Mono-(2-ethylhexyl) Phthalate Exposure Research Articles

Exposure to the phthalate metabolite MEHP impacts survival and growth of human ovarian follicles in vitro

Phthalates are found in everyday items like plastics and personal care products. There is an increasing concern that continuous exposure can adversely affect female fertility. However, experimental data are lacking to establish causal links between exposure and disease in humans. To address this gap, we tested the effects of a common phthalate metabolite, mono-(2-ethylhexyl) phthalate (MEHP), on adult human ovaries in vitro using an epidemiologically determined human-relevant concentration range (2.05 nM – 20.51 mM). Histomorphological assessments, steroid and cytokine measurements were performed on human ovarian tissue exposed to MEHP for 7 days in vitro. Cell viability and gene expression profile were investigated following 7 days of MEHP exposure using the human granulosa cancer cell lines KGN, and COV434, the germline tumor cell line PA-1, and human ovarian primary cells. Selected differentially expressed genes (DEGs) were validated by RT-qPCR and immunofluorescence in human ovarian tissue. MEHP exposure reduced follicular growth (20.51 nM) and increased follicular degeneration (20.51 mM) in ovarian tissue, while not affecting steroid and cytokine production. Out of the 691 unique DEGs identified across all the cell types and concentrations, CSRP2 involved in cytoskeleton organization and YWHAE as well as CTNNB1 involved in the Hippo pathway, were chosen for further validation. CSRP2 was upregulated and CTNNB1 downregulated in both ovarian tissue and cells, whereas YWHAE was downregulated in cells only. In summary, one-week MEHP exposure of human ovarian tissue can perturb the development and survival of human follicles through mechanisms likely involving dysregulation of cytoskeleton organization and Hippo pathway.

Role of formyl peptide receptor 2 in steatosis of L02 cells exposed to Mono-(2-ethylhexyl) phthalate.

Mono-(2-ethylhexyl) phthalate (MEHP) can accumulate in the liver and then lead to hepatic steatosis, while the underlying mechanism remains unclear. Inflammation plays an important role in the disorder of hepatic lipid metabolism. This study aims to clarify the role of the inflammatory response mediated by formyl peptide receptor 2 (FPR2) in steatosis of L02 cells exposed to MEHP. L02 cells were exposed to MEHP of different concentrations and different time. A steatosis model of L02 cells was induced with oleic acid and the cells were exposed to MEHP simultaneously. In addition, L02 cells were incubated with FPR2 antagonist and then exposed to MEHP. Lipid accumulation was determined by oil red O staining and extraction assay. The indicators related to lipid metabolism and inflammatory response were measured with appropriate kits. The relative expression levels of FPR2 and its ligand were determined by Western blot, and the interaction of them was detected by co-immunoprecipitation. As a result, MEHP exposure could promote the occurrence and progression of steatosis and the secretion of chemokines and inflammatory factors in L02 cells. MEHP could also affect the expression and activation of FPR2 and the secretion of FPR2 ligands. In addition, the promotion effect of MEHP on the secretion of total cholesterol and interleukin 1β in L02 cells could be significantly inhibited by the FPR2 antagonist. We concluded that FPR2 might affect the promotion effect of MEHP on steatosis of L02 cells by mediating inflammatory response.

The Blood-Testis barrier disruption is a prerequisite for Toxicant-Induced peritubular macrophage increases in the testis of peripubertal rats.

Peritubular macrophages (PTMφ) are predominantly localized near spermatogonial stem cells in the testis. We previously revealed that exposure of peripubertal male Fischer rats to mono-(2-ethylhexyl) phthalate (MEHP) leads to increased PTMφs in the testis. The mechanisms that trigger increases in PTMφs in the testis are poorly understood. However, MEHP exposure is known to both induce spermatocyte apoptosis and to perturb the blood-testis barrier (BTB). This study aims to elucidate the association between the disruption of BTB and the increases of PTMφs in the testis by comparing the effects observed with MEHP to two other testicular toxicants with variable effects on the BTB and subtype of germ cell undergoing apoptosis. Methoxyacetic acid (MAA) acts directly on spermatocytes and does not affect BTB function, while cadmium chloride (CdCl2) induces profound injury to BTB. The results indicated that MAA exposure significantly increased spermatocyte apoptosis, while no significant changes in the numbers of PTMφs in the testis occurred. In contrast, CdCl2 exposure disrupted BTB function and increased the abundance of PTMφs in the testis. To further investigate whether MEHP-induced changes in BTB integrity accounted for the increase in PTMφs, a plasmid for LG3/4/5, the functional component of laminin-alpha 2, was overexpressed in the testis to stabilize BTB integrity before MEHP exposure. The results showed that LG3/4/5 overexpression substantially reduced the ability of MEHP to compromise BTB integrity and prevented the increase in PTMφ numbers after MEHP exposure. These results indicate that BTB disruption is necessary to increase PTMφs in the testis induced by toxicants.

The role of the aryl hydrocarbon receptor in mediating the effects of mono(2-ethylhexyl) phthalate in mouse ovarian antral follicles†.

Di(2-ethylhexyl) phthalate (DEHP) is a pervasive environmental toxicant used in the manufacturing of numerous consumer products, medical supplies, and building materials. DEHP is metabolized to mono(2-ethylhexyl) phthalate (MEHP). MEHP is an endocrine disruptor that adversely affects folliculogenesis and steroidogenesis in the ovary, but its mechanism of action is not fully understood. Thus, we tested the hypothesis that the aryl hydrocarbon receptor (AHR) plays a functional role in MEHP-mediated disruption of folliculogenesis and steroidogenesis. CD-1 mouse antral follicles were isolated and cultured with MEHP (0-400μM) in the presence or absence of the AHR antagonist CH223191 (1μM). MEHP treatment reduced follicle growth over a 96-h period, and this effect was partially rescued by co-culture with CH223191. MEHP exposure alone increased expression of known AHR targets, cytochrome P450 (CYP) enzymes Cyp1a1 and Cyp1b1, and this induction was blocked by CH223191. MEHP reduced media concentrations of estrone and estradiol compared to control. This effect was mitigated by co-culture with CH223191. Moreover, MEHP reduced the expression of the estrogen-sensitive genes progesterone receptor (Pgr) and luteinizing hormone/choriogonadotropin receptor (Lhcgr) and co-treatment with CH223191 blocked this effect. Collectively, these data indicate that MEHP activates the AHR to impair follicle growth and reduce estrogen production and signaling in ovarian antral follicles.

Responses of peritubular macrophages and the testis transcriptome profiles of peripubertal and adult rodents exposed to an acute dose of MEHP.

Exposure of rodents to mono-(2-ethylhexyl) phthalate (MEHP) is known to disrupt the blood-testis barrier and cause testicular germ cell apoptosis. Peritubular macrophages (PTMφ) are a newly identified type of testicular macrophage that aggregates near the spermatogonial stem cell niche. We have previously reported that MEHP exposure increased the numbers of PTMφs by 6-fold within the testis of peripubertal rats. The underlying mechanism(s) accounting for this change in PTMφs and its biological significance is unknown. This study investigates if MEHP-induced alterations in PTMφs occur in rodents (PND 75 adult rats and PND 26 peripubertal mice) that are known to be less sensitive to MEHP-induced testicular toxicity. Results show that adult rats have a 2-fold higher basal level of PTMφ numbers than species-matched peripubertal animals, but there was no significant increase in PTMφ numbers after MEHP exposure. Peripubertal mice have a 5-fold higher basal level of PTMφ compared with peripubertal rats but did not exhibit increases in number after MEHP exposure. Further, the interrogation of the testis transcriptome was profiled from both the MEHP-responsive peripubertal rats and the less sensitive rodents via 3' Tag sequencing. Significant changes in gene expression were observed in peripubertal rats after MEHP exposure. However, adult rats showed lesser changes in gene expression, and peripubertal mice showed only minor changes. Collectively, the data show that PTMφ numbers are associated with the sensitivity of rodents to MEHP in an age- and species-dependent manner.

The role of estrogen receptors (ERs)-Notch pathway in thyroid toxicity induced by Di-2-ethylhexyl phthalate (DEHP) exposure: Population data and in vitro studies

BackgroundThis study aimed to assess the exposure level and risk of Di-2-ethylhexyl Phthalate (DEHP) among adults in Jilin Province, China, clarify the impact of DEHP on human thyroid function, and to explore the role of estrogen receptors (ERs)-Notch signaling pathway in the effect of DEHP metabolites on thyroid hormones based on population data and in vitro experiments. Methods312 adults participated in this study. Urinary DEHP metabolites were determined by high performance liquid chromatography coupled to a tandem mass spectrometer (HPLC-MS/MS). Two pharmacokinetic models were used to evaluate the estimated daily intake (EDI) and hazard quotient (HQ) of the adults. Multiple linear regression and mediating effect models were used to evaluate the target associations. In cell experiments, thyroid follicular epithelial (Nthy-ori3–1) cells were exposed to mono (2-ethylhexyl) phthalate (MEHP) for testing. The inhibitions of ERα and Notch pathway were conducted by siRNA and Notch pathway inhibitor DAPT. ResultsThe detection rate of five DEHP metabolites was 97.1∼100.0%. The HQ value of 0.3% of adults was higher than 1. The levels of urinary DEHP metabolites were significantly correlated with thyrotropin (TSH), thyrotropin-releasing hormone (TRH), total triiodothyronine (TT3), total thyroxine (TT4), free triiodothyronine (FT3) and free thyroxine (FT4) and gene (estrogen receptor α (ERα), Notch1, Dll4) levels. The ERα-Notch pathway played a mediating role in the association between DEHP metabolite levels and FT4. The cell results showed, the levels of FT3 and FT4 in cell supernatant decreased after MEHP exposure, and the downward trend was reversed after ERα and notch pathways were inhibited, notch pathway genes also decreased after ERα inhibition. ConclusionAdults in the Jilin Province of China were widely exposed to DEHP. ERs-Notch pathway played an important role in the effect of DEHP metabolites on thyroid hormones.

Association of mono-2-ethylhexyl phthalate with adverse outcomes in chronic hemodialysis patients

Phthalate exposure is widespread and has a global impact. Growing evidence shows that mono-2-ethylhexyl phthalate (MEHP) exposure has a negative impact on human health. However, whether MEHP exposure is associated with mortality and other adverse outcomes in hemodialysis patients remains unknown. This study prospectively enrolled 217 patients on maintenance hemodialysis from June 30, 2021, to August 16, 2022. Baseline serum MEHP, di-2-ethylhexyl phthalate (DEHP), and indoxyl sulfate (IS) concentrations were measured. Primary endpoints were all-cause mortality or composite adverse outcomes, including all-cause death plus hospitalization due to cardiovascular disease, heart failure, stroke, infection, or cancer. Serum MEHP concentrations were positively associated with DEHP but not indoxyl sulfate concentrations in hemodialysis patients. Additionally, serum MEHP concentrations were significantly and independently associated with all-cause mortality and composite adverse outcomes (adjusted hazard ratios [HRs], 1.04 and 1.03 per ng/mL, 95% confidence intervals [CIs], 1.01–1.07 and 1.00–1.05; p = 0.016 and 0.015, respectively). We found a cutoff value of MEHP for predicting both endpoints. Patients with serum MEHP concentrations of ≥ 41.8 ng/mL had much higher risks for all-cause mortality and composite adverse outcomes (adjusted HRs, 39.2 and 13; 95% CIs, 2.44–65.7 and 2.74–61.4; p = 0.011 and 0.001, respectively). MEHP exposure is significantly associated with higher risks for all-cause mortality and composite adverse outcomes. Hemodialysis patients with serum MEHP concentrations above 41.8 ng/mL had much poorer prognoses regarding both outcomes.

Mono-(2-ethylhexyl) phthalate (MEHP) reversibly disrupts the blood-testis barrier (BTB) in pubertal rats.

The blood-testis barrier (BTB) is constituted by tight junctions between adjacent Sertoli cells (SC) that create a specialized adluminal microenvironment to foster the development of spermatocytes and spermatids. The BTB is a well-studied target of numerous environmental toxicants, including di-(2-ethylhexyl) phthalate (DEHP), a compound widely used in various consumer products. MEHP is the active toxic metabolite of DEHP that has long been recognized in postnatal rodents to disrupt SC function. This study evaluates the impact of MEHP on the integrity of the BTB in both pubertal and adult rats and the signal transduction pathways known to be involved in the disruption of the BTB. Treatment of prepubertal rats with 700 mg/kg MEHP for 24 hours functionally disrupted the BTB integrity. A similar treatment of adult rats with MEHP did not disrupt the integrity of the BTB. The observed disruption of the BTB integrity in the MEHP-treated prepubertal rats occurred concomitantly with a decreased expression and mislocalization of both the ZO1 and occludin tight junction-associated proteins, as well as sloughing of spermatocytes and spermatids. At this same time, MEHP treatment induced a transient surge of p44/42 mitogen-activated protein kinase (MAPK) pathway. Interestingly, after a recovery period of 5 weeks, the BTB recovered and was functionally intact. This is the first report to indicate that acute MEHP exposure of prepubertal rats, but not adult rats, disrupts the functional integrity of the BTB and that this effect on the BTB is reversible.

MEHP activates JNK to inhibit the migration of human foreskin fibroblasts

This study aimed to investigate the impact of mono(2-ethylhexyl) phthalate (MEHP) on the proliferation, apoptosis, and migration of human foreskin fibroblast cells (HFF-1) and the role of the JNK signaling pathway in cell migration. HFF-1 cells were randomly assigned to the control group with 0 MEHP exposure (M0) or the experimental groups with 25, 50, 100, 200, and 400 μmol/L MEHP exposure (M25, M50, M100, M200, and M400, respectively). After 24 and 48 h of MEHP exposure, the proliferation of HFF-1 cells in any group had no significant change. However, compared with the M0 group, the M200 and M400 groups presented substantially increased apoptosis of HFF-1 cells. Moreover, cell migration ability significantly decreased in all groups (p < 0.05). Additionally, the transcription and phosphorylated protein activation of JNK kinase in HFF-1 cells were substantially upregulated with the increase in MEHP exposure. Subsequently, HFF-1 cells were randomly divided into three groups: the DMSO blank control group, the 100 μM MEHP experimental group (M100), and the 100 μM MEHP plus 10 μM SP600125 (specific JNK inhibitor) experimental group (S10). The activation of JNK protein in HFF-1 cells was substantially downregulated in the S10 group. HFF-1 cells were also divided into the blank control group (M0). They were treated with 100 μM MEHP and varying concentrations of SP600125 (5, 10, and 15 μM for S5, S10, and S15, respectively). As the concentration of the antagonist increased, the migration ability of HFF-1 cells was returned to normal. Finally, the ROS in HFF-1 cells increased under MEHP exposure. This finding indicates that the regulation of cell migration by the JNK signaling pathway may be important in the occurrence of hypospadias.

Activation of the p62-Keap1-Nrf2 pathway protects against oxidative stress and excessive autophagy in ovarian granulosa cells to attenuate DEHP-induced ovarian impairment in mice

Di-(2-ethylhexyl) phthalate (DEHP) is widely used in various plastics but has been demonstrated to cause female reproductive toxicity. However, the exact mechanism underlying the ovarian damage induced by DEHP remains unclear. In this study, DEHP was administered orally to 5-week-old female mice for 30 days at doses of 0, 250, 500, and 1000 mg/kg/day. The findings demonstrated that DEHP exposure disrupted ovarian function and follicular development as well as induced oxidative stress and autophagy in ovarian granulosa cells (GCs). Further, 200 µM mono-(2-ethylhexyl) phthalate (MEHP), the primary metabolite of DEHP in vivo, induced autophagy in both human ovarian granulosa cells line (KGN) and mouse primary GCs within 24 h in vitro. However, it did not affect the p62-dependent autophagy flux. Furthermore, MEHP-induced autophagy was inhibited by the autophagy inhibitor 3-MA and exacerbated by the autophagy activator rapamycin, indicating that MEHP induces excessive autophagy in GCs. Subsequently, we found that MEHP-induced autophagic cell death was primarily attributed to oxidative damage from elevated intracellular ROS levels. Meanwhile, MEHP exposure induced nuclear translocation of erythroid-derived factor 2-related factor (Nrf2), a key regulator of antioxidant activity resulting in activating antioxidant effects. Interestingly, we also found that MEHP-induced increase in p62 competitively binds Keap1, thereby facilitating nuclear translocation of Nrf2 and establishing a positive feedback loop in antioxidant regulation. Therefore, this study demonstrated that inhibition of Nrf2 could aggravate oxidative damage and enhance excessive autophagy caused by MEHP, while activation of Nrf2 could reverse the trend. These findings have also been reinforced in studies of cultured ovaries in vitro. Our study suggests that the p62-Keap1-Nrf2 pathway may serve as a potential protective mechanism against DEHP-induced oxidative stress and excessive autophagy in mouse GCs.

Wnt10a downregulation contributes to MEHP-induced disruption of self-renewal and differentiation balance and proliferation inhibition in GC-1 cells: Insights from multiple transcriptomic profiling

Di (2-ethylhexyl) phthalate (DEHP), one of phthalic acid esters, has been widely used in daily products. Its main metabolite, mono (2-ethylhexyl) phthalate (MEHP) was reported to possess higher testicular toxicity than DEHP. To explore the precise mechanism in MEHP-induced testis damage, multiple transcriptomic sequencing was employed in spermatogonia cell line GC-1 cells treated with MEHP (0, 100, and 200 μM) for 24 h. Integrative omics analysis and empirical validation revealed that Wnt signaling pathway was downregulated and wnt10a, one of hub genes, may be the key player in this process. Similar results were observed in DEHP-exposed rats. MEHP-induced disturbance of self-renewal and differentiation was dose-dependent. Moreover, self-renewal proteins were downregulated; the differentiation level was stimulated. Meanwhile, GC-1 proliferation was decreased. Stable transformation strain of wnt10a overexpression GC-1 cell line constructed from lentivirus was utilized in this study. The upregulation of Wnt10a significantly reversed the dysfunction of self-renewal and differentiation and promoted the cell proliferation. Finally, retinol, predicted to be useful in CONNECTIVITY MAP (cMAP), failed to rescue the damage caused by MEHP. Cumulatively, our findings revealed that the downregulation of Wnt10a induced the imbalance of self-renew and differentiation, and inhibition of cell proliferation in GC-1 cells after MEHP exposure.

Alteration of the N6-methyladenosine epitranscriptomic profile in synthetic phthalate-treated human induced pluripotent stem cell-derived endothelial cells.

Background: This study aimed to characterize the N6-methyladenosine epitranscriptomic profile induced by mono(2-ethylhexyl) phthalate (MEHP) exposure using a human-induced pluripotent stem cell-derived endothelial cell model. Methods: A multiomic approach was employed by performing RNA sequencing in parallel with an N6-methyladenosine-specific microarray to identify mRNAs, lncRNAs, and miRNAs affected by MEHP exposure. Results: An integrative multiomic analysis identified relevant biological features affected by MEHP, while functional assays provided a phenotypic characterization of these effects. Transcripts regulated by the epitranscriptome were validated with quantitative PCR and methylated RNA immunoprecipitation. Conclusion: The authors' profiling of the epitranscriptome expands the scope of toxicological insights into known environmental toxins to under surveyed cellular contexts and emerging domains of regulation and is, therefore, a valuable resource to human health.

Dose–response mapping of MEHP exposure with metabolic changes of trophoblast cell and determination of sensitive markers

Mono(2-ethylhexyl) phthalate (MEHP) is a metabolite of DEHP which is one of phthalic acid esters (PAEs) widely used in daily necessities. Moreover, MEHP has been proven to have stronger biological toxicity comparing to DEHP. In particular, several recent population-based studies have reported that intrauterine exposure to MEHP results in adverse pregnancy outcomes. To explore the mechanisms and metabolic biomarkers of MEHP exposure, we examined the metabolic status of HTR-8/Svneo cell lines exposed to different doses of MEHP (0, 1.25, 5.0, 20 μM). Global and dose–response metabolomics tools were used to identify metabolic perturbations and sensitive markers associated with MEHP. Only 22 metabolic features (accounted for <1 %) were significantly changed when exposed to 1.25 μM. However, when the exposure dose was increased to 5 or 20 μM, the number of significantly changed metabolic features exceeded 300 (approximately 10 %). In particular, amino acid metabolism, pyrimidine metabolism and glutathione metabolism were widely affected according to the enrich analysis of those significant altered metabolites, which has and have previously been reported to be closely related to fetal development. Moreover, 5′-UMP and N-acetylputrescine with the lowest effective concentrations (EC−10 = 0.1 μM and EC+10 = 0.11 μM, respectively) were identified as sensitive endogenous biomarkers of MEHP exposure.

Mono(2-ethylhexyl) phthalate modulates lipid accumulation and reproductive signaling in Daphnia magna.

Mono(2-ethylhexyl) phthalate (MEHP) is a primary metabolite of di(2-ethylhexyl) phthalate (DEHP), which is widely used in industry as a plasticizer. Both DEHP and MEHP have been identified as endocrine disruptors affecting reproduction systems in natural aquatic environments. However, the effects of MEHP exposure on aquatic invertebrates such as Daphnia magna are still poorly understood. In the present study, lipid alterations caused by MEHP in D. magna were identified by analyzing lipid accumulation and nontarget metabolomics. In addition, reproductive endpoints were investigated. MEHP exposure under any conditions upto 2mg/L was not associated with mortality of D. magna; yet, the number of lipid droplets and the adult female daphnids reproduction rates increased after 96h of exposure and 21days of exposure, respectively. MEHP also enhanced lipid metabolism, as evident from 283 potential lipid metabolites, including glycerolipids, glycerophospholipids, and sphingolipids, identified following 48h of exposure. The MEHP-treated group exhibited significantly higher ecdysone receptor (EcR) and vitellogenin 2 (Vtg2) expression levels at 6 and 24h. At 48h, EcR and Vtg2 expression levels were downregulated in the 1 and 2mg/L MEHP exposure groups. Our data reveal that the EcR pathway changes over MEHP exposure could be associated with lipid accumulation, owing to increased lipid levels and the subsequent increase in the reproduction of MEHP-exposed D. magna.

Chronic inflammation as a potential mediator between phthalate exposure and depressive symptoms

BackgroundA few studies have reported phthalate exposure as a risk factor for depressive symptoms, but the results have been inconsistent. Whether chronic inflammation mediates the relationship between phthalates (PAEs) and depressive symptoms remains unclear. In this study, we establish mediating models of inflammatory factors and explore the mediating role of chronic inflammation in the association between PAEs exposure and depressive symptoms. MethodsThe sample included 989 participants from the Study on Health and Environment of the Elderly in Lu'an City, Anhui Province. Geriatric depression scale (GDS-30) was used to screen depressive symptoms of the elderly. The levels of seven kinds of PAEs in urine samples and four inflammatory factors in serum of the elderly were measured. To establish the mediating effect of inflammatory factors to explore the potential effect of PAEs exposure on the increased odds of depressive symptoms. ResultsAdjusted for multiple variables, the highest tertiles of Mono (2-ethylhexyl) phthalate (MEHP) (95%CI = 1.051–2.112), Mono benzyl phthalate (MBzP) (95%CI = 1.016–2.082) and Mono butyl phthalate (MBP) (95%CI = 1.102–2.262) were positively correlated with depressive symptoms. The mediating effect of IL-6 and generalized inflammation factor between MEHP exposure and depressive symptoms were 15.96% (95%CI=0.0288–0.1971) and 14.25% (95%CI = 0.0167–0.1899). ConclusionsHigh levels of MEHP, MBzP and MBP increased the odds of depressive symptoms in the elderly, and chronic inflammation had a partial mediating effect on the increased odds of depressive symptoms due to MEHP exposure.

MEHP promotes liver fibrosis by down-regulating STAT5A in BRL-3A hepatocytes

ObjectiveAs an environmental endocrine disruptor, mono-2-ethylhexyl phthalate (MEHP) can interfere with liver metabolism and lead to liver diseases. We aimed to investigate the role of MEHP in liver fibrosis and its molecular mechanism. MethodsBRL-3A hepatocytes were exposed to MEHP (0, 10, 50, 100 and 200 μM) for 24 h. STAT5A gene was overexpressed by lentivirus transfection. The reactive oxygen species (ROS) was tested by the flow cytometer. The malondialdehyde (MDA), glutathione peroxidase (GSH-PX), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were detected by commercial kits. Real-Time PCR and Western blot were performed to test the relative mRNA and proteins levels, respectively. ResultsMEHP exposure significantly induced oxidative damage in BRL-3A cells, which inhibited the expression of STAT5A and promoted the expression of fibrosis related proteins MMP2, MMP9, TIMP2 and CTGF. After over-expression of STAT5A gene in BRL-3A cells, the elevated expression levels of CTGF, MMP2, MMP9 and TIMP2 induced by MEHP exposure were significantly reversed. ConclusionThis study demonstrated that MEHP exposure inhibited the expression of STAT5A by causing oxidative damage in BRL-3A hepatocytes, thus accelerating the expression of key molecules in fibrosis and promoting the occurrence of liver fibrosis.

Exposure to di-(2-ethylhexyl) phthalate reduces secretion of GDNF via interfering with estrogen pathway and downregulating ERK/c-fos signaling pathway in astrocytes

Di-(2-ethylhexyl) phthalate (DEHP) is a typical endocrine-disrupting chemical (EDC) that can increase the risk of central nervous system disease. This study aimed to investigate the in vitro and in vivo effects of DEHP exposure on GDNF secretion and the underlying mechanisms. Pregnant Wistar rats were randomly assigned into four groups and administered 0, 30, 300, or 750 mg/kg DEHP daily by oral gavage. In addition, primary astrocytes were exposed to mono-(2-ethylhexyl) phthalate (MEHP), the main metabolite of DEHP. Our results showed that DEHP exposure reduced GDNF levels and downregulated the ERK/c-fos signaling pathway in the cerebral cortex of male, but not female, offspring. Moreover, exogenous estrogen could overcome the decreased GDNF levels in astrocytes caused by MEHP exposure. MEHP also decreased p300 levels and downregulated the ERK/c-fos signaling pathway in primary astrocytes. Honokiol restored GDNF levels following MEHP exposure by activating the ERK/c-fos signaling pathway, while the inhibitor U0126 further reduced the GDNF levels. These results suggested that DEHP exposure could interfere with the normal effects of estrogen in the brain and downregulate the ERK/c-fos signaling pathway to decrease the GDNF secretion from astrocytes in the cerebral cortex.

Role of Notch pathway in effect of mono-2-ethylhexyl phthalate on the proliferation and cell cycle of SH-SY5Y cell.

Neuroblastoma (NB) is an estrogen-dependent tumor. Mono-2-ethylhexyl phthalate (MEHP) has an estrogen-like effect. However, the effects of MEHP on the progression of NB are not well illustrated. This study was to clarify the effect of Notch pathway on proliferation and cell cycle of SH-SY5Y cell induced by MEHP. The viability of SH-SY5Y and BE2C cells were detected by CCK8; cell cycle and apoptosis were detected by flow cytometry; the protein expression levels of Notch pathway and cell cycle related proteins were detected by Western-blot. Results show that MEHP exposure can promote cell proliferation and altered the cell cycle. MEHP exposure can up-regulate the expression of C-MYC, Cyclin D1, Bcl-2 and affected the Notch pathway. In conclusion, MEHP exposure can promote NB cell proliferation and affect the cell cycle and apoptosis. Notch pathway plays a critical role in accelerating the cell cycle and inhibiting the apoptosis of SH-SY5Y cells caused by MEHP.

Low-dose mono(2-ethylhexyl) phthalate promotes ovarian cancer development through PPARα-dependent PI3K/Akt/NF-κB pathway

The plasticizer di(2-ethylhexyl) phthalate (DEHP) and its hydrolysate mono(2-ethylhexyl) phthalate (MEHP) are major toxicants from plastics, but their association with hormone-dependent cancers has been controversial. We treated the human ovarian cancer cell lines SKOV3 and A2780 with low concentrations of DEHP/MEHP, and found that although no significant effect on cell proliferation was observed, ovarian cancer cell migration, invasion, and epithelial-mesenchymal transition (EMT) were promoted by submicromolar MEHP but not DEHP. Next, ovarian cancer patient data from The Cancer Genome Atlas (TCGA) were obtained and subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) supported enrichment and Kaplan-Meier survival analyses, which identified PI3K/Akt pathway as a pivotal signaling pathway in ovarian cancer. We found that 500 nM MEHP treatment significantly increased PIK3CA expression, which could be reversed by the knockdown of peroxisome proliferator-activated receptor alpha (PPARα). Silencing PIK3CA significantly suppressed the MEHP-induced migration, invasion and EMT. In addition, we validated that MEHP treatment promoted phosphorylation of Akt and degradation of IκB-α, thereby activating NF-κB and enhancing NF-κB nuclear translocation. In nude mice, MEHP exposure significantly promoted the metastasis of ovarian cancer xenografts, which could be suppressed by the treatment of PPARα inhibitor GW6471. Our findings showed that low-dose MEHP promoted ovarian cancer progression through activating PI3K/Akt/NF-κB pathway, in a PPARα-dependent manner.

Exposure to mono (2-ethylhexyl) phthalate facilitates apoptosis and pyroptosis of human endometrial microvascular endothelial cells through NLRP3 inflammasome.

Mono (2-ethylhexyl) phthalate (MEHP) is a major metabolite of di (2-ethylhexyl) phthalate (DEHP). This study aimed to observe the toxic effect of MEHP on human endometrial microvascular endothelial cells (HEMECs) and its potential molecular mechanism. HEMECs were exposed to different concentrations of MEHP (0, 50, 100, and 200 nM). Cell viability and apoptosis were assessed by cell counting kit-8 (CCK-8) and flow cytometry assays. Western blot was performed to examine the expression of apoptosis-related proteins (Bcl-2, Bax, and Caspase-3). Moreover, the expression of pyroptosis-related Caspase-1 was detected by western blot and immunofluorescence assays. Lactate dehydrogenase (LDH) release levels were evaluated in HEMECs treated with MEHP and/or Caspase-1 inhibitor Ac-YVAD-CHO. After exposure to MEHP, NLRP3 expression was examined by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blot. LDH release and apoptosis levels were tested in HEMECs induced by MEHP and/or siNLRP3. MEHP significantly induced cell viability and inhibited apoptosis for HEMECs, with a concentration-dependent manner. Furthermore, Bcl-2/Bax ratio was distinctly reduced and Caspase-3 expression was increased in HEMECs after exposure to MEHP. Western blot and immunofluorescence results confirmed that MEHP markedly augmented Caspase-1 expression in HEMECs. Furthermore, LDH release levels were fortified in HEMECs treated with MEHP, which were improved following cotreatment with Ac-YVAD-CHO. At the mRNA and protein levels, NLRP3 expression was prominently increased in HEMECs exposed to MEHP. NLRP3 knockdown markedly ameliorated the increase in LDH release and apoptosis induced by MEHP exposure in HEMECs. Our findings suggested that exposure to MEHP facilitates apoptosis and pyroptosis of HEMECs through NLRP3 inflammasome.