Regulation of mRNA Expression of MDR1, MRP1, MRP2 and MRP3 by Prototypical Microsomal Enzyme Inducers in Primary Cultures of Human and Rat Hepatocytes

doi:10.2133/dmpk.21.297

Drug Metabolism and Pharmacokinetics

Volume 21, Issue 4, 2006, Pages 297-307

https://doi.org/10.2133/dmpk.21.297 Get rights and content

Summary:

The mRNA induction of various transporters by rifampicin (Rif), dexamethasone (Dex) and omeprazole (Ome) was investigated in primary cultures of cryopreserved human and rat hepatocytes. Analysis was performed by quantitative realtime RTPCR using primers and TaqMan probes. In primary cultures of human hepatocytes, mRNA levels of MDR and MRP1 were increased by about 1.5 fold and 1.3 fold, respectively, by exposure to Rif at 2 to 50 μM as compared with 0.1% DMSOtreated controls. MRP2 mRNA levels in the same human hepatocytes were significantly increased by 1.2 to 1.8 fold by exposure to Rif at 50 μM as compared with controls. In primary cultures of rat hepatocytes, Mdr1a and Mdr1b mRNA levels were not increased or only slightly increased at 24 hr by exposure to any of the inducers at 2, 10 or 50 μM. Mrp2 mRNA levels in the same rat hepatocytes were significantly increased by 7 to 45 fold by exposure to Dex at 2 μM as compared with controls. Based on the species differences observed in the present study, primary cultures of cryopreserved hepatocytes from both the human and rat should be useful in preclinical drug development for evaluating candidate drugs for transporter induction.

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Previous findings showed that ESE-3 was expressed in the intestine but not in the liver under physiological conditions in human [14,15]. In addition, the expression levels of CYP3A4 and ABCB1 mRNAs were comparably increased by RIF in human enterocytes or intestine slices [16,17], whereas the effects of RIF on ABCB1 mRNA levels were much smaller than those on CYP3A4 mRNA levels in human hepatocytes or liver slices [18–23]. Therefore, ESE-3 may be involved in the induction of ABCB1 gene by RIF also in human intestine.
Pregnane X receptor (PXR) is involved in the transactivation of ABCB1 gene by rifampicin (RIF). However, we found that increase in ABCB1 mRNA by RIF was observed in LS180 cells but not in HepG2 cells. Since both cell lines expressed PXR equally, we hypothesized that a factor(s) other than PXR is responsible for PXR-mediated transactivation of the ABCB1 gene. Reporter activities of a distal enhancer module containing direct repeat 4 (DR4) motifs were increased by RIF in LS180 cells but not in HepG2 cells. Mutation of the DR4 motifs diminished the increase in reporter activities in LS180 cells. Gene subtraction showed that epithelial-specific ETS factor 3 (ESE-3) is a transcription factor enriched in LS180 cells compared to HepG2 cells. When ESE-3 and PXR were co-expressed in HepG2 cells, reporter activities were increased by RIF, which were completely abolished by mutation of DR4 motifs. Chromatin immunoprecipitation assays showed specific binding of ESE-3 to the region containing the DR4 motifs of the ABCB1 gene. Finally, knock-down of ESE-3 in LS180 cells resulted in a decrease in the induction of ABCB1 mRNA. These results suggest that ESE-3 is a factor responsible for PXR-mediated transactivation of the ABCB1 gene by RIF in LS180 cells.
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Eight microliters of single-stranded cDNA (equivalent to 100 ng of total RNA) was mixed with 10 μl of TaqMan Fast Universal PCR Master Mix (2x, Applied Biosystems). The TaqMan primers and probes for CYP2B2, CYP3A2, Mrp2, Mrp3, and Bsep were prepared by referencing previously published studies [12–14], while the probes and primers for CYP7A, Slco1, Slco2, Ntcp, Ho-1, and GAPDH were purchased from Applied Biosystems. Real-time quantitative RT-PCR was performed with an ABI Prism 7900 HT Sequence Detection System (Applied Biosystems).
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Specific primers for Gapdh, Actb, Abcb1a and Abcg2 were provided by Qiagen (confidential sequences). Published primer sequences were used for Abcc1, Abcc2, Abcc3, Esr1, Esr2 and Pgr (Table 1) (Ceccatelli et al., 2006; Nishimura et al., 2005, 2006; St-Pierre et al., 2004). Abcb1b forward and reverse primers were designed with Primer3 (v. 0.4.0)® software.
The mycotoxin zearalenone (ZEN) is produced by a variety of Fusarium fungi and contaminates numerous cereals, fruits and vegetables. Interacting with the estrogen receptors, ZEN and reduced metabolites zearalenols cause hormonal effects in animals. Few data are available on the effects of repeated exposure to ZEN, particularly during pregnancy. The aim of our work was to assess the impact of this toxin on the expression of ABC transporters and nuclear receptors in fetal liver and pregnant rats that were exposed daily (gestation day 7–20) to 1 mg/kg ZEN. Significant variations were observed, depending on the tissue type, the tissue origin (maternal or fetal), and the time of analysis after the last exposure to ZEN (4 h or 24 h). The modulations of expression were independent of the magnitude of tissue impregnation by ZEN and its metabolites. The maternal uterus was the most sensitive tissue: Abcb1a, Abcb1b and Abcg2 mRNA and protein expressions were induced at both times, while Abcc1, Abcc3 and Esr1 mRNA and protein expressions were inhibited then induced 4 h and 24 h after exposure, respectively. In the fetal liver, Abcb1a and Esr1 protein expression was inhibited at both times, while mRNA expression was induced 24 h after the last exposure to ZEN. These results suggested that ZEN exposure could impact maternal and fetal exposure to ABC transporters substrates, and influence fetus development through nuclear receptor modulation.
Ivermectin induces P-glycoprotein expression and function through mRNA stabilization in murine hepatocyte cell line
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In this context, this study aimed at investigating P-gp regulation in response to acute ivermectin treatment. Hepatocytes were chosen as a suitable in vitro model system for studying the potential of xenobiotics to modulate P-gp gene expression [24] and elucidating the role of specific transcription factors in the regulation of gene involved in the metabolism of xenobiotics [25,26]. The objectives were to evaluate the ability of ivermectin to modulate P-gp gene expression, to determine the functional significance of this modulation and to further establish molecular mechanisms underlying this effect.
Ivermectin is widely used in human and veterinary medicine for the control of helminth infections. Ivermectin is known to interact with P-glycoprotein (P-gp/MDR1), being a good substrate and a potent inhibitor, however, the influence of ivermectin on the expression of the transporter has not been investigated. Expression of P-glycoprotein was investigated in cultured mouse hepatocytes acutely exposed to ivermectin. The two P-glycoprotein murine isoforms, Mdr1a and Mdr1b, mRNA levels were assessed by real-time RT-PCR. Ivermectin induced a clear time- and concentration-dependent up-regulation of Mdr1a and Mdr1b mRNA levels (as early as a 12-h exposure and up to 2.5-fold at 10 μM). Moreover, ivermectin-treated cells displayed enhanced cellular efflux of the P-glycoprotein substrate calcein that was inhibited by the P-glycoprotein blocker valspodar, providing evidence that the ivermectin-induced P-glycoprotein was functional. The mechanisms underlying these effects were investigated. Ivermectin-mediated Mdr1 mRNA induction was independent of the two nuclear receptors CAR and PXR, which are known to be involved in drug transporters regulation. Moreover, by using reporter cell lines that detects specific ligand-activated transcription factors, we showed that ivermectin did not displayed CAR, PXR or AhR ligand activities. However, studies with actinomycin D revealed that the half-life of Mdr1a and Mdr1b mRNA were significantly prolonged by two-fold in ivermectin-treated cells suggesting a post-transcriptional mode of ivermectin regulation. This study demonstrates for the first time that ivermectin induces P-glycoprotein overexpression through post-transcriptional mRNA stabilization, thus offering insight into the mechanism of reduced therapeutic efficacy and development of ivermectin-resistant parasites.
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Akita et al., 2002b; Chandra et al., 2005; Hirohashi et al., 1999). Expression of Mrp3 is highest in the adrenal glands but also shows high levels in the liver, colon and stomach (Nishimura and Naito, 2005, Nishimura et al., 2006). While these and other studies (Le Vee et al., 2009) have focused on the expression of both human and rat MRP3/Mrp3, only a limited number of studies have focused on the function of Mrp3 in vivo and in cultures of hepatocytes.
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We evaluated a novel three-dimensional primary culture system using micro-space plates to determine the expression levels of 61 target (drug-metabolizing enzymes, transporters, and nuclear receptors) mRNAs in human hepatocytes. We measured mRNA expression levels of many target genes in four lots of cryopreserved human hepatocyte primary cells after 120 h of culture and compared differences in mRNA expression levels between cultures using traditional plates and those using micro-space plates. In this study, we show that the mRNA levels of many experimental targets in human hepatocytes before inoculation resemble the levels inside the human liver. Furthermore, we show that the rate of change of expression levels of many target mRNAs relative to the value before inoculation of the hepatocytes into micro-space plates was relatively smaller than the rate of change in hepatocytes inoculated into traditional plates. Pharmacokinetics-related examinations using this system are possible within a time frame of 120 h. We report that this novel three-dimensional culture system reproduces mRNA expression levels that are nearer to those in the liver in vivo and is an excellent platform for maintaining mRNA expression levels of drug-metabolizing enzymes and transporters when compared to common monolayer cultures.

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Regular ArticleRegulation of mRNA Expression of MDR1, MRP1, MRP2 and MRP3 by Prototypical Microsomal Enzyme Inducers in Primary Cultures of Human and Rat Hepatocytes

Summary:

Biochim. Biophys. Acta

Drug Metab. Pharmacokinet.

Drug Metab. Pharmacokinet.

J. Biol. Chem.

J. Biol. Chem.

Arch. Biochem. Biophys.

Chem. Biol. Interact.

J. Biol. Chem.

Drug Metab. Pharmacokinet.

Drug Metab. Pharmacokinet.

Drug Metab. Pharmacokinet.

Methods Cell Biol.

Drug Metab. Pharmacokinet.

Drug Metab. Pharmacokinet.

Drug Metab. Pharmacokinet.

Anal. Biochem.

Mol. Cell Probes

Lab. Invest.

J. Biol. Chem.

Lett.

FEBS Lett.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Biochem. Pharmacol.

J. Biol. Chem.

Regular Article
Regulation of mRNA Expression of MDR1, MRP1, MRP2 and MRP3 by Prototypical Microsomal Enzyme Inducers in Primary Cultures of Human and Rat Hepatocytes