ABSTRACT:Sinusoidal and canalicular hepatic drug transporters constitute key factors involved in drug elimination from liver. Regulation of their expression via activation of xenosensors, such as aryl hydrocarbon receptor (AhR), constitutive androstane receptor (CAR), pregnane X receptor (PXR), and nuclear factor E2-related factor 2 (Nrf2), remains incompletely characterized. The present study was therefore designed to carefully analyze expression of major drug transporters in primary human hepatocytes exposed to dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin, TCDD) (an AhR activator), rifampicin (RIF) (a PXR activator), phenobarbital (PB) (a CAR activator), and oltipraz (OPZ) (a Nrf2 activator), using mainly reverse transcription-real time polymerase chain reaction assays. With a threshold corresponding to a 1.5-fold factor change in mRNA levels, observed in at least three of seven independent human hepatocyte cultures, efflux transporters such as MDR1, MRP2 and BCRP were up-regulated by PB, RIF, and OPZ, whereas MRP3 was induced by OPZ and RIF. MDR1 and BCRP expression was also increased by TCDD-and RIF-augmented mRNA levels of the influx transporter OATP-C. Bile acid transporters, i.e., bile salt export pump and Na ؉ -taurocholate cotransporting polypeptide, and the sinusoidal transporter, OAT2, were down-regulated by all the tested chemicals. Influx transporters such as OCT1, OATP-B, and OATP8 were repressed by PB and TCDD. PB also decreased MRP6 expression, whereas mRNA levels of OCT1 and OATP8 were downregulated by RIF and OPZ, respectively. Taken together, these data establish a complex pattern of transporter regulation by xenobiotics in human hepatocytes, in addition to interindividual variability in responsiveness. This may deserve further attention with respect to drug-drug interactions and adverse effects of hepatic drugs.Hepatic drug transporters constitute important factors in the hepatobiliary elimination of xenobiotics (Chandra and Brouwer, 2004). They belong to the solute carrier (SLC) or the ATP-binding cassette (ABC) superfamilies of transporters (Schinkel and Jonker, 2003). SLC transporters, especially organic cation transporter 1 (OCT1/ SLC22A1) (Jonker and Schinkel, 2004), organic anion-transporting polypeptides (OATP-B/SLCO2B1, OATP-C/SLCO1B1, and OATP8/ SLCO1B3) (Hagenbuch and Meier, 2003), and organic anion transporter 2 (OAT2/SLC22A7) (Kobayashi et al., 2005), located at the sinusoidal membrane of hepatocytes, mediate the uptake of endogenous and foreign compounds from blood. Canalicular ABC transporters, such as P-glycoprotein (ABCB1) encoded by multidrug resistance 1 (MDR1) gene, multidrug resistance protein 2 (MRP2/ABCC2), MRP6 (ABCC6), and breast cancer resistance protein (BCRP/ ABCG2), are involved in secretion of drugs or their metabolites into bile (Schinkel and Jonker, 2003;Fardel et al., 2005). The efflux pump MRP3 (ABCC3) is located at the sinusoidal pole, where it is thought to mediate secretion of drug metabolites into the bloodstream for subsequent urinary elimination (Zelcer e...

ABSTRACT:Tumor necrosis factor (TNF)-␣ and interleukin (IL)-6 are proinflammatory cytokines known to alter expression of drug transporters in rodent liver. However, their effects toward human hepatic transporters remain poorly characterized. Therefore, this study was designed to analyze the effects of these cytokines on drug transporter expression in primary human hepatocytes. Exposure to 100 ng/ml TNF-␣ or 10 ng/ml IL-6 for 48 h was found to down-regulate mRNA levels of major sinusoidal influx transporters, including sodium-taurocholate cotransporting polypeptide (NTCP), organic anion-transporting polypeptide (OATP) 1B1, OATP1B3, OATP2B1, organic cation transporter (OCT) 1, and organic anion transporter 2. TNF-␣ and IL-6 concomitantly reduced NTCP and OATP1B1 protein expression and NTCP, OATP, and OCT1 transport activities. IL-6, but not TNF-␣, was also found to decrease mRNA expression of the canalicular transporters multidrug resistance 1 gene, multidrug resistance gene-associated protein (MRP) 2, and breast cancer resistance protein (BCRP); it concomitantly decreased MRP2 and BCRP protein expression. TNF-␣, unlike IL-6, markedly reduced bile salt export pump mRNA levels and increased BCRP protein expression. Expression of the sinusoidal MRP3 efflux pump was found to be up-regulated at protein level by both TNF-␣ and IL-6. Taken together, these data show that TNF-␣ and IL-6 similarly altered expression of sinusoidal drug transporters and rather differentially that of canalicular efflux transporters. Such pronounced changes in hepatic transporter expression are likely to contribute to both cholestasis and alterations of pharmacokinetics caused by inflammation in humans.

Expression of drug transporters corresponds to a crucial parameter in intestinal Caco-2 cells widely used for investigating drug absorption. In order to characterize it in an accurate, reproducible and comparative manner, we analyzed mRNA levels of 19 influx and efflux drug transporters through real-time quantitative polymerase chain reaction assays combined with the use of a total RNA reference standard. Profiles of transporter expression were found to be significantly correlated in two independent Caco-2 cell clones and in human small intestine, which may support the use of Caco-2 cells for investigating intestinal drug transport. Several transporters were nevertheless quantitatively expressed at higher (MRP2, MRP3, MRP4, MRP5, MRP6, OATP-A, OATP-B, OCT1 and MCT1) or lower (BCRP) levels in Caco-2 cells comparatively to small intestine. Moreover, MDR1, MRP2, OATP-A and PEPT1 mRNA relative expression were found to differ in the two analyzed Caco-2 cell clones by at least a twofold factor, highlighting that some variations in transporter expression may occur in Caco-2 cells depending on cell origin, and therefore underlining the interest of carefully characterizing transporter levels in any Caco-2 cell clone before its use for drug transport assays.

Polycyclic aromatic hydrocarbons (PAHs) such as benzo(a)pyrene (BP) are ubiquitous environmental carcinogenic contaminants exerting deleterious effects toward cells acting in the immune defense such as monocytic cells. To investigate the cellular basis involved, we have examined the consequences of PAH exposure on macrophagic differentiation of human blood monocytes. Treatment by BP markedly inhibited the formation of adherent macrophagic cells deriving from monocytes upon the action of either GM-CSF or M-CSF. Moreover, it reduced expression of macrophagic phenotypic markers such as CD71 and CD64 in GM-CSF-treated monocytic cells, without altering cell viability or inducing an apoptotic process. Exposure to BP also strongly altered functional properties characterizing macrophagic cells such as endocytosis, phagocytosis, LPS-triggered production of TNF-α and stimulation of allogeneic lymphocyte proliferation. Moreover, formation of adherent macrophagic cells was decreased in response to PAHs distinct from BP such as dimethylbenz(a)anthracene and 3-methylcholanthrene, which interact, like BP, with the arylhydrocarbon receptor (AhR) known to mediate many PAH effects. In contrast, benzo(e)pyrene, a PAH not activating AhR, had no effect. In addition, AhR was demonstrated to be present and functional in cultured monocytic cells, and the use of its antagonist α-naphtoflavone counteracted inhibitory effects of BP toward macrophagic differentiation. Overall, these data demonstrate that exposure to PAHs inhibits functional in vitro differentiation of blood monocytes into macrophages, likely through an AhR-dependent mechanism. Such an effect may contribute to the immunotoxicity of these environmental carcinogens owing to the crucial role played by macrophages in the immune defense.

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.

ABSTRACT:Primary hepatocyte cultures are considered as a useful in vitro system for pharmacological/toxicological studies. Although expression of drug-metabolizing enzymes and canalicular drug transporters has been well documented in this cellular model, less information is available about sinusoidal drug transporter activities. This has led us to investigate functional expression of the major sinusoidal transporters in primary human and rat hepatocytes. Membrane transport proteins belonging to the solute carrier (SLC) superfamily of transporters play a major role in the initial sinusoidal influx of drugs to hepatocytes (van Montfoort et al., 2003). Organic cation transporters (OCT1/SLC22A1 and Oct1/Slc22a1) thus mediate the transport of small organic cations such as tetraethylammonium (TEA) (Jonker and Schinkel, 2004). Organic anion-transporting polypeptides (OATP-A/SLCO1A2, OATP-B/SLCO2B1, OATP-C/ SLCO1B1, and OATP8/SLCO1B3 in human hepatocytes and Oatp1/ Slco1a1, Oatp2/Slco1a4 and Oatp4/Slco1b2 in rat hepatocytes) are implicated in sodium-independent uptake of organic anions such as estrone-3-sulfate (ES), biliary acids, the antihistamine fexofenadine, and the cholesterol-lowering pravastatin (Kim, 2003); some of these OATPs also transport amphipathic compounds like digoxin, handled by OATP8 and Oatp4. Organic anion transporters (OAT2/SLC22A7 and Oat2/Slc22a7) mediate the influx of organic anions like paraaminohippurate (PAH) and salicylates (Sekine et al., 1998), whereas the Na ϩ -taurocholic acid-cotransporting polypeptides (NTCP/ SLC10A1 and Ntcp/Slc10a1) are involved in Na ϩ -dependent uptake of biliary acids such as taurocholate (Trauner and Boyer, 2003).Primary hepatocytes represent a major in vitro model for studying the activity and regulation of liver-detoxifying pathways and can therefore contribute to prediction of hepatic elimination of xenobiotics (Modriansky et al., 2000;Gebhardt et al., 2003). Such a liver cell culture system has been extensively characterized with respect to expression and activity of drug-metabolizing enzymes and canalicular transporters Payen et al., 2000;Gomez-Lechon et al., 2003;Annaert and Brouwer, 2005). By contrast, activity of sinusoidal membrane transport proteins has, as yet, been less studied, to our knowledge, in cultured hepatocytes, especially in primary human hepatocytes, although these influx transporters play a crucial role in xenobiotic pharmacokinetic and drug-drug interactions (Chandra and Brouwer, 2004;Shitara et al., 2005). The present study was therefore designed to investigate functional expression of the major sinusoidal transporters in primary hepatocyte cultures. Owing to known interspecies differences in liver-detoxifying pathways, this work was conducted with both human and rat hepatocytes. Materials and Methods Chemicals.[ 3 H(G)]Taurocholic acid (specific activity 1.19 Ci/mmol), [6, Cell Isolation and Culture. Hepatocytes from adult male Sprague-Dawley rats weighing 150 to 200 g were isolated by a perfusion of the liver as previously described ,...

Pyrethroids are widely-used chemical insecticides, to which humans are commonly exposed, and known to alter functional expression of drug metabolizing enzymes. Limited data have additionally suggested that drug transporters, that constitute key-actors of the drug detoxification system, may also be targeted by pyrethroids. The present study was therefore designed to analyze the potential regulatory effects of these pesticides towards activities of main ATP-binding cassette (ABC) and solute carrier (SLC) drug transporters, using transporter-overexpressing cells. The pyrethroids allethrin and tetramethrin were found to inhibit various ABC and SLC drug transporters, including multidrug resistance-associated protein (MRP) 2, breast cancer resistance protein (BCRP), organic anion transporter polypeptide (OATP) 1B1, organic anion transporter (OAT) 3, multidrug and toxin extrusion transporter (MATE) 1, organic cation transporter (OCT) 1 and OCT2, with IC50 values however ranging from 2.6 μM (OCT1 inhibition by allethrin) to 77.6 μM (OAT3 inhibition by tetramethrin) and thus much higher than pyrethroid concentrations (in the nM range) reached in environmentally pyrethroid-exposed humans. By contrast, allethrin and tetramethrin cis-stimulated OATP2B1 activity and failed to alter activities of OATP1B3, OAT1 and MATE2-K, whereas P-glycoprotein activity was additionally moderately inhibited. Twelve other pyrethoids used at 100 μM did not block activities of the various investigated transporters, or only moderately inhibited some of them (inhibition by less than 50%). In silico analysis of structure-activity relationships next revealed that molecular parameters, including molecular weight and lipophilicity, are associated with transporter inhibition by allethrin/tetramethrin and successfully predicted transporter inhibition by the pyrethroids imiprothrin and prallethrin. Taken together, these data fully demonstrated that two pyrethoids, i.e., allethrin and tetramethrin, can act as regulators of the activity of various ABC and SLC drug transporters, but only when used at high and non-relevant concentrations, making unlikely any contribution of these transporter activity alterations to pyrethroid toxicity in environmentally exposed humans.

Oltipraz, a synthetic derivative of the cruciferous vegetable product 1,2-dithiole-3-thione, is considered as one of the most potent chemoprotectants. It modulates both cytochrome P-450 (CYP) and glutathione S-transferase expression and activities in rat tissues. Its effects, however, are variable according to the enzyme, tissue, and species. We show here that, as previously found in rat lung and kidney, CYP1A1 is inducible by oltipraz in both rat intestine and Caco-2 cells, a cell line originated from a human colon adenocarcinoma. In these cells, a 50 M oltipraz treatment increased CYP1A1 mRNA (ϳ30-fold), protein and activity. mRNA level was augmented as early as 2 h after the beginning of treatment, suggesting a transcriptional activation, and was maximal between 8 and 12 h. Transient transfection of Caco-2 cells with constructs containing different sizes of the 5-flanking region of the CYP1A1 gene upstream of the luciferase reporter gene showed an increase in luciferase activity in oltipraz-treated cells, which correlates with the presence of the xenobiotic responsive element (XRE). Furthermore we demonstrated that resveratrol, an antagonist of the aryl hydrocarbon (Ah) receptor, inhibited the induction of both CYP1A1 promoter activity and mRNA by oltipraz, supporting the involvement of the Ah receptor in this induction. In an attempt to further characterize the mechanism of CYP1A1 induction, we showed a rapid increase in intracellular calcium concentration upon treatment of Caco-2 cells with oltipraz. Moreover, the effect of this compound on CYP1A1 was strongly abolished in the presence of BAPTA-AM, a well known chelator of intracellular calcium, and 2-aminoethyl diphenylborate, an inhibitor of store-operated calcium channels. These results bring the first demonstration that oltipraz activates transcription of the CYP1A1 gene through the Ah receptor-XRE pathway in Caco-2 cells and that CYP1A1 induction relies upon an increase of intracellular calcium concentration.Oltipraz, a synthetic derivative of 1,2-dithiole-3-thione, a constituent of cruciferous vegetables, is considered as one of the most promising chemopreventive agents in development, based on preclinical studies (1-5) and a recent Phase IIa clinical trial in China (6, 7). This compound was first claimed to act as a chemopreventive agent by enhancing activities of phase II enzymes such as glutathione S-transferase, UDP-glucuronosyltransferases, NAD(P)H:quinone reductase, aflatoxin B 1 -aldehyde reductase, and epoxide hydrolase (for a review see Ref. 8). Biochemical and genetic studies showed that induction of these detoxifying enzymes is primarily due to transcriptional activation of the genes and is regulated by an enhancer, called an antioxidant-responsive element (ARE) 1 or electrophile-responsive element (9 -11). NF-E2-related factor 2 (Nrf2) has been implicated as an essential component of an ARE-binding transcriptional complex (12)(13)(14), but the signal transduction pathways that relay the chemical signals to the ARE-protein complex remain to...