Abstract
Substance use, especially alcohol and opioids, is a major public health concern with lasting effects on brain function and behaviour. These substances influence gene activity through epigenetic mechanisms, including DNA methylation, histone modifications and non-coding RNA regulation, impacting stress response, mood and reward pathways. While the immediate effects of substance use are well-documented, less is known about how these epigenetic changes may be inherited and affect offspring mental health. This review examines how alcohol and opioids modify the epigenome in key brain regions, contributing to anxiety, depression, post-traumatic stress disorder and addiction vulnerability. We also summarise evidence from animal and human studies demonstrating that these epigenetic alterations can be transmitted across generations, increasing susceptibility to mental health disorders and addictive behaviours. These findings highlight that substance use can leave a lasting biological imprint beyond the individual, emphasising the importance of prevention, early intervention and potential therapeutic strategies to mitigate long-term mental health risks.
Introduction
Every day, millions of people around the world drink alcohol or use opioid drugs without realising that these substances may leave marks not only on their own brains, but also on the health of future generations (Heidari et al., 2024). In recent years, scientists have discovered that alcohol and opioids can change the way our genes work through epigenetic mechanisms, meaning that these effects do not alter the DNA itself but influence how it is expressed. This is important because these epigenetic changes can shape mental health, increasing the risk of disorders such as anxiety, depression, post-traumatic stress disorder (PTSD) and addiction (Fitz-James et al., 2022; Yehuda et al., 2018). Even more worrying is that some of these changes appear to be inheritable, which means that substance use in one generation may silently affect the next.
Although the immediate effects of alcohol and opioids are well known, their deeper biological impact, especially on the epigenome, is still not widely understood outside scientific research (Yehuda et al., 2018). Understanding these mechanisms matters because it helps explain why some individuals or even families are more vulnerable to mental illness and addiction than others (Heidari et al., 2024). It also highlights how substance use is not just a personal choice, but something that can influence long-term health across generations (Yehuda et al., 2018).
This review examines how alcohol and opioids alter epigenetic regulation in the brain, how these changes contribute to mental health disorders and how some of these may be passed on to offspring (Heidari et al., 2024). Alcohol and opioids were chosen because they are two of the most widely used and most harmful addictive substances and both produce significant epigenetic changes linked to stress, emotional regulation and addiction pathways (Heidari et al., 2024; Bohnsack et al., 2024; Liu et al., 2021).
To explore this idea, the paper first examines how alcohol affects epigenetic mechanisms such as DNA methylation, histone modifications and microRNA regulation, and how these changes contribute to mental illness (Heidari et al., 2024; Palmisano et al., 2017). The next section examines how opioid use changes epigenetic regulation and how these changes affect emotional control and stress systems (Liu et al., 2021; Grimm et al., 2023). Finally, evidence showing how both alcohol and opioid-related epigenetic marks can be inherited will be discussed (Fitz-James et al., 2022; Yehuda et al., 2018).
Overall, understanding the epigenetic effects of these substances offers new insight into how alcohol and opioids shape mental health not only in one lifetime, but potentially across generations (Heidari et al., 2024; Yehuda et al., 2018).
Alcohol
Alcohol is a common substance that people abuse and become addicted to. Studies have shown that alcohol leads to alterations in the epigenome which occur in different parts of the brain or blood, specifically in terms of mRNA expression or protein production levels (Heidari et al., 2024). This section will explore how alcohol changes your epigenetics and which mental issues it gives rise to.
DNA methylation and how it causes mental illness
First, alcohol changes DNA methylation, a process in which methyl groups attach to the DNA and influence which genes are being silenced/activated (Moore et al., 2012). When the body breaks down alcohol, it uses up many nutrients – like folate and B vitamins – which are needed to make SAM. SAM is the chemical that makes methyl groups for the DNA that control which genes are activated or silenced. Alcohol reduces the amount of SAM, leading to fewer methyl tags. This results in genes such as CRH and ACTH becoming overactive, sending your brain more stress signals than usual. NR3C1 also becomes improperly regulated due to the low methylation, weakening the brain’s ability to shut down the stress response. The HPA axis, therefore, becomes unstable, which is a key biological factor in anxiety, depression and PTSD. In addition, brain-derived neurotrophic factors (BDNF) become overactive, strengthening fear memories and emotional reactivity, which is seen in PTSD. MAOA becomes incorrectly expressed, which disrupts mood stability, linked to depression and anxiety. These genes being affected can cause your brain to be in a constant state of fight-or-flight. Next, genes such as IL-1β, TNF-α, IL-6, TLR4 and NF-κB become hypomethylated due to constant alcohol consumption, leading to inflammation in the brain, disruption of mood pathways, heightened threat detection and enhanced traumatic memory formation (Goeke et al., 2019). These are all respectively contributors to depression, anxiety and PTSD.
Acetaldehyde’s effects on the packaging of DNA and how it causes mental illness
Second, acetaldehyde can cause DNA damage. Acetaldehyde is a toxic alcohol byproduct created when alcohol breaks down in the body, entering different parts of the body and sticking to DNA. It then forces the cells to repair it, changing how the DNA is wrapped (loose or tight) and which genes are active. The damage and repair signals impact fear and trauma-related genes like BDNF and CREB, which strengthen fear memory circuits. EGR1, which helps encode traumatic events and adrenergic genes that increase fear, is also impacted. These gene changes lead to PTSD. Additionally, emotional regulation genes like GABA receptor genes (GABRA1, GABRB3) are underexpressed, while glutamate genes are overactive. Both relate to our neurotransmitters, and when acting irregularly, can cause depression and anxiety.
Histone acetylation and how it causes mental illness
Third, alcohol increases histone acetylation. A chemical called acetate is broken down when alcohol enters the body. Acetate is then converted into acetyl-CoA, which adds acetyl groups to histones, making DNA looser and activating too many genes. Genes related to fear and memory, like BDNF, CREB, Arc and EGR1, which connect to stronger trauma learning, are overly activated, leading to PTSD. Stress pathway genes like CRH, ACTH and FKBP5 cause more cortisol (the main stress hormone) to be produced from being overly active, leading to chronic stress and anxiety. Dopamine receptor genes and serotonin genes are also dysregulated, causing depression. Therefore, alcohol causes histone acetylation, which activates genes in the body that cause the brain to become more sensitive to fear and less able to regulate emotion, leading to mental difficulties (Goeke et al., 2019).
Micro-RNA levels and how they cause mental illness
Lastly, alcohol alters microRNA levels. These microRNA levels regulate brain development, stress hormones and mood by activating and silencing genes. As mentioned above, alcohol is broken up into acetaldehyde, which increases oxidative stress and inflammation in the brain and liver. Both oxidative stress and inflammation activate certain proteins called transcription factors (like NF-κB and CREB). These proteins then increase or decrease the production of certain microRNAs (miRs), affecting different aspects of our mental health. An increase in miR-34a also increases fear and stress sensitivity, relating to PTSD. miR-9 and miR-153 disrupt dopamine/serotonin, which can lead to depression. MicroRNA changes are long-lasting and can make disorders persistent (Goeke et al., 2019).
All in all, alcohol causes molecular changes in epigenetics that interfere with and negatively impact gene expression. This section examined how alcohol specifically creates irregular behaviour in genes, which can cause symptoms of PTSD, depression and anxiety.
Opioids
Opioids are commonly prescribed analgesics that are often misused and highly addictive. Other than acute pharmacological effects, chronic opioid exposure causes long-term neuroadaptations through epigenetic mechanisms. Epigenetic changes have long lasting effects on transcriptional programs in brain structures, such as the nucleus accumbens (NAc), prefrontal cortex and hippocampus, playing a role in reward, motivation, emotional regulation, memory, learning and stress regulation. Epigenetic alterations are starting to become recognised for their contributions to psychiatric vulnerability, including the development of major depressive disorder (MDD), anxiety and PTSD (Rosoff et al., 2021). This section discusses the implications of opioid-induced epigenetic remodelling and the consequent effects on an individual’s mental health.
Opioids and epigenetic programming
Chronic opioid exposure triggers meaningful epigenetic remodelling in regions of the brain controlling reward and stress regulation. This is done by chromatin modification, including histone acetylation and DNA methylation which affects which genes are being expressed and silenced. After opioid exposure locus specific reductions have been seen in histone H3K27 acetylation, inhibiting transcription and leading to the silencing of the gene, essential for synaptic plasticity and adaptive stress response (Heller et al., 2014). Studies show that opioid use disorder causes thousands of DNA methylation changes in the dorsolateral prefrontal cortex, affecting genes in charge of neuronal development and neurotransmission (Liu et al., 2021).
These epigenetic alterations are not limited to a single region of the brain, they span across the addiction network. Hypoacetylation in the prefrontal cortex correlates to weaker decision making and higher stress sensitivity. Chromatin remodelling in the nucleus accumbens encourages compulsive drug-seeking behaviours. Methylation alterations in the hippocampus, which is responsible for controlling memory and emotions, disturbs the synaptic connections which has a knock-on effect on cognitive and mood dysregulation (Liu et al., 2021).
Other than well known, classical chromatin modification, non-coding DNAs also play a key role in opioid-induced epigenetic changes. Particularly, microRNA-mRNA interactions which control gene expression have been found to become dysregulated in opioid use disorder. This affects MAPK, a critical signalling pathway which plays a part in stress response and cell survival and compromises neurovascular integrity. Besides neurons, these alterations also occur in glial and endothelial cells, which shows the wide scale impact epigenetic remodelling has on the brain (Grimm et al., 2023). This research highlights that opioid use implements changes on more than neurotransmitter dynamics; opioids restructure the brain’s epigenetics creating long-term addiction and psychiatric disorder vulnerability (Grimm et al., 2023).
Opioids and mental health consequences
Opioid-induced epigenetic reprogramming meaningfully impacts the neural circuits which control mood and stress regulation, increasing the likeliness of developing psychiatric disorders. Depression is one of the most commonly seen conditions in individuals struggling with opioid use disorder. The epigenetic modifications are responsible for silencing genes involved in dopaminergic signalling, contributing to anhedonia, a major depressive symptom. Additionally, opioids cause the downregulation of BDNF, which is a protein that is important for plasticity and neuroadaptations. The reduction of BDNF weakens the brain’s ability to adapt, further causing the development of depressive symptoms (Levis et al., 2021).
Similar epigenetic underpinnings impact anxiety disorders. Individuals exposed to opioids have alterations to the expression of stress response genes, such as the FKBP5, combined with hypoacetylation in the prefrontal cortex. These changes interfere with emotional regulation capacity and increase the brain’s reactivity to stress. These epigenetic marks remain present long after opioid use has stopped, which shows how stable chromatin modifications are and their potential long-term influence in psychiatric disorders (Rosoff et al., 2021).
Post-traumatic stress disorder demonstrates that the synergistic impact of trauma and opioid exposure combined can worsen the outcome. Epigenetic remodelling within the hippocampal and amygdalar circuits are linked to fear extinction, which plays a vital role in the recovery from past trauma. Moreover, epigenetic changes caused by opioids interact with trauma-specific marks, which lead to an acceleration of biological ageing and cortical atrophy in parts of the brain that regulate emotions (Katrinli et al., 2020). These findings highlight that opioid-induced epigenetic alterations do not only increase addiction risk, but also establish a molecular framework for long-lasting psychiatric problems.
Epigenetic Mechanisms Linking Alcohol Use to Mental Health Across Generations
A growing body of research shows that the risk for alcohol use disorder (AUD) cannot be explained solely by family behaviour, social learning or environmental pressures. Many studies suggest that epigenetic changes caused by alcohol use in one generation can influence the biological vulnerability of the next generation (Jason et al., 2021; Finegersh et al., 2014). These epigenetic changes, such as DNA methylation, histone modification and non-coding altered RNA, do not change a person’s DNA sequence. Instead, they change how genes are turned on or off. Because of this, alcohol-related physical and psychological effects can be passed from parents to children without direct exposure (Jason et al., 2021; Michaelson et al., 2013). Many of the epigenetic pathways affected by alcohol also play a major role in depression, anxiety disorders, PTSD and memory processes (Michaelson et al., 2013; Calvey, 2019; Palmisano et al., 2017).
Research in both animals and humans shows that alcohol-related traits can be inherited. Animal studies provide strong evidence that alcohol exposure before conception can influence the next generation’s behaviour, stress response and alcohol sensitivity (Jason et al., 2021; Finegersh et al., 2014). Human studies are more difficult to conduct, but similar patterns have been observed (Jason et al., 2021). Chronic alcohol use changes gene expression in brain areas important for reward, stress, fear learning and memory, especially the prefrontal cortex, hippocampus and amygdala (Michaelson et al., 2013; Townsend et al., 2021). If alcohol exposure occurs before conception, these epigenetic marks can be passed through sperm or egg cells, making offspring more vulnerable to emotional dysregulation and substance use (Finegersh et al., 2014; Koch, 2014).
Transgenerational Transmission of Psychopathology: Depresson, Anxiety and PTSD
Depression often occurs together with AUD, and epigenetic research helps explain why this link may continue across generations. Alcohol disrupts the hypothalamic-pituitary-adrenal (HPA) axis, which controls the body’s stress response. Long-term alcohol use changes methylation levels on the NR3C1 gene, which affects how the body regulates cortisol, the main stress hormone (Stephens et al., 2012). If these methylation changes occur in germ cells, children may inherit a more reactive and less stable stress system. This means they may be more likely to develop depression even before facing major life stressors (Stephens et al., 2012; Palmisano et al., 2017). Animal studies show that paternal alcohol exposure can lead to reduced stress resilience, lower sensitivity to pleasure (anhedonia), and changes in serotonin and dopamine systems in offspring (Finegersh et al., 2014; Michaelson et al., 2013). These changes closely resemble depressive symptoms in humans. Human studies also show that children of parents with AUD have a higher risk of depression early in life, and some studies have found epigenetic alterations in genes related to mood regulation (Stephens et al., 2012). Together, these findings show that epigenetic inheritance is an important contributor to the connection between alcohol misuse and depression across generations.
Alcohol-related epigenetic changes also play a role in the development of anxiety disorders. Alcohol affects genes related to GABA and glutamate, the two main neurotransmitters that balance brain activity and emotional stability (Michaelson et al., 2013). Animal research shows that alcohol-related epigenetic changes in these systems can be passed to offspring who have never been exposed to alcohol (Finegersh et al., 2014). These offspring often show higher baseline anxiety, stronger reactions to stress and weaker fear extinction, the ability to “turn off” fear responses after danger has passed (Jason et al., 2021; Michaelson et al., 2013). Because GABA and glutamate are also essential for learning and memory, epigenetic changes in these systems influence long-term emotional patterns. A key molecule in this process is BDNF, which supports brain development, synaptic plasticity and emotional control. Alcohol reduces BDNF expression through histone modifications, and these changes can be inherited (Calvey, 2019). Reduced BDNF in offspring can lead to higher anxiety, lower neuroplasticity and a greater chance of using substances as a copying mechanism (Calvey, 2019; Palmisano et al., 2017).
Epigenetic inheritance is also important for understanding why children of individuals with AUD may be more vulnerable to PTSD. Many people with alcohol use disorder have experienced trauma before or during the development of their addiction. Alcohol and trauma together can strengthen trauma-related epigenetic changes (Yehuda et al., 2016; Palmisano et al., 2017). Both alcohol and trauma impact brain regions related to fear, stress and memory, including the hippocampus, amygdala and medial prefrontal cortex (Michaelson et al., 2013; Yehuda et al., 2016). These epigenetic changes can be passed to offspring, making them biologically more sensitive to stress, more reactive to fear cues and more likely to develop PTSD symptoms such as hyperarousal and intrusive memories (Jason et al., 2021; Yehuda et al., 2016). Although PTSD requires exposure to a traumatic event, inherited epigenetic vulnerabilities can strongly influence how a person responds to trauma and how likely they are to develop the disorder (Yehuda et al., 2016).
Cognitive problems and their effects across generations
Memory and learning abilities are especially sensitive to the epigenetic effects of alcohol. Alcohol disrupts hippocampal neurogenesis (growth of new neurons) and synaptic plasticity, which are mainly controlled through histone acetylation and DNA methylation (Townsend et al., 2021). Long-term alcohol use reduces the expression of genes required for memory creation, such as those involved in glutamate signalling and BDNF regulation (Calvey, 2019; Townsend et al., 2021). These cognitive effects can be inherited, leading to problems with working memory, spatial memory and learning in children who were never exposed to alcohol themselves (Jason et al., 2021; Townsend et al., 2021; Boschen et al., 2018). Memory is closely linked to emotional regulation, so inherited memory impairments can increase the risk of depression, anxiety and PTSD. This creates a cycle where cognitive and emotional vulnerabilities increase susceptibility to substance use (Calvey, 2019; Boschen et al., 2018).
Taken together, the research shows that the transgenerational inheritance of alcohol related traits results from a complex interaction of changes in the brain, stress systems and epigenetic regulation. Alcohol exposure in one generation can leave biological marks that increase the vulnerability of the next generation to depression, anxiety, PTSD and cognitive problems (Jason et al., 2021; Boschen et al., 2018). These inherited changes do not determine a person’s future, but they do create a biological environment in which mental health disorders and substance use are more likely. Understanding these mechanisms highlights the importance of early intervention, trauma-informed care and prevention strategies that focus not only on individuals but on families across generations.
Epigenetic Effects of Opioid Consumption
Long-term use of opioids, such as heroin and morphine, causes epigenetic changes to DNA, such as DNA methylation, histone modifications and non-coding RNAs activity (Browne et al., 2021).
Opioid drugs exert their biological effects by activating opioid receptors, which belong to the G-protein-coupled receptor (GPCR) family. These receptors – mu (μ), delta (Δ) and kappa (κ) – can form both homo- and heterodimers and initiate intracellular signalling through multiple pathways. Among them, the μ-opioid receptor is most strongly implicated in addiction-related processes (Blackwood et al., 2021).
Acetylation decreases the electrostatic attraction between histones and DNA, producing a more relaxed, “open” chromatin structure that enhances gene transcription. In the context of opioid exposure, this modification has been studied most extensively on histone H3 tails. Animal studies show that repeated opioid administration. whether experimenter-delivered or self-administered, elevates global H3 acetylation within the mesolimbic dopamine system. This aligns with postmortem findings from individuals with heroin use histories, who also display increased H3 acetylation in the striatum. Notably, the degree of H3 hyperacetylation in heroin users correlates positively with years of heroin use, suggesting that prolonged exposure may reinforce or stabilise this chromatin modification over time (Browne et al., 2021).
Compared with histone acetylation, much less is understood about how opioids influence histone methylation. The limited studies available have primarily identified alterations at a single histone residue – H3K9 – following opioid exposure. It was reported (Sun et al., 2012) that repeated morphine administration selectively decreases H3K9 di-methylation (H3K9me2) in the nucleus accumbens, without affecting the mono- or tri-methylated forms. A similar reduction in H3K9me2 has also been observed in the central nucleus of the amygdala after chronic opioid treatment (Browne et al., 2021) . This decrease in H3K9me2 appears to enhance transcriptional activity and requires prolonged, rather than acute, drug exposure (Sun et al., 2012).
Genome-wide ChIP-sequencing further revealed that chronic morphine produces differential H3K9me2 enrichment across multiple gene loci in the NAc. Notably, there was reduced H3K9me2 across the FosB gene, a transcription factor strongly implicated in addiction suggesting that chronic opioid use may lift transcriptional repression at this locus. Pathway analyses additionally pointed to methylation changes in genes associated with glutamatergic signalling, highlighting a broader role for H3K9me2 in regulating plasticity-related transcriptional networks (Browne et al., 2021). Opioid-induced changes in H3K9 methylation may extend beyond the NAc: one study reported decreased H3K9 tri-methylation in the VTA and locus coeruleus after one week of withdrawal in animals exposed to escalating opioid doses (Browne et al., 2021).
DNA methylation, most commonly the addition of a methyl group to cytosine residues at CpG sites (5mC), typically represses gene expression by hindering the access of RNA polymerase II to DNA. Other forms of cytosine modification, such as 5-hydroxymethylcytosine (5hmC), which is abundant in the brain, are instead more often associated with transcriptional activation. Research on DNA methylation in opioid addiction has focused mainly on 5mC changes, drawing from studies in blood samples from clinical populations, postmortem human brain tissue and a limited number of rodent models (Browne et al., 2021).
Evidence from genome-wide analyses suggests that chronic heroin use increases overall DNA methylation, as shown by elevated methylation at LINE-1 retrotransposon elements in the blood leukocytes of heroin users compared with controls. In the frontal cortex, neuron-specific analyses reveal altered methylation patterns across intragenic regions in individuals with heroin use histories. Increased methylation has also been observed at CpG-rich regions of the μ-opioid receptor gene, OPRM1, in both blood and brain tissue from heroin users (Browne et al., 2021). According to Chorbov et al. (2011), methadone-stabilised former heroin users exhibited elevated DNA methylation at the +182 and +186 CpG sites within the μ-opioid receptor promoter. Notably, similar hypermethylation of OPRM1 is present in patients receiving long-term opioid analgesic treatment compared with untreated individuals, suggesting that prolonged opioid exposure itself can produce these methylation changes (Browne et al, 2021).
Following chronic opioid exposure, it was reported that heightened DNA methylation at CpG sites within the μ-opioid receptor promoter enhances MeCP2 binding, which subsequently recruits the repressors HDAC1 and mSin3A (Hwang et al., 2007). This mechanism ultimately suppresses μ-opioid receptor gene expression. Their interpretation aligns with later evidence showing that MeCP2 acts as a negative regulator of μ-opioid receptor transcription (Lu et al., 2009; Garcia-Concejo et al., 2016).
Beyond histone modifications and DNA methylation, gene expression is also controlled by non-coding RNAs, such as microRNAs and long non-coding RNAs, which act at both transcriptional and translational levels. Although this area has been more extensively studied in the context of psychostimulants, particularly cocaine (Kenny et al., 2014), several findings indicate that opioid exposure alters miRNA activity. Increases in specific miRNAs, including miR-339-3p and members of the Let-7 family, have been reported in certain brain regions following opioid treatment, while decreases have been observed for miR-154, miR-675 and miR-218 after chronic opioid use. Although changes in long non-coding RNAs have not yet been directly investigated in experimental opioid models, early evidence suggests that they are indeed altered in individuals with heroin addiction (Michellhaugh et al., 2011).
Collectively, these findings highlight non-coding RNAs as emerging contributors to transcriptional regulation in opioid addiction (Browne et al., 2021). Epigenetic modifications and the enzymes that control them are driven by drug-activated intracellular signalling pathways, which link synaptic activity to gene regulation through transcription factors that bind DNA in a sequence-specific manner. As a result, epigenetic changes arise from ongoing feedback between intracellular signalling processes and the marks they generate (Browne et al., 2021).
Immediate early genes regulation in opioid exposure and drug-seeking
IEG induction links rapid synaptic activity and intracellular signalling to long-term neuronal adaptations. Many IEGs encode transcription factors that help shape chromatin through epigenetic modifications, and these epigenetic states can in turn influence IEG responses to drug exposure. Similar to other addictive substances, opioids trigger a rapid rise in numerous IEGs following acute administration. However, the regulation of IEG induction during chronic opioid exposure remains unclear (Browne et al., 2021).
Drug-seeking behaviour is likewise linked to alterations in IEG expression. For instance, Egr1 shows region-specific regulation in the NAc and PFC following cue-triggered heroin seeking (Browne et al., 2021).
Despite current knowledge, additional research is needed to clarify the functional significance of these epigenetic alterations, especially in non-coding RNAs, which have recently been identified as regulators of gene expression in opioid addiction.
Epigenetic effects of alcohol on gene expression and their relation to depression and anxiety-like phenotypes
Adolescent intermittent ethanol (AIE) exposure has been found to cause a substantial and chronic increase in EZH2 (a histone methyltransferase) throughout PKC-δ-positive GABAergic neurons within the central (CeA) and medial (MeA) amygdala (Bohnsack, Zhang & Pandey, 2024). GABAergic neurons inside the amygdala are often involved in regulating fear, anxiety, appetite and the fight-or-flight response. In adult rats, EZH2 expression in the CeA exhibits a statistically drastic elevation (male: t = 5.25, p < 0.001; female: t = 6.52, p < 0.001). When EZH2 was knocked down in the CeA using siRNA (1 µg per side), chronic anxiety-like behaviour was substantially reduced in both sexes. AIE-exposed males and females normally spend significantly less time in the open arms of the elevated plus maze (EPM), indicating an increased stress response and anxiety. Consequently, this reduction is reversed after EZH2 knockdown (two-way ANOVA interaction: males F₁,₁₉ = 24.52, p < 0.001; females F₁,₂₂ = 12.8, p < 0.01), and rats were found to reexhibit normal behavioural patterns. Similarly, in the light-dark box (LDB) test, AIE reduced the percent of time in the light box (males F₁,₂₈ = 12.14, p < 0.01; females F₁,₂₃ = 12.37, p < 0.01), but this behaviour was eradicated once EZH2 siRNA was introduced. EZH2 siRNA is capable of epigenetically repressing the EZH2 gene, consistently allowing for EZH2 levels to return to normal after AIE exposure (Bohnsack, Zhang and Pandey, 2024).
At the molecular level in both sexes, AIE increased EZH2 and its catalytic mark H3K27me3 at the Arc synaptic activity response element (SARE), while reducing H3K27ac at the same site (Bohnsack, Zhang & Pandey, 2024). In males, AIE increased EZH2 occupancy (interaction F₁,₂₆ = 5.1, p = 0.032), boosted H3K27me3 (interaction F₁,₂₆ = 43.5, p < 0.001) and decreased H3K27ac (interaction F₁,₂₆ = 14.9, p < 0.001). In females, the effects were found to be even stronger (EZH2: F₁,₂₃ = 76.8, p < 0.001; H3K27me3: F₁,₂₁ = 8.2, p < 0.01; H3K27ac: F₁,₂₃ = 17.8, p < 0.001). Importantly, knockdown of EZH2 reverses these occupancy changes, restoring both H3K27me3 and H3K27ac levels at the SARE (Bohnsack, Zhang & Pandey, 2024).
These epigenetic modifications directly impact Arc expression, commonly associated with anxiety, depression and other mental illnesses (Bohnsack et al., 2024). Experimentally, AIE significantly decreased Arc mRNA (interaction F₁,₂₄ = 15.4, p < 0.001 in males; F₁,₂₃ = 5.9, p = 0.024 in females) and Arc protein in the CeA (male: F₁,₁₆ = 174.2, p < 0.001; female: F₁,₁₆ = 119.3, p < 0.001) once withdrawal took effect in the mice. Although these effects were chronic among the control group, both mRNA and protein levels returned to normal with EZH2 siRNA treatment. Since low Arc expression often causes a destabilisation in mood and an increase in anxiety, this is yet another path in which alcohol may epigenetically increase the risk of developing anxiety in humans (Bohnsack et al., 2024).
As it is not well understood how alcohol interacts with tissues in the body, its effects on the epigenome are largely debated. A realistic framework by Bohnsack and colleagues suggests that adolescent alcohol exposure increases EZH2 in specific amygdala neurons, consequently increasing H3K27me3 and decreasing H3K27ac at the Arc enhancer. Adolescent alcohol exposure increases EZH2 in specific amygdala neurons, consequently increasing H3K27me3 and decreasing H3K27ac at the Arc enhancer. Chromatin remodelling then likely takes place, leading to a suppressed Arc expression of both mRNA and proteins. Finally, neurons are unable to relay signals as effectively without Arc proteins. This eventually leads to a destabilisation in behavioural patterns and an increase in chronic mental illnesses like depression and especially anxiety. Given the conservation of this mechanism (similar EZH2 increase is seen in human AUD postmortem amygdala), EZH2 is a promising therapeutic target primarily for anxiety that arises after adolescent alcohol exposure, which may be treatable with EZH2 siRNA therapy (Bohnsack et al., 2024).
Other research suggests that alcohol can profoundly alter the developing brain through epigenetic reprogramming, increasing the rate of developing mental illnesses and effectively remodelling nerve synapses (Kyzar et al., 2016). Reprogramming of this nature was found to be particularly high in adolescent and early adult years, while chromatin is highly plastic relative to later stages in life. Epigenetic modifications such as DNA methylation or histone acetylation and methylation regulate gene expression without altering DNA sequence, and are often kept in check in a typical developing body. Because these marks shift rapidly during neural maturation, ethanol exposure can drastically alter the homeostatic environment in which these modifications occur, shifting developmental trajectories in potentially permanent ways. Acute alcohol exposure decreased histone deacetylase (HDAC) activity by ~30–40%, causing an average 40–60% increase in H3K9 acetylation within hours. This acetylation spike upregulated plasticity-related genes such as BDNF and Arc by 50–70%, expressing the altered phenotype of a ~20–30% rise in dendritic spine density and short-term anxiolytic effects. Withdrawal then reversed these effects, increasing HDAC activity by ~50–70%, reducing H3K9 acetylation by 30–50% and lowering BDNF/Arc by 40–60%. This produced an opposite phenotypic expression as anxiety-like behaviour was chronically increased (measured as 25–35% reductions in open-arm exploration) (Kyzar et al., 2016).
Adolescent alcohol exposure, especially in binge-like AIE models (typically 5 g/kg, two days on / two days off), produces recurring, long-term epigenetic repression of genes within nervous tissue (Kyzar et al., 2016). Weeks after exposure, HDAC2 and HDAC4 expression in the amygdala and BNST remained elevated by 30–70%, with corresponding 25–50% decreases in H3K9 acetylation and 20–30% reductions in spine density. These molecular changes paralleled a 30–40% average increase in anxiety-like behaviour overall. It was also found that ethanol can also produce region-specific increases in HAT activity and promoter acetylation of genes such as c-Fos, CDK5 and FOSB, often elevated by 60–90%. Furthermore, activating marks like H3K4me3 in the medial prefrontal cortex (mPFC) were found to have decreased by 25–40%. These shifts were consistent with those present in the functional reorganisation of circuits involved in stress reactivity and cognitive control (Kyzar et al., 2016).
Alcohol-related DNA methylation changes also contribute to psychiatric vulnerability and the development of addictive tendencies (Kyzar et al., 2016). Hypermethylation of PPM1G, associated with a 10–15% lower gene expression, predicts impulsivity and early AUD risk in adolescents, further increasing the severity of alcohol-based epigenetic modification. Long-term consequences of AIE include chronically elevated HDAC activity (by 50–80%) and sustained suppression of H3K9 acetylation at BDNF promoters (reduced 50–70%) alongside 40–60% decreases in BDNF mRNA. These epigenetic deficits are thought to cause 25–35% reductions in hippocampal spine density and 30–50% decreases in neurogenesis markers such as DCX and Ki-67, aligning with persistent anxiety-like and depressive-like phenotypes (Kyzar et al., 2016).
Rescue experiments have strongly suggested that these anxiety and depression-like changes in behaviour are causally rooted in epigenetic alterations (Kyzar et al., 2016). The use of HDAC inhibitors like TSA (2 mg/kg) can restore H3K9 acetylation by 60–80%, return BDNF and Arc expression to near-baseline values (~90–110% of control) and reverse structural deficits. When used shortly after alcohol exposure, the reversal of these physiological effects correlated with a near complete reduction of anxious and depression-like behaviours. Alcohol-induced shifts in histone and DNA methylation also contribute to the expression of these behaviours. AIE was found to reduce neuron-specific LSD1+8a by 35–45%, leading to a 40–60% increase in repressive H3K9me2 in the amygdala, and changing that track with ~30% elevations in anxiety indices. Additionally, DNA methylation at genes such as SYT2 rose by 20–40% with escalating alcohol intake, lowering their expression to about 30–60%. Together, these findings show that adolescent alcohol exposure disrupts neuroepigenetic programming by innately shifting rates of histone acetylation, histone methylation and DNA methylation, consistently producing chronic alterations in synaptic plasticity and increasing lifelong vulnerability to anxiety and depression (Kyzar et al., 2016).
Inherited epigenetic effects based on opioid usage
Opioid addiction is a chronic relapsing disorder that has recently been a rising concern due to the easy accessibility of opioid-based drugs through prescription and illegal markets. Recent studies have shown that the effects of addiction are not only environmentally caused, but addiction is heritable. However, genetic variation alone cannot account for full risk (≈40–60% heritable estimates) (Gilardi et al., 2018). Epigenetic inheritance and DNA methylation may explain intergenerational effects and vulnerability due to opioid use of parents. The importance of this is that behavioural risk can be shaped by parental exposure before conception (Yohn et al., 2015; Zeid et al., 2025; Gao et al., 2025). This has been shown in animal studies, specifically mice, where effects can be observed intergenerationally (F0 → F1 via direct exposure) and transgenerationally (F0 exposure affecting F2+).
Animal model evidence on transgenerational opioid effects
For intergenerational related studies, it was observed that F0 (father) heroin self‑administration increases heroin-seeking behaviour in male F1 offspring via miR-19b downregulation. miR-19b is a small RNA molecule that helps control whether certain genes are turned on or off, so when it is downregulated, cells are making less of it than normal. This was found by measuring the expression of opioid receptors in brain regions in the offspring, finding significant differences from those on the control-line F1 (Gao et al., 2025). This indicates addictive tendencies are passed through germline (sperm and egg DNA), not environment. Heroin-exposed paternal sperm RNA, when injected into fertilised eggs, increases drug-seeking in offspring. In the Li et al. (2025) study, paternal heroin exposure caused downregulation of miR‑19b in the fathers’ sperm and in the offspring’s nucleus accumbens (a part of the brain crucial for motivation, reward and emotion). This small RNA change was identified as a key molecular mediator of increased drug-seeking behaviour in the male offspring. When researchers restored regular miR-19b levels, the offspring’s increased heroin-seeking behaviour was reversed, showing how miR‑19b directly links to paternal opioid exposure to behavioural vulnerability in offspring.
Reversibility of transgenerational effects through abstinence in mice
Extended abstinence from morphine alters sperm smRNA expression and prevents transmission of intergenerational phenotypes (Zeid et al., 2025). Another animal study, crucially shows reversibility of transgenerational effects through abstinence in male rats. Male rats (F0) were allowed to self administer morphine or saline for 60 days, and out of those rats, a group had an abstinence from opioid-related substances for 90 days before breeding, which is the length it takes for spermatogenesis to complete its cycle. Out of the abstinence group, the offspring (F1) show normal morphine intake, normal social play behaviour and normal analgesic responses compared to the non-abstinent groups of F0 males. Sperm had been collected from all groups of F0, where a small RNA sequence was performed to identify which smRNAs differed in expression across groups. Two small RNAs were found specifically changed in sperm of non-abstinent morphine exposed fathers vs saline fathers: rno-miR-150-5p and snoRNA. An important note is that these changes were not identified in the sperm of the abstinent morphine and saline groups. This important research indicates that the altered smRNA expression in sperms are tied to active morphine exposure, and may revert or normalise after a long abstinence.
Human Epigenetic Evidence Related to Opioids
Furthermore, in relation to human-based research, a large meta-analysis looked at DNA methylation in the blood of people using prescribed opioids vs non-users. They found six CpG sites where methylation was significantly different in opioid users. Some of those CpGs are linked to genes that are active in brain tissue, not just blood, suggesting a “cross-tissue” relevance. The implementation of this is that these methylation marks could potentially serve as bio markers of opioid exposure. A prenatal opioid exposure and methylation case studied epigenome-wide association of placental co-methylated regions in newborns exposed to opioids in utero. This analysed placenta DNA from babies born to opioid-using vs non-using mothers. The results were that there were no significant methylation differences after correcting for multiple testing, no epigenetic age difference and no global effects, suggesting that human placenta effects are subtle (Lee et al., 2023).
As the (mis)usage of opioids around the world grows, it is notable that a stronger focus and funding for human research is needed to answer many of the biological based questions we still have. Human paternal opioid studies are needed, and there are no human sperm miRNA studies yet, as well as a lack of studies regarding females and how their bodies are affected from epigenetics, indicating a research gap. There should be an effort to follow opioid-exposed pregnancies into adolescence, and an effort to better understand which marks predict addiction risk. Nonetheless, the current research brings more awareness to the impact of actions and lifestyle choices taken before pregnancy by both men and women, and how this may genetically impact offspring and subsequent generations.
Conclusion
The research reviewed in this paper demonstrates that these substances alter the epigenome through changes in DNA methylation, histone modifications and non-coding RNA activity in brain regions responsible for stress regulation, emotional processing, memory and reward. These epigenetic disruptions contribute to mental health disorders such as anxiety, depression, PTSD and addiction vulnerability. A central thesis of this paper is that these molecular changes not only shape the user’s own brain and behaviour but can also be inherited, influencing the mental health and addiction risk of future generations.
Across studies in both humans and animals, alcohol is shown to destabilise the HPA axis, alter neurotransmitter systems, impair memory pathways and modify key genes like BDNF, CRH, ACTH and NR3C1. These epigenetic effects can be passed to offspring through germ cells, increasing their likelihood of developing depression, anxiety disorders, PTSD symptoms and cognitive impairments. Similarly, opioid use produces long-term changes in histone acetylation and methylation, DNA methylation patterns – particularly in genes such as OPRM1 – and levels of small non-coding RNAs. Animal research shows that these molecular changes can heighten drug-seeking behaviour in offspring, while also demonstrating that some epigenetic effects may be reversible with extended abstinence from substance use before conception.
These inherited epigenetic changes do not determine a person’s fate, but they create a biological environment that increases susceptibility to mental health disorders and substance use. The implications of this research are significant: understanding how alcohol and opioids leave a biological imprint across generations emphasises the importance of prevention, early intervention, trauma-informed care and broader public health strategies that address not only individuals but also families and future generations.
Bibliography
Bohnsack, J.P., Zhang, H. & Pandey, S.C. (2024) EZH2-dependent epigenetic reprogramming in the central nucleus of amygdala regulates adult anxiety in both sexes after adolescent alcohol exposure, Translational Psychiatry, 14(1), p. 197.
Boschen, K E., Keller, S.M., Roth, T.L. & Klintsova, A. Y. (2018) Epigenetic mechanisms in alcohol- and adversity-induced developmental origins of neurobehavioral functioning, Neurotoxicol Teratol, 66, pp. 63–79.
Calvey, T. (2019) Human Self-Domestication and the Extended Evolutionary Synthesis of Addiction: How Humans Evolved a Unique Vulnerability, Neuroscience, 419, pp. 100-107.
Fitz-James, M.H & Cavalli, G (2022) Molecular Mechanisms of Transgenerational Epigenetic Inheritance, Nature Review Genetics, 23, pp. 325-341.
Finegersh, A. & Homanics, G.E. (2014) Paternal Alcohol Exposure Reduces Alcohol Drinking and Increases Behavioral Sensitivity to Alcohol Selectively in Male Offspring, PLOS One, 9(6):e99078.
Gilardi, G., Augsburger, M. & Thomas, A. (2018) Will widespread synthetic opioid consumption induce epigenetic consequences in future generations?, Frontiers in Pharmacology, 9, p. 702.
Grimm, S.L., Mendez, E.F., Stertz, L., Meyer, T.D., Fries, G.R., Gandhi, T., Kanchi, R., Selvaraj, S., Teixeira, A.L., Kosten, T.R., Gunaratne, P., Coarfa, C. & Walss-Bass, C. (2023) MicroRNA–mRNA networks are dysregulated in opioid use disorder postmortem brain: Further evidence for opioid-induced neurovascular alterations, Frontiers in Psychiatry, 13(1025346).
Heidari, N., Hajikarim-Hamedani, A., Heidari, A., Ghane, Y., Ashabi, G., Zarrindast, M.R. & Sadat-Shirazi, M.S (2024) Alcohol: Epigenome alteration and inter/trangenerational effect, Alcohol, 117, pp 27-41.
Heller E.A., Cates H.M., Peña C.J., Sun H., Shao N., Feng J. et al. (2014) Locus-specific epigenetic remodeling controls addiction- and depression-related behaviors, Nature Neuroscience, 17(12), pp. 1720–1727.
Jason, L.A., Salomon-Amend, M., Guerrero, M., Bobak, T., O’Brien, J. & Soto-Nevarez, A. (2021) The Emergence, Role, and Impact or Recovery Support Services, Alcohol Research: Current Reviews, 41(1):04.
Katrinli, S., Stevens, J., Wani, A.H., Lori, A., Kilaru, V., van Rooij, S.J.H. et al. (2020) Evaluating the impact of trauma and PTSD on epigenetic prediction of lifespan and neural integrity, Neuropsychopharmacology, 45(10), pp. 1609–1616.
Koch, L. (2014) Let there be light-new implantable hydrogels for in vivo optical sensing and therapy, Nature Reviews Endocrinology, 10, 2.
Kyzar, E.J. et al. (2016) Adolescent alcohol exposure: burden of epigenetic reprogramming, synaptic remodeling, and adult Psychopathology, Frontiers in Neuroscience, 10, p. 222.
Levis, S.C., Mahler, S.V. & Baram, T.Z. (2021) The Developmental Origins of Opioid Use Disorder and Its Comorbidities, Frontiers in Human Neuroscience, 15(601905).
Liu, A., Dai, Y., Mendez, E.F., Hu, R., Fries, G.R., Najera, K.E., Jiang, S., Meyer, T.D., Stertz, L., Jia, P., Walss-Bass, C. & Zhao, Z. (2021) Genome-Wide Correlation of DNA Methylation and Gene Expression in Postmortem Brain Tissues of Opioid Use Disorder Patients, International Journal of Neuropsychopharmacology, 24(11), pp. 879–891.
Michaelson, S.D., Paulsen, I.M., Kozuska, J.L., Martin, I.L. & Dunn, S.M.J. (2013) Importance of recognition loops B and D in the activation of human 5-HT3 receptors by 5-HT and meta-chlorophenylbiguanide, Neuropharmacology, 73, pp. 398-403.
Palmisano, M. & Pandey, S.C. (2017) Epigenetic mechanisms of alcoholism and stress-related disorders, National Library of Medicine, National Center for Biotechnology Information, 60, pp. 7–18.
Rosoff, D.B., Smith, G.D. & Lohoff, F.W. (2021) Prescription Opioid Use and Risk for Major Depressive Disorder and Anxiety and Stress-Related Disorders: A Multivariable Mendelian Randomization Analysis, JAMA Psychiatry, 78(2), pp. 151–160.
Stephens, M.A.C. & Wand, G. (2012) Stress and the HPA Axis: Role of Glucocorticoids in Alcohol Dependence, Alcohol Research: Current Reviews, 34(4), pp. 468-483.
Townsend, E.A., Negus, S.S. & Banks, M.L. (2021) Medications Development for Treatment of Opioid Use Disorder, Cold Spring Harbor Perspectives in Medicine, 15(12).
Yehuda, R. & Lehrner, A. (2018) Intergenerational transmission of trauma effects: putative role of epigenetic mechanisms, World Psychiatry, 17(3), pp. 243-257.
Yehuda, R., Daskalakis, N.P., Bierer, L.M., Bader, H.N., Klengel, T., Holsboer, F. & Binder, E.B. (2016) Holocaust Exposure Induced Intergenerational Effects on FKBP5 Methylation, Biological Psychiatry: A Journal of Psychiatric Neuroscience and Therapeutics, 80(5), pp. 372-380.
