Triphlorethol-A from Ecklonia cava protects V79-4 lung fibroblast against hydrogen peroxide induced cell damage

In the present study, triphlorethol-A, a phlorotannin, was isolated from Ecklonia cava and its antioxidant properties were investigated. Triphlorethol-A was found to scavenge intracellular reactive oxygen species (ROS) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, and thus prevented lipid peroxidation. The radical scavenging activity of triphlorethol-A protected the Chinese hamster lung fibroblast (V79-4) cells exposed to hydrogen peroxide (H₂O₂) against cell death, via the activation of ERK protein. Furthermore, triphlorethol-A reduced the apoptotic cells formation induced by H₂O₂. Triphlorethol-A increased the activities of cellular antioxidant enzymes like, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx). Hence, from the present study, it is suggestive that triphlorethol-A protects V79-4 cells against H₂O₂ damage by enhancing the cellular antioxidative activity.     

Cocoa procyanidins protect PC12 cells from hydrogen-peroxide-induced apoptosis by inhibiting activation of p38 MAPK and JNK

Oxidative stress induced by reactive oxygen species has been strongly associated with the pathogenesis of neurodegenerative disorders, including Alzheimer's disease. In this study, we investigated the possible protective effects of a cocoa procyanidin fraction (CPF) and procyanidin B2 (epicatechin-(4β-8)-epicatechin) – a major polyphenol in cocoa – against apoptosis of PC12 rat pheochromocytoma (PC12) cells induced by hydrogen peroxide (H₂O₂). CPF (1 and 5 μg/ml) and procyanidin B2 (1 and 5 μM) reduced PC12 cell death caused by H₂O₂, as determined by MTT and trypan blue exclusion assays. CPF and procyanidin B2 attenuated the H₂O₂-induced fragmentation of nucleus and DNA in PC12 cells. Western blot data demonstrated that H₂O₂ induced cleavage of poly(ADP-ribose)polymerase (PARP), downregulated Bcl-X_L and Bcl-2 in PC12 cells. Pretreatment with CPF or procyanidin B2 before H₂O₂ treatment diminished PARP cleavage and increased Bcl-X_L and Bcl-2 expression compared with those only treated with H₂O₂. Activation of caspase-3 by H₂O₂ was inhibited by pretreatment with CPF or procyanidin B2. Furthermore, H₂O₂-induced rapid and significant phosphorylation of c-Jun N-terminal protein kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), and both of these effects were attenuated by CPF or procyanidin B2 treatment. These results suggest that the protective effects of CPF and procyanidin B2 against H₂O₂-induced apoptosis involve inhibiting the downregulation of Bcl-X_L and Bcl-2 expression through blocking the activation of JNK and p38 MAPK.     

The role of H2O2 as a mediator of UVB-induced apoptosis in keratinocytes

Apoptosis is an active form of cell death that is initiated by a variety of stimuli, including reactive oxygen species (ROS) and ultraviolet (UV) radiation. Previously, it has been reported that UVB-irradiation of keratinocytes leads to intracellular generation of hydrogen peroxide (H₂O₂) and that antioxidants can inhibit ROS-induced apoptosis. Although both UVB-irradiation and H₂O₂-incubation led to increased intracellular H₂O₂ levels, the antioxidants catalase and glutathione monoester (GME), inhibited apoptosis only when induced by H₂O₂, not by UVB. Furthermore, extracellular signal-regulated kinase (ERK), a prominent member of the mitogen-activated protein kinase (MAPK) family, was found to be activated by treatment with both UVB and H₂O₂. Inhibition of ERK phosphorylation by pre-treatment with PD98059 resulted in enhanced apoptosis after H₂O₂-exposure. However, no significant difference of apoptosis was observed between cells with and without inhibitor pre-treatment upon UVB-irradiation. DNA damage in the form of cyclobutane pyrimidine dimers was observed after exposure to UVB, but no photoproducts were found in H₂O₂-treated cells. These results suggest a ROS-independent pathway of UVB-induced apoptosis. Although UVB-irradiation causes moderate increase in H₂O₂, the generation of H₂O₂ does not contribute to the induction of apoptosis. Moreover, activation of ERK only blocks H₂O₂-dependent apoptosis but has no impact on UVB-induced apoptosis.     

AP-1 activation through endogenous H(2)O(2) generation by alveolar macrophages

Reactive oxygen species released during the respiratory burst are known to participate in cell signaling. Here we demonstrate that hydrogen peroxide produced by the respiratory burst activates AP-1 binding. Stimulation of the macrophage cell line NR8383 with respiratory burst agonists ADP and C5a increased AP-1 binding activity. Importantly, this increase in binding was blocked by catalase, confirming mediation by endogenous H₂O₂. Moreover, exogenously added H₂O₂ mimicked the agonists, and also activated AP-1. Antibodies revealed that the activated AP-1 complex is composed predominantly of c-Fos/c-Jun heterodimers. Treatment of the cells with ADP, C5a and H₂O₂ (100 μM) all increased the phosphorylation of c-Jun. c-Fos protein was increased in cells treated with C5a or high dose (200 μM) H₂O₂, but not in cells treated with ADP. The MEK inhibitor, PD98059, partially blocked the C5a-mediated increase in AP-1 binding. A novel membrane-permeable peptide inhibitor of JNK, JNKi, also inhibited AP-1 activation. Together these data suggest that C5a-mediated AP-1 activation requires both the activation of the ERK and JNK pathways, whereas activation of the JNK pathway is sufficient to increase AP-1 binding with ADP. Thus, AP-1 activation joins the list of pathways for which the respiratory burst signals downstream events in the macrophage.     

Inhibition of gene expression of heparin-binding epidermal growth factor-like growth factor by extracellular superoxide dismutase in rat aortic smooth muscle cells

Both extracellular superoxide dismsutase (EC-SOD) and heparin binding EGF like growth factor (HB-EGF) are produced in smooth muscle cells of the arterial wall, and are thought to play pathological roles in atherosclerosis with heparin binding characteristics. EC-SOD treatment clearly reduced the H₂O₂ induced expression of HB-EGF in rat aortic smooth muscle cells (RASMC). EC-SOD also inhibited the induction of HB-EGF by 12-O-tetradecanoylphorbol-13-acetate (TPA) in RASMC by 60%. Both H₂O₂ and TPA increased intracellular ROS levels, and EC-SOD inhibited ROS generation only for the case of H₂O₂ but not TPA. Treatment of the cells with heparin alone decreased HB-EGF expression by 20%, whereas EC-SOD alone and a co-incubation with EC-SOD and heparin suppressed the induction by 60 and 70%, respectively. These results suggest that EC-SOD is related to the EGF signaling in two ways, competition for HSPG with HB-EGF and as an ROS scavenger.     

Caspases are reversibly inactivated by hydrogen peroxide

Hydrogen peroxide (H₂O₂) is known to both induce and inhibit apoptosis, however the mechanisms are unclear. We found that H₂O₂ inhibited the activity of recombinant caspase-3 and caspase-8, half-inhibition occurring at about 17 μM H₂O₂. This inhibition was both prevented and reversed by dithiothreitol while glutathione had little protective effect. 100–200 μM H₂O₂ added to macrophages after induction of caspase activation by nitric oxide or serum withdrawal substantially inhibited caspase activity. Activation of H₂O₂-producing NADPH oxidase in macrophages also caused catalase-sensitive inactivation of cellular caspases. The data suggest that the activity of caspases in cells can be directly but reversibly inhibited by H₂O₂.     

DNA Single Strand Breakage by H2O2 and Ferric or Cupric Ions: Its Modulation by Histidine

The role of histidine on DNA breakage induced by hydrogen peroxide (H₂O₂) and ferric ions or by H₂O₂ and cupric ions was studied on purified DNA. L-histidine slightly reduced DNA breakage by H₂O₂ and Fe³⁺ but greatly inhibited DNA breakage by H₂O₂ and Cu²⁺. However, only when histidine was present, the addition of EDTA to H₂O₂ and Fe³⁺ exhibited a bimodal dose response curve depending on the chelator metal ratio. The enhancing effect of histidine on the rate of DNA degradation by H₂O₂ was maximal at a chelator metal ratio between 0.2 and 0.5, and was specific for iron. When D-histidine replaced L-histidine, the same pattern of EDTA dose response curve was observed. Superoxide dismutase greatly inhibited the rate of DNA degradation induced by H₂O₂, Fe³⁺, EDTA and L-histidine involving the superoxide radical.

These studies suggest that the enhancing effect of histidine on the rate of DNA degradation by H₂O₂ and Fe³⁺ is mediated by an oxidant which could be a ferrous-dioxygen-ferric chelate complex or a chelate-ferryl ion.     

Hydroxylamine potentiates the effect of low dose hydrogen peroxide in glioma cells independent of p53

We had earlier shown that higher concentration of hydrogen peroxide (H₂O₂) induced p53-dependent apoptosis in glioma cell line with wild type p53 but had minimal effect on cells with mutated p53. Here we show a potentiating effect of hydroxylamine (HA), an inhibitor of catalase, on a nontoxic dose of H₂O₂ in glioma cells. HA sensitized both p53 wild type and mutated glioma cells to 0.25 mM H₂O₂. Potentiating effect of HA was independent of p53. Higher levels of reactive oxygen species (ROS) generation were observed in cells treated with HA+H₂O₂ as compared to cells treated with each component alone in both the cell lines. Dimethyl sulfoxide (DMSO) protected cells. Cytosolic cytochrome c and activated caspase 3 were detected at 4 h. The results suggest that higher levels of intracellular ROS, generated by HA+H₂O₂ act as a molecular switch in activating a rapidly acting p53-independent mitochondrial apoptotic pathway.     

Inactivation of Rabbit Muscle Creatine Kinase by Hydrogen Peroxide

The effects of xanthine + xanthine oxidase-generated reactive oxygen species (ROS) on rabbit muscle creatine kinase (CK) were studied. Xanthine (0.1 mM) + xanthine oxidase (30 mU/ml) inhibited activity of rabbit muscle CK (1.2mU/ml). Catalase (100/ml), but not SOD (100 U/ml), deferoxamine (100μM) or mannitol (20 mM), protected CK from inactivation; suggesting that H₂O₂ was responsible for inactivation. These results were different from previously reported findings on bovine heart CK that superoxide radicals inactivate the enzyme. Thus, enzymes with homologous structures may have different reactivities to different ROS. H₂O₂-induced inactivation of rabbit muscle CK was accompanied by a decrease in its thiol group content, whereas no significant changes in the protein structure were detected by SDS-PAGE or carbonyl content. These results suggest that oxidation of -SH groups by H₂O₂ seems to be a major mechanism of activation of rabbit muscle CK by xanthine + xanthine oxidase. Such inactivation of CK by H₂O₂ may be important in ROS-induced pathology.     

Oxidant-mediated AA release from astrocytes involves cPLA(2) and iPLA(2)

Excessive generation of reactive oxygen species (ROS) in the central nervous system (CNS) is a leading cause of neuronal injury. Despite yet unknown mechanisms, oxidant compounds such as H₂O₂ have been shown to stimulate the release of arachidonic acid (AA) in a number of cell systems. In this study, H₂O₂ and menadione, a compound known to release H₂O₂ intracellularly, were used to examine the phospholipases A₂ (PLA₂) responsible for AA release from primary murine astrocytes. Both H₂O₂ and menadione dose-dependently stimulated AA release, and the release mediated by H₂O₂ was completely inhibited by catalase. H₂O₂ also stimulated phosphorylation of extracellular signal-regulated kinases (ERK1/2) and cytosolic phospholipase A₂ (cPLA₂). However, complete inhibition of cPLA₂ phosphorylation by U0126, an inhibitor for mitogen-activated protein kinase kinase (MEK) and GF109203x, a nonselective PKC inhibitor preferring the conventional and novel isoforms, only reduced H₂O₂-stimulated AA release by 50%. MAFP, a selective, active, site-directed, irreversible inhibitor of both cPLA₂ and the Ca²⁺-independent iPLA₂, nearly completely inhibited H₂O₂-mediated AA release; but, HELSS, a potent irreversible inhibitor of iPLA₂, only inhibited H₂O₂-mediated AA release by 40%. Along with the observation that H₂O₂-mediated AA release was only partially inhibited upon chelating intracellular Ca²⁺ by BAPTA, these results indicate the involvement of both cPLA₂ and iPLA₂ in H₂O₂-mediated AA release in murine astrocytes.     

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.     

Does the cellular labile iron pool participate in the oxidation of 2',7'-dichlorodihydrofluorescein?

The fluorogenic probe 2',7'-dichlorodihydrofluorescein diacetate (H₂DCF-DA) is widely used for the estimation of oxidative stress in cells. It is known that 2',7'-dichlorodihydrofluorescein (H₂DCF), product of intracellular hydrolysis of H₂DCF-DA, is oxidized to the fluorescent compound, DCF, mainly by hydrogen peroxide (H₂O₂) in the presence of catalysts. The present study was aimed at answering the question whether the labile iron pool (LIP) may contribute to the oxidation of H₂DCF in cellular systems. The membrane-permeable lipophilic iron chelator salicylaldehyde isonicotinoyl hydrazone (SIH) was found to inhibit oxidation of the probe by H₂O₂ dependent on ferrous ions but not by peroxidase or superoxide dismutase in defined in vitro systems. When applied to cells, the probe inhibited considerably oxidation of H₂DCF in V79 Chinese hamster fibroblasts and two murine lymphoma L5178Y(LY) sublines (LY-R, LY-S) differing in LIP level, the extent of inhibition being greater in the LY-R line of higher LIP level. These results demonstrate that LIP is a significant factor determining the rate of intracellular H₂DCF oxidation.     

p38 MAPK and Ca2+ contribute to hydrogen peroxide-induced increase of permeability in vascular endothelial cells but ERK does not

To examine the involvement of p38 mitogen-activated protein kinase (p38 MAPK) and extra-cellular signal-regulated kinase (ERK) in the oxidative stress-induced increase of permeability in endothelial cells, the effects of a p38 MAPK inhibitor (SB203580) and ERK inhibitor (PD90859) on the H₂O₂-induced increase of permeability in bovine pulmonary artery endothelial cells (BPAEC) were investigated using a two-compartment system partitioned by a semi-permeable filter. H₂O₂ at 1 mM caused an increase of the permeation rate of fluorescein isothiocyanate (FITC)-labeled dextran 40 through BPAEC monolayers. SB203580 inhibited the H₂O₂-induced increase of permeability but PD98059 did not, though activation (phosphorylation) of both p38 MAPK and ERK was observed in H₂O₂-treated cells in Western blot analysis. An H₂O₂-induced increase of the intracellular Ca²⁺ concentration ([Ca²⁺]_i) was also observed and an intracellular Ca²⁺ chelator (BAPTA-AM) significantly inhibited the H₂O₂-induced increase of permeability. However, it showed no inhibitory effects on the H₂O₂-induced phosphorylation of p38 MAPK and ERK. The H₂O₂-induced increase of [Ca²⁺]_i was not influenced by SB203580 and PD98059. These results indicate that the activation of p38 MAPK and the increase of [Ca²⁺]_i are essential for the H₂O₂-induced increase of endothelial permeability and that ERK is not.     

Cyanide-resistant alternative respiration is strictly correlated to intracellular peroxide levels in Acremonium chrysogenum

Long-wave ultraviolet radiation (UVA) may cause extensive DNA damage via reactive oxygen species (ROS). In this study we examined whether UVA- and H₂O₂-mediated DNA damage have equivalent effects on the induction of G2/M phase checkpoint and cell cycle progression in a transformed keratinocyte cell line HaCaT. By employing single cell gel electrophoresis (comet assay) we determined the equipotent doses of UVA and H₂O₂ with respect to the induction of alkali-labile sites (an indicator of oxidative DNA decay). However, in contrast to H₂O₂ which caused a pronounced G2/M cell cycle arrest 24h after treatment, UVA irradiation did not affect cell cycle progression. Increasing UVA doses up to 150 kJ/m² did not affect cell cycle and proliferation whereas increasing H₂O₂ concentrations caused a cell cycle block or cell death. Cytometric analysis revealed that G2/M cell cycle arrest took place beyond the cyclin B1 restriction point. We conclude that the DNA damage induced by UVA is easily repaired and does not perturb cell growth, whereas the H₂O₂-induced damage leads ultimately to cell cycle arrest or cell death.     

Oxidant-Induced Activation of Protein Kinase C in Uc11mg Cells

Free radical formation and subsequent lipid peroxidation may participate in the pathogenesis of tissue injury, including the brain injury induced by hypoxia or trauma and cardiac injury arising from ischemia and reperfusion. However, the exact cellular mechanisms by which the initial oxidative insult leads to the ultimate tissue damage are not known. A number of reports have indicated that protein kinase C (PKC) may be activated following oxidative stress and that this enzyme may play an important role in the steps leading to cellular damage. In this work, we have examined in a cell model whether PKC is activated following oxidative exposure. UC11MG cells, a human astrocytoma cell line, were treated with H₂O₂. Incubation with 0.5 mM H₂O₂ increased malondialdehyde levels by as early as 15 minutes. To assess the effects of H₂O₂ treatment on PKC activation, we measured phosphorylation of an endogenous PKC substrate, the MARCKS (myristoylated alanine-rich C kinase substrate) protein. Treatment of cells with 0.2-1.0 mM H₂O₂ resulted in a rapid increase in MARCKS phosphorylation. Phosphorylation was stimulated approximately 2.5-fold following treatment with 0.5 mM H₂O₂ for ten minutes. Treatment with phorbol 12-myristate 13-acetate, a PKC activator, increased MARCKS phosphorylation approximately 4-fold. The H₂O₂-induced MARCKS phosphorylation was inhibited by the addition of the kinase inhibitors H-7 and staurosporine. Furthermore, specific down-regulation of PKC by phorbol ester also inhibited H₂O₂-induced MARCKS phosphorylation. These results indicate that PKC is rapidly activated in cells following an oxidative exposure and that this cell system may be a good model to further investigate the role of PKC in regulating oxidative damage in the cell.     

ERK1/2 and p38-MAPK signalling pathways, through MSK1, are involved in NF-kappaB transactivation during oxidative stress in skeletal myoblasts

Skeletal muscle is highly adapted to respond to oxidative imbalances, since it is continuously subjected to an increased production of reactive oxygen species (ROS) during exercise. Oxidative stress, however, has been associated with skeletal muscle atrophy and damage in many diseases. In this study, we examined whether MAPK and NF-κB pathways participate in the response of skeletal myoblasts to oxidative stress, and whether there is a cross talk between these pathways. H₂O₂ induced a strong activation of ERKs, JNKs and p38-MAPK in a time- and dose-dependent profile. ERK and JNK activation by H₂O₂, but not that of p38-MAPK, was mediated by Src kinase and, at least in part, by EGFR. H₂O₂ also stimulated a mild translocation of NF-κB to the nucleus, as well as a moderate phosphorylation of its endogenous cytoplasmic inhibitor IκB (at Ser32/36), without any significant decrease in IκB total levels. Moreover, oxidative stress induced a strong phosphorylation of NF-κB p65 subunit at Ser536 and Ser276. Inhibition of MAPK pathways by selective inhibitors did not appear to affect H₂O₂-induced nuclear translocation of NF-κB or the phosphorylation of IκB. In contrast, phosphorylation of p65 at Ser276 was found to be mediated by MSK1, a substrate of both ERKs and p38-MAPK. In conclusion, it seems that, during oxidative stress, NF-κB translocation to the nucleus is most likely not related with the MAPK activation, while p65 phosphorylations are in part mediated by MAPKs pathways, probably modifying signal specificity.     

Comparative study of the formation of oxidative damage marker 8-hydroxy-2'-deoxyguanosine (8-OHdG) adduct from the nucleoside 2'-deoxyguanosine by transition metals and suspensions of particulate matter in relation to metal content and redox reactivity

An association between exposure to ambient particulate matter (PM) and increased incidence of mortality and morbidity due to lung cancer and cardiovascular diseases has been demonstrated by recent epidemiological studies. Reactive oxygen species (ROS), especially hydroxyl radicals, generated by PM, have been suggested by many studies as an important factor in the oxidative damage of DNA by PM. The purpose of this study was to characterize quantitatively hydroxyl radical generation by various transition metals in the presence of H₂O₂ in aqueous buffer solution (pH 7.4) and hydroxylation of 2'-deoxyguanosine (dG) to 8-hydroxy-2'-deoxyguanosine (8-OHdG) under similar conditions. The order of metals' redox reactivity and hydroxyl radical production was Fe(II), V(IV), Cu(I), Cr(III), Ni(II), Co(II), Pb(II), Cd(II). Then, we investigated the generation of hydroxyl radicals in the presence of H₂O₂ by various airborne PM samples, such as total suspended particulate (TSP), PM₁₀, PM_2.5 (PM with aerodynamic diameter 10 and 2.5 μm), diesel exhaust particles (DEP), gasoline exhaust particles (GEP) and woodsmoke soot under the same conditions. When suspensions of PMs were incubated with H₂O₂ and dG at pH 7.4, all particles induced hydroxylation of dG and formation of 8-OHdG in a dose-dependent increase. Our findings demonstrated that PM's hydroxyl radical (HO√) generating ability and subsequent dG hydroxylation is associated with the concentration of water-soluble metals, especially Fe and V and other redox or ionizable transition metals and not their total metal content, or insoluble metal oxides, via a Fenton-driven reaction of H₂O₂ with metals. Additionally, we observed, by Electron paramagnetic resonance (EPR), that PM suspensions in the presence of H₂O₂ generated radical species with dG, which were spin-trapped by 2-methyl-2-nitroso-propane (MNP).     

Role of antioxidants in protecting cellular DNA from damage by oxidative stress

We have previously derived 2 V79 clones resistant to menadione (Md1 cells) and cadmium (Cd1 cells), respectively. They both were shown to be cross-resistant to hydrogen peroxide. There was a modification in the antioxidant repertoire in these cells as compared to the parental cells. Md1 presented an increase in catalase and glutathione peroxidase activities whereas Cd1 cells exhibited an increase in metallothionein and glutathione contents. The susceptibility of the DNA of these cells to the damaging effect of H₂O₂ was tested using the DNA precipitation assay. Both Md1 and Cd1 DNAs were more resistant to the peroxide action. In the case of Md1 cells it seems clear that the extra resistance is provided by the increase in the two H₂O₂ scavenger enzymes, catalase and glutathione peroxidase. In the case of Cd1 cells the activities of these enzymes as well as of superoxide dismutases (Cu/Zn and Mn) are unaltered as compared to the parental cells. The facts that parental cells exposed to 100 μM Zn²⁺ in the medium exhibit an increase in metallothionein but not in glutathione and that these cells become more resistant to the DNA-damaging effect of H₂O₂ suggest that this protein might play a protective role in vivo against the OH radical attack on DNA.