Gene expression in Rainbow trout (Oncorhynchus mykiss) fry gonads after a chronic exposure to a range of 17α-ethynylestradiol concentrations
ABSTRACT: Sex steroids play a key role in triggering sec differentiation in fish and the use of exogenous hormone treatment leads to partial or complete sex reversal. This phenomenon has attracted attention since the discovery taht even low environmental doses of exogenous steroids can adversely affect gonad morphology (ovotestis development) and induce reproductive failure. Modern genomic-based technologies have enhanced opportunities to find mechanisms of action (MOA) and dentify biomarkers for the toxic action of a compound. The goal of this study are to improve the understanding of feminization in fish by analyzing gene expression patterns in the gonads of rainbow trout fry after a chronic exposure to several doses (0.01, 0.1, 1 and 10 μg/L) of ethynylestradiol (EE2) and to offer target genes as potential biomarkers of ovotestis development. An all-male population of Rainbow trout fry was exposed during 76 days (from 60 to 136 days post-fertilization (dpf)) to five nominal concentrations of 17α-ethynylestradiol (0-solvent control-, 0.01, 0.1, 1 and 10 μg EE2/L of water), using 3 tanks per condition. In total, 30 samples were analyzed independantly: 6 samples per concentration tested (two samples per tank, three tanks per concentration), each sample being a pool of 10 pairs of gonads.
Project description:Sticklebacks were caught from the River Aire (Yorkshire, UK) and Siblyback reservoir (Cornwall, UK), depurated for 4 months at Exeter University, split into groups of 20 individuals before exposure. Fish were exposed to either control conditions, three concentrations of copper (3.2, 32, 128 µg/L; prepared using copper sulphate), three concentrations of ethinyl-estradiol (EE2) (1, 10, 32 ng/L) and three mixtures (3.2 µg Cu/L and 32 ng EE2/L; 128 µg Cu/L and 1 ng EE2/L; 128 µg Cu/L and 32 ng EE2/L) in duplicate tanks for 4 days. After sampling liver of male fish were stored at -80C for transcriptomics. Total RNA was prepared from fish livers, aliquots of all samples were pooled and labeled with Cy3-dCTP as a common reference, individual samples were labeled with Cy5-dCTP. Each hybridisation to the stickleback PGPS2 cDNA microarray consisited of one Cy5-labeled individual sample and one aliquot of the Cy3 labeled commmon reference sample. Data were captured with an Axon scanner using Genepix software.
Project description:The objective of this study was to gain mechanistic insight into how mummichog are able to produce eggs during exposures to high levels of EE2. Mummichog were exposed to 0, 50 or 250 ng/L of EE2 for 14 d. A mummichog microarray was used to identify genomic responses associated with high EE2 exposure, and effects of EE2 on target genes involved in lipid mobilization and transport and steroidogenesis, two important (EE2 regulated) pathways involved in egg production, were measured using real-time PCR. In addition, changes at higher level biological endpoints such as plasma steroid production and gonad morphology were measured to examine downstream responses to EE2. There were no differences in gonadosomatic index, liversomatic index, gonad development, or plasma estradiol levels after exposure to EE2, although testosterone decreased with EE2 exposures. Similar to other species, mummichog showed alterations to plasma steroid levels, and similar processes at the molecular level were identified as being altered by EE2, including cell processes associated with lipids. However, unlike other species, mummichog showed no differences in GSI or ovarian development. Fatty acids were affected at both the transcript and translation level, which suggests they are a major target of estrogens. Lipids are important for both growth and reproduction, and provide oocytes with nutrients. Interestingly, mummichog showed no effect on apolipoprotein expression, which is important in lipid transport. Therefore, it is hypothesized that mummichog are normally able to transport lipids for energy and nutrients to the ovary, while this is affected in other species. Novel gene regulatory networks for protein modification targets were also constructed to learn more about the potential roles of estrogens in the teleost liver. Although post-translational modifications (PTMs) are important regulatory mechanisms, the roles of PTMs in protein regulation in fish and the susceptibility of PTMs to aquatic pollutants are largely unexplored and may offer novel insight into mechanisms of endocrine disruption. Mummichog were exposed to 0, 50, and 250 ng/L EE2 for 14 days. Microarray analysis was performed with five biological replicated (5 control, 5 EE2 50 ng/L, 5 EE2 250 ng/L). Female liver tissue was used for the analysis.
Project description:17-ethinylestradiol (EE2) is a synthetic estrogen commonly used as an active substance in oral contraceptives. It is frequently found in waste water effluent and raise concern due to its persistent nature. EE2 binds to estrogen receptors with similar affinity to oestradiol and acts as one of the most potent hormone mimics found in the environment. Estrogen is involved in many aspects of the development of the neuroendocrine system influencing both brain structure and behavior. We and others have reported a significant effect on non-reproductive behaviors in adult fish and in recent studies we found that developmental exposure to EE2 resulted in an anxiogenic phenotype as adults even after a long remediation period. In this study we aim to study possible mechanisms behind the behavior alterations of zebrafish developmentally exposed to EE2 by sequencing the whole brain transcriptome. Zebrafish embryos were exposed to 0, 2.14 and 7.34 ng/L EE2 from 1 day to 80 days post fertilization. After the exposure period a remediation period of 120 days followed before the fish were sampled. 3 male brains from the control group (0 ng/L) and the 2.14 ng/L group were sampled and 3 female brains from the control group (0 ng/L) and 7.34 ng/L were sampled.
Project description:17α-Ethinylestradiol (EE2) is a ubiquitous aquatic contaminant shown to decrease fish fertility at low concentrations, especially in fish exposed during development. The mechanisms of the decreased fertility are not fully understood. In this study, we perform transcriptome analysis by RNA sequencing of testes from zebrafish with previously reported lowered fertility due to exposure to low concentrations of EE2 during development. Fish were exposed to 1.2 and 1.6 ng/L (measured concentration) of EE2 from fertilization to 80 days of age, followed by 82 days of remediation in clean water.
Project description:17alpha-ethinylestradiol (EE2) is one of the most potent estrogens that have the ability to interfere with the endocrine system of fish. The objective was to investigate the effects and mechanisms of action caused by 60 days of dietary exposure to 0.2 mg EE2/kg and 0.07 mg EE2/kg feed in female largemouth bass (LMB) during the reproductive season. Microarrays and pathway analyses were performed on hepatic tissues to identify genes and biological processes altered in female LMB by EE2 exposure. The hypothesis was that the two concentrations of EE2 would produce dose-response changes in sensitive genes. Body and ovary weights were measured and blood was collected for measurement of plasma steroid hormones (17beta-estradiol (E2), testosterone (T)) and vitellogenin (VTG) using ELISA. The 0.2 mg EE2/kg feed exposure reduced the gonadosomatic index (GSI) by 75%, and plasma levels of E2 and T were reduced by over 90%. Plasma VTG was increased by approximately 100% (from 4 to 8mg/ml) by the 0.2 mg/kg treatment. T levels, from the 0.07 mg EE2/kg feed, reduced GSI by approximately 30% and circulating E2 and T by ~80% but did not affect VTG concentrations. We found 1,594 and 1,165 genes were significantly affected (p<0.05) by the 0.07 mg EE2/kg feed and 0.2 mg EE2/kg feed, respectively. Gene ontology (GO) analysis revealed that there were different biological processes regulated by the two concentrations of EE2. Pathway analysis showed that the 0.07 mg EE2/kg feed exposure caused differential regulation of genes associated with fatty acid biosynthesis and glycolysis, indicating some metabolic effects. In contrast, the 0.2 mg EE2/kg feed exposure altered transcription of genes involved in immune response and apoptosis, suggesting a toxic response at this concentration. These results suggest that the two concentrations demonstrated distinct physiological responses, with the higher concentration inducing complete endocrine disruption in LMB. These findings demonstrate the usefulness of microarrays to identify possible biomarkers and modes of toxic action to dietary exposure in LMB. Two concentrations of EE2 would produce dose-response changes in sensitive genes. Female LMB were fed 5 days per week for 60 days with floating pellets that contained 0.07 or 0.2 mg/kg of EE2.
Project description:The objective of this study was to investigate pathway signatures altered in the livers of female largemouth bass (LMB) and their potential links with biological responses by dietary exposure to 0.2 mg EE2/Kg (1% of their body weight) over two months using a transcriptomics approach. A high concentration of dietary 17alpha-ethinylestradiol (EE2) can activate key signaling pathways in response to oxidative damage may occur regardless of tumorigenesis and cancer. Female LMB received about 1.2g EE2/day/fish (from EE2-laced feed containing 0.2 mg EE2/Kg) for 60 days.
Project description:To study short term (48h) hepatic transcriptional changes and identify potential modes of action in primary rainbow trout (Oncorhynchus mykiss) hepatocytes exposed to 0.03, 0.3, 3 and 30nM EE2. The transcriptional gene expression analysis involved a high density (60k) custom designed oligonucleotide salmonid microarray in combination with quantitative real-time polymerase chain reaction (qPCR). Differently expressed genes (DEGs) were obtained after application of a one-way analysis of variance (ANOVA) and Tukey posthoc test. Enrichment analysis was performed based on Gene Ontology (GO) to determine the biological roles of the DEGs. The obtained DEGs were further mapped against mammalian orthologs. The successfully mapped DEGs were further subjected to a gene network analysis based on well-curated mammalian protein -protein interactions, followed by a canonical and toxicity pathway analysis. The pathways and network analysis were performed in order to link DEGs to specific and toxicological/biological functions. Isolated primary rainbow trout hepatocytes were exposed to 0.03-30nM ethynylestradiol for 48h. The cells were sampled and used for gene expression analysis. A total of 4 biological replicates were analyzed for each concentration, including solvent (DMSO) control.
Project description:Immature, ovariectomized C57BL/6 mice were treated with sesame oil (vehicle) or with 0.1 mg/kg 17-alpha-ethynylestradiol (EE) for the length of time indicated. Mice received one dose, except for the 3x24hr samples, which received 3 consecutive daily doses and were sacrificed 24hr after the final dose. Two replicates of each sample are provided. In addition, one time=0 sample is included.
Project description:This study assessed the implications of a 14 day sub-chronic exposure of ethinylestradiol (EE2; 1.0 or 10.0 µg/L EE2) on male medaka fertility, testicular histology and testicular gene expression. The findings demonstrate that a 14 day exposure to EE2 induced impaired male reproductive capacity and time- and dose-dependent alterations in testicular morphology and gene expression. The average fertilization rate/day following the exposure for control, 1.0 and 10.0 µg/L EE2 was 91.3% (±4.4), 62.8% (±8.3) and 28.8% (±5.8), respectively. The testicular morphologic alterations observed include increased germ cell apoptosis, decreased germinal epithelium and thickening of the interstitium. The morphologic changes observed were highly associated with gene expression changes observed using a medaka-specific microarray. A pathway analysis of the differentially expressed genes emphasized genes and pathways associated with apoptosis, cell cycle and proliferation, collagen production/extracellular matrix organization, hormone signaling, male reproduction and protein ubiquitination among others. Six month old male medaka were exposed to ethinyl estradiol (EE2) for a 14 day time period. Treatment exposures were completed in triplicate including DMSO (vehicle control), 1.0 µg/L EE2, and 10.0 µg/L EE2. Fish were sampled for gene expression on days 1, 7 and 14 of exposure. Five male fish were placed in 2-liter beaker replicates for each treatment and sampling time point.
Project description:The present study explores the potential of compound-specific gene-upregulation profiles in the ubiquitous purple nonsulfur bacterium Rhodospirillum rubrum S1H as biomarkers for exposure to surface water contaminants, i.e. high production-volume pharmaceuticals. Even though the pharmaceuticals [i.e., acetylsalicylic acid (ASA), diclofenac (DCF), and 17α-ethinylestradiol (EE2)] did not affect the bacterial growth kinetics at environmentally-relevant concentrations (86nM), whole-genome microarray analyses revealed the upregulation of 128, 49, and 47 genes upon exposure to DCF, ASA, and EE2, respectively. A strong overlap (27-48%) was observed between transcriptional responses, but a total of 93 genes were found to be upregulated in a compound-specific manner. Hence, we were able to identify 74 and 15 potential biomarker genes for DCF and ASA, respectively. DCF specifically induced genes involved mainly in stress response, signal transduction, response regulation, the electron transport chain, and transcription, while ASA specifically induced genes predominantly involved in signal transduction, response regulation, and trans-membrane translocation. Moreover, our findings validated triclosan-specific biomarker genes that were identified previously. As only 4 genes were specifically-upregulated for EE2, no representative biomarker profile was identified. This study illustrates that a pollutant-specific molecular response can be generated in R. rubrum S1H, which could become a relevant model-microorganism to screen for the ecological impact of surface water contaminants in situ. KEYWORDS: environmental impact studies, risk assessment, biosensor, wastewater, micropollutant, aspirin Two-condition experiments. Comparing samples after induction of three pharmaceuticals each with a non-induced samples. Biological triplicate. Each array contains 3 technical replicates.