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.