Epigenetics: A link between toxicants and diseases

Summary

Epigenetics is a complex network of molecular mechanisms, including DNA modifications, histone modifications, and non-coding RNA transcripts that influence gene transcription in heritable ways across cells. Over the last decades, epigenetic mechanisms have gained significant attention in the field of toxicology. Various research groups are investigating the epigenetic impact of toxicants due to their role in mediating the effects of environmental factors on cellular function, in complex diseases, and in explaining adverse effects on offspring. Here, we give an overview of these studies, exploring the intersection of epigenetics and toxicology and focusing on how not only environmental pollutants but also recreational substances, such as tobacco smoke and alcoholic beverages, can impact DNA and histone modifications.

Graphical abstract

Disease; Environment; Exposure; Epigenetics; Toxicology.

Introduction

Each individual in the course of their life is exposed to a wide variety of toxicants, either unknowingly or more or less consciously, that can pose a persistent threat to global health. Environmental pollution originates not only from human activity but also from natural phenomena (e.g., wildfires, volcanic eruptions, floods, tsunamis, geothermal activity, dust storms, and biological decay), and contributes significantly to the global disease burden, with an even greater impact in developing nations.¹ Yet, there are also substances, such as alcoholic beverages and tobacco smoke, whose adverse effects have been demonstrated, but remain part of daily life (Figure 1). While the exact mechanisms by which these substances cause toxicity are not completely clear, epigenetics was proposed as having a critical role.² The interest in epigenetics within the field of toxicology arises from the fact that epigenetic mechanisms are susceptible to environmental influences and are implicated in many diseases, including cancer, neurological disorders, and cardiovascular diseases.^3,4,5 Additionally, the dynamic nature of epigenetic mechanisms has attracted significant attention from toxicologists, as these mechanisms may represent potential targets for interventions aimed at reducing or preventing the adverse effects of toxicants. This review will provide an overview of the current knowledge of the roles of two key epigenetic mechanisms, DNA methylation and histone modifications, in the toxicity of the most significant environmental pollutants, such as air pollution and heavy metals, and recreational substances, like cigarette smoke, and alcohol.

Figure 1.

The interplay between epigenetics and toxicology

The image depicts how environmental exposures (heavy metals and air pollution) and recreational substances (alcohol and cigarettes) have an impact on the epigenome, affecting DNA methylation and histone modifications, mediating the onset of diseases in the exposed individual or progeny. Created in BioRender. Romanò, A. (2025) BioRender.com/u11h471.

Epigenetic regulation

Epigenetic mechanisms are a complex network of cellular processes, including DNA modifications, histone modifications, and non-coding RNAs, capable of regulating the transcription state of the cell in a heritable manner. They can act at two levels of gene expression regulation: transcription and post-transcription. The former encompasses DNA methylation and histone modifications.⁶

DNA methylation

The addition of a methyl group (-CH₃) on DNA bases occurs preferentially at the fifth carbon of a cytosine within CpG dinucleotides, which are unevenly distributed in the genome. Genomic regions rich in this dinucleotide are called CpG islands and are commonly located at the 5′ end of genes, including the promoter region, making them important transcription regulators. CpG islands are generally hypomethylated in normal tissues, but this state can be altered by pathologies or in specific biological process, such as X chromosome inactivation.⁷ DNA methylation is mainly associated with transcription repression.⁸ The level of DNA methylation in a genomic region is determined by the interplay of two antagonistic processes: DNA methylation and DNA demethylation.⁹ DNA methylation is catalyzed by a family of enzymes known as DNA methyltransferases (DNMTs).¹⁰ DNA demethylation can occur through “passive” or “active” mechanisms. “Passive” demethylation results from the failure of DNMTs to maintain DNA methylation patterns on newly synthesized DNA strands during cell replication, resulting in the dilution of 5mC. “Active” demethylation involves ten-eleven translocation (TET) enzymes, which catalyze the oxidation of 5-methylcytosine to 5-hydroxymethylcytosine (5hmc), 5-formylcytosine (5fc), and 5-carboxylcytosine (5caC), followed by base excision repair mediated by thymine DNA glycosylase (TDG).¹¹ Interestingly, 5hmc has emerged as an epigenetic mark that promotes transcription. (Figure 2).¹²

Figure 2.

Epigenetic mechanisms

The figure illustrates the main epigenetic processes described in this review. DNA methylation occurs at cytosines in the context of CpG islands and promotes transcriptional repression. Histones are modified in their amino terminal tails: acetylation of residues promotes transcriptional activation, whereas methylation can have different effects on transcription depending on the modified residue. For example, methylation of lysine 4, 79, and 36 promote transcription, in contrast, methylation of lysine 9 and 27 results in chromatin compaction and transcriptional repression. Created in BioRender. Romanò, A. (2025) BioRender.com/v57u863.

Histone modifications

Histones are a family of highly conserved proteins essential for packaging DNA into chromatin. There are five main types of histones: H2A, H2B, H3, H4, and H1. Core histones (H2A, H2B, H3, and H4) form the histone octamer around which 146/147 base pairs of DNA are wrapped to form a nucleosome.¹³ Histone H1, also referred to as the linker histone, contributes to the stabilization of higher-order chromatin structures.¹⁴ Core histones can be subjected to a wide array of chemical post-translational modifications (PTMs), such as acetylation, methylation, phosphorylation, ubiquitination, SUMOylation, carbonylation, and glycosylation.¹⁵ These modifications predominantly occur on lysine and arginine residues present on the amino terminal tails of histones, which protrude from the nucleosome core. This makes them an important mechanism for regulating chromatin-associated biological processes, including gene transcription, DNA replication, and repair, as they influence the degree of chromatin condensation, and the binding of proteins involved in these cellular processes.¹⁶

Among these histone modifications, the best functionally characterized are acetylation and methylation. At the level of transcription regulation, these modifications can act as either activators or repressors. Histone acetylation involves the addition of an acetyl group to the amino terminal tail of a histone, which neutralizes its positive charge and results in the loosening of the DNA-histone interaction. The structure of chromatin with acetylation marks is, therefore, more relaxed and allows the interaction of DNA with transcriptional factors, acting as a transcriptional activator.¹⁷ Additionally, acetylation serves as a docking site for proteins that promote transcription.¹⁸ In contrast, histone methylation can be associated with either activation or repression of transcription, depending on the specific residue methylated (Figure 2).¹⁹

Histone acetylation and methylation, more so than DNA methylation—which is generally more stable—are reversible epigenetic markers. This is because there are enzymes that catalyze addition or removal of acetyl as well as of methyl groups on histones. More than 100 enzymes involved in these processes have been identified, making the network that regulates histone acetylation and methylation particularly complex. Enzymes that perform histone acetylation and methylation require two donor molecules—acetyl-CoA for acetylation and S-adenosylmethionine for methylation—as substrates. These are metabolic intermediates and, thus, their availability is closely dependent on cellular metabolism. Additionally, these histone modifications can regulate the expression of key metabolic genes, meaning that there is an interplay between histone modifications and the metabolic state of a cell.²⁰

Air pollution

Air can be polluted by many different substances, such as nitrogen dioxide (NO₂) and carbon monoxide (CO), another important class of air pollutants is airborne particulate matter (PM), which can be defined as a mixture of chemical species and can be classified on the diameter of the particles: PM10 have a diameter of 10 microns or less, while PM2.5 have a diameter of 2.5 microns or less.²¹

Air pollution is a leading health risk, contributing to the development of various chronic diseases. These include asthma, chronic obstructive pulmonary disease (COPD), chronic bronchitis, cardiovascular disease, metabolic and reproductive disorders, neurodegenerative disorders, and cancer. Because of several factors, children and adolescents are particularly vulnerable to air pollution. Their higher respiratory rate compared to adults leads to increased inhalation of pollutants. Additionally, their immune system, which is not fully developed yet, is less efficient at neutralizing toxic substances than that of adults. Finally, as their organs, including lungs and brain, are still developing, they are susceptible to the toxic effects of air pollution.^22,23 However, the cellular mechanisms by which air pollution exerts its detrimental health effects remain unclear. Several studies suggest that DNA methylation and histone modifications are involved in this process. The current knowledge on the epigenetic effects correlated with air pollutants will be overviewed in this section.

Air pollution and DNA methylation

The involvement of DNA methylation in mediating the toxic effects of air pollution is supported by several findings (Figure 3). First, a correlation has been found between long-term exposure to PM2.5 and PM10 and the loss of DNA methylation in repetitive elements in leukocytes.²⁴ The ability of these pollutants to cause DNA demethylation has been confirmed for short-time exposure through a more accurate measurement of global DNA methylation level in buccal cells.²⁵ Second, genes associated with several diseases were found with an altered DNA methylation pattern after exposure to different PMs.^26,27,28 Blood from workers exposed to high levels of PM10 from electric furnaces exhibited lower methylation in the promoter region of iNOS (inducible nitric oxide synthetase).²⁴ In children, exposure to high levels of PM2.5 was associated with hypomethylation of the NOS2 gene, which encodes for the major enzyme responsible for nitric oxide synthesis in the airway.²⁵ Nitric oxide is a signaling molecule with vasodilatory, antimicrobial, and antitumor properties, and impairments to its synthesis rates are implicated in diseases associated with air pollution, including cardiovascular disease and lung cancer.²⁹ In the peripheral blood mononuclear cells of obese individuals, PM10 exposure was found to be associated with reduced DNA methylation levels in the genes encoding for CD14 and TLR4, both of which play a key role in regulating inflammatory response.³⁰ Third, recent studies described changes in the DNA methylation profile of genes involved in cardiovascular signaling, cytokine signaling, immune response, nervous system signaling, and metabolism in the blood cells of individuals exposed to traffic-related air pollution, a complex mixture that includes fine and ultrafine matter (PM2.5 and ultrafine matter, respectively), black carbon (BC), and gaseous pollutants, including CO and NO₂.^31,32,33 These findings suggest that alterations in DNA methylation profile may underlie the increased risk of various diseases, including cardiovascular and metabolic disorders, associated with traffic-related air pollution. Finally, prenatal exposure to particulates has been associated with alterations of methylation of fetal DNA, opening the possibility that this epigenetic mark might mediate the effects of these pollutants on offspring.^34,35

Figure 3.

The effects of air pollution on the epigenome

The diagram shows the main components of air pollution and their effects on the epigenome, with the resulting health outcomes. In detail, particulate matter (PM) 2.5 causes DNA hypomethylation with subsequent overexpression on the NOS2 gene, involved in lung and cardiovascular diseases. PM 2.5 activates p300, a histone acetyltransferase, resulting in the activation of Per1 and Per2, involved in the regulation of circadian rhythms and has an unclear effect on the expression of GATA4, a transcription factor involved in cardiac development. PM10 causes DNA hypomethylation in fetuses and activates iNOS, ICAM-1, VCAM-1, and C-reactive protein in exposed subjects, resulting in inflammation and cardiovascular complications. The metal particles present in PMs also contribute to the detrimental effects and have been demonstrated to increase H3K4me2 and H3K9ac in leukocytes and A549 human lung carcinoma cells. Created in BioRender. Romanò, A. (2025) BioRender.com/y16v399.

Air pollution and histone modifications

Although research on the impact of major air pollutants on histone modifications remains limited, existing studies provide evidence of their involvement in mediating toxic effects (Figure 3).

A recent study showed that PM2.5 exposure disrupts circadian rhythms and metabolism through epigenetic regulation of circadian genes. PM2.5 exposure increases accessibility to gene-expression-regulating genetic elements that contain E-box sequences (binding sites for Bmal1/Clock, two of the master transcription factors of the circadian clock), and enhances the binding of transcription coactivator P300, a histone acetyltransferase, to two circadian target genes (Per1 and Per2). It was proposed that PM2.5 exert these effects by promoting chromatin acetylation of these genetic elements, interfering with the activity of histone deacetylases.³⁶

Epidemiologic and in vivo studies suggest that the metal components of PM may be responsible for their toxicity.³⁷ A study on steel workers found that levels of the activating histone modifications H3K4me2 and H3K9ac in leukocytes were higher in proportion to the years of employment at the plant, in correlation to higher estimated cumulative exposures to arsenic (As) and nickel present in the PM. These findings align with previous studies showing that these metals increase H3K4me2 levels in A549 human lung carcinoma cells, potentially contributing to the onset of disease.³⁷ The mechanism by which altered histone modification levels might contribute to cancer development caused by air pollution remains unknown and will require further studies.

Exposure to PM2.5 in utero and during early life has been linked to an increased incidence of heart failure.³⁸ Given that epigenetic mechanisms play a key role in heart development and in defining the transcription changes occurring in heart failure, it was investigated whether PM2.5 exposure promotes heart failure by impairing the epigenetic landscape of the heart during development.^38,39 Wu et al. found that in the offspring of mice exposed to PM2.5 for 16 weeks, the expression of hypertrophy-related transcription factors, such as GATA4 and Mef2c, were significantly upregulated and that this was due to increased P300/CBP-mediated histone acetylation at their promoters.³⁹ Li et al. analyzed male and female offspring (10 weeks old) separately to verify whether a sex-dependent response to PM2.5 existed. They observed a slight decline in GATA4 mRNA expression, but only a significant decrease at the protein level in male adult offspring, hinting that GATA4 protein may be more susceptible to PM2.5-induced damage than its transcript.³⁸ Due to the contrasting results of these studies regarding how GATA4 mediates the toxic effect of PM2.5, future studies are needed to clarify the role of GATA4 in PM 2.5-induced cardiac hypertrophy.

Furthermore, SIRT1, a class III histone deacetylase, has been shown to protect against inflammation and thrombosis caused by PM exposure, as demonstrated by a SIRT1 knockout mice model in which PM exposure causes aggravated lung vascular leakage and inflammation compared to control mice.⁴⁰ This protective effect can be explained by the ability of sirtuins to inhibit the activity of nuclear factor kappa B (NF-kB), a transcription factor that promotes inflammation, via its deacetylation. This finding indirectly supports the involvement of histone modifications in gene expression responses induced by air pollution, as NF-kB promotes transcription by recruiting transcriptional coactivators, like the histone acetyltransferase complex p300/CBP.⁴⁰

Heavy metal exposure

Heavy metals are non-biodegradable elements that can accumulate in the body over time. Excessive exposure to these metals poses serious health risks.⁴¹ Metals like copper, lead, zinc, nickel, cobalt, mercury, and arsenic can cause a spectrum of health concerns, ranging from acute symptoms (e.g., local irritation, gastroenteritis, and pneumonia) to long-term effects (e.g., abnormal physical development, cancer, central nervous system damage, and kidney damage).⁴² The extensive use of heavy metals (e.g., lead, As) in various industries poses a dual threat: to workers directly exposed during production, and to the general population through environmental contamination of air, water, and food (e.g., mercury in fish).⁴³

Heavy metal exposure and DNA methylation

The ability of As to promote cancer transformation is linked to its capacity to alter the DNA methylation profile of several genes involved in cancer progression (Figure 4).⁴⁴ Hypermethylation of tumor suppressor genes, such as cyclin-dependent kinase inhibitor 2A (CDKN2A7p16^INK4a), cyclin-dependent kinase inhibitor 1A (CDKN1A/p21), tumor protein p53 (TPp53), Ras association domain family protein 1A (RASSF1A), and death-associated protein kinase (DAPK), has been described in different populations exposed to As.^45,46,47 This finding is supported by studies carried out in rat TRL1215 liver cells and in mice, which showed that treatment with 5-aza-deoxycytidine, an inhibitor of DNA methyltransferase, partially reversed the silencing of CDKN1A/p21 and metallothionein-1 (MT-1) caused by As.⁴⁸ Similarly, chronic exposure to arsenite in mice resulted in DNA hypermethylation of the promoter region in lung tissue and tumor, leading to the silencing of CDKN2A and RASSF1A.⁴⁹ However, exposure can also lead to global DNA hypomethylation, and this can cause aberrant gene expression, including increased expression of oncogenes, such as the estrogen receptor-α (ER- α) and HA-ras.⁵⁰ In addition, it was proposed that DNA methylation is involved in mediating the toxicity of arsenite as well as other metals (e.g., Hg, Cu, and Zn) on fetal tissues. Indeed, maternal exposure to heavy metals in the environment causes alterations in the DNA methylation profile of the fetus. These changes occur in genes or genomic regions where compromised regulation increases the risk of developing diseases. For example, prenatal exposure to As causes a slight increase in the methylation of the promoter region of tumor suppressor gene p53.^51,52 This could help explain the major risk of cancer in children under five years of age born in areas with As-contaminated air.⁵³ These studies led to the hypothesis that the toxic effects of these materials on offspring are mediated by changes in DNA methylation that occur during the embryonic stage.

Figure 4.

The effects of arsenic on the epigenome

The diagram illustrates the effects of arsenic on the epigenome, with the resulting health outcomes. Arsenic causes DNA hypomethylation of the oncogenes ER-α and cyclin D1, and DNA hypermethylation of tumor suppressor genes, such as CDKN2A/p16 INK4a, p53, RASSF1A, and DAPK, ultimately resulting in promoting cancer formation. It also reduces H3K27me3 of several genes involved in the IGF2R-MAPK pathway in keratinocyte stem cells, and of pro-inflammatory genes, such as IL-1α, MCP-1, TNF-α, and VEGF, resulting in their transcriptional activation and skin cancer development. Increased H3K9ac caused by arsenic results in the overexpression of Mef2C, resulting in cardiac malformations in fetuses. Due to increased H3K9ac, Fkbp5 is upregulated in the brain of males and is involved in the stress response, while in female brains Crh is upregulated and has a protective effect, thus, arsenic exposure results in cognitive impairments that are more severe in males. Created in BioRender. Romanò, A. (2025) BioRender.com/g05c471.

It has also been demonstrated that nickel and chromium promote hypermethylation of genes involved in cancer. In lung cancer, nickel exposure causes the DNA methylation and silencing of the O⁶-methylguanine-DNA methyltransferase (MGMT) gene.⁵⁴ Additionally, biopsies from lung cancer patients chronically exposed to chromium show increased methylation in several tumor suppressor genes, including the mismatch repair gene human MutL homolog 1 (hMLH1), p16INK4a, TP53, and adenomatous polyposis coli (APC).⁵⁵

Regarding the mechanisms by which heavy metals promote hypermethylation, recent studies have demonstrated that arsenite and nickel interfere with the activity of TET enzymes, a class of epigenetic enzymes that catalyze the oxidation of 5-methylcytosine, a critical step for active DNA demethylation.⁵⁶ This suggests that one way through which these heavy metals promote hypermethylation could be by blocking demethylation processes. 5-hydroxycytosine, in addition to being an intermediate of active DNA methylation, can function as an epigenetic marker promoting transcription. Therefore, these results also suggest that the effects of these heavy metals on transcription may be due not only to an alteration in the methylation profile but also to changes in the genomic distribution of 5-hydroxycytosine. To clarify this, future studies will need to investigate the impact of these metals on the methylation and hydroxymethylation profiles and compare these with transcription changes.

Heavy metal exposure and histone modifications

In vitro and in vivo studies have demonstrated how As causes changes to several histone modifications at a global level. (Figure 4).^37,57,58 Genes with an altered histone modification profile following exposure to this heavy metal have also been identified. For example, exposure of pregnant rats to As (NaAsO₂) can induce cardiac malformation in the fetuses through the upregulation Mef2C, a key transcription factor involved in cardiac development and heart failure, via H3K9ac: folic acid supplementation ameliorated this toxic effect by reducing the abnormally high levels of H3K9ac, promoting an increased level of the histone deacetylase sirtuin.⁵⁹ These results have led to the hypothesis that the increasing incidence of congenital heart disease in China could be due to exposure to As present in environmental pollutants, which may compromise cardiac development by altering the epigenomic profile of heart cells.⁵⁹

Prenatal As exposure also has sex-dependent effects in the frontal cortex of mice, leading to neuronal damage in males but not in females. Solomon et al. proposed that in females, the increased expression of corticotrophin releasing hormone (Crh) due to an increase in H3K9ac has a protective function on brain development that is not present in males. They also observed opposite trends in the expression of the key stress gene Fkbp5, which increases significantly only in males by increased H3K9ac. This might be one of the causes that leads to executive dysfunction in adult males exposed to As during development.⁶⁰

Chronic As exposure is a well-established co-carcinogen in human skin.^61,62 A study on mice suggested that histone methylation plays a role in this toxic effect. Specifically, As-exposed keratinocyte stem cells had an activation of the IGF2R-MAPK signaling axis, a cellular pathway that regulates cell proliferation and differentiation. This activation is due to increased IGF2R expression, caused by a reduced level of H3K27me3 at its promoter.⁶¹ Additionally, As induces a persistent inflammatory state in the skin of prenatally exposed mice, promoting the expression of pro-inflammatory cytokines through the reduction of H3K27me3 and its histone methyltransferases at the promoters of the genes.⁶² Since As is methylated in the liver primarily through As methyltransferase (AS3MT), this process might deplete the level of S-adenosylmethionine. This depletion can reduce histone methylation and alter the epigenome, leading to changes in gene expression that may contribute to the development of diseases.⁶²

An epigenetic effect has also been demonstrated for mercury and its derivative organic compounds, like methylmercury (MeHg).⁶³ Studies on immortalized cell lines and animal models suggest that the developing nervous system is impaired by MeHg-induced toxicity.⁶⁴ Studies have shown that MeHg can alter the epigenetic profile of the neurotrophin brain-derived neurotrophic factor (BDNF) gene, leading to its transcriptional repression and to decreased mRNA levels: mercury exposure causes an increase in H3K27me3 and a decrease of H3 histone acetylation on the IV promoter of BDNF. The effect of MeHg on the expression of BDNF was lost with treatment with fluoxetine, an antidepressive drug that does not alter hypermethylation of H3K27 but promotes the acetylation of histone H3 at the BDNF promoter.⁶⁵ These studies indicate that exposure to MeHg during development can alter the chromatin state and predispose mice to depression and anxiety-like behaviors.^63,65

It has also been reported that cadmium affects the activity of some epigenetic enzymes. For example, cadmium stimulates the activity in testes of G9a, a histone methyltransferase whose deregulation is involved in several diseases, including cancer and cardiovascular disease.⁶⁶ Additionally, cadmium is associated with high levels of inflammatory cytokines, which are produced by increased acetylation and nuclear localization of the pro-inflammatory transcription factor NF-κB.⁶⁷ Various altered patterns of histone modifications were found in many cadmium-induced cancer cells (e.g., UROtsa human urothelial cells, BEAS-2B cells, MCF-7, and T47-D breast cancer cells), which may contribute to their enhanced proliferation, migration, and invasion abilities.⁵⁷

Finally, it has been demonstrated that epigenetic mechanisms are involved in lead (Pb) toxicity in nervous and striated skeletal muscle tissues. PC12 cells, a rat pheochromocytoma cell line, exposed to a low concentration of Pb—which allowed the researchers to study the toxic effects of the metal without significant cell death—had a reduced expression of EZH2, an enzyme responsible for histone methylation, this reduction led to an increase in the expression of its target genes (Alox15, Notch1, NGFR, EGR2, HFE, and CaMKK2) and resulted in abnormal neurite outgrowth.⁶⁸

Another study aimed to assess the impact of Pb exposure on skeletal myogenesis, showing that the protein and mRNA levels of the main myogenic factors, such as MYOD, MYO, MCK, and MYH4, were decreased in C2C12 immortalized murine myoblasts cells exposed to Pb. This decrease was caused by a reduction in H3K9ac at those genes, due to an increase in histone deacetylase HDAC2 levels.⁶⁹ It is not clear whether Pb has a direct action on the overexpression of HDAC2 or if it is an indirect consequence of other toxic mechanisms.

Both studies showed that restoring correct expression of the epigenetic enzyme, respectively, EZH2 overexpression⁶⁸ and HDAC2 knockdown,⁶⁹ interfered with the toxic effects of Pb.

Finally, our laboratory, through integration of transcriptome and epigenome data, has recently provided evidence of how iron oxide nanoparticles with and without cobalt, and their constituent ions, promote changes in the transcription of genes related to toxicity (e.g., metallothionein 1 and 2, cbl proto-oncogene B, and transforming growth factor β). This occurred through the modulation of enhancer activity, distal transcriptional regulatory elements involved in several biological processes and human diseases, via H3K27ac.⁷⁰

Cigarette smoking

Cigarette smoking is a leading, preventable cause of premature deaths worldwide. A 2017 report by the World Health Organization (WHO) revealed that smoking causes over 7 million deaths annually. It is a primary risk factor for several diseases, including cardiovascular disease (CVD), stroke, diabetes, chronic obstructive pulmonary disease, and cancer.^71,72 The report also highlights the dangers of passive smoking, revealing that it was responsible for 1.5 million deaths from chronic respiratory diseases, 1.2 million deaths from cancer, and 60,000 deaths in children under the age of 5 from respiratory infections.

Exposure to second-hand smoke is also strongly linked to CVD and cancer, particularly lung and breast cancer.⁷³ Cigarette smoke contains approximately 250 chemical species known to be toxic for humans, either directly or indirectly, some are even carcinogenic. One such compound is benzo[a]pyrene (BaP), a polycyclic aromatic hydrocarbon formed during incomplete combustion of organic materials. BaP’s high lipophilicity allows it to easily cross the placenta and blood-brain barrier.⁷⁴

Another compound present in cigarettes is nicotine, which is especially harmful due to its addictive properties. Evidence suggests that this substance can alter the formation of long-lasting memories when associated with learning episodes, and this is reflected in changes in brain regions involved in reward learning. Nicotine can increase neuronal activity in the reward circuitry of the brain by directly interacting with the endogenous cholinergic system via nicotinic acetylcholine receptors (nAChRs).⁷⁵ These effects can have long-term effects and be the cause of nicotine addiction relapses. It has been hypothesized that epigenetics might be involved in the maintenance of appetitive and associative learning processes.⁷⁶

Cigarette smoking and DNA methylation

Epigenome-wide studies (EWASs) have revealed changes in DNA methylation in whole blood of active smoking adults.⁷⁷ These methylation changes were associated with altered expression of genes involved in smoking-related diseases.⁷⁸ These findings, which suggest an involvement of this epigenetic mark in promoting the transcriptional changes underlying smoking-related diseases, are supported by a recent study carried out on mice. This study showed that exposing mice to nicotine at concentrations comparable to those found in heavy smokers resulted in reduced expression of DNMT1, a key enzyme involved in DNA methylation. This reduction led to upregulation of glutamic acid decarboxylase (GAD) 67 in GABAergic neurons, due to loss of DNA methylation at the promoter region encoding this GABA synthesis enzyme.⁷⁹ Alteration of DNA methylation profile in blood cells suggested its potential use as a biomarker to evaluate the biological risk of smoking.⁸⁰

Cigarette smoking and histone modifications

Cigarette smoke induces pro-inflammatory genes through chromatin remodeling in the lung, playing a part in the etiology of chronic obstructive pulmonary disease.⁸¹ A study found that neonatal rats exposed to BaP had a lower testosterone level and a reduced expression of the StAr gene at an adult stage. This reduction was linked to lower levels of histone H3 acetylation at lysine 14 (H3K14ac) at the promoter region of this gene. This finding led to the hypothesis that the ability of BaP to impair spermatogenesis and testosterone production is mediated by histone acetylation.⁸² In another study carried out on HeLa cells, it was demonstrated that BaP promoted reactivation of long interspersed nuclear element-1 (LINE-1) caused by, on one hand, enrichment of H3K4me3 and H3K9ac and, on the other, reduction of CpG methylation within of LINE-1 promoter due to impairment of DNMT1 activity.⁸³ Since LINE-1 reactivation occurs in several human cancers, this mechanism could be involved in BaP-evoked carcinogenesis.⁸⁴ Since prenatal exposure to BaP is correlated with impaired hippocampal synaptic plasticity and cognitive function, they tried to investigate the molecular mechanism in a study on a rat model, finding an increased expression of histone deacetylases and a subsequent reduction in histone acetylation in many genes in the hippocampus.⁷⁴

Studies found that nicotine can promote an overall permissive chromatin state via reduction of histone methylation and an increase in histone acetylation in multiple tissue types and animal models. This chromatin state seems to enable gene expression of pathways necessary for addiction and memory-related plasticity.⁷⁶ A study on zebrafish unveiled the crucial role of H3K9 demethylation via G9a/GLP complex in mediating the long-term effects of nicotine in the brain, which seems to activate genes involved in the reward pathway that leads to nicotine addiction.⁸⁵ A study on rats evaluated histone modifications on the promoters of Cdk-5 and BDNF, as they play critical roles in the neuronal plasticity necessary for long-term memory formation. H3K9me2 and H3K27me3 levels were significantly reduced at the BDNF exon IV promoter, and a similar pattern was observed at the Cdk-5 promoter.⁸⁶

Prenatal nicotine exposure (PNE) is associated with numerous adverse effects in offspring and is responsible for an increase in maternal glucocorticoids (GC), resulting in inheritable impacts on hepatic glucose and lipid metabolic function.^87,88,89 PNE impairs the strength of cartilage in adult offspring up to the second generation, probably through the effect of GC on the expression of extracellular matrix and transforming growth factor β (TGF-β) signaling genes, probably induced by a hypoacetylation of their promoters.⁸⁷ PNE significantly decreased histone H3K9 methylation in PND1 pancreatic and INS-1E cells in offspring, promoting a euchromatic state.⁹⁰ PNE has an impact on the synthesis of steroid hormones in adrenal gland and ovary, by reducing the expression of key genes in these pathways correlated with a reduction in histone acetylation.^91,92 In nicotine-exposed rat testes, H3K9me3 marks were significantly increased and perturbations in the expression of germline responsive genes may affect proper gametogenesis.⁹²

Another study analyzed the effect of in-utero exposure to nicotine and ethanol combined, which resulted in milder effects than single exposure. This finding was potentially explained by cross-tolerance through decreased metabolism and pharmacologic effects of these two substances together.⁹³

Alcohol

The recreational use and abuse of alcohol is widespread in the population starting from a very young age, for this reason, it is important to evaluate its negative effects on human health.⁹⁴ While the direct toxic effects of alcohol and its metabolites have been known for some time,⁹⁵ its impact on the epigenome and the long-term effects are being investigated only recently.

Alcohol and DNA methylation

Chronic alcohol consumption can induce alterations in DNA methylation patterns within the liver and brain, leading to changes in gene expression contributing to impaired function of these organs, ultimately promoting the development of alcohol use disorder (AUD), a complex disease affecting brain regions and peripheral organs.⁹⁶ Notably, the impact of alcohol on DNA methylation patterns differs between the liver and brain. In the liver, alcohol induces a loss of DNA methylation by reducing the availability of methionine, a key methyl donor. This occurs through two mechanisms: (1) downregulation of enzymes involved in the methionine cycle, and (2) impaired folate absorption due to dysfunction and reduced expression of intestinal folate transporter (Figure 5).⁹⁷ The importance of these mechanisms is supported by the fact that in micropigs, oxidative liver injury induced by ethanol and a folate-deficient diet was reverted by a diet high in S-adenosylmethionine.⁹⁸ In addition, it was found that alcohol caused a loss of 5-hydroxymethylcytosine by inhibiting the activity of TET1, this induced hepatocyte apoptosis.⁹⁹ Conversely, studies in mice and humans have shown that alcohol promotes hypermethylation in the brain at different stages of life (prenatal, adolescent, and adult) affecting several genes whose deregulation can be linked to various aspects of AUD.⁹⁶ For example, cognitive and attention-related disorders in children whose mothers consumed alcohol during pregnancy have been liked to hypermethylation of key genes involved in neuronal development, as detected in meconium at birth.¹⁰⁰ Bdnf and neuropeptide Y (Npy) were hypermethylated in the amygdala of adult rats following adolescent exposure to intermittent ethanol consumption. Treatment with the DNMT inhibitor 5-azacytidine (5-azaC) in adulthood restored the normal methylation level of these genes and simultaneously alleviated anxiety-like behaviors and alcohol consumption linked to alcohol.¹⁰¹ Additionally, in adults, alcohol consumption was associated with hypermethylation of several genes related to neuronal disorders in saliva cells and peripheral blood cell.^102,103 Finally, it was observed that hypermethylation of genes encoding for the transporter of dopamine—a neurotransmitter involved in the neuronal circuit responsible for alcohol cravings—was inversely related to the intensity of alcohol cravings.¹⁰⁴ This occurs because the decreased expression of the dopamine transporter, which is responsible for reuptake of extracellular dopamine into presynaptic neurons, leads to an abnormal accumulation of the neurotransmitter in the synaptic cleft.¹⁰⁵ This dysregulation contributes to the neurochemical imbalances underlying alcohol dependence.¹⁰⁶

Figure 5.

The effects of alcohol and its metabolism on the epigenome

The figure shows the impact of alcohol metabolism on the epigenome. Alcohol downregulates enzymes involved in the methionine cycle and impairs folate absorption, reducing the availability of methyl donors, it also inhibits TET1 enzymes, resulting in DNA hypomethylation. When metabolized, alcohol produces Ac-CoA, an acetyl donor, and consumes NAD⁺, which is less available for sirtuins (histone deacetylases) that need it as a cofactor, resulting in increased histone acetylation. Created in BioRender. Romanò, A. (2025) BioRender.com/t60k153.

Alcohol and histone modifications

Acute alcohol exposure produces an anxiolytic response associated with the opening of chromatin, due to increased histone acetylation and inhibition of histone deacetylase. These changes are reversed after chronic ethanol exposure. However, during withdrawal, histone acetylation decreases and is associated with the development of anxiety-like behaviors. The effects of alcohol on the body can be accentuated or reduced by innate and behavioral factors.⁹⁴ The rise in histone acetylation can be attributed to ethanol metabolism, which results in the production of acetyl-CoA, which is the acetyl donor for histone acetyltransferases. Simultaneously, ethanol metabolism depletes NAD⁺, an essential cofactor for the family of histone deacetylases called sirtuins (Figure 5).¹⁰⁷

Ethanol can activate TLR4 receptors in the brain, and the elimination of these receptors in mice protected against ethanol-induced damage and inflammation, demonstrating their role in ethanol-mediated toxicity. TLR4 has an impact on epigenetic remodeling of chromatin, since in wild-type mice there was a decrease in histone H4 acetylation and acetyltransferase activity, while in knockout mice there were no changes in chromatin conformation following ethanol exposure.¹⁰⁸

There is a similar situation in brains of an alcohol-use disorder rat model, in which a general inhibition of histone deacetylases and increase in histone H3 and H4 acetylation were reported.¹⁰⁹

Prenatal alcohol exposure (PAE) in mice seems to preferentially harm female offspring and is correlated to the disruption of genetic pathways controlling cellular respiration and oxidative phosphorylation in the brain. Although affected tissues exhibit widespread increases in H3K9me2, it is not associated with transcriptional suppression nor canonical heterochromatin formation.¹¹⁰ PAE can cause an overexpression of heart development-related genes in mice due to histone H3K9ac, causing cardiac hypertrophy.¹¹¹ PAE increased H3K9ac and H3K27ac levels in genes related to the renin-angiotensin system, leading to fetal bone development inhibition and osteopenia after birth in rats.¹¹² Finally, rat-derived mesenchymal stem cells were influenced by PAE, characterized by a resistance to osteogenic and adipogenic inductions and by an altered deposition of H3K27me3 responsible for abnormal expression in pathways related to adipogenesis and glucose metabolism.¹¹³

Final considerations

While numerous studies have reported epigenetic changes following exposure to toxic substances, the causal relationship between these changes and the toxicity of these substances remains unclear. Except for alcohol, nickel, and arsenic, it is uncertain whether the epigenetic changes described after exposure to a toxic substance are a direct mechanism of toxicity or a secondary consequence of exposure. Addressing this critical question requires determining whether epigenetic modifications alter gene expression involved in toxicity and how toxic substances induce these changes. Genome-wide studies correlating transcriptional alterations with epigenetic modifications, alongside assays identifying enzymes and proteins affected by toxicants, could provide valuable insights to answer these questions.

A fundamental issue for toxicology, namely dose, is rarely addressed in epigenetic studies on toxicity. The absence of such experiments leaves open the possibility that the epigenetic landscape may still be disrupted even at non-effective doses. These doses, while not causing overly toxic effects, could sensitize cells to subsequent exposure to the same toxicant or to others (latent toxicity). Another possibility is that such perturbations could enhance resistance to future exposure¹¹⁴ or contribute to the phenomenon of hormesis, in which certain toxicants can exert a beneficial effect at low doses (biphasic response).¹¹⁵ To clarify these aspects, studies are needed to monitor the dynamics of epigenetic modifications along both dose-response and time-response curves, providing a comprehensive understanding of their role in toxicity.

An intriguing aspect of studies on the epigenetic effects of toxic substances is their potential to explain how these substances can mediate toxic effects on offspring.¹¹⁶ Toxicant exposure could perturb the epigenetic profile of gametes, influencing the epigenome and phenotype of progeny. In some cases, these epigenetic perturbations may persist across multiple generations. Similarly, maternal exposure could alter the epigenetic landscape of embryonic somatic tissues, potentially affecting offspring phenotype.¹¹⁷ However, a fundamental question remains open: how do epigenetic mechanisms act as a kind of “inherited memory”, transmitting the effects of parental exposure to toxic substances across generations? Clarifying these aspects will not only enhance our understanding of the mechanisms underlying toxicity but could also pave the way for developing epigenetic therapies to treat toxic effects.

Resource availability

Lead contact

Requests for further information and resources should be directed to and will be fulfilled by the lead contact, Roberto Papait (roberto.papait@uninsubria.it).

Materials availability

This study did not generate new unique reagents.

Data and code availability

This study did not generate new data or code.

Acknowledgments

This work was supported by the PRIN project by the Ministry of Univeristy and Research (MUR), University and Research through the PRIN 2020 (project number 20205X4C9E) and the National Recovery and Resilience Plan (NRPP) through the Spoke n.4 (National Center for Gene Therapy and RNA-based Drugs), to RP.

Author contributions

A.R and R.P. conceptualized and wrote the manuscript; C.P., R.G., and G.B. contributed to the revision and critical editing of the manuscript.

Declaration of interests

The authors declare no competing interests.