Characterization of free radical generation by xanthine oxidase. Evidence for hydroxyl radical generation

Xanthine oxidase has been hypothesized to be an important source of biological free radical generation. The enzyme generates the superoxide radical, .O2- and has been widely applied as a .O2- generating system; however, the enzyme may also generate other forms of reduced oxygen. We have applied electron paramagnetic resonance (EPR) spectroscopy using the spin trap 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO) to characterize the different radical species generated by xanthine oxidase along with the mechanisms of their generation. Upon reaction of xanthine with xanthine oxidase equilibrated with air, both DMPO-OOH and DMPO-OH radicals are observed. In the presence of ethanol or dimethyl sulfoxide, alpha-hydroxyethyl or methyl radicals are generated, respectively, indicating that significant DMPO-OH generation occurred directly from OH rather than simply from the breakdown of DMPO-OOH. Superoxide dismutase totally scavenged the DMPO-OOH signal but not the DMPO-OH signal suggesting that .O2- was not required for .OH generation. Catalase markedly decreased the DMPO-OH signal, while superoxide dismutase + catalase totally scavenged all radical generation. Thus, xanthine oxidase generates .OH via the reduction of O2 to H2O2, which in turn is reduced to .OH. In anaerobic preparations, the enzyme reduces H2O2 to .OH as evidenced by the appearance of a pure DMPO-OH signal. The presence of the flavin in the enzyme is required for both .O2- and .OH generation confirming that the flavin is the site of O2 reduction. The ratio of .O2- and .OH generation was affected by the relative concentrations of dissolved O2 and H2O2. Thus, xanthine oxidase can generate the highly reactive .OH radical as well as the less reactive .O2- radical. The direct production of .OH by xanthine oxidase in cells and tissues containing this enzyme could explain the presence of oxidative cellular damage which is not prevented by superoxide dismutase.     

DNA damage by superoxide-generating systems in relation to the mechanism of action of the anti-tumour antibiotic adriamycin

1. A mixture of NADPH and ferrodoxin reductase is a convenient way of reducing adriamycin in vitro. Under aerobic conditions the adriamycin semiquinone reacts rapidly with O₂ and superoxide radical is produced. 2. Superoxide generated either by adriamycin:ferredoxin reductase or by hypoxanthine: xanthine oxidase can promote the formation of hydroxyl radicals in the presence of soluble iron chelates. 3. Hydroxyl radicals produced by a hypoxanthine:xanthine oxidase system in the presence of an iron chelate cause extensive fragmentation in double-stranded DNA. Protection is offered by catalase, superoxide dismutase or desferrioxamine. 4. Addition of double-stranded DNA to a mixture of adriamycin, ferredoxin reductase, NADPH and iron chelate inhibits formation of both superoxide and hydroxyl radicals. This is not due to direct inhibition of ferredoxin reductase and single-stranded DNA has a much weaker inhibitory effect. It is concluded that adriamycin intercalated into DNA cannot be reduced.     

Purification and immunochemical studies of pyruvate dehydrogenase complex from rat heart, and cell-free synthesis of lipoamide dehydrogenase, a component of the complex

A mixture of NADPH and ferredoxin reductase is a convenient way of reducing adriamycin in vitro. Under aerobic conditions the adriamycin semiquinone reacts rapidly with O2 and superoxide radical is produced. Superoxide generated either by adriamycin:ferredoxin reductase or by hypoxanthine:xanthine oxidase can promote the formation of hydroxyl radicals in the presence of soluble iron chelates. Hydroxyl radicals produced by a hypoxanthine:xanthine oxidase system in the presence of an iron chelate cause extensive fragmentation in double-stranded DNA. Protection is offered by catalase, superoxide dismutase or desferrioxamine. Addition of double-stranded DNA to a mixture of adriamycin, ferredoxin reductase, NADPH and iron chelate inhibits formation of both superoxide and hydroxyl radicals. This is not due to direct inhibition of ferredoxin reductase and single-stranded DNA has a much weaker inhibitory effect. It is concluded that adriamycin intercalated into DNA cannot be reduced.     

Steady-state kinetics of autoxidation of NAD(P)H initiated by hydroperoxyl radical, the acid form of superoxide anion radical.

Rates of autoxidation of NAD(P)H initiated by hydroperoxyl radical, the acid form of superoxide anion radical which was generated by xanthine/xanthine oxidase, followed a typical autoxidation kinetic equation. Second-order rate constants for the reactions of NADPH and NADH with hydroperoxyl radical were found to be 9.82 +/- 0.13 x 10(4) M-1s-1 and 9.26 +/- 0.58 x 10(4) M-1s-1 at 25 degrees C, respectively. Rates of the reactions between NAD(P)H and superoxide to give degraded products other than NAD(P)+ were also investigated.     

An electron spin resonance study of the reduction of peroxides by anthracycline semiquinones

Direct and spin-trapping electron spin resonance methods have been used to study the reactivity of semiquinone radicals from the anthracycline antibiotics daunorubicin and adriamycin towards peroxides (hydrogen peroxide, t-butyl hydroperoxide and cumene hydroperoxide). Semiquinone radicals were generated by one-electron reduction of anthracyclines, using xanthine/xanthine oxidase. It is shown that the semiquinones are effective reducing agents for all the peroxides. From spin-trapping experiments it is inferred that the radical product is either OH (from H2O2) or an alkoxyl radical (from the hydroperoxides) which undergoes beta-scission to give the methyl radical. The rate constant for reaction of semiquinone with H2O2 is estimated to be approx. 10(4)-10(5) M-1 X s-1. The reduction does not appear to require catalysis by metal ions.     

On the nature of biochemically generated hydroxyl radicals. Studies using the bleaching of p-nitrosodimethylaniline as a direct assay method.

An efficient scavenger for radiolytically generated hydroxyl (OH) radicals, p-nitrosodimethylaniline, was used to try to substantiate the presence of this oxygen radical species in several biochemical systems. Most of these systems which were investigated had previously been assumed to generate OH radicals, e.g. the autoxidation of 6-hydroxydopamine, the hydroxylating system NADH/phenazine methosulfate, and the oxidation of xanthine or acetaldehyde by xanthine oxidase. We did not observe inhibition of the bleaching of p-nitrosodimethylaniline in oxygenated solutions by other scavengers of OH radicals nor, in the case of xanthine/xanthine oxidase, by catalase and superoxide dismutase. We therefore conclude that, under biochemical conditions as opposed to radiolysis or photolysis, no freely diffusable OH radicals are formed. Rather, a strongly oxidizing OH-analogous complex is considered to represent the p-nitrosodimethylaniline-detectable species formed under these conditions.     

Thioureas react with superoxide radicals to yield a sulfhydryl compound. Explanation for protective effect against paraquat

Thiourea and superoxide dismutase were effective antidotes to paraquat toxicity in an HL60 cell culture system, whereas other hydroxyl scavengers were ineffective. The efficacy of thioureas was not due to blockage of intracellular paraquat uptake, inhibition of NADPH-P-450 reductase, or reaction with the paraquat radical. Thiourea also competitively inhibited the reduction of cytochrome c by the xanthine/xanthine oxidase superoxide-generating system, and the release of iron from ferritin by superoxide radicals. The reaction of superoxide with thiourea produced a sulfhydryl compound distinct from products formed by hydrogen peroxide or hydroxyl radicals. Spectrophotometric and chromatographic studies indicated the carbon-sulfide double bond was converted to a sulfhydryl group which reacted with Ellman's reagent. Additional confirmatory evidence for the sulfhydryl compound was obtained with carbon-13 NMR and mass spectroscopies. Thus, thioureas are direct scavengers of superoxide radicals as well as hydroxyl radicals and hydrogen peroxide. The rate constant for the reduction of thiourea by superoxide was estimated at 1.1 x 10(3) M-1 s-1. The implication of this finding on free radical studies, the mechanism of paraquat toxicity, and the metabolism of thioureas is discussed.     

Changes in superoxide dismutase activity in the presence of electron donors and acceptors

The activity of superoxide dismutase (SOD) from bovine erythrocytes was measured by the inhibition of nitrotetrazolium blue reduction rate in superoxide anion radical generation systems--xanthine/xanthine oxidase of NADH/phenazine methasulfate. The enzyme activity increases in the presence of compounds acting as electron donors in radical-involving reactions and decreased in the presence of compounds possessing the properties of electron acceptors. Activation of SOD by electron donors and its inhibition by electron acceptors was dependent on the concentration of the above compounds. In the absence of SOD electron donors and acceptors did not change the rate of tetrazolium blue reduction by superoxide anion radicals. The role of the new type of SOD regulation for the enzyme functioning in the cell is discussed.     

Radical-driven Fenton reactions: studies with paraquat, adriamycin, and anthraquinone 6-sulfonate and citrate, ATP, ADP, and pyrophosphate iron chelates

Using paraquat, adriamycin, and anthraquinone 6-sulfonate, we have investigated the ability of radical-driven Fenton reactions to oxidize formate or deoxyribose when catalyzed by iron complexed with citrate, ADP, ATP, or pyrophosphate. Radicals were generated either radiolytically or enzymatically with xanthine oxidase or ferredoxin reductase. With each radical source, the citrate, ADP, and ATP complexes were at least 50% as active as Fe(EDTA) at catalyzing deoxyribose oxidation, and slightly less active as catalysts of CO2 formation from formate. Fe(pyrophosphate) was less efficient and in some cases inactive. Although it is not possible to definitively identify the oxidant involved, it behaved more like the hydroxyl radical than the proposed ferryl or peroxoferrous species formed in equivalent reactions catalyzed by nonchelated iron, which can oxidize deoxyribose but not formate. Chelator concentrations of 1-2 mM were required for maximum effect, which implies that the major effect of the chelators is on the reactivity of Fe2+ in the Fenton reaction with H2O2. This also suggests that any iron available physiologically could participate in the Fenton reaction in a nonchelated form, and produce a ferryl species rather than the hydroxyl radical. Reactions of the organic radicals contrast with the equivalent reactions of superoxide (Haber-Weiss reaction) for which the same iron chelates are all very inefficient catalysts. Fenton reactions driven by organic reducing radicals may therefore contribute more to the toxicity of redox cycling compounds than equivalent reactions of superoxide.     

Catalysis of the Haber-Weiss reaction by iron-diethylenetriaminepentaacetate.

To help settle controversy as to whether the chelating agent diethylenetriaminepentaacetate (DTPA) supports or prevents hydroxyl radical production by superoxide/hydrogen peroxide systems, we have reinvestigated the question by spectroscopic, kinetic, and thermodynamic analyses. Potassium superoxide in DMSO was found to reduce Fe(III)DTPA. The rate constant for autoxidation of Fe(II)DTPA was found (by electron paramagnetic resonance spectroscopy) to be 3.10 M-1 s-1, which leads to a predicted rate constant for reduction of Fe(III)DTPA by superoxide of 5.9 x 10(3) M-1 s-1 in aqueous solution. This reduction is a necessary requirement for catalytic production of hydroxyl radicals via the Fenton reaction and is confirmed by spin-trapping experiments using DMPO. In the presence of Fe(III)DTPA, the xanthine/xanthine oxidase system generates hydroxyl radicals. The reaction is inhibited by both superoxide dismutase and catalase (indicating that both superoxide and hydrogen peroxide are required for generation of HO.). The generation of hydroxyl radicals (rather than oxidation side-products of DMPO and DMPO adducts) is attested to by the trapping of alpha-hydroxethyl radicals in the presence of 9% ethanol. Generation of HO. upon reaction of H2O2 with Fe(II)DTPA (the Fenton reaction) can be inhibited by catalase, but not superoxide dismutase. The data strongly indicate that iron-DTPA can catalyze the Haber-Weiss reaction.     

Methylene blue competes with paraquat for reduction by flavo-enzymes resulting in decreased superoxide production in the presence of heme proteins

Methylene blue competes 100 to 600 times more effectively than paraquat for reduction by three different flavo-containing enzymes; xanthine oxidase, NADH cytochrome c reductase, and NADPH cytochrome c reductase. Paraquat and methylene blue both interact with deflavo xanthine oxidase, indicating that neither electron acceptor reacted at the FAD site of the enzyme where molecular oxygen is reduced to superoxide. As the paraquat radical also directly reduced acetylated cytochrome c the hemeprotein could not be utilized for measuring superoxide production in the presence of the herbicide. In the presence of cytochrome c the methylene blue caused a sharp decrease in both paraquat-induced superoxide and hydroxyl radical production.     

The reactivity of chicken liver xanthine dehydrogenase with molecular oxygen

The reaction of 6-electron reduced chicken liver xanthine dehydrogenase (XDH) with molecular oxygen was studied using both stopped flow and steady-state turnover techniques at pH 7.8, 4 degrees C. Oxidation of fully reduced XDH proceeded via four phases, three of which were detected with the stopped flow spectrophotometer. The fastest phase was second order in oxygen (1900 M-1 s-1), resulted in the appearance of flavin semiquinone and yielded no superoxide. The next phase was also second order in oxygen (260 M-1 s-1), involved the loss of flavin semiquinone and yielded, on average, 1 mol of superoxide/mol of XDH oxidized. The last 2 electron equivalents were located in the iron-sulfur centers. They were released one equivalent at a time in the form of superoxide. Steady-state kinetics were found to be critically dependent on temperature and oxygen concentration. When these factors were carefully controlled, both the xanthine-oxygen and NADH-oxygen reductase reactions gave linear Lineweaver-Burk plots. The xanthine-oxygen data yielded a turnover number of 43 min-1, which was 42% of that for xanthine-NAD turnover. During turnover, with xanthine and O2, 40-44% of the electron equivalents introduced by xanthine appeared as superoxide. Reduced pyridine nucleotides, NAD and 3-aminopyridine adenine dinucleotide, dramatically reduced the formation of superoxide at levels which did not seriously inhibit oxygen reactivity.     

Possible role for metallothionein in protection against radiation-induced oxidative stress. Kinetics and mechanism of its reaction with superoxide and hydroxyl radicals

Rabbit liver metallothionein-1 (Mr 6500), which contains zinc and/or cadmium ions, appears to scavenge free hydroxyl (.OH) and superoxide (O-.2) radicals produced by the xanthine/xanthine oxidase reaction much more effectively than bovine serum albumin (Mr 65 000) which was used as a control. Kinetic competition studies between metallothionein and either a spin trap for .OH or ferricytochrome c for O-.2 radicals, gave bimolecular rate constants of the order of kOH/MT approximately equal to 10(12) M-1 X s-1 and kO-2/MT approximately equal to 5 X 10(5) M-1 X s-1, respectively. The former value suggests that all 20 cysteine sulfur atoms are involved in this quenching process and that they all act in the diffusion control limit. The aerobic radiolysis of an aqueous solution of metallothionein, generating O-.2 and .OH radicals, induced metal ion loss and thiolate oxidation. These effects could be reversed by incubation of the irradiated protein with reduced glutathione and the appropriate bivalent metal ion. Metallothionein appears to be an extraordinarily efficient .OH radical scavenger even when compared to proteins 10-50-times its molecular weight. Moreover, hydroxyl radical damage to metallothionein appears to occur at the metal-thiolate clusters, which may be repaired in the cell by reduced glutathione. Metallothionein has the characteristics of a sacrificial but renewable cellular target for .OH-mediated cellular damage.     

Inhibition of xanthine oxidase by uric acid and its influence on superoxide radical production.

The inhibition of xanthine oxidase by its reaction product, uric acid, was studied by steady state kinetic analysis. Uric acid behaved as an uncompetitive inhibitor of xanthine oxidase with respect to the reducing substrate, xanthine. Under 50 microM xanthine and 210 microM oxygen, the apparent K(i) for uric acid was 70 microM. Uric acid-mediated xanthine oxidase inhibition also caused an increase in the percentage of univalent reoxidation of the enzyme (superoxide radical production). Steady-state rate equations derived by the King-Altman method support the formation of an abortive-inhibitory enzyme-uric acid complex (dead-end product inhibition). Alternatively, inhibition could also depend on the reversibility of the classical ping-pong mechanism present in xanthine oxidase-catalyzed reactions.     

NADH Induces the Generation of Superoxide Radicals in Leaf Peroxisomes

In peroxisomes isolated from pea leaves (Pisum sativum L.) the production of superoxide free radicals (O₂⁻) by xanthine and NADH was investigated. In peroxisomal membranes, 100 micromolar NADH induced the production of O₂⁻ radicals. In the soluble fractions of peroxisomes, no generation of O₂⁻ radicals was observed by incubation with either NADH or xanthine, although xanthine oxidase was found located predominantly in the matrix of peroxisomes. The failure of xanthine to induce superoxide generation was probably due to the inability to fully suppress the endogenous Mn-superoxide dismutase activity by inhibitors which were inactive against xanthine oxidase. The generation of superoxide radicals in leaf peroxisomes together with the recently described production of these oxygen radicals in glyoxysomes (LM Sandalio, VM Fernández, FL Rupérez, LA del Río [1988] Plant Physiol 87: 1-4) suggests that O₂⁻ generation could be a common metabolic property of peroxisomes and further supports the existence of active oxygen-related rôles for peroxisomes in cellular metabolism.     

Effect of phenobarbital and 3-methylcholanthrene treatment on NADPH- and NADH-dependent production of reactive oxygen intermediates by rat liver nuclei.

The effect of inducing the rat liver nuclear mixed-function oxidase system by phenobarbital or 3-methylcholanthrene on NADPH- and NADH-dependent production of reactive oxygen intermediates was evaluated. The inducing agents produced a 2-fold increase in cytochrome P-450, a 50 to 70% increase in NADPH-cytochrome c reductase activity, and a 20 to 30% increase in NADH-cytochrome c reductase activity. Associated with these increases was a corresponding increase in NADPH- and NADH-dependent production of hydroxyl radical (.OH)-like species and of H2O2. Rates of .OH production were inhibited by catalase and partially sensitive to superoxide dismutase. The increase in nuclear production of .OH-like species after drug treatment appears to be due a corresponding increase in H2O2 generation. In contrast to H2O2 and .OH generation, production of thiobarbituric acid-reactive material by nuclei was not increased by the phenobarbital or 3-methylcholanthrene treatment. Redox cycling agents such as menadione and paraquat increased oxygen radical generation to similar extents in the control and the induced nuclei. These results indicate that induction of the nuclear mixed-function oxidase system by phenobarbital or 3-methylcholanthrene can result in a subsequent increase in production of reactive oxygen intermediates in the presence of either NADPH or NADH.     

Free Radical-mediated Formation of Ethylene from 1-Aminocyclopropane-1-carboxylic Acid: A Spin-frap Study

The ability of free radicals to convert l-aminocyclopropane-l-carboxylicacid (ACC) to ethylene under strictly chemical conditions hasbeen investigated using the aerobic xanthine/xanthine oxidasereaction and the Fenton reaction. Ethylene is formed when 1mM ACC is added to either of these reactions. Ethylene productionby the xanthine/xanthine oxidase system can be stimulated byH₂O₂ and inhibited by both catalase and superoxide dismutase,suggesting that the hydroxyl radical (OH?) formed by the Haber-Weissreaction is reacting with ACC to form ethylene. Ethylene productionfrom ACC by the Fenton reagent, which also produces OH?, showsa strong dependence upon H₂O₂. Involvement of the OH? radicalwas confirmed by spin-trap studies using 5,5-dimethyl-l-pyrroline-l-oxide(DMPO). Only the hydroxyl adduct of DMPO was detectable in boththe xanthine/xanthine oxidase reaction and the Fenton reaction.When ACC was added to the Fenton reaction, an additional adductof DMPO was detectable, which, on the basis of its hyperfinesplitting constants, can be tentatively identified as the DMPOadduct of a carbon-centered free radical. The data are consistentwith the view that formation of ethylene from ACC entails attackby OH? and the resultant formation of a carbon-centered radical,possibly of ACC. The chemical conversion of ACC to ethyleneis less efficient than that characteristic of senescing tissues,in which the reaction is enzymatically mediated. (Received October 1, 1981; Accepted November 17, 1981)     

Release of iron from ferritin by semiquinone, anthracycline, bipyridyl, and nitroaromatic radicals

The cytotoxicity of many xenobiotics is related to their ability to undergo redox reactions and iron dependent free radical reactions. We have measured the ability of a number of redox active compounds to release iron from the cellular iron storage protein, ferritin. Compounds were reduced to their corresponding radicals with xanthine oxidase/hypoxanthine under N₂ and the release of Fe²⁺ was monitored by complexation with ferrozine. Ferritin iron was released by a number of bipyridyl radicals including those derived from diquat and paraquat, the anthracycline radicals of adriamycin, daunorubicin and epirubicin, the semiquinones of anthraquinone-2-sulphonate, 1,5 and 2,6-dihydroxyanthraquinone, 1-hydroxyanthraquinone, purpurin, and plumbagin, and the nitroaromatic radicals of nitrofurantoin and metronidazole. In each case, iron release was more efficient than with an equivalent flux of superoxide. Introduction of air decreased the rate of iron release, presumably because the organic radicals reacted with O₂ to form superoxide. In air, iron release was inhibited by superoxide dismutase. Semiquinones of menadione, benzoquinone, duroquinone, anthraquinone 1,5 and 2,6-disulphonate, 1,4 naphthoquinone-2-sulphonate and naphthoquinone, when formed under N₂, were unable to release ferrin iron. In air, these systems gave low rates of superoxide dismutase-inhibitible iron release. Of the compounds investigated, those with a single electron reduction potential less than that of ferritin were able to release ferritin iron.     

The reaction of arsenite-complexed xanthine oxidase with oxygen. Evidence for an oxygen-reactive molybdenum center

The effects of arsenite on the reaction of reduced xanthine oxidase with oxygen are determined. The kinetics of the reaction monitoring the return of enzyme absorbance are investigated as are the kinetics and stoichiometries of peroxide and superoxide formation. Although some of the effects of arsenite are qualitatively consistent with expectations based on the known perturbation of the molybdenum midpoint potentials by arsenite, several results cannot be so easily explained. Specifically, arsenite introduces a very rapid phase (kobs = 110 s-1 at 125 microM oxygen) to the oxidative half-reaction which is not observed with the native enzyme. Arsenite also diminishes the amount of superoxide produced and eliminates one-electron reduced enzyme as a detectable kinetic intermediate in the reoxidation pathway. These differences appear to result from the ability of arsenite to greatly enhance the oxygen- and/or superoxide-reactivity of the reduced molybdenum center. This is reflected in the observation that reduced forms of arsenite-complexed xanthine oxidase lacking functional FAD (iodoacetamide-alkylated enzyme and deflavo enzyme) react relatively rapidly with oxygen whereas these reactions are quite slow in the absence of arsenite.     

Inactivation of mitochondrial adenosine triphosphatase from Trypanosoma cruzi by oxygen radicals

Incubation of Trypanosoma cruzi mitochondrial ATPase (Fo-F1) with the xanthine oxidase system (XO), Fenton's reagent (Fe2+ + H2O2) and the ascorbate-Cu system, caused gradual loss of enzyme activity, which increased as a function of incubation time and rate of oxygen radical generation. The essential role of OH. radicals for ATPase inactivation was supported by a) the enzyme protection afforded by superoxide dismutase, catalase and mannitol, when using the XO system; b) the similar effect of mannitol and benzoate with Fenton's reagent; c) the similar effect of catalase, EDTA and histidine with the ascorbate-Cu system; d) the increased rate of ATPase inactivation by 1) the XO system supplemented with chelated iron, and 2) the ascorbate-Cu system supplemented with H2O2. Comparison of oxygen radical generators for their action on membrane-bound (Fo-F1) and soluble F1 revealed that ascorbate-Cu was the most effective one, possibly because of its capability of producing OH. radicals that react preferentially with the enzyme at their formation site.