Oxidative DNA damage and mutations induced by a polar photosensitizer, Ro19-8022.

The oxidative DNA damage induced by the polar photosensitizer Ro19-8022 in the presence of light was studied and correlated with the associated mutagenicity. Both in isolated DNA and AS52 Chinese hamster ovary cells, photoexcited Ro19-8022 gave rise to a DNA damage profile that was similar to that caused by singlet oxygen: base modifications sensitive to the repair endonuclease Fpg protein, which according to high-performance liquid chromatography (HPLC) analysis were predominantly 8-hydroxyguanine (8-oxoG) residues, were generated in much higher yield than single-strand breaks, sites of base loss (AP sites) and oxidative pyrimidine modifications sensitive to endonuclease III. Fifty percent…

Determination of steady-state levels of oxidative DNA base modifications in mammalian cells by means of repair endonucleases

The alkaline elution technique in combination with various repair endonucleases (Fpg protein, endonuclease III, exonuclease III, T4 endonuclease V) was used to quantify steady-state (background) levels of oxidative base modifications in various types of mammalian cells. In human lymphocytes the number of base modifications sensitive to Fpg protein, which include 8-hydroxyguanine, was 0.25 +/- 0.05 per 10(6) base pairs. Even lower levels (0.07 +/- 0.02 per 10(6) bp) were observed in HeLa cells. The numbers of sites sensitive to the other repair endonucleases were below the detection limit (0.05 per 10(6) bp). In a direct comparison, the background level of Fpg-sensitive modifications determi…

Oxidative DNA damage induced by visible light in mammalian cells: extent, inhibition by antioxidants and genotoxic effects

The extent of the indirect DNA damage generated in mammalian cells by visible light because of the presence of endogenous photosensitizers was studied by means of repair endonucleases. In immortalized human keratinocytes (HaCaT cells) exposed to low doses of natural sunlight, the yield of oxidative DNA base modifications sensitive to the repair endonuclease formamidopyrimidine-DNA glycosylase (Fpg protein) generated by this indirect mechanism was 10% of that of pyrimidine dimers (generated by direct DNA excitation). A similar yield of Fpg-sensitive modifications, which include 8-hydroxyguanine, was observed in primary keratinocytes. The relative yield of oxidative base modifications decreas…

Steady-state (background) levels of oxidative DNA modifications in mammalian cells

Oxidative DNA Damage Profiles in Mammalian Cells

Reactive oxygen species (ROS) are formed inside cells not only under the influence of exogenous agents (visible light, ionizing radiation, and many oxidants such as peroxides or quinones), but also under normal (physiological) conditions as byproducts of oxygen metabolism and other cellular redox reactions (Pryor 1986; Halliwell and Gutteridge 1986; Sies 1986; Clayson et al. 1994). ROS such as hydroxyl radicals and singlet oxygen are a serious threat to the integrity of the cellular genome, since they efficiently react with DNA to generate many types of DNA modifications, at least some of which are pre- mutagenic (Breimer 1990; Halliwell and Aruoma 1991; Epe 1991; Feig et al. 1994). Steady-…

DNA oxidation products determined with repair endonucleases in mammalian cells: Types, basal levels and influence of cell proliferation

Purified repair endonucleases such as Fpg protein, endonuclease III and IV allow a very sensitive quantification of various types of oxidative DNA modifications in mammalian cells. By means of these assays, the numbers of base modifications sensitive to Fpg protein, which include 8-hydroxyguanine (8-oxoG), were determined to be less than 0.3 per 10(6) bp in several types of untreated cultured mammalian cells and human lymphocytes and less than 10 per 10(6) bp in mitochondrial DNA from rat and porcine liver. Oxidative 5,6-dihydropyrimidine derivatives sensitive to endonuclease III and sites of base loss sensitive to endonuclease IV or exonuclease III were much less frequent than Fpg-sensitiv…