氟调醇的代谢转化机制研究
文献类型：学位论文
| 作者 | 李忠民 |
| 学位类别 | 硕士 |
| 答辩日期 | 2016-05 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 郭良宏 |
| 关键词 | 氟调醇 FTOHs I相代谢 phase I metabolism II相代谢 phase II metabolism 代谢动力学 metabolic kinetics 分子对接 molecular docking |
| 其他题名 | Study on the Biotransformation Mechanisms of Fluorotelomer Alcohols |
| 学位专业 | 郭良宏 |
| 中文摘要 | 多氟和全氟烷基化合物（poly- and perfluoroalkyl substances, PFASs）由于其独特的稳定性、疏水性和疏脂性被广泛用于生产和生活中。PFASs的生产方式有两种：电化学氟化法（electrochemical fluorination, ECF）和调聚反应（telomerization）。由于ECF方法产生的全氟烷基酸（perfluoroalkyl acids, PFAAs）具有持久性、高毒性和生物富集性等特点，发达国家开始逐渐停止或限制这种生产工艺。调聚反应产生的氟调醇（fluorotelomer alcohols, FTOHs）的产量却不断增加。研究表明，FTOHs经生物或非生物转化能够生成全氟烷基羧酸（perfluocarboxylic acids, PFCAs），因而是PFCAs环境污染和人体暴露的间接来源。毒理学研究发现FTOHs的一些中间代谢产物能够与多种生物分子共价结合，影响其正常的功能，产生严重的毒性效应。代谢终产物PFAAs和活性巯基化合物又分别能引起肝毒性和肾毒性。因此，代谢转化对FTOHs生物毒性有重要影响，是代谢活化过程。 研究表明，细胞色素P450酶（cytochrome P450s, CYPs）催化了FTOHs的第一步氧化转化。CYPs包含众多亚型，而人体中催化该过程的P450酶亚型仍未知，I相和II相反应的代谢动力学也未见报道。 在本文中，我们利用重组人源P450酶、人肝微粒体（human liver microsomes, HLMs）及人肝胞液考察了6:2、8:2及10:2 FTOH的I相及II相代谢过程，筛查了催化8:2 FTOH的P450酶亚型并鉴定了代谢产物。结果表明，在所研究的11种CYPs亚型中，只有CYP2C19能够催化8:2 FTOH，其Km和Vmax值分别为18.8 μM和8.52 pmol/min/pmol P450。HPLC-MS/MS分析证明8:2 FTOH的氧化产物为8:2 氟调醛（8:2 fluorotelomer aldehyde, 8:2 FTAL），8:2 FTAL易自发失去HF形成8:2氟调不饱和醛（8:2 fluorotelomer unsaturated aldehyde, 8:2 FTUAL）。HLMs也能够催化8:2 FTOH氧化，其Vmax及CLint值与CYP2C19相当。分子对接结果表明8:2 FTOH的末端羟基与催化中心的结合构象有利于氧化反应的发生。在HLMs代谢反应中，我们发现6:2 FTOH能够发生显著的降解，其CLint值为17.96 pmol/min/mg HLMs/μM，8:2和10:2 FTOH的代谢效率较低，分别为11.04和9.10 pmol/min/mg HLMs/μM，说明碳链长度对FTOHs的代谢效率有重要影响。II相代谢实验发现，8:2 FTOH能够被HLMs中的葡萄糖醛酸转移酶（UDP-glucuronosyltransferases, UGTs）催化生成8:2 FTOH-glucuronide，也能够被肝胞液中硫酸转移酶（sulfotransferases, SULTs）催化生成8:2 FTOH-sulfate。I相和II相代谢的效率大小关系为：sulfation > oxidation > glucuronidation。II相加合物水溶性高、易于排泄，因此，排泄是8:2 FTOH人体暴露的主要归宿。本文筛查了FTOHs代谢的关键酶，并计算了代谢反应的动力学常数，为深入了解FTOHs的人体代谢转化机制和评价FTOHs暴露的环境健康风险提供了依据。 |
| 英文摘要 | Poly- and perfluoroalkyl substances (PFASs) have been widely used in the commercial and industrial applications due to their unique properties of stability, hydrophobicity and lipophobicity. Traditionally, PFASs were produced via two major manufacturing processes: electrochemical fluorination (ECF) and telomerization. Due to their persistence, toxicity and bioaccumulation, the ECF production has been phased out gradually in the developed countries. However, the production of fluorotelomer alcohols (FTOHs) by telomerization has been increasing significantly. Recent studies have demonstrated that the biotransformation and abiotic transformation of FTOHs yield perfluocarboxylic acids (PFCAs). Therefore, FTOHs are the indirect source for the environmental pollution and human exposure of PFCAs. Toxicological studies have demonstrated that some of the FTOHs intermediate metabolites were able to covalently bind with various biomolecules and thereby cause serious toxicity. The final metabolites, including PFAAs and active thiol metabolites, were showed to cause hepatotoxicity and renal toxicity, respectively. Therefore, the biotransformation of FTOHs is a bioactivation process, and has great impact on the toxicity of this group of chemicals. It has been suggested that cytochrome P450 (P450) enzymes were involved in the initial oxidative biotransformation of fluorotelomer alcohols (FTOHs), but the specific isoforms responsible for the process in human body remained unknown. The phase II metabolism of FTOHs in humans has not been studied either. In the present study, we characterized the in vitro metabolism of 6:2, 8:2 and 10:2 FTOH by recombinant human P450 enzymes, human liver microsomes (HLMs) and human liver cytosol, and identified the enzyme(s) that engaged in the phase I metabolism of 8:2 FTOH. The results showed that among the 11 isoforms investigated, CYP2C19 was the only enzyme capable of catalyzing 8:2 FTOH with Km and Vmax values of 18.8 μM and 8.52 pmol/min/pmol P450, respectively.HPLC-MS/MS analysis revealed that the direct metabolite was 8:2 fluorotelomer aldehyde (8:2 FTAL), which then eliminated HF to form 8:2 fluorotelomerunsaturated aldehyde (8:2 FTUAL). HLMs also catalyzed 8:2 FTOH transformation, with the Vmax and intrinsic clearance (CLint) values similar to CYP2C19 after the protein content is taken into account. Molecular docking showed that the hydroxyl group of 8:2 FTOH accesses the heme iron-oxo of CYP2C19 in an energetically favored orientation. In HLMs-mediated metabolism, we found 6:2 FTOH was extensively metabolized with the CLint value of 17.96 pmol/min/mg HLMs/μM, while to a less extent for 8:2 and 10:2 FTOH, with CLint values of 11.04 and 9.10 pmol/min/mg HLMs/μM, respectively, indicating that the carbon-fluorine (C-F) chain length was of great importance for the metabolic efficiency. In phase II metabolism, we found that 8:2 FTOH was also transformed by phase II enzymes to form O-glucuronide and O-sulfate conjugates. The CLint values follow the order of sulfation > oxidation > glucuronidation, suggesting that conjugation was the major fate of 8:2 FTOH. These results shed light on FTOHs biotransformation by identifying the key human CYP isoform and quantifying metabolic kinetics. They will help future studies in the FTOHs metabolic mechanisms and the assessment of the potential environmental health risk of FTOHs. |
| 源URL | [http://ir.rcees.ac.cn/handle/311016/36888]  |
| 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 | 李忠民. 氟调醇的代谢转化机制研究[D]. 北京. 中国科学院研究生院. 2016. |
入库方式： OAI收割
来源：生态环境研究中心
浏览0
下载0
收藏0
其他版本
除非特别说明，本系统中所有内容都受版权保护，并保留所有权利。