Cellular protection of morin against the oxidative stress induced by hydrogen peroxide

Flavonoids are a class of secondary metabolites abundantly found in fruits and vegetables. In addition, flavonoids have been reported as potent antioxidants with beneficial effects against oxidative stress-related diseases such as cancer, aging, and diabetes. The present study was carried out to investigate the cytoprotective effects of morin (2′,3,4′,5,7-pentahydroxyflavone), a member of the flavonoid group, against hydrogen peroxide (H₂O₂)-induced DNA and lipid damage. Morin was found to prevent the cellular DNA damage induced by H₂O₂ treatment, which is shown by the inhibition of 8-hydroxy-2′-deoxyguanosine (8-OHdG) formation (a modified form of DNA base), inhibition of comet tail (a form of DNA strand breakage), and decrease of nuclear phospho histone H2A.X expression (a marker for DNA strand breakage). In addition, morin inhibited membrane lipid peroxidation, which is detected by inhibition of thiobarbituric acid reactive substance (TBARS) formation. Morin was found to scavenge the intracellular reactive oxygen species (ROS) generated by H₂O₂ treatment in cells, which is detected by a spectrofluorometer, flow cytometry, and confocal microscopy after staining of 2′,7′-dichlorodihydrofluorescein diacetate (DCF-DA). Morin also induces an increase in the activity of catalase and protein expression. The results of this study suggest that morin protects cells from H₂O₂-induced damage by inhibiting ROS generation and by inducing catalase activation.     

Americanin B protects cultured human keratinocytes against oxidative stress by exerting antioxidant effects

We evaluated the cytoprotective effects of americanin B, a lignan compound, against hydrogen peroxide (H₂O₂)-induced cell damage. Americanin B decreased the level of DPPH radicals, superoxide anions, hydroxyl radicals, and intracellular reactive oxygen species. Americanin B also attenuated DNA damage induced by H₂O₂ treatment, as shown by the inhibition of formation of comet tails, indicative of DNA strand breakage, and prevented the oxidation of protein and peroxidation of lipid, as determined by protein carbonyls and 8-isoprostane. Furthermore, americanin B protected against H₂O₂-induced apoptotic cell death, as determined by a reduction in the numbers of apoptotic bodies stained with Hoechst 33342. These findings suggest that americanin B protects cells against oxidative damage by exerting antioxidant effects and inhibiting apoptosis.     

A comparison of four assays detecting oxidizing species

Quin2, a fluorescent calcium probe, has a low affinity for calcium in comparison to its affinities for transition metal ions. Chelation of ferric ion with quin2 strongly enhanced the formation of oxidizing species in the presence of bolus H₂O₂ as detected with four assays, electron spin resonance with the spin-trap DMPO, the deoxyribose assay, the DMSO assay, and plasmid DNA strand breakage. In comparison, Fe(III)-EDTA reacted with bolus H₂O₂ only as detected with electron spin resonance and the deoxyribose assay, but not as detected with the two latter assays. The addition of reductants, like ascorbate or superoxide generated by hypoxanthine/xanthine oxidase, to Fe(III)-EDTA in the presence of H₂O₂ produced plasmid DNA strand breakage and strong reactivity in both the DMSO and the deoxyribose assays. Our findings suggest that the main oxidizing species produced in Fenton-type reactions is hydroxyl radical. However, the reaction between Fe(III)-EDTA and bolus H₂O₂ appears to be exceptional and dominated by a nonhydroxyl radical species.     

Nicotinamide "protects" resting lymphocytes exposed to hydrogen peroxide from necrosis but not from apoptosis

The aim of this work was to investigate the relationship between mechanisms of DNA repair and apoptosis induced by oxidative stress (H₂O₂) in human lymphocytes. Using the comet assay, fluorescent microscopy, and DNA electrophoresis, we studied the DNA damage induced by hydrogen peroxide (H₂O₂) treatment, the time and the amount of repair of strand breaks, the type of cell death, and the influence of inhibitors of repair (nicotinamide). When lymphocytes were treated with H₂O₂, we observed an increased in necrosis compared to apoptosis. However, when nicotinamide (which inhibits DNA repair) was added, the mode of death reversed to increased apoptosis. These results indicate that nicotinamide "protects" resting lymphocytes exposed to H₂O₂ from necrosis but not from apoptosis. This revised version was published online in July 2006 with corrections to the Cover Date.     

Urea Hydrogen Peroxide Reduces the Numbers of Lactobacilli, Nourishes Yeast, and Leaves No Residues in the Ethanol Fermentation

Urea hydrogen peroxide (UHP) at a concentration of 30 to 32 mmol/liter reduced the numbers of five Lactobacillus spp. (Lactobacillus plantarum, L. paracasei, Lactobacillus sp. strain 3, L. rhamnosus, and L. fermentum) from ~10⁷ to ~10² CFU/ml in a 2-h preincubation at 30°C of normal-gravity wheat mash at ~21 g of dissolved solids per ml containing normal levels of suspended grain particles. Fermentation was completed 36 h after inoculation of Saccharomyces cerevisiae in the presence of UHP, even when wheat mash was deliberately contaminated (infected) with L. paracasei at ~10⁷ CFU/ml. There were no significant differences in the maximum ethanol produced between treatments when urea hydrogen peroxide was used to kill the bacteria and controls (in which no bacteria were added). However, the presence of L. paracasei at ~10⁷ CFU/ml without added agent resulted in a 5.84% reduction in the maximum ethanol produced compared to the control. The bactericidal activity of UHP is greatly affected by the presence of particulate matter. In fact, only 2 mmol of urea hydrogen peroxide per liter was required for disinfection when mashes had little or no particulate matter present. No significant differences were observed in the decomposition of hydrogen peroxide in normal-gravity wheat mash at 30°C whether the bactericidal agent was added as H₂O₂ or as urea hydrogen peroxide. NADH peroxidase activity (involved in degrading H₂O₂) increased significantly (P = 0.05) in the presence of 0.75 mM hydrogen peroxide (sublethal level) in all five strains of lactobacilli tested but did not persist in cells regrown in the absence of H₂O₂. H₂O₂-resistant mutants were not expected or found when lethal levels of H₂O₂ or UHP were used. Contaminating lactobacilli can be effectively managed by UHP, a compound which when used at ca. 30 mmol/liter happens to provide near-optimum levels of assimilable nitrogen and oxygen that aid in vigorous fermentation performance by yeast.     

The role of intracellular zinc in H2O2-induced oxidative stress in human erythrocytes

In vitro exposure of human erythrocytes to H₂O₂ at concentrations of 30–1000 μM resulted in a dose-dependent increase of the intracellular levels of Zn²⁺ and inhibition of the cytosolic esterase activity, which is a major marker of erythrocyte viability. The observed effect depended on the concentration of H₂O₂ and the duration of exposure of the cells to this compound. An inverse relationship between the changes in the intracellular level of labile zinc ions and esterase activity in the cells exposed to hydrogen peroxide was detected; this was indicative of the role of Zn²⁺ in the programmed death of red blood cells. The combined action of hydrogen peroxide and N',N'-tetrakis-(2-pyridyl-methyl)-ethylenediamine, an intracellular zinc ion chelator, has been found to eliminate the cytotoxic effect of H₂O₂, whereas the addition of Zn²⁺ to the erythrocyte incubation medium enhanced the effects of hydrogen peroxide. The reduction of the concentration of non-protein thiol groups due to a decrease of the level of reduced glutathione was shown to contribute to the release of Zn²⁺ from the intracellular binding sites during oxidative stress induced by H₂O₂ in human erythrocytes.

     

Micromolar concentrations of hydrogen peroxide induce oxidative DNA lesions more efficiently than millimolar concentrations in mammalian cells

Reactive oxygen species produce oxidized bases, deoxyribose lesions and DNA strand breaks in mammalian cells. Previously, we demonstrated that aldehydic DNA lesions (ADLs) were induced in mammalian cells by 10 mM hydrogen peroxide (H₂O₂). Interestingly, a bimodal H₂O₂ dose–response relationship in cell toxicity has been reported for Escherichia coli deficient in DNA repair as well as Chinese hamster ovary (CHO) cells. Furthermore, it has been demonstrated that H₂O₂ causes single-strand breaks in purified DNA in the presence of iron and induces mitochondrial DNA damage in CHO cells with a biphasic dose–response curve. Here we show that H₂O₂ produces ADLs at concentrations as low as 0.06 mM in HeLa cells and that lower concentrations of H₂O₂ were much more efficient at inducing ADLs than higher concentrations. This dose–response curve is strikingly similar to that for cell killing effects in E.coli deficient in DNA repair exposed to H₂O₂. Interestingly, serial treatment of submillimolar levels of H₂O₂ induced a massive accumulation of ADLs. The toxicity arising from H₂O₂ determined by intracellular NAD(P)H in cells correlated well with the formation of ADLs. The addition of dipyridyl, an iron (II)-specific chelator, significantly protected against DNA damage and cell toxicity from submillimolar, but not millimolar, amounts of H₂O₂. These results suggest that ADLs induced by submillimolar levels of H₂O₂ may be due to a Fenton-type reaction between H₂O₂ and intracellular iron ions in mammalian cells.     

Leucocytes isolated from simply frozen whole blood can be used in human biomonitoring for DNA damage measurement with the comet assay

Preservation of human blood cells for DNA damage analysis with the comet assay conventionally involves the isolation of mononuclear cells by centrifugation, suspension in freezing medium and slow freezing to ?80 °C—a laborious process. A recent publication (Al‐Salmani et al. Free Rad Biol Med 2011; 51: 719–725) describes a simple method in which small volumes of whole blood are frozen to ?20 or ?80 °C; on subsequent thawing, the comet assay is performed, with no indication of elevated DNA strand breakage resulting from the rapid freezing. However, leucocytes in whole blood (whether fresh or frozen) are abnormally resistant to damage by H₂O₂, and so a common test of antioxidant status (resistance to strand breakage by H₂O₂) cannot be used. We have refined this method by separating the leucocytes from the thawed blood; we find that, after three washes, the cells respond normally to H₂O₂. In addition, we have measured specific endogenous base damage (oxidized purines) in the isolated leucocytes, using the enzyme formamidopyrimidine DNA glycosylase. In a study of blood samples from 10 subjects, H₂O₂ sensitivity and endogenous damage—both reflecting the antioxidant status of the cells—correlated significantly. This modified approach to sample collection and storage is particularly applicable when the available volume of blood is limited and has great potential in biomonitoring and ecogenotoxicology studies where samples are obtained in the field or at sites remote from the testing laboratory. Copyright © 2013 John Wiley & Sons, Ltd.     

Peroxide reduction by a metal-dependent catalase in Nostoc punctiforme (cyanobacteria)

This study investigated the role of a novel metal-dependent catalase (Npun_R4582) that reduces hydrogen peroxide in the cyanobacterium Nostoc punctiforme. Quantitative real-time PCR showed that npun_R4582 relative mRNA levels were upregulated by over 16-fold in cells treated with either 2 μM added Co, 0.5 μM added Cu, 500 μM Mn, 1 μM Ni, or 18 μM Zn. For cells treated with 60 μM H₂O₂, no significant alteration in Npun_R4582 relative mRNA levels was detected, while in cells treated with Co, Cu, Mn, Ni, or Zn and 60 μM peroxide, relative mRNA levels were generally above control or peroxide only treated cells. Disruption or overexpression of npun_R4582 altered sensitivity to cells exposed to 60 μM H₂O₂ and metals for treatments beyond the highest viable concentrations, or in a mixed metal solution for Npun_R4582⁻ cells. Moreover, overexpression of npun_R4582 increased cellular peroxidase activity in comparison with wild-type and Npun_R4582⁻ cells, and reduced peroxide levels by over 50%. The addition of cobalt, manganese, nickel, and zinc increased the capacity of Npun_R4582 to reduce the rate or total levels of peroxide produced by cells growing under photooxidative conditions. The work presented confirms the function of NpunR4582 as a catalase and provides insights as to how cells reduce potentially lethal peroxide levels produced by photosynthesis. The findings also show how trace elements play crucial roles as enzymatic cofactors and how the role of Npun_R4582 in hydrogen peroxide breakdown is dependent on the type of metal and the level available to cells.

     

Estrogens protect against hydrogen peroxide and arachidonic acid induced DNA damage

The ability of estrogens to protect against DNA damage induced by either hydrogen peroxide or arachidonic acid alone or in combination with Cu²⁺ was investigated. DNA strand breaks were determined by conversion of double stranded supercoiled ØX-174 RFI DNA to double stranded open circular DNA and linear single stranded DNA. Estradiol-17β significantly decreased the formation of single and double strand breaks in DNA induced by H₂O₂ alone or with Cu²⁺. Equilin (an equine estrogen) was more effective than estradiol-17β at the doses tested. Arachidonic acid in the presence of Cu²⁺ caused the formation of high levels of linear DNA which was protected by estrogen with equilen being more effective. These studies suggest that estrogens through this protective effect on DNA damage might contribute to cardioprotection.     

Aldehyde oxidase generates deoxyribonucleic acid single strand nicks in vitro

SUMMARY

We purified aldehyde oxidase (AO) from rabbit livers and found that AO produced deoxyribonucleic acid (DNA) single strand nicks in vitro. Acetaldehyde, benzaldehyde, and certain purine bases were effective substrates for AO catalyzed DNA strand nicking. DNA strand nicking did not occur with the reducing substrates nicotinamide-adenine dinucleotide or dithionite that produce superoxide anion (O_2′^?). Inclusion of electron transport inhibitors, potassium cyanide, ferricyanide or menadione, prevented AO catalyzed nicking. AO induced DNA strand nicking was dependent upon hydrogen peroxide (H₂O₂) formation and most likely generation of hydroxyl radical (HO'). The present observations may be pertinent to the recently proposed involvement of AO in inherited juvenile familial amyotrophic lateral sclerosis (JFALS) and other oxygen radical mediated diseases.     

Opposing effects of nitroxide free radicals in Escherichia coli mutants deficient in DNA repair

Nitroxide free radicals have been previously shown to function as superoxide dismutase (SOD) mimics and to protect bacterial and mammalian cells against oxidative damage, particularly from superoxide and hydrogen peroxide. Although nitroxides are generally considered to be non-toxic nor mutagenic, there is no agreement regarding their potential adverse effect. Some toxic effects were observed upon using high concentration of six-membered ring derivatives. Conflicting evidence has also been reported regarding the mutagenic activity of nitroxides toward Salmonella typhimurium. It was also demonstrated that nitroxides exert two opposing effects on exonuclease III deficient cells of Escherichia coli upon exposure to naphthoquinones. The attempts to use nitroxides as contrast agents in nuclear magnetic resonance imaging (MRI) and as a new class of anti-oxidants underscore the need to examine their potential adverse effects. Since nitroxides protected xthA cells from DNA scission caused by H₂O₂, it was anticipated that they would provide even greater t protection for recA DNA repair-deficient cells of E. coli, which are more sensitive to H₂O₂-induced oxidative stress. The results of the present study showed that: (1) nitroxides exert bactericidal and bacteriostatic effects on recA but not on xthA or wild-type E. coli K12 cells, (b) nitroxides and H₂O₂ act synergistically on recA cells, both under aerobic and hypoxic conditions; (c) the nitroxide-induced toxicity in recA cells and the synergistic effect with H₂O₂ were not accompanied by a decrease in the cellular level of reduced glutathione; (d) TEMPAMINE protected against DNA scission induced by H₂O₂ and 1,10-o-phenanthroline chelate of Cu(II) in xthA cells, but potentiated DNA double-strand breakage in recA cells.     

Induction of DNA double strand breaks in cultured mammalian cells exposed to hydrogen peroxide and histidine

The amino acid histidine was found to increase the toxicity of H₂O₂ in cultured mammalian cells. Histidine also augmented the level of DNA single strand breaks (SSB) detectable in cells exposed to the oxidant and, in addition, resulted in the appearance of DNA double strand breaks (DSB), a lesion which is not produced by H₂O₂ alone.     

A simplified chromatin dispersion (nuclear halo) assay for detecting DNA breakage induced by ionizing radiation and chemical agents

Abstract

Methods for visualizing DNA damage at the microscopic level are based on treatment of cell nuclei with saline or alkaline solutions. These procedures for achieving chromatin dispersion produce halos that surround the nuclear remnants. We improved the fast halo assay for visualizing DNA breakage in cultured cells to create a simplified method for detection and quantitative evaluation of DNA breakage. Nucleated erythrocytes from chicken blood were selected as a model test system to analyze the production of nuclear halos after treatment with X-rays or H₂O₂. After staining with ethidium bromide or Wright's methylene blue-eosin solution, nuclear halos were easily observed by fluorescence or bright-field microscopy, respectively, which permits rapid visualization of DNA breakage in damaged cells. By using image processing and analysis with the public domain ImageJ software, X-ray dose and H₂O₂ concentration could be correlated well with the size of nuclear halos and the halo:nucleus ratio. Our results indicate that this simplified nuclear halo assay can be used as a rapid, reliable and inexpensive procedure to detect and quantify DNA breakage induced by ionizing radiation and chemical agents. A mechanistic model to explain the differences between the formation of saline or alkaline halos also is suggested.     

Continuous production of extracellular antioxidants in suspension cells attenuates the oxidative burst detected in plant microbe interactions

Suspension cells of Solanacearum tuberosum and Nicotiana tabacum placed in fresh buffer rapidly produce and maintain significant pools of extracellular antioxidants. The extracellular antioxidant was detected by first adding a known amount of exogenous H₂O₂ to samples and then immediately measuring the remaining H₂O₂. The difference between the amount added and amount remaining was used to determine the antioxidant capacity of the sample. This extracellular antioxidant pool attenuates levels of hydrogen peroxide produced during plant–bacterial interactions. When tobacco cells were inoculated with an isolate Pseudomonas syringae pv. syringae that causes a hypersensitive response much of the antioxidant capacity had been expended neutralizing the oxidative burst characteristic of such plant–microbe interactions. After a brief delay, the levels of extracellular phenolics increased commensurate to antioxidative capacity in freshly transferred cells within 2–4 h. The strong UV absorbance of these extracellular phenolics within 250 and 350 nm was used to follow oxidation upon reaction with H₂O₂. This extracellular antioxidant pool is an important consideration in cell suspension studies of the plant–microbe oxidative burst. This study demonstrates that the true magnitude and timing of the oxidative burst in cell suspensions is masked by extracellular antioxidants.     

DNA strand breakage induced by CuII and NiII, in the presence of peptide models of histone H2B

In the present study we used the plasmid relaxation assay, a very sensitive method for detection of DNA strand breaks in vitro, in order to evaluate the role of peptide fragments of histone H2B in DNA strand breakage induced by copper and nickel. We have found that in the presence of peptides modeling the histone fold domain (H2B_32-62 and H2B_63-93) as well as the N-terminal tail (H2B_1-31) of histone H2B there is an increased DNA damage by Cu²⁺/H₂O₂ and Ni²⁺/H₂O₂ reaction mixtures. On the contrary, the C-terminal tail (H2B_94-125) seems to have a protective role on the attack of ROS species to DNA. We have rendered our findings to the interactions of the peptides with DNA, as well as with the metal.     

‘Turn‐on’ fluorescence sensing of hydrogen peroxide in marine food samples using a carbon dots–MnO2 probe

A ‘turn‐on’ fluorescence method for detection of hydrogen peroxide (H₂O₂) in marine food samples is presented in this article. Using this method, a carbon dots (CDs)–MnO₂ probe was formed in which fluorescence intensity (FI) of CDs was quenched through fluorescence resonance energy transfer by addition of MnO₂ nanosheets. When H₂O₂ was added into the CDs–MnO₂ solution, the MnO₂ nanosheets formed Mn²⁺ ions due to a redox reaction between H₂O₂ and MnO₂ nanosheets, and CD FI was recovered. Under optimized conditions, the detection limit for H₂O₂ was 0.87 μM, and analytical linear range was 4–100 μM. Furthermore, this developed fluorescence sensing system was successfully used with satisfactory results to determine trace H₂O₂ content in marine food samples.     

Hydrogen Peroxide Metabolism in Yeasts

A catalase-negative mutant of the yeast Hansenula polymorpha consumed methanol in the presence of glucose when the organism was grown in carbon-limited chemostat cultures. The organism was apparently able to decompose the H₂O₂ generated in the oxidation of methanol by alcohol oxidase. Not only H₂O₂ generated intracellularly but also H₂O₂ added extracellularly was effectively destroyed by the catalase-negative mutant. From the rate of H₂O₂ consumption during growth in chemostat cultures on mixtures of glucose and H₂O₂, it appeared that the mutant was capable of decomposing H₂O₂ at a rate as high as 8 mmol · g of cells⁻¹ · h⁻¹. Glutathione peroxidase (EC 1.11.1.9) was absent under all growth conditions. However, cytochrome c peroxidase (CCP; EC 1.11.1.5) increased to very high levels in cells which decomposed H₂O₂. When wild-type H. polymorpha was grown on mixtures of glucose and methanol, the CCP level was independent of the rate of methanol utilization, whereas the level of catalase increased with increasing amounts of methanol in the substrate feed. Also, the wild type decomposed H₂O₂ at a high rate when cells were grown on mixtures of glucose and H₂O₂. In this case, an increase of both CCP and catalase was observed. When Saccharomyces cerevisiae was grown on mixtures of glucose and H₂O₂, the level of catalase remained low, but CCP increased with increasing rates of H₂O₂ utilization. From these observations and an analysis of cell yields under the various conditions, two conclusions can be drawn. (i) CCP is a key enzyme of H₂O₂ detoxification in yeasts. (ii) Catalase can effectively compete with mitochondrial CCP for hydrogen peroxide only if hydrogen peroxide is generated at the site where catalase is located, namely in the peroxisomes.