title: Detection and functional characterization of Pgp1 (ABCB1) and MRP3 (ABCC3) efflux transporters in the PLHC-1 fish hepatoma cell line.
abstract: The PLHC-1 hepatoma cell line derived from topminnow (Poeciliopsis lucida) is one of the most frequently used fish cell lines in aquatic ecotoxicology. These cells have been well characterized regarding the presence of phase I and phase II enzymes involved in the metabolism of xenobiotics. However, the presence of the ABC transport proteins possibly involved in the MultiXenobiotic Resistance (MXR) mechanism as phase III of cellular detoxification has never been described in the PLHC-1 cells. The main goal of this study was the detection and functional characterization of toxicologically relevant xenobiotic efflux transporters from ABCB and ABCC subfamily in the PLHC-1 cells. Using specific primer pairs two PCR products 1769 and 1023bp in length were successfully cloned and sequenced. Subsequent multiple alignment and phylogenetic analysis showed that these sequences share a high degree of homology with the P-glycoprotein (Pgp1; ABCB1) and the MRP3 (ABCC3). Functional experiments with fluorescent model substrates and specific inhibitors were used to verify that transport activities of Pgp- and MRP-related proteins are indeed present in PLHC-1 cells. Accumulation or efflux/retention rates of rhodamine 123, calcein-AM or monochlorbimane were time- and concentration-dependent. Cyclosporine A, MK571, verapamil, reversine 205, indomethacine and probenecid were used as specific inhibitors of Pgp1 and/or MRPs transport activities, resulting in a dose dependent inhibition of related transport activities in PLHC-1 cells. Similar to mammalian systems, the obtained IC(50) values were in the lower micromolar range. Taken together these data demonstrate that: (1) the PLHC-1 cells do express a functional MXR mechanism mediated by toxicologically relevant ABC efflux transporters; and (2) the presence of all three critical phases of cellular detoxification additionally affirms the PLHC-1 cells as a reliable in vitro model in aquatic toxicology.
authors.affiliation: Laboratory for Molecular Ecotoxicology, Division for Marine and Environmental Research, Ruder Bosković Institute, Bijenicka 54, 10000 Zagreb, Croatia.
authors.forename: Roko
authors.initials: R
authors.lastname: Zaja
authors.forename: Roberta Sauerborn
authors.initials: RS
authors.lastname: Klobucar
authors.forename: Tvrtko
authors.initials: T
authors.lastname: Smital
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000235:genetics*; D000595:Amino Acid Sequence; D000704:Analysis of Variance; D000818:Animals; D001483:Base Sequence; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D016000:Cluster Analysis; D017931:DNA Primers; D005399:Fishes / Q000235:genetics*; D005452:Fluoresceins / Q000493:pharmacokinetics; D008969:Molecular Sequence Data; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000235:genetics*; D010802:Phylogeny; D020112:Rhodamine 123 / Q000493:pharmacokinetics; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013997:Time Factors
chemical_list: D018528:ATP-Binding Cassette Transporters; D017931:DNA Primers; D005452:Fluoresceins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D020112:Rhodamine 123; C085925:calcein AM; C114462:multidrug resistance-associated protein 3