Advances in Bioscience and Biotechnology, 2013, 4, 9-15 ABB
http://dx.doi.org/10.4236/abb.2013.410A1002 Published Online October 2013 (http://www.scirp.org/journal/abb/)
Effect of estrogen on manganese-induced toxicity on
embryonic astrocytes*
Tyler T. Huynh, Kimberly J. Baker, Harold L. Komiskey#
Department of Biomedical Sciences, Philadelphia College of Osteopathic Medicine-GA Campus, Suwanee, USA
Received 15 August 2013; revised 15 September 2013; accepted 1 October 2013
Copyright © 2013 Tyler T. Huynh et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
Manganese (Mn) is a natural trace metal that is es-
sential for many physiological functions in the human
body. Astrocytes in the central nervous system are su-
sceptible 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 glu-
tamate transporters and glutamine synthetase, trans-
mitters, and the natural gonadal steroids and their re-
ceptors are known to occur in astrocyte cultures de-
rived from various stages of fetal and postnatal deve-
lopment. Cultures of embryonic (E18) hippocampal
astrocytes were examined in this study for the ability
of 17 β-estradiol (E2) to protect them from Mn toxic-
ity by up regulating gene expression of a glutamate
transporter. Primary rat hippocampal astrocytes were
pretreated with β-Estradiol (E2) in vitro and subse-
quently, Mn sulfate (MnSO4). The amount of toxic
damage to the astrocytes was measured by quantify-
ing glial fibrillary acidic protein (GFAP) with a sand-
wiched Enzyme-Linked Immunosorbent Assay (ELISA).
ELISA analysis indicated Mn exposure at 100 μM,
300 μM, or 600 μM significantly increased GFAP lev-
els. However, E2 concentrations at 10 nM or 30 nM
significantly reduced Mn-induced GFAP concentra-
tions at 100 μM. Cells pretreated with 10 nM or 30
nM of E2 significantly lowered GFAP levels. The Wa-
ter-Soluble Tetrazolium-8 (WST-8) method was util-
ized to determine cell viability. The WST-8 assay show-
ed 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, re-
vealing a dose-dependent effect. However, the dehy-
drogenase 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 com-
pared 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 in-
crease in the GLT-1 gene expression. The experimen-
tal results indicate that E2 can attenuate some Mn-in-
duced toxicity in E18 astrocytes.
Keywords: Astrocytes; Estrogen; Embryonic;
Manganese; Hippocampus
1. INTRODUCTION
Astrocytes are brain cells believed to be adversely af-
fected early by manganese (Mn) toxicity [1,2]. Although
astrocytes perform numerous functions essential for
brain activity, they accumulate up to 50 times higher Mn
concentration compared to neurons [3,4]. The normal in-
tracellular Mn concentrations are between 50 to 75 µM,
serving as a cofactor for glutamine synthetase [3,5]. The
buildup of excessive Mn within astrocytes is argued to
mediate neurotoxicity by oxidative stress and diminish-
ing glutamate transport [6,7]. In fact, Mn has been report-
ed to down regulate gene expression of glutamate transpor-
ters [8]. Estrogen (E) receptor agonists have even been re-
ported to protect astrocytes from Mn-provoked neuroto-
xicity by up regulating the gene expression of glutamate
transporters [9-11]. In addition, Mn alters the glutamate-
*Acknowledgements: We thank CCDA and Philadelphia College o
f
Osteopathic Medicine for funding.
#Corresponding author.

T. T. Huynh et al. / Advances in Bioscience and Biotechnology 4 (2013) 9-15 10
glutamine homeostasis [12,13].
The above studies were conducted with postnatal as-
trocytes. However, differences in expression/inducibi-
lity of glutamate transporters and glutamine synthetase
are known to occur in astrocyte cultures derived from va-
rious stages of fetal and postnatal development [14,15].
Differences exist in transmitter effects on embryonic and
postnatal astrocytes [16,17]. In addition, the natural go-
nadal steroids and their receptors vary during fetal and
postnatal development [18,19]. Cultures of embryonic
(E18) hippocampal astrocytes were examined in this stu-
dy for the ability of 17 β-estradiol (E2) to protect them
from Mn toxicity by up regulating gene expression of a
glutamate transporter.
2. MATERIALS AND METHODS
2.1. Cell Culture
Primary Embryonic (day 18 Sprague/Dawley) rat hippo-
campal astrocytes obtained from Gelantis (San Diego,
CA) were prepared using the NeuroPapain enzyme. Fol-
lowing enzymatic pretreatment, the supernatant (contain-
ing the NeuroPapain solution) was removed, and the as-
trocytes were placed in a 75 cm2 Poly-D-Lysine filter cap
flask containing a complete media mixture of 34% Gibco
DMEM High Glucose, 34% HyClone DME/High Glu-
cose (1X), 25% HANKS, 3.5% Gibco Dialyzed Fetal
Bovine Serum, and 3.5% HyClone Donor Equine Serum
at 37˚C with 5% CO2.
2.2. Trypsinization and Pretreatment
Once cells reached 70% or greater confluence, 0.25%
trypsin was used to remove them from the flask. After a
wash with complete media, the astrocytes were transfer-
red to a Poly-D-Lysine coated 48-well plate. The wells
containing astrocytes and complete media mixture were
treated with media or increasing concentrations of E2 on
the same day. Twenty-four hours later, some of the wells
were treated with increasing dosages of MnSO4.
2.3. Bicinchoninic Acid (BCA) Assay
Pierce BCA protein assay (Rockford, IL) was used to
measure the level of total protein concentration present
in the cell samples. The absorbance was read at 562 nm.
2.4. Cell Viability Assay
Cell viability was determined by measuring a water so-
luble formazan after the addition of highly water soluble
tetrazolium salts called WST-8. Cell Counting Kit-8
(CCK-8, Sigma Chemical) used WST-8 to measure the
dehydrogenase enzyme activity inside the cell to analyze
the viability of the astrocytes. The absorbance was read
at 450 nm.
2.4. Enzyme-Linked Immunosorbent
Assay (ELISA)
The amount of toxicity damage to the astrocytes was
measured by quantifying GFAP in a sandwiched ELISA,
according to O’Callaghan [20]. A 96-well microtiter plate
was coated with 100 μl of polyclonal rabbit anti-cow
GFAP antibody, then incubated for 1 hr at 37˚C with 5%
CO2 and overnight at 4˚C in the refrigerator. The poly-
clonal anti-GFAP solution was washed with 200 μl of
PBS four times. Next, 100 μl of BLOTTO (Bovine Lacto
Transfer Technique Optimizer) was added and incubated
for 1 hr. BLOTTO was removed and the purified GFAP
standards and cell samples were loaded. The plate was
washed four times with PBS and 0.5% Triton X-100
combination. A solution of monoclonal anti-GFAP mouse
antibody was mixed with BLOTTO and 0.5% Triton X-
100 to be loaded and incubated for 1 hr at room tem-
perature. Again, the plate was washed four times with the
PBS and 0.5% Triton X-100 mixture. Alkaline Phospha-
tase-Conjugated anti-mouse IgG was mixed with
BLOTTO and 0.5% Triton X-100, added, and incubated
for 30 mins at room temperature. The plate was washed
four times with the PBS and 0.5% Triton X-100 mixture.
P-Nitrophenyl Phosphate Liquid Substrate was added to
the plate and incubated for 25 min at room temperature.
Finally, 0.4 N NaOH was used to stop the reaction and
the absorbance values were read at 405 nm.
2.5. Reverse Transcriptase-Polymerase Chain
Reaction (RT-PCR)
RT-PCR was used to measure Glutamate Transporter-1
gene expressions by detecting the levels of RNA in astro-
cytes. Each sample was normalized to Glyceraldehyde 3-
phosphate dehydrogenase (GAPDH) [21]. RNeasy Mini
Kit from Qiagen was used to extract RNA from the as-
trocytes by following the manufacture’s protocol. A
High-Capacity cDNA Reverse Transcription Kit (Life Te-
chnologies) was used to transcribe the RNA into a cDNA
by following the manufacturer’s protocol. The first cycle
was ran for 10 min at 25˚C, then 120 min at 37˚C, and
finally, for 5 min at 85˚C. PCR was carried out using a
Platinum PCR SuperMix (Life Technologies) by follow-
ing the manufacturer’s protocol also. Primer sequences
were: 5’-CCT CAT GAG GAT GCT GAA GA-3’ (GLT-1
forward), 5’-TCC AGG AAG GCA TCC AGG CTG-3’
(GLT-1 reverse), 5’-TCC CTC AAG ATT GTC AGC
AA-3’ (GAPDH forward), and 5’-AGA TCC ACA ACG
GAT ACA TT-3’ (GAPDH reverse). Each primer was di-
luted to 0.2 μM with RNase-free water according to the
Certificate of Analysis. Then, the mixture was added to
45 μl of PCR SuperMix to make up a total reaction vo-
lume of 50 μl. Amplification was performed with 30 cy-
cles at 94˚C for 30 s (denaturation), 55˚C for 30 s (an-

T. T. Huynh et al. / Advances in Bioscience and Biotechnology 4 (2013) 9-15 11
neal), and 72˚C for 1 min (extension). The samples were
analyzed on a 1.5% agarose gel electrophoresis and vi-
sualized by using GelRed Nucleic Acid Stain.
2.5. Statistical Analysis
Data was analyzed using One-way Analysis of Variance
(ANOVA) using GraphPad Prism 5 Software with Dun-
can’s new multiple range test [22]. P-values less than
0.05 were considered significant. The number of primer
sequence base pairs used in RT-PCR was analyzed from
National Center for Biotechnology Information (NCBI)
online @ http://www.ncbi.nlm.nih.gov/. The average pi-
xel density (APD) of the agarose gel images was analy-
zed using Image J64 to calculate the GLT-1/GAPDH per-
centiles.
3. RESULTS
3.1. E2 Decreases GFAP Levels
To determine the neuroprotective effects of E2, cells
were pretreated with E2 for 72 hrs. The ELISA showed
that astrocytes pretreated with E2 at 10 nM (p < 0.05) or
30 nM (p < 0.05) significantly lowered GFAP levels when
compared with the control (Figure 1).
3.2. GFAP Levels Compared in Cells Treated
with Mn and Cells with E2 and Mn
To determine Mn’s toxic effect in astrocytes, the cells were
treated with Mn for 48 hrs. The ELISA results showed
that Mn significantly increased GFAP production (100 µM,
p < 0.05; 300 µM, p < 0.05; 600 µM, p < 0.05) when
compared to the control (Figure 2). Results also showed
cells pretreated with 10 nM (p < 0.01) or 30 nM (p < 0.01)
of E2 significantly lowered Mn-induced GFAP level at
100 µM (Figure 2).
3.3. E2 Effect on Dehydrogenase Activity
Although not statistically significant, the WST-8 assay
showed cells pretreated with E2 had a trend of increasing
dehydrogenase activity starting at 10 nM (Figure 3).
3.4. Dehydrogenase Activity Compared in Cells
Treated with Mn and Cells Treated with E2
and Mn
The WST-8 assay showed that Mn significantly reduces
the activity of dehydrogenase enzyme at 100 µM (p <
0.01), 300 µM (p < 0.01), or 600 µM (p < 0.01) (Figure
4). Data also show that the control is significantly diffe-
rent from all treatment groups (p < 0.01) (Figure 4). The
dehydrogenase activity in cells treated with 600 µM Mn
was significantly increased when pretreated with 10 nM
E2 (p < 0.05) (Figure 4). Enzyme activity with 600
Figure 1. E2 effect on GFAP level using ELISA. Cells pre-
treated with E2 at 10 nM (p < 0.05) or 30 nM (p < 0.05) sig-
nificantly lowered GFAP levels when compared with the con-
trol (n = 6).
µM Mn was significantly decreased when compared with
100 µM Mn (p < 0.05), revealing a dose-dependent effect
(Figure 4).
3.5. RT-PCR Results for Cells Treated with 10
nM E2 and Cells with 10 nM E2 and 300
μM Mn
Gel electrophoresis and NCBI analysis showed PCR am-
plified a portion of the GLT-1 gene that was 217 base
pairs and 268 base pairs of the GAPDH gene (Figure
5(a)). Although not statistically significant (n = 2), re-
sults showed an increased expression of GLT-1 mRNA
by 17% when cells were pretreated with 10 nM E2 (Fig-
ure 5(b)). When cells were pretreated with 10 nM E2
and afterward, exposed to 300 µM Mn, there was an in-
creased production of the GLT-1 gene expression by
12% (Figure 5(b)).
4. DISCUSSION
The present study demonstrates that acute exposure to E2
can mitigate some Mn-induced toxicity in E18 astrocytes
concerning inflammation, cell viability, and glutamate
transport system. Mn was shown to induce toxicity and
upregulate inflammation in astrocytes by significantly in-
creasing GFAP production at 100 µM, 300 µM, or 600
µM (Figure 2). Although not statistically significant, the
GFAP levels trended to increase with the Mn concentra-
tion. However, E2 concentrations at 10 nM or 30 nM sig-
nificantly decreased Mn-induced GFAP concentrations at
100 µM (Figure 2). In addition, E2 pretreated cells at 10
nM or 30 nM significantly lowered GFAP levels compar-
ed to the control (Figure 1). E2 could be reversing or in-
hibiting the Mn-induced GFAP level. Rozovsky et al. [23]
demonstrated the attenuation of GFAP via E2 in cortex
astrocytes (1 - 3 day old) from ovariectomized female rats
3 months old. It was also shown that the interaction of
ERα and estrogen response element (ERE) were involved

T. T. Huynh et al. / Advances in Bioscience and Biotechnology 4 (2013) 9-15 12
Figure 2. GFAP levels compared in cells treated with Mn and cells treated with E2 and Mn.
Mn was shown to significantly increase GFAP levels at 100 µM (p < 0.05), 300 µM (p < 0.05),
or 600 µM (p < 0.05) when compared to the control (n = 6). However, cells pretreated with 10
nM (p < 0.01) or 30 nM (p < 0.01) of E2 significantly lowered Mn-induced GFAP level at 100
µM (n = 6).
Figure 3. E2 effect on dehydrogenase activity using
the WST-8 assay Dehydrogenase activity in cells. Cells
pretreated with E2 were not statistically significant,
but did show a tendency of increasing activity with 10
nM and 30 nM (n = 6).
in the gene expression of GFAP [23].
Cell viability depends on many chemicals and enzy-
mes working together to ensure that biochemical reac-
tions properly take place. Dehydrogenase enzyme is one
that catalyzes the transfer of a hydride ion to nicotina-
mide adenine dinucleotide (NAD) or nicotinamide ade-
nine dinucleotide phosphate (NADP) in the mitochondria.
These reactions are particularly important in glycolysis,
citric acid cycle, and electron transport chain. Therefore,
to determine the level of dehydrogenase activity would
reveal the cell’s viability [24]. The WST-8 assay establi-
shed that all treatment groups pretreated with E2 and/or
exposed to Mn significantly affected the dehydrogenase
activity in astrocytes when compared to the control
(Figure 4). This could be due to the age of astrocytes
employed; embryonic astrocytes could be especially sen-
sitive to chemical agents, thus greatly affecting their cell
viability [25]. Mn was shown to significantly reduce the
enzyme’s activity at 100 µM, 300 µM, or 600 µM, there-
by, significantly decreasing the number of viable astrocy-
tes (Figure 4). Once excess Mn has been sequestered in
the mitochondria, it could generate reactive oxygen spe-
cies (ROS) generation, oxidative stress, and ultimately,
apoptosis. Pretreatment of E2 at 10 nM was shown to
significantly attenuate Mn-induced decrease in dehydro-
genase activity at 600 µM (Figure 4). E2 was able to
inhibit ROS and oxidative stress formation stimulated by
Mn. Astrocyte protection afforded by E2 was most likely
mediated through the activation of PI3K/ PKB/Akt and
MAPK signaling pathways. Both pathways are exten-
sively involved in Mn-induced cell death and neuro-
protective effects of E2 [21,26]. Lee et al. (2009) con-
ducted a similar experiment where they demonstrated
Mn-induced cell death in neonatal rat primary astrocytes
by generation of ROS [27]. The WST-8 assay showed
cells pretreated with E2 had a non-significant increase in
dehydrogenase activity starting at 10 nM (Figure 3). Also,
Mn produces a dose-dependent effect when the dehydro-
genase activity was significantly decreased at exposure
to 600 µM vs. 100 µM (Figure 4). This result leads to
the notion that the magnitude of adverse biological re-
sponse in astrocytes caused by Mn is dependent on its
concentration.
Researchers have previously shown that E2 can in-
crease the GLT-1 gene expression in cultures with rat ne-
onatal midbrain and cortical astrocytes [27,28]. One of
the experimental aims was to examine if the outcome
would be similar in rat embryonic hippocampal astro-
cytes. PCR analysis did show that the GLT-1 gene expre-
ssion was upregulated in cells pretreated with 10 nM E2
(Figure 5(b)). Due to the fact that an antagonist is not
present, this could suggest that there was a slow genomic
mechanism with E2 binding t either ERα or β in the nu- o

T. T. Huynh et al. / Advances in Bioscience and Biotechnology 4 (2013) 9-15 13
Figure 4. Dehydrogenase activity compared in cells treated with Mn and cells treated with E2 and Mn
using WST-8 assay. Mn was shown to significantly reduce the enzyme’s activity at 100 µM (p < 0.01),
300 µM (p < 0.01), or 600 µM (p < 0.01), thereby, significantly decreasing the number of viable as-
trocytes (n = 6). The control group was shown to be significantly different from all treatment groups (p
< 0.01) (n = 6). Pretreatment of E2 at 10 nM was shown to significantly decrease Mn-induced dehydro-
genase activity at 600 µM (n = 6). Mn produced a dose-dependent effect when the dehydrogenase ac-
tivity was significantly decreased at exposure to 600 µM vs. 100 µM (n = 6).
(a)
(b)
Figure 5. GLT-1 levels in cells pretreated with 10 nM
E2 compared with cells pretreated with 10 nM E2 and
subsequently exposed to 300 μM Mn. Lanes 1, 3, and 5
contain GLT-1 gene. Lanes 2, 4, and 6 contain GAPDH
gene. PCR amplified a section of the GLT-1 gene that
was 217 bp and 268 bp of the GAPDH gene. (a): Image
J64 analysis showed pretreatment of E2 alone increased
GLT-1 mRNA level by 17%; (b): When cells were pre-
treated with 10 nM E2 and afterward, exposed to 300
µM Mn, there was an increased production of the GLT-
1 gene expression by 12%.
cleus to activate ERE and acquire transcription of the
GLT-1 gene. Another genomic possibility is via GPER
and CRE transcription mechanisms [29]. To determine
which ER contributes to the increased GLT-1 gene, ex-
periments pertaining to the silencing of ERs by siRNA
transfection could be carried out. For example, a knock-
down of the GPER and then, treating astrocytes in cul-
ture with G-1, a GPER agonist, could explain the role
GPER plays. Through the process of elimination, it could
deduce which ER is solely responsible or perhaps, it is
the combination of 1 - 3 ERs [21].
However, with the upregulation of GLT-1 with 10 nM
E2 and 300 μM Mn together (Figure 5(b)), there may be
a more rapid mechanism to reverse Mn-induced decrease
in the GLT-1 mRNA. It has been suggested that trans-
forming growth factor-α (TGF-α) and multiple signaling
pathways, which can include PI3K, MAPK, EGFR, and
PKA, mediate the protective effects of E2 on Mn-induc-
ed glutamate transport systems. The possibility that E2
could be activating multiple signaling pathways simulta-
neously to ensure cell proliferation and survival should
be considered strongly. TGF-α, which mainly acts on as-
trocytes and is upregulated by E2, is possibly a major
component in the protection against the downregulation
of GLT-1 gene expression. TGF-α would bind to its re-
ceptor, EGFR, to activate the PI3K and MAPK signaling
cascades, which would reverse or protect Mn-induced
effect on GLT-1 proteins [30]. The notion that E2 could
be mediating genomic effects in addition to non-genomic
is also likely. When the cells were pretreated with E2 and
Mn, E2 not only compensated for the loss of GLT-1
mRNA caused by Mn, but it could have “overcorrected”
the situation. It was observed that there was about a 12%

T. T. Huynh et al. / Advances in Bioscience and Biotechnology 4 (2013) 9-15 14
increase in the gene expression compared to the control
group (Figure 5(b)). This protective mechanism against
Mn would ensure increased glutamate uptake from the
synaptic cleft and prevent neuroexcitation.
The above evidence supports the hypothesis that E2
can attenuate some Mn-induced toxicity in E18 astrocy-
tes. The study revealed Mn’s degree of neurotoxic effects
on astrocytes with varying concentrations. Mn level can
exceed 350 μM in the brain during toxicity, but this study
indicates a concentration starting at 100 μM can become
dangerous for astrocytes [31]. This finding is pertinent to
the growing concern of the association between delete-
rious effects of excess Mn on astrocytes and develop-
mental neurotoxicology. The Mn-induced levels of GFAP,
dehydrogenase activity, and GLT-1 could be the bridge
that connects this association. The results of this study
indicate that E2 can shield rat fetal astrocytes from some
of the cellular damage brought upon by Mn from day 18
on, but the vulnerability of younger fetal astrocytes to
Mn and possible E2 protection are unknown.
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