Antimutagenic Properties of 3,5-Disbustituted 2-Thiohydantoins

Article abstract of DOI:10.1021/jf980430x

3,5-Disubstituted 2-thiohydantoins, which were prepared by the reaction of allyl isothiocyanate (AITC) or 4-(methylthio)-3-butenyl isothiocyanate (MTBI) with various amino acids, were assayed for their inhibitory effects on the mutagenicity of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) on Salmonella typhimurium TA 98. When the 3,5-disubstituted 2-thiohydantoins (125-500 μg/mL) were simultaneously preincubated with the bacterial strain, IQ (0.5 μg/mL), and rat microsomal fraction (S9 mix) for 20 min, a dose-dependent inhibition of IQ mutagenicity was observed in the tested compounds except one prepared from MTBI and L-methionine. This inhibition ranged from 23% to 86%. The inhibitory effect either disappeared or was largely reduced to 5-19%, when the 3,5-disubstituted 2-thiohydantoins were added after metabolic activation of IQ with S9 mix. These results suggest that the 3,5-disubstituted 2-thiohydantoins probably acted as an inhibitor of the S9 mix-mediated metabolic activation of the mutagen.

Full text of DOI:10.1021/jf980430x

J. Agric. Food Chem. 1998, 46, 5037−5042
5037
An tim u ta gen ic P r op er ties of 3,5-Disu bstitu ted 2-Th ioh yd a n toin s
Asaka Takahashi,† Hiroki Matsuoka,‡ Yoshio Ozawa,‡ and Yasushi Uda*,†
Department of Bioproductive Sciences, Faculty of Agriculture, Utsunomiya University,
Utsunomiya 321-8505, J apan, and Gunma Women’s J unior College, Takasaki 370-0033, J apan
3,5-Disubstituted 2-thiohydantoins, which were prepared by the reaction of allyl isothiocyanate
(AITC) or 4-(methylthio)-3-butenyl isothiocyanate (MTBI) with various amino acids, were assayed
for their inhibitory effects on the mutagenicity of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) on
Salmonella typhimurium TA 98. When the 3,5-disubstituted 2-thiohydantoins (125-500 µg/mL)
were simultaneously preincubated with the bacterial strain, IQ (0.5 µg/mL), and rat microsomal
fraction (S9 mix) for 20 min, a dose-dependent inhibition of IQ mutagenicity was observed in the
tested compounds except one prepared from MTBI and L-methionine. This inhibition ranged from
23% to 86%. The inhibitory effect either disappeared or was largely reduced to 5-19%, when the
3,5-disubstituted 2-thiohydantoins were added after metabolic activation of IQ with S9 mix. These
results suggest that the 3,5-disubstituted 2-thiohydantoins probably acted as an inhibitor of the S9
mix-mediated metabolic activation of the mutagen.
Keyw or d s: 2-Thiohydantoins; allyl isothiocyanate; 4-(methylthio)-3-butenyl isothiocyanate; blocking
agent; Salmonella typhimurium TA 98
INTRODUCTION
2-thiohydantoins that are formed from naturally occur-
ring isothiocyanates and common amino acids in foods.
A variety of isothiocyanates are generated in crucif-
erous vegetables (VanEtten and Wolff, 1973). Of these,
allyl isothiocyanate (AITC) is used as a condiment or a
food additive. Isothiocyanates, due to their electrophilic
nature, react with compounds possessing a hydroxy,
thiol, or amino group to form corresponding thiocar-
bamates, dithiocarbamates, and thioureas (Ashworth,
1972). p-Hydroxybenzyl isothiocyanate in white mus-
tard paste is known to be easily degraded into dibenzyl
sulfide and benzyl alcohol (Kawakishi et al., 1967).
AITC is also unstable in an aqueous medium and
degrades to form a variety of sulfur-containing products
such as diallyl mono-, di-, tri-, and tetrasulfides, 1,2-
dithiolene, and 1,2,3-trithiin (Kawakishi and Namiki,
1969; Chen and Ho, 1998). Radish pungent principle,
4-(methylthio)-(E,Z)-3-butenyl isothiocyanate (MTBI),
has recently been reported to be highly reactive with
water or alcohol to form thioxopyrrolidines such as
3-(hydroxymethylene)-2-thioxopyrrolidine, 3-[(methyl-
thio)methylene]-2-thioxopyrrolidine, and methyl 4-
(methylthio)-(E,Z)-3-butenyl dithiocarbamate (Kosemu-
ra et al., 1993; Matsuoka et al., 1997; Uda et al., 1990).
As for the reaction of isothiocyanates with amino acids,
the Edman degradation of proteins and/or peptides is
well-known (Edman, 1950). The kinetics and mecha-
nism of the formation of the 3,5-disubstituted 2-thio-
hydantoins were described by Drobnica and Augustin
(1965a,b). It is thought that, during the cooking or
processing of food and also in the human digestive
system, isothiocyanates in foods react partly with free
amino acids to form their corresponding 2-thiohydan-
toins. Therefore, it is important to obtain information
about the physiological and biological properties of the
There have been many reports on the biological
activities of 2-thiohydantoins. For example, they can
act as antitumor (Al-Obaid et al., 1996), antiviral (El-
Barbary et al., 1994), antibacterial (Froelich et al.,
1954), antifungal (Marton et al., 1993), and anticonvul-
sant (Cortes et al., 1985) agents. Some hydantoins and
2-thio analogues can induce a lymphoproliferative
popliteal lymph node reaction (Kammueller and Seinen,
1988; Marintchev et al., 1995). To the best of our
knowledge, no information is available about anti-
mutagenic and/or anticarcinogenic activities of 3,5-
disubstituted 2-thiohydantoins that are derived from
naturally occurring isothiocyanates and amino acids. It
is known that a variety of mutagens and carcinogens
are formed in foods during cooking and processing. In
cooked foods such as fried or broiled meat and fish,
several heterocyclic amines including 2-amino-3-meth-
ylimidazo[4,5-f]quinoline (IQ) can be produced via amino
acid pyrolyzation or by reaction of creatin(in)e with
Maillard reaction products (Hatch et al., 1984; J a¨gerstad
et al., 1983; Spingarn et al., 1980; Sugimura et al.,
1977). IQ, one of the common food-born heterocyclic
amines, is activated in mammalian liver by cytochrome
P-450-dependent N-hydroxylation to form proximate
mutagen (Abu-Shakra et al., 1986; Yamazoe et al., 1983)
and is not only mutagenic in the Ames/Salmonella assay
(Kasai et al., 1980) but also carcinogenic to give tumors
at a number of sites in mice and rats (Ohgaki et al.,
1984, 1991; Takayama et al., 1984). On the other hand,
various types of compounds possessing an inhibitory
activity against mutagens and carcinogens also occur
in food systems. In cruciferous vegetables, glucosino-
late-derived isothiocyanates and indoles have been
reported to act as antimutagenic and/or anticarcinogenic
agents. For example, benzyl, 2-phenylethyl (PEITC),
and 4-methylsulfinylbutyl isothiocyanates have been
shown in Salmonella (S. typhimurium strains TA 98,
* To whom correspondence should be addressed [fax, (81)
28-649-5401; e-mail, uda@cc.utsunomiya-u.ac.jp].
† Utsunomiya University.
‡ Gunma Women’s J unior College.
10.1021/jf980430x CCC: $15.00 © 1998 American Chemical Society
Published on Web 11/13/1998

5038 J. Agric. Food Chem., Vol. 46, No. 12, 1998
Takahashi et al.
TA 100, and TA 1535) assays to act as antimutagens
against heterocyclic amines including IQ via an inhibi-
tion of cytochrome P-450-mediated metabolic activation
of the mutagens (Barcelo et al., 1996; Hamilton et al.,
1994; Hamilton and Teel, 1996; Knasmuller et al., 1996).
These isothiocyanates can induce phase II detoxifying
enzymes, glutathione S-transferase, and quinone re-
ductase in various mammal tissues (Zhang and Talalay,
1994). In fact, the isothiocyanates showed a clear
suppressive effect on chemical carcinogen-induced mam-
mary tumor models (Wattenberg, 1977, 1987; Morse et
al., 1989, 1991) and, thus, are considered to be cancer-
protective. Indole-3-carbinol, 3,3′-diindolylmethane, and
indole-3-acetonitrile occurring in edible cruciferous
vegetables have been shown to be inhibitors of either
7,12-dimethylbenz[a]anthracene-induced mammary tu-
mor formation in rats or benzo[a]pyrene-induced neo-
plasia of the forestomach in mice (Wattenberg and Loub,
1978), though indole-3-acetonitrile was converted to a
mutagenic 1-nitrosoindole-3-acetonitrile after nitrite
treatment (Wakabayashi et al., 1985).
It is interesting to investigate whether 3,5-disubsti-
tuted 2-thiohydantoins derived from isothiocyanates and
amino acids have an antimutagenic activity as well as
isothiocyanates. The present paper deals with the
antimutagenic properties of 3,5-disubstituted 2-thiohy-
dantoins that have been prepared from AITC or MTBI
and various amino acids against IQ on S. typhimurium
TA 98. This report also describes the effect of pH on
the formation of the 3,5-disubstituted 2-thiohydantoins
and on the growth of some microorganisms.
F igu r e 1. Formation of 3,5-disubstituted 2-thiohydantoins
(1-6). 1: R1 ) allyl; R2 ) isopropyl. 2: R1 ) allyl; R2 ) benzyl.
3: R1 ) allyl; R2 ) indolylmethyl. 4: R1 ) 4-(methylthio)-3-
butenyl; R2 ) isobutyl. 5: R1 ) 4-(methylthio)-3-butenyl; R2
) benzyl. 6: R1 ) 4-(methylthio)-3-butenyl; R2 ) 2-(methyl-
thio)ethyl.
sulfate, was evaporated under reduced pressure to give a solid
residue, which was then recrystallized in EtOH. The crystal
products were analyzed by UV, IR, EI-MS, and NMR spec-
troscopies to confirm their structures (Figure 1). The spectral
data obtained were as follows.
3-Allyl-5-isop r op yl-2-th ioh yd a n toin (1): UV λmax (EtOH)
267 nm; HR-EI-MS m/z (M+) calcd for C9H14ON2S 198.0796,
found 198.0811; LR-EI-MS m/z (%, rel intens.) 198 (M+, 100),
183 (18), 170 (37), 155 (27), 99 (49), 72 (97), 55(40), 41 (54); IR
ν
max (KBr) cm-1 3295 (NH), 1725 (CdO), 1508 (N-CdS), 1171
(CdS); 1H NMR ((CD3)2CO) δ (ppm) 9.13 (1H, s), 5.51-5.59
(1H, m), 4.76-4.95 (2H, m), 4.18-4.21 (2H, m), 4.17-4.18 (1H,
m), 2.19-2.28 (1H, m), 1.08 (3H, d, J ) 6.8 Hz), 0.92 (3H, d,
J ) 6.8 Hz); 13C NMR ((CD3)2CO) δ (ppm) 184.7, 174.3, 132.6,
117.7, 64.9, 43.2, 31.6, 18.6, 16.6.
EXPERIMENTAL PROCEDURES
Ma ter ia ls. A mixture of (E)- and (Z)-MTBI (about 7:1) was
prepared from radish seedlings by our previously described
method (Uda et al., 1990). AITC and IQ (caution: carcinogen)
were purchased from Wako Pure Chemical Inc. (Tokyo, J apan)
and used without further purification. S. typhimurium TA 98
was provided by the National Institute of Health Sciences
(Tokyo, J apan). S9 fraction and cofactors composed of mag-
nesium chloride hexahydrate (80 µmol), potassium chloride
(330 µmol), glucose-6-phosphate (50 µmol), nicotinamide ad-
enine dinucleotide phosphate reduced form (40 µmol), nico-
tinamide adenine dinucleotide reduced form (40 µmol), di-
sodium hydrogen phosphate (842 µmol), and sodium dihydro-
gen phosphate dihydrate (158 µmol) were obtained from
Oriental Yeast Co. (Tokyo, J apan). The cofactors dissolved in
9.0 mL of distilled water were filtrated with a sterilized
membrane filter (0.22 µm) and mixed with 1.0 mL of the S9
fraction. This was immediately used as S9 mix.
In str u m en ta l An a lysis. Ultraviolet (UV) spectra were
obtained in ethanol (EtOH) using a Hitachi 330 double-beam
spectrophotometer. Infrared (IR) spectra were recorded in
potassium bromide (KBr) on a Horiba 1200 FT-IR spectrom-
eter. High-resolution (HR) and low-resolution (LR) electron
impact mass spectral (EI-MS) analysis was done on a J EOL
AX-500 mass spectrometer at 70 eV. 1H and 13C nuclear
magnetic resonance (NMR) spectra were measured in acetone-
d6 ((CD3)2CO) or chloroform-d (CDCl3) on a J EOL EX-400
nuclear magnetic resonance spectrometer using tetramethyl-
silane (TMS) as the internal standard.
3-Allyl-5-ben zyl-2-th ioh yd a n toin (2): UV λmax (EtOH)
269 nm; HR-EI-MS m/z (M+) calcd for C13H14ON2S 246.0827,
found 246.0823; LR-EI-MS m/z (%, rel intens.) 246 (M+, 99),
155 (15), 91 (100), 77 (13), 65 (30), 51(11); IR νmax (KBr) cm-1
3205 (NH), 1747 (CdO), 1521 (N-CdS), 1174 (CdS); 1H NMR
((CD3)2CO) δ (ppm) 9.16 (1H, s), 7.20-7.28 (5H, m), 5.51-5.59
(1H, m), 4.76-4.95 (2H, m), 4.59 (1H, dd, J ) 4.9 & 5.6 Hz),
4.18-4.21 (2H, m), 3.23 (1H, dd, J ) 4.9 & 14.2 Hz), 3.10 (1H,
dd, J ) 5.9 & 14.2 Hz); 13C NMR ((CD3)2CO) δ (ppm) 184.2,
174.1, 135.9, 132.2, 130.7, 129.2, 127.9, 117.1, 60.8, 43.1, 37.3.
3-Allyl-5-(in d olylm eth yl)-2-th ioh yd a n toin (3): UV λmax
(EtOH) 268 nm; LR-EI-MS m/z (%, rel intens.) 285 (M+, 15),
130 (100); IR νmax (KBr) cm-1 3413 (NH), 1732 (CdO), 1533
(N-CdS), 1174 (CdS); 1H NMR ((CD3)2CO) δ (ppm) 10.10 (1H,
s), 9.13 (1H, s), 7.60 (1H, d, J ) 7.8 Hz), 7.36 (1H, d, J ) 7.8
Hz), 7.19 (1H, d, J ) 2.4 Hz), 7.08 (1H, t, J ) 6.8 Hz),7.01
(1H, t, J ) 6.8 Hz), 5.46-5.55 (1H, m), 4.75-4.80 (2H, m),
4.59 (1H, dd, J ) 4.9 & 5.6 Hz), 4.18-4.19 (2H, m), 3.37 (1H,
dd, J ) 6.4 & 14.6 Hz), 3.27 (1H, dd, J ) 4.9 & 14.7 Hz); 13C
NMR ((CD3)2CO) δ (ppm) 184.3, 174.6, 137.4, 132.2, 128.5,
125.0, 122.2, 119.7, 119.4, 117.0, 112.1, 108.9, 60.7, 43.1, 27.5.
3-[4-(Met h ylt h io)-3-b u t en yl]-5-isob u t yl-2-t h ioh yd a n -
toin (4): UV λmax (EtOH) 267 nm; HR-EI-MS m/z (M+) calcd
for C12H20ON2S2 272.1017, found 272.0969; LR-EI-MS m/z (%,
rel intens.) 272 (M+, 19), 225 (57), 100 (91), 85 (100); IR νmax
(KBr) cm-1 3186 (NH), 1749 (CdO), 1527 (N-CdS), 1163 (Cd
S); 1H NMR ((CD3)2CO) δ (ppm) 9.11 (1H, s), 6.08 (1H, d, J )
15.1 Hz), 5.33 (1H, dt, J ) 7.3 & 14.7 Hz), 4.21-4.25 (1H, m),
3.79 (2H, t, J ) 7.3 Hz), 2.42-2.52 (2H, m), 2.21 (3H, s), 1.89-
1.98 (1H, m), 1.57-1.70 (2H, m), 0.97 (3H, d, J ) 6.8 Hz), 0.96
(3H, d, J ) 6.4 Hz); 13C NMR ((CD3)2CO) δ (ppm) 184.4, 175.7,
127.4, 122.5, 58.3, 41.4, 40.8, 32.1, 25.2, 23.4, 21.9, 14.4.
3-[4-(Me t h ylt h io)-3-b u t e n yl]-5-b e n zyl-2-t h ioh yd a n -
toin (5): UV λmax (EtOH) 269 nm; HR-EI-MS m/z (M+) calcd
P r ep a r a tion a n d Str u ctu r a l Con fir m a tion of 3,5-Di-
su bstitu ted 2-Th ioh yd a n toin s. A mixture of MTBI (con-
taining in total of 1.0 mmol of the isomers) or AITC (1.0 mmol)
and the equivalent amount of amino acid (L-valine (Val),
L-luecine (Leu), L-phenylalanine (Phe), L-methionine (Met), and
L-tryptophan (Trp)) in 50 mL of MacIlvaine (citric acid-
disodium hydrogen phosphate) buffer (pH 8.0) was stirred for
48 h at 36 °C. The mixture was extracted with ethyl acetate.
The extract, after drying on a powder of anhydrous sodium

Antimutagenicity of 3,5-Disubstituted 2-Thiohydantoins
J. Agric. Food Chem., Vol. 46, No. 12, 1998 5039
for C15H18ON2S2 306.0861, found 306.0859; LR-EI-MS m/z (%,
rel intens.) 306 (M+, 6), 259 (27), 100 (43), 91 (15), 85 (100),
77 (4), 65 (4), 51 (2); IR νmax (KBr) cm-1 3203 (NH), 1747 (Cd
O), 1519 (N-CdS), 1162 (CdS); 1H NMR (CDCl3) δ (ppm) 7.70
(1H, s), 7.32 (1H, t, J ) 6.8 Hz), 7.27 (1H, t, J ) 7.3 Hz), 7.20
(1H, d, J ) 6.8 Hz), 6.02 (1H, d, J ) 15.1 Hz), 5.26 (1H, dt, J
) 7.3 & 14.6 Hz), 4.30 (1H, dd, J ) 3.9 & 8.8 Hz), 3.77 (2H, t,
J ) 7.3 Hz), 3.28 (1H, dd, J ) 3.9 & 14.2 Hz), 2.87 (1H, dd, J
) 8.8 & 14.2 Hz), 2.30-2.42 (2H, m), 2.21 (3H, s); 13C NMR
(CDCl3) δ (ppm) 183.6, 173.4, 134.7, 129.2, 129.2, 129.0, 127.6,
126.9, 121.2, 60.4, 40.5, 37.6, 31.2, 14.2.
3-[4-(Meth ylth io)-3-bu ten yl]-5-[2-(m eth ylth io)eth yl]-2-
th ioh yd a n toin (6): UV λmax (EtOH) 268, 228 nm; IR νmax
(EtOH) cm-1 3567 (NH), 1740 (CdO), 1541 (N-CdS), 1157
(CdS); LR-EI-MS m/z (rel intens. %) 290 (M+, 2.6), 243 (15.8),
100 (34.2), 85 (100), 72 (3.8), 61 (19.1); 1H NMR ((CD3)2CO) δ
(ppm) 9.12 (1H, s), 6.09 (1H, d, J ) 15.1 Hz), 5.35 (1H, dt, J
) 7.3 & 14.7 Hz), 4.35-4.38 (1H, m), 3.81 (2H, t, J ) 6.8 Hz),
2.66 (2H, t, J ) 7.3 Hz), 2.42-2.53 (2H, m), 2.21 (3H, s), 2.11-
2.19 (1H, m), 2.09 (3H, s), 1.97-2.03 (1H, m); 13C NMR ((CD3)2-
CO) δ (ppm) 184.6, 175.2, 127.4, 122.6, 58.6, 40.9, 32.1, 31.5,
29.6, 14.9, 14.4.
Mea su r em en t of p H Effect. A mixture of 100 µmol of
AITC and 500 µmol of Val or Phe in 10 mL of 0.1 M MacIlvaine
buffer (pH 3.0-9.0) was stirred for 48 h at 36 °C. The reaction
mixture was extracted with ethyl acetate, and the extract was
then analyzed by high-performance liquid chromatography
(HPLC) using a Shimadzu LC-5A liquid chromatograph with
a UV detector and a LiChrosorb RP-18 column (250-mm ×
4-mm i.d.; Merck, Tokyo, J apan). The mobile phase used was
a mixture of methanol/water (70/30, v/v) and was run at 0.8
mL/min. Detection was made at 270 nm. Amounts of the
2-thiohydantoins formed were measured by comparing their
peak areas with those of the authentic specimens.
F igu r e 2. Effect of pH on the formation of compounds 1 (0)
and 2 (9).
and then they were further incubated for 20 min. Thereafter
the same procedure as described above was followed. Each
sample was assayed using duplicate plates. Spontaneous
revertants were subtracted from the number of revertants in
all plates exposed to IQ in the presence or absence of the test
compounds, and those found in the plates exposed to IQ in
the absence of the test compounds are defined as 100%
mutagenicity (positive control). The data are presented as the
mean ( standard deviation of three independent assays.
Significant difference comparison with the positive control is
based on p value differences among the means. Simple
regression analyses of the data were run to determine the
relationship between the antimutagenic activity of the 3,5-
disubstituted 2-thiohydantoins and their concentrations using
the SAS General Linear Models Procedure (SAS, 1987).
An tim icr obia l Activity Assa y. The paper disk diffusion
method (Heisey and Gorham, 1992) was used with Aspergillus
fumigatus (IFO 4311), Eurotium chevalieri (IFO 4090), Peni-
cillium expansum (IFO 8800), Candida albicans (IFO 1061),
Schizosaccharomyces pombe (IFO 0638), Zygosaccharomyces
rouxii (IFO 1053), Escherichia coli (ATCC 25922), Salmonella
typhimurium (ATCC 13311), Salmonella typhimurium TA 98,
and Staphylococcus aureus (ATCC 12598). The fungi and the
bacteria were cultured, respectively, on a glucose-peptone
agar (pH 5.0; Nissui Co., Tokyo J apan) and a soybean-casein
digest broth agar (pH 7.0). A 10-mm diameter paper disk, to
which was added 25 µL of a dimethyl sulfoxide (DMSO)
solution containing 0, 300, 600, or 1000 µg of the 2-thiohy-
dantoins, was put on the center of an agar plate (90 mm i.d.)
seeded with the fungi ((4-5) × 106 colony forming unit (CFU))
or the bacteria ((2-3) × 108 CFU) and then incubated for 72-
96 h at 25 °C for the fungi and for 48 h at 37 °C for the
bacteria. The growth-inhibition zone was measured. The
antimicrobial assay was also carried out using the bacterial
strains in 2.0 mL of a soybean-casein digest broth (pH 7.0),
in which the bacteria were similarly incubated with the same
amounts of the test compounds as in agar plates, and their
growth was measured photometrically at 660 nm. All assays
were done in triplicate. Antimicrobial activity is expressed
as the difference in the growth-inhibition zone and/or the
optical density at 660 nm recorded with or without the test
compounds.
An tim u ta gen icity Assa y. The modification (Nohmi et al.,
1986) of the procedure of Maron and Ames (1983) was used
with S. typhimurium TA 98 as the bacterial strain and IQ as
the mutagen. A mixture of 0.1 mL of the test compound (0-
500 µg) in DMSO, 0.1 mL of IQ (0 or 0.5 µg) in DMSO, 0.3 mL
of the overnight-cultured bacterial suspension (2 × 109 CFU),
and 0.5 mL of S9 mix (10% S9) was incubated for 20 min at
37 °C. The bacteria were twice rinsed with 0.1 M phosphate
buffer (pH 7.4) to remove the test compound and the mutagen
and were then spread on a plate with 2 mL of top agar. After
a 48-h culture at 37 °C, the revertants that resulted were
counted. In another experiment, the test compounds were
added to the bacterial suspension which had been incubated
with the same amount of IQ and S9 mix for 20 min at 37 °C,
RESULTS AND DISCUSSION
Effect of p H on th e F or m a tion of th e 3,5-Disu b-
stitu ted 2-Th ioh yd a n toin s. To study the effect of pH
on the formation of the 3,5-disubstituted 2-thiohydan-
toins, a model reaction was carried out using AITC, Val,
and Phe. The results are shown in Figure 2. The
2-thiohydantoins 1 and 2 formed best at alkaline pH’s.
Our previous study (Matsuoka et al., 1997) showed that
MTBI was converted into dithiocarbamates by reaction
with methanethiol at pH’s over 6.0. The present study
showed that both compounds 1 and 2 could be formed
at neutral and weakly acidic pH’s (5.0-6.0), suggesting
that a certain amount of 2-thiohydantoin derivatives can
be formed in isothiocyanate-containing foods or season-
ings, and even in the human digestive tract. This
finding needs to be further confirmed, as does the time
required for the 2-thiohydantoin derivatives’ formation.
Effect of th e 3,5-Disu bstitu ted 2-Th ioh yd a n toin s
on th e Gr ow th of Micr oor ga n ism s. Froelich et al.
(1954) reported that synthetic 5-alkyl-2-thiohydantoins
had a growth-inhibitory effect on tubercle bacilli. Mar-
ton et al. (1993) showed that some 5-(arylmethylene)-
2-thiohydantoins were fungicidal and bactericidal against
plant pathogens but that no biological activity occurred
with their 5-(alkylmethyl) analogues. Their results also
suggested that the antimicrobial activity depends on the
structure of the substituted groups of 2-thiohydantoins.
In the present study, the six 3,5-disubstituted 2-thio-
hydantoins (1-6) were examined for their effect on the
growth of 10 strains of microorganisms, including 4
bacteria, 3 yeasts, and 3 molds. This experiment also
aimed to confirm whether the compounds have any
disturbing effect on the antimutagenicity assay with S.
typhimurium TA 98. All compounds examined showed
no antimicrobial activity at a dose range from 300 to

5040 J. Agric. Food Chem., Vol. 46, No. 12, 1998
Takahashi et al.
inhibition; p < 0.005) at the same dose level. At 500
µg/mL, these three compounds were nearly equal in
their antimutagenic activity. The six 3,5-disubstituted
2-thiohydantoins examined appeared to be 4 g 1 g 5 >
2 > 3 > 6 in order of their antimutagenic effects. In
contrast, when the bacterial strain, which was pre-
treated with IQ in the presence of S9 mix for 20 min,
was further incubated for 20 min with the test com-
pounds, disappearance of the antimutagenic effect was
observed in compounds 1, 4, and 5, and a large decrease
in the inhibition was shown with both compounds 2 (5-
16% inhibition; r2 ) 0.63; p < 0.05) and 3 (5-19%
inhibition; r2 ) 0.66; p < 0.05). Compound 6 showed a
weak inhibition (14%; p < 0.05) of the mutagenicity of
IQ at only a 500 µg/mL dose. In these experiments, the
mean number of revertants found in the IQ-treated
plates with S9 mix in the absence of the test compounds
was 826/plate, indicating that IQ was activated by the
S9 mix-mediated metabolic system. These results dem-
onstrate that all compounds could inhibit the S9 mix-
mediated activation of IQ when incubated simulta-
neously with IQ, though compound 6 needed a higher
concentration (500 µg/mL) for the inhibition. S9 fraction
from rat liver contains heterocyclic amine (HCA)-
metabolizable enzymes P-450 1A1 and 1A2 (Kamataki
et al., 1983; Soucek and Gut, 1992). The enzymes
metabolize HCA including IQ to N-hydroxy-HCA, which
is subsequently converted into an ultimate form being
highly active on DNA (Ishii et al., 1980; Hamilton and
Teel, 1996; Yamazoe et al., 1983). Therefore, the
antimutagenic activity of the 3,5-disubstituted 2-thio-
hydantoins might be attributed to the inhibition of the
cytochromes P-450.
Although there has been no available study on the
mutagenicity and/or antimutagenicity of 3,5-disubsti-
tuted 2-thiohydantoins derived from naturally occurring
isothiocyanates and amino acids, such derivatives of
2-thiohydantoins as 5-[(5-bromo-2-thienyl)methylene]-
3-(morpholinomethyl)-2-[(2,3,4,6-tetra-O-acetyl-â-D-glu-
copyranosyl)thio]hydantoin and 5-[(5-bromo-2-thienyl)-
methylene]-2-thiohydantoin were shown to act as
antitumor agents against human cell lines including
leukemia cell lines (Al-Obaid et al., 1996). Their related
hydantoin, 5,5-bis(4-chlorophenyl)-1,3-dichlorohydan-
toin, similarly showed a suppressive effect on P-388
lymphocytic leukemia in mice (Rodgers et al., 1977).
Modes of antitumorigenicity of these 2-thiohydantoins
and the hydantoin were not shown. These literature
references suggest that some 2-thiohydantoins are able
to behave as anticarcinogenic compounds depending on
their substituents at the 3 and/or 5 positions. From the
results in Figure 3, it appears that the antimutagenic
activity of the 3,5-disubstituted 2-thiohydantoins ex-
amined depends on the substituents at position 5 rather
than at position 3.
F igu r e 3. Inhibitory effects of the 3,5-disubstituted 2-thio-
hydantoins on the mutagenicity of IQ: (A) compound 1; (B)
compound 2; (C) compound 3; (D) compound 4; (E) compound
5; (F) compound 6. Symbols: O, the bacterial strain that had
been incubated with IQ (0.5 µg/mL) and S9 mix for 20 min at
37 °C was further incubated with the six test compounds for
20 min; 0, the bacterial strain was simultaneously incubated
with the test compounds for 20 min at 37 °C in the presence
of IQ (0.5 µg/mL) and S9 mix. The mutagenicity is expressed
in terms of the percentage in the revertant population found
in the presence or absence of the test compounds, where the
mutagenicity of IQ is defined as 100%. The number of
revertants found spontaneously was 26 ( 7.
1000 µg/plate toward any of the microorganisms includ-
ing S. typhimurium TA 98 (data not shown). In a
soybean-casein digest broth, the same results were
observed against the four bacterial strains (data not
shown). The results clearly demonstrated that these
3,5-disubstituted 2-thiohydantoins had no antimicrobial
activity at dose levels below 1000 µg/plate and, thus,
suggested that no cytotoxic effect of the 3,5-disubsti-
tuted 2-thiohydantoins on S. typhimurium TA 98 in the
antimutagenicity assay occurs at the dose levels. Mini-
mum inhibitory concentrations of the test compounds
were not determined, because solubility of the com-
pounds was marginal at a concentration of 1000 µg/25
µL in DMSO.
An tim u ta gen ic P r op er ty of th e 3,5-Disu bstitu ted
2-Th ioh yd a n toin s. The six 3,5-disubstituted 2-thio-
hydantoins were assayed for their mutagenic and anti-
mutagenic activity. None of the compounds examined
had mutagenic activity on S. typhimurium TA 98 with
or without S9 mix (data not shown). As shown in Figure
3, however, when the bacterial strain and the test
compounds (125-500 µg/mL) were simultaneously
treated with IQ (0.5 µg/mL) in the presence of S9 mix,
the compounds except for 6 showed a dose-dependent
inhibition of the mutagenicity of IQ. No toxicity on S.
typhimurium TA 98 was microscopically observed in all
compounds at a 125-500 µg/mL dose in the presence
or absence of IQ. In this dose range, percentage of
suppressing mutagenicity of IQ and coefficient of de-
termination (r2) from the simple regressions analyses
were for compound 1, 56-86% (r2 ) 0.94; p < 0.001);
for compound 2, 45-74% (r2 ) 0.90; p < 0.005); for
compound 3, 23-63% (r2 ) 0.81; p < 0.025); for
compound 4, 59-81% (r2 ) 0.71; p < 0.01); and for
compound 5, 54-83% (r2 ) 0.91; p < 0.005). Compound
6 showed no inhibition of the IQ mutagenicity up to 250
µg/mL but was antimutagenic (46% inhibition; p <
0.005) at 500 µg/mL. Compound 4 was the most
suppressive against the mutagenicity of IQ at 125 µg/
mL (59% inhibition; p < 0.001) and was followed by both
compounds 1 (56% inhibition; p < 0.001) and 5 (54%
As for MTBI used in this study, there has been no
available information about mutagenicity and/or anti-
mutagenicity. AITC, another isothiocyanate used for
the preparation of the 3,5-disubstituted 2-thiohydan-
toins, was mutagenic on S. typhimurium TA 100 with
a rat liver homogenate having an activity to convert
AITC to an epoxide intermediate (Neudecker and Hen-
schler, 1985), but its antimutagenic property against
HCA is unknown. Recently, Musk et al. (1995) reported
that AITC was unable to induce either chromosome
aberrations or sister chromatid exchanges even at
highly cytotoxic doses. Thus, it is still unclear whether

Antimutagenicity of 3,5-Disubstituted 2-Thiohydantoins
J. Agric. Food Chem., Vol. 46, No. 12, 1998 5041
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MTBI and AITC changed in their behavior against IQ
by their conversion into the corresponding 2-thiohydan-
toins.
Figure 3 demonstrates that the 3,5-disubstituted
2-thiohydantoins were able to inhibit the IQ mutagen-
icity at concentrations of either 125-500 or 500 µg/mL
when incubated simultaneously with IQ in the presence
of S9 mix. Kada and Shimoi (1987) classified inhibitors
of mutagenesis into desmutagens and bio-antimutagens,
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compounds may have a weak activity to inhibit the S9-
mediated metabolic activation of IQ and a reactivity
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ABBREVIATIONS USED
AITC, allyl isothiocyanate; CFU, colony forming unit;
HR-EI-MS, high-resolution electron impact mass spec-
trometry; HCA, heterocyclic amine; IQ, 2-amino-3-
methylimidazo[4,5-f]quinoline; IR, infrared spectrom-
etry; Leu, L-leucine; LR-EI-MS, low-resolution electron
impact mass spectrometry; Met, L-methionine; MTBI,
4-(methylthio)-3-butenyl isothiocyanate; NMR, nuclear
magnetic resonance spectrometry; PEITC, 2-phenylethyl
isothiocyanate; Phe, L-phenylalanine; S9 mix, a mixture
of rat microsomal fraction and cofactors; Trp, L-tryp-
tophan; UV, ultraviolet spectrometry; Val, L-valine; 1,
3-allyl-5-isopropyl-2-thiohydantoin; 2, 3-allyl-5-benzyl-
2-thiohydantoin; 3, 3-allyl-5-(indolylmethyl)-2-thiohy-
dantoin; 4, 3-[4-(methylthio)-3-butenyl]-5-isobutyl-2-
thiohydantoin; 5, 3-[4-(methylthio)-3-butenyl]-5-benzyl-
2-thiohydantoin; 6, 3-[4-(methylthio)-3-butenyl]-5-[2-
(methylthio)ethyl]-2-thiohydantoin.
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