Oscillating formation of 8-oxoguanine during DNA oxidation

Article abstract of DOI:10.1021/ja0343252

Oxidation of free guanine and guanine in salmon testes ds-DNA by hydroxyl radicals generated with Fenton reagent resulted in oscillating 8-oxoguanine concentrations. These oscillations were superimposed on a general trend of decreasing ratio of [8-oxoguanine]/{[8-oxoguanine] + [guanine]} with time, suggesting that a steady state 8-oxoguanine concentration would not be achieved. Mass spectrometry detected 8-oxoguanine and 5-guanidinohydantoin as products, suggesting that the latter was the product of oxidation of 8-oxoguanine. Guanidinohydantoin and other possible intermediates and products may be involved in a complex mechanism leading to the observed behavior. Oscillatory fluctuations in 8-oxoguanine may need to be considered in assessing its clinical significance as a biomarker for oxidative DNA damage. Copyright

Full text of DOI:10.1021/ja0343252

Published on Web 05/09/2003
Oscillating Formation of 8-Oxoguanine during DNA Oxidation
Bl a´ naid White,†,§ Malcolm R. Smyth, James D. Stuart, and James F. Rusling*
†
§
,†,‡
Department of Chemistry, UniVersity of Connecticut, U-60, 55 North EagleVille Road,
Storrs, Connecticut 06269-3060, Department of Pharmacology, UniVersity of Connecticut Health Center,
Farmington, Connecticut 06032, and National Centre for Sensor Research (NCSR), School of Chemical Sciences,
Dublin City UniVersity, Dublin 9, Ireland
Received January 24, 2003; E-mail: james.rusling@uconn.edu
Genetic material undergoes oxidative stress that damages DNA
1
bases. Although nature has evolved diverse strategies to minimize
2
its effects, such damage can interfere with normal replication.
Oxidative DNA damage occurs from irradiation, chemical reactions,
and oxidation with reactive oxygen species (ROS),3 including
hydroxyl radicals. Reaction of DNA with •OH yields 8-oxoguanine,
8
-oxoadenine, thymine glycol, 8-hydroxycytosine, and strand breaks
4
that may participate in mutagenesis. The goal of the present study
was to measure the rate of 8-oxoguanine formation during DNA
oxidation and to investigate its ultimate fate. We found that
Figure 1. Concentration profiles of guanine (O) and 8-oxoguanine (b)
from reaction of 0.8 mM guanine with 150 µM FeSO4 and 50 mM H2O2 at
37 °C. (Lines in figures are cubic spline fits.)
8
-oxoguanine was formed from oxidation of both DNA and free
guanine in oscillating concentrations reflecting a complex reaction
pathway, most likely involving further oxidation to 5-guanidino-
hydantoin.
Guanine has the lowest oxidation potential of the DNA bases.5
Oxidation product 8-oxoguanine is considered a clinical biomarker
for oxidative DNA damage. 8-Oxoguanine has a lower oxidation
potential than guanine. Thus, when 8-oxoguanine was inserted into
DNA sequences, it was the preferred oxidization site.8
6
7
We began by oxidizing free guanine using Fenton reagent to
generate •OH radicals from Fe and hydrogen peroxide9 and
analyzed the products by liquid chromatography with sequential
UV and electrochemical detectors (LC-UV-EC).6 All reactions
were done in triplicate in pH 5.5 buffer. The UV detector at 254
nm detected guanine, and the electrochemical detector detected
II
,10
Figure 2. Ratio of [8-oxoguanine]/{[8-oxoguanine] + [guanine]} for 0.8
mM guanine reacted with 150 µM FeSO4 + 50 mM H2O2, 37 °C.
8-oxoguanine at +550 mV versus Ag/AgCl.
Oxidation of guanine gave a decrease in guanine concentration
along with maxima in 8-oxoguanine concentrations at 4 and 15
min (Figure 1). The maximum concentration was ∼70 µM,
corresponding to 9% of the initial guanine. Qualitatively similar
results were found at 0.8 mM and 8 µM guanine. Oxidation of
8-oxoguanine with •OH showed a rapid exponential decrease in
concentration with 95% reactant consumed in 5 min.
Figure 2 shows the ratio [8-oxoguanine]/{[8-oxoguanine] +
guanine]}. After a small peak at 5 min, peaks in this ratio repeated
[
Figure 3. Ratio of [8-oxoguanine]/{[8-oxoguanine] + [guanine]} for ST
ds-DNA reacted with 150 µM FeSO4 and 50 mM H2O2, then hydrolyzed.
with a frequency of 15 min. If 8-oxoguanine were the only product,
the denominator would remain constant, and this ratio should
increase, regardless of the oscillations. However, after about 20
min, the overall trend underlying the oscillations was a decrease
with time.
Salmon testes (ST) ds-DNA was then reacted with Fenton’s
reagent. Aliquots from the DNA reaction were acid hydrolyzed
under vacuum at 140 °C for 30 min to give free nucleobases.11
Again the 8-oxoguanine concentration oscillated. Figure 3 shows
LC-MS was used to analyze ST ds-DNA incubated with Fenton
reagent for 60 min, then hydrolyzed. LC-ion chromatograms showed
+
peaks at m/z 152 [G + H ] at a retention time (t
R
) of 2.9 min and
+
m/z 158 [guanidinohydantoin + H ] at t
R
3.1 min (see Supporting
Information). The mass spectrum obtained from the LC eluant
between 2.9 and 3.1 min showed peaks at m/z 152 and m/z 158,
and at m/z 174 and m/z 190. The latter two peaks correspond to
ions [G + Na] (m/z 174) and [8-oxoG + Na]+ (m/z 190),
confirming the presence of 8-oxoguanine.
[8-oxoguanine]/{[8-oxoguanine] + [guanine]} over 90 min. There
+
is an initial peak at 5 min and clear peaks at 20 and 65 min. An
overall decreasing trend underlies the oscillations.
The results described above show that oxidation of guanine free
or in ds-DNA by •OH leads to oscillating concentrations of
8-oxoguanine. The frequency for free guanine (one peak/15 min)
†
University of Connecticut.
‡
University of Connecticut Health Center.
§
Dublin City University.
6604
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10.1021/ja0343252 CCC: $25.00 © 2003 American Chemical Society

C O MMU N I C A T I O N S
is larger than that for DNA (Figures 1-3), consistent with a smaller
reaction rate for ds-DNA in which guanine is partly protected from
oxidation within the double helix. Oscillations in the ratio [8-oxo-
guanine]/{[8-oxoguanine] + [guanine]} were superimposed on a
general decreasing trend at longer times, consistent with the
oxidation of 8-oxoguanine. LC-MS detected 8-oxoguanine and
guanidinohydantoin in oxidized DNA, indicating guanidinohydan-
toin as the oxidation product of 8-oxoguanine, as found previously.7
Our results are consistent with a competitive consecutive process
in which guanine is oxidized to 8-oxoguanine, which is oxidized
to guanidinohydantoin. Here, the common oxidant •OH reacts with
starting reactant as well as the initial reaction product. This simple
mechanism12 is unlikely to lead to oscillating concentrations of
initial product 8-oxoguanine. Oscillatory reactions typically have
very complex pathways featuring interactive catalytic cycles. For
example, the chlorite-iodide reaction has 13 elementary steps,13 and
MS, and full experimental details (PDF). This material is available free
of charge via the Internet at http://pubs.acs.org.
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1
In summary, 8-oxoguanine was formed in oscillating concentra-
tions from the oxidation of free guanine and guanine in ds-DNA
by •OH. In living systems, DNA repair, oxidative inhibition, and
other factors may be involved in controlling amounts of oxidized
nucleobases. Nevertheless, oscillatory pathways may need to be
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Acknowledgment. This work was supported by the National
Centre for Sensor Research (NCSR), Enterprise Ireland (MS), and
U.S. PHS grant no. ES03154 (JR) from NIEHS and NIH. Contents
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Supporting Information Available: Seven figures giving examples
of LC chromatograms, raw data on DNA oxidation and controls, LC-
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