Characterization of a structurally intact in situ lung model and comparison of naphthalene protein adducts generated in this model vs lung microsomes.
Airway epithelial cells are a susceptible site for injury by ambient air toxicants such as naphthalene that undergo P450-dependent metabolic activation. The metabolism of naphthalene in Clara cells to reactive intermediates that bind covalently to proteins correlates with cell toxicity. Although several proteins adducted by reactive naphthalene metabolites were identified in microsomal incubations, new methods that maintain the structural integrity of the lung are needed to examine protein targets. Therefore, we developed a method that involves inflation of the lungs via the trachea with medium containing (14)C-naphthalene followed by incubation in situ. The viability of this preparation is supported by maintenance of glutathione levels, rates of naphthalene metabolism, and exclusion of ethidium homodimer-1 from airway epithelium. Following in situ incubation, the levels of adduct per milligram of protein were measured in proteins obtained from bronchoalveolar lavage, epithelial cells, and remaining lung. The levels of adducted proteins obtained in lavage and epithelial cells were similar and were 20-fold higher than those in residual lung tissue. (14)C-Labeled adducted proteins were identified by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry (MS) and quadrupole-TOF MS/MS. Major adducted proteins include cytoskeletal proteins, proteins involved in folding and translocation, ATP synthase, extracellular proteins, redox proteins, and selenium binding proteins. We conclude that in situ incubation maintains structural integrity of the lung while allowing examination of reactive intermediate activation and interaction with target cell proteins of the lung. The proteins adducted and identified from in situ incubations were not the same proteins identified from microsomal incubations.
Lin
Ching Yu
CY
Department of Molecular Biosciences, School of Veterinary Medicine, Molecular Structure Facility, University of California, Davis, California 95616, USA. clin@ucdavis.edu
Isbell
Margaret A
MA
Morin
Dexter
D
Boland
Bridget C
BC
Salemi
Michelle R
MR
Jewell
William T
WT
Weir
Alison J
AJ
Fanucchi
Michelle V
MV
Baker
Gregory L
GL
Plopper
Charles G
CG
Buckpitt
Alan R
AR
D000818:Animals; D002250:Carbon Radioisotopes; D002352:Carrier Proteins / Q000378:metabolism; D002470:Cell Survival; D003598:Cytoskeletal Proteins / Q000378:metabolism; D005109:Extracellular Matrix / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D008168:Lung / Q000378:metabolism*; D008861:Microsomes / Q000378:metabolism*; D025261:Mitochondrial Proton-Translocating ATPases / Q000378:metabolism; D008954:Models, Biological; D009281:Naphthalenes / Q000378:metabolism*; D010084:Oxidation-Reduction; D011487:Protein Conformation; D011506:Proteins / Q000378:metabolism*; D051137:Selenium-Binding Proteins; D014598:Uteroglobin / Q000378:metabolism
D002250:Carbon Radioisotopes; D002352:Carrier Proteins; D003598:Cytoskeletal Proteins; D009281:Naphthalenes; D011506:Proteins; D051137:Selenium-Binding Proteins; C031721:naphthalene; D014598:Uteroglobin; D025261:Mitochondrial Proton-Translocating ATPases; D005978:Glutathione