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TSRC, Tob. Sci. Res. Conf., 2015, 69, abstr. 24

Nrf2 response to whole smoke in three dimensional (3D) airway cultures

FIELDS W.R.; KEYSER B.M.; BOMBICK B.R.
R.J. Reynolds Tobacco Company, Winston-Salem, North Carolina, USA

Lung diseases are frequently accompanied by molecular changes, including those associated with the Nrf2 signaling pathway. Cigarette smoke has been shown to activate this pathway in lung tissue. The goals of this study were to assess the effect of cigarette smoke on the Nrf2 promoter and on genes associated with oxidative stress, inflammation and metabolism in human 3D EpiAirway™ tissue models (MatTek, Inc.). Whole smoke (WS) exposures (8-64 minutes; 8 minutes/cigarette) with Kentucky Reference 3R4F cigarettes were conducted under ISO conditions using the VITROCELL VC1® smoke exposure system. Viability and tissue integrity were assessed with the lactase dehydrogenase and transepithelial electrical resistance (TEER) assays, respectively. Nrf2 promoter activation was determined by a luciferase assay, while gene expression changes were assessed via QRT/PCR at 6, 12, 18, or 24 hours post-exposure. Dose-dependent decreases in viability and tissue integrity were observed. Cell viability was > 90% for exposures up to 32 minutes (4 cigarettes); while a maximum response of 38% viability and diminished TEER were observed for the 64 minute (8 cigarettes) treatment group. Therefore, luciferase and gene expression studies were conducted between 8 and 32 minutes. Time- and dose-related increases in Nrf2 promoter activation were observed with levels exceeding 200-fold at the 12 and 18 hours post-exposure time-points, and the Nrf2 promoter was also differentially regulated in WS as compared to gas vapor phase exposures. Statistically significant increases (p<0.05) ranging from 2 to >100-fold were observed across the time-course for genes associated with oxidative stress, inflammation and metabolism. Collectively, the data indicate that putative biomarkers of effect in the Nrf2 signaling pathway were responsive to cigarette smoke exposures in human 3D EpiAirway™ tissue models. These models may be useful in evaluating tobacco and aerosol exposures and may further understanding of the biological relevance of the responses.