Cytoprotective effect of green tea extract and quercetin against hydrogen peroxide-induced oxidative stress

In this study, we evaluated the cytoprotective effects of antioxidative substances in hydrogen peroxide (H2O2) treated Mel-Ab melanocytes. Tested substances include selenium, quercetin, green tea (GT) extract, and several vitamins (ascorbic acid, Trolox, and folic acid). Of these, both quercetin and GT extract were found to have strong cytoprotective effects on H2O2-induced cell death. We also examined additive effects, but no combination of two of any of the above substances was found to act synergistically against oxidative damage in Mel-Ab cells. Nevertheless, a multi-combination of GT extract, quercetin, and folic acid appeared to prevent cellular damage in a synergistic manner, which suggests that combinations of antioxidants may be of importance, and that co-treatment with antioxidants offers a possible means of treating vitiligo, which is known to be related to melanocyte oxidative stress.     

洋参抗衰合剂对H2O2诱导HepG2细胞氧化损伤的保护作用

目的 考察洋参抗衰合剂对H₂O₂致HepG2细胞氧化应激损伤的保护作用,并初步分析洋参抗衰合剂对H₂O₂诱导HepG2细胞氧化损伤的保护机制。方法 以H₂O₂诱导培养的HepG2细胞为模型,检测洋参抗衰合剂对HepG2细胞中谷胱甘肽(glutathione,GSH)含量、过氧化氢酶(catalase, CAT)活力和丙二醛(malondialdehyde, MDA)含量的变化,分析洋参抗衰合剂的抗氧化作用。结果 随着洋参抗衰合剂质量浓度的增加,H₂O₂诱导HepG2细胞的谷胱甘肽(GSH) 和过氧化氢酶(CAT)含量上升,丙二醛(MDA)的含量有所下降。结论 洋参抗衰合剂对H₂O₂诱导HepG2细胞损伤有抗氧化作用,其机理可能是通过促进还原物质的合成,增加抗氧化酶的活性进而减少脂质过氧化物。     

No protective effect of curcumin on hydrogen peroxide-induced cytotoxicity in HepG2 cells

Scavenging of intracellular reactive oxygen species (ROS) is one of the potential mechanisms contributing to the protective effects of many antioxidants. Curcumin, a natural product, is an effective ROS scavenger. However, the role of its ROS scavenging ability in its cytoprotective action remains to be clarified. Herein, the protective effects of curcumin on hydrogen peroxide (H₂O₂)- and tert-butyl hydroperoxide-induced ROS formation and HepG2 cell injury were determined. HepG2 cells were pretreated with curcumin for 30 min and then treated with H₂O₂ (500 µM) or tert-butyl hydroperoxide (200 µM) for 24 h. Curcumin pretreatment dramatically decreased H₂O₂- and tert-butyl hydroperoxide-induced ROS production, but failed to suppress cytotoxicity of those compounds. H₂O₂ induced decreases in mitochondrial membrane potential (ΔΨm) and increases in DNA fragmentation could not be reversed by curcumin. Furthermore, curcumin enhanced expression of H₂O₂-induced pro-apoptotic protein Bax expression and inhibited expression of anti-apoptotic proteins Bcl-2 and Bcl-xL. In addition, curcumin significantly decreased p38MAPK and phospho-CDC-2 protein expression and increased phospho-p38MAPK, p42/44MAPK, and phospho-p42/44MAPK protein expression. These results suggest that short pretreatment and subsequent longer co-treatment of low concentrations of curcumin showed no obvious protective effect on H₂O₂-induced HepG₂ cell injury.     

Protective effects of kahweol and cafestol against hydrogen peroxide-induced oxidative stress and DNA damage

There is an increasing evidence that oxidative stress is implicated in the processes of inflammation and carcinogenesis. It has been shown that kahweol and cafestol, coffee-specific diterpenes, exhibit chemoprotective effects. This study investigated the effects of kahweol and cafestol, coffee-specific diterpenes, on the hydrogen peroxide (H(2)O(2))-induced oxidative stress and DNA damage in NIH3T3 cells. When the cells were treated with kahweol or cafestol, cytotoxicity, lipid peroxidation, and reactive oxygen species production induced by H(2)O(2) were markedly reduced in a dose-dependent manner. Moreover, kahweol and cafestol were shown to be highly protected against H(2)O(2)-induced oxidative DNA damage as determined by the Comet (single cell gel electrophoresis) assay and the measurement of 8-oxoguanine content in NIH3T3 cells. Kahweol and cafestol also protected hydroxyl radical-induced 2-deoxy-d-ribose degradation by ferric ion-nitrilotriacetic acid and H(2)O(2). In addition, kahweol and cafestol efficiently removed the superoxide anion generated from the xanthine/xanthine oxidase system. These results suggest that kahweol and cafestol are effective in protecting against H(2)O(2)-induced oxidative stress and DNA damage, probably via scavenging free oxygen radicals, and that kahweol and cafestol act as antioxidants.     

Nitroderivatives of olive oil phenols protect HepG2 cells against oxidative stress

A series of nitroderivatives has been synthetized from natural and synthetic olive oil phenols to increase the assortment of compounds with a putative effect against Parkinson disease. Before considering the potential therapeutical and nutraceutical applications of the new compounds it was critical to assess any cytotoxic effects in the liver. The precursor compounds of the nitroderivatives have shown oxidative stress protective effects, therefore we also assessed if the new compounds counteracted oxidative stress. The antioxidant activity of nitrohydroxytyrosol (NO-HTy), nitrohydroxytyrosyl-acetate (NO-HTy-A) and ethyl-nitrohydroxytyrosyl-ether (NO-HTy-E) at 5–20 μM for 20 h, as well as the protective effects of the nitroderivatives after 20 h against oxidative stress induced by tert-butylhydroperoxide (t-BOOH), were assessed in HepG2 cells. Direct treatment with the three nitroderivatives decreased ROS generation compared to the control and NO-HTy at 20 μM also increased glutathione peroxidase (GPx) activity (p < 0.001). Pretreatment with the three nitroderivatives at 5–20 μM counteracted t-BOOH cell damage by decreasing ROS generation (p < 0.001) and malondialdehyde (MDA) levels (p < 0.001), increasing reduced glutathione (p < 0.001) and disminishing GPx (p < 0.05) activity. NO-HTy, NO-HTy-A and NO-HTy-E decreased glutathione reductase activity (p < 0.05). Conclusion: the nitroderivatives do not present cytotoxic effects in the liver and in addition may protect against the oxidative stress involved in degenerative diseases.     

Microcystin-LR induces oxidative DNA damage in human hepatoma cell line HepG2.

Microcystins are naturally occurring hepatotoxins produced by strains of Microcystis aeruginosa. They are involved in promoting primary liver tumours and a previous study showed that they might also be tumour initiators. In this study we demonstrate that microcystin-LR (MCLR) at doses that were not cytotoxic (0.01-1 microg/ml), induced dose and time dependent DNA strand breaks in human hepatoma cell line HepG2. These DNA strand breaks were transient, reaching a maximum level after 4h of exposure and declining with further exposure. In the presence of the DNA repair inhibitors cytosine arabinoside (AraC) and hydroxyurea (HU), together with MCLR, DNA strand breaks accumulated after prolonged exposure. These results suggest that DNA strand breaks are intermediates, produced during the cellular repair of MCLR induced DNA damage. Digestion of DNA with purified, oxidative DNA damage specific enyzmes, endonuclease III (Endo III) and formamidopyrimidine-DNA glycosylase (Fpg) markedly increased DNA strand breaks in MCLR treated cells, providing evidence that a substantial portion of the MCLR induced DNA strand breaks originate from excision of oxidative DNA adducts. A hydroxyl radical scavenger (DMSO) significantly reduced MCLR induced DNA damage. From these results we conclude that MCLR induces formation of reactive oxygen species that cause DNA damage, and that MCLR may act as an initiator of liver cancer.     

Protective effects of protopine on hydrogen peroxide-induced oxidative injury of PC12 cells via Ca(2+) antagonism and antioxidant mechanisms

Calcium and lipid peroxidation play important roles in oxidative stress-induced cellular injury and apoptosis, which ultimately cause cell death. In this study we examined whether protopine had a neuroprotection against H₂O₂-induced injury in PC12 cells. Pretreatment of PC12 cells with protopine improved the cell viability, enhanced activities of superoxide dismutase, glutathione peroxidase and catalase, and decreased malondialdehyde level in the H₂O₂ injured cells. Protopine also reversed the increased intracellular Ca²⁺ concentration and the reduced mitochondrial membrane potential caused by H₂O₂ in the cells. Furthermore, protopine was able to inhibit caspase-3 expression and cell apoptosis induced by H₂O₂. In summary, this study demonstrates that protopine is able to relieve H₂O₂-induced oxidative stress and apoptosis in PC12 cells, at least in part, by Ca²⁺ antagonism and antioxidant mechanisms.     

Protective effect of Lepidium sativum seed extract against hydrogen peroxide-induced cytotoxicity and oxidative stress in human liver cells (HepG2)

Context: Garden cress [Lepidium sativum (Brassicaceae)] has been widely used to treat a number of ailments in traditional medicine. The pharmacological and preventive potential of Lepidium sativum, such as anti-inflammatory, antipyretic, antihypertensive, anti-ashthamatic, anticancer, and anti-oxidant, are well known.

Objective: The present investigation was designed to study the protective effects of chloroform extract of Lepidium sativum seed (LSE) against oxidative stress and cytotoxicity induced by hydrogen peroxide (H₂O₂) in human liver cells (HepG2).

Materials and methods: Cytotoxicity of LSE and H₂O₂ was identified by (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), neutral red uptake (NRU) assays, and morphological changes in HepG2. The cells were pre-exposed to biologically safe concentrations (5–25?μg/ml) of LSE for 24 h, and then cytotoxic (0.25 mM) concentration of H₂O₂ was added. After 24 h of the exposures, cell viability by MTT, NRU assays, and morphological changes in HepG2 were evaluated. Further, protective effects of LSE on reactive oxygen species (ROS) generation, mitochondrial membrane potential (MMP), lipid peroxidation (LPO), and reduced glutathione (GSH) levels induced by H₂O₂ were studied.

Results: Pre-exposure of LSE significantly attenuated the loss of cell viability up to 48% at 25?µg/ml concentration against H₂O₂ (LD₅₀ value?=?2.5?mM). Results also showed that LSE at 25?µg/ml concentration significantly inhibited the induction of ROS generation (45%) and LPO (56%), and increases the MMP (55%) and GSH levels (46%).

Discussion and conclusion: The study suggests the cytoprotective effects of LSE against H₂O₂-induced toxicity in HepG2. The results also demonstrate the anti-oxidative nature of LSE.     

Scutellarin protects PC12 cells from oxidative stress-induced apoptosis

The present study investigated the effects of scutellarin on oxidative stress-induced cell apoptosis in PC12 cells. Exposure of cells to hydrogen peroxide (H₂O₂) triggered a typical apoptosis, as evidenced by DNA fragmentation, DNA loss and externalization of phosphatidylserine (PS). This treatment also caused significant elevation of oxidative stress characterized by intracellular accumulations of reactive oxygen species (ROS) and malondialdehyde (MDA), a product of lipid peroxidation. Preincubation of cells with scutellarin significantly inhibited the fragmentation and loss of DNA, the externalization of PS, and decreased the percentage of cell apoptosis. Also, intracellular accumulations of ROS and MDA resulting from H₂O₂ exposure were significantly reduced by scutellarin. These findings suggest that scutellarin exerts significant protection against oxidative stress-induced apoptosis, which might be beneficial for the prevention and treatment of oxidative stress-mediated disorders.     

Scutellarin protects PC12 cells from oxidative stress-induced apoptosis

The present study investigated the effects of scutellarin on oxidative stress-induced cell apoptosis in PC12 cells. Exposure of cells to hydrogen peroxide (H₂O₂) triggered a typical apoptosis, as evidenced by DNA fragmentation, DNA loss and externalization of phosphatidylserine (PS). This treatment also caused significant elevation of oxidative stress characterized by intracellular accumulations of reactive oxygen species (ROS) and malondialdehyde (MDA), a product of lipid peroxidation. Preincubation of cells with scutellarin significantly inhibited the fragmentation and loss of DNA, the externalization of PS, and decreased the percentage of cell apoptosis. Also, intracellular accumulations of ROS and MDA resulting from H₂O₂ exposure were significantly reduced by scutellarin. These findings suggest that scutellarin exerts significant protection against oxidative stress-induced apoptosis, which might be beneficial for the prevention and treatment of oxidative stress-mediated disorders.     

左卡尼汀对卵泡颗粒细胞抗氧化应激作用的影响

目的探讨左卡尼汀对卵泡颗粒细胞对抗氧化应激的影响。方法收集人卵泡颗粒细胞进行体外培养24h后按0μmol/L左卡尼汀处理组(对照组),20μmol/L左卡尼汀处理组,40μmol/L左卡尼汀处理组,80μmol/L左卡尼汀处理组进行分组培养24h。应用流式细胞检测技术分别对颗粒细胞内活性氧ROS水平和线粒体膜电位进行检测。结果不同浓度左卡尼汀组间ROS含量有显著性差异(χ2=13.14,P=0.04),ROS含量随左卡尼汀浓度升高而减少。不同浓度左卡尼汀组间颗粒细胞线粒体膜电位有显著性差异(χ2=9.375,P=0.025),膜电位水平随左卡尼汀浓度升高而升高。结论左卡尼汀可能提高卵泡颗粒细胞的抗氧化应激能力。     

人参皂苷保护小鼠精原细胞氧化损伤的研究

目的观察人参皂苷对活性氧引起的小鼠睾丸生殖细胞氧化损伤的保护作用。方法利用体外培养的小鼠精原细胞建立氧化应激模型,通过检测生殖细胞活性、脂质过氧化产物丙二醛(MDA)生成、超氧化物歧化酶(SOD)活性和谷胱甘肽(GSH)水平评价人参皂苷对精原细胞氧化损伤的缓解作用。结果次黄嘌呤/黄嘌呤氧化酶(HX/XO)体系产生的活性氧可引起生殖细胞活性降低、MDA的生成量增加、SOD活性和GSH水平降低,而添加人参皂苷(10mg·L-1)能恢复HX/XO引起的生殖细胞活性、SOD活性和GSH水平的下降以及MDA生成的增加。结论人参皂苷可通过抗氧化作用保护活性氧引起的小鼠精原细胞氧化损伤。     

Isoniazid-induced apoptosis in HepG2 cells: Generation of oxidative stress and Bcl-2 down-regulation

Isoniazid (INH) is a first-line antibiotic used in the treatment of infections caused by Mycobacterium tuberculosis. However it has a serious limitation of being hepatotoxic. Delineating the mechanism underlying INH-induced hepatotoxicity may be beneficial in devising ways to counteract its toxic manifestations. Studies in human hepatoma HepG2 cells have indicated that INH exposure causes induction of apoptosis. This study was aimed at identifying the key components/pathways of the INH-induced apoptotic pathway using HepG2 cells. HepG2 cells were exposed to increasing concentrations of INH (6.5, 13, 26, and 52?mM). Hydrogen peroxide (0.3?mM) served as positive control. After incubating for specific time intervals cells were harvested and evidences of cytotoxicity, oxidative stress, and apoptosis were sought. The findings indicated that INH exposure causes increased ROS generation along with alteration in levels of enzymatic antioxidants such as Superoxide dismutase, Catalase, and Glucose-6-Phosphate dehydrogenase. Altered Bcl-2/Bax content, cytochrome-c translocation, caspase activation, and DNA fragmentation emphasized involvement of apoptosis.     

A novel benzotriazole derivative inhibits proliferation of human hepatocarcinoma cells by increasing oxidative stress concomitant mitochondrial damage

Benzotriazole derivatives have been shown to be able to induce growth inhibition in cancer cells. In the present study, we synthesized bioactive compound, 3-(1H-benzo [d] [1,2,3] triazol-1-yl)-1-(4-methoxyphenyl)-1-oxopropan-2-yl benzoate (BmOB), which is a novel benzotriazole derivative. BmOB displayed anti-proliferative effects on several human tumor cell lines. Human hepatocarcinoma BEL-7402 cell line was selected as a model to illustrate BmOB's inhibition effect and its potential mechanism, since it was the highest susceptible cell line to BmOB. It was shown that treatment with BmOB resulted in generation of reactive oxygen species, disruption of mitochondrial membrane potential (DeltaPsim), and cell death in BEL-7402 cells. BmOB induced cytotoxicity could be prevented by antioxidant vitamin C and mitochondrial permeability transition inhibitor cyclosporine A. cyclosporine A could also protect the BmOB induced collapse of DeltaPsim in BEL7402 cells, while vitamin C did not show similar effects. The results suggest that BmOB could inhibit BEL-7402 cell proliferation, and the cell death may occur through the modulation of mitochondrial functions regulated by reactive oxygen species. It appears that collapse of DeltaPsim prior to intracellular reactive oxygen species arose during the cytotoxic process in our experimental system.     

Protective effect of the extracts from Cnidium officinale against oxidative damage induced by hydrogen peroxide via antioxidant effect

The dried rhizomes of Cnidium officinale are used as herbal drugs in the treatment of pain, inflammation, menstrual disturbance and antivitamin deficiency disease, and also act as a blood pressure depressant. In addition, there are several reports suggesting that they have pharmacological properties to tumor metastasis and angiogenesis, and that they act as an inhibitor of high glucose-induced proliferation of glomerular mesangial cells. However, little has been known about the functional role of the extracts from C. officinale on oxidative DNA damage and apoptosis caused by ROS. In this work, we have investigated the DPPH radical, hydroxyl radical and intracellular ROS scavenging capacity, and Fe²⁺ chelating activity of the extracts from C. officinale. In addition, we evaluated whether the extracts are capable of reducing H₂O₂-induced DNA and cell damage in the human skin fibroblast cell. These extracts showed a dose-dependent free-radical scavenging capacity and a protective effect on DNA damage and the lipid peroxidation causing the cell damage by ROS. These antioxidant activities and inhibitory effects of the extracts on DNA and cell damage may further explain that C. officinale is useful as a herbal medicine for cancer chemoprevention.     

Protective effect of methyl gallate from Toona sinensis (Meliaceae) against hydrogen peroxide-induced oxidative stress and DNA damage in MDCK cells.

Methyl gallate (MG) has been shown to be an effective antioxidant in a variety of acellular experiments. Accordingly, this study was designed to assess the ability of MG, extracting from Toona sinensis to protect cultured Madin-Darby canine kidney (MDCK) cells against hydrogen peroxide (H2O2)-mediated oxidative stress. Trolox, a cell permeable and water-soluble vitamin E analogue, was included for comparison. First, when MDCK cells were pretreated with MG and trolox for 1 h, followed by exposing to H2O2 (0.8 mM) for an additional hour, we found that the intracellular peroxide productions, as reflected by dichlorofluorescein (DCF) fluorescence, were shown to be decreased in a concentration-dependent manner. Furthermore, using C11-BODIPY581/591 as a lipid peroxidation probe, we also found that MG, in a concentration of 100 microM, could alleviate lipid peroxidation of the cells exposed to a short-term H2O2 treatment. In addition, MG-treated cells could prevent intracellular glutathione (GSH) from being depleted following an exposure of H2O2 (8.0 mM) for a 3 h period. Next, we also examined the effect of MG on H2O2-mediated oxidative damage to DNA. Using 8-oxoguanine as an indicator for oxidative DNA damage, we demonstrated that the percentage of MDCK cells containing 8-oxoguanine was drastically increased by exposing to H2O2 (40 mM) for 3 h. However, 8-oxoguanine contents were shown to be significantly decreased in the presence of MG prior to H2O2 exposure. Comparatively, MG was shown to be a better protective agent against oxidative damage to DNA as compared to trolox. Taken together, our data suggest that MG is effective in preventing H2O2-induced oxidative stress and DNA damage in MDCK cells. The underlying mechanisms involved scavenging of intracellular reactive oxygen species (ROS), inhibition of lipid peroxidation and prevention of intracellular GSH depletion.     

Akt-dependent heme oxygenase-1 induction by NS-398 in C6 glial cells: a potential role for CO in prevention of oxidative damage from hypoxia

We investigated whether increased heme oxygenase (HO)-1 activity by NS-398 is responsible for protection against hypoxia-induced damage in C6 cells. The expression of HO-1 was analyzed by Western blot and cell viability was analyzed by lactate dehydroxygease (LDH) activity. NS-398 increased HO-1 expression in a concentration- and time-dependent manner during both normoxia and hypoxia (95% N(2)/5% CO(2)), but the latter was much more sensitive. Because induction of HO-1 occurred due to hypoxia itself, NS-398 seemed to potentiate the expression of HO-1. The reduced cell viability due to hypoxia was significantly reversed by either NS-398 or [Ru(CO)(3)(Cl)(2)](2), a CO-donor. Zinc protophorphrin (ZnPPIX), a HO-1 inhibitor, inhibited the protective effect of NS-398 against hypoxia. Treatment with glucose oxidase (GOX, 20 mU/ml) increased ROS production and caused apoptotic death, as assayed by DCFH-DA and TUNEL, respectively. NS-398 significantly reduced GOX-induced cell death and ROS production; these effects were reversed by pre-treatment with oxyhemoglobin (HbO(2)), a CO/NO scavenger, or ZnPPIX. Finally, NS-398 increased PPAR-gamma luciferase activity in transiently PPAR-gamma transfected C6 cells, which was antagonized by ZnPPIX. NS-398 increased phosphorylation of Akt, and LY-294002, a specific PI(3) kinase inhibitor, inhibited NS-398-induced HO-1 expression. Taken together, we conclude that therapeutic use of NS-398 in the treatment of oxidative stress-oriented neuronal disorders would be beneficial through dual actions: HO-1 induction and COX-2 inhibition.     

Pu-erh black tea supplementation decreases quinocetone-induced ROS generation and oxidative DNA damage in Balb/c mice

Quinocetone (3-methyl-2-quinoxalinbenzenevinylketo-1,4-dioxide, QCT), a new feed antibacterial agent of quinoxaline-1,4-dioxides family, has been used as an animal growth promoter. However, few data about its potential toxicity in vivo were available. In this study, genotoxicity of QCT and the relationship with oxidative stress were investigated. Balb/c mice with both sexes were administrated with QCT (12000, 6000 and 3000 mg/kg/bw, respectively) by gavage acutely. DNA damage, generation of reactive oxygen species (ROS) and activity of antioxidative system (total antioxidative capacity, glutathione, glutathione peroxidase, superoxide dismutase and catalase) in liver and kidney were determined. Moreover, Pu-erh black tea extract (BTE) was co-administrated with QCT to evaluate its protective effect against QCT-induced genotoxicity. The DNA damage was observed in all the groups treated with single QCT except the liver with dose of 3000 mg/kg/bw. ROS was accumulated and antioxidative system was suppressed both in liver and kidney. However, the DNA damage, as well as the ROS, was decreased, while the activity of antioxidative system was increased in mice after co-administration of QCT and BTE. These data demonstrate that oxidative stress mediated the genotoxicity induced by QCT in vivo. Furthermore, this oxidative DNA damage can be attenuated by pre-supplementation of BTE.     

Induction of HepG2 cell apoptosis by Irgarol 1051 through mitochondrial dysfunction and oxidative stresses

In this study, HepG2 cells were exposed to 0.04–40 mg/L Irgarol 1051. Results show that Irgarol 1051 can damage cell morphology and cause a significant decrease in cell viability. Positive staining by Annexin V, caspase-3 activity enhancement, and the damage in cell ultrastructure indicated an apoptotic mode of cell death for 4.0 mg/L Irgarol 1051 treatment. At the same time, caspase-9 was also significantly induced by 0.4 and 4.0 mg/L Irgarol 1051 at 72 h, which suggests that the intrinsic mitochondria pathway was involved in the apoptosis. The mitochondrial membrane potential decreased significantly after the HepG2 cells were exposed to Irgarol 1051 for 6 and 72 h. Especially, the translocation of cytochrome c from mitochondria to cytosol was recorded, supporting the idea that the mitochondrial pathway was involved in the apoptosis signal pathways induced by Irgarol 1051. The significantly increased levels of intracellular reactive oxygen species (ROS) and an immediate ROS burst were also recorded. The results here may imply that Irgarol 1051 induces HepG2 cell apoptosis through mitochondrial dysfunction and oxidative stresses. Although it is possible that this chemical has no detrimental effects on human health at the environmentally relevant concentration, it may cause problems to top coastal predators due to bio-accumulation through the food chain.