A80-2-12 Evaluation of Molecular and Cellular Endpoints After Exposure to Tobacco Constituents in Cell Models Relevant to Oral and Lung Cancers

Abstract Background Smoking is the most common cause of lung cancer and a major risk factor for oral cancers. Approximately half a million deaths occur in the United States every year due to the effects of tobacco product use. Cigarette smoke contains more than 7000 chemicals, of which 250 have been identified as harmful and 70 carcinogenic. The broad objective of our study is to investigate the individual and combined effects of tobacco constituents on cellular and molecular endpoints in oral (OKF4) and bronchial epithelial (HBEC14, HBEC3KT) cell lines that are excellent precursor models for oral and lung cancers in humans. Methods Cigarette smoke condensate (CSC) was derived from the new generation reference cigarette 1R6F from the Tobacco Research and Development Center in the College of Agriculture. Both OKF4 and HBEC14 cell lines were exposed to varying concentrations of CSC over time to determine the viable dose range and examine other cellular and molecular endpoints relevant to carcinogenesis. Results In OKF4, HBEC14, and HBEC3KT cells were mostly viable even at 72 h after CSC treatment for 6 h at concentrations ranging from 0-50 μg/ml. In this dose range, the effects of CSC on cell proliferation and survival were dose-dependent. Higher doses resulted in substantial amounts of cell death in these cells. Alterations in cell morphology were observed starting within 4 hours after CSC treatment, with particularly dramatic morphological changes seen in HBEC14 and HBEC3KT cells at 24 and 48 hours after a single CSC treatment at 30 or 40 μg/ml. To understand these morphological changes more clearly, immunofluorescence and other mechanistic studies are done to assess changes in various protein biomarkers. DNA damage studies were also conducted using the alkaline single-cell gel electrophoresis (Comet) assay protocol. Damage in DNA, as evidenced by changes in the number of cells with detectable Comet tails and the size of these tails, increased in a CSC dose-dependent manner. Conclusion These results provide an initial evaluation of the toxicity and genotoxicity of the new generation reference cigarette, and will serve as a benchmark for other labs using this reference product for studies related to cigarette smoke exposure. This abstract is funded by: Kentucky Tobacco Research and Development Center, University of Kentucky and FDA