【作者】 岳丽君；

【导师】 谢剑炜；

【作者基本信息】 中国人民解放军军事医学科学院 ， 药物分析学， 2011， 博士

【摘要】 DNA加合物是最重要的化学暴露生物标志物之一。它是由具有亲电基团的化合物或体内具有亲电基团的代谢产物与DNA共价键合所形成的化合物,是DNA化学损伤最重要和最普遍的形式。DNA加合物的研究不仅可以准确反映机体暴露毒物的剂量,还有助于毒理机制的阐明,成为近年来的研究热点。众所周知,芥子气(SM)是一种非常重要的糜烂性军用化学毒剂,具有制备简单、毒性大、难防、难治等特点。SM是典型的双功能烷基化试剂,通过锍离子过渡态与生物大分子(蛋白质、DNA、RNA等)的亲核基团进行共价结合形成加合物。DNA含量比较低,在修复机制的影响下半衰期更短,但是DNA加合物具有极为重要的生物学意义。目前已知四种SM-DNA加合物中N7-HETEG丰度最高,大约占DNA加合物总量的60-70%,许多研究表明该加合物和损伤效应之间无太多关联,所以N7-HETEG的研究多用于SM暴露的溯源性检测。O6-HETEG、N3-HETEA及Bis-G这三种含量较低的加合物,与DNA链的断裂、碱基错配、基因突变等生物学终点密切相关,因此对其研究有望阐明芥子气的毒理机制,对芥子气的预防治疗提供技术支持。本论文应用HPLC-MS/MS技术(MRM模式),建立了两种SM -DNA加合物的分析方法。并用于体内体外染毒生物样品中DNA加合物的检测,研究DNA加合物的代谢、分布规律,以期为SM暴露的确证、代谢规律及毒理作用机制的阐明提供技术基础。全文共分为4章。第一章是前言部分。介绍了DNA加合物的生物学意义和检测技术。综述了SM中毒后体内代谢过程,尤其是SM-DNA加合物的研究现状,评述了关于SM -DNA加合物的检测在SM暴露确证和毒理作用机制研究中的重要意义。本章的最后归纳了本研究论文的意义、内容与创新点。第二章是标准品的合成和确证。本章共合成了9种标准品:四种DNA加合物标准品;四种DNA加合物氘代内标以及N7-HETEG检测的内标物N7-苯甲基鸟嘌呤。并用1H NMR和LC-MS进行了结构鉴定和纯度测定。基于鸟嘌呤和腺嘌呤在不同介质条件下,其活性亲核位点的差异,在不同的有机溶剂体系中用毒性较小的半芥代替芥子气作反应原料,均相反应获得N7-HETEG、N3-HETEA。鸟嘌呤O6位的反应活性显著弱于N7位,不能通过半芥与鸟嘌呤或鸟苷简单反应得到O6-HETEG。本文在强碱性体系中,巧妙的运用6-氯鸟嘌呤和无毒的硫二甘醇一步反应即获得目标化合物。双取代加合物的合成参照文献方法进行,但需注意的是在制备色谱纯化之前,需先通过稀酸多次洗涤以去除其主要副产物—N7-HETEG。另外,为合成氘代内标,建立了由氘代半芥合成氘代SM和氘代硫二甘醇的方法。合成标准品的纯度均大于95%,氘代内标的氘代率大于99%。第三章,SM -DNA加合物分析方法研究。一、N7-苯甲基鸟嘌呤作内标,运用高效液相色谱-电喷雾离子阱串联质谱技术,多反应监测模式(MRM),建立生物体内丰度最高的SM-DNA加合物—N7-HETEG检测方法,并对该方法进行了完整的方法学验证。不同生物基质中最低检测限约为几百pg/mL,相当于可检测到每107个核苷中的1个加合物。运用此方法可以检测到2μmol/L SM体外染毒人全血中的N7-HETEG,且加合物含量与染毒剂量呈正相关,表明N7-HETEG可以作为SM暴露的生物标志物。二、采用超高压液相色谱-三重四极杆离子阱串联质谱技术,多反应监测模式(MRM),同位素稀释法,首次实现了四种SM-DNA加合物的同时测定。N7-HETEG、O6-HETEG、Bis-G及N3-HETEA的检测限分别为:1、0.2、5和9 pg/mL,灵敏度达到1个加合物/109-11个核苷,线性范围跨越5-7个数量级。与方法一相比,灵敏度更高,特异性更好,特别适用于低丰度加合物的检测,但是需要较高的合成技术和仪器条件。将该方法应用于体外染毒皮肤样品中SM-DNA加合物的检测,结果显示四种SM-DNA加合物与染毒剂量之间均呈线性相关。第四章建立了成年SD雄性大鼠芥子气经皮和腹腔注射两种染毒模型,并对血液和不同组织中SM-DNA加合物的检测鉴定及代谢行为进行了研究。拟从加合物含量、分布与一般毒理指标、病理结果之间的相关性,为芥子气的损伤机理研究提供基础数据。2LD50皮肤染毒大鼠的体重下降明显,动物在染毒后3-7天内全部死亡,脾脏系数明显减小。两种染毒方式下肺脏病理检查结果均有明显病变,且随染毒剂量加大损伤愈显严重。说明两种染毒途径下,肺是芥子气损伤的主要靶器官。染毒大鼠体内SM-DNA加合物检测结果表明:两种染毒模型各剂量组,大鼠各脏器、皮肤和血液中均可检出DNA加合物,并呈现一定的剂量和时间依赖关系,经14或21天代谢消除后仍可检出明显量的四种DNA加合物,其浓度约为最大浓度时的20%,表明其可作为SM暴露的溯源性检测生物标志物;SM通过血液被传送至全身各处,与肺、肝、脾、肾中的DNA形成加合物。皮肤中加合物含量顺序为:N7-HETEG> Bis-G> N3-HETEA> O6-HETEG,但是血液和组织中O6-HETEG含量比N3-HETEA含量高,尤其在肺和脾中O6-HETEG有较高的比例,说明该加合物水平与SM损伤生物效应密切相关。根据血液和组织中四种SM-DNA加合物的不同比例,结合病理检查及组织系数检测结果,推测N7-HETEG、N3-HETEA和Bis-G与SM损伤生物效应无明显关联,是SM的暴露标志物,而O6-HETEG可作为SM损伤的效应标志物。综上所述,本文建立了两种高灵敏、高选择性的SM-DNA加合物分析方法。方法灵敏度可满足生物样品中痕量SM-DNA加合物的测定,为研究其代谢行为及溯源性检测提供技术基础;四种SM-DNA加合物的同时测定更真实的反映了加合物在生物体内的分布及代谢过程;从分子水平揭示SM-DNA加合物与暴露剂量、损伤效应之间的联系,为SM毒理机制的研究提供了新的思路。更多还原

【Abstract】 DNA adduct is one of the most important biomarkers. It is the product of DNA covalently binding with the electrophilic group of compound, and is the most important and commonly mode of DNA damage. Detection of DNA adduct can not only provide the exact interior dosage but also help to illuminate the toxicology mechanism. The research brings DNA adduct back to the centre of research interest. Sulfur mustard (SM) is well known to be a highly toxic and mutagenic warfare agent, featured with synthesis facile, hypertoxicity, security and therapy difficult.Sulfur mustard possesses two electrophilic carbon atoms and its chemistry and metabolism are dominated by their reactions with macromolecules (protein, DNA, RNA et.al.) via the episulfonium ion. The mechanism of its action with regard to vesicancy is unknown but is assumed to result from its reactions with macromolecules. Despite the lowest content and shorter half-time, DNA adducts show important biological significance.SM reacts with DNA to produce four kinds of mono- and bifunctional adducts. These adducts are formed preferentially at the N7 position of guanine (60-70%), so N7-HETEG is considered to be suitable for the retrospective detection of SM. Even though the other three adducts (O6-HETEG, Bis-G and N3-HETEA) are comparatively rare, they have great relationship with biological endpoints, such as misreplication, stall replication and finally induce double strand breaks. So the research on the DNA adducts may help to clarify the toxicological mechanism and lay a foundation for the research of SM prevetion and therapy.For SM retrospective detection and the toxic mechanism illumination, two sensitivity detection methods were developed using HPLC-MS/MS in MRM mode. The methods were used to the detection of the biological samples in vitro and in vivo. The metabolizing behavior and distribution of adducts were evaluated in SD rats administrated with SM percutaneous and ip.This dissertation consists of four chapters. The first chapter is the introduction. In this chapter, we summarized the biological significant and detection methods of DNA adducts. The research progress of the SM-DNA adducts was focused on. The detection methods and the important role in retrospective detection and toxic mechanism illumination were sumarized. The objectives, contents and new insights of this dissertation were briefly outlined at the end of this chapter.The second chapter was about the synthesis and identification of standards. In this chapter, nine pure standards were obtained, which include four DNA adducts, four deuterium labeled internal standards (IS) and N7-benzylguanine. The structure identification and the purity check were performed with 1H NMR and LC-MS.The reactive sites of guanine and adenine are N7 and N3 respectively, N7-HETEG and N3-HETEA were direct synthesised from less toxic material- half mustard in organic homogeneous system. Reactivity of O6 is remarkably less than that of guanine N7, so O6-HETEG can not get from half mustard simply covalent binding to guanine or guanosine. 6-cholorguanine and nontoxic compound- thiodiglycol were used to synthesis O6-HETEG skillfully. Bifunctional SM adduct was gained according to the procedure of reference. Before purified with preparation chromatography, crude product should be washed with diluted acid repeatedly to get rid of the main byproduct-N7 monofunctional adduct.In order to pave the way for synthesis of deuterium labeled internal standards, synthesis method of deuterium labeled sulfur mustard and thiodiglycol were established.All of the puritis of the nine standards were over 95%, and the deuterate percentage of internal standards were over 99%.The third chapter is about the development and verification of analysis methods of SM-DNA adducts.Two detection methods were developed. The first one aimed at detection of the highest aboundant lesion N7-HETEG, using high performance liquid chromatography– positive electrospray ionization tandem mass spectrometry (HPLC-ESI+-MS/MS), working in multiple reaction monitor (MRM) mode and N7-benzylguanine act as IS. The limit of detection was hundreds pg/mL in various biomatrices, viz. one adducts per 107 nucleotides. Validation results showed that the method has satisfactory sensitivity, precision, and accuracy. N7-HETEG in human whole blood treated with 2μmol/L SM in vitro was detected successfully. There is dose-dependent relationship between N7-HETEG and SM dosages, so N7-HETEG can act as the biomarker of SM exposed.Simultaneity determination method was developed at first time using ultra high pressure liquid chromatography-positive electrospray ionization tandem mass spectrometry (UPLC-ESI+-MS/MS), quadrupole and ion trap tandem working in MRM mode, which provides higher sensitivity. Higher selectivity and specificity were achieved by isotope dilution method. The limits of detection of N7-HETEG, O6-HETEG, Bis-G and N3-HETEA were 1 pg/mL, 0.2 pg/mL, 5 pg/mL and 9 pg/mL respectively, nearly one adduct per 109-11 nucleotides. But it need higher synthesis technic and more expensive instrument。DNA adducts in SD rat derma in vitro were detected with this method, result revealed the linearity dependence between DNA adducts content and exposed dosage.The last chapter is about the development of animal models and the metoblizing behavior of SM-DNA adducts. Adult male SD rat was used as the experiment animal, treated with different SM dosages by percutaneous and intraperitoneal injection (ip) exposure. At the given time postdosing, the rats were euthanized with urethane by intraperitoneal injection, the blood was collected. Livers, lungs, spleens and kidneys were immediately isolated and weighed. A portion of these tissues were prepared for histopathological evaluation, the rest were used to DNA adducts detection.The relative spleen weights of SD rats dermal exposed to 45.0 mg/kg SM decreased sharply and had less volume of blood, because higher dosages exposure led extensive damage of haematogenous system. The histological changes of the lung treated by both of two routes were remarkable.The results of detection revealed that the DNA adducts in derma, tissues and blood were all detectable even at 14 or 21 day postdosing, the dose and time dependence were remarkable, which showed DNA adducts can act as the biomarkers for retrospective detection. The blood maybe perform the main metabolizing medium transmit SM to tissues. The decreased order of content in derma was N7-HETEG, Bis-G, N3-HETEA and O6-HETEG, but the content of O6-HETEG was larger than that of N3-HETEA in blood and tissues. The fact that higher contents of O6-HETEG lied in lung and spleen showed that O6-HETEG has great relationship with SM damage. According to the different percentage of DNA adducts in blood and tissues, the conculsion was achieved: N7-HETEG, N3-HETEA and Bis-G could be the excellent exposed biomarker instead of the effect biomarker. O6-HETEG maybe is the important effect biomarker of SM damage.In conclusion, two sensitively and selectively methods were developed for analysis the SM-DNA adducts. The trace SM-DNA adducts in biosamples can be detected successfully. The metabolizing behavior research and retrospective detection were layed foundation. Detecting four SM-DNA adducts simultaneity reflected the distribution and metabolizing process truthly. The relationships between adducts and exposure dosage, damage effect were opened out in molecular level. New thoughtway was provided for the research of SM toxicologically mechanism.更多还原