Cloning and molecular characterization of apical efflux transporters (ABCB1, ABCB11 and ABCC2) in rainbow trout ( Oncorhynchus mykiss) hepatocytes

• Published in: Aquatic toxicology 2008-12, Vol.90 (4), p.322-332
• Main Authors: Zaja, Roko, Munić, Vesna, Klobučar, Roberta Sauerborn, Ambriović-Ristov, Andreja, Smital, Tvrtko
• Format: Article
• Language: English
• Subjects: ABC genes; ABC transporters; Animal, plant and microbial ecology; Animals; Applied ecology; ATP-Binding Cassette Transporters - antagonists & inhibitors; ATP-Binding Cassette Transporters - genetics; ATP-Binding Cassette Transporters - metabolism; ATP-Binding Cassette, Sub-Family B, Member 1 - antagonists & inhibitors; ATP-Binding Cassette, Sub-Family B, Member 1 - genetics; ATP-Binding Cassette, Sub-Family B, Member 1 - metabolism; Biological and medical sciences; Cells, Cultured; Cloning, Molecular; Ecotoxicology, biological effects of pollution; Fish Proteins - genetics; Fish Proteins - metabolism; Fundamental and applied biological sciences. Psychology; gene expression; Gene Expression Regulation - drug effects; General aspects; genes; hepatocytes; Hepatocytes - cytology; Hepatocytes - drug effects; Hepatocytes - metabolism; immunochemistry; liver; metabolic detoxification; molecular cloning; Molecular Sequence Data; Multidrug Resistance-Associated Proteins - antagonists & inhibitors; Multidrug Resistance-Associated Proteins - genetics; Multidrug Resistance-Associated Proteins - metabolism; Oncorhynchus mykiss; Oncorhynchus mykiss - genetics; Oncorhynchus mykiss - metabolism; Phylogeny; polymerase chain reaction; Real-time RT-PCR; Sequence Homology, Nucleic Acid; trout; Trout hepatocytes
• ISSN: 0166-445X; 1879-1514
• DOI: 10.1016/j.aquatox.2008.09.012

Fish possess similar mechanisms of billiary excretion of xeno(endo)biotics and their metabolites as found in higher vertebrates and various types of ABC efflux proteins expressed in apical membranes of polarized cells appears to be key mediators of this vectorial transport. To test this hypothesis the main goals of this study were identification and cloning of genes coding for different types of ABC transport proteins, determination of the gene transcript (mRNA) levels, and characterization of the related protein transport activities in primary cultured rainbow trout ( Oncorhynchus mykiss) hepatocytes. We have cloned one partial and two full gene sequences, which show high degree of identity with mammalian Pgp1 (ABCB1), BSEP (ABCB11) and MRP2 (ABCC2) efflux transporters. Using real-time RT-PCR expression levels of the mRNA of these genes were determined. Identical relative expression patterns of identified efflux transporters (BSEP ≫ MRP2 > Pgp1) were observed for both liver and primary hepatocytes, with expression of all three transporter mRNAs approximately 3–4-fold lower in primary hepatocytes in comparison to intact liver. In addition, the presence of Pgp1-, BSEP- and MRP-like transport activities were indicated using putative specific fluorescent substrates (rhodamine 123, calcein-AM, bodipy-verapamil and dihydrofluorescein diacetat), model inhibitors (verapamil, cyclosporine A, MK571, reversine 205, taurocholate and taurochenodeoxycholate) and their combinations. Taken together the results of this study showed that primary trout hepatocytes express critical components of detoxification pathways—phase I and II enzymes, as well as the ABC proteins involved in transport of xenobiotics, affirming this in vitro model as a promising tool in (eco)toxicological research.