Detection of single-strand breaks and formamidopyrimidine-DNA glycosylase-sensitive sites in DNA of cultured human fibroblasts

Abstract

Under oxidative stress 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodG), a damaged base with mutagenic potential, and single-strand breaks (SSB) are formed in DNA. Both lesions are frequently used as a parameter for oxidative damage of DNA. Here we report on results from the evaluation of a modified nick translation assay, where 8-oxodG and SSB formation in cellular DNA of cultured human fibroblasts were simultaneously detected. The assay is based on a method previously described by others, with several modifications in reaction conditions and type of substrate. We used formamidopyrimidine-DNA glycosylase (FPG) in our assay in order to measure the formation of FPG-sensitive lesions (which include 8-oxodG) in DNA of human fibroblasts in response to ionising radiation. The quantification of the DNA damage was based on calibration experiments with plasmid DNA pUC19. Dose-response curves of SSB and FPG-sensitive lesion formation in human fibroblasts VH-10 were established. A very low background level of 8-oxodG was detected in unirradiated fibroblasts (approx. 500 residues per cell).