For many years various tetrazolium salts and their formazan products have been employed in histochemistry and for assessing cell viability. For the latter application, the most widely used are 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), and 5-cyano-2,3-di-(p-tolyl)-tetrazolium chloride (CTC) for viability assays of eukaryotic cells and bacteria, respectively. In these cases, the nicotinamide-adenine-dinucleotide (NAD(P)H) coenzyme and dehydrogenases from metabolically active cells reduce tetrazolium salts to strongly colored and lipophilic formazan products, which are then quantified by absorbance (MTT) or fluorescence (CTC). More recently, certain sulfonated tetrazolium, which give rise to water-soluble formazans, have also proved useful for cytotoxicity assays. We describe several aspects of the application of tetrazolium salts and formazans in biomedical cell biology research, mainly regarding formazan-based colorimetric assays, cellular reduction of MTT, and localization and fluorescence of the MTT formazan in lipidic cell structures. In addition, some pharmacological and labeling perspectives of these compounds are also described.

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Cationic lipophilic dyes can accumulate in mitochondria, and especially in mitochondria of tumor cells. We investigated the chemical properties and the processes allowing selective uptake into tumor cells using the Fick-Nernst-Planck equation. The model simulates uptake into cytoplasm and mitochondria and is valid for neutral molecules and ions, and thus also for weak electrolytes. The differential equation system was analytically solved for the steady-state and the dynamic case. The parameterization was for a generic human cell, with a 60 mV more negative potential at the inner mitochondrial membrane of generic tumor cells. The chemical input data were the lipophilicity (log K(OW)), the acid/base dissociation constant (pK(a)) and the electric charge (z). Accumulation in mitochondria occurred for polar acids with pK(a) between 5 and 9 owing to the ion trap, and for lipophilic bases with pK(a)>11 or permanent cations owing to electrical attraction. Selective accumulation in tumor cells was found for monovalent cations or strong bases with log K(OW) of the cation between -2 and 2, with the optimum near 0. The results are in agreement with experimental results for rhodamine 123, a series of cationic triarylmethane dyes, F16 and MKT-077, an anticancer drug targeting tumor mitochondria.

Lysosomes are acidic organelles and are involved in various diseases, the most prominent is malaria. Accumulation of molecules in the cell by diffusion from the external solution into cytosol, lysosome and mitochondrium was calculated with the Fick-Nernst-Planck-equation. The cell model considers the diffusion of neutral and ionic molecules across biomembranes, dissociation to mono-or bivalent ions, adsorption to lipids, and electrical attraction or repulsion. Based on simulation results, high and selective accumulation in lysosomes was found for weak mono-and bivalent bases with intermediate to high log Kow. These findings were validated with experimental results and by a comparison to the properties of antimalarial drugs in clinical use. For ten active compounds, nine were predicted to accumulate to a greater extent in lysosomes than in other organelles, six of these were in the optimum range predicted by the model and three were close. Five of the antimalarial drugs were lipophilic weak dibasic compounds. The predicted optimum properties for a selective accumulation of weak bivalent bases in lysosomes are consistent with experimental values and are more accurate than any prior calculation. This demonstrates that the cell model can be a useful tool for the design of effective lysosome-targeting drugs with minimal off-target interactions.

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