Skip to main content

The In Vitro and In Vivo Comet Assays

  • Protocol
  • First Online:
Genetic Toxicology

Part of the book series: Methods in Molecular Biology ((MIMB,volume 817))

Abstract

The strategy for testing for genotoxicity covers three main areas, namely gene mutation, chromosome aberration or breakage (clastogenicity), and chromosome loss or gain (aneuploidy). The current generalized strategy consists of assays capable of detecting all of these endpoints using in vitro assays such as the Ames test for detecting gene mutations in bacteria, the human peripheral lymphocyte chromosome aberration (CA) test for detecting clastogenicity, and the in vitro micronucleus test for clastogenicity and aneuploidy. The primary in vivo assay, and generally the only in vivo assay required, is the in vivo rodent bone marrow micronucleus assay. However, there are instances when these assays alone are inadequate and further testing is required, especially in vivo. Historically, the preferred second assay has been the rodent liver unscheduled DNA synthesis assay but recently this has been superseded by the rodent single cell gel electrophoresis or Comet assay. This assay has numerous advantages especially in vivo, where virtually any tissue can be examined. The status of the in vitro comet assay in regulatory testing is much less clear although a preliminary review of data from the assay has shown it to be more specific than other in vitro genotoxicity tests and less prone to false positives.

Detailed here are general protocols for both the in vitro and in vivo comet assays which will form the basis of the pending OECD guideline for the assay.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 159.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Ostling, O. and Johanson, K. J. (1984). Microelectrophoretic study of radiation induced DNA damages in individual mammalian cells. Biochem. Biophy.s Res. Commun. 123, 291–298.

    CAS  Google Scholar 

  2. Singh, N. P., McCoy, M. T., Tice, R. R., and Schneider, E. L. (1988). A simple technique for quantitation of low levels of DNA damage in individual cells. Exp. Cell Res. 175, 184 –191.

    Article  PubMed  CAS  Google Scholar 

  3. Tice, R. R., Andrews, P. W., Hirai, O., and Singh, N. P. (1991). The single cell gel (SCG) assay: an electrophoretic technique for the detection of DNA damage in individual cells. In: Witmer, C. R., Snyder, R. R., Jollow, D. J., Kalf, G. F., Kocsis, J. J., Sipes, I. G., editors. Biological reactive intermediates IV. Molecular and cellular effects and their impact on human health. New York: Plenum Press. p157–164.

    Google Scholar 

  4. Tice, R. R. (1995). The single cell gel/comet assay: A microgel electrophoretic technique for the detection of DNA damage and repair in individual cells. In: Phillips, D. H., Venitt, S, editors. Environmental mutagenesis. Oxford: Bios Scientific Publishers. p315–339.

    Google Scholar 

  5. Fairbairn, D. W., Olive, P. L., and O’Neill, K. L. (1995). The Comet assay: a comprehensive review. Mutat. Res. 339, 37–59.

    Article  PubMed  CAS  Google Scholar 

  6. Anderson, D., Yu, T-W., and McGregor, D. B. (1998). Comet assay responses as indicators of carcinogenic exposure. Mutagen 13, 539–555.

    Article  CAS  Google Scholar 

  7. Rojas, E., Lopez, M. C., and Valverde, M. (1999). Single cell gel electrophoresis assay: methodology and applications. J. Chromat. B 722, 225–254.

    Article  CAS  Google Scholar 

  8. Speit, G., and Hartmann, A. (1999). The comet assay (single-cell gel test): a sensitive genotoxicity test for the detection of DNA damage and repair. DNA Repair Protocols 113, 203–212.

    Article  CAS  Google Scholar 

  9. Tice, R. R., Agurell, E., Anderson, D., Burlinson, B., Hartmann, A., Kobayashi, H., Miyamae, Y., Rojas, E., Ryu, J.-C., Sasaki, Y. F. (2000). The single cell gel/comet assay: Guidelines for in vitro and in vivo genetic toxicology testing, Environ. Mol. Mutagen. 35, 206–221.

    Article  PubMed  CAS  Google Scholar 

  10. Hartmann, A., Agurell, E., Beevers, C., Brendler-Schwaab, S., Burlinson, B., Clay, P., Collins, A. R., Smith, A., Speit, G., Thybaud, V., and Tice, R. R. (2003). Recommendations for conducting the in vivo alkaline comet assay, Mutagenesis 18, 45–51.

    Article  PubMed  CAS  Google Scholar 

  11. Brendler-Schwaab, S. Y., Hartmann, A., Pfuhler, S., and Speit, G. (2005). The in vivo comet assay: use and status in genotoxicity testing, Mutagenesis 20 245–254.

    Article  PubMed  CAS  Google Scholar 

  12. Burlinson B, et al., (2007). Fourth International Workgroup on Genotoxicity Testing: result of the in vivo comet assay workgroup. Mutation Res. 627, 31–35.

    Article  PubMed  CAS  Google Scholar 

  13. Hartmann, A., Schumacher, M., Plappert-Helbig, U., Lowe, P., Suter, W., and Mueller, L. (2004). Use of the alkaline in vivo comet assay for mechanistic genotoxicity investigations, Mutagenesis 19 51–59.

    Article  PubMed  CAS  Google Scholar 

  14. COM (2000) (Committee on Mutagenicity of Chemicals in Food, Consumer Products and the Environment), COM guidance on a strategy for testing of chemicals for mutagenicity, United Kingdom, December. (http://www.advisorybodies.doh.gov.uk/com/).

  15. Ames, B. N., McCann, J., Yamasaki, E. (1975). Methods for detecting carcinogens and mutagens with the Salmonella/mammalian microsome mutagenicity test. Mutation Res. 31, 347–364.

    Article  PubMed  CAS  Google Scholar 

  16. Elliot, B. M., Combes, R. D., Elcombe, C. R., Gatehouse, D. G., Gibson, G.G., Mackay, J. M., and Wolf, R. C. (1992). Report of UK Environmental Mutagen Society Working Party: Alternatives to Aroclor 1254-induced S9 in in vitro genotoxicity assays. Mutagenesis 7, 175–177.

    Article  Google Scholar 

  17. Galloway, S. M., Aardema, M. J., Ishidate, M. Jr., Ivett, J. L., Kirkland, D. J., Morita, T., Mosesso, P., Sofuni, T. (1994). Report from working group on in vitro tests for chromosomal aberrations. Mutation Res. 312, 241–261.

    Article  PubMed  CAS  Google Scholar 

  18. Collins, A. R., Ai-guo, A., and Duthie, S. J. (1995). The kinetics of repair of oxidative DNA damage (strand breaks and oxidised pyrimidines) in human cells, Mutation Res. 336, 69–77.

    Article  PubMed  CAS  Google Scholar 

  19. Kumaravel, T. S., and Jha, A. N. (2006). Reliable Comet assay measurements for detecting DNA damage induced by ionising radiation and chemicals, Mutation Res. 605, 7–16.

    Article  PubMed  CAS  Google Scholar 

  20. OECD (1986). (Organisation for Economic Co-operation and Development), OECD Guidelines for Testing of Chemicals. Introduction to the OECD Guidelines on genetic toxicology testing and guidance on the selection and application of assays, OECD, Paris, France.

    Google Scholar 

  21. ICH (1995). (International Cooperation on Harmonization), ICH Harmonized Tripartite Guideline. S2A. Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals, July. (http://www.ich.org).

  22. ICH (1997). (International Cooperation on Harmonization), ICH Harmonized Tripartite Guideline. S2B. Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals, July. (http://www.ich.org).

  23. FDA (2006). Guidance for Industry and Review Staff Recommended Approaches to Integration of Genetic Toxicology Study Results, FDA. http://www.fda.gov/cder/guidance/index.htm.

  24. Amacher, D E, Paillet S and Ray V A (1979) Point mutations at the thymidine kinase locus in L5178Y Mouse Lymphoma cells. I Application to Genetic Toxicological testing. Mutat Res 64: 391–406.

    CAS  Google Scholar 

  25. Lovell, D. P., Thomas, G., and Dubow., G. R. (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  PubMed  CAS  Google Scholar 

  26. Wiklund, S. J., and Agurell, E., (2003). Aspects of design and statistical analysis in the Comet assay. Mutagenesis 18(2), 167–175.

    Article  PubMed  CAS  Google Scholar 

  27. Smith, C. C., Adkins, D. J., Martin E. A., and O’Donovan, M. R., (2008) Recommendations for design of the rat comet assay. Mutagenesis 23(3), 233–240.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Brian Burlinson .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2012 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Burlinson, B. (2012). The In Vitro and In Vivo Comet Assays. In: Parry, J., Parry, E. (eds) Genetic Toxicology. Methods in Molecular Biology, vol 817. Springer, New York, NY. https://doi.org/10.1007/978-1-61779-421-6_8

Download citation

  • DOI: https://doi.org/10.1007/978-1-61779-421-6_8

  • Published:

  • Publisher Name: Springer, New York, NY

  • Print ISBN: 978-1-61779-420-9

  • Online ISBN: 978-1-61779-421-6

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics