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 -886, -974, and -1047, but not -491, 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 [3H]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.

Fig. 1.

Dose-dependent effect of MC on EROD (CYP1A1) activities in HepG2 cells. HepG2 cells were treated with MC at concentrations ranging from 1 to 10 μM or DMSO, as described under Materials and Methods, and EROD activities were determined in the cells 24 h after treatment. Values represent mean ± S.E. (n = 3). *, statistically significant differences between MC- and DMSO-treated cells at P < 0.05.

Fig. 2.

Time course of CYP1A1 induction by MC in HepG2 cells. A, HepG2 cells were treated with MC (2.5 μM) or DMSO, and EROD activities were determined in the cells at different time points (12–96 h) after treatment. Values represent mean ± S.E. (n = 3). *, statistically significant differences between MC- and DMSO-treated cells at P < 0.05, as determined by two-way ANOVA. B, HepG2 cells were treated with MC or DMSO, and CYP1A1 apoprotein expression was analyzed in whole cell protein (10 μg) at the indicated time points by Western blotting. Quantitative analyses of the apoprotein contents were conducted by densitometry of the CYP1A1 bands, which were normalized against β-actin controls. Data represent mean ± S.E. of at least three independent treatments of the cells with MC.

Fig. 3.

Effect of MC on CYP1A1 mRNA in HepG2 cells. HepG2 cells were treated with MC or DMSO, as described under Materials and Methods, and total RNA was isolated in the cells harvested at 6, 12, 24, 48, 72, and 96 h after MC treatment. The RNA was subjected to real-time RT-PCR using CYP1A1-specific primers. Data represent mean ± S.E. of -fold induction versus controls from at least three replicates. *, statistically significant differences between MC- and DMSO-treated cells at P < 0.05.

Fig. 4.

Effect of MC on CYP1A1 promoter activity in HepG2 cells. HepG2 cells were transiently transfected with a plasmid containing the 1.6-kilobase human CYP1A1 promoter (pGL3-1A1) and reporter (luciferase) gene, as described under Materials and Methods, and luciferase expression was determined in the transfected cell lysates at 6 to 96 h after MC or DMSO exposure. Data represent mean ± S.E. of three independent experiments. *, statistically significant differences between MC- and DMSO-treated cells at P < 0.05, as determined by two-way ANOVA.

Fig. 5.

Plasmids containing broad deletions of the CYP1A1 enhancer region. Deletion plasmids were conducted by subjecting MC to exonuclease digestion to perform various nested deletions.

Fig. 6.

Effect of MC on luciferase expression using deletion plasmids. HepG2 cells were transiently transfected with a plasmid containing one of several deletions of the human CYP1A1 promoter (pGL3-1A1) and reported (luciferase) gene was determined, as described under Materials and Methods. Data represent mean ± S.E. of three independent experiments. *, statistically significant differences between MC- and DMSO-treated cells at P < 0.05, as determined by two-way ANOVA.

Fig. 7.

A, representative EMSA of nuclear extracts from DMSO- and MC-treated HepG2 cells. Cells were treated in triplicate with MC or DMSO and harvested at 6 to 96 h after MC treatment. Nuclear protein extracts from treated cells were subjected to EMSA, as described under Materials and Methods. The labeled nuclear proteins were separated by polyacrylamide gel electrophoresis, and the gels were dried. The gels were exposed to autoradiography at −80°C for 24 h. Arrow indicates interaction with the AHREs of an MC-specific nuclear protein that appears to be MC-AHR-ARNT complex, which is competed off in the presence of 50-fold excess of cold AHRE-specific oligonucleotide (CP). B, ChIP assays showing complexing of the MC-AHR-ARNT complex to the CYP1A1 enhancer region. Cells were treated with MC or DMSO for the indicated time points, and ChIP assays were conducted using AHR antibodies as described under Materials and Methods.

Fig. 8.

Metabolism of [³H]MC and its metabolites by radiometric TLC (A) in HepG2 cells (B) and media (C). A, HepG2 cells were treated with [³H]MC (2.5 μM; 1.9 Ci/mmol), and at different time points (6–96 h), the cells were harvested, extensively washed, and then MC and its metabolites were extracted with chloroform/methanol. The total radioactivity, representing MC and its metabolites, was measured, and the material was then subjected to radiometric TLC. B, the parent MC and metabolite zones were then excised from TLC plates, counted, and intracellular levels of MC and its metabolites were estimated. Values represent mean ± S.E. of at least three independent treatments of the cells. C, HepG2 cells were treated with [³H]MC, as described in legend to B, and at the indicated time points, the media were extracted with chloroform/methanol, and MC and its metabolites were quantified by radiometric TLC, as described under Materials and Methods. Values represent mean ± S.E. of at least three independent treatments of the cells.

Fig. 9.

LC-MS/MS analyses of MC. A, multiple reaction monitoring (MRM) transition showing the primary product ion MC at m/z 253.9, which represents M⁺ + 1 −15 (loss of methyl group) fragment. B, MS/MS (B1, B3) and UV (B2, B4) chromatograms of MC sample (B1 and B2) and reference standards (B3 and B4) showing the presence of MC by both UV and MS/MS detection.

Fig. 10.

Determination of intracellular and media levels of MC by LC-MS/MS analyses. HepG2 cells were treated with cold MC (2.5 μM), and at the indicated time points, the cells and media were used to analyze the levels of parent MC by LC-MS/MS, as described under Materials and Methods. Values represent mean ± S.E. of at least three independent treatments of the cells with MC.

Fig. 11.

Effect of MC metabolites on EROD (CYP1A1) activities. HepG2 cells were treated with each of the MC metabolites (1 μM), i.e., 1-hydroxy-3-MC (1-OH-MC), 2-hydroxy-3-MC (2-OH-MC), 11-hydroxy-3-MC (11-OH-MC), 11,12-dihydro-11,12-dihydroxy-3-MC (MC-11,12-diol), 1-one-3-MC (MC-1-one), 11,12 epoxy-11,12 dihydro-3-MC (MC-11,12-DE), 11,12-dione-3-MC (MC-11,12-dione), and 11,12-dialdehyde-3-MC (MC-11,12-dialde). The cells were harvested after 24 h, and EROD activities were measured. Values represent mean ± S.E. (n = 3). *, statistically significant differences between MC metabolites and DMSO-treated cells at P < 0.05, as determined by Student's t test.

Fig. 12.

Cell viability by MTT reduction. HepG2 cells were treated with MC as described under Materials and Methods, and at the indicated time points, the viabilities of the cells were determined by MTT reduction. Values represent mean ± S.E. (n = 3). *, statistically significant differences between MC- and DMSO-treated cells at P < 0.05, as determined by Student's t test.