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Mutagenicity of tetranitroazoxytoluenes: a preliminary screening in Salmonella typhimurium strains TA100 and TA100NR
Environmental Mutagenesis
ELSEVIER
Mutation Research 335 (1995) 207-211
Mutagenicity of tetranitroazoxytoluenes: a preliminary screening in Salmonella typhimurium strains TA100 and TA100NR Ronald J. Spanggord *, Kathleen R. Stewart, Edward S. Riccio SR1 International, 333 Rauenswood Are., Menlo Park, CA 9402, USA
Received 23 January 1995;revised 19 April 1995;accepted 24 May 1995
Abstract
Tetranitroazoxytoluenes are polynitroaromatic compounds that can be produced during the microbial reduction of the explosive, 2,4,6-trinitrotoluene (TNT). The three major tetranitroazoxytoluenes were synthesized and tested in Salmonella typhimurium strains TAI00 and TA100NR. All compounds were mutagenic in TAI00 but not in TA100NR, indicating the need for nitroreductase activity to induce mutagenicity. The most active chemical was 4,4',6,6'-tetranitro-2,2'-azoxytoluene (2735 rev//zmol) followed by 2',4,6,6'-tetranitro-2',4-azoxytoluene (929 rev//zmol) and 2,2',6,6'-tetranitro-4,4'azoxytoluene (320 rev//~mol). These chemicals were more active than the aminodinitrotoluenes (298 rev//zmol for 2-amino-4,6-dinitrotoluene and 115 rev//zmol for 4-amino-2,6-dinitrotoluene) and only 4,4',6,6'-tetranitro-2,2'-azoxytoluene was more active than the parent compound, TNT (1022 rev//zmol). Keywords: Tetranitroazoxytoluenesderived from TNT; Mutagenicity in Salmonella typhimurium strains TAI00 and TAI00NR
I. I n t r o d u c t i o n
The explosive 2,4,6-trinitrotoluene (TNT) can persist in the environment for many years. While the health effects of TNT are well known (Gordon and Hartley, 1992), little is known about the health effects of microbial transformation products that can be generated in soil and water. Extensive investigations are now being conducted to evaluate the use of microorganisms as a technique to bioremediate soils
* Corresponding author. Tel.: (415)859-3822; Fax: (415)8592753.
contaminated with TNT. A general pathway used by many aerobic and anaerobic microbial systems to nullify the toxicity of TNT is to use nitroreductase enzymes to reduce the nitro groups to amino functions, producing aminodinitrotoluenes, diaminonitrotoluenes, and triaminotoluene (McCormick et al., 1976). The mutagenic potential of a number of these compounds has been reported by Spanggord et al. (1982). Intermediate C-nitroso and hydroxylamines formed in the reductive process can condense to form tetranitroazoxytoluenes. Kaplan and Kaplan (1982) have shown these compounds to be generated from TNT in compost piles, and McCormick et al. (1976) have demonstrated their formation from a
0165-1161/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0165- 1161(95)00022-4
R.J. Spanggord et al. / Mutation Research 335 (1995) 207-211
208
variety of aerobic and anaerobic bacteria. Since the majority of environmental organisms possess a nitroreductase capability, the potential to transform TNT into tetranitroazoxytoluene derivatives appears high. For TNT, four tetranitroazoxytoluene derivatives, the structures of which are shown in Fig. 1, can be deposited in the environment from microbial processes. However, 2',4,6,6'-tetranitro-2,4'-azoxytoluene is the least likely derivative to be formed from cross-condensation reactions based on high-energy transition states. Sitzman (1974) found this derivative only to be formed in small amounts from the condensation of oxidation products from 2amino-4,6-dinitrotoluene and 4-amino-2,6-dinitrotoluene which led primarily to 2,4',6,6'-tetranitro2',4-azoxytoluene. The health effects of the tetranitroazoxytoluenes are unknown. In order to identify their mutagenic potential, we synthesized the three primary tetranitroazoxytoluene derivatives in our laboratory and used the Ames Salmonella tester strains TA100 and TAI00NR to evaluate their mutagenic response. TAI00 had previously been identified in our laboratory as a responsive tester strain to nitroaromatic compounds, especially those related to TNT (Spanggord et al., 1982). TAI00NR was selected as an additional tester strain to evaluate the effect of nitroreductase on the mutagenic potential of tetranitroazoxytoluene derivatives.
CHa O"
CH 3
NO 2
NO2
H3C
(3" N=N
2.1. Test chemicals The tetranitroazoxytoluene compounds were synthesized according to the methods reported by Sitzman (1974). Structures were confirmed by nuclear magnetic resonance spectroscopy and melting points. Purity was assessed by liquid chromatography and the following purities were estimated based on peak-area response at 254 nm: 2,2',6,6'-tetranitro4,4'-azoxytoluene, > 99%; 4,4',6,6'-tetranitro-2,2'azoxytoluene, > 99%; 2,4',6,6'-tetranitro-2',4azoxytoluene, 97%. The 2,4,6-trinitrotoluene was obtained from K and K Laboratories and was recrystallized from methanol/water. The 4-amino-2,6-dinitrotoluene and 2-amino-4-6-dinitrotoluene were synthesized according to the method of Zbarskii et al. (1971).
2.2. Mutagenicio, testing The test procedures were basically the same as the plate incorporation assay described by Marion and Ames (1983). Strain TAI00 was supplied by Dr. Bruce Ames (University of California at Berkeley) and the nitroreductase-deficient strain TAI00NR was obtained from Dr. William Speck (Case Western Reserve University, Cleveland, Ohio). To 2 ml of molten top agar supplemented with biotin and lim-
CH 3 O"
NO 2
4,4',6,6'-Tetranitro-2,2'-azoxytoluene
O=N
2. Methods
NO2
NO 2
2',4,6,6'-Tetranitro-2,4'-azoxytoluene
CH 3
"O
CH 3 NO2
2,2',6,6'-Tetranitro-4,4'-azoxytoluene
NO 2
NO 2
2,4',6,6'-Tetranitro-2',4-azoxytoluene
Fig. 1. Chemical structures of the tetranitroazoxytoluenesproduced from 2,4,6-trinitroto]uene.
R.J. Spanggord et al. / Mutation Research 335 (1995) 207-211
209
Table 1 Mutagenicity of tetranitroazoxytoluenes and other polynitroaromatics in Salmonella ~'phimurium strains T A I 0 0 and TA100NR Chemical
/xg/Plate
4,4',6,6'-Tetranitro-2,2'-azoxytoluene
0 7.8 15.6 31.2 62.5 125 250
Rev/p~mol 2,4',6,6'-Tetranitro-2,4'-azoxytoluene
Rev//xmol 2,2',6,6'-Tetranitro-4,4'-azoxytoluene
Rev/~mol 4-Amino-2,6-dinitrotoluene
Rev//xmol 2-Amino-4,6-dinitrotoluene
Rev//zmol 2,4,6-Trinitrotoluene
Rev//~mol Nitrofurazone (positive control)
a Average of three determinations.
Mean revertants per plate _+ S.D. a TA- 100
TA- 100NR ! 34 + 21 150 + 21 142 + 3 149 + I1 138+6 121 + 8 148 + 10
0
120 + 10 296 + 28 566 _ 2 921 + 89 1211 + 5 3 1432 + 52 1684 + 67 2735 139 -4- 10 254 + 8 336 + 22 426 + 50 483 + 40 502 + 18 572 + 44 929 133 + 16 138 + 7 150 + 10 170 + 34 177 + 12 202 + 19 197 + 25 320 160 _ 12 170_+ 13 169 -t- 29 1 5 4 + 12 217 + 6 319 + 17 586 + 26 115 144 _+ 18 170_+ 24 181 _+ 10 251 + 4 418 + 26 801 + 3 1 1515-t-6 298 143 + 5 161 _ 2 180 + 15 220+33 319 + 32 814 + 43 1126 + 67 1022 137 + 6
1
584 + 20
0 7.8 15.6 31.2 62.5 125 250 0 7.8 15.6 31.2 62.5 125 250 0 31.2 62.5 125 250 500 1000 0 31.2 62.5 125 250 500 1000 0 7.8 15.6 31.2 62.5 125 250
156 146 142 132 103 112 113
+ + + + + + +
8 20 17 2 6 4 30
145 135 136 124 121 136 145
+ 20 + 21 + 9 + 11 + 14 + 7 _+ 12
148 + 9 141 _ 11 130 __. 19 121 + 10 114 _+ 12 95 + 7 80 _+ 15 155 _+ 6 148 _+ 19 140 _+ 7 123 _+ 10 133 + 16 111 + 3 9 0 + 19 120 + 145 + 143 _ 116+ 127 + 125 + 231 +
10 4 13 18 23 10 53
153 _ 22 165 + 5
R.J. Spanggord et al. / Mutation Research 335 (1995) 207-211
210
ited histidine were added 0.5 ml sodium phosphate buffer, 50 /zl tester strain, and 50 /zl test chemical solution. All dose solutions were prepared in dimethylsulfoxide (DMSO; Mallinckrodt). The mixture was stirred gently, then poured onto plates containing 25 ml of minimal glucose agar. The plates were incubated at 37°C for about 48 h, and then the revertant colonies were counted. Doses ranged from 7.8 to 250 /xg per plate for 2,4,6-trinitrotoluene and the three tetranitroazoxytoluene derivatives, and 31.2 to 1000 /xg per plate for 2-amino-4,6-dinitrotoluene and 4amino-2,6-dinitrotoluene. Dose selection was based on early studies with these test materials (Spanggord et al., 1982). The positive control chemicals used were nitrofurazone and sodium azide. Testing included controls and was performed using three plates per dose.
3. Results Table 1 shows the response elicited by strains TAI00 and TA100NR to the three tetranitroazoxytoluene derivatives, 2,4,6-TNT, 4-amino-2,6-dinitrotoluene, 2-amino-4,6-dinitrotoluene, and nitrofurazone (positive control). A dose-related response was observed for all chemicals with strain TAI00 but not
2000
2~.'-Azoxy ~
with strain TAI00NR, indicating that mutagenicity was derived from reduced products modulated by nitroreductase enzymes. The mutagenic response data are plotted in Fig. 2. Of the tetranitroazoxytoluene derivatives, the greatest response was observed in 4,4',6,6'-tetranitro-2,2'-azoxytoluene (2735 revertants per /xmol) followed by 2,4',6,6'-tetranitro-2',4azoxytoluene (929 revertants per /xmol) and 2,2',6,6'-tetranitro-4,4'-azoxytoluene (320 revertants per #moi). For these isomers, mutagenicity appears to be driven by the availability of a 4-position nitro group and suggests a selectivity by the nitroreductase enzymes in TAI00. The 2,2'-azoxy derivative has two nitro groups in the 4-positions whereas the 2',4-azoxy derivative has one and the 4,4'-azoxy derivative has zero. On this basis, we would predict that the untested isomer, the 2,4'-azoxy derivative, would possess a similar activity to the 2'4-azoxy isomer because of the availability of one nitro group in the 4-position. This same phenomenon was observed in the aminodinitrotoluenes, with 2-amino4,6-dinitrotoluene eliciting a significantly greater response than 4-amino-2,6-dinitrotoluene. The 2,4,6trinitrotoluene, which is more electron-deficient than the aminodinitrotoluenes, showed a dose-response curve similar to the 2,2'-azoxy derivative, but slightly attenuated possibly due to the presence of only one 4-position nitro group. A mutagenic response for TNT was observed at 250 /xg/plate in TAI00NR, but this effect was not investigated further.
2-ADNT
4. Discussion
Z
1000
,, .
o
4-ADNT
¢,r
4,4'-Azoxy 0
•
0
•
n
200
•
•
|
400
-
,
n
600
-
"
l
800
•
•
|
1000
•
•
1200
lag Nitroaromatic
Fig. 2. Mutagenic response of TNT biotransformation products in TAI00. 2,2'-Azoxy =4,4',6,6'-tetranitro-2,2'-azoxytoluene: TNT = 2,4,6-trinitrotoluene; 2',4-azoxy = 2,4',6,6'-tetranitro-2',-4azoxytoluene; 2-ADNT = 2-amino-4,6-dinitrotoluene; 4-ADNT = 4-amino-2,6-dinitrotoluene; 4,4'-azoxy = 2,2',6,6'-tetranitro-4,4'azoxytoluene.
The tetranitroazoxytoluenes were found to be mutagenic in Salmonella strain TA100 which is consistent with the behavior of related polynitroaromatic compounds. The chemical 4,4',6,6'-tetranitro-2,2'azoxytoluene elicited a 2.7-fold greater response per /xmol than the parent compound, TNT. Although the potential exists to form a more mutagenic chemical in active microbial environments from TNT, environmental impacts may be mitigated by the fact that two moles of TNT are required to form one mole of 4,4',6,6'-tetranitro-2,2'-azoxytoluene and aqueous solubilities are decreased from 120 ppm for TNT to 0.3 ppm for 4,4',6,6'-tetranitro-2,2'-azoxytoluene. The remaining tetranitroazoxytoluenes that we inves-
R.J. Spanggord et al. / Mutation Research 335 (1995) 207-211
tigated exhibit a lower mutagenic response than TNT and also possess lower aqueous solubilities (1.9 ppm for 2,2',6,6'-tetranitro-4,4'-azoxytoluene). In strain TA100, the mutagenic response of tetranitroazoxytoluene derivatives correlated well with the availability of a nitro group in the 4-position. The same correlation was also observed in two isomeric aminodinitrotoluenes, suggesting that the Salmonella T A I 0 0 nitroreductase possesses a selectivity that governs the mutagenic response. Nitroreductase activity varies widely in bacteria. Kinouchi and Ohnishi (1983) purified four types of nitroreductase from Bacteroides fragilis and demonstrated different substrate specificities for various nitro-containing compounds. McCoy et al. (1981) provided evidence for a series of nitroreductases in Salmonella typhimurium TA98, which implies that similar nitroreductases exist in TA100. Whether the selectivity by these enzymes for the 4-position nitro group is based on steric factors or slight differences in redox potential cannot be assessed at this time. Other bacterial systems possess nitroreductase systems that prefer the 2-position in polynitrotoluenes (Naumova et al., 1983), indicating that the mutagenic potential of the tetranitroazoxytoluenes described here can be expressed quite differently in other prokaryotic and eukaryotic cells. Thus, the mutagenic response observed in Salmonella strain T A I 0 0 may be highly selective, based on its nitroreductase content, and may represent only a small portion of the total mutagenic potential of nitroreductase-containing organisms in the environment. If nitroreductase selectivity is not an issue, then the mutagenic response could be controlled by the ability of intermediate C-nitroso, hydroxylamino-, or arylnitreniumazoxy derivatives to form adducts with DNA. In this case, steric factors may favor adduct formation at the 4-position. However, since the 2,2'azoxy derivative forms as readily as the 4,4'-azoxy derivative in chemical condensation reactions (Sitzman, 1974), the nucleophilic nature of reduced intermediates appears to be equivalent. More research is required on the nature of the D N A adducts to resolve
211
this question of nitroreductase selectivity versus intermediate reactivity as the responsible agent in mutation induction. The tetranitroazoxytoluenes possess a mutagenic potential of which the 2,2'-azoxy derivative exceeds that of the parent compound, 2,4,6-trinitrotoluene. Therefore, remediation processes should monitor the formation of these derivatives or minimize this reduction pathway until further studies can identify the health risks that these chemicals may pose.
References Gordon, L. and W.R. Hartley (1992) 2,4,6-Trinitrotoluene(TNT), in: Welford C. Roberts and William R. Hartley (Eds.), Drinking Water Health Advisory: Munitions, Lewis Publishers, Boca Raton, LA. Kaplan, D.L. and A.M. Kaplan (1982) Thermophilic biotransformations of 2,4,6-trinitrotoluene under simulated composting conditions, Appl. Environ. Microbiol., 44, 757-760. Kinouchi, T. and Y. Ohnishi (1983) Purification and characterization of 1-nitropyrene nitroreductases from Bacteroides fragilis. Appl. Environ. Microbiol., 46, 596-604. Marion, D.M. and B.N. Ames (1983) Revised methods for the Salmonella mutagenicity test, Mutation Res., 113, 173-215. McCoy, E.C., H.S. Rosenkrantz 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. McCormick, N.G., F.E. Feeherry and H.S. Levinson (1976) Microbial transformation of 2,4,6-trinitrotoluene and other nitroaromatic compounds, Appl. Environ. Microbiol., 31, 949958. Naumova, R.P., N.N. Amerkhanova and L.M. Zolotukhina (1983) Peculiarities of nitroreduction as a key stage in the microbial destruction of aromatic nitrocompounds, Appl. Biochem. Microbiol., 19, 400-404. Sitzman, M.E. (1974) Chemical reduction of 2,4,6-trinitrotoluene --initial products, J. Chem. Eng. Data, 19(2), 179-181. Spanggord, R.J., K.E. Mortelmans, A.F. Griffin and V.F. Simmon (1982) Mutagenicity in Salmonella ~phimurium and structure-activity relationships of wastewater components emanating from the manufacture of trinitrotoluene,Environ. Mutagen., 4, 163-179. Zbarskii, V.L., M.A. Sonis and E.Yu. Orlova (1971) Dinitrophenylcarboxylic acids in the Schmidt reaction, Zh. Prikl. Khim., 44( 11), 2578-2579.
ELSEVIER
Mutation Research 335 (1995) 207-211
Mutagenicity of tetranitroazoxytoluenes: a preliminary screening in Salmonella typhimurium strains TA100 and TA100NR Ronald J. Spanggord *, Kathleen R. Stewart, Edward S. Riccio SR1 International, 333 Rauenswood Are., Menlo Park, CA 9402, USA
Received 23 January 1995;revised 19 April 1995;accepted 24 May 1995
Abstract
Tetranitroazoxytoluenes are polynitroaromatic compounds that can be produced during the microbial reduction of the explosive, 2,4,6-trinitrotoluene (TNT). The three major tetranitroazoxytoluenes were synthesized and tested in Salmonella typhimurium strains TAI00 and TA100NR. All compounds were mutagenic in TAI00 but not in TA100NR, indicating the need for nitroreductase activity to induce mutagenicity. The most active chemical was 4,4',6,6'-tetranitro-2,2'-azoxytoluene (2735 rev//zmol) followed by 2',4,6,6'-tetranitro-2',4-azoxytoluene (929 rev//zmol) and 2,2',6,6'-tetranitro-4,4'azoxytoluene (320 rev//~mol). These chemicals were more active than the aminodinitrotoluenes (298 rev//zmol for 2-amino-4,6-dinitrotoluene and 115 rev//zmol for 4-amino-2,6-dinitrotoluene) and only 4,4',6,6'-tetranitro-2,2'-azoxytoluene was more active than the parent compound, TNT (1022 rev//zmol). Keywords: Tetranitroazoxytoluenesderived from TNT; Mutagenicity in Salmonella typhimurium strains TAI00 and TAI00NR
I. I n t r o d u c t i o n
The explosive 2,4,6-trinitrotoluene (TNT) can persist in the environment for many years. While the health effects of TNT are well known (Gordon and Hartley, 1992), little is known about the health effects of microbial transformation products that can be generated in soil and water. Extensive investigations are now being conducted to evaluate the use of microorganisms as a technique to bioremediate soils
* Corresponding author. Tel.: (415)859-3822; Fax: (415)8592753.
contaminated with TNT. A general pathway used by many aerobic and anaerobic microbial systems to nullify the toxicity of TNT is to use nitroreductase enzymes to reduce the nitro groups to amino functions, producing aminodinitrotoluenes, diaminonitrotoluenes, and triaminotoluene (McCormick et al., 1976). The mutagenic potential of a number of these compounds has been reported by Spanggord et al. (1982). Intermediate C-nitroso and hydroxylamines formed in the reductive process can condense to form tetranitroazoxytoluenes. Kaplan and Kaplan (1982) have shown these compounds to be generated from TNT in compost piles, and McCormick et al. (1976) have demonstrated their formation from a
0165-1161/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0165- 1161(95)00022-4
R.J. Spanggord et al. / Mutation Research 335 (1995) 207-211
208
variety of aerobic and anaerobic bacteria. Since the majority of environmental organisms possess a nitroreductase capability, the potential to transform TNT into tetranitroazoxytoluene derivatives appears high. For TNT, four tetranitroazoxytoluene derivatives, the structures of which are shown in Fig. 1, can be deposited in the environment from microbial processes. However, 2',4,6,6'-tetranitro-2,4'-azoxytoluene is the least likely derivative to be formed from cross-condensation reactions based on high-energy transition states. Sitzman (1974) found this derivative only to be formed in small amounts from the condensation of oxidation products from 2amino-4,6-dinitrotoluene and 4-amino-2,6-dinitrotoluene which led primarily to 2,4',6,6'-tetranitro2',4-azoxytoluene. The health effects of the tetranitroazoxytoluenes are unknown. In order to identify their mutagenic potential, we synthesized the three primary tetranitroazoxytoluene derivatives in our laboratory and used the Ames Salmonella tester strains TA100 and TAI00NR to evaluate their mutagenic response. TAI00 had previously been identified in our laboratory as a responsive tester strain to nitroaromatic compounds, especially those related to TNT (Spanggord et al., 1982). TAI00NR was selected as an additional tester strain to evaluate the effect of nitroreductase on the mutagenic potential of tetranitroazoxytoluene derivatives.
CHa O"
CH 3
NO 2
NO2
H3C
(3" N=N
2.1. Test chemicals The tetranitroazoxytoluene compounds were synthesized according to the methods reported by Sitzman (1974). Structures were confirmed by nuclear magnetic resonance spectroscopy and melting points. Purity was assessed by liquid chromatography and the following purities were estimated based on peak-area response at 254 nm: 2,2',6,6'-tetranitro4,4'-azoxytoluene, > 99%; 4,4',6,6'-tetranitro-2,2'azoxytoluene, > 99%; 2,4',6,6'-tetranitro-2',4azoxytoluene, 97%. The 2,4,6-trinitrotoluene was obtained from K and K Laboratories and was recrystallized from methanol/water. The 4-amino-2,6-dinitrotoluene and 2-amino-4-6-dinitrotoluene were synthesized according to the method of Zbarskii et al. (1971).
2.2. Mutagenicio, testing The test procedures were basically the same as the plate incorporation assay described by Marion and Ames (1983). Strain TAI00 was supplied by Dr. Bruce Ames (University of California at Berkeley) and the nitroreductase-deficient strain TAI00NR was obtained from Dr. William Speck (Case Western Reserve University, Cleveland, Ohio). To 2 ml of molten top agar supplemented with biotin and lim-
CH 3 O"
NO 2
4,4',6,6'-Tetranitro-2,2'-azoxytoluene
O=N
2. Methods
NO2
NO 2
2',4,6,6'-Tetranitro-2,4'-azoxytoluene
CH 3
"O
CH 3 NO2
2,2',6,6'-Tetranitro-4,4'-azoxytoluene
NO 2
NO 2
2,4',6,6'-Tetranitro-2',4-azoxytoluene
Fig. 1. Chemical structures of the tetranitroazoxytoluenesproduced from 2,4,6-trinitroto]uene.
R.J. Spanggord et al. / Mutation Research 335 (1995) 207-211
209
Table 1 Mutagenicity of tetranitroazoxytoluenes and other polynitroaromatics in Salmonella ~'phimurium strains T A I 0 0 and TA100NR Chemical
/xg/Plate
4,4',6,6'-Tetranitro-2,2'-azoxytoluene
0 7.8 15.6 31.2 62.5 125 250
Rev/p~mol 2,4',6,6'-Tetranitro-2,4'-azoxytoluene
Rev//xmol 2,2',6,6'-Tetranitro-4,4'-azoxytoluene
Rev/~mol 4-Amino-2,6-dinitrotoluene
Rev//xmol 2-Amino-4,6-dinitrotoluene
Rev//zmol 2,4,6-Trinitrotoluene
Rev//~mol Nitrofurazone (positive control)
a Average of three determinations.
Mean revertants per plate _+ S.D. a TA- 100
TA- 100NR ! 34 + 21 150 + 21 142 + 3 149 + I1 138+6 121 + 8 148 + 10
0
120 + 10 296 + 28 566 _ 2 921 + 89 1211 + 5 3 1432 + 52 1684 + 67 2735 139 -4- 10 254 + 8 336 + 22 426 + 50 483 + 40 502 + 18 572 + 44 929 133 + 16 138 + 7 150 + 10 170 + 34 177 + 12 202 + 19 197 + 25 320 160 _ 12 170_+ 13 169 -t- 29 1 5 4 + 12 217 + 6 319 + 17 586 + 26 115 144 _+ 18 170_+ 24 181 _+ 10 251 + 4 418 + 26 801 + 3 1 1515-t-6 298 143 + 5 161 _ 2 180 + 15 220+33 319 + 32 814 + 43 1126 + 67 1022 137 + 6
1
584 + 20
0 7.8 15.6 31.2 62.5 125 250 0 7.8 15.6 31.2 62.5 125 250 0 31.2 62.5 125 250 500 1000 0 31.2 62.5 125 250 500 1000 0 7.8 15.6 31.2 62.5 125 250
156 146 142 132 103 112 113
+ + + + + + +
8 20 17 2 6 4 30
145 135 136 124 121 136 145
+ 20 + 21 + 9 + 11 + 14 + 7 _+ 12
148 + 9 141 _ 11 130 __. 19 121 + 10 114 _+ 12 95 + 7 80 _+ 15 155 _+ 6 148 _+ 19 140 _+ 7 123 _+ 10 133 + 16 111 + 3 9 0 + 19 120 + 145 + 143 _ 116+ 127 + 125 + 231 +
10 4 13 18 23 10 53
153 _ 22 165 + 5
R.J. Spanggord et al. / Mutation Research 335 (1995) 207-211
210
ited histidine were added 0.5 ml sodium phosphate buffer, 50 /zl tester strain, and 50 /zl test chemical solution. All dose solutions were prepared in dimethylsulfoxide (DMSO; Mallinckrodt). The mixture was stirred gently, then poured onto plates containing 25 ml of minimal glucose agar. The plates were incubated at 37°C for about 48 h, and then the revertant colonies were counted. Doses ranged from 7.8 to 250 /xg per plate for 2,4,6-trinitrotoluene and the three tetranitroazoxytoluene derivatives, and 31.2 to 1000 /xg per plate for 2-amino-4,6-dinitrotoluene and 4amino-2,6-dinitrotoluene. Dose selection was based on early studies with these test materials (Spanggord et al., 1982). The positive control chemicals used were nitrofurazone and sodium azide. Testing included controls and was performed using three plates per dose.
3. Results Table 1 shows the response elicited by strains TAI00 and TA100NR to the three tetranitroazoxytoluene derivatives, 2,4,6-TNT, 4-amino-2,6-dinitrotoluene, 2-amino-4,6-dinitrotoluene, and nitrofurazone (positive control). A dose-related response was observed for all chemicals with strain TAI00 but not
2000
2~.'-Azoxy ~
with strain TAI00NR, indicating that mutagenicity was derived from reduced products modulated by nitroreductase enzymes. The mutagenic response data are plotted in Fig. 2. Of the tetranitroazoxytoluene derivatives, the greatest response was observed in 4,4',6,6'-tetranitro-2,2'-azoxytoluene (2735 revertants per /xmol) followed by 2,4',6,6'-tetranitro-2',4azoxytoluene (929 revertants per /xmol) and 2,2',6,6'-tetranitro-4,4'-azoxytoluene (320 revertants per #moi). For these isomers, mutagenicity appears to be driven by the availability of a 4-position nitro group and suggests a selectivity by the nitroreductase enzymes in TAI00. The 2,2'-azoxy derivative has two nitro groups in the 4-positions whereas the 2',4-azoxy derivative has one and the 4,4'-azoxy derivative has zero. On this basis, we would predict that the untested isomer, the 2,4'-azoxy derivative, would possess a similar activity to the 2'4-azoxy isomer because of the availability of one nitro group in the 4-position. This same phenomenon was observed in the aminodinitrotoluenes, with 2-amino4,6-dinitrotoluene eliciting a significantly greater response than 4-amino-2,6-dinitrotoluene. The 2,4,6trinitrotoluene, which is more electron-deficient than the aminodinitrotoluenes, showed a dose-response curve similar to the 2,2'-azoxy derivative, but slightly attenuated possibly due to the presence of only one 4-position nitro group. A mutagenic response for TNT was observed at 250 /xg/plate in TAI00NR, but this effect was not investigated further.
2-ADNT
4. Discussion
Z
1000
,, .
o
4-ADNT
¢,r
4,4'-Azoxy 0
•
0
•
n
200
•
•
|
400
-
,
n
600
-
"
l
800
•
•
|
1000
•
•
1200
lag Nitroaromatic
Fig. 2. Mutagenic response of TNT biotransformation products in TAI00. 2,2'-Azoxy =4,4',6,6'-tetranitro-2,2'-azoxytoluene: TNT = 2,4,6-trinitrotoluene; 2',4-azoxy = 2,4',6,6'-tetranitro-2',-4azoxytoluene; 2-ADNT = 2-amino-4,6-dinitrotoluene; 4-ADNT = 4-amino-2,6-dinitrotoluene; 4,4'-azoxy = 2,2',6,6'-tetranitro-4,4'azoxytoluene.
The tetranitroazoxytoluenes were found to be mutagenic in Salmonella strain TA100 which is consistent with the behavior of related polynitroaromatic compounds. The chemical 4,4',6,6'-tetranitro-2,2'azoxytoluene elicited a 2.7-fold greater response per /xmol than the parent compound, TNT. Although the potential exists to form a more mutagenic chemical in active microbial environments from TNT, environmental impacts may be mitigated by the fact that two moles of TNT are required to form one mole of 4,4',6,6'-tetranitro-2,2'-azoxytoluene and aqueous solubilities are decreased from 120 ppm for TNT to 0.3 ppm for 4,4',6,6'-tetranitro-2,2'-azoxytoluene. The remaining tetranitroazoxytoluenes that we inves-
R.J. Spanggord et al. / Mutation Research 335 (1995) 207-211
tigated exhibit a lower mutagenic response than TNT and also possess lower aqueous solubilities (1.9 ppm for 2,2',6,6'-tetranitro-4,4'-azoxytoluene). In strain TA100, the mutagenic response of tetranitroazoxytoluene derivatives correlated well with the availability of a nitro group in the 4-position. The same correlation was also observed in two isomeric aminodinitrotoluenes, suggesting that the Salmonella T A I 0 0 nitroreductase possesses a selectivity that governs the mutagenic response. Nitroreductase activity varies widely in bacteria. Kinouchi and Ohnishi (1983) purified four types of nitroreductase from Bacteroides fragilis and demonstrated different substrate specificities for various nitro-containing compounds. McCoy et al. (1981) provided evidence for a series of nitroreductases in Salmonella typhimurium TA98, which implies that similar nitroreductases exist in TA100. Whether the selectivity by these enzymes for the 4-position nitro group is based on steric factors or slight differences in redox potential cannot be assessed at this time. Other bacterial systems possess nitroreductase systems that prefer the 2-position in polynitrotoluenes (Naumova et al., 1983), indicating that the mutagenic potential of the tetranitroazoxytoluenes described here can be expressed quite differently in other prokaryotic and eukaryotic cells. Thus, the mutagenic response observed in Salmonella strain T A I 0 0 may be highly selective, based on its nitroreductase content, and may represent only a small portion of the total mutagenic potential of nitroreductase-containing organisms in the environment. If nitroreductase selectivity is not an issue, then the mutagenic response could be controlled by the ability of intermediate C-nitroso, hydroxylamino-, or arylnitreniumazoxy derivatives to form adducts with DNA. In this case, steric factors may favor adduct formation at the 4-position. However, since the 2,2'azoxy derivative forms as readily as the 4,4'-azoxy derivative in chemical condensation reactions (Sitzman, 1974), the nucleophilic nature of reduced intermediates appears to be equivalent. More research is required on the nature of the D N A adducts to resolve
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this question of nitroreductase selectivity versus intermediate reactivity as the responsible agent in mutation induction. The tetranitroazoxytoluenes possess a mutagenic potential of which the 2,2'-azoxy derivative exceeds that of the parent compound, 2,4,6-trinitrotoluene. Therefore, remediation processes should monitor the formation of these derivatives or minimize this reduction pathway until further studies can identify the health risks that these chemicals may pose.
References Gordon, L. and W.R. Hartley (1992) 2,4,6-Trinitrotoluene(TNT), in: Welford C. Roberts and William R. Hartley (Eds.), Drinking Water Health Advisory: Munitions, Lewis Publishers, Boca Raton, LA. Kaplan, D.L. and A.M. Kaplan (1982) Thermophilic biotransformations of 2,4,6-trinitrotoluene under simulated composting conditions, Appl. Environ. Microbiol., 44, 757-760. Kinouchi, T. and Y. Ohnishi (1983) Purification and characterization of 1-nitropyrene nitroreductases from Bacteroides fragilis. Appl. Environ. Microbiol., 46, 596-604. Marion, D.M. and B.N. Ames (1983) Revised methods for the Salmonella mutagenicity test, Mutation Res., 113, 173-215. McCoy, E.C., H.S. Rosenkrantz 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. McCormick, N.G., F.E. Feeherry and H.S. Levinson (1976) Microbial transformation of 2,4,6-trinitrotoluene and other nitroaromatic compounds, Appl. Environ. Microbiol., 31, 949958. Naumova, R.P., N.N. Amerkhanova and L.M. Zolotukhina (1983) Peculiarities of nitroreduction as a key stage in the microbial destruction of aromatic nitrocompounds, Appl. Biochem. Microbiol., 19, 400-404. Sitzman, M.E. (1974) Chemical reduction of 2,4,6-trinitrotoluene --initial products, J. Chem. Eng. Data, 19(2), 179-181. Spanggord, R.J., K.E. Mortelmans, A.F. Griffin and V.F. Simmon (1982) Mutagenicity in Salmonella ~phimurium and structure-activity relationships of wastewater components emanating from the manufacture of trinitrotoluene,Environ. Mutagen., 4, 163-179. Zbarskii, V.L., M.A. Sonis and E.Yu. Orlova (1971) Dinitrophenylcarboxylic acids in the Schmidt reaction, Zh. Prikl. Khim., 44( 11), 2578-2579.