Gastric carcinogenesis: 2-Chloro-4-methylthiobutanoic acid, a novel mutagen in salted, pickled sanma hiraki fish, or similarly treated methionine

Article abstract of DOI:10.1021/tx9500585

The customary salting and pickling of fish in high risk gastric cancer regions were modeled to explore the relevant causative chemicals. The fish Sanma hiraki was treated with sodium chloride and sodium nitrite at pH 3. Previously, it had been found that an extract of the treated fish was mutagenic in Salmonella typhimurium TA 1535 without S9 and also that it induced glandular stomach cancer upon garage to rats. We now demonstrate that the mutagenicity was enhanced by preincubation of the raw meat for several days before salt-nitrite treatment. HPLC techniques showed that three mutagens were present in the fish extract. One of the mutagens was found to be stable over the pH range of 1.0-9.0. This mutagen was purified by silica gel solid phase extraction, followed by a series of reverse phase HPLC steps, and was characterized by low and high resolution MS, NMR, and FT-IR. While N- nitroso compounds were generally believed to be associated with gastric carcinogenesis, it was unexpectedly found that the mutagen has the novel structure 2-chloro-4-methylthiobutanoic acid (CMBA). Based on the structure, it seemed likely that methionine might be the precursor, and this was, indeed, proven. Both salt and nitrite are essential factors for forming this mutagen. The yield of CMBA was linear for chloride concentrations from 0 to 800 mM NaCl. Of 20 amino acids reacted with nitrite and chloride at pH 3, only methionine generated a mutagen for S. typhimurium TA 1535. Tryptophan gave a product mutagenic in S. typhimurium TA 100 and TA 98, but not TA 1535, and in the case of tyrosine, the mutagen was active only for TA 100. These results suggest an important role for salt in gastric carcinogenesis and provide new approaches for exploring the formation of mutagens/carcinogens for specific target organs.

Full text of DOI:10.1021/tx9500585

58
Chem. Res. Toxicol. 1996, 9, 58-66
Ga str ic Ca r cin ogen esis: 2-Ch lor o-4-m eth ylth iobu ta n oic
Acid , a Novel Mu ta gen in Sa lted , P ick led Sa n m a Hir a k i
F ish , or Sim ila r ly Tr ea ted Meth ion in e†
Wei Chen, J ohn H. Weisburger,* Emerich S. Fiala, Thomas E. Spratt,
Steven G. Carmella, Di Chen, and Stephen S. Hecht
American Health Foundation, Valhalla, New York 10595
Received April 11, 1995X
The customary salting and pickling of fish in high risk gastric cancer regions were modeled
to explore the relevant causative chemicals. The fish Sanma hiraki was treated with sodium
chloride and sodium nitrite at pH 3. Previously, it had been found that an extract of the treated
fish was mutagenic in Salmonella typhimurium TA 1535 without S9 and also that it induced
glandular stomach cancer upon gavage to rats. We now demonstrate that the mutagenicity
was enhanced by preincubation of the raw meat for several days before salt-nitrite treatment.
HPLC techniques showed that three mutagens were present in the fish extract. One of the
mutagens was found to be stable over the pH range of 1.0-9.0. This mutagen was purified by
silica gel solid phase extraction, followed by a series of reverse phase HPLC steps, and was
characterized by low and high resolution MS, NMR, and FT-IR. While N-nitroso compounds
were generally believed to be associated with gastric carcinogenesis, it was unexpectedly found
that the mutagen has the novel structure 2-chloro-4-methylthiobutanoic acid (CMBA). Based
on the structure, it seemed likely that methionine might be the precursor, and this was, indeed,
proven. Both salt and nitrite are essential factors for forming this mutagen. The yield of
CMBA was linear for chloride concentrations from 0 to 800 mM NaCl. Of 20 amino acids
reacted with nitrite and chloride at pH 3, only methionine generated a mutagen for S.
typhimurium TA 1535. Tryptophan gave a product mutagenic in S. typhimurium TA 100 and
TA 98, but not TA 1535, and in the case of tyrosine, the mutagen was active only for TA 100.
These results suggest an important role for salt in gastric carcinogenesis and provide new
approaches for exploring the formation of mutagens/carcinogens for specific target organs.
are present in foods so treated, in distinction to the
production of gastric carcinogens in the stomach.
We undertook studies to elucidate the chemistry of
formation of carcinogens during salting-pickling. Ear-
lier approaches were based on the pioneering finding by
Sugimura in 1966 that the nitrosamide N-methyl-N′-
nitro-N-nitrosoguanidine (MNNG)1 was the chemical of
choice for inducing glandular gastric cancer in rats,
identical to the human disease (23). MNNG is a direct
acting mutagen in the Salmonella typhimurium system
of Ames. It alkylates DNA at the N-7and O-6 positions
of guanine, as well as at other sites. Since the 1970s,
there have been numerous studies on MNNG-induced
gastric carcinogenesis (cf. ref 5).
Sanma hiraki2 is a fish consumed extensively in
Northern J apan, a high risk region for gastric cancer.
We demonstrated that an extract of the fish homogenate
pickled with a 2% NaCl/73 mM nitrite solution at pH 3
yielded a direct-acting mutagen in S. typhimurium TA
1535 (24). The mutagen formed rapidly over a broad
concentration range of nitrite. The fish mutagen sample
was stable at pH 5-7 for several days without loss of
mutagenicity and so was essentially different from
MNNG, which decomposes rapidly at neutral pH. Fur-
thermore, the mutagen could be extracted into NaOH
In tr od u ction
Gastric cancer remains one of the most common causes
of cancer deaths throughout the world, although its
incidence is declining, especially in the U.S. Epidemio-
logical studies indicate that a risk of gastric cancer is
linked with high consumption of salted, pickled food, or
elevated intake of salt and nitrate, the main common
components in salted, pickled foods (2-8). Laboratory
results and clinical observations revealed that salt influ-
ences stages of gastric carcinogenesis, such as causing
mucosal damage, inducing atrophic gastritis, and en-
hancing the action of carcinogens. Salt also augments
mucosal damage associated with Helicobacter pylori
infection, detected in high risk populations (9-15).
Nitrate in water or foods can be reduced to nitrite by the
bacterial flora in the oral cavity, or in the stomachs of
individuals with salt-induced atrophic gastritis. Nitrite
can nitrosate constituents in food to potentially mu-
tagenic/carcinogenic N-nitroso compounds that are geno-
toxic carcinogens (16-22). However, the chemical rela-
tionship between salted, pickled food intake and gastric
cancer induction is far from clear. Our approach is based
on the concept that gastric carcinogens form during
traditional salting and pickling of specific foods and thus
† A brief “correspondence” summarizing the results published in
detail here has appeared (1). A poster was presented at the 1995
annual meeting of the American Association for Cancer Research,
Proceedings 36, 116 (Abstract 688).
* To whom correspondence should be addressed. Phone: 914-789-
7141; Fax: 914-592-6317; E-mail: J ohn-Weisburger@NYMC.edu.
X Abstract published in Advance ACS Abstracts, November 15, 1995.
1
Abbreviations: MNNG, N-methyl-N′-nitro-N-nitrosoguanidine;
DMSO, dimethyl sulfoxide; EI-MS, electron impact mass spectrometry,
CMBA, 2-chloro-4-methylthiobutanoic acid; HMBA, 2-hydroxy-4-
methylthiobutanoic acid.
2
Sanma hiraki is a processed, slightly salted fish, also called Pacific
mackerel, Scomber japonicus.
0893-228x/96/2709-0058$12.00/0 © 1996 American Chemical Society
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2-Chloro-4-methylthiobutanoic Acid, Fish Mutagen
Chem. Res. Toxicol., Vol. 9, No. 1, 1996 59
Ch a r t 1. Isola tion of Mu ta gen 3 (CMBA) fr om
solution (pH 13) from organic solvents and then returned
back to organic solvents after adjusting the aqueous
phase to pH 3. Clearly, the mutagen in the salt-nitrite-
treated fish did not display the characteristics of an
N-nitrosamide. Even more importantly, glandular stom-
ach cancer and precancerous lesions were successfully
induced in Wistar strain rats by gavage feeding of the
extract of the mutagenic product over 6 months, followed
by a further 9 month period of observation (25). The
mutagenic extract, therefore, contained a gastric carcino-
gen.
Na Cl-Na NO2-Tr ea ted F ish or Met
Thus, it was important to develop procedures to isolate
and identify the fish mutagen. The extent of purification
was followed by assays of intermediate fractions for
mutagenicity in S. typhimurium TA 1535. Chromatog-
raphy involved preliminary solid phase extraction, pu-
rification through multiple HPLC steps, and, finally,
determination of chemical structure by appropriate meth-
ods. It was found that three mutagens are actually
present in the fish extract. One of these was effectively
purified to homogeneity. An unexpected discovery was
made that this mutagen is not an N-nitroso compound,
but rather a new, not heretofore described chemical,
2-chloro-4-methylthiobutanoic acid (CMBA). The same
product was obtained by using methionine (Met) in the
reaction sequence, and the concentration of chloride
played a key role in its formation. Mutagenic products,
similar to that obtained with Met, were not seen with
19 other amino acids treated in the same way, although
tryptophan yielded mutagenic activity for S. typhimu-
rium TA 100 and TA 98, and tyrosine for TA 100 only.
These experiments and supporting conclusions are pre-
sented in this paper.
Exp er im en ta l P r oced u r es
F ish . Sanma hiraki, a lightly salted and semidried frozen
J apanese fish, was imported from Shimoya Heijiroh Shooten
(2192 Uyematsu-cho, Chohshi-shi, Chiba, J apan), purchased
from a local J apanese supermarket, and kept at -20 °C until
use.
Ch em ica ls. ACS-grade sodium chloride, sodium nitrite,
sodium hydroxide, acetic acid, and ammonium sulfamate were
purchased from Aldrich (Milwaukee, WI), as was CD3OD (99.8%
D). Twenty L-amino acids were acquired from Sigma (St. Louis,
MO), among which alanine, asparagine, cysteine, methionine,
phenylalanine, proline, threonine, tryptophan, tyrosine, and
valine were SigmaUltra grade. HPLC-grade ethyl acetate,
methylene chloride, hexane, methanol, and acetonitrile were
obtained from Scientific Products/Baxter (Edison, NJ ). Con-
centrated H3PO4 (85%) was from Fisher (Springfield, NJ ).
2-Hydroxy-4-methylthiobutanoic acid (HMBA) was a gift of Dr.
W. D. Shermer, Monsanto Co. (Chesterfield, MO).
In str u m en ta tion . A Savant Model SC100 Speed Vac (Sa-
vant Instruments, Inc., Farmingdale, NY) was used to concen-
trate samples. HPLC was carried out on a Waters Model 600
multisolvent delivery system (Millipore Corp., Milford, MA) with
a Hewlett Packard (HP, Avondale, PA) Model 1040 Series II
HPLC detection system. UV spectra were determined with a
Beckman DU 640 spectrometer (Beckman Instruments, Inc.,
Fullerton, CA). GC/MS was performed using a HP Model 5971A
mass-selective detector, interfaced to a HP Model 5890 Series
II gas chromatograph with a HP Model 7673 automatic sampler.
NMR spectra were obtained with a Bruker AM360 WB spec-
trometer using TMS as an internal standard. FT-IR spectra
were acquired on a Polaris spectrometer (Mattson Instruments,
Inc., Madison, WI). The samples were directly applied to Type
61 disposable IR cards (3M, St. Paul, MN) for analysis, and
spectra were acquired with a nitrogen purge through the sample
compartment.
Mu ta gen icity Assa ys. Direct-acting mutagenic activity
(without liver S9) was determined in the American Health
Foundation In Vitro Molecular Analysis Facility, using the
method described by Maron and Ames (26) and Levin et al. (27),
in S. typhimurium strains TA 1535, TA 100, TA 98, and TA
102. Samples for the Ames tests were concentrated to dryness
and redissolved in H2O with 10-20% DMSO, except for the
samples tested for stability as a function of pH, which were
redissolved in small volumes of different pH buffers. For each
test, the assays were done in triplicate at two concentrations of
substrate.
P r ep a r a tion of F ish Extr a ct. Sanma hiraki fillet was
minced and incubated at room temperature (23 °C) for 0-7 days
(Chart 1). The preincubated fish muscle was homogenized with
twice the volume (w/v) of deionized H2O in a blender, and then
solid NaCl was added to a concentration of 2%. After stirring
for 1 h at room temperature, the homogenate was centrifuged
at 21000g for 30 min. The sediment was discarded, and the
supernatant was adjusted to pH 3 by adding 12 N HCl. The
volume was reduced to one-fourth in vacuo on a rotary evapora-
tor. Following another centrifugation under the same condi-
tions, the clear supernatant was incubated with NaNO2 (5 mg/g
fish) at 23 °C in the dark for 1 h, and the pH was kept at 3 with
12 N HCl. The reaction was terminated by adding an equimolar
amount of ammonium sulfamate.
A three step extraction was then carried out. First, the
solution was extracted three times with equal volumes of ethyl
acetate. Second, the combined ethyl acetate fractions were
extracted twice with 0.25 volume of 0.4 N NaOH, and the
aqueous layers were quickly readjusted to pH 3 with 12 N HCl.
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60 Chem. Res. Toxicol., Vol. 9, No. 1, 1996
Chen et al.
Third, the aqueous fraction was extracted three times with 0.8
volume of methylene chloride. The collected extracts were
evaporated to dryness, and the residue, dissolved in a small
volume of MeOH (20%)-H2O water (80%), was used for further
study. Controls were done (1) with 100 mL of supernatant
obtained in the same way, but without nitrite, and (2) with 100
mL of H2O, instead of fish supernatant, treated with salt and
nitrite at pH 3.
Tr ea tm en t of Meth ion in e. NaCl was added to 10 mL of
Met (1 mmol) in H2O to give a concentration of 2%. The solution
was incubated with 0.73 mmol of NaNO2 at 23 °C for 1 h after
adjustment to pH 3 with 0.5 N HCl. Excess nitrite was
quenched by the addition of an equimolar amount of ammonium
sulfamate. The mixture was extracted three times with equal
volumes of ethyl acetate, and the combined organic phases were
evaporated in vacuo. The residue was dissolved in MeOH and
diluted with H2O to give a 1:4 (v/v) MeOH-H2O solution for
determination of mutagenicity, or purification by HPLC. Three
different controls, (1) without Met, (2) without NaCl and with
H2SO4 replacing HCl, and (3) without NaNO2, were prepared,
using the same procedure.
Ch r om a togr a p h ic Sep a r a tion a n d P u r ifica tion . The
fish extract was separated into crude mutagenic fractions using
solid phase silica gel extraction. After the extract was dissolved
in a small volume of hexane-CH2Cl2 (1:2), it was loaded onto a
900 mg silica gel Maxi-Clean cartridge (Alltech 20988). The
cartridge was eluted sequentially with a series of organic
solvents: (1) 100% hexane; and then hexane-CH2Cl2-ethyl
acetate mixtures: (2) 70:25:5; (3) 60:30:10; and (4) 50:30:20.
Each fraction was evaporated to dryness, and the residues were
dissolved in MeOH-H2O (1:9) for mutagenicity assays and
further separation by HPLC.
A three-step reverse phase HPLC was developed to isolate
and purify the mutagen from the crude fraction. For step 1, a
10 × 250 mm Ultrasphere ODS column with a 4.6 × 45 mm
Ultrasphere ODS guard column was employed. The mobile
phase A was 0.34 mM H3PO4 in 10% MeOH, and B was 0.5 mM
H3PO4 in 100% MeOH at a flow rate of 1.5 mL/min. The
gradient program was an initial 8 min at 100% A, then 30 min
to 50% B (linear), and a further 50 min to 100% B. For step 2,
a 4.6 × 250 mm Ultrasphere ODS column with a 4.6 × 45 mm
same type guard column was used. The eluant was H2O (A)-
acetonitrile (B) at a flow rate of 1 mL/min, using a linear
gradient from 1% B to 50% B in 25 min, and to 95% B in 35
min. For step 3, the same columns were used as step 2, but
the mobile phase was 0.1% acetic acid in 10% MeOH (pH 3.5)
(A)-acetonitrile (B). The gradient was linear from 0% to 50%
B over 20 min at a flow rate of 1 mL/min. The fractions eluting
from each step of the chromatographic procedures were col-
lected, were evaporated to dryness, and were used for mutage-
nicity assays, further purification, or instrumental analysis.
Except for the silica gel solid phase extraction, which was
not required in this instance, the same HPLC methods were
applied for purification of the mutagen from NaCl-NaNO2-
treated Met.
MS An a lysis. For GC/MS, the purified mutagenic compound
was dissolved in acetonitrile. A HP Ultra-2 capillary column
(5% phenylmethylsiloxane), 12 m × 0.2 mm i.d., 0.11-µm film
thickness, was used. The injector temperature was 175 °C, the
detector temperature was 280 °C, and helium was the carrier
gas. The initial temperature was 40 °C for 3 min, then increased
to 200 °C at 10 min at a rate of 30 °C/min.
The spectral properties of mutagen 3 (F3) from fish or Met
were as follows: UV (H2O), λmax less than 190 nm, 205 (sh); 1H-
NMR (CD3OD) δ 4.57 (d/d, 1H, 2-CH, J ) 2.0, 1.2 Hz), 2.68 (m,
2H, 4-CH2), 2.34 (m, 1H, 3-CH), 2.20 (m, 1H, 3-CH), 2.17 (s,
1H, CH3); 1H-NMR (CD3OD + NaOD) δ 4.37 (d/d, 1H, 2-CH),
2.67 (m, 2H, 4-CH2), 2.30 (m, 1H, 3-CH), 2.15 (m, 1H, 3-CH),
2.13 (s, 1H, CH3); 13C-NMR (CD3OD) δ 173.0 (s, CO), 57.5 (d, J
) 156 Hz), 31.2 (t, J ) 137 Hz), 35.5 (t, J ) 131 Hz), 15.0 (q, J
) 138 Hz); low resolution GC/EI-MS, 6.83 min, M, m/ z 168 and
M + 2, m/ z 170 (33.3% of M); high resolution FT-MS, calcd for
C5H9ClO2S 168.000628, found 167.996597.
Effect of Na Cl Con cen tr a tion in th e Met-Na Cl-Na NO2
Rea ction . Different amounts of NaCl were added to 0.5 mmol
of Met in 10 mL of H2O to final concentrations of 0, 12.5, 50,
100, 200, 400, 800, or 1600 mM. At room temperature, each
solution, adjusted to pH 3 with 0.5 N H2SO4, was reacted with
0.5 mmol of NaNO2. After 1 h, the reaction was stopped with
0.5 mmol of ammonium sulfamate. The solutions were ex-
tracted three times with equal volumes of ethyl acetate. After
removal of the organic solvent, the residue was dissolved in 500
µL of 0.3 mM H3PO4-20% MeOH for analysis. Every experi-
ment was done in duplicate.
Qu a n tita tion of CMBA P r od u ced fr om th e Rea ction of
Met w ith Nitr ite a n d Differ en t Con cen tr a tion s of Na Cl.
Reverse phase HPLC employing an Ultrasphere ODS 5 µm 4.6
× 250 mm column with a 4.6 × 45 mm guard column was used
to quantitate CMBA. The mobile phase A was 0.85 mM H3PO4
in 10% MeOH (pH 3); B was 0.85 mM H3PO4 in 100% MeOH,
and C was acetonitrile. A linear gradient program started from
100% A to 50% B in 20 min and reached 60% B and 30% C in
25 more min. The homogeneity of each CMBA sample was
determined by monitoring UV spectra with diode array detection
and by MS. Standard curves of peak area, as a function of µmol
of CMBA, as well as of HMBA were prepared. Also, a synthetic
sample of CMBA, obtained by three-step synthesis, beginning
with HMBA (DL-methionine hydroxy analog, Sigma M-0126)
(esterification to benzyl ester, conversion to CMBA benzyl ester
with thionyl chloride, and hydrolysis of the ester), was available
through the courtesy of Drs. S. Amin and Dr. J . Kreminski,
American Health Foundation Organic Synthesis Facility.
Effect of Sa lt-Nitr ite a t p H 3 on Am in o Acid s. Solutions
of 1 mmol each of Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His,
Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, or Val, or of 0.4 mmol of
Trp, were prepared in 10 mL of H2O. Because of limited
solubility, 0.5 mmol Tyr was dissolved in 50 mL H2O. NaCl
was added to each of the solutions to give a concentration of
2% NaCl. These solutions were incubated at 23 °C with 0.73
mmol of NaNO2 at pH 3 in dark for 1 h, and an equal
concentration of ammonium sulfamate was added. The treated
solutions were then extracted with 3 equal volumes of ethyl
acetate. The organic solvent was evaporated, and the residue
of each sample was dissolved in 200 µL of MeOH and diluted
with H2O for determination of mutagenicity.
Resu lts
Dir ect-Actin g Mu ta gen icity. The extract of Sanma
hiraki treated with salt-nitrite displayed mutagenic
activity in S. typhimurium TA 1535 without S9. Deletion
of either fish or nitrite from the incubation, with analo-
gous workup, failed to yield mutagenic activity. The
mutagenicity was not significantly affected by exposure
to light or by incubation of the treated fish extract at 50
°C. The mutagenicity was further enhanced by prein-
cubation of the fish fillet at room temperature for 1-7
days, with a maximum activity observed at day 2 (Figure
1). Extracts of Met that had been reacted with salt and
nitrite under identical conditions also yielded mutage-
nicity. Mutagenicity was not found when Met, NaCl or
NaNO2 were omitted (Table 1).
P r elim in a r y Sep a r a tion of Mu ta gen s. The initial
chromatography of the fish extract resulted in three
separately eluting fractions containing mutagenic activity
(Figure 2A), suggesting the presence of three individual
mutagens, designated mutagen 1, 2, and 3 (F1, F2, and
F3). Mutagens 1 and 2 were relatively polar, eluting
between 23 and 30 min. Mutagen 3 was relatively less
polar, eluting between 46 and 47 min and containing
about 26% of the total mutagenicity. Mutagen 3 could
also be separated from the other two by adsorption onto
a silica solid phase extraction cartridge, from which it
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2-Chloro-4-methylthiobutanoic Acid, Fish Mutagen
Chem. Res. Toxicol., Vol. 9, No. 1, 1996 61
F igu r e 1. Mutagenicity in S. typhimurium TA 1535 as a
function of the amount of the extracts from salt-nitrite-treated
fish fillets (g) that had been preincubated at 23 °C for various
times. Each treatment was performed in duplicate. Some of the
highest levels of fish that had been preincubated for 2 and 7
days were toxic. Error bars show standard deviation of 4
determinations, and * indicates significant differences between
1-7 days and 0 day preincubation.
F igu r e 2. Reverse phase HPLC of the extracts of NaCl-NaNO2
-treated fish on a 10 × 250 mm Ultrasphere ODS column. The
upper curve (-‚-) shows the solvent gradient program used;
0.34 mM H3PO4 in 10% MeOH (pH 3.4) was mobile phase A
and 0.34 mM H3PO4 in 100% MeOH was mobile phase B at a
flow rate of 1.5 mL/min. Three mutagenic fractions (F1, F2, and
F3) were detected in the crude fish extract (boxed area, A). The
fraction containing mutagen 3 (F3), eluting from 47 to 48 min,
was separated on a silica gel solid phase cartridge followed by
HPLC (B). Part C shows that the mutagenic compound from
the extract of NaCl-NaNO2-treated Met eluted with the same
retention time as F3.
Ta ble 1. Dir ect-Actin g Mu ta gen icity in S. typh im u r iu m
TA 1535 In d u ced by Na Cl-Na NO2-Tr ea ted F ish Extr a ct
or Met a n d th e Effect of Tem p er a tu r e a n d Ligh t on th e
Mu ta gen icity of th e F ish Extr a ct
rev/plate ( SD
time
(min)
1 ga or
2 ga or
treatment
8 µmolb
16 µmolb
H2O + Cl- + NO2
fish + Cl-
37 ( 2
27 ( 4
25 ( 4
32 ( 2
-
fish + Cl- + NO2
23 °C + darkc
50 °C + dark
4 °C + light
360 ( 16
362 ( 35
354 ( 6
379 ( 4
26 ( 2
520 ( 18
503 ( 18
505 ( 6
534 ( 10
34 ( 8
-
150
30
150
Met + Cl-
Met + NO2
-
39 ( 3
297 ( 11
44 ( 3
462 ( 17
Met + Cl- + NO2
-
a
Extract equivalent to wet weight grams of fish fillets. b Extract
equivalent to amounts of Met. c Samples of extracts from salt-
nitrite-treated fish were maintained as shown for the times listed.
could be eluted with a mixture of 60% hexane, 30% CH2-
Cl2, and 10% ethyl acetate.
Isola tion a n d P u r ifica tion of Mu ta gen 3. After
preliminary separation of mutagen 3 by solid phase
extraction, followed by the step-1 HPLC system, mutage-
nicity was contained in a fraction eluting at 47-48 min
(Figure 2B). This fraction was collected, concentrated,
and reinjected into the step-2 HPLC system. Upon
elution with H2O-acetonitrile, a peak eluting at 26 min
contained most of the mutagenic activity (Figure 3A).
Since this compound had weak absorption at 254 nm, the
detecting wavelength was adjusted to 220 nm. The final
chromatography yielded a single mutagenic peak eluting
at 16 min (Figure 4A).
F igu r e 3. Step-2 HPLC. (A) After the mutagenic fraction at
47-48 min from the fish extract was concentrated to a small
volume, it was further separated by a 4.6 × 250 mm Ultrasphere
ODS column with H2O (phase A) and acetonitrile (phase B) at
a flow rate of 1.0 mL/min. The fraction eluting from 25 to 26
min contained most of the mutagenic activity and was collected
for further HPLC purification. (B) The Met-derived product
displays an identical elution pattern.
F or m a tion of Mu ta gen 3 fr om Met. The reaction
of salt-nitrite at pH 3 with Met gave only a single
mutagenic peak with an HPLC mobility identical to that
of mutagen 3 from the treated fish extract (Figures 2C,
3B, and 4B).
pH range (1-9), purified mutagen 3 showed no signifi-
cant change in its mutagenic activity. It was stable in
phosphoric acid solution, pH 3, for 26 days. After 48
days, the mutagen retained more than 60% of its activity
(Figure 5).
Sta bility of Mu ta gen 3 a t Differ en t p H Va lu es a n d
Stor a ge Tim e. After storage for 60 min over a broad
Sen sitivity of Differ en t S. typh im u r iu m Str a in s
to Mu ta gen 3. Purified mutagen 3 exhibited a higher
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62 Chem. Res. Toxicol., Vol. 9, No. 1, 1996
Chen et al.
F igu r e 4. Final purification step (base line adjusted). The same
column was used as in step 2, and the mobile phases were 0.1%
acetic acid in 10% MeOH (A) and acetonitrile (B), with a 1 mL/
min flow rate. The main peak, eluting at 17 min and showing
mutagenicity in S. typhimurium TA 1535, was collected and
evaporated to dryness for spectral analysis. (A) Compound from
fish and (B) compound from Met.
F igu r e 6. Mutagenicity in S. typhimurium TA 1535 and TA
100 induced by purified mutagen 3.
F igu r e 5. Mutagenicity induced by mutagen 3 as a function
of pH or storage time. Determination of the effects of storage
time was done with the sample kept in H3PO4 solution, pH 3.4.
mutagenic activity in the base-substitution strain S.
typhimurium TA 1535 than in TA 100 at equal test levels
(Figure 6).
F igu r e 7. GC/EI-MS analysis of the mutagen, CMBA, purified
from treated fish (A) or Met (B). The relative abundance of
fragment M + 2, m/ z 170, is 35% of that of M, m/ z 168,
providing evidence for the presence of a chlorine atom.
Id en tifica tion of Mu ta gen 3. The UV spectrum of
mutagen 3 (F3) in H2O showed a shoulder at 205 nm (ꢀ
1600) with a λmax of less than 190 nm, indicating absence
of aromatic or conjugated structure. Analysis by GC/EI-
MS showed a major peak eluting at 6.83 min with M+
m/ z 168 and 170, the latter at one-third intensity,
suggesting the presence of Cl (Figure 7A). High resolu-
tion FT-MS gave a molecular weight of 167.996597, with
the possible formula C5H9ClO2S (calculated mol wt
168.000628).
The FT-IR spectrum showed a strong absorption band
at 1726 cm-1, providing evidence for the presence of a
carbonyl group. The position of this band is consistent
with that expected of an R-chloro-substituted carboxylic
acid.
Ta ble 2. NMR Da ta for CMBA Obta in ed fr om
Na Cl-Na NO2-Tr ea ted F ish or L-Met
δ 1Hb
δ 1Ha
(CD3OD)
(CD3OD +
δ
13Cc
(CD3OD)
NaOD)
CO
173.0 s
57.5 d (J ) 156 Hz)
31.2 t (J ) 137 Hz)
2-CH,d 1H, d/d
3-CH, 1H, m
3-CH, 1H, m
4-CH2, 2H, m
CH3, 1H, s
4.57
2.34
2.20
2.68
2.17
4.37
2.30
2.15
2.67
2.13
35.5 t (J ) 131 Hz)
15.0 q (J ) 138 Hz)
a
1H-NMR spectra of CMBA obtained from either treated fish
b
or Met. This spectrum of CMBA was obtained from treated fish.
The 1H-NMR spectrum of mutagen 3 was similar to
that of Met, with a methyl resonance at 2.13 ppm, a
methine proton at 4.57 ppm, two protons at 2.68 ppm,
and individual protons at 2.34 and 2.20 ppm (Table 2,
Figure 8B). Decoupling and COSY experiments showed
c 13C-NMR spectrum of CMBA from Met. J ) 2.0, 1.2 Hz.
d
that the signals at 2.34 and 2.20 ppm were coupled to
each other and to the signals at 4.57 and 2.68 ppm. The
coupling pattern of the 1H coupled 13C-NMR spectra
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2-Chloro-4-methylthiobutanoic Acid, Fish Mutagen
Chem. Res. Toxicol., Vol. 9, No. 1, 1996 63
F igu r e 9. Typical reverse phase HPLC (see text for method)
of standard CMBA (A) and Met treated with salt-nitrite (B),
giving CMBA and HMBA.
F igu r e 8. 1H-NMR spectrum of CMBA (B: from treated fish,
C: from Met). Part A shows that the signal at 4.57 ppm was
shifted by adding NaOD, which indicates that the chlorine is
in the R-position to the carboxylic group (see text).
confirmed that there was one CH, two CH2, and one CH3
groups. The chemical shift of the methyl group in the
1H-NMR spectra suggested that it could be attached to
S or to a carbonyl. FT-IR spectroscopy and NMR
spectrometry with NaOD, as described below, indicate
that the carbonyl in the compound is a carboxylic acid.
The above evidence is consistent with a structure of
XYCHCH2CH2Z, in which X,Y, and Z are COOH, Cl, and
CH3S.
F igu r e 10. Effect of NaCl concentration on CMBA formation,
quantitated by reverse phase HPLC. Data ( SD with 3-5
assays.
To identify the position of the carboxylate, the 1H-NMR
spectrum in CD3OD was compared with the one obtained
upon NaOD addition. It would be expected that the
protons closest to the COOH group show the largest
decrease in chemical shift. The signal at 4.75 ppm, which
was assigned to the methine proton, was shifted upfield
0.2 ppm, while the shifts of the other signals were less
than 0.05 ppm (Figure 8A). Therefore, X (or Y) was
determined to be a carboxy group. The chemical shift of
the terminal methylene is consistent with Z as the
thiomethyl but not the chloro moiety. Similarly, the
chemical shift of the methine proton is consistent with
carboxylate and chloro but not carboxylate and thio-
methyl substituents. Evaluation of the combined ana-
lytical data indicated that mutagen 3 is 2-chloro-4-
methylthiobutanoic acid (CMBA), a new compound.
single peak at 14 min (B, Figure 9). This elution pattern
was identical to that of an authentic sample of HMBA
obtained from Dr. W. D. Shermer. The structure was
confirmed by GC/EI-MS and 1H-NMR. The yield of
CMBA was directly proportional to the concentration of
sodium chloride, up to 800 mM, but it leveled off when
excess chloride (1600 mM) was added (Figure 10). The
quantity of the HMBA decreased with increasing con-
centrations of NaCl. In addition to CMBA and the
HMBA, HPLC of Met treated with NaCl-NaNO2 showed
other minor products of unknown structure, but these
had no mutagenicity.
Mu ta gen icity of 20 Am in o Acid s Tr ea ted w ith
Na Cl-Na NO2 a t p H 3. Following reaction with salt and
nitrite at pH 3, only Met yielded direct-acting mutage-
nicity in S. typhimurium TA 1535. The NaCl-NaNO2-
treated Met also showed direct-acting mutagenicity in
TA 100, but the response was lower compared to that
obtained with TA 1535. Mutagenicity was not evident
in S. typhimurium strains TA 102 or TA 98. A dose-
response of mutagenicity in TA 100 and TA 98 was
observed only with NaCl-NaNO2-treated Trp and was
positive only in TA 100 with Tyr. None of the other
amino acids tested gave significant mutagenic products
(Tables 3 and 4).
Mu ta gen 3 fr om Met. In all of the analytical systems
used, the Met-derived mutagen displayed the same
characteristics as mutagen 3 from fish. GC/EI-MS data
1
were essentially identical (Figure 7B), as were the H-
NMR data (Figure 8C). FT-IR, a strong absorption band
at 1726 cm-1, was characteristic of a CdO group. Thus,
these data entirely support the structure of mutagen 3
as CMBA, produced independently from Met.
F or m a tion of CMBA fr om Met Tr ea ted w ith
Nitr ite a n d Differ en t Con cen tr a tion s of Sod iu m
Ch lor id e. Under the HPLC conditions described, stan-
dard CMBA eluted at 27 min as a single peak (Figure
9A). A linear relationship (r ) 0.996) was established
for peak area versus µmol of CMBA injected. Using the
same HPLC system, CMBA from Met treated with nitrite
and different concentrations of NaCl eluted at the same
time as the standard (Figure 9B). This peak was absent
when Met was treated only with nitrite (no NaCl). Under
those conditions, HMBA was formed, which eluted as a
Discu ssion
For decades, N-nitroso compounds have drawn most
attention as the carcinogens associated with gastric
cancer (5, 16-23). Indeed, such chemicals are broadly
acting carcinogens and are among the most potent animal
carcinogens. Since many nonmutagenic/noncarcinogenic
precursor compounds become mutagenic/carcinogenic
+
+

64 Chem. Res. Toxicol., Vol. 9, No. 1, 1996
Chen et al.
Ta ble 3. Dir ect-Actin g Mu ta gen icity in S. typh im u r iu m TA 1535, TA 102, TA 100, a n d TA 98 In d u ced by Am in o Acid s
Tr ea ted w ith Sa lt-Nitr ite, p H 3.0
rev/platea
TA 1535
8 µmol 16 µmol
TA 102
16 µmol
TA 100
8 µmol 16 µmol
TA 98
sample
8 µmol
8 µmol
16 µmol
DMSOb
26 ( 7
1058 ( 116
372 ( 34
128 ( 7
882 ( 4
32 ( 7
controlsc
1111 ( 158
391 ( 41
Met + Cl- + NO2
Phe + Cl- + NO2
297 ( 11
462 ( 17
43 ( 12
36 ( 7
389 ( 11
381 ( 16
100 ( 23
349 ( 17
584 ( 27
660 ( 13
26 ( 1d
310 ( 8
358 ( 6
31 ( 10
146 ( 4
141 ( 5
492 ( 6
21 ( 1d
34 ( 6
40 ( 2
43 ( 1
39 ( 3
120 ( 28
53 ( 7
35 ( 1
-
-
24 ( 3
41 ( 14
48 ( 13
38 ( 6
28 ( 6
426 ( 2
321 ( 8
520 ( 7
539 ( 9
225 ( 66
198 ( 21
145 ( 8
188 ( 3
347 ( 1
263 ( 28
39 ( 1
43 ( 12
32 ( 1
74 ( 6
22 ( 4
Pro + Cl- + NO2
Ser + Cl- + NO2
Trp + Cl- + NO2
Tyr + Cl- + NO2
-
-
54 ( 11
50 ( 10
25 ( 6
-
-
a
b
Every sample was tested in triplicate at 8 and 16 µmol of amino acid, except Trp and Tyr, which were 4 and 8 µmol. DMSO, 100
µL/plate. c Controls: Sodium azide, 5 µg/plate, for TA 1535 and TA 100; cumene hydroperoxide, 150 µg/plate, for TA 102; 2-nitrofluorene,
5 µg/plate, for TA 98. Toxic.
d
Ta ble 4. Dir ect-Actin g Mu ta gen icity in S. typh im u r iu m
is designed to reproduce traditional pickling methods (22,
24, 25). Recipes reviewed by Binkerd and Kolari (22) and
by Lauer (31) called for the use as much as 7.5% crude
salt and as much as 2% crude sodium nitrate/sodium
nitrite.
TA 1535 of Am in o Acid s Tr ea ted w ith Sa lt-Nitr ite a t p H
3.0
rev/platea
sample
DMSOb
8 mmol
16 mmol
The separation of mutagens in the salt-nitrite-treated
fish extract yielded three fractions with mutagenicity in
the first HPLC step. The first two mutagenic compounds,
designated mutagen 1 (F1) and mutagen 2 (F2), were
present in the earlier-eluting more polar fractions, and
mutagen 3 (F3) was contained in a later-eluting fraction.
This paper is the first detailed report of a unique kind
of mutagen, F3, formed under laboratory conditions of
traditional “salting-pickling” of food. The procedure
reproduced the usual custom in the Far East of storing
fish, usually with crude salt, at room temperature for a
period of time to enhance its flavor. This technique
increased the mutagenicity significantly when the raw
fish fillets were kept at 23 °C for 1-7 days. Based on
the chemical structure of mutagen 3, CMBA, it was likely
that Met derived from the fish protein is the precursor.
One of the possible causes of the increased mutagenicity
on standing is that more free precursors such as Met
were released by the hydrolysis of protein, a reaction
possibly affected by the salt added to the fish as part of
the preservation process (32). Thus, the role of Met in
forming the mutagen was, indeed, confirmed.
26 ( 7
1058 ( 116
25 ( 5
b
NaN3
Ala + Cl- + NO2
31 ( 4
40 ( 2
-
-
Arg + Cl- + NO2
Asn + Cl- + NO2
Asp + Cl- + NO2
29 ( 1
30 ( 1
24 ( 1
28 ( 1
33 ( 5
42 ( 1
26 ( 1
26 ( 2
22 ( 3
40 ( 5
23 ( 1
40 ( 5
26 ( 4
-
-
35 ( 3
29 ( 3
Cys + Cl- + NO2
Gln + Cl- + NO2
Glu + Cl- + NO2
-
-
-
43 ( 2
28 ( 1
21 ( 4
27 ( 7
23 ( 1
25 ( 1
30 ( 4
29 ( 1
Gly + Cl- + NO2
His + Cl- + NO2
-
-
Ile + Cl- + NO2
-
Leu + Cl- + NO2
-
Lys + Cl- + NO2
Thr + Cl- + NO2
-
-
Val + Cl- + NO2
-
a
Every sample was tested in duplicate at 8 and 16 µmol of
b
treated amino acid. DMSO solvent (100 µL/plate) and positive
control NaN3 (5 µg/plate).
upon treatment with nitrite at low pH, it has been
hypothesized that gastric cancer in humans may result
from the exposure to N-nitroso compounds formed when
nitrite reacts with specific substrates in food during
traditional methods of preservation with salt and nitrite
(5, 22). Nitrosation, therefore, is extensively used as a
method which imitates the traditional food preservation
process for generating mutagenic/carcinogenic com-
pounds. Through this reaction, some constituents in
different food mixtures were identified as precursors to
potential mutagens/carcinogens. 4-Chloro-6-methoxyin-
dole in fava beans yielded 4-chloro-6-methoxy-2-hydroxy-
1-nitrosoindolin-3-one oxime, a direct-acting mutagen to
S. typhimurium TM 677 (28). Indole-3-acetonitrile from
Chinese cabbage formed a mutagen to S. typhimurium
TA 100 and TA 98 without S9, 1-nitrosoindole-3-aceto-
nitrile (29, 30). Thus, N-nitroso compounds have been
considered as the most probable candidates for gastric
carcinogens. The identification of the mutagen in Sanma
hiraki extract as CMBA opens a new, distinct area of
investigation in gastric cancer and markers associated
therewith.
The reaction of nitrite with the aliphatic amino group
is expected to generate a reactive diazonium ion. In the
presence of H2O, this ion would yield the corresponding
hydroxy acid, but in the presence of sufficient amounts
of chloride ion, the R-chlorocarboxylic acid is formed:
Basically, two levels of nitrite are generally used in
studies on factors bearing on gastric cancer. One is
nitrite in the micromolar range, simulating “realistic”
conditions in the gastric environment with formation of
the carcinogen in the stomach (18). In contrast, nitrite
in the millimolar range, used in the present approach,
Clearly, Cl- is essential for the production of the
mutagen, and the formation of CMBA was linear for Cl-
from 0 to 800 mM. The effect of salt on gastric carcino-
genesis has been studied in terms of a potent enhancing
+
+

2-Chloro-4-methylthiobutanoic Acid, Fish Mutagen
Chem. Res. Toxicol., Vol. 9, No. 1, 1996 65
action through several mechanisms. The mutagenicity
of nitrosated black beans was nearly doubled by adding
salt to this staple food in Costa Rica, a region with a high
gastric cancer rate (33). Our results provide strong
evidence of a chemical relationship between NaCl and
nitrite under acidic pickling conditions which thus influ-
ences gastric carcinogenesis. Wang et al. (34), in an
abstract, reported the formation of reactive products,
suggested to be diazo compounds, that could interact with
3,4-dichlorothiophenol, upon treating amino acids with
nitrite at low pH.
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active in this strain than in strain TA 100. It displayed
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In summary, we have developed a systematic approach
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rium TA 1535, and likely gastric carcinogens in rats, from
an extract of salt-nitrite-treated Sanma hiraki fish.
Instead of the expected N-nitroso compound, a novel
mutagen, CMBA, was isolated and identified. According
to the chemical structure analysis, Met from the fish
protein is proposed as its precursor, and the identical
product was, indeed, obtained from Met. Importantly,
both nitrite and Cl- are essential to its formation during
the salting-pickling process. The formation of a mu-
tagenic R-chloro acid as a product appears to be unique
to Met. Yet, the simplest analog, R-chloroacetic acid, is
not mutagenic or carcinogenic (38). Thus, the entire
CMBA molecule is required for direct-acting mutagenicity
and probable gastric carcinogenicity. Dr. C. Furihata,
who pioneered effective in vivo rapid bioassays for gastric
carcinogens (39), has observed a positive effect with
CMBA. Of the 20 amino acids studied, no other amino
acids yielded any mutagenic products to the Ames tester
strain S. typhimurium TA 1535. The carcinogenic and
genotoxic effect of CMBA is being studying in this
laboratory.
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Ack n ow led gm en t. This research was supported, in
part, by U.S. Public Health Service Grants CA-29602 and
CA-47520, and Cancer Center Support Grant CA-17613,
from the National Cancer Institute. In the past, visiting
scientists in our laboratory, Dr. Hildegard Marquardt,
now in Hamburg, Germany, Dr. Howard Mower, Uni-
versity of Hawaii, on sabbatical leave, and Ms. Graczina
Prokopcyzk, had been active in this research. We are
most grateful for the effective contributions of R. Sodum,
S. Coleman, D. Pullo, J .-M. Lin, F.-Q Lou, and B.
McKinney for expert and dedicated assistance in various
aspects of this investigation. We thank Dr. Sanford L.
Shew, Millipore Co., for performing high resolution MS,
J ames E. Gagnon, 3M (St. Paul, MN), for providing
Disposable IR cards and information on their use, and
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66 Chem. Res. Toxicol., Vol. 9, No. 1, 1996
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30, 617-626.
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reaction of amino acids with nitrite in acidic solution. Agric. Biol.
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(4-chloro-6-methoxy-2-hydroxy-1-nitrosoindolin-3-one oxime) that
forms during nitrosation of the fava bean (Vicia faba). Carcino-
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a
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