Gene expression profiling of fathead minnows (Pimephales promelas) following an acute exposure to pulp and paper mill effluents
ABSTRACT: We evaluated the possible mechanisms by which exposure to a sequentially treated pulp and paper mill effluent affects gene expression in the liver of male and female fathead minnows. Overall design: Sexually mature fathead minnows were exposed to either river water, which served as our control (C), 10% untreated kraft effluent (UTK), 25% treated kraft effluent (TK) or 100% final effluent (CMO) from a multiprocess pulp and paper mill for 6 days. A total of 4 treatments. Each exposure aquarium consisted of a 42.1 L column that contained individual 5.3 L chambers. Each chamber contained a FHM breeding pair. A total of 3 biological replicates for male and female FHM per treatment were sent for microarray analysis resulting in a total of 24 arrays run as a reference design with a pooled sample of the 6 river water exposed fish serving as the reference sample..
Project description:We evaluated the possible mechanisms by which exposure to a sequentially treated pulp and paper mill effluent affects gene expression in the liver of male and female fathead minnows. Sexually mature fathead minnows were exposed to either river water, which served as our control (C), 10% untreated kraft effluent (UTK), 25% treated kraft effluent (TK) or 100% final effluent (CMO) from a multiprocess pulp and paper mill for 6 days. A total of 4 treatments. Each exposure aquarium consisted of a 42.1 L column that contained individual 5.3 L chambers. Each chamber contained a FHM breeding pair. A total of 3 biological replicates for male and female FHM per treatment were sent for microarray analysis resulting in a total of 24 arrays run as a reference design with a pooled sample of the 6 river water exposed fish serving as the reference sample..
Project description:We evaluated the possible mechanisms by which exposures to pulp and paper mill effluents gene expression in the fathead minnow hypothalamus Keywords: Toxicology Overall design: Sexually mature fathead minnows were exposed to 100% pulp and paper mill effluents for 5 days. Tanks contained 4 females and 2 males. A total 4 tanks per effluents were used in this experiment. TM5, TM6, and KM4 represent different pulp and paper mill effluents from different mills coded for by FPInnovations-Paprican.
Project description:We evaluated the possible mechanisms by which exposures to pulp and paper mill effluents gene expression in the fathead minnow hypothalamus Keywords: Toxicology Sexually mature fathead minnows were exposed to 100% pulp and paper mill effluents for 5 days. Tanks contained 4 females and 2 males. A total 4 tanks per effluents were used in this experiment. TM5, TM6, and KM4 represent different pulp and paper mill effluents from different mills coded for by FPInnovations-Paprican.
Project description:Municipal wastewater effluent can impact its receiving environment. In the St. Lawrence River, male fish living downstream from Montreal exhibit increased hepatic vitellogenin, intersex, delayed spermatogenesis and altered immune function. Few studies have examined genome-wide effects associated with municipal effluent exposure in fish to decipher the mechanisms of toxicity. The present objective was to identify hepatic cellular signaling pathways in fathead minnows following exposure to municipal wastewater effluent. Immature minnows were exposed for 21 days to either 0% (Control) or 20% municipal effluent, the highest concentration in the St. Lawrence River. Hepatic RNA was extracted and used to hybridize a fathead minnow oligonucleotide microarray containing approximately 15K gene sequences. Sixteen samples were examined, 8 control samples and 8 exposed samples.
Project description:Masculinized female Eastern Mosquitofish (Gambusia holbrooki) have resided downstream of paper mills in Florida since the 1980's. The potential impacts of this effluent on the mosquitofish endocrine system are unknown. The objective of this study was to evaluate gene expression patterns of endocrine system genes and global gene expression patterns in female G. holbrooki from a paper mill-impacted site. Masculinized female G. holbrooki were collected from a paper mill-impacted site (Fenholloway River) and from a reference site (Econfina River) and microarray analysis in livers was conducted. Hepatic microarray analysis revealed an increase in the expression of metabolic genes at the Fenholloway, with similarities in individual genes and biological processes compared to G. holbrooki exposed to androgens. These data indicate G. holbrooki from the Fenholloway may be impacted by a mixture of endocrine-active chemicals, including androgens. During the summer of 2012, G. holbrooki were captured from one site downstream of the Buckeye Pulp and Paper Mill (Taylor County, Perry, FL, USA) on the Fenholloway River (GPS coordinates: N 30 058.341’, W 83 588.569’) and one site in the Econfina conservation area (GPS coordinates: N 30 08.549', W 83 51.962') . Only sexually mature G. holbrooki (females > 15cm standard length and with the presence of the gravid spot near the vent) were collected. A 1/8 mesh seine was used for sample collection. Female G. holbrooki were transferred to 5 gallon aerated buckets filled with site water and were processed at the site immediately after collection. Fish were anesthetized using Tricaine-S (Western Chemical, Ferndale, USA) and sacrificed via spinal transection. Oocyte development was assessed upon dissection and livers were removed and stored in RNAlater (Qiagen, Hilden, Germany) overnight at 4 C before storage at -80 C. RNA was isolated from the livers using TRIzol (Invitrogen, Grand Island, USA), hydrated using RNAsecure (Ambion, Grand Island, USA), and DNase treated using the Turbo DNA-free kit (Ambion, Grand Island, USA). Four oocyte-development stage-matched RNA samples per treatment were evaluated for RNA integrity using the 2100 BioAnalyzer (Agilent, Santa Clara, USA). The range of RIN values was 7.8-8.9.
Project description:This SuperSeries is composed of the following subset Series: GSE28351: Effects of bisphenol A on ovarian gene expression in fathead minnows and zebrafish [ZF data set] GSE28353: Effects of bisphenol A on ovarian gene expression in fathead minnows and zebrafish [FHM data set] Refer to individual Series
Project description:Evaluating the potential human health and ecological risks associated with exposures to complex chemical mixtures in the environment is one of the main challenges of chemical safety assessment and environmental protection. There is a need for approaches that can help to integrate chemical monitoring and biological effects data to evaluate risks associated with specific chemicals present in the environment. In the present study water samples from five locations near two wastewater treatment plants in the St. Croix River basin on the border of MN and WI, USA were analyzed for 127 contaminants including wastewater indicators, pharmaceuticals, and a number of natural and synthetic steroids. Prior knowledge about chemical-gene interactions was used to develop site-specific knowledge assembly models (KAMs) that were used to formulate hypothesis concerning possible biological effects of exposure to the chemicals detected at each location and suggest assays and endpoints for follow-up investigation. Additionally empirical hepatic gene expression data were collected for fathead minnows (Pimephales promelas) exposed in situ, for 12 d, at each location using a high density oligonucleotide microarray. Empirical gene expression data were analyzed to identify functional annotation terms enriched among the lists of differentially-expressed probes. However, the general nature of many of the terms made hypothesis formulation on the basis of the transcriptome-level response alone difficult. However, integrated analysis of the transcriptome data in the context of the site-specific KAMs allowed for evaluation of the likelihood of specific chemicals contributing to observed biological responses, based on prior knowledge. Overall design: Adult male fathead minnows were exposed in situ to treated wastewater effluent, or water upstream or downstream of two wastewater treatment plants along the St. Croix River in MN and WI. Two replicates of ten fish (20 fish total) were placed into mini-mobile environmental monitoring units and exposed to continually flowing water from the appropriate treatment. Fish were exposed for 12 days and then sampled. Livers from 6-7 randomly chosen fathead minnows were isolated and RNA was extracted for hepatic transciptome analysis using microarray. The transcriptomic profiles were compared to control fish exposed to well water for 12 days.
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. Overall design: 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 manufacturer’s 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.