Study On The Combined Toxicity Of Titanium Dioxide Nanoparticles And Copper Ions

Objective:To study the toxic effects of titanium dioxide nanoparticles?TiO₂-NPs?combined with Cu²⁺ on bovine hemoglobin?BHb?,human bronchial epithelial?16HBE?cells and zebrafish embryos.To evaluate the toxic effects of TiO₂-NPs combined with Cu²⁺ at molecule,cell and animal levels.Methods:The surface morphology,particle size,Zeta potential of TiO₂-NPs and mixture of TiO₂-NPs and Cu²⁺ were characterized by transmission electron microscopy.BHb was used as a protein model.The interaction between BHb and TiO₂-NPs,Cu²⁺,mixture of TiO₂-NPs and Cu²⁺ were investigated by UV-Vis spectroscopy,fluorescence spectroscopy,synchronous fluorescence spectroscopy,time-resolved fluorescence spectroscopy and circular dichroism spectroscopy.The toxic effects on BHb were evaluated.16HBE cells as the research object,the toxicity of TiO₂-NPs,Cu²⁺ and their mixture on 16 HBE cells were determined by MTT assay.Apoptosis of 16 HBE cells were detected by AO/EB fluorescent double staining.Zebrafish embryos as animal model,the hatching rate,mortality rate,malformation rate and normal development rate of the swim bladder were used as indicators to construct the toxic evaluation system of TiO₂-NPs,Cu²⁺ and their combination in vivo.Results:The particle size of TiO₂-NPs was about 20 nm,its Zeta potential was 0.0606 mV,and its average particle size in aqueous solution was 76.44 nm.Spectroscopic results showed that TiO₂-NPs,Cu²⁺ and their mixture could interact with BHb,which resulted in the fluorescence quenching of BHb and their quenching mechanisms were mainly static quenching.The conformation and secondary structure of BHb were changed,and the spiral structure of BHb was looser after reacting with TiO₂-NPs,Cu²⁺ and their mixture.All of them had toxic effects on BHb.TiO₂-NPs,Cu²⁺ and their mixture were toxic to 16 HBE cells in a dose-dependent and time-dependent manner.The results of AO/EB demonstrated that TiO₂-NPs,Cu²⁺ and their mixture could induce apoptosis of 16 HBE cells.TiO₂-NPs?0-800 mg·L-1?combined with 10 mg·L-1 Cu²⁺ had stronger toxicity on 16 HBE cells than that of TiO₂-NPs.When the concentration of TiO₂-NPs was in the range of 0-200 mg·L-1,the mortality of zebrafish embryos was less than 15%,and there were no significant difference between TiO₂-NPs treatment group and control group in the abnormal rate,hatching rate and normal development rate of the swim bladder.There was a dose-dependent relationship when the concentration of Cu²⁺ was more than 0.125 mg·L-1,the hatching rate?< 26%?of zebrafish embryos and the normal development rate?< 27%?of the swim bladder at the end of exposure were lower,and the mortality rate?> 8%?and malformations?> 25%?at the end of exposure were higher.The toxicity of TiO₂-NPs?0-1 mg·L-1?combined with 0.125 mg·L-1 Cu²⁺ on zebrafish embryos was similar to that of Cu²⁺ alone.When the concentration of TiO₂-NPs was in the range of 5-200 mg·L-1,the hatching rate and normal development rate of the swim bladder were increased while the mortality and malformation were decreased.Conclusion:TiO₂-NPs,Cu²⁺ and their mixture had certain toxic effect on BHb.The toxic effect of TiO₂-NPs with the concentration of 0-800 mg·L-1 combined with 10 mg·L-1 Cu²⁺ on16 HBE cells was increased in vitro.The toxic effect of TiO₂-NPs with the concentration of 5-200 mg·L-1 combined with 0.125 mg·L-1 Cu²⁺ on zebrafish embryos was decreased in vivo.