The activity of cytochrome P450IA1 in stable cultured rat hepatocytes

Abstract

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 P50IA1. P450IA1 (ethoxyresorufin O-deethylase) enzymatic activity was measured using the rate of conversion of ethoxyresorufin (ER) to resorufin (R). After 14 days of induction with 10 −6, M TCB, hepatocytes in the double collagen gel configuration exhibited a maximum activity of 180.3 ± 46.8 pmol R/ μg, DNA/hr (3.6 ± 0.9 nmol R/10 6, cells/hr) compared with 34.9 ± 3.8 pmol R/ μg DNA/hr for cells cultured on a single layer of gel. At a TCB level of 10 −5, m, the P450IA1 activity in the double-gel configuration peaked at 220.8 ± 37.0 pmol R/ μg DNA/hr. Cessation of 10 −6, M TCB induction produced a decrease in activity to 25.8 ± 4.1 pmol R/ μg DNA/hr within 4 hr. Subsequent re-application of the inducer caused an increase in activity to 76.5 ± 11.1 pmolR/ μg DNA/hr within 6 hr, reaching a maximal value of 131.0 ± 38.6 pmol R/ μg DNA/hr within 12 hr. Since TCB is rapidly metabolized by hepatocytes, a continuous perfusion culture system was developed to examine the effect of exposure to a constant level of TCB. Continuous perfusion of the cells with 10 −8 or 10 −7, 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 P450IA1 was developed using kinetic parameters estimated from static culture data. The model accurately predicted cyclic variations in P450IA1 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 P50IA1 in cultured hepatocytes by a non-invasive method.