DEHP Research Articles

Calcium homeostasis imbalance mediates DEHP induced mitochondrial damage in cerebellum and the antagonistic effect of lycopene

Phthalates (PAEs), especially di (2-ethylhexyl) phthalate (DEHP), are generally considered to have adverse impact on nervous system. The residue of DEHP in the environment has gradually become a widely concerned environmental problem due to its widespread use in plastic items. Lycopene (LYC) as the readily available natural antioxidant is considered to have the potential to alleviate exogenous poisons-induced nerve damage. However, there is currently a lack of strategies to alleviate the neurotoxicity caused by DEHP, and it is also unknown whether LYC can alleviate the neurotoxicity caused by DEHP. The experiment demonstrated that LYC had the potential to mitigate DEHP-induced mitochondrial damage in cerebellum. DEHP induced the disorder of Ca2+ transport in cerebellum, thereby resulting in the imbalance of protein homeostasis. Such disruption in protein homeostasis further results in the overactivation of mitochondrial unfolded protein response (UPRmt) and mitochondrial injury. Mechanistically, LYC could alleviate the imbalance of calcium homeostasis and protein homeostasis induced by DEHP via regulating inositol 1, 4, 5-trisphosphate receptor type1 (IP3R1) and sarco/endoplasmic reticulum Ca (2+)-ATPase 2 (SERCA2), further alleviating mitochondrial damage in cerebellum. Subsequently, the present study suggested the mechanism of cerebellar injury induced by DEHP, and provided a novel approach to treating DEHP-induced neurotoxicity.

Effects of di-(2-ethylhexyl) phthalate and its metabolites on transcriptional activity via human nuclear receptors and gene expression in HepaRG cells

Di-(2-ethylhexyl) phthalate (DEHP) is widely used as a plasticizer in polyvinyl chloride products. DEHP exposure in humans is of great concern due to its endocrine-disrupting properties. In this study, we characterized the agonistic activities of DEHP and its five metabolites, mono-(2-ethylhexyl) phthalate (MEHP), 5OH-MEHP, 5oxo-MEHP, 5cx-MEPP and 2cx-MMHP against human nuclear receptors, peroxisome proliferator-activated receptor α (PPARα), pregnane X receptor (PXR), and constitutive androstane receptor (CAR) using transactivation assays. In the PPARα assay, the order of the agonistic activity was MEHP >> 5cx-MEPP >5OH-MEHP, 5oxo-MEHP >2cx-MMHP > DEHP, with DEHP significantly inhibiting MEHP-induced PPARα agonistic activity. This finding was compared to the results from in silico docking simulation. In the PXR assay, DEHP showed PXR agonistic activity more potent than that of MEHP, whereas the other metabolites showed little activity. In the CAR assay, none of the tested compounds showed agonistic activity. Moreover, the expression levels of PPARα-, PXR-, and CAR-target genes in HepaRG cells exposed to DEHP or MEHP were investigated using qRT-PCR analysis. As a result, exposure to these compounds significantly upregulated PXR/CAR target genes (CYP3A4 and CYP2B6), but not PPARα target genes (CYP4A11, etc.) in HepaRG cells. Taken together, these results suggest that direct PXR and/or indirect CAR activation by several DEHP metabolites may be involved in the endocrine disruption by altering hormone metabolism.

Combined ecotoxicity of polystyrene microplastics and Di-(2-ethylhexyl) phthalate increase exposure risks to Mytilus coruscus based on the bioaccumulation, oxidative stress, metabolic profiles, and nutritional interferences.

Di-(2-ethylhexyl) phthalate (DEHP) and microplastics (MPs) are emerging contaminants frequently detected in the marine environment. However, the influence of MPs on DEHP bioaccumulation and their combined effects on eco-environmental risks remain underexplored. Mytilus coruscus (M. coruscus) were exposed to DEHP (200.0µg/L), polystyrene (PS) (0.050, 0.50, and 5.0mg/L), and their combination at environmentally relevant concentrations for 15-day, followed by a 7-day depuration period. The amount of DEHP accumulation followed the order of digestive gland >gills >muscles >gonad, with PS dose-dependently amplifying DEHP bioaccumulation in digestive gland. The changes in antioxidant enzyme activity indicated disruptions in oxidative defense. Furthermore, metabolomic analysis revealed that PS and DEHP considerably altered the lipid, energy, and citric acid cycles in digestive gland and gonad. Post-depuration analysis showed combined exposure resulted in persistent effects. Compared with single exposures, combined exposure had a greater adverse effect on the metabolism of essential amino acids, fatty acids, and volatile compounds, potentially influencing edibility and nutritional value of M. coruscus. This study underscores cumulative eco-environmental toxicity of PS and DEHP toward M. coruscus and highlights the potential increased risks of co-pollution.

Occurrence, bioaccumulation, and partitioning of phthalate acid esters in the third largest freshwater lake (Lake Taihu) in China

Phthalate acid esters (PAEs) are a category of plasticizers that are ubiquitous in freshwater environments attributable to extensive utilization. We collected water, suspended particulate matter (SPM), surface sediments, phytoplankton, and zooplankton from 23 sampling sites to investigate and complement the occurrence, bioaccumulation, and partitioning of five PAEs including dimethyl phthalate (DMP), diethyl phthalate (DEP), di-n-butyl phthalate (DBP), butyl benzyl phthalate (BBP), and di (2-ethylhexyl) phthalate (DEHP) in the third largest freshwater lake (Lake Taihu) of China. PAEs were extracted using Soxhlet extraction and solid phase extraction, and determined by gas chromatography-mass spectrometry. The average concentrations of the five PAEs in the water column, SPM, sediments, phytoplankton, and zooplankton of Lake Taihu were 1.93±1.57μgL-1, 765±766μgg-1, 1.68±1.47μgg-1, 1358±1877μgg-1, and 72.7±134μgg-1, respectively. DBP and DEHP were the dominant PAE congeners in the five environment compartments. The logarithmic concentrations of DBP, BBP, and DEHP in the SPM were negatively correlated with the logarithmic content of the SPM. Biodilution significantly impacted the occurrence of PAEs in the plankton. Bioaccumulation of PAEs was found in the plankton with log BCF (bioconcentration factor) in the phytoplankton ranging from 1.78±0.86 to 4.13±1.23 and log BAF (bioaccumulation factor) in the zooplankton varying from -0.10±0.26 to 3.04±0.64. Biomagnification of the PAEs from phytoplankton to zooplankton was not observed. DMP, DEP, and BBP migrated from sediments to water. DBP was in dynamic equilibrium in the sediment-water system. DEHP transferred from water to sediments. Our results provide crucial complementary knowledge on bioaccumulation and transfer of PAEs in planktonic food web, and their partitioning in different compartments of waters.

DEHP regulates ferritinophagy to promote testicular ferroptosis via suppressing SIRT1/PGC-1α pathway

To increase elasticity and flexibility, di-2-ethylhexyl phthalate (DEHP) is used in a variety of industrial products, but excessive exposure to it can pose a threat to human health. In epidemiological studies of population exposure to DEHP, attention has been paid to damage to the male reproductive system. However, the toxicological mechanism of DEHP regarding testicular injury is not well understood. We used Western blot analysis, transmission electron microscopy, fluorescence staining, transient transfection and assay kit to detect relevant indicators, and the results were as follows: After DEHP exposure, the expression levels of ACSL4, COX2, TF, FTH1, LC3, AMPK, p-AMPK, ULK1, p-ULK1, serum iron, tissue iron and MDA in the exposure group were significantly increased. The expression levels of GPX4, NCOA4, p62, SIRT1, and PGC-1α, as well as the contents of GSH and ATP, decreased. Electron microscopy showed that more autophagosomes were observed. Our findings suggest that exposure to DEHP induced ferritinophagy and ferroptosis in the testis. In vitro, the promoting effect of ferritinophagy on ferroptosis was verified by applying the autophagy inhibitor (3-MA) and si-NCOA4. Moreover, Mono-(2-ethylhexyl) phthalate (MEHP) inhibited the mitochondrial regulatory protein SIRT1/PGC-1α, leading to mitochondrial dysfunction. Changes in mitochondrial reactive oxygen species (MtROS) and energy over-activated AMPK/ULK1 autophagy pathway, and then promoted ferritinophagy, which increased the sensitivity of TM4 cells to ferroptosis. This research offers a theoretical framework for the prevention and management of DEHP-induced harm.

Network toxicology and cell experiments reveal the mechanism of DEHP-induced diabetic nephropathy via Wnt signaling pathway

Di(2-ethylhexyl) phthalate (DEHP), a widely recognized endocrine disruptor, has been linked to the pathogenesis of diabetic nephropathy (DN) through its interference with hormonal and metabolic homeostasis. This study integrates network toxicology with cell-based assays to elucidate the molecular mechanisms of DEHP-induced DN, seeking to identify novel targets for toxicity assessment and therapeutic intervention. Through comprehensive screening across multiple toxicology and disease-related databases, six core genes (CTNNB1, EGFR, TNF, CCND1, BCL2, CASP3) were identified as shared mediators of DEHP exposure and DN. These genes are predominantly associated with the Wnt signaling pathway, a pivotal regulator of podocyte function, including cellular adhesion, differentiation, apoptosis, and inflammatory response. Mouse glomerular podocytes (MPC-5) exposed to graded concentrations of DEHP, with or without the Wnt pathway inhibitor XAV-939, displayed significant DEHP-induced disruptions: reduced cell adhesion, proliferation, and differentiation; increased autophagy, apoptosis, and migratory activity; elevated inflammatory mediator release; and pronounced activation of the Wnt signaling pathway, evidenced by upregulation of β-catenin, EGFR, TNF, CCND1, BCL2 and downregulation of CASP3. DEHP exposure further altered transcriptional activity and chromatin structure at key loci (CTNNB1, EGFR, and TNF). XAV-939 effectively mitigated these effects, underscoring the Wnt pathway's central role in DN progression under DEHP influence. These findings highlight the complex multi-target, multi-pathway interactions of DEHP in DN pathophysiology, offering deeper mechanistic insights and potential targets for therapeutic intervention against DEHP-induced nephrotoxicity.

Resveratrol alleviates Mono-2-ethylhexyl phthalate-induced mitophagy, ferroptosis, and immunological dysfunction in grass carp hepatocytes by regulating the Nrf2 pathway

Mono-2-ethylhexyl phthalate (MEHP) is the major biologically active metabolite of Di(2-ethylhexyl) phthalate (DEHP). This MEHP mono-ester metabolite can be transported through the bloodstream into tissues such as the liver, kidneys, fat, and testes and cause corresponding damage. Resveratrol (RSV) has anti-inflammatory, antioxidant, and detoxification characteristics. Our research examined whether RSV alleviates MEHP-induced grass carp hepatocyte (L8824 cell) injury and its relationship with the Nrf2 pathway, mitophagy, ferroptosis, and immune function. Therefore, we treated L8824 cells with 85 μM MEHP and/or 2 μM RSV. The findings indicated that exposing MEHP resulted in increased reactive oxygen species (ROS) content and decreased mitochondrial membrane potential in L8824 cells, which induced an up-regulation of the expression of mitophagy-related indicators (PINK1, Parkin, Beclin1, LC3B, and ATG5) and a down-regulation of P62. An up-regulation of the expression of the ferroptosis-related indicators TFR1 and COX-2, and GPX4 and FTH expression was down-regulated. In addition, there was a decrease in the expression of IL-2 and IFN-γ and an increase in the expression of inflammatory cytokines such as TNF-α, IL-1β, and IL-6 after exposure to MEHP. RSV activates the Nrf2 pathway and effectively alleviates MEHP-induced mitophagy, ferroptosis, and immunologic dysfunction of L8824 cells.

Evaluation of phthalate acid esters (PAEs) in kefir samples by using magnetic chitosan coated with polyaniline (Fe3O4@CHI@PANI)/GC–MS method: A health risk assessment study

In this study, by magnetic chitosan coated with polyaniline (Fe3O4@CHI@PANI) −gas chromatography/mass spectrometry (GC/MS) method was applied to analyze six PAE compounds in kefir samples. The results obtained from kefir samples showed that the lowest and highest mean of PAEs were associated to dimethyl phthalate (DMP was not detected or nd) and di-n-octyl phthalate (DNOP, 2.12 µg/kg), respectively. Also according to results the average of DEHP (Bis(2-ethylhexyl)phthalate) and total PAEs in all samples were 1.31 ± 0.72 and 4.58 ± 1.87 µg/kg, respectively. Furthermore, maximum and minimum mean of total PAEs were detected in the packages with a volume of 1000 mL (5.47 ± 2.03 μg/kg) and packages with a volume of 1500 mL (3.69 ± 1.23 μg/kg), respectively, and the maximum and minimum mean total PAE (μg/kg) were related to samples with a date less than the expiration date (5.42 ± 1.46) and samples with a date greater than the production date (3.73 ± 1.91), respectively. The level of these contaminants was lower than the existing standard levels. The assessment of human health risks, comprising risks of non-carcinogenic and carcinogenic were calculated with Monte Carlo simulation (MCS) and sensitivity analysis under certain removal efficiencies from 5 to 95 %. The result of the carcinogenic risk for DEHP showed that adults are not threatened by the consumption of kefir (1.06E-05), while children are exposed to negligible risk the consumption of kefir (1/48E-04). In all samples, the total non-carcinogenic risk of PAE compounds was less than 1 (TTHQ was 2.03E-04 and 4.33E-05 for children and adults, respectively), this values presenting there would be unlikely risks of non-carcinogenic of PAEs in kefir for consumers. In conclusion, it can be stated it doesn’t pose any threat to Iranian consumers (adults and children).

Exploring the Nephrotoxicity and Molecular Mechanisms of Di-2-ethylhexyl phthalate: A Comprehensive Review.

Di-2-ethylhexyl phthalate (DEHP), a widely applied plasticizer in various products, can be absorbed into the human body through several channels and accumulate in the lungs, liver, testes, and kidneys, potentially impairing the function of these organs. Recently, the nephrotoxicity of DEHP has received heightened attention. Numerous epidemiologic findings have demonstrated that DEHP exposure may contribute to renal damage, leading to structural and functional abnormalities and exacerbating the progression of kidney disease. Recent research has discovered the mechanisms behind DEHP-induced nephrotoxicity may involve a variety of pathways, including apoptosis, autophagy, ferroptosis, oxidative stress, inflammation, DNA damage, and lipid metabolism disorders. This review discusses the impact of DEHP on kidney function and delves into the molecular mechanisms of nephrotoxicity mediated by DEHP in recent years. In addition, the review examines evidence for the antioxidant and anti-inflammatory capacities of lycopene, green tea polyphenols, and quercetin in ameliorating DEHP-induced renal injury is reviewed, providing a basis for further research.

Phthalate esters in baltic lagoons: Spatial distribution, ecological risks, and novel insights into their fate using transcriptomics.

Plasticizers such as phthalate esters (PAEs) are organic compounds widely used in various consumer and industrial products, raising strong environmental concerns due to their pervasive presence and potential adverse effects. Lagoon ecosystems are particularly vulnerable to PAE pollution as they are semi-enclosed and receive high loads of organic materials. The present study investigates the distribution of seven common PAEs in three large European lagoons (Curonian, Vistula and Szczecin) in the southern Baltic Sea. The concentration levels of PAEs in the water column, encompassing both the dissolved and particulate-bound phases, and in sediments were assessed to elucidate distribution patterns and potential ecological risks within these lagoon ecosystems. The average concentration of total PAEs in the water column ranged from 0.03 to 1.45 μg L-1, whereas sediment concentration varied from 0.008 to 1.06 μg g-1, levels comparable to or lower than those found in other European coastal areas. Distribution patterns of PAEs in sediment showed notable similarity across all three lagoons, whereas variations were observed in the water column. Notably, di(2-ethylhexyl) phthalate (DEHP), di-n-octyl phthalate (DOP) and dimethyl phthalate (DMP) emerged as the most concerning congeners in studied lagoons, all of which pose a moderate risk to aquatic organisms. This study applied shotgun transcriptomic analysis to field samples, revealing active microbial communities involved in PAEs degradation in the Baltic lagoons for the first time. The degradation of phthalic acid (PA) into intermediate compounds such as protocatechuate was not identified as a rate-limiting step in the studied environment. The degradation activity was primarily localized in the sediment layers, with Gram-negative bacteria playing a major role, while Gram-positive bacteria appeared incapable of degrading PA. These findings provide valuable insights into the distribution and transformation mechanisms of PAEs in estuarine environments.

Antibacterial Activity, GC MS Analysis and In Silico Validation of Bioactive Compound from Endophytic Fungus Lasiodiplodia pseudotheobromae EF-9.

Secondary metabolites synthesized by endophytic fungi have garnered significant interest for their broad applications in treating various ailments. In this study involving 20 plant samples, 11 endophytic fungi were isolated and cultured, and Lasiodiplodia pseudotheobromae EF-9, derived from Hibiscus rosa-sinensis, demonstrated greater antibacterial efficacy than the other isolated endophytes. Phylogenetic analyses using 18S rRNA gene confirmed the EF-9 identity as L. pseudotheobromae. Following mass production, the active compound was partially purified using column chromatography. The fraction collected at the 60th min exhibited good antibacterial activity against Bacillus coagulans (MTCC 6735) and Shigella flexneri (ATCC 12022), with an inhibition zone of approximately 20 mm in diameter. UV spectral studies revealed a wide absorption band at 430 nm. High Performance Liquid Chromatography (HPLC) of the active fraction showed a distinct peak with a retention time of 4.216 min at 430 nm absorbance. Gas Chromatography-Mass Spectrometry (GC-MS) identified the active compound in the L. pseudotheobromae EF-9 culture broth extract as Bis(2-ethylhexyl) phthalate, which displayed a peak at 16.856 min and covered 66.69% of the area in the spectral analysis.

Dietary exposure to di (2-ethylhexyl) phthalate (DEHP) for 6months alters markers of female reproductive aging in mice.

The female reproductive system ages before any other physiological system, making it a sensitive indicator of aging. Early reproductive aging is associated with the early onset of infertility and an increased risk of several diseases. During aging, systemic and reproductive oxidative stress and inflammation levels increase through inflammasome activation, leading to ovarian follicle loss. Other markers of reproductive aging include increased fibrosis and shortening of telomeres in ovarian cells. The factors that accelerate reproductive aging are unclear, but likely involve exposure to endocrine-disrupting chemicals such as phthalates. Di(2-ethylhexyl) phthalate (DEHP) is a widely used phthalate and humans are exposed to it daily. Several studies show that DEHP induces reproductive toxicity by affecting estrous cyclicity, follicle numbers, and hormone levels. However, little is known about the mechanisms underlying DEHP-induced early onset of reproductive aging. Thus, this study tested the hypothesis that dietary exposure to DEHP induces early reproductive aging by affecting inflammation, fibrosis, and the expression of telomere regulators and antioxidant enzymes. Adult CD-1 female mice were exposed to vehicle (corn oil) or DEHP (0.5, 1.5, or 1500ppm) via the chow for six months. Exposure to DEHP increased the expression of antioxidant enzymes and Casp3, increased expression of telomere-associated genes, and increased fibrosis levels in the ovary. In addition, DEHP exposure for 6months altered ovarian and systemic inflammatory status. Collectively, our novel data suggest that 6-month dietary exposure to DEHP may accelerate reproductive aging by affecting several reproductive aging markers in female mice.

Association of joint exposure to organophosphorus flame retardants and phthalate acid esters with gestational diabetes mellitus: a nested case-control study

BackgroundOrganic phosphate flame retardants (OPFRs) and phthalate acid esters (PAEs) are common endocrine-disrupting chemicals that cause metabolic disorders. This study aimed to assess the association between joint exposure to OPFRs and PAEs during early pregnancy in women with gestational diabetes mellitus (GDM).MethodsSeven OPFRs and five PAEs were detected in the urine of 65 GDM patients and 100 controls using gas chromatography-tandem triple quadrupole mass spectrometry (GC-MS). The association of OPFRs and PAEs with GDM was assessed using logistic regression, weighted quantile sum (WQS) regression, and Bayesian kernel machine regression (BKMR) models.ResultsLevels of dibutyl phthalate (DBP), di-2-ethylhexyl phthalate (DEHP), diethyl phthalate (DEP), dimethyl phthalate (DMP), tris (2-butoxyethyl) phosphate (TBEP), tributyl phosphate (TBP), tris (2-chloroethyl) phosphate (TCEP), tris (1,3-dichloro-2-propyl) phosphate (TDCPP), tri-ortho-cresyl phosphate (TOCP), and triphenyl phosphate (TPHP) increased in the GDM group, and the OPFRs and PAEs, except for BBP and TMCP, were associated with GDM in the logistic regression analysis. In the WQS model, the mixture of OPFRs and PAEs was significantly positively associated with GDM (OR = 3.29, 95%CI = 1.27–8.51, P = 0.014), with TDCPP having the highest WQS index weight. BKMR analysis reinforced these results, showing that the overall association of joint exposure to the OPFRs and PAEs with GDM increased at exposure levels of the 55th to 75th percentiles. Independent exposure to TDCPP (OR = 1.42, 95%CI = 1.09–1.86, P = 0.011) and TBEP (OR = 1.29, 95%CI = 1.04–1.60, P = 0.023) were associated with an increased risk of GDM.ConclusionsEnvironmental exposure to OPFRs and PAEs is significantly associated with GDM. These findings provide evidence for the adverse effects of exposure to OPFRs and PAEs on the health of pregnant women.

Rapid quantitative analysis of semi-volatile organic compounds in indoor surface film using direct analysis in real time mass spectrometry: a case study on phthalates

Abstract. Direct analysis in real time mass spectrometry (DART-MS) has recently emerged as a promising approach for measuring semi-volatile organic compounds (SVOCs) on indoor surface films. However, its broader application in indoor environments is limited by low measurement repeatability and no separation of isomers. Herein we developed a sampling suite of indoor surface films for DART-MS analysis, optimized settings of DART to obtain higher analytical performance, and demonstrated the possibility of separating isomeric compounds using tandem mass spectrometry (MS/MS). Two pairs of isomeric phthalate esters, including di(2-ethylhexyl) phthalate (DEHP) and di-n-octyl phthalate (DnOP) and diisobutyl phthalate (DiBP) and di-n-butyl phthalate (DnBP), were used as examples for method optimization and validation. Under optimized conditions, the instrument responses for all four compounds exhibited good linearity (r>0.992) and acceptable repeatability (intraday relative standard deviation (RSD) < 11.0 %). The limits of quantification for the four phthalate esters ranged from 0.042 to 0.24 ng cm−2. The uncertainty in the separation of isomeric components using MS/MS was < 11.4 %, which is acceptable for real sample analysis. To further assess the developed method, we analyzed 10 film samples collected side by side in an occupied office. DnOP was not detected. The RSD among samples was 6.1 % for DEHP, 4.6 % for DnBP, and 10.4 % for DiBP, indicating the overall good repeatability of the collection and measurement method developed. With improved performance, the developed method increases the feasibility of the DART-MS technique for monitoring the dynamics of chemical composition of indoor surface films.

Environmental endocrine disrupting chemical-DEHP exposure-provoked biotoxicity about microbiota-gut-mammary axis in lactating mice via multi-omics technologies

Plastics, pervasive in humans and nature, often contain Di (2-ethylhexyl) phthalate (DEHP) that enhance plastic’s elasticity. However, DEHP is an environmental endocrine disruptor, affecting organisms upon exposure. Understanding mammary gland development in lactating females is crucial for offspring nourishment and dairy production. Employing multi-omics technology, this study aimed to uncover DEHP’s impact on the microbial–gut–mammary axis. Forty mice were exposed to varying DEHP doses for 18 d. We performed 16S sequencing, metabolomics, mammary tissue observation, and gene expression profiling. Results revealed DEHP’s influence on microbial diversity, with increased Lactobacillus abundance and reduced Proteobacteria, alongside colonic inflammation. Elevated GMP and adenosine 5′-monophosphate levels in the bloodstream were noted, while ascorbic acid, glycitein, and others decreased. MEHP, a DEHP metabolite, damaged mammary tissues, inhibiting ERK1/2 phosphorylation, triggering apoptosis and ferroptosis. These findings unveil potential therapeutic targets for DEHP-induced chronic toxicity in humans and animals, aiding dairy livestock health and human well-being. This study underscores the importance of understanding the adverse effects of DEHP exposure on mammalian systems.

PiRNA array analysis provide insight into the mechanism of DEHP-induced testicular toxicology in pubertal male rats

Di-(2-ethylhexyl) phthalate (DEHP), a widely used plasticizer, could cause male reproductive toxicity by disrupting spermatogenesis. Piwi-interacting RNAs (piRNAs) are a small non-coding RNAs specifically highly expressed in the germline and interact with PIWI proteins to regulate spermatogenesis. Accumulating studies have confirmed that environmental poisons could induce male reproductive injury via altering piRNA expression. However, it remains unclear whether DEHP causes male reproductive dysfunction by perturbing piRNA levels. In this study, we conducted piRNA microarray expression analyses on testes of DEHP-exposed and control male rats and performed some in vitro and in vivo studies to explore the role of piRNA on DEHP-induced male reprouctive toxicity. Our results showed that DEHP exposure leaded to changed expression profile of piRNAs in pubertal male rat testes. And bioinformatics analyses revealed that down-regulated piR-rno-26751 probably targeted Insr mRNA expression regulation. Results from gene and protein expression tests demonstrated that DEHP caused decreased expression of INSR mainly in spermatogonia. Moreover, MEHP, the main metabolite of DEHP resulted in cell apoptosis and down-regulation of INSR and its downstream p-IRS1, p-PI3K, p-AKT and p-FOXO1 in GC-1spg cells. Conversely, overexpression of INSR restored cell apoptosis the down-regulation of the above proteins in GC-1spg cells. In conclusion, these findings suggest that DEHP-induced down-regulation of piR-rno-26751 targets the suppression of INSR, leading to apoptosis of spermatogonia in pubertal male rats.

Evaluation of pine needle oil recycled asphalt: Rheological characterization and molecular dynamics simulation

The regenerant based on bio-oil has the potential to lower carbon emissions, conserve natural resources, and shows promising prospects for application. In this paper, pine needle oil (PNO) and dioctyl phthalate (DOP) were utilized as base materials for asphalt rejuvenator. The primary aim of this research was investigate the impact of the rejuvenator on the rheological characteristics of asphalt and the compatibility between the rejuvenator and aged asphalt. The rheological properties of the asphalt were evaluated by the bending beam rheometer (BBR) and the dynamic shear rheometer (DSR). The glass transition temperature of the asphalt was tested by differential scanning calorimetry (DSC). Finally, the glass transition temperature (Tg), mean square displacement (MSD), cohesive energy density (CED), and solubility parameter (δ) were calculated and analyzed through molecular dynamics simulation to clarify the mechanism of PNO and DOP on asphalt. In the tested temperature range, the creep stiffness modulus of the PNO compound DOP recycled asphalt had decreased by 43.7∼68.4 % compared with the aged asphalt, and the rutting factor had decreased by 59.6∼75.3 %, but it was still better than the original asphalt. This showed that the regenerant had significantly restored the rheological properties of the asphalt. At 413.15 K, the difference of solubility parameters between PNO and DOP was the lowest, which was 1.37 and 0.17 (J/cm3)0.5, respectively. At this time, the compatibility between the PNO regenerant and aged asphalt was the best. The addition of the PNO softened the aged asphalt, reduced the rutting factor, adjusted the composition of the aged asphalt, improved its colloidal structure, and thereby enhanced its low temperature performance. The PNO regenerant also improved the diffusion of the four components of the aged asphalt and positively influenced the diffusion and fusion process of the system. DOP molecules lowered the asphalt's glass transition temperature, increased the amount of free space available for the thermal motion of asphalt molecules, and improved the compatibility of the asphalt with the regenerant.

Lycium barbarum polysaccharide alleviates ferroptosis in Sertoli cells through NRF2/SLC7A11/GPX4 pathway and ameliorates DEHP-induced male reproductive damage in mice

Di-(2-ethylhexyl)phthalate (DEHP) is a common plasticizer that has been shown to significantly negatively affect male reproductive health. On the other hand, Lycium barbarum polysaccharide (LBP) has been shown to improve reproductive function. Therefore, we hypothesized that LBP may ameliorate DEHP-induced male reproductive damage. Herein, we found that LBP could alleviate DEHP-induced testicular damage and sperm abnormalities. Furthermore, histomorphological analysis of mice testis revealed that LBP primarily ameliorated the DEHP-induced male reproductive damage by targeting Sertoli cells. Moreover, the detection of the function-related genes of Sertoli cells confirmed this finding. The serum of mice in the Control, DEHP alone, and DEHP+LBP groups was analyzed using non-targeted metabolomics to further elucidate the mechanism of action of LBP in improving DEHP-induced male reproductive damage. According to the results, the differential metabolites were mainly enriched in the glutamate metabolism pathway, implying that LBP may alleviate the ferroptosis-related DEHP-induced testicular injury. Related ferroptosis markers were also found in mice testis. These findings collectively suggest that LBP may ameliorate DEHP-induced testicular injury via alleviating ferroptosis in Sertoli cells. To clarify the specific mechanism, we constructed a cell model in vitro by treating TM4 cells (the Sertoli cell line) with LBP and MEHP (the in vivo DEHP metabolite). Our findings revealed that LBP can improve the function of DEHP-affected Sertoli cells. Furthermore, the analysis of lipid peroxidation, Fe2+ content, and ferroptosis-related protein expressions demonstrated that LBP could ameliorate MEHP-induced ferroptosis in TM4 cells. To clarify the specific mechanism, glutamate metabolism-related proteins involved in the ferroptosis pathway were detected. According to the results, there were significant changes in the expression of NRF2, SLC7A11 and GPX4 proteins, which are involved in the ferroptosis glutamate metabolism pathway. Furthermore, supplementation of NRF2, SLC7A11, and GPX4 inhibitors (ML385, Erastin, and RSL3, respectively) blocked the therapeutic effect of LBP in alleviating MEHP-induced ferroptosis in TM4 cells, implying that LBP could also ameliorate MEHP-induced ferroptosis via the NRF2/SLC7A11/GPX4 pathway. In summary, these findings show that LBP can alleviate DEHP/MEHP-induced ferroptosis through the NRF2/SLC7A11/GPX4 pathway, ameliorating Sertoli cell dysfunction and improving the DEHP-induced male reproductive damage. Therefore, the clinical administration of LBP could be an effective strategy for preventing DEHP-induced male reproductive injury.

A common phthalate replacement disrupts ovarian function in young adult mice.

Di-2-ethylhexyl terephthalate (DEHTP) is a replacement for its structural isomer di-2-ethylhexyl phthalate (DEHP), a known endocrine disrupting chemical and ovarian toxicant. DEHTP is used as a plasticizer in polyvinyl chloride products and its metabolites are increasingly found in biomonitoring studies at levels similar to phthalates. However, little is known about the effects of DEHTP on the ovary. In this research, we tested the hypothesis that DEHTP is an ovarian toxicant and likely endocrine disrupting chemical like its isomer DEHP. The impact of environmentally relevant exposure to DEHTP and/or its metabolite mono-2-ethylhexyl terephthalate (MEHTP) on the mouse ovary was investigated in vivo and in vitro. For the in vivo studies, young adult CD-1 mice were orally dosed with vehicle, 10µg/kg, 100µg/kg, or 100mg/kg of DEHTP for 10 days. For the in vitro studies, isolated untreated ovarian follicles were exposed to vehicle, 0.1, 1, 10, or 100µg/mL of DEHTP or MEHTP. Follicle counts, hormone levels, and gene expression of steroidogenic enzymes, cell cycle regulators, and apoptosis factors were analyzed. In vivo, DEHTP exposure altered follicle counts compared to control. DEHTP exposure also decreased expression of cell cycle regulators and apoptotic factors compared to control. In vitro, follicle growth was reduced compared to controls, and expression of the cell cycle regulator Cdkn2b was increased. Overall, these results suggest that DEHTP and MEHTP may be ovarian toxicants at low doses and should be subjected to further scrutiny for reproductive toxicity due to their similar structures to phthalates.

Phthalates are detected in the follicular fluid of adolescents and oocyte donors with associated changes in the cumulus cell transcriptome

ObjectiveTo investigate follicular fluid (FF) phthalate levels in adolescents undergoing fertility preservation compared to oocyte donors and explore its association with ovarian reserve and cumulus cell gene expression. DesignRetrospective study, and molecular analysis of cumulus cells (CCs) and FF SubjectsAdolescents (n= 20, 16.7 ± 0.6 years old) undergoing fertility preservation and oocyte donors (n= 24, 26.2 ± 0.4 years old). Main Outcome MeasuresPatient demographics, ovarian stimulation, and oocyte retrieval outcomes were analyzed for each group. FF levels of 9 phthalate metabolites were assessed individually and as molar sums representative of common compounds (all phthalates: ƩPhthalates; di(2-ethylhexyl) phthalate (DEHP) associated phthalate metabolites: ƩDEHP), exposure sources (plastics: ƩPlastic; personal care products: ƩPCP), and modes of action (anti-androgenic: ƩAA) and compared between the two groups. The transcriptome of CC associated with mature oocytes was compared between adolescents and oocyte donors using bulk RNA-sequencing. ResultsFollicular fluid ƩPlastic and ƩPCP levels were significantly higher in adolescents compared to oocyte donors (p<0.05). Follicular fluid ƩDEHP, ƩPlastic, ƩPCP, ƩAA, and ƩPhthalates levels were positively associated with antral follicle count (AFC) (p<0.05) in oocyte donors when adjusted for age, BMI, and race/ethnicity. RNA-seq analysis revealed 248 differentially expressed genes (DEGs) in cumulus cells of adolescents within the top quartile (n=4) of FF ƩPhthalates levels compared to the adolescents within the bottom half (n=9). Genes enriched in pathways involved in cell motility and development were significantly downregulated. ConclusionAdolescents undergoing fertility preservation cycles demonstrate higher levels of phthalate metabolites in their follicular fluid compared to oocyte donors. Phthalate metabolite levels in FF are associated with higher AFC levels in oocyte donors.