Gliotoxin induces apoptosis in macrophages unrelated to its antiphagocytic properties

We have previously shown that the fungal metabolite and immunomodulating agent gliotoxin induces apparently random double-stranded fragmentation of genomic DNA in a variety of cell types and double- and single-stranded scission in isolated plasmid DNA. The in vitro damage to plasmid DNA appears to be mediated by reactive oxygen species, but the mechanism of damage to genomic DNA is not yet known. In this paper we show that treatment of macrophages with gliotoxin and some analogues gives rise to discrete DNA fragments with molecular weight 170 +/- 30 base pairs. This pattern of DNA fragmentation has the characteristics of apoptosis, a programmed form of cell death. Three structural analogues of gliotoxin and two S-acetylated precursors capable of intracellular hydrolysis to the thiol form induce identical DNA degradation patterns. Only those compounds with the epipolythiodioxopiperazine (ETP) bridged disulfide structure or those capable of extracellular conversion to ETP compounds are equipotent with gliotoxin in their effects on macrophage phagocytosis, although all are capable of generating reactive oxygen species intracellularly. These results suggest that the effect of gliotoxin on macrophage function as assessed by adherence to plastic surfaces is unrelated to DNA damage and in addition suggests a new mechanism by which the toxin and other ETP compounds may damage cells.     

DNA fragmentation induced in macrophages by gliotoxin does not require protein synthesis and is preceded by raised inositol triphosphate levels.

We have shown that the immunomodulating agent gliotoxin induces DNA fragmentation in macrophages characteristic of programmed cell death or apoptosis (Waring, P., Eichner, R. D., Mullbacher, A., and Sjaarda, A. (1988) J. Biol. Chem, 263, 18493-18499). In addition, morphological changes and DNA fragmentation characteristic of apoptosis are induced in 48 h concanavalin A-stimulated T blasts by gliotoxin and these changes are inhibited by Zn2+ (Waring, P., Egan, M., Braithwaite, A., Mullbacher, A., and Sjaarda, A. (1990) Int. J. Immunopharmacol., in press). We have studied the effects of actinomycin D and the protein synthesis inhibitor cycloheximide on apoptosis induced by gliotoxin in these cells, and these studies demonstrate no effect on apoptosis induced by gliotoxin. Cycloheximide and actinomycin D alone induce DNA fragmentation in these cells. Gliotoxin itself proved to be a potent inhibitor of protein synthesis. The fragmentation caused by cycloheximide correlated with the extent of protein synthesis inhibition. The toxin ricin also induced DNA fragmentation in T blasts characteristic of apoptosis. These results indicate that protein synthesis is not required for induction of apoptosis in macrophages or T blasts by gliotoxin. Gliotoxin caused elevated levels of inositol triphosphate in treated macrophages which may be related to mobilization of Ca2+ levels during apoptosis.     

DNA damage induces reactive oxygen species generation through the H2AX-Nox1/Rac1 pathway

The DNA damage response (DDR) cascade and ROS (reactive oxygen species) signaling are both involved in the induction of cell death after DNA damage, but a mechanistic link between these two pathways has not been clearly elucidated. This study demonstrates that ROS induction after treatment of cells with neocarzinostatin (NCS), an ionizing radiation mimetic, is at least partly mediated by increasing histone H2AX. Increased levels of ROS and cell death induced by H2AX overexpression alone or DNA damage leading to H2AX accumulation are reduced by treating cells with the antioxidant N-Acetyl-L-Cysteine (NAC), the NADP(H) oxidase (Nox) inhibitor DPI, expression of Rac1N17, and knockdown of Nox1, but not Nox4, indicating that induction of ROS by H2AX is mediated through Nox1 and Rac1 GTPase. H2AX increases Nox1 activity partly by reducing the interaction between a Nox1 activator NOXA1 and its inhibitor 14-3-3zeta. These results point to a novel role of histone H2AX that regulates Nox1-mediated ROS generation after DNA damage.     

Genotoxicity of gliotoxin,a secondary metabolite of Aspergillus fumigatus,in a battery of short-term test systems

The genotoxic effects of gliotoxin, a known fungal secondary metabolite, were studied. Gliotoxin was purified from cultivation medium of Aspergillus fumigatus isolated from the indoor air of a moisture problem house. The genotoxicity of gliotoxin was assessed both in bacterial test systems including bacterial repair assay, Ames Salmonella assay and SOS-chromotest, and in mammalian cells using single cell gel (SCG) electrophoresis assay and sister-chromatid exchange (SCE) test. Gliotoxin was found to be genotoxic in the bacterial repair assay but, not in the Salmonella test or SOS-chromotest. A dose-related increase in DNA damage was observed in mouse RAW264.7 macrophages exposed to gliotoxin for 2h in plain medium in the SCG assay. In contrast to the positive response in the SCG assay, gliotoxin did not induce any clear, dose-related increase in SCEs in Chinese hamster ovary (CHO) cells.     

Inactivation of rabbit muscle creatine kinase by reversible formation of an internal disulfide bond induced by the fungal toxin gliotoxin

The biological activity of gliotoxin is dependent on the presence of a strained disulfide bond that can react with accessible cysteine residues on proteins. Rabbit muscle creatine kinase contains 4 cysteines per 42-kDa subunit and is active in solution as a dimer. Only Cys-282 has been identified as essential for activity. Modification of this residue results in loss of activity of the enzyme. Treatment of creatine kinase with gliotoxin resulted in a time-dependent loss of activity abrogated in the presence of reducing agents. Activity was restored when the inactivated enzyme was treated with reducing agents. Inactivation of creatine kinase by gliotoxin was accompanied by the formation of a 37-kDa form of the enzyme. This oxidized form of creatine kinase was rapidly reconverted to the 42-kDa species by the addition of reducing agents concomitant with restoration of activity. A 1:1 mixture of the oxidized and reduced monomer forms of creatine kinase as shown on polyacrylamide gel electrophoresis was equivalent to the activity of the fully reduced form of the enzyme consistent with only one reduced monomer of the dimer necessary for complete activity. Conversion of the second monomeric species of the dimer to the oxidized form by gliotoxin correlated with loss of activity. Our data are consistent with gliotoxin inducing the formation of an internal disulfide bond in creatine kinase by initially binding and possibly activating a cysteine residue on the protein, followed by reaction with a second neighboring thiol. The recently published crystal structure of creatine kinase suggests the disulfide is formed between Cys-282 and Cys-73.     

A new and rapid bioassay for the detection of gliotoxin and related epipolythiodioxopiperazines produced by fungi

Gliotoxin is an immunosuppressive cytotoxin produced by numerous environmental or pathogenic fungal species. For this reason, it is one of the mycotoxins which must be systematically searched for in samples for biological control. In this study, a new, rapid and sensitive method for detecting gliotoxin has been developed. This bioassay is based on the induction of morphological changes in cultured cells (human KB cell line) by gliotoxin. Interpretation of the assay can be carried out after 1 h of incubation, either by direct microscopic observation, or with an automated microplate-reader at 630 nm. The limit of detection is 18-20 ng of gliotoxin in the well, depending on the used observation method. A high degree of specificity of the detection is brought about by the ability of the reducing reactant dithiothreitol to inhibit the biological activities of epipolythiodioxopiperazines (ETPs), such as gliotoxin, by reducing their polysulfide bridge. The bioassay allows a rapid primary screening of samples and a semi-quantitative evaluation of the gliotoxin concentration in extracts. The method has been used to study the gliotoxin production by different fungal strains, allowing to highlight 3 strains of Aspergillus fumigatus producing gliotoxin in various extracts.     

Protective action of melatonin against oxidative DNA damage: chemical inactivation versus base-excision repair

Melatonin is a hormone-like substance that has a variety of beneficial properties as regulator of the circadian rhythm and as anti-inflammatory and anti-cancer agent. The latter activity can be linked with the ability of melatonin to protect DNA against oxidative damage. It may exert such action either by scavenging reactive oxygen species or their primary sources, or by stimulating the repair of oxidative damage in DNA. Since such type of DNA damage is reflected in oxidative base modifications that are primarily repaired by base-excision repair (BER), we tried to investigate in the present work whether melatonin could influence this DNA-repair system. We also investigated the ability of melatonin to inactivate hydrogen peroxide, a potent source of reactive oxygen species. Melatonin at 50 microM and its direct metabolite N(1)-acetyl-N(2)-formyl-5-methoxykynuramine reduced DNA damage induced by hydrogen peroxide at approximately the same ratio. Melatonin stimulated the repair of DNA damage induced by hydrogen peroxide, as assessed by the alkaline comet assay. However, melatonin at 50 microM had no impact on the activity in vitro of three glycosylases playing a pivotal role in BER: Endo III, Fpg and ANPG 80. On the other hand, melatonin chemically inactivated hydrogen peroxide, reducing its potential to damage DNA. And finally, melatonin did not influence the repair of an a-basic (AP) site by cellular extracts, as was evaluated by a functional BER assay in vitro. In conclusion, melatonin can have a protective effect against oxidative DNA damage by chemical inactivation of a DNA-damaging agent as well as by stimulating DNA repair, but key factors in BER, viz. glycosylases and AP-endonucleases, do not seem to be affected by melatonin. Further study with other components of the BER machinery and studies aimed at other DNA-repair systems are needed to clarify the mechanism underlying the stimulation of DNA repair by melatonin.     

Genotoxicity of gliotoxin, a secondary metabolite of Aspergillus fumigatus, in a battery of short-term test systems

The genotoxic effects of gliotoxin, a known fungal secondary metabolite, were studied. Gliotoxin was purified from cultivation medium of Aspergillus fumigatus isolated from the indoor air of a moisture problem house. The genotoxicity of gliotoxin was assessed both in bacterial test systems including bacterial repair assay, Ames Salmonella assay and SOS-chromotest, and in mammalian cells using single cell gel (SCG) electrophoresis assay and sister-chromatid exchange (SCE) test. Gliotoxin was found to be genotoxic in the bacterial repair assay but, not in the Salmonella test or SOS-chromotest. A dose-related increase in DNA damage was observed in mouse RAW264.7 macrophages exposed to gliotoxin for 2 h in plain medium in the SCG assay. In contrast to the positive response in the SCG assay, gliotoxin did not induce any clear, dose-related increase in SCEs in Chinese hamster ovary (CHO) cells.     

Roles of vitamin C in radiation-induced DNA damage in presence and absence of copper

Exposure to either ionizing radiation or certain transition metals results in generation of reactive oxygen species that induce DNA damage, mutation, and cancer. Vitamin C (a reactive oxygen scavenger) is considered to be a dietary radioprotective agent. However, it has been reported to be genotoxic in the presence of certain transition metals, including copper. In order to explore the capacity of vitamin C to protect DNA from radiation-induced damage, and the influence of the presence of copper on this protection, we investigated vitamin C-mediated protection against radiation-induced damage to calf thymus DNA in vitro in the presence or absence of copper(II). Vitamin C (0.08-8.00 mM, pH 7.0) significantly reduced DNA damage induced by gamma-irradiation (30-150 Gy) by 30-50%, similar to the protective effect of glutathione. However, vitamin C plus copper (50 microM) significantly enhanced gamma-radiation-induced DNA damage. Low levels of added copper (5 microM), or chelation of copper with 1-N-benzyltriethylenetetraine tetrahydrochloride (BzTrien) and bathocuprinedisulfonic acid (BCSA), abolished the enhanced damage without diminishing the protective effect of vitamin C. These results indicate that vitamin C can act as: (1) an antioxidant to protect DNA damage from ionizing radiation; and (2) a reducing agent in the presence of copper to induce DNA damage. These effects are important in assessing the role of vitamin C, in the presence of mineral supplements or radioprotective therapeutic agents, particularly in patients with abnormally high tissue copper levels.     

Synthesis, reactive oxygen species generation and copper-mediated nuclease activity profiles of 2-aryl-3-amino-1,4-naphthoquinones

Here we report a series of 2-aryl-3-amino-1,4-naphthoquinones that generated reactive oxygen species (ROS) such as superoxide and hydrogen peroxide upon incubation in pH 7.4 under ambient aerobic conditions. ROS generation from these compounds was sensitive to structural modifications at the 3-amino position and a 2-aryl substituent promoted ROS generation. A number of these compounds were found to induce DNA damage in the presence of Cu(II) without any added reducing agent. Our data suggests that 2-aryl-3-amino-1,4-naphthoquinones' propensity to produce ROS correlated well with its DNA damage inducing ability. 2-Phenyl-3-pyrrolid-1-yl-1,4-naphthoquinone (22) was found to damage DNA at 1 μM suggesting that these compounds may have therapeutic relevance in targeting cancers which over-express Cu(II).     

Hemoprotein-mediated reduction of nitrated DNA bases in the presence of reducing agents

DNA damages by reactive nitrogen oxide species may contribute to the multistage carcinogenesis processes associated with chronic infections and inflammation. The nitrated DNA adducts 8-nitroguanine (8NG) and 8-nitroxanthine (8NX) have been shown to derive from these reactive nitrogen oxide species, but they are not stable in DNA since they undergo spontaneous depurination. We herein report that hemin and hemoproteins, including hemoglobin and cytochrome c, mediate reduction of 8NG and 8NX to their corresponding amino analogues in the presence of reducing agents under physiologically relevant conditions. This reaction is believed to involve the reduced heme moiety produced from the reduction of oxidized hemoglobin or cytochrome c by reducing agents. The combination of hemoglobin and dihydrolipoic acid generated the reduced products in high yields. Ascorbate, quercetin, and glutathione are also capable of reducing these nitrated DNA adducts. The hemoglobin macromolecule reduces 8NG and 8NX formed in nitryl chloride-treated calf thymus DNA, as evidenced by the formation of the amino adducts using reversed-phase HPLC with photodiode array detection. Hemin is more efficient than equal molar of heme on hemoglobin in reducing 8NG-containing DNA, indicating the role of protein in impeding the reaction. Furthermore, we also show that the reduction product 8-aminoguanine is persistent on DNA. These findings suggest that reduction of nitrated DNA by the heme/antioxidant system might represent a possible in vivo pathway to modify DNA nitration.     

Fungal gliotoxin targets the onset of superoxide-generating NADPH oxidase of human neutrophils

Gliotoxin from Aspergillus, bearing a S&bond;S bond in its structure, prevented the onset of O(-)(2) generation by the human neutrophil NADPH oxidase in response to phorbol myristate acetate (PMA). Gliotoxin affected the activation process harder than the activated oxidase, as shown by its stronger inhibition when added to neutrophils prior to, than post-PMA at maximum enzyme turnover. Decreased O(-)(2) generation persisted even if cells treated with gliotoxin were subsequently washed, with half-inhibition concentrations (IC(50)) of 5.3, and 3.5 microM for treatments of 15 and 30 min, respectively. In addition, gliotoxin made neutrophils reduce cytochrome c regardless of absence of PMA, through its reaction with intracellular reductants in an oxygen-dependent process, named redox cycling. Thus, we next tested whether preincubation of neutrophils with gliotoxin under hypoxic conditions would relieve the inhibition of NADPH oxidase. Instead, this prevention of redox cycling significantly favored damage to the NADPH oxidase with an IC(50) of 0.009 microM. Moreover, conversion of gliotoxin to its dithiol derivative by addition of reduced dithiothreitol during incubation protected cells from losing oxidase activity. These findings support that the disulfide form of gliotoxin targets NADPH oxidase activation.     

Peptide repair of oxidative DNA damage

Guanyl radical species are produced in DNA by electron removal caused by ionizing radiation, photoionization, oxidation, or photosensitization. DNA guanyl radicals can be reduced by electron donation from mild reducing agents. Important biologically relevant examples are the redox active amino acids cysteine, cystine, methionine, tryptophan, and tyrosine. We have quantified the reactivity of derivatives of these amino acids with guanyl radicals located in plasmid DNA. The radicals were produced by electron removal using the single electron oxidizing agent (SCN)(2)(*)(-). Disulfides (cystine) are unreactive. Thioethers (methionine), thiols (cysteine), and phenols (tyrosine) react with rate constants in the range 10(4)-10(6), 10(5)-10(6), and 10(5)-10(6) dm(3) mol(-1) s(-1), respectively. Indoles (tryptophan) are the most reactive with rate constants of 10(7)-10(8) dm(3) mol(-1) s(-1). Selenium analogues of amino acids are over an order of magnitude more reactive than their sulfur equivalents. Increasing positive charge is associated with a ca. 10-fold increase in reactivity. The results suggest that amino acid residues located close to DNA (for example, in DNA binding proteins such as histones) might participate in the repair of oxidative DNA damage.     

Glutathione intensifies gliotoxin-induced cytotoxicity in human neuroblastoma SH-SY5Y cells

Gliotoxin is a fungal second metabolite produced by diverse species that can be found in compost, stored crops, moist animal feed and sawdust. The role of glutathione in gliotoxin-induced toxicity was studied in order to elucidate the toxic mechanisms leading to neurite degeneration and cell death in differentiated human neuroblastoma (SH-SY5Y) cells. After 72 h of exposure to gliotoxin, moderate cytotoxicity was induced at 0.1 μmol/L, which was more severe at higher concentrations. A reduction in the number of neurites per cell was also observed. By decreasing the level of intracellular glutathione with l-buthionine-sulfoxamine (BSO) a specific inhibitor of glutathione synthesis, the cytotoxic effect of gliotoxin was significantly attenuated. The gliotoxin-induced cytotoxicity was also slightly reduced by the antioxidant vitamin C. However, the neurite degenerative effect was not altered by BSO, or by vitamin C. A concentration-dependent increase in the ratio between oxidized and reduced forms of glutathione, as well as the total intracellular glutathione levels, was noted after exposure to gliotoxin. The increase of glutathione was also reflected in western blot analyses showing a tendency for the regulatory subunit of γ-glutamylcysteine synthetase to be upregulated. In addition, the activity of glutathione reductase was slightly increased in gliotoxin-exposed cells. These results indicate that glutathione promotes gliotoxin-induced cytotoxicity, probably by reducing the ETP (epipolythiodioxopiperazine) disulfide bridge to the dithiol form.     

Poly(ADP-ribose) polymerase in plants affects energy homeostasis, cell death and stress tolerance

Plants contain two genes that code for poly(ADP-ribose) polymerase (PARP): parp1 and parp2. Both PARPs are activated by DNA damage caused by, example reactive oxygen species. Upon activation polymers of ADP-ribose are synthesized on a range of nuclear enzymes using NAD(+) as substrate. Here, we show that in plants stresses such as drought, high light and heat activate PARP causing NAD(+) breakdown and ATP consumption. When the PARP activity is reduced by means of chemical inhibitors or by gene silencing, cell death is inhibited and plants become tolerant to a broad range of abiotic stresses like high light, drought and heat. Plant lines with low poly(ADP-ribosyl)ation activity maintain under stress conditions their energy homeostasis by reducing NAD(+) breakdown and consequently energy consumption. The higher energy-use efficiency avoids the need for a too intense mitochondrial respiration and consequently reduces the formation of reactive oxygen species. From these results it can be concluded that breeding or engineering for a high energy-use efficiency under stress conditions is a valuable, but until today nearly unexploited, approach to enhance overall stress tolerance of crops.     

Distinct mechanisms of oxidative DNA damage by two metabolites of carcinogenic o-toluidine

Mechanisms of DNA damage by metabolites of carcinogenic o-toluidine in the presence of metals were investigated by the DNA sequencing technique using (32)P-labeled human DNA fragments. 4-Amino-3-methylphenol, a major metabolite, caused DNA damage in the presence of Cu(II). Predominant cleavage sites were thymine and cytosine residues. o-Nitrosotoluene, a minor metabolite, did not induce DNA damage even in the presence of Cu(II), but addition of NADH induced DNA damage very efficiently. The DNA cleavage pattern was similar to that in the case of 4-amino-3-methylphenol. Bathocuproine and catalase inhibited DNA damage by these o-toluidine metabolites, indicating the participation of Cu(I) and H(2)O(2) in the DNA damage. Typical free hydroxyl radical scavengers showed no inhibitory effects on the DNA damage. o-Toluidine metabolites increased the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine in calf thymus DNA in the presence of Cu(II). UV-visible and ESR spectroscopic studies have demonstrated that 4-amino-3-methylphenol is autoxidized to form the aminomethylphenoxyl radical and o-nitrosotoluene is reduced by NADH to the o-toluolhydronitroxide radical in the presence and absence of Cu(II). Consequently, it is considered that these radicals react with O(2) to form O(-)(2) and subsequently H(2)O(2), and that the reactive species generated by the reaction of H(2)O(2) with Cu(I) participate in the DNA damage. Metal-mediated DNA damage by o-toluidine metabolites through H(2)O(2) seems to be relevant for the expression of the carcinogenicity of o-toluidine.     

Photosensitized damage to calf thymus DNA by a hypocrellin derivative: mechanisms under aerobic and anaerobic conditions

The di-cysteine substituted hypocrellin B (DCHB) derivative has been found to be a potential phototherapeutic agent and exhibit photosensitized damage to DNA. Electronic paramagnetic resonance (EPR) and spectrophotometry demonstrate that one-electron transfer from calf thymus DNA to triplet DCHB induces the generation of the reduced form of DCHB (DCHB*- radical), followed by the second electron transfer from DNA to DCHB*- or the disproportionation of DCHB*- to form the hydroquinone of DCHB (DCHBH2) in anaerobic conditions. This electron transfer process induces the direct damage to DNA in oxygen-free media and contributes partly to the damage of DNA in aerobic media. Superoxide radical and hydroxyl radical are formed with enhanced efficiencies while singlet oxygen is generated with a reduced efficiency from irradiation of DCHB and DNA solution under aerobic conditions as compared with the case in the absence of DNA. All of three reactive oxygen species play an evident role in the photosensitized damage to DNA in aerobic system in addition to the direct electron-transfer damage.     

The contributions of excitotoxicity, glutathione depletion and DNA repair in chemically induced injury to neurones: exemplified with toxic effects on cerebellar granule cells

Six chemicals, 2-halopropionic acids, thiophene, methylhalides, methylmercury, methylazoxymethanol (MAM) and trichlorfon (Fig. 1), that cause selective necrosis to the cerebellum, in particular to cerebellar granule cells, have been reviewed. The basis for the selective toxicity to these neurones is not fully understood, but mechanisms known to contribute to the neuronal cell death are discussed. All six compounds decrease cerebral glutathione (GSH), due to conjugation with the xenobiotic, thereby reducing cellular antioxidant status and making the cells more vulnerable to reactive oxygen species. 2-Halopropionic acids and methylmercury appear to also act via an excitotoxic mechanism leading to elevated intracellular Ca2+, increased reactive oxygen species and ultimately impaired mitochondrial function. In contrast, the methylhalides, trichlorfon and MAM all methylate DNA and inhibit O6-guanine-DNA methyltransferase (OGMT), an important DNA repair enzyme. We propose that a combination of reduced antioxidant status plus excitotoxicity or DNA damage is required to cause cerebellar neuronal cell death with these chemicals. The small size of cerebellar granule cells, the unique subunit composition of their N-methyl-d-aspartate (NMDA) receptors, their low DNA repair ability, low levels of calcium-binding proteins and vulnerability during postnatal brain development and distribution of glutathione and its conjugating and metabolizing enzymes are all important factors in determining the sensitivity of cerebellar granule cells to toxic compounds.     

Oxyl radicals, redox-sensitive signalling cascades and antioxidants

Oxidative stress is an increase in the reduction potential or a large decrease in the reducing capacity of the cellular redox couples. A particularly destructive aspect of oxidative stress is the production of reactive oxygen species (ROS), which include free radicals and peroxides. Some of the less reactive of these species can be converted by oxidoreduction reactions with transition metals into more aggressive radical species that can cause extensive cellular damage. In animals, ROS may influence cell proliferation, cell death (either apoptosis or necrosis) and the expression of genes, and may be involved in the activation of several signalling pathways, activating cell signalling cascades, such as those involving mitogen-activated protein kinases. Most of these oxygen-derived species are produced at a low level by normal aerobic metabolism and the damage they cause to cells is constantly repaired. The cellular redox environment is preserved by enzymes and antioxidants that maintain the reduced state through a constant input of metabolic energy. This review summarizes current studies that have been regarding the production of ROS and the general redox-sensitive targets of cell signalling cascades.     

Dual activity of nitroxides as pro- and antioxidants: catalysis of copper-mediated DNA breakage and H2O2 dismutation

Nitroxide antioxidants can be reduced to hydroxylamines or oxidized to oxoammonium cations. Consequently, nitroxides can modify oxidative damage acting as reducing and/or as oxidizing agents, and in many cases the nitroxides are continuously recycled. They provide protection against oxidative stress via various mechanisms including SOD-mimic activity and detoxification of carbon-, oxygen-, and nitrogen-centered radicals, as well as oxidation of reduced transition metals. In contrast to the common concept, according to which the nitroxides' protective effect takes place via inhibition of the Fenton reaction, there are observations suggesting the opposite. In the present investigation, DNA breakage catalyzed by copper served as an experimental model for studying the anti- and pro-oxidative activity of nitroxides. Nitroxides provided protection in the presence of GSH, which is known to facilitate metal-catalyzed DNA damage. In the absence of a reductant, nitroxides enhanced DNA breakage under aerobic conditions with or without added H(2)O(2) and facilitated H(2)O(2) depletion. The rates of nitroxide-catalyzed DNA breakage and H(2)O(2) depletion increased as the concentrations of copper, H(2)O(2), and nitroxide increased. Although the catalytic activity of nitroxides is low, it is sufficient to induce DNA breakage. The efficacy of DNA breakage by the tested piperidine nitroxides correlated with the nitroxide-induced depletion of H(2)O(2) with the exception of the pyrrolidine nitroxide 3-carbamoylproxyl. The results suggest that the nitroxide and the copper are continuously recycled while catalyzing DNA breakage and depletion of H(2)O(2), which serves both as a source of reducing equivalents and as the electron sink.