Gestational Cadmium Exposure Disrupts Fetal Liver Development Via Repressing Estrogen Biosynthesis In Placenta

Background: Cadmium(Cd)is a widely distributed environmental toxicant,which can damage placental function and fetal development.Physiologically,estrogen promotes embryonic/fetal development.However,the role and mechanism of estrogen in the impairment of fetal liver development by environmental Cd exposure during pregnancy are still unclear.Objective: The aim of this study was to investigate the role and mechanism of placental estrogen synthesis inhibition in fetal liver development damage induced by Cd exposure during pregnancy.Methods: Animal experiments and cell experiments were used in this study.The animal experiment consisted of three parts.Experiment 1: To investigate the effects of Cd exposure during pregnancy on fetal liver development,pregnant CD-1 mice were divided into control group(Ctrl),low dose Cd group(LCd,50 mg/L)and high dose Cd group(HCd,150 mg/L).From GD8 to GD17,pregnant mice were exposed to RO water and different concentrations of cadmium chloride(Cd Cl2)through drinking water.Experiment 2: To investigate the role of estradiol in the development of fetal liver damaged by Cd exposure during pregnancy,pregnant mice were divided into control group(Ctrl),estradiol supplementation group(E2),high dose cadmium group(HCd)and high dose cadmium + estradiol group(HE).From GD8 to GD17,pregnant mice in the E2 group and HE group were given intraperitoneal injection of estradiol(E2,15μg/kg/d).The pregnant rats in the HCd group and the HE group were exposed to Cd Cl2(150 mg/L)through drinking water,and the pregnant rats in the Ctrl group and the HCd group were intraperitoneally injected with normal saline.Experiment 3: To investigate whether Cd exposure during pregnancy impairs fetal liver development by inhibiting ovarian estrogen,pregnant mice were divided into Sham+control group(Sham),ovariectomy group(OVX),Sham+cadmium group(Sham+Cd)and ovariectomy+cadmium group(OVX+Cd).Mice in the OVX group and the OVX+Cd group underwent ovariectomy on GD7.Mice in the Ctrl and Cd groups were subjected to open abdominal sham surgery(without ovariectomy).Mice in the Cd group and OVX+Cd group were exposed to Cd Cl2(150 mg/L)in drinking water from GD8 to GD17.All pregnant mice were killed at GD18,and placenta and fetal liver were collected.Fetal liver proliferation and angiogenesis related indicators were detected by immunohistochemistry,HE staining,immunoblotting and RT assay.Estradiol levels in placenta and fetal liver were detected by ELISA.In experiment 1,human placental trophoblast JEG-3 cells were treated with Cd Cl2(20 μM)for 0,2,8 and 12 h,and estrogen synthase and PERK signal were detected.Experiment 2: To confirm the role of PERK signaling in the inhibition of placental estrogen synthesis by Cd,JEG-3 cells were pretreated with the PERK inhibitor GSK2606414 for 1 h and then treated with Cd Cl2(20 μM)for 8 h.The cells were collected for detection of PERK signaling and estrogen synthase.Results: The results of this study showed that maternal Cd exposure during pregnancy significantly reduced fetal liver weight and fetal liver coefficient,inhibited fetal liver proliferation-related proteins PCNA and Ki67,and down-regulated the expression of key angiogenesis proteins VEGF-A,indicating that maternal Cd exposure during pregnancy impaired fetal liver development.The concentration of estradiol in placenta and fetal liver of Cd-exposed group was significantly lower than that of control group.Subsequently,estradiol supplementation during pregnancy significantly alleviated the Cd-induced reduction of fetal liver weight and fetal liver coefficient,reversed the Cdinduced inhibition of fetal liver proliferation and down-regulation of key angiogenesis proteins,indicating that Cd exposure during pregnancy impairs fetal liver development by reducing estradiol levels.Immunoblotting results showed that Cd exposure significantly reduced the expression of estrogen synthetase CYP17A1 and 17β-HSD in placenta,but had no effect on fetal liver.Based on the ovariectomy experiment in pregnant mice,it was found that there was no difference in fetal liver weight and the expression of PCNA,VEGF-A and ERα between the Cd group and the Cd +ovariectomy group.These results suggest that Cd exposure during pregnancy reduces fetal liver estradiol levels through inhibition of placental-derived estrogen synthesis,independent of maternal ovarian estradiol and fetal liver synthesis.In addition,it was found that Cd exposure activated PERK signaling pathway and inhibited the expression of estrogen synthase CYP17A1 and 17β-HSD in mouse and human placental trophoblast cells.GSK2606414,a PERK inhibitor,significantly reversed the downregulation of estrogen synthase CYP17A1 and 17β-HSD protein expression induced by Cd in placental trophoblast cells.This suggests that maternal Cd exposure during pregnancy may inhibit placental estrogen synthesis by activating PERK signaling.Conclusions: Gestational Cd exposure may inhibit placental estrogen synthesis via activating PERK signaling,and then impair fetal liver development.