Received for publication March 20, 2006.
Revised July 26, 2006.
Accepted for publication July 26, 2006.

A NONFEMINIZING ESTROGEN ANALOGUE PROTECTS AGAINST ETHANOL WITHDRAWAL TOXICITY IN IMMORTALIZED HIPPOCAMPAL CELLS

Abstract

We have shown that estrogen 17-estradiol protects against ethanol withdrawal toxicity in rats. Here, we investigated whether a cellular model of ethanol withdrawal could be developed in a cultured hippocampal cell line (HT22) and whether an adamantyl-containing nonfeminizing estrogen analogue (ZYC26) protects against ethanol withdrawal toxicity. HT22 cells were exposed to ethanol (0 - 500 mM) for 24 hours in the presence or absence of ZYC26 or 17-estradiol. The ethanol solution was then removed from the cells for four hours to create ethanol withdrawal. Samples were collected at the end of a 24 hour-ethanol exposure or at four hours of ethanol withdrawal to assess 1) cell viability using a Calcein assay, 2) lipid peroxidation by measuring malondialdehyde, and 3) protein oxidation by measuring carbonyl contents. When tested, ethanol concentrations were constantly maintained during a 24 hour-ethanol exposure and eliminated at four hours of ethanol withdrawal. Ethanol withdrawal decreased cell viability and increased the levels of malondialdehyde and carbonyls more than ethanol exposure. ZYC26 reduced the cell death and malondialdehyde levels at a lower dose (1 µM) than 17-estradiol (10 µM). The increased carbonyl contents were reduced only by ZYC26 treatment. These data suggest that ethanol withdrawal can be created in HT22 cells in a manner that is more toxic than ethanol exposure and that ZYC26 is a more potent cytoprotectant than 17-estradiol against cell death and oxidative damage induced by ethanol withdrawal. Therefore, ZYC26 can be a potential alternative estrogen therapy for a cellular and oxidative imbalance associated with ethanol withdrawal.

Key words: 17beta-estradiol, HT22 cells, ethanol withdrawal, lipid peroxidation, nonfeminizing estrogen, protein oxidation