Characterization of the properties of luminescent probes for free radicals and oxidative stress using radiation-chemical methods

Fluorescent or luminescent probes used to measure oxidative or nitrosative stress include leuco (reduced) dyes such as dihydrofluoresceins (e.g. DCFH2), vicinal diamines, and lucigenin. These may react with hydrogen peroxide, peroxynitrite, or their radical precursors (superoxide, nitric oxide) or products (nitrogen dioxide, carbonate radical, hydroxyl radical). Only by gaining a detailed, quantitative knowledge of the reaction pathways can these probes be used with confidence. By producing specific radicals and observing directly their reactions, e.g. by pulse radiolysis, we have measured the rate at which the lucigenin radical reacts with oxygen to generate superoxide, the product it is supposed to measure; quinones interfere. DCFH2 does not react with hydrogen peroxide significantly, but we have shown that cytochrome c is a potent catalyst (Biochem. Biophys. Res. Commun, 2001, 282, 329). Since this is released from mitochondria to cytosol in apoptosis, probe oxidation may reflect apoptosis rather than increased superoxide generation. GSH also modulates the oxidation of DCFH2 in a superoxide-generating system containing cytochrome c, the effect varying with the GSH/GSSG ratio and the level of SOD. The rate constants of uncatalysed oxidation of DCFH2 with nitrogen dioxide, glutathione thiyl radicals and other radicals have been measured and indicate other potential pathways must be considered. Similarly, fluorescent probes for nitric oxide actually require prior oxidative activation by e.g. peroxidases or nitrogen dioxide, and the reactions are subject to interference from competing reductants