Carcinogenesis, Vol 18, 2225-2231, Copyright © 1997 by Oxford University Press
M Pflaum, O Will and B Epe
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.
ARTICLES
Determination of steady-state levels of oxidative DNA base modifications in mammalian cells by means of repair endonucleases
Institute of Pharmacy, University of Mainz, Germany.
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