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.     

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.     

Degradation of 2,4,6-trinitrotoluene (TNT) by immobilized microorganism-biological filter

The combined process of immobilized microorganism-biological filter was used to degrade TNT in an aqueous solution. The results showed that the process could effectively degrade TNT, which was not detected in the effluent of the system. GC/MS analysis identified 2-amino-4,6-dinitrotoluene (2-A-4,6-DNT), 4-amino-2,6-dinitrotoluene (4-A-2,6-DNT), 2,4-diamino-6-nitrotoluene (2,4-DA-6-NT) and 2,4-diamino-6-nitrotoluene (2,6-DA-4-NT) as the main anaerobic degradation products. In addition, the Haldane model successfully described the anaerobic degradation of TNT with high correlation coefficients (R² = 0.9803). As the electron donor, ethanol played a major role in the TNT biodegradation. More than twice the theoretical requirement of ethanol was necessary to achieve a high TNT degradation rate (above 97.5%). Moreover, Environment Scan Electron Microscope (ESEM) analysis revealed that a large number of globular microorganisms were successfully immobilized on the surface of the carrier. Further analysis by Polymerase Chain Reaction (PCR)-Denaturing Gradient Gel Electrophoresis (DGGE) demonstrated that the special bacterial for TNT degradation may have generated during the domestication with TNT for 150 days. The dominant species for TNT degradation were identified by comparing gene sequences with Genebank.     

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.     

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.     

Transformation of 2,4,6-trinitrotoluene during oxygen and nitrate respiration in Pseudomonas fluorescens

A study of the metabolic pathway and the rate of 2,4,6-trinitrotoluene (TNT) transformation depending on the nature of the electron acceptor in the electron transport chain of Pseudomonas fluorescens B-3468 revealed that the first reaction of nitroreduction of TNT resulting in formation of 2-amino-4,6-dinitrotoluene (2A) and 4-amino-2,6-dinitrotoluene (4A) became more active in case of nitrate respiration as compared to oxygen respiration; a TNT decrease was 100 and 66%, respectively. The same tendency but much more pronounced was observed at the next stage of nitroreduction that lead to 2,4-diamino-6-nitrotoluene (2,4DA). On the contrary, aerobic conditions are more preferable for the subsequent destruction of 2,4DA. Thus monoamino derivatives, 2A and 4A, predominated under anaerobic conditions, whereas 2,4DA under anaerobic ones (85 and 69% of the total nitrogen-containing metabolites), respectively. Phloroglucinol and pyrogallol accumulated in the culture liquid when the bacteria were grown on a medium containing 2,4DA as a sole source of nitrogen. Their role as intermediates was proved by the results obtained by studying oxidative activity of the cells grown in the presence of 2,4DA and phloroglucinol.     

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     

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 microM for TNT and 400 +/- 94 s(-1), respectively. Cyanide, an inhibitor for the CO/CO(2) oxidation/reduction activity of CODH, inhibited the TNT degradation activity of CODH.     

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.     

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.     

Analysis of nitroaromatic compounds in urine by gas chromatography–mass spectrometry for the biological monitoring of explosives

Organic nitrocompounds are the most frequently used constituents of explosives and some of them have been evaluated to be highly toxic or even carcinogenic. Human contact with explosives may originate from a variety of sources, including occupational exposure during the production of ammunition as well as environmental exposure due to the contamination of soil and ground water reservoirs on former military production sites and training areas. This paper describes two gas chromatography–mass spectrometry–selected ion monitoring methods for the determination of twelve nitroaromatic compounds in urine (nitrobenzene, 1,2-dinitrobenzene, 1,3-dinitrobenzene, 1,3,5-trinitrobenzene, 2-nitrotoluene, 3-nitrotoluene, 4-nitrotoluene, 2,4-dinitrotoluene, 2,6-dinitrotoluene, 2,4,6-trinitrotoluene, 2-amino-4,6-dinitrotoluene, 4-amino-2,6-dinitrotoluene). The analytes are detectable in the lowest μg/l range, with imprecisions of 3–22% within series and 5–29% between series, depending on the compound of interest. Both procedures are rapid and relatively easy to perform and, therefore, are advantageous for the screening of occupationally or environmentally exposed persons. We analysed urine samples obtained from nine workers from an ammunition dismantling workshop and from twelve control persons. 2,4,6-Trinitrotoluene was detected in six samples at concentrations between 4 and 43 μg/l. The main metabolites of 2,4,6-trinitrotoluene, 4-amino-2,6-dinitrotoluene and 2-amino-4,6-dinitrotoluene, were found in a concentration range from 143 to 16 832 μg/l and from 24 to 5787 μg/l, respectively. Nonconjugated aminodinitrotoluenes were present as varying percentages of the total amount. 2,4-Dinitrotoluene and 2,6-dinitrotoluene were found in two samples (2–9 μg/l). Nitroaromatics were not detectable in urine specimens from control persons.     

A Transient Study of Formation of Conjugates during TNT Metabolism by Plant Tissues

The formation of TNT-derived conjugates was investigated in hairy root tissue cultures of Catharanthus roseus and in aquatic plant systems of Myriophyllum aquaticum. The temporal profiles of four TNT-derived conjugates, TNT-1, 2A-1, TNT-2 and 4A-1, were determined over 3 to 16-day exposure durations. When axenic C. roseus roots were exposed separately to 2,4,6 trinitrotoluene, 2-amino-4,6-dinitrotoluene and 4-amino-2,6-dinitrotoluene, the array and levels of conjugates varied. Exposure of axenic roots to either 4-amino-2,6-dinitrotoluene or 2-amino-4,6-dinitrotoluene resulted in the formation of only 4A-1 and 2A-1, respectively, and not TNT-1 and TNT-2. However, amendment of previously unexposed roots with TNT produced all four conjugates. The conjugates were preferentially accumulated within the biomass phase of root cultures. Significantly, conjugates TNT-1 and TNT-2 were observed in the biomass phase of intact M. aquaticum plants exposed to TNT. The results clearly indicate the presence of common TNT transformation products in two diverse plants species and tissue type. The distribution of conjugates formed via monoamine derivatives of TNT, however, may be a function of several factors, including the starting xenobiotic type and/or level. Initial bulk rate constants for disappearance of 2,4,6 trinitrotoluene, 2-amino-4,6-dinitrotoluene, and 4-amino-2,6-dinitrotoluene were also determined. Their magnitude followed the order: TNT >> 4-A-2,6-DNT > 2-A-4,6-DNT.     

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.     

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.     

Differences in the biotransformation of 2,4,6-trinitrotoluene (TNT) between wild and axenically grown isolates of Myriophyllum aquaticum

The aim of this study was to demonstrate the potential for aquatic plants and their associated microbes to bioremediate wetland sites contaminated with 2,4,6-trinitrotoluene (TNT). The transformation of TNT was studied using both wild and axenically grown isolates of Myriophyllum aquaticum (parrot feather). Differences in TNT transformation rates and nitroaromatic metabolites were observed between different plants. The wild isolates, containing a consortium of associated microorganisms, transformed TNT into 2-amino-4,6-dinitrotoluene (2-A-DNT) and 4-amino-2,6-dinitrotoluene (4-A-DNT) via 2- and 4-hydroxylamino-dinitrotoluene, which were detected as intermediates. The wild M. aquaticum also converted the metabolites, 2-A-DNT and 4-A-DNT, into low levels of 2,4-diaminotoluene (2,4-DAT). The axenically grown plants, containing no cultureable microorganisms, also transformed TNT into 2-A-DNT and 4-A-DNT, but at a much lower rate than that observed for the wild isolates. Unlike the wild plants, axenically grown M. aquaticum could not transform either 2-A-DNT or 4-A-DNT into 2,4-DAT over the incubation period. The differences in the performance between these plants could indicate that plant-associated microorganisms assisted in the overall transformation of TNT. For each plant, unidentifiable metabolites were observed and the soluble monoamino-derivatives present in the wild and axenic medium accounted for 14 and 7% of the initial TNT concentration, respectively. Thus, the majority of nitroaromatic derivatives remained associated with the plant tissues. Furthermore, only 7 and 3% of the initial TNT concentration were extracted as monoamino-derivatives from the tissues of the wild and axenically grown plants, respectively.     

Transformation of 2,4,6-Trinitrotoluene (TNT) Reduction Products by Lignin Peroxidase (H8) from the White-Rot Basidiomycete Phanerochaete chrysosporium

White-rot fungi are known to degrade a wide range of xenobiotic environmental pollutants, including the nitroaromatic explosive 2,4,6-trinitrotoluene (TNT). TNT is first reduced by the fungal mycelium to aminodinitrotoluenes and diaminonitrotoluenes. In a second phase, reduced TNT metabolites are oxidatively transformed and mineralized. The extracellular oxidative enzyme of the ligninolytic system of these fungi includes the lignin peroxidases (LiP) and the manganese-dependent peroxidases (MnP). In the present study, we have shown that a cell-free enzymatic system containing fast protein liquid chromatography (FPLC)-purified LiP (H8) from the white-rot fungus Phanerochaete chrysosporium was able to completely transform 50 mg/L of 2,4-diamino-6-nitrotoluene (2,4-DA-6-NT) and 2-amino-4,6-dinitrotoluene (2-A-4,6-DNT) in 1 and 48 h, respectively. Veratryl alcohol (VA), often described as a mediator in the LiP-catalyzed oxidative depolymerization of lignin, was not required for the enzymatic transformation of 2,4-DA-6-NT or 2-A-4,6-DNT. 2,4-DA-6-NT was also shown to be a competitive inhibitor of the LiP activity measured through the oxidation of VA. Experiments using ¹⁴C-U-ring labeled compounds showed that 2-A-4,6-DNT was converted to 2,2'-azoxy-4,4' ,6,6'-tetranitrotoluene. No significant mineralization, measured by the release of ¹⁴CO2, was observed over 5 d.     

Surface plasmon resonance immunosensor for highly sensitive detection of 2,4,6-trinitrotoluene

We have examined the sensing characteristics of a surface plasmon resonance (SPR) immunoassay for the detection of 2,4,6-trinitrotoluene (TNT) using an immunoreaction between 2,4,6-trinitrophenol-ovalbumin (TNP-OVA) conjugate and anti-2,4,6-trinitrophenol antibody (anti-TNP antibody). TNP-OVA conjugate was attached to a SPR-gold sensing surface by means of physical immobilization, which undergoes binding interaction with anti-TNP antibody. Both the immobilization and binding processes were studied from a change in the SPR-resonance angle. The quantification of TNT is based on the principle of indirect competitive immunoassay, in which the immunoreaction between the TNP-OVA conjugate and anti-TNP antibody was inhibited in the presence of free TNT in solution. The decrease in the resonance angle shift is proportional to an increase in concentration of TNT used for incubation. The immunoassay exhibited excellent sensitivity for the detection of TNT in the concentration range from 0.09 to 1000 ng/ml with good stability and reproducibility. The immunosensor developed could detect TNT as low as 0.09 ng/ml, within a response time of approximately 22 min. The sensor surface was regenerated by a brief flow of pepsin solution, which disrupts the antigen-antibody complex without destroying the conjugate biofilm. Cross-reactivity of the SPR sensor to some structurally related nitroaromatic derivative and the detection of TNT in the presence of these nitroaromatic compounds were investigated. The cross-reactivity of the SPR sensor to 2,4-dinitrotoluene (2,4-DNT), 1,3-dinitrobenzene (1,3-DNB), 2-amino-4,6-dinitrotoluene (2A-4,6-DNT) and 4-amino-2,6-dinitrotoluene (4A-2,6-DNT) were very low (< or =1.1%). The analytical characteristics of the proposed immunosensor are highly promising for the development of new field-portable sensors for on-site detection of landmines.     

2,4,6-trinitrotoluene as a trigger of oxidative stress in Fagopyrum tataricum callus cells

Effect of 2,4,6-trinitrotoluene (TNT) on callus cells of Tartar buckwheat (Fagopyrum tataricum (L.) Gaertn.) was accompanied by six-electron reduction of ortho- or para-nitro groups of the xenobiotic with the production of 2-amino-4,6-dinitrotoluene (2-ADNT) and 4-amino-2,6-dinitrotoluene (4-ADNT). It was discovered that the xenobiotic TNT impairs integrity of cell membrane, which apparently results from its one-electron reduction coupled with production of nitro radical-anion and superoxide anion.     

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.