Environmental Cadmium Exposure During Gestation Impairs Fetal Brain And Cognitive Function Of Adult Offspring Via Reducing Placental-derived E2 Level

Background:Early-life exposure to environmental cadmium（Cd）caused fetal growth restriction.Nevertheless,the effect and mechanism of gestational exposure to Cd on the offspring’s cognitive function remained unclear.The group research found that Cd is extremely difficult to penetrate the placental barrier and the placenta is an important target organ for Cd to damage fetal growth and development.Estrogen plays a crucial role in neurodevelopment,and the placenta replaces the ovary as the main source of maternal and fetal estrogen in the second and third trimesters.Therefore,this study further explored the effect of environmental Cd exposure during pregnancy on offspring cognitive function and its corresponding mechanism,as well as the role of placental estrogen synthesis in it.Objective:To investigate the effects of environmental Cd exposure during pregnancy on offspring cognitive function and its corresponding mechanism,as well as the role of placental-derived estrogen in it.Methods:This study explored the effects of environmental Cd exposure during pregnancy on offspring cognitive function and its corresponding mechanism through whole animal experiments and in vitro cell experiments.The overall animal experiment consisted of four experiments.Experiment 1:To investigate the effects of Cd exposure during pregnancy on offspring cognitive function,the pregnant mice drank the Cd Cl₂-contained water（50 and 150 mg/L）in LCd and HCd groups from GD8 to GD17.The section of pregnant mice（n=8）was performed mercy killing on GD18.Corresponding samples were retained on GD18.The rest pregnant mice were delivered naturally.After being adjusted the sex between littermates（female:male,5:5）on postnatal day（PND）4,the offspring continued to be raised to adulthood.Male mice in each group started Morris Water Maze（MWM）test to test their cognitive ability on PND140.After the MWM test,all male mice were performed mercy killing.Corresponding samples were collected and stored.Experiment 2:To investigate whether NAC supplementation during pregnancy can alleviate the cognitive impairment of offspring caused by Cd exposure during pregnancy,the pregnant mice drank the Cd Cl₂-contained water（150 mg/L）in HCd and NAC+HCd groups from GD8 to GD17.The mice in NAC and NAC+HCd groups were supplemented NAC（500 mg/kg/day）from GD7 to GD17.The section of pregnant mice（n=8）were performed mercy killing on GD18.Corresponding samples were retained on GD18.The rest pregnant mice were delivered naturally.After adjusting the sex between littermates（female:male,5:5）on PND4,the offspring continued to be raised to adulthood.Male mice in each group started MWM test to test their cognitive ability on PND140.After the MWM test,all male mice were performed mercy killing.Corresponding samples were collected and stored.Experiment 3:To test whether estrogen supplementation during pregnancy can alleviate the impairment of fetal brain development by Cd by reducing placental estrogen synthesis,the pregnant mice were exposed to Cd Cl₂-polluted water（150 mg/L）from GD8 to GD17 with or without E2（15μg/kg/day,i.p.）.All pregnant mice（n=8）were performed euthanasia on GD18.Corresponding samples were collected and stored.Experiment 4:To examine whether Cd impairs fetal brain development by inhibiting placental estrogen synthesis,the mice drank the Cd Cl₂-contained water（150 mg/L）in Sham+HCd and OVX+HCd groups from GD8to GD17.The maternal mice in OVX and OVX+HCd groups were ovariectomied under terminal anesthesia with 2,2,2-tribromoethanol（250 mg/kg,i.p.）.All pregnant mice（n=8）were performed euthanasia on GD18.Corresponding samples were acquired and stored.The in vitro cell experiments consisted of two experiments.Experiment 1:To investigate the time effect of Cd on the inhibition of estrogen synthesis in human placental trophoblast JEG-3 cells,JEG-3 cells were stimulated by Cd Cl₂（20μM）for 0,2,6,12 and24 h.Experiment 2:To verify the role of GCN2 signaling in the inhibition of placental estrogen synthesis by Cd,JEG-3 cells were cultured with GCN2i B（5μM）for 2 h before Cd administration for 12 h.Results:The Morris water maze test showed that the seventh-day platform latency of adult male offspring in the high-dose Cd exposure group（150 ppm）was higher than the control group（P<0.05）.The results suggested that Cd exposure during pregnancy could impair the memory of adult male offspring ability.Further studies found that Cd exposure during pregnancy significantly reduced estrogen levels and the expression of estrogen receptor alpha（ERα）,brain-derived neurotrophic factor（BDNF）,and synapse-related proteins（PSD95 and Synapsin-1）in the fetal brain.However,E2 supplementation during pregnancy significantly reversed the down-regulated expressions of BDNF,PSD95,and Synapsin-1 in fetal brain,suggesting that Cd exposure during pregnancy impairs fetal brain synaptic plasticity by reducing fetal brain E2 levels.Through cell experiments and animal experiments,we confirmed that Cd exposure during pregnancy can reduce the level of placental estrogen and the expression of estrogen synthase（CYP19,CYP17A1）.We found no significant difference in fetal brain estradiol levels between the high-dose Cd exposure group and the ovariectomized+high-dose Cd exposure group after ovariectomy in GD7.The data suggested that gestational Cd exposure inhibited placenta-derived estrogens synthesized to lower E2 levels in the fetal brain.Furthermore,GCN2activity inhibitor（GCN2i B）was used to intervene in human placental trophoblast JEG-3cells,and it was found that the expression of estrogen synthase inhibited by Cd was reversed,which further confirmed that Cd inhibited estrogen synthesis by activating placental GCN2 signaling.Subsequently,NAC supplementation during pregnancy was found to inhibit the activation of placental GCN2 signaling and reverse the Cd-induced reduction of PSD95,Synapsin-1 and BDNF expression in the fetal brain,thereby alleviating cognitive impairment in adult offspring mice.This result further confirmed that Cd exposure during pregnancy inhibited placenta-derived estrogen synthesis by activating GCN2 signaling,resulting in cognitive impairment in adult offspring mice.Conclusion:In summary,Cd inhibited placental-derived estrogen synthesis via activating GCN2 signaling,and impaired fetal brain development,thereby caused cognitive impairment in adult offspring mice.