Xenobiotica the fate of foreign compounds in biological systems
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Bacterial metabolism of 2,6-dinitrotoluene with Salmonella typhimurium and mutagenicity of the metabolites of 2,6-dinitrotoluene and related compounds M. Sayama, M. Inoue, M.-A. Mori, Y. Maruyama & H. Kozuka To cite this article: M. Sayama, M. Inoue, M.-A. Mori, Y. Maruyama & H. Kozuka (1992) Bacterial metabolism of 2,6-dinitrotoluene with Salmonella typhimurium and mutagenicity of the metabolites of 2,6-dinitrotoluene and related compounds, Xenobiotica, 22:6, 633-640, DOI: 10.3109/00498259209053126 To link to this article: http://dx.doi.org/10.3109/00498259209053126
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Date: 25 April 2016, At: 14:33
XENOBIOTICA,
1992, VOL. 22,
NO.
6, 633-640
Bacterial metabolism of 2,6-dinitrotoluene with Salmonella fyphimurium and mutagenicity of the metabolites of 2,6-dinitrotoluene and related compounds M . SAYAMAT, M. I N O U E t , M.-A. MORISg, Y. MARUYAMA and H. KOZUKAS
t Faculty of Engineering, Toyama University, 3190 Gofuku, Toyama 930, Japan
$ Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-01, Japan
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Received 16 October 1991; accepted 13 February 1992 1. Metabolites produced by the incubation of 2,6-dinitrotoluene (2,6-DNT) with Salmonella typhimurium strains T A 98, T A 98/1,8-DNP6 and T A 98NR were examined. Mutagenicities of bacterial products and related compounds were also examined in the Ames assay using T A 98 and T A 100. 2. 2,6-DNT was converted to 2-nitroso-6-nitrotoluene, 2-hydroxylamino-6nitrotoluene and 2-amino-6-nitrotoluene, with concurrent spontaneous formation of 2,2’dimethyl-3,3’-dinitroazoxybenzene, in the incubation with T A 98 and T A 98/1,I-DNP,. Capacity of T A 98NR to reduce 2,6-DNT was much lower than that of T A 98 and T A 98/1J-DNP,. showed no 3. Bacterial products, including 2,2’-dimethyl-3,3‘-dinitroazoxybenzene, mutagenic activity in the Ames assay. 4. Results indicate that the lack of mutagenic activity of 2,6-DNT is not due to low reductive metabolism of 2,6-DNT by bacteria, but due to the lack of mutagenic activity of the bacterial reductive products of 2,6-DNT.
Introduction 2,6-Dinitrotoluene (2,6-DNT), which is one of the major constituents of technical grade D N T , used in the production of toluene diisocyanates, has been shown to be a non-mutagen (Sayama et al. 1989) or possess almost no mutagenic activity (Couch et al. 1981, Tokiwa et al. 1981, Abernethy and Couch 1982) in the Ames assay using Salmonella typhimurium strains T A 98 or T A 100. Strain T A 98 has been shown to possess nitro reductase catalysing the activation of nitrofuran and niridazole (Rosenkranz et al. 1982). I n addition, the nitroreductase contained in T A 98 has been shown to be capable of reducing both 1-nitropyrene and 1,8dinitropyrene but the rates have been shown to be much lower than with nitrofuran (Bryant et al. 1984). Thus, it is important to determine whether the lack of mutagenicity of 2,6-DNT is due to the low metabolic rate of 2,6-DNT by strain T A 98 or due to low mutagenicity of the bacterial products. I n the present study we examined the metabolites of 2,6-DNT formed by S. typhimurium TA98, and the mutagenicities of bacterial metabolites and related compounds. Bacterial metabolism of 2,6-DNT was examined also in the nitroreductase-deficient (TA 98NR) and 0-acetyiase-deficient ( T A 98jl ,8-DNP6) mutants of S. typhimurium strain T A 98 (Rosenkranz and Speck 1975,1976, McCoy et al. 1982, 1983).
8 T o whom correspondence should be addressed. 0049-8254/92 $3.00 0 1992 Taylor & Francis Ltd.
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Experimental Materials 2,6-DNT, 2-amino-6-nitrotoluene and 2,6-diaminotoluene were obtained from Wako Pure Chemical Industries Ltd and purified by recrystallization from methanol. 2-Hydroxylamino-6-nitrotoluenewas prepared from 2,6-DNT by reduction with NH,Cl/Zn, as described previously (Mori et 01. 1986a). 2Nitroso-6-nitrotoluene was prepared from 2-amino-6-nitrotoluene by oxidation with Caro’s acid according to McIntyer and Simpson (1952). 2,2’-Dirnethyl-3,3’-dinitroazoxybenzene was prepared from 2-amino-6-nitrotoluene by oxidation with m-chloroperoxybenzoic acid according to Sitzman (1 974). 3,3’Diamino-2,2’-dimethylazoxybenzenewas prepared from 2-amino-6-nitrotoluene by reduction with glucose/NaOH according to Galbraith et al. (1951). 3,3’-Diamino-2,2’-dimethylazobenzene was prepared from 2-amino-6-nitrotoluene by reduction with NaOH/Zn according to Shine and Chamness (1963). 3,3’-Diacetylamino-2,2‘-dimethylazoxybenzene and 3,3’-diacetylamino-2,2’-dimethylazobenzene were prepared from 3,3’-diamino-2,2’-dimethylazoxybenzene and 3,3’-diamino-2,2’-dimethylazobenzene by acetylation with acetic acid/acetic anhydride, respectively. Chemical purities were confirmed by melting point measurement, elemental analysis, H-n.m.r. and mass spectrometries (table 1). Other chemicals were obtained from the following sources: sodium phenobarbital, 5,6-benzoflavone, 4-nitroquinoline (reagent grade), benzo[o]pyrene (ultra grade), dimethyl sulphoxide (spectrophotometric grade), histidine and D-biotin from Wako Pure Chemical Industries Ltd; NADPH, NADH and glucose 6-phosphate from Sigma Chemical Co.; Bacto nutrient broth from Difco Laboratories. Solvents used were of the highest grade commercially available. Microbial metabolism S . ryphimurium strains T A 98, T A 98NR and T A 98/1,8-DNP6 were grown in nutrient broth (5 ml) under shaking for 15 h at 37°C. Cultured solutions (0.1ml) were diluted by the addition of nutrient broth (3.8ml). T o 0.1 ml of dimethyl sulphoxide containing 2,6-DNT (3pmol) was added the diluted solution (39ml) of each strain, and the mixtures (4ml) were incubated at 37°C for 0-24 h aerobically. Incubotion mixture was extracted three times with ether (10ml) at pH 7 (neutral fraction) and pH 11 (basic fraction), respectively. Neutral and basic fractions were dried over anhydrous Na2S04, solvents evaporated off under N,, and residues were dissolved in 1-2ml of methanol. An appropriate volume (5-1Opl) of these solutions was injected into a Develosil ODS-BOTM column (Toso Cooperation CO:) linked to a h.p.1.c. (Hitachi model 650). H.p.1.c. operating conditions were as follows: flow rate, 1 mr/min; U.V. monitor, 250 nm; column temperature, ambient. Separation was performed using a gradient from acetonitrile-water (15 : 85 v/v) to acetonitrilewater (65 : 35 v/v), over 70min. Detection of metabolites was carried out by comparing the chromatograms of bacterial samples with those of blank samples. Blank samples were prepared by extracting incubation mixtures uncontaining 2,6-DNT in the same way as the bacterial samples. Identification of metabolites was carried out by the co-chromatography of bacterial samples with authentic standards. Quantities of metabolites were determined from standards curves plotted as peak area calculated automatically by Hitachi 655-61 processor A. Standard curve was made by running authentic standards through the same extraction procedure. A linear relationship between the amount of each compound and peak area was found over the range 10-150 ng. Microbial mutagenicity test The mutation test was done according to the method of Ames et al. (1975) with the suspension assay modification of Yahagi (1975). Compounds were dissolved in 0.1 ml of dimethyl sulphoxide, and tested using S . typhimurium strains T A 98 and T A 100 in the absence or presence of metabolic activation. Metabolic activation was carried out for 20 min at 37°C. The S9 fraction used was prepared from the livers of male Wistar rats (180-200g, Sankyo Laboratories) treated with sodium phenobarbital and 5,6benzoflavone, according to previously described procedures (Mori et al. 1985). Colonies on each plate (histidine revertants) were counted after a 2-day incubation at 37°C. Mutagenicities of 4-nitroquinoline (1 .Opg/plate) and benzo[a]pyrene (3.0 pgglplate) were determined in each experiment to monitor the sensitivity of the tester strain. Triplicate plates were run at each dose level ( 0 5 pmol/plate). Mutagenic activity is given as the number of revertants per pmol of the test compounds determined from the linear part of the dose-response curve.
Results Metabolites formed in the incubation of 2,6-DNTwith T A 98 for 12 h were 2-nitroso-6-nitrotoluene, 2-hydroxylamino-6-nitrotoluene, 2-amino-6-nitrotoluene and 2,2’-dimethyl-3,3’-dinitroazoxybenzene, which were detected in the neutral fraction (figure 1). No products including 2,6-diaminotoluene were detected
C7H6NzO3,, Cl,H,,N40,
c 14H16N40
2-Nitroso-6-nitrotoluene
2,2’-Dimethyl-3,3‘-dinitroazobenzene
3,3’-Diamino-2,2’-dimethylazoxybenzene
C,8H,oN40,
3,3’-Diacetylamino-2,2’-dirnethylazobenzene
16.7 (16.6) 169 16.8) 17.7 (17.8)
4.0 (4.9) 3.6 (3.7) 3.8 (3.8)
50.0 (49.9)
23.3 (23.4) 16.5 (16.3) 17.3 (17.1)
6.7 (6.7) 5.9 (6.0) 6.2 (6.2)
70.0 (70.1) 63.5 (63.4) 66.7 (66.7)
21.9 (21.8)
6.3 (6.2)
65.6 (65.4)
50.6 (50.7) 53.2 (53.1)
N
H
C
324 (M’)
340 (M’)
240 (M’)
256 (M’)
316 (M’)
166 (M’)
168 (M’)
Massa (mb)
2.22 (3H,s, CH,), 5.22 (lH, s, OH), 7.23-7.58 (3H, aromatic) 2.50 (3H, s, CH,), 642-8.43 (3H, aromatic) 2.38 (3H, s, CH,), 2.51 (3H, s, CH,), 7.60-8.25 (6H, aromatic) 1.95, 2.04 (each 3H, s, CH,); 5.02, 5.34 (each 2H, d, NH,); 661-7.08 (6H, aromatic) 241, 2.48 (each 3H, s, CH,); 5.08, 5.11 (each 2H, d, NH,); 677-7.02 (4H, aromatic) 208, 210 (each 3H, s, C3CH3); 212, 2.22 (each 3H, s, CH,); 7.27-7.60 (6H, aromatic) 212 (6H, s, 2 x COCH,); 251 (6H, s, 2 x CH,); 723-758 (6H, aromatic)
(6)
lH-n.m.r.b
‘Mass spectra (electron impact) were recorded on a JMS D-200 at 75 eV ionizing potential. ‘H-n.m.r. (in (CD,),SO) were recorded on a Varian XL-200 with Me,Si (6 = 0) as internal standard: s, singlet; d, doublet.
C,,HzoN403
3,3’-Diacetylamino-2,2’-dimethylazoxybenzene
C14H16N4
C7H8N203
2-Hydroxylamino-6-nitrotoluene
Formula
Elemental analysis calculation (found)
Data of elemental analysis, and mass and ‘H-n.m.r. spectral measurements of synthetic standards.
Compound
Table 1.
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s
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636
0
10
20
30 40 50 Retention time (min)
60
70
Figure 1 .
High-performance liquid chromatogram of neutral fraction from incubating 2,6-DNT with Salmonella typhimurium TA 98 for 12 h. The residue from neutral ether fraction was dissolved in 2 ml of methanol and appropriate volume ( 1 0 ~ 1was ) chromatographed on a reverse phase Develosil ODs-80TM column using a gradient sample; ----, blank. from acetonitrile-water (15 : 85 v/v) to acetonitrile-water (65 : 35 v/v). -, 2,6-DNT, 2,6-dinitrotoluene; 2 N 0 6 N T , 2-nitroso-6-nitrotoluene; 2HA6NT, 2-hydroxylamino6-nitrotoluene; 2A6NT, 2-amino-6-nitrotoluene: 2,2’-DM-3,3’-DNAOB, 2,2‘-dimethyl-3,3’dinitroazoxybenzene.
in the basic fraction from incubating 2,6-DNT with T A 98. Nitroazoxy compound, 2,2’-dimethyl-3,3’-dinitroazoxybenzene, detected in the incubation was thought to be a non-enzymic product, because 2,2’-dimethyl-3,3’-dinitroazoxybenzenewas produced rapidly by mixing 2-nitroso-6-nitrotoluene with 2-hydroxylamino-6nitrotoluene in methanol at room temperature. Figures 2-4 show the time course of the products formed by the incubation of 2,6-DNT with T A 98, T A 98/1,8-DNP6 and T A 98NR, respectively. In the incubation with T A 98 (figure 2) and T A 98/1,8-DNP6 (figure 3)) 2,6-DNT decreased rapidly with time and the level of remaining 2,6-DNT at 24 h was less than 5%. 2-Nitroso-6-nitrotoluene, 2-hydroxylamino-6-nitrotolueneand 2-amino-6nitrotoluene appeared after 4 h of incubation. Formation of 2,2‘-dimethyl-3,3‘dinitroazoxybenzene was seen after 8-h incubation. Results shown in figures 2 and 3 indicate that 2,6-DNT is converted to the amino compound stepwise via nitroso and hydroxylamino derivatives, with concurrent spontaneous formation of nitroazoxy compound. Quantified products accounted for only 40-92% of 2,6-DNT which had disappeared from incubation mixture (figures 2 and 3). Deficit may be due to the bacterial degradation of products formed. Consumption of 2,6-DNT in the incubation with T A 98NR was very slow, with about 70% of D N T remaining at 24 h (figure 4). Only a small amount of 2-amino-6nitrotoluene was detected at 12-h incubation, and even after 24-h incubation only small amounts of 2-nitroso-6-nitrotoluene, 2-hydroxylamino-6-nitrotoluene and 2-amino-6-nitrotoluene were found (figure 4). From the results of figures 2-4 it is
Bacterial metabolism of 2,6-dinitrotoluene
637
P
I CI
50
Q)
‘0 u)
CI
0
U
P a
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0
Figure 2.
4
8
12
16
20
24
Incubation time (h) Time course of metabolism of 2,6-DNT by Salmonella typhimurium T A 98.
Bacteria were incubated aerobically with 3 pmol’of 2,6-DNT in 3.9 ml of defined medium for timed intervals. Neutral ether fractions were subjected to h.p.1.c. Each point represents mean k S D for 2,6-DNT; ( A-A), 2-nitroso-6-nitrotoluene; (0-0), 2three samples. ( 0 - O ) , hydroxylamino-6-nitrotoluene;(M-M), 2-amino-6-nitrotoluene; (AA ) , 2,2’-dimethyl3,3’-dinitroazoxybenzene.
-L -gj
100
P
r
0
s
u
P
2
$ 50
CI
U u)
I
V
J 0
P
a
0
4 4
8
12
16
20
24
Incubation time (h)
Figure 3.
Time course of metabolism of 2,6-DNT by Salmonella typhimurium T A 98/1,8-DNP6. Bacteria were incubated aerobically with 3 pmol of 2,6-DNT in 3.9 ml of defined medium for timed intervals. Neutral ether fractions were subjected to h.p.1.c. Each point represents mean k SD for three samples. ( 0 - O ) , 2,6-DNT; ( A-A), 2-nitroso-6-nitrotoluene; (0-0), 2-hydroxylamino-6-nitrotoluene; (W-W), 2-amino-6-nitrotoluene; ( A-A), 2,Tdimethyl-3,3’-dinitroazoxybenzene.
apparent that T A 98NR does not metabolize 2,6-DNT efficiently, but that T A 98 and T A 98/1,8-DNP6 do metabolize 2,6-DNT. 2,6-DNT, 2-amino-6-nitrotoluene and 2-hydroxylamino-6-nitrotoluene showed almost no mutagenic activity ( < 2 or 3 times of the spontaneous revertants) for strains T A 98 and T A 100 in the absence or presence of S9 mix, whereas 2,6diaminotoluene showed higher mutagenic activity (about 123 his+ revertants/pmoI) only in the presence of S9 mix. These findings are in agreement with the results of earlier investigations (Couch et al. 1981, Tokiwa et al. 1981, Abernethy and Couch 1982, Mori et al. 1986 b, Dybing and Thorgeirsson 1977).
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s2
:
a
0
4
8
12
20
16
24
Incubation time (h)
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Figure 4. Time course of metabolism of 2,6-DNT by Salmonella typhimurium T A 98NR. Bacteria were incubated aerobically with 3 pnol of 2,6-DNT in 3.9ml of defined medium for timed intervals. Neutral ether fractions were subjected to h.p.1.c. Each point represents mean S D for three samples. ( 0 - O ) , 2,6-DNT; (A-A), 2-nitroso-6-nitrotoluene; (0-0), 2hydroxylamino-6-nitrotoluene;( m-m), 2-amino-6-nitrotoluene.
Table 2.
Mutagenicity of bacterial products of 2,6-DNT and its related compounds for Solmonella typhimurium strains T A 98 and T A 100. His’ revertantsjpnol’ T A 98 Compound
-S9 mix
2-Nitroso-6-nitrotoluene 2,2’-Dimethyl-3,3’-dinitroazoxybenzene 3,3’-Diamino-2,2’-dimethylazoxybenzene 3,3’-Diamino-2,2’-dirnethylazobenzene 3,3’-Diacetylamino-2,2’-dimethylazoxybenzene 3,3’-Diacetylamino-2,2‘-dimethylazobenzene
n.d. n.d. n.d. n.d. n.d. n.d.
T A 100
+S9 mix
- S9 mix
+ S9 mix
n.d. n.d.
n.d. n.d. n.d. n.d. n.d. n.d.
n.d. n.d. n.d.
15300+3127 144500k8770 n.d. n.d.
12700+2361 n.d. n.d.
* Mutagenic activity was calculated from linear part of dose-response curves. Values are mean fS D of his’ revertants/pmol from 3 to 5 doses. Spontaneous revertants were subtracted: 25 (TA 98, - S9), 63 (TA 98, +S9), 110 (TA 100, -S9), 143 (TA 100, +S9). n.d., Not detected, means less than twice of the number of spontaneous revertants.
2-Nitroso-6-nitrotoluene, 2,2’-dimethyl-3,3’-dinitroazoxybenzene, 3,3’diacetylamino-2,2‘-dimethylazoxybenzene and 3,3’-diacetylamino-2,2’dimethylazobenzene also showed almost no mutagenic activity for T A 98 and T A 100 (table 2). T h e remaining aminoazoxy and aminoazo compounds, 3,3’-diamino2,2’-dimethylaminoazoxybenzene and 3,3’-diamino-2,2‘-dimethylazobenzene, showed no mutagenic activity in the absence of S9 mix, but with S9 mix these compounds showed higher mutagenic activity for T A 98 or T A 100 (table 2). Results indicate that bacterial products of 2,6-DNT are non-mutagenic for T A 98 and T A
100. Discussion The present results show that 2,6-DNT is converted to 2-nitroso-6-nitrotoluene, 2-hydroxylamino-6-nitrotolueneand 2-amino-6-nitrotoluene, with concurrent spontaneous formation of 2,2’-dimethyl-3,3’-dinitroazoxybenzene,on in-
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Bacterial metabolism of 2,6-dinitrotoluene
639
cubation with Salmonella typhimurium strains T A 98 and T A 98/1,8-DNP6. In addition, results show that the rate of reduction of 2,6-DNT by T A 98NR was much lower compared with those by T A 98 and T A 98/1 ,8-DNP6. The finding that T A 98 and TA 98/1,8-DNP, metabolize 2,6-DNT is consistent with the results of Bryant et al. (1984) who have shown that both T A 98 and T A 98/1,8-DNP6 metabolize dinitropyrene to aminonitropyrene. In addition, biotransformation of 2,6-DNT seen in T A 98 (figure 2) and T A 98jl ,8-DNP6 (figure 3) was similar to the observation that 2,4-DNT, an isomer of 2,6-DNT, was converted to aminonitrotoluenes and nitrosonitrotoluenes by Microsporium spores (McComic et al. 1978) and the products of 2,4-DNT formed by mixed bacteria derived from activated sludge were aminonitrotoluenes and azoxy compounds (Dickson et al. 1984). The marked activity seen in the aerobic incubation of 2,6-DNT with T A 98 and TA 98/1,8-DNP6 is consistent with the result of Tatsumi et al. (1982), who have shown that T A 100 possesses two oxygen-insensitive activities mediating the reduction of nitrofuran: the major one linked to NADPH and the minor one linked to NADH. In this connection, three types of nitroreductases (I, I11 and IV) which mediate specifically the reduction of 1-nitropyrene, 1,8-dinitropyrene and 4nitroquinoline, and a nitroreductase (11) which possesses broad specificity have been purified from the extracts of Bacteroids fragilis (Kinouchi and Ohnishi 1983). However, it remains to be determined which type of nitroreductase is active in the reduction of 1,g-dinitropyrene and 4-nitroquinoline in TA 98. The finding that bacterial products including the nitroazoxy compound showed no mutagenic potency (table 2) and no other aminoazoxy or aminoazo compounds were detected in the bacterial metabolism (figure 2) indicated that the lack of mutagenic activity of 2,6-DNT was not due to poor reductive metabolism of this compound by the bacteria, but due to the lack of mutagenic potency of the reductive intermediates produced. Since 3,3’-diacetylamino-2,2’-dimethylazoxybenzeneand 3,3’-diacetylamino2,2‘-dimethylazobenzene showed no mutagenic activity (table 2), it is suggested that the oxidation of amino groups of 3,3‘-diamino-2,2’-dimethylazoxybenzene and 3,3’-diamino-2,2‘-dimethylazobenzene is an essential step in the activation of these compounds to ultimate mutagens.
Acknowledgements The authors are grateful to Professor H. S. Rosenkranz, Case Western Reserve University for supplying Salmonella typhimurium T A 98NR and T A 98/1,8-DNP6, and also to Professor I. Tomita, Shizuoka College of Pharmacy for supplying Salmonella typhimurium T A 98 and T A 100. References ABERNETHY, D. J., and COUCH, D. B., 1982, Cytotoxicity and mutagenicity of dinitrotoluenes in Chinese hamster ovary cells. Mutation Research, 103, 53-59. AMES,B. N., MCCANN, J . , and YAMASAKI, E., 1975, Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsomesmutagenicity test. Mutation Research, 31,347-364. BRYANT, D. W., MCCALLA, D. R., LULTSCHIK, P., QUILLIAN, M. A,, and MCCARRY, B. E., 1984, Metabolism of 1,8-dinitropyrene by Salmonella typhimurium. Chemico-Biological Interactions, 49, 351-368. COUCH,D. V., ALLEN,P. F., and ABERNETHY, D. J . , 1981, The mutagenicity of dinitrotoluenes in Salmonella typhimurium. Mutation Research, 90, 373-383.
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DICKSON, L., THOMSON, K., and ANDERSON, A. C., 1984, Identification of nitroso compounds from biotransformation of 2,4-dinitrotoluene. Applied and Environmental Microbiology, 47,1295-1298. X. X., 1977, Metabolic activation of 2,4-diaminoanisole, a hair-dye DYBING,E., and THORGEIRSSON, components. I. Role of cytochrome P-450 metabolism in mutagenicity in vitro. Biochemical Pharmacology, 26, 729-734. W., DEGERINC, E. F., and HITCH,E. F., 1951, The alkaline reduction of aromatic nitro GALBRAITH, compounds with glucose. Journal of the American Chemical Society, 73, 1323-1324. KINOUCHI, T., and OHNISHI, Y.,1983, Purification and characterization of 1-nitropyrene nitroreductase from Bacteroides fragilis. Applied and Environmental Microbiology, 46, 596-604. J. H., and KAPLAN, A. M., 1978, Identification of biotransformation MCCOMIC, N. G., CORNELL, products from 2,4-dinitrotoluene. Applied and Environmental Microbiology, 35, 945-948. H. S., 1982, Esterification of arylhydroxylamines: McCoy, E. C., McCoy, G. D., and ROSENKRANZ, evidence for a specific gene product in mutagenesis. Biochemical and Biophysical Research Communication, 108, 1362-1 367. McCoy, E. C., ANDERS, M., and ROSENKRANZ, H. S., 1983, The basis of the insensitivity of Salmonella typhimurium strain TA 98/1 ,8-DNP6 to the mutagenic activation of nitroarenes. Mutation Research, 121, 17-23. MCINTYER, J., and SIMPSON, J. C. E., 1952, Cinnolines and other heterocyclic type in relation to the chemotherapy of trypanosomiasia, part IV. Synthesis of azocinnoline derivatives. Journal of the Chemical Society, 2606-26 15 . MORI,M., MIYAHARA, T., MOTO-0,K., FUKUKAWA, M., KOZUKA, H., MIYAGOSHI, M., and NAGAYAMA, T., 1985, Mutagenicity of urinary metabolites of 2.4-dinitrotoluene to Salmonella typhimurium. Chemical and Pharmaceutical Bulletin, 33, 4 5 5 6 4 6 3 . H., 1986a, Preparation of some MORI,M., INOUE,M., NUNOZAWA, T., MIYAHARA, T., and KOZUKA, acetylated, reduced and oxidized derivatives of 2,4-diaminotoluene and 2,6-dinitrotoluene. Chemical and Pharmaceutical Bulletin, 34, 48594861. MORI, M., SAYAMA, M., FUTAGAMI, M., NAITO,M., MIYAHARA, T., and KOZUKA, H., 1986b, Mutagenicity of some hydroxylaminotoluene derivatives towards Salmonella typhimurium in esterification system. Journal of Pharmacobio-Dynamics, 9, 1036-1039. W. T., 1975, Mutagenicity of metronidazole: activation by mammalian ROSENKRANZ, H. S., and SPECK, liver microsomes. Biochemical and Biophysical Research Communication, 66, 520-525. ROSENKRANZ, H. S., and SPECK,W. T., 1976, Activation of nitrofurantoin to a mutagen by rat nitroreductase. Biochemical Pharmacology, 25, 1555-1 556. R., and ROSENKRANZ, H. S., 1982, Evidence for ROSENKRANZ, E. J., McCoy, E. C., MERMELSTEIN, the existence of distinct nitro reductase in Salmonella typhimurium: roles in mutagenesis. Carcinogenesis, 3, 121-123. T., and KOZUKA,H., 1989, SAYAMA, M., MORI, M., SHIROKAWA, T., INOUE,M., MIYAHARA, Mutagenicity of 2,6-dinitrotoluene and its metabolites, and their related compounds in Salmonella typhimurium. Mutation Research, 226, 181-184. J. T., 1963, The benzidine rearrangement, V, first-order acid dependence SHINE,H. J., and CHAMNESS, Journal of Organic Chemistry, 28, 1232-1236. with 4,4’-divinylhydrazobenzene. SITZMAN, M. E., 1974, Chemical reduction of 2,4,6-trinitrotoluene-initialproducts. Journal of Chemical and Engineering Data, 19, 179-181. TATSUMI, K., Dot, T., YOSHIMURA, H., KOGA,H., and HORIUCHI, T., 1982, Oxygen-insensitive nitrofuran reductases in Salmonella typhimurium T A 100. Journal of Pharmacobio-Dynamics, 5, 423429. TOKIWA, H., NAKAGAWA, R., and OHNISHI, Y., 1981, Mutagenic assay of dinitrotoluenes in Salmonella typhimurium. Mutation Research, 91, 321-325. T., 1975, Screening methods using microbes for the enviropmental carcinogen. Tanpakushitsu YAHAGI, Kakusan Koso (Protein, Nucleic acid and Enzyme), 20, 1178-1189.
ISSN: 0049-8254 (Print) 1366-5928 (Online) Journal homepage: http://www.tandfonline.com/loi/ixen20
Bacterial metabolism of 2,6-dinitrotoluene with Salmonella typhimurium and mutagenicity of the metabolites of 2,6-dinitrotoluene and related compounds M. Sayama, M. Inoue, M.-A. Mori, Y. Maruyama & H. Kozuka To cite this article: M. Sayama, M. Inoue, M.-A. Mori, Y. Maruyama & H. Kozuka (1992) Bacterial metabolism of 2,6-dinitrotoluene with Salmonella typhimurium and mutagenicity of the metabolites of 2,6-dinitrotoluene and related compounds, Xenobiotica, 22:6, 633-640, DOI: 10.3109/00498259209053126 To link to this article: http://dx.doi.org/10.3109/00498259209053126
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Date: 25 April 2016, At: 14:33
XENOBIOTICA,
1992, VOL. 22,
NO.
6, 633-640
Bacterial metabolism of 2,6-dinitrotoluene with Salmonella fyphimurium and mutagenicity of the metabolites of 2,6-dinitrotoluene and related compounds M . SAYAMAT, M. I N O U E t , M.-A. MORISg, Y. MARUYAMA and H. KOZUKAS
t Faculty of Engineering, Toyama University, 3190 Gofuku, Toyama 930, Japan
$ Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-01, Japan
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Received 16 October 1991; accepted 13 February 1992 1. Metabolites produced by the incubation of 2,6-dinitrotoluene (2,6-DNT) with Salmonella typhimurium strains T A 98, T A 98/1,8-DNP6 and T A 98NR were examined. Mutagenicities of bacterial products and related compounds were also examined in the Ames assay using T A 98 and T A 100. 2. 2,6-DNT was converted to 2-nitroso-6-nitrotoluene, 2-hydroxylamino-6nitrotoluene and 2-amino-6-nitrotoluene, with concurrent spontaneous formation of 2,2’dimethyl-3,3’-dinitroazoxybenzene, in the incubation with T A 98 and T A 98/1,I-DNP,. Capacity of T A 98NR to reduce 2,6-DNT was much lower than that of T A 98 and T A 98/1J-DNP,. showed no 3. Bacterial products, including 2,2’-dimethyl-3,3‘-dinitroazoxybenzene, mutagenic activity in the Ames assay. 4. Results indicate that the lack of mutagenic activity of 2,6-DNT is not due to low reductive metabolism of 2,6-DNT by bacteria, but due to the lack of mutagenic activity of the bacterial reductive products of 2,6-DNT.
Introduction 2,6-Dinitrotoluene (2,6-DNT), which is one of the major constituents of technical grade D N T , used in the production of toluene diisocyanates, has been shown to be a non-mutagen (Sayama et al. 1989) or possess almost no mutagenic activity (Couch et al. 1981, Tokiwa et al. 1981, Abernethy and Couch 1982) in the Ames assay using Salmonella typhimurium strains T A 98 or T A 100. Strain T A 98 has been shown to possess nitro reductase catalysing the activation of nitrofuran and niridazole (Rosenkranz et al. 1982). I n addition, the nitroreductase contained in T A 98 has been shown to be capable of reducing both 1-nitropyrene and 1,8dinitropyrene but the rates have been shown to be much lower than with nitrofuran (Bryant et al. 1984). Thus, it is important to determine whether the lack of mutagenicity of 2,6-DNT is due to the low metabolic rate of 2,6-DNT by strain T A 98 or due to low mutagenicity of the bacterial products. I n the present study we examined the metabolites of 2,6-DNT formed by S. typhimurium TA98, and the mutagenicities of bacterial metabolites and related compounds. Bacterial metabolism of 2,6-DNT was examined also in the nitroreductase-deficient (TA 98NR) and 0-acetyiase-deficient ( T A 98jl ,8-DNP6) mutants of S. typhimurium strain T A 98 (Rosenkranz and Speck 1975,1976, McCoy et al. 1982, 1983).
8 T o whom correspondence should be addressed. 0049-8254/92 $3.00 0 1992 Taylor & Francis Ltd.
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Experimental Materials 2,6-DNT, 2-amino-6-nitrotoluene and 2,6-diaminotoluene were obtained from Wako Pure Chemical Industries Ltd and purified by recrystallization from methanol. 2-Hydroxylamino-6-nitrotoluenewas prepared from 2,6-DNT by reduction with NH,Cl/Zn, as described previously (Mori et 01. 1986a). 2Nitroso-6-nitrotoluene was prepared from 2-amino-6-nitrotoluene by oxidation with Caro’s acid according to McIntyer and Simpson (1952). 2,2’-Dirnethyl-3,3’-dinitroazoxybenzene was prepared from 2-amino-6-nitrotoluene by oxidation with m-chloroperoxybenzoic acid according to Sitzman (1 974). 3,3’Diamino-2,2’-dimethylazoxybenzenewas prepared from 2-amino-6-nitrotoluene by reduction with glucose/NaOH according to Galbraith et al. (1951). 3,3’-Diamino-2,2’-dimethylazobenzene was prepared from 2-amino-6-nitrotoluene by reduction with NaOH/Zn according to Shine and Chamness (1963). 3,3’-Diacetylamino-2,2‘-dimethylazoxybenzene and 3,3’-diacetylamino-2,2’-dimethylazobenzene were prepared from 3,3’-diamino-2,2’-dimethylazoxybenzene and 3,3’-diamino-2,2’-dimethylazobenzene by acetylation with acetic acid/acetic anhydride, respectively. Chemical purities were confirmed by melting point measurement, elemental analysis, H-n.m.r. and mass spectrometries (table 1). Other chemicals were obtained from the following sources: sodium phenobarbital, 5,6-benzoflavone, 4-nitroquinoline (reagent grade), benzo[o]pyrene (ultra grade), dimethyl sulphoxide (spectrophotometric grade), histidine and D-biotin from Wako Pure Chemical Industries Ltd; NADPH, NADH and glucose 6-phosphate from Sigma Chemical Co.; Bacto nutrient broth from Difco Laboratories. Solvents used were of the highest grade commercially available. Microbial metabolism S . ryphimurium strains T A 98, T A 98NR and T A 98/1,8-DNP6 were grown in nutrient broth (5 ml) under shaking for 15 h at 37°C. Cultured solutions (0.1ml) were diluted by the addition of nutrient broth (3.8ml). T o 0.1 ml of dimethyl sulphoxide containing 2,6-DNT (3pmol) was added the diluted solution (39ml) of each strain, and the mixtures (4ml) were incubated at 37°C for 0-24 h aerobically. Incubotion mixture was extracted three times with ether (10ml) at pH 7 (neutral fraction) and pH 11 (basic fraction), respectively. Neutral and basic fractions were dried over anhydrous Na2S04, solvents evaporated off under N,, and residues were dissolved in 1-2ml of methanol. An appropriate volume (5-1Opl) of these solutions was injected into a Develosil ODS-BOTM column (Toso Cooperation CO:) linked to a h.p.1.c. (Hitachi model 650). H.p.1.c. operating conditions were as follows: flow rate, 1 mr/min; U.V. monitor, 250 nm; column temperature, ambient. Separation was performed using a gradient from acetonitrile-water (15 : 85 v/v) to acetonitrilewater (65 : 35 v/v), over 70min. Detection of metabolites was carried out by comparing the chromatograms of bacterial samples with those of blank samples. Blank samples were prepared by extracting incubation mixtures uncontaining 2,6-DNT in the same way as the bacterial samples. Identification of metabolites was carried out by the co-chromatography of bacterial samples with authentic standards. Quantities of metabolites were determined from standards curves plotted as peak area calculated automatically by Hitachi 655-61 processor A. Standard curve was made by running authentic standards through the same extraction procedure. A linear relationship between the amount of each compound and peak area was found over the range 10-150 ng. Microbial mutagenicity test The mutation test was done according to the method of Ames et al. (1975) with the suspension assay modification of Yahagi (1975). Compounds were dissolved in 0.1 ml of dimethyl sulphoxide, and tested using S . typhimurium strains T A 98 and T A 100 in the absence or presence of metabolic activation. Metabolic activation was carried out for 20 min at 37°C. The S9 fraction used was prepared from the livers of male Wistar rats (180-200g, Sankyo Laboratories) treated with sodium phenobarbital and 5,6benzoflavone, according to previously described procedures (Mori et al. 1985). Colonies on each plate (histidine revertants) were counted after a 2-day incubation at 37°C. Mutagenicities of 4-nitroquinoline (1 .Opg/plate) and benzo[a]pyrene (3.0 pgglplate) were determined in each experiment to monitor the sensitivity of the tester strain. Triplicate plates were run at each dose level ( 0 5 pmol/plate). Mutagenic activity is given as the number of revertants per pmol of the test compounds determined from the linear part of the dose-response curve.
Results Metabolites formed in the incubation of 2,6-DNTwith T A 98 for 12 h were 2-nitroso-6-nitrotoluene, 2-hydroxylamino-6-nitrotoluene, 2-amino-6-nitrotoluene and 2,2’-dimethyl-3,3’-dinitroazoxybenzene, which were detected in the neutral fraction (figure 1). No products including 2,6-diaminotoluene were detected
C7H6NzO3,, Cl,H,,N40,
c 14H16N40
2-Nitroso-6-nitrotoluene
2,2’-Dimethyl-3,3‘-dinitroazobenzene
3,3’-Diamino-2,2’-dimethylazoxybenzene
C,8H,oN40,
3,3’-Diacetylamino-2,2’-dirnethylazobenzene
16.7 (16.6) 169 16.8) 17.7 (17.8)
4.0 (4.9) 3.6 (3.7) 3.8 (3.8)
50.0 (49.9)
23.3 (23.4) 16.5 (16.3) 17.3 (17.1)
6.7 (6.7) 5.9 (6.0) 6.2 (6.2)
70.0 (70.1) 63.5 (63.4) 66.7 (66.7)
21.9 (21.8)
6.3 (6.2)
65.6 (65.4)
50.6 (50.7) 53.2 (53.1)
N
H
C
324 (M’)
340 (M’)
240 (M’)
256 (M’)
316 (M’)
166 (M’)
168 (M’)
Massa (mb)
2.22 (3H,s, CH,), 5.22 (lH, s, OH), 7.23-7.58 (3H, aromatic) 2.50 (3H, s, CH,), 642-8.43 (3H, aromatic) 2.38 (3H, s, CH,), 2.51 (3H, s, CH,), 7.60-8.25 (6H, aromatic) 1.95, 2.04 (each 3H, s, CH,); 5.02, 5.34 (each 2H, d, NH,); 661-7.08 (6H, aromatic) 241, 2.48 (each 3H, s, CH,); 5.08, 5.11 (each 2H, d, NH,); 677-7.02 (4H, aromatic) 208, 210 (each 3H, s, C3CH3); 212, 2.22 (each 3H, s, CH,); 7.27-7.60 (6H, aromatic) 212 (6H, s, 2 x COCH,); 251 (6H, s, 2 x CH,); 723-758 (6H, aromatic)
(6)
lH-n.m.r.b
‘Mass spectra (electron impact) were recorded on a JMS D-200 at 75 eV ionizing potential. ‘H-n.m.r. (in (CD,),SO) were recorded on a Varian XL-200 with Me,Si (6 = 0) as internal standard: s, singlet; d, doublet.
C,,HzoN403
3,3’-Diacetylamino-2,2’-dimethylazoxybenzene
C14H16N4
C7H8N203
2-Hydroxylamino-6-nitrotoluene
Formula
Elemental analysis calculation (found)
Data of elemental analysis, and mass and ‘H-n.m.r. spectral measurements of synthetic standards.
Compound
Table 1.
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s
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636
0
10
20
30 40 50 Retention time (min)
60
70
Figure 1 .
High-performance liquid chromatogram of neutral fraction from incubating 2,6-DNT with Salmonella typhimurium TA 98 for 12 h. The residue from neutral ether fraction was dissolved in 2 ml of methanol and appropriate volume ( 1 0 ~ 1was ) chromatographed on a reverse phase Develosil ODs-80TM column using a gradient sample; ----, blank. from acetonitrile-water (15 : 85 v/v) to acetonitrile-water (65 : 35 v/v). -, 2,6-DNT, 2,6-dinitrotoluene; 2 N 0 6 N T , 2-nitroso-6-nitrotoluene; 2HA6NT, 2-hydroxylamino6-nitrotoluene; 2A6NT, 2-amino-6-nitrotoluene: 2,2’-DM-3,3’-DNAOB, 2,2‘-dimethyl-3,3’dinitroazoxybenzene.
in the basic fraction from incubating 2,6-DNT with T A 98. Nitroazoxy compound, 2,2’-dimethyl-3,3’-dinitroazoxybenzene, detected in the incubation was thought to be a non-enzymic product, because 2,2’-dimethyl-3,3’-dinitroazoxybenzenewas produced rapidly by mixing 2-nitroso-6-nitrotoluene with 2-hydroxylamino-6nitrotoluene in methanol at room temperature. Figures 2-4 show the time course of the products formed by the incubation of 2,6-DNT with T A 98, T A 98/1,8-DNP6 and T A 98NR, respectively. In the incubation with T A 98 (figure 2) and T A 98/1,8-DNP6 (figure 3)) 2,6-DNT decreased rapidly with time and the level of remaining 2,6-DNT at 24 h was less than 5%. 2-Nitroso-6-nitrotoluene, 2-hydroxylamino-6-nitrotolueneand 2-amino-6nitrotoluene appeared after 4 h of incubation. Formation of 2,2‘-dimethyl-3,3‘dinitroazoxybenzene was seen after 8-h incubation. Results shown in figures 2 and 3 indicate that 2,6-DNT is converted to the amino compound stepwise via nitroso and hydroxylamino derivatives, with concurrent spontaneous formation of nitroazoxy compound. Quantified products accounted for only 40-92% of 2,6-DNT which had disappeared from incubation mixture (figures 2 and 3). Deficit may be due to the bacterial degradation of products formed. Consumption of 2,6-DNT in the incubation with T A 98NR was very slow, with about 70% of D N T remaining at 24 h (figure 4). Only a small amount of 2-amino-6nitrotoluene was detected at 12-h incubation, and even after 24-h incubation only small amounts of 2-nitroso-6-nitrotoluene, 2-hydroxylamino-6-nitrotoluene and 2-amino-6-nitrotoluene were found (figure 4). From the results of figures 2-4 it is
Bacterial metabolism of 2,6-dinitrotoluene
637
P
I CI
50
Q)
‘0 u)
CI
0
U
P a
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0
Figure 2.
4
8
12
16
20
24
Incubation time (h) Time course of metabolism of 2,6-DNT by Salmonella typhimurium T A 98.
Bacteria were incubated aerobically with 3 pmol’of 2,6-DNT in 3.9 ml of defined medium for timed intervals. Neutral ether fractions were subjected to h.p.1.c. Each point represents mean k S D for 2,6-DNT; ( A-A), 2-nitroso-6-nitrotoluene; (0-0), 2three samples. ( 0 - O ) , hydroxylamino-6-nitrotoluene;(M-M), 2-amino-6-nitrotoluene; (AA ) , 2,2’-dimethyl3,3’-dinitroazoxybenzene.
-L -gj
100
P
r
0
s
u
P
2
$ 50
CI
U u)
I
V
J 0
P
a
0
4 4
8
12
16
20
24
Incubation time (h)
Figure 3.
Time course of metabolism of 2,6-DNT by Salmonella typhimurium T A 98/1,8-DNP6. Bacteria were incubated aerobically with 3 pmol of 2,6-DNT in 3.9 ml of defined medium for timed intervals. Neutral ether fractions were subjected to h.p.1.c. Each point represents mean k SD for three samples. ( 0 - O ) , 2,6-DNT; ( A-A), 2-nitroso-6-nitrotoluene; (0-0), 2-hydroxylamino-6-nitrotoluene; (W-W), 2-amino-6-nitrotoluene; ( A-A), 2,Tdimethyl-3,3’-dinitroazoxybenzene.
apparent that T A 98NR does not metabolize 2,6-DNT efficiently, but that T A 98 and T A 98/1,8-DNP6 do metabolize 2,6-DNT. 2,6-DNT, 2-amino-6-nitrotoluene and 2-hydroxylamino-6-nitrotoluene showed almost no mutagenic activity ( < 2 or 3 times of the spontaneous revertants) for strains T A 98 and T A 100 in the absence or presence of S9 mix, whereas 2,6diaminotoluene showed higher mutagenic activity (about 123 his+ revertants/pmoI) only in the presence of S9 mix. These findings are in agreement with the results of earlier investigations (Couch et al. 1981, Tokiwa et al. 1981, Abernethy and Couch 1982, Mori et al. 1986 b, Dybing and Thorgeirsson 1977).
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s2
:
a
0
4
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16
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Incubation time (h)
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Figure 4. Time course of metabolism of 2,6-DNT by Salmonella typhimurium T A 98NR. Bacteria were incubated aerobically with 3 pnol of 2,6-DNT in 3.9ml of defined medium for timed intervals. Neutral ether fractions were subjected to h.p.1.c. Each point represents mean S D for three samples. ( 0 - O ) , 2,6-DNT; (A-A), 2-nitroso-6-nitrotoluene; (0-0), 2hydroxylamino-6-nitrotoluene;( m-m), 2-amino-6-nitrotoluene.
Table 2.
Mutagenicity of bacterial products of 2,6-DNT and its related compounds for Solmonella typhimurium strains T A 98 and T A 100. His’ revertantsjpnol’ T A 98 Compound
-S9 mix
2-Nitroso-6-nitrotoluene 2,2’-Dimethyl-3,3’-dinitroazoxybenzene 3,3’-Diamino-2,2’-dimethylazoxybenzene 3,3’-Diamino-2,2’-dirnethylazobenzene 3,3’-Diacetylamino-2,2’-dimethylazoxybenzene 3,3’-Diacetylamino-2,2‘-dimethylazobenzene
n.d. n.d. n.d. n.d. n.d. n.d.
T A 100
+S9 mix
- S9 mix
+ S9 mix
n.d. n.d.
n.d. n.d. n.d. n.d. n.d. n.d.
n.d. n.d. n.d.
15300+3127 144500k8770 n.d. n.d.
12700+2361 n.d. n.d.
* Mutagenic activity was calculated from linear part of dose-response curves. Values are mean fS D of his’ revertants/pmol from 3 to 5 doses. Spontaneous revertants were subtracted: 25 (TA 98, - S9), 63 (TA 98, +S9), 110 (TA 100, -S9), 143 (TA 100, +S9). n.d., Not detected, means less than twice of the number of spontaneous revertants.
2-Nitroso-6-nitrotoluene, 2,2’-dimethyl-3,3’-dinitroazoxybenzene, 3,3’diacetylamino-2,2‘-dimethylazoxybenzene and 3,3’-diacetylamino-2,2’dimethylazobenzene also showed almost no mutagenic activity for T A 98 and T A 100 (table 2). T h e remaining aminoazoxy and aminoazo compounds, 3,3’-diamino2,2’-dimethylaminoazoxybenzene and 3,3’-diamino-2,2‘-dimethylazobenzene, showed no mutagenic activity in the absence of S9 mix, but with S9 mix these compounds showed higher mutagenic activity for T A 98 or T A 100 (table 2). Results indicate that bacterial products of 2,6-DNT are non-mutagenic for T A 98 and T A
100. Discussion The present results show that 2,6-DNT is converted to 2-nitroso-6-nitrotoluene, 2-hydroxylamino-6-nitrotolueneand 2-amino-6-nitrotoluene, with concurrent spontaneous formation of 2,2’-dimethyl-3,3’-dinitroazoxybenzene,on in-
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Bacterial metabolism of 2,6-dinitrotoluene
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cubation with Salmonella typhimurium strains T A 98 and T A 98/1,8-DNP6. In addition, results show that the rate of reduction of 2,6-DNT by T A 98NR was much lower compared with those by T A 98 and T A 98/1 ,8-DNP6. The finding that T A 98 and TA 98/1,8-DNP, metabolize 2,6-DNT is consistent with the results of Bryant et al. (1984) who have shown that both T A 98 and T A 98/1,8-DNP6 metabolize dinitropyrene to aminonitropyrene. In addition, biotransformation of 2,6-DNT seen in T A 98 (figure 2) and T A 98jl ,8-DNP6 (figure 3) was similar to the observation that 2,4-DNT, an isomer of 2,6-DNT, was converted to aminonitrotoluenes and nitrosonitrotoluenes by Microsporium spores (McComic et al. 1978) and the products of 2,4-DNT formed by mixed bacteria derived from activated sludge were aminonitrotoluenes and azoxy compounds (Dickson et al. 1984). The marked activity seen in the aerobic incubation of 2,6-DNT with T A 98 and TA 98/1,8-DNP6 is consistent with the result of Tatsumi et al. (1982), who have shown that T A 100 possesses two oxygen-insensitive activities mediating the reduction of nitrofuran: the major one linked to NADPH and the minor one linked to NADH. In this connection, three types of nitroreductases (I, I11 and IV) which mediate specifically the reduction of 1-nitropyrene, 1,8-dinitropyrene and 4nitroquinoline, and a nitroreductase (11) which possesses broad specificity have been purified from the extracts of Bacteroids fragilis (Kinouchi and Ohnishi 1983). However, it remains to be determined which type of nitroreductase is active in the reduction of 1,g-dinitropyrene and 4-nitroquinoline in TA 98. The finding that bacterial products including the nitroazoxy compound showed no mutagenic potency (table 2) and no other aminoazoxy or aminoazo compounds were detected in the bacterial metabolism (figure 2) indicated that the lack of mutagenic activity of 2,6-DNT was not due to poor reductive metabolism of this compound by the bacteria, but due to the lack of mutagenic potency of the reductive intermediates produced. Since 3,3’-diacetylamino-2,2’-dimethylazoxybenzeneand 3,3’-diacetylamino2,2‘-dimethylazobenzene showed no mutagenic activity (table 2), it is suggested that the oxidation of amino groups of 3,3‘-diamino-2,2’-dimethylazoxybenzene and 3,3’-diamino-2,2‘-dimethylazobenzene is an essential step in the activation of these compounds to ultimate mutagens.
Acknowledgements The authors are grateful to Professor H. S. Rosenkranz, Case Western Reserve University for supplying Salmonella typhimurium T A 98NR and T A 98/1,8-DNP6, and also to Professor I. Tomita, Shizuoka College of Pharmacy for supplying Salmonella typhimurium T A 98 and T A 100. References ABERNETHY, D. J., and COUCH, D. B., 1982, Cytotoxicity and mutagenicity of dinitrotoluenes in Chinese hamster ovary cells. Mutation Research, 103, 53-59. AMES,B. N., MCCANN, J . , and YAMASAKI, E., 1975, Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsomesmutagenicity test. Mutation Research, 31,347-364. BRYANT, D. W., MCCALLA, D. R., LULTSCHIK, P., QUILLIAN, M. A,, and MCCARRY, B. E., 1984, Metabolism of 1,8-dinitropyrene by Salmonella typhimurium. Chemico-Biological Interactions, 49, 351-368. COUCH,D. V., ALLEN,P. F., and ABERNETHY, D. J . , 1981, The mutagenicity of dinitrotoluenes in Salmonella typhimurium. Mutation Research, 90, 373-383.
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