Cadmium Induced Oxidative Damages Of Placenta Trophoblast Cells And The Underlying Mechaisms

Cadmium is a toxic heavy metal widely used in industrial production that leads to serious public health hazards in both highly industrialized countries and developing countries.Previous studies showed that Cd is enriched through the food chain,occupational exposure and cigarette smoking,and eventually accumulates in blood,brain,kidneys,and livers as well as in the reproductive organs,including ovaries,testes and placentas.In particular,women during pregnancy are at greater risk of Cd toxicity because Cd is more liable to accumulate in placentae,resulting in a series of placental-related disorders,such as preeclampsia（PE）,fetal growth restriction（FGR） and miscarriage.In this study,human placental trophoblast cells（HTR-8/SVneo） were used as research models to explore the molecular mechanism of Cd-induced apoptosis in human placenta trophoblast cells,and provide a theoretical basis for studying the pathogenesis of Cd-induced abortion in mammals.The experimental results are as follows:1.MTT results showed that CdCl₂ suppressesed HTR-8/SVneo cell viability and inhibited cell growth in a time- and dose-dependent manner.It was found that the cell morphology showed obvious rounding and shrinkage with the increase of CdCl₂ concentration and the prolongation of treatment time by optical microscopy.Both DCFH-DA fluorescence staining and flow cytometry showed that CdCl₂ treatment induced HTR-8/SVneo cells to produce large amounts of reactive oxygen species（ROS）.It was found that CdCl₂ treatment inhibited the activity of superoxide dismutase（SOD）,catalase（CAT） and glutathione peroxidase（GSH-Px） in HTR-8/SVneo cells,and damaged the antioxidant system;CdCl₂ also induced significantly lipid peroxidation damage in HTR-8/SVneo cells,which caused an increase in intracellular malondialdehyde（MDA） content.The antioxidant N-Acetyl-cysteine（NAC） inhibited intracellular ROS accumulation and protected antioxidant system in HTR-8/SVneo cells.2.It was found by transmission electron microscopy that the mitochondria in HTR-8/SVneo cells were vacuolated and the matrix density decreased after CdCl₂ treatment,and the sputum appeared to break or even disappear.And JC-1 fluorescence staining revealed a decrease in mitochondrial membrane potential of HTR-8/SVneo cells after CdCl₂ treatment.Alkaline comet experiments showed that CdCl₂ induced DNA damage in HTR-8/SVneo cells in a dose-and time-dependent manner.The formation of γ-H2AX focus was observed of the CdCl₂ treatment group by immunofluorescence.Meanwhile,by western blot analysis,the expression of γ-H2AX protein in HTR-8/SVneo cells was significantly increased after CdCl₂ treatment which confirmed the results of the comet experiment.In addition,CdCl₂ treatment can change the cycle distribution of HTR-8/SVneo cell,arrest the cell cycle in G0/G1 phase,and decrease the number of cells in S phase,and inhibit cell growth.Hoechst 33342 and AO/EB fluorescence staining showed that there were chromatin condensation,nuclear pyknosis and even fragmentation in the nucleus after CdCl₂ treatment.Transmission electron microscopy showed that there were chromatin condensation and edge aggregation in the nucleus after CdCl₂ treatment.Annexin V-FITC/PI double fluorescent staining and flow cytometry showed that the apoptotic rate increased with the increase of CdCl₂ concentration.Agarose gel electrophoresis revealed that the DNA in the cells was gradually broken into 180-200 bp fragments after CdCl₂ treatment,forming a DNA ladder,which is a prominent feature of apoptosis.Western blot analysis also showed that the expression of cleaved caspase-3 and proapoptotic protein Bax increased gradually after CdCl₂ treatment,while the expression of anti-apoptotic protein Bcl-2 decreased gradually,and the ratio of Bax/Bcl-2 gradually increased.This indicated that CdCl₂ treatment induced apoptosis in HTR-8/SVneo cells.NAC could alleviated mitochondrial and DNA damage,cell cycle arrest and apoptosis induced by CdCl₂-induced intracellular ROS accumulation in HTR-8/SVneo cells.3.After established the mouse Cd poisoning model,it was found that theplacental diameter and quality of the mouse were significantly decreased after CdCl₂ treatment,and the trophoblast cells appeared severely nuclear condensation and fragmentation,and the cells were lytic necrotic.Immunofluorescence revealed that CdCl₂ specifically induced DNA damage and apoptosis in trophoblast cells of mouse placenta.In conclusion:Cd triggers oxidative stress mediated mitochondrial injury and apoptosis in human trophoblast HTR-8/SVneo cells.These findings in our study provided new insights in understanding the mechanisms of placental-related disorders caused by Cd exposure.