Date of Graduation


Document Type


Degree Type



School of Pharmacy


Pharmaceutical Sciences

Committee Chair

Jeffrey S Fedan

Committee Co-Chair

James M Antonini

Committee Member

Richard D Dey

Committee Member

Ann F Hubbs

Committee Member

Jamal Mustafa


"Popcorn workers' lung" (PWL) is a bronchiolitis obliterans-like obstructive disease caused by inhalation of butter flavoring vapor during the manufacture of microwave popcorn. Lung function tests of employees at several microwave popcorn plants revealed that the severity of abnormal lung function correlated with increasing cumulative exposure to the butter flavorant, diacetyl. Recently, the diacetyl substitute, 2,3-pentanedione, has also been identified as a respiratory hazard. The National Institute for Occupational Safety and Health (NIOSH) demonstrated in rats that inhalation of occupationally-relevant concentrations of diacetyl and 2,3-pentanedione vapor for 6 h resulted in airway epithelial necrosis and apoptosis in the nasal passages 18 h after exposure. Anatomical differences in the airways exist between animals and humans, which, in turn, give rise to different sites of lung injury after flavoring exposure. However, a consistent feature from these studies is the demonstration that the airway epithelium is a major target of injury after flavoring exposure.;Our hypothesis was, diacetyl and 2,3-pentanedione damage airway epithelium by altering epithelial ion transport and tight junction integrity, and modifying airway smooth muscle (ASM), contributing to initial pathophysiological events leading to flavoring-induced lung disease. The focus of the first specific aim was to investigate concentration-response relationships for flavoring induced toxicity in rats, assessing changes in respiratory mechanics and reactivity to methacholine (MCh). There was negligible change in vivo in basal lung resistance (RL) and basal dynamic compliance (C dyn) 18 h after a 6-h inhalation exposure to diacetyl or 2,3-pentanedione (100--360 ppm). Despite evidence of substantial epithelial damage in upper airways of the rat, reactivity to MCh was not increased after flavoring exposure but was slightly decreased. Other flavorings, such as acetic acid and acetoin, are often present in abundant amounts in butter flavoring vapor (Boylstein et al., 2006; van Rooy et al., 2007, 2009). Reactivity to MCh was also slightly decreased in mixed flavoring exposures diacetyl (250 ppm) + acetoin (150 ppm) + acetic acid (27 ppm). In order to evaluate epithelium function and integrity as well as effects on ASM, the rat isolated, perfused trachea preparation (IPT) was employed. Diacetyl exposure caused essentially no effect on reactivity to mucosally-applied MCh 18 h after 6 h exposure; in contrast, 2,3-pentanedione (320 and 360 ppm) increased reactivity to MCh. To further investigate the effects of flavoring on ASM, we assessed diacetyl and 2,3-pentanedione (≥3 mM) applied to the serosal and mucosal surfaces of intact and denuded tracheas in IPT, which resulted in ASM relaxation independent of the presence of epithelium. The purpose of the second specific aim was to investigate the potential mechanisms of this flavoring-induced relaxation. Using precontracted rat tracheal strips (MCh; 3x10 -5 M), we investigated the potential involvement of bitter taste receptors (TAS2Rs) in denatonium (1 mM) and flavoring-induced relaxant responses of ASM and bioelectric responses of tracheal epithelial cells. For the first time, it was demonstrated that the TAS2R agonist, denatonium, induces ASM relaxation in rat tracheal strips via an iberiotoxin-dependant pathway. Flavoring-induced relaxation and reduced short-circuit current (ISC) were not mediated by TAS2R. The purpose of the third specific aim was to understand better the bioelectric responses caused by diacetyl and 2,3-pentanedione vapor on the airway epithelium and to determine toxic flavoring concentrations. Malfunctions of pulmonary epithelial ion transport processes and, therefore, impairment of the liquid balance in the airways is associated with severe respiratory diseases, such as cystic fibrosis and pulmonary edema. Normal human bronchial/tracheal epithelial cells (NHBEs) were exposed to flavoring vapor using a custom-made apparatus and then assessed for changes in ion transport. Concentrations at 60 ppm and above resulted in cell death. Ussing chamber studies indicated that 6 h of flavoring exposure at 25 ppm significantly reduced amiloride (3.5x10-5 M)-sensitive Na + transport but not NPPB (10-4 M)-sensitive Cl - transport at a 0 h post-exposure time point. Thus, our results indicated that flavoring-induced loss of apical Na+ conductance. These studies helped to clarify the direct effects of the flavorings on the epithelium as well as potentially identifying their initial pathophysiological effects. In addition, we demonstrated that NHBEs metabolize diacetyl and 2,3-pentanedione vapors during exposure.;This dissertation begins with an overall literature review in Chapter 1, with the primary focus on inhalation exposure to diacetyl and 2,3-pentanedione vapor, resulting in PWL. Chapters 2, 3, and 4, focus on each Specific Aim, our results, and the comparison of diacetyl and 2,3-pentanedione as well as possible underlying mechanisms of toxicity. Chapter 5 is an extension of Chapter 4 and describes an emerging study of diacetyl and 2,3-pentanedione metabolism by epithelial cells. Finally, the general discussion is found in Chapter 6. (Abstract shortened by UMI.).