| Di-(2-ethylhexyl)phthalate(DEHP)is the most commonly used plasticizer worldwidely and is ubiquitous in arrounding environment.DEHP is a typical endocrine disrupting chemical,male reproductive and developmental toxicant.Gestational DEHP exposure impaired testis development with reduced anogenital distance and decreased testosterone level in serum.Recently,emerging evidences suggested that prenatal DEHP exposure was associated with lower birth weight,but results from epidemiological studies were controversial.Moreover,the critical window of its effects during pregnancy was still unknown,the sex-selective effect was uncertain and the potential mechanism in placenta had not been examined.In order to clarify these problems,in the present study,we firstly established mouse model of gestational DEHP exposure to investigate the effects of gestational DEHP exposure on fetal intrauterine growth and to identify the critical window of DEHP-induced IUGR.We further investigated the impacts of gestational DEHP exposure on placental thyroid hormone receptor signaling and placental angiogenesis.Finally,by integrating the results from the case-control study,we proveed the role of thyroid hormone receptor signaling on gestational DEHP exposure-induced IUGR and its mechanism.Recently,effects of prenatal DEHP exposure on neurobehavioral development has raised the concern of the public,Previous reports showed that prenatal DEHP exposure disturbed neurobehavioral development in early life,in puberty and even in adulthood,but whether gestational DEHP exposure impaired neurobehavior in senile period was still obscure.First of all,we established a mouse model of gestational DEHP exposure.Then,we performed a series of behavioral test to examine the neurobehavior of the aged offspring and to clarify the long-term effects of gestational DEHP exposure on neurobehavioral development.In current study,we clarified the developmental toxicity of DEHP and the critical window of DEHP-induced IUGR.Also,we investigated the potential mechanism of DEHP-induced IUGR in placenta and revealed the long-term effects of gestational DEHP exposure on neurobehavior of offspring.Study 1: The placental mechanism of gestational DEHP exposure-induced IUGRObjective To investigate whether gestational DEHP exposure causes fetal intrauterine growth restriction in a gender-specific manner,identify the critical window of DEHP-induced fetal IUGR and explore the potential mechanism of placenta.Methods To investigate the effects of gestational DEHP exposure on fetal intrauterine development,pregnant mice were divided into three groups randomly: control group,low-dose group(50 mg/kg/day)and high-dose group(200 mg/kg/day).Pregnant mice were administered with DEHP by gavage daily from GD0 to GD17,all dams were sacrificed on GD18.Electrochemical immunoluminescence was used to detect the concentration of thyroid hormones in maternal and fetal sera.The number of resorption site,dead fetuses and live fetuses of per litter was counted,the gender of each live fetuse was identified,crown-rump length and placenta weight were recorded.To investigate the effects of gestational DEHP exposure at different stages on fetal development,pregnant mice were divided into three groups randomly: early gestational stage DEHP exposure group(GD0 to GD6),middle gestational stage DEHP exposure group(GD7 to GD12)and last gestational stage DEHP exposure group(GD13 to GD17),the dosage selected was 200 mg/kg/day.Pregnant mice were administered with DEHP by gavage daily at different gestational stage,all dams were sacrificed on GD18.The number of resorption site,dead fetuses and live fetuses of per litter were counted,the gender of each live fetuses was identified,crown-rump length and placenta weight were recorded.To explore the potential role of placenta in gestational DEHP exposure-induced fetal IUGR,pregnant mice were divided into three groups randomly.Pregnant mice were administered with DEHP(0,50 or 200 mg/kg/day)by gavage daily from GD0 to GD14,all dams were sacrificed on GD15.Maternal serum and placenta were collected,parts of placenta were used for total RNA extraction to examine the expression of thyroid hormone receptors,placental nutrients transporters and placental growth factors;Parts of placenta were used for western blotting to identify the nuclear translocation of thyroid hormone receptors,and parts of placenta were used for histopathology,HE was performed to estimate the blood sinusoids area in the labyrinthine region and immunohistochemistry was to evaluate the microvessel density in labyrinth among different group.The case-control study was based on Ma'anshan birth cohort study,immunohistochemistry was used to estimate the microvessel density and the protein abundance of thyroid hormone receptors in placenta of AGA and SGA newborns.Results Gestational DEHP exposure had no effects on food consumption and body weight gain of pregnant mice.Gestational DEHP exposure significantly reduced fetal weight and length,the incidence of IUGR fetuses per litter was increased obviously in DEHP-exposed mice in a dose-dependent manner,the incidence in control group was8.9%,in low-dose group was 28.6% and in high-dose group was 67.7%.Analyzed by gender,as compared with control,the body weight and crown-rump length of male fetuses in DEHP-treated group were significantly reduced and the body weight and crown-rump length of female fetuses in DEHP-treated group were also significantly reduced,there was no gender specificity observed.The results from gestational DEHP exposure at different stages indicated that maternal DEHP exposure had no effects on food consumption and body weight gain of pregnant mice.Analyzed according the exposed stage,DEHP exposure at early gestational stage did not affect fetal weight,maternal DEHP exposure at middle gestational stage slightly reduced fetal weight and crown-rump length and maternal DEHP exposure at last gestational stage obviously reduced fetal weight and crown-rump length.Consistently,no gender specificity was observed.These results indicated that maternal DEHP exposure during pregnancy caused fetal IUGR in a gestation-specific but gender-independent manner.Results from electrochemical immunoluminescence suggested that gestational DEHP exposure had no effects on thyroid hormone concentrations in fetal and maternal sera.Moreover,the m RNA levels of placental thyroid hormone receptors,Thr?1 and Thr?1,were significantly reduced in DEHP-exposed mice and the nuclear translocation of THR?1and THR?1 were significantly suppressed in DEHP-treated mice.Placental Vegf,Pgf,Igf1 and Igf2,several THR downstream genes,were down-regulated in DEHP-exposed mice.Unexpectedly,placental weight was unaffected in DEHP-treated mice,histopathology showed that blood sinusoids area in the labyrinthine region was shrunken and the microvessel density in labyrinth was reduced in placenta of DEHP-treated mice.Also,the expressions of placental nutrient transports Glut1 and Fatp1 were also down-regulated in DEHP-exposed mice.In case-control study,as compared with AGA group,a significant decrease in labyrinthine microvessel density was presented in SGA placenta and a significant down-regulation was observed in THR?1 and THR?1 protein abundance.Conclusion Gestational DEHP exposure caused fetal IUGR in a stage-specific but gender-independent manner.Gestational DEHP exposure interrupted placental angiogenesis then leaded to fetal IUGR through disturbing placental THR signaling.Study 2: The long-term effects of prenatal DEHP exposure on neurobehavior of offspringObjective To investigate effects of prenatal DEHP exposure on neurobehaviors of the aged mice.Method Pregnant mice were divided into three groups randomly: control group,middle gestational stage DEHP exposure group(GD7 to GD12)and last gestational stage DEHP exposure group(GD13 to GD17),the dosage selected was 200 mg/kg/day.After spontaneous delivery,gender of all fetuses in a litter was identified and weight was recorded,litter size was adjusted to ten pups(five female and five male).At postnatal day 21,all mice were weaned and housed by gender consistent with the treatment of their mothers received.In senile period,a series of behavioral tests were performed.Anxiety status was evaluated by open field test and elevated plus-maze test,depression status was detected by sucrose preference test and forced swim task,spatial learning and memory ability was examined by Morris water maze test.ResultsGestational DEHP exposure at last stage significantly reduced fetal birth weight and catch-up growth pattern was observed in those fetuses.At weaning,adulthood and senile period,there was no significant difference in body weight among different groups,indicating that gestational DEHP exposure had no long-term effects on physical growth in all offspring.Results from behavioral test suggested that gestational DEHP exposure in last stage but not in middle stage increased anxiety-like behaviors of males with the decreased centre residence time and the reduced centre entries in open field test,the reduced entries to open arm and the shortened staying time in open arms in elevated plus maze test.Moreover,gestational DEHP exposure in last stage but not in middle stage increased depression-like behaviors of males with the prolonged immobility time in forced swim task,but there was no significant difference in the preference index of sucrose preference test.On the other hand,gestational DEHP exposure in middle but not in last stage impaired spatial learning and memory of males with the prolonged escape latency on training day,the reduced platform crossing time and the shortened distance in target quadrant on testing day in water maze test.By contrary,female offspring did not display a significant difference in anxiety-and depressive-like behaviors and spatial learning and memory ability among three groups.ConclusionPrenatal exposure to DEHP impaired neurobehavior of the aged mice in a stage-dependent and gender-specific manner.In a summary,the results from study 1 and study 2 allow us to reach the following three conclusion.First,gestational DEHP exposure caused fetal IUGR in a stage-specific but gender-independent manner.Second,Gestational DEHP exposure interrupted placental angiogenesis,then leaded to fetal IUGR through disturbing placental THR signaling.Third,prenatal exposure to DEHP impaired neurobehavior of the aged mice in a stage-dependent and gender-specific manner... |
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