| Atmospheric fine particulate matter(PM2.5)is a major air pollutant that has been widely studied in recent years,which is closely related to an increased risk of many lung diseases(such as asthma,chronic obstructive pneumonia disease and lung cancer)and was identified as a class 1 carcinogen by the International Agency for Research on Cancer.NPAHs(Nitro-polycyclic aromatic hydrocarbons),one of the important organic components of PM2.5,have received more attention due to their mutagenicity and carcinogenicity.SO2(Sulfur dioxide)is a common air pollutant,which can stimulate the respiratory tract and cause diseases such as pneumonia and asthma.In addition,endotoxin,a kind of biological air pollutant,also called lipopolysaccharide(LPS)is closely associated with asthma and lung injury,which is valued for the harm to public health.In order to prevent these diseases,‘The Healthy China Initiative(2019-2030)'put forward to improve the quality of the environment and ensure people's health.Based on the significant demand,in the present study,PM2.5 is the primary atmospheric pollutant.The toxicological effects of different exposure patterns on lung injury were evaluated,including atmospheric PM2.5 and its organic component NPAHs exposure in Taiyuan,China by intratracheal instillation,real-ambient atmospheric PM2.5 exposure,co-exposure of atmospheric PM2.5in Taiyuan by intratracheal instillation and SO2via dynamic inhalation,co-exposure of real-ambient atmospheric PM2.5in Linfen,China and LPS by intraperitoneal injection.Then the toxicological mechanisms of lung diseases induced by three kinds of atmospheric components were discussed.The research contents include as follows:1.Effects of PM2.5 and PM2.5-bound NPAHs(1-nitropyrene(1-NP)and 9-nitroanthracene(9-NA))on rat lung DNA damage were investigated.It has demonstrated that PM2.5 can cause DNA damage,but whether PM2.5-bound1-NP and 9-NA can cause DNA damage and whether 1-NP and 9-NA contribute on PM2.5 toxicity need to be further explored.In this study,male Wistar rats were received DMSO,PM2.5 suspensions(1.5 mg/kg b.w.),different concentrations(low,medium and high concentration)solutions of1-NP(1-NP-1:1.0×10-5 mg/kg b.w.,1-NP-2:4.0×10-5 mg/kg b.w.and1-NP-3:1.6×10-4 mg/kg b.w.)and different concentrations(low,medium and high concentration)solutions of 9-NA(9-NA-1:1.3×10-5 mg/kg b.w.,9-NA-2:4.0×10-5 mg/kg b.w.and 9-NA-3:1.2×10-4 mg/kg b.w.)by intratracheal instillation respectively for one/two days enduring for 10 days.Comet assay was used to observe DNA strand damage in rat lung cells.SDS-KCl precipitation method was used to determine the contents of DNA-protein crosslink(DPC)in rat lungs and enzyme-linked immunosorbent assay(ELISA)was used to investigate the changes of DNA oxidative damage marker(8-hy-droxydeoxyguanosine,8-OHd G),oxidative stress factors(heme oxyge-nase 1(HO-1),malonaldehyde(MDA)and superoxide dismutase(SOD))and metabolic enzymes(glutathione S-transferase(GST),cytochromes P450(CYP450),CYP1A1 and CYP1A2).The expression of DNA damage and repair relative genes(8-Oxoguanine DNA Glycosylase(OGG1),Mut T Homolog 1(MTH1)and X-ray repair cross-complementing group 1(XRCC1))in rat lungs was measured by Real-Time PCR(RT-PCR)and Western Blot(WB).The results showed that exposure to PM2.5,1-NP and 9-NA could induce DNA strand breaks,8-OHd G formation,DNA-protein cross-linking and alter expression of DNA repair genes(OGG1,MTH1 and XRCC1)in the lungs of rats.These results suggested that PM2.5,1-NP and 9-NA may mediate DNA damage by affecting DNA repair ability,enhancing oxidative stress and biotransformation.Besides,at the approximately equivalent dose level,Pearson'correlation analysis was conducted to compare the correlation between medium concentration 1-NP and PM2.5,medium concentration 9-NA and PM2.5 by,the results demonstrated that 1-NP and 9-NA played a vital role in the PM2.5-induced lung DNA damage.This study provides an experimental basis for the lung toxicological mechanism of NPAHs in PM2.5.2.Effect of DNA methylation on lung DNA damage caused by real-ambient atmospheric PM2.5 exposure in Taiyuan was investigated.It has shown that DNA methylation is involved in DNA damage,but it is unclear that whether real-ambient atmospheric PM2.5 exposure can cause DNA damage need to be further explored.In this study,SD male rats were exposed to a real-ambient atmosphere whole-body PM2.5 inhalation system in Taiyuan for 1 month and 2 months.Hematoxylin-eosin(HE)staining analysis was used to evaluate rat pathological lung damage.The changes of inflammation factors(tumornecrosis factor-?(TNF-?)and interleukin-6(IL-6)),oxidative stress factors(hydroxyl radical(HO·),superoxide anion radical(O2-·),Nitric oxide(NO),inducible nitric oxide synthase(i NOS),MDA,SOD and GST),DNA damage-related factors(DNA damage-inducible gene 153(GADD153),8-OHd G and H2AX phosphorylation(?-H2AX))and cancer-related factors(c-fos,c-jun and PTEN)were determined by ELISA,RT-PCR and WB.In order to explore the mechanism of DNA damage,the DNA methylation levels of DNA repair genes(OGG1 and MTH1)promoter regions were determined by bisulfite sequencing PCR(BSP).Results indicated that1-month and 2-month real-ambient atmospheric PM2.5 exposure could cause lung pathological damage and DNA damage,associated with the changes of inflammation factors and oxidative stress-related markers.In addition,2-month PM2.5 exposure significantly decreased DNA methylation level of the OGG1 promoter region 2.The result indicated that DNA damage was not only related to oxidative stress,but also affected by DNA methylation.It is suggested that DNA methylation is maybe an important mechanism of lung genotoxicity induced by PM2.5.Finally,the abnormal expression of c-fos,c-jun and PTEN revealed that real-ambient atmospheric PM2.5exposure could cause the abnormal expression of cancer-related early genes and tumour suppressor gene and might increase the potential risk of lung cancer.These provide some reference values for the effect of PM2.5 on epigenetic modification.3.This study focused on the toxicological effects of co-exposed to PM2.5and SO2on lung damage by inflammatory pathway.PM2.5 and SO2 are two common air pollutants.And it has been found that PM2.5or SO2 exposure can increase the incidence of lung diseases.Coal smoke pollution is the main air pollution in Taiyuan,Shanxi Province,China.PM2.5 and SO2 are the main coal combustion products.The toxicological research of co-exposure of PM2.5 and SO2 are significant to elucidate the pulmonary toxicity of air pollution.Therefore,in this study,male Wistar rats were randomly divided into 6 groups:control group(saline was instilled on alternate days),SO2inhalation group(5.6 mg/m3 SO2 was dynamically inhaled on alternate days),PM2.5 exposure group(1.5 mg/kg b.w.PM2.5 was instilled on alternate days),SO2 and PM2.5(low,medium and high concentration)co-exposure groups(5.6 mg/m3 SO2 was dynamic inhaled on alternate days,at the same time,1.5mg/kg b.w.,6.0 mg/kg b.w.and 24.0 mg/kg b.w.PM2.5was instilled on alternate days).Intratracheal instillation/dynamically inhalation was performed on the same day and SO2 was inhaled for 6 h/d enduring for 10days.HE staining and Transmission Electron Microscope(TEM)technology were used to observe the histopathologic changes and ultrastructure damage.The inflammatory cells in bronchoalveolar lavage fluid(BALF)were counted with Wright-Giemsa staining.ELISA was performed to measure the levels of inflammatory markers(TNF-?,IL-6 and interleukin-1?(IL-1?))and inflammatory pathway relevant factors(inhibitor of NF-?B?(I?B?)?NF-?B kinase?(IKK?)and i NOS).The expression of intercellular adhesion molecule 1(ICAM-1),nuclear factor?appa B(NF-?B),phosphorylated p38(p-p38)and toll-like receptor 4(TLR4)in the lungs of rats was detected by RT-PCR and WB.The results indicated that SO2 exposure(5.6 mg/m3)did not cause apparent inflammatory responses in rat lungs.The PM2.5 exposure(1.5 mg/kg b.w.)increased inflammatory cell counts in BALF and some inflammation damage.Importantly,SO2 and PM2.5(1.5,6.0,and 24.0 mg/kg b.w.)co-exposure induced more pathological and ultrastructural damage and raised inflammatory cells in BALF compared with the control group or SO2group,in which TLR4/p38/NF-?B pathway activation accompanied by abnormal expression of inflammatory pathway relevant factors,consequently caused lung damage.It provides more useful evidence to understand the possible toxicological mechanism that PM2.5 and SO2co-pollution exacerbates lung disease.4.Effect of lung damage induced by co-exposure of real-ambient atmospheric PM2.5 exposure in Linfen and LPS was studied from ferroptosis and fibrosis perspective.PM2.5 and endotoxin are ubiquitous in the atmosphere.Many investigations have shown that ferroptosis is related to many respiratory diseases,and PM2.5/LPS exposure can cause ferroptosis,but the lung toxicological effects of ferroptosis and fibrosis-related factors induced by co-exposure of PM2.5 and LPS is not fully understood.In this study,BALB/c male mice were randomly divided into 4 groups:clean air group(injected PBS buffer),PM2.5 exposure group(injected PBS buffer),LPS exposure group(injected LPS solution)and PM2.5 and LPS co-exposure group(injected LPS solution).Mice were intraperitoneally injected with PBS buffer or LPS solution(0.12 mg/mL)once a week,50?L at a time.Simultaneously,the mice were exposed to real-ambient atmospheric PM2.5 in Linfen City,Shanxi Province of China,for 23 weeks.The histological changes in the mice lungs in different groups were observed by HE staining.The levels of inflammation factors(TNF-?and IL-6),oxidative stress factors(SOD,glutathione(GSH),CAT,MDA and 4-hydroxynonenal(4-HNE))and iron were measured using ELISA.WB was used to evaluate the expression of ferroptosis markers(glutathione peroxidase 4(GPX4),solute carrier family 7member 11(SLC7A11)and ferroportin 1(FPN1))and fibrosis-related genes(?-smooth muscle actin(?-SMA),collagen type I(collagen I)and collagen type III(collagen III))in mice lungs.Masson staining of the lungs of mice was performed to assess collagen distribution characterized by collagen fibrils.The results showed that co-exposure of PM2.5 and LPS could aggravate pathological lung damage,affect the abnormal expression of inflammatory factors and oxidative stress indexes,and induce iron level,which led to decreased expression of GPX4,SLC7A11 and FPN1,and accumulation of 4-HNE and MDA,finally promoting ferroptosis compared with the control group/LPS/PM2.5 group.In addition,severe hyperemia and collagen deposition were found in the co-exposure of PM2.5 and LPS group,which were involved in the increased expression of fibrosis-related factors(?-SMA,collagen I and collagen III).Exposure to PM2.5/LPS could not cause significant changes in these factors.This study reveals the toxicological mechanism of ferroptosis and fibrosis in mice lung caused by co-exposure of PM2.5 and LPS.Due to the diversity of air pollution in different regions and the complexity of PM2.5 components,the mechanisms of respiratory diseases caused by different pollution components in different regions are different.For this purpose,Taiyuan and Linfen of Shanxi Province,where they are more seriously polluted,were selected as the research sites,and a variety of animal exposure models were established.According to different exposure patterns of PM2.5,the effects of PM2.5 and its component NPAHs on lung DNA damage were firstly studied.Further more,the molecular mechanism of PM2.5-induced lung DNA damage was explored from the perspective of DNA methylation.Then,co-exposure of PM2.5 and SO2-caused inflammatory lung injury was investigated via the classical inflammation pathway.Finally,another exposure model of PM2.5 and LPS was established to clarify the effects of PM2.5 and LPS on ferroptosis and fibrosis in the lungs of mice.In all,the mechanisms of three different pollutants on lung damage were clarified from several aspects,which provides the new ideas and evidence for air pollution control of typical regions and health field in China. |
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