Flow cytometry-based assay for the activity of NAD(P)H oxidoreductases of the outer mitochondrial membrane.
NAD(P)H oxidoreductases of the outer mitochondrial membrane (OMM) are able to activate various xenobiotics and stimulate the production of reactive oxygen species and the opening of the mitochondrial permeability transition pore. However, the role of these systems in the cell damage by xenobiotics and chemotherapeutic drugs is poorly understood because the methods for the selective assessment of their activity have not been elaborated and specific inhibitors are unknown. Here we propose a method for the semiquantitative assessment of the activity of NAD(P)H oxidoreductases of the OMM in intact and permeabilized cells that is based on the flow cytometry detection of dimethylbiacridene, a fluorescent product of two-electron reduction of lucigenin. The method uses the structural feature of mitochondrial organization: the proximity of the sites of one-electron reduction of lucigenin to cation radical (NAD(P)H oxidoreductases of the OMM) to the sites of its subsequent oxidation (cytochrome c oxidase). The inhibition of cytochrome c oxidase by cyanide selectively activates the dimethylbiacridene formation by oxidoreductases of the OMM but not by other cellular oxidoreductases. The proposed protocol allows one to assess the lucigenin reductase (two-electron) activity of NAD(P)H oxidoreductases of the OMM and to compare it with the activity of other cellular systems that can be used for the analysis of the role of these systems in the cell damage by xenobiotics and antitumor drugs.
Kruglov
Alexey G
AG
Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, 142290 Pushchino, Moscow Region, Russia. krugalex@rambler.ru
Solov'eva
Marina E
ME
Teplova
Vera V
VV
D000166:Acridines / Q000737:chemistry; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D004249:DNA Damage; D003576:Electron Transport Complex IV / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods*; D049408:Luminescent Agents / Q000737:chemistry; D008297:Male; D051336:Mitochondrial Membranes / Q000201:enzymology*; D009243:NAD / Q000378:metabolism; D019255:NADPH Oxidases / Q000378:metabolism*; D010084:Oxidation-Reduction; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000378:metabolism; D015262:Xenobiotics / Q000494:pharmacology
D000166:Acridines; D000970:Antineoplastic Agents; D049408:Luminescent Agents; D017382:Reactive Oxygen Species; D015262:Xenobiotics; D009243:NAD; C033472:10,10'-dimethyl-9,9'-biacridinium; D019255:NADPH Oxidases; D003576:Electron Transport Complex IV