Identification of a Novel Methylation Site (CpG 388) 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

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 on the negative regulatory elements (NREs) 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 on the CYP1B1 promoter/enhancer region, which included 3 putative CpG islands. We found that mice treated with BP resulted in partially methylated CpG 388 (83%) in lung DNAs, while co‐administration with EPA/DHA significantly suppressed the methylation of CpG 388 (to 33%). These results suggest that PAHs induce CYP1B1 in part by inducing methylation of CpG 388, which might be putative NREs of CYP1B1. We hypothesize that suppression of methylation by EPA/DHA at CpG 388 leads to restoration of negative regulation, leading to repression of CYP1B1 expression, which in turn leads to inhibition of PAH carcinogenesis. We further verified the role of CpG 388 by mutating it in a luficerase reporter plasmid containing mouse CYP1B1 promoter/enhancer. Preliminary results in transiently transfected H441 lung cell line indicated that both wild type and mutant CYP1B1 promoters were activated about 2.5 fold by BP, and mutation of CpG 384 decreased both basal and inducible promoter activities. Further studies could lead to CYP1B1 as an important molecular target for the prevention and/or treatment of lung carcinogenesis by PAHs in humans.Support or Funding InformationThis work was supported in part by NIEHS grant R01ES029382 to BM.