Wei Y D, Bergander L, Rannug U, Rannug A
Department of Genetic and Cellular Toxicology, Stockholm University, Sweden.
Arch Biochem Biophys. 2000 Nov 1;383(1):99-107. doi: 10.1006/abbi.2000.2037.
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.
一种功能性细胞色素P4501A1（CYP1A1）酶被认为可代谢内源性底物并在小鼠肝癌细胞中自动调节自身转录。在本研究中，已在小鼠Hepa-1细胞系中研究了6-甲酰基吲哚并[3,2-b]咔唑（FICZ）对CYP1A1基因转录的调节作用，FICZ是一种推测的芳烃受体（AhR）内源性配体。色氨酸光产物FICZ先前已被表征为具有非常高的AhR结合亲和力，并能在皮摩尔浓度下瞬时诱导培养细胞中CYP1A1基因表达。本研究结果表明，仅在野生型细胞中观察到低浓度FICZ对CYP1A1 mRNA的瞬时诱导。在缺乏CYP1A1的c37细胞中，FICZ引起持续诱导。有趣的是，我们发现培养基中较高含量的色氨酸增加了c37细胞中CYP1A1 mRNA表达的组成水平，但在野生型细胞中没有增加。这表明培养基中色氨酸衍生的AhR配体调节CYP1A1的基础表达。在用c37细胞制备的S9进行的代谢研究中，未从FICZ形成代谢产物，也未观察到FICZ的损失，而在野生型细胞中FICZ迅速代谢。HPLC分析显示，当与大鼠肝脏S9一起孵育时，FICZ以NADPH依赖的方式至少形成三种代谢产物。CYP1A1抑制剂玫瑰树碱完全阻断了FICZ的代谢。玫瑰树碱还增强了基础和FICZ诱导的CYP1A1 mRNA表达。综上所述，这些结果表明色氨酸是内源性配体的前体，并且推测的色氨酸衍生配体FICZ是CYP1A1酶的底物，并参与CYP1A1转录的自动调节。
