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Sharad S. Singhal, Bernard F. Godley, Animesh Chandra, Utpal Pandya, Gui-Fang Jin, Manjit K. Saini, Sanjay Awasthi, Yogesh C. Awasthi; Induction of Glutathione S-Transferase hGST 5.8 Is an Early Response to Oxidative Stress in RPE Cells. Invest. Ophthalmol. Vis. Sci. 1999;40(11):2652-2659.
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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) andγ
-glutamyl cysteine synthetase (γ-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 γ-GCS
activities are consistent with this idea, because GPX activity is also
expressed by hGST 5.8, and γ-GCS is the rate-limiting enzyme in
biosynthesis of GSH, the substrate for hGST
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