The effects of catechol-O-methyltransferase inhibition on estrogen metabolite and oxidative DNA damage levels in estradiol-treated MCF-7 cells

Many of the major identified risk factors for breast cancer are associated with exposure to endogenous estrogen. In addition to the effects of estrogen as a growth factor, experimental and epidemiological evidence suggest that catechol metabolites of estrogen also contribute to estrogen carcinogenesis by both direct and indirect genotoxic mechanisms. O-Methylation catalyzed by catechol-O-methyltransferase (COMT) is a Phase II metabolic inactivation pathway for catechol estrogens. We and others have found that a polymorphism in the COMT gene, which codes for a low activity variant of the COMT enzyme, is associated with an increased risk of developing breast cancer; therefore, the goal of the current study was to investigate the role of decreased COMT activity on estrogen catechol levels and on oxidative DNA damage, as measured by 8-hydroxy-2′-deoxyguanosine (8-oxo-dG) levels. MCF-7 cells were pre-treated with dioxin as a means to increase estrogen metabolism to catechol estrogens, then treated with estradiol (E₂) ± Ro 41-0960, a COMT-specific inhibitor. After extraction from culture medium, estrogen metabolites were separated using an high-performance liquid chromatography-electrochemical detection method. As expected, dioxin dramatically increased E₂ oxidative metabolism, primarily to its 2-OH and 2-methoxy metabolites. The COMT inhibitor blocked 2-methoxy E₂ formation. This was associated with increased 2-hydroxy E₂ (2-OH E₂) and 8-oxo-dG levels. In the presence of COMT inhibition, increased oxidative DNA damage was detected in MCF-7 cells exposed to as low as 0.1 μM E₂, whereas in the absence of COMT inhibition, no increase in 8-oxo-dG was detected at E₂ concentrations ≤10 μM. This study is the first to show that O-methylation of 2-OH E₂ by COMT is protective against oxidative DNA damage caused by 2-OH E₂, a major oxidative metabolite of E₂.