Project description:Wild stocks of Pacific salmon in the Northwestern United States have declined in recent years, and the major factors contributing to these losses include water pollution and loss of habitat. In salmon, sublethal chemical exposures may impact critical behaviors (such as homing, feeding, predator-avoidance) that are important for species survival. Therefore, understanding the potential for these species to biotransform organic compounds within sensitive target tissues such as liver, gills and olfactory region can help estimate or predict their susceptibility to pollutants. In this study, we used real-time quantitative polymerase chain reaction (Q-PCR), Western blotting, and catalytic assays to characterize the expression of Phase I biotransformation enzymes in coho salmon (Oncorhynchus kisutch), a sensitive species in the Pacific Northwest. Gene expression analysis using Q-PCR assays developed for coho genes revealed the presence of the predominant cytochrome P450 mRNAs (CYP1A, CYP2K1, CYP2M1, CYP3A27) in the olfactory rosettes and provided quantitative mRNA expression levels in coho liver and gills. Q-PCR analysis revealed relatively high expression of the major CYP isoforms in the liver and olfactory rosettes, which was generally confirmed by Western blotting. Extrahepatic CYP expression was generally higher in the olfactory rosettes as compared to the gills. Catalytic studies demonstrated functional CYP1A-dependent ethoxyresorufin-O-deethylase, CYP2-dependent pentoxyresorufin-O-dealkylase, CYP2K1-dependent testosterone 16beta-hydroxylase, and CYP3A27-dependent testosterone 6beta-hydroxylase activities in liver, but not at detectable levels in gills. In contrast, flavin-containing monooxygenase (FMO)-dependent thiourea S-oxidase activity was readily observed in the gills and was substantially higher than that observed in liver. Collectively, the results of this study suggest that the olfactory rosettes are important sites of extrahepatic biotransformation in coho salmon, and that tissue specific-differences in Phase I metabolism may lead to contrasting tissue-specific biotransformation capabilities in this species.

Project description:Most Pacific salmonids undergo smoltification and transition from freshwater to saltwater. Saltwater acclimation requires salmonids to make various adjustments in color, shape, size, metabolism, catabolism, and osmotic and ion regulation. The molecular mechanisms underlying this transition are largely unknown. The present study acclimated coho salmon (Oncorhynchus kisutch) to four different salinities (<0.5, 8, 16, and 32 ppth) and assessed gene expression through microarray analysis of gill, liver and olfactory tissues. Gills are involved in osmotic regulation, liver plays a role in energetics, and olfactory tissues are involved in behavior. Between all salinity treatments, liver had the highest number of differentially expressed genes at 1,616. Gills had 1,074 differentially expressed genes and olfactory tissue had 924. The difference in the number of differentially expressed genes may be due to the higher responsiveness of liver to metabolic changes after salinity acclimation to provide energy to fuel other metabolic and osmoregulatory tissues like gills. Differentially expressed genes were tissue and salinity treatment dependent. There were no genes differentially expressed in all salinity treatments and all three tissues. Five genes were targeted for microarray confirmation by qPCR and included CCAAT/enhancer binding protein ? (CEBPB), calpain 1 (CAPN1), proto-oncogene, serine/threonine kinase (Pim1), aldolase B, fructose-bisphosphate (aldob), and complement component 3 (c3). qPCR expression profiles of these genes matched array outputs. Gene ontology term analysis revealed biological processes, molecular functions, and cellular components that were significant. Most terms were tissue dependent. For liver, oxygen binding and transport terms were highlighted, suggesting possible impacts on metabolism. For gills, muscle and cytoskeleton related terms were emphasized and for olfactory tissues, immune response related genes were accentuated. Interaction networks were examined in combination with GO terms and determined similarities between tissues for potential osmosenors and signal transduction cascades. Overall this study suggests that Pacific salmonids share many salinity acclimation molecular mechanisms with other species, with a few new genes identified, and that although the three tissues shared certain underlying mechanism, many of the differentially expressed genes were tissue-specific.

Project description:The glutathione S-transferases (GSTs) are a family of phase II detoxification enzymes which protect against chemical injury. In contrast to mammals, GST expression in fish has not been extensively characterized, especially in the context of detoxifying waterborne pollutants. In the Northwestern United States, coho salmon (Oncorhynchus kisutch) are an important species of Pacific salmon with complex life histories that can include exposure to a variety of compounds including GST substrates. In the present study we characterized the expression of coho hepatic GST to better understand the ability of coho to detoxify chemicals of environmental relevance. Western blotting of coho hepatic GST revealed the presence of multiple GST-like proteins of approximately 24-26kDa. Reverse phase HPLC subunit analysis of GSH affinity-purified hepatic GST demonstrated six major and at least two minor potential GST isoforms which were characterized by liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI MS-MS) and Fourier transform-ion cyclotron resonance (FT-ICR) MS analyses. The major hepatic coho GST isoforms consisted of a pi and a rho-class GST, whereas GSTs representing the alpha and mu classes constituted minor isoforms. Catalytic studies demonstrated that coho cytosolic GSTs were active towards the prototypical GST substrate 1-chloro-2,4-dinitrobenzene, as well as towards ethacrynic acid and nitrobutyl chloride. However, there was no observable cytosolic GST activity towards the pesticides methyl parathion or atrazine, or products of oxidative stress, such as cumene hydroperoxide and 4-hydroxynonenal. Interestingly, coho hepatic cytosolic fractions had a limited ability to bind bilirubin, reflecting a potential role in the sequestering of metabolic by-products. In summary, coho salmon exhibit a complex hepatic GST isoform expression profile consisting of several GST classes, but may have a limited a capacity to conjugate substrates of toxicological significance such as pesticides and endogenous compounds associated with cellular oxidative stress.

Project description:Most Pacific salmonids undergo smoltification and transition from freshwater to saltwater. Saltwater acclimation requires salmonids to make various adjustments in color, shape, size, metabolism, catabolism, and osmotic and ion regulation. The molecular mechanisms underlying this transition are largely unknown. The present study acclimated coho salmon (Oncorhynchus kisutch) to four different salinities (<0.5, 8, 16, and 32 ppth) and assessed gene expression through microarray analysis of gill, liver and olfactory tissues. Gills are involved in osmotic regulation, liver plays a role in energetics, and olfactory tissues are involved in behavior. Between all salinity treatments, liver had the highest number of differentially expressed genes at 1,616. Gills had 1,074 differentially expressed genes and olfactory tissue had 924. The difference in the number of differentially expressed genes may be due to the higher responsiveness of liver to metabolic changes after salinity acclimation to provide energy to fuel other metabolic and osmoregulatory tissues like gills. Differentially expressed genes were tissue and salinity treatment dependent. There were no genes differentially expressed in all salinity treatments and all three tissues. Five genes were targeted for microarray confirmation by qPCR and included CCAAT/enhancer binding protein ? (CEBPB), calpain 1 (CAPN1), proto-oncogene, serine/threonine kinase (Pim1), aldolase B, fructose-bisphosphate (aldob), and complement component 3 (c3). qPCR expression profiles of these genes matched array outputs. Gene ontology term analysis revealed biological processes, molecular functions, and cellular components that were significant. Most terms were tissue dependent. For liver, oxygen binding and transport terms were highlighted, suggesting possible impacts on metabolism. For gills, muscle and cytoskeleton related terms were emphasized and for olfactory tissues, immune response related genes were accentuated. Interaction networks were examined in combination with GO terms and determined similarities between tissues for potential osmosenors and signal transduction cascades. Overall this study suggests that Pacific salmonids share many salinity acclimation molecular mechanisms with other species, with a few new genes identified, and that although the three tissues shared certain underlying mechanism, many of the differentially expressed genes were tissue-specific. To assess how salinity acclimation in coho salmon (Oncorhynchus kisutch) impacted gene expression in gills, liver, and olfactory tissues.

Project description:The olfactory system of salmonids is sensitive to the adverse effects of metals such as copper and cadmium. In the current study, we analyzed olfactory-mediated alarm responses, epithelial injury and recovery, and a suite of olfactory molecular biomarkers encoding genes critical in maintaining olfactory function in juvenile coho salmon receiving acute exposures to cadmium (Cd). The molecular biomarkers analyzed included four G-protein coupled receptors (GPCRs) representing the two major classes of odorant receptors (salmon olfactory receptor sorb and vomeronasal receptors svra, svrb, and gpr27), as well as markers of neurite outgrowth (nrn1) and antioxidant responses to metals, including heme oxygenase 1 (hmox1), and peroxiredoxin 1 (prdx1). Coho received acute (8-168 h) exposures to 3.7 ppb and 347 ppb Cd, and a subset of fish was analyzed following a 16-day depuration. Coho exposed to 347 ppb Cd over 48 h exhibited a reduction in freeze responses, and an extensive loss of olfaction accompanied by histological injury to the olfactory epithelium. The olfactory injury in coho exposed to 347 ppb Cd was accompanied at the gene level by significant decreases in expression of the olfactory GPCRs and increased expression of hmox1. Persistent behavioral deficits, histological injury and altered expression of a subset of olfactory biomarkers were still evident in Cd-exposed coho following a 16-day depuration in clean water. Exposure to 3.7 ppb Cd also resulted in reduced freeze responses and histological changes to the olfactory epithelium within 48 h of Cd exposure, although the extent of olfactory injury was less severe than observed for fish in the high dose Cd group. Furthermore adverse behavioral effects were present in some coho receiving the low dose of Cd following a 16-day depuration. In summary, acute exposures to environmental levels of Cd can cause olfactory injury in coho salmon that may persist following depuration. Mechanism-based biomarkers of oxidative stress and olfactory structures can augment the evaluation of olfactory injury manifested at the physiological level.

Dataset Information

Effects of salinity acclimation on the expression and activity of Phase I enzymes (CYP450 and FMOs) in coho salmon (Oncorhynchus kisutch).

Publications

Effects of salinity acclimation on the expression and activity of Phase I enzymes (CYP450 and FMOs) in coho salmon (Oncorhynchus kisutch).

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