Dataset Information

Cloning, expression and analysis of the olfactory glutathione S-transferases in coho salmon.

ABSTRACT: The glutathione S-transferases (GSTs) provide cellular protection by detoxifying xenobiotics, maintaining redox status, and modulating secondary messengers, all of which are critical to maintaining olfaction in salmonids. Here, we characterized the major coho salmon olfactory GSTs (OlfGSTs), namely omega, pi, and rho subclasses. OlfGST omega contained an open reading frame of 720bp and encoded a protein of 239 amino acids. OlfGST pi and OlfGST rho contained open reading frames of 627 and 681nt, respectively, and encoded proteins of 208 and 226 amino acids. Whole-protein mass spectrometry yielded molecular weights of 29,950, 23,354, and 26,655Da, respectively, for the GST omega, pi, and rho subunits. Homology modeling using four protein-structure prediction algorithms suggest that the active sites in all three OlfGST isoforms resembled counterparts in other species. The olfactory GSTs conjugated prototypical GST substrates, but only OlfGST rho catalyzed the demethylation of the pesticide methyl parathion. OlfGST pi and rho exhibited thiol oxidoreductase activity toward 2-hydroxyethyl disulfide (2-HEDS) and conjugated 4-hydroxynonenal (HNE), a toxic aldehyde with neurodegenerative properties. The kinetic parameters for OlfGST pi conjugation of HNE were K(M)=0.16 ± 0.06mM and V(max)=0.5 ± 0.1?molmin?¹mg?¹, whereas OlfGST rho was more efficient at catalyzing HNE conjugation (K(M)=0.022 ± 0.008 mM and V(max)=0.47 ± 0.05?molmin?¹mg?¹). Our findings indicate that the peripheral olfactory system of coho expresses GST isoforms that detoxify certain electrophiles and pesticides and that help maintain redox status and signal transduction.

SUBMITTER: Espinoza HM

PROVIDER: S-EPMC3660137 | biostudies-literature | 2013 Mar

REPOSITORIES: biostudies-literature

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Cloning, expression and analysis of the olfactory glutathione S-transferases in coho salmon.

Espinoza Herbert M HM Shireman Laura M LM McClain Valerie V Atkins William W Gallagher Evan P EP

Biochemical pharmacology 20121219 6

The glutathione S-transferases (GSTs) provide cellular protection by detoxifying xenobiotics, maintaining redox status, and modulating secondary messengers, all of which are critical to maintaining olfaction in salmonids. Here, we characterized the major coho salmon olfactory GSTs (OlfGSTs), namely omega, pi, and rho subclasses. OlfGST omega contained an open reading frame of 720bp and encoded a protein of 239 amino acids. OlfGST pi and OlfGST rho contained open reading frames of 627 and 681nt, ...[more]

PMID: 23261526

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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:The glutathione S-transferases (GSTs) are a multifunctional family of phase II enzymes that detoxify a variety of environmental chemicals, reactive intermediates, and secondary products of oxidative damage. GST mRNA expression and catalytic activity have been used as biomarkers of exposure to environmental chemicals. However, factors such as species differences in induction, partial analyses of multiple GST isoforms, and lack of understanding of fish GST gene regulation, have confounded the use of GSTs as markers of pollutant exposure. In the present study, we examined the effect of exposure to cadmium (Cd), a prototypical environmental contaminant and inducer of mammalian GST, on GST mRNA expression in coho salmon (Oncorhynchus kisutch) liver, gill, and olfactory tissues. GST expression data were compared to those for metallothionein (MT), a prototypical biomarker of metal exposure. Data mining of genomic databases led to the development of quantitative real-time PCR (qPCR) assays for salmon GST isoforms encompassing 9 subfamilies, including alpha, mu, pi, theta, omega, kappa, rho, zeta and microsomal GST. In vivo acute (8-48 h) exposures to low (3.7 ppb) and high (347 ppb) levels of Cd relevant to environmental scenarios elicited a variety of transient, albeit minor changes (<2.5-fold) in tissue GST profiles, including some reductions in GST mRNA expression. In general, olfactory GSTs were the earliest to respond to cadmium, whereas, more pronounced effects in olfactory and gill GST expression were observed at 48 h relative to earlier time points. Although evaluation of GSTs reflected a cadmium-associated oxidative stress response, there was no clear GST isoform in any tissue that could serve as a reliable biomarker of acute cadmium exposure. By contrast, metallothionein (MT) mRNA was consistently and markedly induced in all three tissues by cadmium, and among the tissues examined, olfactory MT was the most sensitive marker of cadmium exposures. In summary, coho salmon exhibit a complex GST tissue profile consisting of at least 9 isoforms, all of which are present in the peripheral olfactory system. Short-term exposure to environmental levels of Cd causes transient changes in salmon GST consistent with oxidative stress, and in some cases, includes a loss of GST. In a biomarker context, however, monitoring of tissue MT mRNA expression, especially in the peripheral olfactory system, may be of greater utility for assessing short-term environmental exposures to cadmium.

Project description:Exposure to environmental contaminants, including various pesticides and trace metals, can disrupt critical olfactory-driven behaviors of fish such as homing to natal streams, mate selection, and an ability to detect predators and prey. These neurobehavioral injuries have been linked to reduced survival and population declines. Despite the importance of maintaining proper olfactory signaling processes in the presence of chemical exposures, little is known regarding chemical detoxification in the salmon olfactory system, and in particular, the antioxidant defenses that maintain olfactory function. An understudied, yet critical component of cellular antioxidant defense is phospholipid hydroperoxide glutathione peroxidase (PHGPx/GPx4), an isoform within the family of selenium-dependent glutathione peroxidase (GPx) enzymes that can directly reduce lipid peroxides and other membrane-bound complex hydroperoxides. In this study, we cloned two gpx4 isoforms (gpx4a and gpx4b) from Coho salmon olfactory tissues and compared their modulation in olfactory and liver tissues by cadmium, an environmental pollutant and olfactory toxicant that cause oxidative damage as a mechanism of toxicity. Amino acid sequence comparisons of the two gpx4 isoforms shared 71% identity, and also relatively high sequence identities when compared with other fish GPx4 isoforms. Sequence comparisons with human GPx4 indicated conservation of three important active sites at selenocysteine (U46), glutamine (Q81), and tryptophan (W136), suggesting similar catalytic activity between fish and mammalian GPx4 isoforms. Tissue profiling confirmed the expression of gpx4a and gpx4b in all ten Coho tissues examined. The expression of gpx4 mRNAs in the Coho olfactory system was accompanied by comparably high initial rates of GPx4 enzymatic activity in mitochondrial and cytosolic fractions. Exposure to low (3.7 ppb) and high (347 ppb) environmental Cd concentrations for 24-48 h significantly decreased gpx4a expression in Coho olfactory rosettes, whereas olfactory gpx4b mRNA expression was not modulated by exposures at these concentrations. In summary, Coho salmon express two paralogs of gpx4, a key enzyme in the maintenance of signal transduction processes that protect against cellular oxidative damage. The Cd-associated downregulation of salmon olfactory gpx4a expression in particular, may be associated with the loss of olfactory signal transduction that accompanies metal-associated loss of olfaction in salmonids.

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

Cloning, expression and analysis of the olfactory glutathione S-transferases in coho salmon.

Publications

Cloning, expression and analysis of the olfactory glutathione S-transferases in coho salmon.

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