17β-Oestradiol Stimulation of G-Proteins in Aged and Alzheimer’s Human Brain: Comparison with...

ORIGINAL ARTICLE

17b-Oestradiol Stimulation of G-Proteins in Aged and Alzheimer’s HumanBrain: Comparison with PhytoestrogensV. Jefremov,*� A. Rakitin,*� R. Mahlapuu,* K. Zilmer,* N. Bogdanovic,� M. Zilmer* and E. Karelson*

*Department of Biochemistry, Tartu University, Tartu, Estonia.

�Geriatric Department, Neurotec, Karolinska Institute, Huddinge, Sweden.

�Department of Neurology and Neurosurgery, Tartu University Hospital, Tartu, Estonia.

Over the past decade, it has become apparent that the neuropro-

tective action of oestrogens may result from the oestrogen receptor

(ER)-dependent and -independent mechanisms (1, 2). The ER-

dependent mechanisms may be exerted via genomic way (i.e.

through the activation of intracellular ERa and ERb and oestrogen

target genes) (3, 4). In addition, the nongenomic ER-dependent

mechanisms of oestrogens in target cells and tissues have been

described (5, 6). These steroids may interact with the plasma mem-

brane-located ER-like proteins that can activate the G-protein med-

iated down-stream signalling (e.g. cAMP and protein kinase C

Journal ofNeuroendocrinology

Correspondence to:

Ello Karelson, Department of

Biochemistry, University of Tartu,

Ravila 19, 50411 Tartu, Estonia

(e-mail: [email protected]).

The neuroprotective action of oestrogens and oestrogen-like compounds is in the focus of basic

and clinical research. Although such action has been shown to be associated with neuronal

plasma membranes, the implication of G-proteins remains to be elucidated. This study revealed

that micromolar concentrations (lM) of 17b-oestradiol and phytoestrogens, genistein and daidz-

ein, significantly (P < 0.05) stimulate G-proteins ([35S]GTPcS binding) in the post-mortem hippo-

campal membranes of age-matched control women with the respective maximum effects of 28,

20 and 15% at 10 lM. In the frontocortical membranes, the stimulation of G-proteins did not

differ significantly from that in hippocampal membranes. Although in the hippocampus and

frontal cortex of the Alzheimer’s disease (AD) women’s brain, 10 lM 17b-oestradiol produced

significantly (P < 0.05) lower stimulation of G-proteins than in the control regions, stimulation

by phytoestrogens revealed no remarkable decline. 17b-Oestradiol, genistein and daidzein

revealed a selective effect on various G-proteins (Gas, Gao, Gai1 or Ga11 plus Gb1c2) expressed in

Sf9 cells. At a concentration of 10 lM, 17b-oestradiol suppressed the H2O2 and homocysteine

stimulated G-proteins in the frontocortical membranes of control women to a greater extent

than phytoestrogens. In AD, the suppressing effect of each compound was lower than in the

controls. In the cell-free systems, micromolar concentrations of phytoestrogens scavenged OH•

and the 2.2-diphenyl-1-picrylhydrazyl free radical (DPPH•) more than 17b-oestradiol did. In the

frontocortical membranes of control women, the 20 lM 17b-oestradiol stimulated adenylate

cyclase with 20% maximal effect, whereas, in AD, the effect was insignificant. Genistein did not

stimulate enzyme either in control or AD frontocortical membranes. Our data confirm that the

agents stimulate G-proteins in control and AD women’s brains, although 17b-oestradiol and

phytoestrogens have similarities and differences in this respect. We suggest that, besides the

ER-dependent one, the ER-independent antioxidant mechanism is responsible for the oestrogen

stimulation of G-proteins in the brain membranes. Both of these mechanisms could be involved

in the neuroprotective signalling of oestrogens that contributes to their preventive ⁄ therapeutic

action against postmenopausal neurological disorders.

Key words: 17b-oestradiol, phytoestrogens, antioxidativity, G-proteins, aged, Alzheimer’s disease

brain.

doi: 10.1111/j.1365-2826.2008.01696.x

Journal of Neuroendocrinology 20, 587–596

ª 2008 The Authors. Journal Compilation ª 2008 Blackwell Publishing Ltd

pathways) (7–10). The available evidence suggests that oestrogens,

like traditional growth factors, recognise the membrane receptors

with intrinsic tyrosine kinase activity (11). These and other mem-

brane-associated mechanisms, such as the alteration of membrane

fluidity (12), activation of K+ channels (13) and stimulation of Ca2+

influx (14), were thought to be involved in the nongenomic neuro-

protection by oestrogens (15).

The ER-independent neuroprotective action of oestrogens

appears to be mainly caused by their antioxidant free-radical scav-

enging properties. The hydroxyl group in the C3 position on the A

ring of the steroid molecule is mainly responsible for these proper-

ties (16–18). In addition, the modulation of neurotransmission and

neuronal excitability may contribute to the ER-independent neuro-

protective action of oestrogens (19, 20). Studies with neuronal cell

lines have shown that nano- to micromolar concentrations of

17b-oestradiol reduce neuronal cell death induced by glutamate,

superoxide anion, H2O2, neurotoxic amyloid-b peptides and other

pro-oxidants (21–23). In vivo administered pharmacological doses

(mg ⁄ kg) of 17b-oestradiol are effective in protecting the animal

brain from the ischaemic injury (24) and deposition of amyloid-bpeptides (25). More interestingly, pharmacological doses of oestro-

gens may exert preventive ⁄ therapeutic effects against ischaemic

stroke and neurodegeneration in postmenopausal women (26, 27)

whereas age-matched men may reveal a weaker response (28).

In recent years, an interest has grown in alternative compounds

that act as selective oestrogen receptor modulators, but have no

unfavourable side-effects of oestrogen. One group of such com-

pounds consists of phytoestrogens, plant-derived (iso)flavones with

oestrogenic activity, which may act as oestrogen receptor agonists

or antagonists in various cells and tissues (29). In the nano- to

micromolar range, these polyphenolic compounds have been shown

to exert multiple protective effects (30), including neuroprotective

ones (31), that are mainly derived from their antioxidant properties

(32). In addition to the antioxidant activities, several other mecha-

nisms appear to be involved in the neuroprotective action of plant

polyphenols; the most important one might be their ability to

improve neuronal signalling and communication (33). However,

despite these findings, the mechanisms responsible for the neuro-

protective action of plant-derived and endogenous oestrogens need

to be studied further, particularly in the human brain.

The present study aimed to compare the effect of micromolar

concentrations of 17b-oestradiol and phytoestrogenic isoflavones

(genistein, daidzein) (Fig. 1) on G-proteins in the membranes of

age-matched control and Alzheimer’s disease (AD) hippocampus

and frontal cortex post-mortem. Both regions revealed morpho-

functional alterations typical of brain ageing and neurodegeneration

(34, 35). The second objective was to test whether the compounds

under investigation can discriminate between the different types of

heterotrimeric G-proteins expressed in Sf9 insect cells. The third

objective was to compare the antioxidant activity of the compounds

as a property that might imply their modulatory action on G-pro-

teins. To that purpose, the influence of 17b-oestradiol and phytoes-

trogens was tested regarding their influence on the oxymodified

membrane G-proteins and the ability to scavenge 2.2-diphenyl-1-

picrylhydrazyl (DPPH•) and OH• radicals in vitro. Finally, the modu-

lation of adenylate cyclase activity as a possible signalling mecha-

nism for the improvement of neuronal function by oestrogens

comprised an interesting problem to be resolved.

Materials and methods

Materials

All reagents used were of highest analytical grade, mainly purchased from

Sigma Chemical Co. (St Louis, MO, USA). [35S]-GTPcS (1250 Ci ⁄ mmol) was

obtained from NEN Life Science Products (Boston, MA, USA).

Brain tissue sampling

Hippocampal and frontal cortical tissues of post-mortem human brain

were obtained from Huddinge Brain Bank, Sweden. The work of the Brain

Bank as well as of the Department of Biochemistry at Tartu University

was approved by the Ethical Committees at Karolinska Institute (Stockholm,

Sweden) and Tartu University (Tartu, Estonia). The present study included

the regions from 12 aged-matched control and eight AD female subjects

(aged 78 � 6 years and 84 � 12 years, respectively). In addition, the

frontocortical tissues from four aged and four AD male persons (aged

72 � 4 years and 78 � 5 years, respectively) were used. The post-mortem

delay for aged and AD persons was 16 � 5 h and 14 � 8 h, respectively.

The AD patients met clinical DSM-IV criteria (36) and neuropathological

CERAD criteria (37, 38) for AD. The control group consisted of aged-

matched persons with no history of neurological or psychiatric disorders.

The respective brain regions were rapidly dissected and kept at )70 �C

prior to the experiment.

Preparation of brain membranes

The hippocampal and frontocortical membranes for [35S]-GTPcS binding

measurement and for the adenylate cyclase assay were mainly prepared

according to protocol of Karelson et al. (39). The membrane pellet was

finally resuspended either in the standard TE-buffer consisting of 10 mM

Tris-HCl and of 0.1 mM ethylenediaminetetraacetic acid (EDTA), pH 7.4

([35S]-GTPcS binding) or in the 4 mM HEPES-Na containing 1.5 mM the-

ophylline, 8.25 mM MgCl2, 0.75 ethylene glycol tetraacetic acid (EGTA),

7.5 mM KCl and 100 mM NaCl, pH 7.4 (adenylate cyclase assay) to obtain

a protein concentration 0.6–1 mg ⁄ ml as determined by the method of

Lowry et al. (40).

O

OHOOH

HOO

OHO

HOGenisteinDaidzein

OH

HO

Fig. 1. Structure of 17b-oestradiol, daidzein and genistein.

588 V. Jefremov et al.

ª 2008 The Authors. Journal Compilation ª 2008 Blackwell Publishing Ltd, Journal of Neuroendocrinology, 20, 587–596

Preparation of membranes from Sf9 cells

Sf9 cells (the Spodoptera frugiperda insect cell line) were transfected with

baculovirus vectors carrying the genes for the different G-protein alpha-

subunits (Gas, Gao, Gai1 or Ga11) and for Gb1c2. The transfection procedure

was perfomed by Dr Kulliki Saar (Department of Neurochemistry, University

of Stockholm, Sweden) in accordance with a previously described protocol

(41). The transfected Sf9 cells were stored at )70 �C. For the preparation

of membranes, the cells were thawed, resuspended in five volumes of ice-

cold TE-buffer and homogenised. The homogenate was centrifuged at

500 g for 15 min followed by centrifugation of the supernatant at

40 000 g for 40 min. The obtained pellet was washed in ten volumes of

TE-buffer and recentrifuged in the same conditions. The resulting pellet

was resuspended in TE-buffer to obtain the membrane preparation with

2–4 mg ⁄ ml of protein.

[35S]-GTPcS-binding assay

The brain or cell membranes with the final protein concentration of 0.04–

0.05 mg ⁄ ml were incubated in a reaction cocktail containing TE-buffer, GDP

(1 lM), dithiothreitol (1 mM), MgCl2 (5 mM), NaCl (150 mM) and [35S]-GTPcS

(50–70 000 c.p.m. in an aliquot of the reaction cocktail). Incubation of the

membranes was performed at 26 �C in a total volume of 0.1 ml either in

the absence (basal value) or in the presence of various concentrations of

17b-oestradiol or phytoestrogens. Ten minutes of incubation was used to

detect the effect of oestrogens in the human brain membranes. The

membranes of SF9 cells were incubated with oestrogens for 10–30 min

depending on the type of G-protein expressed in the cells. Bound and free

[35S]-GTPcS were separated by vacuum filtration through GF ⁄ B filters

(Whatman International Ltd, Maidstone, UK), which were washed three times

with 5 ml of ice-cold TE-buffer. Radioactivity was quantified using a

Wallac-1409 liquid scintillation counter (Wallac, Turku, Finland). In the data

analysis, the basal (unaffected) [35S]-GTPcS binding was defined as 100%.

Determination of protective effect on the oxymodifiedG-proteins

The protective effect of 17b-oestradiol and phytoestrogens was determined

via the changes that they produced in [35S]-GTPcS binding after the pre-

treatment of membranes with two metabolic pro-oxidants, homocysteine

(Hcy) and H2O2. The 5-min pretreatment of control and AD membranes by

500 lM Hcy or 10 lM H2O2 was carried out at 26 and 30 �C, respectively.

The pre-treatment was terminated by putting the samples into the ice.

Immediately after that, the oestrogen solution and reaction cocktail were

added and [35S]-GTPcS binding was performed. The protective activity was

estimated as a difference between the effects of Hcy or H2O2 on the radioli-

gand binding in the absence and presence of 10 lM 17b-oestradiol or

phytoestrogens.

Measurement of adenylate cyclase activity

The basal adenylate cyclase activity was assayed by determining the cAMP

formed in the reaction media (final volume of 150 ll), which contained a

reaction buffer (30 mM Tris-HCl buffer, 1.5 mM theophylline, 8.25 mM

MgCl2, 0.75 mM EGTA, 7.5 mM KCl and 0.1 M NaCl, 100 lg ⁄ ml bacitracin,

0.05% bovine serum albumin, 10 mM phosphoenol-pyruvate, 45 lg ⁄ ml

pyruvate kinase) and brain membranes with the final protein concentration

of 0.06–0.08 mg ⁄ ml (39). The oestrogenic compounds, dissolved in the 1%

ethanol ⁄ water, were added 2 min before the reaction was initiated by

10 mM ATP ⁄ 10 lM GTP. The reaction at 30 �C was terminated after

15 min by adding 100 mM EDTA and boiling the samples for 3 min. The

cyclic AMP content in the tubes was measured by a competitive protein

saturation assay (42).

Hydroxyl radical scavenging assay

The ability of 17b-oestradiol and phytoestrogens to scavenge OH• was

determined according to (43), using terephthalic acid (THA) as OH• chemical

dosimeter. In brief, 2.0 ml of 10 mM THA solution in 10 mM sodium phos-

phate buffer (pH 7.5) was mixed in a spectrometric cell with 0.1 ml of the

(phyto)estrogen solution. The neutral salt solution consisting of equal

amounts of 0.1 M HCl and 0.1 M NaOH solutions was used as a solvent of

(phyto)estrogens. The mixture of 2.0 ml of THA and 0.1 ml of neutral salt

solutions was used as a control. OH• was generated via the Fenton reaction

initiated by adding the CuSO4 and H2O2 solutions (final concentrations

10 lM and 1 mM, respectively) to the dosimeter solution. The reaction prod-

uct of THA with OH• (THA-OH•) was measured using a Perkin-Elmer LS50B

Luminescence spectrometer (Perkin-Elmer Life Sciences, Inc., Boston, MA,

USA) with excitation at 312 nm and emission at 426 nm. The radical scav-

enging activity of the investigated compounds was expressed as a percent

inhibition of the THA-OH• peaks. The experimental data were analysed using

the one-site binding model (GraphPad Prism, version 3.0; GraphPad Software

Inc., San Diego, CA, USA).

DPPH free radical scavenging assay

The DPPH (2.2-dipenyl-1-picrylhydrazyl) free radical (DPPH•) scavenging

activity of 17b-oestradiol and phytoestrogens was measured according to

the method of Blois (44), as modified by us. In brief, 0.2 ml of the agent

ethanol solution was mixed in a test tube with 0.2 ml of 100 lM DPPH

dissolved in ethanol. The reaction mixture was maintained at room tem-

perature for 100 min. The absorbance of the mixture was recorded at

517 nM by an ultraviolet-visible spectrophotometer Jenway 6405 (Barlo-

world Scientific Ltd, T ⁄ As Jenway, Dunmow, UK). The DPPH• scavenging

activity of 17b-oestradiol and phytoestrogens was calculated by the fol-

lowing equation:

Scavenging ability ð%Þ ¼ ðAo � AcÞ=A0 � 100

where Ao is the absorbance of the DPPH solution (without tested agent),

Ac is the absorbance of the mixture containing both DPPH and the tested

agent.

Preparation of 17b-oestradiol and phytoestrogens solutions

1 mM stock solutions of 17b-oestradiol and phytoestrogens were prepared

in 1% ethanol ⁄ water followed by dilution of stock solutions in the respec-

tive reaction cocktails (for [35S]-GTPcS-binding and adenylate cyclase activity

assays) to get the respective final concentrations 1–30 lM.

Statistical analysis

Each experiment on the compound’s effect on [35S]-GTPcS binding and

adenylate cyclase activity was made independently with the duplicate sam-

ples. The mean � SEM for each statistical group was calculated. The n used

in the statistical analysis was the number of individual brains in the control

or AD group. An unpaired Student’s t-test or one-way ANOVA was used to

identify significant differences (P < 0.05) between the groups. The experi-

ments on the free radical scavenging activities of the compounds were

repeated three to five times and the means � SEM was calculated for each

concentration. The statistical analysis of the experimental data sets was

made using GraphPad Prism software.

17b-oestradiol stimulation of G-proteins in human brain 589


Results

Effect of 17b-oestradiol, genistein and daidzein on[35S]-GTPcS binding in the hippocampal and frontocorticalmembranes

In the membranes from the hippocampus and frontal cortex of

control and AD females, 17b-oestradiol, genistein and daidzein

showed a capability of stimulation the G-proteins (Figs 2 and 3). In

the control hippocampus, 5–10 lM concentrations of the agents

significantly (P < 0.05) stimulated [35S]-GTPcS binding with the

respective maximum effects of 28%, 20% and 15% over the basal

level (421 � 25 fmol ⁄ mg protein). In the AD hippocampus, 10 lM

17b-oestradiol induced significantly (P < 0.05) weaker stimulation

of the radioligand basal binding (374 � 29 fmol ⁄ mg) than that of

the control, whereas the effect of phytoestrogens revealed no

remarkable decline (Fig. 2). In control frontal cortex, the stimulatory

effect of 17b-oestradiol on [35S]-GTPcS basal binding (391 � 21

fmol ⁄ mg) did not differ significantly from that in the control hip-

pocampus. In the AD frontal cortex, 8 and 10 lM 17b-oestradiol

produced significantly (P < 0.05) less stimulation of basal binding

(358 � 26 fmol ⁄ mg) than in the control region, whereas the effect

of phytoestrogens did not show a significant decrease (Fig. 3). In

both control and AD frontal cortex, the females showed a signifi-

100

105

110

115

120

125

130

135

† †

†

*

†

†

†

† †

†

‡

‡

‡

‡

Co

Genistein Daidzein

Alzheimer's disease

1 3 5 8 10 3 1 5 8 10 1 3 5 8 10 µM

Fig. 2. Effect of 1–10 lM 17b-oestradiol, genistein and daidzein on the [35S]-GTPcS binding in the female age-matched control (Co) and Alzheimer’s disease

(AD) hippocampal membranes (protein 0.05 mg ⁄ ml). 100% corresponds to the basal [35S]-GTPcS binding (means � SEM). In both Co and AD, four to five inde-

pendent experiments with the individual brains were conducted; *P < 0.05 for Co hippocampus versus AD hippocampus; comparison of the stimulated [35S]-

GTPcS binding with the basal level: P < 0.05 in Co (�) and AD (�) hippocampus.

100

105

110

115

120

125

130

†

†

*

*

†

†

††

†

‡‡

‡‡

Co

Genistein Daidzein

Alzheimer's disease

1 3 5 8 10 1 3 5 8 10 1 3 5 8 10 µM

Fig. 3. Effect of 1–10 lM 17b-oestradiol, genistein and daidzein on the [35S]-GTPcS binding in the female control (Co) and Alzheimer’s disease (AD) frontocor-

tical membranes. 100% corresponds to the basal [35S]-GTPcS binding (means � SEM.). In both Co and AD, four to five independent experiments with the indi-

vidual brains were conducted; *P < 0.05 for Co frontal cortex versus AD frontal cortex; comparison of the stimulated [35S]-GTPcS binding with the basal level:

P < 0.05 in Co (�) and AD (�) frontal cortex.



cantly (P < 0.05) higher 17b-oestradiol stimulation of G-proteins

than male subjects, whereas the effect of phytoesterogens showed

no gender difference (Fig. 4).

Protective effect of 17b-oestradiol, genistein and daidzeinon the oxymodified G-proteins in the frontocorticalmembranes

Figure 5(A, B) demonstrates suppressing (antioxidative) effects of the

investigated compounds in 10 lM concentration on the stimulation

of G-proteins by the metabolic pro-oxidants H2O2 and Hcy in con-

trol and AD frontal cortex membranes. In the control region, each

of the compounds significantly (P < 0.05) decreased the 25% stim-

ulation of [35S]-GTPcS binding induced by H2O2 and Hcy. 17b-Oes-

tradiol revealed a notably greater antioxidant effect on the H2O2

and Hcy stimulated activity of G-proteins than that of phytoestro-

gens. In AD frontal cortex, the antioxidant effect of 17b-oestradiol

and genistein towards the H2O2 and Hcy stimulation was less pro-

nounced than in the control region, whereas daidzein revealed no

significant protection against the pro-oxidants.

Effect of 17b-oestradiol on the adenylate cyclase activityin the frontocortical membranes

To understand the downstream signalling events induced by oestro-

gens, we investigated the effect of 1–30 lM of 17b-oestradiol on

the adenylate cyclase activity in the membranes of aged control

and AD frontal cortex (Fig. 6). In the control membranes, 17b-oes-

tradiol produced dose-dependent stimulation of adenylate cyclase,

reaching a maximum (plateau) value of 20% at the 20–30 lM con-

centration. In AD, the maximal stimulation of the enzyme was

remarkably lower than in control. Interestingly, the basal adenylate

cyclase activity in AD frontal cortex revealed a significantly

(P < 0.05) lower value (34 � 3 pmol cyclic AMP ⁄ min ⁄ mg protein)

90

100

110

120

130 Gen †*

† ††

†

‡

‡‡

‡

Daidz

Co

[35S]

GTP

S b

ound

, % o

f ba

sal

90

110

120

130

100

Gen

*Daidz

AD

Fig. 4. Effect of 10 lM 17b-oestradiol (17bE), genistein (Gen) and daidzein (Daidz) on the [35S]-GTPcS binding in the female ($) and male (#) frontocortical

membranes from control (Co) and Alzheimer’s disease (AD) brain. 100% corresponds to the basal [35S]-GTPcS binding (means � SEM). In each group, four

independent experiments with the individual brains were conducted; *P < 0.05 for female versus male values; comparison of the stimulated [35S]-GTPcS bind-

ing with the basal level: P < 0.05 in Co (�) and in AD (�).

100

105

110

115

120

125

130

H2O2 + Gen + Daidz 100

105

110

115

120

125

130

+ Daidz + Gen Hcy

[35S]

GTP

S b

indi

ng, %

of

basa

l

[35S]

GTP

S b

indi

ng, %

of

basa

l

H2O2 Co

Hcy Co

Hcy AD

*

*

*

**

**

**

*

H2O2 AD (A) (B)

Fig. 5. Effect of 10 lM 17b-oestradiol (17bE), genistein (Gen) and daidzein (Daidz) on the [35S]-GTPcS binding to control (Co) and Alzheimer’s disease (AD)

frontocortical membranes after the 5-min pretreatment with 10 lM H2O2 at 30 �C (A) or 500 lM homocysteine (Hcy) at 26 �C (B). 100% = basal [35S]-GTPcS

binding (means � SEM). In both, Co and AD, four independent experiments with the individual brains were conducted; *P < 0.05 between the effects of H2O2

or Hcy in the absence and presence of 17bE, Gen or Daidz.



than in control (46 � 4). The fact that the nonhydrolyzable G-pro-

tein blocker GDPbS significantly reduced the stimulatory effect of

20 lM 17b-oestradiol on the adenylate cyclase in the control mem-

branes (Fig. 7) indicates that G-proteins are directly implicated in

the stimulation. Unlike 17b-oestradiol, the phytoestrogen genistein

revealed no statistically significant effect on the adenylate cyclase

activity, either in control or AD brain membranes (data not shown).

Effect of 17b-oestradiol, genistein and daidzein on[35S]-GTPcS binding in the membranes of Sf9 cellsexpressing different types of G-proteins

To elucidate the type(s) of G-proteins that might be influenced by

17b-oestradiol and phytoestrogens, the effect of 10 lM agents was

studied in the plasma membranes of Sf9 cells expressing Gas, Gao,

Ga11 or Gai1 together with Gb1c2 (Table 1). In the membranes of

Gasb1c2 expressing cells, 17b-oestradiol increased the basal [35S]-

GTPcS binding by 18% whereas genistein and daidzein slightly

decreased the binding (by 13% and 15%, respectively). In the mem-

branes of Gaob1c2 cells, 17b-oestradiol stimulated the radioligand

binding by 20% whereas the effect of phytoestrogens was negligi-

ble. In the membranes of Ga11b1c2 cells, only phytoestrogens could

significantly increase the radioligand binding. Interestingly, in the

membranes of Gaib1c2i cells, the investigated compounds did not

exert a significant effect on [35S]-GTPcS binding.

OH• and DPPH• scavenging activity of 17b-oestradiol,genistein and daidzein

The investigated compounds concentration-dependently scavenged

the OH• and DPPH• generated in the respective cell-free systems

in vitro. The maximum OH• scavenging activity (Fig. 8) of daidzein

and genistein (84 � 5% and 54 � 3%, respectively) was higher

than that of 17b-oestradiol (13 � 3%). Similarly, the maximum

DPPH• scavenging activity (Fig. 9) of genistein and daidzein

(12 � 0.9 and 8.3 � 0.8%, respectively) was higher than the activ-

ity of 17b-oestradiol (6.1 � 0.5%).

Discussion

The neuroprotective activities of oestrogens and their plant derived

analogs appear to be controlled by a wide range of mechanisms,

most being related to their antioxidant properties (3, 45). Various

models were used to specify the neuroprotective mechanism for

the nano- to micromolar concentrations of oestrogens (16, 32, 46).

However, this study is the first to elucidate whether micromolar

concentrations of 17b-oestradiol and dietary phytoestrogens (geni-

stein, daidzein) affect the G-proteins and adenylate cyclase in the

aged and AD human brain membranes. In parallel, the antioxidant

properties of micromolar concentrations of the agents were tested

in different systems.

We found a stimulatory effect of 5–10 lM 17b-oestradiol and

phytoestrogens on [35S]-GTPcS binding to the post-mortem hip-

pocampal and frontocortical membranes of aged women, which

suggests that G-proteins are involved in the transduction of oes-

trogen signals. Although the effect was not impressive, it might

prove to be an important mechanism for the amplifying neuro-

protective cascade of oestrogens in the living brain. In fact,

10 lM 17b-oestradiol stimulated the Gs- and Go-proteins and

the 10 lM phytoestrogens stimulated the G11-proteins

expressed in Sf9 cells (Table 1). In addition, 17b-oestradiol acti-

vated the Gs- and Gq ⁄ 11-proteins via the membrane ER,

expressed in the recombinant cells (47, 48). These data, together

with the fact that hippocampus and frontal cortex of aged and

AD human brain express Gs-, Go- and Gq ⁄ 11-proteins (49–51),

suggest that 17b-oestradiol may stimulate the three types of

G-proteins, and the pytoestrogens stimulate the G11-proteins

in these regions.

100

105

110

115

120

125

1 5 10 20 30

Co

AD

µM

Ade

nyla

te c

ycla

se a

ctiv

ity,

% o

f ba

sal

**

*

*

Fig. 6. Effect of 1–30 lM 17b-oestradiol on the adenylate cyclase activity in

the female control (Co) and Alzheimer’s disease (AD) frontocortical

membranes. 100% corresponds to the basal adenylate cyclase activity

(means � SEM). Six and five independent experiments with the individual

brains were conducted in Co and AD, repectively; *P < 0.05 for Co versus AD.

Fig. 7. Effect of 20 lM 17b-oestradiol (17bE) on the adenylate cyclase

activity in the female control frontocortical membranes treated with 100 lM

of GDPbS. 100% corresponds to the basal adenylate cyclase activity

(means � SEM). Four independent experiments with the individual brains

were conducted; *P < 0.05 for 17bE versus GDPbS + 17bE.



The existing literature proposes at least two mechanisms for the

G-protein mediated signalling by oestrogens. The first one involves

the classical ER, which transduce signals via associating with G-

proteins, caveolins and other membrane components (52). The sec-

ond mechanism is related to the membrane-associated oestrogen

binding proteins with the characteristics of G-protein-coupled

receptors (e.g. GPR30) that can rapidly activate the oestrogen sig-

nalling pathways (9, 53). Yet, the present study suggests a third

antioxidant mechanism for the modulation of G-proteins by oestro-

gens. The plausibility of this suggestion is supported by the data of

previous (54–56) as well as the present study (Figs 8 and 9) show-

ing that micromolar concentrations of 17b-oestradiol, genistein and

daidzein scavenge the OH• and DPPH• in cell-free systems; the OH•

scavenging might serve as a G-protein stimulating mechanism of

oestrogens in the aged and AD brain regions where the overaccu-

mulation of iron and copper (57, 58) accelerates the formation of

OH• via Fenton reaction. Second, in the frontocortical membranes

(Fig. 5), the 10 lM 17b-oestradiol and phytoestrogens counteracted

the stimulation of G-proteins by metabolic oxidants (H2O2, Hcy).

Finally, micro- to millimolar concentrations of classical antioxidants

(e.g. melatonin, glutathione, N-acetyl-cysteine, etc.) have been

shown to slightly stimulate G-proteins in the same human brain

membrane fraction that was used in the present study (59–61).

In the brain regions of aged females, 17b-oestradiol produced

stronger stimulation of G-proteins than phytoestrogens did in the

same concentrations. In the cell lines expressing the human ERa or

ERb, the oestrogenic potency of 17b-oestradiol was also stronger

than that of phytoestrogens (e.g. genistein and daidzein) (29). The

difference might be due to the noticeably stronger molecular rigid-

ity and hydrophobic properties of steroid 17b-oestradiol, compared

to polyphenolic isoflavones (Fig. 1), that can provide higher affinity

of 17b-oestradiol toward ER in the subcellular structure (62). In the

plasma membranes, such a structure ⁄ affinity relationship may

define the interaction of these agents with the oestrogen binding

proteins and transduction of the respective signals to G-proteins. In

addition, the structure ⁄ antioxidant activity relationship has been

demonstrated for oestrogens and polyphenolic compounds, which

reveals differences between the model systems (18, 32, 45, 63).

Correspondingly, the 10 lM 17b-oestradiol displayed a more potent

antioxidant effect on the oxymodified (with H2O2 and Hcy) brain

membrane G-proteins than the 10 lM phytoestrogens did (Fig. 5),

which coincides with a stronger stimulatory effect of 17b-oestra-

diol on G-proteins, compared to phytoestrogens (Figs 2 and 3). The

difference could be interpreted by greater lipophilicity of oestrogens

(64), which enables their better incorporation into the bilayer lipid

membrane. At the same time, the ability of 17b-oestradiol to scav-

enge DPPH• and OH• in the cell-free systems was lower than that

Table 1. Effects of 10 lM 17b-Oestradiol, Genistein and Daidzein on [35S]-GTPcS binding to the plasma membranes of Sf9 cells expressing Gas, Gao, Gai1 or

Ga11 together with Gb1c2 (% of basal; 100% corresponds to the respective Bmax value of basal, i.e. unaffected [35S]-GTPcS binding).

(Phyto)estrogen added Gs Go Gi1 G11

Basal binding (Bmax),

no (phyto)estrogen added

100 � 4%

(305 � 11 fmol ⁄ mg)

100 � 8%

(170 � 13 fmol ⁄ mg)

100 � 11%

(92 � 10 fmol ⁄ mg)

100 � 5%

(56 � 3 fmol ⁄ mg)

17b-oestradiol 118.4 � 4.5* 120.1 � 4.1* 109.3 � 4.9 104.4 � 3.8

Genistein 87.1 � 1.3*� 107.1 � 2.4 114.8 � 8.6 119.0 � 2.6*�

Daidzein 84.9 � 1.6*� 107.7 � 2.1 93.7 � 4.8 120.2 � 6.6*�

Data are mean � SEM; n = 3. *P < 0.05 as compared to the basal value; �P < 0.05 17b-oestradiol versus genistein or daidzein.

0 10 20 30 40 50 0

25

50

75

100

Daidzein

Genistein

17bE

Concentration, µM

OH

• sc

aven

ging

act

ivity

(%)

Fig. 8. The hydroxyl radical (OH•) scavenging activity of 1–50 lM 17b-oes-

tradiol (17bE), genistein and daidzein determined as the percent inhibition

of OH• formation in the Fenton reaction (for experimental conditions,

see text). Data are expressed as means � SEM of three to five different

experiments.

Fig. 9. The 2,2-diphenyl-1-picrylhydrazyl radical (DPPH•) scavenging activity

of 5–50 lM 17b-oestradiol (17bE), genistein and daidzein (for experimental

conditions, see text). Data are expressed as means � SEM of three to five

different experiments.



of phytoestrogens (Figs 8 and 9). Such a discrepancy in the ranking

of antioxidant potency of the compounds might be due to the

model system-induced changes in the polarity and phenoxyl radical

stability of 17b-oestradiol and phytoestrogens, as previously dem-

onstrated for phenol and catechol oestrogens (65).

In the brain regions of AD females, the 10 lM 17b-oestradiol

stimulated G-proteins less than it did in the control regions. Simi-

larly, the decrease in the stimulation of G-proteins by glutathione,

melatonin and other antioxidants was shown in AD-injured brain

regions (59–61). These data, together with reports on the excessive

oxidation of proteins in the AD brain (66–68), suggest that one rea-

son for the decrease in 17b-oestradiol stimulation of G-proteins

might be oxymodification of their redox active functional groups.

Next, the decrease in the level of Go- and Gq11 proteins (49, 51)

and functional impairment of Gs-proteins (69) in the AD brain

might reduce the stimulatory response of G-proteins to 17b-oestra-

diol. Finally, local changes in membrane ERa splicing in the AD

brain (70) may diminish the ERa-mediated oestrogen stimulation of

G-proteins.

The gender difference in the 10 lM 17b-oestradiol action on

G-proteins in both, aged and AD frontocortical membranes (Fig. 4),

might be explained by lower production of oestrogens and expres-

sion of oestrogen binding proteins as well as of the respective

co-activators in the male brain membranes compared to those

in the female (71, 72). In addition, the characteristic effects of

oestrogens on the isolated plasma membranes and matrix vesicles

were shown to depend on the gender-specific composition of

membranes and metabolism of their components (particularly of

lipids) (73, 74), which provides another explanation for the gender-

specific 17b-oestradiol stimulation of G-proteins. The fact that

phytoestrogens have lower lipophilicity and incorporate into the

membrane bilayer less potently than classical oestrogens (64)

might help to explain why 10 lM genistein and daidzein, unlike

17b-oestradiol, revealed no gender-specific stimulation of mem-

brane-associated G-proteins.

Several studies have shown the stimulatory effect of nanomolar

concentrations of oestrogens on adenylate cyclase in the non-neuro-

nal cell lines (8, 9, 53). The effect was mediated by the transmem-

brane oestrogen receptor GPR30 and the coupled Gs-protein.

Supportively, 17b-oestradiol significantly potentiated [35S]-GTPcS

binding to the Gs-enriched Sf9 cell membranes (Table 1) and the

G-protein blocker GDPbS remarkably reduced 17b-oestradiol-induced

stimulation of adenylate cyclase in the frontocortical membranes of

aged females (Fig. 7). However, only micromolar concentrations of

17b-oestradiol could stimulate adenylate cyclase in the frontocortical

membranes (Fig. 6). Also, micro- to millimolar concentrations of clas-

sical antioxidants stimulate adenylate cyclase in the brain membranes

(61, 75). These data suggest that oestrogen stimulation of adenylate

cyclase in the brain membranes is mediated at least by two mecha-

nisms: at the nanomolar level, oestrogens stimulate the enzyme via

membrane receptor Gs-protein pathway and, at the micromolar level,

oestrogens may behave as antioxidants that modify the redox active

groups of the enzyme. Based on the recent hypotheses (1–3), both

of these mechanisms might be involved in the downstream neuro-

protective signalling of oestrogens.

In the frontal cortex of AD females, 17b-oestradiol stimulation

of adenylate cyclase was markedly lower than in control group

(Fig. 6). Such a decline in the effect could be explained by reduction

of adenylate cyclase activity and antioxidant sensitivity in the AD

brain (50, 61, 76). In addition, the weakened interaction between

Gs-proteins and adenylate cyclase in AD-injured brain areas (49, 77)

might serve as a causative factor that reduces the adenylate

cyclase stimulatory response to 17b-oestradiol.

The present study is the first to demonstrate that micromolar

concentrations of 17b-oestradiol and dietary phytoestrogens (geni-

stein, daidzein) stimulate G-proteins in the membranes of aged

and AD human brain and that 17b-oestradiol and phytoestrogens

have similarities and differences in this respect. We suggest that,

besides the ER-dependent one, the ER-independent antioxidant

mechanism is responsible for the stimulation of brain membrane

G-proteins by higher concentrations of oestrogens. Both of these

mechanisms could be involved in the G-protein mediated neuro-

protective signalling of oestrogens that contributes to their

preventive ⁄ therapeutic action against postmenopausal neurological

disorders.

Acknowledgements

We sincerely thank Dr Tatsuya Haga (University of Tokyo, Japan) for the

baculovirus tranfer vectors carrying the genes of G-proteins and Dr Kulliki

Saar for her advice and support. Likewise, our thanks are due to Dr Gerald

Finking (Institute of Occupational, Social and Environmental Medicine,

University of Ulm, Germany) for helpful discussions and to Mrs Inga

Volkman (Huddinge Brain Bank) for her excellent technical support. The

project was supported by The Estonian Scientific Foundation (Grants No

5222 and 6574) and the Swedish Foundation of Dementia.

Received: 22 August 2007,

revised 9 January 2008,

accepted 1 February 2008

References

1 Manthey D, Behl C. From structural biochemistry to expression profiling:

neuroprotective activities of estrogen. Neuroscience 2006; 138: 845–

850.

2 Amantea D, Russo R, Bagetta C, Corasaniti MT. From clinical evidence to

molecular mechanisms underlying neuroprotection afforded by estro-

gens. Pharmacol Res 2005; 52: 119–132.

3 Behl C. Oestrogen as a neuroprotective hormone. Nat Rev Neurosci

2002; 3: 433–442.

4 Miller NR, Jover T, Cohen HW, Zukin S, Etgen M. Etrogen can act via

estrogen receptor a and b to protect hipppocampal neurons against glo-

bal ischemia-induced cell death. Endocrinology 2005; 146: 3070–3079.

5 Levin ER. Cellular functions of plasma membrane estrogen receptors.

Steroids 2002; 67: 471–475.

6 Cato AC, Nesti A, Mink S. Rapid actions of steroid receptors in cellular

signalling pathways. Sci STKE 2002; 138: RE9.

7 Rosner W, Hryb DJ, Khan MS, Nakhla AM, Romas NA. Androgen and

estrogen signaling at the cell membrane via G-proteins and cyclic aden-

osine monophospohate. Steroids 1999; 64: 100–106.

8 Filardo EJ, Quinn JA, Frackelton AR Jr, Bland KI. Estrogen action via the

G protein coupled receptor, GPR30: stimulation of adenylyl cyclase and



cAMP-mediated attenuation of the epidermal growth factor receptor-to-

MAPK signaling axis. Mol Endocrinol 2002; 16: 70–84.

9 Thomas P, Pang Y, Filardo J, Dong J. Identity of an estrogen membrane

receptor coupled to a G-protein in human breast cancer cells. Endocri-

nology 2005; 146: 624–632.

10 Sylvia VL, Boyan BD, Dean DD, Schwartz Z. The membrane effects of

17b-estradiol on chondrocyte phenotypic expression are mediated by

activation of protein kinase C through phospholipase C and G-proteins.

J Steroid Biochem Mol Biol 2000; 73: 211–224.

11 Toran-Allerand CD, Singh M, Setalo G Jr. Novel mechanisms of estrogen

action in the brain: new players in an old story. Front Neuroendocrinol

1999; 20: 97–121.

12 Whiting KP, Restall CJ, Brain PF. Steroid-hormone induced effects on

membrane fluidity and their potential role in non-genomic mechanisms.

Life Sci 2000; 67: 743–757.

13 Kelly MJ, Qiu J, Rønnekleiv OK. Estrogen modulation of G-protein-cou-

pled receptor activation of potassium channels in the central nervous

system. Ann NY Acad Sci 2003; 1007: 6–16.

14 Zhao L, Chen S, MingWang J, Brinton RD. 17b-estradiol induces Ca2+

influx, dendritic and nuclear Ca2+ rise and subsequent cyclic AMP

response element-binding protein activation in hippocampal neurons: a

potential initiation mechanism for estrogen neurotrophism. Neuroscience

2005; 132: 299–311.

15 Suzuki S, Brown CM, Wise PM. Mechanisms of neuroprotection by estro-

gen. Endocr 2006; 29: 209–215.

16 Behl C, Skutella T, Lezoualc’h F, Post A, Widman M, Newton CJ, Holsboer

F. Neuroprotection against oxidative stress by estrogens: structure-activ-

ity relationship. Mol Pharmacol 1997; 51: 535–541.

17 Green PS, Gordon K, Simpkins JW. Phenolic A ring requirement for neu-

roprotective effects of steroids. J Steroid Biochem Mol Biol 1997; 63:

229–235.

18 Prokai L, Simpkins JW. Structure-nongenomic neuroprotection relation-

ship of estrogens and estrogen-derived compounds. Pharmacol Ther

2007; 114: 1–12.

19 Bicknell RJ. Sex-steroid actions on neurotransmission. Curr Opin Neurol

1998; 11: 667–671.

20 Boulware MI, Mermelstein PG. The influence of estradiol on nervous sys-

tem function. Drug News Perspect 2005; 18: 631–637.

21 Keller JN, Germeyer A, Begley JB, Mattson MP. 17b-estradiol attenuates

oxidative impairment of synaptic Na+ ⁄ K+-ATPase activity, glucose trans-

port, and glutamate transport induced by amyloid b-peptide and iron.

J Neurosci Res 1997; 50: 522–530.

22 Sawada H, Ibi M, Kihara T, Urushitani M, Akaike A, Shimohama S. Estra-

diol protects mesencephalic dopaminergic neurons from oxidative stress-

induced neuronal death. J Neurosci Res 1998; 54: 707–719.

23 Sribnick EA, Ray SK, Nowak MW, Li L, Banik NL. 17b-estradiol

attenuates glutamate-induced apoptosis and preserves electrophysiolog-

ic function in primary cortical neurons. J Neurosci Res 2004; 76: 688–

696.

24 Simpkins JW, Rajakumar G, Zhang YQ, Simpkins CE, Greenwald D, Yu CJ,

Bodor N, Day AL. Estrogens may reduce mortality and ischemic damage

caused by middle cerebral artery occlusion in the female rat. J Neuro-

surg 1997; 87: 724–730.

25 Petanceska SS, Nagy V, Frail D, Gandy S. Ovariectomy and 17b-estradiol

modulate the levels of Alzheimer’s amyloid b peptides in brain. Exp Ger-

ontol 2000; 35: 1317–1325.

26 Dhandapani KM, Brann DW. Protective effects of estrogen and selective

estrogen receptor modulators in the brain. Biol Reprod 2002; 67: 1379–

1385.

27 Simpkins JW, Yang SH, Wen Y, Singh M. Estrogens, progestins, meno-

pause and neurodegeneration: basic and clinical studies. Cell Mol Life

Sci 2005; 62: 271–280.

28 Gooren LJ, Toorians AW. Significance of oestrogens in male (patho)phys-

iology. Ann Endocrinol 2003; 64: 126–135.

29 Kuiper GG, Lemmen JG, Carlsson B, Corton JC, Safe SH, van der Saag PT,

van der Burg B, Gustafsson J-A. Interaction of estrogenic chemicals and

phytoestrogens with estrogen receptor. Endocrinology 1998; 139: 4252–

4263.

30 Duncan AM, Phipps WR, Kurzer MS. Phyto-estrogens. Best Pract Res Clin

Endocrinol Metab 2003; 17: 253–271.

31 Zhao L, Chen Q, Diaz Brinton R. Neuroprotective and neurotrophic effi-

cacy of phytoestrogens in cultured hippocampal neurons. Exp Biol Med

2002; 227: 509–519.

32 Mitchell JH, Gardner PT, McPhail DB, Morrice PC, Collins AR, Duthie GG.

Antioxidant efficacy of phytoestrogens in chemical and biological model

systems. Arch Biochem Biophys 1998; 360: 142–146.

33 Ramassamy C. Emerging role of ployphenolic compounds in the treat-

ment of neurodegenerative diseases: a review of their intracellular tar-

gets. Eur J Pharmacol 2006; 545: 51–64.

34 Simic G, Kostovic I, Winblad B, Bogdanovic N. Volume and number of

neurons of the human hippocampal formation in normal aging and Alz-

heimer’s disease. J Comp Neurol 1997; 379: 482–484.

35 Braak H, Braak E, Bohl J, Bratzke H. Evolution of Alzheimer’s disease

related cortical lesions. J Neural Transm Suppl 1998; 54: 97–106.

36 American Psychiatric Association . Diagnostic and Statistical Manual of

Mental Disorders, 4th edn. Washington, DC: American Psychiatric Associ-

ation, 1994.

37 Bogdanovic N, Morris JC. Diagnostic criteria for Alzheimer’s disease in

multicentre brain banking. In: Cruz-Sanchez FF, Ravid R, Cuziner ML,

eds. Neuropathological Diagnostic Criteria for Brain Banking, Biomedical

and Health Research Series, Vol. 10. Amsterdam: IOS Press, 1985: 20–29.

38 Mirra SS, Heyman A, McKeel D. The Consortium to establish a registry

for Alzheimer’s Disease (CERAD). Part II. Standardization of the neuropa-

thologic assessment of Alzheimer’s disease. Neurology 1991; 41: 479–

486.

39 Karelson E, Laasik J, Sillard R. Regulation of adenylate cyclase by galanin,

neuropeptide Y, secretin and vasoactive intestinal polypeptide in rat

frontal cortex, hippocampus and hypothalamus. Neuropeptides 1995;

28: 21–28.

40 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement

with the Folin phenol reagent. J Biol Chem 1951; 93: 265–275.

41 Rezaei K, Saar K, Soomets U, Valkna A, Nasman J, Zorko M, Akerman K,

Schroeder T, Bartfai T, Langel U. Role of third intracellular loop of gala-

nin receptor type 1 in signal transduction. Neuropeptides 2000; 34: 25–

31.

42 Brown BL, Ekins RP, Albano JDM. Saturation assay for cyclic AMP using

endogenous binding protein. Adv Cyclic Nucleotide Res 1972; 2: 25–40.

43 Barreto JC, Smith GS, Strobel NH, McQuillin PA, Miller TA. Terephthalic

acid: a dosimeter for the detection of hydroxyl radicals in vitro. Life Sci

1995; 56: PL89–PL96.

44 Blois MS. Antioxidant determinations by the use of stable free radical.

Nature 1958; 181: 1199–2000.

45 Lee YB, Lee HJ, Sohn HS. Soy isoflavones and cognitive function. J Nutr

Biochem 2005; 16: 641–649.

46 Rufer CE, Kulling SE. Antioxidant activity of isoflavones and their major

metabolites using different in vitro assays. J Agric Food Chem 2006; 54:

2926–2931.

47 Razandi M, Pedram A, Greene GL, Levin ER. Cell membrane and nuclear

estrogen receptors (ERs) originate from a single transcript: studies of

ERa and ERb expressed in Chinese hamster ovary cells. Mol Endocrinol

1999; 13: 307–319.

48 Razandi M, Pedram A, Park ST, Levin ER. Proximal events in signalling by

plasma membrane estrogen receptors. J Biol Chem 2003; 278: 2701–

2712.



49 O’Neill C, Wiehager B, Fowler CJ, Ravid R, Winblad B, Cowburn RF.

Regionally selective alterations in G-protein subunit levels in the Alzhei-

mer’s disease brain. Brain Res 1994; 636: 193–201.

50 Ross BM, McLaughlin M, Roberts M, Milligan G, McCulloch J, Knowler JT.

Alterations in the activity of adenylate cyclase and high affinity GTPase

in Alzheimer’s disease. Brain Res 1993; 622: 35–42.

51 Kelly JF, Storie K, Skamra C, Bienias J, Beck T, Bennett DA. Relation-

ship between Alzheimer’s disease clinical stage and Gq ⁄ 11 in sub-

cellular fractions of frontal cortex. J Neural Transm 2005; 112:

1049–1056.

52 Hewitt SC, Deroo BJ, Korach KS. A new mediator for an old hormone.

Science 2005; 307: 1572–1573.

53 Thomas P, Dong J. Binding and activation of the seven-transmembrane

estrogen receptor GPR30 by environmental estrogens: a potential novel

mechanism of endocrine disruption. J Steroid Biochem Mol Biol 2006;

102: 175–179.

54 Ayres S, Abplanalp W, Liu JH, Ravi Subbiah MT. Mechanisms involved in

the protective effect of estradiol-17b on lipid peroxidation and DNA

damage. Am J Physiol Endocrinol Metab 1998; 274: 1002–1008.

55 Wenly Y, Yaping Z, Bo S. The radical scavenging activities of radix

puerariae isoflavonoids: a chemiluminescence study. Food Chem 2004;

86: 525–529.

56 Lee CH, Yang L, Xu JZ, Yeung SYV, Huang Y, Chen ZY. Relative antioxi-

dant activity of soybean isoflavones and their glycosides. Food Chem

2005; 90: 735–741.

57 Castellani RJ, Honda K, Zhu X, Cash AD, Nunomura A, Perry G, Smith

MA. Contribution of redox active iron and copper to oxidative damage

in Alzheimer’s disease. Ageing Res Rev 2004; 3: 319–326.

58 Zhu X, Su B, Wang X, Smith MA, Perry G. Causes of oxidative stress in

Alzheimer’s disease. Cell Mol Life Sci 2007; 64: 2202–2210.

59 Karelson E, Fernaeus S, Reis K, Bogdanovic N, Land T. Stimulation of G-

proteins in human control and Alzheimer’s disease brain by FAD mutants

of APP714-723: implication of oxidative mechanisms. J Neurosci Res 2005;

79: 368–374.

60 Reis K, Zharkovsky A, Bogdanovic N, Karelson E, Land T. Critical

role of methionin-722 in the stimulation of human brain G-pro-

teins and neurotoxicity induced by London familial Alzheimer’s

disease (FAD) mutated V717G-APP714-723. Neuroscience 2007; 144:

571–578.

61 Karelson E, Mahlapuu R, Zilmer M, Soomets U, Bogdanovic N, Langel U.

Possible signalling by glutathione and its novel analogue through potent

stimulation of frontocortical G proteins in normal aging and in Alzhei-

mer’s disease. Ann NY Acad Sci 2002; 973: 537–540.

62 Turner JV, Agatanovic-Kustrin S, Glass BD. Molecular aspects of phytoes-

trogen selective binding at estrogen receptors. J Pharm Sci 2007; 96:

1879–1885.

63 Moosmann B, Behl C. The antioxidant neuroprotective effects of estro-

gens and phenolic compounds are independent from their estrogenic

properties. Proc Natl Acad Sci USA 1999; 96: 8867–8872.

64 Cunningham AR, Klopmnan G, Rosenkranz HS. A dichotomy in the lipo-

philicity of natural estrogens, xenoestrogens and phytoestrogens. Envi-

ron Health Perspect 1997; 105: 665–668.

65 Ruiz-Larrea MB, Martın C, Martınez R, Navarro R, Lacort M, Miller NJ.

Antioxidant activities of estrogens against aqueous and lipophilic radi-

cals: differences between phenol and catechol estrogens. Chem Phys

Lipids 2000; 105: 179–188.

66 Butterfield DA, Perluigi M, Sultana R. Oxidative stress in Alzheimer’s dis-

ease brain: new insights from redox proteomics. Eur J Pharmacol 2006;

545: 39–50.

67 Newman SF, Sultana R, Perluigi M, Coccia R, Cai J, Pierce WM, Klein JB,

Turner DM, Butterfield DA. An increase in S-glutathionylated proteins in

the Alzheimer’s disease inferior parietal lobule, a proteomics approach.

J Neurosci Res 2007; 85: 1506–1514.

68 Bogdanovic N, Zilmer M, Zilmer K, Rehema A, Karelson E. The Swedish

APP670 ⁄ 671 Alzheimer’s disease mutation: the first evidence for strik-

ingly increased oxidative injury in the temporal inferior cortex. Dement

Geriatr Cogn Disord 2001; 12: 364–370.

69 Hashimoto E, Ozawa H, Saito T, Gsell W, Takahata N, Riederer P, Frolich

L. Impairment of Gsa function in human brain cortex of Alzheimetr’s dis-

ease: comparison with normal aging. J Neural Transm 2004; 111: 311–

322.

70 Ishunina TA, Swaab DF. Age-dependent ERa MB1 splice variant expres-

sion in discrete areas of the human brain. Neurobiol Aging 2007 [Epub

ahead of print].

71 Tischkau SA, Ramirez VD. A specific membrane binding protein for pro-

gesterone in rat brain: sex differences and induction by estrogen. Proc

Natl Acad Sci USA 1993; 90: 1285–1289.

72 Balthazart J, Baillien M, Charlier TD, Cornil CA, Ball GF. Multiple mecha-

nisms control brain aromatase activity at the genomic and non-genomic

level. J Steroid Biochem Mol Biol 2003; 86: 367–379.

73 Schwartz Z, Gates PA, Nasatzky E, Sylvia VL, Mendez J, Dean DD, Boyan

BD. Effect of 17b-estradiol on chondrocyte membrane fluidity and phos-

pholipid metabolism is membrane-specific, sex-specific, and cell matura-

tion-dependent. Biochim Biophys Acta 1996; 1282: 1–10.

74 Schuessel K, Leutner S, Cairns NJ, Muller WE, Eckert A. Impact of gender

on upregulation of antioxidant defence mechanisms in Alzheimer’s dis-

ease brain. J Neural Transm 2004; 111: 1167–1182.

75 Soomets U, Mahlapuu R, Tehranian R, Jarvet J, Karelson E, Zilmer M,

Iverfeldt K, Zorko M, Graslund A, Langel U. Regulation of GTPase and

adenylate cyclase activity by amyloid b-peptide and its fragments in rat

brain tissue. Brain Res 1999; 850: 179–188.

76 Mahlapuu R, Viht K, Balaspiri L, Bogdanovic N, Saar K, Soomets U, Land

T, Zilmer M, Karelson E, Langel U. Amyloid precursor protein carboxy-

terminal fragments modulate G-proteins and adenylate cyclase activity

in Alzheimer’s disease brain. Brain Res Mol Brain Res 2003; 117: 73–82.

77 Cowburn RF, O’Neill C, Bonkale WL, Ohm TG, Fastbom J. Receptor-G-pro-

tein signalling in Alzheimer’s disease. Biochem Soc Symp 2001; 67:

163–175.



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