大气中黑炭细颗粒的遗传与表观遗传毒性分析与分子机制研究

Fine particulate matter (PM2.5) is deemed one of the most dangerous to health and moreover, problems persist with high ambient concentrations. In recent years, accumulating evidence (from considerable amount of toxicological research) has identified black carbon as additional air quality metrics that may be valuable in evaluating the health risks of, for example, primary combustion particles from traffic emissions, which are not fully taken into account with PM2.5 mass. Most of the evidence accumulated so far is for an adverse effect on health of carbonaceous material from traffic. Exposure durations from a few minutes up to a year have been linked with adverse effects. To assess the adverse health effects of black carbons and to understand their underlying mechanisms, we will study the toxicology of black carbon from the perspectives of both genetics and epigenetics at the levels of critical enzymes and proteins, affected cells and model animals (for example, zebrafish, mouse). First, we will develop ultrasensitive analytical approaches for detection of various DNA lesions and a variety of DNA/RNA modifications. Second, by the mean of the developed approaches, we will generate a full profile of DNA lesions, including oxidatively generated DNA damages and DNA adducts, which are induced probably by black carbon or/and the adsorbed chemicals through black carbon exposures. Third, we will identify the key DNA/RNA modifications that are sensitive to the black carbon exposure. Combined with gene editing technologies, we will further explore the roles of DNA/RNA methylases and demethylases and repair enzymes involved in the adverse health effects of black carbon-based particulate matters. By this rational experimental design, we will obtain information regarding whether and how these enzymes protect the cells from the black carbon exposure, or enhance the toxicological effects of black carbon. Fourth, we will investigate whether and how the genetic and epigenetic factors affect the formation of DNA lesions and followed repair upon the exposure of black carbon. At last, combined with genome-wide deep sequencing technology, we will profile the distribution changes of key DNA lesions and sensitive DNA/RNA modifications along whole genomes and explore the functional changes and possible involved mechanisms. The new data obtained will support the scientific conclusions for the Air Quality Guidelines and also provides scientific arguments for taking decisive actions to improve air quality and reduce the global burden of disease associated with air pollution.

大气细颗粒的健康危害严重，已受到人们的广泛关注。本项目以大气细颗粒中重要的毒性黑炭细颗粒及其吸附化学组分作为研究对象，在细胞水平和动物水平，开展其遗传与表观遗传的毒性效应及其分子机制研究，以阐明可能引发的健康危害及作用机制,为黑炭细颗粒的健康风险评估和健康危害预防提供科学依据和技术支撑。为此，首先发展只需少量细胞、微量DNA、高灵敏的遗传和表观遗传修饰分析方法，包括多种DNA损伤、DNA/RNA表观修饰。在此基础上，分析鉴定大气黑炭颗粒物诱导的DNA损伤、DNA/RNA甲基化异常，并进一步研究黑炭颗粒物与关键酶（如DNA/RNA甲基化转移酶、去甲基化酶和DNA修复酶）间的相互作用。发展深度测序技术，获得并解析全基因组范围DNA损伤和表观遗传修饰图谱，实现对特定DNA损伤和表观遗传修饰的全景式分析，阐明黑炭颗粒物如何改变遗传和表观遗传等基础性科学问题，致力于实现环境与健康领域的原创性突破。

针对大气细颗粒物以及黑炭可能引发的健康危害，发展了高灵敏DNA损伤与表观修饰分析方法，并开展了遗传与表观遗传的毒性效应及其分子作用机制研究。在方法学方面主要进展有：（1）建立了典型DNA氧化损伤标志物8-羟基脱氧鸟苷（8-OHdG）的高灵敏分析方法。该方法可应用于研究PM2.5暴露引起的外周血细胞中的DNA氧化损伤；（2）建立多种醛类-DNA加合物的高灵敏分析方法，可研究PM2.5中醛类组分诱导形成的DNA加合物，以及评价各种不同来源PM2.5的基因毒性；（3）建立并发展了多种DNA/RNA表观修饰的分析方法与技术，包括5-羟甲基胞嘧啶(5mC)、5-羟甲基胞嘧啶(5hmC)和5-醛基胞嘧啶(5fC)，以及DNA羟甲基化的单碱基测序新方法。利用所发展的技术，证明了5hmC和5fC是更好的表观遗传效应标志物，预期可应用于PM2.5暴露引发的健康危害研究。利用所发展的分析技术与方法，在毒理机制方面的研究已取得一定的创新性成果：（1）通过人支气管上皮细胞(HBE)的 PM2.5暴露实验，发现表观修饰5hmC和5fC的水平均高于非暴露组(p < 0.05)。同时发现，经过PM2.5暴露可增加HEB细胞中(6S,8S)CrodG和(6R,8R)CrodG加合物。这些初步结果暗示了PM2.5具有表观遗传毒性和遗传毒性效应；（2）首次提出了环境双酚类内分泌干扰物引起乳腺癌细胞增殖的表观遗传负反馈调控信号转导新通路。这也是首次建立了环境污染物暴露-TET2-DNA羟甲基化-细胞增殖之间的关联。由于表观遗传变化的可逆性，这项机制研究也为制订干预环境污染引起的疾病措施和方案提供理论支持；（3）发现DNA聚合酶介导掺入的DNA N6-甲基腺嘌呤(6mA)，是哺乳动物DNA 6mA新来源。该类型6mA是细胞受外源刺激或环境压力的潜在标志物；（4）建立了多种细胞模型，发现了炭黑对干细胞自我更新与分化的毒性作用；并开展了多种表观基因敲除模型小鼠的大气细颗粒物暴露研究，并实现全基因组测序。我们在此过程中，发现了多种潜在的表观遗传毒性标志物。在项目实施期间，已发表标注学术论文29篇，其中Cell Res 1篇，Hepatology 1篇，EHP1篇，Chem Sci 1篇，Chem Res Toxicol 1篇，Anal Chem 7篇，Trends Anal Chem 2篇。

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