Elsevier

Toxicology Letters

Volume 338, 1 March 2021, Pages 21-31
Toxicology Letters

Mitochondrial protein adduct and superoxide generation are prerequisites for early activation of c-jun N-terminal kinase within the cytosol after an acetaminophen overdose in mice

https://doi.org/10.1016/j.toxlet.2020.12.005Get rights and content

Highlights

  • Early APAP-induced mitochondrial protein adduct formation precedes JNK activation.

  • Mitochondrial-derived free radicals in cytosol required for initial JNK activation.

  • Spatially directed mitochondrial superoxide release activates cytosolic JNK.

Abstract

Acetaminophen (APAP) overdose is the most common cause of acute liver failure in the United States and formation of APAP-protein adducts, mitochondrial oxidant stress and activation of the mitogen activated protein (MAP) kinase c-jun N-terminal kinase (JNK) are critical for APAP-induced cell death. However, direct evidence linking these mechanistic features are lacking and were investigated by examining the early temporal course of these changes in mice after 300 mg/kg APAP. Protein adducts were detectable in the liver (0.05−0.1 nmol/mg protein) by 15 and 30 min after APAP, which increased (>500 %) selectively in mitochondria by 60 min. Cytosolic JNK activation was only evident at 60 min, and was significantly attenuated by scavenging superoxide specifically in the cytosol by TEMPO treatment. Treatment of mouse hepatocytes with APAP revealed mitochondrial superoxide generation within 15 min, accompanied by hydrogen peroxide production without change in mitochondrial respiratory function. The oxidant stress preceded JNK activation and its mitochondrial translocation. Inhibitor studies identified the putative source of mitochondrial superoxide as complex III, which released superoxide towards the intermembrane space after APAP resulting in activation of JNK in the cytosol. Our studies provide direct evidence of mechanisms involved in mitochondrial superoxide generation after NAPQI-adduct formation and its activation of the MAP kinase cascade in the cytosol, which are critical features of APAP hepatotoxicity.

Introduction

Acetaminophen (APAP) overdose can cause severe liver injury and acute liver failure, which accounts for about 50 % of all cases of ALF in the United States (Rubin et al., 2018). The metabolic activation of APAP generates the reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI), which is detoxified by cellular glutathione (GSH) at therapeutic doses. However, the excessive amount of NAPQI generated after an APAP overdose causes GSH depletion, leading to binding of NAPQI to cellular proteins, especially mitochondrial proteins including ATP synthase, aldehyde dehydrogenase and glutathione peroxidase (Qiu et al., 1998; Jaeschke et al., 2019). APAP overdose also causes early activation of a MAP kinase cascade within the cytosol, ultimately culminating in activation and mitochondrial translocation of c-jun N-terminal kinase (JNK) to the mitochondria (Hanawa et al., 2008; Ramachandran and Jaeschke, 2020). Sustained JNK activation in the cytosol and its mitochondrial translocation causes excessive formation of mitochondrial superoxide, which can react with nitric oxide to produce peroxynitrite, causing nitration of protein tyrosine residues (Knight et al., 2001; Cover et al., 2005). The importance of superoxide in the process is also re-iterated by the aggravation of APAP-induced liver injury in partial manganese superoxide dismutase (MnSOD)-deficient mice (Ramachandran et al., 2011) and by the protection seen against APAP hepatotoxicity with superoxide dismutase mimetics such as Mito-TEMPO (Du et al., 2017, 2019). These mitochondrial changes, including severe sustained oxidative and nitrosative stress ultimately cause mitochondrial permeability transition pore opening, leading to a breakdown of the proton gradient across the membrane, and consequently cell death (Ramachandran and Jaeschke, 2019a,b).

Thus, while formation of mitochondrial NAPQI-protein adducts, JNK activation and mitochondrial dysfunction have been well studied, they have predominantly been investigated at the time of peak APAP-induced injury. While NAPQI-protein adducts on mitochondria have been identified to be critical to hepatocyte injury (Tirmenstein and Nelson, 1989; Xie et al., 2015a), mechanistic insight into how NAPQI protein adduct formation in mitochondria causes MAP kinase cascade activation in the cytosol is not well understood. Early investigation of reactive metabolite targets on mitochondria identified several proteins, including components of the electron transport chain (Qiu et al., 1998), but were again examined at the peak of APAP injury by which time JNK translocation to mitochondria has already taken place. Hence it would be difficult to determine if these alterations occurred prior to JNK translocation or were a consequence of that event. This is important, since mechanisms of NAPQI-adduct induced mitochondrial superoxide production and activation of cytosolic JNK could be distinct from those mediating enhanced reactive oxygen species production and mitochondrial dysfunction after mitochondrial JNK translocation. Mitochondrial superoxide production can have a variety of physiological roles (Sena and Chandel, 2012) and do not necessarily need to cause mitochondrial dysfunction directly. In addition, mitochondrial antioxidant enzymes such as MnSOD would typically scavenge superoxide generated within mitochondria and convert it to non-radicals such as hydrogen peroxide, which has signaling roles of its own (Bao et al., 2009). Thus, the consequences of superoxide formation -whether it results in generation of hydrogen peroxide formation through MnSOD, for example; or generates peroxynitrite through reaction with nitric oxide- would depend on the mitochondrial milieu when the radical is generated. This environment is likely to be different immediately after APAP overdose when NAPQI-adducts are just forming on the mitochondria compared to later time points when activated JNK translocation induces additional signaling cascades. Thus, our study focuses on the very early temporal course of NAPQI-adduct formation and mitochondrial superoxide formation in comparison to JNK activation to gain insight into these very early mechanistic events which initiate APAP hepatotoxicity.

Section snippets

Animals and experimental design

Male C57BL/6J mice (8–12 weeks old) were purchased from Jackson Laboratories (Bar Harbor, ME) and kept in an environmentally controlled room with 12 h dark/light cycle. Experiments were conducted with male mice because female mice, despite similar mechanisms of APAP-induced liver injury, are less susceptible due to an enhanced response to re-synthesize GSH (Du et al., 2014; Masubuchi et al., 2011). C57BL/6 is the most frequently used mouse strain for drug hepatotoxicity research; both

Formation of mitochondrial protein adducts after APAP overdose precedes JNK activation in the cytosol

Initial experiments evaluated the early temporal course of protein adduct formation in liver homogenate and mitochondrial fractions after a moderate APAP overdose (300 mg/kg). Protein adducts were detectable within 15 min after APAP with a slight elevation especially in hepatic mitochondria by 30 min (Fig. 1A). These levels showed significant further increase by 60 min, with mitochondrial adducts per mg protein being higher than those in the liver homogenate. Adducts in both mitochondria and

Early mitochondrial NAPQI-protein adduct formation after APAP overdose precedes cytosolic JNK activation, without peroxynitrite formation

While mitochondrial NAPQI-protein adduct formation, mitochondrial oxidant stress and cytosolic JNK activation have been recognized to be critical early events after APAP overdose (Hanawa et al., 2008), relevant mechanisms linking these early events have not been investigated in detail. This is in contrast to the information on events subsequent to mitochondrial JNK translocation which have been extensively investigated with insight into the JNK binding partner Sab on the mitochondria (Win et

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was supported in part by the National Institutes of Health grants R01 DK102142 (H.J.), P20 GM103549 (H.J.) and a Pilot Grant (A.R.) from the Mechanisms of Liver Injury and Diseases COBRE (NIH P30GM118247, HJ-PI).

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    Present Address: Department of Medicine, Duke University, Durham, North Carolina, USA.

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