Abstract
The ability to non-invasively assess DNA oxidation and its repair, has significant utility in large-scale, population-based studies. Such studies could include the assessments of: the efficacy of antioxidant intervention strategies, pathological roles of DNA oxidation in various disease states and population or interindividual differences in antioxidant defence and DNA repair. The most popular method, to non-invasively assess oxidative insult to the genome is by the analysis of urine for 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG), using chromatographic techniques or immunoassay procedures. The provenance of extracellular 8-oxodG remains a subject for debate. However, previous studies have shown that factors, such as diet and cell death, do not appear to contribute to extracellular 8-oxodG, leaving processes, such as the repair of DNA and/or the 2′-deoxyribonucleotide pool, as the sole source of endogenous 8-oxodG. The method in this chapter describes a non-invasive approach for assessing oxidative stress, via the efficient extraction of urinary 8-oxodG using a validated solid-phase extraction procedure. Subsequent analysis by liquid chromatography-tandem mass spectrometry provides the advantages of sensitivity, internal standardisation, and robust peak identification, and is widely considered to be the “gold standard”.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Kasai, H. (1997) Analysis of a form of oxidative DNA damage, 8-hydroxy-2’-deoxyguanosine, as a marker of cellular oxidative stress during carcinogenesis. Mutat. Res. 387, 147–163.
Bogdanov, M.B., Beal, M.F., McCabe, D.R., Griffin, R.M., and Matson, W.R. (1999) A carbon column-based liquid chromatography electrochemical approach to routine 8-hydroxy-2′-deoxyguanosine measurements in urine and other biologic matrices: a one-year evaluation of methods. Free Radic. Biol. Med. 27, 647–666.
ESCODD, Gedik, C.M. and Collins, A. (2005). Establishing the background level of base oxidation in human lymphocyte DNA: results of an interlaboratory validation study. FASEB J 1, 82–84.
Loft, S., Svoboda, P., Kasai, H., Tjonneland, A., Vogel, U., Moller, 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, 1245–1250.
Wu, L.L., Chiou, C.-C., Chang, P.-Y., and Wu, J.T. (2004) Urinary 8-OHdG: a marker of oxidative stress to DNA and a risk factor for cancer, atherosclerosis and diabetics. Clin. Chim. Acta 339, 1–9.
Cooke, M. S., Evans, M. D., Dove, R., Rozalski, R., Gackowski, D., Siomek, A., Lunec, J., and Olinski, R. (2005) DNA repair is responsible for the presence of oxidatively damaged DNA lesions in urine. Mutat. Res. 574, 58–66.
Siomek, A., Tujakowski, J., Gackowski, D., Rozalski, R., Foksinski, M., Dziaman, T., Roszkowski, K., and Olinski, R. (2006) Severe oxidatively damaged DNA after cisplatin treatment of cancer patients. Int. J. Cancer 119, 2228–2230.
Cooke, M.S., Lunec, J., and Evans, M.D. (2002) Progress in the analysis of urinary oxidative DNA damage. Free Radic. Biol. Med. 33, 1601–1614.
Haghdoost, S., Sjolander, L., Czene, S., and Harms-Ringdahl, M. (2006) The nucleotide pool is a significant target for oxidative stress. Free Radic. Biol. Med. 41, 620–626.
Loft, S. and Poulsen, H.E. (1996). Cancer risk and oxidative DNA damage in man. J. Mol. Med. 74, 297–312.
ESCULA, Marcus, S.C., Olinski, R. and Loft, S. (2008) Measurement and meaning of oxidatively modified DNA lesions in urine. Cancer Epidemiol. Biomarkers Prev. 1, 3–14.
Haghdoost, S., Maruyama, Y., Pecoits-Filho, R., Heimburger, O., Seeberger, A., Anderstam, B., Suliman, M.E., Czene, S., Lindholm, B., Stenvinkel, P. and Harms-Ringdahl, M. (2006). Elevated serum 8-oxo-dG in hemodialysis patients: a marker of systemic inflammation? Antioxid. Redox Signal. 8(11–12), 2169–2173.
Rozalski, R., Siomek, A., Gackowski, D., Foksinski, M., Gran, C., Klungland, A., and Olinski, R. (2004) Diet is not responsible for the presence of several oxidatively damaged DNA lesions in mouse urine. Free Radic. Res. 11, 1201–1205.
Ravanat, J.L., Guicherd, P., Tuce, Z., and Cadet, J. (1999) Simultaneous determination of five oxidative DNA lesions in human urine. Chem. Res. Toxicol. 12, 802–808.
Hu, C.W., Wang, C.J., Chang, L.W., and Chao, M.R. (2006) Clinical-scale high-throughput analysis of urinary 8-oxo-7,8-dihydro-2′-deoxyguanosine by isotope-dilution liquid chromatography-tandem mass spectrometry with on-line solid-phase extraction. Clin. Chem. 52, 1381–1388.
Weimann, A., Belling, D., and Poulsen, H.E. (2001) Measurement of 8-oxo-2′-deoxyguanosine and 8-oxo-2′-deoxyadenosine in DNA and human urine by high performance liquid chromatography-electrospray tandem mass spectrometry. Free Radic. Biol. Med. 30, 757–764.
Teichert, F., Verschoyle, R.D., Greaves, P., Thorpe, J.F., Mellon, J.K., Steward, W.P., Farmer, P.B., Gescher, A.J., and Singh, R. (2009). Determination of 8-oxo-2′-deoxyguanosine and creatinine in murine and human urine by liquid chromatography-tandem mass spectrometry: application to chemoprevention studies. Rapid Commun. Mass Spectrom. 23, 258–266.
Singh, R., McEwan, M., Lamb, J.H., Santella, R.M., and Farmer, P.B. (2003) An improved liquid chromatography/tandem mass spectrometry method for the determination of 8-oxo-7,8-dihydro-2′-deoxyguanosine in DNA samples using immunoaffinity column purification. Rapid Commun. Mass Spectrom. 17, 126–134.
Miwa, M., Matsumaru, H., Akimoto, Y., Naito, S. and Ochi, H. (2004). Quantitative determination of urinary 8-hydroxy-2′-deoxyguanosine level in healthy Japanese volunteers. Biofactors 22, 249–253
Acknowledgements
RS and PBF are supported by the U.K. Medical Research Council. Some development of this methodology in the laboratory of MDE and MSC was supported by using a University of Leicester miscellaneous income fund held by MSC. RS, MDE, PBF, and MSC are partners of 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). JKS is supported by (ECNIS, Contract No. FOOD-CT-2005-513943); FT is supported by Cancer Research UK (CRUK Programme Grant C325/A6691) and Experimental Cancer Medicine Centre Network (ECMC Grant C325/A7241).
The formation of a European-centred laboratory consortium, the European Standards Committee on Urinary (DNA) Lesion Analysis (ESCULA) is enabling an examination of methodology and issues surrounding the analysis of urinary 8-oxodG in the first instance. Further information about this consortium, including activities and membership can be obtained by contacting Dr. M.S. Cooke (msc5@le.ac.uk; http://escula.org).
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2011 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Mistry, V. et al. (2011). Non-invasive Assessment of Oxidatively Damaged DNA: Liquid Chromatography-Tandem Mass Spectrometry Analysis of Urinary 8-Oxo-7,8-Dihydro-2′-Deoxyguanosine. In: Didenko, V. (eds) DNA Damage Detection In Situ, Ex Vivo, and In Vivo. Methods in Molecular Biology, vol 682. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60327-409-8_20
Download citation
DOI: https://doi.org/10.1007/978-1-60327-409-8_20
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-60327-408-1
Online ISBN: 978-1-60327-409-8
eBook Packages: Springer Protocols