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 pi votal role in elimination of foreign compounds but also contribute to gener ation of toxic intermediates, including reactive oxygen species, that may e licit cellular damage if produced excessively. Expression of several xenobi otic-metabolizing P-450 enzymes is induced by phenobarbital (PB). Pronounce d induction is observed for the rat CYP2B1 isoform. A primary rat hepatocyt e culture system was used to investigate whether reactive oxygen species mi ght modulate PB-dependent CYP2B1 induction. In cells cultivated for 3 days with 1.5 mM PB, substantial CYP2B1 mRNA induction was observed (100%). Addi tion of H2O2 or of the catalase inhibitor 3-amino-1,2,4-triazole (AT) to th e medium repressed induction to approximately 30% (at 1 mM H2O2 and 2 mM AT , respectively). Accordingly, treatment of hepatocytes with PB and the glut athione precursor N-acetylcysteine (NAC) led to enhanced PB-dependent induc tion (to over 1000% at 10 mM NAG). In primary hepatocyte cultures transfect ed with a CYP2B1 promoter-luciferase construct containing approximately 2.7 kilobase pairs of the native CYP2B1 promoter sequence, PB-dependent report er gene activation was repressed by AT and stimulated by N-acetylcysteine, Furthermore, a 263-base pair CYP2B1 promoter fragment encompassing the phen obarbital-responsive enhancer module conferred suppression of PB-dependent luciferase expression by AT and activation by NAC in a heterologous SV40-pr omoter construct. In summary, these data demonstrate a regulatory mechanism that is dependent on the cellular redox status, which modulates CYP2B1 mRN A induction by PB on the transcriptional level, thus representing a feedbac k mechanism preventing further P-450-dependent production of reactive oxyge n intermediates under oxidative stress.