Abstract 2955: The effects of the environmental carcinogen dibenzo[a,l]pyrene on genome-wide methylation and the impact of dietary black raspberry in mouse oral tissues

Abstract

Abstract We had previously reported the carcinogenicity of dibenzo[a,l]pyrene (DB[a,l]P) in mouse oral cavity; we also found that that diets containing black raspberry (BRB) significantly reduced the levels of DNA adducts derived from DB[a,l]P in mouse oral tissues. Epigenetic alternations including DNA methylation have been shown to contribute to carcinogenesis induced by certain carcinogens. Literature data showed that dietary BRB can modulate DNA methylation at certain gene-specific promoters; however, the genome-wide methylation alterations by DB[a,l]P and the impact of BRB are not known. Therefore, to mimic the bioassay employed in our previous carcinogenicity study, B6C3F1 female mice (n = 3/group) were fed either control diet or diet containing BRB (5%) for 2 weeks prior to topical application of DMSO containing DB[a,l]P (24 nmol, 3 times a week for 5 weeks); animals treated with DMSO and control diet were used as a control group. DNA was isolated from oral tissues and subjected to enhanced reduced representation bisulfite sequencing analysis which is a single nucleotide resolution technique used to study DNA methylation in CpG sites and the surrounding regions. Briefly, DNA was digested by MspI followed by end repair, adenylation and adapter ligation with a modification of bead size selection to capture MspI fragments of 70-320 bp size. The resulting libraries were bisulfite-converted followed by PCR amplification and read by 1×50 bp on HiSeq 2500. Base calls of bisulfite treated sequencing reads were mapped to the mm9 mouse assembly and methylation calls were performed using Bismark v0.10.1. The methylKit R package was then used to calculate the differential methylation. Differentially methylated bases with q-value < 0.01 and percent methylation difference > 25% were extracted. Differential methylation of 30 genes was observed in DB[a,l]P compared with control; 12 genes were hypermethylated including β-catenin; hypomethylation of 18 genes was observed. Differential methylation of 960 genes was observed in mice treated with DB[a,l]P and fed BRB compared to DB[a,l]P. Ingenuity Pathways analysis was performed on each of the resulting gene sets in the three groups. Pathway analysis of the genes with altered methylation patterns identified canonical pathways for the involvement of cancer related network for genes involved in Wnt/β-catenin signaling, epithelial-mesenchymal transition (EMT), glycolysis and p53 signaling pathways in mice treated with DB[a,l]P. On the other hand, the canonical pathways identified in mice treated with DB[a,l]P and fed BRB are genes involved in glutamate receptor signaling, IGF-1 signaling, EMT, glycolysis, and protein citrullination. Using this quantitative sequencing-based approach, our work uncovers significant global DNA methylation alterations in mouse oral tissues by DB[a,l]P alone and together with BRB. Support: NCI R01-CA173465 Citation Format: Yuan-Wan Sun, Kun-Ming Chen, Yuka Imamura Kawasawa, Anna Salzberg, Cesar Aliaga, Krishnegowda Gowdahalli, Shantu Amin, Gary Stoner, Karam El-Bayoumy. The effects of the environmental carcinogen dibenzo[a,l]pyrene on genome-wide methylation and the impact of dietary black raspberry in mouse oral tissues. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2955. doi:10.1158/1538-7445.AM2015-2955