Proteomics of flavorings-induced airway disease

描述(申请人提供)：本申请的总体目标是改善因接触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因接触常用的人工调味品而患上职业性呼吸道疾病。特别是，黄油调味品中使用的双乙酰(DA)和戊二酮(PD)与闭塞性毛细支气管炎(BO)的发展有关，BO是一种不可逆转的呼吸道纤维化。因此，NIOSH Nora的目标包括侧重于与工作有关的呼吸道疾病的战略目标，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)和确定毒性机制(目标5.1.3)。该提案直接针对制造业和跨部门呼吸系统疾病的NIOSH目标。它也符合研究到实践(R2P)的目标，将重点领域作为制造业工人面临的主要职业健康问题。该项目的一个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记物。我们的总体假设是，暴露于DA或PD的正常人类支气管上皮(NHBE)细胞的蛋白质信号将为人类生物标记物的开发提供一个有用的发现平台。通过随后验证临床前啮齿动物BO模型中最高表达的蛋白质，我们确定了那些最能转化为相关血液或痰测试的靶点，用于化学调味品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的反应蛋白表达模式，以更好地确定调味细胞毒性的机制，并确定哪些细胞途径被激活以响应这些药物。在支持这一应用的初步研究中，我们已经证实，浓度高达40 mM的DA或PD处理NHBE会导致细胞因子IL-8的产生，并呈剂量依赖关系，对细胞活力的影响最小。我们在DA或PD诱导的职业性BO啮齿动物体内模型中发现肺液中IL-8的平行增加。在当前的应用中，我们将扩展这些初步数据，并1)使用最先进的蛋白质组学分析技术对培养的NHBE对DA或PD做出反应的分泌蛋白质进行全谱量化，并应用生物信息学分析来确定调味剂调节失调的途径；2)测量DA或PD诱导的职业性BO啮齿动物模型中已识别的候选蛋白质，以优先选择工作场所研究的生物标记物。我们提出的基于发现的无偏见方法可能会提供新的生物标记物，可以提高对工作场所风险的早期识别，并为化学调味料毒性的机制提供新的见解，可能为BO的预防或治疗提供新的可操作的靶点。

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.