Original Contribution PHOTOREDUCTION OF THE FLUORESCENT DYE 2'-7'-DICHLOROFLUORESCEIN: A SPIN TRAPPING AND DIRECT ELECTRON SPIN RESONANCE STUDY WITH IMPLICATIONS FOR OXIDATIVE STRESS MEASUREMENTS EMANUELA MARCHESI,* CRISTINA ROTA ² ,YANG C. FANN, ‡ COLIN F. CHIGNELL, ² and RONALD P. MASON ² *Dipartimento di Chimica Organica “A. Mangini,” Universita ` di Bologna, Italy; ² Laboratory of Pharmacology and Chemistry and ‡ Information Technology Support Services, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, USA (Received 16 April 1998; Revised 30 June 1998; Accepted 30 June 1998) 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 (  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 gluta- thione (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. © 1998 Elsevier Science Inc. Keywords—2'-7'-Dichlorofluorescein, Photoreduction, Free radical, Superoxide, Spin-trapping INTRODUCTION Fluorogenic, chemiluminescent, or chromogenic probes have been used extensively to monitor oxidative activity in cells [1]. One of the most popular approaches involves the use of chemically reduced (or “leuco”) nonfluores- cent forms of highly fluorescent dyes such as fluorescein and rhodamine. Upon reaction with oxidizing species, these leuco compounds, which are poor fluorescers, are oxidized back to the parent dye molecule, resulting in a dramatic increase in fluorescence intensity [1]. The use of 2'-7'-dichlorofluorescin (DCFH) for this purpose is shown in Scheme 1. The diacetate of DCFH is added to cells where it diffuses across the cell membrane and is hydrolyzed by intracellular esterases to DCFH which, upon oxidation, yields the highly fluorescent DCF. Ox- idation may be achieved by reaction with H 2 O 2 in the presence of peroxidase, cytochrome c, or Fe 2+ [2–5]. DCFH may also be oxidized by peroxynitrite, but not superoxide or hydrogen peroxide [5,6]. A search of MEDLINE revealed that nearly 400 papers report the use of DCFH to detect oxidative events in cells. In 1971, Leaver showed that in the presence of reduc- ing agents such as trimethylamine and certain indoles, xanthene dyes (fluorescein, eosin Y, and rose bengal) in alcohol solution may be converted to their corresponding semi-reduced forms by visible irradiation [7]. Photore- duction of dyes has also been observed in aqueous solu- tion, and in several cases the ESR spectrum of the resultant radical has been observed [8]. We have previ- ously detected superoxide during the visible irradiation of an aqueous, aerobic solution of gentian violet in the presence of reducing agents such as NADH, glutathione, and cysteine [9]. Gentian violet is a triphenylmethane dye that is structurally similar to fluorescein and DCF. If the same reaction can take place in cells loaded with DCF and irradiated with light during examination in a fluorescence microscope or while exposed to room light, then superoxide may be generated which, upon dismu- Address correspondence to: Cristina Rota, NIH/NIEHS, MD F0-02, PO Box 12233, Research Triangle Park, NC 27709, USA; Tel: (919) 541-5197; Fax: (919) 541-1043. Free Radical Biology & Medicine, Vol. 26, Nos. 1/2, pp. 148 –161, 1999 Copyright © 1998 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/99/$–see front matter PII S0891-5849(98)00174-9 148