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PubMed1979

Histochemical demonstration of a circadian rhythm of succinate dehydrogenase in rat pineal gland. Influence of coenzyme Q10 addition.

Succinate dehydrogenase activity was investigated histochemically in the rat pineal gland. The influence of fixation on the activity pattern, the possible diffusion of enzyme, the nothing dehydrogenase reaction, and the substantivity of the tetrazolium salts and formazans were investigated in control experiments. In rats maintained on a 17/7 h light/dark schedule a distinct circadian rhythm of the succinate dehydrogenase was demonstrated in the pineal gland. Activity was lowest during the day and highest during the night. The dorsocaudal part of the gland showed the highest activity and within the same part of the gland the activity varied between individual pinealocytes. A relative lack of endogenous coenzyme Q, as well as a circadian rhythm of this coenzyme, highly influenced the activity of succinate dehydrogenase. It is concluded that succinate dehydrogenase activity in the pineal gland of the rat is regulated by changing the concentration of the active enzyme itself as well as the level of the endogenous coenzyme Q. Whether this is caused by a circadian rhythm in the synthesis or in the catabolism of the enzyme and the coenzyme was not revealed by the present study .

Animals↗
PubMed1992

Assessment of antineoplastic agents by MTT assay: partial underestimation of antiproliferative properties.

In the present study a slightly modified MTT assay was used in conjunction with cell counting to determine the antiproliferative efficacy of N-(2-chloroethyl)-N-nitroso-N'-2-hydroxyethylurea (HECNU), vinblastine, and hexadecylphosphocholine (HPC) in a panel of six tumor cell lines. This panel consisted of two human (MDA-MB231, MCF-7) and two rodents (1/C2, 1/C32) mammary-carcinoma cell lines as well as of two tumor cell lines of gastrointestinal origin (HT-29, KB). It was shown that the use of acid isopropanol as a solvent of the formazan crystals produced correlations between cell number and absorption that were as good as, if not better than, those seen after dimethylsulfoxide (DMSO) application. The optimal period of incubation with the MTT dye was 2 h. A comparison of the antiproliferative activity of HECNU revealed that the HT-29 cell line was most resistant [50% inhibition concentration (IC50), 138.7 mumol/l], followed by MCF-7 cells (IC50, 127.7 mumol/l), whereas MDA-MB231 cells showed the highest sensitivity (IC50, 6 mumol/l). Vinblastine induced the highest (MCF-7 cells; IC50, 0.68 nmol/l) and the lowest (1/C2 cells; IC50, 7.69 nmol/l) degrees of growth inhibition in cell lines derived from mammary carcinoma. This contrasted with the activity of HPC, which was considerably less effective in the four mammary-carcinoma cell lines (IC50 from 29.4 to 69.9 mumol/l) than in the two cell lines of gastrointestinal origin (IC50, 1.9 and 3.1 mumol/l). Interestingly, treatment with HPC stimulated the growth of 1/C32 cells in the lower dose range. After treatment with HECNU, the average IC50 value determined in the MTT assay was 2.4-fold that disclosed by cell counting, whereas the average values found for HPC and vinblastine by both methods corresponded fairly well, with the respective values obtained using the MTT assay being only 26% and 14% higher than those measured by cell counting. A dose-dependent increase in the mean size of MCF-7 cells was observed after exposure to HECNU, which--if taken into account--considerably reduced underestimation of this parameter by the MTT assay. No variation in cell size was noted following treatment with HPC and vinblastine. Thus, depending on the antitumor agent used, the MTT assay can result in slight or even considerable underestimation of the antitumor efficacy of certain compounds and may need correction by consideration of the effect of the drugs on cell size.

Animals↗
PubMed1994

Trypanocidal activity of a myristic acid analog in axenic cultures of Trypanosoma evansi.

The growth of bloodstream forms of Trypanosoma evansi in axenic culture was inhibited by incubation with 11-oxatetradecanoic acid (O-11), an analog of myristic acid. Parasites isolated from Asia, Africa and South America were affected to a similar extent in measurements using three different assay systems concerned with different aspects of trypanosome growth and metabolism. The concentration of O-11 that inhibited trypanosome growth by 50% (LD50) was 3.7 +/- 0.2 microM as measured by direct counting of survivors using a haemocytometer, 5.1 +/- 2.0 microM in a colorimetric test based on the formation of a formazan product, and 8.8 +/- 3.7 microM by estimation of pyruvate. The activity of the drug was enhanced by the addition of fatty-acid-free bovine serum albumin as a carrier protein to the culture medium at an optimal concentration of 5 mg/ml. Increasing amounts of the donor horse serum used for routine maintenance of these cultures, which is normally the only source of myristic acid for these trypanosomes, also affected the toxicity of the drug, in this case increasing the LD50.

Animals↗
PubMed1978

A quantitative histochemical study of three oxidative enzymes in solar keratoses and Bowen's disease.

Succinate dehydrogenase (SDH), glucose-6-phosphate dehydrogenase (G6PDH) and lactate dehydrogenase (LDH) activities have been studied using quantitative enzyme histochemical techniques in the epidermis of five patients with solar keratoses and Bowen's disease. 'Non sun-exposed' buttock skin was compared with the skin from the actual lesion and adjacent, clinically normal paralesional skin. SDH activity was significantly increased in the basal layer and decreased in the granular layer in the epidermis of both lesion and paralesional skin, although the total epidermal activities were unchanged when compared to 'non-exposed' buttock skin. G6PDH activity was increased in the granular layer of paralesional epidermis and of lesions. No change in LDH activity was detected. Inclusion of phenazine methosulphate in the reaction mixtures resulted in a three-fold increase in formazan deposition without altering the localization. It is concluded that the quantitative changes and alteration in localization of SDH and G6PDH reported in solar keratoses are accompanied by similar changes in adjacent, clinically normal 'sun-exposed' skin and differ from normal 'non-exposed' skin. It is suggested that these changes may precede the development of the solar keratoses and that these findings may indicate a significant metabolic alteration in the events that lead to neoplasia.

Aged↗
PubMed1984

Kinetic behaviour of succinate dehydrogenase of three fibre types in skeletal muscle. I. Effects of temperature and a competitive inhibitor.

The kinetic behaviour of succinate dehydrogenase [EC 1.3.99.1] in three fibre types of rat gastrocnemius was examined by a quantitative histochemical method without disruption of the cellular structure. 2-(2-Benzothiazolyl)-3-(4-phthalhydrazidyl)-5-styryl-t etrazolium chloride (BPST) and phenazine methosulphate were used as electron acceptors. On measurement of the absorbance value at 530 nm of BPST formazan, produced by the succinate dehydrogenase reaction in sections, it was found that the staining intensity of succinate dehydrogenase was linearly proportional to both the incubation time and the thickness of the slice therefore, the initial velocity of the staining could be calculated. By Michaelis-Menten (1913) treatment of the dependence of the initial velocity on the substrate concentration in the absence and the presence of a competitive inhibitor, malonate, the Km and Vmax values for succinate and the Ki value for malonate were obtained. The Km and Ki values of the three fibre types were similar. The ration of the Vmax values of type A, B and C fibres was 1.0:2.0:3.3. The temperature dependence of the kinetic parameters was very similar in the three fibre types. These findings confirm that the differences in the staining intensity of the three fibre types reflect differences in the amounts, but not the properties, of succinate dehydrogenase.

Animals↗
PubMed1984

Histochemical localization of monoamine oxidase in whole-body, freeze-dried sections of mice.

Monoamine oxidase was investigated histochemically in tissues of the mouse by incubating freeze-dried, whole-body sections with tryptamine, serotonin, tyramine, beta-phenylethylamine, or benzylamine as substrate and Nitroblue tetrazolium as the final electron acceptor. The most intense staining with tryptamine was exhibited by intestinal epithelium and adrenal cortex; moderate staining was noted in the epithelium of the nose, bronchi, oesophagus, and upper stomach and in preputial gland, pancreas, nerve, spinal cord and brain. Weak staining was seen in the lung, spleen, liver and kidney. The distribution of the formazan deposition was similar, but much less intense, when serotonin and tyramine were used as the substrates. Only very weak staining was observed when beta-phenylethylamine was the substrate; no staining was seen with benzylamine. Monoamine oxidase activities with tryptamine were greatly inhibited by pretreatment with clorgyline (10 microM), while deprenyl (10 microM) slightly inhibited activities in all tissues except liver. This staining technique should be useful in further studies on the identification of the multiple forms of monoamine oxidase in tissues of the mouse. Nicotine and nitrosonornicotine were not substrates in any of the tissues; consequently, this enzyme system does not appear to produce the proximal carcinogen from this nitrosamine.

Adrenal Cortex↗
PubMed1975

Localization in cardiac muscle of some enzymes related to glutamate metabolism.

A tetrazolium staining medium incorporated in a gel has been used in a histochemical study of enzymes in thin sections of heart muscle. Formazan distribution patterns given by mitochondrial enzymes were inconsistent with the location of these enzymes revealed by the extraction of whole tissue. Similar stain distributions were given by lactate dehydrogenase, glutamate oxaloacetate transaminase and glutamate dehydrogenase. The distribution given by succinate dehydrogenase was not the same as that given by cytochrome oxidase stained by a different technique. Alcohol dehydrogenase added to the tissue assumed a distribution which suggested some adsorption of the enzyme to the tissue. But experiments suggested that this enzyme was not firmly bound to muscle proteins in the manner of some glycolytic enzymes.

Alcohol Oxidoreductases↗
PubMed1975

A permeability test for the study of mitochondrial injury in in vitro cultured heart muscle and endothelioid cells.

A cytochemical permeability test for the detection of injury to in situ mitochondria of cultured heart cells is presented. The test is based on the increased rate at which injured mitochondria stain for succinate dehydrogenase activity. Whereas an intact inner mitochondrial membrane limits the rate at which Nitro Blue tetrazolium and phenazine methosulphate reach succinate dehydrogenase, injured mitochondria allow these reactants to reach the enzyme more rapidly to form microscopically-observable formazan granules. The extent of staining at fixed durations of incubation with the reactants was assessed on a blind basis with pseudo dark-field microscopy, using a standardized rating scale. Differences in the staining of control and treated cells were analysed statistically by a semi-quantitative method. Treatment of the cultures with either vitamin A or chlorpromazine, resulted in more rapid mitochondrial staining. Brief pre-fixation of the cells with cold acetone also labilized the mitochondria as did a delay in the change of culture medium.

Animals↗
PubMed1981

Model film studies in enzyme histochemistry with special reference to glucose-6-phosphate dehydrogenase.

This paper deals with the progress made over the last few years in our understanding of enzyme cytochemical staining methods as studied using a fundamental approach with the aid of a model system of thin gel films. Although model films with a matrix of polyacrylamide have been mostly used, the properties and possible applications of other matrices are also reviewed. The chemical aspects of the entrapment of enzyme molecules into a matrix are summarized. Special attention has been paid in model film studies to the principles of the trapping reaction of a diffusable precursor resulting from the enzymatic conversion of a substrate. They are considered here as they concern the cytochemical demonstration of acid phosphatase activity with a lead salt. The effect of fixatives on different enzyme activities, the diffusion rate of substrates and chromogenic compounds to the enzyme site, and enzyme kinetics under cytochemical conditions are also discussed, since they are factors which influence the final results of the staining procedures. The advantage of model film studies in enabling the direct correlation of cytochemical and biochemical results is outlined with special reference to the cytochemical determination of glucose-6-phosphate dehydrogenase with Tetra Nitro BT. A method for determining enzyme activities in the soluble fraction of isolated cells after incorporation in model films is described for the first time. This method has proved to be highly appropriate for microscopical observations of glucose-6-phosphate dehydrogenase activity in single cells, because it results in a good morphology and no formazan precipitates outside the cells. On the other hand, this type of model film forms a bridge between fundamental model film studies using purified enzyme and quantitative enzyme cytochemistry performed in situ.

Acid Phosphatase↗
PubMed1983

Histochemical localization and quantification of glucose-6-phosphate dehydrogenase in bovine leydig cells.

Some of the critical steps in the qualitative histochemical localization of glucose-6-phosphate dehydrogenase (freezing procedures, incubation techniques and the influence of intermediate electron carriers, respiratory chain inhibitors and different tetrazolium salts) were evaluated in sections of bovine testis as a prerequisite for the microdensitometric estimation of the activity of the enzyme in bovine Leydig cells in situ. A modification of the gel incubation method of Rieder et al. (1978) gave the best results and was used for the quantitative investigations. Quantitative data for the dehydrogenase activity gained from microdensitometry of the formazan final reaction products in Leydig cells in situ were compared with the results of assays of the activity in homogenates of testis. The following apparent kinetic properties of glucose-6-phosphate dehydrogenase were obtained for the enzyme in Leydig cells in situ: Vmax = 0.11 absorbance units/min, Km = 0.37 mM. The quantitative characterization of glucose-6-phosphate activity in Leydig cells in situ appears to be suitable for combined morphological and functional diagnoses of small tissue samples such as testicular biopsies. This would give valuable information of the functional status of Leydig cells in normal and diseased testicular tissue.

Animals↗
PubMed1980

Quantitative aspects of the cytochemical demonstration of glucose-6-phosphate dehydrogenase with tetranitro BT studied in a model system of polyacrylamide films.

The cytochemical determination of the activity of glucose-6-phosphate dehydrogenase (G6PDH) with tetranitro blue tetrazolium (TNBT) was studied with model films of polyacrylamide gel incorporating purified enzyme. This model system enabled a quantitative study to be made of different parameters involved with the cytochemical assay as it is applied to sections or smears. The enzyme activity of G6PDH incorporated in the model films was also assayed biochemically. Optimal conditions for retaining the maximum amount of enzymic activity are described. The behaviour of G6PDH towards enzyme inhibitors was found to be similar in model films and in solution. WIth TNBT, absorbance measurements at a single wavelength (535 nm) were used to estimate the enzyme activity quantitatively. When carried out under standardized conditions, both the cytochemical and biochemical assay showed a linear relation with the time of incubation and obeyed the Beer-Lambert law. The correlation between biochemical and cytochemical data was very high, which enabled cytochemical data to be converted into absolute units of enzyme activity. The data obtained in this way closely resembled the data of enzyme activity calculated from the absorbance of formazan produced inside polyacrylamide model films and afterwards extracted into a suitable solvent.

Acrylamides↗
PubMed1993

Lymphocyte cytotoxicity and erythrocytic abnormalities induced in broiler chicks by fumonisins B1 and B2 and moniliformin from Fusarium proliferatum.

Peripheral blood lymphocytes were isolated from broiler chicks that had ingested feed amended with autoclaved Fusarium proliferatum culture material containing fumonisin B1 (FB1), fumonisin B2 (FB2) and moniliformin. Lymphocyte viability was determined for birds that were placed on amended rations at day 1 or day 7 of age at three different levels of mycotoxins, ranging from 61-546 ppm FB1, 14-94 ppm FB2 and 66-367 ppm moniliformin. Reduction of the tetrazolium salt, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide], to yield MTT formazan, based on mitochondrial metabolic activity, was used to assess cell viability. Lymphocyte cytotoxic effects were observed in all treatment groups on day 21; chicks that started on amended feed at day 1 of age were affected more than those that started at day 7. Abnormal erythrocytes resembling early stages of erythroblasts were observed in peripheral blood from test chicks. Abnormally shaped red cells (poikilocytes) having a spindle-shape with one or both ends pointed were present. Some red cells appeared to be undergoing mitosis. Both reduced lymphocyte viability and abnormal erythrogenesis occurred in chicks given feed amended with F. proliferatum culture material containing FB1, FB2 and moniliformin.

Animal Feed↗
PubMed1994

Cytotoxicity of mycotoxins evaluated by the MTT-cell culture assay.

The application of a modified colorimetric bioassay for the evaluation of the biological effects of mycotoxins is reported. Using three different monolayer cell lines (swine kidney, Madin Darby canine kidney, HeLa) the influence of nine different mycotoxins on the cellular methylthiazoltetrazolium (MTT)-cleavage activity was evaluated. The yellow tetrazolium salt MTT is converted by mitochondrial dehydrogenases of metabolically active cells to an insoluble purple formazan product, which was then solubilized with dimethylsulfoxide. The optical density of this homogeneous solution was suitable for a precise spectrophotometric measurement by a plate reader at a wavelength of 510 nm. Nine mycotoxins were simultaneously tested in all three cell lines, from which the swine kidney cell line proved to be the most sensitive. The effects of additional 35 mycotoxins were therefore tested using swine kidney monolayers as target cells. A total of 28 toxins of the 44 mycotoxins tested proved to be cytotoxic in the MTT-bioassay. Most of them belong to the group of trichothecene mycotoxins. Concentrations ranged between 0.01 micrograms and 100 micrograms/ml of cell culture medium. The MTT cleavage assay was found to be a quick (24 hours) and easy to perform system for the evaluation of the biological activity of many different mycotoxins and may also provide a useful tool for the testing of a large variety of sample materials.

Animals↗
PubMed1994

Gliotoxin inhibits transformation and its cytotoxic to turkey peripheral blood lymphocytes.

Gliotoxin, an epipolythiodioxopiperizine mycotoxin, has been shown to be produced by, among other fungi, Aspergillus fumigatus Fresenius. This organism is the major causative agent of the respiratory disease aspergillosis in avian species, especially turkeys. Because gliotoxin has been shown to be immunosuppressive and has the potential for being involved in the pathogenesis of aspergillosis, the in vitro activity of this compound with avian lymphocytes was investigated. Immunosuppression was investigated using peripheral blood lymphocytes from turkeys in a lymphoblastogenesis assay and a cytotoxicity assay using conversion of the tetrazolium salt MTT to MTT formazan by the mitochondrial succinate dehydrogenase enzyme elaborated only by living cells. Gliotoxin appeared to have a threshold level in both tests because little or no response or stimulation was evident when cells were exposed to concentrations of the toxin below 100 ng/ml, but at 100 ng/ml, all cells appeared to be dead. Using T-2 mycotoxin as a known cytotoxic agent, the response in the MTT bioassay using turkey peripheral lymphocytes was linear with increasing concentrations of toxin. Gliotoxin may potentially cause immunosuppression in turkey poults through action on the lymphocytes or if this toxin were present in low concentrations stimulation could possibly occur.

Animals↗
PubMed1976

Distribution of monoamine oxidase (MAO) in relation to the hypothalamo-hypophyseal system of the teleost Clarias batrachus (L.).

Varying degrees of monoamine oxidase (MAO) activity were noticed all along the hypothalamo-hypophyseal neurosecretory tract in C. batrachus. Intense activity was evident on the wall of the secretory cells of the adenohypophysis, neurons of the nucleus preopticus (NPO) and nucleus lateralis tuberis (NLT). However, MAO activity was also observed in the cytoplasm of some neurons in the ventromedian region of the NLT and groups of cells present in the neurohypophyseal ramifications of the pars intermedia. A strong accumulation of formazan granules in the median eminence (ME) and the neuroadeno interface lends further support to the view that C. batrachus has a ME of both the tetrapodan and the teleostean type. The distributional pattern of MAO in the hypothalamus and hypophysis suggests that monoaminergic fibres are probably involved in the secretory mechanism of the releasing factors and pituitary hormones.

Animals↗
PubMed1996

Ultrastructural distribution of NADPH-diaphorase in the normal hippocampus and after long-term potentiation.

The distribution of the enzyme nitric oxide synthase (NOS) was investigated at the ultrastructural level in synaptic structures of the hippocampal formation in relation to long-term potentiation (LTP), based on the histochemical NADPH-diaphorase (NADPH-d) staining with the tetrazolium salt BSPT. BSPT-formazan, the osmiophilic reaction product, was found to be selectively distributed and predominantly attached to membranes of the endoplasmic reticulum. In synaptic regions mainly the presynaptic sides showed labeling. Although several groups have demonstrated a principal involvement of NO in the LTP-mechanism, we found only a low, statistically insignificant increase in NADPH-d stained presynaptic areas of the dentate gyrus, where LTP was evoked. Postsynaptic elements also did not show any noticeable differences. Based on the present results, the predominantly presynaptic localization of NOS should be preferably considered in models describing a functional role of NO in LTP formation, despite the fact that we failed to reveal any indications for an LTP-related change in synaptically located NADPH-d.

Animals↗
PubMed1993

Location of nitroblue tetrazolium-reducing activity in human colonic mucosa obtained by biopsy.

The location of nitroblue tetrazolium-reducing activity was studied in colonic biopsy specimens from 28 patients with ulcerative colitis and from 23 controls. Nitroblue tetrazolium was reduced by epithelial cells, vascular endothelium, and interstitial mononuclear cells of the colonic mucosa from both groups. Blue formazan, a reduced form of nitroblue tetrazolium, was seen faintly after 30 min of culture; the amount increased up to 2 hr tested. The vascular endothelium of the patients with ulcerative colitis reduced nitroblue tetrazolium significantly more than that of the controls. The reduction of nitroblue tetrazolium was graded from 0 to 3 and was 1.0 +/- 0.6 in the controls, 1.6 +/- 0.8 in ulcerative colitis patients (P < 0.05), 1.9 +/- 0.7 (P < 0.01) in patients with active ulcerative colitis, and 1.4 +/- 0.9 in patients with inactive ulcerative colitis (difference not significant). Aggregates of mononuclear cells that reduced nitroblue tetrazolium were found in 10 of 28 patients with ulcerative colitis but not in the controls.

Adult↗
PubMed1988

Simultaneous histochemical assay of two dehydrogenases in the same cell.

A new approach has been developed for the simultaneous assay of the activities of two enzymes (lactate and succinate dehydrogenases) in the same cell in sections of unfixed liver. The sections, mounted on coverslips, were placed on top of 0.6-mm thick 0.8% low gelling-temperature agarose films containing the substrates of both enzymes (70 mM lactate and 50 mM succinate, respectively) plus 80 mM Tris-HCl buffer (pH 7.5), 5 mM EDTA, 10 mM NaN3, 1.5 mM NAD+, 1.2 mM Nitro BT and 0.26 mM phenazine methosulphate. The integrated absorbance (A) at 585 nm of the final reaction product formazans deposited by the two enzymes in a selected hepatocyte was measured continuously at 37 degrees C as a function of incubation time, using a Vickers M85 microdensitometer. The intercept A0 on the A-axis of the linear regression line of A on time was determined. After a known incubation time t, the absorbance A1, was noted and the section placed on another gel film lacking the substrates in order to estimate the final reaction product either formed in the gel film or lost from the cell. The absorbance A2 of the hepatocyte was remeasured. The reaction velocities (activities) vL and vS of lactate and succinate dehydrogenases, respectively, were calculated from the following equations: vL = [(A1-A2) - A0(1- alpha L)]/(1-alpha L)t and vS = (A2-alpha LA1)/(1-alpha L)t where alpha L = A2/A1 for hepatocytes incubated on gel films containing only lactate as the substrate. This parameter was found to be virtually constant (0.44) over a wide range of vL.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗