High-Content Imaging in Human and Rat Hepatocytes Using the Fluorescent Dyes CLF and CMFDA Is Not Specific Enough to Assess BSEP/Bsep and/or MRP2/Mrp2 Inhibition by Cholestatic Drugs

Abstract

Inhibition of hepatic efflux transporters (BSEP, MRP2) is one of the important mechanisms in drug-induced liver injury and in particular cholestasis. The inhibitory effect of compounds on these two hepatic transporters is commonly delineated using inverted membrane vesicles from Sf9 insect cells overexpressing the BSEP or MRP2 protein. However, due to lack of bioactivation and metabolism of the compound, and the lack of the expression and functionality of other sinusoidal efflux and uptake transporters in the cell-free assay system, it is difficult to fully understand the role of a given hepatic transporter in cholestasis. Therefore, our aim was to characterize and develop a competent cell-based bile canalicular imaging assay for measuring the overall inhibitory effects of compounds on biliary transport. Since species differences have been reported for some compounds, we developed the assay using both rat and human hepatocytes. Two fluorescent dyes, cholyl-lysyl-fluorescein (CLF) and carboxymethyl flourescein diacetate (CMFDA), have been reported in the literature to measure BSEP and MRP2 inhibition. We show in our study that neither CLF nor CMFDA is a specific substrate for either BSEP/Bsep or MRP2/Mrp2 as demonstrated by generating K_m values for each transporter. Most of the drugs tested inhibited CLF accumulation and exhibited less potency toward CMFDA accumulation in hepatocytes. We found the highest concordance between IC₅₀ values for inhibition of CLF accumulation in human hepatoyctes and IC₅₀ values generated in the human BSEP vesicle assay. This suggests that the human vesicle-based assays could suffice as a primary screen to avoid future potential human liver injury.