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

Abstract

Inhaled diacetyl vapors are associated with flavorings-related lung disease, a potentially fatal airway disease. The reactive a-dicarbonyl group in diacetyl causes protein damage in vitro. Dicarbonyl/ L-xylulose reductase (DCXR) metabolizes diacetyl into acetoin, which lacks this a-dicarbonyl group. To investigate the hypothesis that flavorings-related lung disease is caused by in vivo protein damage, we correlated diacetyl-induced airway damage in mice with immunofluorescence for markers of protein turnover and autophagy. Western immunoblots identified shifts in ubiquitin pools. Diacetyl inhalation caused dose-dependent increases in bronchial epithelial cells with puncta of both total ubiquitin and K63-ubiquitin, central mediators of protein turnover. This response was greater in Dcxr-knockout mice than in wild-type controls inhaling 200 ppm diacetyl, further implicating the a-dicarbonyl group in protein damage. Western immunoblots demonstrated decreased free ubiquitin in airway-enriched fractions. Transmission electron microscopy and colocalization of ubiquitin-positive puncta with lysosomal-associated membrane proteins 1 and 2 and with the multifunctional scaffolding protein sequestosome-1 (SQSTM1/p62) confirmed autophagy. Surprisingly, immunoreactive SQSTM1 also accumulated in the olfactory bulb of the brain. Olfactory bulb SQSTM1 often congregated in activated microglial cells that also contained olfactory marker protein, indicating neuronophagia within the olfactory bulb. This suggests the possibility that SQSTM1 or damaged proteins may be transported from the nose to the brain. Together, these findings strongly implicate widespread protein damage in the etiology of flavorings-related lung disease. (Am J Pathol 2016, 186: 2887e2908; http://dx

Pulmonary Impairment and Risk Assessment in a Diacetyl-Exposed Population

“…Current data indicate that acetoin is considerably less hazardous than diacetyl and it does not have the reactive α -dicarbonyl group, which has been implicated in the toxicity of diacetyl and 2,3-pentanedione. 40–42 …”

Criteria for a recommended standard: occupational exposure to diacetyl and 2,3-pentanedione.

“…Phagocytosis of olfactory nerve material and increases in Tnfα mRNA were recently demonstrated in the olfactory bulb of mice one day after a 6-hr diacetyl inhalation exposure. By immunofluorescence, the multifunctional scaffolding protein sequestosome-1 accumulated in the olfactory bulb of these mice and often congregated in the microglial cells that contained phagocytized olfactory neuronal material [Hubbs et al 2016].Although powdered butter flavoring can produce fewer vapors than liquid butter flavorings, the powders have a major respirable component Rigler and Longo 2010]. If powdered butter flavorings are substituted for liquid butter flavorings, diacetyl and 2,3-pentanedione vapor concentrations may well be below exposure limits.…”

“…However, current data indicate that acetoin is considerably less hazardous than diacetyl and it does not have the reactive α-dicarbonyl group, which has been implicated in the toxicity of diacetyl and 2,3-pentanedione [Hubbs et al 2016;National Toxicology Program 2015;Zaccone et al 2013].…”

“…Phagocytosis of olfactory nerve material and increases in Tnfα mRNA were recently demonstrated in the olfactory bulb of mice one day after a 6-hr diacetyl inhalation exposure. By immunofluorescence, the multifunctional scaffolding protein sequestosome-1 accumulated in the olfactory bulb of these mice and often congregated in the microglial cells that contained phagocytized olfactory neuronal material [Hubbs et al 2016].…”

“…The data from the National Toxicology Program 90-day inhalation study are available online and was used for the NIOSH animal-based risk assessment (see Chapter 6). Airway damage in mice one day after diacetyl inhalation was found to correlate with markers of increased protein turnover, implicating protein damage in the etiology of diacetylinduced airway damage [Hubbs et al 2016].Eighteen hours after a 6-hour exposure to inhaled diacetyl (100, 200, 300, or 360 ppm), in anesthetized rats 360 ppm elevated slightly lung resistance and dynamic compliance [Zaccone et al. 2013].…”