Differential Neuroprotective Effects of Equine Estrogens Against Oxidized Low Density Lipoprotein—Induced Neuronal Cell Death

Abstract

Objective: In the present study, the neurotoxic effect of oxidized low density lipoprotein on PC-12 neuronal cells maintained in culture was used to test the neuroprotective effect of several equine estrogens, such as estrone (E₁), 17β-estradiol (17β-E₂), 17α-estradiol (17α-E₂), equilin (Eq), 17β-dihydroequilin (17β-Eq), 17α-dihydroequilin (17α-Eq), equilenin (Eqn), 17β-dihydroequilenin (17β-Eqn), 17α-dihydroequilenin (17α-Eqn), Δ⁸-estrone (Δ⁸-E₁), and Δ⁸, 17β-estradiol (Δ⁸, 17β-E₂).

Methods: The PC-12 cells (10,000 cells/well) were grown on collagen-coated 96-well plates in Dulbecco's Modified Eagle Medium supplemented with 10% horse serum, 5% fetal bovine serum, and 10 mM HEPES. In culture, the cells displayed normal PC-12 morphology and behavior, exhibiting increased dendritic growth and cessation of cell division upon exposure to nerve growth factor. Twenty-four hours after plating, various concentrations (0.1-50 μM) of estrogens were added followed by addition of oxidized low density lipoprotein (5-12.5 μg/well) in a total volume of 100 μL. After 24 hours, cell viability was determined using the MTS (3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2H-tetrazolium, inner salt) cell proliferation assay.

Results: The results indicate that the extent of low density liproprotein oxidation and concentration of oxidized low density lipoprotein is directly proportional to cell toxicity. The mean ± standard deviation cell death obtained using 10.0 μg/well of oxidized low density lipoprotein was 53.6% ± 8.7%. With the exception of 17α-estradiol, all estrogens tested were found to be neuroprotective against oxidized low density lipoprotein toxicity in a typical dose-dependent manner. The order of neuroprotective potency was Δ⁸-E₁ (1.2 μM), Δ⁸, 17β-E₂ (1.3 μM), Eqn (5.3 μM), 17β-Eqn (5.3 μM), Eq (6 μM), 17β-Eq (8.5 μM), E₁ (11 μM), 17β-E₂ (11 μM), 17α-Eq (12 μM), and 17α-Eqn (16 μM) followed by 17α-E₂ which provided less than 50% protection.

Conclusion: Our data indicate that the neurotoxic effects of oxidized low density lipoprotein can be inhibited differentially by various estrogens, with the Δ⁸ estrogens being the most potent neuroprotectors. These novel findings further suggest that some of the neuroprotective benefits associated with estrogen therapy might occur by the suppression of oxidized low density lipoprotein neurotoxicity. Because estrogens such as Δ⁸-E₁ are relatively less uterotropic and potentially less feminizing than the classic estrogen 17β-E₂, they may be useful in the prevention of Alzheimer disease and other neurodegenerative disease in both women and men.