Effects of Developmental Lead and Phthalate Exposures on DNA Methylation in Adult Mouse Blood, Brain, and Liver: A Focus on Genomic Imprinting by Tissue and Sex.

BACKGROUND: Maternal exposure to environmental chemicals can cause adverse health effects in offspring. Mounting evidence supports that these effects are influenced, at least in part, by epigenetic modifications. It is unknown whether epigenetic changes in surrogate tissues such as the blood are reflective of similar changes in target tissues such as cortex or liver. OBJECTIVE: We examined tissue- and sex-specific changes in DNA methylation (DNAm) associated with human-relevant lead (Pb) and di(2-ethylhexyl) phthalate (DEHP) exposure during perinatal development in cerebral cortex, blood, and liver. METHODS: Female mice were exposed to human relevant doses of either Pb (32 ppm) via drinking water or DEHP (5mg/kg-day) via chow for 2 weeks prior to mating through offspring weaning. Whole genome bisulfite sequencing (WGBS) was utilized to examine DNAm changes in offspring cortex, blood, and liver at 5 months of age. Metilene and methylSig were used to identify differentially methylated regions (DMRs). Annotatr and ChIP-enrich were used for genomic annotations and gene set enrichment tests of DMRs, respectively. RESULTS: The cortex contained the majority of DMRs associated with Pb (66%) and DEHP (57%) exposure. The cortex also contained the greatest degree of overlap in DMR signatures between sexes (n=13 and 8 DMRs with Pb and DEHP exposure, respectively) and exposure types (n=55 and 39 DMRs in males and females, respectively). In all tissues, detected DMRs were preferentially found at genomic regions associated with gene expression regulation (e.g., CpG islands and shores, 5' UTRs, promoters, and exons). An analysis of GO terms associated with DMR-containing genes identified imprinted genes to be impacted by both Pb and DEHP exposure. Of these, Gnas and Grb10 contained DMRs across tissues, sexes, and exposures, with some signatures replicated between target and surrogate tissues. DMRs were enriched in the imprinting control regions (ICRs) of Gnas and Grb10, and we again observed a replication of DMR signatures between blood and target tissues. Specifically, we observed hypermethylation of the Grb10 ICR in both blood and liver of Pb-exposed male animals. CONCLUSIONS: These data provide preliminary evidence that imprinted genes may be viable candidates in the search for epigenetic biomarkers of toxicant exposure in target tissues. Additional research is needed on allele- and developmental stage-specific effects, as well as whether other imprinted genes provide additional examples of this relationship. https://doi.org/10.1289/EHP14074.

High concentrations of phthalates affect the early development of the sea urchin Paracentrotus lividus.

The toxicity of three phthalates (PAEs) - butylbenzyl phthalate (BBP), diethyl phthalate (DEP), and di-(2-ethylhexyl) phthalate (DEHP) - was tested on the Mediterranean sea urchin Paracentrotus lividus. Fertilized eggs were exposed to environmental and high PAE concentrations for 72 h. The potential toxic effects on larval development and any morphological anomalies were then assessed to estimate PAEs impact. Environmental concentrations never affected development, while high concentrations induced toxic effects in larvae exposed to BBP (EC50: 2.9 ×103 µg/L) and DEHP (EC50: 3.72 ×103 µg/L). High concentrations caused skeletal anomalies, with a slight to moderate impact for DEP/DEHP and BBP, respectively. PAE toxicity was: BBP>DEHP>DEP. In conclusion, the three PAEs at environmental concentrations do not pose a risk to sea urchins. However, PAE concentrations should be further monitored in order not to constitute a concern to marine species, especially at their early developmental stages.

Maternal Di-(2-ethylhexyl)-Phthalate exposure during pregnancy altered energy metabolism in immature offspring and caused hyperglycemia.

Di-(2-ethylhexyl)-phthalate (DEHP), as distinctive endocrine disrupting chemicals, has become a global environmental pollutant harmful to human and animal health. However, the impacts on offspring and mothers with maternal DEHP exposure are largely unknown and the mechanism remains elusive. We established DEHP-exposed maternal mice to investigate the impacts on mother and offspring and illustrate the mechanism from multiple perspectives. Pregnant mice were administered with different doses of DEHP, respectively. Metagenomic sequencing used fecal and transcriptome sequencing using placentas and livers from offspring have been performed, respectively. The results of the histopathology perspective demonstrated that DEHP exposure could disrupt the function of islets impact placentas and fetus development for maternal mice, and cause the disorder of glucose and lipid metabolism for immature offspring mice, resulting in hyperglycemia. The results of the metagenome of gut microbial communities indicated that the dysbiosis of gut microbiota in mother and offspring mice and the dominant phyla transformed through vertical transmission. Transcriptome analysis found DEHP exposure induced mutations of Ahcy and Gstp3, which can damage liver cells and affect the metabolism of the host. DEHP exposure harms pregnant mice and offspring by affecting gene expression and altering metabolism. Our results suggested that exposure of pregnant mice to DEHP during pregnancy and lactation increased the risk of metabolic disorders by altering key genes in liver and gut microbiota, and these results provided new insights into the potential long-term harms of DEHP.

Assessment of biodegradation and toxicity of alternative plasticizer di(2-ethylhexyl) terephthalate: Impacts on microbial biofilms, metabolism, and reactive oxygen species-mediated stress response.

Although di(2-ethylhexyl) terephthalate (DOTP) is being widely adopted as a non-phthalate plasticizer, existing research primarily focuses on human and rat toxicity. This leaves a significant gap in our understanding of their impact on microbial communities. This study assessed the biodegradation and toxicity of DOTP on microbes, focusing on its impact on biofilms and microbial metabolism using Rhodococcus ruber as a representative bacterial strain. DOTP is commonly found in mass fractions between 0.6 and 20% v/v in various soft plastic products. This study used polyvinyl chloride films (PVC) with varying DOTP concentrations (range 1-10% v/v) as a surface for analysis of biofilm growth. Cell viability and bacterial stress responses were tested using LIVE/DEAD™ BacLight™ Bacterial Viability Kit and by the detection of reactive oxygen species using CellROX™ Green Reagent, respectively. An increase in the volume of dead cells (in the plastisphere biofilm) was observed with increasing DOTP concentrations in experiments using PVC films, indicating the potential negative impact of DOTP on microbial communities. Even at a relatively low concentration of DOTP (1%), signs of stress in the microbes were noticed, while concentrations above 5% compromised their ability to survive. This research provides a new understanding of the environmental impacts of alternative plasticizers, prompting the need for additional research into their wider effects on both the environment and human health.

Maslinic acid supplementation prevents di(2-ethylhexyl) phthalate-induced apoptosis via PRDX6 in peritubular myoid cells of Chinese forest musk deer.

Chinese forest musk deer (FMD), an endangered species, have exhibited low reproductive rates even in captivity due to stress conditions. Investigation revealed the presence of di(2-ethylhexyl) phthalate (DEHP), an environmental endocrine disruptor, in the serum and skin of captive FMDs. Feeding FMDs with maslinic acid (MA) has been observed to alleviate the stress response and improve reproductive rates, although the precise molecular mechanisms remain unclear. Therefore, this study aims to investigate the molecular mechanisms underlying the alleviation of DEHP-induced oxidative stress and cell apoptosis in primary peritubular myoid cells (PMCs) through MA intake. Primary PMCs were isolated and exposed to DEHP in vitro. The results demonstrated that DEHP significantly suppressed antioxidant levels and promoted cell apoptosis in primary PMCs. Moreover, interfering with the expression of PRDX6 was found to induce excessive reactive oxygen species (ROS) production and cell apoptosis in primary PMCs. Supplementation with MA significantly upregulated the expression of PRDX6, thereby attenuating DEHP-induced oxidative stress and cell apoptosis in primary PMCs. These findings provide a theoretical foundation for mitigating stress levels and enhancing reproductive capacity of in captive FMDs.

Thyroid and sex hormone disrupting effects of DEHTP at different life stages of zebrafish (Danio rerio).

Di(2-ethylhexyl) terephthalate (DEHTP) is an alternative plasticizer widely used in numerous consumer products, replacing di(2-ethylhexyl) phthalate (DEHP). Hence, DEHTP has been frequently detected in the environment and humans. As a structural isomer and functional analog of DEHP, DEHTP is a suspected endocrine disruptor. Here, we evaluated thyroid-disrupting effects of DEHTP using embryo-larval and adult male zebrafish. We also investigated its sex hormone disruption potential in the adult zebrafish. After 5- and 7-days of exposure to DEHTP, significant increases in whole-body thyroid hormonal levels were observed in the larval fish. Down-regulation of several thyroid-regulating genes, including trh, tshß, nis, and dio2, was observed, but only after 5-day exposure. Following a 21-day exposure, the adult male zebrafish exhibited a significant decrease in total triiodothyronine and an increase in thyroid-stimulating hormones. Potential changes in the deiodination of thyroid hormones, supported by the up-regulation of two deiodinases, dio1 and dio3a, along with the down-regulation of dio2, could explain the thyroid hormone changes in the adult zebrafish. Moreover, significant trends of decrease in estradiol and 11-ketotestosterone, along with increase of testosterone (T), were observed in the adult zebrafish. Up-regulation of several steroidogenic genes may explain elevated T, while exact mechanisms of action warrant further investigation. Our results demonstrate that DEHTP can cause disruptions of thyroid and sex hormones at different life stages in zebrafish.

Prenatal diethylhexylphthalate exposure disturbs adult Leydig cell function via epigenetic downregulation of METTL4 expression in male rats.

Prenatal exposure to diethylhexyl phthalate (DEHP) has been linked with a decline in testosterone levels in adult male rats, but the underlying mechanism remains unclear. We investigated the potential epigenetic regulation, particularly focusing on N6-methyladenosine (m6A) modification, as a possible mechanism. Dams were gavaged with DEHP (0, 10, 100, and 750 mg/kg/day) from gestational day 14 to day 21. The male offspring were examined at the age of 56 days. Prenatal DEHP administration at 750 mg/kg/day caused a decline in testosterone concentrations, an elevation in follicle-stimulating hormone, a downregulated expression of CYP11A1 HSD3B2, without affecting Leydig cell numbers. Interestingly, Methyltransferase Like 4 (METTL4), an m6A methyltransferase, was downregulated, while there were no changes in METTL3 and METTL14. Moreover, CYP11A1 showed m6A reduction in response to prenatal DEHP exposure. Additionally, METTL4 expression increased postnatally, peaking in adulthood. Knockdown of METTL4 resulted in the downregulation of CYP11A1 and HSD3B2 and an increase in SCARB1 expression. Furthermore, the increase in autophagy protection in adult Leydig cells induced by prenatal DEHP exposure was not affected by 3-methyladenosine (3MA) treatment, indicating a potential protective role of autophagy in response to DEHP exposure. In conclusion, prenatal DEHP exposure reduces testosterone by downregulating CYP11A1 and HSD3B2 via m6A epigenetic regulation and induction of autophagy protection in adult Leydig cells as a response to DEHP exposure.

Phthalate Exposure and Coronary Heart Disease: Possible Implications of Oxidative Stress and Altered miRNA Expression.

The relationship between phthalate exposure and coronary heart disease (CHD) is still unclear. This study aimed to investigate the association between phthalate exposure and CHD and determine the possible atherogenic mechanisms of phthalates by assessing oxidative stress and altering miRNA expression. This case-control study included 110 participants (55 CHD patients and 55 healthy controls). The levels of oxidative stress markers, malondialdehyde (MDA), and superoxide dismutase (SOD), and the expression of miRNA-155 (miR-155) and miRNA-208a (miR-208a), were measured and correlated with the urinary mono-2-ethylhexyl phthalate (MEHP). Highly significant differences were detected between the CHD cases and the control group regarding MEHP, MDA, SOD, miR-155, and miR-208a (p-value < 0.001). Spearman correlations revealed a significant positive correlation between MDA and MEHP in urine (P = 0.001 and rs = 0.316) and a significant negative correlation between SOD and MEHP in urine (P < 0.001 and rs = -0.345). Furthermore, significant positive correlations were observed between miR-155 and urinary MEHP (P = 0.001 and rs = 0.318) and miR-208a and urinary MEHP (P < 0.001 and rs = -0.352). This study revealed an association between phthalate exposure, as indicated by urinary MEHP and CHD; altered expression of miR-155 and miR-208a and oxidative stress could be the fundamental mechanisms.

A novel perspective on di-hexyl phthalate (2-ethylhexyl)-induced reproductive toxicity in females: Lipopolysaccharide synergizes with mono-2-ethylhexyl ester to cause inflammatory apoptosis rather than autophagy in ovarian granulosa cells.

Di-hexyl phthalate (2-ethylhexyl) (DEHP) has been confirmed to cause female reproductive toxicity in humans and model animals by affecting the survival of ovarian granulosa cells (GCs), but the interrelationships between DEHP's on autophagy, apoptosis, and inflammation in GCs are not clear. Our previous study demonstrated that DEHP exposure resulted in the disturbance of intestinal flora associated with serum LPS release, which in turn led to impaired ovarian function. LPS has also been shown to determine cell fate by modulating cellular autophagy, apoptosis, and inflammation. Therefore, this study investigated the role and link between LPS and autophagy, apoptosis, and inflammation of GCs in DEHP-induced ovarian injury. Here, we constructed an in vivo injury model by continuous gavage of 0-1500 mg/kg of DEHP in female mice for 30 days and an in vitro injury model by treatment of human ovarian granulosa cells (KGN) cells with mono-2- ethylhexyl ester (MEHP, an active metabolite of DEHP in vivo). In addition, the expression of relevant pathway molecules was detected by immunohistochemistry, immunofluorescence, qRT-PCR, and Western blotting after the addition of the autophagy inhibitor 3-methyladenine (3-MA), the apoptosis inhibitor Z-VAD- FMK and the NF-κB inhibitor BAY11-7082. The current study found that autophagy and apoptosis were significantly activated in GCs of DEHP-induced atretic follicles in vivo and found that MEHP-induced KGN cells autophagy and apoptosis were independent and potentially cytotoxic of each other in vitro. Further studies confirmed that DEHP exposure resulted in LPS release from the intestinal tract and entering the ovary, thereby participating in DEHP-induced inflammation of GCs. In addition, we found that exogenous LPS synergized with MEHP could activate the NF-κB signaling pathway to induce inflammation and apoptosis of GCs in a relatively prolonged exposure condition. Meanwhile, inhibition of inflammatory activation could rescue apoptosis and estrogen secretion function of GCs induced by MEHP combined with LPS. These results indicated that the increased LPS influenced by DEHP might cooperate with MEHP to induce inflammatory apoptosis of GCs, an important cause of ovarian injury in mice.

Recent advances in toxicological research of di-(2-ethylhexyl)-phthalate: Focus on endoplasmic reticulum stress pathway.

The plasticizer di-(2-ethylhexyl)-phthalate (DEHP) is the most significant phthalate in production, usage, and environmental occurrence. DEHP is found in products such as personal care products, furniture materials, cosmetics, and medical devices. DEHP is noncovalently bind with plastic therefore, repeated uses lead to leaching out of it. Exposure to DEHP plasticizers leads to toxicity in essential organs of the body through various mechanisms. The main objective of this review article is to focus on the DEHP-induced endoplasmic reticulum (ER) stress pathway implicated in the testis, brain, lungs, kidney, heart, liver, and other organs. Not only ER stress, PPAR-related pathways, oxidative stress and inflammation, Ca2+ homeostasis disturbances in mitochondria are also identified as the relative mechanisms. ER is involved in various critical functions of the cell such as Protein synthesis, protein folding, calcium homeostasis, and lipid peroxidation but, DEHP exposure leads to augmentation of misfolded/unfolded protein. This review complies with various recently reported DEHP-induced toxicity studies and some pharmacological interventions that have been shown to be effective through ER stress pathway. DEHP exposure does assess health risks and vulnerability to populations across the globe. This study offers possible targets and approaches for addressing various DEHP-induced toxicity.

Effect of MEHP on testosterone synthesis via Sirt1/Foxo1/Rab7 signaling pathway inhibition of lipophagy in TM3 cells.

Mono-2-ethylhexyl phthalic acid (MEHP) is the most toxic metabolite of the plasticizer di-2-ethylhexyl phthalic acid (DEHP), and studies have shown that MEHP causes serious reproductive effects. However, its exact mechanisms of action remain elusive. In this study, we aimed to investigate the reproductive effects of MEHP and preliminarily explore its underlying molecular mechanisms. We found that TM3 cells gradually secreted less testosterone and intracellular free cholesterol with increasing MEHP exposure. MEHP exposure inhibited lipophagy and the Sirt1/Foxo1/Rab7 signaling pathway in TM3 cells, causing aberrant accumulation of intracellular lipid droplets. Addition of the Sirt1 agonist SRT1720 and Rab7 agonist ML-098 alleviated the inhibition of lipophagy and increased free cholesterol and testosterone contents in TM3 cells. SRT1720 alleviated the inhibitory effect of MEHP on the Sirt1/Foxo1/Rab7 signaling pathway, whereas ML-098 only alleviated the inhibition of Rab7 protein expression by MEHP and had no effect on Sirt1 and Foxo1 protein expression. This suggests that MEHP inhibits lipophagy in TM3 cells by suppressing the Sirt1/Foxo1/Rab7 signaling pathway, ultimately leading to a further decrease in cellular testosterone secretion. This study improves our current understanding of the toxicity and molecular mechanisms of action of MEHP and provides new insights into the reproductive effects of phthalic acid esters.

Modulating responses of indicator genes in cellular homeostasis, immune defense and apoptotic process in the Macrophthalmus japonicus exposed to di(2-ethylhexyl) phthalate as a plastic additive.

Di(2-ethylhexyl) phthalate (DEHP), have been increasingly used as plasticizers to manufacture soft and flexible materials and ubiquitously found in water and sediments in the aquatic ecosystem. The aim of the present study was to evaluate the effect of DEHP exposure on cellular homeostasis (HSF1 and seven HSPs), immune responses (ILF), and apoptotic responses (p53, BAX, Bcl-2). DEHP exposure upregulated the expression of HSF1 and ILF. Moreover, it altered the expression levels of HSPs (upregulation of HSP70, HSP90, HSP40, HSP83, and HSP67B2 and downregulation of HSP60 and HSP21) in conjunction with HSF1 and ILF in the gills and hepatopancreas of M. japonicus exposed to DEHP. At the protein level, DEHP exposure changed apoptotic signals in both tissues of M. japonicus. These findings indicate that chronic exposures to several DEHP concentrations could disturb cellular balance, damage the inflammatory and immune systems, and induce apoptotic cell death, thereby affecting the survival of M. japonicus.

Exposure to the phthalate metabolite MEHP impacts survival and growth of human ovarian follicles in vitro.

Phthalates are found in everyday items like plastics and personal care products. There is an increasing concern that continuous exposure can adversely affect female fertility. However, experimental data are lacking to establish causal links between exposure and disease in humans. To address this gap, we tested the effects of a common phthalate metabolite, mono-(2-ethylhexyl) phthalate (MEHP), on adult human ovaries in vitro using an epidemiologically determined human-relevant concentration range (2.05 nM - 20.51 mM). Histomorphological assessments, steroid and cytokine measurements were performed on human ovarian tissue exposed to MEHP for 7 days in vitro. Cell viability and gene expression profile were investigated following 7 days of MEHP exposure using the human granulosa cancer cell lines KGN, and COV434, the germline tumor cell line PA-1, and human ovarian primary cells. Selected differentially expressed genes (DEGs) were validated by RT-qPCR and immunofluorescence in human ovarian tissue. MEHP exposure reduced follicular growth (20.51 nM) and increased follicular degeneration (20.51 mM) in ovarian tissue, while not affecting steroid and cytokine production. Out of the 691 unique DEGs identified across all the cell types and concentrations, CSRP2 involved in cytoskeleton organization and YWHAE as well as CTNNB1 involved in the Hippo pathway, were chosen for further validation. CSRP2 was upregulated and CTNNB1 downregulated in both ovarian tissue and cells, whereas YWHAE was downregulated in cells only. In summary, one-week MEHP exposure of human ovarian tissue can perturb the development and survival of human follicles through mechanisms likely involving dysregulation of cytoskeleton organization and Hippo pathway.

Unraveling DEHP influence on hemoglobin S polymerization in sickle cell disease: Ex vivo, in vitro and in silico analysis.

Sickle cell disease (SCD) is a hereditary hemoglobinopathy, caused by a mutation at position 6 of the ß-globin chain and patients are frequently exposed to several blood transfusions in order to maintain physiological function. Transfusion blood bags are composed of PVC and phthalates (as DEHP) are often introduced to the material in order to confer malleability. In this sense, DEHP can easily elute to the blood and cause harmful effects. This study aimed to unravel DEHP effect on SCD patient's hemoglobin function. We found that HbS polymerization using whole erythrocytes is decreased by DEHP in ex vivo experiments and this effect might be mediated by the DEHP-VAL6 interaction, evaluated in silico. Isolated HbS exhibited less polymerization at low DEHP concentrations and increased polymerization rate at higher concentration. When analyzing the propensity to aggregate, HbS is more inclined to aggregate when compared to HbA due to the residue 6 mutation. Circular dichroism showed characteristic hemoglobin peaks for oxygenated HbS that are lost when oxygen is sequestered, and DEHP at higher concentration mildly recovers a peak close to the second hemoglobin one. Finally, by transmission electron microscopy we demonstrated that high DEHP concentration increased polymer formation with a more organized structure. These findings show for the first-time the beneficial effect of low-dose DEHP on HbS polymerization.

Role of formyl peptide receptor 2 in steatosis of L02 cells exposed to Mono-(2-ethylhexyl) phthalate.

Mono-(2-ethylhexyl) phthalate (MEHP) can accumulate in the liver and then lead to hepatic steatosis, while the underlying mechanism remains unclear. Inflammation plays an important role in the disorder of hepatic lipid metabolism. This study aims to clarify the role of the inflammatory response mediated by formyl peptide receptor 2 (FPR2) in steatosis of L02 cells exposed to MEHP. L02 cells were exposed to MEHP of different concentrations and different time. A steatosis model of L02 cells was induced with oleic acid and the cells were exposed to MEHP simultaneously. In addition, L02 cells were incubated with FPR2 antagonist and then exposed to MEHP. Lipid accumulation was determined by oil red O staining and extraction assay. The indicators related to lipid metabolism and inflammatory response were measured with appropriate kits. The relative expression levels of FPR2 and its ligand were determined by Western blot, and the interaction of them was detected by co-immunoprecipitation. As a result, MEHP exposure could promote the occurrence and progression of steatosis and the secretion of chemokines and inflammatory factors in L02 cells. MEHP could also affect the expression and activation of FPR2 and the secretion of FPR2 ligands. In addition, the promotion effect of MEHP on the secretion of total cholesterol and interleukin 1ß in L02 cells could be significantly inhibited by the FPR2 antagonist. We concluded that FPR2 might affect the promotion effect of MEHP on steatosis of L02 cells by mediating inflammatory response.

Identification of sensitive endpoints for the assessment of phthalates-induced reproductive and developmental toxicity: A literature mining study.

Dibutyl phthalate (DBP) and di(2-ethylhexyl) phthalate (DEHP), two common types of phthalates, are known to cause reproductive and developmental toxicity in animals and humans. The reference doses (RfD) of DBP and DEHP should be determined by sensitive endpoints. We here aimed to identify sensitive endpoints for DBP- and DEHP-induced such toxicity using published literatures. By examining the impacts of maternal exposure to DBP or DEHP on anogenital distance (AGD) and semen quality of offspring, we discovered that DBP or DEHP caused AGD decline in boys but increase in girls with DBP being more potent and the first 14weeks of pregnancy being more susceptible, suggesting a chemical- and time-dependent phenomenon. We also identified AGD shortening and total sperm count reduction as two sensitive endpoints for DBP- or DEHP-induced reproductive and developmental toxicity, respectively. Based upon these two endpoints and the employment of the Bayesian benchmark dose approach with an uncertainty factor of 3,000, we estimated the RfD values of DBP and DEHP were 15 µg/kg/day and 36 µg/kg/day, respectively. Thus, we uncover previously unrecognized phenomena of DBP- or DEHP-induced reproductive and developmental toxicity and establish new and comparable or more conservative RfDs for the risk assessment of phthalates exposure in humans.

Effects of Lactobacillus salivarius ssp. salicinius SA-03 Supplementation on Reversing Phthalate-Induced Asthma in Mice.

Probiotics may protect against asthma. We want to investigate whether probiotics can reverse the adverse effects of phthalate exposure on asthma. We selected the female offspring of BALB/c mice, born from pregnant female mice fed with diethylhexyl phthalate (DEHP). They were continuously administrated DEHP and Lactobacillus salivarius ssp. salicinius SA-03 when they were 5 weeks old, and ovalbumin (OVA) for asthma induction started at 6 weeks for 32 days. The mice were divided into four groups (n = 6/group): 1. control group (C), 2. OVA/DEHP group (OD), 3. OVA/DEHP/probiotics low-dose group (ODP-1X), and OVA/DEHP/probiotics high-dose group (ODP-5X). We found that the administration of probiotics significantly reduced the asthma severity of the mice, as well as serum IgE and IL-5. In the ODP-5X group, the proportion of CD4+ cells in the lung was reduced, whereas IL-10 in serum and CD8+ cells in BALF were increased. In histopathology, the ODP group showed reduced infiltration of inflammatory cells, bronchial epithelial cell hyperplasia, and tracheal mucus secretion. These results might indicate that high-dose probiotics may affect anti-inflammatory cytokines and reduce asthma-relative indicators. The above results may provide evidence that high-dose probiotics supplementation might play a modulating role in DEHP causes of allergic asthma in the pediatric animal model.

DEHP induces apoptosis and autophagy of the thyroid via Rap1 signaling pathway: In vivo and in vitro study.

OBJECTIVE: DEHP has thyroid toxicity and affects thyroid function. However, the mechanism is unclear. METHODS: The offspring of SD rats were gavaged with different doses of DEHP from in utero to 8 or 12 weeks old. We observed the thyroid morphology with HE and autophagosomes with TEM. The THs levels were tested with ELISA. The apoptosis level was tested by flow cytometry. The levels of apoptosis-related genes, autophagy-related genes and Rap1 pathway genes, were measured with qRT-PCR and Western blot. We established an MEHP-treated Nthy-ori 3-1 cell model and inhibited the Rap1 to verify the mechanism. RESULTS: DEHP could cause pathological damage and ultrastructure damage of thyroids in offspring rats. After DEHP exposure, the THs levels were altered, the apoptosis levels increased, and autophagosomes appeared. DEHP significantly affected the levels of apoptosis-related genes and autophagy-related genes. DEHP also affected the levels of Rap1 pathway, which was correlated with the levels of apoptosis and autophagy. After inhibiting Rap1 in Nthy-ori 3-1 cells, the THs levels were altered. Rap1 pathway was inhibited and the levels of apoptosis and autophagy were down-regulated. CONCLUSION: DEHP could induce the apoptosis and autophagy of the thyroid, and Rap1 signaling pathway may play a significant role.