Transnitrosation of non-mutagenic N-nitrosoproline forms mutagenic N-nitroso-N-methylurea

Article abstract of DOI:10.1016/j.bmc.2015.04.058

Abstract N-Nitroso-N-methylurea (NMU) is a potent carcinogen and suspected as a cause of human cancer. In this study, mutagenic NMU was detected by HPLC after the transnitrosation of non-mutagenic N-nitrosoproline (NP) to N-methylurea in the presence of thiourea (TU) under acidic conditions. The structure of NMU was confirmed by comparing ¹H NMR and IR spectra with that of authentic NMU after fractionation by column chromatography. Furthermore, a fraction containing NMU formed by transnitrosation was mutagenic in Salmonella typhimurium TA1535. NMU was formed in the reaction of NP and N-methylurea in the presence of 1,1,3,3-tetramethylthiourea (TTU) or 1,3-dimethylthiourea in place of TU as an accelerator. The reaction rate constants (k) for NMU formation were correlated with their nucleophilicity of sulfur atom in thioureas. The N-methylurea concentration did not affect the NMU formation, whereas the rate of NMU formation correlated linearly with concentrations of NP, TTU and oxonium ion. The observed kinetics suggests a mechanism by which the nitroso group was transferred directly from the protonated NP to the thiourea then to N-methylurea to form NMU. The rate-determining step was the formation of the complex with the protonated NP and thiourea.

Full text of DOI:10.1016/j.bmc.2015.04.058

Bioorganic & Medicinal Chemistry 23 (2015) 3297–3302
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Transnitrosation of non-mutagenic N-nitrosoproline forms
mutagenic N-nitroso-N-methylurea
b
a
b
a,b
Keiko Inami a,b, , Junko Shiino , Shin Hagiwara , Kei Takeda , Masataka Mochizuki
⇑
a Faculty of Pharmaceutical Sciences, Tokyo University of Science, Yamazaki 2641, Noda-shi, Chiba 278-8510, Japan
b Kyoritsu University of Pharmacy, Shibakoen 1-5-30, Minato-ku, Tokyo 105-8512, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
N-Nitroso-N-methylurea (NMU) is a potent carcinogen and suspected as a cause of human cancer. In this
study, mutagenic NMU was detected by HPLC after the transnitrosation of non-mutagenic N-nitrosopro-
line (NP) to N-methylurea in the presence of thiourea (TU) under acidic conditions. The structure of NMU
was confirmed by comparing 1H NMR and IR spectra with that of authentic NMU after fractionation by
column chromatography. Furthermore, a fraction containing NMU formed by transnitrosation was muta-
genic in Salmonella typhimurium TA1535.
Received 9 February 2015
Revised 17 April 2015
Accepted 18 April 2015
Available online 25 April 2015
Keywords:
NMU was formed in the reaction of NP and N-methylurea in the presence of 1,1,3,3-tetramethylth-
iourea (TTU) or 1,3-dimethylthiourea in place of TU as an accelerator. The reaction rate constants (k)
for NMU formation were correlated with their nucleophilicity of sulfur atom in thioureas. The N-methy-
lurea concentration did not affect the NMU formation, whereas the rate of NMU formation correlated lin-
early with concentrations of NP, TTU and oxonium ion. The observed kinetics suggests a mechanism by
which the nitroso group was transferred directly from the protonated NP to the thiourea then to N-
methylurea to form NMU. The rate-determining step was the formation of the complex with the proto-
nated NP and thiourea.
N-Nitrosoproline
N-Nitroso-N-methylurea
Transnitrosation
Thioureas
Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction
Humans are exposed to NMU exogenously through food.3,4 In addi-
tion, NMU can be formed endogenously in stomach5–8 and intes-
N-Nitroso compounds represent a major class of important
chemical carcinogens and mutagens that have been implicated as
a hazard to human health.1 These compounds and their precursors
are present in the human environment and diet. They are also
found in tobacco products, cosmetics, pharmaceutical products,
and they form endogenously in the human body from dietary
components.1
N-Nitroso compounds are divided into two categories:
nitrosamines, which require activation to exert their genotoxicity,
and nitrosamides including N-nitroso-N-methylurea (NMU), which
spontaneously decompose to form alkylating agents.1
tine.9 In fact, NMU has been formed under acidic conditions from
creatinine, which is present at fairly high concentrations in meat
and fish.8 The microbiota of the intestine can reduce nitrate to
nitrite, which can then react to form NMU.9
There are non-mutagenic and non-carcinogenic N-nitrosamines,
for example, N-nitrosoproline (NP) and N-nitrosothioproline.10,11
NP was formed in stomach12–14 and detected in foods.15,16 The
presence of these compounds in urine is considered as an indicator
of nitrosation exposure in vivo.17
Transnitrosation, in which a nitroso group is transferred from
nitrosamine to another amine, is a reaction characteristic of N-aryl-
and N-alkyl-N-nitrosoureas,18–21 and N-aryl-N-nitrosamines.22–27
Furthermore, transnitrosation by aliphatic nitrosamines under
appropriate conditions have also been reported.23,28–31 Thus a
non-carcinogenic N-nitrosamine can give carcinogenic compound
by transnitrosation under the acidic conditions of the mammalian
stomach. In this study, we report that the non-mutagenic NP
transnitrosated to an alkylurea and formed the mutagenic NMU
in the presence of accelerator under acidic conditions. We also
report on our investigation of the mechanism of this
transnitrosation.
NMU is a potent direct-acting carcinogen that has been shown
to induce cancers in a wide variety of animal species and in various
organs, mainly the forestomach, brain, and the nervous system.2
Abbreviations: DTU, 1,3-dimethylthiourea; h, hour; HPLC, high performance
liquid chromatography; IR, infrared; NMR, nuclear magnetic resonance; NMU, N-
nitroso-N-methylurea; NP, N-nitrosoproline; Rf, retention factor; TLC, thin layer
chromatography; TTU, 1,1,3,3-tetramethylthiourea; TU, thiourea.
⇑
Corresponding author. Tel./fax: +81 4 7121 4436.
E-mail address: inami@rs.noda.tus.ac.jp (K. Inami).
http://dx.doi.org/10.1016/j.bmc.2015.04.058
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.

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K. Inami et al. / Bioorg. Med. Chem. 23 (2015) 3297–3302
dichloromethane (4:3:2), UV 254 nm]. A fraction which included
2. Results
a substance with the same Rf value on TLC as authentic NMU
was collected, the organic solvent evaporated and the residue ana-
lyzed. The retention time by HPLC, Rf value on TLC, 1H NMR and IR
spectra were identical with those of authentic NMU.
2.1. Identification of NMU from NP and methylurea in the
presence of thiourea (TU)
The fraction containing the NMU was mutagenic in Salmonella
typhimurium TA1535 (Fig. 1). The products formed in the absence
of either NP, N-methylurea or TU were not mutagenic (Table S1
in Supplementary data).
To a solution of NP (28 mmol) in acetonitrile was added a solu-
tion of N-methylurea (28 mmol) in 0.1 M HCl aqueous solution (pH
1.5) and a solution of TU (11 mmol) in 0.1 M HCl aqueous solution
(pH 1.5), and then the mixture was stirred at 37 °C for 4 h. The
reaction mixture was extracted with chloroform, and the organic
phase was dried over anhydrous sodium sulfate, and the solvent
subsequently evaporated. The residue was fractionated by silica
2.2. Effect of accelerators on NMU formation from NP and
methylurea under acidic conditions
gel
column
chromatography
[n-hexane/diethyl
ether/
An accelerator was necessary for the formation of NMU from NP
and N-methylurea as described above. Thiocyanate is one of the
accelerators for transnitrosation,21,28,29,31 however, it has been
reported to have lower catalytic activity than thioureas.23,25,27,32
In addition to 1,1,3,3-tetramethylthiourea (TTU) and 1,3-
dimethylthiourea (DTU), other sulfur compounds; thioacetamide,
cysteine, methionine and glutathione, were tested as possible
accelerators for transnitrosation from NP to N-methylurea in this
study. NMU was not detected following the reaction with cysteine,
methionine and glutathione, and only a trace amount of NMU was
detected after 22 h reaction with thioacetamide (data not shown).
The effect of three thioureas on NMU formation was compared,
and the reaction was followed by initial rate methods.33,34 NMU
was formed in a dose- and time-dependent manner in the presence
of three thioureas (Fig. 2). A plot of ln[kin] versus ln[a thiourea]
was linearly correlated with a slope (approximately 1.0), indicating
that the order of the reaction was first-order with respect to
thioureas (Fig. 3).
The reaction rate constant (kin) was obtained from a y-intercept
in plot of ln[kin] versus ln[a thiourea] (Fig. 3). The accelerating
effect increased in the following order: TU (1.51 ꢀ 10ꢁ4
M sꢁ1) < DTU (2.94 ꢀ 10ꢁ4 M sꢁ1) < TTU (5.23 ꢀ 10ꢁ4 M sꢁ1). The
Figure 1. Mutagenicity of reaction extract from NP and N-methylurea in the
presence of TU in Salmonella typhimurium TA1535.
Figure 2. Effect of accelerator on NMU formation by transnitrosation of NP. (A) NMU formation from NP and N-methylurea in the presence of TU at several concentrations
[0.005 M (ꢀ), y = 2.24 x ꢀ 10ꢁ7 (R2 = 0.99); 0.01 M (j), y = 3.66 x ꢀ 10ꢁ7 (R2 = 0.99); 0.02 M (N), y = 7.60 x ꢀ 10ꢁ7 (R2 = 0.99); 0.05 M (d), y = 13.3 x ꢀ 10ꢁ7 (R2 = 0.99)]. (B)
NMU formation from NP and N-methylurea in the presence of DTU at several concentrations [0.005 M (ꢀ), y = 1.33 x ꢀ 10ꢁ7 (R2 = 0.96); 0.01 M (j), y = 2.94 x ꢀ 10ꢁ7
(R2 = 0.95); 0.02 M (N), y = 5.79 x ꢀ 10ꢁ7 (R2 = 0.97); 0.05 M (d), y = 13.8 x ꢀ 10ꢁ7 (R2 = 0.97)]. (C) NMU formation from NP and N-methylurea in the presence of TTU at several
concentrations [0.005 M (ꢀ), y = 3.53 x ꢀ 10ꢁ7 (R2 = 0.99); 0.01 M (j), y = 7.66 x ꢀ 10ꢁ7 (R2 = 1.00); 0.02 M (N), y = 14.6 x ꢀ 10ꢁ7 (R2 = 1.00); 0.05 M (d), y = 31.3 x ꢀ 10ꢁ7
(R2 = 1.00)].

K. Inami et al. / Bioorg. Med. Chem. 23 (2015) 3297–3302
3299
correlated with the slope of approximately 1.08 (r2 = 1.00), indicat-
ing that good first-order plots on NP concentration.
The rate of NMU formation in the reaction with NP and N-
methylurea in the presence of TTU was linearly correlated with
oxonium ion concentration (slope = 1.19, r2 = 1.00), indicating that
the transnitrosation proceeds by specific acid catalysis (Fig. 6).
3. Discussion
Transnitrosation of alicyclic N-nitrosamines has been reported
to occur in the presence of accelerator under acidic condi-
tions.23,28–31 Many reports have shown kinetic data obtained by
decreasing the initial concentration of starting N-nitrosamines,
whereas only a few reports have shown kinetic data obtained by
formation of N-nitrosamines28,29 or S-nitroso compounds35,36 pro-
duced by transnitrosation of N-nitrosamines. Although NMU has
been reported to form by nitrosation in vivo,5–8 there have been
few reports of NMU formation by transnitrosation of non-muta-
genic N-nitrosamine. Herein we report that the nitroso group of
the non-carcinogenic NP is transnitrosated to N-methylurea in
the presence of a thiourea, forming carcinogenic NMU under acidic
conditions. We also report on our investigation of the transnitrosa-
tion mechanism.
Figure 3. Plot of ln[kin] versus ln[a thiourea]. Reaction conditions were as follows:
NP, 0.05 M; N-methylurea, 0.05 M; thioureas [TU (j), y = 0.88 x ꢁ 8.38 (R2 = 0.99);
DTU (N), y = 1.01 x ꢁ 8.13 (R2 = 1.00); TTU (d), y = 0.94 x ꢁ 7.50 (R2 = 1.00)], 0.005–
0.05 M; pH 2.0; 37 °C.
NMU produced in the reaction of NP with N-methylurea in the
presence of TU was identified by HPLC retention time and by Rf
value on TLC. The reaction mixture of NP and N-methylurea with
TU was fractionated by silica gel chromatography, and the fraction
containing NMU was obtained. The 1H NMR and IR spectra for this
fraction were also identical to those of authentic NMU.
Furthermore, this fraction was mutagenic in Salmonella typhimur-
ium TA1535 (Fig. 1).
The NMU formation rate from NP and N-methylurea were com-
pared among accelerators. Methionine, glutathione and cysteine
were not effective in forming NMU in the reaction of NP and
methylurea (data not shown). Three thioureas accelerated the
reaction to form NMU following first order with respect to a
thiourea (Fig. 2). The accelerating effect of thioureas is in the order
TTU > DTU > TU, which was in good agreement with nucleophilic-
ity of sulfur atom in thioureas (Fig. 3). The data indicates that
nucleophilicity of sulfur atom in thioureas plays a key role for
the transnitrosation. When thioacetamide was used instead of a
thiourea, NMU formed in a trace amount after 22 h under acidic
conditions. The results showed that thioamide structure was nec-
essary for the formation of NMU by transnitrosation.
data was in good agreement with the order of nucleophilicity of
sulfur atom in thioureas.
2.3. Mechanism of the transnitrosation for NP and N-
methylurea in the presence of TTU
To elucidate the mechanism of transnitrosation by NP and N-
methylurea in the presence of TTU, the effect of concentration of
N-methylurea, NP and oxonium ion on NMU formation was
investigated.
In the reaction with NP and N-methylurea in the presence of
TTU, the concentration of N-methylurea as NO acceptor did not
affect NMU formation under those conditions (Fig. 4).
The effect of the concentrations of NP on NMU formation was
investigated. NMU was formed in a dose- and time-dependent
manner in the reaction (Fig. 5A). A slope of plot of ln[kin] versus
ln[NP] showed the order of reaction with respect to [NP]
(Fig. 5B). The results of kinetics experiments were linearly
The mechanism for the transnitrosation of NP in the presence of
TTU was investigated. N-Methylurea concentration did not affect
the rate of NMU formation (Fig. 4), whereas oxonium ion and
TTU were both necessary for the transnitrosation to proceed. The
NMU formed in a time- and dose-dependent manner with NP
(Fig. 5A). A plot of ln[kin] versus ln[NP] showed a linear correlation
with a slope of approximately 1.0, which indicated that the order of
reaction was 1 with respect to NP (Fig. 5B). The transnitrosation
reaction preceded by specific acid catalysis, indicating that the pro-
tonated NP was involved in the rate-determining step (Fig. 6). The
experimentally observed rate equation for the transnitrosation is
shown in Eq. 1.
Rate ¼ k ½TTUꢂ½NPꢂ½H3Oþꢂ
ð1Þ
NMU stability in 0.1 M HCl solution (pH 1.5) was measured by
the decrease in UV absorption at 254 nm. NMU decomposed by
approximately 25% over 18 h. Since our kinetics experiments were
typically followed for 5 or 6 h, the decomposition rate of NMU was
negligible.
Figure 4. Effect of N-methylurea concentration on NMU formation by transnitro-
sation of NP. Reaction conditions were as follows: NP, 0.05 M; N-methylurea
[0.05 M (j), y = 69.0 x ꢀ 10ꢁ6 (R2 = 0.99); 0.07 M (N), y = 66.9 x ꢀ 10ꢁ6 (R2 = 0.98);
0.1 M (d), y = 70.9 x ꢀ 10ꢁ6 (R2 = 0.99)]; TTU, 0.01 M; pH 2.0; 37 °C.
A presumptive mechanism is shown in Scheme 1. The mecha-
nisms agree with the observation of Singer et al.31 Protonation of

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K. Inami et al. / Bioorg. Med. Chem. 23 (2015) 3297–3302
Figure 5. Effect of NP concentration on NMU formation by transnitrosation of NP. (A) NMU formation from NP at several concentrations and N-methylurea in the presence of
TTU. Reaction conditions were as follows: NP [0.01 M (ꢀ), y = 1.14 x ꢀ 10ꢁ6 (R2 = 0.99); 0.05 M (j), y = 6.58 x ꢀ 10ꢁ6 (R2 = 1.00); 0.07 M (N), y = 9.37 x ꢀ 10ꢁ6 (R2 = 1.00); 0.1 M
(d), y = 12.8 x ꢀ 10ꢁ6 (R2 = 0.99)]; N-methylurea, 0.05 M; TTU, 0.01 M; pH 2.0; 37 °C. (B) Plot of ln[kin] versus ln[NP]. Reaction conditions were as follows; NP, 0.01–0.1 M; N-
methylurea, 0.05 M; TTU, 0.01 M; pH 2.0; 37 °C; y = 1.08 x (R2 = 1.00).
are used as medicines and pesticides39 and can be easily nitrosated
to become a possible source of endogenous carcinogens.
4. Conclusion
We demonstrated that the reaction of NP and N-methylurea
with thioureas under acidic conditions formed NMU that was iden-
tical to authentic NMU according to instrument data. The fraction
containing NMU was mutagenic in Salmonella typhimurium
TA1535. Although NMU formation rate was very small via transni-
trosation of NP, NMU is highly mutagenic and carcinogenic in
rodents. Our data showed first time that mutagenic NMU was
formed via transnitrosation by non-mutagenic N-nitrosamines
under acidic conditions such as in the stomach.
5. Materials and methods
Figure 6. pH profile for the TTU-catalyzed reaction of NP with N-methylurea.
Reaction conditions were as follows: NP, 0.05 M; N-methylurea, 0.05 M; TTU,
0.01 M; 37 °C.
5.1. Chemicals
L
-Proline was obtained from Tokyo Kasei Kogyo Co., Ltd (Tokyo,
nitrogen atom attaching the nitroso group and protonation of oxy-
gen atom are present at an equilibrium process. In addition, the
amino nitrogen atom has been reported to be protonated predom-
inantly below pH 2.37 The initial step underwent necessarily N
protonation to take place the transnitrosation of N-nitrosamines.31
Ohwada et al. demonstrated that alicyclic nitrosamines formed
nitrosonium ion (+NO) upon acid-catalyzed heterolytic cleavage
of the N–NO bonds.38 The observed kinetics suggest a mechanism
by which +NO is transferred directly from the protonated NP to the
thiourea without being released as nitric oxide, nitrous acid or
covalent nitrosyl chloride, and finally is transferred to N-methy-
lurea to form NMU.
Singer et al. have proposed that carcinogenic N-nitrosamines
formed by the transnitrosation of non-carcinogenic N-nitrosami-
nes under acidic conditions.32 In the present study, we demon-
strated that NMU formed in the presence of TU was mutagenic in
Salmonella typhimurium TA1535. The extract derived from the reac-
tion of NP and N-methylurea without accelerator showed no muta-
genicity. This indicates that the accelerator was essential for the
NMU formation by transnitrosation. Many ureas and carbamates
Japan). Bacto agar and bacto nutrient broth were purchased from
Becton Dickinson Microbiology System (Sparks, USA). Sodium
ammonium hydrogen phosphate tetrahydrate was obtained from
Merck (Darmstadt, Germany). Other reagents were purchased from
Wako Pure Chemical Industries (Osaka, Japan). NP was prepared
and purified by recrystallization from chloroform using the
method of Lijinsky et al. (mp 106 °C).40
5.2. Experimental
Melting points were measured on a Yanagimoto microappara-
tus and were uncorrected. The FAB-MS data were obtained with
a JEOL JMS-700 mass spectrometer. The NMR experiments were
performed with JEOL JNM-LA400 using tetramethylsilane as an
internal standard. HPLC was performed using a Shimadzu LC-6A
system [SPD-6AV UV/vis spectrometric detector]. TLC was per-
formed on precoated Kieselgel 60F254 (Merck), and spots were
visualized under UV light. Column chromatography was performed
on silica gel 60 (0.063–0.200 mm, Merck).

K. Inami et al. / Bioorg. Med. Chem. 23 (2015) 3297–3302
3301
Scheme 1. Proposed transnitrosation mechanism of NP under acidic conditions.
5.3. Identification of NMU from reaction mixture of NP and MU
in the presence of TU
5.5. The conditions of NP and N-methylurea reaction in the
presence of thioureas under acidic conditions
Each solution of N-methylurea [2.07 g (28 mmol)/28 mL) and
TU [0.85 g (11 mmol)/35 mL] in 0.1 M phosphate solution was
adjusted to pH 1.5 by adding 0.1 M HCl, and NP [4.0 g
(28 mmol)/28 mL) was dissolved in acetonitrile. The N-methy-
lurea solution and the NP solution were mixed, and the reaction
started by addition of the thioureas solution. The reaction pro-
ceeded at 37 °C for 4 h. The reaction mixture was extracted three
times with chloroform, dried over anhydrous sodium sulfate, fil-
tered, and the organic solvent evaporated to produce a tan yellow
solid. The crude product was fractionated on silica gel column
N-Methylurea (0.05 M, 0.07 M, 0.1 M), a thiourea (0.005 M,
0.01 M,0.02 M, 0.05 M) and NP (0.01 M,0.05 M, 0.07, 0.1 M) were
mixed and adjusted to the desired pH by adding of 0.1 M HCl.
The reactions were started by adding thioureas, and the incubation
was allowed to proceed at 37 °C for various times, after which ali-
quots were taken and ethyl acetate was added to each to remove
sulfur formed. The mixtures were centrifuged (3000 rpm) for
10 min, and the supernatants were analyzed by HPLC [column;
Lichrosorb RP-18 (10
lm, 4.6 ꢀ 250 mm); eluent, acetonitrile/H2O
(7:3); flow rate, 0.5 mL/min; detection, 254 nm]. The extraction
chromatography
[n-hexane/diethyl
ether/dichloro-
efficacy for NMU by ethyl acetate was 87.5%.
methane = 4:3:2, UV 254 nm] to yield 1.9 mg (yield: 0.09%) of a
white solid. mp 123 °C [lit. 126 °C (decomp)41]. 1H NMR (CDCl3,
400 MHz): d 3.19 (s, 3H, NACH3). IR (neat) cmꢁ1: 1722 (C@O),
1417 (NAN@O). The retention time by HPLC, Rf value on TLC,
1H NMR and IR spectra of the authentic NMU was identical to
that of the isolated compound.
The kinetics of the reactions was followed by monitoring the
formation of the NMU peak by HPLC. The reaction rate for the
appearance of NMU was obtained from the initial rate method
because the NMU formation rate was very small (below 5%).33,34
A plot of ln[kin] versus ln[compound] yields an order of the reac-
tion from the slope and a reaction rate from y-intercept as lnk.
Supplementary data
5.4. Bacterial mutation assay
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2015.04.058.
A residue from the chloroform extract of the reaction mixture
was obtained as described above and was assayed according to
the Ames method with
a
plate-incorporation protocol.42,43
References and notes
Salmonella typhimurium TA1535 was kindly provided by Professor
B.N. Ames (University of California, Berkeley, USA).
The fraction containing NMU was diluted with dimethyl sulfox-
ide. Solutions (each 50 lL in dimethyl sulfoxide) with varying con-
centrations of the NMU fraction were each added to a test tube,
and 0.1 M sodium phosphate buffer (pH 7.4, 0.5 mL) and a culture
of the Salmonella typhimurium TA1535 (0.1 mL) were added to the
tube and thoroughly mixed. Top agar (2 mL) was added, and the
mixture was poured onto a minimal-glucose agar plate. The rever-
tant colonies were counted after incubating at 37 °C for 44 h. Each
sample was assayed using duplicate plates twice in separate
experiments.
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