Contamination and Effects of Perfluorochemicals in Baikal Seal (Pusa sibirica). 2. Molecular Characterization, Expression Level, and Transcriptional Activation of Peroxisome Proliferator-Activated Receptor α

Abstract

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.