Protection of HLE B-3 cells against hydrogen peroxide- and naphthalene-induced lipid peroxidation and apoptosis by transfection with hGSTA1 and hGSTA2

Yusong Yang, Rajendra Sharma, Ji Zhong Cheng, Manjit K. Saini, Naseem Ansari, Usha P. Andley, Sanjay Awasthi, Yogesh C. Awasthi

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Abstract

PURPOSE. To investigate the physiological role of two major α-class glutathione S-transferases (GSTs), hGSTAI-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 α-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 α-class GSTs and GPx-1. HLE B-3 cell membranes were prepared, peroxidized, and used to examine whether hGSTAI-1 and hGSTA2-2 catalyzes the reduction of membrane PL-OOH in situ using the microiodometric and spectrophotometric assays. The protective effects of the α-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 α and π class GSTs. The Michaelis-Menten constant (km) and turnover number (kcat) of purified total GSTs toward phosphatidylcholine hydroperoxide (PC-OOH) were found to be 30 ± 4 μM and 1.95 ± 0.26 seconds, respectively. The α-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 hGSTAI-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 α-class GSTs hGSTAI-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.

Original languageEnglish (US)
Pages (from-to)434-445
Number of pages12
JournalInvestigative Ophthalmology and Visual Science
Volume43
Issue number2
StatePublished - 2002

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Glutathione Transferase
Hydrogen Peroxide
Lipid Peroxidation
Transfection
Apoptosis
Lenses
Oxidative Stress
naphthalene
Membranes
Isoelectric Focusing
Glutathione Peroxidase
Affinity Chromatography
Immunoprecipitation
Caspase 3
Glutathione
Phospholipids
Complementary DNA
Antioxidants
Western Blotting
Epithelial Cells

ASJC Scopus subject areas

  • Ophthalmology

Cite this

Protection of HLE B-3 cells against hydrogen peroxide- and naphthalene-induced lipid peroxidation and apoptosis by transfection with hGSTA1 and hGSTA2. / Yang, Yusong; Sharma, Rajendra; Cheng, Ji Zhong; Saini, Manjit K.; Ansari, Naseem; Andley, Usha P.; Awasthi, Sanjay; Awasthi, Yogesh C.

In: Investigative Ophthalmology and Visual Science, Vol. 43, No. 2, 2002, p. 434-445.

Research output: Contribution to journalArticle

Yang, Yusong ; Sharma, Rajendra ; Cheng, Ji Zhong ; Saini, Manjit K. ; Ansari, Naseem ; Andley, Usha P. ; Awasthi, Sanjay ; Awasthi, Yogesh C. / Protection of HLE B-3 cells against hydrogen peroxide- and naphthalene-induced lipid peroxidation and apoptosis by transfection with hGSTA1 and hGSTA2. In: Investigative Ophthalmology and Visual Science. 2002 ; Vol. 43, No. 2. pp. 434-445.
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abstract = "PURPOSE. To investigate the physiological role of two major α-class glutathione S-transferases (GSTs), hGSTAI-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 α-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 α-class GSTs and GPx-1. HLE B-3 cell membranes were prepared, peroxidized, and used to examine whether hGSTAI-1 and hGSTA2-2 catalyzes the reduction of membrane PL-OOH in situ using the microiodometric and spectrophotometric assays. The protective effects of the α-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 α and π class GSTs. The Michaelis-Menten constant (km) and turnover number (kcat) of purified total GSTs toward phosphatidylcholine hydroperoxide (PC-OOH) were found to be 30 ± 4 μM and 1.95 ± 0.26 seconds, respectively. The α-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 hGSTAI-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 α-class GSTs hGSTAI-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.",
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T1 - Protection of HLE B-3 cells against hydrogen peroxide- and naphthalene-induced lipid peroxidation and apoptosis by transfection with hGSTA1 and hGSTA2

AU - Yang, Yusong

AU - Sharma, Rajendra

AU - Cheng, Ji Zhong

AU - Saini, Manjit K.

AU - Ansari, Naseem

AU - Andley, Usha P.

AU - Awasthi, Sanjay

AU - Awasthi, Yogesh C.

PY - 2002

Y1 - 2002

N2 - PURPOSE. To investigate the physiological role of two major α-class glutathione S-transferases (GSTs), hGSTAI-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 α-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 α-class GSTs and GPx-1. HLE B-3 cell membranes were prepared, peroxidized, and used to examine whether hGSTAI-1 and hGSTA2-2 catalyzes the reduction of membrane PL-OOH in situ using the microiodometric and spectrophotometric assays. The protective effects of the α-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 α and π class GSTs. The Michaelis-Menten constant (km) and turnover number (kcat) of purified total GSTs toward phosphatidylcholine hydroperoxide (PC-OOH) were found to be 30 ± 4 μM and 1.95 ± 0.26 seconds, respectively. The α-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 hGSTAI-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 α-class GSTs hGSTAI-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.

AB - PURPOSE. To investigate the physiological role of two major α-class glutathione S-transferases (GSTs), hGSTAI-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 α-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 α-class GSTs and GPx-1. HLE B-3 cell membranes were prepared, peroxidized, and used to examine whether hGSTAI-1 and hGSTA2-2 catalyzes the reduction of membrane PL-OOH in situ using the microiodometric and spectrophotometric assays. The protective effects of the α-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 α and π class GSTs. The Michaelis-Menten constant (km) and turnover number (kcat) of purified total GSTs toward phosphatidylcholine hydroperoxide (PC-OOH) were found to be 30 ± 4 μM and 1.95 ± 0.26 seconds, respectively. The α-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 hGSTAI-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 α-class GSTs hGSTAI-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.

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