Molecular Mechanisms Of Liver Injury Of Mice Caused By Exposure To Titanium Dioxide Nanoparticles

Due to unique photochemical characteristics of titanium dioxide nanoparticles（TiO₂NPs）, they have been widely applied to all aspects of life such as cosmetics,coatings, printing, medicine and dyes, etc. TiO₂NPs can be through the air and the foodchain into the human body, therefore, the security of naomatterial application has alsobeen attracted extensive attention. Lately, numerous studies have reported that TiO₂NPscould cause biological and toxic effects of liver. But the mechanisms of these effectsneed to be further studied. In view of this, we investigated molecular mechanisms ofhepatitis and hepatocyte apoptosis in mice after intragastric administrations with variousdoses of TiO₂NPs suspensions （5,10, and50mg/kg body weight） for60consecutivedays. In addition, we utilized the whole genome microarray analysis technique todetermine the gene expression profiling of liver of mice exposed to10mg/kg bodyweight TiO₂NPs for90consecutive days, and to understand the molecular mechanismof liver injury in a multiple genes worked together, and to find the potential biomarkersof liver toxicity caused by TiO₂NPs exposure. This study provides an importanttheoretical basis for the safety assessment of nanomaterials. The main results are listedas follows:（1） While the inflammation of the mouse liver induced by TiO₂NPs has beendemonstrated, very littleis known about the molecular mechanisms underlying thismouse liver inflammation. In an effort to examine signaling pathway of inflammation ofthe mouse liver caused by intragastric administrations of TiO₂NPs （5,10, and50mg/kgbody weight） for60consecutive days, we assessed Toll-like receptor-2（TLR2）, TLR-4,IκB kinase （IKK-a, IKK-b）, IκB nucleic factor-κB （NF-κB）, NF-κBP52, NF-κBP65,tumor necrosis factor-α （TNF-α）, NF-κB-inducible kinase（NIK）, interleukin-2（IL-2）,biochemical parameters of liver functions, and histopathological changes in the TiO₂NPs-treated mice. The results showed the titanium accumulation in liver, histopathological changes of mice liver, and the liver function damaged by TiO₂NPs.The real-time quantitative RT-PCR and ELISA showed that TiO₂NPs can significantlyincrease the mRNA and protein expression of TLR2and TLR4and severalinflammatory cytokines, including IKK1, IKK2, NF-κB, NF-κBP52, NF-κBP65, TNF-α,and NIK, and TiO₂NPs can significantly decrease the mRNA and protein expression ofIκB and IL-2. The results of this study added to our understanding of TiO₂NPs-inducedliver toxicity. It implied that the signaling pathway of liver injury in the TiO₂NPs-stimulated mouse liver sequentially might occur via activation ofTiO₂â†'TNF–α↑â†'TLRs（TLR2,TLR4）↑â†'NIK↑â†'IKKs（IKK–α,IKK–β）↑â†'IκB↓â†'NF–κB（NF–κBP52,NF–κBP65）↑â†'inflammation↑â†'apoptosisâ†'liver injury.（2） In order to further understand the hepatocyte apoptosis induced by intragastricadministrations of TiO₂NPs （5,10, and50mg/kg body weight） for60consecutive days,the hepatocyte apoptosis, various oxidative stress parameters and the stress-related geneexpression levels were assayed for the mouse liver.60days of TiO₂NPs exposure,hepatocyte apoptosis in the liver could be observed, which was followed by increasedreactive oxygen species accumulation, and decreased the stress-related gene expressionlevels of superoxide dismutase, catalase, glutathione peroxidase, metallothionein, heatshock protein70, glutathione S transferase, P53, and transferrin; and the significantenhancement of the cytochrome p4501A expression level. It implied that hepatocyteapoptosis, oxidative stresses, and alteration of expression levels of the genes relatedwith TiO₂NPs detoxification/metabolism regulation and radical scavenging action.Therefore, the application of TiO₂NPs and exposure effects especially on human liverfor long-term and low-dose treatment should be cautious.（3） While liver toxicity induced by TiO₂NPs has been demonstrated, very little isknown about the molecular mechanisms of multiple genes working together underlyingthis type of liver injury in mice. In this study, we utilized the whole genome microarrayanalysis technique to determine the gene expression profile in the livers of miceexposed to10mg/kg body weight TiO₂NPs for90consecutive days. The findingsshowed that long-term exposure to TiO₂NPs resulted in obvious titanium accumulationin the liver and TiO₂NPs aggregation in hepatocyte nuclei, an inflammatory response,hepatocyte apoptosis, and liver dysfunction. Furthermore, microarray data showedstriking changes in the expression of785genes related to the immune/inflammatory response, apoptosis, oxidative stress, the metabolic process, response to stress, cellcycle, ion transport, signal transduction, cell proliferation, cytoskeleton and celldifferentiation in TiO₂NPs exposed livers. In particular, a significant reduction incomplement factor D （Cfd） expression following long-term exposure to TiO₂NPsresulted in autoimmune and inflammatory disease states in mice. Therefore, Cfd may bea potential biomarker of liver toxicity caused by TiO₂NPs exposure.