Sources of 2,5-diaminoimidazolone lesions in DNA damage initiated by hydroxyl radical attack

Article abstract of DOI:10.1080/10715762.2020.1808632

The present study reports radiation-chemical yields of 2.5-diaminoimidazolone (Iz) derivatives in X-irradiated phosphate-buffered solutions of guanosine and double-stranded DNA. Various gassing conditions (air, N₂0/O₂ (4:1), N₂O, vacuum) were employed to elucidate the contribution of several alternative pathways leading to Iz in reactions initiated by hydroxyl radical attack on guanine. In all systems, Iz was identified as the second by abundance guanine degradation product after 8-oxoguanine, formed in 1:5 (guanosine) and 1:3.3 (DNA) ratio to the latter in air-saturated solutions. Experimental data strongly suggest that the addition of molecular oxygen to the neutral guanine radical G(-H)^? plays a major in Iz production in oxygenated solutions of double-stranded DNA while in other systems it may compete with recombination of G(-H)^? with superoxide and/or alkyl peroxyl radicals. The production of Iz through hydroxyl radical attack on 8-oxoguanine was also shown to take place although the chemical yield of Iz (ca 6%) in this process is too low to compete with the other pathways. The linearity of Iz accumulation with dose also indicates a negligible contribution of this channel to its yield in all systems.

Full text of DOI:10.1080/10715762.2020.1808632

Free Radical Research
ISSN: 1071-5762 (Print) 1029-2470 (Online) Journal homepage: https://www.tandfonline.com/loi/ifra20
Sources of 2,5-diaminoimidazolone lesions in DNA
damage initiated by hydroxyl radical attack
Caroline Suzanne Thomas, Hannah Catherine Pollard, Yuriy Razskazovskiy &
Marina Roginskaya
To cite this article: Caroline Suzanne Thomas, Hannah Catherine Pollard, Yuriy Razskazovskiy &
Marina Roginskaya (2020): Sources of 2,5-diaminoimidazolone lesions in DNA damage initiated by
hydroxyl radical attack, Free Radical Research, DOI: 10.1080/10715762.2020.1808632
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Sources of 2,5-diaminoimidazolone lesions in DNA damage initiated by hydroxyl radical
attack
a
a
b,*
Caroline SuzanneThomas , Hannah Catherine Pollard , Yuriy Razskazovskiy and Marina
a
Roginskaya
a
Department of Chemistry, East Tennessee State University, Johnson City, TN 37614
Department of Physics and Astronomy, East Tennessee State University, Johnson City, TN
b
3
7614
Contact information:
Caroline Suzanne Thomas: THOMASCS@mail.etsu.edu
Hannah Catherine Pollard: JANSON@mail.etsu.edu
Marina Roginskaya:
ROGINSKA@etsu.edu
*
Corresponding author: Yuriy Razskazovskiy
Department of Physics and Astronomy, East Tennessee State University,
Johnson City, TN 37614
Email: raiskazy@etsu.edu
Phone: (423) 439-6908 (office)
(
423) 943-0683 (cell)
Fax: (423) 439-6905
ORCID
0000-0002-1007-2206 (Yuriy Razskazovskiy)
0
000-0002-0204-7780 (Marina Roginskaya)

Abstract
The present study reports radiation-chemical yields of 2.5-diaminoimidazolone (Iz) derivatives in
X-irradiated phosphate-buffered solutions of guanosine and double-stranded DNA. Various gassing
condition (air, N 0/O (4:1), N O, vacuum) were employed to elucidate the contribution of several
2
2
2
alternative pathways leading to Iz in reactions initiated by hydroxyl radical attack on guanine. In all
systems Iz was identified as the second by abundance guanine degradation product after 8-oxoguanine,
formed in 1:5 (guanosine) and 1:3.3 (DNA) ratio to the latter in air-saturated solutions. Experimental

data strongly suggest that addition of molecular oxygen to the neutral guanine radical G(-H) plays a
major in Iz production in oxygenated solutions of double-stranded DNA while in other systems it may

compete with recombination of G(-H) with superoxide and/or alkyl peroxyl radicals. The production of
Iz through hydroxyl radical attack on 8-oxoguanine was also shown to take place although the chemical
yield of Iz (ca 6%) in this process is too low to compete with the other pathways. The linearity of Iz
accumulation with dose also indicates a negligible contribution of this channel to its yield in all systems.
Keywords: hydroxyl radical, guanine, oxidation, diaminoimidazolone, DNA, radiation
I. Introduction
Oxidative damage to DNA bases has been the subject of active research over the past three
decades with the progress in the area extensively reviewed in von Sonntag’s book (1) and numerous
reviews published thereafter (2-5). Guanine has long been recognized as the most prone to oxidation
DNA base due to its low standard reduction potential of +1.29 V at pH 7 (6), and a variety of potentially
mutagenic products have been shown to originate from this source. The most well-known of them, 8-
oxo-7,8-dihydroguanine (8-oxoG), easily undergoes further oxidation to form a radical cation (7) that
further leads to a large group of derivatives including, among others, deoxyribosyl-substituted
spiroiminohydantoin (Sp) (8), guanidinohydantoin (Gh) (9), isomerization/oxidation products of the
latter (10), 2,5-diaminoimidazolone (Iz) and its hydrolysis product 2,2,4-triaminooxazolone (Z) (11,12).
The latter two products are also formed in a separate oxidation channel that does not involve 8-oxoG as
an intermediate (13-15).
O
H
N
NH 2
N
NH
2
O
O
H
N
O
O
N
O
NH
2
HN
HN
HN
NH
O
HN
H 2
N
N
O
O
N
N N
H 2
N
N
HN
dR
O
H
HN
N
HN
dR
dR
dR
dR
8
-o xo G
S p
G h
Iz
Z
The relative role of the reaction channels leading to Iz and Z, and their overall significance in
oxidative damage to guanine are not well understood. In a quantitative study by Cui et al. (16) oxazolone
Z has been identified as the second by abundance after 8-oxoG in double-stranded DNA, oxidized with
peroxynitrite, singlet oxygen, and -radiation, although in much smaller quantities. In other studies,
however, Iz has been identified as a major reaction product (17,18), and our own preliminary
observations also indicated much higher relative yields of this product in X-irradiated solutions of
double-stranded DNA. Some of the problems associated with comparing Iz yields from different studies

is the poor understanding of reaction conditions that favor its formation, and the uncertainty regarding
the nature of oxidant required in the process. It is generally agreed that the precursor to Iz is a neutral

guanine radical G(-H) , which is further oxidized by molecular oxygen (19-21), or recombines with either

-

O2 (22-24), or alkyl peroxyl radicals RO (25), or both (26). These three forms of oxygen may be present
2
in variable ratios depending on reaction conditions, therefore affecting Iz yields. Second, although
oxidation of 8-oxoG has been shown to lead to Iz among other products as mentioned above, the
efficiency of this pathway and its contribution to Iz production in course of oxidative DNA damage
remain uncertain.
The issues we are addressing in the present publications are i) the absolute and relative yields of Iz
in hydroxyl-radical induced damage to the guanine moiety under various gassing conditions; ii) the roles
of different forms of reactive oxygen species in its production, and iii) the extent to which further
oxidation of 8-oxoG contributes to the production of Iz. Both monomeric guanine derivatives and double
stranded DNA were included in our study. The distinctive features of our approach are the use of more
direct procedures for quantification of Iz that exclude lengthy intermediate workout, and keeping track
of material balance in system whenever it is possible. For the sake of clarity all non-specific derivatives
containing guanine, 8-oxoguanine and 2,5-diaminoimidazolone functional groups will generally be
abbreviated as G, 8-oxoG, and Iz, respectively in the future discussion. Specific derivatives will be
abbreviated as introduced below.
2
. Materials and Methods
Materials. Commercially available guanosine (Guo) from Sigma and 2´-deoxyguanosine (dGuo) (TCI
America) were purified by crystallization from water before use. 8-oxo-2´-deoxyguanosine (8-oxodGuo)
was synthesized through copper(II)-catalyzed oxidation of dGuo with hydrogen peroxide in the presence
of ascorbic acid as described in the literature (27). The product was isolated from the reaction mixture
by solid phase extraction using of Strata-X cartridges (Phenomenex) and purified by reverse-phase HPLC
on a Luna C18 10 mm × 250 mm column (Phenomenex) washed isocratically with 0.1% acetic acid/7%
acetonitrile running phase at a 2 ml/min flow rate. Double-stranded salmon testes DNA (dsDNA,
formerly Type III from Sigma) and all other commercially available reagents mentioned in this
manuscript were used as received.
Sample preparation and irradiation. Room-temperature solutions containing Guo (1 mM), dGuo
(
2-4 mM), 8-oxodGuo (0.6 mM) or dsDNA (2.3 mM in base pairs) in 50 mM phosphate buffer (pH 6.9)
were irradiated by X-rays generated by a Phillips tube with tungsten anode operated at 60 kV and a 20
mA current. The dose rates delivered to the samples were from 4.2 to 9.4 Gy/s depending on the
experimental setup, as determined by Fricke dosimetry. Oxygen-containing samples were either air-
saturated or bubbled with a pre-made N O/O (4:1) mixture for 15 min prior to irradiation, and kept
2
2
equilibrated with the corresponding gas phase during irradiation. Fully degassed samples were prepared
in 1.5-ml glass ampoules by means of freeze-pump-thaw procedure with subsequent sealing. Nitrous
oxide -saturated samples were prepared the same way, except a controlled amount of N O was
2
condensed into the ampoules before the final evacuation and sealing step. The N O pressure in the
2
samples prepared this way was close to 1 bar at room temperature.
Product analysis.

Monomeric precursors. Irradiated solutions of Guo, dGuo and 8-oxodGuo were analyzed
immediately after irradiation by reverse-phase HPLC (Shimadzu UFPC) on a Gemini C18 4.6 mm × 250
o
mm column (Phenomenex) washed with 40 mM ammonium acetate at a 1 ml/min flow rate at 30 C. A
-
1
-1
linear acetonitrile gradient (0-16% MeCN over 15 min) was applied. 5-Fluorouracil (254 = 5500 M cm )
was employed both as an external and in some experiments as an internal reference. Quantification of
8
-oxoGuo and 8-oxodGuo was performed at 293 nm using the literature value of the extinction
-
1
-1
coefficient 293 = 10300 M cm (28). The extinction coefficient of free guanine (Gua) was measured
directly using the authentic compound (254 = 9280 M cm ).
-
1
-1
The well-known UV spectrum of the Iz chromophore with max at 320 nm and 253 nm (14,20,29)
was employed for identification and quantification of all imidazolone derivatives. The extinction
coefficient at 253 nm was estimated from relative band intensities in the UV spectra of two conjugates
containing this and another chromophore with distinct and well-characterized absorption features.
Employed for that purpose were -(4-hydrophenyl)ethyl and -(indol-3-yl)ethyl chromophores,
responsible for the absorption of tyrosine and tryptophan in near UV, respectively. The conjugates were
synthesized by treatment of 2-amino-5-(2´-deoxyribose-1´-yl)aminoimidazolone (dIz) with tyramine (Tyr)
o
and tryptamine hydrochlorides (0.2 M), both from Sigma, in aqueous sodium acetate (0.2 M) at 50 C for
1
5-20 min following the procedure described for aliphatic amines (30,31). The UV spectrum of the Tyr-Iz
conjugate thus obtained, and its reconstruction from the spectra of individual non-interacting
chromophores is shown in Figure 1. A similar result was obtained with tryptamine (data shown in the
Supplementary Material section). Based on these reconstruction and the literature extinction
-
1
-1
-1
-1
coefficients of tyrosine and tryptophan ( = 1480 M cm and 5540 M cm , respectively (32)) the

80
4
-1
-1
averaged value of 253 = 1.5×10 M cm for the Iz chromophore was obtained. We note that this
estimate is somewhat lower than the 19900 M cm value reported previously (29).
-
1
-1
dsDNA. Irradiated solutions of DNA were treated with ethanolamine acetate (0.2 M in final
o
solution) for 30 min at 45 C, followed by precipitation of the DNA with protamine sulfate (ca 220 g/ml
in final mixture) and centrifugation. The supernatant was analyzed as described in the previous section
to determine the concentrations of free bases and 2-amino-5-(-hydroxyethyl)aminoimidazolone (EIz)
formed in reaction of DNA-incorporated Iz with ethanolamine with a 60-80% yield (31). DNA-
incorporated 8-oxoG was quantified as free 8-oxoguanine (8-oxoGua) in the hydrolysates obtained using
o
a standard procedure of formic acid DNA hydrolysis (88% formic acid for 90 min at 150 C under vacuum)
following the same protocol as described previously (30).

3
. Results
Radiation-induced damage to Guo. A typical chromatogram produced by irradiation of Guo (1 mM) in
air-equilibrated phosphate buffer is shown in Figure 2. Three out of four major products are readily
identifiable as 2-amino-5-(ribose-1´-yl)aminoimidazolone (rIz), free guanine (Gua) and 8-oxoGuo by their
characteristic UV spectra and by comparison with an authentic reference compound (Gua). An
unidentified product X has the UV-spectrum identical to that of Guo, and, therefore, likely contains a
modified ribose moiety. Dose response curves for Guo loss and Iz accumulation at different gassing
conditions are shown in Figure 3, and the corresponding radiation-chemical yields G are reported in
Table 1. Dose dependencies for Gua and 8-oxoGuo production were also obtained and can be found in
the Supplementary Materials section. While the release of Gua in all systems is reasonably linear over
the entire dose range, the accumulation of 8-oxoGuo shows a tendency to saturation at higher doses,
which is especially pronounced in N O-containing samples. As a result, only low-dose parts of the dose
2
dependencies, assumed to be linear, were used to determine the yields of 8-oxoGuo in the presence of
N O. The G-values thus obtained are also listed in Table 1.
2
The numbers in the Table clearly identify rIz as the second by abundance product after 8-oxoGuo
with the rIz/8-oxoGuo ratio about 0.2 in samples irradiated under air. It may also be concluded that
altogether the pathways leading to products listed in the Table account for the majority (from 69 to
9
5%) of the processes responsible for Guo destruction. Among them the production of rIz accounts for
up to 18% of the total Guo loss (under N O/O mix) that definitely makes this reaction channel worth of
2
2
paying attention to.
dsDNA. A typical chromatogram showing the peaks of EIz and free DNA bases in the
supernatant obtained by post-irradiation treatment of phosphate-buffered solutions of DNA irradiated
under oxic conditions is shown in Figure 4. An extra peak denoted as Lac belongs to 1-(-hydroxyethyl)-
3
5
-methylene- -pyrrolin-2-one formed in reaction of ethanolamine with C4-oxidized abasic sites (33).
Preliminary tests showed that the release of EIz is below reliable detection limit under anoxic conditions
both in the absence and the presence of N O, and these systems were excluded from further
2
consideration. A representative set of dose response curves for free base, EIz, and 8-oxoG production in
solutions of DNA saturated with air or N O/O mix are shown in the insert. The G-values estimated from
2
2
initial slopes of the curves are reported in Table 1 (the last two entries). The G-value for Iz production in
DNA was calculated from the yield of EIz using the scaling factor of 1.7 obtained in the previous
publication (31). This scaling factor accounts for partial hydrolysis of Iz in parallel with the formation of
EIz upon post-irradiation treatment of irradiated DNA with ethanolamine. The data show that the Iz/8-
oxoG ratios found in dsDNA (0.3 under air, in particular), are not much different from those measured
with Guo as a target. The latter number, however, is roughly 10 times greater than the Z/8-oxoG ratio
(0.03) found by Cui et al. (16) in calf thymus DNA under similar conditions. Since the formation of Z from
Iz is generally considered to be quantitative, the source of such a significant disagreement remains
unclear. Other yet unidentified factors, therefore, may be involved.
Imidazolone yield in reaction of hydroxyl radicals with 8-oxodGuo. Irradiation of 8-oxodGuo in a
phosphate-buffered aerated solution results in accumulation of dIz along with free 8-oxoGua and a set
of products whose identification was not attempted (Figure 5). Previously characterized deoxyribosyl-
substituted Sp (8), Gh (9,10), and other related derivatives are likely present among those with retention
times shorter than 5 min under reverse phase separation conditions.

Dose dependencies for both 8-oxodGuo destruction and dIz accumulation are approximately
-
1
linear (insert in Figure 5) with the corresponding G-values of 138 and 7.8 mol J , respectively. These

results identify dIz a minor product of 8-oxodGuo destruction by HO , formed with  6% chemical yield.
Linearity of the dose response curve for dIz accumulation is also inconsistent with the involvement of a
long lived (on the experimental timescale) intermediate precursor to dIz. This makes neither of the
above mentioned stable oxidation products suitable as intermediates in hydroxyl radical-induced
formation of dIz from 8-oxodGuo.
4
. Discussion
There is a consensus in the literature that the major outcome of hydroxyl radical attack on

guanine (60-70% according to Candeias and Steenken (34)) is the neutral guanine radical G(-H) that
formally results from the loss of N -hydrogen, although different views have been expressed on whether
1
its immediate precursor is a C -hydroxyl radical adduct (34), an ion pair formed by the guanine radical
4
-
cation and HO (35), or an N -aminyl radical (36,37). For the purpose of our discussion it is important to
note that regardless of the intermediates involved the formation of G(-H) is not affected by molecular
oxygen in sub-millimolar concentrations available in air-saturated solutions (34). This fact makes G(-H)
2


the most abundant precursor to imidazolone derivatives under either oxic or anoxic conditions. Another
reaction pathway that accounts roughly for 17% of events, is the C -adduct formation that further leads
8
to 8-oxoguanine in oxygenated media, which may also form imidazolone upon further oxidation. The
non-linear dose response for 8-oxoG accumulation observed in solutions of both Guo and DNA does
-

indicate its further degradation likely in reaction with HO , G(-H) (38), or mobile holes in DNA (39).
However, the linearity of Iz accumulation in these systems is inconsistent with the involvement of a
long-lived intermediate, that makes the participation of 8-oxoG in this process unlikely. In addition, the
low chemical yield of dIz in hydroxyl radical-initiated oxidation 8-oxodGuo (ca.6%) measured directly
makes this particular pathway even less feasible.
As follows from the data presented in Table 1, the production of Iz is obviously enhanced in the
presence of molecular oxygen. However, at least in the case of Guo it also occurs with appreciable
efficacy under strictly anoxic conditions if N O is present in the system. Electron scavengers are known
2
to increase the yield of radiation-produced hydrogen peroxide in aqueous solutions (40), which may act
as a source of superoxide under anoxic conditions. To test this possibility an anoxic solution of Guo (1
mM) in 50 mM phosphate containing H O (0.18 mM) was irradiated and analyzed as other similar
2
2
systems. This amount of H O corresponded to its expected concentration in a medium with the
2
2
6
-1
scavenging capacity of 2×10 s after a 2.7 kGy dose (41). No effect on rIz production was observed,
which remained very low regardless of the presence of H O as shown in the Supplementary Materials
2
2
section. This fact clearly demonstrates the existence of pathways leading to Iz that do not require the
involvement of peroxyl-type intermediates at any step. Direct involvement of N O in oxidation of free
2
radical intermediates may, therefore, be considered. The scheme that summarizes hypothetical reaction
pathways from the literature (20-22,24,25) with some additions and modifications is shown in the
Scheme below.

O
-
+
OOH
O 2 , H
N
N
O 2 , [H ]
N
H 2
N
N
O
dR
O
N
O
N
O
OOR
N
N
[H]
O
N
N
RO 2
N
N
N
NH
N
N
N
-CO
H 2
N
N
H 2
N
H 2 N
N
H 2
N
N
O
dR
-scissio n
dR
dR
G (-H )
dR

H 2
O
-H 2 NCHO
O
N 2
O
ON=N
N
N
N
-N 2
NH
dR
N
N
H 2
N
N
H 2 N
dR
Iz

-
From the top three pathways shown in the Scheme, the recombination of G(-H) with O can
2
hardly play a significant role in air-saturated 1 mM solutions of Guo due to the low expected yield of
10
-1 -1
superoxide anion in this system. The rate constant of electron attachment to dGuo (k = 1.7×10 M s
10
-1 -1
(
42)) is practically the same as to O (1.8×10 M s (43)) and, therefore, dissolved oxygen could
2
scavenge only about 16% of radiation-produced electrons due to a 5-fold difference in concentrations of
the scavengers (1 mM Guo vs 0.2 mM O in air-saturated solutions). The guanine radical anion formed in
2
the process undergoes rapid C-protonation to form a neutral radical, which is unreactive towards O (44)
2

-
and, therefore, cannot give rise to O . The situation, however, should be different in dilute (2.3 mM in
2
base pairs) solutions of DNA since the effective rate of solvated electron attachment to DNA (k =
8
-1
-1
(
1.01.4)×10 M cm , DNA concentration in bases (45,46)), is two orders of magnitude slower than to

-
Guo. As a result, 84% of the solvated electrons must be scavenged by O leading to O . This process,
2
2
however, should be completely suppressed by N O in solutions saturated with N O:O = 4:1 mix, which
2
2
2
contains about 22 mM of N O under ambient temperature and pressure, and converts solvated
2

9
-1 -1
electrons into HO in a diffusion-controlled process (k = 9.1×10 M s (47)). Therefore, the fact that no
suppression in Iz production takes place in the presence of N O (actually the opposite is observed)
2

-
suggests a negligible role of G(-H) + O2 recombination as a source of Iz in polymeric DNA as well.
Regarding the other two oxygen-dependent pathways, it remains uncertain which one (the



recombination of G(-H) with RO or direct addition of O to G(-H) ) plays the major role in the case of
2
2
Guo. Substantial yields of free guanine indicate the presence of a significant source of ribose-based


radicals R , and, therefore, of RO in oxygenated systems. Both pathways are expected to be enhanced
2


in the presence of N O (as observed) due to the simultaneous increase in production of R and G(-H) .
2
The same can take place (and is also observed experimentally) in solutions of polymeric DNA. There is a


reason to believe, however, that recombination of G(-H) with RO in DNA should be far less efficient
2
than in solutions of Guo. Peroxyl radicals formed in this system are polymeric, therefore, are far less
mobile than monomeric Guo-derived species. Based on this logic and the fact that Iz formation in N O-
2
saturated solutions of DNA still occurs with the efficiency similar to that in Guo may serve as an

indication of direct addition of O to G(-H) is a major contributor to Iz production in DNA. A potential
2

6
problem with this pathway is the relatively low reactivity of G(-H) towards molecular oxygen (k  10
M s ) reported by Candeias and Steenken (34) although the possibility of this reaction has not been
-
1 -1

ruled out. In particular, it may become competitive in the absence of reducing agents and
recombination reactions as in the case of polymeric DNA in inert medium.
The H O -independent formation of rIz in N O-saturated solutions of Guo under strictly anoxic
2
2
2

conditions suggests that N O itself can possibly act as an oxidant of G(-H) . Nitrous oxide is known to
2
react with metal ions in low oxidation states through oxygen atom transfer with appreciable rates (for
+
+
+
+
reactions with Zn , Cd , Co and Ni see, for example, ref. (48)). It reacts in a similar way with the
hydrogen atom (49) but there is practically no information on reactivity of N O towards carbon-based
2
free radicals. A few reported examples include the reaction with photolytically generated phenyl radicals
8
-1 -1
(
50), and the oxidation of 2-deoxyuridine-1-yl radical with the rate constant of 1.77×10 M s (51). A
hypothetical pathway for oxidation of carbon-based radicals by N O, shown in the scheme, is through
2

addition-elimination, which in the case of G(-H) would lead to an alkoxyl radical, identical to the

-
precursor to Iz in the process initiated by recombination of G(-H) and O . Alternatively, an iminoxyl-
2

type intermediate R-N=N-O has also been considered (51). In dsDNA, however, the yield of Iz turned
out to be negligible under anoxic conditions even in the presence of N O. The reason for that could be
2

restricted accessibility of dissolved N O to G(-H) in double-stranded DNA.
2
5
. Conclusion
The present study identifies Iz as the second by abundance (after 8-oxoG) product of hydroxyl
radical-induced damage to guanine, although the relative yields were found to be much higher than
reported earlier. Besides recombination with superoxide and peroxyl radicals, experimental data

strongly suggest the involvement of direct addition of molecular oxygen to G(-H) as a source of this end
product, especially in double-stranded DNA. There is an indication that nitrous oxide can modify free
radical processes in DNA, including those leading to imidazolone formation, not only through scavenging
of solvated electrons, but also through direct oxidation of free radical intermediates. Secondary

oxidation of 8-oxoguanine by HO can only be a minor source of Iz in radiation-induced damage to DNA
and its constituents.
6
. Acknowledgement
This work was supported by the ETSU Research Development Committee 2018 Major Grant
Program under Grant number 18-019M awarded to MR.
7
. Disclosure of interest
The authors report no conflict of interest.

References
1
. von Sonntag C. Free-radical-induced DNA damage and its repair. A chemical perspective. Springer-
Verlag, Berlin-Heidelberg-New York; 2006.
2
. Cadet J, Douki T, Ravanat J-L. Oxidatively generated damage to the guanine moiety of DNA:
mechanistic aspects and formation in cells. Acc Chem Res 2008; 41:1075-83.
3
4
. Dizdarouglu M, Jaruga P. Mechanisms of free radical-induced damages to DNA. Free Rad Res 2012;
6:382-419.
4
. Cadet J, Wagner JR. Oxidatively generated base damage to cellular DNA by hydroxyl radical and one-
electron oxidants: Similarities and differences. Arch Biochem Biophys 2014; 557:47-54.
5
. Cadet J, Davies KJA, Madeiros MH, Di Masciod P, Wagner JR. Formation and repair of oxidatively
generated damage in cellular DNA. Free Radic Biol Med 2017; 107:13-34.
6
. Steenken S, Jovanovic SV. How easily oxidizable is DNA? One-electron reduction potentials of
adenosine and guanosine radicals in aqueous solution. J Am Chem Soc 1997; 119:617-618.
7
. Shukla LI, Adhikary A, Pazdro R, Becker D, Sevilla MD. Formation of 8-oxo-7,8-dihydroguanine-radicals
in ?-irradiated DNA by multiple one-electron oxidations. Nucleic Acids Res 2004; 32:6565-74.
8
. Luo W, Muller JG, Rachlin EM, Burrows CJ. Characterization of spiroiminodihydantoin as a product of
one-electron oxidation of 8-oxo-7,8-dihydroguanosine. Org Lett 2000; 2:613-6.
9
. Duarte V, Muller JG, Burrows CJ. Insertion of dGMP and dAMP during in vitro DNA synthesis opposite
an oxidized form of 7,8-dihydro-8-oxoguanine. Nucleic Acids Res 1999; 27:496-502.
1
0. Luo W, Muller JG, Rachlin EM, Burrows CJ. Characterization of hydantoin products from one-electron
oxidation of 8-oxo-7,8-dihydroguanosine in a nucleoside model. Chem Res Toxicol 2001; 14:927-38.
1
1. Kino K, Sugiyama H. Possible cause of GC->CG transversion mutation by guanine oxidation product,
imidazolone. Chem Biol 2001; 8:369-378.
1
2. Hickerson RP, Prat IF, Muller JG, Foote CS, Burrows CJ. Sequence and stacking dependence of 8-
oxoguanine oxidation: comparison of one-electron vs singlet oxygen mechanisms. J Am Chem Soc 1999;
1
21:9423-8.
1
3. Ravanat J-L, Berger M, Benard F, Langlois R, Ouellet R, van Lier JE, Cadet J. Phthalocyanine and
naphthalocyanine photosensitized oxidation of 2'-deoxyguanosine. Photochem Photobiol 1992; 55:809-
1
4.
1
4. Raoul S, Berger M, Buchko GW, Joshi PC, Morin B, Weinfeld M, Cadet J. 1H, 13C and 15N nuclear
magnetic resonance analysis and chemical features of the two main radical oxidation products of 2'-
deoxyguanosine: oxazolone and imidazolone nucleosides. J Chem Soc, Perkin Trans 2 1996:371-81.
1
5. Gasparutto D, Ravanat J-L, Gerot O, Cadet J. Characterization and chemical stability of photooxidized
oligonucleotides that contain 2,2-Diamino-4-[(2-deoxy-b-D-erythro-pentofuranosyl)amino]-5(2H)-
oxazolone. J Am Chem Soc 1998; 120:10283-6.

1
6. Cui L, Ye W, Prestwich EG, Wishnok JS, Taghizadeh K, Dedon PC, Tannenbaum SR. Comparative
analysis of four oxidized guanine lesions from reactions of DNA with peroxynitrite, singlet oxygen, and -
radiation. Chem Res Toxicol 2013; 26:196-202.
1
7. Kino K, Saito I, Sugiyama H. Product analysis of GG-specific photooxidation of DNA via electron
transfer: 2-aminoimidazolone as a major guanine oxidation product. J Am Chem Soc 1998; 120:7373-4.
1
8. Kupan A, Saulière A, Broussy S, Seguy C, Pratviel G, Meunier B. Guanine oxidation by electron
transfer: one- versus two-electron oxidation mechanism. Chembiochem 2006; 7:125-33.
1
9. Buchko GW, Cadet J, Ravanat J-L, Labataille P. Isolation and characterization of a new product
produced by ionizing radiation and type I photosensitization of 2'-deoxyguanosine in oxygen-saturated
aqueous solution: (2S)-2,5'-anhydro-1-(2'-deoxy--D-erythro-pentofuranosyl)-5-guanidinylidene-2-
hydroxy-4-oxoimidazolidene. Int J Radiat Biol 1993; 63:669-676.
2

0. Cadet J, Berger M, Buchko GW, Joshi PC, Raoul S, Ravanat J-L. 2,2-Diamino-4-[(3,5-di-acetyl-2-deoxy-
-D-erythro-pentafuranosyl)amino--5-(2H)-oxazolone: a novel and predominant radical oxidation
product of 3',5'-di-acetyl-2'-deoxyguanosine. J Am Chem Soc 1994; 116:7403-04.
2
1. Douki T, Cadet J. Modification of DNA bases by photosensitized one-electron oxidation. Int J Radiat
Biol 1999; 75:571-581.
2
2. Misiaszek R, Crean C, Joffe A, Geacintov NE, Shafirovich V. Oxidative DNA damage associated with
combination of guanine and superoxide radicals and repair mechanisms via radical trapping. J Biol Chem
2
2
004; 279:32106-15.
3. Fleming AM, Muller JG, Ji I, Burrows CJ. Characterization of 2'-deoxyguanosine oxidation products
observed in the Fenton-like system Cu(II)/H O /reductant in nucleoside and oligodeoxynucleotide
2
2
contexts. Org Biomol Chem 2011; 9:3338-48.
2
4. Alshykhly OR, Fleming AM, Burrows CJ. 5-Carboxamido-5-formamido-2-iminohydantoin, in addition
to 8-oxo-7,8-dihydroguanine, Is the major product of the iron-Fenton or X-ray radiation-induced
oxidation of guanine under aerobic reducing conditions in nucleoside and DNA contexts. J Org Chem
2
015 2015; 80:6996-7007.
2
5. Crean C, Geacintov NE, Shafirovich V. Pathways of arachidonic acid peroxyl radical reactions and
product formation with guanine radicals. Chem Res Toxicol 2008; 21:358-73.
2
6. Rokhlenko Y, Geacintov NE, Shafirovich V. Lifetimes and reaction pathways of guanine radical cations
and neutral guanine radicals in an oligonucleotide in aqueous solutions. J Am Chem Soc 2012; 134:4955-
4
962.
2
7. Peres PS, Valerio A, Martinez GR. Synthesis of 8-oxo-7,8-dihydro-2´-deoxyguanosine from 2´-
deoxyguanosine using Cu(II)/H2O2/ascorbate: A new strategy for an improved yield. BioTechniques
2
016; 58:279-84.
2
8. Kasai H, Nishimura S. Hydroxylation of deoxyguanosine at the C-8 position by ascorbic acid and other
reducing agents. Nucleic Acids Res 1984; 12:2137-46.

2
9. Suzuki T, Masuda M, Friesen MD, Fenet B, Oshima H. Novel products generated from 2'-
-
deoxyguanosine by hypochlorous acid or a myeloperoxidase-H O -Cl system: identification of diimino-
2
2
imidazole and amino-imidazolone nucleotides. Nucleic Acids Res 2002; 30:2555-64.
3
0. Roginskaya M, Ampang-Bouteng D, Mohseni R, Razskazovskiy Y. DNA damage by sulfate radical
anions: hydrogen abstraction from the sugar moiety vs. one-electron oxidation of guanine. Free Radical
Res 2016; 50:756-66.
3
1. Roginskaya M, Janson H, Seneviratne D, Razskazovskiy Y. The reactivity of 2,5- diaminoimidazolone
base modification towards primary amines: nucleophilic substitution at C5 as a potential source of
abasic sites in oxidatively damaged DNA. Res Chem Intermediat 2017; 43:1543-55.
3
2. Mach H, Middaugh CR, Lewis RW. Statistical determination of the average values of the extinction
coefficients of tryptophan and tyrosine in native proteins. Anal Biochem 1992; 200:74-80.
3
3. Roginskaya M, Mohseni R, Moore TJ, Bernhard WA, Razskazovskiy Y. Identification of the C4`-
oxidized abasic site as the most abundant 2-deoxyribose lesion in radiation-damaged DNA using a novel
HPLC-based approach. Radiat Res 2014; 181:131-4.
.
3
4. Candeias LP, Steenken S. Reaction of HO with guanine derivatives in aqueous solution: formation of
two different redox-active OH-adduct radicals and their unimolecular transformation reactions.

Properties of G(-H) Chem Eur J 2000; 6:475-84.
.
3
5. Kumar A, Pottiboyina V, Sevilla MD. Hydroxyl radical (OH ) reaction with guanine in an aqueous
environment: a DFT study. J Phys Chem B 2011; 115:15129-37.
3
6. Chatgilialoglu C, D’Angelantonio M, Guerra M, Kaloudis P, Mulazzani QG. A reevaluation of the
ambident reactivity of the guanine moiety towards hydroxyl radicals. Angew Chem, Int Ed 2009;
4
8:2214-7.
3
7. Chatgilialoglu C, D’Angelantonio M, Kciuk G, Bobrowski K. New insights into the reaction paths of
hydroxyl radicals with 2'-deoxyguanosine. Chem Res Toxicol 2011; 24:2200-6.
3
8. Ravanat J-L, Saint-Pierre C, Cadet J. One-electron oxidation of the guanine moiety of 2-
deoxyguanosine: influence of 8-oxo-7,8-dihydro-2-deoxyguanosine. J Am Chem Soc 2003; 125:2030-1.
3
9. Cai Z, Sevilla MD. Electron and hole transfer from DNA base radicals to oxidized products of guanine
in DNA. Radiat Res 2003; 159:411-419.
4
0. Hiroki A, Pimblott SM, LaVerne, JA. Hydrogen peroxide production in the radiolysis of water with
high radical scavenger concentrations. J Phys Chem A 2002; 106:9352-8.
4
1. von Sonntag C. The Chemical Basis of Radiation Biology. Taylor & Francis, London–New York–
Philadelphia; 1987.
4
2. Arce R, Rivera J. Intermediates produced from the room temperature 266 nm laser photolysis of
guanines. J Photochem Photobiol A 1989; 49:219-37.
4
3. Elliot AJ. A pulse radiolysis study of the temperature dependence of reactions involving H, OH and
eaq- in aqueous solutions. Radiat Phys Chem 1989; 34:753-8.

4
4. Candeias LP, Wolf P, O'Neill P, Steenken S. Reaction of hydrated electrons with guanine nucleosides:
Fast protonation on carbon of the electron adduct. J Phys Chem 1992; 96:10302-7.
4
5. Shragge PC, Michaels HB, Hunt JW. Factors affecting the rate of hydrated electron attack on
polynucleotides. Radiat Res 1971; 47:598-611.
4
6. Whillans DW, Adams GE. Electron transfer oxidation of DNA radicals by paranitroacetophenone. Int J
Radiat Biol Relat Stud Phys, Chem Med 1975; 28:501-10.
4
7. Buxton GV, Greenstock CL, Helman WP, Ross AB. Critical review of rate constants for reactions of
-
hydrated electrons, hydrogen atoms and hydroxyl radicals (·OH/·O ) in aqueous solution. J Phys Chem
Ref Data 1988; 17:513-886.
4
8. Buxton GV, Sellers RM, McCracken DR. Pulse radiolysis study of monovalent cadmium, cobalt, nickel
and zinc in aqueous solution. Part 2. Reactions of the monovalent ions. J Chem Soc, Faraday Trans 1
1
4
976; 72:1464-76.
9. Czapski G, Peled E. On the pH-dependence of Greducing in the radiation chemistry of aqueous
solutions. Isr J Chem 1968; 6:421-36.
5
5
0. Park YT, Kim KW, Song NW, Kim D. Nitrous oxide as a radical scavenger. J Org Chem 1998; 63:4494-6.
1. Sarkar U. Metabolically activated heterocyclic N-oxide compounds for killing and visualizing hypoxic
cancer cells. [Ph.D.]. Columbia: University of MIssouri; 2009. 152 p.

Figure Legends
1
. UV spectrum of the tyrosine-imidazolone conjugate (Tyr-Iz, structure shown in the Figure) and its
reconstruction as a linear combination of spectra of tyrosine and dIz.

2
. Sample chromatogram of Guo (1 mM) X-irradiated (200 Gy) in 50 mM phosphate buffer under air. 5-
Fluorouracil (5FU) was added as an internal standard after irradiation.

3
. Dose response curves for rIz accumulation and Guo loss (shown in the insert) in phosphate-buffered
solutions of Guo under various gassing conditions (specified in the Figure).

4
. Sample chromatogram of the supernatant obtained by X-irradiation (200 Gy) of double-stranded DNA
2.3 mM in base pairs) in 50 mM phosphate under air after treatment with ethanolamine (0.2 M) and
precipitation of the polymeric component with protamine. Dose response curves for free base release,
-oxoG and EIz accumulation are shown in the insert.
(
8

5
. Sample chromatograms of 8-oxodGuo (0.6 mM) X-irradiated in 50 mM phosphate under air. Dose
response curves for 8-oxodGuo loss and dIz accumulation are shown in the insert.

Table 1. Radiation-chemical yields G of starting material (SM)
degradation and product formation in X-irradiated solutions of Guo and
double-stranded DNA
-1
Gassing
G, nmol J
SM loss
Iz
Guo
8-oxoG
Gua
All products
Air
73
126
168
58
5.8
23
29
48
46
11
26
49
65
42
61
120
116
54
N O/O (4:1)
2
2
N O
4.9
0.7
2
Vacuum
DNA
Air
n. d.*
n. d.*
6.6
18
22
74
55**
84
92
N O/O (4:1)
107**
2
2
*
*
Total yield of DNA oxidation, not determined
* Free base release