K.C. Donnelly Externship - Promotion of Translational/Transdisciplinary Efforts in Graduate and Post-Doctoral Research - Colvin

K.C.

• 批准号: 10381310
• 负责人: Robyn L Tanguay
• 金额: $ 1.49万
• 依托单位: OREGON STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-23 至 2022-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10381310
• 关键词: 3-Dimensional; Aromatic Polycyclic Hydrocarbons; Biological Assay; Cell Culture Techniques; Cell Line; Cells; Competence; Cultured Cells; DNA Adducts; DNA Damage; DNA Sequence Alteration; Detection; Diesel Exhaust; Electrophoresis; Enzymes; HepG2; Human; Image; In Vitro; Inhalation Exposure; Learning; Lung; Malignant Neoplasms; Mediating; Metabolic; Methods; Modeling; Nucleotide Excision Repair; Phase; Postdoctoral Fellow; Process; Protocols documentation; Reporting; Research; Techniques; Toxic effect; Training; Wood material; airway epithelium; bronchial epithelium; cigarette smoking; computerized data processing; exposure route; externship; metabolic abnormality assessment; tumor growth

PROJECT DESCRIPTION In brief, polycyclic aromatic hydrocarbons (PAHs) are formed during incomplete combustion processes such as cigarette smoking, diesel exhaust, and wood burning, and some have been associated with several forms of cancer, including lung. PAHs are bioactivated into their reactive metabolites by Phase I metabolizing enzymes in the body to cause DNA damage and mutations leading to tumor growth. The airway epithelium is a primary route of exposure for inhaled PAHs. The use of 3D organotypic culture models are demonstrating promise for use in metabolic studies as compared to traditional in vitro cell culture models that often lack metabolic competency. My proposed project will evaluate the extent of DNA damage mediated by PAH reactive intermediates generated in primary human bronchial epithelial cells cultured in both 2D and 3D organotypic formats as compared to cell lines with reported negative (TK6) and positive (HepG2) results using the newly developed CometChip assay. In particular, we will apply a method for the CometChip that traps single strand breaks formed during nucleotide excision repair enabling detection of PAH-bulky DNA adducts. This project will aid in describing the relevance of the 2D and 3D organotypic models for toxicity studies relating to DNA damage induced by PAH metabolites. The specific objectives of this project are to 1) obtain training in CometChip assay techniques such as chip formation, cell loading, electrophoresis, and imaging protocols using TK6 and HepG2 cells 2) obtain training needed for processing data collected from the CometChip assay and 3) learn to adapt protocols as needed to utilize the assay for 2D and 3D primary NHBE culture models with PAH exposures.

项目描述 简而言之，多环芳烃（PAH）在不完全燃烧过程中形成，例如 吸烟、柴油机尾气和木材燃烧，有些人与几种形式的 癌症，包括肺癌。多环芳烃通过I相代谢酶被生物活化成活性代谢物 在体内引起DNA损伤和突变，导致肿瘤生长。气道上皮细胞是一种 吸入多环芳烃的暴露途径。3D器官型培养模型的使用证明了 与通常缺乏代谢的传统体外细胞培养模型相比， 能力我提议的项目将评估PAH反应性介导的DNA损伤的程度， 在2D和3D器官型培养的原代人支气管上皮细胞中产生的中间体 格式与使用新方法报告阴性（TK 6）和阳性（HepG 2）结果的细胞系相比， 开发了CometChip分析。特别地，我们将应用捕获单链的CometChip的方法 在核苷酸切除修复期间形成的断裂使得能够检测PAH-大体积DNA加合物。该项目将 有助于描述与DNA相关的毒性研究的2D和3D器官型模型的相关性 PAH代谢产物引起的损伤。该项目的具体目标是：1）获得以下方面的培训： CometChip分析技术，如芯片形成、细胞加载、电泳和成像方案 2）获得处理从CometChip测定收集的数据所需的训练 和3）学习根据需要调整方案，以利用2D和3D原代NHBE培养模型的测定， PAH暴露。