柴油機尾氣暴露所致DNA甲基化改變及其與微核組學(xué)指標(biāo)間的關(guān)聯(lián)性研究
發(fā)布時間：2018-09-18 10:03
【摘要】：近些年來,空氣污染物對機體造成的健康危害越來越為公眾所關(guān)注。在眾多空氣污染物中,顆粒物與不良健康效應(yīng)的關(guān)系最為密切,而且先前的研究提示細顆粒物相較于粗顆粒物可能對人體的健康危害更大。柴油發(fā)動機尾氣(Diesel engine exhaust, DEE)是空氣中細顆粒物的主要來源之一。鑒于柴油發(fā)動機在市區(qū)的廣泛應(yīng)用,大量的城市居民已經(jīng)成為DEE最主要的暴露人群。此外,許多職業(yè)人群如礦工、卡車司機也會在工作環(huán)境中暴露于DEE。流行病學(xué)研究表明長期暴露于DEE與肺癌發(fā)生風(fēng)險增高間存在關(guān)聯(lián)。遺傳損傷被認為是癌癥引發(fā)階段的關(guān)鍵分子事件。胞質(zhì)阻滯微核試驗(Cytokinesis-block micronucleus assay, CBMN)常被用來評價暴露于遺傳毒物后機體內(nèi)的遺傳損傷程度,它可以測定外周血淋巴細胞微核(Micronucleus, MN)、核質(zhì)橋(Nucleoplasmic bridge, NPB)、核芽(Nuclear bud, NBUD)率等指標(biāo)。雖然已有人群研究報道過DEE暴露可以引起MN率發(fā)生改變,但是由于存在混合暴露的問題,DEE暴露與微核組學(xué)指標(biāo)改變間的關(guān)系尚不完全清楚。此外,DEE誘導(dǎo)遺傳毒效應(yīng)通路中和遺傳毒效應(yīng)發(fā)生后機體應(yīng)答通路中的調(diào)控模式仍然不清楚。表觀遺傳是指不涉及DNA序列并且可以遺傳的基因表達的改變。越來越多的研究表明表觀遺傳修飾改變作為一種新的調(diào)控基因表達的機制在環(huán)境暴露-毒效應(yīng)-機體應(yīng)答這一連續(xù)模式中發(fā)揮著重要的作用。一方面,特異的表觀遺傳修飾改變可以反映機體接觸環(huán)境暴露的程度。另一方面,表觀遺傳修飾改變能夠參與或者響應(yīng)環(huán)境暴露引起的遺傳毒效應(yīng)。因此,表觀遺傳修飾不僅可能作為介導(dǎo)環(huán)境暴露與癌癥風(fēng)險間關(guān)聯(lián)的生物學(xué)機制,還可能作為環(huán)境暴露和遺傳損傷的生物標(biāo)志物。DNA甲基化是迄今為止研究最為深入的一種表觀遺傳修飾,它指的是CpG位點的胞嘧啶末端共價結(jié)合甲基形成5-甲基胞嘧啶。一般情況下,DNA甲基化的發(fā)生與基因沉默有關(guān)。DNA損傷應(yīng)答(DNA damage response, DDR)相關(guān)基因和重復(fù)序列的甲基化狀態(tài)對于維持基因組的穩(wěn)定性十分重要。有研究在肺癌患者中發(fā)現(xiàn)DDR相關(guān)基因和重復(fù)序列發(fā)生了異常甲基化改變。近年來,關(guān)于DEE暴露和DNA甲基化改變的人群研究逐漸增多,已有研究在哮喘患者中發(fā)現(xiàn)短期暴露于DEE可以引起蛋白激酶和核因子kB通路中基因的甲基化水平發(fā)生改變。然而仍然有許多關(guān)鍵的科學(xué)問題亟待解決,比如長期暴露于DEE能否引起DDR相關(guān)基因和重復(fù)序列的甲基化水平發(fā)生改變,這些甲基化水平改變反過來是否與DEE暴露導(dǎo)致的遺傳毒效應(yīng)間存在關(guān)聯(lián)。為了解決以上科學(xué)問題,本研究選取了117名暴露于單純DEE的柴油機試車工人和112名非DEE暴露工人,以DDR相關(guān)基因和重復(fù)序列甲基化為切入點,結(jié)合微核組學(xué)指標(biāo),探討DEE暴露、DNA甲基化、遺傳毒效應(yīng)三者間的關(guān)聯(lián)。一.DEE的成分解析與表征分別用掃描電遷移率粒徑譜儀和碳分析儀測定了DEE顆粒相的粒徑分布及元素碳和有機碳的含量,用氣相色譜質(zhì)譜儀測定了DEE顆粒相和工人個體暴露樣本中多環(huán)芳烴(Polycyclic aromatic hydrocarbons, PAHs)的含量,用便攜式氣相色譜質(zhì)譜儀測定了DEE氣相有機物的濃度,用被動采氣管測定了DEE氣相二氧化氮和二氧化硫的濃度,用高效液相色譜質(zhì)譜測定了工人尿中6種羥基多環(huán)芳烴(Mono-hydroxylated PAHs, OH-PAHs)的濃度。結(jié)果表明84.3%的DEE顆粒為100nm以下。元素碳和有機碳分別占細顆粒物的百分比平均為28.6±6.3%和36.2±6.5%,有機碳／元素碳平均為1.31±0.31。致癌性PAHs和非致癌性PAHs分別占總PAHs的83.3%和16.7%。DEE氣相有機物主要包括鏈烴及其衍生物、環(huán)烷烴衍生物、苯及其衍生物。元素碳與有機碳呈顯著正相關(guān)(r=0.630,P=0.002),與二氧化氮和二氧化硫呈臨界顯著正相關(guān)(r=0.370,P=0.090和r=0.385,P=0.077)。DEE暴露工人對萘、芴、菲、芘四種PAHs的個體暴露水平及尿中總羥基萘、二羥基芴、總羥基菲、一羥基芘的濃度均高于非DEE暴露工人,差異具有統(tǒng)計學(xué)意義(P0.001)。二.DEE暴露與DNA甲基化間的關(guān)聯(lián)性研究用焦磷酸測序的方法測定了工人外周血淋巴細胞三個DDR相關(guān)基因(p16、RASSF1A、MGMT)和LINE-1重復(fù)序列的甲基化水平。結(jié)果表明DEE暴露工人較非DEE暴露工人p16、RASSFIA、MGMT基因甲基化水平降低,差異具有統(tǒng)計學(xué)意義(P0.001),并未發(fā)現(xiàn)LINE-1甲基化水平在兩組間具有顯著差異。隨著尿總OH-PAHs濃度的增高,p16、RASSF1A、MGMT基因甲基化水平呈顯著降低的趨勢(Ptrend分別為0.018、0.006、0.001)。非吸煙工人中,在校正了混雜因素的影響后,尿總OH-PAHs濃度每上升一個四分位間距,p16、RASSF1A、 MGMT基因經(jīng)logit轉(zhuǎn)換后的甲基化水平平均改變分別為-0.13、-0.18、-0.19。DEE暴露工人中,DEE暴露時長與p16、RASSF1A基因甲基化水平呈顯著正相關(guān)(r=0.293,P=0.001和r=0.409,P0.001)。與溶劑對照組相比,DEE提取物處理組原代淋巴細胞p16、RASSF1A、MGMT基因甲基化水平改變方向大體與人群研究結(jié)果一致。三.DEE暴露與微核組學(xué)指標(biāo)間的關(guān)聯(lián)性研究用CBMN法測定了工人外周血淋巴細胞MN、NPB、NBUD率并求得三者之和計算出基因組不穩(wěn)定指數(shù)。用基因組不穩(wěn)定指數(shù)加上本課題組先前報道的凋亡率和壞死率計算出微核組學(xué)指數(shù)。結(jié)果表明DEE暴露工人較非DEE暴露工人MN、NPB、NBUD率、基因組不穩(wěn)定指數(shù)、微核組學(xué)指數(shù)升高,差異具有統(tǒng)計學(xué)意義(P0.001)。隨著尿總OH-PAHs濃度的增高,MN、NPB、NBUD率、基因組不穩(wěn)定指數(shù)、微核組學(xué)指數(shù)呈顯著升高的趨勢(Ptrend0.001)。非吸煙工人中,在校正了混雜因素的影響后,尿總OH-PAHs濃度每上升一個四分位間距,MN、NPB、NBUD率、基因組不穩(wěn)定指數(shù)、微核組學(xué)指數(shù)平均變化百分比分別為93.48%、467.46%、160.39%、128.64%、121.22%。DEE暴露工人中,在校正了混雜因素的影響后,尿總OH-PAHs濃度每上升一個四分位間距,MN和微核組學(xué)指數(shù)平均變化百分比分別為38.13%和27.63%。四.DEE暴露、DNA甲基化、DNA損傷指標(biāo)、微核組學(xué)指標(biāo)的相互關(guān)系用因子分析、相關(guān)分析、中介效應(yīng)分析、交互作用分析等方法探討了本研究所測指標(biāo)及本課題組先前報道的DNA鏈損傷指標(biāo)[外周血淋巴細胞Olive尾距(Olive tail moment, OTM)]和DNA氧化損傷指標(biāo)[尿1,N6-乙烯基脫氧腺苷(1,N6-ethenodeoxyadenosine, εdA)和3,N4-乙烯基脫氧胞苷(3,N4-ethenodeoxycytidine, εdC)]間的相互關(guān)系。結(jié)果表明p16、RASSF1A、 MGMT基因甲基化與εdA間呈顯著負相關(guān)(r=-0.187,P=-0.008；r=-0.177,P=0.013;r=-0.251,P0.001),與OTM間呈顯著負相關(guān)(r=-0.300,P0.001；r=-0.305,P0.001；r=-0.311,P0.001),與基因組不穩(wěn)定指數(shù)間呈顯著負相關(guān)(r=-0.183,P=0.007; r=-0.212, F=0.002; r=-0.244, P0.001)。p16和RASSFIA基因甲基化與核分裂指數(shù)間呈顯著正相關(guān)(r=0.220,P=0.001;r=0.225, P=0.001)。OTM和基因組不穩(wěn)定指數(shù)分別對核分裂指數(shù)效應(yīng)的24.9%和6.1%可以被p16甲基化所介導(dǎo),26.1%和6.6%可以被RASSFIA甲基化所介導(dǎo)。非吸煙工人中,自然對數(shù)轉(zhuǎn)換后的尿總OH-PAHs每上升一個單位,LINE-1甲基化水平85.6%和≥85.6%的工人基因組不穩(wěn)定指數(shù)平均變化百分比分別為64.38%和45.50%(Pinteraction=0.016)。基因組不穩(wěn)定指數(shù)與εdA和OTM間呈顯著正相關(guān)(r=0.204,P=0.004;r=0.353,P0.001)。用尿總OH-PAHs和εdA的第一和第四分位數(shù)作為分界分別判定DEE暴露水平高低和遺傳損傷大小時,MGMT甲基化對應(yīng)的曲線下面積分別為0.758和0.711。用OTM的第一分位數(shù)和第四分位數(shù)作為分界判定遺傳損傷大小時,p16和RASSFIA甲基化對應(yīng)的曲線下面積分別為0.705和0.724。結(jié)論1)DEE顆粒的粒徑大小大多屬于超細顆粒物的范疇,其上吸附的PAHs主要是致癌性PAHs。DEE氣相有機組分主要包括鏈烴、苯及它們的衍生物。元素碳與其它DEE主要組分間呈顯著或臨界顯著正相關(guān)。DEE暴露工人較非DEE暴露工人尿OH-PAHs濃度顯著增高。隨著DEE暴露工人對PAHs個體暴露水平的增高,其尿中相應(yīng)PAHs代謝產(chǎn)物的濃度也隨之增高。2)DEE暴露可以引起外周血淋巴細胞p16、RASS1F、MGMT基因發(fā)生低甲基化改變。DEE暴露與p16、RASSFIA、MGMT基因甲基化水平間呈顯著負關(guān)聯(lián)。DEE暴露時長與p16、RASSFIA基因甲基化水平間呈顯著正相關(guān)。MGMT基因甲基化水平可以作為DEE暴露水平的生物標(biāo)志物。3)DEE暴露可以導(dǎo)致外周血淋巴細胞MN、NPB、NBUD率、基因組不穩(wěn)定指數(shù)、微核組學(xué)指數(shù)增高。DEE暴露與MN、NPB.NBUD率、基因組不穩(wěn)定指數(shù)、微核組學(xué)指數(shù)呈顯著正關(guān)聯(lián)。DEE暴露水平與MN率和微核組學(xué)指數(shù)間呈顯著正關(guān)聯(lián)。4) εdA、OTM、基因組不穩(wěn)定指數(shù)與p16、RASSF1A、MGMT基因甲基化水平間呈顯著負相關(guān)。εdA和OTM與基因組不穩(wěn)定指數(shù)間呈顯著正相關(guān)。p16和RASSFIA以及MGMT基因甲基化水平可以分別作為DEE暴露所致OTM、εdA水平的生物標(biāo)志物。5)p16和RASSF1A基因甲基化可以介導(dǎo)OTM和基因組不穩(wěn)定指數(shù)與核分裂指數(shù)間的關(guān)聯(lián),LINE-1甲基化可以修飾DEE暴露與基因組不穩(wěn)定指數(shù)間的關(guān)聯(lián)。
[Abstract]:In recent years, more and more attention has been paid to the health hazards caused by air pollutants. Among many air pollutants, particulate matter is the most closely related to adverse health effects. Previous studies have suggested that fine particulate matter may be more harmful to human health than coarse particulate matter. Exhaust (DEE) is one of the main sources of fine particulate matter in the air. In view of the wide use of diesel engines in urban areas, a large number of urban residents have become the main exposures to DEE. In addition, many occupational groups, such as miners and truck drivers, are also exposed to DEE in the working environment. Epidemiological studies have shown that long-term exposure to DEE and lung. Cytokinesis-block micronucleus assay (CBMN) is often used to assess the degree of genetic damage in the body after exposure to genetic toxicants. It can be used to measure micronucleus in peripheral blood lymphocytes. MN, NPB, NBUD and other indicators. Although there have been population studies reported that DEE exposure can cause changes in MN rates, the relationship between DEE exposure and changes in micronucleomic indicators is not completely clear due to the problem of mixed exposure. Epigenetics refers to changes in gene expression that do not involve DNA sequences and can be inherited. A growing number of studies have shown that epigenetic modifications, as a new mechanism for regulating gene expression, are involved in environmental exposure-toxicity-response. Continuous patterns play an important role. On the one hand, specific epigenetic modifications can reflect the degree of exposure to the environment. On the other hand, epigenetic modifications can participate in or respond to the genetic toxicity caused by environmental exposure. DNA methylation is one of the most well-studied epigenetic modifications to date. It refers to the formation of 5-methylcytosine by covalently binding methyl at the cytosine end of the CpG site to form 5-methylcytosine. The methylation of DNA damage response (DDR) related genes and repetitive sequences is important for maintaining genomic stability. Abnormal methylation of DDR related genes and repetitive sequences has been found in lung cancer patients. Population studies on DEE exposure and DNA methylation have been conducted in recent years. Increasing numbers of studies have found that short-term exposure to DEE can cause changes in the methylation levels of genes in protein kinase and nuclear factor kB pathways in asthmatic patients. However, there are still many key scientific issues to be resolved, such as whether long-term exposure to DEE can cause changes in the methylation levels of DDR-related genes and repetitive sequences. In order to solve the above scientific problems, 117 diesel engine test workers and 112 non-DEE exposed workers were selected in this study. DDR-related genes and repetitive sequence methylation were used as the breakthrough point, and micronucleomic finger was combined with DNA methylation. The relationship among DEE exposure, DNA methylation and genetic toxicity was investigated. 1. DEE decomposition and characterization were performed by scanning electromobility particle size spectrometer and carbon analyzer respectively to determine the particle size distribution of DEE and the contents of elemental carbon and organic carbon, and to determine DEE particle phase and individual exposure samples by gas chromatography-mass spectrometry. The contents of polycyclic aromatic hydrocarbons (PAHs) were determined by portable gas chromatography-mass spectrometry (GC-MS). The concentrations of DEE vapor-phase organic compounds (VOCs) were determined by passive recovery pipe. Six kinds of hydroxyl polycyclic aromatic hydrocarbons (PAHs) in workers'urine were determined by high performance liquid chromatography-mass spectrometry (HPLC-MS). OH-PAHs concentration. The results showed that 84.3% of DEE particles were below 100 nm. The average percentage of elemental carbon and organic carbon in fine particles was 28.6 (+ 6.3%) and 36.2 (+ 6.5%) respectively. The average percentage of organic carbon/elemental carbon was 1.31 (+ 0.31%). Carcinogenic PAHs and non-carcinogenic PAHs accounted for 83.3% and 16.7% of total PAHs, respectively. Derivatives, cycloalkane derivatives, benzene and its derivatives. Elemental carbon was positively correlated with organic carbon (r = 0.630, P = 0.002), and positively correlated with nitrogen dioxide and sulfur dioxide (r = 0.370, P = 0.090 and R = 0.385, P = 0.077). DEE exposed workers to naphthalene, fluorene, phenanthrene, pyrene, total hydroxy naphthalene, dihydroxy fluorene and total urine The methylation levels of three DDR-related genes (p16, RASSF1A, MGMT) and LINE-1 repeats in peripheral blood lymphocytes of workers were determined by pyrophosphatic acid sequencing. The methylation levels of p16, RASSFIA and MGMT genes in EE exposed workers were significantly lower than those in non-DEE exposed workers (P 0.001). There was no significant difference in the methylation levels of LINE-1 between the two groups. With the increase of urinary total OH-PAHs concentration, the methylation levels of p16, RASSF1A and MGMT genes were significantly decreased (P tree was 0.018, 0.001, respectively). Among non-smoking workers, the methylation levels of p16, RASSF1A, MGMT genes were - 0.13, - 0.18, - 0.19. The methylation levels of p16, RASSF1A genes were significantly positively correlated with the exposure time of DEE in non-smoking workers after adjusting for the influence of confounding factors. Guan (r = 0.293, P = 0.001 and R = 0.409, P 0.001). Compared with the solvent control group, the methylation levels of p16, RASSF1A and MGMT genes in primary lymphocytes treated with DEE extract were generally consistent with the results of the population study. 3. Correlation between DEE exposure and micronucleomic indices. MN, NPB, NBUD in peripheral blood lymphocytes of workers were measured by CBMN method. The results showed that the MN, NPB, NBUD, genomic instability index and micronucleomic index of DEE exposed workers were higher than those of non-DEE exposed workers. Significance (P 0.001). With the increase of urinary total OH-PAHs concentration, MN, NPB, NBUD rate, genomic instability index, micronucleomic index showed a significant increase trend (Ptrend 0.001). Among non-smokers, after adjusting for the influence of confounding factors, the urinary total OH-PAHs concentration increased by a quartile interval, MN, NPB, NBUD rate, genomic instability index, and micronucleomic index. Among DEE exposed workers, the average change percentages of MN and MN and MN were 38.13% and 27.63% for each quartile increase in urinary total OH-PAHs concentration after adjusting for confounding factors. The correlations of micronucleomic indices were analyzed by factor analysis, correlation analysis, mediating effect analysis and interaction analysis. The indexes measured in this study and the DNA strand damage indices [Olive tail moment (OTM) of peripheral blood lymphocytes] and DNA oxidative damage indices [urine 1, N6-vinyl deoxygland] previously reported by our group were discussed. The results showed that p16, RASSF1A, MGMT gene methylation was significantly negatively correlated with epdA (r = - 0.187, P = - 0.187, P = - 0.008; r = - 0.177, P = 0.013; r = - 0.251, P 0.251, P 0.001), and was negatively correlwith OTM (r = - 300, P = - 300, P = - 300, P = - 0.300, P = - 300, P = - 0.001, P = - 0.001; r = - 0.30, r = - 0.30; r = - 0.30; r = - 0.30; 0.0.0.001, P = - 0.001, P = - 0.001; 0.001, P = - 0.001; 5. P16 and RASSFIA gene methylation were positively correlated with mitotic index (r = 0.220, P = 0.001; r = 0.183, P = 0.007; r = - 0.212, F = 0.002; r = - 0.244, P 0.001). OTM and genomic instability index were positively correlated with mitotic index (r = 0.220, P = 0.001; r = 0.225, P = 0.001), respectively. In non-smoking workers, the average percentage of changes in the genomic instability index of LINE-1 methylation levels of 85.6% and (>85.6%) were 64.38% and 45.50% respectively (Pinteraction = 0.016) for each unit increase in urinary total OH-PAHs after natural logarithmic conversion. Genome instability index was positively correlated with epsilon dA and OTM (r = 0.204, P = 0.004; r = 0.353, P 0.001). When the first and fourth quantiles of urinary total OH-PAHs and epsilon dA were used as the dividing line to determine the level of DEE exposure and the extent of genetic damage, the sub-curves of MGMT methylation were 0.758 and 0.711, respectively. Conclusion 1) The size of DEE particles belongs to the category of ultrafine particles, and the PAHs adsorbed on them are mainly carcinogenic PAHs. DEE vapor phase organic components mainly include chain hydrocarbons, benzene and their derivatives. The concentration of urinary OH-PAHs in DEE exposed workers was significantly higher than that in non-DEE exposed workers. With the increase of individual exposure to PAHs, the concentration of urinary PAHs metabolites in DEE exposed workers also increased. 2) DEE exposure could induce p16 and RAS in peripheral blood lymphocytes. There was a significant negative correlation between DEE exposure and methylation levels of p16, RASSFIA, and MGMT genes. DEE exposure was positively correlated with MN, NPB. NBUD, genomic instability index, genomic instability index and micronucleomic index. There was a significant positive correlation between DEE exposure and MN and micronucleomic index. 4) There was a significant positive correlation between epsilon dA, OTM, genomic instability index and methylation levels of p16, RASSF1A and MGMT genes. The methylation levels of p16, RASSFIA and MGMT genes could be used as biomarkers of OTM, and the methylation levels of epsilon dA and RASSF1A genes could mediate the association between OTM and genomic instability index and mitotic index, respectively. Methylation can modify the association between DEE exposure and genomic instability index.
【學(xué)位授予單位】：中國疾病預(yù)防控制中心
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R114
本文編號：2247561
[Abstract]:In recent years, more and more attention has been paid to the health hazards caused by air pollutants. Among many air pollutants, particulate matter is the most closely related to adverse health effects. Previous studies have suggested that fine particulate matter may be more harmful to human health than coarse particulate matter. Exhaust (DEE) is one of the main sources of fine particulate matter in the air. In view of the wide use of diesel engines in urban areas, a large number of urban residents have become the main exposures to DEE. In addition, many occupational groups, such as miners and truck drivers, are also exposed to DEE in the working environment. Epidemiological studies have shown that long-term exposure to DEE and lung. Cytokinesis-block micronucleus assay (CBMN) is often used to assess the degree of genetic damage in the body after exposure to genetic toxicants. It can be used to measure micronucleus in peripheral blood lymphocytes. MN, NPB, NBUD and other indicators. Although there have been population studies reported that DEE exposure can cause changes in MN rates, the relationship between DEE exposure and changes in micronucleomic indicators is not completely clear due to the problem of mixed exposure. Epigenetics refers to changes in gene expression that do not involve DNA sequences and can be inherited. A growing number of studies have shown that epigenetic modifications, as a new mechanism for regulating gene expression, are involved in environmental exposure-toxicity-response. Continuous patterns play an important role. On the one hand, specific epigenetic modifications can reflect the degree of exposure to the environment. On the other hand, epigenetic modifications can participate in or respond to the genetic toxicity caused by environmental exposure. DNA methylation is one of the most well-studied epigenetic modifications to date. It refers to the formation of 5-methylcytosine by covalently binding methyl at the cytosine end of the CpG site to form 5-methylcytosine. The methylation of DNA damage response (DDR) related genes and repetitive sequences is important for maintaining genomic stability. Abnormal methylation of DDR related genes and repetitive sequences has been found in lung cancer patients. Population studies on DEE exposure and DNA methylation have been conducted in recent years. Increasing numbers of studies have found that short-term exposure to DEE can cause changes in the methylation levels of genes in protein kinase and nuclear factor kB pathways in asthmatic patients. However, there are still many key scientific issues to be resolved, such as whether long-term exposure to DEE can cause changes in the methylation levels of DDR-related genes and repetitive sequences. In order to solve the above scientific problems, 117 diesel engine test workers and 112 non-DEE exposed workers were selected in this study. DDR-related genes and repetitive sequence methylation were used as the breakthrough point, and micronucleomic finger was combined with DNA methylation. The relationship among DEE exposure, DNA methylation and genetic toxicity was investigated. 1. DEE decomposition and characterization were performed by scanning electromobility particle size spectrometer and carbon analyzer respectively to determine the particle size distribution of DEE and the contents of elemental carbon and organic carbon, and to determine DEE particle phase and individual exposure samples by gas chromatography-mass spectrometry. The contents of polycyclic aromatic hydrocarbons (PAHs) were determined by portable gas chromatography-mass spectrometry (GC-MS). The concentrations of DEE vapor-phase organic compounds (VOCs) were determined by passive recovery pipe. Six kinds of hydroxyl polycyclic aromatic hydrocarbons (PAHs) in workers'urine were determined by high performance liquid chromatography-mass spectrometry (HPLC-MS). OH-PAHs concentration. The results showed that 84.3% of DEE particles were below 100 nm. The average percentage of elemental carbon and organic carbon in fine particles was 28.6 (+ 6.3%) and 36.2 (+ 6.5%) respectively. The average percentage of organic carbon/elemental carbon was 1.31 (+ 0.31%). Carcinogenic PAHs and non-carcinogenic PAHs accounted for 83.3% and 16.7% of total PAHs, respectively. Derivatives, cycloalkane derivatives, benzene and its derivatives. Elemental carbon was positively correlated with organic carbon (r = 0.630, P = 0.002), and positively correlated with nitrogen dioxide and sulfur dioxide (r = 0.370, P = 0.090 and R = 0.385, P = 0.077). DEE exposed workers to naphthalene, fluorene, phenanthrene, pyrene, total hydroxy naphthalene, dihydroxy fluorene and total urine The methylation levels of three DDR-related genes (p16, RASSF1A, MGMT) and LINE-1 repeats in peripheral blood lymphocytes of workers were determined by pyrophosphatic acid sequencing. The methylation levels of p16, RASSFIA and MGMT genes in EE exposed workers were significantly lower than those in non-DEE exposed workers (P 0.001). There was no significant difference in the methylation levels of LINE-1 between the two groups. With the increase of urinary total OH-PAHs concentration, the methylation levels of p16, RASSF1A and MGMT genes were significantly decreased (P tree was 0.018, 0.001, respectively). Among non-smoking workers, the methylation levels of p16, RASSF1A, MGMT genes were - 0.13, - 0.18, - 0.19. The methylation levels of p16, RASSF1A genes were significantly positively correlated with the exposure time of DEE in non-smoking workers after adjusting for the influence of confounding factors. Guan (r = 0.293, P = 0.001 and R = 0.409, P 0.001). Compared with the solvent control group, the methylation levels of p16, RASSF1A and MGMT genes in primary lymphocytes treated with DEE extract were generally consistent with the results of the population study. 3. Correlation between DEE exposure and micronucleomic indices. MN, NPB, NBUD in peripheral blood lymphocytes of workers were measured by CBMN method. The results showed that the MN, NPB, NBUD, genomic instability index and micronucleomic index of DEE exposed workers were higher than those of non-DEE exposed workers. Significance (P 0.001). With the increase of urinary total OH-PAHs concentration, MN, NPB, NBUD rate, genomic instability index, micronucleomic index showed a significant increase trend (Ptrend 0.001). Among non-smokers, after adjusting for the influence of confounding factors, the urinary total OH-PAHs concentration increased by a quartile interval, MN, NPB, NBUD rate, genomic instability index, and micronucleomic index. Among DEE exposed workers, the average change percentages of MN and MN and MN were 38.13% and 27.63% for each quartile increase in urinary total OH-PAHs concentration after adjusting for confounding factors. The correlations of micronucleomic indices were analyzed by factor analysis, correlation analysis, mediating effect analysis and interaction analysis. The indexes measured in this study and the DNA strand damage indices [Olive tail moment (OTM) of peripheral blood lymphocytes] and DNA oxidative damage indices [urine 1, N6-vinyl deoxygland] previously reported by our group were discussed. The results showed that p16, RASSF1A, MGMT gene methylation was significantly negatively correlated with epdA (r = - 0.187, P = - 0.187, P = - 0.008; r = - 0.177, P = 0.013; r = - 0.251, P 0.251, P 0.001), and was negatively correlwith OTM (r = - 300, P = - 300, P = - 300, P = - 0.300, P = - 300, P = - 0.001, P = - 0.001; r = - 0.30, r = - 0.30; r = - 0.30; r = - 0.30; 0.0.0.001, P = - 0.001, P = - 0.001; 0.001, P = - 0.001; 5. P16 and RASSFIA gene methylation were positively correlated with mitotic index (r = 0.220, P = 0.001; r = 0.183, P = 0.007; r = - 0.212, F = 0.002; r = - 0.244, P 0.001). OTM and genomic instability index were positively correlated with mitotic index (r = 0.220, P = 0.001; r = 0.225, P = 0.001), respectively. In non-smoking workers, the average percentage of changes in the genomic instability index of LINE-1 methylation levels of 85.6% and (>85.6%) were 64.38% and 45.50% respectively (Pinteraction = 0.016) for each unit increase in urinary total OH-PAHs after natural logarithmic conversion. Genome instability index was positively correlated with epsilon dA and OTM (r = 0.204, P = 0.004; r = 0.353, P 0.001). When the first and fourth quantiles of urinary total OH-PAHs and epsilon dA were used as the dividing line to determine the level of DEE exposure and the extent of genetic damage, the sub-curves of MGMT methylation were 0.758 and 0.711, respectively. Conclusion 1) The size of DEE particles belongs to the category of ultrafine particles, and the PAHs adsorbed on them are mainly carcinogenic PAHs. DEE vapor phase organic components mainly include chain hydrocarbons, benzene and their derivatives. The concentration of urinary OH-PAHs in DEE exposed workers was significantly higher than that in non-DEE exposed workers. With the increase of individual exposure to PAHs, the concentration of urinary PAHs metabolites in DEE exposed workers also increased. 2) DEE exposure could induce p16 and RAS in peripheral blood lymphocytes. There was a significant negative correlation between DEE exposure and methylation levels of p16, RASSFIA, and MGMT genes. DEE exposure was positively correlated with MN, NPB. NBUD, genomic instability index, genomic instability index and micronucleomic index. There was a significant positive correlation between DEE exposure and MN and micronucleomic index. 4) There was a significant positive correlation between epsilon dA, OTM, genomic instability index and methylation levels of p16, RASSF1A and MGMT genes. The methylation levels of p16, RASSFIA and MGMT genes could be used as biomarkers of OTM, and the methylation levels of epsilon dA and RASSF1A genes could mediate the association between OTM and genomic instability index and mitotic index, respectively. Methylation can modify the association between DEE exposure and genomic instability index.
【學(xué)位授予單位】：中國疾病預(yù)防控制中心
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R114
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