Abstract
The promoter activities of the genes for cytochrome P450 2B1 (CYP2B1) and cytochrome P450 2C1 (CYP2C1) have been assayed by direct injection of promoter-luciferase chimeric genes into rat liver. Activities of minimal promoters for CYP2C1 and CYP2B1 were detectable in untreated animals but were not increased by treatment of the animals with phenobarbital. After insertion to the 5' side of the minimal promoters of one to three copies of the CYP2B2 sequence from -2318 to -2155, a phenobarbital-responsive element in primary hepatocyte cultures (Trottier, E., Belzil, A., Stoltz, C., and Anderson, A. (1995) Gene (Amst.) 158, 263-268), phenobarbital treatment induced the activity of the CYP2C1 promoter by 5-15-fold and the CYP2B1 promoter by 2.5-5-fold. Mutation of a basal transcription element-like motif and a CCAAT/enhancer binding protein element in the CYP2B1 proximal promoter region reduced expression, but 3-4-fold induction by phenobarbital was retained. Mutation of the "Barbie box," a putative phenobarbital-responsive element (He, J.-S., and Fulco, A. J. (1991) J. Biol. Chem. 266, 7864-7869) in the CYP2B1 proximal promoter did not reduce the relative response to phenobarbital. These results demonstrate that direct injection of DNA into rat liver may be used to assay phenobarbital responsiveness of cytochrome P450 genes. In this system, a distal CYP2B2 element mediates a response to phenobarbital, and proximal elements, including the Barbie box, are not required for the induction.
Highlights
The induction of drug-metabolizing enzymes by phenobarbital (PB)1 was reported almost 40 years ago [1]
A sequence motif was identified as a PB-response element (“Barbie box”) in CYP genes in Bacillus megaterium, and similar motifs were found in other PB-responsive genes in several species including the proximal promoter of mammalian cytochrome P450 2B1 (CYP2B1)/2 and cytochrome P450 2C1 (CYP2C1) (6 – 8)
We report that direct injection of DNA into rat liver is an effective in situ transient transfection method to assay CYP promoter activity
Summary
The induction of drug-metabolizing enzymes by phenobarbital (PB)1 was reported almost 40 years ago [1]. We report that direct injection of DNA into rat liver is an effective in situ transient transfection method to assay CYP promoter activity.
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