Proteo-metabolomics in elucidating mechanisms of acetaminophen toxicity

What is it about?

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. However, the goal of populating the AOP remains elusive due to a lack of mechanistic understanding of the cellular targets and downstream events, which can include activation of signalling cascades, changes in protein post-translational modifications (PTMs), altered gene or protein expression levels, perturbations in organelle function and morphology, etc. In the absence of definitively identified toxicological targets, multi-omics approaches may provide mechanistic insight to pinpoint critical targets and perturbed pathways.

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Why is it important?

The hepatotoxicant acetaminophen (APAP) has long been known to trigger covalent protein binding, yet these do not result in appreciable alterations in protein function that can be causally linked to the toxicity features elicited by APAP. 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 GluICAT proteomic methodology to globally profile aberrant glutathionylation temporally using human HepaRG 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. 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.

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