Experimental Study Of Exposure To Dehp During Pregnancy Interferes With Ths Transplacental Transport And Affects The Neurobehavioral Development Of Offspring

Background Epidemical and animal experiments have shown that di-(2-ethylhexyl)phthalate(DEHP)exposure during pregnancy can cause neurodevelopmental toxicity in offspring.However,the specific mechanism is not clear.Thyroid hormones(THs)are well known as necessary hormones for the development of human and brain.Especially during pregnancy,the THs required by the fetus depend on the maternal transport through the placenta,which is essential for normal fetal neurodevelopment.DEHP,as an endocrine disruptor of THs,can disrupt thyroid function and THs homeostasis in pregnant women and fetuses.However,it is still unclear whether DEHP can interfere with the transplacental transport of THs during pregnancy.Objective The purpose is to investigate whether the exposure of DEHP during pregnancy damage the neural development of the offspring by affecting the placenta,and to explore its specific mechanism.Methods C57BL/6Jcnc mice are planned to be pregnant and randomly divided into 4groups according to body weight on gestational day 9,GD9(0 mg/kg,5 mg/kg,50mg/kg,200 mg/kg DEHP),15 per group.During the period from GD9(the neural tube of fetal mouse)to GD16(the fetal mouse begins to synthesize THs),it is orally administered by oral administration at a volume of 10 ?L/g body weight,once a day.Weight and food intake were weighed daily during pregnancy.GD16,8 pregnant mice in each group were sacrificed pentobarbital sodium,and serum,amniotic fluid and placenta were collected.LC-MS was used to detect the levels of DEHP metabolites of mono(2-ethyl-5-oxohexyl)phthalate(MEOHP),mono(2-ethyl-5-hydroxylhexyl phthalate(MEHHP),and mono(2-ethylhexyl)phthalate(MEHP)in amniotic fluid and placenta.Electrochemiluminescence Immunoassay(ECLIA)was to detect the levels of THs of triiodothyronine(T3),free triiodothyronine(FT3),thyroxine(T4),free thyroxine(FT4)in serum and amniotic fluid.ELISA immunoassay kits was used to measure the levels of thyroid stimulating hormone(TSH)levels in serum and amniotic fluid.Q-PCR and Western blot were used to detect m RNA and protein levels of placental monocarboxylate transporter 8(MCT8),deiodinase?(DIO2),deiodinase?(DIO3)and Placental thyroid hormone receptor ?,?(TR?,?).The rest of 7pregnant dams in each group gave birth naturally.On the 4th day after birth(postnatal day4,PND4),the intra-oral litter of the group was adjusted to 8 per litter,and gender balance was noted.During the observation and recording of general physiological development indicators [body weight,auricle separation,whole body villi,incisor eruption(calculated above incisor eruption),eye opening] compliance.Surface righting reflex experiment(PND5-7),negative geotaxis reflex experiment(PND7-9)and forelimb grip strength experiment(PND14-16)were used to explore the effects of early neural development(somatosensory motor function and motor coordination ability).The elevated plus maze and open field experiment(PND26-27)were used to explore the effects of autonomic activity and anxiety in mice.Morris water maze experiment(PND36-45)was used to analyze the learning and memory ability of pups.Results It was no effect on body weight and food consumption of pregnant mice after DEHP exposure.In amniotic fluid,the concentrations of the three DEHP metabolites in the control group were below LOD.In addition,significantly higher concentrations of the three DEHP metabolites MEOHP,MEHHP and MEHP in the mouse of DEHP exposure groups were observed compared to untreated control mouse.Concentrations of metabolites increased as the dose of DEHP contamination rose.In placenta,the concentrations of the one DEHP metabolites of MEHP in the control group and the two DEHP metabolites of MEOHP and MEHHP in the four group were below LOD.In addition,significantly higher concentrations of MEHP in the mouse in DEHP exposure groups were observed compared to control mouse.The results showed that the DEHP model was successfully established.It did not affect the early physiological development of the pups,and there was no difference in ear separation,whole body villus,incisor eruption,eye opening time,and early weight gain after birth in each group after DEHP exposure during pregnancy.It was no effect on the labyrinth and body correction mechanism of the offspring,but affected the offspring's ability to coordinate and vestibular nerve development.The effect on the strength of the pups and the development of the vestibular nerve increases with age.there is no difference in the rate of positive turning of the pups in each group,and the rate of negative geotaxis reflex of the pups in the DEHP treatment group decreased.At PN14 and PN15,there was no difference in the suspension time of the pups in each group,but in the DEHP treatment group at PND16,the forelimb suspension time of the pups was shortened.It was no effect on the labyrinth and body correction mechanism of the offspring,but it affected the offspring's coordination ability and vestibular nerve development.The effect on the strength and development of the vestibular nerve of the offspring increases with age.There was no difference in the positive rate,and the rate of negative geotaxis reflection in the DEHP treatment group decreased.There was no difference in the suspension time of the pups in PN14 and PN15.Exposure to DEHP during pregnancy increased anxiety-like behavior in female offspring,which was manifested by a decrease in total distance traveled and a significant reduction in the number of entry into the center in open field experiments.Exposure to DEHP during pregnancy can impair the spatial learning and memory function of male pups,as shown in the Morris water maze experiment,where the escape latency is extended during the training period,and the escape latency is extended during the exploration experiment.However,DEHP exposure during pregnancy had no effect on anxious behavior of male rats and spatial learning and memory function of female rats.The exposure of DEHP during pregnancy did not affect the THs level of the mother but reduced the THs level of the offspring.The experimental results showed that there was no difference in the serum THs level of the pregnant rats,while the levels of T3 and FT3 in the amniotic fluid decreased.After exposure to DEHP during pregnancy,the expression of placental THs transporter MCT8 and its nuclear receptors TR? and TR? was down-regulated,the expression of deiodinase D2 was down-regulated and the expression of deiodinase D3 was up-regulated.Conclusion The dams exposed to DEHP during pregnancy showed abnormal neurodevelopment in offsprings,and did not affect the levels of maternal THs,but reduced the levels of T3 and FT3 in the offspring.This may be due to DEHP down-regulates the expression of placental THs transporter MCT8 and its nuclear receptors TR? and TR?,down-regulates the expression of deiodinase D2 and up-regulates the expression of deiodinase D3.This study suggests that DEHP exposure during pregnancy may mediate the THs homeostasis of offspring by interfering with THs transport in the placenta,impairing the neurodevelopmental behavior of the offspring.