Skip to main content
SpringerLink
Log in

The Fast-Halo Assay for the Assessment of DNA Damage at the Single-Cell Level

  • Protocol
  • First Online:
DNA Replication

Part of the book series: Methods in Molecular Biology ((MIMB,volume 521))

Summary

The detection of breaks in mammalian cell DNA and the measurement of their repair represent primary endpoints for genotoxicity testing. Over the past three decades many techniques sensitive to the presence of DNA breaks have been developed: their availability significantly increased the knowledge in the area of genetic toxicology. In general, these techniques have evolved to become more sensitive and flexible as well as less complicated. The fast-halo assay (FHA) is a very recent method to detect DNA-strand breakage induced either by various genotoxic agents or secondary to apoptotic DNA cleavage, and to study the repair of primary DNA breaks at the single-cell level. In FHA, damaged DNA is separated from intact one by means of solvent gradient, stained with ethidium bromide and visualized under a fluorescence microscope. The level of DNA breaks is then determined with an image analysis software. FHA is as sensitive, reliable, and flexible as the well-established comet assay, but it has the advantage of being, as compared to any other existing method, the most rapid and less expensive one. Taken collectively, these unique features render FHA the ideal method to perform a large number of genotoxicity tests on mammalian cells in a particularly ­cost-effective and time-saving manner.

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

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
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. Moustacchi, E. (2000) DNA damage and repair: consequences on dose-responses, Mutat. Res. 464, 35–40

    PubMed  CAS  Google Scholar 

  2. Furihata, C., and Matsushima, T. (1987) Use of in vivo/in vitro unscheduled DNA synthesis for identification of organ-specific carcinogens, Crit. Rev. Toxicol. 17, 245–277

    Article  PubMed  CAS  Google Scholar 

  3. Erixon, K., and Ahnstrom, G. (1979) Single-strand breaks in DNA during repair of UV-induced damage in normal human and xeroderma pigmentosum cells as determined by alkaline DNA unwinding and hydroxylapatite chromatography: effects of hydroxyurea, 5-fluorodeoxyuridine and 1-beta-d-arabinofuranosylcytosine on the kinetics of repair, Mutat. Res. 59, 257–271

    Article  PubMed  CAS  Google Scholar 

  4. Kohn, K.W., and Grimek-Ewig, R.A. (1973) Alkaline elution analysis, a new approach to the study of DNA single-strand interruptions in cells, Cancer Res. 33, 1849–1853

    PubMed  CAS  Google Scholar 

  5. Ostling, O., and Johanson, K.J. (1984) Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells, Biochem. Biophys. Res. Comm. 123, 291–298

    Article  PubMed  CAS  Google Scholar 

  6. 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 

  7. Tice, R.R., Andrews, P.W., and Singh, N.P. (1990) The single cell gel assay: a sensitive technique for evaluating intercellular differences in DNA damage and repair, Basic Life Sci. 53, 291–301

    PubMed  CAS  Google Scholar 

  8. Sestili, P., and Cantoni, O. (1999) Osmotically driven radial diffusion of single-stranded DNA fragments on an agarose bed as a convenient measure of DNA strand scission, Free Radic. Biol. Med. 26, 1019–1026

    Article  PubMed  CAS  Google Scholar 

  9. Sestili, P., Tommasini, I., and Cantoni, O. (2001) Peroxynitrite promotes mitochondrial permeability transition-dependent rapid U937 cell necrosis: survivors proliferate with kinetics superimposable on those of untreated cells, Free Radic. Res. 34, 513–527

    Article  PubMed  CAS  Google Scholar 

  10. Sestili, P., Guidarelli, A., Dacha, M., and Cantoni, O. (1998) Quercetin prevents DNA single strand breakage and cytotoxicity caused by tert-butylhydroperoxide: free radical scavenging versus iron chelating mechanism, Free Radic. Biol. Med. 25, 196–200

    Article  PubMed  CAS  Google Scholar 

  11. Sestili, P., Diamantini, G., Bedini, A., Cerioni, L., Tommasini, I., Tarzia, G., et al (2002) Plant-derived phenolic compounds prevent the DNA single-strand breakage and cytotoxicity induced by tert-butylhydroperoxide via an iron-chelating mechanism, Biochem. J. 364, 121–128

    PubMed  CAS  Google Scholar 

  12. Sestili, P., Alfieri, R., Carnicelli, D., Martinelli, C., Barbieri, L., Stirpe, F., et al (2005) Shiga toxin 1 and ricin inhibit the repair of H2O2-induced DNA single strand breaks in cultured mammalian cells, DNA Repair (Amst) 4, 271–277

    Article  CAS  Google Scholar 

  13. Guidarelli, A., Sestili, P., Fiorani, M., and Cantoni, O. (2000) Arachidonic acid induces calcium-dependent mitochondrial formation of species promoting strand scission of genomic DNA, Free Radic. Biol. Med. 28, 1619–1627

    Article  PubMed  CAS  Google Scholar 

  14. Brigotti, M., Alfieri, R., Sestili, P., Bonelli, M., Petronini, P.G., Guidarelli, A., et al (2002) Damage to nuclear DNA induced by Shiga toxin 1 and ricin in human endothelial cells, FASEB J. 16, 365–372

    Article  PubMed  CAS  Google Scholar 

  15. Wright, W.D., Lagroye, I., Zhang, P., Malyapa, R.S., and Roti, J.L. (2001) Cytometric methods to analyze ionizing-radiation effects, Methods Cell. Biol. 64, 251–268

    Article  PubMed  CAS  Google Scholar 

  16. Singh, N.P. (2000) A simple method for accurate estimation of apoptotic cells, Exp. Cell. Res. 256, 328–337

    Article  PubMed  CAS  Google Scholar 

  17. Meintieres, S., Nesslany, F., Pallardy, M., and Marzin, D. (2003) Detection of ghost cells in the standard alkaline comet assay is not a good measure of apoptosis, Environ. Mol. Mutagen. 41, 260–269

    Article  PubMed  CAS  Google Scholar 

  18. Kumar, I.P., Namita, S., and Goel, H.C. (2002) Modulation of chromatin organization by RH-3, a preparation of Hippophae rhamnoides, a possible role in radioprotection, Mol. Cell. Biochem. 238, 1–9

    Article  PubMed  Google Scholar 

  19. Grasso, S., Scifo, C., Cardile, V., Gulino, R., and Renis, M. (2003) Adaptive responses to the stress induced by hyperthermia or hydrogen peroxide in human fibroblasts, Exp. Biol. Med. (Maywood) 228, 491–498

    CAS  Google Scholar 

  20. Dwarakanath, B.S., Khaitan, D., and Ravindranath, T. (2004) 2-Deoxy-D-glucose enhances the cytotoxicity of topoisomerase inhibitors in human tumor cell lines, Cancer. Biol. Ther. 3, 864–870

    Article  PubMed  CAS  Google Scholar 

  21. Bacso, Z., and Eliason, J.F. (2001) Measurement of DNA damage associated with apoptosis by laser scanning cytometry, Cytometry 45, 180–186

    Article  PubMed  CAS  Google Scholar 

  22. Sestili, P., Martinelli, C., and Stocchi, V. (2006) The fast halo assay: an improved method to quantify genomic DNA strand breakage at the single-cell level, Mutat. Res. 607, 205–214

    PubMed  CAS  Google Scholar 

  23. Brigotti, M., Carnicelli, D., Ravanelli, E., Vara, A.G., Martinelli, C., Alfieri, R.R., et al (2007) Molecular damage and induction of proinflammatory cytokines in human endothelial cells exposed to Shiga toxin 1, Shiga toxin 2, and alpha-sarcin, Infect Immun 75, 2201–2207

    Article  PubMed  CAS  Google Scholar 

  24. Sestili, P., Martinelli, C., Ricci, D., Fraternale, D., Bucchini, A., Giamperi, L., et al (2007) Cytoprotective effect of preparations from various parts of Punica granatum L. fruits in oxidatively injured mammalian cells in comparison with their antioxidant capacity in cell free systems, Pharmacol. Res. 56, 18–26

    Article  PubMed  CAS  Google Scholar 

  25. Guidi, C., Potenza, L., Sestili, P., Martinelli, C., Guescini, M., Stocchi, L., et al (2008) Differential effect of creatine on oxidatively-injured mitochondrial and nuclear DNA, Biochim. Biophys. Acta 1780, 16–26

    Article  PubMed  CAS  Google Scholar 

  26. Morgan, A.R., Evans, D.H., Lee, J.S., and Pulleyblank, D.E. (1979) Review: ethidium fluorescence assay. Part II. Enzymatic studies and DNA-protein interactions, Nucleic Acids Res. 7, 571–594

    Article  PubMed  CAS  Google Scholar 

  27. Cook, P.R., and Brazell, I.A. (1977) The superhelical density of nuclear DNA from human cells, Eur. J. Biochem. 74, 527–531

    Article  PubMed  CAS  Google Scholar 

  28. McGlynn, A.P., Wasson, G., O’Connor, J., McKerr, G., McKelvey-Martin, V.J., and Downes, C.S. (1999) The bromodeoxyuridine comet assay: detection of maturation of recently replicated DNA in individual cells, Cancer Res. 59, 5912–5916

    PubMed  CAS  Google Scholar 

  29. Eriksson, S., Nygren, J., and Ahnstrom, G. (2002) Matrix association of early- and late-replicating chromatin studied by single-cell electrophoresis, Biochim. Biophys. Acta 1590, 103–108

    Article  PubMed  CAS  Google Scholar 

  30. Sestili, P., Cattabeni, F., and Cantoni, O. (1996) Direct excision of 50 kb pair DNA fragments from megabase-sized fragments produced during apoptotic cleavage of genomic DNA, FEBS Lett. 396, 337–342

    Article  PubMed  CAS  Google Scholar 

  31. Lunn, G., and Sansone, E.B. (1987) Ethidium bromide: destruction and decontamination of solutions, Anal. Biochem. 162, 453–458

    Article  PubMed  CAS  Google Scholar 

  32. Burlinson, B.et al (2007) Fourth International Workgroup on Genotoxicity testing: results of the in vivo Comet assay workgroup, Mutat. Res. 627, 31–35

    PubMed  CAS  Google Scholar 

  33. Ross, G.M., McMillan, T.J., Wilcox, P., and Collins, A.R. (1995) The single cell microgel electrophoresis assay (comet assay): technical aspects and applications. Report on the 5th LH Gray Trust Workshop, Institute of Cancer Research, 1994, Mutat. Res. 337, 57–60

    PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Istituto di Ricerca sull’Attività Motoria, Via Maggetti 26, and Istituto di Farmacologia e Farmacognosia, Via S. Chiara 27 – Università degli Studi di Urbino “Carlo Bo”, 61029, Urbino, Italy

    Piero Sestili

Authors
  1. Piero Sestili
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Piero Sestili .

Editor information

Editors and Affiliations

  1. Marie Curie Research Institute, The Chart, Oxted, RH 8 0TL, United Kingdom

    Sonya Vengrova

  2. Marie Curie Research Institute, The Chart, Oxted, RH 8 0TL, United Kingdom

    Jacob Z. Dalgaard

Rights and permissions

Reprints and permissions

Copyright information

© 2009 Humana Press, a part of Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Sestili, P. (2009). The Fast-Halo Assay for the Assessment of DNA Damage at the Single-Cell Level. In: Vengrova, S., Dalgaard, J. (eds) DNA Replication. Methods in Molecular Biology, vol 521. Humana Press. https://doi.org/10.1007/978-1-60327-815-7_30

Download citation

  • DOI: https://doi.org/10.1007/978-1-60327-815-7_30

  • Published:

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-60327-814-0

  • Online ISBN: 978-1-60327-815-7

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation