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Center for the Neurobiology of Aging (P.S.G., S.-H.Y., J.W.S.), Department of Pharmacodynamics (J.W.S.), and Department of Neurosurgery (S.-H.Y.), University of Florida, Gainesville, Florida 32610; and Department of Molecular Biology and Pharmacology (K.R.N., A.S.K., D.F.C.), Washington University, St. Louis, Missouri 63110
Address all correspondence and requests for reprints to: Pattie S. Green, 629 South Elm Street, Slot 807, Donald W. Reynolds Center on Aging, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205. E-mail: greenpatties{at}uams.edu
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The role of estrogen receptor (ER)-dependent transcription in
estrogen’s neuroprotective activity is controversial (for review, see
7). The neuroprotective activity of
ßE2 in culture models is attenuated by the ER
antagonists tamoxifen or ICI 182,780 in some studies
(13, 14, 15); however, others report no effect of these same
ER antagonists on ßE2-mediated neuroprotection
(16, 17, 18, 19, 20). Our laboratory (16, 21, 22) and
others (17, 23) have reported equipotent (to
ßE2) neuroprotective efficacy of
17
-estradiol, which only weakly activates ER-dependent gene
transcription (24), implicating mechanisms other than
ER-mediated transcription in estrogen-mediated protection of neuronal
cultures. In mouse models of cerebral ischemia, the data are equally
inconclusive. Sampei et al. (25) report no
difference in total lesion size between wild-type and ER-deficient
mice. However, ICI 182,780 administration increases striatal lesion
volume in the wild-type mouse (26). ICI 182,780
administration did not alter neocortical lesion volume in this study.
It is important to note that, although protection of neocortical areas
are consistently reported with ßE2 treatment
(8, 9, 10), ßE2-mediated protection
of striatal infarct is not consistently reported (9).
The present study addresses the requirement for ER- dependent
transcription in the neuroprotective effects of estrogens, both
in vitro and in vivo, using a novel enantiomer
strategy. Ent-17ß-estradiol
(Ent-E2), the enantiomer of the
naturally occurring ßE2 (Fig. 1
), has identical physiochemical
properties as ßE2 except for interactions with
other stereospecific molecules such as ERs. Ent-E is
reported to interact only weakly with uterine-derived ERs (27, 28) and lacks estrogenic effects on reproductive tissues in
rodents (29, 30, 31). Some reports indicate that
Ent-E2 exerts slight antiuterotrophic
activity and can antagonize the uterotrophic effects of
ßE2 (32, 33). In contrast,
Ent-E2 has been reported to elicit
alterations in lipid profiles identical to ßE2,
with similar potency (34). Further,
Ent-E2 is not enzymatically converted
to ßE2 (35) and, therefore, is
more adequately suited than 17-estradiol for evaluating the
role of ERs in estrogen-mediated neuroprotection. This study evaluates
the neuroprotective effects of Ent-E2,
both in culture models of oxidative stress and in a rat transient focal
ischemia model and, further, determines whether
Ent-E2 can exert neuroprotective
effects in the absence of stimulation of peripheral estrogen-responsive
tissues.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,3a,9a,9bß)]-3-(1,1-dimethylethoxy)-1,2,3,3a,4,5,8,9,9a,9b-decahydro-3a-methyl-6-[2-(2-methyl-1,3-dioxalan-2-yl)ethyl-7H-benz[e]inden-7-one
(Chemical Abstracts Registry Number 139973–49-2), which was
prepared by a multistep synthetic pathway as described in the
literature (36). This compound was then converted in
either of two ways (method A or method B) to
Ent-19-nortestosterone (Chemical Abstracts Registry Number
4091–86-5). In the first step of method A, the double bond is reduced,
using lithium in liquid ammonia, and the resulting tricyclic compound
is cyclized to Ent-19-nortestosterone in the second step. In
the first step of method B, the double bond is reduced by catalytic
hydrogenation, and the resulting tricyclic compound is again cyclized
to Ent-19-nortestosterone in the second step. Method B has
been previously used to prepare 19-nortestosterone (37).
The hydroxy group of Ent-19-nortestosterone is then
esterified, and the A-ring of the steroid is aromatized using
CuBr2 in acetonitrile. This reaction has been
reported previously for the conversion of 19-nortestosterone,
17-acetate to 17ß-estradiol, 17-acetate (38). Finally,
the 17-acetate group is removed by saponification to give
Ent-E2 (Chemical Abstracts Registry
Number 3736–22-9). The structure of
Ent-E2 was proven by experimental
data, which showed that the compound had the same melting point
(176–177 C), infrared absorption spectra (3449, 3246, 2936, 2864,
1611, 1587, 1500, 1450, 1283, 1250, 1057, 1012, 930, 874
cm-1), 1H NMR [(300
MHz, CD3OD) 
7.06 (1 H, d, J =
8.7 Hz), 6.54–6.46 (2 H, m), 3.64 (1 H, t, J = 8.4
Hz), 0.75 (3 H)] and 13C NMR [(75 MHZ,
CD3OD) 156.07, 138.98, 132.80, 127.32,
116.18, 113.85, 82.57, 51.32, 45.34, 44.36, 40.50, 38.01, 30.67 (2
x C). 28.48, 27.56, 23.99, 11.62) spectra but opposite optical
rotation ([]D28 = -71.2 (c = 0.99, CH3OH))
as ßE2. Steroids were initially dissolved in ethanol at a 10 mM concentration and then diluted to the appropriate concentration in culture media or assay buffer for cell culture or ex vivo assays, respectively. Steroids were dissolved in corn oil at the concentration necessary to yield the indicated dose in 1 ml/kg injection volume for rodent studies.
Cell culture
SK-N-SH human neuroblastoma cells were obtained from
ATCC(Manassas, VA), and HT-22 cells (immortalized
hippocampal neurons of murine origin) were a generous gift of Dr. David
Schubert (The Salk Institute, San Diego, CA). Cells were maintained in
RMPI-1640 and DMEM media (Life Technologies, Inc.,
Gaithersburg, MD), respectively, supplemented with 10%
charcoal/dextran-stripped FBS (HyClone Laboratories, Inc.,
Logan, UT) and 200 µg/ml gentamycin, according to standard culture
conditions.
Cells were plated, 24 h before initiation of experiment, at a density of 20,000 cells/well (SK-N-SH cells) or 5,000 cells/well (HT-22 cells), in both clear- and white-bottomed Nunc 96-well plates (Fisher Scientific, Orlando, FL). Steroids were added at concentrations ranging from 0.1 nM to 10 µM, either 2 or 24 h before exposure to either glutamate (5 mM) or H2O2 (3–60 µM). Ethanol was used at concentrations of 0.001–0.1% vol/vol as a vehicle control. These concentrations of ethanol had no discernible effect on cell viability. After 24 h of toxin exposure, cells were rinsed with PBS, pH 7.4, and viability was assessed by the addition of 1 µM calcein AM (Molecular Probes, Inc., Eugene, OR) and 1 µg/ml propidium iodide (Sigma, St. Louis, MO) in PBS for 15 min. Calcein AM fluorescence was determined at an excitation of 485 nm and an emission of 538 nm. Percent viability was calculated by normalization of all values to the toxin-free control group (= 100%). Percent protection was calculated as the difference between each experimental value and the average of the toxin-only group normalized to the difference between the toxin-free control and toxin-only groups (= 100% protection). Cells that had been lysed by addition of 1% SDS were used for blank readings. Staining was visualized using a fluorescent Nikon (Melville, NY) microscope, and cells were photographed for qualitative documentation.
Animals
Female Sprague Dawley rats (Harlan,
Indianapolis, IN) were housed in pairs in hanging, stainless steel
cages in a temperature-controlled room (25 ± 1 C) with a daily
light cycle (on from 0700 to 1900 h daily). All rats had free
access to laboratory chow and tap water. All procedures
performed on animals were reviewed and approved by the Institutional
Animal Care and Use Committee of the University of Florida before the
initiation of the study.
Ovariectomy
Female Sprague Dawley rats (220–225 g BW) were given 3–5 days
to acclimate, then were bilaterally ovariectomized using a dorsal
approach. Animals were anesthetized with methoxyflurane (Pitman Moore,
Inc., Crossing, NJ) inhalant anesthesia. A small (1 cm) cut was made
through the skin, facia, and muscle. The ovaries were externalized,
clipped, and removed; then the muscle, facia, and skin were sutured
closed. Ovariectomy was performed 2 weeks before experiments.
Middle cerebral artery (MCA) occlusion
Either oil vehicle or 100 µg/kg ßE2 or
Ent-E2 was administered by sc
injection, 2 h before the onset of MCA occlusion. Animals were
anesthetized by ip injection of ketamine (60 mg/kg) and xylazine (10
mg/kg). MCA occlusion was performed as previously described
(8). Briefly, the left common carotid artery, external
carotid artery, and internal carotid artery were exposed through a
midline cervical incision. A 3–0 monofilament suture was introduced
into the internal carotid artery lumen and gently advanced until
resistance was felt. The suture was kept in place for 60 min and then
withdrawn to allow MCA reperfusion. The procedure was performed within
20 min, with minimal bleeding. Rectal temperature was maintained
between 36.5 and 37.0 C during the entire procedure.
Animals were decapitated and the brain removed 24 h after onset of MCA occlusion. The brain was then dissected coronally into 2-mm sections using a metallic brain matrix (ASI Instruments, Inc., Warren, MI). The sections located 3, 5, 7, 9, and 11 mm posterior to the tip of the olfactory bulb were stained by incubation in a 2% solution of 2,3,5-triphenyltetrazolium chloride in a 0.9% saline solution at 37 C for 30 min. Slices were then fixed in 10% formalin and photographed, and the ischemic lesion area was determined for each slice using Image-Pro Plus software (Media Cybernetics, Silver Spring, MD). Percent ischemic lesion area was calculated as the sum of the ischemic lesion area for the five slices divided by the total cross-sectional area of these five slices.
Plasma levels of ßE2
Ovariectomized female Sprague Dawley rats were injected sc with
either oil vehicle or 100 µg/ml ßE2 or
Ent-E2. Blood samples were obtained by
cardiac puncture, 5 min before injection or 1 h, 4 h, or
24 h post injection. Plasma was stored at -20 C, until assayed
using the ultrasensitive ßE2 RIA kit from
Diagnostic Systems Laboratories, Inc. (Los Angeles, CA)
according to the manufacturer’s instructions.
Ent-E2 showed no cross-reactivity with
the RIA at concentrations up to 10 µM.
Uterotrophic assay
Juvenile (25 days old) female Sprague Dawley rats were injected
sc with oil, ßE2 (0.01–1 µg/rat), or
Ent-E2 (1–100 µg/rat) daily (at
0830 h) for 3 days. On the fourth day, the rats were killed using
methoxyflurane, and the uteri were excised. Extraneous tissue was
gently removed from the uteri before wet weight was determined. Vaginal
opening was assessed before uterine removal.
Ligand competition of ER binding
5 nM
[2,4,6,7-3H]-ßE2
(specific activity, 84.1 Ci/mmol; Amersham Pharmacia Biotech, Piscataway, NJ) and 400 pM recombinant
human ER or ERß (Affinity BioReagents, Inc., Golden,
CO) were incubated in ER binding buffer (20 mM Tris, 1
mM EDTA, 400 mM KCl, 1 mM
dithiothreitol, 10% glycerol, 0.1% BSA, pH 7.8) for 1 h at 25 C
either with no added steroid (total binding), 1.2
µM diethylstilbestrol (nonspecific binding), or 0.1
nM–10 µM ßE2 or
Ent-E2. Bound and unbound radioligand
were separated using Sephadex G-25 (Amersham Pharmacia Biotech) columns (1.5 ml bed volume) with a 1-ml elution volume.
Ten milliliters of scintillation fluid was added, and counts were
determined. This method resulted in greater than 90% receptor recovery
and less than 15% nonspecific binding.
Brain membrane oxidation
The brain was removed from ovariectomized female Sprague Dawley
rat, and the neocortex was homogenized in ice-cold Tris buffer
(100 mM, pH 7.4) with 1% Triton X-100 using a Teflon/glass
tissue homogenizer. The homogenate was centrifuged at 2,000 rpm for 10
min. The resulting supernatant was incubated with
ßE2 or Ent-E2
at concentrations ranging from 0.1–100 µM for
30 min at 37 C. FeSO4 was then added to a final
concentration of 200 µM and incubated for an
additional 30 min at 37 C. Butylated hydroxytoluene (100
µM) and diethylenetriaminepentaacetic
acid (100 µM) were then added. 2-thiobarbituric
acid reactive products (TBARs) were immediately determined by addition
of 0.5% 2-thiobarbituric acid, 3.125% trichloroacetic acid, and 0.2
N HCl; and incubation was performed, at 95 C for
1 h. Samples were centrifuged at 10,000 rpm for 10 min, and the
absorbance of the supernatant at 532 nm was determined.
Statistical analysis
All data are presented as mean ± SEM.
Comparison of ischemic lesion volume was performed using a one-way
ANOVA with a Kruskal-Wallis test for planned comparisons between
groups. For all other experiments, the significance of differences
among groups was determined by one-way ANOVA with a Tukey’s
multiple-comparisons test for planned comparisons between groups when a
significant difference was detected. For all tests, P
< 0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). As previously reported
(40), ßE2 treatment, commencing
2 h before glutamate exposure, conferred significant protection in
this model, with a 10,000 nM concentration protecting
35 ± 4% of the cells. Ent-E2,
performed similarly in this model of neuroprotection, with 100
nM and 10,000 nM
Ent-E2 protecting 16 ± 2% and
56 ± 4% of HT-22 cells, respectively.
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).
H2O2 exposure resulted in a
concentration-dependent toxicity in HT-22 cells with a
30-µM concentration resulting in 21 ± 5%
reduction in viability (Fig. 3) and a 60-µM
concentration resulting in a 97 ± 8% reduction (data not
shown);10 nM of either
ßE2 or Ent-E2
completely attenuated the toxicity of 30 µM
H2O2 and protected 48
± 14% or 63 ± 8% of the cells from 40
µM
H2O2 toxicity, respectively
(Fig. 3). No protection was seen with the 10 nM
concentration of either steroid at
H2O2 concentrations greater
than 40 µM (data not shown). SK-N-SH cells were
more sensitive than HT-22 cells to the toxic effects of
H2O2 exposure, with 3
µM
H2O2 reducing SK-N-SH cell
viability by 32 ± 2% (Fig. 4).
This kill was significantly attenuated by
Ent-E2, with a 1
nM concentration conferring 30 ± 9%
protection (Fig. 4). In other studies, 1 nM
ßE2 prevented 40 ± 5% of
H2O2-induced toxicity in
SK-N-SH cells (data not shown). Neither steroid exerted protective
effects in these low nM concentration ranges with
higher concentrations of
H2O2 (data not shown).
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). Comparable with our previous reports
(8), sc injection of ßE2, 2 h
before onset of ischemia, reduced total lesion area by 60 ± 13%.
Administration of Ent-E2 similarly
reduced total ischemic area by 60 ± 12%. This estrogen-mediated
protection was observed in both neocortical and
subcortical/allocortical regions of the ischemic infarct.
ßE2 and Ent-E2
reduced neocortical lesion size by 77 ± 11% and 59 ± 12%,
respectively. Similarly, subcortical/allocortical lesion volume was
reduced by 48 ± 8% and 47 ± 7% by
ßE2 and Ent-E2
administration, respectively. The subcortical/allocortical ischemic
area includes regions of the caudate/putamen, hypothalamus, and
hippocampus.
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). In
contrast, sc injection of ßE2 resulted in a
rapid rise in plasma ßE2 levels, with values of
5.16 ± 0.94 nM within 1 h, and
returned to near baseline (0.24 ± 0.08 nM)
by 24 h.
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). By contrast,
Ent-E2, at doses of 1–10 µg/rat,
had no effect on uterine wet weight. At a dose of 100 µg/rat (average
dose of 1400 µg/kg), Ent-E2 exerted
a slight antiuterotrophic effect, decreasing uterine wet weight by
23 ± 3%. Ent-E2 also slightly
antagonized the uterotrophic effects of 1 µg/rat
ßE2, with a 100 µg/rat dose reducing the
uterotrophic effect of ßE2 by 27 ± 8%.
These results are comparable with previous reports in immature mice,
where Ent-E2 (doses of about 1200
µg/kg) exerted antiuterotrophic effects (29) and
Ent-E2 antagonized the uterotrophic
effects of ßE2 when
Ent-E2 was present in a 100-fold
excess (32).
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).
Ent-E2 exerted mixed
agonist/antagonist effects on vaginal opening, with a 100-µg/rat dose
causing vaginal opening in 50% of the juvenile rats. This dose of
Ent-E2 prevented
ßE2-induced vaginal opening in 40% of the
rats. No change in body weight was observed with administration of
ßE2, Ent-E2,
or combinations thereof. BW of the juvenile rats averaged 72 ±
1 g.
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and ERß, respectively.
Ent-E2 has been previously reported to
have 0.9–6% of ßE2’s relative binding
affinity to cytosolic uterine ERs (27, 28).
Ent-E2 can attenuate brain lipid oxidation ex
vivo
Because estrogens have been previously reported to reduce
oxidative damage to brain lipids (41, 42, 43), we examined the
potency of both ßE2 and
Ent-E2 in an ex vivo assay
of brain membrane oxidation. Thirty-minute incubation of the
neocortical homogenate resulted in a 16-fold increase in TBAR
formation. ßE2 and
Ent-E2 were equipotent in the
attenuation of FeSO4-induced lipid oxidation, as determined
by TBAR formation (Fig. 8), with a
50-µM concentration of either steroid
significantly attenuating FeSO4-induced TBAR
formation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The neuroprotective effects of Ent-E2 are not likely caused by conversion to the more estrogenically potent ßE2, because the conversion requires isomerization of five individual chiral carbons. Isomerization of the 17-hydroxy group could be facilitated by 17ß-hydroxysteroid dehydrogenase; however, Ent-E2 is not a substrate for this enzyme (35). Further, there was no detectable increase in plasma ßE2 levels during 24 h after sc injection of Ent-E2 in female rats, indicating that Ent-E2 is itself neuroprotective.
The minimal neuroprotective concentration of both ßE2 and Ent-E2 varied with the cell culture model used. High physiological concentrations (low nM) were sufficient to attenuate H2O2-induced toxicity in SK-N-SH cells, but significantly higher supraphysiological concentrations (low µM) were required to lessen glutamate toxicity in HT-22 cells. This difference in the neuroprotective potency of estrogens between these models may be attributable to a number of factors, including differences in culture media and differences in toxicity. Further, the concentration of steroid required for protection may depend on the degree of insult, because low concentrations of Ent-E2 or ßE2 did not protect SK-N-SH or HT-22 cells from H2O2 exposure if viability was reduced by more than 70% (P. S. Green and J. W. Simpkins, unpublished observations).
The high doses of Ent-E2 used in some,
but not all, of the experiments in the present report could show
appreciable ER binding; however, this does not adequately explain the
equipotent neuroprotection conferred by the enantiomer. The 16- to
100-fold-lower affinity of Ent-E2 for
the known ERs (this report, and 27, 28) would be
apparent as a similar 16- to 100-fold-lower potency in effects
mediated by either ER or ERß. This potency difference was seen in
uterotrophic and vaginal opening responses but not in assays of
neuroprotection. Regardless of the minimum dose required for
neuroprotection in each model, Ent-E2
attenuated neuronal death with a potency equivalent to that of
ßE2. This result indicates that enantiospecific
interactions between estrogens and other cellular molecules are not
required for the neuroprotective actions of estrogens.
Several lines of evidence connote that the neuroprotective effects of
estrogens do not require ER-dependent gene transcription, including
potent neuroprotective efficacy of several nonfeminizing estrogens
including Ent-E2 (
Figs. 2–5
) and
17-estradiol (16, 21, 22, 23). Further, functional ERs have
not been found in either HT-22 cells (40, 44) or SK-N-SH
cells (22), although this study (
Figs. 2–4) and others
(16, 21, 22, 23, 40, 44) demonstrate estrogen-mediated
protection of these neuronal cell lines. Similarly,
ßE2-mediated protection can occur in the
presence of ER antagonists (16, 17, 18, 19, 20). Together, these
findings, though not excluding a role for ERs in neuroprotection,
implicate cellular mechanisms other than classical ER activity in the
neuroprotective effects of estrogens.
Antioxidant effects have been proposed as one mechanism for the neuroprotective effects of estrogens (19). Interestingly, the structure-activity relationship for the antioxidant effects of estrogens (42) is identical to the structure-activity relationship for the neuroprotective effects (22, 23). Further, it has been reported that the concentrations of ßE2 that are capable of exerting ex vivo antioxidant effects were required for neuroprotective effects (19). ßE2 attenuation of lipid peroxidation has been shown to require µM concentrations (19, 41, 42, 43). The neuroprotective concentration for ßE2 in culture models ranges from 0.1 nM (21, 45) to 50 µM (18). In this study, ex vivo antioxidant effects of ßE2 and Ent-E2 required a minimum concentration of 50 µM, whereas neuroprotective effects were seen at much lower concentrations.
Neuronal effects of estrogens with weak ER agonist activity are being
increasingly described. The classically inactive estrogen,
17-estradiol, has been shown to be neuroprotective in both culture
(16, 21, 22, 23) and MCA occlusion models (8).
Similarly, the weak ER agonist dihydroequilin has been shown to exert
neurotrophic effects in cultured neurons (46). The
cellular mechanisms for these effects of weak ER agonists is not known;
however, several cellular effects of 17-estradiol have been
described. Exposure to E2 can activate the MAP
kinase pathway (47), and this pathway is implicated in
ßE2-mediated neuroprotection (14).
In addition, E2 has also been shown to have
several other direct effects on neurons, including modulation of the
mitochondrial Na+/K+-ATPase activity
(48), alteration of membrane fluidity (49),
and inhibition of toxin-induced activation of NF
B (P. S. Green
and J. W. Simpkins, unpublished observations). It is unknown
whether Ent-E2 can also interact with
any of these cellular pathways.
A profusion of data indicates that estrogens enhance the survival of neurons both in vitro and in vivo, suggesting that estrogens may be useful in the treatment of neurodegenerative disease or acute neuronal death. Estrogens, such as Ent-E2, may offer the beneficial neuroprotective effects of estrogens without the complicating peripheral estrogenic actions and could be useful in both men and women for whom estrogen therapy is contraindicated.
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Acknowledgments |
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Footnotes |
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Received June 13, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-estradiol exerts neuroprotective effects in SK-N-SH cells. J
Neurosci 17:511–515-deficient mice. Stroke 31:738–744-fetoprotein for a series of estrogen
derivatives: studies using equilibrium and nonequilibrium binding
techniques. Endocrinology 105:743–753
4-3-oxosteroids. Steroids 59:621–627[CrossRef][Medline]
-Dihydroequilenin increases hippocampal dendritic
spine density of ovariectomized rats. Brain Res 758:241–244[CrossRef][Medline]
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