Viability assessment in sandwich-cultured rat hepatocytes after xenobiotic exposure.
Troglitazone, bosentan and glibenclamide inhibit the bile salt export pump (Bsep) which transports taurocholate into bile. Sandwich-cultured rat hepatocytes maintain functional sodium taurocholate co-transporting polypeptide and Bsep transport proteins, and may be useful to study inhibition of transport by xenobiotics at concentrations below the lowest observable adverse effect level (LOAEL). The purpose of this study was to compare viability assessments determined with the neutral red, lactate dehydrogenase (LDH), alamar blue, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and propidium iodide assays in sandwich-cultured rat hepatocytes following exposure to xenobiotics known to inhibit Bsep, and to define the LOAEL for these xenobiotics in this system. The neutral red assay was not amenable to use in this model due to crystal formation on the collagen. Troglitazone decreased viability in every assay examined, with a LOAEL approximately 100 microM. Bosentan also decreased viability as measured by the LDH, MTT and propidium iodide assays, with a LOAEL approximately 200 microM; however, a significant decrease in viability was not observed with the alamar blue assay. Glibenclamide did not decrease viability with any assay at the xenobiotic concentrations examined in this study. Based on the results of this study, the LDH or propidium iodide assays would be the methods of choice to assess viability in sandwich-cultured rat hepatocytes after xenobiotic exposure.
University of North Carolina, Curriculum in Toxicology, Chapel Hill, NC 27599, USA.
Daniel C
DC
Kemp
Kim L R
KL
Brouwer
D000818:Animals; D001681:Biological Assay / Q000379:methods; D018929:Cell Culture Techniques; D002470:Cell Survival; D004396:Coloring Agents / Q000032:analysis; D022781:Hepatocytes / Q000201:enzymology*; D007202:Indicators and Reagents / Q000032:analysis; D007770:L-Lactate Dehydrogenase / Q000032:analysis / Q000494:pharmacology*; D010078:Oxazines / Q000032:analysis; D011419:Propidium / Q000032:analysis; D051381:Rats; D013778:Tetrazolium Salts / Q000032:analysis; D013844:Thiazoles / Q000032:analysis; D014966:Xanthenes / Q000032:analysis; D015262:Xenobiotics / Q000633:toxicity*
D004396:Coloring Agents; D007202:Indicators and Reagents; D010078:Oxazines; D013778:Tetrazolium Salts; D013844:Thiazoles; D014966:Xanthenes; D015262:Xenobiotics; C005843:resazurin; D011419:Propidium; D007770:L-Lactate Dehydrogenase; C022616:thiazolyl blue