Methoxychlor and estradiol induce oxidative stress DNA damage in the mouse ovarian surface epithelium

Estrogenic compounds such as 17β-estradiol (E₂) and methoxychlor (MXC) induce oxidative stress damage in breast cells and mouse ovarian follicles, respectively. However, little is known about whether estrogenic compounds cause oxidative stress in the ovarian surface epithelium (OSE). Thus, this work tested the hypothesis that E₂ and MXC cause oxidative stress in the OSE. To test this hypothesis, we employed an improved mouse tissue culture assay in which OSE cells were treated with hydrogen peroxide (H₂O₂ positive control), MXC, or E₂ ± the anti-oxidant vitamin E, or progesterone. The cells then were subjected to a novel direct immunofluorescent assay in which cells in the microtiter plate were reacted with antibodies that detect oxidative damage to DNA (8-hydroxy-2′-deoxyguanosine). The signal was identified with a tyramide Alexa Fluor fluorescent probe and quantified by microfluorimetry. Correction for cellularity was carried out for each well with a fluorescent DNA dye system (CyQuant) at a different wavelength. After 24 h, the mean Alexa Fluor CyQuant ratio was 11.3 ± 0.9 for controls, 132 ± 15 for H₂O₂ treated positive control cells (p ≤ 0.01 from control), 105 ± 6.6 for E₂ treated cells (p ≤ 0.01 from control), and 64 ± 5.1 for MXC-treated cells (p ≤ 0.01 from control). After 72 h, the mean ratio was 121 ± 10.6 for controls, 391 ± 23 for H₂O₂ treated cells (p ≤ 0.01 from control), 200 ± 15 for E₂ treated cells (p ≤ 0.03), and 228 ± 21 for MXC-treated cells (p ≤ 0.01). Further, vitamin E, but not progesterone, protected OSE cells from E₂- and MXC-induced oxidative damage. This study demonstrates the feasibility of direct immunofluorescent quantitation of DNA adducts in cell cultures without DNA extraction. Moreover, these data indicate that E₂ and MXC produce oxidative DNA damage in the OSE, and that this damage is prevented by the anti-oxidant vitamin E.