Measuring Oxidative Stress in Cell Cultures, Animals and Humans: Analysis and Validation of Oxidatively Damaged DNA

  • Hanna L. Karlsson
  • Lennart MöllerEmail author
Part of the Oxidative Stress in Applied Basic Research and Clinical Practice book series (OXISTRESS)


An association between oxidative stress and different diseases has been described in the literature, but there are obvious shortcomings with methods available to assess oxidative stress. This chapter focuses on the analysis of markers for oxidative stress with special attention on biomarkers for analysis of oxidatively damaged DNA. Such markers have often been used, but the levels in healthy and diseased subjects show high variation in the literature. Therefore, various efforts that have been undertaken, or are ongoing, which aim to validate different methods for analysis of oxidatively damaged DNA, will be discussed. It is concluded that measuring oxidatively damaged DNA is a useful biomarker for oxidative stress and that further validation of this biomarker is important. Oxidatively damaged DNA can be used as a marker to monitor oxidative stress in different diseases or following different exposures. However, it may also have the potential to act as a biomarker of disease development risk and may be used clinically to assess efficacy of therapy.


Comet assay DNA damage Oxidative stress Proteins 



The authors of this paper are also partners in ECNIS (Environmental Cancer Risk, Nutrition and Individual Susceptibility), a network of excellence operating within the European Union 6th framework program, priority 5: “Food Quality and Safety” (Contract No. 513943), as well as ESCODD and ECVAG.


  1. 1.
    Halliwell, B. and Gutteridge, J. M. C. (1999) Free radicals in biology and medicine. Oxford University Press Oxford, 1999.Google Scholar
  2. 2.
    Cooke, M. S., Olinski, R. and Evans, M. D. (2006) Does measurement of oxidative damage to DNA have clinical significance? Clin Chim Acta 365, 30–49.Google Scholar
  3. 3.
    Finkel, T. and Holbrook, N. J. (2000) Oxidants, oxidative stress and the biology of ageing. Nature 408, 239–47.Google Scholar
  4. 4.
    Valko, M., Izakovic, M., Mazur, M., Rhodes, C. J. and Telser, J. (2004) Role of oxygen radicals in DNA damage and cancer incidence. Mol Cell Biochem 266, 37–56.Google Scholar
  5. 5.
    Allen, R. G. and Tresini, M. (2000) Oxidative stress and gene regulation. Free Radic Biol Med 28, 463–99.Google Scholar
  6. 6.
    Li, N., Hao, M., Phalen, R. F., Hinds, W. C. and Nel, A. E. (2003) Particulate air pollutants and asthma. A paradigm for the role of oxidative stress in PM-induced adverse health effects. Clin Immunol 109, 250–65.Google Scholar
  7. 7.
    Li, N., Kim, S., Wang, M., Froines, J., Sioutas, C. and Nel, A. (2002) Use of a stratified oxidative stress model to study the biological effects of ambient concentrated and diesel exhaust particulate matter. Inhal Toxicol 14, 459–86.Google Scholar
  8. 8.
    Møller, P., Jacobsen, N. R., Folkmann, J. K., Danielsen, P. H., Mikkelsen, L., Hemmingsen, J. G., Vesterdal, L. K., Forchhammer, L., et al. Role of oxidative damage in toxicity of particulates. Free Radic Res 44, 1–46.Google Scholar
  9. 9.
    Halliwell, B. and Whiteman, M. (2004) 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, 231–55.Google Scholar
  10. 10.
    Shi, T., Duffin, R., Borm, P. J., Li, H., Weishaupt, C. and Schins, R. P. (2005) Hydroxyl-radical-dependent DNA damage by ambient particulate matter from contrasting sampling locations. Environ Res 16, 16.Google Scholar
  11. 11.
    Cheung, K. L., Polidori, A., Ntziachristos, L., Tzamkiozis, T., Samaras, Z., Cassee, F. R., Gerlofs, M. and Sioutas, C. (2009) Chemical characteristics and oxidative potential of particulate matter emissions from gasoline, diesel, and biodiesel cars. Environ Sci Technol 43, 6334–40.Google Scholar
  12. 12.
    Karlsson, H. L., Nilsson, L. and Möller, L. (2005) Subway particles are more genotoxic than street particles and induce oxidative stress in cultured human lung cells. Chem Res Toxicol 18, 19–23.Google Scholar
  13. 13.
    Karlsson, H. L., Nygren, J. and Möller, L. (2004) Genotoxicity of airborne particulate matter: the role of cell-particle interaction and of substances with adduct-forming and oxidizing capacity. Mutat Res. 565, 1–10.Google Scholar
  14. 14.
    Hofer, T. and Möller, L. (1998) Reduction of oxidation during the preparation of DNA and analysis of 8-hydroxy-2’-deoxyguanosine. Chem Res Toxicol 11, 882–7.Google Scholar
  15. 15.
    Mudway, I. S., Stenfors, N., Duggan, S. T., Roxborough, H., Zielinski, H., Marklund, S. L., Blomberg, A., Frew, A. J., et al. (2004) An in vitro and in vivo investigation of the effects of diesel exhaust on human airway lining fluid antioxidants. Arch Biochem Biophys 423, 200–12.Google Scholar
  16. 16.
    Ayres, J. G., Borm, P., Cassee, F. R., Castranova, V., Donaldson, K., Ghio, A., Harrison, R. M., Hider, R., et al. (2008) Evaluating the toxicity of airborne particulate matter and nanoparticles by measuring oxidative stress potential--a workshop report and consensus statement. Inhal Toxicol 20, 75–99.Google Scholar
  17. 17.
    Qin, Y., Lu, M. and Gong, X. (2008) Dihydrorhodamine 123 is superior to 2,7-dichlorodihydrofluorescein diacetate and dihydrorhodamine 6G in detecting intracellular hydrogen peroxide in tumor cells. Cell Biol Int 32, 224–8.Google Scholar
  18. 18.
    Karlsson, H. L., Cronholm, P., Gustafsson, J. and Möller, L. (2008) Copper oxide nanoparticles are highly toxic: a comparison between metal oxide nanoparticles and carbon nanotubes. Chem Res Toxicol 21, 1726–32.Google Scholar
  19. 19.
    Xia, T., Kovochich, M., Liong, M., Madler, L., Gilbert, B., Shi, H., Yeh, J. I., Zink, J. I., et al. (2008) Comparison of the mechanism of toxicity of zinc oxide and cerium oxide nanoparticles based on dissolution and oxidative stress properties. ACS Nano 2, 2121–34.Google Scholar
  20. 20.
    Risom, L., Dybdahl, M., Bornholdt, J., Vogel, U., Wallin, H., Møller, P. and Loft, S. (2003) Oxidative DNA damage and defence gene expression in the mouse lung after short-term exposure to diesel exhaust particles by inhalation. Carcinogenesis 24, 1847–52.Google Scholar
  21. 21.
    Rahman, I., Kode, A. and Biswas, S. K. (2006) Assay for quantitative determination of glutathione and glutathione disulfide levels using enzymatic recycling method. Nat Protoc 1, 3159–65.Google Scholar
  22. 22.
    Rahman, I. and Biswas, S. K. (2004) Non-invasive biomarkers of oxidative stress: reproducibility and methodological issues. Redox Rep 9, 125–43.Google Scholar
  23. 23.
    Sørensen, M., Daneshvar, B., Hansen, M., Dragsted, L. O., Hertel, O., Knudsen, L. and Loft, S. (2003) Personal PM2.5 exposure and markers of oxidative stress in blood. Environ Health Perspect 111, 161–6.Google Scholar
  24. 24.
    Roberts, L. J. and Morrow, J. D. (2002) Products of the isoprostane pathway: unique bioactive compounds and markers of lipid peroxidation. Cell Mol Life Sci. 59, 808–20.Google Scholar
  25. 25.
    Dalle-Donne, I., Rossi, R., Colombo, R., Giustarini, D. and Milzani, A. (2006) Biomarkers of oxidative damage in human disease. Clin Chem. 52, 601–23.Google Scholar
  26. 26.
    Kasai, H. and Nishimura, S. (1984) Hydroxylation of deoxyguanosine at the C-8 position by ascorbic acid and other reducing agents. Nucleic Acids Res 12, 2137–45.Google Scholar
  27. 27.
    David, S. S., O’Shea, V. L. and Kundu, S. (2007) Base-excision repair of oxidative DNA damage. Nature 447, 941–50.Google Scholar
  28. 28.
    Evans, M. D., Dizdaroglu, M. and Cooke, M. S. (2004) Oxidative DNA damage and disease: induction, repair and significance. Mutat Res 567, 1–61.Google Scholar
  29. 29.
    Collins, A. R., Cadet, J., Möller, L., Poulsen, H. E. and Vina, J. (2004) Are we sure we know how to measure 8-oxo-7,8-dihydroguanine in DNA from human cells? Arch Biochem Biophys 423, 57–65.Google Scholar
  30. 30.
    Hofer, T. and Möller, L. (2002) Optimization of the workup procedure for the analysis of 8-oxo-7,8-dihydro-2’-deoxyguanosine with electrochemical detection. Chem Res Toxicol 15, 426–32.Google Scholar
  31. 31.
    Möller, L., Hofer, T. and Zeisig, M. (1998) Methodological considerations and factors affecting 8-hydroxy-2-deoxyguanosine analysis. Free Radic Res 29, 511–24.Google Scholar
  32. 32.
    Hofer, T., Karlsson, H. L. and Möller, L. (2006) DNA oxidative damage and strand breaks in young healthy individuals: a gender difference and the role of life style factors. Free Radic Res 40, 707–14.Google Scholar
  33. 33.
    Iwai, K., Adachi, S., Takahashi, M., Möller, L., Udagawa, T., Mizuno, S. and Sugawara, I. (2000) Early oxidative DNA damages and late development of lung cancer in diesel exhaust-exposed rats. Environ Res 84, 255–64.Google Scholar
  34. 34.
    Östling, O. and Johanson, K. J. (1984) Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells. Biochem Biophys Res Commun 123, 291–8.Google Scholar
  35. 35.
    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, 184–91.Google Scholar
  36. 36.
    Møller, P., Folkmann, J. K., Forchhammer, L., Brauner, E. V., Danielsen, P. H., Risom, L. and Loft, S. (2008) Air pollution, oxidative damage to DNA, and carcinogenesis. Cancer Lett 266, 84–97.Google Scholar
  37. 37.
    Smith, C. C., O’Donovan, M. R. and Martin, E. A. (2006) hOGG1 recognizes oxidative damage using the comet assay with greater specificity than FPG or ENDOIII. Mutagenesis 21, 185–90.Google Scholar
  38. 38.
    Gedik, C. M., Collins, A. and ESCODD (2005) Establishing the background level of base oxidation in human lymphocyte DNA: results of an interlaboratory validation study. Faseb J 19, 82–4.Google Scholar
  39. 39.
    Nagy, E., Adachi, S., Takamura-Enya, T., Zeisig, M. and Möller, L. (2007) DNA adduct formation and oxidative stress from the carcinogenic urban air pollutant 3-nitrobenzanthrone and its isomer 2-nitrobenzanthrone, in vitro and in vivo. Mutagenesis 22, 135–45.Google Scholar
  40. 40.
    Karlsson, H. L., Holgersson, A. and Möller, L. (2008) Mechanisms Related to the Genotoxicity of Particles in the Subway and from Other Sources. Chem Res Toxicol 21, 726–31.Google Scholar
  41. 41.
    Karlsson, H. L., Gustafsson, J., Cronholm, P. and Möller, L. (2009) Size-dependent toxicity of metal oxide particles – a comparison between nano- and micrometer size. Toxicol Lett 188, 112–8.Google Scholar
  42. 42.
    Arranz, N., Haza, A. I., Garcia, A., Möller, L., Rafter, J. and Morales, P. (2006) Protective effects of isothiocyanates towards N-nitrosamine-induced DNA damage in the single-cell gel electrophoresis (SCGE)/HepG2 assay. J Appl Toxicol 26, 466–73.Google Scholar
  43. 43.
    Johansson, C., Rytter, E., Nygren, J., Vessby, B., Basu, S. and Möller, L. (2008) Down-regulation of oxidative DNA lesions in human mononuclear cells after antioxidant supplementation correlates to increase of gamma-tocopherol. Int J Vitam Nutr Res 78, 183–94.Google Scholar
  44. 44.
    Collins, A. R., Dusinska, M., Horvathova, E., Munro, E., Savio, M. and Stetina, R. (2001) Inter-individual differences in repair of DNA base oxidation, measured in vitro with the comet assay. Mutagenesis 16, 297–301.Google Scholar
  45. 45.
    Collins, A. R., Harrington, V., Drew, J. and Melvin, R. (2003) Nutritional modulation of DNA repair in a human intervention study. Carcinogenesis 24, 511–5.Google Scholar
  46. 46.
    Cooke, M. S., Olinski, R. and Loft, S. (2008) Measurement and meaning of oxidatively modified DNA lesions in urine. Cancer Epidemiol Biomarkers Prev 17, 3  –14.Google Scholar
  47. 47.
    ESCODD. (2002) Comparative analysis of baseline 8-oxo-7,8-dihydroguanine in mammalian cell DNA, by different methods in different laboratories: an approach to consensus. Carcinogenesis 23, 2129–33.Google Scholar
  48. 48.
    ESCODD. (2002) Inter-laboratory validation of procedures for measuring 8-oxo-7,8-dihydroguanine/8-oxo-7,8-dihydro-2’-deoxyguanosine in DNA. Free Radic Res 36, 239–45.Google Scholar
  49. 49.
    ESCODD. (2003) Measurement of DNA oxidation in human cells by chromatographic and enzymic methods. Free Radic Biol Med 34, 1089–99.Google Scholar
  50. 50.
    Johansson, C., Møller, P., Forchhammer, L., Loft, S., Godschalk, R. W., Langie, S. A., Lumeij, S., Jones, G. D., et al. (2009) An ECVAG trial on assessment of oxidative damage to DNA measured by the comet assay. Mutagenesis.Google Scholar
  51. 51.
    Evans, M. D., Olinski, R., Loft, S. and Cooke, M. S. (2009) Toward consensus in the analysis of urinary 8-oxo-7,8-dihydro-2’-deoxyguanosine as a noninvasive biomarker of oxidative stress. Faseb J. In press.Google Scholar
  52. 52.
    Loft, S. and Møller, P. (2006) Oxidative DNA damage and human cancer: need for cohort studies. Antioxid Redox Signal 8, 1021–31.Google Scholar
  53. 53.
    Loft, S., Svoboda, P., Kasai, H., Tjonneland, A., Vogel, U., Møller, P., Overvad, K. and Raaschou-Nielsen, O. (2006) Prospective study of 8-oxo-7,8-dihydro-2’-deoxyguanosine excretion and the risk of lung cancer. Carcinogenesis 27, 1245–50.Google Scholar

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© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Unit for Analytical Toxicology, Department of Biosciences and NutritionNovum, Karolinska InstitutetStockholmSweden

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