The detection of cholestasis-inducing agents in cultured primary rat hepatocytes

Scope of the method

Alternative method relates to
  • Human health
Alternative method is situated in
  • Basic Research
  • Translational - Applied Research
Type of alternative method
  • In vitro - Ex vivo
This method makes use of
  • Animal derived cells / tissues / organs
Species from which cells/tissues/organs are derived
Rat
Type of cells/tissues/organs
Primary rat hepatocytes

Description

Method keywords
  • Sandwich cultures
  • Hepatocytes
  • Bile salt export pump (Bsep) inhibition
  • Cholyl-lysyl-fluorescein (CLF)
  • Cholestasis-inducing potential
Scientific area keywords
  • Toxicology
  • in vitro
  • Drug-induced liver injury (DILI)
  • cholestasis
Method desription

The standard operating procedure describes a method to assess the cholestasis-inducing potential of chemicals, in casu in cultures of primary rat hepatocytes. The procedure relies on the accumulation of the fluorescent bile salt export pump (Bsep) substrate cholyl-lysyl-fluorescein (CLF) in the canalicular network of sandwich-cultured rat hepatocytes either in presence or the absence of Bsep inhibitors.

Lab equipment

Fluorescent microscope (Nikon Eclipse Ti-S, Belgium)

Pros, cons & Future potential

Advantages

The standard operating procedure comprises an easy-to-apply method to detect cholestasis-inducing agents based on Bsep inhibition. Since sandwich cultures of hepatocytes, in contrast to conventional monolayer cultures, exhibit reformation of the canalicular network and polarized excretory functions, this culture systems forms an appropriate experimental setting for studying biliary excretion.

Challenges

Most Bsep substrates, including CLF, cannot undergo efficient cellular translocation without the support of an uptake transporter, such as sodium-dependent taurocholate cotransporting polypeptide (Ntcp). A number of drugs, known to inhibit Bsep activity, also possess the ability to interfere with the Ntcp-mediated uptake of bile salts. This phenomenon should always be taken into account as it may complicate the interpretation of the experimental results.

References, associated documents and other information

References

Dawson, S., Stahl, S., Paul, N., Barber, J. & 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, 130-8

Kaplowtiz, N. 2004. Drug-induced liver injury. Clin Infect Dis, 38 Suppl 2, S44-8

Kis, E., Ioja, E., Rajnai, Z., Jani, M., Méhn, D., Herédi-Szabό, K. & Krajcsi, P. 2011. BSEP inhibition - In vitro screens to assess cholestatic potential of drugs. Toxicol In Vitro

Padda, M. S., Sanchez, M., Akhtar, A. J. & Boyer, J. L. 2011. Drug-induced cholestasis. Hepatology, 53, 1377-87

Schuster, D., Laggner, C. & Langer, T. 2005. Why drugs fail - A study on side effects in new chemical entities. Current Pharmaceutical Design, 11, 3545-59

Swift, B., Pfeifer, N. D. & Brouwer, K. L. 2010. Sandwich-cultured hepatocytes: an in vitro model to evaluate hepatobiliary transporter-based drug interactions and hepatotoxicity. Drug Metab Rev, 42, 446-71

Vinken, M., Elaut, G., Henkens, T., Papeleu, P., Snykers, S., Vanhaecke, T. & Rogiers, V. 2006. Rat hepatocyte cultures: collagen gel sandwich and immobilization cultures. Methods Mol Biol, 320, 247-54

Associated documents
BSEP inhibition.docx

Contact person

Raf Van Campenhout