| Asthma, characterized by chronic inflammation, airway hyper-responsiveness, and airway remodeling, is one of the most common chronic lung diseases around the world. The symptoms of asthma include recurrent episodes of breathlessness, wheezing and tight chest. In the past decades, the prevalence of asthma has dramatically increased globally. World Health Organization estimated that 235million people were suffering from asthma in 2013 and there were about 100 thousand people dying from asthma every year. The prevalence of asthma in China had rapidly increased to 1.2% in 2013. Highest increases of 147.9% and 190.2% for asthma prevalence were reported in Beijing and Shanghai, respectively. Therefore, the research on the causes or mechanism of asthma is needed for prevention and treatment.Asthma is considered to be resulted from the combined effect of genetic predisposition and environmental exposure. Particulate matters, pollen, and other allergens were wildly reported to be associated with asthma. However, few studies reported the association of other environmental factors like metals or polycyclic aromatic hydrocarbons (PAHs) with adult asthma. Some current results on this topic are still inconclusive. Given various exposure sources of metals and PAHs, biomonitoring is a preferred method to determine the internal exposure of exogenous toxins than air monitoring. And urine, as a kind of biologic sample wildly used for the evaluation of internal exposure, is the main excretion pathway for metals and PAHs. In this study, we aimed to determine and compare levels of metals and PAHs metabolites, and analyze the potential associations of adult asthma with exposures to metals and PAHs.Increasing evidences showed that the environmental toxins influenced asthma through inflammatory responses. Numerous cytokines are reported to be involved in asthma, but the mechanism is still poorly understood. With the new technique like high-throughput quantitative protein microarray, we aim to search new asthma-related cytokines whose levels were different between asthma cases and healthy controls, and improve the understanding of the pathogenesis of asthma.The objectives of the study included:(1) to screen and verify new asthma-related cytokines, and analyze those associations with adult asthma and the severity of asthma; (2) to analyze the potential associations of adult asthma with exposure to metals or PAHs. This study was divided into three parts:(1) The expression profile of 280 cytokines were determined in 8 adult asthma cases and 16 healthy controls by high-throughput quantitative protein microarray, and 5 cytokines were subsequently selected from those with differential expressions between asthma cases and healthy controls, according to the existing knowledge. In a large-scale case-control study of 1009 asthma cases and 1009 healthy controls, these five selected cytokines were determined by Enzyme-Linked Immunosorbent Assay (ELISA) and the associations of the five cytokines with asthma were analyzed. (2) The subjects whose urine samples had been collected were recruited from asthma cases and healthy controls in part 1. The concentrations of 22 metals in urine were determined by Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) and those associations with adult asthma were analyzed. (3) The PAHs metabolite levels in urine were determined by Gas Chromatograph-Mass Spectrometer (GC-MS). The associations of urinary PAHs metabolites with adult asthma and plasma cytokines were evaluated.Part 1 The association of plasma cytokines with adult asthmaObjects:To search new cytokines which distinguish the asthma cases from healthy controls through screening 280 cytokines and analyze those associations with adult asthma and the severity of asthmaMethods:From October 2010 through January 2012,1009 asthma cases were recruited in a general hospital in Wuhan. All of them were adults and diagnosed by the qualified physicians. The healthy controls were recruited from same residential districts as asthma cases. We excluded the subjects if they had respiratory, cardiovascular or infective diseases. Among the remaining eligible subjects,1009 healthy controls were matched 1:1 with asthma cases for the age (±5 year) and gender. The questionnaire investigations and health examinations were performed for all the subjects.The expression profile of 280 cytokines were determined by high-throughput quantitative protein microarray in 8 adult asthma cases and 16 healthy controls who were nonsmokers, and cytokines whose expression levels were different in two groups were selected. According to the existing knowledge,5 cytokines were chosen to be determined in 1009 asthma cases and 1009 gender-and age-matched healthy controls by ELISA. The differences between asthma cases and healthy controls in demographic characteristics and lung function were compared by the student t-test or chi-square test as appropriate. The distributions of 5 cytokines in asthma cases and healthy controls were compared by Wilcoxon signed-rank test. The correlations among 5 cytokines in the total population were analyzed by Spearman rank correlation. The associations between 5 cytokines with asthma prevalence were analyzed by the conditional logistic regression. And unconditional logistic regression was used to analyze the associations of 5 cytokines with the severity of asthma.Results:Five cytokines like CA15-3, CCL11, CD14, Adipsin and IL-9 were chosen from 280 cytokines. In the asthma cases and healthy controls, male subjects accounted for 41.53%. The average ages of asthma cases and healthy controls were 44.48±14.71years and 45.15±15.12 years, respectively. The asthma cases were divided into intermittent (59.1%) and persistent (40.9%) group by the severity.Compared with healthy controls, the proportion of the subjects with family history of asthma in asthma cases was significantly higher (10.41%), and proportions of current smokers (11.89%) and drinkers (10.61%) were significantly lower. The lung function indices in asthma cases were significantly lower than in healthy controls (.P<0.0001). The differences between asthma cases and healthy controls in keeping pets, planting and BMI were nonsignificant.The IL-9 levels in the peripheral blood of cases in persistent stage was significantly higher than those of cases in intermittent stage, the latter were significantly higher than those of healthy controls (P<0.0001). The levels of CCL11, CD 14 and Adipsin in the peripheral blood of cases in persistent stage was significantly lower than those of cases in intermittent stage, the latter were significantly lower than those of healthy controls (P<0.0001). There was no significant difference of CA15-3 between asthma cases and healthy controls (P=0.2531). As continuous or categorical variables, the elevated levels of CA15-3 and IL-9 were significantly positively associated with asthma; the elevated levels of CCL11, CD 14 and Adipsin were significantly negatively associated with asthma and the severity.Conclusions:The plasma levels of CA 15-3, CCL11, CD 14, Adipsin, and IL-9 were associated with adult asthma, and the further research on mechanism is needed.Part 2 The association of urinary metals with adult asthmaObjects:To compare the urinary metals levels between asthma cases and healthy control subjects and analyze the association of urinary metals with adult asthma.Methods:The study included 551 asthma cases and 551 gender-and age-matched healthy controls. The questionnaire investigations and health examinations were performed for all the participants. The urinary concentrations of 22 metals [(Aluminum (Al), Vanadium (V), Chromium (Cr), Manganese (Mn), Iron (Fe), Cobalt (Co), Nickel (Ni), Copper (Cu), Zinc (Zn), Arsenic (As), Selenium (Se), Rubidium (Rb), Strontium (Sr), Molybdenum (Mo), Cadmium (Cd), Tin (Sn), Antimony (Sb), Barium (Ba), Tungsten (W), Thallium (T1), Lead (Pb), Uranium (U)] were determined by ICP-MS and adjusted by the urinary concentration of creatine. The differences between asthma cases and healthy controls in demographic characteristics and lung function were compared by the student t-test or chi-square test as appropriate. The distributions of urinary metals in asthma cases and healthy controls were compared by Wilcoxon signed-rank test. The correlations among urinary metals in all the subjects were analyzed by Spearman rank correlation. The associations between urinary metals with asthma prevalence were analyzed by the logistic regression. False discovery rate (FDR) was used to adjust the P value for multiple testing.Results:In asthma cases and healthy controls, male subjects accounted for 43.01%. The average ages of asthma cases and healthy controls were 42.41±12.56 years and 42.74±12.80 years, respectively. The BMI in asthma cases (23.02±3.46 kg/m2) was significantly lower than in healthy controls (24.00±3.66 kg/m2). The asthma cases were divided into cases in the exacerbation stage of asthma (357,64.79%) and cases in the remission stage of asthma (194,35.21%).The proportion of the subjects with family history of asthma in asthma cases was significantly higher (11.25%) than in healthy controls (2.00%), and the proportion of current smokers was significantly lower in asthma cases (12.52%) than in healthy controls (23.96%). The lung function indices in asthma cases were significantly lower than in healthy controls (P<0.0001), except FVC (%pred).Among 22 metals, geometric mean concentrations of 11 metals (Cr, Cu, As, Se, Sn Mo, Cd, Sn, Ti, W and U) were significantly higher in asthma cases than in healthy controls, and geometric mean concentrations of 10 metals (Al, V, Mn, Fe, Ni, Zn, Rb, Ba, TI and Pb) were significantly lower in asthma cases than in healthy controls.Through logistics regression analyses, urinary levels of 13 metals were observed to be associated with adult asthma. The elevated levels of Cr, Cu, Se, Mo, Cd and U were significantly positively associated with asthma, and odds ratios (95%CI) were 4.76(2.97-7.62),5.89(3.18-10.90),10.43(4.74-22.95),3.48(2.15-5.62),1.71(1.03-2.85) and 7.26(4.55-11.59); the elevated levels of Mn, Fe, Ni, Zn, Rb, Ba and Pb were significantly negatively associated with asthma, and odds ratios (95%CI) were 0.25(0.15-0.42), 0.38(0.24-0.59),0.29(0.21-0.40),0.40(0.24-0.65),0.08(0.04-0.16),0.46(0.29-0.74) and 0.44(0.27-0.74). Compared asthma cases in the exacerbation stage of asthma and cases in the remission stage of asthma with healthy controls, similar results were observed.Conclusions:The roles of different metals in adult asthma vary greatly. The elevated levels of Cr, Cu, Se, Mo, Cd and U were significantly positively associated with asthma, and the elevated levels of Mn, Fe, Ni, Zn, Rb, Ba and Pb were significantly negatively associated with asthma.Part 3 The association of polycyclic aromatic hydrocarbon metabolites in urine with adult asthmaObjects:To compare the levels of polycyclic aromatic hydrocarbon metabolites (OH-PAHs) between asthma cases and healthy controls, and analyze the association of PAHs with adult asthma.Methods:The study included 507 asthma cases and 536 gender-and age-matched healthy control subjects. The questionnaire investigations and health examinations were performed for all the subjects. The urinary concentrations of 10 polycyclic aromatic hydrocarbon metabolites (1-hydroxynaphthalene,2-hydroxynaphthalene,2-hydroxyfluorene, 9-hydroxyfluorene,4-hydroxyphenanthrene,3-hydroxyphenanthrene, 1-hydroxyphenanthrene,2-hydroxyphenanthrene and 1-hydroxypyrene) were determined by GC-MS and adjusted by the concentration of creatine. The expressions of CA15-3, CCL11, CD 14, Adipsin and IL-9 were determined by ELISA. The differences between asthma cases and healthy controls in demographic characteristics and lung function were compared by the student t-test or chi-square test as appropriate. The distributions of OH-PAHs and 5 cytokines in asthma cases and healthy controls were compared by Wilcoxon signed-rank test. The correlations among OH-PAHs and cytokines were analyzed by Spearman rank correlation. The associations between OH-PAHs with asthma prevalence were analyzed by the logistic regression. And associations between ?H-PAHs with cytokines were analyzed by the generalized linear model. False discovery rate (FDR) was used to adjust the P value for multiple testing.Results:The male subjects accounted for 43.79% in asthma cases and 43.28%in healthy controls. The average ages of asthma cases and healthy controls were 42.09±12.89 years and 42.76±12.51 years, respectively. The BMI in asthma cases (23.00±3.61 kg/m2) was significantly lower than in healthy controls (24.01±3.67 kg/m2).The proportion of the subjects with family history of asthma in asthma cases was significantly higher (10.45%) than in healthy controls (2.05%), and the proportion of current smokers was significantly lower in asthma cases (12.62%) than in healthy controls (24.07%). The lung function indices in cases were significantly lower than in healthy controls (P<0.0001), except FVC (%pred).The significantly positive correlations between 10 OH-PAHs were observed in the total subjects (P<0.0001). The levels of 2-hydroxyfluorene,4-hydroxyphenanthrene, 1-hydroxyphenanthrene,2-hydroxyphenanthrene and 1-hydroxypyrene were significantly higher in asthma cases than in healthy control subjects (P<0.005), and the levels of 2-hydroxynaphthalene and 9-hydroxyfluorene were significantly lower n asthma cases than in healthy controls (P<0.05). According to logistic regression models, the elevated levels of 2-hydroxyfluorene,4-hydroxyphenanthrene,1-hydroxyphenanthrene, 2-hydroxyphenanthrene and 1-hydroxypyrene were significantly positively associated with asthma, and the elevated levels of 2-hydroxynaphthalene and 9-hydroxyfluorene were significantly negatively associated with asthma. The level of?OH-PAHs was positively, but not significantly, associated with asthma (odds ratio=1.23,95%CI:0.97-1.56). The expression of IL-9 was significantly higher in asthma cases than in healthy control subjects (P<0.0001), and the expressions of CCL11, CD14 and Adipsin were lower in asthma cases than in healthy controls (P<0.0001). There was no significant difference observed in the levels of CA15-3 between asthma cases and healthy control subjects (P=0.0834). The associations of ?H-PAHs in urine with five plasma cytokines were not significant.Conclusions:Results are suggestive of a deleterious effect of PAHs exposure on asthma prevalence in adults. The elevated levels of 2-hydroxyfluorene,4-hydroxyphenanthrene, 1-hydroxyphenanthrene,2-hydroxyphenanthrene and 1-hydroxypyrene were significantly positively associated with asthma, and the elevated levels of 2-hydroxynaphthalene and 9-hydroxyfluorene were significantly negatively associated with asthma.The study found that the plasma cytokines and the exposure to metals or PAHs could contribute to adult asthma prevalence in the large-scale study. The innovations about this study included:a) The new evidences about the positive association of CA15-3 and the negative association of Adipsin with the prevalence and severity of asthma were reported; b) The elevated levels of Cr, Cu, Se, Mo, Cd and U were significantly positively associated with asthma, and the elevated levels of MN, Fe, Ni, Zn, Rb, Ba and Pb significantly negatively associated with asthma; c) The exposure to PAHs may be a risk factor for adult asthma. The elevated levels of 2-hydroxyfluorene,4-hydroxyphenanthrene, 1-hydroxyphenanthrene,2-hydroxyphenanthrene and 1-hydroxypyrene were significantly positively associated with asthma, and the elevated levels of 2-hydroxynaphthalene and 9-hydroxyfluorene were significantly negatively associated with asthma. |
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