Formation of N-nitrosamines and N-nitramines by the reaction of secondary amines peroxynitrite and other reactive nitrogen species: Comparison with nitrotyrosine formation

Article abstract of DOI:10.1021/tx990120o

Reactive nitrogen species, including nitrogen oxides (N₂O₃ and N₂O₄), peroxynitrite (ONOO^-), and nitryl chloride (NO₂Cl), have been implicated as causes of inflammation and cancer. We studied reactions of secondary amines with peroxynitrite and found that both N-nitrosamines and N- nitramines were formed. Morpholine was more easily nitrosated by peroxynitrite at alkaline pH than at neutral pH, whereas its nitration by peroxynitrite was optimal at pH 8.5. The yield of nitrosomorpholine in this reaction was 3 times higher than that of nitromorpholine at alkaline pH, whereas 2 times more nitromorpholine than nitrosomorpholine was formed at pH < 7.5. For the morpholine-peroxynitrite, reaction, nitration was enhanced by low concentrations of bicarbonate, but was inhibited by excess bicarbonate. Nitrosation was inhibited by excess bicarbonate. On this basis, we propose a free radical mechanism, involving one-electron oxidation by peroxynitrite of secondary amines to form amino radicals (R₂N·), which react with nitric oxide (·NO) or nitrogen dioxide (·NO₂) to yield nitroso and nitro secondary amines, respectively. Reaction of morpholine with NO· and superoxide anion (O₂·^-), which were concomitantly produced from spermine NONOate and by the xanthine oxidase systems, respectively, also yielded nitromorpholine, but its yield was < 1% of that of nitrosomorpholine. NO· alone increased the extent of nitrosomorpholine formation in a dose-dependent manner, and concomitant production of O₂·^- inhibited its formation. Reactions of morpholine with nitrite plus HOCl or nitrite plus H₂O₂, with or without addition of myeloperoxidase or horseradish peroxidase, also yielded nitration and nitrosation products, in yields that depended on the reactants. Tyrosine was nitrated easily by synthetic peroxynitrite, by NaNO₂ plus H₂O₂ with myeloperoxidase, and by NaNO₂ plus H₂O₂ under acidic conditions. Nitrated secondary amines, e.g., N-nitroproline, could be identified as specific markers for endogenous nitration mediated by reactive nitrogen species.

Full text of DOI:10.1021/tx990120o

Chem. Res. Toxicol. 2000, 13, 301-308
301
F or m a tion of N-Nitr osa m in es a n d N-Nitr a m in es by th e
Rea ction of Secon d a r y Am in es w ith P er oxyn itr ite a n d
Oth er Rea ctive Nitr ogen Sp ecies: Com p a r ison w ith
Nitr otyr osin e F or m a tion
Mitsuharu Masuda,†,‡ Howard F. Mower, Brigitte Pignatelli, Irena Celan,
†,§
†
†
|
‡
,†
Marlin D. Friesen, Hoyoku Nishino, and Hiroshi Ohshima*
Unit of Endogenous Cancer Risk Factors, International Agency for Research on Cancer,
Lyon, France, Department of Biochemistry, Kyoto Prefectural University of Medicine,
Kyoto, J apan, Department of Biochemistry and Biophysics, J ohn A. Burns School of Medicine,
University of Hawaii at Manoa, Honolulu, Hawaii 96822, and Unit of Gene Environment
Interactions, International Agency for Research on Cancer, Lyon, France
Received J uly 7, 1999
2 3 2 4
O and N O
), peroxynitrite (ONOO-),
Reactive nitrogen species, including nitrogen oxides (N
and nitryl chloride (NO Cl), have been implicated as causes of inflammation and cancer. We
2
studied reactions of secondary amines with peroxynitrite and found that both N-nitrosamines
and N-nitramines were formed. Morpholine was more easily nitrosated by peroxynitrite at
alkaline pH than at neutral pH, whereas its nitration by peroxynitrite was optimal at pH 8.5.
The yield of nitrosomorpholine in this reaction was 3 times higher than that of nitromorpholine
at alkaline pH, whereas 2 times more nitromorpholine than nitrosomorpholine was formed at
pH <7.5. For the morpholine-peroxynitrite reaction, nitration was enhanced by low concentra-
tions of bicarbonate, but was inhibited by excess bicarbonate. Nitrosation was inhibited by
excess bicarbonate. On this basis, we propose a free radical mechanism, involving one-electron
•
oxidation by peroxynitrite of secondary amines to form amino radicals (R
2
N ), which react with
•
•
nitric oxide ( NO) or nitrogen dioxide ( NO
2
) to yield nitroso and nitro secondary amines,
respectively. Reaction of morpholine with NO and superoxide anion (O
•
•-
2
), which were
concomitantly produced from spermine NONOate and by the xanthine oxidase systems,
respectively, also yielded nitromorpholine, but its yield was <1% of that of nitrosomorpholine.
•
NO alone increased the extent of nitrosomorpholine formation in a dose-dependent manner,
•-
and concomitant production of O
plus HOCl or nitrite plus H , with or without addition of myeloperoxidase or horseradish
peroxidase, also yielded nitration and nitrosation products, in yields that depended on the
reactants. Tyrosine was nitrated easily by synthetic peroxynitrite, by NaNO plus H with
myeloperoxidase, and by NaNO plus H under acidic conditions. Nitrated secondary amines,
2
inhibited its formation. Reactions of morpholine with nitrite
2
O
2
2
2 2
O
2
2 2
O
e.g., N-nitroproline, could be identified as specific markers for endogenous nitration mediated
by reactive nitrogen species.
with oxygen and superoxide (O
2
•-), respectively. Recent
In tr od u ction
studies have demonstrated that RNS are also formed by
the reaction of nitrite and hypochlorous acid (HOCl), the
•
Nitric oxide (NO ), produced by nitric oxide synthase,
is an important signal molecule that regulates various
physiological functions in the cardiovascular, nervous,
2 2
latter being generated from hydrogen peroxide (H O )
-
and chloride ion (Cl ) by myeloperoxidase (MPO) (3).
•
and immune systems (1, 2). Excess production of NO ,
Furthermore, oxidases such as MPO and horseradish
peroxidase (HPO) can form RNS in the presence of H O
2 2
and nitrite (4, 5).
RNS can react with biomolecules such as proteins,
DNA, and lipids through nitration and nitrosation reac-
tions, thus altering their functions. The presence of
however, has been implicated as a cause of diverse patho-
physiological conditions such as inflammation, neurode-
generative and cardiovasular diseases, and cancer. These
detrimental effects of NO have been attributed to reac-
tive nitrogen species (RNS) such as NOx and peroxyni-
•
1
-
•
trite (ONOO ), which are formed by the reaction of NO
3
-nitrotyrosine (NTYR) in human tissues and body fluids
has been used as a marker of peroxynitrite formation in
vivo, because peroxynitrite is a strong nitrating agent
*
To whom correspondence should be addressed: International
Agency for Research on Cancer, 150 Cours Albert Thomas, 69372 Lyon
Cedex 08, France. Fax: 33 4 72 73 80 88. Telephone: 33 4 72 73 85
0
9. E-mail: Ohshima@iarc.fr.
Unit of Endogenous Cancer Risk Factors, International Agency
†
1
Abbreviations: carboxy-PTIO, 2-(4-carboxyphenyl)-4,5-dihydro-
for Research on Cancer.
4,4,5,5-tetramethyl-1H-imidazol-1-yloxy 3-oxide, potassium salt; DTPA,
diethylenetriaminepentaacetic acid; HPO, horseradish peroxidase;
MPO, myeloperoxidase; NO-MOR, N-nitrosomorpholine; NO -MOR,
2
N-nitromorpholine; NTYR, 3-nitrotyrosine; RNS, reactive nitrogen
‡
Kyoto Prefectural University of Medicine.
University of Hawaii at Manoa.
§
|
Unit of Gene Environment Interactions, International Agency for
Research on Cancer.
species; TEA, thermal energy analyzer.
1
0.1021/tx990120o CCC: $19.00 © 2000 American Chemical Society
Published on Web 03/25/2000

3
02 Chem. Res. Toxicol., Vol. 13, No. 4, 2000
Masuda et al.
that very easily nitrates some aromatic compounds,
including tyrosine (6, 7). However, other RNS, such as
no further increase or decrease in yields of the products was
observed. On this basis, the 15 min incubation time was used
for all reactions with peroxynitrite. The pH values were adjusted
with HCl or NaOH. The reaction was initiated by adding
peroxynitrite to the mixture, and the final pH was read using
an aliquot of the mixture. The effect of morpholine and peroxy-
nitrite concentrations was studied similarly by reacting 1 mL
of 50 mM sodium phosphate buffer (pH 7.5 or 9.5) containing
0.05 mM DTPA, morpholine (0-10 mM), and peroxynitrite (0-
those formed from nitrous acid, NaNO
2
plus HOCl, and
NaNO plus H in the presence of MPO and HPO, can
2
2 2
O
react with tyrosine to form NTYR (3-5, 8, 9). The relative
importance of different RNS in NTYR formation in vivo
has not been elucidated.
Carcinogenic nitrosamines have been reported to be
•
•
formed by NO , which is produced by inducible NO
2
10 mM). The effect of CO was studied using 1 mM morpholine,
synthase expressed in cultured cells, including macro-
phages, hepatocytes, and neutrophils, upon activation
with lipopolysaccharide and γ-interferon (10-15). In-
creased endogenous nitrosation has been also demon-
strated in humans with chronic infection and inflamma-
tory conditions that have been associated with an
1 mM peroxynitrite, and 0-30 mM sodium bicarbonate in 50
mM sodium phosphate buffer containing 0.05 mM DTPA (pH
•
•
7
.5 or 9.5). Similarly, HO scavengers, antioxidants, an NO
scavenging compound (carboxy-PTIO), and methylurea (final
concentrations of 1 and 10 mM) were examined for their effects
on nitrosation and nitration of morpholine (1 mM) by peroxy-
nitrite (1 mM) at pH 7.5. Various amines and tyrosine (1 mM)
were also reacted with 1 mM peroxynitrite in 50 mM sodium
phosphate buffer and 0.05 mM DTPA (pH 7.5 or 9.5). Reverse
order-of-addition control experiments were performed with
peroxynitrite added to buffer first, followed 5 min later by
amines.
•
increased risk for cancer at various sites (16). NO and
reactive oxygen species produced in infected and/or
inflamed tissues could contribute to the process of car-
cinogenesis by several mechanisms, including the for-
•
mation of carcinogenic nitrosamines from excess NO
•
•
Rea ction of Mor p h olin e w ith NO• a n d O •-. Reactions
(
16-18). NO is oxidized by oxygen to NO
2
which reacts
to form the nitrosating agents
2 4
O , respectively. However, it has not been
2
•
•
further with NO and NO
and N
2
were carried out at 37 °C for 3 h in 50 mM sodium phosphate
buffer, containing 0.05 mM DTPA, 1 mM morpholine, spermine
N
2
O
3
•
-
NONOate (prepared in 0.01 N NaOH), an O
system (xanthine oxidase with either 200 µM acetaldehyde or
00 µM hypoxanthine as a substrate), and an appropriate
2
generation
determined whether peroxynitrite and other RNS react
with amines to yield carcinogenic N-nitroso compounds.
In the study presented here, we found that both
nitrosamines and nitramines are formed by the reaction
of secondary amines with peroxynitrite and other RNS.
On this basis, we propose that nitrated secondary amines,
e.g., N-nitroproline, could be identified as a new and
specific marker for endogenous nitration mediated by
peroxynitrite and other RNS.
2
amount of HCl to neutralize the NaOH in the spermine
NONOate solution (final volume of 1 mL and final pH of 7.5)
(
21-24). The final concentrations of spermine NONOate and
xanthine oxidase were 0-800 µM and 0-800 milliunits/mL,
respectively. Under these conditions, NO and O
•
•-
2
were gener-
ated at rates of 0-16 nmol/min, which were measured spectro-
photometrically as described previously (24, 25).
Rea ction of Mor p h olin e w ith Na NO
Na NO p lu s H . Reactions were carried out at 37 °C for 3 h
by incubating 1 mM morpholine in 50 mM sodium phosphate
buffer (final pH of 7.5) containing 0.05 mM DTPA, NaNO (0-5
mM), and HOCl (0-5 mM). Other reactions were carried out at
7 °C for 3 h by incubating 1 mM morpholine in sodium
phosphate buffer (pH 3.5 or 4.5) containing 0.05 mM DTPA, 1
mM NaNO , and 1 mM H . In some experiments, HPO (1 µM)
or MPO (80 nM) was incubated in the reaction mixture (pH 7.5)
containing 1 mM morpholine, 1 mM NaNO , and 1 mM HOCl
or H
2
p lu s HOCl or
2
2 2
O
Ma ter ia ls a n d Meth od s
Ca u tion : Many nitrosamines and nitramines are carcinogenic
in experimental animals and should be handled with caution.
All reactions were carried out at 37 °C and at least in
duplicate.
2
3
2
2 2
O
Ch em ica ls. We purchased spermine NONOate and 2-(4-
carboxyphenyl)-4,5-dihydro-4,4,5,5-tetramethyl-1H-imidazol-1-
yloxy 3-oxide, potassium salt (carboxy-PTIO), from Cayman
Chemical Co. (Ann Arbor, MI). MPO (human leukocytes) were
from Alexis Co. (San Diego, CA), and xanthine oxidase was from
Boehringer Mannheim (Mannheim, Germany); desferrioxamine
was from Ciba-Geigy Laboratoire (Rueil-Malmaison, France),
and morpholine, aminopyrine, dimethylamine, diethylamine,
methylbenzylamine, proline, N-methylurea, and other chemicals
were from Sigma (St. Louis, MO), Aldrich (Milwaukee, WI), or
Merck (Darmstadt, Germany). Nitrosamines were purchased
from Sigma or synthesized in our laboratory by the reaction of
2
2
2
O .
An a lysis of Nitr osa m in es a n d Nitr a m in es by Ga s Ch r o-
m a togr a p h y Cou p led w ith a Th er m a l En er gy An a lyzer
1
(
GC-TEA). Reactions were terminated by adding
of 20% ammonium sulfamate in 3.6 N H SO
500 ng of N-nitrosodibutylamine in ethanol) was added to the
reaction mixture, which was then extracted three times with
an equal volume of CH Cl . The combined CH Cl extracts were
dried over anhydrous Na SO . In the case of reactions with
/10 volume
2
4
. Internal standard
(
2
2
2
2
2
4
proline, 500 ng of N-nitrosopipecolic acid in ethyl acetate was
added as an internal standard to the reaction mixture, which
was then extracted with ethyl acetate. Nitroso- and nitroproline
were converted to their methyl derivatives with diazomethane
before GC-TEA analysis (26).
secondary amines with sodium nitrite (NaNO
conditions. Nitramines were synthesized from corresponding
nitrosamines using 50% H (a gift from Elf Atochem, Pierre-
B e´ nite, France) and trifluoroacetic acid (19). The structures and
purity of the synthesized nitrosamines and nitramines were
verified by gas chromatography/mass spectrometry (GC/MS) (see
below). Peroxynitrite was synthesized in a quenched-flow reac-
2
) under acidic
2
O
2
Nitrosamines and nitramines were identified by GC-TEA
(model 546, Thermoelectron) (15). A glass column (3 mm in
diameter × 2 m in length), packed with 5% FFAP on chromosorb
W-HP (80-100 mesh), was used with a carrier gas, argon, at a
flow rate of 30 mL/min. The oven temperature was 120-190
2 2
tor, and excess H O was destroyed by granular manganese
dioxide (20).
Rea ction of Secon d a r y Am in es w ith P er oxyn itr ite.
Peroxynitrite solution was prepared in cold 0.5 N NaOH. The
effect of pH was studied by incubating 1 mL of 50 mM sodium
phosphate buffer (pH 3-12) containing 0.05 mM diethylenetri-
aminepentaacetic acid (DTPA, a metal chelator), 1 mM mor-
pholine, and 1 mM peroxynitrite for 15 min. We examined the
effect of incubation time on nitrosation and nitration of mor-
pholine by peroxynitrite. At both pH 7.4 and 9.5, the reactions
were complete within 2 min after addition of peroxynitrite and
°
C, depending on the nitrosamine that was analyzed, and
injection port temperature was set 50 °C higher than that of
the oven temperature. The TEA pyrolyzer temperature was 700
°
C. Under these conditions, not only nitrosamines but also
nitramines could be analyzed by the TEA (8).
GC/MS An a lysis. Nitrosated and nitrated products were
analyzed on an HP-1 column (cross-linked methyl siloxane, 25
m in length × 0.18 mm in diameter, 0.18 µm film thickness)

Reactions of Reactive Nitrogen Species and Amines
Chem. Res. Toxicol., Vol. 13, No. 4, 2000 303
F igu r e 1. Typical GC-TEA chromatogram from the analysis
of reaction products of morpholine with peroxynitrite. Peak 1
is N-nitrosodibutylamine (added as an internal standard), and
peaks 2 and 3 are NO-MOR and NO -MOR, respectively, which
2
are formed by the reaction of morpholine with peroxynitrite.
F igu r e 2. (A) Effect of pH on the yields of NO-MOR (O) and
using a Hewlett-Packard 5890 gas chromatograph with a mass-
selective detector (model 5971A). The GC conditions were as
follows: injection port temperature, 250 °C; detector tempera-
ture, 250 °C; interface temperature, 280 °C; GC oven temper-
ature, 50 °C for 1 min, up to 230 °C at a rate of 20 °C/min; and
splitless injection.
NO -MOR (b) in the peroxynitrite-morpholine reaction. (B)
2
Yield of N-nitrosodimethylamine for the reaction of aminopyrine
with peroxynitrite (4) and decomposed peroxynitrite (9).
An a lysis of NTYR by HP LC. Reactions were carried out
using L-tyrosine and various RNS. NTYR was analyzed by a
Spectraphysics HPLC system (model SP 8810) equipped with a
reverse-phase column (4.6 mm × 250 mm Ultrasphere ODS
column, 5 µm, Beckman) under isocratic conditions with a
mobile phase of 15% methanol in 20 mM ammonium formate
buffer (pH 6.0) at a flow rate of 1 mL/min. NTYR was detected
by a Waters LC spectrophotometer (model 481) at 390 nm.
Resu lts
F or m a tion of Nitr osa m in es a n d Nitr a m in es by
th e Rea ction of Secon d a r y Am in es w ith P er oxyn i-
tr ite. Figure 1 shows a typical GC-TEA chromatogram
obtained from the analysis of reaction products of mor-
pholine with peroxynitrite. In addition to an internal
standard peak (N-nitrosodibutylamine, peak 1), two
peaks (peaks 2 and 3) were observed. Peak 2 coeluted
with authentic N-nitrosomorpholine (NO-MOR). Increase
in the pyrolyzer temperature of the TEA from 500 to 700
F igu r e 3. Effect of varying concentrations of (A) peroxynitrite
and (B) morpholine at pH 7.3 and (C) peroxynitrite and (D)
morpholine at pH 9.2 on the yields of NO-MOR (O) and
°
C increased the response for peak 3, suggesting that it
was an N- or C-nitro compound (8). The products were
analyzed by GC/MS. Peak 2 exhibited ions at m/z 116
2
NO -MOR (b).
(
relative intensity, 12%), 86 (14%), and 56 (54%), whereas
peak 3 exhibited ions at m/z 132 (3%), 86 (16%), and 56
74%). The mass spectra of peaks 2 and 3 were in good
agreement with those of authentic NO-MOR and N-
nitromorpholine (NO -MOR), respectively. As peroxyni-
ure 2B). Between pH 5 and 9.5, peroxynitrite reacted
with aminopyrine to form nitrosodimethylamine, whereas
decomposed peroxynitrite did not yield this compound.
Below pH 4.5, peroxynitrite and decomposed peroxyni-
trite yielded similar amounts of nitrosodimethylamine,
possibly due to acid-catalyzed nitrosation of aminopyrine
by nitrite present in peroxynitrite solutions.
(
2
trite is a strong oxidant, it is possible that nitrosamines
were oxidized to nitramines by peroxynitrite. We incu-
bated NO-MOR with peroxynitrite, but no NO
detected. On this basis, we concluded that NO
2
-MOR was
-MOR was
2
Increased concentrations of peroxynitrite dose-de-
pendently increased the yields of both NO-MOR and
formed by the reaction of morpholine with peroxynitrite.
Conditions for the reaction between secondary amines
and peroxynitrite were varied. Figure 2A shows the effect
of pH on this reaction. The NO-MOR yield was 3 times
NO
a plateau at 2 mM peroxynitrite, and only slight increases
in NO-MOR and NO -MOR yields were observed with >2
2
-MOR at neutral pH (Figure 3A); their yields reached
2
higher than that of NO
2
-MOR under alkaline conditions
mM peroxynitrite at alkaline pH (Figure 3C). When
increased concentrations of morpholine were reacted with
0.5 mM peroxynitrite, the NO-MOR yield increased dose-
dependently at both neutral and alkaline pH; however,
(
pH 8.5-10.5), whereas below pH 7.5, twice as much
2
NO -MOR as NO-MOR was formed (Figure 2A). Ami-
nopyrine reacted with peroxynitrite rapidly to form
nitrosodimethylamine but not nitrodimethylamine (Fig-
2
the yield of NO -MOR reached a maximum with 2 mM

3
04 Chem. Res. Toxicol., Vol. 13, No. 4, 2000
Masuda et al.
tions (pH 7.5). Uric acid [an antioxidant and free radical
scavenger (27)] and glutathione strongly inhibited the
formation of NO- and NO
2
-MOR, the 50% inhibitory
concentrations (IC50) being about 0.05 and 0.08 mM,
respectively. As shown in Figure 5, N-acetylcysteine,
ascorbic acid, selenomethionine, and desferrioxamine at
concentrations of 1 mM strongly inhibited nitration of
morpholine, but inhibited its nitrosation by only 60-80%.
•
In contrast, HO scavengers such as D-mannitol, dimethyl
sulfoxide, and ethanol inhibited nitration weakly and did
•
not inhibit nitrosation. Interestingly, the NO trapping
•
agent carboxy-PTIO (1 or 10 mM), which oxidizes NO
•
to NO
2
(28), enhanced nitrosation 8-fold, but completely
inhibited nitration. Similar stimulation of nitrosation in
the presence of carboxy-PTIO has been reported for the
S-nitrosation of glutathione by peroxynitrite (29). N-
Methylurea (10 mM), which may also be N-nitrosated or
N-nitrated by peroxynitrite, did not affect nitrosation, but
slightly (20-30%) inhibited nitration of 1 mM morpholine
by 1 mM peroxynitrite.
Rea ction of Mor p h olin e w ith NO a n d O
peroxynitrite is formed from NO and O
•
•-. As
, we examined
2
F igu r e 4. Effect of bicarbonate on the yield of NO-MOR (O)
and NO -MOR (b) at (A) pH 7.3 and (B) pH 9.5.
•
•-
2
2
whether concurrent production of these radicals has an
effect similar to that of peroxynitrite itself. We carried
morpholine, and no further increase was observed at
either neutral or alkaline pH (panels B and D of Figure
•
•-
out reactions of morpholine with NO and O
2
which
3
0
). Under neutral conditions (pH 7.3), the presence of
.1-3 mM NaHCO dose-dependently enhanced nitration
were generated concomitantly from spermine NONOate
and xanthine oxidase with acetaldehyde or hypoxanthine
3
•
-
of morpholine up to 3-fold, whereas concentrations of
NaHCO above 10 mM inhibited this reaction (Figure
A). The extent of NO-MOR formation was slightly in-
creased in the presence of low concentrations of NaHCO
as the substrate (21-24). When the rate of O
2
genera-
tion was kept constant at 4.4 nmol/min, the extent of
nitrosation of morpholine increased with increasing
amounts of spermine NONOate (Figure 6A). Under these
conditions, the extent of nitration was highest with a rate
3
4
3
(
1
0.1-3 mM), but was inhibited by concentrations above
•
0 mM. Under alkaline conditions (pH 9.5), NO-MOR
of NO release of 1.5-2 nmol/min, whereas increasing the
•
formation was inhibited dose-dependently by NaHCO
whereas NO
-3 mM NaHCO
Eight secondary and tertiary amines with different pK
3
,
NO flux above 4 nmol/min resulted in a decreased level
•
-MOR formation was enhanced 1.5-fold with
(Figure 4B).
of nitration (Figure 6B). In contrast, when the NO flux
2
•
-
1
3
was constant at 4.4 nmol/min, increasing the O
2
flux
inhibited the nitrosation of morpholine (Figure 6C). The
a
•
-
-
values were reacted with peroxynitrite under neutral and
alkaline conditions (Table 1). At pH 9.5, less basic amines
extent of nitration of morpholine was highest for an O
release rate of 4 nmol/min, whereas increasing the O
2
•
2
(
morpholine and aminopyrine), whose pK
a
values are
flux above 8 nmol/min resulted in a decreased nitration
level (Figure 6D). In general, yields of NO -MOR were
100 times lower than those of NO-MOR. The yield of
NO -MOR was also lower by 50-90% with hypoxanthine
as the substrate for xanthine oxidase than with acetal-
dehyde as the substrate, suggesting that the reaction was
inhibited by hypoxanthine and/or uric acid. Because
lower than 9.5, were more easily nitrosated than they
were nitrated, whereas more basic amines (pyrrolidine,
diethylamine, and dimethylamine), whose pK
higher than 9.5, were more easily nitrated than they were
nitrosated. Methylbenzylamine (pK ) 9.54) was nitro-
sated and nitrated to equal extents. At pH 7.4, the yields
of nitramines were about twice as high as those of
nitrosamines. In general, the sum of the amounts of
nitroso and nitro derivatives formed from the reactions
of secondary amines and peroxynitrite was inversely
related to the basicity of the amines that were tested,
2
a
values are
2
a
NaHCO
3
enhanced nitration of morpholine by peroxyni-
on the reac-
(each 4.4 nmol/
trite, we examined the effects of NaHCO
tion of morpholine with NO and O
3
•
•-
2
min generated from spermine NONOate and xanthine
oxidase/acetaldehyde). The presence of up to 30 mM
although the yields were not directly proportional to pK
a
NaHCO
inhibited nitration dose-dependently (about 10 and 50%
inhibition by 0.1 and 0.3 mM NaHCO , respectively, with
complete inhibition at >1 mM) (data not shown).
Effects of MP O a n d HP O on th e Rea ction of
3 3
had no effect on nitrosation. However, NaHCO
values. Exceptions were proline, triethylamine, and ami-
nopyrine. Proline was less easily nitrosated and nitrated
by peroxynitrite than were other secondary amines,
possibly because it posseses a carboxyl group that af-
fected the reactions. Triethylamine yielded more nitro-
diethylamine than nitrosodiethylamine under both neu-
tral and alkaline conditions. Aminopyrine yielded nitro-
sodimethylamine but not nitrodimethylamine.
3
Mor p h olin e w ith Na NO
2 2
p lu s HOCl or Na NO p lu s
2 2
H O
. Because it has been reported that nitrating agents
•
such as nitrogen dioxide ( NO
chloride (NO Cl) are formed by reactions of NaNO
2 2
H O or HOCl in the presence or absence of oxidases such
2
), peroxynitrite, and nitryl
2
2
with
Effects of Sca ven ger s, An tioxid a n ts, Ca r boxy-
P TIO, a n d N-Meth ylu r ea on F or m a tion of NO- a n d
as MPO and HPO (4, 5), we examined the reaction of
morpholine with these reagents. As summarized in Table
2, small amounts of NO-MOR were formed by the
2
NO -MOR by P er oxyn itr ite. We examined effects of
•
various hydroxyl radical ( HO) scavengers, antioxidants,
•
and an NO trapping agent on the reaction of morpholine
reaction of morpholine with NaNO
2 2 2
and H O under
(1 mM) with peroxynitrite (1 mM) under neutral condi-
neutral conditions. The yield increased 3- or 4-fold in the

Reactions of Reactive Nitrogen Species and Amines
Chem. Res. Toxicol., Vol. 13, No. 4, 2000 305
Ta ble 1. Rela tion sh ip betw een th e Ba sicity of Am in es (p Ka Va lu es) a n d F or m a tion of Th eir N-Nitr osa ted a n d N-Nitr a ted
P r od u cts by P er oxyn itr itea
yield of nitrosated and nitrated products (µM, mean ( SD)
pH 7.4
nitramine or NTYR
pH 9.5
nitramine or NTYR
amino compound
pKab
11.27
10.93
10.75
10.72
10.64 (NH)
9.54
8.7
5.04
nitrosamine
nitrosamine
pyrrolidine
0.63 ( 0.10
0c.69 ( 0.06
+
1.62 ( 0.22
0.61 ( 0.24
1.55 ( 0.23
2.49 ( 0.03
137 ( 12
1.48 ( 0.22
2.35 ( 0.27
0.08 ( 0.02
3.03 ( 0.27
0.10 ( 0.03
2.78 ( 0.32
5d.03 ( 0.31
1.81 ( 0.10
1.06 ( 0.17
0.08 ( 0.05
4.03 ( 2.20
1.86 ( 1.64
10.9 ( 4.6
64.8 ( 6.1
117 ( 5
5.45 ( 1.77
7.75 ( 0.57
0.28 ( 0.01
8.48 ( 0.52
0.31 ( 0.18
11.9 ( 1.4
8d.03 ( 1.75
-
diethylamine
triethylamine
dimethylamine
proline
methylbenzylamine
morpholine
aminopyrine
tyrosine
-
e
9.11 (OH)
66.8 ( 0.5e
46.5 ( 2.62
1
0.13 (NH2)
a
Reactions were carried out at 37 °C for 15 min by incubating 1 mL of 50 mM sodium phosphate buffer (pH 7.4 or 9.5) containing 0.05
b
mM DTPA, 1 mM amine, and 1 mM peroxynitrite. Values are the averages ( SD of triplicate analyses. pKa values were taken from refs
c
4
6 and 47. +, trace amounts (just above the detection limit). d -, not detected. e 3-Nitrotyrosine.
F igu r e 5. Effect of various scavengers and antioxidants on the
formation of NO-MOR (white bars) and NO -MOR (black bars)
2
mediated by peroxynitrite. Hydroxyl radical scavengers, anti-
•
oxidants, an NO scavenger (carboxy-PTIO), and other com-
pounds (final concentrations of 1 and 10 mM) were examined
for their effects on nitrosation and nitration of morpholine (1
mM) by peroxynitrite (1 mM) at pH 7.5. The yields (mean (
2
SD) of NO-MOR and NO -MOR in the absence of scavengers
(
controls) were 3.56 ( 0.52 and 9.63 ( 1.07 µM, respectively.
•
F igu r e 6. Formation of NO-MOR and NO
2
-MOR by NO and
Experiments were carried out in triplicate. A lack of bars
indicates that no nitrosation or nitration products were detected.
superoxide generated simultaneously at varying rates. NO-MOR
•
[
O and 4, formed by NO plus superoxide generated with
xanthine oxidase (XO) and hypoxanthine (Hyp) or acetaldehyde
Acet) as the substrate, respectively] or NO -MOR (b and 2,
formed by NO plus superoxide generated with XO and Hyp or
Acet as the substrate, respectively).
presence of HPO or MPO, respectively. NTYR formation
(
2
•
in the presence of NaNO
fold by MPO, whereas it was enhanced only 3-fold by
HPO. NO- and NO -MOR were also detected after the
reaction of morpholine with NaNO and HOCl. The pres-
ence of MPO or HPO decreased the extent of NO-MOR
formation by NaNO and HOCl (Table 2). As peroxyni-
trite may be formed from NaNO and H under acidic
conditions (20), we examined NO- and NO -MOR forma-
tion under acidic conditions (Table 3). Large amounts of
NO-MOR were formed with NaNO at pH 3.5, whereas
the presence of H inhibited nitrosation. In contrast,
NTYR formation was enhanced 2- and 7-fold at pH 3.5
2
and H
2
O
2
was enhanced 90-
2
more N-nitrosodimethylamine at neutral pH than at
alkaline pH. The optimal pH for morpholine nitration by
peroxynitrite was 8.5, whereas nitrosation occurred more
rapidly at alkaline pH than at neutral or acidic pH
Figure 2A). A similar pH effect on the nitration and
nitrosation of phenol by peroxynitrite has been reported
30). The yields of nitrosation and nitration products
formed by peroxynitrite were related to amine basicity.
More nitramines than nitrosamines were formed at
neutral pH (Table 1). Similarly at pH 9.5, more nitra-
mines than nitrosamines were formed in reactions of
peroxynitrite with secondary amines (pyrrolidine, dieth-
2
2
2
2 2
O
(
2
(
2
2
O
2
and 4.5, respectively, in the presence of H
relative to that in the presence of NaNO
2
O
2
and NaNO
2
,
2
alone.
ylamine, and dimethylamine), whose pK
a
values are
Discu ssion
higher than 9.5. Methylbenzylamine (pK ) 9.5) yielded
a
P er oxyn itr ite-Med ia ted Nitr osa tion a n d Nitr a -
tion of Secon d a r y Am in es. We have shown that
peroxynitrite reacts with secondary amines to form both
nitrosated and nitrated products (Table 1). In general,
yields of both products were higher at alkaline pH than
at neutral and acidic pH, except in the case of aminopy-
rine. This compound reacted with peroxynitrite to form
equal amounts of nitramine and nitrosamine, whereas
less basic amines (morpholine and aminopyrine), whose
pK values are lower than 9.5, were more easily nitro-
a
sated than nitrated. These results suggest that unpro-
tonated amines undergo nitrosation more easily than
nitration. However, under both alkaline and neutral
conditions, yields of nitrosamines and nitramines were

3
06 Chem. Res. Toxicol., Vol. 13, No. 4, 2000
Masuda et al.
Ta ble 2. Effect of Myelop er oxid a se (MP O) a n d Hor ser a d ish P er oxid a se (HP O) on Nitr osa tion a n d Nitr a tion of
Mor p h olin e a n d Tyr osin e by Na NO2 p lu s H2O2 or Na NO2 p lu s HOCla
yield of nitrosated and nitrated products (µM, mean ( SD)
NO-MOR
NO2-MOR
NTYR
with MPO (80 nM)
with HPO (1 µM)
no enzyme
NaNO2 + H2O2
NaNO2 + HOCl
NaNO2 alone
NaNO2 + H2O2
NaNO2 + HOCl
NaNO2 alone
NaNO2 + H2O2
NaNO2 + HOCl
NaNO2 alone
0.08 ( 0.01
0.07 ( 0.04
0.02 ( 0.01
0.06 ( 0.02
0.09 ( 0.02
0.02 ( 0.01
0.02 ( 0.01
0.16 ( 0.04
0.02 ( 0.01
0.002 ( 0.001
60.0 ( 1.00
0.55 ( 0.01
0
2.46 ( 0.13
1.24 ( 0.43
0
0.69 ( 0.10
0.75 ( 0.26
0
0
0
0
0.001 ( 0.002
0
0
0.004 ( 0.004
0
a
Reactions were carried out at 37 °C for 3 h, by incubating 50 mM phosphate buffer (pH 7.45) containing 0.05 mM DTPA, 1 mM
morpholine or tyrosine, and 500 µM NaNO2 with or without 500 µM H2O2 or HOCl. All experiments were carried out in triplicate.
Ta ble 3. F or m a tion of NO-MOR, NO2-MOR, a n d NTYR by
Rea ction of Na NO2 w ith Mor p h olin e or Tyr osin e w ith or
2 3
Furthermore, at alkaline pH, N O can be formed from
peroxynitrite anion (35, 37) and can react with unproto-
nated amines to yield nitrosamines (36). On the other
hand, at pH <7, peroxynitrous acid may decompose
a
w ith ou t H2O2 u n d er Acid ic Con d ition s
yield of nitrosated and nitrated products
(µM, mean ( SD)
•
•
homolytically to generate NO
2
and OH (35). Two mol-
),
, generated by
), has been reported
•
reaction
pH
NO-MOR
NO2-MOR
NTYR
ecules of NO
react to form dinitrogen tetroxide (N
which is a strong nitrosating agent. N
radiolysis of aqueous nitrate (NaNO
O
2
2
4
2 4
O
NaNO2 + H2O2 3.50 2.26 ( 0.42
NaNO2 + H2O2 4.50 0.87 ( 0.06
0
0
0
0
19.3 ( 0.18
6.95 ( 0.79
9.33 ( 0.41
0.98 ( 0.03
3
NaNO2 alone
NaNO2 alone
3.50 9.98 ( 0.27
4.50 1.23 ( 0.11
to react with secondary amines to produce both nitro-
samines and nitramines (39). Nucleophiles, such as
amines, react with the symmetrical and asymmetrical
a
Reactions were carried out at 37 °C for 3 h, by incubating 50
mM phosphate buffer (pH 3.5 or 4.5) containing 0.05 mM DTPA,
tautomers of N
tively) to form both nitrosated and nitrated products (36,
9-42). Furthermore, peroxynitrous acid may decompose
2
O
4
(O
2
N-NO
2
3
and ON-NO , respec-
1
1
mM morpholine or tyrosine, and 1 mM NaNO2 with or without
mM H2O2. All experiments were carried out in triplicate.
3
-
+
heterolytically into HO and NO
2
(43), and the latter
not directly proportional to the pK
a
values of the second-
species may react with amines to form nitramines (eqs 2
and 3).
ary amines (Table 1). Low concentrations of NaHCO
3
(
0.1-3 mM) enhanced peroxynitrite-mediated nitration
of secondary amines at neutral pH (Figure 4), as reported
-
+
ONOOH f HO + NO
(2)
(3)
2
for nitration of tyrosine and guanine (31, 32). In contrast,
high concentrations of NaHCO
3
(>10 mM) inhibited the
+
+
R NH + NO f R NNO + H
2
2
2
2
nitration of secondary amines at neutral pH and their
nitrosation at neutral and alkaline pH. High concentra-
The peroxynitrite-CO
2
adduct (ONOOCO
2
-) may also
2
), which reacts with
tions of NaHCO
3
have been reported to inhibit the
-
form a nitrating agent (O
2
N-OCO
nitrosation of phenol by peroxynitrite (33) as well as the
amines to form nitramines (eqs 4-6) (31, 44).
•
nitrosation of morpholine by NO (34).
On the basis of these observations, as well as those
reported in the literature on mechanisms for the nitro-
sation of phenol by peroxynitrite (30, 33), we propose here
a free radical mechanism for nitrosation and nitration
of secondary amines by peroxynitrite. At neutral and
-
-
ONOO + CO f ONOOCO
(4)
(5)
2
2
ONOOCO - a O N-OCO
2
-
2
2
-
-
R NH + O N-OCO f R NNO + HCO
3
(6)
•
•
2
2
2
2
2
2
acidic pH, NO and OH can be generated by homolytic
decomposition of peroxynitrous acid (35). These radicals
•
Rea ction w ith Con com ita n t Gen er a tion of NO
are capable of one-electron oxidation of secondary amines
•
-
a n d O
2
. Pfeiffer and Mayer (24) recently studied
•
to form amino radicals (R
NO or NO
2
N ), which can then react with
•
•-
nitration of free tyrosine by NO and O
2
generated from
spermine NONOate and from xanthine oxidase with
hypoxanthine as a substrate. They found that nitration
was most efficient with the NO donor alone, and that
inhibited the nitration of
tyrosine. These authors concluded that the species formed
at physiological pH differs from
synthetic peroxynitrite (24). However, because tyrosine
can be nitrated by both nitrosating and nitrating agents
8, 9), it is impossible to distinguish nitrosation and
•
•
2
to yield nitroso and nitro secondary amines,
respectively. Amino radicals could also be formed by the
•
-
reaction of amines with CO
the peroxynitrite-CO adduct (ONOOCO
generated from N and N
could be formed from peroxynitrite by the
3
which is generated from
•
-
2
2
) (31) as well
(36). N
•
-
concomitant generation of O
2
•
as with NO
and N
reactions of NO
2
2
O
3
2
O
4
2 3
O
2
O
4
•
•-
from NO and O
2
•
•
•
2
2
with either NO or NO (35, 37).
In addition to this free radical mechanism, nitro-
samines might be formed by direct nucleophilic nitrosa-
tion of amines by peroxynitrite anion (eq 1) in a manner
similar to that for the formation of S-nitrosoglutathione
by peroxynitrite (38), though direct oxidation of secondary
amino groups may be more difficult than that of thiol
groups.
(
nitration reactions using NTYR formation as a marker.
For these reasons, we have studied nitration and nitro-
sation of morpholine using experimental protocols similar
to those described by Pfeiffer and Mayer (24). We
•
observed nitration of morpholine when the fluxes of NO
•-
and O
2
were similar at 4 nmol/min (Figure 6). Increas-
-
-
R NH + OdN-O-O f R NNO + HOO
(1)
•
•-
2
2
ing the relative flux of either NO or O
2
resulted in a

Reactions of Reactive Nitrogen Species and Amines
Chem. Res. Toxicol., Vol. 13, No. 4, 2000 307
decrease in the extent of nitration. Similar inhibitory
amines. Although NTYR has been identified as a marker
of peroxynitrite formation in vivo, a variety of RNS,
including nitrosating and nitrating agents, can nitrate
tyrosine to form NTYR. In contrast, nitrated secondary
amines (e.g., N-nitroproline) are formed more specifically
by nitrating agents. Therefore, N-nitroproline could be
identified in human urine as a specific marker for
endogenous nitration, analogous to N-nitrosoproline in
human urine, which has been analyzed as a marker for
endogenous nitrosation (26).
•
•-
effects of excess NO and O
oxidation of dihydrorhodamine (22). However, the yield
of NO -MOR was >100 times lower than that of
NO-MOR. These results indicate that NO and O
2
have been reported for the
2
•
•-
2
reacted to form peroxynitrite, but the yield of peroxyni-
trite or its reactivity with amines was low under our
experimental conditions. In contrast, with a fixed flux of
•
-
•
O
2
, increasing the flux of NO resulted in higher yields
•
of NO-MOR. When the flux of NO was fixed, increasing
the flux of O
•
-
2
inhibited NO-MOR formation. These
results suggest that nitrosation is mediated by nitrosat-
Ack n ow led gm en t. We thank Dr. J . Cheney and
Mrs. J . Mitchell for editing the manuscript and Mrs. P.
Collard for secretarial assistance.
•
ing agents (probably N
2
O
3
) formed by the reaction of NO
•
with NO
NO . The presence of O
2
, the latter being formed via autoxidation of
•
•-
2
inhibits the formation of nitro-
•
sating agents due to a rapid reaction between NO and
Refer en ces
•-
O
2
to form peroxynitrite. In general, our results (Figure
(
1) Schmidt, H. H., and Walter, U. (1994) NO at work. Cell 78, 919-
25.
6
) are analogous to those observed for the nitration of
9
tyrosine by Pfeiffer and Mayer (24) and for the nitrosa-
tion of 2,3-diaminonaphthalene by Wink et al. (23).
Nitr osa tion a n d Nitr a tion of Mor p h olin e by Va r i-
ou s RNS. We studied nitration and nitrosation of mor-
pholine by various RNS and compared them with ty-
rosine nitration (Table 2). Under physiological conditions,
(
2) Nathan, C., and Xie, Q. W. (1994) Nitric oxide synthases: roles,
tolls, and controls. Cell 78, 915-918.
(3) Eiserich, J . P., Cross, C. E., J ones, A. D., Halliwell, B., and van
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(4) Eiserich, J . P., Hristova, M., Cross, C. E., J ones, A. D., Freeman,
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NaNO
2
alone did not nitrosate nor nitrate morpholine
well, but the addition of HOCl increased the levels of both
nitrosation and nitration (Table 2). The presence of MPO
and HPO increased the level of formation of nitrosamine
(
(
(
5) Sampson, J . B., Ye, Y., Rosen, H., and Beckman, J . S. (1998)
Myeloperoxidase and horseradish peroxidase catalyze tyrosine
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Biochem. Biophys. 356, 207-213.
2 2 2
mediated by NaNO plus H O . These results indicate
that carcinogenic nitrosamines could be formed in in-
-
flamed tissues, where increased levels of nitrite, OCl ,
6) Ischiropoulos, H., Zhu, L., Chen, J ., Tsai, M., Martin, J . C., Smith,
C. D., and Beckman, J . S. (1992) Peroxynitrite-mediated tyrosine
nitration catalyzed by superoxide dismutase. Arch. Biochem.
Biophys. 298, 431-437.
2 2
and H O could be present and MPO is secreted by
activated neutrophils.
Com p a r ison of Nitr osa tion a n d Nitr a tion of Sec-
on d a r y Am in es w ith Nitr a tion of Tyr osin e. Several
possible mechanisms for NTYR formation have been
reported, including (i) reaction of the tyrosyl radical with
7) Ischiropoulos, H. (1998) Biological tyrosine nitration: a patho-
physiological function of nitric oxide and reactive oxygen species.
Arch. Biochem. Biophys. 356, 1-11.
(8) Ohshima, H., Friesen, M., Brouet, I., and Bartsch, H. (1990)
Nitrotyrosine as a new marker for endogenous nitrosation and
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•
NO , leading to formation of 3-nitroso- or O-nitrosoty-
rosine, followed by their two-electron oxidation and
rearrangement to NTYR (45) and (ii) hydrogen abstrac-
(
9) Ohshima, H., Celan, I., Chazotte, L., Pignatelli, B., and Mower,
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chemical detection: results with various nitrating agents. Nitric
Oxide 3, 132-141.
tion from the aromatic ring by oxidant(s) to yield the
•
tyrosyl radical, followed by addition of NO
2
to form
NTYR. In the case of the peroxynitrite-mediated reaction,
tyrosine is more effectively nitrated at physiological pH
than at alkaline pH, suggesting that the mechanism
involving tyrosyl radical formation and nitration by
peroxynitrous acid may be involved. In contrast, the
reactions of secondary amines with peroxynitrite at
alkaline pH could be mediated by the nucleophilic reac-
tion between unprotonated amines and nitrosating agents,
(
10) Iyengar, R., Stuehr, D. J ., and Marletta, M. A. (1987) Macrophage
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6
369-6373.
(
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2 3
O . Under physiological conditions, free radical
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5
66.
•
•
of NO or NO
and nitration of amines. Among the different RNS that
were tested, NaNO plus H in the presence of MPO
2
) may also be responsible for nitrosation
(
13) Liu, R. H., J acob, J . R., Tennant, B. C., and Hotchkiss, J . H. (1992)
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2
2 2
O
was strikingly effective in the nitration of tyrosine,
compared to the nitrosation and nitration of amines. We
think this occurred because aromatic compounds such as
tyrosine undergo one-electron oxidation easily to form
4
139-4143.
(
14) Grisham, M. B., Ware, K., Gilleland, H. E., J r., Gilleland, L. B.,
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03, 1260-1266.
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(
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•
(
NO
2
Cl, NO
2
, and peroxynitrite) produced by MPO in the
-
presence of NaNO
2
, Cl , and H
2 2
O (4, 5).
Con clu sion
(
We have shown that peroxynitrite and other RNS react
with secondary amines to form nitrated and nitrosated

3
08 Chem. Res. Toxicol., Vol. 13, No. 4, 2000
Masuda et al.
(
(
(
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•
(
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(
(
(
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(
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