EFFECTS OF TRANSFECTION WITH THE CU,ZN-SUPEROXIDE DISMUTASE GENE ON XANTHINE XANTHINE OXIDASE-INDUCED CYTOTOXICITY IN FIBROBLASTS FROM RAT SKIN

Purpose. The effects of transfection with the human Cu, Zn-superoxide dismutase (hSOD)(4) gene on active oxygen-induced cytotoxicity in rat skin fibroblasts (FR) were studied for the purpose of developing the n ovel delivery system of hSOD using hSOD gene. Methods. An expression p lasmid for hSOD, pRc/RSV-SOD, was constructed and used to transfect FR cells. Xanthine (X)/xanthine oxidase (XO) system were used to generat e active oxygen species. The effects of transfection with the hSOD gen e on active oxygen-induced cytotoxicity were assessed by comparing the number of surviving cells and the level of lipid peroxidation in host and transformants after exposure to X/XO system. Results. The cellula r SOD activity in RSV-SOD cells transfected with pRc/RSV-SOD was signi ficantly increased in comparison with host or RSV cells transfected wi th the pRc/RSV plasmid containing no hSOD gene as a control. Furthermo re, Western blot analysis using an anti-hSOD antibody indicated the pr oduction of hSOD in RSV-SOD cells. On the other hand, although the num bers of surviving cells in both host and RSV-SOD cultures after exposu re to X/XO system decreased in a time-dependent manner, the decrease i n number of surviving RSV SOD cells was less than that in host cells. In the presence of catalase, the decreases in number of surviving cell s in both host and RSV-SOD cultures after exposure to the X/XO system were also less than those in the absence of catalase. However, the dec reases in cell survival in RSV-SOD cultures were significantly less th an those in host cells in the presence of catalase. Furthermore, the l evels of lipid peroxidation in RSV-SOD cells exposed to the X/XO syste m in the presence or absence of catalase were lower than those in host cells. These results indicated that the increase in cellular SOD acti vity by transfection with the hSOD gene protects cells from oxidative stress. Conclusions. Human SOD gene therapy may be useful for treatmen t of diseases in which oxidative tissue damage is produced.