DNA Damage Induced in Cells by γ and UVA Radiation As Measured by HPLC/GC−MS and HPLC−EC and Comet Assay

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Abstract
The aim of the work was to measure DNA damage induced within tumoral human monocytes by γ rays, UVA radiation, and exogenous photosensitizers. The accurate HPLC−EC assay was used to determine the level of 8-oxodGuo. The formation of FapyGua and FapyAde was monitored by HPLC/GC−MS analyses after formic acid hydrolysis at room temperature. For this purpose, cells were exposed to relatively high doses of γ rays and UVA radiation. The extent of formation of FapyGua in the DNA of cells exposed to γ rays was estimated to be more than 2-fold higher than that of 8-oxodGuo, i.e., about 0.027 lesion per 106 bases per Gy. The yield of FapyAde was estimated to be 1 order of magnitude lower. The latter results were used to calibrate the alkaline comet assay associated with DNA N-glycosylases. The latter approach allowed the determination of the background level (0.11−0.16 Fpg-sensitive site/106 bases) and the yields of strand breaks and DNA base damage upon low irradiation doses. Insights into the mechanism of radiation-induced DNA damage were gained from these measurements. A major involvement of 1O2 with respect to hydroxyl radicals and type I photosensitization was thus observed within cells exposed to UVA radiation.
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