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 endo nucleases (Fpg protein, endonuclease III, exonuclease III, T4 endonucl ease V) was used to quantify steady-state (background) levels of oxida tive base modifications in various types of mammalian cells, Ire human lymphocytes the number of base modifications sensitive to Fpg protein , which include 8-hydroxyguanine, was 0.25 +/- 0.05 per 10(6) base pai rs, Even lower levels (0.07 +/- 0.02 per 10(6) bp) were observed in He La cells, The numbers of sites sensitive to the other repair endonucle ases were below the detection limit (0.05 per 10(6) bp). In a direct c omparison, the background level of Fpg-sensitive modifications determi ned by alkaline elution was much lower than the background level of 8- hydroxydesoxyguanosine (8-oxodG) determined after enzymatic DNA hydrol ysis by HPLC and electrochemical detection, However, the number of add itional Fpg-sensitive modifications induced by a photosensitizer plus light was similar to the additional number of 8-oxodG residues determi ned by HPLC with electrochemical detection. This indicates that the en zyme assay does not systematically underestimate the number of lesions and points to an artefactual generation of 8-oxodG during DNA isolati on and hydrolysis.