Direct and indirect antioxidant properties of α‐lipoic acid and therapeutic potential

Diabetes has emerged as a major threat to worldwide health. The exact mechanisms underlying the disease are unknown; however, there is growing evidence that the excess generation of reactive oxygen species (ROS) associated with hyperglycemia, causes oxidative stress in a variety of tissues. In this context, various natural compounds with pleiotropic actions like α‐lipoic acid (LA) are of interest, especially in metabolic diseases such as diabetes. LA, either as a dietary supplement or a therapeutic agent, modulates redox potential because of its ability to match the redox status between different subcellular compartments as well as extracellularly. Both the oxidized (disulfide) and reduced (di‐thiol: dihydro‐lipoic acid, DHLA) forms of LA show antioxidant properties. LA exerts antioxidant effects in biological systems through ROS quenching but also via an action on transition metal chelation. Dietary supplementation with LA has been successfully employed in a variety of in vivo models of disease associated with an imbalance of redox status: diabetes and cardiovascular diseases. The complex and intimate association between increased oxidative stress and increased inflammation in related disorders such as diabetes, makes it difficult to establish the temporal sequence of the relationship.     

Intake of Oxidized Proteins and Amino Acids and Causative Oxidative Stress and Disease: Recent Scientific Evidences and Hypotheses

The exposure to reactive oxygen species (ROS) is an inevitable consequence of living in an aerobic world. The species contribute to the occurrence of oxidative stress in humans in which an uncontrolled production of ROS exceeds the endogenous antioxidant defences leading to the oxidative damage to essential cellular components, such as lipids, proteins, and DNA. The influence of diet on the modulation of the systemic redox status is recognized and, while some dietary components are found to be protective (that is, fruits and vegetables), others are recognized as pro‐oxidants (that is, processed meat and other animal‐source protein foods). Oxidized proteins and amino acids are potential promoters of luminal and postprandial oxidative stress; preliminary studies have actually reported noxious effects of these species in cultured cells and in experimental animals. However, the underlying pathological mechanisms remain poorly understood. The application of advanced methodological approaches based on mass spectrometric technologies and OMICS disciplines has enabled the elucidation of the molecular basis of the pathological effects of dietary oxidized proteins and amino acids. The present review collects the most recent evidences of the health risks of dietary protein oxidation and proposes reasonable hypotheses and future perspectives on the field.     

线粒体氧化应激与天然抗氧化剂研究进展

活性氧主要来源于线粒体呼吸,是一柄“双刃剑”,其在生理浓度时广泛参与细胞的信号转导和生命过程,过量时又能引起线粒体氧化应激,从而导致衰老及相关疾病的发生。如何保持细胞内的氧化与抗氧化平衡（氧化还原平衡）,既不过度氧化损伤,也不过度抗氧化而影响正常的信号转导,这对健康极为重要。因此,细胞氧化还原调控与衰老机制不再是简单的“衰老的自由基学说”,也不再是简单的抗氧化延缓衰老。抗氧化剂是身体健康所必需的,富含抗氧化剂的膳食有助于心血管疾病、神经退行性疾病、癌症等重大疾病发病率的降低。本文从线粒体氧化应激、抗氧化剂的必要性和来源、抗氧化防御系统、氧化还原平衡与抗氧化悖论、天然抗氧化剂等方面阐述氧化与抗氧化平衡对健康的重要性,为促进健康并延缓衰老提供新的思路。     

红米稻过氧化物还原酶OsPrx3增强秀丽隐杆线虫抗氧化功能

目的：红米稻中OsPrx3基因预编码一个过氧化物还原酶，探究该基因原核细胞表达产物能否增强动物抗氧化的功能。方法：分子克隆OsPrx3基因，采用大肠杆菌表达目的蛋白，对摄入目的蛋白的模式动物-秀丽隐杆线虫(Caenorhabditis elegans)，鉴别运动能力、体长、抗氧化应激能力、体内活性氧与脂肪含量以及与氧化相关的基因表达水平等。结果：借助大肠杆菌细胞成功表达红米稻过氧化物还原酶基因OsPrx3，分离目的蛋白，体外实验显示该蛋白具有较高酶活性，5 min内1μg/mL OsPrx3对H₂O₂相对清除率可达40%以上；与对照相比，摄入目的蛋白的秀丽隐杆线虫，运动能力和体长虽无明显变化，但抵抗外部氧化应激的能力却在氧化处理的15 min内明显增强，而且体内活性氧、细胞脂肪含量显著降低；另外，部分调节氧化的关键基因表达明显上调。结论：基因工程策略成功表达红米稻过氧化物还原酶OsPrx3基因，其产物OsPrx3体外具有较高抗氧化活性，秀丽隐杆线虫摄入该蛋白能显著增强抗氧化能力。     

Svf1 inhibits reactive oxygen species generation and promotes survival under conditions of oxidative stress in Saccharomyces cerevisiae

Aberrant regulation of apoptosis, or programmed cell death, contributes to the aetiology of several diseases, including cancers, immunodeficiencies and neurodegenerative illnesses. We hypothesized that key features of mammalian cell death regulation may be conserved in single celled organisms such as the budding yeast Saccharomyces cerevisiae. We previously identified the yeast gene SVF1 in a screen for mutations that could be functionally complemented by exogenous expression of the human anti-apoptotic gene Bcl-x(L). Anti-apoptotic Bcl-2 family members have been shown to promote redox stability through upregulation of antioxidant pathways in mammalian cells. Here we demonstrate that the Svf1 protein is required for yeast survival under conditions of oxidative stress, including cold stress. Cells lacking SVF1 are hypersensitive to conditions associated with increased reactive oxygen species (ROS) generation and to direct chemical precursors of ROS, and demonstrate increased levels of ROS under these conditions. Hypersensitivity to oxidative stress can be reversed by treatment with the antioxidant N-acetylcysteine or expression of exogenous SVF1, although exogenous expression of Bcl-x(L) did not protect cells from cold stress. Exogenous SVF1 expression in mammalian cells confers resistance to H(2)O(2) exposure. Our data are consistent with previous observations suggesting a key role of oxidative stress response in mammalian apoptotic regulation and validate the use of S. cerevisiae as a model for studying programmed cell death.     

Salvianolic acid B inhibits low‐density lipoprotein oxidation and neointimal hyperplasia in endothelium‐denuded hypercholesterolaemic rabbits

BACKGROUND: Atherosclerosis and restenosis are inflammatory responses involving free radicals and lipid peroxidation and may be prevented/cured by antioxidant‐mediated lipid peroxidation inhibition. Salvianolic acid (Sal B), a water‐soluble antioxidant obtained from a Chinese medicinal herb, is believed to have multiple preventive and therapeutic effects against human vascular diseases. In this study the in vitro and in vivo inhibitory effects of Sal B on oxidative stress were determined. RESULTS: In human aortic endothelial cells (HAECs), Sal B reduced oxidative stress, inhibited low‐density lipoprotein (LDL) oxidation and reduced oxidised LDL‐induced cytotoxicity. Sal B inhibited Cu²⁺‐induced LDL oxidation in vitro (with a potency 16.3 times that of probucol) and attenuated HAEC‐mediated LDL oxidation as well as reactive oxygen species (ROS) production. In cholesterol‐fed New Zealand White rabbits (with probucol as positive control), Sal B intake reduced Cu²⁺‐induced LDL oxidation, lipid deposition in the thoracic aorta, intimal thickness of the aortic arch and thoracic aorta and neointimal formation in the abdominal aorta. CONCLUSION: The data obtained in this study suggest that Sal B protects HAECs from oxidative injury‐mediated cell death via inhibition of ROS production. The antioxidant activity of Sal B may help explain its efficacy in the treatment of vascular diseases. Copyright © 2010 Society of Chemical Industry     

Significant damage-rescuing effects of wood vinegar extract in living Caenorhabditis elegans under oxidative stress

BACKGROUND: Wood vinegar (WV), a byproduct from the charcoal production process, has been reported to have excellent antioxidant capability by chemical examination. However, the biological effect of WV in living animals is still unknown. In this study, a simple model organism, the nematode Caenorhabditis elegans, was used as an in vivo system to assess the biological effects of wood vinegar through the development, lifespan, brood size, germline cell apoptosis and superoxide dismutase (SOD) level. RESULTS: Wood vinegar extract (WVE) promoted the development, prolonged the lifespan and increased the brood size in reactive oxidative species (ROS)‐sensitive mutant worms. WVE treatment rescued the effects of damage in germline cell apoptosis and SOD upregulation induced by paraquat, an ROS generator, to the control level. Additionally, WVE showed comparative ability in rescuing damage as compared with L ‐ascorbic acid and α‐tocopherol. CONCLUSION: WVE treatment exhibits a remedial/beneficial effect on ROS‐sensitive mutant under normal cultural conditions and on wild‐type worms under oxidative stress. ROS scavenging is involved in the damage‐rescuing mechanism. This study will provide a basal biological and nutritional exploration for the use of WV as a functional food, and for the substitution of chemical antioxidants with side effects in food. Copyright © 2011 Society of Chemical Industry     

Maturity‐dependent chilling tolerance regulated by the antioxidative capacity in postharvest cucumber (Cucumis sativus L.) fruits

BACKGROUND: The aim of the present study was to reveal the effect of fruit maturity on the chilling tolerance of cucumber (Cucumis sativus L.) fruit and the oxidative and antioxidative mechanisms involved. Chinese mini‐cucumber (cv. Hangcui‐1) fruits were harvested at four developmental stages: Immature (3–8 days after anthesis (DAA)), Mature (9–16 DAA), Breaker (17–22 DAA) and Yellow (35–40 DAA). All fruits were stored at 2 °C for 9 days and rewarmed at 20 °C for 2 days. RESULTS: The chilling injury index declined with advancing fruit maturity. High superoxide anion radical production rate and hydrogen peroxide content were observed in Immature fruits after cold storage and rewarming. Under chilling stress, superoxide dismutase showed an early response. Fruits at earlier maturity stages exhibited higher catalase, ascorbate peroxidase and monodehydroascorbate reductase activities and glutathione content as well as its redox state, and lower peroxidase, dehydroascorbate reductase and glutathione reductase activities and ascorbate content as well as its redox state. CONCLUSION: Fruits at the earlier developmental stage are more susceptible to chilling injury, which is related to increased oxidative stress. High peroxidase activity and ascorbate content and maintenance of the latter's redox state appear critical to the chilling tolerance of cucumber fruits at later developmental stages. © 2012 Society of Chemical Industry     

Pretreatment of the yeast antagonist, Candida oleophila, with glycine betaine increases oxidative stress tolerance in the microenvironment of apple wounds

In response to wounding, harvested fruit tissues of apple and citrus exhibit the production of reactive oxygen species (ROS). ROS production is greater when yeast antagonists used as biocontrol agents are applied in the wounds. These phenomena result in an oxidative stress environment for the yeast antagonists. It has been demonstrated that pre-exposure of some of these yeast antagonists to sublethal abiotic stress (heat or hydrogen peroxide), or stress-ameliorating compounds such as glycine betaine (GB) can induce subsequent oxidative stress tolerance in the antagonistic yeast. The increased level of oxidative stress tolerance has been demonstrated in vitro and is characterized by higher levels of antioxidant gene expression, increased production of trehalose, and lower levels of ROS when yeast are exposed to a subsequent oxidative stress. The current study determined whether or not the effects of GB on yeast antagonists determined in vitro persist and are present in planta when yeast are applied to wounded apples. The effect of exogenous GB on the production of ROS in the yeast antagonist, Candida oleophila, was determined after the yeast was placed in apple wounds. Oxidative damage to yeast cells recovered from apple wounds was also monitored. Results indicated that GB treatment improved the adaptation of C. oleophila to apple fruit wounds. Compared to untreated control yeast cells, GB-treated cells recovered from the oxidative stress environment of apple wounds exhibited less accumulation of ROS and lower levels of oxidative damage to cellular proteins and lipids. Additionally, GB-treated yeast exhibited greater biocontrol activity against Penicillium expansum and Botrytis cinerea, and faster growth in wounds of apple fruits compared to untreated yeast. The expression of major antioxidant genes, including peroxisomal catalase, peroxiredoxin TSA1, and glutathione peroxidase was elevated in the yeast by GB treatment. This study supports the premise that activation of antioxidant response in biocontrol yeast can improve biocontrol efficacy.     

The role of two putative nitroreductases,Frm2p and Hbn1p,in the oxidative stress response in Saccharomyces cerevisiae

The nitroreductase family is comprised of a group of FMN‐ or FAD‐dependent enzymes that are able to metabolize nitrosubstituted compounds using the reducing power of NAD(P)H. These nitroreductases can be found in bacterial species and, to a lesser extent, in eukaryotes. There is little information on the biochemical functions of nitroreductases. Some studies suggest their possible involvement in the oxidative stress response. In the yeast Saccharomyces cerevisiae, two nitroreductase proteins, Frm2p and Hbn1p, have been described. While Frm2p appears to act in the lipid signalling pathway, the function of Hbn1p is completely unknown. In order to elucidate the functions of Frm2p and Hbn1p, we evaluated the sensitivity of yeast strains, proficient and deficient in both oxidative stress proteins, for respiratory competence, antioxidant‐enzyme activities, intracellular reactive oxygen species (ROS) production and lipid peroxidation. We found reduced basal activity of superoxide dismutase (SOD), ROS production, lipid peroxidation and petite induction and higher sensitivity to 4‐nitroquinoline‐oxide (4‐NQO) and N‐nitrosodiethylamine (NDEA), as well as higher basal activity of catalase (CAT) and glutathione peroxidase (GPx) and reduced glutathione (GSH) content in the single and double mutant strains frm2Δ and frm2Δ hbn1Δ. These strains exhibited less ROS accumulation and lipid peroxidation when exposed to peroxides, H₂O₂ and t‐BOOH. In summary, the Frm1p and Hbn1p nitroreductases influence the response to oxidative stress in S. cerevisae yeast by modulating the GSH contents and antioxidant enzymatic activities, such as SOD, CAT and GPx. Copyright © 2009 John Wiley & Sons, Ltd.     

数学分析法评估大蒜素对HeLa细胞氧化还原水平的影响

为探究大蒜素调控典型肿瘤细胞中的氧化还原水平,建立了佛波酯(phorbol-12-myristate-13-acetate,PMA)诱导人宫颈癌细胞HeLa中活性氧(reactive oxygen species,ROS)升高的氧化应激模型,通过借助中心响应面模型,探究发现当HeLa细胞在10 mg/L的大蒜素培养液中...     

Cranberries and Wild Blueberries Treated with Gastrointestinal Enzymes Positively Modify Glutathione Mechanisms in Caco‐2 Cells In Vitro

Beneficial health effects of cranberries (CBs) and wild blueberries (BBs), such as reduced levels of oxidative stress, have been demonstrated in feeding studies. These Vaccinium berries contain high levels of flavonoids; however, the bioavailability of flavonoids is generally low. We investigated the in vitro effects of these berries on intestinal cells, focusing on mitigating oxidative stress and associated reactive oxygen species (ROS). First, we simulated the passage of CB and BB through the gastrointestinal (GI) tract by treating berry homogenates to a battery of digestive enzymes. Then, Caco‐2 cells, a model of small intestine epithelial uptake, were exposed to these homogenates for 60 min. Using a cell‐free assay, we found that the antioxidant activity in CB homogenates was not affected by these enzymes, but that BB homogenates treated with gut enzymes had 43% lower free‐radical quenching activity (P < 0.05). However, both of the enzyme‐treated homogenates were still able to counteract the ROS‐generating ability of H₂O₂ added exogenously to Caco‐2 cells. Berry homogenates also increased mitochondrial metabolic rates at 60 min posttreatment, as measured by MTT assays. Enzyme‐treated CB (but not BB) homogenates increased the levels of reduced glutathione (GSH) relative to oxidized glutathione (GSSG), a critical indicator of the cellular redox state (P < 0.05). Our data suggest that CBs do not lose their antioxidant ability when passing through the GI tract, and specifically, digested CB may serve to enhance cytoprotective effects in intestinal cells by reducing potential damage caused by free radicals and ROS derived from other food sources.     

Protective Effects of Merlot Red Wine Extract and its Major Polyphenols in Pc12 Cells under Oxidative Stress Conditions

Abstract: The potential effect of the extracts from free‐run and pressed Merlot red wine has been evaluated in PC12 cells under oxidative stress situation. Comparing both vinification process, pressed Merlot red wine extract possessed higher neuroprotective activity than the free run wine, possibly attributed to the major content in all global polyphenolic families. High performance liquid chromatography determination of individual polyphenols showed that the major compounds found in Merlot red wine extract were quercetin, catechin, epicatechin, tyrosol, gallic acid, and procyanidins. Pretreatments with these polyphenolic compounds (0.25 mM and 0.1 mM, 24 h) significantly increased cell viability of H₂O₂ and Fenton reaction treated cells. Moreover, these polyphenols attenuated ROS production and decreased the Redox Index of glutathione (RI = GSSG/GSH + GSSG) in cells treated only with Fenton reaction. Furthermore, some polyphenols induced antioxidant enzymes activity and protein expression. Quercetin was the most active. These results support the beneficial effects of red wine extracts and some of its polyphenols under oxidative stress conditions. Practical Application: This research provides evidences of the preventive properties of wine extracts and its major polyphenols under oxidative stress conditions.     

Protective effects of enzymatic digest from Ecklonia cava against high glucose‐induced oxidative stress in human umbilical vein endothelial cells

BACKGROUND: Antioxidants can prevent pathological damage caused by hyperglycaemia‐induced oxidative stress associated with diabetes. In the present study, we investigated whether the brown alga Ecklonia cava has protective effects against high glucose‐induced oxidative stress in Human umbilical vein endothelial cells (HUVECs). For that purpose, we prepared an enzymatic digest from E. cava (ECC) by using the carbohydrase, Celluclast. RESULTS: High glucose treatment induced HUVECs cell death, but ECC, at a concentration of 10 or 100 µg mL^?1, significantly inhibited the high glucose‐induced cytotoxicity. Furthermore, treatment with ECC dose‐dependently decreased thiobarbituric acid reactive substances (TBARS), intracellular generation of reactive oxygen species, and the nitric oxide level increased by high glucose. In addition, ECC treatment increased activities of antioxidant enzymes including catalase, superoxide dismutase and glutathione peroxidase in high‐glucose pretreated HUVECs. High glucose levels induced the overexpression of inducible nitric oxide synthase, cyclooxygenase‐2 and nuclear factor‐kappa B proteins in HUVECs, but ECC treatment reduced the overexpression of these proteins. CONCLUSION: These results suggest that ECC is a potential therapeutic agent that will reduce the damage caused by hyperglycaemia‐induced oxidative stress associated with diabetes. Copyright © 2009 Society of Chemical Industry     

菌菇复合多糖对秀丽线虫氧化胁迫抗性的影响

目的:研究菌菇复合多糖(FHP)的抗氧化效果及作用机制.方法:用化学方法评价FHP体外自由基清除效率,通过秀丽线虫的氧化胁迫存活率试验评价FHP体内抗氧化效果;通过线虫体内氧化胁迫水平检测,胁迫应激关键转录因子活性及其效应基因表达检测和抗氧化酶活性试验等研究FHP的抗氧化活性;通过线虫的寿命试验及脂褐素水平检测研究FH...     

The more effective treatment of atrial fibrillation applying the natural compounds; as NADPH oxidase and ion channel inhibitors

Atrial fibrillation (AF) is the most common cardiac arrhythmia that occurs because of several different risk factors, e.g., valvular heart disease, coronary artery disease, age ≥75 years, hypertension and diabetes mellitus. One key risk factor that results in AF, is oxidative stress. Evidence suggests that there is a correlation between oxidative processes and the genesis of AF. Oxidative stress occurs when the generation of reactive oxygen species (ROS) increase due to excessive activity of enzymes including NADPH oxidase (NOX) and xanthine oxidase; or its degradation decrease by dysfunctional antioxidant enzyme systems, such as superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx). Afterwards, elevated ROS may shift ion channel activity to increase AF susceptibility.

The outbreak of AF continues to grow. Unfortunately, current treatment strategies may have limited efficacy or adverse effects. On the other hand, the inhibition of ROS formation and alteration of ion channel activity could be important therapeutic targets for prevention or treatments of AF. Additionally, many studies have been shown that several natural compounds have the ability to inhibit NADPH oxidases directly. This review focuses on natural compounds which specially inhibit NOX isoforms and have direct effects on ion channels, suggesting these compounds can be helpful in AF treatment.     

Role of topical and nutritional supplement to modify the oxidative stress

Background: Evidence suggests that signs of skin ageing such as wrinkling, ragging and actinic lentigines, may be connected to cumulative oxidative damage incurred throughout our lifetimes. To counteract this oxidative injury, skin is equipped with a network on enzymatic and non-enzymatic antioxidant systems, such as tocopherols, ascorbate polyphenols. All these compounds administered topically by cosmetics or by oral route by diet supplements, have been shown to exert an antioxidant/protective effect in skin or skin cells. Objective: The object of this study was to evaluate both in vitro and in vivo the activity performed by different topical antioxidants and nutritional supplements. Methods: A randomized double-blind placebo-controlled study was carried out for 8 weeks on 30 dry-skinned elderly volunteers, women aged between 48 and 59 years, with moderate xerosis and photoageing. Surface skin lipids, skin hydration and MDA determination were topically detected by 3C System. ROS was evaluated on the blood serum and on IL-3 stimulated human leukocytes by ROS Meter System at 505 nm. All the subjects applied twice a day for 2 months a nanocolloidal gel and/or take a diet supplement by oral route at the quantity of two capsules per day. All the formulations used were antioxidant-enriched (ascorbic acid, tocopherol, alpha-lipoic acid, melatonin, emblica). Results: Oxidative stress and consequently lipids peroxidation decreased from 30 to 40% (P < 0.005) in blood serum of all the subjects treated with antioxidant compounds topically and by oral route. Both free radicals recovered in blood serum and on skin (in vivo) and ROS induced by irradiation of leucocytes with UVB light (in vitro), appear sensibly lower in subjects antioxidant-treated. Conclusions: From the obtained data, it seems possible to conclude that all the compounds used play interesting role as topical and systemic photoprotectants, thanks to their interesting antioxidant property. Moreover, the antioxidant treatment seems to be a promising therapeutic approach also in reducing the oxidative stress of people affected by photoaging.     

Hydroxytyrosyl acetate contributes to the protective effects against oxidative stress of virgin olive oil

The effects of virgin olive oil phenols, hydroxytyrosyl acetate (HTy-Ac) and hydroxytyrosol (HTy), on cell integrity and steady-state values of cellular redox status were assessed in HepG2 cells, as well as their potential protective effects against oxidative stress induced by tert-butyl hydroperoxide (t-BOOH). Direct treatment for 20 h with 0.5-10 μM HTy or HTy-Ac reduced ROS generation and increased glutathione peroxidase activity at the higher concentrations. Furthermore, after t-BOOH exposure, pretreatment with HTy-Ac and HTy for 2 or 20 h counteracted cell viability damage from 1 μM, counterbalanced reduced glutathione levels from 0.5 μM, protected against lipid peroxidation from 0.5 μM, decreased ROS generation from 1 μM as well as antioxidant enzyme activities from 1 μM. All these changes were statistically significant.HTy-Ac presents antioxidative stress protective effects at physiological concentrations similar to or even slightly higher than that of HTy, thus contributing to the protective role of virgin olive oil.     

Evaluation of antioxidant activities and phenolic subtype contents of ethanolic bran extracts of Thai pigmented rice varieties through chemical and cellular assays

This study revealed that the ethanolic bran extracts of 11 Thai pigmented (red and purple) and 2 nonpigmented rice varieties exerted scavenging activity against DPPH and ABTS radicals and ROS in HL‐60 cells in the following order: red > purple > nonpigmented rice. These rice extracts also showed the same order of phenolic and flavonoid contents, which were strongly correlated with their scavenging activity. Phenolic subtype analysis further indicated that proanthocyanidins as well as anthocyanins and protocatechuic acid contributed directly to antioxidant capacity in red and purple rice bran, respectively. In contrast, these pigmented rice bran extracts possessed moderate chelating activity partly attributed to their contents of phenolics and flavonoids, especially proanthocyanidins in red rice bran. Moreover, rice bran extracts significantly restored SOD and CAT activities in oxidative stress‐induced A549 cells. This study provides new insights on the intracellular potent antioxidant capacity of pigmented rice bran extracts in the cell‐based systems.     

Antioxidative effects of whey protein on peroxide-induced cytotoxicity

Myoblastic toxicity is a major adverse effect caused by reactive oxygen species (ROS) when exercising heavily. Although protection or alleviation of ROS toxicity can be achieved by administration of antioxidant vitamins such as vitamin E and vitamin C, their protective effect remains controversial. Thus, alternative natural antioxidants may be potential candidates for foods for athletes. In this research, we investigated the antioxidative effect of whey protein against hydrogen peroxide (H₂O₂) toxicity using C₂C₁₂ myoblasts. Whey protein pre-incubation prevented the decrease in cell viability after H₂O₂ treatment. The production of 8-hydroxydeoxyguanosine associated with DNA oxidative damage was also inhibited by the whey protein pre-incubation. Endogenous antioxidant defense, such as glutathione, catalase, and superoxide dismutase activity, was also modulated by the antioxidant. At the same time, enhanced mRNA expression levels of heme oxygenase-1 and NADPH quinone oxidoreductase-1 were observed in cells pre-incubated with whey protein before H₂O₂ abuse. These findings suggest that whey protein improved the antioxidant capacity against acute oxidative stress through multiple pathways and this protein may serve as an alternative source of antioxidants for prevention of athletic injuries caused by ROS.