Journal Article

Cellular background level of 8-oxo-7,8-dihydro-2′-deoxyguanosine: an isotope based method to evaluate artefactual oxidation of DNA during its extraction and subsequent work-up

Jean-Luc Ravanat, Thierry Douki, Pierre Duez, Eric Gremaud, Karl Herbert, Tim Hofer, Lydie Lasserre, Christine Saint-Pierre, Alain Favier and Jean Cadet

in Carcinogenesis

Volume 23, issue 11, pages 1911-1918
Published in print November 2002 | ISSN: 0143-3334
Published online November 2002 | e-ISSN: 1460-2180 | DOI: http://dx.doi.org/10.1093/carcin/23.11.1911
Cellular background level of 8-oxo-7,8-dihydro-2′-deoxyguanosine: an isotope based method to evaluate artefactual oxidation of DNA during its extraction and subsequent work-up

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The measurement of oxidative damage to cellular DNA is a challenging analytical problem requiring highly sensitive and specific methods. In addition, artefactual DNA oxidation during its extraction and subsequent work-up may give rise to overestimated levels of oxidized DNA bases. In the present study, we have used 18O-labelled 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) as an internal standard to evaluate the extent of artefactual DNA oxidation during the critical steps preceding the measurement. The labelled oxidized purine nucleoside was specifically generated in cellular DNA using the recently available generator of 18O-labelled singlet oxygen. Artefactual DNA oxidation that could take place during the work-up increases the level of 8-oxodGuo but not of the 18O-oxidized nucleoside. Therefore, the ratio between the two compounds, as measured by high performance liquid chromatography coupled to tandem mass spectrometry, allows an unambiguous comparison of different methodologies. The comparison of different DNA extraction protocols led to the conclusion that artefactual DNA oxidation during the extraction step could be minimized if: (i) nuclei are isolated after cell lysis; (ii) desferrioxamine, a transition metal chelator is added to the different extraction buffers; and (iii) sodium iodide (or alternatively guanidine thiocyanate) is used for DNA precipitation. It was also demonstrated that sodium iodide does not decompose the targeted oxidized purine nucleoside. In addition, three different DNA digestion protocols were evaluated and they were found to give rise to similar results. Using the best-studied protocol, the steady-state cellular background level of 8-oxodGuo, in a lymphocyte cell line, was determined to be ∼0.5 lesions/106 DNA nucleosides.

Keywords: DHPN18O2, naphthalene derivative; EC, electrochemical detection; GC-MS, gas chromatography-mass spectrometry; GTC, guanidine thiocyanate; HPLC, high performance liquid chromatography; MS/MS, tandem mass spectrometry; 8-oxodGuo, 8-oxo-7,8-dihydro-2′-deoxyguanosine.

Journal Article.  8205 words.  Illustrated.

Subjects: Clinical Cytogenetics and Molecular Genetics

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