JNK in Metabolism of APAP in the liver.
APAP is mainly conjugated with glucuronic acid by enzymes in the UDP-glucuronosyltransferase (UGT) 1A subfamily and sulfated by sulfotransferase enzymes at therapeutic doses, to form hydrophilic compounds in the liver after which are then excreted through bile and urine. In addition, only a small portion of APAP is metabolized by phase I enzymes such as cytochrome P450 enzymes, to form the active metabolite N-acetyl-p-benzoquinone imine (NAPQI). NAPQI is a toxic metabolite that is detoxified by reduced Glutathione (GSH) to prevent it from binding to proteins containing sulfhydryl groups, reduce oxidative stress and prevent liver injury. On the other hand, excess NAPQI generated from an overdose of APAP results in depletion of GSH in the cytoplasm and mitochondria [7, 8]. NAPQI then covalently binds with protein sulfhydryl groups and forms NAPQI-protein adducts. Previous studies report that APAP-protein adducts are the main causes of APAP-induced hepatocyte injury [9]. However, formation of NAPQI-protein adducts does not solely cause severe liver injury because 3’-hydroxyacetanilide (AMAP), a non-toxic regioisomer of APAP in mice, also produces the same amount of covalent conjugates as APAP [10]. The difference between APAP and AMAP is that GSH in the cytoplasm and Endoplasmic Reticulum (ER) and not the mitochondria, is consumed when AMAP is used. As a result, there is no activation of various complex pathways associated with JNK and no liver damage occurs when AMAP is administered[11]. Previous studies report that depletion of mitochondrial GSH induces release of Reactive Oxygen Species (ROS) [12, 13] and activates the JNK signaling pathway[14]. Notably, AMAP is toxic to human liver cells as it forms mitochondrial protein adducts and causes mitochondrial dysfunction [15, 16]. Therefore, formation of NAPQI and protein adducts may initiate liver injury although they may not be the only factors implicated in progression of liver injury. Notably, other downstream events after formation of NAPQI-protein adducts play roles in APAP-induced liver injury.
The process of liver damage caused by APAP is therefore divided into two stages; ① the metabolism phase which comprises formation of NAPQI, depletion of GSH and formation of adducts and ② the oxidative phase that comprises oxidative stress, loss of mitochondrial membrane potential and Mitochondrial Membrane Permeability (MPT)[17, 18]. Notably, APAP or NAPQI are not required for the oxidative phase and studies report that adding them into experimental subjects at this phase does not exacerbate toxicity [18]. Therefore, oxidative stress amplification through JNK signaling pathway is the central mechanism in liver damage induced by APAP.
In vivo studies report that depletion of GSH reached a peak after 2 hours of APAP injection, and similar findings are reported for covalent binding of NAPQI [11]. However, the metabolism phase is important because the metabolism of APAP to NAPQI which is mediated by cytochrome P450 enzymes, is an initial effector of liver injury. Notably, inhibition of CYP2E1 and CYP1A2 or increase in GSH synthesis protects the liver against APAP-induced toxicity [19]. Mitochondrial bioenergetic is inhibited 2 hours after APAP injection [20] which can be attributed to JNK-independent mechanism including depletion of GSH or covalent binding of APAP[21]. Moreover, NAPQI-mitochondrial protein adducts are formed in human hepatocytes and liver non-parenchymal cells during APAP-induced liver injury [22]. Previous studies report that mitochondrial proteins are important sources of oxidative stress and NAPQI indirectly (by binding with mitochondrial respiratory compounds or the electron transfer chain) or directly (by destroying redox cycling through GSH depletion or its redox reaction) leads to release of ROS [23]. In addition, a low-dose (75mg/kg) APAP that dose not form mitochondrial adduct does not cause JNK activation, therefore no amplification of oxidative stress [24]. Furthermore, only the depletion of mitochondrial GSH can induce sustained JNK activation [13] and only sustained and not transient JNK activation is significant. Moreover, transient JNK activation and reversible mitochondrial dysfunction occur when 150mg/kg APAP is injected. However, irreversible mitochondrial injury occurs at a dose of 300mg/kg [25]. Notably, transient activation of JNK does not cause mitochondrial injury. For instance, a previous study reported that pretreatment with DAVA (an extract containing Polyphenols) changed sustained activation of JNK into transient activation, subsequently improving liver injury [26].Therefore, further studies should explore the effect of mitochondrial GSH depletion and formation of protein adducts.
Activation of JNK starts 2 hours after injection of APAP and reaches a peak after 4 hours [13]. Liver damage gradually occurs after 4-6 hours [11]. However, the duration of JNK activation is still controversial. For instance, a previous study reported that JNK activation starts at about 2-4 hours after APAP injection, reaches a peak at 6 hours and remains high for 8 hours [27]. On the contrary a different study reported that activation of JNK starts 1 hour after APAP injection [28]. Moreover, other studies report that activation of JNK starts 0.5-6 hours after APAP injection [26]. A previous study reports that JNK induces changes in the quantity and expression of glutathione S-transferase A1, to promote liver injury, implying that activated JNK affects GSH metabolism [29]. In addition, activation of JNK mediates formation of peroxynitrite and nitrotyrosine, to induce oxidative and nitrosative stress [29]. Therefore, JNK-mitochondrial signaling loop plays a key role in liver injury.
In summary, JNK signaling pathway is a downstream pathway involved in depletion of mitochondrial GSH and formation of protein adducts. Therefore, JNK signaling pathway plays a key role in induction of acute liver injury by enhancing oxidative stress in APAP-induced liver injury.