Characterization of hepatic glutathione S-transferases in coho salmon ( Oncorhynchus kisutch)

Abstract

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 24–26 kDa. 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.