Decreased Lung Function Induced By Diesel Engine Exhaust And Their Associations With The Alternations Of CRP And Let-7

Diesel engine exhaust(DEE)is a common air pollutant resulting from incomplete combustion of diesel fuel.DEE is a complex mixture comprising of gases and particulate matter absorbed with mutagenic and carcinogenic organic matters.Polycyclic aromatic hydrocarbons(PAHs)and nitroarenes are presented within both gas and particle phases of DEE.Moreover,DEE is a prominent source of particulate matter<2.5μm in aerodynamic diameter(PM2.5)in rural and urban areas.For the general population,traffic emission can be major sources of exposure to DEE.Occupational exposure to DEE through use of diesel-powered equipment predominantly occurred in industries including mining,construction,and transportation.The potential health effects of ambient DEE exposure are of great interest both for general and occupational population.Epidemiological studies have shown associations between DEE exposure and several respiratory disorders including airway inflammation,allergic respiratory disease,chronic obstructive pulmonary disease(COPD),and lung cancer.Although DEE is ordinary in both urban and rural areas,it is usually very difficult to pick out the effects from other innumerous fuel combustion exposures which coexist in ambient air.Therefore,the inadequate control of potential confounding exposures and lack of quantification of exposure assessment remains hindered the interpretation of occupational and environmental epidemiological studies of DEE.Human controlled-exposure studies have reported mixed findings with respect to the effects of DEE on lung function,including non-significant effects on lung volumes and statistically significant effects on specific airway resistance.Although controlled exposure studies have the advantage of excluding the effects of confounding exposures,it also have some weaknesses,such as small sample size and inability to study the chronic effects of exposure compared with observational epidemiology.In the current study,all subjects in the DEE-exposed group had inspected heavy-duty diesel engines for at least one year in a diesel engine manufacturing plant,and there was no other major exposure source except DEE in the workplace.The underlying toxicological mechanisms by which DEE induces adverse effects on respiratory system remain unclear.However,there have been some studies indicating that inflammation played an important role in the mechanism by which DEE may exert toxicity recently.PM deposited in the alveoli of the lung can induce local inflammation,which may subsequently elicit a systemic inflammatory state.Creactive protein(CRP),which is considered to be a marker of systemic inflammation,has been widely used in several chronic inflammatory disorders.Circulating levels of CRP are known to increase in response to infection and tissue damage.Higher levels of serum CRP were associated with impaired lung function(a lower FEV1)in healthy subjects and patients suffering from COPD,although there are conflicting results.Epidemiologic and human controlled studies suggest a link between circulating levels of CRP and PM exposure,since the CRP levels of healthy volunteers and women increased after PM exposure.PM-induced CRP responses were consistently found among children,but not consistently in adults with chronic inflammatory conditions.Existing evidence suggests that CRP levels are influenced by both environmental and genetic factors.The single nucleotide polymorphisms(SNPs)of the CRP gene are known to be associated with the baseline serum CRP levels.If higher serum CRP levels reduce lung function,then a CRP polymorphism that was associated with higher levels of serum CRP should also lead to impaired lung function.Moreover,DEE is classified as Group I carcinogen by the International Agency for Research on Cancer in 2012.The DEE exposure cause increased risk of lung cancer,while the previous studies often focused on the genetic damage in the process of lung cancer,whether the epigenetic changes such as microRNA(miRNA)participate in the process,is still lack enough research.In the present study,we recruited 137 workers from a diesel engine manufacturing plant as the DEE-exposed group.The DEE-exposed workers tested heavy-duty diesel engines in the engine assembly workshop.We chose 127 workers from a water supply plant in the same city as the control group.The workers of the control group operated the electric powered water pumps.We performed the lung function tests and also evaluated the serum CRP levels were assayed.Genotyping of four CRP single-nucleotide polymorphisms(SNPs)and expression of miRNA let-7 were measured.We explored the effects of lung function,serum CRP levels,and the expression of let-7 following exposure to DEE.Then we further explore the correlation between circulating CRP levels that were directly related to inflammation and lung function in the workers exposed to DEE.Meanwhile,we examine the association between CRP polymorphisms,and circulating CRP levels,and lung function decline inthe DEE-exposed workers.Finally,we analyzed the association between the expressions of microRNA let-7 and lung function decline in the workers exposed to DEE.1 DEE exposure induced decline in lung functionWe measured PM2.5 in the working environment gravimetrically by a micro-balance.The concentrations of elemental carbon(EC)in collected PMs were analyzed by thermal optical analysis.Quantitative chemical analysis of 16 PAHs from collected PMs were performed by high performance liquid chromatography-mass spectrometry(HPLC)with fluorescence detectors.The urine samples were tested for six OH-PAHs using an High performance liquid chromatography-Mass spectrometry(HPLC-MS/MS)method.Lung function tests were performed using a portable calibrated vitalograph spirometer(CHESTAC-8800,Japan)in accordance with the American Thoracic Society(ATS)standards.The means of the PM2.5 level and EC were 510.4μg/m3 and144.56μg/m3 of DEE.Compared with the control group,total OH-PAHs were significantly higher in the DEE-exposed groups(all p<0.001).There was a significant decrease of forced expiratory volume in 1 second(FEV1),ratio of forced expiratory volume in 1 second to forced vital capacity(FEVi/FVC),maximal mid expiratory flow curve(MMF),forced expiratory flow at 50%of FVC(FEF50%),and forced expiratory flow at 75%of FVC(FEF75%)in DEE-exposed workers than the non-DEE-exposed workers(all p<0.05).Among all study subjects,the decreases of FEF75%were associated with the increasing levels of PAH metabolites(p<0.05).When further stratified by smoking status,all of lung function indexes did not differ significantly between current smokers and non-smokers among both DEE-exposed group and control group.2 Associations of levels of CRP and lung function from DEE exposureThe serum CRP levels were assayed using enzyme-linked immunosorbent assay(ELISA).Genotyping of four CRP single nucleotide polymorphisms were performed by TaqMan SNP Genotyping Assays.Serum CRP levels increased in exposed versus control workers,and were inversely associated with FEV1 and FVC of DEE exposed workers(all p<0.001).In addition,there were evidences of a gene-environment interaction.In the DEE-exposed workers,three CRP polymorphisms were associated with serum CRP levels,the subjects of rs1205 TT genotype had lower serum CRP levels,while the subjects of the rs3093068 GC+CC genotype(p=0.002)and rs3091244 GA+GT genotype(p=0.002)had higher serum CRP levels.Moreover,in the DEE-exposed workers,the CRP rs1205 CC genotype carriers exhibited lower FVC(p=0.004)and FEV1(p=0.016)levels than TT genotype carriers,while rs3093068 GC+CC genotype carriers exhibited lower FVC(p=0.029)and FEV1(p=0.033)levels than GG genotype carriers.Finally,rs3091244 GA+GT genotype carriers exhibited lower FVC(p=0.027)and FEV1(p=0.021)levels than GG genotype carriers.3 Associations of expression of let-7 and lung function from DEE exposureThe real-time quantitative PCR was used to detect the expression of eight miRNAs in the let-7 family.After the adjustment for age,body mass index,smoking status,alcohol use and other confounding factors,the expression of let-7a,let-7b,let-7c,let-7d,let-7e and let-7f in the DEE-exposed workers were higher than the non-DEE-exposed workers(P=0.010,0.003,0.003,0.024,0.002,and 0.027;respectively).The levels of expression of let-7a,let-7b,let-7c,let-7d,let-7e,let-7f,and let-7i were significantly positively with the duration of DEE exposure(P-trend<0.001,<0.001,<0.001,<0.001,<0.001,<0.001 and=0.002,respectively).Furthermore,the expression of let-7i was significantly negatively correlated with FVC and FEV1 in the DEE-exposed workers(β=-0.100,95%CI:-0.191--0.010,P=0.029 for FVC;β=-0.092,95%CI:-0.177--0.001,P=0.034 for FEV1,respectively).ConclusionsOur study found that exposure to DEE could induce decrease in lung function which shows mainly obstructive changes of airways and influences of small airways function.The serum CRP levels were higher in the DEE-exposed worker.The polymorphisms in CRP and circulating CRP involved in the inflammatory process,may play significant roles in human sensitivity to lung function injury caused by DEE exposure.This study will help investigate the underlying mechanisms of adverse respiratory effects induced by DEE.Our study also found that long-term exposure to DEE could induce increases of the expression level of miRNA of let-7 family in the peripheral blood leukocytes.DEE exposure duration was positively correlated with the expression level of let-7.This is the first report to investigate association of the expression of let-7 and lung function,among a highly and exclusively DEE-exposed occupational population,which provided the basis to investigate the role of miRNA in the DEE induced respiratory system damage.