Altered ProteinS-Glutathionylation Identifies a Potential Mechanism of Resistance to Acetaminophen-Induced Hepatotoxicity

Abstract

Acetaminophen (APAP) is the most commonly used over-thecounter analgesic. However, hepatotoxicity induced by APAP is a major clinical issue, and the factors that define sensitivity to APAP remain unclear. We have previously demonstrated that mice nulled for glutathione S-transferase Pi (GSTP) are resistant to APAP-induced hepatotoxicity. This study aims to exploit this difference to delineate pathways of importance in APAP toxicity. We used mice nulled for GSTP and heme oxygenase-1 oxidative stress reporter mice, together with a novel nanoflow liquid chromatography-tandem mass spectrometry methodology to investigate the role of oxidative stress, cell signaling, and protein S-glutathionylation in APAP hepatotoxicity. We provide evidence that the sensitivity difference between wild-type and Gstp1/2 2/2 mice is unrelated to the ability of APAP to induce oxidative stress, despite observing significant increases in c-Jun N-terminal kinase and extracellular signal-regulated kinase phosphorylation in wildtype mice. The major difference in response to APAP was in the levels of protein S-glutathionylation: Gstp1/2 2/2 mice exhibited a significant increase in the number of S-glutathionylated proteins compared with wild-type animals. Remarkably, these S-glutathionylated proteins are involved in oxidative phosphorylation, respiratory complexes, drug metabolism, and mitochondrial apoptosis. Furthermore, we found that S-glutathionylation of the rate-limiting glutathione-synthesizing enzyme, glutamate cysteine ligase, was markedly increased in Gstp1/2 2/2 mice in response to APAP. The data demonstrate that S-glutathionylation provides an adaptive response to APAP and, as a consequence, suggest that this is an important determinant in APAP hepatotoxicity. This work identifies potential novel avenues associated with cell survival for the treatment of chemical-induced hepatotoxicity.

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

“…While McGarry and colleagues suggested that protein glutathionylation confers a protective effect to Gstp1/2 -/-mice against APAP toxicity (75), it is difficult to compare with our findings for the following reasons. Our findings are drawn from a straightforward comparison between APAP-exposed and control HepaRG cells (rather than against a more complicated background of GSTP knockout).…”

MicroRNA-mediated regulation of glutathione and methionine metabolism and its relevance for liver disease

“…The overexpression of GST in mammalian tumor cells has been implicated in the resistance to various anti-cancer agents and chemical carcinogens [17]. Additionally, in acetaminophen-induced hepatotoxicity recent work has shown that downregulation of GST-Pi could prevent toxicity by increasing S-glutathionylation of GCL, protecting its integrity and increasing GSH synthesis [18,19]. For a detailed view of GSH-regulated antioxidant mechanisms and other enzymatic systems related to GSH the readers are referred to Espinosa-Diez et al [20].…”

Glutathione s-transferase p in glucose homeostasis in mice.

“…The authors concluded that whereas GSTP may be an important determinant of protein S-glutathionylation in response to oxidative/nitrosative stress, its role as a fundamental component of the Sglutathionylation process is limited. Compared with wild-type mice, only a subset of proteins with significantly altered peptide ratios were identified in GSTP-null mice [111].…”

“…Significant enrichment of S-glutathionylated mitochondrial and Krebs cycle proteins suggests an involvement of this modification in energy metabolism processes in vivo. However, little overall difference between the hepatic protein S-glutathionylation profiles between wild-type and GSTP-null mice was demonstrated [111]. The authors concluded that whereas GSTP may be an important determinant of protein S-glutathionylation in response to oxidative/nitrosative stress, its role as a fundamental component of the Sglutathionylation process is limited.…”