MENADIONE CYTOTOXICITY TO HEP G2 CELLS AND PROTECTION BY ACTIVATION OF NUCLEAR FACTOR-KAPPA-B

Menadione (vitamin K-3,2-methyl-1,4-naphthoquinone), a redox cycling r eagent, generates reactive oxygen intermediates and causes oxidative i njury. The addition of menadione to Hep G2 cells produced a time-and c oncentration-dependent loss of cell viability. Preincubation of Hep G2 cells with low, nontoxic concentrations of menadione increased the vi ability of the cells against toxic doses of menadione or H2O2. Maximum protection was found with menadione concentrations of similar to 3 mu M and preincubation times of similar to 45 min. This protective effec t could be blocked by the protein synthesis inhibitor cycloheximide an d by a variety of antioxidants. The transcription factor nuclear facto r-kappa F (NF-kappa B) is known to be activated by many compounds, inc luding reactive oxygen intermediates. Menadione activated NF-kappa B a s determined by electrophoretic mobility shift assays. This activation was prevented by the same antioxidants that blocked protection agains t cytotoxicity produced by preincubation with menadione. Anti-p50 IgG prevented the menadione-stimulated binding of NF-kappa B to the oligon ucleotide probe, whereas anti-p65 IgG produced a supershift of the NF- kappa B/oligonucleotide complex. Salicylate prevented the activation o f NF-kappa B by menadione, and under these conditions, salicylate pote ntiated the cytotoxicity of menadione or H2O2. Transfection with a pla smid containing cDNA encoding mouse I kappa B beta, an inhibitor of NF -kappa B, resulted in increased toxicity by menadione. Furthermore, wh en protein kinase C was down-regulated by prolonged treatment with act ive phorbol eater (phorbol-12-myristate-13-acetate), the Hep G2 cells became more sensitive to menadione treatment. However, short term trea tment with PMA, which activated NF-kappa B, resulted in protection aga inst menadione cytotoxicity. Menadione cytotoxicity was enhanced when the Hep G2 cells were depleted of GSH. An increased level of GSH was o bserved after menadione pretreatment; this increase was blocked by sal icylate, thereby linking the GSH increase to activation of NF-kappa B by menadione. The results of the current study suggest that menadione pretreatment protects Hep G2 cells from oxidative injury through an NF -kappa B-related mechanism, which may involve, in part, increased prod uction of GSH.