Cloning and molecular characterization of apical efflux transporters (ABCB1, ABCB11 and ABCC2) in rainbow trout (Oncorhynchus mykiss) hepatocytes.
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.
Zaja
Roko
R
Laboratory for Molecular Ecotoxicology, Division for Marine and Environmental Research, Ruder Bosković Institute, Bijenicka 54, 10 000 Zagreb, Croatia.
Munić
Vesna
V
Klobucar
Roberta Sauerborn
RS
Ambriović-Ristov
Andreja
A
Smital
Tvrtko
T
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D018528:ATP-Binding Cassette Transporters / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002478:Cells, Cultured; D003001:Cloning, Molecular; D029941:Fish Proteins / Q000235:genetics* / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects; D022781:Hepatocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D008969:Molecular Sequence Data; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D017686:Oncorhynchus mykiss / Q000235:genetics* / Q000378:metabolism*; D010802:Phylogeny; D012689:Sequence Homology, Nucleic Acid
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D018528:ATP-Binding Cassette Transporters; D029941:Fish Proteins; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins