芥子气暴露后动物体内生物标志物-DNA加合物的检测及代谢研究

发布时间：2018-06-10 07:38

本文选题：芥子气 + DNA加合物　；参考：《中国人民解放军军事医学科学院》2014年博士论文

【摘要】：芥子气，英文全称sulfur mustard（以下简称SM，美军代号HD），化学式为C4H8Cl2S，是双功能烷基化试剂，也是糜烂性化学战剂中最为重要的一种。芥子气制备过程简单，毒性大且持续时间较长，在战争中被数次使用，至今仍然威胁着世界和平和人民生命安全，有着“毒剂之王”之称。然而，芥子气的化学性质决定了其毒性具有广泛性，目前国际上关于其针对性解毒药物的研究进展缓慢，临床救治也多将其视作烧伤处理。因此，深入研究芥子气的毒理作用机制、筛选和开发其对抗特效治疗药物和临床医学干预方案具有重要现实意义，而这些亟需建立可靠的芥子气溯源性检测和毒理学评价方法。长期以来，国内外学者对芥子气毒理作用机制的研究不断深入，并提出了一系列理论。虽然目前仍未得出确切结论，但是各国学者普遍认同芥子气对DNA分子的烷基化损伤是其毒理作用的始发机制以及细胞毒性与生物毒性的物质基础。因此DNA烷基化损伤及修复过程中产生的内源性与外源性标志物可视为芥子气暴露后生物体损伤及恢复情况的有效评价指标。论文采用新兴的分析毒理学相关方法，通过检测芥子气与DNA的烷基化加合物（以下简称SM-DNA加合物）在体内的时效与量效变化趋势，实现对DNA的受损及修复过程的实时监控，从而定量阐述芥子气细胞毒性的物质基础与其毒性之间的相关性。现有文献报道最主要的SM-DNA加合物有以下四种：N7-（2-羟乙基硫代乙基）鸟嘌呤（N7-HETEG），双[2-(N7-鸟嘌呤)]乙硫醚（Bis-G），O6-（2-羟乙基硫代乙基）鸟嘌呤（O6-HETEG），N3（-2-羟乙基硫代乙基）腺嘌呤（N3-HETEA）。这四种SM-DNA加合物的特点各不相同，如N7-HETEG的丰度最高，N3-HETEA是腺嘌呤上唯一烷基化产物，而根据碱基配对原则，Bis-G与O6-HETEG的反应位点可直接破坏DNA双链结构等。因此同时分析这四类加合物就可以全面监测DNA分子的烷基化程度，获得芥子气暴露后DNA的损伤及修复情况，进而为评价救治方案提供可靠技术方法。论文首先建立了同时检测生物体内不同功能组织以及尿液中四种SM-DNA加合物的同位素稀释超高效液相色谱串级质谱（ID-UPLC-MS/MS）分析方法；其次，建立合理的芥子气皮肤染毒的实验动物模型，取其组织器官和尿液；最后，利用建立的分析方法监测SM-DNA加合物在生物体内不同脏器的时效、量效分布，以便全面了解芥子气在生物体内从局部到整体的分布与代谢规律。论文共分六章。第一章为前言，简要介绍了芥子气的相关理化性质，主要阐述其在体内的代谢途径和毒理机制的研究现状，重点介绍芥子气对DNA分子的损伤机理及其后续影响以及SM-DNA加合物的各种检测手段，提出研究目标和研究内容。第二章重点建立了不同脏器中四种SM-DNA加合物的ID-UPLC-MS/MS同时分析测定方法。由于组织中SM-DNA连接在DNA分子上，为结合态加合物，导致样品前处理过程相对复杂。经过一系列的优化过程，该分析方法采用“酶解蛋白—酚氯仿抽提—乙醇沉淀”法提取DNA分子，回收率较高；甲酸加热法水解碱基，利用ID-UPLC-MS/MS进行分析。方法回收率为83-118%，检测限为0.02-0.1ng/mL，定量限为0.05-0.2ng/mL。本章对组织匀浆、DNA提取到碱基水解等全过程进行详细、系统的优化，建立了高效、稳定的检测方法，为下一章各脏器中DNA损伤的研究提供技术支持。第三章建立了不同剂量下SD大鼠芥子气皮肤染毒的动物模型，对心脏、肝脏、脾脏、肺脏、胰脏、肾脏、全脑等主要组织中SM-DNA加合物进行检测，并对其中变化最显著的组织进行了系统的时效、量效关系研究，全面的考察了不同暴露剂量的条件下，芥子气对不同功能的脏器组织中DNA分子的损伤情况及其自身修复的过程。实验结果表明SM-DNA加合物与染毒剂量和时间之间存在良好的量效、时效相关性；第一次系统证实了体内各脏器组织中Bis-G是丰度比例占第二位的DNA加合物（N7-HETEG，62.5-92.0%；Bis-G，7.9-37.0%；N3-HETEA，0.1-2.0%；O6-HETEG0.1%；），揭示芥子气对DNA的损伤在前期研究中被严重低估；染毒剂量增加时，肺脏所受的损伤更为严重；肝脏可能并不是芥子气损伤的主要器官；发现脂类含量高或被脂肪包裹的组织中SM-DNA加合物含量相对较高，提示我们芥子气可能存在体内“脂肪蓄积”的问题；芥子气可以迅速通过血脑屏障，脑中脂类含量高，更易吸收并储存亲脂性的芥子气原型分子，导致单位DNA形成加合物比例高。第四章单独考察了芥子气对大鼠骨髓组织的损伤。骨髓组织是免疫细胞的产生地，实验将骨髓细胞按功能进行分型，分别观察了芥子气中毒后不同功能骨髓细胞的应答反应，有助于从细胞层面上了解芥子气对免疫系统的影响，并试图以此结果部分解释生物机体的中毒症状。研究结果表明：通过对骨髓细胞中SM-DNA加合物进行检测，发现骨髓中Bis-G不仅丰度高，而且随剂量增加其丰度比例甚至超过50%，成为最主要的DNA加合物，提示芥子气对骨髓中DNA更严重的损伤；芥子气染毒后，，大鼠骨髓中单核细胞呈现先剧烈增殖后快速抑制的趋势，其计数变化幅度明显，而粒细胞等多核细胞则缓慢增殖，同时免疫细胞的变化趋势与动物的中毒体征状况具有高度的相关性；相同细胞计数下，单位单核或多核细胞中DNA加合物的含量近似，表明芥子气对单核或多核细胞中DNA的损伤是等同或没有偏好；而单位数量单核细胞中DNA加合物含量未呈现随时间变化的趋势，表明骨髓单核细胞中SM-DNA加合物含量的升高原因主要是由于细胞数量增加，再次证明芥子气烷烃化作用的本质。第五章选取体内代谢的最终载体—尿液作为检测样本，从整体轮廓层面分析SM-DNA的形成-修复-消失全过程，即观察DNA损伤及修复的全身变化过程，与组织中结果相互补充、相互验证。本部分开发了固相萃取新方法，用于尿液中四种SM-DNA加合物的同时提取。优化ID-UPLC-MS/MS方法，回收率达到87-116%，检测限为2-5pg/mL，定量限为5-10pg/mL，实现了对加合物的高灵敏检测。建立了芥子气皮肤染毒的动物模型，并对尿液中SM-DNA加合物进行分析检测。结果显示了良好的时效、量效关系曲线，并且获得了尿液中四种SM-DNA加合物的真实比例关系。从宏观上监测DNA损伤的全身代谢轮廓，与组织中结果进行比较，发现二者在时效关系以及加合物含量关系方面整体趋势相同，可以实现良好的相互验证。由于尿液检测无痛、无损，而且对于SM-DNA的检测可以一定程度的反映机体DNA损伤情况，可作为芥子气溯源分析以及损伤效应监测的理想检测对象。第六章是本论文建立方法的实际应用。我们得到了四名意外接触芥子气患者的尿液，将已建立的固相萃取-ID-UPLC-MS/MS方法应用于其尿液中四种SM-DNA加合物的同时定量检测。检测结果表明尿液中四种SM-DNA均可检出，不仅更进一步确证芥子气染毒，而且发现加合物的含量与患者的暴露程度和临床中毒症状相互吻合。上述结论证明SM-DNA加合物可以作为理想的芥子气溯源性生物标志物。
[Abstract]:Mustard gas, the full name of sulfur mustard in English (hereinafter referred to as SM, US military code HD), chemical formula C4H8Cl2S, is a double functional alkylating reagent and the most important one in the erosive chemical warfare agent. The preparation of mustard gas is simple, toxic and longer, is used in war several times, and still threatens world peace and people. Life safety is known as the "king of poison". However, the chemical nature of mustard gas determines its toxicity, and the research progress of its targeted detoxification drugs is slow in the world. Clinical treatment also treats it as a burn treatment. Therefore, the mechanism of the toxicological action of mustard gas is deeply studied and its special effect is screened and developed. Therapeutic drugs and clinical interventions have important practical significance, and these need to establish reliable methods for the detection and toxicological assessment of mustard gas.
For a long time, scholars at home and abroad have studied the mechanism of the toxicological action of mustard gas, and put forward a series of theories. Although the exact conclusion has not been reached yet, scholars in various countries generally agree that the alkylation damage of mustard gas to DNA molecules is the starting mechanism of its toxicological action and the material basis of cytotoxicity and biological toxicity. The endogenous and exogenous markers produced in the process of DNA alkylation damage and repair can be considered as an effective evaluation index for the damage and recovery of organisms after the exposure of mustard gas.
This paper uses a new method of analytical toxicology to detect the change trend of aging and quantitative effect of mustard gas and DNA alkylated adducts (hereinafter referred to as SM-DNA adducts) in vivo, to realize the real-time monitoring of the damage and repair process of DNA, so as to quantify the relationship between the material base of the toxicity of mustard gas cell and its toxicity. There are four main SM-DNA adducts in the current literature: N7- (2- hydroxyethyl thioethyl) guanine (N7-HETEG), double [2- (N7- guanine), Bis-G, O6- (2- hydroxyethyl thioethyl) guanine (O6-HETEG), N3 (-2- hydroxyethyl thioethyl) adenine. The characteristics of these four kinds of adducts are different. The abundance of 7-HETEG is the highest, N3-HETEA is the only alkylation product on adenine, and the reaction site of Bis-G and O6-HETEG can directly destroy the double chain structure of DNA according to the base pairing principle. Therefore, the analysis of these four kinds of adducts can monitor the degree of alkylation of DNA molecules and obtain the damage and repair of DNA after the exposure to mustard gas. It provides reliable technical methods for evaluating treatment plan.
The paper first established a method for the simultaneous detection of four SM-DNA adducts in different functional tissues and urine by isotopic dilution super high performance liquid chromatography tandem mass spectrometry (ID-UPLC-MS/MS). Secondly, a reasonable experimental animal model of the skin of mustard gas was established, and its tissues and urine were taken. Finally, the establishment of the model was established. The analysis method is used to monitor the distribution and distribution of SM-DNA adducts in different organs in the organism in order to understand the distribution and metabolism of mustard gas from local to whole in a comprehensive way. The paper is divided into six chapters.
In the first chapter, the related physical and chemical properties of mustard gas are briefly introduced. The research status of metabolic pathways and toxicological mechanisms in the body is mainly described. The damage mechanism and subsequent effects of mustard gas on DNA molecules as well as the various detection methods of SM-DNA adducts are introduced, and the research targets and research contents are presented.
The second chapter focuses on the establishment of ID-UPLC-MS/MS simultaneous determination method for four kinds of SM-DNA adducts in different organs. Because the SM-DNA connection in the tissue is on the DNA molecule, it is a combination of state adducts, which leads to the relative complexity of the sample pretreatment process.
After a series of optimization processes, the method used "enzyme hydrolysin - phenol chloroform extraction - ethanol precipitation" to extract DNA molecules, and the recovery rate was high. Acid heating method was used to hydrolyze base and ID-UPLC-MS/MS was used to analyze the method. The recovery rate was 83-118%, the detection limit was 0.02-0.1ng/mL, and the quantitative limit was 0.05-0.2ng/mL. to tissue homogenate. The whole process of DNA extraction to base hydrolysis is detailed and the system is optimized. A high efficient and stable detection method is established, which provides technical support for the research of DNA damage in the next chapter.
The third chapter established the animal model of SD rats with different doses of mustard gas, and tested the SM-DNA adducts in the main tissues such as heart, liver, spleen, lung, pancreas, kidney and whole brain, and studied the most significant changes in the relationship, and investigated the different exposure doses in a comprehensive way. The damage of mustard gas to DNA molecules in different functional organs and the process of self repair were studied.
The experimental results showed that there was a good dose effect and time correlation between the SM-DNA adducts and the dose and time. The first system confirmed that the Bis-G in the organs of the body was second DNA adducts (N7-HETEG, 62.5-92.0%; Bis-G, 7.9-37.0%; N3-HETEA, 0.1-2.0%; O6-HETEG0.1%;), and revealed that the mustard gas was to DNA. The damage was seriously underestimated in the previous study; the lung injury was more severe when the dose increased; the liver may not be the main organ of the mustard gas damage; it was found that the content of SM-DNA adducts in the fat wrapped tissues was relatively high, suggesting that our mustard gas may have "fat accumulation" in the body. The problem is that mustard gas can quickly pass through the blood brain barrier, the lipid content in the brain is high, and it is easier to absorb and store the lipophilic mustard gas prototype molecules, which leads to the high proportion of the unit DNA adducts.
The fourth chapter investigated the damage of the mustard gas to the bone marrow tissue of rats. The bone marrow tissue was the producing area of the immune cells. The experiment was used to classify the bone marrow cells according to the function. The response of different functional bone marrow cells after the mustard gas poisoning was observed respectively. This result partly explains the toxic symptoms of the organism.
The results showed that by detecting SM-DNA adducts in bone marrow cells, it was found that the abundance of Bis-G in bone marrow was not only high, but also increased with the abundance of more than 50%, which became the most important DNA adduct, suggesting that mustard gas was more serious to DNA in bone marrow. The trend of rapid inhibition after acute proliferation was obvious, while the multinucleated cells like granulocytes proliferated slowly, and the change trend of immune cells was highly correlated with the toxic signs of animals. Under the same cell count, the content of DNA adducts in unit mononuclear or multinuclear cells was similar, indicating that mustard gas is single. The damage of DNA in nuclear or multinuclear cells is the same or no preference, but the content of DNA adducts in unit number mononuclear cells does not show a tendency to change with time, indicating that the increase in the content of SM-DNA adducts in bone marrow mononuclear cells is mainly due to the increase in the number of cells, which proves the essence of the alkylation of mustard ananes again.
In the fifth chapter, the final carrier of metabolism in the body, urine as a sample, is used to analyze the whole process of the formation, repair and disappearance of SM-DNA from the overall outline level, that is to observe the whole body change process of DNA damage and repair, and to complement each other and verify the results of the tissue.
A new solid phase extraction method was developed for the simultaneous extraction of four SM-DNA adducts in urine. The ID-UPLC-MS/MS method was optimized, the recovery rate was up to 87-116%, the detection limit was 2-5pg/mL, and the quantitative limit was 5-10pg/mL. The high sensitivity test of the adduct was realized. The animal model of mustard skin skin skin dye was established and SM-DNA addition in urine was added to the urine. The results showed a good time limitation, a quantitative relationship curve, and a true proportion of four SM-DNA adducts in the urine. The overall metabolic profile of DNA damage was monitored from the macro level, and the results were compared with the results of the tissue. It was found that the overall trend of the aging relationship and the relationship of the adducts content was the same as the overall trend. Good mutual validation can be achieved. Since urine tests are painless and nondestructive, and the detection of SM-DNA can reflect the DNA damage to a certain extent, which can be used as an ideal target for the analysis of mustard gas traceability and the monitoring of damage effect.
The sixth chapter is the practical application of this method. We have obtained the urine of four accidental exposure to mustard gas. The established solid phase extraction -ID-UPLC-MS/MS method is applied to the simultaneous quantitative detection of four kinds of SM-DNA adducts in their urine. The results show that four kinds of SM-DNA in the urine can be detected, not only further corroborate the mustard It was found that the content of the adduct was consistent with the exposure degree of the patients and the symptoms of clinical poisoning. These conclusions have proved that the SM-DNA adduct can be used as an ideal biomarker for the origin of mustard gas.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R994.3;R917

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