Effects of Age and Exposure Duration on the Sensitivity of Early Life Stage Fathead Minnow (Pimephales promelas) to Waterborne Propranolol Exposure.

Propranolol is a heavily prescribed, nonspecific beta-adrenoceptor (bAR) antagonist frequently found in wastewater effluents, prompting concern over its potential to adversely affect exposed organisms. In the present study, the transcriptional responses of 4, 5, and 6 days postfertilization (dpf) ±1 h fathead minnow, exposed for 6, 24, or 48 h to 0.66 or 3.3 mg/L (nominal) propranolol were characterized using RNA sequencing. The number of differentially expressed genes (DEGs) was used as an estimate of sensitivity. A trend toward increased sensitivity with age was observed; fish >7 dpf at the end of exposure were particularly sensitive to propranolol. The DEGs largely overlapped among treatment groups, suggesting a highly consistent response that was independent of age. Cluster analysis was performed using normalized count data for unexposed and propranolol-exposed fish. Control fish clustered tightly by age, with fish ≥7 dpf clustering away from younger fish, reflecting developmental differences. When clustering was conducted using exposed fish, in cases where propranolol induced a minimal or no transcriptional response, the results mirrored those of the control fish and did not appreciably cluster by treatment. In treatment groups that displayed a more robust transcriptional response, the effects of propranolol were evident; however, fish <7 dpf clustered away from older fish, despite having similar numbers of DEGs. Increased sensitivity at 7 dpf coincided with developmental milestones with the potential to alter propranolol pharmacokinetics or pharmacodynamics, such as the onset of exogenous feeding and gill functionality as well as increased systemic expression of bAR. These results may have broader implications because toxicity testing often utilizes fish <4 dpf, prior to the onset of these potentially important developmental milestones, which may result in an underestimation of risk for some chemicals. Environ Toxicol Chem 2024;43:807-820. Published 2023. This article is a U.S. Government work and is in the public domain in the USA.

Characterization of vitellogenin concentration in male fathead minnow mucus compared to plasma, and liver mRNA.

The objective of this study was to characterize vitellogenin (VTG) protein in male fathead minnow (Pimephales promelas) mucus compared with more conventional measures in plasma and mRNA isolated from liver. To assess the intensity and duration of changes in mucus VTG concentrations, male fathead minnows were exposed to 17α-ethinylestradiol (EE2) for 7 days with a subsequent depuration period of 14 days. The experiment was conducted in a flow-through system to maintain a consistent concentration of EE2 at a nominal EC50 concentration of 2.5 ng/L and high concentration of 10 ng/L as a positive control. Mucus, plasma and liver were sampled at regular intervals throughout the study. Relative abundance of vtg mRNA increased after 2 days of exposure and returned to control levels after 4 days of depuration. VTG protein concentration displayed similar induction kinetics in both mucus and plasma, however, it was found to be significantly increased after 2 days of exposure using the mucus-based assays and 7 days with the plasma-based assay. Significantly elevated levels of VTG were detected by both assays throughout the 14-day depuration period. The elimination of the laborious plasma collection step in the mucus-based workflow allowed sampling of smaller organisms where blood volume is limiting. It also resulted in significant gains in workflow efficiency, decreasing sampling time without loss of performance.

De Novo Assembly of the Nearly Complete Fathead Minnow Reference Genome Reveals a Repetitive but Compact Genome.

The fathead minnow is a widely used model organism in environmental toxicology. The lack of a high-quality fathead minnow reference genome, however, has severely hampered its uses in toxicogenomics. We present the de novo assembly and annotation of the fathead minnow genome using long PacBio reads, Bionano and Hi-C scaffolding data, and large RNA-sequencing data sets from different tissues and life stages. The new annotated fathead minnow reference genome has a scaffold N50 of 12.0 Mbp and a complete benchmarking universal single-copy orthologs score of 95.1%. The completeness of annotation for the new reference genome is comparable to that of the zebrafish GRCz11 reference genome. The fathead minnow genome, revealed to be highly repetitive and sharing extensive syntenic regions with the zebrafish genome, has a much more compact gene structure than the zebrafish genome. Particularly, comparative genomic analysis with zebrafish, mouse, and human showed that fathead minnow homologous genes are relatively conserved in exon regions but had strikingly shorter intron regions. The new fathead minnow reference genome and annotation data, publicly available from the National Center for Biotechnology Information and the University of California Santa Cruz genome browser, provides an essential resource for aquatic toxicogenomic studies in ecotoxicology and public health. Environ Toxicol Chem 2022;41:448-461. Published 2021. This article is a U.S. Government work and is in the public domain in the USA.

Development of omics biomarkers for estrogen exposure using mRNA, miRNA and piRNAs.

The number of chemicals requiring risk evaluation exceeds our capacity to provide the underlying data using traditional methodology. This has led to an increased focus on the development of novel approach methodologies. This work aimed to expand the panel of gene expression-based biomarkers to include responses to estrogens, to identify training strategies that maximize the range of applicable concentrations, and to evaluate the potential for two classes of small non-coding RNAs (sncRNAs), microRNA (miRNA) and piwi-interacting RNA (piRNA), as biomarkers. To this end larval Pimephales promelas (96 hpf +/- 1h) were exposed to 5 concentrations of 17α- ethinylestradiol (0.12, 1.25, 2.5, 5.0, 10.0 ng/L) for 48 h. For mRNA-based biomarker development, RNA-seq was conducted across all concentrations. For sncRNA biomarkers, small RNA libraries were prepared only for the control and 10.0 ng/L EE2 treatment. In order to develop an mRNA classifier that remained accurate over the range of exposure concentrations, three different training strategies were employed that focused on 10 ng/L, 2.5 ng/L or a combination of both. Classifiers were tested against an independent test set of individuals exposed to the same concentrations used in training and subsequently against concentrations not included in model training. Both random forest (RF) and logistic regression with elastic net regularizations (glmnet) models trained on 10 ng/L EE2 performed poorly when applied to lower concentrations. RF models trained with either the 2.5 ng/L or combination (2.5 + 10 ng/L) treatments were able to accurately discriminate exposed vs. non-exposed across all but the lowest concentrations. glmnet models were unable to accurately classify below 5 ng/L. With the exception of the 10 ng/L treatment, few mRNA differentially expressed genes (DEG) were observed, however, there was marked overlap of DEGs across treatments. Overlapping DEGs have well established linkages to estrogen and several of the 81 DEGs identified in the 10 ng/L treatment have been previously utilized as estrogenic biomarkers (vitellogenin, estrogen receptor-ß). Following multiple test correction, no sncRNAs were found to be differentially expressed, however, both miRNA and piRNA classifiers were able to accurately discriminate control and 10 ng/L exposed organisms with AUCs of 0.83 and 1.0 respectively. We have developed a highly discriminative estrogenic mRNA biomarker that is accurate over a range of concentrations likely to be found in real-world exposures. We have demonstrated that both miRNA and piRNA are responsive to estrogenic exposure, suggesting the need to further investigate their regulatory roles in the estrogenic response.

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.

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.

Aquatic concentrations of chemical analytes compared to ecotoxicity estimates.

We describe screening level estimates of potential aquatic toxicity posed by 227 chemical analytes that were measured in 25 ambient water samples collected as part of a joint USGS/USEPA drinking water plant study. Measured concentrations were compared to biological effect concentration (EC) estimates, including USEPA aquatic life criteria, effective plasma concentrations of pharmaceuticals, published toxicity data summarized in the USEPA ECOTOX database, and chemical structure-based predictions. Potential dietary exposures were estimated using a generic 3-tiered food web accumulation scenario. For many analytes, few or no measured effect data were found, and for some analytes, reporting limits exceeded EC estimates, limiting the scope of conclusions. Results suggest occasional occurrence above ECs for copper, aluminum, strontium, lead, uranium, and nitrate. Sparse effect data for manganese, antimony, and vanadium suggest that these analytes may occur above ECs, but additional effect data would be desirable to corroborate EC estimates. These conclusions were not affected by bioaccumulation estimates. No organic analyte concentrations were found to exceed EC estimates, but ten analytes had concentrations in excess of 1/10th of their respective EC: triclocarban, norverapamil, progesterone, atrazine, metolachlor, triclosan, para-nonylphenol, ibuprofen, venlafaxine, and amitriptyline, suggesting more detailed characterization of these analytes.

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.

Reproductive effects in fathead minnows (Pimphales promelas) following a 21 d exposure to 17α-ethinylestradiol.

17α-ethinylestradiol (EE2) is a synthetic estrogen that is an active ingredient in oral contraception and hormone replacement therapy. Surveys of wastewater treatment plant effluents and surface waters throughout the world have reported EE2 concentrations in the ng/L range, and these low levels can cause significant reproductive effects in fish. This study tested the effects of three environmentally relevant EE2 concentrations: 0.47, 1.54 and 3.92 ng/L using a 21 d short-term reproductive assay to investigate the effects of EE2 on fathead minnow (Pimephales promelas) reproduction. The two highest EE2 concentrations tested in this study caused significant liver gene expression and induction of vitellogenin plasma protein in male fathead minnows. Exposure to 3.92 ng EE2/L increased the production of plasma vitellogenin in the females. Plasma estradiol concentrations were significantly reduced in females exposed to 1.54 and 3.92 ng EE2/L. All three tested concentrations significantly reduced fathead minnow egg production after a 21 d exposure to EE2. The results of this study indicate that the previously reported no observed adverse effect concentration (NOAEC) for EE2 on fathead minnow egg production (1.0 ng/L) may be too high. Because all three treatments resulted in significantly reduced egg production, the lowest observed adverse effect concentration (LOAEC) for EE2 on fathead minnow egg production is 0.47 ng EE2/L. This research estimates a NOAEC for fathead minnow reproduction at 0.24 ng EE2/L following a 21 d exposure. Additionally, induction of vitellogenin is a sensitive indicator of estrogen exposure but does not appear to be predictive of fathead minnow egg production.

Sensitivity of the vitellogenin assay to diagnose exposure of fathead minnows to 17α-ethynylestradiol.

Vitellogenin is frequently used as a biomarker of exposure to environmental estrogens due to its specificity and sensitivity. Appropriate incorporation of this biomarker into environmental monitoring and assessment necessitates evaluation of its critical performance parameters. In this study, we characterize the sensitivity of both vitellogenin gene (vtg) mRNA transcripts in liver and protein (VTG) in plasma over a range of concentrations and exposure durations. Male fathead minnows were exposed to 17α-ethynylestradiol (EE2) in a flow-through system for 2, 4 and 7 days at multiple EE2 concentrations in order to provide information regarding the sensitivity of each of these biomarkers to diagnose exposure to this representative estrogen. Measurements of the expression of the vitellogenin gene and protein both reliably detected exposures to EE2 at concentrations of 5ng/l and higher at all time points. Vtg mRNA and plasma VTG appear to have similar sensitivities, though the lower variability in VTG in control fish may make it more sensitive to small changes in expression compared to vtg. For lower concentrations, sensitivity may be improved by increasing exposure duration. A sample size of ∼12 fish was sufficient in many cases to produce a statistically significant increase in vitellogenin. Larger sample sizes may provide more sensitivity at low concentrations, but detecting exposure to estrogens in the lower range of environmentally relevant concentrations may need larger sample sizes. These data will assist in designing experiments that have sufficient statistical power necessary to determine if fish have been exposed to estrogens.

Quantification and associated variability of induced vitellogenin gene transcripts in fathead minnow (Pimephales promelas) by quantitative real-time polymerase chain reaction assay.

Ecological risk assessors have a growing need for sensitive and rapid indicators of environmental exposures in aquatic ecosystems resulting from natural and synthetic estrogen-like compounds. Investigators developing subcellular exposure markers in traditional sentinel organisms must be vigilant about inherent variability of analyses, especially regarding regulatory and policy statements. Here, we report a quantitative real-time polymerase chain reaction (QPCR) assay for the detection of vitellogenin transcripts environmentally triggered in fathead minnows (Pimephales promelas). We demonstrate that our QPCR assay exhibits little inter- or intra-assay variability (21.7 and 11.9%, respectively). This method appears to be robust in terms of variability stemming from extrinsic sources, indicating that it may be readily transferable to laboratories having the requisite equipment. Our primary focus in development of this method derived from the observation that transcriptional responses of the vitellogenin gene (vtg) in fathead minnows demonstrated high biological variability between identically treated individuals, even under controlled laboratory conditions (coefficient of variation, > 100%). This variability was not seen in other genes from the same RNA preparations that we examined, suggesting that it is specific to the vitellogenin response. Our data and those of others suggest that variability in vtg expression is common to a number of aquatic vertebrates, which is indicative of genetic causation. Despite a relatively high degree of variability in vtg transcription, this method is sensitive enough to detect exposures of 5.0 ng 17alpha-ethinylestradiol (EE2)/L within 24 h of exposure, and it has the ability to discriminate 10.0 and 5.0 ng EE2/L within 48 h. The vitellogenin QPCR assay is a highly sensitive, comparatively rapid, and inexpensive method for the detection and characterization of exposure to environmental estrogens and estrogen mimics.

Collapse of a fish population after exposure to a synthetic estrogen.

Municipal wastewaters are a complex mixture containing estrogens and estrogen mimics that are known to affect the reproductive health of wild fishes. Male fishes downstream of some wastewater outfalls produce vitellogenin (VTG) (a protein normally synthesized by females during oocyte maturation) and early-stage eggs in their testes, and this feminization has been attributed to the presence of estrogenic substances such as natural estrogens [estrone or 17beta-estradiol (E2)], the synthetic estrogen used in birth-control pills [17 alpha-ethynylestradiol (EE2)], or weaker estrogen mimics such as nonylphenol in the water. Despite widespread evidence that male fishes are being feminized, it is not known whether these low-level, chronic exposures adversely impact the sustainability of wild populations. We conducted a 7-year, whole-lake experiment at the Experimental Lakes Area (ELA) in northwestern Ontario, Canada, and showed that chronic exposure of fathead minnow (Pimephales promelas) to low concentrations (5-6 ng x L(-1)) of the potent 17 alpha-ethynylestradiol led to feminization of males through the production of vitellogenin mRNA and protein, impacts on gonadal development as evidenced by intersex in males and altered oogenesis in females, and, ultimately, a near extinction of this species from the lake. Our observations demonstrate that the concentrations of estrogens and their mimics observed in freshwaters can impact the sustainability of wild fish populations.

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.