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Updated: Jul 5, 2026
Detection of DNA Double-Stranded Breaks in Mouse Oocytes
Published on: June 23, 2023
Methoxychlor and estradiol induce oxidative stress DNA damage in the mouse ovarian surface epithelium
Daniel A Symonds1, Istvan Merchenthaler, Jodi A Flaws
1Department of Epidemiology and Preventive Medicine, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
Abstract:
Estrogenic compounds such as 17beta-estradiol (E(2)) 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 E(2) 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 E(2) +/- 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 < or = 0.01 from control), 105 +/- 6.6 for E(2) treated cells (p < or = 0.01 from control), and 64 +/- 5.1 for MXC-treated cells (p < or = 0.01 from control). After 72 h, the mean ratio was 121 +/- 10.6 for controls, 391 +/- 23 for H2O2 treated cells (p < or = 0.01 from control), 200 +/- 15 for E(2) treated cells (p < or = 0.03), and 228 +/- 21 for MXC-treated cells (p < or = 0.01). Further, vitamin E, but not progesterone, protected OSE cells from E(2)- 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 E(2) and MXC produce oxidative DNA damage in the OSE, and that this damage is prevented by the anti-oxidant vitamin E.
Insights
Estrogenic compounds 17beta-estradiol (E(2)) and methoxychlor (MXC) cause oxidative DNA damage in ovarian surface epithelium (OSE) cells. The antioxidant vitamin E protected OSE cells from this damage, indicating a potential protective role.
Area of Science:
- Endocrinology
- Toxicology
- Cell Biology
Background:
- Estrogenic compounds like 17beta-estradiol (E(2)) and methoxychlor (MXC) are known to induce oxidative stress in various cell types.
- The impact of these estrogenic compounds on the ovarian surface epithelium (OSE) and their potential to cause oxidative stress remains largely unexplored.
Purpose of the Study:
- To investigate whether estrogenic compounds E(2) and MXC induce oxidative stress and DNA damage in the ovarian surface epithelium (OSE).
- To evaluate the protective effects of antioxidants, specifically vitamin E and progesterone, against E(2)- and MXC-induced oxidative damage in OSE cells.
Main Methods:
- An improved mouse OSE cell tissue culture assay was utilized.
- Cells were treated with hydrogen peroxide (H2O2), E(2), or MXC, with or without vitamin E or progesterone.
- A novel direct immunofluorescent assay quantified oxidative DNA damage (8-hydroxy-2'-deoxyguanosine) using fluorescent probes and microfluorimetry, with cellularity corrected by CyQuant.
Main Results:
- E(2) and MXC significantly increased oxidative DNA damage in OSE cells after 24 and 72 hours compared to controls.
- Hydrogen peroxide also induced significant oxidative DNA damage, serving as a positive control.
- Vitamin E demonstrated a protective effect against E(2)- and MXC-induced oxidative damage, while progesterone did not.
Conclusions:
- The study successfully demonstrated a novel method for quantifying DNA adducts in cell cultures without DNA extraction.
- Estrogenic compounds E(2) and MXC induce significant oxidative DNA damage in the ovarian surface epithelium.
- The antioxidant vitamin E can prevent oxidative DNA damage in OSE cells caused by E(2) and MXC, suggesting a potential therapeutic or preventative strategy.
