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Poster Session B26 Cigarettes, E-Cigarettes, Marijuana: Epidemiology and Mechanisms
Monday, May 22 9:15 am – 11:15 am Room 150 B, Walter E. Washington Convention Center
Detection of reactive chemicals and oxidants using an organotypic human airway model with Nrf2 reporter activity: Application to evaluation of tobacco products Abstract 213 Poster A3063
Patrick J. Hayden, George R. Jackson Jr., Amy Hunter, Steven Caughlin, Olivia O’Connell, Jaclyn Foisy and Anna Maione
Introduction/rationale: The Nrf2 transcription factor controls expression of enzymes including glutathione-S-transferase and others that are involved in defense against electrophilic and oxidative damage. In the current work, an organotypic model of human airway epithelium containing a luciferase reporter for Nrf2 activation was developed for use in toxicity screening of inhaled chemicals and mixtures. The Nrf2 reporter model was characterized with 12 reference chemicals. The Nrf2 airway reporter model was also used to investigate electrophilic/oxidant stress induced by whole tobacco smoke and electronic cigarette vapor.
Methods: Primary normal human tracheobronchial epithelial cells (NHBE) from 2 donors were tranduced with a lentiviral vector containing an Nrf2 luciferase reporter with puromycin resistance elements. Stably transduced cells were selected by puromycin resistance, expanded and cyropreserved to produce large pools of reporter-expressing cells which were then utilized to produce well differentiated organotypic airway epithelial models. Organotypic structure and barrier properties of models produced from reporter cells were found to be similar to models produced from untransduced cells, as determined by histological evaluation and barrier assessment. The airway reporter models were exposed to test chemicals by apical application of aqueous solutions containing up to 0.6% DMSO. A smoking machine was utilized to expose the models to whole tobacco smoke or e-cigarette vapor. Luciferase activity was evaluated using a commercial kit and a microplate luminometer. Toxicity was evaluated by LDH release. Dose response experiments were performed to determine a range that spanned non-toxic to moderately toxic concentrations. Test chemicals included: isothiocyanates (sulforaphane), oxidants (H2O2, menadione) and electrophilic chemicals (acrolein, iodoacetamide, nitrobenzylbromide, cinnamaldehyde, dinitrochlorobenze, t-butylhydroquinone), parthenolide, quercetin, and cyclophosphamide (precursor of acrolein metabolite).
Results: Little to no Nrf2 activity was obtained following treatment with sulforaphane, a reversible thiol binding chemical. H2O2 and menadione produced only weak activation over the entire span of doses. However, strongly electrophilic chemicals known to form covalent adducts with cellular biomolecules (acrolein, iodoacetamide, nitrobenzylbromide, cinnamaldehyde, dinitrochlorobenze, t-butylhydroquinone) elicited strong induction of Nrf2. Whole tobacco smoke (3R4F, 1 cigarette) also induced strong Nrf2 activation. E-cigarette vapor (e-Blu, 400 puffs) produced only weak activation.
Conclusions: The results demonstrate that the Nrf2 airway reporter model is a highly sensitive detector of reactive electrophilic chemicals or mixtures including whole tobacco smoke. The model may prove useful for safety evaluation of new generation nicotine delivery products.
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