Severe Airway Epithelial Injury, Aberrant Repair and Bronchiolitis Obliterans Develops after Diacetyl Instillation in Rats

Abstract

BackgroundBronchiolitis obliterans (BO) is a fibrotic lung disease that occurs in a variety of clinical settings, including toxin exposures, autoimmunity and lung or bone marrow transplant. Despite its increasing clinical importance, little is known regarding the underlying disease mechanisms due to a lack of adequate small animal BO models. Recent epidemiological studies have implicated exposure to diacetyl (DA), a volatile component of artificial butter flavoring, as a cause of BO in otherwise healthy factory workers. Our overall hypothesis is that DA induces severe epithelial injury and aberrant repair that leads to the development of BO. Therefore, the objectives of this study were 1) to determine if DA, delivered by intratracheal instillation (ITI), would lead to the development of BO in rats and 2) to characterize epithelial regeneration and matrix repair after ITI of DA.Methods and Main ResultsMale Sprague-Dawley rats were treated with a single dose of DA (125 mg/kg) or sterile water (vehicle control) by ITI. Instilled DA resulted in airway specific injury, followed by rapid epithelial regeneration, and extensive intraluminal airway fibrosis characteristic of BO. Increased airway resistance and lung fluid neutrophilia occurred with the development of BO, similar to human disease. Despite rapid epithelial regeneration after DA treatment, expression of the normal phenotypic markers, Clara cell secretory protein and acetylated tubulin, were diminished. In contrast, expression of the matrix component Tenascin C was significantly increased, particularly evident within the BO lesions.ConclusionsWe have established that ITI of DA results in BO, creating a novel chemical-induced animal model that replicates histological, biological and physiological features of the human disease. Furthermore, we demonstrate that dysregulated epithelial repair and excessive matrix Tenacin C deposition occur in BO, providing new insights into potential disease mechanisms and therapeutic targets.

Accumulation of Ubiquitin and Sequestosome-1 Implicate Protein Damage in Diacetyl-Induced Cytotoxicity

“…81,82 A decrease in Scgb1a1 transcripts has also been described in rats during the development of bronchiolitis obliteranselike changes after intratracheal instillation of diacetyl. 83 We believe that in our study, the decrease in Scgb1a1 mRNA results from a loss of club cells (previously known as Clara cells) in the airways of the diacetyl-exposed Dcxr ko2 mice. Consistent with that interpretation, ultrastructural documentation of autophagy was most prominent in club cells in our study.…”

Popcorn Flavoring Effects on Reactivity of Rat Airways in Vivo and in Vitro

“…This contrasts with intratracheally instilled diacetyl, which resulted in significantly increased airway R L and decreased C dyn in rats 7 d after exposure (Palmer et al, 2011). Even more surprisingly, acute inhalation of the flavorings did not lead to airway hyperreactivity.…”

“…Histological investigation in animal models demonstrated that a 6-h exposure inhalation exposure to diacetyl and 2,3-pentanedione causes some epithelial damage 0 h postexposure, which progresses in intensity by 18 to 20 h postexposure (Hubbs et al, 2002(Hubbs et al, , 2008(Hubbs et al, , 2010(Hubbs et al, , 2012Morris and Hubbs, 2009;Morgan et al, 2008Morgan et al, , 2012aMorgan et al, , 2012bPalmer et al, 2011). In addition, inhaled 2,3-pentanedione was found to result in olfactory neuroepithelial injury in rats (Hubbs et al, 2012), and, interestingly, diacetyl may induce long-lasting neurotoxicity by binding to β-amyloid and interfering with pleated sheet structures (More et al, 2012a).…”

“…The rat has been used to model the bronchiolitis-like changes in the lung following exposures to diacetyl or 2,3-pentanedione (Morgan et al, 2012a;Palmer et al, 2011). Understanding the pulmonary function changes after acute diacetyl exposure in the rat is important because it is an established model of flavorings-related lung disease and the spectrum of flavorings-related lung disease recently expanded to include functionally important pulmonary conditions in addition to bronchiolitis obliterans and pulmonary function (Kreiss et al, 2002;Sahakian et al, 2008).…”

“…While intratracheal instillation of diacetyl is known to alter pulmonary function in rats at 7 d postexposure when aberrant epithelial repair and intraluminal airway fibrosis have developed (Palmer et al, 2011), little is known regarding changes in rat pulmonary function and airway reactivity after acute inhalation exposures to diacetyl, 2,3-pentanedione, or diacetyl-containing mixtures of flavorings. This information is critical to understanding the full spectrum of toxicities associated with these flavorings.…”