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...

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.

The pregnane X receptor (PXR) initially isolated as a nuclear receptor regulating xenobiotic and drug metabolism and elimination, seems to play an endobiotic role by affecting lipid homeostasis. In mice, PXR affects lipid homeostasis and increases hepatic deposit of triglycerides. In this study, we show that, in human hepatocyte, PXR activation induces an increase of de novo lipogenesis through the up-regulation of S14. S14 was first identified as a thyroid-responsive gene and is known to transduce hormone-related and nutrient-related signals to genes involved in lipogenesis through a molecular mechanism not yet elucidated. We demonstrate that S14 is a novel transcriptional target of PXR. In addition, we report an increase of fatty acid synthase (FASN) and adenosine triphosphate citrate lyase genes expression after PXR activation in human hepatocyte, leading to an increase of fatty acids accumulation and de novo lipogenesis. RNA interference of the expression of S14 proportionally decreases the FASN induction, whereas S14 overexpression in human hepatic cells provokes an increase of fatty acids accumulation and lipogenesis. These results demonstrate for the first time that xenobiotic or drug-activated PXR promote aberrant hepatic de novo lipogenesis via activation of the nonclassical S14 pathway. In addition, these data suggest that the up-regulation of S14 by PXR may promote aberrant hepatic lipogenesis and hepatic steatosis in human hepatocytes. (HEPATOLOGY 2009;49:2068-2079 L ipid homeostasis is achieved by complex physiological mechanisms. Disruptions of lipid formation and catabolism have been implicated in various metabolic diseases such as obesity and diabetes. Hepatic lipid homeostasis is tightly maintained by balanced lipid synthesis, catabolism (␤-oxidation), and uptake/secretion. The liver is a major organ for lipogenesis and expresses high levels of lipogenic enzymes, such as fatty acid synthase (FASN), adenosine triphosphate citrate lyase, and stearoyl-CoA desaturase-1. Several receptors have been implicated in lipid homeostasis, such as the liver X receptor alpha and beta isoforms (LXR␣ and LXR␤ 1 ) or thyroid hormone receptor (TR 2 ). The effect of LXR on lipogenesis involves both direct and indirect mechanisms. LXR/retinoid X receptor (RXR) heterodimers bind lipogenic gene promoters, such as FASN, or regulate lipogenic gene expression by controlling levels of sterol regulatory element binding protein (SREBP)-1c, a transcriptional factor known to regulate the expression of a battery of lipogenic enzymes. [3][4][5] In addition, thyroid hormone (T3) is known to regulate hepatic lipogenesis after binding to TR, which binds to the target DNA sequence thyroid response element (TRE), leading to an increased transcription of several genes involved in lipogenesis. 6,7 The pregnane X receptor (PXR), initially isolated as a nuclear receptor regulating xenobiotic and drug metabolism and elimination, 8 plays an endobiotic role by affect-

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.

Macrophages represent major cellular targets of various drugs, especially antibiotics and anti-viral drugs. Factors that may govern intracellular accumulation of drugs in these cells, especially those related to activity of drug transporters, are consequently likely important to consider. The present study was therefore designed to extensively characterize expression of solute carrier (SLC) and ATP-binding cassette (ABC) transporters in primary human macrophages generated from blood monocytes. Using quantitative polymerase chain reaction assays, these cells were found to exhibit very high or high levels of mRNA expression of concentrative nucleoside transporter (CNT) 3, equilibrative nucleoside transporter 3, monocarboxylate transporter (MCT) 1, MCT4, peptide/histidine transporter (PHT) 1, PHT2, organic anion transporting polypeptide transporter 2B1 and ABC pumps multidrug resistance protein (MRP) 1/ABCC1 and MRP3/ABCC3. By contrast, other transporters, including the efflux pump ABCB1/P-glycoprotein, were found at lower levels or were not expressed. Concomitantly, human macrophages displayed notable uptake of the MCT substrate lactate and of the CNT substrate uridine and also exhibited cellular efflux of the MRP substrate carboxy-2',7'-dichlorofluorescein. Such a functional expression of these transporters has likely to be considered with respect to cellular pharmacokinetics of drugs targeting macrophages.

Background and Purpose An influx drug/proton antiporter of unknown structure has been functionally demonstrated at the blood–brain barrier. This transporter, which handles some psychoactive drugs like diphenhydramine, clonidine, oxycodone, nicotine and cocaine, could represent a new pharmacological target in drug addiction therapy. However, at present there are no known drugs/inhibitors that effectively inhibit/modulate this transporter in vivo. Experimental Approach The FLAPpharm approach was used to establish a pharmacophore model for inhibitors of this transporter. The inhibitory potency of 44 selected compounds was determined against the specific substrate, [3H]‐clonidine, in the human cerebral endothelial cell line hCMEC/D3 and ranked as good, medium, weak or non‐inhibitor. Key Results The pharmacophore model obtained was used as a template to screen xenobiotic and endogenous compounds from databases [Specs, Recon2, Human Metabolome Database (HMDB), human intestinal transporter database], and hypothetical candidates were tested in vitro to determine their inhibitory capacity with [3H]‐clonidine. According to the transporter database, 80% of the proton antiporter inhibitor candidates could inhibit P‐glycoprotein/MDR1/ABCB1 and specificity is improved by reducing inhibitor size/shape and increasing water solubility. Virtual screening results using HMDB and Recon2 for endogenous compounds appropriately scored tryptamine as an inhibitor. Conclusions and Implications The pharmacophore model for the proton‐antiporter inhibitors was a good predictor of known inhibitors and allowed us to identify new good inhibitors. This model marks a new step towards the discovery of this drug/proton antiporter and will be of great use for the discovery and design of potent inhibitors that could potentially help to assess and validate its pharmacological role in drug addiction in vivo.

A wide range of fluorescent dyes is now available for use in various aspects of drug transporter studies. The use of these dyes for transporter analyses may, however, be hampered by classic pitfalls of fluorescence technology, such as quenching. Transporter-independent processes such as passive diffusion of dyes through plasma membrane or dye sequestration into subcellular compartments must also be considered, as well as the redundant handling by various distinct transporters of some fluorescent probes. Finally, standardization of dye-based transport assays remains an important on-going issue.

(2017) Direct and quantitative evaluation of the human CYP3A4 contribution (f m ) to drug clearance using the invitro SILENSOMES model, Xenobiotica, 47:7, 562-575, DOI: 10.1080/00498254.2016 In this article, we propose an original strategy, called Silensomes TM , to produce human liver microsomes silenced for one specific CYP450, thanks to specific mechanism-based inhibitors (MBI). 2. Using azamulin as a specific CYP3A4 MBI, we demonstrated the proof of concept that CYP3A4 can be totally, specifically (even against 3A5) and permanently (at least for six years) inhibited by our process. Thus, comparing clearance in control and CYP3A4-Silensomes TM , CYP3A4 contributions were determined for 11 CYP3A4 substrates which correlated with known in vivo contributions and revealed accuracy with less than 10% error. In comparison, contributions determined using recombinant human CYP450 (rhCYP450s) were less accurate (more than 10% error for 30% of the tested CYP3A4 substrates). 3. This easy and ready-to-use in vitro method combines the advantages of existing models (specificity of rhCYP450s and representativeness of HLM) without their drawbacks. The same strategy could be used to silence other major CYP450s one-by-one to provide a complete direct CYP450 quantitative phenotyping kit.