Identification of proteins adducted by reactive metabolites of naphthalene and 1-nitronaphthalene in dissected airways of rhesus macaques.

Naphthalene and 1-nitronaphthalene are ambient air pollutants, which undergo P450-dependent bioactivation in the lung. Reactive metabolites of naphthalene and 1-nitronaphthalene covalently bind to proteins, and the formation of covalent adducts correlates with airway epithelial cell injury in rodent models. These studies were designed to identify protein adducts generated from these reactive metabolites within distal respiratory airways. Distal bronchioles and parenchyma from rhesus monkeys were incubated with [(14)C]naphthalene or [(14)C]1-nitronaphthalene. Proteins were separated by 2-DE, blotted to PVDF membranes, and adducted proteins imaged by storage phosphor analysis. MS of in-gel tryptic digests identified numerous adducted proteins including: eight cytoskeletal proteins, two chaperone proteins, seven metabolic enzymes, one redox protein, two proteins involved in ion balance and cell signaling, and two extracellular proteins. While many proteins are adducted by both naphthalene and 1-nitronaphthalene, some are unique to the individual toxicant and airway subcompartment. Although the role which adduction of these proteins plays in cytotoxicity was not evaluated, these studies provide candidate proteins for future work designed to determine the importance of protein adducts in the mechanisms of toxicity and for developing biomarkers useful in determining the relevance of findings in animal models to exposed human populations.

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.

In vivo effects of ozone exposure on protein adduct formation by 1-nitronaphthalene in rat lung.

The incidence of serious photochemical smog events is steadily growing in urban environments around the world. The electrophilic metabolites of 1-nitronaphthalene (1-NN), a common air pollutant in urban areas, have been shown to bind covalently to proteins. 1-NN specifically targets the airway epithelium, and the toxicity is synergized by prior long-term ozone exposure in rat. In this study we investigated the formation of 1-NN protein adducts in the rat airway epithelium in vivo and examined how prior long-term ozone exposure affects adduct formation. Eight adducted proteins, several involved in cellular antioxidant defense, were identified. The extent of adduction of each protein was calculated, and two proteins, peroxiredoxin 6 and biliverdin reductase, were adducted at high specific activities (0.36-0.70 and 1.0 nmol adduct/nmol protein). Furthermore, the N-terminal region of calreticulin, known as vasostatin, was adducted only in ozone-exposed animals. Although vasostatin was adducted at relatively low specific activity (0.01 nmol adduct/nmol protein), the adduction only in ozone-exposed animals makes it a candidate protein for elucidating the synergistic toxicity between ozone and 1-NN. These studies identified in vivo protein targets for reactive 1-NN metabolites that are potentially associated with the mechanism of 1-NN toxicity and the synergistic effects of ozone.