ARTIFACTS ASSOCIATED WITH THE MEASUREMENT OF OXIDIZED DNA BASES

In this paper we review recent aspects of the measurement of oxidized DNA basest currently a matter of debate. There has long been an intere st in the determination of the level of oxidized bases in cellular DNA under both normal and oxidative stress conditions. In this respect, t he situation is confusing because variations that may be as large as t wo orders of magnitude have been reported for the yield of the formati on of 8-oxo-7,8-dihydroguanine (8-oxoGua) in similar DNA samples. Howe ver, recent findings clearly show that application of several assays l ike gas chromatography-mass spectrometry (GC-MS) and [P-32]-postlabeli ng may lead to a significant over-estimation of the level of oxidized bases in cellular DNA. In particular, the silylation step, which is re quired to make the samples volatile for the GC-MS analysis, has been s howvn to induce oxidation of normal bases at the level of about one ox idized base per 10(4) normal bases. This has been found to be a genera l process that applies in particular to 8-oxoGua, 8-oxo-7,8-dihydroade nine, 5-hydroxycytosine, 5-(hydroxymethyl)uracil, and 5-formyluracil. Interestingly, prepurification of the oxidized bases from DNA hydrolys ate prior to the derivatization reaction prevents artefactual oxidatio n. Under these conditions, the level of oxidized bases measured by GC- MS is similar to that obtained by HPLC associated with electrochemical detection (HPLC-EC). It should be added that the level of 8-oxo-7,8-d ihydro-2'-deoxyguanosine in control cellular DNA has been found to be about fivefold lower than in earlier HPLC-EC measurements by using app ropriate conditions of extraction and enzymatic digestion of DNA. Simi lar conclusions were reached by measuring formamidopyrimidine-DNA glyc osylase sensitive sites as revealed by the single cell gel electrophor esis (comet) assay.