Proteomics of flavorings-induced airway disease

描述（由申请人提供）：本申请的总体目标是改善由于暴露于人工香料而在工作场所发生的闭塞性细支气管炎（BO）的诊断和治疗。食品制造行业的工人由于接触常用的人工调味料，患职业性呼吸道疾病的风险很大。特别是，黄油调味剂中使用的二乙酰（DA）和戊二酮（PD）与闭塞性毛细支气管炎（BO）的发展有关，BO是一种不可逆的气道纤维化。因此，NIOSH NORA的目标包括关注与工作有关的气道疾病的战略目标，特别是针对“双乙酰和其他潜在有害的人工香料”的研究，以改进工作场所风险评估（目标5.2.2）并确定毒性机制（目标5.1.3）。该提案直接针对制造业和呼吸系统疾病跨部门的NIOSH目标。它也符合研究到实践（r2p）的目标，针对制造业工人面临的主要职业健康问题的优先领域。本项目的主要目标是确定暴露于人工香料DA和PD后早期肺损伤的新生物标志物。我们的总体假设是，暴露于DA或PD的正常人支气管上皮（NHBE）细胞的蛋白质特征将为人类生物标志物的开发提供一个有用的发现平台。通过随后在临床前啮齿动物BO模型中验证大多数过表达蛋白，我们确定了那些可以最好地转化为相关血液或痰测试的目标，以用于化学香料暴露工人的风险评估。作为第二个目标，我们将使用生物信息学途径分析NHBE细胞对DA或PD的蛋白表达模式，以更好地定义调味剂细胞毒性的机制，并确定哪些细胞途径在响应这些药物时被激活。在支持这一应用的初步研究中，我们已经确定，用浓度高达40毫米的DA或PD处理NHBE会以剂量依赖的方式产生细胞因子白细胞介素(IL)-8，对细胞活力的影响最小。我们证明了DA或pd诱导的啮齿动物职业性BO体内模型中肺液中IL-8的平行增加。在目前的应用中，我们将扩展这一初步数据，1)使用最先进的蛋白质组学分析技术量化培养NHBE对DA或PD的反应的分泌蛋白的全谱，并应用生物信息学分析来确定由调味化学品失调的途径，2)测量血液和肺液中已确定的候选蛋白，DA或PD诱导的职业BO啮齿动物模型优先选择生物标志物进行工作场所研究。我们提出的基于无偏见发现的方法可能提供新的生物标志物，可以提高工作场所风险的早期识别，并为化学调味剂毒性的机制提供新的见解，从而为BO的预防或治疗提供新的可操作目标。

DESCRIPTION (provided by applicant): The overall goal of this application is to improve the diagnosis and treatment of bronchiolitis obliterans (BO) that occurs in the workplace as a result of exposure to artificial flavors. Workers in the food manufacturing industry are at significant rik for occupational airway disease due to exposure to commonly used artificial flavorings. In particular, diacetyl (DA) and pentanedione (PD), components used in butter flavoring, have been linked to the development of bronchiolitis obliterans (BO), an irreversible airway fibrosis. As a result, NIOSH NORA objectives include strategic goals focused on work-related airway diseases, specifically targeting studies of "diacetyl and other potentially harmful artificial flavorings" to improve workplace risk assessment (Goal 5.2.2) and define mechanisms of toxicity (Goal 5.1.3). This proposal directly addresses the NIOSH objectives for the Manufacturing Sector and Respiratory Disease Cross-sector. It is also consistent with the research-to-practice (r2p) goals by targeting an area prioritized as a major occupational health issue facing manufacturing workers. A primary goal of this project is to identify novel biomarkers of early lung injury after exposure to the artificial flavors DA and PD. Our overall hypothesis is that the protein signature from normal human bronchial epithelial (NHBE) cells exposed to DA or PD will provide a useful discovery platform for human biomarker development. By subsequently validating the most overexpressed proteins in a pre-clinical rodent BO model, we identify those targets that can best be translated into relevant blood or sputum tests for risk assessment in chemical flavoring exposed workers. As a secondary goal, we will use bioinformatic pathway analysis of the protein expression pattern of the NHBE cells in response to DA or PD to better define mechanisms of flavoring cellular toxicity, and determine which cellular pathways are activated in response to these agents. In preliminary studies in support of this application we have established that treatment of NHBE with DA or PD in concentrations up to 40 mm results in production of the cytokine interleukin (IL)-8 in a dose dependent manner, with minimal effects on cell viability. We demonstrated parallel increases in IL-8 in the lung flui in DA or PD-induced in vivo models of occupational BO in rodents. In the current application we will extend this preliminary data and 1) quantify the full spectrum of secreted proteins from cultured NHBE in response to DA or PD using state-of-the-art proteomic analysis technology, and apply bioinformatics analysis to determine pathways dysregulated by flavoring chemicals and 2) measure identified candidate proteins in the blood and lung fluid DA or PD-induced rodent models of occupational BO to prioritize selection of biomarkers for workplace studies. Our proposed unbiased discovery based approach is likely to deliver novel biomarkers that could improve early recognition of risk in the workplace and to provide new insights into the mechanisms of chemical flavoring toxicity that could suggest new actionable targets for BO prevention or treatment.