Kinetics of superoxide scavenging by glutathione: an evaluation of its role in the removal of mitochondrial superoxide.

Superoxide radicals are produced in trace amounts by the mitochondrial respiratory chain. Most are removed rapidly by superoxide dismutase in the matrix. Superoxide is also known to react with glutathione. Reported values of the rate constant for this reaction range from 10(2) to in excess of 10(5) M(-1).s(-1). The magnitude of this rate constant has important physiological implications because, if it is at the upper end of the reported range, a significant proportion of mitochondrial superoxide will evade removal by superoxide dismutase, and will oxidize glutathione to the potentially harmful glutathionyl radical. Using EPR spectroscopy to monitor competition between glutathione and the spin trap 5,5-dimethyl-1-pyrroline N-oxide for reaction with superoxide, we have estimated that the rate constant for the reaction between superoxide and glutathione is only approximately 200 M(-1).s(-1). Hence superoxide dismutase will always out-compete glutathione for reaction with the superoxide radical, thereby preventing formation of the glutathionyl radical.

A critical overview of the chemistry of copper-dependent low density lipoprotein oxidation: roles of lipid hydroperoxides, alpha-tocopherol, thiols, and ceruloplasmin.

The mechanisms by which low-density lipoprotein (LDL) particles undergo oxidative modification to an atherogenic form that is taken up by the macrophage scavenger-receptor pathway have been the subject of extensive research for almost two decades. The most common method for the initiation of LDL oxidation in vitro involves incubation with Cu(II) ions. Although various mechanisms have been proposed to explain the ability of Cu(II) to promote LDL modification, the precise reactions involved in initiating the process remain a matter of contention in the literature. This review provides a critical overview and evaluation of the current theories describing the interactions of copper with the LDL particle. Following discussion of the thermodynamics of reactions dependent upon the decomposition of preexisting lipid hydroperoxides, which are present in all crude LDL preparations, attention is turned to the more difficult (but perhaps more physiologically-relevant) system of the hydroperoxide-free LDL particle. In both systems, the key role of alpha-tocopherol is discussed. In addition to its protective, radical-scavenging action, alpha-tocopherol can also behave as a prooxidant via its reduction of Cu(II) to Cu(I). Generation of Cu(I) greatly facilitates the decomposition of lipid hydroperoxides to chain-carrying radicals, but the mechanisms by which the vitamin promotes LDL oxidation in the absence of preformed hydroperoxides remain more speculative. In addition to the so-called tocopherol-mediated peroxidation model, in which polyunsaturated fatty acid oxidation is initiated by the alpha-tocopheroxyl radical (generated during the reduction of Cu(II) by alpha-tocopherol), an evaluation of the role of the hydroxyl radical is provided. Important interactions between copper ions and thiols are also discussed, particularly in the context of cell-mediated LDL oxidation. Finally, the mechanisms by which ceruloplasmin, a copper-containing plasma protein, can bring about LDL modification are discussed. Improved understanding of the mechanisms of LDL oxidation by copper ions should facilitate the establishment of any physiological role of the metal in LDL modification. It will also assist in the interpretation of studies in which copper systems of LDL oxidation are used in vitro to evaluate potential antioxidants.

Cytochrome C is a potent catalyst of dichlorofluorescin oxidation: implications for the role of reactive oxygen species in apoptosis.

The generation of reactive oxygen species has been suggested to occur at increased rates during apoptosis, but the validity and significance of this remain contentious. In several key studies levels of reactive oxygen species have been monitored using the intracellular probe dichlorofluorescin (DCFH(2)), which undergoes oxidation to the fluorescent dichlorofluorescein (DCF). We report here that cytochrome c, which is released from mitochondria during cell death, is a potent catalyst of DCF formation. In a model system using xanthine oxidase to generate superoxide radicals, the rate of DCF formation was insensitive to changes in the rate of superoxide production over a 17-fold range, but extremely sensitive to nanomolar concentrations of cytochrome c. Thus we conclude that the DCF fluorescence observed in dying cells is a reflection of increased cytosolic cytochrome c. Moreover, we suggest that the suppression of DCF formation by the anti-apoptotic oncoprotein Bcl-2, which has been suggested to have antioxidant properties, can be explained on the basis of its prevention of mitochondrial cytochrome c release.

A simple, highly sensitive and improved method for the measurement of bleomycin-detectable iron: the 'catalytic iron index' and its value in the assessment of iron status in haemochromatosis.

In the presence of ferrous ions (Fe(2+)), the anti-tumour agent bleomycin will induce DNA degradation. Degradation of DNA into substances detectable by the thiobarbituric acid test has been used previously for the detection of iron in a form that is capable of catalysing the formation of the potentially harmful hydroxyl free radical. In the present paper, we describe the application of the ethidium-binding assay of DNA damage to the measurement of bleomycin-detectable iron, comparing its performance with the conventional method in the assessment of iron standard solutions and plasma samples from haemochromatosis patients. The ethidium-binding assay proved to be more responsive than the thiobarbituric acid test in the detection of DNA damage induced by very low concentrations of iron, but became saturated at higher iron concentrations. Agreement between the two versions of the assay in the identification of plasma samples containing bleomycin-detectable iron was good, but agreement on the actual concentrations of such iron in the positive samples was poor. This discrepancy is believed to be due to interference with the thiobarbituric acid assay by plasma. Consequently, it was not possible to obtain reliable estimates of free iron concentrations in plasma when using the conventional version of the bleomycin assay. We have devised a parameter of iron status called the catalytic iron index. For healthy, non-haemochromatotic individuals, the mean value of this parameter was found to be 0.81 (range 0.78-0.84; n=20). Elevated values were observed in some plasma samples from haemochromatosis patients, but these showed no correlation with serum ferritin levels. In contrast, correlations were seen with both serum iron and transferrin saturation levels, but only when these were above the normal range.

Effects of trans-resveratrol on copper-dependent hydroxyl-radical formation and DNA damage: evidence for hydroxyl-radical scavenging and a novel, glutathione-sparing mechanism of action.

Resveratrol (3,5,4'-trihydroxy-trans-stilbene) is a natural product occurring in grapes and various other plants with medicinal properties. The phenolic antioxidant has been identified as a potential cancer chemopreventative agent and its presence in red wine has been suggested to be linked to the low incidence of heart disease in some regions of France. Recently, however, resveratrol was reported to promote DNA fragmentation in the presence of copper ions (K. Fukuhara and N. Miyata, 1998, Bioorg. Med. Chem. Lett. 8, 3187-3192), prompting us to investigate this phenomenon in mechanistic detail. By acting as a reducing agent, resveratrol was found to promote hydroxyl-radical (*OH) formation by DNA-bound Cu(H) ions. However, in the presence of either ascorbic acid or glutathione (i.e., under more physiological conditions), the phenolic lost this property and behaved as an antioxidant. In the ascorbate system, resveratrol had no effect on the rate of *OH formation, but protected DNA from damage by acting as a radical-scavenging antioxidant. In contrast, in the glutathione system, resveratrol inhibited *OH formation via a novel mechanism involving the inhibition of glutathione disulfide formation. We have concluded, therefore, that the DNA-damaging properties of resveratrol, identified recently by Fukuhara and Miyata, will be of no significance under physiological conditions. To the contrary, we have demonstrated that the phenolic behaves as a powerful antioxidant, both via classical, hydroxyl-radical scavenging and via a novel, glutathione-sparing mechanism.

trans-resveratrol protects embryonic mesencephalic cells from tert-butyl hydroperoxide: electron paramagnetic resonance spin trapping evidence for a radical scavenging mechanism.

In recent years, the antioxidant and other pharmacological properties of resveratrol, a natural product present in grapes and wine, have attracted considerable interest from the biomedical research community. In an examination of the potential neuroprotective properties of the compound, we have investigated the ability of resveratrol to protect rat embryonic mesencephalic tissue, rich in dopaminergic neurones, from the prooxidant tert-butyl hydroperoxide. Using the electron paramagnetic resonance (EPR) spin-trapping technique, the main radicals detected in cell suspensions were the tert-butoxyl radical and the methyl radical, indicating the one-electron reduction of the peroxide followed by a beta-scission reaction. The appearance of EPR signals from the trapped radicals preceded the onset of cytotoxicity, which was almost exclusively necrotic in nature. The inclusion of resveratrol in incubations resulted in the marked protection of cells from tert-butyl hydroperoxide. In parallel spin-trapping experiments, we were able to demonstrate the scavenging of radicals by resveratrol, which involved direct competition between resveratrol and the spin trap for reaction with the radicals. To our knowledge, this is the first example in which cytoprotection by resveratrol has been demonstrated by EPR spin-trapping competition kinetics to be due to its scavenging of the radicals responsible for the toxicity of a prooxidant.

Extent of incorporation of hydroxycinnamaldehydes into lignin in cinnamyl alcohol dehydrogenase-downregulated plants.

Down-regulation of cinnamyl alcohol dehydrogenase leads to an accumulation of cinnamaldehydes available for incorporation into the developing lignin polymer. Using electron spin resonance spectroscopy we have demonstrated that the parent radical of 4-hydroxy-3-methoxycinnamaldehyde is generated by peroxidase catalysed oxidation. The extent of radical generation is similar to that of 4-hydroxy-3-methoxycinnamyl alcohol and is increased by further aromatic methoxylation. From the distribution of the electron-spin density, it was predicted that the regiochemistry of 4-hydroxy-3-methoxycinnamaldehyde coupling would be similar to that of the corresponding alcohol, with the possibility of a higher degree of 8-O-4 linkages occurring. These predictions were confirmed by polymerisation studies, which also showed that after radical coupling the alpha,beta-enone structure was regenerated. This suggests that, although the cross-linking and physical properties of cinnamaldeyde rich lignins differ from that of normal lignins, cinnamaldehydes are incorporated into the lignin polymer under the same controlling factors as the cinnamyl alcohols.

Alpha-tocopherol induces oxidative damage to DNA in the presence of copper(II) ions.

There is currently much interest in the possibility that dietary antioxidants may confer protection from certain diseases, such as atherosclerosis and cancer. The importance of alpha-tocopherol (vitamin E) as a biological antioxidant is widely recognized. However, pro-oxidant properties of alpha-tocopherol have been observed in chemical systems, and it has been reported that the vitamin can induce tumor formation and act as a complete tumor promotor in laboratory animals. In the present communication, we find that alpha-tocopherol can act as a potent DNA-damaging agent in the presence of copper(II) ions, using a simplified, in vitro model. alpha-Tocopherol was found to promote copper-dependent reactive oxygen species formation from molecular oxygen, resulting in DNA base oxidation and backbone cleavage. Neither alpha-tocopherol nor Cu(II) alone induced DNA damage. Bathocuproine, a Cu(I)-specific chelator, and catalase inhibited the DNA damage, whereas free hydroxyl radical scavengers did not. The order of DNA cleavage sites was thymine, cytosine > guanine residues. Examinations using an oxygen electrode and cytochrome c indicate that molecular oxygen was consumed in the reaction of alpha-tocopherol and Cu(II) and that superoxide was formed. Stoichiometry studies showed that two Cu(II) ions could be reduced by each alpha-tocopherol molecule. Electron spin resonance spin-trapping investigations were then used to demonstrate that hydrogen peroxide interacts with Cu(I) to generate the reactive species responsible for DNA damage, which is either the hydroxyl radical or a species of similar reactivity. These findings may be of relevance to the tumorigenic properties of the vitamin reported in the literature. However, further studies are required to establish the significance of these reactions under in vivo conditions.

Dithiocarbamate toxicity toward thymocytes involves their copper-catalyzed conversion to thiuram disulfides, which oxidize glutathione in a redox cycle without the release of reactive oxygen species.

We have reported previously that diethyldithio-carbamate (DDC) and pyrrolidine dithiocarbamate (PDTC) induce apoptosis in rat thymocytes. Apoptosis was shown to be dependent upon the transport of external Cu ions into the cells and was accompanied by the oxidation of intracellular glutathione, indicating the inducement of pro-oxidative conditions (C. S. I. Nobel, M. Kimland, B. Lind, S. Orrenius, and A. F. G. Slater, J. Biol. Chem. 270, 26202-26208, 1995). In the present investigation we have examined the chemical reactions underlying these effects. Evidence is presented to suggest that dithiocarbamates undergo oxidation by CuII ions, resulting in formation of the corresponding thiuram disulfides, which are then reduced by glutathione, thereby generating the parent dithiocarbamate and oxidized glutathione (glutathione disulfide). Although DDC and PDTC were found to partially stabilize CuI ions, limited redox cycling of the metal ion was evident. Redox cycling did not, however, result in the release of reactive oxygen species, which are believed to be scavenged in situ by the dithiocarbamate. DDC and PDTC were, in fact, shown to prevent copper-dependent hydroxyl radical formation and DNA fragmentation in model reaction systems. The thiuram disulfide disulfiram (DSF) was found to induce glutathione oxidation, DNA fragmentation, and cell killing more potently than its parent dithiocarbamate, DDC. Of particular importance was the finding that, compared with DDC, the actions of DSF were less prone to inhibition by the removal of external copper ions with a chelating agent. This observation is consistent with our proposed mechanism of dithiocarbamate toxicity, which involves their copper-catalyzed conversion to cytotoxic thiuram disulfides.

Mechanism of dithiocarbamate inhibition of apoptosis: thiol oxidation by dithiocarbamate disulfides directly inhibits processing of the caspase-3 proenzyme.

Dithiocarbamates (DCs) have been reported to be potent inhibitors of apoptosis in several different model systems, which suggests a target common to the apoptotic machinery. Without further investigation, this has been assumed to reflect an antioxidant activity of the DCs. However, we have recently shown that DCs exert prooxidant effects on T cells [Nobel et al. (1995) J. Biol. Chem. 270, 26202-26208], which are dependent on their transfer of external copper into the cells and can be inhibited by the inclusion of high-affinity external copper chelators in the medium. Investigating antiapoptotic actions of DCs, we found that inclusion of a membrane-impermeable copper chelator severely compromised the inhibitory activity of reduced DCs. Since copper can promote DC oxidation to the respective DC disulfides, the inhibitory effect on lymphocyte apoptosis might be mediated by the DC disulfides. In agreement with this we observed that DC disulfides were more potent inhibitors of T cell apoptosis than their reduced counterparts. Inhibition of apoptosis by DC disulfides correlated with the inhibition of caspase-3 proenzyme processing and activation. Similar results were obtained in a cell-free model system of caspase-3 activation. Significantly, dithiothreitol reduction of the DC disulfide abolished its inhibition of in vitro proenzyme processing, thereby demonstrating thiol-disulfide exchange between the DC disulfide and a free thiol group on an activator(s) of caspase-3. Since T cell apoptosis involves the generation of mature caspase-3 and requires caspase-3-like activity, we propose that (1) DC disulfides are the active agents behind DC inhibition of apoptosis and (2) their site of action is the proteolytic activation of this enzyme. These findings also reveal the potential for other thiol-oxidizing toxicants to inhibit apoptosis by preventing the proteolytic activation of caspases.

Oxidative coupling during lignin polymerization is determined by unpaired electron delocalization within parent phenylpropanoid radicals.

The high degree of selectivity observed in the incorporation of phenylpropanoids into lignin may be a consequence of the influence exerted by methoxyl substituents on the ambident radicals generated during biosynthesis. Since unpaired electron distribution may be regarded as an important factor in determining positional selectivity during oxidative coupling, electron spin resonance spectroscopy and Austin Model 1 molecular computation were used to study the effects of methoxyl substitution on unpaired electron distribution in lignin precursor radicals. The data obtained were used to predict the effect of substitution on coupling and were compared with the linkage types observed in complementary dehydrogenation polymerization studies employing each of the lignin precursors. We report that methoxyl substitution increases unpaired electron density on the phenolic oxygen of the precursor phenylpropanoid radicals and that this subsequently determines the nature of the bond formation during polymerization.

Research communication copper-1,10-phenanthroline induces internucleosomal DNA fragmentation in HepG2 cells, resulting from direct oxidation by the hydroxyl radical.

In view of the current speculation regarding the possible role of reactive oxygen species (ROS) in apoptosis, both under physiological conditions and in response to chemicals that promote their intracellular formation, the present investigation was undertaken to examine whether DNA fragmentation during oxidative stress results from endonuclease activity (apoptosis) or from direct attack by ROS. We report that the incubation of HepG2 cells (a human-derived hepatoma cell line) with the copper(II) complex of 1,10-phenanthroline, CuII(OP)2, results in internucleosomal DNA fragmentation, which is widely recognized as being a hallmark of apoptosis. DNA fragmentation did not occur at low temperature, but activity was restored by the addition of ascorbic acid. It is proposed that DNA fragmentation results from the direct attack of hydroxyl radicals upon DNA. Hydroxyl radicals are produced from oxygen by the redox-cycling of CuII(OP)2, which is supported by metabolic processes at normal temperature. At low temperature ascorbic acid provides an artificial cellular reducing environment, thereby restoring hydroxyl radical formation. These findings were confirmed by the detection of internucleosomal DNA fragmentation following the exposure of isolated chromatin to a biomimetic CuII(OP)2 redox-cycling system. We conclude that DNA laddering, the widely employed hallmark of apoptosis, is not unique to endonuclease activity and may also result from direct attack upon DNA by the hydroxyl radical.

1,10-Phenanthroline stimulates internucleosomal DNA fragmentation in isolated rat-liver nuclei by promoting the redox activity of endogenous copper ions.

Isolated rat-liver nuclei were incubated with a series of membrane-permeable metal-ion-complexing agents and examined for DNA damage. Of the reagents tested, only 1,10-phenanthroline (OP) and neocuproine (NC) were found to induce DNA fragmentation. Agarose-gel electrophoresis of the DNA fragments generated in the presence of OP revealed internucleosomal cleavage, which is widely considered to be a hallmark for the enzymic DNA digestion that occurs during apoptosis. Ascorbate, particularly in the presence of hydrogen peroxide, increased the levels of fragmentation induced by OP. As well as undergoing fragmentation, the DNA from nuclei was also found to contain 8-hydroxydeoxyguanosine, which indicates attack (oxidation) by the hydroxyl radical. Complementary experiments in vitro involving ESR determinations of hydroxyl radical formation and measurements of DNA oxidation under biomimetic conditions demonstrated that Cu2+, but not Fe3+, forms a complex with either OP or NC (but not the other complexing agents tested) that stimulates hydroxyl radical formation and DNA damage in the presence of hydrogen peroxide and ascorbate. It is therefore proposed that OP in the nuclei incubations binds to Cu2+, which exists naturally in chromosomes, forming a complex that promotes hydroxyl-radical-dependent DNA fragmentation. These findings demonstrate the promotion of hydroxyl-radical-mediated DNA damage by endogenous Cu2+ and, perhaps more significantly, demonstrate that the internucleosomal DNA 'laddering' that is often used as an indicator of apoptosis may also result from DNA fragmentation by non-enzymic processes.

Hydroxyl radical formation from Cu(II)-trolox mixtures: insights into the pro-oxidant properties of alpha-tocopherol.

Although widely recognised as the most important chain-breaking antioxidant of the lipid phase, alpha-tocopherol has also been reported to exert pro-oxidant activity, particularly during the Cu(II)-stimulated oxidation of low density lipoproteins (LDL). In the present communication, we demonstrate that hydroxyl radicals are generated following the interaction of Cu(II) with the alpha-tocopherol model compound Trolox, involving the reduction of Cu(II) by Trolox and the subsequent reduction of molecular oxygen by Cu(I). We suggest, therefore, that the hydroxyl radical may be the species responsible for the initiation of fatty acid oxidation during the Cu(II)-stimulated oxidation of hydroperoxide-free LDL.

Generation of 5,5-dimethyl-1-pyrroline N-oxide hydroxyl and scavenger radical adducts from copper/H2O2 mixtures: effects of metal ion chelation and the search for high-valent metal-oxygen intermediates.

A metal-catalyzed nucleophilic addition mechanism for the formation of radical adducts of the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) has been described recently (K. Makino, T. Hagiwara, A. Hagi, M. Nishi, and A. Murakami, 1990, Biochem. Biophys. Res. Commun. 172, 1073-1080; P. M. Hanna, W. Chamulitrat, and R. P. Mason, 1992, Arch. Biochem. Biophys. 296, 640-644). In the present investigation, we have demonstrated that the recently reported inhibition of copper-dependent hydroxyl radical formation by the complexing agent 1,10-phenanthroline (OP), which appears to contradict the well-known chemical nuclease properties of CuI(OP)2, is an artifact resulting from an inhibition of the nucleophilic addition of water to DMPO by OP (A. C. Mello-Filho and R. Meneghini, 1991, Mutat. Res. 251, 109-113). Copper bound to OP was found to be a good catalyst of hydroxyl radical formation: the CuII(OP)2 complex can be reduced by H2O2 and the CuI(OP)2 generated reacts with the peroxide to form .OH. In contrast, no evidence could be obtained for oxidant formation from the CuII(aq)/H2O2 reaction system, despite the detection of a prominent signal from the DMPO hydroxyl radical adduct (DMPO/.OH) (the formation of which was due solely to the nucleophilic addition of water to DMPO). The failure to generate an oxidant in this reaction mixture was attributed to the failure of hydrogen peroxide to reduce CuII(aq), as hydroxyl radical formation did occur when CuI(aq) was added directly to H2O2. However, in order to account for the high concentration of alpha-hydroxyethanol radicals detected when ethanol was included in the CuI(aq)/H2O2 reaction, the possibility that an oxidant in addition to .OH (e.g., CuO+) is generated is discussed.

Iron supplementation generates hydroxyl radical in vivo. An ESR spin-trapping investigation.

Electron spin resonance (ESR) spectroscopy has been used to investigate hydroxyl radical generation in rats with chronic dietary iron loading. A secondary radical spin-trapping technique was used where hydroxyl radical forms methyl radical upon reaction with DMSO. The methyl radical was then detected by ESR spectroscopy as its adduct with the spin trap alpha-phenyl-N-t-butylnitrone (PBN). This adduct was detected in the bile of rats 10 wk after being fed an iron-loading diet and 40 min after the i.p. injection of the spin trap PBN dissolved in DMSO. Bile samples were collected into a solution of the ferrous stabilizing chelator 2,2'-dipyridyl in order to prevent the generation of radical adducts ex vivo during bile collection. Identification of the ESR spectrum of the major radical adduct as that of PBN/.CH3 provides evidence for the generation of the hydroxyl radical during iron supplementation. Desferal completely inhibited in vivo hydroxyl radical generation stimulated by high dietary iron intake. No radical adducts were detected in rats which were fed the control diet for the same period of time. This is the first evidence of hydroxyl radical generation in chronic iron-loaded rats.

Mitochondrial metabolism of a hydroperoxide to free radicals in human endothelial cells: an electron spin resonance spin-trapping investigation.

Oxidative damage to the vascular endothelium may be an important event in the promotion of atherosclerosis. Several lines of evidence suggest that lipid hydroperoxides may be responsible for the induction of such damage. Hydroperoxides cause loss of endothelial cell integrity, increase the permeability of the endothelium to macromolecules, and compromise its ability to control vascular tone via the secretion of vasoactive molecules in response to receptor stimulation. The molecular mechanisms responsible for these effects are, however, poorly understood. In this paper, we describe an e.s.r. spin-trapping investigation into the metabolism of the model hydroperoxide compound tert-butylhydroperoxide to reactive free radicals in intact human endothelial cells. The hydroperoxide is shown to undergo a single electron reduction to form free radicals. Experiments with metabolic poisons indicate that the mitochondrial electron-transport chain is the source of electrons for this reduction. The metal-ion-chelating agent desferrioxamine was found to prevent cell killing by tert-butylhydroperoxide, but did not affect free radical formation, suggesting that free metal ions may serve to promote free-radical chain reactions involved in cell killing following the initial conversion of the hydroperoxide to free radicals by mitochondria. These processes may well be responsible for many of the reported effects of hydroperoxides on endothelial cell integrity and function.

Detection of oxygen-derived radicals in biological systems using electron spin resonance.

Oxygen-centered radicals, particularly the hydroxyl and superoxide radicals, have been postulated in many biochemical reactions and have been implicated in many adverse reactions in vivo. This article begins with a review of spin-trapping detection of oxygen-centered radicals in vitro and concludes with a presentation of our approach to the detection of the hydroxyl radicals in models of acute iron and copper poisoning.

Calcium indicator dye Quin2 inhibits hydrogen peroxide-induced DNA strand break formation via chelation of iron.

Exposure of HeLa cells to H2O2 at 4 degrees C caused DNA strand breakage which was prevented by the metal chelator 1,10-phenanthroline, but not by neocuproine. This is believed to indicate the participation of iron, rather than copper, in the formation of reactive hydroxyl radicals (.OH) from H2O2. The calcium indicator dye Quin2 also prevented H2O2-induced DNA fragmentation. The inhibition of oxidant-induced DNA fragmentation at 37 degrees C by Quin2 is often presented as evidence for the involvement of Ca(2+)-dependent endonucleases in damage. However, our finding that Quin2 inhibits DNA fragmentation at 4 degrees C led us to investigate the possibility that Quin2 may also inhibit DNA damage via its effects on metal-dependent .OH formation. Using ESR spin trapping techniques and in vitro DNA oxidation measurements, we found that the binding of Fe by Quin2 does not prevent .OH formation, but inhibits DNA damage. This is believed to reflect the prevention of iron ion binding to DNA by Quin2, directing .OH generation to the bulk solution, thereby preventing damage. The Cu-Quin2 complex was found to be a poor catalyst of both .OH formation and in vitro DNA damage. These findings suggest that the frequently reported protective effect of Quin2 toward DNA in cells exposed to oxidants may be due to the chelation of metal ions rather than the prevention of Ca(2+)-dependent endonuclease activation.