Effect Of Di-(2-ethylhexyl) Phthalate Exposure During Pregnancy On Female Fetal Neurodevelopment

Background Phthalates（PAEs）are Endocrine disrupting chemicals（EDCs）which are commonly used as plasticizers in children’s toys,medical devices,cosmetics and so on.DEHP is one of the representatives of PAEs in the environment and has the highest content.DEHP has the effects of estrogen and antiandrogen,and can also interfere with the homeostasis of Thyroid hormones（THs）.Delivery of THs through the placenta during pregnancy is the main source of fetal rats.Previous studies have found that DEHP can interferes with THs transporters and THs receptors in the placenta of male fetal rats,and affects the migration and proliferation of neurons in the cerebral cortex of male fetal rats.However,there are few studies on whether DEHP can cause neurodevelopmental toxicity in female fetuses.It is hypothesized that the effect of DEHP on the neurodevelopment of female fetuses may be consistent with that of male fetuses,that is,the neurodevelopmental toxicity of female fetuses is caused by affecting the transport and synthesis of placental THs. Objective To observe the effects of PAEs represented by DEHP during pregnancy on the growth of female fetal rat neurons,and to explore the possible mechanisms affecting the synthesis and transport of placental THs.Methods ICR mice were planned to be randomly divided into control group（Tween 80and 1%corn oil emulsion）,10 mg/kg,50 mg/kg and 200 mg/kg DEHP treatment group（200 mg/kg/d Less than 1%of the oral LD50 in mice）,During the 9.5th day of pregnancy（gestation day 9.5,GD9.5）to GD15.5,oral administration was performed according to the volume of 1%of the weight of pregnant mice（10 per group）,once a day.During the exposure period,the weight,food and water consumption of the pregnant rats in each group were recorded every morning,and the general conditions such as fur and activity of the experimental animals were observed.At GD15.5,7 pregnant rats were randomly selected from each group and then anesthetized with chloral hydrate（0.04 mL/g）.The levels of thyroid hormone（FT3,FT4,T3,T4）in serum and amniotic fluid were detected by chemiluminescence method.Western blotting was used to detect thyroid hormone receptor alpha 1（TRα1）,thyroid hormone receptor beta1（TRβ1）,and monocarboxylate transporter 8（MCT8）,organic anion transporting polypeptide 1C1（OATP1C1）,type II deiodinase（DIO2）and type III deiodinase （deiodinase III,DIO3）in female placenta.The remaining three pregnant mice in each group were sacrificed in GD18,and the fetal brains of female rats were taken.The fluorescent signal of BDNF in the cerebral cortex of female fetal rats was detected by immunofluorescence histochemistry.The protein levels of growth-associated protein 43（Gap-43）and brain-derived neurotrophic factor（BDNF）in fetal brain were detected by Western blotting,and the growth of brain neurons was observed.Results（1）There was no difference in daily water intake,food consumption,and weight gain in pregnant rats in each dose group.At GD 15.5,there was no difference in body weight,brain weight,and brain body ratio between the groups of fetuses.There was no change in THs levels in the pregnant rats in each dose group.Amniotic fluid FT3 levels were significantly down-regulated in the 50 mg/kg and 200 mg/kg DEHP dose groups（P<0.05,compared with the control group）.（2）The protein levels of Gap-43 and BDNF in fetal fetuses of the fetuses at 50 mg/kg and200 mg/kg were decreased（P<0.05,compared with the control group）.The fluorescent signal of BDNF in the cerebral cortex were decreases.（3）200 mg/kg DEHP decreased the protein level of MCT8 in the placenta of female mices,and the protein level of female placenta OATP1C1 in the DEHP group of 50 mg/kg and200 mg/kg decreased（P<0.05,compared with the control group）.（4）The protein level of DIO₃ in the female placenta decreased in the 200 mg/kg DEHP group,and the protein expression of DIO₂ in the female placenta decreased in all dose groups（P<0.05,compared with the control group）.（5）There was no difference in the protein level of placental TRβ1 in female mices in each dose group,and the protein level of placental TRα1 in female mices in the 200 mg/kg DEHP group decreased（P<0.05,compared with the control group）.（6）Comparison of the difference between the results of exposure to DEHP during pregnancy and the results of female and male mices.It was found that DIO₃ did not change significantly in the placenta of male fetus,but it was in the 50 mg/kg and 200mg/kg dose groups of female fetal placenta.The median was decreased（P<0.05,compared with the control group）.Conclusion Exposure to 200 mg/kg of DEHP during pregnancy did not cause maternal toxicity,nor did it affect FT₄ levels in amniotic fluid,but reduced FT₃ in amniotic fluid.It also affects the growth of neurons in the cerebral cortex of female fetal rats,which may be through lowering the nuclear receptor TRα1 of THs and the protein levels of its transporters MCT8 and OATP1C1.Exposure to DEHP during pregnancy can affect the neurodevelopment of female mices by interfering with the transport of placental THs.In addition,we found that DIO₃ in female placenta was more susceptible to 50 mg/kg and200 mg/kg DEHP compared to male mices,which may be the cause of the more insidious neurotoxicity in female fetuses,providing new support for subsequent studies.