Equine estrogens differentially inhibit DNA fragmentation induced by glutamate in neuronal cells by modulation of regulatory proteins involved in programmed cell death

BACKGROUND: Recent data indicate that excitotoxicity of high levels of neurotransmitter glutamate may be mediated via programmed cell death (apoptosis) and that it can be prevented in HT22 mouse hippocampal cells by various equine estrogens with Δ(8),17β-estradiol (Δ(8),17β-E(2)) being the most potent. In order to delineate the mechanism(s), glutamate-induced cell death of HT22 cells was assessed by measuring (a) DNA fragmentation in the presence or absence of 11 equine estrogens (components of the drug CEE); (b) cell death and (c) levels of anti-apoptotic (Bcl-2) and proapoptotic (Bax) proteins in the presence or absence of two equine estrogens, Δ(8),17β-E(2 )and 17β-estradiol (17β-E(2)) by LDH release assay and Western blot analysis respectively. RESULTS: Glutamate treatment induced cell death was time and dose-dependent. After 18 to 24 h, glutamate induced DNA fragmentation and morphological characteristics of apoptotic cell death. DNA fragmentation and morphological changes induced by 10 mM glutamate were completely inhibited by some equine estrogens. Exposure of cells to various concentrations of glutamate, resulted in a significant increase in cell death associated LDH release that was time-dependent. Both Δ(8),17β-E(2 )and 17β-E(2 )inhibited the glutamate-induced LDH release and cell death in a dose-dependent manner with Δ(8),17β-E(2 )being 10 times more potent than 17β-E(2). Western blot analysis indicated that glutamate also significantly decreased the levels of Bcl-2 and increased Bax levels. This glutamate-induced change in the ratio of Bcl-2 to Bax was reversed by estrogens with Δ(8),17β-E(2 )being more potent. CONCLUSIONS: In HT22 mouse hippocampal cells, glutamate induced apoptosis that was associated with DNA fragmentation, morphological changes and up-regulation of the pro-apoptotic protein Bax and down-regulation of the anti-apoptotic protein Bcl-2. This apoptotic process was differentially prevented by some equine estrogens with Δ(8),17β-E(2 )being more potent than 17β-E(2). Since HT22 cells lacked both glutamate and estrogen receptors, the neuroprotective effects of estrogens most likely involve both genomic and non-genomic mechanisms. Since Δ(8)-estrogens are less feminizing estrogens than 17β-E(2), further chemical modifications of these Δ(8)-estrogens may provide more selective estrogens that will be useful in the prevention of neurodegenerative diseases such as Alzheimer's and Parkinson's in both aging men and women.