Rapid oxidation of dichlorodihydrofluorescin with heme and hemoproteins: formation of the fluorescein is independent of the generation of reactive oxygen species.
Oxidative stress and the generation of reactive oxygen species (ROS) have been implicated in the pathogenesis of cellular damage. These events have usually been reported in terms of oxidation of a reporter molecule such as 2',7'-dichlorodihydrofluorescin diacetate (DCFH-DA). Treatment of HeLa cells with hemin or metalloporphyrins resulted in a rapid oxidation of DCFH in a time- and dose-dependent manner. This oxidation was inhibited by treatment of the cells with a large amount of superoxide dismutase and catalase, which is different from observations that these enzymes had no effect on the induction of heme oxygenase-1, a stress-induced protein, in hemin-treated cells. To examine the possibility that the oxidation of DCFH is independent of the generation of ROS, the oxidation was measured using hemoglobin-synthesizing erythroleukemia K562 cells. When K562 cells were treated with delta-aminolevulinic acid, a precursor of heme, oxidation of DCFH increased depending on the heme content in cells. Then DCFH-DA was oxidized directly with heme, hemoglobin, myoglobin and cytochrome c. These results suggest that oxidation of DCFH is not always related to the generation of ROS but may be related to heme content in cells.
Ohashi
Tomoko
T
Department of Biotechnology, Kyoto Institute of Technology, Kyoto, Japan.
Mizutani
Atsushi
A
Murakami
Akira
A
Kojo
Shosuke
S
Ishii
Tetsuro
T
Taketani
Shigeru
S
D000622:Aminolevulinic Acid / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects; D003574:Cytochrome c Group / Q000378:metabolism; D005452:Fluoresceins / Q000378:metabolism*; D006367:HeLa Cells; D006419:Heme Oxygenase (Decyclizing) / Q000235:genetics / Q000378:metabolism; D006420:Hemeproteins / Q000378:metabolism* / Q000494:pharmacology; D006427:Hemin / Q000378:metabolism* / Q000494:pharmacology; D006454:Hemoglobins / Q000378:metabolism; D006801:Humans; D020014:K562 Cells; D018613:Microscopy, Confocal; D009211:Myoglobin / Q000378:metabolism; D010084:Oxidation-Reduction / Q000187:drug effects; D012333:RNA, Messenger / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism*; D012235:Rhodamines / Q000378:metabolism
D003574:Cytochrome c Group; D005452:Fluoresceins; D006420:Hemeproteins; D006454:Hemoglobins; D009211:Myoglobin; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D012235:Rhodamines; C058319:dihydrorhodamine 123; C037631:2',7'-dichlorofluorescein; D006427:Hemin; D000622:Aminolevulinic Acid; D006419:Heme Oxygenase (Decyclizing)