Mitochondrial ascorbic acid prevents mitochondrial O 2 . − formation, an event critical for U 937 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 ( O 2 . − ) formation and inhibition of the activity of aconitase, aO 2 . −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 ofO 2 . −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 ofO 2 . −formation, including AA, and were only partially blunted by inhibitors of autophagy. As a final note, the toxic effects mediated byO 2 . −were entirely dependent on its conversion to H 2 O 2 . AA‐sensitive mitochondrialO 2 . −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, 42(2):190–200, 2016