Phthalates and epigenetics: An emerging public health concern

Graphical abstract

Highlights

• •Phthalates induce organ-specific epigenetic changes in hormone-related genes.
• •Evidence supports transgenerational inheritance in phthalate-exposed animal models.
• •Mechanisms involve altered DNMT, HAT, and androgen receptor signaling.
• •Epigenetic changes associate with ADHD, infertility, and metabolic disorders.
• •Epigenetic therapies show promise but remain limited against phthalate toxicity.

Abstract

Phthalates are a group of phthalic acid esters that are commonly used as plasticizers in many consumer products to improve elasticity, transparency, durability, and toughness. Phthalates are also ubiquitously found throughout our environment. In recent years, research has indicated a growing concern over the potential negative health effects that phthalates have on the human body. Considering their presence in a wide range of consumer goods, including food packaging, household goods, medical equipment, and personal hygiene products, humans are continuously exposed to many phthalates in their everyday lives. More strikingly, exposure to phthalates has been shown to induce abnormal epigenetic changes in noncoding RNA expression, DNA methylation, and histone modification. Epigenetic changes are critical in governing gene expression while leaving the DNA sequence intact. Previous studies have established the role of aberrant epigenetic changes in the pathogenesis of many diseases, including cancer and endocrine diseases related to phthalate exposure. The purpose of this review is to provide insight into the mechanisms by which phthalates may affect epigenetic processes and the potential adverse health consequences of these interactions.

1. Introduction

Over the past few decades, there has been a significant increase in the global use of plasticizers in industrial products. Phthalates are synthetic chemical plasticizers that are commonly used as additives in plastic manufacturing to soften or increase the flexibility and durability of plastics (Perestrelo et al., 2021). They are synthesized by reacting alcohol with phthalic anhydrite and are used in the production of several consumer goods (Koch et al., 2013). Phthalates are stable, colorless, odorless, and flavorless chemicals that can remain in the liquid state at temperatures ranging from 25 °C to 50 °C (Tran et al., 2022). Some consumer products that contain phthalates include but are not limited to medical equipment (Hannon and Flaws, 2015), such as blood bags, dialysis machines, disposable surgical gloves, and medical devices (Talsness et al., 2009). Phthalates are also common in products such as single-use food and beverage packaging, solvents and detergents (Clausen et al., 2012); personal care items such as (Buckley et al., 2016) perfumes and cosmetics; and even toys (Xu et al., 2010). Because phthalates are ubiquitously utilized in everyday products, during the production and disposal phases of plastic products, they are introduced into the environment, and owing to their capacity to easily leach into their surroundings, the adverse health effects of phthalates are well described (Erythropel et al., 2014). Owing to these leaching properties, phthalates are found in many different parts of the environment, such as water, food, dust, and even in the air (Chou et al., 2009). Over the past few years, concerns related to the health effects of phthalates, particularly their effects on development and the reproductive system, have increased, as phthalates have been shown to possess endocrine-disrupting properties (Ghosh et al., 2022, Wang and Qian, 2021). Furthermore, increasing evidence suggests that phthalates may induce abnormal epigenetic changes to cause other deleterious health effects (Dutta et al., 2020).

Humans and other living organisms are consistently exposed to phthalates as a result of anthropogenic activities. In model system-based studies, exposure to phthalates has adverse effects on health within the respiratory and reproductive systems (Ghosh et al., 2022, Yu and Wang, 2022). Further studies, including clinical studies, indicate that phthalates can cause endocrine disruption and dysfunction, particularly in children and adolescents (Bowman et al., 2019); in vivo, in vitro, and clinical samples indicate that phthalates can also cause cardiovascular disease, neurodevelopment disorders, asthma, infertility, diabetes, and certain forms of cancer (Akingbemi et al., 2001, Kabekkodu et al., 2024, Khan et al., 2022). In humans, phthalate exposure has been shown to be a risk factor for hormonal imbalance and diseases in newborns, children, and adults (Ahn et al., 2006); with children having greater sensitivity to the toxic effects of phthalates (Chou et al., 2009). Because phthalates are associated with lower testosterone and estrogen levels and inhibit the function of thyroid hormones, they may be regarded as reproductive toxicants. The global production of phthalate esters (PAEs) is estimated to be approximately 8.1 million tons annually (Khoshmanesh et al., 2024). In 2021, the annual consumption of PAEs was approximately 3.6 million tons (Khoshmanesh et al., 2024). Mechanistic studies have investigated the potential mechanisms and signaling pathways affected by phthalate exposure (Wang and Wang, 2025, Xiong et al., 2025, Zhang et al., 2025, Zhang et al., 2025, Zhang et al., 2024). In vitro and in vivo studies have shown that exposure to phthalates may modify epigenetic marks to alter the expression of noncoding RNAs and protein-coding genes (Dutta et al., 2020). These alterations in epigenetic marks may have a deleterious impact on human health. The following sections provide a detailed review of epigenetic modifications, such as DNA methylation, histone modification, and noncoding RNA, in relation to phthalates. Furthermore, we discuss the possibility of epigenetic therapy.

2. Phthalate classification, human exposure and metabolism

Phthalates are classified into two categories: low-molecular-weight phthalates and high-molecular-weight phthalates. These categories are based on the number of carbon atoms present. Dimethyl phthalate (DMP), dibutyl phthalate (DBP), and diethyl phthalate (DEP) are examples of low-molecular-weight phthalates with 3--8 carbon atoms. Di(2-ethylhexyl) phthalate (DEHP), diisodecyl phthalate (DIDP), diisononyl phthalate (DINP), and di(2-propylheptyl) phthalate (DPHP) are examples of high-molecular-weight phthalates with 9--13 carbon atoms. The most important class of phthalate plasticizers found in various consumer goods are phthalate diesters such as DEHP, DBP, and DEP. DEHP is extensively utilized in flooring, wall covering, consumer goods, medical equipment, and food contact applications (Adibi et al., 2003, Cheng et al., 2021, Cirillo et al., 2013, Miura et al., 2021). Conversely, DBP and DEP are applied in the manufacturing of lacquers, varnishes, personal care products (including lotions, cosmetics, and perfumes), and coatings of medications (Giuliani et al., 2020). Phthalates are not covalently attached to plastics; therefore, they can easily leach out via repeated exposure to fluctuations in temperature (Henkel et al., 2023, Rose et al., 2012). Phthalates can contaminate the environment by leaking into groundwater from landfills (Battersby and Wilson, 1989); especially as a significant proportion of DEHP-containing plastic is buried in landfills, increasing the risk of groundwater pollution (Chang et al., 2005). Phthalate esters (PAEs) released from residential and commercial wastewater may enter the food chain, thus creating an unintentional source for human introduction (Chang et al., 2021, Liang et al., 2008). Humans are also exposed to phthalates through the use of personal care products via different routes, including ingestion, inhalation, dermal absorption, and parenteral administration. Oral exposure is the result of the use of teething toys, contaminated food and water consumption, and other liquids contaminated with phthalates (Frederiksen et al., 2012). Phthalates evaporated from the PVC, nail polish, and hair spray can enter the human body via inhalation (Guo and Kannan, 2011). It can also enter the human body via skin contact absorption from cosmetics, clothing and medical devices (Araki et al., 2014).

Phthalates are exposed to humns through ingestion, inhalation, and dermal absorption (Rusyn et al., 2006). Once it enters the human body, it is metabolized through two main steps: phase I hydrolysis and phase II conjugation (Frederiksen et al., 2007). In the initial step, diester phthalates are hydrolyzed by intestinal and parenchymal lipases to monoester phthalates as the primary metabolites, which are often more biologically active than the parent compounds are (Zhang et al., 2021). Phthalates have short biological half-lives, approximately 12 h (Hoppin et al., 2002). Short-chain phthalates are mainly excreted in urine as monoesters, whereas long-chain phthalates undergo further oxidation (hydroxylation and oxidation) followed by glucuronidation in phase II. This reaction is catalyzed by uridine 5′-diphosphoglucuronyl transferases, resulting in hydrophilic glucuronide derivatives that are excreted via urine and feces (Frederiksen et al., 2007, Zhang et al., 2021).

3. Phthalate and human health

Phthalates are metabolized through multistep processes such as de-esterification and glucuronide conjugation, followed by excretion through urine, sweat, or feces. Both phthalates and their metabolites can result in inappropriate activation of molecular signaling pathways that can have an impact on human health. Exposure to plasticizers such as phthalates and BPA has been shown to increase the risk of endocrine, cardiac, and metabolic disorders in all age groups. Phthalate exposure is linked to reproductive dysfunction (Land et al., 2025, Radke et al., 2018, Thurston et al., 2016); kidney disease (Kang et al., 2021, Tao et al., 2024); diabetes (Mariana and Cairrao, 2023, Zhang et al., 2022); thyroid disease (Chen et al., 2021, Maia and Vieira-Coelho, 2024); neurological disorders (Ejaredar et al., 2015, Safarpour et al., 2022); obesity (Peng et al., 2023, Wu et al., 2023); respiratory disorders (Ketema et al., 2025, Yu and Wang, 2024); and cardiovascular diseases (Mariana et al., 2023) (Fig. 1). Moreover, they are also linked to pediatric obesity (Kim and Park, 2014); insulin resistance, and obesity through interference with thyroid hormones (Bowman et al., 2019, Ghassabian and Trasande, 2018, Núñez-Sánchez et al., 2023). Phthalates suppress the immune system, exacerbating allergies and asthma (Wang et al., 2021). Studies indicate that phthalate exposure harms reproductive development, contributing to issues such as low sperm counts and genital abnormalities. Phthalates may also accelerate the progression of many cancers (Khan et al., 2022, Kim et al., 2022).

Fig. 1.

Association of phthalates with human diseases.

Recent studies on urine samples revealed detectable amounts of phthalate metabolites, suggesting that urinary phthalate metabolites can be valuable in epidemiological studies (Yang et al., 2022). Individuals taking medications containing phthalates are particularly exposed to high concentrations of these contaminants. For example, those using mesalamine have shown urinary levels of mono-butyl phthalate (Asimakopoulos et al., 2016); a metabolite of DBP, approximately 60 times greater than those not using the drug and exposed only through environmental means (Ahern et al., 2019). Mesalamine, the active ingredient in Asacol, is used to treat inflammatory bowel disease (Iacucci et al., 2010). Men taking Asacol presented phthalate urinary concentration levels that were 1,000 times higher than those of those who were not taking Asacol (Nassan et al., 2019). The extensive use of phthalates and their ubiquitous presence in daily life has raised significant concerns regarding human health. Studies have linked phthalates to various health issues, particularly reproductive disorders (Lin et al., 2011). Infertility, decreased fecundity, and hormone-dependent cancers are among the most common reproductive disorders worldwide (Hlisníková et al., 2020, Kabekkodu et al., 2020). According to a WHO report from 2023, approximately 17.5 % of the adult population, approximately 1 in 6 worldwide, experience infertility (World Health Organization, 2023). Over the past 50 years, there has been a significant decline in the sperm concentration in men, highlighting the increasing prevalence of reproductive disorders (Sengupta et al., 2018). Addressing these increasing reproductive health issues, it is essential to understand in depth the mechanisms by which phthalates exert their effects. While the evidence linking phthalates to cardiovascular, thyroid, respiratory, and other diseases is mixed, further large-scale studies are needed to understand the long-term health impacts of phthalate exposure.

4. Epigenetic changes caused by phthalates

Research on the impact of phthalates has gained increasing attention, particularly in the field of epigenetics (Dutta et al., 2020, Dolinoy and Jirtle, 2008, Swanson et al., 2023). Phthalates, including DBP/BBP/MBP and DEHP/MEHP, can alter epigenetic markers (e.g., DNA methylation, histone modifications, and noncoding RNAs). Research has shown that exposure to low doses of phthalate may also cause epigenetic changes. Epigenetic alterations, such as DNA methylation or histone modification, can upregulate or downregulate gene expression without altering the DNA sequence. These stable changes can persist throughout life and may contribute to adverse outcomes such as cancer or other diseases by disrupting normal gene regulation (Singh and Li, 2012); therefore, it is important for us to understand how these epigenetic changes translate to human health as well as gain an understanding of how one change could impact another. Here, we review the epigenetic effects of environmental phthalate pollutants on human health (Fig. 2).

Fig. 2.

Effects of the epigenetic alterations induced by phthalate exposure on human health.

4.1. Overview of epigenetics

Epigenetic changes such as DNA methylation, noncoding RNAs, and histone modifications are critical for controlling gene expression in prokaryotes and eukaryotic organisms (Adiga et al., 2023, Adiga et al., 2024, Gibney and Nolan, 2010). DNA methylation involves the addition of a methyl group to the fifth carbon of a cytosine base, predominantly occurring inside a CpG dinucleotide. This modification is catalyzed by DNA methyltransferases (DNMTs) (Jang et al., 2017, Arand et al., 2012). This modification plays a critical role in genomic imprinting, chromosomal inactivation, gene regulation and development (SanMiguel and Bartolomei, 2018). In addition to 5-methylcytosine (5mC), different types of DNA modifications occur in human cells. In addition to 5-methylcytosine (5mC), which is the most extensively studied DNA modification, human cells harbor other epigenetic marks, such as 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC), generated through iterative oxidation of 5mC by TET enzymes (Wu and Zhang, 2011, Pfeifer et al., 2020). More recently, N6-methyladenine (6 mA) has also been reported in mammalian genomes, adding another layer of complexity to DNA epigenetic regulation (Shen et al., 2022, Feng and He, 2023). Many chemicals, including phthalates, have been shown to induce epigenetic changes (Dutta et al., 2020, Akanbi et al., 2025). Noncoding RNAs (ncRNAs) are a group of functional RNA molecules that do not encode proteins that regulate gene expression at both the transcriptional and posttranscriptional levels. ncRNAs play an active role in controlling transcription, gene expression, and protein synthesis (Kaikkonen et al., 2011, Kugel and Goodrich, 2012, Eldakhakhny et al., 2024). Histones are basic proteins that help package DNA (Vijayalakshmi et al., 2025). Histones interact with DNA to form nucleosomes, which serve as the fundamental building blocks of chromatin. These structures not only help package DNA into a compact form but also control its accessibility. The flexible regulation of chromatin accessibility plays a crucial role in maintaining genome integrity and supporting various cellular functions. Histones undergo posttranslational modifications that affect their function (Vijayalakshmi et al., 2025). These aberrant epigenomic changes may impact health and contribute to the development and progression of several disorders and diseases (Farsetti et al., 2023). Some of these epigenome-driven diseases and disorders associated with phthalate exposure include decreased pregnancy and high miscarriage rates, anemia, toxemia, preeclampsia, early menopause, and abnormal sex steroid hormone levels (Lucaccioni et al., 2021, Rani et al., 2025, Sathyanarayana et al., 2014, Zhao et al., 2024). We discuss the impact of phthalate exposure on the epigenome, particularly DNA methylation, histone modifications, and microRNA (miRNA) expression, and how these different changes can work in tandem with one another.

4.1.1. DNA methylation in relation to phthalates

DNA methylation is the process by which a methyl group is added to the 5th position of cytosine in a CpG dinucleotide context within a CpG island (Fernández-Sanlés et al., 2017). DNA methylation is critical for chromatin organization, gene expression regulation, the silencing of parasitic sequences, and X chromosome inactivation (Hackett and Surani, 2013). Studies have reported an association between phthalate exposure and alterations in DNA methylation (Wang et al., 2021, Wang et al., 2015) (Table 1). In a cohort study (Bowman et al., 2019); urine and blood samples were collected from mothers in their first (T1), second (T2), and third (T3) trimesters and from the children of the enrolled mothers during follow-up visits from birth until 5 years of age and perimenadolescence (ages 9 and 17 years). DNA methylation was measured by pyrosequencing (at 4 CpG sites for H19 and 5 CpG sites for HSD11B2). The analysis revealed that exposure to the urine phthalate metabolites MBP, MIBP, and MBzP during pregnancy induced DNA methylation in the H19 gene and repetitive elements. In periaadolescent children, a correlation was found between the methylation of growth-regulating genes (H19 and HSD11B2) and phthalate exposure during pregnancy and early development. The H19 and HSD11B2 genes are critical for growth, the regulation of adiposity, and the inhibition of the growth-inhibiting functions of cortisol in peri-adolescent children. Furthermore, the same study indicated that phthalates may influence adiposity by affecting the methylation of adipose pathway genes (Bowman et al., 2019).

Table 1.

Epigenetic impact of phthalate-induced DNA methylation.

Phthalate	Model used	Concentration	Exposure Duration	Genes	Hypomethylation	Hypermethylation	References
DEHP	MCF-7 (ERα-dependent), MDA-MB-231 (ERα-independent)	100 μM	48 hrs.	Esr1, CDH1, EPHA2, ERBB2	Esr1, CDH1	EPHA2, ERBB2	(Chou et al., 2019)
DEHP	Pregnant mice	40 μg	0.5 to 18.5 days post coitum	Igf2r, Peg3 and H19	Igf2 and Peg3	−	(Li et al., 2014)
DBP, BBP	MCF7, MCF10A	10 μM	−	ERα	ERα	−	(Kang and Lee, 2005)
DEHP, MMP, MEP, MBP, MEHP, MEOHP	Human (Sperm and urine sample)	Urine phthalate exposure metabolites	48 hrs. (Urine sample)	LINE-1, H19 and L1T1	LINE-1, H19	L1T1	(Tian et al., 2019)
MEHP	Female CD-1 mice	100 μM	6 days	Tet3	Tet3	−	(Arcanjo et al., 2023)
DEHP	Wild type FVB/N and C57BL/6J mice	300 mg/kg/day	gestation day 9 to 19	Tmem125, Piwil2, Fkbp1a and Smim8	Tmem125	Piwil2, Fkbp1a and Smim8	(Prados et al., 2015)
DEHP	Forty ICR mice (21 day-old)	125, 250, and 375 mg/kg/day	28 days	JNK/p38MAPK/p53	p53	−	(Huang et al., 2019)
DEHP	Juvenile and adult mice	25 mg/kg	2 weeks	Pou2f3, Hrh1, Eng, Rhox11-ps2-201, Slc6a9; Ccdc24, and Cldn14	Pou2f3, Rhox11-ps2-201, Slc6a9; Ccdc24, and Cldn14	Hrh1, Eng	(Liu et al., 2021)
DEHP	Three-month-old male zebrafish	10, 33, 100 mg/L	3 months	cyp19a1a, cyp17a1 and hsd17b3	Downregulated cyp19a1a	Upregulated cyp17a1, hsd17b3	(Ma et al., 2018)
DEHP	Kunming mice	500 mg/kg/d	90 days	INSL3	Downregulated INSL3	−	(Wu et al., 2010)
DEHP	Wistar rats	5, 50, and 500 mg/kg/d	8 weeks	JAK3, STAT5, e STAT5, PPARγ	Downregulated e STAT5, PPARγ	Upregulated JAK3, STAT5	(Xu et al., 2020)

Animal studies have shown that exposure to phthalates during development is linked to alterations in DNA methylation, resulting in adverse effects on offspring (Manikkam et al., 2013, Martinez-Arguelles and Papadopoulos, 2015, Wu et al., 2010). Using either placental or cord blood samples, several human cohort studies have demonstrated a correlation between prenatal phthalate exposure and changes in the DNA methylation of specific genes (Huang et al., 2018, Huen et al., 2016, Montrose et al., 2018). Understanding this correlation contributes to identifying potential risks of prenatal phthalate exposure. A study investigating the effects of phthalates and their metabolites on selected epigenetic parameters in human peripheral blood mononuclear cells (PBMCs) revealed a significant reduction in the global DNA methylation level in PBMCs exposed to BBP, MBP and MBzP. Phthalate exposure not only induces global hypomethylation but also alters the methylation of p16, p53, BCL2, and CCND1 and downregulates the expression of p16 and p53 while increasing the expression of BCL2 and CCND1. p16 and p53 are tumor suppressor genes, and both play critical roles in regulating cell cycle progression. BCL2 and CCND1 play important roles in cell survival and proliferation by inhibiting apoptosis and driving the cell cycle. Both genes, when dysregulated, can contribute to tumor development and cancer progression. These changes have been reported to be associated with the progression of many different cancer types (Sicińska et al., 2022). Phthalate exposure has been correlated with DNA methylation alterations during pregnancy, which impact the genome-wide DNA methylation of offspring at birth (Miura et al., 2021, Huen et al., 2016). Analysis of cord blood from 64 infant‒mother pairs revealed an association between the methylation level of 25 CpG sites and DEHP phthalate exposure, resulting in overall changes in DNA methylation patterns (Chen et al., 2018). Furthermore, exposure to phthalates during pregnancy can lead to alterations in key signaling pathways, such as the p53 signaling pathway and its E27 target genes (Chen et al., 2018). MEOHP, MEHHP, MnBP, and DEHP concentrations were measured in 274 maternal urine samples collected during late pregnancy and 102 neonatal urine samples collected at birth. These samples were used to assess DNA methylation levels via the Illumina HumanMethylationEPIC BeadChip Kit, and CpG sites near the CHN2, CUL3, OR2A2 and MEGF11 genes were found to have methylation levels that were positively correlated with phthalate concentrations in neonatal urine and in female infants. These findings suggest that prenatal exposure to phthalates is significantly associated with DNA methylation changes at multiple CpG sites (Lee et al., 2023).

Association studies suggest a potential role for phthalate exposure in asthma. A previous study reported that the association between urinary metabolites and DNA methylation is related to asthma and that lower methylation of the 5′ CpG island of TNFα is correlated with asthma in children (Wang et al., 2015). Interestingly, the promoter methylation of TNFα was inversely correlated with the urinary concentration of 5OH-MEHP in 256 children with asthma. These findings suggest a potential correlation between phthalate exposure and aberrant DNA methylation (Wang et al., 2015). In a prostate cancer cell line (LNCaP), MEHP exposure resulted in global hypomethylation. Exposing male and female zebrafish to environmentally relevant doses (0.5 µg/L) of DEHP significantly decreased their body length and weight (Kwan et al., 2021). Chronic exposure to DEHP reduces spermatogenesis and the capacity to fertilize oocytes. Furthermore, DEHP exposure reduced circulating testosterone and increased estradiol. CYC19A1 (cyp19b) plays a critical role in the biosynthesis of estrogens. The exposure of human cumulus granulosa cells (hCGCs) to DEHP suppresses the cAMP and ERK1/2 signaling pathways, resulting in reduced steroid hormone production in cells stimulated with FSH (Tesic et al., 2023). DEHP-exposed zebrafish embryos presented increased expression of cyp19b. This dysregulation was the result of an increase in global DNA methylation and hypermethylation of the CYP17A1 and HSD17B3 promoters in DNA isolated from gonads as a result of phthalate exposure (Kwan et al., 2021). CYP17A1 is an enzyme involved in the production of steroid hormones, including testosterone, mineralocorticoids, and glucocorticoids. HSD17B3, which is predominantly expressed in the testis, specifically catalyzes the conversion of androstenedione to testosterone, playing a crucial role in male steroidogenesis. Changes in CYP19B activity, due to genetic mutations or endocrine disruptors such as phthalates, can impact estrogen production, potentially leading to reproductive disorders and fertility issues.

Urinary levels of phthalate metabolites were assessed via ultraperformance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS). Male children with a tenfold increase in the MMP or MBzP level presented greater body fat. Additionally, methylation of exon 2 of the POLG gene is correlated with increased fat deposits in the body and trunk (Chang et al., 2020). DNA methylation of the JAK3/STAT5/PPARγ genes affects metabolic dysfunction caused by DEHP and a high-fat diet (Xu et al., 2020). When two groups of Wistar rats were exposed to 50 or 500 mg/kg/d DEHP for 8 weeks along with a high-fat diet or control diet, the control group presented lower DNA methylation levels of peroxisome proliferator-activated receptor gamma (PPARγ) and JAK3 in the liver and lower STAT5 in adipose tissue than did the high-fat group (Xu et al., 2020). The IGF2 and H19 genes play critical roles in regulating placental development and fetal growth. To determine the impact of DEHP on breast cancer chemical therapy, MCF-7 cells (a breast cancer cell line) were exposed to DEHP, which resulted in hypermethylation of 700 genes and hypomethylation of 221 genes. BBP and DEHP treatment of MCF-7 cells resulted in demethylation of the ESR1 gene (Chou et al., 2019). These epigenetic changes disrupt Wnt/β-catenin signaling activation. Exposure of MCF-7 cells to DEHP results in a global increase in DNA methylation, accompanied by the upregulation of DNA methyltransferase 1 (DNMT1) and methyl-CpG-binding protein 2 (MECP2) expression (Ghosh et al., 2022). These methyltransferases are enzymes that add methyl groups to the fifth carbon of cytosine residues, mainly within CpG sites, and play a key role in epigenetic gene regulation. Coincidently, increased DNMT1 levels are strongly associated with the loss of p21 expression and increased cyclin D1 levels (Ghosh et al., 2022). An epigenome-wide association study consisting of 152 maternal–infant pairs reported changes in placental DNA methylation of genes, particularly those related to endocrine hormone activity, immune pathways, DNA damage, and neurodevelopment (Nakamura et al., 2023). MEHHP and MEOHP can affect placental DNA methylation, which may affect fetal health through the identification of an inverse correlation between IGF2 methylation in the placenta and urinary concentrations of MEHHP and MEOHP (Zhao et al., 2016). Phthalate exposure has been shown to affect DNA methylation in multiple tissues (Morgan et al., 2024). Interestingly, the sex of an organism may influence the pathways affected by phthalate exposure. There is also a known association between adiposity, phthalate exposure, and DNA methylation (Bowman et al., 2019). Overall, evidence from human, animal, and in vitro studies indicates that phthalate exposure alters DNA methylation in key pathways, including imprinting, endocrine signaling, and cancer-related genes. Human studies provide population-level relevance, animal studies confirm dose-dependent effects, and in vitro work offers mechanistic insight through direct impacts on DNA methyltransferase activity. Although the strength of evidence varies, the convergence across models supports DNA methylation as a central mechanism linking phthalates to adverse health outcomes. However, the exact mechanism has yet to be identified and established. Mechanistic studies are needed to dissect the underlying mechanisms that influence how phthalates and their metabolites interact with DNA methyltransferases and DNA demethylating enzymes. Furthermore, the long-term and transgenerational effects of aberrant DNA methylation and health consequences require more detailed investigations.

4.1.2. Noncoding RNAs in relation to phthalates

ncRNAs that are known to play significant roles in human disease are long noncoding RNAs (lncRNAs) and microRNAs (miRNAs). lncRNAs are functional transcripts that are more than 200 nt in length and play major roles in a myriad of regulatory functions, such as gene expression and chromatin remodeling (Dempsey and Cui, 2017). miRNAs are short transcripts ranging from 22 to 24 nt in length that bind to mRNAs, either repressing gene expression or degrading the mRNAs (Chorley et al., 2020). ncRNAs, including lncRNAs and miRNAs, are RNA molecules that do not encode proteins but play critical roles in regulating gene expression at the transcriptional and posttranscriptional levels (Kaikkonen et al., 2011). Unlike mRNAs, which serve as templates for protein synthesis, ncRNAs can modulate chromatin structure, recruit epigenetic modifiers, and influence the stability or translation of target mRNAs (Kumar et al., 2020, Peschansky and Wahlestedt, 2014). In the context of phthalate exposure, changes in ncRNA expression can contribute to altered DNA methylation, histone modifications, and downstream gene regulatory networks, thereby impacting cellular function and disease susceptibility. Exposure to environmental agents such as phthalates has been shown to change ncRNA expression, which has been implicated in numerous human diseases and conditions (Table 2). In contrast, general mRNA regulation primarily reflects changes in protein-coding transcript levels without directly altering epigenetic landscapes (Kan et al., 2022).

Table 2.

Phthalates that target noncoding RNA and histone modifications.

Phthalate used	Model used	Concentration	Exposure Duration	Genes	Downregulation	Upregulated	Reference
DEHP	Pregnant mice	0.1, 0.5, 2.5, 12.5, 62.5 μmol/L	6 days	RP24	RP24	−	(Liu et al., 2023)
DEHP	Adult male Sprague-Dawley rats	300  mg/kg	12 weeks	Abcg5 and Abcg5/8	Abcg5 and Abcg5/8	−	(Li et al., 2022)
DEHP	SHE cells from Syrian hamster embryos	12.5, 25 and 50 μM	24 hrs.	nrp2, coro1C, kif23, cdh3, and ctnnbip1	nrp2	coro1C, kif23, cdh3, ctnnbip1	(Landkocz et al., 2011)
DEHP/MEHP	Human HCT116	10 μM	48 hrs.	ST8SIA6	ST8SIA6	−	(Shih et al., 2023)
DEHP	Zebrafish embryos (AB wild-type strain)	2, 10, 50, 100, and 200 μg/L	24 hrs.	PRPF3, miR-375	miR-375	PRPF3	(Yu et al., 2023)
DEHP	Pregnant rats	2, 10 or 50 mg/kg	24 hrs.	Esr2, Cyp19a1, Grin2a, Avpr1a, kiss1r, Tac3r, Arntl, Clock, Dbp, Mtnr1a, Per2, Crhr1, Drd2	Esr2, Cyp19a1, Grin2a, Avpr1a, kiss1r, Tac3r, Arntl, Clock, Dbp, Mtnr1a, Per2	Crhr1, Drd2	(Gao et al., 2018)
DEHP	ApolipoproteinE gene knockout (ApoE−/−) C57BL/6 mice	100 μM DEHP (5 %w/w)	6 weeks	PCSK9, ABCA1, ABCG5, ABCG8, SR-B1, miR-145–5p, GAS5, ApoA2, LC3-I, LC3-II, and SQSTM1	PCSK9, ABCA1, ABCG5, ABCG8, SR-B1, miR-145–5p	GAS5, ApoA2, LC3-I, LC3-II, and SQSTM1	(Liu et al., 2022)
DEHP	C57BL/6J mice	300 mg/kg/day	9 days	Svs5, FOXA3, ESR1, AR	Svs5, FOXA3,	ESR1, AR	(Stenz et al., 2019)
DEHP, MEOHP, MEHP, MHBP, MiBP, MCOP and	Human (and urine sample)	Human urinary phthalate metabolites	−	DNM1, GTF2I	DNM1, GTF2I	−	(Oluwayiose et al., 2023)
DEHP	Male and female mice	300 mg/kg/day	21 days	Rbp2, mir615, C1qtnf5, Gzmk, Gm8994, Gm6329, Fbxw15, Svs2, Svs3a, Svs3b, Svs4, Svs6, Pate4, and Sva 231002L09Rik, 1700044K03Rik,	Svs2, Svs3a, Svs3b, Svs4, Svs6, Pate4, and Sva	Rbp2, mir615, C1qtnf5, Gzmk, Gm8994, Gm6329, Fbxw15, 231002L09Rik, and 1700044K03Rik	(Stenz et al., 2017)
DEHP	Newborn mice ovaries	10 and 100 μM	72 hrs.	miR-19a-3p	miR-32-5p miR-19a-3p miR-141-3p miR-29b-3p miR-29c-3p miR-335-5p miR-181a-5p miR-181b-5p miR-181c-5p	miR-449a-5p miR-449b-5p miR-449c-5p miR-34a-5p miR-34b-5p miR-34c-5p	(Zhang et al., 2019)
MEHP	Adult male C57BL/6 mice,	Single dose of 1 g/kg	−	Mta1, Timp2	−	Mta1	(Chen et al., 2013)

Phthalates have been shown to impact the expression levels of lncRNAs. A study assessed the correlation between urinary phthalate metabolite concentrations and placenta noncoding RNA, and mono-(carboxynonyl) phthalate demonstrated consistently strong correlations with most lncRNAs (Machtinger et al., 2018). Some of the metabolic phthalates identified in maternal urine include MCNP, MEHP, MEHHP, MECPP, and MEOHP, the majority of which are positively correlated with an overall increase in lncRNA expression. Among these favorable correlations, there is a link with the reduction in DNA methylation of two major lncRNAs that are responsible for fatal growth and development (Machtinger et al., 2018). DHEP, another commonly used phthalate, has been shown to mediate the upregulation of the lncRNA GAS5, which is responsible for controlling cell division and apoptosis, in atherosclerosis mouse models. MEHP has been shown to enhance the migration, invasive properties, and epithelial–mesenchymal transition (EMT) of SKOV3 ovarian cancer cells (Leng et al., 2021). Exposure to DEHP and MEHP in mouse models induced testicular damage by altering lncRNA expression in TM3 Leydig cells (Wu et al., 2021). Whole transcriptome-based studies have suggested that the enrichment of lncRNAs is particularly related to autophagy pathways in the genital tubercles of DBP-exposed rats. The gene expression profile of the genital tubercles of DBP-induced hypospadiac rats revealed that lncRNAs regulate autophagy, demonstrating that ectopic expression of NONRATT008453.2 inhibited the proliferation of genital tubercle fibroblasts (Feng et al., 2021).

BBP at a concentration of 50 μM significantly reduced H19 expression in the C3H10T1/2 cell line. The upregulation of miRNA-103/107 and let-7 family members has also been reported. These changes were observed on day two of BBP exposure, suggesting that changes in ncRNA expression could be one of the earliest changes in response to phthalate exposure (Zhang and Choudhury, 2021). Treatment of a mouse macrophage line (RAW 264.7 cells) with MEHP promoted m6A modification and upregulated the expression of several miRNAs, namely, miR16-1-3p, miR101a-3p, miR362-3-, miR501-5p, miR532-3p, and miR542-3p, which influence RNA stability and translation and upregulate the expression of specific miRNAs that are likely to modulate the expression of genes involved in inflammatory and cellular stress responses. Daily exposure to phthalates altered miRNA biogenesis in mouse ovary cells (Patiño-García et al., 2020). Phthalates can affect the expression of miRNAs (such as miR-96-5p, miR-200b-3p, miR-365-3p, miR-378a-3p and miR-503-5p), which can target steroidogenic pathways and may contribute to reproductive dysfunction. In mouse spermatogenesis pathway cell lines, DBP induced germ cell toxicity and increased miR-29b expression. miR-29b plays a critical role in various biological processes, including cell differentiation, and miR-29b targets and downregulates DNMTs, such as DNMT3b, leading to the demethylation of genes such as the PTEN promoter, which influences gene expression (Li et al., 2019). The phthalate metabolites in the urine correlated with the upregulation of 23 ncRNA transcripts in extracellular vesicles (Oluwayiose et al., 2023).

4.1.3. Histone modification and phthalates

Histone modifications can impact nucleosome positioning, chromatin structure, and DNA accessibility, ultimately affecting gene expression (Liu et al., 2023). Phthalate exposure has been shown to alter histone modifications, which can impact gene expression and increase susceptibility to several disorders.

Exposure to DEHP has been associated with an increased incidence of reproductive problems in males. Exposure during puberty contributes to a reduction in sperm counts and testosterone levels through the downregulation of G9a-mediated histone methylation in males (Liu et al., 2016). A decrease in the protein expression of G9a, H3K9me1, and H3K9me2 upon DEHP exposure may contribute to the impairment of self-renewal, differentiation, and meiotic abnormalities in spermatogonial stem cells (Liu et al., 2016).

Epigenetic regulation of immune cells by phthalates affects the expression of genes related to asthma. For example, DEHP exposure suppressed IRF7 expression by reducing histone H3K4 trimethylation at its promoter region by restricting the nuclear translocation of WDR5, a known H3K4-specific trimethyl transferase. This led to reduced CpG plus IL-3-induced histone H3K4 trimethylation at the IRF7 promoter (Xiao et al., 2023). This histone modification may suppress the MKK1/2-ERK-ELK1, AHR, IRF7, and NFjB-p65 pathways (Kuo et al., 2013). Histone deacetylases (HDACs) are key enzymes that promote heterochromatinization by promoting histone deacetylation. Phthalates have the capacity to induce inflammatory pathway reprogramming in peritoneal macrophages. MEHP exposure altered H3K36 acetylation and demethylation levels and upregulated histone modification enzymes (HDAC3, HDAC4, and HDAC6) in peritoneal macrophages (Li et al., 2018). A549 cells exposed to DEHP and MEHP promoted IL33 expression through histone modifications. Higher concentrations of DEHP or MEHP enhance the acetylation of H3 and H4 and contribute to the trimethylation of H3K4, H3K36, and H3K79 surrounding the IL33 promoter, hence increasing the expression of IL33 (Tsai et al., 2023).

In MCF7 cells, DEHP exposure modulates MECP2 expression. MECP2 expression is linked to chromatin remodeling and HDAC activity (Ghosh et al., 2022). In utero DEHP exposure affects insulin signaling through epigenetic changes in a Wistar rat model. DEHP treatment enhances the interaction of HDAC2 with Glut4 promoter regions, resulting in metabolic dysregulation downstream (Rajesh and Balasubramanian, 2014). Phthalate-mediated EMT in breast epithelial stem cells induces HDAC6 expression, resulting in metastasis in the lungs (Hsieh et al., 2012). A few reports have proposed that phthalate exposure can induce obesity. Mesenchymal stem cells (MSCs, specifically C3H10T1/2) were exposed to BBP. BBP treatment increases lipid accumulation and adipogenesis in MSCs. BBP treatment increased H3K9 acetylation, reduced H3K9 demethylation, upregulated histone acetyltransferase and downregulated HDAC3, HDAC10 and histone methyltransferase, which resulted in the epigenetic regulation of adipogenesis as a result of phthalate exposure (Sonkar et al., 2016).

Taken together, phthalate exposure during critical periods of development has been shown to alter histone modifications, resulting in multigenerational effects in animal models. In vitro models suggest that phthalate exposure can increase the aggressiveness of certain cancer types by affecting histone modifications. Additionally, research has indicated that exposure to phthalates alters histone modifications, which in turn promote important oncogenic pathways such as EMT and WNT activation. However, more detailed studies are needed using animal models and epidemiological data to map histone modifications and design specific epigenetic therapies.

Importantly, the mechanisms by which phthalates cause these epigenetic disruptions may be both direct and indirect. Direct mechanisms include phthalate metabolites interacting with DNA methyltransferases, histone-modifying enzymes, or chromatin-binding proteins, thereby influencing DNA and histone modifications (Swanson et al., 2023, Singh and Li, 2012). Indirect mechanisms may occur through phthalate-induced oxidative stress, metabolic dysregulation, or inflammatory signaling cascades, which secondarily alter epigenetic marks (Dutta et al., 2020, Zhao et al., 2016, Pigini et al., 2022). Furthermore, some phthalates and their metabolites act as endocrine disruptors through androgen receptor (AR)-binding activity (Beg and Sheikh, 2020). This AR-mediated pathway may involve crosstalk with chromatin modifiers, contributing to changes in gene expression and epigenetic regulation. Thus, both receptor-mediated and enzyme-targeted mechanisms likely underlie the epigenetic effects of phthalate exposure.

Overall, phthalate exposure is associated with diverse health effects, including metabolic disorders, reproductive impairments, neurodevelopmental changes, and cancer. Some of these outcomes are linked to epigenetic alterations, such as changes in DNA methylation of imprinted or growth-related genes, histone modifications influencing gene expression, or dysregulation of noncoding RNAs. Other effects may result from nonepigenetic mechanisms, including direct endocrine disruption, oxidative stress, or receptor-mediated signaling. Distinguishing between these pathways provides a clearer perspective on the role of epigenetics and helps prioritize future research aimed at understanding phthalate-induced health effects.

4.2. Potential of epigenetic therapies

There are numerous reports of epigenetic modifications following exposure to environmental toxicants (Baccarelli and Bollati, 2009, Hou et al., 2012, Klukovich et al., 2019, Qin et al., 2021). However, these studies have yet to directly connect the epigenetic changes associated with phthalates and their disease endpoints. There are numerous examples that show a strong correlation between phthalate-induced epigenetic changes and various human illnesses. Aberrant epigenetic modifications are reversible enzymatic processes. Restoration of the silenced gene is achievable with epigenetic treatment since epigenetic modifications are reversible and enzyme-mediated processes. Epigenetic therapy employs inhibitors that target the epigenetic machinery, resulting in the reversal of abnormal epigenetic changes and the re-establishment and restoration of silent genes. These modifications can be reversed through the use of epigenetic drugs that target the enzymes associated with these modifications. The use of drugs or other epigenome-editing methods to treat diseases in humans is referred to as epigenetic therapy. The present epigenetic treatment mostly uses inhibitors of histone deacetylation and DNA demethylation. There are few epidrugs, which include DNA methyltransferase inhibitors (DNMTis), histone deacetylase inhibitors (HDACis), histone methyltransferase inhibitors (HMTis), and histone demethylase inhibitors (Abdel-Hameed et al., 2016). DNMTis irreversibly inhibit the enzymatic activities of DNMTs and trigger proteasomal degradation (Ahuja et al., 2014, Kelly et al., 2010).

A study by Alfardan et al tested the ability of 5-aza-2′-deoxycytidine (DNMT inhibitor) to reduce the DEHP-induced worsening of psoriasiform inflammation in a mouse model (Alfardan et al., 2024). This study revealed that DEHP exposure increased DNMT expression and global methylation in imiquimod-induced psoriasis mice. Exposure to 5-aza-2′-deoxycytidine significantly reduces inflammation in the skin of psoriatic mice by simultaneously increasing Nrf2 signaling and lowering the global DNA methylation level. This study provides compelling evidence that DEHP-induced DNMT1 contributes significantly to the worsening of psoriasiform inflammation in mice and the utility of epigenetic therapy in reversing this phenomenon (Alfardan et al., 2024). Another study demonstrated that MCF-7 cells exposed to 50 nM, 100 nM, or 500 nM DEHP exhibited increased proliferation. The same study revealed an increase in global hypermethylation and the upregulation of DNMT1 and MECP2 in response to DEHP exposure (Ghosh et al., 2022). Furthermore, a reduction in p53 levels and increased occupancy of SP1 and E2F1 at the DNMT1 promoter are also observed in response to DEHP exposure in MCF7 cells (Ghosh et al., 2022). In C2C12 myotubes, treatment with 5-azacytidine decreased Dnmt3a levels and facilitated insulin −stimulated uptake of glucose. The same study proposed that miR-17 downregulation by DEHP could be partially related to Dnmt3a-mediated miR-17 promoter methylation (Wei et al., 2020). However, another study investigated the relationship between the global DNA methylation level and cell proliferation in TM-4 cells. TM-4 cell proliferation was greater than 20 % upon treatment with 5-aza-2′-deoxycytidine. Endocrine disruptors such as DEHP have been shown to negatively affect spermatogenesis and fertility in males, partly by decreasing H3K9 di-methylation. Furthermore, treatment with melatonin protected prepubertal testes from the harmful effects of DEHP by maintaining H3K9 methylation (Cescon et al., 2020). HDACs alter the balance of acetylation and deacetylation of histone lysines, with the latter playing an important role in aberrant gene suppression in cancer (Ahuja et al., 2014, Bose et al., 2014). Intriguingly, the suppression of DNMT activity has been demonstrated for certain drugs. Furthermore, it has been reported that some approved drugs may work via uncharacterized epigenetic processes. On the basis of ongoing preclinical research, the combination of DNMTis and HDACis, particularly in conjunction with other drugs, shows more potential for epigenetic therapy than their use alone (Ahuja et al., 2016). Phthalate exposure has been shown to affect the epigenome of people in all age groups. Abnormal epigenetic changes affect not only the original individual subject to these changes but also their future progeny owing to the known multigenerational inheritance of these changes. Epigenetic therapies can be used to reverse the aberrant changes associated with phthalate exposure before its introduction to the next generation. However, it is important to note the limitations of current epigenetic therapies. None of the available inhibitors specifically target phthalate-induced epigenetic changes. Instead, these drugs broadly modify epigenetic marks across the genome in a uniform manner, which may inadvertently introduce additional epimutations. This nonspecific action can affect genes and pathways unrelated to phthalate exposure, potentially resulting in unintended consequences. Therefore, while epigenetic therapies have promising potential, their application requires caution, and further research is needed to develop more targeted strategies.

More detailed animal model-based mechanistic studies are needed to understand the beneficial effects of epigenetic therapy, as they are related to targeting the adverse health effects associated with phthalate-induced epigenetic changes. Thus, epigenetic therapy can be used to restore the histone modifications induced by phthalate exposure. These data suggest that phthalate exposure may induce aberrant DNA methylation, histone modifications, and altered miRNA expression at the cellular level and may contribute to the development and progression of various diseases. Epigenetic therapy may be beneficial for overcoming or preventing the deleterious health effects of phthalates.

5. Conclusion

The development and progression of many diseases have been linked to epigenetic changes within the epigenome. A large portion of these changes have been identified as the result of certain environmental exposures. Owing to the prevalence and known toxic effects of phthalates, particularly the effects associated with known, aberrant epigenetic alterations, these chemicals may be the cause of numerous diseases. As previously mentioned, phthalates can be associated with a multitude of health problems, such as cancers, reproductive and hormonal issues, and cardiovascular disease, all of which have also been shown to be associated with altered epigenetic patterns. With respect to how prevalent phthalates are globally, more research is needed to understand not only how they alter our epigenome but also how to identify and treat these changes prior to the onset of disease. While epigenetic therapy is still in its infancy, it is one of the most promising solutions for treating toxins that we are unable to avoid.

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This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.