Popcorn flavoring effects on reactivity of rat airways in vivo and 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 (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.

FIGURE 1

Top, typical recordings illustrating stable diacetyl and 2,3-pentanedione levels during a 6-h inhalation exposure. Bottom, typical recordings illustrating stable diacetyl and acetoin and acetic acid levels during a 6-h inhalation exposure. Numbers above the tracings are time-weighted values.

FIGURE 2

Basal values of R_L (left panel) and C_dyn (right panel) after inhalation exposure of rats to air or diacetyl. Left panel: 18 h following 6-h exposure. Air, n = 24; diacetyl, n = 6–9 for each concentration. Right panel: C_dyn. Air: n = 26; diacetyl, n = 6–10 for each concentration. Asterisk indicates significantly different from air-exposed (p < .05).

FIGURE 3

Effect of diacetyl inhalation 18 h following 6-h exposure of rats on reactivity to inhaled MCh. Left panels: diacetyl on effect on R_L responses. Right panels: diacetyl exposure on C_dyn. Asterisk indicates significantly different from air-exposed (p < .05).

FIGURE 4

Effect of 2,3-pentanedione inhalation 18 h following 6-h exposure of rats on reactivity to inhaled. Left panel: R_L responses to MCh after exposure to 120, 240, and 320 ppm 2,3-pentanedione. Right panels: effect on C_dyn. Air: n = 24; 2,3-pentanedione: n = 4–10. Asterisk indicates significantly different from air-exposed (p < .05).

FIGURE 5

Effect of epithelium removal on reactivity to IL-applied MCh in IPT. Left panel: comparison of EL and IL MCh concentration-response curves obtained from epithelium-containing tracheas. EL, n = 6; IL, n = 6. Right panel: responses to MCh in tracheas from which the epithelium was removed. EL, n = 5; IL, n = 5. Asterisk indicates significant difference between EL and IL responses (p < .05).

FIGURE 6

Effect of diacetyl inhalation on reactivity of IPT to IL MCh. MCh EC50 values and maximum responses are summarized in Table 3. Control, n = 50; 100 ppm, n = 10; 200 ppm, n = 9; 300 ppm, n = 14; 360 ppm, n = 8. Asterisk indicates significantly different from air-exposed (p < .05).

FIGURE 7

Effect of 2,3-pentanedione inhalation on reactivity IPT to IL MCh. MCh EC50 values and maximum responses are summarized in Table 3. Control, n = 29; 120 ppm, n = 7; 240 ppm, n = 6; 320 ppm, n = 5; 360 ppm, n = 8. Asterisk indicates EC50s were significantly different from air-exposed (p < .05).

FIGURE 8

Effects of flavorings in MCh-contracted IPT. (A) Representative responses to 1, 3, 10, and 30 mM 2,3-pentanedione added intraluminally (at dots). Note that relaxation followed contraction at 3 mM 2,3-pentanedione. The rat perfused trachea develops spontaneous tone; therefore, the relaxations to both flavorings reduced the level of △P below baseline before MCh addition. Contraction and relaxation responses were also elicited after diacetyl administration and resembled those depicted in (A). (B) The results following IL and EL diacetyl additions are summarized. Diacetyl administered to either EL or IL bath resulted in similar relaxation. IL, n = 8; EL, n = 8. (C) The results following IL and EL 2,3-pentanedione additions are summarized. (D) Comparison of relaxant responses to EL diacetyl and 2,3-pentanedione.

FIGURE 9

Responses of epithelium-denuded IPT to flavorings. (A) Diacetyl applied to the IL bath elicited responses that were similar to what had been observed in intact trachea. (B) Responses to IL 2,3-pentanedione in the absence of the epithelium were not different from responses to IL 2,3-pentanedione in intact trachea; see (A). (C) Comparison of responses to IL diacetyl and 2,3-pentanedione. Diacetyl, n = 5; 2,3-pentanedione, n = 6.

FIGURE 10

Basal values of R_L (left panel) and C_dyn (right panel) after inhalation exposure of rats to air, diacetyl (250 ppm), acetoin (150 ppm), and acetic acid (27 ppm) alone and in combinations involving the three flavorings present in low (“three low”) or high (“hree high”) concentrations. The “three low” concentrations were: 167 ppm diacetyl + 100 ppm acetoin + 18 ppm acetic acid. The “three high” concentrations were: 250 ppm diacetyl + 150 ppm acetoin + 27 ppm acetic acid. Air control animals from the same shipment were run in parallel with the flavoring-exposed animals. The n values for the exposed animals and air controls, respectively, were: diacetyl (250 ppm) alone, 6 and 6; acetoin (150 ppm) alone, 7 and 7; acetic acid (27 ppm) alone, 3 and 7; “three low,” 7 and 7; and “three high,” 6 and 6. Diacetyl (250 ppm) decreased R_L; “three high” increased C_dyn. Asterisk indicates significantly different from air-exposed (p < .05).

FIGURE 11

Effect of inhalation of diacetyl (250 ppm), acetoin (150 ppm) and acetic acid (27 ppm) 18 h following 6-h exposure of rats on reactivity to inhaled MCh. Left panels: diacetyl decreased R_L responses; acetic acid increased R_L responses. Asterisk indicates significantly different from air-exposed (p < .05).

FIGURE 12

Effect of “three low” (top two panels) and “three high” (bottom two panels) mixed exposures 18 h following a 6-h exposure of rats on reactivity to inhaled MCh. The “three high” exposure reduced R_L responses to MCh; both the “three low” and the “three high” exposures increased C_dyn. The n values for the exposed animals and air controls, respectively, were: “three low,” 6 and 6; “three high,” 7 and 7. Asterisk indicates significantly different from air-exposed (p < .05).