Hubbs Ann F, Fluharty Kara L, Edwards Rebekah J, Barnabei Jamie L, Grantham John T, Palmer Scott M, Kelly Francine, Sargent Linda M, Reynolds Steven H, Mercer Robert R, Goravanahally Madhusudan P, Kashon Michael L, Honaker John C, Jackson Mark C, Cumpston Amy M, Goldsmith William T, McKinney Walter, Fedan Jeffrey S, Battelli Lori A, Munro Tiffany, Bucklew-Moyers Winnie, McKinstry Kimberly, Schwegler-Berry Diane, Friend Sherri, Knepp Alycia K, Smith Samantha L, Sriram Krishnan
Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention, Morgantown, West Virginia.
Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention, Morgantown, West Virginia.
Am J Pathol. 2016 Nov;186(11):2887-2908. doi: 10.1016/j.ajpath.2016.07.018.
Inhaled diacetyl vapors are associated with flavorings-related lung disease, a potentially fatal airway disease. The reactive α-dicarbonyl group in diacetyl causes protein damage in vitro. Dicarbonyl/l-xylulose reductase (DCXR) metabolizes diacetyl into acetoin, which lacks this α-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 α-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.
吸入双乙酰蒸汽与调味剂相关的肺部疾病有关，这是一种潜在致命的气道疾病。双乙酰中的反应性α-二羰基基团在体外会导致蛋白质损伤。二羰基/L-木酮糖还原酶（DCXR）将双乙酰代谢为乙偶姻，乙偶姻缺乏这种α-二羰基基团。为了研究调味剂相关的肺部疾病是由体内蛋白质损伤引起的这一假说，我们将双乙酰诱导的小鼠气道损伤与蛋白质周转和自噬标志物的免疫荧光进行了关联。蛋白质免疫印迹法确定了泛素池的变化。吸入双乙酰导致支气管上皮细胞中总泛素和K63-泛素（蛋白质周转的核心介质）的斑点呈剂量依赖性增加。在吸入200 ppm双乙酰的Dcxr基因敲除小鼠中，这种反应比野生型对照更强烈，这进一步表明α-二羰基基团与蛋白质损伤有关。蛋白质免疫印迹法显示气道富集组分中的游离泛素减少。透射电子显微镜以及泛素阳性斑点与溶酶体相关膜蛋白1和2以及多功能支架蛋白sequestosome-1（SQSTM1/p62）的共定位证实了自噬。令人惊讶的是，免疫反应性SQSTM1也在大脑的嗅球中积累。嗅球中的SQSTM1常常聚集在也含有嗅觉标记蛋白的活化小胶质细胞中，表明嗅球内存在噬神经细胞现象。这表明SQSTM1或受损蛋白质可能从鼻子转运到大脑的可能性。总之，这些发现强烈表明广泛的蛋白质损伤与调味剂相关的肺部疾病的病因有关。
