MEHP induces alteration of mitochondrial function and inhibition of steroid biosynthesis in MA-10 mouse tumor Leydig cells.

Di-(2-ethylhexyl) phthalate (DEHP) is a plasticizer that is widely used in manufacturing. Previous studies have shown that mono-(2-ethylhexyl) phthalate (MEHP), the active metabolite of DEHP, has inhibitory effects on luteinizing hormone (LH)-stimulated steroid biosynthesis by Leydig cells. The molecular mechanisms underlying its effects, however, remain unclear. In the present study, we examined the effects of MEHP on changes in mitochondrial function in relationship to reduced progesterone formation by MA-10 mouse tumor Leydig cells. Treatment of MA-10 cells with MEHP (0-300 µM for 24 h) resulted in dose-dependent inhibition of LH-stimulated progesterone biosynthesis. Biochemical analysis data revealed that the levels of the mature steroidogenic acute regulatory protein (STAR), a protein that works at the outer mitochondrial membrane to facilitate the translocation of cholesterol for steroid formation, was significantly reduced in response to MEHP exposures. MEHP also caused reductions in MA-10 cell mitochondrial membrane potential (ΔΨm) and mitochondrial respiration as evidenced by decreases in the ability of the mitochondria to consume molecular oxygen. Additionally, significant increases in the generation of mitochondrial superoxide were observed. Taken together, these results indicate that MEHP inhibits steroid formation in MA-10 cells at least in part by its effects on mitochondrial function.

A comparative study based on activity, conformation and computational analysis on the inhibition of human salivary aldehyde dehydrogenase by phthalate plasticizers: Implications in assessing the safety of packaged food items.

Phthalate plasticizers are commonly used in various consumer-end products. Human salivary aldehyde dehydrogenase (hsALDH) is a detoxifying enzyme which defends us from the toxic aldehydes. Here, the effect of phthalates [Di-2-ethylhexyl phthalate (DEHP), Diethyl phthalate (DEP) and Dibutyl phthalate (DBP)] on hsALDH has been investigated. These plasticizers inhibited hsALDH, and the IC50 values were 0.48 ± 0.04, 283.20 ± 0.09 and 285.00 ± 0.14 µM for DEHP, DEP and DBP, respectively. DEHP was the most potent inhibitor among the three plasticizers. They exhibited mixed-type linear inhibition with inclination towards competitive-non-competitive inhibition. They induced both tertiary and secondary structural changes in the enzyme. Quenching of intrinsic hsALDH fluorescence in a constant manner was observed with a binding constant (Kb) of 8.91 × 106, 2.80 × 104, and 1.31 × 105 M-1, for DEHP, DEP and DBP, respectively. Computational analysis showed that these plasticizers bind stably in the proximity of hsALDH catalytic site, reciprocating via non-covalent interactions with some of the amino acids which are evolutionary conserved. Therefore, exposure to these plasticizers inhibits hsALDH which increases the risk of aldehyde induced toxicity, adversely affecting oral health. The study has implications in assessing the safety of packaged food items which utilize phthalates.

Prenatal and breastfeeding exposure to low dose of diethylhexyl phthalate induces behavioral deficits and exacerbates oxidative stress in rat hippocampus.

Diethylhexyl phthalate (DEHP) is one of the most important derivatives of phthalate that has devastating effects on nervous system function. In this study, the effects of exposure with low doses of DEHP during pregnancy and lactation periods have been evaluated in rat's puppies. DEHP at doses 5, 40, 400 µg/kg/day and 300 mg/kg/day was given to mothers by gavage during pregnancy and lactation. The spatial and working memories were evaluated by Morris water maze test and Y maze, respectively. Oxidative stress levels were measured by biochemical tests. Histopathology of hippocampal tissue was assessed using hematoxylin and eosin, Nissl staining, and immunohistofluorescence in 60-days-old puppies. Behavioral data showed that low doses of DEHP decreased the working and spatial memories of male rats. Increased oxidative stress and decreased antioxidant activity were also observed in the hippocampus of rats which received the low doses of DEHP. However, neuronal damage, inflammation, and astrocyte activation were not significantly increased in the hippocampus of rats. Overall, exposure of mothers to low doses of DEHP during pregnancy and lactation cause behavioral deficits, especially in male newborn. The destructive effects of low doses of DEHP might be mediated through increased levels of oxidative stress in the brain.

Di-(2-ethylhexyl) phthalate-induced tumor growth is regulated by primary cilium formation via the axis of H2O2 production-thymosin beta-4 gene expression.

Di-(2-ethylhexyl) phthalate (DEHP) that is one of the most commonly used phthalates in manufacturing plastic wares regulates tumorigenesis. Thymosin beta-4 (TB4), an actin-sequestering protein, has been reported as a novel regulator to form primary cilia that are antenna-like organelles playing a role in various physiological homeostasis and pathological development including tumorigenesis. Here, we investigated whether DEHP affects tumor growth via primary cilium (PC) formation via the axis of TB4 gene expression and the production of reactive oxygen species (ROS). Tumor growth was increased by DEHP treatment that enhanced TB4 expression, PC formation and ROS production. The number of cells with primary cilia was enhanced time-dependently higher in HeLa cells incubated in the culture medium with 0.1% fetal bovine serum (FBS). The number of cells with primary cilia was decreased by the inhibition of TB4 expression. The incubation of cells with 0.1% FBS enhanced ROS production and the transcriptional activity of TB4 that was reduced by ciliobrevin A (CilioA), the inhibitor of ciliogenesis. ROS production was decreased by catalase treatment but not by mito-TEMPO, which affected to PC formation with the same trend. H2O2 production was reduced by siRNA-based inhibition of TB4 expression. H2O2 also increased the number of ciliated cells, which was reduced by siRNA-TB4 or the co-incubation with CilioA. Tumor cell viability was maintained by ciliogenesis, which was correlated with the changes of intracellular ATP amount rather than a simple mitochondrial enzyme activity. TB4 overexpression enhanced PC formation and DEHP-induced tumor growth. Taken together, data demonstrate that DEHP-induced tumor growth might be controlled by PC formation via TB4-H2O2 axis. Therefore, it suggests that TB4 could be a novel bio-marker to expect the risk of DEHP on tumor growth.

Metabolic, reproductive and thyroid effects of bis(2-ethylhexyl) phthalate (DEHP) orally administered to male and female juvenile rats at dose levels derived from children biomonitoring study.

Bis(2-ethylhexyl) phthalate (DEHP) is a plasticizer used in several items, non-covalently bound to plastics and easily released, since metabolites were found in human matrices. DEHP is an endocrine disrupter and children are particularly vulnerable and susceptible to DEHP effects due to higher exposure levels and developmental stage. A juvenile toxicity study was performed to identify DEHP hazard and mode of action in Sprague-Dawley rats of both sexes during peri-pubertal period - corresponding to childhood phase - from weaning, post-natal day (PND) 23, to full sexual maturity (PND60); the dose levels of 0, 9, 21 and 48 mg/kg bw/day were derived from LIFE PERSUADED biomonitoring study in children. DEHP was administered by gavage for 28 days (5 days/week); timing of preputial separation and vaginal opening was observed during treatment. Histopathological analysis was performed on: adrenals, spleen, liver, thyroid and reproductive organs. The following serum biomarkers were assessed: estradiol, testosterone, anti-Mullerian hormone, tetraiodothyronine, thyroid stimulating hormone, adiponectin and leptin. Gene expression on hypothalamic-pituitary area was focused on follicle stimulating, luteinizing, and thyroid stimulating hormones. The results showed that main targets of DEHP during juvenile period were liver and metabolic system in both sexes, while sex-specific effects were recorded in reproductive system (male rats) and in thyroid (female rats). DEHP exposure during peri-pubertal period at dose levels derived from biomonitoring study in children can induce sex-specific imbalances identifying the juvenile animal model as a sound tool to identify hazards for a reliable risk assessment targeted to children.

The dynamic assessment of toxicity and pathological process of DEHP in germ cells of male Sprague Dawley rats.

Di-(2-ethylhexyl) phthalate is representative of Phthalate esters (PAEs), which is one of the most widely used plasticizer and known to act as a reproductive toxicant. However, little is known about the toxicity and pathological process of DEHP exposure in male reproductive system in terms of different concentrations and time points. In this study, peripubertal male Sprague Dawley rats were continually exposed to different DEHP doses (100 mg/kg, 500 mg/kg, and 900 mg/kg) and periods (7 days, 14 days, 21 days, 28 days, and 35 days) during critical periods for sexual maturity. The reproductive parameters have been investigated, including testicular morphology, serum testosterone level, and testicular P450scc, 3ß-HSD, and PCYP17 levels. We observed disarrangement of testicular spermatogenic epithelium coupled with decrease of serum testosterone, testicular P450scc, 3ß-HSD, and PCYP17 levels, and these changes were more obvious with increase of both the exposure time and dosage. Then trend of the time-dose response to DEHP exposure and the pathological process in germ cells were estimated. The results of this study suggested that DEHP exposure could affect the male reproductive system and the degree of adverse effect depended on the dose and extent of exposure.

The role of STAT3/p53 and PI3K-Akt-mTOR signaling pathway on DEHP-induced reproductive toxicity in pubertal male rat.

Di (2-ethylhexyl) phthalate (DEHP) is a known environmental endocrine disruptor that impairs development of testis and spermatogenesis. This study aims to explore the effects of STAT3/p53 and PI3K-Akt-mTOR signaling pathway on DEHP-induced reproductive toxicity in pubertal male rat. 24 6-week-old male Sprague-Dawley rats were randomly divided into 4 groups (Control, low-dose, middle-dose and high-dose group) and were treated with increasing concentration of DEHP (0, 250, 500, 1000 mg/kg/day) respectively for 28 consecutive days by intragastric administration. Our results showed that DEHP exposure induced obvious morphological changes of testis, decreased organ coefficient of testis and sperm count, and increased testicular cell apoptosis in the 500 and 1000 mg/kg/day DEHP groups (p < .05). The serum testosterone decreased in a dose-dependent manner after treatment with DEHP. Furthermore, the exposure of DEHP elevated the levels of oxidative stress accompanied by upregulated expression of p53 and reduced expression of STAT3. In addition, compared with the control group, the expression of PI3K, p-Akt and p-mTOR proteins significantly decreased, whereas the downstream autophagy-related proteins phosphorylated ULK1, Beclin-1, Atg7, LC3-II obviously increased in the 250 mg/kg/day DEHP group (p < .05). The expression of p62 was reduced in DEHP-treated groups. Our data indicated that autophagy could be activated to protect testes from DEHP-induced reproductive damage by inhibiting PI3K-Akt-mTOR signaling pathway in the 250 mg/kg/day DEHP group. STAT3/p53-mediated mitochondrial apoptosis pathway might play a major role to cause testis injury and reproductive dysfunction in the 500 and 1000 mg/kg/day DEHP groups.

Comparison of aggregated exposure to di(2-ethylhexyl) phthalate from diet and personal care products with urinary concentrations of metabolites using a PBPK model - Results from the Norwegian biomonitoring study in EuroMix.

Phthalates are widely used as plasticisers in flexible plastics and containers for food and personal care products (PCPs) and contaminates foods and PCPs. A human biomonitoring (BM) study was performed to study exposure of chemicals from foods and PCPs. For two 24-h periods, adult volunteers (n = 144) in Norway kept diaries on food eaten and usage of PCPs, and collected 24-h urine. Aggregated exposure to di(2-ethylhexyl) phthalate (DEHP) from dietary and PCPs was estimated by Monte-Carlo simulation using Oracle Crystal Ball©. Simulated urinary concentrations using physiologically based pharmacokinetic (PBPK) models were compared with measured urinary metabolites of DEHP, mono-2-ethylhexyl phthalate (MEHP), mono-2-ethyl-5-hydroxyhexyl phthalate (MEHHP), mono-2-ethyl-5-oxohexyl phthalate (MEOHP) and mono-2-ethyl 5-carboxypentyl phthalate (MECCP). DEHP exposure from food are approximately 10 times higher than exposure than from PCPs. The main contributors to dietary exposure are dairy, grain, fruits and vegetables, meat and fish. Body lotion contribute most to the exposure of DEHP from PCPs. Forward-dosimetry gives good convergence with 24-h urinary concentrations of simulated and measured BM data. The measured concentration of the MECCP metabolite correlated well with simulated high exposure, while the measured concentrations of MEHP, MEHHP and MEOHP partly overlapped with both simulated low, medium and high metabolite exposure.

Effects of intravenous and oral di(2-ethylhexyl) phthalate (DEHP) and 20% Intralipid vehicle on neonatal rat testis, lung, liver, and kidney.

The highest human exposures to the plasticizer di(2-ethylhexyl) phthalate (DEHP) occur through intravenous (iv) exposure from medical procedures. Rodent toxicity studies, mainly using oral exposures, have identified male reproductive toxicity after developmental exposure to DEHP as the primary concern. Other organs are also affected by DEHP and route may influence the degree of target organ involvement. Cammack et al. (2003) reported a critical study focused on testicular toxicity using oral and iv exposures of neonatal Sprague-Dawley rats to 60, 300, or 600 mg/kg body weight/day DEHP in Intralipid vehicle. The present study followed the same dosing paradigm and included assessment of additional organs to evaluate the potential utility of this design for DEHP alternatives. Reduction of testis weight was observed in all DEHP treatment groups and germ cell and Sertoli cell toxicity was observed at the two highest doses with both routes. Lung granulomas occurred in all iv DEHP groups, possibly related to increased fat particle size in DEHP lipid emulsions. Lung alveolar development was inhibited after both oral and iv high dose DEHP. Toxicity of oral Intralipid vehicle was observed in germ and Sertoli cells. The lack of such effects after iv vehicle exposure suggested that this may be a gut-mediated effect.

Hepatotoxicity study of combined exposure of DEHP and ethanol: A comprehensive analysis of transcriptomics and metabolomics.

Both di(2-ethylhexyl)phthalate (DEHP) and ethanol have toxic effects on the liver, and the oral exposure to DEHP in combination with ethanol may exist in the alcohol consumers, however, current food safety risk assessments based solely on the toxicity data of DEHP may underestimate the health impacts of phthalates, especially in population that continually consume alcoholic beverages. Here we performed a comprehensive analysis of transcriptomics and metabolomics to study if there is a synergistic effect of DEHP and ethanol on the liver of mice. We found that the expression of genes associated with lipid accumulation and oxidation elevated simultaneously when exposed to DEHP alone or combined with ethanol, based on the results of pathophysiological tests, we considered that the elevated level of lipid accumulation was more significant which caused hepatic lipid accumulation ultimately. We observed no difference in the transcriptome profiles between combined exposure group and DEHP group, but metabolites pathway enrichment analysis showed that ethanol and DEHP may have a synergistic effect on the unsaturated fatty acid metabolism. In summary, our results suggest that DEHP exposed alone or combined with ethanol caused hepatic lipid accumulation, ethanol and DEHP may have a synergistic effect on the unsaturated fatty acid metabolism.

Glycerin Monostearate Aggravates Male Reproductive Toxicity Caused by Di(2-ethylhexyl) Phthalate in Rats.

Human beings are increasingly exposed to phthalates, which are a group of chemicals used to make plastics more flexible and harder to break, and simultaneously ingesting abundant food emulsifiers via daily diet. The purpose of this study was to investigate the effect of the food emulsifier glycerin monostearate (GMS) on male reproductive toxicity caused by di(2-ethylhexyl) phthalate (DEHP, one of the phthalates) and explore the underlying mechanism. Thirty male Sprague-Dawley rats were randomly divided into control group, DEHP group and DEHP+GMS group. Rats in the DEHP group and DEHP+GMS group were orally administered with 200 mg/kg/d DEHP with or without 20 mg/kg/d GMS. After 30 days of continuous intervention, it was found that the serum testosterone level was significantly lowered in DEHP group and DEHP+GMS group than that in control group (P<0.01). The serum testosterone level and the relative testis weight were significantly decreased in the DEHP+GMS group as compared with those in the DEHP group and control group (P<0.05). More spermatids were observed to be shed off in DEHP+GMS group than in DEHP group. The expression levels of cell cycle checkpoint kinase 1 (Chk1), cell division cycle gene 2 (Cdc2), and cyclin-dependent kinase 2 (CDK2) were down-regulated in DEHP group, and this tendency was more significant in DEHP+GMS group (P<0.05 or P<0.01). There was no significant difference in the P-glycoprotein (P-gp) expression between DEHP group and control group. However, P-gp was markedly down-regulated in DEHP+GMS group (P<0.01). The results indicated that the food emulsifier GMS aggravated the toxicity of DEHP on male reproduction by inhibiting the cell cycle of testicular cells and the expression of P-gp in testis tissues.

Di-(2-ethylhexyl)-phthalate interferes with T-follicular helper cell differentiation and cytokine secretion through signaling lymphocytic activation molecule family member-1.

Exposure to the widely-used phthalate plasticizer di-(2-ethylhexyl)-phthalate (DEHP) has been shown to be closely related to an increased prevalence of allergic diseases in infants and juveniles. Earlier work in our laboratory found that DEHP-related anaphylactic responses could be ascribed to T-follicular helper (Tfh) cell hyperfunction directly. The Tfh cell, a newly identified CD4+ TH cell subset, until recently has been considered as a key player in humoral immunity. Tfh cells can respond to stimulation through various receptors. Signaling lymphocytic activation molecule family member-1 (SLAMF1, CD150) is a surface co-stimulatory receptor that can bind to an intracytoplasmic adaptor signaling lymphocytic activation molecule-associated protein (SAP) to initiate downstream signaling cascades, regulating some events of immune response. The present study explored the role of SLAMF1 in Tfh cell differentiation and cytokine secretion under the condition of DEHP exposure. Using a weanling mice model of DEHP gavage with ovalbumin (OVA) sensitization, it was found that DEHP acted as an immunoadjuvant to elevate SLAMF1 and SAP expression in host Tfh cells. Ex vivo studies of effects from DEHP exposure on Tfh cells from OVA-sensitized hosts showed that DEHP acted in an adjuvant-like manner to promote the expression of adaptor protein SAP, transcription factors Bcl-6 and c-MAF, and cytokines interleukin (IL)-21 and IL-4 in Tfh cells. Transfection of these Tfh cells with Slamf1 small interfering RNA prior to exposure to the DEHP attenuated the over-expression of these molecules that was caused by the DEHP. In conclusion, this study demonstrated that DEHP, via a SLAMF1-mediated pathway, can impact on Tfh cell differentiation and their ability to form select cytokines.

Prenatal exposures to bisphenol A and di (2-ethylhexyl) phthalate disrupted seminiferous tubular development in growing male rats.

Endocrine-disrupting compounds (EDCs) are found in the environment due to their use in industrial and manufacturing activities. Exposure of the population to bisphenol A (BPA) and di (2-ethylhexyl) phthalate (DEHP) is significant because they are present in many consumer products. EDCs target the reproductive tract because they express high levels of steroid hormone receptors, which act as transcriptional factors to regulate reproductive development. In the present study, timed-pregnant Long-Evans female rats (n = 8-10) were administered BPA and DEHP by oral gavage at 2.5 or 25 µg/kg body weight and 5 or 50 µg/kg body weight, respectively. Exposures to chemicals were limited to the period between gestational days 12 and 21 followed by assessment of testicular development in male offspring in the postnatal period. Leydig cells and Sertoli cells are the two major somatic cells present in the testis. The 17ß-hydroxysteroid dehydrogenase (17ß-HSD) steroidogenic enzyme is a marker for Leydig cell maturation, whereas transferrin is a marker for Sertoli cell differentiation. At day 10 post-partum, testes were obtained from cohorts of control and chemical-exposed male rats and processed to measure 17ß-HSD and transferrin expression levels in western blots. Compared to control, 17ßHSD enzyme protein was increased in BPA-treated rats but levels were decreased in animals exposed to DEHP (P < 0.05). Transferrin protein was decreased in male rats exposed to both BPA and DEHP compared to control animals (P < 0.05). To assess qualitative cellular changes within the spermatogenic epithelium, testes were obtained from separate cohorts of male rats at 35 days of age and processed for histopathological analysis. Results showed that prenatal exposures of male rats to BPA and DEHP caused disruption of the spermatogenic epithelium evident as disorganization and atrophy of seminiferous tubules as well as desquamation of germ cells into the tubular lumen. Together, results from the present study support the view that developmental exposures to environmentally relevant levels of BPA and DEHP are associated with disruptions of testicular cell development, which have implications for endocrine and exocrine functions of testis.

Exposure to Mixed Phthalates in Czech Preschool and School Children.

Knowledge of population exposure to phthalates based on the urinary metabolite levels is of the highest importance for health risk assessment. Such data are scarce in the Czech population. In the study conducted in 2016, six urinary phthalate metabolites were analysed in a total of 370 first morning urine samples of healthy children aged 5 and 9 years, namely mono(2-ethylhexyl) phthalate (MEHP), mono(2-ethyl-5-hydroxyhexyl) phthalate (5OH-MEHP), mono(2-ethyl-5-oxohexyl) phthalate (5oxo-MEHP), mono-benzyl phthalate (MBzP), mono-iso-butyl phthalate (MiBP), and mono-n-butyl phthalate (MnBP). The two latter mono-butyl phthalate isoforms dominated among all samples with geometric means of 63.0 µg/L (MnBP) and 44.1 µg/L (MiBP), followed by 5OH-MEHP (20.6 µg/L), 5oxo-MEHP (12.9 µg/L), MBzP (3.65 µg/L), and MEHP (2.31 µg/L). Daily intake (DI) of the parent phthalates was estimated using the creatinine-based model. The highest DI values were found for di-n-butyl phthalate (DnBP) (median 2.5 µg/kg bw/day; 95th percentile 7.8 µg/kg bw/day) and di-2-ethylhexyl phthalate (DEHP) (median 2.3 µg/kg bw/day; 95th percentile 8.9 µg/kg bw/day) in 5-year-old children. The tolerable daily intake (TDI) set by the European Food Safety Authority (EFSA) was exceeded in case of DnBP (in 1% of 9-year-olds and in 3% of 5-year-olds). Exposure risk was assessed based on hazard quotients calculation and cumulative approach for similar health effect. The combined exposure to four phthalates expressed by hazard index (HI) for reprotoxicity revealed exceeding of HI threshold in 14% of 5-year-olds and in 9% of 9-year-olds. These findings strongly support the need to reduce the burden of children by phthalates.

Exposure to cadmium and mono-(2-ethylhexyl) phthalate induce biochemical changes in rat liver, spleen, lung and kidney as determined by attenuated total reflection-Fourier transform infrared spectroscopy.

Attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy is a label-free, non-destructive analytical technique for biochemical analysis of macromolecular components within tissue samples. Cadmium (Cd) and mono-(2-ethylhexyl) phthalate (MEHP), a primary metabolite of di-(2-ethylhexyl) phthalate, are present ubiquitously in the environment and in organisms, and have adverse impacts on ecosystems and human health. Herein we employed ATR-FTIR analysis to identify biomolecular changes in rat liver, spleen, lung and kidney after prepubertal exposure to Cd and MEHP. Our results showed clear segregations between the 3 mg/kg Cd-, 10 mg/kg, 50 mg/kg, 250 mg/kg MEHP- and binary mixture-treated groups vs. the solvent control group. Following principal components analysis coupled with linear discriminant analysis, biochemical alterations associated with different doses of Cd and MEHP were attributed mainly to lipids, proteins, phosphates and carbohydrates. In addition, the ratios of lipid/protein, C=O stretching/CH2 methylene (lipid oxidation level), amide I/amide II, α-helix/ß-sheet and CH3 methyl/CH2 methylene (acetylation level) in target organs were affected by these toxicants. There seems to be no dose-response effect of Cd and MEHP on target organs. We observed hardly any joint toxic action of these toxicants. This is the first study showing the application of ATR-FTIR spectroscopy to the assessment of toxicity of Cd and MEHP. Possibly, destruction of cell membrane structure and integrity could be the common mechanism of Cd and MEHP toxicity in liver, spleen, lung and kidney.

In utero exposure to di(2-ethylhexyl)phthalate suppresses blood glucose and leptin levels in the offspring of wild-type mice.

Exposure of pregnant mice to di(2-ethylhexyl)phthalate (DEHP) induces maternal lipid malnutrition and decreases the number of live fetuses/pups. In this study, we aimed to clarify the relationship between maternal lipid malnutrition and the nutritional status of the neonatal, lactational, and adult offspring, as well as the role of peroxisome proliferator-activated receptor α (PPARα) in these relationships. Sv/129 wild-type (mPPARA), Ppara-null, and PPARα-humanized (hPPARA) mice were fed diets containing 0, 0.01, 0.05, or 0.1% DEHP in utero and/or during the lactational stage. The male offspring were killed on postnatal day 2 or 21, or after 11 weeks. Exposure to either 0.05% or 0.1% DEHP during both the in utero and lactational periods decreased serum glucose concentrations in 2-day-old mPPARA offspring. These dosages also decreased both serum and plasma leptin levels in both 2- and 21-day-old mPPARA offspring. In contrast, exposure to DEHP only during the lactational period did not decrease leptin levels, suggesting the importance of in utero exposure to DEHP. Exposure to 0.05% DEHP during the in utero and lactational periods also increased food consumption after weaning in both mPPARA and hPPARA mice; this was not observed in Ppara-null offspring. In conclusion, in utero exposure to DEHP induces neonatal serum glucose malnutrition via PPARα. DEHP also decreases serum and plasma leptin concentrations in offspring during the neonatal and weaning periods, in association with PPARα, which presumably results in increased of food consumption after weaning.

Gestational di-(2-ethylhexyl) phthalate exposure causes fetal intrauterine growth restriction through disturbing placental thyroid hormone receptor signaling.

Previous study reported that gestational Di-(2-ethylhexyl) phthalate (DEHP) exposure caused fetal intrauterine growth restriction (IUGR). We aimed to investigate the role of placental thyroid hormone receptor (THR) signaling in DEHP-induced IUGR. Dams were treated with DEHP (50 or 200 mg/kg) by gavage daily throughout pregnancy. As expected, gestational DEHP exposure dose-dependently caused fetal IUGR. The mRNA levels of placental Thrα1 and Thrß1 were reduced and nuclear translocation of placental THRα1 and THRß1 were suppressed in DEHP-exposed mice even though thyroid hormones in maternal and fetal sera were unaffected. Correspondingly, Vegf, Pgf, Igf1 and Igf2, several THR downstream genes essential for placental angiogenesis, were down-regulated in placenta of DEHP-exposed mice. Histopathology showed that vascular space in the labyrinthine region was shrunken in placenta of DEHP-treated mice. The microvessel density in labyrinthine region was reduced in DEHP-treated mice. A nested case-control study based on MABC suggested that microvessel density was decreased in placenta of SGA cases. Moreover, protein abundance of placental THRα1 and THRß1 were lower in SGA cases. In conclusion, gestational DEHP exposure increases fetal IUGR incidence through disturbing placental THR signaling. The present study, at least partially, elucidate the underlying mechanism of DEHP-induced fetal IUGR.

Impact of the Di(2-Ethylhexyl) Phthalate Administration on Trace Element and Mineral Levels in Relation of Kidney and Liver Damage in Rats.

Di(2-ethylhexyl) phthalate (DEHP) is a widely used synthetic polymer in the industry. DEHP may induce reproductive and developmental toxicity, obesity, carcinogenesis and cause abnormal endocrine function in both human and wildlife. The aim of this study was to investigate trace element and mineral levels in relation of kidney and liver damage in DEHP-administered rats. Therefore, prepubertal male rats were dosed with 0, 100, 200, and 400 mg/kg/day of DEHP. At the end of the experiment, trace element and mineral levels, glucose-6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (6-PGD), glutathione reductase (GR) and glutathione S-transferase (GST) enzyme activities were evaluated in the serum, liver, and kidney samples of rats. Furthermore, serum clinical biochemistry parameters, organ/body weight ratios and histological changes were investigated to evaluate impact of DEHP more detailed. Our data indicated that sodium (Na), calcium (Ca), potassium (K), lithium (Li), rubidium (Rb) and cesium (Cs) levels significantly decreased, however iron (Fe) and selenium (Se) concentrations significantly increased in DEHP-administered groups compared to the control in the serum samples. On the other hand, upon DEHP administration, selenium concentration, G6PD and GR activities were significantly elevated, however 6-PGD activity significantly decreased compared to the control group in the kidney samples. Decreased G6PD activity was the only significant change between anti-oxidant enzyme activities in the liver samples. Upon DEHP administration, aberrant serum biochemical parameters have arisen and abnormal histological changes were observed in the kidney and liver tissue. In conclusion, DEHP may induce liver and kidney damage, also result abnormalities in the trace element and mineral levels.

Derivation of an oral reference dose (RfD) for di 2-ethylhexyl cyclohexan-1,4-dicarboxylate (DEHCH), an alternative to phthalate plasticizers.

Di 2-ethylhexyl cyclohexane-1,4-dicarboxylate (DEHCH, CAS 84731-70-4) is an ester of polycarboxylic acid assessed in a variety of mammalian toxicity assays as a substitute for phthalate ester-type plasticizers. An OECD 422 combined systemic toxicity study with a reproductive/developmental toxicity screening test in SD rats found minimal effects on the liver, spleen, and thyroid and no indication that DEHCH is a developmental or reproductive toxicant. In a 90-day feeding study in SD rats, no toxicologically relevant effects were noted. Low genotoxic potential of DEHCH is indicated by the lack of mutagenicity or clastogenicity in vitro. No studies assessing mode of action were identified. Where data gaps exist for DEHCH, a read-across approach was used to assess other toxicological endpoints of interest. Di-ethylhexyl terephthalate (DEHT, CAS 6422-86-2) and 1,2-cyclohexane dicarboxylic acid, diisononyl ester (DINCH, CAS 474919-59-0) have higher tiered studies to supplant the data lacking for health-based standard setting. DEHT and DINCH were chosen as the source substances due to similar physical/chemical properties and thus anticipated metabolism and toxicological characteristics. An oral reference dose (RfD) for DEHCH was calculated using the human equivalent NOAEL from the OECD 422 study. A total uncertainty factor of 100 was comprised of interspecies (3x), intraspecies (10x), subchronic to chronic (1x), LOAEL to NOAEL uncertainty (1x) and database uncertainty (3x) factors, resulting in an RfD of 0.3 mg/kg-day.

Intervention effect of gamma aminobutyric acid on anxiety behavior induced by phthalate (2-ethylhexyl ester) in rats.

BACKGROUND: Di(2-ethylhexyl) phthalate (DEHP) is one of the most widely used phthalate esters. The application of DEHP has caused serious environmental pollution and posed a threat to human health. METHODS: A total of 30 male Sprague-Dawley rats were randomly divided into control group, DEHP group (500 mg/kg DEHP), low GABA (Gama-aminobutyric acid) group (500 mg/kg DEHP and 1 mg/kg GABA), medium GABA group (500 mg/kg DEHP and 2 mg/kg GABA) and high GABA group (500 mg/kg DEHP and 4 mg/kg GABA). The interventions continued for 30 consecutive days. Open-field test and elevated plus-maze test were used to detect behavioral changes of rats before and after interventions. RESULTS: The levels of nitric oxide and nitric oxide synthase in prefrontal cortex of rats were measured using enzyme-linked immunosorbent assay. DEHP and GABA treatment had no significant effects on the body weight of rats. GABA restored food utilization rate of rats impaired by DEHP to the level of healthy rats. According to open-field test and elevated plus-maze test, GABA alleviated the effects of DEHP on rat behaviors. Enzyme-linked immunosorbent assay showed that GABA was effective in reducing the levels of nitric oxide and nitric oxide synthase in rats treated with DEHP. CONCLUSION: DEHP exposure induced anxiety in rats, which may be achieved through elevating nitric oxide and nitric oxide synthase levels in prefrontal cortex of rats. However, the effects caused by DEHP could be alleviated by GABA.