DNA damage induced in cells by gamma and UVA radiation as measured by HPLC/GC-MS and HPLC-EC and Comet assay.

ABSTRACT

The aim of the work was to measure DNA damage induced within tumoral human monocytes by gamma 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 gamma rays and UVA radiation. The extent of formation of FapyGua in the DNA of cells exposed to gamma rays was estimated to be more than 2-fold higher than that of 8-oxodGuo, i.e., about 0. 027 lesion per 10(6) 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/10(6) 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 (1)O(2) with respect to hydroxyl radicals and type I photosensitization was thus observed within cells exposed to UVA radiation.