Biomarkers of radiation or oxidative damage to DNA in cells

Major efforts have been devoted during the last two decades to the development of chemical and biochemical assays aimed at monitoring oxidized bases within DNA. Until recently, the level of oxidized bases in cellular DNA was overestimated by factors varying from one to three orders of magnitude. The reasons of these inconsistencies are now identified. Thus, artifactual oxidation of nucleobases may occur during the silylation reaction prior to gas chromatography mass spectrometry analysis and to a lesser extent during DNA extraction. The use of the so-called chaotropic DNA extraction method together with chelating agents was found to significantly prevent spurious DNA oxidation to occur. HPLC separations coupled to either electrochemical detection or versatile electrospray ionization tandem-mass spectrometry appear to be appropriate analytical tools when at least 20 μg of DNA is available. Interestingly, up to 11 modified nucleosides and nucleobases including the four cis and trans diastereomers of 5,6- dihydroxy-5,6-dihydrothymidine, 5-formyl-2'-deoxyuridine, 5-(hydroxymethyl)-2'-deoxyuridine, and 5-hydroxy-2'-deoxyuridine, 8-oxo-7,8-dihydro-2'-deoxyguanosine, 8-oxo-7,8-dihydro-2'-deoxyadenosine together with related formamidopyrimidine derivatives were measured in the DNA of neoplastic human monocytes exposed to ionizing radiation using the latter assay. The association of base excision DNA repair enzymes including bacterial formamidopyrimidine glycosylase (Fpg) and endonuclease III (endo III) with the comet assay represents a better alternative for assessing low levels of base damage. The basal level of Fpg- and endo III-sensitive sites was found to be similar, close to 2.1 per 107 bases, within the DNA of human monocytes. The yields of the different classes of damage per Gy and 107 bases are the following: 0.48 Fpg-sensitive sites, 0.53 endo III-sensitive sites and 1.30 strand breaks (direct nicks and alkali-labile sites). Interestingly, the above three classes of damage to cellular DNA were measured for doses of ionizing radiation as low as 0.2 Gy using an improved version of the modified comet assay