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:Production, usage and disposal of the munitions constituent (MC) cyclotrimethylenetrinitramine (RDX) has led to environmental releases on military facilities. The chemical attributes of RDX are conducive for leaching to surface water which may put aquatic organisms at risk of exposure. Because RDX has been observed to cause aberrant neuromuscular effects across a wide range of animal phyla, we assessed the effects of RDX on central nervous system (CNS) function in the representative aquatic ecotoxicological model species, fathead minnow (Pimephales promelas). A brain-tissue based cDNA library enriched for transcripts differentially expressed in response to RDX exposure was developed for fathead minnow and was transitioned to custom cDNA-based microarrays. All 4,128 cDNAs were sequenced, quality filtered and assembled yielding 3,018 unique sequences and 945 significant blastx matches (E ≤ 10-5). Bioassays were conducted exposing fathead minnows to RDX at 0.625, 1.25, 2.5, 5, 10 mg/L or an acetone-spike control for 10d. Overt toxicity of RDX in fathead minnow occurred only at the highest exposure concentration resulting in 50% mortality. Conversely, Bayesian analysis of microarray data indicated significant changes in transcript expression in fathead minnow brain tissue at RDX concentrations as low as 0.625 mg/L. In total, 154 microarray targets representing 44 unique transcript identities were differentially expressed in RDX exposures, the majority of which were validated by RT-qPCR. Investigation of molecular pathways, gene ontology and individual gene functions indicated that RDX exposures affected metabolic processes involved in: oxygen transport, neurological function, calcium binding / signaling, energy metabolism, cell cycle / cell proliferation, oxidative stress and ubiquitination. In total, our study indicated that RDX exposure affected molecular processes critical to CNS function in fathead minnow. 10 Day RDX Exposure, Brain Tissue Investigation: Sub-adult fathead minnows were exposed to RDX in a 10d dose-series experiment (0.625, 1.25, 2.5, 5.0, or 10 mg/L RDX) which included an acetone-spike control (1% acetone). Each experimental treatment included 8 replicate fish (48 total fish) and endpoints included mortality, total weight and neurotoxicogenomics. The 1.25mg/L dose was not included in the microarray experiment. Please see attached PDF file for detailed 'Balanced, Interwoven Loop Design'.

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Pimephales promelas

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