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Introduction: Drug-mediated inhibition of bile salt efflux transporters may cause liver injury. In vitro prediction of drug effects toward canalicular and/or sinusoidal efflux of bile salts from human hepatocytes is therefore a major issue, which can be addressed using liver cell-based assays., Area Covered: This review, based on a thorough literature search in the scientific databases PubMed and Web of Science, provides key information about hepatic transporters implicated in bile salt efflux, the human liver cell models available for investigating functional inhibition of bile salt efflux, the different methodologies used for this purpose, and the modes of expression of the results. Applications of the assays to drugs are summarized, with special emphasis to the performance values of some assays for predicting hepatotoxicity/cholestatic effects of drugs., Expert Opinion: Human liver cell-based assays for evaluating drug-mediated inhibition of bile acid efflux transporters face various limitations, such as the lack of method standardization and validation, the present poor adaptability to high throughput approaches, and some pitfalls with respect to interpretation of bile acid biliary excretion indexes. Hepatotoxicity of drugs is additionally likely multifactorial, highlighting that inhibition of hepatic bile salt efflux by drugs provides important, but not full, information about potential drug hepatotoxicity.

Micro- and nanoplastics (MPs/NPs) constitute emerging and widely-distributed environmental contaminants to which humans are highly exposed. They possibly represent a threat for human health. In order to identify cellular/molecular targets for these plastic particles, we have analysed the effects of exposure to manufactured polystyrene (PS) MPs and NPs on in vitro activity and expression of human membrane drug transporters, known to interact with chemical pollutants. PS MPs and NPs, used at various concentrations (1, 10 or 100 µg/mL), failed to inhibit efflux activities of the ATP-binding cassette (ABC) transporters P-glycoprotein, MRPs and BCRP in ABC transporter-expressing cells. Furthermore, PS particles did not impair the transport of P-glycoprotein or BCRP substrates across intestinal Caco-2 cell monolayers. Uptake activities of solute carriers (SLCs) such as OCT1 and OCT2 (handling organic cations) or OATP1B1, OATP1B3, OATP2B1, OAT1 and OAT3 (handling organic anions) were additionally not altered by PS MPs/NPs in HEK-293 cells overexpressing these SLCs. mRNA expression of ABC transporters and of the SLCs OCT1 and OATP2B1 in Caco-2 cells and human hepatic HepaRG cells were finally not impaired by a 48-h exposure to MPs/NPs. Altogether, these data indicate that human drug transporters are unlikely to be direct and univocal targets for synthetic PS MPs/NPs., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Triazole pesticides are widely used fungicides, to which humans are rather highly exposed. They are known to activate drug-sensing receptors regulating expression of hepatic drug metabolizing enzymes and drug transporters, thus suggesting that the hepatic drug detoxification system is modified by these agrochemicals. To investigate this hypothesis, the effects of 9 triazole fungicides towards expression of drug metabolizing enzymes and transporters were characterized in cultured human HepaSH cells, that are human hepatocytes deriving from chimeric humanized liver TK-NOG mice. Most of triazoles used at 10 μM were found to act as inducers of cytochrome P-450 (CYP) 1A1, CYP1A2, CYP2B6, CYP3A4 and UDP-glucuronosyltransferase 1A1 mRNA levels and of CYP3A4 protein; some triazoles also enhanced mRNA expression of the canalicular transporters P-glycoprotein/MDR1, multidrug resistance-associated protein 2 and breast cancer resistance protein. Triazoles however concomitantly inhibited CYP2B6 and CYP3A4 activities and thus appeared as dual regulators of these CYPs, being both inducers of their expression and inhibitors of their activity. The inducing effect however predominated, at least for bromuconazole, propiconazole and tebuconazole. Bromuconazole was moreover predicted to enhance CYP2B6 and CYP3A4 expression in humans exposed to this fungicide in a chronic, acute or occupational context. These data demonstrate that key-actors of the human hepatic detoxification system are impacted by triazole pesticides, which may have to be considered for the risk assessment of these agrochemicals. They additionally highlight that the use of human HepaSH cells as surrogates to primary human hepatocytes represents an attractive and promising way for studying hepatic effects of environmental chemicals., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The identification of substrates for solute carriers (SLCs) handling drugs is an important challenge, owing to the major implication of these plasma membrane transporters in pharmacokinetics and drug-drug interactions. In this context, the competitive counterflow (CCF) assay has been proposed as a practical and less expensive approach than the reference functional uptake assays for discriminating SLC substrates and non-substrates. The present article was designed to summarize and discuss key-findings about the CCF assay, including its principle, applications, challenges and limits, and perspectives. The CCF assay is based on the decrease of the steady-state accumulation of a tracer substrate in SLC-positive cells, caused by candidate substrates. Reviewed data highlight the fact that the CCF assay has been used to identify substrates and non-substrates for organic cation transporters (OCTs), organic anion transporters (OATs), and organic anion transporting polypeptides (OATPs). The performance values of the CCF assay, calculated from available CCF study data compared with reference functional uptake assay data, are, however, rather mitigated, indicating that the predictability of the CCF method for assessing SLC-mediated transportability of drugs is currently not optimal. Further studies, notably aimed at standardizing the CCF assay and developing CCF-based high-throughput approaches, are therefore required in order to fully precise the interest and relevance of the CCF assay for identifying substrates and non-substrates of SLCs., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Succinate dehydrogenase inhibitors (SDHIs) are widely-used fungicides, to which humans are exposed and for which putative health risks are of concern. In order to identify human molecular targets for these environmental chemicals, the interactions of 15 SDHIs with activities of main human drug transporters implicated in pharmacokinetics were investigated in vitro. 5/15 SDHIs, i.e., benzovindiflupyr, bixafen, fluxapyroxad, pydiflumetofen and sedaxane, were found to strongly reduce activity of the renal organic anion transporter (OAT) 3, in a concentration-dependent manner (with IC 50 values in the 1.0-3.9 μM range), without however being substrates for OAT3. Moreover, these 5/15 SDHIs decreased the membrane transport of estrone-3 sulfate, an endogenous substrate for OAT3, and sedaxane was predicted to inhibit in vivo OAT3 activity in response to exposure to the acceptable daily intake (ADI) dose. In addition, pydiflumetofen strongly inhibited the renal organic cation transporter (OCT) 2 (IC 50  = 2.0 μM) and benzovindiflupyr the efflux pump breast cancer resistance protein (BCRP) (IC 50  = 3.9 μM). Other human transporters, including organic anion transporting polypeptide (OATP) 1B1 and OATP1B3 as well as multidrug and toxin extrusion protein (MATE) 1 and MATE2-K were moderately or weakly inhibited by SDHIs, whereas P-glycoprotein, multidrug resistance-associated protein (MRP), OCT1 and OAT1 activities were not or only marginally impacted. Then, some human drug transporters, especially OAT3, constitute molecular targets for SDHIs. This could have toxic consequences, notably with respect to levels of endogenous compounds and metabolites substrates for the considered transporters or to potential SDHI-drug interactions. This could therefore contribute to putative health risk of these fungicides., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Organophosphate flame retardants (OPFRs) are environmental pollutants of increasing interest, widely distributed in the environment and exerting possible deleterious effects towards the human health. The present study investigates in vitro their possible interactions with human drug transporters, which are targets for environmental chemicals and actors of their toxicokinetics. Some OPFRs, i.e., tris(2-butoxyethyl) phosphate (TBOEP), tris(1,3-dichloroisopropyl) phosphate (TDCPP), tri-o-cresyl phosphate (TOCP) and triphenyl phosphate (TPHP), were found to inhibit activities of some transporters, such as organic anion transporter 3 (OAT3), organic anion transporting polypeptide (OATP) 1B1, OATP1B3, organic cation transporter 2 (OCT2) or breast cancer resistance protein (BCRP). These effects were concentration-dependent, with IC 50 values ranging from 6.1 µM (for TDCPP-mediated inhibition of OCT2) to 51.4 µM (for TOCP-mediated inhibition of BCRP). OPFRs also blocked the transporter-dependent membrane passage of endogenous substrates, notably that of hormones. OAT3 however failed to transport TBOEP and TPHP. OPFRs additionally repressed mRNA expressions of some transporters in cultured human hepatic HepaRG cells, especially those of OAT2 and OCT1 in response to TOCP, with IC 50 values of 2.3 µM and 2.5 µM, respectively. These data therefore add OPFRs to the expanding list of pollutants interacting with drug transporters, even if OPFR concentrations required to impact transporters, in the 2-50 µM range, are rather higher than those observed in humans environmentally or dietarily exposed to these chemicals., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Human membrane drug transporters are recognized as major actors of pharmacokinetics; they also handle endogenous compounds, including hormones and metabolites. Chemical additives present in plastics interact with human drug transporters, which may have consequences for the toxicokinetics and toxicity of these widely-distributed environmental and/or dietary pollutants, to which humans are highly exposed. The present review summarizes key findings about this topic. In vitro assays have demonstrated that various plastic additives, including bisphenols, phthalates, brominated flame retardants, poly-alkyl phenols and per- and poly-fluoroalkyl substances, can inhibit the activities of solute carrier uptake transporters and/or ATP-binding cassette efflux pumps. Some are substrates for transporters or can regulate their expression. The relatively low human concentration of plastic additives from environmental or dietary exposure is a key parameter to consider to appreciate the in vivo relevance of plasticizer-transporter interactions and their consequences for human toxicokinetics and toxicity of plastic additives, although even low concentrations of pollutants (in the nM range) may have clinical effects. Existing data about interactions of plastic additives with drug transporters remain somewhat sparse and incomplete. A more systematic characterization of plasticizer-transporter relationships is needed. The potential effects of chemical additive mixtures towards transporter activities and the identification of transporter substrates among plasticizers, as well as their interactions with transporters of emerging relevance deserve particular attention. A better understanding of the human toxicokinetics of plastic additives may help to fully integrate the possible contribution of transporters to the absorption, distribution, metabolism and excretion of plastics-related chemicals, as well as to their deleterious effects towards human health., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Chimeric mice with humanized liver are thought to represent a sustainable source of isolated human hepatocytes for in vitro studying detoxification of drugs in humans. Because drug transporters are now recognized as key-actors of the hepatic detoxifying process, the present study was designed to characterize mRNA expression and activity of main hepatic drug transporters in cryopreserved human hepatocytes isolated from chimeric TK-NOG mice and termed HepaSH cells. Such cells after thawing were shown to exhibit a profile of hepatic solute carrier (SLC) and ATP-binding cassette (ABC) drug transporter mRNA levels well correlated to those found in cryopreserved primary human hepatocytes or human livers. HepaSH cells used either as suspensions or as 24 h-cultures additionally displayed notable activities of uptake SLCs, including organic anion transporting polypeptides (OATPs), organic anion transporter 2 (OAT2) or sodium-taurocholate co-transporting polypeptide (NTCP). SLC transporter mRNA expression, as well as SLC activities, nevertheless fell in HepaSH cells cultured for 120 h, which may reflect a partial dedifferentiation of these cells with time in culture in the conventional monolayer culture conditions used in the study. These data therefore support the use of cryopreserved HepaSH cells as either suspensions or short-term cultures for drug transport studies., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

HepaRG cells are highly-differentiated human hepatoma cells, which are increasingly recognized as a convenient cellular model for in vitro evaluation of hepatic metabolism, transport, and/or toxicity of drugs. The present study was designed to evaluate whether HepaRG cells can also be useful for studying drug-mediated inhibition of canalicular and/or sinusoidal hepatic efflux of bile acids, which constitutes a major mechanism of drug-induced liver toxicity. For this purpose, HepaRG cells, initially loaded with the bile acid taurocholate (TC), were reincubated in TC-free transport assay medium, in the presence or absence of calcium or drugs, before analysis of TC retention. This method allowed us to objectivize and quantitatively measure biliary and sinusoidal efflux of TC from HepaRG cells, through distinguishing cellular and canalicular compartments. In particular, time-course analysis of the TC-free reincubation period of HepaRG cells, that is, the efflux period, indicated that a 20 min-efflux period allowed reaching biliary and sinusoidal excretion indexes for TC around 80% and 60%, respectively. Addition of the prototypical cholestatic drugs bosentan, cyclosporin A, glibenclamide, or troglitazone during the TC-free efflux phase period was demonstrated to markedly inhibit canalicular and sinusoidal secretion of TC, whereas, by contrast, incubation with the noncholestatic compounds salicylic acid or flumazenil was without effect. Such data therefore support the use of human HepaRG cells for in vitro predicting drug-induced liver toxicity (DILI) due to the inhibition of hepatic bile acid secretion, using a biphasic TC loading/efflux assay., (© 2022 The Authors. Biopharmaceutics & Drug Disposition published by John Wiley & Sons Ltd.)

Because of a narrow therapeutic index and a wide inter- and intra-patient variability, therapeutic drug monitoring of the immunosuppressant drug tacrolimus (TAC) based on whole-blood concentrations (C blood ) is mandatory in solid organ transplant recipients. Using peripheral blood mononuclear cells concentrations (C PBMC ) could improve patient outcomes. The poor correlation between C blood and C PBMC makes hypothesize that drug transporters are implicated in the intracellular accumulation of TAC. The aim of this work was therefore to clinically study: i) the role of genetic variants and ii) the effect of mRNA and protein expression of 4 drug transporters on the TAC C PBMC/blood ratio. In addition, functional in vitro experiments were performed to mechanistically validate the clinical observations. Genetic variants of ABCB1/P-gp and SLC28A3/CNT3 did not influence TAC C PBMC in liver transplant recipients (LTR). ABCC2/MRP2 at the mRNA level; ABCB1/P-gp, SLC28A3/CNT3 and SLC29A1/ENT1 at the protein level; correlated with the C PBMC/blood in kidney and LTR. In vitro results suing transporter-expressing cells confirmed that TAC is substrate of P-gp but not MRP2, whereas experiments remained inconclusive for CNT3 and ENT1. In conclusion, the genetic-transcription-protein-functional approach presented in this work provides new insights in the understanding of TAC transport at the T lymphocyte plasma membrane., Competing Interests: Declaration of competing interest Gwendal Coste's Ph.D. has been partly funded by Astellas Pharma. Florian Lemaitre has been invited to participation in congresses by Chiesi and Sandoz., (Copyright © 2022 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.)

Chimeric mice with humanized livers constitute an attractive emergent experimental model for investigating human metabolism and disposition of drugs. The present review was designed to summarize key findings about the use of this model for studying human hepatic drug transporters, which are now recognized as important players in pharmacokinetics and consequently have to be considered from a regulatory perspective during pharmaceutical drug development. The reviewed data indicate that chimeric mice with humanized livers have been successfully used for analysing the implications of human hepatic drug transporters for drug hepatobiliary elimination, drug-drug interactions and drug-induced cholestasis. Such transporter studies have been performed in vivo with chimeric mice and/or in vitro with human hepatocytes isolated from humanized liver and used either in suspension or in culture. The residual presence of mouse hepatocytes and the potential morphological/histological alterations of the humanized liver, as well as its immunodeficient mouse environment, have, however, to be considered when using chimeric mice with humanized livers for transporter studies. Finally, if the proof of concept of applying chimeric mice with humanized livers to hepatic drug transport is established, more experimental data on this topic, including from standardization approaches, are likely required to completely and accurately demonstrate the robustness, convenience and added value of this chimeric mouse model for drug transporter studies., (© 2022. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Endotoxemia-induced inflammation may impact the activity of hepatocyte transporters, which control the hepatobiliary elimination of drugs and bile acids. 99m Tc-mebrofenin is a non-metabolized substrate of transporters expressed at the different poles of hepatocytes. 99m Tc-mebrofenin imaging was performed in rats after the injection of lipopolysaccharide (LPS). Changes in transporter expression were assessed using quantitative polymerase chain reaction of resected liver samples. Moreover, the particular impact of pharmacokinetic drug-drug interactions in the context of endotoxemia was investigated using rifampicin (40 mg/kg), a potent inhibitor of hepatocyte transporters. LPS increased 99m Tc-mebrofenin exposure in the liver (1.7 ± 0.4-fold). Kinetic modeling revealed that endotoxemia did not impact the blood-to-liver uptake of 99m Tc-mebrofenin, which is mediated by organic anion-transporting polypeptide (Oatp) transporters. However, liver-to-bile and liver-to-blood efflux rates were dramatically decreased, leading to liver accumulation. The transcriptomic profile of hepatocyte transporters consistently showed a downregulation of multidrug resistance-associated proteins 2 and 3 (Mrp2 and Mrp3), which mediate the canalicular and sinusoidal efflux of 99m Tc-mebrofenin in hepatocytes, respectively. Rifampicin effectively blocked both the Oatp-mediated influx and the Mrp2/3-related efflux of 99m Tc-mebrofenin. The additive impact of endotoxemia and rifampicin led to a 3.0 ± 1.3-fold increase in blood exposure compared with healthy non-treated animals. 99m Tc-mebrofenin imaging is useful to investigate disease-associated change in hepatocyte transporter function.

The search of substrates for solute carriers (SLCs) constitutes a major issue, owing notably to the role played by some SLCs, such as the renal electrogenic organic cation transporter (OCT) 2 ( SLC22A2 ), in pharmacokinetics, drug-drug interactions and drug toxicity. For this purpose, substrates have been proposed to be identified by their cis -inhibition and trans -stimulation properties towards transporter activity. To get insights on the sensitivity of this approach for identifying SLC substrates, 15 various exogenous and endogenous OCT2 substrates were analysed in the present study, using 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (DiASP) as a fluorescent OCT2 tracer substrate. All OCT2 substrates cis -inhibited DiASP uptake in OCT2-overexpressing HEK293 cells, with IC 50 values ranging from 0.24 µM (for ipratropium) to 2.39 mM (for dopamine). By contrast, only 4/15 substrates, i.e., acetylcholine, agmatine, choline and metformin, trans -stimulated DiASP uptake, with a full suppression of the trans -stimulating effect of metformin by the reference OCT2 inhibitor amitriptyline. An analysis of molecular descriptors next indicated that trans -stimulating OCT2 substrates exhibit lower molecular weight, volume, polarizability and lipophilicity than non- trans -stimulating counterparts. Overall, these data indicated a rather low sensitivity (26.7%) of the trans -stimulation assay for identifying OCT2 substrates, and caution with respect to the use of such assay may therefore be considered.

Interactions of the Janus kinase (JAK) inhibitor ruxolitinib with solute carriers (SLCs) remain incompletely characterised. The present study was therefore designed to investigate this issue.The interactions of ruxolitinib with SLCs were analysed using transporter-overexpressing human embryonic kidney HEK293 cells. Substrate accumulation was detected by spectrofluorimetry, liquid chromatography coupled to tandem mass spectrometry or scintillation counting.Ruxolitinib was found to potently inhibit the activities of organic anion transporter 3 (OAT3), organic cation transporter 2 (OCT2), multidrug and toxin extrusion 1 (MATE1) and MATE2-K (half maximal inhibitory concentration (IC 50 ) < 10 µM). It blocked OAT1, OAT4, OATP1B1, OATP1B3, OATP2B1 and OCT3, but in a weaker manner (IC 50 > 10 µM), whereas OCT1 was not impacted. No time-dependent inhibition was highlighted. When applying the US Food and Drug Administration (FDA) criteria for transporters-related drug-drug interaction risk, OCT2 and MATE2-K, unlike MATE1 and OAT3, were predicted to be in vivo inhibited by ruxolitinib. Cellular uptake studies additionally indicated that ruxolitinib is a substrate for MATE1 and MATE2-K, but not for OAT3 and OCT2.Ruxolitinib in vitro blocked activities of most of SLC transporters. Only OCT2 and MATE-2K may be however clinically inhibited by the JAK inhibitor, with the caution for OCT2 that in vitro inhibition data were generated with an FDA-non recommended fluorescent substrate. Ruxolitinib MATEs-mediated transport may additionally deserve attention for its possible pharmacological consequences in MATE-positive cells.

Introduction : Janus kinase inhibitors (JAKinibs) constitute an emerging and promising pharmacological class of anti-inflammatory or anti-cancer drugs, used notably for the treatment of rheumatoid arthritis and some myeloproliferative neoplasms. Areas covered : This review provides an overview of the interactions between marketed JAKinibs and major uptake and efflux drug transporters. Consequences regarding pharmacokinetics, drug-drug interactions and toxicity are summarized. Expert opinion : JAKinibs interact in vitro with transporters in various ways, as inhibitors or as substrates of transporters or as regulators of transporter expression. This may theoretically result in drug-drug interactions (DDIs), with JAKinibs acting as perpetrators or as victims, or in toxicity, via impairment of thiamine transport. Clinical significance in terms of DDIs for JAKinib-transporter interactions remains however poorly documented. In this context, the in vivo unbound concentration of JAKinibs is likely a key parameter to consider for evaluating the clinical relevance of JAKinibs-mediated transporter inhibition. Additionally, the interplay with drug metabolism as well as possible interactions with transporters of emerging importance and time-dependent inhibition have to be taken into account. The role drug transporters may play in controlling cellular JAKinib concentrations and efficacy in target cells is also an issue of interest.

The β-lactam penicillin antibiotic cloxacillin (CLX) presents wide inter-individual pharmacokinetics variability. To better understand its molecular basis, the precise identification of the detoxifying actors involved in CLX disposition and elimination would be useful, notably with respect to renal secretion known to play a notable role in CLX elimination. The present study was consequently designed to analyze the interactions of CLX with the solute carrier transporters organic anion transporter (OAT) 1 and OAT3, implicated in tubular secretion through mediating drug entry at the basolateral pole of renal proximal cells. CLX was first shown to block OAT1 and OAT3 activity in cultured OAT-overexpressing HEK293 cells. Half maximal inhibitory concentration (IC 50 ) value for OAT3 (13 µm) was however much lower than that for OAT1 (560 µm); clinical inhibition of OAT activity and drug-drug interactions may consequently be predicted for OAT3, but not OAT1. OAT3, unlike OAT1, was next shown to mediate CLX uptake in OAT-overexpressing HEK293 cells. Kinetic parameters for this OAT3-mediated transport of CLX (K m  = 10.7 µm) were consistent with a possible in vivo saturation of this process for high CLX plasma concentrations. OAT3 is consequently likely to play a pivotal role in renal CLX secretion and consequently in total renal CLX elimination, owing to the low plasma unbound fraction of the antibiotic. OAT3 genetic polymorphisms as well as co-administered drugs inhibiting in vivo OAT3 activity may therefore be considered as potential sources of CLX pharmacokinetics variability., (© 2020 Société Française de Pharmacologie et de Thérapeutique.)

Interleukin (IL)-22 is a cytokine up-regulated in inflammatory situations and known to exert various hepatic effects. The potential impact of IL-22 towards liver drug detoxifying proteins remains nevertheless unknown, but may be important to determine owing to the well-established alterations of liver detoxification occuring during inflammation. The present study was therefore designed to analyze the effects of IL-22 towards drug metabolizing enzyme and drug transporter expression and activity in cultured human hepatic cells. Exposure of differentiated hepatoma HepaRG cells or primary human hepatocytes to 10 ng/mL IL-22 was found to repress mRNA expression of cytochrome P-450 (CYP) 1A2, CYP3A4, CYP2B6 and CYP2C9 and of the sinusoidal sodium-taurocholate co-transporting polypeptide (NTCP); such IL-22 effects were concentration-dependent for CYP3A4 (IC 50  = 1.7 ng/mL), CYP2B6 (IC 50  = 0.9 ng/mL) and NTCP (IC 50  = 1.8 ng/mL). Activity of CYP1A2 (phenacetin O-deethylation), CYP3A4 (midazolam hydroxylation) and CYP2B6 (bupropion hydroxylation), as well as that of NTCP (taurocholate uptake) were concomitantly decreased in IL-22-treated HepaRG cells; by contrast, activity of organic anion transporter polypeptides (OATPs) (estrone-3-sulfate uptake) and of organic cation transporter (OCT) 1 (tetra-ethylammonium uptake) remained unchanged. IL-22 was next found to activate the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) 3 pathway, whose inhibition by the JAK inhibitor ruxolitinib fully prevented the IL-22-mediated CYP3A4, CYP2B6 and NTCP repression in HepaRG cells. This JAK-dependent down-regulation of hepatic drug detoxifying proteins, notably of CYPs, by IL-22 may contribute to alteration of pharmacokinetics in patients suffering from acute and chronic inflammatory diseases and may be the source of drug-drug interactions., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Human membrane drug transporters are recognized as major actors of pharmacokinetics. Pesticides also interact with human drug transporters, which may have consequences for pesticide toxicokinetics and toxicity. The present review summarizes key findings about this topic. In vitro assays have demonstrated that some pesticides, belonging to various chemical classes, modulate drug transporter activity, regulate transporter expression and/or are substrates, thus bringing the proof of concept for pesticide-transporter relationships. The expected low human concentration of pesticides in response to environmental exposure constitutes a key-parameter to be kept in mind for judging the in vivo relevance of such pesticide-transporter interactions and their consequences for human health. Existing data about interactions of pesticides with drug transporters remain, however, rather sparse; more extensive and systematic characterization of pesticide-transporter relationships, through the use of high throughput in vitro assays and/or in silico methods, is, therefore, required. In addition, consideration of transporter polymorphisms, pesticide mixture effects and physiological and pathological factors governing drug transporter expression may help to better define the in vivo relevance of pesticide-transporter interactions in terms of toxicokinetics and toxicity for humans. © 2019 Society of Chemical Industry., (© 2019 Society of Chemical Industry.)

The interactions of six neonicotinoid pesticides and one neonicotinoid metabolite with drug transporters have been characterized in vitro. Acetamiprid, clothianidin, imidacloprid, nitenpyram, thiacloprid and its metabolite thiacloprid amide, and thiamethoxam, each used at 100 µM, did not impair activity of the efflux pumps P-glycoprotein, multidrug resistance-associated proteins, and breast cancer resistance protein. They also did not inhibit that of the uptake transporters OATP1B1, OATP1B3, OAT4, and MATE1, whereas that of OATP2B1, OAT1, and MATE2-K was affected by only one of the seven neonicotinoids. Activity of OCT1 was moderately stimulated (up to 1.5-fold) by several neonicotinoids. By contrast, that of OAT3 and OCT2 was inhibited by most (OAT3), if not all (OCT2), neonicotinoids, with IC 50 values in the 20 to 60 µM range for thiacloprid, likely not relevant to environmental exposure. Thiacloprid was moreover not transported by OAT3 and OCT2. Overall, these data suggest that neonicotinoid pesticides rather poorly interact with drug transporter activities., (© 2019 Wiley Periodicals, Inc.)

Diesel exhaust particles (DEPs) are common environmental air pollutants known to impair expression and activity of drug detoxifying proteins, including hepatic ATP-binding cassette (ABC) drug transporters. The present study was designed to determine whether organic DEP extract (DEPe) may also target ABC drug transporters in bronchial cells. DEPe (10 μg/mL) was demonstrated to induce mRNA and protein expression of the multidrug resistance-associated protein (MRP) 3 in cultured bronchial epithelial BEAS-2B cells, whereas mRNA levels of other MRPs, multidrug resistance gene 1 or breast cancer resistance protein were unchanged, reduced or not detected. DEPe also increased MRP3 mRNA expression in normal human bronchial epithelial cells. Inhibition of the aryl hydrocarbon receptor (AhR) pathway by AhR antagonist or AhR silencing, as well as the silencing of nuclear-factor-E2-related factor 2 (Nrf2) repressed DEPe-mediated MRP3 induction. This underlines the implication of the AhR and Nrf2 signaling cascades in DEPe-mediated MRP3 regulation. DEPe was additionally demonstrated to directly inhibit MRP activity in BEAS-2B cells, in a concentration-dependent manner. Taken together, these data indicate that DEPs may impair expression and activity of MRPs, notably MRP3, in human bronchial cells, which may have consequences in terms of lung barrier and toxicity for humans exposed to diesel pollution., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Carcinogenic heterocyclic aromatic amines (HAAs) interact with some drug transporters, like the efflux pump BCRP and the organic anion transporters OAT1 and OAT3. The present study was designed to determine whether they can also target activities of the organic cation transporters (OCTs), using mainly OCT1-, OCT2- and OCT3-overexpressing HEK293 cells. Fifteen HAAs were demonstrated to differently alter OCT activities; with a cut-off of at least 50% reduction of transporter activity by 100 μM HAAs, 5/15 HAAs, including Trp-P-1 and Trp-P-2, inhibited activities of OCT1, OCT2 and OCT3, whereas 7/15 HAAs, including PhIP and MeIQx, blocked those of OCT2 and OCT3, 1/15 HAAs reduced those of OCT1 and OCT2 and 2/15 HAAs, including AαC, only that of OCT2. IC 50 values of Trp-P-1 and Trp-P-2 towards OCT activities were found to be in the 2-6 μM range, likely not relevant for human exposure to HAAs through smoking or the diet. Trp-P-1 and Trp-P-2 additionally failed to trans-stimulate OCT1 and OCT2 activities and exhibited similar accumulation in OCT1/2-transduced HEK293 cells and control HEK293-MOCK cells. These data demonstrate that HAAs, notably Trp-P-1 and Trp-P-2, interact with OCT1/2, without however being transported, thus likely discarding a major role for OCT1/2 in HAA systemic toxicokinetics., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Evaluation of hepatobiliary transport of drugs is an important challenge, notably during the development of new molecular identities. In this context, sandwich-cultured human hepatocytes (SCHH) have been proposed as an interesting and integrated tool for predicting in vitro biliary excretion of drugs. The present review was therefore designed to summarize key findings about SCHH, including their establishment, their main functional features and their use for the determination of canalicular transport and the prediction of in vivo biliary clearance and hepatobiliary excretion-related drug-drug interactions. Reviewed data highlight the fact that SCHH represent an original and probably unique holistic in vitro approach to predict biliary clearance in humans, through taking into account sinusoidal drug uptake, passive drug diffusion, drug metabolism and sinusoidal and canalicular drug efflux. Limits and proposed refinements for SCHH-based analysis of drug biliary excretion, as well as putative human alternative in vitro models to SCHH are also discussed.

In animals, cigarette smoke may alter pharmacokinetics by altering activity and expression of ABC drug transporters. We previously demonstrated that cigarette smoke condensate (CSC) impairs activity and expression of several hepatic ABC drug transporters which mediate toxicant efflux. However, CSC effects on efflux transporters are still unknown in Saccharomyces cerevisiae which resists diverse chemical stresses, by inducing pleiotropic drug resistance (PDR) genes among others. The yeast ABC transporters are functionally and structurally homologous to the mammalian ones. In this study, Saccharomyces cerevisiae exposure to CSC for 15 min caused a dose-dependent inhibition of rhodamine 123 efflux, whereas a longer exposure (3 h) induced mRNA expression of the ABC PDR efflux pumps Pdr5, Snq2, Pdr 10 and Pdr15, and of Tpo1, a member of the major facilitator superfamily (MFS). CSC also increased toxicity of caffeine, which is handled by two PDR transporters, Pdr5 and Snq2. Taken together, these data demonstrated that yeast efflux transporters are targets of cigarette smoke chemicals, and that Saccharomyces cerevisiae may cope with CSC-induced stress, including the initial efflux inhibition, by induction of the mRNA of several plasma membrane PDR and MFS efflux transporters. Saccharomyces cerevisiae is therefore a valid model to investigate pollutant effects on ABC and MFS transporters., (Copyright © 2018 Elsevier B.V. All rights reserved.)

HepaRG is an original human hepatoma cell line, acquiring highly differentiated hepatic features when exposed to dimethylsulfoxide (DMSO). To search alternatives to DMSO, which may exert some toxicity, we have analyzed the effects of forskolin (FSK), a cAMP-generating agent known to favor differentiation of various cell types. FSK used at 50 µM for 3 days was found to promote polarization of high density-plated HepaRG cells, i.e., it markedly enhanced the formation of functional biliary canaliculi structures. It also increased expressions of various hepatic markers, including those of cytochrome P-450 (CYP) 3A4, of drug transporters like NTCP, OATP2B1 and BSEP, and of metabolism enzymes like glucose 6-phosphatase. In addition, FSK-treated HepaRG cells displayed enhanced activities of CYP3A4, NTCP and OATPs when compared to untreated cells. These polarizing/differentiating effects of FSK were next shown to reflect not only the generation of cAMP, but also the activation of the xenobiotic sensing receptors PXR and FXR by FSK. Co-treatment of HepaRG cells by the cAMP analog Sp-5,6-DCl-cBIMPS and the reference PXR agonist rifampicin reproduced the polarizing effects of FSK. Therefore, FSK may be considered as a relevant alternative to DMSO for getting polarized and differentiated HepaRG cells, notably for pharmacological and toxicological studies.

Introduction: Drug transporters are now recognized as major actors of pharmacokinetics. They are also likely implicated in toxicokinetics and toxicology of environmental pollutants, notably pesticides, to which humans are widely exposed and which are known to exert various deleterious effects toward health. Interactions of pesticides with drug transporters are therefore important to consider. Areas covered: This review provides an overview of the interactions of pesticides with membrane drug transporters, i.e. inhibition of their activity, regulation of their expression, and handling of pesticides. Consequences for toxicokinetics and toxicity of pesticides are additionally summarized and discussed. Expert opinion: Some pesticides belonging to several chemical classes, such as organochlorine, pyrethroid, and organophosphorus pesticides, have been demonstrated to interact with various uptake and efflux drug transporters, including the efflux pump P-glycoprotein (P-gp) and the uptake organic cation transporters (OCTs). This provides proof of the concept that pesticide-transporter relationships merit attention. More extensive and systematic characterization of pesticide-transporter relationships, possibly through the use of in silico methods, is however likely required. In addition, consideration of transporter polymorphisms, pesticide mixture effects, and realistic pesticide concentrations reached in humans may help better define the in vivo relevance of pesticide-transporter interactions in terms of toxicokinetics and toxicity.

The lung is a preferential organ site for accumulation of lipophilic basic amine drugs, so-called pneumophilic drugs and belonging to various pharmacological classes, which can result in lung toxicity. In order to investigate the mechanism involved in such pulmonary accumulation of drugs, uptake of clonidine, used here as a prototypical basic amine drug, was characterized in cultured human lung cells. Clonidine accumulation in lung alveolar A549 cells was found to be temperature- and pH-dependent; it was saturable, with a Michaelis-Menten affinity constant (Km) value of 569.4 μM. Various pneumophilic drugs, including amitriptyline, verapamil, propranolol, chlorpromazine, imipramine, and quinidine, markedly cis-inhibited clonidine uptake in A549 cells, in a dose-dependent manner for at least some of them. They additionally trans-stimulated clonidine efflux from A549 cells, thus suggesting that they are substrates for the putative clonidine transporter. In addition to alveolar A549 cells, bronchial epithelial BEAS-2B cells as well as lung endothelial HULEC-5a cells were found to exhibit clonidine accumulation abrogated by amitriptyline, verapamil, and chlorpromazine. Taken together, these data likely provided evidence for carrier-mediated uptake of clonidine in human lung cells. This carrier, which remains to be molecularly identified, interacts with various pneumophilic drugs, suggesting that it may contribute to lung accumulation of these drugs in a notable way.

The inflammatory cytokine interleukin (IL)-6, which basically activates the Janus kinase (JAK)/ signal transducer and activator of transcription (STAT) signaling pathway, is well known to repress expression of hepatic cytochromes P-450 (P450s) and transporters. Therapeutic proteins, like monoclonal antibodies targeting IL-6 or its receptor, have consequently been demonstrated to restore full hepatic detoxification capacity, which results in inflammatory disease-related drug-drug interactions (idDDIs). In the present study, we investigated whether ruxolitinib, a small drug acting as a JAK1/2 inhibitor and currently used in the treatment of myeloproliferative neoplasms, may also counteract the repressing effects of IL-6 toward hepatic detoxifying systems. Ruxolitinib was found to fully inhibit IL-6-mediated repression of P450 (CYP1A2, CYP2B6, and CYP3A4) and transporter (NTCP, OATP1B1, and OCT1) mRNA levels in primary human hepatocytes and differentiated hepatoma HepaRG cells. Such effects were dose-dependent, with ruxolitinib EC 50 values around 1.0-1.2 μ M and thus close to ruxolitinib plasma levels that can be reached in patients. Moreover, they were associated with concomitant restoration of P450 and drug transporter activities in IL-6-exposed HepaRG cells. By contrast, ruxolitinib failed to suppress the repression of drug-detoxifying protein mRNA levels caused by IL-1 β The JAK inhibitor and anti-rheumatoid arthritis compound tofacitinib was additionally found to reverse IL-6-mediated suppression of P450 and transporter mRNA expressions. Taken together, our results demonstrated that small drugs acting as JAK inhibitors, like ruxolitinib, counteract IL-6-mediated repression of drug-metabolizing enzymes and drug transporters in cultured human hepatocytes. These JAK inhibitors may consequently be hypothesized to restore hepatic detoxification capacity for patients suffering from inflammatory diseases, which may in turn cause idDDIs., (Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.)

Background and Objectives: Gevokizumab is a potent anti-interleukin (IL)-1β neutralizing monoclonal antibody (mAb), which may be used for treating inflammatory or autoimmune diseases. The present study was designed to characterize the potential effects of this mAb towards well-established IL-1β-mediated repression of hepatic drug detoxifying proteins, like cytochrome P450 (CYP) 3A4 and drug transporters., Methods: Primary cultured human hepatocytes were exposed to various concentrations of IL-1β in the absence or presence of gevokizumab (5 µg/mL); mRNA expression and activity of CYP3A4 and transporters were next determined., Results: Gevokizumab was found to down-modulate, but not abolish, the repression of CYP3A4 and drug transporter mRNAs caused by IL-1β in human hepatocytes, through shifting up IL-1β half maximal inhibitory concentration (IC 50 ) values by factors ranging from 6.8 to 10.4. The mAb concomitantly shifted IL-1β IC 50 values towards CYP3A4 activity from 22.0 pg/mL (in the absence of gevokizumab) to 796 pg/mL (in the presence of gevokizumab) and counteracted the decrease of organic anion-transporting polypeptide activity occurring in response to 50 pg/mL IL-1β, but not that occurring at higher IL-1β concentration (1000 pg/mL)., Conclusion: Gevokizumab attenuates, but not abolishes, IL-1β-mediated functional repression of CYP3A4 and drug transporters in human hepatocytes, which agrees with the fact that the mAb is considered as a modulator and not a blocker of IL-1β signaling. This attenuation of IL-1β-mediated down-regulation of hepatic detoxifying proteins by gevokizumab may have to be evaluated in terms of potential therapeutic protein drug-drug interactions when considering future development and therapeutic uses of this IL-1β neutralizing mAb.

Drug transporters are now recognized as major actors in pharmacokinetics, involved notably in drug-drug interactions and drug adverse effects. Factors that govern their activity, localization and expression are therefore important to consider. In the present review, the implications of protein kinases C (PKCs) in transporter regulations are summarized and discussed. Both solute carrier (SLC) and ATP-binding cassette (ABC) drug transporters can be regulated by PKCs-related signaling pathways. PKCs thus target activity, membrane localization and/or expression level of major influx and efflux drug transporters, in various normal and pathological types of cells and tissues, often in a PKC isoform-specific manner. PKCs are notably implicated in membrane insertion of bile acid transporters in liver and, in this way, are thought to contribute to cholestatic or choleretic effects of endogenous compounds or drugs. The exact clinical relevance of PKCs-related regulation of drug transporters in terms of drug resistance, pharmacokinetics, drug-drug interactions and drug toxicity remains however to be precisely determined. This issue is likely important to consider in the context of the development of new drugs targeting PKCs-mediated signaling pathways, for treating notably cancers, diabetes or psychiatric disorders.

Human hepatoma cells may represent a valuable alternative to the use of human hepatocytes for studying hepatic drug transporters, which is now a regulatory issue during drug development. In the present work, we have characterized hepatic drug transporter expression, activity and regulation in human hepatoma HuH-7 cells, in order to determine the potential relevance of these cells for drug transport assays. HuH-7 cells displayed notable multidrug resistance-associated protein (MRP) activity, presumed to reflect expression of various hepatic MRPs, including MRP2. By contrast, they failed to display functional activities of the uptake transporters sodium taurocholate co-transporting polypeptide (NTCP), organic anion-transporting polypeptides (OATPs) and organic cation transporter 1 (OCT1), and of the canalicular transporters P-glycoprotein and breast cancer resistance protein (BCRP). Concomitantly, mRNA expressions of various sinusoidal and canalicular hepatic drug transporters were not detected (NTCP, OATP1B1, organic anion transporter 2 (OAT2), OCT1 and bile salt export pump) or were found to be lower (OATP1B3, OATP2B1, multidrug and toxin extrusion protein 1, BCRP and MRP3) in hepatoma HuH-7 cells than those found in human hepatocytes, whereas other transporters such as OAT7, MRP4 and MRP5 were up-regulated. HuH-7 cells additionally exhibited farnesoid X receptor (FXR)- and nuclear factor erythroid 2-related factor 2 (Nrf2)-related up-regulation of some transporters. Such data indicate that HuH-7 cells, although expressing rather poorly some main hepatic drug transporters, may be useful for investigating interactions of drugs with MRPs, notably MRP2, and for studying FXR- or Nrf2-mediated gene regulation.

In vitro evaluation of P-glycoprotein (P-gp) inhibitory potential is now a regulatory issue during drug development, in order to predict clinical inhibition of P-gp and subsequent drug-drug interactions. Assays for this purpose, commonly based on P-gp-expressing cell lines and digoxin as a reference P-gp substrate probe, unfortunately exhibit high variability, raising thus the question of developing alternative or complementary tests for measuring inhibition of P-gp activity. In this context, the present study was designed to investigate the use of the fluorescent dye rhodamine 123 as a reference P-gp substrate probe for characterizing P-gp inhibitory potential of 16 structurally-unrelated drugs known to interact with P-gp. 14/16 of these P-gp inhibitors were found to increase rhodamine 123 accumulation in P-gp-overexpressing MCF7R cells, thus allowing the determination of their P-gp inhibitory potential, i.e., their half maximal inhibitor concentration (IC50) value towards P-gp-mediated transport of the dye. These IC50 values were in the range of variability of previously reported IC50 for P-gp and can be used for the prediction of clinical P-gp inhibition according to Food and Drug Administration (FDA) criteria, with notable sensitivity (80%). Therefore, the data demonstrated the feasibility of the use of rhodamine 123 for evaluating the P-gp inhibitory potential of drugs.

Hepatic drug transporters play an important role in pharmacokinetics and drug-drug interactions. Among these membrane transporters, the sodium taurocholate cotransporting polypeptide (NTCP/SLC10A1), the organic anion transporting polypeptides (OATPs) 1B1 (SLCO1B1), 1B3 (SLCO1B3) and 2B1 (SLCO2B1), the organic anion transporter 2 (OAT2/SLC22A7) and the organic cation transporter 1 (OCT1/SLC22A1) are likely major ones, notably mediating sinusoidal uptake of various drugs or endogenous compounds, like bile acids, from blood into hepatocytes. Studying putative interactions of drugs, including those in development processes, with these transporters is an important issue. For this purpose, cultured human hepatocytes, that exhibit functional expression of NTCP, OATPs, OAT2 and OCT1, are considered as a relevant in vitro cellular model. This chapter describes a method allowing to accurately analyze NTCP, OATP, OAT2 and OCT1 transport activities in primary human hepatocyte cultures, which can be applied to the determination of potential interactions of drugs with these hepatic uptake transporters.

Background: The aryl hydrocarbon receptor (AhR) is a transcription factor activated by several environmental pollutants, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and involved in carcinogenesis and various physiological processes, including immune response and endocrine functions. Characterization of kinases-related AhR transduction pathway remains an important purpose., Results: We performed a kinome-wide siRNA screen in human mammary MCF-7 cells to identify non redundant protein kinases implicated in the up-regulation of cytochrome P-450 (CYP) 1A1 activity, an AhR referent target, in response to TCDD exposure. To this aim, we monitored CYP1A1-related ethoxyresorufin-O-deethylase (EROD) activity and quantified cell density. This normalization was crucial since it allowed us to focus only on siRNA affecting EROD activity and discard siRNA affecting cell density. Analyses of the cell density data allowed us to identify several hits already well-characterized as effectors of the cell cycle and original hits. Collectively, these data fully validated the protocol and the siRNA library. Next, 22 novel candidates were identified as kinases potentially implicated in the up-regulation of CYP1A1 in response to TCDD, without alteration of cell survival or cell proliferation. The siRNA library screen gave a limited number of hits (approximately 3%). Interestingly, four of them are able to bind calmodulin among which the IP3 kinase A (ITPKA) and pregnancy up-regulated non-ubiquitously expressed CaM kinase (PNCK, also named CaMKIβ). Remarkably, for both proteins, their kinase activity depends on the calmodulin binding. Involvement of ITPKA and PNCK in TCDD-mediated CYP1A1 up-regulation was further validated by screening-independent expression knock-down. PNCK was finally shown to regulate activation of CaMKIα, a CaMKI isoform previously reported to interplay with the AhR pathway., Conclusions: These data fully support a role for both IP3-related kinase and CaMK isoforms in the AhR signaling cascade. More generally, this study also highlights the interest of large scale loss-of-function screens for characterizing the molecular mechanism of action of environmental contaminants.

Background: Sinuosidal and canalicular hepatic drug transporters, involved in drug uptake in the liver and drug secretion in the bile, respectively, play a major role in liver drug clearance. Inflammation is well-known to impair expression of these transporters in rodents; data about this topic have been more recently reported in human hepatocytes., Objective: The present review is designed to summarise the effects of pro-inflammatory cytokines such as IL-1beta, TNF-alpha and IL-6 toward human hepatic drug and bile salt transporters., Methods: Recent studies aimed at analyzing transporter expression and activity in cytokines-exposed primary human hepatocytes and well-differentiated human hepatoma cells are resumed and discussed., Results/conclusion: Exposure to IL-1beta, TNF-alpha or IL-6 markedly alters expression profile of human hepatic transporters. Bile salt transporters as well as sinusoidal solute carrier transporters are usually repressed, whereas ATP-binding cassette drug efflux pumps remain unchanged or are either downregulated or upregulated. These changes are observed at mRNA levels, but also, for some of the transporters, at protein and activity levels. They are likely to contribute to alterations of drug pharmacokinetics, impairment of bile salt secretion and cholestasis caused by inflammation in humans.

Arsenic is a toxic metalloid known to interact with drug-metabolizing enzymes. In the present study, we investigated the effects of arsenic trioxide (As2O3), recently used as an anticancer drug, on the expression of human cytochrome P450 (P450) 1A1, which bioactivates polycyclic aromatic hydrocarbons into mutagenic metabolites. Clinically relevant concentrations (0.25-5 microM) of As2O3 were demonstrated to inhibit CYP1A activity in primary human hepatocytes and hepatoma Hep3B and HepG2 cells coexposed to 3-methylcholanthrene (3MC), benzo(a)pyrene, or dioxin and the metalloid for 24 h. Inhibition reached 50 and 90% in Hep3B cells treated with 1 and 5 microM As2O3, respectively, and was not due to direct interaction of the metalloid with CYP1A1. As2O3 (2.5-5 microM) was demonstrated to markedly reduce induction of CYP1A1 mRNA and apoprotein levels and gene promotor activity in 3MC-treated Hep3B cells, whereas lower concentrations (0.25-1 microM) were ineffective. These effects of As2O3 were abrogated by N-acetylcysteine. Surprisingly, this agent was found 1) to block cellular arsenic uptake when coincubated with the metalloid and 2) to increase arsenic efflux through multidrug resistance-associated proteins. In addition, blockade of these transporters was shown to enhance intracellular amounts of metalloid and to potentiate its effects on CYP1A1 gene. Finally, our results have demonstrated that As2O3, at low concentrations routinely reached in As2O3-treated patients, prevents induction of human CYP1A1 gene expression and that such an effect is increased by blocking multidrug resistance-associated proteins.