Protection of HLE B-3 cells against hydrogen peroxide- and naphthalene-induced lipid peroxidation and apoptosis by transfection with hGSTA1 and hGSTA2.
OBJECTIVE
To investigate the physiological role of two major alpha-class glutathione S-transferases (GSTs), hGSTA1-1 and hGSTA2-2 in protection against oxidative stress and lipid peroxidation (LPO) in human lens epithelial (HLE B-3) cells.


METHODS
Total GSTs were purified from HLE B-3 cells by glutathione (GSH)-affinity chromatography and characterized by Western blot analysis, isoelectric focusing, and kinetic studies. The relative contributions of the alpha-class GSTs and the Se-dependent glutathione peroxidase (GPx)-1 in GSH-dependent reduction of phospholipid hydroperoxide (PL-OOH) were quantitated through immunoprecipitation studies using separately the specific polyclonal antibodies against human alpha-class GSTs and GPx-1. HLE B-3 cell membranes were prepared, peroxidized, and used to examine whether hGSTA1-1 and hGSTA2-2 catalyzes the reduction of membrane PL-OOH in situ using the microiodometric and spectrophotometric assays. The protective effects of the alpha-class GSTs against H2O2- and naphthalene-induced LPO and apoptosis were examined by transfecting HLE B-3 cells with cDNAs of hGSTA1 and hGSTA2. RESULTS. HLE B-3 cells expressed only the alpha and pi class GSTs. The Michaelis-Menten constant (k(m)) and turnover number (k(cat)) of purified total GSTs toward phosphatidylcholine hydroperoxide (PC-OOH) were found to be 30 +/- 4 microM and 1.95 +/- 0.26 seconds, respectively. The alpha-class GSTs accounted for approximately 65% of the total GPx activity of HLE B-3 cells toward PC-OOH. Our results demonstrate for the first time that hGSTA1-1 and hGSTA2-2 effectively catalyzed GSH-dependent reduction of membrane PL-OOH in situ in HLE B-3 cells. Transfection with hGSTA1 or hGSTA2 protected these cells from H2O2- and naphthalene-induced LPO and attenuated H2O2- and naphthalene-induced apoptosis through inhibiting caspase 3 activation.


CONCLUSIONS
These results demonstrate that the alpha-class GSTs hGSTA1-1 and hGSTA2-2 play a major role as antioxidant enzymes and are the main determinants of the levels of LPO caused by oxidative stress in human lens epithelial cells.
Yang
Yusong
Y
Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, Texas 77555-1067, USA.
Sharma
Rajendra
R
Cheng
Ji-Zhong
JZ
Saini
Manjit K
MK
Ansari
Naseem H
NH
Andley
Usha P
UP
Awasthi
Sanjay
S
Awasthi
Yogesh C
YC
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002478:Cells, Cultured; D019610:Cytoprotection; D004847:Epithelial Cells / Q000187:drug effects / Q000201:enzymology* / Q000473:pathology; D005979:Glutathione Peroxidase / Q000378:metabolism; D005982:Glutathione Transferase / Q000302:isolation & purification / Q000502:physiology*; D006801:Humans; D006861:Hydrogen Peroxide / Q000633:toxicity*; D007527:Isoenzymes / Q000302:isolation & purification / Q000502:physiology*; D007908:Lens, Crystalline / Q000187:drug effects / Q000201:enzymology* / Q000473:pathology; D015227:Lipid Peroxidation / Q000187:drug effects*; D008054:Lipid Peroxides / Q000378:metabolism; D008315:Malondialdehyde / Q000378:metabolism; D009281:Naphthalenes / Q000633:toxicity*; D018384:Oxidative Stress; D011233:Precipitin Tests; D011817:Rabbits; D014162:Transfection
D007527:Isoenzymes; D008054:Lipid Peroxides; D009281:Naphthalenes; C031721:naphthalene; D008315:Malondialdehyde; D006861:Hydrogen Peroxide; D005979:Glutathione Peroxidase; D005982:Glutathione Transferase; C119995:glutathione S-transferase alpha; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases