Feasibility of a simple double-layered coculture system incorporating metabolic processes of the intestine and liver tissue: application to the analysis of benzo[a]pyrene toxicity

A simple double-layered coculture system using Caco-2 cell and Hep G2 cell, which mimic metabolic processes occurring in humans such as absorption through the intestine and cytochrome P450 1A1/2 involving biotransformation in both the intestine and liver cells, was used to investigate the toxicity of model chemical, benzo[a]pyrene (B[a]P). It was found that both Caco-2 and Hep G2 cells can metabolize B[a]P to toxic metabolites including B[a]P-7,8-hydrodiol (7,8-diol), an immediate precursor to the highly-reactive ultimate toxicant of B[a]P, B[a]P-7,8-hydrodiol-9,10-epoxide (BPDE), possibly mediated by cytochrome P450 1A1/2 activity. However, in a double-layered coculture system, no significant reduction of Hep G2 cell viability was found, although an approximately 50% reduction in viability was observed in pure Hep G2 cells. HPLC analysis showed that Caco-2 cells transfer B[a]P and its toxic metabolites back to the apical side, thus decreasing the concentrations of toxic metabolites including B[a]P-7,8-hydrodiol (7,8-diol) in cocultured Hep G2 cells. These results appear to be correlated with in vivo data on the effects of orally administered B[a]P, that is, low (10%) bioavailability in the rats and almost no acute lethal toxicity in rats or mice. As such, the simple double-layered coculture system can provide more accurate information regarding the toxic actions of the hazardous chemicals in humans than a pure culture system, as it also gives the final toxicity as a result of many complicated phenomena such as selective permeation in the intestine and biotransformation in the intestine and liver. (C) 2004 Elsevier Ltd. All rights reserved.