Diacetyl (2,3-butanedione), a volatile component of butter and butter flavoring, gives food the flavor and aroma of butter, and has been associated with fixed airways obstruction in the microwave-popcorn and flavoring industries. Inhaled diacetyl vapors cause epithelial necrosis in the rat and mouse upper respiratory tract. Many cytotoxic agents cause both apoptotic and necrotic cell death but the role of apoptosis in diacetyl-induced cell death is unknown. Dicarbonyl/xylulose reductase (DCXR) metabolizes diacetyl to acetoin and appears to play a role in mucosal absorption of diacetyl. In human skin, DCXR localizes to the cytoplasm near the adhesion molecules, e-cadherin and beta-catenin. Therefore, we hypothesized that diacetyl causes apoptosis and changes in epithelial adhesion molecules. Lungs were collected from Sprague Dawley rats one day after a 6 h exposure to inhaled air (n=6) or 317 ppm diacetyl vapor (n=6). Apoptosis was assessed by the TUNEL assay and immunofluorescence for activated caspase-3. DCXR, e-cadherin, and beta-catenin were visualized by immunofluorescence and confocal microscopy. Diacetyl increased the number of TUNEL and activated caspase 3 positive cells in mainstem bronchus epithelium. However, most cells in necrotic foci did not exhibit these apoptotic markers. DCXR staining was most intense in terminal bronchiolar cells morphologically consistent with Clara cells. In some necrotic foci in the mainstem bronchus epithelium, the normal thin linear staining of beta-catenin at intercellular junctions was replaced by more focal globular expression. Detaching necrotic cells also often lost beta-catenin and e-cadherin expression. Such alterations may play a role in, or possibly result from, necrosis, apoptosis, and/or epithelial detachment. These findings indicate that both apoptosis and necrosis contribute to diacetyl-induced epithelial injury and suggest that diacetyl may alter intercellular adhesion complexes of respiratory epithelium.
Airborne-particles; Biological-effects; Biological-factors; Cell-morphology; Cellular-reactions; Cell-biology; Cell-damage; Cytology; Exposure-assessment; Exposure-levels; Exposure-methods; Irritants; Inhalation-studies; Laboratory-animals; Lung-cells; Lung-disorders; Lung-irritants; Microscopic-analysis; Microscopy; Particle-aerodynamics; Particulates; Pulmonary-disorders; Pulmonary-function; Pulmonary-system-disorders; Respiratory-irritants; Respiratory-system-disorders; Respiratory-hypersensitivity; Risk-factors; Toxic-effects; Vapors
The Toxicologist. Society of Toxicology 48th Annual Meeting and ToxExpo, Baltimore, Maryland, March 15-19, 2009