Carcinogenic polycyclic aromatic hydrocarbons induce CYP1A1 in human cells via a p53-dependent mechanism

Abstract

The tumour suppressor gene TP53 is mutated in more than 50 % of human tumours, making it one of the most important cancer genes. We have investigated the role of TP53 in cytochrome P450 (CYP)-mediated metabolic activation of three polycyclic aromatic hydrocarbons (PAHs) in a panel of isogenic colorectal HCT116 cells with differing TP53 status. Cells that were TP53(+/+), TP53(+/-), TP53(-/-), TP53(R248W/+) or TP53(R248W/-) were treated with benzo[a]pyrene (BaP), dibenz[a,h]anthracene and dibenzo[a,l]pyrene, and the formation of DNA adducts was measured by (32)P-postlabelling analysis. Each PAH formed significantly higher DNA adduct levels in TP53(+/+) cells than in the other cell lines. There were also significantly lower levels of PAH metabolites in the culture media of these other cell lines. Bypass of the need for metabolic activation by treating cells with the corresponding reactive PAH-diol-epoxide metabolites resulted in similar adduct levels in all cell lines, which confirms that the influence of p53 is on the metabolism of the parent PAHs. Western blotting showed that CYP1A1 protein expression was induced to much greater extent in TP53(+/+) cells than in the other cell lines. CYP1A1 is inducible via the aryl hydrocarbon receptor (AHR), but we did not find that expression of AHR was dependent on p53; rather, we found that BaP-induced CYP1A1 expression was regulated through p53 binding to a p53 response element in the CYP1A1 promoter region, thereby enhancing its transcription. This study demonstrates a new pathway for CYP1A1 induction by environmental PAHs and reveals an emerging role for p53 in xenobiotic metabolism.

Keywords: Benzo[a]pyrene; Carcinogen metabolism; Cytochrome P450; DNA adducts; Tumour suppressor p53.

Fig. 1

Western blot analysis of p53, p21 (CDKN1A), CYP1A1, AHR and NQO1 protein expression in isogenic HCT116 cells after exposure to various PAHs or BPDE. Cells were exposed to 2.5 μM BaP, 0.5 μM BPDE, 2.5 μM DB[a,h]A or 2.5 μM DB[a,l]P and harvested after the times indicated. a HCT116 TP53(+/+) cells compared to TP53(+/−) and TP53(−/−) cells. b HCT116 TP53(+/+) cells compared to TP53(R248W/+) and TP53(R248W/−) cells. β-Actin protein expression was used as a loading control. Representative images of the Western blotting are shown; at least duplicate analysis was performed from independent experiments

Fig. 2

DNA adduct levels (RAL, relative adduct labelling) detected by ³²P-postlabelling in isogenic HCT116 cells after exposure to various PAHs and their corresponding diol-epoxides. Cells were exposed to a 2.5 μM BaP, b 0.5 μM BPDE, c 2.5 μM DB[a,h]A, d 0.5 μM DB[a,h]ADE, e 2.5 μM DB[a,l]P or f 0.0025 μM DB[a,l]PDE and harvested after the times indicated. The values are the mean ± range of duplicate cell incubations; each DNA sample was analysed in duplicate in separate experiments. Statistical analysis was performed by one-way ANOVA followed by the Tukey post hoc test [***p < 0.005, different from HCT116 TP53(+/+) cells]. Insets Autoradiographic profiles of DNA adducts formed in HCT116 cells after exposure; the origins, at the bottom left-hand corners, were cut off before exposure

Fig. 3

Gene expression of a CYP1A1 and b CYP1B1 in isogenic HCT116 cells after exposure to 2.5 μM BaP for 24 h. Total RNA was extracted and the mRNA levels of the indicated genes were analysed by qRT-PCR. Values represent the mean ± SD of three incubations; each sample was determined by three separate analyses. Statistical analysis was performed by one-way ANOVA followed by the Tukey post hoc test [* p < 0.05; ** p < 0.01; *** p < 0.005, different from HCT116 TP53(+/+) cells]

Fig. 4

a Western blot analysis of p53 and CYP1A1 protein expression in HCT116 TP53(+/+) and TP53(−/−) cells after pre-treatment with nutlin-3a (5 μM) for 6 + 24 h and co-exposure with 2.5 μM BaP for 24 h. Representative images of the Western blotting are shown and duplicate analysis was performed from independent experiments. HPLC analysis of BaP-t-7,8-dihydrodiol (b) and BaP-tetrol-I-1 (c) in the culture medium of HCT116 TP53(+/+) and TP53(−/−) cells after pre-treatment with nutlin-3a (5 μM) for 6 + 24 h and co-exposure with 2.5 μM BaP for 24 h. Values represent the mean ± SD of at least three separate incubations. Statistical analysis was performed by one-way ANOVA followed by the Tukey post hoc test [**p < 0.01; ***p < 0.005, different from BaP treated only]

Fig. 5

Binding of p53 to the DNA-regulatory elements (p53RE) of a CDKN1A and b CYP1A1 in HCT116 TP53(+/+) after exposure to 2.5 μM BaP for 24 h. HCT116 TP53(+/+) were also treated with nutlin-3a (10 μM) for 6 + 24 h. After chromatin immunoprecipitation (ChIP) with anti-p53 antibody, the immunoprecipitated DNA was subjected to qRT-PCR using primers amplifying the indicated p53RE regions as outlined in the “Materials and methods”. Values represent the mean ± SD of at least three separate experiments. Statistical analysis was performed by one-way ANOVA followed by the Tukey post hoc test [*p < 0.05; ***p < 0.005, different from control (untreated)]