| 题名 | 色谱与ICP-MS联用技术在有机锡形态及其生物毒性研究中的应用 |
| 作者 | 孙婧 |
| 学位类别 | 博士 |
| 答辩日期 | 2012 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 江桂斌 |
| 关键词 | 有机锡 Organotin compounds HPLC-HG-ICP-MS HPLC-HG-ICP-MS CE-ICP-MS CE-ICP-MS 二维电泳 2-DE 蛋白表达差异 Protein expression |
| 其他题名 | Speciation and Toxicology Studies of Organotin Compounds by Using Chromatography On-line Coupled with ICP-MS |
| 中文摘要 | 有机锡化合物作为环境中的一类高毒性污染物，对人体内分泌系统、生殖系统及免疫系统等方面具有毒性效应。环境中的有机锡化合物可以通过食物链富集，并最终进入人体。目前，有机锡化合物的毒性研究已有很多，但有机锡化合物在生物体内吸收、富集的过程及其生物毒性机理尚不明确。本文针对有机锡化合物与蛋白的相互作用及其毒性效应，建立了有机锡化合物的形态分析方法，研究了有机锡化合物与蛋白的相互作用，并对有机锡化合物对细胞蛋白表达的影响进行了研究。 建立了HPLC-HG-ICP-MS联用系统，采用反向色谱法分离了5种有机锡化合物（TMT、MMT、TBT、DBT、MBT），优化了色谱分离及氢化衍生条件，改变了ICP-MS载气方向，方法无需增加仪器附件，消除了流动相及基体干扰，可以快速直接的对简单环境介质中的有机锡化合物进行分离测定。本方法可应用于有机锡生产工厂的工人尿液中有机锡化合物的检测。 建立了CE-ICP-MS联用系统，用于分离鉴定有机锡化合物（TBT、TMT、DBT、MBT）。该联用系统采取了半永久性毛细管涂层技术克服了有机锡化合物在毛细管内壁的吸附问题，可实现多种有机锡化合物的基线分离。实验研究了缓冲液组分和浓度对分离以及检测器灵敏度的影响，并且优化了鞘流液中HNO3的浓度以及压力进样模式下的进样时间等条件。该方法成功应用于标准参考物质CRM477及环境样品（水样、葡萄酒、砂海螂等）中有机锡化合物的分离测定。 利用ACE及NECEEM方法测定并比较了有机锡与人血清白蛋白相互作用的平衡结合常数。实验表明有机锡化合物与HSA蛋白的相互作用为非共价性结合，因结合弱于汞类化合物与HSA的结合，故ACE方法所得结果更符合实验观察。实验也指出CE-ICP-MS联用系统可以应用于金属结合复合物的相应研究。 利用二维电泳技术对经有机锡暴露后的H295R细胞进行了蛋白的分离，研究了有机锡化合物引起的H295R细胞的蛋白表达差异。并利用MALDI-TOF-MS技术对差异蛋白点进行定性分析。证明有机锡化合物可以影响激素表达及蛋白表达过程。通过对差异蛋白的分类，进一步阐释有机锡化合物的生物毒性及机制。 |
| 英文摘要 | As a group of the toxic compounds, organotin compounds are widely applied as PVC stabilizers, pesticides and additives in many industrial and chemical areas. The extensive applications of organotin compounds have resulted in extensive distribution of these compounds in environmental matrixes. Organotin compounds can be biomagnified through the food chain up to humans. It is well known that the organotin compounds could cause damage to the neurological system and reproductive system. There are still some gaps in our knowledge about the detailed mechanisms of organotin toxicity, including the intake, accumulation and transformation in living organisms. In this dissertation, we mainly studied analytical methods for speciation and the toxicities of organotin compounds. In order to directly determine the trace levels of organotin compounds in environmental samples, a novel on-line coupled HPLC-hydride generation (HG)-ICP-MS system was developed for the rapid, direct and sensitive analysis of organotin compounds in urine without any pretreatment steps. MMT, DMT, TMT, TBT and MBT were baseline separated within 15 min by reversed phase HPLC. The method increased the sensitivity of the instrument and decreased the effects of the matrix. A novel method using capillary electrophoresis (CE) coupled with inductively coupled plasma mass spectrometry (ICP-MS) for the separation of four organotin compounds has been developed. Semipermanent coating of dioctadecyl dimethyl ammonium chloride (DDAC) was applied and investigated to enhance separation efficiency of trimethyltin (TMT), tributyltin (TBT), dibutyltin (DBT) and monobutyltin (MBT). The electrolyte composition and ionic strength were optimized. The developed method was successfully applied for speciation analysis of four organotin compounds in one certified reference material (CRM 477, mussel tissue), and environmental samples including water samples, grape wine and biological samples (Mya arenaria Linnaeus). Affinity capillary electrophoresis (ACE) and nonequilibrium capillary electrophoresis assays of equilibrium mixtures (NECEEM) were performed and compared to determine the binding constants of organotin compounds and human.serum albumin (HSA) by using ICP-MS as the detector. A good precision of the binding constants (log Kb) using the ACE method was proved, and the values were in the range of 5.68-6.13, showing non-covalent interaction between organotin compounds and HSA. The binding constants are less than the constants of mecury-HSA complexes. Meanwhile, this study also confirms the suitability of CE-ICP-MS method for further studies on organometallic complexation. Investigation of the protein profiles in H295R cells after organometallic compounds exposed was studies by 2-D Electrophoresis (2-DE) and MALDI-TOF-TOF-MS. The LC50s of organotin compounds to H295R cells have been examined. 2-DE and MALDI-TOF-MS were used to investigate the protein express signatures in order to find the toxicology mechanism of organotin compounds. |
| 内容类型 | 学位论文 |
| 源URL | [http://ir.rcees.ac.cn/handle/311016/35159]  |
| 专题 | 生态环境研究中心_环境化学与生态毒理学国家重点实验室 |
| 推荐引用方式 GB/T 7714 | 孙婧. 色谱与ICP-MS联用技术在有机锡形态及其生物毒性研究中的应用[D]. 北京. 中国科学院研究生院. 2012. |
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