INDUCTION BY ESTROGEN METABOLITE 16-ALPHA-HYDROXYESTERONE OF GENOTOXIC DAMAGE AND ABERRANT PROLIFERATION IN MOUSE MAMMARY EPITHELIAL-CELLS

Background: Estrogens are potent mammary tumor promoters influencing post-initiational events via epigenetic mechanisms. The upregulation (i.e., induction) of the C16-alpha-hydroxylation pathway during 17-beta-estradiol (E2) biotransformation has been associated with mammary cell transformation. The action of E2 metabolites on tumorigenic transformation, however, is poorly understood. Purpose: The newly established mammary epithelial cell line C57/MG, derived from the C57BL mouse strain, was used to examine whether E2 or its metabolites, 16-hydroxyestrone (16-alpha-OHE1) and estriol (E3), function as initiators of mammary cell transformation. Methods: DNA repair (hydroxyurea-insensitive thymidine uptake), estrogen metabolism (H-3 exchange to form (H2O)-H-3), hyperproliferation (increased cell number), and acquisition of anchorage-independent growth (soft-agar colonies) were used as quantitative end points to measure the relative extent of transformation. Results: Treatment of cells with 200 ng/mL 16-alpha-OHE1 resulted in a 55.2% increase in DNA repair synthesis, a 23.09% increase in proliferative activity, and a 18-fold increase in the number of soft-agar colonies, relative to the solvent controls (P < .0001). The extent of upregulation of the three end points was similar to that induced by the genotoxic mammary carcinogen 7, 12-dimethylbenz[a]anthracene (DMBA, positive control). DMBA treatment also upregulated the ratio of 16-alpha/C2 hydroxylation of E2 leading to increased formation of 16-alpha-OHE1. E2 and E3 were not effective in upregulating these markers for transformation. Conclusion: These results demonstrate that in nontransformed C57/MG cells, 16-alpha-OHE1 may function as an initiator, perturbing the intermediate biomarkers for preneoplastic transformation.