Hypoxanthine-xanthine Oxidase System Research Articles

Studies on biological damage by active oxygens. III. Generation of hydroxyl radical and inhibition of insulin release in hypoxanthine-xanthine oxidase system in the presence of pancreatic islet cells

The hydroxyl radicals (HO.) spin adduct of 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO) was obtained in the hypoxanthine-xanthine oxidase (HX-X.O.) system in the presence of isolated islet cells by using electron spin resonance spectroscopy. There was a significant increase in the concentration of DMPO-OH spin adduct dependent on the increase in a number of islet cells. In the absence of islet cell, the DMPO-OOH spin adduct formed by superoxide together with a small amount of DMPO-OH spin adduct was detected in HX-X.O. system. These results suggest that HO. is greatly produced in the HX-X.O. system in the presence of islet cells. The generation of HO. was partially inhibited by the addition of superoxide dismutase or catalase and completely by the combined use of both enzymes. Iron-chelator, diethylenetriaminepentaacetic acid and iron-binding protein, apotransferrin also inhibited the generation of HO., suggesting a possible participation of a trace iron in the islet cells. The inhibition of insulin release from islet cell, as well as the amount of DMPO-OH spin adduct, was dependent on the concentration of X.O. These results indicate that the HO. is produced by the site-specific action with islet cells in HX-X.O. system, suggesting a possible involvement of HO. in the oxygen damages of islet cells.

Ultracytochemical Study of Ca2+-ATPase Activity in Isolated, Superoxide-treated Rat Brains

The relationship between changes in Ca2+-adenosine triphosphatase (ATPase) activity and plasma membrane damage was histochemically studied by enzymatic electron microscopy in rat brains with and without superoxide treatment. The brains were obtained from male Wistar rats after decapitation, and the control brains were examined immediately. Brains not treated with superoxide were incubated at 20 degrees C for 3, 6, or 12 hours. The superoxide-treated brains were immersed in a hypoxanthine-xanthine oxidase system for 20, 60, or 120 minutes. Ca2+-ATPase activity in the cerebral cortex and hippocampal CA1 was studied by the lead citrate method. Control brains showed Ca2+-ATPase activity in the membrane of the nerve cell body and dendrites, the basement membrane of endothelial cells, and the erythrocyte membrane. In untreated brains, enzymatic activity gradually decreased but was still detected after 12 hours. In those treated with superoxide, enzymatic activity gradually decreased but was still observed after 120 minutes in fragments of the plasma membrane. These findings show that the plasma membrane is more affected by treatment with superoxide than by decapitation itself, and that plasma membrane damage precedes the disappearance of Ca2+-ATPase activity. Residual Ca2+-ATPase activity does not necessarily imply reversibility of cerebral ischemia.

Highly sensitive and reliable chemiluminescence method for the assay of superoxide dismutase in human erythrocytes

Superoxide dismutase in human erythrocytes was assayed by the inhibition of highly diluted erythrocyte lysates on 2-methyl-6-( p-methoxyphenyl)-3,7-dihydroimidazo[1,2- a]pyrazin-3-one-dependent luminescence induced by the hypoxanthine-xanthine oxidase system. Our chemiluminescence procedure gave 95-times higher sensitivity than the cytochrome c method. The concentration of superoxide dismutase in erythrocytes of Down's syndrome patients was approx. 1.8-times higher than that of normal humans.

Enhancement by streptozotocin of O −2 radical generation by the xanthine oxidase system of pancreatic β-cells

Spin-trapping techniques and electron spin resonance (ESR) spectroscopy were used to study the relationship between the effect of streptozotocin (STZ) on pancreatic β-cells and free radical formation by these cells. Results showed that STZ enhanced generation of the DMPO-OH radical adduct, which is a degradation product of the superoxide anion (O − 2) in the presence of cellular components, in a hypoxanthine-xanthine oxidase (XOD) system with a homogenate of β-cells. This enhancing effect was also observed in a system without cellular components; STZ increased the signal height due to the O − 2 radical in a concentration-dependent manner and caused a maximum of 150% enhancement at a concentration of 1.5 mM. Thus, STZ seemed to enhance the generation of the O − 2 radical in the XOD system, probably by some mechanism of its interaction with XOD. Pancreatic β-cells exhibited a high XOD activity and a very low superoxide dismutase activity. Therefore, the present result supports the possibility that the cytotoxic effect of STZ is closely related to free radical generation in pancreatic β-cells.

Effects of UVB irradiation on epidermal adenylate cyclase responses in vitro: its relation to sunburn cell formation

UVB irradiation augmented the beta-adrenergic adenylate cyclase response of pig skin epidermis in vitro. The effect was observed 2-4 h following the irradiation and lasted at least for 48 h. There was no significant difference in cyclic AMP phosphodiesterase activity between control and UVB-irradiated epidermis at lower irradiation dose (150 mJ/cm2), which is the dose of the most marked beta-adrenergic augmentation effect. The augmentation effect was specific to the beta-adrenergic system; adenosine and histamine adenylate cyclase responses were unchanged or decreased depending on the irradiation dose. Histologically, marked sunburn-cell formation was observed following the UVB irradiation. It has been suggested that oxygen intermediates generated by ultraviolet radiation participate in sunburn-cell formation. The addition of superoxide dismutase (SOD) in the incubation medium significantly inhibited sunburn-cell formation. On the other hand, the beta-adrenergic augmentation effect was not affected by the addition of SOD. Other scavengers of oxygen intermediates (catalase, catalase + SOD, xanthine, or mannitol) did not inhibit the UVB-induced beta-adrenergic augmentation effect. Further, superoxide-anion generating systems (hypoxanthine-xanthine oxidase system and acetaldehyde-xanthine oxidase system) revealed no stimulatory effect on the beta-adrenergic response of epidermis. These results indicate that (a) the UVB-induced beta-adrenergic augmentation effect is inherent to skin and does not depend on systemic factors such as inflammatory infiltrates following UVB irradiation; (b) in contrast to sunburn-cell formation, induction of the beta-adrenergic adenylate cyclase response is not directly associated with oxygen intermediates generated by UVB irradiation.

Measurement of Serum Superoxide Dismutase Activity by Electron Spin Resonance

We measured serum superoxide dismutase activity by electron spin resonance, using 100mM 5, 5-dimethyl-1-pyrroline-N-oxide (DMPO) as a trap. The addition of superoxide dismutase decreased the height of the DMPO-OOH signal produced by the hypoxanthine-xanthine oxidase system. Serum superoxide dismutase activity was determined from a quantitative line for superoxide dismutase. When bovine Cu, Zn-superoxide dismutase (50, 000U/kg) was injected intravenously into rats, the superoxide dismutase activity decreased almost linearly with a half life of about 6min. This method allowed direct detection of superoxide radicals and new measurement of superoxide dismutase activity.

新規抗炎症薬ＫＭＥ‐４の白血球および血小板の活性化反応に対する作用

The biochemical properties of KME-4, α- (3, 5-di-tert-butyl-4-hydroxybenzylidene) -γ-butyrolactone, were evaluated in vitro. Unlike other NSAIDs such as indomethacin and diclofenac, KME-4 or BW-755C significantly inhibited active oxygen productions from plymorphonuclear leukocytes (PMN) stimulated with opsonized zymosan, without change of viabilities. [3H] f-Met-Leu-Phe binding to the PMN membrane was not affected by either KME-4 or BW-755C but inhibited by indomethacin.KME-4 and BW-755C also clearly inhibited an active oxygen generation in a cellfree hypoxanthine-xanthine oxidase system. KME-4 and BW-755C were more potent than indomethacin and diclofenac in inhibiting the secondary aggregation of human platelet induced by platelet activating factor. These results suggest that KME-4, a dual inhibitor of arachidonic acid metabolism, like BW-755C affects the functions of leukocytes and platelets on different mechanisms from those of typical known NSAIDs.

HIGH CONCENTRATIONS OF HYPOXANTHINE (HX) IN VITREOUS HUMOUR OF BABIES WITH RDS. A PATHOGENIC FACTOR IN DEVELOPING ROP?

Oxygen radicals created by the hypoxanthine-xanthine oxidase system might play a role in development of Retinopathy of Prematurity (ROP). To test whether or not babies at risk for developing ROP have elevated hypoxanthine levels in their eyes, vitreous honour was analyzed post mortem for hypoxanthine in 13 premature babies who died of RDS. As controls serve seven newborn babies dying acutely from cardiac or pulmonary failure. HX was measured both with a pO2 and a HPLC method and high correlation between the methods was found. RDS babies required treatment with 100 % O2 33 ± 30.9 hours compared with 4.75 ± 12.0 hours in controls (p < 0.001). RDS babies experienced paO2 < 5.3 kPa 15.2 ± 10.5 hours versus 0.96 ± 1.2 hours in controls (p < 0.001). There was a significant correlation between HX concentration in vitreous humour and duration of paO2 < 5.3 kPa (r= +0.59, p < .012, n = 19). RDS babies had more than eight times higher HX concentrations than controls, 459 ± 171 micromoles/1 versus 54 ± 71 micromoles/l (p < 0.001). Since the combination of 02 and HX is necessary for the production of oxygen radicals by the hypoxanthine - xanthine oxidase system these data demonstrate that babies with RDS who are treated with higji concentrations of O2 and have high concentrations of HX in their eyes, might produce large quantities of oxygen radicals in their eyes. This might play a role for development of ROP.

Acute and chronic effects of xanthine oxidase on lung thorax-compliance in guinea pigs.

Xanthine oxidase was given intratracheally in a single dose to guinea pigs. Lung compliance was measured after 4 h and 14 days respectively. Lung-thorax compliance was significantly lower compared with saline-treated controls both 4 h and 14 days after application of fluid. At 14 days there was a dose-related response between lung-thorax compliance and xanthine oxidase administered in the range 0-1.0 U. Superoxide dismutase (SOD) had a protective effect on xanthine oxidase action at 4 h, but not after 14 days. We suggest that the decreased lung-thorax compliance was caused by superoxide radicals, produced by the hypoxanthine-xanthine oxidase system, damaging lung tissue. We speculate that free oxygen radicals produced by the hypoxanthine-xanthine oxidase system could be an important contributory pathogenetic factor in producing both acute and chronic lung damage in, for instance, premature babies or adults, with respiratory distress syndrome.

Reduction of the metallochromic indicators murexide and tetramethylmurexide to their free radical metabolites by cytoplasmic enzymes and reducing agents

Murexide underwent reduction by rat liver cytosolic fraction or a hypoxanthine-xanthine oxidase system to produce a free radical metabolite. Reduction of murexide by the freshly prepared cytosolic fraction depended upon the presence of ascorbic acid. N 1-Methylnicotinamide, xanthine or hypoxanthine, in that order, could also serve as a source of reducing equivalents for the production of that free radical by the cytosolic fraction. Several thiol compounds (GSH, cysteine, and cysteamine), pyridine nucleotides (NADH, NADPH) and ascorbic acid were also effective in generating the murexide-derived free radical. Tetramethyl murexide was also reduced to its free radical derivative by a hypoxanthine-xanthine oxidase system.

Ultracytochemical studies on the Na+-K+-ATPase activity in decapitated and superoxide-treated rat brains.

Na+-K+-adenosine triphosphatase (Na+-K+-ATPase) was studied ultracytochemically in decapitated and superoxide-treated rat brains in order to elucidate the relationship between enzyme activity and damage of membranous structures. All experiments were performed on male adult Wistar rats. The decapitated group consisted of 12 animal brains removed 3, 6, and 12 hours after decapitation. The normal control consisted of 4 animal brains removed immediately after decapitation. The superoxide-treated group consisted of 9 animal prefixed brains immersed in a hypoxanthine-xanthine oxidase system for 20, 60, and 120 minutes. In both groups, 1 mm sagittal sections of cerebellar vermis were fixed for 50 minutes in a mixed solution of 0.25% glutaraldehyde and 1 % paraformaldehyde (0.1 M cacodylate buffer, pH 7.3) at 0°C. Non-frozen sections, approximately 15 μm thick for light microscopy and 40 μm thick for electron microscopy, were prepared by a microslicer. Histochemical procedure was carried out by incubating specimens in the substrate medium using lead citrate as capturing reagent for 40 minutes at 37°C. Following the incubation, specimens were embedded in Spurr's resin. Ultrathin sections were stained in 2% uranyl acetate for 3 minutes at 20°C. Three different kinds of medium (substrate free, substitution of Na+ for K+, and ouabain addition) were utilized for control in each experiment. In the normal control Na+-K+ATPase reaction was microscopically detected mainly at the cerebellar glomerulus. Electron microscopy revealed the reaction product localized on postsynaptic membranes, plasmalemmas of both axon and dendrite, and filamentous structures of axonal cytoplasm in glomerulus. However, the reaction was negative in mossy fibers and soma of granular cells. Na+-K+-ATPase activity was almost unchanged 3 hours after decapitation. It decreased considerably in 6 hours, and disappeared in 12 hours. In contrast, Na+-K+-ATPase activity changed dynamically after superoxide treatment. 60-minute peroxidation caused considerable increase in the activity as compared with both normal control and 20 minute-treated brains, although apparently irreversible histological changes had already occurred. Such increase in the enzyme activity seemed to be rather larger than the degree of membranous damages. Na+-K+-ATPase had completely diminished by 120-minute peroxidation. These findings show that damage of the plasmalemma precedes the disappearance of Na+-K+-ATPase activity in ischemic brains. The remaining Na+-K+-ATPase activity does not always suggest the reversibility of ischemic lesions.

Histidine increases the frequency of chromosomal aberrations induced by the xanthine oxidasehypoxanthine system in V79 cells

The combined effects of the xanthine oxidase (XO)—hypoxanthine (HX) system and the various kinds of amino acids in Eagle's minimum essential medium on chromosomal aberrations were studied in Chinese hamster V79 cells. Among 13 amino acids tested, only histidine significantly increased the number of aberrant chromosomes and cytotoxicity in combination with the XO-HX system. This enhancing effect of histidine on chromosomal aberrations was dose-dependent at 0.063%–0.25%; it was not affected by Superoxide dismutase, but was strongly inhibited by catalase.

Studies on the oxygen radical mechanism involved in the small intestinal reperfusion damage.

Characteristic mucosal lesions develop in the small intestine during ischaemia and hypotension. This tissue damage can be further aggravated in the immediate reperfusion phase, presumably secondary to the generation of oxygen free radicals which have been proposed to be generated in this situation through the hypoxanthine-xanthine oxidase system. This was further investigated in the cat small intestine using a standardized regional intestinal hypotension model in which the effects of allopurinol (a xanthine oxidase inhibitor) were compared to those of an exogenous supply of inosine. The grade of mucosal damage, the nucleotide levels, the concentrations of hypoxanthine, total and oxidized glutathione, and of conjugated dienes were measured in the intestinal tissue. The results indicate that oxygen radicals generated by xanthine oxidase are very important, but not the only significant factor in the small intestinal reperfusion damage.

Ethanol oxidation by hydroxyl radicals: role of iron chelates, superoxide, and hydrogen peroxide.

Oxygen-derived free radicals such as the hydroxyl radical (.OH) have been shown to mediate the oxidation of ethanol by a variety of oxy radical-generating systems. Among these are microsomal electron transport systems (both intact and purified, reconstituted systems), the coupled oxidation of hypoxanthine or xanthine by xanthine oxidase, and the model iron-ascorbate system. The sequence of reactions leading to the oxy radical-dependent oxidation of ethanol as well as other hydroxyl radical-scavenging agents by these various systems is believed to proceed through the formation of a common intermediate, namely, hydrogen peroxide (H2O2), after dismutation of the superoxide anion radical (O2-.). The presence of iron, especially chelated iron, greatly enhances the production of .OH by serving as an oxidant for O2-. or a reductant for H2O2. Experiments were carried out to evaluate the role of iron, the chelating agent, O2-., and H2O2 in the oxidation of ethanol by a variety of in vitro systems (chemical, enzymatic, and intact membrane bound) that can produce oxy radicals via different mechanisms. The generation of .OH by all the systems studied was sensitive to catalase, which indicates that H2O2 is the precursor of .OH. Superoxide radical appears to be the reducing agent in the hypoxanthine-xanthine oxidase system, indicating an iron-catalyzed Haber-Weiss reaction. In the ascorbate, reductase, and microsomal systems, superoxide radical does not appear to be the reducing agent. However, superoxide radical probably is the precursor of H2O2. While iron plays an important role in the production of .OH by the various systems, the effect of iron depends on the nature of the iron chelate.(ABSTRACT TRUNCATED AT 250 WORDS)

Reduction of the metallochromic indicators arsenazo III and antipyrylazo III to their free radical metabolites by cytoplasmic enzymes

At a concentration much lower than that usually employed for measuring cytosolic ionized Ca 2+ concentrations, arsenazo III underwent a one-electron reduction by rat liver cytosolic fraction or a hypoxanthinexanthine oxidase system to produce an azo anion radical metabolite. NADH, NADPH, N 1-methylnicotinamide, hypoxanthine, and xanthine, in that order, could serve as a source of reducing equivalents for the production of this free radical by the cytosolic fraction. The steady-state concentration of the azo anion radical and the arsenazo III-stimulated O 2 consumption were enhanced by calcium and magnesium. Antipyrylazo III was ineffective in increasing O 2 consumption by rat liver cytosolic fraction and gave a much weaker ESR signal of an azo anion radical with both the liver cytosolic fraction, in the presence of NADH, and the hypoxanthine-xanthine oxidase system.

Treatment of lymphocyte cultures with a hypoxanthine-xanthine oxidase system induces the formation of transferable clastogenic material

Culture medium of lymphocyte cultures that have been exposed to the superoxide generating system hypoxanthine plus xanthine oxidase (XXO) contains substances with chromosome damaging properties. This is demonstrated by the ability of ultrafiltrates of such culture media to induce chromosomal aberrations and sister chromatid exchanges in the lymphocytes of blood test cultures. Culture medium becomes active about 15 hours after the addition of XXO and stimulation by phytohemagglutinin. Concomitant with the accumulation of clastogenic material, assays for conjugated dienes and thiobarbituric acid-reactive material which measure lipid-peroxidation become positive in the culture media. When cells are pretreated with superoxide dismutase or glutathione peroxidase before the addition of XXO neither clastogenic substances nor lipid peroxidation products are detected. Catalase is a less efficient protector.

Protective effect of superoxid dismutase (SOD) on severe lung damage caused by xanthine oxidase (XO)

Dept.of Paed.,The National Hospital of Norway, Oslo,Childrens Hospital,Helsinki and Research Institute for lung diseases, Berlin-Buch,GDR. During post hypoxic resuscitation the hypoxanthine-xanthine oxidase system creates free oxygen radicals. The effect of free oxygen radicals on the lungs were studied by applying 1 U XO into the trachea of guinea pigs. Lung compliance and arterial blood gases were monitored. Compliance and PaO2 decreased (p<0.01) after application of XO in 1 ml saline (3 ml/kg), when compared with saline alone. SOD (20000 U) prevented the effect of XO. When ventilating with a peak pressure of 20 cm H2O 20 minutes after application of the fluid the data were:The data demonstrate that XO potently damages the lungs probably through O2- formation since SOD prevents the effect. The SOD effect lasts about one hour. The data also show that there is a dramatic effect on healthy lungs when saline is applied into the trachea. This raises the question whether randomised studies for surfactant treatment are justified.

Chromosomal aberrations in v79 cells induced by superoxide radical generated by the hypoxanthinexanthine oxidase system

The effect of the Superoxide radical, generated by the hypoxanthine-xanthine oxidase system, on chromosomal mutation was examined in Chinese hamster V79 cells. When cells were treated with this system for 1 h in Hanks' solution, the incidence of metaphases with chromosomal aberrations was increased with hypoxanthine at concentrations of 2.5 to 10 μg/ml. On the other hand, in Eagle's minimum essential medium (MEM) or MEM supplemented with 10% fetal bovine serum, only hypoxanthine at 5 μg/ml plus xanthine oxidase induced chromosomal aberrations and higher concentrations of hypoxanthine were cytotoxic to V79 cells. The increased frequency of chromosomal aberrations and the cytotoxicity of hypoxanthine plus xanthine oxidase were not affected by Superoxide dismutase, but were strongly inhibited by catalase.

Hydroxylation of phenylalanine by the hypoxanthine-xanthine oxidase system.

When phenylalanine was treated with hypoxanthine and xanthine oxidase in citrate buffer (pH 5.5), p-tyrosine, m-tyrosine and o-tyrosine were identified as hydroxylated products. Addition of superoxide dismutase or catalase to this system prevented the hydroxylation, implying that superoxide radical and hydrogen peroxide were essential intermediates in the hydroxylation reaction. Chemical scavengers of hydroxyl radical (·OH) prevented the hydroxylation. In particular, 50 mM potassium iodide completely prevented the hydroxylation. The addition of lactoferrin or Fe3+ accelerated the hydroxylation of phenylalanine. These results indicate that the hydroxylation of phenylalanine is caused by ·OH formed in a hypoxanthine-xanthine oxidase system.

Pathogenetic aspects of respiratory distress syndrome in adults and newborns. Experimental and clinical data.

Activation of the kallikrein-kinin system was demonstrated in experimental lung failure in dogs and respiratory distress syndrome (RDS) in newborns. Further lung damage was found in rats after intravenous infusion of hypoxanthine during exposure to 100% oxygen for 48 h. It is discussed whether such damage was mediated through free oxygen radicals produced by the hypoxanthine-xanthine oxidase system. Newborn babies with RDS had high serum myoinositol levels and the role of myoinositol as regulator of surfactant synthesis is discussed. These three different approaches demonstrate the complexity of pathogenesis of RDS in newborn babies and adults.