Modulation by bicarbonate the protective effects of phenolic antioxidants on peroxynitrite-mediated cell cytotoxicity

Peroxynitrite (ONOO^-), a potent oxidant formed from the rapid reaction of nitrogen monoxide (^.NO) with superoxide radical (O ₂^.-), is implicated in numerous pathologies including inflammation and age-related diseases (e.g., neurodegenerative disorders and cardiovascular disease). Therefore, there is an increasing interest to develop therapeutic peroxynitrite scavengers. Diet-derived phenolics have been suggested to be powerful peroxynitrite scavengers. However, peroxynitrite reactivity can be significantly modulated by physiological concentrations of carbon dioxide/bicarbonate (CO₂/HCO₃^-, an important buffer system in the body, due to the rapid reaction of ONOO^- and CO₂/HCO₃^- to produce nitrosoperoxycarbonate (ONOOCO₂O^-. Therefore, in order to examine the biological activity of phenolics (caffeic acid, catechin, epicatechin and quercetin) as potential peroxynitrite scavengers, the modulation of CO₂/HCO ₃^-3" on the protective effects of phenolics on peroxynitrite-mediated chondrosarcoma cytotoxicity was assessed by using 4 model systems of peroxynitrite cytoxicity: crystal violet-staining, 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazoliumbromide (MTT) reduction, cellular glutathione measurement and intracellular oxidant formation. All phenolics tested (caffeic acid, catechin, epicatechin and quercetin) significantly inhibited chondrosarcoma cytotoxity induced by peroxynitrite, although bicarbonate (25 mM) decreased their cytoprotective effects. This observation suggests that dietary phenolics were able to limit cell cytotoxicity. However, their activities could be substantially modified by physiological concentrations of bicarbonate. Hence, antioxidants for therapeutic use should be tested for peroxynitrite scavenging in the presence of bicarbonate for avoiding misleading results.