Peroxide toxicity in conditioned lens epithelial cells--evaluation of multi-defense systems

Immortal murine lens epithelial cells which were conditioned to survive peroxide stress were found to have a remarkable increase in catalase activity as well as lesser changes in a number of other antioxidative defense systems [Invest. Ophthalmol. Vis. Sci. 43 (2002) 3251]. Furthermore, the gene expression of hundreds of other genes was altered. In order to determine the relative importance of catalase, other enzyme systems which maintain the reducing environment of the cell and the involvement of Fenton chemistry, an analysis of the effect of inhibiting catalase, disruption of the cells' reducing environment by inhibition of GSSG reductase (GR) and chelation of metal ion was investigated. It was found that inhibition of catalase caused peroxide resistant cells to die within 48-72 hr when exposed to normally tolerated concentrations of peroxide. If 1,10-phenanthroline (OP), an effective metal ion chelator was present, the cells were not affected by catalase inhibition and survived peroxide stress. Peroxide vulnerable unconditioned control cells were similarly protected by the chelator. The results demonstrate that H2O2 itself has minimal toxicity and that it is the products resulting from interaction with metal ion that produces lethal toxicity. In stark contrast, however, metal chelation did not protect the cells when GR was inhibited by BCNU. Examination of non-protein thiol (NP-SH), which is primarily GSH, indicated that rapid and extensive oxidation occurred almost immediately after exposure to peroxide under all conditions. However, NP-SH returns to the normal range in the conditioned cells even though later cell death is observed in some cases, suggesting fatal damage during the period when the cell is exposed to an oxidizing environment. Examination of DNA damage by alkaline elution indicated that H2O2 caused little observed strand breakage in peroxide resistant cells even if catalase is inhibited, suggesting that such cells have developed other systems to protect DNA and that H2O2 induced death is probably not related to DNA single strand breaks. In contrast, unconditioned cells (C cells) show extensive H2O2 induced DNA damage which is prevented by OP. Thus, depending on the conditions, DNA damage may contribute to cell death. The overall results indicate that the conditioned cell lines are not simply dependent on catalase activity but have developed a complex defense which includes GSH dependent systems and possibly more effective regulation of metal ion concentrations to resist oxidative stress.