Persistent Induction of Cytochrome P4501A1 in Human Hepatoma Cells by 3-Methylcholanthrene: Evidence for Sustained Transcriptional Activation of the CYP1A1 Promoter

Abstract

Cytochrome P450 (P450)1A1 plays a critical role in the metabolic activation and detoxification of polycyclic aromatic hydrocarbons (PAHs), many of which are potent human carcinogens. In this investigation, we tested the hypothesis that MC elicits persistent induction of CYP1A1 expression in human hepatoma cells (HepG2) and that this phenomenon is mediated by sustained transcriptional activation of the CYP1A1 promoter. Treatment of HepG2 cells with MC resulted in marked induction (8 -20-fold) of ethoxyresorufin O-de-ethylase activities, CYP1A1 apoprotein contents, and mRNA levels, which persisted for up to 96 h. MC also caused sustained transcriptional activation of the human CYP1A1 promoter for up to 96 h, as inferred from transient transfection experiments. Experiments with deletion constructs indicated that Ah response elements located at Ϫ886, Ϫ974, and Ϫ1047, but not Ϫ491, nucleotides from the start site, contributed to the sustained transcriptional activation of the CYP1A1 promoter. Electrophoretic mobility-shift and chromatin immunoprecipitation assays suggested that prolonged CYP1A1 induction was mediated by Ah receptor (AHR)-independent mechanisms. Experiments with [ 3 H]MC and liquid chromatography-tandem mass spectrometry demonstrated rapid elimination of MC and its metabolites from the cells by 12 to 24 h, suggesting that these compounds did not elicit sustained CYP1A1 induction via the classical AHR-mediated pathway. In conclusion, the results of this study support the hypothesis that MC causes persistent induction of CYP1A1 in human hepatoma cells by mechanisms entailing sustained transcriptional activation of the CYP1A1 promoter via AHR-independent mechanisms. These observations have important implications for human carcinogenesis mediated by PAHs.

Establishment of a stable aryl hydrocarbon receptor-responsive HepG2 cell line

“…The time-and concentration-dependent induction of luciferase activity by 3MC was clearly observed in the stable AHRresponsive HepG2 cell line. These effects were comparable to the results obtained using the method for evaluating the CYP1A1 transcriptional activity (Garrison et al 1996;Fazili et al 2010). The difference between transcriptional activity of CYP1A1 and AHRmediated transcriptional activity is not marked when treated with strong AHR-activating compounds such as 3MC.…”

“…The correlation between the induction of CYP1A1 mRNA expression and AHR-mediated transcriptional activity is particularly strong in a low concentration range (0.01-1.0 lM). Multiple XREs are located 1,252-892 bp upstream of the CYP1A1 promoter and play an important biological role in regulating CYP1A1 expression (Fujii-Kuriyama et al 1992;Garrison et al 1996;Fazili et al 2010). Conversely, XREs are located approximately 3,424-3,299 bp upstream of the UGT1A1 promoter and 2,357-2,333 bp upstream of the ABCG2 promoter (Yueh et al 2003;Tompkins et al 2010).…”

“…Time-and concentration-dependent inductions of AHR target gene transcription by 3MC were observed in the stable AHR-responsive HepG2 cell line, and significant positive correlations between relative luciferase activity and mRNA expression of AHR-target genes were also observed. In previous reports, 3MC induced the gene expression of CYP1A1 (Fazili et al 2010;Tsuchiya et al 2003;Stejskalova et al 2011), UGT1A1 (Smith et al 2005), and ABCG2 (Tompkins et al 2010) in many types of cultured tissues and cells. Moreover, it has been reported that 3MC most strongly induces CYP1A1 expression among the three AHRtarget genes, CYP1A1, UGT1A1, and ABCG2 (Stejskalova et al 2011).…”

Antitumor Activity of a Duocarmycin Analogue Rationalized to Be Metabolically Activated by Cytochrome P450 1A1 in Human Transitional Cell Carcinoma of the Bladder

“…Following 24-hour drug exposure, the expression levels of all 3 CYPs were similar to those of the untreated controls (Supplementary Table S2). A previous study has shown that 12 to 24 hours is the optimal time for dose-dependent CYP1A1 induction in HepG2 cells by 3-methylcholanthrene (27). This indicates that ICT2700, at a therapeutically relevant concentration, does not induce expression of the AhR-regulated CYP1 family or adversely affect the CYP1A1 levels required for its oxidative conversion to a potent cytotoxin.…”

Disruption of the Gene forCYP1A2, which Is Expressed Primarily in Liver, Leads to Differential Regulation of Hepatic and Pulmonary Mouse CYP1A1 Expression and Augmented Human CYP1A1 Transcriptional Activation in Response to 3-Methylcholanthrene In Vivo

“…We also reported that MC elicits sustained CYP1A1 and CYP1A2 induction in vivo via transcriptional activation of the corresponding promoters (Jiang et al, 2009). Furthermore, we showed that MC elicits persistent induction of CYP1A1 in HepG2 cells in vitro by mechanisms entailing sustained transcriptional activation of the CYP1A1 promoter (Fazili et al, 2010).…”