Laboratory for Molecular Ecotoxicology, Division for Marine and Environmental Research, Ruđer Bošković Institute, Bijenička 54, 10 000, Zagreb, Croatia.
Aquat Toxicol. 2011 May;103(1-2):53-62. doi: 10.1016/j.aquatox.2011.02.005. Epub 2011 Feb 15.
P-glycoprotein (P-gp; abcb1) is one of the major ABC transport proteins that mediates multixenobiotic resistance (MXR) defense in fish. In order to offer a sound evaluation of its ecotoxicological relevance it is critical to characterize substrate specificity of fish P-gp. Measurement of the ATPase activity is a reliable approach often used to discern type of interaction of various drugs with mammalian P-gp. A similar assay has never been used for characterization of P-gp in aquatic organisms and the main goal of this study was to develop a specific ATPase assay for characterization of fish P-gp. For this purpose we have used P-gp enriched membrane vesicles isolated from fish hepatoma PLHC-1/dox cells characterized by high overexpression of P-gp. As additional demonstration of a P-gp specific phenotype, we have quantified transcript expression of a series of eight ABC efflux transporter genes constitutively expressed in PLHC-1 wild type and PLHC-1/dox cells. Transcript expression analysis confirmed high and specific P-gp transcript overexpression in PLHC-1/dox cells. Provided that the transcript abundance is translated to protein, the development of ATPase assay is enabled. Using this model we determined Km(ATP) of 0.4mM, baseline ATPase activity from 35-50nmol/mg(PROT)/min, and maximal activation of ATPase activity obtained for fish P-gp in our system was 1.8-2.5-fold over baseline. All these values were in good agreement with data previously reported for mammalian P-gp. In order to perform a more detailed characterization of fish P-gp substrate specificity, in the next step of our study we used the developed ATPase assay to test 50 different compounds for their interaction with fish P-gp. The same set of compounds was also tested with calcein-AM (Ca-AM) transport activity assay both using PLHC-1/dox cells and NIH 3T3/MDR1 fibroblast cells overexpressing human P-gp. Our results showed that there is a clear difference for some substances-five compounds specifically interacted only with fish P-gp, while seven compounds exhibited interaction with human P-gp only. Most of the compounds tested in this study showed similar behavior in respect to fish or human P-gp and relatively high correlation in the interaction potency was found between fish and human P-gp. In summary, the described results represent the first in depth insight into substrate specificity of an important xenobiotic efflux transporter in fish. In addition, our study showed that combination of Ca-AM assay and the developed ATPase assay using inside/out vesicles isolated from PLHC-1/dox cells, offers a high-throughput and reliable approach for identification of environmentally relevant pollutants that interact with fish P-gp.
P-糖蛋白（P-gp；abcb1）是介导鱼类多药耐药性（MXR）防御的主要 ABC 转运蛋白之一。为了对其生态毒理学相关性进行合理评估，必须对鱼类 P-gp 的底物特异性进行特征描述。测量 ATP 酶活性是一种常用的方法，用于区分各种药物与哺乳动物 P-gp 的相互作用类型。类似的测定方法从未用于水生生物中 P-gp 的特征描述，本研究的主要目标是开发一种用于鱼类 P-gp 特征描述的特异性 ATP 酶测定方法。为此，我们使用了从 PLHC-1/dox 细胞中分离的富含 P-gp 的膜囊泡进行研究，这些囊泡具有 P-gp 的高过表达特征。作为 P-gp 特异性表型的额外证明，我们定量测定了在 PLHC-1 野生型和 PLHC-1/dox 细胞中组成性表达的一系列 8 种 ABC 外排转运蛋白基因的转录表达。转录表达分析证实了 PLHC-1/dox 细胞中 P-gp 的高特异性转录过度表达。如果转录丰度可转化为蛋白质，则可以进行 ATP 酶测定。使用该模型，我们确定了鱼类 P-gp 的 Km（ATP）为 0.4mM，基础 ATP 酶活性为 35-50nmol/mg（PROT）/min，并且在我们的系统中，鱼类 P-gp 的 ATP 酶活性最大激活为基础水平的 1.8-2.5 倍。所有这些值与以前报道的哺乳动物 P-gp 的数据非常吻合。为了更详细地描述鱼类 P-gp 的底物特异性，在我们研究的下一步中，我们使用开发的 ATP 酶测定法测试了 50 种不同的化合物与鱼类 P-gp 的相互作用。使用 PLHC-1/dox 细胞和过表达人 P-gp 的 NIH 3T3/MDR1 成纤维细胞，还使用相同的化合物组进行了 calcein-AM（Ca-AM）转运活性测定。我们的结果表明，对于某些物质，存在明显差异-五种化合物仅与鱼类 P-gp 特异性相互作用，而七种化合物仅与人 P-gp 相互作用。在这项研究中测试的大多数化合物在鱼类或人类 P-gp 方面表现出相似的行为，并且在鱼类和人类 P-gp 之间发现了相互作用效力的相对高相关性。总之，这些描述的结果代表了对鱼类中重要的外源性 efflux 转运蛋白的底物特异性的深入了解。此外，我们的研究表明，使用从 PLHC-1/dox 细胞中分离的内外囊泡组合 Ca-AM 测定法和开发的 ATP 酶测定法，为鉴定与鱼类 P-gp 相互作用的具有环境相关性的污染物提供了一种高通量且可靠的方法。
