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PubMed1999

Determination of explosives in soil and ground water by liquid chromatography-amperometric detection.

Electrochemical reduction of trinitrotoluene (TNT) and several nitroaromatics has been exploited toward the development of an amperometric detector for liquid chromatography (LC). Up to a ten-fold increase in sensitivity was accomplished for the explosives using amperometric detection instead of conventional UV measurement. A working glassy carbon electrode (poised at -0.80 V vs. Ag/AgCl) offered a detection limit of 9, 44 and 550 nM for trinitrobenzene, TNT and 1,4-dinitrobenzene, respectively. Separation of eleven TNT-related compounds in a mixture was achieved within 15 min using a C18 column and a mobile phase consisting of acetonitrile-50 mM phosphate buffer pH 5 (1:2, v/v) and 18 mM sodium dodecylsulfate. The LC-amperometric detection system was applicable for analyzing soil extracts and ground water and the results obtained agreed well with that of the US Environmental Protection Agency recommended procedure. Extension to analysis of HMX (octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine) and RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) was accomplished with a silver working electrode instead of a glassy carbon electrode installed in a thin channel cell.

Carbon↗

PubMed1983

Cell-surface antigenic modifications with trinitrophenyl sulfonate versus trifluoromethyl-dinitrophenyl sulfonate.

The aim of the work was to justify the use of trifluoromethyl-dinitrobenzene sulfonate (CF3-DNBS) modification rather than trinitrobenzene-sulfonate (TNBS) modification, so as to be able to take advantage of the presence of fluorine atoms in the analogue, which allow the analysis of the hapten-carrier bonds by 19F-NMR nuclear magnetic resonance. Cell-surface antigenic modifications brought about by exposure to TNBS or CF3-DNBS were found to be immunologically cross-reactive, both in cell-mediated lymphocytotoxicity and in indirect immunofluorescence. The extent of haptenic derivatizations was found to be of the same order of magnitude, as appraised both by quantitative-absorption studies or by using radioactive hapten, provided that the less chemically reactive CF3-DNBS was used at the concentration of 10 mM and TNBS at the concentration of 1 mM. However, only TNBS-modified cells were sensitive to destruction by antibody-plus-complement-mediated cytotoxicity.

Animals↗

PubMed2004

Microbiological changes during bioremediation of explosives-contaminated soils in laboratory and pilot-scale bioslurry reactors.

Changes in the microbial community during bioremediation of explosives-contaminated soil in a molasses-fed bioslurry process were examined. Upon addition of molasses to laboratory-scale reactors, total culturable heterotrophs increased rapidly by three to four orders of magnitude. However, heat-shocked heterotrophs and the percentage of gram-positive bacterial isolates did not increase until the soluble concentrations of 2,4,6-trinitrotoluene (TNT) and 2,4,6-trinitrobenzene (TNB) began to decrease. The number of identified phospholipid fatty acids (PLFA) and the total PLFA concentration also exhibited an immediate increase in response to molasses addition, while the concentration of branched PLFA, indicative of the gram-positive population, remained low until soluble TNT and TNB concentrations had significantly decreased. This same general relationship between explosives degradation and gram-positive-specific PLFA was observed during an experiment with a large field-scale bioslurry lagoon reactor. These results indicate that the gram-positive organisms, which have been shown to be severely impacted by even low concentrations of TNT and TNB [Current Microbiol. 35 (1997) 77; Environ. Toxicol. Chem. 17 (1998) 2185], are able to increase in concentrations after explosives compounds are reduced to non-inhibitory levels, and should therefore be able to reestablish themselves in remediated soils.

Biodegradation, Environmental↗

PubMed2001

Use of a Salmonella microsuspension bioassay to detect the mutagenicity of munitions compounds at low concentrations.

Past production and handling of munitions has resulted in soil contamination at various military facilities. Depending on the concentrations present, these soils pose both a reactivity and toxicity hazard and the potential for groundwater contamination. Many munitions-related chemicals have been examined for mutagenicity in the Ames test, but because the metabolites may be present in low environmental concentrations, a more sensitive method is needed to elucidate the associated mutagenicity. RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine), TNT (2,4,6-trinitrotoluene), tetryl (N-methyl-N-2,4,6-tetranitroaniline), TNB (1,3,5-trinitrobenzene) and metabolites were examined for mutagenicity in a microsuspension modification of the Salmonella histidine reversion assay with and without metabolic activation. TNB and tetryl were positive in TA98 (32.5, 5.2revertants/nmole) and TA100 (7.4, 9.5revertants/nmole) without metabolic activation and were more potent than TNT (TA98, 0.3revertants/nmole; TA100, 2.4revertants/nmole). With the exception of the tetranitroazoxytoluene derivatives, TNT metabolites were less mutagenic than TNT. RDX and two metabolites were negative in both strains, however, hexahydro-1,3,5-trinitroso-1,3,5-triazine was positive in TA100 with and without S9. Microsuspension bioassay results tend to correlate well with published Ames test data, however, there are discrepancies among the published data sets and the microsuspension assay results.

Aniline Compounds↗

PubMed1999

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.

Animals↗

PubMedDate not supplied

On-line monitoring of cell growth and cytotoxicity using electric cell-substrate impedance sensing (ECIS).

An on-line and continuous technique based on electric cell-substrate impedance sensing (ECIS) was developed for measuring the concentration and time response function of fibroblastic V79 cells exposed to mercury chloride and 1,3,5-trinitrobenzene (TNB). Attachment, spreading and proliferation of V79 fibroblastic cells cultured on a microarray of small gold electrodes precoated with fibronectin were detected as resistance changes. The response function was derived to reflect the resistance change as a result of cell attachment, spreading, mitosis and cytotoxicity effect. Exposure of V79 cells to mercury chloride or TNB led to alterations in cell behavior, and therefore, chemical cytotoxicity was easily screened by measuring the response function of the attached and spread cells in the presence of inhibitor. The half inhibition concentration, the required concentration to achieve 50% inhibition, was obtained from the response function to provide information about cytotoxicity during the course of the assay. A simple mathematical model was developed to describe the responses of ECIS that were related to the attachment, spreading, and proliferation of V79 fibroblastic cells. The novel results of this paper are mainly characterized by the systematic study of several parameters including the cell number, detection limit, sensor sensitivity, and cytotoxicity, and they may motivate further research and study of ECIS sensors.

Animals↗

PubMed2001

Mechanisms for the adsorption of substituted nitrobenzenes by smectite clays.

To more fully understand the potential for transport of nitroaromatic compounds in soils and subsoils,the adsorption of a series of para- and meta-substituted nitrobenzenes (SNBs) by K-smectite clay was measured. Adsorption isotherms were fit to the Freundlich equation, and the resultant Freundlich adsorption coefficients (log(Kf) were positively correlated with the Hammett substituent constant (r2 = 0.80). This relationship and a positive reaction constant (p = 1.15) indicate that the adsorption reaction is favored by electron-withdrawing substituents. These results are consistent with an electron donor (smectite)-acceptor (substituted nitrobenzene) mechanism offered previously. However, quantum calculations did not reveal any systematic relationship between the Hammett constant and the electron density on the aromatic ring, which would explain a donor-acceptor relationship. Rather, electron density donated by a second substituent on nitrobenzene appears to be appropriated by the nitro group leaving ring electron density unchanged. Fourier transform infrared spectroscopy revealed shifts in the -NO2 vibrational modes of 1,3,5-trinitrobenzene (TNB) upon adsorption to K+-smectite that were consistent with the complexation of K+ by -NO2 groups. Such TNB vibrational shifts were not observed for SWy-1 saturated with more strongly hydrated cations (i.e., Na+, Mg2+, Ca2+, and Ba2+). The simultaneous interaction of multiple -NO2 groups with exchangeable K+ was indicated by molecular dynamic simulations. Adsorption of SNBs by smectite clays appears to result from the additive interactions of -NO2 groups and secondary substituents with interlayer K+ ions. Adsorption occurs to a greater or lesser extent depending on the abilities of substituents to complex additional interlayer cations and the water solubilities of SNBs. We conclude that the adsorption trends of SNBs on K-SAz-1 can be explained without recourse to hypothetical electron donor-acceptor complexes.

Adsorption↗

PubMed2001

Spectroscopic study of nitroaromatic-smectite sorption mechanisms.

Sorption mechanisms of 1,3- and 1,4-dinitrobenzene, 1,3,5-trinitrobenzene (TNB), dinitro-o-creasol, and 6-sec-butyl-2,4-dinitrophenol (DINOSEB) on smectite were investigated using FTIR spectroscopy and HPLC methods. A quantitative method was developed that established a direct link between the HPLC and the FTIR data. Freundlich sorption values ranged from 47 (L g(-1)) for 1,3,5-TNB to 3.7 for DINOSEB and showed that the extent of nitroaromatic compounds (NAC) sorption was strongly dependent on the number and position of the nitro substituents as well as other substituents and steric effects. The amount of 1,3,5-TNB sorbed to smectite was strongly influenced by the nature of the exchangeable cation. Furthermore, the exchangeable cation significantly influenced the positions and relative intensities of the vibrational modes of the -NO2 groups. The strongest perturbations were observed for cations with lower enthalpies of hydration (e.g., K+) and included a red shift of the v(asym)(NO) band, a concomitant blue shift of the v(sym)(NO) band. These changes were accompanied by a 2-fold increase in the relative intensity of the v(asym)(NO) band relative to the intensity of the v(sym) (NO) band. Molecular quantum mechanics calculations were used to rationalize frequency shifts in terms of nitroaromatic interactions with interlayer cations. Results indicate that the sorption of NACs to smectite surfaces is controlled largely by the hydration characteristics of the exchangeable cation, which regulates both cation-nitroaromatic complexation and swelling of the smectite.

2,4-Dinitrophenol↗

PubMed2000

Inhibition of octopus glutathione transferase by Meisenheimer complex analog, S-(2,4,6-trinitrophenyl) glutathione.

The tight binding of Meisenheimer intermediate with octopus digestive gland glutathione transferase was analyzed with 1,3,5-trinitrobenzene, which forms a trapped Meisenheimer complex with glutathione because there is no leaving group at the ipso carbon. By steady-state enzyme kinetic analysis, an inhibition constant of 1.89 +/- 0.17 microM was found for the transient formed, S-(2,4,6-trinitrophenyl) glutathione. The above inhibition constant is 407-fold smaller than the Km value for the substrate (2,4-dinitrochlorobenzene). Thus, S-(2,4,6-trinitrophenyl) glutathione is considered to be a transition-state analog. The tight binding of this inhibitor to the enzyme provides an explanation for the involvement of the biological binding effect on the rate enhancement in the glutathione transferase-catalyzed SNAr mechanism.

Animals↗

PubMed2003

Studies on nitroaromatic compound degradation in modified Fenton reactions by electrospray ionization tandem mass spectrometry (ESI-MS-MS).

Degradation products resulting from modified Fenton reactions with the nitroaromatic compounds trinitrotoluene (TNT) and trinitrobenzene (TNB) were identified by electrospray ionization tandem mass spectrometry (ESI-MS-MS). Several hydroperoxide adducts were tentatively identified as initial, one-electron reduction products of TNT and tandem mass spectrometry confirmed their structure. A transformation pathway of TNT, resulting from reactions with oxygen radical species generated by the modified Fenton reaction, was proposed.

Environmental Pollutants↗

PubMed2004

Ab initio based force field and molecular dynamics simulations of crystalline TATB.

An all-atom force field for 1,3,5-triamino-2,4,6-trinitrobenzene (TATB) is presented. The classical intermolecular interaction potential for TATB is based on single-point energies determined from high-level ab initio calculations of TATB dimers. The newly developed potential function is used to examine bulk crystalline TATB via molecular dynamics simulations. The isobaric thermal expansion and isothermal compression under hydrostatic pressures obtained from the molecular dynamics simulations are in good agreement with experiment. The calculated volume-temperature expansion is almost one dimensional along the c crystallographic axis, whereas under compression, all three unit cell axes participate, albeit unequally.

Chemistry, Physical↗

PubMed1978

H-2-restricted cytotoxic effectors generated in vitro by the addition of trinitrophenyl-conjugated soluble proteins.

Murine spleen cells from normal donors were cultured in vitro with trinitrobenzene sulfonate (TNBS)-conjugated soluble proteins, i.e., bovine gamma globulin (TNP-BGG) or bovine serum albumin (TNP-BSA). Addition of 100 mug of any of these TNP-proteins to the spleen cell cultures led to the generation of cytotoxic T-cell effectors which were H-2-restricted and TNP- specific. The lytic potential of such effectors was comparable to that generated by sensitization with TNBS-modified syngeneic cells, and was restricted to haplotypes shared at the K or K plus I-A, or the D regions of the H-2 complex. Greater effecter cell activity was generated by addition of TNP-BGG against TNBS-modified targets which shared K plus I-A than against modified targets which shared the D region with the responding cells, which suggests that the same immune response genes are involved when the response is generated by the addition of TNP-conjugated soluble proteins or of TNBS- modified cells. H-2-restricted, TNP-specific effecter cells were generated by culturing mouse spleen cells with syngeneic cells which had been preincubated with TNP- BGG or TNP-BSA for 1.5 h. The addition of unconjugated soluble proteins to the cultures did not result in cytotoxic effectors detectable on H-2-matched targets, whether the targets were prepared by modification with TNBS, or by incubation with either the unconjugated or TNP-conjugated proteins. Depletion of phagocytic cells in the tumor preparation by Sephadex G-10 column fractionation before incubation with TNP-BSA had no effect on their lysis by the relevant effector cells. Immunofluorescent staining of tumor target cells with anti-TNP antibodies indicated that TNP could be detected on the tumor cells within 10 rain of incubation with TNP-BSA. The cytotoxic response generated by addition of the TNP-proteins to spleen cell cultures was found to be T-cell dependent at the effector phase, as shown by the sensitivity of the lytic phase to absorbed RAMB and complement. Furthermore, the response did not appear to be attributable to antibody-dependent cellular cytotoxicity. Three mechanisms were considered which could account for the generation of H-2-restricted, TNP-specific, cytotoxic T-cell effectors by the addition of soluble TNP-proteins. These include covalent linkage of activated TNP groups from the soluble proteins to cell surface components, macrophage processing of the soluble conjugates and presentation to the responding lymphocytes in association with H-2-coded self structures, or hydrophobic interaction of the TNP-proteins to cell surfaces. Results obtained from sodium dodecyl sulfate gel patterns indicating that cell-bound TNP was still linked to BSA, and the observation that phagocytic-depleted cells could interact with the soluble TNP-proteins and function as H-2-restricted targets, appear not to favor the first two proposed mechanisms.

Animals↗

PubMed1984

Trinitrophenyl modification of H-2k and H-2b spleen cells results in enhanced serological detection of Kk-like determinants.

Several anti-H-2Kk but not anti-H-2Dd monoclonal antibodies (mAb) exhibited enhanced binding to B10.A murine spleen cells after modification of the cells with trinitrobenzene sulfonate (TNBS). The number of antibody molecules bound to TNP-modified B10.A spleen cells increased by a factor of two or more. The same anti-2Kk mAb that exhibited enhanced binding to modified B10.A cells did not bind to unmodified C57BL/10 spleen cells, as expected, but did bind to TNP-modified C57BL/10 spleen cells. This TNP-dependent binding was not a result of cross-reactions with cell surface TNP groups nor with Fc receptors. TNP modification of a variant cell line that does not express class I H-2 products did not result in enhanced binding by these mAb. These findings can account for preferential recognition of TNP-Kk by B10.A and B10.BR CTL, and also for cross-reactive lysis by C57BL/10 CTL stimulated by C57BL/10-TNP against unmodified H-2Kk targets.

Animals↗

PubMed1992

Peptide-conjugated hapten groups are the major antigenic determinants for trinitrophenyl-specific cytotoxic T cells.

Several trinitrophenyl (TNP)-specific mouse cytotoxic T cell (CTL) clones recognize TNP-conjugated peptides in association with class I MHC molecules ('hapten-peptide determinants'). However, cell modification with trinitrobenzene sulfonic acid (TNBS) also leads to the formation of TNP determinants covalently attached to MHC molecules ('altered self'). To determine the importance of 'peptide' versus 'altered self' determinants, we used the mutant cell line RMA-S which expresses peptide-free ('empty') Kb and Db molecules at 26 degrees C. Additionally, we stabilized Kb molecules on RMA-S cells at 37 degrees C using the Kb binding heptapeptide N53-59 derived from the vesicular stomatitis virus nucleoprotein. Lacking lysine, this peptide remains unmodified by TNBS and, therefore, only allows the formation of 'altered self' TNP determinants on occupied Kb molecules. RMA-S targets, pretreated or untreated with N53-59, upon TNBS modification were only lysed poorly or not at all by four different TNP-specific CTL. In contrast, all of these clones efficiently lysed TNBS-treated, unmutated RMA cells, and three of them strongly reacted with RMA or RMA-S cells in the presence of tryptic TNP-BSA peptides. Moreover, the clone unreactive for TNP-BSA peptides also recognized TNP self-peptides extracted from TNBS-treated syngeneic spleen cells. Taken together, these data clearly show that TNP residues linked to MHC via associated peptides but not by covalent bondage represent the dominant antigenic epitopes for class I MHC-restricted, hapten-specific T cells.

Amino Acid Sequence↗

PubMed1987

Photocross-linking from DNPated SH1 in myosin head. I. Cross-linking to the 50-kDa fragment.

When DNP-SH1-myosin, selectively dinitrophenylated at SH1 by 1,2,4-trinitrobenzene, was irradiated with a high-pressure mercury lamp equipped with a UV cut filter, a new 220-kDa band called the X-band appeared right above the heavy chain band (200 kDa) on SDS-PAGE (Laemmli). The time course of the X-band formation was composed of two phases, the initial one being rapid, and the second slow. Immune reaction experiments using antibodies specific for heavy or light chains indicated that the X-band in the initial phase contained heavy chain alone, but no light chains. Such an extra band (106 kDa) was also observed in the initial phase of photolysis of DNP-SH1-Subfragment-1 (heavy chain: 96 kDa) obtained from DNP-SH1-myosin. Trypsinolysis of the 106-kDa product generated a 83-kDa band. N-Terminal sequence analysis and the amino acid composition of the band revealed that the X-band is an intraheavy chain cross-linking product between the 20- and the 50-kDa fragments. This presents a striking contrast to the other cross-linking from SH1 using benzophenone-4-iodoacetamide which reacted with the 25-kDa fragment alone (Lu, R.C. et al. (1986) Proc. Natl. Acad. Sci. U.S. 83, 6392-6396). Based upon the result obtained, the spatial arrangement of the three tryptic domains around SH1 is discussed.

Amino Acid Sequence↗

PubMed1994

Trinitrophenylation of the reactive lysine residue in double-headed myosin in the presence of PP.

Lys-83 in the heavy chain of rabbit skeletal muscle myosin is rapidly and stoichiometrically modified by trinitrobenzene sulfonate. Other authors claimed that the half-stoichiometric trinitrophenylation of Lys-83 in myosin in the presence of PPi was correlated to a Pro/Ser microheterogeneity at the 78th residue position in the heavy chain [Miyanishi, T., Maita, T., Matsuda, G., & Tonomura, Y. (1982) J. Biochem. 91, 1845-1853]. However, our recent studies with chymotryptic subfragment 1 (S1) instead of myosin showed no such correlation between the half-stoichiometric trinitrophenylation and the Pro/Ser microheterogeneity [Komatsu, H. & Tawada, K. (1993) J. Biol. Chem. 268, 16974-16978]. Since the global structure of the head portion of myosin is different from that of chymotryptic S1 that lacks DTNB light chain, it could be argued that the difference is due to the structural difference between chymotryptic S1 and myosin. We hence reexamined the situation with myosin, and obtained the same results as found with S1: (i) Lys-83 in myosin was half-stoichiometrically trinitrophenylated in the presence of PPi, although it was stoichiometrically modified in the absence of PPi; (ii) there was a Pro/Ser microheterogeneity at the 78th position in the myosin heavy chain, which was not correlated to the half-stoichiometric trinitrophenylation of Lys-83 in the presence of PPi.

Amino Acid Sequence↗

PubMed1984

Photocross-linking from SH1 of the myosin heavy chain to light chains through the dinitrophenyl group attached to SH1.

Trinitrobenzene selectively dinitrophenylates SH1, a specific thiol in the myosin heavy chain which contains 1 mol of this cysteinyl residue. When the SH1-DNP-myosin thus obtained was irradiated with a mercury lamp, a cross-linked product was formed with a molecular weight of about 220K daltons. It was shown that this product was composed of both heavy and light chains by fluorescence labeling of the heavy chain at SH2, another specific thiol, and immune reaction using an anti-light chain antibody, respectively.

Animals↗

PubMed1985

Biochemical characteristics of cardiac myosin: the pH dependence of Ca-ATPase activity, and that of the absorption spectrum of 2,4,6-trinitrophenyl groups attached to myosin.

For both cardiac and skeletal myosin, the Ca-ATPase activity of myosin at acidic pH was shown to be different from that at alkaline pH, in the susceptibility to heat-inactivation, the effects of organic solvents, and the effect of trinitrophenylation of the myosin. It is therefore suggested that there are two different types of Ca-ATPase of both cardiac and skeletal myosin. Differences in the Ca-ATPase activity were also found between cardiac and skeletal myosins. (a) The Ca-ATPase activity of cardiac myosin was more susceptible to heat-inactivation at alkaline pH than at acidic pH. In contrast, the activity of skeletal myosin was more susceptible to heat-inactivation at acidic pH than at alkaline pH. (b) Dioxane weakly stimulated the activity of cardiac myosin at acidic pH, but strongly activated that of skeletal myosin at acidic pH. Acetone very strongly inhibited the activity of cardiac myosin at alkaline pH, but not so strongly that of skeletal myosin at alkaline pH. (c) Trinitrophenylation of the myosin resulted in loss of the activity optimum at acidic pH with skeletal myosin but not with cardiac myosin. As reported by Srivastava et al. (J. Biochem. 86, 725-731, 1979), 1 mol of lysine residue per mol of cardiac myosin quickly reacted with 2,4,6-trinitrobenzene sulfonate (TNBS) either in the absence or presence of inorganic pyrophosphate (PPi). However, trinitrophenyl (TNP) groups bound to cardiac myosin in the presence of PPi were significantly different, in the pH dependence of the absorption spectrum, from those bound (to cardiac myosin) in the absence of PPi.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗