**3.2.3 The effects of ethanol on synaptic structure**

It has been shown that chronic ethanol consumption affects the synaptic structure. The density of dendritic spines was found lower in the nucleus accumbens, and depicted an upregulation of a subunit of the NMDA receptor. The up-regulated NMDA receptor subunit is a splice variant isoform which is required for membrane-bound trafficking or anchoring into a spine synaptic site. These changes, evoked by ethanol, demonstrated an alteration of micro circuitry for glutamate reception (Zhou et al., 2007).

Adermark and Loviger (2006) showed that ethanol inhibits a Ca2+-insensitive K+ channel activity, and affects gap junction coupling, demonstrating that astrocytes play a critical role in brain K+ homeostasis, and that ethanol effects on astrocytic function could influence neuronal activity.

Finally, despite most of the investigations on the effects of ethanol have been performed following its addition to tissue or cell cultures, an interesting study has shown excessive activation of glutamatergic neurotransmission in the cerebral cortex following ethanol withdrawal and its contribution to significant behavioural disturbances and to alcohol craving. These effects were related to the activity of the enzyme glutamine synthetase, which converts released glutamate to glutamine (Miguel-Hidalgo, 2006).

#### **3.2.4 Ethanol and glial oxidative stress**

Brain tissue is particularly vulnerable to oxidative damage, possibly due to its high consumption of oxygen and the consequent generation of high quantities of ROS during

Ethanol Toxicity in the Brain: Alteration of Astroglial Cell Function 617

the pro-inflammatory cytokine MCP-1 (monocyte chemoattractant protein 1) and microglial activation as well as astrogliosis have been demonstrated by postmortem analyses in

Besides ethanol, its primary metabolite acetaldehyde is also able to modulate TNF-alpha and IL-6 secretion from cultured astrocytes. Both compounds showed a biphasic, hormetic effect on the IL-6 secretion after the acute as well as after the long-term exposure. It has been shown that long-term exposure to ethanol and acetaldehyde is more toxic than an acute exposure. The maximum stimulation was reached for 50 mM ethanol and 1 mM acetaldehyde after chronic exposure. In contrast, both compounds reduced the TNF-alpha secretion, where the effect was concentration dependent. Acetaldehyde showed to be more potent toxin than ethanol, and the ethanol's toxicity in the brain is at least partially due to its

Inflammation is primarily a protective response of the target organism to a noxis. On the other hand, excessive or long-lasting inflammation is often followed by degenerative processes. The stimulatory effect of ethanol and acetaldehyde on IL-6 secretion seems to be involved in both neuroregenerative and survival processes as well as in neurodegeneration. The obtained hormetic dose-response relationship indicates that higher concentrations and long-term exposure could lead in a neurodegenerative direction whereas low concentrations may act as neuroprotective. Unlike TNF-alpha, which is responsible for the induction of multiple pro-inflammatory genes, IL-6 often fails to induce these genes. Moreover, IL-6 can down-regulate the expression of TNF-alpha, which correlate with the data, where the first significant decrease in the TNF-alpha level was found at the highest level of IL-6 after a

Apoptosis or programmed cell death is a form of cell death that occurs in multicellular organisms. Apoptosis is a tightly regulated process which engages multiple cell signalling pathways, and involves the altruistic suicide if individual cells in favour of the organism. This process is desirably during organism development and morphological changes, especially at the embryonic stage, as well as during the activation of the immune system. However, defects in apoptosis can result in cancer, autoimmune diseases and neurodegenerative disorders. Studies on Ca2+ signalling in apoptosis showed that ethanol potentiates apoptotic cell death induction by thapsigargin, caffeine, and the protonophore, which separately caused similar increases in Ca2+ levels, and also induces similar apoptotic death. These effects of ethanol are concentration and time-dependent (Hirata et al., 2006).

The effect of ethanol on the induction of apoptosis in astrocytes, and the formation of ceramide as apoptotic signal was investigated by Schatter et al. (2005). Ethanol induced nuclear fragmentation and DNA laddering, and inhibited phospholipase D-mediated formation of phosphatidic acid, which is a mitogenic lipid messenger. The authors concluded that ethanol induced glial apoptosis during brain development via formation of ceramide. Further studies have shown that astrocytes exposed to ethanol, undergo morphological changes associated with anoikis, a programmed cell death induced by loss of anchorage. Astrocytes depicted peripheral reorganisation of both, focal adhesions and actin-myosin system, cell contraction,

membrane blebbing and chromatin condensation (Gonzalez & Salido, 2009).

alcoholic brains (Gonzalez & Salido, 2009; He and Crews, 2008).

primary metabolite, acetaldehyde (Šarc et al., 2011).

long-term exposure to ethanol (Šarc et al., 2011).

**3.2.6 Ethanol and glial cell death** 

oxidative phosphorylation. In addition, several regions of the brain are rich in iron, which promotes the production of ROS. On the other hand, the brain counts wit relatively poor levels of antioxidant enzymes and antioxidant compounds. ROS increase intracellular concentration of Ca2+ ions, inhibit response of astrocytes to physiological agonists, and stimulate glutamate secretion, which in excess is neurotoxic (Gonzalez et al., 2006a). Although glutamate is the principal excitatory neurotransmitter in the mammalian brain, high levels of this neurotransmitter lead to excitotoxic neuronal death, mediated by Ca2+ influx, principally through NMDA-gated channels (Bambrick et al., 2004).

Ca2+ signalling is an important medium for neuron-glia interaction, in the sense that neuronal activity can trigger Ca2+ signals in glial cells and vice versa. Due to its critical importance for the cellular functions, resting intracellular concentration of Ca2+ ions is tightly controlled, and abnormalities in Ca2+ regulation lead to impairment of cellular physiology. Ca2+-ROS interplay can be considered as a push-pull relationship. An elevated level of intracellular concentration of Ca2+ ions can lead to excessive ROS production, whereas excessive ROS production can lead to cytosolic Ca2+ overload (Gonzalez & Salido, 2009). Acute exposure of astrocytes to ethanol increases intracellular concentration of Ca2+ ions, probably due to inhibition of plasma membrane Ca2+-ATPase activity (Sepulveda & Mata, 2004). Other changes, evoked by ethanol are cell swelling, and transformation of actin cytoskeleton (Allansson et al., 2001).

Mitochondria represent the major source of intracellular ROS, and Ca2+ uptake into the organelle can lead to ROS generation (Gonzalez et al., 2006b; Granados et al., 2004). Ethanolevoked ROS production takes place in the mitochondria, and accumulated mitochondrial ROS can be released to the cytoplasm leading to damage of different transport mechanisms, ion channel modification, lipid peroxidation, and DNA damage. Furthermore, damage to mitochondrial metabolism may generate additional damaging radial species, thus activating cellular death pathways (Gonzalez et al., 2006a).

Ethanol evokes a dose-dependent increase in glutamate secretion by an exocytosis mechanism, which was dependent on Ca2+ mobilisation. The secretory effect of ethanol is reduced in the presence of antioxidants, therefore indicating the participation of ROS in ethanol-evoked glutamate secretion by astrocytes. Glutamate and the attendant increase in intracellular Ca2+ play crucial role in triggering excitotoxic cell death in neighbouring cells (Molz et al., 2008). Because astrocytes are the major regulators of glutamate homeostasis, their death can cause and/or aggravate diseases of the CNS.

#### **3.2.5 Ethanol, inflammation and immune response**

Ethanol is able to activate glial cells, which is a critical event in the neuroinflammatory processes. Chronic ethanol intake enhances inflammatory mediators like COX-2, and iNOS in rat cerebral cortex and cultured astrocytes. Astrocytes undergo actin cytoskeleton disorganisation, and there is a stimulation of both, interleukin receptor-associated kinase (IRAK)/extracellular signal-regulated kinases (ERK)/nuclear factor-kappaB (NF-kappaB) pathway and the COX-2 expression, which are associated with the inflammatory responses (Guasch et al., 2007).

Ethanol-induced glial activation is also associated with changes in the expression of inflammatory cytokines like IL-1alpha, TNF-alpha, IL-6. Notably, an increased expression of

oxidative phosphorylation. In addition, several regions of the brain are rich in iron, which promotes the production of ROS. On the other hand, the brain counts wit relatively poor levels of antioxidant enzymes and antioxidant compounds. ROS increase intracellular concentration of Ca2+ ions, inhibit response of astrocytes to physiological agonists, and stimulate glutamate secretion, which in excess is neurotoxic (Gonzalez et al., 2006a). Although glutamate is the principal excitatory neurotransmitter in the mammalian brain, high levels of this neurotransmitter lead to excitotoxic neuronal death, mediated by Ca2+

Ca2+ signalling is an important medium for neuron-glia interaction, in the sense that neuronal activity can trigger Ca2+ signals in glial cells and vice versa. Due to its critical importance for the cellular functions, resting intracellular concentration of Ca2+ ions is tightly controlled, and abnormalities in Ca2+ regulation lead to impairment of cellular physiology. Ca2+-ROS interplay can be considered as a push-pull relationship. An elevated level of intracellular concentration of Ca2+ ions can lead to excessive ROS production, whereas excessive ROS production can lead to cytosolic Ca2+ overload (Gonzalez & Salido, 2009). Acute exposure of astrocytes to ethanol increases intracellular concentration of Ca2+ ions, probably due to inhibition of plasma membrane Ca2+-ATPase activity (Sepulveda & Mata, 2004). Other changes, evoked by ethanol are cell swelling, and transformation of actin

Mitochondria represent the major source of intracellular ROS, and Ca2+ uptake into the organelle can lead to ROS generation (Gonzalez et al., 2006b; Granados et al., 2004). Ethanolevoked ROS production takes place in the mitochondria, and accumulated mitochondrial ROS can be released to the cytoplasm leading to damage of different transport mechanisms, ion channel modification, lipid peroxidation, and DNA damage. Furthermore, damage to mitochondrial metabolism may generate additional damaging radial species, thus activating

Ethanol evokes a dose-dependent increase in glutamate secretion by an exocytosis mechanism, which was dependent on Ca2+ mobilisation. The secretory effect of ethanol is reduced in the presence of antioxidants, therefore indicating the participation of ROS in ethanol-evoked glutamate secretion by astrocytes. Glutamate and the attendant increase in intracellular Ca2+ play crucial role in triggering excitotoxic cell death in neighbouring cells (Molz et al., 2008). Because astrocytes are the major regulators of glutamate homeostasis,

Ethanol is able to activate glial cells, which is a critical event in the neuroinflammatory processes. Chronic ethanol intake enhances inflammatory mediators like COX-2, and iNOS in rat cerebral cortex and cultured astrocytes. Astrocytes undergo actin cytoskeleton disorganisation, and there is a stimulation of both, interleukin receptor-associated kinase (IRAK)/extracellular signal-regulated kinases (ERK)/nuclear factor-kappaB (NF-kappaB) pathway and the COX-2 expression, which are associated with the inflammatory responses

Ethanol-induced glial activation is also associated with changes in the expression of inflammatory cytokines like IL-1alpha, TNF-alpha, IL-6. Notably, an increased expression of

influx, principally through NMDA-gated channels (Bambrick et al., 2004).

cytoskeleton (Allansson et al., 2001).

cellular death pathways (Gonzalez et al., 2006a).

their death can cause and/or aggravate diseases of the CNS.

**3.2.5 Ethanol, inflammation and immune response** 

(Guasch et al., 2007).

the pro-inflammatory cytokine MCP-1 (monocyte chemoattractant protein 1) and microglial activation as well as astrogliosis have been demonstrated by postmortem analyses in alcoholic brains (Gonzalez & Salido, 2009; He and Crews, 2008).

Besides ethanol, its primary metabolite acetaldehyde is also able to modulate TNF-alpha and IL-6 secretion from cultured astrocytes. Both compounds showed a biphasic, hormetic effect on the IL-6 secretion after the acute as well as after the long-term exposure. It has been shown that long-term exposure to ethanol and acetaldehyde is more toxic than an acute exposure. The maximum stimulation was reached for 50 mM ethanol and 1 mM acetaldehyde after chronic exposure. In contrast, both compounds reduced the TNF-alpha secretion, where the effect was concentration dependent. Acetaldehyde showed to be more potent toxin than ethanol, and the ethanol's toxicity in the brain is at least partially due to its primary metabolite, acetaldehyde (Šarc et al., 2011).

Inflammation is primarily a protective response of the target organism to a noxis. On the other hand, excessive or long-lasting inflammation is often followed by degenerative processes. The stimulatory effect of ethanol and acetaldehyde on IL-6 secretion seems to be involved in both neuroregenerative and survival processes as well as in neurodegeneration. The obtained hormetic dose-response relationship indicates that higher concentrations and long-term exposure could lead in a neurodegenerative direction whereas low concentrations may act as neuroprotective. Unlike TNF-alpha, which is responsible for the induction of multiple pro-inflammatory genes, IL-6 often fails to induce these genes. Moreover, IL-6 can down-regulate the expression of TNF-alpha, which correlate with the data, where the first significant decrease in the TNF-alpha level was found at the highest level of IL-6 after a long-term exposure to ethanol (Šarc et al., 2011).

#### **3.2.6 Ethanol and glial cell death**

Apoptosis or programmed cell death is a form of cell death that occurs in multicellular organisms. Apoptosis is a tightly regulated process which engages multiple cell signalling pathways, and involves the altruistic suicide if individual cells in favour of the organism. This process is desirably during organism development and morphological changes, especially at the embryonic stage, as well as during the activation of the immune system. However, defects in apoptosis can result in cancer, autoimmune diseases and neurodegenerative disorders. Studies on Ca2+ signalling in apoptosis showed that ethanol potentiates apoptotic cell death induction by thapsigargin, caffeine, and the protonophore, which separately caused similar increases in Ca2+ levels, and also induces similar apoptotic death. These effects of ethanol are concentration and time-dependent (Hirata et al., 2006).

The effect of ethanol on the induction of apoptosis in astrocytes, and the formation of ceramide as apoptotic signal was investigated by Schatter et al. (2005). Ethanol induced nuclear fragmentation and DNA laddering, and inhibited phospholipase D-mediated formation of phosphatidic acid, which is a mitogenic lipid messenger. The authors concluded that ethanol induced glial apoptosis during brain development via formation of ceramide. Further studies have shown that astrocytes exposed to ethanol, undergo morphological changes associated with anoikis, a programmed cell death induced by loss of anchorage. Astrocytes depicted peripheral reorganisation of both, focal adhesions and actin-myosin system, cell contraction, membrane blebbing and chromatin condensation (Gonzalez & Salido, 2009).

Ethanol Toxicity in the Brain: Alteration of Astroglial Cell Function 619

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Recently, it has been shown that ethanol affect intracellular trafficking. In fact, ethanol could interfere with nucleoplasmic transport in astrocytes, in such a way that ethanol induces a delay in both import and export of proteins to the nucleus (Marin et al., 2008).

Neurodegeneration, brain injury, and neuroinflammation are associated not only with increased cell apoptosis but also with the activation of a key proteolytic enzyme in this process, caspase-3. Immunohistochemical findings in mice, fed chronically with ethanol, reveal that inflammatory processes occur concomitantly with caspase-3 activation, suggesting an increase in programmed cell death. Moreover, it seems that the alcoholinduced toll-like receptor 4 (TLR4) response triggers both, inflammatory processes and apoptosis. A recent study suggests that the TLR4 response can also induce oxidative stress and neuronal injury, which agrees with a role of TLR4 in ethanol-induced brain damage and possibly in neurodegeneration (Alfonso-Loeches et al., 2010).

It has been shown that ethanol can activate or inhibit TLR4 by interacting with membrane lipids. Low/moderate ethanol concentrations (10–50 mM, in the range found in the blood of social drinkers and alcoholics) are capable of promoting translocation and clustering of TLR4 and a surface marker protein CD14, and the signalling molecules, like interleukin receptor-associated kinase (IRAK) and extracellular signal-regulated kinases (ERK), into the lipid rafts (Blanco et al., 2008; Fernandez-Lizarbe et al., 2008). Conversely, high ethanol concentrations or lipid raft-disrupting agents (streptolysin-*O* or saponin) inhibit ethanolinduced activation of the TLR4 signalling pathway (Blanco et al., 2008; Fernandez-Lizarbe et al., 2008). However, the molecular mechanism of ethanol interactions with TLR4 remains unknown.

#### **4. Conclusion**

Astrocytes are essential for maintaining a healthy and well-functioning brain. They face the synapses, send end-foot processes that enwrap the brain capillaries, and form an extensive network interconnected by gap junctions. They have the potential to impact on essentially all aspects of neuronal function through regulation of blood flow, provision of energy substrates, or by influencing synaptic function and plasticity. Moreover, astrocytes also protect and aid the brain in the functional recovery from injuries. The activation of glial cells in the CNS is the first defence mechanism against pathological abnormalities that occur in neurodegenerative diseases.

Ethanol has an extensive array of actions on astrocytes, transforming them into activated, potentially injurious cells with negative consequences to neuronal function and survival, and to brain function.

Therefore, it is a pivotal solution to seek molecular mechanisms and molecules that may inhibit or attenuate ethanol-induced neurotoxicity in astrocytes, thus offering an alternative strategy to prevent or treat neurodevelopmental disorders and mental retardation caused by ethanol.

#### **5. Acknowledgment**

The work was supported by the grant P3-0067 from the Slovenian Research Agency.

#### **6. References**

618 Pharmacology

Recently, it has been shown that ethanol affect intracellular trafficking. In fact, ethanol could interfere with nucleoplasmic transport in astrocytes, in such a way that ethanol induces a

Neurodegeneration, brain injury, and neuroinflammation are associated not only with increased cell apoptosis but also with the activation of a key proteolytic enzyme in this process, caspase-3. Immunohistochemical findings in mice, fed chronically with ethanol, reveal that inflammatory processes occur concomitantly with caspase-3 activation, suggesting an increase in programmed cell death. Moreover, it seems that the alcoholinduced toll-like receptor 4 (TLR4) response triggers both, inflammatory processes and apoptosis. A recent study suggests that the TLR4 response can also induce oxidative stress and neuronal injury, which agrees with a role of TLR4 in ethanol-induced brain damage and

It has been shown that ethanol can activate or inhibit TLR4 by interacting with membrane lipids. Low/moderate ethanol concentrations (10–50 mM, in the range found in the blood of social drinkers and alcoholics) are capable of promoting translocation and clustering of TLR4 and a surface marker protein CD14, and the signalling molecules, like interleukin receptor-associated kinase (IRAK) and extracellular signal-regulated kinases (ERK), into the lipid rafts (Blanco et al., 2008; Fernandez-Lizarbe et al., 2008). Conversely, high ethanol concentrations or lipid raft-disrupting agents (streptolysin-*O* or saponin) inhibit ethanolinduced activation of the TLR4 signalling pathway (Blanco et al., 2008; Fernandez-Lizarbe et al., 2008). However, the molecular mechanism of ethanol interactions with TLR4 remains

Astrocytes are essential for maintaining a healthy and well-functioning brain. They face the synapses, send end-foot processes that enwrap the brain capillaries, and form an extensive network interconnected by gap junctions. They have the potential to impact on essentially all aspects of neuronal function through regulation of blood flow, provision of energy substrates, or by influencing synaptic function and plasticity. Moreover, astrocytes also protect and aid the brain in the functional recovery from injuries. The activation of glial cells in the CNS is the first defence mechanism against pathological abnormalities that occur in

Ethanol has an extensive array of actions on astrocytes, transforming them into activated, potentially injurious cells with negative consequences to neuronal function and survival,

Therefore, it is a pivotal solution to seek molecular mechanisms and molecules that may inhibit or attenuate ethanol-induced neurotoxicity in astrocytes, thus offering an alternative strategy to prevent or treat neurodevelopmental disorders and mental retardation caused by

The work was supported by the grant P3-0067 from the Slovenian Research Agency.

delay in both import and export of proteins to the nucleus (Marin et al., 2008).

possibly in neurodegeneration (Alfonso-Loeches et al., 2010).

unknown.

**4. Conclusion** 

neurodegenerative diseases.

and to brain function.

**5. Acknowledgment** 

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**Part 7** 

**Future Applications** 

