High-dose Di(2-ethylhexyl)phthalate Research Articles

Subacute exposure to DEHP leads to impaired decidual reaction and exacerbates the risk of early miscarriage in mice.

To investigate the effect of subacute exposure of Di (2-ethylhexyl) phthalate (DEHP) on endometrial decidualization and early pregnancy miscarriage in mice. CD1 mice were orally administrated with 300 (low-dose group), 1000 (medium-dose group), or 3000 mg·kg－1·d－1 DEHP (1/10 LD50, high-dose group) for 28 days, respectively. An early natural pregnancy model and an artificially induced decidualization model were established. The uterine tissues were collected on D7 of natural pregnancy and D8 of artificially induced decidualization, respectively. The effects of a subacute exposure to DEHP on the decidualization of mice were detected by HE staining, Masson staining, TUNEL assay, and Western blotting. A model of spontaneous abortion was constructed in mice after subacute exposure to 300 mg·kg－1·d－1 DEHP, and the effect of impaired decidualization on pregnancy was investigated by observing the pregnancy outcome on the 10th day of gestation. Compared with the control group, the conception rate was significantly decreased in the high-dose DEHP subacute exposure group (P<0.05). HE staining showed that, compared with the control group, the decidual stromal cells in the low- and medium-dose exposure groups were disorganized, the nuclei of the cells were irregular, the cytoplasmic staining was uneven, and the number of polymorphonuclear cells was significantly reduced. Masson staining showed that compared with the control group, the collagen fibers in the decidua region of the DEHP low-dose group and the medium-dose group were more distributed, more abundant and more disorderly. TUNEL assay showed increased apoptosis in the decidua area compared to the control group. Western blotting showed that the expression of BMP2, a marker molecule for endometrial decidualization, was significantly reduced (P<0.05 or P<0.01). The abortion rate and embryo resorption rate were increased, and the number of embryos, uterine wet weight, uterine area and placenta wet weight were decreased in DEHP low-dose group compared to the control group stimulated by mifepristone, an abortifacient drug (P<0.05 or P<0.01). Subacute exposure to DEHP leads to impaired endometrial decidualization during early pregnancy and exacerbates the risk of adverse pregnancy outcomes in mice.

Influence of Ascorbic Acid on Di-(2-Ethylhexyl) Phthalate-induced Ovarian Gene Alterations in Pubertal Female Wistar Rats

Background Di-(2-ethylhexyl) phthalate (DEHP), a plasticizer compound affecting female reproduction, leads to scenarios, such as polycystic ovarian syndrome (PCOS) and infertility through oxidative stress (OS) mechanisms. Ascorbic acid (AA) is one of the antioxidants in infertility issues. Objectives The present study investigates the ameliorative effect of AA on DEHP-induced ovarian toxicity in pubertal female Wistar rats. Materials and Methods Thirty female Wistar rats of four weeks of age were stratified into five groups. Group I was treated with corn oil (Vehicle), groups II and III with low and high dose DEHP, and groups IV and V with low and high dose DEHP+AA were administered for 30 days. Results Increased body weight gain was noted in DEHP groups. Estradiol hormone was considerably reduced, whereas progesterone levels were increased in both low- and high-dose DEHP-treated groups. DEHP+AA groups have shown significant ( p &lt; 0.005) protection of these hormone levels as equal to the control group. The high-dose DEHP group shows an increased, ovarian estrogen receptor (ER) alpha, ER-beta, and progesterone receptor gene expression, and DEHP+AA groups have significantly ( p &lt; 0.005) showed expression similar to the control. OS was noted with decreased superoxide dismutase and increased malondialdehyde expression in Group III (GR III) compared to control, whereas the DEHP+AA treated group significantly protected OS by restoring the expression levels. DEHP-treated groups show elevated levels of both Bcl-2 and BAX which is specific to apoptotic expression and restored by AA treatment ( p &lt; 0.005). Conclusion Evidence suggests that AA may protect against DEHP-induced ovarian toxicity by decreasing OS levels, improving folliculogenesis, and restoring the hormonal with receptor level alterations.

Subchronic exposure to di-(2-ethylhexyl) phthalate (DEHP) elicits blood–brain barrier dysfunction and neuroinflammation in male C57BL/6J mice

BackgroundExposure to di-(2-ethylhexyl) phthalate (DEHP) can cause neurotoxicity but the mechanism is not clear. Blood brain barrier (BBB) is one of the most important tissues to protect the brain. However, whether DEHP can disrupt the BBB or not remains unclear. The objective of this study is to investigate the potential effects of subchronic DEHP exposure on BBB integrity and discuss the role of BBB in DEHP inducible neurotoxicity with an emphasis on neuroinflammatory responses. Male adult C57BL/6J mice were orally administered with vehicle or 200 or 750 mg/kg/day DEHP for 90 days. Subchronic exposure to high-dose DEHP increased water intake but decreased body weight and brain weight. The concentrations of DEHP metabolites increased in serum from all DEHP-exposed groups while increased in brain only from the high-dose group. DEHP induced neurobehavioural alterations and damaged hippocampal neurons. DEHP increased BBB permeability by Evans blue (EB) extravasation and decreased tight junction proteins (ZO-1, occludin, and claudin-5) while presenting a neuroinflammatory feature characterized by the upregulated inflammatory mediators TNF-α and the NLRP3/caspase-1/IL-1β inflammasome pathway. Our data provide new insights into neurotoxicity caused by subchronic DEHP exposure, which is probably involved in BBB dysfunction and neuroinflammatory responses.

Preconception and developmental DEHP exposure alter liver metabolism in a sex-dependent manner in adult mouse offspring

Environmental exposure to endocrine disrupting chemicals (EDCs) during critical periods of development is associated with an increased risk of metabolic diseases, including hepatic steatosis and obesity. Di-2-ethylhexyl-phthalate (DEHP) is an EDC strongly associated with these metabolic abnormalities. DEHP developmental windows of susceptibility are unknown yet have important public health implications. The purpose of this study was to identify these windows of susceptibility and determine whether developmental DEHP exposure alters hepatic metabolism later in life. Dams were exposed to control or feed containing human exposure relevant doses of DEHP (50 μg/kg BW/d) and high dose DEHP (10 mg/kg BW/d) from preconception until weaning or only exposed to DEHP during preconception. Post-weaning, all offspring were fed a control diet throughout adulthood. Using the Metabolon Untargeted Metabolomics platform, we identified 148 significant metabolites in female adult livers that were altered by preconception-gestation-lactation DEHP exposure. We found a significant increase in the levels of acylcarnitines, diacylglycerols, sphingolipids, glutathione, purines, and pyrimidines in DEHP-exposed female livers compared to controls. These changes in fatty acid oxidation and oxidative stress-related metabolites were correlated with hepatic changes including microvesicular steatosis, hepatocyte swelling, inflammation. In contrast to females, we observed fewer metabolic alterations in male offspring, which were uniquely found in preconception-only low dose DEHP exposure group. Although we found that preconception-gestational-lactation exposure causes the most liver pathology, we surprisingly found preconception exposure linked to an abnormal liver metabolome. We also found that two doses exhibited non-monotonic DEHP-induced changes in the liver. Collectively, these findings suggest that metabolic changes in the adult liver of offspring exposed periconceptionally to DHEP depends on the timing of exposure, dose, and sex.

Global gene expression analysis reveals a subtle effect of DEHP in human granulosa cell line HGrC1

Di(2-ethylhexyl) phthalate (DEHP) is an endocrine disruptor that exerts anti-steroidogenic effects in human granulosa cells; however, the extent of this effect depends on the concentration of DEHP and granulosa cell models used for exposure. The objective of this study was to identify the effects of low- and high-dose DEHP exposure in human granulosa cells. We exposed human granulosa cell line HGrC1 to 3 nM and 25 μM DEHP for 48 h. The whole genome transcriptome was analyzed using the DNBSEQ sequencing platform and bioinformatics tools. The results revealed that 3 nM DEHP did not affect global gene expression, whereas 25 µM DEHP affected the expression of only nine genes in HGrC1 cells: ABCA1, SREBF1, MYLIP, TUBB3, CENPT, NUPR1, ASS1, PCK2, and CTSD. We confirmed the downregulation of ABCA1 mRNA and SREBP-1 protein (encoded by the SREBF1 gene), both involved in cholesterol homeostasis. Despite these changes, progesterone production remained unaffected in low- and high-dose DEHP-exposed HGrC1 cells. The high concentration of DEHP decreased the levels of ABC1A mRNA and SREBP-1 protein and prevented the upregulation of STAR, a protein involved in progesterone synthesis, in forskolin-stimulated HGrC1 cells; however, the observed changes were not sufficient to alter progesterone production in forskolin-stimulated HGrC1 cells. Overall, this study suggests that acute exposure to low concentration of DEHP does not compromise the function of HGrC1 cells, whereas high concentration causes only subtle effects. The identified nine novel targets of high-dose DEHP require further investigation to determine their role and importance in DEHP-exposed human granulosa cells.

M6A contributes to a pro-survival state in GC-2 cells by facilitating DNA damage repair: Novel perspectives on the mechanism underlying DEHP genotoxicity in male germ cells

Di(2-ethyl-hexyl) phthalate (DEHP), an environmental endocrine disruptor, can destroy the sperm genomic integrity and impairs spermatogenesis. N6-methyladenosine (m6A) is involved in the cellular effects of DEHP. However, the genotoxic effect of DEHP on spermatocytes and the possible role of m6A in this process remain unclear. This study demonstrated that m6A alleviates DEHP genotoxicity in GC-2 cells. In DEHP-treated mice, DNA double-strand breaks (DSBs) were induced in the testis and spermatocytes. To further explore the molecular mechanism of DEHP genotoxicity on spermatocytes, GC-2 cells were exposed to DEHP. DEHP produced distinct genotoxicity and caused DSBs, which led to the inhibition of DNA synthesis and cell cycle arrest. The DNA damage response (DDR) was initiated to repair the DSBs induced by environmentally relevant levels of DEHP (100 μM and 200 μM). During this process, METTL3 upregulated m6A, which facilitated the DDR via stabilizing the DNA damage repair factors (Rad51 and Xrcc5) mRNA to maintain the pro-survival state. Moreover, Mettl3 knockdown partially inhibited DDR. Interestingly, high-dose DEHP (400 μM and 600 μM) directly induced apoptosis rather than the pro-survival state. Altogether: METTL3-mediated m6A participates in maintaining the pro-survival state by upregulating DDR, providing guidance for mitigating the genotoxicity of environment-related level DEHP exposure.

RF22 | PMON04 Sex- and Dose-Dependent Effects of In Utero Endocrine Disruptor Exposure on Offspring Behavior and Brain Monoamines

Abstract Exposure to endocrine disruptors (EDs), including the well-known plasticizers bisphenol A (BPA) and di(2-Ethylhexyl) phthalate (DEHP), during the highly sensitive gestational period, can produce long-lasting behavioral changes in offspring. In this study, we hypothesized that gestational exposure to BPA and/or DEHP at various doses would alter stress-related behaviors and monoaminergic activity in a region-specific manner in exposed offspring. To test this, female Sprague-Dawley dams were orally administered saline (control group - 10 µL/kg BW), BPA (5 µg/kg body weight (BW)), DEHP (5 µg/kg BW or 7.5 mg/kg BW), a combination of BPA and low-dose (LD) DEHP (5 µg BPA + 5 µg DEHP/kg BW), or a combination of BPA and high-dose (HD) DEHP (5 µg BPA + 7.5 mg DEHP/kg BW) during days 6 through 21 of pregnancy. The male and female offspring of these dams underwent a battery of behavioral tests in adulthood. Brains collected from the offspring were microdissected to obtain the medial prefrontal cortex (mPFC), paraventricular hypothalamic nucleus (PVN), and hippocampus (HC), which were analyzed for monoamine and metabolite levels using High-Performance Liquid Chromatography (HPLC). While BPA and B+D (LD) female offspring demonstrated anxiolytic effects in the Open Field Test, DEHP (HD) male offspring showed feminized anxiety-like behavior in the Elevated Plus Maze. The offspring also displayed impaired fear responses in the Shock Probe Defensive Burying (SPDB) test. Males in most ED groups buried less than their control counterparts. Furthermore, low-dose ED treatments led to a robust preference for passive coping strategies in both male and female offspring. Within the brain, male offspring in the BPA, LD and HD DEHP, and B+D (HD) groups had diminished dopamine (DA) levels in the PVN, which corresponded with their reduced SPDB burying levels. Moreover, DEHP (LD) male offspring had elevated DA turnover in the mPFC, whereas B+D (LD) female offspring had enhanced norepinephrine levels in the HC, which may underlie their behavioral outcomes in the SDPB. The findings of this study have implications for sex differences in mood and anxiety disorders following prenatal exposures to BPA, DEHP, and their mixtures. Presentation: Sunday, June 12, 2022 12:36 p.m. - 12:41 p.m., Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Combined effect of co-exposure to di (2-ethylhexyl) phthalates and 50-Hz magnetic-fields on promoting human amniotic cells proliferation

Di (2-ethylhexyl) phthalate (DEHP) and extremely low-frequency electromagnetic fields (ELF-EMFs) exist far and wide in our surroundings. Studies have reported that both of DEHP and ELF-EMFs could promote cell proliferation which is related with adverse bioeffects. In this study, we investigated whether there is the combined effect between DEHP and 50-Hz magnetic fields (MFs) on cell proliferation in human amniotic (FL) cells. Results revealed that the low-concentration DEHP (1 μM) could promote FL cell proliferation, whereas the high-dose DEHP (100 μM) inhibited cell proliferation. When FL cells were treated jointly by a 50-Hz, 0.2-mT MF and 0.1 μM DEHP, the proliferation rate of cells was significantly higher than that of single factor exposure. Additionally, co-exposure to under-threshold MF and DEHP could cooperatively activate protein kinase B (Akt), sphingosine kinase 1 (SphK1) and extracellular signal regulated kinase (ERK) in a cascade manner, and finally mediate cell proliferation. Taken together, the findings of this study indicated that the co-exposure to under-threshold MF and DEHP could jointly promote cell proliferation through activating proliferation-related signal pathway, which warned us that it should be cautious about assessing the underlying health hazards of co-exposure to MFs and DEHP at under-threshold levels.

Effects of Di(2-ethylhexyl)phthalate on Bone Metabolism in Ovariectomized Mice.

BackgroundThe molecular pathways of how endocrine disruptors affect bone mineral density (BMD) and bone remodeling are still unclear. The purpose of this experimental study is to determine the effects of di(2-ethylhexyl)phthalate (DEHP) on bone metabolism in ovariectomized mice.MethodsTwenty-six-month-old female CD-1 mice were divided into 4 groups: control, low-dose DEHP, high-dose DEHP, and estrogen groups (n=5, each group). All mice were subjected to ovariectomy for the induction of artificial menopause and then exposed to corn oil, DEHP, and estrogen for 2 months. Micro-computed tomography (Micro-CT) of the bone and analysis of blood samples for bone markers were performed to observe the changes in bone metabolism.ResultsOsteocalcin level was decreased in the control, low-dose and high-dose DEHP group, the reduction width was greater in the high-dose DEHP group (−0.219 ng/mL) than control group (−0.077 ng/mL, P<0.05). C-terminal telopeptide of type I collagen level was increased in the control, low-dose and high-dose DEHP group, the increase range of low-dose DEHP group (0.329 ng/mL) showed greater than control group (0.093 ng/mL, P<0.05). Micro-CT analysis revealed that the BMD was significantly lower in the high-dose DEHP group (19.8×10−2 g/cm3) than control group (27.2×10−2 g/cm3, P<0.05). The structure model index was significantly higher in the high-dose DEHP group (2.737) than low-dose DEHP group (2.648) and estrogen group (2.63, P<0.05). It means the progression of osteoporosis in the high-dose DEHP group.ConclusionsThese results confirm the negative effects of DEHP on bone health in ovariectomized mice. Further continuous studies on genetic pathways and other endocrine disruptors will be necessary to validate these findings.

The effect of di-2-ethylhexyl phthalate on inflammation and lipid metabolic disorder in rats

BackgroundPlasticizer di-2-ethylhexyl phthalate (DEHP) can induce lipid metabolic disorder. There was a chronic low level inflammatory response in adipose tissue of patients with lipid metabolic disorder. But the effect of inflammation on lipid metabolic disorder induced by DEHP is unclear. The present study was undertaken to explore the effect of di-2-ethylhexyl phthalate on inflammation and lipid metabolic disorder in rats. MethodsEighty healthy 21-day-old Wistar rats were randomly divided into 4 groups and administered DEHP by gavage at 0, 5, 50, and 500 mg/kg/ d for 8 weeks. Morphological changes of adipose tissue, the levels of IL-1β, TNF-α, LEP, and ADP in rat serum and adipose tissue, the serum TC, TG, HDL-C and LDL-C, the mRNA and protein expression levels of lipid metabolism-related gene CEBP/β and inflammation-related gene CD68 were measured. ResultsAfter exposure to DEHP, the weight of rats in the high dose group was significantly higher than that in the control group (p < 0.05). And the number of adipose tissue cells in the medium-dose and high-dose DEHP groups increased, with much more macrophage infiltrated. The levels of LDL-C, HDL-C, TC in serum and LEP in adipose tissue of rats exposed to 500 mg/kg DEHP were significantly higher than those in the control group (p < 0.05); while the level of ADP in adipose tissue in rats exposed to DEHP was significantly lower (p < 0.05). The levels of IL-1β and TNF-α in surum and adipose tissue of rats exposed to DEHP were significantly higher than those in the control group (p < 0.05). The mRNA and protein expression levels of CEBP/β and CD68 in adipose tissue of rats exposed to DEHP were significantly higher than those in the control group. The TC, LEP and ADP Levels of rats were significantly different among different subgroup of IL-1β and TNF-α, and in high level subgroup, the TC, LEP and ADP Levels were increased. The levels of TC and LEP was increased in high level subgroup of CD68. ConclusionDEHP induced more macrophage infiltrated in adipose tissue of rats, promoted the secretion of IL-1β, TNF-α and the formation of inflammation, and disturbed the normal lipid metabolism and lead to lipid metabolic disorders. What is more, the levels of inflammation were associated with the lipid levels.

Long-term di (2-ethylhexyl)-phthalate exposure promotes proliferation and survival of HepG2 cells via activation of NFκB

Di (2-ethylhexyl)-phthalate (DEHP) is the most commonly used plasticizer. DEHP can easily leach out from products into the environment and has become a significant environmental contamination. However, its carcinogenic effects in human remain controversial. In this study, we exposed hepatocellular carcinoma HepG2 cells to different concentrations of DEHP for a period of times, followed by a combination of RNA-seq analysis and molecular analysis for DEHP-altered genes. We observed that long-term and very low-dose DEHP treatment has growth-inducing activity in HepG2 cells, while short-term and high-dose DEHP treatment has little effects on tumor cell proliferation. Bioinformatic analysis further revealed that a large subset of differentially expressed genes affected by DEHP have functional annotations related to inflammation response. Importantly we found that long-term DEHP treatment stimulated nuclear factor-kappa B (NF-κ B) activity in HepG2 cells, while inhibition of NFκB activity could offset the effects of DEHP exposure. In conclusion, continuous DEHP treatment could promote proliferation of HepG2 cells via activating NFκB signaling pathway.

Effect of di-(2-ethylhexyl) phthalate on lung morphology of newborn rats

Objective To investigate the effect of di-(2-ethylhexyl) phthalate (DEHP) on the postnatal lung development in newborn rats. Methods A total of 60 newborn Sprague–Dawley rats (weighing 5.0-8.0 g) in five age groups were studied in the first experiment. The rats were divided based on the different postnatal ages: postnatal day (PND)1, PND4, PND7 and PND14. A total of 45 newborn Sprague–Dawley rats (weighing 5.0-8.0 g) were randomly divided into three groups according to the dosage of DEHP administered in the second experiment. The newborn rats were administered DEHP through intraperitoneal injection at 10 (low-dose subgroup), 100 (medium-dose subgroup ) or 750 ( high-dose subgroup) mg/kg daily from PND1 to PND13. The rats were sacrificed on PND14. Pups were sacrificed with lethal dose injection of pentobarbital sodium. The lung was removed. The right middle lobes were used for analysis. The tissue was processed for histology and lung sections were stained with HE for light microscopic (LM) morphometric measurement. The analysis was performed by means of a digital image analysis system, including pulmonary interstitial area ratio (IAR) and total length density of all segments. One-way ANOVA, LSD and Dunnet T3 methods were used for statistical analysis. Results In the normal controls, IAR decreased significantly by (31.97±5.03) %, (30.05±3.57) %, (25.33±1.83) % and (22.01±2.19) %, respectively, from PND1 to PND14 (P<0.05 or P<0.01). IAR in medium- and high-dose subgroups increased significantly by (24.11±2.78)% and (26.53±3.42)%, respectively on PND14. The total length density of all segments in unit area lung volume increased significantly by 0.047 8±0.003 7, 0.050 0±0.002 9, 0.071 2±0.003 0 and 0.084 4±0.004 3, respectively from PND1 to PND14 (P<0.01). In the DEHP treated animals, when compared with the control group, IAR was significantly higher on PND14 (P<0.05 or P<0.01), while the total length density of all segments in unit area lung volume was significantly decreased (P<0.05 or P<0.01). Length density in medium-and high-dose subgroups were higher than that of low-dose subgroup by 0.082 9±0.001 8, 0.077 2±0.002 0 and 0.071 3±0.003 7, respectively on PND14 (P<0.05 or P<0.01). Conclusions Medium- and high-dose DEHP affect the postnatal lung development in rats in a dose-dependent mode. Key words: Lung; Growth and development; Image Processing, computer-assisted; Diethylhexyl phthalate; Rats

Involvement of hemeoxygenase-1 in di(2-ethylhexyl) phthalate (DEHP)-induced apoptosis of Neuro-2a cells

A widely-used plasticizer di(2-ethylhexyl) phthalate (DEHP) is known to induce apoptosis in neurons, although the mechanisms responsible for DEHP-induced apoptosis is not well explored yet. We recently showed that exposure to DEHP increases the expression of hemeoxygenase (HO)-1, an oxidative stress related enzyme, in the mice brain. In this study, we investigated whether HO-1 is involved in DEHP-induced apoptosis using a mouse neuroblastoma cell line Neuro-2a, which forcibly express SCAT3, a fluorescent indicator of caspase-3 activity. The doses of DEHP at 1, 10 or 100 µM were used in the present study to mimic the level of human exposure to DEHP. Live image analysis of SCAT3-expressing Neuro-2a cells revealed that caspase-3 activity in the cells was significantly increased by DEHP at 100 µM but not 1 or 10 µM. We measured HO-1 mRNA level in Neuro-2a cells exposed to DEHP and found significant increase in HO-1 mRNA level by DEHP at 100 µM but not 1 or 10 µM. Live image analysis of SCAT3-expresisng Neuro-2a cells was further performed to determine the effects of HO-1 siRNA in DEHP-induced apoptosis via caspase-3 activation. We found that knockdown of HO-1 gene nullifies the effects of DEHP to activate caspase-3. These results suggest that HO-1 is involved in DEHP-induced apoptosis. Moreover, this study demonstrates that high-dose DEHP exposure induces caspase-3-dependent apoptosis, which is at least partially mediated by the up-regulation of HO-1 gene, in Neuro-2a cells.

Expression levels of neuroimmune biomarkers in hypothalamus of allergic mice after phthalate exposure

Previously, we demonstrated that maternal exposure to phthalates enhances atopic dermatitis in male mouse offspring. However, whether phthalate exposure affects neuroimmune biomarkers in allergic mice has not yet been studied. Di-(2-ethylhexyl) phthalate (DEHP) and di-isononyl phthalate (DINP) are environmental chemicals that are commonly used as plasticizers. This study was designed to investigate the expression levels of neuroimmune biomarkers in the hypothalamus of a murine model of allergic asthma after phthalate exposure throughout juvenility until adulthood. Six-week-old C3H/HeJ Jcl male mice were treated with DEHP or DINP (0, 0.02, 0.4 or 8 nmol per body per week) and ovalbumin (OVA; 1 µg per body per 2 weeks) for 7 weeks intratracheally. On the day after the completion of the phthalate and OVA treatment, the hypothalamus from each mouse was collected, and the mRNA expression levels of neuroimmune biomarkers were examined using a real-time RT-PCR analysis. The mRNA expression levels of the proinflammatory cytokines interleukin (IL)-1β and tumor necrosis factor (TNF)-α, the chemokine CCL3, the transcription factor nuclear factor (NF)-κB, the oxidative stress marker heme-oxygenase (HO)1, a nerve growth factor, and the microglia marker Iba1 were remarkably up-regulated in the hypothalami of mice treated with 8 nmol of DEHP in the presence of the allergen. However, no significant changes were observed, except for reductions in the TNF-α and CCL2 mRNA levels, in mice exposed to DINP combined with the allergen. This study is the first report to show that high-dose DEHP exposure throughout juvenility until adulthood may induce neuroinflammation by modulating neuroimmune biomarkers in the hypothalami of allergic mice.

Hepatic peroxisome proliferator-activated receptor α may have an important role in the toxic effects of di(2-ethylhexyl)phthalate on offspring of mice

Maternal exposure to di(2-ethylhexyl)phthalate (DEHP) is associated with adverse effects on offspring, and the metabolites are agonists of peroxisome proliferator-activated receptor (PPAR) α, which exhibits species differences in expression and function. This study aimed to clarify the mechanism of DEHP-induced adverse effects on offspring in relation to maternal mouse and human PPARα. Male and female Sv/129 wild-type (mPPARα), Pparα-null and humanized PPARα (hPPARα) mice were treated with diets containing 0%, 0.01%, 0.05% (medium) or 0.1% (high) DEHP. After 4 weeks, males and females were mated. Dams were killed on gestational day 18 and postnatal day (PND) 2. High-dose DEHP decreased the number of total and live fetuses, and increased resorptions in mPPARα mice. In hPPARα mice, resorptions were increased above the medium dose, and the number of births was decreased at the high dose. The number of live pups on PND2 was decreased over the medium dose in mPPARα and at the high dose in hPPARα mice. No such findings were observed in Pparα-null mice. High-dose DEHP decreased plasma triglyceride in pregnant mPPARα mice, but not in Pparα-null and hPPARα ones. Above the medium dose in mPPARα mice significantly reduced hepatic microsomal triglyceride transfer protein (MTP) expression. Medium- and/or high-dose DEHP increased the levels of maternal PPARα target genes in mPPARα and hPPARα mice. Taken together, PPARα expression is required for the toxicity of DEHP in fetuses and pups and altered plasma triglyceride levels, through regulation of MTP may be important in mPPARα mice and not in hPPARα mice.

Perinatal exposure to di-(2-ethylhexyl) phthalate leads to restricted growth and delayed lung maturation in newborn rats

Pregnant women and infants have significant exposures to the most commonly used plasticizer di-(2-ethylhexyl) phthalate (DEHP). This study was designed to evaluate the effects of DEHP exposure on growth and lung maturation in rats and determine if DEHP regulation of 11beta-hydroxysteroid dehydrogenase type 1 gene (Hsd11b1) expression in the lung tissue plays a role in its effects on lung maturation. Pregnant Sprague-Dawley rats were treated from gestational day 12 to postnatal day (PND) 21 with DEHP orally at dosages of 0, 10, 100 or 750 mg/kg/day, respectively (n=8 for each group). Two rat pups (one male and one female) from each litter were sacrificed at PND 1 and 21. Body weight was measured and the lung was processed for histology and calculation of lung interstitial tissue proportion as well as real-time PCR determination of the expressions of Hsd11b1, surfactant associated protein-A1 gene (Sftpa1) and B gene (Sftpb). The perinatal DEHP exposure led to a dose dependent intrauterine and postnatal growth restriction (P<0.001). High dose DEHP (750 mg/kg/day) exposure led to decreased gas-exchange space as evidenced by increased lung interstitial tissue proportion (P<0.001), but did not cause significant changes in Hsd11b1, Sftpa1 or Sftpb gene expression in the rat lung at PND 1. The DEHP-induced change in lung histology remained significant at PND 21 with improvement despite continual exposure to DEHP. Perinatal DEHP exposure leads to growth restriction and delayed lung maturation in newborn rats.

Biphasic Effects of Postnatal Exposure to Diethylhexylphthalate on the Timing of Puberty in Male Rats

Phthalate esters such as di(2-ethylhexyl)phthalate (DEHP), which are commonly found in cosmetics and in flexible plastics distributed by the food, construction, and medical products industries, have been classified as anti-androgens. High-dose DEHP exposure in utero is associated with decreased androgen levels. However, when administered after birth, low doses of DEHP (eg, 10 mg/kg body weight) may stimulate androgen production. In the present study, the potential of phthalate exposure to advance or delay the timing of puberty was assessed. Male Long-Evans rat pups were chronically subjected to low or high doses of DEHP, with the androgen-driven process of preputial separation serving as an index of pubertal timing. Rats were treated with 0, 10, 500, or 750 mg/kg body weight DEHP for 28 days starting at day 21 postpartum. The average age at which the animals completed preputial separation was measured in each group. The age of preputial separation was 41.5 +/- 0.1 days postpartum in controls (vehicle). The 10 mg/kg DEHP dose advanced pubertal onset significantly to 39.7 +/- 0.1 days postpartum, whereas the 750 mg/kg DEHP dose delayed pubertal onset to 46.3 +/- 0.1 days postpartum. The 10 mg/kg DEHP dose also significantly increased serum testosterone (T) levels (3.13 +/- 0.37 ng/mL) and seminal vesicle weights (0.33 +/- 0.02 g) compared with control serum T (1.98 +/- 0.20 ng/mL) and seminal vesicle weight (0.26 +/- 0.02 g), while the 750 mg/kg dose decreased serum T (1.18 +/- 0.18 ng/mL) as well as testes and body weights. Direct action of the DEHP metabolite, monoethylhexylphthalate (MEHP), on Leydig cell steroidogenic capacity was investigated in vitro. MEHP treatment at a low concentration (100 microM) increased luteinizing hormone-stimulated T production, whereas 10 mM concentrations were inhibitory. In conclusion, data from the present study indicate that DEHP has a biphasic effect on Leydig cell function, with low-dose exposure advancing the onset of puberty. High doses of DEHP, which are anti-androgenic, may also be outside the range of real environmental exposure levels.

Gene Expression Changes Induced in Mouse Liver by Di(2-ethylhexyl) Phthalate

Increasing chemical use necessitates a better understanding of how pollutants, such as di(2-ethylhexyl) phthalate (DEHP), a peroxisome proliferator and a phthalate plasticizer, affect human health. To understand the effects of DEHP exposure, we utilized microarray technology to identify novel DEHP targets in livers of male C57BL/6 mice treated with 1.0% dietary DEHP for 13 weeks. We identified 51 DEHP-regulated genes; these genes are involved in, but not limited to, peroxisome proliferation, xenobiotic detoxification, oxidative stress response, immune function, steroid hormone metabolism, testis development, and pheromone transport. The reproductive toxicity mechanism of DEHP may be due to its effects on steroid hormone metabolism and sexual development. Confirmation of microarray results with Northern blots demonstrated that both low- and high-dose DEHP treatments altered the expression of genes associated with testis development and steroid hormone synthesis. Vanin-1, a regulator of testis development, was upregulated after exposure to 0.2 and 1.0% DEHP by 1.8- and 2.9-fold, respectively. Several genes involved in hormone metabolism were also regulated by DEHP. 11βHSDI was downregulated 1.8- and 3.1-fold by 0.2 and 1.0% DEHP, respectively, while HSD3b5 was suppressed similarly by 0.2% DEHP, 1.5-fold, and more severely by 1.0% DEHP, 8.0-fold. Interestingly, food restriction had a similar effect to DEHP on several genes, including HSD3b5. In addition, steroidogenic gene cyp7B1 was downregulated while phospholipid transfer protein and cyp2B9 were induced. Genes peripherally associated with steroid hormones were also affected: ALDH3, GSTθ2, and Id2. Collectively, our data validate the concern for DEHP as a reproductive developmental toxicant.