Research ArticleCHOP is a critical regulator of acetaminophen-induced hepatotoxicity
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 eIF2Ī±, 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.
Section snippets
Mice and induction of APAP toxicity
C57BL/6J and CHOP knockout mice (on a C57BL/6J background) were purchased from Jackson Laboratories. Mice harboring a conditional floxed allele of XBP1 were crossed to the Alb-Cre expressing strain to obtain a liver specific knockout of XBP-1 (Alb-Cre/XBP-1f/f). Animal care and experiments were approved and conducted in accordance with the Hebrew University-Hadassah Animal Authority guidelines. Male mice 10ā12Ā weeks old, 24ā26Ā g were fasted for 10Ā h at night, and then administered 500Ā mg/kg
APAP overdose induces the UPR
C57BL/6J mice were administered APAP orally by gavage. Prior to gavage, animals were fasted, as fasting improves the uniformity of drug administration, reduces APAP glucuronidation, and potentiates hepatotoxicity [26]. Following gavage, animals were fed ad libitum until time of sacrifice. A fast-fed state was previously shown to induce transient activation of the UPR [27]. Vehicle control was therefore used to ensure that, if UPR is induced, it is a direct result of actual APAP administration.
Discussion
APAP is the most common analgesic currently in use in the world. The toxic effect of APAP overdose is well known and has received ample clinical attention. Furthermore, the initiating steps in APAP toxicity, including the production of NAPQI and depletion of GSH, have been known for decades. It is therefore quite surprising that so many controversies still surround our understanding of this process [36]. It was previously shown that ER stress is activated following APAP administration in mice
Financial support
This research was supported by SAF2012-33283 (MINECO, Spain) and Centro de InvestigaciĆ³n BiomĆ©dica en Red de Diabetes y Enfermedades MetabĆ³licas Asociadas (CIBERDEM), ISCIII and Comunidad de Madrid S2010/BMD-2423 and EFSD/Amylin Programme 2011 to AMV Spain to AMV. The Morasha Program of Israel Science Foundation (grant No. 1668/08) to OS, by the BSF (grant No. 2007083) to OS and RX, and by the Israel Science Foundation (grant 78/09) to BT.
Conflict of interest
The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.
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These authors contributed equally.