The role of superoxide radical in TNF-alpha induced NF-kappa B activation

Electron spin resonance (ESR) spin trapping with 5-(diethoxyphosphoryl)-5-m ethyl-1-pyrroline N-oxide (DEPMPO) was utilized to investigate the generati on of oxygen free radicals from macrophages stimulated by tumor necrosis fa ctor-alpha (TNF-alpha). TNF-alpha stimulated macrophages generated hydroxyl (. OH) and superoxide anion (O-2(.-)) radicals. Incubation of TNF-alpha wi th macrophages resulted in an activation of DNA binding activity of the nuc lear transcription factor NF-kappa B. Superoxide dismutase (SOD), but not c atalase or sodium formate, inhibited this NF-kappa B activation, suggesting that O-2(.-) rather than H2O2 or . OH, radicals play the most critical rol e in this induction. beta-Nicotinamide adenine dinucleotide phosphate (NADP H) did not affect the NF-kappa B activation, while allopurinol, an inhibito r of xanthine oxidase, repressed it, suggesting that xanthine/xanthine oxid ase, and not NADPH dependent oxidase, may be a source of O-2(.-) radicals w hich induce NF-kappa B activation. O-2(.-) is generated via reduction of mo lecular oxygen by xanthine and xanthine oxidase, as demonstrated by the oxy gen consumption assay. The results indicate that TNF-alpha induces oxygen r adical generation from macrophages. O-2(.-) seems to play a key role in TNF -alpha-induced NF-kappa B activation macrophages. Xanthine and xanthine oxi dase appears to be a source of O-2(.-) radicals responsible for TNF-alpha-i nduced NF-kappa B activation.