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2,4,6-trinitrotoluene (TNT) and cobalt (Co) contaminants have posed a severe environmental problem in many countries. Phytoremediation is an environmentally friendly technology for the remediation of these contaminants. However, the toxicity of TNT and cobalt limit the efficacy of phytoremediation application. The present research showed that expressing the Acidithiobacillus ferrooxidans single-strand DNA-binding protein gene (AfSSB) can improve the tolerance of Arabidopsis and tall fescue to TNT and cobalt. Compared to control plants, the AfSSB transformed Arabidopsis and tall fescue exhibited enhanced phytoremediation of TNT and cobalt separately contaminated soil and co-contaminated soil. The comet analysis revealed that the AfSSB transformed Arabidopsis suffer reduced DNA damage than control plants under TNT or cobalt exposure. In addition, the proteomic analysis revealed that AfSSB improves TNT and cobalt tolerance by strengthening the reactive superoxide (ROS) scavenging system and the detoxification system. Results presented here serve as strong theoretical support for the phytoremediation potential of organic and metal pollutants mediated by single-strand DNA-binding protein genes. SUMMARIZES: This is the first report that AfSSB enhances phytoremediation of 2,4,6-trinitrotoluene and cobalt separately contaminated and co-contaminated soil., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Although laccase is involved in the biotransformation of 2,4,6-trinitrotoluene (TNT), little is known regarding the effect of E. coli laccase on TNT biotransformation. In this study, E. coli K12 served as the parental strain to construct a laccase deletion strain and two laccase-overexpressing strains. These E. coli strains were used to investigate the effect of laccase together with copper ions on the efficiency of TNT biotransformation, the variety of TNT biotransformation products generated and the toxicity of the TNT metabolites. The results showed that the laccase level was not relevant to TNT biotransformation in the soluble fraction of the culture medium. Conversely, TNT metabolites varied in the insoluble fraction analyzed by thin-layer chromatography (TLC). The insoluble fraction from the laccase-null strain showed fewer and relatively fainter spots than those detected in the wild-type and laccase-overexpressing strains, indicating that laccase expression levels were interrelated determinants of the varieties and amounts of TNT metabolites produced. In addition, the aquatic invertebrate Tigriopus japonicus was used to assess the toxicity of the TNT metabolites. The toxicity of the TNT metabolite mixture increased when the intracellular laccase level in strains increased or when purified E. coli recombinant Laccase (rLaccase) was added to the culture medium. Thus, our results suggest that laccase activity must be considered when performing microbial TNT remediation., (Copyright © 2019 Elsevier Inc. All rights reserved.)

In this study, the bacterial strain Citrobacter youngae strain E4 was isolated from 2,4,6-trinitrotoluene (TNT)-contaminated soil and used to assess the capacity of TNT transformation with/without exogenous nutrient amendments. C. youngae E4 poorly degraded TNT without an exogenous amino nitrogen source, whereas the addition of an amino nitrogen source considerably increased the efficacy of TNT transformation in a dose-dependent manner. The enhanced TNT transformation of C. youngae E4 was mediated by increased cell growth and up-regulation of TNT nitroreductases, including NemA, NfsA and NfsB. This result indicates that the increase in TNT transformation by C. youngae E4 via nitrogen nutrient stimulation is a cometabolism process. Consistently, TNT transformation was effectively enhanced when C. youngae E4 was subjected to a TNT-contaminated soil slurry in the presence of an exogenous amino nitrogen amendment. Thus, effective enhancement of TNT transformation via the coordinated inoculation of the nutrient-responsive C. youngae E4 and an exogenous nitrogen amendment might be applicable for the remediation of TNT-contaminated soil. Although the TNT transformation was significantly enhanced by C. youngae E4 in concert with biostimulation, the 96-h LC50 value of the TNT transformation product mixture on the aquatic invertebrate Tigriopus japonicas was higher than the LC50 value of TNT alone. Our results suggest that exogenous nutrient amendment can enhance microbial TNT transformation; however, additional detoxification processes may be needed due to the increased toxicity after reduced TNT transformation., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Sheepshead minnows (Cyprinodon variegatus) were exposed to radiolabeled isotopes of the explosives 2,4,6-trinitrotoluene (TNT), exahydro-1,3,5-trinitro-1,3,5-triazine (commonly known as RDX), and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (commonly known as HMX), yielding the bioconcentration factors (BCF) of 3.3, 0.7, and 0.1 L kg(-1), respectively. For TNT, the body residue of transformation product exceeded that of the parent compound by factors of 1, 8, and 16 for total aminonitrotoluenes, total extractable compounds, and total transformation products, respectively, with substantial bioaccumulation of both non-identified extractable and unextractable (i.e., tissue-bound), compounds. In comparison, the sum body residues of RDX and HMX transformation products were <4 times higher than for parent compounds. The concentrations of RDX and HMX and their transformation products were similar among liver, viscera (excluding liver), gills, and body remains (integument and muscles), while 46% of the TNT transformation products resided in the liver, and 64% of the parent compound was in the viscera., (Published by Elsevier Inc.)

Lethal effects of trinitrotoluene (TNT) to juvenile sheepshead minnows (JSHM) (Cyprinodon variegatus) were assessed in ten-day water exposures. Ten-day median lethal concentrations (LC50s) were 2.3 and 2.5 mg L(-1), the 10-d median lethal residue value (LR50) was 26.1 micromol kg(-1) wet weight (ww), and bioconcentration factors (BCFs) ranged from 0.7 to 2.4 L kg(-1). The lethal effects of TNT and its transformation products 2-aminodinitrotoluene (2-ADNT), 2,4-diaminonitrotoluene (2,4-DANT) and trinitrobenzene (TNB) to JSHM were compared in 5-d static-renewal exposures. Nitroreduction decreased the toxicity of TNT to SHM, as the 5-d LC50 for 2-ADNT was 8.6 mg L(-1) and the lowest lethal concentration of 2,4-DANT was 50.3 mg L(-1). TNB (5-d LC50=1.2 mg L(-1)) was more toxic than TNT to SHM. The 5-d LR50s were 4.3 mg kg(-1)ww (20.4 micromol kg(-1)) for SumTNT (TNT exposure) and 54.2 mg kg(-1)ww (275.3 micromol kg(-1)) for 2-ADNT and significant mortality occurred at 47.4 mg kg(-1)ww (283.6 micromol kg(-1)). The range of BCF values was from 1.8 to 2.4, 5.6 to 8.0, and 0.6 to 0.9Lkg(-1) for TNT, 2-ADNT, and 2,4-DANT, respectively., (Published by Elsevier Inc.)

The aim of the present study was to investigate the interaction of 2,4,6-trinitrotoluene (TNT) with liver biotransformation enzymes in European eel Anguilla anguilla (Linnaeus, 1758). Eels were exposed to 0.5, 1 and 2.5mg/l nominal concentrations of TNT for 6 and 24h. Modulation of CYP1A1, UDPGT and GST genes was investigated by real-time PCR. Total CYP450 content, NADPH cytochrome c reductase activity, CYP1A and CYP2B-like activities, such as EROD, MROD and BROD, as well as GST and UDPGT activities, were measured by biochemical assays. An in vitro study was performed on EROD in order to evaluate catalytic modulation by TNT. No modulation of the CYP1A1 gene or protein was observed in TNT-exposed eels. On the other hand, a significant decline of EROD and MROD activities was observed in vivo. An increase in NADPH cyt c reductase, and phase II enzymes (UDPGT and GST) were observed at both gene expression and activity levels. The overall results indicated that TNT is a potential competitive inhibitor of CYP1A activities. A TNT metabolic pathway involving NADPH cyt c reductase and phase II enzymes is also suggested.

The ability of eight species of plants to assimilate 2,4,6-trinitrotoluene (TNT) was investigated. Glycine max (soybean), in particular, demonstrated rapid assimilation of high concentrations of this explosive. Penetration and localization of [1-(14)C]-TNT in plant root cells and leaves were studied via electron microscopic autoradiography. TNT was shown to be localized primarily on membrane structures involved in the transportation of nicotinamide coenzymes (membranes of endoplasmic reticulum, mitochondria, plastids). [1-(14)C]-TNT in roots was incorporated mainly in low-molecular-weight metabolites; however, in stems and leaves, the radiocarbon was incorporated in biopolymers. Enzymatic transformation of TNT in roots was studied, and it was found that degradation involved mainly nitroreductase acting on the TNT nitro groups. The process was intensified in the presence of electron donors--NADH and NADPH. Nitroreductase activity was revealed in root cell cytosol and expression was strongly induced by plant cultivation on TNT-containing media. Oxidation of [C(3)H(3)]TNT by peroxidase and phenoloxidase was also studied. In contrast to the strongly induced nitroreductase, levels of these enzymes changed very little with TNT addition. This suggests that the main pathway of TNT transformation in plant cells is nitro group reduction. A plant's nitroreductase activity and its ability to incorporate TNT from aqueous solutions were correlated in four plants that were studied. The results suggest that plant nitroreductase activity may serve as a good biochemical indicator of plants that can be used for phytoremediation of soils contaminated with TNT.

The acetonitrile-sonication extraction method (US EPA SW-846 Method 8330) and aquatic-based toxicity tests were used on laboratory and field samples, to characterize the ecotoxicity of soils contaminated with energetic substances. Spiked soil studies indicated that 2,4, 6-trinitrotoluene (TNT)-dependent soil toxicity could be measured in organic extracts and aqueous leachates using the 15-min Microtox (Vibrio fischeri, IC50=0.27 to 0.94 mg TNT/liter incubation medium) and 96-h Selenastrum capricornutum growth inhibition (IC50=0.62 to 1. 14 mg/liter) toxicity tests. Analyses of leachates of composite soil samples [containing TNT and some TNT metabolites, 1,3,5-trinitro-1,3, 5-triazacyclohexane (RDX), and 1,3,5,7-tetranitro-1,3,5, 7-tetrazacyclooctane (HMX)] from an explosives manufacturing facility, indicated toxicities similar to those found in the TNT-spiked soil studies and pure TNT in solution, and suggested that TNT was the major toxicant. Using TNT as a model toxicant in soils having different moisture contents (20% vs dry) and textures (sandy vs clayey-sandy) but similar organic matter content (3-4%), multi-factorial analyses of Microtox test data revealed that these soil factors significantly influenced the TNT extractability from soil and subsequent toxicity measurements. Taken together, data indicate that the modified Method 8330 may be used in conjunction with ecotoxicity tests to reflect the toxic potential of soils contaminated with energetic substances., (Copyright 1999 Academic Press.)

Composting has been advocated and is being used as an economical method for remediating 2,4,6-trinitrotoluene (TNT)-contaminated soils. However, evidence suggests that TNT is transformed into products of unknown toxicity during the process. This study was undertaken to examine the in vitro cytotoxicity and mutagenicity of TNT and several of its degradation products/metabolites. TNT was equally cytotoxic to H4IIE cells and Chinese hamster ovary-K1 (CHO) cells (LC50 of 4 g/ml vs 24 microg/ml, with overlapping 95% prediction intervals), indicating that TNT does not need to be metabolized to exhibit cytotoxicity. Four metabolites studied, 4-hydroxylamino-2,6-dinitrotoluene; 2-amino-4,6-dinitrotoluene; 4,4',6,6'-tetranitro-2,2'-azoxytoluene; and 2,2',6,6'-tetranitro-4, 4'-azoxytoluene, were equally cytotoxic to both H4IIE and CHO cells. The LC50s were in the 3- to 18-microg/ml range and were not significantly different from TNT cytotoxicity in both cell lines. 4-Amino-2,6-dinitrotoluene (4-A) was moderately less cytotoxic than TNT to H4IIE cells, but was noncytotoxic to CHO cells. This result indicates that 4-A is metabolized to a cytotoxic compound. Both TNT and its metabolites exhibited only slight mutagenicity at high doses in one or both of the mutagenicity assays. While composting may reduce the levels of TNT in composted material, the hazard associated with TNT-contaminated soils is probably lower, but still uncertain.

2,4,6-Trinitrotoluene (TNT) and compounds associated with its production are toxic and phototoxic to a wide range of biota. The planarian Dugesia dorotocephala, but not Daphnia magna, metabolized TNT (1 mg/liter) to 4-amino-2,6-dinitrotoluene (4A; 0.4 mg/liter) and 2-amino-4,6-dinitrotoluene (2A; 0.2 mg/liter). Coexposure to near-ultraviolet (nuv) light enhanced the toxicity of 2A more than that of TNT and 4A. The toxicities of TNT, 4A, and 2A to Du. dorotocephala were all decreased by glutathione (GSH) conjugation. This suggests that all had mechanisms of toxic action involving formation of quinone-GSH conjugates. Dark and light mechanisms for TNT and 2A depended on GSH conjugation, but the specific mechanisms may be different for each compound. The dark and light mechanisms of toxic action for 4A appeared to be fundamentally different in that the dark toxic mechanism of action was less dependent on GSH conjugation. Hemolysis studies using sheep erythrocytes showed that the light-enhanced toxic mechanism of action for TNT, 2A, and/or 4A did not involve cellular membrane damage in response to nuv-induced anions.