Effect Of Prenatal Phthalates Exposure On Preschooler Intelligence And Neuroinflammatory Mechanisms

Background Phthalates is a non-persistent chemical with endocrine-disrupting abilities widely used in a variety of consumer products.The fetal brain is particularly sensitive to chemical exposures due to its rapid growth and complexity.Some studies have reported the association between maternal phthalates exposure and behaviour but few have assessed the impact on cognitive development.There is concern over potential intelligence quotient(IQ)effects of prenatal phthalate exposures,but available data are inconsistent.What’s more,the underlying biological mechanism of phthalate exposure during pregnancy affecting offspring’s intellectual development is unclear.Microglia are resident macrophages of the brain which plays an important role for in the immune and inflammatory responses of the central nerves system.However,excessively activated microglia has an adverse effect on neurons through the secretion of cytokines,inflammatory factors and other substances,and induce inflammatory reactions in the brain.Objective In the present study,population epidemical research and animal experimental research were used to evaluate the effect of prenatal phthalates exposure on cognitive development in offspring.In the population part,the distribution of preschoolers’ IQ was described,and the health risk of prenatal phthalates exposure on IQ was assessed.Prenatal DEHP exposure animal model was established to explore the mechanism of phthalate-induced cognitive impairment via the pathway of the release of these inflammatory factors from activated microglia.Methods Part 1: The present study involved the Ma’anshan Birth Cohort(MABC)in China,which recruited 3474 mother-newborn pairs.Archived first,second,and third trimester urine samples were collected after finished questionnaires administered by trained interviewers during each trimester.Urinary phthalate metabolites were quantified by solid-phase extraction-high-performance liquid chromatography-tandem mass spectrometry.Wechsler preschool and primary scale of intelligence-fourth editionChinese(WPPSI-Ⅳ CN)were used to evaluate the preschoolers’ cognitive development.In the present study,participants who presented with absence of WPPSI-Ⅳ(CN)and without at least one trimester phthalate metabolites levels excluded from this study.Finally,2128 mother-newborn pairs were included in analysis.After the normality test,the scores of each domains are approximately normal distribution,and x ± s is used to describe the distribution of preschoolers’ intellectual development level.In repeated measures analyses,linear mixed models(LMMs)were performed to evaluate the relationships between the repeated measures with subject-specific random intercepts and slopes for gestational age at sample collection to test the effects of the lntransformed phthalates metabolites levels on the preschooler intelligent quotient and to estimate the effect sizes.To determine whether there were periods of vulnerability to phthalates exposure,we stratified the regression models by the time of sample collection during pregnancy and adjusted all the models for the same covariates as those included in the repeated measures analysis.In the trimester-specific analysis,multiple linear regression models were fitted to examine the independent effects of maternal phthalates exposure on preschooler intelligent quotient.The gender-stratified model was performed to explore the gender difference.For further analysis,the preschooler were divided into three groups: lower normal,FSIQ lower than 90;normal,FSIQ between 90 and 110;higher normal were defined as FSIQ higher than 110.In the statistical analyses,normal served as the reference group.We assessed the relationships between the repeat measures of the ln-transformed phthalate metabolite concentrations and categorified IQ using the generalized linear mixed models(GLMMs).For further analysis,we established multiple logistic regression models to assess the relationships between the ln-transformed phthalate metabolite concentrations and the odds of categorified IQ.Notably,each exposure period is adjusted for the same covariates.Part 2: Pregnant mice received oral gavage of DEHP at the doses of 0,50(low)and 200(high)mg/kg/day from gestational day 0 to 18.The body weight and feed consumption of pregnant mice were recorded every day.Adjust the number of pups per litter after delivery,keep 8 pups per litter(half male and female).Their offspring were weaned on postnatal day(PND)21 and then tested in the Morris water maze task or decapitated for brain tissue collection on PND 28.Immunohistochemistry was used to detect the activation of microglia.RT-q PCR and MILLIPLEX? MAP kits were used to measure the levels of IL-17,IL-1β,IL-6,TNF-α,IL-10,IL-4 in serum and hippocampus.Western blot was used to detect the cognitive-related proteins PSD-95,synpsin I,and apoptosis-related proteins casepase-3,casepase-9,Bax,Bcl-2.ACh E activity was measured using a colorimetric acetylcholine analysis kit.Results Part 1: In the present study,263 children aged 3 years to 3 years 11 months and 1 865 children aged 4 years to 6 years participated in WPPSI.The average ages were 3.63 ± 0.27 years and 4.73 ± 0.47 years,separately.The score of FSIQ was 107.43 ± 11.30,106.71 ± 12.28,and 108.20 ± 10.07 in total,boys and girls,separately.The prevalence of lower normal IQ was 6.53%(137/2128).LMMs showed that MBP exposure during pregnancy was associated with decreased VCI,VSI,and FSIQ,while MEP exposure was associated with elevated VSI,FRI,and PSI.Gender stratification analysis showed that MBP exposure during pregnancy was associated with decreased VSI and FSIQ,MEP exposure was associated with decreased FRI and PSI in boys;while MMP exposure was associated with increased PSI in girls.The trimester-specific analysis showed that MMP exposure was related to decreased PSI scores in the first trimester,and MBP exposure was associated with decreased VCI,VSI,and FSIQ scores in the first trimester;MBz P exposure was related to increased VCI in the third trimester.Boys are more sensitive than girls,and the first trimester is more sensitive than the second and third trimesters.GLMMs showed that MBP(OR=1.14;95%CI:1.03-1.25)exposure during pregnancy increased the risk of lower normal,while MEHP(OR=1.06;95%CI:1.01-1.11)exposure decrease the possibility of higher normal.In the gender-specific analysis,we observed that MBP(OR=1.17;95%CI:1.03-1.30)increases the risk of lower normal,while MEHP(OR =0.84;95%CI:0.66-1.00)decrease the risk of lower normal in boys.The correspondence association was close to null in girls.The results of trimester-specific analysis during pregnancy showed that MBP,LWMP,and ΣPhthalates reduced the possibility of intellectual abnormalities in the first trimester,and MEHP increased the possibility of higher normal in the offspring;MEP exposure in the third trimester increased the risk of lower normal.Part 2: In the water maze test,the offspring with high dose prenatal DEHP exposure displayed poor performance during the place navigation and subsequent probe test relative to those received no or low dose DEHP prenatal exposure.The expression of PSD-95 and synpsin I showed a dose-response reduction of 1 to 2 times.The proportion of activated microglia increased in the high-dose exposure group,the levels of hippocampal inflammatory cytokines IL-17 A,IL-1β,TNF-α increased and the level of IL-10 decreased,and the trend of serum inflammatory factors was consistent with the hippocampus.Prenatal DEHP exposure induced apoptosis in the hippocampus as evidenced by the up-regulations of caspase-3,caspase-9 and Bax and the down-regulation of Bcl-2 at PND 28 in a dose-dependent manner.Moreover,there was a small reduction in hippocampal and serum ACh E in offspring with prenatal DEHP exposure.Conclusions 1.Prenatal phthalate exposure can lead to cognitive impairment in offspring.Boys are more sensitive than girls,and the first trimester is more sensitive than the second and third trimester.But the effects of different phthalate metabolites are not consistent.2.High-dose DEHP exposure at during pregnancy induces hippocampal microglia activation,and the NF-κB signalling pathway is involved in microglia activation to increase the secretion of pro-inflammatory factors IL-17 A,IL-1β,and TNF-α,and accelerate brain neuron apoptosis,Inhibit ACh E secretion.