Probing enhanced cytochrome P4502B1/2 activity in rat hepatocyte spheroidsthrough confocal laser scanning microscopy

Cytochrome P450 (CYP450) enzymes are essential for xenobiotic metabolism. A lthough CYP450s are found in many tissues, CYP2B1/2 are primarily expressed in the rat liver. The constitutive expression in vivo of CYP2B1/2 is low b ut it is induced in the presence of various drugs such as phenobarbital (PB ). In this study, CYP2B1/2 activity in cultured hepatocytes was assessed in situ with the introduction of a fluorogenic substrate, pentoxyresorufin. T he product of 7-pentoxyresorufin-O-dealkylation (PROD), which is catalyzed specifically by CYP2B1/2, was detected using confocal laser scanning micros copy (CLSM). Primary hepatocytes cultured as monolayers on collagen-coated surfaces exhibited background PROD activity and minimal PB inducibility aft er 4 days in culture. In contrast, rat hepatocytes organized in compacted a ggregates, or spheroids, exhibited higher levels of PROD activity and retai ned their ability for PB induction. The results from the CLSM analysis were verified by RT-PCR and Western immunoblotting analysis. Furthermore, CLSM in conjunction with image processing techniques and three-dimensional recon struction revealed the localization of enhanced PROD activity in the center of spheroids. The results support the use of CLSM as a powerful tool for i nvestigating CYP2B1/2 activity in cultured rat hepatocytes.