扑热息痛肝损伤机制研究进展

扑热息痛（AAP）肝损伤是药物性肝损伤的常见原因之一。但迄今为止,其肝损伤机制仍不完全清楚。最新研究进展指出活性代谢产物的形成、谷胱甘肽的耗竭、线粒体蛋白的烷化和过氧化亚硝酸盐的形成是主要原因。本文主要描述了AAP过量所致的线粒体功能异常的研究进展,另外也综述了氧化应激和炎症介质在扑热息痛肝损伤机制中的作用。     

Intracellular signaling mechanisms of acetaminophen-induced liver cell death.

Acetaminophen hepatotoxicity is the leading cause of drug-induced liver failure. Despite substantial efforts in the past, the mechanisms of acetaminophen-induced liver cell injury are still incompletely understood. Recent advances suggest that reactive metabolite formation, glutathione depletion, and alkylation of proteins, especially mitochondrial proteins, are critical initiating events for the toxicity. Bcl-2 family members Bax and Bid then form pores in the outer mitochondrial membrane and release intermembrane proteins, e.g., apoptosis-inducing factor (AIF) and endonuclease G, which then translocate to the nucleus and initiate chromatin condensation and DNA fragmentation, respectively. Mitochondrial dysfunction, due to covalent binding, leads to formation of reactive oxygen and peroxynitrite, which trigger the membrane permeability transition and the collapse of the mitochondrial membrane potential. In addition to the diminishing capacity to synthesize ATP, endonuclease G and AIF are further released. Endonuclease G, together with an activated nuclear Ca2+,Mg2+-dependent endonuclease, cause DNA degradation, thereby preventing cell recovery and regeneration. Disruption of the Ca2+ homeostasis also leads to activation of intracellular proteases, e.g., calpains, which can proteolytically cleave structural proteins. Thus, multiple events including massive mitochondrial dysfunction and ATP depletion, extensive DNA fragmentation, and modification of intracellular proteins contribute to the development of oncotic necrotic cell death in the liver after acetaminophen overdose. Based on the recognition of the temporal sequence and interdependency of these mechanisms, it appears most promising to therapeutically target either the initiating event (metabolic activation) or the central propagating event (mitochondrial dysfunction and peroxynitrite formation) to prevent acetaminophen-induced liver cell death.     

Mechanism of protection by metallothionein against acetaminophen hepatotoxicity

Acetaminophen (APAP) overdose is the most frequent cause of drug-induced liver failure in the US. Metallothionein (MT) expression attenuates APAP-induced liver injury. However, the mechanism of this protection remains incompletely understood. To address this issue, C57BL/6 mice were treated with 100 μmol/kg ZnCl₂ for 3 days to induce MT. Twenty-four hours after the last dose of zinc, the animals received 300 mg/kg APAP. Liver injury (plasma ALT activities, area of necrosis), DNA fragmentation, peroxynitrite formation (nitrotyrosine staining), MT expression, hepatic glutathione (GSH), and glutathione disulfide (GSSG) levels were determined after 6 h. APAP alone caused severe liver injury with oxidant stress (increased GSSG levels), peroxynitrite formation, and DNA fragmentation, all of which were attenuated by zinc-induced MT expression. In contrast, MT knockout mice were not protected by zinc. Hydrogen peroxide-induced cell injury in primary hepatocytes was dependent only on the intracellular GSH levels but not on MT expression. Thus, the protective effect of MT in vivo was not due to the direct scavenging of reactive oxygen species. Zinc treatment had no effect on the early GSH depletion kinetics after APAP administration, which is an indicator of the metabolic activation of APAP to its reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI). However, MT was able to effectively trap NAPQI by covalent binding. We conclude that MT scavenges some of the excess NAPQI after GSH depletion and prevents covalent binding to cellular proteins, which is the trigger for the propagation of the cell injury mechanisms through mitochondrial dysfunction and nuclear DNA damage.     

Vascular and hepatocellular peroxynitrite formation during acetaminophen toxicity: role of mitochondrial oxidant stress.

Peroxynitrite may be involved in acetaminophen-induced liver damage. However, it is unclear if peroxynitrite is generated in hepatocytes or in the vasculature. To address this question, we treated C3Heb/FeJ mice with 300 mg/kg acetaminophen and assessed nitrotyrosine protein adducts as indicator for peroxynitrite formation. Vascular nitrotyrosine staining was evident before liver injury between 0.5 and 2 h after acetaminophen treatment. However, liver injury developed parallel to hepatocellular nitrotyrosine staining between 2 and 6 h after acetaminophen. The mitochondrial content of glutathione disulfide, as indicator of reactive oxygen formation determined 6 h after acetaminophen, increased from 2.8 +/- 0.6% in controls to 23.5 +/- 5.1%. A high dose of allopurinol (100 mg/kg) strongly attenuated acetaminophen protein-adduct formation and prevented the mitochondrial oxidant stress and liver injury after acetaminophen. Lower doses of allopurinol, which are equally effective in inhibiting xanthine oxidase, were not protective and had no effect on nitrotyrosine staining and acetaminophen protein adduct formation. In vitro experiments showed that allopurinol is not a direct scavenger of peroxynitrite. We conclude that there is vascular peroxynitrite formation during the first 2 h after acetaminophen treatment. On the other hand, reactive metabolites of acetaminophen bind to intracellular proteins and cause mitochondrial dysfunction and superoxide formation. Mitochondrial superoxide reacts with nitric oxide to form peroxynitrite, which is responsible for intracellular protein nitration. The pathophysiological relevance of vascular peroxynitrite for hepatocellular peroxynitrite formation and liver injury remains to be established.     

Oxidant stress, mitochondria, and cell death mechanisms in drug-induced liver injury: lessons learned from acetaminophen hepatotoxicity

Hepatotoxicity is a serious problem during drug development and for the use of many established drugs. For example, acetaminophen overdose is currently the most frequent cause of acute liver failure in the United States and Great Britain. Evaluation of the mechanisms of drug-induced liver injury indicates that mitochondria are critical targets for drug toxicity, either directly or indirectly through the formation of reactive metabolites. The consequence of these modifications is generally a mitochondrial oxidant stress and peroxynitrite formation, which leads to structural alterations of proteins and mitochondrial DNA and, eventually, to the opening of mitochondrial membrane permeability transition (MPT) pores. MPT pore formation results in a collapse of mitochondrial membrane potential and cessation of adenosine triphosphate synthesis. In addition, the release of intermembrane proteins, such as apoptosis-inducing factor and endonuclease G, and their translocation to the nucleus, leads to nuclear DNA fragmentation. Together, these events trigger necrotic cell death. Alternatively, the release of cytochrome c and other proapoptotic factors from mitochondria can promote caspase activation and apoptotic cell death. Drug toxicity can also induce an inflammatory response with the formation of reactive oxygen species by Kupffer cells and neutrophils. If not properly detoxified, these extracellularly generated oxidants can diffuse into hepatocytes and trigger mitochondrial dysfunction and oxidant stress, which then induces MPT and necrotic cell death. This review addresses the formation of oxidants and the defense mechanisms available for cells and applies this knowledge to better understand mechanisms of drug hepatotoxicity, especially acetaminophen-induced liver injury.     

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

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 200 mg/kg APAP resulted in liver injury as indicated by elevated plasma alanine aminotransferase activities (2870 ± 180 U/L) and centrilobular necrosis at 6 h. 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.     

Acetaminophen-induced oxidant stress and cell injury in cultured mouse hepatocytes: protection by N-acetyl cysteine.

The increase in cellular and mitochondrial glutathione disulfide (GSSG) levels and the GSSG:GSH ratio after acetaminophen (AAP) overdose suggest the involvement of an oxidant stress in the pathophysiology. However, the initial severe depletion of hepatocellular glutathione makes quantitative assessment of the oxidant stress difficult. Therefore, we tested the hypothesis that oxidant stress precedes the onset of cell injury in a cell culture model using 2',7'-dichlorofluorescein (DCF) fluorescence as a marker for intracellular oxidant stress. Cultured primary murine hepatocytes were exposed to 5 mM AAP. DCF fluorescence, XTT reduction, lactate dehydrogenase (LDH) release, and trypan blue uptake were determined from 0 to 12 h. After glutathione depletion at 3 h, DCF fluorescence increased by 16-fold and was maintained at that level up to 12 h. At 1.5 h after AAP, a significant decrease of the cellular XTT reduction capacity was observed, which continued to decline until 9 h. Cell necrosis (LDH release, trypan blue uptake) was detectable in 20% of cells at 6 h, with a significant further increase at later time points. Pretreatment with 20 mM N-acetylcysteine (NAC) 1 h before AAP enhanced cellular glutathione content, prevented or attenuated the AAP-induced decrease of GSH levels and XTT reduction capacity, respectively, and reduced the loss of cell viability. Additionally, treatment with NAC 2 h after AAP exposure prevented further deterioration of XTT reduction at 3 h and later, and attenuated cell necrosis. Thus, AAP-induced oxidant stress precedes cell necrosis and, in cultured hepatocytes, the oxidant stress is involved in the propagation of cell injury.     

Resveratrol prevents protein nitration and release of endonucleases from mitochondria during acetaminophen hepatotoxicity

Overdose of acetaminophen (APAP) is a common cause of acute liver injury and liver failure. The mechanism involves formation of a reactive metabolite, protein binding, oxidative stress and activation of c-Jun N-terminal kinase (JNK), mitochondrial dysfunction, and nuclear DNA fragmentation caused by endonucleases released from damaged mitochondria. Previous work has shown that the natural product resveratrol (RSV) can protect against APAP hepatotoxicity in mice through prevention of lipid peroxidation and anti-inflammatory effects. However, these earlier studies did not take into consideration several fundamental aspects of the pathophysiology. To address this, we treated C57Bl/6 mice with 300 mg/kg APAP followed by 50 mg/kg RSV 1.5 h later. Our results confirmed that RSV reduced liver injury after APAP overdose in mice. Importantly, RSV did not inhibit reactive metabolite formation and protein bindings, nor did it reduce activation of JNK. However, RSV decreased protein nitration after APAP treatment, possibly through direct scavenging of peroxynitrite. Interestingly, RSV also inhibited release of apoptosis-inducing factor and endonuclease G from mitochondria independent of Bax pore formation and prevented the downstream nuclear DNA fragmentation. Our data show that RSV protects against APAP hepatotoxicity both through antioxidant effects and by preventing mitochondrial release of endonucleases and nuclear DNA damage.     

Mechanisms of hepatotoxicity.

This review addresses recent advances in specific mechanisms of hepatotoxicity. Because of its unique metabolism and relationship to the gastrointestinal tract, the liver is an important target of the toxicity of drugs, xenobiotics, and oxidative stress. In cholestatic disease, endogenously generated bile acids produce hepatocellular apoptosis by stimulating Fas translocation from the cytoplasm to the plasma membrane where self-aggregation occurs to trigger apoptosis. Kupffer cell activation and neutrophil infiltration extend toxic injury. Kupffer cells release reactive oxygen species (ROS), cytokines, and chemokines, which induce neutrophil extravasation and activation. The liver expresses many cytochrome P450 isoforms, including ethanol-induced CYP2E1. CYP2E1 generates ROS, activates many toxicologically important substrates, and may be the central pathway by which ethanol causes oxidative stress. In acetaminophen toxicity, nitric oxide (NO) scavenges superoxide to produce peroxynitrite, which then causes protein nitration and tissue injury. In inducible nitric oxide synthase (iNOS) knockout mice, nitration is prevented, but unscavenged superoxide production then causes toxic lipid peroxidation to occur instead. Microvesicular steatosis, nonalcoholic steatohepatitis (NASH), and cytolytic hepatitis involve mitochondrial dysfunction, including impairment of mitochondrial fatty acid beta-oxidation, inhibition of mitochondrial respiration, and damage to mitochondrial DNA. Induction of the mitochondrial permeability transition (MPT) is another mechanism causing mitochondrial failure, which can lead to necrosis from ATP depletion or caspase-dependent apoptosis if ATP depletion does not occur fully. Because of such diverse mechanisms, hepatotoxicity remains a major reason for drug withdrawal from pharmaceutical development and clinical use.     

An epigrammatic (abridged) recounting of the myriad tales of astonishing deeds and dire consequences pertaining to nitric oxide and reactive oxygen species in mitochondria with an ancillary missive concerning the origins of apoptosis

Mitochondria play a central role in the life and death of cells. These organelles serve as the major energy-producing power-house, whereby the generation of ATP is associated with the utilization of molecular oxygen. A significant fraction (2-3%) of molecular oxygen consumed by mitochondria may be reduced in a one-electron fashion to yield a series of reactive oxygen species (ROS) such as superoxide anion radical, hydrogen peroxide, and hydroxyl radical. ROS are capable of damaging components of the electron transport apparatus and can, in turn, disrupt mitochondrial functioning, limiting cellular ATP levels and ultimately resulting in cell death. ROS-induced disruption of electron transport can perpetuate production of deleterious ROS and propagate mitochondrial damage. Consequently, mitochondria are highly enriched with water-soluble and lipid-soluble antioxidants (glutathione, ascorbate, Vitamin E, and coenzyme Q) and antioxidant enzymes, such as superoxide dismutase, glutathione peroxidase, catalase, thioredoxins, and peroxiredoxin. Another important antioxidant acting as a very effective scavenger of reactive lipid (peroxyl, alkoxyl) radicals is nitric oxide (NO) generated by mitochondrial nitric oxide synthase. However, NO can also be very disruptive to mitochondria function, a process facilitated by its high reactivity with superoxide. This interaction results in the formation of peroxynitrite, an oxidant capable of causing oxidative/nitrosative stress, further aggravating mitochondrial dysfunction, causing ATP depletion and damage to cells. Thus, in the most general sense, the effects of NO in mitochondria may be either protective or deleterious depending on specific conditions of local redox environment (redox potential, ratio of oxidized to reduced glutathione, transition metals, and the presence of other oxygen- and nitrogen-centered radicals).     

Acetaminophen-induced inhibition of Fas receptor-mediated liver cell apoptosis: mitochondrial dysfunction versus glutathione depletion

We reported previously that acetaminophen overdose interrupts the signaling pathway of Fas receptor-mediated apoptosis. The aim of our study was to investigate the mechanism of this effect. Male C3Heb/FeJ mice received a single dose of acetaminophen (300 mg/kg ip) and/or anti-Fas antibody Jo-2 (0.6 mg/kg iv). Some animals were treated with allopurinol (100 mg/kg po) 18 and 1 h before acetaminophen injection. After 90 min of Jo treatment, there was processing of procaspase-3 and a significant increase in liver caspase-3 activity, which is consistent with apoptotic cell death. Treatment with acetaminophen 2.5 h before Jo inhibited the increase in hepatic caspase-3 activity by preventing the processing of the proenzyme. When administered alone, acetaminophen did not induce caspase-3 activation but caused significant liver injury. Acetaminophen treatment alone caused mitochondrial cytochrome c release, depletion of the hepatic ATP content by 55%, and a 10-fold increase in mitochondrial glutathione disulfide levels. Pretreatment with allopurinol prevented the mitochondrial oxidant stress and liver injury due to acetaminophen toxicity but had no effect on Jo-mediated apoptosis. Allopurinol did not affect the initial glutathione depletion after acetaminophen. However, allopurinol restored the sensitivity of hepatocytes to Fas receptor signaling in acetaminophen-treated animals. Histochemical evaluation of DNA fragmentation with the TUNEL assay showed that acetaminophen eliminated Fas receptor-mediated apoptosis in all hepatocytes not just in the damaged cells of the centrilobular area. Our data suggest that acetaminophen-induced mitochondrial dysfunction and not the initial glutathione depletion is responsible for the interruption of Fas receptor-mediated apoptotic signaling in hepatocytes.     

Role of the peroxynitrite-poly (ADP-ribose) polymerase pathway in the pathogenesis of liver injury

Oxidative and nitrosative stress triggers DNA strand breakage, which then activates the nuclear enzyme poly(ADP-ribose) polymerase (PARP). One of the key triggers of DNA single strand breakage in pathophysiological conditions is peroxynitrite, a reactive species produced from the reaction of nitric oxide and superoxide. Activation of PARP can dramatically lower the intracellular concentration of its substrate, nicotinamide adenine dinucleotide, thus slowing the rate of glycolysis, electron transport and subsequently ATP formation. This process can result in cell dysfunction and cell death. Here we review the role of PARP in various forms of liver injury.     

Mitochondrial dysfunction induced by sertraline, an antidepressant agent

Sertraline, a selective serotonin reuptake inhibitor, has been used for the treatment of depression. Although it is generally considered safe, cases of sertraline-associated liver injury have been documented; however, the possible mechanism of sertraline-associated hepatotoxicity is entirely unknown. Here, we report that mitochondrial impairment may play an important role in liver injury induced by sertraline. In mitochondria isolated from rat liver, sertraline uncoupled mitochondrial oxidative phosphorylation and inhibited the activities of oxidative phosphorylation complexes I and V. Additionally, sertraline induced Ca(2+)-mediated mitochondrial permeability transition (MPT), and the induction was prevented by bongkrekic acid (BA), a specific MPT inhibitor targeting adenine nucleotide translocator (ANT), implying that the MPT induction is mediated by ANT. In freshly isolated rat primary hepatocytes, sertraline rapidly depleted cellular adenosine triphosphate (ATP) and subsequently induced lactate dehydrogenase leakage; both were attenuated by BA. Our results, including ATP depletion, induction of MPT, inhibition of mitochondrial respiration complexes, and uncoupling oxidative phosphorylation, indicate that sertraline-associated liver toxicity is possibly via mitochondrial dysfunction.     

Role of lipid peroxidation as a mechanism of liver injury after acetaminophen overdose in mice.

Mitochondrial oxidant stress and peroxynitrite formation have been implicated in the pathophysiology of acetaminophen-induced (AAP-induced) liver injury. Therefore, we tested the hypothesis that lipid peroxidation (LPO) might be involved in the injury mechanism. Male C3Heb/FeJ mice fed a diet high in vitamin E (1 g d-alpha-tocopheryl acetate/kg diet) for 1 week had 6.7-fold higher hepatic tocopherol levels than animals on the control diet (8.2 +/- 0.1 nmol/g liver). Treatment of fasted mice with 300 mg/kg AAP caused centrilobular necrosis with high plasma alanine aminotransferase (ALT) activities at 6 h (3280 +/- 570 U/l) but no evidence of LPO (hepatic malondialdehyde, 4-hydroxynonenal). Animals on the vitamin E diet had similar injury and LPO as mice on the control diet. To verify a potential effect of the vitamin E diet on drug-induced liver injury, animals were pretreated with a combination of phorone, FeSO4, and allyl alcohol. We observed, 2 h after allyl alcohol, massive LPO and liver cell injury in the livers of animals on the control diet, as indicated by a 32-fold increase in malondialdehyde levels, extensive staining for 4-hydroxynonenal, and ALT activities of 2310 +/- 340 U/l. Animals on the vitamin E diet had 40% lower hepatic malondialdehyde levels and 85% lower ALT values. Similar results were obtained when animals were treated for 3 days with alpha- or gamma-tocopherol (0.19 mmol/kg, ip). Both treatments reduced LPO and injury after allyl alcohol but had no effect on AAP hepatotoxicity. Thus, despite the previously shown mitochondrial oxidant stress and peroxynitrite formation, LPO does not appear to be a critical event in AAP-induced hepatotoxicity.     

Potential therapeutic effect of antioxidant therapy in shock and inflammation

Oxidative stress results from an oxidant/antioxidant imbalance, an excess of oxidants and/or a depletion of antioxidants. A considerable body of recent evidence suggests that oxidant stress plays a major role in several aspects of acute and chronic inflammation and is the subject of this review. Immunohistochemical and biochemical evidence demonstrate the significant role of reactive oxygen species (ROS) in acute and chronic inflammation. Initiation of lipid peroxidation, direct inhibition of mitochondrial respiratory chain enzymes, inactivation of glyceraldehyde-3-phosphate dehydrogenase, inhibition of membrane Na+/K+ ATP-ase activity, inactivation of membrane sodium channels, and other oxidative protein modifications contribute to the cytotoxic effect of ROS. All these toxicities are likely to play a role in the pathophysiology of shock, inflammation and ischemia and reperfusion. (2) Treatment with either peroxynitrite decomposition catalysts, which selectively inhibit peroxynitrite, or with SODm's, which selectively mimic the catalytic activity of the human superoxide dismutase (SOD) enzymes, have been shown to prevent in vivo the delayed tissue injury and the cellular energetic failure associated with inflammation. ROS (e.g., superoxide, peroxynitrite, hydroxyl radical and hydrogen peroxide) are all potential reactants capable of initiating DNA single strand breakage, with subsequent activation of the nuclear enzyme poly (ADP ribose) synthetase (PARS), leading to eventual severe energy depletion of the cells, and necrotic-type cell death. Antioxidant treatment inhibits the activation of PARS, and prevents the organ injury associated with acute and chronic inflammation.     

Mitochondrial protein thiol modifications in acetaminophen hepatotoxicity: Effect on HMG-CoA synthase

Acetaminophen (APAP) overdose is the leading cause of drug related liver failure in many countries. N-acetyl-p-benzoquinone imine (NAPQI) is a reactive metabolite that is formed by the metabolism of APAP. NAPQI preferentially binds to glutathione and then cellular proteins. NAPQI binding is considered an upstream event in the pathophysiology, especially when binding to mitochondrial proteins and therefore leads to mitochondrial toxicity. APAP caused a significant increase in liver toxicity 3 h post-APAP administration as measured by increased serum alanine aminotransferase (ALT) levels. Using high-resolution mitochondrial proteomics techniques to measure thiol and protein changes, no significant change in global thiol levels was observed. However, 3-hydroxy-3-methylglutaryl coenzyme A synthase 2 (HMG-CoA synthase) had significantly decreased levels of reduced thiols and activity after APAP treatment. HMG-CoA synthase is a key regulatory enzyme in ketogenesis and possesses a number of critical cysteines in the active site. Similarly, catalase, a key enzyme in hydrogen peroxide metabolism, also showed modification in protein thiol content. These data indicate post-translational modifications of a few selected proteins involved in mitochondrial and cellular regulation of metabolism during liver toxicity after APAP overdose. The pathophysiological relevance of these limited changes in protein thiols remains to be investigated.     

Pathophysiological role of the acute inflammatory response during acetaminophen hepatotoxicity

Neutrophils are recruited into the liver after acetaminophen (AAP) overdose but the pathophysiological relevance of this acute inflammatory response remains unclear. To address this question, we compared the time course of liver injury, hepatic neutrophil accumulation and inflammatory gene mRNA expression for up to 24 h after treatment with 300 mg/kg AAP in C3Heb/FeJ and C57BL/6 mice. Although there was no relevant difference in liver injury (assessed by the increase of plasma alanine aminotransferase activities and the areas of necrosis), the number of neutrophils and the expression of several pro-inflammatory genes (e.g., tumor necrosis factor-alpha, interleukin-1beta and macrophage inflammatory protein-2) was higher in C3Heb/FeJ than in C57BL/6 mice. In contrast, the expression of the anti-inflammatory genes interleukin-10 and heme oxygenase-1 was higher in C57BL/6 mice. Despite substantial hepatic neutrophil accumulation, none of the liver sections from both strains stained positive for hypochlorite-modified proteins, a specific marker for a neutrophil-induced oxidant stress. In addition, treatment with the NADPH oxidase inhibitors diphenyleneiodonium chloride or apocynin or the anti-neutrophil antibody Gr-1 did not protect against AAP hepatotoxicity. Furthermore, although intercellular adhesion molecule-1 (ICAM-1) was previously shown to be important for neutrophil extravasation and tissue injury in several models, ICAM-1-deficient mice were not protected against AAP-mediated liver injury. Together, these data do not support the hypothesis that neutrophils aggravate liver injury induced by AAP overdose.     

Pathophysiological role of poly(ADP-ribose) polymerase (PARP) activation during acetaminophen-induced liver cell necrosis in mice.

DNA fragmentation in hepatocytes occurs early after acetaminophen (AAP) overdose in mice. DNA strandbreaks can induce excessive activation of poly(ADP-ribose) polymerases (PARP), which may lead to oncotic necrosis. Based on controversial findings with chemical PARP inhibitors, the role of PARP-1 activation in AAP hepatotoxicity remains unclear. To investigate PARP-1 activation and evaluate a pathophysiological role of PARP-1, we used both PARP inhibitors (3-aminobenzamide; 5-aminoisoquinolinone) and PARP gene knockout mice (PARP-/-). Treatment of C3Heb/FeJ mice with 300 mg/kg AAP resulted in DNA fragmentation and alanine aminotransferase (ALT) release as early as 3 h, with further increase of these parameters up to 12 h. Few nuclei of hepatocytes stained positive for poly-ADP-ribosylated nuclear proteins (PAR) as indicator for PARP-1 activation at 4.5 h. However, the number of PAR-positive cells and staining intensity increased substantially at 6 and 12 h. Pretreatment with 500 mg/kg 3-aminobenzamide before AAP attenuated hepatic glutathione depletion and completely eliminated DNA fragmentation and liver injury. Delayed treatment several hours after AAP was still partially protective. On the other hand, liver injury was not attenuated in PARP-/- mice compared to wild-type animals. Similarly, the specific PARP-1 inhibitor 5-aminoisoquinolinone (5 mg/kg) was not protective. However, 3-aminobenzamide attenuated liver injury in WT and PARP-/- mice. In summary, PARP-1 activation is a consequence of DNA fragmentation after AAP overdose. However, PARP-1 activation is not a relevant event for AAP-induced oncotic necrosis. The protection of 3-aminobenzamide against AAP-induced liver injury was due to reduced metabolic activation and potentially its antioxidant effect but independent of PARP-1 inhibition.