2--7--dichlorodihydrofluorescein and 2--7--dichlorodihydrofluorescein-diacetate

Heme oxygenase-1 (HO-1) degrades heme into biliverdin, iron and CO. The enzyme participates in adaptive and protective responses to oxidative stress and various inflammatory stimuli, and is induced in response to reactive oxygen species (ROS). 2',7'-Dichlorodihydrofluorescin diacetate (DCFH-DA) is a common reagent used to detect ROS by the oxidation of 2',7'-dichlorodihydrofluorescin (DCFH) to fluorescent dichlorodihydrofluorescein. We previously found that rapid oxidation of DCFH occurred with heme-compounds as well as ROS [Ohashi, T. et al. (2002) FEBS Lett. 511, 21-27], and then examined the effect of DCFH-DA on the induction of HO-1 expression by arsenite, cadmium and hemin, which induce oxidative stress and cytotoxicity. We found suppression of the arsenite-, cadmium- and hemin-dependent induction of HO-1 with DCFH-DA. The suppression occurred at the transcriptional level since the promoter activity of the Maf-recognition site of the HO-1 gene decreased with the DCFH-DA treatment. DCFH abolished the phosphorylation of extracellular signal-regulated kinase, the nuclear translocation of a transcriptional activator Nrf2, and cell death. An antioxidant, N-acetylcysteine (NAC), also suppressed the induction by arsenite and cadmium, but not that by hemin, indicating that DCFH blocked a different site in the stress signal pathway from NAC. Considering that the oxidation of DCFH diminishes ROS generated by various stressors, our findings provide a potential strategy for protection of cells from toxic insults using DCFH-like molecules.

Topics: Acetylcysteine; Active Transport, Cell Nucleus; Antioxidants; Arsenites; Cadmium; Cell Nucleus; Cell Survival; Cytoprotection; Enzyme Induction; Extracellular Signal-Regulated MAP Kinases; Fluoresceins; HeLa Cells; Heme Oxygenase-1; Hemin; Humans; NF-E2-Related Factor 2; Oxidative Stress; Phosphorylation; Promoter Regions, Genetic; Reactive Oxygen Species

2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) is a fluorogenic probe commonly used to detect cellular production of reactive oxygen species (ROS), for example in the respiratory burst of granulocytes and mononuclear phagocytes. This method depends on the de-acetylation of H2DCFDA by cellular esterases, to form the oxidant-sensitive compound, 2',7'-dichlorodihydrofluorescein (H2DCF). Importantly, however, not all cells possess sufficient esterase activity to produce the H2DCF needed for accurate measurement of ROS. In this study, we used chemically de-acetylated probe (H2DCF) to assess the phorbol-ester-triggered respiratory burst of rainbow trout macrophages, which, like some mammalian mononuclear phagocytes, appear to have low probe-esterase activity. We compared this approach to the use of intact H2DCFDA and the cytochrome c reduction assay. The H2DCF and cytochrome c reduction assays gave similar portrayals of the kinetics of the macrophage respiratory burst, while H2DCFDA did not. We therefore recommend the use of H2DCF over H2DCFDA for quantification of the production of reactive oxygen species. Additionally, we stress the need to test reaction buffers or culture media used with H2DCF(DA) for their ability to oxidize the probe directly or indirectly. As an example, we have observed that tyrosine combined with ubiquitous metal contaminants of physiological buffers can result in high levels of oxidation, which may be incorrectly interpreted as cellular activity.

Topics: Acetylation; Animals; Cytochrome c Group; Fluoresceins; Fluorescent Dyes; Fluorometry; In Vitro Techniques; Macrophages; Models, Biological; Oncorhynchus mykiss; Oxidation-Reduction; Reactive Oxygen Species; Respiratory Burst; Tetradecanoylphorbol Acetate