Gwinn1, W.M., Flake1, G.P., Bousquet2, R.W. and Morgan1, D.L. 1Respiratory Toxicology, NTP Laboratories, NTP, NIEHS, NIH, DHHS, RTP, NC; 2Alion Science and Technology Corp., RTP, NC...

Inhalation exposure to diacetyl vapor in artificial butter flavoring has been associated with the development of bronchiolitis obliterans (BO) in microwave popcorn factory workers. Inhalation of diacetyl vapor has also been shown to cause inflammation and BO-like lung lesions in animal models. EpiAirway tissue was investigated as an in vitro model system to assess the toxic effects of diacetyl vapor on functional human airway epithelium. EpiAirway tissue inserts were exposed to diacetyl vapor for 1 hr on day 1, 3, 5 and 7 using vapor cups. Specifically, 0 (vehicle only), 6.25, 12.5, 25 or 50 mM diacetyl (50 μl) was applied to the vapor cup which was then inverted and sealed over the tissue insert for 1 hr. This system allowed exposure of the tissues to the diacetyl vapor but not the diacetyl liquid. The apical surface of the tissue was rinsed with PBS on day 3, 5, 7 and 9. LDH activity in the apical wash (an indicator of cellular injury) increased over time in response to diacetyl vapor exposure. On day 9, tissues from the 6.25, 12.5 and 25 mM diacetyl treatment groups were processed for histologic evaluation (the 50 mM treatment group was processed on day 3 due to severe epithelial injury). In some experiments, tissues were also processed earlier on day 7 (after only 3 vapor exposures) for the 25 mM diacetyl treatment group. Histopathologic changes in 25 and 50 mM diacetyl vaportreated tissues included increased apoptosis, intercellular edema with loss of cell cohesion, karyomegaly, detachment as well as rounding up and loss of ciliated cells compared to vehicle-treated tissue. This study shows that direct exposure of fully-differentiated human airway epithelial tissue to diacetyl vapor causes airway epithelial injury and histopathologic changes, which occur in the absence of inflammation/ lymphocytic infiltration. The EpiAirway system can serve as a useful in vitro model for mechanistic studies of diacetyl vapor-induced pulmonary toxicity.


AIR-100, Apical wash, Apoptosis, Apoptotic bodies, Artificial butter flavoring, Bronchiolitis obliterans, Cell cohesion, Diacetyl vapor, EpiAirway, Intercellular edema, Karyomegaly, LDH activity, Spongiosis, Vacuolization, Vapor cup, Vapor cup method

Materials Tested

2.3-butanedione, Diacetyl

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