Mitochondrial and nonmitochondrial reduction of MTT: interaction of MTT with TMRE, JC-1, and NAO mitochondrial fluorescent probes.
BACKGROUND
Bioreduction of water-soluble tetrazolium salts (e.g., MTS, XTT, and MTT) to their respective formazans is generally regarded as an indicator of cell "redox activity." The reaction is attributed mainly to mitochondrial enzymes and electron carriers. However, MTT reduction may also be catalyzed by a number of other nonmitochondrial enzymes. The goal of this work was to establish the sites of MTT reduction in intact HepG2 human hepatoma cells in culture.


METHODS
In order to establish the subcellular localization of the sites of reduction of MTT, we imaged the formation of MTT-formazan deposits using backscattered light confocal microscopy. Mitochondria were visualized in viable cells using fluorescent dyes that bind in a manner dependent (JC-1 and TMRE) or independent (NAO) of mitochondrial electric potential.


RESULTS
Only 25-45% of MTT-formazan was associated with mitochondria after 25 min of incubation. No more than 25% of the mitochondrial area on images was occupied by MTT-formazan. Mitochondrial fluorescence of TMRE, NAO, and the monomeric form of JC-1 decreased rapidly in cells incubated with MTT. However, the intensity of fluorescence of JC-1 aggregates dropped by less than 30% at the onset of incubation and remained constant as reduction of MTT proceeded further.


CONCLUSIONS
(1) Most of MTT-formazan deposits are not coincident with mitochondria. (2) Monomeric JC-1, as well as TMRE and NAO, accumulating in mitochondria may be displaced by MTT. Thus, the presence of positively charged organic compounds (like MTT) may distort measurements of mitochondrial transmembrane electric potential, which are based on accumulation of fluorescent dyes.
Bernas
Tytus
T
Laboratory of Confocal Microscopy and Image Analysis, Department of Biophysics, Institute of Molecular Biology and Biotechnology, Jagiellonian University, Krakow, Poland.
Dobrucki
Jurek
J
D000609:Aminoacridines; D001562:Benzimidazoles; D002232:Carbocyanines; D002451:Cell Compartmentation / Q000187:drug effects / Q000502:physiology*; D004734:Energy Metabolism / Q000187:drug effects / Q000502:physiology*; D005057:Eukaryotic Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D005456:Fluorescent Dyes; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000295:instrumentation / Q000379:methods; D007425:Intracellular Membranes / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods*; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D009942:Organometallic Compounds; D013778:Tetrazolium Salts / Q000378:metabolism* / Q000494:pharmacology; D013844:Thiazoles / Q000378:metabolism* / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D000609:Aminoacridines; D001562:Benzimidazoles; D002232:Carbocyanines; D005456:Fluorescent Dyes; D009942:Organometallic Compounds; D013778:Tetrazolium Salts; D013844:Thiazoles; C110932:tetramethyl rhodamine ethyl ester; C068624:5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine; C041691:10-N-nonylacridinium orange; C022616:thiazolyl blue