Carcinogenesis, Vol 18, 2415-2419, Copyright © 1997 by Oxford University Press
L Moller and T Hofer
8-Hydroxy-2'-deoxyguanosine (8-OH-dG) is a biomarker for oxidative stress
on DNA, a common lesion in mammalian cells. A correlation between increased
levels of 8-OH-dG and diseases like diabetes, infections and cystic
fibrosis has been found in humans. 8-OH-dG levels have been shown to be
decreased by antioxidants, an indication of the importance of dietary
habits. 8-OH-dG is used as a biomarker for oxidative stress in vivo as well
as in vitro and is suggested to be a mutagenic DNA lesion. Different
methods are used for the analyses of 8- OH-dG, i.e. GC-MS, HPLC-EC and
32P-postlabeling. The most commonly used method is HPLC-EC. In the analysis
of 8-OH-dG, the work-up procedure for DNA, as well as the preparation for
analysis, are of critical importance as there is a risk for auto-oxidation
of deoxyguanosine (dG), which would result in false high background levels
and low sensitivity in analysis. 32P-Postlabeling has recently been applied
to the analysis of 8-OH-dG and has shown to be a very sensitive method for
the detection of DNA adducts. It is shown here that after extrapolation to
normal 32P-postlabeling conditions, [32P]ATP generated 8-OH-dG to levels of
25 8-OH-dG/10(5) dG. [32P]ATP mediated the formation of 8-OH- dG from dG in
a dose-dependent manner at all dose levels (0.13-12 microCi). The reaction
occurred immediately and increased with time in a linear dose-response
fashion. At high doses (6.0 and 12 microCi) the dose-response declined
after 24 h, which indicates a possible decomposition or rearrangement of
8-OH-dG. A repeated experiment with 5 microCi [32P]ATP during 2 h resulted
in a linear formation of 8-OH-dG and a level of 19 8-OH-dG/10(5) dG. The
results indicated that awareness of the auto-oxidation generated by
32P[ATP] in the postlabeling assay is of utmost importance and that dG must
be separated before 32P-postlabeling of 8-OH-dG.
ARTICLES
[32P]ATP mediates formation of 8-hydroxy-2'-deoxyguanosine from 2'- deoxyguanosine, a possible problem in the 32P-postlabeling assay
Karolinska Institute, Department of Biosciences, Huddinge, Stockholm, Sweden.
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