The maintenance of the overall environmental health is quite dependent on aquatic ecosystems. Species overexploitation, pollution from urban, industrial and agricultural areas, as well as habitat loss and alteration have been contributing to the decline of aquatic environmental health. Toxic heavy metals resultant from industrial processes, persistent organic pollutants (POPs) from agricultural and industrial sources and polycyclic aromatic hydrocarbons (PAHs) resultant from oil spills are among the sources of major concern regarding water pollution, due to their known toxic effects on the aquatic biota (Van Der Oost et al., 2003; Weber et al., 2008). More recently, a highly diverse group of contaminants, denominated emergent contaminants (EC), substances that are detected in the environment but are not included in regulatory monitoring programs are added as potential threats to ecosystems and human health and safety (Santos et al., 2010; Celander, 2011). They encompass a large number of compounds that include pharmaceuticals, hazardous and noxious substances (HNS), personal care products (PPCPs), nanomaterials, among others (Farré et al., 2008). Some of these EC have been receiving attention, since these compounds are continually introduced into the aquatic environment as complex mixtures via a number of routes, but primarily by both untreated and treated sewage (Daughton and Ternes, 1999). In order to survive, living organisms have developed strategies of protection to the adverse effects of pollutants. Chronically exposed organisms use a well-developed detoxification mechanism in order to reduce the toxic effects of pollutants (Van Der Oost et al., 2003). These mechanisms include the activity of specific proteins to preclude the permanence of toxic compounds or their metabolites in the cells - some members of the ATP-Binding Cassette (ABC) superfamily—and enzymatic systems to transform the chemicals into a more easily excreted form—phase I and phase II biotransformation enzymes. Thus, the knowledge of the functionality of these detoxification pathways is of critical importance, to support the availability of the aquatic resources for future generations.

The main points of this review will be the detoxification mechanisms adopted by fish species, focusing in (1) the regulation and functioning of ABC transporters; (2) the cooperation with phase I and II biotransformation enzymes; and (3) the ecotoxicological relevance of emergent pollutants with ability to modulate ABC transporters expression and activity. Recent studies have raised the possibility of a coordinated action between these groups of proteins, resulting in a powerful and effective mechanism of cellular detoxification.

The ABC genes represent one of the largest family of transmembrane transporter proteins encoded in the human genome (Dean et al., 2001). These proteins bind to ATP and use that energy to drive the transport of a wide variety of molecules across cellular membranes (Table 1) (Dean and Annilo, 2005). So far, 58 members of the ABC family have been described, including 49 human ABC genes and 9 additional genes found in other animal species. From the 58 genes, 68% are present in all vertebrate genomes, suggesting that their structure and functions have been largely conserved throughout the evolution of vertebrate species (Dean and Annilo, 2005). Based on the sequence and the organization of the ATP-binding domains, also known as nucleotide binding domains (NBDs), ABC proteins were grouped into eight subfamilies in eukaryotes (A–H), with seven of these (A–G) present in the human genome (Dean and Annilo, 2005), as shown in Table 1. The ABCH subfamily has been identified so far only in zebrafish and the function is still unknown (Popovic et al., 2010).

Typically, a functional protein contains two NBDs and two membrane spanning domains (MSDs), the latter being composed by 6–10 membrane spanning α-helices that confer the substrate specificity (Figure 1) (Szakács et al., 2008). Eukaryotic ABC proteins are organized either as full transporters (containing two NBDs and two MSDs), or as half transporters (containing one NBD and one MSD), that have to form homo- or heterodimers in order to constitute a functional protein (Dean et al., 2001). Some variation exists in protein structure in the different subfamilies, as it will be described further ahead, but a high degree of structural and sequence homology is shared among all ABC transporter proteins (Linton and Higgins, 2007).

Aquatic organisms are able to survive and thrive in heavily polluted environments, showing surprisingly low accumulation of pollutants in body organs (Kurelec and Pivčević, 1989). Kurelec and co-workers were the first to demonstrate that aquatic organisms adopt strategies of xenobiotic transport, in order to improve adaptation to pollutants in their habitats (Kurelec and Pivčević, 1989, 1991; Kurelec, 1992). To this phenomenon of resistance, Kurelec coined the term of multixenobiotic resistance (MXR) (Kurelec, 1992), and identified its biochemical basis as similar to the one adjacent to the MDR phenotype. The presence of a drug transporter resembling Pgp (ABCB1) sensitive to verapamil (known inhibitor of human Pgp) was described in two bivalves species (Anodonta cygnea and Mytilus galloprovinciallis) (Kurelec and Pivčević, 1989, 1991). This study was the starting point for the identification and characterization of the MXR phenotype in several other species of aquatic organisms, such as sponges (Kurelec, 1992), molluscs (McFadzen et al., 2000; Minier et al., 2000; Smital et al., 2003; Luckenbach and Epel, 2008; Faria et al., 2011), crabs and sea urchins (Toomey and Epel, 1993; Hamdoun et al., 2002). In fish, the presence of ABC efflux transporters have been described for an increasing number of species, such as winter flounder (Chan et al., 1992), antarctic fish (Zucchi et al., 2010), rainbow trout (Zaja et al., 2008a; Fischer et al., 2010; Loncar et al., 2010), zebrafish (Long et al., 2011a,b; Fischer et al., 2013), mullet (Diaz de Cerio et al., 2012), killifish (Paetzold et al., 2009), nile tilapia (Costa et al., 2012b, 2013), catfish (Liu et al., 2013) and seabass (Ferreira et al., 2014) (Table 2).

Identification of ABC transporters and characterization of the MXR mechanism has been achieved by the use of several detection methods, including quantitative or semi-quantitative reverse transcription polymerase chain reaction (qRT-PCR or RT-PCR) and in situ hybridization to evaluate mRNA expression of genes, immunochemical techniques for protein detection applying mammals monoclonal antibodies to ABCB1 (western blotting or immunohistochemistry), northern-blotting and activity assays (through the measurement of efflux or accumulation of fluorescent model substrates).

Most studies directed to the determination of organ distribution pattern of ABC transporters in aquatic organisms have been directed to Pgp by the use of the mammalian anti-Pgp monoclonal antibody (mAb) C219. This mAb which recognizes an epitope common to all known Pgps (in human MDR1 and MDR3) and also to Spgp (ABCB11) (Georges et al., 1990; Childs et al., 1995; van den Elsen et al., 1999). After probing with the mammalian mAb C219, a protein related to the mammalian MDR protein was identified in embryos and gills of a few aquatic invertebrates (Toomey and Epel, 1993; Cornwall et al., 1995). In fish, positive reaction was seen in the hepatic bile canaliculi (Kleinow et al., 2000; Doi et al., 2001; Bard et al., 2002a,b; Klobučar et al., 2010; Costa et al., 2013), apical membrane of enterocytes (Kleinow et al., 2000; Doi et al., 2001; Bard et al., 2002a; Costa et al., 2013), kidney (Kleinow et al., 2000), and in endothelial capillaries of brain (Miller et al., 2002). A negative reaction of the mAb C219 was patent in gills, kidney, gonad, brain, spleen, heart of killifish and blenny (Bard et al., 2002a,b). In summary, the suggested Pgp localization in fish reveals a pattern similar to that described in mammals, with immunoreactive Pgp occurring in epithelial tissues involved in secretion, absorption or serving a barrier function, For other ABC efflux transporters no fish-functional antibodies are available, thus information on their distribution pattern is quite limited and restricted to the mRNA expression among the various fish organs.

ABCB1 expression has been detected in different fish species, and in distinct organs as described in Table 2. These evidences point to the important role that ABCB1can have also in fish species validating the research on this Pgp-like proteins. As in mammals, ABCB11 protein was found to be almost exclusively expressed in liver, with low expression in proximal intestine, and very low expression in other organs as gills, brain, gonads and kidney of trout (Loncar et al., 2010), in trout cell lines (Fischer et al., 2011) and nile tilapia (Costa et al., 2012b), supporting its role in the efflux of bile salts from hepatocytes into the bile. For ABCCs the information available is more limited and efforts in the last years have increased the information available. In trout, low ABCC1 expression was found, and kidney was the only organ showing notable ABCC1 expression while significant expression levels of ABCC2 in major detoxification organs were reported (Loncar et al., 2010). In nile tilapia, ABCC1 and ABCC2 were shown to be expressed in adult organs like liver, gill and intestine (Costa et al., 2012b) and also in early developmental stages of the larvae (Costa et al., 2012a). ABCC1 and ABCC2 were also present in European seabass liver and responsive to a PAH (Ferreira et al., 2014). High ABCC2 expression was previously reported in kidney, intestine and liver of little skate (Cai et al., 2003) and in apical membrane of proximal kidney tubule of killifish (Miller et al., 2007). Regarding ABCG2, Loncar et al. (2010) have examined the organ distribution pattern of this protein in trout, and found high expression levels in gonads and moderate expression in distal part of intestine, kidney and brain: previously, expression of ABCG2 was also reported by the same group in trout liver and primary hepatocytes (Zaja et al., 2008b) (Table 2). Also the studies performed in our group with nile tilapia have shown that ABCG2 is expressed in barrier organs and at different development stages (Costa et al., 2012a,b).

Based on these results and on mammal studies, investigators believe that, in aquatic organisms, ABC efflux transporters should follow a similar distribution pattern and cellular localization, with expression in organs involved in secretion, absorption or serving as a barrier function, like liver and intestine. Thus, ABCB1, ABCC2, and ABCG2 should be localized in the apical membrane of polarized cells, pumping substrates into the intestinal lumen or bile canaliculi, while ABCC1 localization in basolateral membrane results in the export of its substrates into the blood. Additionally, ABCB1 should efflux mostly unmodified compounds, while ABCCs and ABCG2 should efflux organic anions conjugated by phase II enzymes (Epel et al., 2008).

The bulk of studies conducted in aquatic organisms to evaluate the interaction of environmental contaminants with ABC efflux transporters have been mostly performed on Pgp. Some compounds, as Pgp model reversal agents (verapamil and/or cyclosporine A) and xenobiotics (like the insecticide malathion) were shown to inhibit Pgp activity in fishes, resulting in an increase of the bioaccumulation of toxic compounds like 2-aminoanthracene, hydrocarbon-rich Diesel-2 oil, and/or model Pgp substrates like rhodamine B and rhodamine 123 (Smital and Kurelec, 1998; Fischer et al., 2013; Ferreira et al., 2014). Other studies have demonstrated the ability of some xenobiotics to induce Pgp expression in fish, such as cadmium (Zucchi et al., 2010), the organophosphate insecticide chloropyrifos and the carcinogen N-nitrosodiethylamine (Albertus and Laine, 2001). Similarly, resistance-killifish populations from highly polluted sites also demonstrated enhanced Pgp expression (Cooper et al., 1999; Bard et al., 2002a) suggesting a protective role of Pgp. Nevertheless, in another study performed in killifish from a heavily polluted site with polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs) and heavy metals, Paetzold et al. (2009) found no up-regulation of hepatic ABCB1 transcripts. Similarly, after in vivo exposures of fishes to different xenobiotics, such as prochloraz, nonylphenol diethoxylate, ß-naphthoflavone, BaP and 3,4,3',4'-tetrachlorobiphenyl no changes were seen in the levels of hepatic and/or intestinal Pgp (Doi et al., 2001; Sturm et al., 2001; Costa et al., 2012b, 2013). The study of Pgp in fish species have been given some contradictory results. A recent study in zebrafish revealed that ABCB4 acted as the functional multixenobiotic transporter and showed activity as a barrier to chemical uptake (Fischer et al., 2013). Also in seabass the difficulty to assess ABCB1 expression (Ferreira et al., 2014) can be an indication of different transporters to be functional in different fish species.

Few studies have addressed the response of other ABC efflux transporters upon animals' exposure to pollutants in comparison to Pgp. Paetzold et al. (2009) described an up-regulation of hepatic transcripts for ABCC2 and ABCG2 in a multiresistant population of killifish living in a pond heavily polluted with PAHs, PCBs and heavy metals, while no up-regulation was seen in ABCB1 and ABCB11 mRNA. Exposure to metals has induced the expression of ABCC2 in excretory organs of zebrafish including kidney, liver and intestine (Long et al., 2011b) and also Cd-exposed Trematomus bernachii (Zucchi et al., 2010). Moreover, an up-regulation of ABCC2 was seen in red mullet liver from an oil contaminated site (Della Torre et al., 2010). Long and co-workers described an up-regulation of ABCC1 gene in ZF4 cells and embryos of zebrafish exposed to heavy metals (Long et al., 2011a,b). Increased expression of ABCC2 was observed in seabass primary hepatocytes (Ferreira et al., 2014) and in Nile tilapia gill after waterborne exposure to BaP (Costa et al., 2012b). ABCG2 expression was also up-regulated in Nile tilapia intestine upon BaP exposure (Costa et al., 2012b). These recent studies on MRPs and BCRP highlight the important role these ABC transporters may play in the overall detoxification process and cooperation with phase I and II biotransformation enzymes. Nevertheless the contribution of the different ABC proteins in the efflux of xenobiotics has to be conclusively proved.

Although an increased number of compounds have been shown to interact with ABC efflux transporters, data remains fragmented and largely restricted to laboratory observations in comparison to relevant field data. It remains uncertain which classes of environmental toxicants can induce and/or repress the activity of these proteins, and to what extent this MXR mechanism protects aquatic animals from the toxic action of xenobiotics. There is a fundamental need to identify the common features of ABC efflux transporters substrates, but also the characteristics of chemicals that might inhibit their activity leading to an increased toxicity of normally effluxed chemicals. Most ABC efflux transporters are remarkably non-specific with respect to its substrates. This feature can be an important advantage, as it provides protection against many classical and novel anthropogenic products, but it can also render the system vulnerable (Smital et al., 2004; Epel et al., 2008). The presence of multiple substrates that compete for binding sites can saturate these locations, or directly inhibit the transporter activity, in a phenomenon termed chemosensitization (Bard, 2000; Epel et al., 2008). MXR inhibitors, or chemosensitizers, were divided in two main classes: (1) competitive inhibitors with high affinity that prevent the binding and active transport of other substrates; and (2) non-competitive inhibitors that can act in different ways, such as blocking ATPase activity (Smital et al., 2004). The presence of MXR inhibitors can compromise the effectiveness of the defense system, since toxic substances that would normally be excluded, will remain in the cell and exert their toxic effects (Epel et al., 2008; Fischer et al., 2013). Ability to inhibit ABC transporters of aquatic organisms has been described for different types of contaminants such as PPCPs, pharmaceuticals, pesticides, fragrances, among others (Bard, 2000; Smital et al., 2004; Epel et al., 2008; Fischer et al., 2013), however we still do not know the actual consequences that chemosensitization will have at population and ecosystem level. Progress in research of these proteins has been delayed mostly due to the existence of technical limitations, as, for example, the inexistence of specific probes/antibodies for these transporters in aquatic organisms, and the measurement of its functionality. Thus, there is the need to further investigations in the MXR mechanism in order to fully understand the physiological and toxicological functions of ABC transporters in aquatic organisms, including fish.

Biological membranes are barriers for the uptake, distribution and elimination of xenobiotics in the organism (Simkiss, 1995). Bioaccumulation of organic compounds occurs mainly passively, driven by the compound's hydrophobicity, and once absorbed, the xenobiotic may undergo a detoxification pathway, in order to be excreted from the organism before it exerts toxic effects (Van Der Oost et al., 2003). The most studied pathway for cellular detoxification is the biotransformation of xenobiotics by phase I and phase II enzymes. Biotransformation consists on a two-phase process of enzymatic reactions that alter the chemistry of non-polar lipophilic chemicals to polar-water soluble metabolites, leading to the detoxification and elimination of the parent compound (Black and Coon, 1987; Buhler and Williams, 1989). The enzymes of phase I metabolism (oxidation, reduction, hydration, hydrolysis) introduce a functional group (−OH, −COOH, −NO2, etc.) into the xenobiotic (Commandeur et al., 1995). Phase II of the biotransformation process involves the conjugation of the xenobiotic parent compound or its phase I metabolites with an endogenous ligand, thus facilitating the excretion of chemicals by the addition of more polar groups [e.g., glutathione (GSH), glucuronic acid (GA), sulfate] (Schlenk et al., 2008). In recent years it has been proposed that two additional steps of drug disposal, called phase 0 and phase III, are equally important as the biotransformation process (Szakács et al., 2008). These phases involve the modulation of the cellular efflux, by ABC transporters, of either unmodified or metabolized compounds and xenobiotic efflux transporters and biotransformation enzymes may be part of a coordinated defense mechanism that protects cells from xenobiotic insults (Xu et al., 2005). Thus, understanding the underlying facts behind the possible cooperative action of efflux transporters and biotransformation enzymes has critical implications both for human and environmental health. However, despite its relevance, studies focused on the mechanisms important for this coordinated defense that protects cells from xenobiotic insults in non-mammalian models, including fish, are still very scarce (Bard, 2000). Moreover, the studies published so far did not provide a consistent pattern on the cooperation mechanisms, mostly due to the insufficient knowledge of the basic parameters of ABC transcriptional regulation in fish. Intracellular receptors are proteins which, upon binding of specific ligands, have the ability to regulate the mRNA transcription of specific genes, thereby controlling development, homeostasis, and metabolism of the organism (Xu et al., 2005). From mammalian models, several receptors (e.g., Aryl hydrocarbon receptor-AhR, nuclear factor-erythroid 2 p45-related factor-Nrf2, pregnane X receptor-PXR, peroxisome proliferated activated receptor-PPAR, liver X receptor-LXR, farnesoid X receptor-FXR, retinoid X receptor-RXR, constitutive androstane receptor-CAR, glucocorticoid receptor-GR) have been shown to be key mediators in the regulation of the activity of phase I and phase II metabolizing enzymes, and ABC transporters involved in efflux mechanisms (Xu et al., 2005). Some studies in mammalian in vitro models have associated some nuclear receptors to the regulation of expression of ABC transporters (Naspinski et al., 2008; Chisaki et al., 2009; Rigalli et al., 2011), such as the induction of expression of phase I and mdr genes via CAR and PXR pathways (Gu et al., 2013; Wang et al., 2013a,b). Even though in fish there is a fundamental lack of knowledge on the transcriptional regulation of ABC transporters, in zebrafish an association was shown between PXR, CYP3A and MDR1 (Bresolin et al., 2005), and the same pathways of transcriptional regulation of detoxification enzymes and transporters as in mammals are expected.

However, whether phase I and II enzymes inducers co-ordinately regulate ABC efflux transporters genes requires further studies that can shed some light on the role of receptors, transcription factors and signaling cascades of this metabolism/transport of endogenous and exogenous compounds (Xu et al., 2005). Also in mammalian studies, induction of Pgp has been reported after exposure to known inducers of phase I (CYP1A) and phase II enzymes, like BaP and 3-methylcholanthrene (3MC) (Chao Yeh et al., 1992; Fardel et al., 1996). Moreover, ABCC1, ABCC2 and ABCG2 are known to efflux mainly phase II metabolites, xenobiotics conjugated with GSH, GA and/or sulfate in mammalian models (for a review see Leslie et al., 2005), further supporting their role in phase III of the detoxification process.

Considering the characteristics of common regulation mechanisms and complementarity of substrates, it is believed that ABCB1 acts as a first line of defense preventing unmodified compounds from accumulating in the cell (phase 0 of cellular detoxification), while ABCCs and ABCG2 transport phase I and II metabolites, therefore acting in phase III of cellular detoxification (Bard, 2000; Sturm and Segner, 2005; Epel et al., 2008) (Figure 3). Compared to biotransformation enzymes, research on efflux transporters is still very limited and has largely been confined to mammalian models. In aquatic organisms, a few studies have been conducted in an attempt to elucidate this cooperative mechanism (Cooper et al., 1999; Sturm et al., 2001; Bard et al., 2002b; Paetzold et al., 2009; Costa et al., 2012b; Ferreira et al., 2014). The majority of these studies have been confined to the study of Pgp and CYP1A, and results suggest that these two proteins should not be coordinately regulated in fish, but enhance the fact that they may act in complementarity in cellular detoxification. More recently, this cooperative role has been pointed out in nile tilapia exposed to benzo(a)pyrene by showing increase in expression of both phase I and phase II biotransformation enzymes as well as the toxicological relevant efflux proteins in barrier organs (Costa et al., 2012b, 2013). However, the database on the relation between Pgp and biotransformation enzymes in fish is small and dispersed, and the role of other ABC efflux transporters, besides Pgp, is even more poorly documented. Thus, the full characterization of this mechanism of cellular detoxification, where ABC efflux transporters and biotransformation enzymes display title-roles is yet far from being completely understood.

Understanding the underlying facts behind the mechanisms of cellular detoxification has implications both for human and environmental health. The medical relevance of these transporters is evidenced by their MDR abilities and, under this point of view there is a fundamental need to know how to properly block the action of these transporters to assure the targeting efficacy of chemotherapeutic drugs. In addition, the study of ABC efflux transporters is also very important from an environmental point of view, since they are believed to be an integral part of the cellular detoxification system. For environmentalists, the goal is to understand how these efflux transporters keep toxicants out of the cells, and to ensure that they operate optimally in order to protect the cells from environmental contaminants. The effects of ABC transporters on the toxicity of environmental contaminants is still poorly understood, thus efforts must be made to provide insights into the interactions of contaminants in fish species. In particular, interactions with classical and emergent compounds and the environmental relevance of fish ABC transporters is yet to be thoroughly addressed. The search for chemosensitizers among emergent compounds is relevant since some contaminants considered to be weak or moderately toxic at concentrations found in the aquatic environment can be potent inhibitors of ABC transporters and modulate toxicity of other xenobiotics and led to unpredicted effects to exposed species. Therefore, and in particular with emergent compounds, it is important to evaluate interaction of a large array of contaminants with ABC transporters when assessing toxicity of xenobiotics to the aquatic environment. One difficulty to fully address the role of ABC transporters in the detoxification process and the interactions with xenobiotics with the different classes of contaminants is that the information available on fish species is dispersed in different studies with different goals. So in the future, research must be directed to the study of regulation of ABC transporters and to unravel the importance of these efflux proteins to the overall maintenance of the homeostasis of fish species chronically exposed to foreign chemicals.