The Mechanism Of Cadmium-induced Neural Tube Defects And Fetal Growth Restriction

Objective:Previous studies found that maternal Cd exposure on gestational day?GD?9 caused forelimb ectrodactyly and taildeformity,The aim of the present study was to investigate whether maternal Cd exposure on GD8 induces fetal neural tube defects?NTDs?.Methods:The present study consisted of three independent experiments.Experiment 1.To investigate Cd-inducedneural tube defects in mice,pregnant mice were divided randomly into three groups.In Cd group,pregnant mice were intraperitoneally?i.p.?injected a single dose of Cd Cl₂?2.5 or 5.0 mg/kg?between 08:00 and 09:00 h on GD8.The saline-treated pregnant mice served as controls.The doses of Cd Cl₂ used in the present study were determined by preliminary experiments.The critical period of neural tube development is on GD8.In order to establish the mouse model of Cd-induced NTDs,the time of Cd exposure was chosen on GD8.For mechanical studies,a single dose of Cd by intraperitoneal injection was chosen in the current study.All animals were inspected daily for clinical signs and determined whether a pregnancy loss had occurred according to clinical signs and maternal weight.The dams were sacrificed on GD18.The uterine horns were exposed and weighed.Live,dead and resorbed fetuses were counted.Placentas were collected for histological examination.Live fetuses were sexed,weighed,and examined for external morphological malformations.Experiment 2.To investigate the effectsof maternal Cd exposure during pregnancy on placental folate transport,twenty-four pregnant mice were divided randomly into two groups.In Cd group,pregnant mice were ip.injected with a single dose of Cd Cl₂?5.0mg/kg?between 08:00 and 09:00 h on GD8.The saline-treated pregnant mice served as controls.Pregnant mice were sacrificed 24 h after Cd injection.Maternal serum and embryo were collected for measurement of folate contents.Experiment 3.To investigate the effects of maternal Cd exposure during pregnancy on the expression of placental folate transporters,forty-eight pregnant mice were divided randomlyinto eight groups.In Cd group,all pregnant mice were i.p.injected with a single dose of Cd Cl₂?5.0 mg/kg?between 08:00 and 09:00 h on GD8.The saline-treated pregnant mice served as controls.Cd-treated pregnant mice were sacrificed at different time points?2,12,24,48 and 72 h?after Cd injection.Normal saline-treated pregnant mice were killed on GD8,GD9,GD10 and GD11.Placentas were collected for real-time RT-PCR and Western blotting.Results:Maternal Cd exposure on GD8 resulted in the incidence of neural tube defects?NTDs?.Among mice injected with2.5 mg/kg of Cd Cl₂,33.3%?4/12?of litters were with NTDs.Surprisingly,75%?12/16?of litters were with NTDs among mice injected with5.0 mg/kg of Cd Cl₂.Furtheranalysis showed that 3.6% of fetuses per litter were with either anencephaly or exencephaly or encephalomeningocele among dams injectedwith 2.5 mg/kg of Cd Cl₂.In addition,20% of fetuses per litter were withanencephaly,exencephaly or encephalomeningocele among damsinjected with 5.0 mg/kg of Cd Cl₂.Maternal Cd exposureon GD8 reduced fetal weight in a dose-dependent manner.In addition,maternal Cd exposure on GD8 significantly reduced crown-rump length.Maternal Cd exposure on GD8 reduced placental weight in a dose-dependent manner.In addition,maternal Cdexposure on GD8 significantly reduced placental diameter.The internal space of fetal and maternal blood vessels wasreduced in placentas of Cd-treated mice.To test whether maternal Cd exposure on GD8 disturbs placental folate transport from maternal circulation into the embryos,folate content in maternal serum and embryo was measured 24 h after Cdinjection.There was no significant difference on folate content in maternal serum betweenCd-treated mice and controls.Interestingly,embryonic folate content was obviously decreased in Cd-treated mice.The level of placental Pcft m RNA was markedly decreased asearly as 2h after Cd injection.Interestingly,the level of placental Pcftm RNA remained decreased at 24 h after Cd injection.Correspondingly,the protein level of placental PCFT was persistently reduced,beginningat 24 h and remaining decreased at 72 h after Cd injection.Conclusion:Maternal Cd exposure on GD8 resulted in the incidence of NTDs and fetal growth restriction in mice and maternal Cd exposure during organogenesis disturbs placental folate transport from maternal circulation into the fetuses through down-regulatingplacentalfolate transporters.