Mitochondrial ascorbic acid prevents mitochondrial O2.- formation, an event critical for U937 cell apoptosis induced by arsenite through both autophagic-dependent and independent mechanisms

A 16 h exposure of U937 cells to 2.5 µM arsenite promotes superoxide ( O2.-) formation and inhibition of the activity of aconitase, a O2.- sensitive enzyme. Both responses were abolished by the complex I inhibitor rotenone, or by the respiration-deficient phenotype. Interestingly, a similar suppressive effect was mediated by a short term pre-exposure to a low concentration of l-ascorbic acid (AA), previously shown to be actively taken up by the cells and by their mitochondria. The mitochondrial origin of O2.- was confirmed by fluorescence microscopy studies, whereas different approaches failed to detect a contribution of NADPH oxidase. Under similar conditions, arsenite induced autophagy as well as a decline in mitochondrial membrane potential resulting in delayed (48 h) apoptosis. Importantly, all these events turned out to be sensitive to treatments associated with prevention of O2.- formation, including AA, and were only partially blunted by inhibitors of autophagy. As a final note, the toxic effects mediated by O2.- were entirely dependent on its conversion to H2 O2 . AA-sensitive mitochondrial O2.- formation is therefore involved in autophagy and apoptosis induced by arsenite in U937 cells, although part of the lethal response appears mediated by an autophagy-independent mechanism. © 2016 BioFactors, 2016.