Carcinogen DNA adduct biomarkers in formalin fixed tissues

福尔马林固定组织中的致癌物 DNA 加合物生物标志物

• 批准号: 8737541
• 负责人: Robert J. Turesky
• 金额: $ 34.68万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-04 至 2017-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8737541
• 关键词: 4-biphenylamine; Antibodies; Applications Grants; Archives; Aristolochia; Aristolochic Acids; Aromatic Amines; Benzo(a)pyrene; Biological Assay; Biological Markers; Biological Monitoring; Bladder; Bladder Tissue; Butanones; Cancer Etiology; Carbon; Carboxylic Acids; Carcinogen exposure; Carcinogens; Chemical Agents; Chemicals; Clinical; DNA; DNA Adduction; DNA Adducts; DNA Damage; DNA Modification Process; DNA lesion; Development; Diagnosis; Diet; Disease; Dose; Employment; Environment; Environmental Pollution; Epidemiologic Studies; Etiology; Exposure to; Formalin; Freezing; Fresh Tissue; Funding Opportunities; Hazardous Chemicals; Herb; Histocytochemistry; Human; Kidney; Link; Liver; Lung; Malignant Neoplasms; Mass Spectrum Analysis; Measurement; Measures; Methods; Molecular Epidemiology; Monoclonal Antibodies; Mus; Mutagens; National Cancer Institute; Negative Finding; Nitroso Compounds; Organ; Paraffin Embedding; Pathology; Patients; Plants; Process; Rattus; Reagent; Recovery; Reporting; Research; Resources; Retrieval; Risk Assessment; Rodent; Role; Sampling; Site; Smoker; Source; Specificity; Structure of parenchyma of lung; Techniques; Technology; Temperature; Time; Tissue Sample; Tissues; Tobacco; Tobacco smoke; Tobacco smoking; Validation; adduct; analytical method; anticancer research; base; cancer risk; chemical carcinogen; chemical carcinogenesis; clinical Diagnosis; crosslink; epidemiology study; health disparity; liquid chromatography mass spectrometry; new technology; nuclease; prevent; public health relevance; screening; tissue fixing; tumor

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.

描述（由申请人提供）：DNA 加合物代表内部剂量计，用于测量环境和内源性基因毒物的暴露。不幸的是，在流行病学研究中，DNA 加合物的测量常常因无法获得新鲜组织而无法进行。相比之下，具有临床诊断功能的福尔马林固定石蜡包埋 (FFPE) 组织通常很容易获得。当有抗体可用时，FFPE 组织中 DNA 加合物的筛选很大程度上限于免疫组织化学 (IHC)。然而，IHC 的一个重要缺点是，许多抗体（甚至单克隆抗体）对 DNA 加合物的特异性不确定，因为它们可能与其他 DNA 损伤发生交叉反应，导致识别和定量错误。我们最近报道，可以通过质谱 (MS) 方法在人 FFPE 肾脏中测量马兜铃酸-I (AA-I) 的 DNA 加合物，其灵敏度水平与新鲜冷冻组织相当。 AA-I 是在马兜铃草本植物中发现的一种致癌化合物，其中许多马兜铃已在世界范围内用于药用目的。我们建议调整我们的 DNA 检索方法，并证明可以在 FFPE 组织中测量其他环境和饮食遗传毒物的 DNA 加合物。 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类重要的人类致癌物：芳香胺、多环芳香烃 (PAH) 和 N-亚硝基化合物的 DNA 加合物，展示我们的 DNA 修复方法的多功能性。这些化学物质存在于环境中和/或出现在烟草烟雾中。这些致癌物的主要 DNA 加合物已得到充分表征。芳香胺、PAH 和 N-亚硝基化合物在 DNA 碱基的不同亲核位点形成加合物，因此被选择来评估我们从 FFPE 组织中回收 DNA 的方法的多功能性。在目标 1 中，我们将测量接触 4-氨基联苯、苯并[a]芘或 4-(甲基亚硝基氨基)-1-(3-吡啶基)-1-丁酮后的几种新鲜冷冻和 FFPE 啮齿动物组织中的 DNA 加合物。在目标 2 中，将采用 DNA 修复方法来测量新鲜冷冻和 FFPE 膀胱和肺组织以及暴露于这些化学物质的人类吸烟者的癌症靶器官中的这些 DNA 加合物。 IHC 方法将用于筛选人体中的 4-ABP-DNA 加合物，以直接将当前方法与 MS 进行比较。我们提出的研究与国家癌症研究所发布的资助机会公告直接相关。我们的技术将推动很大程度上未开发的生物样本 FFPE 组织的使用，以测量致癌物质的暴露情况。经过 DNA 检索技术的验证，使用 FFPE 组织测量 DNA 加合物可以加速我们对环境、饮食和烟草中遗传毒物暴露的了解，用于评估癌症风险和健康差异的流行病学研究。