Abstract
Acetaminophen (APAP) is a widely used analgesic and is safe at therapeutic doses. However, an overdose of APAP is hepatotoxic and accidental overdoses are increasingly common due to the presence of APAP in several combination medications. Formation of protein adducts (APAP-CYS) is central to APAP-induced liver injury and their removal by autophagy is an essential adaptive response after an acute overdose. Since the typical treatment for conditions such as chronic pain involves multiple doses of APAP over time, this study investigated APAP-induced liver injury after multiple subtoxic doses and examined the role of autophagy in responding to this regimen. Fed male C57BL/6J mice were administered repeated doses (75 mg/kg and 150 mg/kg) of APAP, followed by measurement of adducts within the liver, mitochondria, and in plasma, activation of the MAP kinase JNK, and markers of liver injury. The role of autophagy was investigated by treatment of mice with the autophagy inhibitor, leupeptin. Our data show that multiple treatments at the 150 mg/kg dose of APAP resulted in protein adduct formation in the liver and mitochondria, activation of JNK, and hepatocyte cell death, which was significantly exacerbated by inhibition of autophagy. While repeated dosing with the milder 75 mg/kg dose did not cause mitochondrial protein adduct formation, JNK activation, or liver injury, autophagy inhibition resulted in hepatocyte death even at this lower dose. These data illustrate the importance of adaptive responses such as autophagy in removing protein adducts and preventing liver injury, especially in clinically relevant situations involving repeated dosing with APAP.
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Abbreviations
- ALT:
-
Alanine aminotransferase
- AMAP:
-
N-Acetyl-m-aminophenol
- APAP:
-
N-Acetyl-p-aminophenol, acetaminophen
- APAP-CYS:
-
APAP-cysteine derived from protein adducts
- CYP:
-
Cytochrome P450
- GSH:
-
Glutathione
- H&E:
-
Hematoxylin and eosin
- JNK:
-
C-jun N-terminal kinase
- MPTP:
-
Mitochondrial permeability transition pore
- NAC:
-
N-Acetylcysteine
- NAPQI:
-
N-Acetyl-p-benzoquinone imine
- P-JNK:
-
Phospho-JNK
- TUNEL:
-
Terminal deoxynucleotidyl transferase dUTP nick end labeling
References
Akakpo JY, Ramachandran A, Kandel SE, Ni HM, Kumer SC, Rumack BH, Jaeschke H (2018) 4-Methylpyrazole protects against acetaminophen hepatotoxicity in mice and in primary human hepatocytes. Hum Exp Toxicol 37:1310–1322
Alhelail MA, Hoppe JA, Rhyee SH, Heard KJ (2011) Clinical course of repeated supratherapeutic ingestion of acetaminophen. Clin Toxicol (Phila) 49:108–112
Bajt ML, Cover C, Lemasters JJ, Jaeschke H (2006) Nuclear translocation of endonuclease G and apoptosis-inducing factor during acetaminophen-induced liver cell injury. Toxicol Sci 94:217–225
Chao X, Wang H, Jaeschke H, Ding WX (2018) Role and mechanisms of autophagy in acetaminophen-induced liver injury. Liver Int 38:1363–1374
Curry SC, Padilla-Jones A, Ruha AM, O’Connor AD, Kang AM, Wilkins DG, Jaeschke H, Wilhelms K, Gerkin RD (2019) The Acetaminophen Adduct Study Group. The relationship between circulating acetaminophen-protein adduct concentrations and alanine aminotransferase activities in patients with and without acetaminophen overdose and Toxicity. J Med Toxicol 15:143–155
Du K, Ramachandran A, McGill MR, Mansouri A, Asselah T, Farhood A, Woolbright BL, Ding WX, Jaeschke H (2017) Induction of mitochondrial biogenesis protects against acetaminophen hepatotoxicity. Food Chem Toxicol 108(Pt A):339–350
Hanawa N, Shinohara M, Saberi B, Gaarde WA, Han D, Kaplowitz N (2008) Role of JNK translocation to mitochondria leading to inhibition of mitochondria bioenergetics in acetaminophen-induced liver injury. J Biol Chem 283:13565–13577
Heard KJ, Green JL, James LP, Judge BS, Zolot L, Rhyee S, Dart RC (2011) Acetaminophen-cysteine adducts during therapeutic dosing and following overdose. BMC Gastroenterol 11:20
Hoffmann KJ, Streeter AJ, Axworthy DB, Baillie TA (1985) Identification of the major covalent adduct formed in vitro and in vivo between acetaminophen and mouse liver proteins. Mol Pharmacol 27:566–573
Hu J, Ramshesh VK, McGill MR, Jaeschke H, Lemasters JJ (2016) Low dose acetaminophen induces reversible mitochondrial dysfunction associated with transient c-Jun N-terminal kinase activation in mouse liver. Toxicol Sci 150:204–215
Jaeschke H (2015) Acetaminophen: dose-dependent drug hepatotoxicity and acute liver failure in patients. Dig Dis 33:464–471
Jaeschke H, Duan L, Nguyen N, Ramachandran A (2019) Mitochondrial damage and biogenesis in acetaminophen-induced liver injury. Liver Res 3:150–156
Kang AM, Padilla-Jones A, Fisher ES, Akakpo JY, Jaeschke H, Rumack BH, Gerkin RD, Curry SC (2020) The effect of 4-methylpyrazole on oxidative metabolism of acetaminophen in human volunteers. J Med Toxicol 16:169–176
Kelly JP, Battista DR, Shiffman S, Malone MK, Weinstein RB, Kaufman DW (2018) Knowledge of dosing directions among current users of acetaminophen-containing medications. Sci Pract Res 58:492–498
Kon K, Kim JS, Jaeschke H, Lemasters JJ (2004) Mitochondrial permeability transition in acetaminophen-induced necrosis and apoptosis of cultured mouse hepatocytes. Hepatology 40:1170–1179
Lancaster EM, Hiatt JR, Zarrinpar A (2015) Acetaminophen hepatotoxicity: an updated review. Arch Toxicol 89:193–199
Larson AM (2007) Acetaminophen hepatotoxicity. Clin Liver Dis 11:525–548
McGill MR, Jaeschke H (2013) Metabolism and disposition of acetaminophen: recent advances in relation to hepatotoxicity and diagnosis. Pharm Res 30:2174–2187
McGill MR, Jaeschke H (2015) A direct comparison of methods used to measure oxidized glutathione in biological samples: 2-vinylpyridine and N-ethylmaleimide. Toxicol Mech Methods 25:589–595
McGill MR, Williams CD, Xie Y, Ramachandran A, Jaeschke H (2012) Acetaminophen-induced liver injury in rats and mice: comparison of protein adducts, mitochondrial dysfunction, and oxidative stress in the mechanism of toxicity. Toxicol Appl Pharmacol 264:387–394
McGill MR, Lebofsky M, Norris HR, Slawson MH, Bajt ML, Xie Y, Williams CD, Wilkins DG, Rollins DE, Jaeschke H (2013) Plasma and liver acetaminophen-protein adduct levels in mice after acetaminophen treatment: dose-response, mechanisms, and clinical implications. Toxicol Appl Pharmacol 269:240–249
Muldrew KL, James LP, Coop L, McCullough SS, Hendrickson HP, Hinson JA, Mayeux PR (2002) Determination of acetaminophen-protein adducts in mouse liver and serum and human serum after hepatotoxic doses of acetaminophen using high-performance liquid chromatography with electrochemical detection. Drug Metab Dispos 30:446–451
Nguyen NT, Du K, Akakpo JY, Umbaugh DS, Jaeschke H, Ramachandran A (2021) Mitochondrial protein adduct and superoxide generation are prerequisites for early activation of c-jun N-terminal kinase within the cytosol after an acetaminophen overdose in mice. Toxicol Lett 338:21–31
Ni HM, Bockus A, Boggess N, Jaeschke H, Ding WX (2012) Activation of autophagy protects against acetaminophen-induced hepatotoxicity. Hepatology 55:222–232
Ni HM, Williams JA, Jaeschke H, Ding WX (2013) Zonated induction of autophagy and mitochondrial spheroids limits acetaminophen-induced necrosis in the liver. Redox Biol 1:427–432
Ni HM, McGill MR, Chao X, Du K, Williams JA, Xie Y, Jaeschke H, Ding WX (2016) Removal of acetaminophen protein adducts by autophagy protects against acetaminophen-induced liver injury in mice. J Hepatol 65:354–362
Ramachandran A, Jaeschke H (2019) Acetaminophen hepatotoxicity. Semin Liver Dis 39:221–234
Ramachandran A, Jaeschke H (2020) A mitochondrial journey through acetaminophen hepatotoxicity. Food Chem Toxicol 140:111282
Saito C, Lemasters JJ, Jaeschke H (2010) c-Jun N-terminal kinase modulates oxidant stress and peroxynitrite formation independent of inducible nitric oxide synthase in acetaminophen hepatotoxicity. Toxicol Appl Pharmacol 246:8–17
Tirmenstein MA, Nelson SD (1989) Subcellular binding and effects on calcium homeostasis produced by acetaminophen and a nonhepatotoxic regioisomer, 3’-hydroxyacetanilide, in mouse liver. J Biol Chem 264:9814–9819
Ueno T, Komatsu M (2019) Measuring nonselective and selective autophagy in the liver. Methods Mol Biol 1880:535–540
Wang H, Ni HM, Chao X, Ma X, Rodriguez YA, Chavan H, Wang S, Krishnamurthy P, Dobrowsky R, Xu DX, Jaeschke H, Ding WX (2019) Double deletion of PINK1 and Parkin impairs hepatic mitophagy and exacerbates acetaminophen-induced liver injury in mice. Redox Biol 22:101148
Win S, Than TA, Kaplowitz N (2018) The regulation of JNK signaling pathways in cell death through the interplay with mitochondrial SAB and upstream post-translational effects. Int J Mol Sci 19:E3657
Xie Y, McGill MR, Du K, Dorko K, Kumer SC, Schmitt TM, Ding WX, Jaeschke H (2015) Mitochondrial protein adducts formation and mitochondrial dysfunction during N-acetyl-m-aminophenol (AMAP)-induced hepatotoxicity in primary human hepatocytes. Toxicol Appl Pharmacol 289:213–222
Yoon E, Babar A, Choudhary M, Kutner M, Pyrsopoulos N (2016) Acetaminophen-induced hepatotoxicity: a comprehensive update. J Clin Transl Hepatol 4:131–142
Acknowledgements
This work was supported in part by the National Institutes of Health Grants R01 DK102142 (Co-PIs WXD/HJ), P20 GM103549 (HJ), and the Mechanisms of Liver Injury and Diseases COBRE P30 GM118247 (HJ). JYA was supported by a NIH predoctoral fellowship F31 DK1200194.
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Nguyen, N.T., Akakpo, J.Y., Weemhoff, J.L. et al. Impaired protein adduct removal following repeat administration of subtoxic doses of acetaminophen enhances liver injury in fed mice. Arch Toxicol 95, 1463–1473 (2021). https://doi.org/10.1007/s00204-021-02985-6
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DOI: https://doi.org/10.1007/s00204-021-02985-6