title: Photoreduction of the fluorescent dye 2'-7'-dichlorofluorescein: a spin trapping and direct electron spin resonance study with implications for oxidative stress measurements.
abstract: The photoreduction of 2'-7'-dichlorofluorescein (DCF) was investigated in buffer solution using direct electron spin resonance (ESR) and the ESR spin-trapping technique. Anaerobic studies of the reaction of DCF in the presence of reducing agents demonstrated that during visible irradiation (lambda > 300 nm) 2'-7'-dichlorofluorescein undergoes one-electron reduction to produce a semiquinone-type free radical as demonstrated by direct ESR. Spin-trapping studies of incubations containing DCF, 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and either reduced glutathione (GSH) or reduced NADH demonstrate, under irradiation with visible light, the production of the superoxide dismutase-sensitive DMPO/*OOH adduct. In the absence of DMPO, measurements with 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 dye and forms superoxide.
authors.affiliation: Dipartimento di Chimica Organica A. Mangini, Università di Bologna, Italy.
authors.forename: E
authors.initials: E
authors.lastname: Marchesi
authors.forename: C
authors.initials: C
authors.lastname: Rota
authors.forename: Y C
authors.initials: YC
authors.lastname: Fann
authors.forename: C F
authors.initials: CF
authors.lastname: Chignell
authors.forename: R P
authors.initials: RP
authors.lastname: Mason
mesh_terms: D000332:Aerobiosis; D000693:Anaerobiosis; D002374:Catalase / Q000378:metabolism / Q000494:pharmacology; D004578:Electron Spin Resonance Spectroscopy; D005452:Fluoresceins / Q000737:chemistry* / Q000378:metabolism / Q000528:radiation effects*; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism / Q000528:radiation effects*; D005609:Free Radicals / Q000737:chemistry / Q000378:metabolism / Q000528:radiation effects; D005978:Glutathione / Q000378:metabolism; D066298:In Vitro Techniques; D009243:NAD / Q000378:metabolism; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D010101:Oxygen Consumption / Q000187:drug effects; D010777:Photochemistry; D012996:Solutions; D013482:Superoxide Dismutase / Q000378:metabolism / Q000494:pharmacology
chemical_list: D005452:Fluoresceins; D005456:Fluorescent Dyes; D005609:Free Radicals; D012996:Solutions; D009243:NAD; C037631:2',7'-dichlorofluorescein; D002374:Catalase; D013482:Superoxide Dismutase; D005978:Glutathione