Using metabolomic profiling to inform use of surrogate species in ecological risk assessment practices.

The U.S. EPA frequently uses avian or fish toxicity data to set protective standards for amphibians in ecological risk assessments. However, this approach does not always adequately represent aquatic-dwelling and terrestrial-phase amphibian exposure data. For instance, it is accepted that early life stage tests for fish are typically sensitive enough to protect larval amphibians, however, metamorphosis from tadpole to a terrestrial-phase adult relies on endocrine cues that are less prevalent in fish but essential for amphibian life stage transitions. These differences suggest that more robust approaches are needed to adequately elucidate the impacts of pesticide exposure in amphibians across critical life stages. Therefore, in the current study, methodology is presented that can be applied to link the perturbations in the metabolomic response of larval zebrafish (Danio rerio), a surrogate species frequently used in ecotoxicological studies, to those of African clawed frog (Xenopus laevis) tadpoles following exposure to three high-use pesticides, bifenthrin, chlorothalonil, or trifluralin. Generally, D. rerio exhibited greater metabolic perturbations in both number and magnitude across the pesticide exposures as opposed to X. laevis. This suggests that screening ecological risk assessment surrogate toxicity data would sufficiently protect amphibians at the single life stage studied but care needs to be taken to understand the suite of metabolic requirements of each developing species. Ultimately, methodology presented, and data gathered herein will help inform the applicability of metabolomic profiling in establishing the risk pesticide exposure poses to amphibians and potentially other non-target species.

Cross-Taxa Distinctions in Mechanisms of Developmental Effects for Aquatic Species Exposed to Trifluralin.

Standard ecological risk assessment practices often rely on larval and juvenile fish toxicity data as representative of the amphibian aquatic phase. Empirical evidence suggests that endpoints measured in fish early life stage tests are often sufficient to protect larval amphibians. However, the process of amphibian metamorphosis relies on endocrine cues that affect development and morphological restructuring and are not represented by these test endpoints. The present study compares developmental endpoints for zebrafish (Danio rerio) and the African clawed frog (Xenopus laevis), 2 standard test species, exposed to the herbicide trifluralin throughout the larval period. Danio rerio were more sensitive and demonstrated a reduction in growth measurements with increasing trifluralin exposure. Size of X. laevis at metamorphosis was not correlated with exposure concentration; however, time to metamorphosis was delayed relative to trifluralin concentration. Gene expression patterns indicate discrepancies in response by D. rerio and X. laevis, and dose-dependent metabolic activity suggests that trifluralin exposure perturbed biological pathways differently within the 2 species. Although many metabolites were correlated with exposure concentration in D. rerio, nontargeted hepatic metabolomics identified a subset of metabolites that exhibited a nonmonotonic response to trifluralin exposure in X. laevis. Linking taxonomic distinctions in cellular-level response with ecologically relevant endpoints will refine assumptions used in interspecies extrapolation of standard test effects and improve assessment of sublethal impacts on amphibian populations. Environ Toxicol Chem 2020;39:1797-1812. Published 2020. This article is a US government work and is in the public domain in the USA.

Tissue-Based Mapping of the Fathead Minnow (Pimephales promelas) Transcriptome and Proteome.

Omics approaches are broadly used to explore endocrine and toxicity-related pathways and functions. Nevertheless, there is still a significant gap in knowledge in terms of understanding the endocrine system and its numerous connections and intricate feedback loops, especially in non-model organisms. The fathead minnow (Pimephales promelas) is a widely used small fish model for aquatic toxicology and regulatory testing, particularly in North America. A draft genome has been published, but the amount of available genomic or transcriptomic information is still far behind that of other more broadly studied species, such as the zebrafish. Here, we used a proteogenomics approach to survey the tissue-specific proteome and transcriptome profiles in adult male fathead minnow. To do so, we generated a draft transcriptome using short and long sequencing reads from liver, testis, brain, heart, gill, head kidney, trunk kidney, and gastrointestinal tract. We identified 30,378 different putative transcripts overall, with the assembled contigs ranging in size from 264 to over 9,720 nts. Over 17,000 transcripts were >1,000 nts, suggesting a robust transcriptome that can be used to interpret RNA sequencing data in the future. We also performed RNA sequencing and proteomics analysis on four tissues, including the telencephalon, hypothalamus, liver, and gastrointestinal tract of male fish. Transcripts ranged from 0 to 600,000 copies per gene and a large portion were expressed in a tissue-specific manner. Specifically, the telencephalon and hypothalamus shared the most expressed genes, while the gastrointestinal tract and the liver were quite distinct. Using protein profiling techniques, we identified a total of 4,045 proteins in the four tissues investigated, and their tissue-specific expression pattern correlated with the transcripts at the pathway level. Similarly to the findings with the transcriptomic data, the hypothalamus and telencephalon had the highest degree of similarity in the proteins detected. The main purpose of this analysis was to generate tissue-specific omics data in order to support future aquatic ecotoxicogenomic and endocrine-related studies as well as to improve our understanding of the fathead minnow as an ecological model.

Linking in vitro estrogenicity to adverse effects in the inland silverside (Menidia beryllina).

High-throughput cell assays that detect and integrate the response of multiple chemicals acting via a common mode of action have the potential to enhance current environmental monitoring practices. Establishing the linkage between in vitro and in vivo responses is key to demonstrating that in vitro cell assays can be predictive of ecologically relevant outcomes. The present study investigated the potency of 17ß-estradiol (E2), estrone (E1), nonylphenol (NP), and treated wastewater effluent using the readily available GeneBLAzer® estrogen receptor transactivation assay and 2 life stages of the inland silverside (Menidia beryllina). In vitro estrogenic potencies were ranked as follows: E2 > E1 >> NP. All 3 model estrogens induced vitellogenin and choriogenin expression in a dose-dependent manner in larvae and juveniles. However, apical effects were only found for E2 and E1 exposures of juveniles, which resulted in female-skewed sex ratios. Wastewater effluent samples exhibiting low in vitro estrogenicity (below the 10% effective concentration [EC10]), did not cause significant changes in M. beryllina. Significant induction of estrogen-responsive genes was observed at concentrations 6 to 26 times higher than in vitro responses. Gonadal feminization occurred at concentrations at least 19 to 26 times higher than the in vitro responses. These findings indicated that in vitro cell assays were more sensitive than the fish assays, making it possible to develop in vitro effect thresholds protective of aquatic organisms. Environ Toxicol Chem 2018;37:884-892. © 2017 SETAC.

Overcoming qRT-PCR interference by select carbon nanotubes in assessments of gene expression.

Nanomaterials (NMs) of various types, including carbon nanotubes (CNTs), can interfere with standard quantitative real-time PCR (qRT-PCR) assays, resulting in inaccurate gene expression measurements; however, the precise step in the qRT-PCR pipeline where this interference occurs has not been well described. Here, we investigated where in the process surface-oxidized multi-walled CNTs (oxMWNTs) inhibited qRT-PCR measurement of the expression of the housekeeping gene GAPDH and explored several strategies to minimize such inhibition. We determined that the interference occurred during the reverse transcription (RT) step and found that doubling reaction reagents or adding BSA successfully mitigated the inhibition. We observed assay interference in the presence of CNTs that were surface-oxidized, but pristine CNTs did not cause the same level of interference. These results highlight the importance of monitoring qRT-PCR assays for interference by CNTs that differ by surface chemistry, as these NMs are commonly used in gene expression assays at concentrations that we have shown to be inhibitory.

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.

Influence of the Gastrointestinal Environment on the Bioavailability of Ethinyl Estradiol Sorbed to Single-Walled Carbon Nanotubes.

Recent evidence suggests that, because of their sorptive nature, if single-walled carbon nanotubes (SWCNTs) make their way into aquatic environments, they may reduce the toxicity of other waterborne contaminants. However, few studies have examined whether contaminants remain adsorbed following ingestion by aquatic organisms. The objective of this study was to examine the bioavailability and bioactivity of ethinyl estradiol (EE2) sorbed onto SWCNTs in a fish gastrointestinal (GI) tract. Sorption experiments indicated that SWCNTs effectively adsorbed EE2, but the chemical was still able to bind and activate soluble estrogen receptors (ERs) in vitro. However, centrifugation to remove SWCNTs and adsorbed EE2 significantly reduced ER activity compared to that of EE2 alone. Additionally, the presence of SWCNTs did not reduce the extent of EE2-driven induction of vitellogenin 1 in vivo compared to the levels in organisms exposed to EE2 alone. These results suggest that while SWCNTs adsorb EE2 from aqueous solutions, under biological conditions EE2 can desorb and retain bioactivity. Additional results indicate that interactions with gastrointestinal proteins may decrease the level of adsorption of estrogen to SWCNTs by 5%. This study presents valuable data for elucidating how SWCNTs interact with chemicals that are already present in our aquatic environments, which is essential for determining their potential health risk.

Examination of Single-Walled Carbon Nanotubes Uptake and Toxicity from Dietary Exposure: Tracking Movement and Impacts in the Gastrointestinal System.

Previous studies indicate that exposure of fish to pristine single-walled carbon nanotubes (SWCNTs) by oral gavage, causes no overt toxicity, and no appreciable absorption has been observed. However, in the environment, SWCNTs are likely to be present in dietary sources, which may result in differential impacts on uptake and biological effects. Additionally, the potential of these materials to sorb nutrients (proteins, carbohydrates, and lipids) while present in the gastrointestinal (GI) tract may lead to nutrient depletion conditions that impact processes such as growth and reproduction. To test this phenomenon, fathead minnows were fed a commercial diet either with or without SWCNTs for 96 h. Tracking and quantification of SWCNTs using near-infrared fluorescence (NIRF) imaging during feeding studies showed the presence of food does not facilitate transport of SWCNTs across the intestinal epithelia. Targeting genes shown to be responsive to nutrient depletion (peptide transporters, peptide hormones, and lipases) indicated that pept2, a peptide transporter, and cck, a peptide hormone, showed differential mRNA expression by 96 h, a response that may be indicative of nutrient limitation. The results of the current study increase our understanding of the movement of SWCNTs through the GI tract, while the changes in nutrient processing genes highlight a novel mechanism of sublethal toxicity in aquatic organisms.

Gene networks and toxicity pathways induced by acute cadmium exposure in adult largemouth bass (Micropterus salmoides).

Cadmium is a heavy metal that can accumulate to toxic levels in the environment leading to detrimental effects in animals and humans including kidney, liver and lung injuries. Using a transcriptomics approach, genes and cellular pathways affected by a low dose of cadmium were investigated. Adult largemouth bass were intraperitoneally injected with 20µg/kg of cadmium chloride (mean exposure level - 2.6µg of cadmium per fish) and microarray analyses were conducted in the liver and testis 48h after injection. Transcriptomic profiles identified in response to cadmium exposure were tissue-specific with the most differential expression changes found in the liver tissues, which also contained much higher levels of cadmium than the testis. Acute exposure to a low dose of cadmium induced oxidative stress response and oxidative damage pathways in the liver. The mRNA levels of antioxidants such as catalase increased and numerous transcripts related to DNA damage and DNA repair were significantly altered. Hepatic mRNA levels of metallothionein, a molecular marker of metal exposure, did not increase significantly after 48h exposure. Carbohydrate metabolic pathways were also disrupted with hepatic transcripts such as UDP-glucose, pyrophosphorylase 2, and sorbitol dehydrogenase highly induced. Both tissues exhibited a disruption of steroid signaling pathways. In the testis, estrogen receptor beta and transcripts linked to cholesterol metabolism were suppressed. On the contrary, genes involved in cholesterol metabolism were highly increased in the liver including genes encoding for the rate limiting steroidogenic acute regulatory protein and the catalytic enzyme 7-dehydrocholesterol reductase. Integration of the transcriptomic data using functional enrichment analyses revealed a number of enriched gene networks associated with previously reported adverse outcomes of cadmium exposure such as liver toxicity and impaired reproduction.

Chronic nanoparticulate silver exposure results in tissue accumulation and transcriptomic changes in zebrafish.

Increasing utilization of metallic nanomaterials in recent years implies an increasing rate of release to the environment, with potentially serious adverse effects on environmentally important species. Previously, we demonstrated that exposure to nanoparticulate silver for 24-48 h results in dramatic alterations in global gene expression patterns and increased tissue burdens in zebrafish gills. The present study reports outcomes associated with chronic exposure to nanoparticulate silver in zebrafish. Adult female Danio rerio were exposed to 5, 15, 25, or 50 µg/L nanoparticulate silver in a time course up to 28 days. A soluble silver treatment (5 µg/L) was also included. Results indicate that use of flow-through systems for chronic nanometal studies is a viable concept; measured concentrations of approximately 60% of nominal values over the course of the 28-day exposure were observed. Dissolution of nanoparticulate silver was measured twice weekly throughout the exposure ranging between 0.5 and 1.0 µg/L, and was relatively consistent between nanoparticulate silver tanks, with no differences between treatments. Gill samples from the 28-day time point were analyzed for global gene expression patterns and histopathology. Tissue accumulation in both gill and eviscerated carcass was dose-dependent, and remained elevated 4 days after the silver was removed. Microarray analysis also revealed a dose-dependent response pattern, with the largest number of genes affected in the 50 µg/L AgNP exposure. Pathway analysis of affected genes identified a number of GO terms that were significantly over-represented in the high AgNP dataset. These terms are associated with DNA damage repair, cellular restructuring, and developmental processes.

Behavioral responses of adult male and female fathead minnows to a model estrogenic effluent and its effects on exposure regime and reproductive success.

Laboratory studies of adult male fathead minnows have shown that when they are exposed to estrogens, they lose their ability to compete for access to females and sire young, suggesting that estrogenic effluents may reduce the genetic fitness of populations of wild fishes. However, it is unknown whether wild fish which are exposed to effluent actually compete with unexposed fishes, how long effects of estrogen exposure last, and whether females are affected by estrogens. This study addressed these issues using the fathead minnow (FHM) and effluent from the Metropolitan Wastewater Treatment Plant (MWTP) a well-studied source of environmental estrogens (EEs) in the Mississippi River. Maze tests found that adult FHMs are neither attracted nor repelled by MWTP effluent while previous studies have shown that minnows are attracted to the warmer waters which characterize effluents; it is realistic that previously unexposed fish enter MWTP effluent in the spring and then compete with exposed individuals. Competitive spawning experiments showed that male FHMs exposed to 44ng E2/l (a high but realistic level) for three weeks failed to compete with unexposed males while males exposed to 4ng E2/l outcompeted and sired more young than unexposed males (p<0.05). The effects of estrogen exposure disappeared within a week of moving fish into uncontaminated water. Female FHM reproductive output and behavior were unaffected by exposure to estrogen. Taken together, these experiments suggest that the behavior of wild fishes likely determines their exposure to EEs and that while the effects of this exposure are likely significant to populations of wild fish, they will be location specific because of factors which determine the duration and intensity of male exposure. We conclude that the role of fish behavior in endocrine disruption strongly warrants additional consideration.

Behavioral and genomic impacts of a wastewater effluent on the fathead minnow.

Rivers containing effluents from water treatment plants are complex soups of compounds, ranging from pharmaceuticals to natural hormones. Male fathead minnows (Pimephales promelas) were exposed for 3 weeks to effluent waters from the Metropolitan Wastewater Treatment Plant in St. Paul, MN. Fish were tested for their competitive nest holding behavior. Changes in vitellogenin were measured and these were correlated to changes in gene expression using a 22,000 gene microarray developed specifically for fathead minnows. Significant changes in gene expression were observed in both liver and testis, which correlate to phenotypic changes of vitellogenin induction and reduced competitive behavior. We also compared by real-time PCR the expression changes in key genes related to steroid biosynthesis and metabolism in fish exposed to the effluent as well as in fish exposed to a model estrogen and a model androgen. While the gene expression signature from effluent-exposed fish shared some elements with estrogen and androgen signatures, overall it was different, underscoring the complexity of compounds present in sewage and their different modes of action.