RT Journal Article
SR Electronic
T1 Repression of Phenobarbital-Dependent CYP2B1 mRNA Induction by Reactive Oxygen Species in Primary Rat Hepatocyte Cultures
JF Molecular Pharmacology
JO Mol Pharmacol
FD American Society for Pharmacology and Experimental Therapeutics
SP 1402
OP 1409
DO 10.1124/mol.59.6.1402
VO 59
IS 6
A1 K. I. Hirsch-Ernst
A1 K. Schlaefer
A1 D. Bauer
A1 A. F. Heder
A1 G. F. Kahl
YR 2001
UL http://molpharm.aspetjournals.org/content/59/6/1402.abstract
AB Xenobiotic-metabolizing cytochrome P-450 (P-450) enzymes not only play a pivotal role in elimination of foreign compounds but also contribute to generation of toxic intermediates, including reactive oxygen species, that may elicit cellular damage if produced excessively. Expression of several xenobiotic-metabolizing P-450 enzymes is induced by phenobarbital (PB). Pronounced induction is observed for the rat CYP2B1 isoform. A primary rat hepatocyte culture system was used to investigate whether reactive oxygen species might modulate PB-dependent CYP2B1 induction. In cells cultivated for 3 days with 1.5 mM PB, substantial CYP2B1 mRNA induction was observed (100%). Addition of H2O2 or of the catalase inhibitor 3-amino-1,2,4-triazole (AT) to the medium repressed induction to approximately 30% (at 1 mM H2O2 and 2 mM AT, respectively). Accordingly, treatment of hepatocytes with PB and the glutathione precursor N-acetylcysteine (NAC) led to enhanced PB-dependent induction (to over 1000% at 10 mM NAC). In primary hepatocyte cultures transfected with aCYP2B1 promoter-luciferase construct containing approximately 2.7 kilobase pairs of the native CYP2B1promoter sequence, PB-dependent reporter gene activation was repressed by AT and stimulated by N-acetylcysteine. Furthermore, a 263-base pair CYP2B1 promoter fragment encompassing the phenobarbital-responsive enhancer module conferred suppression of PB-dependent luciferase expression by AT and activation by NAC in a heterologous SV40-promoter construct. In summary, these data demonstrate a regulatory mechanism that is dependent on the cellular redox status, which modulates CYP2B1 mRNA induction by PB on the transcriptional level, thus representing a feedback mechanism preventing further P-450–dependent production of reactive oxygen intermediates under oxidative stress.