Induction of glutathione S-transferase hGST 5.8 is an early response to oxidative stress in RPE cells.
OBJECTIVE
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 microM 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 microM H2O2 for 20 minutes showed a significant increase in TBARS (1.8-fold) and gamma-glutamyl cysteine synthetase (gamma-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 gamma-GCS activities are consistent 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 substrate for hGST 5.8.
Singhal
S S
SS
Department of Human Biological Chemistry and Genetics, The University of Texas Medical Branch, Galveston 77555, USA.
Godley
B F
BF
Chandra
A
A
Pandya
U
U
Jin
G F
GF
Saini
M K
MK
Awasthi
S
S
Awasthi
Y C
YC
D001555:Benzene Derivatives / Q000378:metabolism; D015153:Blotting, Western; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004790:Enzyme Induction; D005982:Glutathione Transferase / Q000096:biosynthesis*; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007527:Isoenzymes / Q000096:biosynthesis*; D015227:Lipid Peroxidation; D008297:Male; D018384:Oxidative Stress; D010088:Oxidoreductases / Q000378:metabolism; D010857:Pigment Epithelium of Eye / Q000187:drug effects / Q000201:enzymology*; D017392:Thiobarbituric Acid Reactive Substances / Q000378:metabolism
D001555:Benzene Derivatives; D007527:Isoenzymes; D017392:Thiobarbituric Acid Reactive Substances; D006861:Hydrogen Peroxide; D010088:Oxidoreductases; D005982:Glutathione Transferase; C007164:cumene hydroperoxide