Carcinogenesis vol.24 no.12 pp.1985±1993, 2003
DOI: 10.1093/carcin/bgg170
Potent genotoxicity of aminophenylnorharman, formed from non-mutagenic
norharman and aniline, in the liver of gpt delta transgenic mouse
Ken-ichi Masumura1, Yukari Totsuka2, Keiji
Wakabayashi2 and Takehiko Nohmi1,3
1
Division of Genetics and Mutagenesis, National Institute of Health Sciences,
1-18-1 Kamiyoga Setagaya-ku, Tokyo 158-8501, Japan and 2Cancer
Prevention Division, National Cancer Center Research Institute, Chuo-ku,
Tokyo 104-0045, Japan
3
To whom correspondence should be addressed
Email: [email protected]
Aminophenylnorharman (APNH) is formed from nonmutagenic norharman and aniline, and is mutagenic to
Salmonella typhimurium TA98 with S9 mix. Norharman
and aniline are present in cigarette smoke and cooked
foods and both compounds are detected in human urine
samples, suggesting that APNH could be a mutagenic and
carcinogenic human risk factor. The purpose of the present
study was to determine the in vivo mutagenicity of APNH.
Male gpt delta transgenic mice were fed a diet containing
10 or 20 p.p.m. APNH for 12 weeks. The gpt mutant frequency (MF) in the liver increased 10-fold in 20 p.p.m.
APNH-treated mice, which was almost equivalent to the
MF observed in the liver of the same transgenic mice
treated with 300 p.p.m. 2-amino-3,8-dimethylimidazo[4,5-f ]
quinoxaline for 12 weeks. In the colon mucosa, the gpt MF
increased ~5-fold in 20 p.p.m. APNH-treated mice. Our
results suggest that APNH is a strong hepatic mutagen in
mice. The APNH-induced gpt mutations in the liver were
dominated by G:C to T:A transversions, followed by G:C
to A:T transitions. They also included single G:C deletions
in G:C run sequences and 2 bp deletions: GCGC to GC and
CGCG to CG. The Spiÿ deletion MF in the liver was
13-fold higher in 20 p.p.m. APNH-treated mice, relative to
the control, and were dominated by single base pair deletions, in particular, in G:C run sequences. Large deletions
were rare. The mutational characteristics induced by
APNH are compared with those induced by other heterocyclic amines, and the human risk of APNH is discussed.
YG1024 with S9 mix in the presence of non-mutagenic aromatic amines such as aniline (4±8). Aminophenylnorharman
[APNH: 9-(40 -aminophenyl)-9H-pyrido[3,4-b]indole], which
is formed from norharman and aniline by metabolic activation
systems and in vivo, was recently identified as a mutagenic
heterocyclic amine (Figure 1) (7,8). Both norharman and aniline are present in cigarette smoke and cooked foods (3,9) and
are detected in human urine and milk samples (10±12). APNH
is detected in the rat urine when norharman and aniline are
administrated by oral gavage (13). In addition, APNH is
detected in the reaction mixture of norharman and aniline in
the presence of human microsome fractions (13). Therefore,
APNH may be a human risk factor if it is mutagenic in vivo. To
examine this possibility, the genotoxicity of APNH was examined using gpt delta transgenic mice. In this mouse model,
point mutations, such as base substitutions and frameshift
mutations, are detected by 6-thioguanine (6-TG) selection
using the Escherichia coli gpt gene and deletions including
frameshifts are detected by Spiÿ selection using the red/gam
genes of lambda phage (14,15). These two genetic selections
are complementary and identify different types of mutations.
In previous studies, we characterized the in vivo mutations
induced by mutagenic and carcinogenic heterocyclic amines
such as 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine
(PhIP) and 2-amino-3,8-dimethylimidazo[4,5-f ]quinoxaline
(MeIQx) in the same mouse model (16±18). The highest
mutant frequencies (MFs) were observed in the colon and
liver, respectively, in the PhIP- and MeIQx-treated mice.
Here, we report for the first time the in vivo mutagenicity of
APNH in the liver and the colon. APNH is more mutagenic in
the liver than in the colon, and its mutagenicity in the liver is
much stronger than that of MeIQx in the same organ. The
possible carcinogenic risk of APNH is discussed.
Material and methods
Treatment of mice and rescue of transgene
APNH was purchased from the Nard Institute (Osaka, Japan). Transgenic mice
gpt delta (C57BL/6 J background) were fed a diet containing 20 or 10 p.p.m. of
Introduction
Humans are exposed to a variety of environmental chemicals.
Some of these chemicals are mutagens and/or carcinogens
associated with human risk (1). A non-mutagenic b-carboline
compound, 9H-pyrido[3,4-b]indole (norharman), is widely
detected in cigarette smoke and cooked foods at much higher
concentrations than those of known mutagenic/carcinogenic
heterocyclic amines (2,3). Norharman itself is not mutagenic
to Salmonella typhimurium TA98 and TA100 with or without
S9 mix. However, norharman becomes mutagenic to TA98 and
Abbreviations: APNH, aminophenylnorharman; MeIQx, 2-amino-3,
8-dimethylimidazo[4,5-f ]quinoxaline; MF, mutant frequency; PhIP, 2-amino1-methyl-6-phenylimidazo[4,5-b]pyridine; 6-TG, 6-thioguanine.
Carcinogenesis vol.24 no.12 # Oxford University Press; all rights reserved.
Fig. 1. Formation of aminophenylnorharman from norharman and aniline
in the presence of S9 mix.
1985
K.Masumura et al.
APNH for 12 weeks. Control animals were fed a basal diet of CE-2 (Japan Clea
Laboratory, Tokyo, Japan). After the treatments, the mice were fed a basal diet
for another 2 weeks and then killed. Each group consisted of five 7-week-old
male mice. Genomic DNA was extracted from liver and colon mucosa using
RecoverEaseTM DNA Isolation Kit (Stratagene, La Jolla, CA). Lambda EG10
phages were rescued from genomic DNA by in vitro packaging method using
TranspackTM packaging extract (Stratagene). The experimental protocol was
approved by the Committee for Ethics of Animal Experimentation of National
Cancer Center Research Institute.
Mutation assay and sequencing analysis
The gpt mutation assay was performed as described previously (15). The
rescued phages were infected to E.coli YG6020 expressing Cre recombinase
to convert the transgene to plasmid. Infected cells were mixed with molten soft
agar and poured onto agar plates containing chloramphenicol (Cm) and 6-TG.
The plates were incubated at 37 C for the selection of colonies harboring
plasmid carrying the mutated gpt gene. Infected cells were also poured on
the plates containing Cm without 6-TG to determine the number of rescued
plasmids. The gpt MF was calculated as described previously (15). The
selected 6-TG-resistant mutants were cultured and collected. A 739 bp DNA
fragment containing the mutated gpt gene was amplified by polymerase chain
reaction (PCR) as described previously (15). DNA sequencing of the gpt gene
was performed with BigDyeTM Terminator Cycle Sequencing Ready Reaction
(Applied Biosystems, Foster City, CA) on ABI PRISMTM 310 Genetic
Analyzer (Applied Biosystems). The sequencing primer was gptA2 primer
(50 -TCTCGCGCAACCTATTTTCCC-30 ).
The Spiÿ mutation assay was performed as described previously (15) with
some modification. We added 10 mM MgSO4 to both agar plates and soft agar
to improve the detection efficiency of Spiÿ plaques as described previously
(19). The rescued phages were infected to E.coli XL1-Blue MRA (P2).
Infected cells were mixed with molten soft agar, poured onto lambda-trypticase
agar plates and incubated at 37 C. The plaques detected on the plates
(Spiÿ candidates) were suspended in 50 ml of SM buffer. The suspension was
spotted on the two types of plates where XL1-Blue MRA (P2) or WL95
(P2) strain was spread. The plates were incubated for 24 h at 37 C. The numbers of mutants that made clear spots on both strains were counted as confirmed Spiÿ mutants. Phage lysates of the recovered Spiÿ mutants were used
as templates for PCR analysis (20,21). The PCR primers were: primer 001
(50 -CTCTCCTTTGATGCGAATGCCAGC-30 ); primer 002 (50 -GGAGTAATTATGCGGAACAGAATCATGC-30 ); primer 005 (50 -CGTGGTCTGAGTGTGTTACAGAGG-30 ); primer 006 (50 -GTTATGCGTTGTTCCATACAACCTCC-30 ), and primer 012 (50 -CGGTCGAGGGACCTAATAACTTCG-30 ).
The appropriate primers for DNA sequencing were selected based on the
results of PCR analysis. The sequencing primers have been described previously (20±22). The entire sequence of lambda EG10 is available at http://
dgm2alpha.nihs.go.jp.
Statistical analysis
All data are expressed as mean SD. Differences between two groups were
tested for statistical significance using Student's t-test. A P value 50.05
denoted the presence of a statistically significant difference.
Results
Mutagenicity of APNH was assayed in the liver and colon of
APNH-treated and untreated male mice (Figure 2). For the gpt
assay, we analyzed 346 000 to 1 098 000 colonies derived from
the rescued phages per organ per mouse. For the Spiÿ assay,
we analyzed 611 000 to 4 110 000 rescued plaques per organ
per mouse. The gpt MF in the liver of the 20 p.p.m. APNHtreated mice was 68 10ÿ6, which was about 10 times higher
than that of untreated mice (6.6 10ÿ6). Furthermore, ~6-fold
increase was observed in the 10 p.p.m. APNH-treated mice
(38 10ÿ6). In the colon, the gpt MFs of the 10 or 20 p.p.m.
APNH-treated mice were 17 10ÿ6 or 29 10ÿ6, respectively. Those were 3 or 5 times higher than that of untreated
mice (6.2 10ÿ6).
To characterize the APNH-induced gpt mutations, we analyzed 73 and 51 mutants recovered from the liver of the
20 p.p.m. APNH-treated mice and untreated mice, respectively
(Tables I and II). The APNH-induced gpt mutations recovered
from the liver were dominated by single base substitutions
1986
Fig. 2. The gpt and Spiÿ mutant frequencies in the liver and colon mucosa
of the mice fed a diet containing APNH for 12 weeks. Data are mean SD.
P 5 0.01, relative to the control.
Table I. Mutation spectra of gpt mutations recovered from the liver of
APNH-treated gpt delta mice
APNH (12 weeks)
0 p.p.m.
n
Base substitution
Transition
G:C to A:T
(at CpG sites)
A:T to G:C
Transversion
G:C to T:A
G:C to C:G
A:T to T:A
A:T to C:G
Deletion
Insertion
Others
Total
20 p.p.m.
%
MF
(10ÿ6)
n
41
2.7
17 (5)
5
10
0.6
7
1
4
2
9
1
1
14
2
8
4
18
2
2
51
100
21 (12)
%
MF
(10ÿ6)
23
15.8
1
1
0.9
0.9
0.1
0.5
0.3
1.2
0.1
0.1
37
1
0
0
12
0
5
51
1
0
0
16
0
7
34.5
0.9
0.0
0.0
11.2
0.0
4.7
6.6
73
100
68.0
(56/73 ˆ 77%): G:C to T:A transversions (37/56 ˆ 66%)
predominated, followed by G:C to A:T transitions (17/56 ˆ
30%). Deletions were also observed (12/73 ˆ 16%). Most of
them were 1 or 2 bp deletions at G:C base pairs (9/12 ˆ 75%).
They included four single G:C deletions in G:C run sequences,
two single G:C deletions without run sequence, two GCGC to
GC and one CGCG to CG deletions. Other types of mutations
included tandem base substitutions and one base deletion with
base substitution. In untreated mice, 78% (40/51) of total
mutations were base substitutions. G:C to A:T transitions predominated (21/40 ˆ 53%) and 57% (12/21) of them occurred
at 50 -CpG-30 sites. G:C to T:A transversions (7/40 ˆ 18%) and
A:T to G:C transitions (5/40 ˆ 13%) were also observed.
Deletions amounted to 18% (9/51). The predominant types
of deletion were single-base deletions at A:T run sequences
(6/9 ˆ 67%). One insertion and one tandem base substitution
were observed.
The Spiÿ MF in the liver of the 10 or 20 p.p.m. APNHtreated mice were 12 10ÿ6 or 16 10ÿ6, respectively, which
were 10 or 13 times higher than that of the untreated mice
(1.2 10ÿ6). To characterize Spiÿ deletion mutations induced
by APNH, 40 and 11 Spiÿ mutants recovered from the livers
Base substitution
Transition
Transversion
Type of mutation
G:C ! T:A
A:T ! G:C
G:C ! A:T
G!T
G!T
C!A
C!A
G!T
G!T
G!T
G!T
G!T
G!T
C!A
G!T
G!T
G!T
G!T
C!A
G!T
C!A
G!T
T!C
T!C
A!G
A!G
56
181
191
419
7
79
108
109
113
143
145
152
176
185
189
208
232
265
281
287
289
320
401
G!A
G!A
C!T
C!T
G!A
G!A
G!A
G!A
G!A
G!A
G!A
G!A
G!A
G!A
C!T
G!A
G!A
G!A
G!A
G!A
G!A
C!T
Sequence
change
26
27
37
64
87
107
110
115
116
128
143
145
176
185
202
274
281
352
401
402
406
409
Position in
gpt gene
Table II. Summary of gpt mutations recovered from the liver of APNH-treated gpt delta mice
Glu ! Stop
Glu ! Stop
Ser ! Arg
Arg ! Ser
Gly ! Val
Arg ! Leu
Glu ! Stop
Gly ! Val
Cys ! Phe
Ser ! Ile
Tyr ! Stop
Glu ! Stop
Glu ! Stop
Asp ! Tyr
Gly ! Val
Thr ! Asn
Ala ! Ser
Ala ! Glu
Trp ! Leu
Leu ! Pro
Ser ! Pro
Asp ! Gly
Asp ! Gly
Trp ! Stop
Trp ! Stop
Gln ! Stop
Arg ! Stop
Trp ! Stop
Ser ! Asn
Arg ! His
Gly ! Ser
Gly ! Asp
Gly ! Asp
Arg ! His
Glu ! Lys
Cys ! Tyr
Ser ! Asn
Gln ! Stop
Asp ! Asn
Gly ! Asp
Gly ! Ser
Trp ! Stop
Trp ! Stop
Glu ! Lys
Gln ! Stop
Amino acid
change
1
1
1
1
1
2
1
2
1
1
1
1
2
3
1
1
2
1
5
1
1
1
0 p.p.m.
3
1
2
3
1
1
1
1
2
1
1
1
1
1
2
1
1
1
1
1
1
1
1
1
1
1
1
1
2
3
2
2
2
20 p.p.m.
No. of mutants
24
04, 23
23
02, 24, 24
01, 02, 23, 23, 24
21, 24
23
04, 04, 04, 04, 23, 23
05,
22
22
25
25
21,
21
21
02
21
24
21
02,
21,
03
22,
25
21,
03,
24
22, 24, 25
23, 25
25
22
22
25
23
01, 02
01
02, 04
04
01
21,
03,
02
24
02,
01,
05,
04
01,
05,
22
02,
22
04
25
02
24
04
24
24
Source of animal (0 p.p.m.:
01±05, 20 p.p.m.: 21±25)
CpG
CpG
CpG
CpG
CpG
CpG
CpG
CpG
CpG
CpG
CpG sites
Genotoxicity of aminophenylnorharman
1987
1988
Total
Other
Insertion
Deletion
Type of mutation
Table II. Continued
A:T ! C:G
A:T ! T:A
G:C ! C:G
Sequence
change
G!T
G!T
C!A
C!A
G!T
G!C
G!C
T!A
T!A
T!A
T!A
T!G
T!G
AAAAA ! AAAA
ÿ41 bp
CC ! C
TG ! del.
ÿ384 bp
CC ! C
GCGC ! GC
CGCG ! CG
GCGC ! GC
ATC ! del.
C ! del
AAAA ! AAA
GGG ! GG
GGG ! GG
C ! del
‡8 bp repeat (TGACCTGGTGACCTGGTGG)
CTG ! TCT
AT ! CA
CG ! TT
AC ! GA
CC ! A
Position in
gpt gene
402
406
409
413
418
3
46
25
173
187
437
56
218
8±12
66±67 ! 108±109
73±74
111±112
158 ! 543
170±171
204±207
205±8
227±230
253±255
290
342±345
416±418
423±425
435
271 ! 264
77±79
178±179
243±244
277±278
332±333
Ser,Glu ! Phe,Stop
Ile ! His
Gly,Glu ! Gly,Stop
Thr ! Asp
Leu ! Arg
Val ! Gly
Trp ! Arg
Val ! Asp
Tyr ! Asn
Val ! Asp
Met ! Ile
Ala ! Pro
Trp ! Cys
Glu ! Stop
Gln ! Lys
Pro ! Gln
Asp ! Tyr
Amino acid
change
51
1
1
1
1
1
5
1
1
1
1
1
1
1
1
0 p.p.m.
1
73
2
1
1
1
1
1
2
1
1
1
1
1
1
2
1
2
1
4
2
20 p.p.m.
No. of mutants
23, 23
24
25
01
23
05
03, 03, 04, 05, 05
25
24
22, 22
05
21
22
21
22
04
05
01
23
25
21, 21
05
03
04
03
03
03
05
22
21, 21
25
22, 25, 25, 25
22, 23
05
Source of animal (0 p.p.m.:
01±05, 20 p.p.m.: 21±25)
CpG
CpG
CpG
CpG
CpG
CpG
CpG
CpG
CpG sites
K.Masumura et al.
Genotoxicity of aminophenylnorharman
Table III. Mutation spectra of Spiÿ mutations recovered from the liver of
APNH-treated gpt delta mice
APNH (12 weeks)
0 p.p.m.
n
One base pair
deletion
In run sequence
at G:C
at A:T
Beside run sequence
With base
substitution
Other
42 bp deletion
Insertion
Total
%
20 p.p.m.
MF
(10ÿ6)
n
%
MF
(10ÿ6)
6
54.5
0.7
34
85.0
13.2
1
2
1
0
9.1
18.2
9.1
0.0
0.1
0.2
0.1
0.0
20
5
3
4
50.0
12.5
7.5
10.0
7.8
1.9
1.2
1.6
2
4
1
11
18.2
36.4
9.1
100.0
0.2
0.4
0.1
1.2
2
5
1
40
5.0
12.5
2.5
100.0
0.8
1.9
0.4
15.6
of the 20 p.p.m. APNH-treated mice and untreated mice,
respectively, were analyzed (Tables III and IV). In the
APNH-treated group, 73% (29/40) were single G:C base pair
deletions. The majority of these events occurred in G:C run
sequences (20/29 ˆ 69%). The mutational hot spots, defined as
nucleotide positions where more than four mutations were
observed in three or more mice, were at nucleotides 188±190,
238±241 and 286±289 in the gam gene; the sequence changes
were GGG to GG and GGGG to GGG. In addition to single
base pair deletions, five deletions and one insertion were
observed. They include ÿ2 (2), ÿ13, ÿ2690 and ÿ3452 bp
deletions and ‡29 bp insertion. In untreated mice, 55% (6/11)
of the sequenced Spiÿ mutants were single base pair deletions.
Other five mutations were ÿ14, ÿ22, ÿ777 and ÿ4061 bp
deletions and ‡149 bp insertion.
Discussion
APNH is a newly identified mutagenic heterocyclic amine
formed by coupling of norharman with aniline in the presence
of S9 mix (7,8). APNH displays acute testicular toxicity in
F344 rats, although neither norharman nor aniline alone
induces such testicular toxicity (23). APNH also induces sister
chromatid exchanges and chromosome aberrations in cultured
mammalian cells (24). In the present study, we investigated the
genotoxicity of APNH and its molecular characteristics in vivo,
by applying the mutagenicity assay using gpt delta transgenic mice.
The gpt and Spiÿ mutations in the liver and colon were
induced in a dose-dependent manner by subchronic exposure
of mice to APNH (Figure 2). The gpt MF increased 10-fold in
the liver of mice treated with 20 p.p.m. APNH for 12 weeks.
This is almost the same level of MF observed in the liver of
mice treated with 300 p.p.m. MeIQx for 12 weeks (Figure 3).
These results demonstrate that APNH is a strong hepatic mutagen in mice. In the colon mucosa, the gpt MF increased ~5-fold
in 20 p.p.m. APNH-treated mice. Considered together, our
results suggest that liver is a more sensitive organ relative to
the colon with respect to the mutagenicity of APNH in mice.
We also compared the gpt MFs in the liver and colon of mice
treated with APNH, MeIQx or PhIP-treated mice (Figure 3).
MeIQx induces hepatocellular carcinoma, adenoma and neoplastic liver nodules in F344 rats and CDF1 mice (25,26). Our
results showed that MeIQx increased gpt MF in the liver,
which is a major target organ for carcinogenesis (18). These
results suggest that APNH may be a potential hepatocarcinogen. In line with this, Kawamori et al. (27) reported that APNH
induced glutathione S-transferase positive placental formpositive foci in the liver of male F344 rats. Induction of MFs
in the colon could also be indicative of the carcinogenicity
of APNH in the colon. However, the organ specificity and
sensitivity of carcinogenesis could also be due to a different
susceptibility or cell division kinetics in the later stages of
carcinogenesis. For example, the colon is a major target of
MF induction for PhIP in both mice and rats (16,28), but PhIP
does not induce colon cancer in mice (29). It induces colon and
prostate cancers in male F344 rats and mammary carcinoma in
female rats (30,31). To investigate the carcinogenicity of
APNH, studies involving long-term exposure of rodents to
APNH are currently underway.
The APNH-induced gpt mutations were dominated by G:C
to T:A transversions, followed by G:C to A:T transitions
(Table I), although single G:C base pair deletions at G:C run
sequences were also observed. The G:C base pair substitutions
dominated by G:C to T:A and the frameshifts at G:C base pairs
are known as typical characteristics of the mutation spectra in
rodents induced by various heterocyclic amines, such as PhIP,
MeIQx, 2-amino-3,4-dimethylimidazo[4,5-f ]quinoline (MeIQ),
2-amino-3-methylimidazo[4,5-f ]quinoline (IQ) and 2-amino9H-pyrido[2,3-b]indole (AaC) (17,18,32±34). There is sufficient evidence indicating that metabolic activation leading to
formation of DNA adducts is critical for the mutagenicity and
carcinogenicity of these amines. The heterocyclic amines are
enzymatically activated by N-oxidations catalyzed by P450 in
the liver of rodents and humans (35±37), and their N-hydroxy
products are further activated by acetyltransferases or sulfotransferases (38,39). They form DNA adducts at C8 and
N2 positions of guanine bases (40,41). Recently, deoxyguanosineC8-APNH adducts was identified as a major product formed in
various tissues of APNH-treated rats by 32P-postlabeling
method (42). This finding suggests that APNH shows mutagenicity in vivo by forming DNA adducts at guanine residues,
like other carcinogenic heterocyclic amines. In the APNHinduced mutation spectrum, G:C to C:G transversion formed
a small percentage (1%) in contrast with that of PhIP (13%)
(17). These results suggest that the different structures of
adducts could result in different preferences of misincorporation opposite adducts on the template strand during DNA
replication. In addition to DNA adduct formation, oxidative
DNA damage may also play a role in the genotoxicity of
APNH (43). APNH-induced deletions in the gpt gene also
contained 2 bp deletions; GCGC to GC and CGCG to CG. These
preferences of APNH-induced frameshift mutations could
explain the high mutagenic activity of APNH in TA98 and
YG1024, indicators of the frameshifts: 2 bp deletions of a GC
or CG within the sequence CGCGCGCG (7,8). In untreated
mice, 78% (40/51) of total mutations were base substitutions.
G:C to A:T transitions were predominant (21/40 ˆ 53%) and
57% (12/21) of such substitutions occurred at 50 -CpG-30 sites.
These results suggest that deamination of the methylated
cytosine at CpG sites contributes to spontaneous mutations
in vivo (44). G:C to T:A transversions (7/40 ˆ 18%) may
reflect oxidative damage such as 8-oxoguanine or abacic
sites in DNA (45,46). Deletions were 18% (9/51); with the
1989
1990
Other 1 bp deletion
With base substitution
Beside run sequence
In run sequence
‡29 bp
‡149 bp
ÿ13 bp
ÿ14 bp
ÿ22 bp
ÿ777 bpa
ÿ2690 bpa
ÿ3452 bpa
ÿ4061 bp
TTG ! TT
AAAG ! AAA
CAAAAAA ! AAAAAA
GTTT ! TTT
GC ! T
CG ! T
C ! del.
C ! del.
C ! del.
GCGC ! GC
CGCG ! CG
151
202
294
315
166±167
314±315
186
264
357
166±169
167±170
324 ! 296
415 ! 267
335 ! 349
265 ! 280
167 ! 190
TT ! T
CC ! C
CCC ! CC
GG ! G
AAAAA ! AAAA
GG ! G
CCCC ! CCC
GGGG ! GGG
AAAAAA ! AAAAA
CCC ! CC
TT ! T
Sequence
change
149±150
177±178
188±190
213±214
227±231
232±233
238±241
286±289
295±300
308±310
329±330
Position in
gam gene
Those mutations have short homologous sequences (2 bp) at the deletion junction.
a
Total
Insertion
>ÿ2 bp deletion
ÿ2 bp deletion
1 bp deletion
Type of deletion
Table IV. Summary of Spiÿ mutations recovered from the liver of APNH-treated gpt delta mice
1
11
1
1
1
1
1
1
1
1
1
0 p.p.m.
No. of mutants
40
1
1
1
1
1
1
1
1
1
3
1
1
1
1
1
5
7
3
1
1
1
1
4
1
0 p.p.m.
21, 22, 23, 23, 24
22, 23, 24, 25, 25, 25
22, 22, 24
23, 23, 25
24
02
21
02
01
04
23
24
04
25
21
05, 23
21
01
23, 23, 24
23
21
25
23
02
22
21
22,
22
03
24
02,
21,
02,
24
22
Source of animal (0 p.p.m.:
01±05, 20 p.p.m.: 21±25)
K.Masumura et al.
Genotoxicity of aminophenylnorharman
Fig. 3. Comparison of gpt mutant frequencies in the liver and colon of mice
treated with the heterocyclic amines. Five male mice were used for each
group of the experiments of APNH and MeIQx and three male mice for each
group of PHIP. APNH was mixed with the diet at 20 p.p.m. for 12 weeks
and the liver and colon mucosa were recovered after the following 2 weeks
on basal diet without APNH (this study). MeIQx was mixed with the diet at
300 p.p.m. for 12 weeks and the liver and colon were recovered (18). PhIP was
mixed with the diet at 400 p.p.m. for 13 weeks and the liver and colon
mucosa were recovered after the following 2 weeks on basal diet without
PhIP (16). Data are mean SD.
predominant types being single base deletions at A:T run
sequences (6/9 ˆ 67%), in contrast to that of APNH-treated
group.
Spiÿ mutations in the liver of APNH-treated mice were
induced in a dose-dependent manner (Figure 2). Spiÿ selection
positively detects deletion mutations, in which both gam and
red gene functions are simultaneously inactivated or frameshifts in the upstream gam gene (15,17,47). The reason for the
appearance of the latter class of single base pair deletions in
the gam gene is that these frameshifts not only inactivate the
gam gene function but also interfere with the start of translation of the downstream redBA genes, thereby functionally
inactivating both the gam and redBA genes. In this study, the
WL95 (P2) strain was used for confirmation of the Spiÿ
candidate plaques (see Materials and methods). This is
because we observed in the previous study that some Spiÿ
candidate plaques that were recovered from the selection
agar plates had no deletions in the gam gene, but base substitutions (17). They occupy ~12% of total Spiÿ mutants
recovered from the colon of PhIP-treated mice (11/96 ˆ
11.5%). Most of these mutants without deletions do not display
Spiÿ phenotype in another P2 lysogen WL95 (P2) (48) and do
not exhibit Fecÿ phenotype; they could make plaques on a
recA strain, whereas other typical types of Spiÿ deletion
mutants could not. We suspect the appearance of such
substitution-type mutants is due to inadequate selectivity of the
XL1-Blue MRA (P2) strain used in the Spiÿ assay. By confirmation test of the candidate plaques using WL95 (P2), we
could eliminate almost all `irregular' substitution-type mutants
before the calculation of Spiÿ MF and further sequencing
analyses.
The Spiÿ MF in the liver increased 13-fold in 20 p.p.m.
APNH-treated mice, and the majority of APNH-induced
Spiÿ mutations were single G:C base pair deletions that
occurred at G:C run sequences rather than large deletions
(Tables III and IV). In addition, nucleotides 166±170 in the
gam gene, 50 -GCGCG-30 sequence, were a target of 1 bp
deletion with base substitutions and 2 bp deletions. Similar
characteristics were observed in the colon of PhIP-treated mice
(17). On the other hand, the second major type of Spiÿ deletions observed in PhIP-treated mice, G:C base pair beside T, C
and A run sequences (24%), was not a prominently induced
class in the liver of APNH-treated mice (8%). This result
suggests that the frameshift mutation beside run sequences
was not a major target in APNH-induced mutagenesis. The
large deletions were not frequently observed in the Spiÿ
mutants recovered from APNH-treated mice, similar to the
finding in PhIP-treated mice (17). PhIP induces sister chromatid exchange, but not chromosomal aberrations in bone marrow and only weakly induces micronucleus in bone marrow
and peripheral blood in C57BL/6 mice (49). Because of the
similarity of the Spiÿ mutation spectra between APNH and
PhIP, we suspect that APNH may not efficiently induce chromosome aberrations or micronucleus in vivo. However, APNH
is reported to potently induce sister chromatid exchanges and
chromosome aberrations in vitro (24). Thus, further studies are
necessary to conduct micronucleus or chromosome aberration
assays in bone marrow or in liver to clarify the apparent
discrepancy regarding the clastogenic activity of APNH
in vivo and in vitro.
Humans are exposed to a variety of chemicals, and hence it
is not difficult to imagine that more than one compound displays combined effects. For example, simultaneous exposure
to two non-mutagenic/carcinogenic compounds may induce
genotoxicity and/or carcinogenicity in vivo. Such combined
effects have not yet been thoroughly investigated, despite the
mechanistic importance. In this study, we demonstrated that
APNH, formed from non-mutagenic norharman and aniline,
showed a strong mutagenicity in the liver and colon of mice.
We suggest that APNH may be a human risk when it is
produced even in a small amount in our body. There are
possible reasons for the strong mutagenicity of APNH. One
is the high efficiency of DNA replication error on the template
strand containing the APNH±DNA adducts [50]. However, the
characteristics of the mutational spectra of APNH observed
were largely similar to those of other heterocyclic amines,
although they somewhat differed from those of PhIP. Another
possible reason is the strong reactivity of APNH and its derivatives, which could contribute to the formation of the higher
amounts of APNH±DNA adducts. Previous studies showed
severe testicular toxicity in APNH-treated F344 rats (23), and
cultured mammalian cells treated with APNH show induction
of sister chromatid exchange and chromosome aberrations
(24). These results suggest the strong reactivity of APNH to
various cellular components including DNA and proteins.
Further studies are important to determine the mechanism of
the strong mutagenicity of APNH by estimating the amount of
DNA-adducts induced by APNH and comparing these levels
with those of other heterocyclic amines such as PhIP.
Acknowledgement
This study was supported by Grants-in-Aid for Cancer Research from the
Ministry of Health, Labor and Welfare of Japan.
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Received July 10, 2003; revised August 29, 2003; accepted September 2, 2003
1993