Constitutive androstane receptor (CAR) as a potential sensing biomarker of persistent organic pollutants (POPs) in aquatic mammal:: Molecular characterization, expression level, and ligand profiling in Baikal seal (Pusa sibirica)
Constitutive androstane receptor (CAR) as a potential sensing biomarker of persistent organic pollutants (POPs) in aquatic mammal:: Molecular characterization, expression level, and ligand profiling in Baikal seal (Pusa sibirica)
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DOI：
10.1093/toxsci/kfl088
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发表时间：
2006-11-01
影响因子：
3.8
通讯作者：
Tanabe, Shinsuke
中科院分区：
文献类型：
--
作者：
Sakai, Hiroki;Iwata, Hisato;Tanabe, Shinsuke
为了研究组成活性雄烷受体(CAR)在水生哺乳动物中的功能，从俄罗斯贝加尔湖的贝加尔湖海豹(Pusa SiBirica)肝脏中克隆了CAR互补DNA(CDNAs)，并研究了野生种群不同组织器官中CAR的表达水平和CAR的配基分布。海豹CAR基因全长1047bp，编码348个氨基酸，与啮齿动物和人类CAR的氨基酸同源性为74-84%。组织/器官的表达谱表明，贝加尔海豹主要在肝脏表达，其次是心脏和肠道。肝脏CAR mRNA的表达与细胞色素P450(CYP)1A、1B、2B、2C和3A样蛋白的表达无相关性，表明Car的表达水平可能不是调控这些CYP在海豹肝脏中表达的唯一决定因素。CAR的表达与任何持久性有机污染物(POPs)水平均无显著相关性。此外，我们还利用Baikal seal CAR表达载体和(NR1)(3)-荧光素酶报告基因表达载体对MCF-7细胞进行了CAR反式激活实验。在Baikal Seal Car的反式激活分析中，既没有检测到雄烯醇和雄烯醇的抑制作用，也没有检测到雌酮和雌二醇的激活作用，这是公认的小鼠和人CAR的内源性配体。另一方面，鹅去氧胆酸、去氧胆酸和石胆酸等胆汁酸也激活了海豹CAR和小鼠CAR。至于外源化学物质，海豹CAR可被人CAR激动剂6-(4-chlorophenyl)imidazo[2，1-b][1，3]thiazole-5-carbaldehyde O-(3，4-二氯苯基)肟反式激活，但不能被小鼠CAR激动剂(1，4-双[2-(3，5-二氯吡氧基)]苯)反式激活。此外，密封车还被多氯联苯(PCBS)激活(卡内克洛-500，国际纯化学和应用化学联合会编号。2，2‘，4，4’，5，5‘-六氯联苯和PCB180；2，2’，3，4‘，5，5’-七氯联苯)和1，1，1-三氯-2，2-二(对氯苯基)乙烷(p，p‘-DDT)及其代谢物1，1-二氯-2，2-双(对氯苯基)乙烯(p，p’-DDE)。海豹车对多氯联苯的反应比老鼠车更灵敏。根据CAR反式激活试验的结果，按湿重计算，Kanechlor500、PCB153、PCB180、p，p‘-DDT和p，p’-DDE在贝加尔海豹中的最低可观测效应水平分别为10、20、20、10和10ppm。这些结果表明，CAR在包括海豹在内的多种哺乳动物中都是保守的。虽然海豹CAR介导的基因转录可能是对某些POPs暴露的敏感反应，但海豹CAR的配基分布可能与其他哺乳动物CAR的不同。这项研究表明，CAR介导的反应可能是评估POPs等外来生物在野生动物中的生态毒理风险的有用信息，但先前来自啮齿动物和人类CAR的结果可能不适用于野生物种的风险评估。
To characterize the function of constitutive active/androstane receptor (CAR) in aquatic mammals, CAR complementary DNA (cDNA) was cloned from the liver of Baikal seal (Pusa sibirica) from Lake Baikal, Russia, and the messenger RNA (mRNA) expression levels in various tissues/organs of the wild population and the CAR ligand profiles were investigated. The seal CAR cDNA had an open reading frame of 1047 bp encoding 348 amino acids that revealed 74-84% amino acid identities with CARs from rodents and human. The mRNA expression profile of tissues/organs represented that Baikal seal CAR was predominantly expressed in the liver followed by heart and intestine. The expression analysis of hepatic CAR mRNA showed no correlation with expression of cytochrome P450 (CYP) 1A, 1B, 2B, 2C, and 3A-like proteins, indicating that the CAR expression level may not be the sole determinant of the regulation of these CYP expressions in the seal liver. There was no significant correlation between CAR expression and any of the persistent organic pollutants (POPs) levels. Furthermore, we performed an in vitro CAR transactivation assay using MCF-7 cells transfected with Baikal seal CAR expression plasmid and (NR1)(3)-luciferase reporter gene plasmid. In the transactivation analysis of Baikal seal CAR, neither repression by androstanol and androstenol, nor activation by estrone and estradiol, which are recognized as endogenous ligands for mouse and human CARs, was detected. On the other hand, bile acids such as chenodeoxycholic acid, deoxycholic acid, and lithocholic acid activated the seal CAR as well as mouse CAR. As for exogenous chemicals, the seal CAR was transactivated by a human CAR agonist, 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime), but not by a mouse CAR agonist, (1,4-bis[2-(3,5-dichloropyridyloxy)]benzene). In addition, the seal CAR was also activated by polychlorinated biphenyls (PCBs) (Kanechlor-500, International Union of Pure and Applied Chemistry No. PCB153; 2,2',4,4',5,5'-hexachlorobiphenyl and PCB180; 2,2',3,4,4',5,5'-heptachlorobiphenyl), and 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (p,p'-DDT) and its metabolite, 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (p,p'-DDE). The seal CAR responded more sensitively to PCBs than the mouse CAR. Based on the results of CAR transactivation assay, the lowest observable effect levels of Kanechlor-500, PCB153, PCB180, p,p'-DDT, and p,p'-DDE in Baikal seal were estimated to be 10, 20, 20, 10, and 10 ppm on wet weight basis, respectively. These results suggest that CAR is conserved in diverse mammalian species including seals. Whereas the seal CAR-mediated gene transcription may potentially be a sensitive response to the exposure of certain POPs, the ligand profile of seal CAR may be different from those of other mammalian CARs. This study indicates that CAR-mediated responses may be useful information to assess the ecotoxicological risk of xenobiotics such as POPs in wildlife but the previous results derived from rodent and human CAR may not be applicable to the risk assessment in wild species.