Analysis of peroxynitrite reactions with guanine, xanthine, and adenine nucleosides by high-pressure liquid chromatography with electrochemical detection: C8-nitration and -oxidation

Article abstract of DOI:10.1021/tx000189s

Peroxynitrite, the reaction product of nitric oxide and superoxide anion, and a powerful oxidant, was found to nitrate as well as oxidize adenine, guanine, and xanthine nucleosides. A highly sensitive reverse-phase HPLC method with a dual-mode electrochemical detector, which reduces the nitro product at the first electrode and detects the reduced product by oxidation at the second electrode, was applied to detect femtomole levels of 8-nitroguanine and 8-nitroxanthine. This method was used to separate and identify the products of nitration and oxidation from the reactions of nucleosides with peroxynitrite. Peroxynitrite nitrates deoxyguanosine at neutral pH to give the very unstable 8-nitrodeoxyguanosine, in addition to 8-nitroguanine. 8-Nitrodeoxyguanosine, with a half-life of ～10 min at room temperature and ≤3 min at 37 °C, hydrolyzes at pH 7 to 8-nitroguanine. A decrease in the reaction pH resulted in a decrease in the level of C8-nitration. Peroxynitrite also oxidizes deoxyguanosine in a pH-dependent manner, to give 8-oxodeoxyguanosine with a maximum yield (0.5-0.7%) at pH 5. Guanosine and xanthosine exhibit reactivity similar to that of deoxyguanosine toward peroxynitrite at neutral pH, producing only the corresponding 8-nitronucleosides as well as 8-nitroguanine and 8-nitroxanthine, respectively. 8-Nitroguanosine at pH 7, with a half-life of several weeks at 5 °C and 5 h at 37 °C, was much more stable than 8-nitrodeoxyguanosine. C8-nitration was confirmed by dithionite reduction to the corresponding amino nucleosides, which cochromatographed with synthesized 8-amino nucleoside standards. In contrast to guanine nucleosides, adenine nucleosides undergo peroxynitrite-mediated C8 oxidation even at neutral pH to give the corresponding 8-oxoadenine nucleosides in ～0.3% yield. Adenine nitration, though minor compared to C8-oxidation, appears to occur at both C2 and C8 positions of the adenine ring. Lowering the reaction pH from 7 to 5 results in 2.4- and 2.2-fold increases in the yields of 8-oxo-dA and 8-oxo-Ado, respectively, but the level of nitration is not altered.

Full text of DOI:10.1021/tx000189s

4
38
Chem. Res. Toxicol. 2001, 14, 438-450
An a lysis of P er oxyn itr ite Rea ction s w ith Gu a n in e,
Xa n th in e, a n d Ad en in e Nu cleosid es by High -P r essu r e
Liqu id Ch r om a togr a p h y w ith Electr och em ica l
Detection : C8-Nitr a tion a n d -Oxid a tion
Rama S. Sodum* and Emerich S. Fiala
Division of Biochemical Pharmacology, American Health Foundation, Valhalla, New York 10595
Received August 25, 2000
Peroxynitrite, the reaction product of nitric oxide and superoxide anion, and a powerful
oxidant, was found to nitrate as well as oxidize adenine, guanine, and xanthine nucleosides.
A highly sensitive reverse-phase HPLC method with a dual-mode electrochemical detector,
which reduces the nitro product at the first electrode and detects the reduced product by
oxidation at the second electrode, was applied to detect femtomole levels of 8-nitroguanine
and 8-nitroxanthine. This method was used to separate and identify the products of nitration
and oxidation from the reactions of nucleosides with peroxynitrite. Peroxynitrite nitrates
deoxyguanosine at neutral pH to give the very unstable 8-nitrodeoxyguanosine, in addition to
8
-nitroguanine. 8-Nitrodeoxyguanosine, with a half-life of ∼10 min at room temperature and
e3 min at 37 °C, hydrolyzes at pH 7 to 8-nitroguanine. A decrease in the reaction pH resulted
in a decrease in the level of C8-nitration. Peroxynitrite also oxidizes deoxyguanosine in a pH-
dependent manner, to give 8-oxodeoxyguanosine with a maximum yield (0.5-0.7%) at pH 5.
Guanosine and xanthosine exhibit reactivity similar to that of deoxyguanosine toward
peroxynitrite at neutral pH, producing only the corresponding 8-nitronucleosides as well as
8
-nitroguanine and 8-nitroxanthine, respectively. 8-Nitroguanosine at pH 7, with a half-life of
several weeks at 5 °C and 5 h at 37 °C, was much more stable than 8-nitrodeoxyguanosine.
C8-nitration was confirmed by dithionite reduction to the corresponding amino nucleosides,
which cochromatographed with synthesized 8-amino nucleoside standards. In contrast to
guanine nucleosides, adenine nucleosides undergo peroxynitrite-mediated C8 oxidation even
at neutral pH to give the corresponding 8-oxoadenine nucleosides in ∼0.3% yield. Adenine
nitration, though minor compared to C8-oxidation, appears to occur at both C2 and C8 positions
of the adenine ring. Lowering the reaction pH from 7 to 5 results in 2.4- and 2.2-fold increases
in the yields of 8-oxo-dA and 8-oxo-Ado, respectively, but the level of nitration is not altered.
In tr od u ction
The formation of peroxynitrous acid (pernitrous acid)
from hydrogen peroxide and nitrous acid, and its nitra-
tion and hydroxylation of aromatic compounds such as
benzene, had been described by Halfpenny and Robinson
many years ago (1, 2). They had also suggested that
peroxynitrous acid exhibited this property by undergoing
researchers because of its potential to modify biological
macromolecules (3-11). The main targets for modifica-
tions by peroxynitrite in biological systems are proteins,
lipids, and nucleic acids. Peroxynitrite-mediated tyrosine
nitration (12-14), guanine nitration (15), and the further
oxidation and nitration of 8-oxodeoxyguanisine (8-oxo-
1
dG) have been extensively investigated (16, 17). How-
2
ever, the reactivity of peroxynitrite toward other nucleic
2
homolytic fission to OH and NO free radicals (2). The
acid components, for instance, guanosine in RNA, or the
adenine nucleosides in RNA or DNA, and studies on
peroxynitrite-induced oxidative damage to nucleic acids,
have been more limited.
report that nitric oxide and superoxide can combine to
form peroxynitrite, which could be responsible for endot-
helial injury (3), stimulated immense interest during the
past decade in the very complex chemistry and biological
chemistry of peroxynitrite, which has been reviewed
Peroxynitrite has been shown to react at neutral
pH with DNA in vitro to give 8-oxo-dG (18-21) and
a
comprehensively (4, 5). The pK of peroxynitrite has been
reported to be 6.6 or 6.8 (6, 7), which is close to neutral,
and the anionic form is relatively stable in alkaline
solutions (pH ∼12). Once protonated, the acid form
isomerizes spontaneously, mostly to nitrate, with a half-
life of ∼1 s at pH 7.0 (7). During this isomerization, it
also produces a highly potent oxidizing and nitrating
species, whose nature has been a great concern to
1
Abbreviations: HPLC-ECD, high-pressure liquid chromatography
with dual-mode electrochemical detection; EC, electrochemical; 8-NG,
-nitroguanine; 8-NX, 8-nitroxanthine; DTPA, diethylenetriaminepen-
8
taacetate; 8-nitro-dG, 8-nitrodeoxyguanosine; 8-oxo-dG, 8-oxodeox-
yguanosine; 8-oxo-Guo, 8-oxoguanosine; 8-nitro-dA, 8-nitrodeoxyade-
nosine; 8-nitro-Guo, 8-nitroguanosine; 8-nitro-Ado, 8-nitroadenosine;
8
8
-oxo-Ado, 8-oxoadenosine; 8-oxo-dA, 8-oxodeoxyadenosine; 8-NH
-aminodeoxyadenosine; 8-NH -Ado, 8-aminoadenosine.
Throughout this article, the phrases “peroxynitrite reactivity” and
2
-dA,
2
2
*
To whom correspondence should be addressed: American Health
Foundation, 1 Dana Road, Valhalla, NY 10595. Telephone: (914) 789-
263. E-mail: r.sodum@worldnet.att.net.
“peroxynitrite reactions” refer to the reactivity and reactions of the
unstable conjugate acid, peroxynitrous acid, respectively, not those of
the peroxynitrite anion itself.
7
1
0.1021/tx000189s CCC: $20.00 © 2001 American Chemical Society
Published on Web 03/09/2001

Peroxynitrite Nitrates and Oxidizes Nucleosides
-oxodeoxyadenosine (8-oxo-dA) (22) in addition to 8-ni-
Chem. Res. Toxicol., Vol. 14, No. 4, 2001 439
8
P r ep a r a tion of 8-Nitr ogu a n in e fr om 8-Am in ogu a n in e.
-Nitroguanine was prepared by the diazotization of 8-ami-
8
troguanine (15, 23). While the nitration of deoxygua-
nosine in DNA was demonstrated through the formation
of 8-nitroguanine (15, 23), the unstable 8-nitrodeoxygua-
nosine (8-nitro-dG) itself has so far not been observed.
In the report on 8-oxo-dA formation, it is not clear at what
pH the peroxynitrite reaction was carried out and
whether the oxidation may have been due to the con-
tamination of the peroxynitrite used with hydrogen
peroxide (22). We describe here a systematic study of the
reactivity of peroxynitrite with guanine, adenine, and
xanthine nucleosides at specific conditions. These studies
became possible by the application of the reverse-phase
HPLC with dual-mode amperometric detection that had
been previously developed for the quantitation of 3-ni-
trotyrosine (24). Two electrodes in series, operated in the
reductive and oxidative modes sequentially, are utilized
in this procedure. The nitro derivatives of nucleosides and
the nucleic acid bases from the reactions of peroxynitrite
are reduced at the first (upstream) electrode, and the
reduced products are oxidized and detected at the second
noguanine and subsequent reaction of 8-diazoguanine with
sodium nitrite in the presence of acid using a published
procedure (26). The yield of crude 8-nitroguanine was ∼40%. It
was purified by HPLC. 8-Aminoguanine was obtained by acid
hydrolysis of 8-aminoguanosine as previously described (27).
P r ep a r a tion of 8-Nitr oxa n th in e fr om Xa n th in e. 8-Ni-
troxanthine was prepared by nitration of xanthine with a
mixture of acetic acid and nitric acid according to the published
procedure (28). Xanthine (0.22 g) and 1.9 mL glacial acetic acid
were heated in an oil bath to 120 °C; concentrated nitric acid
(0.3 mL) was added dropwise, and the mixture was heated at
1
20 °C for 1 h. An additional portion of 0.5 mL of glacial acetic
acid was added, followed by 0.2 mL of concentrated nitric acid,
and the temperature was increased to reflux the reaction
mixture for 1 h. The mixture was then allowed to cool to room
temperature, and the precipitate was collected by filtration,
washed with cold water, and air-dried. Recrystallization from
0
.5 N hydrochloric acid gave 60 mg (20% yield) of the product.
HPLC analysis of the product showed 8-nitroxanthine was the
only product, but was contaminated with some unreacted
xanthine, necessitating HPLC purification. 8-Nitroxanthine
obtained by this method exhibited UV spectral and chromato-
graphic properties that were the same as those of 8-nitroxan-
thine prepared from the reaction of peroxynitrite with xanthine,
and also the same chemical properties, such as reduction by
sodium dithionite to give 8-aminoxanthine.
(
downstream) electrode. The second electrode which is
always in the oxidative mode can, by itself, detect the
-oxo derivatives of the purine nucleosides. When the
8
reducing electrode is placed on standby, peaks corre-
sponding to the nitro derivatives disappear, and only the
H P LC a n d E CD In st r u m en t a t ion . Shimadzu model
LC-600 HPLC pumps controlled by the Shimadzu model SCL-
8
-oxo products are detected, providing an additional proof
for the identity of both types of compounds. Using this
dual-mode electrochemical detection method, femtomole
levels of 8-nitroguanine, 8-nitroxanthine, and probably
other nitrated bases or nucleosides can be detected in
HPLC effluents.
6
B solvent programmer were used for solvent delivery. The
HPLC column effluent was analyzed using the Waters model
96 photodiode array detector (Waters Corp., Millford, MA)
9
downstream from the Bioanalytical Systems (West Lafayette,
IN) BAS LC-4B EC detector; chromatographic data were
processed using the Waters Millennium Chromatography Man-
ager software. All electrodes were obtained from BAS.
In this study, we find that peroxynitrite reacts with
deoxyguanosine or guanosine at neutral pH to give
The dual-mode EC detector, consisting of two LC-4B control-
lers and one CC4 electrochemical cell compartment, was con-
figured to operate in accordance with the manufacturer’s
instructions. All applied potentials were referenced against the
Ag/AgCl/3 M NaCl electrode. The flow cell was equipped with
one of the three working dual-electrode combinations. These
consisted of either (a) glassy carbon and gold/mercury, (b) glassy
carbon and gold, or (c) two glassy carbon electrodes in series,
with the glassy carbon electrode always downstream. The
preparation and maintenance of the Au/Hg electrode was
described in detail in an earlier publication (24). To maintain a
satisfactory sensitivity, all working electrodes must be periodi-
cally refurbished according to the manufacturer’s instructions.
8
-nitro-dG or 8-nitro-Guo, respectively, and that 8-ni-
troguanine is a product in both reactions; some of the
properties of 8-nitro-dG and 8-nitro-Guo are described.
Like guanine nucleosides, adenine nucleosides also react
with peroxynitrite to undergo nitration at C8, and
possibly also at C2. However, unlike guanine nucleosides,
adenine nucleosides undergo extensive C8-oxidation, the
level of which is several times greater than the level of
C8- and C2-nitration, to give 8-oxo-dA or 8-oxo-Ado even
at neutral pH. As the pH of the reaction decreases, the
level of peroxynitrite-mediated C8-oxidation increases in
the cases of both guanine and adenine nucleosides. Our
studies at the purine base and nucleoside level suggest
that the extent of nitration and oxidative damage caused
by peroxynitrite to nucleic acids may have been under-
estimated and should be reexamined.
HP LC Solven t System s. The solvent systems used for
elution consisted of 50 mM sodium acetate (pH adjusted to 4.7
by addition of ∼6 mL of glacial acetic acid) with either 1%
(solvent system 1), 2% (solvent system 2), 5% (solvent system
3
) or 8% methanol (solvent system 4) or of 100 mM sodium
acetate (pH adjusted to 4.5 with ∼20 mL of glacial acetic acid)
containing 5% methanol (solvent system 5). The flow rate was
Exp er im en ta l P r oced u r es
1
mL/min.
Ma ter ia ls a n d Meth od s. Guanine was obtained from Ald-
rich Chemical Co. (Milwaukee, WI); deoxyguanosine was pur-
chased from U.S. Biochemical Corp. (Cleveland, OH). All the
other nucleosides, nucleotides, and xanthine were obtained from
Sigma Chemical Co. (St. Louis, MO). 8-Oxodeoxyadenosine was
a kind gift from S. Emburey of Glen Research (Sterling, VA).
The Beckman Ultrasphere ODS columns used for HPLC were
obtained from Alltech Co. (Allendale, PA). Peroxynitrite was
prepared according to the method of Pryor et al. (25). The
Fischer model 500 ozone generator, purchased from Fischer
Associates (Deerfield, IL), was used to produce ozone for the
preparation of peroxynitrite. UV spectra were obtained using a
GBC model Centra 40 UV/vis spectrophotometer (GBC Scientific
Equipment, Inc., Arlington Heights, IL).
Rea ction of P er oxyn itr ite w ith Gu a n in e a n d Xa n th in e.
Peroxynitrite, prepared by the ozonation of sodium azide
according to the published procedure (25), was stored at -20
or -80 °C. After dilution with 0.1 N NaOH, and just before the
reaction had started, the peroxynitrite concentration was de-
-
1
-1
termined by its absorbance at 301 nm (ꢀ ) 1670 M cm ).
The final concentration of peroxynitrite in the reaction
mixture was 10-12 mM; the purine base concentration was
6-10 mM. The reactions were conducted at 37 °C or room
temperature. The purine base was dissolved or suspended in
0.5 M sodium phosphate buffer in 0.1 mM diethylenetriamine-
pentaacetate (DTPA) at pH 7.0 and equilibrated at either 37
°C or room temperature prior to the addition of peroxynitrite.
Peroxynitrite was added all at once while the mixture was

4
40 Chem. Res. Toxicol., Vol. 14, No. 4, 2001
Sodum and Fiala
vortexed, and the reaction mixture was returned immediately
to a bath maintained at either 37 °C or room temperature.
Incubation with shaking was continued for an additional 5 min.
For control runs, peroxynitrite was decomposed with 1 N HCl
prior to the addition to the reaction mixture.
To compare the reactivity of peroxynitrite with xanthine and
guanine, both reactions were performed under the same condi-
tions, at the same time and with the same batch of peroxynitrite.
The purine bases, 1 mg (∼6 µmol), were dissolved or suspended
in 1 mL of sodium phosphate buffer at pH 7 and 37 °C, and an
equal amount of peroxynitrite (12 µmol) was added to each of
the mixtures. The rest of the procedure was followed as
described above.
of the nucleosides and the bases used in the various reactions
were 3 mM deoxyguanosine, 3.5 mM guanosine, 5 mM deoxy-
adenosine or adenosine, 10 mM xanthosine, or 8.5 mM 9-me-
thylguanine. All reaction mixtures contained 0.1 mM DTPA and
0.5 M phosphate buffer at pH 7.0 or 0.2 M sodium acetate buffer
at pH 5.0. The buffer solutions were prepared with MilliQ-
filtered water and used as such, and no effort was made to
exclude oxygen or carbon dioxide from either the buffer solutions
or the reaction mixtures. Enough peroxynitrite to achieve the
required concentration was added all at one time, using a glass
micropipet or an Eppendorf pipet, while the reaction mixture
was vortexed. The reaction mixtures of adenine and guanine
nucleosides were analyzed by HPLC immediately after the
addition of peroxynitrite and stored on ice, or used immediately
for dithionite reduction or other reactions. In the case of
deoxyguanosine, an aliquot of the reaction mixture should be
injected within at least 20 s of the addition of peroxynitrite to
observe the elution of the nitrated deoxynucleoside without
excessive decomposition. The yields of the nitropurines and
8
-Nitroxanthine and 8-nitroguanine were reduced to the
corresponding 8-amino derivatives by addition of an excess
amount of solid sodium dithionite (3-10 mg) directly to the
appropriate fractions of the HPLC effluent. The mixture was
vortexed for a few seconds and used for further studies.
8
-Aminoguanine and 8-aminoxanthine are quite stable and can
-nucleosides were estimated as the corresponding amino deriva-
be stored in a refrigerator for several months.
tives after reduction with sodium dithionite, using standard
curves obtained with the 8-aminonucleoside and 8-aminopurine
standards (29-32). Reduction was carried out at room temper-
ature or 5 °C by direct addition of excess solid sodium dithionite
to the reaction mixture or to the HPLC-purified product in
solution at pH 7. All yields are expressed in terms of the
percentage of the parent nucleoside concentration.
For the reactions of deoxyguanosine at various pH values, a
mixed buffer of 0.25 M sodium phosphate and 0.2 M sodium
acetate was used for pH 7 and 6. For reactions carried out at
pH 5-3, 0.2 M sodium acetate buffer was used after adjusting
the pH to the desired value by addition of glacial acetic acid.
All reaction mixtures contained 0.1 mM DTPA.
Control runs were conducted under the same conditions, with
the same concentrations of the starting materials for each
experiment, except that peroxynitrite that had been allowed to
decompose in the same buffer was substituted for peroxynitrite.
HP LC-EC An a lysis of th e Nu cleosid e Rea ction Mix-
tu r es. The HPLC system and the UV and ECD instrumentation
were similar to those described above for the hydrodynamic
voltammetry of 8-nitroguanine and 8-nitroxanthine. HPLC
columns were eluted with 50 mM sodium acetate (pH 4.7) with
HP LC An a lysis a n d Hyd r od yn a m ic Volta m m etr y of
-Nit r ogu a n in e a n d 8-Nit r oxa n t h in e. Characterization of
-nitroguanine and 8-nitroxanthine by hydrodynamic voltam-
8
8
metry was performed by HPLC with EC detection, using two
Ultrasphere ODS 3 µm columns (0.46 cm × 7.5 cm) connected
in series and protected by a 0.46 cm × 4.6 cm Ultrasphere ODS
5
µm guard column. The columns were eluted isocratically at a
flow rate of 1 mL/min with a solution of 1 or 2% methanol in 50
mM sodium acetate (pH 4.7), which was continually sparged
with helium. To determine the optimum potential for the
electrochemical reduction of 8-nitroguanine or 8-nitroxanthine,
a fixed amount of the standard nitro derivative was injected
into the HPLC system and analyzed repeatedly at various
reducing potentials while the oxidation potential was kept
constant. To determine the optimum oxidation potential, the
procedure was repeated with a constant reducing potential and
varying oxidation potentials. In all cases, only the current due
to oxidation at the glassy carbon electrode was recorded.
The HPLC system used for the analysis of the products of
reactions of purine bases was the same as that described above
for the hydrodynamic voltammetry. The level of UV absorption
of the HPLC effluent was measured at 253 and 375 nm, and
the dual-electrode EC detector was set up in the sequential
reduction and oxidation modes. After optimization of the EC
detector conditions by hydrodynamic voltammetry, the upstream
Au electrode was used for reduction at -0.7 or -0.9 V and the
downstream glassy carbon electrode was used for oxidation at
5
or 8% MeOH or 100 mM sodium acetate (pH 4.5) with 5%
MeOH, and the flow rate was 1 mL/min for the analyses of all
of the nucleoside reactions.
For detection of the products from the reactions of nucleoside
with peroxynitrite by HPLC-ECD with dual-mode EC detection,
the Au and glassy carbon dual electrode was used, with the
upstream Au electrode reducing at -0.7 V and the downstream
glassy carbon electrode oxidizing at 0.6 V. For the analysis of
adenine nucleoside reaction mixtures only, a potential of 0.8 V
was applied to the downstream oxidizing electrode and -0.7 V
was applied to the upstream reducing electrode.
0
.6 V unless otherwise specified.
P r ep a r a tion of 8-Oxo- a n d 8-Am in op u r in es a n d th e
Cor r esp on d in g Nu cleosid e Sta n d a r d s. 8-Aminoguanosine
was obtained from the Sigma Chemical Co. 8-Aminoguanine and
8
-oxoguanine were prepared by the acid hydrolysis of the
corresponding ribonucleosides following the procedure described
previously (27). 8-Aminoadenine and 8-oxoadenine were pre-
pared by acid hydrolysis of 8-aminodeoxyadenosine and 8-
oxodeoxyadenosine, respectively, in 1 N HCl at 90 °C for 20 min.
Resu lts
HP LC An a lysis. The reverse-phase HPLC method
with dual-mode electrochemical detection as described
here was originally used in our laboratory for the
determination of 3-nitrotyrosine (24). With a minor
modification of the eluting solvent, it was possible to
apply it to the detection of femtomole amounts of 8-ni-
troguanine and 8-nitroxanthine. When the methanol
concentration of the eluting solvent was increased, the
separation and detection of products from the reactions
of various nucleosides with peroxynitrite also became
feasible. Figure 1 was obtained with 8-nitroguanine,
8
-Aminodeoxyguanosine, 8-aminodeoxyadenosine, and 8-ami-
noxanthosine were prepared from the corresponding bromo
derivatives as described in the literature (29). 8-Oxoxanthosine,
8
according to published procedures (30, 31). 8-Aminoadenosine
and 8-oxoadenosine standards were prepared by the sequential
enzymatic hydrolysis of 8-aminoadenosine 3′,5′-cyclic mono-
phosphate or 8-hydroxyadenosine 3′,5′-cyclic monophosphate
-aminoxanthosine, and 8-oxoguanosine were also prepared
(
Sigma Chemical Co.), respectively, with 3′,5′-cyclic nucleo-
tide phosphodiesterase and alkaline phosphatase (Sigma Chemi-
cal Co.).
Rea ction of P u r in e Nu cleosid es w ith P er oxyn itr ite a t
p H 7.0 a n d 5.0. For the reactions of adenine and guanine
nucleosides, the concentration of peroxynitrite was 10-12 mM;
for the reactions of 9-methylguanine and xanthosine, the
peroxynitrite concentration was 7-8 mM. The concentrations
8
8
-nitroxanthine, and several other standard 8-amino- and
-oxoguanine nucleosides that had been prepared accord-
ing to published procedures (26-31). This figure il-
lustrates the separations and the resolution typically

Peroxynitrite Nitrates and Oxidizes Nucleosides
Chem. Res. Toxicol., Vol. 14, No. 4, 2001 441
F igu r e 1. HPLC-ECD profiles of standard nucleoside deriva-
tives. Oxidizing electrode response, with the reducing electrode
W2 (Au electrode) at -0.9 or -0.7 V and the oxidizing electrode
W1 at 0.6 V. 8-NG, 8-nitroguanine; 8-NX, 8-nitroxanthine;
F igu r e 3. HPLC-ECD profiles of products 1 (trace 1), 2 (trace
2), and 3 (trace 3) in Figure 2A, purified by HPLC, after
dithionite reduction. Trace 4 represents cochromatography of
the reduced product 3 with the 8-aminoguanine standard that
was prepared by acid hydrolysis of 8-aminoguanosine. The
traces represent the response of the oxidizing electrode (W1)
with the reducing electrode (W2) on standby. HPLC solvent
system 1 was used at a rate of 1 mL/min.
8
2
-NH -G, 8-aminoguanine; 8-Oxo-G, 8-oxo-guanine. HPLC sol-
vent system 3 was used at a rate of 1.0 mL/min.
products, 1 and 2 in Figure 2A, each with a UV absorp-
tion maximum at approximately 375 nm, were also puri-
fied by HPLC and reduced with sodium dithionite. Only
product 1, which we identified in later experiments as
8
-nitroxanthine, underwent reduction with dithionite to
give a product (8-aminoxanthine) that produced a detec-
tor response using the oxidative mode alone (Figure 3).
With HPLC conditions similar to those used to quan-
titate 3-nitrotyrosine (24), i.e., 50 mM sodium acetate (pH
4
.7) containing 1% methanol as the eluant, 8-nitrogua-
nine elutes with a retention time of 15 min (or an elution
volume of 15 mL). Using dual-mode amperometric detec-
tion, this species could be detected at very low concentra-
tions. When the reducing electrode is turned off or put
on standby, the peak corresponding to the nitro com-
pound disappears from the chromatogram, providing
additional evidence for the presence of the nitro function.
In the work described here, the upstream electrode
consisted of either an Au/Hg or Au electrode programmed
to operate in the reduction mode at -0.9 or -0.7 V, and
the downstream electrode in all cases was a glassy carbon
electrode operating in the oxidation mode at 0.6 V unless
otherwise specified. Under these conditions, a detection
limit of 190 fmol per injection could be achieved with the
F igu r e 2. HPLC profiles of the 8-nitroguanine obtained by
synthesis from 8-aminoguanine as described in Experimental
Procedures: (A) chromatogram monitored for UV absorption at
3
75 nm, (B) chromatogram monitored at 253 nm, showing the
side product guanine, and (C) rechromatography of 8-nitrogua-
nine, peak 3, collected from HPLC. UV, photodiode array
detector response at 375 nm; EC, dual-mode electrochemical
detector response with the reducing electrode W2 (Au/Hg
electrode) at -0.9 V and the oxidizing electrode at 0.6 V. HPLC
solvent system 1 was used at a rate of 1 mL/min.
8
-nitroguanine standard. Because of the characteristic
long-wavelength UV absorption spectrum of nitro com-
pounds, low detection levels with good selectivity can also
be achieved for 8-nitroguanine using only the photodiode
array detector; however, the sensitivity is about 100 times
il on wj e ec rt i ot nh .a n that of dual-mode ECD, or ∼15 pmol per
achieved using the method. Unlike the nitropurines, the
8
-amino- and 8-oxonucleosides do not require reduction
prior to detection at the downstream oxidation electrode;
hence, these species can be detected in femtomole levels
using either the dual- or single-detection mode.
Analysis by hydrodynamic voltammetry at various
oxidation potentials and a fixed reduction potential of
-0.9 V shows that 8-nitroguanine can be detected at
oxidative potentials as low as 0.2 V when the gold
amalgam electrode is used (Figure 4). The use of such
low potentials has the advantage of providing increased
selectivity and enhanced detection limits during the
analysis of biological samples, since fewer interfering
compounds will be oxidized under those conditions.
Figure 4 also shows that 8-nitroguanine can be easily
reduced on a gold or carbon electrode, although at
somewhat higher negative potentials.
P r ep a r a t ion a n d Det ect ion of 8-Nit r ogu a n in e.
The 8-nitroguanine standard was prepared by the dia-
zotization of 8-aminoguanine to 8-diazoguanine and the
subsequent reaction of 8-diazoguanine with sodium
nitrite to give the product (26). Since analysis showed
guanine as a major side product, 8-nitroguanine, desig-
nated as product 3 in Figure 2A, was purified by reverse-
phase HPLC (Figure 2C). Its identity was evident from
its characteristic UV absorption maximum at 385 nm
(33), and by its reduction to 8-aminoguanine (Figure 3,
trace 3), which is a known compound (27). The other

4
42 Chem. Res. Toxicol., Vol. 14, No. 4, 2001
Sodum and Fiala
F igu r e 5. Reactivity of peroxynitrite with xanthine and with
guanine. (A) Chromatograms of the reaction products of perox-
ynitrite and xanthine monitored for UV light absorption at 375
(trace 1) and 253 nm (trace 3) and of the reaction mixture of
peroxynitrite and guanine, monitored at 375 nm (trace 2). (B)
HPLC-ECD profiles of the reaction products of peroxynitrite
with xanthine (traces 1 and 2) and with guanine (trace 3). For
the control run (trace 1), the peroxynitrite was allowed to
decompose prior to being used. The ECD was in the dual mode,
using the conditions described in the legend of Figure 2. HPLC
system 2 was used at a flow rate of 1 mL/min.
F igu r e 4. Hydrodynamic voltammetry of 8-nitroguanine. In
panel A, different electrode materials at various negative
potentials are used for the reducing upstream electrode, while
the oxidation potential is fixed at 0.6 V at the downstream glassy
carbon electrode. In panel B, various positive potentials are
applied to the downstream glassy carbon electrode while the
reduction potential is kept constant at -0.9 V for the Au/Hg
upstream electrode.
P r ep a r a t ion , Hyd r od yn a m ic Volt a m m et r y, a n d
Detection of 8-Nitr oxa n th in e. In general, the HPLC
method described above could be useful for the detection
and estimation of any nitro derivatives that easily
undergo reversible electrochemical reduction. While sev-
eral nitro derivatives of purine bases may be generated
by reaction with peroxynitrite, so far only the production
of 8-nitroguanine has been adequately described (33).
Since guanine can be deaminated in vivo to xanthine,
which can then be oxidized to uric acid, it seems possible
that 8-nitroguanine might also be metabolized in vivo to
8
-nitroxanthine or even to 8-aminoxanthine. If indeed
this is the case, a method for the quantitation of 8-ni-
troxanthine in body fluids could be extremely useful as
an indicator of overall nucleic acid damage specifically
produced by peroxynitrite.
We prepared 8-nitroxanthine from the reaction of
xanthine with peroxynitrite and separated the products
by reverse-phase HPLC using the same conditions that
were used for the analysis of 8-nitroguanine described
above, except that the methanol composition in the eluent
was increased to 2% (Figure 5A, traces 1 and 3). Interest-
ingly, though 8-nitroxanthine is more nonpolar than
F igu r e 6. UV spectra at various pH values of (A) 8-nitrogua-
nine and (B) 8-nitroxanthine, both obtained from the reaction
of peroxynitrite with guanine or xanthine, respectively, and
purified by HPLC.
8
-nitroguanine, it eluted earlier than 8-nitroguanine at
shown). The 8-aminoxanthine standard, which was pre-
pared by acid hydrolysis of 8-aminoxanthosine, exhibits
a UV spectrum similar to that of the dithionite reduction
product of 8-nitroxanthine. 8-Nitroxanthine prepared by
an alternate route from xanthine (28) exhibited UV
spectral, HPLC elution, and electrochemical properties
similar to those of the 8-nitroxanthine obtained from the
peroxynitrite reaction with xanthine. It also underwent
dithionite reduction to give 8-aminoxanthine. Though the
reducing power of sodium dithionite is at its maximum
in slightly alkaline solutions, the nitropurines described
pH 4.7, perhaps because of specific interactions of the
latter with the column packing. The peak corresponding
to 8-nitroxanthine was collected and used for further
characterization. The UV absorption maximum of 8-ni-
troxanthine was observed to shift to a longer wavelength
at basic pH (Figure 6B) as described in a previous report
(28). Reduction of 8-nitroxanthine with sodium dithionite
gives 8-aminoxanthine, as evidenced by the disappear-
ance of the longer-wavelength absorption (λmax ) 380 nm)
due to the nitro functional group and the appearance of
a new absorption maximum at 287 nm (spectrum not

Peroxynitrite Nitrates and Oxidizes Nucleosides
Chem. Res. Toxicol., Vol. 14, No. 4, 2001 443
F igu r e 8. HPLC-ECD profiles of the reaction products of
peroxynitrite with deoxyguanosine. In panel A, the reaction was
carried out at pH 7 and 0-5 °C, with immediate analysis of
products. In panel B, traces 1 and 2, the reaction was carried
out at pH 7 and 37 °C; the mixture was then allowed to shake
gently at 37 °C for 3 min before analysis. Trace 3 shows the
products of the reaction at 0-5 °C after they were allowed to
be shaken at 37 °C for 5 min prior to analysis. In panel C is
shown the control incubation with decomposed peroxynitrite,
carried out under the same conditions described for panel A.
For clarity, only the dual-mode ECD profiles, obtained with the
reducing electrode W2 at -0.7 V and the oxidizing electrode W1
at 0.6 V, are shown. The retention time of deoxyguanosine is
F igu r e 7. Hydrodynamic voltammetry of 8-nitroxanthine. (A)
Different electrode materials were used for the upstream
electrode at various reduction potentials, while the oxidation
potential was fixed at 0.6 V for the downstream glassy carbon
electrode. (B) The downstream glassy carbon electrode was set
to various oxidation potentials, while the reduction potential
was kept constant at -0.7 V at the gold upstream electrode.
here could be reduced even in sodium acetate buffer at
pH 5, or in just water or water/methanol solutions.
8
-Nitroxanthine, prepared by the reaction of peroxyni-
trite with xanthine or by the nitration of xanthine with
nitric acid (28), exhibits a UV spectrum similar to that
of product 1 in Figure 2, one of the side products in the
1
3 min (does not give ECD response). Trace 2 in all figures is a
portion of trace 1 at a higher sensitivity, with the scale as shown
on the right Y-axis. HPLC solvent system 3 was used at a flow
rate of 1 mL/min.
8
-nitroguanine preparation, and also cochromatographs
with peak 1. Peak 1 in Figure 2 undergoes reduction with
sodium dithionite to give a product similar in UV and
HPLC elution properties to 8-aminoxanthine obtained by
the reduction of 8-nitroxanthine. 8-Nitroxanthine, more-
over, is an expected side product formed by hydrolytic
deamination during the diazotization of 8-aminoguanine.
Parenthetically, we found xanthine as a major product
from the reaction of guanine with nitronium tetrafluo-
roborate, which also gave 8-nitroguanine, albeit in a very
poor yield (results not shown).
for reduction of 8-nitroxanthine and the glassy carbon
electrode for the reduction of 8-nitroguanine. As in the
case of 8-nitroguanine, an oxidative potential as low as
0.2 V can be used to detect 8-nitroxanthine without a loss
in the level of sensitivity. For routine analyses, the gold
electrode may be preferred for reduction because of the
lower background noise level that was obtained.
Rea ction of Deoxygu a n osin e a n d Gu a n osin e w ith
P er oxyn itr ite a t p H 7. During vortexing, peroxynitrite
was added to the solution of deoxyguanosine at pH 7 that
had been pre-equilibrated to the desired temperature.
The reaction mixture was analyzed by HPLC with dual-
mode ECD as soon as possible, i.e., within 30 s of addition
of peroxynitrite, to minimize the decomposition of the
nitrodeoxynucleoside product (Figure 8). The late-eluting
peak at 72 min and 8-nitroguanine eluting at ∼8 min
are formed only upon reaction with peroxynitrite; neither
of these products is formed when the reaction is carried
out with decomposed peroxynitrite (Figure 8C). These
species can be detected by the dual-mode electrochemical
detector only when a potential is applied to the reducing
electrode. 8-Oxo-dG formation was not observed in the
reactions of deoxyguanosine with peroxynitrite at pH 7.
When the reaction mixture was allowed to stand at room
temperature, or when the reaction was carried out at 37
°C, the 72 min peak disappeared and the level of
8-nitroguanine increased (Figure 8B), suggesting a pre-
cursor-product relationship, with the 72 min peak likely
representing 8-nitrodeoxyguanosine (8-nitro-dG). Be-
cause the substance responsible for the 72 min peak was
Surprisingly, we find that at neutral pH and 37 °C
peroxynitrite reacts more extensively with xanthine than
with guanine to give a 10-15% yield of 8-nitroxanthine,
as estimated from the initial concentration of xanthine
(Figure 5). One reason for this may be the better
solubility of xanthine compared to guanine. However, we
also observe that xanthosine undergoes nitration by
peroxynitrite to a greater extent than guanosine does.
Under conditions similar to those described above for
8
-nitroguanine, a detection limit of 150 fmol per injection
could be reached for 8-nitroxanthine. In comparison, the
photodiode array detector, at 375 nm, achieved a detec-
tion limit of 7 pmol per injection for this base. The 8-nitro-
guanine and 8-nitroxanthine we prepared were found to
be stable and can be stored in solution at 4-5 °C and
pH 5 for several months.
As shown by hydrodynamic voltammetry in Figure 7,
8
-nitroxanthine could also be detected with high-level
sensitivity using the gold or carbon electrode for reduc-
tion (the glassy carbon electrode was used for oxidation
in all cases). The gold electrode appears to be the best

4
44 Chem. Res. Toxicol., Vol. 14, No. 4, 2001
Sodum and Fiala
F igu r e 10. HPLC-ECD analysis of the reaction products of
guanosine and peroxynitrite at pH 7 and 0-5 °C. Trace 1 was
obtained with the ECD in the single oxidative mode, with the
oxidizing electrode W1 at 0.6 V and the reducing electrode W2
on standby. Trace 2 was obtained with dual-mode ECD with
the reducing electrode W2 at -0.7 V and the oxidizing electrode
W1 at 0.6 V. Trace 3 is the control, run with decomposed
peroxynitrite, using the same dual-mode ECD conditions de-
scribed for trace 2. The retention time of guanosine is ∼10 min
F igu r e 9. HPLC-ECD profiles of the reaction products of
peroxynitrite and deoxyguanosine after reduction with sodium
dithionite (trace 1) and cochromatography with the 8-aminode-
2
oxyguanosine (8-NH -dG) standard (trace 2). Chromatographic
traces that are shown were obtained using ECD in the single
oxidative mode, with the oxidizing electrode W1 at 0.6V and
the reducing electrode W2 on standby. The HPLC solvent
system is the same as that described in the legend of Figure 8.
(
does not give an ECD response). The HPLC solvent system is
the same as that described in the legend of Figure 8.
so unstable and disappeared even when stored at 5 °C
overnight, it was impractical to collect it from HPLC for
further characterization. Therefore, without attempting
its isolation, we studied its properties in reaction mix-
tures that had been placed on ice immediately after the
addition of peroxynitrite. When sodium dithionite was
added to the reaction mixture at 5 °C within 5 min of
the peroxynitrite addition, both the 72 min peak and the
8
-nitroguanine at 8 min disappeared, along with the
appearance of peaks corresponding to 8-aminodeoxygua-
nosine (8-amino-dG), 8-oxo-dG, 8-aminoguanine, and
8
8
-oxoguanine (Figure 9). Since 8-nitroguanine yields
-aminoguanine upon reduction with dithionite (15, 33),
the 8-amino-dG must be the reduction product of 8-nitro-
dG, the latter being responsible for the 72 min peak.
F igu r e 11. Trace 1, rechromatography of 8-nitroguanosine (8-
2
NO -Guo) that had been purified by HPLC; trace 2, the same
after reduction with sodium dithionite; and trace 3, cochro-
matography of the reduced product in trace 2 with the 8-ami-
noguanosine (8NH -Guo) standard. ECD was used only in the
2
single oxidative mode, with the oxidizing electrode W1 at 0.6 V
and the reducing electrode W2 on standby. The HPLC solvent
system is the same as that described in the legend of Figure 8.
The addition of sodium dithionite to the control mix-
ture gave neither 8-amino-dG nor 8-oxo-dG. Therefore,
we assume that the 8-oxo-dG, which is formed upon
treatment of the reaction mixture with dithionite, results
from the reduction of an unknown product of the reaction
of peroxynitrite with deoxyguanosine that could not be
detected with our analytical method. This unknown
either gives no response with the electrochemical detector
under the conditions that were used or may have a very
low elution time and thus cannot be distinguished from
peaks due to salts, which also elute early. The unknown
could be the 4,8-endoperoxide of the guanine nucleoside,
or might be structurally related to the products reported
in the reaction of peroxynitrite with deoxyguanosine,
such as the 4-hydroxy- or 4-nitrosooxy-dG adduct (34, 35).
This highly interesting phenomenon is currently under
further investigation.
respectively. With an increase in the reaction tempera-
ture, the level of 8-nitroguanine increases and that of
8
-nitro-dG decreases.
Unlike 8-nitro-dG, which undergoes depurination very
quickly under mild conditions, the corresponding ribo-
nucleoside, 8-nitro-Guo, should be more stable and allow
for better characterization. Guanosine also reacted with
peroxynitrite to give 8-nitroguanosine, eluting with a
retention time of ∼42 min (8-nitro-Guo), and also 8-ni-
troguanine, eluting at ∼8 min (Figure 10). The yield of
8
-nitro-Guo, estimated by reduction to 8-aminoguanosine,
was ∼0.5 µM, or 0.02%, from 3 mM guanosine. 8-Nitro-
Guo was found to be much more stable than 8-nitro-dG;
the reaction mixture could be stored in the refrigerator
for several days with minimal changes in composition.
Because of its stability, the product could be collected
from the HPLC column and used for studying its proper-
ties, and for reduction with sodium dithionite to 8-ami-
noguanosine to confirm its identity (Figures 10 and 11).
The formation of 8-amino- and 8-oxoguanosine was
observed only when the reaction mixture, but not the
control mixture, was reduced with sodium dithionite, as
was the case with deoxyguanosine. Dithionite reduction
Using HPLC with dual-mode ECD, we were able to
investigate the stability of 8-nitro-dG in the reaction
mixtures. From the time-dependent decreases in the
areas of the 72 min peak, we estimate the half-life of
8
-nitro-dG to be 44 h at -20 °C, 7 h at 0 °C, ∼10 min at
room temperature, and <3 min at 37 °C (Figure 8B).
After reduction to the corresponding 8-amino derivatives,
the levels of formation of 8-nitro-dG and 8-nitroguanine
in the reaction of deoxyguanosine with peroxynitrite at
pH 7 and 5 °C are estimated to be 1.7 and 1.5 µM, or
0
.06 and 0.05% yields, from 3 mM deoxyguanosine,

Peroxynitrite Nitrates and Oxidizes Nucleosides
Chem. Res. Toxicol., Vol. 14, No. 4, 2001 445
F igu r e 12. Stability of 8-nitro-Guo at pH 7 and 37 °C. HPLC-
purified 8-nitro-Guo, as in Figure 11, was incubated at 37 °C,
and aliquots were taken and analyzed at various times by
HPLC-ECD.
of HPLC-purified 8-nitro-Guo gave only 8-amino-Guo,
again suggesting that the source of the 8-oxoguanosine
was not 8-nitro-Guo but some other unidentified product.
While 8-nitro-dG exhibited a half-life of only a few
minutes, 8-nitro-Guo exhibited a half-life of ∼5 h at pH
7
and 37 °C (Figure 12). At higher temperatures, for
instance, 80-90 °C, it hydrolyzed completely to 8-ni-
troguanine within 5-10 min.
Interestingly, the formation of 8-nitroguanine was
observed in the guanosine reaction at 0-5 °C and neutral
pH, at the level of ∼0.85 µM, even when analyzed almost
immediately after the addition of peroxynitrite (Figure
F igu r e 13. HPLC-ECD of the reaction of peroxynitrite with
(
A) xanthosine and (B) 9-methylguanine at pH 7 and 0-5 °C.
HPLC-ECD conditions are as described in the legend of Figure
8.
1
0). Thus, it is unlikely that the source of the 8-nitrogua-
nine is the decomposition of 8-nitro-Guo, which has a
relatively long half-life. Since the guanosine used in the
reactions with peroxynitrite was analyzed by HPLC and
found to contain no trace contamination with guanine,
the 8-nitroguanine also could not be the product of a
direct reaction of peroxynitrite with guanine. We suggest,
as a possible explanation, that the 8-nitroguanine that
was formed must have been the product of the direct
reaction of peroxynitrite with the ribosyl or deoxyribosyl
8-nitro-9-methylguanine exhibits a UV spectrum similar
to that of 8-nitroguanine, with the absorption maximum
of 385 nm at pH 5 and 420 nm at pH 11. Thus,
C8-nitration of purines may be a general reaction of
peroxynitrite.
In contrast to Burney et al. (38), who reported that
8-nitroguanine reacts more rapidly with peroxynitrite
than does 8-oxo-dG, we find that 8-nitroguanine does not
react with peroxynitrite to the same extent as 8-oxo-dG.
In our hands, at pH 7 and an initial concentration of the
8-oxodeoxynucleoside of 100 µM, 1 mM peroxynitrite
reacted completely, with the loss of all of the initial 8-oxo-
dG. In contrast, 8-nitroguanine exhibited much less
reactivity toward peroxynitrite. Peroxynitrite, 0.5 mM,
exhibited no detectable reaction with 100 µM 8-nitrogua-
nine at pH 7, and 10 mM peroxynitrite reacted with only
14 and 18% of the 8-nitroguanine present in the reaction
mixtures at initial concentrations of 100 and 20 µM,
respectively. We have observed that the storage of
reaction mixtures of peroxynitrite with nucleosides after
the reaction is over, even at -20 °C, can result in the
loss of the original products and the formation of ad-
ditional side products, since the nitrite, which is formed
in addition to nitrate during the decomposition of per-
oxynitrite (39, 40), can react further with the nucleoside
products. This might be one possible reason for differ-
ences in interlaboratory results. 8-Nitroguanine, purified
by HPLC, and thus devoid of peroxynitrite breakdown
products, can be stored at 4-5 °C in a sodium acetate
solution at pH 5 for several months without degradation.
p H -Dep en d en t F or m a t ion of 8-Oxod eoxygu a -
n osin e fr om th e Rea ction of Deoxygu a n osin e w ith
P er oxyn itr ite. The reaction of deoxyguanosine with
peroxynitrite at various pH values was conducted at 0-5
°C as described in Experimental Procedures. An aliquot
of the reaction mixture was submitted to HPLC im-
mediately after the addition of peroxynitrite, before side
-
nucleoside. We hypothesize that ONOO , perhaps acting
as a one-electron oxidant (36, 37), oxidizes deoxygua-
nosine or guanosine to a radical cation, which then
undergoes glycosidic bond hydrolysis and, at the same
time, reacts with another peroxynitrite molecule to give
8
-nitroguanine.
We find similarly that the reaction of xanthosine with
peroxynitrite gives 8-nitroxanthine in addition to 8-ni-
troxanthosine (Figure 13A). The extent of nitration by
peroxynitrite at neutral pH is higher in the xanthosine
reaction than in the guanosine reaction. 8-Nitroxan-
thosine was stable when the reaction mixture was stored
at 5 °C; thus, it was possible to collect the product from
HPLC and study its properties. At 37 °C, 8-nitroxan-
thosine exhibited a half-life of more than 12 h at pH 7
and 37 °C (figure not shown). The product of 8-nitrox-
anthosine decomposition was 8-nitroxanthine. As in the
case of 8-nitroguanosine, 8-nitroxanthosine hydrolyzed
to 8-nitroxanthine in 5 min at 90 °C. Upon reduction with
dithionite, it converted to the amino product, which
cochromatographed with the synthesized 8-aminoxan-
thosine standard (not shown in the figure).
We also examined the reaction of peroxynitrite with
9
8
-methylguanine, which does not have a glycosidic bond.
-Nitro-9-methylguanine, the late-eluting peak at 43 min
in Figure 13B, required electrochemical reduction and
subsequent oxidation to be detected, and was found to
be stable even at 90 °C, as expected. The HPLC-purified

4
46 Chem. Res. Toxicol., Vol. 14, No. 4, 2001
Sodum and Fiala
F igu r e 15. (A) HPLC-ECD profiles of the reaction products of
peroxynitrite with deoxyadenosine at pH 7 (trace 1A) and 5
F igu r e 14. (A) HPLC-ECD profiles of the reaction products of
peroxynitrite with deoxyguanosine at pH 5 and 0-5 °C. (B) pH-
dependent formation of 8-oxo-dG (b), and level of C8-nitration
(
trace 2A), both at 0-5 °C. To show the nitrated deoxyadenosine
product more clearly, traces 1A and 2A are redrawn with higher
magnification as traces 1B and 2B, respectively. The reducing
electrode W2 was set at -0.7 V and the oxidizing electrode W1
at 0.8 V. Deoxyadenosine (no ECD response) elutes at a
retention time of ∼22 min and can be observed by its UV trace
at 253 nm (not shown). (B) HPLC-ECD profiles of the reaction
products of peroxynitrite with adenosine at pH 7 (trace 1A) and
(9) estimated from the combined amounts of 8-NG and 8-NO
2
-
dG. The level of 8-NG and 8-NO -dG from the reaction run at
2
pH 7 is considered 100%. HPLC-ECD conditions for panels A
and B are as described in the legend of Figure 8.
reactions could take place, to determine accurately the
yield of 8-oxo-dG. The formation of 8-oxo-dG appeared
to reach a maximum level of 15 µM (∼0.5% yield) at pH
5
(trace 2A) and 0-5 °C. Trace 1B represents increased vertical
magnification of trace 1A. The same ECD conditions were used
that were used for panel A. Adenosine (no ECD response) elutes
at a retention time of ∼19 min and can be observed by its UV
absorption at 253 nm (not shown). HPLC solvent system 5 was
used at a flow rate of 1 mL/min for both panels A and B.
∼
5, as estimated from the initial deoxyguanosine con-
centration (Figure 14). This pH-dependent C8-oxidation
of deoxyguanosine is mediated by peroxynitrite and is
not due to reaction with side products, since control
reactions using decomposed peroxynitrite and run at the
same pH values showed no increase in the level of 8-oxo-
dG. The inability to observe the formation of 8-oxo-dG
at pH 7 is probably due to its faster reaction with
peroxynitrite at pH 7 than at pH 5. While all of the 100
µM 8-oxo-dG was lost in the reaction with 1 mM perox-
ynitrite at pH 7, 10% of the 100 µM 8-oxo-dG remained
unchanged when the reaction pH was lowered to 5. At
pH 7, since 8-oxo-dG reacts faster with peroxynitrite than
does deoxyguanosine (11), even the amount of 8-oxo-dG
normally present as a contaminant decreases by 40-50%
when commercial deoxyguanosine is reacted with perox-
ynitrite. We found that 96% of 100 µM and 100% of 10
µM 8-oxo-dG were destroyed at pH 5 by 10 mM peroxy-
nitrite; the latter is the average concentration of perox-
ynitrite used in our reactions. Therefore, the amount of
with nitrous acid, which is formed from the nitrite at
acidic pH, to give unknown products; the expected
product, 8-oxodeoxyxanthosine, was not observed.
While the peroxynitrite-mediated C8-oxidation of deoxy-
guanosine reached a maximum at pH 5, the level of C8-
nitration decreased as the pH under which the reaction
was carried out decreased. The reaction at pH 7 produced
the maximum level of C8-nitration of deoxyguanosine,
measured as the amount of 8-nitroguanine and 8-nitro-
dG. When compared to that of the pH 7 reaction, the yield
of 8-nitroguanine decreased by 75% at pH 6 and by 90%
at pH 5, while the yield of 8-nitro-dG decreased by only
35% at both pH 6 and 5 (Figure 14B). 8-Nitroguanine
formation in the peroxynitrite reaction with deoxygua-
nosine does not appear to be catalyzed by acidic pH. As
the pH of the reaction was decreased further to 3, neither
8-nitroguanine nor 8-nitro-dG was formed. At this pH,
some formation of 8-oxo-dG (∼0.1% yield) was observed.
Rea ction of Deoxya d en osin e a n d Ad en osin e w ith
P er oxyn itr ite a t p H 7 a n d 5. Unlike guanine nucleo-
sides, 5 mM deoxyadenosine and adenosine react with
peroxynitrite at pH 7 and 5 °C, or room temperature, to
give 16 µM 8-oxo-dA and 17 µM 8-oxo-Ado (∼0.3% yield),
respectively, in addition to thus far unidentified nitration
products (Figure 15). Probably because of their lower
reactivities with peroxynitrite, it is possible to observe
the formation of 8-oxo-dA and 8-oxo-Ado even in reactions
8
-oxo-dG observed is probably less than that actually
formed. In the reaction at pH 5, the initial concentration
of deoxyguanosine decreased by 2% compared to the
control run, while the yield of 8-oxo-dG was ∼0.5% of the
starting nucleoside. When the reaction mixtures of deox-
yguanosine and peroxynitrite were stored frozen at -20
°C, the amount of 8-oxo-dG from the reaction carried out
at pH 5 decreased by half, and in the case of reactions
carried out at an even lower pH, the 8-oxo-dG disap-
peared completely. 8-Oxo-dG might be lost by reacting

Peroxynitrite Nitrates and Oxidizes Nucleosides
Chem. Res. Toxicol., Vol. 14, No. 4, 2001 447
potential. The former possibility is unlikely since both
of the peaks corresponding to nitrated products disappear
completely after dithionite reduction of the reaction
mixture. The latter possibility is also unlikely since
analysis by hydrodynamic voltammetry showed that the
ECD response to 8-NH
potentials of g0.8 V; at <0.7 V, there is no response (data
not shown). The amount of 8-NH -dA obtained from
2
-dA reaches a plateau at oxidizing
2
dithionite reduction of the deoxyadenosine reaction mix-
ture was ∼20 times smaller than the amount of 0.3-0.4
2
µM 8-NH -Ado obtained after reduction of the adenosine
reaction mixture, suggesting that the level of C8 nitration
was correspondingly lower in the case of the deoxy-
nucleoside.
When the product responsible for the 38 min peak from
the adenosine reaction with peroxynitrite was purified
by HPLC, reduced with dithionite, and then analyzed,
we observed the appearance of two new, thus far uni-
dentified, electrochemically active products, which eluted
earlier than adenosine. However, little 8-NH
tion was observed, even though the reduction was
complete. This suggests that the 8-NH -Ado, which was
2
-Ado forma-
2
observed after dithionite reduction of the reaction mix-
ture, did not arise from the material in the 38 min peak.
Therefore, neither the 38 min peak from the adenosine
reaction nor the 60 min peak from the deoxyadenosine
reaction is likely to represent 8-nitroadenosine or 8-ni-
trodeoxyadenosine. It is possible that adenine nucleosides
can be nitrated at the C2 position. Such 2-nitroadenine
derivatives would yield the corresponding 2,6-diaminopu-
rines upon dithionite reduction. However, while 2-ni-
troadenine nucleosides would be expected to produce a
signal at the downstream electrode of the dual-mode
ECD, we find that 2,6-diaminopurine nucleosides are not
electrochemically active under any of the ECD conditions
used in this report. This possibility is therefore not likely.
As perhaps more likely candidates for the unknowns,
F igu r e 16. (A) HPLC-ECD analysis of the products from the
adenosine-peroxynitrite reaction at pH 7 after reduction with
dithionite (trace 1), the products from the adenosine-peroxyni-
trite reaction at pH 7 after reduction with dithionite cochro-
matographed with the 8-NH
HPLC-ECD profile of the 8-NH
2
-Ado standard (trace 2), and the
-Ado standard alone (trace 3).
2
HPLC conditions were like those described in the legend of
Figure 15. 8-Aminoadenine and 8-oxoadenine standards (not
shown) eluted at ∼7 and ∼8 min, respectively. (B) HPLC-ECD
analysis of the products from the reaction of deoxyadenosine
and peroxynitrite at pH 7 after dithionite reduction (trace 1)
and of 8-NH
2
-dA and 8-oxo-dA standards (trace 2). HPLC system
4
was used. Deoxyadenosine (not shown), which does not give
an ECD response, elutes at ∼19 min.
carried out at neutral pH. Indeed, we found that while
2
-nitro-8-oxoadenine and 8-nitro-2-oxoadenine or the
1
00 µM 8-oxo-dG was destroyed completely by reacting
corresponding nucleosides may be expected to produce
an ECD signal in the dual mode prior to dithionite
reduction, and in the single oxidative mode after dithion-
ite reduction. This, in fact, is observed for the 38 min
peak obtained from the adenosine reaction. Therefore,
we tentatively suggest that the late-eluting peaks at 60
and 38 min in the reactions of peroxynitrite with deoxy-
adenosine or adenosine may represent nucleosides of
with 1 mM peroxynitrite at pH 7 and 5 °C, only 20% of
the 100 µM 8-oxo-dA was lost under these conditions. As
evident from the HPLC profiles in Figure 15, the level
of oxidation of adenine nucleosides at C8 was several-
fold higher than that of nitration, even at neutral pH.
Lowering the reaction pH to 5 resulted in 2.4- and 2.2-
fold increases in the yields of 8-oxo-dA and 8-oxo-Ado,
respectively, but did not alter the level of nitration.
The late-eluting peaks, at ∼60 min in the case of the
dAdo reaction with peroxynitrite (Figure 15A) and at ∼38
min in case of the Ado reaction (Figure 15B), were
observed only when a suitable potential was applied to
the reducing electrode, suggesting that they represent
nitrated nucleosides. For the detection of these species,
a reducing potential of -0.7 V and an oxidizing potential
of 0.6 V were sufficient. However, increasing the oxida-
tion potential to 0.8 V at the downstream electrode
enabled the detection of 8-oxoadenine nucleosides to-
gether with the nitration products (Figure 15). Dithionite
reduction of the reaction mixtures led to formation of
2
-nitro-8-oxoadenine or 2-oxo-8-nitroadenine, although
the possibility of 2,8-dinitroadenine nucleoside is also not
excluded. Overall, nitration appears to be a minor route
in the reactions of peroxynitrite with adenine nucleosides;
as estimated from the size of the peaks corresponding to
8
-oxoadenine nucleoside and the late-eluting peaks cor-
responding to nitroadenine nucleosides, nitration is at
least 50-fold less extensive than C8-oxidation. We also
find that while adenine nucleosides react easily with
peroxynitrite, the adenine base itself exhibited little
reactivity and did not yield any significant nitration
products; this, however, may also be due, in part, to its
poor solubility.
8
-aminoadenine nucleosides, which could be detected at
an oxidation potential of 0.8 V (Figure 16A,B) even when
the reducing electrode was turned off. However, com-
pared to the area of the peaks at 60 and 38 min in Figure
Discu ssion
Several studies have been published on the reactivity
of peroxynitrite with deoxyguanosine and 8-oxo-dG, and
on the peroxynitrite-induced formation of 8-nitroguanine
in DNA (11, 15-17, 23, 33, 35, 38, 41). To our knowledge,
1
5, the areas of the peaks corresponding to the amino
derivatives appear to be much smaller, suggesting, at
first, an incomplete reduction or insufficient oxidation

4
48 Chem. Res. Toxicol., Vol. 14, No. 4, 2001
Sodum and Fiala
however, comparative studies on the extents of perox-
ynitrite-induced oxidative damage and nitration have not
been reported, nor, apparently, has the nitration of
ribonucleosides and deoxyribonucleosides been examined.
Central to the present work is the use of a highly
selective and sensitive method for the detection of
nitrated purines and nucleosides, based on HPLC with
a dual-mode amperometric detector. Highly sensitive
dual-mode electrochemical detection involving sequential
reduction and oxidation of biologically important mol-
ecules such as 3-nitrotyrosine, glutathione, glutathione
disulfide, and 8-nitroguanine has been used in the past
peroxynitrite; however, when the rapidity of the 8-oxo-
dG-peroxynitrite reaction is considered, it becomes
evident that the oxidation at C8 is much more extensive
than nitration in the reactions of both guanine and
adenine nucleosides at either pH 7 or 5. In contrast to
8-oxo-dG, 8-oxo-dA is less susceptible to further reactions
with peroxynitrite or its decomposition products. We
propose, therefore, that 8-oxo-dA may prove to be a more
accurate marker of the extent of peroxynitrite-mediated
oxidative damage to DNA than 8-oxo-dG.
To account for the ability of peroxynitrite to oxidize a
wide variety of biological molecules, the formation of a
new intermediate species, the “H-bonded caged radical
pair”, during the isomerization of peroxynitrite to nitrate,
has been proposed on the basis of theoretical calculations
(43). This intermediate species, like hydroxyl radicals,
would be highly reactive and capable of causing oxidative
damage (43). Alternatively, since peroxynitrite is known
to act as both a one-electron and two-electron oxidant
(36, 37, 44), the 8-oxo-dG and 8-oxo-dA observed in the
reactions of deoxyguanosine and deoxyadenosine may
have been produced from the reaction of a one-electron-
oxidized purine ring with a water molecule (45). The
same one-electron-oxidized purine intermediate, after
loss of the ribosyl or deoxyribosyl group, could also have
given rise to the 8-nitropurine base we observe im-
mediately after the addition of peroxynitrite in the
reactions of guanine and xanthine nucleosides.
In contrast to the findings of Byun et al., who reported
a retention time of 9 min for 8-nitro-dG and 11 min for
8-nitroguanine in a reverse-phase HPLC system (46), we
find that the C8-nitro products from the reaction of
peroxynitrite with purine nucleosides elute with signifi-
cantly different retention times, and in a different order,
in our system. For instance, with HPLC system 3,
deoxyguanosine elutes at 13 min (Figure 8), 8-nitrogua-
nine at ∼9 min (Figure 1), and 8-nitro-dG elutes at 72
min (Figure 8). Generally, the introduction of a nitro
group increases the nonpolarity of a compound and thus
increases the elution time of the compound on reverse-
phase columns; the reason for the large discrepancy
between the retention times we obtained and those
reported by Byun et al. (46) is unknown. Unlike the
8-oxonucleosides, the nitronucleosides were not produced
in a sufficiently high yield in our reactions to produce
UV signals or spectra, but could be observed by the dual-
mode ECD when the reducing electrode W2 was on, as
illustrated in Figure 10 for 8-nitro-Guo. At neutral pH
and 37 °C, 8-nitro-Guo exhibited a half-life of 5 h, but
8-nitro-dG, which was reported to exhibit a half-life of 4
h in DNA and of 1 h in an oligonucleotide (15, 23),
exhibited an apparent half-life of only e3 min as the free
nucleoside.
Peroxynitrite-mediated C8-nitration of guanine and
xanthine nucleosides was confirmed directly by purifica-
tion of the nitro nucleosides by HPLC, followed by
dithionite reduction to give the corresponding 8-amino-
nucleosides. In the peroxynitrite reactions with adenine
nucleosides, C8-nitration, which occurred to a much lower
extent than in the case of guanine and xanthine nucleo-
sides, could only be shown indirectly by the formation of
8-aminoadenine nucleoside after dithionite reduction of
the whole reaction mixture. However, the reaction of
peroxynitrite with adenine nucleosides appears to be
more complex, since the adenine ring is capable of
undergoing C8- and C2-nitration, although nitration was
(24, 41, 42). Other workers (41) have used a coulometric
detector equipped with several electrodes, which also
employed reduction and subsequent oxidation, for the
detection of 8-nitroguanine formation in the reactions of
calf thymus DNA with peroxynitrite in the presence of
various antioxidants; however, the parent deoxynucleo-
side, 8-nitro-dG, from which the 8-nitroguanine was
derived, was not detected.
The work presented here shows that amperometric
detection can be used to achieve high selectivity and
sensitivity for compounds, such as 8-nitroguanine and
8
-nitroxanthine, that can undergo reversible electro-
chemical reduction and oxidation at an electrode surface.
While the current due to the reduction of the nitro
compounds at the upstream electrode is ignored because
of its very low signal-to-noise ratio, high sensitivity is
obtained in our system by measuring only the current
produced at the downstream oxidizing electrode, and by
optimizing the ECD conditions for each compound of
interest through hydrodynamic voltammetry. The very
close proximity of the reducing and oxidizing electrodes
in the BAS ECD decreases the possible loss of unstable
products produced at the upstream electrode. In the case
of nitroaromatic and nitroheterocyclic compounds, turn-
ing the upstream reducing electrode off or placing it on
standby results in a loss of the signal at the downstream
oxidizing electrode. Since signals due to the oxidation of
compounds such as 8-oxo-dG and 8-oxo-dA are still
observable under this condition, this feature provides
enhanced selectivity in addition to that furnished by the
high-performance liquid chromatographic separation.
The latter has been optimized, in this work, to give high
resolution of several purine bases and nucleosides with
nonbulky modifications.
Our work shows that peroxynitrite reacts very quickly
with all of the purine nucleosides examined at neutral
pH, resulting in C8-oxidation as well as in nitration.
Although the C8-oxidation of adenine and guanine
nucleosides by peroxynitrite had been reported in an
earlier work by others (22), it is not clear at what pH
the reaction was carried out or whether the oxidation
may have been partially due to contamination with
hydrogen peroxide. In the work presented here, the
peroxynitrite was prepared by the ozonation of sodium
azide (25), excluding the possibility of hydrogen peroxide
contamination. Also, peroxynitrite, which had been al-
lowed to decompose under the same conditions as the
actual runs, was used in all control incubations. Thus,
the formation of 8-oxo-dA and 8-oxo-dG at various pH
values that we describe in the reactions of deoxyadenos-
ine and deoxyguanosine is indeed mediated by peroxyni-
trite. At neutral pH, the inability to observe the formation
of 8-oxo-dG in the reactions of deoxyguanosine with
peroxynitrite is due to its further reactivity toward

Peroxynitrite Nitrates and Oxidizes Nucleosides
Chem. Res. Toxicol., Vol. 14, No. 4, 2001 449
minor when compared to C8-oxidation. Further work to
fully characterize the nitration products of adenine
nucleosides is in progress in our laboratory. The failure
to detect these modifications up to now may simply be
the lack of the proper HPLC system and mode of
detection. We consider that the results of the studies
described here are important for understanding the
reactive nature of peroxynitrite and also as prerequisites
to the search for analogous products, possibly mutagenic,
from the reaction of peroxynitrite with DNA and RNA
in vitro and in vivo.
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(
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TX000189S