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:Interpretation of toxicogenomic experiments conducted with ovary tissue from asynchronous-spawning small fish species are complicated by background variation in the relative abundance and proportion of follicles at different stages within the ovary tissue sample. This study employed both real-time quantitative PCR and a 15,000 gene oligonucleotide microarray to examine variation in the fathead minnow (Pimephales promelas) ovarian transcriptional profile as a function of quantitative and qualitative differences in ovarian histology. Multiple lines of evidence supported the conclusion that variation in the transcriptional profile was primarily dependent on the relative abundance of previtellogenic versus vitellogenic follicles in the ovary tissue. Due to the relatively small proportions of mature ovulated follicles or atretic follicles in the overall follicle population, few putative molecular markers of maturation, ovulation, or atresia could be identified. However, among the 460 differentially expressed genes identified in the study, targets including HtrA serine peptidase 3 (htra3), tissue inhibitor of metalloproteinase 3 (timp3), aquaporin 8 (aqp8), transgelin 2 like (tagln2), Nedd4 family interacting protein 2 (ndfip2), chemokine ligand 12a (cxcl12a), midkine related growth factor (mdka), and jagged 1b (jag 1b) exhibited responses and functional properties that support them as candidate molecular markers of significant shift in gross ovarian stage. Overall, results of this study provide insights into background variation in ovary transcript profiles that should aid and enhance the interpretation of toxicogenomic data generated in experiments conducted with small, asynchronous spawning fish species. Relative abundance of approximately 15,000 RNA transcripts in 26 ovary samples and 4 expelled oocyte (egg) samples, representing the five different histoclasses defined for the present study, was evaluated using fathead minnow oligonucleotide microarrays. Fathead minnow 15,000 gene arrays were purchased from Agilent (Palo Alto, CA, USA). The Agilent one-color microarray hybridization protocol (One-Color Microarray-Based Gene Expression Analysis, version 5.7, Agilent Technologies, Palo Alto, CA) was used for microarray hybridizations following the manufacturerM-bM-^@M-^Ys protocol and recommendations. One ug of total RNA was used for all hybridizations. cDNA synthesis, cRNA labeling, amplification and hybridization were performed following the manufacturerM-bM-^@M-^Ys kits and protocols (Quick Amp Labeling kit; Agilent, Palo Alto, CA). An Axon GenePixM-BM-. 4000B Microarray Scanner (Molecular Devices Inc., city, state) was used to scan microarray images at 5 M-NM-<m resolution.

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.

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Pimephales promelas

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