Selenomethionine increases proliferation and reduces apoptosis in bovine mammary epithelial cells under oxidative stress

The decline in mammary epithelial cell number as lactation progresses may be due, in part, to oxidative stress. Selenium is an integral component of several antioxidant enzymes. The present study was conducted to examine the effect of oxidative stress and selenomethionine (SeMet) on morphology, viability, apoptosis, and proliferation of bovine mammary epithelial cells (BMEC) in primary culture. Cells were isolated from mammary glands of lactating dairy cows and grown for 3 d in a low-serum gel system containing lactogenic hormones and 0 or 100 μM H₂O₂ with 0, 10, 20, or 50 nM SeMet. Hydrogen peroxide stress increased intracellular H₂O₂ to 3 times control concentrations and induced a loss of cuboidal morphology, cell-cell contact, and viability of BMEC by 25%. Apoptotic cell number more than doubled during oxidative stress, but proliferating cell number was not affected. Supplementation with SeMet increased glutathione peroxidase activity 2-fold and restored intracellular H₂O₂ to control levels with a concomitant return of morphology and viability to normal. Apoptotic BMEC number was decreased 76% below control levels by SeMet and proliferating cell number was increased 4.2-fold. These findings suggest that SeMet modulated apoptosis and proliferation independently of a selenoprotein-mediated reduction of H₂O₂. In conclusion, SeMet supplementation protects BMEC from H₂O₂-induced apoptosis and increased proliferation and cell viability under conditions of oxidative stress.     

Cytoprotective effect of fucoxanthin isolated from brown algae <Emphasis Type="Italic">Sargassum siliquastrum</Emphasis> against H<Subscript>2</Subscript>O<Subscript>2</Subscript>-induced cell damage

In this study, the cytoprotective effect of fucoxanthin, which was isolated from Sargassum siliquastrum, against oxidative stress induced DNA damage was investigated. Fucoxanthin, a kind of carotenoid, was pretreated to the medium and the protective effect was evaluated via 2′,7′-dichlorodihydrofluorescein diacetate, 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide, and comet assays. Intracellular reactive oxygen species were over produced by addition of hydrogen peroxide (H₂O₂), but the production was significantly reduced by the treatment with fucoxanthin. The fucoxanthin strongly enhanced cell viability against H₂O₂ induced oxidative damage and the inhibitory effect of cell damage was a dose-dependent manner. Furthermore, a protective effect against oxidative stress-induced cell apoptosis was also demonstrated via nuclear staining with Hoechst dye. These results clearly indicate that fucoxanthin isolated from S. siliquastrum possesses prominent antioxidant activity against H₂O₂-mediated cell damage and which might be a potential therapeutic agent for treating or preventing several diseases implicated with oxidative stress.     

Catechin-7-O-β-d-glucopyranoside scavenges free radicals and protects human B lymphoma BJAB cells on H2O2-mediated oxidative stress

Catechin-7-O-β-d-glucopyranoside (CA-G) was previously isolated from red bean (the seed of Phaseolus calcaratus cv. Roxburgh). This study examined the ability of CA-G to scavenge reactive oxygen species generated by cell-free systems and to protect cells from oxidative stress caused by hydrogen peroxide (H₂O₂). The mechanism by which CA-G exerts its antioxidant and anti-apoptotic action on H₂O₂-exposed cells was also investigated. CA-G treatment prevented H₂O₂-mediated apoptosis and inhibited the formation of single stand breaks in DNA in H₂O₂-exposed BJAB cells. CA-G suppressed mitochondrial stress and caspase activation caused by H₂O₂. Mechanistic experiments revealed that the antioxidant mechanism of CA-G on H₂O₂-mediated oxidative damage was due to the direct scavenging of hydroxyl radicals and/or to the chelation of metal ions that were used to produce hydroxyl radicals from H₂O₂ via the Fenton reaction. Collectively, these findings suggest beneficial roles of CA-G or CA-G-rich red bean on the protection from oxidative damage.     

Evaluation of the Ability of Polyphenol Extracts of Cocoa and Red Grape to Promote the Antioxidant Response in Yeast Using a Rapid Multiwell Assay

Saccharomyces cerevisiae has been used as a model organism to study the capacity of cocoa and red grape extracts to trigger an antioxidant response. A methodology adapted to microtiter plates has been developed to monitor yeast growth after culture preincubation with food ingredients and exposure to oxidative stress by hydrogen peroxide and menadione. This methodology proved effective in measuring the ability of cocoa and red grape extracts to promote an antioxidant response in yeast, and also the prospect of conducting dose–response studies. Additionally, the method has proven useful to perform studies with mutant strains lacking genes that may be related to the mechanism of action underlying the antioxidant properties. Thus, in a single assay, it is possible to elucidate the sensitivity of strains to oxidative stress, the ability of an ingredient to promote an antioxidant response, and the possible implication of certain genes. Results of assays using strain hst3Δ showed that the antioxidant protection provided by exposure to cocoa and red grape extracts was not present in the strain lacking gene HST3 when H₂O₂ and menadione were used as oxidizing agents. This effect was previously reported for cocoa extract only, with H₂O₂ as stressor. Moreover, the results showed that the mutant strain hst3Δ is more resistant to menadione and H₂O₂ in the absence of preincubation with cocoa and red grape extract, hinting at the possible implication of sirtuin Hst3 in the antioxidant cellular response.     

Administration of rosemary essential oil enhances resistance of rat hepatocytes against DNA-damaging oxidative agents

The aim of this paper was to evaluate the potential DNA-protective effects of rosemary essential oil-supplementation on rat hepatocytes damaged with three different genotoxins attacking DNA by oxidative stress. Hydrogen peroxide (H₂O₂) reacts by the generation of hydroxyl radicals, visible light-excited methylene blue forms oxidative DNA lesions via singlet oxygen and 2,3-dimethoxy-1,4-naphthoquinone (DMNQ) induces oxidative stress by participation in redox cycling. Hepatocytes were isolated from rats supplemented with rosemary oil (RO) for 14 days as well as from control rats. The potential protective effects of RO applied in drinking water of animals were tested on the level of DNA using the conventional and modified single cell gel electrophoresis (comet assay). We found out that administration to rats of rosemary oil, exhibiting free radical-scavenging activity measured by DPPH assay, efficiently and significantly decreased the level of DNA damage induced with H₂O₂, visible light-excited methylene blue and DMNQ.     

Inhibitory effect of wheat bran feruloyl oligosaccharides on oxidative DNA damage in human lymphocytes

The present work assessed the protective effect of feruloyl oligosaccharides (FOs), the ferulic acid ester of oligosaccharides from wheat bran, against oxidative DNA damage in normal human peripheral blood lymphocytes induced by hydrogen peroxide (H₂O₂). The DNA damage was measured by using the single cell gel electrophoresis assay (comet assay). Lymphocytes were subjected to DNA damage by exposure to a range of H₂O₂ concentrations (10–200 μmol/l). H₂O₂, at a concentration of 200 μmol/l, resulted in nearly all cells being highly damaged. FOs showed no cytotoxicity and genotoxicity to normal human lymphocytes at the tested concentrations (10–500 μmol/l). In addition, DNA damage in human lymphocytes induced by 100 μmol/l H₂O₂ was inhibited by FOs in a concentration-dependent fashion with 91.1% inhibition of lymphocyte DNA damage at 500 μmol/l as compared with the control. The results suggest that water-soluble FOs from wheat bran are able to enhance the ability of human lymphocytes to resist H₂O₂ induced oxidative damage.     

Effect of polyphenolic compounds from Coriandrum sativum on H2O2-induced oxidative stress in human lymphocytes

Polyphenolic compounds are widely distributed in plants and known to be excellent antioxidants in vitro. They have the capacity to reduce free-radical formation by scavenging free radicals and protecting antioxidant defences. The present study evaluated the antioxidant potencies of polyphenolic compounds from a spice, Coriandrum sativum against hydrogen peroxide-induced oxidative damage in human lymphocytes. Pretreatment with polyphenolic rich fractions protected human lymphocytes against H₂O₂-induced oxidative damage. H₂O₂ treatment significantly decreased the activities of antioxidant enzymes, such as superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, glutathione-S-transferase and caused decreased glutathione content and increased thiobarbituric acid-reacting substances (TBARS). Treatment with polyphenolic fractions (50 μg/ml) increased the activities of antioxidant enzymes and glutathione content and reduced the levels of TBARS significantly. Observed reduction in the level of lipid peroxides showed a decreased tendency of peroxidative damage. We conclude that, under these experimental conditions, polyphenolic compounds effectively suppress hydrogen peroxide-induced oxidative stress.     

Effects of ethanol extracts from stalked sea squirt (Styela clava) on antioxidant potential,oxidative DNA damage and DNA repair

The aim of this study was to assess the total radical trapping antioxidant potential and antigenotoxic effects by comet assay of ethanol extracts of stalked sea squirt, Styela clava, (tunic, substrate, and whole). All extracts of stalked sea squirt effectively scavenged ABTS^{· +} in a dose dependent manner. Pretreatment with each extract of stalked sea squirt produced significant reductions in oxidative DNA damage at concentrations of 1–50 μg/mL, with whole extract of stalked sea squirt showing higher inhibition (16.1 μg/mL) of H₂O₂ induced DNA damage than substrate or tunic extracts based on ED₅₀ values. The addition of 50 μg/mL of stalked sea squirt extracts to human leukocytes after oxidative stimulus (200 μM H₂O₂) for 5 min positively influences the kinetics of DNA repair during 24 hr of incubation. These results indicate that the ethanol extracts of tunic, substrate, and whole stalked sea squirt have significant antioxidant activities that protect against oxidative DNA damage and improve DNA repair capacity.     

Antioxidant and antiproliferative activities of mango (Mangifera indica L.) flesh and peel

The antioxidant and antiproliferative properties of flesh and peel of mango (Mangifera indica L.) were investigated. The cytoprotective effect of mango flesh and peel extracts on oxidative damage induced by H₂O₂ in a human hepatoma cell line, HepG2, were determined, and the underlying mechanism was examined by a single-cell electrophoresis assay (comet assay). Treatment of HepG2 cell with mango peel extract prior to oxidative stress was found to inhibit DNA damage. The free radical scavenging activities of mango flesh and peel extracts were evaluated by electron spin resonance (ESR). The mango peel extract exhibited stronger free radical scavenging ability on 1,1-diphenyl-2-picrylhydrazyl (DPPH) and alkyl radicals than mango flesh extract, regardless of ripeness. Similarly, peel extract exhibited significant antiproliferative effect against all tested cancer cell lines, compared to that of flesh extract, in a dose-dependent manner. The result also showed that the antiproliferative activity of mango flesh and peel extracts correlated with their phenolic and flavonoid contents. Thus, mango peel, a major by-product obtained during the processing of mango product, exhibited good antioxidant activity and may serve as a potential source of phenolics with anticancer activity.     

Reduction of lipid peroxidation and H2O2‐mediated DNA damage by a red alga (Grateloupia filicina) methanolic extract

The antioxidant activity of an extract of Grateloupia filicina was evaluated using linoleic acid and fish oil as substrates in an induct period at 65 °C. Furthermore, the algal extract was subjected to comet assay to evaluate its protecting ability for H₂O₂‐induced DNA damage in rat lymphocytes. Progression of oxidation was examined using weight gain, peroxide value (PV), 2‐thiobarbituric acid reactive substances and conjugated diene data. Effectiveness of the extract at 0.03 and 0.05% levels was superior to that of α‐tocopherol at 0.01% and the activity of 0.05% extract was higher than that of butylated hydroxyanisole and butylated hydroxytoluene at 0.01%. Also, the algal extract significantly inhibited H₂O₂ induced DNA damage in comet assay. The maximum DNA damage inhibition (68.9%) was recorded from the 50 µg ml⁻¹ alga extract when the rat lymphocyte cells were treated with 50 µM H₂O₂. Copyright © 2005 Society of Chemical Industry     

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.     

葡萄籽粗多糖的体内外抗氧化作用

为了研究葡萄籽粗多糖(crude polysaccharides from grape seeds,GSCPs)体外抗氧化作用及对秀丽隐杆线虫的体内抗氧化作用,采用水提醇沉法提取GSCPs,检测GSCPs对1,1-二苯基-2-三硝基苯肼(1,1-diphenyl-2-picrylhydrazyl,DPPH)自由基和羟自由基的清除作用及对DNA氧化损伤的抑制作用;建立RAW 264.7巨噬细胞氧化损伤模型,在细胞水平探讨GSCPs的抗氧化能力;同时利用秀丽隐杆线虫研究GSCPs的体内抗氧化功能。体外实验结果表明:GSCPs可有效清除自由基,抑制DNA的氧化损伤,质量浓度为0.4mg/mL的GSCPs对DPPH自由基的清除率达84%,对羟自由基清除率为89%;GSCPs可以下调H₂O₂诱导的RAW 264.7巨噬细胞活性氧(reactive oxygen species,ROS)水平,正向调节细胞内超氧化物歧化酶(superoxide dismutase,SOD)和谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-Px)活性,质量浓度为0.2mg/mL的GSCPs处理组可使细胞内SOD活性从H₂O₂处理组的81.1%升至96.3%,GSH-Px活性从H₂O₂处理组的92.1%升至99.6%。此外,GSCPs可延长秀丽隐杆线虫寿命,提高其对抗急性氧化应激的能力,有效清除秀丽隐杆线虫体内的ROS。质量浓度为0.8mg/mL GSCPs处理组秀丽隐杆线虫的平均寿命较对照组延长27.67%,将急性氧化应激的秀丽隐杆线虫平均存活时间延长33.58%,秀丽隐杆线虫体内ROS生成量较对照组可降低56.33%。因此,葡萄籽粗多糖在体内外均表现出良好的抗氧化性,可用于抗氧化功能产品的开发。     

Essential rosemary oil protects testicular cells against DNA-damaging effects of H2O2 and DMNQ

Rosemary oil (RO) is popular in the Mediterranean region as a culinary additive which has the ability to protect delicate organs such as liver, brain and heart. We examined the effect of RO consumption on resistance of rat testicular cells (TCs) against DNA-damaging effects of the oxidative agents H₂O₂ and DMNQ and on the activity of the antioxidant enzymes glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD). DNA lesions were detected by conventional and modified comet assay and the activities of GSH-Px and SOD were measured spectrophotometrically. Since TCs represent a mixture of haploid, diploid and tetraploid cells, we used flow cytometry for their differentiation and calculation of DNA-damaging effects of H₂O₂ and DMNQ in cells of different ploidy. The results showed that the oxidative DNA lesions were significantly reduced in TCs from rats administered RO; however, the activity of antioxidant enzymes did not differ in TCs from control and RO-supplemented rats.     

Effect of heat-shock protein B7 on oxidative stress in adipocytes from preruminant calves

Dairy cows with ketosis display excessive lipolysis in adipose tissue. Heat-shock protein B7 (HSPB7), a small heat-shock protein, plays important roles in mediating cytoprotective responses to oxidative stress in rodent adipose tissue. Accordingly, it is assumed that HSPB7 may also play important roles in the antioxidant response in adipose tissue of ketotic cows. Therefore, the aim of this study is to investigate (1) the redox state of adipose tissue in ketotic cows and (2) the role and mechanism of HSPB7 on the regulation of oxidative stress in adipocytes from preruminant calves. An in vivo study consisting of 15 healthy and 15 clinically ketotic cows was performed to harvest subcutaneous adipose tissue and blood samples. In addition, adipocytes isolated from calves were treated with different concentrations of H₂O₂ (0, 12.5, 25, 50, 100, or 200 μM) for 2 h, transfected with adenovirus-mediated overexpression of HSPB7 for 48 h, or transfected with small interfering RNA of HSPB7 for 48 h followed by exposure to H₂O₂ (200 μM) for 2 h. Serum concentrations of nonesterified fatty acids and β-hydroxybutyrate were greater in cows with clinical ketosis, whereas serum concentration of glucose was lower. Compared with healthy cows, the malondialdehyde content was greater but the activity of glutathione peroxidase and superoxide dismutase was lower in adipose tissue of clinically ketotic cows. The abundance of HSPB7 and nuclear factor, erythroid 2 like 2 (NFE2L2) was greater in adipose tissue of clinically ketotic cows. In vitro, H₂O₂ treatment induced the overproduction of reactive oxygen species and malondialdehyde, and inhibited the activity of antioxidant enzymes glutathione peroxidase and superoxide dismutase in adipocytes from preruminant calves. The low concentration of H₂O₂ (12.5, 25, and 50 μM) increased the abundance of HSPB7 and NFE2L2, but high concentrations of H₂O₂ (100 or 200 μM) reduced the abundance of HSPB7 and NFE2L2. The overexpression of HSPB7 improved the H₂O₂-induced oxidative stress in adipocytes via increasing the abundance of NFE2L2 and its downstream target genes heme oxygenase-1 (HMOX1) and NADH quinone oxidoreductase 1 (NQO1). Knockdown of HSPB7 markedly inhibited the expression of NFE2L2, HMOX1, and NQO1 and further exacerbated H₂O₂-induced oxidative stress. Overall, these results indicate that activation of the HSPB7-NFE2L2 pathway increases cellular antioxidant capacity, thereby alleviating oxidative stress in bovine adipocytes.     

Comparison between conjugated linoleic acid and essential fatty acids in preventing oxidative stress in bovine mammary epithelial cells

Some in vitro and in vivo studies have demonstrated protective effects of conjugated linoleic acid (CLA) isomers against oxidative stress and lipid peroxidation. However, only a few and conflicting studies have been conducted showing the antioxidant potential of essential fatty acids. The objectives of the study were to compare the effects of CLA to other essential fatty acids on the thiol redox status of bovine mammary epithelia cells (BME-UV1) and their protective role against oxidative damage on the mammary gland by an in vitro study. The BME-UV1 cells were treated with complete medium containing 50 μM of cis-9,trans-11 CLA, trans-10,cis-12 CLA, α-linolenic acid, γ-linolenic acid, and linoleic acid. To assess the cellular antioxidant response, glutathione, NADPH, and γ-glutamyl-cysteine ligase activity were measured 48 h after addition of fatty acids (FA). Intracellular reactive oxygen species and malondialdehyde production were also assessed in cells supplemented with FA. Reactive oxygen species production after 3 h of H₂O₂ exposure was assessed to evaluate and to compare the potential protection of different FA against H₂O₂-induced oxidative stress. All FA treatments induced an intracellular GSH increase, matched by high concentrations of NADPH and an increase of γ-glutamyl-cysteine ligase activity. Cells supplemented with FA showed a reduction in intracellular malondialdehyde levels. In particular, CLA isomers and linoleic acid supplementation showed a better antioxidant cellular response against oxidative damage induced by H₂O₂ compared with other FA.     

Antioxidant activities of phlorotannins purified from Ecklonia cava on free radical scavenging using ESR and H2O2-mediated DNA damage

The potential antioxidant activities of three phlorotannins (phloroglucinol, eckol and dieckol) purified from Ecklonia cava collected in Jeju Island were investigated to evaluate their potential value as the natural products for foods or cosmetic application. In this study, antioxidant activities were measured by electron spin resonance spectrometry (ESR) technique for scavenging effects of free radicals such as 1,1-diphenyl-2-picrylhydrazyl (DPPH), alkyl, hydroxyl (HO^•) and superoxide anion radical (O₂ ^•−) and by comet assay for protecting effects against H₂O₂-mediated DNA damage. The results show that all the phlorotannins have the potential DPPH, alkyl, hydroxyl and superoxide radical scavenging activities. Especially, eckol samples scavenged around 93% of DPPH at 0.25, 0.5, 1 mg/mL of concentrations and were higher than the other phlorotannins, such as phloroglucinol and dieckol samples. Also, protecting effects of the phlorotannins against H₂O₂-mediated DNA damage increased with increased concentrations of the samples in the L5178 mouse T-cell lymphoma cell lines (L5178Y-R). In conclusion, these results suggest that the three phlorotannins purified from E. cava have the potential inhibitory effect on H₂O₂-mediated DNA damage and harmful free radicals and can be used as antioxidants in cosmetic, foods and drug industry.     

Inhibitory effect of the ethyl acetate fraction from astringent persimmon on H2O2-induced oxidative stress in HepG2 cells

Persimmon cv. Sangjudungsi (Diospyros kaki Thunb.) is a major astringent cultivar in Korea. A phenolic extract of cv. Sangjudungsi was obtained using acetone with homogenization, then sequentially fractionated into n-hexane, chloroform, ethyl acetate, n-butanol, and water fractions. These 5 fractions were used to evaluate levels of total phenolics and total flavonoids, and the antioxidant capacities, and to investigate whether the fractions protected human hepatoma HepG2 cells from deleterious oxidative stress. The ethyl acetate fraction had the highest levels of total phenolics and total flavonoids, and the greatest antioxidant capacity. Under oxidative stress caused by H₂O₂, the ethyl acetate fraction at non-toxic concentrations significantly (p<0.05) restored the viability of HepG2 cells in a dose-dependent manner, compared with a control. The ethyl acetate fraction also alleviated intracellular oxidative stress, possibly due to effective antioxidant activities in cells. Astringent persimmons are a good source of antioxidants for reduction of oxidative stress.     

Bax induces activation of the unfolded protein response by inducing HAC1 mRNA splicing in Saccharomyces cerevisiae

Bax, a multidomain pro‐apoptotic Bcl‐2 protein, localizes to the endoplasmic reticulum (ER), where it regulates ER stress‐induced apoptosis. Adaptation to ER stress depends on the activation of an integrated signal transduction pathway known as the unfolded protein response (UPR). This study examined the death‐inducing activity of Bax and its ability to induce UPR signalling pathways in yeast. We observed that inhibition of global translation in yeast cells expressing Bax correlated with Bax‐induced cell death. Using a lacZ reporter containing several UPR cis‐activating regulatory elements, we also found that Bax directly activated the UPR. Furthermore, this correlated with the splicing of HAC1 mRNA, a gene involved in UPR activation. Bax induced expression of representative UPR target genes such as KAR2, DER1 and GCN4. Finally, we found that Ire1p function is critical for Bax‐induced cell death. Copyright © 2012 John Wiley & Sons, Ltd.