Mustard Gas Plasma Exposure Markers Quantitative Analysis Method And Its Toxicokinetics

Sulfur mustard [bis-(2-chloroethyl) sulfide,SM] is a well-known vesicant agentand also a potent biological alkylating agent. After exposure, SM could enter into thebody and distribute rapidly in blood, tissues and organs. Its biotransformationpathways in body include oxidation, hydrolysis, glutathione conjugation followed byβ-cleavage and reaction with critical nucleophile sites of DNA or proteins,to form anumber of metabolites and macromolecular adducts. These metabolites and adductshave been studied intensively and used as biomarkers of SM. The SM biomarkers canbe divided into two categories. One is early-exposure (or metabolite) biomarkers,which are mainly consist of oxidative and hydrolysis metabolites, as well asβ-cleavage metabolites of glutathione conjugates. The second category can be calledpersistent (or conjugated) biomarkers, as they can stay in the body for a relativelylonger period. This category mainly refers to the macromolecular adducts of SM. Themetabolite biomarkers of SM usually appear in the body very quickly after SMexposure, and they could be used as early exposure biomarkers for monitoring SMcontamination, assessing extent of intoxication, and used to evaluate the effects of theprotection/therapeutic measures.The present study is focused on the SM metabolite biomarkers. By using updatedconcepts and techniques of modern instrumental analysis, analytical toxicology,quantitative toxicology and toxicokinetics, the following research tasks wereaccomplished: synthesis of standard reference substances of SM biomarkers,development of a key technique for quantitatively analysis of these SM biomarkers,study on toxicokinetics of the biomarkers in rat and dog after SM exposure, analysisof relationship between dose and exposure level, obtaining "detection time windows",and application studies. The above established key techniques and new knowledge ofSM plasma biomarkers will play important roles on exposure detection and diagnosisof SM intoxication, and will also contribute to the development of medicalcountermeasures against SM. In the study,9standard reference substances of SM metabolites and1internalstandard were obtained by chemical synthesis under optimized and controlledconditions, using SM as starting material. The structures of these standards wereconfirmed by several modern techniques, such as NMR, IR and MS. Their puritieswere all above95%. With the availability of these standard reference substances, akey technique was developed, for the first time, for simultaneous quantification of7SM metabolite biomarkers (TDG,TDGO,SMO,SBMTE,MSMTESE,SBMSE andSBSNAE) in rat plasma by a LC-MS/MS method. The method was fully validated asper the guidance for bioanalysis. The results showed that the specificity, recovery,matrix effect, stability, precision and accuracy of the method were all meet therequirements for toxicokinetic studies. When compared to the previously reportedanalytical methods for SM metabolites, the sensitivities of newly developed methodfor various metabolites were comparable to or higher than the old methods. Inaddition, the new method has several advantages over the old methods, includingwider linear ranges, a small volume of sample required, simple and rapid one stepsample preparation procedure of protein precipitation allowing high through-putanalysis of larger volume of biological samples. This technique provides an umbrellasolution to cover different needs of SM biomarker analysis.The plasma kinetics of SM metabolite biomarkers were studied comprehensively inthe SM exposed rats. After subcutaneous or skin exposures of SM at three dose levels,7SM metabolites(SMO,TDG,TDGO,SBMTE,SBMSE,MSMTESE and SBSNAE)were all detected in0.08-72h plasma samples. These7metabolites could be used asplasma exposure biomarkers of SM. It was found in the study, that SMO was a majormetabolite in blood circulation, which makes SMO a suitable plasma biomarker. Theplasma exposure levels of SMO, TDGO, SBMSE, MSMTESE and SBSNAE weredose-dependent. Their plasma concentrations were increased proportionally to thedose increase. The quantitative detection windows for these biomarkers were in thedifferent time frames between5min to48h. The results above suggest that SMO,TDGO, SBMSE, MSMTESE and SBSNAE may be used as diagnosis biomarkers forSM intoxication. The toxicokinetics study on SM biomarkers in beagle dogs showed speciesdifferences between rat and dog. The pathway of glutathione conjugation andβ-cleavage for SM in dog was significantly weaker than that in rat, and the formationof SBSNAE was deficient. The oxidative and hydrolysis metabolites (SMO,TDG andTDGO) are the better plasma exposure biomarkers in dog.The established LC-MS/MS quantitative method for SM biomarkers has beenapplied for efficacy assessment of a skin protectant and a decontamination agent,which were developed by the Institute. Using plasma exposure levels of SMbiomarkers as evaluation criteria, the skin protectant and the decontamination agentwere proved to be effective against skin exposed SM. The results indicated that theabsorption of SM was reduced to about4%of the control group, after the rats in thetreatment group were pre-treated with the testing skin protectant. For the rats in theother treatment group, after decontamination using the testing DECON agent at6min,the exposure of the biomarkers was reduced to about20%of the control group. Theabove results also confirmed the feasibility of the biomarker quantification techniqueto be used in the efficiency assessment of protective measures against SM. Thistechnique will be a useful quantitative tool for rapid and accurate evaluation of SMcountermeasures.The quantitative LC-MS/MS method of SM biomarkers was also used for analysis ofbiological specimens from victims of accidently exposed to an unknown chemicalagent. The plasma, skin transudate and urine samples were collected and analyzed.The SM biomarkers were detected in above biological specimens, and the SMexposure of these4patients was confirmed. The analytical data also provided theimportant quantitative information for the diagnosis of their intoxication. Theoutcome of this case demonstrates the promising application for the currentlydeveloped quantitative method in identification and diagnosis of SM exposure.The novel points and new findings of this study are summarized as follows:1. In the present, a novel quantitative bioanalytical method is developed andvalidated for simultaneously quantification of plasma SM biomarkers byLC-MS/MS. The technical specifications of the method are either comparable to the literature methods, or better than those methods. It also reports, for the firsttime, the quantitative analysis of SMO as a SM biomarker, and direct quantitativeanalysis of TDG/TDGO by LC-MS/MS.2. The toxicokinetic study of SM plasma biomarkers and the kinetic analysis of thedata reveal the good relationship between plasma exposure levels of the biomarkerand the SM intoxication doses. The suitable biomarkers together with theirdetection windows are proposed for exposure confirmation and diagnosis of SMintoxication, which provide essential scientific basis for SM plasma biomarkers tobe used as a diagnosis tool.3. It is found in the present study that, SMO is a major circulation metabolite of SMand it is also a suitable plasma SM biomarker. The pathway of glutathioneconjugation and β-cleavage for SM in dog was significantly weaker than that inrat, and the formation of SBSNAE was deficient, due to the deficient expressionof N-acetyltransferase in dog.4. It was first time to apply the quantitative analytical method of SM plasmabiomarkers in assessment of SM protection measures. It is proved that this methodcould provide a reliable, accurate and rapid quantitative tool for the study ofmedical countermeasures against SM intoxication.