Mechanisms Of Zine Oxide Nanoparticle-induced Interleukin 8 Gene Expression In Human Bronchial Epithelial Cells

Background:Nanoparticles refer to the manufactured particles less than 100 nm in diameter. They have been used for a wide variety of applications. Metal oxide nanomaterials (such as zinc oxide nanoparticles, ZnO-NPs) have been produced in high tonnage worldwide. ZnO-NPs can enter the human body through inhalation, skin contact, ingestion, and other routes. The results from human volunteer studies have shown that inhaled ZnO-NPs cause metal fume fever, with increased levels in inflammatory mediators, such as IL-8, and elevated number in polymorphonuclear leukocytes in bronchial-alveolar lavage fluid. The in vitro studies have demonstrated that exposure of human airway epithelial cells to ZnO-NPs results in excessive production of IL-8 protein. IL-8 protein in human airways is mainly derived from epithelial cells and plays a critical role in mediating the pathogenesis of exogenous toxicant-induced pulmonary disorders. Thus far, the mechanisms underlying ZnO-NPs-induced IL-8 gene expression in human bronchial epithelial cells remain unclear.Aims:The aims of this study are to determine the effect of domestic ZnO-NPs (30nm in diameter) on IL-8 expression in human bronchial epithelial cells and to reveal the mechanisms underlying ZnO-NPs-induced IL-8 expression.Methods:The human bronchial epithelial cell line BEAS-2B was used as the in vitro model. The MTT assay was employed to determine the damage levels of ZnO-NPs to BEAS-2B cells. Real-time PCR and ELISA were used to measure the expression levels of IL-8 mRNA and proteins, respectively. The involvement of transcription regulation in ZnO-NPs-induced IL-8 expression was examined using a selective transcription inhibitor and the BEAS-2B cell lines overexpressing recombinant wild-type or NFκB and C/EBPβbinding site mutated IL-8 promoter constructs. mRNA decay assay was used to determine the effect of ZnO-NPs on IL-8 mRNA stability. The role of phagocytosis in ZnO-NPs-induced IL-8 expression was explored using a phagocytosis inhibitor. The dissolution of ZnO-NPs in culture medium was determined using atom absorption spectrometry. Results:Exposure of BEAS-2B cells to ZnO-NPs significantly increased the expression levels of IL-8 mRNA and protein in a dose-and time-dependent fashion (1-4μg/cm2,2-4 h) (p=0.000). Pretreatment of BEAS-2B cells with the transcription inhibitor actinomycin D (1μg/ml) for 30min abrogated IL-8 gene expression induced by ZnO-NPs (4μg/cm2,2 h) (p=0.000), implying the involvement of transcriptional regulation. Using the BEAS-2B cells that overexpress wilt-type or mutated IL-8 promoter constructs, we observed that the expression level of luciferase, the IL-8 promoter reporter gene, in ZnO-NPs-treated cells containing mutated IL-8 promoter constructs reduced markedly in compared to that in the cells containing the wild-type IL-8 promoter constructs, indicating that the transcription factors NFκB and C/EBPβwere required for ZnO-NPs-induced IL-8 gene transcription. mRNA decay assay following actinomycin D pretreatment showed that ZnO-NPs stimulation (4μg/cm2,2 h) delayed IL-8 mRNA degradation in BEAS-2B cells in comparison with the control, which suggested that ZnO-NPs exposure stabilized IL-8 mRNA in BEAS-2B cells at the posttranscriptional level. Pretreatment of BEAS-2B cells with the phagocytosis inhibitor cytochalasin B (10μg/ml) for 30 min blocked ZnO-NPs-induced IL-8 expression by 30.14%, implying that phagocytosis was partially involved in ZnO-NPs-induced IL-8 expression (p=0.000). A 8μg/ml concentration of ZnO-NPs (equivalent to 4μg/cm2 ZnO-NPs in one well of a 24 well plate) was dissolved in culture medium and incubated for 2 h at 37℃, resulting in a soluble or dissolved concentration of 4.3%or 3.57μM of Zn2+.Conclusions:ZnO-NPs exposure increases IL-8 mRNA and protein expression in BEAS-2B cells. The transcription factors NFκB and C/EBPβare required for ZnO-NPs-induced IL-8 gene transcription. In addition, ZnO-NPs treatment can increase IL-8 mRNA stability. The phagocytosis of BEAS-2B cells partially mediates ZnO-NPs-induced IL-8 expression. A small portion of ZnO-NPs can be dissolved into soluble Zn2+ in culture medium.