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Genotoxicity Assessment pp 237–257Cite as

The Comet Assay: Assessment of In Vitro and In Vivo DNA Damage

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 2031))

Abstract

Anthropogenic activities, indiscriminate and rapid industrialization as well as pursuance of a better life has led to an increase in the concentration of chemicals, like pesticides, automobile exhausts, and new chemical entities, in the environment, which have an adverse effect on all living organisms including humans. Sensitive and robust test systems are thus required for accurate hazard identification and risk assessment. The Comet assay has been used widely as a simple, rapid, and sensitive tool for assessment of DNA damage in single cell from both in vitro and in vivo sources as well as in humans. The advantages of the in vivo Comet assay are its ability to detect DNA damage in any tissues, despite having non-proliferating cells, and its sensitivity to detect genotoxicity. The recommendations from the international workshops held for the Comet assay have resulted in establishment of guidelines, and the OECD has adopted a guideline for the in vivo Comet assay as a test for assessing DNA damage in animals. The in vitro Comet assay conducted in cultured cells can be used for screening large number of compounds and at very low concentrations. The in vitro assay has also been automated to provide a high throughput screening method for new chemical entities, as well as in environmental samples. This chapter details the in vitro Comet assay using the 96-well plate and in vivo Comet assay in multiple organs of the mouse.

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References

  1. Ostling O, Johanson KJ (1984) Microelectrophoretic study of radiation-induced DNA damage in individual mammalian cells. Biochem Biophys Res Commun 123:291–298

    Article  CAS  Google Scholar 

  2. Singh NP, McCoy MT, Tice RR et al (1988) A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 175:184–191

    Article  CAS  Google Scholar 

  3. Dhawan A, Bajpayee M, Parmar D (2009) Comet assay: a reliable tool for the assessment of DNA damage in different models. Cell Biol Toxicol 25:5–32

    Article  CAS  Google Scholar 

  4. Glei M, Schneider T, Schlörmann W (2016) Comet assay: an essential tool in toxicological research. Arch Toxicol 90(10):2315–2336

    Article  CAS  Google Scholar 

  5. Moller P (2018) The comet assay: ready for 30 more years. Mutagenesis 33(1):1–7

    Article  Google Scholar 

  6. Pfuhler S, Fellows M, van Benthem J et al (2011) In vitro genotoxicity test approaches with better predictivity: summary of an IWGT workshop. Mutat Res 723(2):101–107

    Article  CAS  Google Scholar 

  7. Collins AR, Oscoz AA, Brunborg G (2008) The comet assay: topical issues. Mutagenesis 23(3):143–151

    Article  CAS  Google Scholar 

  8. Bajpayee M, Pandey AK, Zaidi S et al (2005) DNA damage and mutagenicity induced by endosulfan and its metabolites. Environ Mol Mutagen 47(9):682–692

    Article  Google Scholar 

  9. Pandey AK, Gurbani D, Bajpayee M et al (2009) In silico studies with human DNA topoisomerase-II alpha to unravel the mechanism of in vitro genotoxicity of benzene and its metabolites. Mutat Res 661(1–2):57–70

    Article  CAS  Google Scholar 

  10. Patel S, Bajpayee M, Pandey AK et al (2007) In vitro induction of cytotoxicity and DNA strand breaks in CHO cells exposed to cypermethrin, pendimethalin and dichlorvos. Toxicol In Vitro 21(8):1409–1418

    Article  CAS  Google Scholar 

  11. Di Bucchianico S, Cappellini F, Le Bihanic F (2017) Genotoxicity of TiO2 nanoparticles assessed by mini-gel comet assay and micronucleus scoring with flow cytometry. Mutagenesis 32(1):127–137

    Article  Google Scholar 

  12. Stang A, Witte I (2010) The ability of the high-throughput comet assay to measure the sensitivity of five cell lines toward methyl methanesulfonate, hydrogen peroxide, and pentachlorophenol. Mutat Res 701(2):103–106

    Article  CAS  Google Scholar 

  13. Meghani N, Patel P, Kansara K et al (2018) Formulation of vitamin D encapsulated cinnamon oil nanoemulsion: its potential anti-cancerous activity in human alveolar carcinoma cells. Colloids Surf B Biointerfaces 166:349–357

    Article  CAS  Google Scholar 

  14. Pandey AK, Bajpayee M, Parmar D et al (2008) Multipronged evaluation of genotoxicity in Indian petrol-pump workers. Environ Mol Mutagen 49(9):695–707

    Article  Google Scholar 

  15. Reisinger K, Blatz V, Brinkmann J et al (2018) Validation of the 3D skin comet assay using full thickness skin models: transferability and reproducibility. Mutat Res 827:27–41

    Article  CAS  Google Scholar 

  16. Sharma V, Anderson D, Dhawan A (2011) Zinc oxide nanoparticles induce oxidative stress and genotoxicity in human liver cells (HepG2). J Biomed Nanotechnol 7(1):98–99

    Article  CAS  Google Scholar 

  17. Shukla RK, Kumar A, Gurbani D et al (2011) TiO(2) nanoparticles induce oxidative DNA damage and apoptosis in human liver cells. Nanotoxicology

    Google Scholar 

  18. Sharma V, Anderson D, Dhawan A (2012) Zinc oxide nanoparticles induce oxidative DNA damage and ROS-triggered mitochondria mediated apoptosis in human liver cells (HepG2). Apoptosis 17(8):852–870

    Article  CAS  Google Scholar 

  19. Senapati VA, Kumar A, Gupta GS et al (2015) ZnO nanoparticles induced inflammatory response and genotoxicity in human blood cells: a mechanistic approach. Food Chem Toxicol 85:61–70

    Article  CAS  Google Scholar 

  20. Sharma V, Singh SK, Anderson D et al (2011) Zinc oxide nanoparticle induced genotoxicity in primary human epidermal keratinocytes. J Nanosci Nanotechnol 11(5):3782–3788

    Article  CAS  Google Scholar 

  21. Shukla RK, Sharma V, Pandey AK et al (2011) ROS-mediated genotoxicity induced by titanium dioxide nanoparticles in human epidermal cells. Toxicol In Vitro 25(1):231–241

    Article  CAS  Google Scholar 

  22. COM (United Kingdom Committee on Mutagenicity of Chemicals in Food, Consumer Products and the Environment) (2000) Guidance on a strategy for testing of chemicals for mutagenicity

    Google Scholar 

  23. U.S. Food and Drug Administration (2012) Guidance for industry. www.fda.gov/downloads/Drugs/Guidances/ucm074931.pdf

  24. International Committee on Harmonization (ICH) guideline S2(R1) (2011) Guidance on genotoxicity testing and data interpretation for pharmaceuticals intended for human use www.ich.org/fileadmin/Public_Web/ICH/S2/S2R1_Step4.pdf

  25. Rothfuss AA, Honma MM, Czich AA et al (2011) Improvement of in vivo genotoxicity assessment: combination of acute tests and integration into standard toxicity testing. Mutat Res 723:108–120

    Article  CAS  Google Scholar 

  26. Tice RR, Agurell E, Anderson D et al (2000) Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ Mol Mutagen 35:206–221

    Article  CAS  Google Scholar 

  27. Hartmann A, Agurell E, Beevers C et al (2003) Recommendations for conducting the in vivo alkaline comet assay. Mutagenesis 18:45–51

    Article  CAS  Google Scholar 

  28. Koppen G, Azqueta A, Pourrut B et al (2017) The next three decades of the comet assay: a report of the 11th international comet assay workshop. Mutagenesis 32(3):397–408

    Article  CAS  Google Scholar 

  29. OECD guideline 489, In vivo mammalian Alkaline Comet Assay, September 2016

    Google Scholar 

  30. Sasaki YF, Sekihashi K, Izumiyama F et al (2000) The comet assay with multiple mouse organs: comparison of comet assay results and carcinogenicity with 208 chemicals selected from the IARC monographs and U.S. NTP carcinogenicity database. Crit Rev Toxicol 30(6):629–799

    Article  CAS  Google Scholar 

  31. Patel S, Pandey AK, Bajpayee M et al (2006) Cypermethrin-induced DNA damage in organs and tissues of the mouse: evidence from the comet assay. Mutat Res 607(2):176–183

    Article  CAS  Google Scholar 

  32. Bakare AA, Patel S, Pandey AK et al (2012) DNA and oxidative damage induced in somatic organs and tissues of mouse by municipal sludge leachate. Toxicol Ind Health 28(7):614–623

    Article  CAS  Google Scholar 

  33. Ansari KM, Chauhan LK, Dhawan A (2005) Unequivocal evidence of genotoxic potential of argemone oil in mice. Int J Cancer 112(5):890–895

    Article  Google Scholar 

  34. Das M, Ansari KM, Dhawan A et al (2005) Correlation of DNA damage in epidemic dropsy patients to carcinogenic potential of argemone oil and isolated sanguinarine alkaloid in mice. Int J Cancer 117(5):709–717

    Article  CAS  Google Scholar 

  35. Ansari KM, Dhawan A, Khanna SK et al (2006) Protective effect of bioantioxidants on argemone oil/sanguinarine alkaloid induced genotoxicity in mice. Cancer Lett 244(1):109–118

    Article  CAS  Google Scholar 

  36. Sharma V, Singh P, Pandey AK et al (2012) Induction of oxidative stress, DNA damage and apoptosis in mouse liver after sub-acute oral exposure to zinc oxide nanoparticles. Mutat Res 745(1–2):84–91

    Article  CAS  Google Scholar 

  37. Shukla RK, Kumar A, Vallabani NV et al (2014) Titanium dioxide nanoparticle-induced oxidative stress triggers DNA damage and hepatic injury in mice. Nanomedicine 9(9):1423–1434

    Article  CAS  Google Scholar 

  38. Burlinson B (2012) The in vitro and in vivo comet assays. In: Parry JM, Parry EM (eds) Genetic toxicology: principal and methods. Methods in molecular biology, vol 817. Humana Press, New York, NY, pp 143–163

    Chapter  Google Scholar 

  39. Lovell DP, Thomas G, Dubow GR (1999) Issues related to the experimental design and subsequent statistical analysis of in vivo and in vitro comet studies. Teratog Carcinog Mutagen 19(2):109–119

    Article  CAS  Google Scholar 

  40. Lovell DP, Omori T (2008) Statistical issues in the use of the comet assay. Mutagenesis 23:171–182

    Article  CAS  Google Scholar 

  41. Bright J, Aylott M, Bate S et al (2011) Recommendations on the statistical analysis of the comet assay. Pharm Stat 10(6):485–493

    Article  Google Scholar 

  42. Enciso JM, Sánchez O, López de Cerain A et al (2015) Does the duration of lysis affect the sensitivity of the in vitro alkaline comet assay? Mutagenesis 30(1):21–28

    Article  CAS  Google Scholar 

  43. Azqueta A, Gutzkow KB, Brunborg G et al (2011) Towards a more reliable comet assay: optimizing agarose concentrations, alkaline unwinding time and electrophoresis conditions. Mutat Res 724:41–45

    Article  CAS  Google Scholar 

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Acknowledgments

The authors acknowledge the funding received from CSIR under various projects. AK gratefully acknowledges funding from Department of Biotechnology, Govt. of India under “NanoToF” (BT/PR10414/PFN/20/961/2014) and DST SERB project (EMR/2016/005286). The financial assistance by the Gujarat Institute for Chemical Technology (GICT) for establishment of a facility for environmental risk assessment of chemicals and nanomaterials is also acknowledged.

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Authors and Affiliations

  1. New Delhi, India

    Mahima Bajpayee

  2. Biological and Life Sciences, School of Arts and Sciences, Ahmedabad University, Ahmedabad, Gujarat, India

    Ashutosh Kumar

  3. Nanomaterial Toxicology Group, CSIR-Indian Institute of Toxicology Research, Lucknow, Uttar Pradesh, India

    Alok Dhawan

Authors
  1. Mahima Bajpayee
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  2. Ashutosh Kumar
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  3. Alok Dhawan
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Corresponding author

Correspondence to Alok Dhawan .

Editor information

Editors and Affiliations

  1. CSIR—Indian Institute of Toxicology Research, Lucknow, Uttar Pradesh, India

    Alok Dhawan

  2. New Delhi, Delhi, India

    Mahima Bajpayee

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Bajpayee, M., Kumar, A., Dhawan, A. (2019). The Comet Assay: Assessment of In Vitro and In Vivo DNA Damage. In: Dhawan, A., Bajpayee, M. (eds) Genotoxicity Assessment. Methods in Molecular Biology, vol 2031. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9646-9_12

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  • DOI: https://doi.org/10.1007/978-1-4939-9646-9_12

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9645-2

  • Online ISBN: 978-1-4939-9646-9

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