Regulation of CYP1A1 transcription via the metabolism of the tryptophan-derived 6-formylindolo[3,2-b]carbazole

Abstract

A functional cytochrome P4501A1 (CYP1A1) enzyme has been suggested to metabolize endogenous substrates and to autoregulate its own transcription in mouse hepatoma cells. In the present study, the regulation of CYP1A1 gene transcription by 6-formylindolo[3,2-b]carbazole (FICZ), a suggested endogenous ligand for the aryl hydrocarbon receptor (AhR), has been studied in mouse Hepa-1 cell lines. The tryptophan photoproduct, FICZ, has previously been characterized to possess very high AhR binding affinity and to transiently induce CYP1A1 gene expression in cultured cells at picomolar concentrations. The results from this study show that a transient induction of CYP1A1 mRNA at a low concentration of FICZ was only seen in wild-type cells. In c37 cells, deficient in CYP1A1, FICZ caused a sustained induction. Interestingly, we found that a higher amount of tryptophan in culture medium increased the constitutive level of CYP1A1 mRNA expression in the c37 cells but not in the wild-type cells. This suggests that a tryptophan-derived AhR ligand in the medium regulates the basal CYP1A1 expression. In metabolism studies performed with S9 prepared from c37 cells no metabolites were formed from FICZ and no loss of FICZ was observed, while with wild-type cells FICZ was rapidly metabolized. HPLC analysis revealed that at least three metabolites were formed in an NADPH-dependent manner from FICZ when incubated with rat liver S9. The CYP1A1 inhibitor ellipticine totally blocked the metabolism of FICZ. Ellipticine also enhanced both basal and FICZ-induced CYP1A1 mRNA expression. Taken together, these results indicate that tryptophan is a precursor of the endogenous ligand and that the suggested tryptophan-derived ligand FICZ is a substrate for the CYP1A1 enzyme and is involved in autoregulation of CYP1A1 transcription.