Quantification of Drug-Induced Inhibition of Canalicular Cholyl-l-Lysyl-Fluorescein Excretion From Hepatocytes by High Content Cell Imaging

Toxicol Sci. 2015 Nov;148(1):48-59. doi: 10.1093/toxsci/kfv159. Epub 2015 Jul 27.


We describe the use of a commercially available high content cell imaging algorithm (Cellomics Arrayscan Spot Detector) to quantify biliary excretion of the fluorescent probe substrate cholyl-l-lysyl-fluorescein (CLF) from rat hepatocytes cultured in collagen/matrigel sandwich configuration and to explore inhibition of this process by a variety of test compounds. The method provided robust, reproducible data. Twenty-nine pharmaceuticals inhibited biliary CLF efflux from hepatocytes and a broad range of potencies of inhibition were observed (IC50 values ranged between <1 and 794 µM). Thirteen drugs that inhibited CLF efflux also inhibited hepatocellular uptake of the probe substrate [(3)H]-taurocholate. Although no clear correlation between the potencies of inhibition of the 2 processes was evident, these data highlight the need to consider possible uptake transporter inhibition when interpreting hepatocyte CLF inhibition data. It has been reported that CLF is transported by MRP2. The CLF efflux inhibition data correlated closely with published data on inhibition by the drugs of the bile salt export pump (Bsep), which suggests that the tested drugs inhibit both Bsep and Mrp2. Calculation of the ratios between the maximum human plasma concentrations of the drugs and their CLF efflux inhibition IC50 values raised the possibility that for many, but not all, of them the in vitro effects may be functionally significant in vivo and that Mrp2 inhibition might be a drug-induced liver injury (DILI) risk factor. These data indicate that imaging hepatocyte CLF inhibition is a promising new method for quantification of biliary efflux inhibition by drugs, which could aid assessment of compound-related DILI risk.

Keywords: DILI; cholyl-l-lysyl-fluorescein; drug hepatotoxicity; hepatobiliary transporters.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • ATP Binding Cassette Transporter, Subfamily B, Member 11
  • ATP-Binding Cassette Transporters / antagonists & inhibitors
  • ATP-Binding Cassette Transporters / genetics
  • ATP-Binding Cassette Transporters / metabolism
  • Absorption, Physiological / drug effects
  • Animals
  • Bile Canaliculi / cytology
  • Bile Canaliculi / drug effects*
  • Bile Canaliculi / metabolism
  • Carrier Proteins / antagonists & inhibitors
  • Carrier Proteins / genetics
  • Carrier Proteins / metabolism
  • Cell Polarity / drug effects
  • Cells, Cultured
  • Cholic Acids / metabolism*
  • Down-Regulation / drug effects*
  • Drug Evaluation, Preclinical / methods
  • Drugs, Investigational / adverse effects
  • Drugs, Investigational / pharmacology*
  • Fluoresceins
  • Fluorescent Dyes / metabolism*
  • Gene Expression Regulation / drug effects*
  • Hepatocytes / cytology
  • Hepatocytes / drug effects*
  • Hepatocytes / metabolism
  • Kinetics
  • Male
  • Membrane Glycoproteins / antagonists & inhibitors
  • Membrane Glycoproteins / genetics
  • Membrane Glycoproteins / metabolism
  • Multidrug Resistance-Associated Protein 2
  • Multidrug Resistance-Associated Proteins / antagonists & inhibitors
  • Multidrug Resistance-Associated Proteins / genetics
  • Multidrug Resistance-Associated Proteins / metabolism
  • Rats, Wistar
  • Reproducibility of Results
  • Taurocholic Acid / metabolism


  • ABCC2 protein, human
  • ATP Binding Cassette Transporter, Subfamily B, Member 11
  • ATP-Binding Cassette Transporters
  • Abcb11 protein, rat
  • Carrier Proteins
  • Cholic Acids
  • Drugs, Investigational
  • Fluoresceins
  • Fluorescent Dyes
  • Membrane Glycoproteins
  • Multidrug Resistance-Associated Protein 2
  • Multidrug Resistance-Associated Proteins
  • bile acid binding proteins
  • cholyl-lysylfluorescein
  • Taurocholic Acid