In the present study, we showed that pretreatment with melatonin significantly alleviated APAP-induced hepatic necrosis and congestion. Correspondingly, melatonin significantly attenuated APAP-induced elevation of serum ALT. In addition, melatonin significantly attenuated APAP-induced increase in hepatic TUNEL+ cells. These results are in agreement with those from two earlier reports 
, in which melatonin protected mice from APAP-induced acute liver failure.
Increasing evidence demonstrates that the sustained activation of JNK plays a major role in APAP-induced hepatotoxicity 
. Indeed, the present study showed that the level of phosphorylated JNK was significantly increased in liver of mice administered with APAP. Melatonin exerts differential actions on JNK activation in normal and cancer cells. According to an in vitro study, melatonin induces cycle arrest and apoptosis through induction of JNK phosphorylation in HepG2 cell, a hepatocarcinoma cell line 
. By contrast, two reports from different laboratories showed that melatonin prevented ischemia/reperfusion-induced liver injury through inhibition of toll-like receptor-mediated hepatic JNK activation 
. In addition, melatonin protected against apoptotic liver damage induced by the rabbit hemorrhagic disease virus through inhibition of hepatic JNK phosphorylation 
. Consistent with its differential effects on JNK, melatonin also exerts differential actions on cell death in normal and cancer cells 
. Melatonin increases apoptotic cell death in cancer cells 
. On the other hand, melatonin exerts its anti-apoptotic effect in normal cells 
. In the present study, we investigated the effects of melatonin on APAP-induced hepatic JNK activation. Consistent with its anti-apoptotic effect, melatonin inhibited APAP-induced hepatic JNK phosphorylation in a dose-dependent manner. These results suggest that melatonin protects mice from APAP-induced cell death through its inhibition of hepatic JNK phosphorylation.
How melatonin inhibits APAP-induced hepatic JNK phosphorylation remains to be determined. An earlier study demonstrates that RIP1 is upstream of JNK in poly (ADP-ribose) polymerase-1 (PARP-1)-induced mitochondrial dysfunction, nuclear AIF translocation and subsequent cell death 
. Another report indicates that RIP1-mediated ASK1-interacting protein 1 (AIP1) phosphorylation is essential for the activation of ASK1-JNK/p38 apoptotic signaling 
. Recently, we found that necrostatin-1, a specific RIP1 inhibitor, significantly alleviated APAP-induced hepatic JNK phosphorylation and caspase-independent cell death (unpublished data). Thus, we guess that RIP1 may be upstream of phosphorylated JNK. Indeed, the present study showed that pretreatment with melatonin significantly attenuated APAP-induced hepatic RIP1 activation. These results suggest that melatonin inhibits APAP-induced hepatic JNK phosphorylation through its inhibition of RIP1 activation.
A recent study indicates that the activated JNK induces Bcl-2 phosphorylation and thereby blocks its translocation to mitochondria 
. The present study found that the level of mitochondrial Bcl-2 was significantly increased in liver of mice administered with APAP, indicating that Bcl-2 is not downstream target of the activated JNK during APAP-induced acute liver failure. Several studies have demonstrated that the phosphorylated JNK promotes Bax translocation from cytosol to mitochondria through phosphorylation of 14-3-3, a cytoplasmic anchor of Bax 
. An earlier report observed that the level of hepatic mitochondrial Bax was significantly increased in mice treated with a toxic dose of APAP 
. Moreover, SP600125, a specific inhibitor of JNK, inhibited APAP-induced mitochondrial Bax translocation 
. Indeed, the present study found that pretreatment with melatonin obviously attenuated APAP-induced hepatic mitochondrial Bax translocation. These results suggest that Bax may be a potential downstream target of phosphorylated JNK in the process of APAP-induced acute liver failure. Melatonin does not only inhibit APAP-induced hepatic JNK phosphorylation but also subsequent mitochondrial Bax translocation.
The proapoptotic protein Bax translocated to mitochondria can form pores in the outer mitochondrial membrane 
. Formation of pores in the outer mitochondrial membrane together with formation of the mitochondrial permeability transition (MPT) pores in the inner membrane promotes the early release of AIF, which is thought to trigger DNA cleavage, from the mitochondria to the nuclei. Several reports demonstrate that AIF, which is translocated to the nuclei from the mitochondria, is responsible for the initial DNA fragmentation and subsequent cell death during APAP-induced acute liver failure 
. The present study showed that nuclear AIF translocation was mainly distributed around hepatic sinus, where nuclei were partially digested. Of interest, pretreatment with melatonin obviously attenuated APAP-induced nuclear AIF translocation. Correspondingly, melatonin significantly alleviated APAP-induced DNA strand breaks around hepatic sinus, as determined by TUNEL assay. These results suggest that melatonin protects mice from nuclear AIF translocation and cell death during APAP-induced acute liver failure.
APAP-mediated acute liver failure is initiated by its reactive metabolite, NAPQI, usually catalysed by hepatic CYP2E1 enzyme 
. In the present study, we found that the level of hepatic cyp2e1
mRNA was significantly decreased when mice were administered with APAP. These results are in agreement with others, in which the expression of hepatic CYP2E1 protein was significantly decreased after repeat exposure to incremental doses of APAP. Interestingly, the present study showed that no significant downregulation of hepatic CYP2E1 protein was observed at 4 h after APAP treatment. Indeed, an earlier report also showed no significant difference on the expression of hepatic CYP2E1 protein 6 h after APAP treatment, whereas there was a significant decrease in hepatic CYP2E1 protein 24 h following APAP treatment of rats. A recent study showed that melatonin significantly inhibited the expression of cyp2e1
mRNA in nigrostriatal tissues 
. The present study investigated the effects of melatonin on the expression of hepatic CYP2E1 in mice treated with APAP. We found that a single dose of melatonin did not affect the expression of hepatic CYP2E1 protein. These results suggest that melatonin protects against APAP-induced acute liver failure probably downstream of CYP2E1.
Numerous reports have demonstrated that hepatic GSH depletion is involved in the process of APAP-mediated sustained activation of JNK and subsequent acute liver injury 
. The present study showed that hepatic GSH content was significantly decreased in mice treated with APAP. By contrast, hepatic GSSG/GSH ratio was significantly increased in mice treated with APAP. Several reports found that melatonin significantly alleviated hepatic GSH depletion 
. Indeed, the present study showed that APAP-induced downregulation of hepatic GSHRd and GSHPx1 was partially reversed by melatonin. Thus, we investigated the effects of melatonin on APAP-induced hepatic GSH depletion. Unexpectedly, melatonin had little effect on APAP-induced hepatic GSH depletion. These results suggest that melatonin inhibits APAP-induced sustained JNK activation independent of its antioxidant effect.
In summary, the present study indicates that melatonin protects against AIF-dependent cell death during APAP-induced acute liver failure. Although it has little effect on APAP-evoked hepatic GSH depletion, melatonin is able to inhibit APAP-induced hepatic RIP1 activation, reducing both JNK phosphorylation and mitochondrial translocation of Bax. Correspondingly, melatonin inhibits APAP-induced AIF translocation from the mitochondria to the nuclei, which seems to be responsible for the significant reduction on APAP-induced cell death when melatonin is administered. Thus, melatonin may have potential preventive and therapeutic utilities for protecting against APAP-induced acute liver failure.