Experimental Study Of Developmental Exposure To DEHP Adversely Impacts Synaptic Development Of Hippocampus By Interfering With Thyroid Hormones In Mice

Background Phthalic acid esters(PAEs)are widely used as plasticizer in various productions including food packages,children's toys,medical equipment and cosmetics.They can easily leach out from production because they are bounding within the matrix by non-covalent,resulting in severe population exposure.It has been shown that PAEs are a kind of environmental endocrine disruptors,which can interfere with the normal physiological function of gonadal hormones and induce reproductive and developmental toxicity.In recent years,a number of researches indicated that PAEs are able to disrupt thyroid endocrine.Di(2-ethylexyl)phthalate(DEHP)is one of the most important members of PAEs,the neurotoxicity of DEHP has been aroused people's attention as it can pass both placental barrier and blood-brain barrier.Some studies have indicated that property of gonadal hormones disruption of DEHP may play an important role in its adversely impacts on neurodevelopment.However,little is known with respect to the relationship between thyroid endocrine and neurotoxicity of DEHP.Objective To investigate whether DEHP will harmful impact the development of synapses through thyroid endocrine disruption.Methods Male littermates of ICR mice randomly assigned to five experimental groups(n=14 for each condition)on PND4 to receive distilled water,vehicle and DEHP(10,50 and 200mg/kg·d)from PND5 to PND38 by gavage at 20?L/g body weight once a day,weighing and recording body weight every day.Vehicle is emulsion that consists of distilled water,1% Tween80 and 1% corn oil,average consumption of water and feed of mice were weighed every day after weaning.Open field task were conducted on PND 26 and Morris water maze task were begun from PND30 to PND 37 to evaluate spontaneous exploration activity and emotion,spatial learning and memory performance of pubertal mice,respectively.All animals were sacrificed after anesthesia by received intraperitoneal injection of 10% chloral hydrate solution at 0.04 m L/10 g body weight On PND39,serum were collected to determinate the levels of THs(T3,T4,FT3 and FT4)and TSH by the automatic biochemical analyzer,bilateral testes were collected and weighed,and after infusion by physiological saline through the heart,cerebellums were separated to determinate the level of MEHP within them by LC-MS,hemicerebrums were readied to determinate the levels of T3 and T4 by electrochemiluminescence,and hippocampi were isolated on ice,then total proteins of hippocampus were extracted,followed by determining the proteins expression of PSD95,synapsin I,BDNF,TR?1,TR?1,MCT8,OATP1C1,DIO2 and DIO3 by western blotting.Results There was no significant difference of all indexs between the vehicle control group and the blank control group(all P>0.05).200mg/kg·d DEHP significantly reduced the growth of body weight,decreased the organ coefficient of testicles and increased the concentrations of MEHP in cerebellum of mice(all P<0.05).DEHP does not significantly influence the levels of serum T3,T4,FT3,FT4,TSH and the levels of brain T3,T4(all P>0.05),but 200mg/kg·d DEHP increased the ratio of T4/T3 significantly.There were no significant difference of total distances in open field and in Morris water maze during the probe trial among groups(P>0.05),indicating that dosages of DEHP used in this study have no adversely impact on motor ability of mice.In open field test,200mg/kg·d DEHP significantly reduced the time staying in the central area(P<0.05),but 10 and 50mg/kg·d DEHP did not(P>0.05),indicating that 10 and 50mg/kg·d DEHP did not caused anxiety.In Morris water maze,200mg/kg·d DEHP significantly prolonged the time searching the hidden platform during the spatial acquisition of mice(P<0.05),and 50mg/kg·d DEHP significantly reduced the percent of time and distance in the target quadrant during the probe trial of mice(P<0.05).The results of western blotting showed that DEHP significantly reduced the protein expression of PSD95 in hippocampus of mice with all dose groups(all P<0.05),expression of synapsin I were only reduce in dose group of 200mg/kg·d DEHP.200mg/kg·d DEHP significantly reduced the expression of TR?1 and TR?1(all P<0.05),but it should note that 50mg/kg·d DEHP reduced the expression of TR?1 significantly already.DEHP significantly reduced the expression of OATP1C1 in hippocampus of mice with all dose groups(all P<0.05),but only 10mg/kg·d DEHP reduced the expression of MCT8 significantly(P<0.05).DEHP does not impact the expression of DIO2 and DIO3 in hippocampus of mice(all P>0.05).200mg/kg·d DEHP reduced the protein expression of BDNF significantly(P<0.05).Conclusions ?Developmental exposure to a higher dosage of DEHP(200mg/kg·d)could affect the general development of mice,may cause significant anti-androgen effects,can damage the hippocampal synaptic development,cause anxiety and impair spatial learning ability,can disrupt brain thyroid hormones homeostasis,and may interfere with thyroid hormone receptor signaling.?Developmental exposure to a lower dosage of DEHP(50mg/kg·d)can damage the hippocampal synaptic development and the ability of spatial memory,may interfere with the hippocampal local thyroid hormones homeostasis and its receptor signaling,although it did not significantly affect the general development of mice or caused anxiety.In conclusion,Developmental exposure to DEHP could damage the development of synapse in hippocampus and adversely impacting spatial memory performance of mice at a dose that were insufficient to significantly influence the general development and result in anxiety.DEHP may disturb the hippocampal thyroid hormones homeostasis and receptor signaling by influencing the transport of thyroid hormones and the expression of receptor,resulting in adversely impact on the development of hippocampal synapses.