Nitrogen dioxide as an oxidizing agent of 8-oxo-7,8-dihydro-2′-deoxyguanosine but not of 2′-deoxyguanosine

Article abstract of DOI:10.1021/tx000204t

The redox reactions of guanine and its widely studied oxidation product, the 8-oxo-7,8-dihydro derivative, are of significant importance for understanding the mechanisms of oxidative damage in DNA. Employing 2′-deoxyguanosine 5′-monophosphate (dGMP) and 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dG) in neutral aqueous solutions as model systems, we have used nanosecond laser flash photolysis to demonstrate that neutral radicals, dGMP(-H)?, derived by the one-electron oxidation and deprotonation of dGMP, can oxidize nitrite anions (NO₂^-) to the nitrogen dioxide radical ?NO₂. In turn, we show that ?NO₂ can give rise to a one-electron oxidation of 8-oxo-G, but not of dGMP. The one-electron oxidation of dGMP was initiated by a radical cation generated by the laser pulse-induced photoionization of a pyrene derivative with enhanced water solubility, 7,8,9,10-tetrahydroxytetrahydrobenzo[α]pyrene (BPT). The dGMP(-H)? neutral radicals formed via deprotonation of the dGMP^?+ radical cations and identified by their characteristic transient absorption spectrum (λ_max ～ 310 nm) oxidize nitrite anions with a rate constant of(2.6 ± 0.3) × 10⁶ M^-1 s^-1. The 8-oxo-dG is oxidized by ?NO₂ with a rate constant of (5.3 ± 0.5) × 10⁶ M^-1 s^-1. The 8-oxo-dG(-H)? neutral radicals thus generated are clearly identified by their characteristic transient absorption spectra (λ_max ～ 320 nm). The rate constant of 8-oxo-dG oxidation (k₁₂) by the ?NO₂ one-electron oxidant (the ?NO₂/NO₂^- redox potential, E° ≈ 1.04 V vs NHE) is lower than k₁₂ for a series of oxidizing aromatic radical cations with known redox potentials. The k₁₂ values for 8-oxo-dG oxidation by different aromatic radical cations derived from the photoionization of their parent compounds depend on the redox potentials of the latter, which were in the range of 0.8-1.6 V versus NHE. The magnitude of k₁₂ gradually decreases from a value of 2.2 × 10⁹ M^-1 s^-1 (E° = 1.62 V) to 5.8 × 10⁸ M^-1 s^-1 (E° = 1.13 V) and eventually to 5 × 10⁷ M^-1 s^-1 (E° = 0.91 V). The implications of these results, including the possibility that the redox cycling of the ?NO₂/NO₂^- species can be involved in the further oxidative damage of 8-oxo-dG in DNA in cellular environments, are discussed.

Full text of DOI:10.1021/tx000204t

Chem. Res. Toxicol. 2001, 14, 233-241
233
Nitr ogen Dioxid e a s a n Oxid izin g Agen t of
8
-Oxo-7,8-d ih yd r o-2′-d eoxygu a n osin e bu t Not of
2
′-Deoxygu a n osin e
,
†
‡
‡
Vladimir Shafirovich,* J ean Cadet, Didier Gasparutto,
†
†
Alexander Dourandin, and Nicholas E. Geacintov
Chemistry Department and Radiation and Solid State Laboratory, 31 Washington Place, New York
University, New York, New York 10003-5180, and Laboratoire “L e´ sions des Acides Nucl e´ iques”,
SCIB/ DRFMC and UMR CNRS 5046, CEA/ Grenoble, 17, rue des Martyrs,
F-38054 Grenoble, Cedex 9, France
Received September 14, 2000
The redox reactions of guanine and its widely studied oxidation product, the 8-oxo-7,8-dihydro
derivative, are of significant importance for understanding the mechanisms of oxidative damage
in DNA. Employing 2′-deoxyguanosine 5′-monophospate (dGMP) and 8-oxo-7,8-dihydro-2′-
deoxyguanosine (8-oxo-dG) in neutral aqueous solutions as model systems, we have used
•
nanosecond laser flash photolysis to demonstrate that neutral radicals, dGMP(-H) , derived
-
by the one-electron oxidation and deprotonation of dGMP, can oxidize nitrite anions (NO
2
) to
•
•
2 2
the nitrogen dioxide radical NO . In turn, we show that NO can give rise to a one-electron
oxidation of 8-oxo-G, but not of dGMP. The one-electron oxidation of dGMP was initiated by
a radical cation generated by the laser pulse-induced photoionization of a pyrene derivative
with enhanced water solubility, 7,8,9,10-tetrahydroxytetrahydrobenzo[a]pyrene (BPT). The
•
•+
dGMP(-H) neutral radicals formed via deprotonation of the dGMP radical cations and
identified by their characteristic transient absorption spectrum (λmax ∼ 310 nm) oxidize nitrite
6
-1 -1
•
anions with a rate constant of (2.6 ( 0.3) × 10 M
s
. The 8-oxo-dG is oxidized by NO
. The 8-oxo-dG(-H) neutral radicals thus generated
2
with
6
-1 -1
•
a rate constant of (5.3 ( 0.5) × 10 M
s
are clearly identified by their characteristic transient absorption spectra (λmax ∼ 320 nm). The
•
•
-
2 2 2
rate constant of 8-oxo-dG oxidation (k12) by the NO one-electron oxidant (the NO /NO redox
potential, E° ≈ 1.04 V vs NHE) is lower than k12 for a series of oxidizing aromatic radical
cations with known redox potentials. The k12 values for 8-oxo-dG oxidation by different aromatic
radical cations derived from the photoionization of their parent compounds depend on the redox
potentials of the latter, which were in the range of 0.8-1.6 V versus NHE. The magnitude of
9
-1 -1
8
-1 -1
k
)
12 gradually decreases from a value of 2.2 × 10 M
s
(E° ) 1.62 V) to 5.8 × 10 M
s
(E°
7
-1 -1
1.13 V) and eventually to 5 × 10 M
s
(E° ) 0.91 V). The implications of these results,
including the possibility that the redox cycling of the NO
•
-
2
/NO
2
species can be involved in the
further oxidative damage of 8-oxo-dG in DNA in cellular environments, are discussed.
In tr od u ction
reaction products have been reported with the main
components, such as 8-oxo-7,8-dihydro-2′-deoxyguanosine
Nitric oxide is generated by vascular endothelial cells
to regulate a variety of diverse biochemical and physi-
ological processes, including blood pressure modulation,
signal transduction, smooth muscle relaxation, platelet
activation, and aggregation (1). Various reactive nitrogen
species produced by the reaction of NO in a cellular
environment can induce a variety of DNA lesions, includ-
ing base modifications that, if not repaired, can give rise
1
(
8-oxo-dG) (9, 11) and 8-nitro-2′-deoxyguanosine (8-nitro-
dG) which is spontaneously released from DNA in the
form of 8-nitroguanine (8-nitro-G) by depurination (5-
7
, 11). Furthermore, 8-oxo-dG is more reactive with
respect to strong oxidants than the parent guanine
residues and A, C, and T bases (8, 13-18). These
observations are fully consistent with recent computa-
•
tional studies (19, 20). Another reactive nitrogen species
to mutations. For example, N
2
O
3
gives rise to nitrosation
•
that is known to be mutagenic is NO
2
(21-23). This
and subsequent deamination of DNA bases (2, 3), while
peroxynitrite induces mostly oxidation and nitration of
the nucleic acid bases (4-14). The primary targets of
peroxynitrite attack are guanines (12), the most readily
oxidized of the four natural DNA bases. A variety of
nitrogen dioxide radical can be generated in a cellular
environment by the oxidation of nitrite, NO , a process
2
-
that can be mediated by myeloperoxidase (24, 25) as well
as by other cellular oxidants (26-28). Nitrite itself is an
end product of the reaction of nitric oxide with oxygen
(29) and is normally present at concentrations of <200
*
To whom correspondence should be addressed: Chemistry Depart-
ment and Radiation and Solid State Laboratory, 31 Washington Place,
New York University, New York, NY 10003-5180. Telephone: (212)
1
Abbreviations: 8-oxo-dG, 8-oxo-7,8-dihydro-2′-deoxyguanosine; 8-
9
98-8456. Fax: (212) 998-8421. E-mail: vs5@nyu.edu.
nitro-G, 8-nitroguanine; 8-nitro-dG, 8-nitro-2′-deoxyguanosine; G,
guanine; dG, 2′-deoxyguanosine; dGMP, 2′-deoxyguanosine 5′-mono-
phospate; BPT, 7,8,9,10-tetrahydroxytetrahydrobenzo[a]pyrene.
†
New York University.
‡
CEA/Grenoble.
1
0.1021/tx000204t CCC: $20.00 © 2001 American Chemical Society
Published on Web 01/25/2001

2
34 Chem. Res. Toxicol., Vol. 14, No. 2, 2001
Shafirovich et al.
Sch em e 1
in this work, since we focused on the electron transfer
•
-
reaction steps between G(-H) and NO
2
with the
with 8-oxo-
•
•
generation of NO
2
and the reaction of NO
2
dG in aqueous solutions. Our observations constitute the
•
2
first direct indication that the redox cycling of the NO /
-
NO
dG. In addition, we demonstrate that NO
deprotonated guanine radicals, thus forming the reactive
2
species can be involved in the oxidation of 8-oxo-
-
2
can react with
•
2
oxidizing species NO .
Exp er im en ta l P r oced u r es
Ma ter ia ls. The 8-oxo-dG nucleoside was synthesized and
purified by HPLC methods, as described previously (45). The
other chemicals (g99% pure) were obtained from Sigma-
Aldrich Fine Chemicals and were used as received. The pyrene
derivative BPT was prepared by hydrolysis of racemic anti-
BPDE (7r,8t-dihydroxy-9t,10-epoxy-7,8,9,10-tetrahydrobenzo-
µM in cellular environments (30, 31). However, the NO -
levels are markedly increased to millimolar levels after
2
ingestion of nitrate (32), or during inflammatory events
•
that reflect increased levels of NO (33). These observa-
[a]pyrene, a gift from S. Amin, American Health Foundation,
•
tions suggest that NO
2
might also be generated from
Valhalla, NY) (46). The absorbances of the BPT phosphate buffer
-1
nitrite in significant amounts and that the potential
reactivity of this oxidizing species with cellular bio-
solutions (pH 7) were generally ∼0.1 (308 nm) or 0.07 cm (355
nm), corresponding to a BPT concentration of ∼25 µM [the
concentration was estimated from the molar extinction coef-
macromolecules is worthy of further examination.
4
-1
-1
•
ficient (40) of BPT, ꢀ343 ) 2.9 × 10 M cm ].
The role of NO
2
in potentiating oxidative DNA damage
La ser F la sh P h otolysis. The transient absorption spectra
were recorded using a kinetic spectrometer system (∼7 ns
response time) described previously (40, 47-49). Briefly, this
system consists of a Q-switched Continuum Surelite II Nd:YAG
has received relatively little attention due to the known
low reactivity of nitrogen dioxide with native DNA (34).
•
On the other hand, the reaction of NO
2
with modified
bases such as 8-oxo-dG has not been reported. As the
redox potential of 8-oxo-dG is lower than that of guanine
-1
-2
laser (355 nm, fwhm ) 4 ns, ∼60 mJ pulse cm , 10 Hz) or a
NY81-20 Nd:YAG laser excitation source (266 nm, fwhm ) 6
ns, e200 mJ pulse-1 cm-2, 20 Hz) excitation source and a 75 W
(35, 36), the oxidation of 8-oxo-dG by electron transfer
mechanisms can trigger a cascade of subsequent oxida-
tion steps, thus generating intermediates that are more
reactive than 8-oxo-dG itself (13-18, 35, 37).
xenon pulsed lamp to probe transient absorption kinetics. The
probe flash was passed through a McPherson monochromator
and monitored by a Hamamatsu R928 photomultiplier; its
output was recorded at 5 nm intervals and was digitized using
a Tektronix TDS 620 oscilloscope. All experiments, including
data collection and analysis, were controlled by a computer.
In this work, we examine the electron transfer reac-
•
tions of NO
2
with 8-oxo-dG in aqueous solution using
laser flash photolysis transient absorption spectroscopy
techniques in which the fate of the 8-oxo-dG radicals
formed is monitored as a function of time. The rate
constants of this one-electron oxidation reaction of 8-oxo-
2 2
The sample solutions were saturated with O or N . Using a
positive gas pressure, the sample solutions were forced through
a 0.5 mL quartz flow cell with an optical path length of 1 cm
aligned parallel to the direction of the probe light beam (40,
dG are compared to the rates of oxidation by other species
4
7-49) at a flow rate of 2-3 mL/min. All laser flash photolysis
•
with known redox potentials. The NO
2
was generated
experiments were performed at 20 °C.
-
from NO
2
by two methods. In one method, an aromatic
radical cation was generated by the two-photon excitation
of a pyrene derivative [7,8,9,10-tetrahydroxytetrahy-
drobenzo[a]pyrene (BPT)] with nanosecond UV laser
Resu lts
Gen er a t ion of d GMP (-H )• R a d ica ls b y On e-
Electr on Oxid a tion of d GMP w ith BP T . The radical
cation BPT can be generated in aqueous solution by
•
+
•+
pulses, and the resulting radical cations, BPT , subse-
-
•
•+
quently oxidize NO
2
to NO
2
. In the other method, the
experimental flash photolysis reaction conditions were
excitation and photoionization of BPT with intense Nd:
YAG 355 nm laser pulses (reaction 1 in Table 1). The
•
+
adjusted so that the BPT radical cations generated the
•
•
neutral deprotonated radical (38, 39), G(-H) , resulting
neutral radicals dGMP (-H) are formed by one-electron
•
+
+
from the one-electron oxidation of dGMP (2′-deoxygua-
nosine 5′-monophosphate) (40). In turn, the rates of
transfer reactions (reaction 2 in Table 1) between BPT
and dGMP, followed by the deprotonation of the dGMP
•
-
•
•
oxidation of NO
2
to NO
2
by dGMP(-H) were evaluated.
radical cation (dGMP rather than dG was selected for
the sake of convenience because of its higher solubility
in water; similar results are observed with dG). The
The overall rationale of our approach is summarized
in Scheme 1. While guanine residues in DNA, or dGMP,
•
•+
in aqueous solutions do not react with NO
2
(34), the
deprotonation of dGMP is known to occur on e500 ns
•
-
•
reactions of the radical intermediate G(-H) with NO
2
time scales (38). The dGMP (-H) radicals were identified
in aqueous solutions occur readily. Guanine radical
cations and the deprotonated radical products are widely
believed to be important reaction intermediates in DNA
by their characteristic transient absorption band at 312
nm, a shoulder near 390 nm, and a broad absorbance
centered around 520 nm (38, 39). In oxygen-saturated
solutions, the hydrated electrons generated in reaction
1 are scavenged at a near-diffusion-controlled rate (reac-
•
subjected to ionizing radiation, oxidizing agents ( OH or
+
R in Scheme 1), and oxidative stress in cellular environ-
3
ments (41-44). The reactions of these DNA-bound oxi-
tion 3 in Table 1). The triplet excited states BPT, formed
•
dized guanine bases with NO
2
might represent pathways
by intersystem crossing from the singlet excited state
1
that lead to further chemical transformation of guanine
residues in DNA, e.g., nitration (5-8, 25). However, the
chemical transformation of guanine was not investigated
BPT, are rapidly quenched by O
2
(53).
•
-. The
Deca y of d GMP (-H) by Rea ction s w ith NO
2
•
kinetics of decay of dGMP (-H) can be conveniently

NO
2
as an Oxidizing Agent of 8-Oxo-dG
Chem. Res. Toxicol., Vol. 14, No. 2, 2001 235
Ta ble 1. Oxid a tion of NO2- by d GMP (-H) Ra d ica ls in Aqu eou s Bu ffer Solu tion s (p H 7)
•
k (M- s-1
1
)
ref
reaction
•
+
1
2
3
4
5
6
BPT + 2hν f BPT + e-
•
+
•
+
9
BPT + dGMP f BPT + dGMP(-H) + H
(1.7 ( 0.1) × 10
40
50
51
43
-
•-
10
e
+ O2 f O2
1.9 × 10
•
-
+
5
2O2 + 2H f H2O2 + O2
6 × 10
•
•
<106
dGMP(-H) + O2 f dGMP(-H)OO
•
•-
+
9
dGMP(-H) + O2 + H f dGMP + O2
(1.3 ( 0.3) × 10
this work
(
and/or dGMPOOH)
dGMP(-H) + NO2 + H+ f dGMP + NO2
•
-
•
(2.6 ( 0.3) × 10
(3.4 ( 0.4) × 10
6
8
this work
this work
52
7
8
9
•
+
-
•
BPT + NO2 f dGMP + NO2
•
•-
-
9
NO2 + O2 f O2NOO
4.7 × 10
monitored at 312 nm after pulsed laser excitation of a
2
1
5 µM BPT, 5 mM dGMP aqueous buffer solution (trace
in Figure 1). The 312 nm transient absorbance reaches
a maximum ∼10 ns after the laser pulse, and decays on
time scales of ∼200 µs (Figure 1). The reactions of
•
dGMP (-H) with O
2
(reaction 5 in Table 1) are rather
slow, and these radicals decay instead by reactions with
•
-
O
2
(reaction 6 in Table 1); from a kinetic analysis of
the decay curves such as trace 1 in Figure 1, we found
9
-1
that the rate constant of reaction 6 is ∼1.3 × 10 M
-
1
s
, which is in good agreement with the value of ∼3 ×
F igu r e 1. Kinetics of the transient absorbance at 312 nm
9
-1 -1
1
0 M
s
reported by Candeias and Steenken (43). At
induced by 355 nm Nd:YAG laser pulse excitation (60 mJ
•
-
-1
-2
pH 7, the dismutation of O
2
(reaction 4 in Table 1) is
pulse cm ) of an oxygen-saturated 20 mM phosphate buffer
solution (pH 7) containing 25 µM BPT, 10 mM dGMP, and
various concentrations of NaNO . The solid lines are best fits
2
slow, and thus, reaction 6 is dominant.
In the presence of nitrite, the decay of dGMP(-H) is
accelerated (traces 2 and 3 in Figure 1). This effect is
•
of eq 11 to the experimental data points. [NaNO
(trace 2), and 4 mM (trace 3). The dependence of k
concentration is shown in the inset.
2
] ) 0 (trace 1),
2
7
′ on NaNO
2
•
attributed to the reduction of the dGMP(-H) radical by
-
•
NO
2
with the formation of NO
2
(reaction 7). Since the
in the spectral range
•
molar extinction coefficient of NO
2
reaction in the absence of dGMP, by measuring the rate
-
1
-1
of 300-500 nm is quite small (ꢀ400 ∼ 200 M cm ; 54),
•+
of decay of the transient absorbance of BPT at 455 nm
the transient absorption kinetics at 312 nm reflect the
(
k
2
40) at increasing NaNO concentrations and found that
•
decay of dGMP(-H) . The rate constant, k
7
, can be
8
-1 -1
8
) (3.4 ( 0.4) × 10 M
s
(Table 1). However, at
•
determined from the decay profiles of dGMP(-H) at
dGMP concentrations of >5 mM, reactions of dGMP with
-
different NO
2
concentrations (calculated from the ex-
•+
BPT are dominant, and the addition of 1-4 mM NaNO
2
perimental kinetic traces in Figure 1 using an ꢀ312 of 6.5
results only in a small decrease in the initial amplitude
3
-1
-1
×
10
M
cm ; 47) and from an analysis of the
•
of the dGMP (-H) absorbance (Figure 1). In aqueous
solutions, NO
differential equation
•
2
radicals are unstable and decay via
various bimolecular pathways, including reactions with
•
•
-
d[dGMP(-H) ]/dt ) k ′[dGMP(-H) ] +
•-
7
O
2
(reaction 9 in Table 1), dimerization (54), and
•
k [dGMP(-H)•][O •-] (10)
combination with G(-H) radicals, resulting in the for-
mation nitroguanines (5-7, 25). However, investigations
along these lines were beyond the scope of this work.
Tr a n sien t Absor p tion Stu d ies of On e-Electr on
6
2
•
With the assumption that, at time zero, [dGMP(-H) ]
0
•
-
)
[O
2
]
0
, the solution of eq 10 yields the following
Oxid a tion Rea ction s of 8-Oxo-d G. We found that
analytical expression for the time dependence of the
radical concentrations:
•
dGMP is not oxidized by NO
2
; this lack of reactivity can
be attributed to the relatively high oxidation potential
of the guanine residue (E ) 1.29 V vs NHE; 55).
However, the oxidation potential of 8-oxo-G is lower (36),
•
•
[
dGMP(-H) ] ) k ′[dGMP(-H) ] /
t 7 0
7
•
{
(k ′ + k [dGMP(-H) ] ) exp(k ′t) -
•
7
6
0
7
and we therefore investigated the possibility that NO
2
•
k [dGMP(-H) ] } (11)
is capable of oxidizing 8-oxo-dG. However, we first
determined the feasibility of observing the transient
absorption spectra of 8-oxo-dG by our laser transient
absorption spectroscopy technique, and characterized the
one-electron oxidation reactions of 8-oxo-dG with well-
known aromatic radical cations (Table 2). Recently,
Steenken et al. (36), using pulse radiolysis techniques,
published the transient absorption spectrum of 8-oxo-dG
in aqueous solution. Here we show that the identical
transient absorption spectrum, which can be attributed
to the 8-oxo-dG radical, can be observed using laser pulse
two-photon excitation techniques.
6
0
7
In this equation, the pseudo-first-order rate constant k ′
-
)
k
7
[NO
lineraly with NaNO
yielding a k
2
]. Experimentally, we find that k
7
′ increases
2
concentration (inset in Figure 1),
6
-1
-1
7
of (2.6 ( 0.3) × 10
M
s
(Table 1).
Identical results, within experimental error, were ob-
tained in the case of 2′-deoxyguanosine, indicating that
the phosphate group in dGMP does not influence the
reaction rate to any significant extent.
In the presence of NaNO
the 312 nm absorption signal due to the dGMP(-H)
2
, there is a small decrease in
•
radicals at time zero. We attribute this decrease to
reaction 8 (Table 1), corresponding to the oxidation of
We measured the rates of oxidation of 8-oxo-dG by
aromatic radical cations derived from a series of com-
pounds with different redox potentials. These compounds,
-
•+
NO
2
by BPT . We determined the rate constant of this

2
36 Chem. Res. Toxicol., Vol. 14, No. 2, 2001
Shafirovich et al.
Ta ble 2. Ra te Con sta n ts k12 of 8-Oxo-d G Oxid a tion by
On e-Electr on Oxid a n ts w ith Kn ow n Red ox P oten tia ls, E°,
in Aqu eou s Bu ffer Solu tion s (p H 7)
λobservation
(nm)
E°
k8 (M- s-1
1
)
(V vs NHE)
oxidant
•
+
9
9
9
8
8
anisole
(2.2 ( 0.5) × 10
(2.3 ( 0.3) × 10
(1.5 ( 0.2) × 10
(6.0 ( 0.8) × 10
(9.3 ( 0.9) × 10
525
455
515
455
540
540
445
320
515
1.62 (56)
∼1.5 (53)
1.45 (57)
1.30 (56)
•
+
BPT
thioanisole
,4-DMB
•
+
•
+
1
•
dGMP(-H)
•
8
dG(-H)
8.4 × 10 (34)
1.29 (55)
1.13 (56)
1.04 (58)
0.91 (59)
•
+
8
6
7
1
,2,4-TMB
(5.8 ( 0.6) × 10
(5.3 ( 0.5) × 10
(5.0 ( 0.8) × 10
•
NO2
promethazine
•
+
F igu r e 3. Dependence of the rate constant k12′ (decay of the
radical cation) on the concentration of 8-oxo-dG. The radical
cations were generated by 266 nm Nd:YAG laser pulse excitation
-
1
-2
(
∼40 mJ pulse cm , fwhm ) 6 ns, 20 Hz) of the radical cation
precursors in an oxygen-saturated 20 mM phosphate buffer
solution (pH 7). The decay traces of radical cations were
monitored at the wavelengths listed in Table 2: line 1, 0.5 mM
anisole; line 2, 0.2 mM thioanisole; line 3, 0.2 mM 1,4-DMB;
line 4, 0.1 mM 1,2,4-TMB; and line 5, 0.05 mM promethazine.
These concentrations of the radical cation precursors were
selected on the basis of generating sufficiently intense transient
absorption signals for measurement.
•
dG(-H) neutral radicals without significant changes in
•
+
a
the absorption spectrum (36). The pK of 8-oxo-dG is
F igu r e 2. Transient absorption spectra of 8-oxo-dG (0.1 mM)
and 1,2,4-TMB (0.1 mM) in an oxygen-saturated 20 mM
phosphate buffer solution (pH 7) measured at various delay
equal to 6.6, and at pH 7, the contribution of the 8-oxo-
•
dG(-H) form is ∼70%. Here, we denote the 8-oxo-dG
•+
•
radical species as 8-oxo-dG /8-oxo-dG(-H) in view of the
equilibrium between the protonated and unprotonated
forms at pH 7. The 266 nm laser pulse excitation of
mixtures of 8-oxo-dG and 1,2,4-TMB in oxygenated
times (t
nm Nd:YAG laser pulses (∼40 mJ pulse cm , fwhm ) 6 ns,
0 Hz). The inset shows the transient absorption spectrum of
D
) 1.2, 4, 8, 15, and 30 µs) after excitation with 266
-1 -2
2
pure 1,2,4-TMB (0.1 mM) measured with a delay time of 1 µs.
•
+
•
solutions generates both 8-oxo-dG /8-oxo-dG(-H) and
together with their redox potentials in the range of 0.9-
•+
the 1,2,4-TMB radical cations. However, the latter
1
.6 V versus NHE, are listed in Table 2. The photoexci-
radicals oxidize 8-oxo-dG, and the concentration of the
tation of these compounds with intense 266 nm laser
pulse, generates the related radical cations, together with
hydrated electrons. Both transient species can be easily
monitored by their characteristic transient absorption
spectra. In oxygenated solutions (pH 7), the hydrated
electrons are rapidly scavenged by molecular oxygen, and
the more stable radical cations serve as one-electron
oxidants of 8-oxo-dG under controlled conditions. We first
measured the transient absorption spectra and decay
kinetics of these radical cations in the absence of 8-oxo-
dG, and subsequently in the presence of different con-
centrations of 8-oxo-dG.
•+
1
,2,4-TMB radical cations decreases with increasing
delay time after the laser flash while the 8-oxo-dG radical
concentration increases (Figure 2).
Utilizing this mode of excitation of 8-oxo-dG and the
radical cation precursors in oxygenated solutions (pH 7),
we found that all radical cations with redox potentials
in the range of 0.9-1.6 V versus NHE (Table 2) are
capable of oxidizing 8-oxo-dG.
The kinetics of 8-oxo-dG oxidation occur on micro-
second time scales, and were measured by monitoring
the decay of the radical cations of the different com-
pounds listed in Table 2 at their characteristic, known
absorption maxima (Table 2). The decay of these radical
cations can be described in all cases by pseudo-first-order
kinetics with an observed rate constant k12′. The value
of k12′ increases linearly with 8-oxo-dG concentration
Typical transient absorption spectra of 8-oxo-dG and
1
,2,4-trimethoxybenzene (1,2,4-TMB) recorded at various
delay times after the 266 nm laser pulse excitation are
shown in Figure 2. At a delay time of t of 1 µs, the
transient absorption spectrum is similar but not exactly
D
(Figure 3) according to the equation k12′ ) k12′′ + k12[8-
•
+
identical to the spectrum of the 1,2,4-TMB radical
cation (absorption bands at 310 and 445 nm; 55) recorded
in the absence of 8-oxo-dG (inset in Figure 2). The
difference arises from the contribution to the overall
absorbance of 8-oxo-dG radicals which are also formed
via the photoionization of 8-oxo-dG at the laser fluences
oxo-dG], where k12′′ is the rate constant of decay of the
radical cation by all other reaction pathways that do not
involve reaction with 8-oxo-dG and k12 is the rate
constant of the bimolecular reaction of the radical cation,
•+
R , with 8-oxo-dG:
-1
-2
•+
•+
•
that are utilized (∼40 mJ pulse cm ). The decay of the
8
-oxo-dG + R f 8-oxo-dG /8-oxo-dG(-H) + R
12)
•
+
1
,2,4-TMB radical cation in the presence of 8-oxo-dG
(
is accompanied by the enhancement of the characteristic
transient absorption bands of 8-oxo-dG radicals (absorp-
tion maximum at 320 nm and a shoulder near 400 nm;
The k12 values calculated from the slope of k12′-8-oxo-
dG concentration plots depend on the redox potential of
•
+
3
6). At the longer delay times, e.g., 30 µs, the spectrum
the one-electron oxidants, R (Table 2). The value of k12
9
-1 -1
•+
is quite close in shape to the characteristic absorption
slowly decreases from 2.2 × 10 M
s
for anisole (E°
8
-1 -1
•+
spectrum of 8-oxo-dG radicals (36). Steenken et al. have
) 1.62 V) to 5.8 × 10 M
s
in the case of 1,2,4-TMB
•
+
(E° ) 1.13 V) and decreases further to 5 × 10 M s-1
7
-1
reported that in neutral aqueous solutions 8-oxo-dG
radical cations are unstable and deprotonate to 8-oxo-
•+
for promethazine (E° ) 0.91 V). This trend in reduction

NO
2
as an Oxidizing Agent of 8-Oxo-dG
Chem. Res. Toxicol., Vol. 14, No. 2, 2001 237
•
Ta ble 3. Oxid a tion of 8-Oxo-d G by NO2 Ra d ica ls in Aqu eou s Bu ffer Solu tion s (p H 7)
k (M- s-1
1
)
ref
reaction
+
-
•
8
8
3
4
5
6
7
BPT• + NO2 f dGMP + NO2
(3.4 ( 0.4) × 10
this work
60
60
60
-
-
2-
2-
9
1
1
1
1
1
NO3 + e f NO3
9.7 × 10
-
-
9
NO2 + e f NO2
4 × 10
2
-
-
•
-
2-
8
NO3 + H2PO4 f NO2 + OH + HPO4
5 × 10
•
NO2 + 8-oxo-dG f NO2 + 8-oxo-dG•+/8-oxo-dG(-H)•
-
6
(5 ( 1) × 10
this work
•
•
8
NO2 + NO2 f N2O4
4.5 × 10
54
54
54
•
•
3
-1
-
17
8
N2O4 f NO2 + NO2
6.9 × 10 s
-
3
-1
1
N2O4 + H2O f NO2 + NO3- + 2H+
1 × 10 s
of the k12 values with decreasing E° is consistent with
the redox potential (36) of 8-oxo-dG (E ) 0.74 V vs NHE
7
at pH 7) (Table 2).
The rate constant of 8-oxo-dG with BPT•+ radical
cations was measured using intense nanosecond 355 nm
•
+
laser pulse excitation for the generation of BPT (reac-
9
-1 -1
tion 1 in Table 1). The k12 value of 2.3 × 10 M
s
•
+
calculated from the analysis of the BPT decay profiles
•
+
(
(
40) recorded at 455 nm is close to that for anisole
•
+
Table 2). The high value of k12 in the case of BPT is
consistent with its redox potential (E° ∼ 1.5 V vs NHE)
53).
The dGMP(-H) neutral radicals also oxidize 8-oxo-
dG. In these experiments, the dGMP(-H) radicals were
generated by 355 nm laser pulse excitation of oxygen-
saturated aqueous buffer solutions (pH 7) containing 5
mM dGMP and 25 mM BPT (reaction 2). The k12 value
(
•
F igu r e 4. Kinetics of the transient absorbance at 320 nm
induced by 355 nm Nd:YAG laser pulse excitation (60 mJ
•
-
1
-2
pulse
solution (pH 7) containing 25 µM BPT, 20 mM NaNO
NaNO , and various concentrations of 8-oxo-dG. [8-oxo-dG] )
.75 (trace 1), 0.50 (trace 2), 0.25 (trace 3), and 0.10 mM (trace
4). The dependence of the initial rate of 8-oxo-dG oxidation by
cm ) of a deoxygenated 20 mM phosphate buffer
2
, 50 mM
3
0
8
-1 -1
of 9.3 × 10 M
s
calculated from the analysis of the
•
2
NO on 8-oxo-dG concentration is shown in the inset.
•
dGMP(-H) decay profiles recorded at 540 nm (Table 2)
8
-1 -1
is close to the value of 8.4 × 10 M
s
reported by
was made to distinguish spectroscopically between these
two species. When the transient absorbance due to the
•
Steenken et al. (36) for 8-oxo-dG oxidation by dG(-H)
radicals (Table 2).
Oxid a tion of 8-Oxo-G by NO
•
+
•
formation of 8-oxo-dG /8-oxo-dG(-H) radicals is moni-
•
2
. A similar approach
tored at 320 nm, three components can be distinguished
was utilized to investigate the possibility that 8-oxo-dG
(Figure 4).
•
can be oxidized by NO
oxidation of NO
2
. These species were prepared by
by BPT•+ radical cations generated by
2
-
The prompt component rises within less than 200 ns
and is due to reaction 12, the oxidation of 8-oxo-dG by
laser flash photolysis (reaction 8 in Table 3). The deoxy-
genated aqueous solutions [25 µM or 20 mM NaNO , 50
mM NaNO , and 20 mM sodium phosphate (pH 7)] were
•
+
BPT . This amplitude thus increases with increasing
2
8
-oxo-dG concentration.
3
The second component reflects the slower increase in
excited with intense 355 nm laser pulses. The hydrated
electrons that formed (reaction 1 in Table 1) were
•
+
•
the level of the 8-oxo-dG /8-oxo-dG(-H) radicals which
is negligible at low 8-oxo-dG concentrations (e.g., 0.1 mM
in Figure 4) and is most pronounced at the highest 8-oxo-
scavenged by nitrate anions (reaction 13 in Table 3). The
-
NO
2
anions also scavenge the hydrated electrons (reac-
dG concentrations. This component represents the oxida-
tion 14 in Table 3), but the rate constant k14 ≈ 0.4k13
•
-
tion of 8-oxo-dG by NO according to reaction 16 with
(
Table 3); also, since the NO
3
concentration is greater
2
-
the rate constant k16 (Table 3). The initial rates of the
than the NO
2
concentration, reaction 13 is dominant in
scavenging the hydrated electrons. The NO
2
-
reaction (calculated from the experimental kinetic traces
3
species are
3
-1
-1
•
in Figure 4 using an ꢀ320 of 11.5 × 10 M cm ; 36) in
the ∼1-50 µs range are plotted as a function of the
unstable and are readily transformed into NO
2
by
anions (reaction 15 in Table 3). In
0 mM phosphate buffer solution (pH 7) and using the
-
2 4
reaction with H PO
2
concentration of 8-oxo-dG, and the plot is linear in
•
8
-1 -1
character. The slope of the straight line is k [ NO ] (≈40
rate constant k15 value of 5 × 10 M
that NO
decays within ∼200 ns. The generation of
by both reactions 8 and 15 is complete on sub-
s
, we calculate
16
-1 -1
2 0
-1
6
2
-
s
), from which a k of (5 ( 1) × 10 M
s
is calculated
14
3
•
•
([ NO ] at time zero was ∼7.5 µM). The value of k is
NO
2
2
0
16
relatively small. Thus, even at the higher concentrations
microsecond time scales, and the reactions of nitrogen
dioxide with 8-oxo-dG can be followed on >1 µs time
scales (Figure 4). Thus, photoionization of BPT in the
•
of 8-oxo-dG (Figure 4), the dimerization reaction of NO
radicals (reactions 17 in Table 3), followed by the hy-
drolysis of N O to form stable products (reaction 18 in
2
-
deoxygenated aqueous solutions containing NO
3
pre-
radicals (reaction 3 in Table
) which can induce undesirable side reactions (e.g.,
reaction 9 in Table 1).
2
4
•
-
Table), should be competitive.
vents the formation of O
2
1
Discu ssion
Gen er a tion of •NO
Ra d ica ls by On e-Electr on
•
2
The reaction of NO with 8-oxo-dG was monitored at
the maximum of the transient absorbance of 8-oxo-dG
radicals at 320 nm (Figure 4). The one-electron oxidation
of 8-oxo-dG leads to the radical cation 8-oxo-dG•+ which,
2
-
2
Oxid a tion of NO . Nitrite is a major end product of
•
-
NO metabolism (29), and increased levels of NO
2
can
•
at pH 7, is in equilibrium with its deprotonated form,
serve as markers of enhanced production of NO in
•
8
-oxo-dG(-H) , since its pK ) 6.6 (36). Thus, no attempt
response to the appearance of diverse inflammatory (61)

2
38 Chem. Res. Toxicol., Vol. 14, No. 2, 2001
Shafirovich et al.
and infectious (62) processes. In vivo, NO -
does not
accumulate because it is rapidly oxidized by oxyhemo-
Steenken (43). However, even in the absence of oxygen
2
•
-
•
reactive species (O
2
and O
2
), the dG(-H) or dGMP(-
-
•
globin or oxymyoglobin to NO
3
(29, 63). However,
H) radicals are unstable and decay via multiple bi-
molecular pathways with the formation of various dimer-
ic products, as shown by the analysis of reaction products
following the electrochemical oxidation of dG (71). In
inflammatory oxidants, such as hypochlorous acid (HOCl)
-
and hydrogen peroxide, can convert NO
2
to the highly
•
reactive species NO
dase-dependent pathways (24-28, 64). In NO
by H , the peroxidases act as one-electron oxidants.
Compound I, the primary product of the H and
peroxidase reaction and a potentially two-electron oxi-
2 2
Cl and NO via mammalian peroxi-
-
•
2
oxidation
DNA, the dimerization of G (-H) radicals is not likely
•
2
O
2
and the lifetime of G (-H) is thus quite long, up to ∼5 s
•
2
O
2
(73). Under these conditions, the reaction of G(-H) with
-
NO
2
might become more favorable at the micromolar
-
-
dant (65-67), causes one-electron oxidation of NO
2
with
to millimolar concentrations of NO
2
encountered in
•
formation of compound II and NO
2
. Compound II oxidizes
cellular environments. In healthy humans, the levels of
-
•
-
NO
2
with formation of the second NO
2
radical and
NO
2
are 0.5-4 µM in plasma (30, 31), 0.4-60 µM in
recovering the initial form of the peroxidase (24, 26). The
gastric juice, and 30-210 µM in saliva (33). Much higher
levels of NO
Thus, the reaction with nitrite of DNA-bound G(-H)
radicals formed via oxidative reactions might represent
•
-
NO
2
radicals that are formed can nitrate tyrosine in
2
are detected during inflammation (32).
•
proteins (28, 34, 64); they also participate in the forma-
tion of carcinogenic nitrosamines (68, 69), and 8-nitro-
-
•
-
dG/8-nitro-G (25). Hence, one-electron oxidation of NO
2
important pathways of the generation of NO
2
from NO
2
,
•
with the formation of NO
2
-
can be considered a very
and thus deserves further examination.
•
important pathway of NO
The one-electron oxidation of NO
2
metabolic activation.
Rea ction s of NO
2
Ra d ica ls w ith 8-Oxo-d G. The
-
•
-
2
requires interaction
redox potential E°( NO
lower than that of guanine, E [G (-H)/G] ()1.29 V vs
NHE) (55), the most easily oxidizable DNA base. Using
2 2
/NO ) ()1.04 V vs NHE) (58) is
•
with strong one-electron oxidants with an E° of >1 V
versus NHE (58). Kinetic measurements performed in
this work show that the rate constant of oxidation of
7
pulse radiolysis technique, Pr u¨ tz et al. have shown (34)
-
•+
•
NO
2
by BPT (E° ∼ 1.5 V vs NHE) is lower than the
that in agreement with these potentials, NO
2
radicals
•
+
•
rate constant of oxidation of dGMP by BPT , although
do not react with intact DNA. However, NO
2
can induce
•
the redox potential E
55) is greater than the E°( NO
NHE (58). The rate constant of NO
7
[G (-H)/G] of 1.29 V versus NHE
damage to DNA lesions by reacting with oxidized nucleo-
tides such as 8-oxo-dG. The latter is a stable product of
•
-
(
2 2
/NO ) of 1.04 V versus
-
2
oxidation (2.6 ×
the two-electron oxidation of guanosine. In neutral aque-
6
-1 -1
•
•
1
0 M
s
) by dGMP (-H) is also significantly lower
ous solutions, the redox potential (36) E
7
(8-oxo-dG /8-oxo-
than the rate constant of 1,2,4-trimethoxybenzene (E° )
dG) ()0.74 V vs NHE) is lower than the redox potential
8
-1 -1
•
1
.13 V vs NHE) oxidation (6.3 × 10 M
s
) by dG(-
of NO
2
, and thus, the direct one-electron oxidation of
•
•
H) (55). These results indicate that the reactivities of
8-oxo-dG by NO is feasible.
2
-
NO
2
in reactions with these one-electron oxidants, either
Kinetic measurements performed in this work have
shown that 8-oxo-dG acts as a typical one-electron donor.
The rate constants of 8-oxo-dG oxidation by strong one-
aromatic radical cations or neutral radicals, are lower
than expected from differences in redox potentials alone.
•
-
•+
•+
The detailed analysis of the NO
2
/NO
2
system published
electron oxidants such as BPT , anisole , and thio-
•
+
in the recent review (70) of Stanbury suggests that this
anisole radical cations (E° ) 1.62-1.45 V vs NHE) are
9
-1 -1
system is characterized by a small self-exchange rate
in the range of 2.3-1.5 × 10 M
s
(Table 2). These
-1
-1
constant (0.3 M
s
) and a high internal reorganization
radical cations with E° values of g1.45 V versus NHE
energy (129 kJ /mol) that results in a low reactivity of
can also oxidize the parent guanine base with similar rate
-
9
-1 -1
•+
NO
tions.
2
in bimolecular outer sphere electron transfer reac-
constants of 1.7 × 10 M
s
(BPT ; 40) and 1.45 ×
9
-1 -1
•+
10 M
s
(thioanisole ; 55). However, in contrast to
Oxidized guanine residues are important intermediates
8-oxo-dG which is easily oxidized by radical cations with
•
in alkali-mediated DNA strand cleavage reactions (44)
an E° of 1.13-0.91 V versus NHE, the dG (-H) neutral
•
and are generated by the reactions of DNA with OH
radical (E ) 1.29 V vs NHE) oxidizes 1,2,4-TMB (E° )
7
8
radicals, or strong oxidants and ionizing radiation (41-
1.13 V vs NHE) with a rate constant (55) of 6.3 × 10
•+
-1 -1
4
3). In neutral aqueous solutions, the radical cations dG
M
s . Hence, in aqueous solutions, 8-oxo-dG is indeed
deprotonate on sub-microsecond time scales to form the
neutral radical dG(-H) (38). While Steenken (42) has
more easily oxidizable than the parent guanine base.
The rate constant of 8-oxo-dG oxidation by different
•
8
-1 -1
suggested that in DNA this deprotonation occurs even
more rapidly than in the case of dG in aqueous solution,
this has not been demonstrated directly. The decay
pathways of the guanine radicals in DNA are complex
and can include a variety of bimolecular reactions (43,
radical cations decreases from 5.8 × 10 M
s
in the
•
+
7
-1
case of 1,2,4-TMB (E° ) 1.13 V vs NHE) to 5 × 10 M
-
1
•+
s
for promethazine (E° ) 0.91 V vs NHE). However,
these rate constants are greater by factors of 10-100
6
-1
than the rate constant of 8-oxo-dG oxidation (5 × 10 M
•
6
-1
-1
•
7
s
1). The reaction of G(-H) with O
2
is slow (k < 10 M
s
) by NO
2
radicals. As in the case of the back reaction
-
1
•
-
; 43). The hydration of G(-H) , proposed as an
important step in the formation of 8-oxo-dG in double-
(one-electron oxidation of NO
2
•
), this slow rate of the
electron transfer reaction of NO
2
radicals can be ex-
stranded DNA (72), occurs with an even lower rate
plained in terms of a small self-exchange rate constant
-
1
•
constant (k < 0.1 s ; 43). Another possible reaction
2
and a high internal reorganization energy in the NO /
•
•-
-
pathway of the G(-H) decay is its reaction with O
2
NO
reactivity of NO
electron transfer reactions. The rate constant of 8-oxo-
2
system (70); these factors account for the lower
•
radicals (reaction 6). The value of the rate constant k
6
2
radicals in bimolecular outer sphere
9
-1 -1
(
1.3 × 10 M
s
), calculated here on the assumption
forms with the same yield as dGMP (-H) and
reacts solely with dGMP(-H) , is close to the value (∼3
10 ) recently estimated by Candeias and
•
-
•
•
that O
2
dG oxidation by NO
2
can be compared with the rate
•
constants of oxidation of typical organic electron donors.
9
-1
-1
•
×
M
s
2
Huie and Neta reported (74) that NO radicals oxidize

NO
2
as an Oxidizing Agent of 8-Oxo-dG
Chem. Res. Toxicol., Vol. 14, No. 2, 2001 239
N,N′-dimethylaniline (E° ) 0.87 V vs NHE) and p-
phenylenediamine (E° ) 0.57 V vs NHE) at pH 9.6 with
oxodG): relevance to the formation of 8-oxodG in DNA exposed
to peroxynitrite. Free Radical Biol. Med. 21, 407-411.
9) Kennedy, L. J ., Moore, K., J r., Caulfield, J . L., Tannenbaum, S.
R., and Dedon, P. C. (1997) Quantitation of 8-oxoguanine and
strand breaks produced by four oxidizing agents. Chem. Res.
Toxicol. 10, 386-392.
10) Niles, J . C., Burney, S., Singh, S. P., Wishnok, J . S., and
Tannenbaum, S. R. (1999) Peroxynitrite reaction products of 3′,5′-
di-O-acetyl-8-oxo-7,8-dihydro-2′-deoxyguanosine. Proc. Natl. Acad.
Sci. U.S.A. 96, 11729-11734.
11) Tretyakova, N. Y., Burney, S., Pamir, B., Wishnok, J . S., Dedon,
P. C., Wogan, G. N., and Tannenbaum, S. R. (2000) Peroxynitrite-
induced DNA damage in the supF gene: correlation with the
mutational spectrum. Mutat. Res. 447, 287-303.
(12) Burney, S., Caulfield, J . L., Niles, J . C., Wishnok, J . S., and
Tannenbaum, S. R. (1999) The chemistry of DNA damage from
nitric oxide and peroxynitrite. Mutat. Res. 424, 37-49.
(
7
7
-1
-1
rate constants of 2.6 × 10 and 4.6 × 10
M
s ,
respectively. These rate constants are somewhat greater
than the rate constant of 8-oxo-dG oxidation. In the case
of 8-oxo-dG, the electron abstraction is accompanied by
(
•
+
the partial deprotonation of 8-oxo-dG radical cations.
•
2
Hence, oxidation of 8-oxo-dG by NO might also be
(
considered in terms of a proton-coupled electron transfer
reaction (48, 53), although it is not clear whether depro-
tonation either accompanies the electron transfer step
or occurs subsequently (75).
•
The combination of NO
2
radicals with other radicals
is a very rapid reaction, and in many cases, the rates of
(
13) Tretyakova, N. Y., Niles, J . C., Burney, S., Wishnok, J . S., and
Tannenbaum, S. R. (1999) Peroxynitrite-induced reactions of
synthetic oligonucleotides containing 8-oxoguanine. Chem. Res.
Toxicol. 12, 459-466.
these reactions are close to the diffusion-controlled limit
•
(
76). Nitrogen and/or oxygen atoms of the NO
2
radical
can participate in the formation of chemical bonds with
the target radical, because the unpaired electron is
delocalized on both N and O atoms (77). Recently, Neta
and their co-workers have discussed (78) the combination
(
14) Burney, S., Niles, J . C., Dedon, P. C., and Tannenbaum, S. R.
(1999) DNA damage in deoxynucleosides and oligonucleotides
treated with peroxynitrite. Chem. Res. Toxicol. 12, 513-520.
15) Hickerson, R. P., Prat, F., Muller, J . G., Foote, C. S., and Burrows,
C. J . (1999) Sequence and stacking dependence of 8-oxoguanine
oxidation: Comparison of one-electron vs singlet oxygen mecha-
nisms. J . Am. Chem. Soc. 121, 9423-9428.
(16) Muller, J . G., Duarte, V., Hickerson, R. P., and Burrows, C. J .
(1998) Gel electrophoretic detection of 7,8-dihydro-8-oxoguanine
and 7,8-dihydro-8-oxoadenine via oxidation by Ir(IV). Nucleic
Acids Res. 26, 2247-2249.
(
•
•-
of NO
2 3
and CO . In this reaction, the formation of a
N-O bond results in the formation of stable molecules
-
(
NO
combination with NO
compounds and nitrosooxy derivatives. For instance,
3
and CO
2
). In the case of organic radicals, their
•
2
can result in the formation of nitro
•
2
Pr u¨ tz et al. have reported (34) that NO reacts with
(
(
(
17) Duarte, V., Muller, J . G., and Burrows, C. J . (1999) Insertion of
dGMP and dAMP during in vitro DNA synthesis opposite an
oxidized form of 7,8-dihydro-8-oxoguanine. Nucleic Acids Res. 27,
9
-1
tyrosine radicals with a rate constant of ∼3 × 10 M
-
1
s
and results in the formation of 3-nitrotyrosine. Both
4
96-502.
8
-nitro-G and 4,5-dihydro-5-hydroxy-4-(nitrosooxy)-2′-
18) Tretyakova, N. Y., Wishnok, J . S., and Tannenbaum, S. R. (2000)
Peroxynitrite-induced secondary oxidative lesions at guanine
nucleobases: Chemical stability and recognition by the Fpg DNA
repair enzyme. Chem. Res. Toxicol. 13, 658-664.
deoxyguanosine have been isolated from the reaction
mixtures of dG and peroxynitrite (79). In the case of
•
8
-oxo-dG, reaction with NO
2
may also trigger a cascade
19) Sugiyama, H., and Saito, I. (1996) Theoretical studies of GG-
specific photocleavage of DNA via electron transfer: Significant
lowering of ionization potential and 5′-localization of HOMO of
stacked GG bases in B-form DNA. J . Am. Chem. Soc. 118, 7063-
of consecutive steps, thus generating the degradation
products of 8-oxo-dG.
7
068.
Ack n ow led gm en t. This work was supported by
National Science Foundation Grant CHE-9700429 and
by a grant from the Kresge Foundation.
(20) Prat, F., Houk, K. N., and Foote, C. S. (1998) Effect of guanine
stacking on the oxidation of 8-oxoguanine in B-DNA. J . Am.
Chem. Soc. 120, 845-846.
(21) Kodama, F., Umeda, M., and Tsutsui, T. (1976) Mutagenic effect
of sodium nitrite on cultured mouse cells. Mutat. Res. 40, 119-
1
24.
Refer en ces
(
22) Drake, J . W. (1970) The molecular basis of mutation, Holden-
Day, San Francisco.
(
1) Ignarro, L. J . (1999) Nitric oxide: a unique endogenous signaling
molecule in vascular biology (Nobel lecture). Angew. Chem., Int.
Ed. 38, 1882-1892.
2) Nguyen, T., Brunson, D., Crespi, C. L., Penman, B. W., Wishnok,
J . S., and Tannenbaum, S. R. (1992) DNA damage and mutation
in human cells exposed to nitric oxide in vitro. Proc. Natl. Acad.
Sci. U.S.A. 89, 3030-3034.
(23) Greenblatt, M., Mirvish, S., and So, B. T. (1971) Nitrosamine
studies: induction of lung adenomas by concurrent administration
of sodium nitrite and secondary amines in Swiss mice. J . Natl.
Cancer Inst. 46, 1029-1034.
(24) Van der Vliet, A., Eiserich, J . P., Halliwell, B., and Cross, C. E.
(1997) Formation of reactive nitrogen species during peroxidase-
catalyzed oxidation of nitrite. A potential additional mechanism
of nitric oxide-dependent toxicity. J . Biol. Chem. 272, 7617-7625.
(
(
3) Caulfield, J . L., Wishnok, J . S., and Tannenbaum, S. R. (1998)
Nitric oxide-induced deamination of cytosine and guanine in
deoxynucleosides and oligonucleotides. J . Biol. Chem. 273, 12689-
(
25) Byun, J ., Henderson, J . P., Mueller, D. M., and Heinecke, J . W.
1999) 8-Nitro-2′-deoxyguanosine, a specific marker of oxidation
(
by reactive nitrogen species, is generated by the myeloperoxidase-
hydrogen peroxide-nitrite system of activated human phagocytes.
Biochemistry 38, 2590-2600.
1
2695.
(
4) Douki, T., and Cadet, J . (1996) Peroxynitrite mediated oxidation
of purine bases of nucleosides and isolated DNA. Free Radical
Res. 24, 369-380.
(
26) Eiserich, J . P., Cross, C. E., J ones, A. D., Halliwell, B., and van
der Vliet, A. (1996) Formation of nitrating and chlorinating
species by reaction of nitrite with hypochlorous acid. A novel
mechanism for nitric oxide-mediated protein modification. J . Biol.
Chem. 271, 19199-19208.
27) J iang, Q., and Hurst, J . K. (1997) Relative chlorinating, nitrating,
and oxidizing capabilities of neutrophils determined with phago-
cytosable probes. J . Biol. Chem. 272, 32767-32772.
28) Sampson, J . B., Ye, Y., Rosen, H., and Beckman, J . S. (1998)
Myeloperoxidase and horseradish peroxidase catalyze tyrosine
nitration in proteins from nitrite and hydrogen peroxide. Arch.
Biochem. Biophys. 356, 207-213.
29) Ignarro, L. J ., Fukuto, J . M., Griscavage, J . M., Rogers, N. E.,
and Byrns, R. E. (1993) Oxidation of nitric oxide in aqueous
solution to nitrite but not nitrate: comparison with enzymatically
formed nitric oxide from L-arginine. Proc. Natl. Acad. Sci. U.S.A.
90, 8103-8107.
(
5) Yermilov, V., Rubio, J ., and Ohshima, H. (1995) Formation of
8
-nitroguanine in DNA treated with peroxynitrite in vitro and
its rapid removal from DNA by depurination. FEBS Lett. 376,
07-210.
2
(
(
(
(
6) Yermilov, V., Rubio, J ., Becchi, M., Friesen, M. D., Pignatelli, B.,
and Ohshima, H. (1995) Formation of 8-nitroguanine by the
reaction of guanine with peroxynitrite in vitro. Carcinogenesis 16,
(
2
045-2050.
7) Yermilov, V., Yoshie, Y., Rubio, J ., and Ohshima, H. (1996) Effects
of carbon dioxide/bicarbonate on induction of DNA single-strand
breaks and formation of 8-nitroguanine, 8-oxoguanine and base-
propenal mediated by peroxynitrite. FEBS Lett. 399, 67-70.
8) Uppu, R. M., Cueto, R., Squadrito, G. L., Salgo, M. G., and Pryor,
W. A. (1996) Competitive reactions of peroxynitrite with 2′-
deoxyguanosine and 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-
(

2
40 Chem. Res. Toxicol., Vol. 14, No. 2, 2001
Shafirovich et al.
(
30) Gaston, B., Reilly, J ., Drazen, J . M., Fackler, J ., Ramdev, P.,
Arnelle, D., Mullins, M. E., Sugarbaker, D. J ., Chee, C., Singel,
D. J ., Loscalzo, J ., and Stamler, J . S. (1993) Endogenous nitrogen
oxides and bronchodilator S-nitrosothiols in human airways. Proc.
Natl. Acad. Sci. U.S.A. 90, 10957-10961.
31) Green, L. C., Wagner, D. A., Glogowski, J ., Skipper, P. L.,
Wishnok, J . S., and Tannenbaum, S. R. (1982) Analysis of nitrate,
nitrite, and [15N]nitrate in biological fluids. Anal. Biochem. 126,
(51) Bielski, B. H. J ., Cabelli, D. E., Arudi, R. L., and Ross, A. B. (1985)
Reactivity of perhydroxyl/superoxide radicals in aqueous solution.
J . Phys. Chem. Ref. Data 14, 1041-1100.
(52) Løgager, T., and Sehested, K. (1993) Formation and decay of
peroxynitric acid: a pulse radiolysis study. J . Phys. Chem. 97,
10047-10052.
(
(
53) Shafirovich, V. Y., Courtney, S. H., Ya, N., and Geacintov, N. E.
1995) Proton-coupled photoinduced electron transfer, deuterium
(
1
31-138.
isotope effects, and fluorescence quenching in noncovalent benzo-
[a]pyrenetetraol-nucleoside complexes in aqueous solutions. J .
Am. Chem. Soc. 117, 4920-4929.
(
(
(
32) Tannenbaum, S. R., Weisman, M., and Fett, D. (1976) The effect
of nitrate intake on nitrite formation in human saliva. Food
Cosmet. Toxicol. 14, 549-552.
33) Torre, D., Ferrario, G., Speranza, F., Orani, A., Fiori, G. P., and
Zeroli, C. (1996) Serum concentrations of nitrite in patients with
HIV-1 infection. J . Clin. Pathol. 49, 574-576.
34) Pr u¨ tz, W. A., Monig, H., Butler, J ., and Land, E. J . (1985)
Reactions of nitrogen dioxide in aqueous model systems: oxidation
of tyrosine units in peptides and proteins. Arch. Biochem. Biophys.
(
54) Gr a¨ tzel, M., Henglein, A., Lilie, J ., and Beck, G. (1969) Pulse
radiolytic study of some elementary processes of nitrite ion
oxidation and reduction. Ber. Bunsen-Ges. Phys. Chem. 73, 646-
653.
(55) Steenken, S., and J ovanovic, S. V. (1997) How easily oxidizable
is DNA? One-electron reduction potentials of adenosine and
guanosine radicals in aqueous solution. J . Am. Chem. Soc. 119,
617-618.
2
43, 125-134.
(
(
(
(
(
(
(
(
(
35) Goyal, R. N., J ain, N., and Garg, D. K. (1997) Electrochemical
and enzymic oxidation of guanosine and 8-hydroxyguanosine and
the effects of oxidation products in mice. Bioelectrochem. Bioenerg.
(
56) J onsson, M., Lind, J ., Reitberger, T., Eriksen, T. E., and Merenyi,
G. (1993) Redox chemistry of substituted benzenes: the one-
electron reduction potentials of methoxy-substituted benzene
radical cations. J . Phys. Chem. 97, 11278-11282.
4
3, 105-114.
36) Steenken, S., J ovanovic, S. V., Bietti, M., and Bernhard, K. (2000)
The trap depth (in DNA) of 8-oxo-7,8-dihydro-2′-deoxyguanosine
as derived from electron-transfer equilibria in aqueous solution.
J . Am. Chem. Soc. 122, 2373-2374.
(
57) J onsson, M., Lind, J ., Mer e´ nyi, G., and Eriksen, T. E. (1995) Redox
properties of 4-substituted aryl methyl chalcogenides in water.
J . Chem. Soc., Perkin Trans. 2, 67-70.
(58) Stanbury, D. M. (1989) Reduction potentials involving inorganic
37) Raoul, S., and Cadet, J . (1996) Photosensitized reaction of 8-oxo-
free radicals in aqueous solution. Adv. Inorg. Chem. 33, 69-138.
7
,8-dihydro-2′-deoxyguanosine: Identification of 1-(2-deoxy-â-D-
(
59) J onsson, M., Lind, J ., Eriksen, T. E., and Mer e´ nyi, G. (1994) Redox
and acidity properties of 4-substituted aniline radical cations in
water. J . Am. Chem. Soc. 116, 1423-1427.
erythro-pentofuranosyl)cyanuric acid as the major singlet oxygen
oxidation product. J . Am. Chem. Soc. 118, 1892-1898.
38) Candeias, L. P., and Steenken, S. (1989) Structure and acid-
base properties of one-electron-oxidized deoxyguanosine, gua-
nosine, and 1-methylguanosine. J . Am. Chem. Soc. 111, 1094-
(
60) Mallard, W. G., Ross, A. B., and Hellman, W. P. (1998) NIST
Standard Reference Database 40, version 3.0, National Institute
of Standards and Technology, Gaithersburg, MD.
1
099.
(
61) Galea, E., and Feinstein, D. L. (1999) Regulation of the expression
of the inflammatory nitric oxide synthase (NOS2) by cyclic AMP.
FASEB J . 13, 2125-2137.
39) Candeias, L. P., and Steenken, S. (1992) Ionization of purine
nucleosides and nucleotides and their components by 193-nm
laser photolysis in aqueous solution: model studies for oxidative
damage of DNA. J . Am. Chem. Soc. 114, 699-704.
40) Kuzmin, V. A., Dourandin, A., Shafirovich, V., and Geacintov, N.
E. (2000) Proton-coupled electron transfer in the oxidation of
guanines by an aromatic pyrenyl radical cation in aqueous
solutions. Phys. Chem. Chem. Phys. 2, 1531-1535.
(62) MacMicking, J ., Xie, Q. W., and Nathan, C. (1997) Nitric oxide
and macrophage function. Annu. Rev. Immunol. 15, 323-350.
(
63) Parks, N. J ., Krohn, K. J ., Mathis, C. A., Chasko, J . H., Geiger,
K. R., Gregor, M. E., and Peek, N. F. (1981) Nitrogen-13-labeled
nitrite and nitrate: distribution and metabolism after intra-
tracheal administration. Science 212, 58-60.
41) Steenken, S. (1989) Purine bases, nucleosides, and nucleotides:
(
64) Eiserich, J . P., Hristova, M., Cross, C. E., J ones, A. D., Freeman,
B. A., Halliwell, B., and van der Vliet, A. (1998) Formation of
nitric oxide-derived inflammatory oxidants by myeloperoxidase
in neutrophils. Nature 391, 393-397.
aqueous solution redox chemistry and transformation reactions
of their radical cations and e- and OH adducts. Chem. Rev. 89,
5
03-520.
42) Steenken, S. (1992) Electron-transfer-induced acidity/basicity and
reactivity changes of purine and pyrimidine bases. Consequences
of redox processes for DNA base pairs. Free Radical Res. Commun.
(
65) Marquez, L. A., and Dunford, H. B. (1995) Kinetics of oxidation
of tyrosine and dityrosine by myeloperoxidase compounds I and
II. Implications for lipoprotein peroxidation studies. J . Biol. Chem.
270, 30434-30440.
1
6, 349-379.
•
43) Candeias, L. P., and Steenken, S. (2000) Reaction of HO with
guanine derivatives in aqueous solution: formation of two dif-
ferent redox-active OH-adduct radicals and their unimolecular
(66) J acob, J . S., Cistola, D. P., Hsu, F. F., Muzaffar, S., Mueller, D.
M., Hazen, S. L., and Heinecke, J . W. (1996) Human phagocytes
employ the myeloperoxidase-hydrogen peroxide system to syn-
thesize dityrosine, trityrosine, pulcherosine, and isodityrosine by
a tyrosyl radical-dependent pathway. J . Biol. Chem. 271, 19950-
•
transformation reactions. Properties of G(-H) . Chem. Eur. J . 6,
4
75-484.
(
(
(
44) Burrows, C. J ., and Muller, J . G. (1998) Oxidative nucleobase
1
9956.
modifications leading to strand scission. Chem. Rev. 98, 1109-
1
151.
(67) Hurst, J . K., and Barrette, W. C., J r. (1989) Leukocytic oxygen
activation and microbicidal oxidative toxins. Crit. Rev. Biochem.
Mol. Biol. 24, 271-328.
45) Bourdat, A. G., Gasparutto, D., and Cadet, J . (1999) Synthesis
and enzymatic processing of oligodeoxynucleotides containing
tandem base damage. Nucleic Acids Res. 27, 1015-1024.
46) Geacintov, N. E., Zhao, R., Kuzmin, V. A., Kim, S. K., and Pecora,
L. J . (1993) Mechanisms of quenching of the fluorescence of a
benzo[a]pyrene tetraol metabolite model compound by 2′-deoxy-
nucleosides. Photochem. Photobiol. 58, 185-194.
(68) Van Stee, E. W., Sloane, R. A., Simmons, J . E., Moorman, M. P.,
and Brunnemann, K. D. (1995) Endogenous formation of N-
1
5
2
nitrosomorpholine in mice from NO by inhalation and mor-
pholine by gavage. Carcinogenesis 16, 89-92.
(69) Masuda, M., Mower, H. F., Pignatelli, B., Celan, I., Friesen, M.
D., Nishino, H., and Ohshima, H. (2000) Formation of N-
nitrosamines and N-nitramines by the reaction of secondary
amines with peroxynitrite and other reactive nitrogen species:
comparison with nitrotyrosine formation. Chem. Res. Toxicol. 13,
(
47) Shafirovich, V., Dourandin, A., Huang, W., Luneva, N. P., and
Geacintov, N. E. (1999) Oxidation of guanine at a distance in
oligonucleotides induced by two-photon photoionization of 2-ami-
nopurine. J . Phys. Chem. B 103, 10924-10933.
3
01-308.
(
48) Shafirovich, V., Dourandin, A., Luneva, N. P., and Geacintov, N.
E. (2000) The kinetic deuterium isotope effect as a probe of a
proton coupled electron transfer mechanism in the oxidation of
guanine by 2-aminopurine radicals. J . Phys. Chem. B 104, 137-
(70) Stanbury, D. M. (1997) Nuclear factors in main-group electron
transfer reactions. In Electron Transfer Reactions (Isied, S. S.,
Ed.) Vol. 253, pp 165-182, American Chemical Society, Wash-
ington, DC.
1
39.
(
49) Shafirovich, V., Dourandin, A., Luneva, N. P., and Geacintov, N.
E. (2000) Acid-base equilibria in aqueous solutions of 2-ami-
nopurine radical cations generated by two-photon photoionization.
J . Chem. Soc., Perkin Trans. 2, 271-275.
50) Bielski, B. H. J . (1978) Reevaluation of the spectral and kinetic
properties of hydroperoxo and superoxide anion free radicals.
Photochem. Photobiol. 28, 645-649.
(71) Subramanian, P., and Dryhurst, G. (1987) Electrochemical oxida-
tion of guanosine. Formation of some novel guanine oligonucleo-
sides. Electroanal. Chem. 224, 137-162.
(72) Kasai, H., Yamaizumi, Z., Berger, M., and Cadet, J . (1992)
Photosensitized formation of 7,8-dihydro-8-oxo-2′-deoxyguanosine
(8-hydroxy-2′-deoxyguanosine) in DNA by riboflavin: a nonsinglet
oxygen-mediated reaction. J . Am. Chem. Soc. 114, 9692-9694.
(

NO
2
as an Oxidizing Agent of 8-Oxo-dG
Chem. Res. Toxicol., Vol. 14, No. 2, 2001 241
(
73) Hildenbrand, K., and Schulte-Frohlinde, D. (1990) ESR spectra
(77) Behar, D., and Fessenden, R. W. (1972) Electron spin resonance
studies of inorganic radicals in irradiated aqueous solutions. I.
Direct observation. J . Phys. Chem. 76, 1710-1721.
of radicals of single-stranded and double-stranded DNA in aque-
•
ous solution. Implications for OH-induced strand breakage. Free
Radical Res. Commun. 11, 195-206.
(
78) Alfassi, Z. B., Dhanasekaran, T., Huie, R. E., and Neta, P. (1999)
(
74) Huie, R. E., and Neta, P. (1986) Kinetics of one-electron transfer
reactions involving chlorine dioxide and nitrogen dioxide. J . Phys.
Chem. 90, 1193-1198.
•-
On the reactions of CO
Phys. Chem. 56, 475-482.
3
x
radicals with NO radicals. Radiat.
(79) Douki, T., Cadet, J ., and Ames, B. N. (1996) An adduct between
(
75) Cukier, R. I., and Nocera, D. G. (1998) Proton-coupled electron
transfer. Annu. Rev. Phys. Chem. 49, 337-369.
76) Neta, P., Huie, R. E., and Ross, A. B. (1988) Rate constants for
reactions of inorganic radicals in aqueous solution. J . Phys. Chem.
Ref. Data 17, 1027-1284.
peroxynitrite and 2′-deoxyguanosine: 4,5-dihydro-5-hydroxy-4-
(nitrosooxy)-2′-deoxyguanosine. Chem. Res. Toxicol. 9, 3-7.
(
TX000204T