A Study On The Cytotoxity Of Two Types Of Nanoparticles On Ciliated Protozoa Pseudourostyla Cristata

With the development of nanotechnology,Ag NPs and TiO₂ NPs are increasingly used in people's daily life.Therefore,the number of nanomaterials that flows into the water environment is increasing,which is toxic to the aquatic organisms.Ciliated protozoa are single-cell eukaryotes that are widely distributed in water environments,and they are susceptible to physical or chemical stimuli.In addition,they are at the bottom of the water food chain,so they are the ideal model organism for risk assessment.Among them,the single-celled ciliated protozoa Pseudourostyla cristata has thin membrane and which body is flexible.Due to these features,we chose Pseudourostyla cristata as the testing subject to investigate the acute and chronic toxic effects of spherical Ag NPs and TiO₂ NPs with a particle size of 30 nm in this study.We also used the TEM,AO-PI double fluorescence staining,ATR-FTIR,the DHE fluorescent probe,the JC-1 fluorescent probe,and the measurement of antioxidant enzymatic activity to investigate the cytotoxic mechanism of the two nanoparticles preliminarily.1.Toxic effects of two types of nanoparticles on Pseudourostyla cristataThe cells were subjected to Ag NPs and TiO₂ NPs respectively for 24 h for acute toxicity test.It was found that the cell mortality and the rate of reproductive inhibition were linear with the logarithm of the concentration.We calculated that the 24h-LC₅₀ of Ag NPs and TiO₂ NPs against Pseudourostyla cristata was 1.21×10^-5 mg/L and 138.75mg/L,the value of 24h-LC₀ was 4.62×10^-6 mg/L and 122.59 mg/L,the value of 24h-EC₅₀0 was 3.55×10^-6 mg/L and 88.85 mg/L.It is indicated that the acute toxicity of Ag NPs to Pseudourostyla cristata is greater than that of TiO₂ NPs.In addition,we also found that both of the nanoparticles have an inhibitory effect on the growth and reproduction of cells,and Ag NPs is stronger.2.Preliminary study on the cytotoxic mechanism of the toxicity of two types of nanoparticles on Pseudourostyla cristataObserved by AO-PI double fluorescent staining combined with TEM,we found that 24h-LC₅₀ of Ag NPs induced vast lysosomes at different stage in cells.Both 24h-LC₅₀ and 24h-EC₅₀ of TiO₂ NPs caused damage to the cell membrane structure,but the destruction of cell membrane structure caused by two concentrations of Ag NPs was not observed.We used ATR-FTIR to detect the changes of the biomacromolecules of the constituents of the cell membrane caused by the two nanoparticles,it was found that24h-LC₅₀ of Ag NPs caused the peak of functional group in cell membrane components decreased,such as??=CH?,-COO^-,?_a(PO^2-)and??C-OH?of carbohydrates.In addition,the content of the above functional groups in the TiO₂ NPs treatment also decreased.Besides,the treatment of TiO₂ NPs induced a large number of RHC=O groups,and caused the amideI group completely disappeared.In conclusion,the TiO₂ NPs have a greater damage to the structure and chemical composition of the cell membrane than Ag NPs.The results of the DHE fluorescent probe,the JC-1 fluorescent probe,the TEM and the measurement of antioxidant enzymatic activities shows,the Ag NPs induced more ROS in cells,caused significant decrease of the mitochondrial transmembrane potential?MMP?and induced more damage to the mitochondrial structure than the TiO₂NPs treatment.Furthermore,the effect of Ag NPs on the activities of SOD and CAT was greater than that of the TiO₂ NPs treatment,which indicated that the oxidative stress damage caused by Ag NPs to cells was stronger than TiO₂ NPs.In summary,we first proposed that Pseudourostyla cristata can be used as a model organism for early environmental prediction risks of nanomaterials by comparing the acute and chronic toxic effects of Ag NPs and TiO₂ NPs on Pseudourostyla cristata with other model organisms.For the first time,microscopic observations combined with ATR-FTIR to investigate the mechanism of TiO₂ NPs.In this paper,the toxic mechanism of Ag NPs which caused the organelle and biochemical indexes changes in cells is explored and finally we found it mainly caused cell apoptosis by oxidative stress and mitochondrial destruction.