Mechanisms of DNA damage induced by morin,an inhibitor of amyloid β-peptide aggregation

Morin is a potential inhibitor of amyloid β-peptide aggregation. This aggregation is involved in the pathogenesis of Alzheimer’s disease. Meanwhile, morin has been found to be mutagenic and exhibits peroxidation of membrane lipids concurrent with DNA strand breaks in the presence of metal ions. To clarify a molecular mechanism of morin-induced DNA damage, we examined the DNA damage and its site specificity on ³²P-5′-end-labeled human DNA fragments treated with morin plus Cu(II). The formation of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG), an indicator of oxidative DNA damage, was also determined in calf thymus DNA treated with morin plus Cu(II). Morin-induced DNA strand breaks and base modification in the presence of Cu(II) were dose dependent. Morin plus Cu(II) caused piperidine-labile lesions preferentially at thymine and guanine residues. The DNA damage was inhibited by methional, catalase and Cu(I)-chelator bathocuproine. The typical ?OH scavengers ethanol, mannitol and sodium formate showed no inhibitory effect on DNA damage induced by morin plus Cu(II). When superoxide dismutase was added to the solution, DNA damage was not inhibited. In addition, morin plus Cu(II) increased 8-oxodG formation in calf thymus DNA fragments. We conclude that morin undergoes autoxidation in the presence of Cu(II) via a Cu(I)/Cu(II) redox cycle and H₂O₂ generation to produce Cu(I)-hydroperoxide, which causes oxidative DNA damage.     

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.     

3,4-Dihydroxybenzaldehyde purified from the barley seeds (Hordeum vulgare) inhibits oxidative DNA damage and apoptosis via its antioxidant activity

Barley is a major crop worldwide. It has been reported that barley seeds have an effect on scavenging ROS. However, little has been known about the functional role of the barley on the inhibition of DNA damage and apoptosis by ROS. In this study, we purified 3,4-dihydroxybenzaldehyde from the barley with silica gel column chromatography and HPLC and then identified it by GC/MS. And we firstly investigated the inhibitory effects of 3,4-dihydroxybenzaldehyde purified from the barley on oxidative DNA damage and apoptosis induced by H₂O₂, the major mediator of oxidative stress and a potent mutagen. In antioxidant activity assay such as DPPH radical and hydroxyl radical scavenging assay, Fe²⁺ chelating assay, and intracellular ROS scavenging assay by DCF-DA, 3,4-dihydroxybenzaldehyde was found to scavenge DPPH radical, hydroxyl radical and intracellular ROS. Also it chelated Fe²⁺. In in vitro oxidative DNA damage assay and the expression level of phospho-H2A.X, it inhibited oxidative DNA damage and its treatment decreased the expression level of phospho-H2A.X. And in oxidative cell death and apoptosis assay via MTT assay and Hoechst 33342 staining, respectively, the treatment of 3,4-dihydroxybenzaldehyde attenuated H₂O₂-induced cell death and apoptosis. These results suggest that the barley may exert the inhibitory effect on H₂O₂-induced tumor development by blocking H₂O₂-induced oxidative DNA damage, cell death and apoptosis.     

Index

Hydrogen peroxide (H₂O₂) can induce cell damage by generating reactive oxygen species (ROS), resulting in DNA damage and cell death. The aim of this study is to elucidate the protective effects of fisetin (3,7,3′,4′,-tetrahydroxy flavone) against H₂O₂-induced cell damage. Fisetin reduced the level of superoxide anion, hydroxyl radical in cell free system, and intracellular ROS generated by H₂O₂. Moreover, fisetin protected against H₂O₂-induced membrane lipid peroxidation, cellular DNA damage, and protein carbonylation, which are the primary cellular outcomes of H₂O₂ treatment. Furthermore, fisetin increased the level of reduced glutathione (GSH) and expression of glutamate-cysteine ligase catalytic subunit, which is decreased by H₂O₂. Conversely, a GSH inhibitor abolished the cytoprotective effect of fisetin against H₂O₂-induced cells damage. Taken together, our results suggest that fisetin protects against H₂O₂-induced cell damage by inhibiting ROS generation, thereby maintaining the protective role of the cellular GSH system.     

Protective effects of sulfated chitooligosaccharides against hydrogen peroxide-induced damage in MIN6 cells

Sulfated chitooligosaccharides (COS-S) with different degrees of substitution (DS) were obtained by the chlorosulfuric acid/pyridine method. Protective effects of COS-S against hydrogen peroxide (H₂O₂)-induced damage were investigated in pancreatic β-cells MIN6 cell line. The cell viability, morphology, insulin contents, malondialdehyde (MDA) inhibition, lactate dehydrogenase (LDH) release and the levels of antioxidant enzymes including catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidise (GPx) were evaluated under oxidative damage by 150 μM H₂O₂ for 6 h. COS-S did not show any harmful or inhibitory effect on cell growth at concentrations ranging from 0.1 to 0.5 mg/ml. While COS-S could enhance the cell viability, decrease the production of ROS, and reduce the MDA level as well as LDH level in oxidative damaged β-cells by being an antioxidant. The underlining mechanisms of protective effects of COS-S are partly due to the enhancement of antioxidant enzyme activity and inhibition of intracellular ROS production, along with suppressing MIN6 cell apoptosis subsequent to the amelioration of ROS. Moreover, increased DS might contribute to the defense mechanisms against H₂O₂-induced oxidative damage in MIN6 cells. These results indicated that the antioxidant properties of COS-S hold great potential for the oxidative diseases treatment, and the sulfate content of polysaccharides made great role in regulating antioxidant activities.     

Different effects of genistein and resveratrol on oxidative DNA damage in vitro

Previous studies have demonstrated that phenolic compounds, including genistein (4′,5,7-trihydroxyisoflavone) and resveratrol (3,4′,5-trihydroxystilbene), are able to protect against carcinogenesis in animal models. This study was undertaken to examine the ability of genistein and resveratrol to inhibit reactive oxygen species (ROS)-mediated strand breaks in φX-174 plasmid DNA. H₂O₂/Cu(II) and hydroquinone/Cu(II) were used to cause oxidative DNA strand breaks in the plasmid DNA. We demonstrated that the presence of genistein at micromolar concentrations resulted in a marked inhibition of DNA strand breaks induced by either H₂O₂/Cu(II) or hydroquinone/Cu(II). Genistein neither affected the Cu(II)/Cu(I) redox cycle nor reacted with H₂O₂ suggest that genistein may directly scavenge the ROS that participate in the induction of DNA strand breaks. In contrast to the inhibitory effects of genistein, the presence of resveratrol at similar concentrations led to increased DNA strand breaks induced by H₂O₂/Cu(II). Further studies showed that in the presence of Cu(II), resveratrol, but not genistein was able to cause DNA strand breaks. Moreover, both Cu(II)/Cu(I) redox cycle and H₂O₂ were shown to be critically involved in resveratrol/copper-mediated DNA strand breaks. The above results indicate that despite their similar in vivo anticarcinogenic effects, genistein and resveratrol appear to exert different effects on oxidative DNA damage in vitro.     

Vasoactive intestinal peptide reduces oxidative stress in pancreatic acinar cells through the inhibition of NADPH oxidase

Vasoactive intestinal peptide (VIP) attenuates experimental acute pancreatitis (AP) by inhibition of cytokine production from inflammatory cells. It has been suggested that reactive oxygen species (ROS) as well as cytokines play pivotal roles in the early pathophysiology of AP. This study aimed to clarify the effect of VIP on the oxidative condition in pancreas, especially pancreatic acinar cells (acini). Hydrogen peroxide (H₂O₂)-induced intracellular ROS, assessed with CM-H₂DCFDA, increased time- and dose-dependently in acini isolated from rats. Cell viability due to ROS-induced cellular damage, evaluated by MTS assay, was decreased with ≥100 μmol/L H₂O₂. VIP significantly inhibited ROS production from acini and increased cell viability in a dose-dependent manner. Expression of antioxidants including catalase, glutathione reductase, superoxide dismutase (SOD) 1 and glutathione peroxidase was not altered by VIP except for SOD2. Furthermore, Nox1 and Nox2, major components of NADPH oxidase, were expressed in pancreatic acini, and significantly increased after H₂O₂ treatment. Also, NADPH oxidase activity was provoked by H₂O₂. VIP decreased NADPH oxidase activity, which was abolished by PKA inhibitor H89. These results suggested that VIP affected the mechanism of ROS production including NADPH oxidase through induction of a cAMP/PKA pathway. In conclusion, VIP reduces oxidative stress in acini through the inhibition of NADPH oxidase. These results combined with findings of our previous study suggest that VIP exerts its protective effect in pancreatic damage, not only through an inhibition of cytokine production, but also through a reduction of the injury caused by oxidative stress.     

Nanoparticle-mediated catalase delivery protects human neurons from oxidative stress

Several neurodegenerative diseases and brain injury involve reactive oxygen species and implicate oxidative stress in disease mechanisms. Hydrogen peroxide (H₂O₂) formation due to mitochondrial superoxide leakage perpetuates oxidative stress in neuronal injury. Catalase, an H₂O₂-degrading enzyme, thus remains an important antioxidant therapy target. However, catalase therapy is restricted by its labile nature and inadequate delivery. Here, a nanotechnology approach was evaluated using catalase-loaded, poly(lactic co-glycolic acid) nanoparticles (NPs) in human neuronal protection against oxidative damage. This study showed highly efficient catalase encapsulation capable of retaining∼99% enzymatic activity. NPs released catalase rapidly, and antioxidant activity was sustained for over a month. NP uptake in human neurons was rapid and nontoxic. Although human neurons were highly sensitive to H₂O₂, NP-mediated catalase delivery successfully protected cultured neurons from H₂O₂-induced oxidative stress. Catalase-loaded NPs significantly reduced H₂O₂-induced protein oxidation, DNA damage, mitochondrial membrane transition pore opening and loss of cell membrane integrity and restored neuronal morphology, neurite network and microtubule-associated protein-2 levels. Further, catalase-loaded NPs improved neuronal recovery from H₂O₂ pre-exposure better than free catalase, suggesting possible applications in ameliorating stroke-relevant oxidative stress. Brain targeting of catalase-loaded NPs may find wide therapeutic applications for oxidative stress-associated acute and chronic neurodegenerative disorders.     

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.     

Dehydroepiandrosterone ameliorates H2O2-induced Leydig cells oxidation damage and apoptosis through inhibition of ROS production and activation of PI3K/Akt pathways

Dehydroepiandrosterone (DHEA) is widely used as a nutritional supplement, and administration of DHEA produces a number of beneficial effects in the elderly. Many researchers have suggested that DHEA exerts it function after conversion into more biologically active hormones in peripheral target cells. The actions of DHEA in Leydig cells, a major target cell of DHEA biotransformation in males, are not clear. The present study found that DHEA increased cell viability and decreased reactive oxygen species (ROS) and malondialdehyde contents in H₂O₂-induced Leydig cells. DHEA significantly increased the activities of superoxide dismutase, catalase and peroxidase, and decreased the DNA damage in H₂O₂-induced Leydig cells. Apoptosis was significant decreased in H₂O₂-induced Leydig cells after DHEA treatment. DHEA inhibited the loss of mitochondrial membrane potential (ΔΨm) and the upregulation of the caspase-3 protein level induced by H₂O₂ in Leydig cells. DHEA also reversed the decrease in PI3K and p-Akt protein levels induced by H₂O₂. These data showed that DHEA could ameliorate H₂O₂-induced oxidative damage by increasing anti-oxidative enzyme activities, which resulted in reduced ROS content, and decreased apoptosis, mainly by preventing the loss of ΔΨm and inhibiting caspase-3 protein levels via activation of PI3K/Akt signaling pathways. These results increase our understanding of the molecular mechanism of the anti-ageing effect of DHEA.     

Honokiol ameliorates oxidative stress-induced DNA damage and apoptosis of c2c12 myoblasts by ROS generation and mitochondrial pathway

ABSTRACT

Honokiol is one of the main active components of Magnolia officinalis, and has been demonstrated to have multiple pharmacological activities against a variety of diseases. Recently, this phenolic compound is known to have antioxidant activity, but its mechanism of action remains unclear. The purpose of the current study was to evaluate the preventive effects of honokiol against oxidative stress-induced DNA damage and apoptosis in C2C12 myoblasts. The present study found that honokiol inhibited hydrogen peroxide (H₂O₂)-induced DNA damage and mitochondrial dysfunction, while reducing reactive oxygen species (ROS) formation. The inhibitory effect of honokiol on H₂O₂-induced apoptosis was associated with the up-regulation of Bcl-2 and down-regulation of Bax, thus reducing the Bax/Bcl-2 ratio that in turn protected the activation of caspase-9 and -3, and inhibition of poly (ADP-ribose) polymerase cleavage, which was associated with the blocking of cytochrome c release to the cytoplasm. Collectively, these results demonstrate that honokiol defends C2C12 myoblasts against H₂O₂-induced DNA damage and apoptosis, at least in part, by preventing mitochondrial-dependent pathway through scavenging excessive ROS.     

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.     

Hydrogen peroxide is critical for UV-induced apoptosis inhibition

Abstract

Apoptosis is an important cell death system that deletes damaged and mutated cells, preventing the induction of cancer. We previously have reported that UV irradiation inhibited the apoptosis induced by serum starvation and cell detachment. This phenomenon is suitable for clarifying the relationship between cancer and the dysregulation of apoptosis by UV irradiation. Here, we have studied the factors responsible for this inhibition of apoptosis, focusing on reactive oxygen species (ROS) and DNA damage. Treatment with xanthine oxidase in the presence of hypoxanthine, which is known to produce superoxide anion (O₂^??) and hydrogen peroxide (H₂O₂), inhibited the induction of apoptosis. The xanthine oxidase-induced anti-apoptotic effect was suppressed in the presence of an H₂O₂-eliminating enzyme, catalase, but not in the presence of an O₂^??-eliminating enzyme, superoxide dismutase. Treatment with H₂O₂ itself significantly inhibited the induction of apoptosis. Furthermore, the effect of the inhibition of cell death by UVB irradiation and by H₂O₂ treatment decreased in H₂O₂-resistant cells. Although both UVB and H₂O₂ are known to induce DNA damage, other DNA damaging agents, like γ-irradiation and treatment with cisplatin and bleomycin, showed no inhibition of apoptosis. These findings suggested that H₂O₂ was essential to the inhibition of apoptosis, in which DNA damage had no role.     

Torulene and torularhodin,protects human prostate stromal cells from hydrogen peroxide-induced oxidative stress damage through the regulation of Bcl-2/Bax mediated apoptosis

The current study was designed to elucidate the cytoprotective effects and possible mechanisms of torulene and torularhodin on hydrogen peroxide (H₂O₂)-induced oxidative stress damage in human prostate stromal cells (WPMY-1). After treated with H₂O₂, a notable decrease was appeared in cell viability, yet the decrease was attenuated when cells were pretreated with torulene and torularhodin (0.5–10?μM) as evaluated by WST-1 assay. Pretreatment with these two carotenoids significantly attenuated H₂O₂-induced apoptosis in WPMY-1 cells through the inhibition of intracellular reactive oxygen species (ROS) and malondialdehyde (MDA) overproduction, as well as the activation of the activities in catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px). Finally, pretreatment of cells with carotenoids resulted in the regulation of the mRNA and protein expression of Bcl-2 and Bax in H₂O₂-exposed prostate stromal cells. The present results indicate that both torulene and torularhodin can protect human prostate stromal cells from oxidative stress damage via Bcl-2/Bax mediated apoptosis.     

Strand scission in DNA by Gossypol and Cu(II): Role of Cu(I) and oxygen-free radicals

Gossypol, a polyphenolic binaphthyl dialdehyde found in cotton seeds, is a dietary mutagen and a potential male contraceptive. In the presence of Cu(II), gossypol caused breakage of supercoiled plasmid pBR322 DNA. The products were relaxed circles or a mixture of these and linear molecules. Other metal ions tested [Ni(II), Co(II), Mn(II), and Fe(II)] were ineffective or less effective in the DNA breakage reaction. In the case of gossypol-Cu(II) mediated cleavage, (Cu(I) was shown to be an essential intermediate by using the Cud) sequestering reagent bathocuproine. By using job plots, it was established that in the absence of DNA, eight Cu(II) ions can be reduced by one gossypol molecule. The involvement of active oxygen species, such as singlet oxygen and H₂O₂, was established by the inhibition of DNA breakage by catalase and by sodium azide. It was further shown that gossypol is capable of directly producing H₂O₂.     

Cell cycle stage-specific roles of Rad18 in tolerance and repair of oxidative DNA damage

The E3 ubiquitin ligase Rad18 mediates tolerance of replication fork-stalling bulky DNA lesions, but whether Rad18 mediates tolerance of bulky DNA lesions acquired outside S-phase is unclear. Using synchronized cultures of primary human cells, we defined cell cycle stage-specific contributions of Rad18 to genome maintenance in response to ultraviolet C (UVC) and H₂O₂-induced DNA damage. UVC and H₂O₂ treatments both induced Rad18-mediated proliferating cell nuclear antigen mono-ubiquitination during G₀, G₁ and S-phase. Rad18 was important for repressing H₂O₂-induced (but not ultraviolet-induced) double strand break (DSB) accumulation and ATM S1981 phosphorylation only during G₁, indicating a specific role for Rad18 in processing of oxidative DNA lesions outside S-phase. However, H₂O₂-induced DSB formation in Rad18-depleted G1 cells was not associated with increased genotoxin sensitivity, indicating that back-up DSB repair mechanisms compensate for Rad18 deficiency. Indeed, in DNA LigIV-deficient cells Rad18-depletion conferred H₂O₂-sensitivity, demonstrating functional redundancy between Rad18 and non-homologous end joining for tolerance of oxidative DNA damage acquired during G₁. In contrast with G₁-synchronized cultures, S-phase cells were H₂O₂-sensitive following Rad18-depletion. We conclude that although Rad18 pathway activation by oxidative lesions is not restricted to S-phase, Rad18-mediated trans-lesion synthesis by Polη is dispensable for damage-tolerance in G₁ (because of back-up non-homologous end joining-mediated DSB repair), yet Rad18 is necessary for damage tolerance during S-phase.