Thiourea protects against copper-induced oxidative damage by formation of a redox-inactive thiourea-copper complex

Although thiourea has been used widely to study the role of hydroxyl radicals in metal-mediated biological damage, it is not a specific hydroxyl radical scavenger and may also exert antioxidant effects unrelated to hydroxyl radical scavenging. Thus, we investigated the effects of thiourea on copper-induced oxidative damage to bovine serum albumin (1 mg/ml) in three different copper-containing systems: Cu(II)/ascorbate, Cu(II)/H₂O₂, and Cu(II)/H₂O₂/ascorbate [Cu(II), 0.1 mM; ascorbate, 1 mM; H₂O₂, 1 mM]. Oxidative damage to albumin was measured as protein carbonyl formation. Thiourea (0.1–10 mM) provided marked and dose-dependent protection against protein oxidation in all three copper-containing systems. In contrast, only minor protection was observed with dimethyl sulfoxide and mannitol, even at concentrations as high as 100 mM. Strong protection was also observed with dimethylthiourea, but not with urea or dimethylurea. Thiourea also significantly inhibited copper-catalyzed oxidation of ascorbate, and competed effectively with histidine and 1,10-phenanthroline for binding of cuprous, but not cupric, copper, as demonstrated by both UV-visible and low temperature electron spin resonance measurements. We conclude that the protection by thiourea against copper-mediated protein oxidation is not through scavenging of hydroxyl radicals, but rather through the chelation of cuprous copper and the formation of a redox-inactive thiourea-copper complex.     

Transition metals mediate enzymatic inactivation caused by favism-inducing agents

Enzymatic activity of purified or membrane-bound acetylcholine esterase was lost when incubated aerobically in the presence of both favism-inducing agent (isouramil or divicine) and copper ions. The requirement for oxygen could be substituted by hydrogen peroxide. Chelating agents provided total protection to the proteins. The suggested mechanism of enzymatic inactivation is analogous to that suggested earlier for the effects of superoxide and ascorbate, and involves the site-specific formation of hydroxyl radicals in the metal-mediated Haber-Weiss reaction. These findings may be relevant to the understanding of the pathogenesis of favism.     

Inhibition of ascorbate oxidation by urate

Cupric sulfate is reduced by ascorbate to the cuprous ion. The cuprous ion is then oxidized back to the cupric form by oxygen. A steady state concentration of the cupric ion is thus established to maintain a continuous oxidation of ascorbate in the presence of a trace amount of copper. In the presence of urate there is an instantaneous oxidation of ascorbate by the cupric ion. However, urate complexes with the cuprous ion and thus reduces the steady state concentration of the cupric ion. This decrease in cupric ion concentration interrupts ascorbate oxidation. The interaction of urate and cuprous ion was documented by analysis of uv absorption spectrum and the isolation of urate-Cu⁺ by high-pressure liquid chromatography.     

Cupric-amyloid beta peptide complex stimulates oxidation of ascorbate and generation of hydroxyl radical

A growing body of evidence supports an important role for oxidative stress in the pathogenesis of Alzheimer's disease. Recently, a number of papers have shown a synergistic neurotoxicity of amyloid beta peptide and cupric ions. We hypothesized that complexes of cupric ions with neurotoxic amyloid beta peptides (Abeta) can stimulate copper-mediated free radical formation. We found that neurotoxic Abeta (1-42), Abeta (1-40), and Abeta (25-35) stimulated copper-mediated oxidation of ascorbate, whereas nontoxic Abeta (40-1) did not. Formation of ascorbate free radical was significantly increased by Abeta (1-42) in the presence of ceruloplasmin. Once cupric ion is reduced to cuprous ion, it can be oxidized by oxygen to generate superoxide radical or it can react with hydrogen peroxide to form hydroxyl radical. Hydrogen peroxide greatly increased the oxidation of cyclic hydroxylamines and ascorbate by cupric-amyloid beta peptide complexes, implying redox cycling of copper ions. Using the spin-trapping technique, we have shown that toxic amyloid beta peptides led to a 4-fold increase in copper-mediated hydroxyl radical formation. We conclude that toxic Abeta peptides do indeed stimulate copper-mediated oxidation of ascorbate and generation of hydroxyl radicals. Therefore, cupric-amyloid beta peptide-stimulated free radical generation may be involved in the pathogenesis of Alzheimer's disease.     

Transition Metals Potentiate Paraquat Toxicity

The involvement of transition metal ions in paraquat toxicity was studied in bacterial model system. We show that the addition of micromolar, or lower, concentrations of copper dramatically enhanced the rate of bacterial inactivation. In contrast, the addition of chelating agents totally eliminated the killing of E. coli. No inactivation was observed under anaerobic exposure to paraquat, both in the absence and presence of copper. However, in the presence of copper, the anaerobic addition of hydrogen peroxide resulted in complete restoration of inactivation as under aerobiosis.

Paraquat either produces superoxide ions or directly reduces bound copper ions in a catalytic mode. The reduced cuprous complexes react with hydrogen peroxide to locally form hydroxyl radicals (OH) which are probably responsible for the deleterious effects.

This study indicates the involvement of a site-specific metal-mediated Haber-Weiss mechanism in paraquat toxicity. It is in agreement with earlier observations that copper unusually enhance biological damage induced by either superoxide or ascorbate.     

Iron Enhancement of Ascorbate Toxicity

Iron has been shown to enhance ascorbate-induced damage to both acetylcholine esterase and E. coli B in a manner analogous to previous studies with ascorbate and copper ions. It is suggested that the mechanism of damage entails interaction of iron with biological macromolecules, followed by its reduction by ascorbate. Subsequently, the iron (11) could participate in generating hydroxyl radicals from hydrogen peroxide via the Fenton reaction, which in turn, could damage biomolecules in a site-specific and multiple hit fashion. The high abundance of iron in biological systems, especially in certain storage disorders, may indicate an important toxicological role of the combination of iron and ascorbate.     

Iron Enhancement of Ascorbate Toxicity

Iron has been shown to enhance ascorbate-induced damage to both acetylcholine esterase and E. coli B in a manner analogous to previous studies with ascorbate and copper ions. It is suggested that the mechanism of damage entails interaction of iron with biological macromolecules, followed by its reduction by ascorbate. Subsequently, the iron (11) could participate in generating hydroxyl radicals from hydrogen peroxide via the Fenton reaction, which in turn, could damage biomolecules in a site-specific and multiple hit fashion. The high abundance of iron in biological systems, especially in certain storage disorders, may indicate an important toxicological role of the combination of iron and ascorbate.     

Copper-dependent inhibition and oxidative inactivation with affinity cleavage of yeast glutathione reductase

Effects of copper on the activity and oxidative inactivation of yeast glutathione reductase were analyzed. Glutathione reductase from yeast was inhibited by cupric ion and more potently by cuprous ion. Copper ion inhibited the enzyme noncompetitively with respect to the substrate GSSG and NADPH. The K_i values of the enzyme for Cu²⁺ and Cu⁺ ion were determined to be 1 and 0.35 μM, respectively. Copper-dependent inactivation of glutathione reductase was also analyzed. Hydrogen peroxide and copper/ascorbate also caused an inactivation with the cleavage of peptide bond of the enzyme. The inactivation/fragmentation of the enzyme was prevented by addition of catalase, suggesting that hydroxyl radical produced through the cuprous ion-dependent reduction of oxygen is responsible for the inactivation/fragmentation of the enzyme. SDS-PAGE and TOF–MS analysis confirmed eight fragments, which were further determined to result from the cleavage of the Met17-Ser18, Asn20-Thr21, Glu251-Gly252, Ser420-Pro421, Pro421-Thr422 bonds of the enzyme by amino-terminal sequencing analysis. Based on the kinetic analysis and no protective effect of the substrates, GSSG and NADPH on the copper-mediated inactivation/fragmentation of the enzyme, copper binds to the sites apart from the substrate-sites, causing the peptide cleavage by hydroxyl radical. Copper-dependent oxidative inactivation/fragmentation of glutathione reductase can explain the prooxidant properties of copper under the in vivo conditions.     

Copper catalyzed oxidation of ascorbate (vitamin C). Inhibitory effect of catalase, superoxide dismutase, serum proteins (ceruloplasmin, albumin, apotransferrin) and amino acids

The inhibitory effect of catalase and superoxide dismutase on copper catalyzed oxidation of ascorbate is probably due to a binding of copper ions. Scavengers of hydroxyl ions and singlet oxygen had no effect on the ascorbate oxidation rate. Copper binding serum proteins reduced the oxidation rate; the order of effectiveness being: Ceruloplasmin greater than human albumin = bovine albumin greater than apotransferrin. The excellent protection obtained with catalase and ceruloplasmin is possibly due to a strong affinity for cuprous ions generated during the reaction. Cupric ion binding amino acids (His, Thr, Glu, Gln, Tyr) had considerably weaker protective effect than the proteins studied. Apparently they do not compete favorably with ascorbate for cupric ions.     

Copper ion redox state is critical for its effects on ion transport pathways and methaemoglobin formation in trout erythrocytes.

We have studied the mechanism of copper uptake by the cells, its oxidative action and effects on ion transport systems using rainbow trout erythrocytes. Cupric ions enter trout erythrocytes as negatively charged complexes with chloride and hydroxyl anions via the band 3-mediated Cl-/HCO3- exchanger. Replacement of Cl- by gluconate, and complexation of cupric ions with histidine abolish rapid Cu2+ uptake. Within the cell cupric ions interact with haemoglobin, causing methaemoglobin formation by direct electron transfer from heme Fe2+ to Cu2+, and consecutive proton release. Ascorbate-mediated reduction of cupric ions to cuprous decreases copper-induced metHb formation and proton release. Moreover, cuprous ions stimulate Na+H+ exchange and residual Na+ transport causing net Na+ accumulation in the cells. The effect requires copper binding to an externally facing thiol group. Copper-induced Na+ accumulation is accompanied by K+ loss occurring mainly via K+-Cl- cotransporter. Taurine efflux is also stimulated by copper exposure. However, net loss of osmolytes is not as pronounced as Na+ uptake and modest swelling of the cells occurs after 5 min of copper exposure. Taken together the results indicate that copper toxicity, including copper transport into the cells and its interactions with ion transport processes, depend on the valency and complex formation of copper ions.     

The oxidation of Octopus vulgaris hemocyanin by nitrogen oxides

The reaction of Octopus vulgaris hemocyanin with nitrite was studied under a variety of conditions in which the green half-met derivative is formed. Analytical evidence shows that the amount of chemically detectable nitrite in various samples of the derivative is not proportional to the cupric copper detected by EPR. The kinetics of oxidation of hemocyanin as a function of protein concentration and pH, in the presence of nitrite and ascorbate, is consistent with a scheme in which NO2 is the reactive oxidant. We suggest that the green half-methemocyanin contains a metal center with one cuprous and one cupric copper without an exogenous nitrogen oxide ligand.     

A dileucine signal situated in the C-terminal tail of the lysosomal membrane protein p40 is responsible for its targeting to lysosomes

The suicide inactivation mechanism of tyrosinase acting on its substrates has been studied. The kinetic analysis of the proposed mechanism during the transition phase provides explicit analytical expressions for the concentrations of o-quinone against time. The electronic, steric and hydrophobic effects of the substrates influence the enzymatic reaction, increasing the catalytic speed by three orders of magnitude and the inactivation by one order of magnitude. To explain the suicide inactivation, we propose a mechanism in which the enzymatic form E(ox) (oxy-tyrosinase) is responsible for such inactivation. A key step might be the transfer of the C-1 hydroxyl group proton to the peroxide, which would act as a general base. Another essential step might be the axial attack of the o-diphenol on the copper atom. The rate constant of this reaction would be directly related to the strength of the nucleophilic attack of the C-1 hydroxyl group, which depends on the chemical shift of the carbon C-1 (delta(1)) obtained by (13)C-NMR. Protonation of the peroxide would bring the copper atoms together and encourage the diaxial nucleophilic attack of the C-2 hydroxyl group, facilitating the co-planarity with the ring of the copper atoms and the concerted oxidation/reduction reaction, and giving rise to an o-quinone. The suicide inactivation would occur if the C-2 hydroxyl group transferred the proton to the protonated peroxide, which would again act as a general base. In this case, the co-planarity between the copper atom, the oxygen of the C-1 and the ring would only permit the oxidation/reduction reaction on one copper atom, giving rise to copper(0), hydrogen peroxide and an o-quinone, which would be released, thus inactivating the enzyme.     

γ-Butyrobetaine hydroxylase: A unique protective effect of catalase

Catalase stimulates the activity of homogeneous γ-butyrobetaine hydroxylase by approximately 300-fold. The stimulation of the hydroxylation reaction elicited by catalase is saturable, and although a number of proteins may be substituted for catalase, none is as effective. γ-Butyrobetaine hydroxylase is also irreversibly inactivated in the presence of one of its substrates, oxygen, and its cofactor, ascorbate. This inactivation of the hydroxylase activity may be prevented by (i) the presence of high concentrations (2 mg/ml) of various proteins, (ii) the presence of catalytic concentrations (20 μg/ml) of catalase, or (iii) the presence of 10 mm histidine or dithiothreitol. Oxidized species of ascorbate do not appear to be responsible for the inactivation process. Time-dependent inactivation is also observed when γ-butyrobetaine hydroxylase is preincubated with hydrogen peroxide generated by the glucose oxidase-catalyzed oxidation of glucose. At low concentrations, superoxide dismutase was not as effective as an equivalent protein concentration of catalase in protecting against inactivation, and hydroxyl radical scavengers were completely ineffective. In measurements of γ-butyrobetaine hydroxylase activity, the presence of catalase both stimulates the catalytic activity of the hydroxylase and protects the enzyme from inactivation by a product of the interaction of components in the assay mixture, presumably hydrogen peroxide.     

In vitro lipid peroxidation of human serum catalyzed by cupric ion: antioxidant rather than prooxidant role of ascorbate.

The dual role of ascorbate as an antioxidant and a prooxidant has been clearly documented in the literature. Ascorbate acts as an antioxidant by protecting human serum from lipid peroxidation induced by azo dye-generated free radicals. On the other hand, ascorbate is readily oxidized in the presence of transition metal ions, (especially cupric ion) and accelerates lipid peroxidation in tissue homogenates by producing free radicals. Interestingly, we observed an antioxidant rather than an expected prooxidant role of ascorbate when human serum supplemented with 1.2mmol/L ascorbate underwent lipid peroxidations initiated by 2mmol/L copper sulfate. The antioxidant role of ascorbate was confirmed by studying the conventional thiobarbituric acid reactive substances (TBARS) as well as by observing the protective effect of ascorbate on the copper-induced peroxidation of unsaturated and polyunsaturated fatty acids. The antioxidation protection provided by ascorbate was comparable to that of equimolar alpha-tocopherol when incubated for 24h. However, lipid peroxidation products were lower in serum supplemented with alpha-tocopherol after 48h of incubation. This effect may be attributed to the binding of copper by plpha-tocopherol after serum proteins, thus preventing direct interaction between cupric ions and ascorbate. This proposed mechanism is based on the observation that the concentration of ascorbate decreased more slowly in serum than in phosphate buffer at physiological pH. Our results also indicate an outstanding anti-oxidant property of human serum due to the chelation of transition metal ions (even at high concentrations) by various serum proteins.     

Nonenzymatic cleavage of proteins by reactive oxygen species generated by dithiothreitol and iron

Many enzymes, represented by yeast glutamine synthetase, are inactivated and degraded in the presence of dithiothreitol (DTT), oxygen, and catalytic amounts of iron salts. The roles of DTT and iron can be replaced by ascorbate and copper, respectively. Experimental data suggest that reactive oxygen species, likely hydroxyl radicals, are generated locally around irons bound at specific sites on enzymes, and these species are responsible for the inactivation and degradation. Since many biochemicals are contaminated with metal salts in quantities sufficient for some hydroxyl radical formation to occur, the possibility of oxidative modification and degradation should be considered when an enzyme is exposed to DTT.     

Homocysteine metabolism and the oxidative modification of proteins and lipids

Altered homocysteine metabolism is implicated as a pathogenic factor in atherogenesis, neoplasia, and aging. Hereditary enzymatic deficiencies and nutritional deficiencies of folate, pyridoxine, or cobalamin are associated with elevated blood homocysteine, accelerated atherosclerosis, and manifestations of aging. The failure of malignant cells to metabolize homocysteine thiolactone to sulfate is attributed to deficiency of thioretinaco, a complex containing cobalamin, homocysteine thiolactone, and retinoic acid. The sulfhydryl group of homocysteine is believed to act catalytically with ferric or cupric ions in a mixed function oxidation system to generate hydrogen peroxide, oxygen radicals, and homocysteinyl radicals. These reactive species may interact with the active site of enzyme protein to cause inactivation of catalytic activity. Homocysteine thiolactone is oxidized to sulfae by a process involving ascorbate, thioretinamide, and superoxide, under the control of thyroxine and growth hormone. Thioretinaco is believed to be the active site of adenosine triphosphate (ATP) binding in oxidative phosphorylation with the participation of oxygen, ascorbate, proton gradient, and electron transport. Depletion of thioretinaco from mitochondrial and microsomal membranes may be associated with increased formation and release of radical oxygen species within neoplastic and senescent cells. Specific proposals are made for investigating the importance of homocysteine metabolism in the oxidative modification of proteins and lipids.     

Prooxidant Action of Maltol: Role of Transition Metals in the Generation of Reactive Oxygen Species and Enhanced Formation of 8-hydroxy-2′-deoxyguanosine Formation in DNA

Maltol (3-hydroxy-2-methyl-4-pyrone) produced reactive oxygen species as a complex with transition metals. Maltol/iron complex inactivated aconitase the most sensitive enzyme to oxidative stress. The inactivation of aconitase was iron-dependent, and prevented by TEMPOL, a scavenger of reactive oxygen species, suggesting that the maltol/iron-mediated generation of superoxide anion is responsible for the inactivation of aconitase. Addition of maltol effectively enhanced the ascorbate/copper-mediated formation of 8-hydroxy-2′-deoxyguanosine in DNA. Oxidation of ascorbic acid by CuSO₄ was effectively stimulated by addition of maltol, and the enhanced oxidation rate was markedly inhibited by the addition of catalase and superoxide dismutase. These results suggest that maltol can stimulate the copper reduction coupled with the oxidation of ascorbate, resulting in the production of superoxide radical which in turn converts to hydrogen peroxide and hydroxyl radical. Cytotoxic effect of maltol can be explained by its prooxidant properties: maltol/transition metal complex generates reactive oxygen species causing the inactivation of aconitase and the production of hydroxyl radical causing the formation of DNA base adduct.     

A site-specific mechanism for free radical induced biological damage: the essential role of redox-active transition metals

The metal-mediated site-specific mechanism for free radical-induced biological damage is reviewed. According to this mechanism, cooper- or iron-binding sites on macromolecules serve as centers for repeated production of hydroxyl radicals that are generated via the Fenton reaction. The aberrations induced by superoxide, ascorbate, isouramil, and paraquat are summarized. An illustrative example is the enhancement of double-strand breaks by ascorbate/copper. Prevention of the site-specific free radical damage can be accomplished by using selective chelators for iron and copper, by displacing these redox-active metals with other redox-inactive metals such as zinc, by introducing high concentrations of hydroxyl radicals scavengers and spin trapping agents, and by applying protective enzymes that remove superoxide or hydrogen peroxide. Histidine is a special agent that can intervene in free radical reactions in variety of modes. In biological systems, there are traces of copper and iron that are at high enough levels to catalyze free-radical reactions, and account for such deleterious processes. In the human body Fe/Cu = 80/1 (w/w). Nevertheless, both (free) copper and iron are soluble enough, and the rate constants of their reduced forms with hydrogen peroxide are sufficiently high to suggest that they might be important mediators of free radical toxicity.     

Effect of Additives on the Inactivation of Lysozyme Mediated by Free Radicals Produced in the Thermolysis of 2, 2-Azo-Bis-(2-Amidinopropane)

The inactivation of lysozyme caused by the radicals produced by thermolysis of 2, 2-azo-bis-2-amidino-propane can be prevented by the addition of different compounds that can react with the damaging free radicals. Compounds of high reactivity (propyl gallate, Trolox, cysteine, albumin, ascorbate, and NADH) afford almost total protection until their consumption, resulting in well-defined induction times. The number of radicals trapped by each additive molecule consumed ranges from 3 (propyl gallate) to 0.12 (cysteine). This last value is indicative of chain oxidation of the inhibitor. Uric acid is able to trap nearly 2.2 radicals per added molecule, but even at large (200 μM) concentrations, a residual inactivation of the enzyme is observed, which may be caused by urate-derived radicals.

Compounds of lower reactivity (tryptophan, Tempol, hydroquinone, desferrioxamine, diethylhydroxylamine, methionine, histidine, NAD⁺ and tyrosine) only partially decrease the lysozyme inactivation rates. For these compounds, we calculated the concentration necessary to reduce the enzyme inactivation rate to one half of that observed in the absence of additives. These concentrations range from 9 μM (tryptophan and Tempol) to 5 mM (NAD⁺).     

ESR study of the non-enzymic scission of xyloglucan by an ascorbate-H2O2-copper system: the involvement of the hydroxyl radical and the degradation of ascorbate

Xyloglucan is degraded by a mixture of copper(II), hydrogen peroxide and ascorbate. In the presence of ascorbate and/or hydrogen peroxide, copper(II) species were rapidly reduced to copper(I), which react with hydrogen peroxide. Spin-trapping experiments showed that hydroxyl radicals formed and attacked xyloglucan causing its degradation. The formation of a carbon-centred ascorbyl (C-ascorbyl) radical and its degradation with the formation of oxalate, was also caused by hydroxyl radicals. As a consequence, the features of the bis(oxalate) copper(II) complex clearly appeared in the frozen solution ESR spectra. The formation of carbon-centred radicals on the xyloglucan is the trigger for a series of possible molecular rearrangements which led to its oxidative scission.