| Ambient air is polluted with a mixture of toxicants, both particulate matter and ozone are two common pollutants. The concentration of ambient PM2.5is closely related with heavy traffic in large cities, which can reach higher levels during the traffic rush hour. Ozone, an oxidant gas, is a secondary pollutant produced by nitrogen oxides (NOx), volatile organic compounds (VOCs) and sunlight, thus causing ozone concentration to usually culminate in the afternoon. These two pollutants may occur during the day and maybe induce more serious adverse health effects than single air pollutant. So considering co-exposure of ozone and PM2.5has significance for both toxicological mechanism and epidemiological studies. According to previous studies, the effects of particles on cardiovascular system involve lung, heart and vascular system. Therefor, we detected the changes of lung, heart and artery to evaluate the toxicological effects and mechanisms of ozone and PM2.5.Up to now, there are still no guidelines for evaluating the health risk of human exposure to multiple air pollutants. Subacute exposure design was used in this study to evaluate cardiopulmonary injury. The specific methods were that all rats were exposed according to four different daily exposure protocols. Each protocol applied twice per week for three weeks and the sequence of exposure for each group was followed the order as:For control group,4hr air followed by intratracheal instillation of saline; For single PM2.5-exposed groups,4hr air followed by intratracheal instillation of PM2.5(0.2,0.8,3.2mg/rat); For single ozone-exposed group,4hr ozone followed by intratracheal instillation of saline and for ozone plus PM2.5groups,4hr ozone followed by intratracheal instillation of PM2.5(0.2,0.8or3.2mg/rat). Before exposure and during three exposure weeks, the rats were weighted every week. Heart rate (HR) and electrocardiogram (ECG) was monitored at approximately24-hour both after the3rd exposure and the last (6th) exposure, and blood pressure (BP) was monitored at approximately24-hour after the6th exposure. Rats were sacrificed by drawing blood from the abdominal aorta24hr after the last (sixth) exposure. Then blood and bronchoalveolar lavage fluid (BALF) were collected for biochemical analysis. The collected BALF was analyzed for inflammatory cells and cytokines. In order to examine lung injury, pro-inflammatory factors (IL-6, TNF-α), total leukocyte number and the percentage of macrophages, lymphocytes and neutrophils in BALF, cytotoxicity markers (LDH, AKP) and permeability markers (TP, ALB) in BALF were measured. The activities of SOD and GSH-Px and MDA levels in lung tissue were measured for oxidative stress indicators. To determine whether the PM2.5and ozone affect endothelium system, iNOS, eNOS and ICAM-1mRNA levels in lung were analyzed by real-time PCR and NO in BALF was detected using colorimetric method. Endothelium system indicators of iNOS, eNOS, MMP-2and ICAM-1mRNA levels in heart were also analyzed by real-time PCR. The activities of SOD and GSH-Px and MDA levels in heart tissue were measured for oxidative stress indicators. MMP-2and ICAM-1mRNA levels in artery were analyzed by real-time PCR. Systemic inflammatory factors (IL-6, TNF-a and CRP) and two systemic injury indicators (Serum LDH and CK) were analyzed. Systemic oxidative stress indicators (MDA), endothelium function factors (ET-1, VEGF and NO) were also detected. Main bronchus, lung tissues, heart and artery were processed for light microscopic. At the same time, lung and heart tissues were also processed for transmission electron microscope (TEM) examinations. We analysed the correlation between local lung inflammation and systemic inflammation, between inflammation and oxidative, between inflammation and HRV and between endothelium function and inflammation. In addition, correlation consistency of exposure-response relationship between single PM2.5exposure and ozone plus PM2.5were also analysed for studying the mechanism relationship among molecular pathological pathways. At the same time, we discuss the changes of toxicological effects and toxicological pathways.In our study, ozone and PM2.5exposure could retard the increase of body weight. Total cell number in BALF of PM2.5-exposed groups were higher than control (P<0.05). PM2.5instillation caused dose-trend increase in TNF-a, IL-6, LDH and total protein of BALF. Exposure to ozone alone did not cause significant changes in above mentioned indicators of lung injury. However, ozone could enhance PM2.5-induced inflammatory changes and pathological characters in rat lungs. SOD and GSH-Px activities in lung were reduced in PM2.5-exposed rats with and without prior ozone exposure compared to control. iNOS mRNA levels was up-regulated in rats exposed to PM2.5(3.2mg/rat) alone and ozone plus PM2.5. However, there were no significant changes of eNOS mRNA in rat lung between different exposure groups. We observed marked up-regulation of ICAM-1mRNA in ozone plus PM2.5groups. Main bronchus hisopathological observation were found that all exposure groups except the control, submucosa neutrophil accumulation, inflammation cell hyperplasia and epithelium of mucosa shedding were the main pathological charactors. The histological examinations showed that PM2.5could cause inflammation injury and this injury was more and more noticeable with the increase of dosage. TEM observation showed that the main pathological change occurred in mitochondria of the pneumocytes type II, those mainly were mitochondrial swelling and vacuole and the increasing of number.For the injuries of cardiovascular system, results showed that blood pressure (BP) increased significantly in rats exposed to high-dose PM2.5(3.2mg/rat) when compared to the control group, and heart rate did not change significantly. PM2.5alone exposure could trigger the significant increase of CRP, MDA, CK, ET-1and BP and decrease of heart rate variability (HRV). Ozone alone exposure in rats did not show significant alterations in any indicators. Ozone plus PM2.5exposure, however, induced CRP, IL-6, CK, LDH and MDA increase, SOD and HRV decrease significantly in a dose-response way. Meanwhile, abnormal ECG types were monitored in rats exposed to PM2.5with and without ozone. Heart hisopathological examinations found that there were particles deposition in heart tissue and myocardial inflammation was observed only in high-dose ozone plus PM1.5group. Obvious myocardial ultrastructural changes were observed by TEM in ozone alone exposure, high-dose PM2.5alone exposure and combination exposure groups. Myocardial arrangement disordering, myofilament fracture, mitochondrions swellingand vacuoled were the main ultrastructural changes. On the other hand, both MMP-2and ICAM-1mRNA expression were up-regulated in arteries of rats exposed to PM2.5with and without ozone. This up-regulation implied that transcriptional mechanism was involved in vascular remodeling and endothelium function. However, there were no obvious changes in artery histopathological observation.The correlation analysis showed that there were significant correlations between pulmonary inflammation, cardiovascular system inflammation/oxidative stress and HRV indicators. Although there was no significant correlation between plasma ET-1and cardiac MMP-2mRNA, significant correlation was existed between plasma ET-1and artery MMP-2mRNA. Analysis of exposure-response relationship between single PM2.5exposure and ozone plus PM2.5indicated that LDH, total protein, albumin, iNOS mRNA、SOD、GSH-PX and MDA were more sensitive for detecting lung toxicology induced by PM2.5, but AKP, IL-6, ICAM-1mRNA were more easily affected by ozone exposure. For cardiovascular effects, CRP and ET-1were more sensitive for PM2.5exposure, but TNF-α, IL-6and TV maybe more sensitive for ozone exposure. VEGF was less sensitive no matter for ozone exposure or PM2.5exposure.In conclusion,0.8ppm ozone (twice a week for two weeks) induced slight cardiopulmonary injuries. High-dose PM2.5(3.2mg/rat) alone exposure could cause cytotoxicity, inflammation, oxidative and histopathological changes in lung. Ozone potentiated these effects induced by PM2.5. The same dosage PM2.5could induce cardiovascular inflammation, endothelial function and ANS dysfunction. Ozone and PM2.5could lead to heart and systemic injuries significantly and these adverse effects had dose-response trend. These data demonstrated that inflammation, oxidative stress, endothelium function and autonomic nerves system were involved in toxicological mechanisms of PM2.5and ozone exposures in rats’ cardiopulmonary system. Actually, all these toxicological pathways are connected, it is hard to distinguish each pathway away from the others when evaluate the toxicology caused by ozone and particulate matter. |
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