Styrylphenyl tetrazolium chloride as a location reagent for thiolsteroids on paper chromatograms.
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Chromatography, Paper↗
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Results for “TETRAZOLIUM COMPOUNDS”
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N.B.T. (nitroblue-tetrazolium dye) test--false-negative and false-positive results.
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Adult↗
The role of quality control in a skin bank: tissue viability determination.
New surgical procedures requiring viable skin have increased rapidly over the last few years. The cell viability assessment in allograft skin is a major step forward in burn treatment, since it is well-known that taking is correlated with grafted tissue viability. Various methods, both qualitative and quantitative, are currently used. Although qualitative assays (histomorphology, immunocytochemistry) are routinely performed in our laboratory, there arose a need to set up a standardised quantitative assay in an attempt to obtain a cut-off value so that the skin sample could be determined valid or not for grafting. Therefore, two different tetrazolium salt compounds MTT and WST-1, were compared in order to determine their efficacy in the evaluation of tissue viability. Several experimental conditions were analysed: 1- cellular cultures of keratinocytes and fibroblasts, 2- fresh skin tissue samples, 3- the same specimen tested daily for at least 2 weeks, 4- after cryopreservation and thawing. Viable cells were analysed by the cleavage of tetrazolium salts to formazan by cellular enzymes. The formazan dye produced by metabolically active cells was then quantified by measuring the absorbance of the dye solution at the appropriate wavelength. It was seen that WST-1 is easier to handle, more stable, has a wider linear range, accelerated colour development and is more sensitive than MTT on fresh specimens and cell suspension. However, after 72 hours of storage at 4 degrees C, most of the WST-1 tested specimens no longer gave any absorbance signal, whilst MTT specimens were seen to give a signal for more than two weeks. Moreover, after thawing WST-1 tested samples were almost negative, whilst MTT samples continued to give strong signals. In conclusion, WST-1 assay offers rapid and precise results as to the cell viability of fresh allografts and cell cultures, whilst the MTT method is much more useful in establishing viability after long conservation and cryopreservation. In our clinical experience, allografts transplanted at 72 hr post-harvesting or after cryopreservation showed a mean of take more than of 80%, demonstrating that the MTT system is more reliable for the determination of allograft viability. Studies are ongoing with larger clinical cohorts to establish the precise cut-off value for skin graft validation.
Journal Article↗
Nothing dehydrogenase reaction as an artefact in serum isoenzyme analyses.
A nonspecific staining occurs in serum in isoenzyme methods based on tetrazolium staining reactions. The artefact is pronounced when a large application volume, a prolonged incubation time, distinctly alkaline conditions, or excess of phenazine methosulphate is used. Only phenazine methosulphate and a tetrazolium stain are required for the appearance. Inhibition studies and histochemical staining reactions give evidence that protein-bound sulphydryl groups are responsible for the artefact in serum.
Chloromercuribenzoates↗
A high throughput colorimetric cell proliferation assay for the identification of human cytomegalovirus inhibitors.
A colorimetric assay based on the cleavage of the tetrazolium salt WST-1 has been developed for human cytomegalovirus (HCMV) antiviral susceptibility testing and adapted to a microtiter plate format. Optimal conditions were determined and the standard routine assay was calibrated with a viral input of 0.05-0.10 plaque forming unit (p.f.u.)/cell with a density of 2000 cells/well in a 96-well microtiter plate for an incubation period of 7 days. Ganciclovir (9-(2-hydroxy-1(hydroxymethyl) ethyoxymethyl) guanine; DHPG), and cidofovir ((S)-1-(3-hydroxy-2-phosphonylmethoxypropyl) cytosine; HPMPC) were used as positive control test compounds to validate the assay. The effective EC50 concentration values obtained with the two antiviral compounds in the present assay were in good agreement with plaque reduction assay results performed in parallel experiments. This method presents the advantage of being easy and rapid to perform, reliable, reproducible, and convenient for use in a high throughput screening capacity.
Antiviral Agents↗
Cytotoxicity of S-conjugates of the sevoflurane degradation product fluoromethyl-2,2-difluoro-1-(trifluoromethyl) vinyl ether (Compound A) in a human proximal tubular cell line.
Fluoromethyl-2,2-difluoro-1-(trifluoromethyl)vinyl ether (FDVE) is a fluorinated alkene formed by degradation of the volatile anesthetic sevoflurane in anesthesia machines. FDVE is nephrotoxic in rats but not humans. Rat FDVE nephrotoxicity is attributed to FDVE glutathione conjugation and bioactivation of subsequent FDVE-cysteine S-conjugates, in part by renal beta-lyase. Although FDVE conjugation and metabolism occur in both rats and humans, the mechanism for selective toxicity in rats and lack of effect in humans is incompletely elucidated. This investigation measured FDVE S-conjugate cytotoxicity in cultured human proximal tubular HK-2 cells, and compared this with known cytotoxic S-conjugates. HK-2 cells were incubated with FDVE and its GSH, cysteine S-mercapturic acid, cysteine S-sulfoxide, and mercapturic acid sulfoxide conjugates (0.1-2.7 mM) for 24 h. Cytotoxicity was determined by lactate dehydrogenase (LDH) release, total LDH, and the ability of viable cells to reduce a tetrazolium-based compound (MTT). FDVE was cytotoxic only at concentrations >/=0.9 mM. No increase in LDH release was observed with either FDVE-GSH conjugate. The FDVE-cysteine conjugates S-(1,1-difluoro-2-fluoromethoxy-2-(trifluoromethyl) ethyl)-L-cysteine (DFEC) and (Z)-S-(1-fluoro-2-fluoromethoxy-2-(trifluoromethyl) vinyl)-L-cysteine ((Z)-FFVC) caused significant differences in LDH release and MTT reduction only at 2.7 mM; (Z)-FFVC was slightly more cytotoxic. Both S-(1,1-difluoro-2-fluoromethoxy-2-(trifluoromethyl) ethyl)-L-cysteine sulfoxide (DFEC-SO) and (Z)-N-acetyl-S-(1-fluoro-2-fluoromethoxy-2-(trifluoromethyl) vinyl)-L-cysteine sulfoxide ((Z)-N-Ac-FFVC-SO) caused slightly greater changes in LDH release or total LDH than the corresponding equimolar DFEC and (Z)-N-acetyl-S-(1-fluoro-2-fluoromethoxy-2-(trifluoromethyl) vinyl)-L-cysteine ((Z)-N-Ac-FFVC) conjugates. In contrast to FDVE S-conjugates, S-(1,2-dichlorovinyl)-L-cysteine was markedly cytotoxic, at concentrations as low as 0.1 mM. These results show that human proximal tubular cells are relatively resistant to FDVE and FDVE S-conjugate cytotoxicity. This may partially explain the lack of FDVE nephrotoxicity in humans.
Anesthetics, Inhalation↗
The mechanism of Hg2+ toxicity in cultured human oral fibroblasts: the involvement of cellular thiols.
To study amalgam-related toxicity in a primary target cell type, human oral fibroblasts were grown in a low-serum medium containing 1.25% fetal bovine serum and exposed to Hg2+, a corrosion product of amalgam. A 1-h exposure to various concentrations of Hg2+ resulted in a dose-dependent loss of colony forming efficiency. Removal of the low-molecular-weight thiol cysteine from the medium increased the toxicity of Hg2+ almost 50-fold in comparison with complete medium or medium without fetal bovine serum. Accordingly, fetal bovine serum was not found to contain detectable levels of low-molecular-weight thiols. The levels of cellular free protein thiols were shown to be depleted Hg2+ at significantly lower concentrations of the metal ion than those required to decrease the levels of the major cellular low-molecular weight thiol glutathione. These decreases were dependent on the exposure conditions, i.e. the presence of serum and thiols, in a manner similar to the effect on colony forming efficiency. Other functions commonly related to cell viability, including the accumulation of the vital dye neutral red, the cytosolic retention of deoxyglucose and the mitochondrial reduction of tetrazolium were also inhibited by Hg2+, albeit at higher concentrations. Finally, the depletion of cellular glutathione, by pre-exposure of the cells to the glutathione synthesis inhibitor buthionine sulfoximine, somewhat increased the toxicity of Hg2+ and potentiated the depletion of protein thiols. Taken together, the toxicity of Hg2+ in human oral fibroblasts was demonstrated in several assays of which colony forming efficiency was the most sensitive, cell killing by this agent was related to its high affinity for protein thiols, whereas glutathione showed a significant, but limited, ability to protect the cells from Hg2+ toxicity.
Buthionine Sulfoximine↗
Comparison of tetrazolium colorimetric and 51Cr release assays for cytotoxicity determination of dental biomaterials.
OBJECTIVES: The purpose of this study was to compare a methylthiazole tetrazolium (MTT) dye colorimetric method with the standard 51Cr assay as methods of assessing cytotoxicity of dental materials. METHODS: Two MTT-based colorimetric formats, test tube and 96-well microplate methods, were compared to the 51Cr release assay. A series of eight dental materials were evaluated. Cytotoxicity profiles were determined for each test material. A TC50 value (Toxic Concentration required to kill 50% of the cells) was determined for each biomaterial, and these results were used to make statistical comparisons between the methods. RESULTS: The three methods were statistically correlated (p<0.005) by comparison of the eight samples tested. That is, the same rank in toxicity was given by the two tetrazolium sample formats and the 51Cr method. SIGNIFICANCE: The MTT assay was found to have several advantages in comparison to the current standard 51Cr release assay. Optimized in the 96-well format, complete dose response curves and greater sample comparisons can be made rapidly, making the MTT method more economical in time and cost. Furthermore, the MTT method is based on intracellular biochemical changes, measuring cell viability rather than cell morbidity, and has lower detectable limits than the 51Cr release method. There is also less detector chemical binding interference than encountered in the 51Cr release method.
Animals↗
Monitoring of morphonuclear characteristics of hormone-sensitive and insensitive HCT-15 and LOVO human colorectal cells by means of digital cell image analysis.
We recently set up in vitro several human colorectal neoplastic cell lines that we labelled hormone-sensitive (HS) in comparison with the original cell lines which appeared to be rather "hormone-insensitive" (HI). We describe here the cell proliferation rate and the morphonuclear characteristics of the HS and HI variants of the HCT-15 and LOVO human neoplastic colorectal cell lines which were cultured either in serum-supplemented or chemically-defined media. Morphonuclear characteristics were monitored by means of the digital cell image analyses of Feulgen-stained nuclei, while the proliferation activity of the various cell types was assessed by means of the tetrazolium-based compound (MTT) assay. The results show that it is possible to culture human LOVO and HCT-15 colorectal cells in chemically defined media. This said, growth in a chemically defined medium is difficult for these cells and is markedly less sustained than when they are cultured in serum-supplemented media. The transition of the culture from a serum-supplemented medium to a chemically defined one is accompanied by a very marked drop in cell proliferation and a number of profound changes in terms of morphonuclear characteristics. These changes basically involve chromatin decondensation, which occurs as the result of the drop in the number of large chromatin clumps in the nucleus.
Cell Differentiation↗
Mitochondrial and nonmitochondrial reduction of MTT: interaction of MTT with TMRE, JC-1, and NAO mitochondrial fluorescent probes.
BACKGROUND: Bioreduction of water-soluble tetrazolium salts (e.g., MTS, XTT, and MTT) to their respective formazans is generally regarded as an indicator of cell "redox activity." The reaction is attributed mainly to mitochondrial enzymes and electron carriers. However, MTT reduction may also be catalyzed by a number of other nonmitochondrial enzymes. The goal of this work was to establish the sites of MTT reduction in intact HepG2 human hepatoma cells in culture. METHODS: In order to establish the subcellular localization of the sites of reduction of MTT, we imaged the formation of MTT-formazan deposits using backscattered light confocal microscopy. Mitochondria were visualized in viable cells using fluorescent dyes that bind in a manner dependent (JC-1 and TMRE) or independent (NAO) of mitochondrial electric potential. RESULTS: Only 25-45% of MTT-formazan was associated with mitochondria after 25 min of incubation. No more than 25% of the mitochondrial area on images was occupied by MTT-formazan. Mitochondrial fluorescence of TMRE, NAO, and the monomeric form of JC-1 decreased rapidly in cells incubated with MTT. However, the intensity of fluorescence of JC-1 aggregates dropped by less than 30% at the onset of incubation and remained constant as reduction of MTT proceeded further. CONCLUSIONS: (1) Most of MTT-formazan deposits are not coincident with mitochondria. (2) Monomeric JC-1, as well as TMRE and NAO, accumulating in mitochondria may be displaced by MTT. Thus, the presence of positively charged organic compounds (like MTT) may distort measurements of mitochondrial transmembrane electric potential, which are based on accumulation of fluorescent dyes.
Aminoacridines↗
Toxic effects of human pancreatic and snake and bee venom phospholipases A2 on MCF-7 cells in culture.
The toxicity of phospholipase A2 (PLA2) has been suggested to be involved in the pathology of a number of severe diseases including septic shock and acute pancreatitis. However, testing the toxicity of these substances is difficult in vivo. In the present study we compared the toxicity of PLA2s from three snake venoms, bee venom and human pancreas on MCF-7 cells grown in culture. Tetrazolium microculture assays were developed to test the cytostatic and cytotoxic effects of PLA2 on MCF-7 cells. These tests are based on the ability of viable cells to reduce a tetrazolium-based compound MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] to a blue formazan product. Leakage of lactate dehydrogenase (LD) from the cells into the culture medium was also measured. There were marked differences in the toxicity of the PLA2s tested. Cobra (Naja mosambique mosambique) venom PLA2 was toxic to the cells at a concentration of 4.5 U/ml. Light microscopic changes were seen in the injured cells after 3 hr treatment. Sixty-seven per cent of cells were dead after 24 hr treatment. Treatment for 4 hr caused irreversible changes in the cells. Leakage of LD was noted from 4 hr onwards. Other snake (Crotalus adamanteus and Laticauda semifasciata) venom PLA2s, even after continuous exposure to 4.5 U/ml caused only slight decreases in values obtained in the MTT test. No morphologic changes suggesting a cytotoxic effect were seen. PLA2 from bee (Apis mellifera) venom had no toxic effect, either. Continuous exposure of cells to human pancreatic PLA2 caused a 15% decrease in the MTT-test.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Oxygen insensitivity of the histochemical assay of glucose-6-phosphate dehydrogenase activity for the detection of (pre)neoplasm in rat liver.
Oxygen insensitivity of the histochemical assay to detect glucose-6-phosphate dehydrogenase (G6PD) activity with NT as tetrazolium salt has been proved to be a powerful tool to discriminate various types of adenocarcinoma from normal tissues. Here we investigated whether this phenomenon can also be applied to differentiate between chemically induced hepatocellular (pre)neoplasms and normal liver tissue in rats. Residual activity (percentage of the amount of final reaction product that is generated in oxygen and that is generated in nitrogen) was 60% in (pre)neoplastic cells and 6% in normal liver parenchymal cells. This means that the oxygen insensitivity test is a useful tool to distinguish (pre)neoplasms from normal rat liver tissue. N-Ethylmaleimide, a blocker of SH groups, did not affect G6PD activity in (pre)neoplastic cells, whereas activity in normal cells was reduced by half. Therefore, the absence of essential SH groups in G6PD in (pre)neoplastic cells is held responsible for the oxygen insensitivity phenomenon. We conclude that oxygen insensitivity of the histochemical assay for G6PD activity is a fast, easy, and cheap tool to diagnose (pre)neoplasms in rat liver. Discrimination is likely to be based on altered properties of the enzyme in (pre)neoplastic cells. (J Histochem Cytochem 49:565-571, 2001)
Animals↗
The mycotoxin patulin alters the barrier function of the intestinal epithelium: mechanism of action of the toxin and protective effects of glutathione.
Patulin is a mycotoxin mainly found in apple and apple products. In addition to being toxic for animals, mutagenic, carcinogenic and teratogenic, patulin induces intestinal injuries, including epithelial cell degeneration, inflammation, ulceration, and hemorrhages. In a study of the cellular mechanisms associated with the intestinal toxicity of patulin, two human epithelial intestinal cell lines (HT-29-D4 and Caco-2-14) were exposed to the mycotoxin. Micromolar concentrations of patulin were found to induce a rapid and dramatic decrease of transepithelial resistance (TER) in both cell lines without major signs of toxicity as assessed by the LDH release assay. Since TER reflects the organization of tight junctions, these data indicate that patulin affected the barrier function of the intestinal epithelium. The inhibitory effect of patulin on TER was closely associated with its reactivity for SH groups: (i) cysteine and glutathione prevented the cells from patulin injury; (ii) patulin toxicity was potentiated by buthionine sulfoximine, a specific glutathione-depleting agent; (iii) treatment of the cells with N-ethylmaleimide, a compound known to react with SH groups, resulted in a marked decrease of TER. Moreover, the inhibitory effect of patulin on TER was mimicked and potentiated by phenylarsine oxide, a specific inhibitor of protein tyrosine phosphatase (PTP). This cellular enzyme is a key regulator of intestinal epithelial barrier function. The active site of PTP contains a cysteine residue (Cys215) that is essential for phosphatase activity. Sulfhydryl-reacting compounds such as acetaldehyde decrease TER through covalent modification of Cys215 of PTP. We propose that the toxicity of patulin for intestinal cells involves, among other potential mechanisms, an inactivation of the active site of PTP.
Antidotes↗
Differential properties of cisplatin and tetraplatin with respect to cytotoxicity and perturbation of cellular glutathione levels.
We compared the cytotoxicity and the effects on the levels of cellular glutathione (GSH) of cisplatin and tetraplatin in the mouse mammary tumour cell line EMT6/P and a cisplatin-resistant variant. EMT6/CPR. EMT6/CPR showed a 2.5-fold resistance to cisplatin but no cross-resistance to tetraplatin. Basal cellular GSH levels in the resistant line were 1.7-fold those in the parent cell line. On exposure of cells to cisplatin, cellular GSH levels initially dropped in both cell lines but subsequently rose. At between 1 and 4 h after the onset of drug exposure, GSH levels were up to 2-fold the baseline levels. The cisplatin-resistant subline displayed higher GSH levels than the parent cell line at all time points. Tetraplatin failed to influence cellular GSH levels in either of the cell lines.
Animals↗
Effects of chemotherapeutic and immunosuppressive drugs on the production of erythropoietin in human hepatoma cultures.
The effects of agents used in combination chemotherapy were studied on erythropoietin synthesis in cultures of the human hepatoma cell line, HepG2. Erythropoietin was measured by radioimmunoassay in the culture media after 24-h treatment periods. The RNA synthesis-inhibiting drugs daunorubicin, cyclophosphamide, ifosfamide, and cis-diamine-dichloroplatinum produced a dose-dependent decrease of the production of erythropoietin. Inhibition was also induced by the tubulin-binding agents vincristine and colchicine and the immunosuppressive agents azathioprine and cyclosporin A. The DNA synthesis-inhibiting drugs methotrexate and cytosine arabinoside, the antimycotics 5-fluorocytosine and amphotericin B, and glucocorticoids did not inhibit. Viability studies showed that the inhibition of erythropoietin production was partly correlated with cytotoxicity. These data could be relevant with respect to recombinant erythropoietin replacement therapy in tumor-associated anemia.
Animals↗
Servocontrolled cardiac assistance: effects of left ventricular to aortic and left atrial to aortic assistance on infarct size.
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Animals↗
Studies on the effect of soluble lymphocyte products (lymphokines) on macrophage physiology. I. Early changes in enzyme activity and permeability.
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Animals↗
Nitrogen mustard drug resistant B-cell chronic lymphocytic leukemia as an in vivo model for crosslinking agent resistance.
Acquired resistance is a limiting factor in chemotherapy. We have employed nitrogen mustard resistant B-cell chronic lymphocytic leukemia (B-CLL) as a clinically relevant model to study this phenomenon. Resistance in B-CLL is associated with enhanced repair of nitrogen mustard crosslinks. In order to identify the repair pathway responsible for nitrogen mustard resistance, lymphocytes were screened for cross-resistance to a variety of DNA damaging agents. The MTT assay was used to measure the resistance of B-CLL lymphocytes to various DNA damaging agents, including nitrogen mustards, UV light, methyl methanesulfonate, and mitomycin C. We have shown that B lymphocytes from patients with nitrogen mustard resistant chronic lymphocytic leukemia reflect their clinical status. This assay allows us to classify lymphocytes as nitrogen mustard sensitive or resistant, based on in vitro observations. The resistant population was 5.6 and 4.1 fold more resistant to the nitrogen mustard analogs, chlorambucil and melphalan, respectively. Resistant lymphocytes displayed no increased resistance to either methyl methanesulfonate or UV light, indicating that neither classical base nor nucleotide excision repair is rate-limiting in resistance. Resistant lymphocytes were 6.0 and 2.2 fold more resistant to mitomycin C and cis-diamminedichloroplatinum (II), respectively, suggesting enhanced crosslink repair. Neither glutathione nor glutathione S-transferase levels correlated with resistance. The development of nitrogen mustard drug resistance in B-CLL appears to be associated with cross-resistance to other bifunctional alkylating agents which produce interstrand crosslinks. Our results indicate that resistance to nitrogen mustards in chronic lymphocytic leukemia is associated with enhanced repair of DNA crosslinks which may involve a recombination dependent system. This model should prove very useful in the elucidation of the molecular mechanisms of crosslink repair.
B-Lymphocytes↗