Abstract

Menadione (vitamin K-3,2-methyl-1,4-naphthoquinone), a redox cycling reagent, generates reactive oxygen intermediates and causes oxidative injury. The addition of menadione to Hep G2 cells produced a time- and concentration-dependent loss of cell viability. Preincubation of Hep G2 cells with low, nontoxic concentrations of menadione increased the viability of the cells against toxic doses of menadione or H₂O₂. Maximum protection was found with menadione concentrations of ∼3 μm and preincubation times of ∼45 min. This protective effect could be blocked by the protein synthesis inhibitor cycloheximide and by a variety of antioxidants. The transcription factor nuclear factor-κF (NF-κB) is known to be activated by many compounds, including reactive oxygen intermediates. Menadione activated NF-κB as determined by electrophoretic mobility shift assays. This activation was prevented by the same antioxidants that blocked protection against cytotoxicity produced by preincubation with menadione. Anti-p50 IgG prevented the menadione-stimulated binding of NF-κB to the oligonucleotide probe, whereas anti-p65 IgG produced a supershift of the NF-κB/oligonucleotide complex. Salicylate prevented the activation of NF-κB by menadione, and under these conditions, salicylate potentiated the cytotoxicity of menadione or H₂O₂. Transfection with a plasmid containing cDNA encoding mouse IκBβ, an inhibitor of NF-κB, resulted in increased toxicity by menadione. Furthermore, when protein kinase C was down-regulated by prolonged treatment with active phorbol ester (phorbol-12-myristate-13-acetate), the Hep G2 cells became more sensitive to menadione treatment. However, short term treatment with PMA, which activated NF-κB, resulted in protection against menadione cytotoxicity. Menadione cytotoxicity was enhanced when the Hep G2 cells were depleted of GSH. An increased level of GSH was observed after menadione pretreatment; this increase was blocked by salicylate, thereby linking the GSH increase to activation of NF-κB by menadione. The results of the current study suggest that menadione pretreatment protects Hep G2 cells from oxidative injury through an NF-κB-related mechanism, which may involve, in part, increased production of GSH.