Collagen Type I Gel Cultures of Adult Rat Hepatocytes as a Screening Induction Model for Cytochrome P450-dependent Enzymes

Abstract

Albumin secretion, expression of cytochrome P450-dependent mono-oxygenases (CYPs) and their inducibility by well-known inducers were evaluated during 1 week in collagen type I gel sandwich and immobilisation cultures of adult primary rat hepatocytes. Albumin secretion increased during culture and, following an initial decrease, CYP biotransformation activities remained stable for at least 7 days. Better preservation results were observed in the collagen gel sandwich culture than in the immobilisation model. The inducibility of CYPs by β-naphthoflavone (β-NF), 3-methylcholanthrene (3-MC), phenobarbital (PB) and dexamethasone (DEX) was studied in both collagen gel hepatocyte cultures. Exposure of the cells to either 5μM 3-MC or 25μM β-NF, added to the culture medium, resulted in strong increases of CYP1A1/2 activity in both culture models. Treatment with PB (3.2mM) resulted in an increase in the CYP2B activity and a higher hydroxylation of testosterone in the 16α-position (CYP2B1/2 and CYP2C11), the 7α-position (CYP2A1/2), and the 6β-position (CYP3A1). DEX (10μM) markedly increased testosterone 6β- and 7α-hydroxylation. Expression and induction experiments on CYP proteins exposed to these molecules confirmed the results of the CYP activity measurements. The patterns of CYP induction in collagen gel cultures of rat hepatocytes were similar to those observed in vivo. Consequently, collagen gel cultures and, more specifically, collagen gel sandwich cultures seem to be suitable as in vitro models for evaluating xenobiotics as potential inducers of CYP-enzymes.