Zaja Roko, Munić Vesna, Klobucar Roberta Sauerborn, Ambriović-Ristov Andreja, Smital Tvrtko
Laboratory for Molecular Ecotoxicology, Division for Marine and Environmental Research, Ruder Bosković Institute, Bijenicka 54, 10 000 Zagreb, Croatia.
Aquat Toxicol. 2008 Dec 11;90(4):322-32. doi: 10.1016/j.aquatox.2008.09.012. Epub 2008 Oct 4.
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.
鱼类具有与高等脊椎动物类似的外源性（内源性）生物及其代谢产物的胆汁排泄机制，极化细胞顶端膜中表达的各种类型的ABC外排蛋白似乎是这种向量运输的关键介质。为了验证这一假设，本研究的主要目标是鉴定和克隆编码不同类型ABC转运蛋白的基因，测定基因转录本（mRNA）水平，并表征原代培养的虹鳟（Oncorhynchus mykiss）肝细胞中相关的蛋白质转运活性。我们克隆了一个部分基因序列和两个完整基因序列，它们与哺乳动物的Pgp1（ABCB1）、BSEP（ABCB11）和MRP2（ABCC2）外排转运蛋白具有高度的同源性。使用实时RT-PCR测定了这些基因的mRNA表达水平。在肝脏和原代肝细胞中均观察到所鉴定的外排转运蛋白（BSEP>>MRP2>Pgp1）具有相同的相对表达模式，与完整肝脏相比，原代肝细胞中所有三种转运蛋白mRNA的表达水平大约低3-4倍。此外，使用推定的特异性荧光底物（罗丹明123、钙黄绿素-AM、硼替佐米-维拉帕米和二氢荧光素二乙酸酯）、模型抑制剂（维拉帕米、环孢素A、MK571、瑞弗西丁205、牛磺胆酸盐和牛磺鹅去氧胆酸盐）及其组合，表明存在Pgp1、BSEP和MRP样转运活性。综合本研究结果表明，原代鳟鱼肝细胞表达解毒途径的关键成分——I相和II相酶，以及参与外源性物质转运的ABC蛋白，证实了这种体外模型是（生态）毒理学研究中有前景的工具。
