Assessment of 2-aminothiazoline-4-carboxylic acid (ATCA) formation and identification of robust serum metabolic biomarkers for acute cyanide exposure in mice fed diets with varying methionine and cystine content.

Accurate assessment of cyanide (CN) exposure is challenging because direct CN measurement is unstable, and the formation of the CN metabolite 2-aminothiazoline-4-carboxylic acid (ATCA) may be influenced by dietary sulfur amino acids, potentially confounding its utility as an exposure indicator. Here, we evaluated the effect of dietary methionine/cystine on ATCA formation and investigated diet-robust endogenous metabolic biomarkers of CN exposure using MS-based metabolomics and bioinformatics. Male ICR mice were fed a control diet, a high-methionine/cystine diet, or a low-methionine/cystine diet for 7 days and then administered CN (5 mg/kg, intraperitoneally) or vehicle. Metabolomics showed that the low-methionine/cystine diet induced a distinct baseline metabolome profile; however, serum ATCA concentrations did not differ significantly among dietary groups after CN exposure, indicating a negligible dietary effect on ATCA biosynthesis under the conditions tested. Multivariate analyses demonstrated clear separation between CN-administered and vehicle cohorts regardless of diet, suggesting the presence of metabolic signatures of CN exposure that are relatively less susceptible to dietary variation. Twenty-five metabolites were identified as candidate biomarkers by multivariate and univariate analyses, and their discriminatory performance was evaluated using a random forest classification model, which supported their specificity for CN exposure. Overall, these results suggest that ATCA formation and selected serum endogenous metabolic biomarkers are minimally influenced by variation in methionine/cystine intake, thereby potentially improving biomarker-based CN exposure assessment.