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Induction of Glutathione S-Transferase hGST 5.8 Is an Early Response to Oxidative Stress in RPE Cells

From the Departments of ¹ Human Biological Chemistry and Genetics, ² Internal Medicine, and ³ Ophthalmology and Visual Sciences, The University of Texas Medical Branch, Galveston.

PURPOSE. To delineate the role of the glutathione S-transferase (GST) isozyme hGST 5.8 in protection mechanisms against oxidative stress, the effect of low-level transient exposure of H₂O₂ to retinal pigmented epithelial (RPE) cells on hGST 5.8 and other enzymes involved in defense against oxidative stress was examined.

METHODS. Cultured human RPE cells were exposed to 50 µM H₂O₂ for 20 minutes. Subsequently, the cells were washed and resuspended in the culture media. The cells were pelleted and lysed, and the levels of lipid peroxidation products including thiobarbituric acid–reactive substances (TBARS), glutathione (GSH), glutathione peroxidase (GPX), glucose 6-phosphate dehydrogenase, glutathione reductase, GST, catalase (CAT), and superoxide dismutase (SOD) were determined and compared with levels in control cells. Total GSTs were purified by GSH-affinity chromatography, and the isozymes were separated by isoelectric focusing, characterized, and quantitated. hGST 5.8 was quantitated by an immunologic method as well as by determining activity toward its preferred substrate, 4-hydroxynonenal (4-HNE). Kinetic constants of hGST 5.8 purified from H₂O₂-treated cells were also determined and compared with those of control cells.

RESULTS. Exposure of RPE cells to 50 µM H₂O₂ for 20 minutes showed a significant increase in TBARS (1.8-fold) and -glutamyl cysteine synthetase (-GCS) activity (1.6-fold). A significant increase (1.2-fold) was also observed in GPX activity toward cumene hydroperoxide, but CAT and SOD activities remained unchanged. There was no significant increase in GST activity toward 1-chloro-2, 4-dinitrobenzene but GST activity toward 4-HNE was increased by 1.4- to 1.8-fold. The increase in GST activity toward 4-HNE was associated with a 2.8-fold increase in protein of the isozyme hGST 5.8, which uses 4-HNE as the preferred substrate.

CONCLUSIONS. Results of these studies show that the induction of hGST 5.8, which is involved in the detoxification of the lipid peroxidation products 4-HNE and hydroperoxides, may be an early adaptive response of RPE cells exposed to low levels of transient oxidative stress. It is suggested that this isozyme may be crucial for protecting the RPE from low levels of chronic oxidative stress. Observed increases in GPX and -GCS activities are consistent with this idea, because GPX activity is also expressed by hGST 5.8, and -GCS is the rate-limiting enzyme in biosynthesis of GSH, the substrate for hGST 5.8.

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