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.     

Reactivity of hydroxyl and hydroxyl-like radicals discriminated by release of thiobarbituric acid-reactive material from deoxy sugars, nucleosides and benzoate.

Hydroxyl radicals (OH.) can be formed in aqueous solution by a superoxide (O2.-)-generating system in the presence of a ferric salt or in a reaction independent of O2.- by the direct addition of a ferrous salt. OH. damage was detected in the present work by the release of thiobarbituric acid-reactive material from deoxy sugars, nucleosides and benzoate. The carbohydrates deoxyribose, deoxygalactose and deoxyglucose were substantially degraded by the iron(II) salt and the iron(III) salt in the presence of an O2.- -generating system, whereas deoxyinosine, deoxyadenosine and benzoate were not. Addition of EDTA to the reaction systems producing radicals greatly enhanced damage to deoxyribose, deoxyinosine, deoxyadenosine and benzoate, but decreased damage to deoxygalactose and deoxyglucose. Further, OH. scavengers were effective inhibitors only when EDTA was present. Inhibition by catalase and desferrioxamine confirmed that H2O2 and iron salts were essential for these reactions. The results suggest that, in the absence of EDTA, iron ions bind to the carbohydrate detector molecules and bring about a site-specific reaction on the molecule. This reaction is poorly inhibited by most OH. scavengers, but is strongly inhibited by scavengers such as mannitol, glucose and thiourea, which can themselves bind iron ions, albeit weakly. In the presence of EDTA, however, iron is removed from these binding sites to produce OH. in 'free' solution. These can be readily intercepted by the addition of OH. scavengers.     

Nucleosomal histone protein protects DNA from iron-mediated damage.

Iron promotes DNA damage by catalyzing hydroxyl radical formation. We examined the effect of chromatin structure on DNA susceptibility to oxidant damage. Oxygen radicals generated by H2O2, ascorbate and iron-ADP (1:2 ratio of Fe2+:ADP) extensively and randomly fragmented protein-free DNA, with double-strand breaks demonstrable even at 1 microM iron. In contrast, polynucleosomes from chicken erythrocytes were converted to nucleosome-sized fragments by iron-ADP even up to 250 microM iron. Cleavage occurred only in bare areas where DNA is unassociated with histone. In confirmation, reassembly of nucleosomes from calf thymus DNA and chicken erythrocyte histone also yielded nucleosomes resistant to fragmentation. Protection of DNA by histone was dependent on nucleosome assembly and did not simply reflect presence of scavenging protein. In contrast to this specific cleavage of internucleosomal linker DNA by iron-ADP, iron-EDTA cleaved polynucleosomes indiscriminately at all sites. The hydroxyl radical scavenger thiourea completely inhibited the random cleavage of polynucleosomes by iron-EDTA but inhibited the nonrandom cleavage of polynucleosomes by iron-ADP less completely, suggesting the possibility that the lower affinity iron-ADP chelate may allow association of free iron with DNA. Thus, oxygen radicals generated by iron-ADP indiscriminately cleaved naked DNA but cleaved chromatin preferentially at internucleosomal bare linker sites, perhaps because of nonrandom iron binding by DNA. These findings suggest that the DNA-damaging effects of iron may be nonrandom, site-directed and modified by histone protein.     

Acyl-CoA-binding protein from cow. Binding characteristics and cellular and tissue distribution.

Hydroxyl radicals (OH.) in free solution react with scavengers at rates predictable from their known second-order rate constants. However, when OH. radicals are produced in biological systems by metal-ion-dependent Fenton-type reactions scavengers do not always appear to conform to these established rate constants. The detector molecules deoxyribose and benzoate were used to study damage by OH. involving a hydrogen-abstraction reaction and an aromatic hydroxylation. In the presence of EDTA the rate constant for the reaction of scavengers with OH. was generally higher than in the absence of EDTA. This radiomimetic effect of EDTA can be explained by the removal of iron from the detector molecule, where it brings about a site-specific reaction, by EDTA allowing more OH. radicals to escape into free solution to react with added scavengers. The deoxyribose assay, although chemically complex, in the presence of EDTA appears to give a simple and cheap method of obtaining rate constants for OH. reactions that compare well with those obtained by using pulse radiolysis.     

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.     

Hydrogen peroxide kills Staphylococcus aureus by reacting with staphylococcal iron to form hydroxyl radical

Two lines of investigation supported the premise that killing of Staphylococcus aureus, 502A, by hydrogen peroxide involves formation of the more toxic hydroxyl radical (.OH) through the iron-dependent Fenton reaction. First, growing S. aureus overnight in broth media with increasing concentrations of iron increased their content of iron and dramatically enhanced their subsequent susceptibility to killing by H2O2. Second, in direct relation to their effectiveness as .OH scavengers, thiourea, dimethyl thiourea, sodium benzoate, and dimethyl sulfoxide inhibited H2O2-mediated killing of S. aureus.     

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.     

Free radicals in iron-containing systems

All oxidative damage in biological systems arises ultimately from molecular oxygen. Molecular oxygen can scavenge carbon-centered free radicals to form organic peroxyl radicals and hence organic hydroperoxides. Molecular oxygen can also be reduced in two one-electron steps to hydrogen peroxide in which case superoxide anion is an intermediate; or it can be reduced enzymatically so that no superoxide is released. Organic hydroperoxides or hydrogen peroxide can diffuse through membranes whereas hydroxyl radicals or superoxide anion cannot. Chain reactions, initiated by chelated iron and peroxides, can cause tremendous damage. Chain carriers are chelated ferrous ion; hydroxyl radical .OH, or alkoxyl radical .OR, and superoxide anion O2-. or organic peroxyl radical RO2.. Of these free radicals .OH and RO2. appear to be most harmful. All of the biological molecules containing iron are potential donors of iron as a chain initiator and propagator. An attacking role for superoxide dismutase is proposed in the phagocytic process in which it may serve as an intermediate enzyme between NADPH oxidase and myeloperoxidase. The sequence of reactants is O2----O2-.----H2O2----HOCl.     

Hydroxyl radical formation from the auto-reduction of a ferric citrate complex.

When a ferric citrate complex is prepared from citric acid and ferric chloride, and the pH value left unchanged, a reduction of the iron moiety takes place. Within several hours a substantial yield of ferrous ions can be detected in the solution. When placed in a phosphate buffer pH 7.0 with a suitable detector molecule, oxidative damage to the detector molecule can be observed. Thus, deoxyribose is degraded with the release of thiobarbituric acid-reactive material and benzoate is hydroxylated to form fluorescent dihydroxy products. Damage can be prevented by scavengers of the hydroxyl radical such as mannitol, formate the thiourea, by catalase and by the protein caeruloplasmin, suggesting that Fenton chemistry occurs leading to the formation of hydroxyl radicals.     

Fragmentation of human ceruloplasmin induced by hydrogen peroxide

We investigated the fragmentation of human ceruloplasmin induced by H2O2 to study its oxidative damage. When ceruloplasmin was incubated with H2O2, the frequency of the protein fragmentation increased in a proportion to the concentration of H2O2. It also increased in a time-dependent manner and was accompanied by gradual loss of the oxidase activity. Hydroxyl radical scavengers such as azide and mannitol inhibited the fragmentation of ceruloplasmin. The deoxyribose assay showed that hydroxyl radicals were generated in the reaction of ceruloplasmin with H2O2. Incubation of ceruloplasmin with H2O2 resulted in a time-dependent release of copper ions. The released copper ion may participate in a Fenton-like reaction to produce hydroxyl radical, which enhanced the fragmentation. The protection of the fragmentation by copper chelators such as diethylenetriaminepentaacetic acid and bathocuproine indicates a role for copper ion in the reaction. These results suggest that the fragmentation of ceruloplasmin induced by H2O2 is due to hydroxyl radicals formed by a copper-dependent Fenton-like reaction.     

Sensitive assay of hydroxyl free radical formation utilizing high pressure liquid chromatography with electrochemical detection of phenol and salicylate hydroxylation products

Evidence is presented for a sensitive method useful for the detection of hydroxyl free radical generation in various systems. The methodology employs high pressure liquid chromatography with electrochemical detection (LCED) for the quantification and identification of the hydroxylation products from the reaction of OH with both phenol and salicylate. A detection limit of less than 1 pmol for the hydroxylation products has been achieved with electrochemical detector responses linear over at least three orders of magnitude. Detection and quantitation of the hydroxylation products obtained and formed during OH generation from biologically meaningful systems have been demonstrated. The three systems utilized were ADP/FE(II)/H2O/, hypoxanthine/xanthine oxidase plus chelated iron, and UV photolysis of H2O2.     

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.     

Ferric iron and superoxide ions are required for the killing of cultured hepatocytes by hydrogen peroxide. Evidence for the participation of hydroxyl radicals formed by an iron-catalyzed Haber-Weiss reaction

Cultured hepatocytes pretreated with the ferric iron chelator deferoxamine were resistant to the toxicity of H2O2 generated by either glucose oxidase or by the metabolism of menadione (2-methyl-1,4-naphthoquinone). Ferric, ferrous, or cupric ions restored the sensitivity of the cells to H2O2. Deferoxamine added to hepatocytes previously treated with this chelator prevented the restoration of cell killing by only ferric iron. The free radical scavengers mannitol, thiourea, benzoate, and 4-methylmercapto-2-oxobutyrate protected either native cells exposed to H2O2 or pretreated hepatocytes exposed to H2O2 and given ferric or ferrous iron. Superoxide dismutase prevented the killing of native hepatocytes by either glucose oxidase or menadione. With deferoxamine-pretreated hepatocytes, superoxide dismutase prevented the cell killing dependent upon the addition of ferric but not ferrous iron. Catalase prevented the killing by menadione of deferoxamine-pretreated hepatocytes given either ferric or ferrous iron. Deferoxamine pretreatment did not prevent the toxicity of t-butyl hydroperoxide but did, however, prevent that of cumene hydroperoxide. It is concluded that both ferric iron and superoxide ions are required for the killing of cultured hepatocytes by H2O2. The toxicity of H2O2 is also dependent upon its reaction with ferrous iron to form hydroxyl radicals by the Fenton reaction. The ferrous iron needed for this reaction is formed by the reduction of cellular ferric iron by superoxide ions. Such a sequence corresponds to the so-called iron-catalyzed Haber-Weiss reaction, and the present report documents its participation in the killing of intact hepatocytes by H2O2. Cumene hydroperoxide but not t-butyl hydroperoxide closely models the toxicity of hydrogen peroxide.     

Evidence that hydroxyl radicals mediate auxin-induced extension growth

Reactive oxygen intermediates, i.e. the superoxide radical (O*-)(2), hydrogen peroxide (H2O2) and the hydroxyl radical (*OH), are generally regarded as harmful products of oxygenic metabolism causing cell damage in plants, animals and microorganisms. However, oxygen radical chemistry may also play a useful role in polymer breakdown leading to wall loosening during extension growth of plant cells controlled by the phytohormone auxin. Backbone cleavage of cell wall polysaccharides can be accomplished in vitro by (*OH) produced from H2O2 in a Fenton reaction or in a reaction catalyzed by peroxidase supplied with O2 and NADH. Here, we show that coleoptile growth of maize seedlings is accompanied by the release of reactive oxygen intermediates in the cell wall. Auxin promotes release of (O*-)(2) and subsequent generation of (*OH)when inducing elongation growth. Experimental generation of (*OH) in the wall causes an increase in wall extensibility in vitro and replaces auxin in inducing growth. Auxin-induced growth can be inhibited by scavengers of (O*-)(2), H2O2 or (*OH), or inhibitors interfering with the formation of these molecules in the cell wall. These results provide the experimental background for a novel hypothesis on the mechanism of plant cell growth in which (*OH), produced from (O*-)(2) and H2O2 by cell wall peroxidase, acts as a wall-loosening agent.     

Formation of hydroxyl radicals from hydrogen peroxide in the presence of iron. Is haemoglobin a biological Fenton reagent?

The ability of oxyhaemoglobin and methaemoglobin to generate hydroxyl radicals (OH.) from H2O2 has been investigated using deoxyribose and phenylalanine as 'detector molecules' for OH.. An excess of H2O2 degrades methaemoglobin, releasing iron ions that react with H2O2 to form a species that appears to be OH.. Oxyhaemoglobin reacts with low concentrations of H2O2 to form a 'reactive species' that degrades deoxyribose but does not hydroxylate phenylalanine. This 'reactive species' is less amenable to scavenging by certain scavengers (salicylate, phenylalanine, arginine) than is OH., but it appears more reactive than OH. is to others (Hepes, urea). The ability of haemoglobin to generate not only this 'reactive species', but also OH. in the presence of H2O2 may account for the damaging effects of free haemoglobin in the brain, the eye, and at sites of inflammation.     

Binding of the intramitochondrial ADP and its relationship to adenine nucleotide translocation

Adriamycin under partially anaerobic conditions degrades deoxyribose with the release of thiobarbituric acid-reactive products. This reaction is seen when electrons are transferred to adriamycin by xanthine oxidase or ferredoxin reductase to form the semiquinone free radical. Under the conditions described, damage to deoxyribose was inhibited by hydroxyl radicals scavengers, catalase and iron chelators. When the ratio of iron chelator to iron salt is varied both EDTA and diethylenetriamino penta-acetic acid (DETAPAC) show stimulatory properties whereas desferrioxamine remains a potent inhibitor of all reaction.     

Manganese complexes and the generation and scavenging of hydroxyl free radicals

In a wide variety of biological systems non-enzyme complexes of the metals copper (Cu) and iron (Fe) have been shown to enhance oxygen radical damage by increasing the production of an oxidative species generally believed to be the hydroxyl free radical (.OH) via "Fenton" and possibly "Haber-Weiss" type reactions. However, the behavior of the chemically and biologically similar transition metal manganese (Mn) with .OH is unknown. Unlike Fe and Cu, inorganic complexes of Mn are known to exist in high concentrations in certain cells. Three different oxygen free radical generating systems and four .OH detection methods were used to investigate the activity of biologically relevant inorganic Mn complexes. These complexes were compared to compounds reported to scavenge and generate .OH. The direct and indirect effects of Mn on the .OH flux were compared by attempting to distinguish the effects of hydrogen peroxide (H2O2), superoxide (O2-), and .OH through the use of selective scavengers and generators. Mn-EDTA and biologically relevant Mn-pyrophosphates and polyphosphates, in contrast to Fe-EDTA, do not generate .OH in these systems. The results suggest that Mn in various forms does, indeed, inhibit oxy-radical damage mediated by .OH, but only if the .OH production is dependent on the presence of O2- or H2O2. Thus, with .OH, as with O2- and H2O2, Mn complexes appear to behave in a fundamentally different fashion from Cu and Fe.     

Iron and xanthine oxidase catalyze formation of an oxidant species distinguishable from OH.: comparison with the Haber-Weiss reaction

O2- was produced by gamma irradiation of formate solutions, by the action of xanthine oxidase on hypoxanthine and O2, and by the action of ferredoxin reductase on NADPH and paraquat in the presence of O2. Its reaction with H2O2 and various iron chelates was studied. Oxidation of deoxyribose to thiobarbituric acid-reactive products that was appropriately inhibited by OH. scavengers, or formate oxidation to CO2, was used to detect OH(.). With each source of O2-, and by these criteria, Fe(EDTA) efficiently catalyzed this (Haber-Weiss) reaction, but little catalysis was detectable with iron bound to DTPA, citrate, ADP, ATP, or pyrophosphate, or without chelator in phosphate buffer. O2- produced from xanthine oxidase, but not from the other sources, underwent another iron-dependent reaction with H2O2, to produce an oxidant that did not behave as free OH(.). It was formed in phosphate or bicarbonate buffer, and caused deoxyribose oxidation that was readily inhibited by mannitol or Tris, but not by benzoate, formate, or dimethyl sulfoxide. It did not oxidize formate to CO2. Addition of EDTA changed the pattern of inhibition to that expected for a reaction of OH(.). The other chelators all inhibited deoxyribose oxidation, provided their concentrations were high enough. The results are compatible with iron bound to xanthine oxidase catalyzing production of a strong oxidant (which is not free OH.) from H2O2 and O2- produced by the enzyme.     

Copper salt-dependent hydroxyl radical formation. Damage to proteins acting as antioxidants

Cupric ions (Cu2+) and ferric ions (Fe3+) added to hydrogen peroxide generate hydroxyl radicals (OH) capable of degrading deoxyribose with the formation of thiobarbituric acid-reactive products. This damage can be inhibited by catalase, OH radical scavengers and specific metal ion chelators. All proteins tested nonspecifically inhibited copper-dependent damage but have little effect on the iron-dependent reaction. Copper ions appear to bind to the proteins which prevents formation of OH radicals in free solution. However, OH radicals are still generated at a site-specific location on the protein molecule. Protein damage is detected as fluorescent changes in amino acid residues.     

渗透胁迫下稻苗中铁催化的膜脂过氧化作用

在－０．７ＭＰａ渗透胁迫下,水稻幼苗体内和Ｈ２Ｏ２大量产生,Ｆｅ２＋积累,膜脂过氧化作用加剧。水稻幼苗体内Ｆｅ２＋含量与膜脂过氧化产物ＭＤＡ含量呈极显著的正相关。外源Ｆｅ２＋、Ｆｅ３＋、Ｈ２Ｏ２、Ｆｅ２＋＋Ｈ２Ｏ２、ＤＤＴＣ均能刺激膜脂过氧化作用,而铁离子的螯合剂ＤＴＰＡ则有缓解作用。ＯＨ的清除剂苯甲酸钠和甘露醇能明显地抑制渗透胁迫下Ｆｅ２＋催化的膜脂过氧化作用。这都表明渗透胁迫下水稻幼苗体内铁诱导的膜脂过氧化作用主要是由于其催化Ｆｅｎｔｏｎ型Ｈａｂｅｒ－Ｗｅｉｓｓ反应形成ＯＨ所致。