Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Immuno-spin trapping analyses of DNA radicals

Nature Protocols volume 2, pages 512–522 (2007)Cite this article

Abstract

Immuno-spin trapping is a highly sensitive method for detecting DNA radicals in biological systems. This technique involves three main steps: (i) in situ and real-time trapping of DNA radicals with the nitrone spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), thus forming DMPO–DNA nitrone adducts (referred to here as nitrone adducts); (ii) purification of nitrone adducts; and (iii) analysis of nitrone adducts by heterogeneous immunoassays using Abs against DMPO. In experiments, DMPO is added prior to the formation of free radicals. It diffuses easily through all cell compartments and is present when DNA free radicals are formed as a result of oxidative damage. Due to its low toxicity, DMPO can be used in cells at high enough concentrations to out-compete the normal reactions of DNA radicals, thus ensuring a high yield of DNA nitrone adducts. Because both protein and DNA nitrone adducts are formed, it is important that the DNA be pure in order to avoid misinterpretations. Depending on the model under study, this protocol can be completed in as few as 6 h.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Oxidatively generated damage to DNA and detection of DNA radicals.
Figure 2: Schematic representation of the generation and trapping of DNA radicals with the nitrone spin trap DMPO forming DNA nitrone adducts.
Figure 3: Immuno-spin trapping analysis of DNA radicals.
Figure 4: Immuno-spin trapping of DNA radicals in macrophages.

Similar content being viewed by others

References

  1. Halliwell, B. & Whiteman, M. Measuring reactive species and oxidative damage in vivo and in cell culture: how should you do it and what do the results mean? Br. J. Pharmacol. 142, 231–255 (2004).

    Article  CAS  Google Scholar 

  2. Dröge, W. Free radicals in the physiological control of cell function. Physiol. Rev. 82, 47–95 (2002).

    Article  Google Scholar 

  3. Halliwell, B. & Gutteridge, J.M. The chemistry of oxygen radicals and other oxygen-derived species. In Free Radicals in Biology and Medicine 20–66 (Oxford Univ. Press Inc., New York, USA, 1985).

    Google Scholar 

  4. Gracy, R.W., Talent, J.M., Kong, Y. & Conrad, C.C. Reactive oxygen species: the unavoidable environmental insult? Mutat. Res. 428, 17–22 (1999).

    Article  CAS  Google Scholar 

  5. Winyard, P.G., Moody, C.J. & Jacob, C. Oxidative activation of antioxidant defence. Trends Biochem. Sci. 30, 453–461 (2005).

    Article  CAS  Google Scholar 

  6. Imlay, J.A., Chin, S.M. & Linn, S. Toxic DNA damage by hydrogen peroxide through the Fenton reaction in vivo and in vitro . Science 240, 640–642 (1988).

    Article  CAS  Google Scholar 

  7. Cooke, M.S., Evans, M.D., Dizdaroglu, M. & Lunec, J. Oxidative DNA damage: mechanisms, mutation, and disease. FASEB J. 17, 1195–1214 (2003).

    Article  CAS  Google Scholar 

  8. Cadet, J., Douki, T., Gasparutto, D. & Ravanat, J.-L. Oxidative damage to DNA: formation, measurement and biochemical features. Mutat. Res. 531, 5–23 (2003).

    Article  CAS  Google Scholar 

  9. von Sonntag, C. DNA model systems. In The Chemical Basis of Radiation Biology 116–294 (Taylor & Francis Ltd., London, UK, 1987).

    Google Scholar 

  10. Hall, D.B., Holmlin, R.E. & Barton, J.K. Oxidative DNA damage through long-range electron transfer. Nature 382, 731–735 (1996).

    Article  CAS  Google Scholar 

  11. Purkayastha, S., Milligan, J.R. & Bernhard, W.A. Correlation of free radical yields with strand break yields produced in plasmid DNA by the direct effect of ionizing radiation. J. Phys. Chem. B 109, 16967–16973 (2005).

    Article  CAS  Google Scholar 

  12. von Sonntag, C. The chemistry of free-radical-mediated DNA damage. Basic Life Sci. 58, 287–321 (1991).

    CAS  PubMed  Google Scholar 

  13. Collins, A.R., Cadet, J., Möller, L., Poulsen, H.E. & Viña, J. Are we sure we know how to measure 8-oxo-7,8-dihydroguanine in DNA from human cells? Arch. Biochem. Biophys. 423, 57–65 (2004).

    Article  CAS  Google Scholar 

  14. Fraga, C.G., Shigenaga, M.K., Park, J.-W., Degan, P. & Ames, B.N. Oxidative damage to DNA during aging: 8-hydroxy-2′-deoxyguanosine in rat organ DNA and urine. Proc. Natl. Acad. Sci. USA 87, 4533–4537 (1990).

    Article  CAS  Google Scholar 

  15. European Standards Committee on Oxidative DNA Damage (ESCODD). Measurement of DNA oxidation in human cells by chromatographic and enzymic methods. Free Radic. Biol. Med. 34, 1089–1099 (2003).

  16. Hildenbrand, K. & Schulte-Frohlinde, D. ESR spectra of radicals of single-stranded and double-stranded DNA in aqueous solution. Implications for •OH-induced strand breakage. Free Radic. Res. Commun. 11, 195–206 (1990).

    Article  CAS  Google Scholar 

  17. Cullis, P.M., Jones, G.D.D., Symons, M.C.R. & Lea, J.S. Electron transfer from protein to DNA in irradiated chromatin. Nature 330, 773–774 (1987).

    Article  CAS  Google Scholar 

  18. Khan, N. et al. Spin traps: in vitro toxicity and stability of radical adducts. Free Radic. Biol. Med. 34, 1473–1481 (2003).

    Article  CAS  Google Scholar 

  19. Haseloff, R.F. et al. Cytotoxicity of spin trapping compounds. FEBS Lett. 418, 73–75 (1997).

    Article  CAS  Google Scholar 

  20. Schaefer, C.F., Janzen, E.G., West, M.S., Poyer, J.L. & Kosanke, S.D. Blood chemistry changes in the rat induced by high doses of nitronyl free radical spin traps. Free Radic. Biol. Med. 21, 427–436 (1996).

    Article  CAS  Google Scholar 

  21. Anzai, K. et al. ESR measurement of rapid penetration of DMPO and DEPMPO spin traps through lipid bilayer membranes. Arch. Biochem. Biophys. 415, 251–256 (2003).

    Article  CAS  Google Scholar 

  22. Ho, W.F., Gilbert, B.C. & Davies, M.J. EPR spin-trapping studies of radicals generated from FeII-catalysed degradation of nucleobase, nucleoside, RNA and DNA hydroperoxides. J. Chem. Soc. Perkin Trans. 2, 2525–2531 (1997).

    Article  Google Scholar 

  23. Kuwabara, M., Ohshima, H., Sato, F., Ono, A. & Matsuda, A. Spin-trapping detection of precursors of hydroxyl-radical-induced DNA damage: identification of precursor radicals of DNA strand breaks in oligo(dC)10 and oligo(dT)10 . Biochemistry 32, 10599–10606 (1993).

    Article  CAS  Google Scholar 

  24. Maurel, V., Ravanat, J.-L. & Gambarelli, S. Detection of reactive free radicals derived from nucleosides by liquid chromatography coupled to tandem mass spectrometry of DMPO spin trapping adducts. Rapid Commun. Mass Spec. 20, 2235–2242 (2006).

    Article  CAS  Google Scholar 

  25. Janzen, E.G., West, M.S., Kotake, Y. & DuBose, C.M. Biological spin trapping methodology. III. Octanol-water partition coefficients of spin-trapping compounds. J. Biochem. Biophys. Methods 32, 183–190 (1996).

    Article  CAS  Google Scholar 

  26. Mason, R.P. Using anti-5,5-dimethyl-1-pyrroline N-oxide (anti-DMPO) to detect protein radicals in time and space with immuno-spin trapping. Free Radic. Biol. Med. 36, 1214–1223 (2004).

    Article  CAS  Google Scholar 

  27. Ramirez, D.C. & Mason, R.P. Immuno-spin trapping: detection of protein-centered radicals. In Current Protocols in Toxicology (eds. Costa, L.G., Maines, M.D., Reed, D.J., Sassa, S. & Sipes, I.G.) 17.7.1–17.7.16 (John Wiley & Sons, Hoboken, New Jersey, USA, 2005).

    Google Scholar 

  28. Ramirez, D.C., Gomez-Mejiba, S.E. & Mason, R.P. Immuno-spin trapping of DNA radicals. Nat. Methods 3, 123–127 (2006).

    Article  CAS  Google Scholar 

  29. Ramirez, D.C., Chen, Y.-R. & Mason, R.P. Immunochemical detection of hemoglobin-derived radicals formed by reaction with hydrogen peroxide: involvement of a protein-tyrosyl radical. Free Radic. Biol. Med. 34, 830–839 (2003).

    Article  CAS  Google Scholar 

  30. Detweiler, C.D. et al. Immunological identification of the heart myoglobin radical formed by hydrogen peroxide. Free Radic. Biol. Med. 33, 364–369 (2002).

    Article  CAS  Google Scholar 

  31. Strober, W. Trypan blue exclusion test of cell viability. In Current Protocols in Immunology (eds. Coligan, J.E., Kruisbeek, A.M., Margulies, D.H., Shevach, E.M. & Strober, W.) A3.B1–A3.B2 (John Wiley & Sons, Inc., Hoboken, New Jersey, USA, 1997).

    Google Scholar 

  32. Crowther, J.R. Titration of reagents. In Methods in Molecular Biology: The ELISA Guidebook Vol 149 83–113 (Humana Press Inc., Totowa, New Jersey, USA, 2001).

    Google Scholar 

  33. Hix, S., Kadiiska, M.B., Mason, R.P. & Augusto, O. In vivo metabolism of tert-butyl hydroperoxide to methyl radicals. EPR spin-trapping and DNA methylation studies. Chem. Res. Toxicol. 13, 1056–1064 (2000).

    Article  CAS  Google Scholar 

  34. Latour, I., Demoulin, J.B. & Buc-Calderon, P. Oxidative DNA damage by t-butyl hydroperoxide causes DNA single strand breaks which is not linked to cell lysis. A mechanistic study in freshly isolated rat hepatocytes. FEBS Lett. 373, 299–302 (1995).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by the National Institute of Environmental Health Sciences (NIEHS) Intramural Research Program. D.C.R. is the recipient of a National Institutes of Health Pathway for Independence Award (1 K99 ES015415-01). We acknowledge J. Corbett for her excellent technical assistance, and M. Ehrenshaft and M. Waalkes for their useful comments. We also thank M. Mason and A. Motten for helping in the editing of this manuscript.

Author information

Author notes

  1. ramirez1@niehs.nih.gov

Authors and Affiliations

  1. Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, National Institutes of Health, 111 TW Alexander Drive, Building 101, MD F0-02, Research Triangle Park, 27709, North Carolina, USA

    Dario C Ramirez, Sandra E Gomez-Mejiba & Ronald P Mason

Authors
  1. Dario C Ramirez
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sandra E Gomez-Mejiba
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ronald P Mason
    View author publications

    You can also search for this author in PubMed Google Scholar

Ethics declarations

Competing interests

The anti-DMPO serum has been licensed to the following companies: Cayman Chemicals (cat. no. 10006170-1), Alexis Biochemicals (cat. no. ALX-210-530-R100), Abcam (cat. no. ab23702), and Oxford Biomedical Research (cat. no. RT15). Ronald P. Mason receives a small royalty for this license.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Ramirez, D., Gomez-Mejiba, S. & Mason, R. Immuno-spin trapping analyses of DNA radicals. Nat Protoc 2, 512–522 (2007). https://doi.org/10.1038/nprot.2007.5

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2007.5

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing