Abstract

Epigenetics, the study of heritable changes in gene expression without modifying the DNA sequence, offers valuable insights into alcoholism. This paper examines the roles of DNA methylation, histone modifications, and microRNA (miRNA) regulation in alcohol use disorder (AUD). Alcohol consumption has a significant impact on brain function, often impairing cognitive abilities, and stimulating dopamine production, which reinforces addictive behaviors. Key findings include the presence of hypermethylated genes and regions with differential methylation patterns in AUD, characterized by a higher density of cytosine and guanine nucleotides (CpG sites). Additionally, important histone modifications are critical in the epigenetic regulation associated with AUD. Furthermore, alcohol consumption alters miRNA expression profiles, impacting genes involved in inflammation, stress response, and neuronal signaling pathways. Understanding these epigenetic mechanisms provides a strong molecular foundation for developing targeted therapies and preventive strategies for AUD. By clarifying these mechanisms, this study emphasizes the importance of epigenetic research in addressing the complex nature of AUD and lays the groundwork for innovative approaches to combat this widespread condition.

1. Introduction

Alcohol use disorder (AUD) refers to the inability to control alcohol consumption, influenced by a combination of genetic, epigenetic, and environmental factors. AUD can be categorized as either alcohol abuse and/or alcohol dependence. A diagnosis of dependency requires the satisfaction of at least three criteria, while abuse requires only one criterion involving alcohol use (Longley et al., 2021). This disease affects over 29.5 million people aged over 12 years in the US alone, and in 2019 caused 2.6 million deaths globally (National Institute on Alcohol Abuse and Alcoholism, 2007; World Health Organization [WHO], 2024). This paper delves into the epigenetic mechanisms involved in alcoholism, exploring how alcohol affects gene expression and the broader implications of these changes. 

The consumption of alcohol results in intoxication through a complex biochemical process metabolized in the liver. However, when it is consumed faster than the liver can process, excess alcohol enters the bloodstream, leading to intoxication. This not only impairs brain function but also stimulates dopamine production, which reinforces alcohol consumption and can lead to addiction. Alcohol consumption significantly impacts brain function, often impairing cognitive abilities and stimulating dopamine production, reinforcing addictive behaviors (Koob & Volkow, 2016). 

One of the key epigenetic changes associated with AUD is DNA methylation, which typically suppresses gene expression. This involves methylation at the fifth carbon of cytosine bases, which commonly occurs at 5’- C – phosphate – G – 3’ (CpG sites) dinucleotides (cytosine followed by a guanine) (Longley et al., 2021). Epigenome-wide association studies (EWAS) have further revealed numerous CpG sites with differential methylation patterns in individuals with AUD, underscoring the widespread epigenetic impact of alcohol. Key findings include the presence of hypermethylated genes and regions with differential methylation patterns in AUD, characterized by a higher density of cytosine and guanine nucleotides (CpG sites) (Zhang et al., 2013). 

Histone modifications, another form of epigenetic regulation, play a significant role in AUD by altering chromatin structure and gene expression (Nestler, 2014)​. Additionally, microRNAs (miRNAs), which are small non-coding RNA molecules, regulate gene expression post-transcriptionally, influencing various biological processes (Huang et al., 2011). Furthermore, alcohol consumption alters miRNA expression profiles, impacting genes involved in inflammation, stress response, and neuronal signaling pathways (Pietrzykowski et al., 2008).

The objective of this review is to explore the epigenetic mechanisms underlying alcoholism, with a focus on DNA methylation, histone modifications, and miRNA regulation. By identifying specific epigenetic changes associated with AUD, this study aims to enhance the understanding of the molecular basis of alcohol addiction and its related disorders. The findings carry significant implications for accelerating/advancing the development of targeted therapies and preventive strategies for AUD.

2. Effect of alcohol in the human body

The common drinking alcohol is ethanol, or ethyl alcohol; it is an organic compound with the chemical formula – CH3CH2OH.  This alcohol is a liquid produced by the fermentation of sugars found in fruits. Examples of this include wine and cider, based on grapes and apples respectively. Alcohol also arises from the fermentation of grains and starches: potatoes and cereal grains are transformed into the popular vodkas and whiskeys. Alcoholic drinks are classified according to their alcohol by volume (ABV), or the percentage of alcohol they contain, or the number of fluid mL per 100mL. Beers and wines typically have a concentration between 5-12% alcohol, while stronger liquors contain 30-60% alcohol (ARK Behavioral Health. (n.d.)) The alcohol percentage and the amount of alcohol consumed both contribute to the rate of alcohol intoxication.

Intoxication is a complex process of physical events in the body. The human body perceives alcohol as a toxin, so the digestive tract sends it to the liver in an attempt to break it down. With small amounts of alcohol, the ethanol molecule is dismantled and releases an organic chemical compound, acetaldehyde (aldehyde), which is attached to an enzyme called acetaldehyde dehydrogenase. This process also releases another substance called glutathione, which is attracted to aldehyde due to aldehyde’s large amount of cysteine. Both glutathione and aldehyde have a high concentration of glycine, which is strongly attracted to cysteine. This reaction results in a nontoxic acetate called malonic acid, which has traits similar to vinegar. However, intoxication may occur if the excess alcohol that has yet to be broken down leaks into the bloodstream and blocks nerve receptors all over the body.

This surplus alcohol can lead to internal effects that damage the central nervous system, vision, respiratory system, gastrointestinal system, and liver. Damage to the central nervous system can lead to impaired judgment, reduced coordination, and slower reaction times. Intoxication can cause mood swings, increased sociability, or aggression. Some people may feel relaxed and euphoric, while others may become sad or extremely angry. Coordination and balance are often compromised, leading to difficulties in walking, standing, and performing tasks that require fine motor skills. Slurred speech is a common sign of intoxication, as alcohol affects the muscles involved in speech production. Alcohol can impair vision, leading to blurred or double vision and difficulty focusing. High levels of alcohol can depress the respiratory system, leading to slowed or irregular breathing, which can be quite dangerous. Alcohol can irritate the stomach lining, leading to nausea, vomiting, and potential gastrointestinal bleeding in severe cases. Alcohol is a diuretic, a substance that promotes urine production, which can lead to increased urination and dehydration, contributing to the most prominent hangover symptoms. 

Alcohol is a depressant that slows down brain function and neural activity. More severe intoxication can cause confusion, uncontrolled eye movements, sleepiness, dizziness, delusions and hallucinations, severe difficulty speaking, severe deficits in coordination and psychomotor skills. These are all the result of slowed neurotransmission due to the burden placed on the brain by intoxication. Excessive consumption also can lead to liver damage, fatty liver, hepatitis, or cirrhosis of the liver. Chronic alcohol abuse can lead to addiction, mental health issues, cardiovascular problems, and various other health complications. After a long period of drinking, the brain begins to rely on alcohol to produce certain chemicals. This is what makes it difficult for heavy drinkers to quit and can cause uncomfortable withdrawal symptoms.

Alcohol Use Disorder (AUD) is a medical condition characterized by an individual’s inability to control or stop drinking despite negative consequences. It encompasses a range of behaviors, including a strong craving for alcohol, increased tolerance, and withdrawal symptoms when not drinking. AUD can lead to significant impairment in social, occupational, and interpersonal functioning. It is classified into mild, moderate, and severe categories based on the number of diagnostic criteria met. According to The Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-V), AUD is defined clinically as a pattern of alcohol use that leads to significant impairment or distress, as manifested by at least two of the following criteria occurring within a 12-month period, listed in Table 1.

Table 1: Criteria to diagnose AUD, according to DSM-V.

The severity of AUD is classified as mild (2-3 criteria), moderate (4-5 criteria), or severe (6 or more criteria).  

In 2016, the World Health Organization (WHO) estimated that 283 million people over the age of 15 had an alcohol use disorder worldwide, which is 5.1% of adults. In 2019, the WHO estimated that 400 million people over the age of 15 had an AUD. It is estimated that alcohol contributes to around 3 million deaths worldwide per year, equivalent to 6% of the global death count. The causes of AUD have been studied for at least 50 years.  AUD can stem from many different factors: biological, environmental, and social. 

Biological and genetic factors account for at least 60% of AUD diagnoses. Studies have concluded that alcohol dependence could be related to at least 51 genes, but much is still not known about why genes get expressed in some individuals and not others. Another major biological cause is the dopamine that is released from consuming alcohol (Edenberg, H.J., 2007). While alcohol is a depressant, and can cause many unpleasant symptoms if over consumed, it also stimulates dopamine production in the brain. Dopamine is a type of monoamine neurotransmitter. It acts as a chemical messenger, sending communications between nerve cells in the brain and the rest of the body. Specifically, it is known to create feelings of happiness, contentment, and pleasure. Dopamine is also involved in the brain reward system: human brains are hardwired to seek and enact behaviors that are rewarded with dopamine production (‌HowStuffWorks, 2004). Alcohol causes a synthetic chemical reaction in the brain that produces excess dopamine through brain reward circuitry. When exposed to a rewarding stimulus, the brain responds by releasing more of the neurotransmitter dopamine. This spreads a warm sense of pleasure all over the body. It is this desire for good feelings that is a major contributor to alcohol addiction, or otherwise known as AUD.

Environmental factors contributing to AUD include proximity to, availability of, and cultural attitude toward alcohol. It turns out that advertising impacts the public’s interest in alcohol, increasing purchase of alcoholic products. Living near bars and having access to alcohol in stores increases the occurrence of alcohol abuse. In addition, income impacts AUD: gallup polls in the US report that affluent individuals consume more alcohol than those living below poverty (Alcohol Rehab Guide, 2023).  

Separate consideration is given to how social factors impact AUD. Family, culture, religion, work, and friend groups all impact beliefs and habits of alcohol use. The biggest social factor is family. Children exposed to parents’ excessive drinking behavior are at much higher risk of becoming susceptible to AUD (Alcohol Rehab Guide, 2023). 

3. Epigenetic mechanisms: biological processes causing epigenetic modifications

DNA methylation is one of the most studied epigenetic modifications for alcoholism. In the promoter region, methylation has been shown to either promote or inhibit transcription factor binding, affecting transcription and gene expression (Longley et al., 2021). Studies investigating DNA methylation in AUD have identified specific genes that are hypermethylated in individuals with AUD. Cytosine methylation in non-CpG sites also has physiological relevance, as Hotchkiss suggested that peaks on his chromatogram were due to cytosine methylation (Rodriguez, 2021). Proteins in the DNA methyltransferase family (DNMTs) usually carry out methylation, resulting in hypermethylation (Longley et al., 2021). Mammals have different types of DNMTs, DNMT1, DNMT2, and DNMT3. These generally fall under 2 families: DNMT3A, DNMT3B and DNMT3C (catalyzes de novo methylation of DNA) is one family, and DNMT1 is another (maintenance of DNA methylation). DNMT2 has no role in catalysis of DNA methylation. These DNMTs reach specific areas of the promoter regions or gene bodies for methylation. To maintain the methylation, patterns of methylation after each cell division are conserved (Rodriguez, 2021).

Changes to the structure of histones, such as acetylation, have mechanisms that may be related to alcoholism. Modifications regulated by the cAMP response element-binding protein (CREB) transcription factor are especially critical in the epigenetic regulation associated with AUD (Pandey et al., 2008). There are 5 types of histone: H1, H2A, H2B, H3 and H4. Histone acetylation is a specific covalent and reversible epigenetic modification, where an acetyl group from acetyl-CoA binds to the ε-amino group of lysine residues. This reaction is catalyzed by histone acetyltransferases, or HATs. When basic residues are acetylated, the positive charge is decreased and thus the histone-DNA interaction is weakened, facilitating transcription. Histone methylation is another such modification, catalyzed by histone methylases; this reaction occurs on basic residues on histones H3 and H4. S-adenosyl-methionine is used as the methyl donor. Methylation can trigger different results, based on where it is present. If lysine at position 4 of H3 is methylated, gene activation occurs. But if lysine 9 is methylated instead, gene inactivation occurs instead. Histone phosphorylation takes place mainly at certain residues: serine, threonine and tyrosine. This modification can alter charge and mass of the histone and can affect chromatin structure and function when present with another modification (Rodriguez, 2021). This is illustrated by the example of TRPM6-cleaved kinase; when it phosphorylates histones, methylation of arginine residues also occurs (Utley et al., 2005). 

The final modifications discussed in this paper are miRNA modifications. miRNAs mark 3’ UTR (untranslated) sequences by using complementary base pairing. Initially, miRNAs are created as primary miRNA transcripts, which then undergo modification by nuclear ribonuclease Drosha and cytoplasmic endonuclease Dicer (Rodriguez, 2021). MicroRNAs are capable of targeting DNA methylases’ activity, and they do so by regulating expression of DNA methylase. miR29b does so for the expression of DNMT3 and ten eleven translocation enzymes (TET enzymes) (Zhang et al., 2018). They may also control the expression of certain proteins that modify histones. One example of this is lysine-specific demethylase in rat amygdalas, which are controlled by miR-137 (Rodriguez, 2021).

4. Epigenetic mechanisms affecting alcoholism

4.1 DNA methylation

The first epigenetic factor affecting alcoholism is DNA methylation. There are two categories of studies investigating DNA methylation in AUD which had some replicable results, large sample sizes, and high statistical power (Longley et al., 2021). This therefore has a higher chance of detecting a difference between groups if it exists (Suresh & Chandrashekara, 2012). The category of global methylation in AUD was excluded as the studies had low power and a small sample size (Longley et al., 2021).

The first category was candidate gene methylation studies, which is an experimental approach that examines genetic influences on a complex disease by identifying DNA, and are based on a priori hypotheses (Lipsky & Lin, 2015).  The a priori hypotheses are assumed deductions from previous studies, performed before the data is collected (York Health Economics Consortium, 2021). In these studies, the a priori hypothesis is that the disease AUD is associated with DNA methylation in specific areas of specific genes, namely the promoter region (Longley et al., 2021). This is where necessary proteins bind to begin transcription of a gene (Promoter, n.d.). However, these studies are not replicable for all genes. Replicability means that the results of a study are found to be consistent when the methodology is replicated and tested (National Academies Press (US), 2019). At least two genes are proved to have replicable results, Aldehyde Dehydrogenase 2 Family Member (ALDH2) and Opioid Receptor Mu 1 (OPRM1) (Longley et al., 2021). Both were hypermethylated in candidate gene studies as well as epigenome-wide association study (EWAS) (Longley et al., 2021). A large number of studies were not replicated, including genes such as Glutamate Ionotropic Receptor NMDA Type Subunit 2B (GRIN2B), Somatostatin Receptor 4 (SSTR 4) and Solute Carrier Family 6 Member 3 (SLC6A3) (Longley et al., 2021). 

The second category, EWAS, surveys CpG sites in the genome for methylation. Aggregation of CpG sites which have different methylation statuses revealed 180 sites which had notable results in two studies and four sites which were notable in three studies (Longley et al., 2021). Table 2 provides information about these four sites. However, it is important to note that any variation between the methylation may be due to the different types of tissue noted in each EWAS (Longley et al., 2021). 

Table 2: This states the genes in close proximity of differentially methylated CpG sites that were noted in 3 studies, including the tissue type tested and the authors of the studies.

Looking at these 4 genes in detail, they may have epigenetic links to AUD and alcohol consumption. HNRNPA1 has been noted to be hypomethylated, indicating a decrease in methylation of CpG sites, in AUD in the 3 studies mentioned in Table 2 (Longley et al., 2021). In blood and saliva cells, methylation of HNRNPA1 was inversely correlated with alcohol consumption. LMF1 was also shown to be differentially methylated in 4 studies, 3 of AUD and 1 of alcohol consumption. However, little is known about the other 2 genes, PLEKHG4B and LRRC20; although LRRC20 has notably been differently methylated in some studies of AUD and of alcohol consumption (Longley et al., 2021).

4.2 Histone modifications

Histone modifications are another epigenetic change that may be linked to alcoholism. Histone acetylation and deacetylation are specific modifications that have been studied in detail. Although the mechanism for the effect of alcohol on histone acetylation is not fully understood, it is likely related to a transcription factor, cAMP – response element binding protein (CREB) (Jangra et al., 2016). Being a transcription factor means that CREB controls the rate of transcription (Latchman, 1993). A CREB binding protein (CBP) can acetylate histones, increasing transcription, when CREB instructs it to do so (Jangra et al., 2016). Expression of genes associated with alcoholism, including genes BDNF and neuropeptide Y is associated with CREB (Narayan et al., 2015). A study in rats showed that alcohol-preferring rats without ethanol exposure had lower expression of genes regulated by CREB, such as BDNF and Arc, in certain regions compared to rats without alcohol preference (Sakharkar et al., 2011). Exposure to ethanol also increased t levels of phosphorylation in CREB as well as Arc and BDNF expression in certain areas of the brain for alcohol-preferring rats (Pascual et al., 2009). 

Histone methylation is a mechanism that is affected by AUD. Chronic consumption of alcohol can cause increases in H3K4 trimethylation in the cortex of the brain (Ponomarev et al., 2012). Acute exposure to alcohol has been shown to affect dimethylation levels on lysine 9 and 27 on H3, as well as increase in this mark in exon 1 on G9a (Zhou et al., 2011). Increases in dimethylation of H3K9 and H3K27 mediated by G9a control the proteolytic (breaking down of protein) breakage of histones by enzyme caspase-3 and neurodegeneration in the hippocampus and neocortex, after low-dose ethanol exposure (Stadler et al., 2005). 

A significant number of studies on model organisms, such as rats, have also yielded results that demonstrate epigenetic modifications following alcohol intake. In rodents, DNA methylation has been altered by alcohol intake, with methylation coinciding with an increase in alcohol intake from light drinking to excessive drinking (Longley et al., 2021). A separate study that used acute and rapid intraperitoneal administration of alcohol promoted site-specific phosphorylation of rat liver in vivo. Sprague-Dawley rats were fed ethanol for 4 weeks, followed by binge administrations of ethanol thrice. After 4 hours of the last ethanol intake, histone modifications were looked for in liver samples. An increase in phosphorylation of H3 at ser-10 and ser-28 was noted, as well as higher levels of dually phospho acetylated histone H3 after acute binge ethanol. Histone H3 lysine-9 acetylation was increased after acute binge and chronic ethanol binge (Aroor et al., 2014). Binge drinking is defined as heavy episodic drinking and can lead to chronic drinking over time (Palmera, 2021). Another study found that acute alcohol exposure in rats increased phosphorylation of histone H3 at ser-10 and ser-28, backing the results of Aroor et al. (2014) (Pavlova et al., 2013). Thus, histone phosphorylation is a significant mechanism in the action of alcohol on genes.

4.3 miRNAs and their effect

Another crucial aspect of the epigenetic impact of alcohol is its effect on microRNA (miRNA) regulation activity (Mayfield, 2012). MicroRNAs are small, non-coding RNA molecules that play an important role in the post-transcriptional regulation of gene expression. They achieve this by binding to complementary sequences on target messenger RNAs (mRNAs), leading to mRNA degradation or inhibition of translation. This regulatory mechanism allows for fine-tuning of gene expression, which is essential for maintaining cellular homeostasis and responding to environmental changes.

Research completed by the National Institute of Health has shown that alcohol consumption can significantly alter miRNA expression profiles, leading to widespread changes in gene expression that are linked to various aspects of alcohol-related disorders (Yakovlev, 2023). For instance, alcohol has been found to modulate the expression of miRNAs involved in inflammation, stress response, and neuronal signaling, all of which are critical in the development and progression of alcohol use disorder (Ureña-Peralta, 2018).

When miRNA serum levels are considered, a relationship between the concentration of specific miRNAs in serum and brain structure can be formed and linked to its function (Ignacio et al., 2015). Among rats, exposure to ethanol has been shown to cause Let-7, a family of miRNAs, to regulate the activation of genes that have a function in neuroplasticity, neuroinflammation and chromatin structural rearrangements (Smith et al., 2016). Rats exposed to alcohol in adolescence have higher levels of miRNA miR-137 in adulthood, and the expression of genes associated with this miRNA, such as Lysine demethylase 1 (Lsd1), decreased (Rodriguez, 2021). Nunez et al. (2013) showed that after ethanol exposure, expression of many miRNAs in both mice and the human brain (including families Let-7, miR-101, miR-221, miR-1952) is positively correlated with expression of mRNAs. This helps to discover the interactions between different mechanisms and substances in the epigenetics of AUD (Wang et al., 2013). 

One well-studied miRNA type affected by alcohol is miR-29. MiR-29 is known to be involved in the regulation of genes related to fibrosis and liver damage. Chronic alcohol consumption can lead to changes in the levels of miR-29, which in turn affects the expression of genes involved in liver fibrosis (Novo-Veleiro, 2018). This is particularly relevant as alcohol-induced liver damage is a major health concern, and understanding the role of miR-29 could help in identifying potential biomarkers for early detection and intervention (Torres, 2018).

Similarly, miR-214 has been linked to the modulation of neuronal responses to alcohol. Studies have shown that miR-214 activity can influence the expression of genes associated with neuroplasticity, the ability of neural networks to change and grow, and addiction. Changes in miR-214 levels can affect the way neurons respond to alcohol exposure, potentially altering the reward pathways in the brain and contributing to the development of alcohol dependence.

Furthermore, the impact of alcohol on miRNA expression also extends to its effects on cellular stress responses. Alcohol-induced oxidative stress can alter the expression of various miRNAs, which then influence the regulation of genes involved in oxidative stress pathways and cellular survival. This disruption in miRNA expression can contribute to the pathophysiology of alcohol-related liver disease and other health issues (Jouve, 2023).

The intertwining between alcohol and miRNAs demonstrates the complexity of alcohol’s effects on gene regulation. Changes in miRNA expression can have significant effects on multiple signaling pathways, influencing not only the development of AUD but also the progression of related conditions such as liver disease and neurological disorders. Yakovlev’s (2023) research into these miRNA changes provides valuable insights into the molecular mechanisms underlying alcohol’s impact on the body and highlights potential targets for therapeutic intervention. This aspect of epigenetic research underscores the importance of understanding miRNA regulation in developing comprehensive strategies to address AUD.

5. Therapies and treatments to epigenetically improve AUD and alcoholism in patients

The discovery of epigenetic mechanisms in alcoholism has led to potential targeted therapies to ameliorate AUD and excessive drinking. Some of these treatments aim to reverse changes to the genome, including DNA and histone modifications, while others aim to stop the mechanism causing these modifications. Bohnsack and colleagues attempted to find an epigenetic treatment via the first avenue, by restoring histone acetylation in the SARE region of the gene, Arc, which would increase expression of the gene. The test subjects, rats, were exposed to binge levels of ethanol, leading to increased alcohol consumption and heightened anxiety in adulthood. The method to restore acetylation was the use of a CRISPR insert, dCas9-P300, which was fused with a histone acetyltransferase (including H3K27ac). 4 single guide RNAs (sgRNAs) were targeted at the Arc SARE region, via a lentivirus vector in rat cell culture. There was an increase in Arc mRNA noted at the Arc SARE site. dCas9-P300 and sgRNA infusion reduced anxiety-like behaviors as well as decreased excessive drinking in rats exposed to adolescent intermittent ethanol back to control levels. The same infusion into the central nucleus of the amygdala improved decreases in Arc expression in rats that had decreased expression due to adolescent intermittent ethanol (Bohnsack et al., 2022). 

The second avenue of treatments, which affects mechanisms of epigenetic modifications, are drugs and inhibitors. Some of the enzymes inhibited include histone deacetylases (HDACs) and DNMTs, both of which have a direct role in epigenetic modifications. Inhibiting the action of DNMT using the drug 5-azacytidine inhibits excessive alcohol consumption in mice (Darcq et al., 2014). The inhibitions of HDACs could potentially have an effect on behavioral and reinforcement-related effects of alcoholism; thus targeting HDACs may help to find an epigenetic treatment for alcoholism (Warnault et al., 2013). The mechanism for this is supported by the fact that the presence of HDAC inhibitor (HDACi), valproic acid, reduced ethanol consumption and ethanol-conditioned place preference in rats in the same study (Warnault et al., 2013). Drugs that can regulate histone methylation are a possible therapy that is largely unexplored; however, a prospective drug candidate is BIX-01294, which inhibits histone H3K9-specific methyltransferases G9a and Glp84 (Peter & Akbarian, 2011). 

While all of these studies have been conducted in rats, research into the impact of HDAC and DNMT inhibitors, drugs such as BIX-01294 and histone acetylation methods on human brain cells may produce new therapies for alcoholism and AUD. This is a crucial step as it may provide help to thousands of people suffering from this devastating disease, as well as improve doctors’ understanding of AUD.

6. Conclusion

In summary, alcohol consumption impacts epigenetic regulation through several mechanisms, such as DNA methylation, histone modifications, and microRNA expression. DNA methylation studies have revealed specific genes linked to alcohol use disorder (AUD), with some showing consistent results across different studies. Histone modifications, particularly through the action of CREB, influence gene expression related to alcoholism and neuroplasticity. Finally, changes in miRNA expression further exemplify the complex interactions between alcohol and gene regulation, and their effect on the various biological processes that contribute to addiction and liver damage. Understanding these epigenetic mechanisms provides valuable knowledge into how alcohol affects the body at a molecular level, which is essential for developing targeted interventions and treatments for alcohol-related disorders. Further research for therapies previously explored with rat models and recreating those therapies with human brain cells could be a potential new treatment avenue. 

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