Unknown,Transcriptomics,Genomics,Proteomics

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Identification of a responsive gene set to evaluate the potential impact of seismic exposure on Atlantic salmon (Salmo salar) inner ear

ABSTRACT: Considerable interest and controversy has arisen over the potential effects of seismic surveys carried out during exploration for oil and gas deposits. Regarding fish, there is a concern that intense sound sources, such as seismic airguns, may injure their auditory system. In this study, salmonid cDNA microarrays, reciprocal suppression subtractive hybridization (SSH) cDNA libraries and quantitative reverse transcription – polymerase chain reaction (QPCR) were used to identify and study a responsive gene set in the inner ear of Atlantic salmon (Salmo salar) following seismic airgun exposure. Microarray analyses on pooled seismic exposed inner ear RNA versus pooled control inner ear RNA revealed 79 unique transcripts (passing background threshold) that were greater than 1.75-fold differentially-regulated by acoustic stress on at least 3 of the 4 slides in the study (including at least one dye-swap). QPCR analyses of 8 microarray-identified transcripts of interest revealed a significant up-regulation (P<0.05) of transcripts encoding nicotinamide riboside kinase 2 (1.89-fold) and hemoglobin subunit alpha-4 (3.78-fold), and a significant down-regulation (P<0.05) of a transcript encoding C14orf159 protein (1.35-fold). QPCR analyses also confirmed an overall up-regulation of transcripts encoding growth hormone I (7.78-fold), c-type lectin receptor A (2.20-fold) and retinol binding protein I (1.24-fold), however these differences were not considered to be statistically significant (P<0.05) due to the high biological variability in the seismic exposed group for these transcripts of interest. A total of 683 expressed sequence tags (ESTs) generated from SSH cDNA salmon ear libraries enriched for genes responsive to seismic airgun noise have been deposited in the GenBank dbEST. Targeted gene discovery in salmon ear allowed for the identification of novel transcripts, including some with sensory-relevant functional annotations, and represents a significant contribution to salmonid hearing research. Initial results demonstrate that genomics has the potential to greatly enhance our understanding of the impact of seismic airguns on gene and molecular pathways involved in hearing, and provide valuable molecular biomarkers that can act as an early warning sensor to acoustic stress. Juvenile Atlantic salmon (Salmo salar) smolt were obtained from North Water Products Ltd., Daniel’s Harbour, NL and held at ambient seawater temperature, in a flow-through system supplied with air at the Northwest Atlantic Fisheries Centre, St. John’s, NL. Two weeks prior to seismic airgun exposures, fish were divided into two 1m3 cages, one each for control (non-exposed) and exposed groups, in a 15,000L aquarium at ambient seawater temperature (0.2°C). Each cage was placed the same distance from the water intake and airstones were placed next to each cage. Fish were fasted for two days prior to exposure. Sixteen control (non-exposed) fish from one cage were sampled prior to seismic activity. Immediately following sampling of control fish, seventeen fish in the remaining cage were placed 2m from a 10in3 Texas Instruments airgun. Fish were subjected to 50 exposures, 1 exposure every 10 seconds, at an average sound pressure level of 204 dB peak-to-peak relative to 1µPa; considered to be a worse case scenario within a few hundred meters of a survey vessel. Seventeen seismic exposed fish were sampled 16 h following exposure. The only aquarium available that was suitable for seismic airgun exposures was not designed to have a regulated photoperiod. For this reason the fish were held under a constant daylight regime. Fish were collected by dip-net and euthanized by severing the spinal cord. The inner ears from each salmon were removed, placed immediately in RNase-free 2 ml tubes, and then flash frozen in liquid nitrogen. RNA isolated from the right inner ear of the 12 seismic exposed and 12 control individuals that gave the highest total RNA yields were used to generate 2 mRNA pools (a “seismic” pool and a “control” pool). Each sample contributed 4.0 µg column purified total RNA to each pool. Comparisons were made for the seismic exposed mRNA pool compared to the control (non-exposed) mRNA pool using the consortium for Genomic Research on All Salmonids Project (cGRASP) 16K (salmonid) cDNA array and the 3DNA Array 900 Detection Kit and instructions (Genisphere). Technical quadruplicate slides including 2 dye-swaps were run for the comparison. Slide GG003_011: Cy5-labeled control ear, Cy3-labeled seismic Slide GG003_012: Cy5-labeled control ear, Cy3-labeled seismic Slide GG003_013: Cy3-labeled control ear, Cy5-labeled seismic Slide GG003_014: Cy3-labeled control ear, Cy5-labeled seismic

ORGANISM(S): Salmo salar

SUBMITTER: Catherine Andrews

PROVIDER: E-GEOD-33257 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The rainbow smelt (Osmerus mordax, Mitchill, 1814) is an anadromous teleost that overwinters in the estuaries and inshore waters along the North American Atlantic coastline. In the winter months, smelt avoid freezing in subzero temperatures by the production of antifreeze proteins and high levels of glycerol. Glycerol production (glyceroneogenesis) occurs in the liver via a branch point in glycolysis and gluconeogenesis and is directly activated by low temperature. In these studies, hepatocytes were isolated from the liver of individual warm fish and incubated at either a warm (8ºC; non-glycerol accumulating) or cold (0.4ºC; glycerol accumulating) temperature over a 72 h time course. Functional genomic techniques were used to identify and validate hepatic transcripts that were differentially expressed between the warm and cold cells. Reciprocal suppression subtractive hybridization (SSH) cDNA libraries enriched for cold-responsive liver transcripts were constructed at the 72 h incubation time. Microarray analyses using the consortium for Genomic Research on All Salmonids Project (cGRASP) 16K (salmonid) cDNA array were performed at the 24, 48 and 72 h incubation times. For quantitative reverse transcription – polymerase chain reaction (QPCR) studies, we focused specifically on the non-colligative [type II antifreeze protein (AFPII)] and colligative (glycerol accumulation) freeze prevention strategies. AFPII (SSH identified) and 21 transcripts (SSH and/or microarray identified or selected by the authors based on a conceptual link to glycerol production) involved in the metabolism of glycolytic (glycogen, glucose) and gluconeogenic (amino acids) sources of glycerol and of lipids with a glycerol backbone (triglyceride, phosphoplipid) were analyzed using QPCR. Rainbow smelt were collected by seine netting from Mount Arlington Heights, Placentia Bay, Newfoundland in late October 2007 and transported to the Ocean Sciences Centre, Memorial University of Newfoundland. The fish were held in a 3000 L indoor free-flowing seawater tank maintained at 8°C to 10°C and followed a natural photoperiod with fluorescent lights set by an outdoor photocell. Hepatocyte isolations were performed for individual male fish (i.e. no pooling of cells from different fish) from Nov. 27th to Dec.17th, 2007. Hepatocytes were not pooled as individual smelt produce different amounts of glycerol in response to cold temperature challenge. Each liver was perfused with medium containing collagenase and yielded approximately 300 million cells per individual. Initial or “pre-incubation” samples were collected and the remaining cell suspension was divided into 20 ml glass scintillation vials (6 x106 cells per vial) containing 2 ml of incubation medium and incubated at either a warm (8ºC; non-glycerol accumulating) or cold (0.4ºC; glycerol accumulating) temperature. Duplicate vials from each temperature were sampled at 24, 48 and 72 h incubation times for RNA isolation. RNA isolated from the hepatocytes of the 9 fish with the highest levels of glycerol production at 0.4°C were used to generate 2 mRNA pools (a “cold” pool and a “warm” pool) for each incubation time (24, 48 and 72 h). To summarize, the “cold” pools contained cells from 9 individual fish incubated at 0.4°C for either 24, 48 or 72 h and which exhibited the highest increase in glycerol levels at 72 h relative to pre-incubation levels, and the “warm” pools contained cells from these same 9 individual fish but incubated at 8°C for either 24, 48 or 72 h and therefore exhibited no increase in glycerol levels at 72 h relative to pre-incubation levels. Comparisons were made for the cold compared to warm pools at the 24, 48 and 72 h time points. For each time point, technical quadruplicate slides including two dye swaps were run per comparison. Incubation time: 24 h: Cold_24h_1, Cold_24h_2, Cold_24h_3, Cold_24h_4 Incubation time: 48 h: Cold_48h_1, Cold_48h_2, Cold_48h_3, Cold_48h_4 Incubation time: 72 h: Cold_72h_1, Cold_72h_2, Cold_72h_3, Cold_72h_4 Technical replicate: Cold_24h_1, Cold_24h_2, Cold_24h_3, Cold_24h_4 Technical replicate: Cold_48h_1, Cold_48h_2, Cold_48h_3, Cold_48h_4 Technical replicate: Cold_72h_1, Cold_72h_2, Cold_72h_3, Cold_72h_4

Project description:Sulfur mustard (SM) is a potent alkylating agent. We are developing medical countermeasures to reduce the injury caused by SM exposure. Screening in the mouse ear vesicant model has identified three effective compounds: dimercaprol (British anti-lewisite), indomethacin, and octyl homovanillamide (OHV). To identify gene expression changes that correlate with compound efficacy we used oligonucleotide microarrays to compare gene expression profiles in vehicle-exposed skin, SM-exposed skin, and skin pretreated with each compound before SM exposure. Mice were topically exposed on the inner surface of the right ear to SM alone or pretreated for 15 min with one of the compounds and then exposed to SM. Left ears were vehicle-exposed. Ear tissue was harvested 24 hr later for ear weight determination (an endpoint indicating compound efficacy). The exposure groups were: methylene chloride (sulfur mustard vehicle); ethanol (drug vehicle); 0.08 mg sulfur mustard; 6.25 mg dimercaprol 15 min before 0.08 mg sulfur mustard; 1.34 mg indomethacin 15 min before 0.08 mg sulfur mustard; 0.6 mg octylhomovanillamide 15 min before 0.08 mg sulfur mustard; 6.25 mg dimercaprol alone; 1.34 mg indomethacin alone; 0.6 mg octylhomovanillamide alone. RNA was extracted from the tissues and used to generate oligonucleotide microarray probes. Principal component analysis of the gene expression data revealed partitioning of the samples based on drug treatment and SM exposure. Vehicle-exposed mouse ears clustered away from the other treatment groups. SM-exposed mouse ears pretreated with dimercaprol or OHV clustered more closely with vehicle-exposed ears, while SM-exposed mouse ears pretreated with indomethacin clustered more closely with SM-exposed ears. This clustering of the samples is supported by the ear weight data, in which the indomethacin group has ear weights closer to the SM-exposed group, whereas the dimercaprol and OHV groups have ear weights closer to the vehicle-exposed group. Correlation coefficients were calculated for each gene based on the correlation between gene expression level and ear weight. These data provide the basis for understanding what gene expression changes are important in the development of effective SM medical countermeasures. Experiment Overall Design: Exposure of mouse ears to sulfur mustard alone, sulfur mustard preceded by drug treatment, or vehicle compounds. Naive controls were also included. Biological replicates of at least n=3 were examined for each exposure condition.

Project description:Juvenile rainbow trout were fed Biodiet starter (4% body weight per day) with MeHg added at 0, 0.5, 5 and 50 ppm for six weeks. Atomic absorption spectrometry was applied to measure the level of MeHg in the whole fish body. Trout at six weeks were sampled from each group for gene expression analysis by cGRASP 16K cDNA microarrays. MeHg-exposed rainbow trout did not show overt signs of toxicity, nor were significant differences seen in mortality, length, mass, or condition factor. The chronic accumulation of total Hg in trout exhibited dose- and time-dependent patterns. The dysregulated genes have multiple functional annotations, such as involving metabolism, cellular development, ion binding and homeostasis, stress response, immune response, transcriptional regulation, hemolytic development, and apoptotic pathways. These results show that numerous molecular pathways involved in the growth and development of multiple organ systems are disrupted by exposure to moderate levels of dietary MeHg. The dysregulated genes will be selected by further analysis and used as biomarkers for MeHg exposure in aquatic environments. Juvenile rainbow trout (Oncorhynchus mykiss, average body 0.118g) were fed Biodiet Starter (Bio-Oregon) 4% of body weight per day with MeHg added at 0ppm, 0.5ppm, 5ppm and 50ppm (with ethanol as vehicle). Trout at six weeks were sampled from each group for gene expression analysis. Total RNA from individual fish was isolated using Trizol reagent (Invitrogen) and further purified using the RNeasy MiniElute cleanup kit (Qiagen).RNA of ten fish from control group was pooled as a common reference, and total RNA of five individual fish from control and MeHg-treated groups were randomly selected for microarray experiment. cDNA was synthesized from 1 M-NM-<g pooled RNA using SuperScript II Reverse Transcriptase (Invitrogen) according to the manufacturerM-bM-^@M-^Ys protocol. The common reference cDNA targets were labeled with Cy3 and cDNA of individual fish from each group was labeled with Cy5 using the Array 900 Expression Array Detection kit (Genisphere) according to the manufacturerM-bM-^@M-^Ys protocol. The labeled cDNA targets were hybridized to cGRASP 16K cDNA microarrays at 50M-BM-0C for 16 hours. Following the first hybridization, arrays were washed in 2X SSC, 0.2% SDS solution at 50 C 1 x 15 min, 2X SSC 1 x 15 min at room temperature, then 0.2X SSC for 1 x 15 min at room temperature. The fluorescent labeling hybridization (50 C for 4hr) utilized the Genisphere 3DNA Cy3 and Cy5 capture reagents in formamide hybridization buffer. The slides were washed as described above, and the arrays were dried by centrifugation. were scanned using the ScanArray Express (PerkinElmer) at 10 um resolution. The TIFF images of arrays were generated with ScanArray Express software and the intensities of raw data of two-channel arrays were collected by the ImaGene 6.0 (BioDiscovery). Statistical analysis of microarray data was performed using scripts written in R language with LIMMA package.

Project description:Our main objectives were: 1) to identify transcriptional signatures specific to the exposure to two metals: Ni and Cd; 2) to investigate the mechanisms of toxicity for these two metals; and 3) to develop a predictive tool to identify the sublethal effects of Ni and Cd contaminants in fish sampled from natural environments. The molecular mechanisms underlying nickel (Ni) and cadmium (Cd) toxicity and their specific effects on fish are poorly understood. Documenting gene transcription profiles offers a powerful approach towards identifying the molecular mechanisms affected by these metals and to discover biomarkers of their toxicity. However, confounding environmental factors can complicate the interpretation of the results and the detection of biomarkers for fish captured in their natural environment. In the present study, a 1000 candidate-gene microarray, developed from a previous RNA-seq study on a subset of individual fish from contrasting level of metal contamination, was used to investigate the transcriptional response to metal (Ni and Cd) and non metal (temperature, oxygen, and diet) stressors in yellow perch (Perca flavescens). Specifically, we aimed at 1) identifying transcriptional signatures specific to Ni and Cd exposure, 2) investigating the mechanisms of their toxicity, and 3) developing a predictive tool to identify the sublethal effects of Ni and Cd contaminants in fish sampled from natural environments. A total of 479 genes displayed significantly different transcription levels when temperature varied while 287 and 177 genes were differentially transcribed at different concentrations of Ni and Cd, respectively. These metals were found to mainly affect the transcription level of genes involved in iron metabolism, transcriptional and translational processes, vitamin metabolism, blood coagulation, and calcium transport. In addition, a linear discriminant analysis (LDA) made using gene transcription levels yielded 94% correctly reassigned samples regarding their level of metal contamination, which indicates the potential of the microarray to detect perch response to Cd or Ni effects. Comparison between fish exposed to three temperatures (11, 20, 28M-BM-0C) using a loop design, comparison between control fish and fich exposed to restrictive diet using a pairwise design, comparison between fish exposed to hypoxia and control fish, comparison between fish exposed to two concentrations of cadmium and control fish using a loop design, and comparison between fish exposed to two concentrations of nickel and control fish using a loop design.

Project description:Perfluorooctanoic acid (PFOA) is a potent hepatocarcinogen and peroxisome proliferator (PP) in rodents. Humans are not susceptible to peroxisome proliferation and are thought to be refractory to carcinogenesis by PFOA and other PPs. However, previous studies with rainbow trout have shown that they are also insensitive to peroxisome proliferation by the PP, dehydroepiandrosterone (DHEA), but are still susceptible to enhanced hepatocarcinogenesis after chronic exposure. In this study, we determined whether PFOA is also a tumor promotor in trout and then examined hepatic gene expression profiles to further investigate possible mechanisms of action. Trout were initiated as fry to the hepatocarcinogen, aflatoxin B1, and then fed 200-1800 ppm PFOA in the diet for 30 weeks. Two structurally diverse PPs, clofibrate (CLOF) and DHEA, were included for comparison. Hepatic gene expression profiles were subsequently examined in animals exposed to similar doses of PFOA, DHEA and CLOF along with 5 ppm 17β-estradiol (E2; a known tumor promotor) in the diet. PFOA (1800 ppm) and DHEA treatments resulted in enhanced liver tumor incidence and multiplicity while CLOF showed no effect. Carcinogenesis seemed independent of peroxisome proliferation as no induction of peroxisomal β-oxidation and catalase activity were observed. Alternately, plasma VTG was elevated in fish fed PFOA and DHEA suggesting that estrogenic mechanisms may play a role. Both tumor promotors, PFOA and DHEA, resulted in strong correlation of transcriptional profiles with E2 by Pearson correlation (R=0.81 and 0.78, respectively). In comparison, CLOF regulated no genes in common with E2. Overall, these data suggest that the tumor promoting activities of DHEA and PFOA in trout are independent of peroxisome proliferation and may involve estrogenic mechanisms. Juvenile trout, 12-18 months old, were fed experimental diets containing 500 or 1800 ppm PFOA, 1800 ppm CLOF, 750 ppm DHEA, 5 ppm E2 or 0.15 % dimethyl sulfoxide vehicle control for 14 days. Liver samples were collected for microarray analysis. Hybridizations were performed using standard reference design with dye-swapping. For each sample, equal amounts of RNA (µg) were pooled from five fish per tank for every treatment (n=3 biological replicates per treatment). cDNA from two of the three biological replicates was dye-swapped and hybridized to two slides as technical replicates (5 arrays per treatment).

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.

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Identification of a responsive gene set to evaluate the potential impact of seismic exposure on Atlantic salmon (Salmo salar) inner ear

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