Effects of methylglyoxal on platelet hydrogen peroxide accumulation,aggregation and release reaction

Methylglyoxal generates a slight increase in the basal level of hydrogen peroxide in platelets. The oxidation effect of methylglyoxal significantly potentiated by thrombin, depends on both the ketoaldehyde and the agonist concentrations. A further significant increase in hydrogen peroxide accumulation was obtained in platelets pretreated with the alkylating agent N-ethylmaleimide which depletes GSH and blocks glutathione peroxidase. Resting platelets completely transform the ketoaldehyde into D (?)lactate, whereas stimulated platelets transform about 10–15 per cent of the metabolized methylglyoxal into D (?)lactate. The metabolic modifications generated by methylglyoxal such as the GSH depletion and hydrogen peroxide accumulation induce modifications in platelet function. Methylglyoxal inhibits platelet aggregation induced by several agonists and ATP release induced by thrombin.     

Genetic modification of the manganese superoxide dismutase/glutathione peroxidase 1 pathway influences intracellular ROS generation in quiescent, but not contracting, skeletal muscle cells

Increased amounts of reactive oxygen species (ROS) are generated by skeletal muscle during contractile activity, but their intracellular source is unclear. The oxidation of 2',7'-dichlorodihydrofluorescein (DCFH) was examined as an intracellular probe for reactive oxygen species in skeletal muscle myotubes derived from muscles of wild-type mice and mice that were heterozygous knockout for manganese superoxide dismutase (Sod2(+/-)), homozygous knockout for glutathione peroxidase 1 (GPx1(-/-)), or MnSOD transgenic overexpressors (Sod2-Tg). Myoblasts were stimulated to fuse and loaded with DCFH 5-7 days later. Intracellular DCF epifluorescence was measured and myotubes were electrically stimulated to contract for 15 min. Quiescent myotubes with decreased MnSOD or GPx1 showed a significant increase in the rate of DCFH oxidation whereas those with increased MnSOD did not differ from wild type. Following contractions, myotubes from all groups showed an equivalent increase in DCF fluorescence. Thus the oxidation of DCFH in quiescent skeletal muscle myotubes is influenced by the content of enzymes that regulate mitochondrial superoxide and hydrogen peroxide content. In contrast, the increase in DCFH oxidation following contractions was unaffected by reduced or enhanced MnSOD or absent GPx1, indicating that reactive oxygen species produced by contractions were predominantly generated by nonmitochondrial sources.     

Selenium-dependent and non-selenium-dependent glutathione peroxidase in human tissues of New Zealand residents

Glutathione peroxidase was assayed in human tissues of New Zealand residents by the coupled assay method. Total glutathione peroxidase was assayed using cumene hydroperoxide. The non-selenium-dependent activity was not detected with t-butyl hydroperoxide and thus was determined from the difference between total activity and the selenium-dependent activity using hydrogen peroxide or t-butyl hydroperoxide. Only selenium-dependent activity was found in whole blood, erythrocytes, platelets and biopsy skeletal muscle. A small non-selenium dependent activity was measured in plasma and a larger activity in biopsy liver supernatant and homogenate. Glutathione-S-transferase was detected in all tissues.     

Coupling of Dopamine Oxidation (Monoamine Oxidase Activity) to Glutathione Oxidation Via the Generation of Hydrogen Peroxide in Rat Brain Homogenates

Abstract: Homogenates of perfused rat brain generated oxidized glutathione from reduced glutathione during incubation with dopamine or serotonin. This activity was blocked by pargyline. a monoamine oxidase inhibitor, or by catalase, a scavenger of hydrogen peroxide. These results demonstrate formation of hydrogen peroxide by monoamine oxidase and the coupling of the peroxide to glutathione peroxidase activity. Oxidized glutathione was measured fluorometrically via the oxidation of NADPH by glutathione reductase. In the absence of added dopamine or serotonin, a much smaller amount of reduced glutathione was oxidized: this activity was blocked by catalase, but not by pargyline. Therefore, endogenous production of hydrogen peroxide, not linked to monoamine oxidase activity, was present. These results indicate that glutathione peroxidase (linked to hexose monophosphate shunt activity) can function to eliminate hydrogen peroxide generated by monoamine oxidase and other endogenous sources in aminergic neurons.     

Inhibition of peroxidase and catalase activities and modulation of hydrogen peroxide level by inositol phosphoglycan-like compounds.

Inositol phosphoglycan-like compounds are produced by the hydrolysis of the membrane bound glycosyl phosphoinositides. Besides being short term mediators of insulin action, they inhibit peroxidases and catalase, increasing the concentration of cellular hydrogen peroxide. Although high concentrations of hydrogen peroxide are toxic, moderate increases of its basal level are signals for different metabolic pathways. The inhibitor, localized in the cytosol of the cell, acts on peroxidases and catalase of the same tissue (homologous action) and of other tissues or organisms (heterologous action). The inositol phosphoglycan-like compound inhibits peroxidases with different prosthetic groups, i.e. containing iron such as: thyroid peroxidase, lactoperoxidase, horseradish peroxidase, soy bean peroxidase; and containing selenium such as glutathione peroxidase and 2-cys peroxiredoxin with no prosthetic group. Besides peroxidases, the inositol phosphoglycan-like compound inhibits catalase, another heme enzyme. The inhibition kinetics demonstrates a noncompetitive effect. The site of action is not the prosthetic group, given that the inhibitor does not produce any effect on the peak in the Soret region in the presence or absence of hydrogen peroxide. In conclusion, the inositol phosphoglycan-like compound is the general inhibitor of peroxidases and catalase involved in the modulation of hydrogen peroxide level that acts in different metabolic pathways as a signal transducer.     

Thiol-linked peroxidase activity of human sensitive to apoptosis gene (SAG) protein

SAG (sensitive to apoptosis gene), a novel zinc RING finger protein, which is redox responsive and protects mammalian cells from apoptosis, is a metal chelator and a potential reactive oxygen species (ROS) scavenger, but its antioxidant properties have not been completely defined. Here, we show that SAG possesses a potent peroxidase property to decompose hydrogen peroxide in the presence of dithiothreitol (DTT). However, without DTT as a reducing equivalent, SAG was not able to destroy hydrogen peroxide. The peroxidase activity was completely abolished by the reaction of SAG with N -ethylmaleimide (NEM), a chemical modification agent for the sulfhydryl of proteins. These observations suggested that the sulfhydryl of cysteines in SAG could function as strong nucleophiles to destroy hydrogen peroxide. In addition to the peroxidase activity used to remove hydrogen peroxide, SAG also showed t -butylhydroperoxide ( t -BOOH) and fatty acid hydroperoxide-selective peroxidase activity.     

Cytosolic glutathione peroxidase from liver of pacu (Piaractus mesopotamicus), a hypoxia-tolerant fish of the Pantanal

Pacu (Piaractus mesopotamicus Holmberg, 1887, Characiformes) dwells in waters of Pantanal, in which it has adapted for alternate concentrations of dissolved oxygen. Intracellular antioxidant protection should be vital for such an adaptation. Accordingly, we found that cytosol from liver of pacu has the highest antioxidant glutathione peroxidase activity so far reported for fish and murine species. To clarify whether this activity was due to a selenium independent glutathione S-transferase or to a glutathione peroxidase, we purified it and studied its kinetics. The substrates cumene hydroperoxide and hydrogen peroxide were promptly reduced by the enzyme, but peroxidized phosphatidylcholine had to undergo previous fatty acid removal with phospholipase A(2). Augmenting concentrations (from 2 to 6 mM) of reduced glutathione activated the pure enzyme. Curves of velocity versus different micromolar concentrations of hydrogen peroxide in the presence of 2, 4 or 8 mM reduced glutathione indicated that at least 2.5 mM reduced glutathione should be available in vivo for an efficient continuous destruction of micromolar concentrations of hydrogen peroxide by this peroxidase. Molecular exclusion HPLC and SDS-polyacrylamide gel electrophoresis indicated that the purified peroxidase is a homotetramer. Data from internal sequences showed selenocysteine in its primary structure and that the enzyme was a homologue of the type-1 glutathione peroxidase found in rat, bull, trout, flounder and zebra fish. Altogether, our data establish that in liver cells of pacu, a hypoxia-tolerant fish from South America, there are high levels of a cytosolic GPX-1 capable of quenching hydrogen peroxide and fatty acid peroxides, providing an effective antioxidant action.     

Oxidative Signals in Tobacco Increase Cytosolic Calcium

Tobacco (Nicotiana plumbaginifolia) seedlings genetically transformed to express apoaequorin were incubated in h-coelenterazine to reconstitute the calcium-sensitive luminescent protein aequorin. Treatment of these seedlings with hydrogen peroxide resulted in a transient burst of calcium-dependent luminescence lasting several minutes. Even though the hydrogen peroxide stimulus was persistent, the change in cytosolic free calcium concentration ([Ca2+]cyt) was transient, suggesting the presence of a refractory period. When seedlings were pretreated with hydrogen peroxide, there was no increase in [Ca2+]cyt upon a second application, which confirmed the refractory character of the response. Only when the two treatments were separated by 4 to 8 hr was full responsiveness recovered. However, treatment with hydrogen peroxide did not inhibit mobilization of [Ca2+]cyt induced by either cold shock or touching, suggesting that these three signals mobilize different pools of intracellular calcium. To examine whether [Ca2+]cyt is regulated by the redox state of the cytoplasm, we pretreated seedlings with buthionine sulfoximine (to modify cellular glutathione levels) and inhibitors of ascorbate peroxidase. These inhibitors modify the hydrogen peroxide-induced transients in [Ca2+]cyt, which is consistent with their effects on the cellular prooxidant/antioxidant ratio. Treatment with hydrogen peroxide that elicited [Ca2+]cyt increases also brought about a reduction in superoxide dismutase enzyme activity. This reduction could be reversed by treatment with the calcium channel blocker lanthanum. This indicates that there is a role for calcium in plant responses to oxidative stress.     

Analysis of the oxidative stress response of Penicillium chrysogenum to menadione

The intracellular superoxide and glutathione disulphide concentrations increased in Penicillium chrysogeum treated with 50,250 or 500 μM menadione (MQ). A significant increase in the intracellular peroxide concentration was also observed when mycelia were exposed to 250 or 500 μM MQ. The specific activity of Cu,Zn and Mn superoxide dismutases, glutathione reductase and glutathione S-transferase as well as the glutathione producing activity increased in the presence of MQ while glutathione peroxidase and γ-glutamyltranspeptidase were only induced by high intracellular peroxide levels. The glucose-6-phosphate dehydrogenase and catalase activities did not respond to the oxidative stress caused by MQ.     

Effect of Auranofin on the mitochondrial generation of hydrogen peroxide. Role of thioredoxin reductase

The mitochondrial production of hydrogen peroxide, in the presence of different respiratory substrates (succinate, glutamate, malate and isocitrate), is stimulated by submicromolar concentrations of auranofin, a highly specific inhibitor of thioredoxin reductase. This effect is particularly evident in the presence of antimycin. Auranofin was also able to unmask the production of hydrogen peroxide occurring in the presence of rotenone. However, at variance with whole mitochondria, auranofin does not stimulate hydrogen peroxide production in submitochondrial particles indicating that it does not alter the formation of hydrogen peroxide by the respiratory chain but prevents its removal. As the mitochondrial metabolism of hydrogen peroxide proceeds through the peroxidases linked to glutathione or thioredoxin, the relative efficiency of the two systems and the effects of auranofin were tested. In conclusion, the inhibition of thioredoxin reductase determines an increase of the basal flow of hydrogen peroxide leading to a more oxidized condition that alters the mitochondrial functions.     

Oxidative metabolism of human platelets.

The reducing capacity toward cytochrome c present in human resting platelets increases upon platelet stimulation, and is partially inhibited by superoxide dismutase. This activity therefore represents the generation of superoxide anion. In order to evaluate hydrogen peroxide formation a quantitative assay by mean of dichlorofluorescin (DCFH) has been set up. The DCFH, trapped inside the cell, is oxidized by hydrogen peroxide to the fluorescent compound DCF. Basal DCF increases during activation of platelets by agonists. Arachidonic acid, calcium ionophore A23187 and to a lesser extent PMA and thrombin are the most effective. N-ethylmaleimide induces a dose-dependent DCFH oxidation and potentiates the effect of agonists. NAD(P)H--cytochrome c reductase enzyme, which catalyzes superoxide anion production, is present in platelets at high specific activity, as well as those enzymes who protect the cells from oxygen reactive species.     

Effect of auranofin on the mitochondrial generation of hydrogen peroxide. Role of thioredoxin reductase

The mitochondrial production of hydrogen peroxide, in the presence of different respiratory substrates (succinate, glutamate, malate and isocitrate), is stimulated by submicromolar concentrations of auranofin, a highly specific inhibitor of thioredoxin reductase. This effect is particularly evident in the presence of antimycin. Auranofin was also able to unmask the production of hydrogen peroxide occurring in the presence of rotenone. However, at variance with whole mitochondria, auranofin does not stimulate hydrogen peroxide production in submitochondrial particles indicating that it does not alter the formation of hydrogen peroxide by the respiratory chain but prevents its removal. As the mitochondrial metabolism of hydrogen peroxide proceeds through the peroxidases linked to glutathione or thioredoxin, the relative efficiency of the two systems and the effects of auranofin were tested. In conclusion, the inhibition of thioredoxin reductase determines an increase of the basal flow of hydrogen peroxide leading to a more oxidized condition that alters the mitochondrial functions.     

N-Ethylmaleimide induces disaggregation of platelets preaggregated by ADP and thrombin and decreases cytoplasmic calcium level

The effect of N-ethylmaleimide (NEM, 1-200 microM) on ADP- and thrombin-induced platelet aggregation and thrombin-induced increase in intracellular Ca2+ concentration was studied. Addition of NEM to platelets preaggregated with ADP or thrombin induces platelet disaggregation. The anti-aggregant activity of NEM was different for ADP- and thrombin-induced aggregations. At 200 microM concentration, NEM completely disaggregated ADP-induced aggregates and only partially disaggregated thrombin-aggregated platelets. NEM did not influence the thrombin-induced increase in cytoplasmic Ca2+ and had no effect on the basal level of Ca2+ in the cytosol of non-activated platelets. However, NEM decreased the level of thrombin-mobilized Ca2+ in the cytosol of activated platelets. Thus, NEM can induce disaggregation of ADP- and thrombin-preaggregated platelets by activating a system which removes Ca2+ from the platelet cytosol.     

Different effects of Triton X-100, deoxycholate, and fatty acids on the kinetics of glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase

The effects of Triton X-100, deoxycholate, and fatty acids were studied on the two steps of the ping-pong reaction catalyzed by Se-dependent glutathione peroxidases. The study was carried out by analyzing the single progression curves where the specific glutathione oxidation was monitored using glutathione reductase and NADPH. While the "classic" glutathione peroxidase was inhibited only by Triton, the newly discovered "phospholipid hydroperoxide glutathione peroxidase" was inhibited by deoxycholate and by unsaturated fatty acids. The kinetic analysis showed that in the case of glutathione peroxidase only the interaction of the lipophilic peroxidic substrate was hampered by Triton, indicating that the enzyme is not active at the interface. Phospholipid hydroperoxide glutathione peroxidase activity measured with linoleic acid hydroperoxide as substrate, on the other hand, was not stimulated by the Triton concentrations which have been shown to stimulate the activity on phospholipid hydroperoxides. Furthermore a slight inhibition was apparent at high Triton concentrations and the effect could be attributed to a surface dilution of the substrate. Deoxycholate and unsaturated fatty acids were not inhibitory on glutathione peroxidase but inhibited both steps of the peroxidic reaction of phospholipid hydroperoxide glutathione peroxidase, in the presence of either amphiphilic or hydrophilic substrates. This inhibition pattern suggests an interaction of anionic detergents with the active site of this enzyme. These results are in agreement with the different roles played by these peroxidases in the control of lipid peroxide concentrations in the cells. While glutathione peroxidase reduces the peroxides in the water phase (mainly hydrogen peroxide), the new peroxidase reduces the amphyphilic peroxides, possibly at the water-lipid interface.     

Oxidation pathways underlying the pro-oxidant effects of apigenin

Apigenin, a natural flavone, is emerging as a promising compound for the treatment of several diseases. One of the hallmarks of apigenin is the generation of intracellular reactive oxygen species (ROS), as judged by the oxidation of reduced dichlorofluorescein derivatives seen in many cell types. This study aimed to reveal some mechanisms by which apigenin can be oxidized and how apigenin-derived radicals affect the oxidation of 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein (H₂DCF), a probe usually employed to detect intracellular ROS. Apigenin induced a rapid oxidation of H₂DCF in two different immortalized cell lines derived from rat and human hepatic stellate cells. However, apigenin did not generate ROS in these cells, as judged by dihydroethidium oxidation and extracellular hydrogen peroxide production. In cell-free experiments we found that oxidation of apigenin leads to the generation of a phenoxyl radical, which directly oxidizes H₂DCF with catalytic amounts of hydrogen peroxide. The net balance of the reaction was the oxidation of the probe by molecular oxygen due to redox cycling of apigenin. This flavonoid was also able to deplete NADH and glutathione by a similar mechanism. Interestingly, H₂DCF oxidation was significantly accelerated by apigenin in the presence of horseradish peroxidase and xanthine oxidase, but not with other enzymes showing peroxidase-like activity, such as cytochrome c or catalase. We conclude that in cells treated with apigenin oxidation of reduced dichlorofluorescein derivatives does not measure intracellular ROS and that pro- and antioxidant effects of flavonoids deduced from these experiments are inconclusive and must be confirmed by other techniques.     

Activation of rat liver microsomal glutathione S-transferase by hydrogen peroxide: role for protein-dimer formation.

The mechanism of oxygen radical-dependent activation of hepatic microsomal glutathione S-transferase by hydrogen peroxide was studied. Glutathione S-transferase activity in liver microsomes was increased 1.5-fold by incubation with 0.75 mM hydrogen peroxide at 37 degrees C for 10 min, and the increase in activity was reversed by incubation with dithiothreitol. Purified glutathione S-transferase was also activated by hydrogen peroxide after incubation at room temperature, and the increase in the activity was also reversed by dithiothreitol. Immunoblotting with anti-microsomal glutathione S-transferase antibodies after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of hydrogen peroxide-treated microsomes or purified glutathione S-transferase revealed the presence of a glutathione S-transferase dimer. These results indicate that the hydrogen peroxide-dependent activation of the microsomal glutathione S-transferase is associated with the formation of a protein dimer.     

Role of lipoxygenase in the O2-dependent activation of soluble guanylate cyclase from rat lung.

Guanylate cyclase activity in rat lung supernatant fractions is stimulated 3-4 fold by aerobic incubation at 30 degrees C for approx. 30 min ('O2-dependent activation'). This stimulation was blocked by 20 microM-eicosa-5,8,11,14-tetraynoic acid (ETYA), an inhibitor of lipoxygenase and cyclo-oxygenase, but not by aspirin or indomethacin, which are cyclo-oxygenase inhibitors. The enzyme activator(s) is presumed to be the fatty acid hydroperoxide(s) formed by lipoxygenase. Removal of lipoxygenase from the supernatant fraction by chromatography on Amberlite XAD-4 also prevented activation, which was restored by the addition of soya-bean lipoxygenase. Bovine serum albumin prevented O2-dependent activation or activation by soya-bean lipoxygenase, through its ability to bind the unsaturated fatty acid substrate of lipoxygenase. The lipoxygenase in the supernatant fraction is inhibited by endogenous glutathione peroxidase plus reduced glutathione (GSH); removal of GSH de-inhibits lipoxygenase and activates guanylate cyclase. This was effected by autoxidation, by cumene hydroperoxide (with GSH peroxidase) and by titration with N-ethylmaleimide (NEM). Activation by NEM was inhibited by serum albumin or ETYA, as was activation by low concentrations (less than 50 microM) of cumene hydroperoxide. Activation by higher concentrations was not so inhibited; therefore, cumene hydroperoxide can also activate by a direct effect on guanylate cyclase. A hypothesis for physiological activation is proposed.     

Evidence for glutathione peroxidase activities in cultured plant cells

Extracts from cultured plant cells of spinach, maize and sycamore and from Lemna plants contain detectable glutathione peroxidase activity, using either hydrogen peroxide or t-butyl hydroperoxide as substrates. Using extracts from cultured maize cells, two peaks of glutathione peroxidase activity could be resolved by a combination of gel filtration and ion exchange chromatography. One peak was eluted along with glutathione transferase activity; the second was distinct from both glutathione transferase and ascorbic acid peroxidase, and was active with both hydrogen peroxide and organic hydroperoxides. It seems likely that at least two enzymes with glutathione peroxidase activity exist in higher plant cells.     

Effects of Peroxidase and Hydrogen Peroxide on the Dityrosine Formation and the Mixing Characteristics of Wheat-Flour Dough

The effects of adding hydrogen peroxide and peroxidase to wheat-flour dough on dityrosine formation and mixing characteristics were investigated. Dityrosine in wheat-flour dough was identified by HPLC with a fluorescence detector and by LC/MS/MS. Formation of dityrosine increased with the addition of hydrogen peroxide, and hydrogen peroxide plus peroxidase, to wheat-flour dough, while the addition of peroxidase had no effect on the amount of dityrosine formed. The mixing curve obtained by a doughgraph changed with the addition of hydrogen peroxide, and hydrogen peroxide plus peroxidase; the peak time was significantly delayed and the dough development time was extended. We found that dityrosine cross-links in wheat-flour dough increased with the addition of peroxidase plus hydrogen peroxide. It is thought that these cross-links can lead to polymerization of the proteins in wheat-flour dough.     

Inactivation of Ascorbate Peroxidase by Thiols Requires Hydrogen Peroxide

The hydrogen peroxide-dependent oxidation of ascorbate by ascorbateperoxidase from tea leaves was inhibited by thiols, such asdithiothreitol, glutathione, mercaptoethanol and cysteine. Thesethiols themselves did not inactivate the enzyme. However, theyinactivated the enzyme when hydrogen peroxide was produced bythe metal-catalyzed oxidation of thiols or when exogenous hydrogenperoxide was added. Thiols were oxidized by ascorbate peroxidaseand hydrogen peroxide to thiyl radicals, as detected by theESR spectra of the thiyl radical-5,5'-dimethyll- pyrroline-N-oxidieadducts. Inactivation of ascorbate peroxidase by thiols andhydrogen peroxide is caused by the interaction of the enzymewith the thiyl radicals produced at its reaction center. (Received September 10, 1991; Accepted December 9, 1991)