Mechanisms involved in the protective effect of estradiol-17beta 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

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Previous studies from our laboratory have shown that estrogens can protect against lipoprotein peroxidation and DNA damage. In this study, the mechanism of estradiol-17beta (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

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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-17beta 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 &Odot;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.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

AAPH (2,2'-azobis[2-amidinopropane]dihydrochloride), cupric sulfate, hydrogen peroxide (30% solution), EDTA, ethidium bromide, OX-174 RFI DNA, estradiol-17beta , alpha -tocopherol, beta -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 <A><AC>O</AC><AC>˙</AC></A><SUP>−</SUP><SUB>2</SUB> and cytochrome c react with one another, the calculated values were taken as representative of <A><AC>O</AC><AC>˙</AC></A><SUP>−</SUP><SUB>2</SUB> 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 &Odot;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 &Odot;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
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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-17beta (E2) were added, as described in MATERIALS AND METHODS. Each data point represents a mean of two determinations.

                              
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Table 1.   Effect of selective oxygen species scavengers on lipid peroxidation: role of E2

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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Table 2.   Effect of inhibitors of oxygen species on DNA damage

                              
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Table 3.   Effect of scavengers of oxygen species on DNA damage: role of E2

Effect of E2 on Formation of ROS

The effect of E2 on ROS production (<A><AC>O</AC><AC>˙</AC></A><SUP>−</SUP><SUB>2</SUB>, H2O2, and &Odot;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 <A><AC>O</AC><AC>˙</AC></A><SUP>−</SUP><SUB>2</SUB> 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 &Odot;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 &Odot;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 &Odot;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).

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 <A><AC>O</AC><AC>˙</AC></A><SUP>−</SUP><SUB>2</SUB> 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, <A><AC>O</AC><AC>˙</AC></A><SUP>−</SUP><SUB>2</SUB>, 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 <A><AC>O</AC><AC>˙</AC></A><SUP>−</SUP><SUB>2</SUB> in cell culture. This observation differs from that of Rifici and Khachadurian (19), who could not demonstrate E2's effect on <A><AC>O</AC><AC>˙</AC></A><SUP>−</SUP><SUB>2</SUB> production in utilizing mononuclear cells. However, using BHMEC, we could demonstrate that increasing concentrations of E2 were effective in blocking <A><AC>O</AC><AC>˙</AC></A><SUP>−</SUP><SUB>2</SUB> 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 &Odot;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 &Odot;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 <A><AC>O</AC><AC>˙</AC></A><SUP>−</SUP><SUB>2</SUB> 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
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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Am J Physiol Endocrinol Metab 274(6):E1002-E1008
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