Abstract
Transporters play a crucial role in the uptake of endo- and exogenous molecules in hepatocytes and efflux into the bile. The bile salt export pump (BSEP; ABCB11) is of major importance for efflux of bile salts to the bile and BSEP inhibition frequently provokes drug-induced cholestasis. This chapter describes two assays to determine inhibition of BSEP-mediated bile salt excretion. The first assay uses inside-out membrane vesicles, prepared from BSEP-transfected cell lines. The cholestasis potential of compounds can be determined by specifically investigating the ability to inhibit BSEP-mediated uptake of tauro-nor-THCA-24-DBD, a fluorescent bile salt derivative. For the second assay, relative accumulation of tauro-nor-THCA-24-DBD in sandwich-cultured hepatocytes, which represents a more biorelevant in vitro system, is investigated. Through incubation with standard or Ca2+/Mg2+-free buffer, the substrate signal can be determined in the cells and bile or the cells alone, respectively. Performing this assay in the presence and absence of potentially interfering compounds of interest enables exploration of the relative effect of these compounds on biliary excretion of the probe substrate.
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References
Dawson PA, Lan T, Rao A (2009) Bile acid transporters. J Lipid Res 50:2340–2357
Pauli-Magnus C, Meier PJ (2005) Hepatocellular transporters and cholestasis. J Clin Gastroenterol 39:S103–S110
Slijepcevic D, Roscam Abbing RLP, Katafuchi T et al (2017) Hepatic uptake of conjugated bile acids is mediated by both sodium taurocholate cotransporting polypeptide and organic anion transporting polypeptides and modulated by intestinal sensing of plasma bile acid levels in mice. Hepatology 66:1631–1643
Meier PJ, Stieger B (2002) Bile salt transporters. Annu Rev Physiol 64:635–661
Stieger B, Meier Y, Meier PJ (2007) The bile salt export pump. Pflugers Arch 453:611–620
Perez M-J, Briz O (2009) Bile-acid-induced cell injury and protection. World J Gastroenterol 15:1677–1689
Stieger B (2010) Role of the bile salt export pump, BSEP, in acquired forms of cholestasis. Drug Metab Rev 42:437–445
Kubitz R, Dröge C, Kluge S et al (2014) Genetic variations of bile salt transporters. Drug Discov Today Technol 12:e55–e67
Jansen PL, Strautnieks SS, Jacquemin E et al (1999) Hepatocanalicular bile salt export pump deficiency in patients with progressive familial intrahepatic cholestasis. Gastroenterology 117:1370–1379
Dawson S, Stahl S, Paul N et al (2012) In vitro inhibition of the bile salt export pump correlates with risk of cholestatic drug-induced liver injury in humans. Drug Metab Dispos 40:130–138
Cheng Y, Woolf TF, Gan J et al (2016) In vitro model systems to investigate bile salt export pump (BSEP) activity and drug interactions: a review. Chem Biol Interact 255:23–30
Doige CA, Sharom FJ (1992) Transport properties of P-glycoprotein in plasma membrane vesicles from multidrug-resistant Chinese hamster ovary cells. Biochim Biophys Acta 1109:161–171
Tabas LB, Dantzig AH (2002) A high-throughput assay for measurement of multidrug resistance protein-mediated transport of leukotriene C4 into membrane vesicles. Anal Biochem 310:61–66
Karlsson JE, Heddle C, Rozkov A et al (2010) High-activity p-glycoprotein, multidrug resistance protein 2, and breast cancer resistance protein membrane vesicles prepared from transiently transfected human embryonic kidney 293-epstein-barr virus nuclear antigen cells. Drug Metab Dispos 38:705–714
van Staden CJ, Morgan RE, Ramachandran B et al (2012) Membrane vesicle ABC transporter assays for drug safety assessment. Curr Protoc Toxicol Chapter 23:Unit 23.5
Kis E, Ioja E, Nagy T et al (2009) Effect of membrane cholesterol on BSEP/Bsep activity: species specificity studies for substrates and inhibitors. Drug Metab Dispos 37:1878–1886
Berg JM, Tymoczko JL, Stryer L (2002) Enzymes can be inhibited by specific molecules. W H Freeman, New York, NY
Yamaguchi K, Murai T, Yabuuchi H et al (2010) Measurement of bile salt export pump transport activities using a fluorescent bile acid derivative. Drug Metab Pharmacokinet 25:214–219
De Bruyn T, Sempels W, Snoeys J et al (2014) Confocal imaging with a fluorescent bile acid analogue closely mimicking hepatic taurocholate disposition. J Pharm Sci 103:1872–1881
Breeuwer P, Drocourt JL, Bunschoten N et al (1995) Characterization of uptake and hydrolysis of fluorescein diacetate and carboxyfluorescein diacetate by intracellular esterases in Saccharomyces cerevisiae, which result in accumulation of fluorescent product. Appl Environ Microbiol 61:1614–1619
Oorts M, Richert L, Annaert P (2015) Drug-induced cholestasis detection in cryopreserved rat hepatocytes in sandwich culture. J Pharmacol Toxicol Methods 73:63–71
Swift B, Pfeifer ND, Brouwer KLR (2010) Sandwich-cultured hepatocytes: an in vitro model to evaluate hepatobiliary transporter-based drug interactions and hepatotoxicity. Drug Metab Rev 42:446–471
De Bruyn T, Chatterjee S, Fattah S et al (2013) Sandwich-cultured hepatocytes: utility for in vitro exploration of hepatobiliary drug disposition and drug-induced hepatotoxicity. Expert Opin Drug Metab Toxicol 9:589–616
Holmstock N, Oorts M, Snoeys J et al (2018) MRP2 inhibition by HIV protease inhibitors in rat and human hepatocytes: a quantitative confocal microscopy study. Drug Metab Dispos 46:697–703
Keemink J, Oorts M, Annaert P (2015) Primary hepatocytes in sandwich culture. Methods Mol Biol 1250:175–188
Herédi-Szabó K, Palm JE, Andersson TB et al (2013) A P-gp vesicular transport inhibition assay - optimization and validation for drug-drug interaction testing. Eur J Pharm Sci 49:773–781
Pál A, Méhn D, Molnár E et al (2007) Cholesterol potentiates ABCG2 activity in a heterologous expression system: improved in vitro model to study function of human ABCG2. J Pharmacol Exp Ther 321:1085–1094
Bodo A, Bakos E, Szeri F et al (2003) Differential modulation of the human liver conjugate transporters MRP2 and MRP3 by bile acids and organic anions. J Biol Chem 278:23529–23537
Zelcer N, Huisman MT, Reid G et al (2003) Evidence for two interacting ligand binding sites in human multidrug resistance protein 2 (ATP binding cassette C2). J Biol Chem 278:23538–23544
Pedersen JM, Matsson P, Bergström CAS et al (2008) Prediction and identification of drug interactions with the human ATP-binding cassette transporter multidrug-resistance associated protein 2 (MRP2; ABCC2). J Med Chem 51:3275–3287
Hooiveld GJEJ, Heegsma J, van Montfoort JE et al (2002) Stereoselective transport of hydrophilic quaternary drugs by human MDR1 and rat Mdr1b P-glycoproteins. Br J Pharmacol 135:1685–1694
Heredi-Szabo K, Kis E, Molnar E et al (2008) Characterization of 5(6)-carboxy-2′,7′-dichlorofluorescein transport by MRP2 and utilization of this substrate as a fluorescent surrogate for LTC4. J Biomol Screen 13:295–301
Deng F, Sjöstedt N, Kidron H (2016) The effect of albumin on MRP2 and BCRP in the vesicular transport assay. PLoS One 11:e0163886
Acknowledgments
This work was financially supported by FWO (grant G089515N). We would like to thank the employees of SOLVO biotechnology for teaching us the tips and tricks of the membrane vesicle assay.
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Van Brantegem, P., Deferm, N., Qi, B., De Vocht, T., Annaert, P. (2019). Vesicle- and Hepatocyte-Based Assays for Identification of Drug Candidates Inhibiting BSEP Function. In: Vinken, M. (eds) Experimental Cholestasis Research. Methods in Molecular Biology, vol 1981. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9420-5_4
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DOI: https://doi.org/10.1007/978-1-4939-9420-5_4
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