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1 2 CBP ms.23122 Revised – part C 3 Review. 4 Current advances on ABC drug transporters in fish 5 6 Till Luckenbacha, Stephan Fischerb,c, Armin Sturmd* 7 8 a 9 Research, 04318 Leipzig, Germany Department of Bioanalytical Ecotoxicology, UFZ – Helmholtz Centre for Environmental 10 b 11 and Technology, 8600 Dübendorf, Switzerland 12 c 13 Pollutant Dynamics, 8092 Zürich, Switzerland 14 d 15 Scotland, UK Department of Environmental Toxicology, Eawag, Swiss Federal Institute of Aquatic Science Department of Environmental Systems Sciences, ETH Zürich, Institute of Biogeochemistry and Institute of Aquaculture, School of Natural Sciences, University of Stirling, Stirling FK9 4LA, 16 17 Running title: ABC drug transporters in fish 18 19 * Corresponding author: 20 Dr. Armin Sturm 21 Institute of Aquaculture 22 School of Natural Sciences 23 University of Stirling 24 Stirling, FK9 4LA 25 Scotland, UK 26 Tel. ++44 1786 46 7898; Fax ++44 1786 47 2133 27 [email protected] 28 29 30 31 32 33 34 35 36 37 38 1 Introduction 1.1 Teleosts and other fishes 1.2 The ABC protein family 1.3 ABC drug transporters 2. Genetic evidence for ABC Drug Transporters in fish 2.1 ABCB/Abcb subfamily 2.2 ABCC/Abcc subfamily 2.3 ABCG/Abcg subfamily 3. Functional activity and expression of ABC drug transporters in fish 3.1 Kidney 1 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 3.2 Liver 3.3 Intestine 3.4 Blood-brain barrier and blood-cerebrospinal fluid barrier 3.5 Other tissues (gills, gonads, skin) 3.6 Fish embryos 4. Substrates and inhibitors of teleost ABC transporters 4.1 Results for primary cell culture systems 4.2 Results for cell lines 4.3 Results with ATP-ase assays 4.4 Metabolic costs of chemical interaction with ABC transporters 5. Regulation of teleost ABC drug transporters 5.1 Regulation of ABC transporters in cancer cells 5.2 Regulation of ABC transporters as part of a general cellular stress response 5.3 Roles of nuclear receptors in ABC transporter regulation 5.4 Hormonal effects on ABC transporters 5.5 Chemical effects on ABC transporter expression in fish 5.6 Altered ABC transporter expression in fish from polluted habitats 6. Summary and conclusions 57 58 Abstract 59 Most members of the large ATP-binding cassette (ABC) gene family are transporters involved 60 in substrate translocation across biological membranes. In eukaryotes, ABC proteins functioning 61 as drug transporters are located in the plasma membrane and mediate the cellular efflux of a 62 wide range of organic chemicals, with some transporters also transporting certain metals. As the 63 enhanced expression of ABC drug transporters can confer multidrug resistance (MDR) to 64 cancers and multixenobiotic resistance (MXR) to organisms from polluted habitats, these ABC 65 family members are also referred to as MDR or MXR proteins. In mammals, ABC drug 66 transporters show predominant expression in tissues involved in excretion or constituting 67 internal or external body boundaries, where they facilitate the excretion of chemicals and their 68 metabolites, and limit chemical uptake and penetration into ‘sanctuary’ sites of the body. 69 Available knowledge about ABC proteins is still limited in teleost fish, a large vertebrate group 70 of high ecological and economic importance. Using transport activity measurements and 71 immunochemical approaches, early studies demonstrated similarities in the tissue distribution of 72 ABC drug transporters between teleosts and mammals, suggesting conserved roles of the 73 transporters in the biochemical defence against toxicants. Recently, the availability of teleost 74 genome assemblies has stimulated studies of the ABC family in this taxon. This review 75 summarises the current knowledge regarding the genetics, functional properties, physiological 76 function, and ecotoxicological relevance of teleostean ABC transporters. The available literature 77 is reviewed with emphasis on recent studies addressing the tissue distribution, substrate 78 spectrum, regulation, physiological function and phylogenetic origin of teleostean ABC 2 79 transporters. 80 81 1. Introduction 82 83 When considering the interaction of organisms with the surrounding chemosphere, central 84 questions regard the mechanisms of chemical uptake and elimination, as well as that of chemical 85 distribution between different body compartments (Van Aubel et al., 2002). It is well 86 documented that biotransformation crucially affects chemical fate in fish (Schlenk et al., 2008). 87 In contrast, the impact of active transport across cellular membranes on chemical fate is still 88 incompletely understood in teleosts. While such transport mechanisms likely affect 89 bioaccumulation and toxicity of pollutants in fish (Nichols et al., 2007), an understanding of the 90 specific interactions of environmental chemicals with transport proteins and the ecotoxicological 91 relevance of such interactions is currently only beginning to emerge. 92 In eukaryotes, ABC (ATP binding cassette) proteins comprise one important group of 93 transporters controlling the transition of compounds across internal and external interfaces of 94 compartments of organisms. These proteins were first described as biochemical factors 95 conferring multidrug resistance (MDR) in cancer, i.e., the resistance of tumour cells against 96 structurally and functionally unrelated cytostatic drugs (Gros et al., 1986; Roninson et al., 1984). 97 These MDR conferring proteins are localised in the cell membrane and function as ATP- 98 dependent biochemical pumps mediating the cellular efflux of a diverse array of organic 99 chemicals and some metals (Gottesman et al., 2002). ABC drug transporters also show high 100 expression levels in normal tissues involved in excretion (e.g., kidney, liver) or acting as barriers 101 (gut epithelium, capillary endothelia forming the blood brain barrier) (Fojo et al., 1987; 102 Thiebaut et al., 1987). ABC drug transporters often localise to the apical side of polarised 103 epithelial cells, suggesting their role in limiting chemical uptake and enhancing chemical 104 elimination, thus contributing to the biochemical defence against toxicants (Leslie et al., 2005). 105 In support of such a role, animals lacking certain ABC drug transporters as the result of natural 106 or targeted mutations usually are healthy and viable, but can show marked hypersensitivity to 107 specific toxicants or mild pathophysiological changes reflecting the impaired excretion of 108 endogenous toxicants (Kruh et al., 2007; Lagas et al., 2009; Schinkel et al., 1995, 1994; 109 Wijnholds et al., 1997). Kurelec and co-workers were the first to report the induction of ABC 110 transporters in marine invertebrate populations from polluted habitats (Kurelec and Pivcevic, 111 1991; Kurelec et al., 1995). In analogy to the phenomenon of MDR in cancer cells, Kurelec and 112 colleagues coined the term “multixenobiotic resistance (MXR) proteins” for ABC drug efflux 113 transporters in aquatic animals, reflecting the role of the cellular pumps as protective factors 3 114 against pollutant toxicity (Kurelec and Pivcevic, 1991; Kurelec and Pivčević, 1989; Kurelec, 115 1992). While the term “MXR proteins” is well established in aquatic toxicology, the present 116 review will employ the more general term “ABC drug transporters” in order to avoid using 117 different terminologies when referring to aquatic and terrestrial animal models. 118 First evidence for the ABC drug transporter Abcb11 (P-glycoprotein) in teleost was provided by 119 a molecular genetic study in winter flounder (Pleuronectes americanus) (Chan, 1992). The 120 presence of Abcb1-like proteins in fish was subsequently confirmed in an immunohistochemical 121 study in guppy (Poecilia reticulata) (Hemmer et al., 1995). Moreover, P-glycoprotein-like 122 transport activities were measured in isolated proximal tubules from winter flounder and 123 killifish (Fundulus heteroclitus) (Miller, 1995; Schramm et al., 1995; Sussman-Turner and 124 Renfro, 1995). Subsequently, ABC drug transporters have been studied in a number of tissues of 125 different teleosts, using immunochemical detection (Hemmer et al., 1998; Kleinow et al., 2000; 126 Cooper et al., 1999; Albertus and Laine 2001) and transport assays (Sturm et al. 2001; Doi et al., 127 2001; Miller et al. 2002). Recently, cDNA sequences have been obtained for various ABC drug 128 transporters (Costa et al., 2012; Diaz de Cerio et al., 2012; Fischer et al., 2013, 2011, 2010; 129 Loncar et al., 2010; Paetzold et al., 2009; Popovic et al., 2010; Sauerborn Klobučar et al., 2010; 130 Zaja et al., 2008b), and the ABC gene family has been annotated and analysed phylogenetically 131 in zebrafish (Danio rerio) and channel catfish (Ictalurus punctatus) (Annilo et al., 2006; Liu et 132 al., 2013). Moreover, a teleost cell line showing enhanced expression of a specific teleost ABC 133 drug transporter has been generated, which enables the identification of environmentally 134 relevant compounds that interact with this efflux pump (Caminada et al., 2008; Smital et al., 135 2011; Zaja et al., 2011, 2008a). 136 The aim of the present review article is to summarise recent insights into ABC transporters in 137 teleost fish, focusing on data that have become available since earlier reviews on the subject 138 (Bard 2000; Sturm and Segner 2005). Molecular, physiological and in vitro studies on ABC 139 transporters are reviewed focusing on teleosts, but also taking into account studies on 140 elasmobranchs where available. Since the annotation of ABC drug transporters in zebrafish 141 (Annilo et al., 2006), genome assemblies have become available in further teleost species, 142 allowing to draw a more complete picture of the complement of ABC drug transporters present 143 in this economically and ecologically important vertebrate taxon. To this end, evolutionary 144 relationships between ABC drug efflux transporters from selected tetrapods and the presently 145 seven teleost species with available genome assemblies are presented. 1 our designation of gene and protein names is based on the Zebrafish Nomenclature Guidelines (http://zfin.org/zf info/nomen.html); fish: shh/Shh, human: SHH/SHH, mouse: Shh/SHH (gene/protein) 4 146 147 1.1 Teleosts and other fishes 148 “Fish” have been defined as aquatic vertebrates having gills and fin-shaped limbs (Nelson, 149 2006). This definition comprises more than half of the extant vertebrates, and includes taxa as 150 diverse as jawless (Agnatha), cartilaginous (Chondrichthyes) and bony fishes (Osteichthyes) 151 (Helfman et al., 2009). The first fossil evidence for the Chondrichthyes, which include the 152 elasmobranchs (sharks and rays), dates from the early Devonian (418 mya). Osteichthyes are 153 divided into actinopterygians (ray-finned fish) and sarcopterygians (lobe-finned fish), with the 154 latter division containing the coelancanths, lungfish and tetrapods (Bone and Moore, 2008). The 155 split between actinopterygians and sarcopterygians has been recently dated to have taken place 156 before 419 mya (Zhu et al., 2009). The teleost fish (Teleostei) are the most advanced 157 actinopterygian division containing ~27,000 living species and account for the majority (~96%) 158 of all living fishes, including important fishery species (Helfman et al., 2009). Teleosts have 159 probably emerged in the Late Triassic (~200 mya) from a neopterygian ancestor (Bone and 160 Moore, 2008), and underwent four radiations, producing the osteoglossomorphs (bony tongues), 161 elopomorphs (tarpons and true eels), ostarioclupeomorphs (herrings and minnow relatives) and 162 finally the Euteleostii, which constitute the most advanced and species rich teleost subdivision 163 (Helfman et al., 2009). 164 Based on the observation that mammals often possess multiple copies of genes present only 165 once in invertebrates, it has been suggested that whole genome duplications played an important 166 role in vertebrate evolution (Ohno, 1970). For instance, mammals possess four clusters of Hox 167 genes whereas only one cluster is found in the primitive chordate Amphioxus (Garcia-Fernandez 168 and Holland, 1994). According to current models, the ancestral chordate genome has undergone 169 at least two rounds of duplication in the lineage leading to the jawed vertebrates (Aparicio, 170 2000; Escriva et al., 2002). While no further genome duplications appear to have occurred in the 171 sarcopterygian and Chondrichthyes lineages, a third whole genome duplication took place in a 172 common ancestor of extant teleosts (Amores et al., 1998; Jaillon et al., 2004; Steinke et al., 173 2006; Taylor et al., 2001). As a result teleost fish frequently possess duplicate copies of genes 174 present in mammals only once (Robinson-Rechavi et al., 2001). Complicating the situation 175 further, additional lineage-specific genome duplications have occurred in particular teleost 176 groups (Johnson et al., 1987). 177 Teleosts inhabit almost every imaginable marine or freshwater habitat and pursue a wide range 178 of trophic strategies, feeding on zooplankton, benthic invertebrates, other fishes, mammals, 179 carrion, detritus, phytoplankton, macroalgae or vascular plants (Helfman et al., 2009). In 180 comparison, elasmobranchs are typically large predators and the majority of species is marine 5 181 (Helfman et al., 2009). Ecotoxicological, physiological and/or genomic data are available only 182 for a limited number of teleosts and a few elasmobranchs, usually species that can be bred in 183 captivity or be obtained easily. The aim of the present article is to review the current knowledge 184 on ABC transporters in teleost fish. Where available, data from elasmobranchs are also 185 considered. While at points general patterns seem to emerge, it should be kept in mind that only 186 a small fraction of teleost species have been studied, and prudence is advised regarding 187 generalisations. 188 189 1.2 The ABC protein family 190 Members of the large ABC (ATP binding cassette) gene superfamily are characterised by 191 conserved nucleotide binding domains and are found in all biota (Higgins, 1992). Typical ABC 192 proteins are ATP-dependent transporters mediating the trafficking of diverse substrates across 193 biological membranes (Dean et al., 2001). Some ABC proteins function as drug transporters and 194 have central relevance in the biochemical defence against toxic chemicals (Schinkel and Jonker 195 2003; Leslie et al., 2005). Other ABC superfamily members are not transporters, but have roles 196 as ion channels, receptors or factors involved in transcription and translation (Dean et al., 2001). 197 The 48 human ABC transporters known to date are divided into seven sub-families characterised 198 by a high degree of sequence homology and designated ABCA/Abca to ABCG/Abcg (Dean et 199 al., 2001). In addition, some teleosts possess members of an eighth subfamily called Abch 200 initially identified in the fruitfly (Annilo et al., 2006; Dean et al., 2001; Popovic et al., 2010). 201 Systematic names for ABC transporters consist of the subfamily name (ABCA/Abca, 202 ABCB/Abcb, ABCC/Abcc, …) followed by a number denoting the protein (e.g., 203 ABCB1/Abcb1, ABCC1/Abcc1). In some cases a further letter is added to distinguish multiple 204 paralogues. In addition, earlier non-systematic names given upon first description of ABC 205 proteins are still common. ABC full transporters (FT) possess two nucleotide binding domains 206 (NBD) and two transmembrane domains (TMD) arranged in the N- to C-terminal order TMD1- 207 NBD1-TMD2-NBD2. ABC FTs are found in subfamilies A/a, B/b, and C/c, with some 208 ABCC/Abcc members showing an additional N-terminal TMD (termed TMD0) (Deeley et al., 209 2006). ABC half transporters (HT) have only one TMD and one NBD which are arranged in the 210 N- to C-terminal order TMD-NBD (subfamilies B/b and D/d) or NBD-TMD (subfamilies G/g 211 and h) (Dean et al., 2001). HTs are generally assumed to form homo- or heterodimers to 212 constitute a functional pump (Schinkel and Jonker, 2003). The members of subfamilies E/e and 213 F/f are not transporters and lack TMDs (Dean et al., 2001). 214 215 1.3 ABC drug transporters 6 216 The contribution of ABC drug transporters to the biochemical defence against toxicants is 217 illustrated in a schematic fashion in Figure 1. After the passive uptake of hydrophobic organic 218 chemicals by a hypothetical epithelial cell, a first line of defence is provided by ABC 219 transporters that pump out hydrophobic substrates (Fig. 1). Biotransformation enzymes 220 constitute a second level of defence by converting organic chemicals to products that are usually 221 less toxic (Fig. 1). Phase I biotransformation reactions involve the introduction of polar groups 222 into the chemical, while phase II reactions comprise conjugations with endogenous moieties 223 such as glutathione, sulfate or glucuronic acid. The products of biotransformation metabolism 224 are removed from the cell by drug transporters accepting less hydrophobic substrates, which 225 include other types of ABC proteins (Fig. 1). In polarised epithelia or endothelia, ABC drug 226 transporters generally show a predominantly apical localisation, resulting in directional transport 227 into excreta or away from sanctuary sites (Fig.1). 228 All eukaryotic ABC drug transporters are active efflux pumps, i.e., substrate translocation 229 occurs from the cytosol or the cell membrane to the extracellular space and is energetically 230 linked to the cleavage of ATP (Ambudkar and Cardarelli, 1997; Bouige et al., 2002; van Veen et 231 al., 2001). This enables ABC pumps to transport substrates against a concentration gradient. In 232 contrast, drug transporters of the solute carrier (SLC) superfamily depend on facilitated 233 diffusion, exchange or co-transport for transport (El-Sheikh et al., 2008; Oostendorp et al., 234 2009). While certain SLCs and ABC transporters can show overlaps in substrate specificity, 235 SLC proteins are beyond the scope of the present review. 236 Three ABC subfamilies, namely ABCB/Abcb, ABCC/Abcc and ABCG/Abcg, are known to 237 contain drug transporters, as well as proteins having roles unrelated to drug transport. In human, 238 the most important ABC drug efflux pumps are ABCB1 (also called MDR1 or P-glycoprotein), 239 ABCC1 (also known as the Multidrug resistance associated protein, MRP1), ABCC2 (also 240 known as MRP2 and the canalicular multispecific organic anion transporter cMOAT) and 241 ABCG2 (also known as the breast cancer resistance protein, BCRP) (Leslie et al., 2005; 242 Schinkel and Jonker, 2003). ABCB1 shows high levels of expression in hepatocytes, proximal 243 renal tubules, enterocytes and brain capillary endothelial cells, and adopts an apical localisation 244 in polarised cells (Ambudkar et al., 1999; Gottesman et al., 2002). ABCC members acting as 245 drug transporters are called multidrug resistance proteins (MRPs) (Slot et al., 2011; Deeley et 246 al., 2006). ABCC1 is found in many tissues, showing high levels in lung, testis, kidney, placenta 247 and skeletal and heart muscles (Deeley et al., 2006; Bakos and Homolya 2007). The localisation 248 of ABCC1 in polarised cells is variable, being basolateral in most cell types, but apical in others, 249 such as the capillary endothelial cells of the blood-brain barrier (Deeley et al., 2006; Bakos and 250 Homolya 2007). ABCC2 always shows an apical localisation in polarised cells and is 7 251 predominantly expressed in liver, kidney, small intestine, colon, gallbladder, placenta and lung 252 (Nies and Keppler, 2007). Compared to ABCC1 and ABCC2, less is known about the remaining 253 human MRPs. Data available to date suggest that ABCC3, 4, and 5 may contribute to the 254 biochemical defence against toxicants (Borst et al., 2007; Kruh et al., 2007; Lagas et al., 2009). 255 ABCC4 parallels ABCC1 in that its localisation in polarised cells depends on the cell type, 256 showing a basolateral expression in hepatocytes but an apical expression in renal proximal 257 tubules and endothelial cells of brain capillaries (Leggas et al., 2004; Van Aubel et al., 2002). 258 ABCG2 shows significant levels in liver, kidney and intestine, as well as in the blood-brain and 259 blood-placenta barriers, where it adopts an apical localisation (Robey et al., 2009; Vlaming et 260 al., 2009). 261 ABC drug transporters generally show a broad substrate selectivity, and substrate spectra 262 significantly overlap between among different types of ABC drug pumps (Leslie et al., 2005; 263 Schinkel and Jonker, 2003). In mammals, ABCB1 accepts a broad range of typically uncharged 264 or moderately basic amphipathic substrates (Ambudkar et al., 1999). Typical substrates of 265 ABCC subfamily transporters are organic anions and include chemical conjugates with 266 glutathione, glucuronic acid or sulphate produced in phase II biotransformation metabolism 267 (Deeley et al., 2006; Slot et al., 2011). Despite the low degree of sequence homology between 268 ABCB1 and ABCC1 (~19% amino acid identity) (Cole et al., 1992), MDR phenotypes 269 associated with ABCB1 and ABCC1 widely overlap, and include resistance to anthracyclins, 270 Vinca alkaloids and epipodophyllotoxins (Gottesman et al., 2002). However, ABCC1 differs 271 from ABCB1 in that it provides only very limited protection against taxanes and confers 272 resistance to metalloid oxyanions (Deeley et al., 2006). Both ABCC1 and ABCC2 transport free 273 glutathione (GSH), and GSH can stimulate the transport of other substrates by ABCC1 and 274 ABCC2 (Borst et al., 2006; Deeley and Cole, 2006). Moreover, ABCC1, ABCC2 and 275 homologous proteins from invertebrates can transport metals such as cadmium, mercury and 276 platinum, probably as complexes with GSH (Bosnjak et al., 2009; Bridges et al., 2008; Broeks et 277 al., 1996; Ishikawa et al., 1996). ABCC4 and 5 are able to transport cyclic nucleotides, as well 278 as drugs that are nucleoside and nucleotide analogues (Borst et al., 2007; Deeley et al., 2006). 279 ABCG2 overlaps in substrate selectivity with both ABCB1 and ABCCs (Krishnamurthy and 280 Schuetz, 2006; Robey et al., 2009). In addition, ABCG2 mediates the biliary excretion of 281 porphyrin precursors, limits the gut uptake of phototoxic chlorophyll breakdown products 282 (Jonker et al., 2002) and contributes to renal urate secretion (Woodward et al., 2011). 283 284 2. Genetic evidence for ABC drug transporters in fish 285 8 286 ABC transporters have previously been annotated from the zebrafish genome, the first fish 287 genome that was comprehensively sequenced (Annilo et al., 2006; Dean and Annilo, 2005). 288 Moreover, the ABC gene family has been analysed in a transcriptome of the catfish (Ictalurus 289 punctatus) generated by RNA-seq (Liu et al., 2013). The zebrafish genome contains members of 290 ABC subfamilies A to G known from tetrapods, as well as one transporter of subfamily H 291 (Annilo et al., 2006). While the presence of an abch gene has been also confirmed for Green 292 spotted puffer (Tetraodon nigroviridis), no member of this subfamily has been retrieved in 293 catfish (Liu et al., 2013). The identification of zebrafish homologues to human ABCB1, ABCC1- 294 5 and ABCG2 confirmed the presence of all major vertebrate ABC drug transporters in teleosts. 295 However, for some tetrapod ABC drug transporters, several isoforms were found in zebrafish, 296 complicating the assignment of function (Annilo et al., 2006). Since the annotation of zebrafish 297 ABC transporters (Annilo et al., 2006), assembled genome sequences have become available for 298 a number of further fish species. In order to base the discussion of the presence or absence of 299 specific ABC transporters in fish on the full range of currently available data, we consider here 300 ABC transporter sequences from seven actinopterygian species with available genome 301 assemblies (zebrafish Danio rerio, medaka Oryzias latipes, stickleback Gasterosteus aculeatus, 302 fugu Takifugu rubripes, green spotted puffer Tetraodon nigroviridis, cod Gadus morhua and 303 tilapia Oreochromis niloticus) and the sarcopterygian species coelacanth (Latimeria 304 chalumnae). Fish sequences of ABC subfamilies containing drug transporters were subjected to 305 phylogenetic analyses together with transporters from human and chicken. 306 307 2.1 ABCB/Abcb subfamily 308 The ABCB/Abcb subfamily contains both FTs and HTs, of which the latter locate to 309 intracellular membranes and have evolutionarily conserved physiological roles unrelated to drug 310 transport (Abele and Tampé, 2006; Burke and Ardehali, 2007; Herget and Tampé, 2007), with 311 evidence for drug transport existing only for ABCB/Abcb FTs (Gottesman et al., 2002; Leslie et 312 al., 2005). Therefore, our evolutionary analyses of vertebrate ABCB/Abcb proteins excluded 313 HTs, focusing on ABCB/Abcb FTs. 314 In the obtained tree, ABCB1/Abcb1 and ABCB4/Abcb4 sequences form a well-supported clade, 315 within which the teleost sequences group together in a subcluster of high bootstrap support, 316 opposing sarcopterygian ABCB1/Abcb1 and ABCB4/Abcb4 sequences (Fig. 2). Human 317 ABCB1 is known to constitute a drug efflux pump also known as MDR1 P-glycoprotein, 318 whereas human ABCB4 is a biliary phospholipid transporter (Ambudkar et al., 1999; Oude 319 Elferink and Paulusma, 2007). The topology of the tree suggests that human ABCB1 and 320 ABCB4 arose from a lineage-specific gene duplication and that the teleost sequences are, 9 321 despite their names, not one-to-one orthologues to either ABCB1 or ABCB4, but co-orthologues 322 to both (Fig. 2). All teleost species possess at least one transporter in the teleost Abcb1/Abcb4 323 clade, with individual sequences being named “Abcb1-like”, “Abcb4-like” or “Abcb4” by 324 automatic and/or synteny based annotation. The teleostean Abcb1/Abcb4 clade includes 325 zebrafish Abcb4 (Fischer et al., 2013), previously annotated as Abcb1b (Annilo et al., 2006), but 326 excludes zebrafish Abcb5, previously annotated as Abcb1a (Annilo et al., 2006; Fischer et al., 327 2013) (see below). Some teleost species, such as japanese pufferfish (Takifugu rubripes) and 328 green spotted pufferfish (Tetraodon nigroviridis), possess two P-glycoprotein genes that based 329 on synteny have been designated as Abcb1 and Abcb4 (Fischer et al., 2013) (Fig. 2). 330 An earlier study proposed that ABCB4 arose in the mammalian lineage, and that birds and 331 teleosts lack functional orthologues to ABCB4 (Annilo et al., 2006). Indeed, no phospholipids 332 are detectable in bile fluid from teleosts and elasmobranch fishes, indicating the lack of hepatic 333 ABCB4-like transport activity (Goto et al., 2003; Moschetta et al., 2005; Oude Elferink et al., 334 2004). Phosphatidylcholine translocation thus appears to be a specific, relatively recent function 335 of mammalian ABCB4, whereas the property of transport of a wide range of toxic compounds 336 by mammalian ABCB1 is more ancient. In support of this notion, it is well documented that 337 teleosts possess ABCB1-like transport activities (Miller, 1995; Schramm et al., 1995; Sussman- 338 Turner and Renfro, 1995), which have been shown to coincide with the expression of abcb1/4 339 (Fischer et al., 2013; Tutundjian et al., 2002; Zaja et al., 2008b). Recent studies provide insights 340 into the molecular identity of teleost ABCB1-like transporters. The characterisation of 341 topminnow (Poeciliopsis lucida) Abcb1 using a cell line overexpressing the transporter 342 demonstrated that this ABC pump constitutes a multidrug transporter (Zaja et al., 2011, 2008a). 343 Evidence for a similar function of zebrafish Abcb4 was obtained by morpholino knock-down 344 studies in embryos and recombinant expression studies (Fischer et al., 2013). 345 In the phylogenetic analysis of vertebrate ABCB/Abcb proteins (Fig. 2), ABCB/Abcb5 proteins 346 formed two clusters. The function of ABCB5, the most recently isolated human ABCB protein, 347 is still unclear (Frank and Frank, 2009; Frank et al., 2003). While an initial report found ABCB5 348 homologues to be lacking in zebrafish (Annilo et al., 2006), a later study proposed that the 349 zebrafish gene initially annotated as abcb1a is actually an ABCB5 orthologue, and suggested 350 renaming the gene abcb5 (Fischer et al., 2013). An abcb5 gene has further been reported from 351 catfish (Liu et al., 2013), but orthologues to ABCB5 appear to be absent in medaka, stickleback, 352 fugu, green spotted puffer, cod and tilapia (Fig. 2). While gene knockdown studies in embryos 353 did not provide evidence for a multidrug transporter function of the zebrafish Abcb5 ortholog 354 (Fischer et al., 2013), hepatic transcript expression of abcb5 is induced by the main zebrafish 355 bile acid cyprinol sulphate (Reschly et al., 2007), suggesting potential roles of Abcb5 related to 10 356 biliary excretion. On the other hand, mRNA expression of zebrafish abcb5 in embryo epidermal 357 cells (Thisse and Thisse, 2004) parallels epidermal ABCB5 expression in mammals where it has 358 been suggested that this protein regulates membrane potential and cell fusion of skin progenitor 359 cells (Frank et al., 2003). 360 All vertebrates considered in the evolutionary analysis of the ABCB/Abcb subfamily possess at 361 least one abcb11 gene (Fig. 2). Abcb11 was originally isolated in winter flounder (Chan, 1992) 362 and regarded a potential drug transporter. However, later studies showed that ABCB11/abcb11 363 encodes the hepatic bile salt export pump (BSEP) (Gerloff et al., 1998; Stieger et al., 2007). The 364 cloning and functional characterisation of Abcb11 in the elasmobranch Raja erinacea (Cai et al., 365 2001) demonstrated transport activity with taurocholate, suggesting the functional conservation 366 of Abcb11 across the vertebrates. 367 368 2.2 ABCC/Abcc subfamily 369 The ABCC/Abcc subfamily is large and complex, containing both transporters and proteins with 370 other roles. ABCC1 and ABCC2 are well studied drug efflux transporters (Leslie et al., 2005; 371 Schinkel and Jonker, 2003). While ABCC3 – 5 and ABCC10 and 11 are capable of drug 372 transport in vitro, little further evidence exists for roles of ABCC10 and 11 as factors in the 373 biochemical defence against toxicants (Deeley et al., 2006a; Slot et al., 2011). A number of 374 vertebrates possess a further MRP (Mrp10/Abcc13), which has undergone pseudogenisation in 375 human (Annilo and Dean, 2004; Annilo et al., 2006). ABCC proteins that are not transporters 376 include the cystic fibrosis transmembrane conductance regulator (CFTR, also called ABCC7), 377 which is a chloride channel, and the sulfonylurea receptors (SUR1, SUR2, also called ABCC8 378 and ABCC9, respectively), which are regulators of potassium channels (Riordan et al. 1989; 379 Aleksandrov et al., 2007; Hibino and Kurachi 2006; Bryan et al. 2007). Moreover, ABCC6 and 380 ABCC12 are likely not drug transporters (Slot et al., 2011). Loss-of-function mutations of 381 human ABCC6 are associated with a rare genetic disorder called PXE (pseudoxanthoma 382 elasticum) (Bergen et al., 2007). ABCC12 is a protein of unknown function expressed in 383 testicular germ cells and sperm (Ono et al., 2007). 384 An evolutionary analysis of the vertebrate ABCC/Abcc family was first carried out taking into 385 account all members (Fig. S1). In the obtained phylogenetic tree, ABCC/Abcc proteins grouped 386 into distinct clusters corresponding to individual isoforms (Fig. S1), suggesting that the 387 divergence of different ABCC/abcc paralogs is likely ancient and has occurred in a common 388 ancestor of vertebrates. All teleosts studied had at least one Abcc6, Abcc7 (CFTR), Abcc8 389 (SUR1) and Abcc9 (SUR2) member, and orthologues of each of these non-drug transporter 390 ABCCs/Abccs formed well supported clades (Fig. S1). A distinct clade with high bootstrap 11 391 support was also formed by ABCC10/Abcc10 members, whereas proteins labelled 392 ABCC11/Abcc11 and ABCC12/Abcc12 combined in one clade (Fig. S1). In order to obtain a 393 tree of manageable size, the analysis was re-run with the main drug-transporting ABCC/Abcc 394 isoforms, ABCC1-5/Abcc1-5 (Fig. 3). Each of the available teleost genomes contains one 395 ortholog of each Abcc1-3 and Abcc5, whereas in some teleost species there are multiple 396 isoforms of Abcc4 (Fig. 3). The occurrence of different numbers of isoforms in the different 397 teleost species raises questions about their specific functions, to which extent functions of the 398 isoforms differ and whether functions of certain isoforms are homologous across species. A 399 cDNA encoding an Abcc2 homologue was isolated from rainbow trout liver (Zaja et al., 2008b). 400 mRNA expression profiles of abcc isoforms in rainbow trout (Oncorhynchus mykiss) are 401 generally comparable to those of their counterparts in mammals (Loncar et al., 2010). 402 Previously, an Abcc2 homologue had been cloned from the elasmobranch small skate (Raja 403 erinacea), where it showed apical expression in liver, kidney and intestine, paralleling the tissue 404 distribution and localisation of ABCC2 in mammals (Cai et al., 2003). 405 406 2.3 ABCG/Abcg subfamily 407 The ABCG/Abcg subfamily contains the drug transporter ABCG2/Abcg2, as well as members 408 involved in sterol metabolism (ABCG/Abcg1, 4, 5 and 8) (Hazard and Patel, 2007; Wang et al., 409 2004). In phylogenetical analyses, all teleost Abcg sequences could be clearly affiliated to one 410 of the above ABCG paralogs (Fig. S2). To obtain a tree of manageable size, the analysis was 411 rerun including ABCG2/Abcg2 homologues only (Fig. 4). Teleosts show duplications of Abcg2, 412 with two isoforms being present in medaka, stickleback, tilapia, green puffer and pufferfish, and 413 four in cod and zebrafish (Fig. 4). In synteny analyses, human ABCG2 showed synteny to 414 abcg2a of green puffer and to abcg2d of zebrafish, but not to the remaining Abcg2 homologues 415 of these teleost species (Fig. 5). Results in medaka, stickleback, tilapia, pufferfish and in cod 416 resembled that in green puffer, with Abcg2a being the only Abcg2 isoforms showing synteny to 417 ABCG2 (data not shown). 418 419 3. Functional activity and expression of ABC drug transporters in fish 420 421 The measurement of transport activity of ABC drug transporters usually involves monitoring the 422 translocation of a conveniently detectable model substrate in a suitable in vitro model (Calcagno 423 et al., 2007). Parallel treatments with inhibitors are included to confirm the identity of the 424 involved transporters. Results obtained with model substrates and inhibitors need to be 425 interpreted with caution, as different classes of ABC pumps overlap in substrate and inhibitor 12 426 specificity (Calcagno et al., 2007). Moreover, the assumed specificities of ‘selective’ 427 compounds have usually been established in mammalian systems and may not necessarily apply 428 in fish. The specificities of the most commonly used substrates and inhibitors are briefly 429 reviewed here to provide the background for the understanding of specific findings in fish 430 reviewed below. 431 A number of substrates initially described as selective for ABCB1 have later been shown to 432 interact with other ABC transporters. For instance, the fluorescent ABCB1-substrates rhodamine 433 123 and doxorubicin are also transported by ABCC1 (Barrand et al., 1993; Twentyman et al., 434 1994; Yeheskely-Hayon et al., 2009). Doxorubicin was further shown to be also transported by 435 ABCG2, which further interacts with the ABCB1 substrate Hoechst 33342 (Krishnamurthy and 436 Schuetz, 2006; Robey et al., 2004). Calcein-acetoxymethyl ester (Calcein-AM) is a non-charged 437 non-fluorescent substrate for both ABCB1 and ABCCs. After cellular uptake, calcein-AM 438 undergoes enzymatic hydrolysis to the anionic fluorophore calcein, which is transported by 439 ABCCs but not ABCB1 (Essodaigui et al., 1998). Pheophorbide a is regarded a specific 440 substrate of ABCG2 (Robey et al., 2004). In order to obtain selective probes for particular ABC 441 transporters, fluorescent derivatives of drug substrates have been prepared (Calcagno et al., 442 2007). BODIPY-FL-verapamil, initially regarded as selective for ABCB1, has been shown to 443 also be transported by ABCC1 (Crivellato et al., 2002). Fluorescein-methotrexate is regarded a 444 specific substrate of ABCC transporters (Masereeuw et al., 2000), whereas fluo-cAMP has been 445 described as a specific probe for ABCC4 (Reichel et al., 2007). 446 The different classes of ABC transporters also overlap regarding their specificity to inhibitors. 447 The inhibitors verapamil and cyclosporin A interact with both ABCB1 and ABCCs (Barrand et 448 al., 1993; Zaman et al., 1994), with median effective concentrations only slightly lower in 449 ABCB1-overexpressing cell lines than in cells showing increased levels of ABCC1 (Holló et al., 450 1996). Compared to the parent compound, the cyclosporin A derivative PSC-833 shows an 451 improved but not complete specificity towards ABCB1 (Leier et al., 1994). The compound 452 tariquidar has been shown to inhibit both ABCB1 and ABCG2 (Gardner et al., 2009). Inhibitors 453 described as specific for ABCB1 include LY335979 (Shepard et al., 2003) and the hydrophobic 454 peptides reversin 121 and 205 (Sharom et al., 1999). The leukotriene LTD4 receptor antagonist 455 MK571 is a specific inhibitor of ABCC transporters (Gekeler et al., 1995), whereas 456 fumitremorgin C and Ko134 have been described as selective ABCG2 inhibitors (Allen et al., 457 2002; Robey et al., 2004). 458 Different types of teleost tissue preparations have been used for transport measurements. 459 Polarised epithelia can be mounted in Ussing chambers, allowing to establish rates of 460 basolateral-to-apical and apical-to-basolateral substrate translocation under different conditions 13 461 (Sussman-Turner and Renfro, 1995). In a number of marine teleosts, isolated kidney tubules 462 reseal spontaneously in vitro, so that the uptake of fluorescent substrates from cell culture media 463 and their secretion into the tubular lumen can be observed using confocal microscopy (Miller, 464 1987). In non-differentiated cells, ABC drug transporter activities can be established as the 465 difference in substrate accumulation between parallel treatments differing in the presence or 466 absence of a suitable inhibitor, reflecting that the activity of ABC drug transporters can limit the 467 accumulation of substrates. In this approach, levels of the fluorescent substrate can be 468 determined either on cell monolayers following extraction (Sturm et al., 2001b), or by flow 469 cytometry using cells in suspension (Kobayashi et al., 2008). 470 As reviewed in detail in the subsequent sections, teleost tissues in which the expression and 471 activity of ABC drug transporters has been ascertained include the kidneys, the liver, the 472 intestine and the capillary endothelia forming the blood brain barrier (Fig. 6). Selected model 473 substrates and inhibitors used in transport activity measurements in teleost tissues are 474 summarised in Table 1. Roles of ABC drug transporters in further teleost tissues are likely to 475 exist but require further investigation. 476 477 3.1 Kidney 478 In teleosts, the kidneys have central roles in the maintenance of the internal electrolyte and acid- 479 base balances (Marshall and Grosell, 2006) and in the excretion of metabolic waste products and 480 chemicals of endogenous or foreign origin (Kleinow et al., 2008). Glomerular ultrafiltration and 481 tubular active secretion contribute to the renal excretion of organic chemicals. Glomerular 482 filtration rates are greatly reduced in euryhaline teleosts adapted to seawater, and some marine 483 teleosts possess nephrons lacking glomeruli (Beyenbach, 2004). Within the nephron, the 484 secretion of organic chemicals occurs mainly in the proximal tubule, which is composed of 485 cuboid epithelial cells rich in mitochondria and possessing a dense layer of microvilli on their 486 apical surfaces. In a number of marine teleosts, nephrons consist mainly of proximal tubules 487 (~90% of nephron length), of which in vitro preparations can be obtained that allow the study of 488 transport of fluorescent model compounds by confocal microscopy (Miller, 1987). 489 Historically, different transport systems have been functionally characterised in the kidney 490 tubule for “organic cations” (including bases) and “organic anions” (including acids) (Wright 491 and Dantzler, 2004), with both types of systems involving two transmembrane transport steps, 492 basolateral uptake and apical secretion. Members of the SLC superfamily have important roles 493 in these systems; however, these transporters are beyond the scope of this article (for reviews, 494 please refer to El-Sheikh et al. (2008); Koepsell et al. (2007); Wright and Dantzler (2004)). In 14 495 addition, ABC transporters mediate the secretion of bulkier organic ions into the renal tubule 496 (Wright and Dantzler, 2004). 497 The transport of the organic base daunomycin (Mw 528 Da) into proximal tubule lumen was 498 inhibited by verapamil and cyclosporin A in winter flounder (Pleuronectes americanus) 499 (Sussman-Turner and Renfro, 1995) and killifish (Fundulus heteroclitus) (Miller, 1995), 500 consistent with a mechanism involving Abcb1 (Table 1). Immunostaining of flounder proximal 501 tubule primary cultures with antibody C219 showed a signal located to apical microvilli 502 (Sussman-Turner and Renfro, 1995). At pH 8.25, luminar daunomycin secretion in killifish 503 proximal kidney tubules was resistant to inhibition by tetraethylammonium (TEA; Mw 130), a 504 “type I” organic cation (Mw < 400) and model substrate for “classical” organic cation transport 505 systems (Miller, 1995). When the pH of the media was decreased to 7.25, however, TEA caused 506 a reduction in cellular and luminal accumulation of daunomycin (Miller, 1995). This finding 507 indicated that at the lower pH “classical” organic cation transport systems became involved in 508 daunomycin transport in addition to the Abcb1-like mechanism, which is in line with the higher 509 proportion of cationic daunomycin expected at pH 7.25 when compared to pH 8.25 (Miller, 510 1995). 511 The secretion of the large organic anion fluorescein-methotrexate (Mw 923 Da) to killifish 512 proximal tubular lumen was inhibited by LTC4, cyclosporin A and verapamil, but remained 513 unaffected by glutarate and ouabain, and was largely sodium independent (Masereeuw et al., 514 1996) (Table 1). Accordingly, basolateral uptake and luminal secretion of fluorescein- 515 methotrexate were by mechanisms distinct from the ouabain-sensitive, sodium-dependent 516 “classical” transport system involving exchange for dicarboxylates that had previously been 517 described for small organic anions such as fluorescein (Mw 130 Da) (Masereeuw et al., 1996). 518 The inhibitory effects of LTC4 and cyclosporin A suggested the luminar secretion of 519 fluorescein-methotrexate by a teleost homologue of ABCC2 (Miller and Pritchard, 1997), an 520 ABC drug transporter known to show apical expression in the mammalian proximal tubule 521 (Table 1). Supporting this interpretation, the luminal membrane of killifish proximal tubule 522 showed immunoreactivity against an antibody raised against rabbit ABCC2 (Masereeuw et al., 523 2000). Examination of the transport of other organic ions suggested the presence of different 524 transport systems in killifish tubules. In addition to activity with the “classical” transport 525 system, transport by an ABCC2-like efflux mechanism could be demonstrated for 526 sulforhodamine 101 (Mw 606 Da) (Masereeuw et al., 1996) and lucifer yellow (Mw 444 Da) 527 (Masereeuw et al., 1999) (Table 1). In addition, the presence of a putative teleost ABCC4 528 homologue in killifish kidney was suggested by the cellular-to-luminar transport of a fluorescent 15 529 cAMP analogue, which was inhibited by the general MRP inhibitors MK571 and LTC4 and the 530 ABCC4-specific compounds cAMP and adefovir (Reichel et al., 2007) (Table 1). 531 The teleost proximal tubule system was subsequently applied to identify classes of renal drug 532 efflux transporters interacting with selected pharmaceuticals by studying custom-made 533 fluorescent analogues of the relevant drugs. ABCB1-like transport was demonstrated for 534 fluorescent derivatives of the anthelminth ivermectin and the immunosuppressants cyclosporin 535 A and rapamycin (Fricker et al., 1999; Miller et al., 1997; Schramm et al., 1995) (Table 1). The 536 HIV protease inhibitors ritonavir and saquinavir inhibited ABCB1- and ABCC2-like transport in 537 killifish kidney tubules, and experiments with a fluorescent analogue of saquinavir further 538 suggested that this compound is transported by both transporters (Gutmann et al., 1999). Using a 539 fluorescent derivative of the somatostatin analogue octreotide, both ABCB1- and ABCC2-like 540 transport of the compound could be measured in killifish kidney tubules (Gutmann et al., 2000). 541 542 3.2 Liver 543 The teleost liver has major roles in the homeostasis of amino acids, carbohydrates and fatty 544 acids. Moreover, numerous proteins are synthesized in the liver and secreted into blood, for 545 instance albumin-like proteins, fibronectins and vitellogenin (Hinton et al., 2008). In addition, 546 the liver plays a key role in the detoxification of endogenous and foreign compounds, including 547 many organic chemicals having relevance as environmental pollutants (Schlenk et al., 2008). 548 Hepatocytes are in contact with blood perfusing the hepatic parenchyma at their sinusoidal 549 (basolateral) membranes, at which blood-borne chemicals can be taken up. In the hepatocyte, 550 chemicals may undergo phase I and II biotransformation metabolism (Schlenk et al., 2008). At 551 the same time, chemicals or their metabolites may be subject to efflux transport by canalicular 552 ABC pumps, resulting in biliary excretion. The importance of the biliary route of excretion is 553 illustrated by the large range of compounds found in fish bile, which includes industrial 554 chemicals such as chlorophenols (Oikari and Kunnamo-Ojala, 1987), combustion products such 555 as polycyclic aromatic hydrocarbons (Collier and Varanasi, 1991) and agricultural compounds 556 such as pesticides (Bradbury et al., 1986; Lech et al., 1973). Alternatively, chemicals or their 557 metabolites can leave the hepatocyte by being transported back into the blood by sinosoidal 558 ABC transporters, after which they may be subject to renal excretion. 559 In mammals, the hepatic ABC drug transporters ABCB1, ABCC2 and ABCG2 localise to the 560 canalicular membrane, while ABCC1 and 3 are expressed in the sinusoidal membrane (Chan, 561 2004). Other canalicular ABC transporters mediate the secretion of bile acids (ABCB11), 562 phospholipids (ABCB4) and cholesterol (ABCG5 / ABCG 8) into the bile fluid (Hazard and 563 Patel, 2007; Oude Elferink and Paulusma, 2007; Stieger et al., 2007). Although not drug 16 564 transporters, these ABC proteins contribute to maintaining bile flow and thus facilitate chemical 565 excretion, and for this reason existing studies on these pumps in fish will be considered in this 566 section. 567 In immunohistochemical investigations of guppy (Poecilia reticulata) tissues using antibodies 568 raised against mammalian P-glycoprotein, monoclonal antibodies C219 and JSB-1 specifically 569 stained bile canaliculi (Hemmer et al., 1995). Subsequently, C219 has been widely used to 570 detect hepatic P-glycoprotein in teleosts, in which it stained canalicular structures in 571 immunohistochemical experiments, and detected protein fractions of the expected molecular 572 mass of P-glycoprotein of ~170 kDa in immunoblots (Albertus and Laine, 2001; Bard et al., 573 2002b; Cooper et al., 1999; Hemmer et al., 1998; Sturm et al., 2001b). However, as the epitope 574 recognised by C219 (VQEALD/VQAALD) is conserved in ABCB4 and ABCB11 (Georges et 575 al., 1990), as well as in Abcb11 from winter flounder (Pleuronectes americanus) (Chan, 1992), 576 C219 can be expected to cross-react with ABC transporters other than Abcb1. The bile salt 577 export pump Abcb11 shows high mRNA expression in teleost liver (Loncar et al., 2010), and 578 could therefore contribute to the hepatic signal detected by C219 in teleosts. 579 Data on the hepatic mRNA expression of ABC drug transporters are available for a number of 580 teleost and elasmobranch species. Tutundjian et al. (2002) cloned a fragment of abcb1 in turbot 581 (Scophthalmus maximus) and demonstrated expression of its mRNA in brain, intestine, kidney 582 and liver by RT-PCR. A cDNA encoding an abcc2 homologue was cloned in the elasmobranch 583 little skate (Raja erinacea), and showed high expression levels in kidney, intestine and liver, 584 where it located to the canalicular membrane (Cai et al., 2003). The group of Smital and co- 585 workers isolated cDNAs of abcb1 and abcc2 from rainbow trout liver (Zaja et al., 2008b), and 586 reported mRNA copy numbers per ng of total liver RNA of 8.61 x 102 for abcb1, 1.29 x 103 for 587 abcc2, 2.55 x 101 for abcc3 and 2.78 x102 for abcg2 (Loncar et al., 2010). Expression of abcc1, 588 abcc4 and abcc5 was also analysed, but found to be low in liver (<5 copies / ng of total RNA) 589 (Loncar et al., 2010). The hepatic mRNA expression of abcb1, different abcc paralogs, and 590 abcg2 has been confirmed in a number of further teleost species in the context of 591 ecotoxicological studies (Bresolin et al., 2005; Costa et al., 2012; Diaz de Cerio et al., 2012; 592 Paetzold et al., 2009; Zucchi et al., 2010), which will be discussed in detail below. 593 The transport activity of hepatic ABC drug transporters in teleosts has been investigated by 594 assessing the effects of selective inhibitors on the accumulation, efflux or transport of 595 fluorescent or radiolabelled model substrates, employing experimental models including whole 596 animals, livers perfused in situ, and primary cultures of isolated hepatocytes and membrane 597 vesicles. The waterborne exposure of common carp (Cyprinus carpio) to 3 mM rhodamine B 598 resulted in a time-dependent accumulation of the dye in liver and bile fluid over three hours 17 599 (Smital and Sauerborn, 2002). When rhodamine B was combined with the ABCB1 inhibitors 600 verapamil or cyclosporin A this resulted in elevated hepatic and biliary dye accumulation 601 compared to fish exposed to the fluorescent probe alone (Smital and Sauerborn, 2002). In 602 channel catfish (Ictalurus punctatus) in situ liver perfusions, transport of rhodamine 123 from 603 perfusate to bile was significantly decreased in the presence of verapamil (Kleinow et al., 2004). 604 Verapamil also inhibited perfusate-to-bile transport of estradiol, which in turn competed with 605 rhodamine 123 transport (Kleinow et al., 2004). Similarly, verapamil enhanced the accumulation 606 of doxorubicin by killifish (Fundulus heteroclitus) hepatocytes (Albertus and Laine, 2001), and 607 verapamil, cyclosporin A, doxorubicin and vanadate increased the accumulation of rhodamine 608 123 by rainbow trout (Oncorhynchus mykiss) hepatocytes (Sturm et al., 2001) (Table 1). While 609 the above findings were interpreted as evidence for the presence of ABCB1-like transporters in 610 the teleost liver, a later study employing further inhibitors (Abcb1: reversin 205, Abcc: MK571) 611 demonstrated that both ABCB1- and ABCC-like transporters are involved in the transport of 612 rhodamine 123 in trout hepatocytes (Zaja et al., 2008b) (Table 1). Two further studies with trout 613 hepatocytes used the inhibitor tariquidar in combination with rhodamine 123 or doxorubicin to 614 measure transport activity attributed to Abcb1 (Bains and Kennedy, 2005; Hildebrand et al., 615 2009). While the ABCB1-inhibitor tariquidar does not inhibit ABCCs, it can interact with 616 ABCG2 (Robey et al., 2004). In the elasmobranch little skate, liver membrane vesicles were 617 used to investigate ABCC-like transport activity (Rebbeor et al., 2000). The uptake of tritiated 618 S-dinitrophenyl-glutathione and glutathione showed an ATP-dependent component that was 619 inhibited by ABCC substrates and GSH (Rebbeor et al., 2000). 620 Relatively little is known on fish ABC transporters involved in the secretion of bile constituents. 621 Conjugated bile salts are found in the bile of all vertebrates, but different vertebrate groups show 622 distinct differences regarding the subclass composition of bile salts present in bile fluid 623 (Moschetta et al., 2005). The abcb11 gene, which encodes the hepatic bile salt export pump 624 (BSEP), was originally isolated in a teleost (Chan, 1992; Childs et al., 1995; Gerloff et al., 625 1998), suggesting a conservation of abcb11 across the vertebrates. In the elasmobranch little 626 skate (Raja erinacea), an antiserum raised against rat ABCB11 reacted with proteins of an 627 apparent mass of 210 kDa in immunoblots and resulted in the immunostaining of bile canaliculi 628 (Ballatori et al., 2000). Subsequently, little skate abcb11 was cloned and shown to transport 629 taurocholate upon transfection into Sf9 cells (Cai et al., 2001). In rainbow trout, abcb11 was 630 highly expressed in liver (Loncar et al., 2010), and BSEP-like activity was detectable in cultured 631 hepatocytes (Zaja et al., 2008b) (Table 1). Cholesterol is found in bile fluid in different 632 vertebrate groups, however can greatly vary in concentration (Moschetta et al., 2005). While the 633 locus encoding the mammalian hepatic cholesterol transporters ABCG5 and ABCG8 is highly 18 634 conserved between mammals and teleosts (Hazard and Patel, 2007), no direct evidence is 635 available regarding the hepatic expression and functional activity of Abcg5 and Abcg8 in fish. 636 Phospholipids, which are absent from the bile of elasmobranchs and reptiles, are lacking or 637 show very low concentrations in the bile of teleosts (Goto et al., 2003), and are found at widely 638 variable levels in the bile of different mammalian species (Moschetta et al., 2005; Oude Elferink 639 et al., 2004). Apparently there is no active translocation of phospholipids into teleost bile 640 indicating the absence of functional homologues to mammalian ABCB4 in teleosts. On the basis 641 of available genomic data, some studies have suggested that teleosts lack an ABCB4 orthologue 642 (Annilo et al., 2006; Liu et al., 2013; Moitra et al., 2011), while others came to the conclusion 643 that teleosts possess an ABCB4 orthologue resembling mammalian ABCB1 in function (Fischer 644 et al., 2013). 645 646 3.3 Intestine 647 The gastrointestinal tract of fish has multiple functions, which include digestion and nutrient 648 absorption, osmoregulation and maintenance of the acid-base balance, and barrier and immune 649 functions (Bakke et al., 2011; Cain and Swan, 2011; Marshall and Grosell, 2006). As in other 650 animals, the intestine is a potential site of uptake of chemical compounds other than nutrients 651 from food (Kleinow et al., 2008), which include anthropogenic contaminants as well as toxic 652 chemicals from natural sources. As an adaptation limiting potential adverse effects of food- 653 borne toxicants, biochemical defence systems, including biotransformation enyzmes and drug 654 transporters, are expressed in the intestinal epithelium (Chan, 2004; Schlenk et al., 2008). In 655 mammals, the intestine is a major site of expression of ABC drug transporters, some of which 656 limit the oral absorption of drugs that are transport substrates (reviewed by Oostendorp et al. 657 (2009)). Relatively little is known about the expression and activity of ABC transporters in the 658 gastrointestinal system of fish. The immunohistochemical staining of lumenal surfaces of the 659 intestinal epithelium in guppy (Poecilia reticulata) suggested the expression of ABCB1-like 660 proteins in the teleost gut (Hemmer et al., 1995). In channel catfish (Ictalurus punctatus) 661 reactivity with mAB C219 in immunoblots was lower in the intestine than in the liver (Doi et 662 al., 2001), whereas immunohistochemistry revealed a more pronounced expression of reactive 663 protein(s) in the distal than the proximal regions of the gut (Kleinow et al., 2000). [3H] 664 vinblastine uptake of membrane vesicles prepared from catfish intestinal mucosa was stimulated 665 by ATP and inhibited by verapamil, consistent with the presence of an ABCB1-like transporter 666 (Doi et al., 2001) (Table 1). Among different rainbow trout (Oncorhynchus mykiss) ABC drug 667 transporters, abcb1, abcc2, abcc3 and abcg2 showed major mRNA expression in the intestine, 668 whereas abcc1, abcc4 and abcc5 mRNAs were present at low abundances (<15 copies per ng of 19 669 total RNA) (Loncar et al., 2010). Of these transporters, abcb1, abcc2, and abcg2 showed higher 670 levels of mRNA expression in the distal than the proximal intestine (Loncar et al., 2010). 671 672 3.4 Blood-brain barrier and blood-cerebrospinal fluid barrier 673 To ensure optimal functioning of the vertebrate central nervous system (CNS), the chemical 674 composition of its extracellular fluids is under tight physiological control and the movement of 675 molecules between blood and CNS is restrained at anatomical interfaces such as the blood-brain 676 barrier and the blood-cerebrospinal fluid barrier (Redzic, 2011). The blood-brain barrier is 677 comprised of the endothelia of the brain capillaries and separates blood from brain interstitial 678 fluid (Cserr and Bundgaard, 1984). Its function is to protect the brain from potentially 679 neurotoxic compounds and prevent its exposure to the physiological fluctuations in 680 concentrations of plasma solutes while at the same time allowing for the exchange of ions, 681 nutrients, metabolic waste products and signalling molecules between blood and brain 682 interstitial fluid (Redzic, 2011). The endothelial cells forming the blood-brain barrier have low 683 pinocytotic activity and possess tight junctions interconnecting adjacent cells, which foreclose 684 the paracellular passage of molecules (Redzic, 2011; Ueno, 2009). In consequence, the capillary 685 brain endothelium shows a high transendothelial electrical resistance in the range of 1500 W 686 cm2, and is in this respect reminiscent of tight epithelia (Crone and Christensen, 1981). Besides 687 constituting an anatomical barrier, brain endothelial cells actively mediate the transport of ions, 688 nutrients, neurotransmitters and metabolic waste products across the endothelium through a 689 multitude of transporters expressed at the apical (blood side) and/or basolateral (brain side) cell 690 membranes (Miller, 2010; Redzic, 2011). In mammals, ABC drug transporters showing an 691 apical expression at the blood-brain barrier comprise ABCB1, ABCC2, ABCC4, ABCC5 and 692 ABCG2 (Redzic, 2011; Ueno, 2009). ABCC1 is present in brain endothelial cells but its 693 subcellular localisation is still a matter of debate (Dallas et al., 2006; Redzic, 2011). 694 Early studies have characterised the teleost blood-brain barrier as tight based on its 695 impermeability for dyes, inulin, iodine and horseradish peroxidase (HRP); however, conflicting 696 results suggesting a less restrictive barrier function were obtained with epinephrine and 697 thiocyanate (Cserr and Bundgaard, 1984). Recent studies confirmed the presence of a fully 698 functional blood-brain barrier in zebrafish using HRP, a biotinylation agent and an ectopically 699 expressed recombinant protein as markers and demonstrated the presence of claudin-5 and other 700 tight-junction proteins in the microvessels (Jeong et al., 2008; Xie et al., 2010). A role of ABC 701 transporters in the teleost blood-brain barrier is suggested by the observation that inhibitors of 702 ABC drug transporters increase the retention of rhodamine 123 in the zebrafish brain (Park et 703 al., 2012). 20 704 The high therapeutic margin of the anti-parasitic drug ivermectin in mammals is partly 705 explained by its selectivity for ecdysozoan GABA- and glutamate-gated ion channels (Lynagh 706 and Lynch, 2012), and partly due to the fact that ABCB1 activity in the brain capillary 707 endothelia limits the brain penetration of ivermectin, preventing interaction with related 708 vertebrate ion channels expressed in the central nervous system (Schinkel et al., 1994). 709 Relatively high brain levels of ivermectin were measured in teleost fish following parenteral 710 administration (Høy et al., 1990; Katharios et al., 2004). While this suggests a limited efficacy 711 of the teleost blood-brain barrier towards ivermectin, co-administration of ivermectin with the 712 ABCB1 inhibitor cyclosporin A resulted in increased adverse behavioural effects compared to a 713 treatment with ivermectin alone (Bard and Gadbois, 2007), suggesting that ABC pumps of the 714 teleost blood-brain barrier provide a partial protection against ivermectin neurotoxicity. 715 Further evidence for roles of ABC transporters in the teleost blood brain barrier is provided by 716 in vitro studies. Isolated killifish brain capillaries accumulated ABC transporters substrates from 717 incubation media and secreted the compounds into the capillary lumen in a concentrative 718 fashion (Miller et al., 2002). The transport of fluorescent derivatives of cyclosporin A and 719 verapamil was inhibited by PSC-833 but not LTC4, which is consistent with an Abcb1- 720 dependent luminal transport mechanism (Miller et al., 2002) (Table 1). In contrast, luminal 721 accumulation of fluorescein-methotrexate and sulforhodamine 101 was inhibited by LTC4 but 722 not PSC-833, suggesting the involvement of Abcc transporters (Miller et al., 2002) (Table 1). 723 Similar luminal accumulation of fluorescent derivatives of cyclosporin A, verapamil and 724 methotrexate was demonstrated in isolated brain capillaries of spiny dogfish shark (Squalus 725 acanthias) (Miller et al., 2002). Immunostaining using mAB C219 and an antibody directed 726 against rabbit ABCC2 localised ABCB1-like and ABCC2-like proteins to the luminal surface of 727 killifish brain capillaries (Miller et al., 2002). In accordance with suggested roles in the blood- 728 brain barrier, ABC transporters showing transcript expression in the rainbow trout brain were, in 729 the order of decreasing abundance expressed as copy numbers per ng of total RNA, abcb1 (1.38 730 x 103 / ng), abcc2 (1.38 x 102 / ng), abcg2 (1.02 x 102 / ng), abcc3 (8.4 x 10 / ng), abcc5 (2.65 / 731 ng), abcc4 (1.68 / ng) and abcc1 (0.72 / ng) (Loncar et al., 2010). 732 The main function of the choroid plexus (CP) epithelium is the secretion of cerebrospinal fluid 733 (Cserr and Bundgaard, 1984). It consists of mitochondria-rich cuboid cells that are 734 interconnected by tight junctions and possess microvilli on their apical sides facing the 735 cerebrospinal fluid. The endothelial cells of the capillary network underlying the CP epithelium 736 on the basolateral side are fenestrated, so that the CP epithelium constitutes the site of the blood- 737 cerebrospinal fluid barrier (Cserr and Bundgaard, 1984; Redzic, 2011). TEER estimates 738 obtained on bullfrog CP explants mounted in Ussing chambers were in the range of 150 W cm2, 21 739 which is similar to values obtained in leaky epithelia (Saito and Wright, 1984). In mammals, 740 ABC transporters expressed in the CP epithelium include ABCC1 and ABCC4, which adopt a 741 basolateral localisation, ABCB1 and ABCG2, which are expressed apically, and ABCC5, the 742 localisation of which remains to be elucidated (Redzic, 2011). While ABCC1 is the most 743 abundant of ABC transporter in the CP epithelial cells, ABCB1 levels in the CP epithelium are 744 low (Redzic, 2011). 745 No studies on the function of the CP exist in teleosts. However, the CP epithelium in the 746 elasmobranch spiny dogfish shark (Squalus acanthias) is anatomically easily accessible and 747 explants of the CP in this species have been used for in vitro transport studies using confocal 748 microscopy (Baehr et al., 2006; Reichel et al., 2008). Fluorescein-methotrexate and 749 sulforhodamine 101 were accumulated by CP cells from incubation media, provided at the 750 cerebrospinal fluid side, and actively secreted into the subepithelial/vascular space (Baehr et al., 751 2006; Reichel et al., 2008). Both cellular accumulation and vascular secretion of the dyes was 752 sensitive to inhibition by the ABCC inhibitors MK571 and LTC4 (Baehr et al., 2006; Reichel et 753 al., 2008). 754 755 3.5 Other tissues 756 In teleosts, the gill is a main site of gas exchange, osmoregulation and excretion of nitrogenous 757 waste products (Evans et al., 2005). Due to the large surface of the gill epithelium, as well as the 758 effective respiratory ventilation of water and circulation of blood, the gills represent a dominant 759 site both for the absorption and the elimination of xenobiotics (Kleinow et al., 2008). Relatively 760 little is known about the expression of ABC drug transporters in the fish gill. A study in guppy 761 using mAB C219, directed against mammalian ABCB1 but also known to crossreact with other 762 ABC transporters, reported a strong staining reaction in gill chondrocytes and an absence of 763 signal in filaments and lamellae (Hemmer et al., 1995). In contrast, in gill tissue from killifish 764 (Fundulus heteroclitus) or high cockscomb blenny (Anoplarchus purpurescens) no 765 immunoreactive bands were visible in Western blots with C219 (Bard et al., 2002a, 2002b). In a 766 study with rainbow trout, ABC drug transporters showing major mRNA expression in gills were 767 abcc2 and abcc3 when data were reported as copy numbers per mass unit of total RNA, whereas 768 mRNAs encoding Abcc3, Abcg2 and to a lesser extent also Abcc5 were the most abundant ABC 769 transcripts based on quantification relative to the reference gene ef1a (Loncar et al., 2010). No 770 data are available on the activity of ABC transporters in the fish gill. 771 The rectal gland is a NaCl-secreting organ of marine elasmobranchs (Marshall and Grosell, 772 2006). In isolated rectal gland tubules from dogfish shark (Squalus acanthias), sulforhodamine 773 101 was taken up from incubation medium and secreted into tubule lumen, suggesting additional 22 774 roles of the rectal gland in xenobiotic excretion (Miller et al., 1998a). Luminal secretion of 775 sulforhodamine 101 in shark rectal gland tubules was saturable, concentrative and sensitive to 776 inhibition by cyclosporin A and LTC4, but not verapamil, p-aminohippurate or TEA, suggesting 777 an involvement of Abcc pumps in the transport mechanism (Miller et al., 1998a). 778 In mammals, a number of ABC drug transporters are expressed in the blood-testis barrier and in 779 the placenta (Leslie et al., 2005). Little is known about blood-gonadal tissue barriers in fish. A 780 number of studies have found marked mRNA expression of ABC transporters in gonads of fish, 781 which could be related to roles of the pumps in blood-tissue barriers. In rainbow trout, 782 transcripts of abcb1, abcc2-4 and abcg2 were abundant in the ovaries (Loncar et al., 2010), 783 whereas in zebrafish abcc1 mRNA showed a marked expression in both male and female 784 gonads and abcc5 mRNA was highly expressed in testis (Long et al., 2011a, 2011b). 785 Evidence for the presence and activity of ABC drug transporters in the teleost epidermis was 786 provided in investigations using a rainbow trout skin primary culture system (Shúilleabháin et 787 al., 2005). In epidermal cell cultures, the efflux of rhodamine 123 was inhibited by verapamil. 788 Moreover, a fraction of the cells in epidermal primary cultures displayed positive 789 immunoreactivity with the mAB C219, which recognises a conserved ABCB1 epitope 790 (Shúilleabháin et al., 2005). Exposure of cultured trout epidermal cells to sediment elutriates 791 from a polluted field site resulted in an increase of the number of intensely JSB1 positive cells 792 and enhanced rhodamine 123 efflux activity (Shúilleabháin et al., 2005). 793 Mammalian stem cells show an increased expression and activity of ABC efflux transporters, 794 which are used as markers of stem cell identification (Bunting, 2002). Side population cells 795 from the hematopoietic tissue of zebrafish kidney were characterised by an increased Hoechst 796 33342 dye efflux activity and showed marked abcg2a expression (Kobayashi et al., 2008). 797 Moreover, zebrafish kidney side population cells were enriched in hematopoietic stem cells 798 (Kobayashi et al., 2008). 799 800 3.6 Fish embryos 801 Most teleosts are oviparous, with the complete embryonic development taking place outside the 802 maternal organism. The developing “orphan” embryo thus is directly exposed to potentially 803 adverse environmental conditions that could affect development and it is a common perception 804 that this ontogenetic life phase is therefore particularly sensitive (McKim, 1985; Oberemm, 805 2000). Correlations of toxicity data for fish embryo and adult fish for various chemicals, 806 however, show that sensitivities of the different life stages are generally highly comparable 807 (Belanger et al., 2013), contradicting the notion of increased vulnerability of teleost embryos to 808 environmental stressors as compared to adult stages. Indeed, despite the lack of differentiated 23 809 organs “orphan” embryos across aquatic animal taxa employ various cellular defence 810 mechanisms enabling them to deal with a range of stressors in development including changes 811 in temperature, hypoxia, pathogens, UV radiation, free radicals, and toxicants (Hamdoun and 812 Epel, 2007). One important component of the suite of defensive mechanisms are multidrug 813 transporters (Hamdoun and Epel, 2007), which are also expressed and active in teleost embryos. 814 It was recently shown that the P-glycoprotein Abcb4 acts as protective barrier against the uptake 815 of toxic compounds dissolved in the water by the zebrafish embryo (Fischer et al., 2013). Fish 816 embryos where expression of functional Abcb4 was disrupted by morpholino knock-down or by 817 co-exposure to ABC transporter inhibitors, such as cyclosporin A or PSC-833, showed increased 818 uptake of fluorescent ABC transporter substrates rhodamine B and calcein-AM (Table 1) and 819 their sensitivity to the toxic impact of the ABC transporter substrate vinblastine was increased 820 (Fischer et al., 2013). By effluxing incoming chemicals Abcb4 keeps cellular levels of those 821 compounds in embryo tissues and cells low and forms a multidrug-resistance type environment- 822 tissue barrier, in analogy to endogenous blood-tissue barriers. Abcb4 transcripts are found in the 823 early embryo before de novo transcription starts, which indicates that they are maternally 824 transferred to the embryo, and efflux activity can be observed already in the very early embryo 825 one hour after fertilization (Fischer et al., 2013). Similarly, transcripts of the multidrug 826 resistance associated transporters abcc1 (Long et al., 2011a) and abcc5 (Long et al., 2011b) are 827 maternally transferred to the zebrafish egg, whereas transcripts of abcc2 occur not before 72 828 hours post fertilization, indicating that this transporter has no relevant function in the early 829 embryo (Long et al., 2011d). The authors related a function of all three transporters Abcc1, 830 Abcc2 and Abcc5 with heavy metal detoxification in zebrafish embryos and larvae (Long et al., 831 2011a, c, d), thus associating them to the suite of molecular detoxification systems in zebrafish 832 early life stages. Constitutive transcript expression levels of ABC transporters were also 833 determined in early life stages of Nile tilapia (Oreochromis niloticus). As in zebrafish, 834 transcripts of P-glycoprotein abcb1b and of abcc1 were found in early developmental stages 835 directly after fertilisation of the egg, whereas abcc2, together with abcg2a and abcb11, occurred 836 in later stages (from pharyngula stage on) (Costa et al., 2012). The function of Abcb11 as bile 837 salt export pump (BESP) in liver has been shown to be highly conserved across vertebrate taxa 838 (Ballatori et al., 2000; Cai et al., 2001) and accordingly high abcb11 transcript levels were found 839 in teleost liver (Loncar et al., 2010); the occurence of abcb11 transcripts in teleost embryos 840 (Costa et al., 2012) may thus be associated with the appearance of the embryonic liver. 841 An interesting question regards whether ABC transporters have specific functions in the 842 developing embryo that are distinct from the function in adult tissues. Indeed, a function 843 essential for development of the zebrafish embryo was found for Abcc6a. The exact function of 24 844 the mammalian ortholog ABCC6 is not clear, but null mutations of ABCC6 are associated with 845 the inherited disorder pseudoxanthoma elasticum (PXE), which is characterised by dystrophic 846 mineralization and fragmentation of soft connective tissues (Pfendner et al., 2007). In zebrafish 847 embryos, expression of abcc6a is located in the Kupffer’s vesicles and in the tail buds; upon 848 knockdown of functional Abcc6a protein expression embryos at one day after fertilisation 849 showed shortening of the body, delay of the development of the head, decreased tail length, and 850 curving of the caudal part and older embryos developed severe heart edema and died at eight 851 days after fertilisation (Li et al., 2010). The Abcc6 knockdown effect in zebrafish embryos was 852 rescued by co-injection of mouse Abcc6 mRNA (Li et al., 2010), which indicates that zebrafish 853 Abcc6a and mouse ABCC6 have similar functional properties, but probably have different 854 functional physiological roles. The knockdown of the Abcb4 multidrug transporter and of 855 Abcb5 did not result in visible developmental effects (Fischer et al., 2013) providing no 856 evidence for developmental roles of these transporters. 857 858 4. Substrates and inhibitors of teleost ABC transporters 859 860 While chemicals have been classified as transport substrates or inhibitors of specific ABC drug 861 transporters, these two categories are not mutually exclusive, as many substrates inhibit the 862 transport of other compounds, while some compounds classified as inhibitors are themselves 863 transported. Chemical interaction has been studied in considerable depth for the mammalian 864 drug transporter ABCB1, and to a lesser extent for other drug transporters. These research 865 efforts have had two main drivers. Firstly, in order to overcome ABC transporter-related MDR 866 of cancers (Gottesman et al., 2002), studies have searched to identify non-cytotoxic inhibitors of 867 ABCB1 and other MDR pumps, which are called reversal agents, resistance modifiers or 868 chemosensitisers (Choi, 2005; Ford and Hait, 1993). Secondly, ABCB1 and possibly other drug 869 transporters can limit oral drug absorption and drug penetration into sanctuary sites of the body. 870 In consequence, ABC drug transporters can provide obstacles for drug delivery and be the basis 871 for drug-drug interactions, so that for drugs other than reversal agents chemical interaction with 872 ABC pumps is generally an undesired trait (Calcagno et al., 2007; Szakács et al., 2008). 873 Mammalian ABC drug transporters accept a wide range of structurally and functionally 874 unrelated pharmaceuticals as transport substrates and are inhibited by a similarly wide range of 875 drugs (Ambudkar et al., 1999; Calcagno et al., 2007; Choi, 2005; Deeley et al., 2006; Ford and 876 Hait, 1990; Krishnamurthy and Schuetz, 2006). Interaction of mammalian ABC drug 877 transporters has further been demonstrated for environmentally relevant compounds including 25 878 surfactants and pesticides (Bain and LeBlanc, 1996; Bain et al., 1997; Lanning et al., 1996; Loo 879 and Clarke, 1998; Oosterhuis et al., 2008; Siegsmund et al., 1994). 880 Substrates and inhibitors of ABC transporters can be identified through vectorial transport 881 studies in cell monolayer systems supporting polarised expression of the ABC transporter of 882 interest (Kim et al., 1998; Polli et al., 1999). However, fish cell lines suitable for this type of 883 approach await being identified. Moreover, transport studies using assay monolayer systems are 884 labour intensive to perform and require analytical quantification of the drugs studied, 885 constraining the usefulness of this methodology for chemical screening. 886 Methodologies to identify chemicals interacting with ABC transporters at comparatively high 887 sample throughput include cytotoxicity and/or dye accumulation assays in drug-resistant cell 888 lines overexpressing specific ABC drug transporters. Such resistant cell lines can be generated 889 by subjecting non-resistant cell lines to a step-wise selection with increasing levels of cytostatic 890 drugs (Riordan and Ling, 1985) or by transfection of suitable cell lines with the transporter in 891 question (Ueda et al., 1987). Cell lines overexpressing specific ABC drug transporters show a 892 decreased cellular accumulation and toxicity of substrates of the relevant pump. Accordingly, 893 transporter substrates can be identified in cytotoxicity assays as compounds showing a 894 decreased toxicity in the resistant when compared to the parental cell line (Szakács et al., 2008). 895 In contrast, in dye accumulation assays with drug-resistant cell lines, both substrates and 896 inhibitors increase the cellular accumulation of fluorescent dyes that are substrates of the studied 897 transporter (Holló et al., 1994; Homolya et al., 1993). 898 Another approach to investigate chemical interaction with ABC pumps at high sample 899 throughput is based on the measurement of transporter ATPase activity in cell membrane 900 fractions obtained from cell lines overexpressing specific transporters (Ambudkar et al., 1992; 901 Doige et al., 1992) or generated in recombinant baculovirus / insect cell expression systems 902 (Germann, 1998; Sarkadi et al., 1992). In these experimental systems, ABCB1 (P-glycoprotein) 903 shows basal ATPase activity in the absence of added substrates of the transporter, which is 904 dependent on the presence of cholesterol in the cell membrane (Garrigues et al., 2002). 905 Chemicals interacting with ABCB1 typically stimulate basal transporter ATPase activity in a 906 concentration-dependent way. For some compounds, ATPase activities follow a biphasic 907 concentration-effect profile, in which increasing levels of the compound provoke increasing 908 ATPase activation up to an optimal concentration beyond which inhibition occurs (Litman et al., 909 1997; Sarkadi et al., 1992). Other chemicals cause inhibition of basal ABCB1 ATPase activity 910 only, with no apparent stimulation at low levels (Litman et al., 1997). 911 The study of chemical interaction with ABC transporters by different methodologies can at 912 times lead to conflicting results, particularly with systems in which transport is not measured 26 913 directly (Szakács et al., 2008). The transport of chemicals by ABC drug transporters is a 914 complex process influenced by various factors, including access and affinity to the transporter’s 915 drug binding site(s) and the ability of the compound to induce ATPase hydrolysis and 916 concomitant conformation changes of the protein effecting transmembrane translocation of the 917 chemical. The recently achieved x-ray structure of ABCB1 in the apo and drug-bound state 918 (Aller et al., 2009) has revealed that ABCB1 possesses a large internal cavity which 919 accommodates distinct drug-binding sites and has portals allowing the entry of substrates from 920 both the cytoplasm and the inner leaflet of the cell membrane. In addition to direct chemical 921 interaction with ABCB1, the chemical’s permeability for the cellular membrane is an important 922 factor determining whether apparent transmembrane transport will occur (Stein, 1997). 923 Compounds effectively extruded by ABC efflux pumps, such as the rhodamine 123 and 924 doxorubicin, typically show low to moderate rates of passive membrane permeation (Eytan et 925 al., 1996b; von Richter et al., 2009). In contrast, reversal agents that are not transported 926 themselves, such as quinidine and verapamil, typically show high membrane permeability 927 (Eytan et al., 1996b; von Richter et al., 2009). 928 The following sections summarise studies providing evidence for the interaction of chemicals 929 with teleost ABC drug transporters based on whole animal studies, as well as studies employing 930 tissue, cellular and sub-cellular models. Subsequently, the metabolic costs of chemical 931 interaction with ABC transporters are addressed. 932 933 4.1 Results for primary cell culture systems 934 As reviewed in detail in section 3, the measurement of the activity of ABC drug transporters in 935 cell or tissue cultures involves the demonstration of effects of specific inhibitors on the 936 accumulation or efflux of model substrates. Model substrates and inhibitors include a number of 937 pharmaceuticals, such as the anthracyclins daunorubicin and doxorubicin, the calcium channel 938 blocker verapamil and the anti-retroviral drug adefovir (Table 1). Studies with isolated kidney 939 tubules have further demonstrated the interaction of renal teleost Abcb1 and/or Abcc 940 transporters with a range of drugs, including the immunosuppressants cyclosporin A and 941 rapamycin (Miller et al., 1997; Schramm et al., 1995), the anthelmintic ivermectin (Fricker et 942 al., 1999), the HIV protease inhibitors ritonavir and saquinavir (Gutmann et al., 1999), and the 943 somatostatin analogue octreotide (see section 3.1 for details). These data suggest that teleost 944 ABC drug transporters resemble their mammalian counterparts in that they interact with a wide 945 range of chemicals. 946 A few studies have used primary cell culture systems or organ perfusions to investigate the 947 interaction of teleost ABC transporters with environmentally relevant chemicals. The industrial 27 948 chemical bisphenol A, which is present in many plastics and the coating of food cans, exerted 949 differential effects on xenobiotic transport in killifish kidney tubules, inhibiting ABCC-like 950 luminal secretion of sulforhodamine but stimulating the efflux of mitoxantrone (Nickel et al., 951 2013). The fungicide prochloraz and, less effectively, the xenoestrogen nonylphenol ethoxylate 952 inhibited efflux of rhodamine 123 from rainbow trout hepatocytes (Sturm et al., 2001a). 953 Similarly, the surfactant linear alkylbenzene sulfonate decreased rhodamine 123 transport into 954 bile in perfused catfish liver (Tan et al., 2010). 955 956 4.2 Results for cell lines 957 Step-wise selection of the topminnow (Poeciliopsis lucida) hepatoma cell line PLHC-1 with 958 increasing levels of doxorubicin was used to generate the subline PLHC-1/dox that shows a 45- 959 fold reduction in doxorubicin sensitivity compared to the parental cell line (Zaja et al., 2008a). 960 Using relative quantification by RT-qPCR, abcb1 expression in PLHC-1/dox compared to 961 PLHC-1 was found to be 42-fold increased when normalised to b-actin (Zaja et al., 2008a) and 962 160-fold increased when normalised to 18S RNA (Zaja et al., 2011), with Abcb1 overexpression 963 in the selected cell line further being apparent from immunoblot analyses (Zaja et al., 2008a). In 964 addition, PLHC-1/dox cells showed small increases (≤ 4.4-fold) in transcript levels of abcc1, 965 abcc2, abcc4 and abcc10, whereas abcg2 mRNA levels were decreased (Zaja et al., 2011). 966 Compared to PLHC-1, cyctotoxicity in PLHC-1/dox cells was decreased for a range of other 967 cytostatic drugs known to be substrates of human ABCB1, including the anthracyclines 968 daunorubicin, the Vinca alkaloids vinblastine and vincristine, and the topoisomerase inhibitor 969 etoposide (Zaja et al., 2008a) (Table 2). However, the resistance spectrum of PLHC-1/dox did 970 not extent to the MRP-substrates methotrexate and cisplatin (Zaja et al., 2008a) (Table 2). A 971 number of pharmaceuticals known to be substrates and/or inhibitors of human ABCB1, 972 including the calcium channel blockers verapamil, nicardipine and diltiazem and the alkaloids 973 quinidine, reserpine and colchicine also interacted with P. lucida Abcb1, as indicated by 974 positive effects on dye accumulation in PLHC-1/dox cells (Zaja et al., 2011) (Table 2). Dye 975 accumulation in the Abcb1-overexpressing cell line was further increased by model inhibitors 976 (Table 2), of which cyclosporin A, PSC-833 and reversin 205 are considered to be specific for 977 ABCB1, whereas MK571 and Ko143 are in mammalian systems selective for MRPs and 978 ABCG2, respectively (Zaja et al., 2011). As would be expected, some of the model inhibitors 979 further had activity as reversal agents capable of abrogating the doxorubicin resistance of 980 PLHC-1/dox cells (Zaja et al., 2008a) (Table 2) . 981 PLHC-1/dox cells were further used to investigate the interaction of environmental pollutants 982 with P. lucida Abcb1. Environmental pharmaceuticals showed positive effects in cellular dye 28 983 accumulation assays in PLHC-1/dox cells and/or acted as reversal agents (Caminada et al., 984 2008) (Table 2). Another experimental approach to reveal chemical interaction with P. lucida 985 Abcb1 involved testing whether the toxicity in PLHC-1/dox cells was enhanced by the co- 986 treatment of cells with the ABCB1-inhibitor cyclosporin A. Again, several environmental 987 pharmaceutics showed positive results, suggesting they represent substrates of P. lucida Abcb1 988 (Caminada et al., 2008) (Table 2). Effects on dye accumulation by PLHC-1/dox cells were 989 further demonstrated for a range of pesticides, suggesting they might be substrates and/or 990 inhibitors of P. lucida Abcb1 (Zaja et al., 2011) (Table 2). Similarly, inhibitory activity on 991 Abcb1-related transport in PLHC-1/dox was demonstrated for a range of waste water 992 components (Smital et al., 2011). 993 In zebrafish, cadmium selection of the fibroblast-like cell line ZF4 was used to generate the 994 cadmium-resistant line ZF4-Cd (Long et al., 2011c). ZF4-Cd cells are cross-resistant to 995 cadmium, mercury, arsenate and arsenite and show an enhanced mRNA expression of abcc2, 996 abcc4 and mt2 genes (Long et al., 2011c). Moreover, ZF4-Cd cells possess elevated levels of 997 glutathione (Long et al., 2011c). 998 A number of permanent fish cell lines have been shown to constitutively express ABC 999 transporters, suggesting it may be feasible to create further cellular models overexpressing ABC 1000 pumps. Lines characterised regarding their complement of ABC transporters include the SAE 1001 Squalus acanthias shark embryo derived cell line (Kobayashi et al., 2007), seven rainbow trout 1002 cell lines (Fischer et al., 2011) and the topminnow (Poeciliopsis lucida) hepatoma cell line 1003 PLHC-1 (Zaja et al., 2007). Relative mRNA levels of ABC transporters were similar among 1004 rainbow trout cell lines RTL-W1, R1, RTH-149, RTgill-W1, RTG-2, RTgutGC and RTbrain, 1005 with mRNA levels abcc1-3 and abcc5 exceeding those of Abcb and Abcg subfamily drug 1006 transporters by 80 to over 1000-fold (Fischer et al., 2011). 1007 1008 4.3 Results with ATPase assays 1009 In teleosts, ATPase assays have been performed with P. lucida Abcb1 and D. rerio Abcb4, 1010 respectively, testing a range of compounds that comprise known substrates and inhibitors of 1011 mammalian ABC transporters and environmentally relevant chemicals (Table 2) (Fischer et al., 1012 2013; Zaja et al., 2011). Recently, ATPase assays were also used in effect directed analysis to 1013 detect compounds interacting with P. lucida Abcb1 in environmental samples (Zaja et al., 2013). 1014 For ATPase assays, cell membranes enriched in P. lucida Abcb1 were prepared from the PLHC- 1015 1/dox cell line overexpressing the transporter (Zaja et al., 2011), whereas membrane fractions 1016 containing D. rerio Abcb4 were obtained following baculovirus/insect cell expression (Fischer 1017 et al., 2013). 29 1018 Chemicals that stimulated the ATPase activity of P. lucida Abcb1 were categorized as substrates 1019 of the transporter, while inhibitors of the basal ATPase activity of P. lucida Abcb1 were 1020 classified as transporter inhibitors (Zaja et al., 2011) (Table 2). In mammalian systems, 1021 chemicals stimulating the ATPase activity of ABCB1 are known to comprise substances that are 1022 substrates in cellular transport assays as well as compounds that are inhibitors of transport 1023 activity in cellular assays (Litman et al., 1997; Sarkadi et al., 1992). Conversely, inhibitors of 1024 ABCB1 ATPase typically constitute inhibitors of transport activity in cellular assays (Litman et 1025 al., 1997; von Richter et al., 2009). Taking this into account, the results from P. lucida Abcb1 1026 ATPase assays are in accordance with earlier findings from cytotoxicity and dye accumulation 1027 assays in PLHC-1/dox cells for many compounds, including calcium channel blockers and 1028 alkaloids, as well as most of the model inhibitors, pesticides and environmental pharmaceutics 1029 (Table 2). 1030 However, diverging results between assays were observed for the cytostatic drugs etoposide and 1031 doxorubicin, the latter of which was used in generating the resistant PLHC-1/dox cell line 1032 (Table 2). The lack of effects of these chemicals on Abcb1 ATPase activity (Table 2) may seem 1033 surprising, considering that Abcb1 overexpression has been suggested as the main molecular 1034 factor behind the resistance of PLHC-1/dox cells to these cytotoxic compounds, which implies 1035 their cellular efflux by this transporter (Zaja et al., 2011, 2008a). However, only small 1036 stimulating effects on the ATPase activity of human ABCB1 have been reported for a number of 1037 confirmed ABCB1 substrates, which include etoposide and doxorubicin (Polli et al., 2001). As 1038 ABCB1 displays a high basal ATPase activity in the absence of added substrate, it has been 1039 hypothesized that substrates stimulating ATPase activity only by a small degree may go 1040 unnoticed due to the high basal ATPase activity of ABCB1 (Eytan et al., 1996a). 1041 Following expression of D. rerio Abcb4 in the baculovirus/insect cell system, a range of 1042 chemicals were examined for effects on ATPase activity of the transporter (Fischer et al., 2013) 1043 (Table 2). The ABCB1 reversal agent verapamil and the ABCB1 substrate rhodamine 123 1044 stimulated basal ATPase activity of D. rerio Abcb4, and were thus classified as substrates of the 1045 transporter (Fischer et al., 2013) (Table 2). The ABCB1 substrate doxorubicin and the 1046 ABCC/Abcc inhibitor MK571 inhibited verapamil-stimulated ATPase activities of D. rerio 1047 Abcb4 and were therefore categorised as inhibitors of the transporter (Fischer et al., 2013) 1048 (Table 2). The remaining compounds tested qualified both as substrates and inhibitors according 1049 to the above criteria and included the Vinca alkaloids vinblastine and vincristine, the model 1050 compounds calcein-AM, cyclosporin A and PSC-833, and the environmental pollutanst 1051 phenanthrene, galaxolide and tonalide (Table 2) (Fischer et al., 2013). (Table 2). 30 1052 The teleostean Abcb1 and Abcb4 proteins and human ABCB1 appear to be largely 1053 corresponding with regard to function and substrate spectra, but ATPase assays reveal subtle 1054 differences among transporter specificities. While the ABCB1 reversal agent cyclosporin A 1055 inhibits ATPase activity of ABCB1 (von Richter et al., 2009) and P. lucida Abcb1 (Table 2) 1056 (Zaja et al., 2011), it provoked a marked stimulation of D. rerio Abcb4 ATPase activity (Table 1057 2) (Fischer et al., 2013). Furthermore, the ABCB1 substrate vinblastine stimulated ATPase 1058 activities of human ABCB1 (Sarkadi et al., 1992) and D. rerio Abcb4 (Fischer et al., 2013) but 1059 had no effects on P. lucida Abcb1 ATPase activity (Zaja et al., 2011) (Table 2). 1060 1061 4.4 Metabolic costs of chemical interaction with ABC transporters 1062 ABC transporters mediate the translocation of their substrates across membranes by an ATP- 1063 dependent mechanism. The energetic costs of transport of rhodamine 123 and doxorubicin were 1064 estimated in two studies with isolated cultured rainbow trout hepatocytes (Bains and Kennedy, 1065 2005; Hildebrand et al., 2009). Exposure of hepatocytes to 5 and 10 mM rhodamine 123 1066 increased respiration by 18.5 and 25.7% over basal rates, respectively (Bains and Kennedy, 1067 2005). The altered respiration rates were not due to direct effects of rhodamine 123 on 1068 mitochondria. Co-treatment of hepatocytes with the ABCB1 inhibitor tariquidar inhibited the 1069 cellular efflux of rhodamine 123 and caused respiration rates to return to basal levels (Bains and 1070 Kennedy, 2005). When doxorubicin-treated hepatocytes were allowed to efflux the 1071 anthracycline for 3 hours, this resulted in an up to 25% decrease of cellular ATP levels 1072 compared to parallel incubations of untreated cells (Hildebrand et al., 2009). In contrast, 1073 following the incubation of doxorubicin-treated hepatocytes in the presence of tariquidar, a 1074 decreased efflux of doxorubicin was observed and cellular ATP levels remained unchanged 1075 (Hildebrand et al., 2009). While these data suggest that ABC drug transporter activity is 1076 associated with significant energy costs, exposure of rainbow trout to restricted feeding intake or 1077 fasting for up to 9 weeks did not result in significant changes in hepatic rates of rhodamine 123 1078 transport (Gourley and Kennedy, 2009). 1079 1080 5 Regulation of expression of teleost ABC drug transporters 1081 1082 Genomic and non-genomic mechanisms contribute to the regulation of ABC transporter activity 1083 in different tissues. Genomic mechanisms of regulation include genetic and epi-genetic effects, 1084 the modulation of transcription rates, effects on mRNA stability and translational silencing by 1085 miRNAs (Masereeuw and Russel, 2012). Mechanisms of non-genomic regulation comprise the 1086 insertion of transporters into and their retrieval from the cell membrane, as well as 31 1087 posttranslational modifications such as phosphorylation and glycosylation and protein-protein 1088 interactions (Masereeuw and Russel, 2012). Comprehensive reviews are available on the 1089 regulation of mammalian ABC transporters in tumour cells (Chen and Sikic, 2012; Chen et al., 1090 2012; Scotto, 2003) and in normal tissues including liver (Chan, 2004; Kipp and Arias, 2002; 1091 Roma, 2008), intestine (Estudante et al., 2013), kidney (Masereeuw and Russel, 2012), testis 1092 (Mruk et al., 2011) and brain (Chan et al., 2013; Miller, 2010). 1093 1094 5.1 Regulation of ABC transporters in cancer cells 1095 MDR in cancer often is based on the enhanced expression of ABCB1 and other ABC drug 1096 transporters in tumour cells (Gottesman et al., 2002). A number of genetic and epi-genetic 1097 changes commonly observed in MDR tumour cells are believed to contribute to enhanced ABC 1098 drug transporter expression (reviewed by Chen and Sikic (2012)). Genetic changes include gene 1099 rearrangements leading to a juxtaposition of other active promoters to the ABCB1 promoter, thus 1100 effecting its de-repression (Chen and Sikic, 2012). Moreover, enhanced expression of ABCB1 1101 following cancerous cellular transformation has been found to be linked to mutations in 1102 oncogenes and tumour suppressor genes such as p53 (Chen and Sikic, 2012). Among epi- 1103 genetic changes affecting ABC drug transporter expression in cancers, de-methylation and 1104 complex changes in acetylation status have been suggested to contribute to the de-repression of 1105 the ABCB1 promoter (Chen and Sikic, 2012). 1106 In a number of marine and freshwater fish, an increased incidence of different hepatic lesions 1107 including neoplasms has been reported from polluted habitats (Black and Baumann, 1991; 1108 Myers et al., 1991; Vethaak and Jol, 1996; Vethaak and Wester, 1996). Epidemiological 1109 analyses of available data strongly support a role of environmental contaminants, particularly 1110 PAHs, in the aetiology of these pathologies (Rotchell et al., 2008). Biochemical changes 1111 associated with hepatocellular carcinogenesis have been studied in European flounder 1112 (Platichthys flesus) from polluted sites (Koehler et al., 2004; Köhler et al., 1998). Compared to 1113 healthy liver tissue, preneoplastic basophilic foci, as well as hepatic adenomas and carcinomas 1114 showed an increase in immunoreactivity with mAB C219, which was interpreted as an 1115 upregulation of Abcb1 (Koehler et al., 2004). In contrast, C219 reactivity was unchanged in 1116 early eosinophilic preneoplastic foci (Koehler et al., 2004). Similarly, in killifish from a creosote 1117 contaminated environment the immunochemical C219 signal was increased in hepatocellular 1118 carcinomas, but not in early proliferative hepatic lesions (Cooper et al., 1999). In addition, 1119 flounder hepatocellular carcinomas showed an increase in glucose-6-phosphate dehydrogenase 1120 activity and glutathion-S-transferase (GST)-A expression, and a decrease in CYP1A1 levels 1121 compared to healthy hepatic tissue (Koehler et al., 2004). Differences in gene expression 32 1122 between normal liver tissue and hepatic adenomas and carcinomas were investigated using 1123 microarray technology in dab (Limanda limanda) (Small et al., 2010). Fifty genes that best 1124 characterised the tumour type and sex of the host included CYP1A and other biotransformation 1125 enzymes, but no ABC transporters (Small et al., 2010). Compared to hepatic tissue from non- 1126 cancer bearing dab, there was a significant decrease in global DNA methylation in 1127 hepatocellular carcinomas and surrounding non-cancerous liver tissue (Mirbahai et al., 2011). 1128 1129 5.2 Regulation of ABC transporters as part of a general cellular stress response 1130 In mammalian tissues, the expression of ABCB1 can be upregulated in response to cellular 1131 stress signals including heat shock, injury, inflammation, hypoxia, and UV and X radiation 1132 exposure (Scotto, 2003). The effects of heat shock on renal drug transport were studied in 1133 primary cultures of Winter flounder (Pleuronectes americanus) renal proximal tubule cells 1134 (Sussman-Turner and Renfro, 1995). As reviewed above, peritubular to luminal daunorubicin 1135 transport in this system involved Abcb1-like transporters. Mild heat shock (5 ¹C elevation for 6- 1136 8 h followed by incubation at normal temperature) stimulated transepithelial transport of the 1137 ABCB1 substrate daunorubicin, with effects being protein synthesis dependent (Sussman- 1138 Turner and Renfro, 1995). 1139 1140 5.3 Roles of transcription factors in the regulation of ABC transporters by chemicals 1141 Different nuclear receptors are involved in the transcriptional regulation of mammalian ABC 1142 drug transporters by endogenous and foreign chemicals (Chen et al., 2012). The pregnane 1143 xenobiotic receptor (PXR) was isolated for its role as a factor mediating the chemical regulation 1144 of isozymes of the CYP3A subfamily (Bertilsson et al., 1998; Blumberg et al., 1998; Kliewer et 1145 al., 1998). PXR is further involved in the regulation of other biotransformation enzymes 1146 (Blumberg et al., 1998; Xie et al., 2003) as well as ABC transporters including ABCB1 and 1147 ABCC2 (Dussault et al., 2001; Geick et al., 2001; Kast et al., 2002; Synold et al., 2001). While 1148 ligands of PXR comprise steroid hormones, bile acids and a wide range of organic chemicals, 1149 pronounced species differences in ligand selectivity exist (Ekins et al., 2008; Jones et al., 2000). 1150 The constitutive androstane receptor (CAR) shows sequence homology to PXR and overlaps 1151 with PXR in ligand spectrum and range of target genes (Maglich et al., 2002; Moore et al., 2000; 1152 Waxman, 1999). Teleosts possess one receptor showing sequence homology to both PXR and 1153 CAR (Handschin et al., 2004; Maglich, 2003), which has been named PXR because functionally 1154 it resembles PXR rather than CAR (Moore et al., 2002). The zebrafish PXR has a more 1155 restrained ligand spectrum than its mammalian counterparts, accepting only a subset of 1156 xenobiotic and steroid agonists of mammalian PXRs (Ekins et al., 2008; Fidler et al., 2012; 33 1157 Moore et al., 2002). Among different bile salts tested, only the main zebrafish bile salt cyprinol 1158 sulphate activated zebrafish PXR (Krasowski et al., 2005; Moore et al., 2002). In contrast, in 1159 green spotted puffer, which shows a more diverse physiological bile salt profile, a variety of bile 1160 salts were agonists of PXR (Krasowski et al., 2011). In zebrafish hepatocyte cultures, PXR 1161 activators induced enzyme activities that are in mammals specific for CYP3As and CYP2Cs 1162 (Reschly et al., 2007). Moreover, cyprinol sulphate treatment increased the abundance of hepatic 1163 Abcb5 transcripts (Reschly et al., 2007). In another study, treatment of zebrafish with the PXR 1164 ligand pregnenolone 16a-carboninitrile provoked increases (1.6- to 1.9-fold) in the mRNAs 1165 levels of PXR, abcb1 (now called abcb4) and CYP3A (Bresolin et al., 2005). The insecticide 1166 chlorpyrifos is a known PXR ligand in zebrafish and human (Ekins et al., 2008). Exposure of 1167 killifish to the metabolically activated oxon form of chlorpyrifos induced hepatic 1168 immunoreactivity to the Abcb1 mAB C218 (Albertus and Laine, 2001). Taken together, the 1169 above data suggest that teleost PXRs are involved in the regulation of detoxification pathways, 1170 which seems to include roles in the chemical induction of certain ABC transporters. 1171 The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor of the bHLH- 1172 PAS (basic Helix-Loop-Helix Per-ARNT-Sim) family of transcriptional regulators (Hahn, 1173 1998). AhR binds a broad spectrum of planar aromatic organic chemicals and acts as a key 1174 transcriptional regulator of a battery of genes that in mammals include the important phase I 1175 biotransformation enzymes CYP1A1 and CYP1A2, as well as phase II enzymes (Nebert et al., 1176 2000). Early studies in rodents have demonstrated induction of hepatic ABCB1 by carcinogens 1177 that are known AhR agonists, such as 2,3,7,8-tetrachlorodibenzodioxin, 2-acetylaminofluorene 1178 and 3-methylcholanthrene (Burt and Thorgeirsson, 1988; Fardel et al., 1996; Gant et al., 1991). 1179 However, subsequent investigations revealed that the mechanism of ABCB1 induction by these 1180 compounds does not involve the AhR (Deng et al., 2001; Teeter et al., 1991). In contrast, a role 1181 of AhR in the regulation of ABCG2 has been suggested based on results obtained in the human 1182 colon adenocarcinoma cell line Caco-2 (Ebert et al., 2005). 1183 In teleost fish, experimental exposures to AhR agonists had variable effects on the expression of 1184 ABC drug transporters. In killifish, injection with 3-methylcholanthrene provoked the expected 1185 induction of CYP1A in liver, but had no effects on hepatic levels of ABCB1-like proteins, 1186 determined in immunoblots with mABC C219 (Bard et al., 2002a; Cooper et al., 1999). 1187 Similarly, injection of cockscomb blennies with b-naphtoflavone did not alter hepatic expression 1188 of ABCB1-like proteins (Bard et al., 2002b). The dietary treatment of catfish with AhR agonists 1189 (b-naphtoflavone, 3,4,3’,4’-tetrachlorobiphenol and benzo[a]pyrene) failed to cause significant 1190 expression changes of ABCB1-like proteins in the intestinal mucosa, measured using mAB 1191 C219 (Doi et al., 2001). In another study, a significant increase in C219 immunostaining in the 34 1192 mucosa of the distal intestine was observed in catfish treated orally with vinblastine or b- 1193 naphthoflavone (Kleinow et al., 2000). Sub-chronic (14 d) waterborne exposure of Nile tilapia 1194 (Oreochromis niloticus) to the AhR agonist benzo[a]pyrene increased the mRNA levels of 1195 abcc2 in gills and of abcg2 in liver and proximal intestine, but had no effects on abcb1 and 1196 abcc1 transcript abundance (Costa et al., 2012). 1197 Peroxisome proliferator-activated receptors (PPARs) bind fatty acids and their metabolites and 1198 participate in the regulation of genes involved in energy metabolism (Feige et al., 2006). A role 1199 of PPARa in regulating ABCB4 expression has been demonstrated in mice (Kok et al., 2003), 1200 while the regulation of ABCG2 by PPARg was shown in myeloid cells (Szatmari et al., 2006). 1201 The farnesol X receptor (FXR) is a bile acid receptor mainly expressed in the liver and intestine 1202 and regulates bile acid synthesis and transport (Kalaany and Mangelsdorf, 2006). Roles of FXR 1203 have been demonstrated in the regulation of ABCB11 and ABCC2 (Ananthanarayanan et al., 1204 2001; Kast et al., 2002). Teleost PPARs and FXRs have been characterised (Krasowski et al., 1205 2011; Leaver et al., 2005; Reschly et al., 2008) but no data are available regarding their potential 1206 roles the regulation of ABC transporters. 1207 1208 5.4 Hormonal effects on ABC transporters 1209 The glucocorticoid receptor (GR) is a nuclear receptor that binds natural and synthetic 1210 glucocorticoids with high affinity and mediates genomic and non-genomic effects of these 1211 steroids (Bamberger et al., 1996). In addition to classical “glucocorticoid” roles related to 1212 metabolism, immune function, development and response to stressors (Charmandari et al., 2005; 1213 Yudt and Cidlowski, 2002), the GR has central roles in the osmoregulation of teleost fish (Bury 1214 and Sturm, 2007; Kiilerich et al., 2011). The effects of glucocorticoids on Abcc2-mediated 1215 transport were investigated in killifish kidney tubules (Prevoo et al., 2011). The rapid 1216 stimulation of Abcc2-dependent transport by the natural hormone cortisol and the synthetic 1217 glucocorticoids dexamethasone was inhibited by the GR antagonist RU486 but was insensitive 1218 to cycloheximide and actinomycin D, suggesting that dexamethasone effects were mediated by 1219 GR in a non-genomic fashion (Prevoo et al., 2011). The mechanism of dexamethasone 1220 activation of renal Abcc2 activity involved the tyrosine receptor kinase c-MET and MEK1/2 1221 (mitogen-activated protein kinase/extracellular signal regulated kinase kinase) (Prevoo et al., 1222 2011). 1223 The vasoconstrictive peptide hormone endothelin reduced the luminal secretion of ABCB1 and 1224 ABCC substrates by killifish proximal tubules (Masereeuw et al., 2000). These effects of ET 1225 were mediated through B-type ET receptor and involved protein kinase C (PKC) (Masereeuw et 1226 al., 2000). Similar inhibitory effects of ET involving PKC were shown on ABCC2-like transport 35 1227 in shark rectal gland (Miller and Masereeuw, 2002). A role of PKC in Abcb1-dependent renal 1228 transport in killifish had previously been shown (Miller et al., 1998b). Different nephrotoxicants 1229 decreased Abcc2-dependent transport activity by the ET -dependent pathway, most probably by 1230 causing an opening of calcium channels that resulted in an increase of intracellular Ca2+ levels 1231 which in turn triggered ET secretion (Terlouw et al., 2001). Subsequent studies demonstrated 1232 the involvement of nitric oxide synthase and guanylyl cyclase signalling in regulation of Abcc2- 1233 activity by ET (Notenboom et al., 2004, 2002). Another study investigated the effects of 1234 calciotropic hormones on Abcc2-dependent transport in teleost kidney (Wever et al., 2007). 1235 Parathyroid hormone (PTH) is a tetrapod hypercalcemic hormone that has recently been shown 1236 to exist in teleost fish, where its roles are unknown. In teleosts, the similar PTH-related protein 1237 (PTHrP) functions as hypercalcemic hormone, stimulating calcium uptake from water, whereas 1238 stanniocalcin (STC) blocks calcium uptake. PTH and PTHrP caused a similar partial inhibition 1239 of Abcc2-activity as ET, and showed additive effects when given together with ET (Wever et 1240 al., 2007). STC reversed the effects of PTHrP, but had no effect when given alone (Wever et al., 1241 2007). 1242 1243 5.5 Chemical effects on ABC transporter expression in fish 1244 A limited number of studies have investigated the inducibility of ABC drug transporters in fish 1245 by organic chemicals. Results obtained with PXR and AhR agonists have been reviewed above. 1246 The effects of the feed adulterants melamine and cyanuric acid on renal ABC transporter 1247 transcript levels were studied in a trial with rainbow trout (Oncorhynchus mykiss) (Benedetto et 1248 al., 2011). While the study does not provide statistical analyses, an apparent stimulation of 1249 abcc2 mRNA levels was observed when both compounds were combined (Benedetto et al., 1250 2011). The anti-parasitic compound emamectin benzoate (EMB), known to be an ABCB1 1251 substrate (Igboeli et al., 2012), was investigated regarding its effects on ABC transporters in 1252 rainbow trout following a standard seven-day administration of medicated feed (Cárcamo et al., 1253 2011). Abcc1 transcripts were up-regulated in all tissues investigated (liver, muscle, gill, kidney, 1254 intestine), with effects being most pronounced in the intestine (Cárcamo et al., 2011). In 1255 addition, a slight increase in hepatic and a small decrease in intestinal abcb1 transcript 1256 abundances was observed (Cárcamo et al., 2011). Waterborne exposure of juvenile rainbow 1257 trout to the cholesterol-lowering drug atorvastatin provoked the up-regulation of abcb1 and 1258 abcc1 transcripts in gill tissue (Ellesat et al., 2012). The effects of heavy fuel oil and 1259 perfluorooctane sulfonate (PFOS) on transcript levels of different ABC transporters (abcb1, 11, 1260 abcc2, 3, abcg2) were studied in thicklip grey mullet (Chelon labrosus) using a semi- 1261 quantitative RT-PCR method (Diaz de Cerio et al., 2012). The authors observed moderate 36 1262 increases of abcb1, abcb11 and abcg2 mRNA levels in liver and of abcb11 mRNA levels in 1263 brain following exposure to PFOS, as well as complex changes in transporter expression in oil 1264 and combined oil and PFOS treatments (Diaz de Cerio et al., 2012). 1265 A number of studies provide evidence for a regulation of teleost ABC drug efflux transporters 1266 by heavy metals. Exposure of killifish to sodium arsenite for 4 to 14 days increased Abcc2 1267 expression and activity in renal proximal tubules, but did not affect Abcc2 mRNA levels (Miller 1268 et al., 2007). Pre-exposure of fish to sodium arsenite provided protection against adverse effects 1269 of the metal on mitochondrial function in renal tubules (Miller et al., 2007). Cadmium (Cd), 1270 mercury (Hg), lead (Pb) and arsenic (As) stimulated the expression of abcc1 and abcc5 1271 transcripts in the zebrafish cell line ZF4 (Long et al., 2011a, 2011b). Overexpression of Abcc1 1272 in zebrafish embryos reduced the toxicity of Cd, Hg and As (Long et al., 2011a). In contrast, 1273 abcc5 overexpression had protective effects only with respect to Cd toxicity, but did not affect 1274 adverse effects of Hg or As (Long et al., 2011b). Exposure of zebrafish to Hg and Pb stimulated 1275 abcc2 transcription in intestine, liver and kidney (Long et al., 2011d). Overexpression of abcc2 1276 in ZF4 cells and embryos decreased the cellular accumulation of Cd, Hg and Pb (Long et al., 1277 2011d). The interaction of toxic metals (Cd, Cr, Hg, As) with ABC drug transporters was 1278 studied in the fish cell lines PLHC-1 (wild type) and PLHC-1/dox (see above) (Della Torre et 1279 al., 2012). All metals upregulated mRNA expression of abcc2, abcc3 and abcc4, and inhibited 1280 Abcb1- and Abcc-like transport activities (Della Torre et al., 2012). Prolonged exposure to Hg 1281 caused increased Abcc-like transport activity but did not affect mRNA levels of any of the 1282 transporters studied (Della Torre et al., 2012). Abcb10, a mitochondrial ABC transporter 1283 associated with the cellular response to oxidative stress, was significantly upregulated in muscle 1284 tissue of zebrafish kept for five days in tanks with sediments spiked with Cu and Cd. Abcb10 1285 transcript levels were also increased in brain, gill and intestinal tissues, albeit to a lesser extent 1286 (Sabri et al., 2012). 1287 1288 5.6 Altered ABC transporter expression in fish from polluted habitats 1289 A number of studies have found altered expression of ABC drug transporters in fish from 1290 polluted habitats. Using immunodetection with the mAB C219, an elevated hepatic expression 1291 of ABCB1-like proteins has been reported in killifish at a creosote-contaminated site when 1292 compared to a control site (Cooper et al., 1999). In another study, killifish from New Bedford 1293 Harbor (MA, USA), a habitat contaminated with planar halogenated aromatic hydrocarbons, 1294 showed decreased hepatic and increased intestinal levels of ABCB1-like proteins when 1295 compared to fish from a reference site (Bard et al., 2002a). However, when fish were kept in 1296 uncontaminated water in the laboratory for 11 weeks, hepatic levels of ABCB1-like proteins 37 1297 decreased in both populations, whereas intestinal levels decreased only in fish from New 1298 Bedford Harbor (Bard et al., 2002a). In another study with the intertidal species cockscomb 1299 blenny (Anoplarchus purpurescens), hepatic levels of ABCB1-like proteins were elevated at 1300 sites affected by pulp mill effluents, but there was no correlation between transporter expression 1301 and the distance to the pollution source (Bard et al., 2002b). ABCB1-like protein levels in liver 1302 decreased after maintenance of fish under controlled laboratory conditions, but attempts to 1303 induce hepatic transporters by exposure of blennies to contaminated sediment or prey items 1304 from the field were unsuccessful (Bard et al., 2002b). As mentioned above, experimental 1305 treatment of killifish with AhR agonists was without effect on ABCB1-like expression in both 1306 killifish and cockscomb blennies (Bard et al., 2002a, 2002b; Cooper et al., 1999). Another study 1307 investigated ABC transporter expression in killifish from the Sydney Tar Ponds, Nova Scotia, 1308 Canada, which are highly contaminated with PAHs, PCBs and heavy metals (Paetzold et al., 1309 2009). Compared to reference site animals, Sydney Tar Pond killifish showed increased hepatic 1310 transcript levels of abcc2, abcg2, cyp1a1 and gst-mu, whereas abcb1 and abcb11 mRNA levels 1311 were unchanged (Paetzold et al., 2009). Together, the above studies demonstrate that 1312 environmental pollutants can affect ABC transporter expression in fish; however, at present, the 1313 exact identity of chemical inducers remains unclear. Complicating the elucidation of possible 1314 links between pollutant exposure and the expression of ABC pumps in the tissues of feral fish, 1315 the possibility exists that chemicals of non-anthropogenic origin may have modulated ABC 1316 transporter expression in fish, as exemplified by the induction of abcb1 mRNA levels and 1317 ABCB1-like protein in the gills and liver of the teleost Jenynsia multidentata (Amé et al., 2009). 1318 1319 6. Summary and conclusions 1320 1321 In the last years, research on ABC drug transporters in teleost fish has substantially advanced. 1322 Progress in the field has been significantly facilitated by the completion of different teleost 1323 genome projects, through which ABC transporter sequences have become available and which 1324 have enabled the annotation of the ABC gene family in zebrafish and catfish; in addition, 1325 partially annoted ABC gene sequences of further teleost species are publically available at the 1326 Ensembl Genome Browser (www.ensembl.org). The comparison of the ABC family between 1327 genomes of teleosts and tetrapods revealed a number of clear orthologous relationships that are 1328 consistent with the hypothesis of conserved function. However, the presence of lineage-specific 1329 gene duplications, particularly in teleosts, complicates the picture in some subfamilies and 1330 further research is needed to elucidate the functional significance of the duplicated ABC 1331 transporter genes in teleosts. 38 1332 A number of studies have investigated the effects of exposure to chemicals on mRNA levels of 1333 ABC transporters in teleost tissues using quantitative RT-qPCR. While these works provide 1334 valuable insights into the regulation of ABC drug transporters in teleosts, it needs to be stressed 1335 that the relationship between mRNA abundance, transporter protein expression and transporter 1336 activity is not necessarily straightforward, reflecting the complexity of the different modes of 1337 genomic and non-genomic regulation of ABC proteins. In other words, the challenge is to link 1338 mRNA levels to protein expression and transport activity. While some progress has been 1339 achieved regarding transport assays of improved specificity, the very limited availability of 1340 antibodies allowing to detect specific ABC transporters in teleosts still represents a significant 1341 obstacle in understanding the regulation of ABC drug pumps in fish. 1342 About two decades ago, results obtained by transport activity measurements and 1343 immunochemical methods revealed that the tissue distribution of the ABC drug transporter P- 1344 glycoprotein/Abcb1 in teleosts resembles that in mammals, suggesting similar functional 1345 properties and physiological roles of ABC pumps in both vertebrate groups. Since then, ABC 1346 protein-dependent transport activities have been characterised in a number of organ culture 1347 models from teleosts, employing a range of fluorescent probes and transporter inhibitors of 1348 known specificity in mammals. In particular, isolated kidney tubules of certain marine teleosts 1349 retain epithelial polarity and transport activities during short-term culture, and have proven a 1350 very useful model for the investigation of renal drug transport in teleosts. 1351 However, in vitro models are not available for all teleost tissues of interest, which currently 1352 restricts feasible approaches to study ABC transporter function in situ. E.g., very little is known 1353 on ABC transporter-mediated processes at teleost branchial and intestinal epithelia. Given the 1354 high importance of both the gills and the gut as surfaces forming external body boundaries in 1355 fish, studies elucidating the role of ABC transporters in chemical uptake and elimination at these 1356 sites are urgently needed to improve current models of chemical fate in fish. The activity of 1357 hepatic ABC drug transporters has been studied in a number of teleosts, usually employing 1358 isolated cultured hepatocytes. However, the common cell monolayer system of hepatocyte 1359 primary culture is not ideally suited for transport studies, as cellular polarity is lost during cell 1360 isolation and usually does not re-establish in culture. A number of alternative primary culture 1361 systems has been proposed but these experimental model systems remain as yet unexplored 1362 regarding their suitability for studies of ABC transporters. 1363 While isolated or cultured tissues are experimental systems depicting the in vivo situation with a 1364 certain degree of realism, such primary culture models usually contain a range of drug 1365 transporters, which limits their usefulness in studies aiming at the characterisation of specific 1366 ABC transporters. Systems allowing functional studies of individual ABC drug transporters 39 1367 have recently become available in teleosts and include drug-selected cell lines overexpressing 1368 specific ABC drug transporters, as well as insect cell / baculovirus systems suitable for the 1369 recombinant expression of ABC pumps. Another approach to address the functions of specific 1370 ABC transporters is provided by reverse genetic methodologies such as gene-knock down 1371 studies in zebrafish embryos. 1372 Considering that certain fish species, such as zebrafish, are increasingly used as vertebrate 1373 model systems in biomedical and ecotoxicological research, it is of utmost importance to 1374 understand the biochemical and cellular factors driving the toxicokinetic and toxicodynamic 1375 properties of chemicals. While the relevance of active transport mediated by ABC transporters is 1376 well accepted in this regard in pharmacology, ABC proteins have so far attracted only limited 1377 attention in aquatic toxicology. It is hoped that the overview of the current state of ABC 1378 transporter related research in teleost fish provided in this article will stimulate research further 1379 elucidating organ-specific roles of these transporters as factors affecting the fate and biological 1380 effects of chemicals in fish. 1381 In summary, while many details regarding the physiological function and ecotoxicological 1382 relevance of ABC transporters await being clarified in teleosts, there has been significant 1383 progress in characterising the complement of teleostean ABC pumps. Moreover, methodologies 1384 have become available that allow addressing both fundamental questions of transporter 1385 regulation and function in teleosts, and applied aspects such as the interaction of ABC pumps 1386 with pharmacologically and ecotoxicologically relevant chemicals. The aspects of chemical 1387 transporter interaction need to be taken into account in studies addressing toxicokinetic and 1388 toxicodynamic processes of chemicals in fish. 1389 1390 Acknowledgements. TL and SF acknowledge support with travel grants from the European 1391 Network on Fish Biomedical Models (EuFishBioMed; COST Action BM0804) that enabled a 1392 meeting of the authors in Stirling to complete a first draft of the review article. AS gratefully 1393 acknowledges support from the MASTS pooling initiative (The Marine Alliance for Science and 1394 Technology for Scotland). 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The oldest articulated osteichthyan reveals mosaic gnathostome characters. Nature 458, 469–74. Zucchi, S., Corsi, I., Luckenbach, T., Mala, S., Regoli, F., Focardi, S., 2010. Identification of five partial ABC genes in the liver of the Antarctic fish Trematomus bernacchii and sensitivity of ABCB1 and ABCC2 to Cd exposure. Environ. Pollut. 158, 2746–56. 56 2156 Legends 2157 2158 2159 2160 2161 2162 2163 2164 2165 2166 2167 2168 2169 Figure 1. ABC drug efflux and biotransformation metabolism in a hypothetical epithelial cell. (1) Hydrophobic chemicals (filled circles) can enter cells by passive diffusion (dashed arrows). (2) Hydrophobic chemicals may be extruded from the cell by the active transport activity (filled arrows) of ABC transporters (T) such as ABCB1 (MDR1 P-glyoprotein) or ABCG2 (Breast cancer related protein), which in polarised epithelia usually localise to the apical side. (3) Hydrophobic chemicals may undergo biotransformation metabolism, in which phase I usually leads to more polar products (open circles), while phase II involves conjugation with endogenous anionic moieties (triangles). (4) ABC drug transporters of the ABCC subfamily (Multidrug resistance associated proteins) mediate the cellular efflux of organic anions and conjugated metabolites. ABCC pumps can show an apical or basolateral localisation, depending on the isoform. The schematic is based on the mammalian intestine and was modified from (Chan, 2004). 2170 2171 2172 2173 2174 2175 2176 2177 2178 2179 2180 Figure 2. Phylogenetic analysis of vertebrate ABCB/Abcb subfamily full transporters. Amino acid sequences of transporters from teleosts (Danio rerio, Gasterosteus aculeatus, Gadus morhua, Oreochromis niloticus Oryzias latipes, Takifugu rubripes, Tetraodon nigroviridis) and further vertebrates (Latimeria chalumnae, Xenopus tropicalis, Gallus gallus and Homo sapiens) (see Table S1 for accession numbers) were aligned using the programme TCoffee (Notredame et al., 2000) and a phylogenetic tree constructed using the neighbor-joining as implemented in the software MEGA5 (Tamura et al., 2011). The percentage concordance based on 1000 bootstrap iterations is shown at the nodes. Trees obtained with the alternative maximum likelihood and minimum evolution methods had very similar topologies (data not shown), indicating that the results are robust. 2181 Figure 3. Phylogenetic analysis of vertebrate ABCC1-5/Abcc1-5 transporters. Included in 2183 the analysis are transporter amino acid sequences from teleosts (Danio rerio, Gasterosteus 2184 aculeatus, Gadus morhua, Oreochromis niloticus Oryzias latipes, Takifugu rubripes, Tetraodon 2185 nigroviridis) and other vertebrates. For further details see the legend of Fig. 2. 2182 2186 Figure 4. Phylogenetic analysis of vertebrate ABCG2/Abcg2 transporters. Included in the 2188 analysis are transporter amino acid sequences from teleosts (Danio rerio, Gasterosteus 2189 aculeatus, Gadus morhua, Oreochromis niloticus Oryzias latipes, Takifugu rubripes, Tetraodon 2190 nigroviridis) and other vertebrates. For further details see the legend of Fig. 1. 2187 2191 Figure 5. Conserved synteny of the human (Homo sapiens) ABCG2 region to homologous 2193 regions in teleost genomes. Dotted lines illustrate that the human ABCG2 region is syntenic to 2194 green spotted pufferfish (Tetraodon nigroviridis) abcg2a and zebrafish (Danio rerio) abcg2d. 2192 57 2195 2196 2197 2198 2199 2200 2201 2202 2203 Figure 6. Expression and activity of ABC drug transporters in teleost tissues. Currently available data support the view that ABC drug transporters are involved in the renal (1) and biliary (2) excretion of organic chemicals and limit the uptake of food-borne chemicals in the gut (3). Moreover, ABC drug transporters in the endothelial capillaries constituting the blooodbrain barrier contribute to the low penetration of foreign chemicals into the brain (4). ABC drug transporters could potentially have roles in further teleost tissues including the gill epithelium and the blood-gonadal barriers; however, more research is required to substantiate or refute such roles. 2204 Table 1. Measurement of ABC transporter-related drug efflux activities in teleost tissues. The 2206 table provides an overview of systems in which the activity of teleost ABC transporters has been 2207 measured, providing details regarding the tissue preparation and species used, the transporter 2208 activity recorded and the model substrates and inhibitors applied. 2205 2209 2210 2211 2212 2213 2214 2215 2216 2217 2218 2219 2220 2221 2222 2223 2224 2225 2226 2227 Table 2 Interactions of chemicals with teleostean ABC efflux transporters. Different types of cytotoxicity assays, as well as fluorescent dye accumulation and ATPase activity assays, have been used to reveal chemical interaction with teleost ABC transporters. In these assays, chemicals can interact with ABC transporters as substrates (S) and/or inhibitors (I), or fail to show such interaction (non-interacting compounds, N). Teleost transporters studied to date include Poeciliopsis lucida Abcb1, which is overexpressed in the cell line PLHC-1/dox (Caminada et al., 2008; Zaja et al., 2008a; 2011), and Danio rerio Abcb4, which has been expressed in baculovirus-transfected insect cells (Fischer et al., 2013). Compounds showing decreased cytotoxicity in PLHC-1/dox as compared to the parental cell line PLHC-1 are putative transport substrates of P. lucida Abcb1, as are chemicals the cytotoxicity of which is potentiated by the ABCB1/Abcb1 inhibitor cyclosporin A. Chemicals reversing the doxorubicin resistance of PLHC-1/dox or enhancing the accumulation of the fluorescent ABCB1/Abcb1 substrate calcein-AM are regarded inhibitors of P. lucida Abcb1 transport activity. In ATPase assays, substrates have been defined as chemicals stimulating ATPase activity, while inhibitors have been defined as substances decreasing basal ATPase activity in a study with P. lucida Abcb1 (Zaja et al., 2011) and as compounds decreasing verapamil-stimulated activities in a study with D. rerio Abcb4 (Fischer et al., 2013). Please see text for further explanations. 58 2228 Table 1. Preparation Species Transporter Substrates Inhibitors Study Isolated renal proximal tubules Killifish (Fundulus heteroclitus) ABCB1-‐like Daunomycin, NBD-‐ cyclosporine A, BODIPY-‐ Ivermectin Verapamil, cyclosporin A, vinblastine, PSC-‐833 Miller 1995; Schramm et al, 1995; Fricker et al., 1999 ABCC2-‐like Fluorescein-‐ methotrexate, lucifer yellow Probenecid, bromosulfophtalein Masereeuw et al., 1996; 1999 ABCC4-‐like Fluo-‐cAMP MK571, LTC4, cAMP, adefovir Reichel et al., 2007 Isolated Rainbow trout ABCB1-‐like hepatocytes (Oncorhynchus mykiss) Rhodamine 123, Verapamil, cyclosporin calcein-‐AM, A, vinblastine, reversin BODIPY-‐verapamil, 205 doxorubicin ABCB11-‐ like Dihydrofluorescein taurocholate, Zaja et diactetate taurochenodeoxycholate al.,2008 ABCC2-‐like Rhodamine 123, calcein-‐AM MK571, verapamil Sturm et al., 2001; Zaja et al., 2008 Zaja et al., 2008 Intestine membrane vesicles Channel catfish (Ictalurus punctatus) ABCB1-‐like [3H] vinblastine Verapamil Doi et al., 2001 Isolated brain capillaries Killifish (Fundulus heteroclitus) ABCB1-‐like NBD-‐cyclosporin A, BODIPY-‐ verapamil PSC-‐833 Miller et al., 2002 ABCC2-‐like Fluorescein-‐ methotrexate LTC4 Miller et al., 2002 Embryo Zebrafish (Danio rerio) Abcb4 (ABCB1-‐ like) rhodamine B, calcein-‐AM, BODIPY-‐ vinblastine cyclosporin A, PSC-‐833, MK571 Fischer et al., 2013 59 2229 2230 Table 2 Substance Poeciliopsis lucida Abcb1 Cytotoxicity assays Cytostatic drugs Doxorubicin Daunorubicin Vinblastine Vincristine Etoposide Mitoxantrone 5-‐Fluorouracil Methotrexate Cisplatin Calcium channel blockers Verapamil 3 S 3 S 3 S 3 S 3 S n.d. n.d. 3 N 3 N I 6 Nicardipine n.d. Diltiazem n.d. Alkaloids Quinidin n.d. Reserpine n.d. 3 Colchicine S Fluorescent model substrates Rhodamine 123 n.d. Rhodamine B n.d. Calcein-‐AM n.d. Hoechst 3334 n.d. Model inhibitors 6 Cyclosporin A I 6 PSC-‐833 I 6 MK571 N Reversine 205 n.d. Ko143 n.d. Environmental Pharmaceuticals Pravastatin n.d. 7 Atorvastatin S 6 Sildenafil I 7 Propranolol S 6 Tamoxifen I 6 Fenofibrate I 7 Acebututolol S Pesticides Calcein-‐AM assay 4 ATPase assay 1 ATPase assay 5 N n.d. 4 N n.d. 4 N 4 N 4 N n.d. n.d. N n.d. N n.d. N I I N N I n.d. 5 I, S 5 5 I , S n.d. n.d. n.d. n.d. n.d. 4 S S 4 I 4 I S S n.d. n.d. I 4 I 4 I 4 S I S n.d. n.d. n.d. n.d. n.d. n.d. 4 I S n.d. n.d. I n.d. 5 S I, S n.d. 4 I I I S N I, S I, S I n.d. n.d. S I S S S n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. I I 4 I 4 I 4 I 4 I 4 N 4 I 4 I 4 I 4 I n.d. n.d. 60 Danio rerio Abcb4 2 4 2231 2232 2233 2234 2235 2236 2237 2238 2239 2240 2241 Endosulfan n.d. I I n.d. 4 Diazinon n.d. I S n.d. 4 Fenoxycarb n.d. I N n.d. 4 Chlorpyrifos n.d. N S n.d. 4 Malathion n.d. I N n.d. 4 Fosalon n.d. I S n.d. Dichlorodiphenyl-‐ 4 n.d. I I n.d. dichloroethylene Polycyclic musks Galaxolide n.d. n.d. n.d. I, S 5 Tonalide n.d. n.d. n.d. I , S Polycyclic aromatic hydrocarbons 4 Benzo[a]pyrene n.d. N N n.d. 5 Phenanthrene n.d. n.d. n.d. I, S Toxic Metals 4 As2O3 n.d. N S n.d. n.d.: not determined. 1 Inhibition effects were determined on basal ATPase activities (Zaja et al., 2011). 2 Inhibition effects were determined on verapamil-‐stimulated ATPase activities (Fischer et al., 2013). 3 S: The cytotoxicity in PLHC-‐1/dox was decreased as compared to that in PLHC-‐1; N: The cytotoxicity was similar in both cell lines (Zaja et al., 2008a). 4 Zaja et al., 2011. 5 Effects were statistically not significant (Fischer et al., 2013). 6 I: The compound enhanced the cytotoxicity of doxorubicin in PLHC-‐1/dox; N: The compound had no effects on doxorubicin cytotoxicity (Caminada et al., 2008; Zaja et al., 2008a). 7 The cytotoxic effects of the compound were enhanced by the ABCB1/Abcb1 inhibitor cyclosporin A (Caminada et al., 2008). 61 2242 Fig. 1 2243 4 2 1 apical T T Phase 1 Phase 2 3 Biotransformation 4 basolateral 1 2244 2245 62 Takifugu rubripes Abcb1 like 100 Tetraodon nigroviridis Abcb1like 100 Takifugu rubripes Abcb4 like 68 100 Tetraodon nigroviridis Abcb4 like Gadus morhua ABCB4 100 Oreochromis niloticus Abcb4 100 Oryzias latipes Abcb4 100 Danio rerio Abcb4 Latimeria chalumnae Abcb1/Abcb4 like Xenopus tropicalis ABCB1 49 Gallus gallus ABCB1 99 84 Abcb1/Abcb4 Gasterosteus aculeatus Abcb4 68 64 Gallus gallus ABCB4 100 Homo sapiens ABCB1 100 Homo sapiens ABCB4 Latimeria chalumnae Abcb5 100 Homo sapiens ABCB5 100 86 100 Xenopus tropicalis ABCB5b Danio rerio Abcb5 Gallus gallus ABCB5 100 Abcb5 Xenopus tropicalis ABCB5a Xenopus tropicalis ABCB5c 100 47 Xenopus tropicalis ABCB5d Gadus morhua Abcb11b 100 100 Oreochromis niloticus Abcb11 95 Danio rerio Abcb11a Xenopus tropicalis Abcb11 100 Gallus gallus ABCB11 50 95 Gadus morhua Abcb11a 88 Danio rerio Abcb11b 92 100 Takifugu rubripes Abcb11 38 Tetraodon nigroviridis Abcb11 Gasterosteus aculeatus Abcb11 98 58 Oreochromis niloticus Abcb11a 39 Oryzias latipes Abcb11 Homo sapiens ABCC1 Outgroup 2246 Fig. 2 0.05 2247 63 Abcb11 Homo sapiens ABCB11 Latimeria chalumnae Abcb11 Gasterosteus aculeatus Abcc1 86 100 Oreochromis niloticus Abcc1 Oryzias latipes Abcc1 99 100 Tetraodon nigroviridis Abcc1 100 Danio rerio Abcc1 Xenopus tropicalis ABCC1 Abcc1 Takifugu rubripes Abcc1 100 Latimeria chalumnae Abcc1 90 100 Gallus gallus ABCC1 97 65 Homo sapiens ABCC1 Homo sapiens ABCC3 Gallus gallus ABCC3 95 100 Xenopus tropicalis ABCC3 Danio rerio Abcc3 84 100 Oryzias latipes Abcc3 97 100 Oreochromis niloticus Abcc3 99 Abcc3 Gadus morhua Abcc3 Gasterosteus aculeatus Abcc3 36 Takifugu rubripes Abcc3 100 Tetraodon nigroviridis Abcc3 Gallus gallus ABCC2 69 80 Xenopus tropicalis ABCC2 51 Homo sapiens ABCC2 Latimeria chalumnae Abcc2 Abcc2 Danio rerio Abcc2 100 Gadus morhua Abcc2 100 Takifugu rubripes Abcc2 100 99 Tetraodon nigroviridis Abcc2 Gasterosteus aculeatus Abcc2 57 Oreochromis niloticus Abcc2 56 99 Oryzias latipes Abcc2 Gallus gallus ABCC5 100 100 Homo sapiens ABCC5 100 Xenopus tropicalis ABCC5 Oryzias latipes Abcc5 100 Gasterosteus aculeatus Abcc5 91 100 Oreochromis niloticus Abcc5 87 Abcc5 Danio rerio Abcc5 Takifugu rubripes Abcc5 100 Tetraodon nigroviridis Abcc5 Oryzias latipes Abcc4b Gasterosteus aculeatus Abcc4c 99 Oreochromis niloticus Abcc4c 100 Takifugu rubripes Abcc4c 100 100 Tetraodon nigroviridis Abcc4d Gallus gallus ABCC4 75 100 Homo sapiens ABCC4 99 Xenopus tropicalis ABCC4 Gasterosteus aculeatus Abcc4a 84 Oreochromis niloticus Abcc4b Oryzias latipes Abcc4a 100 67 Takifugu rubripes Abcc4a 100 100 Tetraodon nigroviridis Abcc4c Gadus morhua Abcc4b 52 Gadus morhua Abcc4a Danio rerio Abcc4 83 Gasterosteus aculeatus Abcc4b 79 99 Oreochromis niloticus Abcc4a Takifugu rubripes Abcc4b 100 100 Tetraodon nigroviridis Abcc4a 100 Tetraodon nigroviridis Abcc4b Homo sapiens ABCB1 Outgroup 2248 Fig. 3 0.05 2249 64 Abcc4 98 2250 Fig. 4 Gadus morhua Abcg2b 36 Oreochromis niloticus Abcg2b 84 Gasterosteus aculeatus Abcg2b 46 58 Oryzias latipes Abcg2b Takifugu rubripes Abcg2b 59 68 Tetraodon nigroviridis Abcg2b 73 Danio rerio Abcg2a Danio rerio Abcg2d Gadus morhua Abcg2a 91 Oreochromis niloticus Abcg2a 85 100 Oryzias latipes Abcg2a 82 Gasterosteus aculeatus Abcg2a 100 Takifugu rubripes Abcg2a 62 100 Tetraodon nigroviridis Abcg2a Gallus gallus ABCG2 66 Homo sapiens ABCG2 100 94 Xenopus tropicalis ABCG2 52 Latimeria chalumnae Abcg2a Latimeria chalumnae Abcg2b 100 Gadus morhua Abcg2c Danio rerio Abcg2c Gadus morhua Abcg2d 100 Danio rerio Abcg2b 92 Oryzias latipes Abcg2c 74 Gasterosteus aculeatus Abcg2c 54 100 Oreochromis niloticus Abcg2c 69 Takifugu rubripes Abcg2c 76 Tetraodon nigroviridis Abcg2c Homo sapiens ABCB1 Outgroup 0.05 2251 2252 65 2253 Fig. 5 2254 2255 66 2256 Fig. 6 1 4 2 3 2257 2258 2259 67 2260 2261 Supplemental material Table S1. ABC tranporter nucleotide and amino acid sequence accession nos. from Ensembl of 2263 Danio rerio, Gasterosteus aculeatus, Gadus morhua, Oreochromis niloticus Oryzias latipes, 2264 Takifugu rubripes, Tetraodon nigroviridis, Latimeria chalumnae, Homo sapiens, Gallus gallus 2265 and Xenopus tropicalis. Sequences were used for phylogenetic and sequence identity analyses. 2262 2266 2267 2268 2269 2270 2271 2272 2273 Figure S1. Phylogenetic tree based on the multiple alignments (T-Coffee) of Abcc1, Abcc2, Abcc3, Abcc4, Abcc5, Abcc6, Abcc7, Abcc8, Abcc9, Abcc10, Abcc11, Abcc12 and Abcc13 ABC transporter amino acid sequences of available teleosts sequences (Ensembl) of Danio rerio, Gasterosteus aculeatus, Gadus morhua, Oreochromis niloticus Oryzias latipes, Takifugu rubripes, Tetraodon nigroviridis and other vertebrates. The tree was generated with MEGA5 using the neighbor-joining method with percentage concordance based on 1000 bootstrap iterations is shown at the nodes. Protein accession no. (Ensembl) are listed in Table S1. 2274 2275 2276 2277 2278 2279 2280 2281 Figure S2. Phylogenetic tree based on the multiple alignments (T-Coffee) of Abcg1, Abcg2, Abcg4, Abcg5 and Abcg8 ABC transporter amino acid sequences of available teleosts sequences (Ensembl) of Danio rerio, Gasterosteus aculeatus, Gadus morhua, Oreochromis niloticus Oryzias latipes, Takifugu rubripes, Tetraodon nigroviridis and other vertebrates. The tree was generated with MEGA5 using the neighbor-joining method with percentage concordance based on 1000 bootstrap iterations is shown at the nodes. Protein accession no. (Ensembl) are listed in Table S1. 2282 68 Table S1 Species Danio rerio (Zebrafish) Gasterosteus aculeatus (Stickleback) Gadus morhua ABC transporter gene / protein name abcb4 / Abcb4 abcb5 / Abcb5 abcb11a / Abcb11a abcb11b / Abcb11b abcc1 / Abcc1 abcc2 / Abcc2 abcc3 / Abcc3 abcc4 / Abcc4 abcc5 / Abcc5 abcc6a / Abcc6a abcc6b / Abcc6b abcc6c / Abcc6c abcc7 / Abcc7 abcc8a / Abcc8a abcc8b / Abcc8b abcc8c / Abcc8c abcc9 / Abcc9 abcc10 / Abcc10 abcc12 / Abcc12 abcc13 / Abcc13 abcg1 /Abcg1 abcg2a / Abcg2a abcg2b / Abcg2b abcg2c / Abcg2c abcg2d / Abcg2d abcg4a / Abcg4a abcg4b / Abcg4b abcg5 / Abcg5 abcg8 / Abcg8 abcb4 / Abcb4 abcb11 / Abcb11 abcc1 / Abcc1 abcc2 / Abcc2 abcc3 / Abcc3 abcc4a / Abcc4a abcc4b / Abcc4b abcc4c / Abcc4c abcc5 / Abcc5 abcc6a / Abcc6a abcc6b / Abcc6b abcc7 / Abcc7 abcc8 / Abcc8 abcc9 / Abcc9 abcc10 / Abcc10 abcc11 / Abcc11 abcg2a / Abcg2a abcg2b / Abcg2b abcg2c / Abcg2c abcg4a / Abcg4a abcg4b / Abcg4b abcg5 / Abcg5 abcg8 / Abcg8 abcb4 / Abcb4 Nucleotide sequence accession no. ENSDART00000148456 ENSDART00000141616 ENSDART00000139631 ENSDART00000102557 ENSDART00000083659 ENSDART00000016604 ENSDART00000114196 ENSDART00000065500 ENSDART00000087070 ENSDART00000065433 ENSDART00000042812 ENSDART00000131340 ENSDART00000060243 ENSDART00000149455 ENSDART00000098668 ENSDART00000088191 ENSDART00000013990 ENSDART00000115249 ENSDART00000149382 ENSDART00000090514 ENSDART00000092809 NSDART00000063180 ENSDART00000091798 ENSDART00000032322 ENSDART00000022733 ENSDART00000141444 ENSDART00000115051 ENSDART00000091845 ENSDART00000147541 ENSGACT00000012310 ENSGACT00000018438 ENSGACT00000000566 ENSGACT00000009869 ENSGACT00000007846 ENSGACT00000005464 ENSGACT00000018513 ENSGACT00000018532 ENSGACT00000023167 ENSGACT00000025400 ENSGACT00000004009 ENSGACT00000011967 ENSGACT00000014622 ENSGACT00000001433 ENSGACT00000026226 ENSGACT00000019659 ENSGACT00000022934 ENSGACT00000024623 ENSGACT00000022003 ENSGACT00000007022 ENSGACT00000027032 ENSGACT00000014133 ENSGACT00000014147 ENSGMOT00000016707 1 Amino acid sequence accession no. ENSDARP00000123836 ENSDARP00000121883 ENSDARP00000118702 ENSDARP00000093333 ENSDARP00000078094 ENSDARP00000025026 ENSDARP00000099145 ENSDARP00000065499 ENSDARP00000081504 ENSDARP00000065432 ENSDARP00000042811 ENSDARP00000118198 ENSDARP00000060242 ENSDARP00000124481 ENSDARP00000089439 ENSDARP00000082624 ENSDARP00000014386 ENSDARP00000101288 ENSDARP00000123782 ENSDARP00000084947 ENSDARP00000087241 ENSDARP00000063179 ENSDARP00000086231 ENSDARP00000033222 ENSDARP00000024285 ENSDARP00000116517 ENSDARP00000102351 ENSDARP00000086278 ENSDARP00000120245 ENSGACP00000012286 ENSGACP00000018402 ENSGACP00000000566 ENSGACP00000009848 ENSGACP00000007827 ENSGACP00000005448 ENSGACP00000018477 ENSGACP00000018496 ENSGACP00000023123 ENSGACP00000025351 ENSGACP00000003995 ENSGACP00000011943 ENSGACP00000014596 ENSGACP00000001432 ENSGACP00000026175 ENSGACP00000019621 ENSGACP00000022891 ENSGACP00000024574 ENSGACP00000021962 ENSGACP00000007004 ENSGACP00000026980 ENSGACP00000014108 ENSGACP00000014122 ENSGMOP00000016292 (Cod) Oreochromis niloticus (Tilapia) Oryzias latipes (Medaka) abcb11a / Abcb11a abcb11b / Abcb11b abcc2 / Abcc2 abcc3 / Abcc3 abcc4a / Abcc4a abcc4b / Abcc4b abcc6 / Abcc6 abcc7 / Abcc7 abcc8 / Abcc8 abcc9 / Abcc9 abcc10 / Abcc10 abcc11 / Abcc11 abcg2a / Abcg2a abcg2b / Abcg2b abcg2c / Abcg2c abcg2d / Abcg2d abcg4 / Abcg4 abcg5 / Abcg5 abcg8 / Abcg8 abcb4 / Abcb4 abcb11a / Abcb11a abcb11b / Abcb11b abcc1 / Abcc1 abcc2 / Abcc2 abcc3 / Abcc3 abcc4a / Abcc4a abcc4b / Abcc4b abcc4c / Abcc4c abcc5 / Abcc5 abcc6 / Abcc6 abcc7 / Abcc7 abcc8 / Abcc8 abcc9 / Abcc9 abcc10 / Abcc10 abcc12 / Abcc12 abcg1 / Abcg1 abcg2a / Abcg2a abcg2b / Abcg2b abcg2c / Abcg2c abcg4a / Abcg4a abcg4b / Abcg4b abcg5 / Abcg5 abcg8 / Abcg8 ENSGMOT00000011098 ENSGMOT00000015568 ENSGMOT00000020053 ENSGMOT00000011036 ENSGMOT00000009590 ENSGMOT00000002180 ENSGMOT00000006327 ENSGMOT00000003159 ENSGMOT00000005414 ENSGMOT00000001393 ENSGMOT00000007808 ENSGMOT00000011684 ENSGMOT00000017881 ENSGMOT00000010794 ENSGMOT00000004369 ENSGMOT00000008528 ENSGMOT00000005930 ENSGMOT00000009159 ENSGMOT00000009161 ENSONIT00000007736 ENSONIT00000014922 ENSONIT00000011102 ENSONIT00000009864 ENSONIT00000011890 ENSONIT00000024679 ENSONIT00000014972 ENSONIT00000010639 ENSONIT00000014996 ENSONIT00000003049 ENSONIT00000023776 ENSONIT00000004931 ENSONIT00000013201 ENSONIT00000022648 ENSONIT00000000146 ENSONIT00000003880 ENSONIT00000021747 ENSONIT00000014693 ENSONIT00000022022 ENSONIT00000004019 ENSONIT00000013235 ENSONIT00000006751 ENSONIT00000001152 ENSONIT00000001157 ENSGMOP00000010800 ENSGMOP00000015178 ENSGMOP00000019576 ENSGMOP00000010738 ENSGMOP00000009337 ENSGMOP00000002113 ENSGMOP00000006151 ENSGMOP00000003062 ENSGMOP00000005256 ENSGMOP00000001344 ENSGMOP00000007591 ENSGMOP00000011376 ENSGMOP00000017450 ENSGMOP00000010507 ENSGMOP00000004238 ENSGMOP00000008293 ENSGMOP00000005762 ENSGMOP00000008913 ENSGMOP00000008915 ENSONIP00000007731 ENSONIP00000014910 ENSONIP00000011093 ENSONIP00000009858 ENSONIP00000011881 ENSONIP00000024658 ENSONIP00000014960 ENSONIP00000010630 ENSONIP00000014984 ENSONIP00000003048 ENSONIP00000023755 ENSONIP00000004928 ENSONIP00000013191 ENSONIP00000022628 ENSONIP00000000147 ENSONIP00000003879 ENSONIP00000021728 ENSONIP00000014681 ENSONIP00000022003 ENSONIP00000004018 ENSONIP00000013225 ENSONIP00000006746 ENSONIP00000001153 ENSONIP00000001158 abcb4 / Abcb4 abcb11 / Abcb11 abcc1 / Abcc1 abcc2 / Abcc2 abcc3 / Abcc3 abcc4a / Abcc4a abcc4b / Abcc4b abcc5 / Abcc5 abcc6 / Abcc6 abcc7 / Abcc7 abcc8 / Abcc8 abcc9 / Abcc9 abcc10 / Abcc10 abcc11 / Abcc11 ENSORLT00000011623 ENSORLT00000004295 ENSORLT00000021452 ENSORLT00000010370 ENSORLT00000025691 ENSORLT00000022220 ENSORLT00000004555 ENSORLT00000000960 ENSORLT00000016844 ENSORLT00000024332 ENSORLT00000020722 ENSORLT00000021374 ENSORLT00000000121 ENSORLT00000018788 ENSORLP00000011622 ENSORLP00000004294 ENSORLP00000021451 ENSORLP00000010369 ENSORLP00000025690 ENSORLP00000022219 ENSORLP00000004554 ENSORLP00000000959 ENSORLP00000016843 ENSORLP00000024331 ENSORLP00000020721 ENSORLP00000021373 ENSORLP00000000121 ENSORLP00000018787 2 Takifugu rubripes (Japanese pufferfish) Tetraodon nigroviridis (Green spotted pufferfish) abcg1 / Abcg1 abcg2a / Abcg2a abcg2b / Abcg2b abcg2c / Abcg2c abcg4a / Abcg4a abcg4b / Abcg4b abcg5 / Abcg5 abcg8 / Abcg8 abcb1 / Abcb1 like abcb4 / Abcb4 like abcb11 / Abcb11 abcc1 / Abcc1 abcc2 / Abcc2 abcc3 / Abcc3 abcc4a / Abcc4a abcc4b / Abcc4b abcc4c / Abcc4c abcc5 / Abcc5 abcc6 / Abcc6 abcc7 / Abcc7 abcc8 / Abcc8 abcc9 / Abcc9 abcc10 / Abcc10 abcc11 / Abcc11 abcg1 / Abcg1 abcg2a / Abcg2a abcg2b / Abcg2b abcg2c / Abcg2c abcg4a / Abcg4a abcg4b / Abcg4b abcg5 / Abcg5 abcg8 / Abcg8 abcb1 / Abcb1 like abcb4 / Abcb4 like abcb11 / Abcb11 abcc1 / Abcc1 abcc2 / Abcc2 abcc3 / Abcc3 abcc4a / Abcc4a abcc4b / Abcc4b abcc4c / Abcc4c abcc4d / Abcc4d abcc5 / Abcc5 abcc6 / Abcc6 abcc7 / Abcc7 abcc8 / Abcc8 abcc9 / Abcc9 abcc10 / Abcc10 abcc11 / Abcc11 abcg1 / Abcg1 abcg2a / Abcg2a abcg2b / Abcg2b abcg2c / Abcg2c abcg4a / Abcg4a abcg4b / Abcg4b abcg5 / Abcg5 abcg8 / Abcg8 ENSORLT00000025749 ENSORLT00000008770 ENSORLT00000003412 ENSORLT00000011474 ENSORLT00000009371 ENSORLT00000019248 ENSORLT00000005857 ENSORLT00000005822 ENSTRUT00000014104 ENSTRUT00000014108 ENSTRUT00000027283 ENSTRUT00000030776 ENSTRUT00000023561 ENSTRUT00000005585 ENSTRUT00000010491 ENSTRUT00000015188 ENSTRUT00000011240 ENSTRUT00000033438 ENSTRUT00000029179 ENSTRUT00000044068 ENSTRUT00000014610 ENSTRUT00000023281 ENSTRUT00000019563 ENSTRUT00000021154 ENSTRUT00000007627 ENSTRUT00000029962 ENSTRUT00000041435 ENSTRUT00000032303 ENSTRUT00000010907 ENSTRUT00000042674 ENSTRUT00000023802 ENSTRUT00000023046 ENSTNIT00000000709 ENSTNIT00000000556 ENSTNIT00000001567 ENSTNIT00000003269 ENSTNIT00000019842 ENSTNIT00000006943 ENSTNIT00000009033 ENSTNIT00000009032 ENSTNIT00000022945 ENSTNIT00000013433 ENSTNIT00000019049 ENSTNIT00000015231 ENSTNIT00000000462 ENSTNIT00000001610 ENSTNIT00000000547 ENSTNIT00000009220 ENSTNIT00000015055 ENSTNIT00000014347 ENSTNIT00000010802 ENSTNIT00000014177 ENSTNIT00000023283 ENSTNIT00000017614 ENSTNIT00000014895 ENSTNIT00000010462 ENSTNIT00000010461 3 ENSORLP00000025748 ENSORLP00000008769 ENSORLP00000003411 ENSORLP00000011473 ENSORLP00000009370 ENSORLP00000019247 ENSORLP00000005856 ENSORLP00000005821 ENSTRUP00000014039 ENSTRUP00000014043 ENSTRUP00000027174 ENSTRUP00000030659 ENSTRUP00000023464 ENSTRUP00000005551 ENSTRUP00000010433 ENSTRUP00000015119 ENSTRUP00000011180 ENSTRUP00000033312 ENSTRUP00000029063 ENSTRUP00000043921 ENSTRUP00000014542 ENSTRUP00000023184 ENSTRUP00000019484 ENSTRUP00000021067 ENSTRUP00000007580 ENSTRUP00000029845 ENSTRUP00000041291 ENSTRUP00000032179 ENSTRUP00000010849 ENSTRUP00000042530 ENSTRUP00000023704 ENSTRUP00000022950 ENSTNIP00000000891 ENSTNIP00000000474 ENSTNIP00000002504 ENSTNIP00000000842 ENSTNIP00000019612 ENSTNIP00000006792 ENSTNIP00000008862 ENSTNIP00000008861 ENSTNIP00000022706 ENSTNIP00000013241 ENSTNIP00000018821 ENSTNIP00000015029 ENSTNIP00000003150 ENSTNIP00000001758 ENSTNIP00000001086 ENSTNIP00000009049 ENSTNIP00000014854 ENSTNIP00000014150 ENSTNIP00000010621 ENSTNIP00000013982 ENSTNIP00000023041 ENSTNIP00000017396 ENSTNIP00000014695 ENSTNIP00000010281 ENSTNIP00000010280 Latimeria chalumnae (Coelacanths) Homo sapiens (Human) Gallus gallus (Chicken) abcb1/4 / Abcb1/4 like abcb5 / Abcb5 abcb11 / Abcb11 abcc1 / Abcc1 abcc2 / Abcc2 abcc6 / Abcc6 abcc7 / Abcc7 abcc8 / Abcc8 abcc9 / Abcc9 abcc10 / Abcc10 abcg1 / Abcg1 abcg2a / Abcg2a abcg2b / Abcg2b abcg4 / Abcg4 abcg5 / Abcg5 abcg8 / Abcg8 ABCB1 / ABCB1 ABCB4 / ABCB4 ABCB5 / ABCB5 ABCB11 / ABCB11 ABCC1 / ABCC1 ABCC2 / ABCC2 ABCC3 / ABCC3 ABCC4 / ABCC4 ABCC5 / ABCC5 ABCC6 / ABCC6 ABCC7 / ABCC7 ABCC8 / ABCC8 ABCC9 / ABCC9 ABCC10 / ABCC10 ABCC11 / ABCC11 ABCC12 / ABCC12 ABCG1 / ABCG1 ABCG2 / ABCG2 ABCG4 / ABCG4 ABCG5 / ABCG5 ABCG8 / ABCG8 Abcb1 / ABCB1 Abcb4 / ABCB4 Abcb5 / ABCB5 Abcb11 / ABCB11 Abcc1 / ABCC1 Abcc2 / ABCC2 Abcc3 / ABCC3 Abcc4 / ABCC4 Abcc5 / ABCC5 Abcc6 / ABCC6 Abcc7 / ABCC7 Abcc8 / ABCC8 Abcc9 / ABCC9 Abcc10 / ABCC10 Abcg1 / ABCG1 Abcg2 / ABCG2 Abcg4 / ABCG4 Abcg5 / ABCG5 Abcg8 / ABCG8 ENSLACT00000009772 ENSLACT00000002302 ENSLACT00000000142 ENSLACT00000001658 ENSLACT00000009537 ENSLACT00000025865 ENSLACT00000003365 ENSLACT00000018718 ENSLACT00000002238 ENSLACT00000002789 ENSLACT00000004137 ENSLACT00000001343 ENSLACT00000004952 ENSLACT00000020472 ENSLACT00000018080 ENSLACT00000017034 ENST00000265724 ENST00000359206 ENST00000404938 ENST00000576612 ENST00000399410 ENST00000370449 ENST00000285238 ENST00000376887 ENST00000334444 ENST00000205557 ENST00000003084 ENST00000389817 ENST00000261201 ENST00000372530 ENST00000356608 ENST00000311303 ENST00000398449 ENST00000237612 ENST00000307417 ENST00000260645 ENST00000272286 ENSGALT00000014467 ENSGALG00000008912 ENSGALT00000017732 ENSGALT00000038467 ENSGALT00000010749 ENSGALT00000011965 ENSGALT00000012164 ENSGALT00000027309 ENSGALT00000013625 ENSGALT00000040027 ENSGALT00000015182 ENSGALT00000009964 ENSGALT00000021626 ENSGALT00000016963 ENSGALT00000026046 ENSGALT00000009304 ENSGALT00000010990 ENSGALT00000016182 ENSGALT00000016186 4 ENSLACP00000009697 ENSLACP00000002284 ENSLACP00000000141 ENSLACP00000001645 ENSLACP00000009465 ENSLACP00000022639 ENSLACP00000003335 ENSLACP00000018585 ENSLACP00000002220 ENSLACP00000002767 ENSLACP00000004101 ENSLACP00000001331 ENSLACP00000004909 ENSLACP00000020332 ENSLACP00000017950 ENSLACP00000016915 ENSP00000265724 ENSP00000352135 ENSP00000384881 ENSP00000459250 ENSP00000382342 ENSP00000359478 ENSP00000285238 ENSP00000366084 ENSP00000333926 ENSP00000205557 ENSP00000003084 ENSP00000374467 ENSP00000261201 ENSP00000361608 ENSP00000349017 ENSP00000311030 ENSP00000381467 ENSP00000237612 ENSP00000304111 ENSP00000260645 ENSP00000272286 ENSGALP00000014451 ENSGALG00000008912 ENSGALP00000017711 ENSGALP00000037672 ENSGALP00000010735 ENSGALP00000011951 ENSGALP00000012150 ENSGALP00000027258 ENSGALP00000013610 ENSGALP00000039234 ENSGALP00000015166 ENSGALP00000009950 ENSGALP00000021593 ENSGALP00000016944 ENSGALP00000025999 ENSGALP00000009290 ENSGALP00000010976 ENSGALP00000016163 ENSGALP00000016167 Xenopus tropicalis (Western clawed frog) Abcb1 / ABCB1 Abcb5a / ABCB5a Abcb5b / ABCB5c Abcb5c / ABCB5c Abcb5d / ABCB5d Abcb11 / ABCB11 Abcc1 / ABCC1 Abcc2 / ABCC2 Abcc3 / ABCC3 Abcc4 / ABCC4 Abcc5 / ABCC5 Abcc6 / ABCC6 Abcc7 / ABCC7 Abcc9 / ABCC9 Abcc10 / ABCC10 Abcg1 / ABCG1 Abcg2 / ABCG2 Abcg4 / ABCG4 Abcg5 / ABCG5 Abcg8 / ABCG8 ENSXETT00000005311 ENSXETT00000016218 ENSXETT00000005310 ENSXETT00000013172 ENSXETT00000016186 ENSXETT00000035384 ENSXETT00000042600 ENSXETT00000008096 ENSXETT00000026744 ENSXETT00000023948 ENSXETT00000031284 ENSXETT00000042610 ENSXETT00000047145 ENSXETT00000001948 ENSXETT00000023222 ENSXETT00000060302 ENSXETT00000065208 ENSXETT00000046632 ENSXETT00000020415 ENSXETT00000020423 5 ENSXETP00000005311 ENSXETP00000016218 ENSXETP00000005310 ENSXETP00000013172 ENSXETP00000016186 ENSXETP00000035384 ENSXETP00000042600 ENSXETP00000008096 ENSXETP00000026744 ENSXETP00000023948 ENSXETP00000031284 ENSXETP00000042610 ENSXETP00000047145 ENSXETP00000001948 ENSXETP00000023222 ENSXETP00000064040 ENSXETP00000062067 ENSXETP00000046632 ENSXETP00000020415 ENSXETP00000020423 Figure S1. Phylogenetic tree based on the multiple alignments (T-Coffee) of Abcc1, Abcc2, Abcc3, Abcc4, Abcc5, Abcc6, Abcc7, Abcc8, Abcc9, Abcc10, Abcc11, Abcc12 and Abcc13 ABC transporter amino acid sequences of available teleosts sequences (Ensembl) of Danio rerio, Gasterosteus aculeatus, Gadus morhua, Oreochromis niloticus Oryzias latipes, Takifugu rubripes, Tetraodon nigroviridis and other vertebrates. The tree was generated with MEGA5 using the neighbor-joining method with percentage concordance based on 1000 bootstrap iterations is shown at the nodes. Protein accession no. (Ensembl) are listed in Table S1. 1 Figure S2. Phylogenetic tree based on the multiple alignments (T-Coffee) of Abcg1, Abcg2, Abcg4, Abcg5 and Abcg8 ABC transporter amino acid sequences of available teleosts sequences (Ensembl) of Danio rerio, Gasterosteus aculeatus, Gadus morhua, Oreochromis niloticus Oryzias latipes, Takifugu rubripes, Tetraodon nigroviridis and other vertebrates. The tree was generated with MEGA5 using the neighbor-joining method with percentage concordance based on 1000 bootstrap iterations is shown at the nodes. Protein accession no. (Ensembl) are listed in Table S1. 2