Contamination and effects of perfluorochemicals in Baikal Seal (Pusa sibirica).: 2.: Molecular characterization, expression level, and transcriptional activation of peroxisome proliferator-activated receptor α
Contamination and effects of perfluorochemicals in Baikal Seal (Pusa sibirica).: 2.: Molecular characterization, expression level, and transcriptional activation of peroxisome proliferator-activated receptor α
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DOI：
10.1021/es0720558
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发表时间：
2008-04-01
影响因子：
11.4
通讯作者：
Petrov, Evgeny A.
中科院分区：
文献类型：
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作者：
Ishibashi, Hiroshi;Iwata, Hisato;Petrov, Evgeny A.
为了研究全氟化合物 (PFC) 的生物效应并确定暴露于 PFC 的生物标志物，本研究重点关注贝加尔海豹 (Pusa sibirica) 中过氧化物酶体增殖物激活受体 α (PPAR α) 介导的影响。我们从贝加尔海豹的肝脏中克隆了编码 PPAR α 的全长 cDNA，其推导的开放阅读框由 468 个氨基酸残基组成，预测分子量为 52.2 kDa。贝加尔海豹 PPAR α 的氨基酸序列与其他哺乳动物 PPAR α 的氨基酸序列比较显示，与狗 (97%)、人 (95%)、大鼠 (92%) 和小鼠 (91%) 的 PPAR α 具有相当大的相似性。对贝加尔海豹组织的实时定量 RTPCR 分析表明，PPAR α mRNA 主要在肝脏、肾脏、心脏和肌肉中表达。肝脏中 PPAR α mRNA 的表达水平与免疫化学检测到的细胞色素 P450 (CYP) 4A 样蛋白的表达水平呈正相关，表明贝加尔海豹中的 PPAR α-CYP4A 信号通路可能是保守的。本研究还开发了一种体外 PPAR α 报告基因测定方法，使用瞬时转染贝加尔海豹 PPAR α cDNA 表达载体和含有过氧化物酶体增殖物反应元件的报告载体的非洲绿猴肾 CV-1 细胞。体外报告基因检测显示对安妥明（clofibrate）有显着的反应，安妥明是人类和啮齿动物中已知的 PPAR α 激动剂。用全氟辛酸 (PFOA)、全氟壬酸 (PFNA)、全氟癸酸 (PFDA)、全氟十一烷酸 (PFUnDA) 或全氟辛烷磺酸 (PFOS) 处理以剂量依赖性方式诱导 PPAR α 介导的转录活性，显示观察到的最低效应浓度为 62.5、125、125、62.5 和分别为125μM。在野生贝加尔海豹的肝脏中，PPAR α mRNA 的表达水平与 PFNA 水平呈显着正相关。此外，肝脏CYP4A样蛋白的表达与肝脏PFNA和PFDA浓度显着相关。这些结果表明野生贝加尔海豹中 PFC 可以调节 PPAR α-CYP4A 信号通路。我们的研究表明，PPAR α 介导的反应可能是评估 PFC 对野生动物潜在生物效应的有用生物标志物。
To investigate the biological effects of perfluorochemicals (PFCs) and to identify biomarkers of exposure to PFCs, this study focused on the effects mediated by peroxisome proliferator-activated receptor alpha (PPAR alpha) in Baikal seals (Pusa sibirica). We cloned a full-length cDNA encoding PPAR alpha from the liver of Baikal seal, which has a deduced open reading frame of 468-amino acid residues with a predicted molecular mass of 52.2 kDa. Comparison of the amino-acid sequence of Baikal seal PPAR alpha with that of other mammalian PPAR alpha showed considerable similarities with PPAR alpha of dog (97%), human (95%), rat (92%), and mouse (91%). The quantitative real-time RTPCR analyses of tissues from Baikal seals revealed that PPAR alpha mRNAs were primarily expressed in the liver, kidney, heart, and muscle. The hepatic expression levels of PPAR alpha mRNA showed a positive correlation with the expression levels of immunochemically detected cytochrome P450 (CYP) 4A-like protein, indicating that the PPAR alpha-CYP4A signaling pathway in Baikal seal is likely conserved. This study also developed an in vitro PPAR alpha reporter gene assay using African green monkey kidney CV-1 cells transiently transfected with Baikal seal PPAR alpha cDNA expression vector and a reporter vector containing a peroxisome proliferator-responsive element. The in vitro reporter gene assay displayed significant response to clofibrate, which is a known PPAR alpha agonist in humans and rodents. Treatment with perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), perfluorodecanoic acid (PFDA), perfluoroundecanoic acid (PFUnDA), or perfluorooctane sulfonate (PFOS) induced PPAR alpha-mediated transcriptional activity in a dose-dependent manner, showing the lowest-observed-effect concentrations of 62.5, 125, 125, 62.5, and 125 mu M, respectively. In the livers of wild Baikal seals, expression levels of PPAR alpha mRNA showed a significant positive correlation with PFNA levels. Moreover, expression of hepatic CYP4A-like protein was significantly correlated with the hepatic concentrations of PFNA and PFDA. These results suggest modulation of the PPAR alpha-CYP4A signaling pathway by PFCs in the wild Baikal seals. Our study demonstrates that the PPAR alpha-mediated response may be a useful biomarker to evaluate potential biological effects of PFCs in wildlife.