A Preliminary Study On The Effect Of Arsenic Exposure During Pregnancy On Learning And Memory Ability Of Offspring And Induced Placental Ferroptosis

Objective The present study was aimed to confirm the effect of maternal arsenic exposure during pregnancy on learning and memory ability of offspring and preliminarily explore the molecular mechanism of placental ferroptosis induced by arsenic exposure during pregnancy.Methods In this study,C57BL/6 female mice were used to establish in vivo model and HTR-8/SVneo cells were used in vitro model.First,the arsenic exposure model during pregnancy was established via the way that female mice drank ultrapure water containing Na As O₂（0 mg/L,1 mg/L,4 mg/L and 16 mg/L）throughout pregnancy.Morris water maze test was performed to evaluate the learning and memory ability of offspring mice（6～7 weeks,sexual maturity）to explore the damaging effect of maternal arsenic exposure during pregnancy on the learning and memory ability of offspring mice.Second,maternal arsenic exposure model during pregnancy was established again.The female mice drank ultrapure water containing Na As O₂（0 mg/L and 16 mg/L）throughout pregnancy.then pregnant mice were killed on Gestational day 18（GD18）.The whole blood samples of pregnant mice were collected and stored in the refrigerator at-80℃for the determination of total arsenic content.After blood collection,the mice were sacrificed by cervical dislocation method,and the number of fetal births,fetal weight and fetal length of each pregnant mouse were recorded to evaluate the general condition of fetuses.Placenta tissues were collected,the diameter of placenta was measured with digital vernier-caliper,and the weight of placenta was weighed with an electronic analytical balance.The intact placenta was selected separately and fixed with 4%paraformaldehyde,and pathological injuries of placenta tissues were observed by hematoxylin-eosin staining（H&E）.The remaining placental tissues were collected into enzyme-free EP tubes and stored at-80℃for the determination of GSH,SOD,MDA and total iron levels as well as some protein levels,so as to investigate whether arsenic exposure during pregnancy can cause ferroptosis in placental tissues.In vitro models with HTR-8 cells,the effects of different concentrations of Na As O₂（0μM,1μM,2.5μM,5μM,8μM,10μM,15μM and 20μM）at different time points（12 h,24 h,36 h and 48 h）on the cell viability were determined via CCK-8 assay.Subsequently,HTR-8 cells were administrated with Na As O₂（0μM,1μM,5μM and 15μM）for 24 h to detect the levels of intracellular oxidative stress related indexes（GSH,SOD,MDA,ROS,HO-1 and NQO1）and ferroptosis related indexes(GPX4,x-CT,FTH1 and Fe²⁺).The morphological and structural changes of mitochondria were observed by transmission electron microscopy to verify Na As O₂ can induce ferroptosis in HTR-8 cells.Finally,antioxidant NAC（2 m M）and ferroptosis inhibitor Fer-1（10μM）were administrated together with Na As O₂（15μM）,respectively,so as to explore the protective effects of NAC and Fer-1 on oxidative stress and ferroptosis in HTR-8 cells.Results In the first part of the in vivo experiment,results from Morris water maze experiment showed that maternal arsenic exposure during pregnancy significantly affected spatial learning and memory ability of offspring mice,which were mainly manifested as the following:in the first stage of positioning navigation experiment,compared with the offspring mice in control group,the latency of offspring mice in arsenic exposure group was significantly increased（P<0.05）;in the second stage of space exploration experiment,compared with the offspring mice in control group,the latency of offspring mice in arsenic exposure group about finding the platform for the first time was longer and the frequency of crossing the platform was reduced（P<0.05）,suggesting that arsenic exposure during pregnancy can affect the learning and memory ability of offspring.In the second part of the in vivo experiment which aimed to explore the relationship between arsenic exposure during pregnancy and the occurrence of placental ferroptosis,results showed that arsenic exposure during pregnancy displayed no significant effect on general condition of the placenta and the growth and development of the fetus（P>0.05）,obviously,the total arsenic level in whole blood and placental tissue of pregnant mice in arsenic exposure group were significantly higher than those in the control group;arsenic exposure can significantly change the morphological structure of trophoblast cells in placental tissue,additionally,arsenic reduced the levels of GSH,SOD and increased the level of MDA as well as increased the levels of HO-1 and NQO1（oxidative stress-related proteins）,decreased the levels of GPX4,x-CT and FTH1（ferroptosis related proteins）and increased the levels of total iron in placental tissue.These results suggested that arsenic exposure during pregnancy can lead to the destruction of oxidation-antioxidant system and induce ferroptosis in placental tissue.In vitro experiments,the results showed that Na As O₂ decreased the cell viability of HTR-8,decreased the levels of GSH and SOD,increased the level of MDA,increased the levels of HO-1,NQO1 and ROS,and decreased the levels of GPX4,x-CT and FTH1,led accumulation of Fe²⁺,and the structure of mitochondria showed obvious changes such as shrinkage,volume reduction,membrane rupture or ridge disappearance,suggesting that Na As O₂ can lead to lipid peroxidation,iron level imbalance and eventual ferroptosis in HTR-8.Finally,HTR-8 cells were treated with antioxidant NAC and ferroptosis inhibitor Fer-1 combined with Na As O₂,respectively.The results showed that the intervention agent could effectively relieve the oxidative stress response（upregulated the levels of GSH and SOD reduced by arsenic,reduced the levels of MDA,down-regulated the expression levels of HO-1 and NQO1 and decreased ROS levels）and reversed the occurrence of iron death(i.e.,GPX4,x-CT,and FTH1 expression were upregulated,Fe²⁺levels were significantly reduced,and intracellular mitochondrial shrinkage,volume reduction,membrane rupture,or ridge loss were significantly alleviated)in HTR-8 cells.Conclusions Maternal arsenic exposure during pregnancy can cause impairment of learning and memory ability of offspring.Arsenic exposure during pregnancy can cause oxidative damage in placenta,resulting in lipid peroxidation,then GPX4 depletion and Fe²⁺accumulation leading to ferroptosis in placenta.In the future,it is necessary to further explore the potential association mechanism between placental ferroptosis induced by arsenic exposure during pregnancy and impairment of learning and memory ability of offspring.