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...

An X-ray crystal structure of Kelch-like ECH-associated protein (Keap1) co-crystallised with (1S,2R)-2-[(1S)-1-[(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl]-1,2,3,4-tetrahydroisoquinolin-2-carbonyl]cyclohexane-1-carboxylic acid (compound (S,R,S)-1 a) was obtained. This X-ray crystal structure provides breakthrough experimental evidence for the true binding mode of the hit compound (S,R,S)-1 a, as the ligand orientation was found to differ from that of the initial docking model, which was available at the start of the project. Crystallographic elucidation of this binding mode helped to focus and drive the drug design process more effectively and efficiently.

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 ,...

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