Role of Estrogen-DNA Adducts in Fuchs Endothelial Corneal Dystrophy

Abstract

Purpose : Fuchs Endothelial Corneal Dystrophy (FECD) is a late-onset oxidative stress disorder characterized by progressive loss of corneal endothelial cells (CEnCs). FECD has a greater incidence in women at a ratio of 4:1 suggesting the possible role of sex hormones accounting for predominance in the females. The aim of this study is to investigate the role of reactive estrogen metabolites in initiating an estrogen genotoxic pathway in FECD.

Methods : Cell viability assayed by Cell Titer Glo, apoptotic cell population analyzed by Annexin-V (Ann) and propidium iodide (PI) staining using flow cytometry, and estrogen metabolites by ultraperformance liquid chromatography/tandem mass spectrometry (UPLC/MS) were investigated in immortalized normal (HCEnC-21T and -SV) and FECD (FECD-SV1 and -SV2) human CEnC lines treated with and without Menadione (MN) and 4-hydroxyestradiol (4-OHE2). FECD-SV1 (F, 73y, expansion of TCF4) and –SV2 (F, 61y, no expansion of TCF4) were generated from FECD specimens. Protein expression for estrogen-metabolizing enzymes (CYP1B1, NQO1 and COMT) was investigated in normal and FECD post-keratoplasty specimens by western blotting.

Results : HCEnC-21T cells treated with both 50mM MN and 10mM 4-OHE2 resulted in 92% loss of cell viability at 24h (p=0.001). MN treatment resulted in 53% (p=0.001) increase of Ann/ PI-positive population representative of apoptotic cells by flow cytometry, whereas the combination of both MN and 4-OHE2 further increased the apoptotic population to 75% (p=0.004). UPLC/MS-based analysis detected increased N3Ade and N7Gua adducts in 4-OHE2 treated HCEnC-21T supernatants (3.6±0.3pg/ml) that further increased with MN co-treatment (7.6±1.3pg/ml) at 24h (p<0.01). FECD-SV1 and –SV2 showed increased susceptibility to 4-OHE2 at 30, 40, and 50mM compared to HCEnC-SV (p<0.001). FECD ex vivo specimens showed 5.7-fold lower expression of NQO1 (p=0.009) and 4.4-fold higher CYP1B1 compared to normal specimens (p=0.001).

Conclusions : We detected an alteration in estrogen metabolizing enzymes involved in estrogen genotoxic pathway in FECD. The combination of altered catechol estrogens and oxidative stress increases susceptibility of CEnCs to apoptosis. This study highlights a possible role of estrogen metabolism in pathogenesis of FECD.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.