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

Abstract

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 −.