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The BBB is a physical and enzymatic barrier that separates the CNS from the systemic vascular environment, shielding the CNS from exposure to circulating potentially harmful compounds. BBB is composed of a monolayer of brain capillary endothelial cells characterized by polarized nature apical and basal membranes, where transporters, including efflux systems such as P-gp and MRPs, are asymmetrically distributed , the paucity of both pinocytotic vesicles and fenestrations and the presence of tight junctions, metabolizing enzymes including cytochrome P hemoproteins and UDP-glucuronosyltransferases and SLC and ABC transporters [ 7 , 50 , 99 , , , ].

Tight junctions form a continuous impermeable cellular barrier, preventing the entrance of large and hydrophilic compounds into the brain. Small and lipophilic molecules gain access to the brain by passive diffusion or active transport [ 7 , 50 , 99 , , , ].

Several studies were performed in order to establish the level of expression of ABC transporters at the BBB of different species, including humans [ , , ]. Differences between species were found.

At the cellular level, most of the published data demonstrate that P-gp, MRP1, 2, 4, 5 and 6 and BCRP are highly expressed in the apical membrane of the brain capillary endothelial cells [ 7 , 51 , 52 , 99 , , , , ]. ABC efflux transporters at the BBB minimize or avoid neurotoxic adverse effects of drugs that otherwise would penetrate into the brain.

However, ABC efflux transporters may also limit the central distribution of drugs that are beneficial to treat CNS diseases [ 99 ]. Schematic overview of the main drug transporters expressed in brain capillary endothelial cells, as well as their localization. Adapted from [ 2 , 11 ]. In vitro cellular models of the BBB started to emerge in the early s, presenting multiple advantages and being complementary to in vivo studies.

Cell-based BBB models can be established with any type of cell source human, animal, or cell line derivative , including the availability of BBB endothelial cells and astrocytes freshly isolated from human brain tissue, fact that allows a considerable degree of reproducibility, both in physiological and pathological scenarios [ ].

Dauchy et al. Ohtsuki et al. P-gp, BCRP and MRP4 expression was detected, although with distinct relative expression level patterns from those found in freshly isolated human brain microvessels [ ]. There are static models and static two-dimensional models of the BBB, using endothelial cells monocultures and co-culture of endothelial cells and glia, respectively [ ].

In the first case, a simple monolayer of highly specialized brain microvascular endothelial cells is used. The cells, obtained from various sources e. Brain vascular endothelial cells grow to confluence on the luminal surface of the membrane, immersed in a specific growth media. This BBB model, with potential for using pure cell populations, allows drug permeability testing and binding affinity. However, in order to circumvent the limitations related to this model, namely its simplicity related to the absence of physiological stimuli, a two-dimensional model containing both endothelial and glia cells was developed [ ].

The addition of abluminal astrocytes, in juxtaposition to the endothelial monolayer, facilitates the formation of more stringent tight junctions and the overall expression of BBB features. In addition, the exposure to glia and induced glial-endothelial interactions increases the expression of brain endothelial marker enzymes, transporters such as P-gp and MRPs and tight junctions, and induces a phenotype more closely mimicking that found in vivo.

Transendothelial electrical resistance TEER , a functional parameter to monitor the quality of cells cultured on filter supports, namely the integrity of the cell monolayer, is higher in co-culture of endothelial cells and glia than in endothelial cell monocultures, indicating the formation of a more stringent and selective vascular bed [ ].

In addition to the cellular and static BBB models just referred, isolated brain microvessels have been extensively used to study BBB since the s [ ]. They have been successfully used for the identification of mechanisms and biochemical signals that play a role in regulating BBB functions in health and disease conditions, allowing the maintenance of the structural and cellular characteristics and properties in ex vivo experimentations.

As such, ABC transporters, that function as efflux pumps limiting the entry of numerous xenobiotics into the brain, have been studied in isolated brain capillaries providing reliable information on the transport processes mediated by different carriers [ , , ]. However, due to technical and functional limitations related to the use of isolated brain microvessels, computational models, artificial membranes, and in vitro cell culture BBB models have been gaining particular relevance.

One of the most used computational models is the in silico model, which, knowing the physicochemical properties of novel molecules, predicts their efficacy and bioavailability in relation to BBB permeability, considering both passive diffusion and active transport processes.

Consequently, computer-assisted structure-based drug design model makes the drug development process faster, predicting drug effectiveness [ ]. The cardiac endothelial cells are characterized by expression of uptake and efflux transporters, which control the transport of a wide range of compounds, including drugs and toxins, into and out of the heart, respectively [ ]. At the end of the s and in the s, the expression of P-gp in several tissues was extensively studied.

Studies using human heart tissues showed P-gp to be expressed in the heart, although generally at relatively low levels, when compared to tissues such as the intestine, the liver, the brain and the placenta [ 14 ]. Particularly, Meissner and colleagues observed, by immunohistochemistry and in situ hybridization, P-gp expression and location in human endothelial cells of the capillaries and arterioles of the ventricles and atria [ ].

Additionally, a few years later, the authors observed the expression of BCRP in the vasculature of human heart, both in health and ischemic conditions [ ]. As such, ABC transporters may provide a functional barrier between the blood and cardiomyocytes, limiting the entry of xenobiotics into the heart, namely those that are cardiotoxic, such as the anticancer drugs mitoxantrone and anthracyclines [ 9 , 14 , , ].

It should be noted that, despite distinct ABC transporters have been identified, P-gp appears to be the most relevant to cardiovascular medicine, where it modulates the efficacy and toxicity of cardioactive agents [ 9 ]. Indeed, many cardiovascular active compounds are subject to drug transport by P-gp, as it is exemplified by digoxin [ ]. In addition to studies demonstrating the presence of P-gp in luminal membranes of the vascular endothelium isolated from the rat heart [ ], the study performed by Estevez and colleagues demonstrated, for the first time, the presence of P-gp in primary cultures of rat heart myocytes [ ].

Similarly to P-gp, other ABC carriers have been studied in the cardiovascular system. Almost all studies demonstrated the expression of MRP1 in both human and other species heart [ , ]. Other multidrug resistance proteins, such as MRP1—3 and MRP5, were identified in human heart, with higher expression in ventricular samples [ , ].

MRP5 was found to be present in cardiomyocytes and in both vascular smooth muscle and endotelial cells [ ]. Its expression was found in both mouse and human hearts [ , ]. Eilers et al. Here, the authors observed the presence of both P-gp and MRP1—5, proteins responsible for the efflux of the main anti-retroviral drugs.

The presence of the carriers was confirmed by the suppression of the transport induced by the ABC transporters inhibitors verapamil and MK 5- 3- 2- 7-Chloroquinolinyl ethenyl phenyl dimethylcarbamyl-4,6-dithiaoctanoic acid , respectively.

In another study, Higashikuni et al. The hMVECs are microvascular endothelial cells and, therefore, probably a cardiac cell model more representative of the heart transporters. The liver is an important tissue involved in the synthesis and secretion of bile acids, metabolism and transport of cholesterol, as well as in the metabolism and efflux of endogenous and exogenous substances [ , ].

As the major organ responsible for drug metabolism, the liver contributes to the first-pass elimination of drugs and for the plasma clearance of systemically distributed therapeutic compounds [ , ].

Therefore, together with the kidneys, the liver is an important detoxifying organ [ ]. These, organized in plates, have a polarized nature, apical and basolateral membranes, with different composition and functions. Hepatocytes are separated by tight junctions, which allow the vectorial transport of compounds with endogenous or exogenous origins from the blood into the bile [ , , , ].

The basolateral membrane is in contact with the sinusoidal blood and the canalicular membrane represents the excretory pole of hepatocytes [ ].

It is known that there is a differentiated functional expression of both sinusoidal and canalicular hepatic drug transporters. Sinusoidal transporters mediate the initial step of hepatic elimination, i.

On the other hand, efflux transporters are located in both the canalicular and basolateral membranes, where they mediate excretion into bile or into the systemic circulation, respectively [ , , ]. Major hepatic canalicular apical and sinusoidal basolateral efflux transporters are ABC proteins Figure 5. Schematic overview of the main drug transporters expressed in hepatocytes, as well as their localization. Those transporters located on the basolateral membrane, such as MRP1 and MRP3—6, perform the removal of endogenous compounds organic anions and bile acids and xenobiotics from the hepatocytes into the sinusoidal blood, for subsequent urinary elimination [ , , , ].

Additionally, Meyer zu Schwabedissen and Kroemer demonstrated the involvement of hepatic BCRP in the biliary excretion of some therapeutically important drugs, such as methotrexate, the 3-hydroxymethylglutaryl-coenzyme A HMG-CoA -reductase inhibitors pitavastatin and rosuvastatin, and fluoroquinolones [ ].

It should be noted, however, that the MATE1 transporter belongs to the SLC transporters SLC47 , is predominantly expressed in the canalicular membrane of hepatocytes and functions as a secondary transport system, utilizing the electrochemical gradient of cations across the membrane for substrate transport.

Organic cation transporter 1 OCT1 may function in concert with MATE1 to mediate the hepatic uptake and biliary excretion, respectively, of cationic drugs and their metabolites [ ]. Many different in vitro liver models have been employed over the years in toxicological field with the aim to predict in vivo responses. Immortalized cell lines and primary isolated liver cells are widely used in vitro models for liver toxicity testing.

HepG2 and HepaRG cell lines are common immortalized liver-derived cell lines used in laboratory protocols [ ]. HepG2 cells express many liver-specific genes but the expression profile of genes involved in phase I and phase II metabolism vary between passages [ ]. HepaRG cells also express aldolase B that is a specific marker of adult hepatocytes. These cells have a high proliferative capacity, being able to differentiate in both hepatocytes and biliary cells.

In fact, when seeded at low density, HepaRG cells acquire an elongated undifferentiated morphology, actively divide and, after having reached confluency, form typical hepatocyte-like colonies surrounded by biliary epithelial-like cells [ ]. This differentiation takes place by treatment with dimethyl sulfoxide DMSO.

Moreover, after differentiation, the expression of the different mentioned proteins remains stable for 6 weeks by treatment with DMSO [ ]. However, since these cells were isolated from a grade I differentiated liver tumor of a single female patient suffering from hepatocellular carcinoma and chronic hepatitis C virus infection, their predictive value for the human population is limited [ , ].

Primary cultures of hepatocytes represent a good model for the study of hepatic drug transporters in vitro. Rodent primary hepatocyte cultures, however, may undergo the so-called de-differentiation process, which consists on changes in cell morphology, structure, polarity, gene expression and liver-specific functions e. For this reason, a sandwich-based culture technique was developed [ , ].

In this system, primary hepatocytes are placed between two layers of a gelled matrix, in a sandwich configuration, retaining the in vivo-like properties. As such, cell morphology, enzymes activity, albumin production and transferrin, fibrinogen and bile salt secretion are kept close to the physiological status over a longer period of time [ ]. This model is suitable for studies of hepatic drug transport, metabolism, biliary excretion and toxicity [ , , ].

Several studies using a sandwich-cultured hepatocyte model have demonstrated enhanced morphology and viability of hepatocytes, normal levels of secretion of liver-specific transporters and CYP enzymes and organic compounds, facilitated formation of gap junctions and functional bile canalicular networks over days in culture. On the other hand, the main sandwich-hepatocyte model disadvantage is the decrease of genes expression, responsible for many liver-specific functions, over time, although keeping itself more useful for the mechanistic studies of hepatobiliary toxicity than primary hepatocytes [ ].

Sandwich-cultured human hepatocytes are considered the gold standard for the in vitro research of human hepatic transporters. However, human primary hepatocytes remain stable with time in culture, with a polarized status. Thus, monolayer-cultured human hepatocytes are also a valuable tool for the study of hepatic transporters since, contrary to that referred for rodent monolayer primary-cultured hepatocytes, the de-differentiation process is not expected to occur [ ].

Due to difficulties in maintaining long-term functionality of primary hepatocytes, immortalized cells and even sandwich-hepatocytes cultures, as well as in an attempt to circumvent problems related to the small predictive value of 2D models in pharmacokinetics processes, new models have been being developed. Within these, particular attention is given to the three-dimensional models that best mimic the processes that occur in vivo. Particularly, hepatocytes have multiple apical and basolateral surfaces and, thus, their polarity is essential to safely predict, in vitro, the processes that may occur in vivo.

In fact, drug uptake and diffusion in 2D systems does not accurately replicate the complexity found in a 3D multicell layer system. There are several distinct 3D hepatocyte models, which vary greatly in complexity [ , ]. Hepatocyte spheroids present a very well defined and uniform size and geometry and, although they can be differently obtained, they can replicate, in a consistent way, the biological complexities of the 3D in vivo environment, allowing a greater maintenance of functionality than that observed in the two-dimensional models.

Particularly, the expression levels of enzymes of phase I metabolism are found at levels close to the physiological. In the toxicokinetics context, 3D hepatocyte spheroids allow the study of ABC proteins by quantifying drug uptake and diffusion, providing an uniform uptake by the entire surface area and avoiding complex experimental and analytical procedures [ ].

Other more complex three-dimensional cultures can be used, namely systems involving porous materials, packed-bed reactors, hollow fibers and perfusion flow [ ]. However, a fully functional liver culture model, where the entire in vivo dynamics can be observed, is still missing and efforts need to be carried out in order to accomplish that purpose. The kidney is responsible for maintaining fluid and electrolyte homeostasis, maintaining the essential nutrients and eliminating both potentially toxic compounds and metabolic waste products from the body.

These functions occur in the physiologic units of the kidney, the nephrons, composed by glomerulus and renal tubules [ , ]. The renal tubules consist of a monolayer of epithelial cells that play reabsorptive and secretory functions due to the presence of membrane transporters, which, in turn, significantly contribute to renal drug handling and for the variability in drug disposition.

ABC carrier proteins are predominantly located in proximal tubules where they use the energy provided by ATP hydrolysis to move substrates across the membrane [ , ]. In fact, MRP members in proximal tubular cells function as extrusion pumps for organic anions across the apical membrane.

Molecular biology techniques have shown that the renal cortical expression of MRP4 is much higher than that of MRP2 [ ]. Schematic overview of main drug transporters expressed in renal epithelial cells, as well as their localization. HK-2 Human Kidney-2 cell line is an immortalized proximal tubule epithelial cell line derived from adult human normal kidney and retains many of the phenotypic and functional characteristics of renal proximal tubular cells in vivo [ , , ].

At the molecular level, the products of E6 and E7 genes bind to the DNA regulatory proteins, resulting in facilitated cell proliferation [ , ]. Phenotypically, the HK-2 cell line has the same characteristics of normal well differentiated adult proximal tubular cells. It was shown that the HK-2 cells maintain the brush border typical enzymatic activities acid and alkaline phosphatase, leucine aminopeptidase and gamma-glutamyl transpeptidase [ ].

Several studies were carried out using HK-2 cells to evaluate, in vitro, the renal transport processes, namely those mediated by the ABC and SLC families of transporters. In fact, HK-2 cells retain the constitutive expression of a functional P-gp in their membranes and its activity and expression may be modulated by drugs and many commonly ingested substances [ , , ].

According to the referred above, and despite the expression of some ABC transporters in HK-2 cells, the absence of several other transporters points to the current lack of relevant cellular models for the study of drug transport at the kidney level. Nomura and colleagues used surgically removed renal tissue and compared the ABC mRNA expression levels in human renal cell carcinomas and normal kidney tissue.

The intestine, in addition to the liver, is an important tissue that regulates the extent of absorption of orally administered drugs [ , ]. The majority of drug absorption occurs at the enterocytes in the small intestine, especially in the duodenum and jejunum, due to the large surface area, which is dependent on the presence of villi and microvilli [ , ].

Moreover, the intestine is known for its absorptive role due to the presence of uptake and efflux transporters, located at the apical and basolateral membranes Figure 7 , apart from the presence of cytochrome P 3A CYP3A4 in humans and conjugation enzymes [ , ]. Schematic overview of main drug transporters expressed in enterocytes, as well as their localization.

P-gp, MRP2, MRP4 and BCRP are located at the apical membrane of enterocytes, causing the drug efflux into the lumen and reducing, in consequence, the drug concentration within the enterocytes. These ABC efflux transporters are the major barrier to intestinal absorption of substrate drugs [ 5 , 9 , 10 , , , , , , , , , ]. Moreover, the pattern of longitudinal expression of several intestinal transporters is not homogeneous along the human intestine, which may has functional implications on the preferable site of intestinal drug absorption.

Additionally, their precise location basolateral or apical is a subject of interest and often controversial [ , ]. In fact, the expression levels of efflux transporters can vary along the small intestine.

Particularly, P-gp is expressed at high levels in the ileum and colon, but it presents the lowest constitutive expression levels in the jejunum and duodenum [ 10 , ]. BCRP is expressed in the small and large intestine but, unlike P-gp, BCRP expression does not vary significantly along the length of the small intestine [ 10 ]. P-gp, BCRP and MRP2 are located at the apical membrane, driving compounds from inside the cell back into the intestinal lumen [ , ]. Since P-gp, BCRP and MRP2 are able to bind to several structurally distinct and unrelated compounds, due to the lack of substrate specificity, they can decrease the absorption of many clinically relevant drugs, such as antibiotics, statins, HIV protease inhibitors, cardiac drugs calcium channel blockers, digitalic , immunossupressants and anticancer agents [ 10 ].

On the contrary, MRP1 and MRP3-MRP5 are expressed at the basolateral side of enterocytes where they pump their substrates from the intracellular compartment into the systemic circulation, thereby benefiting oral bioavailability [ 10 , , , , ]. MRP1 is highly expressed in the small and large intestine, being located at the basolateral membrane of enterocytes where it functions as an absorptive carrier, avoiding the accumulation of chemicals in the enterocytes [ 10 , ].

However, Han and colleagues showed the presence of OCT1 in the apical membrane of both enterocytes and Caco-2 cell monolayers [ ]. Additionally, the OATP2B1 expression at the basolateral membrane of neonatal, infantile and adolescent enterocytes was recently revealed by Mooij and co-authors [ ]. One of the best in vitro models of human intestinal epithelial cells available for studies of drug intestinal absorption and excretion and drug-drug interactions is the Caco-2 cell line [ 16 , 19 , 21 , , , , ].

In , the Caco-2 cell line was established in culture from a human colon adenocarcinoma [ ]. Caco-2 cells exhibit morphological as well as functional similarities to the human enterocytes [ 1 ].

When cultured under specific conditions, Caco-2 cells grow exponentially and, when in confluency, they undergo enterocytic differentiation, which is complete within 21 days in culture [ ]. During their differentiation, they form a polarized monolayer and develop a well-defined and typical brush border with a regular microvilli on the apical surface, as well as tight cellular junctions [ 1 , ]. These brush-border microvilli are very similar to those observed in normal small intestine and colon, with a double-leaflet plasma membrane, a core of microfilaments extending into the cytoplasm and an associated glycocalix.

Caco-2 cells are indeed very similar to the small intestine enterocytes with respect to its structure and to the presence of brush-border-associated hydrolases [ , ]. Caco-2 cells have been extensively characterized and it is known that they are able to express tight junctions and very low amounts of cytochrome enzymes, making them particularly suitable as a model for examining various substrates transport properties [ ].

P-gp and MRP2 expression levels seem to be similar in jejunum and Caco-2 cells, while BCRP expression levels in Caco-2 cells are low when compared with those found in the human jejunum, in vivo [ , ]. The apparent permeability coefficients measured for reference compounds across Caco-2 cells monolayers have shown good correlation with their in vivo absorption [ ]. Hilgendorf et al. The best agreement between human tissue and the cell line was observed for the human jejunum and Caco-2 cells [ ].

Intestinal peptide-associated transporter 1 HPT1 was identified as the most abundantly expressed transporter in the intestinal mucosa. Caco-2 cells can be cultured on semi-permeable inserts, allowing the evaluation of the transport of molecules between the apical and basolateral chambers [ ].

Appropriate in vitro assays for transport studies can be divided in two major groups: membrane-based assays and cell-based assays. The study of the function of the ABC efflux transporters and the identification of their substrates and inhibitors has been performed by using membranes, prepared from cells expressing ABC transporters.

Similar methods can be applied in the identification of inducers and activators. Currently, there are 3 available membrane-based assays: ATPase assays, membrane vesicular transport assays and photoaffinity labeling assays [ 1 ]. Compared to cell-based assays, the membrane-based assays have several advantages, including: 1 the ability to be used to characterize the xenobiotic effects on one specific efflux transporter; 2 the ability to be easily employed in a high throughput mode; 3 the easy with which they are maintained after preparation and 4 the easy with which the assays are performed Table 2 [ 1 ].

Main advantages versus disadvantages of the described in vitro and ex vivo assays adapted from [ 1 ]. The determination of the ABC transporters ATPase activity can be performed either in isolated membranes containing the desired transporter insect or mammalian cell membranes , or in reconstituted ABC protein preparations [ 32 ].

ATPase activity assays are commonly used in P-gp, MRPs and BCRP studies, representing a method for identification of compounds that interact with these efflux transporters [ , ]. The ATPase activity of the efflux transporters is vanadate sensitive and can be changed in the presence of substrates or modulators.

These can directly interact with ABC transporters, leading to stimulation or inhibition of the formation of an intermediate state of ATPase reduction [ 1 , , ]. The efflux transporters can be kept in an intermediate state due to the reaction with inorganic vanadate V i and ATP.

ATP hydrolysis leads to P i dissociation from the transporter and is replaced by V i. Therefore, the ATPase activity at the active sites is completely inhibited [ 1 ]. Compounds that interact with ABC transporters can be identified as stimulators or inhibitors of their ATPase activity. The effect of the test compound on the ATPase activity of the efflux transporter is analyzed by the difference in the amount of phosphate released or, alternatively, in the remaining unmetabolized ATP, using ABC transporter expressing membranes, in the presence or absence of vanadate [ 1 , ].

The released P i levels are determined by a colorimetric reaction under mild acidic conditions, being the released P i amount directly proportional to the ATPase activity of the ABC transporters. Using the other experimental approach, the quantity of unmetabolized ATP is evaluated by a luciferase-generated luminescence signal, and is inversely proportional to the ATPase activity of the ABC transporters.

The assay relies on the ATP dependence of the light-generating reaction of firefly luciferase. Therefore, a decrease in luminescence corresponds to a higher ATP consumption by the transporters, thus, the greater the decrease in luminescence signal, the higher the ATPase activity. Accordingly, samples containing compounds that stimulate the P-gp ATPase will have significantly lower signals than untreated samples.

On the opposite, compounds that act as P-gp inhibitors will trigger less ATP consumption and, in consequence, the luminescence signal will be greater since the amount of unmetabolized ATP is higher.

By comparing the results obtained for the basal activity and for the activity in the presence of the test compound, it can be classified into substrate, activator, inhibitor or without effect on the basal ATPase activity of the ABC transporters [ 1 , 32 , ].

Furthermore, these ATPase assays can also be applied to assess kinetic parameters, such as IC 50 for inhibitors [ 1 ]. Two different protocols can be used to study the interactions between ABC transporters and test compounds, i. In the stimulation assay, the stimulation of the basal ATPase activity of the ABC transporter is measured in the presence of the test compound. The transporter substrates significantly stimulate the basal ATPase activity. In the inhibition assay, the transporter ATPase activity is analyzed with a known substrate and a specific inhibitor.

This last protocol is useful to identify inhibitory compounds and slowly transported compounds that do not change the ATPase activity [ ]. Although ATPase assays allow the screening for ABC transporter substrates that can potentially act as competitive inhibitors, such as verapamil in what concerns to P-gp, resulting in the stimulation of the transporter ATPase activity, the screening for ABC transporter activators may be a tricky issue. Indeed, since this concept of a compound that immediately activates these proteins, inducing a conformational change that increases the transport of a substrate bound to another binding site, is relatively new [ 3 ], it remains unclear whether these activators are, or not, necessarily ABC transporters substrates.

Therefore, two different approaches could be undertaken: the evaluation of the effect of the potential activator, alone, in the transporters ATPase activity; and the evaluation of the potential activator effect on a stimulated ATPase activity, i. Thereby, a P-gp activator should increase the verapamil-mediated stimulation of its ATPase activity by increasing P-gp-mediated verapamil transport ; while a P-gp inhibitor should make the opposite effect.

Furthermore, when evaluating the effect of the potential activator alone, it will be possible to evaluate if such compound is also a substrate, thus providing more information on the activation mechanism, namely if a co-transport of both activator and substrate might be occurring [ 3 ]. Although ATPase assays are simple, reproducible and used to detect transporter-compound interactions, these techniques are not always suitable for distinguishing among potential ABC transporter substrates and modulators, due to the presence of high intra- and inter-assay variability [ 1 , 32 , ].

The ATPase assays may give false negative results for compounds, when they are studied in only one concentration, due to their low affinity and solubility. Compounds can stimulate and inhibit ABC transporters at either low or high concentrations [ 1 ]. These assays can be applied in the: a quantification of the compound transported across the cell membrane; b kinetic analysis of the transported compound, including determination of the affinity constant K m and maximal velocity V max ; c study of the test compound interaction with a known substrate of the efflux transporter, to obtain the inhibitory constant K i and the half maximal inhibitory concentration IC 50 for inhibitors; and d study of the transport driving force or the requirement for the presence of co-transported molecules [ 1 ].

Therefore, these assays, although not allowing the identification of ABC transporters inducers since the increased de novo synthesis of the proteins is needed , are useful for the identification of activators, as well as substrates and inhibitors. The membranes used in these assays are prepared under suitable conditions and are from different sources, such as baculovirus-infected insect ovary cells, transfected or selected mammalian cell lines from the brush border membrane of intestine, kidney and choroids plexus; hepatic sinusoidal and canalicular membranes; and luminal and abluminal membranes of the brain , transfected yeast cells and artificial membrane vesicles [ 1 , , ].

These contain inside-out-oriented vesicles, with both ATP- and ligand-binding sites facing the buffer outside. A rapid filtration method using glass fiber filters or nitrocellulose membranes is used to separate the vesicles from the incubation solution [ 1 , ].

Alternatively, the compounds can be radiolabeled, fluorescent or have a fluorescent tag, being quantified the radioactivity or fluorescence retained on the filter [ ]. Differences detected at level of the substrate uptake, in the presence or absence of ATP, can be attributed to transport mediated by efflux or uptake transporters, respectively [ 1 , ].

The membrane vesicular transport assays are advantageous techniques to measure the disposition of substrates across cell membranes, including compounds with low membrane permeability and low non-specific binding [ 32 ]. The membrane vesicles expressing efflux transporters are commercially available, making it possible for the routine use of these techniques [ 1 ].

However, there are also some disadvantages associated to these assays. Namely, false-negative results can be obtained in the study of compounds with medium-to-high passive permeability or highly lipophilic, due to their high nonspecific binding to the lipid membranes. Additionally, the preparation and purification protocols of the membrane vesicles are time consuming and technically complicated [ 1 , 32 , , ].

The first mentioned technique has been used in the study of the ABC transporters function, including evaluation of the binding sites, binding affinities and structural details of the substrates and modulators [ 1 , 32 ]. Membranes expressing ABC transporters or isolated proteins are incubated with labeled photoaffinity compounds [ 1 ].

The ABC transporters radioactively labeled are solubilized and separated by gel electrophoresis. The protein labeling drug-binding is visualized and quantitated by autoradiography. Another type of photolabeling assays, mentioned above and first documented for P-gp, corresponds to the use of a radioactively labeled ATP analog, 8-azido-ATP [ ]. Labeled 8-azido-ATP binding, under non hydrolytic conditions, can be followed by UV-irradiation, size fractionation and autoradiography.

Under hydrolytic conditions, ATP hydrolysis takes place and the binding and release of an ATP analog is too rapid to be followed. For this reason, a phosphate-mimicking transport inhibitor e. The rate of the formation of this transition state can be assessed stopping the catalytic reaction by excess ATP and UV cross-linking. This formation is proportional to the rate of transport.

When the substrates are efficiently transported, there is an increase in the formation of the trapped nucleotide [ 32 ]. Since both direct photoaffinity labeling and nucleotide trapping experiments are complicated techniques associated with complex protocols and are not routinely applied in the pharmaceutical industry, these techniques are important tools for studying details of the molecular mechanism.

Direct photolabeling is generally not adequate for distinguishing between substrates and inhibitors [ 1 , 32 ]. On the other hand, ABC transporters form low-affinity interactions with a wide variety of hydrophobic compounds. The interaction sites and intensities may directly depend on the test drug and actual conformation of the transporter [ 32 ].

Cell-based assays may provide more clear information about the interaction between compounds and ABC transporters, applied in the evaluation of the following kinetic parameters: K m and V max for substrates, and K i and IC 50 for inhibitors Table 2. The cytotoxicity assay is, by far, the most widely applied cell-based approach for investigating ABC transporters function.

This test compound can be an inhibitor, activator or inducer of the ABC carrier under study. These assays allow a high-throughput screening of compounds due to reduced time consumption and cost, when compared, for example, with the in vivo assays, which have a high cost, are time-consuming, and have ethical restrictions. However, cell-based assays are more labor and time consuming than the membrane-based assays.

It is important to consider the following features: a particular cell line can express multiple transporters, although there are modified cell lines expressing one specific transporter; the culture conditions and number of cell passages may change the transporters expression levels; and the cells need to be maintained under culture conditions prior to use Table 2 [ 1 ].

Tissue localization and changes in gene expression after cells stimulation can be monitored by Northern blot analysis, dot-blot analysis, competitive PCR, RNase protection assays or in situ hybridization. Although these methods require large RNA amounts and starting material, not allowing a rapid analysis of multiple genes and large sample numbers, they are widely accepted and reliable and can be applied to the evaluation of ABC transporters gene expression [ ].

Real-time RT-PCR is commonly used in molecular biology for mRNA analysis, including detection and quantitation, by the use of fluorescent probes [ ]. This technique is sensitive enough to enable precise and reproducible mRNA quantitation both rare and abundant from a single cell [ ].

The evaluation of the gene expression is based on cycle threshold Ct values rather than end-point detection [ ]. There are two main classes of chemistry compounds, i. The PCR product accumulation corresponds to an increase in the fluorescence intensity. Although requiring extensive optimization, this is the most economical and the easiest method. The need of optimization is related to the SYBR Green ability for binding to any double-stranded DNA during reaction, including primer-dimers and other non-specific reaction products, resulting in an overestimation of the target gene concentration.

On the other hand, there are hydrolysis and hybridization FRET-based probes [ ]. The proximity of the dyes, during unhybridized state, does not completely quench the fluorescence, being possible to observe a background fluorescence.

During the PCR reaction, the probe anneals specifically between the primers forward and reverse to the desired target region of the gene. Then, the polymerase carries out the extension of the primer and replicates the template. This process is repeated in every cycle and fluorescence increases in proportion to the amount of probe cleavage. TaqMan probe does not need extensive optimization.

The second FRET-based technique is based on two probes, one labeled with a fluorescent donor dye and other labeled with an acceptor dye. Once in close vicinity 3 to 5 base pairs , the donor dye emits energy that excites the acceptor dye. Consequently, there is emission of fluorescence at a different wavelength, which is monitored with a specific equipment. After each cycle, additional hybridization probes anneal, increasing the fluorescence intensity, which is measured during the exponential phase of the PCR reaction.

The fluorescence intensity is proportional to the amount of input target DNA [ ]. Real-time PCR allows sample processing in a multi-well plate, automatically and with high-throughput. Glyceraldehyde 3-phosphate dehydrogenase GAPDH is used as a reference gene for expression analysis in human tissues, but alternative reference genes can be used for other cell systems [ ].

Langmann and colleagues developed a rapid, accurate and highly sensitive real-time PCR method for detection and quantification of all ABC transporters using a TaqMan probe. The method allows a rapid and complete analysis of all ABC transporters in obtained RNA samples, from twenty different human tissues. As a result, authors identified tissues involved in secretory adrenal gland , metabolic liver and kidney , barrier lung, trachea and small intestine and reproductive and tropic placenta, uterus, prostate and testis functions with high transcriptional activity for ABC transporters [ ].

Flow cytometry is a rapid and specific technique that provides complete cellular analysis, being used as a tool for understanding the regulation and interaction of cell systems, mainly based in the use of fluorescent antibodies. Light emitted from these antibodies allow the identification of a wide array of cell surface and even cytoplasmic antigens [ ]. Flow cytometry provides quantitative measurements of cells and other particles at a high speed, being suitable for the study of single mammalian cells in suspension by measuring their optical and fluorescence characteristics [ ].

Some physical properties, such as cell size and internal complexity, can be measured by flow cytometry [ ]. Additionally, antibodies conjugated with fluorescent dyes can bind to specific proteins on cell membranes intact cells or inside cells permeabilized cells.

Also, the use of fluorescent substrates, such as rhodamine , may be useful for the evaluation of membrane transporters activity. The labeled cells are passed by a light source and the fluorescent molecules are excited to a state of higher energy. When returning to their resting states, the fluorochromes emit light energy at higher wavelengths. The emitted fluorescence is collected using a flow cytometer, spectrally filtered and detected using photomultiplier tubes.

It is possible to simultaneously measure several cell properties, using multiple fluorochromes, each one emitting light at different wavelengths, although being excited with similar wavelengths.

Propidium iodide, phycoerythrin and fluorescein are commonly used dyes [ ]. Flow cytometry assays can be applied to the study of ABC transporters, allowing the characterization of the interactions between drugs and ABC carriers, and usually involve the use of fluorescent transporter substrates, such as rhodamine and calcein acetoxymethyl ester calcein-AM for P-gp [ ].

Vilas-Boas and colleagues evaluated the influence of aging in P-gp expression and activity, in human lymphocytes isolated from whole blood samples of 65 healthy caucasian male donors, comparing two different methodologies. P-gp expression was analyzed using an anti-P-gp monoclonal antibody UIC2 , in the presence and absence of vinblastine. P-gp activity was studied by measuring the efflux rate of the P-gp fluorescent substrate, rhodamine , and by using the UIC2 shift assay.

The results obtained in both studies were compared and showed a significant age-dependent increase in mean P-gp expression and no differences were found in P-gp activity. Moreover, the UIC2 shift assay proved to be more selective than the rhodamine efflux assay, in the analysis of P-gp activity [ ].

The researchers also used flow cytometry to study, in RBE4 cells, the putative modulatory effect of rifampicin and three rifampicin derivatives over P-gp function, using rhodamine as a fluorescent substrate [ 20 ]. Recently, Silva and co-authors have been using a flow cytometry-based approach to study the ability of different compounds, such as doxorubicin, colchicine, X and TX, to modulate P-gp expression and activity, using the Caco-2 cell model.

In these studies, the UIC2 monoclonal antibody conjugated with fluorescein isothiocyanate was used to study P-gp expression, and rhodamine was used to evaluate P-gp activity [ 16 , 21 , 22 , ]. Despite flow cytometry usefulness in expression and functional studies of ABC transporters in live cells, most dyes used as indicators have limited applicability as they do not simultaneously detect all types of ABC carriers [ ].

Beyond flow cytometry, other accumulation and efflux assays are suitable for the screening of compounds that interfere with efflux transporters.

These assays can be performed using cell suspensions, cell monolayers or membrane vesicle preparations [ ]. Upon loading of the cells with lipophilic dye s , with diffusion capacity across cell membranes, the resulting fluorescence intensity of the cell s will depend upon the activity of the ABC transporters [ ]. The accumulation of the fluorescent substrates can be measured in the presence and absence of specific inhibitors or activators, in order to understand the effect of the transporters activity [ ].

The intracellular accumulation of the dye is inversely proportional to the ABC carrier activity and can be measured by fluorescence spectrophotometry [ ].

Therefore, an increased intracellular accumulation of a given substrate higher intracellular fluorescence can be observed in the presence of an inhibitor, while the opposite decreased intracellular accumulation is characteristic of an ABC transporter inducer and activator. However, the discrimination between an inducer and an activator is only related with the time of contact of such compounds with the cells.

On the other hand, the effect of an inducer in the pump activity requires an increased incubation period, since the de novo synthesis of the protein is needed. Moreover, to note that although an increased expression could be observed after incubation with an inducer, it will not necessarily be translated in an increased activity of a given transporter [ 3 , 16 ].

The efflux studies comprise the pre-load of the cells with the dye of interest. The amount of dye in the extracellular environment is measured under various conditions known to influence the transporter activity. In the presence of an inhibitor of the efflux transporter, the amount of dye expelled from the cells will be smaller than that observed for control cells. The change in the intracellular accumulation of the fluorescent compounds when co-administered with inhibitors, inducers or activators, is considered to be mainly due to their effect on the efflux pumps located in the cellular membrane, such as P-gp.

It is important to notice that the analysis of the inhibition of P-gp may depend on the nature of the used substrate, since at least two binding sites, H and R, are considered to exist and inhibitors may differently interact with them. Consequently, inhibition assays may be performed with various P-gp substrates [ 38 , , ].

The analysis of the efflux transporters activity may be based on the evaluation of the dye accumulation, efflux or both. For example, one protocol routinely used for the evaluation of the effect of inducers or activators consists in two phases: i the accumulation phase, in the presence of the dye, and in which the ABC transporter activity is blocked with an inhibitor of energy production e.

The first phase results in maximum substrate accumulation inside the cells. The second phase consists in restoring the normal function of the transporter, which is now able to transport the fluorescent substrate out of the cells.

By analyzing the cells both after the inhibited accumulation phase and after the efflux phase, is possible to infer the amount of substrate transported by the pump. For transfected cells or drug-induced cells that over-express a particular drug efflux transporter, accumulation or efflux studies can be compared to the wild-type or parental cell line that does not have as high a level of drug efflux transporter expression [ ].

It is important the selection of specific inhibitors and specific fluorescent substrates. In P-gp activity studies, rhodamine is frequently used as a fluorescent substrate, and cyclosporine A or PSC as P-gp inhibitors [ 16 , 19 , 20 , , , , , ]. Western blotting or protein blotting or immunoblotting is an important technique used for the immunodetection of proteins post-electrophoresis, particularly those at low abundance [ ].

Western blotting analysis is commonly performed in ABC proteins expression studies [ 22 , , ]. Western blotting is characterized by the following specific advantages: a wet membranes are flexible and of easy handling; b the proteins immobilized on the membrane are easily accessible to different ligands; c only a small amount of reagents is required for transfer analysis; d it is possible to obtain multiple replicas of a gel; e it is possible to storage transferred patterns, prior to use; f the same protein transfer can be used in multiple successive analysis [ ].

Transport assays are the most direct tool for the evaluation of transporter function and permeability of the test compound [ 1 ]. When cells reach confluency, they differentiate and become ready to be used in permeability studies. The two compartments are designated as apical and basolateral, denoting the membrane orientation of polarized cell layers.

These two chambers are connected only through the cells monolayer and their semipermeable support. The transport differences between the basolateral-to-apical and the apical-to-basolateral compartments are easily measured. The calculated ratio is referred to as efflux ratio and for results greater than 2 the test compound is considered substrate of the active efflux transporters [ 1 , 32 , , , , , ]. The experimental protocol is initiated by the addition of a solution containing the test compound to either the apical upper chamber or basolateral lower chamber compartment, for the study of the apical-to-basolateral A-to-B or basolateral-to-apical B-to-A transport, respectively [ 1 , , , , ].

On the other side is added a buffer. At desired time points, aliquots of added solution are removed from the lower chamber for studies of A-to-B transport or from the upper chamber for studies of B-to-A transport. In the presence of efflux transporters expression on the apical membrane, P app, A-to-B is smaller than P app, B-to-A. These results will be contradicted if the transporter is localized on the basolateral cell membrane [ 1 , ]. Passively diffused compounds present P app values that are independent on its concentration.

The flux rate is linearly correlated with the concentration of the compound. The flux rate of actively transported compounds is saturable with increasing of its concentration. The determination of kinetic parameters, such as K m and V max , is possible [ 1 ]. Primary cultured cells, such as primary cultured brain endothelial cells, conjunctiva and alveola epithelial cells are cell types used in these studies [ 1 , ].

The cell type suitable for these assays must be polarized [ 1 ]. During transport assays several points should be taken into consideration, such as the selected cell line, pore size, pore density and filter material [ 32 ]. Many ABC carriers are constitutively expressed at the apical membrane of epithelial cells of different organs, including those that function as body barriers, such as the liver, brain, kidney and intestinal tract [ , ].

In the small intestine and colon, P-gp is one of the most important efflux proteins and may play a major contribution for several orally administered drugs bioavailability [ ]. Ex vivo methodologies are an experimental approach where an organ or tissue is removed from the animal and placed in chambers where physiological conditions found in the living body are mimicked, namely the access to nutrients and oxygen, allowing the viability of the organ or tissue during the experimentation time.

ABC function can be accurately evaluated by using ex vivo approaches Table 2. Serosal to mucosal transport of the fluorescent substrate, in the presence or absence of the putative ABC carrier modulator, is evaluated in each intestinal sac by determining the substrate concentration, by spectrofluorometry, in samples of mucosal medium, over time. Rhodamine is a dye usually used as P-gp substrate [ , , ].

Given the relevance of the ABC transporters in the toxicokinetics and pharmacokinetics, namely in the absorption, distribution BBB permeation and excretion processes, as well as their involvement in diverse pathophysiological conditions, the search for new modulators of these carrier proteins is of particular importance in both pharmacological and toxicological fields.

Thereby, computational models are very valuable tools, allowing the identification of new putative ligands and, at the same time, being a relevant alternative to excessive animal testing and a preliminary approach to the in vitro and ex vivo experiments, very often expensive, laborious and time-consuming. In silico models provide rapid and inexpensive screening platforms, and can include the development of quantitative structure-activity relationship QSAR models, as well as docking studies for ligand-carrier interactions prediction, and also the development of pharmacophores for ABC transporters inducers and activators [ 3 ].

Docking studies have long been used to predict the interaction of compounds with their potential targets proteins, nucleic acids, carbohydrates and lipids. Several docking models were developed to map potential modulators of P-gp, BCRP and MRP1, thus allowing to evaluate the potential binding modes of such compounds in a given transporter [ 20 , 21 , 22 , , , , , , ].

Indeed, newly synthetized thio xanthonic derivatives demonstrated the ability to immediately increase P-gp activity after a short incubation period, an effect compatible with P-gp activation, resulting in a significant decrease in the toxicity of a P-gp substrate, PQ. The possibility of a co-transport mechanism between TXs and PQ was further supported by docking studies using a validated P-gp model [ 22 ].

However, although numerous computational models, based on QSAR analysis, pharmacophore modelling and molecular docking techniques, have been developed to predict ABC transporters substrates and inhibitors, particularly in what concerns to P-gp, the search for new inducers and activators has been mainly performed by random screening [ 21 ].

Noteworthy, and in an attempt to address this gap, pharmacophores for P-gp inducers and activators were recently developed, which can be of utmost importance, in the future, in predicting new ligands [ 22 , ]. In fact, based on the in vitro P-gp activation ability of newly synthetized thioxanthonic derivatives [ 22 ] and on a set of known P-gp activators described in the literature, the authors developed and validated common feature pharmacophore models for P-gp activation. The best ranked pharmacophore reported was composed of three features one hydrophobic feature, one aromatic ring, and one hydrogen bond acceptor group and can be a very useful tool to efficiently and rapidly predict new ligands with the ability to activate P-gp.

Additionally, pharmacophore construction was also performed for P-gp inducers. Briefly, the pharmacophores were validated using known P-gp inducers and can be used to map new compounds, as it was the case of newly synthetized TXs, for which there was previous indication from data of in vitro assays about their potential to activate and induce P-gp. However, since many signalling transduction pathways can be considered in regulating the expression of a given transporter, fact that is particular evident for ABC transporters, and given the structural diversity of the compounds, finding a pharmacophore for P-gp inducers can be a challenging task.

Noteworthy, by using such pharmacophores for P-gp inducers and activators, a perfect match between in silico and in vitro studies was observed [ 21 , 22 ], thus further reinforcing the idea that the use of such in silico strategies can help to predict the P-gp modulatory effects of new drugs that can be initially screened through these newly developed pharmacophores. Also, in vitro data on the ability of newly synthetized dihydroxylated xanthones to activate P-gp and protect Caco-2 cells against the cytotoxicity induced by a P-gp substrate, PQ, triggered the development of a 2D QSAR model, which demonstrated that the maximal partial charge for oxygen atoms is related with the P-gp activation ability of such compounds [ 21 ].

Furthermore, a perfect match was again observed, with both the docking studies and the QSAR model being in accordance with the reported in vitro data [ 21 ]. Taken together, the in silico models disclose new possibilities in drug discovery and can be a valuable and complementary tool in the prediction of new ligands, allowing a more rational use of in vitro, ex vivo and in vivo assays.

In vitro and in vivo studies with inducers and activators of the ABC transporters have shown that the use of these compounds may be an effective antidotal pathway against xenobiotic-induced toxicity. The action mechanisms of both are not clear. Therefore, it is important to conduct more research involving putative inducers and activators of the ABC transporters, in order to understand: 1 their mechanism of action; 2 their specificity and 3 their toxicity in tissues with toxicological relevance.

During the assessment of new modulators of the ABC transporters it is important to use adequate in vitro assays, high throughput and low-cost alternatives to excessive animal testing, evaluating their main effects on the expression and activity of the ABC transporters.

Using only one technique or one concentration of the test compound could lead to false results. To all financing sources the authors are greatly indebted. Published online Apr 8. Author information Article notes Copyright and License information Disclaimer. Received Jan 30; Accepted Mar Abstract Adenosine triphosphate ATP -binding cassette ABC transporters are highly expressed in tumor cells, as well as in organs involved in absorption and secretion processes, mediating the ATP-dependent efflux of compounds, both endogenous substances and xenobiotics, including drugs.

Keywords: inducers, activators, ATP-binding cassette transporters, cellular models, membrane assays, cell-based assays, in vitro assays, P-glycoprotein, multidrug resistance-associated protein 1, breast cancer resistance protein. Introduction The bioavailability of a wide variety of compounds that cannot permeate the membrane by passive diffusion e.

Open in a separate window. Figure 1. Figure 2. Figure 3. Overview of Modulators of the ABC Transporters: Activators and Inducers Compounds that interact with ABC transporters can act as substrates being moved across membranes via the transporter , inhibitors impairing the transporter-mediated efflux of other compounds , inducers enhancing the transporter expression levels or activators enhancing the transporter activity , but one compound can also have overlapping modes of action [ 9 ].

Study Models for ABC Transporters According to in vivo and in vitro results obtained, inducers and activators of the ABC transporters can represent an important protection tool against xenobiotic-induced toxicity and an antidotal pathway to be explored [ 3 , 15 , 16 , 19 , 20 , 21 , 22 ].

Cellular Models 3. Figure 4. Cardiovascular System The cardiac endothelial cells are characterized by expression of uptake and efflux transporters, which control the transport of a wide range of compounds, including drugs and toxins, into and out of the heart, respectively [ ].

Liver The liver is an important tissue involved in the synthesis and secretion of bile acids, metabolism and transport of cholesterol, as well as in the metabolism and efflux of endogenous and exogenous substances [ , ]. Figure 5. Kidney The kidney is responsible for maintaining fluid and electrolyte homeostasis, maintaining the essential nutrients and eliminating both potentially toxic compounds and metabolic waste products from the body.

Figure 6. Intestine The intestine, in addition to the liver, is an important tissue that regulates the extent of absorption of orally administered drugs [ , ]. Figure 7. In Vitro Assays Appropriate in vitro assays for transport studies can be divided in two major groups: membrane-based assays and cell-based assays. Membrane-Based Assays The study of the function of the ABC efflux transporters and the identification of their substrates and inhibitors has been performed by using membranes, prepared from cells expressing ABC transporters.

Table 2 Main advantages versus disadvantages of the described in vitro and ex vivo assays adapted from [ 1 ]. Advantages Disadvantages In vitro assays Cell-based assays Allows to screen for P-gp inducers, activators, inhibitors and substrates. Cell-based transport assays are a classic assay to determine substrates or inhibitors and, more recently, activators.

However, to note that an increased expression of a given transporter may not necessarily result in an increase in its transport activity. May provide more information on the interaction between xenobiotics and transporters, due to the intact cell structure. Can be employed to assess kinetic parameters, such as the half maximal inhibitory concentration IC 50 for inhibitors.

Can be easily adapted to a high throughput mode with automation and cell culture in multi-well plates. Additional information may be obtained, such as information on the xenobiotic permeability and transporter localization in cells. It is more difficult to characterize the xenobiotic effects on one specific efflux transporter, given the expression of multiple transporters in a particular cell line including cell lines that have been engineered to express a given transporter.

The transporters expression levels can change according to the cell culture conditions and number of passages in culture. Cell culture media can be expensive, according to the specific supplementation requirements of a given cell line. These assays are more laborious and time consuming than the ATPase assay and membrane vesicular transport studies.

In the transport assays, polarized epithelium cells with well-defined tight junctions are needed. In the particular case of Caco-2 cells, the development of a proper polarized cell monolayer requires a long-time culture and the cells have multiple efflux transporters expressed.

False negative results can be obtained in the transport assays for xenobiotic with high passive diffusion. ATPase assays can be used as a high throughput screening tool to identify ligands for ABC transporters—a positive result either stimulation or inhibition indicates that the test xenobiotic is a ligand for a specific efflux pump.

The membrane vesicular transport assays, contrarily to the ATPase assays, are functional assays and, thus, can be used to distinguish a transporter inhibitor from a substrate. Do not allow to screen for P-gp inducers, since de novo synthesis of these proteins cannot be detected.

ATPase assays are not functional assays and cannot be used to distinguish between substrates and inhibitors. In the ATPase assays, the xenobiotics effects should be evaluated at several concentrations to avoid false negative results, since the stimulation or inhibition can occur at either low or high concentrations. False negative results may also be observed for low affinity ligands, since the concentration tested can be limited by the xenobiotic solubility. Membrane-based assays aiming the evaluation of membrane vesicular transport mediated by a given transporter may also give false negative results for lipophilic xenobiotics, which have high nonspecific binding and high passive diffusion.

A more accurate determination of the transporter functions in absorption, biliary elimination, renal excretion and brain penetration can be obtained by using isolated perfused intestine, liver, kidney or brain. The use of a perfused organ assay allows a much simpler understanding of the role of a transporter in a given organ, when compared with the use of the whole animal, since the concentration of the drug in the target organ can be controlled and the effect from other organs can be avoided.

It is more difficult to characterize the xenobiotic effects on one specific efflux transporter. The organ integrity and enzyme activity may become fragile and compromised during long-term perfusions. Important to evaluate the potential interspecies differences in transporters when extrapolating data from animal to humans.

ATPase Assays The determination of the ABC transporters ATPase activity can be performed either in isolated membranes containing the desired transporter insect or mammalian cell membranes , or in reconstituted ABC protein preparations [ 32 ]. Cell-Based Assays Cell-based assays may provide more clear information about the interaction between compounds and ABC transporters, applied in the evaluation of the following kinetic parameters: K m and V max for substrates, and K i and IC 50 for inhibitors Table 2.

ABC Transporter Gene Expression Tissue localization and changes in gene expression after cells stimulation can be monitored by Northern blot analysis, dot-blot analysis, competitive PCR, RNase protection assays or in situ hybridization. Flow Cytometry Assays Flow cytometry is a rapid and specific technique that provides complete cellular analysis, being used as a tool for understanding the regulation and interaction of cell systems, mainly based in the use of fluorescent antibodies.

Accumulation and Efflux Assays Beyond flow cytometry, other accumulation and efflux assays are suitable for the screening of compounds that interfere with efflux transporters. Western Blotting Western blotting or protein blotting or immunoblotting is an important technique used for the immunodetection of proteins post-electrophoresis, particularly those at low abundance [ ].

Transport Assays Across Polarized Cell Monolayers Transport assays are the most direct tool for the evaluation of transporter function and permeability of the test compound [ 1 ]. Ex Vivo Assays Many ABC carriers are constitutively expressed at the apical membrane of epithelial cells of different organs, including those that function as body barriers, such as the liver, brain, kidney and intestinal tract [ , ]. In Silico Studies for ABC Transporters Inducers and Activators Given the relevance of the ABC transporters in the toxicokinetics and pharmacokinetics, namely in the absorption, distribution BBB permeation and excretion processes, as well as their involvement in diverse pathophysiological conditions, the search for new modulators of these carrier proteins is of particular importance in both pharmacological and toxicological fields.

Conclusions In vitro and in vivo studies with inducers and activators of the ABC transporters have shown that the use of these compounds may be an effective antidotal pathway against xenobiotic-induced toxicity.

Conflicts of Interest The authors declare no conflicts of interest. References 1. Xia C. Evaluation of drug-transporter interactions using in vitro and in vivo models. Drug Metab. DeGorter M. Drug transporters in drug efficacy and toxicity. Silva R. Modulation of P-glycoprotein efflux pump: Induction and activation as a therapeutic strategy.

Hesselson S. Genetic variation in the proximal promoter of ABC and SLC superfamilies: Liver and kidney specific expression and promoter activity predict variation. Sharom F. ABC multidrug transporters: Structure, function and role in chemoresistance.

Huls M. The role of ATP binding cassette transporters in tissue defense and organ regeneration. Leslie E. Cheepala S. Cyclic nucleotide compartmentalization: Contributions of phosphodiesterases and ATP-binding cassette transporters. Wessler J. The P-glycoprotein transport system and cardiovascular drugs. Estudante M. Close suggestions Search Search. User Settings. Skip carousel. Carousel Previous. Carousel Next.

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Several in vitro and in vivo studies have been performed to evaluate both inducers and activators applicability in the detoxification of potentially harmful compounds. Previous studies performed by our group using known P-gp inducers confirmed that P-gp induction is an effective antidotal pathway against paraquat PQ -induced toxicity [ 15 , 16 , 19 , 21 , 22 , 59 ].

It was also observed a significant decrease in lung damage, with reduction of lipid peroxidation and carbonyl group levels, as well as normalization of myeloperoxidase activity, which resulted in a significant increase in the animals survival rate. Protection provided by dexamethasone was explained by P-gp overexpression in the cytoplasmic membrane of pneumocytes as dexamethasone induced de novo synthesis of P-gp in these cells , causing an increased PQ elimination from lungs [ 59 ].

Two in vitro studies performed by Silva and colleagues [ 16 , 19 ] showed that P-gp induction by doxorubicin in human epithelial intestinal cells Caco-2 cell line resulted in a significant reduction of the PQ-induced cytotoxicity. Using the same in vitro model, hypericin, one of the major active compounds of St. Additionally, Arias and co-authors recently reported the effect of pharmacological concentrations of both ethynylestradiol EE, 0.

Furthermore, in vitro studies performed by our research group showed that newly synthesized thio xanthonic derivatives prevent PQ cytotoxicity. Five thioxanthones TX1—5 and five dihydroxylated xanthones X1—5 were shown to be, in the Caco-2 cell model, P-gp inducers and activators [ 3 , 21 , 22 ]. Given the promising results obtained with these new compounds in the referred in vitro studies, ex vivo and in vivo experiments are being carried out in order to evaluate TX toxicity, toxicokinetics and antidotal ability in a living body.

Moreover, other studies from our group have identified novel inducers and activators of P-gp with therapeutic potential. Vilas-Boas et al.

According to the results obtained, rifampicin causes a significant increase in P-gp expression at 72 h of exposure. The reduced derivative RedRif leads to an increase in both P-gp expression and activity at 24 h and 72 h. Recently, new perspectives for clinical application of ABC inducers were presented. Very importantly, Haslam and colleagues [ 63 , 64 ] suggested a therapeutic protection approach for chemotherapy-induced alopecia based on P-gp upregulation in hair follicle, which may reduce or prevent permanent hair loss following chemotherapy.

It is proposed that ABC transporters upregulation and activation may protect melanocytes precursors in the hair follicle bulge, reducing, in consequence, the graying impact of chemotherapy [ 63 ]. In addition to the ability for carrying exogenous substrates, ABC transporters can also carry endogenous substances, such as cholesterol. This can play a role in some cholesterol-mediated pathologies in which lipids are accumulated inside cells [ 65 ]. Furthermore, several lines of research have been pointing the involvement of ABC transporters, mainly P-gp, in distinct neurodegenerative diseases, such as Alzheimer, Parkinson and epilepsy [ 66 , 67 ].

Because of the involvement of ABC transporters in both physiological and pathophysiological processes, there is much interest in modulating their efflux function.

It has been shown that AB accumulates during aging due to a disturbance in its clearance from the brain, rather than the increase in its production. Moreover, despite distinct clearance pathways have been identified, the AB active efflux across the BBB seems to be the most important one [ 73 ].

P-gp, as a major efflux pump at the BBB, seems to be involved in the AB brain-to-blood transport, which could constitute an important mechanism in the pathogenesis and therapy of AD [ 74 , 75 ]. Thereby, it is proposed that the reduction in AB elimination from the brain into the blood can contribute to AD pathogenesis. Also, P-gp activity is decreased in brain regions important for memory formation in AD patients and, in a transgenic mouse model of the disease, P-gp protein level is reduced, and restoring the expression of P-gp decreases AB accumulation [ 68 ].

Cirrito and colleagues observed an increase in AB levels in the brain of P-gp-knockout mice which, according to the authors, was due to the absence of P-gp-mediated efflux when compared with wild-type mice [ 77 ].

Other researchers showed that both Mdr1a -knockout mice and a strain resulted from crossing Mdr1a -knockout mice with Tg amyloid precursor protein APP transgenic mice a line that routinely accumulates AB in the brain accumulate AB in a greater extent than their respective controls [ 68 ].

Recently, it was reported a decrease in P-gp function in brains from patients with AD, as shown by positron emission tomography [ 78 ].

Taken together these data, the upregulation of P-gp at BBB may constitute a valid therapeutic approach for AD patients, as proposed by Abuznait and colleagues [ 79 ]. Xiong and co-authors showed an increase in the expression pattern of BCRP in the cerebral vessels of both AD and cerebral amyloid angiopathy patients, as well as in transgenic AD mice models [ 80 ].

By using optical imaging in vivo, the authors also showed, after intravenous administration of labeled AB peptides, a significantly greater accumulation of those peptides in the brains of Abcg2 -knockout mice comparing to the wild-type animals.

This observation led the authors to purpose the upregulation of BCRP as a biomarker of amyloid angiopathy in AD patients [ 80 ]. The involvement of the mentioned ABC proteins in the above-mentioned pathology processes raises the hypothesis that these transporters could constitute potential drug targets for the treatment of distinct neurodegenerative disturbances. It has been already observed, in a clinical trial, that rifampin, a drug known to induce P-gp expression, can improve cognitive ability of patients with mild to moderate AD [ 83 ].

One recent example is given by Manda and colleagues, who identified a marine-derived alkaloid as a potent P-gp inducer, establishing its structure-activity relationship [ 84 ]. Particularly, as fascaplysin induces P-gp, inhibits acetylcholinesterase and reveals a good safety profile, the authors consider it a promising anti-Alzheimer agent [ 84 ]. Other natural and synthetic compounds have been tested for P-gp induction purposes, since P-gp induction is one of the recently targeted strategy to increase AB clearance from Alzheimer brains.

Phenyl benzenesulfonamides have shown ability to induce P-gp in human adenocarcinoma LS cells, with an excellent therapeutic window [ 85 ].

A new compound, MC80, was shown to act as a P-gp inducer both in vitro, ex vivo and in vivo [ 86 ]. Similarly, Contino and colleagues, aiming at developing compounds able to up-regulate P-gp expression in order to reach a detoxification effect of the central nervous system CNS caused by AB accumulation, used an ex vivo model and identified a new benzopyrane derivative as a P-gp inducing agent [ 87 ].

Particularly, several studies were carried out in order to evaluate the putative implications of polymorphisms in ABC genes in PD [ 90 , 91 , 92 ]. From these, P-gp have been receiving particular attention and a number of single-nucleotide polymorphisms in MDR1 , the gene encoding P-gp, have been studied [ 93 ].

However, in many cases, the reported effects of these polymorphisms have been conflicting. However, significantly greater expressions of P-gp and BCRP were detected in PD rat cerebral microvessel endothelial cells when comparing to the physiological model, and the efflux of a novel anti-PD candidate agent was exclusively attributed to P-gp [ 94 ].

By contrast, it is described an overexpression of P-gp in epileptic brain tissues, fact that would be responsible for the greater efflux of anticonvulsivant drugs, which contributes to antiseizure drug resistance [ 95 , 96 ]. Indeed, studies show that BBB is altered in animal models of epilepsy and in epileptic patients and P-gp is overexpressed in both in vivo and ex vivo conditions [ 96 ].

Yu and colleagues, using a rat model of epilepsy, developed a nanoparticle-infrared-Pepstatin A-based methodology to detect, by both magnetic resonance and optical imaging, P-gp in rat brains. The authors suggest this methodology as an useful tool for both the understanding of the mechanisms underlying neurological disorders and the use of P-gp-targeted therapies [ 97 ].

According to in vivo and in vitro results obtained, inducers and activators of the ABC transporters can represent an important protection tool against xenobiotic-induced toxicity and an antidotal pathway to be explored [ 3 , 15 , 16 , 19 , 20 , 21 , 22 ]. Available cellular models and in vitro assays for the initial screening and selection of safe and specific inducers and activators of P-gp, MRP1 and BCRP have been proposed as high throughput and low-cost alternatives to excessive animal testing.

In the following sections in vitro study models are presented. The BBB is a physical and enzymatic barrier that separates the CNS from the systemic vascular environment, shielding the CNS from exposure to circulating potentially harmful compounds.

BBB is composed of a monolayer of brain capillary endothelial cells characterized by polarized nature apical and basal membranes, where transporters, including efflux systems such as P-gp and MRPs, are asymmetrically distributed , the paucity of both pinocytotic vesicles and fenestrations and the presence of tight junctions, metabolizing enzymes including cytochrome P hemoproteins and UDP-glucuronosyltransferases and SLC and ABC transporters [ 7 , 50 , 99 , , , ].

Tight junctions form a continuous impermeable cellular barrier, preventing the entrance of large and hydrophilic compounds into the brain. Small and lipophilic molecules gain access to the brain by passive diffusion or active transport [ 7 , 50 , 99 , , , ]. Several studies were performed in order to establish the level of expression of ABC transporters at the BBB of different species, including humans [ , , ].

Differences between species were found. At the cellular level, most of the published data demonstrate that P-gp, MRP1, 2, 4, 5 and 6 and BCRP are highly expressed in the apical membrane of the brain capillary endothelial cells [ 7 , 51 , 52 , 99 , , , , ]. ABC efflux transporters at the BBB minimize or avoid neurotoxic adverse effects of drugs that otherwise would penetrate into the brain. However, ABC efflux transporters may also limit the central distribution of drugs that are beneficial to treat CNS diseases [ 99 ].

Schematic overview of the main drug transporters expressed in brain capillary endothelial cells, as well as their localization. Adapted from [ 2 , 11 ]. In vitro cellular models of the BBB started to emerge in the early s, presenting multiple advantages and being complementary to in vivo studies. Cell-based BBB models can be established with any type of cell source human, animal, or cell line derivative , including the availability of BBB endothelial cells and astrocytes freshly isolated from human brain tissue, fact that allows a considerable degree of reproducibility, both in physiological and pathological scenarios [ ].

Dauchy et al. Ohtsuki et al. P-gp, BCRP and MRP4 expression was detected, although with distinct relative expression level patterns from those found in freshly isolated human brain microvessels [ ]. There are static models and static two-dimensional models of the BBB, using endothelial cells monocultures and co-culture of endothelial cells and glia, respectively [ ].

In the first case, a simple monolayer of highly specialized brain microvascular endothelial cells is used. The cells, obtained from various sources e. Brain vascular endothelial cells grow to confluence on the luminal surface of the membrane, immersed in a specific growth media. This BBB model, with potential for using pure cell populations, allows drug permeability testing and binding affinity.

However, in order to circumvent the limitations related to this model, namely its simplicity related to the absence of physiological stimuli, a two-dimensional model containing both endothelial and glia cells was developed [ ]. The addition of abluminal astrocytes, in juxtaposition to the endothelial monolayer, facilitates the formation of more stringent tight junctions and the overall expression of BBB features.

In addition, the exposure to glia and induced glial-endothelial interactions increases the expression of brain endothelial marker enzymes, transporters such as P-gp and MRPs and tight junctions, and induces a phenotype more closely mimicking that found in vivo.

Transendothelial electrical resistance TEER , a functional parameter to monitor the quality of cells cultured on filter supports, namely the integrity of the cell monolayer, is higher in co-culture of endothelial cells and glia than in endothelial cell monocultures, indicating the formation of a more stringent and selective vascular bed [ ]. In addition to the cellular and static BBB models just referred, isolated brain microvessels have been extensively used to study BBB since the s [ ].

They have been successfully used for the identification of mechanisms and biochemical signals that play a role in regulating BBB functions in health and disease conditions, allowing the maintenance of the structural and cellular characteristics and properties in ex vivo experimentations.

As such, ABC transporters, that function as efflux pumps limiting the entry of numerous xenobiotics into the brain, have been studied in isolated brain capillaries providing reliable information on the transport processes mediated by different carriers [ , , ]. However, due to technical and functional limitations related to the use of isolated brain microvessels, computational models, artificial membranes, and in vitro cell culture BBB models have been gaining particular relevance.

One of the most used computational models is the in silico model, which, knowing the physicochemical properties of novel molecules, predicts their efficacy and bioavailability in relation to BBB permeability, considering both passive diffusion and active transport processes. Consequently, computer-assisted structure-based drug design model makes the drug development process faster, predicting drug effectiveness [ ].

The cardiac endothelial cells are characterized by expression of uptake and efflux transporters, which control the transport of a wide range of compounds, including drugs and toxins, into and out of the heart, respectively [ ]. At the end of the s and in the s, the expression of P-gp in several tissues was extensively studied.

Studies using human heart tissues showed P-gp to be expressed in the heart, although generally at relatively low levels, when compared to tissues such as the intestine, the liver, the brain and the placenta [ 14 ].

Particularly, Meissner and colleagues observed, by immunohistochemistry and in situ hybridization, P-gp expression and location in human endothelial cells of the capillaries and arterioles of the ventricles and atria [ ]. Additionally, a few years later, the authors observed the expression of BCRP in the vasculature of human heart, both in health and ischemic conditions [ ].

As such, ABC transporters may provide a functional barrier between the blood and cardiomyocytes, limiting the entry of xenobiotics into the heart, namely those that are cardiotoxic, such as the anticancer drugs mitoxantrone and anthracyclines [ 9 , 14 , , ]. It should be noted that, despite distinct ABC transporters have been identified, P-gp appears to be the most relevant to cardiovascular medicine, where it modulates the efficacy and toxicity of cardioactive agents [ 9 ]. Indeed, many cardiovascular active compounds are subject to drug transport by P-gp, as it is exemplified by digoxin [ ].

In addition to studies demonstrating the presence of P-gp in luminal membranes of the vascular endothelium isolated from the rat heart [ ], the study performed by Estevez and colleagues demonstrated, for the first time, the presence of P-gp in primary cultures of rat heart myocytes [ ]. Similarly to P-gp, other ABC carriers have been studied in the cardiovascular system.

Almost all studies demonstrated the expression of MRP1 in both human and other species heart [ , ]. Other multidrug resistance proteins, such as MRP1—3 and MRP5, were identified in human heart, with higher expression in ventricular samples [ , ]. MRP5 was found to be present in cardiomyocytes and in both vascular smooth muscle and endotelial cells [ ]. Its expression was found in both mouse and human hearts [ , ]. Eilers et al. Here, the authors observed the presence of both P-gp and MRP1—5, proteins responsible for the efflux of the main anti-retroviral drugs.

The presence of the carriers was confirmed by the suppression of the transport induced by the ABC transporters inhibitors verapamil and MK 5- 3- 2- 7-Chloroquinolinyl ethenyl phenyl dimethylcarbamyl-4,6-dithiaoctanoic acid , respectively. In another study, Higashikuni et al. The hMVECs are microvascular endothelial cells and, therefore, probably a cardiac cell model more representative of the heart transporters. The liver is an important tissue involved in the synthesis and secretion of bile acids, metabolism and transport of cholesterol, as well as in the metabolism and efflux of endogenous and exogenous substances [ , ].

As the major organ responsible for drug metabolism, the liver contributes to the first-pass elimination of drugs and for the plasma clearance of systemically distributed therapeutic compounds [ , ]. Therefore, together with the kidneys, the liver is an important detoxifying organ [ ]. These, organized in plates, have a polarized nature, apical and basolateral membranes, with different composition and functions. Hepatocytes are separated by tight junctions, which allow the vectorial transport of compounds with endogenous or exogenous origins from the blood into the bile [ , , , ].

The basolateral membrane is in contact with the sinusoidal blood and the canalicular membrane represents the excretory pole of hepatocytes [ ]. It is known that there is a differentiated functional expression of both sinusoidal and canalicular hepatic drug transporters.

Sinusoidal transporters mediate the initial step of hepatic elimination, i. On the other hand, efflux transporters are located in both the canalicular and basolateral membranes, where they mediate excretion into bile or into the systemic circulation, respectively [ , , ]. Major hepatic canalicular apical and sinusoidal basolateral efflux transporters are ABC proteins Figure 5. Schematic overview of the main drug transporters expressed in hepatocytes, as well as their localization.

Those transporters located on the basolateral membrane, such as MRP1 and MRP3—6, perform the removal of endogenous compounds organic anions and bile acids and xenobiotics from the hepatocytes into the sinusoidal blood, for subsequent urinary elimination [ , , , ]. Additionally, Meyer zu Schwabedissen and Kroemer demonstrated the involvement of hepatic BCRP in the biliary excretion of some therapeutically important drugs, such as methotrexate, the 3-hydroxymethylglutaryl-coenzyme A HMG-CoA -reductase inhibitors pitavastatin and rosuvastatin, and fluoroquinolones [ ].

It should be noted, however, that the MATE1 transporter belongs to the SLC transporters SLC47 , is predominantly expressed in the canalicular membrane of hepatocytes and functions as a secondary transport system, utilizing the electrochemical gradient of cations across the membrane for substrate transport.

Organic cation transporter 1 OCT1 may function in concert with MATE1 to mediate the hepatic uptake and biliary excretion, respectively, of cationic drugs and their metabolites [ ]. Many different in vitro liver models have been employed over the years in toxicological field with the aim to predict in vivo responses.

Immortalized cell lines and primary isolated liver cells are widely used in vitro models for liver toxicity testing. HepG2 and HepaRG cell lines are common immortalized liver-derived cell lines used in laboratory protocols [ ].

HepG2 cells express many liver-specific genes but the expression profile of genes involved in phase I and phase II metabolism vary between passages [ ]. HepaRG cells also express aldolase B that is a specific marker of adult hepatocytes. These cells have a high proliferative capacity, being able to differentiate in both hepatocytes and biliary cells.

In fact, when seeded at low density, HepaRG cells acquire an elongated undifferentiated morphology, actively divide and, after having reached confluency, form typical hepatocyte-like colonies surrounded by biliary epithelial-like cells [ ]. This differentiation takes place by treatment with dimethyl sulfoxide DMSO. Moreover, after differentiation, the expression of the different mentioned proteins remains stable for 6 weeks by treatment with DMSO [ ].

However, since these cells were isolated from a grade I differentiated liver tumor of a single female patient suffering from hepatocellular carcinoma and chronic hepatitis C virus infection, their predictive value for the human population is limited [ , ].

Primary cultures of hepatocytes represent a good model for the study of hepatic drug transporters in vitro. Rodent primary hepatocyte cultures, however, may undergo the so-called de-differentiation process, which consists on changes in cell morphology, structure, polarity, gene expression and liver-specific functions e.

For this reason, a sandwich-based culture technique was developed [ , ]. In this system, primary hepatocytes are placed between two layers of a gelled matrix, in a sandwich configuration, retaining the in vivo-like properties. As such, cell morphology, enzymes activity, albumin production and transferrin, fibrinogen and bile salt secretion are kept close to the physiological status over a longer period of time [ ]. This model is suitable for studies of hepatic drug transport, metabolism, biliary excretion and toxicity [ , , ].

Several studies using a sandwich-cultured hepatocyte model have demonstrated enhanced morphology and viability of hepatocytes, normal levels of secretion of liver-specific transporters and CYP enzymes and organic compounds, facilitated formation of gap junctions and functional bile canalicular networks over days in culture.

On the other hand, the main sandwich-hepatocyte model disadvantage is the decrease of genes expression, responsible for many liver-specific functions, over time, although keeping itself more useful for the mechanistic studies of hepatobiliary toxicity than primary hepatocytes [ ].

Sandwich-cultured human hepatocytes are considered the gold standard for the in vitro research of human hepatic transporters. However, human primary hepatocytes remain stable with time in culture, with a polarized status. Thus, monolayer-cultured human hepatocytes are also a valuable tool for the study of hepatic transporters since, contrary to that referred for rodent monolayer primary-cultured hepatocytes, the de-differentiation process is not expected to occur [ ].

Due to difficulties in maintaining long-term functionality of primary hepatocytes, immortalized cells and even sandwich-hepatocytes cultures, as well as in an attempt to circumvent problems related to the small predictive value of 2D models in pharmacokinetics processes, new models have been being developed. Within these, particular attention is given to the three-dimensional models that best mimic the processes that occur in vivo.

Particularly, hepatocytes have multiple apical and basolateral surfaces and, thus, their polarity is essential to safely predict, in vitro, the processes that may occur in vivo. In fact, drug uptake and diffusion in 2D systems does not accurately replicate the complexity found in a 3D multicell layer system.

There are several distinct 3D hepatocyte models, which vary greatly in complexity [ , ]. Hepatocyte spheroids present a very well defined and uniform size and geometry and, although they can be differently obtained, they can replicate, in a consistent way, the biological complexities of the 3D in vivo environment, allowing a greater maintenance of functionality than that observed in the two-dimensional models. Particularly, the expression levels of enzymes of phase I metabolism are found at levels close to the physiological.

In the toxicokinetics context, 3D hepatocyte spheroids allow the study of ABC proteins by quantifying drug uptake and diffusion, providing an uniform uptake by the entire surface area and avoiding complex experimental and analytical procedures [ ]. Other more complex three-dimensional cultures can be used, namely systems involving porous materials, packed-bed reactors, hollow fibers and perfusion flow [ ].

However, a fully functional liver culture model, where the entire in vivo dynamics can be observed, is still missing and efforts need to be carried out in order to accomplish that purpose.

The kidney is responsible for maintaining fluid and electrolyte homeostasis, maintaining the essential nutrients and eliminating both potentially toxic compounds and metabolic waste products from the body. These functions occur in the physiologic units of the kidney, the nephrons, composed by glomerulus and renal tubules [ , ]. The renal tubules consist of a monolayer of epithelial cells that play reabsorptive and secretory functions due to the presence of membrane transporters, which, in turn, significantly contribute to renal drug handling and for the variability in drug disposition.

ABC carrier proteins are predominantly located in proximal tubules where they use the energy provided by ATP hydrolysis to move substrates across the membrane [ , ]. In fact, MRP members in proximal tubular cells function as extrusion pumps for organic anions across the apical membrane.

Molecular biology techniques have shown that the renal cortical expression of MRP4 is much higher than that of MRP2 [ ]. Schematic overview of main drug transporters expressed in renal epithelial cells, as well as their localization. HK-2 Human Kidney-2 cell line is an immortalized proximal tubule epithelial cell line derived from adult human normal kidney and retains many of the phenotypic and functional characteristics of renal proximal tubular cells in vivo [ , , ]. At the molecular level, the products of E6 and E7 genes bind to the DNA regulatory proteins, resulting in facilitated cell proliferation [ , ].

Phenotypically, the HK-2 cell line has the same characteristics of normal well differentiated adult proximal tubular cells. It was shown that the HK-2 cells maintain the brush border typical enzymatic activities acid and alkaline phosphatase, leucine aminopeptidase and gamma-glutamyl transpeptidase [ ].

Several studies were carried out using HK-2 cells to evaluate, in vitro, the renal transport processes, namely those mediated by the ABC and SLC families of transporters.

In fact, HK-2 cells retain the constitutive expression of a functional P-gp in their membranes and its activity and expression may be modulated by drugs and many commonly ingested substances [ , , ]. According to the referred above, and despite the expression of some ABC transporters in HK-2 cells, the absence of several other transporters points to the current lack of relevant cellular models for the study of drug transport at the kidney level.

Nomura and colleagues used surgically removed renal tissue and compared the ABC mRNA expression levels in human renal cell carcinomas and normal kidney tissue. The intestine, in addition to the liver, is an important tissue that regulates the extent of absorption of orally administered drugs [ , ].

The majority of drug absorption occurs at the enterocytes in the small intestine, especially in the duodenum and jejunum, due to the large surface area, which is dependent on the presence of villi and microvilli [ , ]. Moreover, the intestine is known for its absorptive role due to the presence of uptake and efflux transporters, located at the apical and basolateral membranes Figure 7 , apart from the presence of cytochrome P 3A CYP3A4 in humans and conjugation enzymes [ , ].

Schematic overview of main drug transporters expressed in enterocytes, as well as their localization. P-gp, MRP2, MRP4 and BCRP are located at the apical membrane of enterocytes, causing the drug efflux into the lumen and reducing, in consequence, the drug concentration within the enterocytes. These ABC efflux transporters are the major barrier to intestinal absorption of substrate drugs [ 5 , 9 , 10 , , , , , , , , , ]. Moreover, the pattern of longitudinal expression of several intestinal transporters is not homogeneous along the human intestine, which may has functional implications on the preferable site of intestinal drug absorption.

Additionally, their precise location basolateral or apical is a subject of interest and often controversial [ , ]. In fact, the expression levels of efflux transporters can vary along the small intestine. Particularly, P-gp is expressed at high levels in the ileum and colon, but it presents the lowest constitutive expression levels in the jejunum and duodenum [ 10 , ]. BCRP is expressed in the small and large intestine but, unlike P-gp, BCRP expression does not vary significantly along the length of the small intestine [ 10 ].

P-gp, BCRP and MRP2 are located at the apical membrane, driving compounds from inside the cell back into the intestinal lumen [ , ]. Since P-gp, BCRP and MRP2 are able to bind to several structurally distinct and unrelated compounds, due to the lack of substrate specificity, they can decrease the absorption of many clinically relevant drugs, such as antibiotics, statins, HIV protease inhibitors, cardiac drugs calcium channel blockers, digitalic , immunossupressants and anticancer agents [ 10 ].

On the contrary, MRP1 and MRP3-MRP5 are expressed at the basolateral side of enterocytes where they pump their substrates from the intracellular compartment into the systemic circulation, thereby benefiting oral bioavailability [ 10 , , , , ]. MRP1 is highly expressed in the small and large intestine, being located at the basolateral membrane of enterocytes where it functions as an absorptive carrier, avoiding the accumulation of chemicals in the enterocytes [ 10 , ].

However, Han and colleagues showed the presence of OCT1 in the apical membrane of both enterocytes and Caco-2 cell monolayers [ ]. Additionally, the OATP2B1 expression at the basolateral membrane of neonatal, infantile and adolescent enterocytes was recently revealed by Mooij and co-authors [ ]. One of the best in vitro models of human intestinal epithelial cells available for studies of drug intestinal absorption and excretion and drug-drug interactions is the Caco-2 cell line [ 16 , 19 , 21 , , , , ].

In , the Caco-2 cell line was established in culture from a human colon adenocarcinoma [ ]. Caco-2 cells exhibit morphological as well as functional similarities to the human enterocytes [ 1 ]. When cultured under specific conditions, Caco-2 cells grow exponentially and, when in confluency, they undergo enterocytic differentiation, which is complete within 21 days in culture [ ].

During their differentiation, they form a polarized monolayer and develop a well-defined and typical brush border with a regular microvilli on the apical surface, as well as tight cellular junctions [ 1 , ]. These brush-border microvilli are very similar to those observed in normal small intestine and colon, with a double-leaflet plasma membrane, a core of microfilaments extending into the cytoplasm and an associated glycocalix. Caco-2 cells are indeed very similar to the small intestine enterocytes with respect to its structure and to the presence of brush-border-associated hydrolases [ , ].

Caco-2 cells have been extensively characterized and it is known that they are able to express tight junctions and very low amounts of cytochrome enzymes, making them particularly suitable as a model for examining various substrates transport properties [ ]. P-gp and MRP2 expression levels seem to be similar in jejunum and Caco-2 cells, while BCRP expression levels in Caco-2 cells are low when compared with those found in the human jejunum, in vivo [ , ].

The apparent permeability coefficients measured for reference compounds across Caco-2 cells monolayers have shown good correlation with their in vivo absorption [ ]. Hilgendorf et al. The best agreement between human tissue and the cell line was observed for the human jejunum and Caco-2 cells [ ]. Intestinal peptide-associated transporter 1 HPT1 was identified as the most abundantly expressed transporter in the intestinal mucosa.

Caco-2 cells can be cultured on semi-permeable inserts, allowing the evaluation of the transport of molecules between the apical and basolateral chambers [ ]. Appropriate in vitro assays for transport studies can be divided in two major groups: membrane-based assays and cell-based assays. The study of the function of the ABC efflux transporters and the identification of their substrates and inhibitors has been performed by using membranes, prepared from cells expressing ABC transporters.

Similar methods can be applied in the identification of inducers and activators. Currently, there are 3 available membrane-based assays: ATPase assays, membrane vesicular transport assays and photoaffinity labeling assays [ 1 ]. Compared to cell-based assays, the membrane-based assays have several advantages, including: 1 the ability to be used to characterize the xenobiotic effects on one specific efflux transporter; 2 the ability to be easily employed in a high throughput mode; 3 the easy with which they are maintained after preparation and 4 the easy with which the assays are performed Table 2 [ 1 ].

Main advantages versus disadvantages of the described in vitro and ex vivo assays adapted from [ 1 ]. The determination of the ABC transporters ATPase activity can be performed either in isolated membranes containing the desired transporter insect or mammalian cell membranes , or in reconstituted ABC protein preparations [ 32 ].

ATPase activity assays are commonly used in P-gp, MRPs and BCRP studies, representing a method for identification of compounds that interact with these efflux transporters [ , ]. The ATPase activity of the efflux transporters is vanadate sensitive and can be changed in the presence of substrates or modulators.

These can directly interact with ABC transporters, leading to stimulation or inhibition of the formation of an intermediate state of ATPase reduction [ 1 , , ].

The efflux transporters can be kept in an intermediate state due to the reaction with inorganic vanadate V i and ATP. ATP hydrolysis leads to P i dissociation from the transporter and is replaced by V i.

Therefore, the ATPase activity at the active sites is completely inhibited [ 1 ]. Compounds that interact with ABC transporters can be identified as stimulators or inhibitors of their ATPase activity.

The effect of the test compound on the ATPase activity of the efflux transporter is analyzed by the difference in the amount of phosphate released or, alternatively, in the remaining unmetabolized ATP, using ABC transporter expressing membranes, in the presence or absence of vanadate [ 1 , ].

The released P i levels are determined by a colorimetric reaction under mild acidic conditions, being the released P i amount directly proportional to the ATPase activity of the ABC transporters. Using the other experimental approach, the quantity of unmetabolized ATP is evaluated by a luciferase-generated luminescence signal, and is inversely proportional to the ATPase activity of the ABC transporters.

The assay relies on the ATP dependence of the light-generating reaction of firefly luciferase. Therefore, a decrease in luminescence corresponds to a higher ATP consumption by the transporters, thus, the greater the decrease in luminescence signal, the higher the ATPase activity.

Accordingly, samples containing compounds that stimulate the P-gp ATPase will have significantly lower signals than untreated samples. On the opposite, compounds that act as P-gp inhibitors will trigger less ATP consumption and, in consequence, the luminescence signal will be greater since the amount of unmetabolized ATP is higher. By comparing the results obtained for the basal activity and for the activity in the presence of the test compound, it can be classified into substrate, activator, inhibitor or without effect on the basal ATPase activity of the ABC transporters [ 1 , 32 , ].

Furthermore, these ATPase assays can also be applied to assess kinetic parameters, such as IC 50 for inhibitors [ 1 ]. Two different protocols can be used to study the interactions between ABC transporters and test compounds, i. In the stimulation assay, the stimulation of the basal ATPase activity of the ABC transporter is measured in the presence of the test compound.

The transporter substrates significantly stimulate the basal ATPase activity. In the inhibition assay, the transporter ATPase activity is analyzed with a known substrate and a specific inhibitor. This last protocol is useful to identify inhibitory compounds and slowly transported compounds that do not change the ATPase activity [ ].

Although ATPase assays allow the screening for ABC transporter substrates that can potentially act as competitive inhibitors, such as verapamil in what concerns to P-gp, resulting in the stimulation of the transporter ATPase activity, the screening for ABC transporter activators may be a tricky issue.

Indeed, since this concept of a compound that immediately activates these proteins, inducing a conformational change that increases the transport of a substrate bound to another binding site, is relatively new [ 3 ], it remains unclear whether these activators are, or not, necessarily ABC transporters substrates. Therefore, two different approaches could be undertaken: the evaluation of the effect of the potential activator, alone, in the transporters ATPase activity; and the evaluation of the potential activator effect on a stimulated ATPase activity, i.

Thereby, a P-gp activator should increase the verapamil-mediated stimulation of its ATPase activity by increasing P-gp-mediated verapamil transport ; while a P-gp inhibitor should make the opposite effect. Furthermore, when evaluating the effect of the potential activator alone, it will be possible to evaluate if such compound is also a substrate, thus providing more information on the activation mechanism, namely if a co-transport of both activator and substrate might be occurring [ 3 ].

Although ATPase assays are simple, reproducible and used to detect transporter-compound interactions, these techniques are not always suitable for distinguishing among potential ABC transporter substrates and modulators, due to the presence of high intra- and inter-assay variability [ 1 , 32 , ]. The ATPase assays may give false negative results for compounds, when they are studied in only one concentration, due to their low affinity and solubility. Compounds can stimulate and inhibit ABC transporters at either low or high concentrations [ 1 ].

These assays can be applied in the: a quantification of the compound transported across the cell membrane; b kinetic analysis of the transported compound, including determination of the affinity constant K m and maximal velocity V max ; c study of the test compound interaction with a known substrate of the efflux transporter, to obtain the inhibitory constant K i and the half maximal inhibitory concentration IC 50 for inhibitors; and d study of the transport driving force or the requirement for the presence of co-transported molecules [ 1 ].

Therefore, these assays, although not allowing the identification of ABC transporters inducers since the increased de novo synthesis of the proteins is needed , are useful for the identification of activators, as well as substrates and inhibitors. The membranes used in these assays are prepared under suitable conditions and are from different sources, such as baculovirus-infected insect ovary cells, transfected or selected mammalian cell lines from the brush border membrane of intestine, kidney and choroids plexus; hepatic sinusoidal and canalicular membranes; and luminal and abluminal membranes of the brain , transfected yeast cells and artificial membrane vesicles [ 1 , , ].

These contain inside-out-oriented vesicles, with both ATP- and ligand-binding sites facing the buffer outside. A rapid filtration method using glass fiber filters or nitrocellulose membranes is used to separate the vesicles from the incubation solution [ 1 , ].

Alternatively, the compounds can be radiolabeled, fluorescent or have a fluorescent tag, being quantified the radioactivity or fluorescence retained on the filter [ ]. Differences detected at level of the substrate uptake, in the presence or absence of ATP, can be attributed to transport mediated by efflux or uptake transporters, respectively [ 1 , ].

The membrane vesicular transport assays are advantageous techniques to measure the disposition of substrates across cell membranes, including compounds with low membrane permeability and low non-specific binding [ 32 ]. The membrane vesicles expressing efflux transporters are commercially available, making it possible for the routine use of these techniques [ 1 ]. However, there are also some disadvantages associated to these assays.

Namely, false-negative results can be obtained in the study of compounds with medium-to-high passive permeability or highly lipophilic, due to their high nonspecific binding to the lipid membranes. Additionally, the preparation and purification protocols of the membrane vesicles are time consuming and technically complicated [ 1 , 32 , , ]. The first mentioned technique has been used in the study of the ABC transporters function, including evaluation of the binding sites, binding affinities and structural details of the substrates and modulators [ 1 , 32 ].

Membranes expressing ABC transporters or isolated proteins are incubated with labeled photoaffinity compounds [ 1 ].

The ABC transporters radioactively labeled are solubilized and separated by gel electrophoresis. The protein labeling drug-binding is visualized and quantitated by autoradiography. Another type of photolabeling assays, mentioned above and first documented for P-gp, corresponds to the use of a radioactively labeled ATP analog, 8-azido-ATP [ ]. Labeled 8-azido-ATP binding, under non hydrolytic conditions, can be followed by UV-irradiation, size fractionation and autoradiography.

Under hydrolytic conditions, ATP hydrolysis takes place and the binding and release of an ATP analog is too rapid to be followed. For this reason, a phosphate-mimicking transport inhibitor e. The rate of the formation of this transition state can be assessed stopping the catalytic reaction by excess ATP and UV cross-linking. This formation is proportional to the rate of transport. When the substrates are efficiently transported, there is an increase in the formation of the trapped nucleotide [ 32 ].

Since both direct photoaffinity labeling and nucleotide trapping experiments are complicated techniques associated with complex protocols and are not routinely applied in the pharmaceutical industry, these techniques are important tools for studying details of the molecular mechanism. Direct photolabeling is generally not adequate for distinguishing between substrates and inhibitors [ 1 , 32 ].

On the other hand, ABC transporters form low-affinity interactions with a wide variety of hydrophobic compounds. The interaction sites and intensities may directly depend on the test drug and actual conformation of the transporter [ 32 ]. Cell-based assays may provide more clear information about the interaction between compounds and ABC transporters, applied in the evaluation of the following kinetic parameters: K m and V max for substrates, and K i and IC 50 for inhibitors Table 2.

The cytotoxicity assay is, by far, the most widely applied cell-based approach for investigating ABC transporters function. This test compound can be an inhibitor, activator or inducer of the ABC carrier under study. These assays allow a high-throughput screening of compounds due to reduced time consumption and cost, when compared, for example, with the in vivo assays, which have a high cost, are time-consuming, and have ethical restrictions.

However, cell-based assays are more labor and time consuming than the membrane-based assays. It is important to consider the following features: a particular cell line can express multiple transporters, although there are modified cell lines expressing one specific transporter; the culture conditions and number of cell passages may change the transporters expression levels; and the cells need to be maintained under culture conditions prior to use Table 2 [ 1 ].

Tissue localization and changes in gene expression after cells stimulation can be monitored by Northern blot analysis, dot-blot analysis, competitive PCR, RNase protection assays or in situ hybridization. Although these methods require large RNA amounts and starting material, not allowing a rapid analysis of multiple genes and large sample numbers, they are widely accepted and reliable and can be applied to the evaluation of ABC transporters gene expression [ ].

Real-time RT-PCR is commonly used in molecular biology for mRNA analysis, including detection and quantitation, by the use of fluorescent probes [ ]. This technique is sensitive enough to enable precise and reproducible mRNA quantitation both rare and abundant from a single cell [ ].

The evaluation of the gene expression is based on cycle threshold Ct values rather than end-point detection [ ]. There are two main classes of chemistry compounds, i. The PCR product accumulation corresponds to an increase in the fluorescence intensity. Although requiring extensive optimization, this is the most economical and the easiest method. The need of optimization is related to the SYBR Green ability for binding to any double-stranded DNA during reaction, including primer-dimers and other non-specific reaction products, resulting in an overestimation of the target gene concentration.

On the other hand, there are hydrolysis and hybridization FRET-based probes [ ]. The proximity of the dyes, during unhybridized state, does not completely quench the fluorescence, being possible to observe a background fluorescence.

During the PCR reaction, the probe anneals specifically between the primers forward and reverse to the desired target region of the gene. Then, the polymerase carries out the extension of the primer and replicates the template. This process is repeated in every cycle and fluorescence increases in proportion to the amount of probe cleavage.

TaqMan probe does not need extensive optimization. The second FRET-based technique is based on two probes, one labeled with a fluorescent donor dye and other labeled with an acceptor dye. Once in close vicinity 3 to 5 base pairs , the donor dye emits energy that excites the acceptor dye.

Consequently, there is emission of fluorescence at a different wavelength, which is monitored with a specific equipment. After each cycle, additional hybridization probes anneal, increasing the fluorescence intensity, which is measured during the exponential phase of the PCR reaction. The fluorescence intensity is proportional to the amount of input target DNA [ ]. Real-time PCR allows sample processing in a multi-well plate, automatically and with high-throughput. Glyceraldehyde 3-phosphate dehydrogenase GAPDH is used as a reference gene for expression analysis in human tissues, but alternative reference genes can be used for other cell systems [ ].

Langmann and colleagues developed a rapid, accurate and highly sensitive real-time PCR method for detection and quantification of all ABC transporters using a TaqMan probe. The method allows a rapid and complete analysis of all ABC transporters in obtained RNA samples, from twenty different human tissues. As a result, authors identified tissues involved in secretory adrenal gland , metabolic liver and kidney , barrier lung, trachea and small intestine and reproductive and tropic placenta, uterus, prostate and testis functions with high transcriptional activity for ABC transporters [ ].

Flow cytometry is a rapid and specific technique that provides complete cellular analysis, being used as a tool for understanding the regulation and interaction of cell systems, mainly based in the use of fluorescent antibodies. Light emitted from these antibodies allow the identification of a wide array of cell surface and even cytoplasmic antigens [ ]. Flow cytometry provides quantitative measurements of cells and other particles at a high speed, being suitable for the study of single mammalian cells in suspension by measuring their optical and fluorescence characteristics [ ].

Some physical properties, such as cell size and internal complexity, can be measured by flow cytometry [ ]. Additionally, antibodies conjugated with fluorescent dyes can bind to specific proteins on cell membranes intact cells or inside cells permeabilized cells.

Also, the use of fluorescent substrates, such as rhodamine , may be useful for the evaluation of membrane transporters activity. The labeled cells are passed by a light source and the fluorescent molecules are excited to a state of higher energy. When returning to their resting states, the fluorochromes emit light energy at higher wavelengths. The emitted fluorescence is collected using a flow cytometer, spectrally filtered and detected using photomultiplier tubes.

It is possible to simultaneously measure several cell properties, using multiple fluorochromes, each one emitting light at different wavelengths, although being excited with similar wavelengths. Propidium iodide, phycoerythrin and fluorescein are commonly used dyes [ ]. Flow cytometry assays can be applied to the study of ABC transporters, allowing the characterization of the interactions between drugs and ABC carriers, and usually involve the use of fluorescent transporter substrates, such as rhodamine and calcein acetoxymethyl ester calcein-AM for P-gp [ ].

Vilas-Boas and colleagues evaluated the influence of aging in P-gp expression and activity, in human lymphocytes isolated from whole blood samples of 65 healthy caucasian male donors, comparing two different methodologies. P-gp expression was analyzed using an anti-P-gp monoclonal antibody UIC2 , in the presence and absence of vinblastine. P-gp activity was studied by measuring the efflux rate of the P-gp fluorescent substrate, rhodamine , and by using the UIC2 shift assay.

The results obtained in both studies were compared and showed a significant age-dependent increase in mean P-gp expression and no differences were found in P-gp activity. Moreover, the UIC2 shift assay proved to be more selective than the rhodamine efflux assay, in the analysis of P-gp activity [ ].

The researchers also used flow cytometry to study, in RBE4 cells, the putative modulatory effect of rifampicin and three rifampicin derivatives over P-gp function, using rhodamine as a fluorescent substrate [ 20 ]. Recently, Silva and co-authors have been using a flow cytometry-based approach to study the ability of different compounds, such as doxorubicin, colchicine, X and TX, to modulate P-gp expression and activity, using the Caco-2 cell model.

In these studies, the UIC2 monoclonal antibody conjugated with fluorescein isothiocyanate was used to study P-gp expression, and rhodamine was used to evaluate P-gp activity [ 16 , 21 , 22 , ]. Despite flow cytometry usefulness in expression and functional studies of ABC transporters in live cells, most dyes used as indicators have limited applicability as they do not simultaneously detect all types of ABC carriers [ ].

Beyond flow cytometry, other accumulation and efflux assays are suitable for the screening of compounds that interfere with efflux transporters. These assays can be performed using cell suspensions, cell monolayers or membrane vesicle preparations [ ].

Upon loading of the cells with lipophilic dye s , with diffusion capacity across cell membranes, the resulting fluorescence intensity of the cell s will depend upon the activity of the ABC transporters [ ]. The accumulation of the fluorescent substrates can be measured in the presence and absence of specific inhibitors or activators, in order to understand the effect of the transporters activity [ ]. The intracellular accumulation of the dye is inversely proportional to the ABC carrier activity and can be measured by fluorescence spectrophotometry [ ].

Therefore, an increased intracellular accumulation of a given substrate higher intracellular fluorescence can be observed in the presence of an inhibitor, while the opposite decreased intracellular accumulation is characteristic of an ABC transporter inducer and activator. However, the discrimination between an inducer and an activator is only related with the time of contact of such compounds with the cells.

On the other hand, the effect of an inducer in the pump activity requires an increased incubation period, since the de novo synthesis of the protein is needed. Moreover, to note that although an increased expression could be observed after incubation with an inducer, it will not necessarily be translated in an increased activity of a given transporter [ 3 , 16 ].

The efflux studies comprise the pre-load of the cells with the dye of interest. The amount of dye in the extracellular environment is measured under various conditions known to influence the transporter activity. In the presence of an inhibitor of the efflux transporter, the amount of dye expelled from the cells will be smaller than that observed for control cells.

The change in the intracellular accumulation of the fluorescent compounds when co-administered with inhibitors, inducers or activators, is considered to be mainly due to their effect on the efflux pumps located in the cellular membrane, such as P-gp. It is important to notice that the analysis of the inhibition of P-gp may depend on the nature of the used substrate, since at least two binding sites, H and R, are considered to exist and inhibitors may differently interact with them.

Consequently, inhibition assays may be performed with various P-gp substrates [ 38 , , ]. The analysis of the efflux transporters activity may be based on the evaluation of the dye accumulation, efflux or both.

For example, one protocol routinely used for the evaluation of the effect of inducers or activators consists in two phases: i the accumulation phase, in the presence of the dye, and in which the ABC transporter activity is blocked with an inhibitor of energy production e.

The first phase results in maximum substrate accumulation inside the cells. The second phase consists in restoring the normal function of the transporter, which is now able to transport the fluorescent substrate out of the cells. By analyzing the cells both after the inhibited accumulation phase and after the efflux phase, is possible to infer the amount of substrate transported by the pump. For transfected cells or drug-induced cells that over-express a particular drug efflux transporter, accumulation or efflux studies can be compared to the wild-type or parental cell line that does not have as high a level of drug efflux transporter expression [ ].

It is important the selection of specific inhibitors and specific fluorescent substrates. In P-gp activity studies, rhodamine is frequently used as a fluorescent substrate, and cyclosporine A or PSC as P-gp inhibitors [ 16 , 19 , 20 , , , , , ]. Western blotting or protein blotting or immunoblotting is an important technique used for the immunodetection of proteins post-electrophoresis, particularly those at low abundance [ ]. Western blotting analysis is commonly performed in ABC proteins expression studies [ 22 , , ].

Western blotting is characterized by the following specific advantages: a wet membranes are flexible and of easy handling; b the proteins immobilized on the membrane are easily accessible to different ligands; c only a small amount of reagents is required for transfer analysis; d it is possible to obtain multiple replicas of a gel; e it is possible to storage transferred patterns, prior to use; f the same protein transfer can be used in multiple successive analysis [ ].

Transport assays are the most direct tool for the evaluation of transporter function and permeability of the test compound [ 1 ]. When cells reach confluency, they differentiate and become ready to be used in permeability studies. The two compartments are designated as apical and basolateral, denoting the membrane orientation of polarized cell layers.

These two chambers are connected only through the cells monolayer and their semipermeable support. The transport differences between the basolateral-to-apical and the apical-to-basolateral compartments are easily measured. The calculated ratio is referred to as efflux ratio and for results greater than 2 the test compound is considered substrate of the active efflux transporters [ 1 , 32 , , , , , ].

The experimental protocol is initiated by the addition of a solution containing the test compound to either the apical upper chamber or basolateral lower chamber compartment, for the study of the apical-to-basolateral A-to-B or basolateral-to-apical B-to-A transport, respectively [ 1 , , , , ].

On the other side is added a buffer. At desired time points, aliquots of added solution are removed from the lower chamber for studies of A-to-B transport or from the upper chamber for studies of B-to-A transport. In the presence of efflux transporters expression on the apical membrane, P app, A-to-B is smaller than P app, B-to-A.

These results will be contradicted if the transporter is localized on the basolateral cell membrane [ 1 , ]. Passively diffused compounds present P app values that are independent on its concentration. The flux rate is linearly correlated with the concentration of the compound. The flux rate of actively transported compounds is saturable with increasing of its concentration.

The determination of kinetic parameters, such as K m and V max , is possible [ 1 ]. Primary cultured cells, such as primary cultured brain endothelial cells, conjunctiva and alveola epithelial cells are cell types used in these studies [ 1 , ]. The cell type suitable for these assays must be polarized [ 1 ].

During transport assays several points should be taken into consideration, such as the selected cell line, pore size, pore density and filter material [ 32 ]. Many ABC carriers are constitutively expressed at the apical membrane of epithelial cells of different organs, including those that function as body barriers, such as the liver, brain, kidney and intestinal tract [ , ]. In the small intestine and colon, P-gp is one of the most important efflux proteins and may play a major contribution for several orally administered drugs bioavailability [ ].

Ex vivo methodologies are an experimental approach where an organ or tissue is removed from the animal and placed in chambers where physiological conditions found in the living body are mimicked, namely the access to nutrients and oxygen, allowing the viability of the organ or tissue during the experimentation time.

ABC function can be accurately evaluated by using ex vivo approaches Table 2. Serosal to mucosal transport of the fluorescent substrate, in the presence or absence of the putative ABC carrier modulator, is evaluated in each intestinal sac by determining the substrate concentration, by spectrofluorometry, in samples of mucosal medium, over time.

Rhodamine is a dye usually used as P-gp substrate [ , , ]. Given the relevance of the ABC transporters in the toxicokinetics and pharmacokinetics, namely in the absorption, distribution BBB permeation and excretion processes, as well as their involvement in diverse pathophysiological conditions, the search for new modulators of these carrier proteins is of particular importance in both pharmacological and toxicological fields.

Thereby, computational models are very valuable tools, allowing the identification of new putative ligands and, at the same time, being a relevant alternative to excessive animal testing and a preliminary approach to the in vitro and ex vivo experiments, very often expensive, laborious and time-consuming.

In silico models provide rapid and inexpensive screening platforms, and can include the development of quantitative structure-activity relationship QSAR models, as well as docking studies for ligand-carrier interactions prediction, and also the development of pharmacophores for ABC transporters inducers and activators [ 3 ]. Docking studies have long been used to predict the interaction of compounds with their potential targets proteins, nucleic acids, carbohydrates and lipids.

Several docking models were developed to map potential modulators of P-gp, BCRP and MRP1, thus allowing to evaluate the potential binding modes of such compounds in a given transporter [ 20 , 21 , 22 , , , , , , ]. Indeed, newly synthetized thio xanthonic derivatives demonstrated the ability to immediately increase P-gp activity after a short incubation period, an effect compatible with P-gp activation, resulting in a significant decrease in the toxicity of a P-gp substrate, PQ.

The possibility of a co-transport mechanism between TXs and PQ was further supported by docking studies using a validated P-gp model [ 22 ]. However, although numerous computational models, based on QSAR analysis, pharmacophore modelling and molecular docking techniques, have been developed to predict ABC transporters substrates and inhibitors, particularly in what concerns to P-gp, the search for new inducers and activators has been mainly performed by random screening [ 21 ].

Noteworthy, and in an attempt to address this gap, pharmacophores for P-gp inducers and activators were recently developed, which can be of utmost importance, in the future, in predicting new ligands [ 22 , ].

In fact, based on the in vitro P-gp activation ability of newly synthetized thioxanthonic derivatives [ 22 ] and on a set of known P-gp activators described in the literature, the authors developed and validated common feature pharmacophore models for P-gp activation.

The best ranked pharmacophore reported was composed of three features one hydrophobic feature, one aromatic ring, and one hydrogen bond acceptor group and can be a very useful tool to efficiently and rapidly predict new ligands with the ability to activate P-gp.

Additionally, pharmacophore construction was also performed for P-gp inducers. Briefly, the pharmacophores were validated using known P-gp inducers and can be used to map new compounds, as it was the case of newly synthetized TXs, for which there was previous indication from data of in vitro assays about their potential to activate and induce P-gp.

However, since many signalling transduction pathways can be considered in regulating the expression of a given transporter, fact that is particular evident for ABC transporters, and given the structural diversity of the compounds, finding a pharmacophore for P-gp inducers can be a challenging task. Noteworthy, by using such pharmacophores for P-gp inducers and activators, a perfect match between in silico and in vitro studies was observed [ 21 , 22 ], thus further reinforcing the idea that the use of such in silico strategies can help to predict the P-gp modulatory effects of new drugs that can be initially screened through these newly developed pharmacophores.

Also, in vitro data on the ability of newly synthetized dihydroxylated xanthones to activate P-gp and protect Caco-2 cells against the cytotoxicity induced by a P-gp substrate, PQ, triggered the development of a 2D QSAR model, which demonstrated that the maximal partial charge for oxygen atoms is related with the P-gp activation ability of such compounds [ 21 ]. Furthermore, a perfect match was again observed, with both the docking studies and the QSAR model being in accordance with the reported in vitro data [ 21 ].

Taken together, the in silico models disclose new possibilities in drug discovery and can be a valuable and complementary tool in the prediction of new ligands, allowing a more rational use of in vitro, ex vivo and in vivo assays. In vitro and in vivo studies with inducers and activators of the ABC transporters have shown that the use of these compounds may be an effective antidotal pathway against xenobiotic-induced toxicity.

The action mechanisms of both are not clear. Therefore, it is important to conduct more research involving putative inducers and activators of the ABC transporters, in order to understand: 1 their mechanism of action; 2 their specificity and 3 their toxicity in tissues with toxicological relevance. During the assessment of new modulators of the ABC transporters it is important to use adequate in vitro assays, high throughput and low-cost alternatives to excessive animal testing, evaluating their main effects on the expression and activity of the ABC transporters.

Using only one technique or one concentration of the test compound could lead to false results. To all financing sources the authors are greatly indebted. Published online Apr 8. Author information Article notes Copyright and License information Disclaimer. Received Jan 30; Accepted Mar Abstract Adenosine triphosphate ATP -binding cassette ABC transporters are highly expressed in tumor cells, as well as in organs involved in absorption and secretion processes, mediating the ATP-dependent efflux of compounds, both endogenous substances and xenobiotics, including drugs.

Keywords: inducers, activators, ATP-binding cassette transporters, cellular models, membrane assays, cell-based assays, in vitro assays, P-glycoprotein, multidrug resistance-associated protein 1, breast cancer resistance protein. Introduction The bioavailability of a wide variety of compounds that cannot permeate the membrane by passive diffusion e.

Open in a separate window. Figure 1. Figure 2. Figure 3. Overview of Modulators of the ABC Transporters: Activators and Inducers Compounds that interact with ABC transporters can act as substrates being moved across membranes via the transporter , inhibitors impairing the transporter-mediated efflux of other compounds , inducers enhancing the transporter expression levels or activators enhancing the transporter activity , but one compound can also have overlapping modes of action [ 9 ].

Study Models for ABC Transporters According to in vivo and in vitro results obtained, inducers and activators of the ABC transporters can represent an important protection tool against xenobiotic-induced toxicity and an antidotal pathway to be explored [ 3 , 15 , 16 , 19 , 20 , 21 , 22 ].

Cellular Models 3. Figure 4. Cardiovascular System The cardiac endothelial cells are characterized by expression of uptake and efflux transporters, which control the transport of a wide range of compounds, including drugs and toxins, into and out of the heart, respectively [ ]. Liver The liver is an important tissue involved in the synthesis and secretion of bile acids, metabolism and transport of cholesterol, as well as in the metabolism and efflux of endogenous and exogenous substances [ , ].

Figure 5. Kidney The kidney is responsible for maintaining fluid and electrolyte homeostasis, maintaining the essential nutrients and eliminating both potentially toxic compounds and metabolic waste products from the body. Figure 6. Intestine The intestine, in addition to the liver, is an important tissue that regulates the extent of absorption of orally administered drugs [ , ]. Figure 7. In Vitro Assays Appropriate in vitro assays for transport studies can be divided in two major groups: membrane-based assays and cell-based assays.

Membrane-Based Assays The study of the function of the ABC efflux transporters and the identification of their substrates and inhibitors has been performed by using membranes, prepared from cells expressing ABC transporters.

Table 2 Main advantages versus disadvantages of the described in vitro and ex vivo assays adapted from [ 1 ]. Advantages Disadvantages In vitro assays Cell-based assays Allows to screen for P-gp inducers, activators, inhibitors and substrates. Cell-based transport assays are a classic assay to determine substrates or inhibitors and, more recently, activators. However, to note that an increased expression of a given transporter may not necessarily result in an increase in its transport activity.

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