Effect of Alcohol on Brain Development Effect of Alcohol on Brain Development

In the world, 3.3 million deaths occur every year due to harmful use of alcohol; this represents 5.9% of all deaths. Ethanol metabolites’ production and their post-translation modification are one of the proposed mechanisms that lead to neuronal toxicity. The projected neurochemical changes in chronic alcohol drinkers may be due to an imbalance between excitatory and inhibitory neurotransmitters. Interaction of alcohol with GABA and glutamate receptors (NMDA and AMPA) resulted in diverse adaptive changes in gene expression through neuronal pathways leading to alcohol toxicity. Alcohol con - sumption in an individual leads to biochemical changes that are correlated with complex inflammatory signaling pathways such as phosphorylation of proteins, synthesis of nitric oxide (NO), NF-kappaB and MAP kinase pathways in certain regions of the brain. Ethanol exposure activates neurons and microglial cells that lead to release of neu - roimmune factors like high-mobility group box 1 (HMGB1), toll-like receptor 4 (TLR4) and certain cytokines involved in immune responses leading to neuroimmune signaling in the brain. Epigenetic modification of DNA and histones may lead to neuronal gene expression, thus regulating ethanol toxicity. Researchers attempt to modulate therapies that can help to foil alcohol toxicity and support the development of original neuronal cells that have been injured or degenerated by alcohol exposure. synaptic plasticity and synaptic formation. Downregulation of genes by ethanol includes protein synthesis, myelination and the ubiquitin-proteasome pathway. Chronic ethanol exposure increases HMGB1–TLR4 and NF-κB signaling which leads to improved NF-κB target genes’ expression. This results in determining neuroimmune responses to ethanol toxicity that releases HMGB1 or directly stimulates TLR and/or NMDA receptors. An epigenetic mecha nism will show potential towards drug dependence by changing the DNA protein structure. Microglial cells will arbitrate the effect of alcohol toxicity on neurogenesis. The progression towards the neurobiologic techniques including micro array, QTL and proteomics will pro vide some anticipation for researching the molecular and cellular mechanisms that act as a keystone for the understanding of neuronal toxicity and enlightening new therapeutic gene targets for this public health burden.


How alcohol is absorbed into the body?
Some people drink socially and do not get addicted while others do. The ground behind the alcohol drinking is related to psychological, physiological, genetics and social factors.
When people consume alcohol, about 20% is absorbed in the stomach and almost 80% is absorbed in the small intestine. Alcohol absorption is related to the two main factors: a. concentration of alcohol and b. heavy meal consumption before drinking. An empty stomach will fasten the alcohol absorption.
Absorbed alcohol enters the blood stream and is carried all through the body. Upon reaching the body, simultaneously the body works to eliminate it. The 10% of alcohol is removed by the kidneys (urine) and lungs (breath). Left-out alcohol is oxidized by the liver, converting alcohol into acetaldehyde first and then further converted to acetic acid.

Alcohol metabolism and distribution
Alcohol is absorbed in the stomach and intestines through the blood stream and crosses the blood-brain barrier (BBB) after consumption. Alcohol is metabolized through the mitochondrial cytochrome P450 (CYP2E1) and catalase in the liver or the brain. In the brain, alcohol metabolism by CYP2E1 produces acetaldehyde and alcohol is considered to be the only source of acetaldehyde. The rest of the acetaldehyde may go through the brain by an enzyme called alcohol dehydrogenase (ADH) which is found in the liver and helps in conversion of alcohol to acetaldehyde. Target of alcohol is considered to be ADH. Alcohol binds through its hydroxyl (─OH) group and zinc atom on ADH [1]. Disulfiram (antabuse), one of the first approved treatments for alcoholism, showed its mechanism by inhibiting the ADH enzyme. When taken regularly, disulfiram decreases the drinking capacity because of the aversive effects [2].

Alcohol and the brain 2.1. How does alcohol act at the neurological level?
Brain chemistry is affected by alcohol through alteration of neurotransmitters. Neurotransmitters are chemical messengers that send out the signals all through the body and control thought processes, behavior and sensation processes. Neurotransmitters are either excitatory (excite brain electrical motion) or inhibitory (decrease brain electrical motion). Alcohol increases the effects of the inhibitory neurotransmitter GABA in the brain. GABA causes the lethargic movements and garbled speech that often occur in alcoholics. At the same time, alcohol inhibits the excitatory neurotransmitter glutamate, which results in a suppression of a similar type of physiological slowdown. In addition, alcohol also increases the amount of chemical dopamine in the brain center, which creates the feeling of pleasure after drinking alcohol. Just after a few drinks, the physical effects of alcohol become perceptible. These effects are linked to blood alcohol concentration (BAC) ( Table 1). The level of BAC rises when the body takes up alcohol faster than it can release it.
In the brain, various centers have been affected due to alcohol, both upper and lower order. As the BAC increases, more centers of the brain are affected (Figure 1) [3]. The order in which brain centers are affected by alcohol consumption is as follows: They become more selfassured or brave.
They become lethargic. They become woozy and may walk unsteadily.
They can barely move at all.
They are unconscious.
The person usually stops breathing and dies.
Their awareness period shortens.
They have a problem in understanding or recalling things (even recent events).
They may be highly emotional, destructive, reserved or overly loving.
They may lapse in and out of consciousness.
Their reflexes are depressed.
They may look flushed (red).
They do not respond to situations as quickly They cannot observe clearly.
They cannot respond to stimuli.
They feel cold.
Their conclusive power is not as good.
Their body movements are uncoordinated.
They are drowsy. They cannot stand or walk.
Their breathing is slower and shallower.
They have trouble with fine movements, such as writing.
They start to lose their sense of balance easily.
They have garbled speech.
They may vomit. Their heart rate may slow.
Their vision becomes dim.
They have clumsy movements.
They may die.
They may have problems in sensing things (hearing, tasting, emotion, etc.).
They may not feel tenderness as readily as a sober person. Heavy or continuous consumption of alcohol can lead to the liver injury. The liver is the chief organ responsible for converting alcohol into nontoxic byproducts and taking them out of the body. Excessive alcohol consumption leads to prolonged liver dysfunction which may also harm the brain, leading to a severe fatal brain disorder known as hepatic encephalopathy [5]. Studies have confirmed that at least two toxic substances, ammonia and manganese, play an important role in the progress of hepatic encephalopathy.
Treatment-Strategies that have been used to treat or prevent the development of hepatic encephalopathy are as follows: 1. L-ornithine L-aspartate: it lowers the concentration of blood ammonia 2. Artificial livers: it clears patients' harmful toxins present in the blood [6] 3. Liver transplantation [7]

Alcohol and the developing brain
Alcohol consumption during pregnancy can lead to changes in the physical, learning and behavioral effects in the developing brain and it is known as fetal alcohol syndrome (FAS) [8].The brains of these people may have less size (i.e., microencephaly) and also a small amount of brain cells (i.e., neurons) that function accurately resulting in long-lasting problems in learning and behavior.

Treatment:
Researchers are looking forward to treat or prevent brain damage, such as associated with FAS.

In-vivo studies yielded a result showing antioxidant therapy and vitamin E which is used
for treating FAS.

2.
Treatment with l-octanol (paradoxically an alcohol itself) on developing mouse embryos significantly reduced the rigorousness of alcohol's effects [8].
3. NAP and SAL have the same property as octanol. They both help in protecting nerve cells against a variety of toxins [9].

4.
A compound called MK-801 that blocks glutamate that helps with alcohol withdrawal [10].
During alcohol drinking, there is a release of excessive glutamate leading to neuronal cell death. This occurs through two pathways: 1. Activation of glutamate receptors causes toxicity [12], resulting into Ca +2 ion influx, Na+ influx, nitric oxide (NO) generation and depolarization of mitochondria. This will lead to untenable elevations in ATP, mitochondrial collapse, dendritic beading and depolymerization of microtubule [13].
A study has been carried out on 13 abstinent young alcoholics showing a major increase in glutamate to creatine ratio by proton magnetic resonance spectroscopy and magnetic resonance imaging [15]. Furthermore, an alteration in glutamate is linked to altered short memory loss. Immediate administration of alcohol (acute dose) prior to the microdialysis experiment would end up into an increased glutamate release. Earlier studies showed that administration of either 2 or 3 g/kg ethanol to immature rats elicits a decrease or no modification in the release of glutamate into the N-acetyl cysteine (NAC) [16]. Researchers showed that there is a genetic component that probably contributes to the brain injury occurring in "binge drinking" alcoholics [17]. In binge drinking, alcohol models, there are no reports of increased NMDA receptors [18]. In one study, "binge-drinking" individuals with compensated alcoholic cirrhosis, dosing 80 g of ethanol, showed a transient increase in serum nitrates and nitrite resulting in an increase in NO production in certain tissues (liver and brain) [19]. During the period of chronic alcohol toxicity, basal concentration of glutamate seems to be normal in various regions of the brain though blood alcohol levels are high as 2 g/l [20].
During chronic alcohol consumption, NMDA receptor (NMDAR1 and NMDAR2B) levels seem to be increasing in numbers and decreasing in sensitivity [21] in the nucleus [22], the striatum [23] and the hippocampus [20].

Gamma-amino-butyric acid
It is a chief inhibitory neurotransmitter. GABA binds to GABA A receptors, resulting in hyperpolarization of the cell membrane and inhibition of neural activity. Increased GABA release upon alcohol administration results due to inhibiting its degradation [24]. Alcohol intoxicity and alcohol's anti-anxiety reduce due to decrease in GABA A receptor activity. Chronic alcohol exposure decreases extrasynaptic GABA-mediated tonic current recorded from neurons in the hippocampus and cortex [25], and this corresponds to a decrease in extrasynaptic GABA A receptors containing the d subunit in hippocampus [26]. Benzodiazepines (the positive allosteric modulator of GABA A ) are considered to be a standard for treating alcohol detoxification owing to their anticonvulsant and anxiolytic pharmacological profile. Improper alcohol interactions have shown some concern about their abuse and dependence responsibility. Therefore, researchers are trying to find out potential anticonvulsant agents as alternatives, that is, gabapentin and topiramate [27,28].

Dopamine
Transmission of dopamine is linked via two groups of G-protein-linked receptors: D1-like (D1 and D5 receptors) and D2-like (D2, D3 and D4 receptors). These receptors are classified on the basis of adenylatecyclase activity (stimulation or inhibition). Dopamine plays a central role in mediating alcohol compensation through mesocortical and mesolimbic pathways [29].
During chronic alcohol consumption, the release of dopamine depends on the large amount of the alcohol consumption, which gives pleasurable effect of alcohol intake. Reduction in dopamine release is observed during alcohol withdrawal. This ultimately reduces noticeable neuronal cells, leading to dysphoria and depression as a major part in the motivational and behavioral changes [30]. Chronic alcohol drinking has been reported to produce constant neurological changes in transmission of dopamine within the mesoaccumbens reward circuitry, including increased basal extracellular levels of dopamine in the NAC [31], increased firing rate in the ventral tegmental area (VTA) dopamine neurons [32] and changes in the function of dopamine receptor [33]. In comparison, withdrawal from chronic alcohol drinking due to increased dopamine uptake levels resulted in decreased VTA dopamine neuronal activity [34], and reduced basal levels of dopamine in the ventral and dorsal subregions of the striatum, possibly due to enhanced dopamine uptake [35]. The aripiprazole (D2 dopamine receptor agonist) has shown some effectiveness in treating dependence of alcohol [36].

Serotonin
Serotonin exerts its known role in regulating various behaviors (e.g., feeding, sleep/arousal, aggression), mood and emotional behavior [37] via several metabotropic (5-HT1 and 5-HT2 subtypes) and ionotropic (5-HT3) receptors throughout the brain depending on the consumption of alcohol [38]. Alcohol elevates serotonin release in the central nervous system (CNS) affecting emotion, temper and thoughts. The 5-HT3 receptor function is altered by ethanol consumption through its actions on receptor proteins [39]. Chronic alcohol exposure reduces serotonin levels in several brain regions [40]. Researchers have focused on the treatment for alcohol dependence and comorbid depression [41], post-stress disorder [42] and anxiety [43].
Inhibition of serotonin reuptake was done through fluoxetine. The milnacipran blocks both serotonin and norepinephrine reuptake. Both fluoxetine and milnacipran are found to be effective in reducing alcohol consumption in the rats model [44]. Consumption of alcohol (5%) every third day for 18 days leads to disturbance in serotonin function within the nucleus accumbens [45] (Tables 2 and 3).

Biochemical changes associated with alcohol intoxicity
Alcohol consumption in an individual leads to biochemical changes that are correlated with complex signaling pathways such as phosphorylation of proteins, synthesis of nitric oxide (NO), NFκBand MAP kinase pathways in certain regions of the brain [48].

Generation of free radicals
Alcohol-induced brain damage may occur due to generation of free radicals [49] by acetaldehyde intoxicity [50] or microsomal ethanol oxidizing system (MEOS) [51]. This can be minimized, if there would be a reduction in glutathione (cytoprotection) in the specific regions of the brain which is affected.

Changes in phosphorylation
Cell signaling events depend on phosphorylation of proteins (phosphorylation: protein kinase and dephosphorylation: protein phosphatases). During intoxication in hippocampal dentate gyrus,
Effect of Alcohol on Brain Development http://dx.doi.org/10.5772/intechopen.73693 the total phosphorylated c-AMP response element-binding protein (CREB) immunoreactivity is reduced in both chronic or binge drinking. In rat cerebellum, the dose of 3 g/kg alcohol (acute) shows an increase in a phosphorylated form of CREB. Research showed that protein phosphatase inhibitor (DARPP-32), when it undergoes phosphorylation changes, plays a crucial role in reducing ethanol inhibition of NMDA receptors [52].

Transcription factors
They are the first signaling proteins which are regulated through sensitive cysteine residues, which need to be reduced to its function. Earlier studies showed that production of reactive oxygen species (ROS) is responsible for the activation of NFκB [53]. Later in vitro studies showed that exogenous hydrogen peroxide is responsible for NFκB activation [54]. In the cortex, one study reported activation of NFκB in chronically alcoholized rats [55]. It was quite clear that NFκB was down-regulated in comparison to acute alcohol drinking. One study showed no NFκB activation in binge alcohol drinking models, when given anti-oxidants (furosemide and butylatedhydroxytoluene) to prevent neuronal degeneration [56].

Epigenetic mechanism
It is an efficient mechanism for gene regulation by altering the packaging of DNA within chromatin through interactions with histones. CREB requires phosphorylation to initiate transcription of pro-survival neuronal factors (Figure 2). A study revealed an increase in CREB (a regulator for plasticity of neurons), increase in H3-H4 (a central histone tetramer) acetylation and inhibition of HDAC (histone deacetylases) activity in a rat amygdala when given an acute dose of ethanol for 15 days. A trichostatin (HDAC inhibitor) was used to treat the rats, resulting in deacetylation of H3-H4, CREB inhibition, reduced NPY expression and HDAC activation in the rat amygdala [57].

Genome profiling of alcohol-responsive genes
With recent technologies like direct sequencing and gene microarray, studying individual genes along with entire genome of an organism has been made possible.

Microarray study
First genome microarray studies were performed in post-mortem alcoholics' human brain; results revealed that myelin related genes expressed in oligodendrites (from frontal cortex) showed downregulation in the brains of human alcoholics compared with brains from control individuals [58], suggesting neuronal dysfunction. In the prefrontal cortex area of the brain, there were 54 genes which were upregulated and those belong to the class of heat shock protein, including HSP70-2, CRYAB, HSP27-1 and HSP40-1 in alcoholic abusive individuals [59]. An old study revealed that 100 mM ethanol (2 days) given to NG108-15 hybrid cell line determined that a number of genes were induced by heat shock and a few were induced by ethanol only. Heat-shock cognate proteins Hsc 70 and Hsc 110 were recognized as ethanol-inducible genes [60]. When C. elegans was given a high dose of ethanol, it showed activation of glr-2, a gene that encodes a subunit of the AMPA glutamate receptor subunit (homologous to mammalian GluR2) within 15 min [61]. Cultured cortical neurons given 75 mM of ethanol for 5 days showed increased levels of gene expression of Hsp84, Hspa8 and Hsp70 [62]. In a whole brain, when C57BL/6J and DBA/2J mouse strains were given acute doses of different ethanol preferences, they revealed downregulation of Erbb3, Mobp and Nkx2-2 (myelin-related genes) [63]. The researcher performed a genomic level along with a microarray experiment on neurons of mouse corticals given 60 mM ethanol or heat treatment at 42°C. Microarray results showed upregulation of a large number of genes by ethanol and heat shock [64]. Among the pool of genes, there were nine genes which showed greater than 50% stimulation. It includes.

Analysis of quantitative trait loci
A quantitative trait loci (QTL) refers to the trait that varies in degree and can be attributed to the interactions between two or more genes and their environment. Possible QTLs have been identified through microarray studies for alcohol uptake. An analysis is done across inbred mouse strains identified a group of genes for alcohol preference QTL on chromosome 9. These genes include Arhgef12, Carm1, Cryab (heat shock protein), Cox5a, Dlat, Fxyd6, Limd1, Nicn1, Nmnat3, Pknox2, Rbp1, Sc5d, Scn4b, Tcf12, Vps11 and Zfp291 [65].

Activation of multiple neuroimmune genes in human alcoholic brains
In human brain-slice cultures, multiple ethanol-induced cytokines are released. Among all, monocyte chemotactic protein-1 (MCP-1) showed increased levels in the amygdala, nucleus accumbens, VAT and hippocampus [66]. In alcoholic brains, elevated levels of TLR 2, TLR3, TLR4 and HMGB1 (high-mobility group box 1) were found in the orbital frontal cortex (OTC) [67]. Release of HMGB1 leads to disruption of synaptic plasticity which causes hyperexcitability of neurotransmitters due to ethanol exposure. There is an increased level of IL-1β inflammatory marker in an alcoholic brain (hippocampus) causing neuro-degeneration [68]. RAGE (HMGB1 receptor) has shown an increased level in an alcoholic brain [69].

Role of microglia
Exposure to alcohol causes activation of microglia along with the proinflammatory cytokines, leading to neuronal inflammation and toxicity [70]. Alcohol exposure causes accumulation of microglia in the brain which occurs through activation of TLRs leading to increased HMGB1 expression [71]. Alcohol-induced neuronal apoptosis leads to stimulation of the transcription factor AP-1 and release of IL-1β, IL-6 and transforming growth factor β (TGF-β1) [71]. An in vitro study revealed that microglial TNF-α production plays an important role in neuronal toxicity [72]. Neuronal cell death occurred due to chronic alcohol exposure which leads to upregulation of the NF-κB expression, which in turn leads to release of TNF-α resulting in neuronal apoptosis [73].
In vivo studies in the cortical and hippocampus region of the brain showed increased levels of NADPH oxidase, superoxide and microglial activation, which is correlated with alcoholinduced ROS production [74]. In vitro studies revealed that upon alcohol exposure, microgliaconditioned cells showed increased ROS production and induced oxidative stress in cultured hypothalamic neuronal cells; this leads to neuronal apoptosis [75]. Alcohol-induced elevation of TGF-β1 levels in neuronal cells is accompanied by a host of molecular and chemical changes such as increase in E2F1 protein expression, mitochondrial proapoptotic proteins bak, bad and bcl-xs and E2F1 protein expression and simultaneously decrease in cyclin D1, cyclin-dependent kinase-4 expression and antiapoptotic protein bcl-2 leading to neuronal apoptosis [76] (Figure 3).

The brain and immune responses
The interface between the brain and the immune system is bidirectional. Recent findings have revealed that alcohol causes the release of HMGB1 in the gut, which in turn activates TLR4 resulting in an increased gut permeability. As a result, there is activation of proinflammatory cytokines in the liver, which leads to induction of TNF-α and other cytokines in the blood. Researchers found that LPS induced increases in serum TNF-α as well as proinflammatory cytokines, leading to induction of the gene in the brain [77]. This proinflammatory cytokines in the blood are then transported across the blood-brain barrier (BBB) by their receptors [78]. For example, 2 to 3 g/kg ethanol when administered into the stomach results in the activation of innate immune response in the gut [79]. This damages the tight junction present in the gut resulting in an opening of the sites where gut bacteria and their endotoxins (LPS) can easily enter the blood stream leading to the liver, where they can activate proinflammatory cytokines (Figure 4). Increased proinflammatory cytokines responses, which affects the brain through TNF-α and other cytokines [80]. The brain response to the proinflammatory cytokine MCP-1 is quite longer than that found in the liver and blood [81]. An in vivo study showed an increased LPS induction of proinflammatory cytokines (TNF-α, IL-1β and MCP-1) only in the brain but not in the liver and blood after ten daily doses of alcohol [81]. In the liver, the researcher suggested that the anti-inflammatory cytokine (IL-10) inhibits NF-κB which was increased after 1 week of ethanol treatment but decreased in the brain [81]. After 10 days of ethanol (5g/kg/day) administration to mice model, this study showed sensitization to TLR3 agonist Poly:IC which induces proinflammatory cytokines in the brain for 24 hours [74].

Conclusion(s)
Alcohol is an anxiolytic and soothing drug. Chronic alcohol consumption leads to determined molecular and cellular modification in the brain system. It is comprehensible that GABA and glutamate neurotransmitters play a crucial role in alcohol toxicity, neuronal toxicity and neuronal cell death. Ethanol exposure triggers the activation of various gene expressions involved in apoptosis, in oxidative stress and in the cell cycle. Upregulation of genes by ethanol includes heat shock proteins and proteins related to synaptic neurotransmission, synaptic plasticity and synaptic formation. Downregulation of genes by ethanol includes protein synthesis, myelination and the ubiquitin-proteasome pathway. Chronic ethanol exposure increases HMGB1-TLR4 and NF-κB signaling which leads to improved NF-κB target genes' expression. This results in determining neuroimmune responses to ethanol toxicity that releases HMGB1 or directly stimulates TLR and/or NMDA receptors. An epigenetic mechanism will show potential towards drug dependence by changing the DNA protein structure. Microglial cells will arbitrate the effect of alcohol toxicity on neurogenesis. The progression towards the neurobiologic techniques including micro array, QTL and proteomics will provide some anticipation for researching the molecular and cellular mechanisms that act as a keystone for the understanding of neuronal toxicity and enlightening new therapeutic gene targets for this public health burden.