and electrochemistry. Using a ruthenium tris(bipyridine)-conjugated IgG (IgG-Ru) as the surface-bound redox probe, the highest electrochemical signal was obtained on GPTMS electrodes with 1 h modification. Under the optimized conditions for ITO modification, antibody immobilization and tracer concentration, competition curves for benzo[a]pyrene and pyrenebutyric acid were obtained with a detection limit of 2.4 and 10 ng mL−1, respectively. The redox-labeled electrochemical immunoassay with signal amplification mechanism offers a potential analytical method for the simultaneous detection of multiple environmental organic pollutants on antibody biochips.

Binding of five perfluoroalkyl acids with human serum albumin (HSA) was investigated by site-specific fluorescence. Intrinsic fluorescence of tryptophan-214 in HSA was monitored upon addition of the chemicals. Although perfluorobutyl acid (PFBA) and perfluorobutane sulfonate (PFBS) did not cause fluorescence change, perfluorooctanoic acid (PFOA), perfluorooctane sulfonate (PFOS), and perfluorododecanoic acid (PFDoA) induced fluorescence quenching, from which binding constant of 2.7 × 105 M−1 for PFOA and 2.2 × 104 M−1 for PFOS was calculated. Two fluorescent probes, dansylamide (DA) and dansyl-l-proline (DP), were employed in fluorescence displacement measurements to study the interaction at two Sudlow’s binding sites. At Site I, both PFBA and PFBS displaced DA with binding constants of 1.0 × 106 M−1 and 2.2 × 106 M−1. At Site II, PFBS and PFDoA displaced DP with binding constants of 6.5 × 106 M−1 and 1.2 × 106 M−1, whereas PFBA did not bind. The data were compared with fatty acids to evaluate the potential toxicological effect of these environmental chemicals.

The binding interaction of many organic carcinogens such as polycyclic aromatic hydrocarbons with DNA is the key step in their genotoxic effect. In this work, an electrochemical displacement method was developed to study such interaction. In the method, a DNA film is deposited on an indium tin oxide electrode surface by layer-by-layer assembly, and a redox-active DNA intercalator Ru(bpy)2(dppz)(BF4)2 (bpy = 2,2′-bipyridine, dppz = dipyrido [3,2-a:2′,3′-c] phenazine) is employed as an electrochemical indicator. If an organic compound competes with the indicator for the same binding site on DNA in the film, it would displace the ruthenium complex from DNA, resulting in a reduction in the measured electrochemical signal. From the titration curve, the binding constant of the organic compound with DNA can be calculated. With the use of oxalate as an electron donor to chemically amplify the oxidation current of the indicator, chemicals can be tested at low micromolar concentrations. Five well-known DNA binding polycyclic organic compounds, thiazole orange, 4,6-diamidine-2-phenylindole, H33258, ethidium bromide, and quinacrine, were investigated by the displacement method. The binding constants obtained in our experiments fall in the range of (4.3 × 105) to (1.2 × 107) M−1, which are generally consistent with those reported in the literature by some established methods. The electrochemical method provides a general tool that complements the commonly used spectroscopic methods for the study of DNA/small molecule interactions.

Photoelectrochemical sensors were developed for the rapid detection of oxidative DNA damage induced by Fe2+ and H2O2 generated in situ by the enzyme glucose oxidase. The sensor is a multilayer film prepared on a tin oxide nanoparticle electrode by layer-by-layer self-assembly and is composed of separate layers of a photoelectrochemical indicator, DNA, and glucose oxidase. The enzyme catalyzes the formation of H2O2 in the presence of glucose, which then reacts with Fe2+ and generates hydroxyl radicals by the Fenton reaction. The radicals attack DNA in the sensor film, mimicking metal toxicity pathways in vivo. The DNA damage is detected by monitoring the change of photocurrent of the indicator. In one sensor configuration, a DNA intercalator, Ru(bpy)2(dppz)2+ (bpy = 2,2′-bipyridine, dppz = dipyrido[3,2-a:2′,3′-c]phenazine), was employed as the photoelectrochemical indicator. The damaged DNA on the sensor bound less Ru(bpy)2(dppz)2+ than the intact DNA, resulting in a drop in photocurrent. In another configuration, ruthenium tris(bipyridine) was used as the indicator and was immobilized on the electrode underneath the DNA layer. After oxidative damage, the DNA bases became more accessible to photoelectrochemical oxidation than the intact DNA, producing a rise in photocurrent. Both sensors displayed substantial photocurrent change after incubation in Fe2+/glucose in a time-dependent manner. And the detection limit of the first sensor was less than 50 µM. The results were verified independently by fluorescence and gel electrophoresis experiments. When fully integrated with cell-mimicking components, the photoelectrochemical DNA sensor has the potential to become a rapid, high-throughput, and inexpensive screening tool for chemical genotoxicity.

Protein phosphorylation plays an important role in many significant cellular processes, and has thus gained tremendous interest in the field of proteomics. The electro-active tyrosine residue, as an important receptor of phosphorylation in proteins, exhibits electro-inactivity after being phosphorylated on the hydroxy group of its aromatic ring. In this study, the electrochemical oxidation of tyrosine on indium tin oxide (ITO) electrodes was catalyzed with an electron mediator Os(bpy)32+ (bpy = 2,2′-bipyridine) and was employed as a signal reporter to differentially detect non-phosphorylated and phosphorylated peptides. A short, tyrosine-containing peptide glu-glu-glu-glu-glu-tyr (EY-6) was immobilized on an ITO surface using the layer-by-layer self-assembly method, and was detected by cyclic voltammetry in an Os(bpy)32+ solution. The limit of detection was about 0.23 μg mL−1 EY-6 in solution. The phosphorylated peptide glu-glu-glu-glu-glu-tyr-OP (EY-6P) did not produce an appreciable oxidation current on the electrode. Surface plasmon resonance measurements revealed that the amount of EY-6 and EY-6P adsorbed on the sensor chip surface was 269 and 378 pg mm−2, respectively. The poly(glu, tyr) (4 : 1) peptide, a protein tyrosine kinase substrate, was also detected by the same approach, with a detection limit of 0.65 μg mL−1. This new approach offers the possibility of label-free and on-chip detection of protein tyrosine kinase activity.

•Two genes encoding glutathione S-transferases are cloned from Anodonta woodiana.•AwGST1 and AwGST2 have a widely tissues distribution.•Expressions of AwGST1 are significantly induced by pentachlorophenol.•Expressions of AwGST2 are significantly up-regulated by pentachlorophenol.Glutathione S-transferases (GST) play a prominent role in protecting cells against oxidative stress. Our previous study showed that the reactive oxygen species (ROS) generated from pentachlorophenol (PCP) could cause an acute impact on freshwater bivalve Anodonta Woodiana, but its chronic toxicity remain unclear. In order to investigate the chronic effect of PCP, clams A. Woodiana were randomly grouped into PCP treated group in which animals were administrated with 13.9 μg/L concentrations of PCP, and control group those with similar volume dimethyl sulfoxide. In addition, two complete GST sequences were isolated from A. Woodianaa and respectively named AwGST1 and AwGST2. The full-length cDNA of AwGST1 was consisted of a 5′ untranslated region (UTR) of 132 bp, a 3′ UTR of 80 bp and an open reading frame (ORF) of 609 bp encoding a polypeptide of 203 amino acids. The full-length cDNA of AwGST2 contained a 5′ UTR of 57 bp, a 3′ UTR of 291 bp and an ORF of 678 bp encoding a polypeptide of 226 amino acids. The constitutive expression levels of AwGST1 and AwGST2 were examined in different tissues including foot, mantle, adductor muscle, heart, hepatopancreas, hemocytes and gill. Administration of PCP could result in a significant increase of AwGST1 and AwGST2 expression in the hepatopancreas, gill and hemocytes. In the hepatopancreas, AwGST1 mRNA levels of PCP treated group increased more than 28.73% at day 1, then 70.37% (P < 0.05) at day 3, reach to 6.64 times (P < 0.01) at day 15 in contrasted with that of control group. AwGST2 increased more 18.18%, 82.88% (P < 0.05) and 2.43 times (P < 0.01) at day 1, 3 and 15, respectively. In the gill, AwGST1 expression showed a significant up-regulation in the PCP treated group during experiment observed compared with that of control group, mRNA level of AwGST2 increased more than 1.44 times (P < 0.05). In addition, expressions of AwGST1 and AwGST2 were significantly induced after PCP treatment in the hemocytes. These results indicated that up-regulations of AwGST1 and AwGST2 expression in bivalve A. woodiana are contribute to against oxidative stress derived from PCP treatment during experiment observed.

•UV irradiation mediated the aggregation behavior of TiO2 nanoparticle in aquatic system.•Long-term UV irradiation accelerated TiO2 aggregation rate in salt solutions.•The photochemical transformation of TiO2 changed their adsorption toward SRHA molecules.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.Download high-res image (292KB)Download full-size image

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 TRβ, with TDCPP being the most potent one. Moreover, molecular docking revealed that all the tested OPEs could fit into the ligand binding pocket of TRβ, 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.Download full-size image

•Amino acid sequence of AwSeGPx shows a high similarity with that of other Se-GPx.•Expression of AwSeGPx is significantly up-regulated by 2,4-dichlorophenol.•Expression of AwSeGPx is significantly up-regulated by 2,4,6-trichlorophenol.•Expression of AwSeGPx is significantly up-regulated by pentachlorophenol.2,4-dichlorophenol (2,4-DCP), 2,4,6-trichlorophenol (2,4,6-TCP), and pentachlorophenol (PCP) pose a health risk to aquatic organism and humans, and are recognized as persistent priority pollutants. Selenium dependent glutathione peroxidase (Se-GPx) belongs to the family of selenoprotein, which acts mainly as an antioxidant role in the cellular defense system. In the current study, a Se-GPx full length cDNA was cloned from Anodonta woodiana and named as AwSeGPx. It had a characteristic codon at 165TGA167 that corresponds to selenocysteine(Sec) amino acid as U44. The full length cDNA consists of 870 bp, an open reading frame (ORF) of 585 bp encoded a polypeptide of 195 amino in which conserved domain (68LGFPCNQF75) and a glutathione peroxide-1 GPx active site (32GKVILVENVASLUGTT47) were observed. Additionally, the eukaryotic selenocysteine insertion sequence (SECIS) was conserved in the 3′UTR. The AwSeGPx amino acid sequence exhibited a high similarity with that of other Se-GPx. Real-time PCR analysis revealed that AwSeGPx mRNA had a widely distribution, but the highest level was observed in hepatopancreas. AwSeGPx mRNA expression was significantly up-regulated in hepatopancreas, gill and hemocytes after 2,4-DCP, 2,4,6-TCP and PCP exposure. Under similar environment, clams A. woodiana showed a more sensitive to PCP than that of 2,4-DCP and 2,4,6-TCP. These results indicate that AwSeGPx plays a protective role in eliminating oxidative stress derived from 2,4-DCP, 2,4,6-TCP and PCP treatment.

•Full-lengths of Cu/Zn SOD and CAT were first obtained from bivalve Anodonta woodiana.•Protein sequence of AwSOD showed a high similarity with Cu/Zn SODs of other species.•Protein sequence of AwCAT showed a high identify with CATs of other species.•Expressions of AwSOD and AwCAT were significantly affected by PBDE-47 and -209.Polybrominated diphenyl ethers-47 (PBDE-47) and -209 are significant components of total PBDEs in water and can catalyze the production of reactive oxygen species (ROS) in the organisms. Anti-oxidant enzymes play an important role in scavenging the high level of ROS. In the current study, two full-length cDNAs of Cu/Zn superoxide dismutase (CuZnSODs) and catalase (CAT) were isolated from freshwater bivalve Anodonta woodiana by rapid amplification of cDNA ends approach and respectively named as AwSOD and AwCAT. The nucleotide sequence of AwSOD cDNA had an open reading frame (ORF) of 465 bp encoding a polypeptide of 155 amino acids in which signature 1 GKHGFHVHEFGDNT and signature 2 GNAGARSACGVI of SODs were observed. Deduced amino acid sequence of AwSOD showed a significant similarity with that of CuZnSODs. AwCAT had an ORF 1536 bp encoding a polypeptide of 512 amino acids which contains a conserved catalytic site motif, and a proximal heme-ligand signature motif of CATs. The time-course expressions of AwSOD and AwCAT in hepatopancreas were measured by quantitative real-time PCR. Expressions of AwSOD and AwCAT showed a significant up-regulation in groups at a low concentration treatment of PBDE-47, a biphasic pattern in groups with a high concentration treatment. Administration of PBDE-209 could result in an up-regulation of AwSOD and AwCAT expressions with time- and dose-dependent matter. These results indicate that up-regulations of AwSOD and AwCAT expression of hepatopancreas of freshwater bivalve A. woodiana contribute to eliminate oxidative stress derived from PBDE-47 and -209 treated.

•Full-lengths of Cu/Zn SOD and CAT were first obtained from bivalve Anodonta woodiana.•Protein sequence of AwSOD showed a high similarity with Cu/Zn SODs of other species.•Protein sequence of AwCAT showed a high identify with CATs of other species.•Expressions of AwSOD and AwCAT were significantly affected by PBDE-47 and -209.Polybrominated diphenyl ethers-47 (PBDE-47) and -209 are significant components of total PBDEs in water and can catalyze the production of reactive oxygen species (ROS) in the organisms. Anti-oxidant enzymes play an important role in scavenging the high level of ROS. In the current study, two full-length cDNAs of Cu/Zn superoxide dismutase (CuZnSODs) and catalase (CAT) were isolated from freshwater bivalve Anodonta woodiana by rapid amplification of cDNA ends approach and respectively named as AwSOD and AwCAT. The nucleotide sequence of AwSOD cDNA had an open reading frame (ORF) of 465 bp encoding a polypeptide of 155 amino acids in which signature 1 GKHGFHVHEFGDNT and signature 2 GNAGARSACGVI of SODs were observed. Deduced amino acid sequence of AwSOD showed a significant similarity with that of CuZnSODs. AwCAT had an ORF 1536 bp encoding a polypeptide of 512 amino acids which contains a conserved catalytic site motif, and a proximal heme-ligand signature motif of CATs. The time-course expressions of AwSOD and AwCAT in hepatopancreas were measured by quantitative real-time PCR. Expressions of AwSOD and AwCAT showed a significant up-regulation in groups at a low concentration treatment of PBDE-47, a biphasic pattern in groups with a high concentration treatment. Administration of PBDE-209 could result in an up-regulation of AwSOD and AwCAT expressions with time- and dose-dependent matter. These results indicate that up-regulations of AwSOD and AwCAT expression of hepatopancreas of freshwater bivalve A. woodiana contribute to eliminate oxidative stress derived from PBDE-47 and -209 treated.

Polybrominated diphenyl ethers (PBDEs) have been shown to alter thyroid hormone level in experimental animals. One of the possible mechanisms for hormone disruption is the competitive binding of hydroxylated PBDEs (OH-PBDEs) with hormone transport proteins. In this study, binding interaction of 14 diversely structured OH-PBDEs with two thyroxine transport proteins was investigated by fluorescence displacement assay, circular dichroism, and molecular docking. Binding affinity of the 14 OH-PBDEs with transthyretin (TTR) and thyroxine-binding globulin (TBG) was measured by competitive fluorescence displacement assay. The binding constant was found to fall in the range of 1.4 × 107 M−1 and 6.9 × 108 M−1 for TTR, and between 6.5 × 106 M−1 and 2.2 × 108 M−1 for TBG. Binding affinity increased significantly with bromination number from 1 to 4, whereas 5- and 6-brominated diphenyl ethers did not show any further increase. Protein secondary structural change of TTR and TBG upon binding with 5-OH-BDE-047 was investigated by circular dichroism. The spectral change displayed a pattern similar to the one with thyroxine, suggesting that the environmental chemical binds to the two proteins at the same sites as the hormone. In molecular docking analysis, a ligand-binding channel in TTR was revealed for OH-PBDEs binding, which appeared to be mostly hydrophobic inside but guarded by positively charged residue Lys15 at the entrance. Binding affinity of the 14 OH-PBDEs with TTR could be rationalized reasonably well by their pocket binding mode and hydrophobic characteristics. Based on the binding constant obtained in this work, possibility of in vitro competitive displacement of thyroid hormones from the transport proteins by OH-PBDEs was evaluated.

Polybrominated diphenyl ethers (PBDEs) have been shown to affect the estrogen receptor (ER) signaling pathway, and one of the proposed disruption mechanisms is direct binding of hydroxylated PBDE (OH-PBDE) to ER. In this paper, the binding affinity of 22 OH-PBDEs with different degrees of bromination to ER was assessed quantitatively using a surface plasmon resonance biosensor technique. Seven OH-PBDEs were found to bind directly with ER with KD ranging from 1.46 x 10−7 M to 7.90 x 10−6 M, and the affinity is in the order of 6-OH-BDE-047 ≧ 4′-OH-BDE-049 > 4′-OH-BDE-017 > 6′-OH-BDE-099 ≧ 5′-OH-BDE-099 > 2′-OH-BDE-007 > 3′-OH-BDE-028. In MVLN luciferase gene reporter assays, 10 low-brominated OH-PBDEs induced luciferase activity alone, but are 105 to 107 fold less potent than E2. Their estrogenic activity is in the order of 4′-OH-BDE-049 > 4′-OH-BDE-017 > 2′-OH-BDE-007 > 3′-OH-BDE-028 > 3-OH-BDE-047 ≧ 3′-OH-BDE-007. The good correlation between estrogenic activity and ER binding affinity of the low-brominated OH-PBDEs strongly suggest that these compounds induce ER transcriptional activity by binding directly with ER. The other 12 high-brominated OH-PBDEs inhibited luciferase activity of E2 to various degrees, demonstrating their antagonistic activity. Molecular docking analysis of the ER/OH-PBDE complexes revealed two distinctive binding modes between low- and high-brominated OH-PBDEs which provided rationale for the difference in their ER activity.Download full-size image

Binding of polycyclic aromatic hydrocarbons (PAHs) with DNA is one of the key steps in their mutagenic process. In this work, a previously established electrochemical displacement method was utilized to measure the binding constants (Kb) of 26 hydroxylated PAHs (OH-PAHs) with DNA. Eighteen OH-PAHs induced more than 50% signal reduction in the displacement measurement with calf thymus DNA, and Kb calculated from the EC50 value is between 8.3 × 104 and 3.0 × 105 M−1. Other eight OH-PAHs induced less than 50% signal reduction. For the latter compounds, EC50 values were obtained by fitting the displacement curve with a bi-exponential decay function and extrapolating it to 50% signal. Kb was then calculated, and was found to be in the range of 4.5 × 104 and 7.6 × 104 M−1. Measurements with polydG·polydC (pdGC) and polydA·polydT (pdAT) demonstrate that the OH-PAHs have no obvious selectivity toward DNA base pairs. In atomic force microscopy, a clear morphological change of calf thymus DNA from linear type to condensation form was observed after binding with 9-hydroxyfluorene. The change is similar to the one observed with the DNA-intercalating electrochemical indicator, suggesting that 9-hydroxyfluorene binds with DNA also by intercalation. An examination of the relationship between the molecular structure characteristics of the 26 OH-PAHs and their DNA-binding affinity revealed that the most positive net atomic charge on a hydrogen atom (q+H) correlated significantly with Kb at 0.05 level, with sig. (2-tailed) of 0.015. The correlation suggests that hydrogen bonding may play an important role in DNA/OH-PAH binding interaction. It was further revealed that Connolly accessible area (CAA), Connolly molecular area (CMA), and Connolly solvent-excluded volume (CSEV) of the nine hydroxybenzo[a]pyrenes correlated significantly with Kb at 0.01 or 0.05 level, with sig. (2-tailed) 0.019, 0.012 and 0.009, implying that steric effect might be an important factor in the binding of differently substituted OH-PAH with DNA.

Polybrominated diphenyl ethers (PBDEs) are used in large quantities as flame retardant additives in commercial products. Bio-monitoring data show that PBDE concentrations have increased rapidly in the bodies of wildlife and human over the last few decades. Based on the studies on experimental animals, the toxicological endpoints of exposure to PBDEs are likely to be thyroid homeostasis disruption, neuro-developmental deficits, reproductive ineffectiveness and even cancer. Unfortunately, the available molecular toxicological evidence for these endpoints is still very limited. This review focuses on the recent studies on the molecular mechanisms of PBDE toxicities carried out through the hormone receptor pathways, including thyroid hormone receptor, estrogen receptor, androgen receptor, progesterone receptor and aryl hydrocarbon receptor pathways. The general approach in the mechanistic investigation is to examine the in vitro direct binding of a PBDE with a receptor, the in vitro recruitment of a co-activator or co-repressor by the ligand-bound receptor, and the participation of the ligand in the receptor-mediated transcription pathways in cells. It is hoped that further studies in this area would provide more insights into the potential risks of PBDEs to human health.

The binding interactions between ligands and receptors play a vital role in their biological functions. In this work, a label-free indirect competitive binding assay based on surface plasmon resonance (SPR) detection was developed, using fatty acid–human serum albumin (HSA) as a model system. In the assay, a fatty acid analog was immobilized on the surface of an SPR gold chip by self-assembly, and was allowed to bind with HSA in solution. Addition of a ligand under investigation into the solution set up a competition with the analog for the protein, causing a reduction in the SPR signal. Due to the asymmetric structure of the ligand binding channel, HSA exhibited remarkable selectivity for the orientation of the fatty acid analog on a gold surface. When the carboxylic acid group of the analog was exposed on the sensor surface, the amount of bound HSA was very low. However, it increased by more than 7 fold when the alkyl chain was exposed. Dilution of the surface coverage of the fatty acid analog also enhanced HSA binding by reducing steric hindrance on the surface. Using a 1:4 mercaptoundecanoic acid–mercaptohexanol modified surface derivatized with octylamine, competitive binding assays for octanoic acid and perfluorooctane sulfonate with HSA were conducted. Using a derived kinetic equilibrium model, the equilibrium association constant was determined to be 4.57 × 105 M−1 and 2.11 × 105 M−1, respectively, which are consistent with the values measured by other established methods. The engineered sensor chip surface can be used to study the binding interactions of other chemicals with this important serum transport protein.

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.