Characteristics of xyloglucan after attack by hydroxyl radicals.

It has been proposed that plant cell-wall polysaccharides are subject in vivo to non-enzymic scission mediated by hydroxyl radicals (-*OH). In the present study, xyloglucan was subjected in vitro to partial, non-enzymic scission by treatment with ascorbate plus H(2)O(2), which together generate -*OH. The partially degraded xyloglucan appeared to contain ester bonds within the backbone, as indicated by an irreversible decrease in viscosity upon alkaline hydrolysis. Aldehyde and/or ketone groups were also introduced into the polysaccharide by -*OH-attack, as indicated by staining with aniline hydrogen-phthalate and by reaction with NaB(3)H(4). The introduction of ester and oxo groups supports the proposed sequence of reactions: (a) -*OH-mediated H-abstraction to produce a carbon-centred carbohydrate radical; (b) reaction of the latter with O(2); and (c) elimination of a hydroperoxyl radical (HO(2)*-). When the partially degraded xyloglucan was reduced with NaB(3)H(4) followed by acid hydrolysis, several 3H-aldoses were detected ([3H]galactose, [3H]xylose, [3H]glucose, [3H]ribose and probably [3H]mannose), in addition to unidentified 3H-products (probably including anhydroaldoses). 3H-Alditols were undetectable, showing that few or no conventional reducing termini were introduced. Digestion of the NaB(3)H(4)-reduced, partially degraded xyloglucan with Driselase released 25 times more [3H]Xyl-alpha-(1-->6)-Glc than Xyl-alpha-(1-->6)-[3H]Glc, suggesting that the xylose side-chains of the xyloglucan had been more heavily attacked by -*OH than the glucose residues of the backbone. The radioactive xyloglucan was readily digested by cellulase, yielding 3H-products in the hepta- to nonasaccharide range. A fingerprinting strategy for identifying -*OH-attacked xyloglucan in plant cell walls is proposed.     

Hydroxylation of p-coumaric acid by horseradish peroxidase. The role of superoxide and hydroxyl radicals.

1. In the presence of dihydroxyfumarate, horseradish peroxidase catalyses the conversion of p-coumaric acid into caffeic acid at pH 6. This hydroxylation is completely inhibited by superoxide dismutase. 2. Dihydroxyfumarate cannot be replaced by ascorbate H2O2, NADH, cysteine or sulphite. Peroxidase can be replaced by high (10 mM) concentrations of FeSO4, but this reaction is almost unaffected by superoxide dismutase. 3. Hydroxylation by the peroxidase/dihydroxyfumarate system is completely inhibited by low concentrations of Mn2+ or Cu2+. It is proposed that this is due to the ability of these metal ions to react with the superoxide radical O2--. 4. Hydroxylation is partially inhibited by mannitol, Tris or ethanol and completely inhibited by formate. This seems to be due to the ability of these reagents to react with the hydroxyl radical -OH. 5. It is concluded that O2-- is generated during the oxidation of dihydroxyfumarate by peroxidase and reacts with H2O2 to produce hydroxyl radicals, which then convert p-coumaric acid into caffeic acid.     

The generation of hydroxyl and alkoxyl radicals from the interaction of ferrous bipyridyl with peroxides.

Reaction conditions by which the iron-chelate ferrous bipyridyl can be used as a Fenton reagent to generate specifically alkoxyl radical (.OR) from its corresponding alkyl hydroperoxide (ROOH) without producing hydroxyl radical (.OH) as a result of autoxidation are described. In this manner, the relative ability of common .OH-scavenging agents to react with .OH and various .OR species could be assessed. When .OH was generated from H2O2, 4-methylmercapto-2-oxobutyrate, ethanol and benzoate all were oxidized. When .OR (cumoxyl radical, t-butoxyl radical or ethoxyl radical) was generated specifically, each was found to oxidize 4-methylmercapto-2-oxobutyrate and ethanol. In contrast with .OH, however, none of the .OR radicals mediated the decarboxylation of benzoate. Cross-competition studies with the scavengers showed that, in contrast with the .OH-dependent reaction, the .OR-dependent oxidation of 4-methylmercapto-2-oxobutyrate and ethanol was not inhibited by benzoate. Rate constants for ferrous bipyridyl oxidation by ROOH and by H2O2 were found to be essentially the same, and therefore the differential oxidation of the various scavengers was not a reflection of iron-peroxide interaction, but rather an interaction between generated oxy radicals and the scavengers. In contrast with the H2O2 system, catalase did not inhibit the oxidation of 4-methylmercapto-2-oxobutyrate or ethanol by either the cumene hydroperoxide or the t-butyl hydroperoxide system, suggesting that the oxidizing species was not derived from H2O2. These results suggest that benzoate decarboxylation might serve as a more specific probe to detect the presence of .OH than either 4-methylmercapto-2-oxobutyrate or ethanol, which react readily with .OR.     

Lipid peroxides, spermatozoa quality and activity of glutathione peroxidase in bull semen

In bull semen spontaneous lipid peroxidation measured by the level of endogenous lipid peroxides and the consequences of this process for morphological and biochemical changes was studied. Glutathione peroxidase activity as protective enzyme against peroxidative damage was also determined. Obtained results showed that approximately two thirds of GSH-Px activity in bull semen was non Se-dependent glutathione peroxidase activity. Malonaldehyde (MDA) level was negative correlated with selenium-dependent GSH-Px activity (r = = -0.38, P less than 0.01). Spermatozoa with acrosome entirely lost appeared to increase as the MDA level increased (r = 0.18, P less than 0.05). The negative correlation between Se GSH-Px activity and spermatozoa with acrosome separation from head (r = -0.28, P less than 0.01) and entirely lost (r = -0.21, P less than 0.05) suggest that selenium-dependent GSH-Px plays role in protecting the acrosome against disruption of the acrosomal membrane. The total glutathione peroxidase activity was unrelated to studied variables of bull semen.     

Generation of hydrogen peroxide, superoxide and hydroxyl radicals during the oxidation of dihydroxyfumaric acid by peroxidase.

1. Dihydroxyfumarate slowly autoxidizes at pH6. This reaction is inhibited by superoxide dismutase but not by EDTA. Mn2+ catalyses dihydroxyfumarate oxidation by reacting with O2 leads to to form Mn3+, which seems to oxidize dihydrofumarate rapidly. Cu2+ also catalyses dihydroxyfumarate oxidation, but by a mechanism that does not involve O2 leads to. 2. Peroxidase catalyses oxidation of dihydroxyfumarate at pH6; addition of H2O2 does not increase the rate. Experiments with superoxide dismutase and catalase suggest that there are two types of oxidation taking place: an enzymic, H2O2-dependent oxidation of dihydroxyfumarate by peroxidase, and a non-enzymic reaction involving oxidation of dihydroxyfumarate by O2 leads to. The latter accounts for most of the observed oxidation of dihydroxyfumarate. 3. During dihydroxyfumarate oxidation, most peroxidase is present as compound III, and the enzymic oxidation may be limited by the low rate of breakdown of this compound. 4. Addition of p-coumaric acid to the peroxidase/dihydroxyfumarate system increases the rate of dihydroxyfumarate oxidation, which is now stimulated by addition of H2O2, and is more sensitive to inhibition by catalase but less sensitive to superoxide dismutase. Compound III is decomposed in the presence of p-coumaric acid. p-Hydroxybenzoate has similar, but much smaller, effects on dihydroxyfumarate oxidation. However, salicylate affects neither the rate nor the mechanism of dihydroxyfumarate oxidation. 5. p-Hydroxybenzoate, salicylate and p-coumarate are hydroxylated by the peroxidase/dihydroxyfumarate system. Experiments using scavengers of hydroxyl radicals shown that OH is required. Ability to increase dihydroxyfumarate oxidation is not necessary for hydroxylation to occur.     

Selenium modifies glutathione peroxidase activity and glutathione concentration in mice exposed to ozone-provoked oxidative stress

The aim of this study was to show the direct effect of selenium on glutathione peroxidase (GSH-Px) activity and GSH/GSSG concentrations in 3- and 6-month-old mice. An ozone-oxygen mixture was used to provoke an oxygen stress. To measure the Se-effect mice were gavaged with sodium selenite. GSH-Px activity and total glutathione concentrations were determined in serum and in the postnuclear fraction of liver and lungs. Additionally glutathione concentrations were determined in whole blood. Both ozone and selenium, administered separately, reduced GSH-Px activity in lungs of 6-month-old animals, while in young mice an opposite effect of Se was observed. Ozone administered jointly with Se did not influence GSH-Px activity in 6-month-old mice, while in young, 3-month-old mice, a stimulatory effect in lungs was observed. There were no significant changes in GSH-Px activity in the liver of 6-month-old mice, but the stimulatory effect occurred in young mice treated with Se and Se & ozone jointly. In young mice, ozone (also ozone with Se) augmented glutathione concentrations. The response to ozone and selenium strictly depended on age and the antagonism between selenium and ozone was observed only in a few cases.     

Knockout of cellular glutathione peroxidase gene renders mice susceptible to diquat-induced oxidative stress.

Two experiments were conducted to determine the protection and the underlying mechanisms of cellular glutathione peroxidase (GPX1) against lethal, acute oxidative stress induced by an intraperitoneal injection of 24 mg diquat/kg body weight. In experiment 1, mortality and survival times were compared among selenium (Se)-adequate or deficient GPX1 knockout mice [GPX1(-/-)] and wild-type mice (WT). In experiment 2, mice from these four groups were euthanized at 0, 1, 2, and 3 h after the injection of diquat to elucidate the time course of oxidative events. The stress produced 100% mortality in all of the groups except for the Se-adequate WT, which were euthanized on day 7 for analysis. The Se-deficient WT and the Se-adequate GPX1(-/-) had similar survival times (4.1 and 3.9 h), which were longer (p < .05) than that of the Se-deficient GPX1(-/-) (2.4 h). However, these three GPX1-deficient groups had higher levels (p < .05) of hepatic F2-isoprostanes and carbonyl contents and/or plasma alanine aminotransferase activities than those of the Se-adequate WT. The diquat-induced formations of hepatic F2-isoprostanes in these animals peaked at 1 h and preceded the rise of plasma alanine aminotransferase in the Se-adequate GPX1(-/-). Responses of hepatic superoxide dismutase activities to the diquat treatment were affected by the GPX1 level. In conclusion, GPX1 is the major selenoprotein to protect mice against the lethal oxidative stress induced by diquat.     

Evidence for suitability of glutathione peroxidase as a protective enzyme: studies of oxidative damage, renaturation, and proteolysis

The stability of glutathione peroxidase was assessed in vitro via oxidative inactivation by peroxides and a peroxidizing fatty acid and by renaturation and proteolysis. The stability of glutathione peroxidase to methyl ethyl ketone peroxide, H2O2, linoleic acid hydroperoxide, and peroxidizing methyl linolenate was compared with the stability of several other enzymes. Sulfhydryl enzymes were the most labile to all four treatments. Some of the enzymes tested were very stable to methyl ethyl ketone peroxide but very labile to linoleic acid hydroperoxide treatment. Glutathione peroxidase in the absence of glutathione was relatively slowly inactivated by each treatment. Linoleic acid hydroperoxide damage to glutathione peroxidase was characterized by release of a nonstoichiometric amount of selenite from the protein. Glutathione peroxidase samples lost all of their activity when (i) acidified to pH 2, (ii) heated 5 min at 100 degrees C, and (iii) treated with 6 M guanidinium hydrochloride or 8.5 M urea and heated 5 min at 100 degrees C. When the pH 2 sample was neutralized or the guanidinium hydrochloride-treated sample was diluted 101-fold, about 80% of the original activity was recovered in 30 min. The samples treated with urea and heat recovered no activity when diluted 101-fold. No loss of glutathione peroxidase occurred during treatment for 24 h within trypsin or thermolysin. Based on these results, glutathione peroxidase appears to be a relatively stable enzyme, and thus is is well-suited to perform its role in peroxide detoxification and prevention of oxidative deterioration of cells.     

A mechanistic study of the formation of hydroxyl radicals induced by horseradish peroxidase with NADH

During the oxidation of NADH by horseradish peroxidase (HRP-Fe(3+)), superoxide (O(-)(2)) is produced, and HRP-Fe(3+) is converted to compound III. Superoxide dismutase inhibited both the generation of O(-)(2) and the formation of compound III. In contrast, catalase inhibited only the generation of O(-)(2). Under anaerobic conditions, the formation of compound III did not occur in the presence of NADH, thus indicating that compound III is produced via formation of a ternary complex consisting of HRP-Fe(3+), NADH and oxygen. The generation of hydroxyl radicals was dependent upon O(-)(2) and H(2)O(2) produced by HRP-Fe(3+)-NADH. The reaction of compound III with H(2)O(2) caused the formation of compound II without generation of hydroxyl radicals. Only HRP-Fe(3+)-NADH (but not K(+)O(-)(2) and xanthine oxidase-hypoxanthine) was able to induce the conversion of metmyoglobin to oxymyoglobin, thus suggesting the participation of a ternary complex made up of HRP-Fe(2+…)O(2)(…)NAD(.) (but not free O(-)(2) or H(2)O(2)) in the conversion of metmyoglobin to oxymyoglobin. It appears that a cyclic pathway is formed between HRP-Fe(3+), compound III and compound II in the presence of NADH under aerobic conditions, and a ternary complex plays the central roles in the generation of O(-)(2) and hydroxyl radicals.     

Susceptibility of cord blood antioxidant enzymes glutathione reductase,glutathione peroxidase and glutathione S-transferase to different antibiotics: in vitro approach

Cord blood has numerous facilities for life and used in many different areas. Cord blood contains many different catalytic proteins including antioxidant enzymes. Here we purified human cord blood glutathione reductase (hcbGR), glutathione S-transferase (hcbGST) and human cord blood glutathione peroxidase (hcbGPx) from human cord blood erythrocytes and analyzed the inhibition effects of the antibiotics incorporating cefuroxime, ceftriaxone, ceftizoxime and cefoperazone, on these enzymes. K_I values for the drugs ranged from 10.42 to 28.72 µM for hcbGR, 32.7 to 244.8 µM for hcbGPx, and 32.39 to 267.3 µM for hcbGST. Cefuroxime caused the highest inhibition on all enzymes with K_I values of 10.42, 32.39, 32.7 µM for hcbGR, hcbGST, and hcbGPx, respectively. All drugs displayed non-competitive inhibition regardless of their structures. Since these drugs are often used during pregnancy, identification of possible undesired impacts on various parameters has a great importance for pharmacological and medical applications.     

Susceptibility of cultured rat hepatocytes to oxidative stress by peroxides and iron. The extracellular matrix affects the toxicity of tert-butyl hydroperoxide

The aim of this study was to set up an in vitro model for studying the importance of an altered extra-cellular matrix composition and its importance for the resistance to oxidative stress, in hepatocytes from normal and iron loaded rats. Primary cultures of hepatocytes from iron loaded and normal rats were plated on a laminin rich extracellular matrix or on collagen type I, and incubated with tert-butyl hydroperoxide (TBH). Malon dialdehyde (MDA) and the activities of lactate dehydrogenase (LDH) in cell culture medium were analyzed. The protein synthesis, the concentrations of glutathione and the expression of manganese-superoxide dismutase and ferritin genes were measured. All hepatocytes contained lower concentrations of glutathione when plated on collagen than on EHS. Ferritin H and Mn-SOD gene expression showed no difference. The rate of lipid peroxidation in iron loaded hepatocytes exposed to TBH was higher on collagen than in those plated on EHS (0.95 +/- 0.28 microM MDA vs. 1.62 +/- 0.22 microM MDA, p < 0.05). Iron loaded cells were in general more susceptible to TBH than were normal hepatocytes (MDA, LDH, protein synthesis and glutathione content). Lipid peroxidation could be prevented by adding desferrioxamine. In conclusion, we show that the combination of iron overload and collagen matrix in rat hepatocytes leads to an increased susceptibility to oxidative stress. These findings may be of interest for the further studies on effects of iron overload and the altered matrix composition in liver fibrosis.     

4-Hydroxynonenal inhibits glutathione peroxidase: protection by glutathione.

4-Hydroxy-2,3-trans-nonenal, a lipid peroxidation product, inhibits glutathione peroxidase in a concentration-dependent manner. The concentration providing 50% inhibition is 0.12 mM. This inhibition can be almost completely (89%) prevented by 1 mM glutathione added to the incubation mixture 30 min before 4-hydroxy-2,3-trans-nonenal or 2,3-trans-nonenal, but not by other thiol-containing antioxidants such as 0.5 mM dithiothreitol or beta-mercaptoethanol. Again the addition of 1 mM glutathione, and not of 0.5 mM dithiothreitol or beta-mercaptoethanol, to the enzyme 30 min after incubation with 4-hydroxy-2,3-trans-nonenal restores activity to the same extent as does the preincubation with GSH. In view of the known reactivity of 4-hydroxy-2,3-trans-nonenal with lysine residues and the reversibility of the inhibition, the involvement of a lysine residue in GSH binding to glutathione peroxidase is proposed. The potential relevance of the inhibition of glutathione peroxidase by 4-hydroxy-nonenal to oxidative tissue damage is discussed with particular emphasis on neurological disorders.     

Cysteine protects freshly isolated cardiomyocytes against oxidative stress by stimulating glutathione peroxidase

Cysteine has been implicated in myocardial protection, although this is controversial and constrained by limited knowledge about the effects of cysteine at the cellular level. This study tested the hypothesis that a physiologically relevant dose of l-cysteine could be safely loaded into isolated cardiomyocytes leading to improved protection against oxidative stress. Freshly isolated adult rat ventricular cardiomyocytes were incubated for 2 h at 37°C with (cysteine incubated) or without (control) 0.5 mM cysteine prior to washing and suspension in fresh cysteine-free media. Cysteine incubated cells had higher intracellular cysteine levels compared to controls (9.6 ± 0.78 vs. 6.5 ± 0.65 nmol/mg protein, P < 0.02, n = 6 ± SE). Cell homeostasis indicators were similar in the two groups. Cysteine incubated cells had significantly higher glutathione peroxidase (GPx) activity (1.11 ± 0.23 vs. 0.54 ± 0.1 U/mg protein, P < 0.05, n = 5 ± SE) and significantly greater expression of GPx-1 (5.01 ± 0.48 vs. 3.01 ± 0.25 OD units/mm², P < 0.05, n = 4 ± SE) compared to controls. Upon exposure to H₂O₂, cysteine incubated cells generated fewer reactive oxygen species and took longer to show contractile changes and undergo hypercontracture. However, when cells were exposed to H₂O₂ in the presence of 0.05 mM of the GPx inhibitor mercaptosuccinic acid, this increased the control cells’ susceptibility to H₂O₂ and completely abolished the cysteine mediated protection. These results suggest a new role for cysteine in myocardial protection involving stimulation of glutathione peroxidase.     

Effect of bleomycin on lipid peroxides, glutathione peroxidase and collagenase in cultured lung fibroblasts

Bleomycin-Cu(II) complex tended to increase the lipid peroxide level in cultured lung fibroblasts, though neither free bleomycin nor free cupric ion increased the level. Simultaneous addition of DL-alpha-tocopherol decreased the level significantly. Bleomycin-Cu(II) complex decreased glutathione peroxidase activity remarkably, though free bleomycin reduced the activity only slightly. Collagenase activity was not decreased but rather increased by both free bleomycin and bleomycin-Cu(II) complex. Accordingly, the accumulation of collagen induced by bleomycin could be explained not by a decrease in collagenase activity, but by the occurrence of cross-linking of collagen due to the increased lipid peroxides.     

Genetic analysis of glutathione peroxidase in oxidative stress response of Saccharomyces cerevisiae.

Three glutathione peroxidase homologs (YKL026C, YBR244W, and YIR037W/HYR1) were found in the Saccharomyces Genome Database. We named them GPX1, GPX2, and GPX3, respectively, and we investigated the function of each gene product. The gpx3Delta mutant was hypersensitive to peroxides, whereas null mutants of the GPX1 and GPX2 did not show any obvious phenotypes. Glutathione peroxidase activity decreased approximately 57 and 93% in the gpx3Delta and gpx1Delta/gpx2Delta/gpx3Delta mutants, respectively, compared with that of wild type. Expression of the GPX3 gene was not induced by any stresses tested, whereas that of the GPX1 gene was induced by glucose starvation. The GPX2 gene expression was induced by oxidative stress, which was dependent upon the Yap1p. The TSA1 (thiol-specific antioxidant) gene encodes thioredoxin peroxidase that can reduce peroxides by using thioredoxin as a reducing power. Disruption of the TSA1 gene enhanced the basal expression level of the Yap1p target genes such as GSH1, GLR1, and GPX2 and that resulted in increases of total glutathione level and activities of glutathione reductase and glutathione peroxidase. However, expression of the TSA1 gene did not increase in the gpx1Delta/gpx2Delta/gpx3Delta mutant. Therefore, de novo synthesis and recycling of glutathione were increased in the tsa1Delta mutant to maintain the catalytic cycle of glutathione peroxidase reaction efficiently as a backup system for thioredoxin peroxidase.     

Reactions of glutathione and glutathione radicals with benzoquinones.

The reactions of glutathione (GSH) and glutathione radicals with a series of methyl-substituted 1,4-benzoquinones and 1,4-benzoquinone have been studied. It was found that by mixing excess benzoquinone with glutathione at pH above 6.5, the products formed were complex and unstable. All of the other experiments were carried out at pH 6.0, where the main product was stable for several hours. Stopped-flow analysis allowed the measurement of the rates of the rapid reactions between GSH and the quinones, and the products were monitored by High Performance Liquid Chromatography (HPLC). The rates of the reactions vary by five orders of magnitude and must be influenced by steric factors as well as changes in the redox states. It was observed that simple hydroquinones were not formed when the different benzoquinones were mixed with excess GSH and suggests that the initial reaction is addition/reduction rather than electron transfer. In the presence of excess quinone, the hydroquinone of the glutathione conjugate is oxidized back to its quinone. The rates of the reaction were measured. By using the technique of pulse radiolysis, it was possible to measure the reduction of the quinones by GSSG.- and the oxidation of hydroquinones by GS(.). It is proposed that the appearance of GSSG in reactions of quinones with glutathione could be due to oxidation of the hydroquinone by oxygen and the subsequent superoxide or H2O2 promoting the oxidation of GSH to GSSG.     

Dps proteins prevent Fenton-mediated oxidative damage by trapping hydroxyl radicals within the protein shell

Dps (DNA-binding proteins from starved cells) proteins belong to a widespread bacterial family of proteins expressed under nutritional and oxidative stress conditions. In particular, Dps proteins protect DNA against Fenton-mediated oxidative stress, as they catalyze iron oxidation by hydrogen peroxide at highly conserved ferroxidase centers and thus reduce significantly hydroxyl radical production. This work investigates the possible generation of intraprotein radicals during the ferroxidation reaction by Escherichia coli and Listeria innocua Dps, two representative members of the family. Stopped-flow analyses show that the conserved tryptophan and tyrosine residues located near the metal binding/oxidation center are in a radical form after iron oxidation by hydrogen peroxide. DNA protection assays indicate that the presence of both residues is necessary to limit release of hydroxyl radicals in solution and the consequent oxidative damage to DNA. In general terms, the demonstration that conserved protein residues act as a trap that dissipates free electrons generated during the oxidative process brings out a novel role for the Dps protein cage.     

Superoxide-dependent formation of hydroxyl radicals: Detection of hydroxyl radicals by the hydroxylation of aromatic compounds

A mixture of xanthine or hypoxanthine and xanthine oxidase generates the superoxide radical, O₂^$\text{?}$, and H₂O₂. In the presence of iron salts, O₂^$\text{?}$ and H₂O₂ can interact to produce the hydroxyl radical, OH·. Superoxide-dependent formation of OH· can be measured by its ability to hydroxylate salicylate as followed by an improved colorimetric assay described in this paper. A more accurate analysis of OH· can be obtained using its ability to hydroxylate phenol, the hydroxylated products being separated and measured after derivatization using gas-liquid chromatography and electron-capture detection. The derivatization and separation techniques are described.