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Evaluating biomarkers of stress in amphibians is critical to conservation, yet current techniques are often destructive and/or time-consuming, which limits ease of use. In the present study, we validate the use of dermal swabs in spotted salamanders (Ambystoma maculatum) for biochemical profiling, as well as glutathione (GSH) stress response following pesticide exposure. Thirty-three purchased spotted salamanders were acclimated to laboratory conditions at Washington College (Chestertown, MD, USA) for 4 weeks. Following acclimation, salamanders were randomly sorted into three groups for an 8-h pesticide exposure on soil: control with no pesticide, 2,4-dichlorophenoxyacetic acid (2,4-D), or chlorpyrifos. Before and after exposure, mucus samples were obtained by gently rubbing a polyester-tipped swab 50 times across the ventral and dorsal surfaces. Salamanders were humanely euthanized and dissected to remove the brain for acetylcholinesterase and liver for GSH and hepatic metabolome analyses, and a whole-body tissue homogenate was used for pesticide quantification. Levels of GSH were present in lower quantities on dermal swabs relative to liver tissues for chlorpyrifos, 2,4-D, and control treatments. However, 2,4-D exposures demonstrated a large effect size increase for GSH levels in livers (Cohen's d = 0.925, p = 0.036). Other GSH increases were statistically insignificant, and effect sizes were characterized as small for 2,4-D mucosal swabs (d = 0.36), medium for chlorpyrifos mucosal swabs (d = 0.713), and negligible for chlorpyrifos liver levels (d = 0.012). The metabolomics analyses indicated that the urea cycle, alanine, and glutamate metabolism biological pathways were perturbed by both sets of pesticide exposures. Obtaining mucus samples through dermal swabbing in amphibians is a viable technique for evaluating health in these imperiled taxa. Environ Toxicol Chem 2024;43:1126-1137. © 2024 SETAC., (© 2024 SETAC.)

Metabolic reprogramming in cancer and immune cells occurs to support their increasing energy needs in biological tissues. Here we propose Single Cell Spatially resolved Metabolic (scSpaMet) framework for joint protein-metabolite profiling of single immune and cancer cells in male human tissues by incorporating untargeted spatial metabolomics and targeted multiplexed protein imaging in a single pipeline. We utilized the scSpaMet to profile cell types and spatial metabolomic maps of 19507, 31156, and 8215 single cells in human lung cancer, tonsil, and endometrium tissues, respectively. The scSpaMet analysis revealed cell type-dependent metabolite profiles and local metabolite competition of neighboring single cells in human tissues. Deep learning-based joint embedding revealed unique metabolite states within cell types. Trajectory inference showed metabolic patterns along cell differentiation paths. Here we show scSpaMet's ability to quantify and visualize the cell-type specific and spatially resolved metabolic-protein mapping as an emerging tool for systems-level understanding of tissue biology., (© 2023. The Author(s).)

Spatially resolved RNA and protein molecular analyses have revealed unexpected heterogeneity of cells. Metabolic analysis of individual cells complements these single-cell studies. Here, we present a three-dimensional spatially resolved metabolomic profiling framework (3D-SMF) to map out the spatial organization of metabolic fragments and protein signatures in immune cells of human tonsils. In this method, 3D metabolic profiles were acquired by time-of-flight secondary ion mass spectrometry to profile up to 189 compounds. Ion beams were used to measure sub-5-nanometer layers of tissue across 150 sections of a tonsil. To incorporate cell specificity, tonsil tissues were labeled by an isotope-tagged antibody library. To explore relations of metabolic and cellular features, we carried out data reduction, 3D spatial correlations and classifications, unsupervised K-means clustering, and network analyses. Immune cells exhibited spatially distinct lipidomic fragment distributions in lymphatic tissue. The 3D-SMF pipeline affects studying the immune cells in health and disease., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Adverse reproductive effects associated with gonadal intersex among freshwater fish could hold considerable implications for population sustainability. Presence of testicular oocytes (TO) is the most common form of intersex and is widespread among centrarchids (sunfishes) of North America and other freshwater teleosts. Placing TO within the toxicological context of adverse outcome pathways (AOPs) to assess ecological risk is a priority for ecotoxicologists due to the association of TO with harmful chemical exposure and adverse reproductive effects in some cases. However, key event relationships between EDC exposure, incidence of TO, and apical outcomes have yet to be fully elucidated - in part due to a lack of knowledge of relationships between intersex gonad physiology and fish health. Understanding the physiological status of intersex fish is critical to assess ecological risk, understand mechanisms of induction, and to establish biomarkers of intersex in fish. In the present study, features of gonad metabolite profiles associated with TO in largemouth bass (LMB, Micropterus salmoides) from an impoundment in Georgia (USA) were determined using GC-MS-based metabolomics. Clinical blood biochemical screens were used to evaluate markers of fish health associated with TO. Results suggest that physiological changes in energy expenditure as well as relatively 'feminized' gonad lipid and protein metabolism may be related to the occurrence of TO in male LMB, and highlight the need to understand relationships between intersex and physical stressors such as elevated temperature and hypoxia. These results provide novel insight to AOPs associated with TO and identify candidate analytes for biomarker discovery., (Copyright © 2020 Elsevier Inc. All rights reserved.)

To assess the potential risks of human exposure to endocrine active compounds (EACs), the mechanisms of toxicity must first be identified and characterized. Currently, there are no robust in vitro models for identifying the mechanisms of toxicity in germ cells resulting from EAC exposure. Human embryonic stem cells can differentiate into numerous functional cell types including germ-like cells (GLCs). These cells possess characteristics indicative of a germ cell state, suggesting they offer a novel system to investigate the consequences of chemical exposure on normal germ cell processes. To characterize these processes, a metabolomic-based approach was employed to determine the response of GLCs following exposure to 0.001, 0.01, 0.1, 1, 10, or 100µM estradiol, testosterone, or progesterone for 48h. Following exposure, cellular extracts underwent gas chromatography coupled with mass spectrometry analysis. Models were then constructed using principal component analysis on acquired spectra to discriminate among steroid hormones as well as doses for each hormone. t-test comparisons generated a preliminary list of metabolites that were statistically significant in GLC's biochemical response to these steroid hormones. Steroid hormone exposures caused fluxes in intracellular pathways such as amino acid synthesis and metabolism, fatty acid synthesis, as well as cholesterol and lipoprotein metabolism. Further pathway analysis, based on these identified metabolites, will aid in modeling the response of GLCs to endogenous steroid hormones and allow for identification of biomarkers delineating germ cell-based developmental and reproductive pathways.

Development of in vitro new approach methodologies has been driven by the need for developmental neurotoxicity (DNT) hazard data on thousands of chemicals. The network formation assay characterizes DNT hazard based on changes in network formation but provides no mechanistic information. This study investigated nervous system signaling pathways and upstream physiological regulators underlying chemically induced neural network dysfunction. Rat primary cortical neural networks grown on microelectrode arrays were exposed for 12 days in vitro to cytosine arabinoside, 5-fluorouracil, domoic acid, cypermethrin, deltamethrin, or haloperidol as these exposures altered network formation in previous studies. RNA-seq from cells and gas chromatography/mass spectrometry analysis of media extracts collected on days in vitro 12 provided gene expression and metabolomic identification, respectively. The integration of differentially expressed genes and metabolites for each neurotoxicant was analyzed using ingenuity pathway analysis. All 6 compounds altered gene expression that linked to developmental disorders and neurological diseases. Other enriched canonical pathways overlapped among compounds of the same class; eg, genes and metabolites altered by both cytosine arabinoside and 5-fluorouracil exposures are enriched in axonal guidance pathways. Integrated analysis of upstream regulators was heterogeneous across compounds, but identified several transcriptomic regulators including CREB1, SOX2, NOTCH1, and PRODH. These results demonstrate that changes in network formation are accompanied by transcriptomic and metabolomic changes and that different classes of compounds produce differing responses. This approach can enhance information obtained from new approach methodologies and contribute to the identification and development of adverse outcome pathways associated with DNT. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The eastern oyster (Crassostrea virginica) supports a large aquaculture industry and is a keystone species along the Atlantic seaboard. Native oysters are routinely exposed to a complex mixture of contaminants that increasingly includes pharmaceuticals and personal care products (PPCPs). Unfortunately, the biological effects of chemical mixtures on oysters are poorly understood. Untargeted gas chromatography‐mass spectrometry metabolomics was utilized to quantify the response of oysters exposed to fluoxetine, N,N‐diethyl‐meta‐toluamide, 17α‐ethynylestradiol, diphenhydramine, and their mixture. Oysters were exposed to 1 µg/L of each chemical or mixture for 10 d, followed by an 8‐d depuration period. Adductor muscle (n = 14/treatment) was sampled at days 0, 1, 5, 10, and 18. Trajectory analysis illustrated that metabolic effects and class separation of the treatments varied at each time point and that, overall, the oysters were only able to partially recover from these exposures post‐depuration. Altered metabolites were associated with cellular energetics (i.e., Krebs cycle intermediates), as well as amino acid metabolism and fatty acids. Exposure to these PPCPs also affected metabolic pathways associated with anaerobic metabolism, osmotic stress, and oxidative stress, in addition to the physiological effects of each chemical's postulated mechanism of action. Following depuration, fewer metabolites were altered, but none of the treatments returned them to their initial control values, indicating that metabolic disruptions were long‐lasting. Interestingly, the mixture did not directly cluster with individual treatments in the scores plot from partial least squares discriminant analysis, and many of its affected metabolic pathways were not well predicted from the individual treatments. The present study highlights the utility of untargeted metabolomics in developing exposure biomarkers for compounds with different modes of action in bivalves. Environ Toxicol Chem 2020;39:419–436. © 2019 SETAC [ABSTRACT FROM AUTHOR]

Environmental context: Metabolomics can be used to provide a snapshot of an organism's physiology as the organism is exposed to varying environmental conditions. In this study, laboratory-reared amphibians were exposed to multiple pesticides, analogous to field exposures, resulting in an impact to both pesticide body concentrations and the amphibians' hepatic metabolome. These data can be used in the environmental and ecological risk assessment of multiple pesticides in non-target species. Pesticide mixtures are frequently co-applied throughout an agricultural growing season to maximise crop yield. Therefore, non-target ecological species (e.g. amphibians) may be exposed to several pesticides at any given time on these agricultural landscapes. The objectives of this study were to quantify body burdens in terrestrial phase amphibians and translate perturbed metabolites to their corresponding biochemical pathways affected by exposure to pesticides as both singlets and in combination. Southern leopard frogs (Lithobates sphenocephala) were exposed either at the maximum or 1/10th maximum application rate to single, double or triple pesticide mixtures of bifenthrin (insecticide), metolachlor (herbicide) and triadimefon (fungicide). Tissue concentrations demonstrated both facilitated and competitive uptake of pesticides when in mixtures. Metabolomic profiling of amphibian livers identified metabolites of interest for both application rates; however, the magnitude of changes varied for the two exposure rates. Exposure to lower concentrations demonstrated downregulation in amino acids, potentially owing to their usage for glutathione metabolism and/or increased energy demands. Amphibians exposed to the maximum application rate resulted in upregulation of amino acids and other key metabolites likely owing to depleted energy resources. Coupling endogenous and exogenous biomarkers of pesticide exposure can be used to form vital links in an ecological risk assessment by relating internal dose to pathophysiological outcomes in non-target species. Environmental context. Metabolomics can be used to provide a snapshot of an organism's physiology as the organism is exposed to varying environmental conditions. In this study, laboratory-reared amphibians were exposed to multiple pesticides, analogous to field exposures, resulting in an impact to both pesticide body concentrations and the amphibians' hepatic metabolome. These data can be used in the environmental and ecological risk assessment of multiple pesticides in non-target species. [ABSTRACT FROM AUTHOR]

Traumatic brain injury (TBI) is a leading cause of death and long-term disability in the United States. The heterogeneity of the disease coupled with the lack of comprehensive, standardized scales to adequately characterize multiple types of TBI remain to be major challenges facing effective therapeutic development. A systems level approach to TBI diagnosis through the use of metabolomics could lead to a better understanding of cellular changes post-TBI and potential therapeutic targets. In the current study, we utilize a GC-MS untargeted metabolomics approach to demonstrate altered metabolism in response to TBI in a translational pig model, which possesses many neuroanatomical and pathophysiologic similarities to humans. TBI was produced by controlled cortical impact (CCI) in Landrace piglets with impact velocity and depth of depression set to 2m/s;6mm, 4m/s;6mm, 4m/s;12mm, or 4m/s;15mm resulting in graded neural injury. Serum samples were collected pre-TBI, 24 hours post-TBI, and 7 days post-TBI. Partial least squares discriminant analysis (PLS-DA) revealed that each impact parameter uniquely influenced the metabolomic profile after TBI, and gray and white matter responds differently to TBI on the biochemical level with evidence of white matter displaying greater metabolic change. Furthermore, pathway analysis revealed unique metabolic signatures that were dependent on injury severity and brain tissue type. Metabolomic signatures were also detected in serum samples which potentially captures both time after injury and injury severity. These findings provide a platform for the development of a more accurate TBI classification scale based unique metabolomic signatures. [ABSTRACT FROM AUTHOR]

ABSTRACT Tamoxifen is an endocrine-active pharmaceutical (EAP) that is used world-wide. Because tamoxifen is a ubiquitous pharmaceutical and interacts with estrogen receptors, a case study was conducted with this compound to (1) determine effects on reproductive endpoints in a nontarget species (i.e., a fish), (2) compare biologically-active metabolites across species, (3) assess whether in vitro assays predict in vivo results, and (4) investigate metabolomic profiles in tamoxifen-treated fish to better understand the biological mechanisms of tamoxifen toxicity. In reproductive assays, tamoxifen exposure caused a significant reduction in egg production and significantly increased ovarian aromatase activity in spawning adult cunner fish ( Tautogolabrus adspersus). In plasma from tamoxifen-exposed cunner, the predominant metabolite was 4-hydroxytamoxifen, while in rats it was N-desmethyltamoxifen. Because 4-hydroxytamoxifen is a more biologically active metabolite than N-desmethyltamoxifen, this difference could result in a different level of risk for the two species. The results of in vitro assays with fish hepatic microsomes to assess tamoxifen metabolism did not match in vivo results, indicating probable differences in excretion of tamoxifen metabolites in fish compared with rats. For the first time, a complete in vitro characterization of the metabolism of tamoxifen using fish microsomes is presented. Furthermore, a metabolomic investigation of cunner gonad extracts demonstrates that tamoxifen alters the biochemical profile in this nontarget species. Understanding the consequence of tamoxifen exposure in nontarget species, and assessing the discrepancies between sex- and species-mediated endpoints, is a step toward understanding how to accurately assess the risks posed by EAPs, such as tamoxifen, in the aquatic environment. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1834-1850, 2016. [ABSTRACT FROM AUTHOR]

Carbon nanotubes (CNTs) have great potential in industrial, consumer, and mechanical applications, based partly on their unique structural, optical and electronic properties. CNTs are commonly oxidized or treated with surfactants to facilitate aqueous solution processing, and these CNT surface modifications also increase possible human and ecological exposures to nanoparticle-contaminated waters. To determine the exposure outcomes of oxidized and surfactant-wrapped multiwalled carbon nanotubes (MWCNTs) on biochemical processes, metabolomics-based profiling of human liver cells (C3A) was utilized. Cells were exposed to 0, 10, or 100 ng/mL of MWCNTs for 24 and 48 h; MWCNT particle size distribution, charge, and aggregation were monitored concurrently during exposures. Following MWCNT exposure, cellular metabolites were extracted, lyophilized, and buffered for 1 H NMR analysis. Acquired spectra were subjected to both multivariate and univariate analysis to determine the consequences of nanotube exposure on the metabolite profile of C3A cells. Resulting scores plots illustrated temporal and dose-dependent metabolite responses to all MWCNTs tested. Loadings plots coupled with t -test filtered spectra identified metabolites of interest. XPS analysis revealed the presence of hydroxyl and carboxyl functionalities on both MWCNTs surfaces. Metal content analysis by ICP-AES indicated that the total mass concentration of the potentially toxic impurities in the exposure experiments were extremely low ( i.e. [Ni] ≤ 2 × 10 − 10 g/mL). Preliminary data suggested that MWCNT exposure causes perturbations in biochemical processes involved in cellular oxidation as well as fluxes in amino acid metabolism and fatty acid synthesis. Dose-response trajectories were apparent and spectral peaks related to both dose and MWCNT dispersion methodologies were determined. Correlations of the significant changes in metabolites will help to identify potential biomarkers associated with carbonaceous nanoparticle exposure. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • Larval D. rerio and X. laevis tadpoles were acutely exposed to high-use pesticides. • Metabolomic profiling was used to identify pesticide-perturbed metabolites. • Larval D. rerio elicited a more perturbed metabolic response than X. laevis tadpoles. • Cellular processes and functions are impacted in both species following exposures. • Variously affected pathways can result in protective acute fish surrogacy for tadpoles. [ABSTRACT FROM AUTHOR]

Pesticide use in agricultural areas requires the application of numerous chemicals to control target organisms, leaving non-target organisms at risk. The present study evaluates the hepatic metabolomic profile of one group of non-target organisms, amphibians, after exposure to a single pesticide and pesticide mixtures. Five common-use pesticide active ingredients were used in this study, three herbicides (atrazine, metolachlor and 2,4- d ), one insecticide (malathion) and one fungicide (propiconazole). Juvenile green frogs ( Lithobates clamitans ) were reared for 60–90 days post-metamorphosis then exposed to a single pesticide or a combination of pesticides at the labeled application rate on soil. Amphibian livers were excised for metabolomic analysis and pesticides were quantified for whole body homogenates. Based on the current study, metabolomic profiling of livers support both individual and interactive effects where pesticide exposures altered biochemical processes, potentially indicating a different response between active ingredients in pesticide mixtures, among these non-target species. Amphibian metabolomic response is likely dependent on the pesticides present in each mixture and their ability to perturb biochemical networks, thereby confounding efforts with risk assessment. [ABSTRACT FROM AUTHOR]