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)

Abstract

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 m...

Toxicological Assessment of Polychlorinated Biphenyls and Their Metabolites in the Liver of Baikal Seal (Pusa sibirica)

“…49 Our previous study found that CAR mRNA expression was abundant in the liver of the Baikal seal. 25 In addition, our in vitro assay demonstrated that Baikal seal CAR was activated by treatment with PCBs. 25,26 These results imply that exposure to OH-PCBs may alter 5′-deiodinase activities via CAR and/or via production of ROS, leading to low serum T 3 /rT 3 ratios in this species.…”

“…25 In addition, our in vitro assay demonstrated that Baikal seal CAR was activated by treatment with PCBs. 25,26 These results imply that exposure to OH-PCBs may alter 5′-deiodinase activities via CAR and/or via production of ROS, leading to low serum T 3 /rT 3 ratios in this species. In addition, it is possible that T 3 -regulated gene expression in the Baikal seals might be repressed by increased rT 3 levels, as observed in mice.…”

DDT, Chlordane, Toxaphene and PCB Residues in Newport Bay and Watershed: Assessment of Hazard to Wildlife and Human Health

“…The authors point out that levels in nursing females were much lower than those in male adults, due to lactation transfer of DDT from mother to newborns. Sakai et al ( 2006 ) reported on an assay for androstane receptor activity in Russian seals. The authors concluded that DDT was active in their assay and that the lowest observable effect level was comparable to a 10 ppm tissue level.…”

Non-coplanar polychlorinated biphenyls (PCBs) are direct agonists for the human pregnane-X receptor and constitutive androstane receptor, and activate target gene expression in a tissue-specific manner

“…PCB153 has been shown to also be an agonist for the PXR paralogue CAR (Sakai et al, 2006), although again this was only evident at relatively high concentrations, with significant activations occurring above 20ppm. Whilst our current data may initially appear contradictory to the suggestion that non-coplanar PCBs act as inhibitors of human PXRmediated transcriptional events.…”