Methodologies to study the induction of rat hepatic and intestinal cytochrome P450 3A at the mRNA, protein, and catalytic activity level

Studies were conducted to characterize assays for the isolation and quantit ation of rat cytochrome P450 (CYP) 3A isoforms from hepatic and intestinal tissues. Isolated intestinal microsomes were analyzed for their alkaline ph osphatase activity and CYP 3A immunoreactivity. The involvement of CYP 3A i n the in vitro hydroxylation of midazolam (MDZ) was also evaluated using is oform specific chemical and antibody inhibitors. The effect of glycerol (a common constituent of the microsomal reconstitution buffer) concentration o n in vitro MDZ hydroxylation was also investigated. Additionally, to verify that the intestinal preparation was adequate for use in studies investigat ing the induction of CYP 3A at the mRNA, protein, and catalytic activity wi thin a single animal, a separate induction study was carried out with the C YP 3A inducer dexamethasone (DEX). A reverse transcription-polymerase chain reaction (RT-PCR) assay and a quantitative Western blotting method were us ed to reliably detect differences in CYP 3A mRNA and immunoreactivity betwe en DEX- and vehicle (VH)-treated tissues. The in vitro hydroxylation of MDZ evaluated CYP 3A catalytic activity and identified increases in CYP 3A act ivity caused by DEX in comparison to VH. Collectively, these described tech niques provide an experimental model to study xenobiotic induction of rat h epatic and intestinal CYP 3A from the molecular to the catalytic level in i ndividual rats without the need for pooling of tissue. (C) 2000 Elsevier Sc ience Inc. All rights reserved.