TNFα Enhances the DNA Single-Strand Breakage Induced by the Short-Chain Lipid Hydroperoxide Analogue tert-Butylhydroperoxide via Ceramide-Dependent Inhibition of Complex III Followed by Enforced Superoxide and Hydrogen Peroxide Formation

Treatment of U937 cells with nontoxic concentrations of TNFα increased the DNA strand scission induced by a short-chain lipid hydroperoxide analogue, tert-butylhydroperoxide. The following lines of evidence suggest that the enhancing effects of TNFα are mediated by inhibition of complex III and by the ensuing formation of superoxides and hydrogen peroxide: (a) the effects of TNFα were mimicked by the complex III inhibitor antimycin A; (b) the effects of TNFα, or antimycin A, were abolished by the complex I inhibitor rotenone, or by myxothiazol, an agent which inhibits the electron flow from the reduced coenzyme Q to cytochrome c1 and therefore prevents ubisemiquinone formation; (c) the effects of TNFα, or antimycin A, were not observed in respiration-deficient cells; and (d) the effects of TNFα, or antimycin A, were sensitive to catalase. The TNFα-dependent inhibition of complex III appears to be mediated by ceramide. Three lines of evidence support this inference: (a) a synthetic cell-permeable ceramide analogue reproduced all the effects of TNFα, (b) TNFα promoted the formation of ceramide via a mechanism sensitive to inhibition of sphingomyelinases by tricyclodecan-9-yl-xanthogenate and imipramine, and (c) the TNFα-mediated enhancement of the tert-butylhydroperoxide-induced DNA-damaging response was prevented under conditions in which ceramide formation was inhibited.