Phenoxyl free radical formation during the oxidation of the fluorescent dye 2 ',7 '-dichlorofluorescein by horseradish peroxidase - Possible consequences for oxidative stress measurements

The oxidation of the fluorescent dye 2',7' dichlorofluorescein (DCF) by hor seradish peroxidase was investigated by optical absorption, electron spin r esonance (ESR), and oxygen consumption measurements. Spectrophotometric mea surements showed that DCF could be oxidized either by horseradish peroxidas e-compound I or -compound II with the obligate generation of the DCF phenox yl radical (DCF'), This one-electron oxidation was confirmed by ESR spin-tr apping experiments. DCF' oxidizes GSH, generating the glutathione thiyl rad ical (GS'), which was detected by the ESR spin-trapping technique. In this case, oxygen was consumed by a sequence of reactions initiated by the GS' r adical. Similarly, DCF' oxidized NADH, generating the NAD' radical that red uced oxygen to superoxide (O-2(radical anion)), which was also detected by the ESR spin-trapping technique. Superoxide dismutated to generate H2O2, wh ich reacted with horseradish peroxidase, setting up an enzymatic chain reac tion leading to H2O2 production and oxygen consumption. In contrast, when a scorbic acid reduced the DCF phenoxyl radical back to its parent molecule, it formed the unreactive ascorbate anion radical. Clearly, DCF catalyticall y stimulates the formation of reactive oxygen species in a manner that is d ependent on and affected by various biochemical reducing agents, This study , together with our earlier studies, demonstrates that DCFH cannot be used conclusively to measure superoxide or hydrogen peroxide formation in cells undergoing oxidative stress.