Research On Metabolism Of Sulfur Mustard And Oxidation Products In Vivo And In Vitro

Sulfur mustard(SM) is a vesicant agent and also a typical bifunctional alkylation agent, known as ―king of warfare agents‖. Due to it is simple to synthesize, easy to save and difficulty to protect, SM still poses a threat to civilian security and environmental safety due to the legacy of stockpiles buried underground since World War II.SM is a vesicant agent with di-alkylation function towards most biomolecules everywhere in vivo. After entering the body, SM undergoes several biochemical reaction paths including hydrolysis, oxidation(on its sulfur atom), alkylation with DNA, glutathione and protein. Compared with hydrolysis product, SM-DNA adducts, protein adducts and β-Lysate of glutathione adducts, there were little research about the oxidation path and products of SM.It has been suggest that the oxidation products mustard sulfoxide(SMO) and mustard sulfone(SMO2, LD50:50mg/kg) might cause potential toxicity by the experiment in vitro. But there was little direct experimental evidence to support the oxidation path of SM after entering body. To solve this problem, our lab conducts a serials research about the metabolism of SM in vivo. LC-MS/MS technology was applied for direct determination of the metabolites of SM in plasma, And the results suggest that oxidation product mustard sulfoxide: SMO occupied a dominant abundance among all kinds of metabolites. Due to the alkylation of mustard sulfone, direct analysis is inappropriate for it. So there was still no direct evidence to support the presence of mustard sulfone in vivo after SM exposed. In addition, we found that the structure of the β-Lysate products have the sulfone characteristics. It needs further study to illustrate whether the oxidation products involved in the glutathione pathways of SM.The present study via the method of modern instrumental analysis and analytical toxicology to research the oxidize paths of SM. And the following works was completed: development of a simultaneous determination method for SM and its oxidation products(SMO and SMO2), monitoring the oxidation paths of SM, study on toxicokinetics of SM and its oxidation products, discovering of the new metabolism of SM in vivo. The above study provides a novel detection technology for research of SM, and further improves the metabolism of SM in vivo. The possible toxic metabolism of oxidation products and a new metabolic pathway were discovered.In the study, a derivatization approach for simultaneous determination of SM, SMO and SMO2 in biological matrix was established. The derivatives were suitable for UPLC-MS/MS analysis with good selectivity and sensitivity. The derivatization conditions containing reaction time, reaction temperature, p H and concentration of DSH were systematically optimized for a high performance. The method was easy to manipulate, the analysis could be completed within 90 mins after optimizing. Then the method was fully validated according to the guideline. Method validation was performed in a blood matrix, the method has a good linearity between 0.2 and 1000 μg/L, the lower limits of quantitation for SM, SMO and SMO2 were 0.2- 1 μg/L, the recoveries were ranged from 68% to 92%, the precious and trueness were all meet the requirement. This method contributes the detection of SM and related oxidation products, and also helps the research of oxidation metabolism of SM in vivo.The animal model of cutaneous exposure to SM was developed. Then a systematic study of oxidation metabolic process in blood after SM exposed was conducted, using the established LC-MS/MS method. All three targeted analytes rapidly appeared in blood. SM was detected in 5min to 6h after SM exposed, while SMO and SMO2 were detected in 5min to 9h after SM exposed. The result suggests that SM undergo oxidation pathway immediately after entering body.After cutaneous exposure to SM, remarkable difference in the value of Cmax was also observed between SMO and SMO2. For SMO2 the Cmax was similar to SM, while the Cmax for SMO was 3143.9 ± 146.3 μg/L. The concentration of SMO was significant higher than other metabolites, suggesting SMO was the major metabolite in blood after SM entering body. This result indicates oxidation is the major metabolic pathway of SM after entering body. To further investigate the oxidation process of SM, the study of metabolic kinetics after intravenous exposure to SMO was conducted. The results show that sulfone can be detected in blood after exposure to SMO, suggesting SMO can continue oxidizes to form SMO2. This result improved the oxidation process of SM in vivo.By comparing the toxic kinetic parameters, we found that the Tmax for SM was 0.5 h. For SMO and SMO2, the Tmax were around 3 h post SM exposure, which was a little bit later than SM, indicating that there is a delayed period in the oxidation metabolism of SM. For the- 11-aspect of mean retention time, the mean retention time(MRT) of SMO2 was 5.3 ± 0.4 h, which was also larger than these of SM and SMO. Considering that SMO2 still owns somewhat high toxicity(LD50 of rat: sc, 50 mg/kg), has a longer residence time and can form by oxidizing of SMO, it could be predicted that co-existence of SM, SMO and SMO2 in vivo would lead to combined toxic effects and unneglected damage to target organs after SM began its oxidation metabolism processes.To further investigate the novel metabolic pathway of SM and the toxicity metabolism of oxidation products, the cellular level and whole animal level experiments were conducted. Combined previous work, a more stable compound divinyl sulfone(DVS) was chosen as the representative of the oxidation products of SM to screening the adducts of the oxidation products with glutathione. The result shows that both SM and its oxidation products can generate glutathione adducts in vivo. According to the currently accepted view, SM adduct with glutathione firstly after entering body then the sulfur atom oxidize to sulfone structure. In view of the structure feature of the adducts that identified in experiment, the GSH detoxification pathway of SM in vivo also includes a neglected pathway that SM was prior oxidized to mustard sulfone, then the oxidation products bind with glutathione(and further β-Lysate). The results above suggesting that oxidation product of SM participate the glutathione detoxification pathway of SM. It’s another path of the glutathione detoxification pathway. At same time the depletion of glutathione is the injury mechanism of SM, so the oxidation products can produce toxicity by binding with glutathione.The innovation and discoveries of this study are listed as following: 1) A novel UPLC-MS/MS method coupled with pre-column derivatization for simultaneous determination of SM and its two related oxidation products in blood was developed. The established method helps the research of the oxidation process of SM. 2) The concentration-time relationship of SM and its oxidation products and the toxicokinetic were studied. The result reveals the oxidation process of SM in vivo. The result also indicates that SMO was the major circulation metabolite and important biomarker after SM exposed. It also the first time, mustard sulfone, the toxic products of SM was reported. The result provides a new thought for the research of metabolic pathways and injury metabolism of SM. 3) By analyzing the active site of SM, oxidation products of SM and glutathione, combined with the structure characteristics of β-lysate, the structure of glutathione adducts was predicted. Then adducts that glutathione combined with SM and oxidation products of SM were all identified in vivo. The identification of glutathione related structure, not only improves the glutathione pathway of SM, but also found a new metabolic pathway of SM. This study also found the toxic metabolism of the oxidation products of SM.