Double null of selenium-glutathione peroxidase-1 and copper, zinc-superoxide dismutase enhances resistance of mouse primary hepatocytes to acetaminophen toxicity

This study was conducted to determine the impact of knockout of selenium (Se)-dependent glutathione peroxidase-1 (GPX1-/-) or double knockout of GPX1 and copper, zinc (Cu,Zn)-super-oxide dismutase (SOD1) on cell death induced by acetaminophen (APAP) and its major toxic metabolite N-acetyl-P-benzoquinoneimine (NAPQI). Primary hepatocytes were isolated from GPX1-/-, double knockout of GPX1 and SOD1 (DKO), and their wild-type (WT) mice and were treated with 5 mM APAP or 100 microM NAPQI for 0, 6, and 12 hrs. Compared with the WT cells, the GPX1-/- and DKO hepatocytes were more resistant (P < 0.05) to the APAP-induced cell death but less resistant to the NAPQI-induced cell death. The APAP-mediated glutathione (GSH) depletion was greater (P < 0.05) at 6 hrs in the WT cells than in the GPX1-/- and DKO cells, whereas there was no genotype effect on the NAPQI-mediated GSH depletion. The DKO cells had lower (P < 0.05) microsomal cytochrome P450 2E1 activities, but higher (P < 0.05) glutathione reductase and thioredoxin reductase activities than the WT cells at 0 hrs, and they responded differently to the APAP and NAPQI treatments. Glutathione-S-transferase activity was not affected by genotypes or treatments. Neither APAP nor NAPQI induced nitric oxide production or protein nitration in cells of any genotype. However, the GPX1-/- and DKO cells were more resistant to peroxynitrite-mediated protein nitration than were the WT cells. In conclusion, double null of GPX1 and SOD1 enhanced the resistance of mouse primary hepatocytes to APAP toxicity by affecting events prior to or at NAPQI formation. While the double knockout attenuated the peroxynitrite-mediated protein nitration in hepatocytes, no protein nitration was detected in these cells treated with APAP or NAPQI.     

Impact of Cu, Zn-superoxide dismutase and Se-dependent glutathione peroxidase-1 knockouts on acetaminophen-induced cell death and related signaling in murine liver

There is increasing evidence showing dual functions of antioxidant enzymes in coping with reactive oxygen species (ROS) versus reactive nitrogen species (RNS). The objective of this study was to compare the impacts of knockout of Cu, Zn-superoxide dismutase (SOD1) and Se-dependent glutathione peroxidase-1 (GPX1) on cell death and related signaling mediated by acetaminophen (APAP), a RNS inducer in liver. Two groups of young adult knockout mice (SOD1(-/-) and GPX1(-/-)), along with their wild types (WT), were killed 5 hrs after an ip injection of saline or APAP (300 mg/kg body wt). While the WT mice showed more hepatic necrosis and DNA breakage than the GPX1(-/-) mice, the SOD1(-/-) mice had essentially no positive response compared with their saline-injected controls. The APAP treatment activated liver c-jun N-terminal kinase (JNK) in the WT and GPX1(-/-) mice, but not in the SOD1(-/-) mice. The APAP-induced changes in other cell death-related signal proteins such as p21, caspase-3, and poly(ADP-ribose) polymerase (PARP) also were obviated in the SOD1(-/-) mice. In conclusion, knockout of GPX1 did not potentiate APAP-induced cell death and related signaling, whereas the SOD1 null blocked APAP-induced hepatic JNK phosphorylation and cell death.     

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.     

Acetaminophen induced acute liver failure via oxidative stress and JNK activation: Protective role of taurine by the suppression of cytochrome P450 2E1

Abstract

The present study was carried out to investigate whether taurine plays any beneficial role in acetaminophen (APAP)-induced acute hepatotoxicity. APAP exposure increased the plasma levels of ALT, ALP, LDH, TNF-α and NO production. Moreover, APAP treatment reduced the glutathione level and antioxidant enzyme activities, increased lipid peroxidation and caused hepatic DNA fragmentation which ultimately leads to cellular necrosis. Also, incubation of hepatocytes with APAP reduced cell viability, enhanced ROS generation and increased CYP2E1 activity. APAP overdose caused injury in the hepatic tissue and hepatocytes via the upregulation of CYP2E1 and JNK. Taurine treatment was effective in counteracting APAP-induced hepatic damages, oxidative stress and cellular necrosis. Results indicate that APAP overdose caused hepatic injury due to its metabolism to hepatotoxic NAPQI (N-acetyl-p-benzoquinone imine), usually catalysed by CYP2E1, and via the direct activation of JNK-dependent cell death pathway. Taurine possesses prophylactic as well as therapeutic potentials against APAP-induced hepatic injury.     

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.     

Protection against paracetamol-induced hepatic injury by prazosin pre-treatment in CD-1 mice

A synergistic depletion of glutathione has been suggested to be one critical factor in the hepatic injury in mice induced by non-toxic doses of paracetamol (APAP) when co-administered with -adrenergic agonists. Prazosin (an -adrenergic antagonist) could confer hepatoprotection following a toxic APAP dose (530 mg/kg) by increasing glutathione levels and enhancing bioinactivation by glucuronidation and glutathione conjugation. The effect of prazosin pre-treatment on APAP-induced gluthathione depletion and bioinactivation in vivo was assessed. Prazosin (15 mg/kg) pre-treatment provided protection against APAP-induced hepatic injury as evidenced by a significant decrease in serum transaminase (ALT) levels after 5 h (p < 0.05). Interestingly, prazosin pre-treatment did not prevent the dramatic depletion of glutathione by high dose APAP and it had no effect on the quantity of the glutathione conjugate formed. However, prazosin pre-treatment caused a significant increase in recovery of the administered dose (530 mg/kg) as the glucuronide metabolite (p < 0.05). UDP-glucuronosyltransferase (UGT) is involved in the bioinactivation of APAP by glucuronidation and we showed that prazosin had no effect on microsomal UGT kinetics. Thus, prazosin had no effect on either APAP-mediated glutathione depletion or the extent of APAP-glutathione conjugate formation and may be affecting other mechanisms to reduce oxidative stress caused by a toxic dose of APAP.     

Identification of novel toxicity-associated metabolites by metabolomics and mass isotopomer analysis of acetaminophen metabolism in wild-type and Cyp2e1-null mice

CYP2E1 is recognized as the most important enzyme for initiation of acetaminophen (APAP)-induced toxicity. In this study, the resistance of Cyp2e1-null mice to APAP treatment was confirmed by comparing serum aminotransferase activities and blood urea nitrogen levels in wild-type and Cyp2e1-null mice. However, unexpectedly, profiling of major known APAP metabolites in urine and serum revealed that the contribution of CYP2E1 to APAP metabolism decreased with increasing APAP doses administered. Measurement of hepatic glutathione and hydrogen peroxide levels exposed the importance of oxidative stress in determining the consequence of APAP overdose. Subsequent metabolomic analysis was capable of constructing a principal components analysis (PCA) model that delineated a relationship between urinary metabolomes and the responses to APAP treatment. Urinary ions high in wild-type mice treated with 400 mg/kg APAP were elucidated as 3-methoxy-APAP glucuronide (VII) and three novel APAP metabolites, including S-(5-acetylamino-2-hydroxyphenyl)mercaptopyruvic acid (VI, formed by a Cys-APAP transamination reaction in kidney), 3,3'-biacetaminophen (VIII, an APAP dimer), and a benzothiazine compound (IX, originated from deacetylated APAP), through mass isotopomer analysis, accurate mass measurement, tandem mass spectrometry fragmentation, in vitro reactions, and chemical treatments. Dose-, time-, and genotype-dependent appearance of these minor APAP metabolites implied their association with the APAP-induced toxicity and potential biomarker application. Overall, the oxidative stress elicited by CYP2E1-mediated APAP metabolism might significantly contribute to APAP-induced toxicity. The combination of genetically modified animal models, mass isotopomer analysis, and metabolomics provides a powerful and efficient technical platform to characterize APAP-induced toxicity through identifying novel biomarkers and unraveling novel mechanisms.     

Lipopolysaccharide-induced hepatic oxidative injury is not potentiated by knockout of GPX1 and SOD1 in mice

Knockout of copper, zinc-superoxide dismutase (SOD1) and (or) cellular glutathione peroxidase (GPX1) has been reported to have dual impacts on coping with free radical-induced oxidative injury. Because bacterial endotoxin lipopolysaccharide (LPS) triggers inflammatory responses involving the release of cytokines, nitric oxide and superoxide in targeted organs such as liver, in this study we used SOD1 knockout (SOD1−/−), GPX1 knockout (GPX1−/−), GPX1 and SOD1 double-knockout (DKO) and their wild-type (WT) mice to investigate the role of these two antioxidant enzymes in LPS-induced oxidative injury in liver. Mice of the four genotypes (2 month old) were killed at 0, 3, 6 or 12 h after an i.p. injection of saline or 5 mg LPS/kg body weight. The LPS injection caused similar increase in plasma alanine aminotransferase among the four genotypes. Hepatic total glutathione (GSH) was decreased (P < 0.05) compared with the initial values by the LPS injection at all time points in the WT mice, but only at 6 and 12 h in the other three genotypes. The GSH level in the DKO mice was higher (P < 0.05) than in the WT at 6 h. Although the LPS injection resulted in substantial increases in plasma NO in a time-dependent manner in all genotypes, the NO level in the DKO mice was lower (P < 0.05) at 3, 6 and 12 h than in the WT. The level in the GPX1−/− and SOD1−/− mice was also lower (P < 0.05) than in the WT at 3 h. The LPS-mediated hepatic protein nitration was detected in the WT and GPX1−/− mice at 3, 6 or 12 h, but not in the SOD1−/−. In conclusion, knockout of SOD1 and (or) GPX1 did not potentiate the LPS-induced liver injury, but delayed the induced hepatic GSH depletion and plasma NO production.     

Cyclophilin D deficiency protects against acetaminophen-induced oxidant stress and liver injury

Acetaminophen (APAP) hepatotoxicity is the main cause of acute liver failure in humans. Although mitochondrial oxidant stress and induction of the mitochondrial permeability transition (MPT) have been implicated in APAP-induced hepatotoxicity, the link between these events is unclear. To investigate this, this study evaluated APAP hepatotoxicity in mice deficient of cyclophilin D, a protein component of the MPT. Treatment of wild type mice with APAP resulted in focal centrilobular necrosis, nuclear DNA fragmentation and formation of reactive oxygen (elevated glutathione disulphide levels) and peroxynitrite (nitrotyrosine immunostaining) in the liver. CypD-deficient (Ppif(-/-)) mice were completely protected against APAP-induced liver injury and DNA fragmentation. Oxidant stress and peroxynitrite formation were blunted but not eliminated in CypD-deficient mice. Thus, mitochondrial oxidative stress and induction of the MPT are critical events in APAP hepatotoxicity in vivo and at least part of the APAP-induced oxidant stress and peroxynitrite formation occurs downstream of the MPT.     

Cellular glutathione peroxidase protects mice against lethal oxidative stress induced by various doses of diquat

This study was to determine if cellular glutathione peroxidase (GPX1) protects against acute oxidative stress induced by diquat. Lethality and hepatic biochemical indicators in GPX1 knockout mice [GPX1(-/-)] were compared with those of wild-type mice (WT) after an intraperitoneal injection of diquat at 6, 12, 24, or 48 mg/kg of body weight. Although the WT survived all the doses, the GPX1(-/-) survived only 6 mg diquat/kg and were killed by 12, 24, and 48 mg diquat/kg at 52, 4.4 and 3.9 hr, respectively. Compared with those of surviving mice that were sacrificed on Day 7, the dead GPX1(-/-) had diquat dose-dependent increases (P < 0.05) in plasma alanine aminotransferase (ALT) activities. The GPX1(-/-) also had higher (P < 0.05) liver carbonyl contents than those of the WT, but the differences were irrespective of diquat doses. Whereas hepatic total GPX and phospholipid hydroperoxide glutathione peroxidase activities or hepatic GPX1 protein was not significantly affected by the diquat treatment, liver thioredoxin reductase and catalase activities were lower (P < 0.05) in the GPX1(-/-) injected with 12 mg diquat/kg than those of other groups. In conclusion, normal GPX1 expression is necessary to protect mice against the lethality, hepatic protein oxidation, and elevation of plasma ALT activity induced by 12-48 mg diquat/kg.     

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.     

Knockout of SOD1 promotes conversion of selenocysteine to dehydroalanine in murine hepatic GPX1 protein

Se-dependent glutathione peroxidase-1 (GPX1) and Cu,Zn-superoxide dismutase (SOD1) are two major intracellular antioxidant enzymes. The purpose of this study was to elucidate the biochemical mechanisms for the 40% loss of hepatic GPX1 activity in SOD1^−/− mice. Compared with the wild type (WT), the SOD1^−/− mice showed no change in the total amount of GPX1 protein. However, their total enzyme protein exhibited 31 and 38% decreases (P < 0.05) in the apparent k_cat for hydrogen peroxide and tert-butylperoxide (at 2 mM GSH), respectively. Most striking, mass spectrometry revealed two chemical forms of the 47th residue of GPX1: the projected native selenocysteine (Sec) and the Se-lacking dehydroalanine (DHA). The hepatic GPX1 protein of the SOD1^−/− mice contained 38% less Sec and 77% more DHA than that of WT and showed aggravated dissociation of the tetramer structure. In conclusion, knockout of SOD1 elevated the conversion of Sec to DHA in the active site of hepatic GPX1, leading to proportional decreases in the apparent k_cat and activity of the enzyme protein as a whole. Our data reveal a structural and kinetic mechanism for the in vivo functional dependence of GPX1 on SOD1 in mammals and provide a novel mass spectrometric method for the assay of oxidative modification of the GPX1 protein.     

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.     

Role of copper,zinc-superoxide dismutase in catalyzing nitrotyrosine formation in murine liver

The only known function of Cu,Zn-superoxide dismutase (SOD1) is to catalyze the dismutation of superoxide anion into hydrogen peroxide. Our objective was to determine if SOD1 catalyzes murine liver protein nitration induced by acetaminophen (APAP) and lipopolysaccharide (LPS). Liver and plasma samples were collected from young adult SOD1 knockout mice (SOD1(-/-)) and wild-type (WT) mice at 5 or 6 h after an ip injection of saline, APAP, or LPS. Hepatic nitrotyrosine formation was induced by APAP and LPS only in the WT mice. The diminished hepatic protein nitration in the SOD1(-/-) mice was not directly related to plasma nitrite and nitrate concentrations. Similar genotype differences were seen in liver homogenates treated with a bolus of peroxynitrite. Adding only the holo-, and not the apo-, SOD1 enzyme into the liver homogenates enhanced the reaction in an activity-dependent fashion and nearly eliminated the genotype difference at the high doses. Mass spectrometry showed four more nitrotyrosine residues in bovine serum albumin and 10 more nitrated protein candidates in the SOD1(-/-) liver homogenates by peroxynitrite with added SOD1. In conclusion, the diminished hepatic protein nitration mediated by APAP or LPS in the SOD1(-/-) mice is due to the lack of SOD1 activity per se.     

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.     

Melatonin Protects against Apoptosis-Inducing Factor (AIF)-Dependent Cell Death during Acetaminophen-Induced Acute Liver Failure

Acetaminophen (APAP) overdose is the most frequent cause of acute liver failure and is primarily caused by cytochrome P450 (CYP) 2E1-driven conversion of APAP into hepatotoxic metabolites. Several reports showed that melatonin attenuated APAP-induced acute liver failure. Nevertheless, the exact mechanism remains obscure. In the present study, we investigated the effects of melatonin on apoptosis-inducing factor (AIF)-dependent cell death in APAP-induced acute liver failure. Mice were intraperitoneally (i.p.) injected with different doses of melatonin (1.25, 5, 20 mg/kg) 30 min before APAP (300 mg/kg, i.p.). As expected, melatonin significantly alleviated APAP-induced cell death, as determined by TdT-mediated dUTP-biotin nick end labeling (TUNEL) assay. Further analysis showed that melatonin significantly attenuated APAP-induced activation of the serine/threonine kinase receptor interacting protein 1 (RIP1). In addition, melatonin inhibited APAP-induced hepatic c-Jun N-terminal kinase (JNK) phosphorylation and mitochondrial Bax translocation. Correspondingly, melatonin inhibited APAP-induced translocation of AIF from mitochondria to nuclei. Interestingly, no changes were induced by melatonin on hepatic CYP2E1 expression. In addition, melatonin had little effect on APAP-induced hepatic glutathione (GSH) depletion. In conclusion, melatonin protects against AIF-dependent cell death during APAP-induced acute liver failure through its direct inhibition of hepatic RIP1 and subsequent JNK phosphorylation and mitochondrial Bax translocation.     

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.