5-cyano-2-3-ditolyltetrazolium-chloride and Liver-Neoplasms

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.

Topics: Carcinoma, Hepatocellular; Cell Membrane; Coloring Agents; Formazans; Humans; Liver Neoplasms; Oxazines; Oxidation-Reduction; Propidium; Spectrometry, Fluorescence; Spectrophotometry; Tetrazoles; Tetrazolium Salts; Tumor Cells, Cultured

Redox activities associated with plasma membranes of nonphagocytic animal and plant cells have been reported by several authors. However, the natural substrates, structure and biological role of these putative enzyme systems are not known. Data indicating extracellular reduction of a nitroxide free radical Cat1 (1-oxy-4-trimethylamine-2,2,6,6,tetramethyl-piperidine) by hepatocytes were thought to be artefactual. We report evidence in support of a notion that Cat1 as well as a tetrazolium salt, CTC (5-cyano-2,3-ditolyl tetrazolium chloride), are reduced extracellularly, probably at the cell surface, by human HepG2 hepatoma cells. These data provide evidence confirming the existence of a yet unidentified reducing activity associated with outer surface of plasma membranes of transformed human hepatocytes.

Topics: 4-Chloromercuribenzenesulfonate; Ascorbic Acid; Calcium Channels; Carcinoma, Hepatocellular; Catalase; Cell Line, Transformed; Cell Membrane; Doxorubicin; Extracellular Matrix; Free Radicals; Humans; Liver Neoplasms; NAD; Oxidation-Reduction; Tetrazoles; Tetrazolium Salts; TRPV Cation Channels