3-Nitrobenzanthrone, a powerful bacterial mutagen and suspected human carcinogen found in diesel exhaust and airborne particulates

Article abstract of DOI:10.1021/es961067i

3-Nitrobenzanthrone (3-nitro-7H-benz[d,e]anthracen-7-one) was isolated from the organic extracts of both diesel exhaust and airborne particles and was identified as a new class of powerful direct mutagen. Its mutagenicity by Ames Salmonella assay is very high (208 000 revertants/nmol in Salmonella typhimurium TA98 and 6 290 000 revertants/nmol in YG1024) and compares with that of 1,8-dinitropyrene, which is the direct mutagen of strongest activity (257 000 revertants/nmol in TA98 and 4 780 000 revertants/nmol in YG1024) so far reported in the literature. The new mutagen was also shown to induce micronuclei in mouse peripheral blood reticulocytes after intraperitoneal administration (micronucleated reticulocytes, 0.64% against 25 mg/kg dose after 48 h), suggesting its potential genotoxicity to mammalians. 3- Nitrobenzanthrone is most likely to be formed not only during the combustion process of fossil fuels but also from the atmospheric reaction between benzanthrone and lower oxides of nitrogen, since the latter ketone was found to be nitrated quite easily under an artificial atmosphere containing gaseous NO₂ (10 ppm) and O₃ (5 ppm) to produce the powerfully mutagenic 3-nitro derivative as the major product, along with several other isomeric mononitrobenzanthrones and dinitro descendants as minor products. 3-Nitrobenzanthrone (3-nitro-7H-benz[d,e]anthracen-7-one) was isolated from the organic extracts of both diesel exhaust and airborne particles end was identified as a new class of powerful direct mutagen. Its mutagenicity by Ames Salmonella assay is very high (208 000 revertants/nmol in Salmonella typhimurium TA98 and 6 290 000 revertants/nmol in YG1024) and compares with that of 1,8-dinitropyrene, which is the direct mutagen of strongest activity (257 000 revertants/nmol in TA98 and 4 780 000 revertants/nmol in YG1024) so far reported in the literature. The new mutagen was also shown to induce micronuclei in mouse peripheral blood reticulocytes after intraperitoneal administration (micronucleated reticulocytes, 0.64% against 25 mg/kg dose after 48 h), suggesting its potential genotoxicity to mammalians. 3-Nitrobenzanthrone is most likely to be formed not only during the combustion process of fossil fuels but also from the atmospheric reaction between benzanthrone and lower oxides of nitrogen, since the letter ketone was found to be nitrated quite easily under an artificial atmosphere containing gaseous NO₂ (10 ppm) and O₃ (5 ppm) to produce the powerfully mutagenic 3-nitro derivative as the major product, along with several other isomeric mononitrobenzanthrones and dinitro descendants as minor products.

Full text of DOI:10.1021/es961067i

Environ. Sci. Technol. 1997, 31, 2772-2776
of these mutagens is believed to be car emissions, and the
3-Nitrobenzanthrone, a Powerful
Bacterial Mutagen and Suspected
Human Carcinogen Found in Diesel
Exhaust and Airborne Particulates
increase of cars at a tremendous rate, especially in developing
countries, has been posing a serious burden in some regions
on maintaining a clean atmosphere devoid of respirable
particulate organic matters that are possibly linked with lung
cancer (2).
Interestingly enough, the direct mutagenicity of known
nitro-PAHs such as nitrofluoranthenes and nitropyrenes has
been shown to account for only up to 10-20% of the total
mutagenic activity of ambient particulate organic matters
(11, 12), and the possible presence of other unknown
mutagenic species in polar fractions of organic extracts has
been strongly suggested (13-15). On the basis of this
consideration, Helmig et al. (16) have recently been successful
in disclosing the presence of an additional type of strong
mutagen, nitrolactones, in polar fractions of the extract of
airborne particulate organic matters.
T A K E J I E N YA A N D H I T O M I S U Z U K I * , †
Department of Chemistry, Graduate School of Science,
Kyoto University, Sakyo-ku, Kyoto 606-01, Japan
T E T S U S H I W A T A N A B E A N D
T E R U H I S A H I R A YA M A * , ‡
Department of Public Health, Kyoto Pharmaceutical
University, Yamashina-ku, Kyoto 607, Japan
We herein report a remarkably high mutagenic aromatic
nitroketone, 3-nitrobenzanthrone (3-nitro-7H-benz[d,e]an-
thracen-7-one) 2 as a new class of mutagen present in the
polar extracts from diesel exhaust and airborne particulates
(Scheme 1). We confirmed the facile formation of this
compound as the major product in the reaction of benzan-
throne with lower oxides of nitrogen, especially in the presence
of ozone. In addition, we observed that the new mutagen 2
can induce considerable structural and/ or numerical chro-
mosomal aberrations in mammalian cells.
YO S H I H A R U H I S A M A T S U * , §
Department of Community Environmental Science,
National Institute of Public Health, Shirokanedai,
Minato-ku, Tokyo 108, Japan
3-Nitrobenzanthrone (3-nitro-7H-benz[d,e]anthracen-7-
one) was isolated from the organic extracts of both diesel
exhaust and airborne particles and was identified as a
new class of powerful direct mutagen. Its mutagenicity by
Ames Salmonella assay is very high (208 000 revertants/
nmol in Salmonella typhimurium TA98 and 6 290 000 revertants/
nmol in YG1024) and compares with that of 1,8-dinitropyrene,
which is the direct mutagen of strongest activity (257 000
revertants/nmol in TA98 and 4 780 000 revertants/nmol in
YG1024) so far reported in the literature. The new mutagen
was also shown to induce micronuclei in mouse peripheral
blood reticulocytes after intraperitoneal administration
(micronucleated reticulocytes, 0.64% against 25 mg/kg dose
after 48 h), suggesting its potential genotoxicity to mam-
malians. 3-Nitrobenzanthrone is most likely to be formed not
only during the combustion process of fossil fuels but also
from the atmospheric reaction between benzanthrone and
lower oxides of nitrogen, since the latter ketone was found
to be nitrated quite easily under an artificial atmosphere
containing gaseous NO (10 ppm) and O (5 ppm) to produce
Experimental Section
Instrum entation. Melting points were determined on a
Yanagimoto hot stage apparatus and are uncorrected. IR
spectra were recorded on a Shimadzu FTIR DR 8000/ 8100
infrared spectrophotometer. 1H-NMR spectra were obtained
with a Varian Gemini-200 (200 MHz) spectrometer in CDCl3
with tetramethylsilane as an internal standard. J values are
given in Hz. All high-performance liquid chromatograph
(HPLC) analyses were performed with a Shimadzu LC-10AS
equipped with a SPD 10A UV detector using a Shimpack CLC
Sil(m) column (4.6 mm i.d. × 250 mm). Preparative HPLC
for prefractionation and purification of the samples was
performed with a Senshu Model SSC-3100 equipped with a
Model SSC-3000AII UV detector, using a Pegasil Silica 60-5
semipreparative column (10 mm i.d. × 250 mm). Gas
chromatography/ mass spectrometry (GC/ MS) analysis for the
characterization of the samples was performed by using a
HP5890/ JEOL, JMS-HX100A instrument; ionization current
300 A; ionization voltage 70 eV; chamber temperature 280-
290 °C; accelerating voltage 10 kV. GC quantification of
3-nitrobenzanthrone from environmental samples was per-
formed with a HP 5890 Series II equipped with a NPD detector
(GC/ NPD), using a 5% phenylmethyl silicone (HP-5) column
of 0.32 i.d. and 300 cm length. The temperature program for
GC/ NPD analysis was as follows: 70 °C for 1 min; increase
7 °C/ min to 300 °C; isothermal for 10 min. 3-nitrobenzan-
throne was eluted at 34.7 min after injection.
2
3
the powerfully mutagenic 3-nitro derivative as the major
product, along with several other isomeric mononitroben-
zanthrones and dinitro descendants as minor products.
Introduction
In recent years, nitrated polycyclic aromatic hydrocarbons
(nitro-PAHs) have caused grave environmental concern
because of their potent mutagenic activity (1) and carcino-
genicity (1, 2). They abundantly exist in the particulated
matters emitted from diesel and gasoline engines (3, 4) and
also on the surface of airborne particulates and are most
likely to be formed during the combustion process of fossil
fuels as well as by the reaction of parent hydrocarbons with
nitrogen oxides in ambient air (5-10). An important source
Materials. Reagent grade benzanthrone was purchased
from Tokyo Kasei Kogyo Co. Ltd. and used after recrystal-
lization from ethanol (purity >99.9%). Isomeric mono- and
dinitrobenzanthrones used as reference compounds were
prepared from appropriately substituted methyl nitro-2-
iodobenzoates 9 and nitro-1-iodonaphthalenes 10 via the
reaction sequence involving the Ullmann coupling and
intramolecular Friedel-Crafts ring closure (17-19) as il-
lustrated in Scheme 2. Details of the preparation as well as
physical properties of these and related compounds are
reported elsewhere (20). This synthetic route excludes
possible formation of other isomers, and the purity of all
products was above 99.99% on HPLC and GC/ MS.
* Authors to whom correspondence should be addressed.
† Phone: +81-075-753-4041; fax: +81-075-751-2085; e-m ail:
suzuki@kuchem.kyoto-u.ac.jp.
‡ Phone: +81-075-595-4650; fax: +81-075-595-4769.
§
Phone: +81-03-3441-7111; fax: +81-03-3446-4314; em ail:
hisa@iph.go.jp.
9
2 7 7 2 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 31, NO. 10, 1997
S0013-936X(96)01067-X CCC: $14.00
1997 Am erican Chem ical Society

reticulocytes (MNRETs) from peripheral blood after a single
intraperitoneal administration with 12.5, 25, or 50 mg/ kg of
3-nitrobenzanthrone was scored up for each mouse. Sta-
tistical analysis was performed by using the method of
Kastenbaum and Bowman (27).
SCHEME 1
Reaction of Benzanthrone with Nitrogen Dioxide in the
Absence or Presence of Ozone. Hom ogeneous Conditions.
Into a stirred solution of benzanthrone (230 mg; 1 mmol) in
dichloromethane (50 mL) cooled to 0 °C, a stream of nitrogen
dioxide (40 mmol/ h) alone or admixed with ozonized oxygen
was passed. Ozone was introduced at a rate of 9 mmol/ h
under an oxygen flow of 10 L/ h. The identification of product
was made by GC/ MS analysis, and the isomer distribution
was determined by HPLC using a 100:3:1:3 mixture of
n-hexane, dichloromethane, THF, and ethyl acetate as the
solvent at a flow rate 1.0 mL/ min. Any other solvent systems
examined were not satisfactory for the separation of isomers.
Retention volumes of benzanthrone and 3-, 9-, and 11-
nitrobenzanthrones were 5.9, 8.7, 17.1, and 8.0, respectively.
Two other yet unidentified mononitrobenzanthrones eluted
as minor components at 7.8 and 12.3, respectively.
Heterogeneous Conditions. Nitration of benzanthrone
adsorbed on a Teflon-coated glass fiber filter was carried out
under an artificial atmosphere containing nitrogen dioxide
(10 ppm) and ozone (5 ppm) in a glass chamber of 6.0-L
volume size. Products were extracted with dichloromethane
and immediately analyzed by GC/ MS and GC/ NPD. Each
isomer was identified by direct comparison of its MS pattern
with an authentic specimen synthesized via an unambiguous
route shown in Scheme 2.
3-Nitrobenzanthrone 2: yellow powder; mp 256-257 °C
(lit. mp 252 °C (21)). IR (KBr): ν ) 1653, 1518, 1508, 1338
cm-1 1H-NMR (CDCl3): δ ) 7.7 (dt, J ) 7.3, 1.1, 1H), 7.8 (dt,
.
J ) 7.6, 1.6, 1H), 8.0 (dd, J ) 8.7, 7.4, 1H), 8.3-8.5 (m, 4H),
8.9 (dd, J ) 7.4, 1.2, 1H), 9.0 (dd, J ) 8.7, 1.2, 1H). MS: m/ z
(%) ) 275 (M+, 97), 245 (M+ - NO, 100), 201 (51) 200 (77).
Sam pling. Diesel exhaust particulate matter was collected
from an Isuzu engine Model 6HEL 7127 cc (maximum power
250 sp/ 2700 rpm) working under various loading conditions.
Exhaust gas containing particle matters was diluted with clean
air up to 10 times in volume in dilution tunnel, and exhaust
particulate was collected using a high-volume air sampler
(sampling rate, 70 m3/ h) with a quartz fiber filter Pallflex 2500
QAT-UP (Pallflex Product Corporation). Sampling volume
was 5-20 m3. Airborne particle organic matter was collected
at the roof top of a five-story building facing a traffic-heavy
thoroughfare of the central Tokyo area (Minato-ku) during
November 30-December 1, 1994, using the same air sampler.
Day sample collection was made during 10 h from 8 A.M. to
Results and Discussion
Environm ental Occurrence and Mutagenicity of 3-Ni-
trobenzanthrone. During the course of GC/ MS analysis of
the mutagenic HPLC fractions from diesel emission and
airborne particle extracts, we noticed the presence of an
extremely powerful mutagenic substance of a composition
C17H9NO3. This compound was identified as 3-nitroben-
zanthrone 2 by direct comparison of its HPLC behavior as
well as mass spectrum pattern with those of the authentic
specimen synthesized from methyl 2-iodobenzoate and
1-iodo-4-nitronaphthalene. Mutagenic assay of authentic 2
showed 208 000 revertants/ nmol of mutagenicity in S. ty-
phimurium TA98. This value is highest among the reported
values of known mononitro PAHs and compares with that of
1,8-dinitropyrene (8) (250 000 revertants/ nmol), which is the
most potent mutagen reported to date in the literature (1,
25). In strain YG 1024 (-S9 mix), the former compound
exhibited the value 1.3 times as high as the latter (4 780 000
revertants/ nmol) (Table 1) (1, 25).
The concentration of 3-nitrobenzanthrone 2 in diesel
exhaust particle extracts was found to be 0.6-6.6 µg/ g (Table
2). This value is as high as the reported value of 1,8-
dinitropyrene 8, 0.3-5.2 µg/ g (1). In view of the fact that the
mutagenic activity of the former compound is comparable
with that of the latter, it is easily understandable that the
nitroketone 2 would contribute considerably to the total
mutagenic activity of diesel exhaust particle extracts.
Noteworthy is a remarkable increase of 3-nitrobenzan-
throne 2 in the emission from a diesel engine working under
high loading conditions, which may warn us of the potential
danger of engine overloading, suggesting a need for stronger
regulation over the load limit of diesel trucks. A possible
connection between the engine loading and concentration
of nitroarenes in emission has not been reported previously,
so the effect of loading conditions on the concentration of
the resulting nitroarenes is subject to further investigation.
Mutagenic Activity of Other Isom eric Nitrobenzan-
thrones. Mutagenic activity of 9- and 11-nitrobenzanthrones
3 and 4 and 3,9- and 3,11-dinitrobenzanthrones 5 and 6 was
also investigated. These nitrobenzanthrones arose as minor
6
P.M. of the same day, and night sample collection was
similarly from 8 P.M. to 6 A.M. of the next day. The samples
from diesel exhaust particulate were extracted with dichlo-
romethane using a Soxhlet extractor, while the samples from
airborne particles were extracted with same solvent under
sonication. The extracts were prefractionated by preparative
HPLC using the solvent gradient: 5 min, 100% n-hexane, 5
min linear gradient to 5% dichloromethane, 15 min linear
gradient to 100% dichloromethane and hold for 5 min, and
finally 5 min linear gradient to 100% acetonitrile and hold for
10 min. Fractions during 12-27 min were collected and
evaporated to an adequate volume for GC/ MS analysis.
Mutagenicity Assay. Mutagenic assay of mono- and
dinitrobenzanthrones was carried out by the method of Ames
et al. (22) (Salmonella typhimurium - S9 mix) with slight
modifications including preincubation step (23). S. typh-
imurium his- strains YG 1021 and 1024 were produced by
transferring a plasmid carrying the nitroreductase or acetyl-
transferase gene into cell TA98. These strains exhibit the
enhanced NRase and O-ATase activity 3-100 times as high
as the original TA98 and were kindly provided by Dr. Nohmi,
National Institute of Hygienic Sciences, Tokyo (24, 25).
Assays were performed for each compound three times,
and the results were averaged. For 3-nitrobenzanthrone, the
assay was carried out five times to make sure of the data
obtained.
Micronucleus Test. Micronucleus test was performed for
3-nitrobenzanthrone suspended in olive oil according to the
method of Hayashi et al. (26). Five ICR male mice (8 weeks
old) were used for each test. One thousand peripheral blood
reticulocytes were analyzed per mouse using the acridine
orange staining method. The number of micronucleated
9
VOL. 31, NO. 10, 1997 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 2 7 7 3

SCHEME 2a
2
a
Reagents and conditions: (a) copper bronze, 150 °C; (b) LiOH, MeOH-THF-H2O; (c) SOCl2, PhH, reflux; (d) AlCl3, CHCl3.
TABLE 1. Direct-Acting Mutagenic Activities (Revertants/nmol) of Nitrobenzanthrones and 1,8-Dinitropyrene
Salmonella typhimurium strain (revertants/nmol ( SD) (-S9 mix)
TA98
TA100
YG1021
YG1024
3-nitrobenzanthrone (2)a
9-nitrobenzanthrone (3)
11-nitrobenzanthrone (4)
3,9-dinitrobenzanthrone (5)
3,11-dinitrobenzanthrone (6)
1,8-dinitropyrene (8)
208 000 ( 22 000
84 000 ( 6 000
6 ( 0
29 700 ( 2 600
3 270 ( 200
6 ( 0
129 000 ( 34 000
26 100 ( 300
10 ( 0
25 100 ( 3 300
3 110 ( 900
285 000c
6 290 000 ( 490 000
490 000 ( 51 000
46 ( 1
46 000 ( 5 200
3 000 ( 450
4 300 ( 400
360 ( 30
223 000 ( 38 000
12 200 ( 1700
257 000b (283 000d)
55 400b
4 780 000c (4 110 000d)
a
Dose response of 2 (dose/revertants): in TA98 (0.125 ng/95 revertants, 0.25 ng/187 revertants, 0.5 ng/369 revertants, 1 ng/785 revertants); in
TA100 (1 ng/124 revertants, 2.5 ng/258 revertants, 5.0 ng/609 revertants, 10.0 ng/1091 revertants); in YG1021 (0.25 ng/118 revertants, 0.50 ng/261
revertants, 1.0 ng/451 revertants, 2 ng/860 revertants); in YG 1024 (0.00625 ng/143 revertants, 0.0125 ng/268 revertants, 0.025 ng/618 revertants, 0.05
b
c
d
ng/1123 revertants). From ref 1. From ref 25. This work.
TABLE 2. Concentration of 3-Nitrobenzanthrone
Section A: Diesel Exhaust Particlea
load of engine
rpm
concn (µg/g)
0% (idling)
6%
80%
600
1670
1696
<0.001
0.647
6.61
Section B: Airborne Particlea
concentration (pg/m3)
sampling date
daytime
nighttime
Nov 30, 1994
Dec 1, 1994
NDb
5.2
7.7
11.5
a
b
Relative SD <6%. ND, not determ ined.
products from the reaction of benzanthrone and nitrogen
dioxide. However, all these compounds proved to be less
mutagenic than 2 (Table 1). In the mononitro series,
9-nitrobenzanthrone 3 was also a mutagen of high activity,
but its value was much lower than that of the 3-nitro isomer
2. Quite interestingly, 11-nitrobenzanthrone 4 was devoid
of mutagenicity. Such a marked dependence of mutagenicity
on the position of the nitro group was not observed in the
nitropyrene and nitrofluoranthene series (1, 28). In the dinitro
series, 3,9-dinitrobenzanthrone 5 showed higher activity in
mutagenicity than the 3,11-dinitro isomer 6, but both
compounds were less potent than the 3-nitro derivative 2. It
has been known that the extent of nitration of PAHs is
generally paralleled by an increase in mutagenicity (29, 30).
However, this trend was reversed in the nitrobenzanthrone
series. In view of the marked difference in mutagenic behavior
from the known series of nitro-PAHs, we may classify the
nitrobenzanthrone series as a new class of mutagens present
in atmospheric environment.
FIGURE 1. Mean frequencies of micronucleated reticulocytes
(MNRETs) from peripheral blood treated with 3-nitrobenzanthrone.
SD value of each point (h ( SD) is as follow; 0 mg dose (0 ( 0.04;
24 ( 0.07; 48 ( 0.11; 72 ( 0.04; 96 ( 0.04); 12.5 mg dose (0 ( 0.08;
24 ( 0.10; 48 ( 0.10; 72 ( 0.19; 96 ( 0.07); 25 mg dose (0 ( 0.10; 24
( 0.10; 48 ( 0.12; 72 ( 0.08; 96 ( 0.14); 50 mg dose (0 ( 0.09; 24 (
0.11; 48 ( 0.12; 72 ( 0.07; 96 ( 0.14).
indicate that 3-nitrobenzanthrone induced not only point
mutations in bacteria but also structural and/ or numerical
chromosomal aberrations in mammalian cells (31-34).
Kyodai-Nitration of Benzanthrone and Its Relevance
to Atm ospheric Form ation of Mutagenic Nitroketone 2.
Many environmental nitroarenes are known to arise from
emissions in various combustion systems as well as from
atmospheric reaction between parent arenes and lower oxides
of nitrogen (5-7). Compound 1 is one of the major
components of the aromatic ketone fractions from ambient
airborne particle extracts (35-38). Universal occurrence of
this ketone 1 in ambient air is well established; it exists in
airborne particles, diesel exhaust fumes, street dust, and
emissions from a variety of combustion sources (35). In busy
urban traffic areas, the concentration of this ketone 1 often
compares with those of pyrene and fluoranthene (36). The
Micronucleus Assay of 3-Nitrobenzanthrone. 3-Ni-
trobenzanthrone 2 was also found to induce micronuclei in
mouse peripheral blood reticulocytes after intraperitoneal
administration of 25-50 mg/ kg (Figure 1). A significant
increase in frequency of micronucleated reticulocytes was
observed at 24-48 h after treatment. The results clearly
9
2 7 7 4 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 31, NO. 10, 1997

FIGURE 2. (a) Nitration of benzanthrone with NO gas alone (square) or with NO -O (circle) in a solution at 0 °C. (b) Heterogeneous nitration
2
2
3
of benzanthrone adsorbed on a Teflon-coated glass fiber filter with NO gas alone (square) or with a NO -O mixture (circle) at room
2
2
3
temperature.
exhaust from gasoline engine contains more ketone 1 than
that from diesel engine (37, 38).
has been developed (kyodai-nitration) (43), for which the
nonclassical nitration mechanism involving the nitrogen
trioxide (NO3) as the initial electrophile has been proposed
(44). We are now inclined to think that the mechanism
proposed for the kyodai-nitration may be largely responsible
for the atmospheric formation of nitroarenes during the night
time. The study of the mechanistic aspect of this atmospheric
nitration is underway.
Among all ring sites in benzanthrone 1, the 3-position is
most susceptible to the attack of electrophile such as
nitronium ion (39). Compound 1 was easily converted to the
3-nitro derivative 2 by treatment with nitric acid (40), so the
atmospheric formation of the title mutagen 2 may reasonably
be attributed to the reaction of ketone 1 with nitrogen oxides
or nitric acid on the surface of airborne particulates during
transport in air. In order to confirm this, we examined the
reaction of compound 1 and nitrogen dioxide under a variety
of conditions with or without ozone.
When benzanthrone 1 was reacted with gaseous NO2 in
dichloromethane at 0 °C, the formation of nitro compounds
was comparatively slow (Figure 2a). The product obtained
was found to contain 3-nitro derivative 2 as the major
component (80-90%); the accompanying minor products
included 9- and 11-nitro derivatives 3 and 4 and two other
yet unidentified mononitro isomers. Under these conditions,
the dinitro compounds were not formed in any significant
amounts. The reaction was appreciably facilitated when a
small amount of iron-impregnated silica gel was added to
the reaction system, suggesting a possible role of the metal-
contaminated particulates from motor engines as catalyst
for the nitration of arenes in atmospheric environment (41).
In the presence of ozone, however, the nitration of ketone 1
with NO2 proceeded very rapidly even at 0 °C to afford the
3-nitro derivative 2 as the main initial product which, on
further reaction, was easily converted to 3,9- and 3,11-
dinitrobenzanthrones 5 and 6.
Under the heterogeneous conditions where benzanthrone
1 adsorbed on a Teflon-coated glass fiber filter was reacted
with gaseous NO2 in the presence or absence of O3, we
obtained the results closely similar to those observed in the
homogeneous nitration reaction carried out in a dichlo-
romethane solution (Figure 2b).
The reaction of polycyclic arenes with NO2 and O3 under
atmospheric conditions has been well studied (5-10). It
mostly occurs in the nighttime when the concentration of
ozone becomes high. So the amount of nitroarenes in the
atmosphere is known to increase during the night (10). This
is in accord with our observation (Table 2, section B). In the
atmospheric environment, the nighttime reaction of ben-
zanthrone 1 with NO2-O3 would occur preferentially on the
surface of airborne particulates, not in a gaseous phase, to
give mutagenic 3-nitrobenzanthrone 3, because more than
96% of the total amount of benzanthrone in atmospheric
environment is known to exist in the adsorbed state (42). In
recent years, a highly efficient method for the nuclear nitration
of arenes by the combined use of nitrogen dioxide and ozone
Acknowledgments
This work has been supported in part by the Grant-in-Aid for
Specially Promoted Scientific Research of the Ministry of
Education, Science, Sports and Culture of Japan (08101003).
We are grateful to Dr. S. Fukuoka of the Tokyo Metropolitan
Research Institute for Environmental Protection for a gift of
diesel exhaust particulate sample.
Literature Cited
(1) Tokiwa, H.; Ohnishi, Y. CRC Crit. Rev. Toxicol. 1986, 17, 23-60.
(2) WHO. World Health Statistics Annual; WHO: Geneva, 1995.
(3) Paputa-Peck, M. C.; Marano, R. S.; Schuetzle, D.; Riley, T. L.;
Hampton, C. V.; Prater, T. J.; Skewes, L. M.; Jensen, T. E.; Ruehle,
P. H.; Bosch, L. C.; Duncan, W. P. Anal. Chem. 1983, 55, 1946-
1954.
(4) Kamiura, T.; Kawaraya, T.; Tanaka, M.; Nakadoi, T. Anal. Chim.
Acta 1991, 254, 27-31.
(5) Pitts, J. N., Jr.; Sweetman, J. A.; Zielinska, B.; Atkinson, R.; Winer,
A. M.; Harger, W. P. Environ. Sci. Technol. 1985, 19, 1115-1121.
(6) Pitts, J. N., Jr.; Zielinska, B.; Sweetman, J. A.; Atkinson, R.; Winer,
A. M. Atmos. Environ. 1985, 19, 911-915.
(7) Arey, J.; Zielinska, B.; Atkinson, R.; Winer, A. M.; Ramdahl, T.;
Pitts, J. N., Jr. Atmos. Environ. 1986, 20, 2339-2345.
(8) Zielinska, B.; Arey, J.; Atkinson, R.; Ramdahl, T.; Winer, A. M.;
Pitts, J. N., Jr. J. Am. Chem. Soc. 1986, 108, 4126-4132.
(9) Zielinska, B.; Arey, J.; Atkinson, R.; Winer, A. M. Atmos. Environ.
1989, 23, 223-229.
(10) Ciccioli, P.; Cecinato, A.; Cabella, R.; Brancaleoni, E.; Buttini, P.
Atmos. Environ. 1993, 27A, 1261-1270.
(11) Arey, J.; Zielinska, B.; Harger, W. P.; Atkinson, R.; Winer, A. M.
Mutat. Res. 1988, 207, 45-51.
(12) Tokiwa, H.; Kitamori, S.; Nakagawa, R.; Horikawa, K.; Matamala,
L. Mutat. Res. 1983, 121, 107-116.
(13) Kamens, R.; Bell, D.; Dietrich, A.; Perry, J.; Goodman, R.; Claxton,
L.; Tejada, S. Environ. Sci. Technol. 1985, 19, 63-69.
(14) Schuetzle, D.; Lewtas, J. Anal. Chem. 1986, 58, 1061A-1075A.
(15) Nisioka, M. G.; Howard, C. C.; Contos, D. A.; Ball, L. M.; Lewtas,
J. Environ. Sci. Technol. 1988, 22, 908-915.
(16) Helmig, D.; Arey, J.; Harger, W. P.; Atkinson, R.; Lo´ pez-Cancio,
J. Environ. Sci. Technol. 1992, 26, 622-624.
(17) Tucker, S. H.; Whalley, M. J. Chem. Soc. 1949, 3213-3216.
(18) Hawkins, J.; Tucker, S. H. J. Chem. Soc. 1950, 3286-3288.
(19) Corey, E. J.; Sze´kely, I.; Shiner, C. S. Tetrahedron Lett. 1977, 40,
3529-3532.
(20) Suzuki, H.; Enya, T. Synthesis in press (KK-022-03-97).
(21) Shioda, H.; Kato, S. Yuki Gosei Kagaku 1957, 15, 361-365.
9
VOL. 31, NO. 10, 1997 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 2 7 7 5

(22) Ames, B. N.; McCann, J.; Yamasaki, E. Mutat. Res. 1975, 31, 347-
364.
(23) Yahagi, T.; Nagao, M.; Seino, Y.; Matsushima, T.; Sugimura, T.;
Okada, M. Mutat. Res. 1977, 48, 121-130.
(24) Watanabe, M.; Ishidate, M., Jr.; Nohmi, T. Mutat. Res. 1989, 216,
211-220.
(25) Watanabe, M.; Ishidate, M., Jr.; Nohmi, T. Mutat. Res. 1990, 234,
337-348.
(26) Hayashi, M.; Morita, T.; Kodama, Y.; Sofuni, T.; Ishidate, M., Jr.
Mutat. Res. 1990, 245, 245-249.
(27) Kastenbaum, M. A.; Bowman, K. O. Mutat. Res. 1970, 9, 527-
549.
(28) Zielinska, B.; Arey, J.; Harger, W. P.; Lee, R. W. K. Mutat. Res.
1988, 206, 131-140.
(29) Klopman, G.; Tonucci, D. A.; Holloway, M.; Rosenkranz, H. S.
Mutat. Res. 1984, 126, 139-144.
(30) Nakagawa, R.; Horikawa, K.; Sera, N.; Kodera, Y.; Tokiwa, H.
Mutat. Res. 1987, 191, 85-91.
H.; Sutou, S.; Suzuki, T.; Wakata, A.; Sofuni, T.; Hayashi, M. Mutat.
Res. 1997, 389, 3-122.
(35) Spitzer, T.; Takeuchi, T. J. Chromatogr. A 1995, 710, 109-116.
(36) Yang, Y.; Baumann, W. Analyst 1995, 120, 243-248.
(37) Handa, T.; Yamauchi, T.; Sawai, K.; Yamamura, T.; Koseki, Y.;
Ishii, T. Environ. Sci. Technol. 1984, 18, 895-902.
(38) Rogge, W. F.; Hildemann, L. M.; Mazurek, M. A.; Cass, G. R.;
Simoneit, B. R. T. Environ. Sci. Technol. 1993, 27, 636-651.
(39) Hida, M. Yuki Gosei Kagaku 1967, 25, 735-749.
(40) Pandit, P. N.; Tilak, B. D.; Venkataraman, F. A. Sc. Proc. Indian
Acad. Sci. 1952, 35A, 159-165.
(41) Linton, R. W.; Loth, A.; Natusch, D. F. S.; Evans, C. A., Jr.; Williams,
P. Science 1976, 191, 852-854.
(42) Ligocki, M. P.; Pankow, J. F. Environ. Sci. Technol. 1989, 23,
75-83.
(43) For a brief account, see Mori, T.; Suzuki, H. Synlett 1995, 383-
392.
(44) Suzuki, H.; Mori, T. J. Chem. Soc., Perkin Trans. 2 1996, 667 and
references cited therein.
(31) Heddle, J. A.; Hite, M.; Kirkhart, B.; Mavournin, K.; MacGregor,
J. T.; Newell, G. W.; Salamone, M. F. Mutat. Res. 1983, 123, 61-
118.
(32) Shelby, M. D. Mutat. Res. 1988, 204, 3-15.
(33) Mavournin, K. H.; Blakey, D. H.; Cimino, M. C.; Salamone, M.
F.; Heddle, J. A. Mutat. Res. 1990, 239, 29-80.
(34) Morita, T.; Ansano, N.; Awogai, T.; Sasaki, Y. F.; Sato, S.; Shimada,
Received for review December 30, 1996. Revised manuscript
received April 28, 1997. Accepted June 12, 1997.X
ES961067I
X Abstract published in Advance ACS Abstracts, August 1, 1997.
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