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Toxic Metabolites of Acetaminophen Cause Protein Adducts and Oxidative Stress
- 1. Toxic Metabolites of Acetaminophen Maryl Wright
- 2. Acetaminophen (APAP) Acetaminophen (APAP) is an analgesic and fever reducer. Initially classified as NSAID APAP largely lacks peripheral anti- inflammatory properties Weak anti inflammatory properties--IV From Propublica.org
- 3. APAP is largely metabolized in the liver CYP2E1 is main Cytochrome P450 enzyme involved in APAP metabolism CYP3A4 CYP1A2 CYP2D6
- 5. Acetaminophen Protein Adducts Complex that forms when NAPQI binds to protein Confined to centrilobular hepatocytes Don’t occur in periportal hepatocytes At least 20 molecular adducts have been detected inside mitochondria, nucleus, and hepatocyte cytosol From slideshare.net, occupational-liver-disorders
- 6. Evidence for Protein Adducts in Hepatocytes with Toxic Levels of APAP J.A. Hinson, Adverse Drug Reactions, 2010 • Acetaminophen cysteine adduct • At 2–6 h hepatocytes undergoing necrosis • By 24 h all the necrotic cells contained adducts.
- 7. Evidence for Protein Adducts in Hepatocytes with Toxic Levels of APAP J.A. Hinson, Adverse Drug Reactions, 2010 acetaminophen nitrotyrosine adducts
- 8. Protein Adducts Cause Oxidative Stress in Hepatocytes J.A. Hinson, Chemical Research in Toxicology, 1998 • Acetaminophen nitrotyrosine adducts • Serum samples were analyzed for ALT (marker of hepatic cell lysis) and nitrate plus nitrite levels (marker of Nitric oxide synthesis).
- 9. Protein Adducts Trigger Mitochondria Dysfunction in Hepatocytes Yuchao Xie, Toxicological Applied Pharmacology, 2015 • Human hepatocytes • Acetaminophen cysteine adducts
- 10. Toxic Rescue Only approved antidote for APAP toxicity is oral or intravenous administration of N-acetyl-cysteine (NAC). NAC is a precursor in the synthesis of GSH. Restores the intracellular pool of GSH and neutralizes any residual NAPQI still present in the liver. Very effective if administered within the first 8 hours after toxic APAP ingestion. Many patients wait too long after overdosing. Liver transplant. Activation of autophagy may protect against APAP liver toxicity.
- 11. Conclusion • APAP is largely metabolized in the liver, predominantly by sulfation and glucuronidation. • Approximately 5%–10% of APAP is metabolized primarily by CYP2E1 to the toxic metabolite N-acetyl-p-benzoquinone imine (NAPQI). • At therapeutic doses of APAP, NAPQI is immediately inactivated by conjugation with glutathione (GSH). • At toxic does of APAP, NAPQI depletes GSH, modifies cellular proteins, and forms protein adducts. • Hepatotoxicity caused by liver cell necrosis. • NAC is only approved antidote for early hepatoxicity,
- 12. Questions?
Editor's Notes
- Weak—in IV- avoid 1st pass effect—can’t get PO
- Hepatic enzymes
- APAP is largely metabolized in the liver, predominantly by sulfation and glucuronidation Approximately 5%–10% of APAP is metabolized by cytochrome P450 (CYP), primarily by CYP2E1,11–13 to the toxic metabolite N-acetyl-p-benzoquinone imine (NAPQI) NAP-KEY. At therapeutic doses, NAPQI (and oxidant) is immediately inactivated by conjugation with reduced glutathione (GSH). GSH antioxidant. With over dosage of APAP (NAPQI) depletes GSH and modifies cellular proteins and forms protein adducts Hepatotoxicity caused by liver cell necrosis While the glucuronide and sulfate conjugates of APAP are directly excreted in urine, APAP-GSH is initially excreted in bile, degraded in other organs including the kidney (63–64), and the degradation products are ultimately excreted in urine (65).
- is a time course for formation of the acetaminophen protein adducts and development of toxicity in individual murine hepatocytes (Roberts et al. 1991). In this assay, the adducts are stained red. Adducts are visible in the liver sections within 15 min of dosing. By 1 h, staining intensity is maximal and adducts are confined to the centrilobular hepatocytes. Adducts do not occur in the periportal hepatocytes. At 2–6 h, hepatocytes containing adducts are undergoing necrotic changes as evidenced by vacuolization and pycnotic changes in the nuclei. Note that the only hepatocytes observed to develop necrosis were those containing acetaminophen-protein adducts. By 24 h, all the necrotic cells contained adducts. The majority of hepatocytes that had acetaminophen-protein adducts were reported to develop necrosis Acetaminophen cysteine adduct 3-cys-A
- Immunohistochemical comparison of cellular localization of acetaminophen-protein adducts to nitrotyrosine in hepatic proteins of mice treated with a toxic dose of acetaminophen. Mice were treated with acetaminophen (300 mg kg−1) or saline and livers removed at 4 h. (b) Liver section was immunochemically stained for nitrotyrosine in protein using an antinitrotyrosine antiserum. (c) Liver section from a saline-treated mouse stained for nitroytrosine in protein using antinitrotyrosine antiserum (Hinson et al. 1998) Nitrotyrosine is identified as an indicator or marker of cell damage, inflammation as well as NO production.
- Immunochemical analysis of liver sections for nitrotyrosine adducts or acetaminophen adducts—found in centrilobular areas of liver Comparison of acetaminophen-induced hepatotoxicity to nitric oxide formation in mice treated with a toxic dose of acetaminophen. Mice (n ) 6) were treated with acetaminophen (300 mg/kg) and sacrificed at 4 h. Serum samples were analyzed for ALT (marker of hepatic cell lysis) and nitrate plus nitrite levels (marker of Nitric oxide synthesis). The data points are serum levels of ALT and nitrate plus nitrite in the individual mice
- APAP triggers GSH depletion, mitochondria dysfunction and protein adduct formation in PHH( A) Time course of cellular glutathione depletion after 10mM APAP. (B) Loss of mitochondria membrane potential over 24 hours after 10mM APAP, as indicated by decrease of red/green fluorescence ratio using the JC-1 assay. APAP-protein adduct formation (C) in the whole cell and (D) in the mitochondria fraction over 15 hours after 10mM APAP. Data represent mean ± SE from experiments using cells from 3–8 donors. * P < 0.05 (compared with time 0). Mitochondria generate ATP
- Autophagy nutrient starvation lysosomal degradation of cellular contents removal of damaged mitochondria