Cardiovascular Toxicity Of Silicon Dioxide Nanoparticles

Background and objectiveAt present, nanotechnology is developing rapidly in the world. Along with many new nanomaterials are being applied to many domains, the exposure opportunity of the researchers, the producers, the consumers, the nano-waste workers and the ecological environment to the nanomaterials increased a lot. It's uncertainty of the safety of the nanotechnology, and then theresearches have received the widespread attention of the experts come from various countries, the related administrants and the public.Some nanomaterials could penetrate the blood-brain barrier, the blood-eye barrier and the blood-testis barrier, thus harms the the security of organism possibly, such as single-walled carbon nanotube (SWCNT) and Fe₂O₃-Glu nano-pellet. These nanomaterials may transfer to kinds of organs of the animals, such as eye, heart, liver, spleen, lung, kidney, stomach, brain, skeleton, muscle, intestine, skin, blood and germ cells. Researches have indicated the nanomaterials may harm the organisms in the level of cell, sub-cell, genome, proteome and the animal. Compares with the micro-level of homogeneous particles, the nanoparticles show a higher biological activity, and the potential toxicity may be also bigger. Considered from the system toxicology, the eyes of the researches focus on the lung toxicity at present.Lots of epidemiology researches indicated that the incidence rate and mortality of cardiovascular disease may be related with the air pollutants, and the cardiovascular disease threatens the human health seriously. Therefore, the cardiovascular effects of the nanoparticles have been the focus of public. Until now, we not yet find the report of the cardiovascular toxicity of nano-SiO₂. In view of the fact that nanomaterials mayplay an important role in the cardiovascular toxicity, this project selects the nano-SiO₂ as the research object, uses the methods of in vitro, in vivo and the population studies, then gives a more comprehensive observation of the effect of nano-SiO₂ on cardiovascular system. So, we could not only observe the effect of nano-SiO₂ on the cardiovascular system in the animal and population level, but understand the molecular mechanism of the vessel endothelial cell damage in vitro.Methods and resultsThe project will be separated into three parts as follows:Study I: The toxicity of nano-SiO₂ particles in the human umbilical vein endothelial cells (HUVECs).Methods: Prepared the nanomaterial, then take HUVECs as the experimental object. The value of IC₅₀ was measured by the trypan blue assay. After the dosages was definited, we use the Annexin V and PI double dyeing method and cell cycle kit to observe the effects of nano-SiO₂ on cell apoptosis and cell cycle by FCM. DNA damage examination: the foci of γH2AX of each group were measured by FCM and immunofluorescence technology. Inflammation cytokines: the release of TNF-α, IL-6 and hsCRP in the culture was determined by ELISA.Results: Probit analysis was used to calculate the 24h IC50 of nano-SiO₂ in HUVECs, and the value was 194.9μg/ml approximately. In this experiment, 100μg/ml (approximately 50% IC50) used as the highest dose of nano-SiO₂. 25μg/ml, 50μg/ml and 100μg/ml nano-SiO₂ could induce the HUVECs cell apoptosis (10.5%,17.9% and 15.3%) and causes the capture of cell cycle at G1 period remarkably, All the nano-SiO₂ groups could cause significant DNA damage, the percentage of positive cells were 14.2% (25μg/ml), 23.9% (50μg/ml) and 25.9% (100 μg/ml) alternately. All the nano-SiO₂ groups could increase the release of the inflammation cytokine TNF-α remarkably, and IL-6 was induced remarkably on 50μg/ml and 100μg/mlnano-SiO₂ besides 25μg/ml. The above results elicit the cytotoxicity of nano-SiO₂ in HUVECs. The effect which nanometer SiO₂ causes was more serious than micro-SiO₂ besides the cell cycle, it is demonstrated that the toxicity of nano-SiO₂ was mainly due to the particle nanometer size.Study II: Cardiovascular toxicity of nano-SiO₂ particles in rats Methods: The effect on mouse blood pressure and heart rate of nano-SiO₂: the left common carotid artery was separated (approximately 2 cm) after anaesthesia. Then the artery was jointed with pressure transducer, the signal of blood pressure and heart rate could be aquired by Medlab-E biology gathering system. The establishing of thrombus model: the method was carried on the improvement of the protocols using by Kura and Tang Yinghong, separated the left common carotid artery to monitor the blood pressure and heart rate as above. MedLab-E biology signal gathering processingsystem monitor blood pressure and heart rate. Placed two pieces of filter paper that was added with 20μl 2.16 mol/L FeCl₃ on the up and down face, recorded the time of the blood pressure curve turn to the straight line after the use of filter paper (namely thrombus form time). Blood platelet functional analysis: some minutes after the intratracheal instillation of nano-SiO₂ into the lungs of rats (200μl/rat), 5ml whole blood was collected from the under cavity vein (3.8% citric acid three sodium 1: 9). Using the automatic blood analyzer to measure the blood platelet number, and after the separation of blood platelets, the gather situation was analyzed by the blood platelet aggregater.Results: Only the highest dosage of nano-SiO₂ (10 mg/kg) group decreased the systolic pressure and the diastolic pressure after 2 h and 4 h treatment compared with that at 0h, and heart rate had no difference between all groups. Similarly, only 1h,2h and 4h after the administration of the highest dosage nano-SiO₂ induced the formation of thrombus remarkably. After 24 h, the micro-SiO₂ group and variousdosage nano-SiO₂ group could not affect the platelet quantity compared with the NS control group, but the medium dosage and the highest dosage of nano-SiO₂ group could increased the platelet aggregation of rat remarkably.Study III: The cardiovascular toxicity of nano-SiO₂ in occupational exposure population.Methods: Chosen 9 nano-SiO₂ occupational workers from a nano-SiO₂ factory with high production. Because the population is less, we select the control workers match with age, sex, stature and the life habits. Then we also choose 9 workers who had no exposure history of nano-SiO₂. The field epidemiology includes the investigation of workplace sanitary, occupation and the exposure history, health situation and blood examination. Blood examination issues included blood platelet (PLT, MPV, PCT and PDW), hemoglutination function, fibrinolytic function (PT, APTT, TT, Fib, AT-III and D- dimer), blood lipids, lipoprotein mearsurement (TC, TG, HDL-C, LDL-C, ApoA-I and ApoB), sICAM-1, TM and inflammation cytokines(TNF-α,IL-6 and hsCRP)Results: The nano-SiO₂ exposure group compared with the control group, the values of PLT, MPV, PCT and PDW has no difference; the four items of hemoglutination function (PT, APTT, TT, Fib) and AT-III also have no difference. Only the plasmas D-dimer has a significant difference between the two groups; TC, HDL-C, LDL-C, ApoA-I, ApoB and ApoA-I/ApoB does not have significant difference, only TG have a significant difference between the two groups; Plasma sICAM-1 has a significant difference, but TM and inflammation cytokines have no significant difference between the two groups.Conclusion1. In vitro: Nano-SiO₂ has the remarkable HUVECs cytotoxicity, and promotes the release inflammation factor remarkably. Besides the cell cycle, the effect which nano-SiO₂ causes is more serious than micro-SiO₂, show that the SiO₂ particle size has the remarkable influence to its toxicity.2. In vivo: Compared with result of in vitro assay, nano-SiO₂ shows a weak cardiovascular toxicity in rat. Only the higher dosage (medium or highest) of the nano-SiO₂ group could decrease the systolic pressure, the diastolic pressure, the thrombus formation, and platelet aggregation rate significantly.3. Population study: Nano-SiO₂ increases the level of plasma D-dimer and sICAM-1 significantly, and decreases the TG significantly. Maybe nano-SiO₂ have the potential remote harm effect on cardiovascular system? We could not give the answer, we should increase the sample size to confirm the effects of nano- SiO₂ in population study, and do fellow-up study in the future.