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 teactive 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(2)O(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(2)O(2), FeSO(4) alone, and a mixture of FeSO(4) and H(2)O(2). The hydroxyl radical (z.rad;OH) scavenger formate and the iron ion chelator deferoxamine reduced the DCF formation induced by FeSO(4) in combination with H(2)O(2). DCFH was insensitive to NO and H(2)O(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(2)O(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(2)O(2). In conclusion, we suggest that the DCF assay is only suitable for measurements of ONOO(-), H(2)O(2) in combination with cellular peroxidases, and z.rad;OH. Luminol is sensitive towards HOCl, while lucigenin is oxidized by O(2)z.rad;(-).