Photoreduction of the fluorescent dye 2 '-7 '-dichlorofluorescein: A spin trapping and direct electron spin resonance study with implications for oxidative stress measurements

The photoreduction of 2'-7'-dichlorofluorescein (DCF) was investigated in b uffer solution using direct electron spin resonance (ESR) and the ESR spin- trapping technique. Anaerobic studies of the reaction of DCF in the presenc e of reducing agents demonstrated that during visible irradiation (lambda > 300 nm) 2'-7'-dichlorofluorescein undergoes one-electron reduction to prod uce a semiquinone-type free radical as demonstrated by direct ESR. Spin-tra pping studies of incubations containing DCF, 5,5-dimethyl-1-pyrroline N-oxi de (DMPO) and either reduced glutathione (GSH) or reduced NADH demonstrate, under irradiation with visible light, the production of the superoxide dis mutase-sensitive DMPO/-OOH adduct. In the absence of DMPO, measurements wit h a Clark-type oxygen electrode show that molecular oxygen is consumed in a light-dependent process. The semiquinone radical of DCF, when formed in an aerobic system, is immediately oxidized by oxygen, which regenerates the d ye and forms superoxide. (C) 1998 Elsevier Science Inc.