Page 1 of 25 Toxicological Sciences 1

2Abstract Estrogenic compounds such as 17-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 celluarity was carried out for each well with a fluorescent DNA dye system (CyQuant™) at a different wavelength. After 24 hours, the mean Alexa Fluor: CyQuant ratio was 11.3±0.9 for controls, 132±15 for H2O2 treated positive-control cells (p0.0