Maternal Di-(2-ethylhexyl) Phthalate Exposure Research Articles

Prenatal exposure to Di(2-ethylhexyl) phthalate and high-fat diet synergistically disrupts gonadal function in male mice†.

Prenatal exposure to Di (2-ethylhexyl) phthalate (DEHP) impairs the reproductive system and causes fertility defects in male offspring. Additionally, high-fat (HF) diet is a risk factor for reproductive disorders in males. In this study, we tested the hypothesis that prenatal exposure to a physiologically relevant dose of DEHP in conjunction with HF diet synergistically impacts reproductive function and fertility in male offspring. Female mice were fed a control or HF diet 7days prior to mating and until their litters were weaned on postnatal day 21. Pregnant dams were exposed to DEHP or vehicle from gestational day 10.5 until birth. The male offspring's gross phenotype, sperm quality, serum hormonal levels, testicular histopathology, and testicular gene expression pattern were analyzed. Male mice born to dams exposed to DEHP + HF had smaller testes, epididymides, and shorter anogenital distance compared with those exposed to HF or DEHP alone. DEHP + HF mice had lower sperm concentration and motility compared with DEHP mice. Moreover, DEHP + HF mice had more apoptotic germ cells, fewer Leydig cells, and lower serum testosterone levels than DEHP mice. Furthermore, testicular mRNA expression of Dnmt1 and Dnmt3a was two to eight-fold higher than in DEHP mice by qPCR, suggesting that maternal HF diet and prenatal DEHP exposure additively impact gonadal function by altering the degree of DNA methylation in the testis. These results suggest that the combined exposure to DEHP and high-fat synergistically impairs reproductive function in male offspring, greater than exposure to DEHP or HF dietalone.

Maternal di-(2-ethylhexyl) phthalate exposure elicits offspring IFN-λ upregulation: Insights from birth cohort, murine model, and in vitro mechanistic analysis.

Maternal exposure to di-(2-ethylhexyl)-phthalate (DEHP), an environmental endocrine disruptor, may lead to developmental immunotoxicity in offspring. The causal relationship and underlying mechanism require further study. A subset of Taiwan Maternal and Infant Cohort Study data (n=283) was analyzed and found a significant association between urinary DEHP metabolite levels from the third trimester of pregnancy and plasma levels of IL-28A and IL-29, named IFNλs, in cord blood. A trans-maternal murine model mimicking human DEHP exposure way showed that bone marrow-derived dendritic cells from maternal DEHP-exposed F1 offspring secreted higher IL-28A levels than control cells, indicating a potential causal relationship. Human bronchial epithelial cell lines treated with DEHP or its primary metabolite, mono-(2-ethyl-5-hexyl) phthalate (MEHP), expressed significantly higher levels of IFNλs mRNA or protein than controls. MEHP's effect on IFNλs expression was blocked by peroxisome proliferator-activated receptor α (PPARα) and PPARγ antagonists, and inhibited by a histone acetyltransferase inhibitor or a histone methyltransferase inhibitor. Chromatin immunoprecipitation assay showed that MEHP treatment promoted histone modifications at H3 and H4 proteins at the promoter regions of Il28a and Il29 genes. These results suggest maternal DEHP exposure could result in high IFNλ expression in offspring, and the health risk of early-life exposure requires further investigation.

Maternal Di-(2-Ethylhexyl) Phthalate (DEHP) Exposure Impairs Insulin Signal in the Liver and Gastrocnemius Muscles of Female Offspring Rats

Di-(2-Ethylhexyl) Phthalate (DEHP) is a potent endocrine disruptor that is commonly present in consumer products and cosmetics. Exposure to DEHP during gestational and lactational periods can adversely affect glucoregulation and lead to the onset of diabetes in progeny. The liver and gastrocnemius muscles play an important role in regulating glucose metabolism and insulin action. This study was designed to investigate the effect of maternal DEHP exposure on insulin signaling molecules in the liver and gastrocnemius muscles of adult female offspring rats. Rat dams were given DEHP (10 and 100 mg/kg b.wt./day) by oral gavage from gestation day 9 (GD 9) to the end of the lactation period Postnatal Day (PND) 21. On PND 80, female offspring rats in diestrus were euthanized and found reduced body weight, organ (liver and gastrocnemius) weight, and hyperglycemia in DEHP-exposed rats. Western blots revealed a dose-dependent reduction in the expression of Insulin Receptor - (IR), IRS, Akt, and GSK-3β proteins as well as their phosphorylated forms in the liver and gastrocnemius muscles of DEHP-exposed offspring rats. Maternal DEHP exposure reduced the levels of GLUT2 and GLUT4 level in the liver and gastrocnemius muscles, respectively. Liver and renal function markers were dose-dependently increased in the serum of offspring female rats born to DEHP exposed mother during gestation and lactation. Thus, the study revealed that maternal DEHP exposure impaired the expression of insulin signaling molecules in the two important tissues involved in glucose metabolism, the liver and gastrocnemius muscles, suggesting that phthalates exposure during development may contribute to the onset of diabetes in female offspring.

Di(2-ethylhexyl) phthalate mediates IL-33 production via aryl hydrocarbon receptor and is associated with childhood allergy development.

Few studies assess cord blood biomarkers to predict prenatal exposure to di(2-ethylhexyl) phthalate (DEHP) on the development of allergic diseases later in childhood. IL-33 has been indicated to play an important role in allergic diseases. We evaluated the association of prenatal DEHP exposure and IL-33 in cord blood on the development of allergic diseases. We also investigated the mechanism of DEHP in human lung epithelial cells and asthma animal models. 66 pregnant women were recruited, and their children followed when they were aged 3 years. Maternal urinary DEHP metabolites were determined using liquid chromatography-electrospray-ionization-tandem mass spectrometry. The effect of DEHP on IL-33 production was investigated in human lung epithelial cells and club cell-specific aryl hydrocarbon receptor (AhR) deficiency mice. ELISA and RT-PCR, respectively, measured the IL-33 cytokine concentration and mRNA expression. The concentrations of maternal urinary DEHP metabolites and serum IL-33 in cord blood with childhood allergy were significantly higher than those in the non-childhood allergy group. DEHP and MEHP could induce IL-33 production and reverse by AhR antagonist and flavonoids in vitro. Enhanced ovalbumin-induced IL-4 and IL-33 production in bronchoalveolar lavage fluid (BALF) by DEHP exposure and suppressed in club cell-specific AhR null mice. Kaempferol has significantly reversed the DEHP effect in the asthma animal model. Cord blood IL-33 level was correlated to childhood allergy and associated with maternal DEHP exposure. IL-33 might be a potential target to assess the development of DEHP-related childhood allergic disease. Flavonoids might be the natural antidotes for DEHP.

Colonic mechanism of serum NAD+ depletion induced by DEHP during pregnancy

Di (2-ethylhexyl) phthalate (DEHP) is a widely used plasticizer in polyvinyl chloride products such as feed piping, packing bag, and medical consumable. Our previous studies have demonstrated that DEHP exposure reduced the concentration of nicotinamide adenine dinucleotide (NAD+) in pregnant mice serum, which cuts off the source of NAD+ to placenta and results fetal growth restriction. However, the mechanism of serum NAD+ depletion by DEHP remains elusive. This study investigated the intestinal mechanism of NAD+ shortage-induced by DEHP in pregnant mice. The transcriptome results implicated that the mRNA level of oxidative response genes Cyp1a1, Gsto2, Trpv1 and Trpv3 were upregulated in colon. These changes induced intestinal inflammation. Transmission Electron Microscopy results displayed that DEHP destroyed the tight junctions and cell polarity of colonic epithelial cells. These dysfunctions diminished the expression of NAD+ precursor transporters SLC12A8, SLC5A8, SLC7A5, and the NAD+ biosynthetic key enzymes NAMPT, NMNAT1–3, and TDO2 in colonic epithelial cells. Analysis of the gut microbiota showed that DEHP led to the dysbiosis of gut microbiota, reducing the relative abundance of Prevotella copri which possesses the VB3 biosynthetic pathway. Therefore, maternal DEHP exposure during pregnancy decreased the transportation of NAD+ precursors from enteric cavity to colonic epithelial cells, and inhibited the synthesis of NAD+ in colonic epithelial cells. Meanwhile, DEHP reduced the NAD+ precursors provided by gut microbiota. Eventually, serum NAD+ content was lowered. Taken together, our findings provide a new insight for understanding the intestinal mechanisms by which DEHP affects serum NAD+ levels.

Maternal exposure to di(2-ethylhexyl) phthalate (DEHP) causes multigenerational adverse effects on the uterus of F1 and F2 offspring rats.

Phthalates are one of the ubiquitous chemicals found in day-to-day products like food packaging, children's toys, and other consumer commodities. There is rising concern that repeated exposure to phthalates during pregnancy and lactation could have long-term effects on maternal and fetal health. We hypothesize that exposure to DEHP during the developmental windows might affect the expression of molecules that regulate uterine function and that this effect would be passed on to further generations. Rat dams were treated with olive oil (vehicle) or DEHP (100 mg/kg b.wt./day) orally from gestational day 9 (GD 9) to the end of lactation (PND 21). F0 maternal DEHP exposure resulted in multigenerational (F1 and F2) reproductive toxicity, as evidenced by an extended estrous cycle, decreased mating, fertility, and fecundity indices. Serum progesterone and estradiol levels were decreased and their cognate receptors (PR and ERα) in the uterus were decreased in the DEHP-exposed offspring rats. Further analysis of the expression of estrogen and progesterone regulatory genes such as Hox a11, VEGF A, Ihh, LIFR, EP4, PTCH, NR2F2, BMP2, and Wnt4 were reduced in the uteri of adult F1 and F2 generation rats born from DEHP-exposed F0 dams. Decreased expression of these crucial proteins due to DEHP exposure may lead to defects in epithelial proliferation and secretion, uterine receptivity, and decidualization in the uteri of successive generations. This study showed that maternal DEHP exposure impairs the expression of molecules that regulate uterine function and this multigenerational effect is transmitted via maternal lineage.

Maternal rodent exposure to di-(2-ethylhexyl) phthalate decreases muscle mass in the offspring by increasing myostatin.

Di-(2-ethylhexyl) phthalate (DEHP) and its metabolites can cross the placenta and may cause birth defects and developmental disorders. However, whether maternal DEHP exposure affects skeletal muscle development in the offspring and the pathways involved are unknown. This study investigated the effects of maternal DEHP exposure and the contribution of myostatin (MSTN) to skeletal muscle development in the offspring. Pregnant wild-type and muscle-specific myostatin knockout (MSTN KO) C57BL/6 mice were randomized to receive vehicle (corn oil) or 250mg/kg DEHP by gavage every other day until their pups were weaned (postnatal day 21 [PND21]). Body weights of the offspring mice were measured longitudinally, and their hindleg muscles were harvested at PD21. Also, C2C12 cells were treated with mono-2-ethylhexyl phthalate (MEHP), the primary metabolite of DEHP, and proteolysis, protein synthesis, and myogenesis markers were measured. The contribution of myostatin to maternal DEHP exposure-induced muscle wasting in the offspring was determined. Maternal DEHP exposure reduced body weight growth, myofibre size, and muscle mass in the offspring compared to controls (Quad: 2.70±0.1 vs. 3.38±0.23, Gastroc: 2.29±0.09 vs. 2.81±0.14, Tibialis: 1.01±0.07 vs. 1.25±0.11, mg/tibial length in mm, all P<0.01, n=35). Maternal DEHP exposure significantly increased Myostatin expression (2.45±0.41 vs. 0.03±0.00 DEHP vs. controls, P<0.01, n=5), Atrogin-1(2.68±0.65 vs. 0.63±0.01, P<0.05, n=5), MuRF1 (1.56±0.51 vs. 0.31±0.01, P<0.05, n=5), and Smad2/3 phosphorylation (4.12±0.35 vs. 0.49±0.18, P<0.05), and decreased MyoD (0.27±0.01 vs. 1.52±0.01, P<0.05, n=5), Myogenin (0.25±0.03 vs. 1.95±0.56, P<0.05, n=5), and AKT phosphorylation (4.12±0.35 vs. 1.00±0.06, P<0.05, n=5), in skeletal muscle of the offspring in MSTNflox/flox , but not in MSTN KO mice. Maternal DEHP exposure resulted in up-regulation of CCAAT/enhancer-binding protein δ (C/EBPδ, 4.12±0.35 vs. 1.00±0.19, P<0.05, n=5) in skeletal muscle of the offspring in MSTNflox/flox and MSTN KO mice (4.12±0.35 vs. 4.35±0.28, P>0.05, n=5). In vitro, C/EBPδ silencing abrogated the MEHP-induced increases in Myostatin, MuRF-1, and Atrogin-1 and decreases in MyoD and Myogenin expression. Maternal DEHP exposure impairs skeletal muscle development in the offspring by enhancing the C/EBPδ-myostatin pathway in mice.

Maternal exposure to di-2-ethylhexyl phthalate (DEHP) depresses lactation capacity in mice.

Increasing evidence shows that di-2-ethylhexyl phthalate (DEHP), mostly commonly used phthalate for the production of flexible polyvinyl chloride (PVC), has the potential to induce serious health risks in humans. However, the understanding of DEHP-induced lactation performance remains largely unknown. We sought to investigate the adverse effects of DEHP on lactation and examine the underlying mechanism linking DEHP exposure with the lactation alterations. We successfully adapted a maternal DEHP exposure model in female pregnant/lactating mice. Then we determined effects of DEHP exposure on food intake, body weight and milk production as well as the alterations in endocrine factors in lactating mice. The integrated metabonomic and transcriptomic analyses of the mammary gland were performed to measure the changed metabolites and genes related to DEHP exposure-induced lactation alterations. We observed the reduced food intake with elevated blood leptin and the decreased milk yield as well as the reduced levels of serum prolactin, growth hormone, insulin-like growth factor 1 and insulin after exposed to DEHP. Furthermore, 208 metabolites and 3452 genes were separately identified as differentially expressed features associated with DEHP exposure. Integrated metabonomic and transcriptomic analyses demonstrated that DEHP caused lactation depression mainly through impairing energy generation, inducing stress responses along with the hypoactivation of inflammation, reducing the production of antioxidants, disrupting hormone homeostasis and repressing the synthesis of milk constituents (the lower glucose availability for lactose synthesis; the disruption of milk fat globule membrane for lipid droplet formation; the ribosomal dysfunction and disruption of post-modifications for milk protein synthesis). We demonstrated that DEHP disrupted several lactation-related hormone homeostasis and multiple processes like energy insufficiency, inflammation activation, oxidative stress aggravation and disturbance of milk production in the mammary gland of female lactating mice. Our results provide valuable information for the health risk of plastic additive (DEHP) on female lactation dysfunction.

Single-cell transcriptomic profiling to evaluate the effects of Di(2-ethylhexyl)phthalate exposure on early meiosis of female mouse germ cells

Di(2-ethylhexyl)phthalate (DEHP) has proven characteristics of an endocrine-disrupting compound (EDC), which can threaten the reproductive health of humans and other animals. In mammals, a series of chromosomal events occur during the meiotic stage of oocytes. External toxins may enter the body and cause infertility and other related diseases. Therefore, it is crucial to explore the influence of DEHP exposure on the molecular mechanism of germ cell meiosis. We used single-cell RNA sequencing (scRNA-seq) to analyse the ovaries of foetal mice at embryonic day 12.5 (E12.5) and E14.5 after maternal DEHP exposure. DEHP exposure further activated the pathways related to DNA repair in germ cells, increased the expression of genes related to DNA damage and changed the developmental trajectory of germ cells. DEHP exposure may affect the proliferation of pregranulosa (PG) cells. Moreover, DEHP exposure altered the signal transduction between PG cells and germ cells. We showed that DEHP affects meiosis by causing DNA damage in oocytes and disrupting the signal transduction between PG cells and germ cells. These results provide a strong theoretical basis for the prevention and treatment of DEHP-mediated female reproductive health problems.

Di (2-ethyl-hexyl) phthalate disrupts placental growth in a dual blocking mode

Di (2-ethyl-hexyl) phthalate (DEHP) is a widely used plasticizer. Maternal DEHP exposure inhibits cell proliferation and reduces placentas size, which associates with fetal growth restriction and adulthood diseases. However, the mechanism of placental cell proliferation inhibition by DEHP remains elusive. This study investigated the effect of DEHP on placental cell proliferation from cell cycle arrest. Utilizing in vitro and in vivo experiments, we investigated cell cycle arrest, DNA double-strand break (DSB) repair, genotoxic stress response, and micronuclei formation. Most DEHP metabolizes to mono (2-Ethylhexyl) phthalate (MEHP) and distributes to organs quickly, so MEHP and DEHP were used in cultured cell and animal experiments, respectively. Here, a double blocking mode for the proliferation inhibition of the placental cell was revealed. One is that the classical DSB repair pathways were suppressed, which arrested the cell cycle at the G2/M phase. The other is that DEHP stimulated an elevated level of progesterone, which blocked the cell cycle at metaphase by disrupting chromosome arrangement. These two sets of events facilitated micronuclei formation and resulted in cell proliferation inhibition. This findings provide a novel mechanistic understanding for DEHP to inhibit placental cell proliferation.

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.

Maternal Exposure to Di-(2-ethylhexyl) Phthalate Impairs Hippocampal Synaptic Plasticity in Male Offspring: Involvement of Damage to Dendritic Spine Development.

Exposure to di-(2-ethylhexyl) phthalate (DEHP), a widely used kind of plasticizer, can result in neurodevelopment impairments and learning and memory disorders. We studied the effects and possible mechanisms of maternal DEHP treatment on hippocampal synaptic plasticity in offspring. Pregnant Wistar rats were randomly divided into four groups and received 0, 30, 300, 750 (mg/kg)/d DEHP by gavage from gestational day (GD) 0 to postnatal day (PN) 21. Our data showed that DEHP exposure impaired hippocampal synaptic plasticity, damaged synaptic ultrastructure, and decreased synaptic protein levels in male pups. Furthermore, DEHP decreased the density of dendritic spines, affected F-actin polymerization, and downregulated the Rac1/PAK/LIMK1/cofilin signaling pathway in male offspring. However, the alterations in the hippocampi of female offspring were not observed. These results illustrate that maternal DEHP exposure could impair hippocampal synaptic plasticity by affecting synaptic structure and dendritic spine development in male offspring, which may be attributed to altered cytoskeleton construction induced by downregulation of the Rac1/PAK/LIMK1/cofilin signaling pathway.

Single-cell transcriptome dissection of the toxic impact of Di (2-ethylhexyl) phthalate on primordial follicle assembly.

Rationale: Accumulated evidence indicates that environmental plasticizers are a threat to human and animal fertility. Di (2-ethylhexyl) phthalate (DEHP), a plasticizer to which humans are exposed daily, can trigger reproductive toxicity by acting as an endocrine-disrupting chemical. In mammals, the female primordial follicle pool forms the lifetime available ovarian reserve, which does not undergo regeneration once it is established during the fetal and neonatal period. It is therefore critical to examine the toxicity of DEHP regarding the establishment of the ovarian reserve as it has not been well investigated.Methods: The ovarian cells of postnatal pups, following maternal DEHP exposure, were prepared for single cell-RNA sequencing, and the effects of DEHP on primordial follicle formation were revealed using gene differential expression analysis and single-cell developmental trajectory. In addition, further biochemical experiments, including immunohistochemical staining, apoptosis detection, and Western blotting, were performed to verify the dataset results.Results: Using single-cell RNA sequencing, we revealed the gene expression dynamics of female germ cells and granulosa cells following exposure to DEHP in mice. Regarding germ cells: DEHP impeded the progression of follicle assembly and interfered with their developmental status, while key genes such as Lhx8, Figla, and others, strongly evidenced the reduction. As for granulosa cells: DEHP likely inhibited their proliferative activity, and activated the regulation of cell death. Furthermore, the interaction between ovarian cells mediated by transforming growth factor-beta signaling, was disrupted by DEHP exposure, since the expression of GDF9, BMPR1A, and SMAD3 was affected. In addition, DNA damage and apoptosis were elevated in germ cells and/or somatic cells.Conclusion: These findings offer substantial novel insights into the reproductive toxicity of DEHP exposure during murine germ cell cyst breakdown and primordial follicle formation. These results may enhance the understanding of DEHP exposure on reproductive health.

Reversal of association between prenatal phthalate levels and birth size when measuring phthalates in placental tissue versus maternal urine

Di (2-Ethylhexyl) phthalate (DEHP) is a widely used plasticizer worldwide and is a potent endocrine disruptor. It is unclear which biospecimen from pregnancy best represents fetal exposure when estimating the relationship between maternal DEHP exposure and child health. Therefore, we compared the association between DEHP metabolites and birth size when biomarkers of exposure were measured in placental tissue versus maternal urine.This study included fifty mother-infant dyads from the CANDLE study. Samples were analyzed for DEHP metabolites (MEHP, MEOHP, MEHHP, MECPP), summarized as a molar sum (DEHP-oxo). Associations of placental and urinary DEHP metabolites were calculated, considering fetal sex, maternal race, and BMI as confounders and effect modifiers of maternal-placental transfer and metabolism. Fetal sex differences in DEHP pharmacokinetics are conceptualized as genetic/epigenetic in origin; whereas race and BMI could represent cultural, dietary, or other sources of variation in exposures and/or pharmacokinetics. In the second aim, multiple regression models were used to assess associations of DEHP oxidized metabolites and birth length and weight z scores, and head circumference (HC). DEHP metabolites in the two tissues were uncorrelated in univariate models. A negative effect was found between MEHP in placenta and MECPP in urine, interacted on race (β=-0.36 log units MEHP [95% CI -0.67, -0.05] in Black vs. White women per log MECPP). The association was positive for the effect of urinary DEHP-oxo on birth size; and negative for the effect of placental tissue DEHP-oxo. The highest precision was observed in the association between placental DEHP-oxo and HC (β= -0.94 cm per log DEHP-oxo; 95% CI=-2.03, 0.15). Maternal urine might provide different information than the placenta on prenatal exposure to phthalates. Future work will compare MEHP in these two tissue types, to better understand the pharmacokinetics and to clarify if these results indicate a measurement error or a biologic inference problem.

Exposure to DEHP or its metabolite MEHP promotes progesterone secretion and inhibits proliferation in mouse placenta or JEG-3 cells

Di (2-ethyl-hexyl)phthalate (DEHP) is an environmental endocrine disruptor and commonly used as plasticizer. Maternal DEHP exposure during pregnancy reduces placental size and destroys placental structure. However, the underlying mechanisms were unclear. In this study, we supposed that DEHP disturbs endocrine function of placenta to inhibit the proliferation of placental cell. Using radioimmunoassay and ELISA, we found that DEHP and its active metabolite mono (2-ethyl-hexyl) phthalate (MEHP) promoted progesterone secretion in pregnant mouse and in JEG-3 cells, respectively. Therefore, placental endocrine function was altered by DEHP. The mRNA and protein level of progesterone synthetase 3β-HSD1 was elevated by DEHP, which is conducive to the synthesis of progesterone. The level of progesterone receptor was down-regulated by DEHP and MEHP in mouse placenta and in JEG-3 cells, respectively. PR deficiency further promoted the level of 3β-HSD1, which leads to a higher progesterone level. In turn, higher concentration of progesterone further inhibited the expression of PGR (PR gene). Therefore, higher progesterone down-regulated the level of its receptor PR. Meanwhile, decreased PR induced more progesterone secretion. There is a feedback loop between progesterone and PR. PR deficiency down-regulated the protein level of Cyclin D1 which plays an important role in cell proliferation. Accordingly, DEHP and its active metabolite MEHP can restrain proliferation of placental cell and disturb the progesterone secretion via decreasing the level of PR.

A benchmark dose study of prenatal exposure to di(2-ethylhexyl) phthalate and behavioral problems in children

Prenatal exposure to di(2-ethylhexyl) phthalate (DEHP) has been reported to be associated with adverse effects on neurodevelopment that yield behavior syndromes in young children with an estimated median exposure lower than the currently recommended tolerable daily intake (TDI) and reference dose (RfD). Our aim was to derive the benchmark dose for prenatal exposure to DEHP for the neurodevelopmental health in children. A total of 122 mother-child pairs from the Taiwan Maternal and Infant Cohort Study were analyzed for the dose-response relationship between maternal exposure to DEHP and children's behavioral syndromes evaluated at 8 years (n = 122, 2009), 11 years (n = 96, 2012), and 14 years (n = 78, 2015) of age. We employed a multivariate regression model to assess the statistical associations between the estimated maternal average daily intake of DEHP and child's individual CBCL scores for boys and girls at each separate age, followed by a mixed model for all the children across three ages accounting for individual variations. We then employed structural equation models by combining the children's specific behavioral problem scores at different ages and obtained a simulated overall latent score in relation to maternal exposure. Based on the established dose-response relationship, we derived the benchmark dose (BMD) and the lower limit (BMDL). Associations of maternal DEHP exposure (median 4.54μg/kg_bw/day) with the Child Behavior Checklist (CBCL) scores were all significant, except for somatic complaints, adjusting for child's age, gender, IQ, and family income. The BMDL, given a benchmark response of 0.10 (0.05) and a background response of 0.05, was 6.01 (2.16) μg/kg_bw/dayfor an integrated CBCL score. The current TDI (RfD) of 50 (20) μg/kg_bw/day for DEHP might not protect pregnant women for their children from behavioral problems. There remains the lack of comparable toxicological data. Further investigations are needed.

Maternal di-(2-ethylhexyl) phthalate exposure alters hepatic insulin signal transduction and glucoregulatory events in rat F1 male offspring.

Di-(2-ethylhexyl) phthalate (DEHP) is a commonly used plasticizer with endocrine disrupting properties. Its widespread use resulted in constant human exposure including fetal development and postnatal life. Epidemiological and experimental data have shown that DEHP has a negative influence on glucose homeostasis. However, the evidence regarding the effect of maternal DEHP exposure on hepatic glucose homeostasis is scarce. Hence, we investigated whether DEHP exposure during gestation and lactation disrupts glucose homeostasis in the rat F1 male offspring at adulthood. Pregnant rats were divided into three groups and administered with DEHP (10 and 100mg/kg/day) or olive oil from gestational day 9 to postnatal day 21 (lactation period) through oral gavage. DEHP-exposed offspring exhibited hyperglycemia, impaired glucose and insulin tolerances along with hyperinsulinemia at postnatal day 80. DEHP exposure significantly reduced the levels of insulin signaling molecules such as insulin receptors, IRS1, Akt and its phosphorylated forms. GSK3β and FoxO1 proteins increased in DEHP-exposed groups whereas its phosphorylated forms decreased. Treated groups showed decreased glycogen synthase activity and glycogen concentration. Glucose-6-phosphatase and phosphoenolpyruvate carboxykinase mRNA level and enzyme activity increased in DEHP-treated groups. The interaction between FoxO1-glucose-6-phosphatase and FoxO1-phosphoenolpyruvate carboxykinase was also increased. This study suggests that DEHP exposure impairs insulin signal transduction and alters glucoregulatory events leading to the development of type 2 diabetes in F1 male offspring.

Maternal di-(2-ethylhexyl) phthalate exposure inhibits cerebellar granule precursor cell proliferation via down-regulating the Shh signaling pathway in male offspring

Di-(2-ethylhexyl) phthalate (DEHP) is an endocrine disrupting chemical (EDC) widely used as a plasticizer in many materials. Epidemiological investigations have shown that DEHP exposure during early development is related to cerebellar-related adverse neurodevelopmental outcomes. However, animal studies involving the effect of DEHP exposure on cerebellar development have rarely been reported and the potential mechanisms are unclear. The aim of this study was to investigate the effect of maternal DEHP exposure on the proliferation of cerebellar granule cell precursor cells (GCPs) and the mechanisms involved. Wistar rats were randomly assigned to four exposure groups and given 0, 30, 300, or 750 mg/kg/d DEHP by intragastric administration from gestational day (GD) 0 to postnatal day (PN) 21. Exposure to 300 and 750 mg/kg/d DEHP restrained GCPs proliferation and impaired neurodevelopment for males. Furthermore, exposure to 300 and 750 mg/kg/d DEHP decreased male pups protein expressions and mRNA levels of molecules related to proliferation, including Shh, Gli1, N-Myc, CyclinD1. In addition, the estrogen level and aromatase expression also reduced in male pups after maternal exposure to DEHP. However, effects on females were not obvious. These results suggested that 300 and 750 mg/kg/d DEHP exposure inhibit the proliferation of GCPs in male offspring and ultimately contribute to the impairment of neuromotor development. This, may be caused by the down-regulation of Shh signaling. And the susceptibility of male offspring to DEHP exposure may be attributed to the decreased estrogen level and aromatase expression in male pup's cerebellum.

Exposure to Di-(2-ethylhexyl) Phthalate During Perinatal Period Gender-Specifically Impairs the Dendritic Growth of Pyramidal Neurons in Rat Offspring.

Di-(2-ethylhexyl) phthalate (DEHP), as a prevalent xenoestrogen endocrine disrupter, is omnipresent in the environment and commonly used in polyethylene plastic products. Although DEHP has potential adverse effects on multisystem organs, damage to the central nervous system is more significant. However, the consequences and mechanisms of DEHP exposure remain to be explored. The aim of this study was to investigate the effects and related mechanisms of maternal DEHP exposure on dendritic development of hippocampal pyramidal neurons in a rat model. Pregnant Wistar rats were intragastrically administrated either vehicle or DEHP (30, 300, and 750 mg/kg/d) from gestation day 0 to postnatal day (PN) 21. The dendritic length and complexity of dendritic arbors’ pattern in pyramidal neurons of the hippocampus were measured using Golgi–Cox staining and Sholl analysis. The expression of dendritic development-related proteins was detected using western blot and immunofluorescence staining. DEHP-treated male but not female pups showed an obvious decrease in the total length and branching numbers of basal dendrites on PN7, PN14, and PN21. The phosphorylation of MAP2c, stathmin, and JNK1 in the male pup hippocampus was significantly decreased in DEHP treatment groups compared to controls. However, protein expression alteration in the hippocampus of female offspring was not observed. In summary, our study indicated that DEHP has a gender-specific negative impact on the dendritic growth of CA1 pyramidal neurons in male offspring of a rat model of DEHP exposure. The adverse impact may be related to the dysregulation of phosphorylated and total MAP2c and stathmin mediated by JNK1.

Transcriptomics and metabonomics analyses of maternal DEHP exposure on male offspring.

The objectives of this study were to evaluate the effect of maternal Di-2-ethylhexyl phthalate (DEHP) exposure on male offspring and to explore the mechanism of changes with the metabolic alterations and differential genes. Pregnant female Sprague-Dawley (SD) rats were intragastrically administered with 600mg/kg body weight of DEHP or corn oil (CON) throughout pregnancy and lactation. The growth of male offspring was investigated until 14weeks old, the indices of blood were detected, and mechanism was studied using metabonomics and transcriptomics. Compared with the CON group, body weight, body length, food intake, body fat weight, Lee's index, organ coefficient, blood lipids, and oral glucose tolerance test (OGTT) of male offspring were not significantly changed in maternal DEHP group. However, serum biochemical indexes such as alanine transaminase (ALT), total protein (TP), albumin (ALB), blood urea nitrogen (BUN), and creatinine (CREA) were markedly reduced in maternal DEHP group (p < 0.05). In addition, insulin level was elevated and catalase (CAT) level was decreased notably in maternal DEHP group compared with the CON group (p < 0.05). Furthermore, thyroxine (T4) level was lower and thyroid stimulating hormone (TSH) level was higher in maternal DEHP group (p < 0.05). Metabonomics revealed seven principal metabolites were identified, including increased L-allothreonine, creatine, uric acid, retinyl ester, L-palmitoylcarnitine, and decreased glycocholic acid and LysoPC (18:3). Transcriptomics displayed 35 differential genes were involved in the mechanism of maternal DEHP exposure. Therefore, this research confirms the effect of a certain dose of maternal DEHP exposure on male offspring and understands exactly the mechanism of these changes with metabonomics and transcriptomics.