Study On Cyto-phototoxicity And Mechanism Of Titanium Dioxide Nanoparticles Under UVA Radiation

With the development of nanotechnology and the widespread application of nanomaterials,the biosafety of nanomaterials has attracted much attention.Titanium dioxide nanoparticles(nano-TiO₂)are a critical nanomaterial,widely used in cosmetics,food,medicine,environmental protection,construction materials,painting,and other fields.Large-scale production and application make the safety of nano-TiO₂ increasingly concerned.Especially under ultraviolet A(UVA,320?400 nm)irradiation,the potential phototoxic hazards of nano-TiO₂ with photochemical activity are of concern.At present,the studies on the phototoxicity of nano-TiO₂ mainly focus on the environmental ecosystem.Moreover,there is a lack of systematic and in-depth research on its phototoxicity at the cellular and molecular levels.This paper investigated the effects of nano-TiO₂ and UVA on sialic acids and genes,using glycans specific recognition,transcriptomics,and three-dimensional(3D)cell culture to explore the phototoxicity and its mechanism of nano-TiO₂ and UVA from different perspectives.The main contents and results of this research are as follows:(1)Sialylation is an essential glycosylation modification process and is involved in a variety of physiological processes.In this paper,the effects of different crystallines of nano-TiO₂(rutile R25,anatase A25,and mixture crystalline P25)on sialic acid levels in human keratinocytes(HaCaT)under UVA irradiation and the mechanism were explored.The results showed that the mixture crystalline nano-TiO₂ P25 significantly reduced the viability of HaCaT cells under UVA irradiation,with the strongest phototoxicity,followed by A25,and the R25 was weakest.The lectin staining results showed that 50?g/m L nano-TiO₂ P25 and UVA caused the alterations in the level of?2,6-linked sialic acids on HaCaT cells,while A25 and R25 did not change the level of sialylation.Under UVA irradiation,nano-TiO₂ P25 induced the production of reactive oxygen species(ROS)in HaCaT cells,which broke the intracellular Ca²⁺balance,disrupted the stability of mitochondrial membrane potential,and led to the death of HaCaT cells.It was found that ROS caused an increase of?2,6-linked sialic acids.The antioxidant vitamin C could remove the excess ROS,reverse the abnormal alteration of?2,6-linked sialic acids,and reduce HaCaT cell death.Western blotting results showed that under UVA irradiation,nano-TiO₂ P25 may affect the level of?2,6-linked sialic acids by increasing the expression of ST6Gal-?.This work investigated the phototoxic mechanism of nanomaterials based on the effect of nano-TiO₂ on sialic acids.(2)Transcriptomics was used to quantify gene expression changes to investigate the phototoxic mechanism of nano-TiO₂ and UVA.Compared with the control group,there were 841 differentially expressed genes in the HaCaT cells treated with nano-TiO₂ and UVA,among which 499 genes were up-regulated,and 342 genes were down-regulated.While there were 169 and 114 differentially expressed genes in the nano-TiO₂ group and UVA group,respectively.KEGG Pathway analysis showed that under UVA irradiation,nano-TiO₂ led to a significant difference in gene expression related to signal transduction pathways.The enrichment level of differentially expressed genes involved in receptor interaction,cell adhesion,Fox O signaling pathway and TGF-?signaling pathway was the most significant.GO functional analysis indicated that nano-TiO₂ and UVA would significantly enrich differentially expressed genes related to cell process,metabolic process,biological regulation process,molecular binding,and catalytic function.Real-time PCR results confirmed that nano-TiO₂ and UVA significant upregulated the expression of genes related to the inflammation,cancer and cell survival(IL1A,MYC,CDKN1A,JUN and THBS1),leading to potential health risks.The results of transcriptomics provided a strong basis for the subsequent studies on the phototoxic mechanism of nano-TiO₂.(3)3D fibrin gel culture technology was used to study the phototoxic mechanism of nano-TiO₂ and UVA.The results showed that the H22 cells were treated with 100?g/m L nano-TiO₂ under UVA irradiation and cultured in 3D fibrin gel for 7 days,resulting in a reduction in cell spheroid diameter by about 25%.However,nano-TiO₂ or UVA treatment alone did not significantly affect the size of 3D cell spheroids.Through cell senescence staining and apoptosis detection,it was found that cell cycle arrest at G0/G1 phase caused cell spheroid size reduction instead of cell senescence and apoptosis.Western blotting and real-time PCR experiments showed that under UVA irradiation,nano-TiO₂ activated the TGF-?/Smad signaling pathway and increased the expression of TGF-?1,Smad3,Cdkn1a,and Cdkn2b both at the m RNA and protein levels,leading to cell cycle arrest at G0/G1phase.DCFH-DA probe and H₂O₂ detection showed that ROS produced by nano-TiO₂under UVA irradiation was involved in regulating the cell cycle by inducing TGF-?1expression.This paper reveals the phototoxicity of nano-TiO₂ and its molecular mechanism,which is of great significance to guide the safe application of nano-TiO₂.It provides the theoretical and experimental basis for the toxicological study of photochemical active nanomaterials.