Detection And Metabolism Of Biomark - DNA Adduct In Animals Exposed To Mustard

Sulfur mustard (SM, U.S. Code HD, chemical name bis(2-chloroethyl)sulfide,molecular formula C4H8Cl2S), is known as an important bifunctional, alkylating,vesicant chemical warfare agent. Because of its ease of preparation, low volatility, andlack of antidotes, SM has been used in wars and military conflicts, a so-calling “kingof the chemical warfare agents”, which continually threatened the public safety.However, considering its extensive toxicity, there is no specific antidote for sulfurmustard, and people suffered from SM have been almost treated as burn mostly inclinic. Therefore, it is necessary for the research of the toxicology mechanism as wellas the therapeutic drug of SM, which, certainly, depends on a reliable and sensitiveanalysis method for the traceability detection and toxicity evaluation of SM.Despite the research into SM for decades, and a series of theory being developed,there is no consistently exact conclusion on it. However, it is widely accepted that theSM destructive effect on the alkylation of DNAs in vivo contributes to the most lesionmechanism and the material basement of its biological toxicity and cytotoxicity,considering that DNAs have large susceptibility towards alkylating agents. As a result,endogenous and exogenous biomarkers generated from the process of DNA alkylatedlesion and repairmen would be detected as significant indexes to indicate the situationof organism after exposure.Since the development of quantification toxicology, this thesis resorted to itsspirit, and researched on the distribution, dose and time relationship of these fourkinds of SM-DNA adducts in vivo, and realize the real-time monitoring of DNA lesionand repair, to explain the relationship between the material base of SM cytotoxicityand its toxicity. According to the previous research, the main positions on DNA molecule of SM alkylating are the N7, O6position of guanine and N3position ofadenine. Until now, four kinds of SM-DNA adducts have been recognized andemployed as good retrospective biomarkers, i.e.,N7-(2-hydroxyethylthioethyl)-2’-guanine (N7-HETEG),bis(2-ethyl-N7-guanine)thioether (Bis-G), which is formed by reacting with each N7position of two guanines in double-stranded DNA (dsDNA),N3-(2-hydroxyethylthioethyl)-2’-adenine (N3-HETEA), andO6-(2-hydroxyethylthioethyl)-2’-guanine (O6-HETEG), respectively. For N7-HETEG,which possesses the highest abundance, there is one guanine reacting withchloromethane group of SM in the N7position, with no hydrogen bonds ofWatson-Crick type destroyed,while for Bis-G, two guanines reacting with eachchloromethane groups of SM in the N7position so as to affect the space structure ofDNAs. And for O6-HETEG, once formed during alkylation with SM, the hydrogenbonds between the bases are cleaved and double-strand DNAs are thus broken down.Therefore, although it occupies the minimum percentage, O6-HETEG is still acceptedas the main product responsible for the DNA damages by SM.Based on thebase-pairing principle, the analysis of SM-DNA adducts could completely survey thedegree of alkylation, obtain the DNA damage and repair condition after SM exposure,which could provide effectively evidences for the evaluation of treatment regime.This work includes the following three items, first of all, the novel and sensitivemethod of isotope-dilution ultrahigh performance liquid chromatography-tandemmass spectrometry (UPLC-MS/MS) was developed for the simultaneousdetermination of four SM-DNA adducts in tissues and urine samples. Then,considering that the percutaneous adsorption is the main path for SM exposure, theanimal models with dermal exposure to SM were developed, and the tissues and urine samples were collected in schedule and analyzed by the methods mentioned above.Lastly, we can learn the content, distribution, dose and time relationship of these fourkinds of SM-DNA adducts in vivo to understand the regularity of SM distribution andmetabolism in organism from part to whole.The following six chapters compose this thesis.The first chapter is the introduction of SM, which displayed the physicochemicalproperty and stated in detail of the using history. There was briefly introduce of themetabolic pathway in vivo and the possible toxicity mechanisms of SM, in which theDNA alkylated lesion and analysis methods of biomarkers were highlighted in thischapter. Finally, the specific study objectives and innovations points were proposed.In the second chapter, a sensitive method of ID-UPLC-MS/MS was developed forthe simultaneous determination of four SM-DNA adducts in tissues. For the adductswere bound metabolites in DNA molecule, it was inevitably complicate for thepretreatment.After a series of optimization process in pretreatment,the method was decided asa process of protein enzymolysis, DNA extraction by phenol-chloroform and DNAsedimentation with alcohol to extract DNA molecule, and followed by basic grouphydrolysis from DNA using formic acid. The SM-DNA adducts uncombined weredetermined by UPLC-MS/MS. The recovery of the method was83-118%, with LODof0.02-0.1ng/mL and LOQ of0.05-0.2ng/mL. This method was high-efficient andstable to make the results receivable in the analysis of practical samples in the thirdchapter.In the third chapter, the animal model of SD rats dermal exposed to SM indifferent dosage was developed. The main tissues of heart, lung, spleen, liver, kidney, pancreas and brain were collected and the SM-DNA adducts were determined in thosetissues with the method mentioned in chapter two, to systematically learn the content,distribution, dose and time relationship of these four kinds of SM-DNA adducts,which could completely display the SM influence on the different function organs andtheir repair process under different toxicant dosage.According the results, we found the significant dose and time dependentresponses of these SM-DNA adducts. For the first time, the sequence of four adductswas achieved as N7-HETEG (62.5-92.0%), Bis-G (7.9-37.0%), N3-HETEA (0.1-2.0%),O6-HETEG (<0.1%), which proofed that the DNA damage by SM alkylation wasunderestimated seriously. Meanwhile, it was also presumed that liver may be not themainly targeting organ of SM, while the lung was injured more severely as SMdosage increases, and it was believed that SM could reach through the Blood-BrainBarrie, since there are high content of lipid to absorb SM and store it more easily,because of the lipotropism of SM.In the fourth chapter, we investigated the damage of bone marrow after exposureto SM, which is an essential central immune organ and produces immune cells withdifferent feature. In this part, we separated the bone marrow cells base on theirfeatures, and observed the different responses between the separated cells afterexposed to SM, which could be helpful to understand the effect on immune system ofSM in cellular levels.It was found that the quantity of monocyte, especially the lymphocyte had anacute increase and followed by decreasing rapidly, while the multinuclear cells wereunchanged. At the same time, the variation trend of immune cells was highly relevantto the clinical manifestation of animals. Then, we determined the SM-DNA adducts in the bone marrow cells, to study the correlation with damage of DNA and variation ofimmune system. It was found that the content of Bis-G was high, about50%of fourSM-DNA adducts, prompting the critical damage of DNA in bone marrow. There wasa similar content of SM-DNA adducts in monocyte and multinuclear cells, whichdeclared the equivalent trend of attack by SM between monocyte and multinuclearcells. It was showed that the content of SM-DNA adduct in monocyte increasedowing to its added amount, which demonstrated the principle of SM alkylationprocess.In the fifth chapter, the urine samples were chosen to be detected, since urine isthe carrier of the final process of metabolism, it was possible to understand the wholeprocess from incept of DNA damage to termination of DNA repair in the entire bodyby holistic approach of SM-DNA in urine.First of all, a novel and sensitive method of isotope-dilution ultrahighperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS)combining with solid phase extraction was developed for the simultaneousdetermination of four SM-DNA adducts. A lower limit of detection of2-5ng·L-1, anda lower limit of quantitation of5-10ng·L-1were achieved, respectively, and therecoveries ranged from87%to116%. Then we applied this method in thedetermination of four SM-DNA adducts in urine of SD rats and rabbits after dermalexposure by SM in three dose levels, so as to investigate the related metabolicbehavior in vivo. For the first time, in SM exposed SD rat and rabbit urine, our resultsrevealed a similar relative accumulation abundance of four SM-DNA adducts, whichwas similar trend with the result in tissue. Meanwhile, the metabolic process of DNAinjuries by SM was surveyed macroscopically. In the sixth chapter, a practical application was introduced. We received someurine samples from four patients who accidently exposed to SM, and applied themethod of isotope dilution-SPE-UPLC-MS/MS mentioned in fifth chapter todetermine the SM-DNA adducts in urine. The four SM-DNA adducts were detectablein the urine samples of all the patients, to confirm the SM poisoning. What’s more, wefound the content of SM-DNA adducts had positive correlation with the clinicaldiagnosis of four patients. It proved that the SM-DNA adducts could serve as the idealtraceability and effect biomarker of SM exposure.