Abstract LB-331: Identification of a novel CpG 388 on the cytochrome P450 (CYP)1B1 promoter in vivo: Its role in attenuation of polycyclic aromatic hydrocarbon (PAH)-mediated carcinogenesis by omega-3 fatty acids

Abstract

Abstract 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 negative regulatory elements in the CpG islands of the promoter. Lung DNA samples from BP-treated mice were subjected to bisulfite deamination, followed by Methylation-Specific PCR (MPP) with primers 5'-TTGATTTTGCGGGG TTTTAG-3' and 5'-CCTACCCAACTCAACCTCCA-3'. ClustalW multiple sequence alignment determined the CpG sites that were methylated. There were a total of 13 putative CpG sites in CYP1B1 promoter region, out of which 7 were methylated. PAH-treated mice showed methylation of 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 388, which is a putative negative regulatory element of CYP1B1. Suppression of methylation by EPA/DHA leads to activation of nucleotide 388, thereby resulting in 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. Citation Format: Bhagavatula Moorthy, Weiwu Jiang, Lihua Wang, Guodong Zhou, Sudha R. Kondraganti, Chun Chu. Identification of a novel CpG 388 on the cytochrome P450 (CYP)1B1 promoter in vivo: Its role in attenuation of polycyclic aromatic hydrocarbon (PAH)-mediated carcinogenesis by omega-3 fatty acids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-331.