The Long-term Neurotoxicity Effects Of Specific Period DEHP Exposure In Utero And The Disruption Mechanism Of Fetal Brain Thyroxine Signaling System

Background The research group found that the prenatal di-(2-ethylhexyl)phthalate(DEHP)exposure didn’t effect the offspring physiological development,but it impair adolescence offspring development neurobehavior.Maternal circulation thyroid hormone levels(thyroxines,THs)weren’t affected,yet amniotic fluid THs were reduced.The results suggested that the placental THs transport system was disrupted.In summery,we further observed long-term neurotoxicity effects on specific period DEHP exposure in utero and detected disruption mechanism of the fetal brain thyroid hormone signaling system.Objective The purpose of this study is to observe the long-term neurotoxicity effects of specific period DEHP exposure in utero,and to explore mechanism from the perspective of fetal brain thyroxine signal disruption.Methods 8 weeks female C57BL/6J mice were assigned to the same cage and randomly divided into 4 groups(0,5,50 and 200 mg/kg DEHP),with 22 mice in each group.DEHP was administered orally once every morning from gestational day 9.5(GD9.5,fetal neural tube closure)to GD16.5(fetal mice start to synthesize THs),according to 10μL/g body weight.12 pregnant mices per group were sacrificed on GD16.5,and collected fetal brains.Liquid Chromatograph Mass Spectrometer(LC-MS)method was used to detect DEHP metabolites.ELISA kit was used to detect triiodothyronine(T3),free triiodothyronine(FT3),tetraiodothyroxine(T4)and free tetraiodothyroxine(FT4)levels.Real Time PCR(RT-PCR)and Western blotting were used to detect the m RNA and protein levels including fetal brain thyroid hormone transportes,metabolic enzymes,thyroid hormone receptor α(TRα)and β(TRβ),as well as brain derived neurotrophic factor(BDNF).The remaining 10 pregnant mice in each group gave birth naturally.The fetals were adjusted to 8 mices per litter,with half males and half males on postnatal day4(PND4).Behavioural tests will be performed in the juvenile period(PND 26)[work completed before the period],adult period(PND 89),middle age(PND 210)[work on this subject],and elderly period(PND 365)[work to be completed after the period].The elevated plus maze and open field experiments was used to test the spontaneous activities and anxiety behaviors.The forced swimming test was used to test depression behaviors.The Morris water maze test was used to test the learning and memory ability.Results Under the conditions of this experiment,the research group found that DEHP the pregnant mice general phenotype was not affected.The results verified that the DEHP metabolites could cross the placenta.Further research the results found that the MEHP,MEHHP and MEOHP increased with DEHP exposure dose in the fetal brain,whice confirmed that DEHP metabolites can cross the developing blood-brain barrier.Fetal length and weight were also not effected.Behavioral results showed that prenatal DEHP exposure increased the female offspring anxiety behaviors in juvenile [work completed before maturity],and continued into adulthood and middle age;however,male offspring developed anxiety behavior in middle age.The total distance,the number and times enter into the center is significantly reduced.Prenatal DEHP exposure impired male offspring learning and memory ability in juvenile [work completed before term],and continued to adult and middle age.The latency to target was extended in the exploratory experiment in the Morris water maze.Prenatal DEHP exposure increased depression behavior in the middle-aged male offspring,but it was not observed in the female mice.The swimming time was decreased in the forced swimming.The behavioral results showed that prenatal DEHP exposure has a long-term effect on offspring’s neurotoxicity and gender differences.The research group also observed that maternal circulating thyroid hormone levels were not affected,but fetal THs levels were reduced.Further research found that the FT3 level was decreased in the fetal brain.The results suggested that fetal brain THs transporter MCT8 and OATP1C1,THs metabolizing enzyme Dio2 were decreased,yet Dio3 was increased.And fetal brain thyroid hormone receptors TRα1,TRβ1 and BDNF also decreased.After sex compared,The results found that male offspring were more susceptible to the prenatal DEHP exposure effects.Conclusion This study provides new evidence for the long-term effects and interference mechanisms of intergenerational neurodevelopmental impairment in a sex-distinct fashion,indicating that prenatal exposure to DEHP could interfere with the fetal brain thyroid hormone signaling systems,and may cause long-term brain damage.