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-mediated Lipid Peroxidation Processes in Photosynthetic Membranes

The phytotoxic effect of Cu via the photosynthetic electron transport system was studied with isolated spinach chloroplasts. Cu(II) ions induce a light-driven peroxidation of membrane lipids leading to ethylene formation, the latter dominating over a concurrent ethane production. Seemingly, the hydroxyl radical originating from superoxide anion is the starting reactive O₂ species. Cu ions inhibit photosynthetic electron transport and apparently catalyze the formation of hydroxyl radical and Fenton-type reactions that result in destruction of unsaturated membrane fatty acids. The concept on the mode of action of Cu(II) and Cu(I) ions in lipid peroxidation as presented here suggests the influence of Cu on different reactions. Two sites are in the photosynthetic redox system; Cu participates in two Fenton-type reactions and in the conversion of ethyl radical to ethylene and ethane.     

Nox4 NADPH Oxidase Mediates Peroxynitrite-dependent Uncoupling of Endothelial Nitric-oxide Synthase and Fibronectin Expression in Response to Angiotensin II: ROLE OF MITOCHONDRIAL REACTIVE OXYGEN SPECIES*

Activation of glomerular mesangial cells (MCs) by angiotensin II (Ang II) leads to extracellular matrix accumulation. Here, we demonstrate that, in MCs, Ang II induces endothelial nitric-oxide synthase (eNOS) uncoupling with enhanced generation of reactive oxygen species (ROS) and decreased production of NO. Ang II promotes a rapid increase in 3-nitrotyrosine formation, and uric acid attenuates Ang II-induced decrease in NO bioavailability, demonstrating that peroxynitrite mediates the effects of Ang II on eNOS dysfunction. Ang II rapidly up-regulates Nox4 protein. Inhibition of Nox4 abolishes the increase in ROS and peroxynitrite generation as well as eNOS uncoupling triggered by Ang II, indicating that Nox4 is upstream of eNOS. This pathway contributes to Ang II-mediated fibronectin accumulation in MCs. Ang II also elicits an increase in mitochondrial abundance of Nox4 protein, and the oxidase contributes to ROS production in mitochondria. Overexpression of mitochondrial manganese superoxide dismutase prevents the stimulatory effects of Ang II on mitochondrial ROS production, loss of NO availability, and MC fibronectin accumulation, whereas manganese superoxide dismutase depletion increases mitochondrial ROS, NO deficiency, and fibronectin synthesis basally and in cells exposed to Ang II. This work provides the first evidence that uncoupled eNOS is responsible for Ang II-induced MC fibronectin accumulation and identifies Nox4 and mitochondrial ROS as mediators of eNOS dysfunction. These data shed light on molecular processes underlying the oxidative signaling cascade engaged by Ang II and identify potential targets for intervention to prevent renal fibrosis.     

A novel anti-oxidant and anti-cancer strategy: a peptoid anti-inflammatory drug conjugate with SOD mimic activity

Activation of reactive oxygen and nitrogen species (RONS) by redox-active metal ions has been proposed to contribute to oxidative damage in inflamed tissues. Here, we report a dual-function anti-oxidant conjugate comprising an anti-inflammatory agent (5-aminosalicylic acid) and a chelator with potential as a superoxide dismutase mimic. The conjugate ethylenediaminetetraacetic acid bis-(5-aminosalicylic acid methyl ester) [EBAME] chelates Cu(II) ions in a 1:1 ratio, as assessed spectrophotometrically using Job's method. Superoxide dismutase (SOD) activity was determined for the Mn(II)-conjugate as 0.758+/-0.130 U at a concentration of 0.99 microM. In inflamed tissues, peptidase mediated release of active 5-ASA would also release the EDTA chelator which has significant SOD mimic activity when complexed to Cu(II) ions. Thus, EBAME has potential as a dual-function anti-inflammatory agent with reduced gastric irritability.     

Photoilluminated riboflavin/riboflavin-Cu(II) inactivates trypsin: Cu(II) tilts the balance

Riboflavin (RF) upon irradiation with fluorescent light generates reactive oxygen species like superoxide anion, singlet and triplet oxygen, flavin radicals and substantial amounts of hydrogen peroxide (H2O2). H2O2 can freely penetrate cell membrane and react with a transition metal ion like Cu(ll), generating hydroxyl radical via the modified metal-catalyzed Haber-Weiss reaction. Earlier, it was reported that trypsin-chymotrypsin mixture served as an indirect antioxidant and decreased free radical generation. Thus, in the present study, we used photoilluminated RF as a source of ROS to investigate the effect of free radicals on the activity of trypsin. We also compared the damaging effect of photoilluminated RF and RF-Cu(ll) system using trypsin as a target molecule. RF caused fragmentation of trypsin and the effect was further enhanced, when Cu(II) was added to the reaction. Results obtained with various ROS scavengers suggested that superoxide radical, singlet and triplet oxygen were predominantly responsible for trypsin damage caused by photoilluminated RF. On the other hand, when Cu(ll) was added to the reaction, hydroxyl radical was mainly responsible for trypsin damage. A mechanism of generation of various ROS in the reaction is also proposed. Trypsin did not show any antioxidant effect with RF alone or with RF-Cu(II) combination.     

Asp-Ala-His-Lys (DAHK) inhibits copper-induced oxidative DNA double strand breaks and telomere shortening

Both DNA and the telomeric sequence are susceptible to copper-mediated reactive oxygen species (ROS) damage, particularly damage attributed to hydroxyl radicals. In this study, ROS-induced DNA double strand breaks and telomere shortening were produced by exposure to copper and ascorbic acid. Asp-Ala-His-Lys (DAHK), a specific copper chelating tetrapeptide d-analog of the N-terminus of human albumin, attenuated DNA strand breaks in a dose dependent manner. d-DAHK, at a ratio of 4:1 (d-DAHKCu), provided complete protection of isolated DNA from double strand breaks and, at a ratio of 2:1 (d-DAHKCu), completely protected DNA in Raji cells exposed to copper/ascorbate. Southern blots of DNA treated with copper/ascorbate showed severe depletion and shortening of telomeres and Raji cell treated samples showed some conservation of telomere sequences. d-DAHK provided complete telomere length protection at a ratio of 2:1 (d-DAHKCu). The human albumin N-terminus analog, d-DAHK, protects DNA and telomeres against copper-mediated ROS damage and may be a useful therapeutic adjunct in ROS disease processes.     

NAD(H) enhances the Cu(II)-mediated inactivation of lactate dehydrogenase by increasing the accessibility of sulfhydryl groups

Copper ions are known to inactivate a variety of enzymes, and lactate dehydrogenase (LDH) is exceptionally sensitive to the presence of this metal. We now found that NADH strongly enhances the Cu(II)-mediated loss of LDH activity. Surprisingly, NADH was not oxidized in this process and also NAD⁺ promoted the Cu(II)-dependent inactivation of LDH. Catalase only partly protected the enzyme, whereas hypoxia even enhanced LDH inactivation. NAD(H) accelerated sulfhydryl (SH) group oxidation of LDH by 5,5-dithio-bis(2-nitrobenzoic acid) (DTNB), and, vice versa, LDH-mediated Cu(II) reduction. LDH activity was preserved by thiol donators and pyruvate and partially preserved by lactate and oxamate. Our results suggest that reactive oxygen species (ROS) are of minor importance for the inactivation of LDH induced by Cu(II)/NADH. We propose that conformational changes of the enzymes' active sites induced by NAD(H)-binding increase the accessibility of active sites' cysteine residues to Cu(II) thereby accelerating their oxidation and, consequently, loss of catalytic activity.     

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.     

In situ assessment of oxidant and nitrogenic stress in bleomycin pulmonary fibrosis

Reactive oxygen species (ROS) and nitric oxide (NO) have a role in the development of pulmonary fibrosis after bleomycin administration. The ROS production induces an antioxidant response, involving superoxide dismutases (SODs), catalase, and glutathione peroxidases. We compared in situ oxidative burden and antioxidant enzyme activity in bleomycin-injured rat lungs and normal controls. ROS expression and catalase, glucose-6-phosphate-dehydrogenase (G6PHD), and NOS/NADPH-diaphorase activity were investigated by using histochemical reactions. Nitric oxide synthase (e-NOS and i-NOS) and SOD (MnSOD, Cu/ZnSOD, ECSOD) expression was investigated immunohistochemically. After treatment ROS production was enhanced in both phagocytes and in type II alveolar epithelial cells. Mn, Cu/Zn, and ECSOD were overexpressed in parenchymal cells, whereas interstitium expressed ECSOD. Catalase and G6PHD activity was moderately increased in parenchymal and inflammatory cells. NOS/NADPH-d activity and i-NOS expression increased in alveolar and bronchiolar epithelia and in inflammatory cells. It can be suggested that the concomitant activation of antioxidant enzymes is not adequate to scavenge the oxidant burden induced by bleomycin lung damage. Inflammatory cells and also epithelial cells are responsible of ROS and NO production. This oxidative and nitrosative stress may be a substantial trigger in TGF-β1 overexpression by activated type II pneumocytes, leading to fibrotic lesions.     

Lipophilic ionophore complexes as superoxide dismutase mimetics

A wide range of metal ion complexes exhibit superoxide dismutase like activities as detected by inhibition of nitroblue tetrazolium reduction. Mn(II) and Cu(II) complexes of EDTA, EHPG, and EGTA exhibit SOD like activities commensurate with many of the purpose-built SOD mimics. Here, we report analogous lipophilic chelators that localise metal ions (Cu(II), Mn(II), and Fe(III)) in the lipid membranes and lipoproteins to protect them from superoxide mediated oxidative damage. Spectroscopic titrations and Jobs method confirm that both 1:1 and 2:1 metal ion monensin complexes form. The cupric complexes are the most active exhibiting IC(50) values of 0.09 and 0.18 microM for 2Cu(II)-monensin and Cu(II)-monensin, respectively, for superoxide destruction. In addition, the IC(50) value for Mn(II)-monensin is 0.31 microM. In conclusion, Mn(II) and Cu(II) complexes of the ionophore monensin exhibit considerable superoxide scavenging activities and represent a novel class of catalytic antioxidants for the protection of lipid structures.     

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).     

Tetracycline-Cu(II) photo-induced fragmentation of serum albumin

The protein-damaging potential of photosensitized tetracycline hydrochloride alone and in combination with the metal ion Cu(II) was assessed using serum albumin as a model protein. Exposure of tetracycline to white light in an aqueous solution triggered the generation of significant amounts of reactive oxygen species (ROS) and engendered substantial protein damage. The appearance of distinct low-molecular-mass protein bands on 10% SDS-polyacrylamide gel ascertained the tetracycline concentration-dependent fragmentation of albumin. Photoexcited tetracycline in combination with 100 microM Cu(II) enhanced the protein fragmentation process with concurrent increase in free radical production. The significant release of acid-soluble amino groups and carbonyl groups from treated albumin provided quantitative estimation of protein fragmentation at 0-1.0 mM concentrations of tetracycline. Cu(II) ions per se did not cause any perceptible protein damage. The results with free radical quenchers suggested the role of hydroxyl radicals (*OH) in tetracycline-Cu(II)-induced protein fragmentation, as no superoxide dismutase (SOD)-mediated quenching effect was noted. The generation of free radicals upon tetracycline photoexcitation and consequent protein fragmentation may be considered as important factors in augmentation of tetracycline-induced phototoxic responses.     

Effect of metals on nucleoside hydroperoxide, a product of ionizing radiation in DNA

Ionizing radiation causes formation of thymine hydroperoxides in DNA. Their decomposition generates more stable products and active oxygen species which may oxidize other DNA bases. We have determined the effects of free and chelated metal ions on the degradation of 5-hydroperoxymethyl-2'-deoxyuridine (HPMdU). Two products were formed as analyzed by HPLC: 5-hydroxymethyl-2'-deoxyuridine (HMdU) and 5-formyl-2'-deoxyuridine (FdU). Sn(II) and Fe(II) caused instantaneous HPMdU degradation; Sn(II) generated only HMdU, whereas Fe(II) formed about equal amounts of both. Sn(IV) and Fe(III) were inactive. Cu(I), Cu(II), and Co(II) caused a time-dependent formation of both products, with FdU predominating. In the presence of Cu(I), Cu(II), and Fe(II), formate inhibited formation of HMdU but enhanced that of FdU. EDTA abolished Cu(I)-induced decomposition of HPMdU but only decreased that which was mediated by Cu(II). In contrast, EDTA enhanced the activity of Fe(III) with a time-dependent formation of FdU. EDTA and diethylenetriaminepentaacetic acid (DTPA) caused an instantaneous Fe(II)-mediated decomposition of HPMdU to FdU. Only desferal partially inhibited the activity of Fe(II), whereas the activities of Cu(I), Cu(II), and Fe(III) were blocked by desferal and DTPA. Possible mechanisms of HPMdU degradation by metal ions in the absence or presence of formate or chelators as well as formation of the .OH are discussed.     

Involvement of cytoplasmic membrane damage in the copper (II)-dependent cytotoxicity of a novel naturally occurring tripyrrole

In the presence of a nonlethal concentration of Cu(II), washed Escherichia coli ATCC8739 cells were killed by a novel tripyrrole 1, isolated as a red pigment from the Serratia sp. Cell killing was accompanied by a depletion in the potassium pools of the cells due to the damage to the cytoplasmic membrane, without any detectable DNA damage as revealed by the transformed plasmid DNA and phage induction assay. This revealed that the bactericidal activity of compound 1 in the presence of Cu(II) results from membrane damage. Induction of endogenous catalase in the E. coli cells increased their resistance against the combination of compound 1 and Cu(II). Although compound 1 alone generated large amount of reactive oxygen species (ROS), it did not show any cell killing against E. coli in the absence of Cu(II). The Cu(II)-dependent bactericidal activity of compound 1 was suppressed by ethylenediaminetetraacetate, bathocuproine, catalase and superoxide disumutase (SOD), but not by dimethyl sulfoxide. These findings suggest that recycling redox reactions between Cu(II) and Cu(I), involving compound 1 and hydrogen peroxide on the cell surface, must be important in the mechanism of the killing. Compound 1 alone showed selective bactericidal activity against the gram positive bacterium, Bacillus cereus ATCC 6630, possibly due to its differential cellular transport.     

Lens proteins block the copper-mediated formation of reactive oxygen species during glycation reactions in vitro.

The formation of advanced glycation endproducts (AGEs) from glucose in vitro requires both oxygen and a transition metal ion, usually copper. These elements combine to produce reactive oxygen species (ROS) which degrade glucose to AGE-forming compounds. We measured the ability of Cu(2+) to accelerate ROS formation, and the effect of added lens proteins on these reactions. Increasing levels of Cu(2+) accelerated the formation of superoxide anion with glucose and fructosyl-lysine, but the addition of 2.0 mg/ml calf lens proteins completely blocked superoxide formation up to 100 microM of added Cu(2+). Lens proteins, however, had no effect on superoxide generated by the hypoxanthine/xanthine oxidase system. The oxidation of ascorbic acid was increased 170-fold by the addition of 10 microM Cu(2+), but was also completely prevented by added lens proteins. Hydroxyl radical formation, as measured by the conversion of benzoate to salicylate, was increased to 30 nmoles/ml after 18 h by the addition of 100 microM Cu(2+) and 2.5 mM H2O2. This increase was also blocked by the addition of lens proteins. However, hydroxyl radical formation, as estimated by the crosslinking and fragmentation of lens proteins, was observed in the presence of 100 microM Cu(2+), likely at the sites of Cu(2+) binding. Since the ratio of lens proteins to Cu(2+) in human lens is at least 1000-fold higher than those used here, the data argue that Cu(2+) in the lens would be tightly bound to protein, preventing ROS-mediated AGE formation from glucose in vivo.     

Dihydrolipoic acid lowers the redox activity of transition metal ions but does not remove them from the active site of enzymes

Alpha-lipoic acid (LA) and its reduced form, dihydrolipoic acid (DHLA), have been suggested to chelate transition metal ions and, hence, mitigate iron- and copper-mediated oxidative stress in biological systems. However, it remains unclear whether LA and DHLA chelate transition metal ions in a redox-inactive form, and whether they remove metal ions from the active site of enzymes. Therefore, we investigated the effects of LA and DHLA on iron- or copper-catalyzed oxidation of ascorbate, a sensitive assay for the redox activity of these metal ions. We found that DHLA, but not LA, significantly inhibited ascorbate oxidation mediated by Fe(III)-citrate, suggesting that reduced thiols are required for iron binding. DHLA also strongly inhibited Cu(II)(histidine)(2)-mediated ascorbate oxidation in a concentration-dependent manner, with complete inhibition at a DHLA:Cu(II) molar ratio of 3:1. In contrast, no inhibition of copper-catalyzed ascorbate oxidation was observed with LA. To investigate whether LA and DHLA remove copper or iron from the active site of enzymes, Cu,Zn superoxide dismutase and the iron-containing enzyme aconitase were used. We found that neither LA nor DHLA, even at high, millimolar concentrations, altered the activity of these enzymes. Our results suggest that DHLA chelates and inactivates redox-active transition metal ions in small-molecular, biological complexes without affecting iron- or copper-dependent enzyme activities.     

Chemistry, physiology and pathology of free radicals

The superoxide anion radical and other reactive oxygen species (ROS) are formed in all aerobic organisms by enzymatic and nonenzymatic reactions. ROS arise in both physiological and pathological processes, but efficient mechanisms have evolved for their detoxification. Similarly, reactive nitrogen intermediates (RNI) have physiological activity, but can also react with different types of molecules, including superoxide, to form toxic products. ROS and RNI participate in the destruction of microorganisms by phagocytes, as in the formation of a myeloperoxidase-hydrogen peroxide-chloride/iodide complex which can destroy many cells, including bacteria. It is known that the cellular production of ROS and RNI is controlled by different mechanisms. These free radicals can react with key cellular structures and molecules, thus altering their biological function. An imbalance between the systems producing and removing ROS and RNI may result in pathological consequences.     

Antioxidant defenses and biochemical changes in pacu (Piaractus mesopotamicus) in response to single and combined copper and hypoxia exposure

The effect of combined-factors (hypoxia+copper) on the biochemical parameters and antioxidant defenses were studied in the neotropical fish Piaractus mesopotamicus. Fish were exposed for 48 h to 0.4 mg Cu(2+) L(-1) (0.4Cu), hypoxia=50 mm Hg (Hpx), and 0.4 mg Cu(2) L(-1)+hypoxia=50 mm Hg (0.4CuHpx). The exposure to 0.4Cu increased the reactive oxygen species (ROS) in the liver, accompanied by increases in superoxide dismutase (SOD) and decreases in catalase (CAT) activity, showing the influence of copper in this protection. The exposure to Hpx decreased the activity of glutathione peroxidase (GSH-Px) and CAT. Exposure to a combined-factor caused an increase in the ROS production followed by an increase in SOD and a decrease in GSH-Px and CAT. At 0.4Cu, fish presented a reduction in CAT, while in Hpx decreases in SOD, CAT and GSH-Px were observed in red muscles. Single-factors were insufficient to cause ROS production. In combined-factors, increased ROS formation and decreased SOD, CAT and GSH-Px were observed. RBC increased in all groups, but only under combined-factors was there an increase in hemoglobin. Copper plasma concentration increased in groups exposed to copper. Na(+)/K(+)-ATPase activity in gills decreased in 0.4Cu and 0.4CuHpx, and increased in Hpx. Metallothionein concentration in gills increased under combined-factors. Combined-factors caused significant disturbances in the antioxidant defenses and biochemical parameters than single-factors.     

Superoxide,Hydrogen Peroxide and Hydroxyl Radical in D1/D2/cytochrome b-559 Photosystem II Reaction Center Complex

Spin-trapping electron spin resonance (ESR) was used to monitor the formation of superoxide and hydroxyl radicals in D1/D2/cytochrome b-559 Photosystem II reaction center (PS II RC) Complex. When the PS II RC complex was strongly illuminated, superoxide was detected in the presence of ubiquinone. SOD activity was detected in the PS II RC complex. A primary product of superoxide, hydrogen peroxide, resulted in the production of the most destructive reactive oxygen species, *OH, in illuminated PS II RC complex. The contributions of ubiquinone, SOD and H(2)O(2) to the photobleaching of pigments and protein photodamage in the PS II RC complex were further studied. Ubiquinone protected the PS II RC complex from photodamage and, interestingly, extrinsic SOD promoted this damage. All these results suggest that PS II RC is an active site for the generation of superoxide and its derivatives, and this process protects organisms during strong illumination, probably by inhibiting more harmful ROS, such as singlet oxygen.     

Thiols can either enhance or suppress DNA damage induction by catecholestrogens

The estrogen metabolites catecholestrogens (or hydroxyestrogens) are involved in carcinogenesis and the development of resistance to methotrexate. This induction of drug resistance correlates with the relative efficiency of catecholestrogens in the generation of reactive oxygen species (ROS) and the induction of DNA strand breaks. Although antioxidants can neutralize ROS, the generation of these reactive species by catecholestrogens can be enhanced by electron donors like NADH. Therefore, this study was undertaken to determine the ability of different thiol agents (GSH, NAC, DTT, DHLA) to either inhibit or enhance the level of DNA damage induced by the H(2)O(2) generating system 4-hydroxyestradiol/Cu(II). Our results show that GSH, DTT, and DHLA inhibited the induction of the 4-hydroxyestradiol/Cu(II)-mediated DNA damage, with GSH showing the best potential. In contrast, the GSH precursor NAC at low concentrations was able to enhance the level of oxidative damage, as observed with NADH. NAC can reduce Cu(II) to Cu(I) producing the radical NAC&z.rad;, which can generate the superoxide anion. However, the importance of this pathway appears to be relatively minor since the addition of NAC to the 4-hydroxyestradiol/Cu(II) system generates about 15 times more DNA strand breaks than NAC and Cu(II) alone. We suggest that NAC can perpetuate the redox cycle between the quinone and the semiquinone forms of the catecholestrogens, thereby enhancing the production of ROS. In conclusion, this study demonstrates the crucial importance of the choice of antioxidant as potential therapy against the negative biological effects of estrogens.