Characterization of the cellular reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT): subcellular localization, substrate dependence, and involvement of mitochondrial electron transport in MTT reduction.
The MTT assay, which is widely used to measure cell proliferation and to screen for anticancer drugs, is based on reduction of the tetrazolium salt, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) by actively growing cells to produce a blue formazan product. Despite broad acceptance of this assay, neither the subcellular localization, nor the biochemical events involved in MTT reduction are known. Mitochondrial involvement in MTT reduction has been inferred from studies with respiratory inhibitors using succinate as a substrate, but the contribution of this activity to overall cellular MTT reduction is unknown. Using the bone marrow-derived cell line, 32D, we investigated the subcellular localization of MTT reduction using succinate, NADH, and NADPH as substrates. At optimum substrate concentrations, MTT reduction by whole cell homogenates was greatest with NADH and least with succinate, which accounted for less than 10% of the combined activities. Using succinate, 96% of recoverable MTT reducing activity was in particulate fractions of the cell and 77% in the mitochondrial and light mitochondrial/lysosomal fractions. When NADH and NADPH were used as substrates, increased amounts of MTT reducing activity were associated with soluble fractions of the cell and association with mitochondrial fractions was less pronounced. To further characterize MTT reduction by the mitochondrial fraction, respiratory chain inhibitors were used to explore involvement of electron transport in MTT reduction. Succinate-dependent mitochondrial MTT reduction was inhibited by 80% with chlorpromazine, 70% by antimycin A, and 85-90% by thenoyltrifluoracetone (TTFA), but inhibition was not observed with rotenone at < or = 2 microM, Amytal, or azide. These results suggest that when succinate is used as an electron donor, 70-80% of mitochondrial MTT reduction occurs subsequent to transfer of electrons from cytochrome c to cytochrome oxidase, but prior to the point of azide inhibition. In contrast to succinate, NADPH-dependent mitochondrial MTT reduction was not affected by any of the respiratory inhibitors tested, and NADH-dependent reduction was only inhibited by chlorpromazine (40-50% at plateau concentrations). These results suggest that most cellular MTT reduction occurs outside the mitochondrial inner membrane and involves NADH and NADPH-dependent mechanisms that are insensitive to respiratory chain inhibitors. This interpretation is supported by whole cell studies in which rotenone failed to affect basal and interleukin-3-stimulated MTT reduction at times up to 4 h but strongly inhibited DNA synthesis. We conclude that most cellular reduction of MTT occurs extramitochondrially and probably involves the pyridine nucleotide cofactors NADH and NADPH.
Malaghan Institute of Medical Research, Wellington School of Medicine, New Zealand.
M V
MV
Berridge
A S
AS
Tan
D000818:Animals; D000968:Antimycin A / Q000494:pharmacology; D001853:Bone Marrow / Q000378:metabolism / Q000648:ultrastructure; D002460:Cell Line; D002729:Chloromercuribenzoates / Q000494:pharmacology; D002746:Chlorpromazine / Q000494:pharmacology; D004396:Coloring Agents; D003574:Cytochrome c Group / Q000378:metabolism; D004579:Electron Transport; D003576:Electron Transport Complex IV / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism; D051379:Mice; D008809:Mice, Inbred C3H; D008928:Mitochondria / Q000378:metabolism*; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D013386:Succinates / Q000378:metabolism; D019802:Succinic Acid; D013778:Tetrazolium Salts / Q000378:metabolism*; D013804:Thenoyltrifluoroacetone / Q000494:pharmacology; D013844:Thiazoles / Q000378:metabolism*; D020245:p-Chloromercuribenzoic Acid
D002729:Chloromercuribenzoates; D004396:Coloring Agents; D003574:Cytochrome c Group; D013386:Succinates; D013778:Tetrazolium Salts; D013844:Thiazoles; D009243:NAD; D013804:Thenoyltrifluoroacetone; D009249:NADP; D020245:p-Chloromercuribenzoic Acid; D000968:Antimycin A; D019802:Succinic Acid; D003576:Electron Transport Complex IV; C022616:thiazolyl blue; D002746:Chlorpromazine