Mechanism whereby arsenite promotes mitochondrial superoxide formation: critical regulation by upstream events resulting in Ca2+ mobilization and mitochondrial accumulation

Arsenite is a widely distributed carcinogen and toxic compound that mediates various deleterious effects via the intermediate formation of reactive oxygen species (ROS). Recent work performed in our laboratory has demonstrated that prolonged (hours) exposure of human myeloid leukemia U937 cells to low micromolar concentrations of arsenite elicits Ca2+ release from the inositol-1,4,5-trisphosphate receptor (IP3R) and the crosstalk between this pool and the ryanodine receptor (RyR). The fraction of Ca2+ derived from the RyR was then critical to promote the mitochondrial accumulation of the cation and the ensuing mitochondrial superoxide (mitoO2-.) formation. It was interesting to observe that these time and concentration requirements were dramatically lowered under conditions in which the mitochondrial accumulation of Ca2+ was enforced by IP3 releasing or RyR agonists. Thus, very low concentrations of arsenite rapidly target the mitochondrial respiratory chain, with however significantly greater time and concentration requirements for Ca2+ mobilization from the ER. Based on the above findings, this second event appears critical and likely restricted to cell types concomitantly expressing the IP3R and a RyR. This notion was clearly established in studies showing that arsenite fails to promote mitochondrial Ca2+ accumulation and mitoO2. formation in cells uniquely expressing the IP3R. Arsenite instead elicited these events when cells were manipulated to gain expression of the RyR and lost these abilities under conditions in which cells expressing both Ca2+ channels were manipulated to downregulate the RyR. RyR expression therefore represents a critical requirement for mitochondrial Ca2+ accumulation and mitoO2 -. formation induced by arsenite. The second requirement is instead represented by the recruitment phase of this channel, apparently located in close apposition with the mitochondria. We found that, under the above conditions, arsenite causes an ER stress response associated with a threefold increase in ER oxidoreductin 1 (ERO1) levels in cells expressing both the IP3R and RyR. EN460, an inhibitor of ERO1, recapitulated all the effects associated with RyR inhibition or downregulation, including prevention of RyR-induced Ca2+ accumulation in mitochondria and the resulting mitoO2 -. formation. Moreover, ERO1 knockout cells responded to arsenite as their wild type counterpart supplemented with EN460. As a final note, arsenite enhanced the expression of ERO1 via a mechanism mediated by Ca2+ release from both the IP3R and RyR. We therefore conclude that arsenite activates a positive feedback amplification cycle between Ca2+ levels and ERO1 in the ER, by which IP3R-dependent Ca2+ induces ERO1 and ERO1 promotes Ca2+ release via RyR, thereby amplifying the initial Ca2+ load and causing the mitochondrial accumulation of the cation, critical for mitoO2 -.formation.