NQO1 downregulation generates genotoxic DNA adducts in in vitro FECD model

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 females. The aim of this study is to investigate the role of NAD(P)H: quinone oxidoreductase 1(NQO1) downregulation in generating genotoxic estrogen metabolites in FECD.

Methods : Cell viability by Cell Titer Glo, apoptotic cell population 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, HCEnC-SV-67F-16, NQO1-WT and KO) and FECD (FECD-SV-73F-74 and -61F-18) human CEnC lines treated with and without Menadione (MN) and 4-hydroxyestradiol (4-OHE2). Protein expression for an estrogen-metabolizing enzyme (NQO1) was investigated in normal and FECD post-keratoplasty specimens by western blotting.

Results : FECD ex vivo specimens showed 5.7-fold lower expression of NQO1 (p=0.009). HCEnC-21T cells treated with both 50μM MN and 10μM 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). NQO1-KO cells showed increased susceptibility to 4-OHE2 at 5,10 and 20μM compared to NQO1-WT cells (p<0.05). UPLC/MS-based analysis detected increased N3Ade and N7Gua adducts in 4-OHE2 treated NQO1-KO supernatants (35.3±3.2pg/ml) that further increased with MN co-treatment (72.4±16.3pg/ml) at 24h compared to NQO1-WT (16.7±2.7, 36.9±7.5pg/ml, p<0.01). FECD-SV-73F-74 and -61F-18 showed increased susceptibility to 4-OHE2 at 30, 40, and 50μM compared to HCEnC-SV (p<0.001).

Conclusions : We detected an alteration in estrogen metabolizing enzymes involved in estrogen genotoxic pathway in FECD.NQO1 downregulation seen in FECD renders CEnCs susceptible to catechol estrogen and estrogen DNA adducts formation. This study highlights a possible role of estrogen metabolism and NQO1 in pathogenesis of FECD.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.