Ishibashi Hiroshi, Iwata Hisato, Kim Eun-Young, Tao Lin, Kannan Kurunthachalam, Tanabe Shinsuke, Batoev Valeriy B, Petrov Evgeny A
Center for Marine Environmental Studies, Ehime University, Matsuyama 790-8577, Japan.
Environ Sci Technol. 2008 Apr 1;42(7):2302-8. doi: 10.1021/es0720558.
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 (PPARalpha) in Baikal seals (Pusa sibirica). We cloned a full-length cDNA, encoding PPARalpha 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 PPARalpha with that of other mammalian PPARalpha showed considerable similarities with PPARalpha of dog (97%), human (95%), rat (92%), and mouse (91%). The quantitative real-time RT-PCR analyses of tissues from Baikal seals revealed that PPARalpha mRNAs were primarily expressed in the liver, kidney, heart, and muscle. The hepatic expression levels of PPARalpha mRNA showed a positive correlation with the expression levels of immunochemically detected cytochrome P450 (CYP) 4A-like protein, indicating that the PPARalpha-CYP4A signaling pathway in Baikal seal is likely conserved. This study also developed an in vitro PPARalpha reporter gene assay using African green monkey kidney CV-1 cells transiently transfected with Baikal seal PPARalpha 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 PPARalpha agonist in humans and rodents. Treatmentwith perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), perfluorodecanoic acid (PFDA), perfluoroundecanoic acid (PFUnDA), or perfluorooctane sulfonate (PFOS) induced PPARalpha-mediated transcriptional activity in a dose-dependent manner, showing the lowest-observed-effect concentrations of 62.5, 125, 125, 62.5, and 125 microM, respectively. In the livers of wild Baikal seals, expression levels of PPARalpha 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 PPARalpha-CYP4A signaling pathway by PFCs in the wild Baikal seals. Our study demonstrates that the PPARalpha-mediated response may be a useful biomarkerto evaluate potential biological effects of PFCs in wildlife.
为了研究全氟化合物（PFCs）的生物学效应并确定PFCs暴露的生物标志物，本研究聚焦于贝加尔湖海豹（Pusa sibirica）中过氧化物酶体增殖物激活受体α（PPARα）介导的效应。我们从贝加尔湖海豹的肝脏中克隆了一个编码PPARα的全长cDNA，其推导的开放阅读框为468个氨基酸残基，预测分子量为52.2 kDa。贝加尔湖海豹PPARα的氨基酸序列与其他哺乳动物PPARα的氨基酸序列比较显示，与狗的PPARα（97%）、人类的PPARα（95%）、大鼠的PPARα（92%）和小鼠的PPARα（91%）有相当高的相似性。对贝加尔湖海豹组织的定量实时RT-PCR分析表明，PPARα mRNA主要在肝脏、肾脏、心脏和肌肉中表达。PPARα mRNA的肝脏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，这表明贝加尔湖海豹中的PPARα - CYP4A信号通路可能是保守的。本研究还利用非洲绿猴肾CV-1细胞瞬时转染贝加尔湖海豹PPARα 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，开发了一种体外PPARα报告基因检测方法。体外报告基因检测对氯贝丁酯有显著反应，氯贝丁酯是人类和啮齿动物中已知的PPARα激动剂。用全氟辛酸（PFOA）、全氟壬酸（PFNA）、全氟癸酸（PFDA）、全氟十一酸（PFUnDA）或全氟辛烷磺酸（PFOS）处理以剂量依赖的方式诱导PPARα介导的转录活性，最低观察到效应浓度分别为62.5、125、125、62.5和125 μM。在野生贝加尔湖海豹的肝脏中，PPARα mRNA的表达水平与PFNA水平呈显著正相关。此外，肝脏CYP4A样蛋白的表达与PFNA和PFDA的肝脏浓度显著相关。这些结果表明，PFCs在野生贝加尔湖海豹中对PPARα - CYP4A信号通路有调节作用。我们的研究表明，PPARα介导的反应可能是评估PFCs对野生动物潜在生物学效应的有用生物标志物。
