Exposure to phthalates enhances estrogen and beta-catenin signaling pathways, leading to endometrial hyperplasia in mice

Abstract

Phthalates, synthetic chemicals widely utilized as plasticizers and stabilizers in various consumer products, present a significant concern due to their persistent presence in daily human life. Although past research predominantly focused on individual phthalates, real-life human exposure typically encompasses complex mixtures of these compounds. The cumulative effects of prolonged exposure to phthalate mixtures on uterine health remain poorly understood. To address this knowledge gap, we conducted studies utilizing adult female mice exposed chronically to a mixture of phthalates for 12 mo through ad libitum chow consumption. Our studies revealed that continuous exposure to this phthalate mixture led to uterine hyperplasia with a significant increase in gland-to-stroma ratio. Endometrial hyperplasia is commonly caused by heightened estrogenic action and inflammatory response in the uterus, leading to increased proliferation of endometrial epithelial cells. Indeed, we observed a marked upregulation of several known estrogen-regulated genes, proinflammatory chemokines, elevated homing of macrophages, and increased KI67 staining in the endometrial epithelial cells upon phthalate exposure. Several signaling pathways, including the MAPK/ERK and Wnt/β-Catenin pathways, promote cell proliferation, leading to the hyperproliferative state of the endometrial cells. Our studies revealed no alteration of the MAPK/ERK pathway but a marked enhancement of the Wnt/β-Catenin signaling pathway in phthalate-exposed uteri. Collectively, this study underscores the significance of understanding the exposure to environmental factors in the pathogenesis of endometrial disorders.

Phthalates, which are esters of phthalic acid, are synthetic chemicals extensively utilized as plasticizers in a variety of consumer products (Pagoni et al. 2022). High molecular weight phthalates with long chains include di(2-ethylhexyl) phthalate (DEHP), di-iso-nonyl phthalate (DiNP), di-iso-decyl phthalate, di-n-octyl phthalate, and di(2-propylheptyl) phthalate. These are incorporated into polyvinyl chloride. Conversely, short-chain phthalates are employed in the production of personal care items such as perfumes, nail polish, deodorants, and lotions. This group comprises dimethyl phthalate, diethyl phthalate (DEP), benzylbutyl phthalate, di-n-butyl phthalate (DnBP), and di-iso-butyl phthalate (DiBP). A significant quantity of phthalates is utilized globally each year (Halden 2010). As phthalates are not covalently bound to plastics, they easily leach into the environment, resulting in continuous human exposure through inhalation, ingestion, and dermal contact (Zhang and Chen 2014). Ingestion is the most prevalent exposure pathway (Wu et al. 2021). Research reveals that phthalate metabolites are detected in nearly 100% of human urine samples tested (Silva et al. 2004). Notably, phthalate metabolite concentrations are higher in women than in men, likely due to the greater use of personal care products by women (James-Todd et al. 2012).

Growing scientific evidence associates phthalate exposure with adverse health effects due to the endocrine-disrupting nature of these chemicals. Epidemiological studies suggest that phthalate exposure correlates with reduced pregnancy rates, increased miscarriage rates, and preeclampsia in women (Heudorf et al. 2007). Additionally, phthalate exposure is linked to adverse pregnancy outcomes, such as low birth weight and compromised intellectual development and growth in children (Zhang et al. 2009; Factor-Litvak et al. 2014). Previous studies using rodents have shown that prenatal exposure to phthalates affects folliculogenesis, delays puberty, and reduces fertility (Schmidt et al. 2012; Brehm et al. 2018). It has also been reported that exposure to DEHP results in uterine abnormalities in mice, including decreased proliferation of luminal epithelium and an increased number of abnormally dilated blood vessels in the endometrium (Richardson et al. 2018). Previous studies demonstrated that while exposure to DiNP impairs the ability of mice to maintain pregnancy, mice exposed to DEHP did not experience pregnancy loss unless fed a high-fat diet (Chiang and Flaws 2019; Chiang et al. 2020; Kannan et al. 2021). Although these studies have primarily focused on exposure to a single phthalate over a limited timeframe, humans encounter a mixture of phthalates daily throughout their lives.

Hence, we designed a study in which female mice were chronically exposed to a phthalate mixture via chow ad libitum and analyzed the effects of this exposure on the uterus. The composition of the phthalate mixture was DEHP, DiNP, BzBP, DBP, DiBP, and DEP, which was derived from the Illinois Kids Development Study (I-KIDS), where the levels of phthalate metabolites were determined in the urine samples from pregnant women (Yazdy et al. 2018). Long-term dietary exposure to this mixture adversely affects estrous cyclicity (Laws et al. 2023) and promotes uterine fibrosis (Shukla et al. 2023). In this study, we report that exposure to the phthalate mixture for 1 yr causes endometrial hyperplasia accompanied by enhanced estrogen-dependent signaling, inflammatory response, and activated Wnt/β-Catenin signaling pathway.

Materials and methods

Chemicals

The phthalates, which were 98% pure, were obtained from Sigma-Aldrich (St Louis, MO). Corn oil from Columbus Vegetable Oils (Des Plaines, IL) served as the vehicle control.

Animals

Female CD-1 mice aged 33 d and male CD-1 mice aged 7 wk were obtained from Charles River Laboratories (Wilmington, MA) and housed in the College of Veterinary Medicine vivarium at the University of Illinois Urbana-Champaign (Urbana, IL). As previously described, the female mice were group housed in polysulfone cages (Allentown, Allentown, NJ) at a density of 3 mice per cage, with 1/8 corn cob bedding (Shepherd Specialty Papers), environmental enrichment (iso-BLOX, Catalog No. 6060, Envigo), and reverse osmosis purified water (Laws et al. 2023). All animal handling, housing, and procedures were approved by the University of Illinois Institutional Animal Care and Use Committee.

Study design and dosing

Phthalates were administered to mice ad libitum in the rodent chow as previously described (Laws et al. 2023). The base chow was a modified version of the AIN-93G formulation (TD.94045), where soybean oil was replaced with corn oil from Columbus Vegetable Oils (Des Plaines, IL). Chow containing 7% corn oil served as the vehicle control group. Phthalates, mixed in corn oil, were provided to Envigo for chow preparation. The phthalate mixture used in this study consisted of 35% DEP, 21% DEHP, 15% DiNP, 15% DBP, 8% DiBP, and 5% BzBP, designed based on phthalate metabolite concentrations in the urine of pregnant women in central Illinois (Yazdy et al. 2018). High phthalate levels are typically found in food contact materials like plastic wrap and containers, making ingestion a significant exposure route for humans (Wu et al. 2021). To replicate human exposure, the phthalate mixture was administered via rodent chow at doses of 0.15 and 1.5 ppm. Each treatment group comprised 12 to 14 mice. Based on previous studies, the doses were selected assuming that a 25-g mouse consumes ∼5 g of food per day (Neier et al. 2020). Therefore, the 0.15-ppm dose equates to about 20 µg phthalate/kg body weight/day, and the 1.5 ppm dose to about 200 µg phthalate/kg body weight/day. These doses fall within the range of daily human, infant, and occupational exposure (Kavlock et al. 2002; Koo and Lee 2005). Beginning at 6 wk of age, the mice were continuously treated with the chow for 12 mo. This continuous daily exposure to phthalates closely mimics human exposure patterns.

As described previously, before the beginning of the experiment, mice were weighed and randomly assigned to treatment groups. For the duration of the experiment, mice were weighed once a week, and their weight was recorded to the nearest tenth of a gram. Cage food hoppers were filled once a week with treatment chow in excess. Mice were permitted to eat the rodent chow ad libitum. To the nearest tenth of a gram, food added each week was weighed, and food left from the previous week was weighed to calculate how much food had been consumed each week per cage. Because mice were group housed (3 per cage), the food consumed per cage each week was divided by the number of mice in the cage to estimate the amount of food consumed per mouse per week.

The urinary phthalate metabolites (MEHP, MEOHP, MEHHP, MECPP, MBP, MiBP, MMP, MBzP, MCPP, and MEP) were measured after adult female CD-1 mice were fed chow containing vehicle control (corn oil) or phthalate mixture containing DEHP, DiNP, benzyl butyl phthalate, DnBP, diisobutyl phthalate, and DEP. Measurements of urinary phthalate metabolites confirmed the effective delivery of phthalates. This study uses uterine tissue from the same mice previously analyzed for phthalate metabolites (Laws et al. 2023).

Tissue collection and analysis

At 1 yr of age, the mice were euthanized during diestrus, and the middle part of their uterine horns was collected. One portion of the uterine horn was fixed in 10% neutral buffered formalin (NBF), whereas the other portion was flash frozen in liquid nitrogen for subsequent gene expression analysis. For histological examination, the fixed uteri were dehydrated through a graded series of ethanol concentrations (from 70% to 100%). The samples were then embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E). For histological analysis, uteri from 3 mice for each treatment group were randomly chosen. As described previously (Kannan et al. 2021), we consistently use the middle section of the uterine horn for histological assessments. We have found this to be a reliable and reproducible region for analysis. Specifically, gland counts in this section serve as a robust proxy for overall glandular density. Sample sizes of 3 mice per study will achieve >80% power to detect a 1.5 SD difference with a 0.05 2-sided significance level based on the t-test. The sample identity was blinded to researchers evaluating histochemical data.

Immunohistochemistry

Uterine tissues were processed and subjected to immunohistochemistry as described previously (Kannan et al. 2021). Briefly, paraffin-embedded tissues were sectioned at 5 μm and mounted on microscopic slides. Sections were deparaffinized in xylene, rehydrated through a series of ethanol washes, and rinsed in water. Antigen retrieval was performed by immersing the slides in 0.1M citrate buffer solution, pH 6.0, followed by microwave heating for 25 min. The slides were allowed to cool and endogenous peroxidase activity was blocked by incubating sections in 0.3% hydrogen peroxide in methanol for 15 min at room temperature. After washing with PBS for 15 min, the slides were incubated in a blocking solution for 1 h. This was followed by incubation overnight at 4 °C with antibodies specific for KI67 and CTNNB1. Pictures were taken using the Olympus BX51 microscope equipped for fluorescent imaging and connected to a Jenoptik ProgRes C14 digital camera with c-mount interface containing a 1.4 Megapixel CCD sensor. Fluorescent images were processed and merged using Adobe Photoshop Extended CS6 (Adobe Systems). The sample identity was blinded to researchers evaluating immunostaining data.

Quantitative real-time PCR analysis

For gene expression analysis, RNA was converted to cDNA, and real-time quantitative PCR was performed using SYBR-green master mix (Applied Biosystems) on a QuantStudioTM 3 Real-time PCR instrument (Applied Biosystems). The mean threshold cycle (Ct) for each sample was calculated from Ct values obtained from 3 replicates. The normalized ΔCt in each sample was determined by subtracting the mean Ct of the reference gene from the mean Ct of the target gene. ΔΔCt was then calculated as the difference between the ΔCt values of the control and treated samples. The fold change in gene expression relative to the control was calculated using the 2−ΔΔCt method for relative quantification. The mean fold induction and SEM were calculated from at least 3 independent experiments.

Results

To investigate the effects of long-term dietary phthalate exposure on the uterus, adult mice were fed chow containing a vehicle control (corn oil) or a mixture of phthalates at doses 0.15 and 1.5 ppm, as previously described (Laws et al. 2023). Uterine sections obtained from control and mice exposed to the phthalate mixture for 1 yr were analyzed by H&E staining to study changes in uterine histology. Interestingly, we observed a dramatic increase in the number of uterine glands in the 0.15 ppm treatment group compared with controls (Fig. 1, middle panel). We found that exposure to the higher dose, 1.5 ppm, did not show a pronounced effect on the glands-to-stroma ratio compared with the 0.15 ppm exposure level. Thus, to gain insights into the mechanisms underlying the development of glandular hyperplasia in the uterus, we focused on the 0.15 exposure level.

Fig. 1.

Histological examination of uterine sections. Upper: Serial sections of uterine horns of control (a), 0.15 ppm (b), and 1.5 ppm (c) of phthalate mixture exposed mice at 12 mo were examined by H&E staining. The representative images from the control and phthalate mixture treated groups are shown (N = 6). Lower: The number of glands in the uterus was quantified using the multi-point tool in ImageJ software. Each data point represents the number of glands present in a single uterine cross-section from one mouse. Statistical analysis was performed using Welch’s corrected 1-way ANOVA to assess statistical differences, followed by the Brown–Forsythe test. Data were presented as mean±SEM from 6 separate samples. Statistical significance was set at P < 0.05.

Elevated estrogen signaling and chronic inflammation are key factors in the development of endometrial hyperplasia. Thus, we determined the expression level of several known estrogen-regulated genes involved in oncogenic pathways in the uterus with or without phthalate exposure (Hewitt et al. 2003; Laws et al. 2008; Zhao et al. 2014; Neff et al. 2019; Bhurke et al. 2020; Vasquez et al. 2020). As shown in Fig. 2, we observed a marked upregulation of mRNA levels corresponding to Cx43, Muc1, Hif2α, Fgf9, Ccn1, Pbx3, Wt1, and Six1 in uterine samples of 0.15 ppm groups, indicating that long-term exposure to the phthalate mixture enhances estrogen signaling (Hewitt et al. 2003; Laws et al. 2008; Li et al. 2011; Zhao et al. 2014; Neff et al. 2019; Bhurke et al. 2020; Vasquez et al. 2020). Since enhanced estrogen signaling affects the expression of CC chemokine ligands (CCL), we determined the levels of a subset of proinflammatory chemokines in the uterus. We observed significant upregulation of Ccl2, Ccl17, Ccl12, Ccl21a, Ccl7, and Ccl8 in phthalate-exposed uteri compared with unexposed controls (Fig. 3A). Consistent with this observation, we noted a marked increase in the homing of macrophages as indicated by immunofluorescence (IF) staining for F4/80, a specific marker for macrophages in the endometrium in response to phthalate exposure (Fig. 3B). Interestingly, most of the macrophages were localized in the subepithelial stroma of phthalate-exposed uteri. This is consistent with the observation that blood vessels are beneath the luminal epithelial cells in the nonpregnant mouse uterus (Rockwell et al. 2002). We next investigated if phthalate-mediated increased estrogen-dependent signaling and inflammation promote epithelial proliferation to cause endometrial hyperplasia. Hence, we performed IF staining for KI67, a well-known marker for cell proliferation, and found a dramatic increase in the number of KI67-positive cells in the uterine glandular epithelium (Fig. 4). Taken together, these results indicated that long-term exposure to the phthalate mixture enhances estrogen-dependent signaling, inflammation, and epithelial cell proliferation.

Fig. 2.

Exposure to a phthalate mixture leads to elevated expression of estrogen-regulated genes in the uterus. Uterine RNA from control and 0.15 ppm groups were subjected to quantitative real-time PCR analysis using primers specific for Hif2α, Cx43, Muc1, Fgf9, Ccn1, Pbx3, Six1, and Wt1. 36b4 was used as the internal control. Statistical analysis was performed using Student’s t-test. Data represent mean±SEM from 6 separate samples. Asterisks indicate statistically significant differences (^P < 0.1, *P < 0.05, **P < 0.01).

Fig. 3.

Exposure to a phthalate mixture causes heightened inflammation in the uterus. A) Uterine RNA from control and 0.15 ppm groups were subjected to quantitative real-time PCR analysis using primers specific for Ccl2, Ccl7, Ccl8, Ccl12, Ccl17, and Ccl21. Statistical analysis was performed using Student’s t-test. Data represent mean±SEM from 6 separate samples. Asterisks indicate statistically significant differences (^P < 0.1, *P < 0.05, **P < 0.01). B) Uterine sections from control and 0.15 ppm treated groups were subjected to IF using macrophage-specific F4/80 antibodies (left panels). Immuno-positive cells for F4/80 were analyzed by ImageJ software (right panel). The experiment was repeated with N = 3 mice, and 2 sections were examined for each uterine sample. Statistical analysis was performed using Student’s t-test.

Fig. 4.

Enhanced proliferation of glandular epithelial cells in the uterus by long-term exposure to phthalates. Immunohistochemical localization of KI67 in the uterine sections of mice with or without exposure to 0.15 ppm phthalate mixture. Left: Upper panels indicate lower magnification (20×), and lower panels indicate higher magnification (40×). G indicates glands. Right: Immuno-positive cells for KI67 were analyzed by ImageJ software. The experiment was repeated with N = 3 mice, and 2 sections were examined for each uterine sample. Statistical analysis was performed using Student’s t-test.

Several signaling pathways play a role in the development and progression of endometrial hyperplasia, including MAPK/ERK and Wnt/β-Catenin pathways (Kim et al. 2014; Pavlidou and Vlahos 2014; McMellen et al. 2020; Fatima et al. 2021). These pathways promote cell proliferation, leading to the hyperproliferative state of the endometrial cells. To identify the pathway that mediates the phthalate mixture-induced enhanced proliferation of endometrial epithelial cells, we investigated the expression levels of the regulatory proteins involved in each pathway. Although phospho-ERK pathway remained unchanged (Fig. 5), we observed a marked elevation in phospho-β-Catenin expression (Fig. 6A). We then determined the levels of a subset of Wnt ligands known to be associated with cancer, such as Wnt4, Wnt 5b, Wnt7a, Wnt8b, and Wnt9a (Fig. 6B), and found them to be upregulated in phthalate-exposed uteri indicating an upregulation of Wnt/β-Catenin pathway upon exposure to phthalates.

Fig. 5.

MAPK/ERK signaling pathway is not altered in the uterus by chronic phthalate exposure. Left: IF analysis of phospho ERK in uterine sections of mice with or without exposure to 0.15 ppm phthalate mixture. Right: Immuno-positive cells for phospho ERK were analyzed using ImageJ software. The experiment was repeated with N = 3 mice, and 2 sections were examined for each uterine sample. Statistical analysis was performed using Student’s t-test.

Fig. 6.

Activation of β-Catenin signaling pathway in the uterus by chronic phthalate exposure. A) IF analysis of phospho CTNNB1 in uterine sections of a control mouse (left) and 0.15 ppm group mouse (right). Immuno-positive cells for phospho CTNNB1 were analyzed by ImageJ software. The experiment was repeated with N = 3 mice, and 2 sections were examined for each uterine sample. Statistical analysis was performed using Student’s t-test. B) Uterine RNA from control and 0.15 ppm groups were subjected to quantitative real-time PCR analysis using primers specific for Wnt4, Wnt5b, Wnt7a, Wnt8a, and Wnt9b. 36b4 was used as the internal control. Statistical analysis was performed using Student’s t-test. Data represent mean±SEM from 6 separate samples. Asterisks indicate statistically significant differences (^P < 0.1, *P < 0.05, **P < 0.01).

Discussion

Phthalate exposure in the general population is ubiquitous, and humans are repeatedly and continually exposed to phthalates (Silva et al. 2004; Heudorf et al. 2007; Hogberg et al. 2008; James-Todd et al. 2012). Studies indicate that phthalate metabolites are present in nearly 100% of tested human urine samples (Silva et al. 2004; Heudorf et al. 2007; Hogberg et al. 2008; James-Todd et al. 2012). These observations highlight the importance of understanding adverse health outcomes associated with phthalate exposure. Previous research indicated that prenatal exposure to a phthalate mixture results in multigenerational and transgenerational effects on uterine morphology in mice (Li et al. 2020). However, humans experience daily phthalate exposure throughout their lives. In this study, 6-wk-old mice were continuously administered the phthalate mixture for 1 yr. Moreover, the phthalate exposure occurred via food chow, mimicking the route of human exposure. Our results show that chronic exposure to a phthalate mixture comparable to what the EPA deems safe for human exposure can lead to undesirable uterine pathologies such as endometrial hyperplasia.

Aberrant proliferation is a hallmark of hyperplasia, a condition characterized by an increase in cell number without changes to cell morphology. Hyperplasia is not itself cancer but can develop into cancer with further genetic mutations or environmental insults (Sanderson et al. 2017). Here, we report that exposure to a mixture of phthalates leads to aberrant proliferation of the uterine epithelium, as indicated by KI67 staining. Notably, proliferation was particularly robust in the epithelium of the uterine glands, which are predominantly the site of origin for endometrial hyperplasia. Previous studies have examined the association between phthalate exposure and uterine conditions, including endometriosis and fibroids (Kim and Kim 2020; Iizuka et al. 2022). Our study shows that chronic exposure to a mixture of phthalates in adults can have deleterious effects on the uterine glands, causing an increased glands-to-stroma ratio suggestive of endometrial hyperplasia. The impact on the uterine glands is pronounced at a low dose. This is not surprising since endocrine-disrupting chemicals such as phthalates have been shown to exhibit nonmonotonic dose responses (Inman and Flaws 2024). In light of these observations, it is concerning that exposure to a low concentration of phthalates, which may not be readily detectable in humans, is causing endometrial hyperplasia.

We report here that chronic phthalate exposure causes an upregulation of estrogen-regulated genes, many of which are involved in epithelial cell proliferation in uterine tissue (Hewitt et al. 2003; Laws et al. 2008; Zhao et al. 2014; Neff et al. 2019; Bhurke et al. 2020; Vasquez et al. 2020). The significance of this finding is underscored by the fact that endometrial hyperplasia is often caused by prolonged exposure to estrogen without adequate progesterone to balance estrogen action (Kim et al. 2013). Prominent estrogen-regulated genes elevated by phthalate exposure are Muc1, Fgf9, Ccn1, Cx43, and Hif2α. Mucin 1, or Muc1, is a glycoprotein expressed on the uterine epithelium, and overexpression of Muc1 has been linked to endometrial cancers (Morrison et al. 2007; Engel et al. 2016). Fgf9 promotes cell proliferation and survival in various tissues (Turner and Grose 2010). In the endometrium, elevated Fgf9 expression might contribute to excessive cellular proliferation, leading to endometrial hyperplasia. Ccn1 is part of the Ccn family of matricellular proteins, which play a crucial role in various cellular processes, including cell proliferation, adhesion, migration, and angiogenesis (Jia et al. 2021). In endometrial cancer, overexpression of Ccn1 is linked to enhanced tumor growth, angiogenesis, and metastasis (Jia et al. 2021). Connexin 43 (Cx43), is a gap junction protein that plays a critical role in cell-to-cell communication by forming channels that allow the exchange of ions, metabolites, and signaling molecules between adjacent cells. The role of Cx43 in endometrial cancer is not known. Hypoxia-inducible factor 2 alpha or Hif2α is a transcription factor that plays a crucial role in the cellular response to hypoxia, a common feature in rapidly growing tumors, including endometrial cancer (Wicks and Semenza 2022). Thus, dysregulated estrogen-dependent genes by long-term phthalate exposure drive the hallmarks of endometrial hyperplasia and underscore the importance of phthalate exposure on uterine homeostasis.

Our study also revealed an elevation in Pbx3, Six1, and Wt1 expression, commonly overexpressed in various epithelial adenocarcinomas, including endometrial cancer. Pbx3 is a transcription factor that plays a critical role in the pathogenesis of endometrial cancer by promoting cancer cell proliferation, migration, and invasion (Morgan and Pandha 2020). Six1 is a homeobox transcription factor involved in embryonic development and organogenesis (Suen et al. 2019). Six1 is known to regulate epithelial proliferation and is overexpressed in endometrial carcinoma. It plays a significant role in endometrial cancer by driving processes such as epithelial–mesenchymal transition and tumor progression. Wilms’ Tumor 1, or Wt1, is a gene that plays a role in the development of certain types of cancer, including endometrial cancer. It is a transcription factor that is typically involved in the regulation of cell growth and differentiation. In endometrial cancer, WT1 can be overexpressed or mutated, which may contribute to tumorigenesis (McEachron et al. 2022).

Persistent inflammation is a well-known risk factor for cancer. Activated immune cells release cytokines and chemokines, potentially playing a crucial role in the development of endometrial hyperplasia (40). Consistent with this notion, we found a marked upregulation of several chemokines, including Ccl2, Ccl7, Ccl12, Ccl17, and Ccl21a, in the uterus upon long-term treatment of phthalates. The influx of macrophages was also significantly elevated in the uterus in response to phthalate exposure. Endometrial macrophages exist along a continuum of pro- and anti-inflammatory states, with their phenotype finely tuned to endometrial function. This dynamic regulation is essential for cyclic remodeling, breakdown, and repair in the nonpregnant endometrium. However, in reproductive health disorders, endometrial macrophages become dysregulated, displaying abnormal phenotypes often marked by excessive pro-inflammatory signaling, as seen in endometriosis, adenomyosis, and heavy menstrual bleeding (Berbic et al. 2009; Cousins et al. 2016; An et al. 2017; Vallve-Juanico et al. 2019). These dysfunctional macrophages contribute to disease pathology by altering the inflammatory microenvironment and exhibiting deficits in scavenging, phagocytosis, and cytokine production, which disrupt key processes like tissue remodeling. Previous studies have shown that an increase in tissue-resident macrophages can drive organ fibrosis by reducing vascular endothelial growth factor and triggering hypoxia. The hypoxic environment induces HIFs that push fibroblasts to overproduce ECM components like collagen, resulting in fibrosis (Murdoch et al. 2008). Interestingly, our studies revealed that Hif2α is significantly upregulated in the uterus upon chronic exposure to phthalates.

Notably, the results from this study show that chronic exposure to phthalates enhances the proinflammatory state in the endometrium and leads to hyperplasia. Previous studies have shown that phthalates can disrupt macrophage polarization by interfering with estrogen signaling and peroxisome proliferator-activated receptor (PPAR) activity, both of which are critical for macrophage function (Li et al. 2018; Xu et al. 2022; Wang et al. 2024). Estrogen typically promotes anti-inflammatory M2 polarization, but phthalates can impair this process, potentially shifting macrophages toward a proinflammatory M1 state. Similarly, PPARγ, a key regulator of M2 polarization, is inhibited by phthalate metabolites like MEHP, reducing anti-inflammatory responses (Li et al. 2018; Xu et al. 2022; Wang et al. 2024). However, the effects vary and depend on various factors such as phthalate type, exposure duration, and biological context. Future studies will address how chronic exposure to phthalates impacts macrophage polarization in the endometrium. Studies will also determine whether the measurement of inflammatory cytokines could be biomarkers for inflammation associated with phthalate exposure and endometrial hyperplasia.

An important finding of this study was the dramatic activation of the β-catenin pathway when mice were exposed long-term to a mixture of phthalates. Several Wnt ligands are induced in the uterus in response to phthalate exposure. Notably, previous studies have shown that Wnt4 is regulated by estrogen in the uterus, and it is upregulated in endometrial cancer (Hou et al. 2004; Katayama et al. 2006). Upon activation of Wnt signaling, β-catenin accumulates in the nucleus and initiates the transcription of genes that promote cell proliferation. Indeed, aberrant activation of this pathway leads to uncontrolled cell proliferation and plays a significant role in the development and progression of endometrial cancer (Fatima et al. 2021). It has been previously reported that a genetic mouse model with stabilized β-catenin expression displays endometrial glandular hyperplasia and not cancer, indicating that alteration in the β-catenin pathway is not sufficient for the development of endometrial cancer (Jeong et al. 2009). These findings are consistent with our results, which show glandular hyperplasia in the phthalate-exposed mouse model displaying upregulation of the Wnt/β-catenin signaling pathway.

In summary, chronic exposure to the phthalate mixture results in the dysregulation of estrogen and Wnt/β-Catenin signaling pathways, ultimately leading to the development of endometrial hyperplasia. Activation of these pathways contributes to abnormal glandular cell proliferation and inflammation in the uterus. Future studies will address how estrogen and β-catenin signaling pathways converge to promote endometrial hyperplasia in phthalate-exposed uteri. Insights into these signaling pathways highlight the potential mechanism by which phthalates induce endometrial hyperplasia and underscore the significance of understanding environmental factors in the pathogenesis of endometrial disorders.

Funding

This work was supported by NIH (R01 ES032163, R01 ES034112, and T32 ES007326).

Conflicts of interest. None declared.