Activation of NFκB and MnSOD gene expression by free radical scavengers in human microvascular endothelial cells

The effect of nonprotein thiol (NPT) free radical scavengers WR-1065 (SH) and WR-33278 (SS), the active thiol and disulfide metabolites of amifostine, N-acetylcysteine (NAC; both L- and D- isomers), mesna, captopril, and dithiothreitol (DTT) on NF kappaB activation in human microvascular endothelial cells (HMEC) was investigated and contrasted to TNF alpha. The use of each of these NPTs at millimolar concentrations independent of oxidative damage-inducing agents resulted in a marked activation of NF kappaB, with the maximum effect observed between 30 min and 1 h after treatment. Only the SH and SS forms of amifostine, however. were effective in activating NF kappaB when administered at micromolar levels. Using a supershift assay, SH and SS equally affected the p50-p65 heterodimer, but not homodimers or heterodimers containing p52 or c-Rel subunits of NF kappaB. Neither catalase nor pyruvate when added to the culture medium to minimize hydrogen peroxide production had an effect on NF kappaB activation by SH. Thus, while oxidative damage is known to activate NF kappaB, the intracellular redox environment may also be affected by the addition of free radical scavenging agents such as NPT, and these in turn are capable of activating the redox sensitive transcription factor NF kappaB. There does not appear to be a significant role, if any, for the production of H2O2 as an intermediate step in the activation of NF kappaB by either the SH or the SS form of amifostine. Rather, the underlying mechanism of action, especially for the SS form, may be related to the close structural and functional similarities of these agents to polyamines, which have been reported to be capable of activating NF kappaB. In contrast to TNF alpha, exposure of cells to either 40 muM or 4 mM of SH for 30 min did not induce intercellular adhesion molecule-1 (ICAM-1) gene expression, but did increase manganese superoxide dismutase (MnSOD) gene expression. MnSOD expression rose by 2-fold and remained elevated from 4 to 22 h following SH exposure. (C) 2001 Elsevier Science Inc.