Equine estrogens differentially prevent neuronal cell death induced by glutamate.
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, 17beta-estradiol, 17alpha-estradiol, equilin, 17beta-dihydroequilin, 17alpha-dihydroequilin, equilenin, 17beta-dihydroequilenin, 17alpha-dihydroequilenin, Delta(8)-estrone(,) and Delta(8),17beta-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 microM) and glutamate (1-10 mM) were added in a total volume of 100 microL. After 24 hours, cell viability was determined using the MTS cell proliferation assay. Results 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 glutamate-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 Delta(8)-ring B unsaturated estrogens were the most neuroprotective, whereas the classic estrogens 17beta-estradiol, estrone, and 17alpha-estradiol were the least potent. The order of potency was Delta(8),17beta-estradiol > Delta(8)-estrone > 17beta-dihydroequilenin > 17alpha-dihydroequilenin > equilenin > 17beta-dihydroequilin = equilin > 17alpha-dihydroequilin > 17beta-estradiol > estrone > 17alpha-estradiol in PC12 cells and Delta(8),17beta-estradiol > Delta(8)-estrone > equilenin = 17beta-dihydroequilenin > 17beta-dihydroequilin > equilin > 17alpha-dihydroequilenin > 17alpha-dihydroequilin > 17alpha-estradiol = 17beta-estradiol > estrone in HT22 cells.


CONCLUSIONS
Our data indicate that the neurotoxic effects of glutamate can be inhibited differentially by various equine estrogens. The less estrogenic (uterotropic) Delta(8) 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.
Bhavnani
Bhagu R
BR
Department of Obstetrics and Gynecology, Institute of Medical Sciences, University of Toronto, and St. Michael's Hospital, Toronto, Ontario, Canada.
Berco
Mauricio
M
Binkley
Julie
J
D000818:Animals; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D004855:Equilenin / Q000494:pharmacology; D004857:Equilin / Q000031:analogs & derivatives* / Q000494:pharmacology; D004958:Estradiol / Q000494:pharmacology; D045166:Estradiol Congeners / Q000737:chemistry / Q000494:pharmacology*; D004970:Estrone / Q000494:pharmacology; D018698:Glutamic Acid / Q000494:pharmacology*; D006624:Hippocampus; D009474:Neurons / Q000187:drug effects*; D018696:Neuroprotective Agents / Q000494:pharmacology*; D016716:PC12 Cells; D051381:Rats; D013329:Structure-Activity Relationship
D045166:Estradiol Congeners; D018696:Neuroprotective Agents; D004857:Equilin; D004970:Estrone; D018698:Glutamic Acid; D004958:Estradiol; C014482:dihydroequilin; D004855:Equilenin