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.

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

“…78 Thus, it seems likely that the protein damage, protein aggregates, ubiquitin depletion, and autophagy seen in our study explain the cytotoxicity in airway epithelium that was seen in this and in previous diacetyl inhalation studies. 3,4 However, we saw increased ubiquitin, SQSTM1, and autophagy in intrapulmonary airways with low levels of significant apoptosis and without significant necrosis, suggesting that ubiquitination and autophagy may have controlled the nature of the proteotoxic cell death. Indeed, autophagy is more frequently a protective biological response than a major contributor to a pathological response.…”

“…4 The respiratory and transitional epithelia of airexposed mice were within normal histological limits (Figure 2A). In contrast, necrosis ( Figure 2B) was significantly increased in the respiratory and transitional epithelia Protein Damage and Diacetyl Cytotoxicity…”

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

“…Current evidence implicates diacetyl as being a critical chemical in toxicity following butter flavoring inhalation, and the pulmonary toxicity of diacetyl has by now been clearly demonstrated (Hubbs et al, 2002(Hubbs et al, , 2008(Hubbs et al, , 2010Morris and Hubbs, 2009;Morgan et al, 2008Morgan et al, , 2012bPalmer et al, 2011). One or more of the other flavorings in the mixture, however, may contribute to the overall toxicity of butter flavoring.…”

“…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).…”

European Tobacco Product Directive: How to address characterizing flavors as a matter of attractiveness?

“…Importantly, it remains to be clarified whether additives that might trigger novel risks which are usually not associated with tobacco products, should be regulated under these provisions. One important example is diacetyl (i.e., butane-2,3-dione), a flavor that can trigger certain respiratory diseases (e.g., bronchiolitis obliterans) when being inhaled (Kreiss et al 2002;Morgan et al 2008Morgan et al , 2012). Yet, until now there are no such studies for tobacco products available which could prove or exclude measureable effects at the stage of consumption.…”