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.     

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.     

H2O2 and Src-dependent transactivation of the EGF receptor mediates the stimulatory effect of leptin on renal ERK and Na+, K+-ATPase

We examined the mechanism through which leptin increases Na⁺, K⁺-ATPase activity in the rat kidney. Leptin was infused under anaesthesia into the abdominal aorta proximally to the renal arteries and then Na⁺, K⁺-ATPase activity was measured in the renal cortex and medulla. Leptin (1 μg/kg min) increased Na⁺, K⁺-ATPase activity after 3 h of infusion, which was accompanied by the increase in urinary H₂O₂ excretion and phosphorylation level of extracellular signal regulated kinase (ERK). The effect of leptin on ERK and Na⁺, K⁺-ATPase was abolished by catalase, specific inhibitors of epidermal growth factor (EGF) receptor, AG1478 and PD158780, as well as by ERK inhibitor, PD98059, and was mimicked by both exogenous H₂O₂ and EGF. The effect of leptin was also prevented by the inhibitor of Src tyrosine kinase, PP2. Leptin and H₂O₂ increased Src phosphorylation at Tyr⁴¹⁸. We conclude that leptin-induced stimulation of renal Na⁺, K⁺-ATPase involves H₂O₂ generation, Src kinase, transactivation of the EGF receptor, and stimulation of ERK.     

Enhancement of radiation-induced apoptosis by 6-formylpterin

Radiation-induced apoptosis and its possible enhancement in the presence of 6-formylpterin (6-FP), a metabolite of folic acid, were examined in human myelomonocytic lymphoma U937 cells. When cells were treated with 6-FP at a nontoxic concentration of 300 μM, and then exposed to X-rays at a dose of 10 Gy, significant enhancement of radiation-induced apoptosis as determined by nuclear morphological change, phosphatidylserine (PS) externalization and DNA fragmentation were observed. Flow cytometry for the detection of intracellular hydrogen peroxide (H₂O₂) revealed that 6-FP increased the formation of intracellular H₂O₂, which further increased when the cells were irradiated. Decrease of mitochondria trans-membrane potential (MMP), release of cytochrome c from mitochondria, and activation of caspase-3 were enhanced after the combined treatment. Remarkable activation of protein kinase C δ (PKC δ) and its translocation from cytosol to mitochondria were detected in combined treatment. Increase of intracellular Ca₂₊ concentrations ([Ca₂₊]i) was also observed, however, neither calpain I nor calpain II could inhibit the apoptosis. In addition, c-Jun NH₂-terminal kinase ( JNK) activation was not enhanced in the combined treatment. A protein involved in a caspase-independent apoptosis pathway, apoptosis inducing factor (AIF), remained unchanged even 3 h after treatment. These results indicate that intracellular H₂O₂ generated by 6-FP enhances radiation-induced apoptosis via the mitochondria-mediated caspase-dependent pathway, with the active involvement of PKC δ.     

Polypeptide from Chlamys farreri protects HaCaT cells from UVB-induced apoptosis

A natural polypeptide from marine Chlamys farreri (a kind of scallop) (PCF), has been recently been found to be an effective photoprotective agent against ultraviolet rays B (UVB)-induced mitochondria damage in normal human fibroblasts. To investigate whether PCF has the antiapoptotic effect on human keratinocytes, in the present study, we established an apoptotic model on HaCaT cell line by means of UVB radiance of 30 mJ/cm(2) and compared the effect of different PCF treatments on UVB-radiated cells. Flow cytometry analyses showed that PCF treatment before UVB-irradiation inhibited UVB-induced apoptosis, the loss of mitochondrial membrane potential (Deltapsim) and the increase of free Ca(2+) level in HaCaT cells. In parallel with these results, UVB-irradiation enhanced activities of caspases-3, 8, 9, while this enhancement was inhibited by PCF treatment prior to irradiation. PCF added after irradiation neither reduced UVB-induced activities of the three caspases nor synergized the effect of pre-added PCF. Cellular ultrastructural features obtained from transmission electron microscopy further confirmed the antiapoptotic effect of PCF pre-treatment. It is concluded that the antiapoptotic effect of PCF is not therapeutic but prophylactic. Caspases-3, 8, 9, Deltapsim and calcium are involved in UVB-induced apoptosis, while prophylactic PCF inhibits apoptosis of UVB-irradiated HaCaT cells by blocking the caspases activities, the Deltapsim lost and the elevation of intracellular free Ca(2+) level.     

Effects of Respiratory Burst Inhibitors on Nitric Oxide Production by Human Neutrophils

Human neutrophils (PMN) activated by N-formyl-methionyl-leucyl-phenylalanine (fMLP) simultaneously release nitric oxide (.NO), superoxide anion (O₂-) and its dismutation product, hydrogen peroxide (H₂O₂). To assess whether NO production shares common steps with the activation of the NADPH oxidase, PMN were treated with inhibitors and antagonists of intracellular signaling pathways and subsequently stimulated either with fMLP or with a phorbol ester (PMA). The G-protein inhibitor, pertussis toxin (1-10 μg/ml) decreased H₂O₂ yield without significantly changing. NO production in fMLP-stimulated neutrophils; no effects were observed in PMA-activated cells. The inhibition of tyrosine kinases by genistein (1-25 μg/ml) completely abolished H₂O₂ release by fMLP-activated neutrophils; conversely, NO production increased about 1.5- and 3-fold with fMLP and PMA, respectively. Accordingly, orthovanadate, an inhibitor of phosphotyrosine phosphatase, markedly decreased -NO production and increased O_2;^- release. On the other hand, inhibition of protein kinase C with staurosporine and the use of burst antagonists like adenosine, cholera toxin or dibutyryl-cAMP diminished both H₂O₂ and NO production. The results suggest that the activation of the tyrosine kinase pathway in stimulated human neutrophils controls positively O₂^- and H₂O₂ generation and simultaneously maintains -NO production in low levels. In contrast, activation of protein kinase C is a positive modulator for O_2;^-and *NO production.     

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.     

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.     

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.     

Mechanisms of H2O2-induced oxidative stress in endothelial cells

Hydrogen peroxide, produced by inflammatory and vascular cells, induces oxidative stress that may contribute to endothelial dysfunction. In smooth muscle cells, H₂O₂ induces production of O₂⁻ by activating NADPH oxidase. However, the mechanisms whereby H₂O₂ induces oxidative stress in endothelial cells are poorly understood. We examined the effects of H₂O₂ on O₂⁻ levels on porcine aortic endothelial cells (PAEC). Treatment with 60 μmol/L H₂O₂ markedly increased intracellular O₂⁻ levels (determined by conversion of dihydroethidium to hydroxyethidium) and produced cytotoxicity (determined by propidium iodide staining) in PAEC. Overexpression of human manganese superoxide dismutase in PAEC reduced O₂⁻ levels and attenuated cytotoxicity resulting from treatment with H₂O₂. L-NAME, an inhibitor of nitric oxide synthase (NOS), and apocynin, an inhibitor of NADPH oxidase, reduced O₂⁻ levels in PAEC treated with H₂O₂, suggesting that both NOS and NADPH oxidase contribute to H₂O₂-induced O₂⁻ in PAEC. Inhibition of NADPH oxidase using apocynin and NOS rescue with L-sepiapterin together reduced O₂⁻ levels in PAEC treated with H₂O₂ to control levels. This suggests interaction-distinct NOS and NADPH oxidase pathways to superoxide. We conclude that H₂O₂ produces oxidative stress in endothelial cells by increasing intracellular O₂⁻ levels through NOS and NADPH oxidase. These findings suggest a complex interaction between H₂O₂ and oxidant-generating enzymes that may contribute to endothelial dysfunction.     

穿心莲内酯衍生物抗H2O2诱导胰岛RIN-mβ细胞凋亡的蛋白组学研究

采用蛋白组学方法筛选穿心莲内酯衍生物(AL-1)抗过氧化氢诱导胰岛RIN-mβ细胞凋亡的差异蛋白质分子并探讨其作用分子机制.结果显示:AL-1浓度依赖性地提高H₂O₂处理的胰岛RIN-mβ细胞的存活率.经蛋白组学研究分析,成功地鉴定了18个与凋亡、应激等相关的蛋白,包括Prohibitin、Shmt2、RhoGDP-dissociationinhibitor-1、Galectin-1、Cyt b5、Hsps等;与对照组(H₂O₂)相比,处理组(AL-1+H₂O₂)中,有9个表达上调的蛋白和9个表达下调的蛋白.AL-1通过调控与细胞凋亡、应激等相关的蛋白发挥其抗H₂O₂诱导的凋亡作用.     

Singlet oxygen-induced attenuation of growth factor signaling: possible role of ceramides

Singlet oxygen, an electronically excited form of molecular oxygen, is a primary mediator of the activation of stress-activated protein kinases elicited by ultraviolet A (UVA; 320-400 nm). Here, the effects of singlet oxygen (₁O₂) on the extracellular signal-regulated kinase (ERK) 1/2 and Akt/protein kinase B pathways were analyzed in human dermal fibroblasts. While basal ERK 1/2 phosphorylation was lowered in cells exposed to either ₁O₂, UVA or photodynamic treatment, Akt was moderately activated by photochemically generated ₁O₂ in a phosphoinositide 3-kinase (PI3K)-dependent fashion, resulting in the phosphorylation of glycogen synthase kinase-3 (GSK3). The activation of ERK 1/2 and Akt as induced by stimulation with epidermal growth factor (EGF) or platelet-derived growth factor (PDGF) was inhibited by ₁O₂ generated intracellularly upon photoexcitation of rose Bengal (RB). Photodynamic therapy (PDT)-induced apoptosis is known to be associated with increased formation of ceramides. Likewise, both ₁O₂ and UVA induced ceramide generation in human skin fibroblasts. The attenuation of EGF- and PDGF-induced activation of ERK 1/2 and Akt by ₁O₂ was mimicked by stimulation of fibroblasts with the cell-permeable C₂-ceramide. Interestingly, EGF-induced tyrosine phosphorylation of the EGF receptor was strongly attenuated by ₁O₂ but unimpaired by C₂-ceramide, implying that, although ceramide formation may mediate the above attenuation of ERK and Akt phosphorylation induced by ₁O₂, mechanisms beyond ceramide formation exist that mediate impairment of growth factor signaling by singlet oxygen. In summary, these data point to a novel mechanism of ₁O₂ toxicity: the known ₁O₂-induced activation of proapoptotic kinases such as JNK and p38 is paralleled by the prevention of activation of growth factor receptor-dependent signaling and of anti-apoptotic kinases, thus shifting the balance towards apoptosis.     

Characterization of H2O2-induced acute apoptosis in cultured neural stem/progenitor cells

In the present study, we characterized hydrogen peroxide (H₂O₂)-induced cell apoptosis and related cell signaling pathways in cultured embryonic neural stem/progenitor cells (NS/PCs). Our data indicated that H₂O₂ induced acute cell apoptosis in NS/PC in concentration- and time-dependent manners and selectively, it transiently increased PI3K-Akt and Mek-Erk1/2 in a dose-dependent manner. Inhibition of PI3K-Akt with wortmannin, a PI3-K inhibitor, was found to significantly increase H₂O₂-induced acute apoptosis and dramatically decrease basal pGSK3β levels. The level of pGSK3β remained unchanged with H₂O₂ exposure. We conclude that the transient activation of PI3K-Akt signaling delays the H₂O₂-induced acute apoptosis in cultured NS/PCs in part through maintaining the basal pGSK3β level and activating other downstream effectors.     

Oxidative stress underlies the mechanism for Ca(2+)-induced permeability transition of mitochondria

Recent studies demonstrated that the generation of intracellular reactive oxygen species (ROS) was enhanced prior to the onset of mitochondrial membrane permeability transition (MPT), a critical step for the induction of DNA fragmentation and apoptosis. Although Ca₂₊ induces typical MPT that involves depolarization and swelling of mitochondria and finally releases cytochrome c into cytosol, the mechanism by which ROS induce MPT remains unclear. In the presence of inorganic phosphate, Ca₂₊ increased the oxygen consumption and ROS production by isolated mitochondria as determined by a chemiluminescence (CHL) method using L-012. Ca₂₊ increased the generation of H₂O₂ by some mechanism that was inhibited by cyclosporin A but not by superoxide dismutase (SOD) and trifluoperazine. Ca₂₊ decreased the content of free thiols in adenine nucleotide translocase (ANT) in mitochondrial membranes with concomitant increase in ROS generation. The presence of cyclosporin A, trifluoperazine, or SOD inhibited the Ca₂₊-induced increase of L-012 CHL and decrease in the free thiols of ANT. These results indicate that Ca₂₊ increases the generation of ROS which oxidize the free thiol groups in mitochondrial ANT, thereby inducing MPT to release cytochrome c.     

水杨酸诱发的丹参悬浮培养细胞内H2O2迸发与其培养基碱化的关系

水杨酸(salicylic acid,SA)处理可诱导丹参悬浮培养细胞内H2O2产生及其培养基碱化。利用NADPH氧化酶抑制剂咪唑(imidazole,IMD)、H2O2淬灭剂二甲基硫脲(dimethylthiourea,DMTU)、质膜H+-ATPase抑制剂钒酸钠(Na3VO4)及激活剂壳梭孢菌素(fusicoccin,FC)处理丹参悬浮培养细胞,探讨SA诱导的H2O2迸发与培养基碱化之间的关系。结果表明,H2O2可促发培养基碱化,IMD和DMTU抑制SA诱发的培养基碱化,说明H2O2参与SA诱发的培养基碱化过程;SA抑制质膜H+-ATPase活性,Na3VO4引发培养基碱化并使H2O2迸发时间提前,FC处理逆转了SA诱导的培养基碱化及H2O2迸发,说明质膜H+-ATPase调控培养基pH值变化,培养基碱化促进了H2O2产生。因此,丹参悬浮培养细胞内H2O2水平与其培养基碱化程度之间相互关联、共同作用,协同响应SA的诱导。     

Lethality of hydrogen peroxide in wild type and superoxide dismutase mutants of Escherichia coli. (A hypothesis on the mechanism of H2O2-induced inactivation of Escherichia coli)

The toxicity of H₂O₂ in Escherichia coli wild type and superoxide dismutase mutants was investigated under different experimental conditions. Cells were either grown aerobically, and then treated in M9 salts or K medium, or grown anoxically, and then treated in K medium. Results have demonstrated that the wild type and superoxide dismutase mutants display a markedly different sensitivity to both modes of lethality produced by H₂O₂ (i.e. mode one killing, which is produced by concentrations of H₂O₂ lower than 5 mM, and mode two killing which results from the insult generated by concentrations of H₂O₂ higher than 10 mM). Although the data obtained do not clarify the molecular basis of H₂O₂ toxicity and/or do not explain the specific function of superoxide ions in H₂O₂-induced bacterial inactivation, they certainly demonstrate that the latter species plays a key role in both modes of H₂O₂ lethality. A mechanism of H₂O₂ toxicity in E. coli is proposed, involving the action of a hypothetical enzyme which should work as an O₂^-• generating system. This enzyme should be active at low concentrations of H₂O₂ (<5 mM) and high concentrations of the oxidant (>5 mM) should inactivate the same enzyme. Superoxide ions would then be produced and result in mode one lethality. The resistance at intermediate H₂O₂ concentrations may be dependent on the inactivation of such enzyme with no superoxide ions being produced at levels of H₂O₂ in the range 5–10 mM. Mode two killing could be produced by the hydroxyl radical in concert with superoxide ions, chemically produced via the reaction of high concentrations of H₂O₂ (>10 mM) with hydroxyl radicals. The rate of hydroxyl radical production may be increased by the higher availability of Fe²⁺ since superoxide ions may also reduce trivalent iron to the divalent form.     

Role of thiocyanate, bromide and hypobromous acid in hydrogen peroxide-induced apoptosis

We have previously reported that H₂O₂-induced apoptosis in HL-60 human leukemia cells takes place in the presence of chloride, requires myeloperoxidase (MPO), and occurs through oxidative reactions involving hypochlorous acid and chloramines. We now report that when chloride is replaced by the pseudohalide thiocyanate, there is little or no H₂O₂-induced apoptosis. Furthermore, thiocyanate inhibits H₂O₂-induced apoptosis when chloride is present at physiological concentrations, and this occurs at thiocyanate concentrations that are present in human serum and saliva. In contrast, bromide can substitute for chloride in H₂O₂-induced apoptosis, but results in a lower percent of the cells induced into apoptosis. Hypobromous acid is likely a short-lived intermediate in this H₂O₂/MPO/bromide apoptosis, and reagent hypobromous acid and bromamines induce apoptosis in HL-60 cells. We conclude that the physiologic concentrations of thiocyanate found in human plasma could modulate the cytototoxicity of H₂O₂ and its resulting highly toxic MPO-generated hypochlorous acid by competing with chloride for MPO. Furthermore, the oxidative products of the reaction of thiocyanate with MPO are relatively innocuous for human leukemic cells in culture. In contrast, bromide can support H₂O₂/MPO/halide apoptosis, but is less potent than chloride and it has no effect in the presence of physiological levels of chloride.     

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.