Tryptophan photoproducts : regulation of CYP1A1 transcription and modulation of cell growth

Sammanfattning: The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor involved in the regulation of many drug-metabolizing enzymes. The regulation of cytochrome P4501A1 (CYP1A1) has been most intensively studied. A large number of highly toxic environmental compounds, such as dioxins and PAHs are ligands for the AhR and give rise to CYP1A1 induction. Also, UV-light induces CYP1A1. Accumulating data indicate the existence of endogenous ligands for the AhR which, however, remain to be identified. UV-irradiation of the amino acid tryptophan produces photoproducts that possess very high binding affinities for the AhR. These photoproducts have therefore been suggested as possible endogenous ligands for the AhR. The biologically most active compound has been identified as 6-formylindolo[3,2-b]carbazole (FICZ).The aim of the present study was to further investigate these compounds, focusing on FICZ , with respect to the regulation of CYP1A1 transcription and the modulation of cell growth, as well as their possible formation in biological systems. The study was carried out using different cultured mammalian cells, including two types of human cells (a keratinocyte cell line and peripheral blood mononuclear cells) as well as mouse hepatoma Hepa-1 cell lines.Experiments with dose- and time-dependent induction and comparison with other inducers showed that FICZ is a potent inducer of CYP1A1 mRNA and that the induction appears very rapidly and is transient in nature. Metabolism studies using Aroclor-induced rat liver S9 and S9 from mouse Hepa-1 cells with HPLC analysis revealed that the transient induction of CYP1A1 mRNA by FICZ was due to its metabolism by the CYP1A1 enzyme. These results, together with gene expression studies, indicated that the level of CYP1A1 mRNA induced by FICZ is auto-negatively controlled by the induced CYP1A1 enzyme and that FICZ thus behaves as an endogenous substrate involved in the autoregulation mechanism. Additionally, the studies in human cells showed that tryptophan facilitates UV-induced CYP1A1 gene expression, indicating that the tryptophan photoproducts may be formed in the cells upon UV-irradiation.The role of CYP1A1 in the regulation of cell growth was characterized. Observations of a slower cell proliferation in wild-type Hepa-1 cells compared to cells lacking CYP1A1 activity indicated that CYP1A1 could reduce cell growth most likely by metabolizing an endogenous substrate that stimulates growth. The results were confirmed by restoring the CYP1A1 activity in the deficient cells by transfection. FICZ was shown to enhance cell proliferation and modulate the expression of genes involved in signal transduction.In conclusion, the results provide evidence for tryptophan-derived substances as being endogenous AhR ligands which autoregulate CYP1A1 transcription and activate cell growth. These findings will form a basis for a better understanding of the physiological processes that are regulated by the AhR and CYP1A1 and thereby also a better understanding of the mechanisms by which xenobiotics disrupt these processes.