Inhibition of nitrous acid-dependent tyrosine nitration and DNA base deamination by flavonoids and other phenolic compounds

Article abstract of DOI:10.1021/tx980163p

Exposure of tyrosine or DNA bases to acidic nitrite at low pH results in the nitration of tyrosine and the formation of base deamination products, respectively. At pH 1, hypoxanthine and xanthine are formed from the deamination of adenine and guanine, respectively, whereas under the same conditions, uracil is not detected. The yield of 3-nitrotyrosine derived from interaction of equimolar nitrite and tyrosine at pHI is approximately 50% of that obtained from equimolar peroxynitrite-tyrosine interactions at pH 7.4. The ability of a range of plant phenolic constituents to prevent damage mediated by acidic nitrite was also examined in comparison with the activity of vitamin C. The epicatechin/gallate family of flavonols, constituents of green tea, red wine, etc., demonstrates the most extensive inhibitory properties against both tyrosine nitration and base deamination. The results also show that ascorbic acid is a poor inhibitor of nitration or deamination under acidic conditions such as those of the stomach. The ability of plant phenolics to scavenge reactive nitrogen species derived from acidic nitrite may contribute to the protective effects of tea polyphenols against gastric cancer.

Full text of DOI:10.1021/tx980163p

1
574
Chem. Res. Toxicol. 1998, 11, 1574-1579
In h ibition of Nitr ou s Acid -Dep en d en t Tyr osin e Nitr a tion
a n d DNA Ba se Dea m in a tion by F la von oid s a n d Oth er
P h en olic Com p ou n d s
†
‡
†
‡
Ceri Oldreive, Kaicun Zhao, George Paganga, Barry Halliwell, and
,
†
Catherine Rice-Evans*
International Antioxidant Research Centre at UMDS-Guy’s Hospital, London SE1 9RT, and
International Antioxidant Research Centre at King’s College, Manresa Road,
London SW3 6LX, U.K.
Received J uly 10, 1998
Exposure of tyrosine or DNA bases to acidic nitrite at low pH results in the nitration of
tyrosine and the formation of base deamination products, respectively. At pH 1, hypoxanthine
and xanthine are formed from the deamination of adenine and guanine, respectively, whereas
under the same conditions, uracil is not detected. The yield of 3-nitrotyrosine derived from
interaction of equimolar nitrite and tyrosine at pH 1 is approximately 50% of that obtained
from equimolar peroxynitrite-tyrosine interactions at pH 7.4. The ability of a range of plant
phenolic constituents to prevent damage mediated by acidic nitrite was also examined in
comparison with the activity of vitamin C. The epicatechin/gallate family of flavonols,
constituents of green tea, red wine, etc., demonstrates the most extensive inhibitory properties
against both tyrosine nitration and base deamination. The results also show that ascorbic
acid is a poor inhibitor of nitration or deamination under acidic conditions such as those of the
stomach. The ability of plant phenolics to scavenge reactive nitrogen species derived from
acidic nitrite may contribute to the protective effects of tea polyphenols against gastric cancer.
In tr od u ction
Exposure of humans to excess nitrite from the diet or
arising from overproduction of endogenous nitric oxide
e.g., at sites of chronic inflammation) may play a role in
The purpose of this study was to model events that
may occur in the human stomach after ingestion of
nitrite. We examined the ability of plant phenolics
(Figure 1) to prevent damage by acidic nitrite in vitro,
(
in comparison with that of vitamin C. Two aspects of
damage were examined: nitration of tyrosine and deami-
nation of DNA bases. Both might be involved in gastric
damage and cancer development (18, 19)
the etiology of cancers of the GI tract, such as stomach
cancer (1, 2). Overproduction of reactive nitrogen species
1
(
RNS) derived from nitrite, e.g., HNO
2
and N
2
O
3
, can
cause damage in several ways. For example, they can
lead to N-nitrosation of amines, nitration of aromatic
compounds, and deamination of DNA bases (3-10).
Exp er im en ta l Section
Many studies have shown that the phenolic constitu-
ents of the diet are efficient scavengers of reactive oxygen
species (11, 12) and reactive chlorine species (13), and
there is considerable interest in the possibility that they
can protect against cancer through such mechanisms.
However, they may also act by scavenging RNS. In
particular, several phenolic compounds are potent inhibi-
tors of the nitration of tyrosine in vitro by peroxynitrite
Ferulic acid and rutin (HPLC grade) were purchased from
Extrasynthese. Epicatechin, catechin (g98%), 3-nitrotyrosine,
caffeic acid, quercetin, gallic acid, 3,4-dihydroxyphenylacetic
acid, xanthine, hypoxanthine, and all DNA bases were from
Sigma. Epigallocatechin (EGC), epicatechin gallate (ECG), and
epigallocatechin gallate (EGCG) were gifts from Unilever
Research Laboratories (Colworth, U.K.). Ultrapure water (18.2
MΩ) was used throughout.
Nitr otyr osin e F or m a tion fr om Acid ic Nitr ite. The time
dependency of the reaction between tyrosine and acidic nitrite
was followed spectrophotometrically using a Beckman DU7500
spectrophotometer. Stock solutions of 3 mM tyrosine and
NaNO2 were prepared in pure water. Tyrosine (2.5 mL) was
(
(
14, 15), and they may also scavenge nitric oxide itself
16, 17). It is widely thought that ascorbic acid is also
an important agent protecting against RNS in vivo, since
it can, under certain circumstances, inhibit nitrosation
and nitration reactions (18), and inhibit nitration by
peroxynitrite (7).
2
added to a solution containing NaNO in 0.5 M HCl to a total
volume of 10 mL (final concentration of 750 µM) and the mixture
incubated at 37 °C. Aliquots (1 mL) were removed every hour
for 7 h for spectrophotometric analysis. A sample was removed
after 4.5 h for analysis by HPLC as described below.
*
To whom correspondence should be addressed: International
Antioxidant Research Centre, UMDS-Guy’s Hospital, St. Thomas’s
Street, London SE1 9RT, U.K. Phone: 0171-955 4240. Fax: 0171-955
In h ibition of Tyr osin e Nitr a tion . Stock solutions of 2.5
mM ascorbic acid and phenolics (ferulic acid, epicatechin,
catechin, EGC, ECG, and EGCG) were prepared in pure water.
Rutin (75 mM) was initially solubilized in DMSO prior to
dilution to 2.5 mM in pure water. The final concentration of
DMSO was 0.3%, and appropriate controls and blanks were
4
983.
†
UMDS-Guy’s Hospital.
‡
1
King’s College.
Abbreviations: RNS, reactive nitrogen species; EGC, epigallocat-
echin; ECG, epicatechin gallate; EGCG, epigallocatechin gallate;
DMSO, dimethyl sulfoxide.
1
0.1021/tx980163p CCC: $15.00 © 1998 American Chemical Society
Published on Web 11/25/1998

Inhibition of Nitrous Acid-Dependent Tyrosine Nitration
Chem. Res. Toxicol., Vol. 11, No. 12, 1998 1575
mM) for pH 3 and 7.4 or in sodium carbonate buffer (50 mM)
for pH 9. The bases were preincubated for 10 min at 37 °C,
and aqueous solutions of sodium nitrite (50 µL) were added,
making the final concentration range 0.05-3.2 mM. For studies
of the time course and pH dependence, a final nitrite concentra-
tion of 0.45 mM was used. After incubation for 1 h at 37 °C,
the mixture (0.5 mL) was neutralized with 1 M KOH (10 µL)
and then mixed with 0.5 mL of phosphate buffer (50 mM, pH
3
.0). This preparation was subjected to HPLC analysis.
HP LC An a lysis of Dea m in a t ed P r od u ct s of t h e F r ee
Ba ses. A Gynkotek HPLC system consisting of a model 480
solvent delivery pump, a GINA 50 autosampler, a UVD 340S
photodiode array UV detector, and an INTRO electrochemical
detector was used. The separation of DNA bases and their
deaminated products was achieved on a reverse phase Hypersil
1
8
C
column (250 mm × 4.6 mm, 5 µm) which was eluted at a
flow rate of 1 mL/min with an isocratic mobile phase, a
potassium phosphate buffer (50 mM, pH 3) containing 2 mM
triethylamine and 0.05 M EDTA. The deaminated products
hypoxanthine, uracil, and xanthine were detected by UV
spectroscopy at 250 and 265 nm, respectively. Xanthine was
also detected by the electrochemical detector at a potential of
0
.9 V.
In h ibition of th e Dea m in a tion of th e F r ee Ba ses. The
stock solutions of the phenolic compounds were prepared by
dissolving in water at a concentration of 20 mM, and then
dilution was carried out to obtain 10, 5, 2.5, and 1.25 mM
solutions. An aqueous solution of sodium nitrite (9 mM) was
used. To DNA base solutions (0.9 mL) were added the phenolics
(50 µL) to final concentrations of 1, 0.5, 0.25, 0.125, and 0.065
mM. The mixture was preincubated at 37 °C for 10 min, and
then sodium nitrite (50 µL) was added to the mixture to a final
concentration of 0.45 mM. After incubation for 1 h at 37 °C,
F igu r e 1. Structures of the phenolic compounds.
included. Each compound was added (final concentration of
0
(
.5 mL of the incubation mixture was neutralized with 1 M KOH
10 µL), mixed with 0.5 mL of phosphate buffer (50 mM, pH 3),
and subjected to HPLC analysis.
0
-400 µM) to a solution containing sodium nitrite and tyrosine
(400 µM final concentration for each) in 0.5 M HCl (final
concentration) to a total volume of 1 mL (final pH of <1). The
reaction was allowed to continue at 37 °C for 4.5 h and then
stopped by placing the mixture on ice. Each sample was diluted
Resu lts
2
-fold with the mobile phase prior to HPLC analysis.
HP LC An a lysis of In h ibition of Nitr otyr osin e F or m a -
tion . An isocratic reverse phase HPLC method was developed
on a Waters HPLC system for the analysis of tyrosine and
nitrotyrosine (calibration ranges of 25-200 and 2.5-15 µM,
respectively) and the phenolics, ferulic acid, epicatechin, cat-
echin, ECG, EGC, EGCG, and rutin (10-200 µM calibration
ranges). It consisted of an autosampler with a peltier temper-
ature controller, a model 626 pump with a model 600S control-
ler, a model 996 photodiode array detector, and the Millennium
software system which controlled all the equipment and carried
Nitr a tion of Tyr osin e by Acid ic Nitr ite. Lowering
the pH of a sodium nitrite solution to <1 changes its
spectrum to give multiple peaks in the region of 357 nm,
consistent with the formation of nitrosating/nitrating
2
species arising from HNO . The interaction between
tyrosine at equimolar (400 µM) concentrations and acidi-
fied nitrite at 37 °C results in the formation of 3-nitro-
L-tyrosine, which has an enhanced absorbance at 357 nm.
The time course of this reaction demonstrates that
completion is reached after 4.5 h with a maximum yield
of 15 µM. No product is formed in the absence of acid.
When analyzed by HPLC, tyrosine and nitrotyrosine have
sharp reproducible peaks with retention times of 6 ( 0.2
and 8 ( 0.5 min, respectively.
1
8
out data processing. A Nova-Pak C column (4.6 mm × 250
mm) with a 4 µm particle size was used and the temperature
maintained by the column oven set at 30 °C. Injections (10 µL)
were made by an autosampler with a 100 µL fixed loop.
-
1
The solvent system (flow rate of 0.5 mL min ) consisted of
8% (v/v) solvent A [50 mM KCl and 7.8 mM HCl (pH 2.2) in
0% methanol] and 2% (v/v) solvent B (acetonitrile) for the
9
2
Dir ect Rea ction s of P h en olics w ith Acid ic Nitr ite.
Figure 2 shows the UV/visible spectra of epicatechin,
epigallocatechin (EGC), ferulic acid, and rutin in HCl and
the resulting changes in their absorbance spectra after
reaction with acidic nitrite. In the case of epicatechin,
after interaction for 10 min with acidic nitrite, a pro-
nounced shoulder appears at 350 nm on the 280 nm band.
On prolonged interaction, the shoulder disappears with
a concomitant further increase in absorbance at 280 nm.
Epigallocatechin responds in a similar way with a
dramatic transformation of the major shoulder at 280 nm
to a peak at 280 nm with a 4-fold increase in absorbance,
accompanied by a pronounced shoulder at 350 nm as for
epicatechin. The spectrum of pure rutin exhibits two
bands: band I with a peak at 350 nm due to the phenolic
phenolics studied, with the exception of rutin. Rutin was
analyzed by a linear gradient system where solvent A was held
at 98% for 10 min and then decreased linearly to 80% solvent
A at 15 min and held at these conditions for a further 20 min
before returning to 98% solvent A at 45 min, being held at these
conditions for a further 5 min. Detection was by diode array at
3
3
57 and 275 nm. Intra- and interassay variations were 0.4 and
.2% for tyrosine and 0.6 and 9.9% for nitrotyrosine, respec-
tively. None of the phenolics interfered with the HPLC analysis
of nitrotyrosine. Sinapic acid (2 µg/mL) was used as an external
standard.
In cu ba tion of F r ee Ba ses w ith Sod iu m Nitr ite. Solu-
tions of DNA bases were prepared by dissolving adenine,
guanine, and cytosine in 0.1 M HCl to a concentration of 1.4
mM. For the pH dependence study, the bases at the same
concentration were dissolved in potassium phosphate buffer (50

1
576 Chem. Res. Toxicol., Vol. 11, No. 12, 1998
Oldreive et al.
Ta ble 1. IC50 Va lu es of Dieta r y P h en olics w ith Resp ect
to Th eir Action on Acid ic Nitr ite-Med ia ted Tyr osin e
Nitr a tion a
compound
IC50b
rutin
ferulic acid
epigallocatechin
epicatechin
catechin
75.8 ( 23.6
73.0 ( 15.1
64.8 ( 12.8
50.2 ( 8.2
41.6 ( 3.7
38.4 ( 7.3
35.8 ( 9.0
epicatechin gallate
epigallocatechin gallate
a
Results are means ( SD. The concentration of tyrosine and
nitrite was 400 µM (for details, see the text). b IC50 is the
concentration (micromolar) of the compound giving 50% inhibition
of nitrotyrosine formation.
catechin completely suppressed tyrosine nitration, whereas
1
00 µM epicatechin or EGC decreased it by about 90%.
At this concentration, rutin and ferulic acid produced 55-
6
0% inhibition.
In all cases, HPLC analysis showed complete loss of
the native polyphenol in the mixture by incubation for
.5 h accompanied by the appearance of new peaks with
4
different absorbance spectra. In the case of ferulic acid,
a peak appears with a retention time of 16.7 min, the
absorbance of which increases as the initial ferulic acid
concentration increases. This was spectrally identical to
nitroferulic acid (15). With rutin a large number of new
bands appeared (1-12), with the absorbance increasing
as the initial rutin concentration increased. An unidenti-
fied peak appears in the solvent front with all the
catechins.
F igu r e 2. Spectral changes on the interaction between a 25
µM phenolic and 250 µM acidic nitrite at 37 °C: (1) epicatechin,
(
2) epigallocatechin, (3) rutin, and (4) ferulic acid at (s) 0, (- -)
0
.5, (---) 10, and (---) 270 min.
B ring and band II with a maximum at 270 nm repre-
senting the A ring (Figure 1). When band I is exposed
to acidic nitrite for 10 min, its intensity declines and its
profile changes, and on prolonged incubation for up to
The loss of the phenolics with the catechol structure
is consistent with inhibition of tyrosine nitration through
a mechanism of oxidation (15, 20).
Dea m in a tion of DNA Ba ses a n d Th eir In h ibition .
Sodium nitrite in acid (pH 1) caused deamination of
adenine and guanine, forming hypoxanthine and xan-
thine as measured by HPLC. Figure 4 shows a repre-
sentative separation. The extent of formation of xanthine
and hypoxanthine increased linearly in the range of
0.056-0.9 mM sodium nitrite (Figure 5). At equimolar
concentrations of free bases and sodium nitrite, about 3%
of the guanine and 0.4% of the adenine were converted
to their deaminated products, suggesting that the reac-
tion between acidified nitrite and guanine is favored over
that with adenine. No uracil, the deamination product
of cytosine, was detected until a very high concentration
of acidified nitrite (7 mM) was used, at which only about
0.01% of the cytosine was converted to uracil, suggesting
that cytosine is a poor substrate for deamination by
acidified nitrite. A time course study showed that the
deamination of the free bases was complete at about 2 h
for hypoxanthine and at about 4 h for xanthine (data not
shown).
The deamination of the free bases was pH-dependent.
About 30- and 2-fold more xanthine and hypoxanthine,
respectively, were formed at pH 1 than at pH 3, while
no deaminated products were detectable at pH >7.4.
Examination of the ability of phenolic compounds to
inhibit deamination showed that all were effective. As
indicated by the IC50 values (Table 2), catechin com-
pounds and caffeic acid exhibited a similar efficacy in
inhibiting the deamination of free bases by the acidified
nitrite with quercetin being significantly less effective.
However, the inhibitory effects of catechol, gallic acid,
and 3,4-dihydroxyphenylacetic acid (DHPAA) were found
2
70 min, the intensity of band I decreases further. For
band II, there is a transient small increase in the
absorbance at 10 min, which has returned to the original
position at 270 min. For ferulic acid, reaction with acidic
nitrite leads to the replacement of the main peak at 320
nm (with its characteristic shoulder at 290 nm) by a
single peak with a lower intensity at 295 nm, ac-
companied by the formation of a broad shoulder at 350
nm. Ascorbic acid also reacted directly with acidic nitrite,
resulting in the rapid (<1 min) loss of the peak at 245
nm.
In h ibition of Tyr osin e Nitr a tion . To investigate
the ability of the phenolics to inhibit nitrotyrosine
formation mediated by acidic nitrite, ferulic acid (4-
hydroxy-3-methoxycinnamic acid), rutin (quercetin-3-
rutinoside), epicatechin, catechin, EGC, ECG, and EGCG
(final concentrations of 0-400 µM) or ascorbic acid (400
µM) were added to nitrite/tyrosine solutions (final con-
centrations of 400 µM). HPLC analysis gave good base-
line separation (Figure 3) and reproducible retention
times for all the compounds tested; there was no inter-
ference with the HPLC detection of 3-nitrotyrosine. All
inhibited the formation of nitrotyrosine, the concentra-
tions of compound giving 50% inhibition (Table 1) being
in the following order of effectiveness: EGCG ≈ ECG ≈
catechin > epicatechin > EGC ≈ ferulic acid ≈ rutin.
Ascorbic acid had no inhibitory effect on tyrosine nitra-
tion by acidic nitrite up to concentrations of 400 µM. At
5
0 µM, EGCG and ECG exerted a 2-fold greater inhibi-
tory effect than ferulic acid and rutin. At 100 µM (25%
of that of the tyrosine substrate), EGCG, ECG, and

Inhibition of Nitrous Acid-Dependent Tyrosine Nitration
Chem. Res. Toxicol., Vol. 11, No. 12, 1998 1577
F igu r e 3. Chromatograms and the corresponding spectra of (A) standard solutions of tyrosine (400 µM), nitrotyrosine (30 µM), and
ferulic acid (400 µM) and (B and C) tyrosine (400 µM) and the resulting nitrotyrosine in the absence and presence of ferulic acid (150
µM), respectively, after exposure to acidic nitrite (400 µM) at 37 °C for 4.5 h.
F igu r e 5. Nitrite-induced conversion of adenine and guanine
to hypoxanthine and xanthine (for conditions, see the text).
F igu r e 4. Chromatogram of HPLC separation of DNA bases
for conditions, see the text).
(
Discu ssion
The nitrosating species arising from aqueous acidic
nitrite are depicted in the following equations (10):
to be much weaker, equally so for the inhibition of
xanthine and hypoxanthine formation.
NO2- + H a HNO
+
The effect of ascorbic acid on the deamination was also
investigated. Ascorbic acid did not inhibit the formation
of xanthine and hypoxanthine significantly. A weak
inhibitory effect on the deamination of free bases was
observed only when the concentration of ascorbic acid
used was raised to 1 mM, when about 40% inhibition was
achieved.
2
+
+
+
HNO + H a H NO a H O + NO
2
2
2
2
2
HNO a N O + H O
2
2
3
2
+
-
N O a NO -NO
2
2
3

1
578 Chem. Res. Toxicol., Vol. 11, No. 12, 1998
Oldreive et al.
Ta ble 2. IC50 Va lu es of th e P h en olics for th e In h ibition
of F or m a tion of Hyp oxa n th in e a n d Xa n th in e by th e
erential reaction with nitrite or nitrite-derived species
such as nitrosyl cation (NO ). Inhibition of nitrosation
+
Nitr ite-Dep en d en t Dea m in a tion of Ad en in e a n d Gu a n in e,
by the hydroxycinnamates, caffeic acid, ferulic acid, and
coffee (rich in caffeic acid esters) and tea (rich in catechin/
gallates) in vivo has also been demonstrated (28, 29).
Furthermore, tannins such as gallic acid (30) appear to
exert a protective effect against nitrosation. For example,
ferulic acid, a disubstituted monohydroxycinnamate,
decreases urinary N-nitrosoproline levels by 14-45% in
vivo in humans, suggesting inhibition of nitrosation (28,
a
Resp ectively
IC50 (µM)
compound
hypoxanthine
xanthine
epigallocatechin gallate
caffeic acid
catechin
epicatechin
epigallocatechin
quercetin
44.67 ( 10.56 31.19 ( 7.67
53.01 ( 2.15
67.22 ( 7.64
69.91 ( 6.20
73.97 ( 1.24
85.30 ( 2.49
62.91 ( 14.58
67.89 ( 2.82
64.98 ( 9.19
63.99 ( 2.48
75.67 ( 5.69
2
9). In addition, the related dihydroxycinnamate chlo-
rogenic acid (an ester of caffeic acid and quinic acid)
suppresses N-nitrosation (by nitrite in acidic solution) of
a model amine in vitro (31). Other dihydroxyphenols
such as dopamine, 3,4-dihydroxybenzoic acid, and cat-
echol also inhibit nitrosation, whereas monosubstituted
hydroxy compounds such as tyrosine, p-hydroxybenzoic
acid, salicylic acid, and p-hydroxyphenylacetic acid do not
(31).
catechol
gallic acid
144.14 ( 12.94 274.53 ( 3.40
150.52 ( 19.55 226.18 ( 4.25
3
,4-dihydroxyphenylacetic acid 171.90 ( 33.58 247.29 ( 11.46
a
Results are means ( SD.
Several phenolic antioxidants have been shown to de-
crease the extent of nitration of tyrosine by peroxynitrite
in vitro (14, 15) and that of nitrosation and nitration of
several amino acids in vivo (3). The results reported in
this study demonstrate that tyrosine is nitrated by nitrite
at acidic pH (<1) to form 3-nitrotyrosine. The yield of
The results described here show that ascorbic acid may
be a poor inhibitor of nitration or deamination by nitrite
under acidic conditions such as those of the stomach. In
neutral aqueous media, ascorbic acid reacts very slowly
with nitrite, forming dehydroascorbic acid via a nitrosa-
tion reaction (32). The rate-limiting step is the formation
of the nitrosating agent which is proposed to be either
3
-nitrotyrosine derived from equimolar interactions of
nitrite and tyrosine is about 8%, increasing to 14% with
a nitrite:tyrosine molar ratio of 2:1. Equivalent yields
of 3-nitrotyrosine from peroxynitrite-tyrosine inter-
actions at equimolar concentrations at pH 7.4 are re-
ported to be in the region of 14% (7). While the
concentration of nitrite (400 µM) is relatively high,
studies suggest that these levels can be achieved in
gastric juice derived from humans consuming 100 g of
spinach (which can contain up to 500 mg of nitrate)
+
N O or H NO (32). The reaction is highly pH-depend-
2
3
2
2
ent, suggesting that nitrite accelerates vitamin C decay
in fruit and vegetables under acidic storage conditions
(32). Dahn et al. (33) have observed that the rate of
reaction between nitrite and ascorbic acid is slow at
physiological concentrations of nitrite in acidic media due
to the formation of N O . It is also known that under
(
21, 22)
2
3
All the phenolic compounds tested were effective in
the acidic conditions of the stomach, and possibly in the
saliva, ascorbic acid can reduce nitrite to nitric oxide,
thus regenerating the nitrosating agent (33-35). The full
cycle involves reduction of nitrite to nitric oxide which
inhibiting acidic nitrite-induced tyrosine nitration, epi-
gallocatechin gallate, epicatechin gallate, epicatechin,
epigallocatechin, and catechin being almost maximally
effective at 25% of the tyrosine concentration and ferulic
acid and rutin at 50% of the tyrosine concentration. For
ferulic acid, the results suggest a competitive nitration,
the identification of nitroferulic acid being consistent with
the findings of Pannala et al. (15) in studies on peroxy-
nitrite-induced nitration of phenolics. Spectral studies
on the reactions of the phenolics with acidic nitrite
suggest that the mechanism of action with the catechin
family and rutin may proceed via oxidation of flavonols
and flavone in scavenging reactive nitrogen species,
whereas for the hydroxycinnamate, ferulic acid, nitration
is the mode of action (14, 15).
is then oxidized to NO2 and N O3 which regenerates
2
nitrite on hydrolysis (27).
Many studies have shown that nitric oxide reacts with
molecular oxygen to yield a variety of reactive nitrogen
2 2 3
species such as NO and N O (36). These species are
potent nitrosating agents and may react with phenolics
to form nitroso or nitrophenolics and with primary and
secondary aromatic amines to form potentially mutagenic
and carcinogenic nitrosamines or to cause deamination
of DNA bases via diazotization of the amino group
followed by hydrolysis (8, 9). It is known that acidified
+
nitrite can produce NO and N
2
O
3
. The deamination of
The results presented here also demonstrate that
acidified nitrite can cause damage to DNA bases, forming
xanthine and hypoxanthine from guanine and adenine,
respectively, and that this can be inhibited by plant
phenolics, epigallocatechin gallate again being the most
effective. These deamination products could conceivably
play an important role in mutagenic events in cells (18,
DNA bases by acidified nitrite may therefore share the
same reaction mechanism as proposed previously for NO
and its derived reactive nitrogen species, deamination
proceeding through the following potential mechanism:
+
+
+
ArNH + NO f ArN + H O f ArOH + N + H
2
2
2
2
1
9, 23). The deamination of cytosine and its mutagenic
+
-
ArNH + N O f ArN + NO2 + H O f
2
2
3
2
2
consequence have been extensively studied (19). How-
ever, in this study, cytosine was shown to be a poor
substrate for the acidified nitrite-mediated deamination.
This finding suggests that the genotoxcity caused by
nitrite or its derived reactive species may not involve
deamination of cytosine.
Ascorbic acid and R-tocopherol have been shown to
inhibit nitrosation in some human studies and animal
models (24-27), a suggested mechanism being by pref-
2
ArOH + HNO + N2
The ability of plant phenolics to scavenge reactive
nitrogen species may account for the protective effects
of tea polyphenols against gastric cancer (37, 38), and
indeed, the studies of Kuenzig et al. (39) suggest that
dietary hydroxycinnamates (constituents of fruit and
grains) may play a role in the body’s defense against

Inhibition of Nitrous Acid-Dependent Tyrosine Nitration
Chem. Res. Toxicol., Vol. 11, No. 12, 1998 1579
carcinogenesis by inhibiting the formation of N-nitroso
compounds.
(20) Kerry, N., and Rice-Evans, C. (1998) FEBS Lett. (in press).
(
21) McKnight, G. M., Smith, L. M., Drummond, R. S., Duncan, C.
W., Golden, M., and Benjamin, N. (1997) Chemical synthesis of
nitric oxide in the stomach from dietary nitrate in humans. Gut
40, 211-214.
Ack n ow led gm en t. We thank the Ministry of Agri-
culture, Fisheries and Food (U.K.) for research support
(22) Van Maanen, J . M. S., Pachen, D. M. F. A., Dallinga, J . W., and
Kleinjans, J . C. S. (1998) Formation of nitrosamines during
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of the use of mouthwashes. Cancer Detect. Prev. 22, 204-212.
(Grant FS1729) and Dr. G. Murphy (Department of
Gastroenterology, Guy’s Hospital) for his advice and
comments.
(
23) Suzuki, T., Matsumura, Y., Ide, H., Kanaori, K., Tajima, K., and
Makino, K. (1997) Deglycosylation susceptibility and base pairing
stability of 2′-deoxyoxanosine in oligodeoxynucleoside. Biochem-
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