Coordinated induction of drug transporters and phase I and II metabolism in human liver slices

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Abstract

Although regulation of phase I drug metabolism in human liver is relatively well studied, the regulation of phase II enzymes and of drug transporters is incompletely characterized. Therefore, we used human liver slices to investigate the PXR, CAR and AhR-mediated induction of drug transporters and phase I and II metabolic enzymes. Precision-cut human liver slices were incubated for 5 or 24 h with prototypical inducers: phenobarbital (PB) (50 μM) for CAR, β-naphthoflavone (BNF) (25 μM) for AhR, and rifampicin (RIF) (10 μM) for PXR, and gene expression of the phase I enzymes CYP1A1, 1A2, 3A4, 3A5, 2B6, 2A6, the phase II enzymes UGT1A1 and 1A6, and the transporters MRP2, MDR1, BSEP, NTCP and OATP8 was measured. BNF induced CYP1A1, UGT1A1 and UGT1A6 and MRP2, NTCP and MDR1. RIF induced CYP3A4, 3A5, 2B6, 2A6, UGT1A1, UGT1A6 and BSEP, MRP2 and MDR1 and slightly downregulated OATP8. PB induced CYP3A4, 3A5, 2B6 and 2A6, UGT1A1 and all transporters.

Large interindividual differences were found with respect to the level of induction.

Enzyme activity of CYP3A4, measured by testosterone metabolism, was increased after 24 h by RIF. 7-Ethoxycoumarin O-deethylation activity, mediated predominantly by CYP 1A1/1A2 but also by other CYPs, was increased after 24 h with PB.

We have shown that regulation of all phases of the (in)activation of a drug via the CAR, AhR and the PXR pathways can be studied in human liver slices. The concomitant induction of metabolic enzymes and transporters shows that also in the human liver transporters and metabolic enzymes are regulated coordinately.

Introduction

The different isoenzymes of the hepatic cytochrome P450 and UDP-glucuronyltransferase family play a major role in phase I and II metabolism of drugs and other xenobiotics (Miners et al., 2004). Recently, it has become appreciated that influx and efflux drug transporters also play an important role in the disposition (in)activation and detoxification of drugs in coordination with the drug metabolism enzymes (Wagner et al., 2005, Xu et al., 2005). Efflux of metabolites by transporters is now commonly described as phase III (Xu et al., 2005). Induction of drug metabolism enzymes and transporters may contribute to an increased detoxification function, but can also lead to undesirable drug–drug interactions by affecting the (metabolic) clearance and thereby the efficacy of (co-administered) drugs and thus may lead to increased toxicity. Induction of the proteins involved in metabolism and transport has been described as being a result of a tangle of networks where a number of nuclear receptors share ligands, partners (such as RXR), DNA responsive elements and target organs (Kullak-Ublick et al., 2004, Xu et al., 2005). Moreover, the level of induction may also depend on the presence of activators and repressors and the rate of proteasomal degradation of the nuclear receptors (NR). Therefore, the net effect of a drug on gene transcription may be complex and difficult to predict from NR reporter assays or cell lines that do not express the physiological levels of NR, chaperones, activators and repressors. In human liver, the regulation of metabolic enzymes is investigated intensively, but the knowledge of the regulation of transporters is far from complete.

To predict the induction of drug metabolism and drug transporters by newly developed drugs in man and especially to determine the clinical implications, in vitro systems as close as possible to the in vivo situation are needed. Human hepatocytes are often used with success. However, in cultured human hepatocytes the metabolic capacity is attenuated (Aninat et al., 2006, Meunier et al., 2000) and the expression of transport proteins is reduced during culturing although not to the same extent as in rat hepatocytes (Jigorel et al., 2006, Payen et al., 2000). Moreover, the apical transport proteins lose their specific localisation (Groothuis et al., 1981, Liang et al., 1993). This may be due to the fact that the hepatocytes are isolated from their natural environment and the interaction with other liver cell types and extracellular matrix is missing. As induction experiments in cultured hepatocytes can only be performed 24–48 h after plating (the FDA prescribes 3 days) to recover the full induction potential (Maurel, 1996, Hewitt et al., 2007), changes in expression levels are inevitable. In contrast, liver slice experiments can start immediately after preparation of the slices at a time point where changes in expression are minimal. In addition, in liver slices the original architecture of the liver is retained and the cell sociology existing in the liver in vivo is preserved. The precision-cut liver slice system has already been applied successfully in metabolism and toxicity studies (De Graaf et al., 2000, De Kanter et al., 2002, Olinga et al., 2001, Van De Bovenkamp et al., 2005, Vickers et al., 1992). Also induction of phase I metabolism was reported in human liver slices (Edwards et al., 2003, Martin et al., 2003, Persson et al., 2006), but regulation of phase II and III was not described before.

Although in slices drug metabolism also decreases upon incubation, this decrease is less than in isolated cultured hepatocytes (Glöckner et al., 2003) and most importantly, transporter expression remains constant during at least 24 h (Elferink et al., 2004, Jung et al., 2007).

In this study, we aimed to investigate the regulation of transporters and phase I and II metabolic enzymes in human liver slices using three model inducers phenobarbital (PB), β-naphthoflavone (BNF) and rifampicin (RIF). Phenobarbital induces drug metabolism predominantly via the orphan nuclear receptor, constitutive androstane receptor (CAR), β-naphthoflavone mainly by the aryl hydrocarbon receptor (AhR) and rifampicin mostly via the pregnane X receptor (PXR) (Xu et al., 2005). In addition, these receptors are also involved in the induction of drug transporters (Kullak-Ublick et al., 2004, Xu et al., 2005). Induction of several phase I drug metabolism enzymes (CYP 1A1, 1A2, 2A6, 2B6, 3A4, 3A5), phase II drug metabolism enzymes (UGT1A1 and 1A6), phase III drug efflux transporters (BSEP, MDR1 and MRP2) and the influx transporters (NTCP and OATP8) were assessed by gene expression analysis. In addition, the effect of the inducers on CYP and UGT metabolic activity was determined using lidocaine, testosterone and 7-ethoxycoumarin as substrates.

This study, for the first time, investigates the induction of all three phases of drug metabolism and transport in the same human liver in vitro, which allows the study of the coordinated regulation of drug transporters and metabolic enzymes.

Section snippets

Materials

Williams’ medium E and Trizol reagent were obtained from Gibco BRL (Paisley, Scotland). Six-well culture plates and PCR tubes were obtained from Greiner (Alphen a/d Rijn, The Netherlands). Reverse Transcription System was obtained from Promega (Madison, WI, USA) and DNAse Treatment & Removal Reagents DNA-free™ was from Ambion. All reagents were of analytical grade.

Human liver tissue

Human liver tissue remaining after reduced-size or split liver transplantation was used. The donor liver was perfused with cold

Results

Precision-cut human liver slices were incubated for 5 or 24 h with the inducers 50 μM phenobarbital (PB), 25 μM β-naphthoflavone (BNF) and 10 μM rifampicin (RIF).

In general, induction of both phase I and II enzymes and the transporters could already be observed at 5 h of incubation. The extent of induction appeared highly variable between the individual livers as was also described by others using isolated human hepatocytes and human liver slices (Jigorel et al., 2006, Glöckner et al., 1999,

Discussion

The aim of the current study was to investigate the regulation via PXR, CAR and AhR activation of all three phases of drug metabolism and transport in the human liver in vitro.

In this study we show that gene expression and enzymatic activity of phase I and II iso-enzymes and gene expression of phase III drug transporters is induced in human liver in vitro by RIF, BNF and PB. These model inducers were clearly capable of inducing target CYP iso-enzymes as previously described (Xu et al., 2005)

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