Unknown,Transcriptomics,Genomics,Proteomics

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

ABSTRACT: 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.

ORGANISM(S): Pimephales promelas

SUBMITTER: Ignacio Rodriguez-Jorquera

PROVIDER: E-GEOD-54506 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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.

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).

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:Perfluorooctane sulfonate (PFOS) has been manufactured for over 50 years in increasing quantities and has been used for several industrial and commercial aims. Due to the persistence and the bioaccumulation of this pollutant, it can be found worldwide in wildlife and humans. Biochemical effects of PFOS exposure are mainly studied in mammalian model species and information about effects on fish species remain largely scarce. This lack of toxicity data points out that there is an urgent need for the mechanistic molecular understanding of the mode of action of this pollutant. In the present study, common carp (Cyprinus carpio) was exposed through water for 14 days at concentrations of 0.1; 0.5 and 1 mg/l PFOS. Liver was selected as target tissue. Custom microarrays were constructed from cDNA libraries obtained with Suppression Subtractive Hybridization-Polymerase chain reaction (SSH-PCR) experiments. Microarray data revealed that the expression of several genes in the liver was influenced by PFOS exposure and real-time PCR was used to confirm these gene expression changes. The affected genes were mainly involved in energy metabolism, reproduction and stress response. Furthermore, the relative condition factor and the hepatosomatic index of the exposed fish were significantly lower after 14 days of exposure as well as the available glycogen reserves. At all levels of biological organization, indications of a trade-off between the metabolic cost of toxicant exposure on one hand and processes vital to the survival of the organism on the other hand were seen. Our results support the prediction that increases in energy expenditure negatively affects processes vital to the survival of an organism, such as growth. Keywords: PFOS, common carp, microarray, condition factor, energy reserves, metabolic cost There were 3 biological replicates for each exposure concentration. For each biological replicate control versus exposed hybridizations were carried out. The mean of the biological replicates was calculated for the differentially expressed genes.

Project description:Despite recent knowledge of the potential environmental impact that compounds present in municipal wastewater effluents, including contaminants of emerging concern (CECs), may have, the implications of fish exposure to this contaminant mixtures are not completely understood. The effects caused by effluent CECs may be subtle and diverse, thus the need for sensitive and comprehensive tools such as gene expression to detect such responses. In this study, we conducted laboratory exposures that examined plasma concentrations of vitellogenin (VTG), changes in secondary sexual characteristics and gene expression in sexually mature male fathead minnows (Pimephales promelas) exposed to environmentally realistic (0.5%) and higher (5%) concentrations of municipal wastewater effluents. Secondary and primary treated effluents were used. Several of the 32 CECs investigated were detected, including pharmaceuticals, personal care products, hormones, current use pesticides and industrial compounds. The percent of males with detectable levels of VTG was higher in fish exposed to effluent treatments. An increased number of males with changes in secondary sexual characteristics (e.g. development of ovipositors), was observed in fish exposed to 5% effluent treatments. Gene expression data indicated that overall expression patterns were characteristic to each effluent. Higher numbers of differentially expressed genes were observed in fish exposed to primary treated effluent when compared to controls. Differentially expressed genes belonged to several functional categories, including xenobiotic metabolism, estogenicity and energy/metabolism processes. Gene expression data provided information to understand some of the mechanisms behind the effects observed at higher biological levels. To investigate gene expression responses resulting from exposure to POTW effluents, two laboratory experiments were conducted using effluent from San Diego (Point Loma; SD) and Los Angeles (Hyperion; LA). The LA effluent received secondary treatment and the SD effluent received advanced primary treatment. Treatments used during exposures consisted of negative controls (moderately hard water), positive controls (E2), and 0.5% and 5% effluent concentrations. The 0.5% concentration of effluent represented an environmentally realistic exposure level. The 5% effluent concentration represented a higher level at which we expected biological responses. The exposures lasted 14 days. Treatments: EFFHa = 5% primary treated effluent EFFHb = 5% secondary treated effluent EFFLa = 0.5% primary treated effluent E2a = Estradiol, positive control for primary effluent E2b = Estradiol, positive control for secondary effluent CTRLa = Moderately hard water, negative control for primary effluent CTRLb = Moderately hard water, negative control for secondary effluent

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

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