Reduction of a tetrazolium salt, CTC, by intact HepG2 human hepatoma cells: subcellular localisation of reducing systems

• Published in: Biochimica et biophysica acta Vol. 1451; no. 1; pp. 73 - 81
• Main Authors: Bernas, Tytus, Dobrucki, Jurek
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 12-08-1999
• Subjects: Carcinoma, Hepatocellular; Cell Membrane - chemistry; Cell Membrane - enzymology; Coloring Agents; CTC; Formazans - chemistry; Humans; Liver Neoplasms; Oxazines; Oxidation-Reduction; Plasma membrane; Propidium; Reductase; Spectrometry, Fluorescence; Spectrophotometry; Tetrazoles - chemistry; Tetrazolium salt; Tetrazolium Salts; Tumor Cells, Cultured; DMEM, Dulbecco’s modified minimal essential medium; NBT, 3,3′-(3,3′-dimethoxy-4,4′-biphenylene)-bis [2-(4-nitrophenyl-5-phenyl)-2 H-tetrazolium chloride; MB, 8-dimethylamino 2,3-benzophenoxazine (Meldola’s Blue); MPMS, 1-methoxy phenazine methosulphate; PI, propidium iodide; NTV, nitro-tetrazolium violet; CTC, 5-cyano-2,3-ditolyl tetrazolium chloride; Tetrazolium salt; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide; XTT, sodium 3′-[1-[(phenylamino) carbonyl]-3,4-tetrazolium]bis(4-methoxy-6-nitro) benzensulphonic acid hydrate; CTC; INT, 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2 H-tetrazolium chloride; Plasma membrane; MTS, 5-(3-carboxymethoxyphenyl)-2-(4,5-dimethylthiazolyl)-3-(4-sulphophenyl) tetrazolium, inner salt; PMS, phenazine methosulphate; Reductase
• ISSN: 0167-4889; 0006-3002; 1879-2596
• DOI: 10.1016/S0167-4889(99)00071-3

Cell-mediated reduction of tetrazolium salts, including MTT, XTT, MTS, NBT, NTV, INT, in the presence or absence of intermediate electron carriers is used as a convenient test for animal or bacterial cell viability. Bioreduction of tetrazolium is considered an alternative to a clonogenic assay and a thymidine incorporation assay. However, correlation between clonogenic potential and capacity to reduce tetrazolium has not been demonstrated convincingly. Moreover, despite a wide use of tetrazolium viability assays, the mechanism and subcellular localisation of reducing systems or species in viable intact cells have not been fully elucidated. We report evidence indicating that a tetrazolium salt CTC can be reduced in the presence as well as in the absence of an electron carrier by viable HepG2 human hepatoma cells. CTC-formazan is formed within or at the outer surface of plasma membranes. We hypothesise that in the presence of an electron carrier the electron donors active in the reduction of CTC are located in the intracellular compartment, as well as in plasma membranes. However, in the absence of an electron carrier, the reduction occurs primarily via a plasma membrane-associated enzymatic system or species.