Abstract 4645: Identification of two methylation sites (CpG 388 and CpG 1664) in the promoter/enhancer region of cytochrome P450 (CYP)1B1: Possible role of promoter methylation in attenuation of CYP1B1 expression and DNA adducts by omega 3- fatty acids in mouse lungin vivo

3-Methylcholanthrene (MC) and benzo[a]pyrene (BP) are polycyclic aromatic hydrocarbons (PAHs) carcinogens. Cytochrome P450 (CYP)1B1 enzymes plays a key role in the activation of PAHs to carcinogenic metabolites, which initiate carcinogenesis by binding covalently to DNA, and the adducts, if not repaired, could lead to tumorigenesis. In this study we tested the hypothesis that pre-treatment of mice with omega-3-fatty acids, i.e. [eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) will lead to attenuation of PAH-mediated pulmonary DNA adduct formation in part by inhibiting CYP1B1. Twelve week old male and female A/J mice received EPA (60 mg/kg) and DHA (40 mg/kg) from day 1 to day 24. Control mice were treated with vehicle corn oil. On day 3, mice were treated with BP (40 µmol/kg) or 3-methylcholanthrene (MC, 40 µmol/kg) by i.p. In the short-term experiment (DNA adduct studies), 3-5 mice from each group were terminated at day 10 (7 days after BP or MC treatment). EPA/DHA significantly suppressed formation of BP-DNA and MC-DNA adducts in lung and liver of both male and female mice. CYP1B1 expression in lung at protein and mRNA levels was induced significantly by PAHs, but was suppressed by about 70% in the lungs of EPA/DHA-treated mice. We tested the hypothesis that PAHs in part induce CYP1B1 by methylating specific CpG sites in the negative regulatory elements of the promoter. Mouse lung DNA samples were subjected to bisulfite deamination, Methylation-Specific PCR (MSP), and a sequencing primer-flanking nested PCR. ClustalW multiple sequence alignment determined the methylation state of a total of 103 putative CpG sites in the CYP1B1 promoter/enhancer region, which included 3 putative CpG islands. We found that CpG 1664, which is located in a putative CpG island, was methylated by BP, and the extent of methylation was decreased by co-treatment with BP and EPA/DHA. BP partially methylated CpG 388 (83%), while EPA/DHA-treated animals showed significantly decreased methylation (33%) at this site. These results suggest that PAHs induce CYP1B1 in part by inducing methylation of CpG 1664 and CpG 388, which might be putative negative regulatory elements of CYP1B1. We hypothesize that suppression of methylation by EPA/DHA at sites CpG 388 and 1664 leads to repression of CYP1B1 expression, which in turn leads to inhibition of PAH carcinogenesis. This is the first report that links in vivo CYP1B1 promoter methylation to modulation of carcinogenesis by PAHs. Further studies could lead to CYP1B1 as an important molecular target for the prevention and/or treatment of lung carcinogenesis by PAHs in humans. Note: This abstract was not presented at the meeting. Citation Format: Bhagavatula Moorthy, Weiwu Jiang, Lihua Wang, Sudha R. Kondraganti, Guodong Zhou, Chun Chu. Identification of two methylation sites (CpG 388 and CpG 1664) in the promoter/enhancer region of cytochrome P450 (CYP)1B1: Possible role of promoter methylation in attenuation of CYP1B1 expression and DNA adducts by omega 3- fatty acids in mouse lung in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4645.