Toxicant Transport by P‐Glycoprotein

Lisa J. Bain1

1 University Of Texas at El Paso, El Paso, Texas
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 5.6
DOI:  10.1002/0471140856.tx0506s15
Online Posting Date:  May, 2003
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Abstract

Toxicant Transport by P‐Glycoprotein (Lisa J. Bain, University of Texas at El Paso, El Paso, Texas).This unit describes several different methods of measuring active transport of toxicants by P‐glycoprotein uptake of a radiolabeled toxicant, uptake of an unlabeled toxicant with detection by HPLC or GC, uptake of radiolabeled toxicant in membrane vesicles; movement of radiolabeled toxicant from the basal to apical compartment using polarized epithelial cells grown in Transwell culture. P‐glygoprotein is an important protein for limiting entry into the brain through the blood‐bain barrier. It also alters the rates of uptake and elimination.

     
 
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Table of Contents

  • Basic Protocol 1: Celluar Efflux of Radiolabeled Toxicants
  • Basic Protocol 2: Accumulation of Toxicants into Celluar Membrane Vesicles
  • Basic Protocol 3: Apical Transport of Toxicants in Caco‐2 Cells Cultured on Transwell Inserts
  • Basic Protocol 4: Inhibition of Doxorubicin Uptake by Toxicants
  • Support Protocol 1: Preparation of Membrane Vesicles
  • Reagents and Solutions
  • Commentary
     
 
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Materials

Basic Protocol 1: Celluar Efflux of Radiolabeled Toxicants

  Materials
  • Transfected cultured cells expressing P‐gp after transfection with a vector containing P‐gp cDNA along with a mock‐transfected control or tissue, e.g., liver or intestine
  • Appropriate cell culture medium (e.g., RPMI 1640 supplemented with 10% fetal bovine serum)
  • [14C] or [3H]‐labeled toxicant, diluted sufficiently with unlabeled toxicant to provide a 1 to 20 µM solution having 100,000 to 300,000 dpm per 5 µl
  • 5 mM verapamil stock (see recipe)
  • PBS ( appendix 2A), ice cold
  • 30% scintillation cocktail (e.g., Scintisafe or equivalent)
  • 12‐well culture plates
  • 20‐ml scintillation vials
  • Scintillation counter
  • Additional reagents and equipment for counting cells ( appendix 3B)

Basic Protocol 2: Accumulation of Toxicants into Celluar Membrane Vesicles

  Materials
  • Membrane vesicles (see protocol 5)
  • ATP and AMP solutions (see recipe)
  • 1000 U/ml creatine kinase in recipeTris‐sucrose buffer (store 150‐µl aliquots in 0.5‐ml microcentrifuge tubes for up to 1 month at −20°C)
  • Tris‐sucrose buffer (see recipe), ice cold
  • 0.5 µCi of 418.2 nM [3H]vincristine (see recipe)
  • [14C] or [3H]‐labeled toxicant, diluted sufficiently with unlabeled toxicant to provide a 10 nm to 10 µM solution having 0.5 µCi per 350 µl Tris‐sucrose buffer
  • 22 µM verapamil in Tris‐sucrose buffer (see recipe)
  • 50% scintillation cocktail (Scintisafe Plus or equivalent)
  • 0.22‐µm membrane filters, 25‐mm diameter (e.g., Millipore)
  • Sampling vacuum manifold for 25‐mm filters (e.g., Millipore)
  • Vacuum pump
  • Flat‐tipped forceps
  • 7‐ml scintillation vials
  • Scintillation counter
IMPORTANT NOTE: Ensure that everything is set up before beginning the experiment as there is a very short period between each sampling time point.

Basic Protocol 3: Apical Transport of Toxicants in Caco‐2 Cells Cultured on Transwell Inserts

  Materials
  • Caco‐2 cells
  • Cell culture medium, such as DMEM with high glucose (4.5 g/liter) supplemented with 10% fetal bovine serum
  • Hank's balanced salt solution ( appendix 2A) containing 25 mM HEPES, pH 7.4 (HBSS/HEPES)
  • [3H]‐ or [14C]‐labeled toxicant of interest diluted with unlabeled toxicant to provide a final solution of 1 to 100 µM containing 0.5 µCi/2.5 ml transport medium
  • 5 mM verapamil (see recipe)
  • 50% scintillation cocktail (e.g., Scintisafe Plus or equivalent)
  • 6‐well transwell plate (Costar) with 0.45‐µm polyester inserts
  • 37°C, 5% CO 2 humidified incubator
  • 7‐ml scintillation vials
  • Scintillation counter

Basic Protocol 4: Inhibition of Doxorubicin Uptake by Toxicants

  Materials
  • P‐glycoprotein expressing cultured cells, such as Caco‐2 cells, or transfected cells expressing P‐gp after transfection with a vector containing P‐gp cDNA and a mock‐transfected control
  • 10 mM doxorubicin‐HCl stock (see recipe)
  • Toxicant of interest dissolved in PBS, saline, ethanol, or acetone at a 200‐fold concentrate of the final concentration for the assay
  • 5 mM verapamil stock (see recipe)
  • PBS ( appendix 2A), ice cold
  • Extraction solution (see recipe)
  • 1.5‐ml microcentrifuge tubes
  • 96‐well plates
  • 96‐well fluorescence spectrophotometer (e.g., SpectraMax Gemini or equivalent; or a single‐cuvette fluorimeter)
  • Additional reagents and equipment for counting cells ( appendix 3B)
NOTE: Doxorubicin is a suspected carcinogen. Use appropriate precautions when handling.

Support Protocol 1: Preparation of Membrane Vesicles

  Materials
  • P‐glycoprotein expressing cultured cells in 100‐cm2 culture dishes, such as Caco‐2 cells, or transfected cells expressing P‐gp after transfection with a vector containing P‐gp cDNA and a mock‐transfected control or tissue such as liver or intestine
  • PBS ( appendix 2A), ice cold
  • 0.05% collagenase (Sigma type I) in PBS
  • Hypotonic lysis buffer containing protease inhibitors (see recipe)
  • 0.4% trypan blue (Sigma or equivalent)
  • Tris‐sucrose buffer (see recipe)
  • 38% (w/v) sucrose (see recipe)
  • Bio‐Rad protein assay kit
  • 40‐µm mesh nylon screen
  • Hemacytometer
  • Confocal microscope
  • Motor‐driven Potter‐Elvehjem homogenizer (e.g., Dyna‐Mix Stirrer, Fisher, or equivalent)
  • Dounce homogenizer
  • 12‐ml polycarbonate round‐bottom tubes
  • Centrifuge with a Beckman SW‐40 rotor or equivalent
  • 1‐ml pipets
  • 27‐G needle and 1‐ml syringe
  • 1.5‐ml cryovials
NOTE: After step , perform all procedures at 4°C.
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Figures

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Literature Cited

Literature Cited
   Anderle, P., Niederer, E., Rubas, W., Hilgendorf, C., Spahn‐Langguth, H., Wunderli‐Allenspach, H., Merkle, H.P. and Langguth, P. 1998. P‐glycoprotein (P‐gp) mediated efflux in Caco‐2 cell monolayers: The influence of culturing conditions and drug exposure on P‐gp expression levels. J. Pharm. Sci. 87:757‐762.
   Artursson, P., Palm, K. and Luthman, K. 1996. Caco‐2 monolayers in experimental and theoretical predications of drug transport. Adv. Drug Deliv. Rev. 22:67‐84.
   Bain, L.J. and LeBlanc, G.A. 1996. Interaction of structurally diverse pesticides with the human MDR1 gene product P‐glycoprotein. Toxicol. Appl. Pharmacol. 141:288‐298.
   Bain, L.J., McLachlan, J.B. and LeBlanc, G.A. 1997. Structure‐activity relationships for xenobiotic transport substrates and inhibitory ligands of P‐glycoprotein. Environ. Health Perspect. 105:812‐818.
   Cordon‐Cardo, C., O'Brien, J.P., Casais, D., Rittman‐Grauer, L., Biedler, J.L., Melamed, M.R. and Bertino, J.R. 1989. Multidrug‐resistance gene (P‐glycoprotein) is expressed by endothelial cells at blood‐brain barrier sites. Proc. Natl. Acad. Sci. U.S.A. 86:695‐698.
   Egorin, M.J., Hildebrand, R.C., Cimino, E.F. and Bachur, N.R. 1974. Cytofluorescence localization of adriamycin and daunorubicin. Cancer Res. 40:2243‐2254.
   Fojo, A.T., Akiyama, S.‐I., Gottesman, M.M. and Pastan, I. 1985. Reduced drug accumulation in multiply drug‐resistant human KB carcinoma cell lines. Cancer Res. 45:3002‐3007.
   Gottesman, M.M. and Pastan, I. 1993. Biochemistry of multidrug resistance mediated by the multidrug transporter. Annu. Rev. Biochem. 62:385‐427.
   Hilgers, A.R., Conradi, R.A. and Burton, P.S. 1990. Caco‐2 cell monolayers as a model for drug transport across the intestinal mucosa. Pharm. Res. 7:902‐10.
   Horio, M., Gottesman, M.M. and Pastan, I. 1988. ATP‐dependent transport of vinblastine in vesicles from human multidrug‐resistant cells. Proc. Natl. Acad. Sci U.S.A. 85:3580‐3584.
   Ishikawa, T. 1989. ATP/Mg2+‐dependent cardiac transport system for glutathione S‐conjugates. A study using rat heart sarcolemma vesicles. J. Biol. Chem. 264:17343‐17348.
   Ishikawa, T., Muller, M., Klunemann, C., Schaub, T. and Keppler, D. 1990. ATP‐dependent primary active transport of cysteinyl leukotrienes across the liver canalicular membrane. J. Biol. Chem. 265:19279‐19286.
   Juliano, R.L. and Ling, V. 1976. A surface glycoprotein modulating drug permeability in Chinese hamster ovary cell mutants. Biochim. Biophys. Acta. 455:152‐162.
   Krishna, G., Chen, K., Lin, C. and Nomeir, A.A. 2001. Permeability of lipophilic compounds in drug discovery using in‐vitro human absorption model, Caco‐2. Int. J. Pharm. 222:77‐89.
   Leier, I., Jedlitschky, G., Buchholz, U., Cole, S.P., Deeley, R.G. and Keppler, D. 1994. The MRP gene encodes on ATP‐dependent export pump for leuokotriene C4 and structurally related conjugates. J. Biol. Chem. 268:27807‐27810.
   Schinkel, A.H., Smit, J.J.M., van Tellingen, O., Beijnen, J.H., Wagenaar, E., van Deemter, L., Mol, C.A.A.M., van der Valk, M.A., Robanus‐Maandag, E.C., te Riele, H.P.J., Berns, A.J.M. and Borst, P. 1994. Disruption of the mouse mdr1a P‐glycoprotein gene leads to a deficiency in the blood‐brain barrier and increased sensitivity to drugs. Cell. 77:491‐502.
   Thiebaut, F., Tsuruo, T., Hamada, A.H., Gottesman, M.M., Pastan, I. and Willingham, M.C. 1987. Cellular localization of the multidrug resistance gene product in normal human tissues. Proc. Natl. Acad. Sci. U.S.A. 84:7735‐7738.
   Theibaut, F., Tsuruo, T., Hamada, H., Gottesman, M.M., Pastan, I. and Willingham, M.C. 1989. Immunohistochemical localization in normal tissues of different epitopes in the multidrug transport protein P170: Evidence for localization in brain capillaries and cross‐reactivity of one antibody with a muscle protein. J. Histochem. Cytochem. 37:159‐164.
   Tsuruo, T., Iida, H., Tsukagoshi, S. and Sakurai, Y. 1981. Overcoming of vincristine resistance in P388 leukemia in vivo and in vitro through enhanced cytotoxicity of vincristine and vinblastine by verapamil. Cancer Res. 41:1967‐1972.
   van Tellingen, O. 2001. The importance of drug‐transporting P‐glycoproteins in toxicology. Toxicol. Lett. 120:31‐41.
   Ueda, K., Cardarelli, C., Gottesman, M.M. and Pastan, I. 1987. Expression of a full length cDNA for the MDR1 gene confers resistance to colchicine, doxorubicin, and vinblastine. Proc. Natl. Acad. Sci. U.S.A. 84:3004‐3008.
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Key References
   Bain and LeBlanc, 1996. See above.
   Describes the toxicant efflux method described in , as well as adaptations of this protocol for detecting unlabeled toxicants by HPLC.
   Egorin et al., 1974. See above.
   Describes the doxorubicin extraction and fluorescence spectroscopy methods. A modification of this method is described in .
   Ishikawa, 1989. See above.
   Describes the transport of compounds by membrane vesicles using the rapid filtration technique. A modification of this method is described in .
   Ishikawa et al., 1990. See above.
   Describes the preparation of cellular membrane vesicles. A modification of this method is described in
   Krishna et al., 2001. See above.
   A good review article describing the transport of compounds by Caco‐2 cells grown on Transwell inserts, as described in .
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