Mass isotopomer-guided decluttering of metabolomic data to visualize endogenous biomarkers of drug toxicity

Metabolomics offers the opportunity to uncover endogenous biomarkers that can lead to metabolic pathways and networks and that underpin drug toxicity mechanisms. A novel protocol is presented and discussed that is applicable to drugs which generate urinary metabolites when administered to mice sensitive to its toxicity. The protocol would not apply to drugs that are not metabolized or eliminated by a different route. Separate stable isotope-labeled and unlabeled drug administration to mice is made together with collection of urines from control animals. Untargeted mass spectrometry-based metabolomic analysis of these three urine groups is conducted in addition to principal components analysis (PCA). In the case of unlabeled acetaminophen and [acetyl-²H₃]acetaminophen, each given at a hepatotoxic dose (400 mg/kg i.p.) to the sensitive mouse strain (wild-type 129), the PCA loadings plot showed a distribution of ions in the shape of a “fallen-Y” with the deuterated metabolites in one arm and the paired nondeuterated metabolites in the other arm of the fallen-Y. Ions corresponding to the endogenous toxicity biomarkers sat in the mouth of the fallen-Y. This protocol represents an innovative means to separate endogenous biomarkers from drug metabolites, thereby aiding the identification of biomarkers of drug toxicity. For acetaminophen, increased hepatic oxidative stress, mitochondrial damage, Ca²⁺ signaling, heme catabolism, and saturation of glucuronidation, together with decreased fatty acid β-oxidation and cellular energy dysregulation were all implied from the discovered biomarkers. The protocol can be applied to other drugs and may now be translated to clinical studies.