Alvine C. Mehinto

Benchmarking Organic Micropollutants in Wastewater, Recycled Water and Drinking Water with In Vitro Bioassays

Thousands of organic micropollutants and their transformation products occur in water. Although often present at low concentrations, individual compounds contribute to mixture effects. Cell-based bioassays that target health-relevant biological endpoints may therefore complement chemical analysis for water quality assessment. The objective of this study was to evaluate cell-based bioassays for their suitability to benchmark water quality and to assess efficacy of water treatment processes. The selected bioassays cover relevant steps in the toxicity pathways including induction of xenobiotic metabolism, specific and reactive modes of toxic action, activation of adaptive stress response pathways and system responses. Twenty laboratories applied 103 unique in vitro bioassays to a common set of 10 water samples collected in Australia, including wastewater treatment plant effluent, two types of recycled water (reverse osmosis and ozonation/activated carbon filtration), stormwater, surface water, and drinking water. Sixty-five bioassays (63%) showed positive results in at least one sample, typically in wastewater treatment plant effluent, and only five (5%) were positive in the control (ultrapure water). Each water type had a characteristic bioanalytical profile with particular groups of toxicity pathways either consistently responsive or not responsive across test systems. The most responsive health-relevant endpoints were related to xenobiotic metabolism (pregnane X and aryl hydrocarbon receptors), hormone-mediated modes of action (mainly related to the estrogen, glucocorticoid, and antiandrogen activities), reactive modes of action (genotoxicity) and adaptive stress response pathway (oxidative stress response). This study has demonstrated that selected cell-based bioassays are suitable to benchmark water quality and it is recommended to use a purpose-tailored panel of bioassays for routine monitoring.

Uptake and Biological Effects of Environmentally Relevant Concentrations of the Nonsteroidal Anti-inflammatory Pharmaceutical Diclofenac in Rainbow Trout (Oncorhynchus mykiss)

Diclofenac, a nonsteroidal anti-inflammatory drug, is widely detected in surface waters and can potentially cause deleterious effects in fish. Here, we investigated the biological effects of 21-day exposure to waterborne diclofenac at environmentally relevant concentrations (0, 0.5, 1, 5, and 25 μg/L) in rainbow trout Accumulation of diclofenac in the bile was measured and responses in selected tissues were assessed via changes in the expression of selected genes (cytochrome P450 (cyp) 1a1, cyclooxygenase (cox) 1 and 2, and p53) involved in metabolism of xenobiotics, prostaglandin synthesis, and cell cycle control, respectively, together with histopathological alterations in these tissues. Diclofenac accumulated in the bile by a factor of between 509 ± 27 and 657 ± 25 and various metabolites were putatively identified as hydroxydiclofenac, diclofenac methyl ester, and the potentially reactive metabolite hydroxydiclofenac glucuronide. Expression levels of both cox1 and cox2 in liver, gills, and kidney were significantly reduced by diclofenac exposure from only 1 μg/L. Expression of cyp1a1 was induced in the liver and the gills but inhibited in the kidney of exposed fish. Diclofenac exposure induced tubular necrosis in the kidney and hyperplasia and fusion of the villi in the intestine from 1 μg/L. This study demonstrates that subchronic exposure to environmental concentrations of diclofenac can interfere with the biochemical functions of fish and lead to tissue damage, highlighting further the concern about this pharmaceutical in the aquatic environment.

Applications for next-generation sequencing in fish ecotoxicogenomics

The new technologies for next-generation sequencing (NGS) and global gene expression analyses that are widely used in molecular medicine are increasingly applied to the field of fish biology. This has facilitated new directions to address research areas that could not be previously considered due to the lack of molecular information for ecologically relevant species. Over the past decade, the cost of NGS has decreased significantly, making it possible to use non-model fish species to investigate emerging environmental issues. NGS technologies have permitted researchers to obtain large amounts of raw data in short periods of time. There have also been significant improvements in bioinformatics to assemble the sequences and annotate the genes, thus facilitating the management of these large datasets.The combination of DNA sequencing and bioinformatics has improved our abilities to design custom microarrays and study the genome and transcriptome of a wide variety of organisms. Despite the promising results obtained using these techniques in fish studies, NGS technologies are currently underused in ecotoxicogenomics and few studies have employed these methods. These issues should be addressed in order to exploit the full potential of NGS in ecotoxicological studies and expand our understanding of the biology of non-model organisms.

Transcriptomic effects-based monitoring for endocrine active chemicals: assessing relative contribution of treated wastewater to downstream pollution.

The present study investigated whether a combination of targeted analytical chemistry information with unsupervised, data-rich biological methodology (i.e., transcriptomics) could be utilized to evaluate relative contributions of wastewater treatment plant (WWTP) effluents to biological effects. The effects of WWTP effluents on fish exposed to ambient, receiving waters were studied at three locations with distinct WWTP and watershed characteristics. At each location, 4 d exposures of male fathead minnows to the WWTP effluent and upstream and downstream ambient waters were conducted. Transcriptomic analyses were performed on livers using 15,000 feature microarrays, followed by a canonical pathway and gene set enrichment analyses. Enrichment of gene sets indicative of teleost brain-pituitary-gonadal-hepatic (BPGH) axis function indicated that WWTPs serve as an important source of endocrine active chemicals (EACs) that affect the BPGH axis (e.g., cholesterol and steroid metabolism were altered). The results indicated that transcriptomics may even pinpoint pertinent adverse outcomes (i.e., liver vacuolization) and groups of chemicals that preselected chemical analytes may miss. Transcriptomic Effects-Based monitoring was capable of distinguishing sites, and it reflected chemical pollution gradients, thus holding promise for assessment of relative contributions of point sources to pollution and the efficacy of pollution remediation.

Interlaboratory comparison of in vitro bioassays for screening of endocrine active chemicals in recycled water

In vitro bioassays have shown promise as water quality monitoring tools. In this study, four commercially available in vitro bioassays (GeneBLAzer(®) androgen receptor (AR), estrogen receptor-alpha (ER), glucocorticoid receptor (GR) and progesterone receptor (PR) assays) were adapted to screen for endocrine active chemicals in samples from two recycled water plants. The standardized protocols were used in an interlaboratory comparison exercise to evaluate the reproducibility of in vitro bioassay results. Key performance criteria were successfully achieved, including low background response, standardized calibration parameters and high intra-laboratory precision. Only two datasets were excluded due to poor calibration performance. Good interlaboratory reproducibility was observed for GR bioassay, with 16-26% variability among the laboratories. ER and PR bioactivity was measured near the bioassay limit of detection and showed more variability (21-54%), although interlaboratory agreement remained comparable to that of conventional analytical methods. AR bioassay showed no activity for any of the samples analyzed. Our results indicate that ER, GR and PR, were capable of screening for different water quality, i.e., the highest bioactivity was observed in the plant influent, which also contained the highest concentrations of endocrine active chemicals measured by LC-MS/MS. After advanced treatment (e.g., reverse osmosis), bioactivity and target chemical concentrations were both below limits of detection. Comparison of bioassay and chemical equivalent concentrations revealed that targeted chemicals accounted for ≤5% of bioassay activity, suggesting that detection limits by LC-MS/MS for some chemicals were insufficient and/or other bioactive compounds were present in these samples. Our study demonstrated that in vitro bioassays responses were reproducible, and can provide information to complement conventional analytical methods for a more comprehensive water quality assessment.

Correlation of gene expression and contaminant concentrations in wild largescale suckers: a field-based study.

Toxic compounds such as organochlorine pesticides (OCs), polychlorinated biphenyls (PCBs), and polybrominated diphenyl ether flame retardants (PBDEs) have been detected in fish, birds, and aquatic mammals that live in the Columbia River or use food resources from within the river. We developed a custom microarray for largescale suckers (Catostomus macrocheilus) and used it to investigate the molecular effects of contaminant exposure on wild fish in the Columbia River. Using Significance Analysis of Microarrays (SAM) we identified 72 probes representing 69 unique genes with expression patterns that correlated with hepatic tissue levels of OCs, PCBs, or PBDEs. These genes were involved in many biological processes previously shown to respond to contaminant exposure, including drug and lipid metabolism, apoptosis, cellular transport, oxidative stress, and cellular chaperone function. The relation between gene expression and contaminant concentration suggests that these genes may respond to environmental contaminant exposure and are promising candidates for further field and laboratory studies to develop biomarkers for monitoring exposure of wild fish to contaminant mixtures found in the Columbia River Basin. The array developed in this study could also be a useful tool for studies involving endangered sucker species and other sucker species used in contaminant research.

A tiered, integrated biological and chemical monitoring framework for contaminants of emerging concern in aquatic ecosystems

The chemical-specific risk-based paradigm that informs monitoring and assessment of environmental contaminants does not apply well to the many thousands of new chemicals that are being introduced into ambient receiving waters. We propose a tiered framework that incorporates bioanalytical screening tools and diagnostic nontargeted chemical analysis to more effectively monitor for contaminants of emerging concern (CECs). The framework is based on a comprehensive battery of in vitro bioassays to first screen for a broad spectrum of CECs and nontargeted analytical methods to identify bioactive contaminants missed by the currently favored targeted analyses. Water quality managers in California have embraced this strategy with plans to further develop and test this framework in regional and statewide pilot studies on waterbodies that receive discharge from municipal wastewater treatment plants and stormwater runoff. In addition to directly informing decisions, the data obtained using this framework can be used to construct and validate models that better predict CEC occurrence and toxicity. The adaptive interplay among screening results, diagnostic assessment and predictive modeling will allow managers to make decisions based on the most current and relevant information, instead of extrapolating from parameters with questionable linkage to CEC impacts. Integr Environ Assess Manag 2016;12:540-547.

Gene expression changes in the human diaphragm after cardiothoracic surgery

OBJECTIVE We examined the effects of cardiothoracic surgery, including cardiopulmonary bypass and controlled mechanical ventilation, on messenger RNA gene expression in human diaphragm. We hypothesized that genes responsible for stress response, redox regulation, protein turnover, energy metabolism, and contractile function would be altered by cardiothoracic surgery. METHODS Paired diaphragm biopsy samples were obtained from 5 male patients (67 ± 11 years) during cardiothoracic surgery, the first as soon as the diaphragm was exposed and the second as late in surgery as possible (4.9 ± 1.8 hours between samples). We profiled messenger RNA from 5 specimen pairs with microarray analysis (Hu U133 plus 2.0; Affymetrix UK Ltd, High Wycombe, UK). Quantitative reverse transcriptase polymerase chain reaction was performed with a select set of genes exhibiting differential expression for validation. RESULTS Microarray analysis identified 779 differentially expressed (early vs late samples) unique gene products (P < .005). Postoperatively, genes related to stress response and redox regulation were upregulated. Additionally, we found significantly upregulated expression of cathepsin C (2.7-fold), cathepsin L1 (2.0-fold), various ubiquitin-conjugating enzymes (E2, approximately 1.8-fold), proinflammatory cytokine interleukin 6 (15.6-fold), and muscle-specific ubiquitin ligase (MuRF-1, 2.6-fold). Comparison of fold change values obtained by quantitative reverse transcriptase polymerase chain reaction and microarray yielded significant correlation (r = 0.95, P < .0001). CONCLUSIONS Cardiothoracic surgery results in rapid changes in human diaphragm gene expression in the operating room, including genes related to stress response, inflammation, redox regulation, and proteolysis. These results may provide insight into diaphragm muscle biology after prolonged cardiothoracic procedures.

Ecotoxicogenomics: Microarray interlaboratory comparability.

Transcriptomic analysis can complement traditional ecotoxicology data by providing mechanistic insight, and by identifying sub-lethal organismal responses and contaminant classes underlying observed toxicity. Before transcriptomic information can be used in monitoring and risk assessment, it is necessary to determine its reproducibility and detect key steps impacting the reliable identification of differentially expressed genes. A custom 15K-probe microarray was used to conduct transcriptomics analyses across six laboratories with estuarine amphipods exposed to cyfluthrin-spiked or control sediments (10 days). Two sample types were generated, one consisted of total RNA extracts (Ex) from exposed and control samples (extracted by one laboratory) and the other consisted of exposed and control whole body amphipods (WB) from which each laboratory extracted RNA. Our findings indicate that gene expression microarray results are repeatable. Differentially expressed data had a higher degree of repeatability across all laboratories in samples with similar RNA quality (Ex) when compared to WB samples with more variable RNA quality. Despite such variability a subset of genes were consistently identified as differentially expressed across all laboratories and sample types. We found that the differences among the individual laboratory results can be attributed to several factors including RNA quality and technical expertise, but the overall results can be improved by following consistent protocols and with appropriate training.

Derivation and Evaluation of Putative Adverse Outcome Pathways for the Effects of Cyclooxygenase Inhibitors on Reproductive Processes in Female Fish

Cyclooxygenase (COX) inhibitors are ubiquitous in aquatic systems and have been detected in fish tissues. The exposure of fish to these pharmaceuticals is concerning because COX inhibitors disrupt the synthesis of prostaglandins (PGs), which modulate a variety of essential biological functions, including reproduction. In this study, we investigated the effects of well-characterized mammalian COX inhibitors on female fathead minnow reproductive health. Fish (n = 8) were exposed for 96 h to water containing indomethacin (IN; 100 µg/l), ibuprofen (IB; 200 µg/l) or celecoxib (CX; 20 µg/l), and evaluated for effects on liver metabolome and ovarian gene expression. Metabolomic profiles of IN, IB and CX were not significantly different from control or one another. Exposure to IB and CX resulted in differential expression of comparable numbers of genes (IB = 433, CX = 545). In contrast, 2558 genes were differentially expressed in IN-treated fish. Functional analyses (canonical pathway and gene set enrichment) indicated extensive effects of IN on PG synthesis pathway, oocyte meiosis, and several other processes consistent with physiological roles of PGs. Transcriptomic data were congruent with PG data; IN-reduced plasma PG F2α concentration, whereas IB and CX did not. Five putative AOPs were developed linking the assumed molecular initiating event of COX inhibition, with PG reduction and the adverse outcome of reproductive failure via reduction of: (1) ovulation, (2) reproductive behaviors mediated by exogenous or endogenous PGs, and (3) oocyte maturation in fish. These pathways were developed using, in part, empirical data from the present study and other publicly available data.