Cardiovascular Toxicity And Molecular Mechanisms Induced By Long-term And Low-dose Titanium Dioxide Nanoparticles Exposure In Mice

Titanium dioxide nanoparticles（TiO₂ NPs）, with a diameter of 1-100 nm, are defined as new types of inorganic nanomaterials. In recent years, TiO₂ NPs have been applied in a wide range including printing, food additives, cosmetics and medicine, etc., which are closely related to our daily life, due to their unique properties（high stability, anticorrosion, and efficient photocatalysis）. TiO₂ NPs can access to the body via various routes（inhalation, injection, dermal deposition and gastrointestinal tract absorption, etc.）, subsequently enter into the cardiovascular system through blood circulation, potentially inducing some cardiovascular impacts. Currently, researches have demonstrated that people for long-term exposure to particulate matter can induce a series of cardiovascular events, such as thrombosis, coronary heart disease, and myocardial infarction. However, studies regarding the correlation between long-term and low-dose exposure to TiO₂ NPs and cardiac inflammatory response and atherosclerosis（AS）, as well as its toxic molecular mechanisms have yet to be understood deeply. In view of this, when the mice were exposed to TiO₂ NPs（anatase; 5 nm） at different doses of 2.5, 5, and 50 mg/kg BW by intragastric administration for 90 days, we investigated the cardiac damage（inflammatory response） induced by NPs and its molecular mechanisms. In addition, we investigated the formation of AS in mice following intranasal instillation with different doses（1.25, 2.5, and 5 mg/kg BW） of TiO₂ NPs（anatase; 5 nm） for 9 months. This study will provide an important theoretical basis for the risk assessment of long-term NPs exposure on cardiovascular system.The two main results are listed as follows:（1） Recent researches have demonstrated that long-term exposure to air particulate matter can induce cardiovascular disease, but whether cardiovascular events, such as cardiac damage, are induced by long-term and low-dose TiO₂ NPs exposure and its toxic molecular mechanisms, are rarely evaluated. In the present study, mice were continuously exposed to TiO₂ NPs with various concentrations（2.5, 5 or 10 mg/kg BW） by intragastric administration for 90 days. The expression of key inflammatory cytokines and signalling pathway proteins, as well as ion channel enzyme activities in the heart, were investigated. We found that TiO₂ NPs were accumulated in the heart, causing obvious cardiac damage and inflammatory response. Histopathological changes including cardiac fragmentation or disordered myocardial fibre arrangement, tissue necrosis, myocardial haemorrhage, and inflammatory cell infiltration in the myocardial interstitium, etc. were observed. In addition, the expression levels of nuclear factor-κB（NF-κB） and its inhibitor I-κB, and pro-inflammatory factors tumour necrosis factor-α（TNF-α）, interleukin-6（IL-6）, interleukin-1β（IL-1β） and interferon-α（IFN-α） were changed significantly. The activities of Ca2+-ATPase, Ca2+/Mg2+-ATPase and Na+/K+-ATPase were greatly decreased. Moreover, TiO₂ NPs considerably up-regulated the expression of Na+/Ca2+ exchanger-1（NCX-1）, calcium/calmodulin-dependent protein kinase II（CAMK II）, α1-adrenergic receptor（α1-AR）, and β1-adrenergic receptor（β1-AR）, and increased the levels of phosphorylated kinase C ε（p-PKCε） or phosphorylated extracellular signal-regulated kinase（p-ERK1/2）. Our results suggested that long-term and low-dose exposure to TiO₂ NPs induced cardiac damage and the inflammatory response which was potentially mediated by NF-κB activation via the PKCε or ERK1/2 signaling cascades in mice.（2） The cardiovascular system is mainly composed of heart and blood vessels. Recent researches have demonstrated that exposure to TiO₂ NPs can led to cardiac oxidative damage, microvascular dysfunction and endothelial dysfunction, etc., but the evaluation regarding whether long-term and low-dose TiO₂ NPs exposure could promote the progression of AS, is rarely conducted. Therefore, to further explore the effect of TiO₂ NPs on vessels, we investigated the related parameters in the serum, and evaluated the systemic inflammation（pulmonary inflammation） in mice after continuously intranasal instillation with different doses（1.25, 2.5, and 5 mg/kg BW） of TiO₂ NPs for 9 months. The results indicated that chronic exposure to TiO₂ NPs resulted in AS coupling with pulmonary inflammation. The pulmonary histopathological examinations showed emphysema, fibrotic thickening of the alveolar septae, cuffing of alveolar blood vessels, and infiltration of inflammatory cells; the coronary artery exhibited histopathological changes including obvious foamy cell and inflammatory cell infiltration, and fibrous cap formation. The numbers of inflammatory cells, including macrophages, lymphocytes, neutrophils, and eosinophils in the bronchoalveolar lavage fluid（BALF） were significantly increased by TiO₂ NP exposure, and the inflammatory cytokine levels（IL-6 and TNF-α） in BALF were also greatly elevated. The levels of total cholesterol（TC）, triglycerides（TG）, glucose（GLU）, and low-density lipoprotein cholesterol（LDL-C）, parameters of lipid and cholesterol in serum were enhanced, whereas high-density lipoprotein cholesterol（HDL-C） level was reduced. Additionally, exposure to TiO₂ NPs altered the serum AS parameters, i.e. elevated the levels of advanced glycation end products（AGEs）, reactive oxygen species（ROS）, NAD（P）H oxidases 4（Nox4）, C-reaction protein（CRP）, E-selectin（E-sel）, endothelin-1（ET-1）, tissue factor（TF）, intercellular adhesion molecule-1（ICAM-1）, vascular cell adhesion molecule-1（VCAM-1）, monocyte chemoattractant protein-1（MCP-1）, and plasminogen activator inhibitor-1（PAI-1）, whereas decreased the tissue plasminogen activator（t-PA） and nitric oxide（NO） levels. It suggested a close association between AS formation mediated by long-term exposure to TiO₂ NPs and pulmonary inflammatory response.