3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) causes Akt phosphorylation and morphological changes in intracellular organellae in cultured rat astrocytes

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) is widely used for cell viability and cytotoxicity assays, but cell biological effects of MTT itself have not been investigated. In this paper we show that MTT induces a morphological change in an intracellular membranous compartment labeled with anti-Rab5 antibody, dissociation of early endosomal auto-antigen (EEA1) from the membrane fraction, and phosphorylation of Akt probably through a phosphatidylinositol-3-OH kinase [PI(3)K] pathway in cultured rat astrocytes. These findings suggest that MTT affects cellular functions and conditions to some extent, and such effects of MTT may cause some discrepancies of measurement of cell viability using MTT assay and other assays. That is, the effects of MTT on cells could influence the results of cell viability assay. Moreover, MTT or other tetrazolium salts could be used as interesting activators of Akt to investigate the mechanism by which Akt or PI(3)K is activated.     

The role of plasma membrane in bioreduction of two tetrazolium salts, MTT, and CTC

Despite widespread use of various tetrazolium assays, the mechanisms of bioreduction of these compounds have not been fully elucidated. We investigated the capacity of tetrazolium salts to penetrate through intact cell plasma membranes. 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) tetrazolium salts appear to represent examples of species that are reduced by different mechanisms. We provide evidence suggesting that MTT readily crosses intact plasma membranes and is reduced intracellularly. MTT appears to be reduced by both plasma membrane and intracellular reductases; reducing cells are not damaged and remain metabolically active for at least 45 min. In contrast, CTC remains extracellular with respect to viable cells and thus requires plasma membrane permeable electron carrier to be reduced efficiently. However, reduction of CTC in the presence of an electron carrier inflicts damage on plasma membranes. The intracellular vs extracellular sites of reduction of tetrazolium salts were established on the basis of deposition of formazans. Crystals of formazan were detected using fluorescence or backscattered light confocal laser microscopy. We postulate that the capacity of a tetrazolium salt to cross intact plasma membranes constitutes an important experimental variable which needs to be controlled in order to correctly interpret the outcome of tetrazolium assays designed to measure cellular production of oxygen radicals, activity of mitochondrial, cytosolic, or outer membrane reductases, etc.     

Potential of a soluble tetrazolium/formazan assay for the evaluation of filarial viability

Using female Acanthocheilonema viteae we have investigated the bioreduction of the tetrazolium reagent XTT (2,3-bis(2-methoxy-4-nitro-sulphonyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide). Unlike the formazan formed by other tetrazolium salts, that derived from XTT readily diffuses out of A. viteae in vitro. Formazan formation can therefore be quantified by direct absorbance reading of the incubation medium, eliminating the need for a DMSO solubilization step. Optimum assay conditions involved a 4 h incubation, in the presence of the electron coupling agent phenazine methosulphate (PMS). Repeat 4 h incubations with XTT-PMS were well tolerated by worms for 5 consecutive days. This confirmed the low toxicity of XTT formazan and its usefulness in the semi-continuous assessment of filarial viability. In comparison to our previously reported MTT (3-(4, 5 dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide)-reduction assay XTT-PMS reduction showed comparable drug sensitivity and accuracy, however XTT-PMS appears to be at least 10-15 times less efficiently reduced by A. viteae females. A possible application of the XTT assay using female Onchocerca volvulus is discussed.     

Applicability of tetrazolium salts for the measurement of respiratory activity and viability of groundwater bacteria

A study was undertaken to measure aerobic respiration by indigenous bacteria in a sand and gravel aquifer on western Cape Cod, MA using tetrazolium salts and by direct oxygen consumption using gas chromatography (GC). In groundwater and aquifer slurries, the rate of aerobic respiration calculated from the direct GC assay was more than 600 times greater than that using the tetrazolium salt 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl tetrazolium chloride (INT). To explain this discrepancy, the toxicity of INT and two additional tetrazolium salts, sodium 3'-[1-(phenylamino)-carbonyl]-3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzenesulfonic acid hydrate (XTT) and 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), to bacterial isolates from the aquifer was investigated. Each of the three tetrazolium salts was observed to be toxic to some of the groundwater isolates at concentrations normally used in electron transport system (ETS) and viability assays. For example, incubation of cells with XTT (3 mM) caused the density of four of the five groundwater strains tested to decline by more than four orders of magnitude. A reasonable percentage (>57%) of cells killed by CTC and INT contained visible formazan crystals (the insoluble, reduced form of the salts) after 4 h of incubation. Thus, many of the cells reduced enough CTC or INT prior to dying to be considered viable by microscopic evaluation. However, one bacterium (Pseudomonas fluorescens) that remained viable and culturable in the presence of INT and CTC, did not incorporate formazan crystals into more than a few percent of cells, even after 24 h of incubation. This strain would be considered nonviable based on traditional tetrazolium salt reduction assays. The data show that tetrazolium salt assays are likely to dramatically underestimate total ETS activity in groundwater and, although they may provide a reasonable overall estimate of viable cell numbers in a community of groundwater bacteria, some specific strains may be falsely considered nonviable by this assay due to poor uptake or reduction of the salts.     

Serum albumin leads to false-positive results in the XTT and the MTT assay

Tetrazolium salts like 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) or sodium 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) that form formazans after reduction are widely used to investigate cell viability. Besides cellular enzymes, some constituents of cell media and other substances reduce tetrazolium salts, thereby interfering with these assays. We describe here that different preparations of serum albumin from bovine or human origin can lead to a concentration-dependent increase in the signals of the XTT assay; therefore leading to an overestimation of cell numbers and to an underestimation of potential cytotoxic effects of compounds to be tested. The same effect was seen in the MTT assay with human serum albumin (HSA). We demonstrate that this reductive activity cannot be inactivated by proteolytic digestion, but that it is due to the free cysteine residue in albumin, and is also observed when cysteine or glutathione (GSH) are used. Binding of N-ethylmaleimide (NEM) to the free cysteine residue leads to a decrease of the albumin interference in the XTT assay.     

Superoxide anion radical-independent pathway for reduction of tetrazolium salts in aerobic mixtures consisting of NADH and 5-methylphenazinium methyl sulfate in the presence of aqueous micelles of nonionic and cationic detergents

The effect of Triton X-100 and certain other nonionic as well as cationic detergents on 5-methyl-phenazinium methyl sulfate (PMS)-mediated reduction of tetrazolium salts was studied under aerobic conditions using an exogenous source of reducing equivalents, such as NADH or by generating NADPH through an enzymatic reaction. In the absence of detergents, 5,10-dihydro-5-methylphenazine (MPH), formed on reduction of 5-methylphenazinium cation (MP+) of PMS by NAD[P]H, was reoxidized allowing first the univalent reduction of molecular oxygen (O2) to the superoxide anion radical (O2-.) which, in turn, reduced tetrazolium salts. In the presence of detergents, however, a significant fraction of the PMS-mediated reduction of tetrazolium salts appeared to proceed without the intervention of O2-. The reasons for this were examined experimentally and it was suggested that the reduced phenazine (i.e., MPH), which is sparingly soluble in aqueous solutions, migrates into detergent micelles where tetrazolium salts are reduced in preference to O2. By lowering the pH and thereby facilitating the H+-mediated dismutation of O2-., it was possible to obtain the reduction of tetrazolium salts, mediated selectively and directly by MPH in the micellar pseudophase. Employing the technique of saturation analysis, further evidence was obtained that lends support for preferential reduction of tetrazolium salts (e.g., nitroblue tetrazolium chloride) to that of O2 by the micelle-bound MPH.     

Apoptotic effect of eugenol in human colon cancer cell lines

Eugenol, a natural compound available in honey and various plants extracts including cloves and Magnoliae flos, is exploited for various medicinal applications. Since most of the drugs used in the cancer are apoptotic inducers, the apoptotic effect and anticancer mechanism of eugenol were investigated against colon cancer cells. Antiproliferative effect was estimated using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay]. Earlier events like MMP (mitochondrial membrane potential), thiol depletion and lipid layer break were measured by using flow cytometry. Apoptosis was evaluated using PI (propidium iodide) staining, TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling) assay and DNA fragmentation assay. MTT assay signified the antiproliferative nature of eugenol against the tested colon cancer cells. PI staining indicated increasing accumulation of cells at sub-G1-phase. Eugenol treatment resulted in reduction of intracellular non-protein thiols and increase in the earlier lipid layer break. Further events like dissipation of MMP and generation of ROS (reactive oxygen species) were accompanied in the eugenol-induced apoptosis. Augmented ROS generation resulted in the DNA fragmentation of treated cells as shown by DNA fragmentation and TUNEL assay. Further activation of PARP (polyadenosine diphosphate-ribose polymerase), p53 and caspase-3 were observed in Western blot analyses. Our results demonstrated molecular mechanism of eugenol-induced apoptosis in human colon cancer cells. This research will further enhance eugenol as a potential chemopreventive agent against colon cancer.     

Use of MTT assay for determination of the biofilm formation capacity of microorganisms in metalworking fluids

Biofilm formation is a well-known problem in management of metalworking fluid systems. Due to persistence of microorganisms within biofilms, the reappearance of various species of bacteria, including nontuberculous mycobacteria is often observed after the use of biocides and/or cleaning of delivery systems and replacement of cooling fluid. The aim of this study was to determine the usefulness of the tetrazolium salt assay (MTT assay) for assessing the viability of bacteria in biofilms formed in vitro in fresh and used cutting oils, as well as their susceptibility to antimicrobial biocides. Biofilms were established in the microtiter plate format. The results showed that quantification of formazan, a product of the tetrazolium salt reduction by electron transport system could be used for determination of the propensity of bacteria to form biofilms in these complex media. The use of the assay allows also determination of antimicrobial activity of biocides against biofilms in fresh and used metalworking fluids. Biofilms produced by Gram-negative isolates recovered from field metalworking fluids as well as the wild bacterial communities differed in metabolic activity depending on the type of fresh coolants. The MTT assay has high-throughput potential and can be efficiently used for determination of biofilm-forming capacity of microorganisms from individual machines in metalworking industry. The use of the assay may also guide the selection of the most appropriate biocide to fight these microorganisms.     

Effects of high glucose concentration on survival and respiration of human endothelial cells.

To determine whether or not endothelial cell survival was decreased after incubation with high glucose concentrations in culture media, we studied the influence of D-glucose or L-glucose (a non-metabolizable stereoisomer of D-glucose) on cell survival using the trypan-blue exclusion test. Simultaneously, the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide) assay was used to measure both the mitochondrial respiratory chain activity and cell viability. Respiratory chain activity per cell increased when D-glucose concentrations rose but at the same time trypan-blue excluded cells were decreased. Comparison with data in the literature showed that the MTT assay was not reliable for studies involving endothelial cell survival when glucose reduction was affected on these cells. It seems important to check MTT assay reliability carefully when it is used for drugs affecting glucose metabolism, or with other cell types.     

Assessment of bovine sperm viability by MTT reduction assay

The MTT reduction assay depends on the ability of metabolically active cells to reduce the tetrazolium salt (3[4,5-dimethylthiazol-2-y1]-2,5-diphenyltetrazolium bromide) to formazan. This study was conducted to examine and validate a simple and less costly MTT test to determine bovine sperm viability and compare the efficiency of this test with a flow cytometer. Fresh ejaculates from eight bulls were included in this study. Semen sample was diluted to 30x10(6) sperms/ml in a Hepes 0.1% BSA. The rates of MTT reduction were measured in microtiter plates after incubation for 1h at 37 degrees C using spectrophotometer (MS2 Reader) at wave length 550nm. Simultaneously split samples of the same semen were tested, using a flow cytometer for sperm viability, mitochondrial activity, and acrosomal integrity using SYBR-14, Rhodamine 123 and LysoTracker Green DNA-26, respectively. The correlation between the results of these tests was calculated using the Pearson correlation coefficients. The results revealed a strong correlation (P<0.001) between the results of MTT reduction rate and the results that simultaneously determined by flow cytometer, yielding correlation coefficients of r=0.950 for sperm viability, of r=0.926 for mitochondrial activity and of r=0.959 for acrosomal integrity. The same correlation coefficient was observed between the values of sperm viability calculated on the basis of MTT reduction rates and the results of flow cytometer. In conclusion, the MTT reduction test was found to be a reliable method in evaluating bovine semen viability and can be used successfully, especially in routine analysis, where practical aspects such as time, costs and practicability are important.     

Macelignan protects HepG2 cells against tert-butylhydroperoxide-induced oxidative damage

In this study, we investigated the protective effect of macelignan, isolated from Myristica fragrans Houtt. (nutmeg) against tert-butylhydroperoxide (t-BHP)-induced cytotoxicity in a human hepatoma cell line, HepG2. The tetrazolium dye colorimetric test (MTT test) and lactate dehydrogenase (LDH) assay were used to monitor cell viability and necrosis, respectively. Lipid peroxidation [malondialdehyde (MDA) formation] was estimated by the fluorometric method. Intracellular reactive oxygen species (ROS) formation was measured using a fluorescent probe 2',7'-dichlorofluorescein diacetate (DCFH-DA), and DNA damage was detected using single cell gel electrophoresis (comet assay). The results showed that macelignan significantly reduced the cell growth inhibition and necrosis caused by t-BHP. Furthermore, macelignan ameliorated lipid peroxidation as demonstrated by a reduction in MDA formation in a dose-dependent manner. It was also found that macelignan reduced intracellular ROS formation and DNA damaging effect caused by t-BHP. These results strongly suggest that macelignan has significant protective ability against oxidative damage caused by reactive intermediates.     

Investigation of different modifications to the tetrazolium based colorimetric viability assay

Summary Three commonly used solvents for the 3-(4,5-dimethylthiazolyl-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) based viability assay for mammalian cells were compared: Acid/propan-2-ol, dimethyl sulfoxide (DMSO) and a lysing buffer containing sodium dodecylsulphate (SDS). Acid/propan-2-ol and DMSO could only be used when most of the medium was removed from the cell suspensions, whereas the lysing buffer was found to perform satisfactorily for both hybridomas and fibroblast cell lines without medium removal for cell densities up to 10⁶ cells mL⁻¹. Furthermore a newly synthesized tetrazolium salt was investigated, which forms a water soluble formazan upon reduction and thus eliminates the need for a solvent. However this salt adds a new complication to the method: the need for an electron carrier. For this reason we do not find that the new tetrazolium salt has any practical advantages over MTT.     

WST-1-based cell cytotoxicity assay as a substitute for MTT-based assay for rapid detection of toxigenic Bacillus species using CHO cell line

Bacillus cereus continues to be one of the important foodborne pathogens due to its ability to produce various heat-labile and -stable toxins. Several methods have been developed to assess the pathogenicity of the B. cereus strains; however, most of these take more than 2–3 days to provide confirmatory results. In this study we standardized a one-step cytotoxicity assay using WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) and compared with the traditional MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide)-based assay for rapid detection of cytotoxic Bacillus spp. using Chinese hamster ovary (CHO) cell line. Crude toxin preparations from 50 isolates of Bacillus spp. were exposed to CHO cell line for 1 h or 24 h and the cytotoxicity was determined by using WST-1 and MTT-based methods. Most B. cereus strains and some strains of other Bacillus species from our collection or from food sources showed comparably high cytotoxicity using either of the methods (P = 0.81); however, WST-1 assay provided results in only 3 h while MTT assay in 44–52 h. A positive correlation (R² = 0.93) between WST-1 and MTT assays strongly suggests that the WST-1-based cytotoxicity assay could be used as an alternative method to MTT assay for rapid (3 h) confirmation of toxigenic Bacillus species in foods prior to their retail distribution or consumption.     

Colorimetric quantitation of filarial viability

A simple three-step colorimetric assay based on the tetrazolium salt MTT (3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) has been developed for quantifying filarial viability. Living (but not dead) filariae take up MTT and rapidly reduce it to formazan, so staining themselves dark blue. This colour change which is easily seen provides a rapid qualitative test for filarial viability. Quantitative data can be obtained by solubilizing formazan out of the worm with DMSO and measuring the absorbance of the resulting solution at 510 nm. To date the technique has been demonstrated in several species of filariae including Onchocerca volvulus. MTT reduction is thought to be selective for NADH-dependent dehydrogenase activity in viable worms. The reaction occurs readily in all developmental stages of Dipetalonema viteae including fragments of filarial tissue. Enzyme activity in viable intact D. viteae appears to be primarily associated with the hypodermis/muscle cells, with minimal formazan formation in the gut and reproductive tracts. The application of this MTT assay as a parameter for quantifying in vitro drugs effects is described. Assay procedures have been developed and optimized with D. viteae and Brugia pahangi for the assessment of effects of macrofilariae and microfilarial release, and the activity of a range of antifilarial standards reported. Several potential applications of the technique to studies on filarial biology are discussed.     

Polarographic investigation on tetrazolium reductase activity

Summary An attempt was made to elaborate a simple method of tetrazolium reductase activity assay in biological material. The method is based on recording of changes in the amperage of threshold current, occuring in course of the reduction of tetrazolium salts. — The method could be employed by histochemists working on the reduction of tetrazolium salts.     

Colorimetric cell proliferation assay for microorganisms in microtiter plate using water-soluble tetrazolium salts

A colorimetric method to assay cell proliferation of microorganisms in 96-well microtiter plates using water-soluble tetrazolium salts and electron mediators was developed. Combinations of 6 kinds of water-soluble tetrazolium salts and 27 kinds of electron mediators that considered the metabolic efficiency of microorganisms and the influence with medium components were investigated. 2-Methyl-1,4-naphthoquinone (NQ) was reduced most effectively by various species of microorganisms, and a combination of WST-8 as a water-soluble tetrazolium salt with 2-methyl-1,4-NQ repressed the increase in background due to medium components. In the presence of 2-methyl-1,4-NQ, WST-8 was reduced by microbial cells to formazan, which exhibited maximum absorbance at 460 nm. The proposed tetrazolium method could be applied to measure proliferations of various microbial cells including 3 kinds of yeast, 9 kinds of Gram-positive bacteria, and 10 kinds of Gram-negative bacteria. Linear relationships between the absorbance and viable microbial cell density were obtained in all microorganisms, suggesting that the absorbance change reflected the microbial cell proliferation.     

One step forward to improve the latest method of antibacterial susceptibility testing of vitro-cultured bacteria: an implication for antibacterial efficacy of Enrofloxacine on <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis>

Recently a new method of antibacterial susceptibility testing has been developed, which based on the reduction of the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) by bacteria. This study aimed to improve the latest developed method in terms of the period of time, concentration of bacterium, accuracy of assay and minimizing the toxic effect of the MTT solution. The commonly used broth microdilution method was compared in this study, as well. The minimum inhibition concentration (MIC) of the enrofloxacine against Aeromonas hydrophila as an opportunistic and ubiquitous bacterium was tested by using the improved MTT method. Using both methods showed that enrofloxacine exerts an excellent antibacterial effect against A. hidrophyla with MIC value of 62.5 ng/ml. The simplicity of the improved MTT method was shown with performing all steps of the assay in one plate, using rather low concentration of the bacterium (500 CFU/well) and shortening of the incubation time to 9 h. Moreover, 5–30 min incubation of bacteria with MTT solution excludes any toxic effect of tetrazolium salt against bacteria. Thus, our results suggest that enrofloxacine might be considered as a potent antibacterial agent against A. hydrophila induced contaminations. Moreover, MTT method with current improved characteristics such as short incubation time, low concentration of bacteria, and high sensitivity could be more practical alternative for the broth microdilution method in antibacterial susceptibility testing protocol.     

Menadiol diphosphate, a new substrate for non-specific alkaline phosphatase in histochemistry and immunohistochemistry.

Menadiol diphosphate was introduced as a new substrate for nonspecific alkaline phosphatase, following a search for new and less expensive substrates, which give a more sensitive response and are easily synthesized in the laboratory. Menadiol released by phosphatase action can be assayed by its reduction of tetrazolium salts, or it can be coupled with diazonium salts; alternatively, the phosphate can be trapped by metal ions. The synthesis and purification of menadiol diphosphate are described, and it was shown to be sufficiently stable for qualitative and semiquantitative histochemistry, as well as for the immunohistochemistry of enzymes and cytoskeletal proteins with nonspecific alkaline phosphatase as the enzyme label. For qualitative as well as semiquantitative histochemistry and immunohistochemistry, the best results were obtained by applying the method with nitro-blue tetrazolium (NBT) to acetone-chloroform pretreated cryostat sections. Tetranitro-blue tetrazolium (TNBT), benzothiazolylphthalhydrazidyl tetrazolium (BSPT) and various diazonium salts were less suitable. Fast Blue BB and VB produced satisfactory results. Ce3+ ions and the DAB-Ni-H2O2 procedure yielded better results than Ca2+ ions in the Co-(NH4)2S visualization method. The NBT method with menadiol diphosphate is superior to existing methods employing azo, azoindoxyl or tetrazolium salts and to metal precipitation methods. The Ce3+ technique and the NBT/menadiol diphosphate method give similar results, and appear to be of equal value. In qualitative histochemistry and immunohistochemistry the NBT/menadiol diphosphate method resulted in higher quantities of precisely localized stain. Semiquantitative histochemistry with minimal incubation revealed more favorable kinetics for the menadiol diphosphate method, especially when using NBT.     

A model for NADH-tetrazolium reductase

Summary In the presence of light, reduced nicotinamide adenine dinucleotide (NADH) and riboflavin formed a complex which was able to reduce certain tetrazolium salts. Neither NADH (10^–3 M) nor riboflavin (10^–4 M) alone was able to induce tetrazolium reduction in the presence of oxygen, but in a nitrogen atmosphere photoreduction of riboflavin induced reduction of tetrazolium salts. Only electrophilic nitro and thiazolyl substituted tetrazolium salts with more positive redox potentials were reduced by the NADH-riboflavin complex, and only monoformazans were produced from the ditetrazolium salts. The reduction kinetics of these tetrazolium salts are given, and the spectral area capable for induction of electron transfer in the NADH-riboflavin complex is screened. It is concluded that the electron transfer in flavin nucleotide dependent dehydrogenase systems will probably proceed without direct interference with the apoenzyme. This may have practical implications for the histochemistry of tetrazolium reductases especially as regards fixation. The catalytic action of light on tetrazolium reduction should also be taken into consideration when tetrazolium salts are used as electron acceptors in a histochemical reaction.