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Inhaled diacetyl vapors are associated with flavorings-related lung disease, a potentially fatal airway disease. The reactive α-dicarbonyl group in diacetyl causes protein damage in vitro. Dicarbonyl/l-xylulose reductase (DCXR) metabolizes diacetyl into acetoin, which lacks this α-dicarbonyl group. To investigate the hypothesis that flavorings-related lung disease is caused by in vivo protein damage, we correlated diacetyl-induced airway damage in mice with immunofluorescence for markers of protein turnover and autophagy. Western immunoblots identified shifts in ubiquitin pools. Diacetyl inhalation caused dose-dependent increases in bronchial epithelial cells with puncta of both total ubiquitin and K63-ubiquitin, central mediators of protein turnover. This response was greater in Dcxr-knockout mice than in wild-type controls inhaling 200 ppm diacetyl, further implicating the α-dicarbonyl group in protein damage. Western immunoblots demonstrated decreased free ubiquitin in airway-enriched fractions. Transmission electron microscopy and colocalization of ubiquitin-positive puncta with lysosomal-associated membrane proteins 1 and 2 and with the multifunctional scaffolding protein sequestosome-1 (SQSTM1/p62) confirmed autophagy. Surprisingly, immunoreactive SQSTM1 also accumulated in the olfactory bulb of the brain. Olfactory bulb SQSTM1 often congregated in activated microglial cells that also contained olfactory marker protein, indicating neuronophagia within the olfactory bulb. This suggests the possibility that SQSTM1 or damaged proteins may be transported from the nose to the brain. Together, these findings strongly implicate widespread protein damage in the etiology of flavorings-related lung disease., (Copyright © 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

"Popcorn workers' lung" is an obstructive pulmonary disease produced by inhalation of volatile artificial butter flavorings. In rats, inhalation of diacetyl, a major component of butter flavoring, and inhalation of a diacetyl substitute, 2,3-pentanedione, produce similar damage to airway epithelium. The effects of diacetyl and 2,3-pentanedione and mixtures of diacetyl, acetic acid, and acetoin, all components of butter flavoring, on pulmonary function and airway reactivity to methacholine (MCh) were investigated. Lung resistance (RL) and dynamic compliance (Cdyn) were negligibly changed 18 h after a 6-h inhalation exposure to diacetyl or 2,3-pentanedione (100-360 ppm). Reactivity to MCh was not markedly changed after diacetyl, but was modestly decreased after 2,3-pentanedione inhalation. Inhaled diacetyl exerted essentially no effect on reactivity to mucosally applied MCh, but 2,3-pentanedione (320 and 360 ppm) increased reactivity to MCh in the isolated, perfused trachea preparation (IPT). In IPT, diacetyl and 2,3-pentanedione (≥3 mM) applied to the serosal and mucosal surfaces of intact and epithelium-denuded tracheas initiated transient contractions followed by relaxations. Inhaled acetoin (150 ppm) exerted no effect on pulmonary function and airway reactivity in vivo; acetic acid (27 ppm) produced hyperreactivity to MCh; and exposure to diacetyl + acetoin + acetic acid (250 + 150 + 27 ppm) led to a diacetyl-like reduction in reactivity. Data suggest that the effects of 2,3-pentanedione on airway reactivity are greater than those of diacetyl, and that flavorings are airway smooth muscle relaxants and constrictors, thus indicating a complex mechanism.