New biomonitoring methodologies to measure DNA adducts in human tissues

 描述（由申请人提供）：膀胱癌（BC）是男性中第四大常见癌症，2010年有超过70，000例新发病例和14，680例死亡。BC的主要风险因素包括吸烟和染料和橡胶工业中的芳香胺职业暴露。这些暴露被认为占膀胱病例的约50%。几项流行病学研究表明，食用含有结构相关的杂环芳香胺（哈斯）的全熟熟肉与BC风险有关。内源性脂质过氧化产物也可能导致膀胱突变。然而，导致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生物标本，用于癌症生物标志物的分析。例如，尿液中分泌的囊泡（外泌体）可作为遗传信息的载体和癌症生物标志物的新来源。我们建议，脱落的尿细胞也可以作为一个非侵入性的生物标本，以评估DNA损伤的上，下尿路。这项资助更新的目标是“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细胞作为评估化学品暴露的标本来源，其中一些可能导致上尿路上皮癌（UUC）和BC。我们将检查尿脱落细胞和尿路上皮组织中形成的DNA加合物，这些加合物来自环境、饮食、药物和内源性遗传毒物。我们的初步数据表明，马兜铃酸（AA），尿路上皮致癌物中发现的中药（TCM）或作为污染物的面粉，DNA加合物可以量化脱落的尿细胞，超高效液相色谱/多级扫描质谱（UPLC/MSn），一种具有上级灵敏度的方法，常用的，但非特异性32 P-后标记法。我们建议使用脱落的尿细胞作为生物标本的非侵入性来源，以评估DNA损伤，使用UPLC/MSn，在上尿路上皮和/或膀胱的受试者在发展UUC和BC的风险。目的1：检测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的尿路上皮细胞中AA的DNA加合物。在目标2中，我们建立了技术来测量其他遗传毒物的DNA加合物，可以损害膀胱，包括芳香胺，哈斯，和脂质过氧化物的小鼠暴露于这些化学品或烟草烟雾，使用有针对性的和“内收体”扫描方法的尿道和尿细胞。在目的3中，应用MS方法来测量在已经经历了BC的膀胱切除术的患者的尿道上皮和脱落的尿细胞中形成的DNA加合物，以及非吸烟者和吸烟者的尿细胞。我们提出的研究与NIEHS的使命高度相关，即了解环境暴露如何影响健康结果。脱落的尿细胞是一种未开发的生物标本，可用于筛选导致BC病因学的化学物质引起的DNA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.