雌激素样化合物对人细胞色素氧化酶2A6基因的转录调控  

Induction of human cytochrome P450 2A6 transcription by estrogenic compounds

作　　者：黄春玲[1] 邓寒[1] 柯雪娥 韩霜雪[2] 何晓阳[1] 

机构地区：[1]深圳大学生命科学学院深圳市海洋生物资源与生态环境重点实验室,广东深圳518060 [2]深圳大学生命科学学院深圳市微生物基因工程重点实验室,广东深圳518060

出　　处：《中国药理学与毒理学杂志》2014年第1期88-96,共9页Chinese Journal of Pharmacology and Toxicology

基　　金：国家自然科学基金(31070731);国家自然科学基金(21271131)~~

摘　　要：目的建立尼古丁代谢酶CYP2A6报告基因高容量筛选系统,分析环境和天然雌激素样化合物对CYP2A6基因转录的影响。方法构建3种CYP2A6基因转录上游-2460^+1全长、增强子-启动子串联和3倍增强子-启动子串联的荧光素酶报告基因重组体pGL3-2A6 3UP,GL3-2A6 EP和pGL3-2A63EP,分别与雌激素受体α(ERα)及海肾荧光蛋白表达载体三重共转染人肝HepG2细胞。用双荧光素酶报告基因分析技术,选择确认对雌二醇诱导应答最强的CYP2A6报告基因系统;分别用氰戊菊酯、2,2',4,4'-四溴联苯醚、4-二羟基二苯基丙烷、全氟辛酸铵、淫羊藿苷、大豆苷元和槲皮素等7种拟雌激素化合物处理转染报告基因的HepG2细胞,同时设置雌二醇10 nmo·lL-1阳性和0.1%DMSO阴性对照,48 h后测定各组细胞相对荧光强度,分析评估拟雌激素物质对CYP2A6基因转录的诱导作用。结果 3种CYP2A6报告基因重组体中pGL3-2A6 3EP对雌二醇诱导应答最强,且呈显著量效依赖关系。7种拟雌激素化合物对CYP2A6报告基因均具显著诱导作用,其中,2,2',4,4'-四溴联苯醚1.0~100.0 nmol·L-1诱导倍变值为1.45±0.13~3.30±0.13(P<0.01),诱导效应最强。表现出相似的量效诱导关系,淫羊藿苷、大豆苷元和槲皮素10μmo·lL-1时的诱导倍变值为2.41~2.83(P<0.01)。结论建立灵敏可靠、重复性强的CYP2A6报告基因高容量筛选系统,证实多种拟雌激素物质通过激活ERα诱导CYP2A6报告基因转录。提示环境拟雌激素污染物和药材食材雌激素样活性物质可诱导个体尼古丁代谢酶基因表达,从而干扰个体尼古丁代谢清除活性及其对环境化合物致癌代谢活化水平。OBJECTIVE To develop a CYP2A6 gene transcriptional high-capacity screening system and to evaluate the regulatory effects of four environmental estrogenic compounds and three phytoestrogens on CYP2A6 gene transcription. METHODS Three luciferase reporter plasmids containing the CYP2A6gene- 2460 ~ ＋ 1 5＇-flanking region（ pGL3-2A6 UP）,the potential enhancer region host estrogen response element（ ERE）-like tandem in the 5＇ of promoter（ pGL3-2A6 EP）,and three copies of this ERE-like tandem in the 5＇ of promoter（ pGL3-2A6 3EP） were constructed. Each reporter plasmid DNA was co-transfected into HepG2 cells with receptor pcDNA3. 0-ERα and pRL-TK. Dual-luciferase assay was performed after 48 h treatment by different estrogenic compounds,including four environmental pollutants 2,2＇,4,4＇-tetrabromodiphenyl ether（ BDE-47）,bisphenol（ ABPA）,fenvalerate（ FEN）,perfluorooctanoic acid（ PFOA） and three phytoestogens icariin（ ICA）,daidzein（ DAI） and quercetin（ QUE）,as well as 0. 1% DMSO（ negative control） and 10 nmol·L- 1estradiol（ positive control）. RESULTS pGL3-2A6 3EP reporter construct represented the strongest concentration-dependent induction response to the estradiol treatment. CYP2A6 transcription activity was significantly up-regulated by all the seven estrogenic compounds. Among these chemicals,BDE-47 was of particular interest with remarkable induction fold changes from 1. 45 ± 0. 13 to 3. 30 ± 0. 13（ P 0. 01） corresponding to concentrations ranging from 1. 0 to 100 nmo·lL- 1. Phytoestrogens ICA,DAI and QUE represented a similar concentrationinduction pattern. 241% to 283 % increases compared with DMSO control in CYP2A6 promoter activity were observed under phytoestrogens 10 μmo·lL- 1treatment. CONCLUSION CYP2A6 promoter activity is induced by estrogen and estrogenic compounds in an estrogen receptor-dependent manner,implying the biological effects of common environmental endocrine disrupting the chemicals＇ regulation of CYP2A6 gene transcription

关 键 词：细胞色素P450 CYP2A6 转录水平调控 拟雌激素化合物 

分 类 号：R96[医药卫生—药理学]