The role of superoxide radical in TNF-α induced NF-κB activation

Electron spin resonance (ESR) spin trapping with 5- (diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO) was utilized to investigate the generation of oxygen free radicals from macrophages stimulated by tumor necrosis factor-α (TNF-α). TNF-α stimulated macrophages generated hydroxyl (·OH) and superoxide anion (O₂^.-) radicals. Incubation of TNF-α with macrophages resulted in an activation of DNA binding activity of the nuclear transcription factor NF-κB. Superoxide dismutase (SOD), but not catalase or sodium formate, inhibited this NF-κB activation, suggesting that O₂^.- rather than H₂O₂ or ·OH, radicals play the most critical role in this induction. β-Nicotinamide adenine dinucleotide phosphate (NADPH) did not affect the NF-κB activation, while allopurinol, an inhibitor of xanthine oxidase, repressed it, suggesting that xanthine/xanthine oxidase, and not NADPH dependent oxidase, may be a source of O₂^.- radicals which induce NF-κB activation. O₂^.- is generated via reduction of molecular oxygen by xanthine and xanthine oxidase, as demonstrated by the oxygen consumption assay. The results indicate that TNF- α induces oxygen radical generation from macrophages. O₂^.- seems to play a key role in TNF-α-induced NF-κB activation in macrophages. Xanthine and xanthine oxidase appears to be a source of O₂^.- radicals responsible for TNF-α-induced NF-κB activation.