Reduction Of Tetrazolium Salt Research Articles

2-D Gel analysis of protein synthesis in fish hepatocytes exposed to Pb: Development of stress proteins as biomarkers of sublethal chemical exposure : JUDITH GLAVEN, MIRIAM AKKERMAN & BRUCE A. FOWLER. University-wide Program in Toxicology, University of Maryland, Baltimore, Maryland 21201, USA

There is a need in aquatic toxicology to develop alternative assays for the assessment of toxicity in fish. In recent years, in vitro assays have been developed for scientific, ethical and economical reasons. In this study, the fish hepatoma cell line PLHC-1 (Hightower & Renfro, 1988) was employed for assessing the cytotoxicity of environmental chemicals, to derive structure activity relationships and to compare in vitro with in vivo toxicity. The cytotoxicity of several important organic compounds, including chloro- and nitrophenols, estrogenic alkylphenols and sulphonic acids, as well as organotin compounds (Brüschweiler et al., 1995), was assessed. Two cytotoxicity assays were performed: inhibition of neutral red uptake (NR) into lysosomes based on cell membrane damage; and the tetrazolium salt reduction (MTT) assay to determine inhibition of mitochondrial succinate dehydrogenase. A high correlation between the two assays was found. Of the various compounds, organotins were most toxic, followed by higher substituted phenols, lower substituted phenols and sulphonic acids. The in vitro cytotoxicity showed a similar trend as the in vivo acute toxicity of fish for organotins and substituted phenols. Furthermore, a positive trend between cytotoxicity and n-octanol-water partition coefficients of the chemicals was found. The results indicate that cytotoxicity assays using this metabolically active hepatoma cell line are a promising tool in the first evaluation and toxicity screening of chemicals prone to contaminate aquatic systems.

Regulation of mitogen-driven lymphoreticular cell activation by human corneal cells and interleukin-1.

Keratin-positive fibroblast-like epithelial cells (FLE), isolated from human corneo-scleral-conjunctival rims, were shown to inhibit mitogen-driven (concanavalin A) DNA synthesis by murine thymocytes and splenocytes [lymphoreticular cells (LRC)]. The effect exerted by live cells in culture and by their supernatants was caused by factors active across species barriers. Paraformaldehyde-fixed or irradiated cells also suppressed mitogen-induced thymocyte DNA synthesis, but their supernatants manifested no such activity. Interaction between FLE cells and LRC in the presence of the mitogen resulted in suppressed cellular activation as evidenced by significantly lowered tetrazolium salt (MTT) reduction in murine thymocytes and splenocytes, suggesting reduced mitochondrial activity. The suppressive effect was seen with live and paraformaldehyde-fixed FLE cells. There was a good correlation between MTT assays and [3H]thymidine uptake experiments. Suppression of MTT reduction in murine thymocytes and splenocytes by intact FLE cells could be reversed by the addition of interleukin-1 (IL-1). Indomethacin prevented FLE-conditioned medium-induced suppression but failed to relieve suppression by whole FLE cells. Thus, suppression of LRC function by FLE cells and their secretions appeared to operate by different mechanisms. One mechanism related to prostaglandins present in FLE cell-conditioned medium, whereas another mechanism appeared to involve cell-membrane-associated factor(s). The findings not only provide additional information on the capability of corneal cells to regulate lymphoreticular cells but suggest an important role for IL-1 in the regulation of LRC function and corneal inflammation and immunity.

Genistein inhibits tumour cell growth in vitro but enhances mitochondrial reduction of tetrazolium salts: A further pitfall in the use of the MTT assay for evaluating cell growth and survival

The natural isoflavone genistein inhibits the growth of a number of tumour cell lines in vitro. During investigations on the antiproliferative effects of genistein we observed that, with respect to direct cell counting, a tetrazolium (MTT) colorimetric assay consistently underestimated the growth inhibitory activity of the substance. Cell proliferation was markedly inhibited by genistein in three tumour cell lines (MCF-7, human breast tumour; Jurkat cells, human T-cell leukaemia; L-929, mouse transformed fibroblasts) when cell number was evaluated by direct counting, whereas a 72-h MTT assay failed to reveal any growth-inhibitory effect. Cell cycle analysis by propidium iodide staining and flow-cytometry revealed a G2/M cell cycle arrest after genistein treatment. Genistein-treated cells displayed an increase in cell volume and in mitochondrial number and/or activity, as revealed by enhanced formazan generation and increased uptake of the vital mitochondrial dye rhodamine 123. These results suggest that alterations in cell cycle phase redistribution of tumour cells by genistein may significantly influence mitochondrial number and/or function and, consequently, MTT reduction to formazan. This may constitute an important bias in analysing the effects of genistein, and possibly other drugs that block the G2/M transition, on growth and viability of cancer cells in vitro by MTT assay.

A new approach for measurement of cytotoxicity using colorimetric assay

Using in vitro established tumour cell lines attempts were made to assess the suitability of tetrazolium salt reduction (MTT) assay to replace the conventional radioactive base techniques for measuring cell proliferation and cell killing. The optimum conditions for MTT loading time, concentration of MTT and the time for colour development were found to be 4 h, 5 mg/ml and 30 min respectively, conditions which were used for subsequent experiments. Analysis of the correlation between increasing cell numbers and optical densities (OD) showed a direct relationship with correlation of coefficient values of r > 0.98 and 10,000 cells/well was found to provide an accurate ODs for a wide variety of cell types. The accuracy of replicate readings of the assay was investigated by setting a wide range of cell numbers and the variation among seven replicates was calculated and found to be less that 6% of the mean values. The reproducibility of the assay for two cell lines was tested using the lines on four different occasions. The ODs for Jar and Fen cell lines were 0.80 ± 0.01, 0.82 ± 0.02, 0.90 ± 0.02, 0.79 ± 0.05 and 0.56 ± 0.01, 0.58 ± 0.03, 0.60 ± 0.02 and 0.61 ± 0.02 respectively giving maximum variation of less than 11% of mean on repeated testings. Comparison between the results of MTT with 3H-Tdr or 51Cr release assays showed a high degree of correlation over a wide range of cell numbers and cell types. The r values between the results of MTT with 3H-Tdr (for cell number ranging from 1.8 to 60 × 10 3/well) or 51Cr release assays (for E/ T ratios of between 5:1 and 40:1) were 0.89 ( p = 0.001) and 0.96 ( p < 0.03) respectively. These results demonstrate that it is possible to use the MTT assay interchangeably with radioactive base techniques to measure cell proliferation and cytotoxicity. The ease of its execution, safety and its suitability for analysing as few as 3000 cells makes this method a serious contender for replacing the conventional radioactive techniques.

Nerve growth factor effects on pyridine nucleotides after oxidant injury of rat pheochromocytoma cells

Neurotrophic factors regulate neuronal survival and neurite growth in development and following injury. Oxidative stress produced in neurons as a consequence of primary injury, or during reperfusion following ischemia, may contribute to cell death. Here, the effects of nerve growth factor (NGF) on the response to H 2O 2 injury were examined in the PC12 rat pheochromocytoma cell line. Specifically, the effect of NGF on cell viability after H 2O 2 injury was measured. Pretreatment with NGF enhanced survival after H 2O 2 treatment, as measured by Trypan blue dye exclusion, radiolabeled amino acid incorporation, tetrazolium salt reduction, or cytoplasmic enzyme release. One early event associated with H 2O 2 treatment was a rapid decrease in NAD +. Although initial decreases in NAD + levels were similar in control and NGF-treated cells, the latter recovered more rapidly and extensively. The decline in total NAD observed after NGF treatment was almost equal in magnitude to the measured increase in NADP. Inhibition of poly(ADP-ribose) polymerase also enhanced viability following H 2O 2 injury. Treatment with both NGF and an inhibitor of this enzyme resulted in a greater reduction of H 2O 2 toxicity than was observed with either agent alone. These data suggest that NGF protection is multifactorial and that a significant component of the NGF effect is due to its regulatory role in the metabolism of pyridine nucleotides.

Islet cell metabolism is reflected by the MTT (tetrazolium) colorimetric assay

Insulin secretion depends critically on glucose metabolism. We investigated whether a rapid viability test could be established for assessing glucose metabolism in insulin secreting cells. The MTT (C,N-diphenyl-N'-4,5-dimethyl thiazol-2-yl tetrazolium bromide) colorimetric assay (reduction of tetrazolium salt to formazan) was applied to rat islets and rat insulinoma cell lines. It was found that the rate of formazan production correlated with glucose oxidation and glucose utilization at glucose concentrations which also stimulated insulin secretion. In differentiated insulinoma INS-1 cells, salt reduction paralleled the insulin release at glucose concentrations of up to 8.3 mmol/l. The glucose-induced formazan production in INS-1 cells and islets was abolished by exposure to the Beta-cell cytotoxic agents, streptozotocin or alloxan. The MTT assay thus provides a convenient tool for the rapid assessment of Beta-cell metabolism and viability.

Calibration of a flow cytometric assay of glucose-6-phosphate dehydrogenase activity.

The reduction of tetrazolium salts to colored formazans is a reaction which has been exploited both in histo- and cytochemistry. Tetrazolium salts forming fluorescent formazans prove suitable for measuring defined cellular dehydrogenase activities in automated processes. This study considers an important aspect of formazan measurement in flow cytometry, namely, calibration. Calibration is performed by correlating the number (and fluorescence intensity) of formazan-bearing cells measured by flow cytometry with simultaneously performed biochemical analyses of the same material. The method is demonstrated by an example of glucose-6-phosphate dehydrogenase. Using the data of a typical experiment, the enzyme activity is expressed in femtomol of hydrogen transferred per cell during incubation time. Furthermore, through spatially resolved double excitation of formazan and nuclear DAPI fluorescence, an independent analysis of cell cycle and cellular enzymatic activity is established.

An In Vitro Cytotoxicity Assay Using Rat Hepatocytes and MTT and Coomassie Blue Dye as Indicators

Isolated hepatocytes from male Sprague-Dawley rats suspended in culture medium supplemented with either 0.2 or 2% bovine serum albumin (BSA) were allowed to attach to collagen coated 96-well dishes. Ten test chemicals from the MEIC list and salicylic acid were added individually to the dishes, and at the end of 24 and 48 hours, cytotoxicity was determined by measuring MTT (tetrazolium salt) reduction (mitochondrial integrity) and total cellular protein using Coomassie blue dye (reflecting cell number). Total cellular lactate dehydrogenase activity was also determined in some experiments, as an indicator of plasma membrane integrity. The relative toxicities of the test chemicals were quantified by the estimation of EC10, EC20 and EC50 values for each parameter. Except for one chemical, digoxin, in the MTT assay, cytotoxic potency increased with incubation time. The hepatocytes tended to be more sensitive to the chemicals in medium containing 0.2% BSA than in medium containing 2% BSA. Simple linear regression analyses of the log transformed data from the MTT assay versus log oral LD50 in rats for the test chemicals gave the best results using EC10 at 24 hours (r2 = 0.86). With protein as the cytotoxic indicator, the best results were obtained with EC values in the medium containing 2% BSA, again at 24 hours (r2 = 0.83). These results suggest that the MTT and Coomassie blue dye assays could be useful indicators for testing the cytotoxic potential of chemicals in rat hepatocyte cultures.

Suppression of cell-mediated immunity by purified aflatoxin B 1 in broiler chicks

The effect of purified aflatoxin B 1 on cell-mediated immunity (CMI) in broiler chicks was assessed using doses of 0.3 and 1 mg/kg feed from hatching to 6 weeks of age. Total lymphocyte and T lymphocyte counts and the 2,4-dinitrofluorobenzene skin sensitivity test, graft-versus-host reaction and nitroblue tetrazolium salt reduction tests were used to evaluate CMI. Both doses of aflatoxin B 1, including the apparently nontoxic dose of 0.3 mg/kg feed, caused a significant ( P < 0.05) decline in CMI. The functional activity of splenic macrophages was decreased significantly ( P < 0.05) by both doses of the toxin.

Biocompatibility evaluation of glass ionomer cement using cell culture techniques

Cell culture techniques have been employed to carry out a preliminary investigation into the biocompatibility of a glass ionomer cement (GIC). A modification of the commonly used Agar Overlay test and a rapid, colorimetric assay based on the reduction of tetrazolium salt were used. The GIC is shown to leach a cytotoxic agent, possibly fluoride. This agent is effectively removed by an extraction procedure, and re-examination of the GIC demonstrates its cytocompatibility.

Effects of the prolyl 4-hydroxylase proinhibitor HOE 077 on human and rat hepatocytes in primary culture.

Alterations in extracellular matrix occur in many chronic liver diseases leading to the formation of hepatic fibrosis. We have studied the effects of the putative hepatoselective fibrosuppressive compound HOE 077, a proinhibitor of prolyl 4-hydroxylase, on normal adult human and rat hepatocytes in primary culture. In human hepatocyte cultures, the cytotoxicity of HOE 077 was assessed after a 20-h treatment at concentrations ranging from 0.125 to 2 mg/ml of medium. No significant change was found in cell morphology, neutral red uptake, red oil staining, lactate dehydrogenase release, tetrazolium salt reduction, ethoxyresorufin O-deethylase activity and protein synthesis; however, HOE 077 slightly decreased DNA synthesis at 2 mg/ml. In rat hepatocyte cultures, the cytotoxicity of the compound was assessed by testing the same parameters after a daily exposure of cultures for 2 days or 4 days, at concentrations ranging from 0.25 to 4.5 mg/ml of medium. Whatever the concentration, the compound had no obvious morphological effect. However, hepatocytes were less spread at the concentration of 4.5 mg/ml. HOE 077 at 2 mg/ml slightly decreased neutral red uptake but was without obvious effect on protein synthesis after 2 days. By contrast, on day 4, protein synthesis was markedly reduced in hepatocyte cultures exposed to HOE 077 at 4.5 mg/ml. Hydroxyproline content determination in media from 4-day-old hepatocyte cultures incubated with HOE 077 at 0.5 to 4.5 mg/ml, showed a dose-dependent decrease in the hydroxyproline/proline ratio in acetic acid soluble material. By indirect immunoperoxidase, intracellular collagen IV was found to be inhibited in hepatocyte cultures after 4 days of exposure to 4.5 mg/ml HOE 077.(ABSTRACT TRUNCATED AT 250 WORDS)

Induction of granulocytic maturation in acute myeloid leukemia by G-CSF and retinoic acid

AML cells were cultured free of serum with G-CSF in combination with all- trans-retinoic acid (RA), prostaglandin E2 or 8-bromocyclic AMP to see whether the maturation blockade of these cells could be overcome. The combination G-CSF + RA was most effective in inducing morphologic maturation, i.e. in 7/10 cases. Morphological alterations in response to G-CSF + RA indicated progression of the cells along the granulocytic pathway towards metamyelocytes and granulocytes. However, morphologically mature AML cells remained negative for myeloperoxidase and Sudan black stainings, indicators of granulocytic maturation. Chloracetate esterase positivity and CD15 membrane antigens became expressed on cultured AML cells, i.e. on unstimulated and G-CSF/RA exposed blasts. Ingestion of latex beads and reduction of nitroblue tetrazolium salt occurred in cultured AML cells regardless of the presence of inducers. In almost all cases clonogenic cells persisted after exposure to G-CSF + RA suggesting that subpopulations of immature cells escaped the action of these inducers. Thus although G-CSF + RA were capable of inducing maturation of AML cells along the granulocytic lineage, maturation was incomplete and the effect was evident in a subfraction of the cells only.

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.

Detection of malignant and potentially malignant cells in the colon by means of an oxygen-sensitive tetrazolium salt reduction

Detection of malignant and potentially malignant cells in the colon by means of an oxygen-sensitive tetrazolium salt reduction

A recycling assay for alkaline phosphatase applied to studies on its transport in E. coli K12

A recycling assay for alkaline phosphatase, based on its ability to hydrolyse NADP to NAD +, is presented. The product NAD + is recycled in a coupled assay consisting of NADH regeneration and reduction of a nitroblue tetrazolium salt. This assay is 10–12 times more sensitive than the conventional assay. We demonstrate the role of energy poisons in transport of this protein into the periplasm by combining the improved detection with phase separation of the periplasmic and cytoplasmic alkaline phosphatase pools.

Toxic interactions between carbon tetrachloride and chloroform in cultured rat hepatocytes

Primary cultures of adult rat hepatocytes were incubated (1.5–16 hr) with various concentrations of CCl 4 (≤0.5 m m) and/or CHCl 3 (≤2.5 m m). Agent-dependent alterations in hepatocyte functions were assessed by measuring (1) [ 3H]choline incorporation into phosphatidylcholine (endoplasmic reticulum), (2) MTT (tetrazolium salt) reduction (mitochondria), and (3) AST release into medium (plasma membrane). Cultured hepatocytes incubated with 0.5 m m CCl 4 displayed a significant ( p ≤ 0.001) and rapid (1.5 hr) reduction (40%) in endoplasmic reticulum function that preceded significant ( p ≤ 0.001) alterations in mitochondria (6–16 hr) and plasma membrane (6–16 hr) functions. CCl 4-dependent alterations in liver cell functions are a result of CCl 4 bioactivation since metyrapone inhibits the CCl 4-mediated changes in cell functions. Response surface methods (RSM) were used to determine the influence of combinations of CCl 4 and CHCl 3 on liver cell MTT reduction and [ 3H]choline incorporation. Regression coefficients were determined for CCl 4, CHCl 3, and CCl 4-CHCl 3. All results were significant ( p < 0.0001) and implied that CCl 4 was a more potent hepatotoxin in vitro than CHCl 3. The RSM analysis also suggested that combinations of CHCl 3 and CCl 4 have greater than additive effects on MTT reduction and [ 3H]choline incorporation. These effects of CCl 4 and/or CHCl 3 on liver cell functions in vitro are consistent with liver alterations observed in vivo. Therefore, primary cultures of adult rat hepatocytes may be an appropriate model in vitro to assess the hepatotoxic potential of agents alone or in combination.

The ‘nothing dehydrogenase’ reaction and the detection of ischaemic damage

The 'nothing dehyrogenase' reaction is defined as the reduction of tetrazolium salts in media lacking specific substrates for dehydrogenases. In this investigation, the kinetics of the 'nothing dehydrogenase' reaction were studied in cryostat sections of rat heart and liver with the use of various polyvinyl alcohol-containing incubation media. Formazan production was measured at 585 nm with a cytophotometer. The 'nothing dehydrogenase' reaction was substantially lower in the heart than in the liver which was due to low levels of endogenous lactate and the absence of proteins containing thiol groups, such as albumin, in the heart. In vitro ischaemia resulted in a reduced 'nothing dehydrogenase' reaction due to loss of NAD+, possibly as a consequence of its breakdown by glycohydrolase activity. One hour reperfusion following one hour ischaemia caused a decreased 'nothing dehydrogenase' reaction in certain areas of the liver. This reduction was a result of leakage of lactate dehydrogenase and thiol-containing molecules. It appeared at the ultrastructural level that parenchymal and endothelial cells were heavily damaged in the areas containing a low 'nothing dehydrogenase' activity. In conclusion, early ischaemic damage in liver can be detected with the 'nothing dehydrogenase' reaction.

Protection of crystal-induced polymorphonuclear leukocyte membranolysis by phosphocitrate

The protection afforded by phosphocitrate, a phosphorylated polycarboxylic acid, against crystal-induced membrane damage to polymorphonuclear leukocytes was studied in vitro. Membranolysis was assessed by nitro blue tetrazolium salt reduction, lactate dehydrogenase release, and scanning electron microscopy. Phosphocitrate protected strongly against hydroxyapatite crystal-induced damage, an action attributable to crystal surface binding of phosphocitrate rather than to the membrane. The ability of phosphocitrate to prevent hydroxyapatite crystallization, together with its membrane protective effect against preformed crystals, would suggest that the compound might have a useful future role against crystal-induced arthropathies.

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 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-dihydor-5-methylphenazine (MPH), formed on reduction of 5-methyl-phenazinium cation (MP +) of PMS by NAD[P]H, was reoxidized allowing first the univalent reduction of molecular oxygen (O 2) to the superoxide anion radical O 2 ⪰ which 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 O 2 ⪰. 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 O 2. By lowering the pH and thereby facilitating the H +-mediated dismutation of O 2 ⪰, 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 O 2 by the micelle-bound MPH.

Reduction of tetrazolium salts by sulfate-reducing bacteria

The reduction of tetrazolium salts by the sulfate-reducing bacteria, Desulfovibrio desulfuricans and Desulfotomaculum orientis, was examined. D. desulfuricans and D. orientis reduced triphenyltetrazolium chloride (TTC) and 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium chloride (INT) forming intracellular formazan deposits. The reduction rate of INT was higher than that of TTC. INT reduction was not inhibited by the addition of sulfate or molybdate, and sulfate uptake was inhibited by the addition of both INT and molybdate. The ratio of intracellular formazan forming cells to acridine orange direct counts in both strains decreased with culture age and starvation time.