The Role Of ATF3 In ZnO Nanoparticles-induced Toxicity In Human Bronchial Epithelial Cells

Nanoparticles(NPs)are engineered structures with less than 100 nm in at least one dimension.These materials are increasingly being used for diverse industrial and biomedical applications as well as in consumer products.Among these NPs,zinc oxide nanoparticles(Zn O NPs)are one of the most important metal oxide nanoparticles,which have raised serious concerns about their safety for human health and the environment.As airway represents the predominant exposure route for Zn O NPs,it becomes imperative to understand the potential toxic effects of Zn O NPs on the human respiratory system and the underlying molecular mechanisms.However,the interplay between Zn O NPs and cellular components is still limited.Our previous study demonstrated Zn O NPs induced the expression of ATF3.In this study,we evaluated the role of ATF3 in Zn O NPs induced toxicity in human bronchial epithelial cells.Firstly,we evaluated the characterization of Zn O NPs.Data showed that the Zn O NPs we used exhibited a hexagonal structure with the average diameter of 34.0 nm.Then we determined the cytotoxicity of Zn O NPs.CCK-8 assay revealed that exposure to 5 ?g/m L of Zn O NPs for 24 h significantly reduced cell viability.And Zn O NPs decreased cell viability in a dose-dependent manner with an estimated LC50 at about 7.5 ?g/m L.Induction of oxidative stress has been ascribed as the main cause of Zn O NPs toxicity.We observed that 6 h exposure to 5 ?g/m L of Zn O NPs was enough to induce significant elevation of intracellular ROS level in HBE cells.To obtain insights into the possible Zn O NPs genotoxicity,we analyzed DNA-double strand breaks using ?H2AX foci staining.Data showed that both the average number of ?H2AX foci per cell and the percentage of ?H2AX foci positive cells significantly increased in cells treated with Zn O NPs at the concentration of 5 ?g/m L.These data indicated that Zn O NPs induce cytotoxicity and genotoxicity in HBE cells.Since ATF3 plays important roles in various stresses,we evaluated the response of ATF3 to Zn O NPs.Data showed Zn O NPs increased ATF3 level and ROS scavenger NAC almost reduced ATF3 expression to the basal level,implying that the induction of ATF3 relies on ROS signaling.Nrf2 is a master transcription factor regulating antioxidant responses.To determine the role of Nrf2 in regulating ATF3 expression,we silenced the endogenous Nrf2 by si RNA and found that knockdown of Nrf2 resulted in a significant reduction of ATF3 expression under Zn O NPs treatment,indicating that ATF3 transcription is under the control of Nrf2.To determine the direct regulation of Nrf2 on ATF3 expression,we used the luciferase reporters containing ATF3 promoters with either wild-type or AREs-deleted sequences.Data indicated that ATF3 promoter activity is Nrf2-dependent.Given the induction of ATF3 by Zn O NPs,we investigated the role of ATF3 in Zn O NPs-induced genotoxicity.By down-regulating the expression of ATF3 in HBE cells,we found that the ?H2AX foci significantly increased after ATF3 knockdown,implying that ATF3 played an essential role in DNA repair response during Zn O NPs treatment.Cell apoptosis is one way for the cells to prevent the damaged DNA from being passed down to its progeny.Therefore,we evaluated the function of ATF3 in regulating cell apoptosis during Zn O NPs-induced genotoxicity.Data showed that the apoptosis in ATF3-deficient cells was significantly induced after Zn O NPs treatment.These results demonstrated that ATF3 is essential in mediating cell apoptosis during Zn O NPs-induced genotoxicity.As p53 is a key factor in regulating DNA repair and apoptosis,we explored the relationship between ATF3 and p53 to explore the mechanism of action of ATF3.Data showed that Zn O NPs treatment increased p53 protein expression,while knockdown of ATF3 decreased p53 level.Besides,Co-immunoprecipitation(Co-IP)assay showed that p53 was co-immunoprecipitated by FLAG-ATF3,indicating an interaction between ATF3 and p53.These data suggested that ATF3 binds to p53 and stabilizes p53 protein during Zn O NP treatment.In summary,the main findings of this dissertation include that Zn O NP treatment dramatically induced the expression of ATF3 in human bronchial epithelial(HBE)cells,which was mediated by the nuclear factor erythroid 2-related factor 2(Nrf2).ATF3 was required for the repair of Zn O NP-induced DNA damage as gamma foci number increased when endogenous ATF3 was silenced.Moreover,ATF3 also contributed to Zn O NP-induced cell apoptosis.Mechanistic study revealed that ATF3 interacted with the p53 protein and upregulated its expression under Zn O NP treatment.Collectively,our findings demonstrated ATF3 as an important regulator of epithelial homeostasis by promoting both DNA repair and the death of damaged cells under Zn O NP-induced genotoxic stress.