Research On Biomarkers Of Sulfur Mustard Exposure-the Identification, Determination And Application Of Hemoglobin Adduct And Urinary Metabolites

Sulfur mustard (HD, 2, 2′-dichlorodiethyl sulfide) is a vesicant that can injure skin, eyes, respiratory system, etc. It is one of the most hazardous chemical warfare agents (CWAs), and it has been extensively used in World War I and more recent conflicts in the Middle East. A number of chemical weapons were forced on the Chinese army and civilian by the Japanese army in World War II, and then abandoned in China when they defeated. These weapons contain abundant HD which is still a serious threat to the safety and health of local residents up to nowSulfur mustard possesses two electrophilic carbon atoms and is a typical bifunctional alkylating agent. Its chemistry and metabolism are dominated by their reactions with nucleophiles. It can react with amino, sulfhydryl, hydroxyl, carboxyl, phosphate and imidazole under physiological conditions that induce complex biological effects. Despite studied for a long time, the toxicological mechanism of HD has not yet been fully elucidated. And the victims are vulnerable to genetic toxicity after HD exposure and require a long recovery period. It has important practical significance to research on the mechanism of HD toxicity. Skin symptoms after exposure to HD are similar to those for burn and other chemical injuries, and therefore, exact diagnostic methods for HD exposure should be established.HD can attack and react with several targets such as organisms, leaving many biomarkers produced in the body. Free metabolites in urine and blood can be validated as diagnostic biomarkers of HD exposure, but the major disadvantage lies in their relatively rapid elimination from the body, as ideal biomarkers should be stable and exist for several months after exposure. Covalent adducts with macromolecules, such as proteins and DNA, offer the potential for much longer-lived biomarkers of exposure in comparison with free metabolites. These adducts are formed in nucleophilic sites on macromolecules when reacted with HD. They can remain in blood and tissue for a very long period of time and provide definite biomarkers of HD exposure. On the other hand, biomarkers analysis offer a greater challenges because of the low concentrations of analytes likely to be encountered, the greater complexity of biological matrices and the reference chemicals are difficult to be prepared.From the pointview of biomarkers chosen in this dissertation, HD adducts with hemoglobin are generally stable and most appear to have lifetimes in humans similar to the native protein (approximately 120 days). Consequently, the hemoglobin adducts may be detectable for a long period after HD exposure. TDG and TDGO in urine are mainly generated from the hydrolysis or oxidation of HD especially in the early stage after HD exposure. For its relatively simple conversion process, the contents of TDG and TDGO can reflect the degree of HD injuries more accurately than other free metabolites. Most HD adducts can release TDG and TDGO during the metabolic process in the later times after HD exposure. So it is important to study TDG and TDGO in order to understand the metabolic behavior after HD exposure. In this dissertation, sensitive isotope-dilution-NCI-GC/MS methods for determining HETE-Val, TDG and TDGO were established. The distribution and metabolism were studied after in vitro and in vivo HD exposure.This dissertation consists of six chapters.The first chapter is the introduction. In this chapter, we summarized four types HD biomarkers: hydrolysis and oxidation products, HD adducts with glutathione, proteins and DNA. The detection methods, distribution and metabolism were reviewed focusing on these biomarkers. The objectives, contents and new insights of this dissertation were briefly outlined at the end of this chapter.In chapter two, the synthetic route of HETE-Val was improved and the synthetic routes of HETE-Val-P and HETE-Val-P-P were established. The products were characterized based on GC, GC/MS, LC/MS and NMR data. In order to get more accurate and stable methods, the isotope internal standard was needed. First, we established the microsynthetic routes of 2-(2-chlordeuterethylthio) ethanol (half-mustard-d4) and HD-d4. The purity of them were larger than 96% as determined through GC/MS, and the deuterium labeling ratio were almost 100%. Then the isotope internal standard for HETE-Val was prepared through HD-d4 and HETEG-d4 was synthesized by half-mustard-d4. In chapter three, based on the synthesized products, the modified Edman degradation, solid-phase extraction (SPE) and derivatization procedure were investigated and optimized, and a sensitive bioanalytical method was established. The minimum detectable exposure level of human blood (in vitro) to HD was 20 nmol/L (S/N>3) and the the sensitivity was improved by almost tenfold through our method. The limit of quantitation (LOQ) was 100 nmol/L (S/N>10) and the baseline of the chromatogram was clean.Globin samples isolated from human blood, which had been exposed to various concentrations of HD (0.1μmol/L–120μmol/L), were analyzed according to the procedure based on isotope-dilution-NCI-GC/MS analysis after a modified Edman degradation. The results show a nearly linear dose-effect relationship between HEVE-Val and HD exposure concentrations which show the reaction ratio between HD and N-terminal valine residue of hemoglobin in blood was stable. Through conversion, we can know that the percentage of HD that reacted with the N-terminal valine in globin was about 1%–2% of total exposed HD. In chapter four, taking it into account that the LD50 of rabit is the same as human which is 100 mg/kg after skin exposure to HD, the rabbit skin exposure model was established.Blood samples were collected from the ears of exposed rabbits. Globin was isolated and analyzed by using the modified Edman degradation for determination of HETE-Val. The results show that there are obviously exposure-dose-response relationships and exposure-time-response relationships after HD exposure. HETE-Val was generated much quickly and could be detected only 15 minutes after HD exposure. The contents of HETE-Val in blood were increased in the early two days and were stable from the second to ninth day. And then it gradually decreased in the next days. HETE-Val could still be detected at the 103rd day. Through calculation, we can know that about 0.15‰of total exposed HD reacted with the N-terminal valine in globin, and the percentage of HD that reacted with blood was about 1%.In chapter five, through investigating and optimizing SPE steps, the extraction and purification procedures of TDG and TDGO from urine were established. And because the reaction of TDG and pentafluorobenzoyl chloride did not give a clean product, the derivative product was also cleaned up. Then the sensitive methods for TDG and TDGO detection were established based on isotope-dilution-NCI-GC/MS analysis and the limited of detection (LOD) was 0.1 ng/mL. The sensitivity was improved tenfold compared to the references using the same instruments.The urine samples of rabbits were collected after HD exposure. TDG and TDGO were extracted and monitored based on isotope-dilution-NCI-GC/MS analysis. The results show that the concentrations of TDG and TDG+TDGO in the urine increased quickly after HD exposure and also decreased fastly in the early two days after exposure. The concentrations of TDG+TDGO continued to decrease from the third to the ninth day. From the results of the experiments, we can conclude that TDG and TDGO are diagnosis indicators for HD exposure and the concentrations of them are important references for the degree of HD injures. The contents of TDGO in urine are much higher than that of TDG. The excreted amount of TDG in urine represents approximately 2%-3% of the amount of TDG+TDGO, among which the glucuronated and sulfated TDG and TDGO conjugates are much little.In chapter six, the analysis methods of TDG and TDG+TDGO in urine were applied on four victims after accidental chemical exposure which happened in HeBei province. The results show that the concentrations of TDG and TDG+TDGO in urine were much higher than that of the background levels, and HD exposure was asserted. The concentrations was coincide with the symptoms of victims, which show that the concentrations of TDG and TDG+TDGO can indicate the degree of injures after human exposed by HD. The following researches also show that the metabolic trends of TDG and TDG+TDGO in the urine of the victims are similar to that of the rabbits after HD exposure. And the concentrations of TDGO in urine are much higher than that of TDG. The conjugated TDG and TDGO are also much little in the urine of the victims. It is concluded that the the determination methods established in chapter 5 has a good application on the diagnosis of victims injured by HD, and the HD esposure model of rabbit skin is a good reference for the researches on the toxicological mechanism of human HD exposure.