17-β Estradiol Preserves Endothelial Cell Viability in an In Vitro Model of Homocysteine-Induced Oxidative Stress

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Abstract
High levels of homocysteine (Hcy) accelerate endothelial cell damage by producing hydrogen peroxide (H2O2). We investigated whether 17-β estradiol may prevent the accelerated rate of endothelial cell detachment and reduced cell viability in cultured endothelial cells challenged with Hcy. Cultured bovine aortic endothelial cells (BAEC) were incubated for 72-h with either vehicle (alcohol) or different concentrations of 17-β estradiol (1 nM [1E2] and 10 nM [10E2]) before being challenged with 0.5 mM of Hcy. Cell viability and H2O2 levels were evaluated at 30 min, 1-, 2-, 4-, 8-, and 24-h after adding Hcy. Cell suspensions were frozen in liquid nitrogen and used later for spectrophotometric measurement of intracellular glutathione (GSH) levels. Cell viability 24 h after Hcy administration was significantly lower in vehicle versus 1 nM and 10 nM 17-β estradiol (44 ± 5% vs. 70.66 ± 4%, [p < 0.001] and 79 ± 5% respectively, [p < 0.001]). H2O2 levels were higher in vehicle (1 ± 0.05 μM) compared with 1E2 and 10E2 (0.8 ± 0.1 μM, p = 0.02 and 0.1 ± 0.05 μM, respectively, p < 0.001), whereas GSH content was increased in 1E1 and 10E2 versus control (27.69 ± 4.6 nM/106 cells and 43.49 ± 5.5 nM/106 cells vs. 13.33 ± 1.5 nM/106 cells, p < 0.001). Bovine aortic endothelial cells treatment with 17-β estradiol (0, 1, and 10 nM) and 0.1 mmol buthionine sulfoximine, an inhibitor of gamma-glutamylcysteine synthase, abolished the beneficial effects of estradiol alone on cell viability, GSH content, and H2O2 generation. Estradiol prevents Hcy-induced endothelial cell injury by increasing the intracellular content of GSH.