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In this study, respiration-proficient (RP) and -deficient (RD) cells were exposed to 2.5 or 10 μM arsenite to generate superoxide (O2-.) respectively in the mitochondrial respiratory chain or via NADPH oxidase activation. These treatments, while causing similar, although mitochondrial permeability transition-dependent (RP-cells) or independent (RD-cells), delayed apoptosis, surprisingly generated identical kinetics and levels of dihydrorhodamine oxidation, indicative of O2-. formation. These similarities were attributable to the involvement of a common upstream event resulting in activation of the two O2-.-generating systems, and to intrinsic features of the cells. Both mechanisms required an initial and sequential mobilization of Ca2+ from the inositol-1,4,5-trisphosphate receptor and the ryanodine receptor (RyR), with however different implications. The close contacts existing between the RyR and the mitochondria created optimal conditions for the Ca2+ clearance, and the ensuing formation of O2-. in RP-cell mitochondria. Exposure to low concentrations of l-ascorbic acid (AA) transported by high affinity mechanisms in cells and mitochondria, suppressed O2-. formation. Much more Ca2+, and hence more arsenite, was necessary to promote NADPH oxidase activation in RD-cells, as a consequence of the cytosolic dilution and mitochondrial clearance of Ca2+. For the same reasons, an exposure to high concentrations of AA was required to suppress O2-. formation under these conditions.Low arsenite promotes efficient mitochondrial Ca2+ accumulation and O2-. formation.Low AA accumulates in RP-cell mitochondria and scavenges mitoO2-..High arsenite is required for efficient NADPH oxidase activation in RD-cells.High arsenite mobilizes large amounts of Ca2+ needed for NADPH oxidase activation.High AA is necessary to scavenge NADPH oxidase-derived O2-.