Inhalation of butter flavoring by workers in the microwave popcorn industry may result in “popcorn workers' lung.” In previous in vivo studies rats exposed for 6 h to vapor from the flavoring agents, diacetyl and 2,3-pentanedione, acquired flavoring concentration-dependent damage of the upper airway epithelium and airway hyporeactivity to inhaled methacholine. Because ion transport is essential for lung fluid balance, we hypothesized that alterations in ion transport may be an early manifestation of butter flavoring-induced toxicity. We developed a system to expose cultured human bronchial/tracheal epithelial cells (NHBEs) to flavoring vapors. NHBEs were exposed for 6 h to diacetyl or 2,3-pentanedione vapors (25 or ≥60 ppm) and the effects on short circuit current and transepithelial resistance (Rt) were measured. Immediately after exposure to 25 ppm both flavorings reduced Na+ transport, without affecting Cl− transport or Na+,K+-pump activity. Rt was unaffected. Na+ transport recovered 18 h after exposure. Concentrations (100–360 ppm) of diacetyl and 2,3-pentanedione reported earlier to give rise in vivo to epithelial damage, and 60 ppm, caused death of NHBEs 0 h post-exposure. Analysis of the basolateral medium indicated that NHBEs metabolize diacetyl and 2,3-pentanedione to acetoin and 2-hydroxy-3-pentanone, respectively. The results indicate that ion transport is inhibited transiently in airway epithelial cells by lower concentrations of the flavorings than those that result in morphological changes of the cells in vivo or in vitro.

Abstract"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 (R L ) and dynamic compliance (C dyn ) 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.Employees at microwave popcorn manufacturing factories have developed "popcorn workers' lung" (Department

COREXIT EC9500A (COREXIT) was used to disperse crude oil during the 2010 Deepwater Horizon oil spill. While the environmental impact of COREXIT has been examined, the pulmonary effects are unknown. Investigations were undertaken to determine whether inhaled COREXIT elicits airway inflammation, alters pulmonary function or airway reactivity, or exerts pharmacological effects. Male rats were exposed to COREXIT (mean 27 mg/m(3), 5 h). Bronchoalveolar lavage was performed on d 1 and 7 postexposure. Lactate dehydrogenase (LDH) and albumin were measured as indices of lung injury; macrophages, neutrophils, lymphocytes, and eosinophils were quantified to evaluate inflammation; and oxidant production by macrophages and neutrophils was measured. There were no significant effects of COREXIT on LDH, albumin, inflammatory cell levels or oxidant production at either time point. In conscious animals, neither breathing frequency nor specific airway resistance were altered at 1 hr, 1 d and 7 d postexposure. Airway resistance responses to methacholine (MCh) aerosol in anesthetized animals were unaffected at 1 and 7 d postexposure, while dynamic compliance responses were decreased after 1 d but not 7 d. In tracheal strips, in the presence or absence of MCh, low concentrations of COREXIT (0.001% v/v) elicited relaxation; contraction occurred at 0.003-0.1% v/v. In isolated, perfused trachea, intraluminally applied COREXIT produced similar effects but at higher concentrations. COREXIT inhibited neurogenic contractile responses of strips to electrical field stimulation. Our findings suggest that COREXIT inhalation did not initiate lung inflammation, but may transiently increase the difficulty of breathing.

Nerve growth factor (NGF) is overexpressed in patients with inflammatory lung diseases, including virus infections. Airway surface liquid (ASL), which is regulated by epithelial cell ion transport, is essential for normal lung function. No information is available regarding the effect of NGF on ion transport of airway epithelium. To investigate whether NGF can affect ion transport, human primary air‐interface cultured epithelial cells were placed in Ussing chambers to obtain transepithelial voltage (−7.1 ± 3.4 mV), short‐circuit current (Isc, 5.9 ± 1.0 μA), and transepithelial resistance (750 Ω·cm2), and to measure responses to ion transport inhibitors. Amiloride (apical, 3.5 × 10−5 mol/L) decreased Isc by 55.3%. Apically applied NGF (1 ng/mL) reduced Isc by 5.3% in 5 min; basolaterally applied NGF had no effect. The response to amiloride was reduced (41.6%) in the presence of NGF. K‐252a (10 nmol/L, apical) did not itself affect Na+ transport, but it attenuated the NGF‐induced reduction in Na+ transport, indicating the participation of the trkA receptor in the NGF‐induced reduction in Na+ transport. PD‐98059 (30 μmol/L, apical and basolateral) did not itself affect Na+ transport, but attenuated the NGF‐induced reduction in Na+ transport, indicating that trkA activated the Erk 1/2 signaling cascade. NGF stimulated phosphorylation of Erk 1/2 and the β‐subunit of ENaC. K‐252a and PD‐98059 inhibited these responses. NGF had no effect on Isc in the presence of apical nystatin (50 μmol/L). These results indicate that NGF inhibits Na+ transport through a trkA‐Erk 1/2‐activated signaling pathway linked to ENaC phosphorylation.

RATIONALE“Popcorn workers’ lung” is a life‐threatening, fixed obstructive pulmonary disease caused by inhalation of butter flavoring vapor. Histological evidence from animals exposed by inhalation to the butter flavorings, diacetyl (2,3‐butanedione), and a diacetyl substitute, 2,3‐pentanedione (acetyl proprionyl), demonstrated vapor‐concentration‐dependent damage to epithelial cells. Because nothing is known about the effects of diacetyl and 2,3‐pentanedione on airway smooth muscle (ASM), we investigated the pharmacological activity of these two flavorings on isolated airways.METHODSIn rat isolated, perfused tracheas, the contractile and relaxant effects of diacetyl and 2,3‐pentanedione applied to the serosal and mucosal surfaces of intact and denuded contracted (MCh; 3×10−5 M) tracheas were evaluated.RESULTSConcentrations of both agents greater than 3 mM resulted in ASM relaxation. Relaxation of the trachea by diacetyl and 2,3‐pentanedione was not dependent upon the presence of the epithelium.CONCLUSIONSOur findings indicate that both flavorings are similar in their effects as ASM relaxants.

COREXIT® EC9500A (CE) was used to disperse crude oil during the 2010 Deepwater Horizon oil spill response. While the environmental impact of CE has been examined, the pulmonary effects of CE are unknown. Here we investigated whether inhaled CE elicits airway inflammation, alters pulmonary function, or affects airway reactivity to methacholine (MCh). Rats were exposed to CE (mean 27 mg/m3, 5 h) and examined 1 and 7 d post‐exposure. Under anesthesia, the lung lavage fluid was harvested. Lactate dehydrogenase (LDH) and albumin (A) were measured as indices of lung injury, recovered lung cells were differentiated (macrophages, neutrophils, lymphocytes, and eosinophils) to evaluate inflammation, and oxidant production by macrophages was measured. There were no significant differences in LDH, A, inflammatory cell levels or oxidant production between air‐ and CE‐exposed groups at either time point. Pulmonary function assessed in conscious animals indicated that neither breathing frequency nor specific airway resistance were altered at 1 and 7 d post‐exposure. Airway resistance responses to MCh aerosol in anesthetized animals were unaffected at 1 and 7 d post‐exposure, while dynamic compliance responses were decreased at 1 d but not at 7 d post‐exposure. Our findings suggest that CE inhalation does not initiate lung inflammation, but may transiently increase the difficulty of breathing.

Oil dispersant COREXIT® EC9500A (CE) was used to reduce the environmental impact of the Deepwater Horizon oil spill in 2010. Little is known of the pulmonary effects of CE on airway smooth muscle and nerves. We investigated in vitro the effects of CE on rat tracheal preparations. In tracheal strips, CE (0.001 %v/v) elicited relaxation. Contraction occurred at higher concentrations (0.003–0.1 %v/v; EC50 0.0031 %v/v). CE (>0.1 %v/v) elicited relaxation of methacholine (MCh; 3×10−5 M)‐contracted strips (EC50 0.39 %v/v). CE (0.003 %v/v) had no effect on MCh‐induced tone but inhibited neurogenic contractile responses of strips to electrical field stimulation (10 Hz). In the isolated, perfused trachea preparation (IPT), in which agents may be applied separately to the mucosal or serosal surface, CE (0.3–10 %v/v) administered to the mucosal bath resulted in contraction. In MCh‐contracted perfused tracheas, mucosal CE (0.1–10 %v/v) elicited contraction followed by relaxation. Thus, CE was less potent in the IPT compared to strips. CE produced a progressive precipitate at >0.1 %v/v. Our results indicate that CE contracts airway smooth muscle and inhibits excitatory neurotransmission. Relaxation at high CE concentrations cannot be distinguished from precipitate formation or osmotic effects.

Nerve growth factor (NGF) is over‐expressed in airways of patients with inflammatory lung diseases. Ion transport by the epithelial cells of the lung is responsible for controlling the periciliary liquid layer, and changes in ion transport can accompany disease. To investigate NGF’s effects on airway epithelium, human primary cultured epithelial cells were placed in an Ussing chamber to measure short‐circuit current (Isc). NGF (1 ng/ml), applied apically, reduced Isc (4.5%) and reduced amiloride (apical, 3.5×10‐5 M) ‐sensitive Na+ transport (control, 52%; NGF‐treated, 38%). To investigate whether the NGF receptor, trkA, is responsible for mediating the responses to NGF, cells were incubated with either the tyrosine kinase inhibitor K‐252a (10 nM, apical) or DMSO (0.004 %) in the absence or presence of NGF. K‐252a did not affect Na+transport, but attenuated the NGF‐induced reduction in Na+ transport. To investigate whether trkA activates the Erk1/2 signaling cascade, which results in the down‐regulation of ENaC, cells were incubated with PD‐98059 (30 µM, apical and basolateral) or DMSO (0.06 %) in the absence or presence of NGF. PD‐98059 did not affect Na+ transport, but attenuated the NGF‐induced reduction in Na+ transport. These results indicate that NGF reduces Na+ transport through a trkA‐Erk1/2 mediated signaling pathway. Grant Funding Source: Supported by NIOSH