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Single-cell Gel Electrophoresis (single-cell + gel_electrophoresis)
Selected AbstractsDNA damage and repair measurements from cryopreserved lymphocytes without cell culture,A reproducible assay for intervention studiesENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 7 2006Jyh-Lurn Chang Abstract Single-cell gel electrophoresis (the Comet assay) can be used to measure DNA damage and DNA repair capacity (DRC). However, to test DRC of cryopreserved lymphocytes, published methods include steps for cell culturing and phytohemagglutinin stimulation, which may limit use of this assay in intervention studies. We developed a modified Comet assay protocol that allows us to measure DRC from cryopreserved lymphocytes without these in vitro manipulations. Assay reproducibility was evaluated by performing the assay six times on different dates using six aliquots from one blood draw of one individual. The interindividual variation was assessed by performing the assay using one aliquot from six individuals. When ,-irradiation was used as the mutagen, intra-assay coefficients of variation (CVs.) for baseline DNA damage, damage after ,-irradiation exposure, and DRC,measured as tail moment,were 8, 31, and 10%, respectively. Interindividual CVs. were higher. When H2O2 was used as the mutagen, intra-assay CVs. for damage measurements were lower for a protocol modification that included damage and repair at 37°C (CVs. ranging from 8 to 35%) than for the more standard 4°C protocol. Analyzing moment arm,the average distance of DNA migration within the tail,yielded similar results. DNA repair was successfully detected in each experiment. Comparing freshly isolated lymphocytes to cryopreserved lymphocytes from the same individuals' blood draw indicated that DRC was highly correlated when determined using moment arm values. This modified protocol extends the use of the Comet assay to measuring DRC in intervention studies (e.g., dietary interventions) in that it assesses cellular response after cryopreservation without cell culture or other extensive manipulation. Environ. Mol. Mutagen., 2006. © 2006 Wiley-Liss, Inc. [source] The relationship between obesity and markers of oxidative stress in dogsJOURNAL OF ANIMAL PHYSIOLOGY AND NUTRITION, Issue 2 2009M. G. Cline Obesity, a serious epidemic affecting much of our pet population, increases the risk of developing numerous diseases. It has been demonstrated that obesity increases oxidative stress in obese children, cats and other species. Oxidative stress can result in DNA damage with subsequent alterations in gene expression, cell signaling, mutations, cell death or cell transformation. These effects of oxidative damage predispose animals and humans to numerous disease processes and cancer. The objective of the study was to demonstrate that obese dogs are under oxidative stress resulting in DNA damage and decreased endogenous antioxidant protection measured by serum glutathione levels and the ratio of reduced (GSH) to oxidized (GSSG) glutathione. In this case,control study, 10 obese dogs were compared with aged-matched healthy control dogs. Dogs with BCS of 7 or greater (9 pt scale) were considered obese. Dogs were evaluated by history, physical exam, body condition score, CBC, serum biochemical analysis and total T4, with both groups showing no significant differences in CBC, serum biochemical or T4 analysis. Single-cell gel electrophoresis (Comet assay) was used to measure DNA damage, and high performance liquid chromatography was used to measure serum glutathione. Reduced glutathione levels were significantly higher in the obese group (p = 0.012). The results of this pilot study suggest that obesity is associated with an increase in antioxidant potential, therefore justifying a larger study with antioxidant supplementation to determine how antioxidants in weight loss diets effects endogenous antioxidant capabilities. [source] Single-cell gel electrophoresis: a tool for investigation of DNA protection or damage mediated by dietary antioxidantsJOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 13 2007Yim Tong Szeto Abstract One method of assessing DNA damage is the comet assay, which was developed in 1988. The comet assay enables the detection of DNA strand breaks in individual cells. This test has also been used to study the in vitro and in vivo genotoxic or genoprotective effects of certain agents such as dietary antioxidants. This paper aims to consolidate the antioxidant and pro-oxidant effects of a series of dietary agents which have been evaluated by comet assay. Copyright © 2007 Society of Chemical Industry [source] DNA damage in mice treated with sulfur dioxide by inhalationENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 3 2005Ziqiang Meng Abstract Sulfur dioxide (SO2) is a ubiquitous air pollutant produced by the burning of fossil fuels. In this study, single-cell gel electrophoresis (the Comet assay) was used to evaluate the DNA damage produced by inhalation exposure of mice to SO2. Male and female mice were housed in exposure chambers and treated with 14.00 ± 1.25, 28.00 ± 1.98, 56.00 ± 3.11, and 112.00 ± 3.69 mg/m3 SO2 for 6 hr/day for 7 days, while control groups were exposed to filtered air. Comet assays were performed on blood lymphocytes and cells from the brain, lung, liver, spleen, kidney, intestine, and testicles of the animals. SO2 caused significant, dose-dependent increases in DNA damage, as measured by Olive tail moment, in all the cell types analyzed from both sexes of mice. The results indicate that inhalation exposure to SO2 damages the DNA of multiple organs in addition to the lung, and suggests that this damage could result in mutation, cancer, and other diseases related to DNA damage. Further work will be required to understand the ultimate toxicological significance of this damage. These data also suggest that detecting DNA damage in blood lymphocytes, using the Comet assay, may serve as a useful tool for evaluating the impact of pulmonary SO2 exposure in human biomonitoring studies. Environ. Mol. Mutagen., 2005. © 2005 Wiley-Liss, Inc. [source] DNA damage assessment by comet assay of human lymphocytes exposed to jet propulsion fuelsENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 1 2002Shawna M. Jackman Abstract Exposure to jet fuel damages DNA and results in a number of physiological changes in liver, lung, immune, and neurological tissue. In this study the single-cell gel electrophoresis assay or comet assay was used to compare the DNA damage in human peripheral lymphocytes produced by three jet propulsion fuels: JP-8, JP-5, and JP-8+100. These fuels consist of complex mixtures of aliphatic, aromatic, and substituted naphthalene hydrocarbons. Two exposure times were investigated which correspond to estimated occupational exposure times and concentrations of fuels were used that were based on previous fuel toxicity studies. Analysis of samples for the extent of DNA damage as determined by tail moment and percent tail DNA was performed on exposed cells following a brief recovery time. All fuels produced significant increases in DNA damage; however, only JP-8+100 was genotoxic at the lowest exposure concentration (1:500). At the highest exposure concentration (1:75), the mean tail moments for JP-8 and JP-8+100 (32.041 ± 2.599 and 45.774 ± 4.743, respectively) were significantly greater than for JP-5 (1.314 ± 0.474). These results indicate that JP-8+100 is the most potent inducer of DNA damage in human peripheral lymphocytes and that both JP-8+100 and JP-8 are capable of damaging lymphocyte DNA to a greater extent than JP-5. Environ. Mol. Mutagen. 40:18,23, 2002. © 2002 Wiley-Liss, Inc. [source] Genotoxicity related to transfer of oil spill pollutants from mussels to mammals via foodENVIRONMENTAL TOXICOLOGY, Issue 4 2004Sébastien Lemiere Abstract Heavy fuel oils containing high levels of polycyclic aromatic hydrocarbons (PAHs) were released into the marine environment after the Erika oil spill on the Atlantic coast. As highly condensed PAH pollutants can bioaccumulate in invertebrates, their transfer to vertebrates through the food chain was of concern. This study aimed to estimate potential genotoxic effects in rats fed for 2 or 4 weeks with the marine mussel Mytilus edulis contaminated by oil pollutants. Two levels of PAH contamination were studied, around 100 and 500 ,g of total PAHs/kg dry weight (d.w.) in mussels. Genotoxic damage in rats was investigated by single-cell gel electrophoresis (Comet assay) and micronucleus assays in liver, bone marrow, and peripheral blood. DNA damage was observed in the liver of rats fed with the most contaminated mussels (500 ,g PAHs/kg d.w.).DNA damage also was observed in the bone marrow but less than that in the liver. A small increase in micronuclei frequency was registered as well. This work underlines the bioavailability of pollutants in fuel-oil-contaminated mussels to consumers and the usefulness of the Comet assay as a sensitive tool in biomonitoring to analyze responses to PAH transfer in food. The occurrence of substituted PAHs and related compounds such as benzothiophenes in addition to nonsubstituted PAHs in fuel oils and mussels raised the question of whether they were implicated in the genotoxic effects registered in rats. © 2004 Wiley Periodicals, Inc. Environ Toxicol 19: 387,395, 2004. [source] Oral malodorous compound causes apoptosis and genomic DNA damage in human gingival fibroblastsJOURNAL OF PERIODONTAL RESEARCH, Issue 4 2008K. Yaegaki Background and Objective:, Volatile sulfur compounds are the main cause of halitosis. Hydrogen sulfide is one of these volatile sulfur compounds and the principal malodorous compound in physiological halitosis. Periodontally pathogenic activities of hydrogen sulfide have been previously reported. Hydrogen sulfide induces apoptotic cell death in aorta smooth muscle cells and in other tissues. Apoptosis plays an important role in the onset and progress of periodontitis. The objective of this study was to determine whether hydrogen sulfide causes apoptosis in human gingival fibroblasts. Material and methods:, Necrotic cells were detected using a lactate dehydrogenase assay. Apoptosis was ascertained using a histone-complexed DNA fragment assay and flow cytometry. The level of caspase 3, a key enzyme in apoptotic signaling, was also measured, and the effects of hydrogen sulfide on reactive oxygen species and superoxide dismutase were assessed. DNA damage caused by hydrogen sulfide was examined by means of single-cell gel electrophoresis. Results:, After 72 h of incubation with 100 ng/mL of hydrogen sulfide, necrosis was found in less than 10% of human gingival fibroblasts, whereas apoptosis was significantly increased (p < 0.05). Superoxide dismutase activity was strongly inhibited, and reactive oxygen species production was enhanced, after 48 and 72 h of incubation. Caspase 3 activity was also increased after 72 h of incubation (p < 0.01). Tail length, percentage of DNA in tail, and tail moment, measured by single-cell gel electrophoresis, were also intensified after 72 h of incubation (p < 0.001). Conclusion:, Hydrogen sulfide caused apoptosis and DNA damage in human gingival fibroblasts. An increased level of reactive oxygen species stimulated by hydrogen sulfide may induce apoptosis and DNA strand breaks. [source] Ethanol Can Modify the Effects of Certain Free Radical-Generating Systems on AstrocytesALCOHOLISM, Issue 4 2004B. Gonthier Abstract: The central nervous system is vulnerable to oxidative stress, especially when a toxicant can modify the physiological balance between anti- and pro-oxidant mechanisms. Among brain cells, astrocytes seem less vulnerable than neurons, but their impairment can dramatically affect neurons because of their protective role toward neurons. Ethanol is able to stimulate the formation of reactive oxygen species and modify the activity of most of the antioxidant agents. However, ethanol can react with the OH· radical to form the ,-hydroxyethyl radical, which is considered to be less toxic. Ethanol also can stimulate H2O2 degradation through catalase activation. This study, therefore, sought to determine whether ethanol affected the sensitivity of astrocytes exposed to various free radical-generating systems. The cellular impact of such exposure was assessed by assays exploring cytotoxicity (i.e., NR (neutral red) and MMT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetiazolium bromide) reduction assays) and genotoxicity (comet assay) induced by these treatments. DNA alterations were evaluated by single-cell gel electrophoresis (comet assay), considered a precocious biomarker of intracellular alterations. After concomitant exposure to H2O2 and ethanol, the viability of astrocytes decreased significantly whereas the mean percentage of DNA in the tail increased, reflecting DNA damage (H2O2 was either directly added to the culture medium or endogenously produced from menadione). Ethanol also reduced the loss of viability and DNA alterations after exposure to OH· radicals produced by a Fenton system. The exposure to a xanthine/xanthine oxidase system had the same effect. [source] Proapoptotic Nitric Oxide Production in Amyloid , Protein-Treated Cerebral Microvascular Endothelial CellsMICROCIRCULATION, Issue 2 2007CHIWAKA KIMURA ABSTRACT Objective: The objective of this study was to investigate the effects of amyloid , protein (A,) on cerebral microvascular endothelium, and their possible involvement in A,-induced apoptosis in the neighboring cells. Methods: Cultured bovine brain microvascular endothelial cells (BBECs) were incubated with A, for 24 h. Production of nitric oxide (NO) was assessed by nitric oxide-sensitive fluorescent dye, DAF-2, and the expression of NO synthase (NOS) proteins was examined by Western blotting. Effects of A,-treated microvascular endothelium on the DNA damage of the neighboring cells were assessed by single-cell gel electrophoresis. Results: A, increased the expression of iNOS protein, but did not affect eNOS and nNOS expressions in BBECs. A,-treated BBECs showed spontaneous NO production in the presence of L-arginine. The neural cell line PC12 showed marked apoptosis after being co-cultured with A,-treated BBECs for 48 h, and the apoptosis was as potent as that induced by the inflammatory stimuli lipopolysaccharide and interferon-,. The DNA damage of PC12 cells evoked by co-culture with A,-treated BBECs was prevented by L- NG -nitroarginine methyl ester, an inhibitor of NOS. Conclusions: These results indicate that A, induces the expression of iNOS in BBECs, and that microvascular endothelium-derived NO may induce apoptosis in neighboring neural cells. [source] Effect of Bupleuri Radix Extracts on the Toxicity of 5-Fluorouracil in HepG2 Hepatoma Cells and Normal Human LymphocytesBASIC AND CLINICAL PHARMACOLOGY & TOXICOLOGY, Issue 4 2008Su Jin Kang We sought to assess whether Bupleuri Radix extract enhances 5-fluorouracil-induced cytotoxicity in HepG2 hepatoma cells, while protecting normal blood lymphocytes. Bupleuri Radix, used for treatment of liver disease in oriental medicine, possesses antitumour properties; it induces apoptosis through cell arrest in tumour cells, but does not affect normal lymphocytes. In this study, we evaluated the protective and enhancing effects of Bupleuri Radix on 5-fluorouracil-induced cytotoxicity in HepG2 cells and normal lymphocytes. Treatment with Bupleuri Radix increased the micronuclei frequency and DNA damage, resulting from 5-fluorouracil treatment. However, when human lymphocytes were cotreated with Bupleuri Radix and 5-fluorouracil, the frequency of 5-fluorouracil-induced micronuclei decreased. Although the extent of 5-fluorouracil-induced DNA damage, determined by single-cell gel electrophoresis, increased after treating HepG2 cells with Bupleuri Radix, it decreased in normal lymphocytes. When cells were treated with 20 µM 5-fluorouracil and 200 µg/ml Bupleuri Radix simultaneously, Bax protein increased in HepG2 cells at 24 hr; however, p21 and p53 proteins were up-regulated in normal human lymphocytes. Cotreatment with 200 µg/ml Bupleuri Radix and 20 µM 5-fluorouracil resulted in cell arrest at the late G1/early S phase in HepG2 cells (55.80 ± 0.19%) and normal lymphocytes (97.19 ± 0.27%). In addition, Bupleuri Radix and 5-fluorouracil treatment increased mitochondria membrane potential collapse only in HepG2 cells (19.02%), while it was not changed in lymphocytes. In conclusion, our findings suggest that Bupleuri Radix may be effective as a therapeutic agent to treat hepatomas. [source] A combination of soy isoflavone supplementation and exercise improves lipid profiles and protects antioxidant defense-systems against exercise-induced oxidative stress in ovariectomized ratsBIOFACTORS, Issue 4 2007Hea Young Oh Abstract Menopause is often accompanied with weight gain, metabolic lipid abnormalities, and oxidative stress. In this study, we investigated the combined effects of exercise and soy isoflavone supplemention on the lipid profiles and antioxidant capacities of ovariectomized rats. Twenty-five female Sprague-Dawley rats were divided into 5 groups: sham-operated, ovariectomized (OVX), OVX with exercise (OVX + EX), OVX with soy isoflavone supplementation (OVX + ISO), and OVX with both soy isoflavones and exercise (OVX + ISO + EX). After 12 weeks of intervention, antioxidant status was evaluated in collected blood samples by the ferric reducing ability of plasma (FRAP), glutathione (GSH) content, and sodium oxide dismutase (SOD) activity. DNA damage in the lymphocytes was determined using alkaline single-cell gel electrophoresis (the Comet assay). Although there were no significant differences in weight gain and food intake, weight gain was lower in OVX + EX, OVX + ISO, and OVX + ISO + EX than in OVX. OVX + EX, OVX + ISO, and OVX + ISO + EX showed a significant decrease in total cholesterol, triglycerides, and LDL-cholesterol compared to OVX. The soy isoflavone supplemented group had significantly increased FRAP values and GSH contents in contrast to no changes in the exercised group, whereas exercise markedly increased SOD activity and H2O2 -induced DNA tail length and tail moment. Exercise with soy isoflavone supplementation significantly increased FRAP values and had no difference on SOD activity, including DNA damage. These results demonstrate that a combined treatment of moderate exercise and soy isoflavone supplementation could exert a beneficial effect on weight control and lipid profiles, and offer protection from exercise-induced oxidative stress in postmenopausal women. [source] | |||||||||||||