Neuroprotective effects of an estratriene analog are estrogen receptor independent in vitro and in vivo

Abstract

Estrogens are potent neuroprotectants both in vitro and in vivo. In the present study, we compared the potency and efficacy of a non-feminizing estrogen, 2-(1-adamantyl)-4-methylestrone (ZYC-26), with its parent estrogen, estrone, and an expected non-neuroprotective 3-O-methyl analog of (17β)-2-(1-adamantyl)estradiol (ZYC-23). These estratriene derivatives were tested for their ability to protect in an in vitro lipid peroxidation model, to neuroprotect against oxidative stress in cell culture models, to bind the estrogen receptors (ERα and ERβ), to elicit uterotrophic effects, and to affect brain damage from transient middle cerebral artery occlusion. We observed that in contrast to estrone, neither ZYC-26 nor ZYC-23 bound to either estrogen receptors (ER) and both failed to elicit a uterotrophic response. In vitro, the active estrogen analogue ZYC-26 was more potent that estrogen in its ability to inhibit lipid peroxidation and to protect HT-22 cells from either glutamate or iodoacetic acid (IAA) toxicity. Further, ZYC-26 was as active in preventing brain damage from transient middle cerebral artery occlusion (MCAO) as was estrone. Collectively, these studies suggest that the antioxidant activity, rather than ER binding of non-feminizing estrogens such as ZYC-26, mediates their potent neuroprotective activity. Further, in view of the now known toxicities of chronic feminizing estrogen use in older women, non-feminizing estrogens may be a useful alternative for estrogen-induced brain protection.

Introduction

Sex differences in the incidence and outcome of stroke in human subjects suggest that hormonal factors may influence stroke occurrence and damage [12], [28]. The severity of ischemic damage in spontaneously hypertensive rats is dependent upon estrogen status, i.e., rats subjected to MCAO during proestrous periods showed less damage than rats injured during metestrus times [4], [5]. Exogenously administered E2 is also able to reduce ischemic lesions in animals subjected to middle cerebral artery occlusion [8], [32], [36], [44]; estradiol-mediated protection against cerebral ischemia is seen in young [20], [36], middle-aged [7], and diabetic [38] rats, as well as in mice [9] and gerbils [33]. Injury due to MCAO is decreased in rats whether with pretreatment with physiological levels of E2 [8] or with pharmacological post-treatment [25], [44] methods. Further, other estrogens and nonsteroidal estrogens (17αE2, estrone, diethylstilbestrol) are also protective in a gerbil model of global ischemia [33]. Finally, protective effects of estrogens have been widely reported in different types of neuronal cells against various toxicities including serum deprivation, oxidative stress, amyloid β peptide, and excitotoxicity [13], [14], [15], [26].

The recent Women's Health Initiative (WHI) reports indicate that in older women, chronic exposure to feminizing estrogens alone [1] or in combination with a synthetic progestin [23], [41] leads to an increase in pro-thrombotic and pro-mitotic side effects. These side effects of feminizing estrogens are believed to be mediated by estrogen receptor α (ERα) in estrogen responsive tissues, like the liver, uterus, and breast. Needed, but currently not available, are compounds that retain the neuroprotective effects of estrogens, but which are devoid of their feminizing effects.

In the present study, we assessed estratrienes with bulky groups flanking the 3-hydroxyl group in the phenolic A ring of estrone (ZYC-26), a 3-O-methyl derivative of 17β-estradiol bearing a bulky substituent at steroid position C-2 (ZYC-23) and estrone (E1) (Fig. 1). These estratriene derivatives were tested for their ability to protect in an in vitro lipid peroxidation model, to neuroprotect against oxidative stress in cell culture models, to bind the estrogen receptors (ERα and ERβ), to elicit uterotrophic effects, and to affect brain damage from transient middle cerebral artery occlusion.