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Phytoestrogens are very important in human and animal
nutrition[1]. Isoflavones are abundant in soybeans, and they
are present in appreciable concentrations in a variety of beans,
spouts and legumes[2,3]. The major and most relevant
property of soybean isoflavones is their modest agonist effect
on the b estrogen receptor (approximately one-third as
potent as estradiol) and their weak effect on the
a estrogen receptor (with 0.001 of the potency of
estradiol)[4]. Genistein is also a potent inhibitor of protein tyrosine
kinase[5-7]. Protein tyrosine kinase is known to play a key role in growth
factor-related signal modulation and in programmed cell
death, known as apoptosis[7]. In cells exposed to genistein,
the number of cells undergoing apoptosis increase
in vitro and in vivo[7,8]. In aortic artery and atrial fibers, genistein
decreases the contractile response and arterial blood
pressure[9,10].
In postmenopausal women, estrogen therapy significantly
lowers the risk of Alzheimer disease. Estrogens also
alter certain specific pathologies such as the epileptic seizure
threshold and Parkinson’s
disease[11-13].
Recently, it has been reported that there is a cross-talk
between steroid hormones and neurotransmitters. Estrogen
induces structural and functional changes in excitatory
input to hippocampal CA1 pyramidal cells in adult female rats.
Estrogen increases the density of dendritic spines and spine
synapses on CA1 pyramidal cells[14-16] concomitant with an
increase in peak Ca2+ levels, an effect that was blocked by an
N-methyl-D-aspartate receptor antagonist. The
estrogen-modulated signaling would presumably lead to an increase
in downstream calcium-dependent
responses[17]. However, the modulation of intracellular calcium is very important in
long-term treatment with estrogen or isoflavones because a
sustained increase in calcium may induce neuronal death. A
calcium-binding protein, parvalbumin (PV), participates in
the regulation of calcium homeostasis and acts as an
intra-cellular calcium buffer that regulates the concentration of
the ion during neuronal activity[18]. In the present study,
therefore, we investigated the long-term effects of soybean
isoflavones on PV immunoreactivity in the hippocampus in
normal female, ovariectomized (OVX) female, and normal male
rats.
Materials and methods
Extraction of soybean isoflavones Isoflavones were
extracted from soybean hypocotyls with 10 volumes of 80%
aqueous methanol by stirring for 4 h at room temperature.
The methanol extract was condensed in a rotary evaporator
at 50 °C, and the soybean isoflavone extract was obtained
by freeze-drying the concentrated methanol extract. A high
performance liquid chromatography (HPLC) analysis of 3
isoflavones and their 9 derivatives was performed using a
diode array detector (HP1100 system, Agilent, Palo Alto,
CA,USA) and an eclipse XDB C-18 column (Agilent).
Ultraviolet detection was performed at a wavelength of 260 nm, and
the injection volume was 5 µL. The mobile phases used for
analysis were 0.1% acetic acid in H2O (solvent A) and 0.1%
acetic acid in acetonitrile (solvent B). A flow rate of 1.2
mL/min under an initial condition of 93:7 (A:B) was held for 25
min, then brought to 15% B in 25 min, to 20% B in 5 min, and
to 25% B in 15 min, all with a linear gradient. Genistein,
daidzein, and glycitein were purchased from Sigma (St Louis,
MO, USA), and the isoflavone derivatives (the malonyl-,
acetyl-, and glycoside forms) were purchased from Fujico
(Tokyo, Japan) for use as standards in the HPLC
analyses[19].
Experimental animals The present study used the
progeny of male and female Sprague-Dawley rats (3-4 months
old) purchased from Bio-Genomics (Seoul, South Korea).
The animals were housed at constant temperature (23 ºC) and
relative humidity (60%) with a fixed 12 h light/dark cycle and
free access to food and water. Procedures involving animals
and their care conformed to guidelines, which are in
compliance with current international laws and policies (NIH Guide
for the Care and Use of Laboratory Animals, NIH Publication
No 85-23, 1985, revised 1996). All experiments were
conducted to minimize the number of animals used and suffering
caused.
When the rats were 10 months old, they were assigned
to one of 9 groups (n=7 in each group) based on body weight
using a randomized complete-block design. The groups were:
control diet-treated normal females, OVX females, and males;
0.3 g/kg soy isoflavone-treated normal females, OVX females,
and males; and 1.2 g/kg soy isoflavone-treated females, OVX
females, and males. The rats’ isoenergetic and isonitrogenous
diets were based on the AIN76A formulation and the
isoflavone extract was blended with the basal diet at the
expense of fiber. These experimental diets were fed to the rats
for 16 weeks. Distilled water was always available to the rats.
Tissue processing and immunohistochemistry
All the animals were anesthetized with pentobarbital sodium, and
perfused transcardially with 0.1 mol/L phosphate-buffered
saline (PBS, pH 7.4) followed by 4% paraformaldehyde in
0.1 mol/L PBS (pH 7.4). The rats’ brains were removed, and
postfixed in the same fixative for 6 h. Brain tissues were
cryoprotected by infiltration with 30% sucrose overnight.
Thereafter the brain tissues were frozen and sectioned with
a cryostat at 30 mm and consecutive sections were collected
in 6-well plates containing PBS.
To ensure that immunohistochemistry data were
comparable between groups, free-floating sections of the brains of
experimental animals from each group were processed for
immunohistochemical analysis simultaneously. The sections
were sequentially treated with 0.3% hydrogen peroxide
(H2O2) in PBS for 30 min and 10% normal horse serum in 0.05 mol/L
PBS for 30 min. The sections were next incubated with
diluted mouse anti-PV (Sigma, USA; 1:1000) overnight at room
temperature. Thereafter the tissues were exposed to
biotinyl-ated goat anti-mouse IgG and streptavidin peroxidase
complex (Vector, USA). The sections were visualized with
3,3’-diaminobenzidine in 0.1 mol/L Tris buffer and mounted on
gelatin-coated slides[20,21].
Quantification of data and statistical analysis
All measurements were performed in order to ensure objectivity, by
2 observers for each experiment, who were blinded to the
treatments received by the rats, carrying out the
measurements for the control and experimental sections
simultane-ously.
At magnifications of 25-50, the mid-point areas of the
hippocampal CA1 region were measured. Images of all
PV-immunoreactive structures taken from the 3 layers (striata
oriens, pyramidale and radiatum in the hippocampus proper,
and molecular, granule cell and polymorphic layers in the
dentate gyrus) were obtained through an Axiophot light
microscope (Carl Zeiss, Germany) connected via a Charge
Coupled Device (CCD) camera to a computer monitor. Video
images were digitized into an array of 512×512 pixels
corresponding to a tissue area of 140 µm×140 µm (40×primary
magnification). To evaluate the PV-immunoreactive fibers
and neurons, the PV-immunolabeled fibers or neurons were
selected by using the ImageJ software (RSB, Bethesda,
Maryland, USA) by interactively determining each cell limit.
The resolution of each pixel was 256 gray levels. The
intensity of PV immunoreactivity was evaluated by means of
a relative optical density (ROD) value. ROD was obtained
after transformation of mean gray values into ROD using the
formula: ROD=log(256/mean gray value). The values of
background staining were obtained and subtracted from the ROD
values of all immunoreactive structures before statistically
processing the obtained values. ROD values were presented
as ROD units.
The number of PV-immunoreactive neurons was
calculated by using an image analyzing software system (Optimas
6.5, CyberMetrics, USA). The mid-point of the hippocampal
CA1 region (500 µm2 ) and the hilar region of the dentate
gyrus were selected by the image analyzing system. Cell
counts were obtained by averaging the counts from 30 sections taken at the same level of the hippocampal CA region
and dentate gyrus.
Four to 6 separate immunohistochemical experiments were
run for each primary antibody. Each individual experiment
was composed of 6-10 tissue sections of each animal from
each group. Five to ten fields were measured for each brain
area in each section of each animal. Inter-animal differences
in each group, as well as interexperiment differences, were
not statistically significant. The values shown here
represent the means of experiments performed for each
hippocampal area. Differences among the means were statistically
analyzed by one-way analysis of variance followed
by Duncan’s new multiple range method or the Newman-Keuls
test to elucidate the effect of isoflavones in PV
immunoreactivity or to investigate the differences with respect to PV
immunoreactivity in different sexes. P<0.05 was considered
statistically significant.
Results
Isoflavone concentrations in the diets A yield of 5018.2 mg
isoflavone/100 g isoflavone extract was obtained (Table 1).
The isoflavone concentrations of the 0.3 g/kg isoflavone-treated
group and 1.2 g/kg isoflavone diets were 0.321 g/kg diet and
1.284 g/kg, respectively. Glycoside and malonyl forms were
dominant in the extract. The daily soy isoflavone intake in
the 0.3 g/kg isoflavone-treated group was 10.89 mg/kg body
weight per day, and that in the 1.2 g/kg isoflavone group
was 38.53 mg/kg body weight per day.
Food consumption and body weight Food consumption
and body weight did not differ among the groups (data not
shown).
Changes in PV immunoreactivity PV immunoreactivity
was significantly changed in the hippocampal CA1 region
and in the dentate gyrus, but not in the hippocampal CA2/3
region in any of the experimental groups after long-term
treatment with isoflavones.
In the CA1 region, PV immunoreactivity in the control
diet females was detected in non-pyramidal cells located near
the striatum pyramidale (Figure 1A). PV immunoreactivity in
the control diet OVX females and control diet males was
higher than that in the control diet females (Figure 1B, 1C).
PV immunoreactivity in the control diet OVX females
was similar to that in the control diet males. PV immunoreactivity
in all the 0.3 g/kg isoflavone-treated groups was decreased
compared with all the control diet-treated groups (Figure
1D,1F, 2). PV-immunoreactive neuronal processes in these
groups were poor compared with those of all the control
diet-treated groups. PV immunoreactivity in all the 1.2 g/kg
isoflavone-treated groups was decreased compared with all
the 0.3 g/kg isoflavone-treated groups (Figure 1G,1H, 2). In
particular, PV immunoreactivity in the 1.2 g/kg
isoflavone-treated males was negligible (Figure 1I).
In the dentate gyrus, PV-immunoreactive neurons in the
control diet females were distributed in the polymorphic layer
(Figure 3A). These PV-immunoreactive neurons were
non-granule cells and generally located in the subgranular zone
in the polymorphic layer. Some PV-immunoreactive neurons
were detected at the border between the granule cell layer
and molecular layer. PV immunoreactivity in the control diet
OVX females was similar to that of the control diet males,
and was greater than that in the control diet females (Figure
3B, 4). PV immunoreactivity was decreased in all the 0.3 g/kg
isoflavone-treated groups compared with rats in all the
control diet-treated groups (Figure 3D-F, 4). In all the 1.2
g/kg isoflavone-treated groups, PV immunoreactivity was
decreased compared with rats in all the 0.3 g/kg
isoflavone-treated groups (Figure 3G-I, 4).
Changes in the number of PV-immunoreactive neurons
In the hippocampal CA1 region, the number of
PV-immunoreactive neurons in the control diet females was
approximately 2.6 in a 500 µm diameter region of the CA1 (Figure 5).
The number in the control diet OVX females was
significantly increased compared with that of the control diet
females, and similar to that for the control diet males (Figure
5). The number of PV-immunoreactive neurons in all the
isoflavone-treated groups decreased dose-dependently. In
the 1.2 g/kg isoflavone-treated males, there were negligible
numbers of PV-immunoreactive neurons in the CA1 region.
In the dentate gyrus, the number of PV-immunoreactive
neurons in the control diet females was approximately 15 in a
500 µm diameter region of the dentate gyrus (Figure 5). The
number of PV-immunoreactive neurons in the control diet
OVX females and males was greater than that in the control
diet females (Figure 5). This finding is similar to that in the
hippocampal CA1 region. The number of
PV-immunoreactive neurons in all the isoflavone-treated groups decreased
dose-dependently.
Discussion
It has been reported that a reduction in PV
immunoreactivity occured in the hippocampal CA1, CA3 and dentate
gyrus following environmental enrichment. And the enriched
housing animal also represents the also represents the
positive effect on behavior and
learning[22]. Because PV-expressing chandelier cells play an important role in controlling
pyramidal cell excitability[23], the reduced expression of PV in
the hippocampal CA1 region may be associated with
protection against memory deficits during the aging process. This result coincides with that of our previous study, in which we
found that soy isoflavones could ameliorate deficits in
memory tasks resulting from the loss of cholinergic input to
the hippocampus or cholinergic degeneration in elderly male
rats[19].
In the present study, the formulation of the isoflavone
diet was based on that used in previous
studies[24,25]. Pan et al defined a dose suitable for a woman and added certain
amounts of estradiol or soy isoflavones to the experimental
diet[24]. The soy isoflavone content of the 0.3 g/kg
isoflavone-treated group was determined by considering the
caloric density of the diet, and making the dose equivalent to
that for a woman. Lund et al investigated the neurobehavioral
effects of isoflavones on rats[25]; they used a diet containing
soy isoflavones (0.6 g/kg diet). This diet produced an
improvement in cognitive function in female rats, but tests with
higher concentrations of soy isoflavones were not performed.
In the present study, we tried to evaluate the effects of very
large amounts of isoflavone on the brains of female rats.
In the present study, we investigated changes in PV
immunoreactivity in the hippocampus of normal female,
ovariectomized female and normal male rats after long-term
treatment with soybean isoflavones. PV immunoreactivity was
found in non-pyramidal cells in the hippocampus proper and
in polymorphic cells in the dentate gyrus. PV
immunoreactivity and immunoreactive neurons in the control diet OVX
females were more pronounced than in the control diet
females. PV immunoreactivity and the number of
immunoreactive neurons in the control diet OVX females were similar
to those in the control diet males. In all the
isoflavone-treated groups, PV immunoreactivity and the number of
immunoreactive neurons decreased dose-dependently after
isoflavone treatment. This result shows that ovariectomy
may increase PV immunoreactivity in the hippocampus.
Choi and Lee investigated the cytotoxic effects of
genistein in vivo, and found that a high intake of genistein
(20 mg/day) induced cytotoxicity in rat brain, but that a lower
intake (2 mg/day) produced no such
effect[26]. The daily soy isoflavone intake in the 1.2 g/kg isoflavone-treated groups
was 29.74 mg. However, the proportion of the soy
isoflavones in the diet represented by genistein was 15%, and the daily
genistein intake was only 4.461 mg/day. This amount is
much lower than the level of genistein that was found to
produce cytotoxic effects on rat
brain[26]. Therefore, the dose of soy isoflavones used in the present study produces no
cytotoxic or apoptotic effects in rat brain.
It has been reported that estrogen modulates neuronal
excitability either by affecting the presynaptic release of
neurotransmitters or by direct actions on the postsynaptic
membrane[27]. Estrogen transiently disinhibits hippocampal CA1
pyramidal cells in adult female rats and prolongs the decay
time of inhibitory postsynaptic currents in these cells, thus
excitatory input to CA1 pyramidal cells is enhanced,
dendritic spine and synapse density are
increased[28], and spatial working memory is
improved[29,30].
In neurons pretreated with isoflavones, the excitotoxic
glutamate-induced rise in intracellular
Ca2+ concentration is decreased to non-toxic
levels[31]. In the present study, the decrease of PV immunoreactivity in the hippocampus may
be associated with the sustained decrease of calcium in this
region. The sustained decrease of calcium may induce the
downregulation of PV in this region. The attenuation of the
excitotoxic glutamate-induced rise in intracellular calcium to
near the non-toxic range provides a probable mechanism by
which isoflavones reduce glutamate cytotoxicity and
promote neuronal survival.
In aging OVX rats, long-term hormone replacement with
oestrogen or oestrogen plus progesterone produces
substantial reductions in the levels of choline acetyltransferase
(ChAT) and tyrosine receptor kinase A (trkA) mRNA in the
medial septum and nucleus basalis magnocellularis, relative
to much younger OVX controls[32]. However, the number
and size of the cholinergic neurons were not significantly
altered. This result suggests that long-term hormone
replacement had no apparent effect on the number of
ChAT-positive neurons, and did not prevent the reduction in ChAT
and trkA mRNA associated with ovariectomy and
aging[32].
In conclusion, long-term administration of soybean
isoflavones induces the reduction of PV immunoreactivity
in the hippocampus, and the reduction of PV in this region
suggests that the long-term administration of isoflavones
may be related to calcium homeostasis in the hippocampal
CA1 region and in the hilar region of the dentate gyrus,
indicating the neuronal plasticity of these neurons.
Acknowledgements
The authors would like to thank Mr Suek HAN,
Seung-uk LEE and Ms Hyun-sook KIM for their technical help in
this study.
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