Deoxyribonucleic acid-protein and deoxyribonucleic acid interstrand cross-links induced in isolated chromatin by hydrogen peroxide and ferrous ethylenediaminetetraacetate chelates

DNA-protein and DNA interstrand cross-links were induced in isolated chromatin after treatment with H2O2 and ferrous ethylenediaminetetraacetate (EDTA). Retention of DNA on membrane filters after heating of chromatin in a dissociating solvent indicated the presence of a stable linkage between DNA and protein. Treatment of protein-free DNA with H2O2/Fe2+-EDTA did not result in enhanced filter retention. Incubation of cross-linked chromatin with proteinase K completely eliminated filter retention. Resistance to S1 nuclease after a denaturation-renaturation cycle was used to detect DNA interstrand cross-links. Heating the treated chromatin at 45 degrees C for 16 h and NaBH4 reduction enhanced the extent of interstrand cross-linking. The following data are consistent with, but do not totally prove, the hypothesis that cross-links are induced by hydroxyl radicals generated in Fenton-type reactions: (1) cross-linking was inhibited by hydroxyl radical scavengers; (2) the degree of inhibition of DNA interstrand cross-links correlated very closely with the rate constants of the scavengers for reaction with hydroxyl radicals; (3) cross-linking was eliminated or greatly reduced by catalase; (4) the extent of cross-linking was directly related to the concentration of Fe2+-EDTA. Partial inhibition of cross-linking by superoxide dismutase indicates that superoxide-driven Fenton chemistry is involved. The data indicate that DNA cross-linking may play a role in the manifestation of the biological activity of agents or systems that generate reactive hydroxyl radicals.     

Transient Ca2+ changes in endothelial cells induced by low doses of reactive oxygen species: Role of hydrogen peroxide

Cultured human and rat endothelial cells were used to study cellular toxicity and Ca2+ signalling upon exposure to reactive oxygen species. Superoxide and hydrogen peroxide (O2·–/H2O2) were produced by the hypoxanthine/xanthine oxidase system (HX/XO) and caused intracellular Ca2+ concentration ([Ca2+]i) to rise steadily when activities above 2 mU/ml were used. These Ca2+ increases were also measured when the glucose/glucose oxidase (G/GO) system above 5 mU/ml was used to produce hydrogen peroxide (H2O2). Gross morphological changes appeared to parallel elevated [Ca2+]i levels preceding cell death. However, when HX/XO or G/GO were used at non toxic doses rapid and transient changes in [Ca2+]i were measured. These treatments did not alter subsequent receptor mediated Ca2+ signalling induced by ATP (10 M) or histamine (100 M). Superoxide dismutase (50 U/ml), which dismutates O2·minus; into H2O2 al ient [Ca2+]i responses. H2O2 added directly was able to induce similar Ca2+ transients when concentrations of at least 500 M were used. Buffering trace amounts of iron (o-phenanthroline; 200 M) in order to inhibit úOH radical formation was not effective to alter Ca2+ changes. Experiments performed in Ca2+-free buffer showed a similar rise in [Ca2+]i and readdition of Ca2+ to the extracellular medium indicated the activation of store operated Ca2+ entry. Blocking Ca2+-ATPases of the endoplasmatic reticulum with thapsigargin (1 M) inhibited ROS induced transient increases and cells preincubated with pertussis toxin (200 nM) showed unchanged Ca2+ transients after exposure to both enzyme systems. Phospholipase C inhibitor U73122 (2 M) effectively reduced hydrogen peroxide induced emptying of intracellular stores. Taken together, we demonstrate that enzymatically produced non-toxic H2O2 rather than O· ndash; or · OH causes calcium signalling from thapsigargin sensitive stores, and activates store operated Ca2+ entry at least partially by activating phospholipase C. These changes clearly differ from pathological oxidative stress associated with a progressive increase in [Ca2+]i.     

Abscisic acid mediates hydrogen peroxide production in peanut induced by water stress

Peanut plants exposed to water stress induced by polyethylene glycol (PEG) accumulated abscisic acid (ABA) and hydrogen peroxide (H₂O₂), the increase being significant at 12 and 24 h after addition, respectively. To address the question whether the increase in H₂O₂ production was related to ABA accumulation, the peanut leaves were pretreated with ABA biosynthesis inhibitor (sodium tungstate) and then exposed to water stress. Under these conditions, a decrease of ABA and H₂O₂ content were found after 12 h. The addition of 100 μM ABA restored H₂O₂ content reaching values similar to those under water stress at 12 h. We concluded that ABA accumulation is the first signal that triggers the H₂O₂ generation in peanut during first 12 h but its subsequent production is partially ABA-independent.     

Non-oxygen-forming pathways of hydrogen peroxide degradation by bovine liver catalase at low hydrogen peroxide fluxes

Heme catalases are considered to degrade two molecules of H₂O₂ to two molecules of H₂O and one molecule of O₂ employing the catalatic cycle. We here studied the catalytic behaviour of bovine liver catalase at low fluxes of H₂O₂ (relative to catalase concentration), adjusted by H₂O₂-generating systems. At a ratio of a H₂O₂ flux (given in μM/min^- 1) to catalase concentration (given in μM) of 10 min^- 1 and above, H₂O₂ degradation occurred via the catalatic cycle. At lower ratios, however, H₂O₂ degradation proceeded with increasingly diminished production of O₂. At a ratio of 1 min^- 1, O₂ formation could no longer be observed, although the enzyme still degraded H₂O₂. These results strongly suggest that at low physiological H₂O₂ fluxes H₂O₂ is preferentially metabolised reductively to H₂O, without release of O₂. The pathways involved in the reductive metabolism of H₂O₂ are presumably those previously reported as inactivation and reactivation pathways. They start from compound I and are operative at low and high H₂O₂ fluxes but kinetically outcompete the reaction of compound I with H₂O₂ at low H₂O₂ production rates. In the absence of NADPH, the reducing equivalents for the reductive metabolism of H₂O₂ are most likely provided by the protein moiety of the enzyme. In the presence of NADPH, they are at least in part provided by the coenzyme.     

Oxidative modification of ferritin induced by hydrogen peroxide

Excess free iron generates oxidative stress that may contribute to the pathogenesis of various causes of neurodegenerative diseases. In this study, we assessed the modification of ferritin induced by H(2)O(2). When ferritin was incubated with H(2)O(2), the degradation of ferritin L-chain increased with the H(2)O(2) concentration whereas ferritin H-chain was remained. Free radical scavengers, azide, thiourea, and N-acetyl-(L)-cysteine suppressed the H(2)O(2)-mediated ferritin modification. The iron specific chelator, deferoxamine, effectively prevented H(2)O(2)-mediated ferritin degradation in modified ferritin. The release of iron ions from ferritin was increased in H(2)O(2) concentration-dependent manner. The present results suggest that free radicals may play a role in the modification and iron releasing of ferritin by H(2)O(2). It is assumed that oxidative damage of ferritin by H(2)O(2) may induce the increase of iron content in cells and subsequently lead to the deleterious condition.     

Fragmentation of human ceruloplasmin induced by hydrogen peroxide

We investigated the fragmentation of human ceruloplasmin induced by H2O2 to study its oxidative damage. When ceruloplasmin was incubated with H2O2, the frequency of the protein fragmentation increased in a proportion to the concentration of H2O2. It also increased in a time-dependent manner and was accompanied by gradual loss of the oxidase activity. Hydroxyl radical scavengers such as azide and mannitol inhibited the fragmentation of ceruloplasmin. The deoxyribose assay showed that hydroxyl radicals were generated in the reaction of ceruloplasmin with H2O2. Incubation of ceruloplasmin with H2O2 resulted in a time-dependent release of copper ions. The released copper ion may participate in a Fenton-like reaction to produce hydroxyl radical, which enhanced the fragmentation. The protection of the fragmentation by copper chelators such as diethylenetriaminepentaacetic acid and bathocuproine indicates a role for copper ion in the reaction. These results suggest that the fragmentation of ceruloplasmin induced by H2O2 is due to hydroxyl radicals formed by a copper-dependent Fenton-like reaction.     

Copper ions mediate the lethality induced by hydrogen peroxide in low iron conditions in Escherichia coli

Iron ions mediate the formation of lethal DNA damage by hydrogen peroxide. However, when cells are depleted of iron ions by the treatment with iron chelators, DNA damage can still be detected. Here we show that the formation of such damage in low iron conditions is due to the participation of copper ions. Copper chelators can inhibit cell inactivation, DNA strand breakage and mutagenesis induced by hydrogen peroxide in cells pre-treated with iron chelators. The Fpg and UvrA proteins play an important role in the repair of DNA lesions formed in these conditions, as suggested by the great sensitivity of the uvrA and fpg mutant strains to the treatment when compared to the wild type strain.     

Microsatellite instability induced by hydrogen peroxide in Escherichia coli

Damage to DNA by reactive oxygen species may be a significant source of endogenous mutagenesis in aerobic organisms. Using a selective assay for microsatellite instability in E. coli, we have asked whether endogenous oxidative mutagenesis can contribute to genetic instability. Instability of repetitive sequences, both in intronic sequences and within coding regions, is a hallmark of genetic instability in human cancers. We demonstrate that exposure of E. coli to low levels of hydrogen peroxide increases the frequency of expansions and deletions within dinucleotide repetitive sequences. Sequencing of the repetitive sequences and flanking non-repetitive regions in mutant clones demonstrated the high specificity for alterations with the repeats. All of the 183 mutants sequenced displayed frameshift alterations within the microsatellite repeats, and no base substitutions or frameshift mutations occurred within the flanking non-repetitive sequences. We hypothesize that endogenous oxidative damage to DNA can increase the frequency of strand slippage intermediates occurring during DNA replication or repair synthesis, and contribute to genomic instability.     

Mechanism of oxyhemoglobin oxidation induced by hydrogen peroxide]

The process of oxyhemoglobin oxidation initiated by hydrogen peroxide in low (10(-7) M) concentrations was investigated. It was found, that H2O2 in this concentration is able to induce the process of chain oxidation of oxyhemoglobin to methemoglobin. The following observations indicate that the process is essentially the chain reaction: 1) The amount of the methemoglobin in haem groups, produced in the reaction, exceed by 20 times the quantity of hydrogen, added initially, to induce the oxidation. 2) Catalase stopped this process at any stage of the reaction. This fact implies that the chain process involves generation of new molecules of H2O2 in the course of oxidation of oxyhemoglobin. The chain reaction proceeded only in the presence of oxygen. But if oxygen was introduced into hemoglobin solution, preincubated with H2O2 in vacuum, than again the oxidation of hemoglobin developed. Apparently, H2O2 in low concentrations appears, mainly, as an inductor of the oxyhemoglobin autooxidation.     

The ability of campylobacter media supplements to neutralize photochemically induced toxicity and hydrogen peroxide

B olton , F.J. C oates D. & H utchinson , D.N. 1984. The ability of campylobacter media supplements to neutralize photochemically induced toxicity and hydrogen peroxide. Journal of Applied Bacteriology 56 , 151–157.
Nutrient agar plates stored in light and air for 48 h became inhibitory for Campylobacter jejuni, C. coli and nalidixic acid-resistant, therrnophilic campylobacter (NARTC) strains. All five campylobacter test strains showed > 5 log reduction in counts on media which had been stored in light and air. Media stored in the dark and/or in a reduced atmosphere did not become inhibitory and supported the growth of campylobacters. Ferrous sulphate, sodium pyruvate, blood, charcoal or sodium metabisulphite, compounds frequently used as supplements in campylobacter media, were added to nutrient agar prior to storage of media in light and air. All additives except sodium metabisulphite prevented the accumulation of photochemically generated toxic oxygen derivatives and allowed growth of test strains. In qualitative tests to determine the ability of supplements to neutralize hydrogen peroxide, blood was the most active, charcoal and sodium pyruvate slightly less active and ferrous sulphate and sodium metabisulphite the least active. The results of this study confirm that supplements in campylobacter media act as quenching or detoxifying agents and not as enrichment factors.     

Turning point in apoptosis/necrosis induced by hydrogen peroxide

The turning point between apoptosis and necrosis induced by hydrogen peroxide (H₂O₂) have been investigated using human T-lymphoma Jurkat cells. Cells treated with 50 μM H₂O₂ exhibited caspase-9 and caspase-3 activation, finally leading to apoptotic cell death. Treatment with 500 μM H₂O₂ did not exhibit caspase activation and changed the mode of death to necrosis. On the other hand, the release of cytochrome c from the mitochondria was observed under both conditions. Treatment with 500 μM H₂O₂, but not with 50 μM H₂O₂, caused a marked decrease in the intracellular ATP level; this is essential for apoptosome formation. H₂O₂-reducing enzymes such as cellular glutathione peroxidase (cGPx) and catalase, which are important for the activation of caspases, were active under the 500 μM H₂O₂ condition. Prevention of intracellular ATP loss, which did not influence cytochrome c release, significantly activated caspases, changing the mode of cell death from necrosis to apoptosis. These results suggest that ATP-dependent apoptosome formation determines whether H₂O₂-induced cell death is due to apoptosis or necrosis.     

Ceruloplasmin enhances DNA damage induced by hydrogen peroxide in vitro

Ceruloplasmin (Cp) was found to promote the oxidative damage to DNA, as evidenced by the formation of 8-hydroxy-2'-deoxyguanosine and strand breaks, when incubated with H₂O₂ in vitro. The capacity of Cp to enhance oxidative damage to DNA was inhibited by hydroxyl radical scavengers such as sodium azide and mannitol, a metal chelator, diethylenetriaminepenta-acetic acid, and catalase. Although the oxidized protein resulted in an increase in the content of carbonyl groups, the ferroxidase activity and the proteolytic susceptibility were not significantly altered. The release of a portion of Cu from Cp was observed, and conformational alterations were indicated by the changes in fluorescence spectra. Based on these results, we suggest that damage to DNA is mediated in the H₂O₂/Cp system via the generation of ^·OH by released Cu²⁺ and/or loosely bound Cu exposed from oxidatively damaged Cp through the conformational change. The release of Cu from Cp during oxidative stress could enhance the formation of reactive oxygen species and could also potentiate cellular damage.     

Caspase-dependent and -independent events in apoptosis induced by hydrogen peroxide

To define the role of caspase-3 in H2O2-induced apoptosis, we introduced caspase-3 cDNA into MCF-7 breast carcinoma cells that otherwise lack caspase-3 expression. H2O2 treatment induced DNA fragmentation and nuclear condensation in the caspase-3-expressing cells, but not in the caspase-3-deficient cells. This indicated that caspase-3 is essential for nuclear events. However, H2O2 induced an externalization of membrane phosphatidylserine (PS) and cell death regardless of caspase-3 expression. These events were not suppressed by Ac-DEVD-CHO and Z-VAD-fmk, which inhibit DEVD-specific caspases and a broad spectrum of caspases, respectively. In Jurkat T cells, these inhibitors abolished H2O2-induced PS relocalization, but not cell death. Therefore, caspases appear to be dispensable for lethality by H2O2, but required for PS redistribution in a cell-type-specific manner.     

Turning point in apoptosis/necrosis induced by hydrogen peroxide

The turning point between apoptosis and necrosis induced by hydrogen peroxide (H₂O₂) have been investigated using human T-lymphoma Jurkat cells. Cells treated with 50 μM H₂O₂ exhibited caspase-9 and caspase-3 activation, finally leading to apoptotic cell death. Treatment with 500 μM H₂O₂ did not exhibit caspase activation and changed the mode of death to necrosis. On the other hand, the release of cytochrome c from the mitochondria was observed under both conditions. Treatment with 500 μM H₂O₂, but not with 50 μM H₂O₂, caused a marked decrease in the intracellular ATP level; this is essential for apoptosome formation. H₂O₂-reducing enzymes such as cellular glutathione peroxidase (cGPx) and catalase, which are important for the activation of caspases, were active under the 500 μM H₂O₂ condition. Prevention of intracellular ATP loss, which did not influence cytochrome c release, significantly activated caspases, changing the mode of cell death from necrosis to apoptosis. These results suggest that ATP-dependent apoptosome formation determines whether H₂O₂-induced cell death is due to apoptosis or necrosis.