Di-(2-Ethylhexyl)-Phthalate (DEHP) Causes Impaired Adipocyte Function and Alters Serum Metabolites

Di-(2-ethylhexyl)-phthalate (DEHP), an ubiquitous environmental contaminant, has been shown to cause adverse effects on glucose homeostasis and insulin sensitivity in epidemiological studies, but the underlying mechanisms are still unknown. We therefore tested the hypothesis that chronic DEHP exposure causes impaired insulin sensitivity, affects body weight, adipose tissue (AT) function and circulating metabolic parameters of obesity resistant 129S6 mice in vivo. An obesity-resistant mouse model was chosen to reduce a potential obesity bias of DEHP effects on metabolic parameters and AT function. The metabolic effects of 10-weeks exposure to DEHP were tested by insulin tolerance tests and quantitative assessment of 183 metabolites in mice. Furthermore, 3T3-L1 cells were cultured with DEHP for two days, differentiated into mature adipocytes in which the effects on insulin stimulated glucose and palmitate uptake, lipid content as well as on mRNA/protein expression of key adipocyte genes were investigated. We observed in female mice that DEHP treatment causes enhanced weight gain, fat mass, impaired insulin tolerance, changes in circulating adiponectin and adipose tissue Pparg, adiponectin and estrogen expression. Serum metabolomics indicated a general increase in phospholipid and carnitine concentrations. In vitro, DEHP treatment increases the proliferation rate and alters glucose uptake in adipocytes. Taken together, DEHP has significant effects on adipose tissue (AT) function and alters specific serum metabolites. Although, DEHP treatment led to significantly impaired insulin tolerance, it did not affect glucose tolerance, HOMA-IR, fasting glucose, insulin or triglyceride serum concentrations. This may suggest that DEHP treatment does not cause impaired glucose metabolism at the whole body level.     

Di (2-ethylhexyl) Phthalate Exposure Impairs Growth of Antral Follicle in Mice

Di (2-ethylhexyl) phthalate (DEHP) is a widely used plastic additive. As an environmental endocrine disruptor, it has been shown to be harmful to the mammalian reproductive system. Previous studies indicated that DEHP inhibited the development of mouse ovarian follicles. However, the mechanisms by which DEHP affects ovarian antral follicle development during the pre-puberty stage are poorly understand. Thus, we investigated the effects of direct DEHP exposure on antral follicle growth in pre-pubescent mice by use of intraperitoneal injection. Our results demonstrated that the percentage of large antral follicles was significantly reduced when mice were exposed to 20 or 40 μg/kg DEHP every 5 days from postnatal day 0 (0 dpp) to 15 dpp. In 20 dpp, we performed microarray of these ovaries. The microarray results indicated that mRNA levels of apoptosis related genes were increased. The mRNA levels of the apoptosis and cell proliferation (negative) related genes Apoe, Agt, Glo1 and Grina were increased after DEHP exposure. DEHP induced the differential gene expression of Hsp90ab1, Rhoa, Grina and Xdh which may play an important role in this process. In addition, TUNEL staining and immunofluorescence showed that DEHP exposure significantly increased the number of TUNEL, Caspase3 and γH2AX positive ovarian somatic cells within the mouse ovaries. Flow cytometer analyses of redox-sensitive probes showed that DEHP caused the accumulation of reactive oxygen species. Moreover, the mRNA expression of ovarian somatic cell antioxidative enzymes was down-regulated both in vivo and in vitro. In conclusion, our data here demonstrated that DEHP exposure induced oxidative stress and ovarian somatic cell apoptosis, and thus may impact antral follicle enlargement during the pre-pubertal stage in mice.     

Identification of nucleoporin 93 (Nup93) that mediates antiviral innate immune responses

RIG-I-like receptors (RLRs) are cytoplasmic sensors for viral RNA that elicit antiviral innate immune responses. RLR signaling culminates in the activation of the protein kinase TBK1, which mediates phosphorylation and nuclear translocation of IRF3 that regulates expression of type I interferon genes. Here, we found that Nucleoporin 93 (Nup93), components of nuclear pore complex (NPC), plays an important role in RLR-mediated antiviral responses. Nup93-deficient RAW264.7 macrophage cells exhibited decreased expression of Ifnb1 and Cxcl10 genes after treatment with a synthetic RLR agonist stimulation as well as Newcastle Disease Virus infection. Silencing Nup93 in murine primary macrophages and embryonic fibroblasts also resulted in reduced expression of these genes. IRF3 nuclear translocation during RLR signaling was impaired in Nup93-deficient RAW264.7 cells. Notably, the activation of TBK1 during RLR signaling was also decreased in Nup93-deficient cells. We found that Nup93 formed a complex with TBK1, and Nup93 overexpression enhanced TBK1-mediated IFNβ promoter activation. Taken together, our findings suggest that Nup93 regulates antiviral innate immunity by enhancing TBK1 activity and IRF3 nuclear translocation.     

Effects of diethylhexyl phthalate (DEHP) given neonatally on spermatogenesis of mice

We previously demonstrated that the effects of diethylhexyl phthalate (DEHP) alter reproduction function on male mice. Immature male mice were treated daily with DEHP from postnatal day 7–21, 7–35, 7–49, in a dose-dependent manner. As results, both the quality and quantity of spermatozoa were decreased in 60-day-old mice. The results by RT-PCR analysis indicated that DDx3Y, Usp9Y, RBM, E1F1AY, EGF, FSHR and EGFR genes were down-regulated, and LHR, Cyp17a1 and Cyp19a1 were down-regulated in response to DEHP. These genes were selected based on their markedly increased or decreased expression levels. However, DEHP had no effect on the meiotic process and recombination levels in male mouse germ cells. Treatment with DEHP induced histopathological changes in the testes. Taken together, these results provide a new insight into the molecular mechanisms underlying the detrimental impacts of DEHP in humans and wildlife.     

Exposure to diethylhexyl phthalate (DEHP) results in a heritable modification of imprint genes DNA methylation in mouse oocytes

Diethylhexyl phthalate (DEHP) is an estrogen-like compound widely used as a plasticizer in commercial products and is present in medical devices, and common household items. It is considered an endocrine disruptor since studies on experimental animals clearly show that exposure to DEHP can alter epigenetics of germ cells. This study was designed to assess the effects of DEHP on DNA methylation of imprinting genes in germ cells from fetal and adult mouse. Pregnant mice were treated with DEHP at doses of 0 and 40 μg DEHP/kg body weight/day from 0.5 to 18.5 day post coitum. The data revealed DEHP exposure significantly reduced the percentage of methylated CpG sites in Igf2r and Peg3 differentially methylated regions (DMRs) in primordial germ cells from female and male fetal mouse, particularly, in the oocytes of 21 dpp mice (F1), which were produced by the pregnant micetreated with DEHP. More surprisingly, the modification of the DNA methylation of imprinted genes in F1 mouse oocytes was heritable to F2 offspring which exhibit lower percentages of methylated CpG sites in imprinted genes DMRs. In conclusion, DEHP exposure can affect the DNA methylation of imprinting genes not only in fetal mouse germ cells and growing oocytes, but also in offspring’s oocytes.     

Toxicogenomic Screening of Replacements for Di(2-Ethylhexyl) Phthalate (DEHP) Using the Immortalized TM4 Sertoli Cell Line

Phthalate plasticizers such as di(2-ethylhexyl) phthalate (DEHP) are being phased out of many consumer products because of their endocrine disrupting properties and their ubiquitous presence in the environment. The concerns raised from the use of phthalates have prompted consumers, government, and industry to find alternative plasticizers that are safe, biodegradable, and have the versatility for multiple commercial applications. We examined the toxicogenomic profile of mono(2-ethylhexyl) phthalate (MEHP, the active metabolite of DEHP), the commercial plasticizer diisononyl cyclohexane-1,2-dicarboxylate (DINCH), and three recently proposed plasticizers: 1,4-butanediol dibenzoate (BDB), dioctyl succinate (DOS), and dioctyl maleate (DOM), using the immortalized TM4 Sertoli cell line. Results of gene expression studies revealed that DOS and BDB clustered with control samples while MEHP, DINCH and DOM were distributed far away from the control-DOS-BDB cluster, as determined by principle component analysis. While no significant changes in gene expression were found after treatment with BDB and DOS, treatment with MEHP, DINCH and DOM resulted in many differentially expressed genes. MEHP upregulated genes downstream of PPAR and targeted pathways of cholesterol biosynthesis without modulating the expression of PPAR’s themselves. DOM upregulated genes involved in glutathione stress response, DNA repair, and cholesterol biosynthesis. Treatment with DINCH resulted in altered expression of a large number of genes involved in major signal transduction pathways including ERK/MAPK and Rho signalling. These data suggest DOS and BDB may be safer alternatives to DEHP/MEHP than DOM or the commercial alternative DINCH.     

Expression of Chironomus riparius serine-type endopeptidase gene under di-(2-ethylhexyl)-phthalate (DEHP) exposure

Environmental stressors can induce changes in gene expression that can be useful as biomarkers. To identify potential biomarkers of water quality, we characterized full-length cDNA sequences of the serine-type endopeptidase (SP) gene from Chironomus riparius. Their expression was analyzed during different life-history stages and in response to treatment with various concentrations of di(2-ethylhexyl) phthalate (DEHP) for short and long periods of time. A comparative molecular and phylogenetic investigation was then conducted among different orders of insects using sequence database analysis. The sequence of the C. riparius SP gene was found to be most closely related to the sequence of SPs isolated from Aedes aegypti. In addition, the basal level of C. riparius SP mRNA was more highly expressed in larvae than in other life-history stages. However, the expression of C. riparius SP was primarily limited to the gut in larvae. When the effects of short-term exposure to DEHP were evaluated, C. riparius SP gene expression decreased within 1 h of treatment, regardless of dose. We also investigated expression of the C. riparius SP gene following long-term DEHP exposure (10 days) and found that it decreased significantly across all DEHP dosages. Finally, the response of the SP gene was more sensitive in C. riparius that were exposed to low concentrations of DEHP than in those that were exposed to high concentrations. These results show that suppression of the C. riparius SP gene by DEHP is as a potential biomarker that could be useful for monitoring aquatic quality.     

Degradation of di-ethylhexyl phthalate (DEHP) in bioslurry phase reactor and identification of metabolites by HPLC and MS

Di-ethylhexyl phthalate (DEHP) belongs to the class of phthalate esters and is used as an additive in many products including plastics, paints and inks or as a solvent in industrial formulations. However, it is used mostly for its plasticizing ability in polyvinyl chloride (PVC) products, in which it is often added in concentrations exceeding 40% by mass. DEHP is one of the more recalcitrant phthalate esters, which has xeno-estrogenic, carcinogenic and mutagenic effects. Five different bioslurry reactors were operated under different conditions to study the degradation of DEHP (1 mg g⁻¹ soil) in soil. The process performance was assessed by monitoring DEHP concentration periodically using high performance liquid chromatography (HPLC). The ongoing biological process was monitored by analyzing pH, oxidation–reduction potential (ORP), dissolved oxygen (DO), oxygen uptake rate (OUR) and colony forming units (CFU) for every 24 h. More than 90% degradation was observed within 12 days of the cycle period in the augmented reactors. Metabolites formed during the degradation of DEHP in the slurry phase reactor were identified and the pathway was also established. The degradation process was found to follow zero-order kinetic model.     

Differentially enhanced gene expression in hemocytes from Macrobrachium rosenbergii challenged in vivo with lipopolysaccharide

In order to better understand the immune response in prawns after treatment with the immunostimulant lipopolysaccharide (LPS), in this study, the differential gene expression of the hemocytes from LPS-injected versus non-injected prawns (Macrobrachium rosenbergii) were isolated and identified using suppression subtractive hybridization (SSH). The hemocytes were extracted after treatment for 1, 6, and 12 h. The upregulated genes (i.e., where gene expression was elevated) were identified and could be divided into four classes on the basis of physiological function: genes concerning defense-related molecules, genes involved in energy-production (respiration), genes related to protein synthesis and folding, and genes with unknown function. The time-course for gene expression indicated that, except for expression of the gene anti-microbial peptide (amp), which was increased at 12 h after LPS treatment, the expression of the other two immune-related genes was much earlier (at 1 h), including alpha-2-macroglobulin (α2-M) and Mas-like protein (mas). These results suggest that in the early phase of LPS stimulation some immune reactions regulated by α-2M and Mas may be induced, such as the activation of prophenoloxidase activating system, opsonization, and anti-microbial activity. In addition, six unigenes with unproven function were particularly interesting and worthy of further study because their expression in LPS-treated hemocytes was clearly enhanced.     

Molecular dissection of the response of a rice leucine-rich repeat receptor-like kinase (LRR-RLK) gene to abiotic stresses

Leucine-rich repeat (LRR) receptor-like kinase (RLK) proteins play key roles in a variety of biological pathways. In a previous study, we analyzed the members of the rice LRR-RLK gene family using in silico analysis. A total of 23 LRR-RLK genes were selected based on the expression patterns of a genome-wide dataset of microarrays. The Oryza sativa gamma-ray induced LRR-RLK1 (OsGIRL1) gene was highly induced by gamma irradiation. Therefore, we studied its expression pattern in response to various different abiotic and phytohormone treatments. OsGIRL1 was induced on exposure to abiotic stresses such as salt, osmotic, and heat, salicylic acid (SA), and abscisic acid (ABA), but exhibited downregulation in response to jasmonic acid (JA) treatment. The OsGIRL1 protein was clearly localized at the plasma membrane. The truncated proteins harboring juxtamembrane and kinase domains (or only harboring a kinase domain) exhibited strong autophosphorylation. The biological function of OsGIRL1 was investigated via heterologous overexpression of this gene in Arabidopsis plants subjected to gamma-ray irradiation, salt stress, osmotic stress, and heat stress. A hypersensitive response was observed in response to salt stress and heat stress, whereas a hyposensitive response was observed in response to gamma-ray treatment and osmotic stress. These results provide critical insights into the molecular functions of the rice LRR-RLK genes as receptors of external signals.     

Effect of DEHP exposure in Calliphora vicina (Diptera: Calliphoridae)

Analysis of a possible delay between a person’s death and insect colonization plays a crucial role in forensic entomology and could be affected by numerous factors. Di- (2-ethylhexyl) phthalate (DEHP) is an endocrine disruptor with the ability to target various organs, intervene with their normal function and cause harmful impact on organisms. The current paper points out the effects of exposure to DEHP and/or its metabolites on the larval stages of Calliphora vicina. We observed significantly lower larval length of the second instars (p = 0.04) and width of the third instars (p = 0.04) exposed directly by parent DEHP. Length of the first instars on the board of significance (p = 0.057) was also recorded. We found discrepancy in development, but development rate and larval weight did not significantly differ from the control group. In the experiment focusing on the effect of DEHP metabolites throughout mice cadavers exposed to this diester during life, we detected a statistically significantly increased larval weight of the post-feeding third instars in the experimental group (p = 0.04). Hypothetically, our preliminary results indicate a disruptive effect of DEHP and/or its metabolites on the metric parameters of larvae of the forensically important Calliphora vicina species, but further studies are needed.     

The Mechanism of Environmental Endocrine Disruptors (DEHP) Induces Epigenetic Transgenerational Inheritance of Cryptorchidism

Discussion on the role of DEHP in the critical period of gonadal development in pregnant rats (F0), studied the evolution of F1-F4 generation of inter-generational inheritance of cryptorchidism and the alteration of DNA methylation levels in testis. Pregnant SD rats were randomly divided into two groups: normal control group and DEHP experimental group. From pregnancy 7d to 19d, experimental group was sustained to gavage DEHP 750mg/kg bw/day, observed the incidence of cryptorchidism in offspring and examined the pregnancy rate of female rats through mating experiments. Continuous recording the rat’s weight and AGD value, after maturation (PND80) recording testis and epididymis’ size and weight, detected the sperm number and quality. Subsequently, we examined the evolution morphological changes of testicular tissue for 4 generation rats by HE staining and Western Blot. Completed the MeDIP-sequencing analysis of 6 samples (F1 generation, F4 generation and Control). DEHP successfully induced cryptorchidism occurrence in offspring during pregnancy. The incidence of cryptorchidism in F1 was 30%, in F2 was 12.5%, and there was no cryptorchidism coming up in F3 and F4. Mating experiment shows conception rate 50% in F1, F2 generation was 75%, the F3 and F4 generation were 100%. HE staining showed that the seminiferous epithelium of F1 generation was atrophy and with a few spermatogenic cell, F2 generation had improved, F3 and F4 generation were tend to be normal. The DNA methyltransferase expression was up-regulated with the increase of generations by Real Time-PCR, immunohistochemistry and Western Blot. MeDIP-seq Data Analysis Results show many differentially methylated DNA sequences between F1 and F4. DEHP damage male reproductive function in rats, affect expression of DNA methyltransferase enzyme, which in turn leads to genomic imprinting methylation pattern changes and passed on to the next generation, so that the offspring of male reproductive system critical role in the development of imprinted genes imbalances, and eventually lead to producing offspring cryptorchidism. This may be an important mechanism of reproductive system damage.