title: Determination of steady-state levels of oxidative DNA base modifications in mammalian cells by means of repair endonucleases.
abstract: 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 determined by alkaline elution was much lower than the background level of 8-hydroxydesoxyguanosine (8-oxodG) determined after enzymatic DNA hydrolysis by HPLC and electrochemical detection. However, the number of additional Fpg-sensitive modifications induced by a photosensitizer plus light was similar to the additional number of 8-oxodG residues determined by HPLC with electrochemical detection. This indicates that the enzyme assay does not systematically underestimate the number of lesions and points to an artefactual generation of 8-oxodG during DNA isolation and hydrolysis.
authors.affiliation: Institute of Pharmacy, University of Mainz, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Pflaum
authors.forename: O
authors.initials: O
authors.lastname: Will
authors.forename: B
authors.initials: B
authors.lastname: Epe
mesh_terms: D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000378:metabolism*; D004260:DNA Repair; D004563:Electrochemistry; D004720:Endonucleases / Q000378:metabolism*; D006147:Guanine / Q000031:analogs & derivatives / Q000378:metabolism; D006801:Humans; D010084:Oxidation-Reduction
chemical_list: C024829:8-hydroxyguanine; D006147:Guanine; D004247:DNA; D004720:Endonucleases