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.     

Intra- and intermolecular oxidation of oxymyoglobin and oxyhemoglobin induced by hydroxyl and carbonate radicals

The mechanism of the reactions of myoglobin and hemoglobin with *OH and CO3*- in the presence of oxygen was studied using pulse and gamma-radiolysis. Unlike *NO2, which adds to the porphyrin iron, *OH and CO3*- form globin radicals. These secondary radicals oxidize the Fe(II) center through both intra- and intermolecular processes. The intermolecular pathway was further demonstrated when BSA radicals derived from *OH or CO3*- oxidized oxyhemoglobin and oxymyoglobin to their respective ferric states. The oxidation yields obtained by pulse radiolysis were lower compared to gamma-radiolysis, where the contribution of radical-radical reactions is negligible. Full oxidation yields by *OH-derived globin radicals could be achieved only at relatively high concentrations of the heme protein mainly via an intermolecular pathway. It is suggested that CO3*- reaction with the protein yields Tyr and/or Trp-derived phenoxyl radicals, which solely oxidize the porphyrin iron under gamma-radiolysis conditions. The *OH particularly adds to aromatic residues, which can undergo elimination of H2O forming the phenoxyl radical, and/or react rapidly with O2 yielding peroxyl radicals. The peroxyl radical can oxidize a neighboring porphyrin iron and/or give rise to superoxide, which neither oxidize nor reduce the porphyrin iron. The potential physiological implications of this chemistry are that hemoglobin and myoglobin, being present at relatively high concentrations, can detoxify highly oxidizing radicals yielding the respective ferric states, which are not toxic.     

Evaluation of the ability of the angiotensin-converting enzyme inhibitor captopril to scavenge reactive oxygen species.

Captopril, an inhibitor of angiotensin-converting enzyme, has been suggested to have additional cardioprotective action because of its ability to act as an antioxidant. The rates of reaction of captopril with several biologically-relevant reactive oxygen species were determined. Captopril reacts slowly, if at all, with superoxide (rate constant less than 10(3) M-1 s-1) or hydrogen peroxide (rate constant less than M-1 s-1). It does not inhibit peroxidation of lipids stimulated by iron ions and ascorbate or by the myoglobin/H2O2 system. Indeed, mixtures of ferric ion and captopril can stimulate lipid peroxidation. Captopril reacts rapidly with hydroxyl radical (rate constant greater than 10(9) M-1 s-1) but might be unlikely to compete with most biological molecules for OH because of the low concentration of captopril that can be achieved in vivo during therapeutic use. Captopril did not significantly inhibit iron ion-dependent generation of hydroxyl radicals from hydrogen peroxide. By contrast, captopril is a powerful scavenger of hypochlorous acid: it was able to protect alpha 1-antiproteinase (alpha 1 AP) against inactivation by this species and to prevent formation of chloramines from taurine. We suggest that the antioxidant action of captopril in vivo is likely to be limited, and may be restricted to protection against damage by hypochlorous acid derived from the action of neutrophil myeloperoxidase.     

Action of biologically-relevant oxidizing species upon uric acid. Identification of uric acid oxidation products

Uric acid is an end-product of purine metabolism in Man, and has been suggested to act as an antioxidant in vivo. Products of attack upon uric acid by various oxidants were measured by high performance liquid chromatography. Hypochlorous acid rapidly oxidized uric acid, forming allantoin, oxonic/oxaluric and parabanic acids, as well as several unidentified products. HOCl could oxidize all these products further. Hydrogen peroxide did not oxidize uric acid at detectable rates, although it rapidly oxidized oxonic acid and slowly oxidized allantoin and parabanic acids. Hydroxyl radicals generated by hypoxanthine/xanthine oxidase or Fe2(+)-EDTA/H2O2 systems also oxidized uric acid to allantoin, oxonic/oxaluric acid and traces of parabanic acid. Addition of ascorbic acid to the Fe2(+)-EDTA/H2O2 system did not increase formation of oxidation products from uric acid, possibly because ascorbic acid can 'repair' the radicals resulting from initial attack of hydroxyl radicals upon uric acid. Mixtures of methaemoglobin or metmyoglobin and H2O2 also oxidized uric acid: allantoin was the major product, but some parabanic and oxonic/oxaluric acids were also produced. Caeruloplasmin did not oxidize uric acid under physiological conditions, although simple copper (Cu2+) ions could, but this was prevented by albumin or histidine. The possibility of using oxidation products of uric acid, such as allantoin, as an index of oxidant generation in vivo in humans is discussed.     

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 antioxidant action of human extracellular fluids. Effect of human serum and its protein components on the inactivation of alpha 1-antiproteinase by hypochlorous acid and by hydrogen peroxide.

The elastase-inhibitory capacity of purified human alpha 1-antiproteinase is inactivated by low concentrations of the myeloperoxidase-derived oxidant hypochlorous acid, but much higher concentrations are required to inhibit the elastase-inhibitory capacity of serum samples. The protective effect of serum appears to be largely due to albumin. High concentrations of H2O2 also inactivate the elastase-inhibitory capacity of alpha 1-antiproteinase, by a mechanism not involving formation of hydroxyl radicals. Serum offers protection against H2O2 inactivation of alpha 1-antiproteinase. The relevance of these results to the tissue damage produced by activated phagocytes is discussed.     

A time-resolved electron spin resonance study of the oxidation of ascorbic acid by hydroxyl radical.

Time-resolved electron spin resonance (ESR) spectroscopy for the study of radicals produced by pulse radiolysis is illustrated by a study of the oxidation of ascorbic acid by OH radical in aqueous solution. In basic solution, the direct oxidation product, the ascorbate mono-anion radical, is formed within less than 2 mus of the radiolysis pulse. In acid solutions (pH 3(-4.5), N(2)O:saturated) three radicals are initially formed, the ascorbate mono-anion radical, an OH adduct seen also in steady-state ESR experiments, and an OH adduct at C2 with the main spin density at C3 of the ring. The first OH adduct decays with an initial half-life of about 100 mus, probably by biomolecular reaction. The second OH adduct, which shows one hyperfine splitting about a(H) = 24.4 +/- 0.3 G and g = 2.0031 +/- 0.0002, decays with a half-life of about 10 mus. On this same time scale the concentration of the ascorbate radical approximately doubles. It is concluded that the adduct at C2, but not the other adduct, loses water rapidly to form the ascorbate radical.     

A pulse-radiolysis study of the catalytic mechanism of the iron-containing superoxide dismutase from Photobacterium leiognathi.

The mechanism of the enzymic reaction of an iron-containing superoxide dismutase purified from the marine bacterium Photobacterium leiognathi was studied by using pulse radiolysis. Measurements of activity were done with two different preparations of enzyme containing either 1.6 or 1.15 g-atom of iron/mol. In both cases, identical values of the second-order rate constant for reaction between superoxide dismutase and the superoxide ion in the pH range 6.2-9.0 (k=5.5 X 10(8) M-1-S-1 at pH 8.0) were found. As with the bovine erythrocuprein, there was no evidence for substrate saturation. The effects of reducing agents (H2O2, sodium ascorbate or CO2 radicals) on the visible and the electron-paramagnetic-resonance spectra of the superoxide dismutase containing 1.6 g-atom of ferric iron/mol indicate that this enzyme contains two different types of iron. Turnover experiments demonstrate that only that fraction of the ferric iron that is reduced by H2O2 is involved in the catalysis, being alternately oxidized and reduced by O2; both the oxidation and the reduction steps have a rate constant equal to that measured under turnover conditions. These results are interpreted by assuming that the superoxide dismutase isolated from the organism contains 1 g-atom of catalytic iron/mol and a variable amount of non-catalytic iron. This interpretation is discused in relation to the stoicheiometry reported for iron-containing superoxide dismutases prepared from several other organisms.     

Superoxide-dependent and -independent mechanisms of iron mobilization from ferritin by xanthine oxidase. Implications for oxygen-free-radical-induced tissue destruction during ischaemia and inflammation.

Xanthine oxidase is able to mobilize iron from ferritin. This mobilization can be blocked by 70% by superoxide dismutase, indicating that part of its action is mediated by superoxide (O2-). Uric acid induced the release of ferritin iron at concentrations normally found in serum. The O2(-)-independent mobilization of ferritin iron by xanthine oxidase cannot be attributed to uric acid, because uricase did not influence the O2(-)-independent part and acetaldehyde, a substrate for xanthine oxidase, also revealed an O2(-)-independent part, although no uric acid was produced. Presumably the amount of uric acid produced by xanthine oxidase and xanthine is insufficient to release a measurable amount of iron from ferritin. The liberation of iron from ferritin by xanthine oxidase has important consequences in ischaemia and inflammation. In these circumstances xanthine oxidase, formed from xanthine dehydrogenase, will stimulate the formation of a non-protein-bound iron pool, and the O2(-)-produced by xanthine oxidase, or granulocytes, will be converted by 'free' iron into much more highly toxic oxygen species such as hydroxyl radicals (OH.), exacerbating the tissue damage.     

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.     

Superoxide-dependent and ascorbate-dependent formation of hydroxyl radicals from hydrogen peroxide in the presence of iron. Are lactoferrin and transferrin promoters of hydroxyl-radical generation?

Apo-lactoferrin and apo-transferrin protect against iron-ion-dependent hydroxyl-radical (.OH) generation from H2O2 in the presence of superoxide radicals or ascorbic acid at pH 7.4, whether the necessary iron is added as ionic iron or as ferritin. Iron-loaded transferrin and lactoferrin [2 mol of Fe(III)/mol] show no protective ability, but do not themselves accelerate .OH production unless chelating agents are present in the reaction mixture, especially if the proteins are incorrectly loaded with iron. At acidic pH values, the protective ability of the apoproteins is diminished, and the fully iron-loaded proteins can release some iron in a form able to accelerate .OH generation. The physiological significance of these observations is discussed.     

Radical intermediates involved in the bleaching of the carotenoid crocin. Hydroxyl radicals, superoxide anions and hydrated electrons

The participation of the primary radicals in the bleaching of aqueous solutions of the carotenoid crocin by ionizing radiation was investigated, employing both X-radiolysis and pulse radiolysis. The pulse-radiolytic data demonstrated a very rapid diffusion-controlled attack by both hydroxyl radicals (.OH) and hydrated electrons (eaq-), while superoxide anions (O2-) did not react at all. The site of the initial reaction of these radicals was not limited to the polyene chromophore. Slower secondary reactions involving crocin alkyl or peroxy radicals contribute mainly to the overall bleaching, in particular during steady-state irradiation.     

The deoxyribose method: a simple "test-tube" assay for determination of rate constants for reactions of hydroxyl radicals

Hydroxyl radicals, generated by reaction of an iron-EDTA complex with H2O2 in the presence of ascorbic acid, attack deoxyribose to form products that, upon heating with thiobarbituric acid at low pH, yield a pink chromogen. Added hydroxyl radical "scavengers" compete with deoxyribose for the hydroxyl radicals produced and diminish chromogen formation. A rate constant for reaction of the scavenger with hydroxyl radical can be deduced from the inhibition of color formation. For a wide range of compounds, rate constants obtained in this way are similar to those determined by pulse radiolysis. It is suggested that the deoxyribose assay is a simple and cheap alternative to pulse radiolysis for determination of rate constants for reaction of most biological molecules with hydroxyl radicals. Rate constants for reactions of ATP, ADP, and Good's buffers with hydroxyl radicals have been determined by this method.     

Salivary thiocyanate/nitrite inhibits hydroxylation of 2-hydroxybenzoic acid induced by hydrogen peroxide/Fe(II) systems under acidic conditions: possibility of thiocyanate/nitrite-dependent scavenging of hydroxyl radical in the stomach

Formation of OH radicals in the stomach is possible by Fenton-type reactions, as gastric juice contains ascorbic acid (AA), iron ions and H2O2. An objective of the present study is to elucidate the effects of salivary SCN- and NO2- on the hydroxylation of salicylic acid which was induced by H2O2/Fe(II) and AA/H2O2/Fe(II) systems. Thiocyanate ion inhibited the hydroxylation of salicylic acid by the above systems in acidic buffer solutions and in acidified saliva. The inhibition by SCN- was deduced to be due to SCN- -dependent scavenging of OH radicals. Nitrite ion could enhance the SCN- -dependent inhibition of the hydroxylation induced by AA/H2O2/Fe(II) systems. The enhancement was suggested to be due to scavenging of OH radicals by NO which was formed by the reactions among AA, HNO2 and SCN- contained in the reaction mixture. The concentrations of SCN- and NO2-, which were effective for the inhibition, were in ranges of their normal salivary concentrations. These results suggest that salivary SCN- can cooperate with NO2- to protect stomach from OH radicals formed by AA/H2O2/Fe(II) systems under acidic conditions.     

Homogentisic acid autoxidation and oxygen radical generation: implications for the etiology of alkaptonuric arthritis

The metabolic disorder, alkaptonuria, is distinguished by elevated serum levels of 2,5-dihydroxyphenylacetic acid (homogentisic acid), pigmentation of cartilage and connective tissue and, ultimately, the development of inflammatory arthritis. Oxygen radical generation during homogentisic acid autoxidation was characterized in vitro to assess the likelihood that oxygen radicals act as molecular agents of alkaptonuric arthritis in vivo. For homogentisic acid autoxidized at physiological pH and above, yielding superoxide (O2-)2 and hydrogen peroxide (H2O2), the homogentisic acid autoxidation rate was oxygen dependent, proportional to homogentisic acid concentration, temperature dependent and pH dependent. Formation of the oxidized product, benzoquinoneacetic acid was inhibited by the reducing agents, NADH, reduced glutathione, and ascorbic acid and accelerated by SOD and manganese-pyrophosphate. Manganese stimulated autoxidation was suppressed by diethylenetriaminepentaacetic acid (DTPA). Homogentisic acid autoxidation stimulated a rapid cooxidation of ascorbic acid at pH 7.45. Hydrogen peroxide was among the products of cooxidation. The combination of homogentisic acid and Fe3+-EDTA stimulated hydroxyl radical (OH.) formation estimated by salicylate hydroxylation. Ferric iron was required for the reaction and Fe3+-EDTA was a better catalyst than either free Fe3+ or Fe3+-DTPA. SOD accelerated OH. production by homogentisic acid as did H2O2, and catalase reversed much of the stimulation by SOD. Catalase alone, and the hydroxyl radical scavengers, thiourea and sodium formate, suppressed salicylate hydroxylation. Homogentisic acid and Fe3+-EDTA also stimulated the degradation of hyaluronic acid, the chief viscous element of synovial fluid. Hyaluronic acid depolymerization was time dependent and proportional to the homogentisic acid concentration up to 100 microM. The level of degradation observed was comparable to that obtained with ascorbic acid at equivalent concentrations. The hydroxyl radical was an active intermediate in depolymerization. Thus, catalase and the hydroxyl radical scavengers, thiourea and dimethyl sulfoxide, almost completely suppressed the depolymerization reaction. The ability of homogentisic acid to generate O2-, H2O2 and OH. through autoxidation and the degradation of hyaluronic acid by homogentisic acid-mediated by OH. production suggests that oxygen radicals play a significant role in the etiology of alkaptonuric arthritis.     

Production of superoxide radicals with pulse radiolysis of water with high linear energy transfer

The radiolysis of water with heavy ions of high linear energy transfer (LET) (-dE/dx) is characterized, in deaerated medium, by the production of superoxide anions, the radiolytic yields of which increase with the LET. Radiobiological interest in such radical species comes from the oxidative stress which may be generated by their dismutation in O2 and H2O2 in anoxic medium (radiotherapy with heavy ions). A brief review of the measurements of superoxide free radicals in aqueous solution by indirect or direct methods is presented. Moreover, some experimental results obtained by pulse radiolysis with Ar18+ ions (TEL = 290 keV x microm(-1)), are described. The interpretation of the kinetics takes into account the superoxide absorbance and that of hydrogen peroxide, which is present at the millisecond time scale.     

Reactions of reactive oxygen species (ROS) with curcumin analogues: Structure-activity relationship

Three curcumin analogues viz., bisdemethoxy curcumin, monodemethoxy curcumin, and dimethoxycurcumin that differ at the phenolic substitution were synthesized. These compounds have been subjected for free radical reactions with DPPH radicals, superoxide radicals (O(2)(?-)), singlet oxygen ((1)O(2)) and peroxyl radicals (CCl(3)O(2)(?)) and the bimolecular rate constants were determined. The DPPH radical reactions were followed by stopped-flow spectrometer, (1)O(2) reactions by transient luminescence spectrometer, and CCl(3)O(2)(?) reactions using pulse radiolysis technique. The rate constants indicate that the presence of o-methoxy phenolic OH increases its reactivity with DPPH and CCl(3)O(2)(?), while for molecules lacking phenolic OH, this reaction is very sluggish. Reaction of O(2)(?-) and (1)O(2) with curcumin analogues takes place preferably at β-diketone moiety. The studies thus suggested that both phenolic OH and the β-diketone moiety of curcumin are involved in neutralizing the free radicals and their relative scavenging ability depends on the nature of the free radicals.     

Problems concerning the biochemical action of superoxide dismutase (erythrocuprein).

The decay of the tetraperoxochromate- (V) complex (CrO83theta) was examined to study the substrate specificity of erythrocuprein (super-oxide dismutase). The decay of CrO83theta proved rather complex in aqueous solutions. Apart from the two known oxygen species O2theta and singlet oxygen (1 deltagO2), H2O2 and probably OH radicals were formed. No unequivocal evidence for the appearance of superoxide was obtained. The possible electron transfer from Cr5 to Fe3 (cytochrome c) was also discussed. In Tris buffer, pH 7.8, there were absolutely no signs of superoxide or OH radical formation. In fact, pulse radiolysis measurements employing a homogeneous OH source demonstrated that the Tris and OH radicals react with each other. One mol of H2O2 was generated from 1 mol of CrO83theta in Tris buffer. By contrast, only 0.5 mol H2O2 could be determined when the CrO83theta decay was carried out in 2-[4-(2-hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid (HEPES) buffer, pH 7.8. The phenomenon of reducing oxidized cytochrome c could not fully be assigned to a superoxide-mediated reduction, since erythrocuprein was unable to inhibit this cytochrome c reduction efficiently. The energetic oxygen species (1deltag O2, OH etc.) appearing during the CrO83theta decay gave rise to a clearly detectable chemiluminescence. In this system, erythrocuprein was very active regardless of which buffer was used. Even in the absence of a chemiluminescent mediating agent, which might have interferred with the enzyme, erythrocuprein proved capable of inhibiting the CrO83theta-induced chemiluminescence in a rather specific way. No such specificity was seen in the presence of low molecular weight Cu-chelates including Cu(Tyr)2, Cu(Lys)2 and Cu(His)2. The ability to suppress chemiluminescence was approximately 3 orders of magnitude less pronounced than that of the native enzyme. It is presumed that erythrocuprein reacts with oxygen species other than the superoxide radical.     

Inhibition of the iron-catalysed formation of hydroxyl radicals from superoxide and of lipid peroxidation by desferrioxamine.

The peroxidation of membrane phospholipids induced in vitro by ascorbic acid or by dialuric acid (hydroxybarbituric acid) does not occur in the absence of traces of metal ions. Peroxidation induced by adding iron salts to phospholipids can either be promoted or inhibited by the chelators EDTA, diethylenetriaminepenta-acetic acid and bathophenanthrolinesulphonate, depending on the ratio [chelator]/[iron salt]. The iron chelator desferrioxamine inhibits peroxidation at all concentrations tested, and it also inhibits the iron-catalysed formation of hydroxyl radicals (OH.) from superoxide (O2-.). Since desferrioxamine is approved for clinical use, it might prove a valuable tool in the treatment of inflammation, poisoning by autoxidizable molecules and radiation damage.     

Oxygen-dependent antagonism of lipid peroxidation

Measurements of the rates for formation of conjugated dienes, malonylaldehyde, and lipid hydroperoxides show that increasing the concentration of O2 from 0.11 mM to 0.35 mM or 0.69 mM can slow the rate of linoleic acid peroxidation in a xanthine oxidase/hypoxanthine system. This effect is seen at pH 7.0 but not 7.4 and depends on the presence of monounsaturated fatty acids (oleic, cis, or trans vaccenic acid). Oxygen antagonism of ascorbic acid-iron-EDTA mediated lipid peroxidation is similarly dependent on fatty acid mixtures and occurs at pH 5.0 and 6.0 but not 7.0. The efficiency of initiation of peroxidation in the xanthine oxidase system is unaffected by monounsaturated fatty acids and O2 concentration. Increasing the O2 concentration increases the rate of superoxide radical production, but there is no change in salicylate hydroxylation (e.g., OH. production) or ferrous ion concentration. Oxygen-mediated slower rates of lipid peroxidation are associated with either increased H2O2 production or, based on an indirect assay, singlet O2 production. Increased O2 concentrations increase the rate of azobisisobutyronitrile-initiated lipid peroxidation as expected but addition of exogenous superoxide radicals slows the rate. Under similar conditions superoxide reacts with fatty acids to produce singlet O2. Overall, the data suggest that O2-mediated antagonism occurs because of termination reactions between hydroperoxyl (HO2.) and organic radicals, and singlet O2 or H2O2 are products of these reactions.