title: 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 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.
authors.affiliation: Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention, Morgantown, West Virginia. Electronic address: ahubbs@cdc.gov.
authors.forename: Ann F
authors.initials: AF
authors.lastname: Hubbs
authors.affiliation: Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention, Morgantown, West Virginia.
authors.forename: Kara L
authors.initials: KL
authors.lastname: Fluharty
authors.affiliation: Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention, Morgantown, West Virginia; Department of Forensic and Investigative Science, West Virginia University, Morgantown, West Virginia.
authors.forename: Rebekah J
authors.initials: RJ
authors.lastname: Edwards
authors.affiliation: Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention, Morgantown, West Virginia; College of Veterinary Medicine, University of Georgia, Athens, Georgia.
authors.forename: Jamie L
authors.initials: JL
authors.lastname: Barnabei
authors.affiliation: Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention, Morgantown, West Virginia; School of Medicine, West Virginia University, Morgantown, West Virginia.
authors.forename: John T
authors.initials: JT
authors.lastname: Grantham
authors.affiliation: Duke University School of Medicine, Durham, North Carolina.
authors.forename: Scott M
authors.initials: SM
authors.lastname: Palmer
authors.affiliation: Duke University School of Medicine, Durham, North Carolina.
authors.forename: Francine
authors.initials: F
authors.lastname: Kelly
authors.affiliation: Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention, Morgantown, West Virginia.
authors.forename: Linda M
authors.initials: LM
authors.lastname: Sargent
authors.affiliation: Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention, Morgantown, West Virginia.
authors.forename: Steven H
authors.initials: SH
authors.lastname: Reynolds
authors.affiliation: Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention, Morgantown, West Virginia.
authors.forename: Robert R
authors.initials: RR
authors.lastname: Mercer
authors.affiliation: Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention, Morgantown, West Virginia; Centers for Neuroscience, West Virginia University, Morgantown, West Virginia.
authors.forename: Madhusudan P
authors.initials: MP
authors.lastname: Goravanahally
authors.affiliation: Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention, Morgantown, West Virginia.
authors.forename: Michael L
authors.initials: ML
authors.lastname: Kashon
authors.affiliation: Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention, Morgantown, West Virginia.
authors.forename: John C
authors.initials: JC
authors.lastname: Honaker
authors.affiliation: Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention, Morgantown, West Virginia.
authors.forename: Mark C
authors.initials: MC
authors.lastname: Jackson
authors.affiliation: Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention, Morgantown, West Virginia.
authors.forename: Amy M
authors.initials: AM
authors.lastname: Cumpston
authors.affiliation: Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention, Morgantown, West Virginia.
authors.forename: William T
authors.initials: WT
authors.lastname: Goldsmith
authors.affiliation: Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention, Morgantown, West Virginia.
authors.forename: Walter
authors.initials: W
authors.lastname: McKinney
authors.affiliation: Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention, Morgantown, West Virginia.
authors.forename: Jeffrey S
authors.initials: JS
authors.lastname: Fedan
authors.affiliation: Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention, Morgantown, West Virginia.
authors.forename: Lori A
authors.initials: LA
authors.lastname: Battelli
authors.affiliation: Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention, Morgantown, West Virginia.
authors.forename: Tiffany
authors.initials: T
authors.lastname: Munro
authors.affiliation: Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention, Morgantown, West Virginia.
authors.forename: Winnie
authors.initials: W
authors.lastname: Bucklew-Moyers
authors.affiliation: Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention, Morgantown, West Virginia.
authors.forename: Kimberly
authors.initials: K
authors.lastname: McKinstry
authors.affiliation: Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention, Morgantown, West Virginia.
authors.forename: Diane
authors.initials: D
authors.lastname: Schwegler-Berry
authors.affiliation: Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention, Morgantown, West Virginia.
authors.forename: Sherri
authors.initials: S
authors.lastname: Friend
authors.affiliation: Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention, Morgantown, West Virginia; Department of Forensic and Investigative Science, West Virginia University, Morgantown, West Virginia.
authors.forename: Alycia K
authors.initials: AK
authors.lastname: Knepp
authors.affiliation: Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention, Morgantown, West Virginia; Department of Forensic and Investigative Science, West Virginia University, Morgantown, West Virginia.
authors.forename: Samantha L
authors.initials: SL
authors.lastname: Smith
authors.affiliation: Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention, Morgantown, West Virginia.
authors.forename: Krishnan
authors.initials: K
authors.lastname: Sriram
mesh_terms: D000818:Animals; D001343:Autophagy; D003931:Diacetyl / Q000009:adverse effects*; D004847:Epithelial Cells / Q000378:metabolism / Q000473:pathology; D005421:Flavoring Agents / Q000009:adverse effects*; D006801:Humans; D019570:Inhalation Exposure; D008171:Lung Diseases / Q000139:chemically induced / Q000209:etiology* / Q000378:metabolism / Q000473:pathology; D051907:Lysosomal Membrane Proteins / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D017628:Microglia / Q000378:metabolism / Q000473:pathology; D009830:Olfactory Bulb / Q000378:metabolism / Q000473:pathology; D051842:Olfactory Marker Protein / Q000235:genetics / Q000378:metabolism; D012137:Respiratory System / Q000378:metabolism / Q000473:pathology; D000071456:Sequestosome-1 Protein / Q000235:genetics / Q000378:metabolism*; D013401:Sugar Alcohol Dehydrogenases / Q000235:genetics* / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism*
chemical_list: D005421:Flavoring Agents; D051907:Lysosomal Membrane Proteins; D051842:Olfactory Marker Protein; D000071456:Sequestosome-1 Protein; C101343:Sqstm1 protein, mouse; D025801:Ubiquitin; D013401:Sugar Alcohol Dehydrogenases; C023300:L-xylulose reductase; D003931:Diacetyl