Antioxidant Effects

The studies discussed above show a clear role of CBF enhancement in explaining the relative neuroprotection seen in females in some ischemic injury models. On the other hand, Hall et al.15 failed to find a difference in pre-, intra-, or postischemic cortical CBF between male and female gerbils subjected to a 3-h unilateral carotid occlusion and reperfusion. Females showed significantly less cortical neuronal loss at 24 h postreperfusion compared to males. However, CBF before, during, and for the first 2 h after ischemia in females was not different from that measured in male animals, as shown in Figure 1.3. Thus, improved CBF cannot be the sole explanation for female-associated neuroprotection.

Another important mechanism by which estrogen may provide neuroprotection is by limiting an important component of the secondary injury cascade, free radical-induced lipid peroxidation. Lipid peroxidation is a destructive process initiated by free radicals that has been shown to produce significant damage following acute brain ischemia or traumatic injury (see references 70 and 71 for reviews). Lipid peroxidation of cell membranes is a geometrically progressing process that spreads over the surface of the membrane and on to surrounding cells. Free-radical scavengers such as superoxide dismutase69 and lipid antioxidants, such as the glucocorticoid steroid methylprednisolone,70 U-72099E,71 the 21-aminosteroid tirilazad mesylate,72

CORTICAL Ca<J+AND CBF DURING AND AFTER ISCHEMIA (3 HR. UCO)

CORTICAL Ca<J+AND CBF DURING AND AFTER ISCHEMIA (3 HR. UCO)

ISCHEMIA (HRS) REPERFUSION (HRS)

ISCHEMIA (HRS) REPERFUSION (HRS)

ISCHEMIA (HRS) REPERFUSION (HRS)

figure 1.3 Comparison of cortical blood flow in male vs. female gerbils before, during, and for 2 h after a 3-h period of unilateral carotid occlusion as measured by hydrogen clearance. (Originally published in Hall et al., J. Cereb. Blood Flow Metab, 11:292-298, 1991. Reproduced with permission.)

ISCHEMIA (HRS) REPERFUSION (HRS)

figure 1.3 Comparison of cortical blood flow in male vs. female gerbils before, during, and for 2 h after a 3-h period of unilateral carotid occlusion as measured by hydrogen clearance. (Originally published in Hall et al., J. Cereb. Blood Flow Metab, 11:292-298, 1991. Reproduced with permission.)

and the nonsteroidal 2-methylaminochroman U-78517F,73 have been reported to attenuate posttraumatic pathophysiology and/or to promote survival and recovery in experimental head injury. Tirilazad and U-78517F have also been found protective in models of focal cerebral ischemia.74 Thus, there is extensive evidence that antioxidant compounds are neuroprotective.

Estrogens have been shown to be powerful antioxidants15,75-78 and more potent inhibitors of lipid peroxidation than ^-carotene, superoxide dimutase,76 and vitamin E.15,76 The chemical structure of estrogen allows for the donation of an electron in the form of a hydrogen atom from the hydroxyl in the 3 position of the aromatic A ring to a lipid peroxyl radical, thus neutralizing its ability to react with neighboring polyunsaturated fatty acids. A schematic of estrogen's antioxidant activity is shown in Figure 1.4. This antioxidant property of estrogen allows for inhibition of the propagation of lipid peroxidation reactions.76,79,80 Indeed, efforts have been undertaken to improve upon the antioxidant properties of 17^-estradiol by introduction of additional phenolic hydroxyl moieties into the estrogen structure.81

Scavenging Mechanism for Estradiol

GSHPx

17ß-estradiol 17ß-estradiol

17ß-estradiol 17ß-estradiol

CH OH radical CH OH

Ascorbate radical

Ascorbate figure 1.4 Chemical basis for 17p-estradiol's lipid peroxyl radical scavenging chemical antioxidant action.

CH OH radical CH OH

Ascorbate radical

Ascorbate figure 1.4 Chemical basis for 17p-estradiol's lipid peroxyl radical scavenging chemical antioxidant action.

Lipid peroxidation is initiated when a free radical species such as an hydroxyl radical (•OH) steals an electron from an allylic carbon of a polyunsaturated fatty acid (LH) esterified to a membrane phospholipid. The resulting alkyl radical (L^) then reacts with molecular oxygen (O2) to form a lipid peroxyl radical (LOO^). The peroxyl can react with another LH creating a second L^ representing propagation of the lipid peroxidation chain reactions. Alternatively, 17^-estradiol can step in and donate an electron from the phenolic hydroxyl moiety (3-OH) to the lipid peroxyl radical. As a result, the membrane chain reaction is stopped. The resulting lipid hydroperoxide (LOOH) can be decomposed by the antioxidant enzyme glutathione peroxidase (GSHPx) to an innocuous lipid alcohol (LOH). While uncertain, the resulting 17P-estradiol radical can be reduced by acceptance of an electron from ascorbate in the same way that ascorbate regenerates vitamin E from vitamin E radical.

The antioxidant effects of estrogen have been mainly demonstrated in neural tissues. Hall et al.15 showed that 17^-estradiol potently inhibits iron-catalyzed lipid peroxidation in rat brain homogenates and is more potent in that regard than vitamin E. Behl et al.82 assessed oxidative cell death caused by P amyloid (AP), H2O2, and glutamate in mouse clonal hippocampal cells and found that preincubation of the cells with 17^-estradiol prevented oxidative stress-induced cell damage and cell death (measured as cell viability and cell lysis). Goodman et al.,83 using primary rat hippocampal cultures, likewise found a 17P-estradiol attenuation of the oxidative neurotoxicity produced by Ap. Culmsee et al.39 found that 17P-estradiol and 2-OH-estradiol reduced the percentage of damaged chick embryonic neurons when treated with FeSO4, an initiator of lipid peroxidation. In this primary neuron culture, reactive oxygen species were also found to be diminished after treatment with 17^-estradiol or 2-OH-estradiol, suggesting that protection was due to an antioxidant effect of the estrogens used. Vedder et al.84 found 17^-estradiol reduced iron-induced lipid peroxidation in four cell systems: rat brain homogenates, hippocampal HT22 cells, rat primary neocortical cultures, and human brain homogenates. The incubation of primary neuronal cultures with 17^-estradiol showed that the inhibitory effect on lipid peroxidation was paralleled by an increase in survival of cultured cells.

Several in vivo studies of ischemic brain injury have addressed the question of estrogen's ability to act as an antioxidant and reduce lipid peroxidative damage. Hall et al.15 measured brain vitamin E levels before and after unilateral carotid occlusion in male and female gerbils. While they found no difference in baseline levels in brain vitamin E, a difference in vitamin E loss was seen after injury — with much more depletion in males than in females, as shown in Figure 1.5. This suggests that a greater level of oxygen radical-induced lipid peroxidative damage was experienced by the male, resulting in lowered levels of vitamin E. Similarly, Ferris et al.85 examined ascorbate levels and loss after decapitation ischemia in male and female rats.

MALE FEMALE COMBINED MALE FEMALE SHAM SHAM SHAM ISCHEMIC ISCHEMIC N=4 N=5 N=9 N=6 N=8

figure 1.5 Comparison of vitamin E levels in the ischemic hemisphere of male vs. female gerbils 2 hours after a 3-hour period of unilateral carotid occlusion. P values were obtained using student's t tests.

MALE FEMALE COMBINED MALE FEMALE SHAM SHAM SHAM ISCHEMIC ISCHEMIC N=4 N=5 N=9 N=6 N=8

figure 1.5 Comparison of vitamin E levels in the ischemic hemisphere of male vs. female gerbils 2 hours after a 3-hour period of unilateral carotid occlusion. P values were obtained using student's t tests.

They found significant ascorbate loss in the males, but not the females, again suggesting higher levels of oxidative damage in the males. In a follow-up study, Kume-Kick et al.86 demonstrated that gonadectomy of the female eliminated this sex difference, suggesting that female gonadal hormones are responsible for the effect. Gonadectomy of the male, on the other hand, did not alter ascorbate loss after ischemia. The same group more recently demonstrated that, in some brain regions at least, chronic estrogen replacement (170-estradiol) prevented the ovariectomy-in-duced enhanced ascorbate loss associated with ischemia.87

Additional evidence for estrogen's in vivo antioxidant neuroprotective effect comes from Hall and Sutter,20 who compared infarct size after unilateral carotid occlusion in male and female transgenic mice that overexpress the antioxidant enzyme Cn, Zn superoxide dismutase (Cu, Zn SOD) with wild type males and females. In the nontransgenics, lesion size was greater in males than females, hypothesized to be due to an antioxidant neuroprotective effect of estrogen in the females. In male trans-genics, lesion size was smaller than in nontransgenic males, presumably because of the overexpressed Cu, Zn SOD. Female transgenic mice, however, had lesions the same size as females of the wild type. Thus, while overexpression of Cu, Zn SOD provided protection to the males, it did not provide any additional protection in the females, likely because the females already have the benefit of the antioxidant effects of endogenous estrogen.

Estrogen may also exert an indirect antioxidant action secondary to its ability to increase NO^ (vide supra). Increased NO^ levels decrease lipid peroxidative damage because NO^ can act as an inhibitor of the lipid peroxidation chain reactions by scavenging lipid peroxyl radicals by the reaction LOO + NO^ ^ LOONO.88-90

Finally, Wang et al.91 demonstrated that estrogen limits 3-nitrotyrosine (3-NT) levels after transient forebrain ischemia in rats. 3-NT is a marker of peroxynitrite, which is now thought to play a critical role in postischemic oxidative neuronal damage. Greater levels of 3-NT immunoreactivity were found in the hippocampus and intermediate cortical layers in ovariectomized compared to intact female rats after unilateral carotid occlusion plus hemorrhagic hypotension, suggesting endogenous estrogen reduces peroxynitrite production in vulnerable brain regions. As noted earlier, peroxynitrite is formed by the reaction of NO^ with O2-. Whether NO^ reacts with O2- to form peroxynitrite or with peroxyl radicals to halt lipid peroxidation depends on the balance of O2- and NO^ concentrations.89 When NO^ concentration is lower or equal to O2- concentration, NO^ reacts with O2-. When NO^ concentration is higher than O2-, NO^ remains free to react with and neutralize lipid peroxyl radicals. Pathological conditions such as stroke tend to enhance local release of O2-, leading to increased peroxynitrite and oxidative tissue injury. Enhancement of NO^ production, however, allows NO^ to play a protective role and interrupt the propagation of the lipid peroxidation reactions down the membrane. Thus, an estrogen-associated increase in NO^ levels is neuroprotective.

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