ABSTRACT
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 pretreated 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 microM E2, whereas in the absence of COMT inhibition, no increase in 8-oxo-dG was detected at E2 concentrations < or =10 microM. 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.
Subject(s)
Breast Neoplasms/metabolism , Catechol O-Methyltransferase Inhibitors , DNA Damage , Deoxyguanosine/analogs & derivatives , Estradiol/metabolism , Estradiol/pharmacology , Estrogens, Catechol/metabolism , 8-Hydroxy-2'-Deoxyguanosine , Benzophenones/pharmacology , Breast Neoplasms/enzymology , Breast Neoplasms/genetics , DNA, Neoplasm/metabolism , Deoxyguanosine/metabolism , Enzyme Inhibitors/pharmacology , Humans , Oxidative Stress/physiology , Tumor Cells, CulturedABSTRACT
Ethinyl estradiol (EE) is a strong promoter of hepatocarcinogenesis in the rat. Treatment with EE and other hepatic promoters induces transient growth stimulation followed by growth inhibition (mitosuppression) in hepatocytes. Previously, we identified several genes whose transcript levels were increased during EE-induced mitosuppression, including transforming growth factor beta (TGF-beta), which inhibits growth and induces apoptosis in hepatocytes. Various hepatic promoters, including phenobarbital and several peroxisomal proliferators, have been shown to inhibit TGF-beta-induced apoptosis in rat hepatocytes. The goal of this study was to investigate whether EE is also an inhibitor of TGF-beta-induced apoptosis in rat hepatocytes. Several approaches to detect apoptosis were used, including the TUNEL assay, detection of high molecular weight DNA fragmentation by field inversion gel electrophoresis and determination of cytosolic cytochrome c levels by western analysis. TGF-beta-induced apoptosis in cultured, precision cut liver slices and hepatocytes of female rats. EE (