Discovery And Verification Of Accumulation Of Sulfur Mustard In Adipose Tissue & Screening And Evaluation Of Lipophilic Scavengers

Sulfur Mustard(SM,US military code: HD)is a powerful vesicant chemical agent,which is known as �king of the chemical warfare agents�.The exposure of SM can cause the injury of eyes,skin,lungs and other organs and mucous membranes,and the disease is long-coursed and difficult to cure.As the SM has simple preparation process,strong combat cascualties ability and is extremely difficult to cure,it has been used repeatedly in the war.For China,SM is still a huge terrorist threat.SM is a major component of the abandoned chemical weapons by Japan(JACW).Especially in the northeast region,the issue of abandoned chemical weapons has occurred from time to time.For example,people were injured by the abandoned SM in Qiqihar in 2003.In recent years,with the increasingly serious anti-terrorism situation worldwide,and with the complicated peripheral security environment of our country,the possibility of SM to be used in terrorist attacks is increasing.As an extensive alkylating agent,SM can cause damages in multiple organs and targets in vivo.Due to the complicated toxicological mechanism of SM,traditional toxicological studies could not fully explain the process of transformation and material balance in vivo,and the toxicological mechanism has not yet fully elucidated.There is no specific antitoxic agent for now,so people can only take combined treatment methods with decontamination to improve symptoms in clinical.The current medical intervention applied clinically treats the SM poisoning simply as burn,and there are certain limits such as misdiagnosis at early stage,delayed diagnosis of skin injury,recurrent fester and erosion of skin even after decontamination,lacking parameters for early diagnosis of organic damage and none specific antitoxic agents.In this paper,the strategy of "chemical conversion" was used to break the bottleneck of accurate quantitative analysis of highly reactive compounds such as SM,and at the same time,isotope-dilution LC-MS/MS was coupled with chemical conversion to study the content,distribution and metabolic process of intact SM and its oxidation products.These efforts led to the discovery and verification of that intact SM tended to accumulate in adipose tissues.Furthermore,based on the distribution characteristics of SM,the lipophilic scavengers were designed,and then,with the concentration changes and kinetics parameters of intact SM and its oxidation products as evaluation indicators,the appropriate in vivo SM scavengers were selected.This provides new ideas for the clinical treatment of SM exposure and the screening of therapeutic drugs.This paper is divided into four chapters.In the first chapter,the current situation of SM was introduced,which mainly introduces the research advances of the toxicological mechanism after SM exposure,the current developments of therapeutic drugs,the detective methods of intact SM and its oxidation products,and the opportunity provided by the adipose accumulation effect.This chapter puts forward the research significance,research content and innovative points.In the second chapter,LC-MS/MS method for the detection of intact SM and its oxidation products in different biological matrices was established,and the pre-processing procedure of the simultaneous quantitative analysis method of intact SM and its oxidation products in biological matrix was optimized.Then method-validated detection method was used to measure the contents of intact SM and its oxidation products in the rats with dermal exposed with SM.At present,the detection methods of intact SM are mostly classical gas chromatography or isotope tracer method.However,due to its high reactivity,it is difficult to obtain the real concentration of intact SM in biological samples accurately,thus it is difficult to study the metabolic behaviors in vivo.In this paper,based on the isotope-dilution LC-MS/MS,the chemical conversion method was used to transform SM into stable derivative product with good chromatographic and mass spectrometric behavior.Then,the contents of SM in the complex biological sample were quantified.This method possesses the advantages of simple pre-processing procedure,high sample throughput,high recovery rate,good precision and accuracy,and can be used to trace and analyze the intact SM in biological matrices.In this paper,the simultaneous quantitative analysis method of SM and its oxidation products was optimized,and solid phase extraction(SPE)was added to the pre-processing procedure,which reduced the interference of matrix effect of biological samples,and the method could detect the contents of mustard sulfone in the organ tissues of rats accurately and quantitatively.Through establishment of rat cutaneous exposure model,we have detected the contents of SM and its oxidation products in the blood,adipose tissues and some organs.The results showed that intact SM could be detected at 5 min in the blood after exposure,with peak time at 30-60 min,and then decreased rapidly.But the level of mustard sulfoxide and mustard sulfone reached the peak at 3 h,which lagged far behind SM,and this showed that there was an oxidation process of SM to mustard sulfoxide and mustard sulfone.The result of tissue distribution studies showed that the level of intact SM in adipose tissues was considerably higher than that in non-adipose tissues,with peak time at 1-3 h,and then decreased rapidly,which showed dose-and time-effect relationships.This result indicated that intact SM accumulated significantly in adipose tissues,and the monitoring window was extended greatly.In the other side,the level of mustard sulfoxide and mustard sulfone in adipose tissues was much lower than that in other tissues,which showed that the adipose tissues provided a relatively inert environment and the active alkylation reactions were inhibited and the rate of oxidative metabolism was slowed down.Though the level of intact SM in organ tissues was low,there was high level of mustard sulfoxide in the kidney and high level of mustard sulfone in the liver and spleen,and the level of intact SM and mustard sulfoxide decreased over time,while the level of mustard sulfone was increased to the peak at 6 h.It indicated that the oxidative metabolism of SM might take place in liver with CYP450 involved.In the third chapter,on the basis of the distribution characteristics of SM,a series of lipophilic scavengers were designed specifically.Then through the established detection method,the effects of lipophilic scavengers were evaluated taking concentrations of SM,mustard sulfoxide and mustard sulfone as assessment indexes.The results showed that after applying RSDL to the exposed skin areas,the concentration of intact SM and its oxidation products in the rats' blood and adipose tissues decreased obviously,and the peak time,elimination half-life and mean residence time was shorten.In the other side,after applying RSDL to the unexposed skin areas,the concentration of intact SM in blood and adipose tissues did not decrease,but the concentration of mustard sulfoxide and mustard sulfone were lower than that of the control,and peak time,elimination half-life and mean residence time of the two oxidation products decreased.We learned that applying RSDL to the exposed skin area was effective,while applying RSDL to the unexposed parts showed limited effects,which indicated that the external removal effect of RSDL was far better than internal.Through the evaluation of fat-solublederivatives of N-acetyl-cysteine,we found that most testing compounds can reduce the intact SM and its oxidation products,but a few of compounds showed the opposite effects,which improved the level of the three substances.After tail vein injection of sodium thiosulfate solution and glutathione isopropyl ester solution,we found that glutathione isopropyl ester played a better role than sodium thiosulfate in reducing SM and mustard sulfone in blood through comparing the kinetic parameters of intact SM and its oxidation products.Comprehensive research results showed that based on the quantitative detection method of SM and its oxidation products,the changes of the three substances could be quantified accurately,and they could be used as indexes to evaluate the effect of removal.Application of RSDL to the exposed skin areas was more effective than unexposed areas.Intravenous injection of glutathione isopropyl ester had better effect than sodium thiosulfate solution.It indicated that lipophilic compounds had advantages in removal of intact SM and its oxidation products,and the feasibility of quantitative assessmentof drugs was affirmed.In the fourth chapter,the survival condition of the rats that were administeredRSDL by different ways was studied,and the result showed that the survival condition of the rats was improved significantlywhen RSDL was applied to the exposed areas after SM poisoning,and the survival rates increased obviously.Application of RSDL to unexposed areas could not improve the survival rates of the rats,but it could alleviate the symptoms caused by SM exposure.Application of RSDL to the skin showed no obvious therapeutic effect after tail-veinpoisoning.The statistical results of the survival conditionshowed that the therapeutic effect of RSDL mainly reflected in the decontamination of skin surface,and transdermal absorption of RSDL showed no obvious scavenging effect,which could only alleviate the symptoms such as diarrhea.