Sulfur-to-nitrogen transnitrosation: Transfer of nitric oxide from S-nitroso compounds to diethanolamine and the role of intermediate sulfur-to-sulfur transnitrosation

Article abstract of DOI:10.1016/S0300-483X(01)00388-2

S-Nitrosothiols are formed in vivo and are involved in NO signaling. We investigated the sulfur-to-nitrogen transnitrosation activity of S-nitrosocysteine, S-nitrosoglutathione, S-nitrosohomocysteine, S-nitrosocysteinylglycine and S-nitroso-N-acetylcysteine in their reaction with the secondary amine diethanolamine in vitro. The resulting N-nitrosodiethanolamine, a strong carcinogen, was formed in yields of up to 11% from S-nitrosocysteine and S-nitrosocysteinylglycine, whereas the transnitrosation activity of the other S-nitroso compounds was weak. However, the addition of L-cysteine to a solution of S-nitrosohomocysteine and diethanolamine accelerated the decomposition of S-nitrosohomocysteine and resulted in a significant formation of N-nitrosodiethanolamine accompanied by the intermediate generation of S-nitrosocysteine. Thus, reactive nitrosothiols can be formed from less reactive analogs via sulfur-to-sulfur transnitrosation. We suggest that this affects regulation of NO trafficking in vivo. The reaction provides an alternative mechanism for the generation of carcinogenic N-nitroso derivatives.

Full text of DOI:10.1016/S0300-483X(01)00388-2

Toxicology 163 (2001) 127–136
www.elsevier.com/locate/toxicol
Sulfur-to-nitrogen transnitrosation: transfer of nitric oxide
from S-nitroso compounds to diethanolamine and the role
of intermediate sulfur-to-sulfur transnitrosation
Ahmed H. Al-Mustafa, Helmut Sies, Wilhelm Stahl *
Institut fu¨r Physiologische Chemie I, Heinrich-Heine-Uni6ersita¨t Du¨sseldorf, Postfach 101007, D-40001 Du¨sseldorf, Germany
Received 17 January 2001; received in revised form 3 April 2001; accepted 3 April 2001
Abstract
S-Nitrosothiols are formed in vivo and are involved in NO signaling. We investigated the sulfur-to-nitrogen
transnitrosation activity of S-nitrosocysteine, S-nitrosoglutathione, S-nitrosohomocysteine, S-nitrosocysteinylglycine
and S-nitroso-N-acetylcysteine in their reaction with the secondary amine diethanolamine in vitro. The resulting
N-nitrosodiethanolamine, a strong carcinogen, was formed in yields of up to 11% from S-nitrosocysteine and
S-nitrosocysteinylglycine, whereas the transnitrosation activity of the other S-nitroso compounds was weak. However,
the addition of L-cysteine to a solution of S-nitrosohomocysteine and diethanolamine accelerated the decomposition
of S-nitrosohomocysteine and resulted in a significant formation of N-nitrosodiethanolamine accompanied by the
intermediate generation of S-nitrosocysteine. Thus, reactive nitrosothiols can be formed from less reactive analogs via
sulfur-to-sulfur transnitrosation. We suggest that this affects regulation of NO trafficking in vivo. The reaction
provides an alternative mechanism for the generation of carcinogenic N-nitroso derivatives. © 2001 Elsevier Science
Ireland Ltd. All rights reserved.
Keywords: Nitrosamine; Transnitrosation; Secondary amine; S-nitroso-cysteine; S-nitroso-glutathione; S-nitroso-homocysteine;
Peroxynitrite
al., 1997; Kashiba et al., 1999; Hogg, 2000), in-
cluding S-nitroso derivatives of the most promi-
nent endogenous thiols, such as S-nitrosocysteine,
S-nitrosoglutathione, S-nitrosohomocysteine or S-
nitrosoalbumin. A role for S-nitrosothiols in NO-
dependent signaling and the preservation of the
biological activity of NO has been suggested (Ig-
narro et al., 1981; Stamler et al., 1992; Mathews
and Kerr, 1993; Stamler, 1995). NO can be stabi-
lized by an intermediate molecular species like
1. Introduction
S-nitrosothiols are formed in the reaction of
nitric oxide with thiol groups. Various S-nitro-
sothiol compounds have been identified in body
fluids and tissues (Upchurch et al., 1995; Kluge et
* Corresponding author. Tel.: +49-211-8112711; fax: +49-
211-8113029.
E-mail address: wilhelm.stahl@uni-duesseldorf.de (W.
Stahl).
0300-483X/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved.
PII: S0300-483X(01)00388-2

128
A.H. Al-Mustafa et al. / Toxicology 163 (2001) 127–136
nitrosothiols, thus increasing its physiological
half-life. S-nitrosoalbumin exhibits NO-like prop-
erties in vivo, including vasorelaxation and
platelet inhibition. Biological activities related to
NO effects have been attributed to low-molecular-
weight S-nitroso compounds also used as NO
donors in experimental drug development
(Janero, 2000). S-nitrosothiols interact with other
thiols in sulfur-to-sulfur transnitrosation reac-
tions, a process involved in the regulatory net-
work of NO signaling. Enzymes are also involved
in the homeostasis of S-nitroso-compounds. The
glutathione-dependent formaldehyde dehydroge-
nase exhibits S-nitrosoglutathione reductase activ-
ity catalyzing the denitrosation of this
S-nitrosothiol (Liu et al., 2001).
NO also reacts with secondary amines to yield
relatively stable nitrosamines, some of which are
potent carcinogenic agents (Tamir and Tannen-
baum, 1996). Nitrosamines can be taken up from
exogenous sources like contaminated food, cos-
metics or tobacco smoke or are formed in the
stomach in the reaction of nitrite with secondary
amines under acidic conditions. Transnitrosation
from S-nitroso compounds to secondary amines
has been described as a possible mechanism of
nitrosamine formation in meat (Dennis et al.,
1979).
glutathione,
sulfanilamide,
N-(1-naphthyl)-
ethylenediamine and bathocuproine disulfonic
acid were from Sigma (Deisenhofen, Germany).
Diethanolamine was from Aldrich-Sigma (Deisen-
hofen, Germany).
2.2. Methods
N-nitrosodiethanolamine was synthesized from
diethanolamine according to Preussmann (1962).
The compound was identified by UV-spec-
troscopy and mass-spectrometry; purity was
checked by HPLC.
Peroxynitrite was prepared by reaction of H2O2
and sodium nitrite using a quenched-flow reactor
(Koppenol et al., 1996) and stored at −80°C
until use. The final concentration was determined
spectrophotometrically at 302 nm (‡=1670/M
cm).
S-nitrosocysteine, S-nitrosohomocysteine, S-ni-
trosocysteinylglycine, S-nitrosoglutathione, and S-
nitroso-N-acetylcysteine
according to Mathews and Kerr (1993). The con-
centration of the nitrosothiols was determined by
measuring the absorbance at 330 nm as described
(Mathews and Kerr, 1993); their purity was
checked by means of HPLC and was \95%. The
compounds were stored at −20°C and stable for
more than 8 weeks.
were
synthesized
N-nitrosodiethanolamine is one of the most
prevalent nitrosamines in the environment. It in-
duces liver and nasal cavity tumors in mice and
can be formed from its precursor diethanolamine
which is found in a number of consumer products
(Lijinsky et al., 1980; Preussmann et al., 1982;
Denkel et al., 1986; Loeppky, 1999). In the
present paper we investigated the formation of
N-nitrosodiethanolamine in vitro from the reac-
tion of S-nitroso compounds or peroxynitrite with
diethanolamine.
2.3. Incubation of diethanolamine with
S-nitrosothiols and peroxynitrite
The reactions of nitrosothiols or peroxynitrite
with diethanolamine were carried out in 3.5 ml 10
mM phosphate buffer pH 7.0, in a glass tube
under stirring at 37°C. The reaction mixtures
contained 1.25 mM nitrosothiol (control), 1.25
mM nitrosothiol plus 1.25 mM diethanolamine,
or 1.25 mM nitrosothiol plus 2.5 mM
diethanolamine.
2. Materials and methods
In an additional set of experiments S-nitrosoho-
mocysteine (1.25 mM) was incubated with 1.25
mM and 2.5 mM diethanolamine in the presence
of cysteine (1 mM) under the same conditions to
make an examplary study of the influence of
thiols on the stability of S-nitroso compounds.
2.1. Chemicals
Sodium nitrite and N-acetyl-
purchased from Merck (Darmstadt, Germany).
DL-homocysteine, -cysteine, cysteinylglycine,
L-cysteine were
L

A.H. Al-Mustafa et al. / Toxicology 163 (2001) 127–136
129
Peroxynitrite at final concentrations of 1 or 2
3. Results
mM was incubated with 1 mM diethanolamine
in 10 mM phosphate buffer pH 7.0 at 37°C; 100
mM stock solution of peroxynitrite was used.
The pH was 7.4 after addition of peroxynitrite.
During the incubations of diethanolamine with
S-nitrosothiols and peroxynitrite the pH did not
change.
In separate experiments five different nitro-
sothiols, including S-nitrosocysteine, S-nitrosog-
lutathione, S-nitrosohomocysteine, S-nitrosocy-
steinylglycine and S-nitroso-N-acetylcysteine or
peroxynitrite were incubated with diethanol-
amine. The decomposition of S-nitroso com-
pounds and the formation of N-nitrosodi-
ethanolamine generated via sulfur-to-nitrogen
transnitrosation were followed by HPLC. Typi-
cal chromatograms obtained from an incubation
mixture of S-nitrosocysteine and diethanolamine
at different time points are shown in Fig. 1. The
compounds of interest are baseline-separated and
are assigned in the chromatograms. The signal at
a retention time of 2.8 min originates from the
sample solvent front and from cystine, which
was formed during incubation; no free thiols
were detectable in the mixtures using Ellman’s
reagent. Other disulfides also eluted with the
sample solvent front. The formation of disulfides
upon decomposition of S-nitrosothiols has been
described in the literature as a major reaction
pathway (Barnett et al., 1994). N-nitrosodi-
ethanolamine was identified by comparison of
retention times with the synthetic reference com-
pound and by UV spectroscopy. The peak as-
signed to N-nitrosodiethanolamine was isolated
and further characterized by mass spectrometry.
The mass spectrum obtained from the isolated
fraction was identical with the spectrum of a
synthetic reference compound (MH+ at m/z 135)
and exhibited a similar fragmentation pattern.
Characteristic fragments were observed at m/z
117 (parent ion–H2O), m/z 104 (parent ion–NO)
and m/z 74 (parent ion–NO–OH–CH2).
2.4. Analysis of N-nitrosodiethanolamine and
S-nitrosothiols
Aliquots (20 ml) of the reaction mixtures were
taken at various time points and analyzed for
N-nitrosodiethanolamine and S-nitrosothiol con-
tent by means of HPLC with a Merck-Hitachi
HPLC system (pump 655A-12, UV–Vis-detector
L-4200, Chromato-Integrator D2500; Merck,
Darmstadt, Germany). For separation a reversed
phase C-30 column 4.6×250 mm (YMC-
carotenoid, YMC Ltd., Japan) was used apply-
ing a flow rate of 1 ml/min. The detection
wavelength
was
236
nm.
N-nitrosodi-
ethanolamine and S-nitrosothiols were quantified
according to the external standard method.
Various mobile phases were applied for the
separation of N-nitrosodiethanolamine and S-ni-
trosothiols in different incubation mixtures. Sep-
aration
of
N-nitrosodiethanolamine
and
S-nitrosocysteine: 10 mM phosphate buffer pH
7.0; or S-nitrosocysteinylglycine: 10 mM phos-
phate buffer pH 2.5, 0.05% trifluoro-acetic acid,
6% methanol; or S-nitrosohomocysteine: 10 mM
phosphate buffer pH 3.0, 0.1% trifluoro-acetic
acid; or S-nitrosoglutathione: 10 mM phosphate
buffer pH 2.5, 0.1% trifluoro-acetic acid, 6%
methanol; or S-nitroso-N-acetylcysteine: 10 mM
phosphate buffer pH 7.0, 6% methanol.
3.1. Transnitrosation from S-nitrosocysteine to
diethanolamine
The transnitrosation from S-nitrosothiols to
diethanolamine yielding N-nitrosodiethanolamine
was investigated at pH 7.0 in 10 mM phosphate
buffer at 37°C. The concentration of added S-ni-
troso compounds was 1.25 mM in all experi-
ments; diethanolamine was added at 1.25 mM or
at 2.5 mM.
2.5. Nitrite and nitrate analysis
Aliquots of the incubation mixtures were with-
drawn at various time points and analyzed for
nitrite and nitrate concentration according to
Marzinzig et al. (1997).

130
A.H. Al-Mustafa et al. / Toxicology 163 (2001) 127–136
Without any diethanolamine present in the mix-
after 80 min, a transnitrosation efficacy of about
4%. Nitrite levels also increased during incubation
and accounted for about 45% of NO-equivalents
of S-nitrosocysteine after complete decomposi-
tion; no nitrate was detected. When the concen-
tration of diethanolamine was increased to 2.5
mM, the half-life of S-nitrosocysteine was further
shortened (Fig. 2B). After 30 min the compound
was fully consumed, and about 140 mM of N-ni-
trosodiethanolamine had formed (transnitrosation
efficacy of 11%).
ture (control), S-nitrosocysteine decomposed
spontaneously with a half-life of about 35 min
(Fig. 2A); after 60 min of incubation the concen-
tration of the parent compound had decreased to
0.36 mM. When diethanolamine (1.25 mM) was
present (Fig. 2A), S-nitrosocysteine decomposed
more rapidly; the half-life then was 25 min, and
only 0.21 mM was left at 60 min (Table 1). In the
presence of diethanolamine (1.25 mM), N-ni-
trosodiethanolamine was formed to yield 51 mM
Fig. 1. HPLC traces obtained from an incubation mixture of S-nitrosocysteine and diethanolamine. S-nitrosocysteine and
diethanolamine were 1.25 mM in 10 mM phosphate buffer pH 7.0 at 37°C. Samples were taken at 0, 10 and 60 min. Separation was
achieved with a reversed phase C-30 column 4.6×250 mm (YMC-carotenoid, YMC Ltd., Japan); flow rate of 1 ml/min, detection
wavelength 236 nm. 10 mM phosphate buffer pH 7.0 was used as mobile phase.

Table 1
Transnitrosation from S-nitrosothiols to diethanolamine yielding N-nitrosodiethanolaminea
Mixture
Molar ratio Time
(mM)
0 min
60 min
210 min
RSNO (mM) N-DEA
NO−2 (mM) RSNO (mM) N-DEA
(mM)
NO−2 (mM) RSNO (mM) N-DEA
NO−2 (mM)
(mM)
CysNO/DEA (1:0)
1.1990.02
1.0190.11
1.0590.05
1.1390.15
1.0990.03
1.0390.03
1.1890.04
–
36.293.2
31.996.5
32.393.6
36.395.8
36.294.5
36.297.6
10.293.3
0.3690.05
0.2190.04
–
0.2590.03
0.0990.01
–
–
29193.3
489914.7
n.m.
238911.3
53899.8
n.m.
n.m.
n.m.
n.m.
n.m.
n.m.
n.m.
1.1890.03
n.m.
n.m.
n.m.
n.m.
n.m.
n.m.
-
n.m.
n.m.
n.m.
n.m.
n.m.
n.m.
18.891.2
(1:1)
(1:2)
7.091.1
28.490.2
–
0.590.10
1.490.00
–
48.592.0
138.092.9
–
38.091.2
89.098.4
–
/
CGNO/DEA (1:0)
(1:1)
(1:2)
HcysNO/DEA (1:0)
1.1790.06
16.791.7
163
(1:1)
(1:2)
1.1990.04
1.1890.12
1.2390.03
1.1990.04
1.1890.05
1.2590.01
–
–
–
–
–
–
8.991.1
1.1890.05
1.1590.13
1.2290.06
1.1990.01
1.1590.03
1.2590.02
0.4090.06
1.7190.60
–
0.5390.14
0.8890.22
–
26.693.4
35.992.1
13.491.3
16.792.4
17.892.0
13.191.4
1.1690.03
1.1290.05
1.2290.02
1.1890.01
1.1490.02
1.2590.01
2.0990.24 45.893.6
4.4091.02 79.995.0
(
14.892.9
10.591.3
10.391.4
11.091.3
12.592.1
GSNO/DEA (1:0)
(1:1)
–
20.393.6
0.9390.09 31.291.9
1.7490.35 43.591.6
127
(1:2)
136
NACNO/DEA(1:0)
–
16.792.2
(1:1)
(1:2)
1.2590.03
1.2490.02
–
–
12.391.6
12.390.6
1.2490.02
1.1590.03
0.1590.01
0.2590.08
17.191.4
20.491.1
1.2490.01
1.2390.01
0.4390.07 21.191.2
1.2290.04 29.290.5
a The mixtures contained 1.25 mM S-nitrosothiols (RSNO) in 10 mM phosphate buffer, pH 7.0, at 37°C, either no DEA (1:0 molar ratio), 1.25 mM DEA (1:1 molar
ratio) or 2.5 mM DEA (1:2 molar ratio). RSNOs and N-DEA concentrations were determined by HPLC. Results represent means of three independent experiments
9SD-: below detection limit; n.m.: not measured; DEA, diethanolamine; N-DEA, N-nitrosodiethanolamine; CysNO, S-nitrosocysteine; CGNO, S-nitrosocysteinyl-
glycine; HcysNO, S-nitrosohomocysteine; GSNO, S-nitrosoglutathione; NACNO, S-nitroso-N-acetylcysteine.

132
A.H. Al-Mustafa et al. / Toxicology 163 (2001) 127–136
practically fully consumed, and 38 mM N-nitrosodi-
ethanolamine had formed (transnitrosation efficacy
3%) (Fig. 3); nitrite accounted for 43% of the NO
equivalents in the mixture. The decomposition of
S-nitrosocysteinylglycine was accelerated at 2.5
mM diethanolamine (Table 1). The yield of N-ni-
trosodiethanolamine was 89 mM after 60 min
(transnitrosation efficacy 8%) (Table 1).
Other S-nitrosothiols investigated were more
stable under the same conditions (Table 1). Neither
in the presence nor absence of diethanolamine a
statistically significant decomposition of S-nitroso-
homocysteine, S-nitrosoglutathione and S-nitroso-
N-acetylcysteine was observed within 210 min of
incubation. Differences between the levels of nitro-
sothiols at t=0 and 210 min are within the
coefficient of variation of the analytical method,
which is 4%. It took several days until the com-
pounds were decomposed (data not shown). Less
than 4.4 mM N-nitrosodiethanolamine was found
after 210 min which corresponds to a transnitrosa-
tion efficacy of less than 0.4%; only small amounts
of nitrite where detected.
Fig. 2. Formation of N-nitrosodiethanolamine and loss of
S-nitrosocysteine. (A): Reaction mixtures contained 1.25 mM
S-nitrosocysteine in 10 mM phosphate buffer pH 7.0 at 37°C;
S-nitrosocysteine and N-nitrosodiethanolamine were measured
as shown in Fig. 1. S-nitrosocysteine (control) (ꢀ), S-nitroso-
cysteine with 1.25 mM diethanolamine (ꢁ). N-nitrosodi-
ethanolamine (ꢂ) formed from S-nitrosocysteine and 1.25
mM diethanolamine. (B): As in A; but concentration of di-
ethanolamine was 2.5 mM. Results are given as means of three
independent experiments 9SD.
3.2. Transnitrosation from
S-nitrosocysteinylglycine to diethanolamine
Fig. 3. Formation of N-nitrosodiethanolamine and loss of
S-nitrosocysteinylglycine. Reaction mixtures contained 1.25
mM S-nitrosocysteinylglycine in 10 mM phosphate buffer pH
7.0 at 37°C; S-nitrosocysteinylglycine and N-nitrosodi-
ethanolamine were measured at different time points as de-
scribed in experimental procedure. S-nitrosocysteinylglycine
(control) (ꢀ), S-nitrosocysteinylglycine with 1.25 mM di-
ethanolamine (ꢁ) and N-nitrosodiethanolamine (ꢂ) formed
from 1.25 mM diethanolamine. Results are given as means of
three independent experiments 9SD.
Very similar results were obtained when S-nitro-
socysteinylglycine was used as a transnitrosating
agent (Fig. 3). The half-life of the compound
without diethanolamine (control) was also about 30
min; upon addition of diethanolamine (1.25 mM)
the half-life was shortened to about 15 min. After
50 min of incubation S-nitrosocysteinylglycine was

A.H. Al-Mustafa et al. / Toxicology 163 (2001) 127–136
133
3.4. Nitrosation of diethanolamine with
peroxynitrite
For comparison, the nitrosating activity of per-
oxynitrite was investigated, measuring the yield of
N-nitrosodiethanolamine when diethanolamine
was incubated with 1 or 2 mM peroxynitrite for
15 min at pH 7 and 37°C. At a ratio of 1 mM
diethanolamine and 1 mM peroxynitrite the yield
of N-nitrosodiethanolamine was about 3.5 mM
(transnitrosation efficacy of 0.35%). The yield of
N-nitrosodiethanolamine increased to 46 mM with
2 mM peroxynitrite (transnitrosation efficacy of
2.3%). The reaction was completed within
seconds.
Fig. 4. Formation of N-nitrosodiethanolamine and loss of
S-nitrosohomocysteine in the presence of cysteine. Reaction
mixture contained 1.25 mM S-nitrosohomocysteine, 1.25 mM
diethanolamine and 1 mM cysteine in 10 mM phosphate
buffer pH 7.0 at 37°C; S-nitrosohomocysteine, S-nitrosocys-
teine and N-nitrosodiethanolamine were measured at different
time points as described in experimental procedure. S-nitroso-
homocysteine. (ꢀ), S-nitrosocysteine (ꢁ), N-nitrosodi-
ethanolamine (ꢂ). Results are given as means of three
independent experiments 9SD.
4. Discussion
The present study demonstrates that S-nitro-
sothiols which are found in body fluids and tissues
can react with the secondary amine di-
ethanolamine in a transnitrosation reaction to
generate the carcinogenic N-nitrosodiethanol-
amine. Among the compounds investigated, S-ni-
3.3. Transnitrosation from S-nitrosohomocysteine
to diethanolamine in the presence of cysteine
trosocysteine
and
S-nitrosocysteinylglycine
S-nitrosohomocysteine was incubated with 1.25
mM diethanolamine in the presence of 1 mM
showed the highest sulfur-to-nitrogen transnitro-
sation activity. At equimolar levels they reacted
with diethanolamine to yield N-nitrosodi-
ethanolamine at yields of 3–4% within 60 min. In
contrast, S-nitrosoglutathione, S-nitrosohomocys-
teine and S-nitroso-N-acetylcysteine were much
more stable in the presence or in the absence of
diethanolamine. Differences in the stability of S-
nitroso derivatives have been described earlier
(Mathews and Kerr, 1993). The stability of the
S-nitrosothiols was found to decrease in the order:
S-nitrosoglutathione \S-nitrosohomocysteine \
S-nitrosocysteine (Mathews and Kerr, 1993), in
accordance with the observations in the present
study. The highly reactive S-nitrosocysteine and
S-nitrosocysteinylglycine carry a primary amine
group spaced by a methylene group from the
S-nitroso function. In the less reactive S-nitroso-
homocysteine, the amine group is separated from
the S-nitroso function by two methylene groups.
S-nitrosoglutathione and S-nitroso-N-acetylcys-
L
-cysteine, and the loss of S-nitrosohomocysteine
as well as the formation of N-nitrosodi-
ethanolamine was followed (Fig. 4). When -cys-
L
teine was present in the mixture, the level of
S-nitrosohomocysteine decreased rapidly with a
half-life of 45 min; after 150 min all S-nitrosoho-
mocysteine was consumed. The level of N-ni-
trosodiethanolamine in the mixture increased up
to 30 mM at 150 min (transnitrosation efficacy of
2.4%). S-nitrosocysteine was formed as an inter-
mediate in the reaction, reaching maximum con-
centration of 0.55 mM at 30 min (Fig. 4). In the
presence of
L-cysteine and 2.5 mM di-
ethanolamine, S-nitrosohomocysteine decom-
posed with a similar time course, and the
intermediate formation of S-nitrosocysteine was
also comparable between both experiments; how-
ever; more N-nitrosodiethanolamine (59 mM) was
generated (transnitrosation efficacy of 4.7%).

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A.H. Al-Mustafa et al. / Toxicology 163 (2001) 127–136
trosations (Barnett et al., 1994) allow the transfer
of the NO group from a stable S-nitrosothiol to a
less stable analog.
The system presented here provides a method
to investigate the transnitrosation activity of S-ni-
trosothiols under various conditions. The result-
teine do not possess a free primary amine close to
the S-nitrosothiol structure; here the nitrogen is
part of an amide bond with an electron-withdraw-
ing carbonyl group attached. Such structural
properties are involved in the stabilization of S-ni-
trosothiol functions and may have impact on the
transnitrosating activity.
ing N-nitrosodiethanolamine is
a
stable
compound and can be readily analyzed. Whether
sulfur-to-nitrogen transnitrosation plays a biologi-
cal role in the generation of nitrosamines needs
further research. The concentrations used in the
present model are higher than expected in vivo.
However, the present study demonstrates that the
formation of nitrosamines from S-nitroso deriva-
tives is possible at pH 7.0. Another possibility for
the formation of nitrosamines is the reaction of
thiols with peroxynitrite, a compound capable of
nitrosating thiols and amines (van der Vliet et al.,
1998; Masuda et al., 2000).
Sulfur-to-sulfur transnitrosations between S-ni-
troso compounds and other thiols are thought to
play an important role in the transfer of NO
signals in the organism (Garthwaite and Boulton,
1995; Butler and Rhodes, 1997). Differences in the
stability of endogenous S-nitrosothiols and varia-
The decompositon of S-nitrosothiols is influ-
enced by various factors, including metal ions
(Singh et al., 1996), thioredoxin reductase (Niki-
tovic and Holmgren, 1996), ascorbate reductase
(Kashiba-Iwatsuki et al., 1996), superoxide anion
(Aleryani et al., 1998) or protein thiols (Scorza et
al., 1997). The stability of S-nitrosocysteine and
S-nitrosocysteinylglycine also decreased in the
presence of secondary amines.
Even more interesting is the observation that
the stability of S-nitrosohomocysteine was dra-
matically affected by the addition of cysteine. As
shown in Fig. 4, the stable S-nitrosohomocysteine
rapidly transferred its NO group to cysteine yield-
ing the reactive S-nitrosocysteine as an intermedi-
ate, which was capable of transferring the NO to
diethanolamine yielding N-nitrosodiethanolamine
(Fig. 5). Such interthiol (sulfur-to-sulfur) transni-
Fig. 5. Reaction scheme for the formation of N-nitrosodiethanolamine formed from S-nitrosohomocysteine and diethanolamine in
the presence or absence of cysteine.

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Acknowledgements
A.A.-M. is a Stipendiat of the ‘Katholischer
Akademischer Ausla¨nder-Dienst’ (KAAD), Bonn,
Germany. H.S. is a Fellow of the National Foun-
dation for Cancer Research, Bethesda, MD.
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