Repression of phenobarbital-dependent CYP2B1 mRNA induction by reactive oxygen species in primary rat hepatocyte cultures.
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 H(2)O(2) or of the catalase inhibitor 3-amino-1,2,4-triazole (AT) to the medium repressed induction to approximately 30% (at 1 mM H(2)O(2) 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 a CYP2B1 promoter-luciferase construct containing approximately 2.7 kilobase pairs of the native CYP2B1 promoter 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.
Hirsch-Ernst
K I
KI
Institute of Pharmacology and Toxicology, Department of Toxicology, University of Göttingen, Robert-Koch-Strasse 40, D-37075 Göttingen, Germany. khirsche@med.uni-goettingen.de
Schlaefer
K
K
Bauer
D
D
Heder
A F
AF
Kahl
G F
GF
D000111:Acetylcysteine / Q000494:pharmacology; D000640:Amitrole / Q000494:pharmacology; D000818:Animals; D019362:Cytochrome P-450 CYP2B1 / Q000096:biosynthesis* / Q000187:drug effects / Q000235:genetics; D004347:Drug Interactions; D004790:Enzyme Induction / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D020868:Gene Silencing / Q000502:physiology; D022781:Hepatocytes / Q000187:drug effects* / Q000201:enzymology; D006861:Hydrogen Peroxide / Q000494:pharmacology; D066298:In Vitro Techniques; D008297:Male; D010634:Phenobarbital / Q000494:pharmacology*; D011401:Promoter Regions, Genetic / Q000187:drug effects; D012333:RNA, Messenger / Q000096:biosynthesis / Q000187:drug effects; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000502:physiology*; D015533:Transcriptional Activation
D004791:Enzyme Inhibitors; D018691:Excitatory Amino Acid Antagonists; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D006861:Hydrogen Peroxide; D019362:Cytochrome P-450 CYP2B1; D000111:Acetylcysteine; D010634:Phenobarbital; D000640:Amitrole