Analysis of the sulphur mustard metabolites thiodiglycol and thiodiglycol sulphoxide in urine using isotope-dilution gas chromatography-ion trap tandem mass spectrometry

A sensitive method has been developed for the trace analysis of the sulphur mustard metabolite thiodiglycol (TDG) in urine, and its oxidation product thiodiglycol sulphoxide (TDGO) after reduction to thiodiglycol. Thiodiglycol was extracted from urine by solid phase extraction onto a polymeric cartridge and, after isolation, converted to its bis-heptafluorobutyryl derivative with heptafluorobutyryl imidazole. An ion trap mass spectrometer in selected reaction monitoring mode detected spiked concentrations down to 0.2 ng/ml with a signal to noise ratio>3:1. Urine, from human volunteers with no known exposure to sulphur mustard, contained detectable but very low concentrations (<0.2 ng/ml) of thiodiglycol, consistent with previous observations using different methodologies. Combined concentrations of thiodiglycol and thiodiglycol sulphoxide were determined after reduction of the latter with titanium trichloride. In this case higher background levels (up to 3 ng/ml) were observed, consistent with the sulphoxide being the major excretion product of the two metabolites. The method was applied to urine samples, stored frozen for 13 years, from two casualties of accidental mustard poisoning. Levels of thiodiglycol were 1 and 3 ng/ml, which increased to 78 and 104 ng/ml after treatment of the urine with titanium trichloride.     

The Bioutilization of Thiodiglycol (a Detoxication Product of Mustard Gas): Isolation of Degrading Strains and Investigation of Degradation Conditions

The Alcaligenes xylosoxydans subsp.denitrificans strain TD1 capable of degrading thiodiglycol (TDG), a product of mustard gas hydrolysis, was isolated from soil contaminated with breakdown products of this chemical warfare agent. The selected stable variant of TD1 (strain TD2) can grow on TDG with a lag phase of 4–8 h and a specific growth rate of 0.04–0.045 h^–1. Optimal conditions for the biodegradation of TDG (pH, the concentration of TDG in the medium, and specific substrate loading) were determined. TDG was found to be degraded with the formation of diglycolsulfoxide and thiodiglycolic acid as intermediate products. The data obtained can be used to develop approaches to the bioremediation of mustard gas–contaminated soils.     

Improved methodology for the detection and quantitation of urinary metabolites of sulphur mustard using gas chromatography-tandem mass spectrometry

Gas chromatography-tandem mass spectrometry (GC-MS-MS) with selected-reaction monitoring was applied to the analysis of urinary metabolites of sulphur mustard, derived from the β-lyase pathway and from hydrolysis. In the case of β-lyase metabolites, a limit of detection of 0.1 ng/ml was obtained, compared to 2–5 ng/ml using single stage GC-MS with selected-ion monitoring. GC-MS-MS methodology was less useful when applied to the analysis of thiodiglycol bis(pentafluorobenzoate) using negative-ion chemical ionisation although selected-reaction chromatograms were cleaner than selected-ion chromatograms. The advantage of using GC-MS-MS was demonstrated by the detection of low levels of β-lyase metabolites in the urine of casualties who had been exposed to sulphur mustard.     

Thiodiglycol Metabolism in Alcaligenes xylosoxydans subsp. denitrificans

The investigation of the degradation of thiodiglycol (the major product of mustard gas hydrolysis) by Alcaligenes xylosoxydans subsp. denitrificans strain TD2 showed that thiodiglycol is metabolized through the oxidation of its primary alcohol groups and the subsequent cleavage of C–S bonds in the intermediate products, thiodiglycolic and thioglycolic acids. The end products of these reactions are SO₄ ^2– ions and acetate, the latter being involved in the central metabolism of strain TD2. The oxidation of the sulfur atom gives rise to diglycolsulfoxide, which is recalcitrant to further microbial degradation. Based on the data obtained, a metabolic pathway of thiodiglycol transformation by A. xylosoxydans subsp. denitrificans strain TD2 is proposed.     

New insights into the biodegradation of thiodiglycol, the hydrolysis product of Yperite (sulfur mustard gas)

Aims:  To isolate thiodiglycol (TDG)-degrading bacteria, the mustard gas hydrolysis product, and to characterize the metabolites formed and the enzymes involved in the degradation.
Methods and Results:  Two strains, identified as Achromobacter xylosoxydans G5 and Paracoccus denitrificans E4, isolated from a petroleum-contaminated soil, utilized TDG as sole carbon and sulfur source. During the degradation of TDG by strain E4 [(2-hydroxyethyl)thio] acetic acid (HETA), thiodiglycolic acid (TDGA) and bis -(2-hydroxyethyl)disulfide (BHEDS) were identified by gas chromatography–mass spectrometry analysis, while HETA and TDGA were identified for strain G5. Two-dimensional isoelectric focussing-gel electrophoresis (2-D IEF/SDS–PAGE) maps of protein extracts of P. denitrificans E4 grown on TDG showed a spot identified as a methanol dehydrogenase. Increased expression of a putative iscS gene, involved in sulfur assimilation, was observed in TDG-grown cells of A. xylosoxydans G5.
Conclusions:  TDG degradation by P. denitrificans E4 occurred through two pathways: one involved cleavage of the C–S bond of HETA, yielding BHEDS and the other, oxidation of the alcoholic groups of TDG, yielding TDGA. The cleavage of the C–S bond of TDGA gave mercaptoacetic acid, further oxidized to acetate and sulfate.
Significance and Impact of the Study:  Increased knowledge of TDG-degrading bacteria and the possibility of using them in a tailored-two-stage mustard gas destruction process.     

In vitro oxidation of the hydrolysis product of sulfur mustard, 2,2′-thiobis-ethanol,by mammalian alcohol dehydrogenase

The mechanism of vesication from sulfur mustard remains unknown in spite of 80 years of investigation. We recently reported sulfur mustard–related inhibition of one or more protein (serine/threonine) phosphatases in tissue cytosol in vitro, suggesting a mechanism common to other vesicants such as cantharidin and Lewisite. Our investigation showed that this inhibition was related to the concentration of 2,2′-thiobis-ethanol (thiodiglycol), the hydrolysis product of sulfur mustard, rather than to the concentration of mustard itself. Related work showed an increase in the rate of NAD (but not NADP) reduction upon the addition of thiodiglycol to mouse liver cytosol. This result provided evidence that metabolism beyond thiodiglycol may be contributing to protein phosphatase inhibition. This observation indicated that metabolism involving one or more dehydrogenases may be necessary to produce the ultimate inhibitor of the protein phosphatases. We report here that thiodiglycol is a substrate for horse liver alcohol dehydrogenase (K_m = 3.68 ± 0.45 mM and V_max = 0.22 ± 0.01 μmol min⁻¹ mg protein⁻¹) and for pyridine nucleotide-linked enzymes in mouse liver and human skin cytosol. The alcohol dehydrogenase-specific inhibitor 4-methylpyrazole inhibited the oxidation of thiodiglycol by the pure horse liver enzyme as well as by the enzymes in human skin and mouse liver cytosol, indicating that the activity in the tissue preparations is also alcohol dehydrogenase. © 1998 John Wiley & Sons, Inc. J Biochem Mol Toxicol 12: 361–369, 1998     

Complete degradation of Yperite, a chemical warfare agent, by basidiomycetes

The complete degradation of Yperite (bis(2-chloroethyl) sulfide), a chemical warfare agent, was achieved by two basidiomycetous cultures. Two distinct metabolic pathways were detected in each fungus during degradation of Yperite. The major path involved a non-enzymatic hydrolysis to generate thiodiglycol. In the minor path, the sulfide bond was cleaved prior to the hydrolytic dechlorination reaction, yielding chloroethanol and chloromercaptoethane, both of which were then metabolized completely.     

Soil bacterium Pseudomonas sp.: destroyer of mustard gas hydrolysis products

A bacterial culture capable of utilizing products of mustard gas hydrolysis as a source of carbon was isolated from soil. This culture was tolerant to organochlorine substances in the hydrolysate. The bacterium was identified as Pseudomonas sp. The bacterium utilizes the major product of mustard gas hydrolysis, thiodiglycol, through two pathways. One involves the oxidation of the primary alcoholic groups in thiodiglycol, yielding thiodiglycolic and thioglycolic acids. The cleavage of the C-S bonds in these acids gives rise to acetate, which is then used further in the cell metabolism. The other pathway involves the cleavage of the C-S bond in the thiodiglycol molecule, yielding beta-mercaptoethanol, which is transformed by Pseudomonas sp. into thioglycolic acid. The results show the promise of this bacterium for the bioremediation of mustard gas-contaminated soils.     

Thiodiglycol metabolism in Alcaligenes xylosoxydans subsp. denitrificans

The investigation of the degradation of thiodiglycol (the major product of mustard gas hydrolysis) by Alcaligenes xylosoxydans subsp. denitrificans strain TD2 showed that thiodiglycol is metabolized through the oxidation of its primary alcohol groups and the subsequent cleavage of C-S bonds in the intermediate products, thiodiglycolic and thioglycolic acids. The end products of these reactions are SO4(2-) ions and acetate, the latter being involved in the central metabolism of strain TD2. The oxidation of the sulfur atom gives rise to diglycolsulfoxide, which is recalcitrant to further microbial degradation. Based on the data obtained, a metabolic pathway of thiodiglycol transformation by A. xylosoxydans subsp. denitrificans strain TD2 is proposed.     

Specific and sensitive quantitation of 2,2′-dichlorodiethyl sulphide (sulphur mustard) in water, plasma and blood: application to toxicokinetic study in the rat after intravenous intoxication

A sensitive and specific capillary gas chromatographic method has been developed to measure trace amounts of 2,2′-dichlorodiethyl sulphide (sulphur mustard) in environmental or biological samples. Sulphur mustard was isolated from water or plasma by a solid-phase extraction procedure and from blood by liquid—liquid extraction. The accuracy and precision of the methods were demonstrated using replicate analyses of spiked water, plasma or blood: within-run and between-run variabilities were less than 20%. These analytical methods were used to evaluate the rate of sulphur mustard degradation in water or plasma. Good linear calibration curves, with a detection limit of 45 ng/ml, were obtained for quantitation and determination of sulphur mustard in blood following its intravenous administration to rats. Initial toxicokinetic data were obtained.     

The bioutilization of thiodiglycol (a breakdown product of mustard gas): isolation of degraders and investigation of degradation conditions

The Alcaligenes xylosoxydans subsp. denitrificans strain TD1 capable of degrading thiodiglycol (TDG), a breakdown product of mustard gas, was isolated from soil contaminated with breakdown products of this chemical warfare agent. The selected stable variant of TD1 (strain TD2) can grow on TDG with a lag phase of 4-8 h and a specific growth rate of 0.04-0.045 h-1. Optimal conditions for the biodegradation of TDG (pH, the concentration of TDG in the medium, and its relative content with respect to the bacterial biomass) were determined. TDG was found to be degraded with the formation of diglycolsulfoxide and thiodiglycolic acid. The data obtained can be used to develop approaches to the bioremediation of mustard gas-contaminated soils.     

Solid-phase extraction and reversed-phase high-performance liquid chromatographic determination of sulphur mustard in blood

A reversed-phase high-performance liquid chromatographic method is reported for the analysis of sulphur mustard in blood with the aid of solid-phase extraction sample preparation. Sulphur mustard is extracted from blood samples (both in vitro and in vivo) of rats with a solution of 0.05 M sodium dodecyl sulphate and pre-concentrated over Sep-Pak C₁₈cartridges pre-coated with Tween-20. A Polygosil C₁₈ column is used with acetonitrile—water (52:48, v/v) as mobile phase for separation and sulphur mustard was detected at 200 nm.     

Nitrogen and sulphur relations in effecting yield and quality of cereals and oilseed crops

Nitrogen and sulphur, both vital structural elements, are especially needed for the synthesis of proteins and oils. Investigations revealed the required application of sulphur is one half to one third the amount of nitrogen, and the ratio becomes narrower in mustard (Brassica juncea L.), followed by wheat and rice. The efficiency of an increased level of nitrogen required a proportionately higher amount of sulphur. A critical investigation on the effective utilization of applied vis-à-vis absorbed nitrogen in wheat and mustard envisaged accumulation of NO3-N in vegetative parts when sulphur remained proportionately low. Application of sulphur hastened the chemical reduction of absorbed NO3- for its effective utilization. The effect was more pronounced in mustard than in wheat. Easily available forms of sulphur, like ammonium sulphate and gypsum, as compared to pyrite or elemental sulphur, maintained adequate N to S ratio in rice, resulting in a reduction in the percent of unfilled grain, a major consideration in rice yield. A narrow N to S ratio, with both at higher levels, increased the oil content but raised the saponification value of the oil, a measure of free fatty acids. Whereas, a proportionately narrow N to S ratio at moderate dose resulted in adequately higher seed and oil yield with relatively low saponification value, associated with increased iodine value of the oil, indicating respectively low free fatty acids and higher proportion of unsaturated fatty acids, an index for better quality of the oil.     

Activity and function in lung injuries due to sulphur mustard

Aim. Pulmonary complications are known to occur in over half the patients exposed to sulphur mustard. Many studies have focused on the clinical complications, often ignoring the pathogenesis of sulphur mustard. Also, the reasons for the variable severity of lung injuries caused by sulphur mustard are unclear. Hence, the current study was performed to evaluate the correlation between superoxide dismutase (SOD) and catalase (CAT) activity and pulmonary function in patients exposed to sulphur mustard. Methods. Our study was a comparative cross-sectional survey. Two hundred and fifty incident survivors were selected from the Sardasht population who were exposed to sulphur mustard in 1987. A control group from non-exposed civilians was also selected. We used a pulmonary function test, and SOD and CAT activity was measured in these groups. Results. The mean SOD activity in the healthy control group (70.5±10.8 U ml^?1) was higher than in the moderate-to-severe group (67.0±6.1 U ml^?1) (p <0.001, one-tail ANOVA, least significant difference (LSD) post hoc). The mean activity in the mild group (72.5±6.9 U ml^?1) was no higher than in the healthy control group (70.5±10.8 U ml^?1) (p=0.095 one-tail ANOVA, LSD post hoc). The mean CAT activity in the healthy control group (4.9±1.5 U ml^?1) was lower than in the moderate-to-severe group (8.0±1.8 U ml^?1) (p <0.001, one-tail ANOVA, LSD post hoc) and in the mild group (7.5±1.5 U ml^?1) (p=0.012 one-tail ANOVA, LSD post hoc). Conclusion. According to our findings, it is reasonable to hypothesize that re-establishment of the activation–inactivation or oxidant–antioxidant balance in favour of the activation and antioxidant balances would be useful as a therapeutic strategy to suppress pathological mechanisms underlying lung injuries.     

Cytotoxicity of alkylating agents towards sensitive and resistant strains of Escherichia coli in relation to extent and mode of alkylation of cellular macromolecules and repair of alkylation lesions in deoxyribonucleic acids

1. A quantitative study was made of the relationship between survival of colony-forming ability in Escherichia coli strains B/r and B(s-1) and the extents of alkylation of cellular DNA, RNA and protein after treatment with mono- or di-functional sulphur mustards, methyl methanesulphonate or iodoacetamide. 2. The mustards and methyl methanesulphonate react with nucleic acids in the cells, in the same way as found previously from chemical studies in vitro, and with proteins. Iodoacetamide reacts only with protein, principally with the thiol groups of cysteine residues. 3. The extents of alkylation of cellular constituents required to prevent cell division vary widely according to the strain of bacteria and the nature of the alkylating agent. 4. The extents of alkylation of the sensitive and resistant strains at a given dose of alkylating agent do not differ significantly. 5. Removal of alkyl groups from DNA of cells of the resistant strains B/r and 15T(-) after alkylation with difunctional sulphur mustard was demonstrated; the product di(guanin-7-ylethyl) sulphide, characteristic of di- as opposed to mono-functional alkylation, was selectively removed; the time-scale of this effect suggests an enzymic rather than a chemical mechanism. 6. The sensitive strain B(s-1) removed alkyl groups from DNA in this way only at very low extents of alkylation. When sensitized to mustard action by treatment with iodoacetamide, acriflavine or caffeine, the extent of alkylation of cellular DNA corresponding to a mean lethal dose was decreased to approximately 3 molecules of di(guanin-7-ylethyl) sulphide in the genome of this strain. 7. Relatively large numbers of monofunctional alkylations per genome can be withstood by this sensitive strain. Iodoacetamide had the weakest cytotoxic action of the agents investigated; methyl methanesulphonate was significantly weaker in effect than the monofunctional sulphur mustard, which was in turn weaker than the difunctional sulphur mustard. 8. Effects of the sulphur mustards on nucleic acid synthesis in sensitive and resistant strains were studied. DNA synthesis was inhibited in both strains at low doses in a dose-dependent manner, but RNA and protein synthesis were not affected in this way. 9. DNA synthesis in E. coli B(s-1) was permanently inhibited by low doses of mustards. In the resistant strains 15T(-) and B/r a characteristic recovery in DNA synthesis was observed after a dose-dependent time-lag. This effect could be shown at low doses in the region of the mean lethal dose. 10. Cellular DNA was isotopically prelabelled and the effect of mustards on stability of DNA was investigated. With resistant strains a dose-dependent release of DNA nucleotide material into acid-soluble form was found; this was much more extensive with the difunctional mustard (about 400 nucleotides released per DNA alkylation) than with the monofunctional mustard (about 10 nucleotides per alkylation). With the sensitive strain no dose-dependent release was found, though the DNA was less stable independent of cellular alkylation. 11. The results are discussed in terms of the concepts that alkylation of cellular DNA induces lesions which interfere with DNA replication, but which can be enzymically ;repaired'. The possible nature of these lesions is discussed in terms of the known reactions of the alkylating agents with DNA.     

Biocatalytic transformation of [(2-Hydroxyethyl)thio]acetic acid and thiodiglycolic acid from thiodiglycol by Alcaligenes xylosoxydans ssp. xylosoxydans (SH91)

A Gram-negative bacterium, Alcaligenes xylosoxydans ssp. xylosoxydans (SH91), consumed thiodiglycol (TDG), the nontoxic hydrolysis product of sulfur mustard, as a primary carbon source and transformed TDG to commercially relevant chemical precursors, [(2-hydroxyethyl)thio]acetic acid (HETA) and thiodiglycolic acid (TDGA). Aerobic fed batch and repeated batch experiments were run to compare the molar yields of HETA and TDGA that result under different operating policies. In repeated batch experiments, 35% of the TDG was converted to HETA. Under the conventional batch process and a repeated fed batch process, the HETA yields were reduced (21% and 18%, respectively), while the yield of TDGA was increased (47% and 31%,respectively). This work demonstrated that cell growth associated biocatalytic transformations were manipulated to achieve a desired byproducts profile through an understanding of the specific reaction and cell growth kinetics and by altering the reaction operating policy accordingly.     

The action of azimexone on the cells of the hemopoietic system in mice,especially after damage with X-rays

Mice were exposed to single doses of whole body X-irradiation (1 - 2 - 4 Gy) or were treated with sulphur mustard (15 mg/kg body weight i.p.). This treatment caused a reduction of the pluripotent stem cells in the bone marrow, of the total count of nucleated bone marrow cells in the femora and of the WBC in the peripheral blood. The size distribution of the bone marrow cells showed three separate peaks. From the histological examination of the bone marrow of X-irradiated mice it was deduced that the first peak represents erythrocytes, the second lymphocytes and the third peak the precursors of red and white blood cells. Multiple doses (25 - 50 - 100 mg/kg body weight) of azimexone, an immunomodulating substance, led after moderate doses of X-rays (2 Gy) or sulphur mustard to a more rapid recovery of the various parameters. In particular a stimulant action of azimexone on the pluripotent stem cells of mice not subjected to the injurious agents could also be demonstrated.     

Historical perspective on effects and treatment of sulfur mustard injuries

Sulfur mustard (2,2′-dichlorodiethyl sulfide; SM) is a potent vesicating chemical warfare agent that poses a continuing threat to both military and civilian populations. Significant SM injuries can take several months to heal, necessitate lengthy hospitalizations, and result in long-term complications affecting the skin, eyes, and lungs. This report summarizes initial and ongoing (chronic) clinical findings from SM casualties from the Iran–Iraq War (1980–1988), with an emphasis on cutaneous injury. In addition, we describe the cutaneous manifestations and treatment of several men recently and accidentally exposed to SM in the United States.     

Biosynthesis and urinary excretion of methyl sulfonium derivatives of the sulfur mustard analog, 2-chloroethyl ethyl sulfide, and other thioethers

Thioether methyltransferase was previously shown to catalyze the S-adenosylmethionine-dependent methylation of dimethyl selenide, dimethyl telluride, and various thioethers to produce the corresponding methyl onium ions. In this paper we show that the following thioethers are also substrates for this enzyme in vitro: 2-hydroxyethyl ethyl sulfide, 2-chloroethyl ethyl sulfide, thiodiglycol, t-butyl sulfide, and isopropyl sulfide. To demonstrate thioether methylation in vivo, mice were injected with [methyl-3H]methionine plus different thioethers, and extracts of lungs, livers, kidneys, and urine were analyzed by high-performance liquid chromatography for the presence of [3H]methyl sulfonium ions. The following thioethers were tested, and all were found to be methylated in vivo: dimethyl sulfide, diethyl sulfide, methyl n-propyl sulfide, tetrahydrothiophene, 2-(methylthio)ethylamine, 2-hydroxyethyl ethyl sulfide, and 2-chloroethyl ethyl sulfide. This supports our hypothesis that the physiological role of thioether methyltransferase is to methylate seleno-, telluro-, and thioethers to more water-soluble onium ions suitable for urinary excretion. Conversion of the mustard gas analog, 2-chloroethyl ethyl sulfide, to the methyl sulfonium derivative represents a newly discovered mechanism for biochemical detoxification of sulfur mustards, as this conversion blocks formation of the reactive episulfonium ion that is the ultimate alkylating agent for this class of compounds.