Intracellular ascorbic acid enhances the DNA single-strand breakage and toxicity induced by peroxynitrite in U937 cells

Abstract

A well-established protocol to increase the intracellular content of ascorbic acid was used to investigate the effects of the vitamin on DNA single-strand breakage and toxicity mediated by authentic peroxynitrite (ONOO-) in U937 cells. This protocol involved exposure for 60 min to 100 muM dehydroascorbic acid, which was taken up by the cells and converted into ascorbic acid via a GSH-independent mechanism. At the time of exposure to ONOO-, which was performed in fresh saline immediately after loading with dehydroascorbic acid, the vitamin present in the cells was all in its reduced form. It was found that, in cells that are otherwise ascorbate-deficient, an increase in their ascorbic acid content does not prevent, but rather enhances, the DNA-damaging and lethal responses mediated by exogenous ONOO-. These results therefore suggest that acute supplementation of ascorbic acid can be detrimental for individuals with pathologies associated with a decrease in ascorbic acid and in which ONOO- is known to promote deleterious effects

Mitochondrial ascorbic acid is responsible for enhanced susceptibility of U937 cells to the toxic effects of peroxynitrite

“…DHA mitochondrial uptake through hexose transporters, however, most likely also takes place in our cells and becomes significant at greater extracellular concentrations. Preliminary results indicate that this is indeed the case (data not shown), in agreement with the very old data from our laboratory [35,36], showing that DHA, at nonphysiological concentrations (e.g., 100 mM), reproduces the enhancing effects of peroxynitrite herein obtained with AA.…”

Ascorbate-mediated enhancement of reactive oxygen species generation from polymorphonuclear leukocytes: modulatory effect of nitric oxide

“…ONOO .-, a powerful oxidant, reacts with protein and nonprotein thiol residues [45], inhibits some enzymes of the mitochondrial electron transport chain [46], promotes lipid peroxidation [47], and finally, leads to mitochondrial permeability transition and apoptosis [46,48]. Although ascorbic acid has been shown to attenuate the deleterious effect of this oxidant [49], our study exhibited that in PMN ascorbate, enhanced AA and E. coli induced ROS generation, as also observed by Guidarelli et al [50] in U937 cells. It seems that supplementation of ascorbate in vitro to the PMNs increased the oxidative and nitrosative stress, which might be used by PMNs for effective bactericidal activity.…”

Enhancing effects of intracellular ascorbic acid on peroxynitrite-induced U937 cell death are mediated by mitochondrial events resulting in enhanced sensitivity to peroxynitrite-dependent inhibition of complex III and formation of hydrogen peroxide

“…First, it is important to emphasize that our previous work [18] demonstrated that DHA is promptly reduced to AsA and that, at the time of ONOO − exposure, all the vitamin was present in its reduced form. Secondly, exposure to DHA does not appear to cause significant changes in the redox state of U937 cells, since GSH levels [18] and mitochondrial membrane potential (Table 1) were unaffected, formation of H 2 O 2 ( Table 2) and MPT (Figure 1F) did not take place, and the cells remained viable ( Table 2) and capable of proliferating with rates superimposable on those of untreated cells [18]. Thirdly, extracellular formation of H 2 O 2 , which may occur via dismutation of superoxide generated by autoxidation of AsA present as a contaminant of DHA, did not take place (results not shown).…”

“…damage was observed in patients with β-thalassaemia major [13], idiopathic haemochromatosis [14], dietary iron overload [15] and severe/acute inflammation caused by eccentric exercise [16] or sepsis [17] upon AsA supplementation. We recently focused our attention on the effects of intracellular AsA in cells exposed to peroxynitrite (ONOO − ), the coupling product of superoxide and nitric oxide, and found that DHA-preloaded U937 cells are hypersentive to its DNA-damaging and toxic effects [18]. Along the same lines, Puskas et al [19] have shown that Jurkat T-cells exposed to DHA for 20-120 min and then immediately challenged with H 2 O 2 were killed more efficiently than cells which did not receive DHA before the oxidant exposure.…”