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Mutagenesis Advance Access published online on May 13, 2008
Mutagenesis, doi:10.1093/mutage/gen023
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© The Author 2008. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org.

Evaluation of the mutagenicity of nitration products derived from phenalenone (1H-phenalen-1-one)

Kentaro Misaki1,2,3, Yoshiharu Hisamatsu4,6, Hitomi Suzuki3 and Takeji Takamura-Enya3,5,*

1Department of Environmental Engineering, Graduate School of Engineering 2Department of Technology and Ecology, Graduate School of Global Environmental Studies, Kyoto University, Yoshida-honmachi, Sakyo-ku, Kyoto 606-8501, Japan 3Department of Chemistry, Graduate School of Science, Kyoto University, Kitashirakawa-cho, Sakyo-ku, Kyoto 606-8502, Japan 4Department of Community Environmental Science, National Institute of Public Health, 2-3-6 Minami, Wako, Saitama 351-0197, Japan 5Department of Applied Chemistry, Kanagawa Institute of Technology, 1030 Shimo-Ogino, Atsugi, Kanagawa 243-0292, Japan

1H-Phenalen-1-one (phenalenone) is one of the major oxygenated polyaromatic compounds present in the atmospheric environment. In order to gain detailed information regarding the mutagenicity and physicochemical properties of the nitration products of phenalenone, we measured Ames Salmonella mutagenicity, lower LUMO (lowest unoccupied molecular orbital) energy and octanol–water partition coefficient of the products obtained from the nitration reaction of phenalenone. Both nitration reactions of phenalenone, i.e. with mixed inorganic acids (a mixture of nitric acid and sulphuric acid) and with NO2-O3 in an aprotic solvent, preferentially afforded the nitration products 2-nitrophenalenone and 5-nitrophenalenone. Formation of a 6-nitro derivative of phenalenone was, however, only observed in the nitration reaction with sulphuric acid. Moreover, dinitro derivatives of phenalenone and also two oxidatively decomposed products of nitrophenalenone, i.e. 3-nitro- and 4-nitronaphthalic anhydride, were isolated from the reaction mixture. The mutagenicities of the six nitro compounds obtained from the nitration reactions were tested with the Salmonella strains TA98, TA100, YG1021 and YG1024 in the absence of S9 mix. Among these products, 2-nitrophenalenone exhibited the most potent mutagenic activity against TA98, TA100 and YG1024 (160, 230 and 2800 revertants/nmol for strains TA100, TA98 and YG1024, respectively), whereas 2,5-dinitrophenalenone exerted the highest mutagenicity against YG1021. Semi-empirical calculation showed that among the mononitrophenalenone series, the mononitro derivatives possessing lower LUMO energy tended to exhibit greater mutagenic activity than those with higher LUMO energy. This tendency, however, did not extend to the compounds with different aromatic ring systems due to the considerable differences in the hydrophobicities of these compounds.


   Introduction
Top
 Introduction
Materials and methods
 Results
 Discussion
 Funding
 References
 
In the atmospheric environment, various mutagens and carcinogens are present in exhaust gas and particulate matter, which are emitted from internal combustion engines such as those used in industrial factories, combustion furnaces and automobiles (1Go,2Go). Certain wildlife species and humans are continuously exposed to these mutagens/carcinogens. Nitrated polycyclic aromatic compounds (nitro-PACs) are well-known bacterial direct-acting mutagens and/or rodent carcinogens that are present in such gaseous and particulate matter (2Go–5Go). Nitro-PACs are originally emitted from various combustion engines and are more likely to be formed by the reaction of parental compounds with nitrogen dioxide in ambient air (6Go–9Go). Although many nitro-PACs have been identified and detected in the atmospheric environment, the combined mutagenicity of these known nitro-PACs only explains 10–20% of the total mutagenicity of atmospheric samples (7Go). It has also been reported that the mutagenicity of the extracts of diesel and gasoline exhaust particulates, and also that of airborne particulates, is concentrated in chromatographically isolated polar fractions (1Go,9Go–12Go). Since only a small number of mutagens have been identified in these polar fractions, the presence of other unknown compounds in the polar fractions has always been debated (13Go,14Go).

In a previous study, we identified 3-nitrobenzanthrone (3-NBAO) as a novel potent mutagen in the polar fraction of diesel exhaust particulates (15Go). It was further shown that 3-NBAO universally existed in the atmospheric environment (16Go), and its oral administration induced lung cancer in F344 rats via reductive metabolic activation resulting in the formation of DNA adducts (17Go–19Go). The correlation between the mutagenicities of the selected nitrated benzanthrones and their physicochemical properties has also been discussed in detail elsewhere (20Go).

Compounds containing nitro substituents liable to be reduced to hydroxylamines generally exhibit potent mutagenic activity (2Go,4Go). This leads to the prediction that nitro compounds possessing carbonyl substituents are enzymatically reduced to hydroxylamine more efficiently than nitro compounds without carbonyl substituents; this is due to the electron-withdrawing effect of these carbonyl substituents. However, data concerning the mutagenicity of such compounds—defined as nitropolycyclic aromatic ketones (NPAKs) and nitropolycyclic aromatic lactones—are very few, even though these compounds are predicted to be present in the polar fractions of environmental samples (14Go,15Go). In the present study, we selected 1H-phenalen-1-one (phenalenone, PhO) as a target compound, the nitration products of which would be identified as novel environmental mutagens possessing a carbonyl substituent. Although nitrophenalenones (NPhOs), nitration products of PhO, have hitherto been unidentified in an atmospheric environment, its presence in natural environments could be highly possible. Considerable amounts of PhO have been detected in oil (106 mg/l) (21Go), exhaust particulates of both diesel and non-catalytic gasoline engines (~15 µg/km, respectively) (11Go,22Go) and also in airborne particulates (~90 to 180 ng/mg equivalent organic carbon) (23Go,24Go). In an atmospheric environment, nitration of PhO adsorbed on airborne particulates with gaseous NOx or nitric acid mist (gas–solid disproportional reaction) probably occurs more preferentially than nitration of gaseous PhO with them (gas phase reaction) because it was reported that 90% of PhO in tunnel particulates emitted from automobiles was adsorbed with the solid phase despite its facile sublimation property (25Go,26Go). The conjectured nitration products of PhO are expected to possess low reduction potential due to the presence of a carbonyl group; this suggests that the nitrated products of PhO would be potentially mutagenic in the Ames Salmonella assay. Moreover, NPhO shares a common moiety with the NBAOs that exhibit extremely potent mutagenic activity. A comparison of the mutagenicity and physicochemical properties of these nitrated products will provide useful information for predicting the mutagenicity of nitro-PACs containing a carbonyl functional group.

Therefore, in this study, we prepared the nitration products derived from the reaction of PhO with various nitrating agents. The mutagenic activities of the nitro compounds thus obtained were determined in the Ames Salmonella assay using various test strains. Moreover, the correlation between the mutagenicity of the nitro derivatives of PhO and their physicochemical properties are discussed, and the data are compared with those previously obtained for the mutagenic and physicochemical properties of NBAOs.


   Materials and methods
Top
 Introduction
 Materials and methods
Results
 Discussion
 Funding
 References
 
General
Melting points were determined on a Yanagimoto hot-stage apparatus and are uncorrected. Infrared (IR) spectra were recorded on a Shimadzu FTIR DR 8000/8100 IR spectrophotometer and prominent peaks in the 2000–700/cm region were recorded. 1H-nuclear magnetic resonance (NMR) spectra were recorded with a Varian Gemini-200 (200 MHz) spectrometer in CDCl3 with trimethylsilyl as an internal standard. J values are shown in hertz. Electron impact mass spectra (EI-MS) were recorded on an HP5890/JEOL, JMS-HX100A (ionization current, 300 A; ionization voltage, 70 eV; chamber temperature, 280–290°C and accelerating voltage, 70 eV).

Chemicals
The isomeric NPhOs and nitrated naphthalic anhydrides (NNAs) used in this study are shown in Figure 1. PhO, naphthalic anhydride (NA), 3-NNA and 4-NNA were supplied by Aldrich Chemical Co. (St. Louis, MO, USA). Fuming nitric acid (HNO3, 94%) and concentrated sulphuric acid (H2SO4, 97%) were supplied by Nacalai Tesque Co. (Kyoto, Japan). Nitrogen dioxide (99% pure) was obtained from Sumitomo Seika Co. and used after transfer distillation. Ozone was generated by a type ON-1-2 apparatus manufactured by Nippon Ozone Co., which produced ozone at a rate of 10 mmol/h under an oxygen flow of 10 l/h at an applied voltage of 65 V. Methanol [high-performance liquid chromatography (HPLC) grade], hexane, ethyl acetate, chloroform and tetrahydrofuran (THF) were purchased from Wako Chemical Co. (Osaka, Japan).


Figure 1
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  		Fig. 1. Structure of NPhOs, NNAs and NBAOs.

 
Mutagenicity assay
The Salmonella typhimurium strains TA98, TA100, YG1021 and YG1024 were used as the bacterial test strains for the Ames assay. The latter two strains were kindly provided by T. Nohmi of the National Institute of Health Sciences, Tokyo (27Go,28Go). The mutagenicity tests were carried out according to the method of Ames et al. (29Go) with a slight modification, including a pre-incubation step (30Go). Ampicillin (25 µg/ml) and tetracycline (6.25 µg/ml) were added to the broth used to culture YG1021 and YG1024. All mutagenicity assays were performed in the absence of a rat metabolic activation system (S9 mix). Furylfuramide (0.01 µg per plate) was used as positive controls for the TA98 and TA100 strains, respectively, whereas 2-nitrofluorene (0.1 and 0.01 µg per plate) was used for both YG1021 and YG1024. The assays were performed three times for each compound with duplicate samples, and the results were averaged. For three mononitrophenalenones (MNPhOs) tested with the TA100 and YG1021 strains, the assays were carried out two times with duplicate samples, and the results were averaged. Mutagenic activities of test samples were calculated from the linear portions of dose–response curves obtained with three to five doses with duplicate plates in two independent experiments.

Quantum data
The lowest unoccupied molecular orbital (LUMO) energy levels of the NPhOs and NNAs were calculated by means of the semi-empirical PM3 quantum-mechanical method using MOPAC version 5.0. The initial geometries were constructed using the standard bond lengths and angles. The geometry was then completely optimized using algorithms in the MOPAC programme.

Measurement of the octanol–water partition coefficient (Kow)
Measurements of the octanol–water partition coefficient (Kow) were performed by the reversed-phase HPLC method, as described by Sarna et al. (31Go). The HPLC system comprised a Shimadzu Shimpack MRC-ODS column (ø4.6 x 250 mm), an LC-10AS pump, an SPD-10A UV detector at 254 nm and a C-R7A system controller (Shimadzu, Kyoto, Japan). The mobile phase used was methanol:water (60:40) at a flow rate of 1.0 ml/min. A standard curve was prepared using a series of standard chemicals with published log Kow values in the range 0.95–2.99. The log Kow values of NPhOs and NNAs were obtained from the retention volumes, using a standard calibration curve.

Isolation of the nitration products of PhO, obtained using various nitrating agents
Nitration with fuming HNO3 and 97% H2SO4. Several MNPhOs were prepared using the mixed acid method (the nitric acid-concentrated sulphuric acid system) described by Dokunikhin et al. (32Go). PhO (210 mg, 1.2 mmol) was dissolved in 1 ml of 97% sulphuric acid and cooled to 0°C. A mixture of 0.6 ml of sulphuric acid monohydrate and 0.06 ml of fuming nitric acid was then added. The mixture was stirred for 20 min and added to water and dichloromethane. This solution was neutralized with sodium hydroxide and sodium bicarbonate and then extracted with dichloromethane. The organic phase was dried over sodium sulphate and evaporated to dryness in vacuo. The residue was chromatographed using the low-pressure liquid chromatography (LPLC) method. The LPLC system comprised a Yamazen Si-40-S column (ø37 x 300 mm), an LPLC Model 540, a Model SSC-3000AII UV detector at 254 nm and an FR50N fraction collector (Yamazen, Osaka, Japan). The first separation for half of the total amount was repeated two times using hexane:THF (step gradient, 20–100% for THF) as the mobile phase. Fractions at prominent UV absorbance peaks were collected. Using this method, pure samples of 6-NPhO and 2-NPhO were obtained as the second and third fractions, respectively. The first fraction containing a mixture of 5-NPhO and PhO was subject to further chromatographic separation by using a mixed solvent of hexane and ethyl acetate (4:1):chloroform (step gradient, 29–100% for chloroform). At this second separation, PhO and 5-NPhO were perfectly separated and obtained as the first and second fractions, respectively. The yields of 2-NPhO, 5-NPhO and 6-NPhO were 20, ~55 and 24%, respectively (Table I, entry 2). Further purification was performed for the respective isomers using repeated LPLC.


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  		Table I. Product distribution in the nitration of phenalenone

 
2-NPhO: yellow powder, mp 243–245°C [lit. mp 240–241°C (33Go)]. IR (KBr): {nu} = 1649, 1582, 1520, 1352/cm. 1H-NMR (CDCl3): {delta} = 7.7 (dd, 1H, J = 8.2, 7.2, H5), 7.9 (t, 1H, J = 7.8, H8), 8.1 (d, 1H, J = 7.3, H4), 8.3 (d, 1H, J = 8.4, H6), 8.3 (d, 1H, J = 8.1, H7), 8.5 (s, 1H, H3), 8.8 (dd, 1H, J = 7.4, 1.2, H9). MS: m/z (%) = 225 (M+, 100), 195 (M+-NO, 10), 179 (18), 167 (63), 151 (89), 150 (43), 139 (26). HR-EI-MS C13H7NO3: calculated value, 225.0426; found 225.0406.

5-NPhO: yellow powder, mp 226–227.5°C [lit. mp 227–228°C (33Go)]. IR (CHCl3): {nu} = 1727, 1464, 1262/cm. 1H-NMR (CDCl3): {delta} = 6.9 (d, 1H, J = 9.8, H2), 7.8 (d, 1H, J = 9.8, H3), 8.0 (t, 1H, J = 7.6, H8), 8.4 (d, 1H, J = 7.8, H9), 8.5 (d, 1H, J = 2.2, H4), 8.8 (d, 1H, J = 7.4, H7), 9.0 (d, 1H, J = 2.0, H6). MS: m/z (%) = 225 (M+, 100), 195 (M+-NO, 17), 179 (46), 167 (14), 151 (44), 150 (25), 139 (15). HR-EI-MS C13H7NO3: calculated value, 225.0426; experimentally determined value, 225.0425.

6-NPhO: yellow powder, mp 228–230°C [lit. mp 229–230°C (32Go)]. IR (KBr): {nu} = 1653, 1516, 1507, 1339/cm. 1H-NMR (CDCl3): {delta} = 6.8 (d, 1H, J = 9.9, H2), 7.7–7.9 (m, 2H, H3 and H9), 8.0 (dd, 1H, J = 8.6, 7,4, H8), 8.3 (d, 1H, J = 7.9, H7), 8.7 (dd, 1H, J = 7.4, 1.2, H4), 8.9 (dd, 1H, J = 8.7, 1.2, H5). MS: m/z (%) = 225 (M+, 100), 195 (M+-NO, 38), 179 (26), 167 (30), 151 (58), 150 (39), 139 (45). HR-EI-MS C13H7NO3: calculated value, 225.0426; experimentally determined value, 225.0405.

Nitration with fuming HNO3 and 70% H2SO4. Nitration of PhO using the mixed acid method was also performed as described by Dokunikhin et al. (32Go). PhO (200 mg, 1.1 mmol) was dissolved in 5 ml of 70% sulphuric acid, and 0.06 ml of fuming nitric acid was added at room temperature. The mixture was heated to 85°C, stirred for 6.5 h, then cooled and added to water and dichloromethane. This solution was neutralized with sodium hydroxide and sodium bicarbonate and extracted with dichloromethane. The organic phase was dried with sodium sulphate and evaporated to dryness in vacuo. The presence of these nitro derivatives and NA was confirmed by separating the mixture with LPLC under the same conditions as those for nitration with fuming HNO3 and 97% H2SO4. The conversion was 74%, and the yields of 2-NPhO, 5-NPhO and 6-NPhO; 2,5-dinitrophenalenone (2,5-DNPhO) and NA, as determined by comparing the NMR spectra of the mixture and these products, were 32, 5, 22, trace and 5%, respectively (Table I, entry 2). An unidentified compound was also isolated and its yield was estimated to be ~10% by comparing the integration of the proton signal of this compound with that of known NPhOs in the 1H-NMR spectra of the reaction mixture.

Nitration with the NO2-O3 system (Kyodai nitration). A solution of PhO (190 mg, 1.0 mmol) in 25 ml of dichloromethane was mixed with liquid nitrogen dioxide (0.5 ml, 7.5 mmol) at 0°C, and ozonized oxygen (5.0 mmol) was passed slowly into this solution at the same temperature (34Go). After 30 min, the reaction mixture was added to water. This solution was neutralized with sodium bicarbonate and extracted with dichloromethane. The organic phase was dried with sodium sulphate and evaporated to dryness in vacuo. The residue was divided into several portions and each portion was chromatographed using the LPLC method. The first separation for each of the constituent portions was performed several times using hexane:THF (step gradient, 20–100% for THF) as the mobile phase. The third UV peak fraction was collected, and the second separation was performed using a mixed solvent of hexane and ethyl acetate (4:1):chloroform (step gradient, 29–100% for chloroform). In this second separation, 5-PhO, 6-NPhO and 2,5-NPhO were included in the first, second and third fractions, respectively. The conversion was 92%, and the yields of 2-NPhO, 5-NPhO and 2,5-DNPhO were 33, 23 and 8%, respectively. It was also confirmed with NMR that the yields of NA and 6-NPhO were 2% and trace, respectively (Table I, entry 3). 2,5-NPhO was purified using repeated LPLC.

2,5-DNPhO: yellow powder, mp 275–278°C [lit. mp 283–284°C (33Go)]. IR (KBr): {nu} = 1655, 1590, 1534, 1343/cm. 1H-NMR (CDCl3): {delta} = 8.1 (t, 1H, J = 7.7, H8), 8.5–8.6 (m, 2H, H3 and H9), 8.8 (d, 1H, J = 2.1, H4), 8.9 (dd, 1H, J = 7.5, 1.2, H7), 9.2 (d, 1H, J = 2.0, H6). MS: m/z (%) = 270 (M+, 100), 240 (M+-NO, 12), 212 (67), 194 (8), 178 (48), 166 (28), 150 (75), 138 (19). HR-EI-MS C13H6N2O3: calculated value, 270.0277; experimentally determined value, 270.0265.


   Results
Top
 Introduction
 Materials and methods
 Results
Discussion
 Funding
 References
 
Identification and isomer distribution of the nitration products of PhO, obtained using various nitrating agents
In order to obtain precise information regarding the preferential nitration positions of phenalenone, we performed three nitration reactions with a fuming nitric acid–sulphuric acid (97%) system, a fuming nitric acid–sulphuric acid (70%) system and a NO2-O3 system. Nitration of PhO with fuming nitric acid in 97% sulphuric acid at 0°C resulted in the formation of three nitration products including 5-NPhO (~55%), 6-NPhO (24%) and 2-NPhO (20%), in descending order of yield (Table I, entry 1). When a considerably lower concentration of sulphuric acid (HNO3/70% H2SO4) was used for nitration, the conversion yields were decreased to 74% and the formation of 5-NPhO was depressed to a yield of 5%. Regardless of the lower rate of conversion, other nitro compounds, i.e. 2-NPhO and 6-NPhO, were obtained at higher yields of 32 and 22%, respectively (Table I, entry 2). Under this condition, trace amounts of 2,5-DNPhO were also observed. Moreover, NA was formed with a yield of 5%. An unidentified compound with a yield of 10% was also detected by NMR studies.

‘Kyodai’ nitration (an ozone-mediated nitration system) also efficiently nitrated PhO at a 92% conversion within 30 min, giving 33 and 23% yields of 2-NPhO and 5-NPhO, respectively (Table I, entry 3). In this reaction, formation of NA and 6-NPhO at yields of 2% and trace, respectively, were confirmed by NMR studies. Moreover, small amounts (8%) of the dinitro derivative, 2,5-DNPhO and other polar compounds—probably the peroxidized or decomposed products of PhO—were also observed.

Mutagenicity of the nitration products of PhO
All six products obtained from the nitration of PhO exhibited mutagenicity in all test bacterial strains in the absence of an S9 mix (Figure 1, Table II). 2-NPhO exhibited the most potent mutagenicity in the TA98 and TA100 strains (230 and 160 revertants/nmol, respectively). Other nitro products also exhibited mutagenic activity against TA98 comparable to that against TA100; this suggests that every product induced a frameshift-type mutation at the same level as base pair substitution-type mutations. The order of the mutagenicity of the NPhOs and NNAs in TA98 in the absence of the S9 mix was 2-NPhO > 6-NPhO > 2,5-DNPhO > 5-NPhO > 4-NNA > 3-NNA (35Go). The 2-NPhOs exhibited more potent mutagenicity than 2,5-DNPhO in TA98; however, their activities towards YG1021 strains were reversed in order.


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  		Table II. Direct-acting mutagenic activities of NPhOs and NNAsa

 
The YG1021 and YG1024 strains are derivatives of TA98, which possess enhanced nitroreductase (N-rase) and O-acetyltransferase levels, respectively (27Go,28Go). All compounds tested in this study showed enhanced mutagenicity in YG1021 and YG1024. The mutagenicity of 2-PhO was detected ~12 times more efficiently in YG1024 than in TA98. The mutagenicity of 2,5-DNPhO was detected ~41 and 18 times more efficiently in YG1021 and YG1024, respectively, than in TA98. Other tested nitro compounds exhibited four to nine times greater mutagenic activity in YG1024 compared to TA98.

LUMO energy
The calculated LUMO energy values of the NPhOs ranged from –2.0 to –2.6 eV and those for the NNAs ranged from –2.4 to –2.5 eV (Table III, Figure 2). LUMO energy is known to correlate with the polarographic half-wave reduction potential, which in turn has been demonstrated to correspond to the reduction potential or electron affinities of nitroarenes (20Go,36Go).


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  		Table III. Physicochemical parameters of NPhOs, NNAs and NBAOs

 


Figure 2
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  		Fig. 2. Relationship between LUMO energy and TA98 mutagenic activity. LUMO energy was calculated using the self-consistent field (PM3) method. Data for NBAOs are from ref. (20Go): diamonds, NPhOs; triangles, NNAs and circles, NBAOs.

 
Among the MNPhOs tested in this study, 6-NPhO exhibited the lowest LUMO energy (–2.23 eV), followed by 2-NPhO (–2.12 eV) and 5-NPhO (–2.03 eV). The dinitro derivative of PhO, i.e. 2,5-DNPhO, exhibited lower LUMO energy (–2.64 eV) than the MNPhOs. This tendency is predicted in view of the fact that an increase in the number of nitro group possessing electron-withdrawing activity facilitates the reduction of this group.

Hydrophobicity
Hydrophobicity was demonstrated as the octanol–water partition coefficient (log Kow) calculated by the reversed-phase HPLC method (Table III, Figure 3) (31Go). Within the NPhO series, 6-NPhO was the most hydrophobic (log Kow = 2.41) and 2-NPhO was the most hydrophilic (log Kow = 1.96), whereas in the NNA series, 3-NNA was more hydrophilic (log Kow = 0.13) than 4-NNA (log Kow = –0.55).


Figure 3
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  		Fig. 3. Relationship between log Kow and TA98 mutagenic activity. Kow is the octanol–water partition coefficient. Data for NBAOs are from ref. (20Go): diamonds, NPhOs; triangles, NNAs and circles NBAOs.

 

   Discussion
Top
 Introduction
 Materials and methods
 Results
 Discussion
Funding
 References
 
Analysis of the nitration products derived from the reaction of PhO with various nitrating agents
Nitration of PhO has already been described by several investigators (25Go,32Go,33Go). Positional selectivity of PhO in the nitration reaction completely depended on the reaction conditions. In a strong acid solvent like 100.13% sulphuric acid, formation of 5-NPhO was reported to be predominant, i.e. above 80%, whereas in much more milder conditions like 70% sulphuric acid, ~60 and 10% yields of 2-NPhO and 6-NPhO, respectively, were obtained (32Go). Nitration of the second position of PhO was mainly observed with a solvent of acetic acid (32Go,33Go). These drastic changes in nitration position depending on the experimental conditions were also observed in our studies. Nitration of PhO using 97% sulphuric acid gave 5-NPhO efficiently, whereas nitration of PhO using 70% sulphuric acid efficiently yielded 2-NPhO and 6-NPhO, while 5-NPhO was formed to lesser extent (Table I, entry 1). Treatment with three equivalents of fuming nitric acid, instead of one equivalent of nitric acid in 70% sulphuric acid, resulted in the formation of only 4-NNA and 3-NNA with decreasing amounts of MNPhOs (data not shown), although conversion was increased to 100 from 70% in the original system. Oxidation of 6-NPhO and 5-NPhO or the nitration of NA formed by the oxidation of PhO and 2-NPhO might occur more readily than the nitration of PhO.

Kyodai nitration—ozone-mediated nitration with nitrogen dioxide in dichloromethane—is known to be an analogue of the gas–solid disproportional reaction in the atmospheric environment and in internal combustion engines (34Go,37Go–40Go). Kyodai nitration of PhO afforded very different results in the product ratio compared to sulphuric acid systems. The nitration reaction of PhO by the Kyodai nitration system gave 2-NPhO and 5-NPhO at yields of 33 and 23%, respectively, with 92% conversion of PhO (Table I, entry 3). Interestingly, the yield of 6-NPhO, which is efficiently formed by nitration at lower sulphuric acid concentrations, is negligible, and a large amount of 5-NPhO was observed. Moreover, the yield of 2,5-DNPhO (8%) in this reaction was much higher than that obtained using the usual mixed acid reaction. When the nitration was performed with an elevated concentration of ozone, efficient formation of 4-NNA and 3-NNA was observed (data not shown). Therefore, it is possible that NPhOs are readily oxidized to form 4-NNA and 3-NNA in an atmospheric environment. If the oxidation of NPhOs proceeds, the mutagenic risk will decrease owing to the lower mutagenicity of NNAs compared to NPhOs.

Relationship between mutagenicity of the nitro derivatives of PhO and their physicochemical properties
The mutagenicities of six products formed from the laboratory nitration of PhO, namely 3 MNPhOs, 1 DNPhO and 2 mononitronaphthalic anhydrides (MNNAs), were tested with the Salmonella strains TA98, TA100, YG1021 and YG1024 in the absence of the S9 mix (Table II). The mutagenicity of all test compounds was increased in the N-rase-overproducing strain, YG1021, and in the acetyltransferase-overproducing strain, YG1024. Apparently, the nitroreduction step and O-acetylation of the resulting hydroxylamine are crucial for enhancing the mutagenicity of these compounds (17Go–20Go). The mutagenic activity of 2-NPhO was the most potent among the MNPhOs in the strains TA98 and YG1024 (230 and 2800 revertants/nmol, respectively). In the case of YG1021, 2,5-DNPO is the most mutagenic compound whose activity was detected ~41 times more efficiently in YG1021 than in TA98.

Lopez de Compadre et al. suggested that the mutagenicity of nitro compounds is highly dependent on their LUMO energy and hydrophobicity (41Go–46Go). The LUMO energy is indicative of the reductivity of the nitro substituent—a simple linear correlation is generally observed between LUMO energy and the first reductive potential (Ered) of nitro compounds (20Go,39Go,41Go,42Go). In the case of NPhOs, both the nitro group and carbonyl group were suspected of being chemically reduced. However, it appears that the carbonyl group in the NPhOs is less reductive than the nitro group since only the nitro group of NPhO is reduced by reducing agents such as hydrazine hydrate/palladium carbon, sodium sulphide and the Zn/glacial acetic acid system (25Go,32Go,33Go). This was also the case with the NNAs: only the nitro group was reduced by hydrogen/palladium carbon and SnCl2/HCl (47Go,48Go). The calculated LUMO energy values for the MNPhOs ranged from –2.0 to –2.2 eV; those for the MNNAs ranged from –2.4 to ––2.5 eV. These values are considerably lower than those of mononitropolycyclic aromatic hydrocarbons such as mononitropyrenes and mononitrofluoranthenes, whose values ranged from –1.5 to –1.8 eV, and almost comparable to the values of the dinitropolycyclic aromatic hydrocarbons such as dinitropyrenes (approximately –2.5 eV) (Table III, Figure 2) (41Go–43Go,45Go,46Go).

It is predicted that nitro compounds possessing carbonyl substituents are more readily reduced to hydroxylamine because of the electron-withdrawing activity of these substituents. The mutagenicity of the mononitro-PACs tested in this study was found to tend to increase with decreasing LUMO energy; 2-ZPhO and 6-NPhO were more potent mutagens than 5-NPhO, and 4-NNA was more potent than 3-NNA (35Go). Curiously enough, in the comparison between 2-NPhO and 6-NPhO, the correlation between mutagenicity and LUMO energy was contrary to this tendency. 2-NPhO has a unique structure wherein the nitro group is adjacent to the carbonyl group. Due to steric impulsion, orientation of the nitro group of such a compound is expected to be perpendicular to the aromatic ring plane (49Go–53Go). This estimation was also supported by the PM3 calculation, which showed that the nitro group of 2-NPhO is almost perpendicular to the aromatic ring plane (data not shown). Moreover, the LUMO atomic {pi}-orbital was found not to overlap with C-NO2 nuclei (54Go). Regardless of the results of these calculations, 2-NPhO contains a nitro group that can be more easily reduced chemically than that of the other MNPhOs, as stated above. It is probable that the orientation of the nitro group may be altered due to efficient protonation of the carbonyl group in an acidic medium, resulting in the ease of nitroreduction, as illustrated in Figure 4. Moreover, the resulting hydroxylamine and/or an aryl nitrenium ion at the 2-position is stabilized by hydrogen bond formation with the oxygen atom in the carbonyl group and/or by the electron negativity of the oxygen atom in the carbonyl group present at the ortho position of the nitro group (25Go).


Figure 4
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  		Fig. 4. Predicted stereochemistry of protonated 2-NPhO.

 
Generally, the mutagenicity of dinitro compounds is greater than that of their mononitro counterparts marked with dinitropyrenes (2Go,4Go,27Go–29Go,36Go,55Go). However, this tendency was not observed in the NPhO series in TA98. The relationship between mutagenicity in TA98 and the above-mentioned LUMO energy was only observed for compounds with the same number of nitro substituents and could not be extended to compounds with a different number of nitro groups; 2,5-DNPhO possessing considerably lower LUMO energy than 2-NPhO was a less potent mutagen. The decrease of mutagenicity from dinitropyrenes to trinitropyrenes was also observed to be independent of LUMO energy. However, mutagenicity in YG1021 did not follow these trends. Every compound tested in this study enhanced its mutagenicity in YG1021 compared to TA98. In particular, 2,5-DNPhO exerted 40 times higher mutagenicity in YG1021 than in TA98. This indicates that the LUMO energy of 2,5-DNPhO is sufficiently low; however, the compound was not efficiently reduced by the original amount of classical N-rase in TA98, probably due to low affinity of the enzyme to NPhO. Therefore, when a sufficient amount of reductase is present in strains, a linear relationship between LUMO energy and mutagenicity would be observed. In the YG1021 series, we obtained a simple linear correlation in the form of an equation: LogYG1021 = 1.93LUMO + 1.6, R2 = 0.64.

Comparison of the mutagenicity of NPhOs with that of structurally related NBAOs
Although the nitroreduction potential affected mutagenicity in the same aromatic ring system, this tendency was not observed in different aromatic ring systems. The range of LUMO energy values for MNNAs (–2.36 to –2.53 eV) fell between the range for the MNPhOs (–2.03 to –2.23 eV) and the value for 2,5-DNPhO (–2.64 eV) (Table III, Figure 2). 2-NPhO, possessing lower LUMO energy than the MNBAOs, was considerably less mutagenic than 3-NBAO, 9-NBAO and 1-NBAO. Furthermore, the biological behaviour of NPhO and NBAO was completely different. NPhO enhanced the mutagenicity in YG1021, whereas 3-NBAO decreased its mutagenicity in the same strains. In our previous report, NBAO is estimated to be easily reduced to form a corresponding amino derivative without the formation of a hydroxylamino intermediate. This phenomenon is prominent in the YG1021 strain, which is a N-rase overproducing strain. Substrate specificity of the classical N-rase may explain the difference in the alteration of mutagenicity in YG1021 compared to TA98, between NBAO and NPhO. Another nitro compound, 4-NNA, possessing lower LUMO energy than the MNPhOs, was less mutagenic than 2-NPhO and 6-NPhO (35Go).

Clear differences in the physicochemical properties of these compounds are apparent in their hydrophobicities—the NBAOs (log Kow = 3.60–3.99) are more hydrophobic than the NPhOs (log Kow = 1.96–2.41), and the MNPhOs are more hydrophobic than the MNNAs (log Kow = –0.55 to 0.13) (Table III, Figure 3) (20Go). Hydrophobicity is associated with the ease of penetration into cells: the more hydrophilic a compound, the weaker is its penetration and therefore the lower its mutagenicity (36Go,41Go–46Go). Moreover, we believe that to some extent, hydrophobicity is also a crucial determinant of accessibility of the compound to the enzyme activity pocket; however, much more detailed experiments are required to prove this. The hydrophobicity of the nitro-PACs remarkably depended on their parental PACs; therefore, the number of aromatic rings can be regarded as a good indicator of the hydrophobicity as well as the mutagenicity of these compounds. A recent finding that expansion of the ring system of NBAO enhanced their mutagenicity in TA98 also supports that hydrophobicity is the main determinant of the mutagenicity of NPAK (56Go).

In conclusion, of the six products formed by the nitration of PhO, 2-NPhO exhibited the most potent mutagenic activity against three Salmonella strains (TA98, TA100 and YG1024). The mononitro compounds possessing lower LUMO energy tended to exhibit greater mutagenic activity than other isomers containing the same aromatic ring system. This tendency, however, did not extend to the compounds with different aromatic ring systems due to the considerable differences in the hydrophobicities of these compounds.

In the Kyodai nitration reaction, 2-NPhO, 5-NPhO and 2,5-DNPhO were the major reaction products; therefore, it is plausible that these nitro compounds exist in the atmospheric environment. Based on the observed mutagenicity of the nitro compounds in the present study and other data on the concentrations of parental PACs in the atmospheric environment (11Go,15Go,16Go,21Go–24Go), 2-NPhO may, to some extent, contribute to the mutagenicity of atmospheric samples. The concentrations of these nitro derivatives of PhO remain to be examined. Moreover, it is necessary to further examine the mutagenicities and environmental concentrations of NPAKs; these studies are currently being conducted by our group.


   Funding
Top
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
References
 
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, 19510073).


   Acknowledgments
 
Conflict of interest statement: None declared.


   Notes
 
* To whom correspondence should be addressed. Tel: +81 46 291 3072; Fax: +81 46 242 8760; E-mail: takamura{at}chem.kanagawa-it.ac.jp

6 Present address: Tokyo University of Agriculture and Technology, Field Science Center, 3-5-8 Saiwai-cho, Fuchu, Tokyo 183-8509, Japan Back


   References
Top
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 

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Received on July 26, 2007; revised on March 19, 2008; accepted on April 11, 2008.


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