Carcinogen DNA adduct biomarkers in formalin fixed tissues

描述（由申请人提供）：DNA加合物代表内部剂量计，用于测量暴露于环境和内源性基因毒物。不幸的是，在流行病学研究中，DNA加合物的测量常常由于缺乏新鲜组织而被排除在外。相比之下，临床上诊断的福尔马林固定石蜡包埋（FFPE）组织通常是可获得的。在FFPE组织中DNA加合物的筛选主要局限于免疫组织化学（IHC），当抗体可用时。然而，免疫组化的一个重要缺点是，许多抗体，甚至单克隆抗体，对DNA加合物的特异性是不确定的，因为它们可以与其他DNA病变交叉反应，导致鉴定和定量错误。我们最近报道了马兜铃酸- i （AA-I）的DNA加合物可以通过质谱（MS）方法在人FFPE肾脏中以与新鲜冷冻组织相当的灵敏度水平进行测量。AA-I是一种在马兜铃属草本植物中发现的致癌化合物，其中许多已在世界范围内用于药用。我们建议调整我们的DNA检索方法，并证明其他环境和饮食基因毒物的DNA加合物可以在FFPE组织中测量。FFPE组织中致癌物质DNA加合物的鉴定可能为怀疑环境原因的人类癌症的起源提供线索。本提案的目的是通过分析三种重要的人类致癌物：芳香胺，多环芳烃（PAHs）和n -亚硝基化合物的DNA加合物来展示我们的DNA检索方法的多功能性。这些化学物质存在于环境中和/或产生于烟草烟雾中。这些致癌物的主要DNA加合物有很好的特征。芳香胺、多环芳烃和N-亚硝基化合物在DNA碱基的不同亲核位点形成加合物，因此，我们选择它们来评估我们从FFPE组织中提取DNA的方法的通用性。在目的1中，我们将在暴露于4-氨基联苯、苯并[a]芘或4-（甲基亚硝胺）-1-（3-吡啶基）-1-丁酮后，测量几种新鲜冷冻和FFPE啮齿动物组织中的DNA加合物。在Aim 2中，DNA检索方法将用于测量新鲜冷冻和FFPE膀胱和肺组织中的这些DNA加合物，这些组织是暴露于这些化学物质的人类吸烟者的癌症靶器官。一种免疫组化方法将用于筛选人类4- ABP-DNA加合物，从而直接将现有方法与ms进行比较。我们提出的研究与美国国家癌症研究所发布的资助机会公告直接相关。我们的技术将促进大量未开发的生物标本FFPE组织的使用，以测量致癌物质的暴露。在DNA检索技术得到验证后，利用FFPE组织来测量DNA加合物可以加速我们对环境、饮食和烟草中基因毒物暴露的理解，从而用于评估癌症风险和健康差异的流行病学研究。

DESCRIPTION (provided by applicant): DNA adducts represent internal dosimeters to measure exposure to environmental and endogenous genotoxicants. Unfortunately, in epidemiologic studies, measurement of DNA adducts often is precluded by the un- availability of fresh tissue. In contrast, formalin-fixed paraffin embedded (FFPE) tissues with clinical diagnosis are frequently accessible. The screening of DNA adducts in FFPE tissue has been largely limited to immuno- histochemistry (IHC), when antibodies are available. However, an important drawback of IHC is that the specificity of many antibodies, even monoclonal antibodies, for DNA adducts is uncertain as they can cross-react with other DNA lesions, leading to errors in identification and quantification. We recently reported that DNA adducts of aristolochic acid-I (AA-I), can be measured, by mass spectrometric (MS) methods, in human FFPE kidney at a level of sensitivity comparable to that of freshly frozen tissue. AA-I is a carcinogenic compound found in Aristolochia herbaceous plants, many of which have been used worldwide for medicinal purposes. We propose to adapt our method of DNA retrieval and demonstrate that DNA adducts of other environmental and dietary genotoxicants can be measured in FFPE tissues. The identification of carcinogen DNA adducts in FFPE tissue may provide clues to the origin of human cancers for which an environmental cause is suspected. The objective of this proposal is to show the versatility of our DNA retrieval method through the analysis of DNA adducts of three important classes of human carcinogens: aromatic amines, polycyclic aromatic hydro- carbons (PAHs), and N-nitroso compounds. These chemicals occur in the environment and/or arise in tobacco smoke. The major DNA adducts of these carcinogens are well characterized. Aromatic amines, PAHs and N- nitroso compounds form adducts at different nucleophilic sites of DNA bases and thus, have been selected to evaluate the versatility of our method of DNA retrieval from FFPE tissue. In Aim 1, we will measure DNA adducts in several freshly frozen and FFPE tissues of rodents, following exposure to 4-aminobiphenyl, benzo[a]pyrene, or 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. In Aim 2, the DNA retrieval method will be employed to measure these DNA adducts in freshly frozen and FFPE urinary bladder and lung tissues, target organs of cancer in human smokers exposed to these chemicals. An IHC method will be used to screen for 4- ABP-DNA adducts in humans to directly compare current methods to MS. Our proposed research is directly relevant to the Funding Opportunity Announcement issued by the National Cancer Institute. Our technology will advance the usage of a largely untapped biospecimen, FFPE tissue, to measure exposures to carcinogens. Upon validation of the DNA retrieval technology, the employment of FFPE tissue to measure DNA adducts can accelerate our understanding of exposures to genotoxicants in the environment, diet, and tobacco for epidemiologic studies assessing cancer risk and health disparities.