Global transcriptomic profiling of microcystin-LR or -RR treated hepatocytes (HepaRG).

The canonical mode of action (MOA) of microcystins (MC) is the inhibition of protein phosphatases, but complete characterization of toxicity pathways is lacking. The existence of over 200 MC congeners complicates risk estimates worldwide. This work employed RNA-seq to provide an unbiased and comprehensive characterization of cellular targets and impacted cellular processes of hepatocytes exposed to either MC-LR or MC-RR congeners. The human hepatocyte cell line, HepaRG, was treated with three concentrations of MC-LR or -RR for 2 h. Significant reduction in cell survival was observed in LR1000 and LR100 treatments whereas no acute toxicity was observed in any MR-RR treatment. RNA-seq was performed on all treatments of MC-LR and -RR. Differentially expressed genes and pathways associated with oxidative and endoplasmic reticulum (ER) stress, and the unfolded protein response (UPR) were highly enriched by both congeners as were inflammatory pathways. Genes associated with both apoptotic and inflammatory pathways were enriched in LR1000. We present a model of MC toxicity that immediately causes oxidative stress and leads to ER stress and the activation of the UPR. Differential activation of the three arms of the UPR and the kinetics of JNK activation ultimately determine whether cell survival or apoptosis is favored. Extracellular exosomes were enrichment of by both congeners, suggesting a previously unidentified mechanism for MC-dependent extracellular signaling. The complement system was enriched only in MC-RR treatments, suggesting congener-specific differences in cellular effects. This study provided an unbiased snapshot of the early systemic hepatocyte response to MC-LR and MC-RR congeners and may explain differences in toxicity among MC congeners.

Multigene Biomarkers of Pyrethroid Exposure: Exploratory Experiments.

We describe initial development of microarray-based assays for detecting 4 pyrethroid pesticides (bifenthrin, cypermethrin, esfenvalerate, and permethrin) in water. To facilitate comparison of transcriptional responses with gross apical responses, we estimated concentration-mortality curves for these pyrethroids using flow-through exposures of newly hatched Daphnia magna, Pimephales promelas adults, and 24 h posthatch P. promelas. Median lethal concentration (LC50) estimates were below most reported values, perhaps attributable to the use of flow-through exposures or of measured rather than nominal concentrations. Microarray analysis of whole P. promelas larvae and brains from exposed P. promelas adults showed that assays using either tissue type can detect these pyrethroids at concentrations below LC50 values reported for between 72 and 96% of aquatic species, depending on the pesticide. These estimates are conservative because they correspond to the lowest concentrations tested. This suggests that the simpler and less expensive whole-larval assay provides adequate sensitivity for screening contexts where acute aquatic lethality is observed, but the responsible agent is not known. Gene set analysis (GSA) highlighted several Gene Ontology (GO) terms consistent with known pyrethroid action, but the implications of other GO terms are less clear. Exploration of the sensitivity of results to changes in data processing suggests robustness of the detection assay results, but GSA results were sensitive to methodological variations. Environ Toxicol Chem 2019;38:2436-2446. Published 2019 Wiley Periodicals, Inc. on behalf of SETAC. This article is a US government work, and as such, is in the public domain in the United States of America.

Tools to minimize interlaboratory variability in vitellogenin gene expression monitoring programs.

The egg yolk precursor protein vitellogenin is widely used as a biomarker of estrogen exposure in male fish. However, standardized methodology is lacking and little is known regarding the reproducibility of results among laboratories using different equipment, reagents, protocols, and data analysis programs. To address this data gap we tested the reproducibility across laboratories to evaluate vitellogenin gene (vtg) expression and assessed the value of using a freely available software data analysis program. Samples collected from studies of male fathead minnows (Pimephales promelas) exposed to 17α-ethinylestradiol (EE2) and minnows exposed to processed wastewater effluent were evaluated for vtg expression in 4 laboratories. Our results indicate reasonable consistency among laboratories if the free software for expression analysis LinRegPCR is used, with 3 of 4 laboratories detecting vtg in fish exposed to 5 ng/L EE2 (n = 5). All 4 laboratories detected significantly increased vtg levels in 15 male fish exposed to wastewater effluent compared with 15 male fish held in a control stream. Finally, we were able to determine that the source of high interlaboratory variability from complementary deoxyribonucleic acid (cDNA) to quantitative polymerase chain reaction (qPCR) analyses was the expression analysis software unique to each real-time qPCR machine. We successfully eliminated the interlaboratory variability by reanalyzing raw fluorescence data with independent freeware, which yielded cycle thresholds and polymerase chain reaction (PCR) efficiencies that calculated results independently of proprietary software. Our results suggest that laboratories engaged in monitoring programs should validate their PCR protocols and analyze their gene expression data following the guidelines established in the present study for all gene expression biomarkers. Environ Toxicol Chem 2017;36:3102-3107. Published 2017 Wiley Periodicals Inc. on behalf of SETAC. This article is a US government work and, as such, is in the public domain in the United States of America.

Initial development of a multigene 'omics-based exposure biomarker for pyrethroid pesticides.

Omics technologies have long since promised to address a number of long standing issues related to environmental regulation. Despite considerable resource investment, there are few examples where these tools have been adopted by the regulatory community, which is in part due to a focus of most studies on discovery rather than assay development. The current work describes the initial development of an omics based assay using 48h Pimephales promelas (FHM) larvae for identifying aquatic exposures to pyrethroid pesticides. Larval FHM were exposed to seven concentrations of each of four pyrethroids (permethrin, cypermethrin, esfenvalerate and bifenthrin) in order to establish dose response curves. Then, in three separate identical experiments, FHM were exposed to a single equitoxic concentration of each pyrethroid, corresponding to 33% of the calculated LC50. All exposures were separated by weeks and all materials were either cleaned or replaced between runs in an attempt to maintain independence among exposure experiments. Gene expression classifiers were developed using the random forest algorithm for each exposure and evaluated first by cross-validation using hold out organisms from the same exposure experiment and then against test sets of each pyrethroid from separate exposure experiments. Bifenthrin exposed organisms generated the highest quality classifier, demonstrating an empirical Area Under the Curve (eAUC) of 0.97 when tested against bifenthrin exposed organisms from other exposure experiments and 0.91 against organisms exposed to any of the pyrethroids. An eAUC of 1.0 represents perfect classification with no false positives or negatives. Additionally, the bifenthrin classifier was able to successfully classify organisms from all other pyrethroid exposures at multiple concentrations, suggesting a potential utility for detecting cumulative exposures. Considerable run-to-run variability was observed both in exposure concentrations and molecular responses of exposed fish across exposure experiments. The application of a calibration step in analysis successfully corrected this, resulting in a significantly improved classifier. Classifier evaluation suggested the importance of considering a number of aspects of experimental design when developing an expression based tool for general use in ecological monitoring and risk assessment, such as the inclusion of multiple experimental runs and high replicate numbers.

A new approach for the laboratory culture of the fathead minnow, Pimephales promelas.

Fathead minnows are routinely cultured for use in aquatic toxicology studies. A new mass culture system described in the present study consisted of 6 stainless steel tanks, each containing 68 fish and 20 spawning substrates. Spawning results are compared with a previous system of 22 individual glass aquaria, which contained 16 fish and 4 spawning substrates per tank. During a 19-mo period, the new system produced an average of 4105 eggs/d, compared with an average of 2465 eggs/d with the previous system. Labor and maintenance were reduced with the new system. The stainless steel tanks eliminated aquaria glass breakage, and daily water use was reduced by 45%. Analysis of reference toxicant data from fish cultured using both systems indicated no change in the sensitivity of the test animals. Analyses of 2009 egg production data determined that a 6:1 to 7:1 female to male ratio had a significantly positive impact on egg production levels and that 6-mo-old breeding stock should be introduced to the spawning tanks in mid-spring for optimal egg production during the rest of the year. Implementing a stainless steel mass culture system significantly increased efficiency of egg production; reduced turnaround delay of mature animal availability for toxicity and molecular testing; and reduced labor time, costs, and inherent safety hazards, compared with glass aquaria systems.

An integrated assessment of sediment remediation in a midwestern U.S. stream using sediment chemistry, water quality, bioassessment, and fish biomarkers.

A comprehensive biological, sediment, and water quality study of the lower Little Scioto River near Marion, Ohio, USA, was undertaken to evaluate the changes or improvements in biotic measurements following the removal of creosote-contaminated sediment. The study area covered 7.5 river miles (RMs), including a remediated section between RMs 6.0 and 6.8. Fish and macroinvertebrate assemblages, fish biomarkers (i.e., polycyclic aromatic hydrocarbon [PAH] metabolite levels in white sucker [Castostomus commersoni] and common carp [Cyprinus carpio] bile and DNA damage), sediment chemistry, and water quality were assessed at five locations relative to the primary source of historical PAH contamination-upstream (RM 9.2), adjacent (RM 6.5), and downstream (RMs 5.7, 4.4, and 2.7). Overall, the biomarker results were consistent with the sediment PAH results, showing a pattern of low levels of PAH bile metabolites and DNA damage at the upstream (reference or background location), as well as the remediated section, high levels at the two immediate downstream sites, and somewhat lower levels at the furthest downstream site. Results show that remediation was effective in reducing sediment contaminant concentrations and exposure of fish to PAHs and in improving fish assemblages (60% increase in index of biotic integrity scores) in remediated river sections. Additional remedial investigation and potentially further remediation is needed to improve the downstream benthic fish community, which is still heavily exposed to PAH contaminants.

Assessing impacts of land-applied manure from concentrated animal feeding operations on fish populations and communities.

Concentrated animal feeding operation (CAFO) manure is a cost-effective fertilizer. In the Midwest, networks of subsurface tile-drains expedite transport of animal hormones and nutrients from land-applied CAFO manure to adjacent waterways. The objective of this study was to evaluate impacts of land-applied CAFO manure on fish populations and communities. Water chemistry including hormone, pesticide, and nutrient concentrations was characterized from study sites along with fish assemblage structure, growth, and endocrine disruption assessed in selected fish species. Although most CAFO water samples had hormone concentrations <1 ng/L, equivalent concentrations for 17ß-E2 and 17α-TB peaked at >30 ng/L each during the period of spawning, hatching, and development for resident fishes. CAFO sites had lower fish species richness, and fishes exhibited faster somatic growth and lower reproductive condition compared to individuals from the reference site. Fathead minnows (Pimephales promelas) exposed to CAFO ditchwater during early developmental stages exhibited significantly skewed sex ratios toward males. Maximum observed hormone concentrations were well above the lowest observable effect concentrations for these hormones; however, complexities at the field scale make it difficult to directly relate hormone concentration and impacts on fish. Complicating factors include the consistent presence of pesticides and nutrients, and the difference in temperature and stream architecture of the CAFO-impacted ditches compared to the reference site (e.g., channelization, bottom substrate, shallow pools, and riparian cover).

Effects of eutrophication on vitellogenin gene expression in male fathead minnows (Pimephales promelas) exposed to 17alpha-ethynylestradiol in field mesocosms.

This study evaluated the effect of aquatic secondary nutrient supply levels (nitrogen and phosphorus) on the subcellular response of adult male fathead minnows (Pimephales promelas) exposed to a single nominal concentration of 17alpha-ethynylestradiol (EE2), a potent synthetic estrogen, under quasi-natural field conditions. Outdoor mesocosms were maintained under low, medium, and high nutrient supply conditions as categorized by total phosphorus (TP) level (nominal 0.012, 0.025, and 0.045 mg TP/L, respectively), and treated with EE2 with and without a carrier solvent. Using reverse transcription-polymerase chain reaction methods, vitellogenin gene (Vg) expression was determined in the fish collected at 0 h, 8 h, 24 h, 4 d, 7 d, and 14 d post-exposure. Induction of Vg was detected as early as 8h post-exposure, with and without the carrier solvent, and persisted through Day 14. Results showed Vg to be significantly greater at low nutrient levels (p<0.05), suggesting that EE2 bioavailability to the fish was likely greater under less-turbid water conditions.

Responses of molecular indicators of exposure in mesocosms: common carp (Cyprinus carpio) exposed to the herbicides alachlor and atrazine.

Common carp (Cyprinus carpio) were treated in aquatic mesocosms with a single pulse of the herbicides atrazine or alachlor to study the bioavailability and biological activity of these herbicides using molecular indicators: Liver vitellogenin gene expression in male fish for estrogenic activity, liver cytochrome P4501A1 gene expression, and DNA damage in blood cells using the single-cell gel electrophoresis method. Both alachlor and atrazine showed dose-related increases in DNA strand breaks at environmentally relevant concentrations (<100 ppb). Gene expression indicators showed that neither herbicide had estrogenic activity in the carp, whereas atrazine at concentrations as low as 7 ppb induced cytochrome P4501A1. These results support the study of molecular indicators for exposure in surrogate ecosystems to gauge relevant environmental changes following herbicide treatments.

17alpha-ethynylestradiol-induced vitellogenin gene transcription quantified in livers of adult males, larvae, and gills of fathead minnows (Pimephales promelas).

We have applied a method for quantifying relative levels of messenger RNA (mRNA) transcription to assess chemically induced gene expression in fathead minnows (Pimephales promelas). Synthetic oligonucleotides designed for the fathead minnow vitellogenin gene transcription product were used in a reverse transcription polymerase chain reaction (RT-PCR) protocol. This sensitive and rapid strategy detected vitellogenin gene transcription in livers of male fathead minnows exposed to concentrations as low as 2 ng/L of the endocrine-disrupting compound 17alpha-ethynylestradiol for 24 h. Surprisingly, vitellogenin transcription products also were detected in gill tissue and in 48-h-old posthatch fathead minnow larvae. Relative levels of vitellogenin gene induction among individuals were quantified in a single-step reaction (PCR multiplex) with 18S rRNA universal primers and Competimers concurrently with fathead minnow vitellogenin oligonucleotides. This quantitative approach will markedly enhance detection of the first cellular event of estrogenic exposure to aquatic ecosystems in both field and laboratory systems. Use of the model provides sensitivity of detection at a concentration below those that cause mortality or visible signs of stress in fish or other aquatic organisms. The model may also provide an in vivo screening method for estrogenlike endocrine-disrupting compounds.