Histochemical study of monoamine oxidase latency in the liver of the rat.

In a histochemical test system with adrenaline as substrate and nitroblue tetrazolium (NBT) as electron acceptor, an increase of NBT reduction in rat liver sections was found microspectrophotometrically following short hypotonic treatment. Investigations with iproniazide, a monoamine oxidase inhibitor, and non-enzymatic NBT reduction showed that the increased formazan formation was related to the presence of monoamine oxidase. It is suggested that the reason for the observed increase of formazan formation is due to increased permeability of the inner mitochondrial membrane to NBT. Consequently, the increase of monoamine oxidase observed in the histochemical test system does not represent mobilization of a latent activity, but rather complete assessment of activity that is normally present.     

四唑(Tetrazolium)对固氮鱼腥藻固氮和氢代谢的影响

固氮鱼腥藻(Anabaena azotica Ley)细胞能还原无色的TTC和NBT分别成为红色或蓝色的甲(月朁)(formazan)沉淀。异形胞还原TTC的速率高于营养细胞。前异形胞及异形胞附近的营养细胞对NBT的还原作用最强。而异形胞对NBT不起还原作用。无论在异形胞形成红色甲(月朁)或在营养细胞形成蓝色甲(月朁)后都抑制固氮酶活性。NBT甲(月朁)对固氮酶活性的抑制作用大于TTC甲(月朁),因为NBT氧化还原电位低于TTC。 TTC和NBT两者都明显地抑制固氮鱼腥藻完整细胞的放氢。因鱼腥藻的放氢是由固氮酶催化的结果。四唑抑制放氢推想是由于它截取了固氮酶催化系统中的电子的缘故。固氮微生物(包括蓝色细菌和根瘤菌)对四唑还原与吸氢酶之间有无相关是一个争论的问题。一些学者认为分离豆科植物体的一些根瘤菌株培养于含有TTC的琼脂培养基,如还原,便可证明这些根瘤菌株能氧化氢;换言之,应用TTC的还原可作为一些根瘤菌的菌落具有吸氢酶的验证。相反,我们发现固氮鱼腥藻还原TTC和NBT之后,都没有影响吸氢的能力。因此,我们推想固氮鱼腥藻对四唑之还原与吸氢酶是没有直接的关系。     

Characteristics of Eliciting Effects of Furostanol Glycosides on Cultured Yam Cells

To unravel mechanisms of elicitor action of furostanol glycosides (FGs), the formation of superoxide anion after the addition of FGs to a suspension culture of yam (Dioscorea deltoidea Wall. ex Griseb) cells was studied. The substantial increase in superoxide level, evaluated by nitroblue tetrazolium (NBT) reduction to formazan, was found at the exponential phase of cell growth. The involvement of NADPH oxidase in the superoxide generation was revealed by means of inhibitory analysis. Diphenyliodonium chloride (DPI), the inhibitor of NADPH oxidase, compromised the action of FGs. Meanwhile, the elimination of apoplastic peroxidase did not affect the accumulation of formazan, which suggests the involvement of NADPH oxidase but not peroxidase in the superoxide generation. In addition to NBT-test, the superoxide formation was judged by changes in activity of superoxide dismutase (SOD). Exogenous FGs activated the enzyme due to the increased production of superoxide anion. In this case, DPI decreased SOD activity that conforms to the NADPH oxidase involvement in the superoxide generation. The analysis of antioxidant activity of FGs by inhibition of radicals of 2,2-diphenyl-1-picrylhydrazyl showed that FGs are weak reductants in comparison with ascorbic acid. The results of the work allow for the suggestion that, supposing a weak reducing capacity of FGs, the special feature of their exogenous action on cultured yam cells is the increase in the level of superoxide anion radical mainly produced by NADPH oxidase.     

Quantitative determination of superoxide in plant leaves using a modified NBT staining method

Introduction – In plants, the ROS (reactive oxygen species) level is tightly regulated because their accumulation produces irreversible damage leading to cell death. However, ROS accumulation plays a key role in plant signaling under biotic or abiotic stress. Although various methods were reported to evaluate ROS accumulation, they are restricted to model plants or provide only qualitative information. Objective – Develop a simple method to quantify superoxide radicals produced in plant tissues, based on the selective extraction of the formazan produced after nitroblue tetrazolium (NBT) reduction in histochemical staining. Methodology – Plant leaves were stained with a standard NBT method and the formazan precipitated in tissues was selectively extracted using chloroform. The organic phase was dried and formazan residue dissolved in dimethylsulfoxide–potassium hydroxide and quantified by spectrophotometry. The method was tested in strawberry plant leaves under different stressing conditions. Results – Formazan extracted from leaves subjected to stress conditions showed similar absorption spectra to those obtained from standard solutions using pure formazan. Calibration curves showed a linear relationship between absorbance and formazan amounts, within the range 0.5–8 µg. Outcomes suggested that formazan was retained in the solid residue of leaf tissues. This protocol allowed us to quantify superoxide radicals produced under different stress conditions. Conclusions – Chloroform allowed a selective formazan extraction and removal of potential endogenous, exogenous or procedural artefacts that may interfere with the quantitative determination. This protocol can be used to quantify the superoxide produced in plant tissues using any traditional qualitative NBT histochemical staining method.     

Improvement in Soluble Dehydrogenase Histochemistry by Nitroblue Tetrazolium Preuptake in Sections: A Qualitative and Quantitative Study

An improvement in the histochemical demonstration of soluble dehydrogenase enzymes has been obtained by preincubating frozen sections in a nitroblue tetrazolium (NBT)/ acetone solution, followed by routine incubation in polyvinyl alcohol (PVA) enriched media. Tissue binding properties of NBT were shown clearly to be decreased in histochemical media containing the colloid PVA for soluble enzymes, thus causing loss of the final reaction product (formazan) from the sections. The preincubation step in NBT/acetone allows tetrazolium salt to bind firmly to tissue lipoprotein (substantivity) and diminishes the loss of reduced formazan from heavily reacting tissue sections. The time course of NBT substantivity was examined and it was found that NBT binds rapidly to tissues (liver, kidney, heart) during preincubation, so that a preincubation of 30-60 seconds at room temperature is sufficient to improve the final morphological results greatly. Microspectrophotometric measurements of matched controls and NBT/acetone preincubated sections show that the preincubation step may slightly decrease lactate dehydrogenase (LDH) and glucose-6-phosphate dehydrogenase (G6PDH) activities. This decrease was probably due to increased binding efficiency of formazan to cell lipoproteins but was judged, however, to be irrelevant compared to the morphological advantages produced by the NBT/acetone preincubation procedure.     

Histochemical evidence of monoamine oxidase activity in rats of different ages

Summary The histochemically detectable monoamine oxidase activity in certain organs of young and old rats is compared. Regardless of age, the activity is strong in the liver, faint in the skeletal muscle, and absent in the kidney. In the myocardium, however, the quantity of monoamine oxidase increases strongly with age. Its activity is manifest in the form of granular and diffuse formazan precipitates; both disappear after a preliminary treatment of the animals with a monoamine oxidase inhibitor. This finding indicates that the diffuse as well as the previously identified granular precipitates represent monoamine oxidase.     

Improvement in soluble dehydrogenase histochemistry by nitroblue tetrazolium preuptake in sections: a qualitative and quantitative study

An improvement in the histochemical demonstration of soluble dehydrogenase enzymes has been obtained by preincubating frozen sections in a nitroblue tetrazolium (NBT)/acetone solution, followed by routine incubation in polyvinyl alcohol (PVA) enriched media. Tissue binding properties of NBT were shown clearly to be decreased in histochemical media containing the colloid PVA for soluble enzymes, thus causing loss of the final reaction product (formazan) from the sections. The preincubation step in NBT/acetone allows tetrazolium salt to bind firmly to tissue lipoprotein (substantivity) and diminishes the loss of reduced formazan from heavily reacting tissue sections. The time course of NBT substantivity was examined and it was found that NBT binds rapidly to tissues (liver, kidney, heart) during preincubation, so that a preincubation of 30-60 seconds at room temperature is sufficient to improve the final morphological results greatly. Microspectrophotometric measurements of matched controls and NBT/acetone preincubated sections show that the preincubation step may slightly decrease lactate dehydrogenase (LDH) and glucose-6-phosphate dehydrogenase (G6PDH) activities. This decrease was probably due to increased binding efficiency of formazan to cell lipoproteins but was judged, however, to be irrelevant compared to the morphological advantages produced by the NBT/acetone preincubation procedure.     

Quantitative aspects of the histochemical tetrazolium salt reaction on monoamine oxidase activity in rat liver

Summary The tetrazolium method for the histochemical detection of monoamine oxidase (MAO) activity in rat liver cryostat sections has been tested for its specificity and its possible use in quantification. The tetrazolium salt tetranitro blue tetrazolium is recommended for the localization of MAO activity, rather than nitro blue tetrazolium or BPST [2-(2-benzothiazolyl)-3(4-phthalhydrazidyl)-5-styryl-tetrazolium]. Hardly any formazan was produced in the absence of the substrate tryptamine and Marsilid, a specific inhibitor of MAO activity, prevented formazan production almost completely. A linear relationship between the integrated absorbance measured with a microdensitometer and either the incubation period or section thickness was obtained. We conclude that the method described in this paper can be used for the quantitative analysis of MAO activity in tissue sections of rat liver. MAO activity was found to be 20–25% higher in the periportal zone of rat liver than in the perivenous zone.     

Quantitative histochemical determination of succinic dehydrogenase activity in skeletal muscle fibres

Summary A histochemical technique was developed for the quantitative determination of succinic dehydrogenase (SDH) activity in muscle cross-sections using 1-methoxyphenazine methosulphate (mPMS) as the exogenous electron carrier, and azide as an inhibitor of cytochrome oxidase. The optimal composition of the incubation medium for the SDH reaction was determined. This histochemical procedure was compared to one using phenazine methosulphate (PMS) instead of mPMS and cyanide instead of azide. The substitution of mPMS and azide resulted in a substantial decrease in the non-specific reduction of nitroblue tetrazolium (NBT; the reaction indicator), i.e., nothing dehydrogenase activity. With mPMS and azide in the reaction medium, the production of NBT formazan was linear for at least 9 min during the enzymic reaction. This compared to a non-linear reduction of NBT during the initial stages of the reactions (SDH and nothing dehydrogenase) when using PMS and cyanide. The use of both mPMS and azide also eliminated the production of NBT monoformazan which occurred with PMS and cyanide. This procedure was shown to meet various criteria established for the quantification of histochemical reactions.     

Re-evaluation of superoxide scavenging capacity of xanthohumol

The chemopreventive chalcone xanthohumol (Xh) has been reported to decrease xanthine oxidase (XOD) catalysed formation of formazan from nitroblue tetrazolium (NBT) and is discussed as a potent scavenger of superoxide. Re-evaluation of the scavenging capacity indicated that Xh disturbed detection of superoxide with NBT, in case of an insufficient NBT/Xh ratio. Xh lacked superoxide scavenging activity in contrast to the Xh-derivative 3'-hydroxy-Xh with catechol substructure, used as positive control. This was shown by the use of sufficient concentration of NBT and other detectors such as hydroxylamine, XTT, cytochrome c and hydroethidine. HPLC analysis of reaction products in a xanthine/XOD/peroxidase system demonstrated beside enhanced inhibition of NBT-formazan by Xh that NBT even prevented oxidation of Xh. p-coumaric acid or ferulic acid could replace Xh in that system, indicating that superoxide detection using NBT is likely jeopardized by interference of phenoxyl-radicals. Furthermore, this study provides evidence that Xh can moderately generate superoxide via auto-oxidation.     

Superoxide-dependent and superoxide-independent pathways for reduction of nitroblue tetrazolium in isolated rat cardiac myocytes.

Spectroscopic studies indicated that nitroblue tetrazolium (NBT) could be reduced to blue formazan by several distinct reactions in suspensions of isolated rat cardiac myocytes. Both NADPH- and NADH-linked pathways for reduction of NBT were observed. NADPH-linked NBT reduction showed little activity in the absence of digitonin, but could be stimulated an average of 9.5-fold by digitonin permeabilization of the plasma membrane. NADH-linked NBT reduction occurred in the absence of digitonin, and could be increased an average of 3.5-fold by digitonin treatment. Analysis of the effects of cell viability on the extent of digitonin stimulation with these substrates suggested that the NADPH-linked reaction involved a cytosolic component, while the NADH-linked reaction involved an intracellular membrane enzyme system. With either NADPH or NADH, NBT reduction was completely inhibited by dicoumarol (100 microM). Dicoumarol-insensitive NBT reduction could subsequently be observed following the addition of 2 mM cyanide, a level of cyanide known to inhibit cytosolic superoxide dismutase. Cyanide-stimulated, dicoumarol-insensitive NBT reduction was augmented by the presence of either antimycin or doxorubicin, two agents which enhance superoxide formation by different mechanisms. The results indicate the existence of multiple pathways for both superoxide-independent and superoxide-dependent reduction of NBT. Dicoumarol-insensitive, cyanide-stimulated NBT reduction may be useful as a spectroscopic probe for intracellular superoxide formation.     

Re-evaluation of superoxide scavenging capacity of xanthohumol

Abstract

The chemopreventive chalcone xanthohumol (Xh) has been reported to decrease xanthine oxidase (XOD) catalysed formation of formazan from nitroblue tetrazolium (NBT) and is discussed as a potent scavenger of superoxide. Re-evaluation of the scavenging capacity indicated that Xh disturbed detection of superoxide with NBT, in case of an insufficient NBT/Xh ratio. Xh lacked superoxide scavenging activity in contrast to the Xh-derivative 3′-hydroxy-Xh with catechol substructure, used as positive control. This was shown by the use of sufficient concentration of NBT and other detectors such as hydroxylamine, XTT, cytochrome c and hydroethidine. HPLC analysis of reaction products in a xanthine/XOD/peroxidase system demonstrated beside enhanced inhibition of NBT-formazan by Xh that NBT even prevented oxidation of Xh. p-coumaric acid or ferulic acid could replace Xh in that system, indicating that superoxide detection using NBT is likely jeopardized by interference of phenoxyl-radicals. Furthermore, this study provides evidence that Xh can moderately generate superoxide via auto-oxidation.     

Cyanide-resistant reduction of nitroblue tetrazolium and hydrogen peroxide production by the rabbit blastocyst.

The ability of the rabbit blastocyst to reduce nitroblue tetrazolium (NBT) to formazan in the presence of cyanide was assayed as an indicator of extramitochondrial oxidase activity capable of generating the superoxide radical. A cytochemical method initially developed for the detection and localization of hydrogen peroxide production at the ultrastructural level in phagocytosing leukocytes (Briggs et al.: J Cell Biol 67:566, 1975) was also applied to the blastocyst. The results demonstrate that the rabbit blastocyst acquires the ability to reduce NBT by a cyanide-insensitive process and to generate hydrogen peroxide between the fourth and fifth days postcoitum. The enzymatic activity responsible is apparently an NAD(P)H-dependent oxidase in the outer, microvillous plasma membrane of the trophoblast.     

Differentiation of HL-60 promyelocytic leukemia cells monitored by flow cytometric measurement of nitro blue tetrazolium (NBT) reduction

Reduction of nitro blue tetrazolium (NBT) to insoluble blue formazan granules occurs during the stimulus-induced respiratory burst of mature granulocytes and is routinely used as an indicator of the extent of granulocytic differentiation of HL-60 acute promyelocytic leukemia cells. In the present study, the differentiation of HL-60 leukemia cells induced by dimethylsulfoxide (DMSO) or retinoic acid was monitored by flow cytometric (FCM) measurement of forward and 90 degree light scatter of NBT treated cells. Two-parameter correlated analysis permitted a distinction between cells with increased forward and decreased 90 degree light scatter (NBT-), and cells with decreased forward and increased 90 degree light scatter (NBT+). Fixation of NBT treated cells with 1% paraformaldehyde facilitated flow cytometric analysis, and allowed differences in NBT reduction to be quantitated. DMSO-induced cells expressed an all-or-none reduction of NBT to formazan, compared with retinoic acid treated cells that exhibited a graded response. Three parameter flow cytometric analysis of HL-60 leukemia cells stained with propidium iodide in combination with NBT allowed the determination of the cell cycle distribution of NBT-treated cells.     

植物冻害和氧化胁迫——甘蓝叶冻-融循环中超氧的产生

为检验植物冻害的发生和氧化胁迫这一假说,在冰冻前把氮蓝四唑（NBT）真空渗入到甘蓝叶圆片中,在叶圆片冻－融循环中NBT被还原为甲Zan,把其中的单甲Zan用乙醇提取出来,在分光光度计上比色,可作为冻融循环中产生的氧化胁迫的定量指标,NBT本身作为氧化剂,使冻害稍有增加,作为冰冻保护剂的二甲基亚砜真空渗入叶圆片使其抗冻性显著增加,而NBT还原则显著减少,表明二甲基亚砜在保护叶组织免受冻害上的作用和它减缓植物组织氧化胁迫的作用有关。实验结果支持植物冻害的发生和氧化胁迫有关这一假说。实验还表明还原NBT的还原剂很可能是超氧阴离子自由基。     

Reduction of mercuric ion in vitro by superoxide anion

The reduction rate of mercuric ion to metallic mercury by a superoxide anion produced by a xanthine-xanthine oxidase system increased with an increased concentration of xanthine oxidase in the presence of enough xanthine. The reduction rate of mercuric ion by a superoxide anion in the presence of nitroblue tetrazolium (NBT) was proportional to the concentration of NBT. The result suggests that NBT was reduced to diformazan by a superoxide anion produced by a xanthine-xanthine oxidase system and that mercuric ion will be reduced to metallic mercury by diformazan. The reduction rate of mercuric ion was also indicative that a superoxide anion produced by an NADH-phenazine methosulfate (PMS) system increased with an increased concentration of PMS.     

Flow cytometric evaluation of nitro blue tetrazolium (NBT) reduction in human polymorphonuclear leukocytes

Oxidative metabolic burst of activated human polymorphonuclear leukocytes (PMN) is most commonly investigated in clinical practice by evaluating nitroblue tetrazolium (NBT) reduction at the single cell level. Reduced NBT precipitates where the redox reaction has taken place and can be visualized as PMN-associated dark blue granules of formazan in light microscopy. Although widely used and not technically demanding, this method remains subjective and labor intensive, especially when large numbers of samples need to be investigated. We developed a new flow cytometry technique in which PMN membrane was rendered fluorescent by a short incubation with fluorescein-conjugated Concanavalin A. PMN were then incubated with NBT and increasing doses of a suitable stimulus, such as phorbol myristate acetate (PMA). Formazan has a distinct peak of absorption at 520 nm that represents the peak of emission of fluorescein. As a consequence, formazan quenches the PMN-associated fluorescence. Data show that a dose-dependent reduction of fluorescence can be obtained using graded amounts of PMA in normal PMN cultures. PMN-associated fluorescence remains unchanged in control patients with chronic granulomatous (CGD) disease, a disorder characterized by a selective impairment of PMN oxidative metabolism. Electronic cell size increases upon PMA incubation in normal PMN, irrespective of the presence of NBT. Conversely, forward light scatter intensity decreases in the presence, but not in the absence, of NBT indicating that the phenomenon is due to the capacity of formazan to absorb/scatter the incident light. The present method for easily detecting NBT reducing activity at single cell level by flow cytometry makes use of commonly available, inexpensive reagents and standard instrumentation. It could become a useful test for clinical purposes.     

Developmental changes in mitochondria during the transition into lactation in the mouse mammary gland. II. Membrane marker enzymes and membrane ultrastructure

The activity of cytochrome oxidase (an inner mitochondrial membrane marker) in mouse mammary gland homogenates was found to increase five- to sixfold from late pregnancy to day 8 of lactation, while that of monoamine oxidase (an outer membrane marker) increased only about 25%. The specific activity of cytochrome oxidase in the isolated mitochondria decreased slightly over the same period while the specific activity of monoamine oxidase decreased fivefold. This reflects the fact that both cytochrome oxidase and mitochondrial protein are increasing at a much greater rate than is monoamine oxidase activity. Mixing experiments preclude the possibility that the release or removal of an inhibitor or stimulator produces the changes in enzymatic activity. The cytochrome oxidase to monoamine oxidase ratio was followed throughout the pregnancy-lactation cycle in total mammary homogenates, isolated mammary parenchymal cells, and isolated mammary mitochondria. In each preparation the pattern was the same with little change in the ratio until late pregnancy; and then a three- to fourfold increase occurred and the values reached a maximum by day 8 of lactation. These experiments were interpreted as demonstrating that the observed enzymatic changes are reflective of alterations in the mitochondria of the mammary parenchymal cell population. Electron micrographs of mid-pregnant and mid-lactating mammary parenchymal cells in situ were prepared, and distinct changes in the mitochondrial morphology noted. The most significant and obvious change is the large increase in the number of inner membrane cristae and an increase in matrix density in the lactating gland cell. Therefore, both enzymatic and morphological studies support the concept of an expansion of the mitochondrial inner membrane during presecretory differentiation in the mouse mammary parenchymal cell.     

Generation of Superoxide Anion and Localization of CuZn-Superoxide Dismutase in the Vascular Tissue of Spinach Hypocotyls: Their Association with Lignification

The sites of generations of superoxide anions and hydrogen peroxidein cross sections of hypocotyls from spinach seedlings werelocated by staining with nitroblue tetrazolium (NBT) and withstarch-iodide, respectively. Formazan, produced upon the reductionof NBT by superoxide, was observed mainly in the vascular tissueonly in the presence of inhibitors of CuZn-superoxide dismutase(CuZn-SOD), and its formation was suppressed under anaerobicconditions. Thus, NBT was reduced to formazan specifically bythe superoxide anions generated in vascular tissue. The reductionof NBT was suppressed by inhibitors of NAD(P)H oxidase, butneither by cyanide nor azide, indicating the involvement ofNAD(P)H oxidase in the generation of superoxide anions in thevascular tissue. Starch-I₂ complex also was formed in the vasculartissue, but not in the presence of either the CuZn-SOD inhibitoror the NAD(P)H oxidase inhibitor, indicating that the hydrogenperoxide is produced via the catalytic disproportionation withCuZn-SOD of the superoxide generated by NAD(P)H oxidase. Generationsof superoxide anions and hydrogen peroxide in the vascular tissuewere particularly apparent in the xylem and associated withthe sites of distribution of CuZn-SOD as determined by an immunohistochemicalmethod, and also with the location of lignin as determined bythe phloroglucin-HCI reaction. ⁴Present address: Chemical Research Laboratories, Toray IndustriesInc., 9–1 Oe-cho, Minato-ku, Ngagoya, 455 Japan     

A fluorescent redox dye. Influence of several substrates and electron carriers on the tetrazolium salt-formazan reaction of Ehrlich ascites tumour cells

Summary This study was performed to elaborate the best conditions for measuring the redox activity of Ehrlich ascites tumour cells by using a new tetrazolium salt, cyantolyl tetrazolium chloride (CTC). This tetrazolium salt forms a fluorescent water-insoluble formazan on reduction on the surface of intact vital cells. The influences of fixation and of various substrates and electron carriers on the cellular reduction of CTC were investigated quantitatively using an elution technique. The amount of formazan obtained after incubating vital cells with Meldola Blue as electron carrier was greater than that obtained with Methylene Blue, menadione, 2,6-dichloroindophenol, 1-methoxyphenazine methosulphate or phenazine methosulphate. Using flow cytometry, the formazan production per cell and, after staining the nuclear DNA, the distribution of the redox activity in the cell population can be visualized with satisfactory resolution. We conclude from our findings that dehydrogenases are only partially involved in the reduction of tetrazolium salts by intact cells and that a redox activity, probably related to a cell membrane-bound NAD(P)H—oxidase system, is mainly measured.