Evidence for the involvement of N-methylthiourea, a ring cleavage metabolite, in the hepatotoxicity of methimazole in glutathione-depleted mice: Structure-toxicity and metabolic studies

Article abstract of DOI:10.1021/tx990155o

In mice depleted of GSH by treatment with buthionine sulfoximine (BSO), methimazole (2-mercapto-1-methylimidazole, MMI) causes liver injury characterized by centrilobular necrosis of hepatocytes and an increase in serum alanine transaminase (SALT) activity. MMI requires metabolic activation by both P450 monooxygenase and flavin-containing monooxygenase (FMO) before it produces the hepatotoxicity. MMI and its analogues were examined for the ability to increase SALT activity in GSH-depleted mice. Saturation of the C- 4,5 double bond in MMI resulted in a complete loss of hepatotoxicity. Similarly, ring fusion of a benzene nucleus to the C-4,5 double bond, forming 2-mercapto-1-methylbenzimidazole, abolished the toxic potency. As for MMI, 2- mercapto-1,4,5-trimethylimidazole, and 2-mercapto-1-methyl-4,5-di-n- propylimidazole, the toxic potency decreased with the increasing bulk of the 4- and 5-alkyl substituents. Furthermore, methylation of the thiol group of MMI totally reduced its toxicity. These structural requirements and the known toxicity of thiono-sulfur compounds led us to the hypothesis that MMI would undergo epoxidation of the C-4,5 double bond by P450 enzymes and, after being hydrolyzed, the resulting epoxide would be then decomposed to form N- methylthiourea, a proximate toxicant. Before N-methylthiourea would produce toxicity, it would be further biotransformed to its S-oxidized metabolites mainly by FMO. Evidence for this hypothesis was provided by the facts that N- methylthiourea and glyoxal as the accompanying fragment were identified as urinary metabolites in mice treated with MMI and that N-methylthiourea caused a marked increase in SALT activity when administered to mice in combination with BSO.

Full text of DOI:10.1021/tx990155o

170
Chem. Res. Toxicol. 2000, 13, 170-176
Evid en ce for th e In volvem en t of N-Meth ylth iou r ea , a
Rin g Clea va ge Meta bolite, in th e Hep a totoxicity of
Meth im a zole in Glu ta th ion e-Dep leted Mice:
Str u ctu r e-Toxicity a n d Meta bolic Stu d ies
Tamio Mizutani,* Kaoru Yoshida, Mihoko Murakami, Mutsuko Shirai, and
Sadahiro Kawazoe
Department of Food Sciences and Nutritional Health, Kyoto Prefectural University,
Shimogamo, Kyoto 606-8522, J apan
Received August 31, 1999
In mice depleted of GSH by treatment with buthionine sulfoximine (BSO), methimazole (2-
mercapto-1-methylimidazole, MMI) causes liver injury characterized by centrilobular necrosis
of hepatocytes and an increase in serum alanine transaminase (SALT) activity. MMI requires
metabolic activation by both P450 monooxygenase and flavin-containing monooxygenase (FMO)
before it produces the hepatotoxicity. MMI and its analogues were examined for the ability to
increase SALT activity in GSH-depleted mice. Saturation of the C-4,5 double bond in MMI
resulted in a complete loss of hepatotoxicity. Similarly, ring fusion of a benzene nucleus to the
C-4,5 double bond, forming 2-mercapto-1-methylbenzimidazole, abolished the toxic potency.
As for MMI, 2-mercapto-1,4,5-trimethylimidazole, and 2-mercapto-1-methyl-4,5-di-n-propyl-
imidazole, the toxic potency decreased with the increasing bulk of the 4- and 5-alkyl
substituents. Furthermore, methylation of the thiol group of MMI totally reduced its toxicity.
These structural requirements and the known toxicity of thiono-sulfur compounds led us to
the hypothesis that MMI would undergo epoxidation of the C-4,5 double bond by P450 enzymes
and, after being hydrolyzed, the resulting epoxide would be then decomposed to form
N-methylthiourea, a proximate toxicant. Before N-methylthiourea would produce toxicity, it
would be further biotransformed to its S-oxidized metabolites mainly by FMO. Evidence for
this hypothesis was provided by the facts that N-methylthiourea and glyoxal as the
accompanying fragment were identified as urinary metabolites in mice treated with MMI and
that N-methylthiourea caused a marked increase in SALT activity when administered to mice
in combination with BSO.
GSH synthesis. The injury is characterized by centri-
lobular necrosis of hepatocytes and an increase in serum
alanine transaminase (SALT) activity. Moreover, the
previous results have suggested that MMI is activated
by reactions mediated by both P450 monooxygenase and
flavin-containing monooxygenase (FMO) to reactive me-
tabolite(s) and that the inadequate rates of detoxification
of the metabolite(s) are responsible for the hepatotoxicity
in GSH-depleted mice.
A variety of drugs, such as acetaminophen (8), adren-
ergic agonists (9), adriamycin (10), and morphine (11),
and conditions, such as dietary protein deficiency (12) and
exercise (13), depress hepatic GSH levels in animals. In
particular, hyperthyroidism itself also significantly re-
duces hepatic GSH levels as shown in the hyperthyroid
rat model (14). Although these data cannot necessarily
be extrapolated to humans, interactions between MMI
metabolite(s) and cell constituents following hepatic GSH
depletion could also be a mechanism for the liver damage
observed in humans during MMI therapy.
In tr od u ction
Methimazole (2-mercapto-1-methylimidazole, MMI1) is
widely used clinically in the treatment of hyperthyroid-
ism. The adverse effects caused by this drug include
dermal and gastrointestinal disorders, loss of taste,
lupoid-like syndrome, and bone marrow depression, with
agranulocytosis as the most serious complication (1-3).
Hepatic injury is another reaction that occurs during
MMI therapy, although its frequency is considered low
(1, 4). In most cases of MMI-induced liver injury, chole-
static jaundice, without evidence of hepatic necrosis on
liver biopsy, has been reported (5). However, Floreani et
al. (6) have observed a case in which anicteric hepato-
cellular damage developed and suggested that the liver
damage induced by MMI therapy may be more frequent
than so far documented on the basis of jaundice cases.
Our previous study (7) has shown that MMI causes
severe liver injury in mice depleted of GSH by pretreat-
ment with buthionine sulfoximine (BSO), an inhibitor of
We designed this study to elucidate the structural
requirements of MMI analogues for the hepatotoxicity
in GSH-depleted mice, to suggest the chemical identity
of the postulated reactive metabolite, and then to provide
positive support for the suggestion produced by metabolic
studies.
* To whom correspondence should be addressed: Department of
Food Sciences and Nutritional Health, Kyoto Prefectural University,
Shimogamo, Kyoto 606-8522, J apan. Telephone: +81-75-703-5408.
Fax: +81-75-721-8023. E-mail: mizutani@kpu.ac.jp.
1
Abbreviations: BSO, buthionine sulfoximine; FMO, flavin-con-
taining monooxygenase; MMI, methimazole; SALT, serum alanine
transaminase.
10.1021/tx990155o CCC: $19.00 © 2000 American Chemical Society
Published on Web 03/02/2000

N-Methylthiourea: A Toxic Species of Methimazole
Chem. Res. Toxicol., Vol. 13, No. 3, 2000 171
Na2SO4 and concentrated. The residue was recrystallized from
EtOH to give 1.69 g (72%) of 11 as a yellow solid: mp 187-189
°C; 1H NMR (60 MHz, Me2SO-d6) δ 2.40 (br s, 1H, NH), 2.85 (d,
J ) 4.3 Hz, 3H, NHCH3), 7.30 (s, 1H, thiazolyl H-5), 7.90 (d,
J ) 8.8 Hz, 2H, phenyl H), 8.11 (d, J ) 8.8 Hz, 2H, phenyl H).
Anal. Calcd for C10H9O2N3S: C, 51.05; H, 3.86; N, 17.86.
Found: C, 51.09; H, 3.80; N, 17.96.
Ch a r t 1. Str u ctu r es of Com p ou n d s Exa m in ed for
Hep a totoxicity
An im a ls. Six-week-old male ICR mice were obtained from
Charles River J apan, Inc. (Kanagawa, J apan) and acclimatized
to our laboratory conditions for 1 week before being used. Mice
were housed in aluminum boxes on a wood-chip bedding (White
Flake, Charles River J apan, Inc.) at a constant temperature
(23 ( 2 °C) and under a 12 h light/dark cycle. Except when
stated otherwise, mice received food (Funabashi F-2, Funabashi
Farms, Chiba, J apan) and water ad libitum.
Assessm en t of Hep a totoxicity. Mice were treated ip with
BSO (3 mmol/kg of body weight) in water (20 mL/kg of body
weight). One hour later, the treated animals received po doses
of MMI analogues (1a -c and 2-4) or thiourea analogues
(5a -c). MMI, 3, and thioureas 5a -c were dissolved in water
(5 mL/kg of body weight) and 1b , 1c, 2, and 4 in olive oil
(5 mL/kg of body weight). The animals were fasted for 16 h
before dosing with test compounds and then for 2 h after
administration. Blood was collected by cardiac puncture under
pentobarbital anesthesia. The blood was allowed to clot at 37
°C for 1 h, and serum was prepared by centrifugation (900g).
SALT activity was measured with a commercial kit, GPT-UV
test Wako (Wako Pure Chemical Industries, Osaka, J apan).
Stu d y on th e Ur in a r y Meta bolites of MMI. Four mice
received MMI (0.2 mmol/kg of body weight) in water (5 mL/kg
of body weight) by po intubation and were placed collectively
in a metabolic cage. The urine was collected for 32 h. The cage
was rinsed with a small volume of water; the wash and collected
urine were combined, and the total volume was adjusted to 30
mL with water. After removal of particulate materials by
centrifugation, the urine sample was passed through an octa-
decylsilica cartridge (Bond Elute LRC C18, Varian) to remove
hydrophobic materials and then subjected to analysis for the
presence of N-methylthiourea (5b) and glyoxal (8).
Exp er im en ta l P r oced u r es
Ch em ica ls. The compounds examined for hepatotoxicity are
shown in Chart 1. All the compounds were chromatographically
pure. MMI (1a ), thiourea (5a ), N-methylthiourea (5b), and
N-ethylthiourea (5c) were purchased from Aldrich Chemical Co.
(Milwaukee, WI). 1-Methyl-2-methylthioimidazole (2) (15),
2-mercapto-1-methylbenzimidazole (4) (16), and BSO (17) were
synthesized according to published procedures.
Syn th esis of 2-Mer ca p to-1,4,5-tr im eth ylim id a zole (1b).
A mixture of acetoin (3.52 g, 40 mmol), N-methylthiourea (5b)
(2.89 g, 32 mmol), and EtOH (3 mL) was heated to 150 °C in a
sealed glass tube for 9 h, then allowed to cool, and diluted with
ether (20 mL). A white solid separated, which was washed with
ether and recrystallized from EtOH to give 1.4 g (31%) of 1b:
mp 222-224 °C; 1H NMR (60 MHz, CDCl3) δ 2.06 (s, 3H, 4-CH3
or 5-CH3), 2.08 (s, 3H, 5-CH3 or 4-CH3), 3.50 (s, 3H, NCH3). Anal.
Calcd for C6H10N2S: C, 50.67; H, 7.09; N, 19.70. Found: C,
50.70; H, 7.07; N, 19.87.
(A) Id en tifica tion of N-Meth ylth iou r ea (5b). A portion
(13 mL) of the urine sample was added to a solution of
p-nitrophenacyl bromide (10 mg) in EtOH (15 mL), and the
mixture was heated for 2 h under reflux. The reaction mixture
was diluted with water (20 mL), made alkaline with aqueous
ammonia, and extracted with ether (2 × 50 mL). The extract
was dried over Na2SO4 and evaporated to dryness by using a
Syn th esis of 2-Mer ca p to-1-m eth yl-4,5-d i-n -p r op ylim id -
a zole (1c). This compound was prepared according to the
procedure (18) described previously for 2-mercapto-4,5-di-n-
propylimidazole. A mixture of butyroin (3.61 g, 25 mmol),
N-methylthiourea (5b) (1.80 g, 20 mmol), and EtOH (3 mL) was
heated to 150 °C in a sealed glass tube for 9 h, then allowed to
cool, and diluted with ether (20 mL), causing a white solid to
separate. The solid was collected by filtration, washed with
ether, and recrystallized from EtOH to give 1.54 g (39%) of 1c:
mp 140-142 °C; 1H NMR (60 MHz, CDCl3) δ 0.77-1.10 (m, 6H,
two CH3CH2CH2), 1.13-1.83 (m, 4H, two CH3CH2CH2), 2.27-
2.53 (m, 4H, two CH3CH2CH2), 3.53 (s, 3H, NCH3). Anal. Calcd
for C10H18N2S: C, 60.56; H, 9.15; N, 14.13. Found: C, 60.29; H,
9.07; N, 14.29.
Syn th esis of 2-Mer ca p to-1-m eth yl-4,5-d ih yd r oim id a zole
(3). This compound was prepared according to the procedure
(19) described earlier for 2-mercapto-4,5-dihydroimidazole. To
a solution of N-methylethylenediamine (8.9 g, 120 mmol) in
EtOH/water (1:1) (40 mL) was added carbon disulfide (80 mL)
by portions over a period of 1 h. The mixture was heated to
reflux for 1 h and then treated with HCl (1.0 mL). The mixture
was heated under reflux for an additional 8 h and allowed to
cool in a refrigerator. The white solid which separated was
collected by filtration, washed with acetone, and recrystallized
from EtOH to give 5.9 g (42%) of 3: mp 128-129 °C [lit. mp
(20) 128 °C].
Kuderna-Danish concentrator and finally with
a nitrogen
stream. The residue was dissolved in benzene (1 mL) and
applied to a silica gel dry column [Silica Gel 60, 70-230 mesh,
Merck, deactivated with 5% (w/w) water; 3 g, 7.5 mm i.d.]. The
column was washed with 10 mL of hexane, followed by 14 mL
of hexane/ether (6:4), and the derivatized metabolite was eluted
with an additional 10 mL of hexane/ether (6:4). The eluate was
analyzed by GC/MS for the presence of 2-(methylamino)-4-(p-
nitrophenyl)thiazole (11) derivatized from N-methylthiourea
(5b).
(B) Id en tifica tion of Glyoxa l (8). To a portion (13 mL) of
the urine sample was added o-phenylenediamine (50 mg) in 1
mL of EtOH, and the mixture was heated at 80 °C for 30 min.
The mixture was extracted with ether (2 × 40 mL). The organic
phase was washed with 0.08 N HCl (2 × 10 mL), dried over
Na2SO4, and evaporated to a volume of 0.5 mL. The residue was
diluted with hexane (2 mL) and applied to a silica gel dry column
[Silica Gel 60, 70-230 mesh, Merck, deactivated with 5%
(w/w) water; 3 g, 7.5 mm i.d.]. The column was washed with
10 mL of hexane, followed by 6 mL of hexane/ether (8:2), and
the derivatized metabolite was eluted with an additional 2 mL
of hexane/ether (8:2). The eluate was analyzed by GC/MS for
the presence of quinoxaline (12) derivatized from glyoxal (8).
Syn th esis of 2-(Meth yla m in o)-4-(p-n itr op h en yl)th ia zole
(11). A solution of N-methylthiourea (5b) (1.26 g, 14 mmol) in
EtOH (40 mL) was added to a solution of p-nitrophenacyl
bromide (2.44 g, 10 mmol) in EtOH (40 mL), and the mixture
was heated to reflux for 2 h. EtOH was removed under reduced
pressure, and the residue was partitioned between CHCl3
(100 mL) and H2O (100 mL). The CHCl3 extract was dried over
Qu a n t it a t ion of N-Met h ylt h iou r ea (5b ),
a Ur in a r y
Meta bolite of MMI a n d 1c. MMI (dissolved in water, 5 mL/
kg of body weight) and 1c (in olive oil, 5 mL/kg of body weight)

172 Chem. Res. Toxicol., Vol. 13, No. 3, 2000
Mizutani et al.
Ta ble 1. SALT Activity 6 h a fter Ad m in istr a tion of MMI
An a logu es in Mice Dep leted of GSH by Tr ea tm en t w ith
BSOa
were each administered by po intubation to four groups of four
mice at a dose of 0.2 mmol/kg of body weight. Mice were placed
in groups of four in metabolic cages, and the urine was collected
for 32 h.
SALT (Karmen units/mL)
For metabolite quantitation, pooled urine samples from four
sets of four mice were analyzed individually. Each urine sample
was derivatized with p-nitrophenacyl bromide according to
essentially the same procedure as that used for the identification
of the MMI metabolite. HPLC analysis of the derivatized
metabolite was performed on a Shim-pack CLC-ODS column
(6 mm × 150 mm) (Shimadzu, Kyoto, J apan) on a Shimadzu
LC-6A solvent delivery system. The column was eluted with
CH3CN/water [7:3 (v/v)] at a flow rate of 1 mL/min. Under these
compound
0.2 mmol/kg 0.6 mmol/kg
control
MMI (1a )
20.8 ( 2.7
5980 ( 3120b
2-mercapto-1,4,5-trimethyl-
imidazole (1b)
2-mercapto-1-methyl-4,5-di-n-propyl-
imidazole (1c)
1-methyl-2-methylthioimidazole (2)
2-mercapto-1-methyl-4,5-dihydro-
imidazole (3)
103 ( 19.1b 2050 ( 266b
58.9 ( 8.6b
132 ( 22.6b
29.9 ( 4.6
30.5 ( 4.8
conditions, an authentic sample of 11 was eluted with
a
retention time of 5.7 min. The samples were monitored by UV
absorbance at 275 nm using a Shimadzu SPD-6A detector. The
peak area responses were linearly related to the amounts of the
reference compound in the range 0.02-0.1 µg.
2-mercapto-1-methylbenzimidazole (4)
20.0 ( 7.7
a
Mice were treated with BSO (3 mmol/kg of body weight, ip)
and received MMI analogues (po) 1 h later. Values are means (
b
SE from 5-12 mice. Significantly different from control group
GC/MS. Analyses of the derivatized metabolites were carried
out by GC/MS using a model 5890 series II gas chromatograph
(Hewlett-Packard, Palo Alto, CA) coupled to a model J MS-
SX102A mass spectrometer (J EOL Ltd., Tokyo, J apan). A 0.2
mm i.d. × 25 m fused silica capillary column (HP-5, Hewlett-
Packard) was used for GC separations. The injector and the GC/
MS interface temperatures were set at 210 and 230 °C,
respectively. The samples were injected in the splitless mode
with an initial column temperature of 80 °C for 1 min. The
column temperature was programmed at a rate of 15 °C/min to
210 °C and then kept at 210 °C for 18 min. The carrier gas was
helium with a flow rate of 0.7 mL/min. The mass spectrometer
was operated in the electron impact ionization mode and
scanned from 50 to 500 amu every 0.5 s with an ionization
energy of 70 eV.
(p < 0.05).
Sch em e 1. Der iva tiza tion of N-Meth ylth iou r ea (5b)
w ith p-Nitr op h en a cyl Br om id e
as judged by SALT activity. None of the compounds
(1a -c and 2-4) produced a sign of hepatotoxicity in the
absence of BSO pretreatment (data not shown).
Sta tistica l An a lysis. Data from the SALT activity assay
were analyzed by Kruskal-Wallis nonparametric analysis of
variance followed by a Mann-Whitney’s U test. For data of
urinary N-methylthiourea, comparison was made with a Stu-
dent’s t test. Differences were considered significant if p < 0.05.
Id en tifica tion of N-Meth ylth iou r ea (5b) a s a Ur i-
n a r y Meta bolite of MMI. The presence of N-methyl-
thiourea (5b ) in the urine of mice treated with MMI
(1a ) was determined by condensing 5b with p-nitro-
phenacyl bromide, forming 2-(methylamino)-4-(p-nitro-
phenyl)thiazole (11) (Scheme 1). This procedure is based
on Hantzsch’s method for the synthesis of thiazoles (21)
and, as previously reported (22, 23), is effective for the
isolation and identification of unstable and/or nonvolatile
thioamide metabolites. Panel A in Figure 1 shows the
reconstructed ion chromatograms for the urinary sample
derivatized with p-nitrophenacyl bromide. The selected
ion chromatograms at m/z 235 and 189 equally showed
a peak (M - 1) with a retention time (19.2 min) identical
with that of the authentic standard, 2-(methylamino)-4-
(p-nitrophenyl)thiazole (11). The mass spectrum of peak
M - 1 (Figure 1B) was very similar to that of the
authentic sample (Figure 1C), with a molecular ion at
m/z 235, an M - NO peak at m/z 205, an M - NO2 peak
at m/z 189, an M - NO2 - CH3 peak at m/z 174, and an
M - NO - CH3NHCN peak at m/z 149. These results
thus unambiguously demonstrate the presence of N-
methylthiourea (5b) in the urine as a metabolite of MMI
(1a ).
Resu lts
Hep a totoxicity of MMI a n d Str u ctu r a lly Rela ted
Com p ou n d s in Mice Dep leted of GSH by BSO. MMI
and several structurally related compounds were evalu-
ated for their hepatotoxicity in mice depleted of GSH by
pretreatment with BSO. Our previous study has shown
that MMI in combination with BSO produced injury in
the liver, as evidenced by centrilobular necrosis of hepato-
cytes and an increase in SALT activity (7). Therefore,
changes in SALT activity at 6 h after the administration
of test compounds (0.2 and/or 0.6 mmol/kg of body weight)
were used as an indication of hepatotoxicity. Mice were
pretreated with BSO according to the same regimen that
was used in the previous study (7). We have reported in
detail on the tissue GSH depletion produced by the BSO
pretreatment regimen (7). Briefly, the pretreatment with
BSO reduces hepatic GSH concentrations to 33 and 37%
of the control after 2 and 4 h, respectively. As shown in
Table 1, MMI (1a ) caused a severe increase (300-fold) in
SALT activity at a dose of 0.2 mmol/kg of body weight.
At the lower dose (0.2 mmol/kg of body weight), com-
pound 1b resulted in a minor, but statistically significant,
increase (5-fold) in SALT activity, whereas it produced
a marked increase (100-fold) in SALT activity at the
higher dose (0.6 mmol/kg of body weight). Compound 1c
produced only a marginal increase (6-fold) in SALT
activity even at the higher dose. In contrast with com-
pounds 1a -c, compounds 2-4 (up to 0.6 mmol/kg of body
weight) were totally ineffective in inducing hepatotoxicity
Id en tifica tion of Glyoxa l (8) a s a Ur in a r y Meta bo-
lite of MMI. The production of glyoxal (8) as a urinary
metabolite of MMI (1a ) was examined by GC/MS after
derivatization to its quinoxaline derivative (12) (Scheme
2). Panel A in Figure 2 shows the reconstructed ion
chromatograms for the urinary extract obtained after
derivatization with o-phenylenediamine. The selected ion
traces at m/z 130 and 103 both exhibited a peak (M - 2)
with a retention time (7.6 min) identical with that of

N-Methylthiourea: A Toxic Species of Methimazole
Chem. Res. Toxicol., Vol. 13, No. 3, 2000 173
F igu r e 1. (A) GC/MS-reconstructed ion chromatogram of the
derivatized urinary extract from mice treated with MMI (1a ).
The arrow indicates the tR of authentic 8. (B and C) Electron
impact mass spectra of (B) the derivatized metabolite M - 1
and (C) the 4-(p-nitrophenyl)thiazole derivative (11) of authentic
N-methylthiourea (5b).
F igu r e 2. (A) GC/MS-reconstructed ion chromatogram of the
derivatized urinary extract from mice treated with MMI (1a ).
The arrow indicates the tR of authentic 12. (B and C) Electron
impact mass spectra of (B) the derivatized metabolite M - 2
and (C) the quinoxaline derivative (12) of authentic glyoxal (8).
Ta ble 2. SALT Activity 6 h a fter Ad m in istr a tion of
Th iou r ea An a logu es in Mice Dep leted of GSH by
Tr ea tm en t w ith BSOa
Sch em e 2. Der iva tiza tion of Glyoxa l (8) w ith
o-P h en ylen ed ia m in e
dose (mmol/kg
SALT
compound
control
of body weight) (Karmen units/mL)
28.6 ( 3.7
N-methylthiourea (5b)
0.2
0.6
1.0
1.0
1.0
241 ( 11.1b
1840 ( 1570b
3870 ( 1620b
1050 ( 146b
authentic 12. The mass spectra of peak M - 2 (Figure
2B) and the authentic sample (Figure 2C) were in good
agreement with respect to typical peaks, a molecular ion
at m/z 130, an M - HCN peak at m/z 103, and an M -
2HCN peak at m/z 76. Thus, the formation of glyoxal (8)
as a urinary metabolite of MMI was confirmed.
thiourea (5a )
N-ethylthiourea (5c)
11900 ( 6130b
a
Mice were treated with BSO (3 mmol/kg of body weight, ip)
and received thiourea analogues (po) 1 h later. Values are
b
means ( SE from 5-12 mice. Significantly different from control
group (p < 0.05).
Ur in a r y E xcr et ion of N-Met h ylt h iou r ea a s a
Met a b olit e of MMI a n d 2-Mer ca p t o-1-m et h yl-4,5-
d i-n -p r op ylim id a zole (1c). After administration of
equimolar doses (0.2 mmol/kg of body weight) of MMI
(1a ) and 2-mercapto-1-methyl-4,5-di-n-propylimidazole
(1c), N-methylthiourea (5b) excreted in the urine was
derivatized to its thiazole derivative for quantitation by
HPLC. The derivatized metabolite (11) was eluted with
a retention time of 5.7 min under conditions described
in Experimental Procedures. The levels of urinary excre-
tion of 5b 32 h after treatment with MMI and 1c were
6.3 ( 2.1 and 1.3 ( 0.3% (mean ( SD of four experi-
ments), respectively, of the dose; these values were
significantly different (p < 0.005). This indicates that 1c
was metabolized to 5b to a smaller extent than MMI.
Hep a totoxicity of Th iou r ea Hom ologu es in Mice
Dep leted of GSH by BSO. Thiourea (5a ), N-methyl-
thiourea (5b), and N-ethylthiourea (5c) were examined
for their ability to increase SALT activity in mice
depleted of GSH by treatment with BSO. The results
are shown in Table 2. Treatment with 5b produced
dose-dependent increases in SALT activity at doses up
to 1.0 mmol/kg of body weight. At a dose of 1.0 mmol/kg
of body weight, 5a and 5c also produced marked in-
creases in SALT activity; the rank order of hepatotoxicity

174 Chem. Res. Toxicol., Vol. 13, No. 3, 2000
Sch em e 3. P r op osed P a th w a y of Meta bolic Activa tion of MMI in Mice
Mizutani et al.
was 5c > 5b > 5a as judged by SALT activity. Treatment
with 5b (up to at least 1.0 mmol/kg) in the absence of
BSO produced no significant changes in SALT activity
(data not shown).
enzymes to form the 4,5-epoxide (6). The enzymatic or
nonenzymatic hydrolysis of the epoxide (6) would produce
an unstable hemiketal-like intermediate (7), which is
expected to undergo spontaneous ring cleavage to form
glyoxal (8) and N-methylthiourea (5b). The metabolism
of thioureas is complex, but it is believed that sulfur
oxidation, mediated mainly by FMO, primarily to the
sulfenic acids and then possibly to the sulfinic acids is a
necessary step in the bioactivation of thioureas resulting
in protein binding, enzyme inactivation, and organ toxic-
ity (24-26). Therefore, 5b, if formed, would be further
biotransformed to its S-oxidized metabolites (9 and 10)
before it produces hepatotoxicity. In an earlier paper, we
reported the breakdown of hepatotoxic thiazoles, such as
thiabendazole, in a manner similar to that proposed here
for MMI which yields thioamide metabolites as proximate
toxicants (22, 23). The metabolic pathways shown in
Scheme 3 may suggest another possibility that MMI-
induced hepatotoxicity is mediated by epoxide 6 as a
reactive intermediate. However, this hypothesis seems
incompatible with the result of structure-toxicity study
whereby the free thiol substituent of MMI plays an
important role in the development of hepatotoxicity.
Discu ssion
To understand the chemical nature of the postulated
toxic metabolite of MMI, we studied structure-toxicity
relations for a number of MMI derivatives in mice
depleted of GSH by BSO. The results presented in Table
1 are informative for considering the structural require-
ments for the hepatotoxic effect of MMI in GSH-depleted
mice. The saturation of the C-4,5 double bond in MMI,
forming 2-mercapto-1-methyl-4,5-dihydroimidazole (3),
resulted in a complete loss of the hepatotoxicity. Simi-
larly, ring fusion of a benzene nucleus to the 4 and 5
positions of MMI to give 2-mercapto-1-methylbenz-
imidazole (4) abolished the toxic potency. As for MMI
analogues 1a -c, the hepatotoxic potency appears to
decrease with the increasing bulk of the 4- and 5-alkyl
substituents (1a > 1b > 1c). Collectively, these results
suggest that the presence of a C-4,5 double bond is
critically important for obligatory metabolic activation
of MMI and that the rate of activation may be affected
by the type of substitution at the 4 and 5 positions.
Furthermore, the total loss of toxicity observed by meth-
ylation of the thiol group of MMI, forming 1-methyl-2-
methylthioimidazole (2), clearly shows that the free thiol
also has a critical role in hepatotoxicity caused by MMI.
These structural requirements for the hepatotoxicity
can satisfactorily be explained by assuming that before
the development of toxicity MMI undergoes a ring cleav-
age reaction involving the 4 and 5 positions to yield some
thiono-sulfur-containing species including the C-2 unit
of parent MMI. The well-known toxic properties of a
number of thiono-sulfur compounds such as thioamides
and thioureas (24) lead us to suggest this idea. A possible
mechanism for the postulated ring cleavage is shown in
Scheme 3. Initially, MMI (Ia ) may be oxidized by P450
Our previous study has shown that P450-dependent
monooxygenase inhibitors such as methoxsalen, piperon-
yl butoxide, and isosafrole prevent the hepatotoxicity
caused by MMI in combination with BSO (7); these
observations are consistent with suggesting the involve-
ment of epoxidation by P450 in the toxification processes
of MMI. In addition, the substitution effect observed in
the hepatotoxic potency of 4- and 5-substituted MMI
analogues can be explained on the basis of the steric
effect of the substituents on the epoxidation of the C-4,5
double bond. The increasing bulk of the substituents in
the 4 and 5 positions is likely to reduce the rate of
epoxidation catalyzed by P450 enzymes (27). Further-
more, the previous results that competitive substrates
of FMO, N,N-dimethylaniline and ethyl methyl sulfide,
result in remarkable suppression of the hepatotoxicity

N-Methylthiourea: A Toxic Species of Methimazole
Chem. Res. Toxicol., Vol. 13, No. 3, 2000 175
are consistent with the suggestion that N-methylthiourea
(5b) as the proximate toxicant is further biotransformed
mainly by FMO to its S-oxidized metabolites (9 and 10)
before it produces hepatotoxicity. In contrast, the effec-
tiveness of the FMO inhibitors in preventing MMI-
induced toxicity cannot reasonably be understood on the
basis of the alternative hypothesis that epoxide 6 itself
is the toxic species of MMI. The structure-toxicity study
presented here showed requirement of the free thiol
group of MMI for toxicity; this is also in accord with the
structural feature that is essential for the existence of
S-oxidized intermediates (9 and 10).
On the other hand, previous studies have shown that
MMI is ultimately metabolized to N-methylimidazole and
sulfite both in vivo (28) and in vitro (29). S-Oxidation
products of MMI, the sulfenic (13) and sulfinic (14) acids,
are believed to be reactive metabolites produced during
the metabolism of MMI to N-methylimidazole (29);
several events such as the loss of rat liver microsomal
P450 during MMI metabolism (30) and the olfactory
toxicity induced by MMI in rats (31) are attributed to
these intermediates. These results may suggest the
possibility that metabolic activation by direct oxidation
of the thiol group of MMI is responsible for the hepato-
toxicity induced by MMI in combination with BSO.
Seemingly, requirement of the free thiol group for
the toxicity is also likely to be consistent with this
mechanism. However, the major metabolic pathways
of a variety of cyclic thiocarbamides other than MMI,
including 2-mercapto-4,5-dihydroimidazole (32) and 2-
mercaptobenzimidazole (33), compounds structurally
closely related to 3 and 4, respectively, and 4 itself (33),
are also known to involve oxidation at their thiol groups,
giving the corresponding sulfenates. Nevertheless, 3 and
4 were totally ineffective in inducing the hepatotoxicity
(Table 1); this strongly suggests that the direct S-
oxidation pathway is not responsible for the hepato-
toxicity of MMI in GSH-depleted mice. Moreover, the
inhibitory effect of substituents at the 4 and 5 positions
on the toxic potency cannot reasonably be explained by
the direct S-oxidation mechanism.
To provide experimental evidence for the postulated
metabolic pathways, we studied the metabolism of MMI.
When MMI was administered to mice, a ring cleavage
product, N-methylthiourea (5b), and the accompanying
fragment, glyoxal (8), were identified as urinary metabo-
lites after being trapped as 2-(methylamino)-4-(p-nitro-
phenyl)thiazole (11) and quinoxaline (12), respectively
(Figures 1 and 2). A 4,5-disubstituted analogue of MMI,
2-mercapto-1-methyl-4,5-di-n-propylimidazole (1c), also
formed a ring cleavage product 5b as a urinary metabo-
lite; however, the extent of formation of 5b from 1c was
considerably lower than that from MMI. On the other
hand, the structure-toxicity study clearly showed that
1c is much less hepatotoxic than MMI (Table 1). Col-
lectively, these findings support the idea that the hepato-
toxicity caused by MMI is mediated by 5b as a proximate
toxicant. Furthermore, the observation that when exter-
nally administered, 5b is hepatotoxic to mice depleted
of GSH by BSO (Table 2) strengthens the hypothesis
(Scheme 3) that this product is a proximate metabolite
responsible for the toxicity of MMI. Unexpectedly, 5b, a
putative proximate hepatotoxicant, was found to be less
toxic than parent MMI (Tables 1 and 2). However, this
discrepancy may be ascribed to possible differences
between 5b and MMI in the extent of absorption and
distribution to the target organ because 5b is much less
lipophilic than MMI. This idea may be supported by the
present observation that there are marked differences
in the hepatotoxicity of a series of thiourea homologues
(5c > 5b > 5a ) (Table 2) because this result suggests
that the lipophilicity of compound plays an important
part in determining the toxic potency of thioureas
externally administered.
In the study presented here, N-methylthiourea (5b),
as well as MMI, required GSH depletion to produce
hepatotoxicity when administered to mice. This suggests
that GSH plays a protective role toward MMI by in-
activating reactive metabolite(s) generated by further
metabolism of 5b; this metabolite can attack the cellular
constituents which results in the hepatotoxicity in GSH-
depleted mice. As mentioned above, for many thioureas
the sulfenic and/or sulfinic acid metabolites have been
postulated as the reactive intermediates responsible for
their protein binding, enzyme inactivation, and organ
toxicity (24-26). In metabolic studies with microsomes,
a number of thioureas were shown to bind to microsomes
and to inactivate P450 enzymes; the addition of GSH
completely inhibited the enzyme inactivation with the
concomitant formation of GSSG. Similarly, Krieter et al.
(34) have observed an increase in the biliary efflux of
GSSG upon perfusion of rat livers with thiourea, phenyl-
thiourea, and MMI. It is reported that the sulfenic acids
generated from the thioureas oxidize GSH to GSSG
nonenzymatically. Thus, GSH can be involved in the
detoxification of thioureas as a cellular reductant for their
reactive intermediates. In agreement with such a role of
GSH, GSH conjugates were not identified in many
metabolic studies on thioureas, although many studies
demonstrated that the addition of GSH to thiourea
microsomal reactions prevents covalent protein binding.
Recently, the existence of a novel GSH conjugate formed
from N-(5-chloro-2-methylphenyl)-N′-(2-methylpropyl)-
thiourea, a thiourea developed for the treatment of
atherosclerosis, has been demonstrated in rat microsomal
incubations (35). In rat liver microsomes, the major
metabolite of this thiourea is an S-oxide. The NADPH-
dependent oxidation of the thiourea resulted in covalent
binding to microsomal protein; addition of GSH to the
incubations decreased the extent of protein binding and
resulted in the formation of a novel GSH conjugate. These
observations suggest another possibility that GSH plays
a role as a nucleophile in the detoxication of reactive
intermediates formed from thioureas. In the case pre-
sented here, however, the exact nature of the ultimate
toxic species formed via 5b (Scheme 3) and the chemical
mechanism of its interaction with GSH remain to be
elucidated.
Ack n ow led gm en t. We thank Rika Shimizu, Yumi
Nagura, Maki Tanaka, and Kaori Tanaka for their
excellent technical assistance.
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