Project description:Paper Abstract. Extrapolating toxicological sensitivity information across species can require the use of highly conservative safety factors as prescribed by ecological risk assessment protocols. The objective of our study was to use multipule factors to understand the dynamics of stress responses by comparing ecotoxicological, developmental, and genomic data to quantitatively determine the relative sensitivity of two common model aquatic vertebrates to assess the potential for developing refined safety factors among species. Larval (3-d old) zebrafish (Danio rerio) and fathead minnows (Pimephales promelas) were concurrently exposed in 96-h static exposures to the widely-used military energetic compound cyclotrimethylenetrinitramine (RDX). Endpoints assessed included mortality, incidence of vertebral deformities, swimming behavior, and differential gene (transcript) expression. Analytically verified treatment concentrations ranged from 0.9 to 27.7 mg/L RDX. While RDX concentrations prepared for exposures included 0, 0.9, 1.8, 3.5, 7.5, 15.0, and 30.0 mg/L, analytically verified treatment concentrations were 0, 0.88, 1.75, 3.46, 6.96, 13.79, and 27.7 mg/L, respectively. Changes in prepared RDX concentrations and analytically verified treatment concentrations are a result of chemical breakdown, a well known characteristic of RDX Our results indicated a higher sensitivity to RDX in P. promelas than in D. rerio in both mortality (LOECs 13.79 and 27.7 mg/L, respectively) and vertebral deformity (LOECs 13.79 and >13.79, respectively) endpoints. Swimming behavior was assessed in 10 min behavioral trials using Noldus EthoVision® digital tracking software. Swimming behavior trials indicated non-significant trends of decreased velocity, distance moved, and increased meander (change in direction per cm traveled) for P. promelas. No such trends were apparent in D. rerio swimming behavior. A comprehensive genomic analysis is in progress to determine the genes, gene ontology elements, metabolic pathways and metabolic networks affected in common among species in the RDX exposure. Taken as a whole, the ecotoxicological, developmental and genomic results will provide valuable insights into the similarity in response among these fish species to RDX. Three-day-old fathead minnow (FHM, Pimephales promelas) or Zebrafish (ZF, Danio rerio) fry were exposed to either control water or one of six concentrations of RDX (0.9, 1.9, 3.8, 7.5, 15, or 27.7 mg/L) in 96-h exposures. However, microarray analysis was only performed on FHM exposed to 0, 0.9, 1.9, 7.5, or 15 mg/L RDX and ZF exposed to 0, 0.9, 1.9, 3.8, 7.5, or 15 mg/L RDX due to insufficient tissue for microarray analysis due to animal death at the other RDX concentrations. The experimental design was based on the U.S. EPA (2002) acute testing method for P. promelas, test method 2000.0 and included four replicates per treatment with ten fish per replicate. Whole fish (10 fish per replicate) were investigated for differential expression in response to RDX exposure.

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'.

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:Paper Abstract. Extrapolating toxicological sensitivity information across species can require the use of highly conservative safety factors as prescribed by ecological risk assessment protocols. The objective of our study was to use multipule factors to understand the dynamics of stress responses by comparing ecotoxicological, developmental, and genomic data to quantitatively determine the relative sensitivity of two common model aquatic vertebrates to assess the potential for developing refined safety factors among species. Larval (3-d old) zebrafish (Danio rerio) and fathead minnows (Pimephales promelas) were concurrently exposed in 96-h static exposures to the widely-used military energetic compound cyclotrimethylenetrinitramine (RDX). Endpoints assessed included mortality, incidence of vertebral deformities, swimming behavior, and differential gene (transcript) expression. Analytically verified treatment concentrations ranged from 0.9 to 27.7 mg/L RDX. While RDX concentrations prepared for exposures included 0, 0.9, 1.8, 3.5, 7.5, 15.0, and 30.0 mg/L, analytically verified treatment concentrations were 0, 0.88, 1.75, 3.46, 6.96, 13.79, and 27.7 mg/L, respectively. Changes in prepared RDX concentrations and analytically verified treatment concentrations are a result of chemical breakdown, a well known characteristic of RDX Our results indicated a higher sensitivity to RDX in P. promelas than in D. rerio in both mortality (LOECs 13.79 and 27.7 mg/L, respectively) and vertebral deformity (LOECs 13.79 and >13.79, respectively) endpoints. Swimming behavior was assessed in 10 min behavioral trials using Noldus EthoVision® digital tracking software. Swimming behavior trials indicated non-significant trends of decreased velocity, distance moved, and increased meander (change in direction per cm traveled) for P. promelas. No such trends were apparent in D. rerio swimming behavior. A comprehensive genomic analysis is in progress to determine the genes, gene ontology elements, metabolic pathways and metabolic networks affected in common among species in the RDX exposure. Taken as a whole, the ecotoxicological, developmental and genomic results will provide valuable insights into the similarity in response among these fish species to RDX.

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.

Dataset Information

Natural Variation in Fish Transcriptomes: Comparative Analysis of the Fathead Minnow (Pimephales promelas) and Zebrafish (Danio rerio)

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure