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One of the major challenges in environmental science is monitoring and assessing the risk of complex environmental mixtures. In vitro bioassays with limited key toxicological end points have been shown to be suitable to evaluate mixtures of organic pollutants in wastewater and recycled water. Omics approaches such as transcriptomics can monitor biological effects at the genome scale. However, few studies have applied omics approach in the assessment of mixtures of organic micropollutants. Here, an omics approach was developed for profiling bioactivity of 10 water samples ranging from wastewater to drinking water in human cells by a reduced human transcriptome (RHT) approach and dose-response modeling. Transcriptional expression of 1200 selected genes were measured by an Ampliseq technology in two cell lines, HepG2 and MCF7, that were exposed to eight serial dilutions of each sample. Concentration-effect models were used to identify differentially expressed genes (DEGs) and to calculate effect concentrations (ECs) of DEGs, which could be ranked to investigate low dose response. Furthermore, molecular pathways disrupted by different samples were evaluated by Gene Ontology (GO) enrichment analysis. The ability of RHT for representing bioactivity utilizing both HepG2 and MCF7 was shown to be comparable to the results of previous in vitro bioassays. Finally, the relative potencies of the mixtures indicated by RHT analysis were consistent with the chemical profiles of the samples. RHT analysis with human cells provides an efficient and cost-effective approach to benchmarking mixture of micropollutants and may offer novel insight into the assessment of mixture toxicity in water. [ABSTRACT FROM AUTHOR]

There are thousands of chemicals used by humans and detected in the environment for which limited or no toxicological data are available. Rapid and cost-effective approaches for assessing the toxicological properties of chemicals are needed. We used CRISPR-Cas9 functional genomic screening to identify the potential molecular mechanism of a widely used antimicrobial triclosan (TCS) in HepG2 cells. Resistant genes at IC50 (the concentration causing a 50% reduction in cell viability) were significantly enriched in the adherens junction pathway, MAPK signaling pathway, and PPAR signaling pathway, suggesting a potential role in the molecular mechanism of TCS-induced cytotoxicity. Evaluation of the top-ranked resistant genes, FTO (encoding an mRNA demethylase) and MAP2K3 (a MAP kinase kinase family gene), revealed that their loss conferred resistance to TCS. In contrast, sensitive genes at IC10 and IC20 were specifically enriched in pathways involved with immune responses, which was concordant with transcriptomic profiling of TCS at concentrations of

It was hypothesized that polychlorinated diphenyl sulfides (PCDPSs) can potentially interact with an aryl hydrocarbon receptor (AHR) and thereby cause adverse effects in wildlife like birds. A recently developed avian AHRl-luciferase report gene (LRG) assay was used to assess the interaction between avian AHR1 and 18 PCDPSs and to compare the interspecies sensitivity among chicken, ring-necked pheasant, and Japanese quail by PCDPSs. Most of the tested PCDPSs could activate the AHR1-mediated pathways in avian species, and the relative potency (ReP) of the PCDPSs increased with the increasing number of substituted Cl atoms. The rank orders of PCDPSs potency were generally similar among birds, although the ReP varied. In addition, not all the sensitivity rank orders of avian AHR1 constructs for PCDPSs were consistent with that of TCDD. ReP values of PCDPSs suggested that some PCDPSs like 2,3,3',4,5,6-hexa-CDPS and 2,2',3,3',4,5,6-hepta-CDPS are higher than the avian WHO-TEFs of OctaCDD, OctaCDF, and most of the coplanar PCBs. Our results report for the first time the activation of an AHR1-mediated molecular toxicological mechanism by PCDPSs, and provide the ranking of ReP and relative sensitivity values of different congeners, which could guide the further toxicity test of this group of potential high priority environmental pollutants. [ABSTRACT FROM AUTHOR]

Cytotoxicity of 6-HO-BDE-47 and its two analogues, BDE-47 and 6-MeO-BDE-47, and the associated molecular mechanisms were assessed by use of a live cell reporter assay system which contains a library of 1820 modified green fluorescent protein (GFP) expressing promoter reporter vectors constructed from E. coli K12 strains. 6-HO-BDE-47 inhibited growth of E. coli with a 4 h median effect concentration (EC50) of 22.52 ± 2.20 mg/L, but neither BDE-47 nor 6-MeO-BDE-47 were cytotoxic. Thus, 6-HO-BDE-47 might serve as an antibiotic in some living organisms. Exposure to 6-HO-BDE-47 resulted in 65 (fold change >2) or 129 (fold change >1.5) genes being differentially expressed. The no observed transcriptional effect concentration (NOTEC) and median transcriptional effect concentration (TEC50) based on transcriptional end points, of 6-HO-BDE-47 were 0.0438 and 0.580 mg/L, respectively. The transcriptional responses were 514- and 39-fold more sensitive than the acute EC50 to inhibit cell growth. Most of the genes that were differentially expressed in response to 6-HO-BDE-47 were not modulated by BDE-47 or 6-MeO-BDE-47. These results suggest that cytotoxicity of 6-HO-BDE-47 to E. coli was via a mechanism that was different from that of either BDE-47 or 6-MeO-BDE-47. Gene expression associated with metabolic pathways was more responsive to 6-HO-BDE-47, which suggests that this pathway might be the primary target of this compound. [ABSTRACT FROM AUTHOR]

Mixtures of naphthenic acids (NAs), which include cyclopentyl and cyclohexyl carboxylic acids, have been suggested to be toxic components in oils spills, effluents from the petrochemical industry and in oil sands process waters (OSPW). The present study demonstrated, for the first time, an application of a high throughput live bacterial cell array in a genome-scale investigation of the toxic mechanisms of environmental chemicals, a commercial NAs technical mixture extracted from crude oil. Real time gene profiling of time- and concentration- dependent responses of live cells exposed to NAs for three hours was conducted using a library of 1800 fluorescent transcriptional reporters for Escherichia coli (E coli) growing in 384-well plates. The response patterns obtained after exposure to NAs suggested that the primary cellular responses were up-regulation of genes in the pentose phosphate pathway, involved in the molecular function of NADP or NADPH binding, and down-regulation of the ATP-binding cassette (ABC) transporter complex Transcriptional networks that were significantly modulated by NAs included those that were regulated by transcriptional factors such as CRP-, RecA-, and GadE. Down-regulation of the SOS response pathway suggested that DNA damage might not be the direct result of NAs within the first three hours of exposure. However, CRP-dependent genes modulated by exposure to NAs indicated that the cellular level of cyclic AMP was altered immediately upon exposure of cells to NAs. Furthermore, the linear range of the concentration-response curve of the selected gene reporters encompassed a range of concentrations between 10 and 1000 mg NAs/L, which covers concentrations typically observed in the environment and makes this assay system ideal for the detection of environmental NAs. [ABSTRACT FROM AUTHOR]

The objective of this study was to assess chemical-induced effects on cross-talk among the hypothalamic-pituitary-gonad (HPG), hypothalamic-pituitary-adrenal (HPA), and hypothalamic-pituitary-thyroid (HPT) axes of fish. Adult female zebrafish were exposed to 300 μg/L prochloraz (PCZ) or 100 mg/L propylthiouracil (PTU), and the transcriptional profiles of the HPG, HPA, and HPT axes were examined. Exposure to PCZ decreased plasma testosterone (T) and 17β-estradiol (E2) concentrations and affected HPA and HPT axes by down-regulating corticotrophin-releasing hormone (CRH) after 12 and 48 h. By using correlation analyses, it was found that the decrease in E2 plasma concentrations caused by PCZ was correlated with the down-regulation of CRH mRNA expression. Exposure to PTU resulted in lesser concentrations of thyroxine (T4) and triiodothyronine (T3), greater concentrations of follicle stimulating hormone (FSH) and luteinizing hormone (LH) peptides, and increase in steroidogenic gene expression after 12 and 48 h. Concentrations of FSH and LH were negatively correlated with concentrations of T4 and T3. These results are consistent with the hypothesis that increased steroidogenic gene expression after PTU exposure resulted from a reduction in T4 and T3 concentrations, which resulted in greater secretion of FSH and LH. [ABSTRACT FROM AUTHOR]

Concentration-dependent response relationships provide essential information on the characteristics of chemical-induced effects on toxicological end points, which include effect (inhibition or induction), potency, and efficacy of the chemical. Recent developments in systems biology and high through put technologies have allowed simultaneous examination of many chemicals at multiple end point levels. While this increase in the quantity of information generated offers great potential, it also poses a significant challenge to environmental scientists to efficiently manage and interpret these large data sets. Here we present a novel method, ToxClust, that allows clustering of chemicals on the basis of concentration-response data derived with single or multiple end points. This method utilizes a least distance-searching algorithm (LOSA) to measure the pattern dissimilarity of concentration-response curves between chemicals and their relative toxic potency. ToxCiust was tested using simulated data and chemical test data collected from the human H295R cell-based in vitro steroidogenesis assay. ToxClust effectively identified similar patterns of simulated data and responses to the exposure with the five model chemicals and separated them into different groups on the basis of their dissimilarities. These observations demonstrate that ToxClust not only provides an effective data analysis and visualization tool, but also has value in hypothesis generation and mechanism-based chemical classification. [ABSTRACT FROM AUTHOR]

Effects of two model imidazole-type fungicides, prochloraz (PCZ) and ketoconazole (KTC), on the hypothalamic-pituitary-gonadal (HPG) axis of the Japanese medaka (Oryzias latipe) were examined by use of real time PCR (RT-PCR) array. Fourteen-week-old Japanese medaka were exposed for seven days to concentrations of PCZ or KTC from 3.0 to 300 μg/L Exposure to KTC or PCZ caused significant reduction of fecundity of Japanese medaka and down-regulated expression of estrogen receptor (ER)-γ and egg precursors in livers of males and females. However, PCZ was more potent than KTC both in modulating transcription and causing lesser fecundity. Exposure to nominal 30 μg PCZ/L resulted in 50% less fecundity and significant down-regulation of vitellogenin // expression, but KTC did not cause such effects at this concentration. Exposure to PCZ caused a compensatory up-regulation in cytochrome P450 c17αhydroxylase, 17,20-lyase (CYP17) and aromatase (CYP19) expression in the ovary, while KTC did not Furthermore, the ecologically relevant end point, fecundity was log-log related to mRNA level of six genes in livers of females. [ABSTRACT FROM AUTHOR]

Gene expression profiles show considerable promise for the evaluation of the toxic potential of environmental contaminants. For example, any alterations in the pathways of steroid synthesis or breakdown have the potential to cause endocrine disruption. Therefore monitoring these pathways can provide information relative to a chemical's ability to impact endocrine function. One approach to monitoring these pathways has been to use a human adrenocortical carcinoma cell line (H295R) that expresses all the key enzymes necessary for steroidogenesis. In this study we have further developed these methods using accurate and specific quantification methods utilizing molecular beacon-based quantitative RT-PCR (Q-RT-PCR). The assay system was used to analyze the expression patterns of 11 steroidogenic genes in H295R cells. The expression of gene transcripts was measured using a real- time PCR system and quantified based on both a standard curve method using a dilution series of RNA standards and a comparative Ct method. To validate the optimized method, cells were exposed to specific and nonspecific model compounds (inducers and inhibitors of various steroidogenic enzymes) for gene expression profiling. Similar gene expression profiles were exhibited by cells treated with chemicals acting through common mechanisms of action. Overall, our findings demonstrated that the present assay can facilitate the development of compound- specific response profiles, and will provide a sensitive and integrative screen for the effects of chemicals on steroidogenesis. [ABSTRACT FROM AUTHOR]