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Quantification and mapping of alkylation in the human genome reveal single nucleotide resolution precursors of mutational signatures

ABSTRACT: Benzo[a]pyrene (BaP) is a known human carcinogen (IARC Group 1) found in food, coal tar, as well as cigarettes and other smoke. Its diol-epoxide metabolites (Benzo[a]pyrene diol-epoxide [BPDE]) react with DNA forming DNA adducts, predominantly N2-BPDE-deoxyguanosine (N2-BPDE-dG). While the capacity of BPDEs to alkylate DNA and induce mutations is well known, little is known about how the genomic features influence the accumulation of DNA damage at a genome-wide level. To bridge this gap, we developed a single-nucleotide resolution damage sequencing method to map N2-BPDE-dG in a BPDE exposed human lung cell line, and combined this analysis with mass spectrometry to quantify the total absolute levels of the adduct in the genome. Comparing damage abundance with DNase hypersensitive sites, transcription levels, and other genome annotation data showed that although the overall adduct levels increased with increasing concentration of BPDE, the genomic distribution patterns of N2-BPDE-dG were stable and correlated with the genomic features related to chromatin state and transcriptional activities. In addition, we extracted the preferred local DNA sequence contexts for N2-BPDE-dG, i.e., its DNA damage signature, and found that it was highly similar to the mutational signatures identified from smoking-related lung cancers. These results suggest that genomic features and sequence contexts are important in shaping the landscape of DNA damage arising from chemical exposures and provide an effective strategy for linking single-nucleotide resolution damage sequencing data with cancer mutations.

ORGANISM(S): Homo sapiens

PROVIDER: GSE224001 | GEO | 2023/02/01

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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Quantification and mapping of alkylation in the human genome reveal single nucleotide resolution precursors of mutational signatures

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