Airway Epithelial Injury, Fibroblast Changes and Cytokine/Chemokine Profiles Induced by Exposure of Human EpiAirway-FT Tissues to Diacetyl Vapors

1W.M. Gwinn, 1G.P. Flake, 2R.W. Bousquet and 1D.L. Morgan, 1NTP Laboratory, DNTP, NIEHS, NIH, DHHS, RTP, NC; 2Alion Science and Technology Corp., RTP, NC

Inhalation exposure to diacetyl (DA) vapors in artificial butter flavorings has been associated with the development of obliterative bronchiolitis (OB) in microwave popcorn factory workers. Inhalation of DA vapors has also been shown to cause OB-like lesions in rats. EpiAirway-full thickness (FT) tissue was investigated as an in vitro model to assess the toxicity of DA vapors on functional human airway epithelium with a sub-epithelial collagen matrix containing fibroblasts. EpiAirway-FT tissues were exposed to DA vapors at the air-liquid interface for 1 hr on day 0, 2 and 4. Specifically, 0 (vehicle) or 25 mM DA (50 μl) was applied to vapor cups in order to provide vapor exposure (~1000 ppm) for 1 hr to the apical surface. Culture media was collected every 24 hr in order to measure changes in the production of cytokines/chemokines via multiplex assay in response to DA vapor exposure. On day 6, the tissues were processed for histologic evaluation. Degenerative and regenerative histopathologic changes including decreased ciliated cells and goblet cells, increased apoptosis/necrosis, atrophy/erosion and karyomegaly were observed within the epithelial layer of tissues exposed to DA vapors compared to vehicle-treated controls as well as basal spongiosis. DA vapor-induced stromal changes were also observed which included fibroblast hyperchromasia suggestive of increased fibroblast activity in response to DA vapor treatment. TGFa, MIP-1a, IL-1a, sIL-1Ra, IL-6, IL-8 and MCP-3 were > 3- fold increased in culture media samples collected on day 5 from DA vapor exposed tissues compared to controls. In summary, acute DA vapor exposure induced airway epithelial injury, increased fibroblast activity and alterations in cytokine/chemokine production, all of which occurred in the absence of lymphocyte responses and inflammation. The EpiAirway-FT system can serve as a useful in vitro model for mechanistic studies of diacetyl vapor-induced OB.


Obliterative bronchiolitis, diacetyl (DA) vapors, EpiAirway-full thickness, vapor cups, TGFa, MIP-1a, IL-1a, IL-1Ra, IL-6, IL-8, MCP-3, LDH, IL-10, rantes, eotaxin, VEGF, MCP-1, IL-17a, IL-15, IFNg, GM-SCF, G-CSF, EGF, Alcian blue, basal spongiosis, karyomegaly, hyperchromasia, human 41-plex, vapor exposure

Materials Tested

Diacetyl, 2,3-butanedione, artificial butter flavoring, 2,3-hexanedione

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