Vol. 27, Issue 12, 1512-1518, December 1999

Induction of the Metabolism of Midazolam by Rifampin in Cultured Porcine Hepatocytes: Preliminary Evidence for CYP3A Isoforms in Pigs¹

Vinayak P. Hosagrahara,² Linda K. Hansen, and Rory P. Remmel

Departments of Medicinal Chemistry (V.P.H., R.P.R.), and Laboratory Medicine and Pathology (L.K.H.), University of Minnesota, Minneapolis, Minnesota

The induction of putative CYP3A isoforms in cultured porcine hepatocytes was evaluated by measurement of midazolam metabolism, a model substrate of the CYP3A family. The induction was also studied at the molecular level by quantitation of mRNA and protein levels, by Northern blotting and Western blotting, respectively. Pretreatment with rifampin (50 µM) resulted in a 5.5- to 9-fold higher rate of midazolam metabolism when compared with control cultures. No induction was observed when the cultures were pretreated with 50 µM dexamethasone. A 12-fold increase in the CYP3A mRNA signal (~2.4 kB) was observed in induced cultures over control cultures. Microsomal proteins were separated by SDS-polyacrylamide gel electrophoresis and detected by immunoblotting with a polyclonal antibody raised against human CYP3A4. The immunoblots showed the presence of four bands in microsomes prepared from pig livers, with two bands (51.5 and 52 kD) that showed intense staining. Microsomes prepared from a pig pretreated with rifampin showed marked induction of these two bands. Immunoblotting of microsomes from rifampin-induced cultures also showed significantly greater intensity than in control cultures. Our results indicate that rifampin, but not dexamethasone, is an inducer of midazolam hydroxylase in pig hepatocytes. This induction may be regulated at the transcriptional level as detected by an increase in mRNA with a CYP3A oligonucleotide probe. Finally, there appears to be a multiplicity of the CYP3A isoforms in pig hepatocytes, similar to that observed in humans and rats.