Reactive Metabolite-induced Protein Glutathionylation: A Potentially Novel Mechanism Underlying Acetaminophen Hepatotoxicity*

James Chun Yip Chan, Alex Cheow Khoon Soh, Dorinda Yan Qin Kioh, Jianguo Li, Chandra Verma, Siew Kwan Koh, Roger Wilmer Beuerman, Lei Zhou, Eric Chun Yong Chan

Although covalent protein binding is established as the pivotal event underpinning acetaminophen (APAP) toxicity, its mechanistic details remain unclear. In this study, we demonstrated that APAP induces widespread protein glutathionylation in a time-, dose- and bioactivation-dependent manner in HepaRG cells. Proteo-metabonomic mapping provided evidence that APAP-induced glutathionylation resulted in functional deficits in energy metabolism, elevations in oxidative stress and cytosolic calcium, as well as mitochondrial dysfunction that correlate strongly with the well-established toxicity features of APAP. We also provide novel evidence that APAP-induced glutathionylation of carnitine O-palmitoyltransferase 1 (CPT1) and voltage-dependent anion-selective channel protein 1 are respectively involved in inhibition of fatty acid β-oxidation and opening of the mitochondrial permeability transition pore. Importantly, we show that the inhibitory effect of CPT1 glutathionylation can be mitigated by PPARα induction, which provides a mechanistic explanation for the prophylactic effect of fibrates, which are PPARα ligands, against APAP toxicity. Finally, we propose that APAP-induced protein glutathionylation likely occurs secondary to covalent binding, which is a previously unknown mechanism of glutathionylation, suggesting that this post-translational modification could be functionally implicated in drug-induced toxicity.