Respiratory toxicity of diacetyl in C57BL/6 mice

Abstract

Diacetyl, a component of artificial butter flavoring, is a potential etiological agent of obliterative bronchiolitis (OB); however, the toxic dose and mechanisms of toxicity remain controversial. We evaluated the respiratory toxicity of diacetyl in a murine model using several exposure profiles relevant to workplace conditions at microwave popcorn packaging plants. Male C57Bl/6 mice were exposed to inhaled diacetyl across several concentrations and duration profiles, or by direct oropharyngeal aspiration. Effects of diacetyl on the respiratory tract were evaluated by histopathology and BALF analyses. Subacute exposure to 200 or 400 ppm diacetyl for 5 days caused deaths, necrotizing rhinitis, necrotizing laryngitis and bronchitis. Reducing the exposure to 1 h/day (100, 200, 400 ppm) for 4 weeks resulted in less nasal and laryngeal toxicity, but led to peribronchial and peribronchiolar lymphocytic inflammation. A similar pattern was observed with intermittent high-dose exposures at 1200 ppm (15 min, twice a day, 4 weeks). Subchronic exposures to 100 ppm (6 h/day, 12 weeks) caused moderate nasal injury, and peribronchial lymphocytic inflammation accompanied by epithelial atrophy, denudation, and regeneration. Treatment with 400 mg/kg by oropharyngeal aspiration to bypass the nose caused foci of fibrohistiocytic proliferation with little or no inflammation at the junction of the terminal bronchiole and alveolar duct. Depending on the route and duration of exposure, diacetyl causes significant epithelial injury, peribronchial lymphocytic inflammation, or fibrohistiocytic lesions in the terminal bronchioles. Collectively these results indicate that clinically relevant diacetyl exposures result in a pattern of injury that replicates features of human OB.

FIG. 1

Subacute exposure. Inhalation exposure to 400 ppm diacetyl for 4 days (6 h/day) resulted in necrosis of nasal, laryngeal, and mainstem bronchial mucosa. (a) Respiratory epithelium of nasal cavity, Level 1, is inflamed and necrotic (arrows); squamous epithelium (arrowhead) is intact, 4×, hematoxylin and eosin (H&E). (b) Inflammation, necrosis and sloughing of nasal respiratory epithelium, 20×, H&E. (c) Diffuse mucosal necrosis (arrows) of extrapulmonary right mainstem bronchus, 4×, H&E.

FIG. 2

Intermittent low concentration exposure, 1 h/day, 5 days/week for 4 weeks. Diacetyl toxicity for the nasal cavity was reduced by decreasing the exposure duration to 1 h/day for 4 weeks (0, 100, 200, or 400 pm). (a) Acute rhinitis, with inflammatory cells in nasal submucosa (arrows) and suppurative exudate (asterisk), was present in the Level I nasal cavity of mice exposed to 400 ppm, 10×, hematoxylin and eosin (H&E). (b) Moderately severe lymphocytic bronchitis (arrow) was present in the lungs of mice exposed to 400 ppm diacetyl, 10×, H&E.

FIG. 3

Intermittent high concentration exposure (1200 ppm) 15 min two times per day, 5 days/week for 2 weeks. Peribronchiolar lymphocytic inflammation (arrows), preterminal bronchiole, 10×, hematoxylin and eosin.

FIG. 4

Subchronic exposure. Body weights of mice exposed to diacetyl 6 h/day, 5 days/week for 12 weeks and at 6 weeks after termination of the exposure (week 18). Body weights of mice exposed to 100 ppm were significantly decreased from air-exposed (0 ppm) controls (p < 0.05) throughout the study.

FIG. 5

Subchronic exposure. Pulmonary function (Buxco) was measured in a subgroup of mice after 3, 6, and 12 weeks of exposure and at 6 weeks after termination of the exposures (week 18). *Significantly decreased from airexposed (0 ppm) controls (p < 0.05).

FIG. 6

Subchronic exposure. Mice were exposed to 0, 25, 50, 100 ppm diacetyl for 12 weeks (6 h/day, 5 days/week), (a) Mainstem bronchus exhibiting mixed pattern of epithelial atrophy (arrow), denudation (arrowhead), and regenerative hyperplasia with karyomegaly (asterisk). 100 ppm, 10×, hematoxylin and eosin (H&E). (b) Lymphocytic bronchitis was present in some smaller airway branches of the lung. 100 ppm, 10×, H&E.

FIG. 7

Oropharyngeal aspiration. Diacetyl was administered by a single oropharyngeal aspiration. (a) Focal fibrohistiocytic lesions with minimal inflammation were observed at the junction of the terminal bronchiole and alveolar duct (arrows) at 4 days after treatment with 400 mg/kg diacetyl, 10×, hematoxylin and eosin (H&E). (b) The fibrohistiocytic lesions were usually composed of a mixture of spindle cells and histiocyte-like cells (arrow), 20×, H&E. (c) Masson’s trichrome stain for collagen. Within this fibrohistiocytic focus, a few blue fibers suggestive of collagen may be seen. 40×, H&E.