New biomonitoring methodologies to measure DNA adducts in human tissues

 描述(申请人提供)：膀胱癌(BC)是男性第四大常见癌症，2010年新增病例超过70,000例，死亡14,680例。BC的主要危险因素包括吸烟以及在染料和橡胶行业中职业暴露于芳香胺。这些暴露被认为约占膀胱病例的50%。一些流行病学研究已经将食用含有结构上相关的杂环芳胺(HAAs)的熟肉与BC风险联系在一起。内源性脂质过氧化产物也可能导致膀胱的突变。然而，造成BC的具体化学物质在很大程度上是未知的。尿液是一种很有前途的分析癌症生物标志物的生物标记物。例如，尿液中排出的小泡(外切体)是遗传信息的载体，也是癌症生物标记物的新来源。我们认为，尿脱落细胞也可以作为一种无创的生物标本来评估上、下尿路的DNA损伤。这一拨款续期的目标是“测量人体组织中DNA加合物的新生物监测方法”，目的是验证脱落的尿液细胞作为评估化学物质暴露的样本来源，其中一些化学物质可能导致上尿路上皮癌(UUC)和BC。我们将检测在环境、饮食、药物和内源性基因毒物的作用下，尿液脱落细胞和尿路上皮组织中形成的DNA加合物。我们的初步数据表明，马兜铃酸(AA)的DNA加合物可以通过超高效液相色谱/多级扫描质谱仪(UPLC/MSN)在尿脱落细胞中定量，该方法比常用的非特异性32P后标记方法具有更高的灵敏度。马兜铃酸是一种在中药中发现的尿路上皮致癌物质，也是面粉的污染物。我们建议使用尿脱落细胞作为生物显微镜的非侵入性来源，使用UPLC/MSN来评估UUC和BC风险人群的上尿路上皮和/或膀胱中的DNA损伤。在目标1中，我们检测了通过中药或污染面包接触AA的受试者的尿路上皮组织和脱落细胞中AA的DNA加合物。在目标2中，我们建立了测量其他基因毒物的DNA加合物的技术，这些基因毒物可以损害膀胱，包括暴露于这些化学物质或烟草烟雾中的小鼠尿路上皮和尿细胞中的芳香胺、HAAs和过氧化脂质，使用靶向和“内收切除”扫描方法。在目的3中，应用MS方法检测膀胱癌切除患者尿路上皮和脱落细胞以及非吸烟者和吸烟者尿细胞中形成的DNA加合物。我们提议的研究与NIEHS的使命高度相关，该使命是为了了解环境暴露如何影响健康结果。尿脱落细胞是一种未开发的生物标本，可用于筛选导致BC病因学的化学物质对DNA的损伤。

 DESCRIPTION (provided by applicant): Bladder cancer (BC) is the fourth most common cancer in men with more than 70,000 new cases and 14,680 deaths in 2010. Major risk factors of BC include tobacco smoking and occupational exposure to aromatic amines in the dye and rubber industries. These exposures are thought to account for about 50% of bladder cases. Several epidemiological studies have linked the consumption of well-done cooked meat containing structurally related heterocyclic aromatic amines (HAAs) with BC risk. Endogenous lipid peroxidation products also may contribute to bladder mutagenesis. However, the specific chemicals that contribute to BC are largely unknown. Urine is a promising biospecimen for the analysis of cancer biomarkers. For example, vesicles (exosomes) excreted in urine serve as carriers of genetic information and a novel source of cancer biomarkers. We propose that exfoliated urinary cells also may serve as a non-invasive biospecimen to assess DNA damage of the upper and lower urinary tract. The goal of this grant renewal, "New biomonitoring methodologies to measure DNA adducts in human tissues" is to validate exfoliated urinary cells as a source of specimen to assess exposures to chemicals, some of which may contribute to upper urothelial cancer (UUC) and BC. We will examine DNA adducts formed in exfoliated urinary cells and in urothelial tissues from exposure to environmental, dietary, medicinal, and endogenous genotoxicants. Our preliminary data demonstrate that DNA adducts of aristolochic acid (AA), a urothelial carcinogen found in traditional Chinese medicine (TCM) or as a contaminant of flour, can be quantified in exfoliated urinary cells, by ultraperformance liquid chromatography/multistage scan mass spectrometry (UPLC/MSn), a method with superior sensitivity to that of the commonly used but non-specific 32P-postlabeling assay. We propose to use exfoliated urinary cells as a noninvasive source of biospecimen to assess DNA damage, using UPLC/MSn, in the upper urothelium and/or bladder of subjects at risk of developing UUC and BC. In Aim 1, we examine DNA adducts of AA in urothelial tissues and exfoliated urothelial cells of subjects exposed to AA via TCM or contaminated bread. In Aim 2, we establish techniques to measure DNA adducts of other genotoxicants, that can damage bladder, including aromatic amines, HAAs, and lipid peroxides in the urothelium and urinary cells of mice exposed to these chemicals or to tobacco smoke, using targeted and "adductomics" scanning approaches. In Aim 3, MS methods are applied to measure DNA adducts formed in urothelium and exfoliated urinary cells of patients who have undergone cystectomy for BC, and urinary cells of non-smokers and smokers. Our proposed research is highly relevant to the NIEHS mission, which is to understand how environmental exposures contribute to health outcomes. Exfoliated urinary cells are an untapped biospecimen that can be used to screen for damage to DNA induced by chemicals that contribute to the etiology of BC.