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J Pharmacol Toxicol Methods. 2000 Jan-Feb;43(1):41-54.

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

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Department of Pharmacology and Experimental Therapeutics, Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA 02111, USA.


Studies were conducted to characterize assays for the isolation and quantitation of rat cytochrome P450 (CYP) 3A isoforms from hepatic and intestinal tissues. Isolated intestinal microsomes were analyzed for their alkaline phosphatase activity and CYP 3A immunoreactivity. The involvement of CYP 3A in the in vitro hydroxylation of midazolam (MDZ) was also evaluated using isoform specific chemical and antibody inhibitors. The effect of glycerol (a common constituent of the microsomal reconstitution buffer) concentration on in vitro MDZ hydroxylation was also investigated. Additionally, to verify that the intestinal preparation was adequate for use in studies investigating the induction of CYP3A at the MRNA, protein, and catalytic activity within a single animal, a separate induction study was carried out with the CYP 3A inducer dexamethasone (DEX). A reverse transcription-polymerase chain reaction (RT-PCR) assay and a quantitative Western blotting method were used to reliably detect differences in CYP 3A mRNA and immunoreactivity between DEX- and vehicle (VH)-treated tissues. The in vitro hydroxylation of MDZ evaluated CYP 3A catalytic activity and identified increases in CYP 3A activity caused by DEX in comparison to VH. Collectively, these described techniques provide an experimental model to study xenobiotic induction of rat hepatic and intestinal CYP 3A from the molecular to the catalytic level in individual rats without the need for pooling of tissue.

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