Arsenic Metabolism and Toxicity Influenced by Ferric Iron in Simulated Gastrointestinal Tract and the Roles of Gut Microbiota.

Iron (Fe) is a common trace element in drinking water. However, little is known about how environmental concentrations of Fe affect the metabolism and toxicity of arsenic (As) in drinking water. In this study, influence of Fe at drinking water-related concentrations (0.1, 0.3, and 3 mg Fe (total)/L) on As metabolism and toxicity, and the roles of gut microbiota during this process were investigated by using in vitro Simulator of the Human Intestinal Microbial Ecosystem (SHIME). Results showed that Fe had ability to decrease bioaccessible As by coflocculation in small intestine. 0.1 and 0.3 mg/L Fe significantly increased As methylation in simulated transverse and descending colon. Gut microbiota played an important role in alteration of As species, and Fe could affect As metabolism by changing the gut microbiota. Bacteroides, Clostridium, Alistipes, and Bilophila had As resistance and potential ability to methylate As. Cytotoxicity assays of effluents from simulated colons showed that the low levels of Fe decreased As toxicity on human hepatoma cell line HepG2, which might be due to the increase of methylated As. When assessing the health risk of As in drinking water, the residual Fe should be considered.

Pyrosequencing analysis of bacterial community and assembly in activated sludge samples from different geographic regions in China.

In order to investigate if there are geographic differences of bacterial community in the activated sludge collected from different geographic regions (eastern, northwestern, and northern parts) of China and to determine the co-occurrence patterns of bacterial community, activated sludge samples were collected from 10 municipal wastewater treatment plants located in 8 cities in China. High-throughput pyrosequencing combined with the bioinformatics analysis were used to examine the bacterial community compositions in the activated sludge samples. The result of taxonomy classifier indicated that a total of 76 genera were commonly shared by more than 7 samples, which accounted for 62 to 96 % of the classified sequences in each sample. Even though some core genera existed in all examined activated sludge samples regardless of the sampling geographic location and treatment process, significant geographic differences of bacterial community compositions among the activated sludge samples were revealed by the nonmetric multidimensional scaling (NMDS) and analyses of similarity (ANOSIM) analysis. A total of 165 pairs of significant and robust correlations (positive and negative) were identified from 61 bacterial genera based on the network analysis. The data obtained in this study could provide useful information to understand the bacterial community composition in geographically distributed wastewater treatment plants and discern the co-occurrence patterns of bacterial community.

Mouse organ coefficient and abnormal sperm rate analysis with exposure to tap water and source water in Nanjing reach of Yangtze River.

Organ coefficients (including kidney, testis, liver and spleen coefficient) and abnormal sperm rate were used in our study to reflect the exposure to the Yangzte River water. The concentrations of total dissolved metals and semi-volatile organic compounds in tap and source water were measured by ICP-OES and GC-MS, respectively. After mice were fed with purified water (CK), Nanjing tap water (NJT) and Nanjing source water (NJS) for 90 day, the individual and organs (including kidney, testis, liver and spleen) of each mouse were weighted. And abnormal sperm types (such as hook less, banana-like form, amorphous, folded and two tails) were determined by microscope. The results showed that significant differences of liver coefficient between experimental group (NJT, NJS) and control group (CK) were observed; furthermore liver coefficient is positive correlation with the concentrations of total dissolved metals. However, no significant differences of abnormal sperm rates between experimental group (NJT, NJS) and control group (CK) were noted. So liver coefficient might be more sensitive than other organ coefficients to reflect the exposure to tap water and source water, while abnormal sperm rate could not be used to reveal the exposure to them.

Identifying health effects of exposure to trichloroacetamide using transcriptomics and metabonomics in mice ( Mus musculus ).

Microarray-based transcriptomics and one-dimensional proton nuclear magnetic resonance ((1)H NMR) based metabonomics approaches were employed to investigate the health effects of nitrogenous disinfection byproducts (N-DBPs) of trichloroacetamide (TCAcAm) on mice. Mice were exposed to TCAcAm at concentrations of 50, 500, and 5000 µg/L for 90 days, and hepatic transcriptome and serum metabonome and histopathological parameters were detected in comparison with those of control. TCAcAm esposures resulted in liver inflammation, weight loss (in 5000 ug/L TCAcAm group), and alterations in hepatic transcriptome and serum metabonome. Based on the differentially expressed genes and altered metabolites, several significant pathways were identified, which are associated with lipid, xenobiotics, amino acid and energy metabolism, and cell process. Moreover, integrative pathway analyses revealed that TCAcAm exposure in this study induced hepatotoxicity and cytotoxicity. These results also highlight the noninvasive prospect of transcriptomic and metabonomic approaches in evaluating the health risk of emerging N-DBPs.

Hepatic gene expression analysis of mice exposed to raw water from Meiliang Bay, Lake Taihu, China.

Lake water is a micro-polluted water system, and characterization of its toxicity remains difficult. Microarray-based determination of altered gene expression might be an alterative approach. We chose raw water from Meiliang Bay, Lake Taihu, China as the target water. Male mice were exposed to the lake water for 90 days. Total hepatic RNA was applied to interrogate the Affymetrix Mouse Genome 430A 2.0 array. Gene ontology analysis, pathway analysis and gene network analysis were used to identify biological effects of differently expressed genes. The results showed that the expressions of 170 genes were altered. Nine biological processes and nine biological pathways were significantly perturbed (P ≤ 0.01), mainly linked to the regulation of cell processes, DNA repair, chromatin modification, oxidative reduction and carbohydrate metabolism. Important genes, such as Prkca, Pik3r1, Fgfr1 and Zbtb16, were identified by gene network analysis. This study provided excellent insights into early toxicological effects related to raw Lake Taihu water, and illustrated that the toxicogenomic approach might be a useful tool to evaluate potential environmental health effects of raw lake water.

Metagenomic analysis of cadmium and copper resistance genes in activated sludge of a tannery wastewater treatment plant.

In order to comprehensively characterize the copper and cadmium resistance in activated sludge of a tannery wastewater treatment plant, a resistance protein database of the two heavy metals was manually created by retrieving annotated sequences and related information from the public databases and published literatures. The metagenomic DNA was extracted from the activated sludge for Illumina high-throughput sequencing, and the obtained 11,973,394 clean reads (1.61 Gb) were compared against the established databases using BLAST tool. Annotations of the BLAST hits showed that 222 reads (0.019 per thousand) and 197 reads (0.016 per thousand) were identified as copper and cadmium resistance genes, respectively. Among the identified cadmium resistance genes, czcA encoding cobalt-zinc-cadmium resistance protein had the highest abundance (83 reads, 0.0069 per thousand), which was further confirmed by annotation of the open reading frames predicted with the assembly contigs. Among the copper resistance genes, copA (66 reads, 0.0055 per thousand) was most abundant, followed by copK and cusR. Alignment against the Clusters of Orthologous Groups (COG) database also suggested that 87.26% of the matched reads were grouped in COG0474 (cation transport ATPase). This study may be practically helpful for exploring various functional genes in the environment using high-throughput sequencing and bioinformatics methods.

Sewage treatment plant serves as a hot-spot reservoir of integrons and gene cassettes.

This study investigated the occurrence and abundance of class 1 integrons and related antibiotic resistance genes (ARGs) in a sewage treatment plant (STP) of China. Totally, 189 bacterial strains were isolated from influent, activated sludge and effluent, and 40 isolates contained the integons with a complete structure. The intl1-carrying isolates were found to harbor two types of gene cassettes: dfr17-aadA5 and aadA2, conferring resistances to trimethoprim and streptomycin, which were further confirmed by antimicrobial susceptibility analysis. Many other gene cassettes were carried on integron, including qnrVC1, catB-8-blaoxa-10-aadA1-aac(6'), aadB-aacA29b, aadA2, aac(6')-1b, aadA6 and aadA12, which were detected using DNA cloning. Quantitative real time PCR showed that over 99% of the integrons was eliminated in activated sludge process, but average copy number of integrons in given bacterial cells was increased by 56% in treated sewage. Besides integrons, other mobile gene elements (MGEs) were present in the STP with high abundance. MGEs and the associated ARGs may be wide-spread in STPs, which constitute a potential hot spot for selection of antibiotic resistant bacteria and horizontal transfer of ARGs.

Evaluating the transcriptomic and metabolic profile of mice exposed to source drinking water.

Transcriptomic and metabonomic methods were used to investigate mice's responses to drinking source water (DSW) exposure. After mice were fed with DSW for 90 days, hepatic transcriptome was characterized by microarray and serum metabonome were determined by (1)H nuclear magnetic resonance (NMR) spectroscopy. A total of 243 differentially expressed genes (DEGs) were identified, among which 141 genes were up-regulated and 102 genes were down-regulated. Metabonomics revealed significant changes in concentrations of creatine, pyruvate, glutamine, lysine, choline, acetate, lipids, taurine, and trimethylamine oxide. Four biological pathways were identified by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis where both gene expression and metabolite concentrations were altered in response to DSW exposure. These results highlight the significance of combined use of transcriptomic and metabonomic approaches in evaluating potential health risk induced by DSW contaminated with various hazardous materials.

Responses of mouse liver to dechlorane plus exposure by integrative transcriptomic and metabonomic studies.

Dechlorane plus (DP), a chlorinated flame retardant, has been widely detected in different environmental matrices and biota. However, toxicity data for DP have seldom been reported. In the present study, we investigated hepatic oxidative stress, DNA damage, and transcriptomic and metabonomic responses of male mice administered 500 mg/kg, 2000 mg/kg, and 5000 mg/kg of DP by gavage for 10 days. The results showed that DP exposure increased the level of superoxide dismutase (SOD) and 8-hydroxy-2-deoxyguanosine (8-OHdG). The microarray-based transcriptomic results demonstrated that DP exposure led to significant alteration of gene expression involved in carbohydrate, lipid, nucleotide, and energy metabolism, as well as signal transduction processes. The NMR-based metabonomic analyses corroborated these results showing changes of metabolites associated with the above altered mechanisms. Our results demonstrate that an oral exposure to DP can induce hepatic oxidative damage and perturbations of metabolism and signal transduction. These observations provide novel insight into toxicological effects and mechanisms of action of DP at the transcriptomic and metabonomic levels.

Microcystin-LR promotes melanoma cell invasion and enhances matrix metalloproteinase-2/-9 expression mediated by NF-κB activation.

This study aimed to explore the molecular mechanisms behind the stimulation effects of microcystin-LR (a well-known cyanobacterial toxin produced in eutrophic lakes or reservoirs) on cancer cell invasion and matrix metalloproteinases (MMPs) expression. Boyden chamber assay showed that microcystin-LR exposure (>12.5 nM) evidently enhanced the invasion ability of the melanoma cells (MDA-MB-435). Tumor Metastasis PCR Array demonstrated that 24 h microcystin-LR treatment (25 nM) caused overexpression of eight genes involved in tumor metastasis, including MMP-2, MMP-9, and MMP-13. Quantitative real-time PCR, Western blotting and gelatin zymography consistently demonstrated that mRNA and protein levels of MMP-2/-9 were increased in the cells after microcystin-LR exposure (P < 0.05 each). Immunofluorescence assay and electrophoretic mobility shift assay revealed that microcystin-LR could activate nuclear factor kappaB (NF-κB) by accelerating NF-κB translocation into the nucleus and enhancing NF-κB binding ability. Furthermore, addition of NF-κB inhibitor in culture medium could suppress the invasiveness enhancement and MMP-2/-9 overexpression. This study indicates that microcystin-LR can act as a NF-κB activator to promote MMP-2/-9 expression and melanoma cell invasion, which deserves more environmental health concerns.

Effects of microcystin-LR exposure on matrix metalloproteinase-2/-9 expression and cancer cell migration.

This study assessed the effects of microcystin-LR (MC-LR) exposure on matrix metalloproteinases (MMPs) expression and cancer cell migration. After male mice were orally administered with different concentrations of MC-LR for 270 d, histopathologic observation revealed an obvious hepatic lymphocyte infiltration or fatty degeneration. Immunohistochemical staining and enzyme-linked immunosorbent assay demonstrated that MC-LR treatment (even at 1 nM) caused up-regulated expressions of hepatic MMP-2/-9. Quantitative reverse-transcriptase PCR showed that the exposure to 80 nM MC-LR induced an increase of MMP-2/-9 mRNA levels by 1.0 and 1.9 fold. Breast cancer cells (MDA-MB-435s) were also cultured with MC-LR solutions and a wound healing assay demonstrated that MC-LR posed a time/dose-dependent stimulation effect on migration of the cancer cells. Gelatin electrophoresis and quantitative PCR showed significant increases in cellular MMP-2/-9 expressions after MC-LR exposure. This study indicated that chronic exposure to MC-LR could alter MMP-2/-9 expressions and stimulate cancer cell migration.

Preliminary evaluation of gene expression profiles in liver of mice exposed to Taihu Lake drinking water for 90 days.

Differential gene expression profiling was performed via DNA microarray in the liver tissue of Mus musculus mice after exposure to drinking water of Taihu Lake for 90 days. A total of 75 differentially expressed candidate genes (DEGs) were identified (1.5-fold, p ≤ 0.05), among which the expression of 29 genes was up-regulated and that of 46 genes was down-regulated. Most DEGs were involved in biological process based on gene ontology mapping analysis. The drinking water of Taihu Lake significantly influenced the expression of genes related to cell proliferation, apoptosis, amino acid metabolism, development and immune responses. Long-term exposure to the Taihu drinking water may result in increased carcinogenic risk.

Risk assessment of polycyclic aromatic hydrocarbons in aquatic ecosystems.

A probability risk assessment of anthracene, benzo(a)pyrene, chrysene, fluorene, phenanthrene and pyrene was carried out to examine the ecological risk of these six polycyclic aromatic hydrocarbons (PAHs) in aquatic ecosystems in China. The literature on PAH concentrations in surface water in China was collected to evaluate the environmental exposure concentrations (EEC). The 10th percentile of predicted no observed effect concentration (PNEC(10%)) of PAHs, calculated according to the data from the USEPA AQUIRE database and regulatory reviews, was applied as the toxicity assessment endpoint. The ratio of EEC and PNEC(10%), expressed as a risk quotient (RQ), was used to characterize the risk value. Bootstrapping method and Monte Carlo simulation were utilized to calculate the distribution of EEC, PNEC(10%), RQ and associated uncertainties. Risk assessment showed that reliable maximum RQs of anthracene, benzo(a)pyrene, chrysene, fluorene and phenanthrene were in the range of 0.064-0.755, lower than the acceptable value of 1. However, the reliable maximum RQ of pyrene was 1.39, indicating its potential ecological risk. Notwithstanding the uncertainty, these results suggest that the aquatic ecosystems with high PAH concentrations might pose potential ecological risks, and concerted efforts are required to ensure that surface water is protected.

Genome-wide screening of indicator genes for assessing the potential carcinogenic risk of Nanjing city drinking water.

Effects of all pollutants existing in the Nanjing city drinking water (DWNC) on mouse gene transcription levels were measured to assess the DWNC carcinogenic risks and to identify candidate indicator genes for assessing and early warning the cancer risks. Transcriptional expression levels of 14,000 hepatic genes for the treatment group mice (Mus musculus, ICR) fed with DWNC for 90 days were detected using the GeneChip(®) Mouse Genome 430A 2.0 array. The analysis indicated that the transcriptional levels of 294 genes were up-regulated and 542 ones were down-regulated. Of these genes, 12 ones identified to be involved in at least five different types of cancers were further analyzed. An interrogation by Kyoto Encyclopedia of Genes and Genomes (KEGG) revealed that three (including ITGAV, CCND1 and SMAD2) of the 12 genes were mapped to pathway in cancer. Gene Ontology (GO) function annotation also showed that they were associated with the functional categories of cell cycle regulation, adhesion, apoptosis, signal transduction and so on which are closely implicated in tumorigenesis and progression. The correlations between the aberrant expressions of them and the genesis and progression of cancers have been further documented by a number of scientific researches. These results might demonstrate that the potential toxicity and carcinogenic risks were associated with DWNC. Moreover, ITGAV, CCND1 and SMAD2 were identified as the most likely candidate indicator genes for the assessment of the combined carcinogenic risk of all pollutants existing in DWNC.

NMR-based metabolic profiling for serum of mouse exposed to source water.

(1)H nuclear magnetic resonance (NMR) based metabonomic method was used to characterize the profile of low-molecular-weight endogenous metabolites in mouse (Mus musculus) serum following exposure to Taihu Lake source water for 90 days. The (1)H NMR spectra of mice sera were recoded and a total of 21 metabolites were identified. Data reduction and latent biomarkers identification were processed by pattern recognition (PR) analysis. The principal component analysis (PCA) and partial least square discriminant analysis (PLS-DA) identified differences in metabolic profiles between control and treatment groups. A number of serum metabolic perturbations were observed in sera of source water treatment mice compared to control mice, including decreased levels of acetone, pyruvate, glutamine, lysine and citrate. These results indicated that Taihu Lake source water could induce energy metabolism changes in mouse related to fatty acid ß-oxidation, tricarboxylic acid (TCA) cycle, citric acid cycle, and metabolism of some amino acids. (1)H NMR-based metabolic profiling provides new insight into the toxic effect of Taihu Lake source water, and suggests potential biomarkers for noninvasive monitoring of health risk.

Integration of gene chip and topological network techniques to screen a candidate biomarker gene (CBG) for predication of the source water carcinogenesis risks on mouse Mus musculus.

Screening of a candidate biomarker gene (CBG) to predicate the carcinogenesis risks in the Yangtze River source of drinking water in Nanjing area (YZR-SDW-NJ) on mouse (Mus musculus) was conducted in this research. The effects of YZR-SDW-NJ on the genomic transcriptional expression levels were measured by the GeneChip(®) Mouse Genome and data treated by the GO database analysis. The 298 genes discovered as the differently expressed genes (DEGs) were down-regulated and their values were ≤-1.5-fold. Of the 298 DEGs, 25 were cancer-related genes selected as the seed genes to build a topological network map with Genes2Networks software, only 7 of them occurred at the constructed map. Smad2 gene was at the constructed map center and could be identified as a candidate biomarker gene (CBG) primarily which involves the genesis and development of colorectal, leukemia, lung and prostate cancers directly. Analysis of the gene signal pathway further approved that smad2 gene had the relationships closely to other 16 cancer-related genes and could be used as a CBG to indicate the carcinogenic risks in YZR-SDW-NJ. The data suggest that integration of gene chip and network techniques may be a way effectively to screen a CBG. And the parameter values for further judgment of the CBG through signal pathway relationship analysis also will be discussed.

Assessing the toxicity of ingested Taihu Lake water on mice via hepatic histopathology and matrix metalloproteinase expression.

Water from Taihu Lake (China) is used as a drinking source. The frequency of contamination in Taihu Lake has increased over the past decade and the bloom-forming cyanobacteria are the dominant species during eutrophication. Cyanobacteria can produce various harmful secondary substances including microcystins capable of endangering human health and ecological safety. This study investigated toxicity of ingested Taihu Lake water on mice via hepatic histopathology and matrix metalloproteinase (MMP) expression. Water was sampled from four Taihu Lake locations, Meiliang Bay 2 group (M2), Meiliang Bay 1 group (M1), Lake Center (H) and Xukou Bay (X), along a gradient of decreasing degree of eutrophication. The experimental design consists of five groups of male mice (Mus musculus, ICR): one control and four groups ingesting water from the four sampling sites for 90 days. Compared to control, M1 and M2 mice showed hepatic histopathological changes including swollen, vacuolar degeneration or inflammatory. Immunohistochemical staining demonstrated a higher expression of MMP-2 proteins in M2 group and a lower expression of MMP-9 in M1. Enzyme-linked immunosorbent assay indicated that MMP-9 concentration was significantly reduced from 0.55 to 0.28 ng/g liver weight in M2 (p < 0.05). Real time PCR revealed a down-regulation of MMP-9 mRNA by 2.2 fold in M1 and an up-regulation of MMP-2 mRNA by 1.73 fold in H. Using this mouse model as a gauge of water toxicity, our results revealed that potential health risks induced by Taihu Lake water might arise from the use of this source water by local resident.

Occurrence, abundance and elimination of class 1 integrons in one municipal sewage treatment plant.

Integrons are elements that encode a site-specific recombination system that recognizes and captures mobile gene cassettes and are closely related to multiple resistances of environmental microorganisms. This study was undertaken to determine the efficiency of an activated sludge process to remove integrons. The prevalence and characteristics of class 1 integrons were investigated for bacterial species isolated from the activated sludge of Nanjing Jiangxinzhou sewage treatment plant (STP, China). A total of 189 bacterial strains were isolated from influent water, activated sludge and effluent water, and PCR-RFLP (Polymerase chain reaction--restriction fragment length polymorphism) of 16S rRNA gene showed that the isolated bacteria were Escherichia coli, Aeromonas veronii, Klebsiella spp., Aeromonas salmonicida and Aeromonas media. PCRs showed that 57 isolates contained class 1 integronase gene intI1. The integron detection frequency in the isolated strains was 20.4% for influent, 30.9% for activated sludge and 38.9% for effluent. Quantitative real-time PCR assay showed that the abundance of integrons in effluent was higher than that in influent. This study indicates that class 1 integrons are wide-spread in STPs which might be involved in multiple resistances in the activated sludge characterized by high biomass and biodiversity.

Comparative analysis of binding affinities between styrene and mammalian CYP2E1 by bioinformatics approaches.

Cytochrome P450 2E1 (CYP2E1) is a cytochrome P450 enzyme involved in styrene metabolism. This study compared the binding affinities between styrene and 11 mammalian CYP2E1 systems using bioinformatics methods. Firstly, amino acid sequences of CYP2E1s were obtained from the Swiss-Prot database. Then, taking the crystal structure of human CYP2E1 as a template, 3D models of the CYP2E1s of other mammals were constructed using the SWISS-MODEL program. Finally, the generated homology models were applied to calculate their docking capacities against styrene and polystyrene using the Surflex-Dock program, which could automatically dock ligands into a receptor's ligand binding site using a protomol based approach and assess the affinity by an empirically derived scoring function. Docking experiments showed that the studied mammalian CYP2E1s had high binding affinities with styrene. For polystyrene, the dimmer of styrene has high binding affinities with CYP2E1s, however, trimer and other high polymers were found hard to be docked into the CYP2E1s. The results of this study indicated that bioinformatics approaches might be useful tools to predict styrene and polystyrene affinities with mammalian CYP2E1s.

Identification of trace organic pollutants in freshwater sources in Eastern China and estimation of their associated human health risks.

Concentrations of polycyclic aromatic hydrocarbons (PAHs) and organochlorine pesticides (OCPs) in ten water samples (four pairs of tap water with source water and two additional tap water) were analyzed and a US EPA algorithm was used to estimate their associated human health risks. Organochlorine pesticides were found in most samples analyzed. Concentrations of total PAHs ranged from 4.8 to 84.4 and 0.7 to 53.8 ng/l, for source and tap water, respectively. Carcinogenic and noncarcinogenic risk assessments were employed for various trace organic pollutants in the tap water. The results indicated that carcinogenic risk for male and female lifetime of tap water from XuZh (XZ) city was higher than the others. XZ located in the northern part of Jiangsu province, which takes ground water as the water source. Children were more vulnerable to the carcinogenic chemicals than adults. Carcinogenic risks for male children (0-14 years old) in XZ were the highest, reaching 3.68 × 10(-6). Contribution analysis showed that dibenz[a,h]anthracene contributed most to the carcinogenic risk in XZ city, and α-HCH, ß-HCH and γ-HCH posed the most carcinogenic risk in tap water from GoHu (GH) in Eastern Taihu Lake. Additionally, noncarcinogenic risks posed by the detected chemicals to local people were negligible. Risk alleviation strategies should be adopted, taking into account the results of these health risk assessments.