Induction of glutathione S-transferase hGST 5.8 is an early response to oxidative stress in RPE cells

PURPOSE. To delineate the role of the glutathione S-transferase (GST) isozy me hGST 5.8 ill protection mechanisms against oxidative stress, the effect of low-level transient exposure of H2O2 to retinal pigmented epithelial (RP E) cells on hGST 5.8 and other enzymes involved in defense against oxidativ e stress was examined. METHODS. Cultured human RPE cells were exposed to 50 mu M H2O2 for 20 minut es. Subsequently, the cells were washed and resuspended in the culture medi a. The cells were pelleted and lysed, and the levels of Lipid peroxidation products including thiobarbituric acid-reactive substances (TBARS), glutath ione (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 wer e purified by GSH-affinity chromatography, and the isozymes were separated by isoelectric focusing, characterized, and quantitated, hGST 5.8 was quant itated by an immunologic method as well as by determining activity toward i ts preferred substrate, 4-hydroxynonenal (4-HNE). Kinetic constants of hGST 5.8 purified from H2O2-treated cells were also determined and compared wit h those of control cells. RESULTS. Exposure of RPE cells to 50 mu M H2O2 for 20 minutes showed a sign ificant increase in TEARS (1.8-fold) and gamma-glutamyl cysteine synthetase (gamma-GCS) activity (1.6-fold), A significant increase (1.2-fold) was als o observed in GPX activity toward cumene hydroperoxide, but CAT an SOD acti vities remained unchanged. There was no significant increase in GST activit y toward 1-chloro-2, 4-dinitrobenzene but GST activity toward 4-HNE was inc reased by 1.4- to 1.8-fold. The increase in GST activity toward 4-HNE was a ssociated with a 2.8-fold increase in protein of the isozyme hGST 5.8, whic h 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 ex posed to low levels of transient oxidative stress. It is suggested that thi s isozyme may be crucial for protecting the RPE from low levels of chronic oxidative stress. Observed increases in GPX and gamma-GCS activities are co nsistent with this idea, because GPX activity is also expressed by hGST 5.8 , and gamma-GCS is the rate-limiting enzyme in biosynthesis of GSH, the sub strate for hGST 5.8.