m6A RNA methylation regulates mitochondrial function.

RNA methylation of N6-methyladenosine (m6A) is emerging as a fundamental regulator of every aspect of RNA biology. RNA methylation directly impacts protein production to achieve quick modulation of dynamic biological processes. However, whether RNA methylation regulates mitochondrial function is not known, especially in neuronal cells which require a high energy supply and quick reactive responses. Here we show that m6A RNA methylation regulates mitochondrial function through promoting nuclear-encoded mitochondrial complex subunit RNA translation. Conditional genetic knockout of m6A RNA methyltransferase Mettl14 (Methyltransferase like 14) by Nestin-Cre together with metabolomic analysis reveals that Mettl14 knockout-induced m6A depletion significantly downregulates metabolites related to energy metabolism. Furthermore, transcriptome-wide RNA methylation profiling of wild type and Mettl14 knockout mouse brains by m6A-Seq shows enrichment of methylation on mitochondria-related RNA. Importantly, loss of m6A leads to a significant reduction in mitochondrial respiratory capacity and membrane potential. These functional defects are paralleled by the reduced expression of mitochondrial electron transport chain complexes, as well as decreased mitochondrial super-complex assembly and activity. Mechanistically, m6A depletion decreases the translational efficiency of methylated RNA encoding mitochondrial complex subunits through reducing their association with polysomes, while not affecting RNA stability. Together, these findings reveal a novel role for RNA methylation in regulating mitochondrial function. Given that mitochondrial dysfunction and RNA methylation have been increasingly implicate in neurodegenerative disorders, our findings not only provide insights into fundamental mechanisms regulating mitochondrial function, but also open up new avenues for understanding the pathogenesis of neurological diseases.

Verification of In Vivo Estrogenic Activity for Four Per- and Polyfluoroalkyl Substances (PFAS) Identified as Estrogen Receptor Agonists via New Approach Methodologies.

Given concerns about potential toxicological hazards of the thousands of data-poor per- and polyfluorinated alkyl substances (PFAS) currently in commerce and detected in the environment, tiered testing strategies that employ high-throughput in vitro screening as an initial testing tier have been implemented. The present study evaluated the effectiveness of previous in vitro screening for identifying PFAS capable, or incapable, of inducing estrogenic responses in fish exposed in vivo. Fathead minnows (Pimephales promelas) were exposed for 96 h to five PFAS (perfluorooctanoic acid [PFOA]; 1H,1H,8H,8H-perfluorooctane-1,8-diol [FC8-diol]; 1H,1H,10H,10H-perfluorodecane-1,10-diol [FC10-diol]; 1H,1H,8H,8H-perfluoro-3,6-dioxaoctane-1,8-diol [FC8-DOD]; and perfluoro-2-methyl-3-oxahexanoic acid [HFPO-DA]) that showed varying levels of in vitro estrogenic potency. In agreement with in vitro screening results, exposure to FC8-diol, FC10-diol, and FC8-DOD caused concentration-dependent increases in the expression of transcript coding for vitellogenin and estrogen receptor alpha and reduced expression of insulin-like growth factor and apolipoprotein eb. Once differences in bioconcentration were accounted for, the rank order of potency in vivo matched that determined in vitro. These results provide a screening level benchmark for worst-case estimates of potential estrogenic hazards of PFAS and a basis for identifying structurally similar PFAS to scrutinize for putative estrogenic activity.

Evaluation of Complex Mixture Toxicity in the Milwaukee Estuary (WI, USA) Using Whole-Mixture and Component-Based Evaluation Methods.

Anthropogenic activities introduce complex mixtures into aquatic environments, necessitating mixture toxicity evaluation during risk assessment. There are many alternative approaches that can be used to complement traditional techniques for mixture assessment. Our study aimed to demonstrate how these approaches could be employed for mixture evaluation in a target watershed. Evaluations were carried out over 2 years (2017-2018) across 8-11 study sites in the Milwaukee Estuary (WI, USA). Whole mixtures were evaluated on a site-specific basis by deploying caged fathead minnows (Pimephales promelas) alongside composite samplers for 96 h and characterizing chemical composition, in vitro bioactivity of collected water samples, and in vivo effects in whole organisms. Chemicals were grouped based on structure/mode of action, bioactivity, and pharmacological activity. Priority chemicals and mixtures were identified based on their relative contributions to estimated mixture pressure (based on cumulative toxic units) and via predictive assessments (random forest regression). Whole mixture assessments identified target sites for further evaluation including two sites targeted for industrial/urban chemical mixture effects assessment; three target sites for pharmaceutical mixture effects assessment; three target sites for further mixture characterization; and three low-priority sites. Analyses identified 14 mixtures and 16 chemicals that significantly contributed to cumulative effects, representing high or medium priority targets for further ecotoxicological evaluation, monitoring, or regulatory assessment. Overall, our study represents an important complement to single-chemical prioritizations, providing a comprehensive evaluation of the cumulative effects of mixtures detected in a target watershed. Furthermore, it demonstrates how different tools and techniques can be used to identify diverse facets of mixture risk and highlights strategies that can be considered in future complex mixture assessments. Environ Toxicol Chem 2023;42:1229-1256. © 2023 SETAC.

Case Study in 21st-Century Ecotoxicology: Using In Vitro Aromatase Inhibition Data to Predict Reproductive Outcomes in Fish In Vivo.

To reduce the use of intact animals for chemical safety testing, while ensuring protection of ecosystems and human health, there is a demand for new approach methodologies (NAMs) that provide relevant scientific information at a quality equivalent to or better than traditional approaches. The present case study examined whether bioactivity and associated potency measured in an in vitro screening assay for aromatase inhibition could be used together with an adverse outcome pathway (AOP) and mechanistically based computational models to predict previously uncharacterized in vivo effects. Model simulations were used to inform designs of 60-h and 10-21-day in vivo exposures of adult fathead minnows (Pimephales promelas) to three or four test concentrations of the in vitro aromatase inhibitor imazalil ranging from 0.12 to 260 µg/L water. Consistent with an AOP linking aromatase inhibition to reproductive impairment in fish, exposure to the fungicide resulted in significant reductions in ex vivo production of 17ß-estradiol (E2) by ovary tissue (≥165 µg imazalil/L), plasma E2 concentrations (≥74 µg imazalil/L), vitellogenin (Vtg) messenger RNA expression (≥165 µg imazalil/L), Vtg plasma concentrations (≥74 µg imazalil/L), uptake of Vtg into oocytes (≥260 µg imazalil/L), and overall reproductive output in terms of cumulative fecundity, number of spawning events, and eggs per spawning event (≥24 µg imazalil/L). Despite many potential sources of uncertainty in potency and efficacy estimates based on model simulations, observed magnitudes of apical effects were quite consistent with model predictions, and in vivo potency was within an order of magnitude of that predicted based on in vitro relative potency. Overall, our study suggests that NAMs and AOP-based approaches can support meaningful reduction and refinement of animal testing. Environ Toxicol Chem 2023;42:100-116. © 2022 SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

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.

Male fathead minnow transcriptomes and associated chemical analytes in the Milwaukee estuary system.

Contaminants of Emerging Concern (CECs) can be measured in waters across the United States, including the tributaries of the Great Lakes. The extent to which these contaminants affect gene expression in aquatic wildlife is unclear. This dataset presents the full hepatic transcriptomes of laboratory-reared fathead minnows (Pimephales promelas) caged at multiple sites within the Milwaukee Estuary Area of Concern and control sites. Following 4 days of in situ exposure, liver tissue was removed from males at each site for RNA extraction and sequencing, yielding a total of 116 samples from which libraries were prepared, pooled, and sequenced. For each exposure site, 179 chemical analytes were also assessed. These data were created with the intention of inviting research on possible transcriptomic changes observed in aquatic species exposed to CECs. Access to both full sequencing reads of animal samples as well as water contaminant data across multiple Great Lakes sites will allow others to explore the health of these ecosystems in support of the aims of the Great Lakes Restoration Initiative.

Corrosion Resistance of Electrochemically Synthesized Modified Zaccagnaite LDH-Type Films on Steel Substrates.

Modified zaccagnaite layered double hydroxide (LDH) type films were synthesized on steel substrates by pulsed electrochemical deposition from aqueous solutions. The resulting films were characterized by X-ray diffraction, scanning electron microscopy/X-ray dispersive spectroscopy, and Fourier transform infrared spectroscopy. Structural characterization indicated a pure layered double hydroxide phase; however, elemental analysis revealed that the surface of the films contained Zn:Al ratios outside the typical ranges of layered double hydroxides. Layer thickness for the deposited films ranged from approximately 0.4 to 3.0 µm. The corrosion resistance of the film was determined using potentiodynamic polarization experiments in 3.5 wt.% NaCl solution. The corrosion current density for the coatings was reduced by 82% and the corrosion potential was shifted 126 mV more positive when 5 layers of modified LDH coatings were deposited onto the steel substrates. A mechanism was proposed for the corroding reactions at the coating.

Assessing effects of aromatase inhibition on fishes with group-synchronous oocyte development using western mosquitofish (Gambusia affinis) as a model.

Exposure to certain anthropogenic chemicals can inhibit the activity to cytochrome P450 aromatase (CYP19) in fishes leading to decreased plasma 17ß-estradiol (E2), plasma vitellogenin (VTG), and egg production. Reproductive dysfunction resulting from exposure to aromatase inhibitors has been extensively investigated in several laboratory model species of fish. These model species have ovaries that undergo asynchronous oocyte development, but many fishes have ovaries with group-synchronous oocyte development. Fishes with group-synchronous oocyte development have dynamic reproductive cycles which typically occur annually and are often triggered by complex environmental cues. This has resulted in a lack of test data and uncertainty regarding sensitivities to and adverse effects of aromatase inhibition. The present study used the western mosquitofish (Gambusia affinis) as a laboratory model to investigate adverse effects of chemical aromatase inhibition on group-synchronous oocyte development. Adult female western mosquitofish were exposed to either 0, 2, or 30 µg/L of the model nonsteroidal aromatase inhibiting chemical, fadrozole, for a complete reproductive cycle. Fish were sampled at four time-points representing pre-vitellogenic resting, early vitellogenesis, late vitellogenesis/early ovarian recrudescence, and late ovarian recrudescence. Temporal changes in numerous reproductive parameters were measured, including gonadosomatic index (GSI), plasma sex steroids, and expression of selected genes in the brain, liver, and gonad that are important for reproduction. In contrast to fish from the control treatment, fish exposed to 2 and 30 µg/L of fadrozole had persistent elevated expression of cyp19 in the ovary, depressed expression of vtg in the liver, and a low GSI. These responses suggest that completion of a group-synchronous reproductive cycle was unsuccessful during the assay in fish from either fadrozole treatment. These adverse effects data show that exposure to aromatase inhibitors has the potential to cause reproductive dysfunction in a wide range of fishes with both asynchronous and group-synchronous reproductive strategies.

Case Study in 21st Century Ecotoxicology: Using In Vitro Aromatase Inhibition Data to Predict Short-Term In Vivo Responses in Adult Female Fish.

The present study evaluated whether in vitro measures of aromatase inhibition as inputs into a quantitative adverse outcome pathway (qAOP) construct could effectively predict in vivo effects on 17ß-estradiol (E2) and vitellogenin (VTG) concentrations in female fathead minnows. Five chemicals identified as aromatase inhibitors in mammalian-based ToxCast assays were screened for their ability to inhibit fathead minnow aromatase in vitro. Female fathead minnows were then exposed to 3 of those chemicals: letrozole, epoxiconazole, and imazalil in concentration-response (5 concentrations plus control) for 24 h. Consistent with AOP-based expectations, all 3 chemicals caused significant reductions in plasma E2 and hepatic VTG transcription. Characteristic compensatory upregulation of aromatase and follicle-stimulating hormone receptor (fshr) transcripts in the ovary were observed for letrozole but not for the other 2 compounds. Considering the overall patterns of concentration-response and temporal concordance among endpoints, data from the in vivo experiments strengthen confidence in the qualitative relationships outlined by the AOP. Quantitatively, the qAOP model provided predictions that fell within the standard error of measured data for letrozole but not for imazalil and epoxiconazole. However, the inclusion of measured plasma concentrations of the test chemicals as inputs improved model predictions, with all predictions falling within the range of measured values. Results highlight both the utility and limitations of the qAOP and its potential use in 21st century ecotoxicology. Environ Toxicol Chem 2021;40:1155-1170. © 2020 SETAC. This article has been contributed to by US Government employees and their work is in the public domain in the USA.

Adverse Outcome Pathway Network-Based Assessment of the Interactive Effects of an Androgen Receptor Agonist and an Aromatase Inhibitor on Fish Endocrine Function.

Predictive approaches to assessing the toxicity of contaminant mixtures have been largely limited to chemicals that exert effects through the same biological molecular initiating event. However, by understanding specific pathways through which chemicals exert effects, it may be possible to identify shared "downstream" nodes as the basis for forecasting interactive effects of chemicals with different molecular initiating events. Adverse outcome pathway (AOP) networks conceptually support this type of analysis. We assessed the utility of a simple AOP network for predicting the effects of mixtures of an aromatase inhibitor (fadrozole) and an androgen receptor agonist (17ß-trenbolone) on aspects of reproductive endocrine function in female fathead minnows. The fish were exposed to multiple concentrations of fadrozole and 17ß-trenbolone individually or in combination for 48 or 96 h. Effects on 2 shared nodes in the AOP network, plasma 17ß-estradiol (E2) concentration and vitellogenin (VTG) production (measured as hepatic vtg transcripts) responded as anticipated to fadrozole alone but were minimally impacted by 17ß-trenbolone alone. Overall, there were indications that 17ß-trenbolone enhanced decreases in E2 and vtg in fadrozole-exposed fish, as anticipated, but the results often were not statistically significant. Failure to consistently observe hypothesized interactions between fadrozole and 17ß-trenbolone could be due to several factors, including lack of impact of 17ß-trenbolone, inherent biological variability in the endpoints assessed, and/or an incomplete understanding of interactions (including feedback) between different pathways within the hypothalamic-pituitary-gonadal axis. Environ Toxicol Chem 2020;39:913-922. © 2020 SETAC.

Resilience and cortical thickness: a MRI study.

Resilience is defined as the psychological resistance which enables the processing of stress and adverse life events and thus constitutes a key factor for the genesis of psychiatric illness. However, little is known about the morphological correlates of resilience in the human brain. Hence, the aim of this study is to examine the neuroanatomical expression of resilience in healthy individuals. 151 healthy subjects were recruited and had to complete a resilience-specific questionnaire (RS-11). All of them underwent a high-resolution T1-weighted MRI in a 3T scanner. Fine-grained cortical thickness was analyzed using FreeSurfer. We found a significant positive correlation between the individual extent of resilience and cortical thickness in a right hemispherical cluster incorporating the lateral occipital cortex, the fusiform gyrus, the inferior parietal cortex as well as the middle and inferior temporal cortex, i.e., a reduced resilience is associated with a decreased cortical thickness in these areas. We lend novel evidence for a direct linkage between psychometric resilience and local cortical thickness. Our findings in a sample of healthy individuals show that a lower resilience is associated with a lower cortical thickness in anatomical areas are known to be involved in the processing of emotional visual input. These regions have been demonstrated to play a role in the pathogenesis of stress and trauma-associated disorders. It can thus be assumed that neuroanatomical variations in these cortical regions might modulate the susceptibility for the development of stress-related disorders.

Quantitative Response-Response Relationships Linking Aromatase Inhibition to Decreased Fecundity are Conserved Across Three Fishes with Asynchronous Oocyte Development.

Quantitative adverse outcome pathways (qAOPs) describe quantitative response-response relationships that can predict the probability or severity of an adverse outcome for a given magnitude of chemical interaction with a molecular initiating event. However, the taxonomic domain of applicability for these predictions is largely untested. The present study began defining this applicability for a previously described qAOP for aromatase inhibition leading to decreased fecundity developed using data from fathead minnow (Pimephales promelas). This qAOP includes quantitative response-response relationships describing plasma 17ß-estradiol (E2) as a function of plasma fadrozole, plasma vitellogenin (VTG) as a function of plasma E2, and fecundity as a function of plasma VTG. These quantitative response-response relationships simulated plasma E2, plasma VTG, and fecundity measured in female zebrafish (Danio rerio) exposed to fadrozole for 21 days but not these responses measured in female Japanese medaka (Oryzias latipes). However, Japanese medaka had different basal levels of plasma E2, plasma VTG, and fecundity. Normalizing basal levels of each measurement to equal those of female fathead minnow enabled the relationships to accurately simulate plasma E2, plasma VTG, and fecundity measured in female Japanese medaka. This suggests that these quantitative response-response relationships are conserved across these three fishes when considering relative change rather than absolute measurements. The present study represents an early step toward defining the appropriate taxonomic domain of applicability and extending the regulatory applications of this qAOP.

Differential Sensitivity to In Vitro Inhibition of Cytochrome P450 Aromatase (CYP19) Activity Among 18 Freshwater Fishes.

There is significant concern regarding potential impairment of fish reproduction associated with endocrine disrupting chemicals. Aromatase (CYP19) is a steroidogenic enzyme involved in the conversion of androgens to estrogens. Inhibition of aromatase by chemicals can result in reduced concentrations of estrogens leading to adverse reproductive effects. These effects have been extensively investigated in a small number of laboratory model fishes, such as fathead minnow (Pimephales promelas), Japanese medaka (Oryzias latipes), and zebrafish (Danio rerio). But, differences in sensitivity among species are largely unknown. Therefore, this study took a first step toward understanding potential differences in sensitivity to aromatase inhibitors among fishes. Specifically, a standard in vitro aromatase inhibition assay using subcellular fractions of whole tissue homogenates was used to evaluate the potential sensitivity of 18 phylogenetically diverse species of freshwater fish to the nonsteroidal aromatase inhibitor fadrozole. Sensitivity to fadrozole ranged by more than 52-fold among these species. Five species were further investigated for sensitivity to up to 4 additional nonsteroidal aromatase inhibitors, letrozole, imazalil, prochloraz, and propiconazole. Potencies of each of these chemicals relative to fadrozole ranged by up to 2 orders of magnitude among the 5 species. Fathead minnow, Japanese medaka, and zebrafish were among the least sensitive to all the investigated chemicals; therefore, ecological risks of aromatase inhibitors derived from these species might not be adequately protective of more sensitive native fishes. This information could guide more objective ecological risk assessments of native fishes to chemicals that inhibit aromatase.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains.

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Rapid effects of the aromatase inhibitor fadrozole on steroid production and gene expression in the ovary of female fathead minnows (Pimephales promelas).

Cytochrome P450 aromatase catalyzes conversion of C19 androgens to C18 estrogens and is critical for normal reproduction in female vertebrates. Fadrozole is a model aromatase inhibitor that has been shown to suppress estrogen production in the ovaries of fish. However, little is known about the early impacts of aromatase inhibition on steroid production and gene expression in fish. Adult female fathead minnows (Pimephales promelas) were exposed via water to 0, 5, or 50µg fadrozole/L for a time-course of 0.5, 1, 2, 4, and 6h, or 0 or 50µg fadrozole/L for a time-course of 6, 12, and 24h. We examined ex vivo ovarian 17ß-estradiol (E2) and testosterone (T) production, and plasma E2 concentrations from each study. Expression profiles of genes known or hypothesized to be impacted by fadrozole including aromatase (cytochrome P450 [cyp] 19a1a), steriodogenic acute regulatory protein (star), cytochrome P450 side-chain cleavage (cyp11a), cytochrome P450 17 alpha hydroxylase/17,20 lyase (cyp17), and follicle stimulating hormone receptor (fshr) were measured in the ovaries by quantitative real-time polymerase chain reaction (QPCR). In addition, broader ovarian gene expression was examined using a 15k fathead minnow microarray. The 5µg/L exposure significantly reduced ex vivo E2 production by 6h. In the 50µg/L treatment, ex vivo E2 production was significantly reduced after just 2h of exposure and remained depressed at all time-points examined through 24h. Plasma E2 concentrations were significantly reduced as early as 4h after initiation of exposure to either 5 or 50µg fadrozole/L and remained depressed throughout 24h in the 50µg/L exposure. Ex vivo T concentrations remained unchanged throughout the time-course. Expression of transcripts involved in steroidogenesis increased within the first 24h suggesting rapid induction of a mechanism to compensate for fadrozole inhibition of aromatase. Microarray results also showed fadrozole exposure caused concentration- and time-dependent changes in gene expression profiles in many HPG-axis pathways as early as 4h. This study provides insights into the very rapid effects of aromatase inhibition on steroidogenic processes in fish.

Prioritization of Contaminants of Emerging Concern in Wastewater Treatment Plant Discharges Using Chemical:Gene Interactions in Caged Fish.

We examined whether contaminants present in surface waters could be prioritized for further assessment by linking the presence of specific chemicals to gene expression changes in exposed fish. Fathead minnows were deployed in cages for 2, 4, or 8 days at three locations near two different wastewater treatment plant discharge sites in the Saint Louis Bay, Duluth, MN and one upstream reference site. The biological impact of 51 chemicals detected in the surface water of 133 targeted chemicals was determined using biochemical endpoints, exposure activity ratios for biological and estrogenic responses, known chemical:gene interactions from biological pathways and knowledge bases, and analysis of the covariance of ovary gene expression with surface water chemistry. Thirty-two chemicals were significantly linked by covariance with expressed genes. No estrogenic impact on biochemical endpoints was observed in male or female minnows. However, bisphenol A (BPA) was identified by chemical:gene covariation as the most impactful estrogenic chemical across all exposure sites. This was consistent with identification of estrogenic effects on gene expression, high BPA exposure activity ratios across all test sites, and historical analysis of the study area. Gene expression analysis also indicated the presence of nontargeted chemicals including chemotherapeutics consistent with a local hospital waste stream. Overall impacts on gene expression appeared to be related to changes in treatment plant function during rain events. This approach appears useful in examining the impacts of complex mixtures on fish and offers a potential route in linking chemical exposure to adverse outcomes that may reduce population sustainability.

Impaired swim bladder inflation in early life stage fathead minnows exposed to a deiodinase inhibitor, iopanoic acid.

Inflation of the posterior and/or anterior swim bladder is a process previously demonstrated to be regulated by thyroid hormones. We investigated whether inhibition of deiodinases, which convert thyroxine (T4) to the more biologically active form, 3,5,3'-triiodothyronine (T3), would impact swim bladder inflation. Two experiments were conducted using a model deiodinase inhibitor, iopanoic acid (IOP). First, fathead minnow embryos were exposed to 0.6, 1.9, or 6.0 mg/L or control water until 6 d postfertilization (dpf), at which time posterior swim bladder inflation was assessed. To examine anterior swim bladder inflation, a second study was conducted with 6-dpf larvae exposed to the same IOP concentrations until 21 dpf. Fish from both studies were sampled for T4/T3 measurements and gene transcription analyses. Incidence and length of inflated posterior swim bladders were significantly reduced in the 6.0 mg/L treatment at 6 dpf. Incidence of inflation and length of anterior swim bladder were significantly reduced in all IOP treatments at 14 dpf, but inflation recovered by 18 dpf. Throughout the larval study, whole-body T4 concentrations increased and T3 concentrations decreased in all IOP treatments. Consistent with hypothesized compensatory responses, deiodinase-2 messenger ribonucleic acid (mRNA) was up-regulated in the larval study, and thyroperoxidase mRNA was down-regulated in all IOP treatments in both studies. These results support the hypothesized adverse outcome pathways linking inhibition of deiodinase activity to impaired swim bladder inflation. Environ Toxicol Chem 2017;36:2942-2952. 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.

Re-evaluating the Significance of Estrone as an Environmental Estrogen.

Studies worldwide have demonstrated the occurrence of feminized male fish at sites impacted by human and animal wastes. A variety of chemicals could contribute to this phenomenon, but those receiving the greatest attention in terms of research and monitoring have been 17ß-estradiol (ß-E2) and 17α-ethinylestradiol, due both to their prevalence in the environment and strong estrogenic potency. A third steroid, estrone (E1), also can occur at high concentrations in surface waters but generally has been of lesser concern due to its relatively lower affinity for vertebrate estrogen receptors. In an initial experiment, male fathead minnow (Pimephales promelas) adults were exposed for 4-d to environmentally relevant levels of waterborne E1, which resulted in plasma ß-E2 concentrations similar to those found in reproductively active females. In a second exposure we used 13C-labeled E1, together with liquid chromatography-tandem mass spectrometry, to demonstrate that elevated ß-E2 measured in the plasma of the male fish was indeed derived from the external environment, most likely via a conversion catalyzed by one or more 17ß-hydroxysteroid dehydrogenases. The results of our studies suggest that the potential impact of E1 as an environmental estrogen currently is underestimated.

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.

Effects of the antimicrobial contaminant triclocarban, and co-exposure with the androgen 17ß-trenbolone, on reproductive function and ovarian transcriptome of the fathead minnow (Pimephales promelas).

Triclocarban (TCC) is an antimicrobial agent routinely detected in surface waters that has been hypothesized to interact with the vertebrate endocrine system. The present study examined the effects of TCC alone and in combination with the model endocrine disruptor 17ß-trenbolone (TRB) on fish reproductive function. Adult Pimephales promelas were continuously exposed to either 1 µg TCC/L or 5 µg TCC/L, to 0.5 µg TRB/L, or to a mixture (MIX) of 5 µg TCC/L and 0.5 µg TRB/L for 22 d, and a variety of reproductive and endocrine-related endpoints were examined. Cumulative fecundity was significantly reduced in fathead minnows exposed to TRB, MIX, or 5 µg TCC/L. Exposure to 1 µg TCC/L had no effect on reproduction. In general, both TRB and MIX treatments caused similar physiological effects, evoking significant reductions in female plasma vitellogenin, estradiol, and testosterone, and significant increases in male plasma estradiol. Based on analysis of the ovarian transcriptome, there were potential pathway impacts that were common to both TRB- and TCC-containing treatment groups. In most cases, however, those pathways were more plausibly linked to differences in reproductive status than to androgen-specific functions. Overall, TCC was reproductively toxic to fish at concentrations at or near those that have been measured in surface water. There was little evidence that TCC elicits reproductive toxicity through a specific mode of endocrine or reproductive action, nor that it could augment the androgenic effects of TRB. Nonetheless, the relatively small margin of safety between some measured environmental concentrations and effect concentrations suggests that concern is warranted. Environ Toxicol Chem 2017;36:231-242. 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.