Project 4: Urinary Protein Biomarkers for Assessing the Potential Toxicity

肺部疾病是美国人口发病率和死亡率的重要原因。接触空气、水和食物中的化学物质会导致这些疾病。许多化学物质经过生物活化，在啮齿类动物中产生选择性肺损伤。这个项目的重点是存在于超级基金垃圾场的萘（NA）和1-硝基萘（NN），它们代表了一类对环境重要的化合物。这两种化学物质都经过代谢成为亲电中间体，这些中间体与靶细胞中的蛋白质共价结合。蛋白质结合是细胞毒性的关键组成部分。我们已经证明，目标蛋白在啮齿动物和灵长类动物的鼻上皮中是相似的。这强调了开发与毒性紧密相关的生物标志物的必要性。核心假设是：靶呼吸组织中NA/NN反应性代谢物形成关键蛋白加合物与细胞毒性有因果关系；测量尿液和鼻刷中的内合肽/蛋白质提供了暴露和影响的高度敏感的分子特征。/这项提议的工作建立在最近的发现之上：1)在接受过na治疗的小鼠的尿液中有高比活性加合物，2)在小鼠、大鼠和猴子的肺部和鼻腔中有许多已确定的蛋白质加合物。拟议的研究将利用项目3中的抗体从尿液中捕获和浓缩加合肽，并评估动物模型系统中的加合物水平。这些方法将通过加速器质谱法（Core A）对暴露于少量14C-NA后的加合物水平进行评估，并将利用质量对其进行验证

Lung diseases are a significant cause of morbidity and mortality in the US population. Exposure to chemicals in air, water and food contributes to these diseases. A number of chemicals undergo bioactivation to produce selective lung injury in rodents. This project focuses on naphthalene (NA) and 1-nitronaphthalene (NN), which are present in superfund waste sites and which represent a class of environmentally important compounds. Both chemicals undergo metabolism to electrophilic intermediates which become bound covalently to proteins in target cells. Protein binding is a key component of cytotoxicity. We have demonstrated that the proteins targeted are similar in rodents and primate nasal epithelium. This underscores the need to develop biomarkers which are tightly tied to toxicity. The central hypothesis is: /the formation of key protein adducts with reactive metabolites of NA/NN in target respiratory tissue is causally related to cytotoxicity; measurement of adducted peptides/proteins in urine and nasal brushings provides a highly sensitive molecular signature of exposure and effect./ The proposed work builds on recent findings showing 1) high specific activity adducts in urine of NA-treated mice, and 2) numerous identified protein adducts in lungs and nasal cavity of mice, rats and monkeys. The proposed studies will utilize antibodies from project 3 to trap and concentrate adducted peptides from the urine and to evaluate adduct levels in animal model systems. These approaches will be validated for their ability to assess adduct levels following exposures to small amounts of 14C-NA by accelerator mass spectrometry (Core A), will utilize the mass spectrometry facilities of cores A and B for analysis of adducted peptides and will depend heavily on the proteomics services offered by Core B. Overall, these studies are expected to: 1) yield fundamental information on how adducted proteins, formed in respiratory tissues, are handled in the whole animal, 2) explore concentration-response relationships at environmentally relevant concentrations, 3) provide methods which can be applied to exposed human populations, which will be useful-with modification-for assessing risk of other metabolically activated chemicals and 4) provide methods which can clarify the importance of genetic polymorphisms in genes responsible for the biodisposition of chemicals like NA and NN.