Evidence for free radical formation during the oxidation of 2 '-7 '-dichlorofluorescin to the fluorescent dye 2 '-7 '-dichlorofluorescein by horseradish peroxidase: Possible implications for oxidative stress measurements

The oxidation of 2'-7'-dichlorofluorescin (DCFH) to the fluorescent 2'-7'-d ichlorofluorescein (DCF) by horseradish peroxidase (HRP) was investigated b y fluorescence, absorption, and electron spin resonance spectroscopy (ESR). As has been previously reported, HRP/H2O2 oxidized DCFH to the highly fluo rescent DCF. However, DCF fluorescence was still observed when H2O2 was omi tted, although its intensity was reduced by 50%. Surprisingly, the fluoresc ence increase, in the absence of exogenous H2O2, was still strongly inhibit ed by catalase, demonstrating that H2O2 was present and necessary for DCF f ormation. H2O2 was apparently formed during either chemical or enzymatic de acetylation of 2'-7'-dichlorofluorescin diacetate (DCFH-DA), probably by au to-oxidation. Spectrophotometric measurements clearly showed that DCFH coul d be oxidized either by HRP-compound I or HRP-compound II with the obligate generation of the DCF semiquinone free radical (DCF.-). Oxidation of DCF.- to DCF by oxygen would yield superoxide (O-2(.-)). ESR spectroscopy in con junction with the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) reveale d the presence of both superoxide and hydroxyl radicals in the DCFH/H2O2/HR P system. Both radicals were also detected in the absence of added H2O2, al though the intensities of the resultant adducts were decreased. This work d emonstrates that DCF fluorescence cannot be used reliably to measure O-2(.- ) in cells because O-2(.-) itself is formed during the conversion of DCFH t o DCF by peroxidases. The disproportionation of superoxide forms H2O2 which , in the presence of peroxidase activity, will oxidize more DCFH to DCF wit h self-amplification of the fluorescence. Because the deacetylation of DCFH -DA, even by esterases, can produce H2O2, the use of this probe to measure H2O2 production in cells is problematic. (C) 1999 Elsevier Science Inc.