Ligand dependence in the copper-catalyzed oxidation of hydroquinones

Transition metal-mediated oxidation of hydroquinones is an important physiologic reaction, and copper(II) effectively catalyzes the reaction in phosphate-buffered saline (PBS). Studies reported herein in phosphate buffer alone demonstrate that copper(II) is an ineffective catalyst in the absence of coordinating ligands, but that 1,10-phenanthroline and histamine facilitate the copper(II)-mediated oxidation of hydroquinone and its 2,5- and 2,6-di-tert-butyl analogs to the corresponding benzoquinones. The high concentration of chloride in PBS is the key element that allows copper(II) to work in this system. Although the bis-bathocuproine disulfonate complex of Cu(II), (BC)₂Cu(II), is a strong stoichiometric oxidant, stoichiometric amounts of copper(II) in the presence of ligands other than BC oxidize hydroquinones very slowly under anaerobic conditions. Thus, the rapid copper(II)-catalyzed reaction operating aerobically does not involve a simple ping-pong reduction of copper(II) to copper(I) by hydroquinone and reoxidation of copper(I) by O₂.     

Reaction of bovine-liver copper-zinc superoxide dismutase with hydrogen peroxide. Evidence for reaction with H2O2 and HO2- leading to loss of copper

The reaction of hydrogen peroxide with the copper-zinc bovine-liver superoxide dismutase at low molar ratios (0.2-20.0) of H2O2/active site between pH 7.3-10.0 leads to the loss of native enzyme as a distinct form monitored by electrophoresis. The pH dependence of the loss of native enzyme between 7.3 and 9.0 indicates the involvement of a conjugate base on the enzyme of pKa of 8.7 +/- 0.1. The rate of loss of the native enzyme is first order with respect to the concentration of both enzyme and hydrogen peroxide between pH 7.3 and 9.0 with no evidence for binding of peroxide. A second-order rate constant of 3.0 +/- 1.0 M-1 s-1 is obtained from these data. At pH 10.0 the reaction is first order with respect to enzyme concentration but saturable in H2O2. All data are consistent with the interpretation that H2O2 reacts with the enzyme at the lower pH where the reaction is dependent upon the conjugate base of a functional group on the enzyme. At the higher pH, the data are consistent with the reaction of HO2- and H2O2 with the dismutase. The dissociation constant for HO2- calculated from the kinetic data at pH 10.0 is between 25-50 microM and the rate constant for the breakdown of the HO2- dismutase complex is 1.10 + 0.05 x 10(-2) s-1. The change in the electrophoretic pattern at all pH values is accompanied by the loss of the ability of the enzyme to bind copper. Weakly bound or free copper can be detected using bathocuproine disulfonate. Furthermore copper-defficient forms of the enzyme can be detected by staining gels of the peroxide-treated dismutase with diethyldithiocarbamate.     

Direct evidence for inhibition of free radical formation from Cu(I) and hydrogen peroxide by glutathione and other potential ligands using the EPR spin-trapping technique.

Copper-induced oxidative damage is generally attributed to the formation of the highly reactive hydroxyl radical by a mechanism analogous to the Haber-Weiss cycle for Fe(II) and H2O2. In the present work, the reaction between the Cu(I) ion and H2O2 is studied using the EPR spin-trapping technique. The hydroxyl radical adduct was observed when Cu(I), dissolved in acetonitrile under N2, was added to pH 7.4 phosphate buffer containing 100 mM 5,5-dimethyl-1-pyrroline N-oxide (DMPO). Formation of the hydroxyl radical was dependent on the presence of O2 and subsequent formation of H2O2. The kscav/kDMPO ratios obtained were below those expected for a mechanism involving free hydroxyl radical and reflect the interference of nucleophilic addition of H2O to DMPO to form the DMPO/.OH adduct in the presence of nonchelated copper ion. Addition of ethanol or dimethyl sulfoxide to the reaction suggests that a high-valent metal intermediate, possibly Cu(III), was also formed. Spin trapping of hydroxyl radical was almost completely inhibited upon addition of Cu(I) to a solution of either nitrilotriacetate or histidine, even though the copper was fully oxidized to Cu(II) and H2O2 was formed. Bathocuproinedisulfonate, thiourea, and reduced glutathione all stabilized the Cu(I) ion toward oxidation by O2. Upon addition of H2O2, the Cu(I) in all three complexes was oxidized to varying degrees; however, only the thiourea complex was fully oxidized within 2 min of reaction and produced detectable hydroxyl radicals. No radicals were detected from the bathocuproinedisulfonate or glutathione complexes. Overall, these results suggest that the deleterious effects of copper ions in vivo are diminished by biochemical chelators, especially glutathione, which probably has a major role in moderating the toxicological effects of copper.     

Reaction of copper-zinc superoxide dismutase with diethyldithiocarbamate.

Diethyldithiocarbamate reacted with superoxide dismutase from bovine erythrocytes. Changes in both optical and esr spectra, which accompanied this reaction, indicated involvement of the Cu(II). The reaction was accelerated by raising the concentrations of the reactants, elevating the temperature, and lowering the pH, in the range 10.2 to 5.5, and it was independent of the presence of oxygen. During the first phase of this reaction the Cu(II).diethyldithiocarbamate complex remained bound to the enzyme and the catalytic activity did not diminish. There followed a second and slower process which was accompanied by the appearance of colloidal Cu(II).chelate complex and by a loss of activity which could be restored by the addition of CuSO4. All of the observations are accomodated by a model in which 1 diethyldithiocarbamate molecule reacts/copper center, with retention of activity, in Phase I, while a second diethyldithiocarbamate displaces the copper, with a loss of activity, in Phase II.     

Relationship between copper biosorption and microbial inhibition of hydroxyl radical formation in a copper(II)–hydrogen peroxide system

The microbial retardation of the spin adduct, DMPO-OH, formed in a copper(II)–hydrogen peroxide–DMPO (5,5-dimethyl-1-pyrroline N-oxide) solution was examined in relation to copper biosorption. A hydroxyl radical is formed in the solution through two steps, the reduction of Cu(II) to Cu(I) by H₂O₂ and the Fenton-type reaction of Cu(I) with H₂O₂. The resultant radical is trapped by DMPO to form DMPO-OH. Microbial cells retarded the DMPO-OH in the Cu(II)–H₂O₂–DMPO far more significantly than in the UV-irradiated H₂O₂–DMPO solution. Egg albumin showed a higher DMPO-OH retardation than microbial cells both in the Cu(II)–H₂O₂–DMPO and the UV-irradiated H₂O₂–DMPO solutions. These results indicated that the retardation effect is related to organic matter and not to microbial activity. Microorganisms having higher affinities for copper ion retarded DMPO-OH more significantly. The linear relationship between the amounts of copper biosorption and the inverse of the median inhibitory doses for DMPO-OH indicated that the microbial cells inhibited the reduction of Cu(II) to Cu(I) by H₂O₂, followed by the decrease of hydroxyl radical formation and the retardation of DMPO-OH. These results also suggest that the coupling between microbial cells and Cu(II) ion can be estimated from their ability to retard DMPO-OH.     

Reconstitution of Cu,Zn-superoxide dismutase by the Cu(I).glutathione complex

The reconstitution of Cu,Zn-superoxide dismutase from the copper-free protein by the Cu(I).GSH complex was monitored by: (a) EPR and optical spectroscopy upon reoxidation of the enzyme-bound copper; (b) NMR spectroscopy following the broadening of the resonances of the Cu(I).GSH complex after addition of Cu-free,Zn-superoxide dismutase; and (c) NMR spectroscopy of the Cu-free,Co(II) enzyme following the appearance of the isotropically shifted resonances of the Cu(I), Co enzyme, Cu(I).GSH was found to be a very stable complex in the presence of oxygen and a more efficient copper donor to the copper-free enzyme than other low molecular weight Cu(II) complexes. In particular, 100% reconstitution was obtained with stoichiometric copper at any GSH:copper ratio between 2 and 500. Evidence was obtained for the occurrence of a Cu(I).GSH.protein intermediate in the reconstitution process. In view of the inability of copper-thionein to reconstitute Cu,Zn-superoxide dismutase and of the detection of copper.GSH complexes in copper-over-loaded hepatoma cells (Freedman, J.H., Ciriolo, M.R., and Peisach, J. (1989) J. Biol. Chem. 264, 5598-5605), Cu(I).GSH is proposed as a likely candidate for copper donation to Cu-free,Zn-superoxide dismutase in vivo.     

Degradation of DMPO Adducts from Hydroxyl and 1-Hydroxyethyl Radicals by Rat Liver Microsomes

Hydroxyl and 1-hydroxyethyl radical adducts of 5, 5-dimethylpyrroline N-oxide (DMPO) were prepared by photolysis, and mechanisms for loss of their EPR signals in rat liver microsomal suspensions were evaluated. Rates of NADPH-dependent EPR signal loss were more rapid in phosphate buffer than in Tris buffer. Addition of superoxide dismutase (SOD) partially protected the adducts when Tris was used as a buffer, but was relatively ineffective in the presence of phosphate. The ferrous iron chelator bathophenanthrolene partially protected the spin adducts in the presence and absence of phosphate, but complete protection was observed when SOD was also added. The spin adducts were unstable in the presence of Fe⁺² and K₃Fe(CN)₆, but Fe⁺³ alone had little effect on the EPR signals. The data are consistent with two mechanisms for microsomal degradation of DMPO spin adducts under these conditions. Microsomes form superoxide in the presence of oxygen and NADPH, which attacks these DMPO spin adducts directly. The spin adducts are also degraded in the presence of Fe⁺², and phosphate stimulates this iron-dependent destruction of DMPO spin adducts.     

A solution study of the interaction of the Cu(II) ions with HisGlyGlyTrp tetrapeptide and its evaluation as superoxide dismutase mimetic complex

The superoxide anion radical is a highly reactive toxic species produced during the metabolic processes. A number of copper (II) complexes with amino acids and peptides are known to show superoxide dismutase (SOD) like activity. The design and application of synthetic low molecular weight metal complexes as SOD mimics have received considerable attention during the last decade. A variety of di- and tri-peptides containing histidyl residue in different positions have been employed to bind Cu(II) and to show the activity. But reports on Cu(II) complex with tetra-peptide having histidine amino acid in this regard are limited. As the HGGGW peptide having His at its N-terminal is reported to be a potential moiety for Cu(2+) binding, in the present work the synthesis of HisGlyGlyTrp peptide and its complexation with copper (II) ions has been reported. The interaction of synthesized peptide with Cu(II) was studied by electron spray ionization-mass spectrometer (ESI-MS) and UV-Vis spectroscopic methods. The species distribution was studied by combined spectrophotometric and potentiometric methods. The studies were performed at 25 ± 0.1 °C with constant ionic strength (μ = 0.1 M NaNO(3)) in aqueous solution using Bjerrum-Calvin's pH-titration technique as adopted by Irving and Rossotti for binary systems. The solution studies suggested that the pH of the medium play important role in the different species formation of the copper complexes. Species distribution curves indicate that Cu complexation takes place at all physiological pH values from 3-11. The resultant copper (II) peptide complex at physiological pH was tested for superoxide dismutase activity using standard NBT method. The complex has SOD activity with the IC(50) value of 1.32 μM.     

Copper complexes of 1,10-phenanthroline and related compounds as superoxide dismutase mimetics

In a preliminary study we tested CuSO4.5H2O, (Cu(II]2[3,5-diisopropylsalicylate]4.2H2O and a number of copper complexes of substituted 1,10-phenanthrolines for superoxide anion dismutase activity. It appeared that this activity depends on the ligands involved and might be governed by the redox potential of the Cu(I) complex/Cu(II) complex couple. The strong superoxide anion dismutase activity of Cu(II)[DMP]2 complex can be expected considering its high redox potential. Rather surprisingly is the superoxide anion dismutase activity of the Cu(I)[DMP]2 complex since it involves oxidation to Cu(II)[DMP]2 complex. From regression analysis it was established that steric and field effects of the substituents of the investigated phenanthrolines play an important role in SOD activity and therefore it is concluded that complex formation is important for the superoxide dismutase-like activity.     

Studies on the gamma-glutamyl Cu-binding peptide from Schizosaccharomyces pombe

The gamma-glutamyl peptide induced in Schizosaccharomyces pombe in response to metal stress has been purified following exposure of the organism to cadmium and copper salts. Induction of the peptide enables S. pombe to proliferate in media containing high concentrations of cadmium and copper. Two Cd-gamma-Glu peptide complexes are produced which differ in the content of acid-labile sulfur. One Cu-gamma-Glu peptide complex is induced, and it lacks acid-labile sulfur in the metal-binding cluster. The peptides are composed of repeating dipeptide units of gamma-Glu-Cys with a carboxyl-terminal glycine with heterogeneity observed in the repeat unit n. The number of repeats averages 3.2 and 3.8 for the Cd-peptides I and II and 3.6 for the Cu-peptide, in the case of the Cu-complex peptides with n values from 2 to 4 were separated by reverse phase high pressure liquid chromatography. The Cu-gamma-Glu peptide complex is oligomeric, but the exact number of peptide units per complex is not known. The copper binding stoichiometry averages 2.3 g atoms of Cu/mol of peptide, whereas Cd-peptides I and II average 1.8 and 2.7 mol eq of Cd(II)/peptide unit. The pH of half-dissociation of Cu ions from the gamma-Glu peptide is near 1.3, whereas pH values of 4 and 5.4 are sufficient for half-displacement of Cd ions from the sulfide-containing and -lacking peptides II and I, respectively. In the Cu-peptide complex copper is bound as Cu(I) as the complex exhibits luminescence characteristic of Cu(I)-S chelation. The luminescence emission peaks at 619 nm with a corrected excitation peak centered at 290 nm. The luminescence of the Cu-complex indicates the clustering of Cu(I) ions within a solvent-inaccessible complex. The complex is air-labile as the luminescence emission is gradually lost upon air exposure.     

Degradation of DMPO Adducts from Hydroxyl and 1-Hydroxyethyl Radicals by Rat Liver Microsomes

Hydroxyl and 1-hydroxyethyl radical adducts of 5, 5-dimethylpyrroline N-oxide (DMPO) were prepared by photolysis, and mechanisms for loss of their EPR signals in rat liver microsomal suspensions were evaluated. Rates of NADPH-dependent EPR signal loss were more rapid in phosphate buffer than in Tris buffer. Addition of superoxide dismutase (SOD) partially protected the adducts when Tris was used as a buffer, but was relatively ineffective in the presence of phosphate. The ferrous iron chelator bathophenanthrolene partially protected the spin adducts in the presence and absence of phosphate, but complete protection was observed when SOD was also added. The spin adducts were unstable in the presence of Fe⁺² and K₃Fe(CN)₆, but Fe⁺³ alone had little effect on the EPR signals. The data are consistent with two mechanisms for microsomal degradation of DMPO spin adducts under these conditions. Microsomes form superoxide in the presence of oxygen and NADPH, which attacks these DMPO spin adducts directly. The spin adducts are also degraded in the presence of Fe⁺², and phosphate stimulates this iron-dependent destruction of DMPO spin adducts.     

Golgi membranes from liver express an ATPase with femtomolar copper affinity, inhibited by cAMP-dependent protein kinase

Copper-stimulated P-type ATPases are essential in the fine-tuning of intracellular copper. In the present work we characterized a copper-dependent ATPase hydrolysis in a native Golgi-enriched preparation from liver and investigated its modulation by cyclic AMP-dependent protein kinase (PKA). The very high-affinity Atp7b copper pump presented here shows a K(0.5) for free copper of 2.5×10(-17) M in bathocuproine disulfonate/copper buffer and ATP hydrolysis was inhibited 50% upon stimulation of PKA pathway, using forskolin, cAMP or cholera toxin. Incubation with PKA inhibitor (PKAi(5-24) peptide) raises Cu(I)-ATPase activity by 50%. Addition of purified PKA α-catalytic subunit increases K(0.5) for free copper (6.2×10(-17) M) without modification in the affinity for ATP in the low-affinity range of the substrate curve (～1 mM). The Hill coefficient for free copper activation also remains unchanged if exogenous PKA is added (2.7 and 2.3 in the absence and presence of PKA, respectively). The results demonstrate that this high-affinity copper pump in its natural environment is a target of the liver PKA pathway, being regulatory phosphorylation able to influence both turnover rate and ion affinity.     

Superoxide dismutase-like activities of copper(II) complexes tested in serum

The superoxide dismutase-like activities of a series of coordination complexes of copper were evaluated and compared to the activities of bovine erythrocyte superoxide dismutase (superoxide: superoxide oxidoreductase, EC 1.15.1.1) in serum using the nitroblue tetrazolium chloride (NBT)-reduction assay and electron paramagnetic resonance (EPR) spectroscopy. A 40% inhibition was observed for the initial rate of the NBT reduction by superoxide dismutase in serum, but more than 40% inhibition was achieved with CuSO4, Cu(II)-dimethylglyoxime, Cu(II)-3,8-dimethyl-4,7-diazadeca-3,7-dienediamide, Cu2[N,N'-(2-(O-hydroxy-benzhydrylidene)amino)ethyl]2-1,2-ethane dia mine), Cu(II)-(diisopropylsalicylate)2, Cu(II)-(p-bromo-benzoate)2, Cu(II)-(nicotinate)2 and Cu(II)-(1,2-diamino-2-methylpropane)2. The electron paramagnetic resonance technique of spin trapping was used to detect the formation of superoxide (O2-.) and other free radicals in the xanthine-xanthine oxidase system under a variety of conditions. Addition of the spin trapping agent 5,5-dimethylpyrroline 1-oxide (DMPO) to the xanthine-xanthine oxidase system in fetal bovine serum produced the O2-.-spin adduct of DMPO (herein referred to as superoxide spin adduct, DMPO-OOH) as the well known short-lived nitroxyl whose characteristic EPR spectrum was recorded before its rapid decay to undetectable levels. The hydroxyl radical (HO.) adduct of the spin trap DMPO (herein referred to as DMPO-OH) was detected to a very small extent. When CuSO4, or the test complexes of copper, were added to the xanthine-xanthine oxidase system in serum containing the spin trap, the yield of DMPO-OOH was negligible. In addition to their superoxide dismutase-like activity, CuSO4 and the copper complexes also behaved as Fenton-type catalysts as seen by the accumulation of varying amounts of the hydroxyl spin adduct DMPO-OH. Both the Fenton-type catalysis and the superoxide dismutase-like action of these compounds were lost when a chelator such as EDTA was included in the xanthine-xanthine oxidase incubation mixture. Addition of superoxide dismutase instead of the copper compounds to this enzyme system abolished the formation of superoxide adduct DMPO-OOH, and no hydroxyl adduct DMPO-OH was detected. This effect of superoxide dismutase remained unaltered by EDTA.     

Quinolinic acid — Iron(II) complexes: Slow autoxidation,but enhanced hydroxyl radical production in the Fenton reaction

Quinolinate (pyridine-2,3-dicarboxylic acid, Quin) is a neurotoxic tryptophan metabolite produced mainly by immune-activated macrophages. It is implicated in the pathogenesis of several brain disorders including HIV-associated dementia. Previous evidence suggests that Quin may exert its neurotoxic effects not only as an agonist on the NMDA subtype of glutamate receptor, but also by a receptor-independent mechanism. In this study we address ability of ferrous quinolinate chelates to generate reactive oxygen species. Autoxidation of Quin-Fe(II) complexes, followed in Hepes buffer at pH 7.4 using ferrozine as the Fe(II) detector, was found to be markedly slower in comparison with iron unchelated or complexed to citrate or ADP. The rate of Quin-Fe(II) autoxidation depends on pH (squared hydroxide anion concentration), is catalyzed by inorganic phosphate, and in both Hepes and phosphate buffers inversely depends on Quin concentration. These observations can be explained in terms of anion catalysis of hexaaquairon(II) autoxidation, acting mainly on the unchelated or partially chelated pool of iron. In order to follow hydroxyl radical generation in the Fenton chemistry, electron paramagnetic resonance (EPR) spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) was employed. In the mixture consisting of 100 mM DMPO, 0.1 mM Fe(II), and 8.8 mM hydrogen peroxide in phosphate buffer pH 7.4, 0.5 mM Quin approximately doubled the yield of DMPO-OH adduct, and higher Quin concentration increased the spin adduct signal even more. When DMPO-OH was pre-formed using Ti³⁺/hydrogen peroxide followed by peroxide removal with catalase, only addition of Quin-Fe(II), but not Fe(II), Fe(III), or Quin-Fe(III), significantly promoted decomposition of pre-formed DMPO-OH. Furthermore, reaction of Quin-Fe(II) with hydrogen peroxide leads to initial iron oxidation followed by appearance of iron redox cycling, detected as slow accumulation of ferrous ferrozine complex. This phenomenon cannot be abolished by subsequent addition of catalase. Thus, we propose that redox cycling of iron by a Quin derivative, formed by initial attack of hydroxyl radicals on Quin, rather than effects of iron complexes on DMPO-OH stability or redox cycling by hydrogen peroxide, is responsible for enhanced DMPO-OH signal in the presence of Quin. The present observations suggest that Quin-Fe(II) complexes display significant pro-oxidant characteristics that could have implications for Quin neurotoxicity.     

Quinolinic acid-iron(ii) complexes: slow autoxidation, but enhanced hydroxyl radical production in the Fenton reaction

Quinolinate (pyridine-2,3-dicarboxylic acid, Quin) is a neurotoxic tryptophan metabolite produced mainly by immune-activated macrophages. It is implicated in the pathogenesis of several brain disorders including HIV-associated dementia. Previous evidence suggests that Quin may exert its neurotoxic effects not only as an agonist on the NMDA subtype of glutamate receptor, but also by a receptor-independent mechanism. In this study we address ability of ferrous quinolinate chelates to generate reactive oxygen species. Autoxidation of Quin-Fe(II) complexes, followed in Hepes buffer at pH 7.4 using ferrozine as the Fe(II) detector, was found to be markedly slower in comparison with iron unchelated or complexed to citrate or ADP. The rate of Quin-Fe(II) autoxidation depends on pH (squared hydroxide anion concentration), is catalyzed by inorganic phosphate, and in both Hepes and phosphate buffers inversely depends on Quin concentration. These observations can be explained in terms of anion catalysis of hexaaquairon(II) autoxidation, acting mainly on the unchelated or partially chelated pool of iron. In order to follow hydroxyl radical generation in the Fenton chemistry, electron paramagnetic resonance (EPR) spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) was employed. In the mixture consisting of 100 mM DMPO, 0.1 mM Fe(II), and 8.8 mM hydrogen peroxide in phosphate buffer pH 7.4, 0.5 mM Quin approximately doubled the yield of DMPO-OH adduct, and higher Quin concentration increased the spin adduct signal even more. When DMPO-OH was pre-formed using Ti³⁺/hydrogen peroxide followed by peroxide removal with catalase, only addition of Quin-Fe(II), but not Fe(II), Fe(III), or Quin-Fe(III), significantly promoted decomposition of pre-formed DMPO-OH. Furthermore, reaction of Quin-Fe(II) with hydrogen peroxide leads to initial iron oxidation followed by appearance of iron redox cycling, detected as slow accumulation of ferrous ferrozine complex. This phenomenon cannot be abolished by subsequent addition of catalase. Thus, we propose that redox cycling of iron by a Quin derivative, formed by initial attack of hydroxyl radicals on Quin, rather than effects of iron complexes on DMPO-OH stability or redox cycling by hydrogen peroxide, is responsible for enhanced DMPO-OH signal in the presence of Quin. The present observations suggest that Quin-Fe(II) complexes display significant pro-oxidant characteristics that could have implications for Quin neurotoxicity.     

A comparison of cobalt(II) and iron(II) hydroxyl and superoxide free radical formation

We have employed the electron spin resonance spin-trapping technique to study the reaction of Co(II) with hydrogen peroxide in a chemical system and in a microsomal system. In both cases, we employed the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and were able to detect the formation of DMPO/.OH and DMPO/.OOH. DMPO/.OOH was the predominant radical adduct formed in the chemical system, while the two adducts were of similar concentrations in the microsomal system. The formation of both of these adducts in either reaction system was inhibited by the addition of superoxide dismutase or catalase, and by chelating the cobalt with either ethylenediaminetetraacetic acid (EDTA) or diethylenetriaminepentaacetic acid (DTPA). The incorporation of the hydroxyl radical scavengers ethanol, formate, benzoate, or mannitol inhibited the formation of DMPO/.OH in both systems. We also repeated the study using Fe(II) in place of Co(II). In contrast to the Co(II) results, Fe(II) reacted with hydrogen peroxide to yield only DMPO/.OH, and this adduct formation was relatively insensitive to the presence of added superoxide dismutase. In addition, Fe(II)-mediated DMPO/.OH formation increased when the iron was chelated to either EDTA or DTPA rather than being inhibited as for Co(II). Thus, we propose that Co(II) does not react with hydrogen peroxide by the classical Fenton reaction at physiological pH values.     

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.     

Effect of chelating agents and superoxide on human neutrophil NAD(P)H oxidation

NAD(P)H oxidation is frequently measured to assay the activity of the neutrophil O-2-generating oxidase. It was found that 10(-4) M ethylene glycol bis (beta-aminoethyl ether)-N-N'-tetraacetic acid (EGTA) increased NAD(P)H oxidation by the 27,000 g granule fraction of resting and stimulated human neutrophils without altering net O-2 production. The commonly used chelating agents EDTA and diethylene triamine pentaacetic acid had similar effects. The addition of superoxide dismutase eliminated the effect of the chelating agents and thus demonstrated that the stimulated reaction was dependent upon O-2. KCN and bathophenanthroline disulfonate, an iron-chelating agent, prevented O-2-dependent NADPH oxidation by neutrophil granule fractions in the presence of EGTA. In contrast, bathocuproine disulfonate, a copper-chelating agent, mimicked the EGTA effect. The effects of both bathophenanthroline disulfonate and bathocuproine disulfonate were completely abolished when the agents were saturated with iron and copper, respectively. All the chelating agents studied, except bathophenonthroline disulfonate, also promoted O-2-dependent NADPH oxidation in a system wherein O-2 was generated by xanthine oxidase. Thus, commonly used chelating agents, by interacting with available iron and copper, may alter the apparent stoichiometry of the neutrophil O-2-generating oxidase and artifactually increase NADPH oxidation in other systems where O-2 is present.     

Metal-independent production of hydroxyl radicals by halogenated quinones and hydrogen peroxide: an ESR spin trapping study

The metal-independent production of hydroxyl radicals (*OH) from H(2)O(2) and tetrachloro-1,4-benzoquinone (TCBQ), a carcinogenic metabolite of the widely used wood-preservative pentachlorophenol, was studied by electron spin resonance methods. When incubated with the spin trapping agent 5,5-dimethyl-1-pyrroline N-oxide (DMPO), TCBQ and H(2)O(2) produced the DMPO/*OH adduct. The formation of DMPO/*OH was markedly inhibited by the *OH scavenging agents dimethyl sulfoxide (DMSO), ethanol, formate, and azide, with the concomitant formation of the characteristic DMPO spin trapping adducts with *CH(3), *CH(CH(3))OH, *COO(-), and *N(3), respectively. The formation of DMPO/*OH and DMPO/*CH(3) from TCBQ and H(2)O(2) in the absence and presence, respectively, of DMSO was inhibited by the trihydroxamate compound desferrioxamine, accompanied by the formation of the desferrioxamine-nitroxide radical. In contrast, DMPO/*OH and DMPO/*CH(3) formation from TCBQ and H(2)O(2) was not affected by the nonhydroxamate iron chelators bathophenanthroline disulfonate, ferrozine, and ferene, as well as the copper-specific chelator bathocuproine disulfonate. A comparative study with ferrous iron and H(2)O(2), the classic Fenton system, strongly supports our conclusion that *OH is produced by TCBQ and H(2)O(2) through a metal-independent mechanism. Metal-independent production of *OH from H(2)O(2) was also observed with several other halogenated quinones.     

Enhancement of cytotoxicity of bleomycin by dithiocarbamates: formation of bis(dithiocarbamato) cu(II).

Properties of the reactions of dithiocarbamates and their Cu(II) or Fe(III) complexes with Ehrlich cells were determined and related to their effects on the inhibition of cell proliferation caused by bleomycin and Cu bleomycin. In complete culture medium containing Eagle's minimal essential medium plus Earles salts and 2.5% fetal calf serum, dimethyl- and diethyldithiocarbamates and their copper complexes inhibit cell proliferation and cause cell death. The copper complexes are more effective agents. Ferric tris-diethyldithiocarbamate is also a cytotoxic species. In contrast, when cells are exposed to dimethyldithiocarbamate or its copper complex in Ringer's buffer under metal-restricted condition, washed, and then placed in complete medium, the copper complex is much more active in inhibiting cell growth. The difference is magnified when dihydroxyethyldithiocarbamate and N-methylglucamine dithiocarbamate and their copper complexes are compared in complete media. Incubation of bleomycin or copper bleomycin with Ehrlich cells in Ringer's buffer with or without dimethyldithiocarbamate or bis-dimethyldithiocarbamato Cu(II) leads to no enhancement of cytotoxicity from combinations of agents, except when the two copper complexes are present. Diethyl- or dimethyldithiocarbamate readily extracts copper from Cu(II)bleomycin and iron from Fe(III)bleomycin when ethylacetate is present to remove the tris-dithiocarbamato Fe(III) complex from aqueous solution. When bis-dimethyldithiocarbamato Cu(II) is incubated with Ehrlich cells, copper is released from the complex and bound to high molecular weight and metallothionein fractions. A reductive mode of dissociation of the copper complexes in cells is supported by ESR experiments. Reactions of diethyl- and dimethyldithiocarbamato Cu(II) with thiol compounds demonstrates one possible mechanism of reduction of these complexes.