An optimized comet-based in vitro DNA repair assay to assess base and nucleotide excision repair activity.
Journal
Nature protocols
ISSN: 1750-2799
Titre abrégé: Nat Protoc
Pays: England
ID NLM: 101284307
Informations de publication
Date de publication:
12 2020
12 2020
Historique:
received:
16
03
2020
accepted:
17
08
2020
pubmed:
18
11
2020
medline:
2
2
2021
entrez:
17
11
2020
Statut:
ppublish
Résumé
This optimized protocol (including links to instruction videos) describes a comet-based in vitro DNA repair assay that is relatively simple, versatile, and inexpensive, enabling the detection of base and nucleotide excision repair activity. Protein extracts from samples are incubated with agarose-embedded substrate nucleoids ('naked' supercoiled DNA) containing specifically induced DNA lesions (e.g., resulting from oxidation, UVC radiation or benzo[a]pyrene-diol epoxide treatment). DNA incisions produced during the incubation reaction are quantified as strand breaks after electrophoresis, reflecting the extract's incision activity. The method has been applied in cell culture model systems, human biomonitoring and clinical investigations, and animal studies, using isolated blood cells and various solid tissues. Once extracts and substrates are prepared, the assay can be completed within 2 d.
Identifiants
pubmed: 33199871
doi: 10.1038/s41596-020-0401-x
pii: 10.1038/s41596-020-0401-x
doi:
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.
Langues
eng
Sous-ensembles de citation
IM
Pagination
3844-3878Références
Ostling, O. & Johanson, K. J. Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells. Biochem. Biophys. Res. Commun. 123, 291–298 (1984).
pubmed: 6477583
Cook, P. R., Brazell, I. A. & Jost, E. Characterization of nuclear structures containing superhelical DNA. J. Cell Sci. 22, 303–324 (1976).
pubmed: 1002771
Collins, A. R. & Azqueta, A. Single cell gel electrophoresis combined with lesion-specific enzymes to measure oxidative damage to DNA. in Laboratory Methods in Cell Biology, Vol. 112 (eds Anderson, C. T., Howell, E. S. & Dixit, R.) 69–92 (Elsevier, 2012).
Stefanini, M. et al. Novel Chinese hamster ultraviolet-sensitive mutants for excision repair form complementation groups 9 and 10. Cancer Res. 51, 3965–3971 (1991).
pubmed: 1855213
Lorenzo, Y. et al. The carotenoid beta-cryptoxanthin stimulates the repair of DNA oxidation damage in addition to acting as an antioxidant in human cells. Carcinogenesis 30, 308–314 (2009).
pubmed: 19056931
Collins, A. R., Fleming, I. M. & Gedik, C. M. In vitro repair of oxidative and ultraviolet-induced DNA damage in supercoiled nucleoid DNA by human cell extract. Biochim. Biophys. Acta 1219, 724–727 (1994).
pubmed: 7948034
Langie, S. A. et al. Development and validation of a modified comet assay to phenotypically assess nucleotide excision repair. Mutagenesis 21, 153–158 (2006).
pubmed: 16556641
Collins, A. R. et al. Inter-individual differences in repair of DNA base oxidation, measured in vitro with the comet assay. Mutagenesis 16, 297–301 (2001).
pubmed: 11420396
Møller, P. et al. Searching for assay controls for the Fpg- and hOGG1-modified comet assay. Mutagenesis 33, 9–19 (2018).
pubmed: 28992346
Borghini, A., Roursgaard, M., Andreassi, M. G., Kermanizadeh, A. & Moller, P. Repair activity of oxidatively damaged DNA and telomere length in human lung epithelial cells after exposure to multi-walled carbon nanotubes. Mutagenesis 32, 173–180 (2017).
pubmed: 27530331
Jensen, D. M. et al. Telomere length and genotoxicity in the lung of rats following intragastric exposure to food-grade titanium dioxide and vegetable carbon particles. Mutagenesis 34, 203–214 (2019).
pubmed: 30852617
Lohr, M. et al. Association between age and repair of oxidatively damaged DNA in human peripheral blood mononuclear cells. Mutagenesis 30, 695–700 (2015).
pubmed: 25925070
Gaivao, I., Piasek, A., Brevik, A., Shaposhnikov, S. & Collins, A. R. Comet assay-based methods for measuring DNA repair in vitro; estimates of inter- and intra-individual variation. Cell Biol. Toxicol. 25, 45–52 (2009).
pubmed: 18058031
Herrera, M. 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. Clin. Cancer Res. 15, 5466–5472 (2009).
pubmed: 19690199
van Dyk, E., Steenkamp, A., Koekemoer, G. & Pretorius, P. J. Hereditary tyrosinemia type 1 metabolites impair DNA excision repair pathways. Biochem. Biophys. Res. Commun. 401, 32–36 (2010).
pubmed: 20828540
Langie, S. A. et al. The effect of oxidative stress on nucleotide-excision repair in colon tissue of newborn piglets. Mutat. Res. 695, 75–80 (2010).
pubmed: 20015477
Mikkelsen, L. et al. Aging and defense against generation of 8-oxo-7,8-dihydro-2’-deoxyguanosine in DNA. Free Radic. Biol. Med. 47, 608–615 (2009).
pubmed: 19500668
Langie, S. A. et al. Measuring DNA repair incision activity of mouse tissue extracts towards singlet oxygen-induced DNA damage: a comet-based in vitro repair assay. Mutagenesis 26, 461–471 (2011).
pubmed: 21355044
Slyskova, J. et al. Functional, genetic, and epigenetic aspects of base and nucleotide excision repair in colorectal carcinomas. Clin. Cancer Res. 18, 5878–5887 (2012).
pubmed: 22966016
Azqueta, A., Slyskova, J., Langie, S. A., O’Neill Gaivao, I. & Collins, A. Comet assay to measure DNA repair: approach and applications. Front. Genet. 5, 288 (2014).
pubmed: 25202323
pmcid: 4142706
Yauk, C., Lambert, I., Marchetti, F. & Douglas, G. AOP 15. Alkylation of DNA in male pre-meiotic germ cells leading to heritable mutations. AOPWiki https://aopwiki.org/aops/15
Pottenger, L. H., Schoeny, R., Moore, M. & Simon, T. W. AOP 46. AFB1: mutagenic mode-of-action leading to hepatocellular carcinoma (HCC). AOPWiki https://aopwiki.org/aops/46
Silva, J. P., Gomes, A. C. & Coutinho, O. P. Oxidative DNA damage protection and repair by polyphenolic compounds in PC12 cells. Eur. J. Pharmacol. 601, 50–60 (2008).
pubmed: 18996367
Ramos, A. A., Azqueta, A., Pereira-Wilson, C. & Collins, A. R. Polyphenolic compounds from Salvia species protect cellular DNA from oxidation and stimulate DNA repair in cultured human cells. J. Agric. Food Chem. 58, 7465–7471 (2010).
pubmed: 20486687
Ramos, A. A., Pereira-Wilson, C. & Collins, A. R. Protective effects of ursolic acid and luteolin against oxidative DNA damage include enhancement of DNA repair in Caco-2 cells. Mutat. Res. 692, 6–11 (2010).
pubmed: 20659486
Azqueta, A., Costa, S., Lorenzo, Y., Bastani, N. E. & Collins, A. R. Vitamin C in cultured human (HeLa) cells: lack of effect on DNA protection and repair. Nutrients 5, 1200–1217 (2013).
pubmed: 23571651
pmcid: 3705343
Silva, J. P., Gomes, A. C., Proenca, F. & Coutinho, O. P. Novel nitrogen compounds enhance protection and repair of oxidative DNA damage in a neuronal cell model: comparison with quercetin. Chem. Biol. Interact. 181, 328–337 (2009).
pubmed: 19665457
Sliwinski, T. et al. STI571 reduces NER activity in BCR/ABL-expressing cells. Mutat. Res. 654, 162–167 (2008).
pubmed: 18602021
Folkmann, J. K. et al. Oxidatively damaged DNA in rats exposed by oral gavage to C60 fullerenes and single-walled carbon nanotubes. Environ. Health Perspect. 117, 703–708 (2009).
pubmed: 19479010
Langie, S. A. et al. Maternal folate depletion and high-fat feeding from weaning affects DNA methylation and DNA repair in brain of adult offspring. FASEB J. 27, 3323–3334 (2013).
pubmed: 23603834
Langie, S. A. et al. Redox and epigenetic regulation of the APE1 gene in the hippocampus of piglets: the effect of early life exposures. DNA Repair (Amst.) 18, 52–62 (2014).
Langie, S. A. et al. The ageing brain: effects on DNA repair and DNA methylation in mice. Genes (Basel) 8, https://doi.org/10.3390/genes8020075 (2017).
Setayesh, T. et al. Impact of weight loss strategies on obesity-induced DNA damage. Mol. Nutr. Food Res. 63, e1900045 (2019).
pubmed: 31141317
Gaivao, I. & Sierra, L. M. Drosophila comet assay: insights, uses, and future perspectives. Front. Genet. 5, 304 (2014).
pubmed: 25221574
pmcid: 4148904
Dusinska, M., Dzupinkova, Z., Wsolova, L., Harrington, V. & Collins, A. R. Possible involvement of XPA in repair of oxidative DNA damage deduced from analysis of damage, repair and genotype in a human population study. Mutagenesis 21, 205–211 (2006).
pubmed: 16613913
Slyskova, J. et al. Relationship between the capacity to repair 8-oxoguanine, biomarkers of genotoxicity and individual susceptibility in styrene-exposed workers. Mutat. Res. 634, 101–111 (2007).
pubmed: 17855160
Dusinska, M. et al. Are glutathione S transferases involved in DNA damage signalling? Interactions with DNA damage and repair revealed from molecular epidemiology studies. Mutat. Res. 736, 130–137 (2012).
pubmed: 22450146
Staruchova, M. et al. Occupational exposure to mineral fibres. Biomarkers of oxidative damage and antioxidant defence and associations with DNA damage and repair. Mutagenesis 23, 249–260 (2008).
pubmed: 18281292
Azqueta, A. et al. DNA repair as a human biomonitoring tool: comet assay approaches. Mutat. Res. 781, 71–87 (2019).
pubmed: 31416580
Dusinska, M. et al. Genotoxic effects of asbestos in humans. Mutat. Res. 553, 91–102 (2004).
pubmed: 15288536
Dusinska, M. et al. Does occupational exposure to mineral fibres cause DNA or chromosome damage? Mutat. Res. 553, 103–110 (2004).
pubmed: 15288537
Vodicka, P. et al. Cytogenetic markers, DNA single-strand breaks, urinary metabolites, and DNA repair rates in styrene-exposed lamination workers. Environ. Health Perspect. 112, 867–871 (2004).
pubmed: 15175174
pmcid: 1242014
Jensen, A. et al. Influence of the OGG1 Ser326Cys polymorphism on oxidatively damaged DNA and repair activity. Free Radic. Biol. Med. 52, 118–125 (2012).
pubmed: 22019439
Collins, A. R., Harrington, V., Drew, J. & Melvin, R. Nutritional modulation of DNA repair in a human intervention study. Carcinogenesis 24, 511–515 (2003).
pubmed: 12663512
Caple, F. et al. Inter-individual variation in DNA damage and base excision repair in young, healthy non-smokers: effects of dietary supplementation and genotype. Br. J. Nutr. 103, 1585–1593 (2010).
pubmed: 20082738
Stoyanova, E. et al. Base excision repair capacity in chronic renal failure patients undergoing hemodialysis treatment. Cell Biochem. Funct. 32, 177–182 (2014).
pubmed: 23873307
Fikrova, P. et al. DNA crosslinks, DNA damage and repair in peripheral blood lymphocytes of non-small cell lung cancer patients treated with platinum derivatives. Oncol. Rep. 31, 391–396 (2014).
pubmed: 24154806
Slyskova, J. et al. Differences in nucleotide excision repair capacity between newly diagnosed colorectal cancer patients and healthy controls. Mutagenesis 27, 225–232 (2012).
pubmed: 22294771
Slyskova, J. et al. Post-treatment recovery of suboptimal DNA repair capacity and gene expression levels in colorectal cancer patients. Mol. Carcinog. 54, 769–778 (2015).
pubmed: 24585457
Vodenkova, S. et al. Base excision repair capacity as a determinant of prognosis and therapy response in colon cancer patients. DNA Repair (Amst.) 72, 77–85 (2018).
Slyskova, J., Langie, S. A., Collins, A. R. & Vodicka, P. Functional evaluation of DNA repair in human biopsies and their relation to other cellular biomarkers. Front. Genet. 5, 116 (2014).
pubmed: 24904630
pmcid: 4033188
Singh, N. P., McCoy, M. T., Tice, R. R. & Schneider, E. L. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp. Cell Res. 175, 184–191 (1988).
pubmed: 3345800
Collins, A. R., Ord, M. J. & Johnson, R. T. Correlations of DNA damage and repair with nuclear and chromosomal damage in HeLa cells caused by methylnitrosamides. Cancer Res. 41, 5176–5187 (1981).
pubmed: 7307013
Crebelli, R. et al. Biomonitoring of primary aluminium industry workers: detection of micronuclei and repairable DNA lesions by alkaline SCGE. Mutat. Res. 516, 63–70 (2002).
pubmed: 11943612
Vande Loock, K., Decordier, I., Ciardelli, R., Haumont, D. & Kirsch-Volders, M. An aphidicolin-block nucleotide excision repair assay measuring DNA incision and repair capacity. Mutagenesis 25, 25–32 (2010).
pubmed: 19843590
Figueroa-Gonzalez, G. & Perez-Plasencia, C. Strategies for the evaluation of DNA damage and repair mechanisms in cancer. Oncol. Lett. 13, 3982–3988 (2017).
pubmed: 28588692
pmcid: 5452911
Lambert, B., Ringborg, U. & Skoog, L. Age-related decrease of ultraviolet light-induced DNA repair synthesis in human peripheral leukocytes. Cancer Res. 39, 2792–2795 (1979).
pubmed: 445484
Athas, W. F., Hedayati, M. A., Matanoski, G. M., Farmer, E. R. & Grossman, L. Development and field-test validation of an assay for DNA repair in circulating human lymphocytes. Cancer Res. 51, 5786–5793 (1991).
pubmed: 1933849
Redaelli, A., Magrassi, R., Bonassi, S., Abbondandolo, A. & Frosina, G. AP endonuclease activity in humans: development of a simple assay and analysis of ten normal individuals. Teratog. Carcinog. Mutagen. 18, 17–26 (1998).
pubmed: 9586767
Elliott, R. M., Astley, S. B., Southon, S. & Archer, D. B. Measurement of cellular repair activities for oxidative DNA damage. Free Radic. Biol. Med. 28, 1438–1446 (2000).
pubmed: 10924862
Roldan-Arjona, T. et al. Molecular cloning and functional expression of a human cDNA encoding the antimutator enzyme 8-hydroxyguanine-DNA glycosylase. Proc. Natl Acad. Sci. USA 94, 8016–8020 (1997).
pubmed: 9223306
Sauvaigo, S. et al. An oligonucleotide microarray for the monitoring of repair enzyme activity toward different DNA base damage. Anal. Biochem. 333, 182–192 (2004).
pubmed: 15351295
Paz-Elizur, T. et al. DNA repair activity for oxidative damage and risk of lung cancer. J. Natl Cancer Inst. 95, 1312–1319 (2003).
pubmed: 12953085
Paz-Elizur, T. et al. Reduced repair of the oxidative 8-oxoguanine DNA damage and risk of head and neck cancer. Cancer Res. 66, 11683–11689 (2006).
pubmed: 17178863
Paz-Elizur, T. et al. Development of an enzymatic DNA repair assay for molecular epidemiology studies: distribution of OGG activity in healthy individuals. DNA Repair (Amst.) 6, 45–60 (2007).
Leitner-Dagan, Y. et al. N-methylpurine DNA glycosylase and OGG1 DNA repair activities: opposite associations with lung cancer risk. J. Natl Cancer Inst. 104, 1765–1769 (2012).
pubmed: 23104324
pmcid: 3502197
Leitner-Dagan, Y. et al. Enzymatic MPG DNA repair assays for two different oxidative DNA lesions reveal associations with increased lung cancer risk. Carcinogenesis 35, 2763–2770 (2014).
pubmed: 25355292
pmcid: 4303808
Azqueta, A. & Collins, A. R. The essential comet assay: a comprehensive guide to measuring DNA damage and repair. Arch. Toxicol. 87, 949–968 (2013).
pubmed: 23685795
Riso, P. et al. DNA damage and repair activity after broccoli intake in young healthy smokers. Mutagenesis 25, 595–602 (2010).
pubmed: 20713433
Danielsen, P. H. et al. Oxidatively damaged DNA and its repair after experimental exposure to wood smoke in healthy humans. Mutat. Res. 642, 37–42 (2008).
pubmed: 18495177
Collins, A. R. & Azqueta, A. DNA repair as a biomarker in human biomonitoring studies; further applications of the comet assay. Mutat. Res. 736, 122–129 (2012).
pubmed: 21459100
Guarnieri, S. et al. DNA repair phenotype and dietary antioxidant supplementation. Br. J. Nutr. 99, 1018–1024 (2008).
pubmed: 17925050
Gaivão, I., Rodríguez, R. & Sierra, L. M. Use of the comet assay to study DNA repair in Drosophila melanogaster. in Genotoxicity and DNA Repair: A Practical Approach (eds Sierra, L. M. & Gaivão, I.) Ch. 23 (Humana Press, 2014).
Gorniak, J. P. et al. Tissue differences in BER-related incision activity and non-specific nuclease activity as measured by the comet assay. Mutagenesis 28, 673–681 (2013).
pubmed: 24097409
Muruzabal, D., Langie, S. A. S., Pourrut, B. & Azqueta, A. The enzyme-modified comet assay: enzyme incubation step in 2 vs 12-gels/slide systems. Mutat. Res. 845, 402981 (2019).
pubmed: 31561901
Azqueta, A., Langie, S. & Collins, A. R. The effect of extract concentration and time of incubation in the comet based in vitro DNA repair assay. Abstracts of the 12th International Comet Assay Workshop held at the University of Navarra, Pamplona, Spain, 29–31 August 2017. Mutagenesis 32, e24 https://academic.oup.com/mutage/article/32/6/e1/4844756#121612377 (2018).
Boer, K., Isenmann, S. & Deufel, T. Strong interference of hemoglobin concentration on CSF total protein measurement using the trichloroacetic acid precipitation method. Clin. Chem. Lab. Med. 45, 112–113 (2007).
pubmed: 17243927
Roman, Y., Bomsel-Demontoy, M. C., Levrier, J., Chaste-Duvernoy, D. & Jalme, M. S. Effect of hemolysis on plasma protein levels and plasma electrophoresis in birds. J. Wildl. Dis. 45, 73–80 (2009).
pubmed: 19204337
Brodersen, R. Bilirubin. Solubility and interaction with albumin and phospholipid. J. Biol. Chem. 254, 2364–2369 (1979).
pubmed: 429290
Kjellin, K. G. Bilirubin compounds in the CSF. J. Neurol. Sci. 13, 161–173 (1971).
pubmed: 5579629
Moller, P. et al. On the search for an intelligible comet assay descriptor. Front. Genet. 5, 217 (2014).
pubmed: 25101109
pmcid: 4101262
Forchhammer, L. et al. Variation in assessment of oxidatively damaged DNA in mononuclear blood cells by the comet assay with visual scoring. Mutagenesis 23, 223–231 (2008).
pubmed: 18326868
Azqueta, A. et al. The influence of scoring method on variability in results obtained with the comet assay. Mutagenesis 26, 393–399 (2011).
pubmed: 21227901
Brunborg, G., Rolstadaas, L. & Gutzkow, K. B. Electrophoresis in the comet assay. in Electrophoresis: Life Sciences Practical Applications (eds Boldura, O-M. & Baltă, C.) 526–652 (IntechOpen, 2018).
Shaposhnikov, S. et al. Twelve-gel slide format optimised for comet assay and fluorescent in situ hybridisation. Toxicol. Lett. 195, 31–34 (2010).
pubmed: 20188804
Collins, A. R. et al. The comet assay: topical issues. Mutagenesis 23, 143–151 (2008).
pubmed: 18283046
Forchhammer, L. et al. Variation in the measurement of DNA damage by comet assay measured by the ECVAG inter-laboratory validation trial. Mutagenesis 25, 113–123 (2010).
pubmed: 19910383
Azqueta, A., Langie, S. A., Slyskova, J. & Collins, A. R. Measurement of DNA base and nucleotide excision repair activities in mammalian cells and tissues using the comet assay—a methodological overview. DNA Repair (Amst.) 12, 1007–1010 (2013).
Gungor, N. et al. Lung inflammation is associated with reduced pulmonary nucleotide excision repair in vivo. Mutagenesis 25, 77–82 (2010).
pubmed: 19917589
Azqueta, A. et al. A comparative performance test of standard, medium- and high-throughput comet assays. Toxicol. Vitr. 27, 768–773 (2013).
Brauner, E. V. et al. Exposure to ultrafine particles from ambient air and oxidative stress-induced DNA damage. Environ. Health Perspect. 115, 1177–1182 (2007).
pubmed: 17687444
pmcid: 1940068
Moller, P. et al. Measurement of oxidative damage to DNA in nanomaterial exposed cells and animals. Environ. Mol. Mutagen. 56, 97–110 (2015).
pubmed: 25196723
Hasplova, K. et al. DNA alkylation lesions and their repair in human cells: modification of the comet assay with 3-methyladenine DNA glycosylase (AlkD). Toxicol. Lett. 208, 76–81 (2012).
pubmed: 22019460
Dusinska, M. et al. Testing strategies for the safety of nanoparticles used in medical applications. Nanomed. (Lond.) 4, 605–607 (2009).
Choi, S. W., Yeung, V. T., Collins, A. R. & Benzie, I. F. Redox-linked effects of green tea on DNA damage and repair, and influence of microsatellite polymorphism in HMOX-1: results of a human intervention trial. Mutagenesis 30, 129–137 (2015).
pubmed: 25527735
Brevik, A. et al. Supplementation of a Western diet with golden kiwifruits (Actinidia chinensis var.‘Hort 16A’): effects on biomarkers of oxidation damage and antioxidant protection. Nutr. J. 10, 54 (2011).
pubmed: 21586177
pmcid: 3118331
Hanova, M. et al. Modulation of DNA repair capacity and mRNA expression levels of XRCC1, hOGG1 and XPC genes in styrene-exposed workers. Toxicol. Appl. Pharmacol. 248, 194–200 (2010).
pubmed: 20692273
Humphreys, V. et al. Age-related increases in DNA repair and antioxidant protection: a comparison of the Boyd Orr Cohort of elderly subjects with a younger population sample. Age Ageing 36, 521–526 (2007).
pubmed: 17913757
Langie, S. A. et al. Modulation of nucleotide excision repair in human lymphocytes by genetic and dietary factors. Br. J. Nutr. 103, 490–501 (2010).
pubmed: 19878615
Al-Serori, H. et al. Mobile phone specific electromagnetic fields induce transient DNA damage and nucleotide excision repair in serum-deprived human glioblastoma cells. PLoS One 13, e0193677 (2018).
pubmed: 29649215
pmcid: 5896905
Soares, J. P. et al. Effects of combined physical exercise training on DNA damage and repair capacity: role of oxidative stress changes. Age (Dordr.) 37, 9799 (2015).