Carcinogenesis, Vol 18, 2367-2371, Copyright © 1997 by Oxford University Press
D Schuler, M Otteneder, P Sagelsdorff, E Eder, RC Gupta and WK Lutz
8-Oxo-2'-deoxyguanosine (8-oxo-dG) is emerging as a useful marker for
oxidative DNA damage. Reported basal levels determined by 32P- postlabeling
(PPL) method were 10-fold or more higher than those obtained with
HPLC/electrochemical detection (ECD). This discrepancy was investigated. In
commercial calf thymus DNA, levels of 4 +/- 1 and 64 +/- 14 8-oxo-dG per
10(6) 2'-deoxynucleosides (dN) were measured by the standard HPLC/ECD and
PPL methods, respectively. DNA digestion by micrococcal nuclease/spleen
phosphodiesterase and nuclease P1 (as used in the standard PPL method),
followed by ECD analysis resulted in a level of 8 +/- 3. In calf thymus DNA
spiked with chemically synthesized 8-oxo-dGp to give an increment of 9
8-oxo-dG/10(6) dN, the added standard produced a significant increase with
HPLC/ECD but not PPL. After spiking the DNA with 90 8-oxo-dG/10(6) dN, the
added 8-oxo-dGp was detectable also with PPL, with a labeling efficiency of
65%. In order to investigate the role of ionizing radiation from 32P for
the higher 8-oxo-dG levels in PPL, incubation times and amounts of
radioactivity in the phosphorylation reaction with commercial dGp were
increased, and external irradiation of commercial dG with 32P was
investigated. All modifications resulted in higher values of 8-oxo-dG
measured, but the effect was not large enough to fully explain the
discrepancy between PPL and HPLC/ECD. Using [gamma-33P]ATP instead of
[gamma-32P]ATP or adding [33P]phosphate to a 32P-PPL assay resulted in even
higher levels of 8-oxo-dG measured. The increase in 8-oxo-dG levels during
the PPL workup is attributed to the presence and oxidation of unmodified
dGp in the reaction mixture. For a determination of true basal levels, the
PPL method will have to be modified, including the removal of dGp prior to
the phosphorylation reaction.
ARTICLES
Comparative analysis of 8-oxo-2' -deoxyguanosine in DNA by 32P- and 33P- postlabeling and electrochemical detection
Department of Toxicology, University of Wurzburg, Germany.
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