8-Nitroxanthine, an adduct derived from 2′-deoxyguanosine or DNA reaction with nitryl chloride

Article abstract of DOI:10.1021/tx0002334

Activated phagocytic cells generate reactive nitrogen species, including nitryl chloride and peroxynitrite, for host defense against invading pathogens. It has been proposed that these reactive nitrogen species may cause DNA damage and thus contribute to the multistage carcinogenesis process associated with chronic infections and inflammation. Previous studies showed that peroxynitrite reacted with guanine, 2′-deoxyguanosine, or DNA forming 8-nitroguanine. We herein report formation of 8-nitroxanthine as the major nitration product in reactions of 2′-deoxyguanosine or calf thymus DNA with nitryl chloride produced by mixing nitrite with hypochlorous acid, and 8-nitroguanine was a minor product in these reactions. 8-Nitroxanthine was characterized by its NMR and laser desorption ionization mass spectra and by deamination of 8-nitroguanine. Formation of 8-nitroxanthine was also detected by xanthine reaction with various reactive nitrogen species, including nitryl chloride, peroxynitrite, nitronium tetrafluoroborate, and heated nitric and nitrous acid. The identity of 8-nitroxanthine in nitryl chloride-treated dG and DNA was confirmed by co-injection with synthetic 8-nitroxanthine and by its reduction to 8-aminoxanthine. Levels of 8-nitroxanthine and 8-nitroguanine in these reactions were quantified by reversed-phase HPLC with photodiode array detection. Once formed, 8-nitroxanthine was spontaneously removed from DNA with a half-life of 2 h at 37 °C and pH 7.4. Therefore, 8-nitroxanthine might be an important DNA lesion derived from reactive nitrogen species in vivo.

Full text of DOI:10.1021/tx0002334

5
36
Chem. Res. Toxicol. 2001, 14, 536-546
8
-Nitr oxa n th in e, a n Ad d u ct Der ived fr om
2
′-Deoxygu a n osin e or DNA Rea ction w ith Nitr yl Ch lor id e
,
†
†
†
Hauh-J yun Candy Chen,* Yuan-Mao Chen, Tze-Fan Wang,
†
‡
Kuang-Sian Wang, and J entaie Shiea
Department of Chemistry, National Chung Cheng University, 160 San-Hsing, Ming-Hsiung,
Chia-Yi 621, Taiwan, and Department of Chemistry, National Sun Yat-Sen University,
Kaohsiung, 80424, Taiwan
Received November 8, 2000
Activated phagocytic cells generate reactive nitrogen species, including nitryl chloride and
peroxynitrite, for host defense against invading pathogens. It has been proposed that these
reactive nitrogen species may cause DNA damage and thus contribute to the multistage
carcinogenesis process associated with chronic infections and inflammation. Previous studies
showed that peroxynitrite reacted with guanine, 2′-deoxyguanosine, or DNA forming 8-nitro-
guanine. We herein report formation of 8-nitroxanthine as the major nitration product in
reactions of 2′-deoxyguanosine or calf thymus DNA with nitryl chloride produced by mixing
nitrite with hypochlorous acid, and 8-nitroguanine was a minor product in these reactions.
8
-Nitroxanthine was characterized by its NMR and laser desorption ionization mass spectra
and by deamination of 8-nitroguanine. Formation of 8-nitroxanthine was also detected by
xanthine reaction with various reactive nitrogen species, including nitryl chloride, peroxynitrite,
nitronium tetrafluoroborate, and heated nitric and nitrous acid. The identity of 8-nitroxanthine
in nitryl chloride-treated dG and DNA was confirmed by co-injection with synthetic 8-nitro-
xanthine and by its reduction to 8-aminoxanthine. Levels of 8-nitroxanthine and 8-nitroguanine
in these reactions were quantified by reversed-phase HPLC with photodiode array detection.
Once formed, 8-nitroxanthine was spontaneously removed from DNA with a half-life of 2 h at
3
7 °C and pH 7.4. Therefore, 8-nitroxanthine might be an important DNA lesion derived from
reactive nitrogen species in vivo.
In tr od u ction
decomposition (10, 11). It is now well established that
peroxynitrite can be formed under oxidative stress in
several disease states (12-16) and in cigarette smoking
Chronic infections and inflammation are important
risk factors in cancer development (1). It has been
postulated that peroxynitrite may cause DNA and tissue
damage and contribute to the multistage carcinogenesis
associated with chronic infections and inflammation (2).
Peroxynitrite (and peroxynitrous acid, its conjugate acid)
was formed from the concomitant release of superoxide
(17, 18). Peroxynitrite has been reported to induce DNA
strand breaks and is mutagenic (19). The products of
peroxynitrite reaction with DNA bases include 8-nitro-
guanine (8NG), 8-oxoguanine, base propenals (20, 21),
secondary oxidation products of 8-oxoguanine (22, 23),
xanthine (24), and 4,5-dihydro-5-hydroxy-4-(nitrosooxy)-
1
anion and nitric oxide (NO) by activated macrophages
2
′-deoxyguanosine (25). Among them, 8NG induced GC
and neutrophils in inflamed tissues (3, 4). Many of the
damaging effect of NO are attributed to the formation of
to TA transversion mutations, the same mutations
induced by peroxynitrite (19).
-
peroxynitrite and/or nitrosoperoxycarbonate (ONO
the reactive product of peroxynitrite with CO
abundant in physiological fluids (5-7). Peroxynitrite is
a relatively stable reactive oxygen species with a half-
life of ca. one second at physiological pH (8, 9). Therefore,
it can diffuse and cross cell membranes before its
2
CO
2
),
Another source of 8NG is 2′-deoxyguanosine (dG)
2
, which is
reaction with nitryl chloride (NO
2
Cl) and/or nitrogen
•
dioxide radical (NO
2
), produced in activated phagocytes
at the inflammation sites (26). Nitryl chloride is capable
of nitrating guanine, tyrosine, and lipids as well as
chlorinating and oxidizing tyrosine (26-28). Suzuki and
co-workers also reported that 8NG was formed in reaction
*
To whom correspondence should be addressed. Fax: (886) 5-272-
040. E-mail: chehjc@ccunix.ccu.edu.tw.
National Chung Cheng University.
National Sun Yat-Sen University.
Abbreviations: 8AG, 8-aminoguanine; 8AX, 8-aminoxanthine; dG,
of dG with NO/O
2
gas mixture under physiological
1
†
‡
1
condition (29).
In this study, 8-nitroxanthine (8NX) and 8NG were
2
formed in reaction of calf thymus DNA or dG with NO -
i
2
′-deoxyguanosine; Pr
2
EtN, diisopropylethylamine; DMF, dimethyl-
, dinitrogen pentoxide;
, hydrogen peroxide; LDI, laser
formamide; DMSO, dimethyl sulfoxide; N
, dinitrogen tetroxide; H
desorption ionization; NO, nitric oxide; NO
2 5
O
Cl generated in vitro by mixing nitrite with hypochlorous
acid in the absence of an enzyme (Scheme 1). Previously
found as a side product in reaction of nitrous acid with
guanosine and xanthosine (30), 8NX was the major
nitration product in these two reactions and 8NG was
instead a minor product. Formation of 8NX from other
N
2
O
4
2 2
O
•
2
, nitrogen dioxide radical;
+
-
+
-
4
,
8
NG, 8-nitroguanine; NO
nitrosonium tetrafluoroborate; N
troxanthine; Na , sodium hydrosulfite or sodium dithionite;
NaOCl, sodium hypochlorite; NaNO , sodium nitrite; NO Cl, nitryl
, peroxynitrite; PDA, photodiode array.
2
BF
4
, nitronium tetrafluoroborate; NO BF
2
O
3
, nitrous anhydride; 8NX, 8-ni-
2
2 4
S O
2
2
-
2
chloride; ONO
1
0.1021/tx0002334 CCC: $20.00 © 2001 American Chemical Society
Published on Web 04/26/2001

8
-Nitroxanthine in Nitryl Chloride-Treated DNA
Chem. Res. Toxicol., Vol. 14, No. 5, 2001 537
Sch em e 1. Rea ction of Nitr yl Ch lor id e w ith DNA
or d G F or m in g 8NX a n d 8NG
potassium phosphate buffer, pH 12.8) and 4.5 µL of a solution
of NaOCl (0.29 M in 0.4 M potassium phosphate buffer, pH 12.8)
with stirring. Final pH of the reaction mixtures was between 7
and 7.4. The reaction mixture was filtered through a 0.22 µm
nylon syringe filter (Advantec, MFS, Inc.) and collected from
1
7 to 20 min by HPLC using system 1. The collected fractions
were combined and evaporated to dryness. The mixture was
reconstituted in water and injected to HPLC using system 2
collecting from 14 to 16 min to obtain salt-free 8NX. UVmax (pH
reactive nitrogen species was also investigated. Depuri-
nation of 8NX from DNA was spontaneous with a half-
life of 2 h under physiological conditions. Therefore, 8NX
might be an important DNA damage due to the physi-
ological roles of nitrite and hypochlorous acid in inflam-
mation and in cancer development.
-1
-1
5
.5) λ 205, 376 nm; ꢀ382 ) 7673 M cm (pH 7.0); MS (LDI-)
); H NMR (DMSO-d ) δ 7.0 (broad s, 1H, N9-
-
1
196 ([M - H]
H), 10.2 (s, 1H, N3-H), 10.9 (s, 1H, N1-H).
6
Syn th esis of 8-Nitr ogu a n in e (8NG). 8-Nitroguanine was
synthesized by a modified method (36). Briefly, a solution
containing guanine (0.3 mg, 0.53 µmol) in 0.95 mL of 0.2 N HCl
and 50 mM ammonium formate was added 38 µL of peroxyni-
trite (200 mM in 0.67 N NaOH and 50 mM ammonium formate)
and 962 µL of 0.67 N NaOH containing 50 mM ammonium
formate with vigorous stirring at room temperature for 2 min.
Final pH of the reaction was between 6.8 and 7.0. The reaction
mixture was concentrated to ca. 1 mL, filtered through a 0.22
µm Nylon syringe filter, and collected repeatedly from 15 to 16
min, by HPLC using system 1. The collected fractions were
combined and evaporated to dryness. The mixture was recon-
stituted in water, injected to HPLC using system 2, and collected
from 5 to 7 min to obtain salt-free 8NG. MS (LDI-) m/z 195 ([M
Ma ter ia ls a n d Meth od s
Ma ter ia ls. Calf thymus DNA and 8-amimoguanosine were
from Sigma Chemical Co. (St. Louis, MO). Guanine, diethylene-
triamine pentaacetic acid (DTPA), manganese dioxide, nitro-
nium tetrafluoroborate, nitrosonium tetrafluoroborate, sodium
2 2 4
hydrosulfite (sodium dithionite, Na S O ), and sodium hypochlo-
ride (NaOCl) were obtained from Aldrich Chemical Co. (Mil-
waukee, WI). Isoamylnitrite was from T.C.I. Co. (Tokyo, J apan).
Hydrogen peroxide was purchased from Acros Organic Chemical
Co. (Geel, Belgium) and quantified based on the absorbance at
-
-
- H] ), 179 ([M-17] ); UVmax (pH 5.5) λ 210, 231, 258, 393 nm.
1
-1
-1
-1
2
40 nm (ꢀ ) 43.6 M- cm ) (31). Peroxynitrite was synthesized
ꢀ
400 ) 9144 M cm (pH 7.0).
according to the previously described procedures using isoamylni-
trite and hydrogen peroxide (32) and was stored at -80 °C. The
concentration of peroxynitrite was determined by the absorbance
Syn th esis of 8AX. 8NX (1.4 mg, 8.4 µmol) in 1.0 mL of water
was added sodium hydrosulfite (5.85 mg, 33.6 µmol) at room
temperature with stirring for 10 min. The reaction mixture was
concentrated to ca. 1 mL, filtered through a 0.22 µm Nylon
syringe filter, adjusted to pH 5.5, and collected repeatedly from
3.5 to 14.5 min, by HPLC using system 1. The collected
fractions were combined and evaporated to dryness. The mixture
was reconstituted in water and injected to HPLC using system
-
1
-1
at 302 nm in 1N NaOH (ꢀ ) 1670 M
cm ) (33). The
concentration of hypochlorite was determined by the absorbance
-
1
-1
at 292 nm (pH 12, ꢀ ) 350 M cm ) (34). Standard 8-amino-
1
guanine was obtained by acid hydrolysis of 8-aminoguanosine
(
35).
In str u m en ts. NMR spectra were recorded on a Bruker DPX
00 MHz (Billerica, MA) instrument. Laser desorption ionization
LDI) mass spectra was performed on a HP G2025A LD-TOF
2 collecting from 5 to 7 min to obtain salt-free 8AX. UV
max
2
(H O)
4
(
λ 207, 288 nm. MS (MALDI+) 168 ([M + H]
([M - H] ).
+
); MS (LDI-) 166
-
mass spectrometer equipped with a 337 nm nitrogen laser. 8NX
and 8AX were dissolved in double distilled water and 1 N HCl,
respectively. One microliter of the sample solution was applied
on a direct sample probe and dried under reduced pressure. The
probe was subsequently sent into the ion source of the mass
spectrometer. The accelerating voltage in the ion source was
Red u ction of 8NG to 8AG. 8NG (2.0 mg, 10.2 µmol) in 1.0
mL of water was added sodium hydrosulfite (3.55 mg, 20.4 µmol)
at room temperature with stirring for 10 min as described (37).
The reaction mixture was adjusted to pH 5.5 and analyzed by
HPLC using system 3. The retention time and UV spectrum
were compared with standard 8-aminoguanine obtained from
2
8 kV. Fast-atom bombardment (FAB) mass spectrum was
acid hydrolysis of 8-aminoguanosine (35). UVmax (pH 5.5) λ 226,
performed on a VG Trio-2000 spectrometer. HPLC Chromatog-
raphy was performed by a Hitachi L-7000 pump system with
D-7000 interface, a Rheodyne injector, and a L-7450A photo-
diode array (PDA) detector.
HP LC Con d ition s: (1) System 1. A Prodigy ODS (3) 250
mm × 10 mm, 5 µm column (Phenomenex, Torrance, CA) was
used with the following isocratic conditions: 50 mM ammonium
formate (pH ) 5.5) buffer at a flow rate of 4.0 mL/min.
+
2
50, 291 nm. MS (FAB+) 167 ([M + H] ).
8
NG Rea ction w ith ter t-Bu tyln itr ite. A solution of 8NG
t
(
2.5 µmol) in 4.0 mL of 3 N HCl was added BuONO (30 µL, 250
µmol) with stirring at room temperature for 1 h. The reaction
mixture was adjusted to pH 5.5 and analyzed by HPLC system
3
. A portion of the reaction mixture (80 µL) was co-injected with
synthetic 8NX (35 nmol) described above and analyzed by HPLC
system 3. Another portion of the reaction mixture (3.0 mL) was
collected by HPLC system 3 and reduced by sodium hydrosulfite
(
2) System 2. A Prodigy ODS (3) 250 mm × 10 mm, 5 µm
column was eluted with H O at a flow rate of 4.0 mL/min.
2
(500 µg). One thirtieth of the reduced mixture with added
(
3) System 3. A Prodigy ODS (3) 250 mm × 4.6 mm, 5 µm
synthetic 8AX (6.3 nmol) described above and analyzed by HPLC
system 3.
column was used with the following isocratic conditions: 50 mM
ammonium formate (pH ) 5.5) buffer at a flow rate of 1.0 mL/
min.
Effect of p H on Nitr a tion of Xa n th in e or Gu a n in e.
Xanthine or guanine (0.1 mg, 0.66 µmol in 0.1 mM HCl) was
(
4) System 4. A Prodigy ODS (3) 250 mm × 4.6 mm, 5 µm
added 0.4 M potassium phosphate buffer (pH 3.0-10.0), NaNO
2
column was eluted with 50 mM ammonium formate (pH ) 4.0)
from 0 to 40 min, followed by a linear gradient of 0-100% MeOH
from 40 to 60 min at a flow rate of 1.0 mL/min.
(
0.66 µmol), and NaOCl (0.66 µmol) at room temperature with
vortexing for 2 min and the final volume was 0.4 mL. The
reaction mixture was adjusted to pH 5.5 and analyzed by HPLC
system 3. Quantification of 8NX and 8NG was monitored at 376
and 393 nm, respectively.
Syn th esis of 8NX (1) fr om P er oxyn itr ite. Xanthine (2.0
mg, 13.2 µmol) dissolved in 0.5 mL of 0.2 N HCl was added
peroxynitrite (200 mM in 56 mM NaOH and 50 mM ammonium
formate) and 962 µL of 56 mM NaOH containing 50 mM
ammonium formate with vigorous stirring at room temperature
for 2 min. (2) fr om Nitr yl Ch lor id e. A solution containing
xanthine (0.2 mg, 1.31 µmol) dissolved in 0.2 N HCl (0.2 mL)
Xa n th in e or Gu a n in e Rea ction w ith Nitr ic or Nitr ou s
Acid . Xanthine or guanine (7.4 mg, 49 µmol) was added 5.0 mL
of 1.0 M nitric acid or nitrous acid and heated at 90 °C for 3 h.
An aliquot (100 µL) of the reaction mixture was adjusted to pH
5.5 and analyzed by HPLC system 3.
2
was added 298 µL of a solution of NaNO (4.4 mM in 0.4 M

5
38 Chem. Res. Toxicol., Vol. 14, No. 5, 2001
Chen et al.
Xa n th in e or Gu a n in e Rea ction w ith Nitr on iu m Tetr a -
flu or obor a te or Nitr oson iu m Tetr a flu or obor a te. Xanthine
or guanine (1.2 mg, 7.9 µmol) dissolved in 3.0 mL of anhydrous
+
-
+
-
4
DMF was added NO
92 mg, 790 µmol) with stirring at room temperature for 5 min.
An aliquot (50 µL) of the reaction mixture was adjusted to pH
2
BF
4
(250 mg, 1.88 mmol) or NO BF
(
5
.5 and analyzed by HPLC system 3.
d G Rea ction w ith Nitr yl Ch lor id e. A solution containing
2
2
′-dG (0.6 mg, 2.25 µmol) and NaNO (7.8 mg, 113 µmol)
dissolved in 0.5 mL of 0.2 M potassium phosphate buffer (pH
7
1
.0) was added a solution containing 470 µL of NaOCl (0.24 M,
13 µmol) with stirring at room temperature for 2 min. The
reaction mixture was adjusted to pH 4.0 and an aliquot (50 µL)
was analyzed by HPLC using system 4.
Rea ction of DNA w ith Nitr yl Ch lor id e. Calf thymus DNA
(0.4 mg in 0.4 mL 0.1 M potassium phosphate, pH 7.0) was
added 520 µL of a solution containing NaNO (0.52 M) and 340
2
µL of a solution of NaOCl (66 mM) with vortexing for 2 min.
The final pH of the reaction mixture was 7.0. The reaction
mixture was added 3 mL cold ethanol and centrifuged at 15 000
rpm at 4 °C for 5 min. The precipitated DNA was washed with
F igu r e 1. UV spectrum of 8NX at pH 1, 7, and 10.
Sch em e 2. Red u ction of 8NG a n d 8NX
7
0% ethanol (3 mL × 2) and evaporated to dryness.
H yd r olysis of Nit r yl Ch lor id e-Tr ea t ed Ca lf Th ym u s
DNA. After calf thymus DNA reacted with nitryl chloride, the
reaction mixture was divided into two equal portions. Both
portions were precipitated, centrifuged, washed, and evaporated.
One portion of DNA was added 0.3 mL of 0.1 N HCl and heated
at 100 °C (acid hydrolysis). The other portion of DNA was
2
hydrolyzed in 0.3 mL of H O heating at 100 °C (neutral
hydrolysis). After 30, 60, 90, 120, and 180 min, an aliquot (50
µL) of each of the hydrolysate was removed, adjusted to pH 4.0,
and analyzed by HPLC system 4 at 376 nm.
Id en tity of 8NX in Nitr yl Ch lor id e-Tr ea ted d G a n d Ca lf
Th ym u s DNA. (1) d G. One milliliter solution containing 2′-
dG (final concentration 2.32 mM), NaNO
2
(final concentration
1
16 mM), and 0.1 M potassium phosphate buffer (pH 7.0) was
added a solution of NaOCl (final concentration 116 mM) with
stirring at room temperature for 2 min. 2. DNA. Calf thymus
DNA (2.0 mg in 2.0 mL 0.2 M potassium phosphate buffer, pH
Resu lts
Ch a r a cter iza tion of 8NX. Like 8NG, 8NX has a
characteristic UV-vis absorption above 350 nm. At
neutral pH, 8NX and 8NG exhibited an absorbance
maximum at 383 and 400 nm, respectively. A hypsochro-
mic shift was observed for 8NX with decreasing pH
7
.0) was added 770 µL of a solution containing NaNO
and 372 µL of a solution of NaOCl (0.30 M). After vortexing for
min, DNA was precipitated, washed, and hydrolyzed with 0.1
2
(0.58 M)
2
N HCl (400 µL) at 100 °C for 30 min. Both of the reaction
mixtures of dG and DNA were adjusted to pH 5.5 and a 100 µL
aliquot from each reaction mixture was analyzed by HPLC using
system 3. The remaining reaction mixtures were collected from
(
Figure 1) as was reported for 8NG (36). As reduction
with sodium hydrosulfite converted 8NG to 8-aminogua-
nine (8AG), same reagent reduced 8NX to 8-aminoxan-
thine (8AX), which was evident by mass spectroscopic
analysis (Scheme 2).
The negative laser desorption ionization (LDI) mass
spectra of 8NX and 8AX showed the m/z 196 and 166 as
2
0 to 22 min by HPLC using system 3. The collected 8NX
fraction was concentrated and reconstituted in 400 µL of water.
A portion of the aliquot (100 µL) was analyzed by HPLC system
3
8
at 376 nm and another portion (100 µL) was added synthetic
NX (1.0 µg) and analyzed under the same condition. The
-
remaining aliquot (200 µL) was reduced by sodium hydrosulfite
and 100 µL of the aliquot was analyzed by HPLC system 3 at
the [M - H] ions, respectively (Figure 2). The odd
molecular weight of 8NX and 8AX determined by LDI
mass spectrometry was supported by the nitrogen rule
of odd number of molecular weight for compounds
containing odd nitrogen atoms. Reduction of the nitro
groups turned the yellow 8NX and 8NG to the colorless
8AX and 8AG, respectively. The 8AG produced from 8NG
reduction was identical to that from acid hydrolysis of
commercially available 8-aminoguanosine as evidenced
by their mass and UV spectra, their retention times and
coelution on reversed-phase HPLC. The UV spectrum of
2
87 nm. The rest of the reduction mixture (100 µL) was added
8
AX (48 ng) and analyzed by HPLC system 3 at 287 nm.
Dep u r in a tion of 8NX in Nitr yl Ch lor id e-Tr ea ted Ca lf
Th ym u s DNA. After calf thymus DNA (4.8 mg in 4.8 mL of
.1 M potassium phosphate, pH 7.0) was added 2.4 mL of NaNO
2.24 M) and 7.9 mL of NaOCl (0.17 M) at room temperature,
0
2
(
the reaction mixture was incubated at 37 °C. An aliquot (1.26
mL) of the reaction mixture was removed at various time points.
DNA was precipitated with cold ethanol (2.5 mL), centrifuged,
washed with 2.5 mL of 70% cold ethanol twice, and evaporated.
DNA was reconstituted in 400 µL of 0.1 M phosphate buffer
and hydrolyzed at 100 °C for 3 h and pH of the mixture was
adjusted to 4.0, filtered through a 0.22 µm Nylon syringe filter,
and analyzed by HPLC system 4 at 376 nm. The ethanol
solutions were combined, evaporated, adjusted pH to 4.0, filtered
through a 0.22 µm Nylon syringe filter, and analyzed by HPLC
system 4 at 376 nm for 8NX released in the medium.
8
AX has the absorption maximum at 206 and 287 nm as
compared to that for 8AG at 250 and 291 nm at pH 5.5.
To further confirm that the nitro group of 8NX was at
the C-8 position as that in 8NG, 8NG was treated with
acidic tert-butylnitrite, which was known to deaminate
guanine to xanthine (38). In the presence of the deami-
nation agent, 8NG was converted to 8NX (Scheme 3 and

8
-Nitroxanthine in Nitryl Chloride-Treated DNA
Chem. Res. Toxicol., Vol. 14, No. 5, 2001 539
F igu r e 2. Laser desorption ionization mass spectrum of (a) 8NX and (b) 8AX.
Figure 3a). The identity of the product in this reaction
was confirmed by their UV spectra and by coelution in
reversed-phase HPLC with the authentic 8NX synthe-
sized from xanthine reaction with peroxynitrite (Figure
Sch em e 3. Syn th esis of 8NX
3
b). Furthermore, the product was collected from reversed-
phase HPLC, reduced by sodium hydrosulfite, and co-
eluted with 8AX synthesized from reduction of 8NX
obtained from xanthine reaction with peroxynitrite (Fig-
ure 3c). The reduced product also has identical UV
spectrum with synthetic 8AX. The sharp singlets at 10.9
1
and 10.2 ppm in H NMR of 8NX in DMSO-d
6
are the
N1- and N3-protons and the N9-proton is the broad peak
around 7.0 ppm. The absence of the C8-H around 8.0
ppm, as that of xanthine, indicated that the C8-H is
substituted with the nitro group.

5
40 Chem. Res. Toxicol., Vol. 14, No. 5, 2001
Chen et al.
t
F igu r e 3. HPLC Chromatogram of reaction of 8NG with BuONO to form 8NX. (a) A solution of 8NG (2.5 µmol) in 4.0 mL of 3 N
t
HCl was added BuONO (250 µmol) with stirring at room temperature for 1 h; (b) a portion of the reaction mixture (80 µL) was
co-injected with synthetic 8NX (35 nmol); (c) a portion of the reaction mixture (3.0 mL) was collected and reduced by sodium hydrosulfite
(
500 µg). One thirtieth of the reduced mixture was added synthetic 8AX (6.3 nmol) and analyzed by HPLC as described in the
Materials and Methods.
F or m a tion of 8NX a n d 8NG. The pH-dependent
nitration of xanthine by nitryl chloride was examined.
In reaction of nitryl chloride with equal amount of
xanthine, maximum yield of 8NX was achieved at slightly
alkaline pH (Figure 4), which is physiologically signifi-
cant. The pH profile was consistent with that in tyrosine
nitration by mixing NaNO
2
and NaOCl (27). This result
suggests that hypochlorous acid (HOCl) and nitrite ion

8
-Nitroxanthine in Nitryl Chloride-Treated DNA
Chem. Res. Toxicol., Vol. 14, No. 5, 2001 541
F igu r e 4. Effect of pH on nitration of xanthine or guanine with equal amount of nitryl chloride forming the respective 8NX ([) or
(0.66
8
NG (0). Xanthine or guanine (0.66 µmol in 0.1 mM HCl) was added 0.4 M potassium phosphate buffer (pH 3.0-10.0), NaNO
2
µmol), and NaOCl (0.66 µmol) at room temperature with a final volume of 0.4 mL. Results are expressed as mean ( SD of triplicate
experiments.
Ta ble 1. Rea ction s of Xa n th in e or Gu a n in e w ith Va r iou s Rea ctive Nitr ogen Sp ecies
reaction
yield of 8NXa
reaction
yield of 8NX
yield of 8NGa
b
G + NO2Cl (1:1)b
NDe
ND
0.1%
13%
ND
X + NO2Cl (1:1)
7.3%
9.2%
4.0%
2.5%
0.4%
7.3%
8.9%
3.7%
ND
-
b
G + ONO2 (1:1)b
-
X + ONO2 (1:1)
c
c
c
X + HNO3 (1:100)
X + HNO2 (1:100)
G + HNO3 (1:100)c
G + HNO2 (1:100)
+
-
d
G + NO2 BF4 (1:9)d
+
-
X + NO2 BF4 (1:240)
1.5%
a
Average of duplicate experiments. Room temperature, pH 7.0, 5 min. c 90 °C, 3 h. d Room temperature, 5 min. Prolonged reaction
b
e
time led to decrease of the product. Not detectable.
-
-
(
(
NO
2
), rather than hypochlorite (OCl ) and nitrous acid
), are involved in formation of this nitration species
of HOCl and HNO is 7.5 and 3.4, respectively.
Synthesis of 8NX was achieved by reactions of xanthine
with excessive amount of NO Cl or peroxynitrite (Scheme
). To isolate pure 8NX, it was collected from the reaction
deamination product of guanine, also present in the
reaction mixture. Heating nitrous acid generates nitrous
HNO
2
•
•
•
since pK
a
2
anhydride (N
is a nitrating agent, but in the presence of O
oxidized to NO
of nitrating aromatic compounds (39, 40). On the other
2
O
3
), NO
2
, and NO . Neither NO nor N
2
•
O
3
2
, NO is
•
6
-2 -1
(k ) 2 × 10 M
s ), which is capable
2
2
3
mixture by reversed-phase HPLC eluting with a buffer
at pH 5.5 to separate it from unreacted xanthine, followed
by removal of the salt with a second HPLC eluting with
water. In reaction of xanthine with peroxynitrite, 8NX
was the only product, whereas other side products were
present in the reaction mixture of xanthine with nitryl
chloride. Therefore, collection of 8NX from the reaction
mixture of xanthine with peroxynitrite gave large quan-
tity of the pure product more efficiently than from the
nitryl chloride reaction.
2 3
hand, nitration of xanthine by the nitrosating agent N O
is presumably via oxidation of the intermediate 8-nitro-
soxanthine (41). In reaction of guanine with nitrous acid,
all the guanine was deaminated to xanthine and high
yield (13%) of 8NX was obtained without detectable
amount of 8NG. This result suggested that formation of
the diazonium salt at the N2-amino enhanced yield of
nitration at the C8 position and that 8NG was not an
intermediate in formation of 8NX.
Formation of 8NX was observed when xanthine was
+
-
8
-Nitroxanthine was also formed as the major product
2 4
treated with nitronium tetrafluoroborate (NO BF )
in reaction of xanthine with nitric or nitrous acid heated
(Scheme 3), presumably via electrophilic attack of the
nitronium ion. Nitration of xanthine or guanine by
NO BF was less effective than other nitrating agents
2 4
at 90 °C for 3 h, a condition that reactive nitrogen oxides,
•
+
-
such as nitrogen dioxide (NO
2
), dinitrogen tetroxide
) were generated.
(N
2
O
4
), and dinitrogen pentoxide (N
2
O
5
described (Table 1), possibly due to the rapid hydrolysis
of nitronium ion. On the other hand, reaction of xanthine
Nitric acid gave 8NX in a yield (4.0%) slightly higher than
nitrous acid (2.5%) (Table 1). Upon heating, nitric acid
+
-
4
with nitrosonium tetrafluoroborate (NO BF ) gave a
generates NO
rium with each other. The anhydride of nitric acid, N
is also generated upon heating HNO . Among these
reactive nitrogen species, NO , N , and N are
nitrating agents. When heated nitric acid reacted with
guanine, yield of 8NG (3.7%) was slightly less than its
reaction with xanthine. Only trace amount of 8NX (0.1%)
was obtained as a result of nitration of xanthine, the
2
and its dimer N
2
O
4
, which are in equilib-
product with UV spectrum and retention time in HPLC
distinct from those of 8NX. These findings further
confirmed that 8NX is a result of nitration, not nitrosy-
lation. The identity of 8NX in reactions of xanthine with
2
5
O ,
3
2
2
O
4
2 5
O
+
-
2 4
heated nitric or nitrous acid or NO BF were confirmed
by comparing their UV spectrum and by coelution with
8NX synthesized from xanthine with peroxynitrite. Fur-
thermore, 8NX in each reaction mixture was isolated

5
42 Chem. Res. Toxicol., Vol. 14, No. 5, 2001
Chen et al.
Ta ble 2. 8NX a n d 8NG F or m a tion in Rea ction s of d G or
DNA w ith Nitr yl Ch lor id e or P er oxyn itr ite
depurination. The half-life of 8NX in nitryl chloride-
treated calf thymus DNA was 2 h as measured by the
time-dependent release of 8NX in the medium and 8NX
remained in DNA at various intervals. The latter was
analyzed after DNA precipitation followed by hydrolysis.
The sum of 8NX in the medium and 8NX remained in
DNA was nearly constant, indicating that the adduct was
completely released after 24 h (Figure 6).
reaction
dG + NO2Cl (1: 50)b
DNA + NO2Cl (1: 250)
a
yield of 8NX
yield of 8NGa
2.5%
0.71%
0.50%
0.30%
b
Average of duplicate experiments. b Room temperature, pH
7
.0, 5 min.
from reversed phase HPLC, reduced by sodium hydro-
sulfite, and the reduced adduct coeluted with authentic
Discu ssion
8
AX obtained from reduction of synthetic 8NX.
Rea ction of d G or DNA w ith Nitr yl Ch lor id e.
Since chronic infections and inflammation is implicated
in cancer development, it is of interest to study the
chemistry of these reactive species with DNA. Among the
reactive species, peroxynitrite and nitryl chloride are
capable of nitrating biomolecules. The nitration product
of guanine, 8NG, can be derived from peroxynitrite
reaction with guanine or DNA (21, 36). It is also formed
in the myeloperoxidase-hydrogen peroxide-nitrite system
Reactivity of nitryl chloride toward dG or calf thymus
DNA was much lower as compared to its reaction with
xanthine or guanine, and the product yield was depend-
ent on the amount of nitryl chloride. Both 8NX and 8NG
were formed in reaction of dG with nitryl chloride. On
the other hand, only 8NG, but not 8NX was detected in
reaction of dG or DNA with peroxynitrite. Molar yields
of 8NX and 8NG in reaction of dG with nitryl chloride
of activated human neutrophils with dG (26), presumably
•
by NO
2
Cl or NO
2
formed via the one-electron oxidation
5
0 times in molarity were much higher than in DNA
of the nitrite ion by compound I or compound II of
peroxidases (42).
reaction with 250 times of nitryl chloride (Table 2). (The
adducts were merely detectable in reaction of DNA with
In the absence of an enzyme, our results demonstrate
the formation of 8NX, in addition to 8NG, as the major
nitration product in reaction of 2′-deoxyguanosine or calf
thymus DNA with nitryl chloride, generated in vitro from
sodium nitrite and sodium hypochlorite under neutral
pH. 8-Nitroxanthine was also detected in xanthine reac-
tion with various reactive nitrogen species, including
nitryl chloride, peroxynitrite, nitronium tetrafluorobo-
rate, and heated nitric or nitrous acid. Multiple lines of
experiments suggested that the reaction pathway of
forming 8NX was independent of 8NG formation. First,
addition of excess nitryl chloride to purified 8NG pro-
duced negligible amount of 8NX. Second, a much higher
yield of 8NX was obtained in reaction of nitrous acid with
guanine as compared to the same reaction with xanthine,
indicating that formation of the diazonium ion at the N2-
amino group of guanine may play a key role to enhance
the nitration yield at the C8 position. Another supple-
mentary support can be rendered by the ratio of 8NX
versus 8NG in nitryl chloride-treated DNA, which was
lower than that in nitryl chloride reaction with dG,
suggesting that the Watson-Crick type hydrogen bond-
ings of guanine in DNA hindered the diazonium ion
formation to yield 8NX. Furthermore, yield of 8NX in
nitryl chloride-treated dG or DNA exceeded that of 8NG,
while xanthine was not detectable in the reaction mix-
tures. Collectively, these results indicate that formation
of 8NX in nitryl chloride-treated dG or DNA is not
through the intermediacy of 8NG, but rather the forma-
tion of 8NX and 8NG is ascribed to two competing
processes.
5
0 times of nitryl chloride.) In both reactions, 8NX was
the major product, and the ratio of 8NX versus 8NG was
.0 and 2.3 in reaction of dG and DNA, respectively. The
5
ratio of 8NX versus 8NG indicated that hydrogen bond-
ings of guanine in DNA protected it from deamination.
Depurination of 8-nitro-2′-deoxyxanthosine in reaction of
dG with nitryl chloride, appeared to be very fast since
that the 8-nitro nucleoside was not detected even when
HPLC analysis was performed immediately after the
reaction.
Formation of xanthine was not detected in reaction of
dG with nitryl chloride nor in the hydrolysate of nitryl
chloride-treated DNA, indicating that formation of 8NX
in dG and in DNA was not through the intermediacy of
xanthine. When isolated 8NG was added excess nitryl
chloride, only very small amount (1.8%) of 8NX was
detected (data not shown), suggesting that nitryl chloride
is not a good agent for deamination. With the preferred
formation of 8NX over 8NG in reactions of nitryl chloride
with dG or DNA, the possibility that 8NX detected in
these reactions being the end product of 8NG deamina-
tion is ruled out.
Yield of 8NX in nitryl chloride-treated DNA was not
affected by time of acid (0.1 N HCl, 100 °C, 30-180 min)
or neutral thermal hydrolysis (0.1 M phosphate buffer,
pH 7.0, 100 °C, 1-24 h) (data not shown). These results
indicate that nitryl chloride is short-lived and that 8NX
is stable under these hydrolysis conditions. There were
more peaks in HPLC chromatogram of the DNA hydroly-
sate obtained after acid hydrolysis than neutral hydroly-
sis. The identity of 8NX in DNA hydrolysate and in dG
reaction was confirmed by coelution of fractions collected
from the reaction mixtures with 8NX synthesized from
xanthine reaction with peroxynitrite and analyzed by
reversed-phase HPLC with photodiode array detection.
In addition, the 8NX elution of these two reactions were
collected, reduced, and co-injected with standard 8AX
obtained from reduction of synthetic 8NX (Figure 5).
Dep u r in a tion of 8NX in DNA. The stability and half-
life of 8NX formed in nitryl chloride-treated calf thymus
DNA was investigated under physiological conditions.
When nitryl chloride-treated calf thymus DNA was
incubated at 37 °C, pH 7.0, it underwent spontaneous
A tentative mechanism proposed for the formation of
8NX and 8NG is depicted in Scheme 4. Upon reaction of
nitryl chloride with dG or the dG moiety in DNA, a
•
neutral radical, dG(-H) , is obtained through the one-
electron oxidation and simultaneous deprotonation at the
C-8 position. Additional nitryl chloride can attack this
radical at C-8, resulting in 8-nitro-dG which subsequently
depurinates to give 8NG. Alternatively, nitryl chloride
can react with the N-2 amino group forming diazonium
ion radical intermediate I. This intermediate I reacts
rapidly with another nitryl chloride at C-8 to form the
diazonium intermediate II, which is subsequently hy-
drolyzed to yield 8-nitro-2′-deoxyxanthosine and 8NX

8
-Nitroxanthine in Nitryl Chloride-Treated DNA
Chem. Res. Toxicol., Vol. 14, No. 5, 2001 543
F igu r e 5. 8NX in reaction of calf thymus DNA with nitryl chloride. (A) Hydrolysate of calf thymus DNA reaction with nitryl chloride.
Calf thymus DNA (2.0 mg in 2.0 mL 0.2 M potassium phosphate buffer, pH 7.0) was added 770 µL of a solution containing NaNO
0.58 M) and 372 µL of a solution of NaOCl (0.30 M). After vortexing for 2 min., DNA was precipitated, washed, and hydrolyzed with
2
(
acid as described under the Materials and Methods. (B) Reduction mixture of 8NX collected from hydrolysate of nitryl chloride-
treated DNA. (C) Co-injection of 8AX (48 ng) with the reduction mixture of 8NX collected from hydrolysate of nitryl chloride-treated
calf thymus DNA.
after depurination. Nitration of intermediate I to form
intermediate II is expected to be a highly exergonic, low
energy barrier process due to the possible resonance
induction via the electron-withdrawing group (i.e., the
diazonium ion). Accordingly, the bimolecular rate con-
mediate I, i.e., d[I]/dt ≈ 0, while the k
2
and k
3
are rate
•
determining steps for the decomposition of dG(-H) . In
reaction of dG, the yield of 8NX is much greater than
8NG, indicating k
the rate of formation of intermediate I, k
3
> k
2
. Whereas in reaction of DNA,
, is relatively
3
stant k
intermediate II should be much greater than k
resulting in a steady-state approximation for the inter-
4
for the decomposition of intermediate I forming
slow due to the hydrogen bonding formation at the N-2
amino position, resulting in an increase of the relative
yield of 8NG. Since intermediate I is likely to reach a
2
and k
3
,

5
44 Chem. Res. Toxicol., Vol. 14, No. 5, 2001
Chen et al.
F igu r e 6. Release of 8NX from DNA under physiological conditions. Calf thymus DNA (1.0 mg/mL) was treated with nitryl chloride
at room temperature, followed by incubation at 37 °C, pH 7.0. At various time intervals, an aliquot (0.4 mL) of the reaction mixture
was removed, precipitated with cold ethanol, and washed twice with 70% cold ethanol. The ethanol solutions were combined and
evaporated for analysis of 8NX released in the medium. DNA was dissolved in phosphate buffer (pH 7.0) and hydrolyzed at 100 °C
for 3 h. ([) 8NX in DNA; (O) 8NX in the medium; (4) total 8NX. Results are expressed as mean ( SD of duplicate experiments.
Sch em e 4. A P r op osed Mech a n ism for th e F or m a tion of 8NX a n d 8NG
steady state due to the large bimolecular rate constant
, it qualitatively explains the fact that xanthine, its
reactive nitrogen species generated in vivo by phagocytes
at sites of inflammation.
k
4
hydrolysis product, was not detected in DNA hydrolysate.
Although nitryl chloride has weaker nitrating activity
toward phenolics, such as tyrosine and acetaminophen,
than peroxynitrite (43, 44), it might be significant in
nitration, chlorination, and oxidation of many biomol-
ecules, including nucleic acids, lipids, and sterols. Nitrite
ion, being a substrate and inhibitor of myeloperoxidase
(45), plays an important role in the antimicrobial activity
of reactive nitrogen species (46). Concentrations of nitrite
in extracellular fluids vary between sub-micromolar in
plasma to ca. 200 µM in saliva, which can be increased
The glycosidic linkage of 8NX in DNA is more labile
than that of 8NG. Once formed in DNA, 8NX underwent
spontaneous depurination with a half-life of 2 h when
incubated under physiological conditions. Therefore, 8NX
might be a more mutagenic lesion than 8NG in forming
apurinic sites since the half-life of 8NG in peroxynitrite-
treated calf thymus DNA was 4 h (21). Although the role
of 8NX in carcinogenesis remains to be determined, it
might be an important DNA damage due to nitration by

8
-Nitroxanthine in Nitryl Chloride-Treated DNA
Chem. Res. Toxicol., Vol. 14, No. 5, 2001 545
to millimolar range after intake of nitrate from diet (47)
or due to inflammation (48, 49). The elevated level of
nitrite during inflammation appears to be a result of
overproduction of NO by inducible nitric oxide synthase
induced alterations in synaptosomal membrane proteins: insight
into oxidative stress in Alzheimer’s disease. J . Neurochem. 72,
3
10-317.
(
17) Pennathur, S., J ackson-Lewis, V., Przedborski, S., and Heinecke,
J . W. (1999) Mass spectrometric quantification of 3-nitrotyrosine,
ortho-tyrosine, and o,o′-dityrosine in brain tissue of 1-methyl-4-
phenyl-1,2,3,6-tetrahydropyridine-treated mice, a model of oxida-
tive stress in Parkinson’s disease. J . Biol. Chem. 274, 34621-
(50). Since at least 28% of the oxygen consumed by
human neutrophils is converted to HOCl (51, 52), pro-
duction of nitryl chloride in vivo is highly plausible when
HOCl meets nitrite in the biological fluids (53). Therefore,
increased levels of both HOCl and nitrite during inflam-
mation might contribute significantly to endogenous
nitration of biomolecules. Detection of nitrated and
chlorinated tyrosine residues of protein in phagocyte
activation of human neutrophils (54, 55) indicated that
elevated production of NO and HOCl at inflammatory
sites and that nitryl chloride is at least one of the reactive
species (56). Thus, 8NX and 8NG may be useful bio-
markers for the exposure of DNA to endogenous nitrating
species, including nitryl chloride and peroxynitrite.
3
4628.
(18) Aoyama, K., Matsubara, K., Fujikawa, Y., Nagahiro, Y., Shimizu,
K., Umegae, N., Hayase, N., Shiono, H., and Kobayashi, S. (2000)
Nitration of manganese superoxide dismutase in cerebrospinal
fluids is a marker for peroxynitrite-mediated oxidative stress in
neurodegenerative diseases. Ann. Neurol. 47, 524-527.
(19) J uedes, M. J ., and Wogan, G. N. (1996) Peroxynitrite-induced
mutation spectra of pSP189 following replication in bacteria and
in human cells. Mutat. Res. 349, 51-61.
20) 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.
21) 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,
207-210.
Ack n ow led gm en t. This work was supported by
grants from National Science Council of Taiwan and from
National Chung Cheng University (to H.-J .C.C.). The
authors thank Ms. May-J in Lee (NSYSU) for performing
the LDI mass spectrometry experiments.
(
22) 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.
23) 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.
(
Refer en ces
(
(
(
24) Muller, T., Haussmann, H. J ., and Schepers, G. (1997) Evidence
for peroxynitrite as an oxidative stress-inducing compound of
aqueous cigarette smoke fractions. Carcinogenesis 18, 295-301.
25) Pryor, W. A., and Stone, K. (1993) Oxidants in cigarette smoke.
Radicals, hydrogen peroxide, peroxynitrate, and peroxynitrite.
Ann. N. Y. Acad. Sci. 686, 12-27.
(
1) Ohshima, H., and Bartsch, H. (1994) Chronic infections and
inflammatory processed as cancer risk factors: possible role of
nitric oxide in carcinogenesis. Mutat. Res. 305, 253-264.
2) Gordon, L. I., and Weitzman, S. A. (1993) Inflammation and
Cancer. Cancer J . 6, 257-261.
(
(
26) Byun, J ., Henderson, J . P. Mueller, D. M., and Heinecke, J . W.
3) Koppenol, W. H., Moreno, J . J ., Pryor, W. A., Ischiropoulos, H.,
and Beckman, J . S. (1992) Peroxynitrite, a cloaked oxidant formed
by nitric oxide and superoxide. Chem. Res. Toxicol. 5, 834-842.
4) Ischiropoulos, H., Zhu, L., and Beckman, J . S. (1992) Peroxynitrite
formation from macrophage-derived nitric oxide. Arch. Biochem.
Biophys. 298, 446-451.
(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.
(
(
(
(
(
(
(
27) 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.
5) Lymar, S. V., and Hurst, J . K. (1995) Rapid reaction between
peroxynitrite ion and carbon dioxide: Implications for biological
activity. J . Am. Chem. Soc. 117, 8867-8868.
6) Lymar, S. V., and Hurst, J . K. (1996) Carbon Dioxide: Physi-
ological Catalyst for Peroxynitrite-Mediated Cellular Damage or
Cellular Protectant? Chem. Res. Toxicol. 9, 845-850.
7) Lymar, S. V., J iang, Q., and Hurst, J . K. (1996) Mechanism of
carbon dioxide-catalyzed oxidation of tyrosine by peroxynitrite.
Biochemistry 35, 7855-7861.
8) Lemercier, J . N., Squadrito, G. L., and Pryor, W. A. (1995) Spin
trap studies on the decomposition of peroxynitrite. Arch. Biochem.
Biophys. 321, 31-39.
9) Beckman, J . S., Beckman, T. W., Chen, J ., Marshall, P. A., and
Freeman, B. (1990) Apparent hydroxyl radical production by
peroxynitrite: Implications for endothelial injury from nitric oxide
and superoxide. Proc. Natl. Acad. Sci. U.S.A. 87, 1620-1624.
(
28) Schmitt, D., Shen, Z., Zhang, R., Colles, S. M., Wu, W., Salomon,
R. G., Chen, Y., Chisolm, G. M., Hazen, S. L. (1999) Leukocytes
utilize myeloperoxidase-generated nitrating intermediates as
physiological catalysts for the generation of biologically active
oxidized lipids and sterols in serum. Biochemistry 38, 16904-
1
6915.
(
29) Suzuki, T., Yamada, M., Kanaori, K., Tajima, K., Morii, T., and
Makino, K. (1999) Formation of 8-nitroguanine from 2′-deoxygua-
nosine by NO/O
2
system. Nucleic Acids Symp. Ser. 42, 155-156.
(
30) Shapiro, R., and Pohl, S. H. (1968) The reaction of ribonucleosides
with nitrous acid. Side products and kinetics. Biochemistry 7,
4
48-455.
(
(
(
10) Marla, S. S., Lee, J ., and Groves, J . T. (1997) Peroxynitrite rapidly
(31) Nelson, D. P., and Kiesow, L. A. (1972) Enthalpy of decomposition
of hydrogen peroxide by catalase at 25 degrees C. Anal. Biochem.
49, 474-478.
permeates phospholipid membranes. Proc. Natl. Acad. Sci. U.S.A.
9
4, 14243-14248.
11) Denicola, A., Souza, J . M., and Radi, R. (1998) Diffusion of
peroxynitrite across erythrocyte membranes. Proc. Natl. Acad.
Sci. U.S.A. 95, 3566-3571.
(32) Uppu, R. M., and Pryor, W. A. (1996) Biphasic synthesis of high
concentrations of peroxynitrite using water-insoluble alkyl nitrite
and hydrogen peroxide. Method Enzymol. 269, 322-329.
(33) Beckman, J . S., Chen, J ., Ischiropoulos, H., and Crow, J . P. (1994)
Oxidative chemistry of peroxynitrite. Methods Enzymol. 233,
229-240.
12) Spencer, J . P. E., Wong, J ., J enner, A., Aruoma, O. I., Cross, C.
E., and Halliwell, B. (1996) Base modification and strand break-
age in isolated calf thymus DNA and in DNA from human skin
epidermal keratinocytes exposed to peroxynitrite or 3-morpholi-
nosydnonimine. Chem. Res. Toxicol. 9, 1152-1158.
(34) Morris, J . C. (1966) The acid ionization constant of HOC1 from 5
to 35 degrees. J . Phys. Chem. 70, 3798-3805.
(
13) Douki, T., Cadet, J ., and Ames, B. N. (1996) An adduct between
peroxynitrite and 2′-deoxyguanosine: 4,5-dihydro-5-hydroxy-4-
(35) Sodum, R. S., Nie, G., and Fiala, E. S. (1993) 8-Aminoguanine:
a base modification produced in rat liver nucleic acids by the
hepatocarcinogen 2-nitropropane. Chem. Res. Toxicol. 6, 269-276.
(36) Yermilov, V., Rubio, 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,
2045-2050.
(37) Redemann, C. T., and Redemann, C. E. (1955) 5-Amino-2,3-
dihydro-1,4-phthalazinedione. Org. Synth. Coll. 3, 69-70.
(38) Wu, K. Y., Scheller, N., Ranasinghe, A., Yen, T. Y., Sangaiah, R.,
Giese, R, and Swenberg, J . A. (1999) A gas chromatography/
electron capture/negative chemical ionization high-resolution
(nitrosooxy)-2′-deoxyguanosine. Chem. Res. Toxicol. 9, 3-7.
(
14) Arteel, G. E., Kadiiska, M. B., Rusyn, I., Bradford, B. U., Mason,
R. P., Raleigh, J . A., and Thurman, R. G. (1999) Oxidative stress
occurs in perfused rat liver at low oxygen tension by mechanisms
involving peroxynitrite. Mol. Pharmacol. 55, 708-715.
15) Wu, G. S., Zhang, J ., and Rao, N. A. (1997) Peroxynitrite and
oxidative damage in experimental autoimmune uveitis. Invest.
Ophthalmol. Visual Sci. 38, 1333-1339.
(
(
16) Koppal, T., Drake, J ., Yatin, S., J ordan, B., Varadarajan, S.,
Bettenhausen, L., and Butterfield, D. A. (1999) Peroxynitrite-

5
46 Chem. Res. Toxicol., Vol. 14, No. 5, 2001
Chen et al.
mass spectrometry method for analysis of endogenous and exog-
enous N7-(2-hydroxyethyl)guanine in rodents and its potential
for human biological monitoring. Chem. Res. Toxicol. 12, 722-
(48) Leone, A. M., Francis, P. L., Rhodes, P., and Moncada, S. (1994)
A rapid and simple method for the measurement of nitrite and
nitrate in plasma by high performance capillary electrophoresis.
Biochem. Biophys. Res. Commun. 200, 951-957.
7
29.
(
39) Olah, G. A., Malhotra, and Narang, S. C. (1989) Nitration.
Methods and Mechanisms, p 92-132, VCH Publishers, New York.
40) Lewis, R. S., and Deen, W. M. (1994) Kinetics of the reaction of
nitric oxide with oxygen in aqueous solutions. Chem. Res. Toxicol.
(
49) Green, L. C., Wagner, D. A., Glogowski, J ., Skipper, P. L.,
Wishnok, J . S., and Tannenbaum, S. R. (1982) Analysis of nitrate,
(
15
nitrite, and [ N]nitrate in biological fluids. Anal. Biochem. 126,
1
31-138.
7
, 568-574.
(
50) Kharitonov, S. A., Chung, K. F., Evans, D., O’Connor, B. J ., and
Barnes, P. J . (1996) Increased exhaled nitric oxide in asthma is
mainly derived from the lower respiratory tract. Am. J . Respir.
Crit. Care Med. 153, 1773-1780.
(41) Lewis, R. S., Tannenbaum, S. R., and Deen, W. M. (1995) Kinetics
of N-nitrosation in oxygenated nitric oxide solutions at physi-
ological pH: Role of nitrous anhydride and effects of phosphate
and chloride. J . Am. Chem. Soc. 117, 3933-3939.
(
(
42) van der Vliet, A., Eiserich, J . P., Halliwell, B., and Cross, C. E.
(
51) Weiss, S. J ., Klein, R., Slivka, A., and Wei, M. (1982) Chlorination
of taurine by human neutrophils. Evidence for hypochlorous acid
generation. J . Clin. Invest. 70, 598-607.
(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.
43) Ohshima, H., Celan, I., Chazotte, L., Pignatelli, B., and Mower,
H. F. (1999) Analysis of 3-nitrotyrosine in biological fluids and
protein hydrolyzates by high-performance liquid chromatography
using a postseparation, on-line reduction column and electro-
chemical detection: Results with various nitrating agents. Nitric
Oxide 3, 132-141.
(
52) Foote, C. S., Goyne, T. E., and Lehrer, R. I. (1983) Assessment of
chlorination by human neutrophils. Nature 301, 715-716.
(53) Whiteman, M., Spencer, J . P., J enner, A., and Halliwell, B. (1999)
Hypochlorous acid-induced DNA base modification: potentiation
by nitrite: Biomarkers of DNA damage by reactive oxygen species.
Biochem. Biophys. Res. Commun. 257, 572-576.
(
44) Lakshmi, V. M., Hsu, F. F., Davis, B. B., and Zenser, T. V. (2000)
Nitrating reactive nitric oxygen species transform acetaminophen
to 3-nitroacetaminophen. Chem. Res. Toxicol. 13, 891-899.
45) van Dalen, C. J ., Winterbourn, C. C., Senthilmohan, R., and
Kettle, A. J . (2000) Nitrite as a substrate and inhibitor of
myeloperoxidase. Implications for nitration and hypochlorous acid
production at sites of inflammation. J . Biol. Chem. 275, 11638-
(
54) Hazen, S. L., Hsu, F. F., Mueller, D. M., Crowley, J . R., and
Heinecke, J . W. (1996) Human neutrophils employ chlorine gas
as an oxidant during phagocytosis. J . Clin. Invest. 98, 1283-1289.
(
(
55) Evans, T. J ., Buttery, L. D., Carpenter, A., Springall, D. R., Polak,
J . M., and Cohen, J . (1996) Cytokine-treated human neutrophils
contain inducible nitric oxide synthase that produces nitration
of ingested bacteria. Proc. Natl. Acad. Sci. U.S.A. 93, 9553-9558.
1
1644.
(
46) Klebanoff, S. J . (1993) Reactive nitrogen intermediates and
(56) Eiserich, J . P., Hristova, M., Cross, C. E., J ones, A. D., Freeman,
B. A., Halliwell, B., and van der Vliet, A. (1999) Formation of
nitric oxide-derived inflammatory oxidants by myeloperoxidase
in neutrophils. Nature 391, 393-397.
antimicrobial activity: Role of nitrite. Free Radical Biol. Med.
1
4, 351-360.
(
47) 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.
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