Reduced flavins promote oxidative DNA damage in non-respiring Escherichia coli by delivering electrons to intracellular free iron

When cells are exposed to external H(2)O(2), the H(2)O(2) rapidly diffuses inside and oxidizes ferrous iron, thereby forming hydroxyl radicals that damage DNA. Thus the process of oxidative DNA damage requires only H(2)O(2), free iron, and an as-yet unidentified electron donor that reduces ferric iron to the ferrous state. Previous work showed that H(2)O(2) kills Escherichia coli especially rapidly when respiration is inhibited either by cyanide or by genetic defects in respiratory enzymes. In this study we established that these respiratory blocks accelerate the rate of DNA damage. The respiratory blocks did not substantially affect the amounts of intracellular free iron or H(2)O(2), indicating that that they accelerated damage because they increased the availability of the electron donor. The goal of this work was to identify that donor. As expected, the respiratory inhibitors caused a large increase in the amount of intracellular NADH. However, NADH itself was a poor reductant of free iron in vitro. This suggests that in non-respiring cells electrons are transferred from NADH to another carrier that directly reduces the iron. Genetic manipulations of the amounts of intracellular glutathione, NADPH, alpha-ketoacids, ferredoxin, and thioredoxin indicated that none of these was the direct electron donor. However, cells were protected from cyanide-stimulated DNA damage if they lacked flavin reductase, an enzyme that transfers electrons from NADH to free FAD. The K(m) value of this enzyme for NADH is much higher than the usual intracellular NADH concentration, which explains why its flux increased when NADH levels rose during respiratory inhibition. Flavins that were reduced by purified flavin reductase rapidly transferred electrons to free iron and drove a DNA-damaging Fenton system in vitro. Thus the rate of oxidative DNA damage can be limited by the rate at which electron donors reduce free iron, and reduced flavins become the predominant donors in E. coli when respiration is blocked. It remains unclear whether flavins or other reductants drive Fenton chemistry in respiring cells.     

Intracellular copper does not catalyze the formation of oxidative DNA damage in Escherichia coli

Because copper catalyzes the conversion of H(2)O(2) to hydroxyl radicals in vitro, it has been proposed that oxidative DNA damage may be an important component of copper toxicity. Elimination of the copper export genes, copA, cueO, and cusCFBA, rendered Escherichia coli sensitive to growth inhibition by copper and provided forcing circumstances in which this hypothesis could be tested. When the cells were grown in medium supplemented with copper, the intracellular copper content increased 20-fold. However, the copper-loaded mutants were actually less sensitive to killing by H(2)O(2) than cells grown without copper supplementation. The kinetics of cell death showed that excessive intracellular copper eliminated iron-mediated oxidative killing without contributing a copper-mediated component. Measurements of mutagenesis and quantitative PCR analysis confirmed that copper decreased the rate at which H(2)O(2) damaged DNA. Electron paramagnetic resonance (EPR) spin trapping showed that the copper-dependent H(2)O(2) resistance was not caused by inhibition of the Fenton reaction, for copper-supplemented cells exhibited substantial hydroxyl radical formation. However, copper EPR spectroscopy suggested that the majority of H(2)O(2)-oxidizable copper is located in the periplasm; therefore, most of the copper-mediated hydroxyl radical formation occurs in this compartment and away from the DNA. Indeed, while E. coli responds to H(2)O(2) stress by inducing iron sequestration proteins, H(2)O(2)-stressed cells do not induce proteins that control copper levels. These observations do not explain how copper suppresses iron-mediated damage. However, it is clear that copper does not catalyze significant oxidative DNA damage in vivo; therefore, copper toxicity must occur by a different mechanism.     

Superoxide and the production of oxidative DNA damage.

The conventional model of oxidative DNA damage posits a role for superoxide (O2-) as a reductant for iron, which subsequently generates a hydroxyl radical by transferring the electron to H2O2. The hydroxyl radical then attacks DNA. Indeed, mutants of Escherichia coli that lack superoxide dismutase (SOD) were 10-fold more vulnerable to DNA oxidation by H2O2 than were wild-type cells. Even the pace of DNA damage by endogenous oxidants was great enough that the SOD mutants could not tolerate air if enzymes that repair oxidative DNA lesions were inactive. However, DNA oxidation proceeds in SOD-proficient cells without the involvement of O2-, as evidenced by the failure of SOD overproduction or anaerobiosis to suppress damage by H2O2. Furthermore, the mechanism by which excess O2- causes damage was called into question when the hypersensitivity of SOD mutants to DNA damage persisted for at least 20 min after O2- had been dispelled through the imposition of anaerobiosis. That behavior contradicted the standard model, which requires that O2- be present to rereduce cellular iron during the period of exposure to H2O2. Evidently, DNA oxidation is driven by a reductant other than O2-, which leaves the mechanism of damage promotion by O2- unsettled. One possibility is that, through its well-established ability to leach iron from iron-sulfur clusters, O2- increases the amount of free iron that is available to catalyze hydroxyl radical production. Experiments with iron transport mutants confirmed that increases in free-iron concentration have the effect of accelerating DNA oxidation. Thus, O2- may be genotoxic only in doses that exceed those found in SOD-proficient cells, and in those limited circumstances it may promote DNA damage by increasing the amount of DNA-bound iron.     

Protective effects of carnosine and homocarnosine on ferritin and hydrogen peroxide-mediated DNA damage

Previous studies have shown that one of the primary causes of increased iron content in the brain may be the release of excess iron from intracellular iron storage molecules such as ferritin. Free iron generates ROS that cause oxidative cell damage. Carnosine and related compounds such as endogenous histidine dipetides have antioxidant activities. We have investigated the protective effects of carnosine and homocarnosine against oxidative damage of DNA induced by reaction of ferritin with H(2)O(2). The results show that carnosine and homocarnosine prevented ferritin/H(2)O(2)-mediated DNA strand breakage. These compounds effectively inhibited ferritin/H(2)O(2)-mediated hydroxyl radical generation and decreased the mutagenicity of DNA induced by the ferritin÷H(2)O(2) reaction. Our results suggest that carnosine and related compounds might have antioxidant effects on DNA under pathophysiological conditions leading to degenerative damage such as neurodegenerative disorders.     

Intracellular iron, but not copper, plays a critical role in hydrogen peroxide-induced DNA damage

The role of intracellular iron, copper, and calcium in hydrogen peroxide-induced DNA damage was investigated using cultured Jurkat cells. The cells were exposed to low rates of continuously generated hydrogen peroxide by the glucose/glucose oxidase system, and the formation of single strand breaks in cellular DNA was evaluated by the sensitive method, single cell gel electrophoresis or "comet" assay. Pre-incubation with the specific ferric ion chelator desferrioxamine (0.1-5.0 mM) inhibited DNA damage in a time- and dose-dependent manner. On the other hand, diethylenetriaminepentaacetic acid (DTPA), a membrane impermeable iron chelator, was ineffective. The lipophilic ferrous ion chelator 1,10-phenanthroline also protected against DNA damage, while its nonchelating isomer 1,7-phenanthroline provided no protection. None of the above iron chelators produced DNA damage by themselves. In contrast, the specific cuprous ion chelator neocuproine (2,9-dimethyl-1,10-phenanthroline), as well as other copper-chelating agents, did not protect against H(2)O(2)-induced cellular DNA damage. In fact, membrane permeable copper-chelating agents induced DNA damage in the absence of H(2)O(2). These results indicate that, under normal conditions, intracellular redox-active iron, but not copper, participates in H(2)O(2)-induced single strand break formation in cellular DNA. Since BAPTA/AM (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester), an intracellular Ca(2+)-chelator, also protected against H(2)O(2)-induced DNA damage, it is likely that intracellular Ca(2+) changes are involved in this process as well. The exact role of Ca(2+) and its relation to intracellular transition metal ions, in particular iron, needs to be further investigated.     

A mechanism by which nitric oxide accelerates the rate of oxidative DNA damage in Escherichia coli

The presence of nitric oxide (NO) greatly accelerates the rate at which hydrogen peroxide (H2O2) kills Escherichia coli. Workers have suggested that this effect may be important in the process of bacteriocide by phagocytes. The goal of this study was to determine the mechanism of this synergism. The filamentation of the dead cells, and their protection by cell-permeable iron chelators, indicated that NO/H2O2 killed cells by damaging their DNA through the Fenton reaction. Indeed, the number of DNA lesions was far greater when NO was present during H2O2 exposure. In the Fenton reaction, free intracellular iron transfers electrons from adventitious donors to H2O2, producing hydroxyl radicals. Although NO damaged the [Fe-S] clusters of dehydratases, this did not increase the amount of free iron and was therefore not the reason for acceleration of Fenton chemistry. However, NO also blocked respiration, an event that previous studies have shown can stimulate oxidative DNA damage. The resultant accumulation of NADH accelerates the reduction of free flavins by flavin reductase, and these reduced flavins drive Fenton chemistry by transferring electrons to free iron. Indeed, mutants lacking the respiratory quinol oxidases were sensitive to H2O2, and NO did not have any further effect. Further, mutants that lack flavin reductase were resistant to NO/H2O2, and overproducing strains were hypersensitive. We discuss the possibility that H2O2 and NO synergize when macrophages attack captive bacteria.     

Semi-quantitative evaluation of genotoxic activity of chemical substances and evidence for a biological threshold of genotoxic activity

Oxidative DNA damage caused by a cysteine metal-catalyzed oxidation system (Cys-MCO) comprised of Fe(3+), O(2), and a cysteine as an electron donor was enhanced by copper, zinc superoxide dismutase (CuZnSOD) in a concentration-dependent manner, as reflected by the formation of 8-hydroxy-2'-deoxyguanosine (8-OH-dG) and strand breaks. Unlike CuZnSOD, manganese SOD (MnSOD) as well as iron SOD (FeSOD) did not enhance DNA damage. The capacity of CuZnSOD to enhance damage to DNA was inhibited by a spin-trapping agent, 5, 5-dimethyl-1-pyrroline N-oxide (DMPO) and a metal chelator, diethylenetriaminepentaacetic acid (DETAPAC). The deoxyribose assay showed that hydroxyl free radicals were generated in the reaction of CuZnSOD with Cys-MCO. We found that the Cys-MCO system caused the release of free copper from CuZnSOD. CuZnSOD also caused the two-fold enhancement of a mutation in the pUC18 lacZ' gene in the presence of Cys-MCO when measured as a loss of alpha-complementation. Based on these results, we interpret the effects of CuZnSOD on Cys-MCO-induced DNA damage and mutation as due to reactive oxygen species, probably hydroxyl free radicals, formed by the reaction of free Cu(2+), released from oxidatively damaged CuZnSOD, and H(2)O(2) produced by the Cys-MCO system.     

Protective effect of metallothionein to ras DNA damage induced by hydrogen peroxide and ferric ion-nitrilotriacetic acid

Metallothionein (MT) is a strong antioxidant, due to a large number of thiol groups in the MT molecule and MT has been found in the nucleus. To investigate whether MT can directly protect DNA from damage induced by hydroxyl radical, the effects of MTs on DNA strand scission due to incubation with ferric ion-nitrilotriacetic acid and H2O2 (Fe3+ -NTA/H2O2) were studied. The Fe3+-NTA/H2O2 resulted in a higher rate of deoxyribose degradation, compared to incubation of Fe3+/H2O2, presumably mediated by the formation of hydroxyl radicals (*OH). This degradation was inhibited by either Zn-MT or Cd-MT, but not by Zn2+ or Cd2+ at similar concentrations. The Fe3+ -NTA/H2O2 resulted in a concentration dependent of increase in DNA strand scission. Damage to the sugar-phosphodiester chain was predominant over chemical modifications of the base moieties. Incubation with either Zn-MT or Cd-MT inhibited DNA damage by approximately 50%. Preincubation of MT with EDTA and N-ethylmaleimide, to alkylate sulfhydryl groups of MT, resulted in MT that was no longer able to inhibit DNA damage. These results indicates that MT can protect DNA from hydroxyl radical attack and that the cysteine thiol groups of MT may be involved in its nuclear antioxidant properties.     

Cell killing and DNA damage by hydrogen peroxide are mediated by intracellular iron.

Phenanthroline, a strong iron chelator, prevents both the formation of DNA single-strand breaks and the killing of mouse cells produced by H2O2. These results, taken together with our previous findings, indicate that the DNA damage is produced by hydroxyl radicals formed when H2O2 reacts with chromatin-bound Fe2+ and that this damage is responsible for the killing effect.     

Intracellular-produced hydroxyl radical mediates H2O2-induced Ca2+ influx and cell death in rat beta-cell line RIN-5F

The melastatin-related transient receptor potential channel TRPM2 is a Ca(2+)-permeable channel that is activated by H(2)O(2), and the Ca(2+) influx through TRPM2 mediates cell death. However, the responsible oxidants for TRPM2 activation remain to be identified. In the present study, we investigated the involvement of hydroxyl radical on TRPM2 activation in TRPM2-expressing HEK293 cells and the rat beta-cell line RIN-5F. In both cell types, H(2)O(2) induced Ca(2+) influx in a concentration-dependent manner. However, the addition of hydroxyl radical, which was produced by mixing FeSO(4) and H(2)O(2), to the cells, did not increase intracellular Ca(2+) concentration. Interestingly, when H(2)O(2) was added to the cells under intracellular Fe(2+)-accumulated conditions, Ca(2+) influx was markedly enhanced compared to H(2)O(2) alone. In addition, the H(2)O(2)-induced Ca(2+) influx was reduced by hydroxyl radical scavengers and an iron chelator. Under intracellular Fe(2+)-accumulated conditions, H(2)O(2)-induced RIN-5F cell death through TRPM2 activation was also markedly enhanced. Hydroxyl radical scavengers and an iron chelator suppressed the RIN-5F cell death by H(2)O(2). These results strongly suggest that the intracellular hydroxyl radical plays a key role in the activation of TRPM2 during H(2)O(2) treatment, and TRPM2 activation mediated by hydroxyl radical is implicated in H(2)O(2)-induced cell death in the beta-cell line RIN-5F.     

Nitric oxide-induced resistance to hydrogen peroxide stress is a glutamate cysteine ligase activity-dependent process

Nitric oxide (*NO) is a reactive nitrogen species known to be involved in cytotoxic processes. Cells respond to cytotoxic injury by stress response induction leading to the development of cellular resistance. This report describes an *NO-induced stress response in Chinese hamster fibroblasts (HA1), which leads to glutathione synthesis-dependent resistance to H2O2-mediated oxidative stress. The development of resistance to H2O2 was completely abolished by the inhibition of glutamate cysteine ligase (GCL) during the first 8 h of recovery after *NO exposure. Altered thiol metabolism was observed immediately after *NO exposure as demonstrated by up to 75% decrease in intracellular thiol pools (glutathione, gamma-glutamylcysteine, and cysteine), which then reaccumulated during the *NO-mediated development of resistance. Immunoreactive protein and activity associated with GCL decreased immediately after exposure to *NO and then reaccumulated during the development of resistance to H2O2 challenge. Moreover, compared to N2 controls the activity levels of GCL in *NO-exposed cells increased approximately twofold 24 h after H2O2 challenge. These results demonstrate that *NO exposure is capable of inducing an adaptive response to H2O2-mediated oxidative stress in mammalian cells, which involves alterations in thiol metabolism and is dependent upon glutathione synthesis and increased GCL activity.     

Calcium chelator Quin 2 prevents hydrogen-peroxide-induced DNA breakage and cytotoxicity

Exposure of cultured Chinese hamster ovary (CHO) cells to hydrogen peroxide results in the production of extensive DNA breakage which can be prevented by the intracellular calcium chelator Quin 2. This effect occurs at Quin 2 AM concentrations as low as 0.1 microM and is maximal at 1 microM. Addition of the extracellular calcium chelator, EGTA, does not affect the level of DNA breakage generated by H2O2. Quin 2 also significantly reduces cellular toxicity caused by the oxidant. Experiments with spin-trapping techniques demonstrate that Quin 2 does not affect the formation of hydroxyl radicals generated by the action of Fe2+ on H2O2. Quin 2 at high concentrations, similar to those reached within the cell, actually enhanced generation of hydroxyl radical in the absence of other iron chelators under our experimental conditions. These results suggest that H2O2 or H2O2-derived radicals do not directly induce DNA strand breakage in intact mammalian cells; rather, these radicals may disturb intracellular Ca2+ homeostasis which results in secondary reactions ultimately leading to DNA strand breakage. In addition to strand breakage, membrane and protein oxidation probably contribute to the cytotoxic effect of H2O2.     

Quantitative analysis of gene-specific DNA damage in human spermatozoa

Recent studies have suggested that human spermatozoa are highly susceptible to DNA damage induced by oxidative stress. However, a detailed analysis of the precise nature of this damage and the extent to which it affects the mitochondrial and nuclear genomes has not been reported. To induce DNA damage, human spermatozoa were treated in vitro with hydrogen peroxide (H2O2; 0-5 mM) or iron (as Fe(II)SO4, 0-500 microM). Quantitative PCR (QPCR) was used to measure DNA damage in individual nuclear genes (hprt, beta-pol and beta-globin) and mitochondrial DNA. Single strand breaks were also assessed by alkaline gel electrophoresis. H2O2 was found to be genotoxic toward spermatozoa at concentrations as high as 1.25 mM, but DNA damage was not detected in these cells with lower concentrations of H2O2. The mitochondrial genome of human spermatozoa was significantly (P<0.001) more susceptible to H2O2-induced DNA damage than the nuclear genome. However, both nDNA and mtDNA in human spermatozoa were significantly (P<0.001) more resistant to damage than DNA from a variety of cell lines of germ cell and myoblastoid origin. Interestingly, significant DNA damage was also not detected in human spermatozoa treated with iron. These studies report, for the first time, quantitative measurements of DNA damage in specific genes of male germ cells, and challenge the commonly held belief that human spermatozoa are particularly vulnerable to DNA damage.     

Oxidation of proteinaceous cysteine residues by dopamine-derived H2O2 in PC12 cells.

Cellular metabolism of dopamine (DA) generates H2O2, which is further reduced to hydroxyl radicals in the presence of iron. Cellular damage inflicted by DA-derived hydroxyl radicals is thought to contribute to Parkinson's disease. We have previously developed procedures for detecting proteins that contain H2O2-sensitive cysteine (or selenocysteine) residues. Using these procedures, we identified ERP72 and ERP60, two members of the protein disulfide isomerase family, creatine kinase, glyceraldehyde-3-phosphate dehydrogenase, phospholipase C-gamma1, and thioredoxin reductase as the targets of DA-derived H2O2. Experiments with purified enzymes identified the essential Cys residues of creatine kinase and glyceraldehyde-3-phosphate dehydrogenase, that are specifically oxidized by H2O2. Although the identified proteins represent only a fraction of the targets of DA-derived H2O2, functional impairment of these proteins has previously been associated with cell death. The oxidation of proteins that contain reactive Cys residues by DA-derived H2O2 is therefore proposed both to be largely responsible for DA-induced apoptosis in neuronal cells and to play an important role in the pathogenesis of Parkinson's disease.     

Mammalian cells are not killed by DNA single-strand breaks caused by hydroxyl radicals from hydrogen peroxide

Cell killing by ionizing radiation has been shown to be caused by hydroxyl free radicals formed by water radiolysis. We have previously suggested that the killing is not caused by individual OH free radicals but by the interaction of volumes of high radical density with DNA to cause locally multiply damaged sites (LMDS) (J. F. Ward, Radiat. Res. 86, 185-195, 1985). Here we test this hypothesis using hydrogen peroxide as an alternate source of OH radicals. The route to OH production from H2O2 is expected to cause singly damaged sites rather than LMDS. Chinese hamster V79-171 cells were treated with H2O2 at varying concentrations for varying times at 0 degree C. DNA damage produced intracellularly was measured by alkaline elution and quantitated in terms of Gray-equivalent damage by comparing the rate of its elution with that of DNA from gamma-irradiated cells. The yield of DNA damage produced increases with increasing concentration of H2O2 and with time of exposure. H2O2 is efficient in producing single-strand breaks; treatment with 50 microM for 30 min produces damage equivalent to that formed by 10 Gy of gamma irradiation. In the presence of a hydroxyl radical scavenger, dimethyl sulfoxide (DMSO), the yield of damage decreases with increasing DMSO concentration consistent with the scavenging of hydroxyl radicals traveling an average of 15 A prior to reacting with the DNA. In contrast to DNA damage production, cell killing by H2O2 treatment at 0 degree C is inefficient. Concentrations of 5 X 10(-2) M H2O2 for 10 min are required to produce significant cell killing; the DNA damage yield from this treatment can be calculated to be equivalent to 6000 Gy of gamma irradiation. The conclusion drawn is that individual DNA damage sites are ineffectual in killing cells. Mechanisms are suggested for killing at 0 degree C at high concentrations and for the efficient cell killing by H2O2 at 37 degrees C at much lower concentrations.     

Cystinotic and normal fibroblasts: Differential susceptibility to cysteine toxicity in vitro

Summary Extracellular cysteine concentrations between 0.5 and 2.5 mM resulted in death of normal but not cystinotic cells grown in Eagle's minimal essential medium containing supplemental fetal bovine serum and antibiotics. Differential cell survival was determined by viable cell counting using Trypan Blue dye exclusion. In cocultivation experiments of [³H]thymidine-labelled cystinotic fibroblasts with nonradioactive normal fibroblasts, autoradiography confirmed the selective survival of cystinotic cells in medium containing 1 mM cysteine. At this concentration of 1 mM cysteine, intracellular cystine content increased slightly in surviving normal cells but not in cystinotic cells, which normally contain a high level of intracellular cystine. This comparative resistance of cystinotic fibroblasts to elevated extracellular cysteine concentrations forms the basis for an in vitro selective system for these mutant human cells. Further exploration of this resistance phenomenon may well expand the understanding of the molecular defect in cystinotic cells.     

Low concentrations of acid-soluble thiol (cysteine) in the blood plasma of HIV-1-infected patients

Blood plasma samples from HIV-1-infected persons contain elevated glutamate concentrations up to 6-fold the normal level and relatively low concentrations of acid-soluble thiol (i.e. decreased cysteine concentrations). The intracellular glutathione concentration in peripheral blood-mononuclear cells (PBMC) and monocytes from HIV antibody-positive persons are also significantly decreased. Therapy with azidothymidine (AZT) causes a substantial recovery of the plasma thiol levels; but glutamate levels remain significantly elevated and intracellular glutathione levels remain low. Cell culture experiments with approximately physiological amino-acid concentrations revealed that variations of the extracellular cysteine concentration have a strong influence on the intracellular glutathione level and the rate of DNA synthesis [( 3H]thymidine incorporation) in T cell clones and human and murine lymphocyte preparations even in the presence of several-fold higher cystine and methionine concentrations. Cysteine cannot be replaced by a corresponding increase of the extracellular cystine or methionine concentration. These experiments suggest strongly that the low cysteine concentration in the plasma of HIV-infected persons may play a role in the pathogenetic mechanism of the acquired immunodeficiency syndrome.     

The so-called Listeria innocua ferritin is a Dps protein. Iron incorporation, detoxification, and DNA protection properties

Listeria innocua Dps (DNA binding protein from starved cells) affords protection to DNA against oxidative damage and can accumulate about 500 iron atoms within its central cavity through a process facilitated by a ferroxidase center. The chemistry of iron binding and oxidation in Listeria Dps (LiDps, formerly described as a ferritin) using H(2)O(2) as oxidant was studied to further define the mechanism of iron deposition inside the protein and the role of LiDps in protecting DNA from oxidative damage. The relatively strong binding of 12 Fe(2+) to the apoprotein (K(D) approximately 0.023 microM) was demonstrated by isothermal titration calorimetry, fluorescence quenching, and pH stat experiments. Hydrogen peroxide was found to be a more efficient oxidant for the protein-bound Fe(2+) than O(2). Iron(II) oxidation by H(2)O(2) occurs with a stoichiometry of 2 Fe(2+)/H(2)O(2) in both the protein-based ferroxidation and subsequent mineralization reactions, indicating complete reduction of H(2)O(2) to H(2)O. Electron paramagnetic resonance (EPR) spin-trapping experiments demonstrated that LiDps attenuates the production of hydroxyl radical by Fenton chemistry. DNA cleavage assays showed that the protein, while not binding to DNA itself, protects it against the deleterious combination of Fe(2+) and H(2)O(2). The overall process of iron deposition and detoxification by LiDps is described by the following equations. For ferroxidation, Fe(2+) + Dps(Z)--> [(Fe(2+))-Dps](Z+1) + H(+) (Fe(2+) binding) and [(Fe(2+))-Dps](Z+1) + Fe(2+) + H(2)O(2) --> [(Fe(3+))(2)(O)(2)-Dps](Z+1) + 2H(+) (Fe(2+) oxidation/hydrolysis). For mineralization, 2Fe(2+) + H(2)O(2) + 2H(2)O --> 2Fe(O)OH((core)) + 4H(+) (Fe(2+) oxidation/hydrolysis). These reactions occur in place of undesirable odd-electron redox processes that produce hydroxyl radical.     

Preventing metal-mediated oxidative DNA damage with selenium compounds

Copper and iron are two widely studied transition metals associated with hydroxyl radical (˙OH) generation, oxidative damage, and disease development. Because antioxidants ameliorate metal-mediated DNA damage, DNA gel electrophoresis assays were used to quantify the ability of ten selenium-containing compounds to inhibit metal-mediated DNA damage by hydroxyl radical. In the Cu(I)/H(2)O(2) system, selenocystine, selenomethionine, and methyl-selenocysteine inhibit DNA damage with IC(50) values ranging from 3.34 to 25.1 μM. Four selenium compounds also prevent DNA damage from Fe(II) and H(2)O(2). Additional gel electrophoresis experiments indicate that Cu(I) or Fe(II) coordination is responsible for the selenium antioxidant activity. Mass spectrometry studies show that a 1?:?1 stoichiometry is the most common for iron and copper complexes of the tested compounds, even if no antioxidant activity is observed, suggesting that metal coordination is necessary but not sufficient for selenium antioxidant activity. A majority of the selenium compounds are electroactive, regardless of antioxidant activity, and the glutathione peroxidase activities of the selenium compounds show no correlation to DNA damage inhibition. Thus, metal binding is a primary mechanism of selenium antioxidant activity, and both the chemical functionality of the selenium compound and the metal ion generating damaging hydroxyl radical significantly affect selenium antioxidant behavior.