Cardiovascular Toxicology Of Multi-walled Carbon Nanotubes

BackgroudCardiovascular diseases are the world's largest killers. Numerous epidemiologic studies have shown that increased levels of environmental particulate matter (PM) pollutants are positively associated with cardiovascular diseases. PM exposure may trigger serious cardiovascular disorders, and the smaller particle volumes, the greater harmfulness. Multi-walled carbon nanotubes (MWCNTs) are a kind of nano-particles. They are used in various applications, such as nano-coil, electronic parts, catalysts, and even in medicine field. The chance of CNT exposure to cardiovascular system is increased. However their biosafety evaluation mainly focuses on pulmonary toxicity. A lot of studies showed that MWCNTs induced lung and/or systemic inflammatory response, and activated hemeoxygenase gene and induced oxidative damage. They may cause endothelial dysfunction, forming condensed state of blood. MWCNTs have a certain toxic effects of cardiovascular system. Vascular endothelial injury is the major early event in the pathological changes, and ultimately leads to a series of cardiovascular diseases. Our previous studies showed that MWCNTs induced DNA damage and apoptosis in endothelial cells. So in this study in vivo and in vitro tests were employed to explain the mechanism of endothelial injury, and laied the foundation for the later experiments.After it is clear that MWCNTs can induce endothelial damge, we investigated the cardiovascular toxicity of MWCNTs using high-lipid-diet SD rats. Taking into account the degree of lung inflammation and fatty liver closely related with cardiovascular system, we also observe lung and liver in addition to observation of heart, aorta and blood.To further verify the impact of MWCNTs on atheroclerosis, and compared with other nanocarbons, we use LDLR-/-mice with high fat diet as a model, to comparative study of SWCNTs, MWCNTs and MWCNOs.Aims, methods and results1 To study on endothelial damage including functional and organic changes induced by multi-walled carbon nanotubes (MWCNTs), using immunohistochemistry and western blot to detect the expression of vWF in endothelial cells, and using ELISA to detect plasma vWF and sICAM-1 are to comprehensive evaluation of organic damage of endothelial cells; using resistance tester to detect the resistance of endothelial cells, and using western blot to detect the expression of ZO-1 in endothelial cells are to evaluate the functional damage of endothelial cells; using western blot to detect the expression of procaspase-3, cleaved caspase-3, p62 and atg8 are to explain the molecular mechanism. We found that, after continuous intravenous inject 200?g/kg MWCNTs 7d, vWF expression was increased in endothelial cells and secreted into blood, resulting in injury of endothelial function, leading to sICAM-1 decreased in serum (P?0.05). Different concentrations of MWCNTs treated HUVECs 0-48 h, the cell resistance is time and concentration-dependent lowered. After MWCNTs treated 24 h, ZO-1 expression decreased, endothelial cell permeability increased (P?0.05). Different concentrations of MWCNTs treated HUVECs 24 h, cell viability decreased, procaspase-3, cleaved caspase-3, and p62 expression decreased, vWF and atg 8 expression increased (P?0.05). 2 After different doses of multi-walled carbon nanotubes treated 1-4 months, observing the influence on blood, heart, aorta, liver and lung. Using HE staining to observe heart, aorta, lung, liver and spleen pathology. Using each kit to detect GSH, MDA, or T-SOD in serum or liver, which reflect the oxidative situation. Using each kit to detect GOT, GPT, or?-GT in serum or liver, which reflect liver function. Using oil red O staining and alizarin red staining to observe aortic lipid deposition and aortic calcification. Using fluorescence quantitative PCR to detect plumonary inflammatory cytokines IL-1?and TNF-?mRNA levels. They are shown that MWCNTs elevated serum TG levels.50,100,200?g/kg MWCNTs treated rats after 2-4 months increased aortic intimal lipid depositon areas.50,100,200?g/kg MWCNTs treated rats after 2 or 4 months exacerated the degree of aortic calcification.50,100,200?g/kg MWCNTs treated rats after 2-4 months made different influence on GOT, GPT,?-GT, GSH, T-SOD, MDA level.50,100,200?g/kg MWCNTs treated rats after 2-4 months changed the lipid in the liver.50,100, 200?g/kg MWCNTs treated rats after 2-4 months made lung septal thickening differently, and form pulmonary granuloma in different sizes.3 To comparison of single-walled carbon nanotubes (SWCNTs), multi-walled carbon nanotubes (MWCNTs), and multi-walled carbon nano-onions (MWCNOs) to observe the different influences on blood, heart, aorta, lung, liver and spleen in high-lipid-diet LDLR-/-mice. Using HE staining to observe heart, aorta, lung, liver and spleen pathology.Using each kit to detect serum CHOL, TG, LDL-c or HDL-c, which were reflecting the situation of blood-fat. Using each kit to detect serum GSH, MDA, or T-SOD, which reflect the oxidative situation. Using each kit to detect serum GOT, GPT, or?-GT, which reflect liver function. They are shown that SWCNTs, MWCNTs or MWCNOs induced lung septal thichening and pulmonary granuloma formation. However, MWCNTs had more accumulation in the lung, and increased the lung index, and then form more and bigger granulomas. The heart index and spleen index were significantly decreased by MWCNTs or MWCNOs. SWCNTs, MWCNTs, or MWCNOs decreased blood fat, and the extent of aortic fatty degeneration. The impact of fatty liver, induced by SWCNTs, MWCNTs or MWCNOs, was no significant difference. SWCNTs, MWCNTs or MWCNOs decreased GSH level in serum. The liver functions are affected by MWCNTs or MWCNOs.Conclusion1. MWCNTs induced functional and structural damage in endothelial cells.2. MWCNTs aggregated atherosclerosis.3. SWCNTs, MWCNTs or MWCNOs can be lower blood lipids.