TITLE:
Effect of estrogen on manganese-induced toxicity on embryonic astrocytes
AUTHORS:
Tyler T. Huynh, Kimberly J. Baker, Harold L. Komiskey
KEYWORDS:
Astrocytes; Estrogen; Embryonic; Manganese; Hippocampus
JOURNAL NAME:
Advances in Bioscience and Biotechnology,
Vol.4 No.10A,
October
22,
2013
ABSTRACT: Manganese (Mn) is a natural trace metal that is essential
for many physiological functions in the human body. Astrocytes in the central
nervous system are susceptible reservoirs for Mn accumulation. Estrogen, a
steroidal hormone, has been shown to mitigate Mn-induced toxicity in cultures
of postnatal astrocytes. However, differences in expression/inducibility of glutamate transporters and glutamine synthetase, transmitters, and the natural
gonadal steroids and their receptors are known to occur in astrocyte cultures
derived from various stages of fetal and postnatal development. Cultures of
embryonic (E18) hippocampal astrocytes were examined in this study for the
ability of 17 β-estradiol (E2) to
protect them from Mn toxicity by up regulating gene expression of a glutamate transporter. Primary rat hippocampal astrocytes
were pretreated with β-Estradiol
(E2) in vitro and subsequently, Mn
sulfate (MnSO4). The amount of toxic damage to the astrocytes was
measured by quantifying glial fibrillary acidic protein (GFAP) with a sandwiched Enzyme-Linked Immunosorbent Assay (ELISA). ELISA
analysis indicated Mn exposure at 100 μM, 300 μM, or 600 μM significantly
increased GFAP levels. However, E2 concentrations at 10 nM or 30 nM
significantly reduced Mn-induced GFAP concentrations at 100 μM. 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 μM, 300 μM, or 600 μM significantly reduced the dehydrogenase
activity, thereby decreasing the number of viable astrocytes. Enzyme activity
with 600 μM of Mn was significantly decreased when compared with 100 μM of Mn,
revealing a dose-dependent effect. However, the dehydrogenase activity in
cells treated with 600 μM Mn was significantly increased when pretreated with
10 nM of E2. 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 μM 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 10 nM E2. When
the cells were pretreated with 10 nM E2 and subsequently, 300 μM Mn, there was
an increase in the GLT-1 gene expression. The experimental results indicate
that E2 can attenuate some Mn-induced toxicity in E18 astrocytes.