Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Taking microarrays to the field: Differential hepatic gene expression of caged fathead minnow from Nebraska watersheds

ABSTRACT: This study aimed to evaluate the utility of microarrays as a biomonitoring tool in field studies. A 15,000-oligonucleotide microarray was used to measure the hepatic gene expression of fathead minnows (Pimephales promelas) caged in four Nebraska, USA watersheds - the Niobrara and Dismal Rivers (low-impact agricultural sites) and the Platte and Elkhorn Rivers (high-impact agricultural sites). Gene expression profiles were site specific and fish from the low- and high-impact sites aggregated into distinct groups. Over 1500 genes were differentially regulated between fish from the low- and high-impact sites. Many gene expression differences (1218) were also noted when the Platte and Elkhorn minnows were compared to one another and Platte fish experienced a higher degree of transcript alterations than Elkhorn fish. These findings indicate that there are differences between the low-impact and high-impact sites, as well as between the high-impact sites. Historical water quality data supports these results as only trace levels of agrichemicals have been detected at the low-impact sites, while substantial levels of agrichemicals have been reported at the high-impact sites with agrichemical loads at the Platte generally exceeding those at the Elkhorn. Overall, this study demonstrates that microarrays can be utilized to discriminate sites with different contaminant loads from one another. A 15,000-oligonucleotide microarray was used to measure the hepatic gene expression of fathead minnows (Pimephales promelas) caged in four Nebraska, USA watersheds - the Niobrara and Dismal Rivers (low-impact agricultural sites) and the Platte and Elkhorn Rivers (high-impact agricultural sites).

ORGANISM(S): Pimephales promelas

SUBMITTER: Barbara Carter

PROVIDER: E-GEOD-33436 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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:To assess the impact of a municiple effluent across different environments, a gradient design (upstream, downstream, and at effluent) was set up across three waste water treatment plant outflows in three different regions of MN. These three sites represents vastly different land use and contamination profiles. The upstream location at each site was used as a point of comparison to reduce site specific differences in water. Fish were exposed at three sites in three locations for 4 days. The objectives of the study were to 1). determine if biological impact of the effluent could be detected at the downstream site; 2). If the use of the upstream site would allow point source influence to be detected over the ambient level of biological activity; 3). if using functional analyses of transcriptomic results would show similarities between effleunt and downstream sites. The current series contains n=53 microarrays associated with fish exposed at three locations (upstream, effluent, downstream) at 3 different site R, H, E for 4 days until tissues were collected At the specific locations (UP, EF, DS), sexually mature female fathead minnows (SI-SM2) were exposed to location specific water in mini-mobile environmental monitoring units (MMU)14 for a period of 4d, at each of three sites (R, H, E) during the summer and fall of 2010 (SI-SM3). The MMUs allowed for consistency in aeration, temperature, and feeding. In the case of the riverine R and H sites, the MMU was supplied with a continuous flow of location-specific water. Due to logistical constraints, exposures at the lake site (E) were conducted under static renewal conditions within the MMUs. However, the cumulative number of daily water exchanges were equivalent to those in the flow-through studies. After exposure fish were anesthetized (buffered MS-222), and necropsied at a facility near the site, specific tissues were excised and flash frozen in liquid nitrogen, and stored at -80M-BM-0C. Gonads were removed from female and flash-frozen for gene expression analyses using microarray. Ovarian transcripts from six females per location (three locations per site, three sites) were analyzed using a custom 15,000 feature microarray (GEO Platform Accession GPL9248). Data sets for this phase (n=53 microarrays) were normalized independently using Fastlo (Ballman et al., 2004) implemented in R (http://www.r-project.org/), but analyzed using parallel approaches.

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. Liver tissue was investigated for differential expression in response to RDX exposure.

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Taking microarrays to the field: Differential hepatic gene expression of caged fathead minnow from Nebraska watersheds

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