The pregnane X receptor (PXR) is a ligand-activated nuclear receptor controlling hepatocyte expression of numerous genes. Although expression changes in xenobiotic-metabolizing, lipogenic, gluconeogenic and bile acid synthetic genes have been described after PXR activation, the temporal dynamics of their expression is largely unknown. Recently, 3D spheroids of primary human hepatocytes (PHHs) have been characterized as the most phenotypically relevant hepatocyte model. We used 3D PHHs to assess time-dependent expression profiles of 12 prototypic PXR-controlled genes in the time course of 168 h of rifampicin treatment (1 or 10 µM). We observed a similar bell-shaped time-induction pattern for xenobiotic-handling genes (CYP3A4, CYP2C9, CYP2B6, and MDR1). However, we observed either biphasic profiles for genes involved in endogenous metabolism (FASN, GLUT2, G6PC, PCK1, and CYP7A1), a decrease for SHP or oscillation for PDK4 and PXR. The rifampicin concentration determined the expression profiles for some genes. Moreover, we calculated half-lives of CYP3A4 and CYP2C9 mRNA under induced or basal conditions and we used a mathematical model to describe PXR-mediated regulation of CYP3A4 expression employing 3D PHHs. The study shows the importance of long-term time-expression profiling of PXR target genes in phenotypically stable 3D PHHs and provides insight into PXR function in liver beyond our knowledge from conventional 2D in vitro models.

Department of Biophysics and Physical Chemistry Faculty of Pharmacy in Hradec Kralove Charles University Akademika Heyrovskeho 1203 Hradec Kralove 500 05 Czech Republic

Department of Pharmacology and Toxicology Faculty of Pharmacy in Hradec Kralove Charles University Akademika Heyrovskeho 1203 Hradec Kralove 500 05 Czech Republic

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Acocella G (1978) Clinical pharmacokinetics of rifampicin. Clin Pharmacokinet 3(2):108–127. https://doi.org/10.2165/00003088-197803020-00002 PubMed DOI

Arakawa H, Kamioka H, Jomura T et al (2017) Preliminary evaluation of three-dimensional primary human hepatocyte culture system for assay of drug-metabolizing enzyme-inducing potential. Biol Pharm Bull 40(7):967–974. https://doi.org/10.1248/bpb.b16-00885 PubMed DOI

Bell CC, Hendriks DF, Moro SM et al (2016) Characterization of primary human hepatocyte spheroids as a model system for drug-induced liver injury, liver function and disease. Sci Rep 6:25187. https://doi.org/10.1038/srep25187 PubMed DOI PMC

Bell CC, Lauschke VM, Vorrink SU et al (2017) Transcriptional, functional, and mechanistic comparisons of stem cell-derived hepatocytes, HepaRG cells, and three-dimensional human hepatocyte spheroids as predictive in vitro systems for drug-induced liver injury. Drug Metab Dispos 45(4):419–429. https://doi.org/10.1124/dmd.116.074369 PubMed DOI PMC

Chan CYS, Roberts O, Rajoli RKR et al (2018) Derivation of CYP3A4 and CYP2B6 degradation rate constants in primary human hepatocytes: a siRNA-silencing-based approach. Drug Metab Pharmacokinet 33(4):179–187. https://doi.org/10.1016/j.dmpk.2018.01.004 PubMed DOI

Dixit V, Moore A, Tsao H, Hariparsad N (2016) Application of Micropatterned Cocultured hepatocytes to evaluate the inductive potential and degradation rate of major xenobiotic metabolizing enzymes. Drug Metab Dispos 44(2):250–261. https://doi.org/10.1124/dmd.115.067173 PubMed DOI

Faucette SR, Wang H, Hamilton GA et al (2004) Regulation of CYP2B6 in primary human hepatocytes by prototypical inducers. Drug Metab Dispos 32(3):348–358. https://doi.org/10.1124/dmd.32.3.348 PubMed DOI

Gerets HH, Tilmant K, Gerin B et al (2012) Characterization of primary human hepatocytes, HepG2 cells, and HepaRG cells at the mRNA level and CYP activity in response to inducers and their predictivity for the detection of human hepatotoxins. Cell Biol Toxicol 28(2):69–87. https://doi.org/10.1007/s10565-011-9208-4 PubMed DOI PMC

Godoy P, Hewitt NJ, Albrecht U et al (2013) Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME. Arch Toxicol 87(8):1315–1530. https://doi.org/10.1007/s00204-013-1078-5 PubMed DOI PMC

Goodwin B, Jones SA, Price RR et al (2000) A regulatory cascade of the nuclear receptors FXR, SHP-1, and LRH-1 represses bile acid biosynthesis. Mol Cell 6(3):517–526. https://doi.org/10.1016/s1097-2765(00)00051-4 PubMed DOI

Hakkola J, Rysa J, Hukkanen J (2016) Regulation of hepatic energy metabolism by the nuclear receptor PXR. Biochim Biophys Acta 1859(9):1072–1082. https://doi.org/10.1016/j.bbagrm.2016.03.012 PubMed DOI

Hendriks DFG, Vorrink SU, Smutny T et al (2020) Clinically relevant cytochrome P450 3A4 induction mechanisms and drug screening in three-dimensional spheroid cultures of primary human hepatocytes. Clin Pharmacol Ther 108(4):844–855. https://doi.org/10.1002/cpt.1860 PubMed DOI

Hofmann AF (2002) Rifampicin and treatment of cholestatic pruritus. Gut 51(5):756–757. https://doi.org/10.1136/gut.51.5.756 PubMed DOI PMC

Hukkanen J, Hakkola J, Rysa J (2014) Pregnane X receptor (PXR)–a contributor to the diabetes epidemic? Drug Metabol Drug Interact 29(1):3–15. https://doi.org/10.1515/dmdi-2013-0036 PubMed DOI

Ihunnah CA, Jiang M, Xie W (2011) Nuclear receptor PXR, transcriptional circuits and metabolic relevance. Biochim Biophys Acta 1812(8):956–963. https://doi.org/10.1016/j.bbadis.2011.01.014 PubMed DOI PMC

Istrate MA, Nussler AK, Eichelbaum M, Burk O (2010) Regulation of CYP3A4 by pregnane X receptor: the role of nuclear receptors competing for response element binding. Biochem Biophys Res Commun 393(4):688–693. https://doi.org/10.1016/j.bbrc.2010.02.058 PubMed DOI

Kandel BA, Thomas M, Winter S et al (2016) Genomewide comparison of the inducible transcriptomes of nuclear receptors CAR, PXR and PPARalpha in primary human hepatocytes. Biochim Biophys Acta 1859(9):1218–1227. https://doi.org/10.1016/j.bbagrm.2016.03.007 PubMed DOI

Kanebratt KP, Diczfalusy U, Backstrom T et al (2008) Cytochrome P450 induction by rifampicin in healthy subjects: determination using the Karolinska cocktail and the endogenous CYP3A4 marker 4beta-hydroxycholesterol. Clin Pharmacol Ther 84(5):589–594. https://doi.org/10.1038/clpt.2008.132 PubMed DOI

Kanebratt KP, Janefeldt A, Vilen L et al (2021) Primary human hepatocyte spheroid model as a 3D in vitro platform for metabolism studies. J Pharm Sci 110(1):422–431. https://doi.org/10.1016/j.xphs.2020.10.043 PubMed DOI

Kenny JR, Ramsden D, Buckley DB et al (2018) Considerations from the Innovation and Quality Induction Working Group in Response to Drug-Drug Interaction Guidances from Regulatory Agencies: Focus on CYP3A4 mRNA In Vitro Response Thresholds, Variability, and Clinical Relevance. Drug Metab Dispos 46(9):1285–1303. https://doi.org/10.1124/dmd.118.081927 PubMed DOI

Kliewer SA, Willson TM (2002) Regulation of xenobiotic and bile acid metabolism by the nuclear pregnane X receptor. J Lipid Res 43(3):359–364 DOI

Kodama S, Moore R, Yamamoto Y, Negishi M (2007) Human nuclear pregnane X receptor cross-talk with CREB to repress cAMP activation of the glucose-6-phosphatase gene. Biochem J 407(3):373–381. https://doi.org/10.1042/BJ20070481 PubMed DOI PMC

Kolodkin A, Sahin N, Phillips A et al (2013) Optimization of stress response through the nuclear receptor-mediated cortisol signalling network. Nat Commun 4:1792. https://doi.org/10.1038/ncomms2799 PubMed DOI

Kozyra M, Johansson I, Nordling A, Ullah S, Lauschke VM, Ingelman-Sundberg M (2018) Human hepatic 3D spheroids as a model for steatosis and insulin resistance. Sci Rep 8(1):14297. https://doi.org/10.1038/s41598-018-32722-6 PubMed DOI PMC

Lauschke VM, Vorrink SU, Moro SM et al (2016) Massive rearrangements of cellular MicroRNA signatures are key drivers of hepatocyte dedifferentiation. Hepatology 64(5):1743–1756. https://doi.org/10.1002/hep.28780 PubMed DOI

Li T, Chiang JY (2005) Mechanism of rifampicin and pregnane X receptor inhibition of human cholesterol 7 alpha-hydroxylase gene transcription. Am J Physiol Gastrointest Liver Physiol 288(1):G74-84. https://doi.org/10.1152/ajpgi.00258.2004 PubMed DOI

Li T, Chiang JY (2006) Rifampicin induction of CYP3A4 requires pregnane X receptor cross talk with hepatocyte nuclear factor 4alpha and coactivators, and suppression of small heterodimer partner gene expression. Drug Metab Dispos 34(5):756–764. https://doi.org/10.1124/dmd.105.007575 PubMed DOI

Li D, Gaedigk R, Hart SN, Leeder JS, Zhong XB (2012) The role of CYP3A4 mRNA transcript with shortened 3’-untranslated region in hepatocyte differentiation, liver development, and response to drug induction. Mol Pharmacol 81(1):86–96. https://doi.org/10.1124/mol.111.074393 PubMed DOI PMC

Lin W, Wang YM, Chai SC et al (2017) SPA70 is a potent antagonist of human pregnane X receptor. Nat Commun 8(1):741. https://doi.org/10.1038/s41467-017-00780-5 PubMed DOI PMC

Liu YT, Hao HP, Liu CX, Wang GJ, Xie HG (2007) Drugs as CYP3A probes, inducers, and inhibitors. Drug Metab Rev 39(4):699–721. https://doi.org/10.1080/03602530701690374 PubMed DOI

Luke NS, DeVito MJ, Shah I, El-Masri HA (2010) Development of a quantitative model of pregnane X receptor (PXR) mediated xenobiotic metabolizing enzyme induction. Bull Math Biol 72(7):1799–1819. https://doi.org/10.1007/s11538-010-9508-5 PubMed DOI

Maglich JM, Stoltz CM, Goodwin B, Hawkins-Brown D, Moore JT, Kliewer SA (2002) Nuclear pregnane x receptor and constitutive androstane receptor regulate overlapping but distinct sets of genes involved in xenobiotic detoxification. Mol Pharmacol 62(3):638–646. https://doi.org/10.1124/mol.62.3.638 PubMed DOI

Martin P, Riley R, Back DJ, Owen A (2008) Comparison of the induction profile for drug disposition proteins by typical nuclear receptor activators in human hepatic and intestinal cells. Br J Pharmacol 153(4):805–819. https://doi.org/10.1038/sj.bjp.0707601 PubMed DOI

Meneses-Lorente G, Pattison C, Guyomard C et al (2007) Utility of long-term cultured human hepatocytes as an in vitro model for cytochrome p450 induction. Drug Metab Dispos 35(2):215–220. https://doi.org/10.1124/dmd.106.009423 PubMed DOI

Moreau A, Teruel C, Beylot M et al (2009) A novel pregnane X receptor and S14-mediated lipogenic pathway in human hepatocyte. Hepatology 49(6):2068–2079. https://doi.org/10.1002/hep.22907 PubMed DOI

Nishimura M, Yoshitsugu H, Naito S, Hiraoka I (2002) Evaluation of gene induction of drug-metabolizing enzymes and transporters in primary culture of human hepatocytes using high-sensitivity real-time reverse transcription PCR. Yakugaku Zasshi 122(5):339–361. https://doi.org/10.1248/yakushi.122.339 PubMed DOI

Nishimura M, Koeda A, Suzuki E et al (2006) Regulation of mRNA expression of MDR1, MRP1, MRP2 and MRP3 by prototypical microsomal enzyme inducers in primary cultures of human and rat hepatocytes. Drug Metab Pharmacokinet 21(4):297–307. https://doi.org/10.2133/dmpk.21.297 PubMed DOI

Ohtsuki S, Schaefer O, Kawakami H et al (2012) Simultaneous absolute protein quantification of transporters, cytochromes P450, and UDP-glucuronosyltransferases as a novel approach for the characterization of individual human liver: comparison with mRNA levels and activities. Drug Metab Dispos 40(1):83–92. https://doi.org/10.1124/dmd.111.042259 PubMed DOI

Ourlin JC, Lasserre F, Pineau T et al (2003) The small heterodimer partner interacts with the pregnane X receptor and represses its transcriptional activity. Mol Endocrinol 17(9):1693–1703. https://doi.org/10.1210/me.2002-0383 PubMed DOI

Pavek P, Stejskalova L, Krausova L, Bitman M, Vrzal R, Dvorak Z (2012) Rifampicin does not significantly affect the expression of small heterodimer partner in primary human hepatocytes. Front Pharmacol 3:1. https://doi.org/10.3389/fphar.2012.00001 PubMed DOI PMC

Ramsden D, Zhou J, Tweedie DJ (2015) Determination of a degradation constant for CYP3A4 by direct suppression of mRNA in a novel human hepatocyte model, HepatoPac. Drug Metab Dispos 43(9):1307–1315. https://doi.org/10.1124/dmd.115.065326 PubMed DOI

Raucy JL, Mueller L, Duan K, Allen SW, Strom S, Lasker JM (2002) Expression and induction of CYP2C P450 enzymes in primary cultures of human hepatocytes. J Pharmacol Exp Ther 302(2):475–482. https://doi.org/10.1124/jpet.102.033837 PubMed DOI

Rysa J, Buler M, Savolainen MJ, Ruskoaho H, Hakkola J, Hukkanen J (2013) Pregnane X receptor agonists impair postprandial glucose tolerance. Clin Pharmacol Ther 93(6):556–563. https://doi.org/10.1038/clpt.2013.48 PubMed DOI

Smutny T, Dusek J, Hyrsova L et al (2020) The 3’-untranslated region contributes to the pregnane X receptor (PXR) expression down-regulation by PXR ligands and up-regulation by glucocorticoids. Acta Pharm Sin B 10(1):136–152. https://doi.org/10.1016/j.apsb.2019.09.010 PubMed DOI

Smutny T, Hyrsova L, Braeuning A, Ingelman-Sundberg M, Pavek P (2021) Transcriptional and post-transcriptional regulation of the pregnane X receptor: a rationale for interindividual variability in drug metabolism. Arch Toxicol 95(1):11–25. https://doi.org/10.1007/s00204-020-02916-x PubMed DOI

Stage TB, Damkier P, Christensen MM, Nielsen LB, Hojlund K, Brosen K (2016) Impaired glucose tolerance in healthy men treated with St. John’s Wort. Basic Clin Pharmacol Toxicol 118(3):219–224. https://doi.org/10.1111/bcpt.12486 PubMed DOI

Stott KE, Pertinez H, Sturkenboom MGG et al (2018) Pharmacokinetics of rifampicin in adult TB patients and healthy volunteers: a systematic review and meta-analysis. J Antimicrob Chemother 73(9):2305–2313. https://doi.org/10.1093/jac/dky152 PubMed DOI PMC

Tao R, Xiong X, Harris RA, White MF, Dong XC (2013) Genetic inactivation of pyruvate dehydrogenase kinases improves hepatic insulin resistance induced diabetes. PLoS ONE 8(8):e71997. https://doi.org/10.1371/journal.pone.0071997 PubMed DOI PMC

Tebbens JD, Azar M, Friedmann E, Lanzendorfer M, Pavek P (2018) Mathematical models in the description of pregnane X Receptor (PXR)-regulated cytochrome P450 enzyme induction. Int J Mol Sci 19(6):1785. https://doi.org/10.3390/ijms19061785 DOI

Tebbens JD, Matonoha C, Matthios A, Papacek S (2019) On parameter estimation in an in vitro compartmental model for drug-induced enzyme production in pharmacotherapy. Appl Math 64:253–277. https://doi.org/10.21136/AM.2019.0284-18 DOI

Tolson AH, Wang H (2010) Regulation of drug-metabolizing enzymes by xenobiotic receptors: PXR and CAR. Adv Drug Deliv Rev 62(13):1238–1249. https://doi.org/10.1016/j.addr.2010.08.006 PubMed DOI PMC

Vorrink SU, Ullah S, Schmidt S et al (2017) Endogenous and xenobiotic metabolic stability of primary human hepatocytes in long-term 3D spheroid cultures revealed by a combination of targeted and untargeted metabolomics. FASEB J 31(6):2696–2708. https://doi.org/10.1096/fj.201601375R PubMed DOI PMC

Yamashita F, Sasa Y, Yoshida S et al (2013) Modeling of rifampicin-induced CYP3A4 activation dynamics for the prediction of clinical drug-drug interactions from in vitro data. PLoS ONE 8(9):e70330. https://doi.org/10.1371/journal.pone.0070330 PubMed DOI PMC

Zhang JG, Ho T, Callendrello AL, Crespi CL, Stresser DM (2010) A multi-endpoint evaluation of cytochrome P450 1A2, 2B6 and 3A4 induction response in human hepatocyte cultures after treatment with beta-naphthoflavone, phenobarbital and rifampicin. Drug Metab Lett 4(4):185–194. https://doi.org/10.2174/187231210792928224 PubMed DOI

Zhao P, Rowland M, Huang SM (2012) Best practice in the use of physiologically based pharmacokinetic modeling and simulation to address clinical pharmacology regulatory questions. Clin Pharmacol Ther 92(1):17–20. https://doi.org/10.1038/clpt.2012.68 PubMed DOI

The hypolipidemic effect of MI-883, the combined CAR agonist/ PXR antagonist, in diet-induced hypercholesterolemia model