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 estroge n as a growth factor, experimental and epidemiological evidence suggest tha t catechol metabolites of estrogen also contribute to estrogen carcinogenes is by both direct and indirect genotoxic mechanisms. O-Methylation catalyze d by catechol-O-methyltransferase (COMT) is a Phase II metabolic inactivati on pathway for catechol estrogens. We and others have found that a polymorp hism in the COMT gene, which codes for a low activity variant of the COMT e nzyme, is associated with an increased risk of developing breast cancer; th erefore, the goal of the current study was to investigate the role of decre ased 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 pretreated with dioxin as a means to increase estrogen metabolism to c atechol estrogens, then treated with estradiol (E-2) +/- Ro 41-0960, a COMT -specific inhibitor. After extraction from culture medium, estrogen metabol ites were separated using an high-performance liquid chromatography-electro chemical detection method. As expected, dioxin dramatically increased E-2 o xidative metabolism, primarily to its 2-OH and 2-methoxy metabolites. The C OMT inhibitor blocked 2-methoxy E-2 formation. This was associated with inc reased 2-hydroxy E-2 (2-OH E-2) and 8-oxo-dG levels. In the presence of COM T inhibition, increased oxidative DNA damage was detected in MCF-7 cells ex posed to as low as 0.1 muM E-2, whereas in the absence of COMT inhibition, no increase in 8-oxo-dG was detected at E-2 concentrations less than or equ al to 10 muM. This study is the first to show that O-methylation of 2-OH E- 2 by COMT is protective against oxidative DNA damage caused by 2-OH E-2, a major oxidative metabolite of E-2.