Transfection of HLE B-3 Cells with hGSTA1 or hGSTA2 Protects Against Hydrogen Peroxide and Naphthalene Induced Lipid Peroxidation and Apoptosis*

Abstract

Abstract: : Purpose: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 cells (HLE B-3). 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 1 (GPx-1) in GSH-dependent reduction of phospholipid hydroperoxide (PL-OOH) were quantitated through immnuoprecipitation 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 catalyzed the reduction of membrane PL-OOH in situ using the microidometric 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 Km and kcat values of purified 'total GSTs' toward phosphatidylcholine hydroperoxide (PC-OOH) were found to be 30 ± 4 µM and 1.95 ± 0.26 s-1, respectively. The Alpha class GSTs accounted for about 65% of the total GPx activity of HLE B-3 cells towards 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. Conclusion: 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.

Keywords: 323 apoptosis/cell death • 321 antioxidants • 458 lipids