Effects of hydrogen peroxide, nitric oxide and antioxidants on NF-κB.

Abstract

The NF-κB complex consists of a family of transcription factors which bind to specific sequences present in the regulatory regions of mammalian genes and in the human immunodeficiency virus (HIV) long terminal repeat. It has been suggested that free radicals may play a role in NF-κB activation and in the activation of HIV expression. The effects of H2O2 and nitric oxide (NO(•)) on NF-κB deoxyribonucleic acid (DNA) binding were examined using the electrophoretic mobility shift assay. When nuclear protein extracts containing NF-κB are treated with H2O2 in vitro, DNA binding to the κB consensus element is inhibited, although the NF-κB heterodimer remains intact. This inhibitory effect is concentration- and temperature-dependent and can be reversed by the reducing agent dithiothreitol (DTT). Co-incubation with reduced glutathione protects nuclear extracts from H2O2, while other antioxidants such as vitamin C and the chelators deferoxamine and diethyldithiocarbamate provide no such protection. The thiol blocker iodoacetate also inhibits DNA binding similar to H2O2, suggesting that protein thiols are involved. The nitric oxide generating compound diethylamine NONOate inhibits the binding of NF-κB to DNA in vitro. This DNA binding inhibition may also be due to an interaction with protein thiols, since it is also reversible with DTT. Thus, although H2O2 and NO(•) activate NF-κB in vivo, they inhibit DNA binding in a cell-free system. This paradox suggests the involvement of other factors in the activation of NF-κB mediated transcription. A better understanding of this process will aid in an understanding of the pathogenesis of acquired immune deficiency syndrome (AIDS).