Di-2-ethylhexyl Phthalate Groups Research Articles

Repair mechanism of Wuwei Fuzheng Yijing formula in di-2-ethylhexyl phthalate-induced sperm DNA fragmentation in mice

Context Di-2-ethylhexyl phthalate (DEHP), a known persistent organic pollutant, can increase the sperm DNA fragmentation index (DFI). Objective To investigate the mechanism underlying the repair of DEHP-induced sperm DNA damage in mice by Wuwei Fuzheng Yijing (WFY) formula. Materials and methods The potential targets of WFY and sperm DNA fragment (SDF) were obtained from the TCMSP, BATMAN-TCM, OMIM and GeneCards. The protein–protein interaction (PPI) network, GO and KEGG pathway analyses of WFY-SDF were constructed. An animal model of DEHP-induced sperm DNA damage was replicated by gavage of SPF ICR (CD1) mice DEHP at 1 g/kg/d and treated with WFY at 8.92, 17.84 and 35.67 g/kg, respectively, for 60 d. Sperm DFI of each group was detected and compared. The target genes of WFY identified by transcriptomic and proteomic analyses were validated by qRT-PCR and Western blotting. Results Network pharmacology pathway analysis indicated that PI3K/Akt was the potential target of WFY on SDF. The DFI of the DEHP group (25.48%) was significantly higher than that of the control group (4.02%). The high-dose WFY group (19.05%) exhibited the most significant repairing effect. The related pathways were PI3K/Akt and metabolic. Aass, Aldh1a7, GSTA3, betaine homocysteine S-methyltransferase (Bhmt), Mug2 and Svs1 were screened and Bhmt was validated. Discussion and conclusions WFY can repair sperm DNA damage caused by DEHP, and the mechanism may be related to PI3K/Akt and metabolic pathways, and Bhmt. This provides a new direction for using traditional Chinese medicine to prevent and repair reproductive system injury caused by pollutants.

Metabolomics and Data-Driven Bioinformatics Revealed Key Maternal Metabolites Related to Fetal Lethality via Di(2-ethylhexyl)phthalate Exposure in Pregnant Mice.

We performed serum metabolome analysis of di(2-ethylhexyl)phthalate (DEHP)-exposed and control pregnant mice. Pregnant mice (n = 5) were fed a DEHP-containing diet (0.1% or 0.2% DEHP) or a normal diet (control) from gestational days 0–18. After maternal exposure to 0.2% DEHP there were no surviving fetuses, indicating its strong fetal lethality. There were no significant differences in the numbers of fetuses and placentas between the 0.1% DEHP and control groups, although fetal viability differed significantly between them, suggesting that maternal exposure to 0.1% DEHP could inhibit fetal growth. Metabolomics successfully detected 169 metabolites in serum. Principal component analysis (PCA) demonstrated that the three groups were clearly separated on PCA score plots. The biological interpretation of PC1 was fetal lethality, whereas PC2 meant metabolic alteration of pregnant mice via DEHP exposure without fetal lethality. In particular, the first component was significantly correlated with fetal viability, demonstrating that maternal metabolome changes via DEHP exposure were strongly related to fetal lethality. Levels of some amino acids were significantly increased in the DEHP-exposed groups, whereas those of some fatty acids, nicotinic acid, and 1,5-anhydroglucitol were significantly decreased in the DEHP groups. DEHP-induced increases in glycine levels could cause fetal neurological disorders, and decreases in nicotinic acid could inhibit fetal growth. In addition, a machine-learning Random forest could determine 16 potential biomarkers of DEHP exposure, and data-driven network analysis revealed that nicotinic acid was the most influential hub metabolite in the metabolic network. These findings will be useful for understanding the effects of DEHP on the maternal metabolome in pregnancy and their relationship to fetal lethality.

Di-(2-ethyl hexyl) phthalate induced oxidative stress promotes microplastics mediated apoptosis and necroptosis in mice skeletal muscle by inhibiting PI3K/AKT/mTOR pathway

The plastic decomposition product microplastics (MPs) and the plastic additive Di (2-ethylhexyl) phthalate (DEHP) in the environment can damage various organs of the organism by inducing oxidative stress. The PI3K/AKT/mTOR signaling pathway participate in toxin-induced apoptosis and necroptosis. However, the effects of DEHP/MPs alone and combined exposure on skeletal muscle cell injury in mice and the role of PI3K/AKT/mTOR axis remain unclear. To investigate the effect of DEHP or/and MPs on skeletal muscle in mice and its possible toxicological mechanism, 60 mice were randomly divided into control group, DEHP group (DEHP 200 mg/kg dissolved in 50 mL corn oil mixed with 2.5 kg diet), MPs group (10 mg/L MPs in drinking water) and combined exposure group. In vitro, C2C12 cells were exposed to DEHP 600 μM/MPs 800 μM alone or in combination for 24 h. The results showed that DEHP/MPs exposure alone or in combination increased MDA content, decreased activities of CAT, T-AOC, SOD and GSH-Px, increased mRNA and protein expressions of Caspase-3, BAX, RIPK1, RIPK3 and MLKL, and decreased BCL-2 expression. The expression of PI3K/AKT/mTOR signaling pathway was significantly down-regulated. All the above results showed that the combined exposure group was more toxic, and similar experimental results were obtained by DEHP/MPs exposure test of C2C12 cells in vitro. It is suggested that DEHP/MPs can induce apoptosis and necroptosis by activating oxidative stress and down-regulating PI3K/AKT/mTOR pathway. This study provides new evidence for clarifying the possible mechanism of toxicity of DEHP and MPs to skeletal muscle of mice.

The effects of prenatal and lactational bisphenol A and/or di(2-ethylhexyl) phthalate exposure on female reproductive system

Bisphenol A (BPA) and di(2-ethylhexyl) phthalate (DEHP) are endocrine disrupting chemicals (EDCs) that are abundantly used in polyvinyl chloride plastics, polycarbonates and epoxy resins. Prenatal and early postnatal exposures to EDCs are suggested to be more critical. Such exposures can lead to reprotoxic effects, hormonal and metabolic consequences in adulthood. Moreover, combined exposure to different EDCs can lead to more serious adverse effects, some of which cannot be predicted by examining their individual toxicity profiles. This study aimed to evaluate effects of single and combined prenatal and lactational exposures to BPA and/or DEHP on female reproductive hormones and ovarian follicle development. Pregnant Sprague-Dawley rats were divided randomly to four groups (n = 3/group): Control (received vehicle only); DEHP (30 mg/kg/day); BPA (50 mg/kg/day) and BPA + DEHP (30 mg/kg/day DEHP; 50 mg/kg/day BPA) through 6–21 gestational days and lactation by intra-gastric lavage. Female offspring (n = 6/group) were fed until the end of twelfth postnatal week and then euthanized. Reproductive hormones, ovarian follicle numbers and ovarian development were determined. Plasma testosterone and estradiol levels of BPA and BPA + DEHP groups were significantly lower than control. In BPA group, the number of tertiary ovarian follicles decreased significantly compared to control. In the combined exposure group, the number of corpus luteum (29%), as well as the number of primordial follicles (36%), showed marked decreases compared to control group. It can be suggested that early life exposure to BPA and DEHP may cause late life adverse effects in female reproductive system, especially after combined exposure.

Quercetin ameliorates Di (2-ethylhexyl) phthalate-induced nephrotoxicity by inhibiting NF-κB signaling pathway.

This study aimed to evaluate the possible protective effects of quercetin, a natural flavonoid, against nephrotoxicity induced by Di (2-ethylhexyl) phthalate (DEHP) in kidney tissue of rats and human embryonic kidney (HEK) 293 cell line. The HEK-293 cells were treated with different concentrations of quercetin 24h before treatment with monoethylhexyl phthalate (MEHP). Male rats were treated with 200-mg/kg DEHP, 200-mg/kg DEHP plus quercetin (50 and 100mg/kg), and 200-mg/kg DEHP plus vitamin E (20mg/kg) for 45days by gavage. Quercetin treatment reduced cytotoxicity and oxidative damage inducing by MEHP in HEK-293 cells. The in vivo findings showed that 100-mg/kg quercetin significantly suppressed DEHP-induced kidney damage. For exploring the involved mechanisms, the expressions of nuclear factor E2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), nuclear factor kappa B (NFκB), and tumor necrosis factor alpha (TNFα) genes were determined via real-time Polymerase chain reaction (PCR) assay. High dose of quercetin significantly decreased the gene expressions of NF-κB and TNFα, whereas the alternations of Nrf2 and HO-1 gene expressions were not significant in quercetin groups in compared with DEHP group. These findings suggested that the suppression of DEHP-induced nephrotoxicity via quercetin is correlated, at least in part, with its potential to regulate NF-κB signaling pathway.

Transcriptomic and proteomic characteristics of the di(2-ethylhexyl) phthalate-induced sperm dna damage mouse model.

Di(2-ethylhexyl) phthalate (DEHP) is one of the most common organic pollutants and is added to various plastic products as a plasticizer. DEHP oxidative metabolite content in the human body is associated with DNA damage in sperm and decreased testosterone levels in blood. In this study, a DEHP-induced sperm DNA damage mouse model was replicated and improved, and the transcriptomic and proteomic characteristics of the model were observed. Male mice in the two groups were exposed to DEHP 1g/kg/d or the same amount of normal saline for 60days, and the sperm DNA fragmentation index (DFI) was detected by a sperm chromatin structure assay (SCSA). The mRNA and protein expression profiles of the testis were detected by RNA-seq and data-independent acquisition (DIA). The sperm DFI of the DEHP group was significantly increased. Compared with the control group, 111 differentially expressed genes (DEGs) and 2147 differentially expressed proteins (DEPs), such as Lamb2, Ahnak, Tkt, Dnah8 and Tbl2, were found in the DEHP group. These genes were mainly enriched in metabolic pathways, pathways in cancer and the PI3K-Akt signaling pathway. Our results showed that DEHP 1g/kg/d can induce sperm DNA damage in a male mouse model after 60days of intragastric administration. The reproductive toxicity of DEHP may be related to metabolic pathways in cancer and the PI3K-Akt signaling pathway.

A Chronic-Low-Dose Exposing of DEHP with OECD TG 443 Altered the Histological Characteristics and Steroidogeic Gene Expression of Adrenal Gland in Female Mice.

Phthalates and their metabolites are well-known endocrine disrupting chemicals. Di-(2-ethylhexyl) phthalate (DEHP) has been widely used in industry and the exposing possibility to adult is high. In this study, DEHP was treated (133 μg/L and 1,330 μg/L in drinking water) according to the OECD test guideline 443 to mature female mice and their adrenal gland were examined for histological characteristics and steroidogenic gene expression. The wet weight of the adrenal gland was increased in all administrated groups compared to control. The diameter of zona fasciculata (ZF) was increased by DEHP in both outer ZF and inner ZF but there was no difference in morphology of the cells and arrangements into zona between groups. In addition, the arrangement of extracellular matrix was not different between control and DEHP groups. CYP11B1 was mainly localized at ZF and the intensity was not different between groups. DAX1 was localized in zona glomerulosa (ZG) and ZF, and its expression levels were decreased by DEHP administration. Its level was lower in DEHP133 group than DEHP1330 group. On the other hand, CYP17A1 was localized in ZG of DEHP1330 group. These results suggest that chronic low-dose DEHP exposing may modify the microstructure and function of the adrenal cortical cortex.

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.

DEHP-induced testicular injury through gene methylation pathway and the protective effect of soybean isoflavones in Sprague–Dawley rats

As one of the most important members of Phthalate esters (PAEs), di-(2-ethylhexyl) phthalate (DEHP) is widely used in plastics and known as a male reproductive toxicant. Many studies have shown that soybean isoflavones (SI) can rescue the testicular injury caused by DEHP, but the underlying mechanism is unknown. Because methylation is one of the most important mechanisms for maintaining normal biological functions, we studied whether methylation is involved in testicular injury induced by DEHP and whether SI could counter testicular impairment in peripubertal male Sprague Dawley rats. Compared with the control group, we found that the mRNA levels of testicular Sod2, Gpx1, and Igf-1 significantly decreased in the 900 mg/kg DEHP group (DEHP′ group) (P < 0.01); however, in the DEHP + SI group, the mRNA levels of the genes obviously increased compared with the DEHP’ group (P < 0.01). Simultaneously, the methylation level changes of testicular Sod2, Gpx1, and Igf-1 were similar to the mRNA levels (P < 0.01). Therefore, DEHP may affect testis and leydig cells via inducing methylation of Sod2, Gpx1, and Igf-1, and SI may rescue the impairments at the methylation level. In summary, SI is supposed to be used in DEHP-induced testicular injury treatment.

Differential Effects of Bisphenol A and Di (2-Ethylhexyl) Phthalate Exposure on Crestin and the Expression of Some Genes Related to Apoptosis and Inflammation in Zebrafish Embryos

Objective: Endocrine disrupting chemicals (EDC) in plastics may disrupt proper endocrine system functioning. Zebrafish embryos are formed through external fertilization, and their rapid development, short life cycle, and transparency provide imaging advantages. In zebrafish embryos, neural crest development occurs similarly to other vertebrate embryos and crestin is found in the neural crest during embryogenesis. The aim of our study is to evaluate the effects of bisphenol A (BPA) and Di (2-ethylhexyl) phthalate (DEHP), which are the most widely used EDCs, on the expression of crestin, apoptosis, and inflammation-related parameters in zebrafish embryos. Materials and Methods: The embryos were exposed to either DEHP or BPA in well plates for 72 h post fertilization (hpf). Expressions of crestin were evaluated by in situ hybridization, while the expressions of bax, casp8, casp3a, ifng1, fas, and tp53 were evaluated by RT-PCR. Results: Expressions of bax and casp8 increased and casp3a, ifng1, and fas decreased in BPA and DEHP groups. tp53 expression increased in the BPA group but decreased in the DEHP group compared with the control group. In the DEHP group, casp3a, ifng1, fas, bax, casp8, and tp53 expressions decreased compared with the BPA group. No significant change was observed in the crestin expressions in the groups. When compared with the control group, an inverse relation between ifng1 expression and apoptosis, as evidenced by increased bax and casp8 expressions, was observed in the BPA and DEHP groups. Conclusion: Our study provided important data on the effects of EDCs on the relationship between 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.

Stereological analysis and transcriptome profiling of testicular injury induced by di-(2-ethylhexyl) phthalate in prepubertal rats

Di-(2-ethylhexyl) phthalate (DEHP) is the most common phthalate that can affect the male reproductive system. DEHP exposure at the prepubertal stage could lead to the injury of immature testes, but the mechanism has not been fully clarified. In the present study, we elucidated the possible underlying mechanism of DEHP-induced prepubertal testicular injury through stereological analysis and transcriptome profiling. Compared with the control group, the DEHP-treated rats had lower body weight gain and decreased testicular weight and organ coefficient. Moreover, lower serum levels of testosterone and LH were observed in the DEHP group, in contrast to the increased FSH level. Additionally, the serum level of estradiol had no significant difference after DEHP exposure. Stereological analysis showed significant reduction in volumes of most testicular structures, especially in the seminiferous tubule and seminiferous epithelium, along with a vast decrease of spermatogenic cells and obvious structural damages with substantial pathological signs (germ cracks, cytoplasmic vacuolization, sloughing, multinucleated giant cell formation, chromatolysis desquamation and dissolution, pyknosis of nuclei) in the seminiferous tubule upon DEHP exposure at the prepubertal stage. Furthermore, transcriptome profiling identified 5548 differentially expressed genes (DEGs) upon DEHP exposure. Pathway enrichment analysis revealed several crucial signaling pathways related to retinol metabolism, oxidative phosphorylation, steroid hormone biosynthesis, and cell adhesion molecules (CAMs). In addition, seven DEGs selected from RNA-seq data were validated by quantitative real-time polymerase chain reaction (qRT-PCR), and the results showed the same trends as the RNA-seq results. In conclusion, the above findings provide basic morphological data and lay a foundation for systematic research on transcriptome profiling in prepubertal testicular injury induced by DEHP.

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.

Neuroendocrine disruption by bisphenol A and/or di(2-ethylhexyl) phthalate after prenatal, early postnatal and lactational exposure.

Bisphenol A (BPA) and di(2-ethylhexyl)phthalate (DEHP) are abundant endocrine disrupting chemicals (EDCs). In recent years, studies showed that EDCs may lead to neurodevelopmental diseases. The effects of prenatal exposure to these chemicals may have serious consequences. Moreover, exposure to EDCs as a mixture may have different effects than individual exposures. The present study aimed to determine the toxicity of BPA and/or DEHP on central nervous system (CNS) and neuroendocrine system in prenatal and lactational period in Sprague-Dawley rats. Pregnant rats were randomly divided into four groups: control (received vehicle); BPA group (received BPA at 50mg/kg/day); DEHP group (received DEHP at 30mg/kg/day); and combined exposure group (received both BPA at 50mg/kg/day and DEHP at 30mg/kg/day) during pregnancy and lactation by oral gavage. At the end of lactation, male offspring (n = 6) were randomly grouped. The alterations in the brain histopathology, neurotransmitter levels and enzyme activities in the cerebrum region, oxidative stress markers, and apoptotic effects in the hippocampus region were determined at adulthood. The results showed that exposure to EDCs at early stages of life caused significant changes in lipid peroxidation, total GSH and neurotransmitter levels, and activities of neurotransmitter-related enzymes. Moreover, BPA and/or DEHP led to apoptosis and histopathologic alterations in the hippocampus. Therefore, we can suggest that changes in oxidant/antioxidant status, as well as in neurotransmitters and related enzymes, can be considered as the underlying neurotoxicity mechanisms of BPA and DEHP. However, more mechanistic studies are needed.

Di-2-ethylhexyl phthalate (DEHP) induces apoptosis of mouse HT22 hippocampal neuronal cells via oxidative stress.

Di-2-ethylhexyl phthalate (DEHP) has been widely used as a plasticizer in industry and can affect memory; however, the underlying mechanism remains unclear. In the present study, mouse HT22 cells, an immortalized hippocampal neuronal cell line, was utilized as an in vitro model. We showed that DEHP dramatically inhibited cell viability and increased lactate dehydrogenase (LDH) release from the cells in a dose-dependent manner, suggesting that DEHP could cause cytotoxicity of mouse HT22 cells. The protein levels of cleaved Caspase-8, cleaved Caspase-3, and Bax markedly increased in the DEHP-treated cells, whereas there was a significant decrease in the Bcl-2 protein level, implying that DEHP could induce apoptosis of mouse HT22 cells. DEHP exposure significantly increased the content of malondialdehyde, whereas it markedly decreased the level of glutathione and the activities of glutathione peroxidase and superoxide dismutase, suggesting that DEHP induced oxidative stress of the cells. Compared with the DEHP-treated group, the inhibition of cell viability and the release of LDH were rescued in the N-acetyl-l-cysteine plus DEHP group. Furthermore, inhibition of oxidative stress could rescue the induction of apoptosis by DEHP. Collectively, our results indicated that DEHP could induce apoptosis of mouse HT22 cells via oxidative stress.

Effect of di-(2-ethylhexyl) phthalate (DEHP) on allergic rhinitis

Allergic rhinitis (AR) is a common chronic inflammatory disease of the upper respiratory tract. Di(2-ethylhexyl) phthalate (DEHP) is a widely used plasticizer and belongs to environmental endocrine disruptors (EDCs). It can be entered the human body which is harmful to health. The relationship between DEHP and AR is still inconclusive. This study aims to investigate the effect of environmental pollutants DEHP on AR. By examining DEHP metabolites in the urine of AR patients and building an AR model. 24 BALB/c mice were used as the study subjects, and ovalbumin (OVA) and DEHP (3 mg/kg/body) were used for intragastric administration. They were divided into control group, DEHP group, OVA group and OVA + DEHP group. Examination, behavioral scoring, inflammatory factor testing, oxidative stress testing, detection of aryl hydrocarbon receptor (AhR) and signaling pathways CYP1A1 and CYP1B1 related proteins and mRNA. The concentrations of 3 metabolites of DEHP (MEHHP, MEOHP, and MEHP) in urine of AR patients were higher. And HE-staining showed that for the control group, many chronic inflammatory cell infiltration and nasal mucosal destruction were observed in the OVA + DEHP group and were more severe than the OVA group. Allergic symptom scores were obtained from sneezing, scratching, number of scratching, and nose flow. The scores of the OVA group and the OVA + DEHP group were higher than 7 points. Serum ELISA and nasal mucosal oxidative stress tests are more serious in the OVA + DEHP group. The expression of AhR protein and its mRNA was increased in the DEHP group, OVA group and OVA + DEHP group. The OVA + DEHP group was more significant in the OVA group and DEHP group. And the mRNAs of the AhR-related signaling pathways CYP1A1 and CYP1B1 were also more prominent in the OVA + DEHP group. DEHP may aggravate its inflammatory response through the AhR pathway closely related to the environment. When combined with OVA, DEHP can further aggravate the OVA-induced nasal inflammatory response and make the nasal cavity have undergone severe changes, and many inflammatory cells have infiltrated. DEHP has shown an adjuvant effect, and the AhR-related signaling pathways CYP1A1 and CYP1B1 may be critical.

Combined effects of obesity and di-(2-ethylhexyl) phthalate on testosterone levels and kisspeptin/GPR54 expression in hypothalamus and testes of male mice.

Background:This study evaluated whether obese male mice exposed to di-(2-ethylhexyl) phthalate (DEHP) showed synergistic effects on testosterone levels and the potential underlying mechanism.Methods:Forty-eight male mice were assigned to six groups for 12-week treatments as follows: normal, DEHP100, diet-induced obesity (DIO), DIO + DEHP30, DIO + DEHP100, and DIO + DEHP300. Serum hormone levels, including testosterone (T), luteinizing hormone (LH), and leptin, were detected by ELISA. The levels of Ob-R, kisspeptin, and GPR54 protein expression in hypothalamus and testicular tissues were measured by western blot.Results:There were significantly lower levels of serum T and LH, higher levels of serum leptin and Ob-R, and kisspeptin and GPR54 protein expression were reduced in hypothalamus and testicular tissues in the DIO and DEHP groups compared with controls. Moreover, serum T and leptin levels were more severe in the combined DIO and DEHP exposure group than in the single exposure groups. Serum LH levels and GPR54 expression in the testis were significantly decreased in DIO + DEHP300 mice compared with DIO mice (p < 0.05).Conclusion:Obesity- and DEHP-only exposure had adverse effects on testosterone levels in mice, which may be due to high leptin levels and decreased Ob-R, kisspeptin, and GPR54 expression. Obesity combined with DEHP exposure had an additive adverse effect on testosterone levels in mice. One of the potential mechanisms is higher leptin levels and decreased GPR54 expression in the testes.

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.

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.

Secondary Zinc Deficiency via Exposure to the Environmental Toxicant Di-2-ethylhexyl phthalate Affects Neurogenesis at Embryonic Day 19

Abstract Objectives Zinc deficiency can affect early brain development. We previously found that developmental marginal zinc deficiency affected neurogenesis leading to a lower number of neurons and altered neural specification in the adult rat brain. Zinc deficiency can occur as low dietary zinc intake and secondary to diseases, infections, and exposure to environmental toxicants such as phthalates. This work investigated if gestational exposure to toxicant Di-2-ethylhexyl phthalate (DEHP) could decrease zinc availability to the fetus and altered neurogenesis. Methods Rats were fed an adequate (25 µg zinc/g diet) (C) or a marginal zinc deficient (MZD) (10 µg zinc/g diet), without or with DEHP (300 mg/kg BW) (C + DEHP, MZD + DEHP) from gestational day zero until embryonic day 19 (E19). Zinc concentration was analyzed by atomic absorption spectrometry (AAS). Neurogenesis was evaluated in the offspring at E19 measuring parameters of neural progenitor cells (NPC) proliferation and differentiation by Western blot and/or immunofluorescence. Results Fetal brain zinc concentration was significantly decreased in MZD, C + DEHP and MZD + DEHP than in C. Protein Markers of neurogenesis (NeuN, PAX6, SOX2, TBR2) were lower in MZD and C + DEHP than C, and lowest in MZD + DEHP. The excitatory neuron marker vesicular glutamate transporter 1 (VGLUT1) was lower in C + DEHP, MZD and MZD + DEHP than in C, while the marker of inhibitory neurons glutamic acid decarboxylase (GAD65) level were similar among groups. The ERK1/2 pathway, crucial to neurogenesis, was affected by MZD and DEHP. ERK1/2 activation was lower, and at a similar extent in C + DEHP and MZD groups than in C, while it was markedly lower in the MZD + DEHP group compared to all other groups. Lower ERK1/2 activation could be due to activation of the ERK1/2 phosphatase 2A (PP2A). We found that PP2A activation was higher, in MZD and DEHP than in C, being highest in the MZD + DEHP group. Conclusions Gestational exposure to DEHP in rats causes a secondary zinc deficiency in the fetal brain and altered neurogenesis. This can be due to the inhibition of the ERK1/2 signaling pathway. DEHP exposure can adversely affect the offspring's brain development and result in irreversible consequences to adult brain structure and function. Funding Sources Supported by grants from NIFA CA-D-XXX-7244-H, Packer-Wentz foundation, NIEHS T 32 training grant (T32 ES 0,07059).