不同种属来源活化体系中CYP2A6酶活及其对烟碱代谢的动力学  

CYP2A6 activity and its effect on nicotine metabolic kinetics in different metabolic activation systems

作　　者：王蕙婷 李翔[1] 华辰凤 许良涛 赵阁[1] 尚平平[1] 乔梁峻 赵俊伟[1] 刘惠民[1] 彭桂新 谢复炜[1] WANG Huiting;LI Xiang;HUA Chenfeng;XU Liangtao;ZHAO Ge;SHANG Pingping;QIAO Liangjun;ZHAO Junwei;LIU Huimin;PENG Guixin;XIE Fuwei(Zhengzhou Tobacco Research Institute of CNTC,Zhengzhou 450001,China;Technology Center of China Tobacco Henan Industrial Co.,Ltd.,Zhengzhou 450000,China)

机构地区：[1]中国烟草总公司郑州烟草研究院,郑州高450001 [2]河南中烟工业有限责任公司技术中心,郑州市450000

出　　处：《烟草科技》2019年第7期42-50,共9页Tobacco Science & Technology

基　　金：中国烟草总公司郑州烟草研究院院长科技发展基金项目“烟气暴露-代谢复合体外模型构建及在烟气诱发炎症反应评价中的应用”(322017CA0140)

摘　　要：为探讨10种不同种属来源活化体系中CYP2A6蛋白的表达水平与酶活性之间的关系,包括来自人体的细胞(BEAS-2B、HepG2和巨噬细胞)及肝微粒体(25供体混合性别人肝微粒体及男性人肝微粒体)、实验室常用不同动物肝微粒体或S9(雄性SD大鼠肝微粒体、雄性恒河猴肝微粒体、雄性比格犬肝微粒体及雄性大鼠诱导肝S9)和同源重组酶,通过免疫印迹法测定其CYP2A6蛋白表达水平,并以烟碱的代谢活化为基础,通过HPLC-MS/MS法测定其酶代谢动力学参数。结果表明:①10种活化体系中CYP2A6酶活具有明显差异,烟碱分别与25供体混合性别人肝微粒体、同源重组CYP2A6酶及雄性恒河猴肝微粒体共孵育时其浓度明显降低,另外7种活化体系中无酶活或活性较低;②CYP2A6蛋白表达水平与CYP2A6酶活具有一定的相关性。研究结果可为卷烟烟气体外毒理学评价中代谢活化体系的选择提供参考,也可为进一步了解CYP2A6基因多态性对烟碱代谢的影响提供体外实验数据。The relationship between the expression levels and enzymatic activities of 10 CYP2A6 proteins from different source activation systems were investigated in this work. The systems included cells (BEAS-2B cells, HepG2 cells, and macrophages) from human, human liver microsomes from 25 donors of mix gender, human liver microsomes from males, liver microsomes and S9 from animals commonly used in laboratories (male SD rat liver microsomes and male rhesus monkey liver microsomes, male beagle liver microsomes, induced liver S9 of male rats) and homologous recombinase. The expression levels of CYP2A6 protein were determined by western blot assay, and the metabolic kinetic parameters were assessed on the basis of nicotine metabolism by HPLC-MS/MS method. The results showed that: 1) CYP2A6 from the 10 activation systems presented clear differences in enzymatic activity. The nicotine concentration significantly reduced when incubating with human liver microsomes from 25 donors of mix gender, homologous CYP2A6 recombinase and liver microsomes of male rhesus monkeys separately, however there was no or weak enzymatic activity in the other 7 activation systems. 2) There existed a certain correlation between the content of CYP2A6 protein and the enzyme activity of CYP2A6. The results of this study offers a useful reference for the selection of metabolic activation system used in the toxicological evaluation of cigarette smoke and aerosols from next generation nicotine products in vitro, and provides in vitro experimental data for understanding the effects of CYP2A6 gene polymorphism on nicotine metabolism.

关 键 词：CYP2A6酶 烟碱 酶活 代谢动力学 

分 类 号：TS416[农业科学—烟草工业]