In vitro kinetics of hepatic glutathione S-transferase conjugation in largemouth bass and brown bullheads

The kinetics of glutathione S-transferase (GST) catalysis were investigated in largemouth bass (Micropterus salmoides) and brown bullheads (Amerius nebulosus), two freshwater fish species found in a variety of polluted waterways in the eastern United States. The initial rates of hepatic GST activity toward four GST substrates, including 1-chloro-2,4-dinitrobenzene, ethacrynic acid, Delta 5-androstene-17-dione, and nitrobutyl chloride, were significantly higher in brown bullheads than in largemouth bass. Hepatic GST activity toward 1,2-dichroro-4-nitrobenzene, a mu-class GST substrate in rodents, was not detectable in either species. Liver cytosolic GSTs were more efficient in bullheads than in bass at catalyzing 1-chloro-2,4-dinitrobenzene-reduced glutathione (CDNB-GSH) conjugation over a broad range of electrophile (CDNB) concentrations, including those representative of environmental exposure. In contrast, largemouth bass maintained higher ambient concentrations of GSH, the nucleophilic cofactor for GST-mediated conjugation, than brown bullheads. Biphasic kinetics for GST-CDNB conjugation under conditions of variable GSH concentration were apparent in Eadie-Hofstee plots of the kinetic data, suggesting the presence of at least two hepatic GST isozymes with markedly different K-m values for GSH in both species. The GST-CDNB reaction rate data obtained under conditions of variable GSH were well fitted (R-2 = 0.999) by the two-enzyme Michaelis-Menten equation. In addition, Western blotting experiments confirmed the presence of two different hepatic GST-like proteins in both largemouth bass and brown bullhead liver. Collectively, these findings indicate that largemouth bass and brown bullhead GSTs catalyze the conjugation of structurally diverse, class-specific GST substrates, and that brown bullheads exhibit higher initial rates of GST activity than largemouth bass. The relatively higher rates of in vitro liver GST activity at the low substrate concentrations relevant to environmental exposure is expected to protect brown bullheads from the toxic effects of sediment-associated electrophilic chemicals. The somewhat lower rates of GST activity in largemouth bass liver compared with brown bullhead liver however, may be offset by maintenance of higher ambient hepatic GSH concentrations in largemouth bass.