Aquatic Toxicology 101 (2011) 438–446 Contents lists available at ScienceDirect Aquatic Toxicology journal homepage: www.elsevier.com/locate/aquatox Constitutive mRNA expression and protein activity levels of nine ABC efflux transporters in seven permanent cell lines derived from different tissues of rainbow trout (Oncorhynchus mykiss) Stephan Fischer a,b,c , Jovica Loncar d , Roko Zaja d , Sabine Schnell e , Kristin Schirmer b,c,f , Tvrtko Smital d , Till Luckenbach a,∗ a Department of Bioanalytical Ecotoxicology, UFZ – Helmholtz Centre for Environmental Research, 04318 Leipzig, Germany b Eawag, Swiss Federal Institute of Aquatic Science and Technology, 8600 Dübendorf, Switzerland c ETH Zürich, Institute of Biogeochemistry and Pollutant Dynamics, 8092 Zürich, Switzerland d Rud ¯er Boˇ skovi´ c Institute, Laboratory for Molecular Ecotoxicology, Division for Marine and Environmental Research, Bijeniˇ cka 54, 10001 Zagreb, Croatia e Environmental Chemistry Department, IDAEA-CSIC, Barcelona, Spain f EPF Lausanne, School of Architecture, Civil and Environmental Engineering, 1015 Lausanne, Switzerland article info Article history: Received 18 September 2010 Received in revised form 11 November 2010 Accepted 20 November 2010 Keywords: ABC efflux transporters Multixenobiotic resistance MXR Permanent rainbow trout cell lines Constitutive mRNA expression Efflux transporter activity Oncorhynchus mykiss abstract Permanent fish cell lines have become common model systems for determining ecotoxicological effects of pollutants. For these cell lines little is known on the cellular active transport mechanisms that con- trol the amount of a compound entering the cell, such as the MXR (multixenobiotic resistance) system mediated by ATP binding cassette (ABC) transport proteins. Therefore, for toxic evaluation of chemi- cals with those cells information on MXR is important. We here present data on constitutive mRNA expression and protein activity levels of a series of ABC efflux transporters in seven permanent cell lines derived from liver (RTL-W1; R1) and liver hepatoma (RTH-149), gill (RTgill-W1), gonad (RTG-2), gut (RTgutGC) and brain (RTbrain) of rainbow trout (Oncorhynchus mykiss). In addition to known trans- porters abcb1 (designated here abcb1a), abcb11, abcc1-3, abcc5 and abcg2, we quantified expression levels of a newly identified abcb1 isoform (abcb1b) and abcc4, previously unknown in trout. Quantitative real time PCR (qPCR) indicated that mRNA of the examined ABC transporters was constitutively expressed in all cell lines. Transporter mRNA expression patterns were similar in all cell lines, with expression levels of abcc transporters being 80 to over 1000 fold higher than for abcg2, abcb1a/b and abcb11 (abcc1- 5 > abcg2 > abcb1a/b, 11). Transporter activity in the cell lines was determined by measuring uptake of transporter type specific fluorescent substrates in the presence of activity inhibitors. The combination of the ABCB1 and ABCC transporter substrate calcein-AM with inhibitors cyclosporine A, PSC833 and MK571 resulted in a concentration-dependent fluorescence increase of up to 3-fold, whereas reversin 205 caused a slight, but not concentration-dependent fluorescence increase. Accumulation of the dyes Hoechst 33342 and 2 ′ ,7 ′ -dichlorodihydrofluorescein diacetate was basically unchanged in the presence of Ko134 and taurocholate, respectively, indicating low Abcg2 and Abcb11 activities, in accordance with low abcg2 and abcb11 transcript levels. Our data indicate that transporter expression and activity pat- terns in the different trout cell lines are irrespective of the tissue of origin, but are determined by factors of cell cultivation. © 2010 Elsevier B.V. All rights reserved. 1. Introduction ATP binding cassette (ABC) transporters represent one impor- tant line of cellular defence (Gottesman and Pastan, 1988; Kurelec, 1992; Leslie et al., 2005). These transporters pump a broad array of chemical compounds against a concentration gradient in an active, ATP dependent process out of the cell, mediating the so-called ∗ Corresponding author at: Department of Bioanalytical Ecotoxicology, UFZ – Helmholtz Centre for Environmental Research, 04318 Leipzig, Germany. E-mail address: till.luckenbach@ufz.de (T. Luckenbach). multixenobiotic resistance (MXR) phenotype, equivalent to the multidrug resistance (MDR) of cancer cells and pathogens in a clinical context (Kurelec, 1992). The efflux pumps act as a “first line of defence” by preventing chemicals from entering the cell (phase 0 of cellular detoxification) and pump out phase 1 and phase 2 metabolites, such as oxidized molecules and glutathione, glucuronic or sulfate water soluble conjugates (phase 3). Conse- quently, this efflux activity controls the amount of toxicants in the cells (Sarkadi et al., 2006). In humans, 49 ABC transporters have been identified, which, based on sequence homology and protein organisation, are divided into seven sub-families designated ABCA–ABCG (Dean et al., 2001). 0166-445X/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.aquatox.2010.11.010