Effect of H(2)O(2)on human lens epithelial cells and the possible mechanism for oxidative damage repair by thioltransferase.
Human lens epithelial (HLE) B3 cells were used to study the oxidative damage and cellular repair with respect to the redox homeostasis, the oxidative defense enzymes and the glucose metabolic pathway. The effect of oxidative stress on cell growth was initially analyzed by culturing the cells with a bolus amount (0.02--0.1m M) of hydrogen peroxide (H(2)O(2)) in minimal essential medium (MEM) containing 20% fetal bovine serum (FBS) for 1 week. Concentration of H(2)O(2)greater than 0.03m M showed progressive inhibition of cell growth. However, the cells were also shown to tolerate H(2)O(2)concentrations up to 0.5m M by detoxifying the exogenous oxidant within 3hr without any detectable DNA damage. Therefore, this short-term H(2)O(2)exposure model was chosen to study the effect of oxidative stress on the cellular redox homeostasis. HLE B3 cells were first grown to confluence in MEM with 20% FBS. Approximately 1.6 million cells were gradually weaned off serum by subculturing in 2% FBS overnight, followed by serum-free medium for 30 min before subjecting to a bolus of 0.1m M H(2)O(2)for up to 180 min. These cells were used for biochemical analysis, which included H(2)O(2)detoxification (H(2)O(2)in the medium), glutathione (GSH) level and lactate production. Activity measurements were conducted on the oxidation defense enzymes: glutathione-S-transferase (GST), glutathione reductase (GR) and glutathione peroxidase (GPx); the dethiolating enzyme, thioltransferase (TTase); and a key glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (G-3PD). While the B3 cells were shown to tolerate and detoxify 0.1m M H(2)O(2)within 60 min, the GSH pool was transiently depleted in the first 60 min before fully recovered. GPx suffered more than 80% loss in activity and was unable to recover fully. GST showed slight inactivation but neither GR nor TTase was affected. G-3PD was inactivated to < 50% within 15 min of oxidative stress and was reactivated gradually to 80% of normal at the end of 180 min, concurrent with the transient loss of lactate production in the same cells. The reactivation of G-3PD was both temperature- and GSH-dependent, occurring only at physiological temperature and failing to reactivate when the intracellular GSH pool was depleted by BCNU (GR inhibitor) pretreatment. The inactivated cellular G-3PD in the cell extract could be partially reactivated by DTT (6m M) or by recombinant human lens thioltransferase (RHLT) but not by GSH (1m M), GR or GST. HLE cells cultured in the presence of L-(35)S-cystine and cycloheximide displayed an extra radiolabelled protein band on the autoradiograph in the H(2)O(2)treated cells. The labelled band was positively reacted with G-3PD antibody and could be removed by RHLT, indicating that S-thiolation of G-3PD occurred. The H(2)O(2)pre-exposed cells also transiently accumulated proteins modified by thiolation, including protein-S-S-glutathione (PSSG) and protein-S-S-cysteine (PSSC). It can be concluded that HLE could endure up to 0.1m M of H(2)O(2)oxidative stress since the cell could be protected by its effective repair systems, including dethiolating the inactivated key SH-sensitive enzymes. TTase may play a role in this. One of the mechanisms may be through preserving glucose metabolism and supplying ATP needed for maintaining cell viability.
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Center for Biotechnology and Department of Veterinary and Biomedical Sciences, University of Nebraska--Lincoln, Lincoln, NE 68583, USA.
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D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D004249:DNA Damage; D004305:Dose-Response Relationship, Drug; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D054477:Glutaredoxins; D005978:Glutathione / Q000378:metabolism; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D008658:Inactivation, Metabolic; D007908:Lens, Crystalline / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D018384:Oxidative Stress / Q000502:physiology*; D010088:Oxidoreductases / Q000502:physiology*; D011490:Protein Disulfide Reductase (Glutathione); D013696:Temperature
D054477:Glutaredoxins; D006861:Hydrogen Peroxide; D010088:Oxidoreductases; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D011490:Protein Disulfide Reductase (Glutathione); D005978:Glutathione; D005947:Glucose