Chelated iron-catalyzed OH. formation from paraquat radicals and H2O2: mechanism of formate oxidation

Traces of iron, when complexed with either EDTA or diethylenetriaminepentaacetic acid (DTPA), catalyze an OH.-producing reaction between H2O2 and paraquat radical (PQ+.): H2O2 + PQ+.----PQ++ + OH. + OH-.[1]. Kinetic studies show that oxidation of formate induced by this reaction occurs by a Fenton-type mechanism, analagous to that assumed in the metal-catalyzed Haber-Weiss reaction, in which the rate determining step is H2O2 + Fe2+ (chelator)----Fe3+(chelator) + OH. + OH-,[7]; with k7 = 7 X 10(3) M-1 s-1 for EDTA and 8 X 10(2) M-1 s-1 for DTPA at pH 7.4. PQ+. rapidly reduces both Fe3+ (EDTA) and Fe3+ (DTPA), and hence allows both agents to catalyze [1] with comparable efficiency, in contrast to the much lower efficiency reported for the latter as a catalyst for the Haber-Weiss reaction. The catalytic properties of these chelating agents is attributed to their lowering of E0 (Fe3+/Fe2+) by 0.65 V, thus making [7] thermodynamically possible at pH 7. Approximately 2.5% of the OH. produced is consumed by internal or "cage" reactions, which decompose the chelator and produce CO2; however, the majority (97%) diffuses into the bulk solution and participates in competitive reactions with OH. scavengers.     

Ascorbate and H2O2 induced oxidative DNA damage in Jurkat cells

The effect of vitamin C (ascorbate) on oxidative DNA damage was examined by first incubating cells with dehydroascorbate, which boosts the intracellular concentration of ascorbate, and then exposing cells to H₂O₂. Oxidative DNA damage was estimated by the analysis of 5-hydroxy-2′-deoxycytidine (oh⁵dCyd) and 8-oxo-7,8-dihydro-2′-deoxyguanosine (oxo⁸dGuo). The presence of a high concentration of ascorbate (30 mM), compared to the absence of ascorbate in cells, when exposed to H₂O₂ (200 μM), resulted in a remarkable sensitization of oh⁵dCyd from 2.7 ± 0.6 to 40.8 ± 6.1 lesions /10⁶ dCyd (15-fold). In contrast, the level of oxo⁸dGuo increased from 8.4 ± 0.4 to 12.1 ± 0.5 lesions/10⁶ dGuo (50%). The formation of oh⁵dCyd was also observed at lower concentrations of intracellular ascorbate and exogenous H₂O₂. Additional studies showed that replacement of H₂O₂ with tert-butyl hydroperoxide completely abolished damage, and that preincubation with iron and desferroxamine increased and decreased this damage, respectively. The latter studies suggest that a Fenton reaction is involved in the mechanism of damage. In conclusion, we report a novel model system in which ascorbate sensitizes H₂O₂-induced oxidative DNA damage in cells, leading to elevated levels of oh⁵dCyd and oxo⁸dGuo, with a strong bias toward the formation of oh⁵dCyd.     

The role of interactions between O2, H2O2, .OH,e- and O2- in free radical damage to biological systems

The occurrence of the Haber-Weiss reaction and other interactions between free radicals has been investigated in the effects of mixtures of free radicals on the permeability of resealed erythrocyte ghosts and on the activity of membrane-bound glyceraldehyde-3-phosphate dehydrogenase. The following mixtures were found to induce damage greater than that which could be accounted for by the independent actions of the constituent free radicals: (i) · OH + H₂O₂, and (ii) · OH + H₂O₂ + O₂^?. In contrast, the following mixtures were found to induce less damage than that predicted on the basis of independent actions of constituent free radicals: (i) H₂O₂ + O₂^?, and (ii) oxidizing radicals ( · OH, H₂O₂) + reducing radicals (e^?, H · ). These results suggest a Haber-Weiss-like interaction between H₂O₂ and O₂^?and an interaction between H₂O₂ and · OH to produce a species more potent than either in causing increased permeability. The decrease in damage observed in the simultaneous presence of oxidizing and reducing radicals suggests an antagonistic effect by which each tends to moderate damage by the other. Inactivation of glyceraldehyde-3-phosphate dehydrogenase was found to be more sensitive to radiation than permeability by an order of magnitude, while permeability was more sensitive to the enhancement of damage by oxygen. Comparison of the effectiveness of free radical scavengers in inhibiting the increase in permeability caused by free radicals showed the following order of effectiveness, expressed in terms of percentage protection: formate (90%) > nitrogen (65%) > catalase (60%) > dismutase (32%), and with respect to enzymatic inactivation, nitrogen (100%) > formate (77%) > dismutase (48%) > catalase (44%). The relative rates observed anaerobically and aerobically in the presence and absence of the above scavengers suggest that (at least in the case of radiation damage to the membranes of erythrocyte ghost cells) the “oxygen effect” is due to the interaction of oxygen with e^? and H., producing O₂^? which aggravates damage under conditions which allow consequent Haber-Weiss-like reactions. The further increase in damage when oxygen concentration is raised yet higher is due to the interaction of oxygen with the sites of initial damage.     

Vitamin C modulation of H2O2-induced damage and iron homeostasis in human cells

Vitamin C (ascorbic acid, AA) is an important antioxidant in human plasma. It is clear, however, that AA has other important, nonantioxidant roles in cells. Of particular interest is its involvement in iron metabolism, since AA enhances dietary iron absorption, increases the activity of Fe(2+)-dependent cellular enzymes, promotes Fenton reactions in vitro, and was reported to have deleterious effects in individuals with iron overload. Nevertheless, the ability of AA to modulate iron metabolism and enhance iron-dependent damage in cells, tissues, and organisms has not been fully elucidated. Here we investigated the effect of AA on iron-mediated oxidative stress in normal human fibroblasts. Incubation with physiologically relevant concentrations of AA was not harmful but sensitised cells toward H(2)O(2)-induced, iron-dependent DNA strand breakage and cell death. We also report that AA increased the levels of intracellular catalytic iron and concomitantly modulated the expression of two well-established iron-regulated genes, ferritin and transferrin receptor. In summary, we present evidence of a novel, nonantioxidant role of AA in human cells, where it increases iron availability and enhances ROS-mediated, iron-dependent damage. We suggest that AA may exacerbate the deleterious effects of metals in vivo and promote normal tissue injury in situations associated with elevated ROS production.     

Red blood cells protect endothelial cells against H2O2-mediated but not hyperoxia-induced damage

We studied the effect of intact red blood cells on the exogenous H2O2-mediated damage as well as on the hyperoxia-induced injury of cultured endothelial cells. Red blood cells protected endothelial cells against H2O2-mediated injury efficiently, but had no effect on the hyperoxia-induced damage. Failure of red blood cells to protect endothelial cells against hyperoxia-induced injury was not due to hemolysis. Furthermore, hyperoxia-exposed red blood cells were still capable of protecting endothelial cells against H2O2-mediated damage.     

Protection by tropolones against H2O2-induced DNA damage and apoptosis in cultured Jurkat cells

Tropolones, the naturally occurring compounds responsible for the durability of heartwood of several cupressaceous trees, have been shown to possess both metal chelating and antioxidant properties. However, little is known about the ability of tropolone and its derivatives to protect cultured cells from oxidative stress-mediated damage. In this study, the effect of tropolones on hydrogen peroxide-induced DNA damage and apoptosis was investigated in cultured Jurkat cells. Tropolone, added to the cells 15 min before the addition of glucose oxidase, provided a dose dependent protection against hydrogen peroxide induced DNA damage. The IC₅₀ value observed was about 15 μM for tropolone. Similar dose dependent protection was also observed with three other tropolone derivatives such as trimethylcolchicinic acid, purpurogallin and β-thujaplicin (the IC₅₀ values were 34, 70 and 74 μM, respectively), but not with colchicine and tetramethyl purpurogallin ester. Hydrogen peroxide-induced apoptosis was also inhibited by tropolone. However, in the absence of exogenous H₂O₂ but in the presence of non-toxic concentrations of exogenous iron (100 μM Fe³⁺), tropolone dramatically increased the formation of single strand breaks at molar ratios of tropolone to iron lower than 3 to 1, while, when the ratio increased over 3, no toxicity was observed. In conclusion, the results presented in this study indicate that the protection offered by tropolone against hydrogen peroxide-induced DNA damage and apoptosis was due to formation of a redox-inactive iron complex, while its enhancement of iron-mediated DNA damage at ratios of [tropolone]/[Fe³⁺] lower than 3, was due to formation of a lipophilic iron complex which facilitates iron transport through cell membrane in a redox-active form.     

UVA-induced oxidative damage in retinal pigment epithelial cells after H2O2 or sparfloxacin exposure

Retinal impairment is one of the leading causes of visual loss in an aging human population. To explore a possible cause for retinal damage in the human population, we have monitored DNA oxidation in human retinal pigment epithelial (RPE) cells after exposure to hydrogen peroxide (H₂O₂) or the quinolone antibacterial sparfloxacin. When H₂O₂- or sparfloxacin-exposed cells were further exposed to ultraviolet A (UVA) irradiation, oxidative damage to the DNA of these cells was greatly increased over baseline values. This RPE+pharmaceutical-UVA cell system was developed to mimic in vivo retinal degeneration, seen in mouse studies using quinolone and UVA exposure. DNA damage produced by sparfloxacin and UVA in RPE cells could be remedied by the use of antioxidants, indicating a possible in vivo method for prevention or minimization of retinal damage in humans This revised version was published online in July 2006 with corrections to the Cover Date.     

EphA2 overexpression reduces H2O2-induced damage of lens epithelial cells

Age-related cataract (ARC) is a progressive lens opacification that occurs from middle to old age. Eph-receptor tyrosinekinase-type A2 (EphA2) has been reported to be associated with ARC. This work aims to investigate the molecular mechanism of EphA2 in ARC. We treated human lens epithelial cells (SRA01/04) with different concentration of H₂O₂ to induce lens epithelial cell damage. Then, we found that H₂O₂ treatment significantly suppressed cell viability and enhanced the expression of EphA2 in the SRA01/04 cells. H₂O₂ treatment repressed cell viability and enhanced the levels of reactive oxygen species (ROS) in SRA01/04 cells, which was partly abolished by EphA2 up-regulation. Moreover, EphA2 overexpression reduced H₂O₂-induced apoptosis of SRA01/04 cells. EphA2 up-regulation caused an up-regulation of Bcl-2, and repressed the expression of Bax and Cleaved-caspase-3 in the SRA01/04 cells following H₂O₂ treatment. In conclusion, our data confirm that EphA2 overexpression enhances cell viability and inhibits apoptosis in the H₂O₂-treated SRA01/04 cells, thereby reducing H₂O₂-induced damage of lens epithelial cells. Thus, this work provides new insights into the mechanism of EphA2 in ARC.     

Differential DNA damage induced by H2O2 and bleomycin in subpopulations of human white blood cells

The purpose of this study was to characterize the differential sensitivities of various subpopulations of human white blood cells after exposure to H2O2 (an oxidant agent) and bleomycin (a radiomimetic glycopeptide), in vitro, using single-cell gel electrophoresis (SCGE). Human peripheral blood was fractionated into mononuclear cells, which were further separated into monocytes, CD4+ T-cells, CD8+ T-cells, B-cells and natural killer cells (NK cells). The separated fractions were exposed to different doses of H2O2 and bleomycin, and then used to measure levels of induced and basal DNA damage. There was a significant increase in the amount of DNA damage in CD4+ T-cells, CD8+ T-cells, NK cells and B-cells when treated with H2O2 and bleomycin, whereas monocytes had the lowest sensitivity to H2O2 compared with the other cell fractions, but no lower sensitivity to bleomycin. Furthermore, CD4+ T-cells and CD8+ T-cells had the highest levels of basal DNA damage. When basal DNA damage was taken into account, NK cells tended to show a higher sensitivity to H2O2 than CD4+ T-cells, CD8+ T-cells and monocytes. In addition, B-cells, which showed lower sensitivity to H2O2 than CD4+ T-cells, CD8+ T-cells and NK cells when exposed to lower doses of H2O2 (<10 microM), showed higher sensitivity to H2O2 at higher doses (>20 microM). On the other hand, B-cells showed the highest sensitivity to bleomycin.     

Evolution of DNA damage in irradiated cells

Ionizing radiation damage to a mammalian genome is modeled using continuous time Markov chains. Models are given for the initial infliction of DNA double strand breaks by radiation and for the enzymatic processing of this initial damage. Damage processing pathways include DNA double strand break repair and chromosome exchanges. Linear, saturable, or inducible repair is considered, competing kinetically with pairwise interactions of the DNA double strand breaks. As endpoints, both chromosome aberrations and the inability of cells to form clones are analyzed. For the post-irradiation behavior, using the discrete time Markov chain embedded at transitions gives the ultimate distribution of damage more simply than does integrating the Kolmogorov forward equations. In a representative special case explicit expressions for the probability distribution of damage at large times are given in the form used for numerical computations and comparisons with experiments on human lymphocytes. A principle of branching ratios, that late assays can only measure appropriate ratios of repair and interaction functions, not the functions themselves, is derived and discussed.This work was supported in # DMS-9025103     

Death by protein damage in irradiated cells

A founding concept of radiobiology that deals with X-rays, γ-rays and ultraviolet light is that radiation indiscriminately damages cellular macromolecules. Mounting experimental evidence does not fit into this theoretical framework. Whereas DNA lesion-yields in cells exposed to a given dose and type of radiation appear to be fixed, protein lesion-yields are highly variable. Extremely radiation resistant bacteria such as Deinococcus radiodurans have evolved extraordinarily efficient antioxidant chemical defenses which specifically protect proteins and the functions they catalyze. In diverse prokaryotes, the lethal effects of radiation appear to be governed by oxidative protein damage, which inactivates enzymes including those needed to repair and replicate DNA. These findings offer fresh insight into the molecular mechanisms of radiation resistance and present themselves as new opportunities to study and control oxidative stress in eukaryotes, including mammalian cells and their cancer cell counterparts.     

Radiation damage to histone H2A by the primary aqueous radicals

Histone H2A has been examined for radiation-induced changes in structure and in amino acid composition. The effects of the individual radical intermediates--the hydroxyl radical, solvated electron, and superoxide radical--have been determined by irradiating in dilute aqueous solution under controlled environmental conditions. Amino acid analysis of irradiated histone H2A shows a selective attack on a few residues; only the aromatic residues, phenylalanine and tyrosine, and the heterocyclic residue, histidine, are significantly decreased. A significant increase in aspartic acid is also observed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that the hydroxyl radical is the effective radical for promoting changes in protein structure.     

Protein radicals in the reaction between H2O2-activated metmyoglobin and bovine serum albumin

Hydrogen peroxide activation of MMb with and without the presence of BSA gave rise to rapid formation of hyper-valent myoglobin species, myoglobin ferryl radical (·MbFe(IV)=O) and/or ferrylmyoglobin (MbFe(IV)=O). Reduction of MbFe(IV)=O showed first-order kinetics for a 1-2 times stoichiometric excess of H₂O₂ to MMb while a 3-10 times stoichiometric excess of H₂O₂ resulted in a biphasic reaction pattern. Radical species formed in the reaction between MMb, H₂O₂ and BSA were influenced by [H₂O₂] as measured by electron spin resonance (ESR) spectroscopy and resulted in the formation of cross-linking between BSA and myoglobin which was confirmed by SDS-PAGE and subsequent amino acid sequencing. Moreover, dityrosine was formed in the initial phases of the reaction for all concentrations of H₂O₂. However, initially formed dityrosine was subsequently utilized in reactions employing stoichiometric excess of H₂O₂ to MMb. The observed breakdown of dityrosine was ascribed to additional radical species formed from the interaction between H₂O₂ and the hyper-valent iron-center of H₂O₂-activated MMb.     

Hydroxyl radicals (*OH) are associated with titanium dioxide (TiO(2)) nanoparticle-induced cytotoxicity and oxidative DNA damage in fish cells

TiO(2) nanoparticles (< 100 nm diameter) have been reported to cause oxidative stress related effects, including inflammation, cytotoxicity and genomic instability, either alone or in the presence of UVA irradiation in mammalian studies. Despite the fact that the aquatic environment is often the ultimate recipient of all contaminants there is a paucity of data pertaining to the potential detrimental effects of nanoparticles on aquatic organisms. Therefore, these investigations aimed to evaluate the potential cytotoxic and genotoxic effects of TiO(2) nanoparticles on goldfish skin cells (GFSk-S1), either alone or in combination with UVA. Whilst neutral red retention (NRR) assay (a measure of lysosomal membrane integrity) was used to evaluate cell viability, a modified Comet assay using bacterial lesion-specific repair endonucleases (Endo-III, Fpg) was employed to specifically target oxidative DNA damage. Additionally, electron spin resonance (ESR) studies with different spin traps were carried out for qualitative analysis of free radical generation. For cell viability, TiO(2) alone (0.1-1000 microg ml(-1)) had little effect whereas co-exposure with UVA (0.5-2.0 kJm(-2)) caused a significant dose-dependent decrease which was dependent on both the concentration of TiO(2) and the dose of UVA administered. For the Comet assay, doses of 1, 10 and 100 microg ml(-1) in the absence of UVA caused elevated levels of Fpg-sensitive sites, indicating the oxidation of purine DNA bases (i.e. guanine) by TiO(2). UVA irradiation of TiO(2)-treated cells caused further increases in DNA damage. ESR studies revealed that the observed toxic effects of nanoparticulate TiO(2) were most likely due to hydroxyl radical (OH) formation.     

Protective effects of quercetin and its metabolites on H2O2-induced chromosomal damage to WIL2-NS cells

We investigated chromosomal damage caused by a typical flavonoid, quercetin, and its two conjugates, quercetin-3-O-sulfate and isorhamnetin, and their protective effects against chromosomal damage induced by H2O2. The chromosomal damage was detected by the cytokinesis-block micronucleus (CBMN) assay using a lymphoblastoid cell line, WIL2-NS. We found that quercetin itself induced chromosomal damage at 10 microM, but quercetin-3-O-sulfate and isorhamnetin did not induce damage up to 30 microM. In the medium used for the CBMN assay, quercetin (at 100 microM) generated a high concentration of H2O2, but the two conjugates did not at the same concentration. On the other hand, pretreatment with quercetin (at 1 microM), quercetin-3-O-sulfate (at 10 microM), and isorhamnetin (at 5 microM) prevented H2O2-induced chromosomal damage to WIL2-NS cells. These findings suggest that the induction and prevention of H2O2-induced chromosomal damage are different between quercetin and its metabolites.     

Influence of salicylic acid on H2O2 production, oxidative stress, and H2O2-metabolizing enzymes. Salicylic acid-mediated oxidative damage requires H2O2.

We investigated how salicylic acid (SA) enhances H2O2 and the relative significance of SA-enhanced H2O2 in Arabidopsis thaliana. SA treatments enhanced H2O2 production, lipid peroxidation, and oxidative damage to proteins, and resulted in the formation of chlorophyll and carotene isomers. SA-enhanced H2O2 levels were related to increased activities of Cu,Zn-superoxide dismutase and were independent of changes in catalase and ascorbate peroxidase activities. Prolonging SA treatments inactivated catalase and ascorbate peroxidase and resulted in phytotoxic symptoms, suggesting that inactivation of H2O2-degrading enzymes serves as an indicator of hypersensitive cell death. Treatment of leaves with H2O2 alone failed to invoke SA-mediated events. Although leaves treated with H2O2 accumulated in vivo H2O2 by 2-fold compared with leaves treated with SA, the damage to membranes and proteins was significantly less, indicating that SA can cause greater damage than H2O2. However, pretreatment of leaves with dimethylthiourea, a trap for H2O2, reduced SA-induced lipid peroxidation, indicating that SA requires H2O2 to initiate oxidative damage. The relative significance of the interaction among SA, H2O2, and H2O2-metabolizing enzymes with oxidative damage and cell death is discussed.     

Rat germinal cells require PARP for repair of DNA damage induced by gamma-irradiation and H2O2 treatment

The ability of rat germinal cells to recover from genotoxic stress has been investigated using isolated populations of primary spermatocytes and round spermatids. Using a comet assay at pH 10.0 to assess single strand breakage (SSB) in DNA, it was found that a high level of damage was induced by 5 Gy gamma-irradiation and acute exposure to 50 microM H2O2. This damage was effectively repaired during a subsequent recovery period of 1-3 hours culture in vitro but repair was significantly delayed in the presence of the poly(ADP-ribose)polymerase (PARP) inhibitor 3-aminobenzamide (3-ABA). Immunofluorescence detection of PARP with specific antibodies localised the protein to discrete foci within the nucleus of both spermatocytes and spermatids. Poly(ADP-ribose) (pADPR) could also be detected in spermatid nuclei following gamma-irradiation or H2O2 treatment. Moreover, PARP activation occurs both in spermatocytes and spermatids left to recover after both genotoxic stresses. The NO donors, 3-morpholino-sydnonimine (SIN-1) and S-nitrosoglutathione (SNOG), caused significant SSBs in both spermatocytes and spermatids. The effects of SIN-1 could be prevented by exogenous catalase (CAT), but not superoxide dismutase (SOD), in the cell suspensions. SNOG-induced SSBs were insensitive to both CAT and SOD. It is concluded that DNA in spermatocytes and spermatids is sensitive to damage by gamma-irradiation and H2O2 and that efficient repair of SSBs requires PARP activity.     

Angiopoietin-1 protects H9c2 cells from H2O2-induced apoptosis through AKT signaling

Loss of cardiomyocytes by apoptosis is proposed to cause ventricular remodeling and heart failure. Reactive oxygen species-induced apoptosis of cardiomyocytes has been reported to play an important role in many types of pathological processes of the heart. We investigated whether angiopoietin-1 (Ang1) has direct cytoprotective effects on cardiomyocytes against oxidative stress. Cultured H9c2 cells (cardiomyocytes) were treated with hydrogen peroxide (H(2)O(2)). Apoptosis was evaluated by flow cytometry, TUNEL assay and DNA laddering. The H(2)O(2) treatment caused typical apoptosis of H9c2 cells in a time-dependent manner. Transfection of recombinant adenovirus expressing Ang1 resulted in a sustained phosphorylation of AKT and inhibition of H(2)O(2)-induced apoptosis in H9c2 cells. This effect could be reversed by AKT inhibition. These results suggest that Ang1 protects cardiomyocytes from oxidative stress-induced apoptosis by regulating the activity of AKT.