The effect of up- and downregulation of MnSOD enzyme on oxidative stress in human lens epithelial cells.

Abstract

Gene knockouts serve as useful experimental models to investigate the role of antioxidant enzymes in protection against oxidative stress in the lens. In the absence of gene knockout animals for Mn-containing superoxide dismutase (MnSOD), the effect of this enzyme on oxidative stress was investigated in a human lens epithelial cell line (SRA 01/04) in which the enzyme was up- or downregulated by transfection with sense and antisense expression vectors for MnSOD. Human lens epithelial cells (SRA 01/04) were transfected with plasmids containing sense and antisense human cDNA for MnSOD. The enzyme levels were determined by Western blot analysis and immunocytochemistry. The protective effect of the enzyme against the cytotoxicity of H(2)O(2) was evaluated by cell viability, DNA strand breaks, and morphologic changes observed by transmission electron microscopy. In addition, the protective effect of this enzyme against photochemically induced stress and UVB irradiation in cells was assessed by measuring the damage of cellular DNA. The MnSOD level in upregulated cells was three times higher than in downregulated cells, and the enzyme surrounded the nucleus. Cells with elevated enzyme levels were more resistant to the cytotoxic effect of H(2)O(2) with greater cell viability. MnSOD-deficient cells showed dramatic mitochondrial damage when exposed to 50 micro M H(2)O(2) for 1 hour. Similarly, oxidative challenge by H(2)O(2), photochemically generated reactive oxygen species, or UVB irradiation produced greater DNA strand breaks in MnSOD-deficient cells than in those in which the enzyme was upregulated. These findings demonstrate the protective effect of MnSOD in antioxidant defense of cultured lens epithelial cells. This approach to modulating the enzyme level in cultured cells provides a new experimental model for study of the role of antioxidant enzymes in the lens.