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.     

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.     

ADP-iron as a Fenton reactant: radical reactions detected by spin trapping, hydrogen abstraction, and aromatic hydroxylation

A mixture of ADP, ferrous ions, and hydrogen peroxide (H2O2) generates hydroxyl radicals (OH) that attack the spin trap DMPO (5,5-dimethyl-pyrollidine-N-oxide) to yield the hydroxyl free radical spin-adduct, degrade deoxyribose and benzoate with the release of thiobarbituric acid-reactive material, and hydroxylate benzoate to give fluorescent products. Inhibition studies, with scavengers of the OH radical, suggest that the behavior of iron-ADP in the reaction is complicated by the formation of ternary complexes with certain scavengers and detector molecules. In addition, iron-ADP reacting with H2O2 appears to release a substantial number of OH radicals free into solution. During the generation of OH radicals the ADP molecule was, as expected, damaged by the iron bound to it. Damage to the iron ligand in this way is not normally monitored in reaction systems that use specific detector molecules for OH radical damage. Under certain reaction conditions the ligand may be the major recipient of OH radical damage thereby leading to the incorrect assumption that the iron ligand is a poor Fenton reactant.     

Copper + zinc and manganese superoxide dismutases inhibit deoxyribose degradation by the superoxide-driven Fenton reaction at two different stages. Implications for the redox states of copper and manganese.

When OH. radicals are formed in a superoxide-driven Fenton reaction, in which O2.- is generated enzymically, deoxyribose degradation is effectively inhibited by CuZn- and Mn-superoxide dismutases. The products of this reaction are H2O2 and a Fe3+-EDTA chelate. The mixing of H2O2 and a Fe3+-EDTA chelate also generates OH. radicals able to degrade deoxyribose with the release of thiobarbituric acid-reactive material. This reaction too is inhibited by CuZn- and Mn-superoxide dismutases, suggesting that most of the OH. is formed by a non-enzymic O2.--dependent reduction of the Fe3+-EDTA chelate. Since the reaction between the Fe3+-EDTA chelate and H2O2 leads to a superoxide dismutase-inhibitable formation of OH. radicals, it could suggest a much wider protective role for the superoxide dismutase enzymes in biological systems. Urate produced during the reaction of xanthine oxidase and hypoxanthine limits deoxyribose degradation as well as the effectiveness of the superoxide dismutase enzymes to inhibit damage to deoxyribose by H2O2 and the Fe3+-EDTA chelate. Some of this damage may result from an O2.--independent pathway to OH. formation in which urate reduces the ferric complex.     

The ability of scavengers to distinguish OH. production in the iron-catalyzed Haber-Weiss reaction: comparison of four assays for OH

Kinetic analysis has been used to access how well scavenger inhibition can characterize the reactivity of oxidants produced in the iron-catalyzed reaction of H2O2 with xanthine oxidase-derived O2-.. Formate oxidation to CO2, deoxyribose oxidation, benzoate hydroxylation, and ethylene production from alpha-keto-gamma-methiolbutyric acid (KMB) were measured. With Fe(EDTA) as catalyst, inhibition by most scavengers was quantitatively as expected for OH. involvement. Exceptions were urate and thiourea, which inhibited excessively and appeared to scavenge intermediates of the detection reactions. With nonchelated iron, there was minimal formate oxidation, but benzoate, KMB, and deoxyribose gave, respectively, 17%, 25%, and approximately the same product yield as with Fe(EDTA). Deoxyribose oxidation was not inhibited by some scavengers and excessively inhibited by others. However, scavengers that did not inhibit deoxyribose oxidation did inhibit with KMB and benzoate, and differences in scavenger effects in the presence and absence of EDTA in these assays were relatively minor. The results with formate and deoxyribose, but not KMB and benzoate, can therefore exclude free OH. as a significant oxidant product of the nonchelated iron-catalyzed Haber-Weiss reaction. It is proposed that the different patterns of scavenger inhibition arise in the different assays because scavengers can react with intermediates in the detection reactions, all of which are multistep chains. Thus, inhibition may not signify OH. involvement, and similarities with inhibition expected for OH. my be fortuitous.     

铜锌超氧物歧化酶与过氧化氢反应中羟自由基的形成

应用脱氧核糖降解法研究了离体条件下Cu,Zn-SOD与H2O2反应产生·OH,并对其机理进行了探讨。H2O2可使Cu,Zn-SOD失活,在失活过程中有·OH产生,甲酸钠和苯甲酸钠均能不同程度地保护Cu,Zn-SOD和降低H2O2与Cu,Zn-SOD反应中·OH的产额;热失活SOD也可和H2O2反应生成·OH,且效能高于活性Cu,Zn-SOD;用螫合剂脱去Cu,Zn-SOD的金属辅基后,脱辅基的SOD蛋白不能和H2O2反应产生·OH;Cu2＋和H2O2反应产生·OH的效率很高,而Zn2＋产生·OH的效率很低。实验结果提示Cu,Zn－SOD与H2O2反应产生的·OH可能是SOD活性中心的Cu2＋与H2O2发生Fenton反应的结果．     

Factors that influence the deoxyribose oxidation assay for Fenton reaction products.

The mechanism of oxidation of deoxyribose to thiobarbituric acid-reactive products by Fenton systems consisting of H2O2 and either Fe2+ or Fe2+ (EDTA) has been studied. With Fe2+ (EDTA), dependences of product yield on reactant concentrations are consistent with a reaction involving OH.. With Fe2+ in 5-50 mM phosphate buffer, yields of oxidation products were much higher and increased with increasing deoxyribose concentration up to 30 mM. The product yield varied with H2O2 and Fe2+ concentrations in a way to suggest competition between deoxyribose and both reactants. Deoxyribose oxidation by Fe2+ and H2O2 was enhanced 1.5-fold by adding superoxide dismutase, even though superoxide generated by xanthine oxidase increased deoxyribose oxidation. These results are not as expected for a reaction involving free OH. or site localized OH. product on the deoxyribose. They can be accommodated by a mechanism of deoxyribose oxidation involving an iron(IV) species formed from H2O2 and Fe2+, but the overall conclusion is that the system is too complex for definitive identification of the Fenton oxidant.     

Cobalt(II) ion as a promoter of hydroxyl radical and possible ‘crypto-hydroxyl’ radical formation under physiological conditions. Differential effects of hydroxyl radical scavengers

Co(II) ions react with hydrogen peroxide under physiological conditions to form a ‘reactive species’ that can hydroxylate aromatic compounds (phenol and salicylate) and degrade deoxyribose to thiobarbituric-acid-reactive material. Catalase decreases the formation of this species but superoxide dismutase or low concentrations of ascorbic acid have little effect. EDTA, present in excess over the Co(II), can accelerate deoxyribose degradation and aromatic hydroxylation. In the presence of EDTA, deoxyribose degradation by the reactive species is inhibited competitively by scavengers of the hydroxyl radical (OH), their effectiveness being related to their second-order rate constants for reaction with OH. In the absence of EDTA the scavengers inhibit only at much higher concentrations and their order of effectiveness is changed. It is suggested that, in the presence of EDTA, hydroxyl radical is formed ‘in free solution’ and attacks deoxyribose or an aromatic molecule. In the absence of EDTA, OH radical is formed in a ‘site-specific’ manner and is difficult to intercept by OH scavengers. The relationship of these results to the proposed ‘crypto OH’ radical is discussed.     

Hydroxyl-radical production in physiological reactions. A novel function of peroxidase.

Peroxidases catalyze the dehydrogenation by hydrogen peroxide (H2O2) of various phenolic and endiolic substrates in a peroxidatic reaction cycle. In addition, these enzymes exhibit an oxidase activity mediating the reduction of O2 to superoxide (O2.-) and H2O2 by substrates such as NADH or dihydroxyfumarate. Here we show that horseradish peroxidase can also catalyze a third type of reaction that results in the production of hydroxyl radicals (.OH) from H2O2 in the presence of O2.-. We provide evidence that to mediate this reaction, the ferric form of horseradish peroxidase must be converted by O2.- into the perferryl form (Compound III), in which the haem iron can assume the ferrous state. It is concluded that the ferric/perferryl peroxidase couple constitutes an effective biochemical catalyst for the production of .OH from O2.- and H2O2 (iron-catalyzed Haber-Weiss reaction). This reaction can be measured either by the hydroxylation of benzoate or the degradation of deoxyribose. O2.- and H2O2 can be produced by the oxidase reaction of horseradish peroxidase in the presence of NADH. The .OH-producing activity of horseradish peroxidase can be inhibited by inactivators of haem iron or by various O2.- and .OH scavengers. On an equimolar Fe basis, horseradish peroxidase is 1-2 orders of magnitude more active than Fe-EDTA, an inorganic catalyst of the Haber-Weiss reaction. Particularly high .OH-producing activity was found in the alkaline horseradish peroxidase isoforms and in a ligninase-type fungal peroxidase, whereas lactoperoxidase and soybean peroxidase were less active, and myeloperoxidase was inactive. Operating in the .OH-producing mode, peroxidases may be responsible for numerous destructive and toxic effects of activated oxygen reported previously.     

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.     

Interaction of ferric complexes with rat liver nuclei to catalyze NADH-and NADPH-Dependent production of oxygen radicals

The production of potent oxygen radicals by microsomal reaction systems has been well characterized. Relatively little attention has been paid to generation of oxygen radicals by liver nuclei, or to the interaction of nuclei with different ferric complexes to catalyze NADH- or NADPH-dependent production of reactive oxygen intermediates. Intact rat liver nuclei were capable of catalyzing an iron-dependent production of .OH as reflected by the oxidation of .OH scavenging agents such as 2-keto-4-thiomethylbutyrate, dimethyl sulfoxide, and t-butyl alcohol. Inhibition of .OH production by catalase implicates H2O2 as the precursor of .OH generated by the nuclei, whereas superoxide dismutase had only a partially inhibitory effect. The production of .OH with either cofactor was striking increased by addition of ferric-EDTA or ferric-diethylenetriamine-pentaacetic acid (DTPA) whereas ferric-ATP and ferric-citrate were not effective catalysts. All these ferric complexes were reduced by the nuclei in the presence of either NADPH or NADH. The pattern of iron chelate effectiveness in catalyzing lipid peroxidation by nuclei was opposite to that of .OH production; with either NADH or NADPH, nuclear lipid peroxidation was increased by the addition of ferric ammonium sulfate, ferric-ATP, or ferric-citrate, but not by ferric-EDTA or ferric-DTPA. NADPH-dependent nuclear lipid peroxidation was insensitive to catalase, superoxide dismutase, or .OH scavengers; the NADH-dependent reaction showed a partial sensitivity (30 to 40%) to these additions. The overall patterns of .OH production and lipid peroxidation by the nuclei are similar to those shown by microsomes, e.g., effect of ferric complexes, sensitivity to antioxidants; however, rates with the nuclei are less than 20% those of microsomes, which reflect the lower activities of NADPH- and NADH-cytochrome c reductase in the nuclei. The potential for nuclei to reduce ferric complexes and catalyze production of .OH-like species may play a role in the susceptibility of the genetic material to oxidative damage under certain conditions since such radicals would be produced site-directed and not exposed to cellular antioxidants.     

In vitro production of free oxygen radicals induced by pulsed ultrasounds in whole blood exposed to diagnostical frequencies and intensities.

DNA alkalinization experiments on lymphocytes from sonicated whole blood and on in vitro cultured lymphocytes in presence of free radical scavengers (superoxide dismutase, catalase and mannitol) showed that lesions inflicted upon DNA by pulsed ultrasounds could be ascribed to production of free radicals (O2-, OH.) and H2O2, which could mediate the production of still unidentified organic radicals, likely to be responsible for DNA damage.     

A marked stimulation of Fe(3+)-dependent lipid peroxidation in phospholipid liposomes under acidic conditions

Lipid peroxidation in phosphatidylcholine liposomes induced by Fe(3+) alone, assessed by thiobarbituric acid-reactive substances (TBARS) production, was markedly enhanced as the solution pH was lowered from 7.4 to 5.5. On the other hand, at physiological pH, TBARS production by Fe(3+) was almost negligible. Results of the radical scavenger experiments with superoxide dismutase, catalase and hydroxyl radical ((&z.rad;)OH) scavengers (sodium benzoate, mannitol and dimethylthiourea), deoxyribose degradation and ESR spectrometry suggest that the stimulation of Fe(3+)-dependent lipid peroxidation under acidic conditions is involved in generation of superoxide anion (O(2)(&z.rad;-)), hydrogen peroxide (H(2)O(2)) and (&z.rad;)OH during the reaction. The stimulation of Fe(3+)-dependent TBARS production by increasing the [H(+)] completely disappeared by triphenylphosphine (TPP) treatment of the liposomes, but the reaction was reversible with either incorporation of cumen hydroperoxide (CumOOH) into the TPP-treated liposomes or the addition of CumOOH to the treated liposomes. Incubation of the CumOOH-incorporated TPP-treated liposomes with Fe(3+) at pH 5.5 also resulted in (&z.rad;)OH generation. Based on these results, a possible mechanism of stimulatory effect of Fe(3+) on lipid peroxidation under acidic conditions is discussed.     

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.     

Cobalt(II) ion as a promoter of hydroxyl radical and possible 'crypto-hydroxyl' radical formation under physiological conditions. Differential effects of hydroxyl radical scavengers

Co(II) ions react with hydrogen peroxide under physiological conditions to form a 'reactive species' that can hydroxylate aromatic compounds (phenol and salicylate) and degrade deoxyribose to thiobarbituric-acid-reactive material. Catalase decreases the formation of this species but superoxide dismutase or low concentrations of ascorbic acid have little effect. EDTA, present in excess over the Co(II), can accelerate deoxyribose degradation and aromatic hydroxylation. In the presence of EDTA, deoxyribose degradation by the reactive species is inhibited competitively by scavengers of the hydroxyl radical (.OH), their effectiveness being related to their second-order rate constants for reaction with .OH. In the absence of EDTA the scavengers inhibit only at much higher concentrations and their order of effectiveness is changed. It is suggested that, in the presence of EDTA, hydroxyl radical is formed 'in free solution' and attacks deoxyribose or an aromatic molecule. In the absence of EDTA, .OH radical is formed in a 'site-specific' manner and is difficult to intercept by .OH scavengers. The relationship of these results to the proposed 'crypto .OH' radical is discussed.     

Free-radical generation by copper ions and hydrogen peroxide. Stimulation by Hepes buffer.

Hydroxyl radicals (OH.), generated by a phosphate-buffered Cu2+/H2O2 system, were detected by lucigenin-amplified chemiluminescence, deoxyribose degradation and benzoate hydroxylation. In each system the buffer, Hepes, was found to stimulate radical generation significantly. There are two main reasons for this effect: Hepes increases Cu2+ solubility in phosphate-buffered systems, and forms a complex with Cu2+ that is effective in generating OH. from H2O2. Pipes, a structurally similar buffer, and histidine, a known Cu2+ chelator, were found to have a similar effect. These data suggest that the crucial factor in such free-radical-generating systems is the availability of Cu2+, and that these actions of Hepes should be considered in the design of studies utilizing such systems.     

Deoxyribose breakdown by the adriamycin semiquinone and H2O2: evidence for hydroxyl radical participation

We report our finding that the reaction between the adriamycin semiquinone (produced by reduction of the drug by xanthine oxidase) and H2O2 in N2 causes deoxyribose degradation to a thiobarbituric acid-reactive chromogen. Deoxyribose breakdown was inhibited by scavengers of hydroxyl radicals, providing evidence for the participation of hydroxyl radicals. The reaction was detected in air, but was less efficient in air than in N2. Deoxyribose degradation did not require a metal catalyst, and was inhibited by superoxide dismutase in air, but not N2. A similar reaction with deoxyribose in DNA may be of major importance in the antitumour action of adriamycin.     

Superoxide dismutase inhibits the superoxide-driven Fenton reaction at two different levels. Implications for a wider protective role

Superoxide dismutase (SOD) completely inhibits the damage caused by a ferric-EDTA chelate in the presence of a superoxide-generating system. In this reaction superoxide is enzymically dismuted to hydrogen peroxide. Since hydrogen peroxide and a ferric-EDTA chelate are themselves a hydroxyl radical-generating system, it follows that SOD must also protect against damage done by this reaction. The ability of SOD to inhibit damage to deoxyribose caused by hydrogen peroxide and a ferric-EDTA chelate is experimentally demonstrated in this paper.     

Hydroxyl radical scavenging and singlet oxygen quenching properties of polyamines

Polyamines (cadaverine, putrescine, spermidine, spermine) have been shown to be present in all prokaryotic and eukaryotic cells, and proposed to be important anti-inflammatory agents. Some polyamines at high concentrations are known to scavenge superoxide radicals in vitro. We have investigated the possible antioxidant properties of polyamines and found that polyamines, e.g., cadaverine, putrescine, spermidine and spermine do not scavenge superoxide radicals at 0.5, 1.0 and 2 mM concentrations. However, polyamines were found to be potent scavengers of hydroxyl radicals. Hydroxyl radicals were produced in a Fenton type reaction and detected as DMPO-OH adducts by electron paramagnetic resonance spectroscopic technique. Spermine, spermidine, putrescine and cadaverine inhibited DMPO-OH adduct formation in a dose dependent manner, and at 1.5 mM concentration virtually eliminated the adduct formation. The *OH-dependent TBA reactive product of deoxyribose was also inhibited by polyamines in a dose-dependent manner. Polyamines were also found to inhibit the 1O2-dependent 2,2,6,6-tetramethylpiperidine N-oxy 1 (TEMPO) formation. 1O2 was produced in a photosensitizing system using Rose Bengal or Methylene Blue as photosensitizers, and was detected as TEMP-1O2 adduct by EPR spectroscopy. Spermine or spermidine inhibited the 1O2-dependent TEMPO formation maximally to 50%, whereas putrescine or cadaverine inhibited this reaction only up to 15%, when used at 0.5 and 1 mM concentrations. These results suggest that polyamines are powerful. OH scavengers, and spermine or spermidine also can quench singlet oxygen at higher concentrations.     

Binding of iron(II) ions to the pentose sugar 2-deoxyribose

Iron(II) ions are able to form a weak complex (apparent equilibrium constant about 10(2) at pH 7.4 and 25 degrees C) with 2-deoxyribose over a range of pH values, including pH 7. Evidence for this complex formation has been obtained by spectrophotometric experiments and by studies of Fe(II) oxidation. Iron(II) ions bound to deoxyribose seem to react with H2O2, in a site-specific reaction, to form hydroxyl radicals (.OH) that immediately damage the deoxyribose molecule.