Cytochrome P450 2B enzyme (CYP2B) induction defect following phenobarbital treatment in the fa/fa Zucker rat: molecular characterization.
The present study describes the mechanism of the dampened induction of the CYP2B1 and CYP2B2 genes following phenobarbital treatment in the phenotypically obese fa/fa Zucker rat. The fa/fa Zucker rat demonstrated a threefold lower level of CYP2B1/2B2 enzyme induction, as indicated by reduced testosterone oxidation at the 16 beta position and resorufin formation from pentoxy- and benzyloxyresorufin, protein concentration (Western blot analysis), and steady-state mRNA levels (Northern and slot blot analyses) following in vivo treatment with phenobarbital than the Fa/? littermate controls. A primary hepatocyte cell culture system was used to determine if the dampened induction of the CYP2B1/2B2 enzyme is dependent on hormonal influences. Phenobarbital-treated (0.75 mM) hepatocytes from fa/fa Zucker rats showed approximately a three-fold lower induction response based on measurements of CYP2B1/2B2 (R-17 cDNA probe) and CYP2B1 (oligo probe) mRNAs. In order to evaluate whether this dampened response was at the level of transcriptional activation or initiation, as opposed to altered message stability, we measured the rate of transcription of CYP2B1/2B2 genes in nuclei from cultured hepatocytes during run-off experiments. Compared to Fa/? controls, the fa/fa Zucker rat had a greater than threefold lower nuclear transcription rate of CYP2B1/2B2 mRNA. These results suggest that the defective induction of the CYP2B1 and CYP2B2 genes exists at the transcriptional level in the mutant obese fa/fa Zucker rat. These data provide strong evidence that at least two genes are involved. Multiple gene involvement would suggest that the defect is not due to a mutation of the CYP2B gene cis-acting sequence. Instead, the lack of binding of a trans-acting factor, the presence of a repressor, or a defect in transcriptional activation is more likely the molecular mechanism(s) for this enzyme induction defect.
Division of Pharmacology and Experimental Therapeutics, College of Pharmacy, University of Kentucky, Lexington 40536-0082.
R A
RA
Blouin
A M
AM
Bandyopadhyay
I
I
Chaudhary
L W
LW
Robertson
B
B
Gemzik
A
A
Parkinson
D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D002478:Cells, Cultured; D019362:Cytochrome P-450 CYP2B1; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D015342:DNA Probes; D008099:Liver / Q000187:drug effects / Q000201:enzymology; D008297:Male; D009154:Mutation; D009765:Obesity / Q000201:enzymology*; D010078:Oxazines / Q000378:metabolism; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000235:genetics*; D010634:Phenobarbital / Q000494:pharmacology*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D011924:Rats, Zucker; D013739:Testosterone / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects
D015342:DNA Probes; D010078:Oxazines; D012333:RNA, Messenger; D013739:Testosterone; C014180:resorufin; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D019362:Cytochrome P-450 CYP2B1; D010634:Phenobarbital