Research Project
10381310
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.
