文献类型：期刊文献

中文题名：孕烷X受体双荧光素酶报告基因方法建立及有机磷酸酯阻燃剂的受体激活效应研究

英文题名：Establishment of a Dual Luciferase Reporter Gene Assay for Pregnane X Receptor and Study of Agonistic Activities of Organophosphate Flame Retardants

作者：吴一凡[1,2,3];聂青宇[1,2,3];郑静怡[1,2,3];镇华君[1,2,3,4];修光利[1,2,3,4]

机构：[1]华东理工大学资源与环境工程学院,上海200237;[2]上海市环境保护化学污染物环境标准与风险管理重点实验室,上海200237;[3]国家环境保护化工过程环境风险评价与控制重点实验室,上海200237;[4]上海污染控制与生态安全研究院,上海200092

年份：2023

卷号：18

期号：2

起止页码：33

中文期刊名：生态毒理学报

外文期刊名：Asian Journal of Ecotoxicology

收录：CSTPCD;;北大核心:【北大核心2020】；CSCD:【CSCD_E2023_2024】；

基金：国家自然科学基金资助项目(22276056);上海市浦江人才计划资助项目(20PJ1402700)。

语种：中文

中文关键词：有机磷酸酯阻燃剂;孕烷X受体;报告基因检测;化学品健康风险

外文关键词：organophosphate flame retardant;pregnane X receptor;reporter gene test;chemical health risk

摘要：孕烷X受体(pregnane X receptor,PXR)是一种调控生物体异源性/内源性物质代谢的核受体。环境污染物可介由PXR改变人体代谢稳态进而增加多种代谢性疾病风险,但目前尚缺乏一种灵敏、稳定的检测化学品PXR结合活性的体外实验方法。本研究基于重组基因技术构建了带有海肾荧光素酶基因的pBIND-hPXR表达质粒,将其与插入上游激活序列的荧光素酶报告基因质粒GAL4-UAS-Luc共转染至HEK 293T细胞,最终成功建立了一种可筛查化学品人孕烷X受体激动/拮抗作用的双荧光素酶报告基因检测方法,该方法具有灵敏度高、适用范围广和重复性好的特点。进一步检测了9种典型有机磷酸酯(organophosphate esters,OPEs)阻燃剂的hPXR激活效应,结果表明芳基类OPEs的激活活性强于氯代和烷基类OPEs,其中磷酸三甲苯酯(TCP)的20%最大效应浓度(基于阳性对照)及最大激活效应值和已知的hPXR强激动剂利福平相当。此外,首次发现2-乙基己基二苯基磷酸酯(EHDPP)在略高于人体血液最高检出浓度(80 nmol·L^(-1))的染毒组(316.2 nmol·L^(-1))即表现出显著的hPXR激活效应。本方法有助于研究化学品对PXR受体信号通路的扰动作用,并为OPEs类化学品的健康风险评估提供科学依据。
Pregnane X receptor(PXR)is a nuclear receptor that regulates the metabolism of heterologous/endogenous substances in organisms.Environmental pollutants can alter metabolic homeostasis in humans by PXR-mediated pathways and thus increase the risks of common metabolic diseases,but there is lack of sensitive and stable in vitro assays to detect PXR binding activity of chemicals.In this study,the pBIND-hPXR expression plasmid containing a renilla luciferase reporter gene was constructed based on the recombinant DNA technology,and then co-transfected with a firefly luciferase reporter gene plasmid GAL4-UAS-Luc inserted with the upstream activation sequence into HEK 293T cells.The established dual luciferase reporter gene assay exhibited high sensitivity,broad applicability and good reproducibility when screening chemicals for agonistic/antagonistic activities of human pregnane X receptor(hPXR).The assay was further applied to investigate the hPXR binding activities of nine typical organophosphate esters(OPEs),and the results showed that aryl-substituted OPEs in general exerted stronger agonistic activities than those of chlorinated and alkyl-substituted OPEs.Among these OPEs,tricresyl phosphate(TCP)exhibited the strongest agonistic activity,as exemplified by the comparable values of 20%maximal effective concentration and maximum activity with those of rifampicin,a known strong hPXR agonist.For the first time,2-ethylhexyl diphenyl phosphate(EHDPP)was found to exhibit significant hPXR agonistic activity at a concentration of 316.2 nmol·L^(-1),which was only slightly higher than the reported maximum detectable concentration in human blood samples(80 nmol·L^(-1))from a Chinese population.Overall,this assay could facilitate studying the impact of chemical exposure on the PXR signaling pathway and provide a scientific basis for health risk assessment of OPEs.