Use of a perfusion co-culture system consisting of Caco-2 and Hep G2 cell compartments for the kinetic analysis of benzo[a]pyrene toxicity

Abstract

Conventional cytotoxicity tests cannot usually include various metabolic processes in humans. We therefore developed a physiologically based, multi-compartment perfusion co-culture system, using a Caco-2 cell monolayer on a semi-permeable membrane and a microcarrier-based, three-dimensional culture of Hep G2 cells to mimic permeation across the small intestine and biotransformation of the small intestine and the liver. Stable operations allowed us to maintain various activities of both cells for at least 4 days. Co-cultivation improved the growth of Hep G2 cells and enhanced the cytochrome P450 1A1/2 capacities of both Hep G2 and Caco-2 cells. When benzo[a]pyrene (BaP) was loaded on the apical side of the Caco-2 cell layer, the enhanced P450 capacities produced a higher amount of BaP-7,8-hydrodiol, a precursor of the ultimate carcinogen of BaP, BaP-7,8-dihydrodiol-9,10-epoxide (BPDE). These phenomena led to the initially retarded, but later stronger, expression of BaP toxicity in the co-culture system than in pure cultures, which agreed with the actual load of BaP-7.8-hydrodiol to the Hep G2 cells. Because this kind of system can reproduce such complicated phenomena, including those influenced by organ-organ interactions, it is useful as a new in vitro experimental system, for understanding the unknown mechanisms involved in final toxicity in humans and thereby improving physiologically based pharmacokinetic (PBPK) simulation models.