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Reaction products of hexamethylene diisocyanate vapors with "self" molecules in the airways of rabbits exposed via tracheostomy.

ABSTRACT: 1.?Hexamethylenediisocyanate (HDI) is a widely used aliphatic diisocyanate and a well-recognized cause of occupational asthma. 2.?"Self" molecules (peptides/proteins) in the lower airways, susceptible to chemical reactivity with HDI, have been hypothesized to play a role in asthma pathogenesis and/or chemical metabolism, but remain poorly characterized. 3.?This study employed unique approaches to identify and characterize "self" targets of HDI reactivity in the lower airways. Anesthetized rabbits free breathed through a tracheostomy tube connected to chambers containing either, O2, or O2 plus ?200 ppb HDI vapors. Following 60 minutes of exposure, the airways were lavaged and the fluid was analyzed by LC-MS and LC-MS/MS. 4.?The low-molecular weight (<3?kDa) fraction of HDI exposed, but not control rabbit bronchoalveolar lavage (BAL) fluid identified 783.26 and 476.18 m/z [M+H]+ ions with high energy collision-induced dissociation (HCD) fragmentation patterns consistent with bis glutathione (GSH)-HDI and mono(GSH)-HDI. Proteomic analyses of the high molecular weight (>3?kDa) fraction of exposed rabbit BAL fluid identified HDI modification of specific lysines in uteroglobin (aka clara cell protein) and albumin. 5.?In summary, this study utilized a unique approach to chemical vapor exposure in rabbits, to identify HDI reaction products with "self" molecules in the lower airways.

SUBMITTER: Wisnewski AV

PROVIDER: S-EPMC5863241 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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Reaction products of hexamethylene diisocyanate vapors with "self" molecules in the airways of rabbits exposed via tracheostomy.

Wisnewski Adam V AV Kanyo Jean J Asher Jennifer J Goodrich James A JA Barnett Grace G Patrylak Lyn L Liu Jian J Redlich Carrie A CA Nassar Ala F AF

Xenobiotica; the fate of foreign compounds in biological systems 20170601 5

1. Hexamethylenediisocyanate (HDI) is a widely used aliphatic diisocyanate and a well-recognized cause of occupational asthma. 2. "Self" molecules (peptides/proteins) in the lower airways, susceptible to chemical reactivity with HDI, have been hypothesized to play a role in asthma pathogenesis and/or chemical metabolism, but remain poorly characterized. 3. This study employed unique approaches to identify and characterize "self" targets of HDI reactivity in the lower airways. Anesthetized rabbit ...[more]

PMID: 28489470

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Project description:BackgroundIsocyanates, a leading cause of occupational asthma, are known to induce adaptive immune responses; however, innate immune responses, which generally precede and regulate adaptive immunity, remain largely uncharacterized.ObjectiveThe aim of the study was to identify and characterize the cellular, molecular and systemic innate immune responses induced by hexamethylene diisocyanate (HDI).MethodsHuman peripheral blood mononuclear cells (PBMCs) were stimulated in vitro with HDI-albumin conjugates or control antigen, and changes in phenotype, gene and protein expression were characterized by flow cytometry, microarray, Western blot and ELISA. Cell uptake of isocyanate was visualized microscopically using HDI-albumin conjugates prepared with fluorescently labelled albumin. In vivo, human HDI exposure was performed via a specific inhalation challenge, and subsequent changes in PBMCs and serum proteins were measured by flow cytometry and ELISA. Genotypes were determined by PCR.ResultsHuman monocytes take up HDI-albumin conjugates and undergo marked changes in morphology and gene/protein expression in vitro. The most significant (P-values 0.007-0.05) changes in microarray gene expression were noted in lysosomal genes, especially peptidases and proton pumps involved in antigen processing. Chemokines that regulate monocyte/macrophage trafficking (MIF, MCP-1) and pattern-recognition receptors that bind chitin (chitinases) and oxidized low-density lipoprotein (CD68) were also increased following isocyanate-albumin exposure. In vivo, HDI-exposed subjects exhibited a drastic increase in the percentage of PBMCs with the same HDI-albumin responsive phenotype characterized in vitro (HLA-DR(+)/CD11c(+) with altered light scatter properties). An exposure-dependent decrease (46+/-11%; P<0.015) in serum concentrations of chitinase 3-like-1 was also observed in individuals who lack the major (type 1) human chitinase (due to genetic polymorphism), but not in individuals possessing at least one functional chitinase-1 allele.ConclusionsPreviously unrecognized innate immune responses to HDI and HDI-albumin conjugates could influence the clinical spectrum of exposure reactions.

Project description:The isocyanate monomer 1,6-hexamethylene diisocyanate (HDI) and one of its trimers, HDI isocyanurate, are airway and skin sensitizers contained in polyurethane paint. The toxic response of cultured skin cells to these compounds was measured by evaluating the isocyanate concentrations at which 50% of the cells die (i.e., lethal concentration 50%, LC50) because the relative toxicity of each form of HDI should be considered when exposure limits of HDI-based paints are set. By using a luminescent ATP-viability assay, we compared the cytotoxic effects of HDI monomer and HDI isocyanurate on cultured human skin cells (keratinocytes, fibroblasts, and melanocytes) after 4-h isocyanate exposures using culture media with varying levels of nutrients in order to also determine the effects of media composition on isocyanate toxicity. Before analysis, experimental wells were normalized to controls containing cells that were cultured with the same vehicle and media. The measured mean LC50 values ranged from 5 to 200 µM across the experimental conditions, in which HDI isocyanurate in protein-devoid media was the most toxic to cells, producing the lowest LC50 values. For HDI monomer, keratinocytes were the most resistant to its toxicity and melanocytes were the most susceptible. However, when exposed to HDI isocyanurate, the opposite was observed, with melanocytes being the most resilient and the keratinocytes and fibroblasts were more susceptible. Depending on the type of skin cells, dose-response data indicated that HDI isocyanurate was 2-6 times more toxic than HDI monomer when using protein-devoid media whereas HDI isocyanurate was 4-13 times more toxic than HDI monomer when protein-rich media was used. Therefore, if the protein-devoid saline medium alone were used for these experiments, then a significant under-estimation of their relative toxicities in protein-rich environments would have resulted. This difference is because HDI monomer toxicity was more attenuated by the presence of protein in the culture media than HDI isocyanurate toxicity. Thus, conclusions based on comparative toxicity studies and consequent inference applied to potential human toxicity can be affected by in vitro culture media conditions. The physiochemical difference in reactivity of the two forms of HDI to biological molecules most likely explains the observed toxicity differences and may have implications for skin penetration, adverse effects like skin sensitization, and systemic responses like asthma. Future studies are warranted to investigate differences in the biological availability, cellular toxicity, and immunologic sensitization mechanisms for HDI monomer and HDI isocyanurate.

Dataset Information

Reaction products of hexamethylene diisocyanate vapors with "self" molecules in the airways of rabbits exposed via tracheostomy.

Publications

Reaction products of hexamethylene diisocyanate vapors with "self" molecules in the airways of rabbits exposed via tracheostomy.

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