Studies on the reduction of nitroblue tetrazolium chloride mediated through the action of NADH and phenazine methosulphate.

The general features of the reduction of nitroblue tetrazolium chloride (NBT) by NADH and phenazine methosulphate (PMS) have been studied under aerobic and anaerobic conditions. Under aerobic condition the reduction appears to be mediated through the intermediate formation of the superoxide anion radical O2-.; this reaction is strongly inhibited by superoxide dismutase and by a number of O2-. scavengers such as propyl gallate, (+)-catechin, manganous ions, reduced glutathione and benzoquinone. Cupric ions inhibited the overall reaction by reoxidising reduced PMS. Under anaerobic conditions, superoxide dismutase had only a small inhibitory action and, with the exception of cupric ions, the other substances mentioned above were ineffective as inhibitors. The data presented show that the use of NBT to detect the presence of O2-. is fraught with difficulties due to an equally rapid reduction of NBT by NADH and PMS under anaerobic conditions.     

锰——超氧化物歧化酶活力测定的五种方法比较研究

 本文分别以CN~-抑制和SDS处理区分Mn-SOD与CuZn-SOD,对五种SOD活力测定方法进行了比较研究。结果表明:(1)化学发光法和光化学扩增法不适用于Mn-SOD活力测定,CN~-和SDS对这两种方法有明显的干扰作用。(2)NBT还原、Cyt c还原和亚硝酸盐形成法都能用于Mn-SOD活力测定,用这三种方法测得Mn-SOD每活力单位相当于酶的含量分别为2.93μg、0.11μg和0.028μg。说明NBT还原法灵敏度最低,其次是Cyt c还原法。亚硝酸盐形成法灵敏度高,专一性强,为五种测定方法之首。     

The catalytic effect of the carcinogen "4-nitroquinoline-N-oxide" on the oxidation of vitamin C.

The carcinogen 4-nitroquinoline-N-oxide was found to mediate the reaction between ascorbate and oxygen. The oxidation of ascorbate was initiated by the production of the nitro radical anion which reacted with oxygen to produce the oxygen superoxide radical anion, peroxide and hydroxyl radical. The production of partially reduced oxygen intermediates resulted in additional reactions with ascorbate. The consumption of oxygen could be either completely blocked by reacting the nitro radical with ferricytochrome c or partially blocked by the combined effects of superoxide dismutase and catalase. The consumption of oxygen could be enhanced by reducing the hydroxyl radicals with dimethylsulfoxide.     

Generation of radical anions of nitrofurantoin, misonidazole, and metronidazole by ascorbate

Nitrofurantoin, misonidazole, and metronidazole were reduced to their corresponding nitro anion radicals by ascorbate in anaerobic solutions at high pH. The nitrofurantoin anion radical could be detected at neutral pH. In neutral solutions, the nitro anion radicals of misonidazole and metronidazole were too unstable to be observed by electron spin resonance spectroscopy. At neutral pH, solutions containing ascorbate, nitrofurantoin, or misonidazole consumed oxygen. The addition of superoxide dismutase, catalase, or both superoxide dismutase and catalase decreased the rate of oxygen consumption. These results show that nitro anion radicals are formed by reduction with ascorbate, and superoxide anion radical and hydrogen peroxide are produced by reactions of these radicals with oxygen.     

Vanadate-stimulated NADH oxidation in microsomes

Addition of vanadate, stimulated oxidation of NADH by rat liver microsomes. The products were NAD⁺ and H₂O₂. High rates of this reaction were obtained in the presence of phosphate buffer and at low pH values. The yellow-orange colored polymeric form of vanadate appears to be the active species and both ortho- and meta-vanadate gave poor activities even at mM concentrations.The activity as measured by oxygen uptake was inhibited by cyanide, EDTA, mannitol, histidine, ascorbate, noradrenaline, adriamycin, cytochrome c, Mn²⁺, superoxide dismutase, horseradish peroxidase and catalase. Mitochondrial outer membranes possess a similar activity of vanadate-stimulated NADH oxidation. But addition of mitochondria and some of its derivative particles abolished the microsomal activity. In the absence of oxygen, disappearance of NADH measured by decrease in absorbance at 340 nm continued at nearly the same rate since vanadate served as an electron acceptor in the microsomal system. Addition of excess catalase or SOD abolished the oxygen uptake while retaining significant rates of NADH disappearance indicating that the two activities are delinked. A mechanism is proposed wherein oxygen receives the first electron from NAD radical generated by oxidation of NADH by phosphovanadate and the consequent reduced species of vanadate (V^iv) gives the second electron to superoxide to reduce it H₂O₂. This is applicable to all membranes whereas microsomes have the additional capability of reducing vanadate.     

Oxygen radicals are generated by dye-mediated intracellular photooxidations: a role for superoxide in photodynamic effects

Representative thiazines, xanthenes, acridines, and phenazines photosensitized the oxidation of reduced pyridine nucleotides and reduced glutathione when illuminated with low intensity visible light. Photooxidation resulted in oxygen consumption and in superoxide generation, assayed as the superoxide dismutase (SOD)-inhibitable reduction of ferricytochrome c. The major pathway of electron transfer involved dye reduction rather than singlet oxygen-mediated oxidation of the substrate, as demonstrated by the relative insensitivity of the oxidation to inhibition by sodium azide and by the observable bleaching of the dye. Hydrogen peroxide was a stable end product of photooxidation. Photosensitive dyes were photoreduced intracellularly. These dyes were transported across the membranes of Escherichia coli B and stimulated a light- and concentration-dependent increase in the cyanide-insensitive respiration. Dyes reduced intracellularly subsequently diffused out of the cell where they reduced extracellular cytochrome c. The photosensitive dyes examined in this study exhibited a light-dependent bacteriostatic effect on E. coli B grown in nutrient broth, manifested as an increased lag prior to growth. Restoration of growth coincided with increased levels of SOD, and the intracellular level of SOD correlated with the level of illumination, the dye concentration, and the reactivity of the dye to NADH in vitro. The thiazine dye, toluidine blue o, imposed a light- and oxygen-dependent lethality on E. coli B grown in glucose minimal medium. Toxicity was relieved by hydroxyl radical scavengers, and their ability to protect the cells was proportional to their reactivity with the hydroxyl radical. The results indicate that oxygen radicals and related species mediate photodynamic effects in E. coli B.     

SUPEROXIDE DISMUTASE OF MAMMALIAN NERVOUS SYSTEM

Superoxide dismutase was assayed in portions of the nervous system (a) by inhibition of tetrazolium reduction by oxygen radicals, generated enzymically, and (b) by inhibition of tetrazolium reduction by oxygen radicals generated by oxidation of NADH in presence of phenazine methosulphate. Superoxide dismutase activity was found in beef brain, retina and adrenal medulla, as well as in brain, retina and lungs of adult and of newborn rats. Preliminary experiments with rats exposed to hyperbaric oxygen showed no alteration of enzyme activities in tissues of newborn and adult animals. The possible role of superoxide dismutase in the nervous system is discussed.     

Hydrogen peroxide formation and iron ion oxidoreduction linked to NADH oxidation in radish plasmalemma vesicles

Previously, we showed the presence in radish (Raphanus sativus L.) plasmalemma vesicles of an NAD(P)H oxidase, active at pH 4.5-5.0, which elicits the formation of anion superoxide (Vianello and Macrí (1989) Biochim. Biophys. Acta 980, 202-208). In this work, we studied the role of hydrogen peroxide and iron ions upon this oxidase activity. NADH oxidation was stimulated by ferrous ions and, to a lesser extent, by ferric ions. Salicylate and benzoate, two known hydroxyl radical scavengers, inhibited both basal and iron-stimulated NADH oxidase activity. The iron chelators EDTA (ethylenediaminetetraacetic acid) and DFA (deferoxamine melysate) at high concentrations (2 mM) inhibited the NADH oxidation, whereas they were ineffective at lower concentrations (80 microM); the subsequent addition of ferrous ions caused a rapid and limited increase of oxygen consumption which later ceased. Hydrogen peroxide was not detected during NADH oxidation but, in the presence of salicylate, its formation was found in significant amounts. NADH oxidase activity was also associated to a Fe2+ oxidation which was only partially inhibited by salicylate. Ferrous ion oxidation was partially inhibited by catalase and prevented by superoxide dismutase, while ferric ion reduction was abolished by catalase and unaffected by superoxide dismutase. These results show that during NADH oxidation iron ions undergo oxidoreduction and that hydrogen peroxide is produced and rapidly consumed. As previously suggested, this oxidation appears linked to the univalent oxidoreduction of iron ions by a reduced flavoprotein of radish plasmalemma which is then converted to a radical form. The latter, reacting with oxygen generates the superoxide anion which dismutases to H2O2. Hydrogen peroxide, through a Fenton's reaction, may react with Fe2+ to produce hydroxyl radicals, or with Fe3+ to generate the superoxide anion.     

Changes in superoxide dismutase activity in the presence of electron donors and acceptors

The activity of superoxide dismutase (SOD) from bovine erythrocytes was measured by the inhibition of nitrotetrazolium blue reduction rate in superoxide anion radical generation systems--xanthine/xanthine oxidase of NADH/phenazine methasulfate. The enzyme activity increases in the presence of compounds acting as electron donors in radical-involving reactions and decreased in the presence of compounds possessing the properties of electron acceptors. Activation of SOD by electron donors and its inhibition by electron acceptors was dependent on the concentration of the above compounds. In the absence of SOD electron donors and acceptors did not change the rate of tetrazolium blue reduction by superoxide anion radicals. The role of the new type of SOD regulation for the enzyme functioning in the cell is discussed.     

Properties of 2-nitropropane dioxygenase of Hansenula mrakii. Formation and participation of superoxide.

2-Nitropropane dioxygenase, purified to homogeneity from a yeast, Hansenula mrakii, is significantly inhibited by superoxide dismutase and various scavengers for superoxide anion such as cytochrome c, epinephrine, NADH, thiols, and polyhydric phenols. The reduction of cytochrome c and the oxidation of epinephrine and NADH are concomitant with the inhibition of enzymatic oxygenation. Neither the oxidation nor the reduction occursin the presence of superoxide dismutase or in the absence of 2-nitropropane or oxygen. Superoxide anion added externally induces the oxygenation. These findings indicate the generation of superoxide anion and its participation in the oxygenation of 2-nitropropane. The difference spectrum of the binding of NADH to 2-nitropropane dioxygenase exhibits a negative peak at 353 nm. One mole of NADH is bound to 1 mol of the enzyme and the pro-R hydrogen of the nicotinamide moiety of bound NADH predominantly is transferred to superoxide anion formed enzymatically or given externally. Thus, the diastereotopic hydrogen of NADH is discriminated by the enzyme, although not completely.     

Superoxide production by mitochondria isolated from green bell pepper fruit

Evidence is increasing to suggest that a wide range of environmentally induced plant disorders, including chilling injury, is mediated by reactive oxygen species produced during stress or upon relief from stress. Mitochondria were isolated from pericarp tissue of chilling-sensitive bell pepper fruit and their respiratory activity and ability to produce superoxide when supplied with NADH, succinate or malate-pyruvate were determined. Oxygen uptake rates were greater and less sensitive to cyanide with succinate than with NADH; rates increased and sensitivity to cyanide and respiratory control ratios (RCRs) decreased in fruit stored at 2°C. Disrupting mitochondrial membranes led to increased oxygen consumption with NADH and decreased consumption with succinate. resulting in RCRs of approximately 1 with both substrates. Superoxide production was greater with NADH than with either succinate or malatepyruvate. Superoxide dismutase and cyanide inhibited superoxide production almost completely. Antimycin A did not inhibit superoxide production with NADH, but did partially with succinate, especially in mitochondria sensitive to cyanide. Disrupting mitochondrial membranes enhanced superoxide production with NADH. Superoxide production by mitochondria isolated from fruit stored at 2°C increased with NADH and decreased with succinate. Results provide evidence that mitochondria may be a major source of superoxide in chilling-sensitive plant tissues exposed to low temperatures.     

The role of catalytic superoxide formation in the O2 inhibition of nitroreductase.

Many nitroreductases are strongly inhibited by oxygen. The first intermediate of nitroreductase activity, the nitroaromatic anion free radical, cannot be detected in aerobic microsomal incubations. Even though the nitro compounds are unchanged, both nitrofurantoin and $\text{p}$-nitrobenzoate profoundly increase the NADPH-supported oxygen uptake. This catalytic oxygen consumption is partially reversed by superoxide dismutase, suggesting that superoxide anion free radical is being formed by the rapid air oxidation of the nitroaromatic anion radical.     

A protective function of superoxide dismutase during respiratory chain activity.

(1) Aerobic incubation of heart muscle submitochondrial particles in phosphate buffer after treatment with NADH causes a progressive and substantial inhibition of the NADH oxidation system. Succinate oxidation remains almost unaffected by NADH treatment. (2) The loss of NADH oxidase activity is due to an inhibition of the respiratory chain-linked NADH dehydrogenase. This inhibition of the enzyme is very similar to that caused by combination of the organic mercurial mersalyl with NADH dehydrogenase. (3) The inhibition of NADH oxidation is largely prevented by compounds that are known to react with superoxide ions (02-.), including superoxide dismutase, cytochrome c, tiron and Mn2+. EDTA also has a protective effect, but a number of other metal chelating agents, and several proteins, including catalase, are without effect. (4) It is concluded that the inhibition of NADH oxidation of NADH oxidation by superoxide ions or by mersalyl is reversible and is therefore not due to the loss of oxidoreduction components from the respiratory chain or to an irreversible change in protein conformation. (6) The function of mitochondrial superxide dismutase is discussed in relation to the key role of NADH dehydrogenase in energy-conserving reactions and the formation of hydrogen peroxide during mitochondrial oxidations.     

Interaction of heme nonapeptide derived from cytochrome c with microsomal reductases

The interaction of heme nonapeptide (a proteolytic product of cytochrome c) with purified NADH:cytochrome b5 (EC 1.6.2.2) and NADPH:cytochrome P-450 (EC 1.6.2.4) reductases was investigated. In the presence of heme nonapeptide, NADH or NADPH were enzymatically oxidized to NAD+ and NADP+, respectively. NAD(P)H consumption was coupled to oxygen uptake in both enzyme reactions. In the presence of carbon monoxide the spectrum of a carboxyheme complex was observed during NAD(P)H oxidation, indicating the existence of a transient ferroheme peptide. NAD(P)H oxidation could be partially inhibited by cyanide, superoxide dismutase and catalase. Superoxide and peroxide ions (generated by enzymic xanthine oxidation) only oxidized NAD(P)H in the presence of heme nonapeptide. Oxidation of NAD(P)H was more rapid with O2- than O2-2. We suggest that a ferroheme-O2 and various heme-oxy radical complexes (mainly ferroheme-O-2 complex) play a crucial role in NAD(P)H oxidation.     

Generation of superoxide free radical by neocarzinostatin and its possible role in DNA damage

Spectroscopic analysis of the reduction of both nitro blue tetrazolium and ferricytochrome c induced by neocarzinostatin shows that superoxide free radical is produced during the spontaneous degradation of the antibiotic. The amount of superoxide free radical produced from neocarzinostatin is not affected by the presence of thiol, although earlier work has shown that DNA damage is stimulated at least 1000-fold by thiol. Transition metals are not involved in this reaction. Although superoxide dismutase inhibits the reduction of nitro blue tetrazolium and cytochrome c induced by neocarzinostatin, neither it nor catalase interferes with the action of neocarzinostatin on DNA, whether or not drug has been activated by thiol. The pH profiles for spontaneous base release and alkali-labile base release (a measure of nucleoside 5'-aldehyde formation at a strand break) do not correspond with that for the generation of superoxide free radical from neocarzinostatin. The same holds for supercoiled DNA cutting by neocarzinostatin chromophore in the absence of a thiol, which is an acid-favored reaction. These results indicate that the generation of superoxide free radical by the drug does not correlate with DNA damage activity, whether or not thiol is present. Furthermore, the failure of hydroxyl free-radical scavengers to inhibit drug-induced single-strand breaks in supercoiled DNA in the absence of thiol also indicates that a diffusible hydroxyl free radical is most probably not involved in this reaction.     

A comparative study of superoxide dismutase activity assays in Crocus sativus L. corms

Superoxide dismutase catalyzes the breakdown of superoxide radical anion and provides the first line of defense against oxygen toxicity. Its vital importance has made it the subject of numerous investigations. Several assays have been proposed for the detection and quantitation of superoxide dismutase activity, but their use has remained controversial and no comparative studies have been reported. In this investigation, three commonly used methods for the measurement of superoxide dismutase activity were compared to assay the enzyme in Crocus sativus L. corm extract. The methods, based on the competition between the enzyme itself and a superoxide scavenger, involved cytochrome c reduction, nitro blue tetrazolium reduction, and pyrogallol autooxidation, respectively. Because of its accuracy, reproducibility, simplicity, and cost benefit, the latter method was preferred. The text was submitted by the authors in English.     

Mechanism of thyroxine-mediated oxidation of reduced nicotinamide adenine dinucleotide in peroxidase-H2O2 system.

The oxidation of reduced nicotinamide adenine dinucleotide (NADH) by the horseradish peroxidase (HRP)-H2O2 system is greatly increased by the addition of thyroxine or related compounds. On the basis of a study of the rate of NADH oxidation in the presence of various concentrations of thyroxine, it is clear that thyroxine acts as a catalyst for NADH oxidation. Spectral changes of a HRP-H2O2 complex (compound I) indicate that thyroxine acts as an electron donor to both compounds I and II. The rate of electron donation from thyroxine is much faster than that from NADH. The HRP-H2O2 system requires 0.83 mol of O2 for the oxidation of 1 mol of NADH. Ferricytochrome c is reduced to ferrocytochrome c by the system, and causes an inhibition of O2 consumption which can be abolished by superoxide dismutase. JUDGING FROM THE INHIBITION OF O2 uptake by ferricytochrome c, about 54% of the total flux of electrons from NADH to oxygen appears to proceed by way of O2-. These results suggest that the initial step of thyroxine-mediated NADH oxidation by HRP and H2O2 is the formation of oxidized thyroxine, a phenoxy radical, which attacks NADH to produce NAD.     

Trans-plasma membrane electron transport: A cellular assay for NADH- and NADPH-oxidase based on extracellular, superoxide-mediated reduction of the sulfonated tetrazolium salt WST-1

Summary Plasma membrane NADH-oxidase of mammalian cells is usually assayed biochemically in isolated plasma membranes by measuring its ability to oxidise NADH or to reduce oxygen to water. Lack of a convenient cellular assay has greatly limited the study of NADH-oxidase, the physiological significance of which remains uncertain. Recently, we demonstrated that the novel cell-impermeative sulfonated tetrazolium salt WST-1 (2-[4-iodophenyl]-3-[4-nitrophenyl]-5-[2,4-disulfophenyl]-2H-tetrazolium, monosodium salt), used in conjunction with an intermediate electron acceptor, was reduced extracellularly suggesting involvement of a component of the trans-plasma membrane electron transport system in WST-1 reduction. In this study we provide evidence that WST-1 is reduced at the external surface of the plasma membrane by an NADH-oxidase, and that reduction is primarily mediated by superoxide. Thus, WST-1 reduction was extensively inhibited by superoxide dismutase and by the potent NADH-oxidase inhibitor resiniferatoxin. Dihydrocapsaicin and capsaicin which are less potent inhibitors of NADH-oxidase also inhibited WST-1 reduction, but the impermeative SH-blocking reagentpara-chloromercuriphenylsulfonic acid and trypsin, both of which are known to inhibit NADH-ferricyanide reductase but not NADH oxidase, had little effect on WST-1 reduction. Human peripheral blood neutrophils activated by phorbol myristate acetate efficiently reduced WST-1. This reduction was inhibited by 95% by superoxide dismutase but was unaffected by resiniferatoxin indicating a distinct mechanism of reduction by neutrophil NADPH-oxidase. Metabolic inhibitors were used to investigate putative involvement of cytosolic NADH in WST-1 reduction. Mitochondrial inhibitors such as cyanide and thenoyltrifluoroacetone, and to a lesser extent azide and rotenone, stimulated WST-1 reduction by Jurkat cells whereas inhibitors of glucose uptake and glycolysis were inhibitory. These results are explained by respiratory inhibitors having a sparing effect on cytosolic NADH levels and by glycolytic inhibitors lowering NADH. We conclude that WST-1 is reduced extracellularly by plasma membrane NADH-oxidase by a mechanism involving superoxide production. WST-1 is also efficiently reduced by the plasma membrane NADPH-oxidase of activated neutrophils.Abbreviations WST-1 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt - MTT 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide - XTT 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-carboxanilide-2H-tetrazolium, monosodium salt - MTS 3-(4,5-dimethylthiazol-2-yl)-5-(3-car-boxymemoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt - TTFA thenoyltrifluoroacetone - pCMBS p-chloromercuriphenylsul-fonic acid - SOD Superoxide dismutase - PMOR plasma membrane - NADH oxidoreductase - PMS phenazine methosulfate - PMA phorbol myristate acetate     

Phenazine Methosulfate; its Use in Evaluating Activity of Dehydrogenase Systems of Avian Liver

Lactate dehydrogenase (LDH), malate dehydrogenase (MDH) and suecinate dehydrogenase were demonstrated in livers of 15-day chick embryos. The addition of phenazine methosulfate (PMS) to the LDH and MDH incubation mixtures reduced diformazan deposition in the liver epithelium but not in connective tissue. A 30 sec formalin fixation, absence of PMS, or the addition of sodium azide or potassium cyanide to the PMS-containing incubation mixtures facilitated formazan deposition. These results are explained by assuming that, in the absence of PMS, dehydrogenase activity is demonstrated via endogenous diaphorase. When PMS is present, Nitro BT reduction occurs within the incubation mixture. A side effect of the azide or cyanide is an interference with, the action of PMS, thus allowing diformazan deposition via the endogenous diaphorase when this is present in the tissue.     

Comparative study of superoxide dismutase activity assays in Crocus sativus L. corms

Superoxide dismutase catalyzes the breakdown of the superoxide radical anion and provides the first line of defense against oxygen toxicity. Its vital importance has made it the subject of numerous investigations. Several assays have been proposed for the detection and quantitation of superoxide dismutase activity, but their use has remained controversial and no comparative studies have been reported. In this investigation, three commonly used methods were compared for the measurement of superoxide dismutase activity in Crocus sativus L. corm extract. The methods, based on a competition between the enzyme itself and another superoxide scavenger, involved respectively cytochrome c reduction, nitro blue tetrazolium reduction, and pyrogallol autoxidation. Because of its accuracy, reproducibility, simplicity and cost benefit, the latter method was the most appropriate.