Abstract 4679: A novel assay to predict susceptibility to tobacco-induced disease.

Abstract Background: Tobacco misuse is the leading preventable cause of morbidity and mortality in the world. Tobacco-induced DNA damage is one of the main mechanisms contributing to the pathogenesis of cancer, stroke, heart and pulmonary diseases. Tobacco-induced DNA damage is modulated by genetic and epigenetic factors, as well as life-style choices, and is expected to be a major determinant of the individual susceptibility to tobacco-induced diseases. Technical limitations allow only a few types of DNA damage to be quantified in human samples. Recently, we filled a major methodological gap in the field of DNA damage by developing a novel and highly sensitive primer-anchored DNA damage detection assay (PADDA) to map and quantify in vivo levels of DNA damage. Aims: (1) To standardize PADDA for the detection of oxidative DNA damage, one of the main types of tobacco-induced damage. (2) To define the levels of persistent DNA damage in the oral mucosa of smokers. (3) To determine if persistent nucleotide damage at p53 co-localizes with tobacco-induced cancer mutational hotspots. Methods: To standardize the assay, PADDA was used on a high-throughput setting to quantify DNA damage in oral cell lines exposed to very low doses of hydrogen peroxide. DNA damage was mapped and quantified on the p53 gene by PADDA in oral epithelial cells collected from smokers and non-smokers by oral scrapings. The location of p53 nucleotide damage was compared with reported tobacco-induced p53 cancer mutational hotspots. Saliva cotinine levels were used to confirm smoking status. Data were analyzed by chi-square goodness of fit and exact non-parametric tests. Results: Our data documented PADDA's ability to detect dose-dependent increase in DNA damage in human oral cells. DNA damage was significantly higher in smokers than in non-smokers. Moreover, we documented for the first time that the persistent DNA damage in the oral mucosa of smokers has significantly higher mutagenic potential than that present in non-smokers and persists mainly at p53 nucleotides that are hotspots for mutation in tobacco-induced cancers. Conclusion: PADDA detects dose-dependent DNA damage response, a crucial test of its accuracy and a prerequisite for its use in biomonitoring. PADDA documents the extent of tobacco-induced DNA damage in vivo, and reinforces the importance of smoking cessation. Of major clinical importance, we show, for the first time, that tobacco-induced DNA damage persists preferentially in p53 nucleotides that are hotspots for mutation. Application of this assay to large series of smokers and former smokers has a major potential to establish biomarkers of susceptibility to tobacco-induced disease, which can guide preventive and diagnostic strategies. Funding: This work was supported by the Oklahoma Tobacco Research Center and the Oklahoma Center for the Advancement of Science & Technology. Dr. Queimado holds a Presbyterian Health Foundation Endowed Chair in Otorhinolaryngology. Citation Format: Vengatesh Ganapathy, Wilbur K. Mills, Elangovan Thavathiru, Ilangovan Ramachandran, Leslie Chandler, Antonio Reis, Lurdes VF Queimado. A novel assay to predict susceptibility to tobacco-induced disease. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4679. doi:10.1158/1538-7445.AM2013-4679