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Effects of exposure to the β-blocker propanolol on the reproductive behavior and gene expression of the fathead minnow, Pimephales promelas.

ABSTRACT: Propranolol is a beta-adrenergic receptor antagonist (β-blocker) that has been detected in United States wastewater effluents at concentrations ranging from 0.026 to 1.90 µg/l. In mammals, there is evidence that β-blockers can cause sexual dysfunction, and alter serotonergic pathways which may impact reproductive behavior but little is known about the effects on fish behavior. The present study tested the effects of propranolol on fecundity and on reproductive behavior of the fathead minnow, Pimephales promelas, a fish that exhibits male parental care. Sexually mature fathead minnows were housed at a ratio of one male and two females per tank and exposed to nominal concentrations of 0, 0.1, 1, 10 µg/l for 21 days. Measured concentrations (±SD) of propranolol were 0.05±0.02, 0.88±0.34 and 4.11±1.19 µg/l. There were no statistically significant differences in fecundity, fertilization rate, hatchability and time to hatch. Propranolol exposure was not associated with a change in nest rubbing behavior, time spent in the nest or approaching the females. There was a significant difference in the number of visits to the nest with males receiving low and medium propranolol treatments. The microarray analysis showed that there were 335 genes up-regulated and 400 genes down-regulated in the brain after exposure to the highest dose of propranolol. Among those genes, myoglobin and calsequestrin transcripts (fold change=10.84 and 5.49, respectively) were highly up-regulated. Ontological analyses indicated changes in genes involved in calcium ion transport, transcription, proteolysis and apoptosis/anti-apoptosis. The results showed that exposure to propranolol at concentrations as high as 4.11 µg/l did not significantly impact reproductive behavior or spawning abilities of fathead minnow but did alter the regulation of genes within the brain of fish.

ORGANISM(S): Pimephales promelas

PROVIDER: GSE34947 | GEO | 2012/06/01

SECONDARY ACCESSION(S): PRJNA150961

REPOSITORIES: GEO

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Project description:Propranolol is a beta-adrenergic receptor antagonist (Î²-blocker) that has been detected in United States wastewater effluents at concentrations ranging from 0.026 to 1.90 Âµg/l. In mammals, there is evidence that Î²-blockers can cause sexual dysfunction, and alter serotonergic pathways which may impact reproductive behavior but little is known about the effects on fish behavior. The present study tested the effects of propranolol on fecundity and on reproductive behavior of the fathead minnow, Pimephales promelas, a fish that exhibits male parental care. Sexually mature fathead minnows were housed at a ratio of one male and two females per tank and exposed to nominal concentrations of 0, 0.1, 1, 10 Âµg/l for 21 days. Measured concentrations (Â±SD) of propranolol were 0.05Â±0.02, 0.88Â±0.34 and 4.11Â±1.19 Âµg/l. There were no statistically significant differences in fecundity, fertilization rate, hatchability and time to hatch. Propranolol exposure was not associated with a change in nest rubbing behavior, time spent in the nest or approaching the females. There was a significant difference in the number of visits to the nest with males receiving low and medium propranolol treatments. The microarray analysis showed that there were 335 genes up-regulated and 400 genes down-regulated in the brain after exposure to the highest dose of propranolol. Among those genes, myoglobin and calsequestrin transcripts (fold change=10.84 and 5.49, respectively) were highly up-regulated. Ontological analyses indicated changes in genes involved in calcium ion transport, transcription, proteolysis and apoptosis/anti-apoptosis. The results showed that exposure to propranolol at concentrations as high as 4.11 Âµg/l did not significantly impact reproductive behavior or spawning abilities of fathead minnow but did alter the regulation of genes within the brain of fish. Effects of propanolol exposure were investigated in the brain of adult male fathead minnow exposed to 10 Âµg/L of propanolol or a solvent control solution (0.01% ethanol). For each treatment, the brain of four different fish were analyzed.

Project description:Metformin, along with its biotransformation product guanylurea, are 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 in the literature with regard to effects. To further investigate the potential reproductive toxicity of metformin and guanylurea to fish, a series of experiments were performed with reproductively mature fathead minnows (Pimephales promelas). First, explants of mature fathead minnow ovary tissue were exposed to 0.001-100 µM metformin or guanylurea to investigate whether they can directly perturb steroidogenesis. Second, spawning pairs of fathead minnows were exposed to metformin (0.41, 4.1, 41 µg/L) or guanylurea (1.0, 10, 100 µg/L) for 23 d 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 or guanylurea effected estradiol or testosterone by ovary tissue exposed in vitro. In the 23 d exposure, neither compound significantly impacted transcription of endocrine-related genes in male liver or gonad, circulating steroid concentrations in male or female fish, or fecundity of spawning pairs. In the 96 h time course, 100 µg guanylurea/L elicited more differential gene expression than 41 µg metformin/L , and showed the greatest impacts after 96 h. A number of DEGs up-regulated after 24 h were subsequently down-regulated after 96 h, demonstrating time-dependent impacts of guanylurea on the liver. Overall, metformin and guanylurea did not elicit effects consistent with reproductive toxicity in adult fathead minnows at environmentally relevant concentrations. Where effects were identified using ‘omics approaches, guanylurea induced greater impacts than metformin.

Project description:Fluoxetine (FLX) is the most common selective serotonin reuptake inhibitor pharmaceutical used for the treatment of psychological disorders such as depression and anxiety. Increased prescription use of FLX by human populations has led to its pseudo-persistence in effluent-receiving waterways. Despite emerging concerns of FLX becoming a potential threat to the aquatic environment, there is currently limited information on effects of FLX on aquatic wildlife, including its specific mechanism of action and associated toxicity pathways in fish. The main goal of this study was to identify and validate key molecular toxicity pathways that are predictive of apical endpoints induced by exposure to FLX using the fathead minnow (Pimephales promelas), a model species common to North American freshwater systems. Adult fathead minnows were exposed to three concentrations of FLX (2.42, 10.7, and 56.7 µg/L) and a control for 21 days. After 96 hours, a subset of fish was sampled, and liver and brain tissue were collected to characterize molecular toxicity pathways using whole transcriptome and proteome analyses. In addition, at the end of the 21-day exposure, individuals were assessed for apical outcomes of regulatory relevance including histopathology and fecundity. Differential gene expression observed in the liver of fish exposed to the highest FLX treatment and revealed dysregulation of pathways associated with biosynthesis and metabolism of fatty acids, which may be an upstream molecular response that led to lipid-type vacuolation of hepatocytes, as observed in the histology analysis. Whole proteome analysis of the same fish revealed dysregulation of PPAR signalling, which may be associated with the enrichment of lipid-related pathways observed in the transcriptome. In addition, there was an indication of pathway enrichment of transcription- and translation-related pathways in the proteome. Common dysregulated genes in the brain of the treated fish were related to cellular signalling processes that are influenced by serotonin levels and shown to be involved in reproductive behaviour and, in turn, reproductive success. This was confirmed through a significant decrease in fecundity following the 21-day exposure. Pathways to measurable adverse outcomes are complex, however, this research does provide some important clues to the mechanistic toxicity that FLX inflicts.

Project description:This experiment was conducted as part of an overall study aimed at examining effects of a dopamine receptor antagonist on reproduction, behavior, and gene expression in cyprinid fish. Relative to gene expression, we hypothesized that if haloperidol disrupted normal regulation of the HPG axis, there may be robust transcriptional alterations in the ovary of exposed females that might serve as useful markers of exposure and/or effects. For the purposes of this study, robust transcriptional alterations were defined as those detected in two separate fish species (fathead minnow and zebrafish), exposed to the same concentration of haloperidol for the same duration. There is a companion zebrafish microarray experiment (GSE14861) that these data are compared to. Transcriptional responses in the ovary of the two species are compared in terms of differentially expressed genes, enriched gene ontology categories they represent, and associated pathways. Fathead minnow and zebrafish microarray experiment Samples for microarray analysis were generated in a separate, 96 h, continuous flow-through experiment. Nominal treatment concentrations for the microarray experiment were 0 and 50 Î¼g haloperidol/L. Chemical (or control water) delivery was initiated 24 h prior to adding fish to the tanks. To start the experiment, fathead minnows (4 males, 4 females per tank) were added to each of three tanks per treatment group, while zebrafish (6 males, 6 females per tank) were added to each of two tanks per group. The time of fish addition was staggered by replicate within each treatment such that all samples from a given exposure tank could be collected within 60 min of the intended 96 h exposure duration. After 96 h, male and female zebrafish and female fathead minnows were anesthetized in buffered MS-222 and weighed. Whole gonads were removed, weighed, and preserved in RNAlater. Total RNA was isolated from fathead minnow ovary samples using RNeasy kits (Qiagen). RNA quality was assessed with a Agilent 2100 Bioanalyzer and quantity was determined using a Nanodrop ND-1000 spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA). Aliquots of six fathead minnow ovary RNA samples per treatment group (two from each replicate per treatment) were prepared for microarray analysis.

Project description:Rivers containing effluents from water treatment plants are complex soups of compounds, ranging from pharmaceuticals to natural hormones. Male fathead minnows (Pimephales promelas) were exposed for 3 weeks to effluent waters from the Metropolitan Wastewater Treatment Plant in St. Paul, MN. Fish were tested for their competitive nest holding behavior. Changes in vitellogenin were measured and these were correlated to changes in gene expression using a 22,000 gene microarray developed specifically for fathead minnows. Significant changes in gene expression were observed in both liver and gonad, which correlate to phenotypic changes of vitellogenin induction and reduced competitive behavior. We also compared by real-time PCR the expression changes in key genes related to steroid biosynthesis and metabolism in fish exposed to the effluent as well as in fish exposed to a model estrogen and a model androgen. While the gene expression signature from effluent-exposed fish shared some elements with estrogen and androgen signatures, overall it was different, underscoring the complexity of compounds present in sewage and their different modes of action. Fathead minnow 22,000 gene arrays were designed by EcoArray (Alachua, FL) and were purchased from Agilent. Array hybridizations were performed using a reference design, where each sample was compared to a reference sample. The reference consisted of equal amounts of RNA from control female and male tissues (liver, brain and gonad) and was prepared as a standard for several experiments. Four replicates consisting of four different individuals were analyzed for each of the tissues, liver and gonad for both the control and the effluent-exposed fish. cDNA synthesis, cRNA labeling, amplification and hybridization were performed following the manufacturerM-bM-^@M-^Ys kits and protocols (Agilent Low RNA Input Fluorescent Linear Amplification Kit and Agilent 60-mer oligo microarray processing protocol; Agilent, Palo Alto, CA). Ovarian and liver samples from the fish were labeled with Cy5 while the reference sample was labeled with Cy3.

Project description:Traditional toxicity testing has been unable to keep pace with the introduction of new chemicals into commerce. Consequently, there are limited or no toxicity data upon which to base a risk assessment for many chemicals to which fish and wildlife may be exposed. Per- and polyfluoroalkyl substances (PFAS) are emblematic of this issue in that most the ecological hazards of most PFAS remain uncharacterized. The present study employed a high throughput assay to identify the concentration at which 20 PFAS, with diverse properties, elicited a concerted gene expression response in larval fathead minnows (Pimephales promelas, 5-6 days post-fertilization) exposed for 24 h. Based on a reduced transcriptome approach that measured whole body expression of 1832 genes, the median transcriptomic point of departure (tPOD) for the 20 PFAS tested was 10 µM. Longer chain carboxylic acids (12-13 C-F) and an eight C-F di-alcohol, N-alkyl sulfonamide, and telomer sulfonic acid were among the most potent PFAS, eliciting gene expression responses at concentrations below 1 µM. With a few exceptions, larval fathead minnow tPODs were concordant with those based on whole transcriptome response in human cell lines. However, larval fathead minnow tPODs were often greater than those for Daphnia magna exposed to the same PFAS. The tPODs overlapped concentrations at which other sub-lethal effects have been reported in fish (available for 10 PFAS; including a range of species, life stages, and study designs). Nonetheless, fathead minnow tPODs were all orders of magnitude higher than aqueous PFAS concentrations detected in tributaries of the North American Great Lakes suggesting a substantial margin of safety in those systems, even for PFAS with significant potential for bioaccumulation. Overall, results broadly support the use of a fathead minnow larval transcriptomics assay to derive screening level potency estimates for use in ecological risk-based prioritization. Traditional toxicity testing has been unable to keep pace with the introduction of new chemicals into commerce. Consequently, there are limited or no toxicity data upon which to base a risk assessment for many chemicals to which fish and wildlife may be exposed. Per- and polyfluoroalkyl substances (PFAS) are emblematic of this issue in that most the ecological hazards of most PFAS remain uncharacterized. The present study employed a high throughput assay to identify the concentration at which 20 PFAS, with diverse properties, elicited a concerted gene expression response in larval fathead minnows (Pimephales promelas, 5-6 days post-fertilization) exposed for 24 h. Based on a reduced transcriptome approach that measured whole body expression of 1832 genes, the median transcriptomic point of departure (tPOD) for the 20 PFAS tested was 10 µM. Longer chain carboxylic acids (12-13 C-F) and an eight C-F di-alcohol, N-alkyl sulfonamide, and telomer sulfonic acid were among the most potent PFAS, eliciting gene expression responses at concentrations below 1 µM. With a few exceptions, larval fathead minnow tPODs were concordant with those based on whole transcriptome response in human cell lines. However, larval fathead minnow tPODs were often greater than those for Daphnia magna exposed to the same PFAS. The tPODs overlapped concentrations at which other sub-lethal effects have been reported in fish (available for 10 PFAS; including a range of species, life stages, and study designs). Nonetheless, fathead minnow tPODs were all orders of magnitude higher than aqueous PFAS concentrations detected in tributaries of the North American Great Lakes suggesting a substantial margin of safety in those systems, even for PFAS with significant potential for bioaccumulation. Overall, results broadly support the use of a fathead minnow larval transcriptomics assay to derive screening level potency estimates for use in ecological risk-based prioritization.

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Effects of exposure to the β-blocker propanolol on the reproductive behavior and gene expression of the fathead minnow, Pimephales promelas.

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