Figure 1. Estrogen and its metabolites cause DNA double strand breaks in S/G2 phase cells.

A) Representative images of 53BP1 and γ-H2AX marked DNA DSBs in MCF10A cells 3-hours after mock treatment or treatment with10nM Estradiol (E2). B) Quantification of 53BP1 and γ-H2AX foci in cells described above. Bars represent mean number of foci per cell +/− SEM from three independent experiments (>200 cells were counted per experiment). Significance of changes in foci numbers were assessed using students two-tailed t-test C) Representative images of 53BP1 and γ-H2AX marked DNA DSBs in MCF10A cells 3-hours after mock treatment or treatment with1μM 2-OHE₂ or 4-OHE₂. D) Quantification of 53BP1 and γ-H2AX foci in cells described above. E) Representative comet images of MCF10A cells treated as above and subjected to neutral single cell electrophoresis. Fluorescence intensity mapping of comet images has been applied using CometScore. F) Quantification of tail DNA in comets from above. Tail DNA was quantified using CometScore. Bars represent mean percentage of DNA in comet tails, +/− SEM, from three independent experiments (>100 comets were scored per experiment). G) Frequency distribution of 53BP1 and γ-H2AX foci in MCF10A cells treated with 2-OHE₂ or 4-OHE₂ H) Representative images of γ-H2AX marked DNA DSBs in MCF10A cells 3-hours after mock treatment or treatment with 1μM 2-OHE₂ or 4-OHE₂. Cells were also stained with Cyclin-A antibodies to identify cells in S/G2-phase. I) Quantification of γ-H2AX foci in Cyclin-A positive and negative cells treated as above.