| Sulfur mustard(SM, U.S. Code HD) is a well-known chemical warfare agent not only hard to prevent or treat, but also mainly abandoned by the Japanese army in China. Its structure is very simple and easy to synthesize, and with the probability of terrorist threat. Therefore, based on coping with chemical warfare or terrorist attacks, as well as the complexity environment of the surrounding country, it is urgent to adopt new means to go into the details of poisoning mechanism of SM, under the conditions of that there is no effective prevention or treatment measures.On the level of endogenous substances basis within the SM damage, most of the current research on SM restricted to concern isolated some areas, such as a particular gene or protein changes, and lack of the overall and system- level research. This work focus on the complex toxicology of SM, based on systems toxicology theory, a multidisciplinary set intersection with the features of holistic, dynamic, composite, and the joint between microscopic and macroscopic matter as well as quantitative and qualitative analytical methods simultaneously. Application of integration metabolomics technology to conduct a comprehensive system analysis, analyzing trends of metabolome from different biological samples, describing the toxic effects of metabolomic trajectory as well as dose-effect relationship, drawing the biotransformation spectrum within exposure, ultimately predicting poisoning mechanism of SM, for assistant of clinical diagnosis and proposed medical interventions to guide prevention and treatment.On the other level of exogenous substances basis within the SM damage, Since the present analysis of intact SM with low sensitivity, as well as complex and unatable preparation of biological samples, resulting lack of research data regarding metabolic behavior in vivo and no good explanation for toxicodynamics of SM, while there is only speculation without conclusive experimental data supportting for the throty of "adipose depot accumulation". This research aims to establish a high-sensitivity and robust analysis method based on stable isotope dilution technique, applying for toxicodymics on SM. Through the establishment of exposure model in vitro and in vivo, the time or dose-effect relationship, metabolites mode, pathways and mechanisms, retention time and ways of the accumulation were all investigated from the data of monitoring the progress of distribution and metabolism of SM intact. Meanwhile, the correlation was explored between retention phase or accumulation mode and sustained toxicity injured by SM. Eventually, the relationship was established between the metabolic behavior and the extent of damage as well as the dose of SM, through comparing the metabolic behavior of intact SM and biomarkers.The main results are referred as follows:(1) The discovery, identification and application of endogeous biomarkersMetabolomic analysis of urine samplesCombined with RPLC-MS and HILIC-MS technologies, 34 distinguished metabolites related to SM exposure were identified by comprehensively means of spectral analysis, mainly including nucleic acid metabolites, such as cytosine, deoxycytidine, deoxyadenosine, etc.; tryptophan and its metabolites, such as xanthurenic acid, kynurenic acid, indole carboxylic acid glucuronide, etc.; phenylalanine and its metabolites, such as ferulic acid, hippuric acid, etc.; energy metabolism metabolites such as citric acid, gluconic acid, bile salts, etc.; and some metabolites associated with the digestion of food flavonoids, such as daidzein and glucuronidation daidzein and so on. Metabolic pathways involved in metabolism related with DN A damage repair, metabolism of amino acid associated with inflammation, intestinal flora imbalance, other energy metabolism about sugar and fat, as well as the inhibition of phase II enzyme(such as glucuronidation and sulfation), etc.Metabolomic analysis of plasma samples27 distinguished metabolites of small polar molecule related with SM exposure were identified by comprehensively means of spectral analysis, mainly including metabolites of bile acids, such as cholic acid, deoxycholic acid, glycine acid, etc.; amino acids and their metabolites, such as phenylalanine, tryptophan, benzyl alcohol sulfate, etc.; energy metabolism metabolites, such as citric acid, uric acid and the like;, and phospholipids such as phosphatidylethanolamine and lysophosphatidylcholine.Meanwhile, 163 lipid metabolites were identified through the technology of all information tandem mass spectrometry(MSE), from specific lipid extraction of plasma, and application of targeted lipidomics analysis for these lipids compounds, and found distinguished lipid metabolites of triglycerides, diglycerides and phospholipids related with SM exposure. Combined with the results of blood biochemistry index and metabolome trajectory, both small polar molecule metabolites and lipids metabolites, within skin exposure of SM, elaborated an acute liver injury and the process of lipid peroxidation highly relevant to the unsaturated lipid metabolites.Tissue samples for metabolomic analysisThrough the metabolic profiling trajectory analysis of various organs, including liver, lung, spleen, brain and pancreas, exploration of the direction and effect of the metabolic state changes when SM acting on different organs. Combined metabolomic trajectory analysis of small polar molecule and non-targeted lipidomics analysis of non-polar lipid metabolites in liver, as well as indications of blood biochemistry index analysis, found SM may resulted in liver acute injury during incubation period of 6-12 h, manifested elevation of various transaminases, disruption of synthesis and transport of bile acid, inhibition of metabolizing enzyme system in liver, the rise of lysophospholipid. The q PC R results of liver tissues of SD rats exposed to SM also found that RN A expression levels related bile acid metabolism and transport, including CYP7A1, NTCP, ASBT and BSEP, were all reduced.(2) The analysis method and metabolic behavior of intact SMA new isotope-dilution liquid chromatography-tandem mass spectrometry(LC-MS/MS) method for determination of sulfur mustard has been developed using direct chemical derivatization method by nucleophile potassium thioacetate(PTA) in aqueous solution. The reaction conditions for derivatization, such as reaction temperature, time, solvent and concentration of PTA, were optimized for high performance. Reversed phase liquid chromatography was suitable for analysis of such a PTA derivatized SM in complex environmental samples. Compared with other conventional gas chromatography or gas chromatography- mass spectrometry methods for direct detection on SM, better sensitivity and selectivity were achieved by this pre-column derivatization and LC-MS/MS method. The whole procedure for both derivatization and analysis was quick and simple, and the total time was less than 1 h. This established method has been successfully employed for effective separation and detection of SM in complex matrices, including environmental samples and biological samples. The method is suitable for the aqueous samples and can be widely used in environmental monitoring and scientific research.Results of the metabolic behavior research of intact sulfur mustard in blood and different tissues from SD rats dermal exposure of SM, showed the rapid penetration into blood and then distribution throughout the whole organs with the circulatory system, and there is an obvious evidence of accumulation in adipose tissue. Through toxicokinetic analysis found that SM in adipose tissue reached an average peak within 1-3 h, followed by the rapid elimination, and the mean residence time in the fat within 2.5-5.3 h, while there is a certain correlation between the concentration of intact and exposure dose. So far, there is no research on toxicokinetics of SM against the adipose tissue, which is part of the results of this work will contribute to a comprehensive analysis of disposal in vivo within the whole exposure。In summary, there is a very significant effect with the time course as well as complex under skin exposure of SM. Such as, in the incubation period, based on DNA damage and repair, lipid peroxidation and acute liver injury; during development period, mainly as excessive inflammation response; in the recovery period, performance of energy metabolism with feedback. C linical diagnosis and medical interventions should be targeted genotyping adopted a phased treatment, since multi- target toxic effects of SM during various periods. For instant, In the early, transaminase combined with biochemical markers of DNA damage to assistant the clinical diagnosis of SM exposure, and meanwhile adopting medical intervention for liver damage and free radical scavenging, but in the medium term will conduct anti- inflammatory therapy, late need to focus on the restoration of energy metabolism. In the early stage of SM exposure(within 3 hours), the physical removal or pharmacological scavengers can be targeted against the SM in adipose tissue, so as for timely intervention or blocking damage of SM. |
PDF Full Text Request