Multi-omic responses of fish exposed to complex chemical mixtures in the Shenandoah River watershed.

To evaluate relationships between different anthropogenic impacts, contaminant occurrence, and fish health, we conducted in situ fish exposures across the Shenandoah River watershed at five sites with different land use. Exposure water was analyzed for over 500 chemical constituents, and organismal, metabolomic, and transcriptomic endpoints were measured in fathead minnows. Adverse reproductive outcomes were observed in fish exposed in the upper watershed at both wastewater treatment plant (WWTP) effluent- and agriculture-impacted sites, including decreased gonadosomatic index and altered secondary sex characteristics. This was accompanied with increased mortality at the site most impacted by agricultural activities. Molecular biomarkers of estrogen exposure were unchanged and consistent with low or non-detectable concentrations of common estrogens, indicating that alternative mechanisms were involved in organismal adverse outcomes. Hepatic metabolomic and transcriptomic profiles were altered in a site-specific manner, consistent with variation in land use and contaminant profiles. Integrated biomarker response data were useful for evaluating mechanistic linkages between contaminants and adverse outcomes, suggesting that reproductive endocrine disruption, altered lipid processes, and immunosuppression may have been involved in these organismal impacts. This study demonstrated linkages between human-impact, contaminant occurrence, and exposure effects in the Shenandoah River watershed and showed increased risk of adverse outcomes in fathead minnows exposed to complex mixtures at sites impacted by municipal wastewater discharges and agricultural practices.

Expanding non-invasive approaches for fish-health monitoring: A survey of the epidermal mucous metabolomes of phylogenetically diverse freshwater fish species.

There is a pressing need for more-holistic approaches to fisheries assessments along with growing demand to reduce the health impacts of sample collections. Metabolomic tools enable the use of sample matrices that can be collected with minimal impact on the organism (e.g., blood, urine, and mucus) and provide high-throughput, untargeted biochemical information without the requirement of a sequenced genome. These qualities make metabolomics ideal for monitoring a wide range of fish species, particularly those under protected status. In the current study, we surveyed the relative abundances of 120 endogenous metabolites in epidermal mucus across eight freshwater fish species belonging to seven phylogenetic orders. Principal component analysis was used to provide an overview of the data set, revealing strong interspecies relationships in the epidermal mucous metabolome. Normalized relative abundances of individual endogenous metabolites were then used to identify commonalities across multiple species, as well as those metabolites that showed notable species specificity. For example, taurine was measured in high relative abundance in the epidermal mucus of common carp (Cyprinus carpio), northern pike (Esox lucius), golden shiner (Notemigonus crysoleucas), rainbow trout (Oncorhynchus mykiss), and rainbow smelt (Osmerus mordax), whereas γ-amino butyric acid (GABA) exhibited a uniquely high relative abundance in flathead catfish (Pylodictis olivaris). Finally, hierarchical cluster analysis was used to evaluate species relatedness as characterized by both the epidermal mucous metabolome (phenotype) and genetic phylogeny (genotype). This comparison revealed species for which relatedness in the epidermal mucous metabolome composition closely aligns with phylogenetic relatedness (e.g., N. crysoleucas and C. carpio), as well as species for which these two measures are not well aligned (e.g., P. olivaris and Polyodon spathula). These, and other findings reported here, highlight novel areas for future research with fish, including development of epidermal mucous-based markers for non-invasive health monitoring, sex determination, and hypoxia tolerance.

Effects of Metformin and its Metabolite Guanylurea on Fathead Minnow (Pimephales promelas) Reproduction.

Metformin, along with its biotransformation product guanylurea, is commonly observed in municipal wastewaters and subsequent surface waters. Previous studies in fish have identified metformin as a potential endocrine-active compound, but there are inconsistencies with regard to its effects. To further investigate the potential reproductive toxicity of metformin and guanylurea to fish, a series of experiments was performed with adult fathead minnows (Pimephales promelas). First, explants of fathead minnow ovary tissue were exposed to 0.001-100 µM metformin or guanylurea to investigate whether the compounds could directly perturb steroidogenesis. Second, spawning pairs of fathead minnows were exposed to metformin (0.41, 4.1, and 41 µg/L) or guanylurea (1.0, 10, and 100 µg/L) for 23 days to assess impacts on reproduction. Lastly, male fathead minnows were exposed to 41 µg/L metformin, 100 µg/L guanylurea, or a mixture of both compounds, with samples collected over a 96-h time course to investigate potential impacts to the hepatic transcriptome or metabolome. Neither metformin nor guanylurea affected steroid production by ovary tissue exposed ex vivo. In the 23 days of exposure, neither compound significantly impacted transcription of endocrine-related genes in male liver or gonad, circulating steroid concentrations in either sex, or fecundity of spawning pairs. In the 96-h time course, 100 µg guanylurea/L elicited more differentially expressed genes than 41 µg metformin/L and showed the greatest impacts at 96 h. Hepatic transcriptome and metabolome changes were chemical- and time-dependent, with the largest impact on the metabolome observed at 23 days of exposure to 100 µg guanylurea/L. Overall, metformin and guanylurea did not elicit effects consistent with reproductive toxicity in adult fathead minnows at environmentally relevant concentrations. Environ Toxicol Chem 2022;41:2708-2720. © 2022 SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Untargeted MSn-Based Monitoring of Glucuronides in Fish: Screening Complex Mixtures for Contaminants with Biological Relevance.

The complexity of contaminant mixtures in surface waters has presented long-standing challenges to the assessment of risks to human health and the environment. As a result, novel strategies for both identifying contaminants that have not been routinely monitored through targeted methods and prioritizing detected compounds with respect to their biological relevance are needed. Tracking biotransformation products in biofluids and tissues in an untargeted fashion facilitates the identification of chemicals taken up by the resident species (e.g., fish), so by default ensuring that detected compounds are biologically relevant in terms of exposure. In this study, we investigated xenobiotic glucuronidation, which is arguably the most important phase II metabolism pathway for many pharmaceuticals, pesticides, and other environmental contaminants. The application of an untargeted high-resolution mass spectrometry-based approach tentatively revealed the presence of over 70 biologically relevant xenobiotics in bile collected from male and female fathead minnows exposed to wastewater treatment plant effluents. The majority of these were not targets of conventional contaminant monitoring. These results highlight the utility of biologically based untargeted screening methods when evaluating chemical contaminants in complex environmental mixtures.

The Eco-Exposome Concept: Supporting an Integrated Assessment of Mixtures of Environmental Chemicals.

Organisms are exposed to ever-changing complex mixtures of chemicals over the course of their lifetime. The need to more comprehensively describe this exposure and relate it to adverse health effects has led to formulation of the exposome concept in human toxicology. Whether this concept has utility in the context of environmental hazard and risk assessment has not been discussed in detail. In this Critical Perspective, we propose-by analogy to the human exposome-to define the eco-exposome as the totality of the internal exposure (anthropogenic and natural chemicals, their biotransformation products or adducts, and endogenous signaling molecules that may be sensitive to an anthropogenic chemical exposure) over the lifetime of an ecologically relevant organism. We describe how targeted and nontargeted chemical analyses and bioassays can be employed to characterize this exposure and discuss how the adverse outcome pathway concept could be used to link this exposure to adverse effects. Available methods, their limitations, and/or requirement for improvements for practical application of the eco-exposome concept are discussed. Even though analysis of the eco-exposome can be resource-intensive and challenging, new approaches and technologies make this assessment increasingly feasible. Furthermore, an improved understanding of mechanistic relationships between external chemical exposure(s), internal chemical exposure(s), and biological effects could result in the development of proxies, that is, relatively simple chemical and biological measurements that could be used to complement internal exposure assessment or infer the internal exposure when it is difficult to measure. Environ Toxicol Chem 2022;41:30-45. © 2021 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Untargeted Lipidomics for Determining Cellular and Subcellular Responses in Zebrafish (Danio rerio) Liver Cells Following Exposure to Complex Mixtures in U.S. Streams.

Surface waters often contain a variety of chemical contaminants potentially capable of producing adverse outcomes in both humans and wildlife due to impacts from industrial, urban, and agricultural activity. Here, we report the results of a zebrafish liver (ZFL) cell-based lipidomics approach to assess the potential ecotoxicological effects of complex contaminant mixtures using water collected from eight impacted streams across the United States mainland and Puerto Rico. We initially characterized the ZFL lipidome using high resolution mass spectrometry, resulting in the annotation of 508 lipid species covering 27 classes. We then identified lipid changes induced by all streamwater samples (nonspecific stress indicators) as well as those unique to water samples taken from specific streams. Subcellular impacts were classified based on organelle-specific lipid changes, including increased lipid saturation (endoplasmic reticulum stress), elevated bis(monoacylglycero)phosphate (lysosomal overload), decreased ubiquinone (mitochondrial dysfunction), and elevated ether lipids (peroxisomal stress). Finally, we demonstrate how these results can uniquely inform environmental monitoring and risk assessments of surface waters.

Cell-Based Metabolomics for Untargeted Screening and Prioritization of Vertebrate-Active Stressors in Streams Across the United States.

The U.S. Geological Survey and the U.S. Environmental Protection Agency have assessed contaminants in 38 streams across the U.S., using an extensive suite of target-chemical analysis methods along with a variety of biological effects tools. Here, we report zebrafish liver (ZFL) cell-culture based NMR metabolomic analysis of these split stream samples. We used this untargeted approach to evaluate the sites according to overall impact on the ZFL metabolome and found that neither the total number of organics detected at the sites, nor their cumulative concentrations, were good predictors of these impacts. Further, we used partial least squares regression to compare ZFL endogenous metabolite profiles to values for 455 potential stressors (organics, inorganics, and physical properties) measured in these waters and found that the profiles covaried with at most 280 of the stressors, which were subsequently ranked into quartiles based on the strength of their covariance. While contaminants of emerging concern (CECs) were well represented in the top, most strongly covarying quartile-suggesting considerable potential for eliciting biological responses at these sites-there was even higher representation of various well-characterized legacy contaminants (e.g., PCBs). These results emphasize the importance of complementing chemical analysis with untargeted bioassays to help focus regulatory efforts on the most significant ecosystem threats.

Assessing the impact of wastewater treatment plant effluent on downstream drinking water-source quality using a zebrafish (Danio Rerio) liver cell-based metabolomics approach.

Cell-based metabolomics was used in a proof-of-concept fashion to investigate the biological effects of contaminants as they traveled from a wastewater treatment plant (WWTP) discharge to a drinking water treatment plant (DWTP) intake in a surface-water usage cycle. Zebrafish liver (ZFL) cells were exposed to water samples collected along a surface-water flowpath, where a WWTP was located ∼14.5 km upstream of a DWTP. The sampling sites included: 1) upstream of the WWTP, 2) the WWTP effluent discharging point, 3) a proximal location downstream of the WWTP outfall, 4) a distal location downstream of the WWTP outfall, 5) the drinking water intake, and 6) the treated drinking water collected prior to discharge to the distribution system. After a 48-h laboratory exposure, the hydrophilic and lipophilic metabolites in ZFL cell extracts were analyzed by proton nuclear magnetic resonance (1H NMR) spectroscopy and gas chromatography-mass spectrometry (GC-MS), respectively. Multivariate statistical analysis revealed distinct changes in metabolite profiles in response to WWTP effluent exposure. These effects on the hydrophilic metabolome gradually diminished downstream of the WWTP, becoming non-significant at the drinking water intake (comparable to upstream of the WWTP, p = 0.98). However, effects on the lipophilic metabolome increased significantly as the river flowed from the distal location downstream of the WWTP to the drinking water intake (p < 0.001), suggesting a source of bioactive compounds in this watershed other than the WWTP. ZFL cells exposed to treated drinking water did not exhibit significant changes in either the hydrophilic (p = 0.15) or lipophilic metabolome (p = 0.83) compared to the upstream site, suggesting that constituents in the WWTP effluent were efficiently removed by the drinking water treatment process. Impacts on ZFL cells from the WWTP effluent included disrupted energy metabolism, a global decrease in amino acids, and altered lipid metabolism pathways. Overall, this study demonstrated the utility of cell-based metabolomics as an effective tool for assessing the biological effects of complex pollutant mixtures, particularly when used as a complement to conventional chemical monitoring.

Evaluation of targeted and untargeted effects-based monitoring tools to assess impacts of contaminants of emerging concern on fish in the South Platte River, CO.

Rivers in the arid Western United States face increasing influences from anthropogenic contaminants due to population growth, urbanization, and drought. To better understand and more effectively track the impacts of these contaminants, biologically-based monitoring tools are increasingly being used to complement routine chemical monitoring. This study was initiated to assess the ability of both targeted and untargeted biologically-based monitoring tools to discriminate impacts of two adjacent wastewater treatment plants (WWTPs) on Colorado's South Platte River. A cell-based estrogen assay (in vitro, targeted) determined that water samples collected downstream of the larger of the two WWTPs displayed considerable estrogenic activity in its two separate effluent streams. Hepatic vitellogenin mRNA expression (in vivo, targeted) and NMR-based metabolomic analyses (in vivo, untargeted) from caged male fathead minnows also suggested estrogenic activity downstream of the larger WWTP, but detected significant differences in responses from its two effluent streams. The metabolomic results suggested that these differences were associated with oxidative stress levels. Finally, partial least squares regression was used to explore linkages between the metabolomics responses and the chemical contaminants that were detected at the sites. This analysis, along with univariate statistical approaches, identified significant covariance between the biological endpoints and estrone concentrations, suggesting the importance of this contaminant and recommending increased focus on its presence in the environment. These results underscore the benefits of a combined targeted and untargeted biologically-based monitoring strategy when used alongside contaminant monitoring to more effectively assess ecological impacts of exposures to complex mixtures in surface waters.

High-resolution mass spectrometry of skin mucus for monitoring physiological impacts and contaminant biotransformation products in fathead minnows exposed to wastewater effluent.

High-resolution mass spectrometry is advantageous for monitoring physiological impacts and contaminant biotransformation products in fish exposed to complex wastewater effluent. We evaluated this technique using skin mucus from male and female fathead minnows (Pimephales promelas) exposed to control water or treated wastewater effluent at 5, 20, and 100% levels for 21 d, using an on-site, flow-through system providing real-time exposure. Both sex-specific and non-sex-specific responses were observed in the mucus metabolome, the latter suggesting the induction of general compensatory pathways for xenobiotic exposures. Altogether, 85 statistically significant treatment-dependent metabolite changes were observed out of the 310 total endogenous metabolites that were detected (156 of the 310 were annotated). Partial least squares-regression models revealed strong covariances between the mucus metabolomes and up-regulated hepatic messenger ribonucleic acid (mRNA) transcripts reported previously for these same fish. These regression models suggest that mucus metabolomic changes reflected, in part, processes by which the fish biotransformed xenobiotics in the effluent. In keeping with this observation, we detected a phase II transformation product of bisphenol A in the skin mucus of male fish. Collectively, these findings demonstrate the utility of mucus as a minimally invasive matrix for simultaneously assessing exposures and effects of environmentally relevant mixtures of contaminants. Environ Toxicol Chem 2018;37:788-796. 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.

The Role of Omics in the Application of Adverse Outcome Pathways for Chemical Risk Assessment.

In conjunction with the second International Environmental Omics Symposium (iEOS) conference, held at the University of Liverpool (United Kingdom) in September 2014, a workshop was held to bring together experts in toxicology and regulatory science from academia, government and industry. The purpose of the workshop was to review the specific roles that high-content omics datasets (eg, transcriptomics, metabolomics, lipidomics, and proteomics) can hold within the adverse outcome pathway (AOP) framework for supporting ecological and human health risk assessments. In light of the growing number of examples of the application of omics data in the context of ecological risk assessment, we considered how omics datasets might continue to support the AOP framework. In particular, the role of omics in identifying potential AOP molecular initiating events and providing supportive evidence of key events at different levels of biological organization and across taxonomic groups was discussed. Areas with potential for short and medium-term breakthroughs were also discussed, such as providing mechanistic evidence to support chemical read-across, providing weight of evidence information for mode of action assignment, understanding biological networks, and developing robust extrapolations of species-sensitivity. Key challenges that need to be addressed were considered, including the need for a cohesive approach towards experimental design, the lack of a mutually agreed framework to quantitatively link genes and pathways to key events, and the need for better interpretation of chemically induced changes at the molecular level. This article was developed to provide an overview of ecological risk assessment process and a perspective on how high content molecular-level datasets can support the future of assessment procedures through the AOP framework.

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.

Linking field-based metabolomics and chemical analyses to prioritize contaminants of emerging concern in the Great Lakes basin.

The ability to focus on the most biologically relevant contaminants affecting aquatic ecosystems can be challenging because toxicity-assessment programs have not kept pace with the growing number of contaminants requiring testing. Because it has proven effective at assessing the biological impacts of potentially toxic contaminants, profiling of endogenous metabolites (metabolomics) may help screen out contaminants with a lower likelihood of eliciting biological impacts, thereby prioritizing the most biologically important contaminants. The authors present results from a study that utilized cage-deployed fathead minnows (Pimephales promelas) at 18 sites across the Great Lakes basin. They measured water temperature and contaminant concentrations in water samples (132 contaminants targeted, 86 detected) and used 1 H-nuclear magnetic resonance spectroscopy to measure endogenous metabolites in polar extracts of livers. They used partial least-squares regression to compare relative abundances of endogenous metabolites with contaminant concentrations and temperature. The results indicated that profiles of endogenous polar metabolites covaried with at most 49 contaminants. The authors identified up to 52% of detected contaminants as not significantly covarying with changes in endogenous metabolites, suggesting they likely were not eliciting measurable impacts at these sites. This represents a first step in screening for the biological relevance of detected contaminants by shortening lists of contaminants potentially affecting these sites. Such information may allow risk assessors to prioritize contaminants and focus toxicity testing on the most biologically relevant contaminants. Environ Toxicol Chem 2016;35:2493-2502. Published 2016 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.

Metabolite profiles of repeatedly sampled urine from male fathead minnows (Pimephales promelas) contain unique lipid signatures following exposure to anti-androgens.

The purpose of this study was twofold. First, we sought to identify candidate markers of exposure to anti-androgens by analyzing endogenous metabolite profiles in the urine of male fathead minnows (mFHM, Pimephales promelas). Based on earlier work, we hypothesized that unidentified lipids in the urine of mFHM were selectively responsive to exposure to androgen receptor antagonists, which is otherwise difficult to confirm using established fish toxicity assays. A second goal was to evaluate the feasibility of non-lethally and repeatedly sampling urine from individual mFHMs over the time course of response to a chemical exposure. Accordingly, we exposed mFHM to the model anti-androgens vinclozolin or flutamide. Urine was collected from each fish at 48hour intervals over the course of a 14day exposure. Parallel experiments were conducted with mFHM exposed to bisphenol A or control water. The frequent handling/sampling regime did not cause apparent adverse effects on the fish. Endogenous metabolite profiling was conducted with gas chromatography-mass spectrometry (GC-MS), which exhibited lower variation for the urinary metabolome than was found in earlier work with nuclear magnetic resonance (NMR) spectroscopy. Specifically, for inter- and intra-individual variations, the median spectrum-wide relative standard deviation (RSD) was 32.6% and 33.3%, respectively, for GC-MS analysis of urine from unexposed mFHM. These results compared favorably with similar measurements of urine from other model species, including the Sprague Dawley rat. In addition, GC-MS allowed us to identify several lipids (e.g., certain saturated fatty acids) in mFHM urine as candidate markers of exposure to androgen receptor antagonists.

Integrated approach to explore the mechanisms of aromatase inhibition and recovery in fathead minnows (Pimephales promelas).

Aromatase, a member of the cytochrome P450 superfamily, is a key enzyme in estradiol synthesis that catalyzes the aromatization of androgens into estrogens in ovaries. Here, we used an integrated approach to assess the mechanistic basis of the direct effects of aromatase inhibition, as well as adaptation and recovery processes in fish. We exposed female fathead minnows (Pimephales promelas) via the water to 30 µg/L of a model aromatase inhibitor, fadrozole, during 8 days (exposure phase). Fish were then held in clean water for 8 more days (recovery phase). Samples were collected at 1, 2, 4, and 8 days of both the exposure and the recovery phases. Transcriptomics, metabolomics, and network inference were used to understand changes and infer connections at the transcript and metabolite level in the ovary. Apical endpoints directly indicative of endocrine function, such as plasma estradiol, testosterone, and vitellogenin levels were also measured. An integrated analysis of the data revealed changes in gene expression consistent with increased testosterone in fadrozole-exposed ovaries. Metabolites such as glycogen and taurine were strongly correlated with increased testosterone levels. Comparison of in vivo and ex vivo steroidogenesis data suggested the accumulation of steroidogenic enzymes, including aromatase, as a mechanism to compensate for aromatase inhibition.

Impacts of 17α-ethynylestradiol exposure on metabolite profiles of zebrafish (Danio rerio) liver cells.

Endocrine disrupting chemicals (EDCs) that are frequently detected in bodies of water downstream from sewage treatment facilities can have adverse impacts on fish and other aquatic organisms. To properly assess risk(s) from EDCs, tools are needed that can establish linkages from chemical exposures to adverse outcomes. Traditional methods of testing chemical exposure and toxicity using experimental animals are excessively resource- and time-consuming. In line with EPA's goal of reducing animal use in testing, these traditional screening methods may not be sustainable in the long term, given the ever increasing number of chemicals that must be tested for safety. One of the most promising ways to reduce costs and increase throughput is to use cell cultures instead of experimental animals. In accordance with National Research Council's vision on 21st century toxicity testing, we have developed a cell culture-based metabolomics approach for this application. Using a zebrafish (Danio rerio) liver cell line (ZFL), we have applied NMR-based metabolomics to investigate responses of ZFL cells exposed to 17α-ethynylestradiol (EE2). This analysis showed that metabolite changes induced by EE2 exposure agree well with known impacts of estrogens on live fish. The results of this study demonstrate the potential of cell-based metabolomics to assess chemical exposure and toxicity for regulatory application.

Fishy aroma of social status: urinary chemo-signalling of territoriality in male fathead minnows (Pimephales promelas).

Chemical structures of several urinary reproductive pheromones in fish have been identified, and their role in the chemical communication of reproductive condition is well characterized. On the contrary, the role of chemical communication in signalling of social/territorial status in fish is poorly understood. Fathead minnows are an example of a fish species whose life history traits appear conducive to evolution of chemical communication systems that confer information about social/territorial status. Male reproduction in this species is dependent upon their ability to acquire and defend a high quality nesting territory, and to attract a female to the nest. We hypothesized that fathead minnow males use visual and urine-derived chemical cues to signal territorial status. To test this hypothesis, effects of territorial acquisition on male-specific secondary sex characteristics (SSCs) and urine volumes were first assessed. Second, frequencies of male urination in varying social contexts were examined. Finally, nuclear magnetic resonance-based metabolomics was used to identify urinary metabolites that were differentially excreted in the urine of territorial versus non-territorial males. The expression of SSCs, sperm, and urine volumes increased with territory acquisition, and either remained unchanged or decreased in non-territorial males. Frequency of male urination increased significantly in the presence of females (but not males), suggesting that females are the main target of the urinary signals. Territorial and non-territorial males had distinct urinary metabolomic profiles. An unforeseen finding was that one could discern future territorial status of males, based on their initial metabolomic profiles. Bile acids and volatile amines were identified as potential chemical signals of social status in the fathead minnow. The finding that trimethylamine (a fishy smelling volatile amine) may be a social cue is particularly interesting, because it is known to bind trace amine-associated receptors, indicating that these receptors may play role in chemical signalling of social status in fish.

Push-through direct injection NMR: an optimized automation method applied to metabolomics.

There is a pressing need to increase the throughput of NMR analysis in fields such as metabolomics and drug discovery. Direct injection (DI) NMR automation is recognized to have the potential to meet this need due to its suitability for integration with the 96-well plate format. However, DI NMR has not been widely used as a result of some insurmountable technical problems; namely: carryover contamination, sample diffusion (causing reduction of spectral sensitivity), and line broadening caused by entrapped air bubbles. Several variants of DI NMR, such as flow injection analysis (FIA) and microflow NMR, have been proposed to address one or more of these issues, but not all of them. The push-through direct injection technique reported here overcomes all of these problems. The method recovers samples after NMR analysis, uses a "brush-wash" routine to eliminate carryover, includes a procedure to push wash solvent out of the flow cell via the outlet to prevent sample diffusion, and employs an injection valve to avoid air bubbles. Herein, we demonstrate the robustness, efficiency, and lack of carryover characteristics of this new method, which is ideally suited for relatively high throughput analysis of the complex biological tissue extracts used in metabolomics, as well as many other sample types. While simple in concept and setup, this new method provides a substantial improvement over current approaches.

Use of gene expression, biochemical and metabolite profiles to enhance exposure and effects assessment of the model androgen 17ß-trenbolone in fish.

The impact of exposure by water to a model androgen, 17ß-trenbolone (TRB), was assessed in fathead minnows using an integrated molecular approach. This included classical measures of endocrine exposure such as impacts on testosterone (T), 17ß-estradiol (E2), and vitellogenin (VTG) concentrations in plasma, as well as determination of effects on the hepatic metabolome using proton nuclear magnetic resonance spectroscopy. In addition, the rates of production of T and E2 in ovary explants were measured, as were changes in a number of ovarian gene transcripts hypothesized to be relevant to androgen exposure. A temporally intensive 16-d test design was used to assess responses both during and after the TRB exposure (i.e., depuration/recovery). This strategy revealed time-dependent responses in females (little impact was seen in the males), in which changes in T and E2 production in the ovary, as well as levels in plasma, declined rapidly (within 1 d), followed shortly by a return to control levels. Gene expression measurements revealed dynamic control of transcript levels in the ovary and suggested potential mechanisms for compensation during the exposure phase of the test. Proton nuclear magnetic resonance spectroscopy revealed a number of hepatic metabolite changes that exhibited strong time and dose dependence. Furthermore, TRB appeared to induce the hepatic metabolome of females to become more like that of males at both high test concentrations of TRB (472 ng/L) and more environmentally relevant levels (33 ng/L).

Impacts of an anti-androgen and an androgen/anti-androgen mixture on the metabolite profile of male fathead minnow urine.

Male and female fathead minnows (Pimephales promelas, FHM) were exposed via water to 20 or 200 microg/L of cyproterone acetate (CA), a model androgen receptor (AR) antagonist. FHM were also exposed to 500 ng/L of 17beta-trenbolone (TB), a model AR agonist, and to mixtures of TB with both concentrations of CA. The urine metabolite profile (as measured by 1H NMR spectroscopy) of male FHM exposed to the high concentration of CA was markedly different from that of controls, and this difference was less for males coexposed to the associated TB+CA mixture. The exposure to TB alone had almost no impact on the male urine profile. These results suggest that male FHM urinary metabolite profiling may be useful for directly detecting effects of anti-androgens. In contrast, the urinary profile of male FHM exposed to the lower concentration of CA was not very different from that of controls, but, unexpectedly, this difference was increased when coexposed to the associated TB+CA mixture. This suggests that TB with CA at the lower concentration impacts male FHM through an interactive effect possibly unrelated, or in addition, to AR antagonism. The relative occurrence of male-like nuptial tubercles in female FHM exposed to TB and to the mixtures of TB and CA supported the metabolomics data.