Application of A Dynamic Model of ApoE-/- Mouse Atherosclerosis to Assess the Impact of Cigarette Smoke Exposure on Endothelial Cell Function


Authored by  JND Battey, N Sierro, NV Ivanov

Presented at ISSX 2013     

Abstract

The lung is the predominant target organ for numerous types of chemically-induced damage related to smoking, due to its direct exposure to aerosol constituents. Although the lung has significant capability for biotransforming such compounds with the aim of reducing any potential toxicity, in some instances this biotransformation can result in the generation of more reactive, and often more toxic, metabolites. Knowledge regarding these reactive metabolites will enable the establishment of a link between potentially harmful smoke constituents and disease relevant mechanisms or pathways (i.e., oxidative stress, inflammation, apoptosis…). Due to the instability and therefore short lifetime of reactive electrophilic intermediates, a widely accepted approach for their analysis is by trapping with nucleophiles to generate more stable adducts and subsequent measurement by liquid chromatography coupled to mass spectrometry (LC/MS). Well established procedures using glutathione as a nucleophile for trapping soft electrophiles are often chosen due to a direct relevance for biological systems. However, the use of glutathione has significant limitations associated with the structural elucidation of the conjugated electrophilic compounds, which thereby limits its applicability for generic screening approaches for phase i activation of xenobiotics in complex mixtures. In order to define the most suitable approach for screening, identifying and characterizing reactive intermediates generated by microsomal activation of smoke constituents, four different nucleophiles were evaluated for use within the test system, namely glutathione, cysteine, mercaptoethanol and 3-aminopropan-1-thiol. Reaction kinetics towards Acrolein, applicability for microsomal incubations for phase i oxidation of furan and 2-methylfuran, and the ability to provide structural information using mass-spectrometry were considered for each nucleophile. The nucleophile 3-aminopropan-1-thiol was also assessed for reaction selectivity, demonstrating its ability to distinguish between nonreactive and reactive electrophiles, since there was no information available in the public domain regarding its strength as a nucleophile for this purpose. The ability to perform structural elucidation of reactive intermediates using LC/MS was highly dependent upon the nucleophile used. The structural information that could be derived from each specific type of nucleophile-adduct using high resolution mass spectrometric data was compared. Mass-spectrometric fragmentation patterns, diversity of conjugation reactions, including intramolecular rearrangement reactions, and the feasibility to predict these different nucleophilic conjugates using the in silico metabolite prediction tool meteor nexus were considered.