CHOP is a critical regulator of acetaminophen-induced hepatotoxicity

Background & Aims

The liver is a major site of drug metabolism and elimination and as such is susceptible to drug toxicity. Drug induced liver injury is a leading cause of acute liver injury, of which acetaminophen (APAP) is the most frequent causative agent. APAP toxicity is initiated by its toxic metabolite NAPQI. However, downstream mechanisms underlying APAP induced cell death are still unclear. Endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) have recently emerged as major regulators of metabolic homeostasis. UPR regulation of the transcription repressor CHOP promotes cell death. We analyzed the role of UPR and CHOP in mediating APAP hepatotoxicity.

Methods

A toxic dose of APAP was orally administered to wild type (wt) and CHOP knockout (KO) mice and damage mechanisms were assessed.

Results

CHOP KO mice were protected from APAP induced damage and exhibited decreased liver necrosis and increased survival. APAP metabolism in CHOP KO mice was undisturbed and glutathione was depleted at similar kinetics to wt. ER stress and UPR activation were overtly seen 12 h following APAP administration, a time that coincided with strong upregulation of CHOP. Remarkably, CHOP KO but not wt mice exhibited hepatocyte proliferation at sites of necrosis. In vitro, large T immortalized CHOP KO hepatocytes were protected from APAP toxicity in comparison to wt control cells.

Conclusions

CHOP upregulation during APAP induced liver injury compromises hepatocyte survival in various mechanisms, in part by curtailing the regeneration phase following liver damage. Thus, CHOP plays a pro-damage role in response to APAP intoxication.

Introduction

The liver is a major site for drug metabolism and elimination, and as such is susceptible to drug toxicity [1]. In fact, drug induced liver injury (DILI) has become a leading cause of acute liver failure and transplantation in developed countries [2]. The mechanism of hepatotoxicity associated with most drugs is idiosyncratic and animal models are lacking, making research in this field a challenging task. Only a handful of drugs exhibit type A toxicity, which is characterized by dose dependent tissue damage. Of the various type A hepatotoxic drugs, acetaminophen (N-acetyl-p-aminophenol, APAP) overdose is the leading cause of drug-induced acute liver failure in the western world [3], and one of few hepatotoxic drugs that has been studied in animal models.

APAP toxicity stems from its cytochrome P450-dependent metabolism to N-acetylbenzoquinoneimine (NAPQI). NAPQI is a powerful electrophile and is detoxified by glutathione (GSH). When GSH is depleted, NAPQI accumulates, forming covalent bonds with cysteine groups on hepatocyte macromolecules, thereby interfering with their function [4]. The exact mode of cell death in APAP-induced DILI is still not fully understood. It is clear that APAP toxicity involves massive necrosis of liver parenchyma. However, it is not clear whether apoptosis participates in, and what is the exact role of non-parenchymal cells in the pathogenesis of APAP induced liver injury [5], [6].

While it was initially postulated that following GSH depletion hepatocytes undergo necrosis due to oxidative stress and mitochondrial damage [7], recent evidence indicates the involvement of pro-apoptotic factors in APAP toxicity, such as Bim and CXCR2 [8]. It was further shown that innate immunity via Toll-like receptors (TLRs), dendritic cells, and autophagy are regulators of liver induced damage following APAP overdose [9], [10], [11].

The endoplasmic reticulum (ER) is the major cellular site of protein folding and modification. ER stress occurs when the amount of protein entering the ER exceeds its folding capacity. This imbalance induces a cyto-protective reaction collectively termed the unfolded protein response (UPR) [12]. The mammalian UPR is transduced by three major sensors (IRE1, PERK, and ATF6) that reside in the ER and undergo activation under ER stress conditions. IRE1 and PERK are activated by autophosphorylation, while ATF6 is activated by intra-membrane cleavage, which releases its N-terminal fragment for transcription transactivation. Activated IRE1 splices the mRNA of XBP1 in a non-canonical fashion, yielding the potent transcription factor XBP1s [13], [14]. Activated PERK phosphorylates eIF2α, inducing selective translation of ATF4 and transcription of ATF3 and CCAAT-enhancer-binding protein homologous protein (CHOP) in a sequential manner [15].

Activation of the UPR leads initially to attenuation of protein synthesis and protein translocation into the ER, thus preventing further accumulation of misfolded proteins. This initial step is followed by an increase in the capacity of the ER to handle unfolded proteins. If the stress is not relieved in a timely fashion, cell death is triggered in an intricate mechanism that involves caspase activation, calcium leakage from the ER and mitochondrial damage [16]. One of the main elements in the program of ER stress mediated apoptosis is CHOP, a transcription repressor that is activated downstream of the PERK and IRE1 pathways of the UPR. Once activated, CHOP translocates to the nucleus, inhibits the expression of anti-apoptotic genes, and activates pro-apoptotic genes [17]. CHOP expression in the liver has been demonstrated to respond to various types of stress modules, such as LPS and CCl₄ treatment [18], [19]. Moreover, deletion of CHOP protects mice from various liver-specific challenges, such as diet-induced steatohepatitis, bile duct ligation, and alcohol intoxication [20], [21], [22]. Because CHOP is not restrictively regulated by the UPR, it is not clear whether ER stress is also involved in these types of injury. Nonetheless, these studies implicate CHOP as a factor that mediates liver damage following diverse types of stress responses.

While Nagy et al. observed the induction of ER stress following APAP administration [23], [24], a recent study by Hur et al. did not observe any signs of UPR activation [25]. Both studies administered APAP by i.p. injection, a route not relevant to the clinical use of the drug. Here we followed UPR activation following oral administration of APAP. ER stress and UPR activation were observed as a late event in the cascade of responses activated by APAP and coincided with CHOP upregulation. Furthermore, CHOP knockout, both in vitro and in vivo, conferred a survival advantage, possibly mediated by increased proliferation. Our data implicates CHOP as a critical regulator of APAP toxicity.