Journal Article

Observation and prevention of an artefactual formation of oxidized DNA bases and nucleosides in the GC-EMS method

Thierry Douki, Thierry Dealtour, Franca Bianchini and Jean Cadet

in Carcinogenesis

Volume 17, issue 2, pages 347-353
Published in print February 1996 | ISSN: 0143-3334
Published online February 1996 | e-ISSN: 1460-2180 | DOI: https://dx.doi.org/10.1093/carcin/17.2.347
Observation and prevention of an artefactual formation of oxidized DNA bases and nucleosides in the GC-EMS method

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Gas chromatography coupled to electron impact mass spectrometry (GC-EMS) analysis following hydrolysis of DNA is a widely used assay for the detection of oxidized nucleobases and nucleosides. However, evidence was recently provided for an oxidation of guanine residues of hydrolysed DNA during the silylation prior to GC-EMS analysis. This reaction accounts for the overestimation of the yield of 8-oxo-7,8-dihydroguanine by GC-EMS. In the present work, we showed that adenine, cytosine, thymine and themidine also give rise to oxidized derivatives during the derivatization. This was inferred from the measurement of the amount of 5-OHCyt), 8-oxo-7,8-dihydroadenine formyluracil, 5-hydroxymethyluracil, 5-hydroxycytosine (8-OxoAde) and 5-hydroxymethyl-2'-deoxyuridine (5-HMdUrd) in a series of experiments based on the use of purified bases and nucleosides. Isotopically labelled oxidized bases and 5-HMdUrd were used as internal standards to control the quantitative aspect of the silylation reaction. Support for an artefactual oxidation of hydrolysed DNA was provided by the comparison of the amount of 8-OxoAde and 5-OHCyt detected within native and γ-Sirradiated DNA by HPLC-EC and GC-EIMS. To prevent the artefactual formation of oxidized bases during the dilylation, an approach based on an HPLC prepurifica-tion was developed to remove the precursors of the oxidized bases measured in the DNA sample. The HPLC/GC-EMIS assay was successfully applied to the quantification of 8-OxoAde and 5-OHCyt in calf thymus DNA. In addition, the detection of the dose-dependent formation of 5-HMdUrd in isolated DNA exposed to ionizing radiation was achieved using the same approach.

Journal Article.  0 words. 

Subjects: Clinical Cytogenetics and Molecular Genetics

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