Abstract 4454: Genome-wide analysis of DNA methylation induced by environmental carcinogen dibenzo[def,p]chrysene in ovarian tissues of mice

We and others have demonstrated that the administration of dibenzo[def,p]chrysene [also known as dibenzo[a,l]pyrene (DBP)], a representative example of the class of polycyclic aromatic hydrocarbon (PAH), by ip, oral gavage, or topical application onto the oral cavity induced tumors in multiple organ sites in mice; the ovary was the most susceptible tissue. We further demonstrated that the capacity of the target organs to metabolize DBP to active intermediates that can form DNA adducts may account for its tissue selective tumorigenicity. In addition, formation of DNA adducts by certain chemical carcinogens has been linked to aberrant DNA methylation, including the most extensively studied prototype PAH benzo[a]pyrene (B[a]P). The goal of this study is to examine whether alteration of DNA methylation occurs in the ovarian tissues of mice treated with DBP during the early stage of tumor development. In this study, we employed a previously established animal protocol in which the levels of DBP-DNA adducts as a function of time had been determined in the ovary following the oral administration of DBP (24 nmol, 3×/week for 5 weeks) or vehicle (DMSO) to female B6C3F1 mice (six weeks old, n = 3/group). DNA was isolated from ovary and subjected to enhanced reduced representation bisulfite sequencing (ERRBS) 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 (Babraham Bioinformatcis, UK). The methylKit v0.9.2 R package was then used to calculate the differential methylation. Differentially methylated bases with q-value 25% were extracted. Among 179 differentiated methylation sites (DMS) identified between DBP and vehicle-treated mice, 68 are hypermethylated and 111 are hypomethylated. About 25% of DMS are located in promoter or exon, 32% in intron and 44% in intergenic regions. DMS are located in genes including oocyte specific homeobox 2, transforming growth factor alpha, and tumor necrosis factor. Ingenuity Pathways analysis of the genes with altered methylation patterns identified top canonical pathways as growth hormone signaling, spermine biosynthesis, threonine degradation, trehalose degradation and L-serine degradation. Collectively, our previous results together with those presented here demonstrate that both genetic and epigenetic alterations may account for the carcinogenicity of DBP in the mouse ovary. Support: NIEHS R21ES020411 and NCI R01-CA173465. Citation Format: Yuan-Wan Sun, Karam El-Bayoumy, Yuka Imamura Kawasawa, Anna Salzberg, Cesar Aliaga, Krishnegowda Gowdahalli, Shantu Amin, Kun-Ming Chen. Genome-wide analysis of DNA methylation induced by environmental carcinogen dibenzo[def,p]chrysene in ovarian tissues of mice. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4454.