How consistent are we? Interlaboratory comparison study in fathead minnows using the model estrogen 17α-ethinylestradiol to develop recommendations for environmental transcriptomics.

Fundamental questions remain about the application of omics in environmental risk assessments, such as the consistency of data across laboratories. The objective of the present study was to determine the congruence of transcript data across 6 independent laboratories. Male fathead minnows were exposed to a measured concentration of 15.8 ng/L 17α-ethinylestradiol (EE2) for 96 h. Livers were divided equally and sent to the participating laboratories for transcriptomic analysis using the same fathead minnow microarray. Each laboratory was free to apply bioinformatics pipelines of its choice. There were 12 491 transcripts that were identified by one or more of the laboratories as responsive to EE2. Of these, 587 transcripts (4.7%) were detected by all laboratories. Mean overlap for differentially expressed genes among laboratories was approximately 50%, which improved to approximately 59.0% using a standardized analysis pipeline. The dynamic range of fold change estimates was variable between laboratories, but ranking transcripts by their relative fold difference resulted in a positive relationship for comparisons between any 2 laboratories (mean R2 > 0.9, p < 0.001). Ten estrogen-responsive genes encompassing a fold change range from dramatic (>20-fold; e.g., vitellogenin) to subtle (∼2-fold; i.e., block of proliferation 1) were identified as differentially expressed, suggesting that laboratories can consistently identify transcripts that are known a priori to be perturbed by a chemical stressor. Thus, attention should turn toward identifying core transcriptional networks using focused arrays for specific chemicals. In addition, agreed-on bioinformatics pipelines and the ranking of genes based on fold change (as opposed to p value) should be considered in environmental risk assessment. These recommendations are expected to improve comparisons across laboratories and advance the use of omics in regulations. Environ Toxicol Chem 2017;36:2593-2601. © 2017 SETAC.

Targeted Gene Expression in Zebrafish Exposed to Chlorpyrifos-Oxon Confirms Phenotype-Specific Mechanisms Leading to Adverse Outcomes.

Zebrafish models for mild, moderate, and severe acute organophosphorus poisoning were previously developed by exposing zebrafish larvae to chlopyrifos-oxon. The phenotype of these models was characterized at several levels of biological organization. Oxidative stress and mitochondrial dysfunction were found to be involved in the development of the more severe phenotype. Here we used targeted gene expression to understand the dose-responsiveness of those two pathways and their involvement on generating the different zebrafish models. As the severe phenotype is irreversible after only 3 h of exposure, we also analyzed the response of the oxidative stress pathway at 3 and 24 h. Some of the genes related to oxidative stress were already differentially expressed at 3 h. There was an increase in differentially expressed genes related to both oxidative stress and mitochondrial function from the more mild to the more severe phenotype, suggesting the involvement of these mechanisms in increasing phenotype severity. Temporal data suggest that peroxynitrite leading to lipid peroxidation might be involved in phenotype transition and irreversibility.

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.

Using transcriptomic tools to evaluate biological effects across effluent gradients at a diverse set of study sites in Minnesota, USA.

The aim of this study was to explore the utility of "omics" approaches in monitoring aquatic environments where complex, often unknown stressors make chemical-specific risk assessment untenable. We examined changes in the fathead minnow (Pimephales promelas) ovarian transcriptome following 4-day exposures conducted at three sites in Minnesota (MN, USA). Within each site, fish were exposed to water from three locations along a spatial gradient relative to a wastewater treatment plant (WWTP) discharge. After exposure, site-specific impacts on gene expression in ovaries were assessed. Using an intragradient point of comparison, biological responses specifically associated with the WWTP effluent were identified using functional enrichment analyses. Fish exposed to water from locations downstream of the effluent discharges exhibited many transcriptomic responses in common with those exposed to the effluent, indicating that effects of the discharge do not fully dissipate downstream. Functional analyses showed a range of biological pathways impacted through effluent exposure at all three sites. Several of those impacted pathways at each site could be linked to potential adverse reproductive outcomes associated with the hypothalamic-pituitary-gonadal (HPG) axis in female fathead minnows, specifically signaling pathways associated with oocyte meiosis, TGF-beta signaling, gonadotropin-releasing hormone (GnRH) and epidermal growth factor receptor family (ErbB), and gene sets associated with cyclin B-1 and metalloproteinase. The utility of this approach comes from the ability to identify biological responses to pollutant exposure, particularly those that can be tied to adverse outcomes at the population level and those that identify molecular targets for future studies.

Identification of metabolic pathways in Daphnia magna explaining hormetic effects of selective serotonin reuptake inhibitors and 4-nonylphenol using transcriptomic and phenotypic responses.

The molecular mechanisms explaining hormetic effects of selective serotonin reuptake inhibitors (SSRIs) and 4-nonylphenol in Daphnia magna reproduction were studied in juveniles and adults. Transcriptome analyses showed changes in mRNA levels for 1796 genes in juveniles and 1214 genes in adults (out of 15000 total probes) exposed to two SSRIs (fluoxetine and fluvoxamine) or to 4-nonylphenol. Functional annotation of affected genes was improved by assuming the annotations of putatively homologous Drosophila genes. Self-organizing map analysis and partial least-square regression coupled with selectivity ratio procedures analyses allowed to define groups of genes with specific responses to the different treatments. Differentially expressed genes were analyzed for functional enrichment using Gene Ontology and Kyoto Encyclopaedia of Genes and Genomes databases. Serotonin metabolism, neuronal developmental processes, and carbohydrates and lipid metabolism functional categories appeared as selectively affected by SSRI treatment, whereas 4-nonylphenol deregulated genes from the carbohydrate metabolism and the ecdysone regulatory pathway. These changes in functional and metabolic pathways are consistent with previously reported SSRIs and 4-nonylphenol hormetic effects in D. magna, including a decrease in reserve carbohydrates and an increase in respiratory metabolism.

Neurotoxicogenomic investigations to assess mechanisms of action of the munitions constituents RDX and 2,6-DNT in Northern bobwhite (Colinus virginianus).

Munitions constituents (MCs) including hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), 2,4,6-trinitrotoluene (TNT), and TNT derivatives are recognized to elicit aberrant neuromuscular responses in many species. The onset of seizures resulting in death was observed in the avian model Northern bobwhite after oral dosing with RDX beginning at 8 mg/kg/day in subacute (14 days) exposures, whereas affective doses of the TNT derivative, 2,6-dinitrotoluene (2,6-DNT), caused gastrointestinal impacts, lethargy, and emaciation in subacute and subchronic (60 days) exposures. To assess and contrast the potential neurotoxicogenomic effects of these MCs, a Northern bobwhite microarray was developed consisting of 4119 complementary DNA (cDNA) features enriched for differentially-expressed brain transcripts from exposures to RDX and 2,6-DNT. RDX affected hundreds of genes in brain tissue, whereas 2,6-DNT affected few (

Up-and-down procedure (UDP) determinations of acute oral toxicity of nitroso degradation products of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX).

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), a widely used military explosive and soil and ground water contaminant of munitions manufacturing and artillery training sites, undergoes microbial nitroreductase metabolism to hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX), hexahydro-1,3-dinitroso-5-nitro-1,3,5-triazine (DNX), and hexahydro-1,3,5-trinitroso-1,3,5-triazine (TNX). Human occupational and accidental exposures to RDX, as well as acute oral exposures in rats, result in seizures, but little is known about the toxicity of the RDX degradation products. The main objective of the present study was to determine the oral LD50 of the most potent RDX N-nitroso product in female Sprague-Dawley rats using the recently validated up-and-down procedure (UDP). With only 26 rats, MNX was identified as the most potent metabolite and a maximum likelihood estimate of 187 mg kg(-1) (95% confidence interval 118-491 mg kg(-1)) for its LD50 was established and found equivalent to that of RDX determined with the same protocol. CNS toxicity, manifested as forelimb clonic seizures progressing to generalized clonic-tonic seizures, was the critical adverse effect. Further, confirmation of the UDP LD50 for MNX with a fixed-dose design enabled identification of 94 mg kg(-1) as the highest nonlethal dose. An ED50 of 57 mg kg(-1) was determined for neurotoxicity, while splenic hemosiderosis and decreased blood hematocrit and hemoglobin concentration occurred with a threshold at 94 mg kg(-1) in 14-day survivors. These studies, while providing new toxicity data necessary for the management of RDX-contaminated sites, illustrate the efficiency of the UDP for comparative acute toxicity determinations and its value in guiding further characterization of dose dependency of identified adverse effects.

Treatment of trinitrotoluene by crude plant extracts.

Crude plant extract solutions (spinach and parrotfeather) were prepared and spiked with 2,4,6-trinitrotoluene (TNT) (20 mgl(-1)). 90-h TNT removal by these solutions was compared to controls. Spinach and parrotfeather extract solutions removed 99% and 50% of the initial TNT, respectively; TNT was not eliminated in the controls or in extract solutions where removal activity was deactivated by boiling. A first-order removal constant of 0.052 h(-1) was estimated for spinach extract solutions treating 20 mgl(-1) TNT concentrations, which compared favorably to intact plant removal. Concentration variation was described by Michaelis-Menton kinetics. Detectable TNT degradation products represented only a fraction of the total TNT transformed, and the transformation favored the formation of 4-aminodinitrotoluene. The results indicated that crude plant extracts transform TNT, without the presence of the live plant.

Analysis of CL-20 in environmental matrices: water and soil.

Analytical techniques for the detection of 2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaazatetracyclo(5.5.0.05,9.03,11)dodecane (CL-20) in water and soil are developed by adapting methods traditionally used for the analysis of nitroaromatics. CL-20 (a new explosives compound) is thermally labile, exhibits high polarity, and has low solubility in water. These constraints make the use of specialized sample handling, preparation, extraction, and analysis necessary. The ability to determine the concentrations of this new explosive compound in environmental matrices is helpful in understanding the environmental fate and effects of CL-20; understanding the physical, chemical, and biological fate of CL-20; and can be used in developing remediation technologies and determining their efficiency. The toxicity and mobility of new explosives in soil and groundwater are also of interest, and analytical techniques for quantitating CL-20 and its degradation products in soil and natural waters make these investigations possible.