Structures of mutagens produced by the co-mutagen norharman with o- and m-toluidine isomers

Article abstract of DOI:10.1016/S1383-5718(01)00168-1

Norharman, abundantly present in cigarette smoke and cooked foods, is not mutagenic to Salmonella typhimurium strains. However, norharman shows mutagenicity to S. typhimurium TA98 and YG1024 in the presence of S9 mix when coexisting with aromatic amines,

Full text of DOI:10.1016/S1383-5718(01)00168-1

Mutation Research 493 (2001) 115–126
Structures of mutagens produced by the co-mutagen
norharman with o- and m-toluidine isomers
Noriyasu Hadaa,1, Yukari Totsukaa, Takeji Enyaa, Kumiko Tsurumakia,
Mariko Nakazawaa, Nobuo Kawaharab, Yasuoki Murakamic, Yuusaku Yokoyamac,
Takashi Sugimuraa, Keiji Wakabayashia,∗
a
Cancer Prevention Division, National Cancer Center Research Institute, 1-1 Tsukiji 5-chome,
Chuo-ku, Tokyo 104-0045, Japan
National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-0098, Japan
b
c
School of Pharmaceutical Sciences, Toho University, 2-2-1 Miyama, Funabashi, Chiba 274-0072, Japan
Received 16 January 2001; accepted 12 March 2001
Abstract
Norharman, abundantly present in cigarette smoke and cooked foods, is not mutagenic to Salmonella typhimurium strains.
However, norharman shows mutagenicity to S. typhimurium TA98 and YG1024 in the presence of S9 mix when coexisting with
aromatic amines, including aniline, o- and m-toluidines. We previously reported that the mutagenicity from norharman and ani-
line in the presence of S9 mix was due to the formation of a mutagenic compound, 9-(4ꢀ-aminophenyl)-9H-pyrido[3,4-b]indole
(aminophenylnorharman). In the present study, we analyzed the mutagens produced by norharman with o- or m-toluidine in
the presence of S9 mix. When norharman and o-toluidine were reacted at 37◦C for 20 min, two mutagenic compounds, which
were mutagenic with and without S9 mix, respectively, were produced, and these were isolated by HPLC. The former mutagen
was deduced to be 9-(4ꢀ-amino-3ꢀ-methylphenyl)-9H-pyrido[3,4-b]indole (amino-3ꢀ-methylphenylnorharman) on the basis
of various spectral data, and this new heterocyclic amine was confirmed by its chemical synthesis. The latter mutagen was
identified to be the hydroxyamino derivative. Amino-3ꢀ-methylphenylnorharman induced 41,000 revertants of TA98, and
698,000 revertants of YG1024 per ␮g with S9 mix. Formation of the same DNA adducts was observed in YG1024 when
amino-3ꢀ-methylphenylnorharman or a mixture of norharman plus o-toluidine was incubated with S9 mix. These observations
suggest that norharman reacts with o-toluidine in the presence of S9 mix to produce amino-3ꢀ-methylphenylnorharman, and
this compound is metabolically activated to yield its hydroxyamino derivative. After activation by O-acetyltransferase, it might
bind to DNA and exert mutagenicity in S. typhimurium TA98 and YG1024. When norharman and m-toluidine were reacted
Abbreviations: HCAs, heterocyclic amines; Aminophenylnorharman, 9-(4ꢀ-aminophenyl)-9H-pyrido[3,4-b]indole; DMSO, dimethyl sulfoxide;
Amino-3ꢀ-methylphenylnorharman, 9-(4ꢀ-amino-3ꢀ-methylphenyl) -9H-pyrido[3,4-b]indole; Hydroxyamino-3ꢀ-methylphenylnorharman, 9-(4ꢀ-
hydroxyamino-3ꢀ-methylphenyl)-9H-pyrido[3,4-b]indole; RAL, relative adduct labeling; Amino-2ꢀ-methylphenylnorharman, 9-(4ꢀ-amino-
2ꢀ-methylphenyl)-9H-pyrido[3,4-b]indole; Trp-P-1, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole; Glu-P-1, 2-amino-6-methyldipyrido[1,2-
a:3ꢀ,2ꢀ-d]imidazole
∗Corresponding author. Tel.: +81-3-3547-5271; fax: +81-3-3543-9305.
E-mail address: kwakabay@gan2.ncc.go.jp (K. Wakabayashi).
1Present address: Kyoritsu College of Pharmacy, 1-5-30 Shibakoen, Minato-ku, Tokyo 105-8512, Japan.
1383-5718/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved.
PII: S1383-5718(01)00168-1

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N. Hada et al. / Mutation Research 493 (2001) 115–126
in the presence of S9 mix, 9-(4ꢀ-amino-2ꢀ-methylphenyl)-9H-pyrido[3,4-b]indole (amino-2ꢀ-methylphenylnorharman) was
identified as a mutagen. Thus, the mutagenicity of norharman with m-toluidine may follow a mechanism similar to that with
o-toluidine. © 2001 Elsevier Science B.V. All rights reserved.
Keywords: Norharman; Toluidine isomers; Amino-3ꢀ-methylphenylnorharman; Amino-2ꢀ-methylphenylnorharman
1. Introduction
the presence of S9 mix. In contrast, the combination
of norharman with p-toluidine did not show muta-
genicity in Salmonella strains [6,8,9]. Moreover, good
correlation was found between DNA adduct formation
and the appearance of mutagenicity in Salmonella
strains by norharman with toluidine isomers [9].
As mentioned above, norharman is present in ciga-
rette smoke, and o- and m-toluidine are also detected
at levels of 24–3919 ng per cigarette [11,16]. More-
over, o-toluidine has been found in human urine and
milk samples [13,14,17]. Thus, it is likely that humans
are simultaneously exposed to norharman and tolui-
dine isomers in daily life. Therefore, it is important
to elucidate the mechanisms of the co-mutagenicity
of norharman with toluidine o- and m-isomers to
understand the genotoxic effects of norharman with
toluidine isomers in humans.
In the present study, we isolated mutagenic
compounds produced from norharman with o- or
m-toluidine in the presence of S9 mix, and then deter-
mined their structures by various spectrometric meth-
ods and by comparison with chemically synthesized
authentic compounds. Possible metabolic activation
processes of the mutagens are also discussed.
The development of human cancer is thought to
be mainly associated with diet and smoking-related
factors [1]. A non-mutagenic ␤-carboline compound,
norharman, is widely distributed in our environment,
such as cigarette smoke and cooked foods [2–4].
It is found at levels 11–730-fold higher than those
of known mutagenic and carcinogenic heterocyclic
amines (HCAs) [5]. Norharman is a co-mutagen, as
demonstrated by its mutagenicity toward Salmonella
typhimurium TA98 and YG1024 in the presence of
both S9 mix and aromatic amines, including aniline
[6–9]. Aniline is present in cigarette smoke conden-
sates and some vegetables [10,11]. In addition, it has
been reported that both of norharman and aniline
were detected in human urine [12–14]. In a previous
report, we studied the mechanism of the co-mutagenic
action of norharman with aniline, and mutagenicity
was found to be due to the formation of a mutagenic
coupled compound of norharman with aniline, 9-(4ꢀ-
aminophenyl)-9H-pyrido[3,4-b]indole (aminophenyl-
norharman) [15]. Under the same conditions, an
N-hydroxy derivative of aminophenylnorharman was
also produced. Moreover, the same DNA adducts were
observed in YG1024 when aminophenylnorharman
or a mixture of norharman plus aniline was incu-
bated with S9 mix. The hydroxyamino derivative also
yielded the same DNA adducts in YG1024. Thus, the
mechanism of the co-mutagenic action of norharman
with aniline was suggested to be as follows: in the
presence of S9 mix, a coupled mutagenic compound,
aminophenylnorharman, is formed from norharman
and aniline. This compound is then converted to
the N-hydroxy derivative and forms DNA adducts
after esterification to induce mutations in Salmonella
strains [15].
2. Materials and methods
2.1. Generals
Melting points were determined with a Yanagimoto
micro melting-point apparatus and are uncorrected.
1H and 13C-NMR spectra were recorded with JEOL
GX-400 and ␣600 with micro probe FT-NMR spec-
trometers, respectively. Tetramethylsilane was used as
the internal standard for solutions in CDCl3. EI-MS
was recorded on a JEOL JMS-DX 300 mass spectro-
meter and UV absorbance spectra were measured with
a PD-8020 photodiode array detector (Tosoh, Tokyo,
Japan). Distillation for elemental analysis was
Other aromatic amines, o- and m-toluidine, have
also been shown to react with norharman to show
mutagenicity in S. typhimurium TA98 and YG1024 in

N. Hada et al. / Mutation Research 493 (2001) 115–126
117
performed with a Micro Distillation and Sublimation
apparatus (Yanagimoto CHN coder MT-3, Tokyo,
Japan).
acetonitrile in 25 mM phosphate buffer (pH 2.0) was
pumped in isocratically at a flow rate of 1 ml/min.
The mutagenic fractions eluted at retention times of
15–17 min were further purified on the same TSKgel
ODS-80Ts column. Applied material was eluted with
a mobile phase of 50% acetonitrile in water with 0.1%
diethylamine adjusted to pH 6.0 with acetic acid at a
flow rate of 1 ml/min.
In the absence of S9 mix, mutagenicity was
observed in fractions with retention times of
48–64 min on the semi-preparative TSKgel ODS-120A
column. The mutagenic fractions with retention times
of 56–64 min were combined, and further purified on
an analytical-grade TSKgel ODS-80Ts column with a
mobile phase of 25% acetonitrile in 25 mM phosphate
buffer (pH 2.0) under the same conditions used for
the separation of mutagenic compounds that showed
activity with S9 mix.
Isolation of the mutagen, produced by the reaction
of norharman with m-toluidine in the presence of
S9 mix, was performed with the same procedures
as described above. Norharman·HCl (40 mg) and
m-toluidine (20 mg) were incubated with S9 mix at
37◦C for 20 min, then the mutagen showing activity
with S9 mix was isolated using semi-preparative ODS
column. The major mutagenic activity was observed
in the fractions with retention times of 52–56 min.
Then, these fractions were further purified using an
analytical grade ODS column with the following two
eluent conditions, 25% acetonitrile in 25 mM phos-
phate buffer (pH 2.0) and 50% acetonitrile in water
with 0.1% of diethylamine adjusted to pH 6.0 with
acetic acid.
2.2. Materials
Norharman was purchased from Katsura Chemi-
cal Co. (Tokyo, Japan). o-Toluidine, NADPH, G6P,
5-fluoro-2-nitrotoluene and potassium carbonate
were from Wako Pure Chemical Industries (Osaka,
Japan). Micrococcal nuclease and phosphodiesterase
II were purchased from Worthington Biochemical Co.
(Freehold, NJ). [␥-32P]ATP, T4 polynucleotide kinase,
nuclease P1 and phosphodiesterase I were obtained
from ICN Biochemicals (Irvine, CA), Takara Shuzo
Co. (Kyoto, Japan), Yamasa Shoyu Co. (Choshi,
Japan) and Worthington Biochemical Co. (Freehold,
NJ), respectively. Silica gel 60 for column chromato-
graphy (spherical, 100–210 mesh) was from Kanto
Chemical Co. (Tokyo, Japan) and Kiesel gel GF254
for thin layer chromatography was from Merck Japan
Ltd. (Tokyo, Japan). All other chemicals used were
of analytical grade.
2.3. Isolation of mutagens produced by norharman
with o- or m-toluidine in the presence of S9 mix
Norharman·HCl (40 mg) and o-toluidine (20 mg)
dissolved in 20 ml of water were mixed with 10 ml
of S9 mix, and incubated for 20 min at 37◦C. After
incubation, 30 ml of cooled acetonitrile was added
and the mixture was centrifuged at 10,000 rpm and
4◦C to remove the proteins. The supernatant was
evaporated in vacuo and the residue dissolved in
2 ml of 50% methanol was then separated by HPLC
with a semi-preparative TSKgel ODS-120A column
(10 ␮m particle size, 7.8 mm × 300 mm; Tosoh) as
reported previously [15]. An aliquot of each 4 min
fraction (8 ml) was tested for mutagenicity using
S. typhimurium YG1024 in the presence or absence
of S9 mix.
In the presence of S9 mix, mutagenicity was
mainly observed in fractions with retention times of
48–52 min. These were evaporated and the residue
was dissolved in 2 ml of 50% methanol. An aliquot
of the solution was injected into an analytical-grade
TSKgel ODS-80Ts column (5 ␮m particle size,
4.6 mm×250 mm, Tosoh) and a mobile phase of 25%
All the above HPLC procedures were performed
at the ambient temperature by monitoring of UV
absorbance of the eluate at 254 nm.
2.4. Chemical synthesis of 9-(3ꢀ-methyl-4ꢀ-
nitrophenyl)-9H-pyrido[3,4-b]indole
A mixture of norharman (168 mg, 1 mmol), 5-
fluoro-2-nitrotoluene (465 mg, 3 mmol) and anhydrous
potassium carbonate (140 mg) in N,N-dimethylform-
amide (2 ml) was heated at 100◦C with stirring under
an argon atmosphere for 10 h. The reaction mixture
was poured into 100 ml of water and extracted twice
with 100 ml of ethyl acetate. The organic layer was
washed with water, dried over magnesium sulfate,

118
N. Hada et al. / Mutation Research 493 (2001) 115–126
and evaporated to dryness in vacuo. The residue was
dissolved in dichloromethane and chromatographed
on a silica gel column (2.5 cm × 20 cm) using
dichloromethane–methanol (50:1, v/v) as the eluent,
2.6. Chemical synthesis of 9-(4ꢀ-hydroxyamino-3ꢀ-
methylphenyl)-9H-pyrido[3,4-b]indole
A
mixture of 9-(3ꢀ-methyl-4ꢀ-nitrophenyl)-9H-
and
9-(3ꢀ-methyl-4ꢀ-nitrophenyl)-9H-pyrido[3,4-b]
pyrido[3,4-b]indole (50 mg, 0.16 mmol) and 5% Pd–C
(30 mg) in 5 ml of tetrahydrofuran was stirred under a
hydrogen atmosphere for 30 min at 20◦C. After remo-
ving Pd–C by filtration, the filtrate was concentrated at
30◦C and subjected to preparative thin layer chroma-
tography with silica gel (20 cm×20 cm plate) (develo-
ping solvent; dichloromethane–methanol, 10:1, v/v)
to yield 9-(4ꢀ-hydroxyamino-3ꢀ-methylphenyl)-9H-
pyrido[3,4-b]indole (hydroxyamino-3ꢀ-methylphenyl-
norharman) (20 mg, 43%); silica gel TLC (developing
solvent; dichloromethane–methanol, 10:1, v/v) Rf
0.40; UV λ max (methanol) 251, 300, 388 nm; EI-MS
m/z 289 (M+).
indole was obtained as a yellow powder (242 mg,
80%); m.p. 165–168◦C; silica gel TLC (developing
solvent; dichloromethane–methanol, 10:1, v/v) Rf
0.63; 1H NMR (CDCl3) δ 8.93 (1H, s, H-1), 8.58
(1H, d, J = 5.2 Hz, H-3), 8.30 (1H, d, J = 9.2 Hz,
H-5ꢀ), 8.21 (1H, d, J = 7.9 Hz, H-5), 8.03 (1H, d,
J = 5.2 Hz, H-4), 7.59–7.64 (3H, m, H-2ꢀ, H-6ꢀ,
H-7), 7.55 (1H, d, J = 7.9 Hz, H-8), 7.41 (1H, t,
J = 7.3 Hz, H-6), 2.76 (3H, s, CH3). Anal. calcd. for
C18H13N3O2: C, 71.28; H, 4.32; N, 13.85. Found C,
71.27; H, 4.13; N, 13.83.
2.5. Chemical synthesis of 9-(4ꢀ-amino-3ꢀ-
methylphenyl)-9H-pyrido[3,4-b]indole
2.7. Chemical synthesis of 9-(2ꢀ-methyl-4ꢀ-
nitrophenyl)-9H-pyrido[3,4-b]indole
A
mixture of 9-(3ꢀ-methyl-4ꢀ-nitrophenyl)-9H-
pyrido[3,4-b]indole (70 mg, 0.23 mmol) and 5% Pd–C
(50 mg) in 7 ml of ethanol–acetic acid (5:2, v/v) was
stirred under a hydrogen atmosphere for 1 h at 20◦C.
After the catalyst was removed by filtration, the fil-
trate was condensed under reduced pressure. The
residue was dissolved in dichloromethane and chro-
matographed on a silica gel column (2.5 cm × 20 cm)
using dichloromethane–methanol (20:1, v/v) as an elu-
ent to yield 9-(4ꢀ-amino-3ꢀ-methylphenyl)-9H-pyrido
[3,4-b]indole (amino-3ꢀ-methylphenylnorharman) (55
mg, 87%); m.p. 164–167◦C; silica gel TLC (devel-
oping solvent; dichloromethane–methanol, 10:1, v/v)
Rf 0.56; UV λ max (methanol) 238, 288, 357 nm;
EI-MS m/z 273 (M+); 1H NMR (CDCl3) δ 8.77 (1H,
s, H-1), 8.49 (1H, d, J = 4.5 Hz, H-3), 8.18 (1H, d,
J = 7.5 Hz, H-5), 7.99 (1H, d, J = 4.5 Hz, H-4), 7.52
(1H, t, J = 7.5 Hz, H-7), 7.41 (1H, d, J = 7.5 Hz,
H-8), 7.31 (1H, br.t, J = 7.5 Hz, H-6), 7.21 (1H,
s, H-2ꢀ), 7.19 (1H, d, J = 8.0, H-6ꢀ), 6.86 (1H, d,
J = 8.0 Hz, H-5ꢀ), 3.86 (2H, br.s, NH2), 2.26 (3H,
br.s, CH3); 13C NMR (CDCl3) δ 144.6 (C-4ꢀ), 142.3
(C-8a), 139.3 (C-3), 137.5 (C-9a), 133.4 (C-1), 129.0
(C-2ꢀ), 128.4 (C-4a), 128.3 (C-7), 127.0 (C-1ꢀ), 125.8
(C-6ꢀ), 123.6 (C-3ꢀ), 121.6 (C-5), 121.1 (C-4b), 120.1
(C-6), 115.6 (C-5ꢀ), 114.4 (C-4), 110.6 (C-8). Anal.
calcd. for C18H15N3: C, 79.10; H, 5.53; N, 15.37.
Found C, 79.09; H, 5.31; N, 14.93.
A mixture of norharman (168 mg, 1 mmol), 2-
fluoro-5-nitrotoluene (155 mg, 1 mmol) and anhydrous
potassium carbonate (140 mg) in N,N-dimethylform-
amide (2 ml) was heated at 100◦C with stirring under
an argon atmosphere for 10 h. The reaction mixture
was poured into 100 ml of water and extracted twice
with 100 ml of ethyl acetate. The organic layer was
washed with water, dried over magnesium sulfate,
and evaporated to dryness in vacuo. The residue was
dissolved in dichloromethane and chromatographed
on a silica gel column (2.5 cm × 20 cm) using
dichloromethane–methanol (50:1, v/v) as the eluent,
and
9-(2ꢀ-methyl-4ꢀ-nitrophenyl)-9H-pyrido[3,4-b]
indole was obtained as a yellow powder (268 mg,
88%); m.p. 129–130◦C; silica gel TLC (developing
solvent; dichloromethane–methanol, 10:1, v/v) Rf
1
0.63; H NMR (CDCl3) δ 8.57 (1H, d, J = 5.5 Hz,
H-3), 8.50 (1H, s, H-1), 8.41 (1H, d, J = 2.3 Hz,
H-3ꢀ), 8.31 (1H, dd, J = 2.3, 8.5 Hz, H-5ꢀ), 8.24
(1H, d, J = 7.5 Hz, H-5), 8.06 (1H, d, J = 5.5 Hz,
H-4), 7.61 (1H, d, J = 7.5 Hz, H-6ꢀꢀ), 7.57 (1H,
t, J = 7.5 Hz, H-7), 7.40 (1H, t, J = 7.5 Hz,
H-6), 7.10 (1H, d, J = 7.5 Hz, H-8), 2.16 (3H,
s, CH3). Anal. calcd. for C18H13N3O2: C, 71.28;
H, 4.32; N, 13.85. Found C, 71.27; H, 4.13; N,
13.83.

N. Hada et al. / Mutation Research 493 (2001) 115–126
119
2.8. Chemical synthesis of 9-(4ꢀ-amino-2ꢀ-
methylphenyl)-9H-pyrido[3,4-b]indole
2.10. Preparation of DNA samples from S.
typhimurium YG1024 treated with norharman and
o-toluidine, or amino-3ꢀ-methylphenylnorharman
A
mixture of 9-(2ꢀ-methyl-4ꢀ-nitrophenyl)-9H-
pyrido[3,4-b]indole (106 mg, 0.35 mmol) and 5%
Pd–C (80 mg) in 7 ml of ethanol-acetic acid (5:2, v/v)
was stirred under a hydrogen atmosphere for 1 h at
20◦C. After the catalyst was removed by filtration, the
filtrate was condensed under reduced pressure. The
residue was dissolved in dichloromethane and chro-
matographed on a silica gel column (2.5 cm × 20 cm)
using dichloromethane–methanol (20:1, v/v) as an elu-
ent to give 9-(4ꢀ-amino-2ꢀ-methylphenyl)-9H-pyrido
[3,4-b]indole (amino-2ꢀ-methylphenylnorharman) (80
mg, 84%); m.p. 131–132◦C; silica gel TLC (deve-
loping solvent; dichloromethane–methanol, 10:1, v/v)
Rf 0.55; UV λ max (methanol) 239, 289, 356 nm;
EI-MS m/z 273 (M+); 1H NMR (CDCl3) δ 8.52 (1H,
s, H-1), 8.49 (1H, d, J = 5.2 Hz, H-3), 8.19 (1H, d,
J = 7.9 Hz, H-5), 8.01 (1H, d, J = 5.2 Hz, H-4),
7.52 (1H, dd, J = 7.9, 8.2 Hz, H-7), 7.31 (1H, t,
J = 7.9 Hz, H-6), 7.14 (1H, d, J = 8.2 Hz, H-8), 7.12
(1H, d, J = 8.2 Hz, H-6ꢀ), 6.75 (1H, d, J = 3.5 Hz,
H-3ꢀ), 6.68 (1H, dd, J = 3.5, 8.2 Hz, H-5ꢀ), 2.76 (3H,
s, CH3), 3.89 (2H, br.s, NH2); 13C NMR (CDCl3) d
147.2 (C-4ꢀ), 142.4 (C-8a), 139.3 (C-3), 138.0 (C-2ꢀ),
137.6 (C-9a), 133.3 (C-1), 129.9 (C-6ꢀ), 128.4 (C-7),
128.3 (C-4a), 125.4 (C-1ꢀ), 121.7 (C-5), 121.0 (C-4b),
120.0 (C-6), 117.2 (C-3ꢀ), 114.5 (C-4), 113.6 (C-5ꢀ),
110.6 (C-8), 17.4 (CH3). Anal. calcd. for C18H15N3:
C, 79.10; H, 5.53; N, 15.37. Found C, 79.25; H,
5.62; N, 15.30.
Ten milliliter of an overnight culture of S. typhimu-
rium YG1024 in Oxoid No. 2 (Unipath Ltd., Hamp-
shire, England) were pelleted by centrifugation and
resuspended in 4 ml of 0.1 M sodium phosphate buffer
(pH 7.4). This suspension was mixed with 2 ml of
water containing 8 mg of norharman·HCl plus 4 mg of
o-toluidine or 2 ml of 25% DMSO solution containing
5 ␮g of amino-3ꢀ-methylphenylnorharman with 20 ml
of S9 mix. After incubation for 6 h at 37◦C, the bacte-
ria were collected by centrifugation and the bacterial
pellet was washed with 4 ml of distilled water. Bac-
terial cell clumps were mixed with buffer containing
25 mM Tris–HCl (pH 8.0), 1.7 mM sodium chloride,
4 mM potassium chloride, 0.3 mM disodium hydro-
genphosphate, 10 mM EDTA and 0.4% SDS. DNA
was then obtained by standard procedures involving
the enzymatic digestion of protein and RNA followed
by extraction with phenol and chloroform/isoamyl al-
cohol (24:1, v/v). DNA concentration was estimated
spectrophotometrically at 260 nm and adjusted to
2 mg per milliliter of 0.01 × SSC buffer.
2.11. 32P-postlabeling method
Bacterial DNA was digested with micrococcal nu-
clease and phosphodiesterase II, and the digest was
32P-postlabeled under modified adduct intensification
conditions, as reported previously [9].
2.9. Measurement of mutagenic activity
3. Results
The mutation assay was carried out as described
previously [18], using S. typhimurium TA98, TA100,
YG1024 and YG1029. S. typhimurium YG1024 and
YG1029, produced by introducing plasmids contain-
ing the acetyltransferase gene from TA1538 into TA98
and TA100, respectively [19], were kindly provided
by Dr. T. Nohmi, National Institute of Hygienic Sci-
ences, Tokyo. All test samples except those obtained
by HPLC separation were dissolved in 100 ␮l of
dimethyl sulfoxide (DMSO). For materials at various
steps in HPLC purification, 100 ␮l of 50% DMSO
was used. The S9 mix contained 50 ␮l of S9, unless
otherwise stated, in a total volume of 500 ␮l.
3.1. Isolation and structural determination
of mutagens formed from norharman with
o-toluidine in the presence of S9 mix
The reaction products from norharman with
o-toluidine in the presence of S9 mix were sepa-
rated by HPLC on a semi-preparative ODS column.
Fig. 1 shows HPLC profiles for UV absorbance and
mutagenicity. Mutagenicity of each fraction using
S. typhimurium YG1024 was examined in the pres-
ence of S9 mix, and the major mutagenic activity

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N. Hada et al. / Mutation Research 493 (2001) 115–126
Fig. 2. HPLC chromatogram of mutagenic compound I. Column:
analytical ODS-80Ts, mobile phase: 50% acetonitrile in water with
0.1% diethylamine adjusted to pH 6.0 with acetic acid. The upper
line shows UV absorbance at 254 nm, and the lower bar shows
mutagenicity of each 1 min fraction in S. typhimurium YG1024
with S9 mix.
Fig. 1. Separation of mutagens formed from norharman with
o-toluidine in the presence of S9 mix by HPLC. A sample of the
reaction mixture was applied to a semi-preparative ODS column,
and an aliquot of each 4 min fraction was tested for mutagenicity
in S. typhimurium YG1024 with or without S9 mix. The UV ab-
sorbance of the eluate was monitored at 254 nm. Elution positions
of norharman and o-toluidine under these conditions were 16 and
10 min, respectively.
was repeated more than 10 times and about 30 ␮g of
compound I was collected for the measurement of UV
1
absorption, mass and H-NMR spectra for structural
analysis. The yield of compound I from norharman
was around 0.06%.
The UV-absorption spectrum of the mutagen, com-
pound I, obtained on the ODS column with a photodi-
ode array detector, showed absorption maxima at 238,
288 and 357 nm (Fig. 3). This UV absorption pattern
was similar to that of aminophenylnorharman [15].
The mass spectrum of compound I exhibited a molec-
was recovered in fractions with retention times of
48–52 min. On the other hand, when the mutation
test was carried out in the absence of S9 mix, mu-
tagenicity was observed in fractions with retention
times of 48–64 min. The elution positions of these
mutagenic fractions on HPLC were different from
those of norharman and o-toluidine.
The mutagenic fraction, which showed activity
with S9 mix, was separated by HPLC on a TSKgel
ODS-80Ts column. Several UV absorption peaks
were detected and the mutagenicity was mainly recov-
ered in fractions with retention times of 15–17 min.
This mutagenic fraction was further purified on the
same column with a mobile phase of 50% acetonitrile
in 0.1% diethylamine-water solution adjusted to pH
6.0 with acetic acid, and a single peak fraction with
mutagenicity (compound I) was detected at retention
times of 13–14 min (Fig. 2). The above procedure
1
ular ion peak at m/z 273. The H-NMR spectrum in
CDCl3 is shown in Table 1. It showed the presence of
the following protons: δ (ppm) 2.27 (3H), 6.83 (1H),
7.19 (1H), 7.21 (1H), 7.31 (1H), 7.41 (1H), 7.53 (1H),
8.14 (1H), 8.18 (1H), 8.48 (1H), 8.76 (1H). Among
these, seven protons were assigned to the norharman
moiety, H-1 at 8.76, H-3 at 8.48, H-4 at 8.14, H-5 at
8.18, H-6 at 7.31, H-7 at 7.53 and H-8 at 7.41 ppm,
three to the aromatic protons of the o-toluidine moi-
ety, H-2ꢀ at 7.21, H-5ꢀ at 6.83 and H-6ꢀ at 7.19 ppm,
and the three at 2.27 ppm corresponded to the methyl
1
group. Based on the UV, mass and H-NMR spec-
tra of compound I, its structure was deduced to be
amino-3ꢀ-methylphenylnorharman.

N. Hada et al. / Mutation Research 493 (2001) 115–126
121
UV-absorption peaks were detected, and mutagenic-
ity was only found in a peak fraction (compound II)
with retention times of 14–15 min. The UV absorption
maxima of compound II were observed at 250, 301 and
387 nm. Based on the present and previous observa-
tions [15], the structure of compound II was deduced
to be hydroxyamino-3ꢀ-methylphenylnorharman.
3.2. Structural determination of compound I
To confirm the structure of compound I, we tried to
chemically synthesize amino-3ꢀ-methylphenylnorhar-
man which were the deduced mutagen produced by
norharman and o-toluidine. When norharman was
reacted with three molar equivalents of 5-fluoro-
2-nitrotoluene in the presence of potassium carbonate,
9- (3ꢀ- methyl-4ꢀ -nitrophenyl)-9H-pyrido[3,4-b]indole
was produced at a yield of 80%. This nitro derivative
was then reduced to obtain amino-3ꢀ-methylphenylnor-
harman under a hydrogen atmosphere in the presence
of Pd–C. The yield of amino compound from nitro
compound was 87%. The UV and mass spectra of
the synthetic compound were the same as those of
the mutagenic compound I formed by the reaction
Fig. 3. UV absorption spectrum of compound I. The spectrum was
measured on an ODS column with a photodiode array detector.
The material was eluted with the same mobile phase described for
Fig. 2.
On the other hand, the fractions, which showed
activity without S9 mix, with retention times of 56–
64 min on the semi-preparative ODC column (Fig. 1)
were further purified by HPLC under the same condi-
tions as for compound I. As shown in Fig. 4, several
Table 1
a
1H-NMR spectral data of compound I and chemically synthesized amino-3ꢀ-methylphenylnorharman in CDCl3
Compound I
Synthetic compound
1
8.76 (1H, sb)
8.77 (1H, s)
3
4
5
6
7
8
2ꢀ
5ꢀ
6ꢀ
CH3
NH2
8.48 (1H, br.s)
8.14 (1H, br.s)
8.49 (1H, d, J = 4.5 Hz)
7.99 (1H, d, J = 4.5 Hz)
8.18 (1H, d, J = 7.5 Hz)
7.31 (1H, br.t, J = 7.5 Hz)
7.52 (1H, t, J = 7.5 Hz)
7.41 (1H, d, J = 7.5 Hz)
7.21 (1H, s)
6.86 (1H, d, J = 8.0 Hz)
7.19 (1H, d, J = 8.0 Hz)
2.26 (3H, br.s)
8.18 (1H, d, J = 7.5 Hz)
7.31 (1H, br.t, J = 7.5 Hz)
7.53 (1H, br.t, J = 7.5 Hz)
7.41 (1H, d, J = 7.5 Hz)
7.21 (1H, br.s)
6.83 (1H, d, J = 8.0 Hz)
7.19 (1H, d, J = 8.0 Hz)
2.27 (3H, br.s)
3.86 (2H, br.s)
a 1H-NMR chemical shifts are presented as parts per million.
b s: singlet; d: doublet; t: triplet; and br: broad.

122
N. Hada et al. / Mutation Research 493 (2001) 115–126
mutagenic activity without S9 mix, was tentatively
considered to be hydroxyamino-3ꢀ-methylphenyl-
norharman. To confirm its structure, authentic hydro-
xyamino-3ꢀ-methylphenylnorharman was synthesized
from 9-(3ꢀ-methyl-4ꢀ-nitrophenyl)-9H-pyrido[3,4-b]
indole by its reduction. The elution position and UV
spectrum of this synthetic hydroxyamino-3ꢀ-methyl-
phenylnorharman in HPLC on an ODS column were
the same as those of compound II produced by norhar-
man and o-toluidine in the presence of S9 mix. Based
on these results, compound II was determined to be
hydroxyamino-3ꢀ-methylphenylnorharman.
3.4. Mutagenicity and DNA adduct formation by
amino-3ꢀ-methylphenylnorharman
The mutagenicity of synthesized amino-3ꢀ-methyl-
phenylnorharman was tested in S. typhimurium TA98,
TA100, YG1024 and YG1029 with S9 mix containing
5 ␮l of S9 per plate. Amino-3ꢀ-methylphenylnorhar-
man was mutagenic in all four strains in a dose-
dependent manner. Similar to aminophenylnorharman,
amino-3ꢀ-methylphenylnorharman showed higher mu-
tagenicity in S. typhimurium TA98 and YG1024, de-
tectors of frameshift mutations, than in S. typhimurium
TA100 and YG 1029, detectors of base-pair-change
mutations, in the presence of S9 mix. Amino-3ꢀ-
methylphenylnorharman induced 41,000 revertants of
TA98 and 1,980 revertants of TA100 per microgram.
YG1024 and YG1029, which have high acetyltrans-
ferase activity, were more sensitive to this mutagen,
and 1 ␮g of the compound gave 698,000 revertants
for YG1024 and 8,640 revertants for YG1029. In the
absence of S9 mix, amino-3ꢀ-methylphenylnorharman
was not mutagenic in either strain.
Fig. 4. HPLC profile of mutagenic compound II. The fractions with
mutagenic activity in YG1024 without S9 mix, as shown in Fig. 1,
were combined and applied to an analytical ODS 80Ts column.
The material was eluted with a mobile phase of 25% acetonitrile
in 25 mM phosphate buffer (pH 2.0). The upper line shows UV
absorbance of the eluate at 254 nm, and the lower bar shows
mutagenicity of each 1 min fraction in S. typhimurium YG1024
without S9 mix. A UV absorption peak showing mutagenicity is
indicated by an arrow.
of norharman with o-toluidine in the presence of S9
mix. When the 1H-NMR spectrum of the synthetic
compound in CDCl3 was measured, 15 protons were
observed. Among these, 13 protons indicating aro-
matic protons from the norharman and o-toluidine
moieties and methyl protons coincided with those
of compound I (Table 1). In addition, two protons
at 3.86 ppm assigned to an amino group were
detected in the synthetic compound. Based on
these results, compound I was considered to be
amino-3ꢀ-methylphenylnorharman, formed by cou-
pling of norharman at the N-9 position with o-toluidine
at the para position of an amino group.
A sample of 5 ␮g of amino-3ꢀ-methylphenylnorhar-
man was incubated with an overnight culture of
S. typhimurium YG1024 in the presence of S9 mix
for 6 h at 37◦C, and the resulting DNA adducts were
analyzed by the 32P-postlabeling method under mod-
ified adduct intensification conditions. As shown in
Fig. 5A, three adduct spots, two major (spots 1 and 2)
and one minor (spot 3), were detected, and their RALs
were estimated to be 0.7, 0.4 and 0.1 adducts per 108
nucleotides, respectively, with a total RAL value of
1.2 per 108 nucleotides. A mixture of norharman·HCl
(8 mg) and o-toluidine (4 mg) in the presence of S9
mix was incubated with YG1024, and DNA adducts
3.3. Structure of compound II, showing
activity without S9 mix
Aminophenylnorharman and its N-hydroxy deriva-
tive have been previously reported to be produced
by reacting norharman with aniline in the presence of
S9 mix [15]. Therefore, compound II, which showed

N. Hada et al. / Mutation Research 493 (2001) 115–126
123
Fig. 5. Autoradiograms of DNA adducts in S. typhimurium YG1024 treated with amino-3ꢀ-methylphenylnorharman or a mixture of
norharman and o-toluidine in the presence of S9 mix. Amino-3ꢀ-methylphenylnorharman (A) or a mixture of norharman plus o-toluidine
(B) was incubated with an overnight culture of S. typhimurium YG1024 and S9 mix for 6 h at 37◦C, and DNA adduct formation
was analyzed under modified adduct intensification conditions. The imaging plates were exposed for 24 h. DNA adducts derived from
amino-3ꢀ-methylphenylnorharman and the mixture of norharman and o-toluidine are indicated by arrowheads.
were analyzed under the same conditions as for
amino-3ꢀ-methylphenylnorharman. The combination
of norharman with o-toluidine yielded the same DNA
adduct pattern as amino-3ꢀ-methylphenylnorharman,
with a total RAL value for the three adduct spots (I, II,
III) of 186 per 108 nucleotides (Fig. 5B). No adduct
spots were detected in the absence of S9 mix with
either amino-3ꢀ-methylphenylnorharman or a mixture
of norharman and o-toluidine.
HPLC under the same three conditions as were used
for the separation of amino-3ꢀ-methylphenylnorhar-
man. At the third HPLC step with a TSKgel ODS-80Ts
column, a single UV absorption peak showing mu-
tagenicity in YG1024 with S9 mix was detected at a
retention time of 12.5 min. Under this condition, au-
thentic amino-2ꢀ-methylphenylnorharman was eluted
at the same position. These procedures were repeated
several times and about 50 ng of this mutagen was
collected for measurement of the UV absorption
spectrum on the ODS column with a photodiode
array detector. Fig. 6A shows the UV spectrum of the
mutagen produced from norharman with m-toluidine
in the presence of S9 mix. Absorption maxima of
the mutagen were seen at 238, 283 and 356 nm,
and its spectrum coincided with that of authentic
amino-2ꢀ-methylphenylnorharman (Fig. 6B). Thus, the
mutagen produced by norharman and m-toluidine was
concluded to be amino-2ꢀ-methylphenylnorharman.
The yield of this mutagen from norharman was
0.02%. Amino-2ꢀ-methylphenylnorharman induced
140 revertants of TA98, 16 revertants of TA100, 3000
revertants of YG1024 and 69 revertants of YG1029 per
microgram in the presence of S9 mix. These mutagenic
activities were much lower than those of the 3ꢀ-methyl
3.5. Isolation and structural determination of
mutagen formed from norharman with m-toluidine
in the presence of S9 mix
Based on the above results, it is expected that
amino-2ꢀ-methylphenylnorharman would be produced
when a mixture of norharman and m-toluidine was
incubated in the presence of S9 mix. Thus, authen-
tic amino-2ꢀ-methylphenylnorharman was chemically
synthesized from norharman and 2-fluoro-5-nitroto-
luene as starting materials. Then, the formation of
amino-2ꢀ-methylphenylnorharman in a reaction mix-
ture of norharman and m-toluidine with S9 mix at
37◦C for 20 min was analyzed by HPLC as follows.
The mutagen in the reaction mixture was purified by

124
N. Hada et al. / Mutation Research 493 (2001) 115–126
Fig. 6. UV absorbance spectra of mutagen formed from norharman with m-toluidine, and authentic amino-2ꢀ-methylphenylnorharman.
Mutagen (A) produced by norharman with m-toluidine and authentic amino-2ꢀ-methylphenylnorharman (B) were examined on an analytical
ODS 80Ts column with a photodiode array detector. The materials were eluted with a mobile phase of 50% acetonitrile in water with
0.1% diethylamine adjusted to pH 6.0 with acetic acid.
derivative. Amino-2ꢀ-methylphenylnorharman was
not mutagenic in either strain without S9 mix.
than in S. typhimurium TA100 and YG1029,
detectors of base-pair-change mutations. In addition,
this compound gave higher mutagenic activities in
YG1024 and YG1029, which have high O-acetyltrans-
ferase activity, than in TA98 and TA100, suggesting
that acetyltransferase is required for the mutagenic-
ity of amino-3ꢀ-methylphenylnorharman, as with
aminophenylnorharman and other known carcinogenic
HCAs [5,15,20]. The mutagenic activity of amino-3ꢀ-
methylphenylnorharman is comparable to those of 3-
amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1)
and 2-amino-6-methyldipyrido[1,2-a:3ꢀ,2ꢀ-d]imidazole
(Glu-P-1) [5]. Amino-3ꢀ-methylphenylnorharman
yielded the same DNA adducts in S. typhimurium
YG1024 as those observed with the mixture of
norharman and o-toluidine. Based on these obser-
vations, proposed mechanisms of the co-mutagenic
action of norharman with o-toluidine in the pres-
ence of S9 mix are shown in Fig. 7. First, the N-9
position of norharman couples with o-toluidine at the
para position of the amino group by enzymatic re-
action to produce amino-3ꢀ-methylphenylnorharman.
Next, the exocyclic amino group of this compound
may be oxidized to an N-hydroxyamino derivative by
P-450s, and further metabolically activated to form
the N-acetoxy derivative by the action of acetyltrans-
ferase. The ultimate form reacts with DNA bases to
induce mutations in Salmonella strains. In addition,
4. Discussion
Recently, we reported that the mechanism of the
appearance of mutagenicity by norharman with ani-
line in the presence of S9 mix was suggested to be
as follows [15]; norharman reacts with aniline and
produces the mutagen, aminophenylnorharman. This
compound is metabolically activated by S9 mix and
converts to a hydroxyamino derivative. Then, hydroxy-
aminophenylnorharman is further activated to its
ultimate forms and reacts with DNA bases to induce
mutation in Salmonella strains.
In the present study, we determined the structures
of mutagenic compounds produced from norhar-
man with toluidine isomers. When norharman and
o-toluidine were incubated with S9 mix, two mu-
tagens, showing mutagenicity with and without S9
mix, respectively, were produced. The mutagen that
showed activity with S9 mix was determined to be
a new HCA, amino-3ꢀ-methylphenylnorharman, and
the other was confirmed to be its N-hydroxyamino
derivative. Amino-3ꢀ-methylphenylnorharman exhib-
ited higher mutagenic activities in S. typhimurium
TA98 and YG1024, detectors of frameshift mutations,

N. Hada et al. / Mutation Research 493 (2001) 115–126
125
Fig. 7. Mutagen formation from norharman with o-toluidine in the presence of S9 mix, and its metabolic activation processes to induce
mutation in Salmonella strains.
norharman also reacted with m-toluidine in the
presence of S9 mix to yield amino-2ꢀ-methylphenyl-
norharman. A similar mechanism (Fig. 7) is expected
for the appearance of mutagenicity of the mixture
of norharman with m-toluidine in the presence of
S9 mix.
In conclusion, mutagenicity of a mixture of norhar-
man, o- or m-toluidine and S9 mix was due to the
formation of a new mutagenic HCA compound, either
amino-3ꢀ-methylphenylnorharman or amino-2ꢀ-methyl
phenylnorharman, as in the generation of aminopheny-
lnorharman by the reaction of norharman and aniline
[15]. When aminophenylnorharman was given to F344
rats by i.g. intubation, severe testicular damage was
observed [21]. Moreover, we have recently reported
that the development of glutathione S-transferase
placental form (GST-P)-positive foci, a putative pre-
neoplastic lesion, in the liver of rat was increased
in a dose dependent manner by administration of
aminophenylnorharman in diet at a dose of 10, 20
and 50 ppm for 4 weeks [22]. This suggests that
aminophenylnorharman could induce liver tumors in
rats. Aminomethylphenylnorharman, being an ana-
logue of aminophenylnorharman, might also possess
similar biological activities. As mentioned above,
norharman and toluidine isomers, such as o- and
m-toluidines, are present together in our environment.
Therefore, it is very important to examine the for-
mation of aminomethylphenylnorharman derivatives
from norharman and toluidine isomers in vivo and
to determine what enzymes might be involved in the
reaction of norharman with these aromatic amines.
Moreover, it is also important to evaluate the toxicity
and carcinogenicity of aminomethylphenylnorharman
derivatives or mixtures of norharman with toluidine
isomers in rodents.
We previously reported that the mixture of norhar-
man (200 mg) with o- or m-toluidine (100 mg) induced
6990 and 62 revertants of TA98, respectively, in the
presence of S9 mix [9]. In the present study, the mu-
tagenic activity of amino-3ꢀ-methylphenylnorharman
was more than 200-fold that of amino-2ꢀ-methylphenyl
norharman. Moreover, the yield of amino-3ꢀ-methyl-
phenylnorharman from norharman was three times
larger than that of the 2ꢀ-methyl derivative. These
diversities may be associated with the difference
between in the mutagenicities of norharman and o-
or m-toluidine. In contrast, a mixture of norharman
with p-toluidine in the presence of S9 mix has been
shown to be not mutagenic in S. typhimurium TA98
[9]. Thus, the methyl group of p-toluidine may in-
terfere with the coupling reaction of norharman with
p-toluidine. Moreover, we cannot exclude the possi-
bility that even though coupled compound(s), such as
9-(2ꢀ-amino-5ꢀ-methylphenyl)-9H-pyrido[3,4-b]indole
and 9-(5ꢀ-amino-2ꢀ-methylphenyl)-9H-pyrido[3,4-b]
indole, were formed from norharman with p-toluidine,
they may not show any mutagenicity, although the
mutagenicities of these derivatives have not yet been
examined.

126
N. Hada et al. / Mutation Research 493 (2001) 115–126
[10] IARC (International Agency for Research on Cancer),
Acknowledgements
Aniline and aniline hydrochloride, IARC Monographs on the
Evaluation of Carcinogenic Risks of Chemicals to Human,
Vol. 27, IARC, Lyon, 1982, pp. 39–61.
This study was supported by a Grant-in-Aid for
Cancer Research from the Ministry of Health and
Welfare of Japan, by grants from the Organization for
Pharmaceutical Safety and Research (OPSR) of Japan,
and by the Smoking Research Foundation. N. Hada,
Y. Totsuka and T. Enya were recipients of Research
Resident Fellowships from the Foundation of Cancer
Research during this work.
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