Environ. Sci. Technol. 1997, 31, 2772-2776
3-Nitrobenzanthrone, a Powerful Bacterial Mutagen and Suspected Human Carcinogen Found in Diesel Exhaust and Airborne Particulates T A K E J I E N Y A 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 TETSUSHI WATANABE AND T E R U H I S A H I R A Y A M A * ,‡ Department of Public Health, Kyoto Pharmaceutical University, Yamashina-ku, Kyoto 607, Japan Y O 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-7one) 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.
Introduction In recent years, nitrated polycyclic aromatic hydrocarbons (nitro-PAHs) have caused grave environmental concern because of their potent mutagenic activity (1) and carcinogenicity (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 * Authors to whom correspondence should be addressed. † Phone: +81-075-753-4041; fax: +81-075-751-2085; e-mail:
[email protected]. ‡ Phone: +81-075-595-4650; fax: +81-075-595-4769. § Phone: +81-03-3441-7111; fax: +81-03-3446-4314; email:
[email protected].
2772
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 31, NO. 10, 1997
of these mutagens is believed to be car emissions, and the 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. We herein report a remarkably high mutagenic aromatic nitroketone, 3-nitrobenzanthrone (3-nitro-7H-benz[d,e]anthracen-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 benzanthrone 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 chromosomal aberrations in mammalian cells.
Experimental Section Instrumentation. 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 280290 °C; accelerating voltage 10 kV. GC quantification of 3-nitrobenzanthrone from environmental samples was performed 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-nitrobenzanthrone was eluted at 34.7 min after injection. Materials. Reagent grade benzanthrone was purchased from Tokyo Kasei Kogyo Co. Ltd. and used after recrystallization from ethanol (purity >99.9%). Isomeric mono- and dinitrobenzanthrones used as reference compounds were prepared from appropriately substituted methyl nitro-2iodobenzoates 9 and nitro-1-iodonaphthalenes 10 via the reaction sequence involving the Ullmann coupling and intramolecular Friedel-Crafts ring closure (17-19) as illustrated 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.
S0013-936X(96)01067-X CCC: $14.00
1997 American Chemical Society
SCHEME 1
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). Sampling. 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 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 dichloromethane 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. typhimurium his- strains YG 1021 and 1024 were produced by transferring a plasmid carrying the nitroreductase or acetyltransferase 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
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. Statistical analysis was performed by using the method of Kastenbaum and Bowman (27). Reaction of Benzanthrone with Nitrogen Dioxide in the Absence or Presence of Ozone. Homogeneous 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 11nitrobenzanthrones 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.
Results and Discussion Environmental Occurrence and Mutagenicity of 3-Nitrobenzanthrone. 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-nitrobenzanthrone 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. typhimurium 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 1,8dinitropyrene 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-nitrobenzanthrone 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 Isomeric Nitrobenzanthrones. 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
VOL. 31, NO. 10, 1997 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
2773
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)
3-nitrobenzanthrone (2)a 9-nitrobenzanthrone (3) 11-nitrobenzanthrone (4) 3,9-dinitrobenzanthrone (5) 3,11-dinitrobenzanthrone (6) 1,8-dinitropyrene (8)
TA98
TA100
YG1021
YG1024
208 000 ( 22 000 84 000 ( 6 000 6(0 46 000 ( 5 200 3 000 ( 450 257 000b (283 000d)
29 700 ( 2 600 3 270 ( 200 6(0 4 300 ( 400 360 ( 30 55 400b
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 ( 1 223 000 ( 38 000 12 200 ( 1700 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 ng/1123 revertants). b From ref 1. c From ref 25. d 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
