Catalytic characterization and cytokine mediated regulation of cytochrome P450 4Fs in rat hepatocytes (original) (raw)

Evaluation of Time-Dependent Cytochrome P450 Inhibition Using Cultured Human Hepatocytes

Drug Metabolism and Disposition, 2006

Primary human hepatocytes in culture are commonly used to evaluate cytochrome P450 (P450) induction via an enzyme activity endpoint. However, other processes can confound data interpretation. To this end, the impact of time-dependent P450 inhibition in this system was evaluated. Using a substrate-cassette approach, P450 activities were determined after incubation with the prototypic inhibitors tienilic acid (CYP2C9), erythromycin, troleandomycin, and fluoxetine (CYP3A4). Kinetic analysis of enzyme inactivation in hepatocytes was used to describe the effect of these time-dependent inhibitors and derive the inhibition parameters k inact and K I , which generally were in good agreement with the values derived using recombinant P450s and human liver microsomes (HLMs). Tienilic acid selectively inhibited CYP2C9-dependent diclofenac 4-hydroxylation activity, and erythromycin, troleandomycin, and fluoxetine inhibited CYP3A4-dependent midazolam 1hydroxylation in a time-and concentration-dependent manner.

The activity of cytochrome P450IA1 in stable cultured rat hepatocytes

In this study, a stable, differentiated culture format (primary hepatocytes cultured between two layers of collagen gel) was used to study the effect of the inducer 3,3',4,4'-tetrachlorobiphenyl (TCB) on the activity of cytochrome P4501Al. P450IAI (ethoxyresorufin O-deethylase) enzymatic activity was measured using the rate of conversion of ethoxyresorufin (ER) to resorufin (R). After I4 days of induction with 10mh M TCB, hepatocytes in the double collagen gel configuration exhibited a maximum activity of 180.3 + 46.8 pmol R/fig DNA/hr (3.6 + 0.9 nmol R/IO6 cells/hr) compared with 34.9 + 3.8 pmol R/pg DNA/hr for cells cultured on a single layer of gel. At a TCB level of IOms M, the P450IAI activity in the double-gel configuration peaked at 220.8 + 37.0 pmol R/pg DNA/hr. Cessation of 10e6 M TCB induction produced a decrease in activity to 25.8 + 4.1 pmol R/pg DNA/hr within 4 hr. Subsequent re-application of the inducer caused an increase in activity to 76.5 k-1 I. I pmol R/pg DNA/hr within 6 hr. reaching a maximal value of 131.0 + 38.6 nmol R/u~ DNAihr within I2 hr. Since TCB is ranidlv metabolized bv hepatocytes, a continuous perfusion cul&re system was developed to examine the effect of exposure to a constant level of TCB. Continuous perfusion of the cells with 10-s or IO-'M TCB, resulted in activities significantly higher than those of cultures induced by daily application of induction medium. A mechanistic model of TCB-dependent induction of P4501Al was developed using kinetic parameters estimated from static culture data. The model accurately predicted cyclic variations in P450IAl activity in static culture, and the steady-state activity level of perfusion cultures. This work describes procedures for exposing stable hepatocyte cultures to either continuous or declining levels of consumable inducers and for measuring the activity of cytochrome P450IAl in cultured hepatocytes by a non-invasive method.