Crosstalk between ERO1α and ryanodine receptor in arsenite-dependent mitochondrial ROS formation.

Arsenite, a well-established human carcinogen and toxic compound, promotes the formation of mitochondrial superoxide (mitoO2-) via a Ca2+-dependent mechanism, in which an initial stimulation of the inositol 1, 4, 5-trisphosphate receptor (IP3R) is followed by the activation of the ryanodine receptor (RyR), critical for providing Ca2+ to the mitochondria. We now report that, under the same conditions, arsenite triggers endoplasmic reticulum (ER) stress and a threefold increase in ER oxidoreductin 1α (ERO1 α) levels in proliferating U937 cells. EN460, an inhibitor of ERO1 α, recapitulated all the effects associated with RyR inhibition or downregulation, including prevention of RyR-induced Ca2+ accumulation in mitochondria and the resulting O2-. formation. Quantitatively similar results were obtained in inhibitor studies performed in terminally differentiated wild type C2C12 cells. Moreover, ERO1 α knockout C2C12 myotubes responded to arsenite as their wild type counterpart supplemented with EN460. As a final note, arsenite enhanced the expression of ERO1 α 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 α in the ER, by which IP3R-dependent Ca2+ induces ERO1 α and ERO1 α promotes Ca2+ release via RyR, thereby amplifying the initial Ca2+ load and causing the mitochondrial accumulation of the cation, critical for mitoO2- formation.

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 U937 cells, a versatile promonocytic cellular system that, based on its endoplasmic reticulum (ER)/mitochondria functional relationships, responds 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, which is followed by Ca2+-dependent mitochondrial O2 -. (mitoO2 -.) formation. These events, which were barely detectable after 3 hours, were better appreciated at 6 hours. We found that markedly shorter exposure to arsenite 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 minutes of exposure, and this response was uniquely dependent on the enforced mitochondrial Ca2+ accumulation. The dramatic anticipation of and sensitization to the effects of arsenite caused by the IP3R or RyR agonists were 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 respiration-proficient cells, arsenite causes mitochondrial Ca2+ accumulation and Ca2+-dependent mitochondrial superoxide formation. We now report that the second event requires remarkably lower concentrations of and 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 mitochondrial Ca2+ concentration ([Ca2+]m) was increased by inositol-1,4,5-trisphosphate receptor or ryanodine receptor agonists. Hence, specific substances or conditions enhancing the [Ca2+]m may potentiate the deleterious effects of arsenite by selectively increasing mitochondrial superoxide formation.

Bracciano's Lake waters: an experimental survey on the surface layer pollution.

The chemical and trophic conditions of the surface waters of the lake Bracciano, near Roma, have been investigated all through the year 2005. Electrical conductivity, pH, alkalinity, hardness, total nitrogen, phosphorus content, chloride, fluoride and sulphate ions were measured. The results were compared with similar analyses formerly carried out by different authors, to achieve a right evaluation of the actual quality of the surface waters of the lake Bracciano all over the year 2005.