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In order to supply the tetrazolium salt procedure to flow cytometry we have synthesized monotetrazolium salts which are converted into fluorescent formazans on reduction. In this preliminary study we investigated the "oxygen sensitivity" of four of our compounds and compared them to the commercially available nitrotetrazole blue (NBT). The amount of formazan produced by Ehrlich ascites tumour cells via the succinic dehydrogenase reaction during oxic and anoxic incubations was determined by elution technique. Under oxic conditions no NBT formazan but much cyano-di-chlorophenyl formazan (==CCPC) was generated. The yields of this formazan differed only slightly between oxic and anoxic incubations. Twice as much of the amount of the other three formazans cyano-ditolyl- (==CTC, Stellmach 1984), cyanodiphenyl- (==CPC), and cyano-dianisyl tetrazolium chloride (==CAC), were produced in nitrogen atmosphere as in oxygen. The amount of all formazans investigated increased both in oxic and in anoxic conditions if cyanide was present. All cyano-aryl formazans have the maximum of absorbance between 430 and 470 nm and fluoresce in the spectral range above 580 nm with CTC formazan being the brightest. The tetrazolium salts, i.e. in unreduced state, did not fluoresce in the visible part of the spectrum. The formazans could be applied to flow cytometry. After staining the nuclear DNA with the fluorochrome DAPI, we obtained detailed two-parametric distributions correlating the amount of formazan per cell with the DNA content. The results are a step to establish a method for automated identification of malignant cells.
Amyloid beta peptide (A beta) neurotoxicity is believed to play a central role in the pathogenesis of Alzheimer's disease. An early indicator of A beta toxicity is the inhibition of cellular 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction to MTT formazan, a widely used assay for measuring cell viability. In this report we show that A beta and other cytotoxic amyloid peptides such as human amylin dramatically enhance MTT formazan exocytosis, resulting in the inhibition of cellular MTT reduction. Only the amyloid peptides that are known to be cytotoxic enhanced MTT formazan exocytosis. Basal MTT formazan exocytosis and amyloid peptide-enhanced MTT formazan exocytosis are blocked by several drugs with diverse known effects. These and other data suggest that MTT formazan exocytosis is a multistep process and that cytotoxic amyloid peptides enhance MTT formazan exocytosis through an intracellular signal transduction pathway.
Perhaps the most reproducible early event induced by the interaction of amyloid beta peptide (A beta) with the cell is the inhibition of cellular 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction. We recently demonstrated that cytotoxic amyloid peptides such as A beta and human amylin inhibit cellular MTT reduction by dramatically enhancing MTT formazan exocytosis. We now show the following: (a) Insulin and glucagon, when converted to fibrils with beta-pleated sheet structure, induce MTT formazan exocytosis that is indistinguishable from that induced by A beta. NAC35, an amyloidogenic fragment of alpha-synuclein (or NACP), also induces MTT formazan exocytosis. (b) All protein fibrils with the beta-pleated sheet structure examined are toxic to rat hippocampal neurons. (c) Many sterol sex hormones (e.g., estradiol and progesterone) block amyloid fibril-enhanced MTT formazan exocytosis as well as MTT formazan exocytosis in control cells by acting at a common late step in the exocytic pathway. Steroids fail, however, to protect hippocampal neurons from acute amyloid fibril toxicity. These findings suggest that the ability to enhance MTT formazan exocytosis and to induce neurotoxicity are common biological activities of protein fibrils with beta-pleated sheet structure but that enhanced MTT formazan exocytosis is not sufficient for acute A beta neurotoxicity.
The tetrazolium dye, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), is reduced by live but not dead cells, and this reaction is used as the end point in a rapid drug-screening assay. It can also be used for accurate determinations of drug sensitivity but only if a quantitative relationship is established between cell number and MTT-formazan production. We have shown that reduction of MTT to MTT-formazan by cells is dependent on the amount of MTT in the incubation medium. The concentration required to give maximal MTT-formazan production differs widely between cell lines. The absorption spectrum of MTT-formazan varies with cell number and with pH. At a low cell density or a high pH, the absorption maximum is at a wavelength of 560 to 570 nm. However, at a high cell density or a low pH, there are two absorption maxima; one at 510 nm and a second at about 570 nm. Measurements of absorbance at 570 nm underestimate MTT-formazan production and, hence, cell number at high cell densities. This error can result in a 10-fold underestimation of chemosensitivity. Addition of a buffer at pH 10.5 to the solubilized MTT-formazan product can overcome the effects of both cell density and culture medium on the absorption spectrum. Provided that sufficient MTT is used and the pH of the MTT-formazan product is controlled, dye reduction can be used to estimate cell numbers in a simple chemosensitivity assay the results of which agree well with a commonly used clonogenic assay.
XTT can be metabolically reduced by mitochondrial dehydrogenase in viable cells to a water-soluble formazan product. Thus XTT has been widely used to evaluate cell viability and to screen anti-HIV agents and the cytotoxicity of these agents. The present studies demonstrated that XTT formazan derived from XTT in cell culture significantly inhibits the fusion of HIV-1-infected cells with uninfected cells. Synthetic XTT formazan effectively inhibited the replication of laboratory-adapted and primary HIV-1 isolates and cell-to-cell fusion with low cytotoxicity. It blocks the six-helix bundle formation between peptides derived from the N- and C-terminal heptad repeat regions of the gp41 ectodomain (designated N- and C-peptides, respectively). Analysis by a computer-aided docking program indicates that XTT formazan may bind to the highly conserved hydrophobic pocket on the surface of the central trimeric coiled coil of gp41. These results suggest that XTT formazan inhibits HIV-1 entry by targeting the alpha-helical coiled-coil domain of gp41. This small molecular nonpeptide antiviral compound can be used as a lead for designing more effective HIV-1 entry inhibitors targeting the fusion stage of HIV-1 infection. But because XTT formazan itself has anti-HIV-1 activity, caution should be exercised when XTT is used to evaluate HIV-1 infectivity.
Metabolically active animal and plant cells reduce MTT tetrazolium salt to a corresponding nonfluorescent formazan. Reduction of MTT by viable cells is exploited in a number of tests widely used in biological research. The aim of this study was to optimize a microscopy method of detecting small crystals of MTT-formazan formed in intact cells maintained in in vitro cultures. We examined scattering properties of small intracellular crystals of MTT formazan and found that the efficiency of light scattering was dependent on wavelength. Small (<3 microm) crystals of MTT-formazan, formed in viable cells, scattered red, but not blue, light. Large crystals, which are formed later at a stage when cells begin to lose viability, scattered both red and blue light. We conclude that optimal detection of early stages of crystallization of MTT-formazan in living cells is possible using confocal microscopy of red, but not blue, scattered light. High contrast and resolution of images can be achieved by filtering out interference effects in the frequency domain.
In previous papers, the synthesis and chemical properties of iron-complexed azo and formazan dyes were reported. It was shown that in certain cases iron could be substituted for the traditionally used metals such as chromium and cobalt, without having an adverse effect on dye stability. While these results suggested that the iron analogs were potential replacements for the commercially used chromium and cobalt prototypes, characterization of potentially adverse environmental effects of the new dyes was deemed an essential step in their further development. The present paper provides results from using the Salmonella/mammalian microsome assay to determine the mutagenicity of some important commercial metal complexed dyes, their unmetallized forms, and the corresponding iron-complexed analogs. The study compared the mutagenic properties of six unmetallized azo dyes, six commercial cobalt- or chromium-complexed azo dyes, six iron-complexed azo dyes, six unmetallized formazan dyes, and six iron-complexed formazan dyes. The results of this study suggest that the mutagenicity of the unmetallized dye precursors plays a role in determining the mutagenicity of the iron-complexes. For the monoazo dye containing a nitro group, metal complex formation using iron or chromium decreased or removed mutagenicity in TA100; however, little reduction in mutagenicity was noted in TA98. For the formazan dye containing a nitro group, metal-complex formation using iron increased mutagenicity. Results varied for metal-complexes of azo and formazan dyes without nitro groups, but in general, the metal-complexed dyes based on mutagenic ligands were also mutagenic, while those dyes based on nonmutagenic ligands were nonmutagenic.
The history of the tetrazolium salts and formazans goes back 100 years, to when Friese (1875) reacted benzene diazonium nitrate with nitromethane, to produce a cherry-red "Neue Verbindung". This was the first formazan. 19 years later, Von Pechmann and Runge (1894) oxidised a formazan to produce the first tetrazolium salt. Many hundreds of tetrazolium salts and formazans were prepared in the following years, but only a handful have found applications in biological research. This article has attempted to describe the properties of these compounds, and to illustrate how the tetrazolium salt-formazan reaction has been exploited to serve an extremely wide variety of functions.
The enzyme succinate dehydrogenase was demonstrated histochemically in organs of rats, especially in experimentally infarcted hearts. 88 tetrazolium salts were used as indicators, the resulting formazans were examined for birefringence. Formazans with cristalline shapes show birefringence, but formazans of amorphous character do not. In the atypic species of nitrogroup-substituted monoformazans it is possible to form an oriented association to cell structures in spite of an amorphous precipitation. Therefore, anomalous colours of polarization, and dichroism is observed. Relations are discussed between shape of formazan deposits, polarity of formazans, and the occurrence of optical anisotropism.
Synthesis and biological evaluation of a series of formazans and their corresponding tetrazolium salts are described. Several 1,3,5-trisubstituted formazans were prepared by condensing 4-acetoxy-3-methoxybenzaldehydephenylhydrazone and 2,4-dinitrophenylhydrazone of 2-benzimidazolecarboxaldehyde, with an appropriate phenyldiazonium salt. The formazans derived from 4-acetoxy-3-methoxybenzaldehydephenylhydrazone were further oxidized to their corresponding cyclization products, 2,3,5-trisubstituted tetrazolium salts. Oxidation of 1-aryl-3-(2'-benzimidazolyl)-5-(5-(2',4'-dinitrophenyl) formazans to their corresponding tetrazolium salt did not meet with success. Both the highly coloured formazans and water-soluble tetrazolium salts were screened for their antiviral, activity against vaccinia virus and Ranikhet disease virus in a stationary culture of chorioallantoic membrane of chick embryo. Two of the compounds were found to exhibit significant activity (87% and 83%) against the Ranikhet disease virus. However no activity against vaccinia virus could be observed.
The tetrazolium salt 3(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) is reduced to formazan by the succinate dehydrogenase system of active mitochondria, and hence, specifically used to assay for the viable cells, such as measurement of cell proliferation, cytotoxicity, and cell number. However, in the present study we have shown that some component specifically present in M199 but not in RPMI 1640 media can reduce MTT to formazan in the absence of a living system. Further study revealed that ascorbic acid reduced MTT to formazan, which was profoundly increased by a very small amount of retinol, whereas retinol alone had no effect. Oxidation of ascorbic acid by H(2)O(2) destroyed its ability to reduce MTT. The rate of MTT reduction was directly proportional to the concentration of MTT in the absence of retinol, but approached a zero-order state beyond a certain concentration of MTT in the presence of retinol. Furthermore, retinol remained unchanged after the completion of the reaction. Taken together, these results showed that retinol acts as a reductase that catalyzes the reduction of MTT to formazan using ascorbic acid as the cosubstrate (electron donor).
We describe a novel system for measuring the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction in three-dimensional histoculture which is no longer dependent on colorimetric determination of extracted formazan, but rather is based on a pixel image analysis of formazan crystals, and which allows intratumor heterogeneity to be taken into account. The MTT test is based on the enzymatic reduction of the tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-dipheniltetrazolium bromide to formazan crystals by living, metabolically active cells, but not in dead cells. The reaction was carried out in situ in six-well plates on gel-supported histocultured human tumors. After a 24-h incubation with different drugs the tumors were incubated with a solution of MTT. Frozen sections of the tumor pieces were made and the slides were then stained with a propidium iodide solution, whose fluorescence is proportional to the number of cells present. We demonstrate here that the formazan crystals, formed by MTT reduction, reflect polarized light and that this can be quantified by using an image analysis system based on bright-pixel quantitation directly on a frozen section of the original tissue. Combined with the use of the fluorescent dye propidium iodide, also measured by pixel analysis, we can express a ratio between the total amount of MTT reduction and the total number of cells present in the specimen that expresses the effect of drugs on the histocultured tumors. Since histology is well maintained in histoculture it is possible to take into account the heterogeneity present in the tumor with regard to drug response.(ABSTRACT TRUNCATED AT 250 WORDS)
MTT is taken up by cells by endocytosis and reduced to formazan in the endosomal/lysosomal compartment. Formazan is deposited intracellularly as blue granules and is later exocytosed as needle-like formazan crystals. The present study involves an analysis of the pattern of exocytosis of MTT in different cell types showing clearcut differences in the response that can be associated to their ability to phagocytose. To further assess the characteristics of the exocytic mechanism of MTT/formazan, different experimental conditions were assayed. When culture medium with decreasing serum concentration was used as a metabolic modulator no variations were observed in the proportion of cells with formazan crystals. Conversely, the markedly sensitivity of phagocytic cells to increasing concentrations of genistein constituted a remarkable difference with non-phagocytic cells. These results must be considered when the modulation of MTT exocytosis is used as a signal of the progress of human diseases.
OBJECTIVES: To apply an enzymatic method for assaying uric acid in serum based on the uricase (EC 1.7.3.3)-catalase (EC 1.11.1.6)-formaldehyde dehydrogenase (FADH, EC 1.2.1.46) coupled with the new tetrazolium salt producing a water-soluble formazan dye as an indicator system. Unlike the traditional tetrazolium salts, e.g., iodonitrotetrazolium (INT) and nitrotetrazolium blue (NTB), the corresponding formazan dye produced did not absorb to the test tube and the reaction cells of the analyzer. Moreover, this formazan dye had a higher water solubility and more sensitivity. DESIGN AND METHODS: A two electron reduction of tetrazolium salt with NADH, produced by the uricase-catalase-FADH reaction, was mediated by an electron carrier, 1-methoxy PMS. The generation of formazan, monitored at 440 nm, was proportional to the concentration of uric acid in serum. The sensitivity of this method was about 1.5 times that of the peroxidase-TOOS [N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine] method. The assay was evaluated with TBA-80FR.NEO biochemical analyzer. The average within-run and between-day imprecision (CV) were 0.75-2.44% and 3.20-3.33%, respectively. RESULTS: Results of the proposed method (y) correlated well with those determined by the conventional chromogen method (x): y = 0.970 x - 0.018 mmol/L (Sy l x = 0.019 mmol/L, r = 0.993, n = 67). CONCLUSIONS: We also present data showing that the method is rapid, relatively free of interference, and amenable to automation.
Alzheimer's disease amyloid beta protein (Abeta) inhibits cellular reduction of the dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Kaneko et al. have previously hypothesized that Abeta works by suppressing mitochondrial succinate dehydrogenase (SDH), but Liu and Schubert have recently demonstrated that Abeta decreases cellular MTT reduction by accelerating the exocytosis of MTT formazan in neuronal cells. To ask which is the case in astrocytes, we compared the effects of Abeta and 3-nitropropionic acid (3-NP), a specific SDH inhibitor, on MTT reduction in cultured rat cortical astrocytes. Treatment with 3-NP (10 mM) decreased cellular activity of MTT reduction, regardless of the time of incubation with MTT. On the other hand. Abeta-induced inhibition of cellular MTT reduction was dependent on the time of incubation with MTT. The cells treated with Abeta (0.1-1000 nM) exhibited normal capacity for MTT reduction at an early stage of incubation ( < 30 min), but ceased to reduce MTT at the late stage (> 1 h). Microscopic examination revealed that Abeta treatment accelerated the appearance of needle-like MTT formazan crystals at the cell surface. These observations support that Abeta accelerates the exocytosis of MTT formazan in astrocytes. In addition to inhibition of MTT reduction, Abeta is known to induce morphological changes in astrocytes. Following addition of Abeta (20 microM), polygonal astrocytes changed into process-bearing stellate cells. To explore a possible linkage between these two effects of Abeta, we tested if astrocyte stellation is induced by agents that mimic the effect of Abeta on MTT reduction. Cholesterol (5 5000 nM) and lysophosphatidic acid (0.2-20 microg/ml) were found to accelerate the exocytosis of MTT formazan in a similar manner to Abeta, but failed to induce astrocyte stellation. Therefore, Abeta-induced inhibition of MTT reduction is unlikely to be directly linked to its effect on astrocyte morphology.