Using 2‑(2-Chlorophenyl)thiazolidine-4-carboxylic\nAcid as a Novel Biomarker for 2‑Chlorobenzalmalononitrile Exposure

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