Evaluation of precopulatory pairing behaviour and male fertility in a marine amphipod exposed to plastic additives.

Plastics contain a mixture of chemical additives that can leach into the environment and potentially cause harmful effects on reproduction and the endocrine system. Two of these chemicals, N-butyl benzenesulfonamide (NBBS) and triphenyl phosphate (TPHP), are among the top 30 organic chemicals detected in surface and groundwater and are currently placed on international watchlist for evaluation. Although bans have been placed on legacy pollutants such as diethylhexyl phthalate (DEHP) and dibutyl phthalate (DBP), their persistence remains a concern. This study aimed to examine the impact of plastic additives, including NBBS, TPHP, DBP, and DEHP, on the reproductive behaviour and male fertility of the marine amphipod Echinogammarus marinus. Twenty precopulatory pairs of E. marinus were exposed to varying concentrations of the four test chemicals to assess their pairing behaviour. A high-throughput methodology was developed and optimised to record the contact time and re-pair time within 15 min and additional point observations for 96 h. The study found that low levels of NBBS, TPHP, and DEHP prolonged the contact and re-pairing time of amphipods and the proportion of pairs reduced drastically with re-pairing success ranging from 75% to 100% in the control group and 0%-85% in the exposed groups at 96 h. Sperm count declined by 40% and 60% in the 50 µg/l and 500 µg/l DBP groups, respectively, whereas TPHP resulted in significantly lower sperms in 50 µg/l exposed group. Animals exposed to NBBS and DEHP showed high interindividual variability in all exposed groups. Overall, this study provides evidence that plastic additives can disrupt the reproductive mechanisms and sperm counts of amphipods at environmentally relevant concentrations. Our research also demonstrated the usefulness of the precopulatory pairing mechanism as a sensitive endpoint in ecotoxicity assessments to proactively mitigate population-level effects in the aquatic environment.

Responses of multifunctional immune complement component 1q (C1q) and apoptosis-related genes in Macrophthalmus japonicus tissues and human cells following exposure to environmental pollutants.

Apoptosis is a key defense process for multiple immune system functions, playing a central role in maintaining homeostasis and cell development. The purpose of this study was to evaluate the effects of environmental pollutant exposure on immune-related apoptotic pathways in crab tissues and human cells. To do this, we characterized the multifunctional immune complement component 1q (C1q) gene and analyzed C1q expression in Macrophthalmus japonicus crabs after exposure to di(2-ethylhexyl) phthalate (DEHP) or hexabromocyclododecanes (HBCDs). Moreover, the responses of apoptotic signal-related genes were observed in M. japonicus tissues and human cell lines (HEK293T and HCT116). C1q gene expression was downregulated in the gills and hepatopancreas of M. japonicus after exposure to DEHP or HBCD. Pollutant exposure also increased antioxidant enzyme activities and altered transcription of 15 apoptotic signaling genes in M. japonicus. However, patterns in apoptotic signaling in response to these pollutants differed in human cells. HBCD exposure generated an apoptotic signal (cleaved caspase-3) and inhibited cell growth in both cell lines, whereas DEHP exposure did not produce such a response. These results suggest that exposure to environmental pollutants induced different levels of immune-related apoptosis depending on the cell or tissue type and that this induction of apoptotic signaling may trigger an initiation of carcinogenesis in M. japonicus and in humans as consumers.

Involvement of the peroxisome proliferator-activated receptor gamma (Pparγ) and matrix metalloproteinases-2 and -9 (Mmp-2 and -9) in the mechanism of action of di(2-ethylhexyl)phthalate (DEHP) in cultured mouse brain astrocytes and neurons.

Di(2-ethylhexyl)phthalate (DEHP) is one of the most widely used phthalates in industry. It has been shown that, after entering the body, DEHP has the ability to cross the blood-placenta and blood-brain barriers. One of the proposed mechanisms of action of DEHP is the activation of peroxisome proliferator-activated receptors (PPARs). Many different functions of PPARγ in cells have been demonstrated, one of which is the modulation of the activation of matrix metalloproteinases (MMPs). The aim of this study was to investigate the role of Pparγ, Mmp-2, and Mmp-9 in the mechanism of action of DEHP. The experiments were performed on in vitro primary murine neurons and astrocytes. The results showed that DEHP has a pro-apototic effect on neurons, causing an increase in caspase-3 activity and in the number of apoptotic bodies. However, in astrocytes, the increase in caspase-3 activity was not related to the apoptosis process, as no increase in the formation of apoptotic bodies was observed. Moreover, DEHP increased the proliferation of astrocytes, which was confirmed by the increase in the amount and expression of the Ki-67 protein. In astrocytes, DEHP decreased the expression of the Pparγ and Mmp-9 proteins but increased the expression of the Mmp-2 protein. In DEHP neurons, it increased the expression of the Pparγ protein but decreased the expression of the Mmp-2 and Mmp-9 proteins.

Combined toxicity influence of polypropylene microplastics and di-2-ethylhexyl phthalate on physiological-biochemical characteristics of cucumber (Cucumis sativus L.).

Microplastics and di-2-ethylhexyl phthalate (DEHP) are prevalent and emerging pollutants in agro-ecosystem, raising concerns due to their widespread co-presence. Nevertheless, their combined toxicity on terrestrial plants remains largely unexplored. This study investigated the impact of polypropylene microplastics (MPs), DEHP, and their mixture on the physiological and biochemical characteristics of cucumber seedlings. The changes of membrane stability index (MSI), antioxidase activities, photosynthetic pigments and chlorophyll fluorescence in cucumber seedlings were assessed. The results demonstrated that MPs alone significantly inhibited MSI, photosynthetic pigments (Chl a, Chl b, and Chl a + b), Fm and qp of cucumber seedlings, and significantly promoted the carotene content and antioxidant enzyme activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX) in cucumber seedlings. While DEHP alone significantly inhibited MSI and photosynthetic pigments of cucumber seedlings, and significantly promoted antioxidant enzyme activities in cucumber seedlings. Moreover, the combined toxicity of MPs and DEHP was found to be less pronounced than that of the single action of MPs and DEHP. The interaction between DEHP and MPs may contribute to the reduced toxicity. Abbott's modeling revealed that the combined toxicity systems were all antagonistic (RI < 1). Two-factor analysis and principal component analysis further confirmed that the treatment of MPs alone contributed the most to the toxicological effects of the physiological properties of cucumbers. In summary, this study highlighted the importance of understanding the combined effects of MPs and DEHP on plant physiology, providing insights for the development of effective treatments for emerging pollutants in agricultural ecosystems.

Regulation of the cell cycle and P13K/AKT/mTOR signaling pathway by phthalates in normal human breast cells.

OBJECTIVE: To investigate the impact of phthalates, including Butyl benzyl phthalate (BBP), di(n-butyl) phthalate (DBP), and di(2-ethylhexyl) phthalate (DEHP), in breast carcinogenesis. MATERIALS AND METHODS: MCF-10A normal breast cells were treated with phthalates (100 nM) and 17ß-estradiol (E2, 10 nM), which were co-cultured with fibroblasts from normal mammary tissue adjacent to estrogen receptor positive primary breast cancers. Cell viability was determined using a 3-(4,5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Cell cycles were analyzed using flow cytometry. The proteins involving cell cycles and P13K/AKT/mTOR signaling pathway were then evaluated by Western blot analysis. RESULTS: MCF-10A co-cultured cells treated with E2, BBP, DBP, and DEHP exhibited a significant increase in cell viability using MTT assay. The expressions of P13K, p-AKT, and p-mTOR, as well as PDK1 expression, were significantly higher in MCF-10A cells treated with E2 and phthalates. E2, BBP, DBP, and DEHP significantly increased cell percentages in the S and G2/M phases. The significantly higher expression of cyclin D/CDK4, cyclin E/CDK2, cyclin A/CDK2, cyclin A/CDK1, and cyclin B/CDK1 in MCF-10A co-cultured cells were induced by E2 and these three phthalates. CONCLUSION: These results provide consistent data regarding the potential role of phthalates exposure in the stimulating proliferation of normal breast cells, enhancing cell viability, and driving P13K/AKT/mTOR signaling pathway and cell cycle progression. These findings strongly support the hypothesis that phthalates may play a crucial role in breast tumorigenesis.

Hydrolysis of dibutyl phthalate and di(2-ethylhexyl) phthalate in human liver, small intestine, kidney, and lung: An in vitro analysis using organ subcellular fractions and recombinant carboxylesterases.

Phthalates are widely used plasticizers that are primarily and rapidly metabolized to monoester phthalates in mammals. In the present study, the hydrolysis of dibutyl phthalate (DBP) and di(2-ethylhexyl) phthalate (DEHP) in the human liver, small intestine, kidney, and lung was examined by the catalytic, kinetic, and inhibition analyses using organ microsomal and cytosolic fractions and recombinant carboxylesterases (CESs). The Vmax (y-intercept) values based on the Eadie-Hofstee plots of DBP hydrolysis were liver > small intestine > kidney > lung in microsomes, and liver > small intestine > lung > kidney in cytosol, respectively. The CLint values (x-intercept) were small intestine > liver > kidney > lung in both microsomes and cytosol. The Vmax and CLint or CLmax values of DEHP hydrolysis were small intestine > liver > kidney > lung in both microsomes and cytosol. Bis(4-nitrophenyl) phosphate (BNPP) effectively inhibited the activities of DBP and DEHP hydrolysis in the microsomes and cytosol of liver, small intestine, kidney, and lung. Although physostigmine also potently inhibited DBP and DEHP hydrolysis activities in both the microsomes and cytosol of the small intestine and kidney, the inhibitory effects in the liver and lung were weak. In recombinant CESs, the Vmax values of DBP hydrolysis were CES1 (CES1b, CES1c) > CES2, whereas the CLmax values were CES2 > CES1 (CES1b, CES1c). On the other hand, the Vmax and CLmax values of DEHP hydrolysis were CES2 > CES1 (CES1b, CES1c). These results suggest an extensive organ-dependence of DBP and DEHP hydrolysis due to CES expression, and that CESs are responsible for the metabolic activation of phthalates.

Connexin-43 is a promising target for lycopene preventing phthalate-induced spermatogenic disorders.

INTRODUCTION: Male infertility is a multifactorial pathological condition and may be a harbinger of future health. Phthalates are ubiquitous environmental contaminants that have been implicated in the global decline in male fertility. Among them, di-(2-ethylhexyl) phthalate (DEHP) is the most prevalently used. Lycopene (LYC) is a possible preventive and therapeutic agent for male infertility owing to its antioxidant properties. The blood-testis barrier (BTB) is formed between Sertoli cells where it creates a unique microenvironment for spermatogenesis. OBJECTIVES: We hypothesize that phthalate caused male infertility and LYC plays an important role in phthalate-induced male fertility disorders. METHODS: Hematoxylin-eosin (H&E) staining, ultrastructure observation, and fluorescence microscopy were used to examine the morphological changes. RNA-Seq, and western blotting were conducted to detect gene and protein levels. Routine testing for sperm morphology and sperm-egg binding assay were conducted to examine the morphological structure and function of sperm. Cell scratch assay and transepithelial electrical resistance (TER) were used to detect cell migration capacity and barrier integrity. RESULTS: In vivo experiments, we showed that LYC prevented DEHP-induced impairment of BTB integrity, which provided a guarantee for the smooth progress of spermatogenesis. LYC improved DEHP-induced change in sperm parameters and fertilization ability. Subsequent in vitro experiments, LYC alleviated MEHP-induced disruption of intercellular junctions in mouse Spermatogonia cells (GC-1 cells) and mouse Sertoli cells (TM4 cells). In MEHP-induced BTB impairment models of Sertoli cells, treatment with LYC or overexpressing connexin-43 (Cx43) promoted cell migration capacity and normalized BTB integrity. Cx43 knockdown inhibited cell migration capacity and perturbed BTB reassembly in LYC preventing DEHP-induced BTB impairment. CONCLUSION: Our study provides evidence for the role of LYC in phthalates-induced spermatogenic disorders and points to Cx43 as a potential target for male fertility.

Quercetin inhibits testicular toxicity induced by the mixture of three commonly used phthalates in rats.

BACKGROUND: Phthalates (PEs), such as butyl benzyl phthalate, dibutyl phthalate and di(2-ethylhexyl) phthalate, are one of the most widely used plasticizers, and humans are increasingly exposed to them. Phytochemical quercetin (Que) is a typical flavonoid with several biological effects, such as antioxidative and anti-inflammatory. The present study was designed to explore the effect of Que on testicular toxicity caused by the mixture of three commonly used PEs (MPEs), and the underlying mechanism. Forty male Sprague-Dawley rats were randomly and equally divided into five groups (n = 8). Rats in control the group were orally treated with the excipient. Rats in the MPEs group were orally administered with 900 mg kg-1 day-1 MPEs, whereas rats in the MPEs+L-Que, MPEs+M-Que and MPEs+H-Que groups were simultaneously treated with 900 mg kg-1 day-1 MPEs and, respectively, 10, 30 and 90 mg kg-1 day-1 Que for 30 days. RESULTS: Compared with the control group, the testes weight, epididymides weight, serum testosterone, luteinizing hormone, follicle-stimulating hormone and estradiol levels, and anogenital distance in the MPEs group were significantly decreased (P < 0.05). The testicular tissues were injured with atrophy of seminiferous tubules, hyperplasia of Leydig cells and arrest of spermatogenesis in the MPEs group. Testicular steroidogenic proteins (StAR, P450scc, CYP17A1 and 17ß-HSD, P450arom) were up-regulated, whereas P-element-induced wimpy testis proteins (PIWIL1 and PIWIL2) were down-regulated in the MPEs group (P < 0.05). However, the alterations of these parameters were inhibited in the MPEs+M-Que and MPEs+H-Que groups. CONCLUSION: MPEs disturbed steroid hormone metabolism and caused testicular injuries. Que could inhibit testicular toxicity of MPEs, which might relate to the improved regulation of steroid hormone metabolism. © 2022 Society of Chemical Industry.

Influence on the adult male Leydig cell biomarker insulin-like peptide 3 of maternal exposure to estrogenic and anti-androgenic endocrine disrupting compound mixtures: A retrospective study.

Insulin-like peptide 3 (INSL3) is a peptide biomarker secreted specifically by the mature Leydig cells of the testes. It is constitutive, has low within-individual variance, and effectively measures the functional capacity of Leydig cells to make testosterone. In young adult men there is a large 10-fold range of serum INSL3 concentration, persisting into old age, and implying that later hypogonadal status might be programmed in early life. To determine whether maternal exposure to environmental endocrine disrupting compounds (EDCs) influences adult serum INSL3 concentration, using a retrospective paradigm, INSL3 was measured in young adult male rats (80-90 days) from the F1 generation of females maternally exposed to varied doses of bisphenol A (BPA), butylparaben, epoxiconazole, and fludioxonil as single compounds, as well as estrogenic and anti-androgenic mixtures of BPA and butylparaben, and di(2-ethylhexyl) phthalate and procymidone respectively. A mixture of BPA and butylparaben significantly reduced circulating INSL3 concentration in adult male progeny. The remaining compounds or mixtures tested, though sufficient to induce other effects in the F1 generation were without significant effect. Maternal exposure to low concentrations of some EDCs may be a contributing factor to the variation in the Leydig cell biomarker INSL3 in young adulthood, though caution is warranted translating results from rats to humans.

Effects of phthalate esters on soil microbial community under different planting patterns in Northern China: Case study of Hebei Province.

Soil microorganisms are biological factors involved in the farmland environment. The factors that shape soil microbial communities and how these are influenced by geographic location, planting pattern (open-field or greenhouse), and soil organic pollutants (phthalate esters, PAEs) remain poorly understood at large scales. Using 16 S rRNA gene and ITS sequencing, we characterized the soil microbiota in open-field and greenhouse soils in Hebei Province, China, and correlated their structure and composition to geographic location, planting pattern and PAEs. Compared with geographic location, planting pattern is more decisive for shaping soil microbes and has more significant effects on bacteria, and the effects are shaped by the number and types of core OTUs. PAEs participated in the shaping of soil microbial communities by altering the relative abundances of dominant microorganisms in the two planting patterns, and the effects of PAEs with high Kow were more significant. PAEs have a greater impact on bacteria than fungi in both planting patterns. Bacteria in the greenhouse soil were sensitive to the 9 kinds of PAEs detected, however in the open-field samples, mainly responded to PAEs with high Kow and rarely respond to PAEs with low Kow. DEHP and DBP, as two monomers with the highest concentration, have significant effects on dominant genera of microorganisms under both planting patterns, with inhibiting effect on bacteria and significantly promotion on fungi. Our study clarified the factors that have a substantial impact on soil microorganisms at the provincial scale and the mechanisms involved in shaping soil microbial community structure, as well as the significant impact of PAEs on soil microbial dominant microorganisms.

Icariin promotes mouse Leydig cell testosterone synthesis via the Esr1/Src/Akt/Creb/Sf-1 pathway.

Icariin (ICA), extracted from Epimedium, is a flavonoid used in traditional Chinese medicine. Di(2-ethylhexyl) phthalate (DEHP) is a phthalate used in commercial products as a plasticizer that can influence the human endocrine and reproduction system. We previously found that ICA reversed DEHP-induced damage through the prevention of reactive oxygen species accumulation and promotion of testosterone secretion. Here we investigated the mechanisms of ICA in promoting testosterone secretion from murine Leydig cells. We used ICA, DEHP, the Akt agonist SC-79, the Akt inhibitor MK2206, and the Creb inhibitor KG501 to determine the effect of these treatments on the expression levels of the steroidogenic enzymes, Cyp11a1 and Hsd3b, which play critical roles in androgen production, in Leydig cells. Bioinformatic analysis was used to search for ICA-targeted proteins and their associated pathways. We found that icariin interacted with estrogen receptor on the cell membrane, leading to increased phosphorylation levels of Akt and Creb proteins and enhanced transcription of genes encoding steroidogenic enzymes and testosterone synthesis. We further investigated ICA activity in vivo using male mice pretreated with 100 mg/kg ICA and then treated with 750 mg/kg DEHP. ICA pretreatment reversed the reduced protein expression levels of Cyp11a1 and Hsd3b induced by DEHP in Leydig cells in vivo. Furthermore, while the phosphorylation levels of Akt and Creb were decreased in testes of mice exposed to DEHP alone, these effects were reversed by ICA pretreatment. These findings indicate that ICA promotes testosterone synthesis via the Esr1/Src/Akt/Creb/Sf-1 signaling pathway.

DEHP mediates drug resistance by directly targeting AhR in human breast cancer.

Resistance to chemotherapy and hormonal therapy is a major clinical problem in breast cancer medicine, especially for cancer metastasis and recurrence. Di(2-ethylhexyl)phthalate (DEHP) affects drug resistance by an unknown mechanism of action. Here we analyzed breast cancer patients (N = 457) and found that Σ4MEHP (the sum of MEHP, MEHHP, MECPP and MEOHP concentrations) in urine was significantly higher (P = 0.018) in the recurrent breast cancer group compared with non-recurrent patients. Σ4MEHP-High was positively and significantly correlated with tumor stage (P = 0.005), lymph node status (P = 0.001), estrogen receptor status (P = 0.010), Her2/Neu status (P = 0.004), recurrence (P = 0.000) and tumor size (P = 0.002), as well as an independent prognostic marker (OR = 1.868; 95% CI = 1.424-2.451; P < 0.000) associated with poor survival rates based on a positive Her2/Neu status (P = 0.035). In addition, we found that DEHP inhibited paclitaxel and doxorubicin effects in breast cancer cell lines MCF-7 and MDA-MB-231 and in zebrafish and mouse tumor initiation models. DEHP induced trefoil factor 3 (TFF3) expression through the vinculin/aryl hydrocarbon receptor (AhR)/ERK signaling pathway and induced CYP2D6, CYP2C8 and CYP3A4 expression through the AhR genomic pathway to increase the epithelial-mesenchymal transition (EMT) and doxorubicin metabolism, respectably. DEHP mediated AhR-related alterations in estrogen receptor expression through the ubiquitination system, which decreased tamoxifen effects in AhR knockout mice. These findings suggest a novel therapeutic avenue by targeting AhR in drug-resistant and recurrent breast cancer.

Regulation of TRAF6 by MicroRNA-146a in Zebrafish Embryos after Exposure to Di(2-Ethylhexyl) Phthalate at Different Concentrations.

As an endocrine disruptor, di(2-ethylhexyl) phthalate (DEHP) is ubiquitous in multiple environmental media, causing long-term toxic effects on organisms. MicroRNAs are a class of noncoding RNAs with only 20-24 nucleotides in length, which regulate the expression of many protein-coding genes when organisms are exposed to environmental chemicals. MiR-146a, a differentially expressed miRNA after DEHP exposure, was screened by miRNA sequencing. As its target, TRAF6 was predicted and identified by double fluorescent protein assay and double fluorescent gene reporting assay. It shows the contrary expression pattern with miR-146a when mimics and inhibitors were transfected into ZF4 cells. MiR-146a and TRAF6 were downregulated and upregulated, respectively, in zebrafish embryos exposed to a low-dose concentration gradient of DEHP. These results deepen our understanding of the molecular mechanisms of DEHP toxicity and suggest that miR-146a can serve as a potential biomarker for DEHP exposure.

Epi-mutations for spermatogenic defects by maternal exposure to di(2-ethylhexyl) phthalate.

Exposure to environmental factors during fetal development may lead to epigenomic modifications in fetal germ cells, altering gene expression and promoting diseases in successive generations. In mouse, maternal exposure to di(2-ethylhexyl) phthalate (DEHP) is known to induce defects in spermatogenesis in successive generations, but the mechanism(s) of impaired spermatogenesis are unclear. Here, we showed that maternal DEHP exposure results in DNA hypermethylation of promoters of spermatogenesis-related genes in fetal testicular germ cells in F1 mice, and hypermethylation of Hist1h2ba, Sycp1, and Taf7l, which are crucial for spermatogenesis, persisted from fetal testicular cells to adult spermatogonia, resulting in the downregulation of expression of these genes. Forced methylation of these gene promoters silenced expression of these loci in a reporter assay. These results suggested that maternal DEHP exposure-induced hypermethylation of Hist1h2ba, Sycp1, and Taf7l results in downregulation of these genes in spermatogonia and subsequent defects in spermatogenesis, at least in the F1 generation.

Mono-(2-ethylhexyl) phthalate Promotes Dengue Virus Infection by Decreasing IL-23-Mediated Antiviral Responses.

Exposure to environmental hormones such as di(2-ethylhexyl) phthalate (DEHP) has become a critical human health issue globally. This study aimed to investigate the correlations between DEHP/mono-(2-ethylhexyl) phthalate (MEHP) levels and macrophage-associated immune responses and clinical manifestations in dengue virus (DV)-infected patients. Among 89 DV-infected patients, those with DV infection-related gastrointestinal (GI) bleeding (n = 13, 15% of patients) had significantly higher DEHP exposure than those without GI bleeding (n = 76, 85% of patients), which were 114.2 ng/ml versus 52.5 ng/ml ΣDEHP in urine; p = 0.023). In an in vitro study using cultured human monocyte-derived macrophages (MDMs) to investigate the effects of MEHP, treatment increased IL-1ß and TNF-α release but decreased IL-23 release, with negative correlations observed between urine ΣDEHP and serum IL-23 levels in patients. MEHP-treated MDMs had lower antiviral Th17 response induction activity in mixed T-cell response tests. The in vitro data showed that MEHP increased DV viral load and decreased IL-23 release dose-dependently, and adding IL-23 to MEHP-exposed MDMs significantly reduced the DV viral load. MEHP also suppressed IL-23 expression via the peroxisome proliferator-activated receptor-gamma (PPAR-γ) pathway. Further, the PPAR-γ antagonist GW9662 significantly reversed MEHP-induced IL-23 suppression and reduced the DV viral load. These study findings help to explain the associations between high MEHP levels and the high global burden of dengue disease.

Exposure to the plasticizer, Di-(2-ethylhexyl) phthalate during juvenile period exacerbates autism-like behavior in adult BTBR T + tf/J mice due to DNA hypomethylation and enhanced inflammation in brain and systemic immune cells.

Epigenetic modifications are known to play a crucial role in the behavioral modifications through regulation of gene expression. Environmental factors are known to regulate genetic transcription through DNA methylation which is one of the mechanisms of epigenetic modification. Di-2-ethylhexyl phthalate (DEHP) is one of the most abundant phthalate plasticizers in day-to-day products. Prenatal/postnatal DEHP administration has been reported to cause inflammation as well as behavioral dysregulation, however it is not known if exposure to DEHP during juvenile stage affects peripheral/neuronal inflammation and autism-like symptoms in BTBR mice at adulthood. This study investigated effect of DEHP exposure during juvenile period on DNA methylation (global DNA methylation/DNMT1 expression) and inflammation (IL-17A, IL-6, MCP-1, TNF-α) in CD4 + T cells/CD11c + DCs and cortex, and autism-like symptoms (three-chambered sociability test, self-grooming and marble burying test) in asocial BTBR and social C57 mice at adulthood. Our data reveal that BTBR mice exposed to DEHP during juvenile period have hypomethylated DNA/DNMT1 expression in CD11c + DCs and cortex as compared to vehicle-exposed BTBR mice. It was associated with upregulated inflammation in periphery [plasma IL-6/IL-17A, CD11c + DCs (IL-6/MCP-1/TNF-α), and CD4+ T cells (IL-17A)] and cortex (IL-6, MCP-1, TNF-α), and aggravation in autism-like symptoms in DEHP-treated BTBR mice. These data propose that exposure of DEHP during juvenile period may affect autism-like behavior and inflammation in BTBR mice at adulthood through epigenetic regulation. Therefore, underlying genetic predisposition may play a crucial role in worsening of autistic symptoms in ASD subjects in adulthood if they are exposed to environmental pollutants such as DEHP during juvenile period.

Phthalate inhibits Leydig cell differentiation and promotes adipocyte differentiation.

Studies have shown that phthalates are capable of affecting the development and functions of male reproductive system. The effect of phthalates on Leydig cell functions is well documented. However, little is known about their potential effects on the functions of stem Leydig cells (SLC). In the present study, we have examined the effects of mono-(2-ethylhexyl) phthalate (MEHP) on SLC functions in vitro by culturing seminiferous tubules and isolated SLCs. The results indicate that MEHP can significantly inhibit the proliferation and differentiation of SLCs in both the organ and cell culture systems. Interestingly, the minimal effective concentration that is able to affect SLC function was lower in the tubule culture system (1 µM) than in the isolated cells (10 µM), suggesting a possible involvement of the niche cells. Also, MEHP appeared to affect both the efficiency of SLCs to form Leydig cells and a selected group of Leydig cell-specific genes, including Lhcgr, Scarb1, Hsd3b1, Cyp17a1, Star, Srd5a1, Akr1c14, Insl3, Hao2 and Pah. Since SLCs are multipotent, we also tested the effect of MEHP on the differentiation of SLCs to adipocytes. Though MEHP by itself can not specify SLCs into adipocyte lineage, it indeed significantly increased the adipogenic activity of SLCs if used with an adipocyte inducing medium by up-regulation of multiple adipogenic-related genes, including Pparg and Cebpa. Overall, the results indicate that MEHP inhibits SLCs differentiating into Leydig lineage while stimulates the differentiating potential of SLCs to adipocytes.

Effects of DEHP on the ecdysteroid pathway, sexual behavior and offspring of the moth Spodoptera littoralis.

Bis(2-ethylhexyl) phthalate (DEHP) is a widely produced plasticizer that is considered to act as an endocrine-disrupting chemical in vertebrates and invertebrates. Indeed, many studies have shown that DEHP alters hormonal levels, reproduction and behavior in vertebrates. Few studies have focused on the effects of DEHP on insects, although DEHP is found almost everywhere in their natural habitats, particularly in soils and plants. Here, we investigated the effects of DEHP on the sexual behavior and physiology of a pest insect, the noctuid moth Spodoptera littoralis. In this nocturnal species, olfaction is crucial for sexual behavior, and ecdysteroids at the antennal level have been shown to modulate sex pheromone detection by males. In the present study, larvae were fed food containing different DEHP concentrations, and DEHP concentrations were then measured in the adults (males and females). Hemolymphatic ecdysteroid concentrations, the antennal expression of genes involved in the ecdysteroid pathway (nuclear receptors EcR, USP, E75, and E78 and calmodulin) and sexual behavior were then investigated in adult males. The success and latency of mating as well as the hatching success were also studied in pairs consisting of one DEHP male and one uncontaminated female or one DEHP female and one uncontaminated male. We also studied the offspring produced from pairs involving contaminated females to test the transgenerational effect of DEHP. Our results showed the general downregulation of nuclear receptors and calmodulin gene expression associated with the higher concentrations of DEHP, suggesting peripheral olfactory disruption. We found some effects on male behavior but without an alteration of the mating rate. Effects on offspring mortality and developmental rates in the N + 1 generation were also found at the higher doses of DEHP. Taken together, the results of the study show for the first time that larval exposure to DEHP can induce delayed endocrine-disruptive effects in the adults of a terrestrial insect as well as effects on the next generation. To date, our study is also the first description of an impact of endocrine disrupter on olfaction in insects.

Application of a combined aggregate exposure pathway and adverse outcome pathway (AEP-AOP) approach to inform a cumulative risk assessment: A case study with phthalates.

Advancements in measurement and modeling capabilities are providing unprecedented access to estimates of chemical exposure and bioactivity. With this influx of new data, there is a need for frameworks that help organize and disseminate information on chemical hazard and exposure in a manner that is accessible and transparent. A case study approach was used to demonstrate integration of the Adverse Outcome Pathway (AOP) and Aggregate Exposure Pathway (AEP) frameworks to support cumulative risk assessment of co-exposure to two phthalate esters that are ubiquitous in the environment and that are associated with disruption of male sexual development in the rat: di(2-ethylhexyl) phthalate (DEHP) and di-n-butyl phthalate (DnBP). A putative AOP was developed to guide selection of an in vitro assay for derivation of bioactivity values for DEHP and DnBP and their metabolites. AEPs for DEHP and DnBP were used to extract key exposure data as inputs for a physiologically based pharmacokinetic (PBPK) model to predict internal metabolite concentrations. These metabolite concentrations were then combined using in vitro-based relative potency factors for comparison with an internal dose metric, resulting in an estimated margin of safety of ~13,000. This case study provides an adaptable workflow for integrating exposure and toxicity data by coupling AEP and AOP frameworks and using in vitro and in silico methodologies for cumulative risk assessment.

Differential Expression of DUB Genes in Ovarian Cells Treated with Di-2-Ethylhexyl Phthalate.

Premature ovarian failure (POF) is defined as loss of ovarian function in women less than 40 years of age. The causes of POF are diverse and include environmental factors. Di-2-ethylhexyl phthalate (DEHP) is one factor that may cause POF. The ubiquitin-proteasome system maintains intracellular balance by promoting or inhibiting protein degradation. To investigate the differential expressions of deubiquitinating enzyme (DUB) genes in patients with POF, we developed two in vitro POF models by treating A2780 or OVCAR5 with DEHP. Using these models, a multiplex RT-PCR system for DUB genes was applied to identify biomarkers by comparing expression patterns and DUB mRNA levels; multiplex RT-PCR results were validated by qRT-PCR and Western blotting analyses. Observed differential expression levels of several DUB genes including USP12, COPS5, ATXN3L, USP49, and USP34 in A2780 and OVCAR5 cells at the mRNA and protein levels suggest that they should be investigated as potential biomarkers of POF.