Method development for analysis of 8-oxodG as a biomarker for oxidative stress
Author: Hofer, Tim
Date: 2001-12-13
Location: Hörsalen, plan 4, Novum
Time: 13.30
Department: Biovetenskaper och näringslära / Biosciences and Nutrition
Abstract
Oxidative damage to DNA gives oxidation of DNA bases and strand breaks
that can cause mutations leading to cancer. Various forms of agents are
capable of oxidising DNA in vivo including peroxides, singlet oxygen and
UV- or gamma-irradiation. Oxidation of DNA bases occurs mainly at dG,
giving stable DNA adducts such as 8-oxodG and FapydG.
The use of 8-oxodG as a biomarker (expressed as the ratio 8-oxodG/dG)
requires well- controlled workup conditions because 8-oxodG can be formed
during the workup procedure. This may result in false high levels with
difficulties in determining the true background levels, and differences
between control and exposed tissue as a result. Existing methods require
time consuming and warm (37°C) workup steps to extract and hydrolyse DNA
before analysis. Since natural levels of 8-oxodG are low, a sensitive
analytical tool is required when working with low mg amounts of cells.
In this thesis, methods for workup procedures and analysis of 8-oxodG in
DNA using HPLC/EC/UV and 32P-HPLC were developed. 32p was found to
strongly oxidise dG into 8- oxodG and a HPLC pre-separation step was
developed before 32p- postlabelling of 8-oxodG. Factors such as the
purity of solutions, temperature, incubation time, peroxide removal and
amount of cells during the sensitive workup procedure were found
important, affecting the 8- oxodG/dG ratio. A fast (=10 min) cold (0°C),
high salt non-phenol DNA extraction method procedure was developed to
reduce the artifactual 8-oxodG formation. In addition, the use of
catalase and the electron acceptor TEMPO was found to be protective
against artifactual oxidation.
A new hydroxylation mechanism of carbon compounds by peroxides is
suggested, where transition metals mediate the two-electron reduction of
H202, with one-electron oxidation of the reducing agent and compound
respectively. This produces carbon radical cations, which are
hydroxylated in water. For dG oxidation in a H202ascorbate system, this
mechanism is shown to be thermodynamically favourable to a one-electron
reduction of H202, producing OH° and, in addition, added OH° scavengers
were found to be ineffective. Peroxide oxidation of DNA is likely, if
forming DNA radical cations, to give mainly oxidation of G due to
electron transfer in DNA, compared to systems generating the more
randomly adding OH° (such as gamma- radiolysis of water).
Background 8-oxodG/dG ratios in human lymphocytes were found to be much
lower than previously reported. Oxidation of dG to 8-oxodG during workup
was found to be relatively constant and to fit a mathematically defined
curve that can help in estimating the true background level and the
artifactual formation of 8-oxodG.
As the degree of workup formation of 8-oxodG can vary on different days,
it is important to include control samples during each round of workup
and analysis.
List of papers:
I. Moller L, Hofer T (1997). "[32P]ATP mediates formation of 8-hydroxy-2-deoxyguanosine from 2-deoxyguanosine, a possible problem in the 32P-postlabeling assay. " Carcinogenesis 18(12): 2415-9
Pubmed
II. Hofer T, Moller L (1998). "Reduction of oxidation during the preparation of DNA and analysis of 8-hydroxy-2-deoxyguanosine. " Chem Res Toxicol 11(8): 882-7
Pubmed
III. Moller L, Hofer T, Zeisig M (1998). "Methodological considerations and factors affecting 8-hydroxy-2-deoxyguanosine analysis. " Free Radic Res 29(6): 511-24
Pubmed
IV. Zeisig M, Hofer T, Cadet J, Moller L (1999). "32P-postlabeling high-performance liquid chromatography (32P-HPLC) adapted for analysis of 8-hydroxy-2-deoxyguanosine. " Carcinogenesis 20(7): 1241-5
Pubmed
V. Hofer T (2000). "Oxidation of 2-deoxyguanosine by H202-ascorbate: evidence against free OH and thermodynamic support for two-electron reduction of H2O2." J Chem Soc, Perkin Trans 2: 210-3
VI. Hofer T, Moller L (2001). "Optimization of the workup procedure for the analysis of 8-oxo-7, 8-dihydro-2-deoxyguanosine with electrochemical detection." (Submitted)
I. Moller L, Hofer T (1997). "[32P]ATP mediates formation of 8-hydroxy-2-deoxyguanosine from 2-deoxyguanosine, a possible problem in the 32P-postlabeling assay. " Carcinogenesis 18(12): 2415-9
Pubmed
II. Hofer T, Moller L (1998). "Reduction of oxidation during the preparation of DNA and analysis of 8-hydroxy-2-deoxyguanosine. " Chem Res Toxicol 11(8): 882-7
Pubmed
III. Moller L, Hofer T, Zeisig M (1998). "Methodological considerations and factors affecting 8-hydroxy-2-deoxyguanosine analysis. " Free Radic Res 29(6): 511-24
Pubmed
IV. Zeisig M, Hofer T, Cadet J, Moller L (1999). "32P-postlabeling high-performance liquid chromatography (32P-HPLC) adapted for analysis of 8-hydroxy-2-deoxyguanosine. " Carcinogenesis 20(7): 1241-5
Pubmed
V. Hofer T (2000). "Oxidation of 2-deoxyguanosine by H202-ascorbate: evidence against free OH and thermodynamic support for two-electron reduction of H2O2." J Chem Soc, Perkin Trans 2: 210-3
VI. Hofer T, Moller L (2001). "Optimization of the workup procedure for the analysis of 8-oxo-7, 8-dihydro-2-deoxyguanosine with electrochemical detection." (Submitted)
Issue date: 2001-11-22
Rights:
Publication year: 2001
ISBN: 91-7349-064-4
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