Biomarkers of formaldehyde based on DNA-protein cross-links

描述(申请人提供)：甲醛是一种广泛使用的高产量化学品，也是各种建筑产品燃烧和排放的副产品。甲醛已被国际癌症研究机构列为动物和人类致癌物，可导致鼻咽癌。据报道，甲醛也与白血病的诱发有关。甲醛与DNA和蛋白质高度反应，可引起多种损伤，其中DNA-蛋白质交联物(DPC)的形成是主要的遗传毒性效应。由于缺乏合适的生物标志物，甲醛的内部暴露监测和癌症风险评估一直具有挑战性。这也限制了我们检查远离鼻黏膜的器官中甲醛的远距离影响的能力，这一点仍然存在很大争议。最近，我们建立了第一个甲醛特异的DNA加合物生物标记物，并利用该生物标记物生成的丰富的数据集被广泛用于甲醛的风险评估。然而，在高丰度下形成的DPC在生物学上更重要，因为它们显著干扰DNA修复，因为它们是极其巨大的DNA损伤。然而，由于大量的技术挑战，还没有建立生物标记物来量化甲醛诱导的DPC。以前的测量DPC的方法要么使用灵敏度低的非化学特异性方法，要么依赖放射性甲醛进行曝光。这项应用的目的是开发生物标志物和基于灵敏质谱学的方法来测量甲醛诱导的DPC。中心假说是，含有半胱氨酸的交联物可以作为量化甲醛诱导的DPC的生物标志物，与DNA交联的蛋白质也可以作为评估DPC形成的生物标志物。这一假设是基于申请人实验室的初步数据和我们在开发DNA损伤的质谱分析方面的经验而提出的。我们将首先优化酶解方法，将DPC消化成小的交联物用于质谱学检测，然后开发灵敏的质谱学分析方法来定量含半胱氨酸的交联物，从而验证这一假设。我们还将分离、鉴定和量化甲醛暴露后与DNA交联的蛋白质作为DPC的生物标记物。这种方法是创新的，因为高灵敏质谱学的新应用开发了新的甲醛特异性生物标记物来量化DPC的形成。这项研究意义重大，因为它有望建立一套新的甲醛暴露生物标记物来测量甲醛诱导的DPC，这是目前甲醛生物标记物研究中的一个空白。这项研究的结果也将为未来的研究奠定基础，旨在利用DPC的这些新的生物标志物建立分子剂量学，以更好地了解甲醛的致癌作用及其癌症风险评估。

DESCRIPTION (provided by applicant): Formaldehyde is a widely used high production chemical that is also released as a byproduct of combustion and off-gassing of various building products. Formaldehyde has been classified as an animal and human carcinogen by the International Agency for Research on Cancer, causing nasopharyngeal cancer. Formaldehyde is also reported to be associated with the induction of leukemia. Formaldehyde is highly reactive with DNA and proteins to induce diverse lesions, with the formation of DNA-protein cross-links (DPC) as the primary genotoxic effect. Internal exposure monitoring and cancer risk assessment of formaldehyde have been challenging due to the lack of suitable biomarkers. This also limits our capability of examining formaldehyde distant effects in organs remote to nasal epithelium, which remains highly controversial. Recently, we have established N2-hydroxymethyl-deoxyguanosine as the first formaldehyde-specific DNA adduct biomarker, and the rich dataset generated using this biomarker is widely used in risk assessment of formaldehyde. However, DPC, formed at high abundance, are biologically more important because they significantly interfere with DNA repair as extremely bulky DNA lesions. However, no biomarkers have been established to quantify formaldehyde-induced DPC due to substantial technical challenges. Previous methods of measuring DPC either utilized non-chemical-specific methods with low sensitivity or relied on radioactive formaldehyde for exposure. The objective of this application is to develop biomarkers and sensitive mass spectrometry-based methods to measure formaldehyde-induced DPC. The central hypothesis is that cysteine-containing cross-links can be developed as biomarkers to quantify formaldehyde-induced DPC, and that proteins cross-linked with DNA can also serve as biomarkers to assess the formation of DPC. This hypothesis has been formulated on the basis of preliminary data produced in the applicant's laboratory and our experience on developing mass spectrometry assays for DNA lesions. We will test the hypothesis by first optimizing enzymatic hydrolysis approaches to digest DPC into small cross-links for mass spectrometry detection, and then developing sensitive mass spectrometry assays to quantify cysteine-containing cross-links. We will also isolate, identify and quantify proteins cross-linked with DNA following formaldehyde exposure as biomarkers of DPC. The approach is innovative because of the novel application of highly sensitive mass spectrometry to develop new formaldehyde-specific biomarkers to quantify DPC formation. The proposed research is significant, because it is expected to establish a set of novel biomarkers of formaldehyde exposure to measure formaldehyde- induced DPC, a current void in formaldehyde biomarker research. Results from this study will also lay a foundation for future studies aiming at establishing molecular dosimetry using these novel biomarkers of DPC to better understand formaldehyde carcinogenicity and its cancer risk assessment.