Diacetyl and 2,3-pentanedione exposure of human cultured airway epithelial cells: Ion transport effects and metabolism of butter flavoring agents

Abstract

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.

Keywords: 2,3-Pentanedione; Diacetyl; Dicarbonyl/l-xylulose reductase; Human airway epithelial cells; Ion transport; Vapor exposure.

Fig. 1

Schematic of the apparatus designed and used for exposing ALI NHBEs to vapors of diacetyl delivered to the apical surface. 1: Gas (5% CO₂ in air) for providing oxygenation of cells and maintaining pH of the culture medium. 2: Passage of gas through sterile, distilled water (37 °C) to humidify the gas to 85% relative humidity. 3: The exposure chamber. 4: Chamber temperature was maintained at 37 °C. The exposure chamber mimicked conditions in the incubator in which the cells were grown and differentiated. 5: Cultured cell plate containing NHBEs rested on a porous stainless steel shelf. 6: Fan to ensure mixing of agent in the chamber. 7: Photoionization detector (PID) for measuring vapor level. The PID withdrew air from the chamber and returned it while measuring vapor level in the chamber. The output of the PID was sent to the proprietary computer software (11) for integration. 8: A pump was activated/deactivated by the computer in response to input from the PID. The pump delivered vapors to the chamber. 9: Vapor source from which vapor is pumped to the exposure chamber upon activation of the pump. The vapor was generated from diacetyl or 2,3-pentanedione liquid placed in the bottom of the vessel. 10: The computer software activated/ deactivated a switch to engage or disengage the pump. 11: Proprietary software was used to monitor vapor levels in the chamber and deliver vapor as needed to maintain the level within 5% of the desired level. 12: The temperature-humidity probe was monitored by computer software.

Fig. 2

R_t of NHBE in ALI culture. R_t values increased and stabilized over time. After raising the cultures in air interface, R_t increased from 105 ± 5 Ω·cm² from day 1 to 816 ± 72 Ω·cm² on day 15. The R_t was relatively stable after 1 week and thereafter.

Fig. 3

Characteristics of cultured NHBEs in ALI. (A) Alcian blue staining for apical mucus (objective magnification = 10×). Bar = 100 μm. (B) Immunostaining for apical β-tubulin (objective magnification = 20×). (C) SEM image of apical cilia. (D) TEM image of cilia, demonstrating the 9 + 2 arrangement of ciliary microtubules.

Fig. 4

Representative bioelectric responses of naïve NHBEs to ion transport blockers amiloride (3.5 × 10⁻⁵ M), NPPB (10⁻⁴ M), and ouabain (10⁻⁴ M). Vertical deflections reflect the I_sc responses to the application of 1 mV pulses across the epithelium for calculation of R_t using Ohm's law.

Fig. 5

SEM image of the apical surface of air-exposed and naïve NHBEs and cells exposed to 25 ppm diacetyl or 2,3-pentanedione. (A) SEM images of control cells, (B) 25 ppm diacetyl-exposed cells and (C) 2,3-pentanedione-exposed cells.

Fig. 6

H&E staining (objective magnification = 100×) illustrating similar morphology between control NHBEs and cells viewed after a 6-h diacetyl (25 ppm) or 2,3-pentanedione (25 ppm) exposure. (A) Unexposed, naïve control NHBEs, (B) ECC cells, (C) cells exposed to diacetyl and (D) cells exposed to 2,3-pentanedione. Bar = 10 μm.

Fig. 7

Effects of diacetyl and 2,3-pentanedione (25 ppm) on bioelectric responses of NHBEs to apically-applied amiloride (3.5 × 10⁻⁵ M) at 0 h and 18 h post-exposure. (A) Responses to amiloride after air or diacetyl (25 ppm) exposure at the 0 h post-exposure time point and (B) the 18 h post-exposure time point. (C) Response to amiloride after 2,3-pentanedione (2,3-Pent; 25 ppm) exposure at the 0 h time point and (D) the 18 h time point. *Significantly different from control.

Fig. 8

SEM images of the apical surface of NHBE control cells and cells exposed to diacetyl (60, 100, and 360 ppm). (A) SEM images of control cells (left panel) compared to 60 ppm diacetyl-exposed cells (right panel). (B) Control cells (left panel) compared to 100 pm diacetyl-exposed cells (right panel). (C) Control cells (left panel) compared to 360 ppm diacetyl-exposed cells (right panel).

Fig. 9

H&E images (objective magnification = 100×) illustrating the effects of diacetyl (100, 200, 300 ppm) on NHBE morphology. (A) Unexposed, control epithelial cells demonstrating the presence of a confluent, pseudo-stratified epithelium. (B) Cells exposed to diacetyl at 100 ppm, (C) 200 ppm and (D) 300 ppm. Bar = 10 μM.

Fig. 10

Identification of metabolites of diacetyl (acetoin; A) and 2,3-pentanedione (2-hydroxy-3-pentanone; B) in basolateral chamber samples following exposure of NHBEs to 25 ppm diacetyl or 2,3-pentanedione. The arrows point to the peaks identified as acetoin (A) or 2-hydroxy-3-pentanone (B). The diacetyl and 2,3-pentanedione peaks are not shown. The identity of the other peaks is not known; they could be impurities or other metabolites of the flavorings.