Equine Estrogens Differentially Prevent Neuronal Cell Death Induced by Glutamate

Abstract

Objective: In the present study, neuronal PC12 cells and hippocampal HT22 cells maintained in culture were used to test the neuroprotective effect of equine estrogens estrone, 17β-estradiol, 17α-estradiol, equlin, 17β-dihydroequilin, 17α-dihydroequilin, equilenin, 17β-dihydroequilenin, 17α-dihydroequilenin, Δ⁸-estrone and Δ⁸,17β-estradiol against glutamate toxicity.

Methods: The HT22 and PC12 cells were grown in Dulbecco modified Eagle medium supplemented with 5% horse serum, 10% fetal bovine serum,and 10 mM HEPES. The undifferentiated PC12 cells were plated on collagen-coated, 96-well plastic plates at 10,000 cells per well, and the HT22 cells were plated on uncoated 96-well plates at 2500 cells per well. Twenty-four hours after plating, various concentrations of estrogens (0.1-40 μM) and glutamate (1-10 mM) were added in a total volume of 100 μL. After 24 hours, cell viability was determined using the MTS cell proliferation assay. Reslts were verified in some experiments by using the lactate dehydrogenase cytotoxicity assay.

Results: The results indicate that cell toxicity in both cell lines was directly proportional to the concentration of glutamate. The lowest dose of glutamate that reduced cell viability by 50% under these conditions was 1.8 mM for HT22 cells and 3 mM for PC12 cells. All estrogens tested were neuroprotective against glutanate-induced cell death in a typical dose-related manner. However, these estrogens differed extensively with respect to their neuroprotective potencies. In both cell lines, the Δ⁸-ring B unsaturated estrogens were the most neuroprotective, whereas the classic estrogens 17β-estradiol, estrone, and 17α-estradiol were the least potent. The order of potency was Δ⁸,17β-estradiol > Δ⁸-estrone > 17β-dihydroequilenin > 17α-dihydroequilenin > equilenin > 17β-dihydroequilin = equilin > 17α-dihydroequilin > 17β-estradiol > estrone > 17α-estradiol in PC12 cells and Δ⁷,17β-estradiol > Δ⁸-estrone > equilenin = 17β-dihydroequilenin > 17β-dihydroequilin > equilin > 17α-dihydroequilenin > 17α-dihydroequilin > 17α-estradiol = 17β-estradiol > estrone in HT22 cells.

Conclusion: Our data indicate that the neutrotoxic effects of glutamate can be inhibited differentially by various equine estrogens. The less estrogenic (uterotropic) Δ⁸ estrogens were the most effective neuroprotectors, and further chemical modifications of these estrogens may provide compounds that are useful for preventing neurodegenerative diseases in both women and men.

Keywords

Equine estrogen neuroprotective effects of estrogens glutamate neurotoxicity neurotransmitter neurons neurodegeneration Alzheimer's disease PC12 HT22 cells

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