Zaja Roko, Klobucar Roberta Sauerborn, Smital Tvrtko
Laboratory for Molecular Ecotoxicology, Division for Marine and Environmental Research, Ruder Bosković Institute, Bijenicka 54, 10000 Zagreb, Croatia.
Aquat Toxicol. 2007 Mar 30;81(4):365-76. doi: 10.1016/j.aquatox.2006.12.015. Epub 2007 Jan 14.
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.
源自食蚊鱼（Poeciliopsis lucida）的PLHC-1肝癌细胞系是水生生态毒理学中最常用的鱼类细胞系之一。这些细胞在参与外源性物质代谢的I相和II相酶的存在方面已得到充分表征。然而，作为细胞解毒第三阶段的、可能参与多外源性物质抗性（MXR）机制的ABC转运蛋白在PLHC-1细胞中的存在情况从未被描述过。本研究的主要目标是检测PLHC-1细胞中ABCB和ABCC亚家族的毒理学相关外源性物质流出转运蛋白并对其进行功能表征。使用特异性引物对成功克隆并测序了两条长度分别为1769和1023bp的PCR产物。随后的多重比对和系统发育分析表明，这些序列与P-糖蛋白（Pgp1；ABCB1）和多药耐药相关蛋白3（MRP3；ABCC3）具有高度同源性。使用荧光模型底物和特异性抑制剂进行的功能实验用于验证PLHC-1细胞中确实存在Pgp和MRP相关蛋白的转运活性。罗丹明123、钙黄绿素-AM或单氯联苯胺的积累或流出/保留率呈时间和浓度依赖性。环孢素A、MK571、维拉帕米、瑞弗西丁205、吲哚美辛和丙磺舒用作Pgp1和/或MRPs转运活性的特异性抑制剂，导致PLHC-1细胞中相关转运活性呈剂量依赖性抑制。与哺乳动物系统类似，获得的IC50值处于低微摩尔范围内。综上所述，这些数据表明：（1）PLHC-1细胞确实表达由毒理学相关ABC流出转运蛋白介导的功能性MXR机制；（2）细胞解毒所有三个关键阶段的存在进一步证实PLHC-1细胞是水生毒理学中可靠的体外模型。
