A Review of P-Glycoprotein Function and Regulation in Fish

Abstract

The teleost ATP Binding Cassette (ABC) transporter P-glycoprotein (P-gp) is an active transmembrane transporter that plays a pivotal role in facilitating the movement of both endogenous and xenobiotic substrates (moderately hydrophobic and amphipathic compounds) across cell membranes. P-gp exhibits substrate specificity often shared with other ABC transporters and solute carrier proteins, thereby ensuring the maintenance of chemical homeostasis within cells. These transporters are integral to chemical defense systems in fish, as they actively expel a wide range of substrates, primarily unmodified compounds, from cells. This transport process assists in preventing chemical absorption (e.g., intestine), safeguarding sensitive tissues (e.g., brain and gonads), and effectively excreting substances (e.g., liver and kidney). Upregulated P-gp export activity in aquatic animals results in the multi-xenobiotic resistance (MXR) phenotype that plays an essential protective role in survival in contaminated environments. Pollutants inhibiting P-gp are termed chemosensitizers and heighten fish sensitivity to toxic P-gp substrates. While the known intrinsic functions of P-gp in fish encompass steroid hormone and bile acid processing, relatively little attention has been given to endogenous substrates and inhibitors. Fish P-glycoprotein regulation is orchestrated by pivotal nuclear transcription factors, including pregnane X receptor (PXR) and nuclear factor erythroid 2-related factor 2 (Nrf2). This comprehensive review provides profound insights into P-gp’s significance across diverse fish species, contributing to an enhanced understanding of fish physiology, evolution, and toxicology, and provides information with potential applications, such as environmental monitoring.

1. Introduction

Cell membranes house a diverse array of transporters essential for maintaining cellular homeostasis and functionality. These transporters are categorized into distinct classes based on their mechanism of action and substrate specificity. Solute carrier (SLC) transporters encompass a large superfamily that facilitates the transport of nutrients, ions, and metabolites. Ion channels permit the passive flux of ions across membranes, critical for electrical signaling and osmoregulation. Symporters and antiporters, categorized as secondary active transporters, utilize electrochemical gradients to transport substrates simultaneously in the same direction or in opposite directions, respectively. Finally, vesicular transporters mediate the intracellular trafficking of molecules through vesicle formation and fusion. These transporters control the movement of molecules across membrane barriers and serve to maintain chemical homeostasis. They also export endogenous molecules from sites of production to where they are needed, such as in the transport of morphogenic signaling molecules during embryonic development [1], the movement of gut microbiome metabolites into host intestinal tissue [2,3], and the export of fragrant volatile organic compounds from flowers [4]. ATP-binding cassette (ABC) transporters utilize energy from ATP hydrolysis to efflux a wide range of substrates, including lipids, ions, and xenobiotics. These transporters maintain concentrations of endogenous molecules at appropriate physiological levels and serve to reduce xenobiotic accumulation to prevent potential toxicity as part of the cell’s chemical defense system [1].

Cells have evolved intricate defense mechanisms against the accumulation of xenobiotic compounds, and they process these xenobiotics through a sequence of four phases: (phase 0) unmodified efflux, (phase I) functionalization reactions, (phase II) conjugation reactions, and (phase III) metabolite elimination. Phases I and II are biotransformation reactions that convert hydrophobic compounds into more hydrophilic metabolites with reduced membrane permeability. In phase III, hydrophilic metabolites are actively exported from the cell by transmembrane transport proteins, including the breast cancer-resistance protein (BCRP) and multidrug resistance proteins (MRPs) [5]. In phase 0, unmodified parent compounds are actively exported from the cell by transmembrane transporters, which include the ABC protein P-glycoprotein [6,7,8].

The ATP Binding Cassette (ABC) family of proteins facilitates the translocation of an extensive array of substrates. These transporters engage in critical cellular processes such as nutrient uptake, lipid trafficking, and antigen presentation, and play a major role in limiting the toxic potential of xenobiotics. Their structure is modular, and consists of highly conserved nucleotide binding domains (NBDs), which bind and hydrolyze ATP, and structurally diverse transmembrane domains (TMDs), which anchor the protein in the membrane and bind and transport substrates [9,10]. Full transporters include four components (two NBDs and two TMDs) in one protein, while half transporters include one NBD and one TMD, and must dimerize to function [9]. Eukaryotic ABC proteins are categorized into ten different families: ABCA to ABCJ [9,11]. The ABCA and ABCC families consist entirely of full transporters, the ABCB family includes both full and half transporters, and the ABCD, ABCG, and ABCH families are all half transporters [12]. Transporters in the ABCI family are composed of four separate proteins, with each NBD and TMD encoded by a different gene [9]. The recently proposed ABCJ family consists of DNA repair enzymes. These proteins are composed of half-enzymes, with one NBD and one substrate-interacting domain each, that form dimers around one or two DNA strands [13,14]. ABCE and ABCF proteins contain only a pair of NBDs without any TMDs, and function in the assembly of sub-cellular components by NBD dimerization [12].

ABC transporters are represented in all branches of life; prokaryotes have both importers and exporters, while eukaryotic ABC transporters are mostly exporters with only a few examples of importers [9,10]. Plants have an especially broad set of ABC transporters, with more than 120 members, compared to about 60 in fish and mammals [9,11]. This diversity of plant ABC transporters appears to be related to secondary metabolism, as well as the increased importance of stress responses and detoxification for sessile organisms compared to motile organisms [9]. The physiological functions of ABC transporters are an area of active research, and they tend to fall into the broad categories of homeostasis, signaling, and detoxification.

The permeability glycoprotein (P-glycoprotein [P-gp]) is a transmembrane exporter in the ABCB family. It transports a broad range of substrates, including both endogenous and xenobiotic molecules that tend to be mildly hydrophobic [15,16]. Its expression in excretory (e.g., liver, kidney), absorptive (e.g., intestine), and barrier (e.g., blood–brain, blood–eye, blood–gonad) tissues allows it to modulate its substrates’ absorption, tissue distribution, and elimination [17,18,19]. P-gp’s main identified function is xenobiotic resistance, but its role in regulating endogenous molecules is being increasingly recognized [1,11].

The discovery of P-gp marked a significant milestone in the understanding of cellular drug resistance mechanisms. P-gp was first identified in Chinese hamster ovary cells resistant to colchicine, revealing its role in the efflux of drugs from cells [20]. A major impetus for investigating P-gp stems from its role in multidrug resistance (MDR) observed in some forms of cancer. Tumor cells that overexpress P-gp exhibit an enhanced ability to efflux chemotherapy drugs, rendering them less susceptible to the cytotoxic effects of these agents. This phenomenon, often accompanied by overexpression of other transporters and metabolic enzymes, poses a significant challenge in cancer treatment [21]. The ability of P-gp (sometimes called multidrug resistance protein 1 [MDR1]) to mediate MDR highlights its clinical relevance and the need for a deeper understanding of its underlying mechanisms.

Research efforts into P-gp have been primarily directed towards unraveling the molecular basis of MDR and exploring strategies to circumvent this resistance. The study of mammalian P-gp, in particular, has garnered attention due to its involvement in drug interactions, bioavailability, and treatment outcomes. Investigations into the structural and functional aspects of P-gp have revealed insights into its substrate specificity, drug-binding sites, and the conformational changes associated with substrate transport [22]. These studies provide a foundation for designing targeted interventions aimed at modulating P-gp activity to enhance drug efficacy.

P-gp has received relatively little attention in the context of cancer in fish, although the MDR phenotype has been observed in several fish cancers [23,24,25]. The focus of research on fish and other aquatic animals originated in the discovery that animals living in contaminated aquatic environments over-express the same set of proteins that are overexpressed in MDR cancers, resulting in the multi-xenobiotic resistance (MXR) phenotype that allows them to survive and reproduce despite continuous chemical exposure. Specifically, resistant animals display higher levels of expression and activity of both efflux transporters (especially ABCB, ABCC and ABCG transporters) and biotransformation enzymes (including cytochromes and transferases) [26,27]. The MXR phenotype was first identified in marine and freshwater mussels [28,29], and was reported in fish soon after [30].

The evolution of fish populations towards pollution tolerance or resistance underscores the intricate interplay between natural selection and environmental pressures. Anthropogenic pollution is a recent and novel stressor in aquatic systems, and in response, fish populations have exhibited adaptive changes in chemical defense that, over generations, enhances their ability to reduce the potential of adverse effects [31]. Over time, the frequency of these beneficial genetic variations can increase within a population. For example, stickleback populations exposed to pulp mill effluent over many generations show significant changes in the genotype compared to populations from uncontaminated sites [32]. Pollution-driven selection can lead to shifts in the allele frequencies of certain genes associated with stress response mechanisms. In two North Atlantic eel species, the allele frequencies of genes related to sterol regulation and the response to oxidative stress differed significantly between populations exposed to varying contaminant profiles [33]. Fish populations exposed to pollutants may experience increased selection pressure on genes that code for transporters and enzymes involved in biotransformation and detoxification [33,34,35]. This can result in the evolution of fish with improved abilities to reduce accumulation, biotransform, and eliminate contaminants.

In addition to multi-generational genetic changes, rapid responses, such as phenotypic plasticity, allow fish to adjust their traits within their lifetime in response to pollution-induced stressors. This can include changes in behavior, morphology, or physiology that improve their chances of survival in polluted habitats, including the induction of P-gp and other genes that lead to the MXR phenotype [26,27]. These plastic responses can provide an initial buffer against pollution impacts while genetic adaptations accumulate over longer timeframes. The inhibitors of MXR proteins, including P-gp, are an emerging class of environmental pollutant [36,37] called chemosensitizers, making resistant organisms sensitive to toxic MXR protein substrates by reducing toxic concentration thresholds.

While most research on P-gp’s function in fish has focused on its role in xenobiotic defense and reducing chemical accumulation, the physiological roles of P-gp in fish are also beginning to be explored. This review aims to collect and synthesize the research on P-gp in fish to date, for the purpose of identifying knowledge gaps and providing a solid background for future research.

2. P-Glycoprotein Genes in Fish Species

In humans, P-glycoprotein is encoded by the ABCB1 gene on chromosome 7 [38,39]. Human ABCB4 has a high sequence homology to ABCB1 and is located next to it on the same chromosome due to a gene duplication [40]. ABCB5 also has a high sequence homology to ABCB1 due to an earlier gene duplication, and is located farther away on the same chromosome [40]. All fish species whose P-glycoprotein genes have been investigated thus far have Abcb4 or an Abcb4-like gene [38,41,42,43]. In zebrafish, the protein encoded by Abcb4 is functionally the most similar to the mammalian P-glycoprotein encoded by Abcb1 [38]. Thus, Abcb4 appears to be the primary functional P-glycoprotein in fish. Fish Abcb4 is an ortholog to the gene that gave rise to mammalian Abcb1 and Abcb4 [15]. Multidrug transport is the ancestral function of this gene, which was retained in mammalian Abcb1 [15]. All fish P-gp genes were initially labelled Abcb1 (or a derivation thereof) after their mammalian counterpart, and this nomenclature persists in the literature [38].

Most fish species investigated to date, including Atlantic cod (Gadus morhua), Nile tilapia (Oreochromis niloticus), medaka (Oryzias latipes), and three-spined stickleback (Gasterosteus aculeatus), lack Abcb1 and Abcb5 genes [15,38]. Two pufferfish species (Takifugu rubripes and Tetraodon nigroviridis) have Abcb1 in addition to Abcb4 [38], while African coelacanth (Latimeria chalumnae), channel catfish (Ictalurus punctatus), rainbow trout (Oncorhynchus mykiss), and zebrafish (Danio rerio) have Abcb5 in addition to Abcb4 [38,41,42]. The Abcb1 gene present in pufferfish likely arose from a separate family-specific duplication of Abcb4 [15,38]. The P-gp genes that have been identified in fish species are shown in

Table 1. P-glycoprotein genes in fish species, determined by synteny analysis. + indicates that the gene is present in the species, − indicates that the gene is absent in the species.

Species	Abcb1	Abcb4	Abcb5	References
Danio rerio (Zebrafish)	−	+	+	[38]
Gadus morhua (Atlantic cod)	−	+	−	[15,38]
Gasterosteus aculeatus (Three-spined stickleback)	−	+	−	[15,38]
Ictalurus punctatus (Channel catfish)	−	+	+	[15,42]
Latimeria chalumnae (African coelacanth)	−	+	+	[38,41]
Oncorhynchus mykiss (Rainbow trout)	−	+	+	[41]
Oreochromis niloticus (Nile tilapia)	−	+	−	[41]
Oryzias latipes (Medaka)	−	+	−	[15,38]
Takifugu rubripes (Japanese pufferfish)	+	+	−	[15,38]
Tetraodon nigroviridis (Green spotted pufferfish)	+	+	−	[38]

.

Fish Abcb5 is an ortholog of mammalian Abcb5 [15]. Its function is not entirely clear, but it provides some multidrug transport function in fish and mammals [15,38,44]. It also appears to have endogenous transport functions, perhaps related to biliary excretion, epithelial cell adhesion, and stem cell maturation [15,38,41].

3. P-Glycoprotein Tissue Expression and Function

P-gp tends to be expressed in tissues with excretory (e.g., liver, kidney), absorptive (e.g., intestine), and barrier (e.g., skin, blood–brain, blood–eye and blood–gonad barriers) functions in both fish and mammals [17,18,19]. P-gp activity in excretory and absorptive tissues eliminates P-gp substrates from the body through excretory fluids (urine, bile, feces), while P-gp activity in blood–tissue barriers protect sanctuary tissues from the accumulation of P-gp substrates. The P-gp mRNA and protein expressions reported in fish tissues are shown in Table 2.

Many studies into P-gp expression used samples of only a single tissue, almost exclusively the liver [45,46,47,48]. Studies of this nature confirm the presence of a P-gp gene in a species without examining tissue expression in any detail. When P-gp expression is measured in multiple tissues, expression is usually highest in the kidney, followed by the liver and intestine [19,49,50,51]. P-gp expression tends to be moderate in the brain, eye, and gonads, while gill, muscle, skin, and other tissues generally have low P-gp expression [17,19,49,51,52]. This expression pattern suggests that the primary role of P-gp in fish is the elimination of substrates from the body, and that among sanctuary tissues, the brain, eye, and gonads are the most protected by P-gp.

In fish, as well as in mammals and invertebrates, P-gp can play a role in preventing the absorption of chemicals from the intestinal tract, protecting the organism from potentially harmful substances [53]. P-gp is expressed in the apical membrane of enterocytes, where it actively pumps substrates back into the intestinal lumen, limiting their uptake into the bloodstream [11]. P-gp expression increases along the intestinal tract, allowing for increasing rates of active transport as the lumen contents become more concentrated [11,17,19]. This efflux mechanism significantly contributes to the overall reduced accumulation and subsequent elimination of potentially harmful compounds.

P-gp plays a pivotal role in the excretion of chemicals by both the liver and the kidney, contributing to the elimination of xenobiotics. In the liver, P-gp is expressed in the canalicular membrane of hepatocytes, facilitating the transport of various substances, including drugs and environmental contaminants, from the hepatocyte cytoplasm into the bile canaliculus [54]. This excretion into the bile ultimately leads to the elimination of these substances from the body via feces. Studies using fish hepatocytes have demonstrated the active export of xenobiotic compounds and its impairment by P-gp inhibitors [54,55,56,57].

In the kidney, P-gp is expressed in the proximal tubules, where it is involved in the active secretion of chemicals from the blood into the renal filtrate [58,59]. This process aids in the elimination of xenobiotics, contributing to their excretion in urine. By preventing the reabsorption of these substances from the filtrate, P-gp assists in reducing their systemic exposure and potential toxicity. Isolated renal proximal tubules from flounder (Paralichthys lethostigma, Pseudopleuronectes americanus) and killifish (Fundulus heteroclitus) were an important early model system for studying xenobiotic transport in fish [59,60]. These studies provided evidence for the P-gp-mediated transport of numerous xenobiotics, including cyclosporin A, daunomycin, rapamycin, and ivermectin, into the renal tubule lumen [58,59,61,62], as well as an understanding of the relationship between SLC importers and ABC exporters expressed on opposite sides of the same epithelial cells [58,62,63].

Table 2. Expression of P-glycoprotein mRNA or protein (Abcb1, Abcb4, Abcb5, P-glycoprotein not otherwise specified) in fish tissues.

Species	Tissue	References
Anoplarchus insignis (Slender cockscomb blenny)	Liver	[64]
Anoplarchus purpurescens (High cockscomb blenny)	Liver	[27]
Barbus barbus (Barbel)	Liver	[47]
Carassius auratus gibelio (Silver Prussian carp)	Liver	[47]
Chelon labrosus (Thicklip grey mullet)	Liver, brain	[65]
Chondrostoma nasus (Sneep)	Liver	[47]
Cyprinodon variegatus (Sheepshead minnow)	Liver, kidney, intestine, exocrine pancreas	[66]
Cyprinus carpio (Common carp)	Liver	[47]
Danio rerio (Zebrafish)	Liver, kidney, intestine, ovary, testis, gill, skin, brain, brain vasculature, eye, muscle, heart, embryo (72 hpf), embryonic ionocytes	[52,67,68,69]
Fundulus heteroclitus (Killifish, mummichog)	Liver, distal intestine, brain capillaries	[70,71,72]
Gambusia affinis (Western mosquitofish)	Liver	[46]
Gobiocypris rarus (Chinese rare minnow)	Liver, kidney, intestine, skin, brain, spleen	[51]
Hatcheria macraei (Patagonian catfish)	Liver, gill	[45]
Ictalurus punctatus (Channel catfish)	Liver, intestine	[73]
Jenynsia multidentata (Killifish, one-sided livebearer)	Liver, gill, brain	[74]
Lepomis macrochirus (Bluegill sunfish)	Liver	[46]
Limanda limanda (Common dab)	Liver	[37]
Oncorhynchus mykiss (Rainbow trout)	Liver, kidney, intestine, gonad, gill, skin, brain, larval abdominal viscera, larval skin, larval head, larval muscle, larval yolk sac	[17,19,41,45,54,75,76,77,78]
Oreochromis niloticus (Nile tilapia)	Liver, intestine, zygote, embryo, larva	[79,80]
Poecilia reticulata (Guppy)	Liver, kidney, intestine, gill, muscle, ovary, esophagus, pancreas, branchial blood vessels, adrenal cortical tissue, gas gland	[49,81]
Salmo trutta (Brown trout)	Liver	[45]
Scophthalmus maximus (Turbot)	Liver, kidney, intestine, gill, brain, muscle, esophagus, heart	[50]
Squalius cephalus (European chub)	Liver	[47]
Thunnus albacares (Yellowfin tuna)	Liver, gill, brain, gonad	[43]
Trematomus bernacchii (Emerald rockcod)	Liver	[48]

Table 2. Expression of P-glycoprotein mRNA or protein (Abcb1, Abcb4, Abcb5, P-glycoprotein not otherwise specified) in fish tissues.

Species	Tissue	References
Anoplarchus insignis (Slender cockscomb blenny)	Liver	[64]
Anoplarchus purpurescens (High cockscomb blenny)	Liver	[27]
Barbus barbus (Barbel)	Liver	[47]
Carassius auratus gibelio (Silver Prussian carp)	Liver	[47]
Chelon labrosus (Thicklip grey mullet)	Liver, brain	[65]
Chondrostoma nasus (Sneep)	Liver	[47]
Cyprinodon variegatus (Sheepshead minnow)	Liver, kidney, intestine, exocrine pancreas	[66]
Cyprinus carpio (Common carp)	Liver	[47]
Danio rerio (Zebrafish)	Liver, kidney, intestine, ovary, testis, gill, skin, brain, brain vasculature, eye, muscle, heart, embryo (72 hpf), embryonic ionocytes	[52,67,68,69]
Fundulus heteroclitus (Killifish, mummichog)	Liver, distal intestine, brain capillaries	[70,71,72]
Gambusia affinis (Western mosquitofish)	Liver	[46]
Gobiocypris rarus (Chinese rare minnow)	Liver, kidney, intestine, skin, brain, spleen	[51]
Hatcheria macraei (Patagonian catfish)	Liver, gill	[45]
Ictalurus punctatus (Channel catfish)	Liver, intestine	[73]
Jenynsia multidentata (Killifish, one-sided livebearer)	Liver, gill, brain	[74]
Lepomis macrochirus (Bluegill sunfish)	Liver	[46]
Limanda limanda (Common dab)	Liver	[37]
Oncorhynchus mykiss (Rainbow trout)	Liver, kidney, intestine, gonad, gill, skin, brain, larval abdominal viscera, larval skin, larval head, larval muscle, larval yolk sac	[17,19,41,45,54,75,76,77,78]
Oreochromis niloticus (Nile tilapia)	Liver, intestine, zygote, embryo, larva	[79,80]
Poecilia reticulata (Guppy)	Liver, kidney, intestine, gill, muscle, ovary, esophagus, pancreas, branchial blood vessels, adrenal cortical tissue, gas gland	[49,81]
Salmo trutta (Brown trout)	Liver	[45]
Scophthalmus maximus (Turbot)	Liver, kidney, intestine, gill, brain, muscle, esophagus, heart	[50]
Squalius cephalus (European chub)	Liver	[47]
Thunnus albacares (Yellowfin tuna)	Liver, gill, brain, gonad	[43]
Trematomus bernacchii (Emerald rockcod)	Liver	[48]

The blood–brain barrier (BBB) in fish, much like that in mammals, plays a pivotal role in regulating the exchange of substances between the bloodstream and the brain in order to maintain an environment optimal for neural function. Central to this barrier’s function is the presence of efflux transporters, notably P-gp, which actively transport molecules out of brain endothelial cells, limiting their entry into the brain. The evidence for P-gp’s role in the fish BBB is supported by studies demonstrating its presence in brain capillary endothelial cells, where it contributes to restricting the passage of xenobiotics and potential neurotoxicants into the brain parenchyma [71]. This is particularly important as fish species are frequently exposed to aquatic contaminants that act as neurotoxicants [82,83,84].

The importance of the BBB in fish varies among species and is influenced by ecological and evolutionary factors. Evidence for a strong BBB in certain fish species is shown in studies where P-gp actively prevents the entry of lipophilic compounds. For example, in isolated killifish (Fundulus heteroclitus) and dogfish shark (Squalus acanthias) brain capillaries, the fluorescent analogues of the P-gp substrates verapamil and cyclosporin A were actively exported into the capillary lumen; this transport was inhibited by P-gp inhibitor PSC-833 (valspodar) [71]. Similarly, the exposure of rainbow trout to the P-gp substrate ivermectin revealed limited brain accumulation due to P-gp-mediated efflux, indicating a robust barrier function [85]. Conversely, studies on some fish species suggest a more permeable BBB [86]. Evidence for a weak BBB can be seen in the relatively higher brain uptake of certain molecules or drugs, possibly due to lower P-gp expression levels or less restrictive tight junctions between endothelial cells [86]. The variability in BBB properties across fish species is likely linked to their habitats and ecological niches, as well as to prior chemical exposures.

In addition to the blood–brain barrier, P-gp is expressed with other transmembrane transporters in barriers protecting sensitive tissues. In the mammalian eye, P-gp transports its substrates from the endothelium of ocular capillaries into the blood as part of the blood-retina or blood-aqueous barrier [87,88]. Fish also possess a blood-retina barrier, but the role of P-gp in this barrier has not been investigated in detail [88]. Mammalian gonads are protected by blood-testis and blood-follicle barriers, which include P-gp [89,90]. P-gp is also expressed in fish gonads [17,19,43,49,52], but the existence of blood–gonad barriers has not been investigated in fish.

Conversely, P-gp plays a relatively small role in xenobiotic transport in gills. Fish gills form an important interface between the animal and its environment, allowing for the exchange of gasses, ions, and organic molecules between the water and the blood [91]. P-pg expression is typically lower in the gills than in the liver, kidney, intestine, brain, and gonad [17,19]. In rainbow trout gills, the main xenobiotic transporter form of P-gp (Abcb4) is nearly absent, while Abcb5 is expressed only in interlamellar progenitor cells in contact with neither the water nor the blood [41]. Gills express high levels of biotransformation enzymes, similar to or sometimes higher than expression in the liver and intestine [41,80]. Perhaps due to the prevalence of biotransformed metabolites, phase III transporters are expressed at much higher levels in fish gills than P-gp [19,41,80].

4. P-Glycoprotein in the Chemical Defense System

Proteins perform a wide spectrum of chemical defense functions, and collaboratively direct the movement of endogenous and xenobiotic molecules across bodily barriers, their distribution within an organism, and their subsequent elimination. Playing a key role in this chemical defense system in fish is P-gp, which operates in tandem with other defense components. Fish tissues commonly express P-gp alongside other transporters and enzymes, including phase I functionalization enzymes (e.g., cytochrome 450 oxidases), phase II conjugating enzymes (e.g., glutathione S-transferase, sulfotransferase, UDP-glucuronosyltransferase) [75,92,93,94,95], phase III exporters (e.g., multidrug resistance proteins [MRPs], breast cancer resistance protein [BCRP]) [41,75,93,96]), and solute carrier (SLC) transporters (e.g., organic anion transporting polypeptides [OATPs], organic anion transporters [OATs], organic cation transporters [OCTs], and multidrug and toxin extrusion proteins [MATEs]) [53,97,98]. Although the ABCB (MDR, P-gp) and ABCC (MRP) families of transporters are both referred to as “multidrug resistance proteins”, they are distinct classes of transporters that perform different functions in the chemical defense system. The interrelation of these enzymes and transporters in epithelial cells is illustrated in Figure 1.

In fish, the liver, kidney, and gill express high levels of biotransformation (phase I and II) enzymes [41,80]. In these tissues, biotransformation enzyme mRNA expression is 10- to 150-fold higher than ABC transporter expression [41,80]. These tissues also have higher expressions of phase III transporters (MRPs [Abccs], BCRP [Abcg]) than P-gp, as expected for tissues that have a high concentration of biotransformed metabolites [19,41,80]. Conversely, the fish intestine displays relatively modest biotransformation enzyme expression, with nearly equivalent levels of biotransformation enzymes and ABC transporters [80]. In the proximal intestine, phase III Abcc and Abcg transporters are expressed at similar levels to P-gp, but the distal intestine expresses much more P-gp compared to phase III transporters [19,80]. This transporter expression pattern favors the transport of unmodified parent compounds, which will be the predominant form (as opposed to biotransformed metabolites) due to the lower expression of biotransformation enzymes in this tissue.

Importer proteins tend to be expressed in the same cells as exporters, usually on the opposite sides of barrier tissues. The SLC21 (OATP) and SLC22 (OAT, OCT) families of secondary active transporters in particular are commonly expressed with P-gp in fish tissues [53,97,98]. These transport proteins import both membrane-permeable (lipophilic) and non-membrane permeable (hydrophilic) molecules. Membrane-permeable molecules require import proteins for the same reasons that they require export proteins including P-gp: active transport moves these molecules across barriers much faster than passive diffusion, and allows the organism to maintain substrate concentrations that are substantially different from passive equilibrium concentrations [4,9,99]. These import proteins share a very similar set of substrates and inhibitors with P-gp [1,100,101,102]. This common substrate base is expected for an integrated system that controls the movement of compounds across barriers: each substrate molecule needs to be sequentially imported then exported in order to cross an epithelial cell, thus the importers and exporters in the same cell will transport a shared set of substrates.

The regulation of these defense mechanisms is orchestrated by various signaling pathways and transcription factors. Nuclear receptors, including the aryl hydrocarbon receptor (AhR) and pregnane X receptor (PXR), play a crucial role in coordinating the expression of biotransformation enzymes, P-gp, and MRPs. Upon binding to their ligands, these receptors initiate the transcription of target genes involved in xenobiotic metabolism and transport. For example, AhR activation in fish induces the expression of phase I enzymes [27,70], while PXR activation upregulates both biotransformation enzymes and efflux transporters, including P-gp and MRPs [103,104,105,106].

P-gp performs essential chemical defense functions in different tissues, but it is not essential for life until faced with specific chemical challenges. For example, the dog and cat genomes both include alleles coding for non-functional P-gp proteins, but affected individuals show no adverse health effects unless exposed to a toxic P-gp substrate (e.g., ivermectin) [107]. Similarly, P-gp knockout mice are viable and generally indistinguishable from the wild-type, but are much more sensitive to drugs that are P-gp substrates [108]. In fish, zebrafish embryos subjected to the morpholino knockdown of Abcb4 and Abcb5 are viable and develop normally, but accumulate higher levels of P-gp substrate molecules compared to untreated embryos [38].

5. P-Glycoprotein Structure and Transport Mechanism

P-gp is a full transporter with two transmembrane domains (TMDs) and two nucleotide binding domains (NBDs) (Figure 2). In both fish and mammals, each TMD contains six transmembrane helices [16,38,41,80]. The NBDs contain highly conserved motifs that are a hallmark of all ABC transporters: A-loop, Walker A, Walker B, and the ABC signature [9,41]. P-gp has an atomic mass of 170 kDa in both fish and mammals [16,43,72,74]. The three-dimensional structure has been determined for human and mouse P-gp [109,110], but the structure of fish P-gp has not yet been fully elucidated. There is a 64% amino acid sequence identity between zebrafish Abcb4 and human ABCB1, while zebrafish Abcb5 shares 57% of its amino acid sequence with human ABCB1 [38]. Thus, fish and mammalian P-gp are expected to share most aspects of their structure and function [38,69]. The differences in P-gp function between these animal lineages are found in the finer points of their operation, such as substrate specificities and post-translational regulation [69,111]. These structural disparities might be attributed to differences in evolutionary history, ecological niches, and physiological requirements between mammals and fish.

Substrates enter the drug-binding pocket of P-gp either from the cytoplasm or directly from the hydrophobic region of the plasma membrane [109,110]. The drug-binding pocket is surrounded by the 12 α-helices of the two TMDs, and is lined with hydrophobic, amphipathic, polar, and charged amino acid side chains [109]. Substrate molecules interact with a subset of these amino acid side chains, forming the molecule’s binding site [109]. While the amino acid composition of human and zebrafish Abcb4 and Abcb5 is similar, there are different amino acids in their TMD α-helices, leading to different substrate specificities and affinities [69].

The resting state of the P-gp molecule is the inward-facing configuration [112]. In this state, the outside ends of two TMDs are in contact with each other (closed) on the outer surface of the cell membrane. The intracellular ends of the two TMDs are separated (open) on the cytoplasmic side of the cell membrane, and the two NBDs are separated from each other in the cytoplasm [112]. When a substrate is bound in the drug-binding pocket and an ATP molecule is bound to each of the two NBDs, the transport cycle begins (Figure 3) [112]. First, the cytoplasmic ends of the TMDs come together, and the NBDs dimerize with the two ATP molecules sandwiched between them. One of the bound ATP molecules is then hydrolyzed, and the P-gp molecule transitions to an outward-facing configuration. The outer ends of the TMDs separate, changing the shape of the drug binding site, and releasing the substrate into the extracellular media. Finally, the second ATP molecule is hydrolyzed, and the two NBDs separate. The P-gp molecule transitions back to the inward-facing configuration, from which it is able to begin a new transport cycle [112].

Figure 3. The P-glycoprotein transport cycle. 1. P-gp has an inward-open conformation, with no substrate or ATP bound. 2. A substrate binds in the drug-binding pocket between the transmembrane domains (TMDs), and one ATP molecule binds to each of the two nucleotide binding domains (NBDs). The NBDs dimerize with the two ATP molecules sandwiched between them. 3. One of the ATP molecules is hydrolyzed. The P-gp molecule transitions to an outward-facing conformation and releases the substrate into the extracellular space. 4. The second ATP molecule is hydrolyzed, allowing P-gp to return to an inward-facing conformation [112].

Figure 3. The P-glycoprotein transport cycle. 1. P-gp has an inward-open conformation, with no substrate or ATP bound. 2. A substrate binds in the drug-binding pocket between the transmembrane domains (TMDs), and one ATP molecule binds to each of the two nucleotide binding domains (NBDs). The NBDs dimerize with the two ATP molecules sandwiched between them. 3. One of the ATP molecules is hydrolyzed. The P-gp molecule transitions to an outward-facing conformation and releases the substrate into the extracellular space. 4. The second ATP molecule is hydrolyzed, allowing P-gp to return to an inward-facing conformation [112].

6. P-Glycoprotein Substrates

The substrates of fish P-gp tend to be moderately hydrophobic, amphipathic, and often have a positively charged nitrogen atom and aromatic rings, closely matching the substrates of mammalian P-gp [16]. The known substrates of fish P-glycoprotein are shown in

Table 3. Substrates of fish P-glycoprotein (P-gp), as determined by methods including transport inhibition by model chemosensitizers, decreased accumulation in P-gp-over expressing cells, increased cytotoxicity with model chemosensitizers, and increased ATPase activity in combination with the inhibition of model substrate accumulation.

Substrate	Species	References
Hormones
19-methyl testosterone	Poeciliopsis lucida	[113]
Cortisol	Oncorhynchus mykiss	[114]
Octreotide (synthetic somatostatin)	Fundulus heteroclitus	[115]
Bacterial Toxins
Lipopolysaccharides	Danio rerio	[116]
Microcystin-LR	Danio rerio	[52]
Fluorescent Dyes
BCECF-AM	Danio rerio	[68]
Calcein-AM	Danio rerio, Oncorhynchus mykiss, Poeciliopsis lucida	[38,68,78,117,118,119]
DiOC6	Danio rerio	[68]
Rhodamine 123	Oncorhynchus mykiss, Poeciliopsis lucida	[54,78,113,114,117,119,120]
Rhodamine B	Hatcheria macraei, Salmo trutta, Oncorhynchus mykiss, Oncorhynchus tshawytscha, Danio rerio, Cyprinus carpio	[38,45,67,121]
Metal Compounds
Arsenic trioxide (As2O3)	Poeciliopsis lucida	[113]
Cadmium chloride (CdCl2)	Danio rerio	[122]
Silver nitrate (AgNO3)	Danio rerio	[122]
Polycyclic Aromatic Hydrocarbons
2-aminoanthracene	Leuciscus idus melanotus	[30]
Benzo[a]pyrene	Cyprinus carpio	[123]
Phenanthrene	Danio rerio	[38]
Fragrance Ingredients
α-amylcinnamaldehyde	Danio rerio	[124]
α-hexylcinnamaldehyde	Danio rerio	[124]
Galaxolide	Danio rerio	[38]
Isoeugenol	Danio rerio	[124]
Musk xylene	Danio rerio	[124]
Nerol	Danio rerio	[124]
Tonalide	Danio rerio	[38]
Pesticides
Chlorpyrifos	Poeciliopsis lucida	[113]
Diazinon	Poeciliopsis lucida	[113]
Emamectin benzoate	Oncorhynchus mykiss	[84]
Methyl parathion	Danio rerio	[125]
Phosalone	Poeciliopsis lucida	[113]
Hydrophobic Peptide
Reversin 205	Poeciliopsis lucida	[113]
Immunosuppressants
Cyclosporin A	Fundulus heteroclitus, Squalus acanthias	[71,97,126]
Rapamycin	Fundulus heteroclitus	[61]
Psychoactive Pharmaceuticals
Bupropion	Oncorhynchus mykiss	[127]
Citalopram	Oncorhynchus mykiss	[127]
Clozapine	Oncorhynchus mykiss	[127]
Fluoxetine	Danio rerio	[124]
Venlafaxine	Oncorhynchus mykiss	[127]
Analgesic Pharmaceuticals
Colchicine	Poeciliopsis lucida	[113]
Diclofenac	Danio rerio	[124]
Antiarrhythmics
Acebutolol	Poeciliopsis lucida	[117]
Diltiazem	Poeciliopsis lucida	[113]
Nicardipine	Poeciliopsis lucida	[113]
Prazosin	Poeciliopsis lucida	[113]
Propanolol	Oncorhynchus mykiss, Poeciliopsis lucida	[113,128]
Quinidine	Oncorhynchus mykiss, Poeciliopsis lucida	[113,114]
Verapamil	Danio rerio, Poeciliopsis lucida, Fundulus heteroclitus, Squalus acanthias, Oncorhynchus mykiss	[38,71,78,113]
Antiretroviral
Saquinavir	Fundulus heteroclitus	[129]
Lipid-Lowering Pharmaceuticals
Atorvastatin	Poeciliopsis lucida	[117]
Pravastatin	Poeciliopsis lucida	[113]
Simvastatin	Danio rerio	[124]
Antiparasitic
Ivermectin	Danio rerio, Oncorhynchus mykiss, Fundulus heteroclitus	[62,82,83,84]
Phosphodiesterase Inhibitor
Sildenafil	Poeciliopsis lucida	[113]
Anticancer Pharmaceuticals
AT9283	Danio rerio	[69]
Bisantrene	Danio rerio	[69]
Daunomycin (Daunorubicin)	Fundulus heteroclitus, Pleuronectes americanus	[58,130]
Doxorubicin	Oncorhynchus mykiss, Poeciliopsis lucida, Danio rerio	[54,56,69,117]
Etoposide	Danio rerio	[69]
KW-2478	Danio rerio	[69]
Mitoxantrone	Danio rerio	[69]
Paclitaxel	Danio rerio	[69]
Romidepsin	Danio rerio	[69]
Sepantronium bromide (YM-155)	Danio rerio	[69]
Tanespimycin (17-AAG)	Danio rerio	[69]
Tozasertib (VX-680)	Danio rerio	[69]
Vinblastine	Danio rerio, Ictalurus punctatus	[38,69,73]
Vincristine	Danio rerio	[38]
Vinorelbine	Oncorhynchus mykiss	[57]

.

In studies comparing the substrates of fish P-gp to mammalian P-gp directly, the sets of molecules that are substrates in each animal lineage are similar but not identical. In a high-throughput study of 90 known substrates of human P-gp, zebrafish abcb4-expressing cells closely matched human P-gp-expressing cells in cytotoxicity resistance assays, while zebrafish abcb5-expressing cells showed resistance to fewer substrates in these assays [69]. In a study comparing human P-gp to P-gp from the livebearer fish Poeciliopsis lucida, 16 substrates were common to both species, while 5 substrates were exclusive to fish P-gp and 7 substrates were exclusive to human P-gp [113].

The majority of research efforts have concentrated on substrates of anthropogenic origin, which are materials that have become pervasive environmental contaminants due to human activities, such as fluorescent dyes, pharmaceuticals, pesticides, and industrial chemicals. This emphasis can be attributed to the origins of P-gp’s discovery, which revolved around drug resistance in mammalian cancers and resistance to xenobiotic pollutants in aquatic organisms. Xenobiotic substrates present in the environment throughout evolutionary history likely included such chemicals as bacterial toxins (e.g., doxorubicin, microcystin-LR, lipopolysaccharides) and plant metabolites (e.g., quinidine, nerol, isoeugenol).

P-gp has played an important role in chemical homeostasis in fish predating the onset of anthropogenic pollution, but its physiological roles have received relatively little attention. Few studies have investigated endogenous substrates in fish to any great extent; only two steroid hormones (cortisol and testosterone) have been identified as substrates to date [113,114]. Mammalian P-gp actively transports a diverse array of endogenous substrates including steroid hormones (e.g., cortisol, aldosterone, testosterone), lipids (e.g., phospholipids, cholesterol), and peptides (e.g., enkephalins) [131,132,133,134,135]. By managing intracellular levels of these compounds, P-gp plays a pivotal role in hormone signaling, lipid metabolism, and neurotransmission. While research focus has been on xenobiotic efflux and multidrug resistance, understanding the intricate interplay between P-gp and endogenous substrates provides evidence that it has broader regulatory significance in several physiological pathways [1].

Studies of P-gp expression have provided preliminary evidence that fish P-gp, especially Abcb5, may be involved in processing hormones and growth factors. In zebrafish ovaries, abcb5 is highly expressed in follicles during the early pre-vitellogenic stages of egg development, but is absent during the later vitellogenic and pre-ovulatory stages, while abcb4 is expressed at low levels at all stages of egg development [69]. This expression pattern suggests a possible role for fish P-gp in transporting hormones that stimulate early oocyte maturation, including follicle-stimulating hormone (FSH), luteinizing hormone (LH), and estradiol [136,137], and that abcb5 may have a higher affinity for these substrates than abcb4 does [69]. In rainbow trout gills, abcb5 is highly expressed in immature progenitor cells that mature into epithelial pavement (respiratory) cells, but abcb5 is not expressed in mature gill epithelial cells [41,138]. This suggests that P-gp plays a role in cell maturation in fish gills [41]. In mammals, P-gp has been implicated in stem cell differentiation; the ability of stem cells to differentiate into specialized cell types involves the regulation of multiple transport mechanisms including the P-gp transport of endogenous signaling molecules, such as growth factors and hormones, crucial for directing stem cell fate [139]. Exploring the endogenous substrates of P-gp in fish holds great potential and aligns with similar inquiries into the intrinsic functions of membrane transporters in mammals and other species [1].

7. The Regulation of P-Glycoprotein Expression

Diverse factors including xenobiotic exposure, heat stress, and oxidative stress can increase P-gp expression as part of an adaptive cellular defense response [16]. In mammals, the induction of P-gp has been extensively studied, especially in the context of drug interactions and multidrug resistance. Exposure to xenobiotics activates nuclear transcription factors including the pregnane X receptor (PXR), constitutive androstane receptor (CAR), aryl hydrocarbon receptor (AhR), and nuclear factor erythroid 2-related factor 2 (Nrf2), which bind to specific enhancer sequences in the ABCB1 promoter region, orchestrating the transcriptional upregulation of P-gp [140,141].

In fish, P-gp induction has gained attention due to its role in environmental detoxification and chemical defense. Exposure to aquatic pollutants, heavy metals, and pharmaceuticals can induce P-gp expression in fish, particularly in the liver, gills, and intestines. The regulation of P-gp in fish involves molecular mechanisms similar to those in mammals, including interactions between receptors, genetic response elements, and nuclear transcription factors. A key similarity between fish and mammals is the involvement of the nuclear receptors PXR and Nrf2 [94,122]. These receptors are activated by oxidative stress (Nrf2), or upon binding to specific ligands (PXR), and induce the subsequent transcription of P-gp and other genes. In fish, PXR has been identified as a central regulator of P-gp expression [103,104]. Studies have shown that PXR activation by xenobiotics, including pharmaceuticals (e.g., paracetamol, simvastatin) and plant flavonoids (e.g., quercetin, rotenone), leads to the upregulation of P-gp expression in fish tissues [95,142,143]. Similarly, Nrf2 activation has been linked to the regulation of P-gp in both fish and mammals [94,144]. For example, in zebrafish, the organophosphate pesticide malathion induced oxidative stress in zebrafish livers, leading to an increase in P-gp expression via the Nrf2 pathway [94].

Despite these similarities, there are notable differences in P-gp regulation between fish and mammals. Although AhR has been shown to regulate the expression of cytochrome P450 in fish, it does not affect P-gp expression [27,70]. Fish also lack the constitutive androstane receptor (CAR) that induces P-gp expression in mammals [105]. Mammalian research has provided detailed insights into the role of nuclear receptors and transcription factors in P-gp expression, often involving well-defined pathways. In contrast, fish studies are still evolving, and while the key players are identified, the intricate network of interactions remains to be fully elucidated.

8. Modulators of P-Glycoprotein Expression

P-gp expression in fish tissues can be induced or inhibited by many different chemicals, including both xenobiotic and endogenous compounds. Chemicals that modulate P-gp expression include those of anthropogenic origin such as pharmaceuticals (e.g., morphine, paracetamol, simvastatin), pesticides (e.g., malathion, emamectin benzoate), and industrial chemicals (e.g., crude oil, perfluorooctane sulfonate [PFOS]), and those of naturally-derived origin, such as plant metabolites (e.g., glycyrrhizic acid, quercetin) and bacterial compounds (e.g., microcystin-LR). Endogenous chemical modulators include bile acids and steroid hormones. Compounds that induce P-gp expression in fish are listed in

Table 4. Inducers of P-glycoprotein (Abcb1, Abcb4, Abcb5, P-glycoprotein not otherwise specified) mRNA or protein expression in fish.

Expression Inducer	Species	Tissue	References
Bile Acids and Salts
5α-cyprinol 27-sulfate	Danio rerio	Liver	[105]
Lithocholic acid	Oncorhynchus mykiss	Liver	[146]
Enzyme Cofactor
Lipoic acid	Jenynsia multidentata	Liver	[147]
Hormone
Pregnenolone 16α-carbonitrile (Synthetic steroid)	Oncorhynchus mykiss	Liver	[146]
Vitamin
Vitamin B12	Danio rerio	Liver	[94]
Cyanotoxin
Microcystin-LR	Danio rerio	Embryo	[52]
Microcystin-LR	Jenynsia multidentata	Liver, gill, brain	[74]
Metal Compounds
Arsenic trioxide (As2O3)	Poeciliopsis lucida	Liver	[148]
Cadmium chloride (CdCl2)	Trematomus bernacchii	Liver	[48]
Cadmium chloride (CdCl2)	Danio rerio	Embryo	[122]
Silver nitrate (AgNO3)	Danio rerio	Embryo	[122]
Phytochemicals
Glycyrrhizic acid	Ictalurus punctatus	Kidney	[142]
Quercetin	Ictalurus punctatus	Kidney	[142]
Schisandrin A	Ictalurus punctatus	Kidney	[142]
Schisandrin B	Ictalurus punctatus	Kidney	[142]
Schisandrol A	Ictalurus punctatus	Kidney	[142]
schisandrol B	Ictalurus punctatus	Kidney	[142]
Industrial Chemicals
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)	Dicentrarchus labrax	Liver	[149]
Crude oil (Exxon Valdez)	Anoplarchus purpurescens	Liver	[27]
Heavy fuel oil (fresh)	Chelon labrosus	Liver	[65]
Perfluorooctane sulfonate (PFOS)	Chelon labrosus	Liver	[65]
Pesticides
Carbendazim	Jenynsia multidentata	Gill	[150]
Chlorpyrifos	Jenynsia multidentata	Liver	[151]
Emamectin benzoate	Oncorhynchus mykiss	Liver	[152]
Malathion	Danio rerio	Liver, brain	[94]
Rotenone	Ictalurus punctaus	Kidney	[142]
Triclosan	Xiphophorus helleri (males)	Liver	[153]
Zinc pyrithione	Carassius auratus gibelio	Liver, kidney	[154]
Analgesic Pharmaceuticals
Diclofenac	Gambusia affinis	Liver	[155]
Paracetamol (Acetaminophen)	Mugilogobius chulae	Liver	[95]
Antibiotic
Norfloxacin	Xiphophorus Helleri	Liver	[156]
Antifungal Pharmaceutical
Clotrimazole	Oncorhynchus mykiss	Intestine, brain	[17]
Lipid-Lowering Pharmaceutical
Simvastatin	Mugilogobius abei	Liver	[143]
Simvastatin	Gambusia affinis	Liver	[92]

, and those that inhibit P-gp expression are shown in Table 5. In addition to the specific modulating substances listed, complex mixtures of toxicants in industrial and urban effluents and receiving waters induce P-gp expression, and contribute to the MXR phenotype [11,45,76]. Environmental stressors can also alter P-gp expression. For example, fasted (4 weeks) rainbow trout exhibit induced P-gp expression in intestinal epithelia [145].

Alterations in P-gp expression are intricate in terms of timing, effective dosages, and combinations of chemicals. Certain compounds can induce or inhibit P-gp expression at different time intervals, adding to the complexity of this modulation. For example, in zebrafish brains, malathion induced P-gp expression after 3 d, but inhibited its expression after 7 d [94]. Stress that results from acute exposures to toxicants can differ from long-term exposures [157], and may require different gene expression patterns in response. In addition, early inductions of coordinately regulated genes (e.g., antioxidant enzymes) may alter the condition of the tissue (e.g., oxidative stress), thereby altering later P-gp expression [94].

Some substances affect P-gp expression only when present in mixtures. The pesticides cypermethrin and chlorpyrifos significantly decreased P-gp expression in killifish gill and brain tissue in combination, but had no significant effect in these tissues when present individually [151]. Individually, titanium dioxide nanoparticles and the dioxin TCDD did not affect P-gp expression in sea bass liver, but the combination of both compounds caused a significant reduction in P-gp expression [96]. The pesticide methyl parathion induced P-gp expression in zebrafish liver only in combination with verapamil [125]. These additive or synergistic effects may indicate that each of the compounds in the mixture interacts with different aspects of the mechanisms regulating P-gp expression in fish.

The effects of P-gp expression modulators can be species-specific. For example, the synthetic steroid pregnenolone 16α-carbonitrile (PCN) induced P-gp expression in rainbow trout hepatocytes [146], inhibited P-gp expression in zebrafish livers [104], and had no effect on P-gp expression in killifish hepatoma cells [158]. Likewise, the bile salt 5α-cyprinol 27-sulfate induced P-gp expression in zebrafish hepatocytes, but similar induction effects were not observed in sea lampreys (Petromyzon marinus) [105]. Within a single species, the effects of expression modulators can vary by sex and by tissue. For example, the antimicrobial agent triclosan increased liver P-gp expression in male swordtail fish (Xiphophorus helleri), but decreased expression in females [153]. The fungicide carbendazim induced P-gp expression in killifish gill, but decreased expression in the liver [150]. Emamectin benzoate-treated rainbow trout had increased P-gp expression in the liver, but decreased P-gp expression in the intestine [152]. These differences may arise from variations in exposure profiles between tissues, sexes, and species, which would require different chemical defense strategies.

The modulation of P-gp expression is of great importance in both clinical and environmental contexts. In clinical settings, changes in P-gp expression can alter the bioavailability and pharmacokinetics of therapeutic drugs. In aquatic toxicology, P-gp expression confers resistance to environmental toxicants; thus, alterations can have major impacts on fitness and survival in contaminated environments. Further research into inducers and inhibitors of P-gp expression in fish will therefore provide valuable information relating to veterinary medicine, aquaculture, and environmental risk assessment.

Table 5. Inhibitors of P-glycoprotein (Abcb1, Abcb4, Abcb5, P-glycoprotein not otherwise specified) mRNA or protein expression in fish.

Expression Inhibitor	Species	Tissue	References
Hormone
Pregnenolone 16α-carbonitrile (Synthetic steroid)	Danio rerio	Liver	[104]
Phytochemical
Genistein	Ictalurus punctatus	Kidney	[142]
Carbon Allotrope
Fullerene (nC60)	Cyprinus carpio	Liver	[123]
Industrial Chemicals
Metal mine tailings (Mn, Cd, As, Cu, Cr)	Astyanax lacustris	Liver	[159]
Pesticides
Carbendazim	Jenynsia multidentate	Liver	[150]
Emamectin benzoate	Oncorhynchus mykiss	Intestine	[152]
Malathion (7-day exposure)	Danio rerio	Brain	[94]
Triclosan	Xiphophorus helleri (females)	Liver	[153]
Analgesic Pharmaceutical
Morphine	Danio rerio	Embryo	[116]

Table 5. Inhibitors of P-glycoprotein (Abcb1, Abcb4, Abcb5, P-glycoprotein not otherwise specified) mRNA or protein expression in fish.

Expression Inhibitor	Species	Tissue	References
Hormone
Pregnenolone 16α-carbonitrile (Synthetic steroid)	Danio rerio	Liver	[104]
Phytochemical
Genistein	Ictalurus punctatus	Kidney	[142]
Carbon Allotrope
Fullerene (nC60)	Cyprinus carpio	Liver	[123]
Industrial Chemicals
Metal mine tailings (Mn, Cd, As, Cu, Cr)	Astyanax lacustris	Liver	[159]
Pesticides
Carbendazim	Jenynsia multidentate	Liver	[150]
Emamectin benzoate	Oncorhynchus mykiss	Intestine	[152]
Malathion (7-day exposure)	Danio rerio	Brain	[94]
Triclosan	Xiphophorus helleri (females)	Liver	[153]
Analgesic Pharmaceutical
Morphine	Danio rerio	Embryo	[116]

9. Inhibitors of P-Glycoprotein Transport Activity

P-gp activity inhibitors attenuate the efflux function of the transporter, thereby interfering with its role in actively exporting substrates out of cells. Inhibitors of P-gp transport activity are being recognized as an important class of environmental pollutants that inhibit chemical defense enzymes and transporters [160]. They interfere with MXR mechanisms that contribute to chemical defense in contaminated environments [7,11]. P-gp is the most studied MXR protein, but many transporters and enzymes contribute to the MXR phenotype [15,28,29]. The inhibition of MXR proteins allows substrates to exert harmful effects at lower concentrations than they would in the absence of these inhibitors [160,161,162,163,164]. This increases the organism’s sensitivity to environmental toxicants; thus, these inhibitors are referred to as chemosensitizers. Chemosensitizers in complex mixtures increase the toxicity of the mixture [160]. Inhibitors of MXR transporters including P-gp, MRPs, BCRP, and MATEs interfere with transport activity, which leads to the increased intracellular accumulation of their substrates [7,160]. In clinical contexts, compounds that act as chemosensitizers are major contributors to drug–drug and drug–food interactions, since they can alter the pharmacokinetics of co-administered pharmaceuticals [165].

Chemosensitizers in fish are usually studied in the context of complex mixtures of environmental pollutants: most identified inhibitors of fish P-gp are anthropogenic in origin (e.g., pesticides, industrial chemicals, pharmaceuticals, personal care products). Only one endogenous P-gp inhibitor is known in fish: the bile acid taurochenodeoxycholate [100]. More P-gp inhibitors have been identified in fish than any other type of interacting chemical (inhibitors, substrates, and expression modulators). Chemosensitizers are relatively easy to detect using in vitro methods, especially dye accumulation and ATP consumption assays [113,160]. However, experiments of this nature, especially if they use a high-throughput design, usually provide a simple confirmation of the effect rather than a more thorough concentration–response relationship [160]. The known inhibitors of fish P-gp activity are shown in

Table 6. Inhibitors of fish P-glycoprotein activity, as determined by methods including inhibition of transport of a known P-gp substrate, and inhibition of stimulated ATPase activity.

Inhibitor	Species	References
Fluorescent Dye
Hoechst 3334	Poeciliopsis lucida	[113]
Cyanotoxin
Microcystin-LR	Oncorhynchus mykiss	[166]
Metal Compounds
Arsenic trioxide (As2O3)	Poeciliopsis lucida	[148]
Cadmium chloride (CdCl2)	Poeciliopsis lucida	[148]
Mercury (II) chloride (HgCl2)	Poeciliopsis lucida	[148]
Orthovanadate (VO4)	Poeciliopsis lucida	[113]
Potassium dichromate (K2Cr2O7)	Poeciliopsis lucida	[148]
Hormone
Octreotide (Synthetic somatostatin)	Fundulus heteroclitus	[115]
Bile Acid
Taurochenodeoxycholate	Poeciliopsis lucida	[113]
Fragrance Ingredients
Galaxolide	Danio rerio	[38]
α-hexylcinnamaldehyde	Danio rerio	[124]
Isoeugenol	Danio rerio	[124]
Musk ketone	Danio rerio	[124]
Musk xylene	Danio rerio	[124]
Nerol	Danio rerio	[124]
Tonalide	Danio rerio	[38,67]
Polycyclic Aromatic Hydrocarbon
Phenanthrene	Danio rerio	[38]
Industrial Chemicals
Oil-Sands Process-Affected Water (fresh)	Oryzias latipes	[167]
Polychlorinated Biphenyls (PCBs)	Thunnus albacares	[43]
Surfactants
Alcohol polyethoxylates	Poeciliopsis lucida	[168]
C-12 linear alkylbenzene sulfonate	Ictalurus punctatus	[169]
Nonylphenol diethoxylate	Oncorhynchus mykiss	[77]
Perfluorooctane sulfonate (PFOS)	Danio rerio	[170]
Polypropylene glycols	Poeciliopsis lucida	[168]
Flame Retardants
Polybrominated diphenyl ethers (BDEs)	Thunnus albacares	[43]
Pesticides
Azinphos-methyl	Danio rerio	[67]
Diazinon	Poeciliopsis lucida	[113]
Dichlorodiphenyldichloroethane (DDD)	Thunnus albacares	[43]
Dichlorodiphenyldichloroethylene (DDE)	Thunnus albacares, Poeciliopsis lucida	[43,113]
Dichlorodiphenyltrichloroethane (DDT)	Thunnus albacares	[43]
Dieldrin	Thunnus albacares	[43]
2,4-dinitrophenol	Fundulus heteroclitus	[59]
Emamectin benzoate	Oncorhynchus mykiss	[84]
Endosulfan	Poeciliopsis lucida	[113]
Endrin	Thunnus albacares	[43]
Metazachlor	Danio rerio	[67]
Mirex	Thunnus albacares	[43]
Phosalone	Poeciliopsis lucida	[113]
Prochloraz	Oncorhynchus mykiss	[77]
Terbuthylazine	Danio rerio	[67]
Hydrophobic Peptide
Reversin 205	Oncorhynchus mykiss, Poeciliopsis lucida	[41,113,118,119]
Anticoagulant
Dipyridamole	Danio rerio	[67]
Antimalarial
Quinine	Fundulus heteroclitus	[59]
Antiparasitic
Ivermectin	Danio rerio, Oncorhynchus mykiss, Fundulus heteroclitus	[62,67,84]
Leukotriene Antagonist
MK571	Danio rerio, Poeciliopsis lucida	[38,100,142]
Antihistamine
Cimetidine	Oncorhynchus mykiss	[128]
Antibiotics
Erythromycin	Danio rerio, Poeciliopsis lucida	[97,113]
Troleandomycin	Poeciliopsis lucida	[158]
Analgesic Pharmaceutical
Diclofenac	Danio rerio, Poeciliopsis lucida	[124,158]
Antifungal Pharmaceutical
Clotrimazole	Oncorhynchus mykiss	[75]
Anti-Inflammatories
Indomethacin	Poeciliopsis lucida	[119]
Sulfasalazine	Poeciliopsis lucida	[113]
Antihypertensives
Furosemide	Poeciliopsis lucida	[117]
Reserpine	Poeciliopsis lucida	[113]
Antihyperuricemic
Probenecid	Poeciliopsis lucida	[119]
Phosphodiesterase Inhibitor
Sildenafil	Poeciliopsis lucida	[113,117]
Antiretrovirals
Ritonavir	Fundulus heteroclitus	[129]
Saquinavir	Fundulus heteroclitus	[129]
Lipid-Lowering Pharmaceuticals
Atorvastatin	Poeciliopsis lucida	[113,117]
Gemfibrozil	Poeciliopsis lucida	[117]
Pravastatin	Poeciliopsis lucida	[117]
Simvastatin	Poeciliopsis lucida	[117]
Psychoactive Pharmaceuticals
Carbamazepine	Danio rerio	[67]
Fluoxetine	Danio rerio	[124]
Sertraline	Danio rerio	[124]
Trifluoperazine	Poeciliopsis lucida	[113]
Immunosuppressants
Cyclosporin A	Danio rerio, Oncorhynchus mykiss, Thunnus albacares, Poeciliopsis lucida, Cyprinus carpio, Fundulus heteroclitus, Pleuronectes americanus	[38,41,43,58,61,67,68,77,113,114,115,117,118,119,121,128,130,148,168,171]
Cyclosporin G	Fundulus heteroclitus	[59]
Rapamycin	Fundulus heteroclitus	[61]
Tacrolimus (FK506)	Fundulus heteroclitus	[61]
Antiarrhythmics
Diltiazem	Poeciliopsis lucida	[113]
Nicardipine	Poeciliopsis lucida	[113]
Prazosin	Poeciliopsis lucida	[113]
Propranolol	Poeciliopsis lucida	[113]
Quinidine	Oncorhynchus mykiss, Poeciliopsis lucida	[113,114,128]
Verapamil	Danio rerio, Oncorhynchus mykiss, Thunnus albacares, Hatcheria macraei, Salmo trutta, Oncorhynchus tshawytscha, Poeciliopsis lucida, Scophthalmus maximus, Cyprinus carpio, Ictalurus punctatus, Fundulus heteroclitus, Pleuronectes americanus	[38,43,45,54,59,61,62,67,69,73,77,78,97,113,114,115,119,121,124,130,172]
Anti-Cancer Pharmaceuticals
Doxorubicin	Oncorhynchus mykiss	[54]
Elacridar	Danio rerio	[69]
Fluorouracil	Poeciliopsis lucida	[113]
Mitoxantrone	Poeciliopsis lucida	[113]
Tamoxifen	Poeciliopsis lucida	[113]
Tariquidar (XR9576)	Danio rerio, Oncorhynchus mykiss	[56,69]
Valspodar (PSC833)	Oncorhynchus mykiss, Thunnus albacares, Danio rerio, Poeciliopsis lucida, Fundulus heteroclitus, Squalus acanthias	[38,43,61,62,68,69,71,113,114,115,118,171]
Vinblastine	Danio rerio, Oncorhynchus mykiss, Fundulus heteroclitus, Pleuronectes americanus	[38,54,59,68,77,130]

.

P-gp inhibitors act by one of two general mechanisms: competitive and non-competitive inhibition. Competitive inhibitors are P-gp substrates that inhibit the transport of other substrates by occupying P-gp transport capacity [160]. The most potent competitive P-gp inhibitors have high affinity in the P-gp drug-binding pocket, and high membrane permeability [173,174]. These properties allow for a futile and energetically costly cycle of outward active transport and inward diffusion [15]. Non-competitive inhibitors impede some aspect of the P-gp transport mechanism. Vanadate, for example, inhibits P-gp ATPase activity, while tariquidar blocks the movement of transmembrane helices necessary for substrate translocation [110]. Non-competitive inhibitors reduce ATP consumption by preventing P-gp from actively transporting substrates [15,113]. Many studies do not determine the inhibition mechanism of chemosensitizers; however, if a substance has been identified as both a substrate and an inhibitor, it is likely a competitive inhibitor.

In vivo P-gp inhibition can exacerbate the behavioral effects of substrate exposure, as observed with the neurotoxic P-gp substrate ivermectin (IVM). IVM exposure causes behavioral deficits in fish, including altered swimming performance, poor motor coordination, and lethargy [82,83,84]. During co-exposure to the P-gp inhibitor cyclosporin A, all of these neurotoxic IVM effects were more severe in killifish, rainbow trout, and zebrafish [82,83,84].

Substantial knowledge gaps remain with respect to the magnitude of chemosensitizer effects as environmental pollutants [160,175]. More research is also needed to understand the contribution of the inhibition of other MXR transporters in chemical defense, since nearly all studies of chemosensitizers in aquatic animals have focused on P-gp [160]. A greater diversity of chemical compounds, and combinations of compounds, should be tested for chemosensitizing effects, especially the industrial and agricultural chemicals currently in use. More detailed information regarding effect concentration thresholds is needed as well. Studies of chemosensitizer exposure in aquatic media should report measured concentrations wherever possible, rather than nominal concentrations. Many reported IC50 concentrations for chemosensitizers exceed the water solubility of those chemicals; thus, the observed chemosensitizing effects will have occurred at much lower aqueous concentrations than those reported [160]. Expanding the available information about chemosensitizer effects in these areas will greatly improve our ability to accurately account for chemosensitizers in environmental risk assessments.

10. The Energetic Costs of P-Glycoprotein Activity

P-gp transport activity is powered by ATP hydrolysis, and may be energetically costly in terms of overall energy budgets. P-gp activity increases cellular energy consumption when fish hepatocytes are challenged in vitro with P-gp substrates [55,56]. During in vivo exposure, P-gp induction alone does not increase whole-body respiration rates in rainbow trout [176]. Increased P-gp activity during substrate exposure likewise does not affect whole-animal energy consumption [176]. Only when P-gp expression is induced and the organism is simultaneously challenged with substrate exposure does P-gp activity increase the in vivo respiration rate of rainbow trout [176].

The protective function of P-gp is a high priority in fishes’ energy budgets. P-gp activity is maintained during fasting and starvation in zebrafish and rainbow trout [82,177]. Similarly, four weeks of starvation induced P-gp expression in rainbow trout intestinal epithelia [145]. Thus, the cellular-level activity of P-gp is integrated into protective effects at the organismal level, and this protection is prioritized even during periods of limited resources.

11. Conclusions

In fish, P-gp protects sensitive tissues from its potentially harmful substrates, and helps to remove those substrates from the body. Although P-gp has been studied less extensively in fish than in mammals, it seems to share similar functions in both animal lineages. P-gp expression confers resistance to toxic substrates, and this resistance can be diminished by chemosensitizing P-gp inhibitors. Fish P-gp has been studied primarily in the context of resistance to anthropogenic contamination, so its endogenous functions are only beginning to be explored.

P-gp likely has broad regulatory significance in fish, potentially transporting hormones, growth factors, and other signaling molecules, thereby influencing processes including cell maturation, reproductive development, and energy metabolism. P-gp transport helps to maintain homeostasis at all levels of biological organization, from molecules to the whole organism, and contributes to the health of populations and ecosystems [1,178]. A deeper understanding of the endogenous physiological functions of P-gp will allow for more accurate predictions of the off-target effects of drugs and the identification of sensitive endpoints in ecotoxicology research.

Environmental exposure to P-gp inhibitors and toxic P-gp substrates presents serious risks to the health of aquatic ecosystems. P-gp confers multixenobiotic resistance to fish and invertebrates living in contaminated environments, but chemosensitizing compounds inhibit this resistance. While substantial research efforts have identified a wide range of environmental contaminants that are substrates, inhibitors, and expression modulators of fish P-gp, much remains to be discovered with respect to their effect concentrations, physiological effects, and interactions with other compounds. Continued research into the endogenous functions and xenobiotic protective effects of P-gp in fish will provide valuable information for applications, including those in veterinary medicine, aquaculture, and environmental monitoring.