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: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: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.

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