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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.

ORGANISM(S): Pimephales promelas

PROVIDER: GSE33436 | GEO | 2012/03/01

SECONDARY ACCESSION(S): PRJNA148689

REPOSITORIES: GEO

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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:The Nation’s streams and rivers contain several contaminants in the form of complex mixtures. These cocktails of chemicals are not equivalent in concentrations, some pollutants such as nutrients can be found in the range of mg/L (macro-pollutants) but others components (micro-pollutants) such as endocrine disrupting chemicals (EDCs) are in amounts thousands to millions of times less concentrated ug/L to ng/L. These mixtures hamper the determination of particular effects of contaminants in aquatic biota. Nonetheless, the fact that toxicity is preceded by alteration in gene expression in an organism allows the use of gene expression profiling (from microarray studies) to detect early toxic effects and identify mechanisms of action. The microarray technology, a collection of DNA fragments attached to a solid surface, can be used to measure the expression levels of large numbers of genes. This facilitates establishment of links between toxicants and effects on biota. In urban waters, micropollutants such as EDCs, are known to cause effects at very low concentrations. One common class of EDCs found in low levels in urban waters is the class of perfluorochemicals (PFCs). Previously, we observed that urban waters with wastewater influence containing PFCs in the 300 ng/L range exerted effects in fish by altering the expression of cholesterol metabolism and DNA repair genes in the liver. To determine whether low concentrations in the range of the PFCs found in the environment can elicit gene expression changes, we investigated the impact of 7 different types of PFCs in a controlled laboratory study by exposing fathead minnows for 48h to environmentally relevant concentrations of PFCs. Additionally, we use blood as starting material for microarray analysis in order to explore non-invasive techniques. No fish mortality was observed in any treatment exposures, but gene expression was altred. Surprisingly, low levels of PFCs that we used altered gene expression in fish liver and blood. Several of the same genes were altered in both liver and blood from exposed fish. Micorarray analysis yields information on altered molecular pathways that predict changes at higher levels of biological organization such as survival and reproduction.

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

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