Journal of Biochemistry Advance Access originally published online on September 7, 2006
Journal of Biochemistry 2006 140(4):483-489; doi:10.1093/jb/mvj187
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© 2006 The Japanese Biochemical Society.
ARTICLE |
The Antioxidant Role of a Reagent, 2',7'-Dichlorodihydrofluorescin Diacetate, Detecting Reactive-Oxygen Species and Blocking the Induction of Heme Oxygenase-1 and Preventing Cytotoxicity
1 Department of Biotechnology and 5 Insect Biomedical Center, Kyoto Institute of Technology, Kyoto 606-8585; 2 Departments of Food Science and Nutrition, Nara Women's University, Nara 622; 3 Graduate School of Comprehensive Human Sciences, Tsukuba University, Tukuba 305-8575; and 4 The First Department of Pathology, Kansai Medical University, Moriguchi, Osaka 570-8506
* To whom correspondence should be addressed at: Department of Biotechnology, Kyoto Institute of Technology, Kyoto 606-8585. Phone: +81-75-724-7789, Fax: +81-75-724-7760, E-mail: taketani{at}kit.ac.jp
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.