Chem. Res. Toxicol. 1990, 3, 231-235
231
'H NMR Studies on Mutagenic Nitrofluoranthenes and Exposure Risk Assessment Giuseppe L. Squadrito,tJ Barbara S. Shane,g Frank R. Fronczek,* Daniel F. Church,tJ and William A. Pryor**tti*s Biodynamics Institute, Department of Chemistry, and Institute for Environmental Studies, Louisiana State University, Baton Rouge, Louisiana 70803 Received March 13, 1989
The 'H NMR spectra of the nitrofluoranthene isomers are presented to allow gas chromatographic analysis of environmental samples suspected of containing nitrofluoranthenes. The mutagenic isomers 1-,2-, 3-, 7-, and 8-nitrofluoranthene and 1,2- and 1,3-dinitrofluoranthene have been studied by using nuclear and a nonmutagenic analogue, l-pheny1-4-nitronaphthalene, Overhauser effects and 2D shift-correlated 'H NMR spectroscopy. The X-ray crystal structure of 1-phenyl-4-nitronaphthalene is also reported. Reduction potentials and coplanarity of the nitro group have been used to correlate the mutagenicity of nitrated polycyclic aromatic hydrocarbons (nitro-PAH) measured by the Ames assay, but our data suggest that these parameters are not sufficient for the prediction of mutagenic potency in the nitrofluoranthene series.
Introduction About three million tons of nitrogen oxides (NOx) are discharged into the atmosphere every year as a result of worldwide anthropogenic activities, including the use of combustion engines and residential home heating (1). Similarly, about 1000 tons of polycyclic aromatic hydrocarbons (PAH) are released yearly into the atmosphere as a result of the operation of diesel engines in the United States.' Other industrial activities, such as aluminum smelting the coke processing, can lead to PAH concentrations in the environment of the industrial site that are up to 10000 times higher than those found in urban atmospheres (2). This mixture of NOx and PAH leads to the nitration of PAH, a process of grave concern to the environmentalist because some nitro-PAH are more mutagenic than their parent PAH. Fluoranthene is one of the most abundant PAH in airborne particulate organic matter (POM) (3),and 2-nitro-
8
"0: 5
4
3
Fluoronthene ( F l )
fluoranthene (2NF) has been reported to be the most abundant nitro-PAH in airborne POM ( 4 ) . In addition, 1-, 3-, 7-, and 8-nitrofluoranthene have been observed in diesel engine emissions (5, 6). Gas chromatographic techniques ( 5 , 6 )have been used to identify the genotoxic nitrofluoranthenes (7-13), but nitrofluoranthene isomers have been confused due to their very similar retention times (14-1 7). Recent developments in capillary gas chromatography have made possible the base-line separation of the nitrofluoranthene isomers, but the quality of the separation and the order of elution are highly dependant on the nature of the bonded phase used (18-22). Biodynamics Institute. *Department of Chemistry. 8 Institute for Environmental Studies.
Since the nitrofluoranthene isomers have very different mutagenicities, their unequivocal identification in environmental samples is essential for risk assessment from exposure to these widely disseminated pollutants. Proton NMR is a valuable technique for the identification of the nitrofluoranthenes, even at submilligram levels that minimize risks of exposure. However, the currently available lH NMR data on the nitrofluoranthenes (5,23-25) are incomplete and sometimes incorrect. In this paper we identify isomers of nitrofluoranthenes by 'H NMR. We also include here an analogue of the potent mutagen 3-nitrofluoranthene, namely, 1-phenyl-4-nitronaphthalene, that despite structural and reduction potential similarities is not mutagenic (26). In addition, we discuss the mutagenic potencies of 1,2- and 1,3-dinitrofluoranthene. As we have shown, these dinitrofluoranthenes are formed under nitration conditions that involve free radicals, reaction conditions that appear to occur in environmental nitrations (18).
Experimental Section Chemicals. Caution! The nitrofluoranthenes are mutagens. Samples for NMR studies were purified by HPLC using hexane-methylene chloride mixtures on a Varian 5000 liquid chromatograph equipped with a 25-cm silica gel column (IBM), with the exception of l-phenyl-4-nitronaphthalene, which was purified by column chromatography on silica gel using hexanemethylene chloride mixtures. GC and GC/MS analyses were conducted on a Varian 3700 gas chromatograph provided with a 55 ml0.25 mm i.d. DB-17 J&W capillary column and a flame ionization detector and on a Hewlett-Packard 5890 GC instrument equipped with an HP 5970 mass-selective detector using a 20 ml0.18 mm i.d. DB-17 J&W capillary column. GC analyses indicate a minimum purity of 99.5% for the nitro-PAH studied here. 1-Phenyl-4-nitronaphthalene was synthesized by adding 6 mL of a N02/N204(Matheson) solution (0.14 M as NOz in CClJ to 2 mL of a 1-phenylnaphthalene (Aldrich) solution (0.16 M in CC4) a t 25 OC. After 24 h, the solvent was thoroughly purged with argon and removed in a rotary evaporator, giving >90% yield Approximate value calculated from the 1986 data for discharge of particulate organic matter data from diesel vehicles (39)and the typical PAH content of diesel automobile particulate organic matter (40).
0 1990 American Chemical Society
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Chem. Res. Toxicol., Vol. 3, No. 3, 1990
of 1-phenyl-4-nitronaphthalene(by GC). 'H NMR Experiments. The NMR experiments were conducted on a Bruker AM 400 spectrometer operating a t 400.13 MHz. 'H-lH COSY Experiments. The 2D 'H-lH COSY spectra were conducted a t 25 "C by using the 90°-D2-tl-450-FID pulse sequence, employing the COSYLR.AUR automation program provided with the Bruker software. The COSY experiments were optimized for relatively small couplings (4 Hz) with an average digital resolution of 2-6 Hz prior to zero filling. Zero filling (one) was done in the F2 dimension, the data were 2D transformed, and the magnitude spectra were multiplied by a sine window in each dimension and symmetrized along the diagonal. The samples were 1C-15 mM in the nitro-PAH in CDC13as solvent. The COSY spectrum of 2-nitrofluoranthene is discussed below, and COSY spectra of the other compounds studied here are included in the supplementary material. lH-'H Nuclear Overhauser Effect Experiments. All the nuclear Overhauser effect (NOE)experiments were evaluated as the difference of an irradiation and a control experiment. The difference spectra were obtained by subtracting the free induction decay (FID) signals of the irradiation and control experiments. The difference obtained this way was Fourier-transformed with small line broadening (equal to or lower than the digital resolution employed) to improve the inherent low signal-to-noise ratio of these experiments. The Fourier-transformed difference spectra were then phased. The NOE observed on 2- and &nitrofluoranthene and on 1,2-dinitrofluoranthene are included in the supplementary material. Ames Mutagenic Assay. Salmonella typhimurium TA98, kindly donated by Dr. Bruce Ames, was incubated in Oxoid broth for 12 h at 37 "C. Frozen permanent samples of this culture were then made and utilized for all experiments. On each day on which an experiment was to be performed, crystal violet sensitivity (rfa mutation) and the presence of the pKM 101 plasmid were evaluated by streaking on ampicillin plates (27). The plate incorporation assay as described by Maron and Ames (27) was used in evaluating the mutagenic potential of the individual nitrofluoranthenes. A bacterial suspension was prepared by inoculating 0.1 mL from the frozen culture into 25 mL of Oxoid broth, which was then incubated a t 37 "C for 14 h. A 0.1-mL aliquot of the bacterial suspension was placed in a test tube containing 0.5 mL of phosphate buffer, p H 7.4, and 2 mL of top agar supplemented with a 0.5 mM histidine/biotin solution. The substrates were added in 0.1 mL of dimethyl sulfoxide. Doseresponse curves were constructed by using 1ng-10 pg of substrate, depending on its potency and toxicity. In experiments using S9, a 9OOOg supernatant from the liver of Aroclor-pretreated rats fortified with 0.1 M NADP, 1M glucose 6-phosphate, and 33 mM KCl/MgCl, in 200 mM phosphate buffer, p H 7.4, replaced the phosphate buffer. Protein concentration of the S9 fraction was maintained a t 0.5 mg/plate throughout the assay. The contents of the tubes were well mixed and then poured onto Vogel-Bonner plates, allowed to dry, inverted, and incubated for 72 h a t 37 "C. The number of colonies were enumerated by using a colony counter. The potency (revertants/nmol) of each substrate was determined from the linear part of the dose-response curve by using the Ames-fit program as described by Felton and Moore (28). A 0.1-mL aliquot of DMSO was used as the negative control while 5 pg of 2-nitrofluorene was added for the positive control without S9 and 10 pg of 2-aminofluorene was added with S9. A compound was considered mutagenic if either the number of revertants that were recorded was twice the background value or an increase in response with increasing doses was obtained. X-ray Crystallography. X-ray data for l-phenyl-4-nitronaphthalene were collected by using a crystal of dimensions 0.44 X 0.48 X 0.76 mm on an Enraf-Nonius CAD4 diffractometer equipped with Cu K a radiation ( A = 1.54184 A) and a graphite monochromator. Crystal data are as follows: C16HllN02,MW = 249.3, monoclinic space group Cc, a = 11.878 (4), b = 15.477 (31, c = 7.194 (2) A, /3 = 114.19 (Z)", V = 1206.4 (12) A3, Z = 4, d, = 1.372 g ~ m - T~ = , 25 "C, and p = 7.0 cm-'. One quadrant of data was collected by w - 29 scans within 4' < 29 < 150". Data reduction included corrections for background, Lorentz, poiarization, and absorption by $ scans, with minimum relative transmission coefficient 98.04%. Of 1245 data, 1233 had I > 3a(n
Squadrito et al. Table I. Coordinates for 1-Phenrl-4-nitronaphthalene X 2 atom B , A2 Y 01 0 0.1141 (1) 0 5.92 (4) 02 0.0608 (2) 0.2195 (1) 0.2119 (3) 6.05 (4) N 0.0817 (1) 0.1229 (2) 4.05 (3) 0.1588 (1) c1 0.3812 (1) 0.04508 (9) 0.2076 (2) 2.69 (2) c2 0.4262 (1) -0.0404 (1) 0.2127 (2) 2.99 (2) c3 0.3536 (1) -0.1113 (1) 0.1931 (2) 3.31 (3) c4 0.2306 (2) -0.1001 (1) 0.1697 (2) 3.51 (3) 0.1822 (1) -0.0198 (1) c5 3.36 (3) 0.1596 (2) C6 0.2541 (1) 0.0550 (1) 0.1730 (2) 2.83 (3) c7 0.2109 (1) 0.1415 (I) 0.1613 (2) 3.17 (3) C8 0.2834 (2) 0.2119 (1) 0.1817 (3) 3.69 (3) c9 0.4073 (2) 0.2003 (1) 0.2150 (3) 3.58 (3) c10 0.4576 (1) 0.1194 (1) 0.2317 (2) 2.97 (3) c11 0.5911 (1) 0.11273 (9) 0.2729 (2) 3.09 (3) c12 0.6741 (2) 0.0668 (1) 0.4386 (3) 3.57 (3) C13 0.7996 (2) 0.0659 (1) 0.4798 (3) 4.26 (4) 0.8419 (2) C14 0.1097 (1) 0.3426 (3) 4.35 (4) 0.7601 (2) 0.1546 (1) C15 0.1867 (3) 4.06 (3) C16 0.6354 (1) 0.1572 (1) 0.1478 (2) 3.49 (3) and were used in the refinement. The structure was solved by direct methods and refined by full-matrix least squares, treating non-hydrogen atoms anisotropically. Hydrogen atoms were located by hF and refined isotropically. At convergence, R = 0.030 for 215 variables, and the maximum residual density was 0.14 e Coordinates for l-phenyl-4-nitronaphthalene are listed in Table I, and an ORTEP drawing of its X-ray crystal structure is shown in Figure 2.
Results The results of the 'H-lH COSY experiments were used to assign the members of each three subspectra present in the nitro- and dinitrofluoranthenes. Connectivities via J couplings are usually broken between H-3 and H-4, H-6 and H-7, and H-1 and H-10, respectively, giving rise to the three subspectra, and the results of the NOE experiments were used to provide the missing connectivities by use of through-space couplings between the pairs of "peri" and "bay" protons. The following assignments were than made possible. Relevant 'H NMR data for all the nitrofluoranthenes studied here and for l-phenyl-4-nitronaphthalene are listed in Tables I1 and 111, respectively. 2-Nitrofluoranthene (2NF). The 'H-lH COSY 45 experiment of 2NF (Figure 1) reveals the presence of a two-spin, a three-spin, and a four-spin subspectrum. The NOE's observed upon saturation of H-1 and H-3 (ca. 4% on H-10 and ca. 5% on H-4, respectively) provide their unequivocal assignment, as revealed by their proximity to the four-spin and three-spin subspectra, respectively. The results of the NOE experiments also provide the assignments of H-4, H-5, H-6, H-7, and H-10. H-8 and H-9 can be assigned as tightly coupled partially superimposing multiplets between 7.42-7.48 and 7.44-7.50 ppm, respectively. The assignment of H-8 and H-9 is based on their strong cross-peaks with their ortho protons on the COSY spectrum.
Mutagenicity. The Ames test was used to assess the mutagenic potency of the nitrofluoranthenes and of 1phenyl-4-nitronaphthalene.S. typhimurium strain TA98 was selected as the tester strain because of its sensitivity to frame-shift mutations that are frequently observed with nitro-PAH. The results, shown in Table IV, indicate that the mutagenic potency of the nitrofluoranthenes varies dramatically with the position of nitro substitution. Of the seven nitrofluoranthenes examined for mutagenicity, 3NF is the most potent, followed by 8NF, lNF, ZNF, and 7NF. In l-phenyl-4-nitronaphthalenethe phenyl group is not coplanar with the rest of the molecule, as is true for 3NF, and this nonplanar analogue is not mutagenic. 1,2-
Chem. Res. Toxicol., Vol. 3, No. 3, 1990 233
IH NMR Studies on Mutagenic Nitrofluoranthenes
Table 11. IH NMR Parameters of Nitrofluoranthenes 2NF" 3NF" 7NF" 8NF" 1,2DNF" 8.64, d 7.91, d 8.08, d 8.11, d 8.58, d 7.72, dd 7.75, dd 8.81, d 7.99, d 8.01, d 8.67, s 7.96. d 8.65. d 8.03, d 7.98, d 7.96, d 7.74; dd 7.78, dd 7.76, dd 7.75, dd 7.83, dd 8.04, d 7.93, d 8.68, d 8.09, d 8.09, d 7.88-7.92, m 7.89, br db 8.76, dd 7.90, d 7.42-7.48, mc 7.43-7.47, md 8.22, dd 7.5e7.54, m 7.39-7.43, md 7.54, dd 8.31, dd 7.41-7.45, m 7.44-7.50, me 7.93-7.97, m 7.87, br db 8.10, dd 8.03, dd 7.83, d
lNPa H1
H2
8.21, d 7.91, d 7.83. d 7.72; dd 7.95, d 7.89, br d 7.46-7.50, m 7.41-7.45, m 8.49, br d
H3 H4 H5 H6 H7 H8 H9 H10
1,3DNFa 9.10, s 8.62, br d 7.86, dd 7.96, br d 7.85, d 7.50-7.54, m 7.42-7.46, m 8.45, br d
"Relevant coupling constants are summarized as follows: (INF) J(?,3) = 9 Hz; J(4,5) = 8 Hz; J(5,6) = 7 Hz; 5(7,8) = 8 Hz; J(9,lO) = 8 Hz; (2NF) 5(1,3) = 2 Hz; 5(4,5) = 8 Hz; J(5,6) = 7 Hz; (3NF) J(1,2) = 8 Hz; J(4,5) = 8 Hz; J(5,6) = 7 Hz; J(7,8) = 7 Hz; J(9,lO) = 7 Hz; (7NF) J(1,2) = 8 Hz; 5(2,3) = 8 Hz; J(4,5) = 8 Hz; J(5,6) = 7.5 Hz;J(8,9) = 7 Hz; J(8,lO) = 1 Hz; J(9,lO) = 8 Hz; (8NF) J(1,2) = 6.5 Hz; J(2,3) = 8 Hz; J(4,5) = 8 Hz; J(5,6) = 7 Hz; J(7,9) = 2 Hz; J(7,lO) = 0.3 Hz;J(9,lO) = 8 Hz; (1,PDNF) J(4,5) = 9 Hz; J(5,6) = 7 Hz; J(7,8) = 8 Hz; J(9,lO) = 8 Hz; (1,3DNF) J(4,5) = 9 Hz; J(5,6) = 7 Hz; J(7,8) = 8 Hz;J(9,lO) = 8 Hz. bInterchangeable pair. 'H8 and H9 are partially superimposing in 2NF. Interchangeable pair.
Q?
Table IV. Mutagenic Activity of Different Nitrofluoranthenes and of l-Phenyl-4-nitronaphthalene with S. typhimurium TA98" TA98, TA98 + S9, revertants/ revertants/ compound nmol nmol l-nitrofluoranthene 370 f 24 120 f 14 2-nitrofluoranthene 61 f 15 29 f 6 5982 f 215 3-nitrofluoranthene 590 f 20 4 f l 24 f 1 7-nitrofluoranthene 8-nitrofluoranthene 2223 f 225 462 f 41 1,2-dinitrofluoranthene 1042 f 74 177 f 11 1,3-dinitrofluoranthene 2601 f 80 473 f 34 l-phenyl-4-nitronaphthalene 0.7 f 0.1 0.4 f 0.1 "The specific activity expressed as mean f SD of revertants/ nmol was obtained from the linear region of the dose-response curve. Each value used to construct the dose-response curve is based on at least two experiments using two plates/dose. TA98 was exposed to four nontoxic concentrations of each substrate.
I
I
I
~
0
I
0
1
'
8.8
1
8.6
'
1
'
8.4
1
8.2
'
PPM
1
8.0
'
1
'
7.8
1
7.6
'
1
7.4
Figure 1. 'H-'H COSY 45 experiment of 2-nitrofluoranthene. Table 111. 'HNMR Parameters of l-Phenyl-4-nitronaphthalene proton 6, ppm J , Hz 1 2 3
7.50, d 8.26, d
4 5 6 7 8 2'-6'
8.62, br d 7.73, ddd 7.56, ddd 7.97, br d 7.43-7.56
J(2,3) = 8 J(5,6) = 9 J(6,7) = 8; J(6,8) = 1 J(5,7) = 1 J(7,8) = 8.5
and 1,3-dinitrofluoranthene are potent mutagens, with 1,3DNF being more active than 1,BDNF. In comparison with the mononitrofluoranthenes, the dinitrofluoranthenes have intermediate activity, similar to that of 8NF. In the presence of a microsomal enzyme preparation, deactivation of all nitrofluoranthenes is observed (Table IV). The presence of S9 lowers the response of two nitrofluoranthenes (3NF and 8NF) and two dinitrofluoran-
thenes (1,BDNF and 1,3DNF) to 10-209'0 of the response in TA98 without the microsomal enzyme preparation.
Discussion 'H NMR Analysis, Mutagenic Potencies, and the Importance of Purity and Correct Identification. Paputa-Peck et al. reported some 'H NMR parameters for the five mononitrofluoranthenes (5),Zielinska et al. reported those for 2-nitrofluoranthene (10,23,25)and 1,2dinitrofluoranthene (23,251,and Svendsen et al. studied the 'H NMR spectra of 1-,3-, 7-, and 8-nitrofluoranthene in some detail (24). However, the assignments of the 'H NMR resonances are incomplete, with noticeable disagreements among the authors. In fact, the parameters reported by Paputa-Peck et al. and Svendsen et al. for the nitrofluoranthenes are so dissimilar as to suggest that one of them may have inadvertedly examined derivatized nitrofluoranthenes. In this regard, we have observed that the nitrofluoranthenes are thermally stable but their bright yellow solutions darken after a few days' exposure to laboratory fluorescent light. Aromatic compounds may associate and changes in the chemical shifts may occur (29);however, with the nitrofluoranthenes we noticed only small chemical shift changes (0.01-0.02 ppm) over a 10-fold range of concentrations centered around 10-15 mM. Our NMR parameters for the mononitrofluoranthenes are consistent with those reported by Paputa-Peck et al.; however, some of the assignments must be revised. Thus, H-1 and H-3 were reversed for 2NF by both Paputa-Peck et al. and Zielinska et al. For the 'H NMR spectrum of l,PDNF, our results suggest that the resonances for the pairs H-7, H-10 and H-8, H-9 had been misassigned (23)
234 Chem. Res. Toxicol., Vol. 3, No. 3, 1990
Squadrito et al.
Q NO2
3-Nitrofluoronthene( 3 N F )
Figure 2. ORTEP drawing of the X-ray crystal structure of 1phenyl-4-nitronaphthalene.
and are in agreement with a recent report by Zielinska et al. with revised assignments (25). The difficulties were perhaps due to the near-perpendicular orientation of the C-1 nitro group [revealed by our single-crystal X-ray study (19)]that results in a shielding effect (30-32) on H-10. The anisotropic effects of nitro groups on the 'H NMR spectra of the mononitrofluoranthenes, of 1,3-DNF, and of 1phenyl-4-nitronaphthalene (1P4NN) indicate that they are coplanar or near planar. The position of the nitro group in 1-phenyl-4-nitronaphthalene determined by 'H NMR was confirmed by a single-crystal X-ray analysis that also provides the spatial orientation of the phenyl and nitro substituents, as shown in Figure 2. Mutagenicity. The mutagenic potency of 3NF and 8NF is higher than has been reported for other mononitro-PAH, 1NF and 2NF have intermediate potency, and the mutagenicity of 7NF is negligible. The relative potencies of the mononitrofluoranthenes reported here differ from those in the literature (8,12,25,33),where it has been reported that 8NF is more potent than 3NF. There is an order of magnitude difference in response in the mutagenicity studies of 3NF (8, 12, 25, 33-35), and the disagreement may be due to the variable purity of the compound used by different investigators.2 Structure-Activity Relationships. The mutagenic potencies of many of the nitro-PAH can be correlated with their reduction potentials, leading Klopman et al. to suggest that the ultimate mutagenic species are formed in a rate-determining step that involves the reduction of the nitro group (36). However, our data show that the structurally similar compounds 3NF and l-phenyl-4nitronaphthalene (1P4NN) possess identical reduction potentials (26),but 3NF is a potent mutagen and 1P4NN
NO2
1-Phenyl-4-nitronophthalene (lP4NN)
completely lacks mutagenic activity. In 1P4NN, the benzene ring is not coplanar with the naphthalene system, as can be observed in the ORTEP drawing of its X-ray crystal structure in Figure 2. Thus, our data suggest that the reduction potential correlates well with the mutagenic potencies of nitro-PAH only if the compounds have similar steric requirements. Our results indicate that 1,2- and 1,3-dinitrofluoranthene are about 2 orders of magnitude less potent mutagens than 3,7DNF and 3,9DNF (37), suggesting that steric interactions present when disubstitution occurs in the same ring may reduce mutagenic potency. A similar trend was observed in the dinitropyrene series ( 1 I), where 1,3-dinitropyrene is less mutagenic than is either 1,6- or 1,8-dinitropyrene. The effects of forcing the nitro group into a position nearly perpendicular to the plane of the hydrocarbon skeleton, as occurs in 9-nitroanthracene and other nitro-PAH with nitro groups flanked by two peri protons, have been examined by Fu and his collaborators (38). The fact that 1,2-dinitrofluoranthene is about 50% less mutagenic than 1,3-dinitrofluoranthene may be due to the tilting of the C-1 nitro group of 1,BDNF out of planarity with the ring in 1,2DNF because of steric hindrance, as demonstrated by its X-ray crystal structure (19). Conclusion. The identification of standards for chromatographic analysis of nitrofluoranthenes can be conveniently accomplished by 'H NMR. Regarding structure-biological activity correlations for nitro-PAH, it had been suggested that reduction potentials correlate mutagenicity. However, our data show that striking exceptions to this rule occur if compounds differ in their geometry and/or steric requirements. A clear example of this is the very different mutagenicities of 3NF and 1P4NN despite their similar reduction potentials. Also, all the nitrofluoranthene isomers have similar reduction potentials (26) but very different mutagenic activities. The steric hindrance between the vicinal nitro groups of 1,2DNF turns the nitro group attached to C-1 into a near-perpendicular position and results in reduced mutagenic potency when compared to 1,3DNF. We suggest that binding to the nitroreductase may become rate determining for nonplanar or sterically hindered nitro-PAH. Reduction potentials may be of use in predicting the mutagenic potency of nitro-PAH only in limited series of compounds with similar geometries, steric requirements, and electron densities.
Acknowledgment. This work was supported by a MERIT Award from NIHBL, Grant R37-HL16029, to W.A.P. and by a contract from The National Foundation for Cancer Research.
~~
The importance of confirming the identity and purity of the compounds is illustrated by the studies by Svendsen et al. (24) and Greibrokk et al. (12),who found that 8NF was more mutagenic than 3NF. However, the discrepancies between their reported 8NF 'H NMR spectrum and those reported by Paputa-Peck et al. (5) and by us in this study indicate that Svendsen et al. may have inadvertantly derivatized their sample of 8NF. Furthermore, their synthetic methodology using nitric acid in acetic anhydride can result in the formation of 3,7DNF and 3,9DNF, which may have contaminated the late-eluting 8NF by normal-phase HPLC), resulting in spuriously high mutagenic potency. (Both 3,7DNF and 3,9DNF are extremely potent mutagens that were synthesized under ionic electrophilic nitration conditions (37). The mutagenic potencies of 3,7DNF and 3,9DNF are similar to those of 1,6- and l&dinitropyrene, the most active bacterial mutagens known (33).]
Registry No. 1-Nitrofluoroanthene, 13177-28-1; 2-nitrofluoranthene, 13177-29-2; 3-nitrofluoranthene, 892-21-7; 7-nitrofluoranthene, 13177-31-6; 8-nitrofluoranthene, 13177-32-7; 1,2dinitrofluoranthene, 33611-88-0; 1,3-ditritrofluoranthene, 110419-21-1; l-phenyl-4-nitronaphthalene, 33457-01-1. Supplementary Material Available: Discussion of NMR experiments, six tables giving atomic coordinates for hydrogen atoms, bond distances involving non-hydrogen atoms, bond distances involving hydrogen atoms, bond angles involving nonhydrogen atoms, bond angles involving hydrogen atoms, and anisotropic thermal parameters for l-phenyl-4-nitronaphthalene,
IH NMR Studies on Mutagenic Nitrofluoranthenes respectively, seven figures showing COSY spectra of 1-, 3-, 7-, and fbnitrofluoranthene, 1,2- and 1,3-dinitrofluoranthene,and 1phenyl-4-nitronaphthalene,and three figures containing spectra showing the nuclear Overhauser effects observed on 2- and 8nitrofluoranthene and 1,2-dinitrofluoranthene (22 pages). Ordering information is given on any current masthead page.
References (1) Singh, H. (1987) Reactive nitrogen in the troposphere. Enuiron.
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