Download Review. Current advances on ABC drug transporters in fish

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). MASTS is funded by the Scottish Funding Council (grant reference
1395 HR09011) and contributing institutions.
1396 1397 1398 1399 1400 1401 1402 1403 References
Abele, R., Tampé, R., 2006. Modulation of the antigen transport machinery TAP by friends and enemies.
FEBS Lett. 580, 1156–63.
Albertus, J.A., Laine, R.O., 2001. Enhanced xenobiotic transporter expression in normal teleost
hepatocytes: response to environmental and chemotherapeutic toxins. J. Exp. Biol. 204, 217–27.
Allen, J.D., van Loevezijn, A., Lakhai, J.M., van der Valk, M., van Tellingen, O., Reid, G., Schellens,
40 1404 1405 1406 1407 1408 1409 1410 1411 1412 1413 1414 1415 1416 1417 1418 1419 1420 1421 1422 1423 1424 1425 1426 1427 1428 1429 1430 1431 1432 1433 1434 1435 1436 1437 1438 1439 1440 1441 1442 1443 1444 1445 1446 1447 1448 1449 1450 J.H.M., Koomen, G.-J., Schinkel, A.H., 2002. Potent and specific inhibition of the breast cancer
resistance protein multidrug transporter in vitro and in mouse intestine by a novel analogue of
fumitremorgin C. Mol. Cancer Ther. 1, 417–425.
Aller, S.G., Yu, J., Ward, A., Weng, Y., Chittaboina, S., Zhuo, R., Harrell, P.M., Trinh, Y.T., Zhang, Q.,
Urbatsch, I.L., Chang, G., 2009. Structure of P-glycoprotein reveals a molecular basis for polyspecific drug binding. Science 323, 1718–22.
Ambudkar, S. V, Dey, S., Hrycyna, C.A., Ramachandra, M., Pastan, I., Gottesman, M.M., 1999.
Biochemical, cellular, and pharmacological aspects of the multidrug transporter. Annu. Rev.
Pharmacol. Toxicol. 39, 361–98.
Ambudkar, S. V, Lelong, I.H., Zhang, J., Cardarelli, C.O., Gottesman, M.M., Pastan, I., 1992. Partial
purification and reconstitution of the human multidrug-resistance pump: characterization of the
drug-stimulatable ATP hydrolysis. Proc. Natl. Acad. Sci. USA 89, 8472–8476.
Ambudkar, S. V., Cardarelli, C.O., 1997. Relation between the turnover number for vinblastine transport
and for vinblastine-stimulated ATP hydrolysis by human P-glycoprotein. J. Biol. Chem. 272,
21160–21166.
Amé, M.V., Baroni, M.V., Galanti, L.N., Bocco, J.L., Wunderlin, D.A., 2009. Effects of microcystin-LR
on the expression of P-glycoprotein in Jenynsia multidentata. Chemosphere 74, 1179–86.
Amores, a, Force, a, Yan, Y.L., Joly, L., Amemiya, C., Fritz, a, Ho, R.K., Langeland, J., Prince, V.,
Wang, Y.L., Westerfield, M., Ekker, M., Postlethwait, J.H., 1998. Zebrafish hox clusters and
vertebrate genome evolution. Science 282, 1711–4.
Ananthanarayanan, M., Balasubramanian, N., Makishima, M., Mangelsdorf, D.J., Suchy, F.J., 2001.
Human bile salt export pump promoter is transactivated by the farnesoid X receptor/bile acid
receptor. J. Biol. Chem. 276, 28857–65.
Annilo, T., Chen, Z.-Q., Shulenin, S., Costantino, J., Thomas, L., Lou, H., Stefanov, S., Dean, M., 2006.
Evolution of the vertebrate ABC gene family: analysis of gene birth and death. Genomics 88, 1–
11.
Aparicio, S., 2000. Vertebrate evolution: recent perspectives from fish. Trends Genet. 16, 54–6.
Baehr, C.H., Fricker, G., Miller, D.S., 2006. Fluorescein-methotrexate transport in dogfish shark
(Squalus acanthias) choroid plexus. Am. J. Physiol. Regul. Integr. Comp. Physiol. 291, R464–72.
Bain, L.J., LeBlanc, G.A., 1996. Interaction of structurally diverse pesticides with the human MDR1
gene product P-glycoprotein. Toxicol. Appl. Pharmacol. 141, 288–98.
Bain, L.J., McLachlan, J.B., LeBlanc, G.A., 1997. Structure-activity relationships for xenobiotic
transport substrates and inhibitory ligands of P-glycoprotein. Environ. Health Perspect. 105, 812–
8.
Bains, O.S., Kennedy, C.J., 2005. Alterations in respiration rate of isolated rainbow trout hepatocytes
exposed to the P-glycoprotein substrate rhodamine 123. Toxicology 214, 87–98.
Bakke, A.M., Glover, C., Krogdahl, A., 2011. Feeding, digestion and absorption of nutrients, in: Grosell,
M., Farrell, A.P., Brauner, C. (Eds.), The Multifunctionall Gut of Fish. Elsevier, Amsterdam, The
Netherlands, pp. 57–110.
Bakos, E., Homolya, L., 2007. Portrait of multifaceted transporter, the multidrug resistance-associated
protein 1 (MRP1/ABCC1). Pflugers Arch. 453, 621–41.
Ballatori, N., Rebbeor, J.F., Connolly, G.C., Seward, D.J., Lenth, B.E., Henson, J.H., Sundaram, P.,
Boyer, J.L., 2000. Bile salt excretion in skate liver is mediated by a functional analog of
Bsep/Spgp, the bile salt export pump. Am. J. Physiol. Gastrointest. Liver Physiol. 278, G57–63.
Bamberger, C.M., Schulte, H.M., Chrousos, G.P., 1996. Molecular determinants of glucocorticoid
receptor function and tissue sensitivity to glucocorticoids. Endocr. Rev. 17, 245–261.
Bard, S.M., 2000. Multixenobiotic resistance as a cellular defense mechanism in aquatic organisms.
41 1451 1452 1453 1454 1455 1456 1457 1458 1459 1460 1461 1462 1463 1464 1465 1466 1467 1468 1469 1470 1471 1472 1473 1474 1475 1476 1477 1478 1479 1480 1481 1482 1483 1484 1485 1486 1487 1488 1489 1490 1491 1492 1493 1494 1495 1496 1497 Aquat. Toxicol. 48, 357–389.
Bard, S.M., Bello, S.M., Hahn, M.E., Stegeman, J.J., 2002a. Expression of P-glycoprotein in killifish
(Fundulus heteroclitus) exposed to environmental xenobiotics. Aquat. Toxicol. 59, 237–51.
Bard, S.M., Gadbois, S., 2007. Assessing neuroprotective P-glycoprotein activity at the blood-brain
barrier in killifish (Fundulus heteroclitus) using behavioural profiles. Mar. Environ. Res. 64, 679–
82.
Bard, S.M., Woodin, B.R., Stegeman, J.J., 2002b. Expression of P-glycoprotein and cytochrome p450
1A in intertidal fish (Anoplarchus purpurescens) exposed to environmental contaminants. Aquat.
Toxicol. 60, 17–32.
Barrand, M., Rhodes, T., Center, M., Twentyman, P., 1993. Chemosensitisation and drug accumulation
effects of cyclosporin A, PSC-833 and verapamil in human MDR large cell lung cancer cells
expressing a 190k membrane protein distinct from P-glycoprotein. Eur. J. Cancer 29A, 408–415.
Belanger, S.E., Rawlings, J.M., Carr, G.J., 2013. Use of fish embryo toxicity tests for the prediction of
acute fish toxicity to chemicals. Environ. Toxicol. Chem. 32, 1768–83.
Benedetto, A., Squadrone, S., Prearo, M., Elia, A.C., Giorgi, I., Abete, M.C., 2011. Evaluation of ABC
efflux transporters genes expression in kidney of rainbow trout (Oncorhynchus mykiss) fed with
melamine and cyanuric acid diets. Chemosphere 84, 727–30.
Bertilsson, G., Heidrich, J., Svensson, K., Asman, M., Jendeberg, L., Sydow-Bäckman, M., Ohlsson, R.,
Postlind, H., Blomquist, P., Berkenstam, a, 1998. Identification of a human nuclear receptor
defines a new signaling pathway for CYP3A induction. Proc. Natl. Acad. Sci. USA. 95, 12208–13.
Beyenbach, K.W., 2004. Kidneys sans glomeruli. Am. J. Physiol. Renal Physiol. 286, F811–27.
Black, J.J., Baumann, P.C., 1991. Carcinogens and cancers in freshwater fishes. Environ. Health
Perspect. 90, 27–33.
Blumberg, B., Sabbagh Jr., W., Juguilon, H., Bolado Jr., J., van Meter, C.M., Ong, E.S., Evans, R.M.,
1998. SXR, a novel steroid and xenobioticsensing nuclear receptor. Genes Dev. 12, 3195–3205.
Bone, Q., Moore, R.H., 2008. Biology of fishes, 3rd editio. ed. Taylor & Francis, New York.
Borst, P., de Wolf, C., van de Wetering, K., 2007. Multidrug resistance-associated proteins 3, 4, and 5.
Pflugers Arch. 453, 661–73.
Borst, P., Zelcer, N., van de Wetering, K., Poolman, B., 2006. On the putative co-transport of drugs by
multidrug resistance proteins. FEBS Letts. 580, 1085–93.
Bosnjak, I., Uhlinger, K.R., Heim, W., Smital, T., Franekić-Colić, J., Coale, K., Epel, D., Hamdoun, A.,
2009. Multidrug efflux transporters limit accumulation of inorganic, but not organic, mercury in
sea urchin embryos. Environ. Sci. Technol. 43, 8374–80.
Bouige, P., Laurent, D., Piloyan, L., Dassa, E., 2002. Phylogenetic and functional classification of ATPbinding cassette (ABC) systems. Curr. Protein Pept. Sci. 3, 541–559.
Bradbury, S., Coats, J., Mckim, J.M., 1986. Toxicokinetics of fenvalerate in rainbow trout (Salmo
gairdneri). Environ. Toxicol. Chem. 5, 567–576.
Bresolin, T., de Freitas Rebelo, M., Celso Dias Bainy, A., 2005. Expression of PXR, CYP3A and MDR1
genes in liver of zebrafish. Comp. Biochem. Physiol. C. Toxicol. Pharmacol. 140, 403–7.
Bridges, C.C., Joshee, L., Zalups, R.K., 2008. MRP2 and the DMPS- and DMSA-mediated elimination
of mercury in TR(-) and control rats exposed to thiol S-conjugates of inorganic mercury. Toxicol.
Sci. 105, 211–20.
Broeks, A., Gerrard, B., Allikmets, R., Dean, M., Plasterk, R.H., 1996. Homologues of the human
multidrug resistance genes MRP and MDR contribute to heavy metal resistance in the soil
nematode Caenorhabditis elegans. EMBO J. 15, 6132–43.
Bunting, K., 2002. ABC transporters as phenotypic markers and functional regulators of stem cells. Stem
Cells 11–20.
42 1498 1499 1500 1501 1502 1503 1504 1505 1506 1507 1508 1509 1510 1511 1512 1513 1514 1515 1516 1517 1518 1519 1520 1521 1522 1523 1524 1525 1526 1527 1528 1529 1530 1531 1532 1533 1534 1535 1536 1537 1538 1539 1540 1541 1542 1543 1544 Burke, M.A., Ardehali, H., 2007. Mitochondrial ATP-binding cassette proteins. Transl. Res. 150, 73–80.
Burt, R.K., Thorgeirsson, S.S., 1988. Coinduction of MDR-1 multidrug-resistance and cytochrome P-450
genes in rat liver by xenobiotics. J. Natl. Cancer Inst. 80, 1383–6.
Bury, N.R., Sturm, A., 2007. Evolution of the corticosteroid receptor signalling pathway in fish. Gen.
Comp. Endocrinol. 153, 47–56.
Cai, S., Soroka, C., Ballatori, N., 2003. Molecular characterization of a multidrug resistance-associated
protein, Mrp2, from the little skate. Am. J. Physiol. Regul. Integr. Comp. Physiol. 284, R125–
R130.
Cai, S., Wang, L., Ballatori, N., Boyer, J.L., 2001. Bile salt export pump is highly conserved during
vertebrate evolution and its expression is inhibited by PFIC type II mutations. Am. J. Physiol.
Gastrointest. Liver Physiol. 281, G316–G322.
Cain, K., Swan, C., 2011. Barrier function and immunology, in: Grosell, M., Farrell, A.P., Brauner, C.J.
(Eds.), Fish Physiology: The Multifunctional Gut of Fish. Elsevier, Amsterdam, The Netherlands,
pp. 112–135.
Calcagno, A.M., Kim, I.-W., Wu, C.-P., Shukla, S., Ambudkar, S. V., 2007. ABC drug transporters as
molecular targets for the prevention of multidrug resistance and drug-drug interactions. Curr. Drug
Deliv. 4, 324–333.
Caminada, D., Zaja, R., Smital, T., Fent, K., 2008. Human pharmaceuticals modulate P-gp1 (ABCB1)
transport activity in the fish cell line PLHC-1. Aquat. Toxicol. 90, 214–22.
Cárcamo, J.G., Aguilar, M.N., Barrientos, C.A., Carreño, C.F., Quezada, C.A., Bustos, C., Manríquez,
R.A., Avendaño-Herrera, R., Yañez, A.J., 2011. Effect of emamectin benzoate on transcriptional
expression of cytochromes P450 and the multidrug transporters (Pgp and MRP1) in rainbow trout
(Oncorhynchus mykiss) and the sea lice Caligus rogercresseyi. Aquaculture 321, 207–215.
Chan, G.N.Y., Hoque, M.T., Bendayan, R., 2013. Role of nuclear receptors in the regulation of drug
transporters in the brain. Trends Pharmacol. Sci. 34, 361–72.
Chan, K.M., 1992. P-glycoprotein genes in the winter flounder, Pleuronectes americanus: isolation of
two types of genomic clones carrying 3’ terminal exons. Biochim. Biophys. Acta 1171, 65–72.
Chan, L., 2004. The ABCs of drug transport in intestine and liver: efflux proteins limiting drug
absorption and bioavailability. Eur. J. Pharm. Sci. 21, 25–51.
Charmandari, E., Tsigos, C., Chrousos, G., 2005. Endocrinology of the stress response. Annu. Rev.
Physiol. 67, 259–84.
Chen, K.G., Sikic, B.I., 2012. Molecular pathways: regulation and therapeutic implications of multidrug
resistance. Clin. Cancer Res. 18, 1863–9.
Chen, Y., Tang, Y., Guo, C., Wang, J., Boral, D., Nie, D., 2012. Nuclear receptors in the multidrug
resistance through the regulation of drug-metabolizing enzymes and drug transporters. Biochem.
Pharmacol. 83, 1112–26.
Childs, S., Yeh, R.L., Georges, E., Ling, V., 1995. Identification of a sister gene to P-Glycoprotein.
Cancer Res. 55, 2029–2034.
Choi, C.-H., 2005. ABC transporters as multidrug resistance mechanisms and the development of
chemosensitizers for their reversal. Cancer Cell Int. 5, 30.
Cole, S., Bhardwaj, G., Gerlach, J., Mackie, J., Grant, C., Almquist, K., Stewart, A., Kurz, E., Duncan,
A., Deeley, R., RG., 1992. Overexpression of a transporter gene in a multidrug-resistant human
lung cancer cell line. Science 258, 1650–1654.
Collier, T.K., Varanasi, U., 1991. Hepatic activities of xenobiotic metabolizing enzymes and biliary
levels of xenobiotics in English sole (Parophrys vetulus) exposed to environmental contaminants.
Arch. Environ. Contam. Toxicol. 20, 462–73.
Cooper, P.S., Vogelbein, W.K., Van Veld, P.A., 1999. Altered expression of the xenobiotic transporter
43 1545 1546 1547 1548 1549 1550 1551 1552 1553 1554 1555 1556 1557 1558 1559 1560 1561 1562 1563 1564 1565 1566 1567 1568 1569 1570 1571 1572 1573 1574 1575 1576 1577 1578 1579 1580 1581 1582 1583 1584 1585 1586 1587 1588 1589 1590 1591 P-glycoprotein in liver and liver tumours of mummichog (Fundulus heteroclitus) from a creosotecontaminated environment. Biomarkers 4, 48–58.
Costa, J., Reis-Henriques, M.A., Castro, L.F.C., Ferreira, M., 2012. Gene expression analysis of ABC
efflux transporters, CYP1A and GSTα in Nile tilapia after exposure to benzo(a)pyrene. Comp.
Biochem. Physiol. C. Toxicol. Pharmacol. 155, 469–82.
Crivellato, E., Candussio, L., Rosati, A.M., Bartoli-Klugmann, F., Mallardi, F., Decorti, G., 2002. The
fluorescent probe Bodipy-FL-verapamil is a substrate for both P-glycoprotein and multidrug
resistance-related protein (MRP)-1. J. Histochem. Cytochem. 50, 731–4.
Crone, C., Christensen, O., 1981. Electrical resistance of a capillary endothelium. J. Gen. Physiol. 77,
349–371.
Cserr, H.F., Bundgaard, M., 1984. Blood-brain interfaces in vertebrates  : a comparative approach. Am. J.
Physiol. Regul. Integr. Comp. Physiol. 246, R277–R288.
Dallas, S., Miller, D.S., Bendayan, R., 2006. Multidrug resistance-associated proteins  : Expression and
function in the central nervous system. Pharmacol. Rev. 58, 140–161.
Dean, M., Annilo, T., 2005. Evolution of the ATP-binding cassette (ABC) transporter superfamily in
vertebrates. Annu. Rev. Genomics Hum. Genet. 6, 123–42.
Dean, M., Rzhetsky, A., Allikmets, R., 2001. The human ATP-binding cassette (ABC) transporter
superfamily. Genome Res. 11, 1156–1166.
Deeley, R.G., Cole, S.P.C., 2006. Substrate recognition and transport by multidrug resistance protein 1
(ABCC1). FEBS Letts. 580, 1103–11.
Deeley, R.G., Westlake, C., Cole, S.P.C., 2006. Transmembrane transport of endo- and xenobiotics by
mammalian ATP-binding cassette multidrug resistance proteins. Physiol. Revs. 86, 849–99.
Della Torre, C., Zaja, R., Loncar, J., Smital, T., Focardi, S., Corsi, I., 2012. Interaction of ABC transport
proteins with toxic metals at the level of gene and transport activity in the PLHC-1 fish cell line.
Chem. Biol. Interact.
Deng, L., Lin-Lee, Y.C., Claret, F.X., Kuo, M.T., 2001. 2-acetylaminofluorene up-regulates rat mdr1b
expression through generating reactive oxygen species that activate NF-kappa B pathway. J. Biol.
Chem. 276, 413–20.
Diaz de Cerio, O., Bilbao, E., Cajaraville, M.P., Cancio, I., 2012. Regulation of xenobiotic transporter
genes in liver and brain of juvenile thicklip grey mullets (Chelon labrosus) after exposure to
Prestige-like fuel oil and to perfluorooctane sulfonate. Gene 498, 50–58.
Doi, A.M., Holmes, E., Kleinow, K.M., 2001. P-glycoprotein in the catfish intestine  : inducibility by
xenobiotics and functional properties. Aquat. Toxicol. 55, 157– 170.
Doige, C.A., Yu, X., Sharom, F.J., 1992. ATPase activity of partially purified P-glycoprotein from
multidrug-resistant Chinese hamster ovary cells. Biochim. Biophys. Acta 1109, 149–160.
Dussault, I., Lin, M., Hollister, K., Wang, E.H., Synold, T.W., Forman, B.M., 2001. Peptide mimetic
HIV protease inhibitors are ligands for the orphan receptor SXR. J. Biol. Chem. 276, 33309–12.
Ebert, B., Seidel, A., Lampen, A., 2005. Identification of BCRP as transporter of benzo[a]pyrene
conjugates metabolically formed in Caco-2 cells and its induction by Ah-receptor agonists.
Carcinogenesis 26, 1754–63.
Ekins, S., Reschly, E.J., Hagey, L.R., Krasowski, M.D., 2008. Evolution of pharmacologic specificity in
the pregnane X receptor. BMC Evol. Biol. 8, 103.
Ellesat, K.S., Holth, T.F., Wojewodzic, M.W., Hylland, K., 2012. Atorvastatin up-regulate
toxicologically relevant genes in rainbow trout gills. Ecotoxicology 21, 1841–56.
El-Sheikh, A.A.K., Masereeuw, R., Russel, F.G.M., 2008. Mechanisms of renal anionic drug transport.
Eur. J. Pharmacol. 585, 245–55.
Escriva, H., Manzon, L., Youson, J., Laudet, V., 2002. Analysis of lamprey and hagfish genes reveals a
44 1592 1593 1594 1595 1596 1597 1598 1599 1600 1601 1602 1603 1604 1605 1606 1607 1608 1609 1610 1611 1612 1613 1614 1615 1616 1617 1618 1619 1620 1621 1622 1623 1624 1625 1626 1627 1628 1629 1630 1631 1632 1633 1634 1635 1636 1637 1638 complex history of gene duplications during early vertebrate evolution. Mol. Biol. Evol. 19, 1440–
50.
Essodaigui, M., Broxterman, H.J., Garnier-Suillerot, A., 1998. Kinetic analysis of calcein and calceinacetoxymethylester efflux mediated by the multidrug resistance protein and P-glycoprotein.
Biochemistry 37, 2243–2250.
Estudante, M., Morais, J.G., Soveral, G., Benet, L.Z., 2013. Intestinal drug transporters: An overview.
Adv. Drug Deliv. Rev. 65, 1340–56.
Evans, D.H., Piermarini, P.M., Choe, K.P., 2005. The multifunctional fish gill: Dominant site of gas
exchange, osmoregulation, acid-base regulation, and excretion of nitrogenous waste. Physiol. Rev.
85, 97–177.
Eytan, G.D., Regev, R., Assaraf, Y.G., 1996a. Functional reconstitution of P-glycoprotein reveals an
apparent near stoichiometric drug transport to ATP hydrolysis. J. Biol. Chem. 271, 3172–3178.
Eytan, G.D., Regev, R., Oren, G., Assaraf, Y.G., 1996b. The role of passive transbilayer drug movement
in multidrug resistance and its modulation. J. Biol. Chem. 271, 12897–12902.
Fardel, O., Lecureur, V., Corlu, a, Guillouzo, a, 1996. P-glycoprotein induction in rat liver epithelial cells
in response to acute 3-methylcholanthrene treatment. Biochem. Pharmacol. 51, 1427–36.
Feige, J.N., Gelman, L., Michalik, L., Desvergne, B., Wahli, W., 2006. From molecular action to
physiological outputs: peroxisome proliferator-activated receptors are nuclear receptors at the
crossroads of key cellular functions. Prog. Lipid Res. 45, 120–59.
Fidler, A.E., Holland, P.T., Reschly, E.J., Ekins, S., Krasowski, M.D., 2012. Activation of a tunicate
(Ciona intestinalis) xenobiotic receptor orthologue by both natural toxins and synthetic toxicants.
Toxicon 59, 365–72.
Fischer, S., Klüver, N., Burkhardt-Medicke, K., Pietsch, M., Schmidt, A.-M., Wellner, P., Schirmer, K.,
Luckenbach, T., 2013. Abcb4 acts as multixenobiotic transporter and active barrier against
chemical uptake in zebrafish (Danio rerio) embryos. BMC Biol. 11, 69.
Fischer, S., Loncar, J., Zaja, R., Schnell, S., Schirmer, K., Smital, T., Luckenbach, T., 2011. Constitutive
mRNA expression and protein activity levels of nine ABC efflux transporters in seven permanent
cell lines derived from different tissues of rainbow trout (Oncorhynchus mykiss). Aquat. Toxicol.
101, 438–46.
Fischer, S., Pietsch, M., Schirmer, K., Luckenbach, T., 2010. Identification of multi-drug resistance
associated proteins MRP1 (ABCC1) and MRP3 (ABCC3) from rainbow trout (Oncorhynchus
mykiss). Mar. Environ. Res. 69 Suppl, S7–S10.
Fojo, A.T., Ueda, K., Slamon, D.J., Poplack, D.G., Gottesman, M.M., Pastan, I., 1987. Expression of a
multidrug-resistance gene in human tumors and tissues. Proc. Natl. Acad. Sci. USA 84, 265–269.
Ford, J.M., Hait, W.N., 1990. Pharmacology of drugs that alter multidrug resistance in cancer.
Pharmacol. Revs. 42, 155–199.
Ford, J.M., Hait, W.N., 1993. Pharmacologic circumvention of multidrug resistance. Cytotechnology 12,
171–212.
Frank, N., Frank, M., 2009. ABCB5 gene amplification in human leukemia cells. Leuk. Res. 33, 1303–5.
Frank, N.Y., Pendse, S.S., Lapchak, P.H., Margaryan, A., Shlain, D., Doeing, C., Sayegh, M.H., Frank,
M.H., 2003. Regulation of progenitor cell fusion by ABCB5 P-glycoprotein, a novel human ATPbinding cassette transporter. J. Biol. Chem. 278, 47156–65.
Fricker, G., Gutmann, H., Droulle, A., Drewe, J., 1999. Epithelial transport of anthelmintic ivermectin in
a novel model of isolated proximal kidney tubules. Pharm. Res. 16, 1570–1575.
Gant, T.W., Silverman, J.A., Bisgaard, H.C., Burt, R.K., Marino, P.A., Thorgeirson, S.S., 1991.
Regulation of 2-acetylaminofluorene- and 3-methylcholanthrene-mediated induction of multidrug
resistance and cytochrome P450IA gene family expression in primary hepatocyte cultures and rat
45 1639 1640 1641 1642 1643 1644 1645 1646 1647 1648 1649 1650 1651 1652 1653 1654 1655 1656 1657 1658 1659 1660 1661 1662 1663 1664 1665 1666 1667 1668 1669 1670 1671 1672 1673 1674 1675 1676 1677 1678 1679 1680 1681 1682 1683 1684 1685 liver. Mol. Carcinog. 4, 499–509.
Garcia-Fernandez, J., Holland, P.W., 1994. Archetypal organization of the amphioxus Hox gene cluster.
Nature 370, 563–566.
Gardner, E.R., Smith, N.F., Figg, W.D., Sparreboom, A., 2009. Influence of the dual ABCB1 and
ABCG2 inhibitor tariquidar on the disposition of oral imatinib in mice. J. Exp. Clin. Cancer Res.
28, 99.
Garrigues, A., Escargueil, A.E., Orlowski, S., 2002. The multidrug transporter, P-glycoprotein, actively
mediates cholesterol redistribution in the cell membrane. Proc. Natl. Acad. Sci. USA 99, 10347–
10352.
Geick, A., Eichelbaum, M., Burk, O., 2001. Nuclear receptor response elements mediate induction of
intestinal MDR1 by rifampin. J. Biol. Chem. 276, 14581–7.
Gekeler, V., Ise, W., Sanders, K.H., Ulrich, W.-R., Beck, J., 1995. The leukotriene LTD4 receptor
antagonist MK571 specifically modulates MRP associated multidrug resistance. Biochem.
Biophys. Res. Commun. 208, 345–352.
Georges, E., Bradley, G., Gariepy, J., Ling, V., 1990. Detection of P-glycoprotein by gene-specific
monoclonal antibodies. Proc. Natl. Acad. Sci. USA 87, 152–156.
Gerloff, T., Stieger, B., Hagenbuch, B., Madon, J., Landmann, L., Roth, J., Hofmann, a F., Meier, P.J.,
1998. The sister of P-glycoprotein represents the canalicular bile salt export pump of mammalian
liver. J. Biol. Chem. 273, 10046–50.
Germann, U., 1998. Baculovirus-mediated expression of human multidrug resistance cDNA in insect
cells and functional analysis of recombinant P-glycoprotein. Meths. Enzymol. 292, 427–441.
Goto, T., Holzinger, F., Hagey, L.R., Cerrè, C., Ton-Nu, H.-T., Schteingart, C.D., Steinbach, J.H.,
Shneider, B.L., Hofmann, a F., 2003. Physicochemical and physiological properties of 5alphacyprinol sulfate, the toxic bile salt of cyprinid fish. J. Lipid Res. 44, 1643–51.
Gottesman, M.M., Fojo, T., Bates, S.E., 2002. Multidrug resistance in cancer: Role of ATP-dependent
transporters. Nat. Rev. Cancer 2, 48–58.
Gourley, M.E., Kennedy, C.J., 2009. Energy allocations to xenobiotic transport and biotransformation
reactions in rainbow trout (Oncorhynchus mykiss) during energy intake restriction. Comp.
Biochem. Physiol. Part C Toxicol. Pharmacol. 150, 270–278.
Gros, P., Croop, J., Housman, D., 1986. Mammalian multidrug resistance gene: complete cDNA
sequence indicates strong homology to bacterial transport proteins. Cell 47, 371–380.
Gutmann, H., Fricker, G., Drewe, J., Toeroek, M., Miller, D.S., 1999. Interactions of HIV protease
inhibitors with ATP-dependent drug export proteins. Mol. Pharmacol. 56, 383–9.
Gutmann, H., Miller, D.S., Droulle, A., Drewe, J., Fahr, A., Fricker, G., 2000. P-glycoprotein- and mrp2mediated octreotide transport in renal proximal tubule. Br. J. Pharmacol. 129, 251–6.
Hahn, M.E., 1998. The aryl hydrocarbon receptor: a comparative perspective. Comp. Biochem. Physiol.
C. Pharmacol. Toxicol. Endocrinol. 121, 23–53.
Hamdoun, A., Epel, D., 2007. Embryo stability and vulnerability in an always changing world. Proc.
Natl. Acad. Sci. USA 104, 1745–50.
Handschin, C., Blättler, S., Roth, A., Looser, R., Oscarson, M., Kaufmann, M.R., Podvinec, M., Gnerre,
C., Meyer, U. a, 2004. The evolution of drug-activated nuclear receptors: one ancestral gene
diverged into two xenosensor genes in mammals. Nucl. Recept. 2, 7.
Hazard, S.E., Patel, S.B., 2007. Sterolins ABCG5 and ABCG8: regulators of whole body dietary sterols.
Pflugers Arch. 453, 745–52.
Helfman, G.S., Collette, B.B., Facey, D.E., Bowen, B.W., 2009. The diversity of fishes, 2nd editio. ed.
John Wiley, Oxford, UK.
Hemmer, M., Courtney, L., Benson, W., 1998. Comparison of three histological fixatives on the
46 1686 1687 1688 1689 1690 1691 1692 1693 1694 1695 1696 1697 1698 1699 1700 1701 1702 1703 1704 1705 1706 1707 1708 1709 1710 1711 1712 1713 1714 1715 1716 1717 1718 1719 1720 1721 1722 1723 1724 1725 1726 1727 1728 1729 1730 1731 1732 immunoreactivity of mammalian P-glycoprotein antibodies in the sheepshead minnow,
Cyprinodon variegatus. J. Exp. Zool. 281, 251–259.
Hemmer, M., Courtney, L., Ortego, L., 1995. Immunohistochemical detection of P-glycoprotein in
teleost tissues using mammalian polyclonal and monoclonal antibodies. J. Exp. Zool. 272, 69–77.
Herget, M., Tampé, R., 2007. Intracellular peptide transporters in human--compartmentalization of the
“peptidome”. Pflugers Arch. 453, 591–600.
Higgins, C.F., 1992. ABC transporters: From microorganism to man. Annu. Rev. Cell Biol. 8, 67–113.
Hildebrand, J.L., Bains, O.S., Lee, D.S.H., Kennedy, C.J., 2009. Functional and energetic
characterization of P-gp-mediated doxorubicin transport in rainbow trout (Oncorhynchus mykiss)
hepatocytes. Comp. Biochem. Physiol. C. Toxicol. Pharmacol. 149, 65–72.
Hinton, D.E., Segner, H., Au, D.W., Kullman, S.W., Hardman, R.C., 2008. Liver toxicity, in: Di Giulio,
R., Hinton, D.E. (Eds.), The Toxicology of Fishes. CRC Press, Boca Raton, FL, USA, pp. 327–
400.
Holló, Z., Homolya, L., Davis, C., Sarkadi, B., 1994. Calcein accumulation as a fluorometric functional
assay of the multidrug transporter. Biochim. Biophys. Acta (BBA)-Biomembranes 1191, 384–388.
Holló, Z., Homolya, L., Hegedüs, T., Sarkadi, B., 1996. Transport properties of the multidrug resistanceassociated protein (MRP) in human tumour cells. FEBS Lett.s 383, 99–104.
Homolya, L., Holló, Z., Germann, U., Pastan, I., Gottesman, M.M., Sarkadi, B., 1993. Fluorescent
cellular indicators are extruded by the multidrug-resistance protein. J. Biol. Chem. 268.
Høy, T., Horsberg, E., Nafstad, I., 1990. The disposition of ivermectin in Atlantic salmon (Salmo salar).
Pharmacol. Toxicol. 67, 307–312.
Igboeli, O., Fast, M., Heumann, J., Burka, J., 2012. Role of P-glycoprotein in emamectin benzoate
(SLICE®) resistance in sea lice, Lepeophtheirus salmonis. Aquaculture 344-349, 40–47.
Ishikawa, T., Bao, J.J., Yamane, Y., Akimaru, K., Frindrich, K., Wright, C.D., Kuo, M.T., 1996.
Coordinated induction of MRP/GS-X pump and gamma-glutamylcysteine synthetase by heavy
metals in human leukemia cells. J. Biol. Chem. 271, 14981–14988.
Jaillon, O., Aury, J.-M., Brunet, F., Petit, J.-L., Stange-Thomann, N., et al., 2004. Genome duplication in
the teleost fish Tetraodon nigroviridis reveals the early vertebrate proto-karyotype. Nature 431,
946–57.
Jeong, J.-Y., Kwon, H.-B., Ahn, J.-C., Kang, D., Kwon, S.-H., Park, J.A., Kim, K.-W., 2008. Functional
and developmental analysis of the blood-brain barrier in zebrafish. Brain Res. Bull. 75, 619–628.
Johnson, K.R., Wright, J.E., May, B., 1987. Linkage relationships reflecting ancestral tetraploidy in
salmonid fish. Genetics 116, 579–91.
Jones, S.A., Moore, L.B., Shenk, J.L., Wisely, G.B., Hamilton, G.A., McKee, D.D., Tomkinson, N.C.,
LeCluyse, E.L., Lambert, M.H., Willson, T.M., Kliewer, S.A., Moore, J.T., 2000. The pregnane X
receptor: a promiscuous xenobiotic receptor that has diverged during evolution. Mol. Endocrinol.
14, 27–39.
Jonker, J.W., Buitelaar, M., Wagenaar, E., Van Der Valk, M.A., Scheffer, G.L., Scheper, R.J., Plösch, T.,
Kuipers, F., Oude Elferink, R.P.J., Rosing, H., Beijnen, J.H., Schinkel, A.H., 2002. The breast
cancer resistance protein protects against a major chlorophyll-derived dietary phototoxin and
protoporphyria. Proc. Natl. Acad. Sci. USA 99, 15649–54.
Kalaany, N.Y., Mangelsdorf, D.J., 2006. LXRS and FXR: the yin and yang of cholesterol and fat
metabolism. Annu. Rev. Physiol. 68, 159–91.
Kast, H.R., Goodwin, B., Tarr, P.T., Jones, S.A., Anisfeld, A.M., Stoltz, C.M., Tontonoz, P., Kliewer, S.,
Willson, T.M., Edwards, P.A., 2002. Regulation of multidrug resistance-associated protein 2
(ABCC2) by the nuclear receptors pregnane X receptor, farnesoid X-activated receptor, and
constitutive androstane receptor. J. Biol. Chem. 277, 2908–15.
47 1733 1734 1735 1736 1737 1738 1739 1740 1741 1742 1743 1744 1745 1746 1747 1748 1749 1750 1751 1752 1753 1754 1755 1756 1757 1758 1759 1760 1761 1762 1763 1764 1765 1766 1767 1768 1769 1770 1771 1772 1773 1774 1775 1776 1777 1778 1779 Katharios, P., Pavlidis, M., Iliopoulou-Georgudaki, J., 2004. Accumulation of ivermectin in the brain of
sea bream, Sparus aurata after intraperitoneal administration. Environ. Toxicol. Pharmacol. 17, 9–
12.
Kiilerich, P., Milla, S., Sturm, A., Valotaire, C., Chevolleau, S., Giton, F., Terrien, X., Fiet, J., Fostier,
A., Debrauwer, L., Prunet, P., 2011. Implication of the mineralocorticoid axis in rainbow trout
osmoregulation during salinity acclimation. J. Endocrinol. 209, 221–35.
Kim, R.B., Fromm, M.F., Wandel, C., Leake, B., Wood, A.J.J., Roden, D.M., Wilkinson, G.R., 1998.
The drug transporter P-glycoprotein limits oral absorption and brain entry of HIV-1 protease
inhibitors. J. Clin. Invest. 101, 289–294.
Kipp, H., Arias, I.M., 2002. Trafficking of canalicular ABC transporters in hepatocytes. Annu. Rev.
Physiol. 64, 595–608.
Kleinow, K.M., Doi, A.M., Smith, A.A., 2000. Distribution and inducibility of P-glycoprotein in the
catfish  : immunohistochemical detection using the mammalian C-219 monoclonal. Mar. Environ.
Res. 50, 313–317.
Kleinow, K.M., Hummelke, G.C., Zhang, Y., Uppu, P., Baillif, C., 2004. Inhibition of P-glycoprotein
transport: a mechanism for endocrine disruption in the channel catfish? Mar. Environ. Res. 58,
205–8.
Kleinow, K.M., Nichols, J.W., Hayton, W.L., Mckim, J.M., Barron, M.G., 2008. Toxicokinetics in
fishes, in: The Toxicology of Fishes. CRC Press, Boca Raton, FL, USA, pp. 55–152.
Kliewer, S.A., Moore, J.T., Wade, L., Staudinger, J.L., Watson, M.A., Jones, S.A., McKee, D.D., Oliver,
B.B., Willson, T.M., Zetterström, R.H., Perlmann, T., Lehmann, J.M., 1998. An orphan nuclear
receptor activated by pregnanes defines a novel steroid signaling pathway. Cell 92, 73–82.
Kobayashi, H., Parton, A., Czechanski, A., Durkin, C., Kong, C., Barnes, D., 2007. Multidrug resistanceassociated protein 3 (Mrp3/Abcc3/Moat-D) is expressed in the SAE Squalus acanthias shark
embryo-derived cell line. Zebrafish 4, 261–275.
Kobayashi, I., Saito, K., Moritomo, T., Araki, K., Takizawa, F., Nakanishi, T., 2008. Characterization
and localization of side population (SP) cells in zebrafish kidney hematopoietic tissue. Blood 111,
1131–7.
Koehler, A., Alpermann, T., Lauritzen, B., Van Noorden, C.J.F., 2004. Clonal xenobiotic resistance
during pollution-induced toxic injury and hepatocellular carcinogenesis in liver of female flounder
(Platichthys flesus (L.)). Acta Histochem. 106, 155–70.
Koepsell, H., Lips, K., Volk, C., 2007. Polyspecific organic cation transporters: structure, function,
physiological roles, and biopharmaceutical implications. Pharm. Res. 24, 1227–51.
Köhler, A., Lauritzen, B., Banns, S., 1998. Clonal adaptation of cancer cells in flatfish liver to
environmental contamination by changes in expression of P-gp related MXR, CYP450, GST-A
and G6PDH activity. Mar. Environ. Res. 46, 191–195.
Kok, T., Bloks, V.W., Wolters, H., Havinga, R., Jansen, P.L.M., Staels, B., Kuipers, F., 2003. Perxisome
proliferator-activated receptor a (PPARa)-mediated regulation of multidrug resistance 2 (Mdr2)
expression and function in mice. Biochem. J. 369, 539–547.
Krasowski, M.D., Ai, N., Hagey, L.R., Kollitz, E.M., Kullman, S.W., Reschly, E.J., Ekins, S., 2011. The
evolution of farnesoid X, vitamin D, and pregnane X receptors: insights from the green-spotted
pufferfish (Tetraodon nigriviridis) and other non-mammalian species. BMC Biochem. 12, 5.
Krasowski, M.D., Yasuda, K., Hagey, L.R., Schuetz, E.G., 2005. Evolution of the pregnane x receptor:
adaptation to cross-species differences in biliary bile salts. Mol. Endocrinol. 19, 1720–39.
Krishnamurthy, P., Schuetz, J.D., 2006. Role of ABCG2/BCRP in biology and medicine. Annu. Rev.
Pharmacol. Toxicol. 46, 381–410.
Kruh, G.D., Belinsky, M.G., Gallo, J.M., Lee, K., 2007. Physiological and pharmacological functions of
48 1780 1781 1782 1783 1784 1785 1786 1787 1788 1789 1790 1791 1792 1793 1794 1795 1796 1797 1798 1799 1800 1801 1802 1803 1804 1805 1806 1807 1808 1809 1810 1811 1812 1813 1814 1815 1816 1817 1818 1819 1820 1821 1822 1823 1824 1825 1826 Mrp2, Mrp3 and Mrp4 as determined from recent studies on gene-disrupted mice. Cancer
Metastasis Rev. 26, 5–14.
Kurelec, B., 1992. The multixenobiotic resistance mechanism in aquatic organisms. Crit Rev Toxicol.
22, 12–43.
Kurelec, B., Lucic, D., Pivcevic, B., Krca, S., 1995. Induction and reversion of multixenobiotic
resistance in the marine snail Monodonta turbinata. Mar. Biol. 123, 305–312.
Kurelec, B., Pivcevic, B., 1991. Evidence for a multixenobiotic resistance mechanism in the mussel,
Mytilus galloprovincialis. Aquat. Toxicol. 19, 291–302.
Kurelec, B., Pivčević, B., 1989. Distinct glutathione-dependent enzyme activities and a verapamilsensitive binding of xenobiotics in a fresh-water mussel Anodonta cygnea. Biochem. Biophys.
Res. Commun. 164, 934–940.
Lagas, J.S., Vlaming, M.L.H., Schinkel, A.H., 2009. Pharmacokinetic assessment of multiple ATPbinding cassette transporters: The power of combination knockout mice. Mol. Interv. 9, 136–145.
Lanning, C.L., Sachs, C.W., Rao, U.S., Corcoran, J.J., Abou-Donia, M.B., Fine, R.L., 1996. Chlorpyrifos
oxon interacts with the mammalian multidrug resistance protein, P-glycoprotein. J. Toxicol.
Environ. Health 47, 395–407.
Leaver, M.J., Boukouvala, E., Antonopoulou, E., Diez, A., Favre-Krey, L., Ezaz, M.T., Bautista, J.M.,
Tocher, D.R., Krey, G., 2005. Three peroxisome proliferator-activated receptor isotypes from each
of two species of marine fish. Endocrinology 146, 3150–62.
Lech, J., Pepple, S., Statham, C., 1973. Fish bile analysis - possible aid in monitoring water-quality.
Toxicol. Appl. Pharmacol. 25, 430–434.
Leggas, M., Adachi, M., Scheffer, G.L., Wielinga, P., Du, G., Mercer, K.E., Panetta, J.C., Johnston, B.,
Scheper, R.J., Stewart, C.F., Schuetz, J.D., Sun, D., Zhuang, Y., 2004. Mrp4 confers resistance to
topotecan and protects the brain from chemotherapy. Mol. Cell. Biol. 24, 7612–7621.
Leier, I., Jedlitschky, G., Buchholz, U., Cole, S.P., Deeley, R.G., Keppler, D., 1994. The MRP gene
encodes an ATP-dependent export pump for leukotriene C4 and structurally related conjugates. J.
Biol. Chem. 269, 27807–27810.
Leslie, E.M., Deeley, R.G., Cole, S.P.C., 2005. Multidrug resistance proteins: role of P-glycoprotein,
MRP1, MRP2, and BCRP (ABCG2) in tissue defense. Toxicol. Appl. Pharmacol. 204, 216–37.
Li, Q., Sadowski, S., Frank, M., Chai, C., Váradi, A., Ho, S.-Y., Lou, H., Dean, M., Thisse, C., Thisse,
B., Uitto, J., 2010. The abcc6a gene expression is required for normal zebrafish development. J.
Invest. Dermatol. 130, 2561–8.
Litman, T., Zeuthen, T., Skovsgaard, T., Stein, W.D., 1997. Structure-activity relationships of Pglycoprotein interacting drugs: Kinetic characterization of their effects on ATPase activity.
Biochim. Biophys. Acta - Mol. Basis Dis. 1361, 159–168.
Liu, S., Li, Q., Liu, Z., 2013. Genome-wide identification, characterization and phylogenetic analysis of
50 catfish ATP-binding cassette (ABC) transporter genes. PLoS One 8, e63895.
Loncar, J., Popović, M., Zaja, R., Smital, T., 2010. Gene expression analysis of the ABC efflux
transporters in rainbow trout (Oncorhynchus mykiss). Comp. Biochem. Physiol. C. Toxicol.
Pharmacol. 151, 209–15.
Long, Y., Li, Q., Cui, Z., 2011a. Molecular analysis and heavy metal detoxification of ABCC1/MRP1 in
zebrafish. Mol. Biol. Rep. 38, 1703–11.
Long, Y., Li, Q., Li, J., Cui, Z., 2011b. Molecular analysis, developmental function and heavy metalinduced expression of ABCC5 in zebrafish. Comp. Biochem. Physiol. B. Biochem. Mol. Biol. 158,
46–55.
Long, Y., Li, Q., Wang, Y., Cui, Z., 2011c. MRP proteins as potential mediators of heavy metal
resistance in zebrafish cells. Comp. Biochem. Physiol. C. Toxicol. Pharmacol. 153, 310–7.
49 1827 1828 1829 1830 1831 1832 1833 1834 1835 1836 1837 1838 1839 1840 1841 1842 1843 1844 1845 1846 1847 1848 1849 1850 1851 1852 1853 1854 1855 1856 1857 1858 1859 1860 1861 1862 1863 1864 1865 1866 1867 1868 1869 1870 1871 1872 1873 Long, Y., Li, Q., Zhong, S., Wang, Y., Cui, Z., 2011d. Molecular characterization and functions of
zebrafish ABCC2 in cellular efflux of heavy metals. Comp. Biochem. Physiol. C. Toxicol.
Pharmacol. 153, 381–91.
Loo, T.W., Clarke, D.M., 1998. Nonylphenol ethoxylates, but not nonylphenol, are substrates of the
human multidrug resistance P-glycoprotein. Biochem. Biophys. Res. Commun. 247, 478–80.
Lynagh, T., Lynch, J.W., 2012. Ivermectin binding sites in human and invertebrate Cys-loop receptors.
Trends Pharmacol. Sci. 33, 432–41.
Maglich, J.M., 2003. The first completed genome sequence from a teleost fish (Fugu rubripes) adds
significant diversity to the nuclear receptor superfamily. Nucleic Acids Res. 31, 4051–4058.
Maglich, J.M., Stoltz, C.M., Goodwin, B., Hawkins-Brown, D., Moore, J.T., Kliewer, S.A., 2002.
Nuclear pregnane x receptor and constitutive androstane receptor regulate overlapping but distinct
sets of genes involved in xenobiotic detoxification. Mol. Pharmacol. 62, 638–46.
Marshall, W.S., Grosell, M., 2006. Ion transport, osmoregulation, and acid-base balance, in: Evans, D.H.,
Claiborne, J.B. (Eds.), The Physiology of Fishes. CRC Press, Boca Raton, FL, USA, pp. 177–230.
Masereeuw, R., Moons, M.M., Toomey, B.H., Russel, F.G., Miller, D.S., 1999. Active lucifer yellow
secretion in renal proximal tubule: evidence for organic anion transport system crossover. J.
Pharmacol. Exp. Ther. 289, 1104–11.
Masereeuw, R., Russel, F.G., Miller, D.S., 1996. Multiple pathways of organic anion secretion in renal
proximal tubule revealed by confocal microscopy. Am. J. Physiol. 271, F1173–82.
Masereeuw, R., Russel, F.G.M., 2012. Regulatory pathways for ATP-binding cassette transport proteins
in kidney proximal tubules. AAPS J. 14, 883–94.
Masereeuw, R., Terlouw, S.A., van Aubel, R.A., Russel, F.G., Miller, D.S., 2000. Endothelin B receptormediated regulation of ATP-driven drug secretion in renal proximal tubule. Mol. Pharmacol. 57,
59–67.
McKim, J.M., 1985. Early life stage toxicity tests, in: Rand, G.M., Petrocelli, S.R. (Eds.), Fundamentals
of Aquatic Toxicology. Hemisphere, New York, pp. 58–95.
Miller, D., 1995. Daunomycin secretion by killifish proximal tubules. Am. J. Physiol. 269, R370–R379.
Miller, D., Masereeuw, R., 2002. Regulation of MRP2-mediated transport in shark rectal salt gland
tubules. Am. J. Physiol. Regul. Integr. Comp. Physiol. 282, R774–R781.
Miller, D.S., 1987. Aquatic models for the study of renal transport function and pollutant toxicity.
Environ. Health Perspect. 71, 59–68.
Miller, D.S., 1995. Daunomycin secretion by killfish renal proximal tubules. Am. J. Physiol. Regul.
Integr. Comp. Physiol. 269, R370–R379.
Miller, D.S., 2010. Regulation of P-glycoprotein and other ABC drug transporters at the blood-brain
barrier. Trends Pharmacol. Sci. 31, 246–54.
Miller, D.S., Fricker, G., Drewe, J., 1997. p-Glycoprotein-mediated transport of a fluorescent rapamycin
derivative in renal proximal tubule. J. Pharmacol. Exp. Ther. 282, 440–4.
Miller, D.S., Graeff, C., Droulle, L., Fricker, S., Fricker, G., 2002. Xenobiotic efflux pumps in isolated
fish brain capillaries. Am. J. Physiol. Regul. Integr. Comp. Physiol. 282, R191–8.
Miller, D.S., Masereeuw, R., Henson, J., Karnaky, K.J., 1998a. Excretory transport of xenobiotics by
dogfish shark rectal gland tubules. Am. J. Physiol. 275, R697–705.
Miller, D.S., Pritchard, J.B., 1997. Dual pathways for organic anion secretion in renal proximal tubule. J.
Exp. Zool. 279, 462–70.
Miller, D.S., Shaw, J.R., Stanton, C.R., Barnaby, R., Karlson, K.H., Hamilton, J.W., Stanton, B.A., 2007.
MRP2 and acquired tolerance to inorganic arsenic in the kidney of killifish (Fundulus
heteroclitus). Toxicol. Sci. 97, 103–10.
Miller, D.S., Sussman, C.R., Renfro, J.L., 1998b. Protein kinase C regulation of p-glycoprotein-mediated
50 1874 1875 1876 1877 1878 1879 1880 1881 1882 1883 1884 1885 1886 1887 1888 1889 1890 1891 1892 1893 1894 1895 1896 1897 1898 1899 1900 1901 1902 1903 1904 1905 1906 1907 1908 1909 1910 1911 1912 1913 1914 1915 1916 1917 1918 1919 1920 xenobiotic secretion in renal proximal tubule. Am. J. Physiol. 275, F785–95.
Mirbahai, L., Yin, G., Bignell, J.P., Li, N., Williams, T.D., Chipman, J.K., 2011. DNA methylation in
liver tumorigenesis in fish from the environment. Epigenetics 6, 1319–33.
Moitra, K., Scally, M., McGee, K., Lancaster, G., Gold, B., Dean, M., 2011. Molecular evolutionary
analysis of ABCB5: the ancestral gene is a full transporter with potentially deleterious single
nucleotide polymorphisms. PLoS One 6, e16318.
Moore, L.B., Maglich, J.M., McKee, D.D., Wisely, B., Willson, T.M., Kliewer, S.A., Lambert, M.H.,
Moore, J.T., 2002. Pregnane X receptor (PXR), constitutive androstane receptor (CAR), and
benzoate X receptor (BXR) define three pharmacologically distinct classes of nuclear receptors.
Mol. Endocrinol. 16, 977–86.
Moore, L.B., Parks, D.J., Jones, S.A., Bledsoe, R.K., Consler, T.G., Stimmel, J.B., Goodwin, B., Liddle,
C., Blanchard, S.G., Willson, T.M., Collins, J.L., Kliewer, S.A., 2000. Orphan nuclear receptors
constitutive androstane receptor and pregnane X receptor share xenobiotic and steroid ligands. J.
Biol. Chem. 275, 15122–7.
Moschetta, A., Xu, F., Hagey, L.R., van Berge-Henegouwen, G.P., van Erpecum, K.J., Brouwers, J.F.,
Cohen, J.C., Bierman, M., Hobbs, H.H., Steinbach, J.H., Hofmann, A.F., 2005. A phylogenetic
survey of biliary lipids in vertebrates. J. Lipid Res. 46, 2221–32.
Mruk, D.D., Su, L., Cheng, C.Y., 2011. Emerging role for drug transporters at the blood-testis barrier.
Trends Pharmacol. Sci. 32, 99–106.
Myers, M.S., Landahl, J.T., Krahn, M.M., McCain, B.B., 1991. Relationships between hepatic neoplasms
and related lesions and exposure to toxic chemicals in marine fish from the U.S. West Coast.
Environ. Health Perspect. 90, 7–15.
Nebert, D.W., Roe, A.L., Dieter, M.Z., Solis, W.A., Yang, Y., Dalton, T.P., 2000. Role of the aromatic
hydrocarbon receptor and [Ah] gene battery in the oxidative stress response, cell cycle control, and
apoptosis. Biochem. Pharmacol. 59, 65–85.
Nelson, G., 2006. Fishes of the world, 4th Editio. ed. Wiley, New York.
Nichols, J., Erhardt, S., Dyer, S., James, M., Noore, M., Plotzke, K., Segner, H., Schultz, I., Thomas, K.,
Vasiluk, L., Weisbrod, A., 2007. Use of in vitro absorption, distribution, metabolism, and
excretion (ADME) data in bioaccumulation assessments for fish. Hum. Ecol. Risk Assess. 13,
1164–1191.
Nickel, S., Bernd, A., Miller, D.S., Fricker, G., Mahringer, A., 2013. Bisphenol - A modulates function
of ABC transporters in killifish. MDIBL Bull. 52, 30.
Nies, A.T., Keppler, D., 2007. The apical conjugate efflux pump ABCC2 (MRP2). Pflugers Arch. 453,
643–59.
Notenboom, S., Miller, D.S., Smits, P., Russel, F.G.M., Masereeuw, R., 2002. Role of NO in endothelinregulated drug transport in the renal proximal tubule. Am. J. Physiol. Renal Physiol. 282, F458–
64.
Notenboom, S., Miller, D.S., Smits, P., Russel, F.G.M., Masereeuw, R., 2004. Involvement of guanylyl
cyclase and cGMP in the regulation of Mrp2-mediated transport in the proximal tubule. Am. J.
Physiol. Renal Physiol. 287, F33–8.
Notredame, C., Higgins, D.G., Heringa, J., 2000. T-Coffee: A novel method for fast and accurate
multiple sequence alignment. J. Mol. Biol. 302, 205–17.
Oberemm, A., 2000. The use of a refined zebrafish embryo bioassay for the assessment of aquatic
toxicity. LAB Anim. York - 29, 32–43.
Ohno, S., 1970. Evolution by gene duplication. Springer, New York.
Oikari, A., Kunnamo-Ojala, T., 1987. Tracing of xenobiotic contamination in water with the aid of fish
bile metabolites: A field study with caged rainbow trout (Salmo gairdneri). Aquat. Toxicol. 9,
51 1921 1922 1923 1924 1925 1926 1927 1928 1929 1930 1931 1932 1933 1934 1935 1936 1937 1938 1939 1940 1941 1942 1943 1944 1945 1946 1947 1948 1949 1950 1951 1952 1953 1954 1955 1956 1957 1958 1959 1960 1961 1962 1963 1964 1965 1966 1967 327–341.
Oostendorp, R.L., Beijnen, J.H., Schellens, J.H.M., 2009. The biological and clinical role of drug
transporters at the intestinal barrier. Cancer Treat. Rev. 35, 137–47.
Oosterhuis, B., Vukman, K., Vági, E., Glavinas, H., Jablonkai, I., Krajcsi, P., 2008. Specific interactions
of chloroacetanilide herbicides with human ABC transporter proteins. Toxicology 248, 45–51.
Oude Elferink, R.P.J., Ottenhoff, R., Fricker, G., Seward, D.J., Ballatori, N., Boyer, J., 2004. Lack of
biliary lipid excretion in the little skate, Raja erinacea, indicates the absence of functional Mdr2,
Abcg5, and Abcg8 transporters. Am. J. Physiol. Gastrointest. Liver Physiol. 286, G762–8.
Oude Elferink, R.P.J., Paulusma, C.C., 2007. Function and pathophysiological importance of ABCB4
(MDR3 P-glycoprotein). Pflugers Arch. 453, 601–10.
Paetzold, S.C., Ross, N.W., Richards, R.C., Jones, M., Hellou, J., Bard, S.M., 2009. Up-regulation of
hepatic ABCC2, ABCG2, CYP1A1 and GST in multixenobiotic-resistant killifish (Fundulus
heteroclitus) from the Sydney Tar Ponds, Nova Scotia, Canada. Mar. Environ. Res. 68, 37–47.
Park, D., Haldi, M., Seng, W.L., 2012. Zebrafish: a new in vivo model for identifying P-glycoprotein
efflux modulators, in: McGrath, P. (Ed.), Zebrafish: Methods for Assessing Drug Safety and
Toxicity. Wiley, Hoboken, NJ, USA, pp. 177–190.
Pfendner, E.G., Vanakker, O.M., Terry, S.F., Vourthis, S., McAndrew, P.E., McClain, M.R., Fratta, S.,
Marais, A.-S., Hariri, S., Coucke, P.J., Ramsay, M., Viljoen, D., Terry, P.F., De Paepe, A., Uitto,
J., Bercovitch, L.G., 2007. Mutation detection in the ABCC6 gene and genotype-phenotype
analysis in a large international case series affected by pseudoxanthoma elasticum. J. Med. Genet.
44, 621–8.
Polli, J.W., Jarrett, J.L., Studenberg, S.D., Humphreys, J.E., Dennis, S.W., Brouwer, K.R., Woolley, J.L.,
1999. Role of P-glycoprotein on teh CNS disposition of amprenavir (141W94), an HIV protease
inhibitor. Pharm. Res. 16, 1206–1212.
Popovic, M., Zaja, R., Loncar, J., Smital, T., 2010. A novel ABC transporter: the first insight into
zebrafish (Danio rerio) ABCH1. Mar. Environ. Res. 69 Suppl, S11–3.
Prevoo, B., Miller, D.S., Water, F.M. Van De, Wever, K.E., Russel, F.G.M., Flik, G., Masereeuw, R.,
2011. Rapid, nongenomic stimulation of multidrug resistance protein 2 (Mrp2) activity by
glucocorticoids in renal proximal tubule. J. Pharmacol. Exp. Ther. 338, 362–371.
Rebbeor, J.F., Connolly, G.C., Henson, J.H., Boyer, J.L., Ballatori, N., 2000. ATP-dependent GSH and
glutathione S -conjugate transport in skate liver  : role of an Mrp functional homologue ATPdependent GSH and glutathione S-conjugate transport in skate liver  : role of an Mrp functional
homologue. Am. J. Physiol. Gastrointest. Liver Physiol. 279, G417–G425.
Redzic, Z., 2011. Molecular biology of the blood-brain and the blood-cerebrospinal fluid barriers:
similarities and differences. Fluids Barriers CNS 8, 3.
Reichel, V., Masereeuw, R., van den Heuvel, J.J.M.W., Miller, D.S., Fricker, G., 2007. Transport of a
fluorescent cAMP analog in teleost proximal tubules. Am. J. Physiol. Regul. Integr. Comp.
Physiol. 293, R2382–9.
Reichel, V., Miller, D.S., Fricker, G., 2008. Texas Red transport across rat and dogfish shark (Squalus
acanthias) choroid plexus. Am. J. Physiol. Regul. Integr. Comp. Physiol. 295, R1311–9.
Reschly, E.J., Ai, N., Ekins, S., Welsh, W.J., Hagey, L.R., Hofmann, A.F., Krasowski, M.D., 2008.
Evolution of the bile salt nuclear receptor FXR in vertebrates. J. Lipid Res. 49, 1577–87.
Reschly, E.J., Bainy, A.C.D., Mattos, J.J., Hagey, L.R., Bahary, N., Mada, S.R., Ou, J., Venkataramanan,
R., Krasowski, M.D., 2007. Functional evolution of the vitamin D and pregnane X receptors. BMC
Evol. Biol. 7, 222.
Riordan, J.R., Ling, V., 1985. Genetic and biochemical characterization of multidrug resistance.
Pharmacol. Ther. 28, 51–75.
52 1968 1969 1970 1971 1972 1973 1974 1975 1976 1977 1978 1979 1980 1981 1982 1983 1984 1985 1986 1987 1988 1989 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 Robey, R.W., Steadman, K., Polgar, O., Morisaki, K., Blayney, M., Mistry, P., Bates, S.E., 2004.
Pheophorbide a is a specific probe for ABCG2 function and inhibition. Cancer Res. 64, 1242–
1246.
Robey, R.W., To, K.K.K., Polgar, O., Dohse, M., Fetsch, P., Dean, M., Bates, S.E., 2009. ABCG2: a
perspective. Adv. Drug Deliv. Rev. 61, 3–13.
Robinson-Rechavi, M., Marchand, O., Escriva, H., Bardet, P.L., Zelus, D., Hughes, S., Laudet, V., 2001.
Euteleost fish genomes are characterized by expansion of gene families. Genome Res. 11, 781–
788.
Roma, M.-G., 2008. Dynamic localization of hepatocellular transporters in health and disease. World J.
Gastroenterol. 14, 6786.
Roninson, I., Abelson, A., Housman, D., Howell, N., Varshavsky, A., 1984. Amplification of specific
DNA sequences correlates with multi-drug resistance in Chinese hamster cells. Nature 309, 626–
628.
Rotchell, J.M., Miller, M.R., Hinton, D.E., Di Giulio, R.T., Ostrander, G.K., 2008. Chemical
carcinogenesis in fishes, in: Di Giulio, R.T., Hinton, David E. (Eds.), The Toxicology of Fishes.
CRC Press, Boca Raton, FL, USA, pp. 531–596.
Sabri, D.M., Rabie, T., Ahmed, A.I., Zakaria, S., Bourdineaud, J.-P., 2012. Heavy metals-induced
expression of ABCB10 gene in zebrafish Danio rerio. Egypt. Acad. J. Biol. Sci. 4, 97–106.
Saito, Y., Wright, E.M., 1984. Regulation of bicarbonate transport across the brush border membrane of
the bull frog choroid plexus. J. Physiol. 350, 327–342.
Sarkadi, B., Price, E.M., Boucher, R.C., Germann, U. a, Scarborough, G. a, 1992. Expression of the
human multidrug resistance cDNA in insect cells generates a high activity drug-stimulated
membrane ATPase. J. Biol. Chem. 267, 4854–8.
Sauerborn Klobučar, R., Zaja, R., Franjević, D., Brozović, A., Smital, T., 2010. Presence of
ecotoxicologically relevant Pgp and MRP transcripts and proteins in cyprinid fish. Arh. Hig. Rada
Toksikol. 61, 175–82.
Schinkel, A.H., Jonker, J.W., 2003. Mammalian drug efflux transporters of the ATP binding cassette
(ABC) family: an overview. Adv. Drug Deliv. Rev. 55, 3–29.
Schinkel, A.H., Smit, J.J., van Tellingen, O., Beijnen, J.H., Wagenaar, E., van Deemter, L., Mol, C.A.,
van der Valk, M.A., Robanus-Maandag, E.C., te Riele, H.P., Berns, A.J.M., Borst, P., 1994.
Disruption of the mouse mdr1a P-glycoprotein gene leads to a deficiency in the blood-brain barrier
and to increased sensitivity to drugs. Cell 77, 491–502.
Schinkel, A.H., Wagenaar, E., van Deemter, L., Mol, C.A.A.M., Borst, P., 1995. Absence of the mdr1a
P-Glycoprotein in mice affects tissue distribution and pharmacokinetics of dexamethasone,
digoxin, and cyclosporin A. J. Clin. Invest. 96, 1698–705.
Schlenk, D., Celander, M., Gallagher, E., George, S., James, M., Kullman, S.W., Van den Hurk, P.,
Willett, K., 2008. Biotransformation in fishes, in: Di Giulio, R., Hinton, D.E. (Eds.), The
Toxicology of Fishes. CRC Press, Boca Raton, FL, USA, pp. 153–234.
Schramm, U., Fricker, G., Wenger, R., Miller, D.S., 1995. P-glycoprotein-mediated secretion of a
fluorescent cyclosporin analogue by teleost renal proximal tubules. Am. J. Physiol. Physiol. 268,
F46–F52.
Scotto, K.W., 2003. Transcriptional regulation of ABC drug transporters. Oncogene 22, 7496–511.
Sharom, F.J., Yu, X., Lu, P., Liu, R., Chu, J.W., Szabó, K., Müller, M., Hose, C.D., Monks, a, Váradi, a,
Seprôdi, J., Sarkadi, B., 1999. Interaction of the P-glycoprotein multidrug transporter (MDR1)
with high affinity peptide chemosensitizers in isolated membranes, reconstituted systems, and
intact cells. Biochem. Pharmacol. 58, 571–86.
Shepard, R.L., Cao, J., Starling, J.J., Dantzig, A.H., 2003. Modulation of P-glycoprotein but not MRP1-
53 2015 2016 2017 2018 2019 2020 2021 2022 2023 2024 2025 2026 2027 2028 2029 2030 2031 2032 2033 2034 2035 2036 2037 2038 2039 2040 2041 2042 2043 2044 2045 2046 2047 2048 2049 2050 2051 2052 2053 2054 2055 2056 2057 2058 2059 2060 2061 or BCRP-mediated drug resistance by LY335979. Int. J. Cancer 125, 121–125.
Shúilleabháin, S.N., Davoren, M., Mothersill, C., Sheehan, D., Hartl, M.G.J., Kilemade, M., O’brien,
N.M., O’halloran, J., Van Pelt, F.N.A.M., Lyng, F.M., 2005. Identification of a multixenobiotic
resistance mechanism in primary cultured epidermal cells from Oncorhynchus mykiss and the
effects of environmental complex mixtures on its activity. Aquat. Toxicol. 73, 115–27.
Siegsmund, M.J., Cardarelli, C., Aksentijevich, I., Sugimoto, Y., Pastan, I., Gottesman, M.M., 1994.
Ketoconazole effectively reverses multidrug resistance in highly resistant KB cells. J. Urol. 151,
485–491.
Slot, A.J., Molinski, S. V, Cole, S.P.C., 2011. Mammalian multidrug resistance proteins (MRPs). Essays
Biochem. 50, 179–207.
Small, H.J., Williams, T.D., Sturve, J., Chipman, J.K., Southam, A.D., Bean, T.P., Lyons, B.P.,
Stentiford, G.D., 2010. Gene expression analyses of hepatocellular adenoma and hepatocellular
carcinoma from the marine flatfish Limanda limanda. Dis. Aquat. Organ. 88, 127–41.
Smital, T., Sauerborn, R., 2002. Measurement of the activity of multixenobiotic resistance mechanism in
the common carp Cyprinus carpio. Mar. Environ. Res. 54, 449–53.
Smital, T., Terzic, S., Zaja, R., Senta, I., Pivcevic, B., Popovic, M., Mikac, I., Tollefsen, K.E., Thomas,
K. V, Ahel, M., 2011. Assessment of toxicological profiles of the municipal wastewater effluents
using chemical analyses and bioassays. Ecotoxicol. Environ. Saf. 74, 844–51.
Stein, W.D., 1997. Kinetics of the multidrug transporter and its reversal. Physiol. Rev. 77, 545–590.
Steinke, D., Hoegg, S., Brinkmann, H., Meyer, A., 2006. Three rounds (1R/2R/3R) of genome
duplications and the evolution of the glycolytic pathway in vertebrates. BMC Biol. 4, 16.
Stieger, B., Meier, Y., Meier, P.J., 2007. The bile salt export pump. Pflugers Arch. 453, 611–20.
Sturm, A., Cravedi, J.P., Segner, H., 2001a. Prochloraz and nonylphenol diethoxylate inhibit an mdr1like activity in vitro, but do not alter hepatic levels of P-glycoprotein in trout exposed in vivo.
Aquat. Toxicol. 53, 215–28.
Sturm, A., Segner, H., 2005. P-glycoproteins and xenobiotic efflux transport in fish. Biochem. Mol. Biol.
Fishes (T.P. Mommsen T.W. Moon, Eds) 6, 495–533.
Sturm, A., Ziemann, C., Hirsch-Ernst, K.I., Segner, H., 2001b. Expression and functional activity of Pglycoprotein in cultured hepatocytes from Oncorhynchus mykiss. Am. J. Physiol. Regul. Integr.
Comp. Physiol. 281, R1119–26.
Sussman-Turner, C., Renfro, J.L., 1995a. Heat-shock stimulated transepithelial daunomycin secretion by
flounder renal proximal tubule primary cultures. Am. J. Physiol. 267, F135–F144.
Sussman-Turner, C., Renfro, J.L., 1995b. Heat-shock-stimulated transepithelial daunomycin secretion by
flounder renal proximal tubule primary cultures. Am. J. Physiol. 268, F135–44.
Synold, T.W., Dussault, I., Forman, B.M., 2001. The orphan nuclear receptor SXR coordinately regulates
drug metabolism and efflux. Nat. Med. 7, 584–590.
Szakács, G., Váradi, A., Ozvegy-Laczka, C., Sarkadi, B., 2008. The role of ABC transporters in drug
absorption, distribution, metabolism, excretion and toxicity (ADME-Tox). Drug Discov. Today
13, 379–93.
Szatmari, I., Vámosi, G., Brazda, P., Balint, B.L., Benko, S., Széles, L., Jeney, V., Ozvegy-Laczka, C.,
Szántó, A., Barta, E., Balla, J., Sarkadi, B., Nagy, L., 2006. Peroxisome proliferator-activated
receptor gamma-regulated ABCG2 expression confers cytoprotection to human dendritic cells. J.
Biol. Chem. 281, 23812–23.
Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., Kumar, S., 2011. MEGA5: molecular
evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum
parsimony methods. Mol. Biol. Evol. 28, 2731–9.
Tan, X., Yim, S.-Y., Uppu, P., Kleinow, K.M., 2010. Enhanced bioaccumulation of dietary contaminants
54 2062 2063 2064 2065 2066 2067 2068 2069 2070 2071 2072 2073 2074 2075 2076 2077 2078 2079 2080 2081 2082 2083 2084 2085 2086 2087 2088 2089 2090 2091 2092 2093 2094 2095 2096 2097 2098 2099 2100 2101 2102 2103 2104 2105 2106 2107 2108 in catfish with exposure to the waterborne surfactant linear alkylbenzene sulfonate. Aquat.
Toxicol. 99, 300–8.
Taylor, J.S., Van de Peer, Y., Braasch, I., Meyer, a, 2001. Comparative genomics provides evidence for
an ancient genome duplication event in fish. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 356, 1661–
79.
Teeter, L.D., Petersen, D.D., Nebert, D.W., Kuo, M.T., 1991. Murine mdr1-1, mdr-2, and mdr-3 gene
expression: No coinducion with the Cyp1a-1 and Nmo-1 genes in liver by 2,3,7,8tetrachlorodibenzo-p-dioxin. DNA Cell Biol. 10, 433–441.
Terlouw, S.A., Masereeuw, R., Russel, F.G., Miller, D.S., 2001. Nephrotoxicants induce endothelin
release and signaling in renal proximal tubules: effect on drug efflux. Mol. Pharmacol. 59, 1433–
40.
Thiebaut, F., Tsuruo, T., Hamada, H., Gottesman, M.M., Pastan, I., 1987. Cellular localization of the
multidrug-resistance gene product in normal human tissues. Proc. Natl. Acad. Sci. USA 84, 7735–
7738.
Thisse, B., Thisse, C., 2004. Fast release clones: A high throughput expression analysis. ZFIN direct data
submission. [WWW Document]. URL http://zfin.org/cgi-bin/webdriver?MIval=aaxpatselect.apg&query_results=true&gene_name=abcb5&searchtype=equals
Tutundjian, R., Cachot, J., Leboulenger, F., Minier, C., 2002. Genetic and immunological
characterisation of a multixenobiotic resistance system in the turbot (Scophthalmus maximus).
Comp. Biochem. Physiol. B. Biochem. Mol. Biol. 132, 463–71.
Twentyman, P., Rhodes, T., Rayner, S., 1994. A comparison of rhodamine 123 accumulation and efflux
in cells with P-glycoprotein-mediated and MRP-associated multidrug resistance phenotypes. Eur.
J. Cancer 30A, 1360–1369.
Ueda, K., Cardarelli, C., Gottesman, M.M., Pastan, I., 1987. Expression of a full-length cDNA for the
human “MDR1” gene confers resistance to colchicine, doxorubicin, and vinblastine. Proc. Natl.
Acad. Sci. USA 84, 3004–3008.
Ueno, M., 2009. Mechanisms of the penetration of blood-borne substances into the brain. Curr.
Neuropharmacol. 7, 142–9.
Van Aubel, R.A.M.H., Smeets, P.H.E., Peters, J.G.P., Bindels, R.J.M., Russel, F.G.M., 2002. The
MRP4/ABCC4 gene encodes a novel apical organic anion transporter in human kidney proximal
tubules: putative efflux pump for urinary cAMP and cGMP. J. Am. Soc. Nephrol. 13, 595–603.
Van Veen, H.W., Higgins, C.F., Konings, W.N., 2001. Multidrug transport by ATP binding cassette
transporters: a proposed two-cylinder engine mechanism. Res. Microbiol. 152, 365–74.
Vethaak, A., Jol, J., 1996. Diseases of flounder Platichthys flesus in Dutch coastal and estuarine waters,
with particular reference to environmental stress factors. I. Epizootiology of gross lesions. Dis.
Aquat. Organ. 26, 81–97.
Vethaak, A.D., Wester, P.W., 1996. Diseases of flounder Platichthys flesus in Dutch coastal and
estuarine waters, with particular reference to environmental stress factors. II. Liver histopathology.
Dis. Aquat. Organ. 26, 99–116.
Vlaming, M.L.H., Lagas, J.S., Schinkel, A.H., 2009. Physiological and pharmacological roles of ABCG2
(BCRP): recent findings in Abcg2 knockout mice. Adv. Drug Deliv. Rev. 61, 14–25.
Von Richter, O., Glavinas, H., Krajcsi, P., Liehner, S., Siewert, B., Zech, K., 2009. A novel screening
strategy to identify ABCB1 substrates and inhibitors. Naunyn. Schmiedebergs. Arch. Pharmacol.
379, 11–26.
Wang, N., Lan, D., Chen, W., Matsuura, F., Tall, A.R., 2004. ATP-binding cassette transporters G1 and
G4 mediate cellular cholesterol efflux to high-density lipoproteins. Proc. Natl. Acad. Sci. USA
101, 9774–9.
55 2109 2110 2111 2112 2113 2114 2115 2116 2117 2118 2119 2120 2121 2122 2123 2124 2125 2126 2127 2128 2129 2130 2131 2132 2133 2134 2135 2136 2137 2138 2139 2140 2141 2142 2143 2144 2145 2146 2147 2148 2149 2150 2151 2152 2153 2154 2155 Waxman, D.J., 1999. P450 gene induction by structurally diverse xenochemicals  : central role of nuclear
receptors CAR, PXR, and PPAR. Arch. Biochem. Biophys. 369, 11–23.
Wever, K.E., Masereeuw, R., Miller, D.S., Hang, X.M., Flik, G., 2007. Endothelin and calciotropic
hormones share regulatory pathways in multidrug resistance protein 2-mediated transport. Am. J.
Physiol. Renal Physiol. 292, F38–46.
Wijnholds, J., Evers, R., VanLeusden, M.R., Mol, C., Zaman, G.J.R., Mayer, U., Beijnen, J.H.,
VanderValk, M., Krimpenfort, P., Borst, P., 1997. Increased sensitivity to anticancer drugs and
decreased inflammatory response in mice lacking the multidrug resistance-associated protein. Nat.
Med. 3, 1275–1279.
Woodward, O.M., Köttgen, A., Köttgen, M., 2011. ABCG transporters and disease. FEBS J. 278, 3215–
25.
Wright, S.H., Dantzler, W.H., 2004. Molecular and cellular physiology of renal organic cation and anion
transport. Physiol. Rev. 84, 987–1049.
Xie, J., Farage, E., Sugimoto, M., Anand-Apte, B., 2010. A novel transgenic zebrafish model for bloodbrain and blood-retinal barrier development. BMC Dev. Biol. 10, 76.
Xie, W., Yeuh, M.-F., Radominska-Pandya, A., Saini, S.P.S., Negishi, Y., Bottroff, B.S., Cabrera, G.Y.,
Tukey, R.H., Evans, R.M., 2003. Control of steroid, heme, and carcinogen metabolism by nuclear
pregnane X receptor and constitutive androstane receptor. Proc. Natl. Acad. Sci. USA 100, 4150–
5.
Yeheskely-Hayon, D., Regev, R., Katzir, H., Eytan, G.D., 2009. Competition between innate multidrug
resistance and intracellular binding of rhodamine dyes. FEBS J. 276, 637–648.
Yudt, M.R., Cidlowski, J.A., 2002. The glucocorticoid receptor: coding a diversity of proteins and
responses through a single gene. Mol. Endocrinol. 16, 1719–1726.
Zaja, R., Caminada, D., Lončar, J., Fent, K., Smital, T., 2008a. Development and characterization of Pglycoprotein 1 (Pgp1 , ABCB1) -mediated doxorubicin-resistant PLHC-1 hepatoma fish cell line.
Toxicol. Appl. Pharmacol. 227, 207–218.
Zaja, R., Klobucar, R.S., Smital, T., 2007. Detection and functional characterization of Pgp1 (ABCB1)
and MRP3 (ABCC3) efflux transporters in the PLHC-1 fish hepatoma cell line. Aquat. Toxicol.
81, 365–376.
Zaja, R., Lončar, J., Popovic, M., Smital, T., 2011. First characterization of fish P-glycoprotein (abcb1)
substrate specificity using determinations of its ATPase activity and calcein-AM assay with
PLHC-1/dox cell line. Aquat. Toxicol. 103, 53–62.
Zaja, R., Munic, V., Klobučar, R.S., Ambriovic-Ristov, A., Smital, T., 2008b. Cloning and molecular
characterization of apical efflux transporters (ABCB1, ABCB11 and ABCC2) in rainbow trout
(Oncorhynchus mykiss) hepatocytes. Aquat. Toxicol. 90, 322–332.
Zaja, R., Terzić, S., Senta, I., Lončar, J., Popović, M., Ahel, M., Smital, T., Terzić, S., 2013.
Identification of P-glycoprotein inhibitors in contaminated freshwater sediments. Sci. Technol. 47,
4813–4821.
Zaman, G.J., Flens, M.J., van Leusden, M.R., de Haas, M., Mülder, H.S., Lankelma, J., Pinedo, H.M.,
Scheper, R.J., Baas, F., Broxterman, H.J., 1994. The human multidrug resistance-associated
protein MRP is a plasma membrane drug-efflux pump. Proc. Natl. Acad. Sci. USA 91, 8822–6.
Zhu, M., Zhao, W., Jia, L., Lu, J., Qiao, T., Qu, Q., 2009. 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