title: Analysis of dihydroethidium fluorescence for the detection of intracellular and extracellular superoxide produced by NADPH oxidase.
abstract: All methods used for quantitation of superoxide have limitations when it comes to differentiating between extracellular and intracellular sites of superoxide production. In the present study, we monitored dihydroethidium (DHE)-derived fluorescence at 570 nm, which indicates hydroxyethidium derived from reaction with superoxide produced by human leukemia cells (HL-60) and microvascular endothelial cells (HMEC-1). Phorbol-12-myristate 13-acetate (PMA; 100 ng/ml) caused an increase in fluorescence and lucigenin chemiluminescence in HL-60, which was abolished by superoxide dismutase (SOD; 600 U/ml) indicating that DHE detects extracellular superoxide. Furthermore, both HL-60 cells and HMEC-1 generated a fluorescence signal in the presence of DHE under resting conditions, which was unaffected by SOD, but abolished by polyethylene glycosylated-SOD (PEG-SOD) (100 U/ml) and MnTmPyP (25 microM), indicating that DHE also detects superoxide produced intracellularly. In HMEC-1, silencing of either Nox2 or Nox4 components of NADPH oxidase with small interference RNA (siRNA) resulted in a significant reduction in superoxide detected by both DHE fluorescence (Nox2 siRNA; 71 +/- 6% and Nox4 siRNA 83 +/- 7% of control) and lucigenin chemiluminescence (Nox2; 54 +/- 6% and Nox4 74 +/- 4% of control). In conclusion, DHE-derived fluorescence at 570 nm is a convenient method for detection of intracellular and extracellular superoxide produced by phagocytic and vascular NADPH oxidase.
authors.affiliation: Cytoprotection Pharmacology Laboratory, Bernard O'Brien Institute of Microsurgery, University of Melbourne, Melbourne, Vic., Australia.
authors.forename: Hitesh M
authors.initials: HM
authors.lastname: Peshavariya
authors.forename: Gregory James
authors.initials: GJ
authors.lastname: Dusting
authors.forename: Stavros
authors.initials: S
authors.lastname: Selemidis
mesh_terms: D000166:Acridines / Q000494:pharmacology; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism; D004996:Ethidium / Q000031:analogs & derivatives* / Q000737:chemistry; D005453:Fluorescence; D018922:HL-60 Cells; D006801:Humans; D049449:Luminescence; D049408:Luminescent Agents / Q000494:pharmacology; D008562:Membrane Glycoproteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D000074662:NADPH Oxidase 2; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D034741:RNA, Small Interfering / Q000494:pharmacology; D013482:Superoxide Dismutase / Q000378:metabolism; D013481:Superoxides / Q000032:analysis / Q000378:metabolism*; D019820:Xanthine / Q000494:pharmacology; D014969:Xanthine Oxidase / Q000378:metabolism
chemical_list: D000166:Acridines; D049408:Luminescent Agents; D008562:Membrane Glycoproteins; D034741:RNA, Small Interfering; C067883:dihydroethidium; D013481:Superoxides; D019820:Xanthine; C033472:10,10'-dimethyl-9,9'-biacridinium; D013482:Superoxide Dismutase; D014969:Xanthine Oxidase; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C498910:NOX4 protein, human; D004996:Ethidium