Long non-coding RNA Arid2-IR affects advanced glycation end products-induced human retinal endothelial cell injury by binding to Smad3.

PURPOSE: Long non-coding RNAs (lncRNAs) have been reported to play significant roles in the pathogenesis of diabetic retinopathy (DR). The aim of the present study was to investigate the role of lncRNA Arid2-IR in advanced glycation end product (AGE)-induced human retinal endothelial cells (HRECs) injury. MATERIALS AND METHODS: Cell viability was determined by Cell Counting Kit-8 assay following induction with AGEs. The expression of Arid2-IR and Smad3 was detected by reverse transcription-quantitative PCR or western blotting. A luciferase reporter assay was conducted to determine the interaction between Arid2-IR and Smad3. The levels of inflammation-related and oxidative stress-related factors were evaluated by respective kits. The expression of extracellular matrix (ECM)-related and apoptosis-related proteins was detected by western blotting. Immunofluorescence assay was used to detect the level of vascular endothelial growth factor, and flow cytometry was applied to measure the levels of apoptosis. RESULTS: The results revealed that AGE treatment decreased HREC proliferation and upregulated the expression of Arid2-IR and Smad3. The luciferase assay indicated that Smad3 was able to bind to the promoter region sequence of Arid2-IR. Moreover, Arid2-IR silencing reduced inflammation, oxidative stress and ECM production induced by AGEs in HRECs, and Smad3 inhibition further reduced the levels of the aforementioned factors, while Smad3 overexpression exerted the opposite effect. Furthermore, apoptosis of HRECs induced by AGEs was decreased following Arid2-IR silencing, which was further reduced following treatment with Smad3 inhibitor, but was reversed after transfection with Smad3 pcDNA3.1. CONCLUSION: The findings demonstrated that Arid2-IR affects AGE-induced HREC injury by binding to Smad3.

Microarray analysis of altered long non-coding RNA expression profile in liver cancer cells treated by ginsenoside Rh2.

Microarray expression profiles of lncRNAs and mRNAs were investigated in HepG2 cells treated with 20 µg/ml ginsenoside Rh2 as well as in ginsenoside Rh2-untreated cells. Microarray analysis showed 618 upregulated lncRNAs and 161 downregulated lncRNAs in HepG2 cells treated with ginsenoside Rh2 compared with the control group. Moreover, three differentially expressed lncRNAs were validated by quantitative real-time polymerase chain reaction (qRT-PCR). This may be beneficial to patients as an anti-cancer treatment and potentially provide novel targets for HCC (hepatocellular carcinoma) therapy.

Toward Streamlined Identification of Dioxin-like Compounds in Environmental Samples through Integration of Suspension Bioassay.

Effect-directed analysis (EDA) is a powerful strategy to identify biologically active compounds in environmental samples. However, in current EDA studies, fractionation and handling procedures are laborious, consist of multiple evaporation steps, and thus bear the risk of contamination and decreased recoveries of the target compounds. The low resulting throughput has been one of the major bottlenecks of EDA. Here, we propose a high-throughput EDA (HT-EDA) work-flow combining reversed phase high-performance liquid chromatography fractionation of samples into 96-well microplates, followed by toxicity assessment in the micro-EROD bioassay with the wild-type rat hepatoma H4IIE cells, and chemical analysis of bioactive fractions. The approach was evaluated using single substances, binary mixtures, and extracts of sediment samples collected at the Three Gorges Reservoir, Yangtze River, China, as well as the rivers Rhine and Elbe, Germany. Selected bioactive fractions were analyzed by highly sensitive gas chromatography-atmospheric pressure laser ionization-time-of-flight-mass spectrometry. In addition, we optimized the work-flow by seeding previously adapted suspension-cultured H4IIE cells directly into the microplate used for fractionation, which makes any transfers of fractionated samples unnecessary. The proposed HT-EDA work-flow simplifies the procedure for wider application in ecotoxicology and environmental routine programs.

Effect-Directed Analysis of Aryl Hydrocarbon Receptor Agonists in Sediments from the Three Gorges Reservoir, China.

The construction of the Three Gorges Dam (TGD) in the Yangtze River raises great concern in ecotoxicological research since large amounts of pollutants enter the Three Gorges Reservoir (TGR) water bodies after TGD impoundment. In this work, effect-directed analysis (EDA), combining effect assessment, fractionation procedure, and target and nontarget analyses, was used to characterize aryl hydrocarbon receptor (AhR) agonists in sediments of the TGR. Priority polycyclic aromatic hydrocarbons (PAHs) containing four to five aromatic rings were found to contribute significantly to the overall observed effects in the area of Chongqing. The relatively high potency fractions in the Kaixian area were characterized by PAHs and methylated derivatives thereof and heterocyclic polycyclic aromatic compounds (PACs) such as dinaphthofurans. Benzothiazole and derivatives were identified as possible AhR agonists in the Kaixian area based on nontarget liquid chromatography-high resolution mass spectrometry (LC-HRMS). To our knowledge, this study is the first one applying the EDA approach and identifying potential AhR agonists in TGR.

Yangtze Three Gorges Reservoir, China: A holistic assessment of organic pollution, mutagenic effects of sediments and genotoxic impacts on fish.

Besides obvious benefits, the Three Gorges Dam's construction resulted in new pollution scenarios with the potentials to threaten the Three Gorges Reservoir (TGR) ecosystem. In order to record organic contamination, to find links to ecotoxicological impacts and to serve as reference for ensuing monitoring, several sites in the TGR area were screened applying the triad approach with additional lines-of-evidence as a holistic assessment method. Sediments and the benthic fish species Pelteobagrus vachellii were sampled in 2011 and 2012 to determine organic pollution levels, mutagenic potentials and genotoxic impacts. Two regional hot-spots near the cities of Chongqing and Kaixian were identified and further investigated in 2013. Only polycyclic aromatic hydrocarbons (PAHs) could be detected in sediments in 2011 (165-1653ng/g), emphasizing their roles as key pollutants of the area. Their ubiquity was confirmed at Chongqing (150-433ng/g) and Kaixian (127-590ng/g) in 2013. Concentrations were comparable to other major Chinese and German rivers. However, the immense sediment influx suggested a deposition of 216-636kgPAH/day (0.2-0.6mgPAH/(m(2)·day)), indicating an ecotoxicological risk. PAH source analysis highlighted primary impacts of combustion sources on the more industrialized upper TGR section, whereas petrogenic sources dominated the mid-low section. Furthermore, sediment extracts from several sites exhibited significant activities of frameshift promutagens in the Ames fluctuation assay. Additionally, significant genotoxic impairments in erythrocytes of P. vachellii were detected (Chongqing/Kaixian), demonstrating the relevance of genotoxicity as an important mode of action in the TGR's fish. PAHs, their derivatives and non-target compounds are considered as main causative agents.

Identification of differentially expressed genes induced by aberrant methylation in acute myeloid leukemia using integrated bioinformatics analyses.

Acute myeloid leukemia (AML) is a life-threatening hematological malignant disease. Methylation plays a crucial role in the etiology and pathogenesis of AML. The aim of the present study was to identify the aberrantly methylated differentially expressed genes (DEGs) in AML and determine the underlying mechanisms of tumorigenesis by conducting integrated bioinformatics analyses. Gene expression profiles (GSE109179, GSE142699, GSE49665 and GSE14772) and a gene methylation profile (GSE42042) were analyzed to identify the aberrantly methylated DEGs. Functional enrichment analyses of identified genes were conducted based on the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, and protein-protein interaction networks were established. Finally, the DEGs were validated by the reverse transcription-quantitative PCR analysis of patient samples. A total of seven downregulated hypermethylated genes and eight upregulated hypomethylated genes were validated. The differentially methylated DEGs were enriched in GO biological process terms associated with control of the immune response and the KEGG analysis indicated they were involved in AML, ferroptosis, TGF-ß signaling and necroptosis pathways. Additionally, five downregulated hypermethylated genes that were also tumor suppressor genes (TSGs) were identified. In vitro assays revealed that the overexpression of transcription factor 7 (TCF7) and integrin a M (ITGAM) significantly inhibited the proliferation of HL60 cells; by contrast, the knockdown of TCF7 and CAMK4 promoted HL60 cell proliferation. Overall, the present study identified differentially methylated DEGs and pathways associated with AML, which may enhance the understanding of the underlying molecular mechanisms of AML. In the future, abnormally methylated oncogenes and TSGs may function as biomarkers and treatment targets for the diagnosis and treatment of AML.

Prevalence and factors associated with visual impairment in middle-aged and older Chinese population.

Objective: This study aimed to estimate the prevalence of visual impairment and to identify the factors associated with it in China. Methods: Data for this cross-sectional study were retrieved from the China Health and Retirement Longitudinal Study (CHARLS) for a total of 16,480 subjects who completed the questionnaire between June 2011 and March 2012. The prevalence of visual impairment was estimated considering the complex survey design and response rate. Associated factors were identified using the weighted logistic regression analysis. Results: The overall prevalence of visual impairment among middle-aged and older Chinese adults was 6.22%. Regionally, Qinghai and Gansu provinces showed the highest prevalence of visual impairment, whereas Shanghai showed the lowest prevalence of visual impairment in China. Older age (OR = 1.888; 95% CI: 1.537-2.467) was correlated with a higher likelihood of visual impairment, whereas a non-alcohol intake (OR = 0.072; 95% CI: 0.018-0.246) was correlated with a lower likelihood of visual impairment. Hypertension (OR = 1.299; 95% CI: 1.189-1.467), diabetes (OR = 2.000; 95% CI: 1.163-3.765), lung diseases (OR = 1.795; 95% CI: 1.067-3.019), liver diseases (OR = 1.270; 95% CI: 1.221-2.876), stroke (OR = 1.135; 95% CI: 1.107-3.528), and heart disease (OR = 1.350; 95% CI: 1.104-1.708) were significantly associated with visual impairment. Conclusion: Geographical variations in the prevalence of visual impairment in China were defined, indicating that such variations do exist in China. Age, alcohol intake, hypertension, diabetes, lung diseases, liver diseases, stroke, and heart disease were factors associated with visual impairment.

Dihydromyricetin Alleviates High Glucose-Induced Oxidative Stress and Apoptosis in Human Retinal Pigment Epithelial Cells by Downregulating miR-34a Expression.

BACKGROUND: Diabetic retinopathy (DR) is one of the most common microvascular complications of diabetes mellitus, which leads to neuronal and vascular dysfunction in the retina with a final outcome of complete loss of vision. The aim of the present study was to investigate the effects of dihydromyricetin (DHM), a natural flavanol compound, on diabetic retinopathy (DR) and identify its potential mechanisms. METHODS: Retinal pigment epithelial cell line (ARPE-19) treated with high glucose (HG) was used to simulate the DR model in vitro. After treatment with different concentrations of DHM, the cell viability, production of reactive oxygen species (ROS) and the levels of oxidative stress-related markers in the in vitro model were detected using corresponding kits. Cell apoptosis was determined using terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) staining, and the expression of apoptotic proteins was examined using Western blot analysis. Subsequently, microRNA (miR)-34a expression was measured by reverse transcription-quantitative PCR (RT-qPCR). The levels of oxidative stress and apoptosis were evaluated after miR-34a overexpression. RESULTS: Results indicated that DHM dose-dependently elevated the decreased cell viability induced by HG. Moreover, the content of ROS was significantly reduced in HG-stimulated ARPE-19 cells, accompanied by enhanced activities of superoxide dismutase (SOD) and catalase (CAT) antioxidases, as well as concentration of glutathione (GSH). Furthermore, remarkably decreased apoptosis of ARPE-19 cells induced by HG was observed following DHM intervention. Importantly, HG stimulation notably upregulated miR-34a expression, which was reversed by DHM treatment. Importantly, the inhibitory effects of DHM on HG-induced oxidative stress and apoptosis of ARPE-19 cells were restored following miR-34a overexpression. CONCLUSION: Taken together, this work demonstrated that DHM exerts protective effects on HG-induced oxidative stress and apoptotic damage in ARPE-19 cells via inhibition of miR-34a expression, providing a promising therapeutic agent for the treatment of DR.

Scutellaria barbata D. Don Polysaccharides Inhibit High Glucose-Induced Proliferation and Angiogenesis of Retinal Vascular Endothelial Cells.

PURPOSE: The traditional Chinese medicine Scutellaria barbata D. Don (S. barbata) has been reported to exhibit anti-cancer and anti-inflammation activities. The ethanol extract of S. barbata has been confirmed to attenuate diabetic retinopathy (DR). This study aimed to investigate the effects and underlying mechanisms of the polysaccharides isolated from S. barbata (PSB) on the proliferation and angiogenesis of retinal vascular endothelial cells (RVECs) in DR. METHODS: Human RVECs (HRVECs) were cultured in normal glucose (NG, 5.5 mM), mannitol (MA, 30 mM), high glucose (HG, 30 mM) and HG plus 40 µg/mL PSB, respectively. Then, cell proliferation, migration and angiogenesis were evaluated. The cell proliferation was also estimated in the presence of SLIGKV, which was used to induce the phosphorylation of ERK (p-ERK). RESULTS: PSB reduced normal and HG-induced HRVECs cell viability in a concentration-dependent manner. The protein expression of proliferating cell nuclear antigen (PCNA) and proliferating antigen KI67 (Ki67), the migration rate and tube formation ability, which were increased by HG treatment, were significantly decreased by PSB. PSB also inhibited the phosphorylation of Raf, MEK and ERK in HG-stimulated HRVECs. Moreover, the application of SLIGKV recovered cell viability and the expression of p-ERK, PCNA and Ki67, in HG plus PSB-treated cells. Finally, the HG-enhanced expression of VE-cadherin, Frizzed, ß-catenin, MMP-2 and MMP-9 was all reversed by PSB. CONCLUSION: PSB could inhibit HG-induced HRVECs proliferation, migration and neovascularization, and these effects might work through blocking the activation of MEK/ERK pathway and VEGF/VE-cadherin axis.

Is a liver comparable to a liver? A comparison of different rat-derived S9-fractions with a biotechnological animal-free alternative in the Ames fluctuation assay.

Metabolism has to be considered during the toxicological assessment of chemical and environmental samples because it is an important process in the mammalian liver. It can be assessed in vitro via liver homogenates called S9-fractions, an external metabolic activation system. However, the external metabolic activation systems can vary greatly in their composition due to biological variations among individual animals and animal strains that the S9-fraction are derived as well as the differences in the production treatment. To gain more insight into these variances, three different but commonly used rat-derived S9-fractions were compared in the present study for their variance and performance with a reference compound in the Ames fluctuation assay with Salmonella typhimurium strains TA 98 and TA 100 according to ISO 11350. Severe shortcomings of conventional rat-derived S9-fractions were observed in the present study, such that S9-fractions differed significantly within the same rat strain and for different types of induction procedures in regards to the metabolic capability. An intrinsic mutagenic potential of the three rat-derived S9-fractions were identified in the Ames fluctuation assay with varying S9-fraction concentrations. To address some of the shortcomings of the animal-derived S9-fraction, the present study investigated the use and performance of a biotechnological, animal-free alternative, ewoS9R, in comparison to one of the rat-derived S9-fraction as the others showed a mutagenic potential themselves. Specifically, 12 different chemicals were used as a reference to determine if ewoS9R could serve as an adequate and more consistent replacement of traditional rat-derived metabolic activation systems: 8 pro-mutagenic compounds (i.e., require metabolic activation to show a mutagenic potential), one pro-mutagenic compound but not in the tested strains, one mutagenic compound without metabolic activation and two compounds that are equivocal in the literature. EwoS9R was evaluated as a promising approach in the Ames fluctuation assay with 5 compounds observed to have similar results with both rat-derived S9-fraction and ewoS9R (41%), for 3 compounds ewoS9R was a better metabolization system than the rat-derived S9-fraction (16%). Further research is necessary to determine the full potential of ewoS9R in comparison to rat-derived S9-fractions.

Using a high-throughput method in the micronucleus assay to compare animal-free with rat-derived S9.

This study presents a high-throughput (HTP) micronucleus assay in multi-well plates with an automated evaluation for risk assessment applications. The evaluation of genotoxicity via the micronucleus assays according to international guidelines ISO 21427-2 with Chinese hamster (Cricetulus griseus) V79 cells was the starting point to develop our methodology. A drawback of this assay is that it is very time consuming and cost intensive. Our HTP micronucleus assay in a 48-well plate format allows for the simultaneous assessment of five different sample-concentrations with additional positive, negative and solvent controls with six technical replicates each within a quarter of the time required for the equivalent evaluation using the traditional slide method. In accordance with the 3R principle, animal compounds should be replaced with animal-free alternatives. However, traditional cell culture-based methods still require animal derived compounds like rat-liver derived S9-fraction, which is used to simulate the mammalian metabolism in in vitro assays that do show intrinsic metabolization capabilities. In the present study, a recently developed animal-free biotechnological alternative (ewoS9R) was investigated in the new high-throughput micronucleus assay. In total, 12 different mutagenic or genotoxic chemicals were investigated to assess the potential use of the animal-free metabolization system (ewoS9R) in comparison to a common rat-derived product. Out of the 12 compounds, one compound did not induce micronuclei in any treatment and 2 substances showed a genotoxic potential without the need for a metabolization system. EwoS9R demonstrated promising potential for future applications as it shows comparable results to the rat-derived S9 for 6 of the 9 pro-genotoxic substances tested. The remaining 3 substances (2-Acetamidofluorene, Benzo[a]pyrene, Cyclophosphamide) were only metabolized by rat-derived S9. A potential explanation is that ewoS9R was investigated with an approx. 10-fold lower enzyme concentration and was only optimized for CYP1A metabolization that may be improved with a modified production procedure. Future applications of ewoS9R go beyond the micronucleus assay, but further research is necessary.

Ecological risk assessment of fifty pharmaceuticals and personal care products (PPCPs) in Chinese surface waters: A proposed multiple-level system.

Interest in the risks posed by trace concentrations of pharmaceuticals and personal care products (PPCPs) in surface waters is increasing, particularly with regard to potential effects of long-term, low-dose exposures of aquatic organisms. In most cases, the actual studies on PPCPs were risk assessments at screening-level, and accurate estimates were scarce. In this study, exposure and ecotoxicity data of 50 PPCPs were collected based on our previous studies, and a multiple-level environmental risk assessment was performed. The 50 selected PPCPs are likely to be frequently detected in surface waters of China, with concentrations ranging from the ng L-1 to the low-g L-1, and the risk quotients based on median concentrations ranged from 2046 for nonylphenol to 0 for phantolide. A semi-probabilistic approach screened 33 PPCPs that posed potential risks to aquatic organisms, among which 15 chemicals (nonylphenol, sulfamethoxazole, di (2-ethylhexyl) phthalate, 17ß-ethynyl estradiol, caffeine, tetracycline, 17ß-estradiol, estrone, dibutyl phthalate, ibuprofen, carbamazepine, tonalide, galaxolide, triclosan, and bisphenol A) were categorized as priority compounds according to an optimized risk assessment, and then the refined probabilistic risk assessment indicated 12 of them posed low to high risk to aquatic ecosystem, with the maximum risk products ranged from 1.54% to 17.38%. Based on these results, we propose that the optimized risk assessment was appropriate for screening priority contaminants at national scale, and when a more accurate estimation is required, the refined probability risk assessment is useful. The methodology and process might provide reference for other research of chemical evaluation and management for rivers, lakes, and sea waters.

Optimization of a pre-metabolization procedure using rat liver S9 and cell-extracted S9 in the Ames fluctuation test.

Many environmental pollutants pose a toxicological hazard only after metabolic activation. In vitro bioassays using cell lines or bacteria have often no or reduced metabolic activity, which impedes their use in the risk assessment. To improve the predictive capability of in vitro assays, external metabolization systems like the liver S9 fraction are frequently combined with in vitro toxicity assays. While it is typical for S9 fractions that samples and testing systems are combined in the same exposure system, we propose to separate the metabolism step and toxicity measurement. This allows for a modular combination of metabolic activation by enzymes isolated from rat liver (S9) or a biotechnological alternative (ewoS9R) with in vitro bioassays that lack metabolic capacity. Benzo(a)pyrene and 2-aminoanthracene were used as model compounds to optimize the conditions for the S9 metabolic degradation/activation step. The Ames assay with Salmonella typhimurium strains TA98 and TA100 was applied to validate the set-up of decoupling the S9 activation/metabolism from the bioassay system. S9 protein concentration of 0.25 mgprotein/mL, a supplement of 0.13 mM NADPH and a pre-incubation time of 100 min are recommended for activation of samples prior to dosing them to in vitro bioassays using the regular dosing protocols of the respective bioassay. EwoS9R performed equally well as Moltox S9, which is a step forward in developing true animal-free in vitro bioassays. After pre-incubation with S9 fraction, chemicals induced bacteria revertants in both the TA98 and the TA100 assay as efficiently as the standard Ames assay. The pre-incubation of chemicals with S9 fraction could serve for a wide range of cellular in vitro assays to efficiently combine activation and toxicity measurement, which may greatly facilitate the application of these assays for chemical hazard assessment and monitoring of environmental samples.

Effects of microRNA­217 on high glucose­induced inflammation and apoptosis of human retinal pigment epithelial cells (ARPE­19) and its underlying mechanism.

Diabetic retinopathy is a major complication of diabetes. Increasing evidence has indicated that microRNAs (miRs) serves an important role in diabetic retinopathy. However, the expression and mechanism of miR­217 in high glucose­induced human retinal pigment epithelial cells ARPE­19 is still unclear. Therefore, the aim of this study was to investigate the role of miR­217 in high glucose­induced retinal epithelial cell damage, and further to explore the molecular mechanisms. In our study, we found that compared with control group, miR­217 was upregulated in high glucose­induced ARPE­19 cells. In addition, TargetScan and a dual­luciferase reporter gene assay showed that Sirtuin 1 (SIRT1) was a direct target of miR­217. Then, we performed reverse transcription­quantitative polymerase chain reaction assay and western blot assay to explore the expression of SIRT1 in high glucose­induced ARPE­19 cells. Our results demonstrated that SIRT1 was downregulated at the mRNA and protein levels in high glucose­induced ARPE­19 cells. Then, ARPE­19 cells were transfected with inhibitor control, miR­217 inhibitor or miR­217 inhibitor + SIRT1­small interfering RNA for 6 h, and then the cells were treated with 50 mM D­glucose for 24 h. We then investigated the effects of miR­217 inhibitor on ARPE­19 cell viability and apoptosis. An MTT assay revealed that miR­217 inhibitor significantly increased the viability and decreased the apoptosis of high glucose­induced ARPE­19 cells. ELISA indicated that miR­217 inhibitor significantly reduced the expression of inflammatory factors, such as interleukin (IL)­1ß, tumor necrosis factor­α, and IL­6 in high glucose­treated ARPE­19 cells. Additionally, a western blot assay demonstrated that miR­217 inhibitor suppressed the expression of p­p65. The effects of miR­217 inhibitor on high glucose­treated ARPE­19 cells were significantly reversed by the silencing the SIRT1 gene. Therefore, our findings suggested that miR­217 inhibitor protected against retinal epithelial cell damage caused by high glucose via targeting SIRT1, thereby playing a protective role in diabetic retinopathy. Targeting miR­217 may have therapeutic potential in the treatment of diabetic retinopathy.

Integrated zebrafish-based tests as an investigation strategy for water quality assessment.

Water pollution risks to human health and the environment are emerging as serious concerns in the European Union and worldwide. With the aim to achieve good ecological and chemical status of all European water bodies, the "European Water Framework Directive" (WFD) was enacted. With the framework, bioanalytical techniques have been recognized as an important aspect. However, there are limitations to the application of bioassays directly for water quality assessment. Such approaches often fail to identify pollutants of concern, since the defined priority and monitored pollutants often fail to explain the observed toxicity. In this study, we integrated an effect-based risk assessment with a zebrafish-based investigation strategy to evaluate water sample extracts and fractions collected from the Danube. Four tiered bioassays were implemented, namely RNA-level gene expression assay, protein-level ethoxyresorufin-O-deethylase (EROD) assay, cell-level micronucleus assay and organism-level fish embryo test (FET). The results show that teratogenicity and lethality during embryonic development might be induced by molecular or cellular damages mediated by the aryl hydrocarbon receptor (AhR) -mediated activity, estrogenic activity and genotoxic activity. With the combination of high-throughput fractionation, this effect-based strategy elucidated the major responsible mixtures of each specific toxic response. In particularly, the most toxic mixture in faction F4, covering a log Kow range from 2.83 to 3.42, was composed by 12 chemicals, which were then evaluated as a designed mixture. Our study applied tiered bioassays with zebrafish to avoid interspecies differences and highlights effect-based approaches to address toxic mixtures in water samples. This strategy can be applied for large throughput screenings to support the main toxic compounds identification in water quality assessment.

Toxicological and chemical insights into representative source and drinking water in eastern China.

Drinking water safety is continuously threatened by the emergence of numerous toxic organic pollutants (TOPs) in environmental waters. In this study, an approach integrating in vitro bioassays and chemical analyses was performed to explore toxicological profiles of representative source and drinking water from waterworks of the Yangtze River (Yz), Taihu Lake (Th), and the Huaihe River (Hh) basins in eastern China. Overall, 34 of 96 TOPs were detected in all water samples, with higher concentrations in both source and drinking water samples of Hh, and pollutant profiles also differed across different river basins. Non-specific bioassays indicated that source water samples of Hh waterworks showed higher genotoxicity and mutagenicity than samples of Yz and Th. An EROD assay demonstrated dioxin-like toxicity which was detected in 5 of 7 source water samples, with toxin concentration levels ranging from 62.40 to 115.51 picograms TCDD equivalents per liter of water (eq./L). PAHs and PCBs were not the main contributors to observed dioxin-like toxicity in detected samples. All source water samples induced estrogenic activities of 8.00-129.00 nanograms 17ß-estradiol eq./L, and estrogens, including 17α-ethinylestradiol and estriol, contributed 40.38-84.15% of the observed activities in examined samples. While drinking water treatments efficiently removed TOPs and their toxic effects, and estrogenic activity was still observed in drinking water samples of Hh. Altogether, this study indicated that the representative source water in eastern China, especially that found in Hh, may negatively affect human health, a finding that demonstrates an urgent requirement for advanced drinking water treatments.

In situ microbiota distinguished primary anthropogenic stressor in freshwater sediments.

Conventional assessment and evaluation of sediment quality are based on laboratory-based ecotoxicological and chemical measurements with lack of concern for ecological relevance. Microbiotas in sediment are responsive to pollutants and can be used as alternative ecological indicators of sediment pollutants; however, the linkage between the microbial ecology and ecotoxicological endpoints in response to sediment contamination has been poorly evaluated. Here, in situ microbiotas from the Three Gorges Reservoir (TGR) area of the Yangtze River were characterized by DNA metabarcoding approaches, and then, changes of in situ microbiotas were compared with the ecotoxicological endpoint, aryl hydrocarbon receptor (AhR) mediated activity, and level of polycyclic aromatic hydrocarbons (PAHs) in sediments. PAHs and organic pollutant mixtures mediating AhR activity had different effects on the structures of microbiotas. Specifically, Shannon indices of protistan communities were negatively correlated with the levels of AhR mediated activity and PAHs. The sediment AhR activity was positively correlated with the relative abundance of prokaryotic Acetobacteraceae, but had a negative correlation with protistan Oxytrichidae. Furthermore, a quantitative classification model was built to predict the level of AhR activity based on the relative abundances of Acetobacteraceae and Oxytrichidae. These results suggested that in situ Protista communities could provide a useful tool for monitoring and assessing ecological stressors. The observed responses of microbial community provided supplementary evidence to support that the AhR-active pollutants, such as PAHs, were the primary stressors of the aquatic community in TGR area.

Electrochemical simulation of triclosan metabolism and toxicological evaluation.

Tricolsan (TCS), an antimicrobial agent, is considered as emerging pollutant due to its wide dispersive use in personal care products and high aquatic toxicity. In the present study, phase I metabolism of triclosan was investigated through laboratory electrochemical simulation studies. The products formed in the electrochemical (EC) cell were identified by online and offline coupling with QTRAP and high-resolution FTICR mass spectrometers, respectively. The sequential formation and disappearance of each product, with the continuous increase of voltage from 0 to 3500 mV, was observed to reveal the transformation pathways of TCS. The toxic potential of TCS and the identified products was estimated using Quantitative structure-activity relationship (QSAR) modeling on 16 target proteins. The toxicity change of TCS during simulated metabolism and toxicological effects of reaction mixture were assessed by Fish embryo toxicity (FET) test (Danio rerio) and quantitative real-time polymerase chain reaction (qPCR). Eight metabolites formed during the simulated metabolism of TCS mainly via the mechanisms of hydroxylation, ether-bond cleavage and cyclization. In FET test, the reaction mixture (LC50, 48h=1.28 mg/L) after electrochemical reactions showed high acute toxicity on zebrafish embryos, which was comparable to that of triclosan (LC50, 48h=1.34 mg/L). According to the modeling data, less toxic products formed only via ether-bond cleavage of TCS while the products formed through other mechanisms showed high toxicity. AhR-mediated dioxin-like effects on zebrafish embryos, such as developmental retardation in skeleyton and malformations in cardiovascular system, were also observed after exposure to the TCS reaction mixture in FET test. Activation of the AhR by the reaction mixture in zebrafish embryos was further proved in cyp1a gene expression analysis.

Electrochemical oxidation of fluoroquinolone antibiotics: Mechanism, residual antibacterial activity and toxicity change.

In this paper, we studied the electrochemical oxidation mechanisms of three typical fluoroquinolone antibiotics (FQs), and investigated residual antibacterial activity and toxicity changes after oxidation processes. Electrochemistry coupled to mass spectrometry (EC-MS) was used to study the oxidation processes of ciprofloxacin (CIP), norfloxacin (NOR) and ofloxacin (OFL). Eight oxidation products for each parent compound were identified and their chemical structures were elucidated. The transformation trend of each product, with the continuous increase of voltage from 0 to 3000 mV, was recorded by online EC-MS. The oxidation pathways were proposed based on the structural information and transformation trends of oxidation products. We found the oxidation mechanisms of FQs consisted of the hydroxylation and cleavage of piperazinyl ring via reactions with hydroxyl radicals, while the fluoroquinolone core remained intact. The antibacterial activity of the parent compounds and their oxidation mixtures was estimated using zone inhibition tests for gram-negative bacteria Salmonella typhimurium. It was found that the oxidation mixtures of CIP and NOR retained the antibacterial properties with lower activity compared to their parent compounds, while the antibacterial activity of OFL was almost eliminated after oxidation. Furthermore, the toxicity of the three FQs and their oxidation mixtures were evaluated using algal growth inhibition test (Desmodesmus subspicatus). The median effective concentration (EC50) values for the algal inhibition tests were calculated for the end point of growth rate. The toxicity of CIP and NOR to green algae after electrochemical oxidation, remained unchanged, while that of OFL significantly increased. The results presented in this paper contribute to an understanding of the electrochemical oxidation mechanisms of FQs, and highlight the potential environmental risks of FQs after electrochemical oxidation processes.

The metabolite 3,4,3',4'-tetrachloroazobenzene (TCAB) exerts a higher ecotoxicity than the parent compounds 3,4-dichloroaniline (3,4-DCA) and propanil.

3,4,3',4'-tetrachloroazobenzene (TCAB) is not commercially manufactured but formed as an unwanted by-product in the manufacturing of 3,4-dichloroaniline (3,4-DCA) or metabolized from the degradation of chloranilide herbicides, like propanil. While a considerable amount of research has been done concerning the toxicological and ecotoxicological effects of propanil and 3,4-DCA, limited information is available on TCAB. Our study examined the toxicity of TCAB in comparison to its parent compounds propanil and 3,4-DCA, using a battery of bioassays including in vitro with aryl hydrocarbon receptor (AhR) mediated activity by the 7-ethoxyresorufin-O-deethylase (EROD) assay and micro-EROD, endocrine-disrupting activity with chemically activated luciferase gene expression (CALUX) as well as in vivo with fish embryo toxicity (FET) assays with Danio rerio. Moreover, the quantitative structure activity response (QSAR) concepts were applied to simulate the binding affinity of TCAB to certain human receptors. It was shown that TCAB has a strong binding affinity to the AhR in EROD and micro-EROD induction assay, with the toxic equivalency factor (TEF) of 8.7×10(-4) and 1.2×10(-5), respectively. TCAB presented to be a weak endocrine disrupting compound with a value of estradiol equivalence factor (EEF) of 6.4×10(-9) and dihydrotestosterone equivalency factor (DEF) of 1.1×10(-10). No acute lethal effects of TCAB were discovered in FET test after 96h of exposure. Major sub-lethal effects detected were heart oedema, yolk malformation, as well as absence of blood flow and tail deformation. QSAR modelling suggested an elevated risk to environment, particularly with respect to binding to the AhR. An adverse effect potentially triggering ERß, mineralocorticoid, glucocorticoid and progesterone receptor activities might be expected. Altogether, the results obtained suggest that TCAB exerts a higher toxicity than both propanil and 3,4-DCA. This should be considered when assessing the impact of these compounds for the environment and also for regulatory decisions.