TNFalpha 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 TNFalpha 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 TNFalpha are mediated by inhibition of complex III and by the ensuing formation of superoxides and hydrogen peroxide: (a) the effects of TNFalpha were mimicked by the complex III inhibitor antimycin A; (b) the effects of TNFalpha, 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 c(1) and therefore prevents ubisemiquinone formation; (c) the effects of TNFalpha, or antimycin A, were not observed in respiration-deficient cells; and (d) the effects of TNFalpha, or antimycin A, were sensitive to catalase. The TNFalpha-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 TNFalpha, (b) TNFalpha promoted the formation of ceramide via a mechanism sensitive to inhibition of sphingomyelinases by tricyclodecan-9-yl-xanthogenate and imipramine, and (c) the TNFalpha-mediated enhancement of the tert-butylhydroperoxide-induced DNA-damaging response was prevented under conditions in which ceramide formation was inhibited.