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.
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 (18)O-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 (18)O-labelled singlet oxygen. Artefactual DNA oxidation that could take place during the work-up increases the level of 8-oxodGuo but not of the (18)O-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 approximately 0.5 lesions/10(6) DNA nucleosides.
Ravanat
Jean-Luc
JL
Laboratoire Lésions des Acides Nucléiques, DRFMC/SCIB UMR 5046 CEA Grenoble, 17 Avenue des Martyrs, F-38054 Grenoble Cedex 9, France. jravanat@cea.fr
Douki
Thierry
T
Duez
Pierre
P
Gremaud
Eric
E
Herbert
Karl
K
Hofer
Tim
T
Lasserre
Lydie
L
Saint-Pierre
Christine
C
Favier
Alain
A
Cadet
Jean
J
D000080242:8-Hydroxy-2'-Deoxyguanosine; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D016477:Artifacts; D002417:Cattle; D002458:Cell Fractionation / Q000379:methods*; D002614:Chelating Agents / Q000494:pharmacology; D011232:Chemical Precipitation; D002851:Chromatography, High Pressure Liquid; D003497:Cyclic N-Oxides / Q000494:pharmacology; D004247:DNA / Q000737:chemistry* / Q000302:isolation & purification; D004249:DNA Damage; D004273:DNA, Neoplasm / Q000737:chemistry / Q000302:isolation & purification; D003676:Deferoxamine / Q000494:pharmacology; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000032:analysis*; D007948:Leukemia, Monocytic, Acute / Q000473:pathology; D008099:Liver / Q000737:chemistry; D013058:Mass Spectrometry; D009000:Monocytes / Q000737:chemistry; D010084:Oxidation-Reduction; D010103:Oxygen Isotopes / Q000032:analysis; D051381:Rats; D012015:Reference Standards; D012016:Reference Values; D026082:Singlet Oxygen; D012974:Sodium Iodide / Q000494:pharmacology; D013950:Thymus Gland / Q000737:chemistry; D014407:Tumor Cells, Cultured / Q000737:chemistry
D000975:Antioxidants; D002614:Chelating Agents; D003497:Cyclic N-Oxides; D004273:DNA, Neoplasm; D010103:Oxygen Isotopes; D026082:Singlet Oxygen; D000080242:8-Hydroxy-2'-Deoxyguanosine; D004247:DNA; D012974:Sodium Iodide; D003849:Deoxyguanosine; D003676:Deferoxamine; C003959:TEMPO