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ToxSci Advance Access published online on May 22, 2008
Toxicological Sciences, doi:10.1093/toxsci/kfn100
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© The Author 2008. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Methoxychlor and Estradiol Induce Oxidative Stress DNA Damage in the Mouse Ovarian Surface Epithelium

Daniel A. Symonds1, Istvan Merchenthaler1 and Jodi A. Flaws2

1 Department of Epidemiology and Preventive Medicine, University of Maryland School of Medicine, Baltimore, MD 21201 2 Department of Veterinary Biosciences, University of Illinois, Urbana, IL 61802

Correspondance: Jodi A. Flaws, Ph.D. Department of Veterinary Biosciences University of Illinois 2001 S. Lincoln Ave. Urbana, IL 61802 Email: jflaws{at}uiuc.edu Phone (217) 333-7933 Fax (217) 244-1652

Received March 13, 2008; revision received May 15, 2008; accepted May 19, 2008


  Abstract

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 FluorTM fluorescent probe and quantified by microfluorimetry. Correction for celluarity was carried out for each well with a fluorescent DNA dye system (CyQuantTM) 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 (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 hours, 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.

Key Words: ovarian surface epithelium; oxidative stress damage; DNA adducts; estrogen.


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