Materials & Methods: Human, rat, dog and monkey hepatocytes were incubated in serum free media. 2-4 days after cell isolation a time and concentration dependent uptake of [3H]- estrone-3-sulfate (E3S), -bromosulfophthaleine (BSP), -digoxine (Dig) and -taurocholic acid (TA) at 4°C and 37°C was measured using liquid scintillation counting. Competition assays were performed using rifampicin.
Results: All hepatocytes showed at 37°C a time-dependent and saturable increase in E3S and BSP uptake compared to the uptake at 4°C. By contrast, Dig and TA showed a time-dependent and saturable transport only in hepatocytes of humanoids and rats, but not in dog. E3S revealed a high affinity to human (Km = 12.9 ± 10.1 µmol/l; Vmax = 84.2 ± 30.3 pmol/mg × min), monkey (Km = 8.2 ± 2.3 µmol/l; Vmax = 31.3 ± 3.6 pmol/mg × min) and dog (Km = 3.3 ± 2.3 µmol/l; Vmax = 10.0 ± 2.1 pmol/mg × min) but not to rat hepatocytes. For BSP and Dig no pharmacokinetic data could be calculated and TA was transported only into hepatocytes of monkey (Km = 17.5 ± 12.5 µmol/l; Vmax = 5.8 ± 2.1 pmol/mg × min). Rifampicin clearly inhibited the E3S uptake in monkey and dog hepatocytes while uptake of E3S in rat hepatocytes was not influenced.
Discussion: Our data suggest that from all tested substrates E3S is the most suitable substrate to verify the functionality of uptake transporters in primary hepatocytes of humanoids and dogs, but not of rats. However, none of the tested substrates can be used to verify the functionality of uptake transporters in primary rat hepatocytes. This species specific differences in hepatocellular uptake transporter activities may contribute to species specific differences very often observed when drug metabolism and toxicity are analyzed.