Implication of mitochondrial hydrogen peroxide generation in ceramide-induced apoptosis

The key events implicated in ceramide-triggered apoptosis remain unknown. In this study we show that 25 mu M CG-ceramide induced significant H2O2, production within 60 min, which increased up to 180 min in human myeloid leukemia U937 cells. Inactive analogue dihydro-C6-ceramide had no effect, Furthermore, no H2O2 production was observed in CG-ceramide-treated U937 rho degrees cells, which are mitochondrial respiration-deficient. We also present evidence that ceramide-induced activation of the transcription factors NF-kappa B and AP-I is mediated by mitochondrial derived reactive oxygen species, Both H2O2 production, transcription factor activation as well as apoptosis could be inhibited by rotenone and thenoyltrifluoroacetone (specific mitochondrial complexes I and II inhibitors) and antioxidants, N-acetylcysteine and pyrrolidine dithiocarbamate. These effects could be potentiated by antimycin A (specific complex III mitochondrial inhibitor); H2O2 production was also inhibitable by ruthenium red, suggesting a role of mitochondrial calcium homeostasis alterations in ceramide-induced oxidative stress, Finally, CG-ceramide had no influence on mitochondrial membrane potential within the first 6 h. Altogether, our study points to reactive oxygen species, generated at the ubiquinone site of the mitochondrial respiratory chain, as an early major mediator in ceramide-induced apoptosis.