**5. Estrogen and ethanol withdrawal**

#### **5.1 Introduction**

The distress of alcohol (ethanol) withdrawal is initiated by abruptly removing the inhibitory stimulus of ethanol and thus, is associated with rebound hyper-excitatory stimuli. In general, the overt initial signs of ethanol withdrawal include anxiety, ataxia, muscle incoordination, seizures, coma, and even death (American Psychiatric Association, 2000). While repeating unsuccessful attempts to quit heavy drinking, the brain undergoes random exposure to ethanol and withdrawal, damaging cellular and neuronal integrity (Wober et al., 1998).

The neuronal activity of the brain tends to be hyper-excitable during ethanol withdrawal due to an increase in the level of glutamate, a major excitatory neurotransmitter (Rossetti & Carboni, 1995). This can result in neuronal damage to vulnerable brain areas such as the cortex, hippocampus, and cerebellum. In addition to this well known glutamate neurotransmission, ethanol withdrawal perturbs the homeostasis of redox balance and signaling mechanisms. For instance, ethanol withdrawal provokes the intense generation of reactive oxygen species and activates stress-responding protein kinases (Jung et al., 2009). In addition, ethanol withdrawal inflicts mitochondrial membranes/membrane potential and suppresses mitochondrial enzymes such as cytochrome c oxidase, all of which impair fundamental functions of mitochondria (Jung et al., 2007, 2009). In our recent study, brain aging occurred earlier in ethanol withdrawn animals than in control-diet animals (Jung et al., 2010). These studies indicate that mal-managed ethanol withdrawal can clearly provoke neurodegenerative disorders.

#### **5.2 Oxidative stress**

148 Sex Steroids

(H. Chen et al., 2003; Wahner et al., 2007). A positive correlation was found between antecedent brain injuries, such as trauma or exposure to infectious agents and the development of Parkinson's disease (B. Liu et al., 2003). This correlation implies that the brain inflammatory response to these noxious events, and specifically microglial activation, plays a critical role in Parkinson's disease. In support of this view, researchers have detected pro-inflammatory molecules (e.g. TNF-α) and excessive reactive oxygen species in the nervous system of Parkinson's disease patients (Hunot et al., 1996; Knott et al., 2000). The inflammatory molecules seem to amplify neuroinflammation as well as neuro-toxicity, ultimately leading to a slow and irreversible destruction of dopaminergic neurons. Using estrogen receptor-null mice, several studies have demonstrated that estrogen receptor-α is involved in the anti-inflammatory activity of estrogen (Dubal et al., 2001; Vegeto et al., 2003). Although estrogen receptor-β is expressed widely in brain, it does not seem to mediate the protective effect of estrogen. Or the effects of estrogen receptors on inflammation depend on the brain area (Harris et al., 2003). Whether or which receptor mediates estrogen's protection against inflammatory response still

Collectively, the protective effects of estrogen on Parkinson's disease appear to involve dopaminergic neuroprotection, anti-oxidant activities, anti-inflammatory activities, and estrogen receptors. Considering that Parkinson's disease is more prevalent in male than female patients, how these effects of estrogen can be implemented to clinical usages is an open question. At the very least, estrogen can be used as an interventional tool for a new

The distress of alcohol (ethanol) withdrawal is initiated by abruptly removing the inhibitory stimulus of ethanol and thus, is associated with rebound hyper-excitatory stimuli. In general, the overt initial signs of ethanol withdrawal include anxiety, ataxia, muscle incoordination, seizures, coma, and even death (American Psychiatric Association, 2000). While repeating unsuccessful attempts to quit heavy drinking, the brain undergoes random exposure to ethanol and withdrawal, damaging cellular and neuronal integrity (Wober et

The neuronal activity of the brain tends to be hyper-excitable during ethanol withdrawal due to an increase in the level of glutamate, a major excitatory neurotransmitter (Rossetti & Carboni, 1995). This can result in neuronal damage to vulnerable brain areas such as the cortex, hippocampus, and cerebellum. In addition to this well known glutamate neurotransmission, ethanol withdrawal perturbs the homeostasis of redox balance and signaling mechanisms. For instance, ethanol withdrawal provokes the intense generation of reactive oxygen species and activates stress-responding protein kinases (Jung et al., 2009). In addition, ethanol withdrawal inflicts mitochondrial membranes/membrane potential and suppresses mitochondrial enzymes such as cytochrome c oxidase, all of which impair fundamental functions of mitochondria (Jung et al., 2007, 2009). In our recent study, brain aging occurred earlier in ethanol withdrawn animals than in control-diet animals (Jung et al., 2010). These studies indicate that mal-managed ethanol withdrawal can clearly provoke

mechanistic insight into this neurodegenerative disease.

**5. Estrogen and ethanol withdrawal** 

remains unclear.

**5.1 Introduction** 

al., 1998).

neurodegenerative disorders.

Chronic ethanol consumption and ethanol withdrawal both generate oxidative free radicals and subsequent lipid peroxidation (Nordmann et al., 1990; Montoliu et al., 1994). Lipid peroxidation reflects the interaction between oxygen and the polyunsaturated fatty acids of membrane lipids, generating deteriorating breakdown products. Since the brain consists of a high content of unsaturated membrane lipids, it is a preferred target of both reactive oxygen species and ethanol (Hernandez-Munoz et al., 2000). Ethanol withdrawal-induced oxidative stress was associated with an increase in glutamatergic neurotransmission (Rossetti & Carboni, 1995), the upregulation of calcium channels, and the accumulation of intracellular calcium (Rewal et al., 2005). The functional consequence of prooxidant ethanol withdrawal is shown in several animal and human studies. For instance, enhanced reactive oxygen species concurred with ethanol withdrawal-induced seizure activity in rats (Vallett et al., 1997). The cerebrospinal fluid of patients who underwent ethanol withdrawal showed higher concentrations of excitatory neurotransmitters and oxidative markers (Marotta et al., 1997; Tsai et al., 1998) than control subjects. Higher levels of lipid peroxide and lower levels of superoxide dismutase (antioxidant enzyme) activity were also seen in those patients (Tsai et al., 1998). These studies indicate that the redox imbalance has a causative relationship with ethanol withdrawal insults.

If ethanol withdrawal is a prooxidant stimulus, estrogen treatment should be able to mitigate the stress through its antioxidant property. Our recent findings essentially confirmed the hypothesis using the in vivo and in vitro model of ethanol withdrawal. Estrogen treatment mitigated reactive oxygen species generation, lipid peroxidation, and protein oxidation (Jung et al. 2004, 2006). Estrogen protection against the prooxidant effect of ethanol withdrawal may involve glutamate transmission because glutamate-induced oxidative stress is attenuated by estrogen (Behl & Manthey, 2000) and the quinol derived from estrogen (Prokai et al., 2003). It is also possible that estrogen elevates the levels of endogenous antioxidants, such as glutathione, so that a favorable redox potential for an antioxidant environment is created (Prokai et al., 2003). Since oxidative molecules are generated mainly from mitochondria, these studies suggest that the antioxidant protection of estrogen against ethanol withdrawal is linked to the mitoprotective activity of estrogen.

#### **5.3 Mitochondria**

Indeed, the mitoprotective effects of estrogen are interactive with the antioxidant effect by virtue of the fact that mitochondria are the major source and target of oxidative free radicals. The mitoprotective effect of estrogen has been extended to the ethanol withdrawal model in our recent study in which ethanol withdrawal provokes the oxidation of mitochondrial proteins in rats, in a manner mitigated by estrogen. Since cellular energy ATP is mainly generated in mitochondria, it is not surprising that estrogen protects against mitochondrial respiratory deficit during ethanol withdrawal (Jung et al., 2011). Presumably, estrogen plays a role in alleviating the oxidative burden in mitochondria, thus increasing mitochondrial respiration efficiency (J.Q. Chen & Yager 2004; Jung et al., 2011).

#### **5.4 Signaling pathways**

P38 is referred to as a stress-activated protein kinase because it is often activated in response to a variety of stress. A transient, moderate activation of P38 normally occurs in association

Estrogen and Brain Protection 151

Ajmo, C. T., Jr.; Vernon, D. O.; Collier, L.; Hall, A. A.; Garbuzova-Davis, S.; Willing, A. &

Andersen, K.; Launer, L. J.; Dewey, M. E.; Letenneur, L.; Ott, A.; Copeland, J. R.; Dartigues,

Arvin, M.; Fedorkova, L.; Disshon, K. A.; Dluzen, D. E. & Leipheimer, R. E. (2000). Estrogen

Association, American Psychiatric. (2000). *Diagnostic and Statistical Manual of Mental Disorders Dsm-Iv-Tr*. 4th ed. (Washington, DC: Amer Psychiatric Pub). Azcoitia, I.; Sierra, A.; Veiga, S. & Garcia-Segura, L. M. (2003). Aromatase Expression by

Azcoitia, I.; Sierra, A.; Veiga, S.; Honda, S.; Harada, N. & Garcia-Segura, L. M. (2001). Brain

Barca, O.; Costoya, J. A.; Senaris, R. M. & Arce, V. M. (2008). Interferon-Beta Protects

Bastide, M.; Ouk, T.; Plaisier, F.; Petrault, O.; Stolc, S. & Bordet, R. (2007). Neurogliovascular

Behl, C. & Manthey, D. (2000). Neuroprotective Activities of Estrogen: An Update. *J* 

Benedetti, M. D.; Maraganore, D. M.; Bower, J. H.; McDonnell, S. K.; Peterson, B. J.; Ahlskog,

Beyer, C. & Karolczak, M. (2000). Estrogenic Stimulation of Neurite Growth in Midbrain

*Psychoneuroendocrinology*, Vol. 32 Suppl 1 pp. S36-9, ISSN 0306-4530

*Neurocytol*, Vol. 29, No. 5-6, pp. 351-8, ISSN 0300-4864

*Disord*, Vol. 16, No. 5, pp. 830-7, ISSN 0885-3185

*Res*, Vol. 59, No. 1, pp. 107-16, ISSN 0360-4012

Research Group. *Neurology*, Vol. 53, No. 9, pp. 1992-7, ISSN 0028-3878 Andreassen, O. A.; Ferrante, R. J.; Dedeoglu, A.; Albers, D. W.; Klivenyi, P.; Carlson, E. J.;

*Neuroscience*, Vol. 169, No. 2, pp. 781-6, ISSN 1873-7544

Pennypacker, K. R. (2008). The Spleen Contributes to Stroke-Induced Neurodegeneration. *J Neurosci Res*, Vol. 86, No. 10, pp. 2227-34, ISSN 1097-4547 Amtul, Z.; Wang, L.; Westaway, D. & Rozmahel, R. F. (2010). Neuroprotective Mechanism

Conferred by 17beta-Estradiol on the Biochemical Basis of Alzheimer's Disease.

J. F.; Kragh-Sorensen, P.; Baldereschi, M.; Brayne, C.; Lobo, A.; Martinez-Lage, J. M.; Stijnen, T. & Hofman, A. (1999). Gender Differences in the Incidence of Alzheimer's Disease and Vascular Dementia: The Eurodem Studies. Eurodem Incidence

Epstein, C. J. & Beal, M. F. (2001). Mice with a Partial Deficiency of Manganese Superoxide Dismutase Show Increased Vulnerability to the Mitochondrial Toxins Malonate, 3-Nitropropionic Acid, and MPTP. *Exp Neurol*, Vol. 167, No. 1, pp. 189-

Modulates Responses of Striatal Dopamine Neurons to Mpp(+): Evaluations Using in Vitro and in Vivo Techniques. *Brain Res*, Vol. 872, No. 1-2, pp. 160-71, ISSN 0006-

Reactive Astroglia Is Neuroprotective. *Ann N Y Acad Sci*, Vol. 1007 pp. 298-305,

Aromatase Is Neuroprotective. *J Neurobiol*, Vol. 47, No. 4, pp. 318-29, ISSN 0022-

Astrocytes against Tumour Necrosis Factor-Induced Apoptosis Via Activation of P38 Mitogen-Activated Protein Kinase. *Exp Cell Res*, Vol. 314, No. 11-12, pp. 2231-7,

Unit after Cerebral Ischemia: Is the Vascular Wall a Pharmacological Target.

J. E.; Schaid, D. J. & Rocca, W. A. (2001). Hysterectomy, Menopause, and Estrogen Use Preceding Parkinson's Disease: An Exploratory Case-Control Study. *Mov* 

Dopaminergic Neurons Depends on cAMP/Protein Kinase a Signaling. *J Neurosci* 

**8. References** 

95, ISSN 0014-4886

ISSN 0077-8923

ISSN 1090-2422

8993

3034

with cell survival or differentiation. However, excess activation generally correlates with pathological conditions (Barca et al., 2008). P38 is activated upon phosphorylation (Moriguchi et al., 1996) and thus, pP38 is often measured as an indicator of P38 activation. A previous study reported that the P38 inhibitor SB203580 attenuated ethanol-induced cell death (Ku et al., 2007), suggesting that P38 activation mediates cytotoxic ethanol. Acute ethanol treatment led to P38 activation (Norkina et al., 2007) and augmented endotoxininduced pP38 levels in a manner attenuated by P38 inhibitor in human monocytes (Drechsler et al., 2006). Recently, we have demonstrated that estrogen protected against ethanol withdrawal-induced hyperactivation of P38, suggesting that there is a crucial link between estrogen, P38, and ethanol withdrawal (Jung et al., 2010). In that study, middle-age female rats (12-15 month old) were more vulnerable to the ethanol withdrawal-induced P38 activation than young or older rats (Jung et al., 2010). Importantly, chronic estrogen treatment abolished the age difference in P38 activation. These studies indicate that ethanol withdrawal interferes with signaling pathways, including P38, in a manner that depends on age and that is protected by estrogen.

In conclusion, findings from our and others' laboratories suggest that ethanol withdrawal distress is more than a neurotransmitter disorder. It is attributed to the perturbation of redox balance, protein kinase signaling, and mitochondria, all of which can be mitigated by estrogen treatment. Understanding the interaction between ethanol withdrawal and estrogen may contribute to the improvement of the pharmacological treatment of ethanol withdrawal.
