Study On Mechanisms Of The Cardiovascular Toxicity Of Ambient Fine Particles

Air pollution is one major pollution in perspective of public health.Air particulate matter(PM),varying in different aerodynamic diameters and generally composed of a complex of carbon,nitrogen,sulfur,biological chemicals and heavy metals,is one of the most important air pollutants.Among others,fine particles (aerodynamic diameter≤2.5μm),in comparison with coarse particles,have been considered as a more serious pollutant,due to its high deposition efficiency into human respiratory system as well as cardiovascular system.It is of major concern because of their high propensity to penetrate the epithelium and reach interstitial sites, even transportation to other organs by air exchange in human lungs,resulting in respiratory system diseases,and extrapulmonary injuries,such as in cardiovascular and immunonological system.Epidemiological studies have shown that increases in airborne fine particle concentrations are adversely associated with cardiovascular health,but there is a lack of comprehensive studies on the effects of fine particles on cardiovascular system in toxicological perspectives,as well as the potential toxicity mechanisms.The purpose of this study is to explore the toxicological mechanisms of fine particles on cardiovascular system in respect to autonomic nervous system,inflammation, coagulation,endothelial function and cell apoptosis through animal experiment and cell culture studies.In addition,drug intervention studies by statin were performed to verify the possible toxicological mechanisms of fine particles on vein endothelial cells in vitro.In this study,spontaneously hypertensive(SH) rats were used as a model of human cardiovascular disease and Wistar Kyoto(WKY) rats as a model of healthy human.Ambient fine particles were collected by air sampling in the urban area in Shanghai and prepared into particle solutions in different concentrations.Acute exposure to fine particle solution preparations were performed by intratracheally instillation.The SH model was exposed to a gradient of fine particle concentration in 1.6 mg/kg b.w,8.0 mg/kg b.w and 40.0 mg/kg b.w,respectively,while the control model was exposed to physiological saline.The exposure was performed once a day for three days.In 24h after the last exposure,blood pressure,heart rate and cardiogram were monitored in both types of rats,as well as the biomarkers of inflammation,coagulation,endothelial function and myocardial cells lesion in blood and cardiac muscle tissue.The results showed that blood pressure and heart rate significantly increased both in SH rats and WKY rats after exposure to fine particles with an elevation of ST segment in cardiograms.In blood samples,the levels of blood high sensitive C-response protein(hs-CRP),D-dimer and endothelin-1(ET-1) increased significantly in SHR and WKY rats in a dose-dependent manner,whereas the level of blood nitrous oxide(NO) was decreased.The serum level of myocardial enzyme MB isoenzyme of creatine kinase(CK-MB) and lactate dehydrogenase(LDH) increased both in SHR and WKY rats in a dose-dependent manner.In the cardiac muscle tissue,the mRNA expression of inflammation-related genes IL-1β,IL-6, TNF-α,MIP-2 significantly increased in rats,as well as the endothelial function-related gene ET-1 and coagulation function-related gene ICAM-1.Moreover, expression of apoptosis bax gene increased significantly in rats while the anti-apoptosis gene bcl-2 mRNA expression decreased.The latter result was consistent with the protein expression levels determined by immunohistochemical staining measurement.It was found that in rats,the protein expression of apoptosis bax gene was up-regulated while the anti-apoptosis bcl-2 gene was down-regulated. These results indicated that exposure to fine particle may cause inflammation, coagulation,endothelial dysfunction and apoptosis in rats.Finally,protein expression of transforming growth factor-beta₁(TGF-β₁) measured by the same staining method showed that the TGF-β₁ protein expression obviously increased in SH rats in comparison with WYK rats,which suggested that in the fine particle toxicity mechanisms,in addition to the endothelial dysfunction and the increase of vascular permeability,the extracellular matrix synthesis and secretion and myocardial fibrosis were potentially the additional ways of cardiovascular toxicity effects.Interestingly, when the same exposure level was performed in both SHR and WKY rats,the levels of hs-CRP,D-dimer,ET-1,LDH and CK-MB were significantly higher in SH rats than in WKY rats,which indicated that model rats with the presence of hypertension diseases could be more susceptible to fine particle exposure than normal ones.This is in agreement with the results from epidemiological studies on human population, which prove that after exposure to fine particles,higher rate of cardiopulmonary morbidity is more likely in people with cardiovascular disorders than in healthy people.The cardiovacular effects of fine particle ion components were analyzed on human umbilical vein endothelial cells(EC-304).Particle samples were extracted for specific anion samples including SO₄^2-,NO₃^-,Cl^-,and five metal ion samples in doses of 0.01mmol/L,0.05mmol/L,0.1mmol/L,0.5mmol/L,1.0mmol/L,5.0mmol/L and 10.0mmol/L,respectively.After exposure experiment,it was found that only NO₃^-anions could inhibit cell activity in all the above concentration levels,and all metal ions could inhibit cell activity in different inhibition rates:Ni²⁺＞Cu²⁺＞Zn²⁺＞Fe³⁺＞Mg²⁺.Furthermore,the cardiovascular effects of chemical components,such as organic extracts,soluble and insoluble components of fine particles(exposure doses: 100μg/mL,200μg/mL and 400μg/mL,respectively for 3 kinds of components) were analyzed on EC-304 cells.Malondialdehyde(MDA),LDH,reactive oxygen species (ROS) increased and NO and Superoxide dismutase(SOD) decreased in EC-304 cells for all 3 component extraction exposure.The mRNA expression of IL-4,IL-6,TNF-α, ET-1,P-selectin and fas gene increased in EC-304 exposure cells while mRNA expression of eNOS decreased.Within the 3 different components,the inhibition effects of cell activity,oxidative stress,inflammatory response were more significant in soluble extracts than in organic extracts than in insoluble extracts.However,effects of cell apoptosis and ROS level were higher in organic extracts than in soluble extracts than insoluble extracts.In summary,different components of fine particles could lead to different levels of toxicity to cells.Statin can improve endothelial function,elevate NO biological activity,inhibit inflammation,modulate immune function and reduce oxidation in clinical studies.In this study,atorvastatin was used for intervention studies on the possible toxicological mechanisms of fine particle components on EC-304.EC-304 were exposed to the 400μg/mL organic,soluble and insoluble extracts of fine particles,respectively,followed by an intervention of astorvastatin in concentrations of 0.1μmol/L,1μmol/L and 10μmol/L.The results showed that in comparison with the group of cells with no atorvastatin addition,the levels of ROS,MDA and LDH in EC-304 cells induced by either organic extract,soluble or insoluble components of fine particles,were obviously inhibited after the addition of atorvastatin,while cell activities,SOD and NO levels in EC-304 cells increased significantly.Furthermore,the mRNA expression of IL-4,IL-6,TNF-α,ET-1,P-selectin,TGF-β₁ and fas in cells decreased greatly after treatment with atorvastatin as well,and eNOS increased significantly in a dose-dependent manner.Since atorvastatin is a clinical drug for cure of cardiovascular disorders with the known effects of reducing inflammation and improving endothelial functions,this intervention study consistently suggested that oxidative stress, inflammation,endothelial dysfunction and apoptosis were the potential mechanisms of cardiovascular injuries caused by fine particles.In summary,this study examined the potential biological pathways triggering toxicological effects of fine particles on cardiovascular system.By methods based on animal experiment and cell culture,the results indicated that fine particles increased the risk of cardiovascular disease by increasing autonomic nervous dysfunction, oxidative stress,inflammation,coagulation,endothelial dysfunction and apoptosis. Drug intervention studies further supported the presence of the above mechanisms. These results were in consistence with the main conclusions from epidemiological studies on human population exposed to fine particles.It will contribute in further exploration of the real mechanisms by fine particle on cardiovascular diseases,and will be of importance in understanding the effects of air particulate matter on human health.