Arsenite-induced mitochondrial superoxide formation: time and concentration requirements for the effects of the metalloid on the endoplasmic reticulum and mitochondria

The present study used human myeloid leukemia cells, a versatile promonocytic cellular system which, based on its endoplasmic reticulum (ER)/mitochondria functional relationships, respond to low micromolar concentrations of arsenite with a single, defined mechanism of superoxide (O2-.) formation. Under these conditions, we observe an initial Ca2+ mobilization from the ER associated with the mitochondrial accumulation of the cation, followed by Ca2+-dependent mitochondrial O2-. (mitoO2-.) formation. These events, barely detectable after 3 h were better appreciated at 6 h. We found that remarkably shorter exposure to, and lower concentrations of, arsenite are required to induce extensive O2-. formation in cells supplemented with inositol-1,4,5-trisphosphate receptor (IP3R) or ryanodine receptor (RyR) agonists. Indeed, nanomolar arsenite induced maximal O2-. formation after only 10 min of exposure, and this response was uniquely dependent on the enforced mitochondrial Ca2+ accumulation. The dramatic anticipation of, sensitization to, the effects of arsenite caused by the IP3R or RyR agonists was accompanied by a parallel significant genotoxic response in the absence of detectable mitochondrial dysfunction and cytotoxicity. We conclude that the prolonged, low micromolar arsenite exposure paradigm resulting in mitoO2-. formation is necessary to affect Ca2+ homeostasis and accumulate the cation in mitochondria. The arsenite requirements to promote mitoO2-. formation in the presence of sufficient mitochondrial Ca2+ were instead remarkably lower in terms of both concentration and time of exposure. These conditions were associated with the induction of extensive DNA strand scission in the absence of detectable signs of toxicity. SIGNIFICANCE STATEMENT: In RP-cells, arsenite causes mitochondrial Ca2+ accumulation ([Ca2+]m) and Ca2+-dependent mitochondrial superoxide formation. We now report that the second event requires remarkably lower concentrations of /time of exposure to the metalloid than the former. Indeed, a brief exposure to nanomolar levels of arsenite produced maximal effects under conditions in which the [Ca2+]m was increased by IP3R or RyR agonists. Hence, specific substances or conditions enhancing the [Ca2+]m, may potentiate the deleterious effects of arsenite by selectively increasing mitochondrial superoxide formation.