Research On The Joint Cytotoxicity Of Nano Metallic-oxide&Vitamin C In Food Additives To Gastrointestinal Tract And Nervous System

Nowadays, with the fast development of nanotechnology, metal oxidenanoparticles are receiving increasing attention for a large variety of applications.Major metal oxides nanoparticles: zinc, silica, copper and titanium are showingpromise in food safety and technology. Food and Drug Administration （FDA） havelisted Zinc oxide （ZnO） as a generally recognized as safe （GRAS） material which canbe used as food additive. Titanium dioxide （TiO2） has been used commercially since1900which are widely used in food additives as a white pigment for products such ascheese and white chocolate. So far, Food additive safety evaluation standard wasestablished on the basis of the toxic effects of single additive. But the kinds of foodspeople eat every day, a combined use of additives often exist in a variety of food. Toevaluation the joint toxicity effects of food additives has great significance to ensurethe safe use of food additives. Vitamin C （VC） have been widely studied and appliedin the fields of food additives. VC acts as a potent water-soluble antioxidant inbiological fluids for the synthesis and function of immune system, many foodadditives add VC to enhance human immunity. It is important to know the jointcytotoxicity of metallic nano-oxide&VC in food additives to gastrointestinal tractand nervous system. Herein, we adopted kinds of cytotoxicity assays to research onthe joint toxicity effects by using the gastric epithelial cell line（GES-1）and the neuralstem cell （C17.2） as gastrointestinal tract and nervous system models. The ZnOnanoparticals （ZnO-NPs） and TiO2nanoparticals （TiO2-NPs） in different particle sizesand different properties （hydrophilic or oleophylic） are used in our study.First of all, we use transmission electron microscopy （TEM）, X-ray fluorescenceanalysis, X ray diffraction technology, NTA, Zeta potential and infrared spectrummethod characterized size, crystal structure, purity, particle size and surface potentialin the medium and in the water, sample surface properties for all the samples. Sixkinds of ZnO-NPs were used in this study, namely ZnO-1（nanoparticles with anaverage diameter of20nm, hydrophilic）, ZnO-2（nanoparticles with an average diameter of20nm, oleophylic）, ZnO-3（nanoparticles with an average diameter of100nm, hydrophilic）, ZnO-4（nanoparticles with an average diameter of100nm,oleophylic）, ZnO-5（nanoparticles with an average diameter of100nm, foodgrade）and ZnO-6（nanotubes with an average diameter of100nm, food grade）, ZnO-5and ZnO-6came from different manufacturers. Four kinds of TiO2-NPs were usedin this study, namely TiO2-1（nanoparticles with an average diameter of20nm,hydrophilic）, TiO2-2（nanoparticles with an average diameter of20nm, oleophylic）,TiO2-3（nanoparticles with an average diameter of50nm, highly dispersed foodgrade） and TiO2-4（nanoparticles with an average diameter of50nm, food grade）. TheTEM images show the size and morphology of ZnO-NPs and TiO2-NPs. From TEMimages we can see that the sizes of ZnO-NPs and TiO2-NPs are different. However,there is no big difference of the sizes of ZnO-NPs and TiO2-NPs in water or inmedium. The purities of these NPs are higher than95%according to X-rayfluorescence （XRF） analyses. Based on the X-Ray diffraction （XRD） patterns,ZnO-NPs are in hexagonal type, while the TiO2-NPs are in anatase phase.On this basis, this paper studied the joint cytotoxicity of the composite system:metal nano oxide&VC. Studies have shown that when the concentration of ZnO-NPsis less than15μg/mL or the concentration of VC is less than300μg/mL, there are nosignificant toxicity to GES-1and C17.2. The cell viability is higher than90%and95%respectively. However, when the15μg/mL of ZnO-NPs and300μg/mL of VCare introduce to cells together, we have found that VC can strengthen the cell toxicity（GES1and C17.2） of ZnO-NPs, the cell viability decreased to30%. Based onapoptosis experiment results we find that VC （300μg/mL） and ZnO-5（15μg/mL）are not caused early apoptosis, but the group of ZnO-5（15μg/mL）&VC （300μg/mL）cause early apoptosis significantly compared with control group after co-incubationwith cells for24h. For GES-1cells the apoptosis rate is as high as52.17%, mortalityrate is5.46%; for C17.2cells the apoptosis rate is as high as51.38%, mortality rateis9.44%. Fluo-3probes are used to detect the effect on the concentration of Ca²⁺incells. GES-1cells and C17.2cells are exposed to ZnO-5（15μg/mL）, VC （300μg/mL） and ZnO-5（15μg/mL）&VC （300μg/mL）. The concentration of Ca²⁺in cells keep unchanged after coincubaiton with ZnO-5（15μg/mL） and VC （300μg/mL） for2h. But after cells coinbation with ZnO-5（15μg/mL）&VC （300μg/mL） for thesame time, the concentration of Ca²⁺is increased significantly. From confocal laserscanning study, we further confirme the joint cytotocixity of ZnO-NPs and VC.ZnO-5（15μg/mL） and VC （300μg/mL） has no singnificant effect on the cells, butZnO-NPs （15μg/mL）&VC （300μg/mL） has caused significant deformation of thenucleus, which indicates that most of the cells have died. But when TiO2-NPs and VCcombination, there is no joint toxicity effect on cells.Finally, we have explored the reason why the two metal oxide nanoparticlesproduced different results. Studies have found that ZnO-NPs can easily react withvitamin C, produce large amounts of Zn²⁺, when the concentration of ZnO-NPs is30μg/mL, the solubility of Zn²⁺is17μg/mL, after added VC the solubility of Zn2+increase to29μg/mL, which makes effects on cells. On the other hand, VC reactswith ZnO-NPs made the reunion of ZnO-NPs reduced, and thus it is easier to entercells to affect cells.Our results are essential for the biomedical applications and food safetyassessment of complex system and would stimulate more toxicology evaluations ofcomplex system.