Regulation of Hepatic Drug Transporter Activity ... - Wiley Online Library

J BIOCHEM MOLECULAR TOXICOLOGY Volume 28, Number 3, 2014

Regulation of Hepatic Drug Transporter Activity and Expression by Organochlorine Pesticides Simon Bucher,1 Marc Le Vee,1 Elodie Jouan,1 and Olivier Fardel1,2 1 Institut de Recherches en Sant´ e, Environnement et Travail (IRSET), UMR INSERM U1085, Facult´e de Pharmacie, Universit´e de Rennes 1, Rennes, France 2 Pole ˆ Biologie, Centre Hospitalier Universitaire, Rennes, France; Email: [email protected]

Received 21 September 2013; revised 31 October 2013; accepted 1 November 2013

ABSTRACT: Organochlorine (OC) pesticides constitute a major class of persistent and toxic organic pollutants, known to modulate drug-detoxifying enzymes. In the present study, OCs were demonstrated to also alter the activity and expression of human hepatic drug transporters. Activity of the sinusoidal influx transporter OCT1 (organic cation transporter 1) was thus inhibited by endosulfan, chlordane, heptachlor, lindane, and dieldrine, but not by dichlorodiphenyltrichloroethane isomers, whereas those of the canalicular efflux pumps MRP2 (multidrug resistance-associated protein 2) and BCRP (breast cancer resistance protein) were blocked by endosulfan, chlordane, heptachlor, and chlordecone; this latter OC additionally inhibited the multidrug resistance gene 1 (MDR1)/P-glycoprotein (P-gp) activity. OCs, except endosulfan, were next found to induce MDR1/P-gp and MRP2 mRNA expressions in hepatoma HepaRG cells; some of them also upregulated BCRP. By contrast, expression of sinusoidal transporters was not impaired (organic anion-transporting polypeptide (OATP) 1B1 and OATP2B1) or was downregulated (sodium taurocholate co-transporting polypeptide (NTCP) and OCT1). Such regulations of drug transporter activity and expression, depending on the respective nature of OCs and transporters, may contribute to the toxicity of C 2013 Wiley Periodicals, Inc. J Biochem OC pesticides. Mol Toxicol 28:119–128, 2014; View this article online at wileyonlinelibrary.com. DOI 10.1002/jbt.21543

KEYWORDS: Organochlorines; Pesticides; Drug Transporter; Liver; Influx; Efflux; Regulation

INTRODUCTION Organochlorine (OC) pesticides constitute a major class of lipophilic organic pollutants that have been C

Correspondence to: Olivier Fardel. 2013 Wiley Periodicals, Inc.

largely used as neurotoxic insecticides in the past [1]. Although most of them are now banished in industrialized countries, humans remain widely exposed to OCs because these chemicals are highly resistant to degradation and, therefore, persist in the environment [2]. Moreover, OCs accumulate in organ tissues, including the liver, without essentially undergoing metabolism, and thus showing long half-lives [3, 4]. Chronic exposure to OCs is thought to contribute to various health problems, such as endocrine disruption, metabolic disorders like diabetes, neurodegenerative pathologies, including Parkinson’s disease, cancers, immune dysregulations, and reproductive and birth defects [5–11]. In addition, OCs, like other pesticides, can interact with drug-detoxification pathways, especially at the liver level [12, 13]. They thus upregulate the expression of hepatic drug metabolizing enzymes like cytochrome P-450 (CYP) 3A4 [14] through, at least for some of them, activating the drug-sensing receptor pregnane X receptor (PXR) [15, 16]. Drug transporters, whose major role in xenobiotic elimination and hepatic drug clearance is now well recognized [17], are also targeted by OCs [18]. Indeed, activity of the adenosine triphosphate (ATP)binding cassette (ABC) efflux pump P-glycoprotein (Pgp), encoded by multidrug resistance gene 1 (MDR1) and physiologically expressed at the canalicular pole of hepatocytes [19], is inhibited by various OCs, including 4,4 -dichlorodiphenyltrichloroethane (DDT), endosulfan, and chlordecone [20–22], whereas that of the breast cancer resistance protein (BCRP/ABCG2), an ABC transporter also present at the biliary pole of hepatocytes, is blocked by DDT and endosulfan [22]. Putative interactions of OCs with hepatic transporters are also supported by the fact that OCs, including chlordecone, are likely to undergo enterohepatic circulation [23], which is consistent with their transportersmediated hepatobiliary secretion. OCs may also impair expression levels of drug transporters. Indeed, 119

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treatment by the OC dieldrin stimulates biliary secretion of benzo(a)pyrene polar metabolites, thus suggesting that this pesticide induces the expression of transporter(s) involved in this secretion [24]; in the same way, treatment of rats by chlordecone has been reported to increase biliary efflux of the pesticide, favoring the idea that chlordecone enhances the expression of a liver transporter involved in its own canalicular secretion [25]. Despite these data, potential interactions of OCs with hepatic transporter activity and/or expression remain rather poorly characterized. The present study was, therefore, designed to gain insights about this point. For this purpose, the effects of various OCs toward the activity and expression of human hepatic drug transporters were analyzed in vitro, using highly differentiated human hepatoma HepaRG cells and transporter-overexpressing cell lines. Transporters analyzed in this work were the sinusoidal transporters sodium taurocholate co-transporting polypeptide (NTCP/SLC10A1), organic anion transporting polypeptide (OATP) 1B1 (SLCO1B1), OATP2B1 (SLOC2B1), and organic cation transporter 1 (OCT1/SLC22A1), involved in the uptake of xenobiotics from blood into hepatocytes, and the canalicular transporters MDR1/P-gp, multidrug resistance-associated protein 2 (MRP2/ABCC2), and BCRP, mediating the secretion of drugs or drug metabolites into the bile [26]. Studied OCs were reference OCs, namely the chloroethane derivatives 4,4 -DDT and 2,4 -DDT, the cyclodienes endosulfan, chlordane, heptachlor, and dieldrin, the hexachlorohexane lindane, and the caged OC chordecone; their chemical structures are indicated in Figure S1 in the Supporting Information. Our data highlight differential regulations of hepatic transporter activity and expression by OCs, depending on the nature of transporters and OCs.

MATERIALS AND METHODS

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sulfate (E3S) (sp. act. 57.3 Ci/mmol), and [1–14 C] tetraethylammonium (TEA) (sp. act. 2.4 mCi/mmol) were from Perkin-Elmer (Boston, MA). All other chemicals were commercial products of the highest purity available.

Cell Culture Highly-differentiated human hepatoma HepaRG cells, which exhibit activity of the sinusoidal solute carrier (SLC) transporters NTCP, OATPs, and OCT1 [27], and are now well recognized as surrogates for human hepatocytes in drug-detoxification pathway studies [28], were routinely cultured in Williams’ E medium (Life Technologies) supplemented with 10% (vol/vol) fetal calf serum, 100 IU/mL penicillin, 100 µg/mL streptomycin, 5 µg/mL insulin, 2 mM glutamine, and 5 × 10−5 M hydrocortisone hemisuccinate; additional culture for 2 weeks in the same medium supplemented with 2% (vol/vol) dimethylsulfoxide was performed to get a full hepatocytic differentiation of the cells [29]. For OC treatment, cells were shifted to a dimethylsulfoxide- and fetal calf serum-free medium for 24 h before the beginning of OC exposure. P-gp-overexpressing mammary MCF-7/R cells [30] and MRP2-expressing human hepatoma Huh7 cells [31] were cultured in Dulbecco’s modified Eagle medium (DMEM) (Life Technologies), supplemented with 10% (vol/vol) fetal calf serum, 100 IU/mL penicillin, and 100 µg/mL streptomycin. BCRP-transfected HEK 293 cells [32], kindly gifted by Dr. X. Decleves (Faculty of Pharmacy, University Paris-Descartes, Paris, France), were cultured in DMEM supplemented with 10% (vol/vol) fetal calf serum, 100 IU/mL amoxicillin, 100 µg/mL erythromycin, and 200 µg/mL G418.

Transporter Activity Assays SLC Transporter Activity

Chemicals DDT isomers, endosulfan (used under its commercial form, i.e., a mixture of the two stereoisomers α- and β-endosulfan in a ratio 7:3), chlordane, heptachlor, dieldrin, lindane, chlordecone, rhodamine 123, verapamil, probenecid, fumitremorgin C, 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and G418 were provided by SigmaAldrich (Saint-Quentin Fallavier, France), whereas carboxy-2,7-dichlorofluorescein (CF) diacetate and Hoechst 33342 were from Life Technologies (Saint Aubin, France). [3 H(G)] Taurocholic acid (specific activity (sp. act.) 1.19 Ci/mmol), [6, 7–3 H(N)] estrone-3-

The effects of OC pesticides on NTCP, OATP, and OCT1 activities were analyzed in HepaRG cells, through determining the intracellular accumulation of radiolabeled substrates of these sinusoidal influx transporters in a well-defined transport medium, as previously described [27]. Briefly, for NTCP activity, HepaRG cells were incubated at 37◦ C with 43.4 nM [3 H] taurocholate for 10 min, in the absence or presence of sodium or OC pesticides; for OATP activity, HepaRG cells were incubated at 37◦ C with 3.4 nM [3 H] E3S for 3 min, in the absence or presence of OCs or 2 mM probenecid, used here as a reference OATP inhibitor; for OCT1 activity, HepaRG cells were incubated J Biochem Molecular Toxicology

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ORGANOCHLORINES AND HEPATIC TRANSPORTERS

at 37◦ C with 40 µM [14 C] TEA for 5 min, in the absence or presence of OCs or 50 µM verapamil, used here as a reference OCT1 inhibitor. After washing in phosphatebuffered saline (PBS), cells were lyzed and accumulation of radiolabeled substrates was determined through scintillation counting.

ABC Transporter Activity The effects of OC pesticides on P-gp activity were analyzed in P-gp-expressing MCF-7/R cells, through measuring the intracellular accumulation of the P-gp substrate rhodamine 123 [33]. Briefly, MCF-7/R cells were incubated at 37◦ C with 5.25 µM rhodamine 123 for 30 min, in the presence or absence of OCs or 50 µM verapamil, used here as a reference inhibitor for Pgp. After washing in PBS, cells were lyzed and intracellular accumulation of rhodamine 123 was next determined by spectrofluorimetry using a SpectraMax Gemini SX spectrofluorometer (Molecular Devices, Sunnyvale, CA) (excitation and emission wavelengths were 485 and 535 nm, respectively). The effects of OC pesticides on MRP2 activity were analyzed in MRP2-expressing Huh7 cells, through measuring the intracellular accumulation of the MRP2 substrate CF [34]. Briefly, Huh7 cells were incubated at 37◦ C with 3 µM CF diacetate for 30 min, in the presence or absence of OCs or 2 mM probenecid, used here as a reference inhibitor for MRP2 [34]. After washing in PBS, cells were lyzed and the intracellular accumulation of CF was next determined by spectrofluorimetry using a SpectraMax Gemini SX spectrofluorometer (excitation and emission wavelengths were 485 and 535 nm, respectively). The effects of OC pesticides on BCRP activity were analyzed in BCRP-HEK 293 cells, through measuring the intracellular retention of the BCRP substrate Hoechst 33342 [35]. Briefly, BCRP-HEK 293 cells were first loaded at 37◦ C with 16.2 µM Hoechst 33342 for 30 min. After washing in PBS, cells were reincubated in Hoechst 33342-free medium at 37◦ C for 90 min in the absence or presence of OCs or 10 µM fumitremorgin C, used here as a reference inhibitor for BCRP. After washing in PBS, cells were lyzed and the intracellular retention of Hoechst 33342 was next determined by spectrofluorimetry using a SpectraMax Gemini SX spectrofluorometer (excitation and emission wavelengths were 355 and 460 nm, respectively).

Cell Viability Assay Viability of HepaRG cells exposed to OC pesticides for 48 h was determined using a colorimetric MTT assay, as previously described [36]. J Biochem Molecular Toxicology

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RNA Isolation and Analysis Total RNAs were extracted using the TRI reagent (Sigma-Aldrich). RNA was then subjected to reverse transcription-quantitative polymerase chain reaction (RT-qPCR), using the reverse transcription kit from Applied Biosystems (Foster City, CA), the fluorescent dye SYBR Green methodology and an ABI Prism 7300 detector (Applied Biosystems), as previously described [37]. Gene-specific primers for transporters, CYP3A4 and 18 S were exactly as previously described [27, 38]. The specificity of each gene amplification was verified at the end of qPCR reactions through the analysis of dissociation curves of the PCR products. Amplification curves were next analyzed with ABI Prism 7000 SDS software, using the comparative cycle threshold method. Relative quantification of the steady-state target mRNA levels was calculated after normalization of the total amount of cDNA tested to the 18S mRNA endogenous reference using the 2(-Ct) method.

Statistical Analysis Quantitative data were analyzed using analysis of variance followed by a Dunnett’s post-hoc test. The criterion of significance was p < 0.05. Half maximal inhibitory concentration (IC50 ) or half maximal effective concentration (EC50 ) values were determined using GraphPad Prism software (GraphPad Software, La Jolla, CA).

RESULTS Effects of OC Pesticides on Activity of Hepatic Sinusoidal Transporters NTCP, OATP, and OCT1 activities were determined in HepaRG cells in the presence or absence of OCs used at 100 µM. As indicated in Figure 1A, the various OCs failed to significantly alter taurocholate accumulation in HepaRG cells, thus indicating that NTCP activity was not impaired; by contrast, retrieval of sodium from the transport assay medium markedly inhibited taurocholate accumulation (Figure 1A), which is consistent with the fact that NTCP acts as a sodiumdependent transporter and consequently indicates that NTCP was fully active in HepaRG cells. OCs, unlike the OATP inhibitor probenecid, did not also reduce the accumulation of E3S, a substrate shared by OATP1B1 and OATP2B1 [39], in HepaRG cells (Figure 1A), demonstrating that OATP activity was not impaired by OCs. By contrast, various OCs, such as endosulfan, chordane, heptachlor, dieldrin, and lindane, decreased the

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FIGURE 1. Effects of OC pesticides on the activity of hepatic SLC transporters. (A) Intracellular accumulations of the NTCP substrate taurocholate, the OATP substrate E3S, and the OCT1 substrate TEA were analyzed in HepaRG cells, in the absence (control) or presence of various OCs, each used at 100 µM, as described in Materials and Methods; retrieval of sodium (NTCP activity) and addition of 2 mM probenecid (OATP activity) or 50 µM verapamil (OCT1 activity) were performed in parallel to get referent transporter inhibition. Data are expressed as percentage of substrate accumulation in control cells (indicated by a dashed line) and are Means ± SEM of three independent assays; *p < 0.05 when compared with control; (B) accumulation of the OCT1 substrate TEA was determined in HepaRG cells, in the absence or presence of various concentrations (from 0.01 to 100 µM) of dieldrin and lindane; data are expressed as percentage of TEA accumulation in the absence of OC and are the Means ± SEM of three independent assays. IC50 values are indicated on the top of each graph.

intracellular accumulation of TEA in HepaRG cells (Figure 1A), most likely suggesting that these OCs inhibited OCT1 activity. The well-established OCT1 inhibitor verapamil also markedly reduced TEA uptake into HepaRG cells, whereas, by contrast, 4,4 -DDT, 2,4 -DDT, and chlordecone were without effect (Figure 1A). Inhibitions of TEA accumulation by dieldrin and lindane were moreover found to be dependent on OC concentrations, with estimated IC50 values of 0.90 and 1.52 µM for dieldrin and lindane, respectively (Figure 1B).

Effects of OC Pesticides on Activity of Hepatic Canalicular ABC Efflux Transporters The effects of various OCs used at 100 µM on the activity of the efflux pump P-gp were first determined in P-gp-overexpressing MCF-7/R cells. As shown in Figure 2A, only chlordecone was able to significantly increase the accumulation of the P-gp substrate rhodamine 123 in MCF-7/R cells, thereby supporting the idea that this OC inhibited P-gp-mediated efflux of the dye out of MCF-7/R cells. Verapamil similarly

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probenecid enhanced accumulation of the MRP2 substrate CF in Huh7 cells (Figure 3A), thus indicating that they were able to inhibit MRP2-mediated efflux of the dye out of Huh7 cells. Stimulation of CF accumulation by chlordecone and endosulfan was next demonstrated to be concentration dependent, the concentration of 300 µM being the most active for the two OCs (Figure 3B); these concentration-dependent effects however failed to be modeled and no EC50 value can, therefore, be determined. By contrast, the stimulatory effect of probenecid toward CF accumulation was successfully modeled, allowing to determine an EC50 value of 0.62 mM for probenecid (Figure 3B). The putative effects of OCs used at 100 µM on BCRP activity were finally studied in BCRP-HEK 293 cells. Endosulfan, chlordane, heptachlor, and chlordecone significantly enhanced the cellular retention of Hoechst 33342 (Figure 4A), indicating that these OCs most likely inhibited BCRP-mediated efflux of this dye out of BCRP-HEK 293 cells. Fumitremorgin C was also found to markedly enhance Hoechst 33342 retention (Figure 4A), as expected for this well-established referent BCRP inhibitor [41]. Increases of Hoechst 33342 retention by endosulfan and chlordecone were next shown to be concentration dependent, with EC50 values of 24.8 and 90.4 µM for endosulfan and chlordecone, respectively (Figure 4B).

FIGURE 2. Effects of OC pesticides on the activity of P-gp. (A) Intracellular accumulation of the P-gp substrate rhodamine 123 was analyzed in P-gp-overexpressing MCF-7/R cells, in the absence (control) or presence of various OCs, each used at 100 µM, or of the P-gp inhibitor verapamil (50 µM), as described in Materials and Methods. Data are expressed as percentage of rhodamine 123 accumulation in control cells (indicated by a dashed line) and are Means ± SEM of three independent assays; *p < 0.05 when compared with control; (B) accumulation of rhodamine 123 was determined in MCF-7/R cells, in the absence or the presence of various concentrations of chlordecone (from 1 to 350 µM). Data are expressed as percentage of rhodamine 123 accumulation in the absence of chlordecone and are Means ± SEM of three assays. EC50 value is indicated on the top of the graph.

enhanced rhodamine 123 level in MCF-7/R cells, thus agreeing with the well-known inhibitory effect of verapamil toward P-gp activity [19]. Stimulation of rhodamine 123 accumulation by chlordecone was concentration dependent, with an EC50 value of 138.8 µM (Figure 2B). Activity of the efflux pump MRP2, physiologically expressed at the biliary pole of hepatocytes like P-gp and BCRP [40], was next analyzed in MRP2-expressing hepatoma Huh7 cells, in the absence or presence of various OCs used at 100 µM. Endosulfan, chordane, heptachlor, chordecone, and the referent MRP2 inhibitor J Biochem Molecular Toxicology

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Regulation of Hepatic Drug Transporter Expression by OC Pesticides The effects of OC treatment on transporter mRNA expression were analyzed in HepaRG cells, which have been demonstrated to be convenient for studying regulatory pathways of hepatic detoxifying proteins, including those of transporters [42]. HepaRG cells were exposed to 10 µM OCs for 48 h, knowing that similar treatment conditions have been previously shown to induce the expression of CYPs in primary hepatocytes [43]. We first verified that these exposures to OCs were not deleterious for HepaRG cells. As shown in Figure S2 in the supporting information, 2,4 -DDT, 4,4 DDT, endosulfan, chlordane, heptachlor, dieldrin, and lindane did not exert cytotoxicity, as assessed by MTT viability assay; by contrast, chlordecone, used at 10 µM and also at the lower concentration of 1 µM, reduced HepaRG cell viability and was, therefore, not retained for the transporter regulation studies that followed. Owing to the fact that OCs have been previously shown to induce CYP3A4 expression in human hepatocytes through activating PXR [15], their effects toward CYP3A4 mRNA levels in HepaRG cells were first determined. As indicated in Figure 5A, 4,4 -DDT, 2,4 DDT, chlordane, heptachlor, dieldrin, and lindane markedly

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FIGURE 3. Effects of OC pesticides on activity of MRP2. (A) Intracellular accumulation of the MRP2 substrate CF was analyzed in MRP2expressing Huh7 cells, in the absence (control) or presence of various OCs, each used at 100 µM, or of the MRP2 inhibitor probenecid (2 mM), as described in Materials and Methods. Data are expressed as percentage of CF accumulation in control cells (indicated by a dashed line) and are Means ± SEM of three independent assays; *p < 0.05 when compared with control; (B) accumulation of CF was determined in Huh7 cells, in the absence or the presence of various concentrations of chlordecone (from 1 to 300 µM), endosulfan (from 1 to 300 µM) or probenecid (from 0.01 to 10 mM). Data are expressed as percentage of CF accumulation in control cells and are Means ± SEM of three assays. Probenecid EC50 value is indicated on the top of the corresponding graph.

and significantly enhanced the CYP3A4 mRNA expression by factors ranging from 6.5-fold (for heptachlor) to 11.6-fold (for lindane). Endosulfan treatment also induced the CYP3A4 expression but by a weaker factor (2.8-fold), and this upregulation failed to reach a significant level (Figure 5A). NTCP mRNA expression was next found to be repressed by all OCs, whereas those of OATP1B1 and OATP2B1 mRNAs were not impaired (Figure 5B). OCs also decreased the OCT1 mRNA expression, knowing,

however, that such a downregulation failed to reach a significant level for 4,4 -DDT (Figure 5B). By contrast, OCs, except endosulfan, induced the expression of MDR1/P-gp and MRP2 mRNAs (Figure 5C); the levels of MDR1/P-gp induction ranged from 1.7-fold (for heptachlor) to 2.3-fold (for chlordane), and those of MRP2 induction from 1.5-fold (for 4,4 -DDT and dieldrin) to 1.8-fold (for lindane). Some OCs, that is, 2,4 -DDT, chlordane, dieldrin, and lindane, also significantly enhanced BCRP mRNA levels (Figure 5C). J Biochem Molecular Toxicology

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DISCUSSION

FIGURE 4. Effects of OC pesticides on activity of BCRP. (A) Intracellular retention of the BCRP substrate Hoechst 33342 was analyzed in BCRP-transfected HEK 293 cells, in the absence (control) or presence of various OCs, each used at 100 µM, or of the BCRP inhibitor fumitremorgin C (FTC) (10 µM), as described in Materials and Methods. Data are expressed as percentage of initial CF loading and are Means ± SEM of three independent assays; *p < 0.05 when compared with control. Dashed line indicates Hoechst 33342 retention level in control cells; (B) retention of Hoechst 33342 was determined in BCRP-HEK 293 cells, in the absence or the presence of various concentrations (from 1 to 300 µM) of endosulfan or chlordecone. Data are expressed as percentage of initial loading of Hoechst 33342 and are Means ± SEM of three assays. EC50 values are indicated on the top of each graph. J Biochem Molecular Toxicology

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The data reported in the present study fully confirm that hepatic transporters constitute targets for OC pesticides. The nature of the effects, that is, inhibition of transporter activity or induction or repression of their expression, however, depends on both transporters and OCs. Indeed, with respect to the activity of sinusoidal uptake transporters, only that of OCT1 was inhibited by certain OCs (endosulfan, chlordane, heptachlor, lindane, and dieldrine). In the same way, if chlordecone blocked activities of P-gp, MRP2 and BCRP, endosulfan, chlordane, and heptachlor acted only on MRP2 and BCRP activities, whereas DDT isomers, lindane, and dieldrin were without effects on the ABC transporters. Such data are fully consistent with previously published works demonstrating, for example, that chlordecone, unlike DDT, inhibits P-gp activity [21]. However, some of them do not agree with other studies indicating that 4,4 -DDT inhibits ATPase activity of P-gp and BCRP [22] and that endosulfan blocks transport and ATPase activity due to P-gp [44, 45]; such discrepancies may be due to differences in methods and cellular models used for investigating the transporter activity, as already discussed [44, 46]. With respect to the expression of transporters, some of them, such as MDR1 and MRP2, were induced, whereas, by contrast, others were not impaired (OATP1B1 and OATP2B1) or were downregulated (NTCP or OCT1) by most, if not all, OCs; some OCs were moreover shown to increase the BCRP expression. These regulations were observed at mRNA level, knowing, however, that drug transporters are usually coordinately regulated at mRNA and protein levels by prototypical chemical inducers [38]. With respect to molecular mechanisms involved in OCs-mediated MDR1 and MRP2 induction, PXR is probably involved because (i) PXR is well known to contribute to regulation of these transporters [47, 48], (ii) OCs are wellestablished PXR agonists [16], and (iii) the PXR signaling pathway was efficiently activated in OCs-treated HepaRG cells, as demonstrated by the upregulation of CYP3A4, a referent PXR target [49]. Only endosulfan, that failed to efficiently induce CYP3A4 in HepaRG cells, concomitantly did not increase MDR1 and MRP2 expression, suggesting that this OC may be a poor activator of PXR in HepaRG cells, although endosulfan has been reported to be a potent PXR agonist in other cell systems [15]. Modulation of hepatic drug transporter activity or expression in humans exposed to OCs may be hypothesized to alter the hepatobiliary secretion of xenobiotics, including drugs and environmental chemicals, handled by these transporters. It may also

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FIGURE 5. Effects of OC pesticides on expression of hepatic drug transporters. HepaRG cells were either untreated (control) or exposed to OCs, each used at 10 µM, for 48 h. Expression of (A) CYP3A4 mRNAs, (B) sinusoidal drug transporter mRNAs, and (C) canalicular drug transporter mRNAs was then determined by RT-qPCR. Data are expressed relatively to the mRNA expression level found in untreated control cells, arbitrary set at 1 unit, and indicated by a dashed line; they are Means ± SEM of five independent assays. *p < 0.05 when compared with control cells.


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concomitantly impair biliary elimination of physiological substrates for these transporters, such as neurotransmitters and hormones, therefore contributing to the toxicity of OCs. In this context, it is, however, noteworthy that OC concentrations reached in humans, that commonly range from 1 to 100 nM in the plasma [50,51], are rather lower than those required to in vitro modulate transporters, that range from around 1 µM (for inhibition of OCT1 activity by lindane or dieldrin) to approximately 100 µM or more (for inhibition of Pgp and MRP2 activity by chlordecone), therefore indicating that in vivo plasmatic concentrations of OCs may fail to modulate transporters. Some OCs, especially chlordecone, are nevertheless known to accumulate in the liver [4], and their hepatic concentrations, that ranged from approximately 27 to 350 µM in chlordecone-exposed workers [52], are likely to efficiently impair drug transporters. Further studies are therefore required to precise the real impact of in vivo exposure to OC pesticides on hepatic drug transport.

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SUPPORTING INFORMATION Figures S1 and S2 are available from the corresponding author ([email protected]) on request.

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DOI 10.1002/jbt