M. Pérez López

Glutathione S-transferases from rainbow trout liver and freshly isolated hepatocytes: purification and characterization

Glutathione S-transferases (GST) form an important family of biotransformation enzymes catalyzing the conjugation of glutathione to a great variety of xenobiotic compounds. The objective of this study was to compare the different characteristics of GST from freshly isolated rainbow trout hepatocytes with those corresponding to the total liver of the same fish, in order to establish the similarities. GST was purified by affinity chromatography and enzymatic activity was determined towards two substrates, 1-chloro-2,4-dinitrobenzene (CDNB) and ethacrynic acid (ETHA). The different isoenzymes were determined by HPLC associated with SDS-PAGE. Slight differences between the samples were obtained when the results corresponding to the enzyme activity were compared. HPLC results showed that all GST isoforms present in the total liver samples were represented in the isolated cells too, corresponding to isoforms with molecular masses of approximately 25.5 and 23.0 kDa.

Induction of cytosolic Glutathione S-transferases from Atlantic eel (Anguilla anguilla) after intraperitoneal treatment with polychlorinated biphenyls

Glutathione S-transferases (GST) are phase II biotransformation enzymes influenced by exposure to foreign compounds and are proposed as biomarkers of environmental pollution. The hepatic and renal cytosolic GST isoenzymes from juvenile Atlantic eels were isolated and partially characterized. In both organs, three different GST isoenzymes were purified using HPLC and the final subcellular purified fraction resulted in enzymatic subunits of approximately 24 500 Da. Total GST activity was significantly higher towards the substrate 1-chloro-2,4-dinitrobenzene (CDNB) than the ethacrynic acid (ETHA). Induction of isoenzymes of GST was studied after intraperitoneal treatment with a single dose (25 mg/kg bw) of a polychlorinated biphenyls mixture. In the hepatic cytosol, an inductory effect on the major enzymatic isoform was observed when comparing treated with control eels. This was directly related to statistically significant (P<0.05) differences in total GST activity toward the CDNB between treated and control groups, with an increase of 33.6% of GST total activity measured in the last subcellular purified samples. In the kidney cytosol, PCB treatment did not affect GST activity. Significant differences were observed when analyzing the effect of PCB treatment on the main GST subunit height (retention time of 27 min). These results indicate that hepatic GST profiles could be considered as a good tool in environmental studies as specific biomarkers of contamination.