Comet assay to measure DNA repair: approach and applications

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.

[1]  Chung-Chu Chen,et al.  Is the Oxidative DNA Damage Level of Human Lymphocyte Correlated with the Antioxidant Capacity of Serum or the Base Excision Repair Activity of Lymphocyte? , 2013, Oxidative medicine and cellular longevity.

[2]  K. Brauers,et al.  Development and validation of a modified comet assay to phenotypically assess nucleotide excision repair. , 2006, Mutagenesis.

[3]  Paloma Martin,et al.  Differences in Repair of DNA Cross-links between Lymphocytes and Epithelial Tumor Cells from Colon Cancer Patients Measured In vitro with the Comet Assay , 2009, Clinical Cancer Research.

[4]  J. Mathers,et al.  Maternal folate depletion and high‐fat feeding from weaning affects DNA methylation and DNA repair in brain of adult offspring , 2013, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[5]  Sabine A S Langie,et al.  Measuring DNA repair incision activity of mouse tissue extracts towards singlet oxygen-induced DNA damage: a comet-based in vitro repair assay. , 2011, Mutagenesis.

[6]  A. Berrebi,et al.  Development of an enzymatic DNA repair assay for molecular epidemiology studies: distribution of OGG activity in healthy individuals. , 2007, DNA repair.

[7]  July , 1890, The Hospital.

[8]  A. Collins,et al.  Comet assay-based methods for measuring DNA repair in vitro; estimates of inter- and intra-individual variation , 2009, Cell Biology and Toxicology.

[9]  A. Collins,et al.  Single-cell gel electrophoresis applied to the analysis of UV-C damage and its repair in human cells. , 1992, International journal of radiation biology.

[10]  R. Tice,et al.  A simple technique for quantitation of low levels of DNA damage in individual cells. , 1988, Experimental cell research.

[11]  A. Collins,et al.  Both genetic and dietary factors underlie individual differences in DNA damage levels and DNA repair capacity. , 2014, DNA Repair.

[12]  A. Collins,et al.  In vitro repair of oxidative and ultraviolet-induced DNA damage in supercoiled nucleoid DNA by human cell extract. , 1994, Biochimica et biophysica acta.

[13]  J. Mathers,et al.  Dietary and genetic modulation of DNA repair in healthy human adults , 2007, Proceedings of the Nutrition Society.

[14]  R. Godschalk,et al.  The effect of oxidative stress on nucleotide-excision repair in colon tissue of newborn piglets. , 2010, Mutation research.

[15]  Amaya Azqueta,et al.  Measurement of DNA base and nucleotide excision repair activities in mammalian cells and tissues using the comet assay--a methodological overview. , 2013, DNA repair.

[16]  Remo Guidieri.  Res , 1995, RES: Anthropology and Aesthetics.

[17]  A. Collins,et al.  Combining fluorescent in situ hybridization with the comet assay for targeted examination of DNA damage and repair. , 2011, Methods in molecular biology.

[18]  W. V. Berghe,et al.  Redox and epigenetic regulation of the APE1 gene in the hippocampus of piglets: The effect of early life exposures. , 2014, DNA repair.

[19]  A. Collins,et al.  Protective effects of ursolic acid and luteolin against oxidative DNA damage include enhancement of DNA repair in Caco-2 cells. , 2010, Mutation research.

[20]  J. Mathers,et al.  Tissue differences in BER-related incision activity and non-specific nuclease activity as measured by the comet assay. , 2013, Mutagenesis.

[21]  P. Hanawalt,et al.  Comet-FISH with strand-specific probes reveals transcription-coupled repair of 8-oxoGuanine in human cells , 2013, Nucleic acids research.

[22]  A. Collins,et al.  Functional evaluation of DNA repair in human biopsies and their relation to other cellular biomarkers , 2014, Front. Genet..

[23]  Andrew R Collins,et al.  Nutritional modulation of DNA repair in a human intervention study. , 2003, Carcinogenesis.

[24]  A. Collins,et al.  DNA repair as a biomarker in human biomonitoring studies; further applications of the comet assay. , 2012, Mutation research.

[25]  M. Ruchirawat,et al.  Challenge assay: A functional biomarker for exposure-induced DNA repair deficiency and for risk of cancer. , 2010, International journal of hygiene and environmental health.

[26]  A. Collins,et al.  Vitamin C in Cultured Human (HeLa) Cells: Lack of Effect on DNA Protection and Repair , 2013, Nutrients.

[27]  J. Mathers,et al.  Inter-individual variation in DNA damage and base excision repair in young, healthy non-smokers: effects of dietary supplementation and genotype , 2010, British Journal of Nutrition.

[28]  A. Collins,et al.  Supplementation of a western diet with golden kiwifruits (Actinidia chinensis var.'Hort 16A':) effects on biomarkers of oxidation damage and antioxidant protection , 2011, Nutrition journal.

[29]  A. Naccarati,et al.  Functional, Genetic, and Epigenetic Aspects of Base and Nucleotide Excision Repair in Colorectal Carcinomas , 2012, Clinical Cancer Research.

[30]  R. Malekzadeh,et al.  Variation in PAH‐related DNA adduct levels among non‐smokers: The role of multiple genetic polymorphisms and nucleotide excision repair phenotype , 2013, International journal of cancer.

[31]  K Fujikawa,et al.  [Oxidative DNA damage]. , 2001, Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme.

[32]  A. Naccarati,et al.  Relationship between the capacity to repair 8-oxoguanine, biomarkers of genotoxicity and individual susceptibility in styrene-exposed workers. , 2007, Mutation research.

[33]  A. Collins,et al.  The carotenoid beta-cryptoxanthin stimulates the repair of DNA oxidation damage in addition to acting as an antioxidant in human cells. , 2008, Carcinogenesis.

[34]  G. Koekemoer,et al.  Hereditary tyrosinemia type 1 metabolites impair DNA excision repair pathways. , 2010, Biochemical and biophysical research communications.

[35]  R. Marcos,et al.  Base excision repair capacity in chronic renal failure patients undergoing hemodialysis treatment , 2014, Cell biochemistry and function.

[36]  A. Collins,et al.  Does occupational exposure to mineral fibres cause DNA or chromosome damage? , 2004, Mutation research.

[37]  A. Gomes,et al.  Novel nitrogen compounds enhance protection and repair of oxidative DNA damage in a neuronal cell model: comparison with quercetin. , 2009, Chemico-biological interactions.

[38]  R. Godschalk,et al.  Modulation of nucleotide excision repair in human lymphocytes by genetic and dietary factors , 2009, British Journal of Nutrition.

[39]  P. Møller,et al.  DNA damage and repair activity after broccoli intake in young healthy smokers. , 2010, Mutagenesis.

[40]  A. Collins,et al.  A Standardized Protocol for the In Vitro Comet-Based DNA Repair Assay , 2014 .

[41]  P. Hanawalt,et al.  New applications of the Comet assay: Comet-FISH and transcription-coupled DNA repair. , 2009, Mutation research.

[42]  A. Collins,et al.  In vivo supplementation with coenzyme Q10 enhances the recovery of human lymphocytes from oxidative DNA damage , 2001, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[43]  I. Hällström,et al.  Genetic variation in cytochrome P-450 and xenobiotic metabolism in Drosophila melanogaster. , 1984, Biochemical pharmacology.

[44]  A. Collins,et al.  Effects of micronutrients on DNA repair , 2012, European Journal of Nutrition.

[45]  A. Collins,et al.  Inter-individual differences in repair of DNA base oxidation, measured in vitro with the comet assay. , 2001, Mutagenesis.

[46]  Micheline Kirsch-Volders,et al.  An aphidicolin-block nucleotide excision repair assay measuring DNA incision and repair capacity. , 2010, Mutagenesis.

[47]  A. Collins,et al.  Both base excision repair and nucleotide excision repair in humans are influenced by nutritional factors , 2011, Cell biochemistry and function.

[48]  L. Søndergaard,et al.  Homology between the mammalian liver and the Drosophila fat body. , 1993, Trends in genetics : TIG.

[49]  Stephen M. Mount,et al.  The genome sequence of Drosophila melanogaster. , 2000, Science.

[50]  A. Gomes,et al.  Oxidative DNA damage protection and repair by polyphenolic compounds in PC 12 cells , 2008 .

[51]  I. Gaivão,et al.  Use of the Comet Assay to Study DNA Repair in Drosophila melanogaster , 2014 .

[52]  A. Collins,et al.  Polyphenolic compounds from Salvia species protect cellular DNA from oxidation and stimulate DNA repair in cultured human cells. , 2010, Journal of agricultural and food chemistry.

[53]  P. Cook,et al.  Characterization of nuclear structures containing superhelical DNA. , 1976, Journal of cell science.

[54]  P. Møller,et al.  Aging and defense against generation of 8-oxo-7,8-dihydro-2'-deoxyguanosine in DNA. , 2009, Free radical biology & medicine.

[55]  T. Skorski,et al.  STI571 reduces NER activity in BCR/ABL-expressing cells. , 2008, Mutation research.

[56]  P. Møller,et al.  DNA repair phenotype and dietary antioxidant supplementation , 2008, British Journal of Nutrition.

[57]  V. Spanswick,et al.  Measurement of DNA interstrand crosslinking in individual cells using the Single Cell Gel Electrophoresis (Comet) assay. , 2010, Methods in molecular biology.

[58]  E. Gálová,et al.  DNA alkylation lesions and their repair in human cells: modification of the comet assay with 3-methyladenine DNA glycosylase (AlkD). , 2012, Toxicology letters.

[59]  J. Sekelsky,et al.  DNA Repair in Drosophila , 2000, The Journal of cell biology.

[60]  K J Johanson,et al.  Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells. , 1984, Biochemical and biophysical research communications.

[61]  A. Collins,et al.  Genotoxic effects of asbestos in humans. , 2004, Mutation research.

[62]  A. Collins,et al.  In vitro comet assay for DNA repair: a warning concerning application to cultured cells. , 2009, Mutagenesis.

[63]  Amaya Azqueta,et al.  The essential comet assay: a comprehensive guide to measuring DNA damage and repair , 2013, Archives of Toxicology.

[64]  A. Gomes,et al.  Oxidative DNA damage protection and repair by polyphenolic compounds in PC12 cells. , 2008, European journal of pharmacology.