Estrogen Effect on Manganese-Induced Astrocytic Toxicity

Date of Award

2013

Degree Type

Thesis

Degree Name

Master of Science in Biomedical Sciences

First Advisor

Harold Kominsky, PhD

Second Advisor

Kimberly Baker, PhD

Third Advisor

Richard White, PhD

Abstract

Manganese (Mn) is a natural trace metal that is essential for many physiological functions in the human body. A chronic overexposure to Mn can disrupt normal functions and can lead to a neurodegenerative condition commonly referred to as manganism. Symptoms linked to manganism are emotional instabilities, memory and learning difficulty, and visual coordination with motor and spatial impairment. Astrocytes in the central nervous system are susceptible reservoirs for Mn accumulation. Estrogen, a steroidal hormone, has been shown to mitigate Mn-induced toxicity that contributes to the cause of neurological diseases. We propose that pretreatment with estrogen would attenuate some of Mn's cytotoxic effects in astrocytes. To assess this hypothesis, primary rat hippocampal astrocytes were pretreated with ~-Estradiol (E2) and subsequently, Mn sulfate (MnS04). The amount of toxic damage to the astrocytes was measured by quantifying glial fibrillary acidic protein (GFAP) with a sandwiched Enzyme-Linked lmmunosorbent Assay (ELISA). ELISA analysis indicated Mn exposure at 100 11M, 300 11M, or 600 11M significantly increased GFAP levels. However, E2 concentrations at 10nM or 30 nM significantly reduced Mn-induced GFAP concentrations at 100 11M. Cells pretreated with 10 nM or 30 nM of E2 significantly lowered GFAP levels. The Water-Soluble Tetrazolium-8 (WST-8) method was utilized to determine cell viability. The WST-8 assay showed that Mn concentrations of 100 11M, 300 11M, or 600 11M significantly reduced the dehydrogenase activity, thereby decreasing the number of viable astrocytes. However, the dehydrogenase activity in cells treated with 600 11M was significantly increased when pretreated with 10 nM of E2. Enzyme activity with 600 11M of Mn was significantly decreased when compared with 100 11M of Mn, revealing a dose-dependent effect. Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) was used to measure changes in glutamate transporter-1 gene expression in astrocytes after pretreatment of E2 and subsequently, Mn. PCR analysis showed that when cells were exposed to 300 11M Mn, the GLT-1 gene expression was reduced compared to the control. Data also showed that the GLT-1 mRNA was upregulated in cells pretreated with 10nM E2. When the cells were pretreated with 10 nM E2 and subsequently, 300 11M Mn, there was an increase in the GLT-1 gene expression. The experimental results indicate that E2 can attenuate some Mn-induced toxicity in astrocytes.

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