The roles of thiol-derived radicals in the use of 2',7'-dichlorodihydrofluorescein as a probe for oxidative stress.

Abstract

2',7'-Dichlorodihydrofluorescein (DCFH2) is one of the most widely used probes for detecting intracellular oxidative stress, but requires a catalyst to be oxidized by hydrogen peroxide or superoxide and reacts nonspecifically with oxidizing radicals. Thiyl radicals are produced when many radicals are "repaired" by thiols, but are oxidizing agents and thus potentially capable of oxidizing DCFH2. The aim of this study was to investigate the reactivity of thiol-derived radicals toward DCFH2 and its oxidized, fluorescent form 2',7'-dichlorofluorescein (DCF). Thiyl radicals derived from oxidation of glutathione (GSH) or cysteine (CysSH) oxidized DCFH2 with rate constants at pH 7.4 of approximately 4 or approximately 2x10(7) M(-1) s(-1), respectively. Both the rates of oxidation and the yields of DCF were pH-dependent. Glutathione-derived radicals interacted with DCF, resulting in the formation of DCFH* absorbing at 390 nm and loss of fluorescence; in contrast, cysteine-derived radicals did not cause any depletion of DCF fluorescence. We postulate that the observed apparent difference in reactivity between GS* and CysS* toward DCF is related to the formation of carbon-centered, reducing radicals from base-catalyzed isomerization of GS*. DCF formation from interaction of DCFH2 with GS* was inhibited by oxygen in a concentration-dependent manner over the physiological range. These data indicate that in applying DCFH2 to measure oxidizing radicals in biological systems, we have to consider not only the initial competition between thiols and DCFH2 for the oxidizing radicals, but also subsequent reactions of thiol-derived radicals, together with variables--including pH and oxygen concentration--which control thiyl radical chemistry.