Mechanisms involved in the protective effect of
estradiol-17
on lipid peroxidation and DNA damage
Stacey
Ayres1,
William
Abplanalp2,
James H.
Liu3, and
M. T. Ravi
Subbiah2
1 Interdisciplinary Graduate
Studies Program and 2 Division of
Endocrinology and Metabolism, Department of Internal Medicine, and
3 Department of Obstetrics and
Gynecology, University of Cincinnati Medical Center, Cincinnati, Ohio
45267-0540
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ABSTRACT |
Previous studies from our laboratory have
shown that estrogens can protect against lipoprotein peroxidation and
DNA damage. In this study, the mechanism of estradiol-17
(E2) action was investigated by
comparing E2 with selective
scavengers of reactive oxygen species (ROS) in terms of inhibition of
1) human low-density lipoprotein
(LDL) peroxidation (measured by the diene conjugation method) and
2) DNA damage (measured by the
formation of strand breaks in supercoiled OX-174 RFI DNA). In addition,
the direct effect of E2 on the
generation of individual ROS was also measured. By use of ROS
scavengers, it was determined that lipoprotein peroxidation was
predominantly due to superoxide (39%), with some contributions from
hydrogen peroxide (23%) and peroxy (38%) radicals.
E2 was a more effective inhibitor
of peroxidation than all the ROS scavengers combined. In DNA damage,
scavengers of hydrogen peroxide, hydroxyl, and superoxide radical
offered significant protection (49-65%). E2 alone offered a similar degree
of protection, and no additional effect was evident when it was
combined with ROS scavengers. E2 caused a significant reduction (37%) in the production of superoxide radical by bovine heart endothelial cells in culture but had no effect
on the formation of either hydrogen peroxide or hydroxyl radicals.
These studies show that 1) the
protection offered by E2 in terms
of lipid peroxidation could be due to its ability to inhibit generation
of superoxide radical and prevent further chain propagation, and
2) in DNA damage protection,
E2 mainly appears to inhibit chain
propagation.
reactive oxygen species
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INTRODUCTION |
THERE IS CONSIDERABLE EVIDENCE that oxidative DNA
damage mediated by reactive oxygen radicals and lipid peroxidation may
play a role in carcinogenesis and atherogenesis (2). Reactive oxygen species, especially in the presence of transition metals, are potent
stimulators of lipid peroxidation and DNA damage (10). A variety of
antioxidant defenses, including enzymes, have evolved to protect
against reactive oxygen species, but these defenses are not completely
efficient or effective. As a result, there is an accumulation of
oxidative damage to DNA, lipids, and proteins during the aging process.
Although the chemical mechanisms are still not fully understood,
oxidized lipids and reactive oxygen species have been shown to damage
DNA (3, 27); thus attempts to prevent these damaging effects are
essential. Estrogens have been shown to be powerful antioxidants,
effectively preventing lipid peroxidation (4, 14, 23). In particular,
estrogen decreases oxidative modification of low-density lipoproteins
(LDL), both in vitro and in vivo (9, 10, 14, 19, 21, 23). It is
postulated that this antioxidant activity is considered to be one
mechanism by which estrogen confers cardioprotection (7).
The chemical structure of estrogen allows for donation of a
H+ atom to a peroxyl radical. This
property of estrogen allows free radical scavenging and may exert its
effect by interfering early or during the propagation phase of lipid
peroxidation. Recently, we presented evidence that estradiol-17
or
estrogen (E2) was as effective
as vitamin E in preventing LDL peroxidation and cholesterol oxidation
(4). We postulated that E2 may act
by regenerating or maintaining endogenous antioxidants in lipoproteins
and thus delaying the appearance of peroxidized products. Our studies
suggested that the protection or regeneration of antioxidant vitamin
content in the lipoprotein is not the mechanism of
E2 action, because E2 had no effect on the content of
antioxidants in lipoproteins, and no protective effect was observed
after reisolation of LDL and high-density lipoproteins (HDL) incubated
with E2. These experiments suggest
that E2 may be exerting its effect
by interfering early in the peroxidation process, perhaps by modifying
the reaction pathway responsible for the propagation phase in the
generation of reactive oxygen species (ROS), such as superoxide,
H2O2,
or
H radicals.
In addition to its protective role in preventing lipid peroxidation,
E2 has been shown to protect
against DNA damage. Tang and Subbiah (24), utilizing a chemical model
system consisting of OX-174 RF1 DNA, a supercoiled DNA, demonstrated
that E2 protected against DNA
damage induced by hydrogen peroxide and arachidonic acid, as indicated
by DNA strand breakage. However, the precise mechanism of
E2's protective effect on DNA
damage was not clear.
To further evaluate the antioxidant property and mechanism responsible
for the protective effect of E2 on
lipid peroxidation and DNA damage, studies were conducted
1) to compare the effect of
E2 with that of selective
scavengers of ROS in terms of lipid peroxidation and DNA damage and
2) to determine the direct effect of
E2 on the production of individual
ROS. Our studies suggest that E2
might offer protection from lipid peroxidation and DNA damage by
inhibiting the formation of superoxide radical and also by interfering
with the oxidative chain propagation leading to lipid peroxidation.
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MATERIALS AND METHODS |
AAPH (2,2'-azobis[2-amidinopropane]dihydrochloride),
cupric sulfate, hydrogen peroxide (30% solution), EDTA, ethidium
bromide, OX-174 RFI DNA, estradiol-17
,
-tocopherol,
-carotene,
superoxide dismutase (SOD), catalase, mannitol, and sodium azide were
purchased from Sigma Chemical (St. Louis, MO). Additional chemicals and reagents used in detecting formation of ROS include salicylic acid
(Aldrich Chemical, Milwaukee, WI); 2,3-, 2,4-, and 2,5-dihydroxybenzoic acid (DHBA; Aldrich); HPLC-grade diethyl ether (Aldrich); HPLC-grade methanol (Aldrich); and menadione
(1,2,3,4-tetrahydro-2-methyl-1,4-dioxo-2-naphthalenesulfonic acid
sodium salt; Sigma Chemical). Bovine heart microvessel endothelial cells (BHMEC) were obtained from Gensia (San Diego, CA).
Isolation of Plasma and Lipoproteins
Human plasma was obtained by centrifugation of blood samples collected
from normal fasting donors (both male and female) at 1,500 g for 15 min at 4°C. Preliminary
experiments indicated no differences in response of isolated LDL from
male and female subjects to E2.
All subjects were nonsmokers and were not taking any antioxidants. Plasma lipoproteins were sequentially isolated by ultracentrifugation, dialyzed, and stored at 4°C after being purged with nitrogen as described previously (23). Briefly, very low density lipoproteins, LDL,
and HDL were isolated with solid potassium bromide when density was
<1.006 at 42,000 rpm for 18 h, when density was 1.006-1.063 at
42,000 rpm for 20 h, and when density was 1.063-1.21 at 48,000 rpm
for 24 h, respectively, with a Beckman L3-50 ultracentrifuge and a Ti
50.3 rotor. The lipoproteins were flushed with
N2 and dialyzed Dulbecco's PBS
(0.037 M Na2,
HPO4, 7 H2O; 0.0018 M
K2, HPO4, 0.046 M NaCl, and KCl 0.003 M, pH 7.4) without EDTA for 24 h with two changes of dialyzing
solution. Protein was determined by the method of Lowry et al. (12).
Assessment of Lipid Peroxidation
Effect of E2, vitamins, and free
radical scavengers on lipid peroxidation was evaluated by the diene
conjugation method. The oxidation of lipoproteins was followed by
measuring the absorbency at 234 nm resulting from the formation of
conjugated dienes from unsaturated fatty acids, as described by
Esterbauer et al. (8). The time profile of the 234 nm absorption curve
shows three distinct phases: lag phase (absorption does not increase or
only slightly increases, indicating that the lipoprotein resists
oxidation), propagation phase (absorption rapidly increases to a
maximum value, indicative of the chain reaction lipoprotein
peroxidation process), and decomposition phase (absorption decreases
again as conjugated dienes slowly decrease and decomposition reactions
predominate) (8). The formation of conjugated dienes was measured by
incubating 60 µg LDL with 5.5 mM/l AAPH in 0.985 ml PBS medium. The
absorbency at 234 nm was measured continuously in a
thermostat-controlled (37°C) computerized Beckman DU-64
spectrophotometer equipped with a six-position automatic sampler
changer. The increase in 234 nm absorption was recorded every 5 min
during a 3-h period. The susceptibility of the lipoproteins to
oxidation was assessed on the basis of lag time (min), the time
interval between the addition of AAPH and the intercept of the slope of
the absorbance curve (8). The maximal amount of dienes formed was
determined as described by Esterbauer et al.
In most studies of lipid peroxidation, an
E2 concentration of 54 µM was
used. This concentration was chosen because at this level, significant
inhibition of lipid peroxidation was noted in our previous study (4).
In some experiments, ROS scavengers SOD (0.1 mg/ml), catalase (0.1 mg/ml), and mannitol (5 mM) were added independently or conjointly to
the reaction mixture to evaluate the effect of selective scavengers on
ROS generation.
Assessment of DNA Damage
OX-174 RFI DNA contains ~85-90% double-stranded, covalently
closed, supercoiled DNA molecules, and 10-15% double-stranded, open, circular DNA with no linear DNA molecules. DNA strand breaks were
determined by measuring the conversion of double-stranded supercoiled
OX-174 RFI DNA (form A) to
double-stranded open circular DNA (form
B) and linear DNA (form
C), as described by Li and Trush (11). The
experiments were performed in 1.5-ml Eppendorf tubes. DNA (0.2 µg)
was incubated with
H2O2
(15 µM), copper ion (33 µM), and estrogens (7-36 × 10
6 M) in PBS (final volume
30 µl) at 37°C for 30 min. After incubation, the samples were
loaded in a 1% agarose gel containing 40 mM Tris-acetate buffer with 1 mM EDTA and were subjected to electrophoresis in the same buffer for 2 h. The gels were stained with a solution of ethidium bromide and
destained in distilled water. The gels were photographed, and
quantification of DNA bands was performed by band densitometer tracing
with a computer-assisted imaging system. The percentage of various DNA
forms was calculated by optical density values of each band.
In most studies of DNA damage, the
E2 concentration ranged from 7 to
36 µM. This range was chosen because
1) at these levels significant
inhibition of DNA damage was noted in our previous study (24), and
2) this range allowed manipulations
for the study of mechanisms. In those instances when significant
changes were not evident, concentrations were changed accordingly. To evaluate the effect of selective scavengers on ROS generation and DNA
damage, in some experiments ROS scavengers SOD (0.1 mg/ml), catalase
(0.1 mg/ml), and mannitol (5 mM) were added independently or conjointly
to the reaction mixture (5).
Effect of E2 on Formation of ROS
Superoxide detection by cytochrome c reduction.
BHMEC were grown in minimal essential medium (MEM) supplemented with
10% horse serum and penicillin or streptomycin and were incubated for
30 min at 37°C with menadione (200 µM) under 5% CO2. SOD-inhibitable cytochrome
c reduction experiments as described by McCord and Fridovich (15) were carried out: cells in tissue culture
were washed three times with buffer and incubated in Krebs bicarbonate-buffered perfusate solution [(in mM) 110 NaCl, 2.6 KCl, 1.2 MgSO4, 1.2 KH2PO4,
25 NaHCO3, 25 HEPES, and 11 glucose] containing 20 µM ferricytochrome
c. Pairs of cultures dishes, one with
and the other without SOD, were exposed to the experimental conditions
at 37°C. The range of E2
concentration used was 0-100 µM. The cells were saved for
determination of protein content, the supernatants were collected, and
the absorbance was determined at 550 nm. Subsequently, the
SOD-inhibitable reduction of cytochrome c was calculated using a molar
extinction coefficient of 21,000 with normalization to the amount of
protein in each sample. Because equimolar quantities of
and
cytochrome c react with one another,
the calculated values were taken as representative of
produced
(13). The lower limit of detection of superoxide when this technique is
used is 0.75 mmol/ml (50 mmol/culture plate).
H2O2
detection.
H2O2
production was determined by two approaches. In cell culture,
H2O2
production was assessed according to the method of Pick and Mizel (18)
by use of an automated enzyme immunoassay reader, with the following
modifications. BHMEC were incubated for 2 h (37°C) in the presence
of 1.5 mM menadione, with or without E2, at varying concentrations (0.1 µM-100 µM). In the second approach, we used the chemical reaction
between azobis and LDL. The reaction mixture consisted of 0.6 ml of PRS
buffer [140 mM NaCl, 10 µM potassium phosphate buffer (pH 7.0),
5.5 mM dextrose, 0.28 mM phenol red, and 8.5 U/ml (50 µg/ml)
horseradish peroxidase], 330 µg azobis, and 100 µg LDL, which
were incubated at 37°C for 0-2 h. The sample was then placed
in a cuvette with the addition of 10 µl of 1 M NaOH, and absorption
was read at 610 nm.
Hydroxyl radical detection.
Effect of E2 on hydroxyl radical formation was
examined both in cell culture and by a chemical method. In both cases
the detection and quantification of hydroxyl radicals were performed by
HPLC, as described by Onodera and Ashraf (17) with modifications. The
assay is based on the chemical interaction of
H
with salicylic acid, forming 2,3- and 2,4-DHBAs. The products can be
separated from one another and from salicylic acid by HPLC. Both can be used as an index of
H production, which is
quantitated by integrating the area under the peak using 2,4-DHBA as an
internal standard. The in vitro system of generating hydroxyl radicals
was examined by a modification of the method of Onodera and Ashraf, as
follows: 15 µM H2O2, 33 µM
CuSO4, 10 µM salicylic acid, 10 µM 2,4-DHBA (internal standard), and 50 µl of 1 N HCl were placed in a 5-ml tube
with or without E2 and mixed for 120 s.
E2 concentration ranged from 10 to 100 µM. The
sample was extracted with 4 ml of HPLC-grade diethyl ether on a Vortex
mixer for 90 s. The diethyl ether layer was separated and completely
evaporated under nitrogen. The extraction was carried out a second
time. The dried residue was dissolved in 50 µl of 1 N HCl and 32.5 µl of mobile phase, and 20 µl of this solution were injected into
the HPLC unit. The amount of 2,5-DHBA was calculated and expressed as
nmol/mg protein. A Perkin-Elmer Series 200 LC pump with Applied
Biosystems 785A programmable ultraviolet (UV) detector at a wavelength
of 315 nm and a Hibar RT, Lichrosorb-RP-18 column (10 µm, 25 cm × 0.4 cm) were used. The mobile phase consisted of 80% 0.03 M
citric acid-0.03 M acetic acid buffer (pH of 3.6) and 20% methanol at a flow rate of 1.0 ml/min.
In cell culture, cells were plated in 60-mm wells and grown to
confluence. Cells were washed with PBS and placed in 3-ml Eagle's MEM
(phenol red free, GIBCO/BRL) with 1% fetal bovine serum. Salicylic acid (10 µl of 100 mM) and 200 µl menadione (100 mM) were added for
1 h, and the samples were incubated without and with
E2 (0.01-1 mM) under UV light
for 30 min. The media were collected, and 200 µl of 1 N HCl were
added. 2,4-DHBA (50 µl of 1 mM) was used as an internal standard. The
sample was extracted twice with 5 ml ethyl ether, dried down, and run
on HPLC as described above.
Statistics
Data were expressed as means ± SE. Group comparisons were
done by ANOVA with multiple comparison. A difference of
P < 0.05 was considered significant.
 |
RESULTS |
Lipid Peroxidation
To examine the mechanism of E2
protection, we determined the formation of conjugated dienes during
AAPH-induced oxidation of LDL (Fig. 1). Lag
times for E2 could not be
estimated because of the nature of the curve, as noted in our previous
study (4). LDL peroxidation induced by azobis, a peroxy radical
generator, was significantly inhibited (39.5 ± 0.71%) by SOD, a
scavenger of superoxide (0.1 mg/ml). Incubation with catalase, a
hydrogen peroxide scavenger, at 0.1 mg/ml, resulted in partial
inhibition (23.5 ± 2.1%) of LDL peroxidation. Mannitol (5 mM), a
hydroxyl radical scavenger, had no effect. The quantitative aspects of changes in lipid peroxidation induced by the scavengers of ROS and
their comparison with E2-induced
changes are shown in Table 1. LDL
peroxidation was markedly inhibited (70.5 ± 0.71%) by E2 (54 µM), which was greater
than that of any of the free radical scavengers examined (Table 1). The
combination of catalase or SOD with
E2 did not result in any further
degree of inhibition.

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Fig. 1.
Time course of formation of conjugated dienes during AAPH-induced
oxidation of low-density lipoproteins (LDL). LDL (60 µg) were
incubated with 5.5 mM/l AAPH in 0.985 ml PBS medium. Absorbance at 234 nm was measured continuously in a thermostat-controlled (37°C)
spectrophotometer. Increase in 234 nm absorption was recorded every 5 min for 3 h. Selected oxygen scavengers and estradiol-17
(E2) were added, as described in
MATERIALS AND METHODS. Each data point
represents a mean of two determinations.
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DNA Damage
Supercoiled plasmid OX-174 RFI DNA was examined as a substrate, because
relaxation of the molecule from the supercoiled to the open circular or
linear forms is an indicator of single-stranded or double-stranded
breaks, respectively. Cu2+ and
H2O2
alone did not induce damage to the DNA. The addition of
Cu2+ with
H2O2
induced a significant formation of linear DNA, form C (Fig. 2). With the
independent addition of catalase, mannitol, and SOD, there was 49, 61, and 65% DNA damage, respectively, compared with control (100%) (Table
2). When all three free radical scavengers were added together, 60% damage still occurred. From the data (Table
2), it appears that hydrogen peroxide, superoxide, and hydroxyl
radicals are all contributing to DNA damage. Sodium azide was
completely ineffective in inhibiting damage, suggesting that singlet
oxygen appears to play no role in the generation of DNA damage.
E2 significantly reduced the
formation of linear DNA (Table 3).
E2 alone was just as effective in
inhibiting DNA damage as the other individual scavengers alone or in
combination. When E2 was added in
combination with the radical scavengers, no additional protection was
noted (Table 3).

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Fig. 2.
Agarose gel electophoresis (stained with ethidium bromide) of OX-174
RFI plasmid DNA incubated with
H2O2
and Cu2+ in the presence or
absence of E2 at 37°C for 30 min. A, B, and C represent supercoiled, open circular, and linear forms
of DNA, respectively. Lane 1: control
DNA; lanes 2-4: DNA + H2O2
(15 µM) + Cu2+ (33 µM);
lanes 5, 6: DNA + H2O2
(15 µM) + Cu2+ (33 µM) + E2 (36 µM).
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Effect of E2 on Formation of ROS
The effect of E2 on ROS production
(
,
H2O2,
and
H) is shown in Figs. 3-7. In terms
of superoxide production in cell culture,
E2 at 1 µM concentration was
37% effective in inhibiting
formation, with a
dose-response effect with increasing
E2 concentrations (Fig.
3). E2
at concentrations up to 1 µM had no effect on the production of
H2O2
in cell culture, but a slight inhibition was noted at higher
concentrations of E2 (Fig.
4). In in vitro experiments using LDL,
however, we found marked inhibition of
H2O2
production at 54 µM concentration of
E2 (Fig.
5). E2
concentrations up to 100 µM had no effect on production of
H in cell culture (Fig. 6), but inhibition at a concentration of 10 µM E2 could be seen when the
chemical technique was utilized (Fig. 7).

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Fig. 3.
Effect of E2 on
O 2 production by bovine heart
microvessel endothelial cells (BHMEC). Superoxide dismutase (SOD)
production was measured after incubation with BHMEC with Krebs-Ringer
bicarbonate (KRB) solution containing 20 µM ferricytochrome
c. Pairs of culture dishes with or
without SOD were exposed to experimental conditions at 37°C.
Supernatants were collected and absorbency was determined at 550 nm.
Values are means ± SD of triplicate determinations.
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Fig. 4.
Effect of E2 on
H2O2
formation in BHMEC. BHMEC were incubated for 2 h at 37°C in the
presence of 1.5 mM menadione with or without
E2, and
H2O2
was measured as described in MATERIALS AND
METHODS. Values are means ± SD of triplicate
determinations.
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Fig. 5.
Effect of E2 on
H2O2
formation. LDL (100 µg) were incubated at 37°C in 0.6 ml of PRS
buffer with 330 µg azobis for 0-2 h with or without
E2 (54 µM). Samples were then
placed in a cuvette with addition of 10 µl (1 M NaOH), and absorption
was read at 610 nm. Values are means ± SD of triplicate
determinations.
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Fig. 6.
Effect of E2 on
H formation in BHMEC. Cells were washed with PBS and
placed in 3 ml MEM (phenol red free) (GIBCO/BRL) with 1% fetal bovine
serum. Salicylic acid (10 µl of 100 mM) and 200 µl menadione (100 mM) were added for 1 h, and samples were incubated with and without
E2 under ultraviolet (UV) light
for 30 min. Medium was extracted with 5 ml ethyl ether after addition
of 200 µl of 1 N HCl and was quantitated by HPLC using
2,4-dihydroxybenzoic acid (DHBA; 50 µl of 1 mM) as a standard. DHBA
peak area/internal standard represents total amount of 2,3- and
2,5-DHBA formed in relation to a known amount of 2,4-DHBA (internal
standard).
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Fig. 7.
Effect of E2 on
H radical formation.
H2O2
(15 µM), CuSO4 (33 µM),
salicylic acid (10 µM), 2,4-DHBA (internal standard, 10 µM), and 1 N HCl (50 µl) were placed in a 5-ml tube with or without
E2 and mixed for 120 s. Samples
were extracted with diethyl ether and evaporated under nitrogen.
Residue was dissolved in 50 µl of 1 N HCl and 32.5 µl of mobile
phase, and 20 µl of this solution were injected into HPLC as
described in MATERIALS AND METHODS.
2,3- and 2,5-DHBA represent salicylate derivatives formed. DHBA peak
area/internal standard represents total amount of 2,3 and 2,5-DHBA
formed in relation to a known amount of 2,4-DHBA (internal standard).
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 |
DISCUSSION |
In our present studies, we have examined the antioxidant properties of
E2 by utilizing techniques that
assess lipid peroxidation and DNA damage. On the basis of our studies
examining the mechanism of inhibition of lipid peroxidation and DNA
damage by E2, three important
points may be made. 1) It appears
that lipid peroxidation is most often induced by
and
H2O2,
as indicated by our studies showing that 60% of the damage is
inhibited by SOD and catalase. The remaining amount of damage appears
to be caused by peroxyl radicals, which are the initial radicals
produced by the oxidation initiator azobis (16).
E2 alone collectively blocks 70%
of the damage, and the further addition of the two scavengers provides
no additional inhibition of damage. This indicates that
E2 is acting as a chain-breaking antioxidant, inhibiting the effect of
H2O2,
, and hydroperoxyl
radicals. 2)
E2 action in inhibiting DNA damage supports this view. E2 prevented
DNA strand breaks in a manner similar to the free radical scavengers
catalase, SOD, and mannitol. When all three scavengers were added
together and in combination with
E2, the formation of DNA strand
breaks could not be entirely prevented. Thus the action of
E2 does not selectively restrict itself to specific ROS but prevents the continuation of the propagation phase of free radical attack on its intended target. Because sodium azide had no effect on inhibiting damage, the singlet oxygen did not
account for any of this disparity.
3) The experiments concerned with
the effect of E2 on the formation
of ROS demonstrate that E2 has
significant effect on the formation of
in cell culture.
This observation differs from that of Rifici and Khachadurian (19), who
could not demonstrate E2's effect on
production in
utilizing mononuclear cells. However, using BHMEC, we could demonstrate
that increasing concentrations of
E2 were effective in blocking
production. This
difference could be attributed to the utilization of different kinds of
cells. In cell culture we do not see any significant effect of
E2 on the formation of
either
H2O2
or
H radicals. In the chemical method, high
E2 concentrations (50 µM) seem
to have an effect. At lower concentrations,
E2 appears to have no effect on
H2O2
and
H formation.
Our in vitro studies suggest that
E2 might be preventing oxidative
DNA damage to some extent by inhibiting the formation of superoxides.
The in vivo significance of this finding deserves some discussion in
view of a recent report stating that
E2 decreases apoptosis of
endothelial cells (1). In cellular apoptosis the BCL-2 gene plays a
central role, and a variety of stimuli such as oxidants, toxins,
oncogenes, and some growth factors can modulate expression of this gene
(25). Estrogens are known to modulate the transcription of a number of
genes through their binding to cytosolic estrogen receptors, which
translocate to nucleus. The receptor/estrogen complex binds to specific
palidromic DNA targets (6). It is possible that, in this way, estrogens
can directly or indirectly modulate BCL-2 expression. In amyotrophic
lateral sclerosis, cell death is considered to be due to a mutation in SOD, causing inability to handle oxygen radicals (20). In vitro superoxide-related cell death can be corrected by antioxidants (26).
Therefore, it is possible that the ability of estrogens to decrease
oxidation of DNA damage in vitro might have some in vivo significance
in terms of apoptosis.
On the basis of our studies, we conclude that
E2 might be decreasing lipid
peroxidation and DNA damage either by inhibiting
production and/or by acting as a chain-breaking antioxidant. The
concentration of E2 in our studies
is slightly higher than the plasma levels reached after estrogen
therapy (0.2-1 × 10
6 mol/l). We think that
inhibition of lipid peroxidation and DNA damage by
E2 contributes to the
cardioprotection noted after estrogen therapy. Furthermore, in view of
the potential role of superoxide in lipid peroxidation, attempts to
manipulate SOD in vivo would be beneficial.
 |
ACKNOWLEDGEMENTS |
This study was supported in part by National Heart, Lung and Blood
Institute Grant HL-50881.
 |
FOOTNOTES |
Address for reprint requests: M. T. Ravi Subbiah, Univ. of Cincinnati,
PO Box 670540, Cincinnati, OH 45267-0540.
Received 18 November 1997; accepted in final form 26 February
1998.
 |
REFERENCES |
1.
Alvarez, R. J., Jr.,
S. J. Gips,
N. Moldovan,
C. C. Wilhide,
E. E. Milliken,
A. T. Hoang,
R. H. Hruban,
H. S. Silverman,
C. V. Dang,
and
P. J. Goldschmidt-Clermont.
17
-Estradiol inhibits apoptosis of endothelial cells.
Biochem. Biophys. Res. Commun.
237:
372-381,
1997[Medline].
2.
Ames, B. N.
Endogenous oxidative DNA damage, aging and cancer.
Free Radical Res. Commun.
7:
121-128,
1989[Medline].
3.
Ames, B. N.,
and
L. S. Gold.
Endogenous mutagens and the causes of aging and cancer.
Mutation Res.
250:
3-16,
1991[Medline].
4.
Ayres, S. A.,
M. Tang,
and
M. T. R. Subbiah.
Estradiol-17
as an antioxidant: some distinct features when compared with common fat-soluble antioxidants.
J. Lab. Clin. Med.
128:
367-375,
1996[Medline].
5.
Bhat, R.,
and
S. M. Hadi.
DNA damage by tannic acid and Cu(II): sequence specificity of the reaction and involvement of oxygen species.
Mutation Res.
313:
39-48,
1994[Medline].
6.
Brann, D. W.,
L. B. Hendry,
and
V. B. Mahesh.
Emerging diversities in the mechanism of action of steroid hormones.
J. Steroid Biochem. Mol. Biol.
52:
113-133,
1995[Medline].
7.
Bush, T. L.,
E. Barrett-Connor,
L. D. Cowan,
M. H. Criqui,
R. B. Wallace,
C. M. Suchindran,
H. A. Tyroler,
and
B. M. Rifkind.
Cardiovascular mortality and non-contraceptive estrogen use in women: results from the Lipid Research Clinics Program Follow-up Study.
Circulation
75:
1002-1009,
1987.
8.
Esterbauer, H.,
G. Striegl,
and
H. Puhl.
Continuous monitoring of in vitro oxidation of human low-density lipoprotein.
Free Radical Res. Commun.
6:
67-75,
1989[Medline].
9.
Keaney, J. F., Jr.,
G. T. Shwaery,
A. Xu,
R. J. Nicolosi,
J. Loscalzo,
T. L. Foxall,
and
J. A. Vita.
17-
Estradiol preserves endothelial vasodilator function and limits low-density lipoprotein oxidation in hypercholesterolemic swine.
Circulation
89:
2251-2259,
1994[Abstract].
10.
Lacort, M.,
A. M. Leal,
M. Liza,
C. Martin,
R. Martinez,
and
M. B. Ruiz-Larrea.
Protective effect of estrogens and catecholestrogens against peroxidative membrane damage in vitro.
Lipids
30:
141-146,
1995[Medline].
11.
Li, Y.,
and
M. A. Trush.
DNA damage resulting from the oxidation of hydroquinone by copper: role for a Cu(II)/Cu(I) redox cycle and reactive oxygen generation.
Carcinogenesis
14:
1303-1311,
1993[Abstract].
12.
Lowry, O. H.,
N. J. Rosebrough,
A. L. Farr,
and
R. J. Randall.
Protein measurement with the Folin phenol reagent.
J. Biol. Chem.
193:
265-275,
1951[Free Full Text].
13.
Matsubara, T.,
and
M. Ziff.
Superoxide anion release by human endothelial cells: synergism between a phorbol ester and a calcium ionophore.
J. Cell. Physiol.
127:
207-210,
1986[Medline].
14.
Maziere, C.,
M. Auclair,
M. C. Ronveaux,
S. Salmon,
R. Santus,
and
J. C. Maziere.
Estrogens inhibit copper and cell-mediated modification of low density lipoprotein.
Atherosclerosis
89:
175-182,
1991[Medline].
15.
McCord, J. M.,
and
I. Fridovich.
Superoxide dismutase: an enzymatic function for erythrocuprein (hemocuprein).
J. Biol. Chem.
244:
6049-6063,
1969[Abstract/Free Full Text].
16.
Niki, E.,
M. Saito,
Y. Yoshikawa,
Y. Yamamoto,
and
Y. Kamiya.
2,2'-Azobis[2-amidinopropane]dihydrochloride is a water soluble peroxy-radical generator.
Bull. Chem. Soc. Jpn.
59:
471-477,
1986.
17.
Onodera, T.,
and
M. Ashraf.
Detection of hydroxyl radicals in the post-ischemic reperfused heart using salicylate as a trapping agent.
J. Mol. Cell. Cardiol.
23:
365-370,
1991[Medline].
18.
Pick, E.,
and
D. Mizel.
Rapid microassays for the measurement of superoxide and hydrogen peroxide production by macrophages in culture using an automatic enzyme immunoassay reader.
J. Immunol. Methods
46:
211-226,
1981[Medline].
19.
Rifici, V. A.,
and
A. K. Khachadurian.
The inhibition of low-density lipoprotein oxidation by 17-
estradiol.
Metabolism
41:
1110-1114,
1992[Medline].
20.
Rosen, D. R.,
T. Siddique,
D. Patterson,
D. A. Figlewicz,
P. Sapp,
A. Hentati,
D. Donaldson,
J. Goto,
J. P. O'Regan,
H. Deng,
Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis.
Nature
362:
59-62,
1993[Medline].
21.
Sack, M. N.,
D. J. Rader,
and
R. O. Cannon III.
Oestrogen and inhibition of low-density lipoproteins in postmenopausal women.
Lancet
343:
269-270,
1994[Medline].
22.
Stampfer, M. J.,
G. A. Colditz,
W. C. Willet,
J. E. Manson,
B. Rosner,
F. E. Speizer,
and
C. H. Hennekens.
Postmenopausal estrogen therapy and cardiovascular disease.
N. Engl. J. Med.
325:
756-762,
1991[Abstract].
23.
Subbiah, M. T. R.,
B. Kessel,
M. Agrawal,
R. Rajan,
W. Abplanalp,
and
Z. Rymazewski.
Antioxidant potential of specific estrogens on lipid peroxidation.
J. Clin. Endocrinol. Metab.
77:
1095-1098,
1993[Abstract].
24.
Tang, M.,
and
M. T. R. Subbiah.
Estrogens protect against hydrogen peroxide and arachidonic acid induced DNA damage.
Biochim. Biophys. Acta
1299:
155-159,
1996[Medline].
25.
Thompson, C. B.
Apoptosis in the pathogenesis and treatment of disease.
Science
267:
1456-1462,
1995[Medline].
26.
Troy, C. M.,
and
M. L. Shelanski.
Down-regulation of copper/zinc superoxide dismutase causes apoptotic death in PC12 neuronal cells.
Proc. Natl. Acad. Sci.
91:
6384-6387,
1994[Abstract].
27.
Vaca, C. E.,
J. M. Wilhelm,
and
M. Harms-Kingdahl.
Interaction of lipid peroxidation byproducts with DNA, a review.
Mutat. Res.
195:
137-144,
1988[Medline].
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