The adverse outcomes pathway (AOP) paradigm describes the events beginning from the initial interaction between a toxicant and its cellular target, which activates a cascade of cellular responses, culminating in cellular injury or death.

Additionally, although many downstream events such as opening of the mitochondrial permeability transition pore (MPTP) and DNA fragmentation have been identified, the upstream triggers of these events are still unknown.

Interestingly, secondary to overt covalent binding, APAP can covertly induce aberrant protein glutathionylation, which is a physiological PTM known to functionally modulate protein activity.

To determine if APAP-induced protein glutathionylation is involved in APAP toxicity, we developed and applied the Glu ICAT proteomic methodology to globally profile aberrant glutathionylation temporally using human Hepa RG cells.

APAP was found to cause aberrant glutathionylation of critical proteins involved in energy metabolism, protein turnover, defense against cellular stress, calcium dynamics and mitochondrial permeability transition pore (MPTP) opening.

In summary, this case study highlights the identification of a novel and previously unrecognized mechanism of drug-induced liver injury through proteomic profiling of drug-induced PTM.

Application of a complimentary metabolomics investigation confirmed that this aberrant PTM resulted in functional deficits that could be ameliorated by appropriate treatments designed to correct the perturbation.Carefully designed multi-omics investigations can yield mechanistic insights that populate the AOP and are translatable to therapeutic solutions in combating drug-induced toxicities.A parallel metabolomics investigation permitted integrative proteo-metabolomics and revealed remarkable coherence in the functional deficits elicited by glutathionylation and corresponding metabolic derangements caused by APAP, with particular emphasis on energy metabolism (glycolysis, Krebs cycle, fatty acid β-oxidation and oxidative phosphorylation).Longitudinal analysis indicated these perturbations occurred within 3-6 h and preceded later events such as opening of the mitochondrial permeability transition pore, which suggests a causative, as opposed to an adaptive role.Separately, fibrate pre-treatment and N-acetylcysteine/S-adenosylmethionine supplementation is known to prevent and reverse APAP toxicity respectively.Integrative proteo-metabolomics revealed that these treatments exerted their effects by correcting the deficits in energy metabolism caused by aberrant glutathionylation, underscoring the causative role of impaired energy metabolism in APAP toxicity.