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Early transcriptional responses induced by benzo(a)pyrene diol epoxide in human amnion epithelial FL cells

ABSTRACT: Genotoxic agents cause cellular DNA damage and stress responses, including transcriptional changes. Here we focused on the early transcriptional responses of human cells to benzo(a)pyrene diol epoxide (BPDE), which causes bulky DNA adduct damage. Human amnion epithelial FL cells were exposed to three doses of BPDE (5, 50, and 500 nM) and the vehicle control DMSO, and differential gene expression profiles were obtained 4 h after exposure using oligonucleotide microarrays followed by validation with quantitative real-time RT-PCR. Compared with a few genes affected by the low and medium-dose exposure, extensive and robust changes in gene expression were induced by the high-dose BPDE. We found that the expression of cell cycle-regulators, signaling molecules and transcription factors were significantly altered and important signaling pathways related to cell survival or apoptosis were affected by BPDE. Several genes and related regulatory pathways that were previously not known to be responsive to this genotoxic agent have now been implicated, which helps to draw the whole picture of how cells respond to environmental chemical exposure via transcriptional regulation. Keywords: Dose response

ORGANISM(S): Homo sapiens

PROVIDER: GSE7675 | GEO | 2008/01/01

SECONDARY ACCESSION(S): PRJNA99729

REPOSITORIES: GEO

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Project description:BACKGROUND: Benzo[a]pyrene (BaP) is a widespread environmental genotoxic carcinogen that damages DNA by forming adducts. This damage along with activation of the aryl hydrocarbon receptor (AHR) induces complex transcriptional responses in cells. To investigate whether human cells are more susceptible to BaP in a particular phase of the cell cycle, synchronised breast carcinoma MCF-7 cells were exposed to BaP. Cell cycle progression was analysed by flow cytometry, DNA adduct formation was assessed by 32P-postlabeling analysis, microarrays of 44K human genome-wide oligos and RT-PCR were used to detect gene expression (mRNA) changes and Western blotting was performed to determine the expression of some proteins, including cytochrome P450 (CYP) 1A1 and CYP1B1, which are involved in BaP metabolism. RESULTS: Following BaP exposure, cells evaded G1 arrest and accumulated in S-phase. Higher levels of DNA damage occurred in S- and G2/M- compared with G0/G1-enriched cultures. Genes that were found to have altered expression included those involved in xenobiotic metabolism, apoptosis, cell cycle regulation and DNA repair. Gene ontology and pathway analysis showed the involvement of various signalling pathways in response to BaP exposure, such as the Catenin/Wnt pathway in G1, the ERK pathway in G1 and S, the Nrf2 pathway in S and G2/M and the Akt pathway in G2/M. An important finding was that higher levels of DNA damage in S- and G2/M-enriched cultures correlated with higher levels of CYP1A1 and CYP1B1 mRNA and proteins. Moreover, exposure of synchronised MCF-7 cells to BaP-7,8-diol-9,10-epoxide (BPDE), the ultimate carcinogenic metabolite of BaP, did not result in significant changes in DNA adduct levels at different phases of the cell cycle. CONCLUSIONS: This study characterised the complex gene response to BaP in MCF-7 cells and revealed a strong correlation between the varying efficiency of BaP metabolism and DNA damage in different phases of the cell cycle. Our results suggest that growth kinetics within a target-cell population may be important determinants of susceptibility and response to a genotoxic agent.

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Early transcriptional responses induced by benzo(a)pyrene diol epoxide in human amnion epithelial FL cells

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