Margitta Lebofsky

Plasma and liver acetaminophen-protein adduct levels in mice after acetaminophen treatment: dose-response, mechanisms, and clinical implications.

At therapeutic doses, acetaminophen (APAP) is a safe and effective analgesic. However, overdose of APAP is the principal cause of acute liver failure in the West. Binding of the reactive metabolite of APAP (NAPQI) to proteins is thought to be the initiating event in the mechanism of hepatotoxicity. Early work suggested that APAP-protein binding could not occur without glutathione (GSH) depletion, and likely only at toxic doses. Moreover, it was found that protein-derived APAP-cysteine could only be detected in serum after the onset of liver injury. On this basis, it was recently proposed that serum APAP-cysteine could be used as diagnostic marker of APAP overdose. However, comprehensive dose-response and time course studies have not yet been done. Furthermore, the effects of co-morbidities on this parameter have not been investigated. We treated groups of mice with APAP at multiple doses and measured liver GSH and both liver and plasma APAP-protein adducts at various timepoints. Our results show that protein binding can occur without much loss of GSH. Importantly, the data confirm earlier work that showed that protein-derived APAP-cysteine can appear in plasma without liver injury. Experiments performed in vitro suggest that this may involve multiple mechanisms, including secretion of adducted proteins and diffusion of NAPQI directly into plasma. Induction of liver necrosis through ischemia-reperfusion significantly increased the plasma concentration of protein-derived APAP-cysteine after a subtoxic dose of APAP. While our data generally support the measurement of serum APAP-protein adducts in the clinic, caution is suggested in the interpretation of this parameter.

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

Abstract 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.

Liver-Specific Loss of Atg5 Causes Persistent Activation of Nrf2 and Protects Against Acetaminophen-Induced Liver Injury

Autophagy is an evolutionarily conserved biological process that degrades intracellular proteins and organelles including damaged mitochondria through the formation of autophagosome. We have previously demonstrated that pharmacological induction of autophagy by rapamycin protects against acetaminophen (APAP)-induced liver injury in mice. In contrast, in the present study, we found that mice with the liver-specific loss of Atg5, an essential autophagy gene, were resistant to APAP-induced liver injury. Hepatocyte-specific deletion of Atg5 resulted in mild liver injury characterized by increased apoptosis and compensatory hepatocyte proliferation. The lack of autophagy in the Atg5-deficient mouse livers was confirmed by increased p62 protein levels and the absence of LC3-lipidation as well as autophagosome formation. Analysis of histological and clinical chemistry parameters indicated that the Atg5 liver-specific knockout mice are resistant to APAP overdose (500 mg/kg). Further investigations revealed that the bioactivation of APAP is normal in Atg5 liver-specific knockout mice although they had lower CYP2E1 expression. There was an increased basal hepatic glutathione (GSH) content and a faster recovery of GSH after APAP treatment due to persistent activation of Nrf2, a transcriptional factor regulating drug detoxification and GSH synthesis gene expression. In addition, we found significantly higher hepatocyte proliferation in the livers of Atg5 liver-specific knockout mice. Taken together, our data suggest that persistent activation of Nrf2 and increased hepatocyte proliferation protect against APAP-induced liver injury in Atg5 liver-specific knockout mice.

The impact of partial manganese superoxide dismutase (SOD2)-deficiency on mitochondrial oxidant stress, DNA fragmentation and liver injury during acetaminophen hepatotoxicity

UNLABELLED Acetaminophen (APAP) hepatotoxicity is the most frequent cause of acute liver failure in many countries. The mechanism of cell death is initiated by formation of a reactive metabolite that binds to mitochondrial proteins and promotes mitochondrial dysfunction and oxidant stress. Manganese superoxide dismutase (SOD2) is a critical defense enzyme located in the mitochondrial matrix. The objective of this investigation was to evaluate the functional consequences of partial SOD2-deficiency (SOD2+/-) on intracellular signaling mechanisms of necrotic cell death after APAP overdose. Treatment of C57Bl/6J wild type animals with 200mg/kg APAP resulted in liver injury as indicated by elevated plasma alanine aminotransferase activities (2870±180U/L) and centrilobular necrosis at 6h. In addition, increased tissue glutathione disulfide (GSSG) levels and GSSG-to-GSH ratios, delayed mitochondrial GSH recovery, and increased mitochondrial protein carbonyls and nitrotyrosine protein adducts indicated mitochondrial oxidant stress. In addition, nuclear DNA fragmentation (TUNEL assay) correlated with translocation of Bax to the mitochondria and release of apoptosis-inducing factor (AIF). Furthermore, activation of c-jun-N-terminal kinase (JNK) was documented by the mitochondrial translocation of phospho-JNK. SOD2+/- mice showed 4-fold higher ALT activities and necrosis, an enhancement of all parameters of the mitochondrial oxidant stress, more AIF release and more extensive DNA fragmentation and more prolonged JNK activation. CONCLUSIONS the impaired defense against mitochondrial superoxide formation in SOD2+/- mice prolongs JNK activation after APAP overdose and consequently further enhances the mitochondrial oxidant stress leading to exaggerated mitochondrial dysfunction, release of intermembrane proteins with nuclear DNA fragmentation and more necrosis.

Reduced activities of key enzymes of gluconeogenesis as possible cause of acute toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in rats

Male Sprague--Dawley rats (350-375 g) were injected i.p. with TCDD (25 [sublethal dose] and 125 micrograms/kg [lethal dose], respectively, in corn oil/acetone), or vehicle only; vehicle-treated animals were pair-fed to their TCDD-treated counterparts. 1, 2, 4, 8, 16, and 32 days (28 days for lethal dose) thereafter, animals were sacrificed and activities of two key enzymes of gluconeogenesis determined in livers of rats. In livers of pair-fed rats both enzyme activities were little affected. In the livers of TCDD-treated animals the activity of phosphoenolpyruvate carboxykinase (PEPCK, EC 4.1.1.32) decreased rapidly, exhibiting significant losses by the 2nd day after treatment. Time course and extent of loss of PEPCK activity (about 50%) were similar after either dose. The activity of glucose-6-phosphatase (G-6-Pase, EC 3.1.3.9) decreased more slowly as a result of TCDD treatment; statistically significant losses were observed by 4 or 8 days after the lethal and sublethal dose, respectively. These results confirm the hypothesis that reduced in vivo rates of gluconeogenesis in TCDD-treated rats are due to decreased activities of gluconeogenic enzymes. In an additional set of experiments, rats were treated with 125 micrograms/kg TCDD, 25 micrograms/kg TCDD, or with vehicle alone. The 25 micrograms/kg or vehicle-treated rats were then pair-fed to rats dosed with 125 micrograms/kg of TCDD. Mean time to death and body weight loss at the time of death were essentially identical in all groups, lending additional support to the hypothesis that reduced feed intake is the major cause of TCDD-induced death in male Sprague--Dawley rats. Both appetite suppression and reduced total PEPCK activity in whole livers occurred in the same dose-ranges of TCDD, suggesting the possibility of a cause-effect relationship.

Apoptosis-Inducing Factor Modulates Mitochondrial Oxidant Stress in Acetaminophen Hepatotoxicity

Acetaminophen (APAP) overdose causes liver injury in humans and mice. DNA fragmentation is a hallmark of APAP-induced cell death, and nuclear translocation of apoptosis-inducing factor (AIF) correlates with DNA fragmentation after APAP overdose. To test the hypothesis that AIF may be a critical mediator of APAP-induced cell death, fasted male AIF-deficient Harlequin (Hq) mice and respective wild-type (WT) animals were treated with 200 mg/kg APAP. At 6 h after APAP, WT animals developed severe liver injury as indicated by the increase in plasma alanine aminotransferase (ALT) activities (8600 ± 1870 U/l) and 61 ± 8% necrosis. This injury was accompanied by massive DNA strand breaks in centrilobular hepatocytes (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling [TUNEL] assay) and release of DNA fragments into the cytosol (anti-histone ELISA). In addition, there was formation of reactive oxygen (increase in liver glutathione disulfide (GSSG) levels and mitochondrial protein carbonyls) and peroxynitrite (nitrotyrosine [NT] staining) together with mitochondrial translocation of activated c-jun-N-terminal kinase (P-JNK) and release of AIF from the mitochondria. In contrast, Hq mice had significantly less liver injury (ALT: 330 ± 130 U/l; necrosis: 4 ± 2%), minimal nuclear DNA damage, and drastically reduced oxidant stress (based on all parameters) at 6 h. WT and Hq mice had the same baseline levels of cyp2E1 and of glutathione. The initial depletion of glutathione (20 min after APAP) was the same in both groups suggesting that there was no relevant difference in metabolic activation of APAP. Thus, AIF has a critical function in APAP hepatotoxicity by facilitating generation of reactive oxygen in mitochondria and, after nuclear translocation, AIF can be involved in DNA fragmentation.

Key enzymes of gluconeogenesis are dose-dependently reduced in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-treated rats

Male Sprague-Dawley rats (240–245 g) were dosed ip with 5, 15, 25, or 125 μg/kg -,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in corn oil. Ad libitumfed and pair-fed controls received vehicle (4 ml/kg) alone. Two or 8 days after dosing five rats of each group were sacrificed, their livers removed and assayed for the activities of three gluconeogenic enzymes [phosphoenol-pyruvate carboxykinase (PEPCK; EC 4.1.1.32), pyruvate carboxylase (PC; EC 6.4.1.1.), and glucose-6-phosphatase (G-6-Pase, EC 3.1.3.9)], and one glycolytic enzyme [pyruvate kinase (PK; EC 2.7.1.40)] by established procedures. The activity of PK was not affected by TCDD at either time point. The activity of G-6-Pase tended to be decreased in TCDD-treated animals, as compared to pair-fed controls, but the decrease was variable without an apparent dose-response. The activity of PEPCK was significantly decreased 2 days after dosing, but a clear dose-response was apparent only at the 8-day time point. Maximum loss of activity at the highest dose was 56% below pair-fed control levels. PC activity was slightly decreased 2 days after TCDD treatment and displayed statistically significant, dose-dependent reduction by 8 days after dosing with a 49% loss of enzyme activity after the highest dose. It is concluded that inhibition of gluconeogenesis by TCDD previously demonstrated in vivo is probably due to decreased activities of PEPCK and PC. The data also support the prevailing view that PEPCK and PC are rate-determining enzymes in gluconeogenesis.

Oxidant stress-induced liver injury in vivo: role of apoptosis, oncotic necrosis, and c-Jun NH2-terminal kinase activation

Oxidant stress is critically involved in various liver diseases. Superoxide formation causes c-Jun NH2-terminal kinase (JNK)- and caspase-dependent apoptosis in cultured hepatocytes. To verify these findings in vivo, male Fisher rats were treated with diquat and menadione. The oxidant stress induced by both compounds was confirmed by increased formation of glutathione disulfide and 4-hydroxynonenal protein adducts. Plasma alanine aminotransferase activities increased from 46+/-4 U/l in controls to 955+/-90 U/l at 6 h after diquat treatment. Hematoxylin and eosin staining of liver sections revealed large areas of necrotic cells at 3 and 6 h. DNA strandbreaks, evaluated with the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, showed clusters of TUNEL-positive cells, where the staining was predominantly cytosolic and the cells were swollen, indicating oncotic necrosis. There was no significant increase in caspase-3 activities or relevant release of DNA fragments into the cytosol at any time between 0 and 6 h after diquat treatment. Despite the activation of JNK after high doses of diquat, the JNK inhibitor SP-600125 did not protect against diquat-induced necrosis. Menadione alone did not cause liver injury, but, in combination with phorone and FeSO4, induced moderate oncotic necrosis. On the other hand, if animals were treated with galactosamine/endotoxin as positive control for apoptosis, caspase-3 activities were increased by 259%, the number of TUNEL-positive cells with apoptotic morphology was increased 103-fold, and DNA fragmentation was enhanced 6-fold. The data indicate that liver cell death initiated by diquat-induced superoxide formation in vivo is mediated predominantly by oncotic necrosis and is independent of JNK activation.

Purinergic receptor antagonist A438079 protects against acetaminophen-induced liver injury by inhibiting p450 isoenzymes, not by inflammasome activation.

Acetaminophen (APAP) overdose is the most frequent cause of acute liver failure in the western world. Controversy exists regarding the hypothesis that the hepatocyte injury is amplified by a sterile inflammatory response, rather than being the result of intracellular mechanisms alone. A recent study suggested that the purinergic receptor antagonist A438079 protects against APAP-induced liver injury by preventing the activation of the Nalp3 inflammasome in Kupffer cells and thereby preventing inflammatory injury. To test the hypothesis that A438079 actually affects the intracellular signaling events in hepatocytes, C57Bl/6 mice were treated with APAP (300 mg/kg) and A438079 (80 mg/kg) or saline and GSH depletion, protein adduct formation, c-jun-N-terminal kinase (JNK) activation, oxidant stress, and liver cell necrosis were determined between 0 and 6 h after APAP administration. APAP caused rapid GSH depletion, extensive protein adduct formation in liver homogenates and in mitochondria, JNK phosphorylation and mitochondrial translocation of phospho-JNK within 2 h, oxidant stress, and extensive centrilobular necrosis at 6 h. A438079 significantly attenuated GSH depletion, which resulted in a 50% reduction of total liver and mitochondrial protein adducts and substantial reduction of JNK activation, mitochondrial P-JNK translocation, oxidant stress, and liver injury. The same results were obtained using primary mouse hepatocytes. A438079 did not directly affect JNK activation induced by tert-butyl hydroperoxide and GSH depletion. However, A438079 dose-dependently inhibited hepatic P450 enzyme activity. Thus, the protective effect of A438079 against APAP hepatotoxicity in vivo can be explained by its effect on metabolic activation and cell death pathways in hepatocytes without involvement of the Nalp3 inflammasome.

The strength of the Fas ligand signal determines whether hepatocytes act as type 1 or type 2 cells in murine livers.

The BH3‐interacting domain death agonist Bid has been shown to be critical for Fas‐induced hepatocellular apoptosis. Furthermore, some studies have suggested that phosphorylation of Bid may determine its apoptotic function and may act as a switch to nonapoptotic functions. The aim of this study was to evaluate the role of Bid and phosphorylated Bid for Fas ligand (FasL)‐induced apoptosis in murine livers. The monoclonal antibody Jo2 and a hexameric form of sFasL (MegaFasL) were used to induce apoptosis in wild‐type, Bid‐deficient (Bid−/−), Bid transgenic mice expressing a nonphosphorable form of Bid and Fas receptor‐deficient lpr mice. Apoptosis sensitivity was determined in healthy mice and in mice following bile duct ligation, partial hepatectomy, or suramin pretreatment. As previously reported, loss of Bid protects mice against Jo2‐induced liver failure. Remarkably however, Bid−/− mice are highly sensitive to MegaFasL‐induced apoptosis. MegaFasL‐treated Bid−/− mice showed a typical type I cell signaling behavior with activation of caspase‐3 without Bax translocation to the mitochondria and no cytochrome C/Smac release into the cytosol. In contrast to previous in vitro findings, phosphorylation of Bid does not affect the sensitivity of hepatocytes to Fas receptor‐mediated apoptosis in vivo. Conclusion: Our data suggest that Bid mainly amplifies a weak death receptor signal in quiescent and nonquiescent hepatocytes rendering the liver more sensitive to FasL‐induced apoptosis. Thus, depending on the efficacy of Fas receptor activation, hepatocytes and nonparenchymal cells can either behave as type I or type II cells. (HEPATOLOGY 2009.)