Experiments Study Of Sub-chronic Exposure To DEHP Elicits Neurotoxicity And The Mechanism Of Blood Brain Barrier Dysfunction

Background Epidemical and animal experiments have shown that exposure to di-(2-ethylhexyl)phthalate(DEHP)can cause neurotoxicity,but the mechanism is not clear.Blood brain barrier(BBB)is one of the most important tissues to protect the brain.It has the ability to maintain the normal function of the neuronal microenvironment in the brain for strictly limiting the entry of neurotoxic substances and pathogens in blood vessels into the brain,and controlling the transport of oxygen,nutrients and few necessary molecules and ions inside or outside the brain.As a neurotoxicant,DEHP can interfere with the homeostasis of nervous function and lead to neurobehavioral dysfunction.However,whether DEHP can disrupt the BBB or not remains unclear.Objective To investigate whether DEHP exposure can induce neurotoxicity and damage the BBB in adulthood,and to explore its specific mechanism.Methods Adult C57BL/6Jcnc mice were randomly divided into 3 groups according to body weight(0 mg/kg,200 mg/kg,750 mg/kg DEHP),with 12 mice per group.The mice were administered at 10 μL/g body weigh after 8 weeks when the mice are the onset of sexual maturation through gavage.The mice were weighed their weight,water intake and food intake every day.After 90-day administration,the behavior experiment was taken: the elevate plus maze(EPM)and open field(OF)experiments were used to investigate the effects of DEHP exposure on autonomous mobility and anxiety in adult male mice;Morris water maze(MWM)was used to analyze the effect of DEHP exposure on the learning and memory ability of adult male mice;Forced swimming(FS)was used to analyze the effect of DEHP exposure on depression in adult male mice.Evans blue(EB)dye was injected intravenously and then let circulate in the body for 2 h.Serum was taken and the brain was dissected and weighed.The intracerebral permeability of EB was determined by spectrophotometry and immunofluorescence,and meantime the permeability of BBB was tested.Nissl staining and electron microscopy(EM)was used to observe the damage of neurons in hippocampal CA1 and CA3 after DEHP exposure.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 serum and brain tissues.Western blot were used to detect the expression levels of tight junction(TJ)proteins including occluding,claudin-5 and ZO-1 in BBB;the expression levels of neuroinflammatory cytokines including NLRP3,caspase-1 and IL-1β were detected as well.Results DEHP exposure in high-dose group resulted in increased water intake and decreased body weight and brain weight in adult male mice.High-dose exposure to DEHP impaired spatial learning and memory in adult male mice,which was manifested by prolonged latency of reference memory and working memory in MWM;high-dose exposure to DEHP increased depressive behavior,manifested as a significant reduction in the number of struggles during the FS test;however,DEHP exposure had no effect on anxious behavior in adult male mice.High-dose DEHP exposure reduced the number of neurons in CA1 and CA3 regions of hippocampus,leading to neuron cell edema,organelles decreased,significant expansion of endoplasmic reticulum and vacuolation,mitochondria swelling,structural destruction,disorganized and sparse cristae,and massive disappearance of ribosomes.The concentrations of MEHP and MEOHP in serum of adult male mice exposed to DEHP increased with the increase of dose,but the concentrations of MEHP and MEOHP in brain tissue increased only in the high-dose group.Exposure to DEHP increased the amount of EB permeability into the brain and resulted in a significant down-regulation of BBB-TJ protein including occludin and claudin-5.Exposure to DEHP resulted in upregulated expression of inflammasome marker protein TNF-α,NLRP3,cleaved-caspase-1 and cleaved-IL-1β,inducing activation of neuroinflammatory pathway。Conclusion Under the present experimental conditions,DEHP could not penetrate the BBB,but with the increase of exposure time and the accumulation of exposure dose,the level of tight junction protein was down-regulated by the activation of TNF-α/NLRP3/caspase-1/IL-1β neuroinflammatory pathway,resulting in BBB damage and increased permeability,induced the spatial learning and memory impairment and the depressive manifestation,the hippocampal CA1 and CA3 region neuron reduction and other neurotoxic effects.