IDH2 deficiency exacerbates acetaminophen hepatotoxicity in mice via mitochondrial dysfunction-induced apoptosis

Acetaminophen (APAP)-induced hepatotoxicity is a major factor in liver failure and its toxicity is associated with the generation of reactive oxygen species (ROS), decreased levels of reduced glutathione (GSH) and overall oxidative stress. Mitochondrial NADP⁺-dependent isocitrate dehydrogenase (IDH2) was demonstrated as an essential enzyme for mitochondria to maintain their antioxidant system by generating NADPH, which is an essential reducing equivalent for GSH turnover in mitochondria. Here, we investigated the role of IDH2 in APAP-induced liver injury with IDH2 deficient (idh2^−/−) mice. Hepatotoxicity was promoted through apoptotic cell death following APAP administration in IDH2 deficient hepatocytes compared to that in wild-type hepatocytes. Apoptosis was found to result from the induction of ER stress and mitochondrial dysfunction as shown by the blocking the effect of phenylbutyrate and Mdivi1, respectively. In addition, mito-TEMPO, a scavenger of mitochondrial ROS, was seen to ameliorate APAP-induced hepatotoxicity in idh2^−/− mice. In conclusion, IDH2 deficiency leads to a fundamental shortage of GSH that increases susceptibility to ROS generation and oxidative stress. This leads to excessive mitochondrial dysfunction and ER stress induction in response to APAP administration. Our study provides further evidence that IDH2 has a protective role against APAP-induced liver injury and emphasizes the importance of the elaborate linkages and functions of the antioxidant system in liver health.     

P2X7 receptor-mediated purinergic signaling promotes liver injury in acetaminophen hepatotoxicity in mice

Inflammation contributes to liver injury in acetaminophen (APAP) hepatotoxicity in mice and is triggered by stimulation of immune cells. The purinergic receptor P2X7 is upstream of the nod-like receptor family, pryin domain containing-3 (NLRP3) inflammasome in immune cells and is activated by ATP and NAD that serve as damage-associated molecular patterns. APAP hepatotoxicity was assessed in mice genetically deficient in P2X7, the key inflammatory receptor for nucleotides (P2X7-/-), and in wild-type mice. P2X7-/- mice had significantly decreased APAP-induced liver necrosis. In addition, APAP-poisoned mice were treated with the specific P2X7 antagonist A438079 or etheno-NAD, a competitive antagonist of NAD. Pre- or posttreatment with A438079 significantly decreased APAP-induced necrosis and hemorrhage in APAP liver injury in wild-type but not P2X7-/- mice. Pretreatment with etheno-NAD also significantly decreased APAP-induced necrosis and hemorrhage in APAP liver injury. In addition, APAP toxicity in mice lacking the plasma membrane ecto-NTPDase CD39 (CD39-/-) that metabolizes ATP was examined in parallel with the use of soluble apyrase to deplete extracellular ATP in wild-type mice. CD39-/- mice had increased APAP-induced hemorrhage and mortality, whereas apyrase also decreased APAP-induced mortality. Kupffer cells were treated with extracellular ATP to assess P2X7-dependent inflammasome activation. P2X7 was required for ATP-stimulated IL-1β release. In conclusion, P2X7 and exposure to the ligands ATP and NAD are required for manifestations of APAP-induced hepatotoxicity.     

Chronic Deletion and Acute Knockdown of Parkin Have Differential Responses to Acetaminophen-induced Mitophagy and Liver Injury in Mice

We previously demonstrated that pharmacological induction of autophagy protected against acetaminophen (APAP)-induced liver injury in mice by clearing damaged mitochondria. However, the mechanism for removal of mitochondria by autophagy is unknown. Parkin, an E3 ubiquitin ligase, has been shown to be required for mitophagy induction in cultured mammalian cells following mitochondrial depolarization, but its role in vivo is not clear. The purpose of this study was to investigate the role of Parkin-mediated mitophagy in protection against APAP-induced liver injury. We found that Parkin translocated to mitochondria in mouse livers after APAP treatment followed by mitochondrial protein ubiquitination and mitophagy induction. To our surprise, we found that mitophagy still occurred in Parkin knock-out (KO) mice after APAP treatment based on electron microscopy analysis and Western blot analysis for some mitochondrial proteins, and Parkin KO mice were protected against APAP-induced liver injury compared with wild type mice. Mechanistically, we found that Parkin KO mice had decreased activated c-Jun N-terminal kinase (JNK), increased induction of myeloid leukemia cell differentiation protein (Mcl-1) expression, and increased hepatocyte proliferation after APAP treatment in their livers compared with WT mice. In contrast to chronic deletion of Parkin, acute knockdown of Parkin in mouse livers using adenovirus shRNA reduced mitophagy and Mcl-1 expression but increased JNK activation after APAP administration, which exacerbated APAP-induced liver injury. Therefore, chronic deletion (KO) and acute knockdown of Parkin have differential responses to APAP-induced mitophagy and liver injury in mice.     

Enhanced Production of Adenosine Triphosphate by Pharmacological Activation of Adenosine Monophosphate-Activated Protein Kinase Ameliorates Acetaminophen-Induced Liver Injury

The hepatic cell death induced by acetaminophen (APAP) is closely related to cellular adenosine triphosphate (ATP) depletion, which is mainly caused by mitochondrial dysfunction. Adenosine monophosphate (AMP)-activated protein kinase (AMPK) is a key sensor of low energy status. AMPK regulates metabolic homeostasis by stimulating catabolic metabolism and suppressing anabolic pathways to increase cellular energy levels. We found that the decrease in active phosphorylation of AMPK in response to APAP correlates with decreased ATP levels, in vivo. Therefore, we hypothesized that the enhanced production of ATP via AMPK stimulation can lead to amelioration of APAP-induced liver failure. A769662, an allosteric activator of AMPK, produced a strong synergistic effect on AMPK Thr172 phosphorylation with APAP in primary hepatocytes and liver tissue. Interestingly, activation of AMPK by A769662 ameliorated the APAP-induced hepatotoxicity in C57BL/6N mice treated with APAP at a dose of 400 mg/kg intraperitoneally. However, mice treated with APAP alone developed massive centrilobular necrosis, and APAP increased their serum alanine aminotransferase and aspartate aminotransferase levels. Furthermore, A769662 administration prevented the loss of intracellular ATP without interfering with the APAP-mediated reduction of mitochondrial dysfunction. In contrast, inhibition of glycolysis by 2-deoxy-glucose eliminated the beneficial effects of A769662 on APAP-mediated liver injury. In conclusion, A769662 can effectively protect mice against APAP-induced liver injury through ATP synthesis by anaerobic glycolysis. Furthermore, stimulation of AMPK may have potential therapeutic application for APAP overdose.     

Comparative evaluation of N-acetylcysteine and N-acetylcysteineamide in acetaminophen-induced hepatotoxicity in human hepatoma HepaRG cells

Acetaminophen (N-acetyl-p-aminophenol, APAP) is one of the most widely used over-the-counter antipyretic analgesic medications. Despite being safe at therapeutic doses, an accidental or intentional overdose can result in severe hepatotoxicity; a leading cause of drug-induced liver failure in the U.S. Depletion of glutathione (GSH) is implicated as an initiating event in APAP-induced toxicity. N-acetylcysteine (NAC), a GSH precursor, is the only currently approved antidote for an APAP overdose. Unfortunately, fairly high doses and longer treatment times are required due to its poor bioavailability. In addition, oral and intravenous administration of NAC in a hospital setting are laborious and costly. Therefore, we studied the protective effects of N-acetylcysteineamide (NACA), a novel antioxidant, with higher bioavailability and compared it with NAC in APAP-induced hepatotoxicity in a human-relevant in vitro system, HepaRG. Our results indicated that exposure of HepaRG cells to APAP resulted in GSH depletion, reactive oxygen species (ROS) formation, increased lipid peroxidation, mitochondrial dysfunction (assessed by JC-1 fluorescence), and lactate dehydrogenase release. Both NAC and NACA protected against APAP-induced hepatotoxicity by restoring GSH levels, scavenging ROS, inhibiting lipid peroxidation, and preserving mitochondrial membrane potential. However, NACA was better than NAC at combating oxidative stress and protecting against APAP-induced damage. The higher efficiency of NACA in protecting cells against APAP-induced toxicity suggests that NACA can be developed into a promising therapeutic option for treatment of an APAP overdose.     

Silencing Glycogen Synthase Kinase-3�� Inhibits Acetaminophen Hepatotoxicity and Attenuates JNK Activation and Loss of Glutamate Cysteine Ligase and Myeloid Cell Leukemia Sequence 1

Previously we demonstrated that c-Jun N-terminal kinase (JNK) plays a central role in acetaminophen (APAP)-induced liver injury. In the current work, we examined other possible signaling pathways that may also contribute to APAP hepatotoxicity. APAP treatment to mice caused glycogen synthase kinase-3β (GSK-3β) activation and translocation to mitochondria during the initial phase of APAP-induced liver injury (∼1 h). The silencing of GSK-3β, but not Akt-2 (protein kinase B) or glycogen synthase kinase-3α (GSK-3α), using antisense significantly protected mice from APAP-induced liver injury. The silencing of GSK-3β affected several key pathways important in conferring protection against APAP-induced liver injury. APAP treatment was observed to promote the loss of glutamate cysteine ligase (GCL, rate-limiting enzyme in GSH synthesis) in liver. The silencing of GSK-3β decreased the loss of hepatic GCL, and promoted greater GSH recovery in liver following APAP treatment. Silencing JNK1 and -2 also prevented the loss of GCL. APAP treatment also resulted in GSK-3β translocation to mitochondria and the degradation of myeloid cell leukemia sequence 1 (Mcl-1) in mitochondrial membranes in liver. The silencing of GSK-3β reduced Mcl-1 degradation caused by APAP treatment. The silencing of GSK-3β also resulted in an inhibition of the early phase (0–2 h), and blunted the late phase (after 4 h) of JNK activation and translocation to mitochondria in liver following APAP treatment. Taken together our results suggest that activation of GSK-3β is a key mediator of the initial phase of APAP-induced liver injury through modulating GCL and Mcl-1 degradation, as well as JNK activation in liver.     

Mice deficient in Cu,Zn-superoxide dismutase are resistant to acetaminophen toxicity

Although antioxidants are used to treat an overdose of the analgaesic/antipyretic drug APAP (acetaminophen), roles of antioxidant enzymes in APAP-induced hepatotoxicity remain controversial. Our objective was to determine impacts of knockout of SOD1 (superoxide dismutase; Cu,Zn-SOD) alone or in combination with selenium-dependent GPX1 (glutathione peroxidase-1) on APAP-induced hepatotoxicity. All SOD1-null (SOD1-/-) and SOD1- and GPX1-double-knockout mice survived an intraperitoneal injection of 600 mg of APAP per kg of body mass, whereas 75% of WT (wild-type) and GPX1-null mice died within 20 h. Survival time of SOD1-/- mice injected with 1200 mg of APAP per kg of body mass was longer than that of the WT mice (934 compared with 315 min, P<0.05). The APAP-treated SOD1-/- mice had less (P<0.05) plasma ALT (alanine aminotransferase) activity increase and attenuated (P<0.05) hepatic glutathione depletion than the WT mice. The protection conferred by SOD1 deletion was associated with a block of the APAP-mediated hepatic protein nitration and a 50% reduction (P<0.05) in activity of a key APAP metabolism enzyme CYP2E1 (cytochrome P450 2E1) in liver. The SOD1 deletion also caused moderate shifts in the APAP metabolism profiles. In conclusion, deletion of SOD1 alone or in combination with GPX1 greatly enhanced mouse resistance to APAP overdose. Our results suggest a possible pro-oxidant role for the physiological level of SOD1 activity in APAP-mediated hepatotoxicity.     

Post-treatment with glycyrrhizin can attenuate hepatic mitochondrial damage induced by acetaminophen in mice

Overdose of acetaminophen (APAP) is responsible for the most cases of acute liver failure worldwide. Hepatic mitochondrial damage mediated by neuronal nitric oxide synthase- (nNOS) induced liver protein tyrosine nitration plays a critical role in the pathophysiology of APAP hepatotoxicity. It has been reported that pre-treatment or co-treatment with glycyrrhizin can protect against hepatotoxicity through prevention of hepatocellular apoptosis. However, the majority of APAP-induced acute liver failure cases are people intentionally taking the drug to commit suicide. Any preventive treatment is of little value in practice. In addition, the hepatocellular damage induced by APAP is considered to be oncotic necrosis rather than apoptosis. In the present study, our aim is to investigate if glycyrrhizin can be used therapeutically and the underlying mechanisms of APAP hepatotoxicity protection. Hepatic damage was induced by 300 mg/kg APAP in balb/c mice, followed with administration of 40, 80, or 160 mg/kg glycyrrhizin 90 min later. Mice were euthanized and harvested at 6 h post-APAP. Compared with model controls, glycyrrhizin post-treatment attenuated hepatic mitochondrial and hepatocellular damages, as indicated by decreased serum glutamate dehydrogenase, alanine aminotransferase, and aspartate aminotransferase activities as well as ameliorated mitochondrial swollen, distortion, and hepatocellular necrosis. Notably, 80 mg/kg glycyrrhizin inhibited hepatic nNOS activity and its mRNA and protein expression levels by 16.9, 14.9, and 28.3%, respectively. These results were consistent with the decreased liver nitric oxide content and liver protein tyrosine nitration indicated by 3-nitrotyrosine staining. Moreover, glycyrrhizin did not affect the APAP metabolic activation, and the survival rate of ALF mice was increased by glycyrrhizin. The present study indicates that post-treatment with glycyrrhizin can dose-dependently attenuate hepatic mitochondrial damage and inhibit the up-regulation of hepatic nNOS induced by APAP. Glycyrrhizin shows promise as drug for the treatment of APAP hepatotoxicity.     

New Therapeutic Approach: Diphenyl Diselenide Reduces Mitochondrial Dysfunction in Acetaminophen-Induced Acute Liver Failure

The acute liver failure (ALF) induced by acetaminophen (APAP) is closely related to oxidative damage and depletion of hepatic glutathione, consequently changes in cell energy metabolism and mitochondrial dysfunction have been observed after APAP overdose. Diphenyl diselenide [(PhSe)₂], a simple organoselenium compound with antioxidant properties, previously demonstrated to confer hepatoprotection. However, little is known about the protective mechanism on mitochondria. The main objective of this study was to investigate the effects (PhSe)₂ to reduce mitochondrial dysfunction and, secondly, compare in the liver homogenate the hepatoprotective effects of the (PhSe)₂ to the N-acetylcysteine (NAC) during APAP-induced ALF to validate our model. Mice were injected intraperitoneal with APAP (600 mg/kg), (PhSe)₂ (15.6 mg/kg), NAC (1200 mg/kg), APAP+(PhSe)₂ or APAP+NAC, where the (PhSe)₂ or NAC treatment were given 1 h following APAP. The liver was collected 4 h after overdose. The plasma alanine and aspartate aminotransferase activities increased after APAP administration. APAP caused a remarkable increase of oxidative stress markers (lipid peroxidation, reactive species and protein carbonylation) and decrease of the antioxidant defense in the liver homogenate and mitochondria. APAP caused a marked loss in the mitochondrial membrane potential, the mitochondrial ATPase activity, and the rate of mitochondrial oxygen consumption and increased the mitochondrial swelling. All these effects were significantly prevented by (PhSe)₂. The effectiveness of (PhSe)₂ was similar at a lower dose than NAC. In summary, (PhSe)₂ provided a significant improvement to the mitochondrial redox homeostasis and the mitochondrial bioenergetics dysfunction caused by membrane permeability transition in the hepatotoxicity APAP-induced.     

Arjunolic acid,a triterpenoid saponin,prevents acetaminophen (APAP)-induced liver and hepatocyte injury via the inhibition of APAP bioactivation and JNK-mediated mitochondrial protection

Acetaminophen (APAP) is a widely used analgesic and antipyretic drug and is safe at therapeutic doses but its overdose frequently causes liver injury. In earlier studies, we demonstrated that arjunolic acid (AA) has a protective effect against chemically induced hepatotoxicity. The purpose of this study was to explore whether AA plays any protective role against APAP-induced acute hepatotoxicity and, if so, what molecular pathways it utilizes for the mechanism of its protective action. Exposure of rats to a hepatotoxic dose of acetaminophen (700 mg/kg, ip) altered a number of biomarkers (related to hepatic oxidative stress), increased reactive oxygen species production, reduced cellular adenosine triphosphate level, and induced necrotic cell death. Arjunolic acid pretreatment (80 mg/kg, orally), on the other hand, afforded significant protection against liver injury. Arjunolic acid also prevented acetaminophen-induced hepatic glutathione depletion and APAP metabolite formation although arjunolic acid itself did not affect hepatic glutathione levels. The results suggest that this preventive action of arjunolic acid is due to the metabolic inhibition of specific forms of cytochrome P450 that activate acetaminophen to N-acetyl-p-benzoquinone imine. In addition, administration of arjunolic acid 4 h after acetaminophen intoxication reduced acetaminophen-induced JNK and downstream Bcl-2 and Bcl-xL phosphorylation, thus protecting against mitochondrial permeabilization, loss of mitochondrial membrane potential, and cytochrome c release. In conclusion, the data suggest that arjunolic acid affords protection against acetaminophen-induced hepatotoxicity through inhibition of P450-mediated APAP bioactivation and inhibition of JNK-mediated activation of mitochondrial permeabilization.     

Targeting autophagy for drug-induced hepatotoxicity

Autophagy is a lysosomal degradation pathway for bulk cytosolic proteins and damaged organelles, and is well known to act as a cell survival mechanism. Acetaminophen (APAP) overdose can cause liver injury in animals and humans by inducing necrosis due to mitochondrial damage. We recently found that pharmacological induction of autophagy by rapamycin protects against, whereas pharmacological suppression of autophagy by chloroquine exacerbates, APAP-induced liver injury in mice. Autophagy is induced to remove APAP-induced damaged mitochondria and thus attenuates APAP-induced hepatocyte necrosis. To our surprise, we found that liver-specific Atg5 knockout mice are not more susceptible, but are resistant to APAP-induced liver injury due to compensatory effects. Our work suggests that pharmacological modulation of autophagy is a novel therapeutic approach to ameliorate APAP-induced liver injury. Moreover, our work also suggests that caution needs to be exercised when using genetic autophagy gene knockout mice for pathophysiological studies.     

Current issues with acetaminophen hepatotoxicity--a clinically relevant model to test the efficacy of natural products

There is a significant need to evaluate the therapeutic potential of natural products and other compounds purported to be hepatoprotective. Acetaminophen-induced liver injury, especially in mice, is an attractive and widely used model for this purpose because it is both clinically relevant and experimentally convenient. However, the pathophysiology of liver injury after acetaminophen overdose is complex. This review describes the multiple steps and signaling pathways involved in acetaminophen-mediated cell death. The toxicity is initiated by the formation of a reactive metabolite, which depletes glutathione and binds to cellular proteins, especially in mitochondria. The resulting mitochondrial oxidant stress and peroxynitrite formation, in part through amplification by c-jun-N-terminal kinase activation, leads to mitochondrial DNA damage and opening of the mitochondrial permeability transition pore. Endonucleases from the mitochondrial intermembrane space and lysosomes are responsible for nuclear DNA fragmentation. Despite the oxidant stress, lipid peroxidation is not a relevant mechanism of injury. The mitochondrial dysfunction and nuclear DNA damage ultimately cause oncotic necrotic cell death with release of damage-associated molecular patterns that trigger a sterile inflammatory response. Current evidence supports the hypothesis that innate immune cells do not contribute to injury but are involved in cell debris removal and regeneration. This review discusses the latest mechanistic aspects of acetaminophen hepatotoxicity and demonstrates ways to assess the mechanisms of drug action and design experiments needed to avoid pitfalls and incorrect conclusions. This review should assist investigators in the optimal use of this model to test the efficacy of natural compounds and obtain reliable mechanistic information.     

Targeting autophagy for drug-induced hepatotoxicity

Autophagy is a lysosomal degradation pathway for bulk cytosolic proteins and damaged organelles, and is well known to act as a cell survival mechanism. Acetaminophen (APAP) overdose can cause liver injury in animals and humans by inducing necrosis due to mitochondrial damage. We recently found that pharmacological induction of autophagy by rapamycin protects against, whereas pharmacological suppression of autophagy by chloroquine exacerbates, APAP-induced liver injury in mice. Autophagy is induced to remove APAP-induced damaged mitochondria and thus attenuates APAP-induced hepatocyte necrosis. To our surprise, we found that liver-specific Atg5 knockout mice are not more susceptible, but are resistant to APAP-induced liver injury due to compensatory effects. Our work suggests that pharmacological modulation of autophagy is a novel therapeutic approach to ameliorate APAP-induced liver injury. Moreover, our work also suggests that caution needs to be exercised when using genetic autophagy gene knockout mice for pathophysiological studies.     

Selenium: Mercury Molar Ratios in Freshwater Fish in the Columbia River Basin: Potential Applications for Specific Fish Consumption Advisories

Overdoses of acetaminophen (APAP), a famous and widely used drug, may have hepatotoxic effects. Nanoscience is a novel scientific discipline that provides specific tools for medical science problems including using nano trace elements in hepatic diseases. Our study aimed to assess the hepatoprotective role of selenium nanoparticles (Nano-Se) against APAP-induced hepatic injury. Twenty-four male rats were classified into three equal groups: a control group that received 0.9 % NaCl, an APAP-treated group (oral administration), and a group treated with Nano-Se (10–20 nm, intraperitoneal (i.p.) injection) and APAP (oral administration). APAP overdose induced significant elevations in liver function biomarkers, hepatic lipid peroxidation, hepatic catalase, and superoxide dismutase (SOD), decreased the reduced glutathione (GSH) content and glutathione reductase (GR) activity, and stimulated significant DNA damage in hepatocytes, compared to control rats. Nano-Se administration improved the hepatic antioxidant protection mechanism and decreased cellular sensitivity to DNA fragmentation. Nano-Se exhibits a protective effect against APAP-induced hepatotoxicity through improved liver function and oxidative stress mediated by catalase, SOD, and GSH and decreases hepatic DNA fragmentation, a hepatic biomarker of cell death. Nano-Se could be a novel hepatoprotective strategy to inhibit oxidative stress.     

Kava extract, an herbal alternative for anxiety relief, potentiates acetaminophen-induced cytotoxicity in rat hepatic cells

The widely used over-the-counter analgesic acetaminophen (APAP) is the leading cause of acute liver failure in the United States and due to this high incidence, a recent FDA Advisory Board recommended lowering the maximum dose of APAP. Kava herbal dietary supplements have been implicated in several human liver failure cases leading to the ban of kava-containing products in several Western countries. In the US, the FDA has issued warnings about the potential adverse effects of kava, but kava dietary supplements are still available to consumers. In this study, we tested the potential of kava extract to potentiate APAP-induced hepatocyte cytotoxicity. In rat primary hepatocytes, co-treatment with kava and APAP caused 100% loss of cell viability, while the treatment of kava or APAP alone caused ∼50% and ∼30% loss of cell viability, respectively. APAP-induced glutathione (GSH) depletion was also potentiated by kava. Co-exposure to kava decreased cellular ATP concentrations, increased the formation of reactive oxygen species, and caused mitochondrial damage as indicated by a decrease in mitochondrial membrane potential. In addition, similar findings were obtained from a cultured rat liver cell line, clone-9. These observations indicate that kava potentiates APAP-induced cytotoxicity by increasing the magnitude of GSH depletion, resulting in oxidative stress and mitochondrial dysfunction, ultimately leading to cell death. These results highlight the potential for drug-dietary supplement interactions even with widely used over-the-counter drugs.     

Protective effect of diallyl sulfone against acetaminophen-induced hepatotoxicity in mice

Diallyl sulfone (DASO₂) is a metabolite of diallyl sulfide, a compound derived from garlic. The present study investigated the effect of DASO₂ as a protective agent against acetaminophen (APAP)-induced hepatotoxicity in mice. Oral administration of DASO₂ protected mice against the APAP-induced hepatotoxicity in a dose- and time-dependent manner. When administrated 1 hour prior to, immediately after, or 20 minutes after a toxic dose of APAP, DASO₂ at a dose of 25 mg/kg completely protected mice from development of hepatotoxicity, as indicated by liver histopathology and serum lactate dehydrogenase levels. Protective effect was observed when DASO₂ at a dose as low as 5 mg/kg was given to mice 1 hour prior to APAP administration. Oral administration of DASO₂ to mice 1 hour prior to a toxic dose of APAP significantly inhibited the APAP-induced glutathione depletion in the liver. DASO₂ treatment also decreased the levels of oxidative APAP metabolites in the plasma without affecting the concentrations of nonoxidative APAP metabolites. In liver microsomes, 0.1 mM of DASO₂ caused a 60% decrease in the rate of APAP oxidation to N-acetyl-p-benzoquinone imine, which was determined as glutathione conjugate. This inhibitory effect is mainly due to its inhibition of cytochrome P450 2E1 activity; with an IC₅₀ value equal to 0.11 mM. DASO₂ also slightly inhibited the activities of P450s 3A and 1A, with IC₅₀ values >5 mM. Furthermore, a single oral dose of DASO₂ inactivated P450 2E1- and P450 1A-dependent activities in liver microsomes. The results suggest that the protective effect of DASO₂ against APAP-induced hepatotoxicity is due to its ability to block acetaminophen bioactivation mainly by the inactivation and inhibition of P450 2E1. © 1996 John Wiley & Sons, Inc.     

Role of JNK translocation to mitochondria leading to inhibition of mitochondria bioenergetics in acetaminophen-induced liver injury

Previously, we demonstrated JNK plays a central role in acetaminophen (APAP)-induced liver injury (Gunawan, B. K., Liu, Z. X., Han, D., Hanawa, N., Gaarde, W. A., and Kaplowitz, N. (2006) Gastroenterology 131, 165-178). In this study, we examine the mechanism involved in activating JNK and explore the downstream targets of JNK important in promoting APAP-induced liver injury in vivo. JNK inhibitor (SP600125) was observed to significantly protect against APAP-induced liver injury. Increased mitochondria-derived reactive oxygen species were implicated in APAP-induced JNK activation based on the following: 1) mitochondrial GSH depletion (maximal at 2 h) caused increased H2O2 release from mitochondria, which preceded JNK activation (maximal at 4 h); 2) treatment of isolated hepatocytes with H2O2 or inhibitors (e.g. antimycin) that cause increased H2O2 release from mitochondria-activated JNK. An important downstream target of JNK following activation was mitochondria based on the following: 1) JNK translocated to mitochondria following activation; 2) JNK inhibitor treatment partially protected against a decline in mitochondria respiration caused by APAP treatment; and 3) addition of purified active JNK to mitochondria isolated from mice treated with APAP plus JNK inhibitor (mitochondria with severe GSH depletion, covalent binding) directly inhibited respiration. Cyclosporin A blocked the inhibitory effect of JNK on mitochondria respiration, suggesting JNK was directly inducing mitochondrial permeability transition in isolated mitochondria from mice treated with APAP plus JNK inhibitor. Addition of JNK to mitochondria isolated from control mice did not affect respiration. Our results suggests that APAP-induced liver injury involves JNK activation, due to increased reactive oxygen species generated by GSH-depleted mitochondria, and translocation of activated JNK to mitochondria where JNK induces mitochondrial permeability transition and inhibits mitochondria bioenergetics.     

Toll Like Receptor 3 Plays a Critical Role in the Progression and Severity of Acetaminophen-Induced Hepatotoxicity

Toll-like receptor (TLR) activation has been implicated in acetaminophen (APAP)-induced hepatotoxicity. Herein, we hypothesize that TLR3 activation significantly contributed to APAP-induced liver injury. In fasted wildtype (WT) mice, APAP caused significant cellular necrosis, edema, and inflammation in the liver, and the de novo expression and activation of TLR3 was found to be necessary for APAP-induced liver failure. Specifically, liver tissues from similarly fasted TLR3-deficient (tlr3^−/−) mice exhibited significantly less histological and biochemical evidence of injury after APAP challenge. Similar protective effects were observed in WT mice in which TLR3 was targeted through immunoneutralization at 3 h post-APAP challenge. Among three important death ligands (i.e. TNFα, TRAIL, and FASL) known to promote hepatocyte death after APAP challenge, TNFα was the only ligand that was significantly reduced in APAP-challenged tlr3^−/− mice compared with APAP-challenged WT controls. In vivo studies demonstrated that TLR3 activation contributed to TNFα production in the liver presumably via F4/80⁺ and CD11c⁺ immune cells. In vitro studies indicated that there was cooperation between TNFα and TLR3 in the activation of JNK signaling in isolated and cultured liver epithelial cells (i.e. nMuLi). Moreover, TLR3 activation enhanced the expression of phosphorylated JNK in APAP injured livers. Thus, the current study demonstrates that TLR3 activation contributes to APAP-induced hepatotoxicity.