Protection against nuclear DNA damage offered by flavonoids in cells exposed to hydrogen peroxide: the role of iron chelation

The ability of a number of flavonoids belonging to the flavone, flavonol, flavanone, and flavan-3-ol subclasses to protect cellular DNA from H₂O₂-induced single-strand breaks and the underlying molecular mechanisms were investigated in this work. Formation of single-strand breaks on nuclear DNA, after exposure of Jurkat cells to continuously generated H₂O₂ in the presence or absence of the flavonoid compounds, was evaluated by the comet assay (single-cell gel electrophoresis). The results indicate the following structural requirements of flavonoids for effective DNA protection: (a) the ortho-dihydroxy structure in either ring A or ring B, (b) the hydroxyl moiety on position 3 in combination with the oxo group at position 4, and (c) the presence of a C₂, C₃ double bond in ring C. In contrast to free flavonoids, the ability of complexes of [Fe²⁺]/[flavonoid] to protect nuclear DNA was decreased as the ratio increased, and the complex was completely inactive when the ratio reached a certain value. Moreover, it was observed that several of the flavonoids tested were able to remove iron from calcein loaded into cells and that this property was in excellent correlation with their ability to protect DNA (Spearman's correlation coefficient, ρ = 0.9, p = 0.005). The antioxidant (electron donating) capacities of the same flavonoids were also evaluated by a conventional method, but no relation with their DNA-protective ability could be established even when their membrane-penetrating abilities were taken into account (p = 0.64). In conclusion, the results presented in this work strongly support the notion that intracellular binding of iron is responsible for the protection offered by flavonoids against H₂O₂-induced DNA damage.     

Methylmercury and H(2)O(2) provoke lysosomal damage in human astrocytoma D384 cells followed by apoptosis

Methylmercury (MeHg) is a neurotoxic agent acting via diverse mechanisms, including oxidative stress. MeHg also induces astrocytic dysfunction, which can contribute to neuronal damage. The cellular effects of MeHg were investigated in human astrocytoma D384 cells, with special reference to the induction of oxidative-stress-related events. Lysosomal rupture was detected after short MeHg-exposure (1 μM, 1 h) in cells maintaining plasma membrane integrity. Disruption of lysosomes was also observed after hydrogen peroxide (H₂O₂) exposure (100 μM, 1 h), supporting the hypothesis that lysosomal membranes represent a possible target of agents causing oxidative stress. The lysosomal alterations induced by MeHg and H₂O₂ preceded a decrease of the mitochondrial potential. At later time points, both toxic agents caused the appearance of cells with apoptotic morphology, chromatin condensation, and regular DNA fragmentation. However, MeHg and H₂O₂ stimulated divergent pathways, with caspases being activated only by H₂O₂. The caspase inhibitor z-VAD-fmk did not prevent DNA fragmentation induced by H₂O₂, suggesting that the formation of high-molecular-weight DNA fragments was caspase independent with both MeHg and H₂O₂. The data point to the possibility that lysosomal hydrolytic enzymes act as executor factors in D384 cell death induced by oxidative stress.     

TRPA1 receptor stimulation by hydrogen peroxide is critical to trigger hyperalgesia and inflammation in a model of acute gout

Acute gout attacks produce severe joint pain and inflammation associated with monosodium urate (MSU) crystals leading to oxidative stress production. The transient potential receptor ankyrin 1 (TRPA1) is expressed by a subpopulation of peptidergic nociceptors and, via its activation by endogenous reactive oxygen species, including hydrogen peroxide (H₂O₂), contributes to pain and neurogenic inflammation. The aim of this study was to investigate the role of TRPA1 in hyperalgesia and inflammation in a model of acute gout attack in rodents. Inflammatory parameters and mechanical hyperalgesia were measured in male Wistar rats and in wild-type (Trpa1^+/+) or TRPA1-deficient (Trpa1^−/−) male mice. Animals received intra-articular (ia, ankle) injection of MSU. The role of TRPA1 was assessed by receptor antagonism, gene deletion or expression, sensory fiber defunctionalization, and calcitonin gene-related peptide (CGRP) release. We found that nociceptor defunctionalization, TRPA1 antagonist treatment (via ia or oral administration), and Trpa1 gene ablation abated hyperalgesia and inflammatory responses (edema, H₂O₂ generation, interleukin-1β release, and neutrophil infiltration) induced by ia MSU injection. In addition, we showed that MSU evoked generation of H₂O₂ in synovial tissue, which stimulated TRPA1 producing CGRP release and plasma protein extravasation. The MSU-elicited responses were also reduced by the H₂O₂-detoxifying enzyme catalase and the reducing agent dithiothreitol. TRPA1 activation by MSU challenge-generated H₂O₂ mediates the entire inflammatory response in an acute gout attack rodent model, thus strengthening the role of the TRPA1 receptor and H₂O₂ production as potential targets for treatment of acute gout attacks.     

Inhibition of succinate-linked respiration and complex II activity by hydrogen peroxide

Hydrogen peroxide produced from electron transport chain derived superoxide is a relatively mild oxidant, and as such, the majority of mitochondrial enzyme activities are impervious to physiological concentrations. Previous studies, however, have suggested that complex II (succinate dehydrogenase) is sensitive to H₂O₂-mediated inhibition. Nevertheless, the effects of H₂O₂ on succinate-linked respiration and complex II activity have not been examined in intact mitochondria. Results presented indicate that H₂O₂ inhibits succinate-linked state 3 mitochondrial respiration in a concentration dependent manner. H₂O₂ has no effect on complex II activity during state 2 respiration, but inhibits activity during state 3. It was found that conditions which prevent oxaloacetate accumulation during state 3 respiration, such as inclusion of rotenone, glutamate, or ATP, blunted the effect of H₂O₂ on succinate-linked respiration and complex II activity. It is concluded that H₂O₂ inhibits succinate-linked respiration indirectly by sustaining and enhancing oxaloacetate-mediated inactivation of complex II.     

The antioxidant action of N-acetylcysteine: Its reaction with hydrogen peroxide, hydroxyl radical, superoxide, and hypochlorous acid

N-acetylcysteine has been widely used as an antioxidant in vivo and in vitro. Its reaction with four oxidant species has therefore been examined. N-acetylcysteine is a powerful scavenger of hypochlorous acid (H---OCl); low concentrations are able to protect ₁-antiproteinase against inactivation by HOCl. N-acetylcysteine also reacts with hydroxyl radical with a rate constant of 1.36 × 10¹⁰ M⁻¹s⁻¹, as determined by pulse radiolysis. It also reacts slowly with H₂O₂, but no reaction of N-acetylcysteine with superoxide (O₂⁻) could be detected within the limits of our assay procedures.     

The effect of iron and agar on production of hydrogen peroxide by stimulated and activated mouse peritoneal macrophages

The effect of iron on H₂O₂ production by mouse peritoneal macrophages exposed to opsonised zymosan has been investigated. Macrophages elicited with thioglycollate broth produced less H₂O₂ than macrophages activated by Corynebacterium parvum, and levels were not affected by prior incubation of the cells with 0.1 mM iron nitrilotriacetate. However, preincubation with the iron chelator desferrioxamine (1 mM) reduced H₂O₂ production by both types of macrophages. Incubation of macrophages with agar, a component of thioglycollate broth, also reduced H₂O₂ production, particularly by C. parvum-activated macrophages. The results indicate that although iron appears to be necessary for H₂O₂ production by macrophages, the low level of production by thioglycollate-elicited macrophages is not due to an inadequate level of metabolically utilisable iron, but may be a result of prior ingestion of agar present in the broth.     

氧对口腔链球菌产生过氧化氢的影响

目的观察环境中氧含量对口腔链球菌过氧化氢产生速率的影响。方法采用ABTS-HRP微量板法测定在不同氧含量条件下口腔链球菌过氧化氢产生的速率。结果口腔链球菌在严格厌氧条件下过氧化氢产生速率为9.29nmol/(min×109细胞);当氧含量增高,口腔链球菌过氧化氢合成速率加快;在不同氧含量的环境中口腔链球菌产生过氧化氢的速率差异存在显著性:有氧振荡培养>有氧静置培养>厌氧(P<0.05)。结论环境中氧含量是影响口腔链球菌过氧化氢产生速率的重要因素。     

Action of 1,1-dimethylhydrazine on bacterial cells is determined by hydrogen peroxide

Seven different recombinant bioluminescent strains of Escherichia coli containing, respectively, the promoters katG and soxS (responsive to oxidative damage), recA (DNA damage), fabA (membrane damage), grpE, and rpoE (protein damage) and lac (constitutive expression) fused to the bacterial operon from Photorhabdus luminescens, were used to describe the mechanism of toxicity of 1,1-dimethylhydrazine (1,1-DMH) on bacteria, as well as to determine whether bacteria can sensitively detect the presence of this compound. A clear response to 1,1-DMH was observed only in E. coli carrying the katG’::lux, soxS’::lux, and recA’::lux-containing constructs. Preliminary treatment with catalase of the medium containing 1,1-DMH completely diminished the stress-response of the P_katG, P_recA, and P_soxS promoters. In the strain E. coli (pXen7), which contains a constitutive promoter, the level of cellular toxicity caused by the addition of 1,1-DMH was dramatically reduced in the presence of catalase.It is suggested that the action of 1,1-DMH on bacterial cells is determined by hydrogen peroxide, which is formed in response to reduction of the air oxygen level.     

Perspectives on hydrogen peroxide and drug-induced hemolytic anemia in glucose-6-phosphate dehydrogenase deficiency

G-6-PD-deficiency is a genetic disorder of erythrocytes in which the inability of affected cells to maintain NAD(P)H levels sufficient for the reduction of oxidized glutathione results in inadequate detoxification of hydrogen peroxide through glutathione peroxidase. Although a variety of free-radical species may be produced during the interaction of xenobiotic agents with erythrocytes and hemoglobin, the inability to destroy peroxides seems to be the hallmark of the disease. Colloid osmotic hemolysis is seldom observed in this disorder and it is possible that hydroxyl radicals derived from peroxide damage both lipid and protein constituents of the plasma membrane so that its intrinsic mechanical properties are altered. Erythrocytes with damaged membranes become less deformable and may be subjected to mechanical entrapment in the microcirculation. Ultimate recognition of damaged cell and sequestration by phagocytes leads to anemia.     

Adaptive response of Schizosaccharomyces pombe to hydrogen peroxide

Abstract Schizosaccharomyces pombe becomes resistant to killing by high concentration of hydrogen peroxide and other severe stresses including oxidants, high temperature and high concentration of ethanol when pretreated with nonlethal levels of hydrogen peroxide. In the presence of the protein synthesis inhibitor, cycloheximide, during hydrogen peroxide pretreatment, the cell obtained partial resistance to a higher level of hydrogen peroxide. The partial resistance to hydrogen peroxide in the presence of cycloheximide was acquired within 30 min of pretreatment but complete resistance obtained with de novo protein synthesis was not attained before 45 min of pretreatment. During adaptation to hydrogen peroxide, at least 15 polypeptides are induced, as analyzed by two-dimensional gel electrophoresis. Catalase activity is induced eight-fold by treatment with a nonlethal level of hydrogen peroxide.     

Role of intracellular labile iron,ferritin, and antioxidant defence in resistance of chronically adapted Jurkat T cells to hydrogen peroxide

To examine the role of intracellular labile iron pool (LIP), ferritin (Ft), and antioxidant defence in cellular resistance to oxidative stress on chronic adaptation, a new H₂O₂-resistant Jurkat T cell line “HJ16” was developed by gradual adaptation of parental “J16” cells to high concentrations of H₂O₂. Compared to J16 cells, HJ16 cells exhibited much higher resistance to H₂O₂-induced oxidative damage and necrotic cell death (up to 3 mM) and had enhanced antioxidant defence in the form of significantly higher intracellular glutathione and mitochondrial ferritin (FtMt) levels as well as higher glutathione-peroxidase (GPx) activity. In contrast, the level of the Ft H-subunit (FtH) in the H₂O₂-adapted cell line was found to be 7-fold lower than in the parental J16 cell line. While H₂O₂ concentrations higher than 0.1 mM fully depleted the glutathione content of J16 cells, in HJ16 cells the same treatments decreased the cellular glutathione content to only half of the original value. In HJ16 cells, H₂O₂ concentrations higher than 0.1 mM increased the level of FtMt up to 4-fold of their control values but had no effect on the FtMt levels in J16 cells. Furthermore, while the basal cytosolic level of LIP was similar in both cell lines, H₂O₂ treatment substantially increased the cytosolic LIP levels in J16 but not in HJ16 cells. H₂O₂ treatment also substantially decreased the FtH levels in J16 cells (up to 70% of the control value). In contrast in HJ16 cells, FtH levels were not affected by H₂O₂ treatment. These results indicate that chronic adaptation of J16 cells to high concentrations of H₂O₂ has provoked a series of novel and specific cellular adaptive responses that contribute to higher resistance of HJ16 cells to oxidative damage and cell death. These include increased cellular antioxidant defence in the form of higher glutathione and FtMt levels, higher GPx activity, and lower FtH levels. Further adaptive responses include the significantly reduced cellular response to oxidant-mediated glutathione depletion, FtH modulation, and labile iron release and a significant increase in FtMt levels following H₂O₂ treatment.     

The periodontal pathogen Porphyromonas gingivalis harnesses the chemistry of the mu-oxo bishaem of iron protoporphyrin IX to protect against hydrogen peroxide

The major haem component in the black pigment of Porphyromonas gingivalis is the mu-oxo bishaem of iron protoporphyrin IX and formation and cell-surface binding of this haem species is proposed as an extracellular buffer against reactive oxidants [Smalley, J.W. et al. (1998) Biochem. J. 331, 681-685]. P. gingivalis cells grown in the presence of the mu-oxo bishaem were protected against H(2)O(2) compared to control cells grown without it. When added to the growth medium, soluble mu-oxo bishaem inactivated H(2)O(2) and supported cell growth. Cells carrying a surface layer of mu-oxo bishaem were less susceptible to peroxidation by H(2)O(2). Cell-surface haems were slowly destroyed during reaction with H(2)O(2). Binding of mu-oxo bishaem by P. gingivalis may aid survival during neutrophil attack through inactivation of hydrogen peroxide.     

The kinetics of the complex formation between iron(III)-ethylenediaminetetraacetate and hydrogen peroxide in aqueous solution

The kinetics of the formation of the purple complex [Fe^III(EDTA)O₂]³⁻, between Fe^III-EDTA and hydrogen peroxide was studied as a function of pH (8.22-11.44) and temperature (10-40 °C) in aqueous solutions using a stopped-flow method. The reaction was first-order with respect to both reactants. The observed second-order rate constants decrease with an increase in pH and appear to be related to deprotonation of Fe^III-EDTA ([Fe(EDTA)H₂O]⁻ ⇔ Fe(EDTA)OH]²⁻ + H⁺). The rate law for the formation of the complex was found to be d[Fe^IIIEDTAO₂]³⁻/dt=[(k₄[H⁺]/([H⁺] + K₁)][Fe^III-EDTA][H₂O₂], where k₄=8.15±0.05×10⁴ M⁻¹ s⁻¹ and pK₁=7.3. The steps involved in the formation of [Fe(EDTA)O₂]³⁻ are briefly discussed.     

The centennial of the Fenton reaction

A short account is given of Fenton's life and research, with special emphasis on the Fenton reactions.     

产H_2O_2的乳酸杆菌分离培养基的改良

对MRS培养基的使用方法进行了改良。将MRS(含0.25 mg/ml TMB)融化后倾倒入平皿,待其凝固后,在琼脂表明加入200μl(0.01 mg/ml)HRP,涂匀后进行乳酸杆菌划线分离。37℃、厌氧培养48~72 h。结果表明,产生H2O2的乳酸杆菌呈蓝色菌落,和原来的培养方法相比,改良的方法简便经济、HPR的使用量为原来的1%,分离产H2O2的乳酸杆菌的效果良好。     

Reaction of iron(III)-polyaminocarboxylate complexes with hydrogen peroxide: Correlation between ligand structure and reactivity

Reactions of iron(III) complexes with five polyaminocarboxylates and hydrogen peroxide in an alkaline solution were investigated. Iron(III) complexes of which the ring including two nitrogen and iron atoms is five-membered formed a well-known stable side-on peroxo adduct. On the other hand, iron(III) complexes which have a six-membered ring formed a short-lived side-on peroxo adduct and then changed to iron(II) complex and superoxide. Electrochemical measurements showed that the redox potentials of the iron complexes having a six-membered ring are higher than those of the complexes having a five-membered ring. These results indicate that the chelate size is an important factor for tuning the redox potential of the iron center and for the reactivity toward hydrogen peroxide.     

Antioxidant action of sodium diethyldithiocarbamate: Reaction with hydrogen peroxide and superoxide radical

The oxidation of sodium diethyldithiocarbamate (DDC) by hydrogen peroxide or superoxide radicals has been investigated. Hydrogen peroxide oxidizes DDC, leading to the formation of a hydrated form of disulfiram, a dimer of DDC having a disulfide group. In equimolar conditions, the overall process appears as a first-order reaction (k = 0.025±0.005 s⁻¹), the first step being a second-order reaction (k = 5.0±0.1mol⁻¹.1. s⁻¹). No radical intermediate was observed in this process. In the presence of an excess of any of the reagents, the hydrated form of disulfiram transforms into different products corresponding to the fixation of oxygen by sulfur atoms or replacement of C = S group by ketone function, in the presence of an excess of hydrogen peroxide. Superoxide anions (produced by steady-state ⁶⁰Co γ-radiolysis) oxidize DDC, yielding similar products to those obtained with hydrogen peroxide with a maximum oxidation G-value of 0.3 μmol.J⁻¹. The rate constant k(O₂^·− + DDC) is equal to 900 mol⁻¹. 1. s⁻¹.     

Proteomic analysis of the proteins expressed by hydrogen peroxide treated cultured human dermal microvascular endothelial cells

Reactive oxygen species (ROS) have been traditionally regarded as toxic by-products of aerobic metabolism. However, ROS also act as intracellular signaling molecules and can mediate phenotypes in vascular endothelial cells, which may be physiological or pathological in nature. To clarify the molecular mechanisms of ROS signaling, we examined hydrogen peroxide (H(2)O(2))-responsive proteins in cultured human dermal microvascular endothelial cells (HMVEC) using proteomic tools. Protein expression in HMVEC was studied after they had been exposed to low- and high-levels of H(2)O(2) for various times, and intracellular ROS production was examined by flow cytometer and UV spectrophotometer. Proteins obtained from dose- and time-dependent series were separated by two-dimensional gel electrophoresis and tentatively identified by matrix-assisted laser desorption-time of flight mass spectrometry, by matching the tryptic mass maps obtained with entries in the NCBI and Swiss-Prot protein sequence database. At least 163 proteins were changed by H(2)O(2), and 60 proteins were identified. Oxidative stress triggered dramatic change in the expression of proteins in primary microvessel endothelial cells, and their mapping to cellular process provided a view of the ubiquitous cellular changes elicited by H(2)O(2). These results could provide a framework for the understanding of the mechanisms of cellular redox homeostasis and H(2)O(2) metabolism in microendothelium environment in various biological processes as well as pathological conditions.     

Oxygen and hydrogen peroxide enhance light-induced carotenoid synthesis in Neurospora crassa

Previously, we found that intracellular reactive oxygen species (ROS) affect photomorphogenesis in Neurospora crassa. In this study, we investigated the physiological roles of ROS in the response to light and found that the exposure of mycelia to air was important for the light-induced carotenogenesis. Mycelia treated with a high concentration of O(2) gas and H(2)O(2) to release ROS showed an enhancement of light-induced carotenoid accumulation and the expression of gene related to light-inducible carotenogenesis. These results suggested that stimuli caused by the exposure of the mycelia to air containing O(2) gas triggered the light-induced carotenoid synthesis.