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      Comet assay to measure DNA repair: approach and applications

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          Abstract

          Cellular repair enzymes remove virtually all DNA damage before it is fixed; repair therefore plays a crucial role in preventing cancer. Repair studied at the level of transcription correlates poorly with enzyme activity, and so assays of phenotype are needed. In a biochemical approach, substrate nucleoids containing specific DNA lesions are incubated with cell extract; repair enzymes in the extract induce breaks at damage sites; and the breaks are measured with the comet assay. The nature of the substrate lesions defines the repair pathway to be studied. This in vitro DNA repair assay has been modified for use in animal tissues, specifically to study the effects of aging and nutritional intervention on repair. Recently, the assay was applied to different strains of Drosophila melanogaster proficient and deficient in DNA repair. Most applications of the repair assay have been in human biomonitoring. Individual DNA repair activity may be a marker of cancer susceptibility; alternatively, high repair activity may result from induction of repair enzymes by exposure to DNA-damaging agents. Studies to date have examined effects of environment, nutrition, lifestyle, and occupation, in addition to clinical investigations.

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          Most cited references55

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          A simple technique for quantitation of low levels of DNA damage in individual cells.

          Human lymphocytes were either exposed to X-irradiation (25 to 200 rads) or treated with H2O2 (9.1 to 291 microM) at 4 degrees C and the extent of DNA migration was measured using a single-cell microgel electrophoresis technique under alkaline conditions. Both agents induced a significant increase in DNA migration, beginning at the lowest dose evaluated. Migration patterns were relatively homogeneous among cells exposed to X-rays but heterogeneous among cells treated with H2O2. An analysis of repair kinetics following exposure to 200 rads X-rays was conducted with lymphocytes obtained from three individuals. The bulk of the DNA repair occurred within the first 15 min, while all of the repair was essentially complete by 120 min after exposure. However, some cells demonstrated no repair during this incubation period while other cells demonstrated DNA migration patterns indicative of more damage than that induced by the initial irradiation with X-rays. This technique appears to be sensitive and useful for detecting damage and repair in single cells.
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            Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells.

            Mammalian cells were after irradiation suspended in melted agarose, and casted on microscope slides. The slides were after gelling at 0 degree C immersed in a neutral detergent solution which lysed the cells. A weak electric field (5 V/cm) was then applied over the gel for 5 minutes. The DNA in the gel was stained with the fluorescent dye acridine orange and gives a green emission in a microscope photometer. DNA had migrated towards the anode and this migration was more pronounced in irradiated than in control cells. The differences in migration pattern were quantitatively measured. The lower detection limit was below 0.5 Gy and a plateau in the dose-effect curve was reached at about 3 Gy. In repair experiments residual DNA damage could be observed after postirradiation incubation for 60 minutes. The advantages of the method is: no radioactive labelling and only a few number of cells is required.
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              Nutritional modulation of DNA repair in a human intervention study.

              DNA oxidation is a potential cause of cancer in humans. It is well-known that fruits and vegetables protect against cancer, and this may be in part because they contain antioxidants, which decrease the level of oxidation of DNA. However, there are other possible mechanisms, such as an enhancement of cellular repair of this damage. A randomized cross-over study was carried out on healthy human subjects, who were given kiwifruit as a supplement to their normal diet, for 3-week periods at different 'doses', with 2-week washout periods between doses. Endogenous oxidation of bases in lymphocyte DNA, and the resistance of the DNA to oxidation ex vivo, were assessed using single cell gel electrophoresis (the 'comet assay'). The capacity to repair DNA base oxidation was measured with an in vitro test, and levels of expression of repair-related genes OGG1 and APE1 were assessed by semi-quantitative RT-PCR. Concentrations of dietary antioxidants were measured in plasma. The antioxidant status of plasma and of lymphocytes was increased by consumption of kiwifruit. Levels of endogenous oxidation of pyrimidines and purines in DNA were markedly decreased, and DNA repair measured on a substrate containing 8-oxo-7,8-dihydroguanine was substantially increased (without change in levels of OGG1 or APE1 mRNA). The magnitude of these effects was generally not related to the number of kiwifruits consumed per day. Kiwifruit provides a dual protection against oxidative DNA damage, enhancing antioxidant levels and stimulating DNA repair. It is probable that together these effects would decrease the risk of mutagenic changes leading to cancer.
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                Author and article information

                Contributors
                Journal
                Front Genet
                Front Genet
                Front. Genet.
                Frontiers in Genetics
                Frontiers Media S.A.
                1664-8021
                03 July 2014
                25 August 2014
                2014
                : 5
                : 288
                Affiliations
                [1] 1Department of Pharmacology and Toxicology, Faculty of Pharmacy, University of Navarra Pamplona, Spain
                [2] 2Department of Molecular Biology of Cancer, Institute of Experimental Medicine, Academy of Science of the Czech Republic Prague, Czech Republic
                [3] 3Environmental Risk and Health Unit, Flemish Institute of Technological Research Mol, Belgium
                [4] 4Department of Genetics and Biotechnology, Animal and Veterinary Research Centre, University of Trás-os-Montes and Alto Douro Vila Real, Portugal
                [5] 5Department of Nutrition, University of Oslo Oslo, Norway
                Author notes

                Edited by: H. Steven Wiley, Pacific Northwest National Laboratory, USA

                Reviewed by: James M. Ford, Stanford University School of Medicine, USA; Wei Xu, Northwestern University, USA

                *Correspondence: Amaya Azqueta, Department of Pharmacology and Toxicology, Faculty of Pharmacy, University of Navarra, C/Irunlarrea 1, 31008 Pamplona, Spain e-mail: amazqueta@ 123456unav.es

                This article was submitted to Genomic Assay Technology, a section of the journal Frontiers in Genetics.

                Article
                10.3389/fgene.2014.00288
                4142706
                601ab075-1b9e-40b1-a098-a51e48ed7ba3
                Copyright © 2014 Azqueta, Slyskova, Langie, O’Neill Gaivão and Collins.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                History
                : 29 May 2014
                : 04 August 2014
                Page count
                Figures: 1, Tables: 1, Equations: 0, References: 61, Pages: 8, Words: 0
                Categories
                Genetics
                Review Article

                Genetics
                dna repair,animal studies,human biomonitoring,occupational studies,clinical studies,base excision repair (ber),nucleotide excision repair (ner),comet assay

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