Acetaminophen (paracetamol, N-acetyl-p-aminophenol; APAP) is widely used as analgesic and antipyretic drug. At therapeutic doses, acetaminophen is safe to be used. Acetaminophen other than aspirin and ibuprofen has only weak anti-inflammatory properties. At higher doses, acetaminophen produces a centrilobular hepatic necrosis that can be fatal. Acetaminophen-lnduced Hepatotoxicity Observation/ Microscopic Examination Sign and Symptoms 1. Indicated fulminating hepatic necrosis 2. Eosinophilic degeneration of cells with pyknosis of nuclearmaterial in hepatocytes 3.
Vacuolization and early degenerative changes surrounding the portal areas 4. Mild polymorphonuclear leukocytic infiltration 5. Necrosis in the cells of the proximal tubules of the kidney 1. Nausea and vomiting within 2-3 hour of ingestion 2. Abdominal pain in the right upper quadrant 3. Liver dysfunction occurred within 24 hour 4. Dramatic increase in serum alanine aminofiansferase (ALT) and asparatate aminotransferase (AST) levels 5. Mild hyperbilirubinemia 6. Increased prothrombin time nephrotoxicity Histological Observation 1.
Glycogen loss and vacuolization of centrilobular hepatocytes by 2 hours 2. Clear demarcation of the centrilobular areas from the rest of the liver 3. Nuclear changes in centrilobular hepatocytes and single cell necrosis with pycnotic cells by 3 hours 4. Gross necrosis of the entire centrilobular areas by 6 hours 5. Hepatic congestion . Metabolism in Acetaminophen Toxicity 1. Drug metabolizing enzymes convert acetaminophen to a reactive metabolite that bound to proteins covalently 2. Glutathione detoxify the metabolite and form acetaminophen-glutathione conjugate at nontoxic dose.
However, the metabolite depleted hepatic glutathione and covalently bound to protein at toxic dose. Since diethylmaleate depleted hepatic glutathione without causing toxicity, it was postulated that glutathione depletion was not the mechanism of toxicity 3. N-acetyl-7r benzoquinoneimine (NAPQI), the reactive metabolite of acetaminophen is formed by cytochrome p-450 (CYP) by a direct two electron oxidation of acetaminophen. The CYP isoforms important in acetaminophen metabolism included CYP2El, CYP lA2, CYP 3A4, and CYP2D6.
Reaction of NAPQI with glutathione occurs by conjugation to form 3-glutathion-s-ylacetaminophen and by reduction to acetaminophen 4. The reaction could be catalyzed by glutathione transferase pi. Detoxification of NAPQI is extremely rapid, and the rapid rate explained why covalent binding to proteins was not observed in hepatocytes until glutathione was almost completed depleted Figure 1: Mechanism determinants in acetaminophen-induced hepatic necrosis Covalent Binding Covalent binding of acetaminophen to protein is correlated with acetaminophen induced hepatotoxicity.
The appearance of acetaminophen-protein adducts in serum correlated with increases of ALT and AST and act as a biomarker for the formation of hepatic acetaminophen-piotein adducts and acetaminophen toxicity. By using hepatocyte suspension assays, it suggested that covalent binding is not the mechanism of toxicity. Researchers found that acetaminophen toxicity in freshly isolated hamster hepatocytes occurred in two phases. Incubation of the hepatocyes with acetaminophen for 90 minutes resulted in glutathione depletion and covalent binding, but no toxicity.
Subsequent washing of the hepatocytes to remove acetaminophen and re-incubation of the hepatocytes with media alone resulted in significant toxicity in the re-incubation phase. Addition of N- acetylcysteine or dithiothreitol to the media protected the hepatocytes against development of toxicity. Alterations in Hepatic Blood Flow in Acetaminophen Toxicity 1. Occurs with hepatic congestion from the accumulation of red blood cells within endocytic vacuoles and the space of Disse with a collapse of the sinusoidal lumens 2.
Significant increase in liver weight at 1-5 hour and is approximately two fold over baseline levels at 6 hour, subsequently decreased by 24 h 3. Increased liver hemoglobin and decreased by 24h 4. Albumin studies indicated a blockage of blood flow 5. Liver weight decreased between 6 and 24 hour and large increased in serum ALT and AST levels indicates hepatocytes lysis 6. Intrahepatic pressure and portal vein pressure decreased dramatically 7. Acetaminophen damaged hepatic microvascular (sinusoidal endothelial cells) and produce hepatocellular injury 8.
The injury consists of swelling of endothelial cells and penetration of erythrocytes into the sinusoidal space of Disse 9. Endothelial cell damage play a role in the toxicity and the biochemical events associated with toxicity Oxidative Stress in Acetaminophen Toxicity 1. Iron-mediated oxidative stress (Fenton mechanism) Initiated by cellular superoxide formation and its dis-mutation to form increased hydrogen peroxide Superoxide is formed by uncoupling of cytochrome p-+sb2gt or other enzymes and mitochondria or activation of NADPH oxidase Glutathione is the cofactor for glutathione peroxidase.
Detoxification of peroxides is compromised in acetaminophen-induced toxicity Glutathione depletion increased intracellular peroxide levels and increased oxidative stress via Fenton mechanism Involves reduction of peroxide by ferrous ions forming highly reactive hydroxyl radical which oxidize lipids leading to initiation of lipid peroxidation and oxidation of proteins and nucleic acids Mediates protein aldehyde formation.
2. Nitric oxide (Nitrogen Stress) Reacts with superoxide at an extremely rapid rate to form peroxynitrite (both an oxidizing and a nitrating agent) Detoxified by glutathione which is depleted by NAPQI in acetaminophen-induced hepatotoxicity Peroxynitrite nitrates tyrosine lead to formation of the unique biomarker 3- nitrotyrosine, and nitrated proteins have been used as unique biomarkers of nitrogen stress 3. CYP2EI.
Plays a role in acetaminophen toxicity in vivo and is a major CYP contributing to the metabolism of acetaminophen to NAPEI Other CYPs including CYPlA2 and CYP3A4 also metabolize acetaminophen to NAPQI CYP2EI catalytic activity with characteristic coupling is a source of increased oxidative stress in the hepatocyte NADPH oxidase is the major respiratory burst enzyme that generates superoxide formation in activated Kupffer cells 4. Neutrophil-induced Oxidant stress.
Hypochlorite (hypochlorous acid) is produced by neutrophils involving myelo-peroxidase utilization of hydrogen peroxide and chloride ions The resultant hypochlorite reacts with tyrosine residues to form 3-chlorotyrosine Depletion of neutrophils by treatment with anti-Gr-l antibody (RB6-8c5) protected against acetaminophen induced liver injury, as evidenced by markedly reduced serum ALT level centrilobular hepatic necrosis, and improved mouse survival Mitochondrial lnjury as a Critical Alteration in Acetaminophen Toxicity 1.
Mitochondria are the target for acetaminophen reactive metabolite and a number of arylated proteins were found in it 2. Inhibition of mitochondrial respiration occurs at complexes I and II, but not complex III 3. Mitochondrial permeability transition (MPT) is the mechanism used in acetaminophen-induced hepatotoxicity 4. It represents an abrupt increase in the permeability of the inner mitochondrial membrane to ions and small molecular weight solutes 5. Oxidants such as peroxides and Ca++ promote MPT, and hepatocyte levels of peroxides increased in acetaminophen toxicity and alterations in calcium homeostasis 6.
The permeability changes are inner mitochondrial membrane depolarization, uncoupling of oxidative phosphorylation, release of intra-mitochondrial ions and metabolic intermediates, mitochondrial swelling, and decreased ATP synthesis 7. MPT is promoted by oxidative stress and is associated with large increase in oxidative stress 8.
Cyclosporine A blocks MPT at protein channel or pore that transports both anionic and cationic solutes of masses less than 1,500 Da, which may be the same structure as the voltage-dependent anion channel (VDAC), an essential element of the pore 9. Proteins in the MPT pore: adenine nucleotide translocator, cyclophilin D, and voltage-dependent anion channel 10.
Necrosis is mediated by opening of the MPR pore in the inner mitochondrial membrane lead to loss of ability to produce ATP 11. Apoptosis is mediated by opening of a pore or channel in the outer mitochondrial membrane, the mitochondrial apoptosis-induced channel 12. This pore release pro-apoptotic factors including cytochrome c, endonuclease G, smac/Diablo, and apoptosis-inducing factor (AIF) from the membrane space into the cytosol 13. Low ATP levels are associated with necrosis whereas adequate ATP levels favor apoptosis.
lnflammation, cytokines and chemokines in Devetopment of Acetaminophen Toxicity 1. Acetaminophen toxicity occurred with activation of Kupffer cells (hepatic macrophages) 2. Kupffer cell activation increased both pro-inflammatory and anti-inflammatory cytokines 3. Kupffer cell inactivators, gadolinium chloride and dextran sulfate, able to decrease acetaminophen toxicity 4. Interleukin 10, 11, and 13 are known to modulate the pro-inflammatory response in hepatic injury 5. Cyclooxygenase-2 (COX-2) derived prostaglandins are recognized for their critical role in female reproduction, bone resorption, renal function, and mucosal defense 6.
Prostaglandins play a protective role in various hepatotoxicities lntracellular Signaling Mechanisms in Acetaminophen Toxicity 1. C-Jun N terminal kinases (JNKs), a subfamily of the mitogen-activated protein (MAp) kinases, is activated by phosphorylation both in vitro and in vivo 2. JNK activation is mediated by reactive oxygen species and by TNF-O 3. JNK activation is associated with the initiation of mitochondrial permeability transition (MPT) 4. MPT leads to additional oxidative stress with loss of mitochondrial membrane potential and loss of the ability of the hepatocyte to synthesize ATP 5.
The JNK inhibitor did not alter GSH depletion but blocked JNK activation in homogenate, JNK translocation to mitochondria, and toxicity 6. DNA fragmentation is another mechanism that has been implicated in acetaminophen-induced hepatotoxicity 7. The presence of ladder-like DNA fragments indicated the involvement of a calcium-dependent endo-nuclease 8. Aurintricarboxylic acid, a calcium-endonuclease inhibitor, and EGTA, a chelator of calcium required for endo-nuclease activation, significantly decreased DNA fragmentation and toxicity 9.
Calcium-calmodulin antagonist chlorpromazine and calcium channel blocker verapamil decreased acetaminophen-induced hepatic necrosis and deceased DNA fragmentation 10. Endonuclease G is important in acetaminophen induced nuclear fragmentation and is present in the mitochondria and is released under conditions of outer mitochondrial membrane permeabilization Repairing Mechanisms of Acetaminophen Liver Toxicity 1. Proliferation of mature cell and hepatocytes are mediated by TNF-O and interleukin IL-6 2. STAT3, signal transduction factor that activates a large number of genes are important in hepatocyte regeneration 3.
PCNA is an auxiliary protein for DNA polymerase delta and a biomarker of increased cellular proliferation 4. Vascular endothelial growth factor (VEGF) is expressed by endothelial cells and is a critical mitogen and survival factor for endothelial cells 5. It is also the major regulator of angiogenesis during organ development and differentiation during embryogenesis and a critical mediator of angiogenesis in cancer 6. VEGF promotes the survival of endothelial cells by induces the expression of anti-apoptotic proteins in human endothelial cells 7.
Hypoxia, cytokines, iNOS, and hyperglycemia regulate VECF 8. VEGF has two primary receptors, VEGFRI and VEGFR2. VEGFR2 appears to mediate the angiogenic properties of this growth factor, whereas VEGRI may have pro-mitotic effects 9. VEGF have mitogenic effects on hepatocytes by orchestrating interactions between endothelial cells and hepatocytes 10. Activation of VEGFRI in vitro increased the release of mitogens (growth factor (HGF) and IL-6) by endothelial cells Figure 2: Mechanisms determinants in repair of acetaminophen-induced hepatic necrosis.