Long-term Exposure To Titanium Dioxide Nanoparticles Induced Pulmonary Toxicity In Mice And Its Molecular Mechanisms

Titanium dioxide nanoparticles (TiO2NPs) are one of the most importantnanomaterials, which sizes between1and100nm, because of its unique surface effect,quantum size effect and the volume effect and so on, they are widely used in variousfields, such as paints, dyes, printing, pharmaceuticals, cosmetics, food and other dailylife. TiO2NPs could get into the human body through the air and food chain. Due to it’ssmaller than red blood cells, It can reach any part of the organism, so its applicationsecurity has caused widespread concern in world. Respiration is the major route oftaking nanoparticles, thus cause acute and chronic pulmonary disease, such as,inflammation, fibrosis, and even cause lung caner. At present, TiO2NPs have beencarried out on pulmonary toxicity studies, but its molecular mechanism is still unclear.In view of this, we investigated molecular mechanisms of inflammatory and oxidativestress in mice after nasal administrations with various doses of TiO2NPs suspensions(2.5、5and10mg/kg BW) for90consecutive days. Furthermore, we utilized the wholegenome microarray analysis technique to determine the gene expression profiling oflung and to explore multi-factor mechanisms of mice after nasal administration of10mg/kg BW TiO2NPs for90consecutive days. Our findings will provide an importanttheoretical basis for evaluating lung toxicity underlying effects of nanomaterials onanimals and human and let people pay more attention to the potential toxicity ofnanoparticles.The main results are listed as follows:(1) Exposure to TiO2NPs has been demonstrated to result in pulmonaryinflammation in animals; however, very little is known about the molecular mechanismsof pulmonary injury due to TiO2NPs exposure. The aim of this study was to evaluatethe oxidative stress and molecular mechanism associated with pulmonary inflammationin chronic lung toxicity caused by the intratracheal instillation of TiO2NPs for90 consecutive days in mice. Our findings suggest that TiO2NPs are significantlyaccumulated in the lung, leading to an obvious increase in lung indices, inflammationand bleeding in the lung. Exposure to TiO2NPs significantly increased theaccumulation of reactive oxygen species (ROS) and the level of lipid peroxidation, anddecreased antioxidant capacity in the lung. Furthermore, TiO2NPs exposure activatednuclear factor–κB, increased the levels of tumor necrosis factor-α, cyclooxygenase-2,heme oxygenase-1, interleukin-2, interleukin-4, interleukin-6, interleukin-8,interleukin-10, interleukin-18, interleukin-1β, and CYP1A1expression. However, TiO2NPs exposure decreased NF-κB-inhibiting factor and heat shock protein70expression.Our results suggest that the generation of pulmonary inflammation caused by TiO2NPsin mice is closely related to oxidative stress and the expression of inflammatorycytokines.(2) Exposure to TiO2NPs elicits an adverse response such as oxidative damage.The molecular targets of TiO2NPs remain largely unidentified. In the present study, thefunction and signal pathway of nuclear factor erythroid2related factor2(Nrf2) inprotection against TiO2NP-induced oxidative stress in mouse lung were investigated.Mice were exposed to10mg/kg body weight by an intratracheal administration for15days~90days. With increasing exposed terms, TiO2NPs were significantlyaccumulated and increased the ROS production in lung, which resulted in severepulmonary edema, inflammatory response and pneumonocyte apoptosis for90days.Furthermore, TiO2NPs exposure could greatly induce expression of Nrf2, hemeoxygenase1(HO-1) and glutamate-cysteine ligase catalytic subunit (GCLC) from15-day to75-day exposure, while90-day exposure caused significant decreases of threefactors expression levels in the lung. Our findings imply that the induction of Nrf2expression is an adaptive intracellular response to TiO2NP-induced oxidative stress inthe mouse lung, and that Nrf2is protective against TiO2NP-induced pulmonarydamages during certain exposure terms.(3) The pulmonary damage induced by TiO2NPs is of great concern, but themechanism of how this damage may be incurred has yet to be elucidated. Here, weexamined how multiple genes may be affected by TiO2NPs exposure to contribute tothe observed damage. The results suggest that long-term exposure to TiO2NPs led tosignificant increases in inflammatory cells, and levels of lactate dehydrogenase, alkalinephosphate, and total protein, and promoted production of ROS and peroxidation of lipid, protein and DNA in mouse lung tissue. Specifically, microarray analysis showedsignificant alterations in the expression of847genes in the TiO2NP-exposed lungtissues. Of521genes with known functions,361were up-regulated and160down-regulated, which were associated with the immune/inflammatory responses,apoptosis, oxidative stress, the cell cycle, stress responses, cell proliferation, thecytoskeleton, signal transduction, and metabolic processes. In this study, we speciallyelaborate the expressions of immune/inflammatory responses (Defb4, H2-Oa, IL-10,Alox5ap, Chi3l3), apoptosis (Pdi2, Niacr1, v-erb-b2, Spk2, Ada), oxidative stress(Cryαb, Alkbh7) and the cell cycle (Cdkn1a, Cdkn1c), Therefore, the application ofTiO2NPs should be carried out cautiously, especially in humans.