Effects Of Di(2-ethylhexyl) Phthalate Research Articles

Prenatal exposure to di (2-ethylhexyl) phthalate causes autism-like behavior through inducing Nischarin expression in the mouse offspring

Epidemiologic evidence has suggested a relationship between di (2-ethylhexyl) phthalate (DEHP) prenatal exposure and autism spectrum disorders (ASD), but the underlying mechanisms are still at large unknown. In this study, pregnant mice were intragastrically administered with DEHP once a day from GD 3 to GD 17 and the neurobehavioral changes of offspring were evaluated. In addition to the repetitive stereotyped behaviors, DEHP at the concentration of 50 mg/kg/day and above significantly impaired the sociability of the offspring (P < 0.05) and decreased the density of dendritic spines of pyramidal neurons in the prefrontal cortex (P < 0.05). At the same time, the expression of Nischarin protein in prefrontal lobe increased (P < 0.05). Similarly, after 12-h incubation of DEHP at the concentration of 100 nM, the total spine density, especially the mushroom and stubby spine populations, significantly decreased in the primary cultured prefrontal cortical neurons (P < 0.05). However, the inhibitory effect of DEHP were reversed by knockdown of Nischarin expression. Collectively, these results suggest that prenatal DEHP exposure induces Nischarin expression, causes dendritic spine loss, and finally leads to autism-like behavior in mouse offspring.

Perinatal DEHP exposure modulates pituitary estrogen receptor α and β expression altering lactotroph and somatotroph cell growth in prepuberal and adult male rats

Phthalates are synthetic chemicals widely used to make polyvinylchloride (PVC) soft and flexible. Of these, Di-(2-ethylhexyl) phthalate (DEHP) is the most commonly used, with high human exposure occurring as early as the fetal developmental stage and affecting the endocrine system. We focused on the perinatal DEHP effects on pituitary estrogen receptor (ER) expression in male rats, explored their impact on lactotroph and somatotroph cell growth, and evaluated the direct effects of this phthalate on pituitary cell cultures. Our results showed that DEHP perinatal exposure was unable to modify the ERα+ pituitary cell number from prepuberal rats, but increased ERβ+ cells. In adulthood, the pituitary ERα+ cells underwent a slight decrease with ERβ showing the greatest changes, and with a significant increase observed in somatotroph cells. Also, in vitro, DEHP reduced the ERα+ cells, increased the percentage of ERβ+ pituitary cells and modified the Ki67 index, as well as decreasing the lactotrophs and increasing the somatotroph cells. In conclusion, the present study showed that DEHP induced ER expression changes in normal pituitary glands from male rats in in vivo and in vitro conditions, suggesting that DEHP could differentially modulate lactotroph and somatotroph cell growth, possibly as a consequence of ER imbalance.

Disruption of Intestinal Homeostasis Through Altered Responses of the Microbial Community, Energy Metabolites, and Immune System in Zebrafish After Chronic Exposure to DEHP.

Di-(2-ethylhexyl) phthalate (DEHP) is ubiquitously reported in global water bodies and exhibits various environmental and human health risks. However, the effects of DEHP chronic exposure on the intestinal microbiota and associated host health concerns in aquatic species are still largely unexplored. In this study, chronic exposure to DEHP at environmental levels significantly increased the body weight, length, and body mass index (BMI), especially in male fish. The microbial community was disrupted with the relative abundance of phylum Firmicutes and genera diversity for Prevotella-7, Deefgea, PeM15, Halomonas, Akkermansia, Chitinibacter, and Roseomonas, which are significantly activated in zebrafish after exposure to DEHP. The height of the gut villus, the thickness of muscularis layer, and the number of goblet cells per villus were significantly decreased, as well as showed differences between female and male zebrafish. Further, the levels of energy-related metabolites in gut tissues were increased, compared to the control group. The expression levels of immune-related genes (interleukin 8, il-8, also referred to as cxcl8a), microbial defense-related genes (lysozyme, lyz, interleukin 10, and il-10), and obesity-related genes (aquaporin 8a, aqp8, mucin 2.1, muc2.1, fibroblast growth factor 2, fgf2, and proopiomelanocortin a, pomca) were significantly up-regulated in zebrafish, except the down-regulated expressions of toll-like receptor-5 (tlr-5) and interleukin 1β (il-1β) in the females and pomca in the males, respectively. Importantly, Spearman’s correlation analyses revealed that the levels of metabolites and gene expressions in the gut were closely related to the dominant microbial genera, such as Aeromonas, Deefgea, Akkermansia, PeM15, Mycobacterium, and Rhodobacter. Taken together, chronic exposure to DEHP at environmental levels disturbed bacterial composition accompanied by the altered expressions of intestinal metabolites and the critical immune and intestinal function-related genes, which provided novel insights into DEHP effects on perturbation of gut microbiota and metabolic homeostasis in zebrafish.

EFFECT OF DI-(2-ETHYLHEXYL) PHTHALATE (DEHP) EXPOSURE ON MICROARCHITECTURE OF FEMORAL BONE IN MALE LABORATORY MOUSE: PRELIMINARY RESULTS

Di-2-ethylhexyl phthalate (DEHP) is a toxic and hazardous endocrine disruptor with adverse effects on animal and human health. However, its impact on bone tissue has not been sufficiently investigated. Therefore, the purpose of our preliminary study was to examine the effects of DEHP on compact bone structure in two 57-days-old male mice. Daily oral administration of DEHP (4.5 mg/kg body weight dissolved in 500 µl of peanut oil per 15 days) was studied, compared to a control. We observed a significant effect of DEHP exposure on macroscopic bone characteristics. Similarly, we identified differences in qualitative characteristics, such as the presence of resorption lacunae and absence of non-vascular and primary vascular radial bone tissue near the endosteal border, compared to the control. On the contrary, quantitative analysis showed no demonstrable alterations in morphometric parameters. Our preliminary findings support the hypothesis about the negative impact of DEHP on bone tissue. However, further investigation is needed to understand this issue better and more precisely.

The novel role of the aquaporin water channel in lycopene preventing DEHP-induced renal ionic homeostasis disturbance in mice

Di-(2-ethylhexyl) phthalate (DEHP), an extensively used plasticizer, can cause environmental pollution and organ injury. Lycopene (LYC) is a natural carotene that has the potential to prevent chronic diseases. To reveal the effect of DEHP and/or LYC on the kidney, male mice were treated with LYC (5 mg/kg) and/or DEHP (500 mg/kg or 1000 mg/kg) by gavage for 28 days. The study indicated that DEHP caused glomerular atrophy, tubular expansion, disappearance of the mitochondrial membrane, and cristae rupture. DEHP exposure can increase the expression of aquaporin (AQP) subunits and the activity of Ca2+-Mg2+-ATPase and decrease the activity of Na+-K+-ATPase, which results in ion disorder. However, LYC can relieve kidney injury by regulating the activity of ATPase, the expression of ATPase subunits, and AQP subunit expression. The results indicated that AQP was a target for LYC in antagonizing the disturbance of DEHP-induced renal damage.

Di-(2-ethylhexyl) phthalate enhances cytokine release from group 2 innate lymphoid cells in the presence of interleukin-33

Epidemiological and experimental studies have shown that di-(2-ethylhexyl) phthalate (DEHP), a plasticizer, can aggravate allergic diseases. DEHP promotes adaptive immune responses, although its effect on the innate immune system remains largely unknown. The present study investigated the effects of DEHP on group 2 innate lymphoid cells (ILC2) that produce Th2 cytokines in response to epithelial cell-derived cytokines, such as interleukin (IL)-33. ILC2 (lineage-negative, CD45.2+, Sca1+, KLRG1+) were isolated from the lungs of C57BL/6 J mice. Co-exposure to DEHP and IL-33 significantly increased IL-5 release from ILC2, whose level was higher than that of the vehicle and IL-33 alone. The effects of DEHP in the presence of IL-33 showed an inverted-U dose-response. The present is the first report showing that DEHP exacerbates allergy through the innate immune system.

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.

Effects of Di-2-ethylhexyl phthalate on growth, lipid peroxidation and antioxidative enzyme activity in Dunaliella salina

Di-2-ethylhexyl phthalate (DEHP) is a toxic organic compound. It has been shown to exert negative effects on non-targeted aquatic organisms. In order to study the toxicological effects of DEHP on marine microalgae, we investigated cell growth, oxidative damage and antioxidant enzyme activities of Dunaliella salina under DEHP treatment. After exposure to DEHP of 10 to 800 mg/L for 7 days, D. salina growth was gradually inhibited with the increase in DEHP concentration. After D. salina was exposed to 400 mg/L DEHP for 4 days, the contents of malondialdehyde and hydrogen peroxide in D. salina were significantly increased as compared with those in the control group. After 6 days of exposure to DEHP at 10 to 400 mg/L, glutathione peroxidase activity in D. salina was significantly reduced in the 10 and 40 mg/L DEHP groups. Exposure of D. salina to 100 to 400 mg/L DEHP resulted in significantly higher inhibition of total superoxide dismutase in D. salina (p<0.05), and copper-zinc superoxide dismutase of D. salina was significantly inhibited compared with that of the control group (p<0.01). The results showed that DEHP exposure can damage the antioxidant defense system and cause cell death in D. salina.

Protective effect of N-acetylcysteine (NAC) against Di-ethylhexyl phthalate (DEHP) induced pulmonary toxicity in male albino rats (histological and immunohistochemical study)

Di-ethylhexyl phthalate (DEHP) is a global environmental pollutant. Human exposure to DEHP occurs through environmental sources. Community exposure (food, air, water) as well as medical settings’ exposure impose crucial effects on the human health. DEHP had been reported to have cytotoxic, immunotoxic, genotoxic, and reproductive toxic properties. This work aims to assess the possible toxic effects of DEHP on adult albino rats' lungs and to evaluate the possible protective effects of N-acetylcysteine (NAC) using body weight and relative lung weight parameters. Assessment of DHEP toxicity is measured by biochemical, histopathological and immunohistochemical methods. Fifty male adult albino rats were divided into five equal groups as follows: Group Ι (Negative control group), Group ΙΙ (Positive control group), Group IΙΙ (NAC-treated group): was given NAC orally (200 mg/kg/day), Group IV (DEHP-treated group): was given DEHP orally (3gm/kg once daily for 4 weeks) and Group V: (DEHP + NAC-treated group): was treated with DEHP concomitantly with NAC at the same previous doses. The results of the present study revealed that DEHP has significantly increased the lipid peroxidation level and significantly reduced glutathione content (GSH), superoxide dismutase (SOD) activity and catalase activity. The histological results of group IV showed inflammatory cellular infiltration of the lungs associated with interstitial edema, hemorrhage and inter-alveolar septal thickening that were markedly reduced in group V. Also, group V, showed a significant decrease in the collagen fibers accumulation and in caspase-3 expression as compared to group IV. Conclusion: treatment with NAC can protect against DEHP induced pulmonary toxicity in rats by decreasing oxidative stress, inflammation and apoptosis.

Di-2-ethylhexyl phthalate induces heart looping disorders during zebrafish development.

Di-2-ethylhexyl phthalate (DEHP) is a type of plasticizer widely used in industry. It is well-known for its toxic effects to endocrine and reproductive systems and has been detected in amniotic fluid and placenta. In the present study, we explored the effects of DEHP on heart development by using zebrafish as a model organism. DEHP (0.02 pg) was injected into the yolk sac of zebrafish embryos at the one-cell stage. No significant difference was found in embryonic lethality between control and DEHP groups at 1-day postfertilization (dpf), but mortality significantly increased in DEHP groups at 2 and 3 dpf. The average heart rate was significantly reduced in the surviving DEHP-treated zebrafish larvae at 3 and 4 dpf. In addition, massive pericardial edema was found in DEHP-treated zebrafish (12.6 ± 1.5%), which was significantly higher than that of the control group. Serious heart looping disorder was also observed in DEHP-treated larvae, mainly manifested with an elongated atrial-ventricular distance. Moreover, the expression of heart development transcription factors was affected by DEHP injection. Real-time polymerase chain reaction confirmed that five transcription factors (hand2, tp53, mef2c, esr1, and tbx18) were significantly downregulated in the DEHP group at 2 dpf, and three transcription factors (zic3, tcf21, and gata4) were significantly upregulated. Our results emphasize the need for the development of a nontoxic plasticizer to prevent possible deleterious effects on humans and other life-forms.

Dose-Dependent Effects of Di-(2-Ethylhexyl) Phthalate (DEHP) in Mussel Mytilus galloprovincialis

Phthalic acid esters (PAEs) are environmental organic pollutants that are ubiquitous in the ocean, and di-(2-ethylhexyl) phthalate (DEHP) is the most widely used PAE. The environmental concentration of DEHP was reported to be up to 42.52 μg/L in seawater in the estuaries located in Jiaozhou Bay along the Yellow Sea. DEHP has been investigated with respect to its toxicity in marine organisms. However, evidence on the dose-dependent effects of DEHP remains contradictory and limited. We used marine mussel Mytilus galloprovincialis as the experimental animal to study the dose-dependent effects of various levels of exposure to DEHP (concentrations of 4, 12, 36, 108, and 324 μg/L). These effects and the underlying mechanisms were elucidated by the levels of antioxidant enzyme activity, gene expression, and metabolite. The results indicated that, at environmentally relevant concentrations (12 and 36 μg/L), DEHP induced significant hormetic effects. This was indicated by the U-shaped or inverted U-shaped responses of the gene expression levels related to stress response (CAT, GST, and MgGLYZ) and antioxidant enzyme activities (SOD and CAT). The metabolic profiles revealed that DEHP generally caused monophasic response in osmotic regulation (homarine) and biphasic response (hormesis) in energy metabolism (glucose, glycogen, and amino acids), respectively. These findings can aid in ecological risk assessment with respect to DEHP and the determination of hormetic dose responses.

Early-life chronic di(2-ethylhexyl)phthalate exposure worsens age-related long-term associative memory decline associated with insulin/IGF-1 signaling and CRH-1/CREB in Caenorhabditis elegans

The ubiquitous contamination of di(2-ethylhexyl)phthalate (DEHP) in the environment, biota, and food poses potential ecological and human health risks. DEHP exposure can adversely affect learning and memory, yet the underlying mechanisms remain unclear. In this study, Caenorhabditis elegans was used to investigate the effect of early-life DEHP exposure on age-related long-term associative memory (LTAM) decline, as well as the associations with the cAMP-responsive element-binding protein (CREB) transcription factor and insulin/IGF-1 signaling (IIS). We showed that early-life exposure to DEHP reduced LTAM in wild-type worms at day-0 adulthood. Chronic exposure to DEHP from the L1 stage to day-5 adulthood worsened the age-dependent decline of LTAM. Moreover, the effect of DEHP on age-related LTAM requires CRH-1, a homolog of CREB. Mutations in daf-2, the sole receptor of C. elegans IIS, ameliorated the inhibition of LTAM by DEHP, and the effect depended on daf-16. In addition, daf-2 mutation restored the CRH-1 level in DEHP-exposed worms, and the effect required daf-16. Our study suggests that early-life chronic exposure to DEHP worsens age-related LTAM decline and the effect is associated with CRH-1 and IIS in C. elegans. The evolutionary conservation of IIS and CREB implies possible adverse effects by DEHP across species.

Prenatal Exposure to the Endocrine Disrupting Chemical DEHP Impacts Reproduction-Related Gene Expression in the Pituitary

Abstract Phthalates are chemicals used in various common products including plastics and medical devices, leading to widespread contact. Phthalate exposure during embryonic development can cause changes in puberty timing, reduced fertility and genital abnormalities. Previous studies on prenatal exposure to Di-(2-ethylhexyl) phthalate (DEHP) in mice indicated that it disrupts pituitary-gonadal feedback and alters reproductive performance in the offspring, however, the mechanism behind this is unknown. We hypothesize that prenatal exposure to DEHP during a critical period of embryonic development (e15.5 to e18.5) will cause sex-specific disruptions in reproduction-related functions in the pituitary in offspring due to interference with androgen and aryl hydrocarbon receptor (AhR) signaling. In order to discover the direct effects of DEHP on the reproduction-related functions in the pituitary, we performed both in vivo dosing and in vitro pituitary culture experiments. First, we dosed pregnant CD-1 mice with corn oil, the antiandrogen flutamide or DEHP from gestational day 15.5 to 18.5, then collected the pituitaries of the offspring on postnatal day 0. We found that prenatal DEHP exposure caused a significant increase in Fshb specifically in males, and flutamide caused significant increases in both Lhb and Fshb in males. Besides, DEHP exposure significantly increased AhR pathway related gene Cyp1b1 in both males and females. In the in vitro experiment, we took whole pituitaries from e16.5 embryos and cultured them in media containing DEHP, MEHP and/or AhR antagonist for 72hrs. We found that the DEHP metabolite MEHP was actually the chemical that exerted the effects directly at the level of the pituitary. Similar to in vivo experiments, Cyp1a1 and Cyp1b1 mRNA level were increased in pituitaries treated with MEHP in both sexes and the induction could be reduced by co-treatment with AhR antagonist. The mRNA level of Lhb, Fshb and Gnrhr were significantly decreased in both sexes by MEHP and co-treatment with AhR antagonist did not restore mRNA levels. The induction of Cyp1a1/Cyp1b1 gene in both in vivo and in vitro experiments indicates the possible activation of AhR by DEHP/MEHP. The in vitro experiment with AhR antagonist further proved that the induction of Cyp1a1/Cyp1b1 was indeed due to AhR activation directly at the level of the pituitary. The difference between in vivo and in vitro experiments in terms of gonadotropin gene expression indicates multiple mechanisms should be involved in the regulation of gonadotropin gene expression in vivo including androgen-related pathways and possibly AhR-related pathways. In summary, our data suggest that phthalates can directly affect the function of the pituitary in terms of regulation of reproductive- related genes. This indicates that pituitary impacts of phthalates could contribute to reproductive dysfunction observed in exposed mice and humans.

The Endocrine‐Disrupting Chemical Di‐(2‐ethylhexyl) phthalate (DEHP) Increases Corticosteroid Secretion in Human Adrenocortical Cells

Di-(2-ethylhexyl) phthalate (DEHP) is an endocrine-disrupting chemical that is widely used as a plasticizer in the production of polyvinyl chloride containing plastics. DEHP is a component of many common household items (toys, packaging, furniture, etc.) and medical devices, resulting in significant and chronic human exposure. Human exposure to DEHP has been linked to androgen disruption and developmental toxicity, including cryptorchidism, decreased anogenital distance, and decreased testosterone levels. Other adrenal corticosteroids are also susceptible to the endocrine-disrupting effects of DEHP. One study has demonstrated that fetal exposure to DEHP results in altered adrenal aldosterone production in adult rats. Due to these potential effects of DEHP on adrenal cortex steroidogenesis, the focus of our studies is to characterize the effect of DEHP on aldosterone secretion in a human adrenocortical cell line. HAC15 cells were exposed to vehicle or various concentrations of DEHP (10 nM-30 μM). After 72 h, cell viability, aldosterone secretion, and gene expression of enzymes and cofactors involved in aldosterone synthesis was examined. No effects on cell viability were observed with the concentrations studied. Basal aldosterone secretion was significantly increased from 100 nM-30 μM DEHP, with maximal secretion at 10 μM (in pg/mL: vehicle, 75.6±3.5; 10 μM DEHP, 7735.2±151.3; n=8). Ang II-stimulated aldosterone secretion was significantly increased from 100 nM-30 μM DEHP, with maximal secretion at 10 μM (in pg/mL: vehicle, 4278.9±110.2; 10 μM DEHP, 18651.7±572.2; n=8). ACTH-stimulated aldosterone secretion was significantly increased from 100 nM-30 μM DEHP, with maximal secretion at 10 μM (in pg/mL: vehicle, 124.0±3.7; 10 μM DEHP, 23455.8±272.7; n=8). Gene expression of most enzymes and cofactors involved in aldosterone synthesis were increased by 10 μM DEHP. Aldosterone synthase (CYP11B2) had the greatest induction at 175.2±2.0 fold. These data support the evidence that DEHP disrupts the regulation of aldosterone secretion and provides further evidence that DEHP is an endocrine disruptor.

Antagonistic effect of nano-selenium on hepatocyte apoptosis induced by DEHP via PI3K/AKT pathway in chicken liver

Di-(2-ethylhexyl) phthalate (DEHP) is a common plasticizer which is mainly used as a kind of plastic additive to increase the flexibility of plastic products. Given the widespread use of plastic products, DEHP, as a ubiquitous artificial pollutant, are widely present in the environment. In addition, DEHP could cause biological damage in various organs through oxidative stress. Nano-Selenium, a novel form of selenium, has a wide variety of biomedical applications as an antioxidant, anticancer and anti-inflammatory agent. Nevertheless, researches on the toxicity of DEHP in chicken hepatocyte lines is insufficient. In particular, researches on the interaction between DEHP and nano-selenium is insufficient in chicken cell. Therefore, the innovation of this study is to explore the theoretical mechanism of DEHP toxicity in hepatocytes and the antagonistic effect of nano-selenium on a series of damage in chicken hepatocytes caused by DEHP. Our results showed that, after DEHP exposure, oxidative stress levels in hepatocytes increased, and the mRNA and protein levels of apoptosis-related genes p53, Capsase9, Caspase3 and Bax increased significantly except Bcl-2. The protein levels of apoptosis markers cleaved-Caspase9 and cleaved-Caspase3 also increased significantly. Moreover, the result of TUNEL assay also showed that the level of apoptotic cells increased after DEHP exposure. Meanwhile, the mRNA and protein levels of PI3K, AKT and p-AKT decreased. Therefore, DEHP is able to enhance the degree of oxidative damage and apoptosis of chicken liver cells. Nevertheless, the addition of nano-selenium can reverse the above changes. Experimental results revealed that nano-selenium antagonizes the toxic effects of DEHP via the PI3K/AKT pathway.

Promotion effects of DEHP on hepatocellular carcinoma models: up-regulation of PD-L1 by activating the JAK2/STAT3 pathway.

Di(2-ethylhexyl) phthalate (DEHP), as an endocrine disruptor, is often used as a plasticizer in various polyvinyl chloride plastic products and medical consumables. Epidemiological studies have shown that long-term large intake of DEHP may be a risk factor for liver dysfunction. Long-term exposure to DEHP is associated with liver disease and aggravates the progression of chronic liver injury. However, the effects of DEHP on hepatocellular carcinoma (HCC) are rarely studied. In this study, we sought to determine the effects of DEHP on HCC induced by carbon tetrachloride combined with diethylnitrosamine, and further study its molecular mechanism. It was found that DEHP exposure significantly promotes tumor immune escape and activates signaling pathways involved in related protein expression of tumor immune escape, including PD-L1, JAK2, and STAT3. In addition, the trends observed in the HepG2 cells assay are consistent with vivo conditions. In summary, DEHP may play a tumor-promoting role in HCC mice and IFN-γ stimulated HepG2 cells, which may be related to the JAK2/STAT3 signaling pathway.

DEHP Down-Regulates Tshr Gene Expression in Rat Thyroid Tissues and FRTL-5 Rat Thyrocytes: A Potential Mechanism of Thyroid Disruption.

BackgroundDi-2-ethylhexyl phthalate (DEHP) is known to disrupt thyroid hormonal status. However, the underlying molecular mechanism of this disruption is unclear. Therefore, we investigated the direct effects of DEHP on the thyroid gland.MethodsDEHP (vehicle, 50 mg/kg, and 500 mg/kg) was administered to Sprague-Dawley rats for 2 weeks. The expression of the thyroid hormone synthesis pathway in rat thyroid tissues was analyzed through RNA sequencing analysis, quantitative reverse transcription-polymerase chain reaction (RT-PCR), and immunohistochemical (IHC) staining. DEHP was treated to FRTL-5 rat thyroid cells, and an RT-PCR analysis was performed. A reporter gene assay containing the promoter of thyroid stimulating hormone receptor (TSHR) in Nthy-ori 3-1 human thyroid cells was constructed, and luciferase activity was determined.ResultsAfter DEHP treatment, the free thyroxine (T4) and total T4 levels in rats significantly decreased. RNA sequencing analysis of rat thyroid tissues showed little difference between vehicle and DEHP groups. In the RT-PCR analysis, Tshr expression was significantly lower in both DEHP groups (50 and 500 mg/kg) compared to that in the vehicle group, and IHC staining showed that TSHR expression in the 50 mg/kg DEHP group significantly decreased. DEHP treatment to FRTL-5 cells significantly down-regulated Tshr expression. DEHP treatment also reduced luciferase activity in a reporter gene assay for TSHR.ConclusionAlthough overall genetic changes in the thyroid hormone synthesis pathway are not clear, DEHP exposure could significantly down-regulate Tshr expression in thyroid glands. Down-regulation of Tshr gene appears to be one of potential mechanisms of thyroid disruption by DEHP exposure.

In vitro evaluation of cytotoxic effects of di (2-ethylhexyl) phthalate (DEHP) produced by Bacillus velezensis strain RP137 isolated from Persian Gulf

Phthalates are widely used in polymer science and have potential toxicity related to their chemical structures. However, lots of evidence indicate that phthalate derivatives are undoubtedly produced as secondary metabolites by organisms, including plants, animals, and microorganisms. In the present study, Bacillus velezensis strain RP137 was cultured under optimized conditions. Its biomass was extracted with ethyl acetate with one fraction showing cytotoxic properties. A pure compound was isolated from the active fraction using combined silica gel and LH20 size exclusion column chromatography. Structural evaluation including FT-IR, 1H NMR, 13C NMR, HR-MS and CHN analysis identified the purified compound as di(2-ethylhexyl)phthalate (DEHP) with the formula C24H38O4 and the molecular weight of 389.29 Da. The microorganism-derived (stereospecific) DEHP was strongly reduced the proliferation and induced cytotoxic effects on various eukaryotic cell lines in compare to the synthetic racemic mixture of the compound when assessed by MTT assay. Furthermore, crystal violet assay and morphological changes confirmed the cytotoxic effect of DEHP. Interestingly, non-malignant SV40-immortalized fibroblast cells were less affected by the purified DEHP. Further evaluation on the antibacterial activity of DEHP documented no effect toward Gram-positive (S. aureus) and Gram-negative (E. coli and P. aeruginosa) pathogens even at a high concentration of 100 μM. In conclusion, existence of DEHP as byproduct of microorganism's metabolism can seriously be considered as a warning to human health.

Diethylhexyl phthalate (DEHP) regulates the proliferation and chemosensitivity of esophageal squamous cell carcinoma cells via regulation of PTEN.

As one of the most prevalent and deadly cancers worldwide, esophageal squamous cell carcinoma (ESCC) can be directly exposed to endocrine-disrupting chemical (EDC). As a potential EDC, diethylhexyl phthalate (DEHP) can trigger the development of various cancers, while the potential effect of DEHP on the ESCC progression was not clear. Our present study revealed that DEHP can trigger the proliferation of ESCC cells and decrease the cisplatin (CDDP) and fluorouracil (5-FU) sensitivity. Mechanistical studies indicated that DEHP can decrease the transcription of PTEN, a well-characterized tumor suppressor, in ESCC cells. Over expression of PTEN can reverse DEHP-regulated ESCC cell proliferation and chemosensitivity. Further, DEHP can increase the expression of HES-1, which can bind with the promoter of PTEN to inhibit its transcription. Collectively, DEHP can increase proliferation while decrease chemosensitivity of ESCC cells via regulation of HES-1/PTEN axis. Further, daily expression of DEHP may be a potent risk factor for ESCC development.

The Effect of Di (2-Ethylhexyl) Phthalate on Hair Trace Mineral Levels in Rats.

We studied the effect of di (2-ethylhexyl) phthalate (DEHP) on rat hair deposition of Iron (Fe), Copper (Cu), Manganese (Mn), and Zinc (Zn). Four groups, each of eight of female Wistar rats weighing 250-300 g, were randomly distributed to (1) control (corn oil-based diet), (2) DEHP 20 (20 mg DEHP per kg body weight (bw), (3) DEHP 100 (100mg DEHP kg/bw, and (4) DEHP 500 (500 mg DEHP kg/bw). The diets were fed daily for 14 days by gastric gavage before the rats were sacrificed. Hair content of Fe, Cu, Mn, and Zn was analyzed with atomic absorption spectrophotometry. There were no significant effect of DEHP on hair Fe content. However, hair Cu, Mn, and Zn were increased after DEHP 20 exposure (p<0.001). After administering DEHP 100 and DEHP 500, both Mn and Zn were decreased (p<0.001), respectively. Hair deposition of Cu, Mn, and Zn was affected by DEHP.