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1,8-DNP6
Mutation Research, 206 (1988) 131-140 Elsevier
131
MTR 01307
Mutagenic activities of selected nitrofluoranthene derivatives in Salmonella typhimurium strains TA98, T A 9 8 N R and T A 9 8 / 1 , 8 - D N P 6 Barbara Zielinska 1, Janet Arey 1, William P. Harger 1 and Robert W.K. Lee 2 Statewide Air Pollution Research Center, University of California, Riverside, CA 92521, and 2 Chemistry Department, University of California, Riverside, CA 92521 (U.S.A.) (Received 10 August 1987) (Revision received 8 February 1988) (Accepted 15 February 1988)
Keywords: Nitrofluoranthenes; Dinitrofluoranthenes; Trinitrofluoranthenes; NMR; Mutagenicity; Salmonella typhimurium, TA98, TA98NR, TA98/1,8-DNP6
Summary The mutagenic activities of novel nitrofluoranthene derivatives in Salmonella strains TA98, TA98NR and TA98/1,8-DNP 6 (with and without $9 addition) are given. These derivatives were produced from the reactions of fluoranthene (FL) and its directly mutagenic 2- and 3-nitro derivatives with covalent dinitrogen pentoxide (N205) in CC14 solution at ambient temperature. The influence of the addition of a nitro group on the observed activity of the resulting di- and tri-nitrofluoranthenes is discussed.
2-Nitrofluoranthene (2-NF) has recently been identified as a major nitrated polycyclic aromatic hydrocarbon (nitro-PAH) in ambient particulate organic matter (POM) collected at various locations in the U.S.A. and Europe (Pitts et al., 1985; Arey et al., 1986, 1987; Sweetman et al., 1986; Ramdahl et al., 1986; Nielsen and Ramdahl, 1986). This particular nitro-PAH has been observed to account for up to 5% of the overall directly acting mutagenicity of ambient POM (Ramdahl et al., 1988a; Arey et al., 1988) in the Salmonella test. One possible formation pathway for the 2-NF has been identified as the gas-phase reaction of fluoranthene (FL) with dinitrogen pentoxide (N205) (Sweetman et al., 1986; Zielinska et al., 1986), a
Correspondence: Dr. B. Zielinska, Statewide Air Pollution Research Center, University of California, Riverside, CA 92521 (U.S.A.).
species expected to the present in the ambient nighttime atmosphere (Atkinson et al., 1986). We have studied the mechanism of the reaction of FL with N205 in CC14 solution (Zielinska et al., 1986), where, as in the gas phase, N 2 0 5 exists in the covalent state (Schofield, 1980). At ambient temperature the solution-phase reaction produced 2-NF as the sole mononitrofluoranthene isomer, together with 1,2-dinitrofluoranthene (1,2-DNF) and several oxygenated nitro products. To explain the observed solution-phase products, a radical mechanism involving molecule-assisted homolysis has been postulated for this reaction of FL with N 2 0 5 (Zielinska et al., 1986). The reaction of FL and its 2- and 3-nitro isomers with covalent N20 s in CC14 solution produces nitro derivatives distinct from those formed by electrophilic nitration reactions (Zielinska et al., 1986). We report here the mutagenic activities of these novel nitrofluoranthene derivatives in
0165-1218/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
132
Salmonella typhimurium strain TA98 and its nitro reductase- and transacetylase-deficient strains TA98NR and TA98/1,8-DNP6, respectively, with and without $9 microsomal activation.
Experimental ~H N M R spectra were recorded with a Nicolet 300 pulsed Fourier transform N M R spectrometer with an NIC 1280 computer. Typically 200-300 /~g of sample were dissolved in 0.5 ml CDC13 or CD3OD. The proton resonance shifts have been expressed in ppm on the 8 scale. Mass spectra were recorded by using a Finnigan 3200 gas chromatograph/mass spectrometer ( G C / M S ) operated in the electron impact (EI) mode. A Spectra-Physics Model 8100 high-performance liquid chromatograph (HPLC) equipped with a 250 mm x 10 mm Ultrasphere ODS or Si column was employed for compound purifications.
Syntheses The reactions of FL, 2-NF and 3-NF with N205 in C C l 4 solution at ambient temperature and the isolation of products from these reactions were carried out as described previously (Zielinska et al., 1986). The products produced in low abundance were additionally purified by semi-preparative HPLC, using an Altex Ultrasphere Si column eluted with n-hexane/CHzC12 or an Altex Ultrasphere ODS column eluted with C H 3 O H / H 2 0 (for 2-hydroxy-l-nitrofluoranthene, 2,1-HNF). 2,3-Dinitrofluoranthene (2,3-DNF) was synthesized using methods described in the literature (Kloetzel et al., 1956; Hodgson et al., 1947). 3Aminofluoranthene (Aldrich Chemical Co.) was converted to 3-amino-2-nitrofluoranthene (3amino-2-NF) according to the method described by Kloetzel et al. (1956) with an overall yield of 85%. 3-Amino-2-NF was reacted with cupro-cupri sulphite, as described by Hodgson et al. (1947) for the synthesis of dinitronaphthalenes. However, the main product from this reaction was 2-NF, together with lesser amounts of 2,3-DNF ( - 10% of the 2-NF yield). The 2,3-DNF was separated from the 2-NF by chromatography on silica-gel 60 (Merck), using CC14/CHzC12 elution (9:1 v/v) and was then recrystallized from ethanol, m.p. 227-228°C. MS (main ions): m / z (rel. int.) 292
([M] +, 100%), 204 ([M-2NO-CO] +, 43%), 200 ([M-2NO21 +, 95%). The 3- and 8-nitrofluoranthenes were synthesized according to the method of Radner (1983), using NzO 4 in CH2C12 with methanesulfonic acid as a catalyst. The isomers were separated by semi-preparative HPLC, employing an Ultrasphere Si column eluted with n-hexane/CH2C12. The nitrofluoranthene derivatives investigated were > 99% pure, as established by GC a n d / o r HPLC analyses with the exception of 2,5-DNF, 2,3,5-TNF and 2,1-HNF which were > 95% pure.
Mutagenicity assays The Salmonella plate-incorporation mutagenicity tests were performed according to the standard procedure (Ames et al., 1975) with modifications to improve precision (Belser et al., 1981). The Salmonella strains used were TA98, which is sensitive to nitroarene mutagenicity, and strains TA98NR and TA98/1,8-DNP 6, which are deficient in nitroreductase (Rosenkranz and Speck, 1975; McCoy et al., 1981) and transace~clase enzymes (McCoy et al., 1983; Orr et al., 1985; Saito et al., 1985), respectively. Cultures of these strains were grown for 12 h in LB broth, diluted to approximately 1 x 10 9 cells/ml, and maintained at ice temperature until testing. All samples, including positive controls, were dissolved in dimethyl sulfoxide (DMSO) and tested in triplicate at 8 doses. Mutagenic activity, as reported here, was the slope of the linear region of the dose-response curve and was determined by a least-squares linear regression analysis. Positive control mutagens were quercetin, 2-nitrofluorene, 1,8-dinitropyrene and benzo[a]pyrene (with rat liver metabolic activation). A single test was conducted for all compounds with metabolic activation. The $9 mix (2% v/v) was prepared from Aroclor 1254-induced rat liver $9 (Litton Bionetics, Inc.) containing 25 m g / m l protein. Benzo[a] pyrene was utilized as the control mutagen and gave the following values (rev./nmole _+ S.E.) in strains TA98 (+$9), TA98NR ( + $ 9 ) and TA98/1,8-DNP 6 ( + $9), respectively: 92 _+ 3, 106 _+ 4, 92 _+ 4. Spontaneous reversions for this test as determined from 6 DMSO blanks per strain were (rev./plate + S.D.): TA98 (71 + 7), TA98NR (49 _+ 7), TA98/1,8-DNP 6 (47 + 7).
133 The majority o f the compounds were tested without $9 in a single experiment; those compounds tested in a separate second test were 3-NF, 8-NF and 1,2-DNF. The values for the control mutagens in these two tests were, for 2-nitrofluorene (rev./nmole + S.E.): TA98 (104 ___3, 91 + 5); TA98NR (16 + 1, 12 + 0.7); TA98/1,8-DNP 6 (20 + 1, 16 + 1), for quercetin (rev./nmole + S.E.): TA98 (5.3 ___0.3, 4.8 + 0.1), TA98NR (5.7 ___0.2, 5.2 + 0.2), TA98/1,8-DNP 6 (5.0 + 0.2, 5.0 + 0.2), and for 1,8-dinitropyrene ( r e v . / n m o l e + S.E.): TA98 (3.0 X 105 + 1 X 104, 2.7 X 105 + 8 X 103), TA98NR ( 3 . 2 X 1 0 5 + 1 X 1 0 4 , 2 . 7 x 1 0 5 + 9 X 103), TA98/1,8-DNP 6 (9900 + 600, 6700 +_ 100). Spontaneous reversions for these two tests were (rev./plate + S.D. of 6 replicates): TA98 (50 + 4, 3 9 + 4 ) , TA98NR ( 4 0 + 4 , 30_+3) and T A 9 8 / 1,8-DNP 6 (43 + 4, 46 _+ 5).
Results
The structures of FL, 2- and 3-NF and the products identified from their reaction with N205 in CC14 solution at ambient temperature, are shown in Fig. 1. Note that FL has been drawn to illustrate its axis of symmetry and that IUPAC rules dictate that the substituents are numbered beginning with the lowest possible number. Thus, for example, the nitration of 3-NF in the 5-position produces 2,4-DNF. As noted above, 2-NF and 1,2-DNF were the major products from the reaction of FL with N205 in CC14 at ambient temperature (Zielinska et al., 1986). In addition, this reaction yielded lower amounts of 2,3-DNF ( - 5% of the 1,2-DNF yield), and oxygenated nitro products including 2-hydroxy-l-nitrofluoranthene (2,1-HNF) and 10bnitrato-l-nitro-l,2,3,10b-tetrahydrofluoranthene2,3-oxide (NNF-oxide). To prove its structure, 2,3-DNF was also synthesized by an independent method (see Experimental). All products were identified on the basis of their MS and 1H N M R data (Zielinska et al., 1986, 1987a). The reactions of 2-NF and 3-NF with N205 in CC14 at 25 °C produced nitro products analogous to those formed from FL (Zielinska et al., 1986). Thus, 2-NF yielded 2,5-DNF and 1,2,5-TNF with
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a lower yield of 2,3,5-TNF while 3-NF formed 2,4-DNF and 1,2,4-TNF (2,3,4-TNF was not formed in detectable amounts, possibly due to steric considerations). The D N F and TNF products from these reactions were identified using MS and 1H N M R data (Zielinska et al., 1986, 1987a; Ramdahl et al., 1988b). These N205 nitration products are distinct from those produced by electrophilic nitrations. Thus, electrophilic nitration of FL has been shown to produce 3-, 8-, 7- and 1-NF (in order of decreasing yield), while dinitration of FL and nitration of 3-NF produce mainly 3,9- and 3,7-DNF with lower amounts of 3,8- and 3,4-DNF (Ruehle et al., 1985; Nakagawa et al., 1987). In contrast, the reaction of FL with N205 gives substitution in the 2-position and further nitration of 2- and 3-NF does not produce nitration in the C ring (see Fig. 1), but rather in the B ring. Furthermore, dinitration of FL and 2- and 3-NF with N205 gives unique ortho-substituted D N F and TNF isomers, respectively.
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135 TABLE 2 MUTAGENIC ACTIVITIES (rev./nmole) a OF NITROFLUORANTHENE DERIVATIVES IN Salmonella typhimurium Compound
TA98
TA98NR
TA98/1,8-DNP 6
-$9
+$9 b
-$9
+$9 b
-$9
1,2-Dinitrofluoranthene 2,3-Dinitrofluoranthene 2,4-Dinitrofluoranthene 2,5-Dinitrofluoranthene
1290+_ 40 420_+ 20 6 000 +_200 211+_ 7
190+_10 80+_10 550 _+50 540+_30
570+_ 20 180+_ 10 5 700 +_300 105+_ 7
97_+ 3 80+_20 360 +_30 280+_10
1010+_40 369_+ 7 2 000 +_100 67___ 2
1,2,4-Trinitrofluoranthene 1,2,5-Trinitrofluoranthene 2,3,5-Trinitrofluoranthene
3600+-500 1440+ 80 2750+- 90
136+ 9 69+- 3 720+_30
2600+200 770+ 40 1100+100
90+-10 24+- 3 404+_ 9
1350_+60 560+_ 30 56_+ 4
52 +_5 22 +_2 6.4 +_ 0.9
71+
7
53+- 3
34+_ 2
8.5 +_ 0.5
2-Hydroxy-l-nitrofluoranthene
180+ 20
110+- 8
450 +_ 50
63 +_ 3
330 +_ 20
63 + 3
220 _+ 20
1030+_ 20 7 700 +_600 18 200 +-500 500 47
760+_20 ---
250+_ 20 3 800 +_200 6 000 +_300 230 18
230+_20 --
180_+ 9 1020 + 20 5 400 +_300 ---
+$9 b 180 33 180 92
_+20 _+2 _+30 +_6
lOb-Nitrato-l-nitro-l,2,3-1Ob-tetrahydrofluoranthene-2,3-oxide 2-Nitrofluoranthene ~ 3-Nitrofluoranthene 8-Nitrofluoranthene 1-Nitrofluoranthene d 7-Nitrofluoranthene d
35 +_3 < 20
a Slope of a least-squares regression of dose-response curve+ S.E. of slope. b 2% v/v mix was used. From Zielinska et al. (1987b). d From Vance and Levin (1984).
T h e 1H N M R data for the n i t r o f l u o r a n t h e n e derivatives shown in Fig. 1 are given i n T a b l e 1 *. T h e p r o t o n chemical shifts were assigned o n the basis of h o m o n u c l e a r decoupling experiments and, where possible, n u c l e a r Overhauser e n h a n c e m e n t ( N O E ) experiments (Zielinska et al., 1987a). Analysis of the 1H N M R data s u m m a r i z e d i n T a b l e 1 reveals steric interactions between the adjacent NO2 groups i n 1,2- a n d 2 , 3 - D N F a n d 1,2,4-, 1,2,5- a n d 2,3,5-TNF. Thus, in the s p e c t r u m of 2 , 3 - D N F , the chemical shift of the p r o t o n peri to the 3-NO 2 group (H-4) is upfield of that of H-6 (A = 0.22 ppm). This is in contrast to the spect r u m of 3-NF, in which H-4 is shifted - 0.76 p p m downfield relative to H-6. Thus, although a p r o t o n peri to a p l a n a r N O 2 group (e.g., H-4 in 3 - N F ) will be shifted downfield relative to its shift i n the
* Although some of these data were reported previously (Zielinska et al., 1986) not all chemical shifts were unequivocally assigned. In addition, since only one of these TNF isomers was available at that time, the 1H NMR spectrum of 2,3,5-TNF was incorrectly interpreted as that of 1,2,5-TNF.
p a r e n t P A H , a p r o t o n peri to a n o n p l a n a r N O 2 group will be shifted upfield (for a discussion of the m a g n e t i c a n i s o t r o p i c effect of the N O 2 group, see Miller et al., 1985). C o m p a r i s o n of the 1H N M R spectrum of 2,3,5T N F with that of 2 , 4 - D N F reveals a n analogous steric effect for 2,3,5-TNF. The 2 , 4 - D N F spect r u m shows n o evidence for steric i n t e r a c t i o n between the n i t r o group in the C-4 position with the peri h y d r o g e n (H-3). T h e signal c o r r e s p o n d i n g to H-3 is shifted - 0 . 9 p p m downfield i n the spect r u m of 2 , 4 - D N F w h e n c o m p a r e d with the peri h y d r o g e n in the 3 - N F spectrum, thus showing the influence of b o t h the m a g n e t i c a n i s o t r o p y effect of the N O 2 group in p o s i t i o n C-4 a n d the inductive effect of the N O 2 in p o s i t i o n C-2 o n the chemical shift of H-3. I n the s p e c t r u m of 2 , 3 , 5 - T N F the peri p r o t o n (H-4) is shifted - 0 . 9 p p m further upfield t h a n the peri p r o t o n (H-3) in the spectrum of 2 , 4 - D N F , thus i n d i c a t i n g that the presence of a n a d j a c e n t n i t r o group forces the N O 2 group in the 3-position of 2 , 3 , 5 - T N F out of the p l a n e of the a r o m a t i c rings. Conversely, the nearly identical
136
chemical shifts of the peri protons (H-3) in the 2,4-DNF and 1,2,4-TNF spectra indicate that the NO 2 group in the 4-position of 1,2,4-TNF is in the plane of the aromatic rings. A comparison of 1H N M R data for 1-NF (Svendsen et al., 1983) and 1,2-DNF (Table 1) indicates that the NO 2 group in the 1-position of 1,2-DNF adopts a conformation more out-of-plane of the aromatic rings than in 1-NF since the H-10 proton in the bay region is shifted - 0 . 5 ppm upfield in the spectrum of 1,2-DNF relative to the spectrum of 1-NF. The essentially identical chemical shifts for these bay region protons (H-10) in the spectra of 1,2-DNF, 1,2,4-TNF and 1,2,5-TNF indicate that the NO 2 groups in the 1-position of both TNF are also twisted out-of-plane owing to crowding by the NO 2 groups in the 2-position. Table 2 shows the mutagenic activities of the N F derivatives toward Salmonella strains TA98, TA98NR and TA98/1,8-DNP 6 tested with ( + $9) and without ( - $ 9 ) metabolic activation. In the absence of $9, as seen from Table 2, all of the NF derivatives tested were mutagenic in TA98 and all but 2,4-DNF showed significantly reduced activity in the nitroreductase-deficient strain TA98NR in comparison with that in TA98. The largest reduction in activity in strain TA98NR (76%) was observed for 2-nitrofluoranthene. In strain TA98/1,8-DNP 6 which is deficient in transacetylase activity, all of the nitro derivatives with the exception of 1,2-DNF and 2,3-DNF exhibited mutagenic responses less than half of those in the transacetylase competent strain, TA98. The addition of 2% (v/v) of $9 generally resuited in highly diminished activity. The exceptions were the response of 2,5-DNF in all 3 strains and the response of 2-NF in strain TA98NR. Discussion
It is generally accepted that the direct mutagenic activity (i.e. without $9 addition) of nitroarenes in the Salmonella reversion assay is a consequence of enzymatic reduction of the nitro group to an hydroxylamine, possibly occurring through a nitroso intermediate (Rosenkranz and Mermelstein, 1983, and refs. therein; Beland et al., 1985, and refs. therein; Heflich et al., 1985a). This is consistent with the reduced activity observed for
many nitroarenes in the nitroreductase-deficient strain TA98NR (Rosenkranz and Mermelstein, 1983; Beland et al., 1985). The hydroxylamine is postulated to be a proximate mutagen which requires further activation to form an arylnitrenium ion, the reactive electrophile which serves as the ultimate mutagen (Vance and Levin, 1984). For some nitroarenes formation of the arylnitrenium ion occurs through enzymatic O-acetylation of the hydroxylamine as evidenced by the significantly reduced mutagenic response of these nitroarenes in strain TA98/1,8-DNP 6 (which is deficient in acetyltransferase activity) compared to strain TA98 (McCoy et al., 1982, 1983; Saito et al., 1985, 1986; Orr et al., 1985). In Salmonella, binding of arylnitrenium ions to DNA has been shown to occur at the C-8 of guanine for 1-nitropyrene and 1,8-dinitropyrene (Howard et al., 1983; Heflich et al., 1985b). It has been suggested (Rosenkranz et al., 1985) that, for steric reasons, all nitroarenes having more than 2 rings will form DNA adducts with the C-8 position of guanine. It was first demonstrated by Klopman and co-workers (1984) that the direct mutagenicity of certain nitroarenes, and in particular the nitropyrenes, could be correlated with the ease of their nitro reduction as measured by their halfwave potentials or estimated from their calculated energies of the lowest unoccupied molecular orbital (LUMO). Subsequent studies (Vance and Levin, 1984; Fu et al., 1985) showed that nitroarenes with the nitro substituent sterically forced out of the plane of the aromatic ring exhibited only little or no direct-acting bacterial mutagenicity and that for these nitroarenes the inverse correlation of the L U M O energy (ELuMO) with activity did not hold (Fu et al., 1985). However, ELUMO calculations which take the nitro group orientation into account may correlate better with mutagenic activity (Maynard et al., 1986). The direct-acting mutagenicities in TA98 of 4 of the 5 nitro isomers of fluoranthene (1-, 3-, 7and 8-) have been discussed previously (Vance and Levin, 1984; Maynard et al., 1986), while the activity of the remaining isomer, 2-nitrofluoranthene, has only recently been reported (Zielinska et al., 1987b). The mutagenic activities of the nitrofluoranthene isomers (see Table 2) decrease in the following order: 8-NF > 3-NF > 2-NF > 1-
137 N F > 7-NF. Although the 1H N M R data suggest that the 1-, 3-, and 7-isomers are planar (no upfield shift is observed for the peri or bay region hydrogens relative to fluoranthene) (Vance and Levin, 1984), recent calculations suggest that the nitro group of the 1- and 7-NF isomers will interact with the hydrogen in the bay region (Maynard et al., 1986). Taking the predicted rotation of the nitro groups in 1- and 7-NF into account, Maynard and co-workers (1986) calculated the ELUMO energies of the N F isomers and found them to decrease in the order 7-NF > 1-NF > 8-NF >/3NF, thus predicting reasonably well the trend in their observed mutagenicities, although 8-NF is more mutagenic than 3-NF by at least a factor of 2 (see Table 2). The addition of a strongly electron-withdrawing group (such as a nitro group) to a nitrofluoranthene molecule should presumably lower the redox potential of the NO2 group (Bock and Lechner-Knoblauch, 1985), and would be expected to increase the nitroarene's mutagenic potency (Klopman et al., 1984). Indeed, the mutagenic activities of 3,7- and 3,9-DNF have been reported to be - 2 0 times higher than that of 3-NF (Nakagawa et al., 1987) and 1,3-, 1,6-, and 1,8-dinitropyrene are among the most powerful bacterial mutagens tested to date, being from 300- to 500-fold more mutagenic than 1-nitropyrene in the Salmonella test (Rosenkranz and Mermelstein, 1983). However, none of the mutagenic activities of the D N F and T N F isomers listed in Table 2 approaches those of 3,7- or 3,9-DNF. The D N F tested can be viewed as having a second NO 2 group added to a parent mononitrofluoranthene isomer as follows: to 1-NF (i.e., 1,2DNF), to 2-NF (i.e., 1,2-; 2,3-; 2,4- and 2,5-DNF), to 3-NF (i.e., 2,3- and 2,4-DNF). Only 1,2-DNF has a somewhat higher mutagenic activity (TA98, -$9) than either of its parent N F isomers. The activity of 2,4-DNF resembles that of 3-NF, while the activities of both 2,3-DNF and 2,5-DNF are significantly reduced in TA98 ( - $ 9 ) relative to the parent 2- or 3-NF isomers. Steric considerations may explain the highly reduced activity of 2,3-DNF since, as discussed above, the 1H N M R data show that in 2,3-DNF the nitro group at C-3 is out of the plane of the aromatic ring and as
observed previously (Vance and Levin, 1984; Fu et al., 1985) such nitroarenes exhibit little direct activity. The remaining activity of 2,3-DNF may be largely due to metabolism of the nitro group at C-2, which presumably could be planar with the aromatic ring. The addition of another nitro group to 2,3-DNF at position C-5 increased the mutagenicity of the resulting 2,3,5-TNF in TA98 and TA98NR ( - 6fold) but decreased it in T A 9 8 / 1 , 8 - D N P 6. This activity may indicate that the nitro group at C-5 is preferentially metabolized by the Salmonella bacteria and, as with 2-NF, O-acetylation of the hydroxylamine is an important step in the bacterial activation of 2,3,5-TNF. Although the 1H N M R data for 1,2-DNF (see above) indicate that the nitro groups are sterically constrained, the mutagenic activity of 1,2-DNF is comparable to that of 2-NF. As suggested above for 2,3-DNF, the nitro group in position C-2 may be coplanar with the aromatic ring and may be activated by the bacterial nitroreductase. It is interesting to note, however, that the mutagenic activities of 1,2-DNF and 2,3-DNF, in contrast to 2-NF, are not significantly reduced in strain T A 9 8 / 1 , 8 - D N P 6, indicating that O-acetylation of the hydroxylamine is not an important step in their activation. The addition of another nitro group to the 1,2-DNF molecule either increases (for 1,2,4-TNF, - 3-fold in TA98) or does not significantly change the mutagenic activity (for 1,2,5-TNF) relative to 1,2-DNF. Since the increased activity occurs for 1,2,4-TNF (which can also be viewed as 3-NF with 2 additional N O 2 substituents) rather than for 1,2,5-TNF (which can be viewed as 2-NF with the same additional N O 2 substituents), it suggests that activation of the single N O 2 group (i.e., that on the B ring) contributes to the observed activity. The mutagenic activity of 2,4-DNF towards strain TA98 approaches that of 3-NF, but unlike 3-NF, is not significantly reduced in strain TA98NR. This indicates that 2,4-DNF (similar to the 1,8- and 1,6-dinitropyrenes) does not depend on the "classical" nitroreductase for expressing its maximal activity (Rosenkranz and Mermelstein, 1983). The addition of another nitro group to 2,4-DNF in the C-1 position resulted in a lowering of the
138 mutagenic response in strain TA98 and an increase in the ratio of mutagenic activities T A 9 8 / T A 9 8 N R for 1,2,4-TNF, again suggesting that the N O 2 present in the B-ring may be activated. Neither nitro group in 2,5-DNF is sterically crowded and, due to the symmetrical structure of this molecule, both nitro groups will be indistinguishable to the nitroreductase enzyme, however, its mutagenic activity in strain TA98 is only - 2 0 % of that of 2-NF. In contrast to the other D N F tested, the nitro groups of 2,5-DNF are not resonance-conjugated, which may influence the stability of the nitrofluoranthenyl-nitrenium ion (Vance and Levin, 1984). In addition, the S9mediated mutagenic activity of 2,5-DNF is higher than its direct activity in all strains tested, which may indicate that importance of metabolic pathways other than nitro reduction, for example, ring oxidation (Beland et al., 1985, and refs. therein). An additional nitro group either in the C-1 (1,2,5T N F ) or C-3 (2,3,5-TNF) position substantially increases the mutagenic activity in all strains tested and the mutagenicities of these two T N F isomers are again lower in the presence of $9. The effect of a conjugated hydroxy group on the mutagenic activity of nitroarenes is usually to reduce their potency, due to the formation of stable quinoneimine structures, which do not react with D N A (Vance et al., 1985). This is in accord with the lower mutagenic activity of 2,1-HNF in comparison with that of 1-NF in all 3 strains tested. Finally, the NNF-oxide expressed significant mutagenic activity in strain TA98. However, since this compound is unstable and may decompose forming the less mutagenic 2,1-HNF (Zielinska et al., 1986), the activity listed for this compound in Table 2 is probably a lower limit. In summary, the data presented here for a unique series of D N F and T N F indicate that the addition of a second or third N O 2 group to the naphthalene moiety of the FL molecule does not enhance significantly the mutagenic activity of the resulting compound in Salmonella strain TA98 over those of the mononitrofluoranthenes. This is consistent with the report that the mutagenicity of 3,4-DNF is comparable to that of 3-NF in strain TA98 (Nakagawa et al., 1987). As discussed above,
the observed relatively low mutagenic activities of certain di- and tri-nitrofluoranthenes (Table 2) m a y be rationalized by steric a n d / o r inductive/ resonance effects presumed to ultimately influence bacterial D N A adduct formation. Recent data have shown that the n u m b e r of reversions induced for each D N A adduct formed is not the same for different chemical species even when only C-8 guanine adducts are formed (Beland et al., 1983). Possible effects of the structure of the D N A adducts formed from the D N F and T N F examined here on their efficiency to induce frameshift reversions in TA98 remain to be explored.
Acknowledgments The authors thank Drs. William A. Vance and Roger Atkinson for helpful discussions. Dr. Arthur M. Winer is thanked for encouraging this work and Ms. Patricia A. McElroy for very able technical assistance. The financial support of the Califomia Air Resources Board, Contract No. A4-081-32 is gratefully acknowledged.
References Ames, B.N., J. McCann and E. Yamasaki (1975) Methods for detecting carcinogens and mutagens with the Salmonella/ mammalian-microsome mutagenicity test, Mutation Res., 31, 347-364. Arey, J., B. Zielinska, R. Atkinson, A.M. Winer, T. Ramdahl and J.N. Pitts Jr. (1986) The formation of nitro-PAH from the gas-phase reaction of fluoranthene and pyrene with the OH radical in the presence of NOx, Atmos. Environ., 20, 2339-2345. Arey, J., B. Zielinska, R. Atkinson and A.M. Winer (1987) Polycyclic aromatic hydrocarbons and nitroarene concentrations in ambient air during a wintertime high-NOx episode in the Los Angeles basin, Atmos. Environ., 21, 1437-1444. Arey, J., B. Zielinska, W.P. Harger, R. Atkinson and A.M. Winer (1988) The contribution of nitrofluoranthenes and nitropyrenes to the mutagenic activity of ambient particulate organic matter in Southern California, Mutation Res., 207, 45-51. Atkinson, R., A.M. Winer and J.N. Pitts Jr. (1986) Estimation of nighttime N205 concentrations from ambient N O 2 and N O 3 radical concentrations and the role of N205 in nighttime chemistry, Atmos. Environ., 20, 331-339. Beland, F.A., D.T. Beranek, K.L. Dooley, R.H. Heflich and F.F. Kadlubar (1983) Arylamine-DNA adducts in vitro and in vivo: their role in bacterial mutagenesis and urinary
139 bladder carcinogenesis, Environ. Health Perspect., 49, 125-134. Beland, F.A., R.H. Heflich, P.C. Howard and P.P. Fu (1985) The in vitro metabolic activation of nitro polycyclic aromatic hydrocarbons, in: R.G. Harvey (Ed.), Polycyclic Hydrocarbons and Carcinogenesis, American Chemical Society, Washington, DC, pp. 371-396. Belser Jr., W.L., S.D. Shaffer, R.D. Bliss, P.M. Hynds, L. Yamamoto, J.N. Pitts Jr. and J.A. Winer (1981) A standardized procedure for quantification of the Ames Salmonella/mammalian-microsome mutagenicity test, Environ. Mutagen., 3, 123-139. Bock, H., and U. Lechner-Knoblauch (1985) The electrochemical reduction of aromatic nitrocompounds in aprotic solution, Z. Naturforsch., 40B, 1463-1475. Fu, P.P., M.W. Chou, D.W. Miller, G.L. White, R.H. Heflich and F.A. Beland (1985) The orientation of the nitro substituent predicts the direct-acting bacterial mutagenicity of nitrated polycyclic aromatic hydrocarbons, Mutation Res., 143, 173-181. Heflich, R.H., P.C. Howard and F.A. Beland (1985a) 1-Nitrosopyrene: an intermediate in the metabolic activation of 1-nitropyrene to a mutagen in Salmonella typhimurium TA1538, Mutation Res., 149, 25-32. Heflich, R.H., E.K. Filer, Z. Djuric and F.A. Beland (1985b) DNA-adduct formation and mutation induction by nitropyrenes in Salmonella and Chinese hamster ovary cells: relationships with nitroreduction and acetylation, Environ. Health Perspect., 62, 135-143. Hodgson, H.H., A.P. Mahadevan and E.R. Ward (1947) The replacement of the diazonium by the nitro-group, III. Decompositions by cupro-cupri sulphite, J. Chem. Soc., 1392-1394. Howard, P.C., R.H. Heflich, F.E. Evans and F.A. Beland (1983) Formation of DNA-adducts in vitro and in Salmonella typhimurium upon metabolic reduction of the environmental mutagen 1-nitropyrene, Cancer Res., 43, 2052-2058. Kloetzel, M.C., W. King and J.H. Menkes (1956) Fluoranthene derivatives, III. 2-Nitrofluoranthene and 2-aminofluoranthene, J. Am. Chem. Soc., 78, 1165-1168. Klopman, G., D.A. Tonucci, M. Holloway and H.S. Rosenkranz (1984) Relationship between polarographic reduction potential and mutagenicity of nitroarenes, Mutation Res., 126, 139-144. Maynard, A.T., L.G. Pedersen, H.S. Posner and J.D. McKinney (1986) An ab initio study of the relationship between nitroarene mutagenicity and electron affinity, Mol. Pharmacol., 29, 629-636. McCoy, E.C., H.S. Rosenkranz and R. Mermelstein (1981) Evidence for the existence of a family of bacterial nitroreductases capable of activating nitrated polycyclics to mutagens, Environ. Mutagen., 3, 421-427. McCoy, E.C., G.D. McCoy and H.S. Rosenkranz (1982) Esterification of arylhydroxylamines, Evidence for a specific gene product in mutagenesis, Biochem. Biophys. Res. Commun., 108, 1362-1367. McCoy, E.C., M. Anders and H.S. Rosenkranz (1983) The basis of the insensitivity of Salmonella typhimurium strain
TA98/1,8-DNP 6 to the mutagenic action of nitroarenes, Mutation Res., 121, 17-23. Miller, D.W., F.E. Evans and P.P. Fu (1985) Effect of a nitro group geometry on the proton NMR chemical shifts of nitro aromatics, Spectros. Int. J., 4, 91-94. Nakagawa, R., K. Horikawa, N. Sera, Y. Kodera and H. Tokiwa (1987) Dinitrofluoranthene induction, identification and gene mutation, Mutation Res., 191, 85-91. Nielsen, T., and T. Ramdahl (1986) Discussion on "Determination of 2-nitrofluoranthene and 2-nitropyrene in ambient particulate matter: evidence for atmospheric reactions", Atmos. Environ., 20, 1507. Orr, J.C., D.W. Bryant, D.R. McCalla and M.A. Quilliam (1985) Dinitropyrene-resistant Salmonella typhimurium are deficient in an acetyl-CoA acetyltransferase, Chem.-Biol. Interact., 54, 281-288. Pitts Jr., J.N., J.A. Sweetman, B. Zielinska, A.M. Winer and R. Atkinson (1985) Determination of 2-nitrofluoranthene and 2-nitropyrene in ambient particulate organic matter: evidence for atmospheric reactions, Atmos. Environ., 19, 1601-1608. Radner, F. (1983) Nitration of polycyclic aromatic hydrocarbons with dinitrogen tetroxide, a simple and selective synthesis of mononitro derivatives, Acta Chem. Scand., B 37, 65-67. Ramdahl, T., B. Zielinska, J. Arey, R. Atkinson, A.M. Winer and J.N. Pitts Jr. (1986) Ubiquitous occurrence of 2-nitrofluoranthene and 2-nitropyrene in air, Nature (London), 321,425-427. Ramdahl, T., J.A. Sweetman, B. Zielinska, W.P. Harger, A.M. Winer and R. Atkinson (1988a) Determination of nitrofluoranthenes and nitropyrenes in ambient air and their contribution to direct mutagenicity, in: M. Cook and A.J. Dennis (Eds.), Polynuclear Aromatic Hydrocarbons: a Decade of Progress, Battelle, Columbus, OH, pp. 745-759. Ramdahl, T., B. Zielinska, J. Arey and R.W. Kondrat (1988b) The electron-impact mass spectra of di- and trinitrofluoranthenes, Biomed. Environ. Mass Spectrom., in press. Rosenkranz, H.S., and R. Mermelstein (1983) Mutagenicity and genotoxicity of nitroarenes: all nitro-containing chemicals were not created equal, Mutation Res., 114, 217-267. Rosenkranz, H.S., and W.T. Speck (1975) Mutagenicity of metronidazole: activation by mammalian liver microsomes, Biochem. Biophys. Res. Commun., 66, 520-525. Rosenkranz, H.S., E.C. McCoy, M. Frierson and G. Klopman (1985) The role of DNA sequence and structure of the electrophile on the mutagenicity of nitroarenes and arylamine derivatives, Environ. Mutagen., 7, 645-653. Ruehle, P.H., L.C. Bosch and W.P. Duncan (1985) Synthesis of nitrated Polycyclic aromatic hydrocarbons, in: C.M. White (Ed.)., Nitrated Polycyclic Aromatic Hydrocarbons, Dr. Alfred Huethig Verlag, Heidelberg, pp. 169-235. Saito, K., A. Shinohara, T. Kamataki and R. Kato (1985) Metabolic activation of mutagenic N-hydroxylarylamines by O-acetyltransferase in Salmonella typhimurium TA98, Arch. Biochem. Biophys., 239, 286-295. Saito, K., A. Shinohara, T. Kamataki and R. Kato (1986) Reznikoff, C.A., J.S. Bertram, D.S. Brankow and C. Heidel-
140 N-Hydroxylarylamine O-acetyltransferase in hamster liver: identity with arylhydroxamic acid N,O-acetyltransferase and arylamine N-acetyltransferase, J. Biochem., 99, 1689-1697. Schofield, K. (1980) Aromatic Nitration, Cambridge University Press, Cambridge, pp. 73-76. Svendsen, H., H.-P. Ronningsen, L.K. Sydnes and T. Greibrokk (1983) Separation and characterization of mononitro derivatives of phenanthrene, pyrene, chrysene, fluoranthene and triphenylene, Acta Chem. Scand., B 37, 833-844. Sweetman, J.A., B. Zielinska, R. Atkinson, T. Ramdahl, A.M. Winer and J.N. Pitts Jr. (1986) A possible formation pathway for the 2-nitrofluoranthene observed in ambient particulate organic matter, Atmos. Environ., 20, 235-238. Vance, W.A., and D.E. Levin (1984) Structural features of nitroaromatics that determine mutagenic activity in Salmonella typhimurium, Environ, Mutagen., 6, 797-811.
Vance, W.A., Y.Y. Wang and H.S. Okamoto (1985) Bifunctional nitrofluorenes: structure-activity relationships in Salmonella typhimurium, Mutation Res., 143, 213-218. Zielinska, B., J. Arey, R. Atkinson, T. Ramdahl, A.M. Winer and J.N. Pitts Jr. (1986) Reaction of dinitrogen pentoxide with fluoranthene, J. Am. Chem. Soc., 108, 4126-4132. Zielinska, B., J. Arey and W.P. Harger (1987a) The synthesis and mutagenic properties of selected polynitro-PAH derivatives, presented at the XI International Symposium on Polynuclear Aromatic Hydrocarbons, September 23-25, Gaithersburg, MD. Zielinska, B., W.P. Harger, J. Arey, A.M. Winer, R.A. Haas and C.V. Hanson (1987b) The mutagenicity of 2nitrofluoranthene and its in vitro hepatic metabolites, Mutation Res., 190, 259-266.
131
MTR 01307
Mutagenic activities of selected nitrofluoranthene derivatives in Salmonella typhimurium strains TA98, T A 9 8 N R and T A 9 8 / 1 , 8 - D N P 6 Barbara Zielinska 1, Janet Arey 1, William P. Harger 1 and Robert W.K. Lee 2 Statewide Air Pollution Research Center, University of California, Riverside, CA 92521, and 2 Chemistry Department, University of California, Riverside, CA 92521 (U.S.A.) (Received 10 August 1987) (Revision received 8 February 1988) (Accepted 15 February 1988)
Keywords: Nitrofluoranthenes; Dinitrofluoranthenes; Trinitrofluoranthenes; NMR; Mutagenicity; Salmonella typhimurium, TA98, TA98NR, TA98/1,8-DNP6
Summary The mutagenic activities of novel nitrofluoranthene derivatives in Salmonella strains TA98, TA98NR and TA98/1,8-DNP 6 (with and without $9 addition) are given. These derivatives were produced from the reactions of fluoranthene (FL) and its directly mutagenic 2- and 3-nitro derivatives with covalent dinitrogen pentoxide (N205) in CC14 solution at ambient temperature. The influence of the addition of a nitro group on the observed activity of the resulting di- and tri-nitrofluoranthenes is discussed.
2-Nitrofluoranthene (2-NF) has recently been identified as a major nitrated polycyclic aromatic hydrocarbon (nitro-PAH) in ambient particulate organic matter (POM) collected at various locations in the U.S.A. and Europe (Pitts et al., 1985; Arey et al., 1986, 1987; Sweetman et al., 1986; Ramdahl et al., 1986; Nielsen and Ramdahl, 1986). This particular nitro-PAH has been observed to account for up to 5% of the overall directly acting mutagenicity of ambient POM (Ramdahl et al., 1988a; Arey et al., 1988) in the Salmonella test. One possible formation pathway for the 2-NF has been identified as the gas-phase reaction of fluoranthene (FL) with dinitrogen pentoxide (N205) (Sweetman et al., 1986; Zielinska et al., 1986), a
Correspondence: Dr. B. Zielinska, Statewide Air Pollution Research Center, University of California, Riverside, CA 92521 (U.S.A.).
species expected to the present in the ambient nighttime atmosphere (Atkinson et al., 1986). We have studied the mechanism of the reaction of FL with N205 in CC14 solution (Zielinska et al., 1986), where, as in the gas phase, N 2 0 5 exists in the covalent state (Schofield, 1980). At ambient temperature the solution-phase reaction produced 2-NF as the sole mononitrofluoranthene isomer, together with 1,2-dinitrofluoranthene (1,2-DNF) and several oxygenated nitro products. To explain the observed solution-phase products, a radical mechanism involving molecule-assisted homolysis has been postulated for this reaction of FL with N 2 0 5 (Zielinska et al., 1986). The reaction of FL and its 2- and 3-nitro isomers with covalent N20 s in CC14 solution produces nitro derivatives distinct from those formed by electrophilic nitration reactions (Zielinska et al., 1986). We report here the mutagenic activities of these novel nitrofluoranthene derivatives in
0165-1218/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
132
Salmonella typhimurium strain TA98 and its nitro reductase- and transacetylase-deficient strains TA98NR and TA98/1,8-DNP6, respectively, with and without $9 microsomal activation.
Experimental ~H N M R spectra were recorded with a Nicolet 300 pulsed Fourier transform N M R spectrometer with an NIC 1280 computer. Typically 200-300 /~g of sample were dissolved in 0.5 ml CDC13 or CD3OD. The proton resonance shifts have been expressed in ppm on the 8 scale. Mass spectra were recorded by using a Finnigan 3200 gas chromatograph/mass spectrometer ( G C / M S ) operated in the electron impact (EI) mode. A Spectra-Physics Model 8100 high-performance liquid chromatograph (HPLC) equipped with a 250 mm x 10 mm Ultrasphere ODS or Si column was employed for compound purifications.
Syntheses The reactions of FL, 2-NF and 3-NF with N205 in C C l 4 solution at ambient temperature and the isolation of products from these reactions were carried out as described previously (Zielinska et al., 1986). The products produced in low abundance were additionally purified by semi-preparative HPLC, using an Altex Ultrasphere Si column eluted with n-hexane/CHzC12 or an Altex Ultrasphere ODS column eluted with C H 3 O H / H 2 0 (for 2-hydroxy-l-nitrofluoranthene, 2,1-HNF). 2,3-Dinitrofluoranthene (2,3-DNF) was synthesized using methods described in the literature (Kloetzel et al., 1956; Hodgson et al., 1947). 3Aminofluoranthene (Aldrich Chemical Co.) was converted to 3-amino-2-nitrofluoranthene (3amino-2-NF) according to the method described by Kloetzel et al. (1956) with an overall yield of 85%. 3-Amino-2-NF was reacted with cupro-cupri sulphite, as described by Hodgson et al. (1947) for the synthesis of dinitronaphthalenes. However, the main product from this reaction was 2-NF, together with lesser amounts of 2,3-DNF ( - 10% of the 2-NF yield). The 2,3-DNF was separated from the 2-NF by chromatography on silica-gel 60 (Merck), using CC14/CHzC12 elution (9:1 v/v) and was then recrystallized from ethanol, m.p. 227-228°C. MS (main ions): m / z (rel. int.) 292
([M] +, 100%), 204 ([M-2NO-CO] +, 43%), 200 ([M-2NO21 +, 95%). The 3- and 8-nitrofluoranthenes were synthesized according to the method of Radner (1983), using NzO 4 in CH2C12 with methanesulfonic acid as a catalyst. The isomers were separated by semi-preparative HPLC, employing an Ultrasphere Si column eluted with n-hexane/CH2C12. The nitrofluoranthene derivatives investigated were > 99% pure, as established by GC a n d / o r HPLC analyses with the exception of 2,5-DNF, 2,3,5-TNF and 2,1-HNF which were > 95% pure.
Mutagenicity assays The Salmonella plate-incorporation mutagenicity tests were performed according to the standard procedure (Ames et al., 1975) with modifications to improve precision (Belser et al., 1981). The Salmonella strains used were TA98, which is sensitive to nitroarene mutagenicity, and strains TA98NR and TA98/1,8-DNP 6, which are deficient in nitroreductase (Rosenkranz and Speck, 1975; McCoy et al., 1981) and transace~clase enzymes (McCoy et al., 1983; Orr et al., 1985; Saito et al., 1985), respectively. Cultures of these strains were grown for 12 h in LB broth, diluted to approximately 1 x 10 9 cells/ml, and maintained at ice temperature until testing. All samples, including positive controls, were dissolved in dimethyl sulfoxide (DMSO) and tested in triplicate at 8 doses. Mutagenic activity, as reported here, was the slope of the linear region of the dose-response curve and was determined by a least-squares linear regression analysis. Positive control mutagens were quercetin, 2-nitrofluorene, 1,8-dinitropyrene and benzo[a]pyrene (with rat liver metabolic activation). A single test was conducted for all compounds with metabolic activation. The $9 mix (2% v/v) was prepared from Aroclor 1254-induced rat liver $9 (Litton Bionetics, Inc.) containing 25 m g / m l protein. Benzo[a] pyrene was utilized as the control mutagen and gave the following values (rev./nmole _+ S.E.) in strains TA98 (+$9), TA98NR ( + $ 9 ) and TA98/1,8-DNP 6 ( + $9), respectively: 92 _+ 3, 106 _+ 4, 92 _+ 4. Spontaneous reversions for this test as determined from 6 DMSO blanks per strain were (rev./plate + S.D.): TA98 (71 + 7), TA98NR (49 _+ 7), TA98/1,8-DNP 6 (47 + 7).
133 The majority o f the compounds were tested without $9 in a single experiment; those compounds tested in a separate second test were 3-NF, 8-NF and 1,2-DNF. The values for the control mutagens in these two tests were, for 2-nitrofluorene (rev./nmole + S.E.): TA98 (104 ___3, 91 + 5); TA98NR (16 + 1, 12 + 0.7); TA98/1,8-DNP 6 (20 + 1, 16 + 1), for quercetin (rev./nmole + S.E.): TA98 (5.3 ___0.3, 4.8 + 0.1), TA98NR (5.7 ___0.2, 5.2 + 0.2), TA98/1,8-DNP 6 (5.0 + 0.2, 5.0 + 0.2), and for 1,8-dinitropyrene ( r e v . / n m o l e + S.E.): TA98 (3.0 X 105 + 1 X 104, 2.7 X 105 + 8 X 103), TA98NR ( 3 . 2 X 1 0 5 + 1 X 1 0 4 , 2 . 7 x 1 0 5 + 9 X 103), TA98/1,8-DNP 6 (9900 + 600, 6700 +_ 100). Spontaneous reversions for these two tests were (rev./plate + S.D. of 6 replicates): TA98 (50 + 4, 3 9 + 4 ) , TA98NR ( 4 0 + 4 , 30_+3) and T A 9 8 / 1,8-DNP 6 (43 + 4, 46 _+ 5).
Results
The structures of FL, 2- and 3-NF and the products identified from their reaction with N205 in CC14 solution at ambient temperature, are shown in Fig. 1. Note that FL has been drawn to illustrate its axis of symmetry and that IUPAC rules dictate that the substituents are numbered beginning with the lowest possible number. Thus, for example, the nitration of 3-NF in the 5-position produces 2,4-DNF. As noted above, 2-NF and 1,2-DNF were the major products from the reaction of FL with N205 in CC14 at ambient temperature (Zielinska et al., 1986). In addition, this reaction yielded lower amounts of 2,3-DNF ( - 5% of the 1,2-DNF yield), and oxygenated nitro products including 2-hydroxy-l-nitrofluoranthene (2,1-HNF) and 10bnitrato-l-nitro-l,2,3,10b-tetrahydrofluoranthene2,3-oxide (NNF-oxide). To prove its structure, 2,3-DNF was also synthesized by an independent method (see Experimental). All products were identified on the basis of their MS and 1H N M R data (Zielinska et al., 1986, 1987a). The reactions of 2-NF and 3-NF with N205 in CC14 at 25 °C produced nitro products analogous to those formed from FL (Zielinska et al., 1986). Thus, 2-NF yielded 2,5-DNF and 1,2,5-TNF with
7
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NO2 3-NF
1,2,5-TNF
OzN~NO2 NO2 2,3,5-TNF
2,1-HNF NNF-OXIDE ]Fig. 1. Structures of compounds investigated.
a lower yield of 2,3,5-TNF while 3-NF formed 2,4-DNF and 1,2,4-TNF (2,3,4-TNF was not formed in detectable amounts, possibly due to steric considerations). The D N F and TNF products from these reactions were identified using MS and 1H N M R data (Zielinska et al., 1986, 1987a; Ramdahl et al., 1988b). These N205 nitration products are distinct from those produced by electrophilic nitrations. Thus, electrophilic nitration of FL has been shown to produce 3-, 8-, 7- and 1-NF (in order of decreasing yield), while dinitration of FL and nitration of 3-NF produce mainly 3,9- and 3,7-DNF with lower amounts of 3,8- and 3,4-DNF (Ruehle et al., 1985; Nakagawa et al., 1987). In contrast, the reaction of FL with N205 gives substitution in the 2-position and further nitration of 2- and 3-NF does not produce nitration in the C ring (see Fig. 1), but rather in the B ring. Furthermore, dinitration of FL and 2- and 3-NF with N205 gives unique ortho-substituted D N F and TNF isomers, respectively.
134
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135 TABLE 2 MUTAGENIC ACTIVITIES (rev./nmole) a OF NITROFLUORANTHENE DERIVATIVES IN Salmonella typhimurium Compound
TA98
TA98NR
TA98/1,8-DNP 6
-$9
+$9 b
-$9
+$9 b
-$9
1,2-Dinitrofluoranthene 2,3-Dinitrofluoranthene 2,4-Dinitrofluoranthene 2,5-Dinitrofluoranthene
1290+_ 40 420_+ 20 6 000 +_200 211+_ 7
190+_10 80+_10 550 _+50 540+_30
570+_ 20 180+_ 10 5 700 +_300 105+_ 7
97_+ 3 80+_20 360 +_30 280+_10
1010+_40 369_+ 7 2 000 +_100 67___ 2
1,2,4-Trinitrofluoranthene 1,2,5-Trinitrofluoranthene 2,3,5-Trinitrofluoranthene
3600+-500 1440+ 80 2750+- 90
136+ 9 69+- 3 720+_30
2600+200 770+ 40 1100+100
90+-10 24+- 3 404+_ 9
1350_+60 560+_ 30 56_+ 4
52 +_5 22 +_2 6.4 +_ 0.9
71+
7
53+- 3
34+_ 2
8.5 +_ 0.5
2-Hydroxy-l-nitrofluoranthene
180+ 20
110+- 8
450 +_ 50
63 +_ 3
330 +_ 20
63 + 3
220 _+ 20
1030+_ 20 7 700 +_600 18 200 +-500 500 47
760+_20 ---
250+_ 20 3 800 +_200 6 000 +_300 230 18
230+_20 --
180_+ 9 1020 + 20 5 400 +_300 ---
+$9 b 180 33 180 92
_+20 _+2 _+30 +_6
lOb-Nitrato-l-nitro-l,2,3-1Ob-tetrahydrofluoranthene-2,3-oxide 2-Nitrofluoranthene ~ 3-Nitrofluoranthene 8-Nitrofluoranthene 1-Nitrofluoranthene d 7-Nitrofluoranthene d
35 +_3 < 20
a Slope of a least-squares regression of dose-response curve+ S.E. of slope. b 2% v/v mix was used. From Zielinska et al. (1987b). d From Vance and Levin (1984).
T h e 1H N M R data for the n i t r o f l u o r a n t h e n e derivatives shown in Fig. 1 are given i n T a b l e 1 *. T h e p r o t o n chemical shifts were assigned o n the basis of h o m o n u c l e a r decoupling experiments and, where possible, n u c l e a r Overhauser e n h a n c e m e n t ( N O E ) experiments (Zielinska et al., 1987a). Analysis of the 1H N M R data s u m m a r i z e d i n T a b l e 1 reveals steric interactions between the adjacent NO2 groups i n 1,2- a n d 2 , 3 - D N F a n d 1,2,4-, 1,2,5- a n d 2,3,5-TNF. Thus, in the s p e c t r u m of 2 , 3 - D N F , the chemical shift of the p r o t o n peri to the 3-NO 2 group (H-4) is upfield of that of H-6 (A = 0.22 ppm). This is in contrast to the spect r u m of 3-NF, in which H-4 is shifted - 0.76 p p m downfield relative to H-6. Thus, although a p r o t o n peri to a p l a n a r N O 2 group (e.g., H-4 in 3 - N F ) will be shifted downfield relative to its shift i n the
* Although some of these data were reported previously (Zielinska et al., 1986) not all chemical shifts were unequivocally assigned. In addition, since only one of these TNF isomers was available at that time, the 1H NMR spectrum of 2,3,5-TNF was incorrectly interpreted as that of 1,2,5-TNF.
p a r e n t P A H , a p r o t o n peri to a n o n p l a n a r N O 2 group will be shifted upfield (for a discussion of the m a g n e t i c a n i s o t r o p i c effect of the N O 2 group, see Miller et al., 1985). C o m p a r i s o n of the 1H N M R spectrum of 2,3,5T N F with that of 2 , 4 - D N F reveals a n analogous steric effect for 2,3,5-TNF. The 2 , 4 - D N F spect r u m shows n o evidence for steric i n t e r a c t i o n between the n i t r o group in the C-4 position with the peri h y d r o g e n (H-3). T h e signal c o r r e s p o n d i n g to H-3 is shifted - 0 . 9 p p m downfield i n the spect r u m of 2 , 4 - D N F w h e n c o m p a r e d with the peri h y d r o g e n in the 3 - N F spectrum, thus showing the influence of b o t h the m a g n e t i c a n i s o t r o p y effect of the N O 2 group in p o s i t i o n C-4 a n d the inductive effect of the N O 2 in p o s i t i o n C-2 o n the chemical shift of H-3. I n the s p e c t r u m of 2 , 3 , 5 - T N F the peri p r o t o n (H-4) is shifted - 0 . 9 p p m further upfield t h a n the peri p r o t o n (H-3) in the spectrum of 2 , 4 - D N F , thus i n d i c a t i n g that the presence of a n a d j a c e n t n i t r o group forces the N O 2 group in the 3-position of 2 , 3 , 5 - T N F out of the p l a n e of the a r o m a t i c rings. Conversely, the nearly identical
136
chemical shifts of the peri protons (H-3) in the 2,4-DNF and 1,2,4-TNF spectra indicate that the NO 2 group in the 4-position of 1,2,4-TNF is in the plane of the aromatic rings. A comparison of 1H N M R data for 1-NF (Svendsen et al., 1983) and 1,2-DNF (Table 1) indicates that the NO 2 group in the 1-position of 1,2-DNF adopts a conformation more out-of-plane of the aromatic rings than in 1-NF since the H-10 proton in the bay region is shifted - 0 . 5 ppm upfield in the spectrum of 1,2-DNF relative to the spectrum of 1-NF. The essentially identical chemical shifts for these bay region protons (H-10) in the spectra of 1,2-DNF, 1,2,4-TNF and 1,2,5-TNF indicate that the NO 2 groups in the 1-position of both TNF are also twisted out-of-plane owing to crowding by the NO 2 groups in the 2-position. Table 2 shows the mutagenic activities of the N F derivatives toward Salmonella strains TA98, TA98NR and TA98/1,8-DNP 6 tested with ( + $9) and without ( - $ 9 ) metabolic activation. In the absence of $9, as seen from Table 2, all of the NF derivatives tested were mutagenic in TA98 and all but 2,4-DNF showed significantly reduced activity in the nitroreductase-deficient strain TA98NR in comparison with that in TA98. The largest reduction in activity in strain TA98NR (76%) was observed for 2-nitrofluoranthene. In strain TA98/1,8-DNP 6 which is deficient in transacetylase activity, all of the nitro derivatives with the exception of 1,2-DNF and 2,3-DNF exhibited mutagenic responses less than half of those in the transacetylase competent strain, TA98. The addition of 2% (v/v) of $9 generally resuited in highly diminished activity. The exceptions were the response of 2,5-DNF in all 3 strains and the response of 2-NF in strain TA98NR. Discussion
It is generally accepted that the direct mutagenic activity (i.e. without $9 addition) of nitroarenes in the Salmonella reversion assay is a consequence of enzymatic reduction of the nitro group to an hydroxylamine, possibly occurring through a nitroso intermediate (Rosenkranz and Mermelstein, 1983, and refs. therein; Beland et al., 1985, and refs. therein; Heflich et al., 1985a). This is consistent with the reduced activity observed for
many nitroarenes in the nitroreductase-deficient strain TA98NR (Rosenkranz and Mermelstein, 1983; Beland et al., 1985). The hydroxylamine is postulated to be a proximate mutagen which requires further activation to form an arylnitrenium ion, the reactive electrophile which serves as the ultimate mutagen (Vance and Levin, 1984). For some nitroarenes formation of the arylnitrenium ion occurs through enzymatic O-acetylation of the hydroxylamine as evidenced by the significantly reduced mutagenic response of these nitroarenes in strain TA98/1,8-DNP 6 (which is deficient in acetyltransferase activity) compared to strain TA98 (McCoy et al., 1982, 1983; Saito et al., 1985, 1986; Orr et al., 1985). In Salmonella, binding of arylnitrenium ions to DNA has been shown to occur at the C-8 of guanine for 1-nitropyrene and 1,8-dinitropyrene (Howard et al., 1983; Heflich et al., 1985b). It has been suggested (Rosenkranz et al., 1985) that, for steric reasons, all nitroarenes having more than 2 rings will form DNA adducts with the C-8 position of guanine. It was first demonstrated by Klopman and co-workers (1984) that the direct mutagenicity of certain nitroarenes, and in particular the nitropyrenes, could be correlated with the ease of their nitro reduction as measured by their halfwave potentials or estimated from their calculated energies of the lowest unoccupied molecular orbital (LUMO). Subsequent studies (Vance and Levin, 1984; Fu et al., 1985) showed that nitroarenes with the nitro substituent sterically forced out of the plane of the aromatic ring exhibited only little or no direct-acting bacterial mutagenicity and that for these nitroarenes the inverse correlation of the L U M O energy (ELuMO) with activity did not hold (Fu et al., 1985). However, ELUMO calculations which take the nitro group orientation into account may correlate better with mutagenic activity (Maynard et al., 1986). The direct-acting mutagenicities in TA98 of 4 of the 5 nitro isomers of fluoranthene (1-, 3-, 7and 8-) have been discussed previously (Vance and Levin, 1984; Maynard et al., 1986), while the activity of the remaining isomer, 2-nitrofluoranthene, has only recently been reported (Zielinska et al., 1987b). The mutagenic activities of the nitrofluoranthene isomers (see Table 2) decrease in the following order: 8-NF > 3-NF > 2-NF > 1-
137 N F > 7-NF. Although the 1H N M R data suggest that the 1-, 3-, and 7-isomers are planar (no upfield shift is observed for the peri or bay region hydrogens relative to fluoranthene) (Vance and Levin, 1984), recent calculations suggest that the nitro group of the 1- and 7-NF isomers will interact with the hydrogen in the bay region (Maynard et al., 1986). Taking the predicted rotation of the nitro groups in 1- and 7-NF into account, Maynard and co-workers (1986) calculated the ELUMO energies of the N F isomers and found them to decrease in the order 7-NF > 1-NF > 8-NF >/3NF, thus predicting reasonably well the trend in their observed mutagenicities, although 8-NF is more mutagenic than 3-NF by at least a factor of 2 (see Table 2). The addition of a strongly electron-withdrawing group (such as a nitro group) to a nitrofluoranthene molecule should presumably lower the redox potential of the NO2 group (Bock and Lechner-Knoblauch, 1985), and would be expected to increase the nitroarene's mutagenic potency (Klopman et al., 1984). Indeed, the mutagenic activities of 3,7- and 3,9-DNF have been reported to be - 2 0 times higher than that of 3-NF (Nakagawa et al., 1987) and 1,3-, 1,6-, and 1,8-dinitropyrene are among the most powerful bacterial mutagens tested to date, being from 300- to 500-fold more mutagenic than 1-nitropyrene in the Salmonella test (Rosenkranz and Mermelstein, 1983). However, none of the mutagenic activities of the D N F and T N F isomers listed in Table 2 approaches those of 3,7- or 3,9-DNF. The D N F tested can be viewed as having a second NO 2 group added to a parent mononitrofluoranthene isomer as follows: to 1-NF (i.e., 1,2DNF), to 2-NF (i.e., 1,2-; 2,3-; 2,4- and 2,5-DNF), to 3-NF (i.e., 2,3- and 2,4-DNF). Only 1,2-DNF has a somewhat higher mutagenic activity (TA98, -$9) than either of its parent N F isomers. The activity of 2,4-DNF resembles that of 3-NF, while the activities of both 2,3-DNF and 2,5-DNF are significantly reduced in TA98 ( - $ 9 ) relative to the parent 2- or 3-NF isomers. Steric considerations may explain the highly reduced activity of 2,3-DNF since, as discussed above, the 1H N M R data show that in 2,3-DNF the nitro group at C-3 is out of the plane of the aromatic ring and as
observed previously (Vance and Levin, 1984; Fu et al., 1985) such nitroarenes exhibit little direct activity. The remaining activity of 2,3-DNF may be largely due to metabolism of the nitro group at C-2, which presumably could be planar with the aromatic ring. The addition of another nitro group to 2,3-DNF at position C-5 increased the mutagenicity of the resulting 2,3,5-TNF in TA98 and TA98NR ( - 6fold) but decreased it in T A 9 8 / 1 , 8 - D N P 6. This activity may indicate that the nitro group at C-5 is preferentially metabolized by the Salmonella bacteria and, as with 2-NF, O-acetylation of the hydroxylamine is an important step in the bacterial activation of 2,3,5-TNF. Although the 1H N M R data for 1,2-DNF (see above) indicate that the nitro groups are sterically constrained, the mutagenic activity of 1,2-DNF is comparable to that of 2-NF. As suggested above for 2,3-DNF, the nitro group in position C-2 may be coplanar with the aromatic ring and may be activated by the bacterial nitroreductase. It is interesting to note, however, that the mutagenic activities of 1,2-DNF and 2,3-DNF, in contrast to 2-NF, are not significantly reduced in strain T A 9 8 / 1 , 8 - D N P 6, indicating that O-acetylation of the hydroxylamine is not an important step in their activation. The addition of another nitro group to the 1,2-DNF molecule either increases (for 1,2,4-TNF, - 3-fold in TA98) or does not significantly change the mutagenic activity (for 1,2,5-TNF) relative to 1,2-DNF. Since the increased activity occurs for 1,2,4-TNF (which can also be viewed as 3-NF with 2 additional N O 2 substituents) rather than for 1,2,5-TNF (which can be viewed as 2-NF with the same additional N O 2 substituents), it suggests that activation of the single N O 2 group (i.e., that on the B ring) contributes to the observed activity. The mutagenic activity of 2,4-DNF towards strain TA98 approaches that of 3-NF, but unlike 3-NF, is not significantly reduced in strain TA98NR. This indicates that 2,4-DNF (similar to the 1,8- and 1,6-dinitropyrenes) does not depend on the "classical" nitroreductase for expressing its maximal activity (Rosenkranz and Mermelstein, 1983). The addition of another nitro group to 2,4-DNF in the C-1 position resulted in a lowering of the
138 mutagenic response in strain TA98 and an increase in the ratio of mutagenic activities T A 9 8 / T A 9 8 N R for 1,2,4-TNF, again suggesting that the N O 2 present in the B-ring may be activated. Neither nitro group in 2,5-DNF is sterically crowded and, due to the symmetrical structure of this molecule, both nitro groups will be indistinguishable to the nitroreductase enzyme, however, its mutagenic activity in strain TA98 is only - 2 0 % of that of 2-NF. In contrast to the other D N F tested, the nitro groups of 2,5-DNF are not resonance-conjugated, which may influence the stability of the nitrofluoranthenyl-nitrenium ion (Vance and Levin, 1984). In addition, the S9mediated mutagenic activity of 2,5-DNF is higher than its direct activity in all strains tested, which may indicate that importance of metabolic pathways other than nitro reduction, for example, ring oxidation (Beland et al., 1985, and refs. therein). An additional nitro group either in the C-1 (1,2,5T N F ) or C-3 (2,3,5-TNF) position substantially increases the mutagenic activity in all strains tested and the mutagenicities of these two T N F isomers are again lower in the presence of $9. The effect of a conjugated hydroxy group on the mutagenic activity of nitroarenes is usually to reduce their potency, due to the formation of stable quinoneimine structures, which do not react with D N A (Vance et al., 1985). This is in accord with the lower mutagenic activity of 2,1-HNF in comparison with that of 1-NF in all 3 strains tested. Finally, the NNF-oxide expressed significant mutagenic activity in strain TA98. However, since this compound is unstable and may decompose forming the less mutagenic 2,1-HNF (Zielinska et al., 1986), the activity listed for this compound in Table 2 is probably a lower limit. In summary, the data presented here for a unique series of D N F and T N F indicate that the addition of a second or third N O 2 group to the naphthalene moiety of the FL molecule does not enhance significantly the mutagenic activity of the resulting compound in Salmonella strain TA98 over those of the mononitrofluoranthenes. This is consistent with the report that the mutagenicity of 3,4-DNF is comparable to that of 3-NF in strain TA98 (Nakagawa et al., 1987). As discussed above,
the observed relatively low mutagenic activities of certain di- and tri-nitrofluoranthenes (Table 2) m a y be rationalized by steric a n d / o r inductive/ resonance effects presumed to ultimately influence bacterial D N A adduct formation. Recent data have shown that the n u m b e r of reversions induced for each D N A adduct formed is not the same for different chemical species even when only C-8 guanine adducts are formed (Beland et al., 1983). Possible effects of the structure of the D N A adducts formed from the D N F and T N F examined here on their efficiency to induce frameshift reversions in TA98 remain to be explored.
Acknowledgments The authors thank Drs. William A. Vance and Roger Atkinson for helpful discussions. Dr. Arthur M. Winer is thanked for encouraging this work and Ms. Patricia A. McElroy for very able technical assistance. The financial support of the Califomia Air Resources Board, Contract No. A4-081-32 is gratefully acknowledged.
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