Toxicity Of Nickel Nanoparticles On Human Placental Trophoblast Cells And Underlying Molecular Mechanism

Objective:To provide a scientific basis for the health protection of occupational and environmental exposure on pregnant women,the mitochondrial toxicity of nickel nanoparticles（Ni NPs）on human placental trophoblast cells and underlying molecular mechanism were studied.Methods:MTT test was used to detect the cell viability under different concentrations（0,2.5,5,7.5,10,12.5μg/cm²）of Ni NPs on human placental trophoblast cells（HTR-8/SVneo）;Flow cytometry was used to detect the content of intracellular reactive oxygen species（ROS）,mitochondrial membrane potential（JC-1）,and the rate of cell apoptosis and cell cycle;Fluorescence microscope was used to observe ROS and JC-1 level at various concentrations after fluorescent staining;Luminometer was used to measure RLU,and then value of intracellular ATP content was calculated;Fluorescence microplate reader was used to measure intracellular MDA,SOD,and Cu Zn/Mn-SOD activities;q RT-PCR was used to detect mitochondrial DNA（mt DNA）content to evaluate the mitochondrial DNA replication;Western Blotting was used to detect protein expression levels in the placental trophoblast cells related with the Nrf2-regulated antioxidant signaling pathway,MAPK signaling pathway and Cytochrome c apoptosis signal pathway at different exposure concentrations.Results:1.MTT assay showed that Ni NPs caused HTR-8/SVneo cell shrinkage and decreased the cell viability after 24 hour treatment.With the increase of the Ni NPs concentration,the number of living cells and the cell density gradually decreased and the black particles in the cells were observed.The vacuoles in the cells could be found under the microscope,and a large number of suspended cells could be found at the higher Ni NPs concentration groups（10,12.5μg/cm²）.2.Flow cytometry analysis showed that,compared with the control group,the FITC level in the Ni NPs treated cells gradually increased,the level of ROS were also simultaneously increased,in a significant dose-dependent relationship;Fluorescence microscopy showed that with the increase of the Ni NPs concentration,the proportion of cells emitting green fluorescence gradually increased.3.Compared with the control group,the ATP level in the Ni NPs treated cells significantly decreased at the dose of 5,7.5,10,and 12.5μg/cm² groups;While MDA levels significantly increased,in a significant dose-dependent manner.4.Flow cytometry analysis showed that,compared with the control group,the Ni NPs treated cells showed a rapid decrease in the fluorescence ratio of JC-1 polymer/JC-1monomer,indicating that the mitochondrial membrane potential was significantly decreased.Fluorescence microscopy showed that with the increase of the Ni NPs concentration the red fluorescence gradually decreased,and only very little red fluorescence could be observed under the high Ni NPs concentration,while the green fluorescence gradually increased.5.Compared with the control group,the total SOD activity of cells decreased significantly with the increase of the Ni NPs concentration.At the dose of 5,7.5,10,12.5μg/cm² group,Cu Zn/Mn-SOD activity decreased significantly.6.Flow cytometry analysis showed that,compared with the control group,the proportion of Ni NPs treated cells in the Q2 quadrant increased significantly,up to 32.7%at the dose of10μg/cm² group,while the proportion of apoptotic cells in the 12.5μg/cm² group decreased;the histogram and statistical analysis showed that the apoptosis rate of HTR-8/SVneo cells increased significantly with the increase of Ni NPs concentration.7.Flow cytometry detection showed that,compared with the control group,there was no obvious change in the proportion of HTR-8/SVneo cells in G₀/G₁ phase at the low Ni NPs concentration group,the proportion of cells in G₀/G₁ phase decreased significantly in the dose of10μg/cm² group.Compared with the control group,the percentage of S-phase increased significantly at the dose of 2.5,5,7.5,10,and 12.5μg/cm² groups.The percentage of cells in the G₂/M phase significantly decreased under the Ni NPs treatment.8.q RT-PCR analysis showed that the ND1 andβ2M primers were well designed.Compared with the control group,the relative copy number of mt DNA at 2.5,5,7.5,10,and 12.5μg/cm²concentration groups decreased significantly.9.Compared with the control group,the expression level of Nrf2 and P62 significantly decreased at 5,7.5,10,and 12.5μg/cm²,while the expression levels of Keap1,NQO1 and HO-1significantly increased;Ni NPs showed up-regulation of the phosphorylation level of MAPK signaling pathway.Compared with the control group,the expression level of p-Erk,p-P38,and p-JNK significantly increased;Ni NPs could activate Cytochrome c apoptosis signal pathway indicated by significant increase of the ratio of Bax/Bcl-2,Cleaved Caspase-9,Cleaved Caspase-3,and Cleaved PARP-1.Conclusion:Ni NPs induced the reduction of viability of HTR-8/SVneo cells and inhibit cell proliferation;Ni NPs can disturb the HTR-8/SVneo cells’oxidation/antioxidant balance,and then to activate the MAPK protein signaling pathway,which in turn mediates Nrf2 transcription factors into the nucleus and up-regulated NQO1 and HO-1 protein expression levels.At the same time,Ni NPs activated cytochrome c apoptosis signal pathway,up-regulated cleavage of Capase-9,Caspase-3,and PARP protein,thereby induce apoptosis.Furthermore,Ni NPs can induce cell S-phase arrest to inhibit cell restore undergo the apoptotic process.In addition,the Ni NPs caused a significant decrease in the relative copy number of mt DNA and affected mitochondrial function.In conclusion,our results suggest that Ni NPs show significant toxicity on human placental trophoblast cells,therefore,the potential health hazards on pregnant women after occupational or environmental exposure should not be ignored.