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: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:Prochloraz is a fungicide known to cause endocrine disruption through effects on the hypothalamic-pituitary-gonadal (HPG) axis. To determine the short-term impacts of prochloraz on gene expression and steroid production, adult female fathead minnows (Pimephales promelas) were exposed to the chemical (0 or 300 M-NM-<g/L) for a time-course of 6, 12 and 24 h. Consistent with inhibition of cytochrome P450 17M-NM-1-hydroxylase/17,20-lyase (CYP17) and aromatase (CYP19), known molecular targets of prochloraz, plasma 17M-NM-2-estradiol (E2) was reduced within 6 hours. Ex vivo E2 production was significantly reduced at all time-points, while ex vivo testosterone (T) production remained unchanged. Consistent with the decrease in E2 levels, plasma concentrations of the estrogen-responsive protein vitellogenin were significantly reduced at 24 h. Genes coding for CYP19, CYP17, and steroidogenic acute regulatory protein were up-regulated in a compensatory manner in ovaries of the prochloraz-treated fish. In addition to targeted quantitative real-time polymerase chain reaction analyses, a 15k feature fathead minnow microarray was used to determine gene expression profiles in ovaries. From time-point to time-point, the microarray results showed a relatively rapid change in the differentially expressed gene (DEG) profiles associated with the chemical exposure. Functional analysis of the DEGs indicated changes in expression of genes associated with cofactor and coenzyme binding (GO: 0048037 and 0050662), fatty acid binding (GO:0005504) and organelle organization and biogenesis (GO: 0006996). Overall, the results from this study are consistent with compensation of the fish HPG axis to inhibition of steroidogenesis by prochloraz, and provide further insights into relatively rapid, system-wide, effects of a model chemical stressor on fish. RNA samples for each treatment/time point combination were used for microarray analysis (total of 23 arrays). For the microarray work, total RNA was isolated from ovary samples using RNeasy kits (Qiagen, Valencia, CA, USA). The RNA quality was assessed with an Agilent 2100 Bioanalyzer (Agilent, Wilmington, DE, USA) and quantity was determined using a NanodropM-BM-. ND-1000 spectrophotometer. Total RNA was stored at -80oC until analyzed with oligonucleotide microarrays. Microarray analysis was conducted at the US Army Engineer Research and Development Center (Vicksburg, MS, USA) using a 15k feature fathead minnow microarray (GEO: http://www.ncbi.nlm.nih.gov/geo/; Accession platform number GPL9248) designed by Dr. Nancy Denslow (University of Florida, Gainesville, FL, USA) and manufactured by Agilent Technologies (Palo Alto, CA, USA). The Agilent one-color microarray hybridization protocol (One-Color Microarray-Based Gene Expression Analysis, version 5.7, Agilent Technologies) was used for microarray hybridizations. One M-BM-5g of total RNA was used for all hybridizations. The cDNA synthesis, cRNA labeling, amplification and hybridization were performed following the manufacturerM-bM-^@M-^Ys kits and protocols (Quick Amp Labeling kit; Agilent Technologies). An Axon GenePixM-BM-. 4000B Microarray Scanner (Molecular Devices Inc., Sunnyvale, CA, USA) was used to scan microarray images at 5 M-NM-<m resolution. Data were resolved from microarray images using Agilent Feature Extraction software (Agilent Technologies). Statistical analyses were conducted using Statistica 8 (StatSoft Inc., Tulsa, OK, USA) and GraphPad Instat v. 3.01 (GraphPad Software, San Diego, CA, USA). Data were tested for normality and homogeneity of variance. When data conformed to parametric assumptions, data were analyzed using a parametric one-way ANOVA with chemical treatment as the independent variable. When data did not conform to parametric assumptions, they were either transformed (log 10) or analyzed using a non-parametric KruskallM-bM-^@M-^SWallis test (pM-bM-^IM-$ 0.05). The data are presented as means with standard errors of the mean. Differences were considered significant at p M-bM-^IM-$ 0.05. Microarray data were imported into the Rosetta Resolver 7.2 system for gene expression analysis (Rosetta Biosoftware, Kirkland, WA, USA). Data were normalized using the default, text loader, intensity profile builder settings in the Rosetta Resolver system. Significantly DEGs were identified using one-way ANOVA (p < 0.01) with no multiple testing correction

Project description:Effects of bisphenol A (BPA) on ovarian transcript profiles as well as targeted endpoints with endocrine/reproductive relevance were examined in two fish species, fathead minnow (Pimephales promelas) and zebrafish (Danio rerio), exposed in parallel using matched experimental designs. Four days of waterborne exposure to 10 µg BPA caused significant vitellogenin induction in both species. However, zebrafish were less sensitive to effects on hepatic gene expression and steroid production than fathead minnow and the magnitude of vitellogenin induction was more modest. The concentration-response at the ovarian transcriptome level was non-monotonic and violated assumptions that underlie proposed methods for estimating hazard thresholds from transcriptomic results. However, the non-monotonic profile was consistent among species and there were nominal similarities in the functions associated with the differentially expressed genes, suggesting potential activation of common pathway perturbation motifs in both species. Overall, the results provide an effective case study for considering the potential application of ecotoxicogenomics to ecological risk assessments and provide novel comparative data regarding effects of BPA in fish. Fish were exposed to 0, 0.01, 0.1, 1.0, 10, or 100 µg BPA/L delivered in a continuous flow (45 ml/min) of sand filtered, UV treated, Lake Superior Water, without the use of carrier solvents. The design employed six replicate tanks per treatment. Three of the replicates were loaded with three male and three female zebrafish per tank, while the remaining three replicates were loaded with three male and three female fathead minnows. Addition of fish to the exposure tanks was staggered by replicate such that all sampling could be completed within 60 min of the desired 96 h exposure duration. After 96 h of exposure, fish were anesthetized in a buffered solution of tricaine methanesulfonate (MS-222; Finquel; Argent, Redmond, WA, USA) and weighed. Blood was collected from the caudal vasculature using microhematocrit tubes, centrifuged to separate the plasma, and plasma was stored at -80oC until analyzed. Liver, gonad, and brain tissues (including pituitary gland) were snap frozen in liquid nitrogen and stored at -80oC until extracted. Total RNA was isolated from ovary tissue of both fathead minnow and zebrafish using RNeasy kits (Qiagen, Valencia, CA, USA). RNA quality was evaluated using an Agilent 2100 Bioanalyzer (Agilent, Wilmington, DE, USA) and RNA was quantified spectrophotometrically (Nanodrop). Total RNA samples were stored at -80oC until used for microarray analyses. Ovarian transcripts from 32 fathead minnows (n=5 per treatment, except for control and 1.0 µg BPA/L, n=6) were analyzed using a custom, 15,000 gene microarray (GEO Platform Accession GPL9248) purchased from Agilent Technologies (Palo Alto, CA, USA). Ovarian transcripts from 34 zebrafish (n=6 per treatment, except 0.01 and 0.1 µg BPA/L, n=5) were analyzed using a commercial 44,000 feature microarray (Agilent design 019161; GEO Platform Accession GPL6457). The same hybridization and scanning procedures were used for both platforms. Briefly, microarrays were hybridized following the manufacturer’s guidelines for One-Color Microarray-Based Gene Expression Analysis (version 5.7; Agilent). Hybridizations were conducted with 1 µg of total RNA. Complementary DNA synthesis, cRNA labeling, amplification, and hybridizations were performed following the manufacturer’s kits and protocols (Quick Amp labeling kit; Agilent). Scanning was conducted at 5 µm resolution using an Axon GenePix® 4000B Microarray Scanner (Molecular Devices Inc., Concord, Ontario, Canada). Data were extracted from microarray array images using Agilent Feature Extraction Software (Agilent; Palo Alto, CA, USA).

Project description:We investigated ecotoxicological effects and toxicogenomic responses in fathead minnows (Pimephales promelas) exposed to an environmentally-relevant concentration (0.83 mg/L) of the munitions compound cyclotrimethylenetrinitramine (RDX) in one year and multi-generational assays. In the one year assay, RDX effects were discerned by comparing breeding groups reared in control or RDX-exposure conditions for one year. RDX had no detectable effect on gonad-somatic index, or condition factor in females assayed at 1 day and at 1, 3, 6, 9, and 12 months, however the liver-somatic index was significantly increased versus controls only at the 12 month time point. RDX had no effect on live-prey capture rates at all time points assayed and no significant impacts on egg production, fertilization or hatch success in the 1-year exposure trial. Genomic analyses indicated that RDX exposure caused limited differential expression of transcripts within time points and no functional conservation of effects indicative of RDX exposure among time points for either brain or liver tissues in the one year exposure. In the multi-generational assay, the effects of acute (96h) exposure to RDX were compared in fish reared to the F2 generation in either control or RDX-exposure conditions. The RDX-reared fish were not observed to have appreciably enhanced RDX tolerance versus the control-reared fish. However, significant differences in gene expression were observed among the control and RDX-reared fish related to neuro-excitatory glutamate metabolism, sensory signaling and processes in neurological development. In total, our results indicate that exposure to an RDX concentration approximating maximum levels observed in the field (0.83 mg/L) caused limited impacts in fathead minnows in a one year exposure, however caused altered expression in genes involved in neural function in a multi-generational exposure. Adult fathead minnows were exposed to 0.83 mg/L (0.15 mg/L standard deviation) in experimental units including 2 male and 4 fish. Five replicate males were randomly sampled with replacement from each of 8 control and 8 RDX-exposed experimental units at 1d, 1mo, 3mo, 6mo, 9mo and 12mo sampling periods. Brain tissue was investigated for differential expression in response to RDX exposure. Data were analyzed in 3 separate investigations. First, the Reference vs Reference data were analysed to determine an empirical false positive detection rate. Next, in order to meet milestones set up by our upper management, we were forced to generate results prior to the end of the bioassay. Therefore, we investigated gene expression among Control and RDX-exposed fish in the 1 day - 6 month sampling periods. Investigation of gene expression among the control and RDX-exposed fish for the 9 month and 12 month time periods were investigated separately.

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Effects of trenbolone, flutamide and a mix of both in fathead minnow ovaries

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