Micronucleus induction and chromosomal aberration of 1- and 3-nitroazabbenzo[a]pyrene and their N-oxides

Article abstract of DOI:10.1093/mutage/16.3.183

Nitro-azabenzo[a]pyrenes, 1- or 3-nitro-azabenzo[a]pyrene and their N-oxides are nitrated derivatives of azabenzo[a] pyrene (ABP) containing nitrogen in the 6-position of benzo[a]pyrene (B[a]P). The nitro-ABP-N-oxides (ABPOs) were formed by reaction of ABP with excess HNO₃. These derivatives were noteworthy as potent mutagens for Salmonella strains, and were present in fine particles of diesel particulates. In this study, micronucleus induction in mice and chromosomal aberrations due to means of Chinese hamster lung fibroblast (CHL) cells were investigated to determine genotoxicity in order to define the relationship with the mutagenic potency of these derivatives. The induction of micronucleus polychromatic erythrocytes (MNPCEs) was dependent on the dose response of 10-40 mg for 3-N-6-ABP, and of 10-40 mg for 1-N-6-ABP, and in addition, 1- and 3-N-6-ABPOs markedly induced MNPCEs in a dose range of 10-400 mg and from 1 to 80 mg, respectively, when the compound was intraperitoneally administered in two mice at each dose. The results show that of the four compounds, 3-N-6-ABPO demonstrated a marked increase in MNPCEs. On the other hand, chromosomal aberrations of the four compounds were investigated by the duplicate tests using CHLs. The results after a 48 h treatment induced aberrations of the chromatid type, chromatid breaks and exchanges for 1-and 3-N-6-ABP, and mainly chromatid exchanges for 1- and 3-N-6-ABPO. The frequency of chromosomal aberrations associated with nitro substitution on the ABPO structure. Chromosomal aberrations of nitro derivatives of ABPO substituted at the 3-position on the structure were more potent than those at the 1-position. N-oxide derivatives have been found to be reduced to anion radicals much more easily than azaB[a]P and its nitro derivatives. This suggests that the electrochemical reduction of the chemicals plays an important role in the metabolic activation of nitrated B[a]P derivatives.

Full text of DOI:10.1093/mutage/16.3.183

Mutagenesis vol.16 no.3 pp.183–187, 2001
Micronucleus induction and chromosomal aberration of 1- and
3-nitroazabenzo[a]pyrene and their N-oxides
Nobuyuki Sera, Kiyoshi Fukuhara1, Naoki Miyata1 and
Hiroshi Tokiwa2,3
be potent mutagens as well as nitrated derivatives of most
PAHs for Salmonella tester strains (Sera et al., 1992). In
airborne particulate matter, the nitrated ABPs were present in
a semivolatile phase of the fine particle matter, mostly as
combustion products from diesel and gasoline emissions (Sera
et al., 1994). These semivolatile organics were adsorbed onto
the XAD-4 resin, and chemicals were purified on a silica
gel-packed column. The presence of these chemicals was
chemically-ignored for mutagens from the organic phase of
particulate matter. However, the behavior of mutagens inhaled
into the lung alveolar is important to evaluate carcinogenic
action and biochemical reactions due to by phagocytosis in
alveolar macrophages (Tokiwa et al., 1999).
It has been reported that particulate air pollution was
associated with lung cancer mortality and cardiopulmonary,
and in particular, exposure to fine particles in air pollutants
resulted in increased mortality and an increase in respiratory
symptoms and respiratory hospitalizations (Pope et al., 1995).
Of the various mutagens present in fine particles, nitrated ABP
and its N-oxides may play an important role in mutagenesis
in lung cells and also may be associated with declines of lung
function because the chemicals are more mutagenic and
genotoxic than other nitroarenes.
Fukuoka Institute of Health and Environmental Science, Dazaifu, Fukuoka,
Japan, 1National Institute of Health Sciences, 1-18-1 Kamiyoga,
Setagaya-ku, Tokyo, Japan and 2Department of Environmental Health
Science, Kyushu Women’s University, Jiyugaoka Yahatanishiku, Kitakyushu,
Japan
Nitro-azabenzo[a]pyrenes, 1- or 3-nitro-azabenzo[a]pyrene
and their N-oxides are nitrated derivatives of azabenzo[a]
pyrene (ABP) containing nitrogen in the 6-position of
benzo[a]pyrene (B[a]P). The nitro-ABP-N-oxides (ABPOs)
were formed by reaction of ABP with excess HNO3.
These derivatives were noteworthy as potent mutagens for
Salmonella strains, and were present in fine particles of
diesel particulates. In this study, micronucleus induction
in mice and chromosomal aberrations due to means of
Chinese hamster lung fibroblast (CHL) cells were
investigated to determine genotoxicity in order to define
the relationship with the mutagenic potency of these
derivatives. The induction of micronucleus polychromatic
erythrocytes (MNPCEs) was dependent on the dose
response of 10–40 mg for 3-N-6-ABP, and of 10–40 mg for
1-N-6-ABP, and in addition, 1- and 3-N-6-ABPOs markedly
induced MNPCEs in a dose range of 10–400 mg and
from 1 to 80 mg, respectively, when the compound was
intraperitoneally administrated in two mice at each dose.
The results show that of the four compounds, 3-N-6-ABPO
demonstrated a marked increase in MNPCEs. On the other
hand, chromosomal aberrations of the four compounds
were investigated by the duplicate tests using CHLs. The
results after a 48 h treatment induced aberrations of the
chromatid type, chromatid breaks and exchanges for 1-
and 3-N-6-ABP, and mainly chromatid exchanges for
1- and 3-N-6-ABPO. The frequency of chromosomal aberra-
tions associated with nitro substitution on the ABPO
structure. Chromosomal aberrations of nitro derivatives of
ABPO substituted at the 3-position on the structure were
more potent than those at the 1-postion. N-oxide derivatives
have been found to be reduced to anion radicals much
more easily than azaB[a]P and its nitro derivatives. This
suggests that the electrochemical reduction of the chemicals
plays an important role in the metabolic activation of
nitrated B[a]P derivatives.
In the present study, the genotoxic activity of 1- and 3-N-
6-ABP or ABPO were determined for induction of micronuclei
in polychromatic erythrocytes in mice, and chromosomal
aberrations in Chinese hamster lung fibroblast (CHL) cells.
Materials and methods
Chemicals
1- and 3-N-6-ABP and 1- and 3-N-6-ABPO were synthesized according to
the methods mentioned in a previous report (Fukuhara et al., 1992).
Micronucleus induction in mice
Eight-week-old ddY mice were used for determination of micronuclei. The
animals were intraperitoneally treated with the test chemicals suspended in
olive oil or dimethylsulfoxide (DMSO) at dose levels of 10–150 mg/kg for
1- and 3-N-6-ABP, and 25–400 mg/kg and 3–50 mg/kg for 1- and 3-N-6-
ABPO, respectively. Twenty four hours after inoculation the animals were
then killed by cervical dislocation. The test was replicated with the same age
mice, and the mean results were calculated. The femoral marrow cells were
flushed out with fetal bovine serum and smeared on clean slides. Some of the
smeared cells were fixed with methanol for 5 min and stained according to
the May–Grunwald–Giemsa technique. Other preparations were stained with
¨
acridine orange. The acridine orange (0.24 mM in 1/15 M of So¨rensens
phosphate buffer pH 6.8) was used as a working solution. The fixed slides
were rinsed in the buffer three times for 1–3 min each time. The preparations
were mounted with the same buffer, and sealed with Balsam paraffin or
another suitable medium. Observations were made within 24 h. The slides
were analyzed by one microscopist from coded slides; 1000 polychromatic
and normochromatic erythrocytes with or without micronuclei were scored per
animal. The ratio of polychromatic to normochromatic cells was simultaneously
recorded by counting the number of cells until the score for one cell type
reached 1000.
Introduction
The mutagenicity and genotoxicity of various polycyclic
aromatic hydrocarbons (PAHs) and their nitrated derivatives
have been widely investigated for bacterial and mammalian
cells (Tokiwa et al., 1986), but those of azabenzo[a]pyrene
(ABP) and its N-oxide derivatives containing nitrogen in the
parent compound have not been clarified. According to our
previous results, nitrated ABP and its N-oxides were found to
Chromosomal aberration test in vitro
The chromosomal aberration test was carried out according to the protocol
previously reported by Matsuoka et al. (1991, 1993). Briefly, CHLs were
3To whom correspondence should be addressed. Tel: ϩ81 93 693 3088; Email: tokiwa@kwuc.ac.jp
© UK Environmental Mutagen Society/Oxford University Press 2001
183

N.Sera et al.
cultured in Eagle’s minimal essential medium (EMEM; Gibco BRL) supple-
mented with 10% heat-inactivated calf serum. In the direct method, cells were
seeded at a density of 2.2ϫ104 cells per dish with 5 ml of medium. On the
third day, the cells were treated with each compound for 6, 24, or 48 h and
then harvested. In the metabolic activation method, the cells were treated for
6 h with 0.5 ml of rat liver S9 mix, 2.5 ml of medium and 0.015 ml of the
test compound dissolved in DMSO, 3 days after seeding. The final S9
concentration was 5%. The S9 fraction, prepared from the livers of male SD
rats pretreated with phenobarbital and 5,6-benzoflavone, was purchased
from Kikkoman. After the treatment, the reaction mixture was replaced with
fresh medium and cells were harvested after an additional culture for 18 h.
Chromosome preparation cultures were made and stained with Giemsa
solution. The test was repeated three times and the data were summarized as
the mean number of chromosome aberrations.
The slides were coded, but not scored, blind. One observer scored for
aberrations. The number of cells with chromosomal aberrations among 100
well-spread metaphases was recorded. The types of aberrations were divided
into six groups: chromatid and chromosome gaps (ctg), chromatid breaks
(ctb), chromatid exchanges (cte), fragmentation (frg), chromosome breaks
(csb) and chromosome exchanges (cse). In our criteria, a gap was a chromatic
lesion whose length was longer than or equal to the width of a chromatid,
suggesting a discontinuity at the DNA level. Thus, the evaluation of results
was carried out including gaps in the present study. The incidence of polyploid
cells among the 100 metaphases was also recorded. Solvent-treated cells
served as negative controls.
using the May–Gru¨nwald–Giemsa technique for the preparation
from chemically-treated mice using DMSO, and by the acridine
orange technique for the preparation using olive oil as the
solvent. With DMSO as a solvent, the spontaneous induction
of micronuclei was 31 (0.51%) per 6000 PCE. The induction
of MNPCE in mice treated with 1- and 3-N-6-ABP was dose-
dependent at dose levels from 10 to 40 mg, corresponding to
3.8 to ~31 nM 1- and 3-N-6-ABP/kg of mouse (Figure 2). On
the other hand, the micronucleus induction of 3-N-6-ABPO in
mice was more potent than that of 1-N-6-ABPO (Figure
3): MNPCEs in mice treated with 3-N-6-ABPO were dose-
dependent at smaller doses (from 10 to 80 mg, corresponding
to 2 to ~16 nM 3-N-6-ABPO), and MNPCEs in mice treated
with 1-N-6-ABPO were observed at larger doses (from 20 to
400 mg, corresponding to 7.7 to ~248 nM 1-N-6-ABPO/kg of
mouse). Of the four compounds, therefore, micronucleus
induction of 3-N-6-ABPO was clearly observed at dose levels
Ͻ50 mg (16 nM) (Figure 3). The results indicate that only 3-
N-6-ABPO showed a marked induction of the appearance of
micronuclei.
Our historical database showed that the frequency of CHL cells with
structural or numerical aberrations in both untreated and solvent-treated
negative controls did not exceed 4%. We therefore decided a result was
positive (ϩ) if the frequency of aberrant cells or polyploidy was ~2-fold
greater than that of the control, that is, м10%. The uncertain range between
negative and positive, i.e. 5–9%, we termed inconclusive (Ϯ). Overall
evaluation for each chemical was made by judging individual results in
different dose groups. When the outcome was evaluated as inconclusive, the
experiment was repeated.
Chromosomal aberrations of 1- and 3-N-6-ABP or ABPO
Chromosomal aberration tests for 1- and 3-N-6-ABP or ABPO
were carried out using the direct method for 6, 24 and 48 h,
and the results after 48 h of treatment are summarized in
Tables I and II. No cytotoxicity was seen in almost all
derivatives: there was no clear decrease in surviving cells
and analyzable metaphases. Both 1- and 3-N-6-ABP induced
chromosomal aberrations in the absence of exogenous meta-
bolic activation after continuous treatment for 48 h (Table
I). The 48 h treatments of both compounds induced potent
chromatid exchanges. Generally, dose–response relationships
were not clearly shown. This phenomenon could be explained
by the insolubility of these chemicals in the culture medium,
although we did not analyze the concentration of chemicals in
it. The chromosome aberrations induced by both 1- and 3-N-
6-ABP were mainly of the chromatid type, such as chromatid
Statistical analysis was conducted using the Cochran–Armitage test.
Results
Incidence of micronucleus polychromatic erythrocytes
(MNPCEs) with 1- and 3-N-6-ABP, and 1- and 3-N-6-ABPO
The chemical structures of 1- or 3-N-6-ABP, and 1- or 3-N-
ABPO are illustrated in Figure 1. In experimental nitration,
6-azaB[a]P formed 1-N-6-ABP and 3-N-6-ABP at rates of
50 and 37%, respectively, when it was nitrated under excess
HNO3. In order to determine the micronucleus induction, mice
were inoculated intraperitoneally with 1- and 3-N-6-ABP or
ABPO suspended in olive oil or DMSO as described in the
Materials and methods. MNPCEs were scored after staining
Fig. 2. Dose–response curves of micronucleous induction of 1-N-6-ABP
(open circles) and 3-N-6-ABP (closed circles).
Fig. 1. Chemical structure of nitro ABP and its N-oxide.
184

Micronucleus induction and chromosomal aberration
breaks and exchanges (Table I). The results of chromosomal
aberrations for 1- and 3-N-6-ABPO are summarized in Table
II, showing that chromatid exchanges were mainly induced.
The frequency of chromatid breaks and exchanges, and
chromosome breaks were found in 3-N-6-ABPO rather than
in 1-N-6-ABPO, but their dose-dependency for aberrations
was not defined except for chromatid exchanges: chromatid
exchanges were found at a significant level with a dose of
0.2 mg for 1-N-6-ABPO, and both 0.01 and 0.005 mg for 3-
N-6-ABPO/kg of mouse. The induction of chromatid exchanges
for both chemicals was potent, corresponding to the same
magnitude at the smaller dose of 0.04 µg/ml of mitomycin C
as a positive control. These positive results were not found
using the metabolic activation method (Table III). In the
presence of the rat liver S9 preparation, chromosome aberra-
tions of 1- and 3-N-ABP, or ABPO were lower than seen for
the 48 h treatment using the direct method. This fact indicates
that chromosome aberrations of these chemicals were not
activated by the rat liver S9 preparation, whereas those of
benzo[a]pyrene (B[a]P), as a positive control, were dependant
on the metabolic activation (Table III).
Discussion
In this study, the results of micronucleus induction in
mouse femoral marrow cells and chromosomal aberrations
in CHLs showed the same structure–activity relationship for
nitro-substitution of B[a]P, as in the results for Salmonella
mutagenicity (Sera et al., 1992). The results for micronucleus
induction were revealed to be more potent in nitro derivatives
substituted at the 3-position than at the 1-position in ABP
N-oxides but not ABP. The induction resulted in a marked
increase in N-oxide derivatives of 3-N-6-ABPO. Basically,
6-azaB[a]P and its N-oxides were inactive without the S9
mix for TA98 and TA100, while they showed moderate
mutagenicity with the S9 mix (Sera et al., 1992). As for
chemical properties, it was suggested that the increase in
hydrophilicity of the azaarenes compared to the deaza analogue
was due to the decrease in mutagenicity (Fukuhara et al.,
1992). Micronucleus induction of 3-N-6-ABPO was potent as
observed in a previous study with 3,7- and 3,9-dinitrofluor-
anthenes (Tokiwa et al., 1988). This result may be due to
nitroreduction of nitroreductase and acetyltransferase in the
in vivo test, i.e., the reduction ability of the derivative substi-
tuted at the 3-position of the nitro function was more mutagenic
in Salmonella strains and tumorigenic in rats than for the
substitution at the 1- or 6-position (Horikawa et al., 1998). In
addition, nitroreduction has been demonstrated to be more
potent in 3,6-dinitroB[a]P than in 1,6-dinitroB[a]P (Sera
et al.,1991, 1992).
On the other hand, chromosomal aberrations of nitro aza-
B[a]P and its N-oxides were mainly observed as chromatid
breaks and exchanges. These aberrations were similar to the
results for 3,7- and 3,9-dinitrofluoranthenes (Tokiwa et al.,
1988). However, chromosome aberrations of nitro azaB[a]P
and its N-oxides were not dependant on metabolic activation
of rat liver S9 preparation whereas those of B[a]P were
significantly induced in the presence of rat liver S9. These
results suggest that there was a possibility of metabolic
activation due to nitroreductase being present in CHL cells,
Fig. 3. Dose–response curves of micronucleous induction of 1-N-6-ABPO
(open circles) and 3-N-6-ABPO (closed circles).
Table I. Frequency of chromosomal aberrations induced by 1- and 3-N-6-ABP
Chemical
Dose
(mg/ml)
Cells
observed
Polyploid
(%)
Cells with chromosome aberrations (%)
Total
Judgement
ctg
ctb
cte
frg
csb
cse
1-N-6-ABP
3-N-6-ABP
0.023
0.045
0.09
100
100
100
100
100
100
100
100
0
0
1
0
0
1
1
1
0
0
1
0
0
1
0
1
1
3
2
1
3
9
18a
21a
9
0
0
0
0
0
0
0
0
1
1
2
1
2
2
0
0
0
0
1
0
0
1
0
0
11
ϩ
ϩ
ϩ
ϩ
ϩ
ϩ
ϩ
Ϫ
20a
24a
11
0.023
0.045
0.09
18a
22a
12a
0
21a
25a
17a
2
2
Mitomycin C
DMSO
0.04b
10a
0
ctg, chromosome and chromatid gaps; ctb, chromatid breaks; cte, chromatid exchanges; frg, fragmentation; csb, chromosome breaks; cse, chromosome
exchanges.
aP м 0.01.
bµg/ml.
185

N.Sera et al.
Table II. Frequency of chromosomal aberrations induced by 1- and 3-N-6-ABPO
Chemical
Dose
(mg/ml)
Cells
observed
Polyploid
(%)
Cells with chromosome aberrations (%)
Total
Judgement
ctg
ctb
cte
frg
csb
cse
1-N-6-ABPO
3-N-6-ABPO
0.05
0.1
0.2
0.0025
0.005
0.01
0.04b
100
100
100
100
100
100
100
100
0
0
0
0
0
0
1
2
0
0
1
0
0
1
0
1
1
1
1
2
1
2
10
0
5
9
0
0
0
0
0
0
0
0
1
1
1
2
2
2
0
0
0
0
1
1
0
1
0
0
7
11
ϩ
ϩ
ϩ
ϩ
ϩ
ϩ
ϩ
Ϫ
10a
10
19a
39a
13a
0
13a
13
21a
42a
18a
2
Mitomycin C
DMSO
ctg, chromosome and chromatid gaps; ctb, chromatid breaks; cte, chromatid exchanges; frg, fragmentation; csb, chromosome breaks; cse, chromosome
exchanges.
aP м 0.01.
bµg/ml.
Table III. Chromosomal aberrations induced by 1- and 3-N-6-ABP, and 1-and 3-N-6-ABPO in the presence of S9 mix
Chemical
Dose
(mg/ml)
Cells
observed
Polyploid
(%)
Cells with chromosome aberrations (%)
Total
Judgement
ctg
ctb
cte
frg
csb
cse
1-N-6-ABP
3-N-6-ABP
1-N-6-ABPO
3-N-6-ABPO
0.023
0.045
0.09
0.023
0.045
0.09
0.05
0.1
0.2
100
100
100
100
100
100
100
100
100
100
100
100
100
100
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
2
2
1
0
0
1
1
1
1
2
6
0
0
0
1
0
0
1
0
1
2
0
1
1
0
1
2
0
1
2
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
1
1
0
0
0
0
0
1
0
0
0
0
0
0
1
0
0
0
2
4
2
3
4
0
3
5
Ϫ
Ϫ
Ϯ
Ϫ
Ϫ
Ϯ
Ϫ
Ϫ
Ϯ
Ϫ
Ϯ
Ϯ
ϩ
Ϫ
0.0025
0.005
0.01
0
2
0
2
1
5
1
2
7
B[a]P
DMSO
0.01
27a
0
34a
0
37a
0
ctg, chromosome and chromatid gaps; ctb, chromatid breaks; cte, chromatid exchanges; frg, fragmentation; csb, chromosome breaks; cse, chromosome
exchanges; Ϯ, inconclusive.
aP м 0.01.
as well as Salmonella mutagenicity (Sera et al., 1992), but not
in rat liver microsomes.
activity and the electrochemical reduction of related com-
pounds.
As for the chemical properties of the four derivatives, two
reversible waves involving a one-electron transfer process
were observed (Fukhara et al., 1992). The first half-wave
redox potential of nitrated 6-ABPs, such as 1- or 3-N-6-ABP,
was higher than that of mononitroB[a]Ps, such as 1- or 3-
nitroB[a]P, and, in addition, that of their N-oxides was also
higher: 1- and 3-N-6-ABPO were reduced to anion radicals
much more easily than mononitroB[a]P (Fukhara et al., 1992).
As indicated previously, the mutagenicity of 3-N-6-ABP and
3-N-6-ABPO for Salmonella strains TA98 and YG1024, an
acetyltransferase-rich mutant of TA98, showed 348 and 7670,
and 1260 and 30 500 revertants/ng without the S9 mix,
respectively. The 3-N-6-ABP was more mutagenic than 1-N-
6-ABP (352 and 2750 revertants/ng, respectively), and 3-N-6-
ABPO was more mutagenic than 1-N-6-ABPO (115 and 1930
revertants/ng, respectively). It has also been found that the
mutagenicity and tumorigenicity of 3,6-dinitroB[a]P were more
potent than those of 1,6-dinitroB[a]P (Horikawa et al., 1998;
Sera et al., 1991): the difference in the activity may cause
metabolic pathways dependent on nitro reduction (Sera et al.,
1992). There was also an association between biological
On the other hand, nitro-azaB[a]P was detected from semi-
volatile materials passed through a 0.45 µm filter on airborne
and diesel particulate matter (Sera et al., 1994). It is likely
that related compounds may be present as a coating chemical
on elemental carbon or as a semivolatile form involved with
smaller particles inhaled into lung alveoli, and were deposited
in lung tissues (Tokiwa et al., 1993, 1998). Normally, most
organic chemicals are identified by gas-chromatography and
mass spectrometry, but these compounds could be directly
determined by mass spectrometric analysis after purification
using liquid column chromatography. The compounds were
detected on the column at an oven temperature programmed
from 80 to 310°C (Sera et al., 1994). Under these conditions,
1- and 3-N-6-ABP or ABPO were detected at the levels from
0.8 to 7.7 ng/g of material. In our earlier study on the ABPs
and ABPOs, the production of 8-hydroxyguanosine as a result
of oxidative damage in cellular DNA, resulted in a marked
increase compared with other related nitroarenes, although this
was in vivo data from formed mouse lungs (unpublished data).
These results suggest that these ABPs and ABPOs generated
oxygen free radicals (or hydroxyl radicals) in the mouse lung,
186

Micronucleus induction and chromosomal aberration
and led to mutation at the deoxyguanosine residue in the DNA.
These results indicate that the ABPs and ABPOs induced not
only bacterial mutation and genotoxic action in vitro, but also
pulmonary injury as a result of oxidative damage.
The sources and formation of these chemicals in the environ-
ment have not been chemically-revealed in the present study.
However, these are serious mutagens that elevate cardio-
pulmonary and lung cancer incidence.
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Received on October 16, 2000; accepted on October 20, 2000
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