Skip to main content
SpringerLink
Log in

Alkaline Nuclear Dispersion Assays for the Determination of DNA Damage at the Single Cell Level

  • Protocol
  • First Online:
Functional Analysis of DNA and Chromatin

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1094))

Abstract

Over the past three decades the development of methods for visualizing at the cell level the extent of DNA breakage significantly contributed to genotoxicity testing: their availability greatly improved the knowledge in the field of genetic toxicology. These procedures are based on the separation and visualization of DNA fragments resulting from cleavage of nuclear DNA. The separation process can be obtained either electrically (comet assay, linear migration of DNA fragments) or chemically (alkaline dispersion assays, radial diffusion of DNA fragments). Once separated and stained, intact and fragmented DNA can be observed with fluorescence or light microscope. Appropriate computer-assisted image analysis allows quantitative determination of the extent of DNA breakage. These procedures have been proven to be sensitive, flexible, and reliable, and, as compared to former methods, they are simpler, are less time and money consuming, and have the unique capability of detecting DNA damage at the single cell level. This last feature has the additional advantage of allowing the identification of cellular subpopulations characterized by different sensitivity to the damaging agent. The fast halo assay (FHA) is currently the simplest and quickest nuclear dispersion assay; recent modifications of FHA have further improved the assay and pave the way to a full exploitation of its analytical potential. In this chapter the development, procedures, applications, and limits of these dispersion assays, with a particular focus on FHA, will be illustrated.

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 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 109.99
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. Moustacchi E (2000) DNA damage and repair: consequences on dose-responses. Mutat Res 464:35–40

    Article  PubMed  CAS  Google Scholar 

  2. Lou Z, Chen J (2005) Mammalian DNA damage response pathway. Adv Exp Med Biol 570:425–455

    Article  PubMed  CAS  Google Scholar 

  3. Furihata C, 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 

  4. Erixon K, 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 

  5. Kohn KW, Grimek-Ewig RA (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 

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

    Article  PubMed  CAS  Google Scholar 

  7. 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  PubMed  CAS  Google Scholar 

  8. Tice RR, Andrews PW, Singh NP (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 

  9. Sestili P, 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 

  10. Singh NP (2000) A simple method for accurate estimation of apoptotic cells. Exp Cell Res 256:328–337

    Article  PubMed  CAS  Google Scholar 

  11. Meintieres S, Nesslany F, Pallardy M et al (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 

  12. Dwarakanath BS, Khaitan D, 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 

  13. Sestili P, Martinelli C, 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

    Article  PubMed  CAS  Google Scholar 

  14. Cantoni O, Sestili P, Cattabeni F et al (1990) Comparative effects of doxorubicin and 4'-epi-doxorubicin on nucleic acid metabolism and cytotoxicity in a human tumor cell line. Cancer Chemother Pharmacol 27:47–51

    Article  PubMed  CAS  Google Scholar 

  15. Sestili P, Cantoni O, Cattabeni F et al (1995) Evidence for separate mechanisms of cytotoxicity in mammalian cells treated with hydrogen peroxide in the absence or presence of L-histidine. Biochim Biophys Acta 1268:130–136

    Article  PubMed  Google Scholar 

  16. Burattini S, Ferri P, Battistelli M et al (2009) Apoptotic DNA fragmentation can be revealed in situ: an ultrastructural approach. Microsc Res Tech 72:913–923

    Article  PubMed  CAS  Google Scholar 

  17. Singh NP (2005) Apoptosis assessment by the DNA diffusion assay. Methods Mol Med 111:55–67

    PubMed  CAS  Google Scholar 

  18. Roti Roti JL, Wright WD (1987) Visualization of DNA loops in nucleoids from HeLa cells: assays for DNA damage and repair. Cytometry 8:461–467

    Article  PubMed  CAS  Google Scholar 

  19. Fernández JL, Muriel L, Goyanes V et al (2005) Simple determination of human sperm DNA fragmentation with an improved sperm chromatin dispersion test. Fertil Steril 84:833–842

    Article  PubMed  Google Scholar 

  20. Galaz-Leiva S, Perez-Rodriguez G, Blázquez-Castro A et al (2012) A simplified chromatin dispersion (nuclear halo) assay for detecting DNA breakage induced by ionizing radiation and chemical agents. Biotech Histochem 87:208–217

    Article  PubMed  CAS  Google Scholar 

  21. Qiao Y, Wang C, Su M et al (2012) Single cell DNA damage/repair assay using HaloChip. Anal Chem 84:1112–1116

    Article  PubMed  CAS  Google Scholar 

  22. Sestili P, Cattabeni F, 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 

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

    Article  PubMed  CAS  Google Scholar 

  24. Burlinson B, Tice RR, Speit G et al (2007) Fourth International Workgroup on genotoxicity testing: results of the in vivo comet assay workgroup. Mutat Res 627:31–35

    Article  PubMed  CAS  Google Scholar 

  25. Ross GM, McMillan TJ, Wilcox P et al (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

    Article  PubMed  CAS  Google Scholar 

  26. Sestili P, Paolillo M, Lenzi M et al (2010) Sulforaphane induces DNA single strand breaks in cultured human cells. Mutat Res 689:65–73

    Article  PubMed  CAS  Google Scholar 

  27. Godard T, Deslandes E, Lebailly P et al (1999) Early detection of staurosporine-induced apoptosis by comet and annexin V assays. Histochem Cell Biol 112:155–161

    Article  PubMed  CAS  Google Scholar 

  28. Bacso Z, Eliason JF (2001) Measurement of DNA damage associated with apoptosis by laser scanning cytometry. Cytometry 45:180–186

    Article  PubMed  CAS  Google Scholar 

  29. Guidarelli A, Sestili P, Fiorani M et al (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 

  30. Trivedi PP, Tripathi DN, Jena GB (2011) Hesperetin protects testicular toxicity of doxorubicin in rat: role of NFkappaB, p38 and caspase-3. Food Chem Toxicol 49:838–847

    Article  PubMed  CAS  Google Scholar 

  31. Vivek Kumar PR, Cheriyan VD, Seshadri M (2009) Could a strong alkali deproteinization replace the standard lysis step in alkaline single cell gel electrophoresis (comet) assay (pH > 13)? Mutat Res 678:65–70

    Article  PubMed  CAS  Google Scholar 

  32. Vorob'eva N, Antonenko AV, Osipov AN (2011) Particularities of blood lymphocyte response to irradiation in vitro in breast cancer patients. Radiats Biol Radioecol 51:451–456

    PubMed  Google Scholar 

  33. Chaudhary P, Shukla SK, Sharma RK (2011) REC-2006-A fractionated extract of Podophyllum hexandrum protects cellular DNA from radiation-induced damage by reducing the initial damage and enhancing its repair in vivo. Evid Based Complement Alternat Med 2011:473953

    Article  PubMed  Google Scholar 

  34. Guidi C, Potenza L, Sestili P 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 

  35. Sestili P, Alfieri R, Carnicelli D 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 

  36. Crimella C, Cantoni O, Guidarelli A et al (2011) A novel nonsense mutation in the APTX gene associated with delayed DNA single-strand break removal fails to enhance sensitivity to different genotoxic agents. Hum Mutat 32:E2118–E2133

    Article  PubMed  Google Scholar 

  37. Sestili P, Martinelli C, Ricci D 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 

  38. Grasso S, Scifo C, Cardile V et al (2003) Adaptive responses to the stress induced by hyperthermia or hydrogen peroxide in human fibroblasts. Exp Biol Med 228:491–498

    CAS  Google Scholar 

  39. Guidarelli A, Palomba L, Fiorani M et al (2008) Susceptibility of rat astrocytes to DNA strand scission induced by activation of NADPH oxidase and collateral resistance to the effects of peroxynitrite. Free Radic Biol Med 45:521–529

    Article  PubMed  CAS  Google Scholar 

  40. Di Pietro A, Baluce B, Visalli G et al (2011) Ex vivo study for the assessment of behavioral factor and gene polymorphisms in individual susceptibility to oxidative DNA damage metals-induced. Int J Hyg Environ Health 214:210–218

    Article  PubMed  Google Scholar 

  41. Cantoni O, Guidarelli A (2008) Indirect mechanisms of DNA strand scission by peroxynitrite. Methods Enzymol 440:111–120

    Article  PubMed  CAS  Google Scholar 

  42. Guidarelli A, Cerioni L, Cantoni O (2007) Inhibition of complex III promotes loss of Ca2+ dependence for mitochondrial superoxide formation and permeability transition evoked by peroxynitrite. J Cell Sci 120:1908–1914

    Article  PubMed  CAS  Google Scholar 

  43. Guidarelli A, Cerioni L, Tommasini I et al (2005) Role of Bcl-2 in the arachidonate-mediated survival signaling preventing mitochondrial permeability transition-dependent U937 cell necrosis induced by peroxynitrite. Free Radic Biol Med 39:1638–1649

    Article  PubMed  CAS  Google Scholar 

  44. Guidarelli A, Sciorati C, Clementi E et al (2006) Peroxynitrite mobilizes calcium ions from ryanodine-sensitive stores, a process associated with the mitochondrial accumulation of the cation and the enforced formation of species mediating cleavage of genomic DNA. Free Radic Biol Med 41:154–164

    Article  PubMed  CAS  Google Scholar 

  45. Guidarelli A, Palomba L, Cantoni O (2000) Peroxynitrite-mediated release of arachidonic acid from PC12 cells. Br J Pharmacol 129:1539–1541

    Article  PubMed  CAS  Google Scholar 

  46. Guidarelli A, Cerioni L, Fiorani M et al (2009) Differentiation-associated loss of ryanodine receptors: a strategy adopted by monocytes/macrophages to prevent the DNA single-strand breakage induced by peroxynitrite. J Immunol 183:4449–4457

    Article  PubMed  CAS  Google Scholar 

  47. Kadioglu E, Sardas S, Erturk S et al (2009) Determination of DNA damage by alkaline halo and comet assay in patients under sevoflurane anesthesia. Toxicol Ind Health 25:205–212

    Article  PubMed  CAS  Google Scholar 

  48. Brigotti M, Alfieri R, Sestili P 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 

  49. Brigotti M, Carnicelli D, Ravanelli E 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 

  50. Mondal NK, Bhattacharya P, Ray MR (2011) Assessment of DNA damage by comet assay and fast halo assay in buccal epithelial cells of Indian women chronically exposed to biomass smoke. Int J Hyg Environ Health 214:311–318

    Article  PubMed  CAS  Google Scholar 

  51. Potenza L, Martinelli C, Polidori E et al (2010) Effects of a 300 mT static magnetic field on human umbilical vein endothelial cells. Bioelectromagnetics 31:630–639

    Article  PubMed  CAS  Google Scholar 

  52. Garcia-Alonso FJ, Guidarelli A, Periago MJ (2007) Phenolic-rich juice prevents DNA single-strand breakage and cytotoxicity caused by tert-butylhydroperoxide in U937 cells: the role of iron chelation. J Nutr Biochem 18:457–466

    Article  PubMed  CAS  Google Scholar 

  53. Guidarelli A, Clementi E, De Nadai C et al (2001) TNFalpha enhances the DNA single-strand breakage induced by the short-chain lipid hydroperoxide analogue tert-butylhydroperoxide via ceramide-dependent inhibition of complex III followed by enforced superoxide and hydrogen peroxide formation. Exp Cell Res 270:56–65

    Article  PubMed  CAS  Google Scholar 

  54. Guidarelli A, De Sanctis R, Cellini B et al (2001) Intracellular ascorbic acid enhances the DNA single-strand breakage and toxicity induced by peroxynitrite in U937 cells. Biochem J 356:509–513

    Article  PubMed  CAS  Google Scholar 

  55. Palomba L, Guidarelli A, Scovassi AI et al (2001) Different effects of tert-butylhydroperoxide-induced peroxynitrite-dependent and -independent DNA single-strand breakage on PC12 cell poly(ADP-ribose) polymerase activity. Eur J Biochem 268:5223–5228

    Article  PubMed  CAS  Google Scholar 

  56. Chaudhary P, Shukla SK, Kumar IP et al (2006) Radioprotective properties of apple polyphenols: an in vitro study. Mol Cell Biochem 288:37–46

    Article  PubMed  CAS  Google Scholar 

  57. Shukla SK, Chaudhary P, Kumar IP et al (2006) Protection from radiation-induced mitochondrial and genomic DNA damage by an extract of Hippophae rhamnoides. Environ Mol Mutagen 47:647–656

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

The authors wish to thank Prof. Alfonso Blázquez-Castro and Prof. Juan C. Stockert for critically reading and commenting on this chapter. The experimental work has been funded by M.I.U.R., PRIN 2009, 200974K3JC_002.

Author information

Authors and Affiliations

  1. Dipartimento di Scienze Biomolecolari, Università degli Studi di Urbino “Carlo Bo”, Urbino, Italy

    Piero Sestili

  2. Dipartimento di Farmacologia, Alma Mater Studiorum, Università di Bologna, Bologna, Italy

    Carmela Fimognari

Authors
  1. Piero Sestili
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Carmela Fimognari
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Department of Biology, Autonomous University of Madrid Faculty of Sciences, Madrid, Spain

    Juan C. Stockert

  2. Departamento de Biología, Facultad de Ciencias, Universidad Autónoma de Madrid, Madrid, Spain

    Jesús Espada

  3. Department of Biology, Autonomous University of Madrid Faculty of Sciences, Madrid, Spain

    Alfonso Blázquez-Castro

Rights and permissions

Reprints and permissions

Copyright information

© 2014 Springer Science+Business Media, New York

About this protocol

Cite this protocol

Sestili, P., Fimognari, C. (2014). Alkaline Nuclear Dispersion Assays for the Determination of DNA Damage at the Single Cell Level. In: Stockert, J., Espada, J., Blázquez-Castro, A. (eds) Functional Analysis of DNA and Chromatin. Methods in Molecular Biology, vol 1094. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-706-8_5

Download citation

  • DOI: https://doi.org/10.1007/978-1-62703-706-8_5

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-705-1

  • Online ISBN: 978-1-62703-706-8

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation