Metabolism of [¹⁴C]-2,4,6-Trinitrotoluene in Tobacco Cell Suspension Cultures

Abstract

The metabolism of 2,4,6-trinitrotoluene (TNT) was investigated in tobacco cell suspension cultures amended with [14C]-TNT. Five metabolites were purified and characterized. Temporal evolution of metabolites was monitored during a 120 h incubation period. Metabolites structure was identified by acid and enzymatic hydrolysis, followed by electrospray ionization mass spectrometry and 1H and 13C NMR spectroscopy analyses. The majority of metabolites were conjugates formed by glycose conjugation on the hydroxylamine group of either 2-hydroxylamino-4,6-dinitrotoluene (2-HADNT) or 4-hydroxylamino-2,6-dinitrotoluene (4-HADNT), which led to monoglycoside then to diglycoside. Various diglycosides were observed with gentiobioside or sophoroside formation. Bound residues represented a small fraction (<10% of initial 14C) irrespective of the interval after TNT amendment. Free ADNT was detected only in the medium. This study highlights the central role played by HADNT in the TNT metabolic pathway in tobacco cell suspension culture, and the key role of these compounds and of glycosyltransferases in TNT phytoremediation processes.

Mineralization of 2,4,6-Trinitrotoleune (TNT) in Coastal Waters and Sediments

“…The types of whole plants that have been studied include the bush bean (Harvey et al 1990), hybrid poplar (Thompson et al 1998), soybean (Adamia et al 2006), switchgrass (Peterson et al 1998), ryegrass (Sung et al 2004), yellow nutsedge (Palazzo and Leggett 1986) and a variety of wetland species (Best et al 1999a, b, Bhadra et al 1999a, b, Burken et al 2000, Gorge 1994, Paquin et al 2004, Pavlostathis et al 1998. Some investigators have used cell cultures , Vanek et al 2002, Vila et al 2005 or plant extracts (Medina et al 2004) to differentiate between degradation by the plant cells vs the associated microflora. Commensal bacteria are known to enhance TNT metabolism , Van Aken et al 2004) especially those bacteria found in the rhizosphere (Siciliano et al 2000).…”

Phytoremediation of Explosive-Contaminated Soils

“…In Nicotiana tabacum (tobacco) strain BY2 cell suspension cultures, monosaccharides such as glucose were identified as common conjugates. This occurs through a glycosidic bond, which was thought to form at the nitroso group in the case of HADNTs [33,34,39] (Fig. 1).…”

Application of plant tissue cultures in phytoremediation research: Incentives and limitations

“…Five metabolites purified and characterized; almost complete transformation to hydroxylamino-4,6-dinitrotoluene Vila et al (2005) Cell suspension Hamill and Lidgett (1997). The general properties, methods for initiation, and applications of shooty teratomas are described by Subroto et al (1996Subroto et al ( , 1997.…”

“…The release of polar metabolites that are unable to pass through the cytoplasmic membrane by diffusion has been attributed to an active membrane transporter (Schmidt et al, 1994), or to uncoupling of the cell membranes as a toxic effect of the xenobiotic compound applied . It has been suggested that metabolite release is a consequence of lower enzyme affinities or less efficient conjugation mechanisms for those compounds within the plant cells (Lao et al, 2003;Owen and Donzel, 1986;Vila et al, 2005), or because the metabolites are not sequestered effectively in the vacuosle (Schmidt et al, 1994). Malonylation and glucosylation of natural plant products are known to influence the transport and storage of metabolites and these conjugations may act as signals to direct xenobiotic metabolites to either the vacuole for sequestration within the cell or to the extracellular space (Lao et al, 2003).…”