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This study addressed the development of a novel biomarker for 2-chlorobenzalmalononitrile (CS) gas exposure. Using liquid chromatographic and mass spectrometric techniques, we found that CS underwent rapid hydrolysis into 2-chlorobenzaldehyde (2-CBA), a highly reactive intermediate that reacted swiftly with endogenous cysteine (Cys) and Cys residues in proteins, producing a stable 2-(2-chlorophenyl)thiazolidine-4-carboxylic acid adduct (ClPh-SPro) in high yield, which may be used as a CS exposure dosimeter. In particular, it was found that most CS was rapidly hydrolyzed under physiologically relevant conditions, with over 90% of CS being converted into 2-CBA in as short as 20 min. The resultant 2-CBA then reacted swiftly with Cys ( k = 0.086 M -1 s -1 ), forming the stable thiazolidine-4-carboxylic acid adduct, which was detected both in the intracellular fluid and in the cell-isolated proteins of CS-exposed lung cells, as well as in purified human serum albumin. It is expected that the results of this study will facilitate exposure assessment for bystanders who may have been exposed to high levels of CS gas unwillingly.

Formaldehyde (FA) is a human carcinogen that is ubiquitous in the ambient environment and also generated endogenously in oxidatively stressed cells. There is accumulated evidence that FA is an etiological agent of leukemia development in humans. To develop a biomarker for FA exposure, we have, in this study, developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) coupled with stable isotope-dilution method to explore the reactivity of FA with glutathione (GSH) in physiologically relevant conditions. Interestingly, analysis of the reaction mixture by LC-MS/MS revealed exposure concentration- and duration-dependent formation of thioproline-glycine (SPro-Gly) produced by reaction of FA with cysteinyl-glycine (Cys-Gly) as a novel metabolite. Furthermore, dose-dependent formation of the thioproline adduct was observed in human cells separately exposed to FA and Fe 2+ -EDTA, a hydroxyl radical source. To the best of our knowledge, this is the first study reporting a thiazolidine carboxylic acid formed by reaction of FA and Cys-Gly is a major metabolite of FA. The results suggest a variety of GSH-derived thiazolidine metabolites may serve as potential biomarkers for FA and oxidative stress exposure, and the developed LC-MS/MS method provides a means for accurate determination of SPro-Gly as a dosimeter of oxidative stress and formaldehyde exposure.