Effects Of Di-(2-ethylhcxyl) Phthalate Exposure During Mid To Late Pregnancy On Fetal Development And Related Mechanism

Di-(2-ethylhcxyl) phthalate is the most common type of plasticizer widely used in food packaging, cosmetics, building materials, children’s toys and other fields, they can increase the ductility and pliability of plastic products. Studies have shown that DEHP is an environmental endocrine disruptors, they have estrogen-like effects that may interfere with the synthesis,release, transport and metabolic of the body’s normal endocrine processes when into the body. Thereby DEHP will damage the internal environment homeostasis. A large number of epidemiological data and animal experiments confirmed that DEHP has reproductive toxicity, embryo development toxicity, neurotoxicity and immunotoxicity and will cause multiple organ damage when into the body. We have large amount of DEHP using in a wide range, so the cause of environmental and health issues can not be ignored. In this study, Wistar famale rats were given to different doses of DEHP by the way of gavage from the second trimester, to establishment of a simple and effective trimester DEHP toxicity evaluation system and observed the fetal growth and development situation. The rats placenta were used as a key organization,many related molecules were detected in gene and protein levels.To provide a theoretical basis for clarifying the mechanism of embryonic development toxicity of DEHP.Objective1、Wistar rats were given to different doses of DEHP solution by oral administration during the mid to late pregnancy, to establish the DEHP exposure model.2、Observed the morphological and ultrastructural changes of rat placental tissue in different doses of DEHP exposure group during mid to late pregnancy.3、To investigate different doses of DEHP exposure on fetal growth and development4、To find out the mechanism of embryonic development of DEHP.Method1、60 female Wistar rats were randomly divided into four groups: control group(corn oil), low-dose group(100mg / kg), medium-dose group(500mg / kg) and high-dose groups(1000mg / kg). Pregnant rats were fed with DEHP solution from the 11 th day, dams were sacrificed on the 19 th day. Observed the general condition of rats during pregnancy.2、Recorded the fetuses number of each dose group, count the number of live births, stillbirth and teratogenic, measure fetal weight, length, tail length and calculate fetal heart, liver, kidneys visceral index.3、Measured the placental weight, placental tissue were taken for HE staining and electron microscopy,observed the morphological and ultrastructural changes in different groups4、Using rat whole genome expression microarray to detect different genes expressed between high-dose and the control group.According to the classification of gene function and pathway.5、RT-PCR technology to test partly of differentially expressed genes WTI, PPARα, FX, HNF1 a, SFRP2, SPP1 and AREG in m RNA levels.6、Select peroxisome proliferators-activated receptors three subtypes PPARα, PPARβ, PPARγ, to detect their gene and protein levels changes.7、Select PPARs related molecules FATP4, FABPs, FAT/CD36, CPT1 A, ACOX1,SCD-1, LPL and EL,which are in the downstream pathway of lipid metabolism, to detect their gene and protein levels changes.Result1、Pregnant rats in each group after administration DEHP were in good condition, no miscarriage and premature birth, no animal deaths. The weight of pregnant rats increased slowly in medium and high dose groups.2、Light microscopy showed that the placenta tissue in low-dose group had no significant changes.The structure of placenta in mid and high-dose groups mainly change in the labyrinth and sponge trophoblast. We can see the sponge trophoblast cell were degeneration, necrosis, labyrinth was expansion of congestion. The placenta tissue in high dose group has the most significant changed.3、Placenta tissue in each dose group mainly changed in microvilli, mitochondria and endoplasmic reticulum of ultrastructural. The structural of placental trophoblast cells were damage in high dose group, apoptotic bodies were appearance.4、Fetal malformations and mortality in medium and high dose groups were significantly increased. Fetal weight were lower, body length and tail length were smaller, growth restriction. Liver and kidneys visceral index were decreased.5、Different gene expression were detected between high-dose group and the control group using microarray analysis. More than two times greater of differentially expressed genes were 951, including 424 upregulated genes, 527 genes were down-regulated.6、Differentially expressed genes were analysis according to GO functional, Up-regulated genes mainly involved include: organizational development, wound healing, steroid hormone metabolism, stress reactions. Down-regulated genes mainly involved in steroid metabolism, transport of organic matter and lipid metabolism.7、Use KEGG pathway database to analysis the differentially expressed genes, the most significant increase pathway is stimulate the nerve pathway ligand- receptor and cytokines-cytokine receptors. The most significant reduction pathway is the complement and the coagulation cascade, fat digestion and absorption.8、Real time-PCR verification, The m RNA levels of WTI, PPARα and AREG were upregulation, FX, HNF1 a,SFRP2, SPP1 were downregulation. Consistent with the microarray results.9、Gene and protein levels of Lipid metabolism related molecules RXRα,PPARα and PPARγ were increasd in medium and high dose groups. The expression of PPARβ were not changed.10、The expression of FATP4, FABPs, FAT/CD36, CPT1 A, ACOX-1,SCD-1,LPL and EL m RNA have a increasing trend in the four groups,the high-dose group increased significantly. The expression of FATP4, FABPs and FAT/CD36 protein increased significantly in high dose group.Conclusion1、DEHP exposure during mid to late pregnancy can affect the embryos development, resulting in adverse pregnancy outcomesd.2、DEHP toxicity to placenta and fetus has significant dose- response relationship, 1000mg/kg DEHP has the most influenced.3、The influence of DEHP on embryonic development involves in multiple genes, multiple paths, were many factors working together. The function of each gene does not exist independently,each gene was links and interact together to form a complex network.4、DEHP may activate PPARs to affect placenta lipid metabolism pathways, ultimately resulting in embryonic development abnormal.