Abstract
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.
Footnotes
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Send reprint requests to: Dr. Rory P. Remmel, Department of Medicinal Chemistry, University of Minnesota, 8–101 Weaver-Densford Hall, 308 Harvard St. S.E., Minneapolis, MN 55455. E-mail:remme001{at}tc.umn.edu
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↵1 These results were in partial fulfillment of the doctoral degree requirements of V.H. Preliminary results were reported at the 8th North American ISSX Meeting, Oct, 26–30, 1997, Hilton Head, NC.
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↵2 Present address: Bristol Myers Squibb, P.O. Box 4000, F13–02, Princeton, NJ 08543.
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This work was supported by the National Institutes of Health Grant R01 DK45371–02.
- Abbreviations used are::
- P-450
- cytochrome P-450
- MDZ
- midazolam
- 1′-OH MDZ
- 1′-hydroxymidazolam
- 4-OH MDZ
- 4-hydroxymidazolam
- 1′,4-diOH MDZ
- 1′,4-dihydroxymidazolam
- SSC
- saturated sodium citrate
- DMSO
- dimethyl sulfoxide
- Received June 7, 1999.
- Accepted September 21, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
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Induction of the Metabolism of Midazolam by Rifampin in Cultured Porcine Hepatocytes: Preliminary Evidence for CYP3A Isoforms in Pigs
