Effects of transfection with the Cu, Zn-superoxide dismutase gene on xanthine/xanthine oxidase-induced cytotoxicity in fibroblasts from rat skin.
OBJECTIVE
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 novel delivery system of hSOD using hSOD gene.


METHODS
An expression plasmid for hSOD, pRc/RSV-SOD, was constructed and used to transfect FR cells. Xanthine (X)/xanthine oxidase (XO) system were used to generate active oxygen species. The effects of transfection with the hSOD gene 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 cellular SOD activity in RSV-SOD cells transfected with pRc/RSV-SOD was significantly increased in comparison with host or RSV cells transfected with the pRc/RSV plasmid containing no hSOD gene as a control. Furthermore, Western blot analysis using an anti-hSOD antibody indicated the production of hSOD in RSV-SOD cells. On the other hand, although the numbers of surviving cells in both host and RSV-SOD cultures after exposure to X/XO system decreased in a time-dependent manner, the decrease in number of surviving RSV-SOD cells was less than that in host cells. In the presence of catalase, the decreases in number of surviving cells 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 decreases in cell survival in RSV-SOD cultures were significantly less than those in host cells in the presence of catalase. Furthermore, the levels of lipid peroxidation in RSV-SOD cells exposed to the X/XO system in the presence or absence of catalase were lower than those in host cells. These results indicated that the increase in cellular SOD activity by transfection with the hSOD gene protects cells from oxidative stress.


CONCLUSIONS
Human SOD gene therapy may be useful for treatment of diseases in which oxidative tissue damage is produced.
Nishiguchi
K
K
Department of Hospital Pharmacy, School of Medicine, Kobe University, Japan.
Ishida
K
K
Nakajima
M
M
Maeda
T
T
Komada
F
F
Iwakawa
S
S
Tanigawara
Y
Y
Okumura
K
K
D000704:Analysis of Variance; D000818:Animals; D001358:Avian Sarcoma Viruses / Q000235:genetics*; D001483:Base Sequence; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects* / Q000235:genetics; D015316:Genetic Therapy; D006801:Humans; D008969:Molecular Sequence Data; D018384:Oxidative Stress; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics; D051381:Rats; D012867:Skin / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D013482:Superoxide Dismutase / Q000235:genetics* / Q000378:metabolism; D017392:Thiobarbituric Acid Reactive Substances / Q000032:analysis; D014162:Transfection; D019820:Xanthine; D014969:Xanthine Oxidase / Q000633:toxicity*; D014970:Xanthines / Q000633:toxicity*
D018076:DNA, Complementary; D017392:Thiobarbituric Acid Reactive Substances; D014970:Xanthines; D019820:Xanthine; D013482:Superoxide Dismutase; D014969:Xanthine Oxidase