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Comparative Transcriptomic Response from Fathead Minnow Liver and Blood Exposed to Environmentally Relevant Perfluorochemicals Concentrations

ABSTRACT: 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.

ORGANISM(S): Pimephales promelas

PROVIDER: GSE54506 | GEO | 2014/01/30

SECONDARY ACCESSION(S): PRJNA236739

REPOSITORIES: GEO

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Project description:The NationM-bM-^@M-^Ys 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. Gene expression after 48 hours of exposure to prefluorochemicals in male liver of FHM Exposure Water Preparation Four different treatment were prepared. A control (0 ug/L); two PFOS concentrations: PFOS high (25 ug/L), PFOS low (0.5 ug/L); and a PFCs mixture, consisting in 7 types of PFCs at concentrations similar than those found in a previous research downstream to a wastewater treatment plant (Rodriguez-Jorquera et al., in prep.) in an attempt to mimic the mixtures usually found in waters with wastewater influence. The preparation of all exposure treatments was completed dissolving the needed amount of PFCs for each concentration first on a 50 ml polypropylene falcon tube and 1, 9 ul of TEG (Triethylene glycol) as vehicle. For the control group, Milli-Q water and TEG was mixed in a falcon tube. Then, the 50 ml preparation were poured into a pre cleaned fiberglass cylinder containing 38 liters of carbon filtered and de-chlorinated city water. All PFCs were purchased from Wellington labs. Water samples were collected at the end of the exposures from the distribution cylinders. 1 L sample was collected for each treatment in a polypropylene bottles for PFCs analysis of 7 types of perfluorinated carboxylic acids (including PFOA) using EPA Method 537. Fish Exposure and Tissue Collection Thirty two males were separated from the common tank two weeks before the experiment and placed in the treatment aquaria for 48 h. The exposure system consisted of 10 L glass aquaria. Each exposure was conducted in quadruplicate and each aquarium contained two male FHM in 4 L of treatment water. The water used in the control treatment was carbon filtered, dechlorinated tap water. The positions of the treatment tanks were randomized and test initiation times were staggered to ensure an exposure/sampling interval of 48 h. The fish were not fed during the experiment. The temperature range of the water was 24- 26 M-BM-0C with a photoperiod of 16 h light: 8 h dark. At the conclusion of the exposures, fish were anesthetized with MS-222 and weighed to the nearest 0.1 g .The testes were removed and preserved for histological analysis to check sex and sexual stage. Liver tissue and whole blood was flash frozen using liquid nitrogen and stored at -80 M-BM-0C until RNA extraction. Liver and blood were isolated from all males. The liver and blood from same individuals (four for each treatment) were used for RNA extraction. All procedures involving live fish were reviewed and approved by the University of Florida Institutional Animal Care and Use Committee (IACUC). After the exposure of fish to each treatment, four biological replicates were used to isolate RNA from FHM livers and blood using the same individuals in order to diminish variability among fish.Then microarrays were washed according to the Agilent protocol and, kept in the dark until scanning.

Project description:The diverse mixture of contaminants frequently present in estuarine wetlands complicates their assessment by routine chemical or biological analyses. We investigated the use of gene expression to assess contaminant exposure and the condition of southern California (USA) estuarine fish. Liver gene expression, plasma estradiol concentrations and gonad histopathology were used to investigate the biological condition of longjaw mudsuckers (Gillichthys mirabilis). A wide array of metals, legacy organochlorine pesticides, PCBs and contaminants of emerging concern were detected in sediments and whole fish. Overall gene expression patterns were characteristic to each of four sites investigated in this study. Differentially expressed genes belonged to several functional categories including xenobiotic metabolism, detoxification, disease and stress responses. In general, plasma estradiol concentrations were similar among fish from all areas. Some fish gonads had pathologic changes (e.g. infection, inflammation) that could indicate weakened immune systems and chronic stress. The differential expression of some genes involved in stress responses correlated with the prevalence of histologic gonad lesions. This study indicates that sentinel fish gene expression data is a promising tool for assessing the biological condition of fish exposed to environmental contaminants. Key Words: Gene expression, fish, contaminants, estuaries. This abstract belongs to a manuscript that has been submitted to Environmental Science and Technology. The manuscript has been invited as part of an especial Omics Issue which is expected to be published in 2012. In this study, we used hepatic gene expression in wild longjaw mudsuckers (Gillichthys mirabilis) to assess biological responses from anthropogenically influenced wetlands. We investigated the relationships among gene expression responses, chemical exposure and additional biological responses in this species. We studied estuarine wetlands that had diverse contaminant characteristics and received three main types of contaminant inputs in different proportions: agricultural runoff, urban runoff and municipal wastewater.

Project description:Three surface waters in Gainesville, Florida were used in a 48 hour whole effluents exposure to assess gene expression profiles of male fathead minnow liver. Microarray analysis was used to determine changes in gene expression of exposed fish to waters from a site downstream of a wastewater treatment plant (streamwater), a wastewater treatment plant (wastewater), and a lake (stormwater). Differences in gene expression between fish exposed to collected waters and controls were observed. Number of altered genes and biological processes were 1028 and 18 for stormwater; 787 and 19 for streamwater; and: 575 and 12 for wastewater. In general, the effects observed in all exposed fish were related with fatty acid metabolism, DNA repair, oxidation-reduction process, cell wall catabolic process and apoptosis. All exposed fish showed altered expression of genes related with DNA damage repair. In particular fish exposed to stormwater and streamwater showed downregulation of several key intermediates transcripts of cholesterol. The presence and environmental persistence of perfluorinated chemicals (PFCs) in these waters, the resemblance in known effects on transcripts with those found in this study, suggest that the set of genes differentially regulated in fathead minnows after 48 hours of exposure may be attributed to exposure to PFCs. Three surface water sites were chosen for effluent collection in Gainesville, Florida: A lake (stormwater), surface water downstream of a wastewater treatment plant (streamwater), and a wastewater treatment plant effluent used for landscaping irrigation (wastewater). Water from each site was collected two days prior to the fish exposure experiment using Chemfluor ® tubing and a 120 liters steel barrels coated with polyester resin (gel coat) to avoid cross-contamination. Three barrels for each effluent were collected during day 1. Water from the barrel was transported to the laboratory and pumped into four fiberglass cylinders in the aquatic toxicology facility. Water from each cylinder was then pumped into four replicate aquariums per treatment and kept for 1 day without fish (pre-treatment). On day 2, four male fathead minnows from a common tank were transferred to each replicate aquarium and kept for 48 hours, with one 75% water change after first 24 hours. The exposure system consisted of 40 L glass aquaria. Each exposure was conducted in quadruplicate and each aquarium contained the four male fish in 25 L of treatment water . The water used in the control treatment was carbon filtered, dechlorinated tap water. The positions of the treatment tanks were randomized and test initiation times were staggered to ensure an exposure/sampling interval of 48 h. The fish were not fed during the experiment. The temperature range of the water was 24-26 °C with a photoperiod of 16 h light: 8 h dark. Liver was isolate from 4 males indviduals for each treatment except for control group (3 individuals).

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Comparative Transcriptomic Response from Fathead Minnow Liver and Blood Exposed to Environmentally Relevant Perfluorochemicals Concentrations

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