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 gene expression of a series of eight ABC efflux transporters constitutively expressed in PLHC-1 wild type and PLHC-1/dox cells. Gene expression analysis confirmed high and specific P-gp overexpression in PLHC-1/dox cells, enabling development of the ATPase assay. Using this model we determined KmATP of 0.4 mM, baseline ATPase activity from 35 – 50 nmol/mgPROT/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 – 5 compounds specifically interacted only with fish P-gp, while 7 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.

Biology,Earth science