High concentrations of peroxynitrite elicit delayed formation of DNA-damaging species through a mechanism dependent on mitochondrial Ca2+ accumulation and inhibition of complex III. A second mechanism, requiring remarkably lower peroxynitrite concentrations, is observed in the presence of bona fide complex III inhibitors and is Ca2+ independent. We now report evidence for a third mechanism, also operative with low peroxynitrite concentrations, independent of electron transport, and entirely based on mitochondrial Ca2+ accumulation. This concept was established by using permeabilized respiration-proficient and-deficient U937 cells supplemented with Ca2+, inhibitors of mitochondrial Ca2+ accumulation, and specific respiratory-chain inhibitors. The results obtained were validated by experiments performed with intact cells, by using caffeine (Cf) to promote mitochondrial Ca2+ accumulation. Under these conditions, low concentrations of peroxynitrite, otherwise unable to generate detectable DNA cleavage, caused maximal DNA strand scission through a mechanism insensitive to respiratory-chain inhibitors or to the respiration-deficient phenotype. The effects of Cf were mimicked by other ryanodine receptor agonists, were suppressed by ryanodine, and were not observed in cells failing to express the ryanodine receptor, as differentiated U937 cells or human monocytes. This study provides evidence for a novel mechanism whereby peroxynitrite may indirectly mediate DNA strand scission under inflammatory conditions.
A novel mechanism, uniquely dependent on mitochondrial calcium accumulation, whereby peroxynitrite promotes formation of superoxide/hydrogen peroxide and the ensuing strand scission of genomic DNA
GUIDARELLI, ANDREA;FIORANI, MARA;AZZOLINI, CATIA;CANTONI, ORAZIO
2010
Abstract
High concentrations of peroxynitrite elicit delayed formation of DNA-damaging species through a mechanism dependent on mitochondrial Ca2+ accumulation and inhibition of complex III. A second mechanism, requiring remarkably lower peroxynitrite concentrations, is observed in the presence of bona fide complex III inhibitors and is Ca2+ independent. We now report evidence for a third mechanism, also operative with low peroxynitrite concentrations, independent of electron transport, and entirely based on mitochondrial Ca2+ accumulation. This concept was established by using permeabilized respiration-proficient and-deficient U937 cells supplemented with Ca2+, inhibitors of mitochondrial Ca2+ accumulation, and specific respiratory-chain inhibitors. The results obtained were validated by experiments performed with intact cells, by using caffeine (Cf) to promote mitochondrial Ca2+ accumulation. Under these conditions, low concentrations of peroxynitrite, otherwise unable to generate detectable DNA cleavage, caused maximal DNA strand scission through a mechanism insensitive to respiratory-chain inhibitors or to the respiration-deficient phenotype. The effects of Cf were mimicked by other ryanodine receptor agonists, were suppressed by ryanodine, and were not observed in cells failing to express the ryanodine receptor, as differentiated U937 cells or human monocytes. This study provides evidence for a novel mechanism whereby peroxynitrite may indirectly mediate DNA strand scission under inflammatory conditions.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
