The antioxidant action of ergothioneine.

Ergothioneine is a product of plant origin that accumulates in animal tissues. Its suggested ability to act as an antioxidant has been evaluated. Ergothioneine is a powerful scavenger of hydroxyl radicals (.OH) and an inhibitor of iron or copper ion-dependent generation of .OH from hydrogen peroxide (H2O2). It is also an inhibitor of copper ion-dependent oxidation of oxyhaemoglobin, and of arachidonic acid peroxidation promoted by mixtures of myoglobin (or haemoglobin) and H2O2. Ergothioneine is a powerful scavenger of hypochlorous acid, being able to protect alpha 1-antiproteinase against inactivation by this molecule. By contrast, it does not react rapidly with superoxide (O2-) or hydrogen peroxide (H2O2) and it does not inhibit microsomal lipid peroxidation in the presence of iron ions. Overall, our results show that ergothioneine at the concentrations present in vivo could act as an antioxidant.     

Chemical and biochemical aspects of superoxide radicals and related species of activated oxygen

The spectrum of biological processes in which oxygen is used by living systems is quite large, and the products include some damaging species of activated oxygen, particularly the superoxide radical (O-.2) and hydrogen peroxide (H2O2). Superoxide radicals and hydrogen peroxide, in turn, can lead to the formation of other damaging species: hydroxyl radicals (.OH) and singlet oxygen (1O2). Hydroxyl radicals react with organic compounds to give secondary free radicals that, in the presence of oxygen, yield peroxy radicals, peroxides, and hydroperoxides. Formation, interconversion, and reactivity of O-.2 and related activated oxygen species, methods available for their detection, and the basis of their biological toxicity are briefly reviewed.     

Synergistic damage from H2O2 and OH radicals in irradiated cells

The anoxic sensitization of bacterial spores by added H2O2 has been studied. Two mechanistic pathways for damage from H2O2 were found; one of these requires the presence of OH radicals. For this kind of damage, the relationship between H2O2 and OH appears to be that they are reactants. O-2 (and/or HO2), the product of such a reaction, is likely the agent which actually causes damage. These results with reagent H2O2 are compared with results of experiments in which H2O2 and OH are present as radiolytic products.     

Nitric oxide protects Cu,Zn-superoxide dismutase from hydrogen peroxide-induced inactivation

Reaction of Cu,Zn-superoxide dismutase (SOD1) and hydrogen peroxide generates a putative oxidant SOD-Cu2+-.OH that can inactivate the enzyme and oxidize 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO) to DMPO-.OH. In the presence of nitric oxide (.NO), the SOD1/H2O2 system is known to produce peroxynitrite (ONOO-). In contrast to the proposed cytotoxicity of .NO conferred by ONOO-, we report here a protective role of .NO in the H2O2-induced inactivation of SODI. In a dose-dependent manner, .NO suppressed formation of DMPO-.OH and inactivation of the enzyme. Fragmentation of the enzyme was not affected by .NO. Bicarbonate retarded formation of ONOO-, suggesting that .NO competes with bicarbonate for the oxidant SOD-Cu2+-.OH. We propose that .NO protects SOD1 from H2O2-induced inactivation by reducing SOD-Cu2+.OH to the active SOD-Cu2+ with concomitant production of NO+ which reacts with H2O2 to give ONOO-.     

The oxygen-centered radicals scavenging activity of sulfasalazine and its metabolites. A direct protection of the bowel.

Oxygen-centered radicals, such as superoxide (O2-) and hydroxyl radicals (.OH) generated by phagocytes have been suggested to be involved in the pathogenesis of chronic inflammations of the bowel, such as Crohn's disease and colitis ulcerosa. Recently, sulfasalazine (SASP) and its metabolites have been reported to exert their effects as a direct scavenger of oxygen-centered radicals in the bowel. To scavenge oxygen-centered radicals in vivo, however, SASP and its metabolites have to react with O2- and/or .OH in vitro very rapidly, furthermore they have to reach an appropriate (possible millimolar) concentration range at the site of inflammation. To test this possibility, we investigated the direct O2- and .OH scavenging activity of SASP and its metabolites using the specific electron paramagnetic resonance/spin trapping method, and we compared the 50% inhibition rates of SASP and its metabolites with their known concentrations in the bowel and in the human plasma. It was found that SASP and its metabolites, such as 5-amino-salicylic acid (5-ASA), and acetyl-5-amino-salicylic acid (AC-5-ASA), but not sulfapyridine (SP) and acetyl-sulfapyridine (Ac-SP) have a direct O2- and .OH scavenging activity in vitro systems. Among the compounds, SASP and 5-ASA can reach a concentration which is appropriate to scavenge oxygen-centered radicals in the bowel but not in the human plasma. It was concluded that the in vivo antiinflammatory effects of SASP and its metabolites are, at least partly, due to the direct oxygen-centered scavenging activity of these drugs.     

Flavoenzymes reduce vanadium(V) and molecular oxygen and generate hydroxyl radical.

ESR spectroscopic evidence is presented for the formation of vanadium(IV) in the reduction of vanadium(V) by three typical, NADPH-dependent, flavoenzymes: glutathione reductase, lipoyl dehydrogenase, and ferredoxin-NADP+ oxidoreductase. The vanadium(V)-reduction mechanism appears to be an enzymatic one-electron reduction process. Addition of superoxide dismutase (SOD) showed that the generation of vanadium(IV) does not involve the superoxide (O2-) radical significantly. Measurements under anaerobic atmosphere showed, however, that the enzymes-vanadium-NADPH mixture can cause the reduction of molecular oxygen to generate H2O2. The H2O2 and vanadium(IV) thus formed react to generate hydroxyl (.OH) radical. The .OH formation is inhibited strongly by catalase and to a lesser degree by SOD, but it is enhanced by exogenous H2O2, suggesting the occurrence of a Fenton-like reaction. The inhibition of vanadium(IV) formation by N-ethylmaleimide indicates that the SH group on the flavoenzyme's cystine residue plays an important role in the enzyme's vanadium(V) reductase function. These results thus reveal a new property of the above-mentioned, NADPH-dependent flavoenzymes--their function as vanadium(V) reductases, as well as that as generators of .OH radical in the vanadium(V) reduction mechanism.     

One-electron reduction of chromate by NADPH-dependent glutathione reductase

Electron spin resonance (ESR) measurements provide evidence for the formation of Cr(V) intermediates in the enzymatic reduction of Cr(VI) by glutathione reductase (GSSG-R) in the presence of NADPH, indicating an initial single-electron transfer step in the reduction mechanism. Depending on the pH, at least two different Cr(V) species are generated which are relatively long-lived. In addition, we have detected the hydroxyl (.OH) radical formation during the GSSG-R catalyzed reduction of Cr(VI) by spin trapping, employing 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) as spin traps. Superoxide dismutase (SOD) causes only a minor effect on the .OH radical and Cr(V) formation, indicating that the O2- is not significantly involved in the reaction mechanism. Catalase enhances the Cr(V) formation and substantially inhibits the .OH radical formation, indicating the involvement of hydrogen peroxide (H2O2) in the reaction mechanism. Addition of H2O2 suppresses Cr(V) and enhances the .OH radical formation. Measurements involving N-ethylmaleimide show that the Cr(V) species, produced enzymatically by the reduction of Cr(VI) by GSSG-R, react with H2O2 to generate .OH radicals, which might participate in the initiation of Cr(VI) carcinogenicity.     

Superoxide dismutase and Fenton chemistry. Reaction of ferric-EDTA complex and ferric-bipyridyl complex with hydrogen peroxide without the apparent formation of iron(II).

A ferric-EDTA complex, prepared directly from FeCl3 or from an oxidized ferrous salt, reacts with H2O2 to form hydroxyl radicals (.OH), which degrade deoxyribose and benzoate with the release of thiobarbituric acid-reactive material, hydroxylate benzoate to form fluorescent dihydroxy products and react with 5,5-dimethylpyrrolidine N-oxide (DMPO) to form a DMPO-OH adduct. Degradation of deoxyribose and benzoate and the hydroxylation of benzoate are substantially inhibited by superoxide dismutase and .OH-radical scavengers such as formate, thiourea and mannitol. Inhibition by the enzyme superoxide dismutase implies that the reduction of the ferric-EDTA complex for participation in the Fenton reaction is superoxide-(O2.-)-dependent, and not H2O2-dependent as frequently implied. When ferric-bipyridyl complex at a molar ratio of 1:4 is substituted for ferric-EDTA complex (molar ratio 1:1) and the same experiments are conducted, oxidant damage is low and deoxyribose and benzoate degradation were poorly if at all inhibited by superoxide dismutase and .OH-radical scavengers. Benzoate hydroxylation, although weak, was, however, more effectively inhibited by superoxide dismutase and .OH-radical scavengers, implicating some role for .OH. The iron-bipyridyl complex had available iron-binding capacity and therefore would not allow iron to remain bound to buffer or detector molecules. Most .OH radicals produced by the iron-bipyridyl complex and H2O2 are likely to damage the bipyridyl molecules first, with few reacting in free solution with the detector molecules. Deoxyribose and benzoate degradation appeared to be mediated by an oxidant species not typical of .OH, and species such as the ferryl ion-bipyridyl complex may have contributed to the damage observed.     

Superoxide,Hydrogen Peroxide and Hydroxyl Radical in D1/D2/cytochrome b-559 Photosystem II Reaction Center Complex

Spin-trapping electron spin resonance (ESR) was used to monitor the formation of superoxide and hydroxyl radicals in D1/D2/cytochrome b-559 Photosystem II reaction center (PS II RC) Complex. When the PS II RC complex was strongly illuminated, superoxide was detected in the presence of ubiquinone. SOD activity was detected in the PS II RC complex. A primary product of superoxide, hydrogen peroxide, resulted in the production of the most destructive reactive oxygen species, *OH, in illuminated PS II RC complex. The contributions of ubiquinone, SOD and H(2)O(2) to the photobleaching of pigments and protein photodamage in the PS II RC complex were further studied. Ubiquinone protected the PS II RC complex from photodamage and, interestingly, extrinsic SOD promoted this damage. All these results suggest that PS II RC is an active site for the generation of superoxide and its derivatives, and this process protects organisms during strong illumination, probably by inhibiting more harmful ROS, such as singlet oxygen.     

The response of Azotobacter chroococcum to oxygen: superoxide-mediated effects.

Nitrogenase in Azotobacter chroococcum whole cells was inhibited by enzymically generated superoxide anion (O2-), hydrogen peroxide, and ethyl hydrogen peroxide. The degree of inhibition produced by O2- was related to the quantity of oxygen supplied to the organisms in continuous cultures. O2- also inhibited oxygen uptake by whole cells. These O2- mediated inhibitions were prevented by bovine superoxide dismutase. The quantities of superoxide dismutase (SOD), and catalase associated with cells grown under varying oxygen concentrations were determined. The role of hydrogen peroxide, and of the hydroxyl radical (.OH) in nitrogenase inhibition was examined. The response of Azotobacter chroococum to oxygen was evaluated with respect to the observed effects of O2- on the organism, and some explanation is given to account for nitrogenase sensitivity to oxygen.     

Superoxide and hydrogen peroxide formation during enzymatic oxidation of DOPA by phenoloxidase

Generation of superoxide anion and hydrogen peroxide during enzymatic oxidation of 3-(3,4-dihydroxyphenyl)-DL-alanine (DOPA) has been studied. The ability of DOPA to react with O2*- has been revealed. EPR spectrum of DOPA-semiquinone formed upon oxidation of DOPA by O2*- was observed using spin stabilization technique of ortho-semiquinones by Zn2+ ions. Simultaneously, the oxidation of DOPA by O2*- was found to produce hydrogen peroxide (H2O2). The analysis of H2O2 formation upon oxidation of DOPA by O2*- using 1-hydroxy-3-carboxy-pyrrolidine (CP-H), and SOD as competitive reagents for superoxide provides consistent values of the rate constant for the reaction between DOPA and O2*- being equal to (3.4+/-0.6)x10(5) M(-1) s(-1).The formation of H2O2 during enzymatic oxidation of DOPA by phenoloxidase (PO) has been shown. The H2O2 production was found to be SOD-sensitive. The inhibition of H2O2 production by SOD was about 25% indicating that H2O2 is produced both from superoxide anion and via two-electron reduction of oxygen at the enzyme. The attempts to detect superoxide production during enzymatic oxidation of DOPA using a number of spin traps failed apparently due to high value of the rate constant for DOPA interaction with O2*-.     

沙棘果皮多糖清除氧自由基的活性研究

沙棘(Hippophae rhamnosides)果皮经80℃恒温水浴提取, 乙醇沉淀得粗多糖。Sevag法去蛋白,经50%和70%乙醇分级, 得3种级分沙棘多糖H1、H2和H3; 以Fenton 反应, 即H2O2/Fe2+/水杨酸为.OH产生和检测体系; 以邻苯三酚/EDTA/Tris-HCl为O2 -. 产生体系, 对沙棘多糖H1、H2和H3进行抗氧自由基活 性研究。结果表明, 沙棘多糖对.OH和O2 -. 有较显著的清除能力。不同级分多糖H1、H2和H3浓度达200mg.mL-1时, 对.OH的清除率分别为44.9%、49.0%和26.4%, 抗O2-. 活性分别为36.9%,15.4%和23.1%。多糖质量浓度增大时,两种自由基清除率增加, 且呈量效关系。     

Elevation of hydrogen peroxide after spinal cord injury detected by using the Fenton reaction.

To reveal whether reactive oxygen species (ROS) play a role after spinal cord injury, we developed a unique method for assaying hydrogen peroxide (H2O2) and determined the time course of its concentration changes following impact injury to the rat spinal cord. Microdialysis was used to sample H2O2 in the extracellular space and the dialysates were collected into a vial containing salicylate and ferrous chloride (FeCl2). H2O2 collected in the vial was converted to hydroxyl radicals (*OH) by FeCl2 catalysis. 2,3- and 2,5-dihydroxybenzoic acid produced by reaction of *OH with salicylate in the collecting vial were measured by HPLC and calibrated to H2O2 concentrations. The postinjury levels of H2O2 were significantly increased (p = 0.02) for over 11 h. FeCl2 administered through the dialysis fiber catalyzes H2O2 conversion in the cord to *OH. This *OH does not reach the collecting vial due to its extremely short lifetime (nanoseconds). The reduced H2O2 levels in the vials validate the measurement of H2O2. The relatively long-lasting formation of H2O2 and superoxide reported herein and previously suggests that ROS may be important in secondary spinal cord damage and that removal of ROS may be a realistic treatment strategy for reducing injury caused by free radicals.     

The interaction between Cu(I) superoxide dismutase and hydrogen peroxide

The interaction between superoxide dismutase (SOD) and peroxide, under anaerobic conditions in the presence of an OH radical scavenger, formate, and an indicator, nitro blue tetrazolium, involves five reactions and an equilibrium: (table; see text) Reaction 3 occurs at a rate that is proportional to both peroxide and enzyme with no kinetic evidence for any intermediate peroxide-enzyme complex. Rate studies as a function of pH corroborate previously published work (Fuchs, H. J. R., and Borders, C. L., Jr. (1983) Biochem Biophys. Res. Commun. 116, 1107-1113; Blech, D. M., and Borders, C. L., Jr. (1983) Arch. Biochem. Biophys. 224, 579-586) suggesting that HO2-, and not H2O2, is the active species in this system: k(HO2- + superoxide dismutase-Cu+) = 2.6 x 10(3) M-1 s-1. Evidence is presented which suggests that HO2-, like O2-, reacts at rates that are affected by the electrostatic forces of the enzyme.     

Susceptibility of micro-organisms to active oxygen species: sensitivity to the xanthine-oxidase-mediated antimicrobial system

Xanthine oxidase with acetaldehyde as substrate (the XOA system) generated superoxide anion and hydrogen peroxide, but this system had only weak bactericidal activity. Addition of Fe2+ and EDTA to the XOA system (XOA-Fe-EDTA system) increased bactericidal activity against Staphylococcus aureus, Escherichia coli, Listeria monocytogenes and Salmonella typhimurium, although both Mycobacterium tuberculosis and Candida albicans remained highly resistant. Catalase (H2O2 scavenger) and mannitol (.OH scavenger) almost completely inhibited the bactericidal activity of the XOA-Fe-EDTA system whereas SOD (O2- scavenger) was less inhibitory. Azide (1O2 scavenger) caused no such inhibition. The results suggest the possible role of .OH, H2O2 and O2- in the XOA-Fe-EDTA-mediated antimicrobial system, as effector molecules. There was no correlation between resistance of a given bacterium to active oxygen and the level of endogenous active oxygen-scavengers.     

The antioxidant action of taurine, hypotaurine and their metabolic precursors.

It has been suggested that taurine, hypotaurine and their metabolic precursors (cysteic acid, cysteamine and cysteinesulphinic acid) might act as antioxidants in vivo. The rates of their reactions with the biologically important oxidants hydroxyl radical (.OH), superoxide radical (O2.-), hydrogen peroxide (H2O2) and hypochlorous acid (HOCl) were studied. Their ability to inhibit iron-ion-dependent formation of .OH from H2O2 by chelating iron ions was also tested. Taurine does not react rapidly with O2.-, H2O2 or .OH, and the product of its reaction with HOCl is still sufficiently oxidizing to inactivate alpha 1-antiproteinase. Thus it seems unlikely that taurine functions as an antioxidant in vivo. Cysteic acid is also poorly reactive to the above oxidizing species. By contrast, hypotaurine is an excellent scavenger of .OH and HOCl and can interfere with iron-ion-dependent formation of .OH, although no reaction with O2.- or H2O2 could be detected within the limits of our assay techniques. Cysteamine is an excellent scavenger of .OH and HOCl; it also reacts with H2O2, but no reaction with O2.- could be measured within the limits of our assay techniques. It is concluded that cysteamine and hypotaurine are far more likely to act as antioxidants in vivo than is taurine, provided that they are present in sufficient concentration at sites of oxidant generation.     

The oxidative inactivation of mitochondrial electron transport chain components and ATPase

Bovine heart submitochondrial particles (SMP) were exposed to continuous fluxes of hydroxyl radical (.OH) alone, superoxide anion radical (O2-) alone, or mixtures of .OH and O2-, by gamma radiolysis in the presence of 100% N2O (.OH exposure), 100% O2 + formate (O2- exposure), or 100% O2 alone (.OH + O2- exposure). Hydrogen peroxide effects were studied by addition of pure H2O2. NADH dehydrogenase, NADH oxidase, succinate dehydrogenase, succinate oxidase, and ATPase activities (Vmax) were rapidly inactivated by .OH (10% inactivation at 15-40 nmol of .OH/mg of SMP protein, 50-90% inactivation at 600 nmol of .OH/mg of SMP protein) and by .OH + O2- (10% inactivation at 20-80 nmol of .OH + O2-/mg of SMP protein, 45-75% inactivation at 600 nmol of .OH + O2-/mg of SMP protein). Importantly, O2- was a highly efficient inactivator of NADH dehydrogenase, NADH oxidase, and ATPase (10% inactivation at 20-50 nmol of O2-/mg of SMP protein, 40% inactivation at 600 nmol of O2-/mg of SMP protein), a mildly efficient inactivator of succinate dehydrogenase (10% inactivation at 150 nmol of O2-/mg of SMP protein, 30% inactivation at 600 nmol of O2-/mg of SMP protein), and a poor inactivator of succinate oxidase (less than 10% inactivation at 600 nmol of O2-/mg of SMP protein). H2O2 partially inactivated NADH dehydrogenase, NADH oxidase, and cytochrome oxidase, but even 10% loss of these activities required at least 500-600 nmol of H2O2/mg of SMP protein. Cytochrome oxidase activity (oxygen consumption supported by ascorbate + N,N,N',N'-tetramethyl-p-phenylenediamine) was remarkably resistant to oxidative inactivation, with less than 20% loss of activity evident even at .OH, O2-, OH + O2-, or H2O2 concentrations of 600 nmol/mg of SMP protein. Cytochrome c oxidase activity, however (oxidation of, added, ferrocytochrome c), exhibited more than a 40% inactivation at 600 nmol of .OH/mg of SMP protein. The .OH-dependent inactivations reported above were largely inhibitable by the .OH scavenger mannitol. In contrast, the O2(-)-dependent inactivations were inhibited by active superoxide dismutase, but not by denatured superoxide dismutase or catalase. Membrane lipid peroxidation was evident with .OH exposure but could be prevented by various lipid-soluble antioxidants which did not protect enzymatic activities at all.(ABSTRACT TRUNCATED AT 400 WORDS)     

Mechanism of NADH-sensitized formation of DNA breaks during irradiation with near UV light]

NADH-photosensitized in vitro formation of single-stranded breaks in plasmid DNA pBR322 depends on both the concentration of the sensitizer and the influence of near-UV radiation (320-400 nm). Scavengers and inhibitors of different activated oxygen species (sodium azide, sodium benzoate, catalase and superoxide dismutase) prevent the formation of breaks in full or partly. The data obtained show that hydroxyl radical (.OH) and singlet oxygen (1O2) are directly involved in the induction of breaks. In this process hydrogen peroxide (H2O2) plays the role of an intermediate in the reaction of .OH formation from superoxide anion-radical (O2-.) which is the first NAD.H-photogenerated product.     

The effect of oxygen on the radiolysis of tyrosine in aqueous solutions

The effect of oxygen on the radiolysis of tyrosine in aqueous solutions was investigated by using gamma and pulsed electron irradiation. Steady-state radiolysis was reexamined and extended to include the effect of pH and determination of hydrogen peroxide. The loss of tyrosine, G(-Tyr), during irradiation and yields of 3,4-dihydroxyphenylalanine, G(DOPA), and hydrogen peroxide, G(H2O2), are determined in the pH range from 1 to 9. In the whole pH range used G(-Tyr) equals G(DOPA), and a higher G(H2O2) than expected was observed. In slightly acid and neutral media, both G(-Tyr) and G(DOPA) equal the yield of hydroxyl radicals, GOH, formed in the radiolysis of water, while the excess of hydrogen peroxide equals 1/2 GOH. Hence it was concluded that all tyrosine OH-adducts react with oxygen yielding peroxy radicals. In acid and alkaline media all measured yields decrease. This is caused by formation of tyrosine phenoxyl radicals (TyrO), which react with superoxide anion (O2-) and hydroperoxy (HO2) radicals regenerating tyrosine. By using pulse radiolysis K(TyrO + O2) less than or equal to 2 X 10(5) mol-1 dm3 s-1 and k(TyrO + O2-) = (1.7 +/- 0.2) X 10(9) mol-1 dm3 s-1 were determined. On the basis of the results, a reaction mechanism is proposed.     

The roles of free radicals in amyotrophic lateral sclerosis: reactive oxygen species and elevated oxidation of protein, DNA, and membrane phospholipids.

To explore whether reactive oxygen species (ROS) play a role in the pathogenesis of amyotrophic lateral sclerosis (ALS), a unique microdialysis or microcannula sampling technique was used in mice transfected with a mutant Cu,Zn-superoxide dismutase (SOD1) gene from humans with familial ALS, mice transfected with the normal human SOD1 gene, and normal mice. We demonstrate for the first time that the levels of hydrogen peroxide (H(2)O(2)) and the hydroxyl radical ((.)OH) are significantly higher, and the level of the superoxide anion (O(2)(.-)) is significantly lower in ALS mutant mice than in controls, supporting by in vivo evidence the hypothesis that the mutant enzyme catalyzes (.)OH formation by the sequence: O(2)(.-) --> H(2)O(2) --> (.)OH. This removes doubts regarding the relevance of elevated ROS in FALS raised by in vitro experiments. The levels of oxidation products are also significantly higher in the mutant mice than in controls, consistent with some previous reports. Only the superoxide concentration differs between two controls among all the measurements. Our findings correlate in vivo a gene mutation to both elevated H(2)O(2) and (.)OH and increased oxidation of cellular constituents. The elevated H(2)O(2) in mutant mice indicates impairment of its detoxification pathways, perhaps by changed interactions between SOD1 and H(2)O(2) detoxification enzymes.-Liu, D., Wen, J., Liu, J., Li, L. The roles of free radicals in amyotrophic lateral sclerosis: reactive oxygen species and elevated oxidation of protein, DNA, and membrane phospholipids.