Evaluation of the probes 2′,7′-dichlorofluorescin diacetate, luminol, and lucigenin as indicators of reactive species formation

• Published in: Biochemical pharmacology Vol. 65; no. 10; pp. 1575 - 1582
• Main Authors: Myhre, Oddvar, Andersen, Jannike M., Aarnes, Halvor, Fonnum, Frode
• Format: Journal Article
• Language: English
• Published: New York, NY Elsevier Inc 15.05.2003; Elsevier Science
• Subjects: 2′,7′-Dichlorofluorescin diacetate; Acridines - metabolism; Biological and medical sciences; Cell-free DCFH system; Fluoresceins - analysis; Fluoresceins - metabolism; Free Radicals - analysis; Humans; Hydrogen Peroxide - analysis; Hydroxyl Radical - analysis; Indicators and Reagents - metabolism; Lucigenin; Luminescent Measurements; Luminol; Luminol - metabolism; Medical sciences; Neutrophils; Reactive Oxygen Species - analysis; DDC; RONS; DCF; 2′,7′-Dichlorofluorescin diacetate; HRP; Neutrophils; A1242; SNAP; SHA; Lucigenin; Cell-free DCFH system; SIN-1; DCFH-DA; 3,4-DHBA; NOS; ROS; Luminol; HBSS; 4-HBA; 2',7'-Dichlorofluorescin diacetate
• ISSN: 0006-2952; 1873-2968
• DOI: 10.1016/S0006-2952(03)00083-2

This study attempts to provide a critical assessment of three different common approaches to identifying reactive species formed in biological systems: the 2′,7′-dichlorofluorescin diacetate (DCFH-DA) assay, and the luminol- and lucigenin-amplified chemiluminescence assays. There have been several contradictory reports about the specificity of these methods. Our results show that DCFH is oxidized to the fluorescent compound 2′,7′-dichlorofluorescin (DCF) in human neutrophils exposed to the following compounds: Aroclor (A)1242, hydrogen peroxide (H 2O 2), nitric oxide (NO), and FeSO 4. Use of a cell-free DCFH system showed increased formation of DCF by peroxynitrite (ONOO −), horseradish peroxidase (HRP) alone, and HRP in combination with H 2O 2, FeSO 4 alone, and a mixture of FeSO 4 and H 2O 2. The hydroxyl radical ( OH) scavenger formate and the iron ion chelator deferoxamine reduced the DCF formation induced by FeSO 4 in combination with H 2O 2. DCFH was insensitive to NO and H 2O 2 in the cell-free system. In the presence of neutrophils, the A1242-induced luminol chemiluminescence was decreased by the superoxide dismutase inhibitor diethyldithiocarbamic acid (DDC) and the myeloperoxidase inhibitor salicylhydroxamic acid (SHA). Exposure of the neutrophils to NO, FeSO 4, or H 2O 2 alone did not have any effect. A1242-induced lucigenin chemiluminescence in the neutrophils was increased slightly by DDC, but was not affected by SHA, NO, FeSO 4, or H 2O 2. In conclusion, we suggest that the DCF assay is only suitable for measurements of ONOO −, H 2O 2 in combination with cellular peroxidases, and OH. Luminol is sensitive towards HOCl, while lucigenin is oxidized by O 2 −.