2,4,6-Trinitrotoluene Reduction by Carbon Monoxide Dehydrogenase from Clostridium thermoaceticum

Purified CO dehydrogenase (CODH) from Clostridium thermoaceticum catalyzed the transformation of 2,4,6-trinitrotoluene (TNT). The intermediates and reduced products of TNT transformation were separated and appear to be identical to the compounds formed by C. acetobutylicum, namely, 2-hydroxylamino-4,6-dinitrotoluene (2HA46DNT), 4-hydroxylamino-2,6-dinitrotoluene (4HA26DNT), 2,4-dihydroxylamino-6-nitrotoluene (24DHANT), and the Bamberger rearrangement product of 2,4-dihydroxylamino-6-nitrotoluene. In the presence of saturating CO, CODH catalyzed the conversion of TNT to two monohydroxylamino derivatives (2HA46DNT and 4HA26DNT), with 4HA26DNT as the dominant isomer. These derivatives were then converted to 24DHANT, which slowly converted to the Bamberger rearrangement product. Apparent K_m and k_cat values of TNT reduction were 165 ± 43 μM for TNT and 400 ± 94 s⁻¹, respectively. Cyanide, an inhibitor for the CO/CO₂ oxidation/reduction activity of CODH, inhibited the TNT degradation activity of CODH.     

Transformation of 2,4,6-Trinitrotoluene by Pseudomonas pseudoalcaligenes JS52

Pseudomonas pseudoalcaligenes JS52 grows on nitrobenzene via partial reduction of the nitro group and enzymatic rearrangement of the resultant hydroxylamine. Cells and cell extracts of nitrobenzene-grown JS52 catalyzed the transient formation of 4-hydroxylamino-2,6-dinitrotoluene (4HADNT), 4-amino-2,6-dinitrotoluene (4ADNT), and four previously unidentified metabolites from 2,4,6-trinitrotoluene (TNT). Two of the novel metabolites were identified by liquid chromatography/mass spectrometry and (sup1)H-nuclear magnetic resonance spectroscopy as 2,4-dihydroxylamino-6-nitrotoluene (DHANT) and 2-hydroxylamino-4-amino-6-nitrotoluene (2HA4ANT). A polar yellow metabolite also accumulated during transformation of TNT by cells and cell extracts. Under anaerobic conditions, extracts of strain JS52 did not catalyze the production of the yellow metabolite or release nitrite from TNT; moreover, DHANT and 2HA4ANT accumulated under anaerobic conditions, which indicated that their further metabolism was oxygen dependent. Small amounts of nitrite were released during transformation of TNT by strain JS52. Sustained transformation of TNT by cells required nitrobenzene, which indicated that TNT transformation does not provide energy. Transformation of TNT catalyzed by enzymes in cell extracts required NADPH. Transformation experiments with (sup14)C-TNT indicated that TNT was not mineralized; however, carbon derived from TNT became associated with cells. Nitrobenzene nitroreductase purified from strain JS52 transformed TNT to DHANT via 4HADNT, which indicated that the nitroreductase could catalyze the first two steps in the transformation of TNT. The unusual ability of the nitrobenzene nitroreductase to catalyze the stoichiometric reduction of aromatic nitro compounds to the corresponding hydroxylamine provides the basis for the novel pathway for metabolism of TNT.     

Rapid separation of reduction products of 2,4,6-trinitrotoluene using TLC

Silica gel TLC methods were developed for the separation of 2,4,6-trinitrotoluene (TNT) in mixtures with possible reduction products. The methods employed repeated elutions with simple binary or ternary solvent systems in either one or two dimensional modes. The resolved analytes include TNT, selected amino derivatives (2-amino-4,6-di-nitrotoluene, 4-amino-2,6-dinitrotoluene, 2,4-diamino-6-nitrotoluene) and known hydroxylamino derivatives (2-hydroxyl-amino-4,6-dinitrotoluene, 4-hydroxylamino-2,6-dinitrotoluene and 2,4-dihydroxylamino-6-nitrotoluene).     

Biotransformation of 2,4,6-trinitrotoluene with Phanerochaete chrysosporium in agitated cultures at pH 4.5.

The biotransformation of 2,4,6-trinitrotoluene (TNT) (175 microM) by Phanerochaete chrysosporium with molasses and citric acid at pH 4.5 was studied. In less than 2 weeks, TNT disappeared completely, but mineralization (liberated 14CO2) did not exceed 1%. A time study revealed the presence of several intermediates, marked by the initial formation of two monohydroxylaminodinitrotoluenes (2- and 4-HADNT) followed by their successive transformation to several other products, including monoaminodinitrotoluenes (ADNT). A group of nine acylated intermediates were also detected. They included 2-N-acetylamido-4,6-dinitrotoluene and its p isomer, 2-formylamido-4, 6-dinitrotoluene and its p isomer (as acylated ADNT), 4-N-acetylamino-2-amino-6-nitrotoluene and 4-N-formylamido-2-amino-6-nitrotoluene (as acetylated DANT), 4-N-acetylhydroxy-2,6-dinitrotoluene and 4-N-acetoxy-2, 6-dinitrotoluene (as acetylated HADNT), and finally 4-N-acetylamido-2-hydroxylamino-6-nitrotoluene. Furthermore, a fraction of HADNTs were found to rearrange to their corresponding phenolamines (Bamberger rearrangement), while another group dimerized to azoxytoluenes which in turn transformed to azo compounds and eventually to the corresponding hydrazo derivatives. After 30 days, all of these metabolites, except traces of 4-ADNT and the hydrazo derivatives, disappeared, but mineralization did not exceed 10% even after the incubation period was increased to 120 days. The biotransformation of TNT was accompanied by the appearance of manganese peroxidase (MnP) and lignin-dependent peroxidase (LiP) activities. MnP activity was observed almost immediately after TNT disappearance, which was the period marked by the appearance of the initial metabolites (HADNT and ADNT), whereas the LiP activity was observed after 8 days of incubation, corresponding to the appearance of the acyl derivatives. Both MnP and LiP activities reached their maximum levels (100 and 10 U/liter, respectively) within 10 to 15 days after inoculation.     

Biotransformation of 2,4,6-Trinitrotoluene with Phanerochaete chrysosporium in Agitated Cultures at pH 4.5

The biotransformation of 2,4,6-trinitrotoluene (TNT) (175 μM) by Phanerochaete chrysosporium with molasses and citric acid at pH 4.5 was studied. In less than 2 weeks, TNT disappeared completely, but mineralization (liberated ¹⁴CO₂) did not exceed 1%. A time study revealed the presence of several intermediates, marked by the initial formation of two monohydroxylaminodinitrotoluenes (2- and 4-HADNT) followed by their successive transformation to several other products, including monoaminodinitrotoluenes (ADNT). A group of nine acylated intermediates were also detected. They included 2-N-acetylamido-4,6-dinitrotoluene and its p isomer, 2-formylamido-4,6-dinitrotoluene and its p isomer (as acylated ADNT), 4-N-acetylamino-2-amino-6-nitrotoluene and 4-N-formylamido-2-amino-6-nitrotoluene (as acetylated DANT), 4-N-acetylhydroxy-2,6-dinitrotoluene and 4-N-acetoxy-2,6-dinitrotoluene (as acetylated HADNT), and finally 4-N-acetylamido-2-hydroxylamino-6-nitrotoluene. Furthermore, a fraction of HADNTs were found to rearrange to their corresponding phenolamines (Bamberger rearrangement), while another group dimerized to azoxytoluenes which in turn transformed to azo compounds and eventually to the corresponding hydrazo derivatives. After 30 days, all of these metabolites, except traces of 4-ADNT and the hydrazo derivatives, disappeared, but mineralization did not exceed 10% even after the incubation period was increased to 120 days. The biotransformation of TNT was accompanied by the appearance of manganese peroxidase (MnP) and lignin-dependent peroxidase (LiP) activities. MnP activity was observed almost immediately after TNT disappearance, which was the period marked by the appearance of the initial metabolites (HADNT and ADNT), whereas the LiP activity was observed after 8 days of incubation, corresponding to the appearance of the acyl derivatives. Both MnP and LiP activities reached their maximum levels (100 and 10 U/liter, respectively) within 10 to 15 days after inoculation.     

Implication of manganese (III), oxalate, and oxygen in the degradation of nitroaromatic compounds by manganese peroxidase (MnP)

The fungal ligninolytic enzyme manganese peroxidase (MnP) is known to function by oxidizing Mn(II) to Mn(III), a powerful oxidant. In this work, an abiotic system consisting of Mn(III) in oxalate buffer under aerobic conditions (Mn(III)/oxalate/O2 system) was shown to be capable of extensively transforming 2-amino-4,6-dinitrotoluene (2A46DNT)--one of the main reduction products of 2,4,6-trinitrotoluene (TNT). No significant transformation occurred in the presence of other organic acids or under anaerobic conditions. The Mn(III)/oxalate/O2 system was also able to transform other nitroaromatic compounds such as 2-nitrotoluene, 4-nitrotoluene, 2,4-dinitrotoluene, TNT - the latter to a lesser extent -, and their reduction derivatives. The Mn(III)/oxalate/O2 system mineralized 14C-U-ring labeled 2A46DNT slightly, while no significant mineralization of 14C-U-ring labeled TNT was observed. Unidentified 14C-transformation products were highly polar. Electron spin resonance experiments performed on the Mn(III)/oxalate/O2 system revealed the generation of formyl free radicals (*COO-). The oxygen requirement for the transformation of nitroaromatic compounds suggests the involvement of superoxide free radicals (O2-*). produced through autoxidation of *COO- by molecular oxygen. The implication of such a Mn(III)/oxalate/O2 system in the MnP-catalyzed degradation of nitroaromatic pollutants by white-rot fungi is further discussed.     

TNT Biotransformation and Detoxification by a Pseudomonas Aeruginosa Strain

Successful microbial-mediated remediation requires transformationpathways that maximize metabolism and minimize the accumulation of toxic products. Pseudomonas aeruginosa strain MX, isolated from munitions-contaminated soil, degraded 100 mg TNT L^-1 in culture medium within 10 h under aerobic conditions. The major TNT products were 2-amino-4,6-dinitrotoluene (2ADNT, primarily in the supernatant) and 2,2'-azoxytoluene (2,2'AZT, primarily in the cell fraction), which accumulated as major products via the intermediate2-hydroxylamino-4,6-dinitrotoluene (2HADNT). The 2HADNT and2,2'AZT were relatively less toxic to the strain than TNT and 2ADNT. Aminodinitrotoluene (ADNT) production increased when yeast extract was added to the medium. While TNT transformation rate was not affected by pH, more HADNTs accumulated at pH 5.0 than at pH 8.0 and AZTs did not accumulate at the lower pH. The appearance of 2,6-diamino-4-nitrotoluene (2,6DANT) and 2,4-diamino-6-nitrotoluene (2,4DANT); dinitrotoluene (DNT) and nitrotoluene (NT); and 3,5-dinitroaniline (3,5DNA) indicated various routes of TNT metabolism and detoxification by P. aeruginosa strain MX.     

Anaerobic transformation of 2,4,6-TNT by bovine ruminal microbes

Degradation of TNT by bovine rumen fluid, a novel source of anaerobic microbes, was investigated. Whole rumen fluid contents were spiked with TNT and incubated for a 24h time period. Supernatant samples taken at 0, 1, 2, 4, and 24h were analyzed by reverse-phase HPLC with diode array detection. Within 1h, TNT was not detectable and reduction products of TNT including 2-hydroxyl-amino-4,6-dinitrotoluene, 4-hydroxylamino-2,6-dinitrotoluene, and 4-amino-2,6-dinitrotoluene were present with smaller amounts of diamino-nitrotoluenes. Within 2h, only the diamino and dihydroxyamino-nitrotoluene products remained. After 4h, 2,4-diamino-6-nitrotoluene and 2,4-dihydroxyamino-6-nitrotoluene were the only known molecular species left. At 24h known UV absorbing metabolites were no longer detected, suggesting further transformation such as complete reduction to triaminotoluene or destruction of the aromatic ring of TNT may have occurred. TNT was not transformed at 24h in autoclaved and buffered controls. This study presents the first direct evidence of biodegradation of TNT by ruminal microbes.     

NAD(P)H:flavin mononucleotide oxidoreductase inactivation during 2,4,6-trinitrotoluene reduction

Bacteria readily transform 2,4,6-trinitrotoluene (TNT), a contaminant frequently found at military bases and munitions production facilities, by reduction of the nitro group substituents. In this work, the kinetics of nitroreduction were investigated by using a model nitroreductase, NAD(P)H:flavin mononucleotide (FMN) oxidoreductase. Under mediation by NAD(P)H:FMN oxidoreductase, TNT rapidly reacted with NADH to form 2-hydroxylamino-4,6-dinitrotoluene and 4-hydroxylamino-2,6-dinitrotoluene, whereas 2-amino-4,6-dinitrotoluene and 4-amino-2,6-dinitrotoluene were not produced. Progressive loss of activity was observed during TNT reduction, indicating inactivation of the enzyme during transformation. It is likely that a nitrosodinitrotoluene intermediate reacted with the NAD(P)H:FMN oxidoreductase, leading to enzyme inactivation. A half-maximum constant with respect to NADH, K(N), of 394 microM was measured, indicating possible NADH limitation under typical cellular conditions. A mathematical model that describes the inactivation process and NADH limitation provided a good fit to TNT reduction profiles. This work represents the first step in developing a comprehensive enzyme level understanding of nitroarene biotransformation.     

Biodegradation of 2,4,6-trinitrotoluene (TNT) by the white-rot basidiomycete Phlebia radiata

Phlebia radiatatransformed 2,4,6-trinitrotoluene (TNT), as well as its first reduction products, the aminodinitrotoluenes, into 4-hydroxylamino-2,6-dinitrotoluene (4-OHA-2,6-DNT) and 4-amino-2,6-dinitrotoluene (4-A-2,6-DNT). No extracellular peroxidases were involved in this step. The ligninolytic extracellular fluid, assumed to contain peroxidases, did not reduce TNT. However, ligninolytic peroxidases are implicated in the transformation of the first reduction products of TNT.     

Protein engineering of the archetypal nitroarene dioxygenase of Ralstonia sp. strain U2 for activity on aminonitrotoluenes and dinitrotoluenes through alpha-subunit residues leucine 225, phenylalanine 350, and glycine 407

Naphthalene dioxygenase (NDO) from Ralstonia sp. strain U2 has not been reported to oxidize nitroaromatic compounds. Here, saturation mutagenesis of NDO at position F350 of the alpha-subunit (NagAc) created variant F350T that produced 3-methyl-4-nitrocatechol from 2,6-dinitrotoluene (26DNT), that released nitrite from 23DNT sixfold faster than wild-type NDO, and that produced 3-amino-4-methyl-5-nitrocatechol and 2-amino-4,6-dinitrobenzyl alcohol from 2-amino-4,6-dinitrotoluene (2A46DNT) (wild-type NDO has no detectable activity on 26DNT and 2A46DNT). DNA shuffling identified the beneficial NagAc mutation G407S, which when combined with the F350T substitution, increased the rate of NDO oxidation of 26DNT, 23DNT, and 2A46DNT threefold relative to variant F350T. DNA shuffling of NDO nagAcAd also generated the NagAc variant G50S/L225R/A269T with an increased rate of 4-amino-2-nitrotoluene (4A2NT; reduction product of 2,4-dinitrotoluene) oxidation; from 4A2NT, this variant produced both the previously uncharacterized oxidation product 4-amino-2-nitrocresol (enhanced 11-fold relative to wild-type NDO) as well as 4-amino-2-nitrobenzyl alcohol (4A2NBA; wild-type NDO does not generate this product). G50S/L225R/A269T also had increased nitrite release from 23DNT (14-fold relative to wild-type NDO) and generated 2,3-dinitrobenzyl alcohol (23DNBA) fourfold relative to wild-type NDO. The importance of position L225 for catalysis was confirmed through saturation mutagenesis; relative to wild-type NDO, NDO variant L225R had 12-fold faster generation of 4-amino-2-nitrocresol and production of 4A2NBA from 4A2NT as well as 24-fold faster generation of nitrite and 15-fold faster generation of 23DNBA from 23DNT. Hence, random mutagenesis discovered two new residues, G407 and L225, that influence the regiospecificity of Rieske non-heme-iron dioxygenases.     

Biotransformation of 2,4,6-trinitrotoluene (TNT) by the fungus Fusarium oxysporum

The fungus Fusarium oxysporum was isolated and identified from the aquatic plant M. aquaticum. The capability of this fungus to transform 2,4,6-trinitrotoluene (TNT) in liquid cultures was investigated TNT was added to shake flask cultures and transformed into 2-amino-4,6-dinitrotoluene (2-A-DNT), 4-amino-2,6-dinitrotoluene (4-A-DNT), and 2,4-diamino-6-nitrotoluene (2,4-DAT) via 2- and 4-hydroxylamino-dinitrotoluene derivatives, which could be detected as intermediate metabolites. Transformation of TNT, 2-A-DNT, and 4-A-DNT was observed by whole cultures and with isolated mycelium. Cell-free protein extracts from the extracellular, soluble, and membrane-bound fractions were prepared from this fungus and tested for TNT-reducing activity. The concentrated extracellular culture medium was unable to transform TNT; however, low levels of TNT transformation were observed by the membrane fraction in the presence of nicotinamide adenine dinucleotide phosphate in an argon atmosphere. A concentrated extract of soluble enzymes also transformed TNT, but to a lesser extent. When TNT toxicity was studied with this fungus, a 50% decrease in the growth of F. oxysporum mycelium was observed when exposed to 20 mg/L TNT.     

Anaerobic transformation of 2,4,6-TNT and related nitroaromatic compounds by Clostridium acetobutylicum

The transformation of TNT and related aminated nitrotoluenes by Clostridium acetobutylicum was investigated. 2,4,6-trinitrotoluene (TNT) was rapidly reduced (537 nM min⁻¹ mg protein⁻¹) to undetermined end products via monohydroxylamino derivatives. TNT reduction was more rapid than that of 2-amino-4,6-dinitrotoluene, 4-amino-2,6-dinitrotoluene and 2,4-diamino-6-nitrotoluene. The metabolic phase of clostridial cultures affected rates and extents of transformation of TNT and its intermediates. Acidogenic cultures showed rapid transformation rates and the ability to transform TNT and its primary reduction products to below detection limits; solventogenic cultures did not transform TNT completely, and showed accumulation of its hydroxylamino derivatives. Carbon monoxide-induced solventogenesis was capable of slowing the transformation of TNT and intermediates. Studies employing [ring-U-¹⁴C]-TNT demonstrated that no significant mineralization occurred and that products of transformation were water-soluble. Received 06 November 1995/ Accepted in revised form 15 August 1996     

NAD(P)H:Flavin Mononucleotide Oxidoreductase Inactivation during 2,4,6-Trinitrotoluene Reduction

Bacteria readily transform 2,4,6-trinitrotoluene (TNT), a contaminant frequently found at military bases and munitions production facilities, by reduction of the nitro group substituents. In this work, the kinetics of nitroreduction were investigated by using a model nitroreductase, NAD(P)H:flavin mononucleotide (FMN) oxidoreductase. Under mediation by NAD(P)H:FMN oxidoreductase, TNT rapidly reacted with NADH to form 2-hydroxylamino-4,6-dinitrotoluene and 4-hydroxylamino-2,6-dinitrotoluene, whereas 2-amino-4,6-dinitrotoluene and 4-amino-2,6-dinitrotoluene were not produced. Progressive loss of activity was observed during TNT reduction, indicating inactivation of the enzyme during transformation. It is likely that a nitrosodinitrotoluene intermediate reacted with the NAD(P)H:FMN oxidoreductase, leading to enzyme inactivation. A half-maximum constant with respect to NADH, K_N, of 394 μM was measured, indicating possible NADH limitation under typical cellular conditions. A mathematical model that describes the inactivation process and NADH limitation provided a good fit to TNT reduction profiles. This work represents the first step in developing a comprehensive enzyme level understanding of nitroarene biotransformation.     

Reduction and Acetylation of 2,4-Dinitrotoluene by a Pseudomonas aeruginosa Strain

Aerobic and anoxic biotransformation of 2,4-dinitrotoluene (DNT) was examined by using a Pseudomonas aeruginosa strain isolated from a plant treating propellant manufacturing wastewater. DNT biotransformation in the presence and absence of oxygen was mostly reductive and was representative of the type of cometabolic transformations that occur when a high concentration of an easily degradable carbon source is present. P. aeruginosa reduced both nitro groups on DNT, with the formation of mainly 4-amino-2-nitrotoluene and 2-amino-4-nitrotoluene and small quantities of 2,4-diaminotoluene. Acetylation of the arylamines was a significant reaction. 4-Acetamide-2-nitrotoluene and the novel compounds 2-acetamide-4-nitrotoluene, 4-acetamide-2-aminotoluene, and 2,4-diacetamidetoluene were identified as DNT metabolites. The biotransformation of 2,4-diaminotoluene to 4-acetamide-2-aminotoluene was 24 times faster than abiotic transformation. 2-Nitrotoluene and 4-nitrotoluene were also reduced to their corresponding toluidines and then acetylated. However, the yield of 4-acetamidetoluene was much higher than that of 2-acetamidetoluene, demonstrating that acetylation at the position para to the methyl group was favored.     

Purification and characterization of NAD(P)H-dependent nitroreductase I from Klebsiella sp. C1 and enzymatic transformation of 2,4,6-trinitrotoluene

Three NAD(P)H-dependent nitroreductases that can transform 2,4,6-trinitrotoluene (TNT) by two reduction pathways were detected in Klebsiella sp. C1. Among these enzymes, the protein with the highest reduction activity of TNT (nitroreductase I) was purified to homogeneity using ion-exchange, hydrophobic interaction, and size exclusion chromatographies. Nitroreductase I has a molecular mass of 27 kDa as determined by SDS-PAGE, and exhibits a broad pH optimum between 5.5 and 6.5, with a temperature optimum of 30–40°C. Flavin mononucleotide is most likely the natural flavin cofactor of this enzyme. The N-terminal amino acid sequence of this enzyme does not show a high degree of sequence similarity with nitroreductases from other enteric bacteria. This enzyme catalyzed the two-electron reduction of several nitroaromatic compounds with very high specific activities of NADPH oxidation. In the enzymatic transformation of TNT, 2-amino-4,6-dinitrotoluene and 2,2′,6,6′-tetranitro-4,4′-azoxytoluene were detected as transformation products. Although this bacterium utilizes the direct ring reduction and subsequent denitration pathway together with a nitro group reduction pathway, metabolites in direct ring reduction of TNT could not easily be detected. Unlike other nitroreductases, nitroreductase I was able to transform hydroxylaminodinitrotoluenes (HADNT) into aminodinitrotoluenes (ADNT), and could reduce ortho isomers (2-HADNT and 2-ADNT) more easily than their para isomers (4-HADNT and 4-ADNT). Only the nitro group in the ortho position of 2,4-DNT was reduced to produce 2-hydroxylamino-4-nitrotoluene by nitroreductase I; the nitro group in the para position was not reduced.     

Urinary metabolites of workers exposed to nitrotoluenes.

Nitrotoluenes are important intermediates in the chemical industry. 2,6-Dinitrotoluene (26DNT), 2,4-dinitrotoluene (24DNT) and 2-nitrotoluene (2NT) are carcinogenic in animals and possibly carcinogenic in humans. Thus, it is important to develop methods to biomonitor workers exposed to such chemicals. The authors have monitored the air and urine metabolite levels for a group of workers in China exposed to 24DNT, 26DNT, 2NT and 4-nitrotoluene (4NT). The metabolites 2,4-dinitrobenzylalcohol (24DNBAlc), 2-amino-4-nitrobenzoic acid (2A4NBA), 4-amino-2-nitrobenzoic acid (4A2NBA) and 2,4-dinitrobenzoic acid (24DNBA) resulting from exposure to 24DNT were found in 89, 88, 91 and 78% of the exposed workers, respectively. The metabolites 2,6-dinitrobenzylalcohol (26DNBAlc) and 2,6-dinitrobenzoic acid resulting from 26DNT exposure were found in 99 and 86% of the exposed workers, respectively. Quantitatively, 2A4NBA, 4A2NBA and 26DNBAlc were the major metabolites. The nitrobenzoic acids were the major metabolites resulting from exposure to 2NT and 4NT and were present in 96 and 73% of the exposed workers, respectively. Air concentrations of DNT and 2NT did not correlate with the levels of metabolites in the urine. In conclusion, the dinitrobenzyl alcohols and aminonitrobenzoic acids determined in the urine provided a good marker for recently absorbed dose and were intrinsically related to the bioactivation and detoxification pathways of DNT. Air measurements were not a good measure to predict internal exposure.     

Urinary metabolites of workers exposed to nitrotoluenes

Nitrotoluenes are important intermediates in the chemical industry. 2,6-Dinitrotoluene (26DNT), 2,4-dinitrotoluene (24DNT) and 2-nitrotoluene (2NT) are carcinogenic in animals and possibly carcinogenic in humans. Thus, it is important to develop methods to biomonitor workers exposed to such chemicals. The authors have monitored the air and urine metabolite levels for a group of workers in China exposed to 24DNT, 26DNT, 2NT and 4-nitrotoluene (4NT). The metabolites 2,4-dinitrobenzylalcohol (24DNBAlc), 2-amino-4-nitrobenzoic acid (2A4NBA), 4-amino-2-nitrobenzoic acid (4A2NBA) and 2,4-dinitrobenzoic acid (24DNBA) resulting from exposure to 24DNT were found in 89, 88, 91 and 78% of the exposed workers, respectively. The metabolites 2,6-dinitrobenzylalcohol (26DNBAlc) and 2,6-dinitrobenzoic acid resulting from 26DNT exposure were found in 99 and 86% of the exposed workers, respectively. Quantitatively, 2A4NBA, 4A2NBA and 26DNBAlc were the major metabolites. The nitrobenzoic acids were the major metabolites resulting from exposure to 2NT and 4NT and were present in 96 and 73% of the exposed workers, respectively. Air concentrations of DNT and 2NT did not correlate with the levels of metabolites in the urine. In conclusion, the dinitrobenzyl alcohols and aminonitrobenzoic acids determined in the urine provided a good marker for recently absorbed dose and were intrinsically related to the bioactivation and detoxification pathways of DNT. Air measurements were not a good measure to predict internal exposure.     

Construction of a Pseudomonas hybrid strain that mineralizes 2,4,6-trinitrotoluene.

A bacterium, Pseudomonas sp. strain C1S1, able to grow on 2,4,6-trinitrotoluene (TNT), 2,4- and 2,6-dinitrotoluene, and 2-nitrotoluene as N sources, was isolated. The bacterium grew at 30 degrees C with fructose as a C source and accumulated nitrite. Through batch culture enrichment, we isolated a derivative strain, called Pseudomonas sp. clone A, which grew faster on TNT and did not accumulate nitrite in the culture medium. Use of TNT by these two strains as an N source involved the successive removal of nitro groups to yield 2,4- and 2,6-dinitrotoluene, 2-nitrotoluene, and toluene. Transfer of the Pseudomonas putida TOL plasmid pWW0-Km to Pseudomonas sp. clone A allowed the transconjugant bacteria to grow on TNT as the sole C and N source. All bacteria in this study, in addition to removing nitro groups from TNT, reduced nitro groups on the aromatic ring via hydroxylamine to amino derivatives. Azoxy dimers probably resulting from the condensation of partially reduced TNT derivatives were also found.     

Cytotoxic and genotoxic effects of energetic compounds on bacterial and mammalian cells in vitro.

The mutagenicity and toxicity of energetic compounds such as 2,4, 6-trinitrotoluene (TNT), 1,3,5-trinitrobenzene (TNB), hexahydro-1,3, 5-trinitro-1,3,5-triazine (RDX) and octahydro-1,3,5,7-tetranitro-1,3, 5,7-tetrazocine (HMX), and of amino/nitro derivatives of toluene were investigated in vitro. Mutagenicity was evaluated with the Salmonella fluctuation test (FT) and the V79 Chinese hamster lung cell mutagenicity assay. Cytotoxicity was evaluated using V79 and TK6 human lymphoblastic cells. For the TK6 and V79 assays, TNB and 2, 4,6-triaminotoluene were more toxic than TNT, whereas RDX and HMX were without effect at their maximal aqueous solubility limits. The primary TNT metabolites (2-amino-4,6-dinitrotoluene, 4-amino-2, 6-dinitrotoluene, 2,4-diamino-6-nitrotoluene and 2, 6-diamino-4-nitrotoluene) were generally less cytotoxic than the parent compound. The FT results indicated that TNB, TNT and all the tested primary TNT metabolites were mutagenic. Except for the cases of 4-amino-2,6-dinitrotoluene and 2,4-diamino-6-nitrotoluene in the TA98 strain, addition of rat liver S9 resulted in either no effect, or decreased activity. None of the tested compounds were mutagenic for the V79 mammalian cells with or without S9 metabolic activation. Thus, the FT assay was more sensitive to the genotoxic effects of energetic compounds than was the V79 test, suggesting that the FT might be a better screening tool for the presence of these explosives. The lack of mutagenicity of pure substances for V79 cells under the conditions used in this study does not preclude that genotoxicity could actually exist in other mammalian cells. In view of earlier reports and this study, mutagenicity testing of environmental samples should be considered as part of the hazard assessment of sites contaminated by TNT and related products.