The Vesicular Transport Assay: Validated In Vitro Methods to Study Drug‐Mediated Inhibition of Canalicular Efflux Transporters ABCB11/BSEP and ABCC2/MRP2

Krisztina Herédi‐Szabó1, Emese Kis1, Peter Krajcsi1

1 Solvo Biotechnology, Szeged, Hungary
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 23.4
DOI:  10.1002/0471140856.tx2304s54
Online Posting Date:  November, 2012
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Abstract

The canalicular membrane of hepatocytes contains several transport proteins that use the energy of ATP to efflux potentially toxic molecules to the bile. Probably the two most important proteins at this location are MRP2 and BSEP, which transport phase II conjugates of xenobiotics and endobiotics and conjugated bile salts, respectively. The impaired function of either of these transporter proteins reduces the clearance of the toxic conjugates, resulting in their accumulation in the hepatocytes and eventually the plasma. Conjugated bile salts and phase II metabolites are compounds with low passive permeability; therefore, the most commonly used test system to investigate MRP2‐ and BSEP‐mediated transport processes is the vesicular transport assay. The concentration of probe substrates and inhibitors used in the experiment is close to their free concentration in the hepatocytes, providing an advantage when calculating kinetic parameters (Km, Ki, Vmax). The protocols aim to assist scientists to set up a transport assay for a known or potential substrate and test small molecule inhibition of the transporters. Curr. Protoc. Toxicol. 54:23.4.1‐23.4.16. © 2012 by John Wiley & Sons, Inc.

Keywords: ABCB11/BSEP; ABCC2/MRP2; taurocholate; CDCF; canalicular membrane

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Characterization of Substrate Transport by BSEP and MRP2
  • Support Protocol 1: Generation of CDCF Calibration Curve
  • Basic Protocol 2: Inhibition of BSEP‐ and MRP2‐Mediated Transport
  • Support Protocol 2: Data Analysis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Characterization of Substrate Transport by BSEP and MRP2

  Materials
  • Transporter‐overexpressing and control membrane vesicles (5 mg total protein/ml), as described in Table 23.4.1, row 1
  • Assay mix for BSEP (see recipe)
  • Assay mix for MRP2 (see recipe)
  • Substrate stock solution in MilliQ water or in DMSO, as described in Table 23.4.1, row 2
  • 0.2 M Mg‐ATP (see recipe)
  • 0.2 M AMP (see recipe)
  • Washing mix for BSEP, ice cold (see recipe)
  • Washing mix for MRP2, ice cold (see recipe)
  • Scintillation cocktail (OptiPhase SuperMix from PerkinElmer or equivalent)
  • Detector solution: 1 N NaOH
  • Inhibitor stock solution in DMSO, as described in Table 23.4.1, row 3
  • 96‐well tissue culture plates
  • Plate incubator/shaker
  • 96‐well filter plates (Millipore MSFBN6B10 or equivalent)
  • 96‐well rapid‐filtration apparatus (Millipore Multiscreen HTS Vacuum Manifold (MSVMHTS00) or equivalent)
  • Repeater pipet with corresponding tips
  • Multichannel pipet (8 or 12 channels)
  • Hair dryer (optional)
  • Liquid scintillation counter capable of reading 96‐well plates
  • Fluorimeter suitable for 96‐well format

Support Protocol 1: Generation of CDCF Calibration Curve

  Materials
  • 500 µM CDCF stock solution (see recipe)
  • Detector solution: 1 N NaOH
  • 96‐well filter plates (Millipore MSFBN6B10 or equivalent)
  • 96‐well rapid‐filtration apparatus (Millipore Multiscreen HTS Vacuum Manifold (MSVMHTS00) or equivalent)
  • 96‐well tissue culture plate
  • Fluorimeter suitable for 96‐well format

Basic Protocol 2: Inhibition of BSEP‐ and MRP2‐Mediated Transport

  Materials
  • Transporter‐overexpressing and control membrane vesicles (5 mg total protein/ml) as described in Table 23.4.1, row 1
  • Assay mix for BSEP (see recipe)
  • Assay mix for MRP2 (see recipe)
  • Substrate stock solution in MilliQ water or in DMSO as described in Table 23.4.1, row 2
  • Test compound stock solution in DMSO (concentration should be selected based on the potency of the compound. A general concentration range is shown in Figure . The stock is diluted 100‐fold in the assay.)
  • 0.2 M Mg‐ATP (see recipe)
  • 0.2 M AMP (see recipe)
  • Washing mix for BSEP, ice cold (see recipe)
  • Washing mix for MRP2, ice cold (see recipe)
  • Scintillation cocktail (OptiPhase SuperMix from PerkinElmer or equivalent)
  • Detector solution: 1 N NaOH
  • Inhibitor stock solution in DMSO as described in Table 23.4.1, row 3
  • 5‐ml test tube
  • 96‐well tissue culture plates
  • 96‐well filter plates (Millipore MSFBN6B10 or equivalent)
  • Plate incubator/shaker
  • 96‐well rapid filtration apparatus [Millipore Multiscreen HTS Vacuum Manifold (MSVMHTS00) or equivalent]
  • Repeater pipet with corresponding tips
  • Multichannel pipet (8 or 12 channels)
  • Hair dryer (optional)
  • Liquid scintillation counter capable of reading 96‐well plates
  • Fluorimeter suitable for the 96‐well format
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Figures

Videos

Literature Cited

   Akita, H., Suzuki, H., Ito, K., Kinoshita, S., Sato, N., Takikawa, H., and Sugiyama, Y. 2001. Characterization of bile acid transport mediated by multidrug resistance associated protein 2 and bile salt export pump. Biochim. Biophys. Acta 1511:7‐16.
   Ansede, J.H., Smith, W.R., Perry, C.H., St Claire, R.L. 3rd, and Brouwer, K.R. 2010. An in vitro assay to assess transporter‐based cholestatic hepatotoxicity using sandwich‐cultured rat hepatocytes. Drug Metab. Dispos. 38:276‐280.
   Bodo, A., Bakos, E., Szeri, F., Varadi, A., and Sarkadi, B. 2003. Differential modulation of the human liver conjugate transporters MRP2 and MRP3 by bile acids and organic anions. J. Biol. Chem. 278:23529‐23537.
   Byrne, J.A., Strautnieks, S.S., Mieli‐Vergani, G., Higgins, C.F., Linton, K.J., and Thompson, R.J. 2002. The human bile salt export pump: Characterization of substrate specificity and identification of inhibitors. Gastroenterology 123:1649‐1658.
   Cosgrove, B.D., King, B.M., Hasan, M.A., Alexopoulos, L.G., Farazi, P.A., Hendriks, B.S., Griffith, L.G., Sorger, P.K., Tidor, B., Xu, J.J., and Lauffenburger, D.A. 2009. Synergistic drug‐cytokine induction of hepatocellular death as an in vitro approach for the study of inflammation‐associated idiosyncratic drug hepatotoxicity. Toxicol. Appl. Pharmacol. 237:317‐330.
   Dawson, S., Stahl, S., Paul, N., Barber, J., and Kenna, J.G. 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.
   Gerloff, T., Stieger, B., Hagenbuch, B., Madon, J., Landmann, L., Roth, J., Hofmann, A.F., and Meier, P.J. 1998. The sister of P‐glycoprotein represents the canalicular bile salt export pump of mammalian liver. J. Biol. Chem. 273:10046‐10050.
   Glavinas, H., Mehn, D., Jani, M., Oosterhuis, B., Heredi‐Szabo, K., and Krajcsi, P. 2008. Utilization of membrane vesicle preparations to study drug‐ABC transporter interactions. Exp. Opin. Drug Metab. Toxicol. 4:721‐732.
   Hartman, J.C., Brouwer, K., Mandagere, A., Melvin, L., and Gorczynski, R. 2010. Evaluation of the endothelin receptor antagonists ambrisentan, darusentan, bosentan, and sitaxsentan as substrates and inhibitors of hepatobiliary transporters in sandwich‐cultured human hepatocytes. Can. J. Physiol. Pharmacol. 88:682‐691.
   Hayashi, H., Takada, T., Suzuki, H., Onuki, R., Hofmann, A.F., and Sugiyama, Y. 2005. Transport by vesicles of glycine‐ and taurine‐conjugated bile salts and taurolithocholate 3‐sulfate: A comparison of human BSEP with rat Bsep. Biochim. Biophys. Acta 1738:54‐62.
   Heredi‐Szabo, K., Kis, E., Molnar, E., Gyorfi, A., and Krajcsi, P. 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.
   Heredi‐Szabo, K., Glavinas, H., Kis, E., Mehn, D., Bathori, G., Veres, Z., Kobori, L., von Richter, O., Jemnitz, K., and Krajcsi, P. 2009. Multidrug resistance protein 2‐mediated estradiol‐17beta‐D‐glucuronide transport potentiation: in vitro‐in vivo correlation and species specificity. Drug Metab. Dispos. 37:794‐801.
   Jemnitz, K., Heredi‐Szabo, K., Janossy, J., Ioja, E., Vereczkey, L., and Krajcsi, P. 2010a. ABCC2/Abcc2: A multispecific transporter with dominant excretory functions. Drug Metab. Rev. 42:402‐436.
   Jemnitz, K., Veres, Z., and Vereczkey, L. 2010b. Contribution of high basolateral bile salt efflux to the lack of hepatotoxicity in rat in response to drugs inducing cholestasis in human. Toxicol. Sci. 115:80‐88.
   Keppler, D., Jedlitschky, G., and Leier, I. 1998. Transport function and substrate specificity of multidrug resistance protein. Methods Enzymol. 292:607‐616.
   Kis, E., Ioja, E., Nagy, T., Szente, L., Heredi‐Szabo, K., and Krajcsi, P. 2009a. Effect of membrane cholesterol on BSEP/Bsep activity: Species specificity studies for substrates and inhibitors. Drug Metab. Dispos. 37:1878‐1886.
   Kis, E., Rajnai, Z., Ioja, E., Heredi Szabo, K., Nagy, T., Mehn, D., and Krajcsi, P. 2009b. Mouse Bsep ATPase assay: A nonradioactive tool for assessment of the cholestatic potential of drugs. J. Biomol. Screen. 14:10‐15.
   Kis, E., Ioja, E., Rajnai, Z., Jani, M., Mehn, D., Heredi‐Szabo, K., and Krajcsi, P. 2011. BSEP inhibition ‐ In vitro screens to assess cholestatic potential of drugs. Toxicol. In Vitro. In press.
   Kostrubsky, S.E., Strom, S.C., Kalgutkar, A.S., Kulkarni, S., Atherton, J., Mireles, R., Feng, B., Kubik, R., Hanson, J., Urda, E., and Mutlib, A.E. 2006. Inhibition of hepatobiliary transport as a predictive method for clinical hepatotoxicity of nefazodone. Toxicol. Sci. 90:451‐459.
   Krahenbuhl, S., Talos, C., Fischer, S., and Reichen, J. 1994. Toxicity of bile acids on the electron transport chain of isolated rat liver mitochondria. Hepatology 19:471‐479.
   Letschert, K., Komatsu, M., Hummel‐Eisenbeiss, J., and Keppler, D. 2005. Vectorial transport of the peptide CCK‐8 by double‐transfected MDCKII cells stably expressing the organic anion transporter OATP1B3 (OATP8) and the export pump ABCC2. J. Pharmacol. Exp. Ther. 313:549‐556.
   Maillette de Buy Wenniger, L. and Beuers, U. 2010. Bile salts and cholestasis. Dig. Liver Dis. 42:409‐418.
   Matsushima, S., Maeda, K., Kondo, C., Hirano, M., Sasaki, M., Suzuki, H., and Sugiyama, Y. 2005. Identification of the hepatic efflux transporters of organic anions using double‐transfected Madin‐Darby canine kidney II cells expressing human organic anion‐transporting polypeptide 1B1 (OATP1B1)/multidrug resistance‐associated protein 2, OATP1B1/multidrug resistance 1, and OATP1B1/breast cancer resistance protein. J. Pharmacol. Exp. Ther. 314:1059‐1067.
   Mita, S., Suzuki, H., Akita, H., Stieger, B., Meier, P.J., Hofmann, A.F., and Sugiyama, Y. 2005. Vectorial transport of bile salts across MDCK cells expressing both rat Na+‐taurocholate cotransporting polypeptide and rat bile salt export pump. Am. J. Physiol. Gastrointest. Liver Physiol. 288:G159‐G167.
   Mita, S., Suzuki, H., Akita, H., Hayashi, H., Onuki, R., Hofmann, A.F., and Sugiyama, Y. 2006. Vectorial transport of unconjugated and conjugated bile salts by monolayers of LLC‐PK1 cells doubly transfected with human NTCP and BSEP or with rat Ntcp and Bsep. Am. J. Physiol. Gastrointest. Liver Physiol. 290:G550‐G556.
   Morgan, R.E., Trauner, M., van Staden, C.J., Lee, P.H., Ramachandran, B., Eschenberg, M., Afshari, C.A., Qualls, C.W. Jr., Lightfoot‐Dunn, R., and Hamadeh, H.K. 2010. Interference with bile salt export pump function is a susceptibility factor for human liver injury in drug development. Toxicol. Sci. 118:485‐500.
   Noe, J., Stieger, B., and Meier, P.J. 2002. Functional expression of the canalicular bile salt export pump of human liver. Gastroenterology 123:1659‐1666.
   Pessayre, D., Berson, A., Fromenty, B., and Mansouri, A. 2001. Mitochondria in steatohepatitis. Semin. Liver Dis. 21:57‐69.
   Rius, M., Hummel‐Eisenbeiss, J., and Keppler, D. 2008. ATP‐dependent transport of leukotrienes B4 and C4 by the multidrug resistance protein ABCC4 (MRP4). J. Pharmacol. Exp. Ther. 324:86‐94.
   Swift, B., Pfeifer, N.D., and Brouwer, K.L. 2010. Sandwich‐cultured hepatocytes: An in vitro model to evaluate hepatobiliary transporter‐based drug interactions and hepatotoxicity. Drug Metab. Rev. 42:446‐471.
   Zelcer, N., Huisman, M.T., Reid, G., Wielinga, P., Breedveld, P., Kuil, A., Knipscheer, P., Schellens, J.H., Schinkel, A.H., and Borst, P. 2003. Evidence for two interacting ligand binding sites in human multidrug resistance protein 2 (ATP binding cassette C2). J. Biol. Chem. 278:23538‐23544.
Key References
   Glavinas et al., 2008. See above.
  Discusses utilization of membrane assays.
   Kis et al., 2009a. See above.
  Describes development and characterization of cholesterol‐enriched BSEP‐Sf9 membranes [high‐activity membranes (HAM)].
   Heredi‐Szabo et al., 2008. See above.
  Introduces CDCF as a surrogate for LTC4.
Internet Resources
  http://www.solvo.com
  This is the Web page of Solvo Biotechnology, which provides information on available BSEP and MRP2 membranes and kits as well as additional scientific literature and technical bulletins.
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