Abstract
Acetaminophen (APAP) overdose is the leading cause of acute liver failure in western countries. APAP can cause extensive hepatocellular necrosis, which triggers an inflammatory response involving neutrophil and monocyte recruitment. Particularly the role of neutrophils in the injury mechanism of APAP hepatotoxicity has been highly controversial. Thus, the objective of the current study was to assess whether a potential contribution of neutrophils was dependent on the APAP dose and the sex of the animals. Male and female C57BL/6 J mice were treated with 300 or 600 mg/kg APAP and the injury and inflammatory cell recruitment was evaluated between 6 and 48 h. In both male and female mice, ALT plasma levels and the areas of necrosis peaked at 12–24 h after both doses with more severe injury at the higher dose. In addition, Ly6g-positive neutrophils started to accumulate in the liver at 6 h and peaked at 6–12 h after 300 mg/kg and 12–24 h after 600 mg/kg for both sexes; however, the absolute numbers of hepatic neutrophils in the liver were significantly higher after the 600 mg/kg dose. Neutrophil infiltration correlated with mRNA levels of the neutrophil chemoattractant Cxcl2 in the liver. Treating mice with an anti-Cxcl2 antibody at 2 h after APAP significantly reduced neutrophil accumulation at 24 h after both doses and in both sexes. However, the injury was significantly reduced only after the high overdose. Thus, neutrophils, recruited through Cxcl2, have no effect on APAP-induced liver injury after 300 mg/kg but aggravate the injury only after severe overdoses.
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Data sharing not applicable to this article as no datasets were generated or analysed during the current study. All data generated or analysed during this study are included in this published article [and its supplementary information files].
References
Akakpo JY, Ramachandran A, Curry SC, Rumack BH, Jaeschke H (2022) Comparing N-acetylcysteine and 4-methylpyrazole as antidotes for acetaminophen overdose. Arch Toxicol 96:453–465
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
Bajt ML, Farhood A, Lemasters JJ, Jaeschke H (2008) Mitochondrial bax translocation accelerates DNA fragmentation and cell necrosis in a murine model of acetaminophen hepatotoxicity. J Pharmacol Exp Ther 324:8–14
Bankhead P, Loughrey MB, Fernández JA, Dombrowski Y, McArt DG, Dunne PD, McQuaid S, Gray RT, Murray LJ, Coleman HG, James JA, Salto-Tellez M, Hamilton PW (2017) QuPath: Open source software for digital pathology image analysis. Sci Rep 7:16878
Bhushan B, Apte U (2019) Liver Regeneration after Acetaminophen Hepatotoxicity: Mechanisms and Therapeutic Opportunities. Am J Pathol 189:719–729
Bhushan B, Walesky C, Manley M, Gallagher T, Borude P, Edwards G, Monga SP, Apte U (2014) Pro-regenerative signaling after acetaminophen-induced acute liver injury in mice identified using a novel incremental dose model. Am J Pathol 184:3013–3025
Chang L, Xu D, Zhu J, Ge G, Kong X, Zhou Y (2020) Herbal therapy for the treatment of acetaminophen-associated liver injury: recent advances and future perspectives. Front Pharmacol 11:313
Chauhan A, Sheriff L, Hussain MT, Webb GJ, Patten DA, Shepherd EL, Shaw R, Weston CJ, Haldar D, Bourke S, Bhandari R, Watson S, Adams DH, Watson SP, Lalor PF (2020) The platelet receptor CLEC-2 blocks neutrophil mediated hepatic recovery in acetaminophen induced acute liver failure. Nat Commun 11:1939
Corcoran GB, Racz WJ, Smith CV, Mitchell JR (1985) Effects of N-acetylcysteine on acetaminophen covalent binding and hepatic necrosis in mice. J Pharmacol Exp Ther 232:864–872
Cover C, Liu J, Farhood A, Malle E, Waalkes MP, Bajt ML, Jaeschke H (2006) Pathophysiological role of the acute inflammatory response during acetaminophen hepatotoxicity. Toxicol Appl Pharmacol 216:98–107
Dai G, He L, Chou N, Wan YJ (2006) Acetaminophen metabolism does not contribute to gender difference in its hepatotoxicity in mouse. Toxicol Sci 92:33–41
Dambach DM, Watson LM, Gray KR, Durham SK, Laskin DL (2002) Role of CCR2 in macrophage migration into the liver during acetaminophen-induced hepatotoxicity in the mouse. Hepatology 35:1093–1103
Du K, Williams CD, McGill MR, Jaeschke H (2014) Lower susceptibility of female mice to acetaminophen hepatotoxicity: Role of mitochondrial glutathione, oxidant stress and c-jun N-terminal kinase. Toxicol Appl Pharmacol 281:58–66
Du K, Ramachandran A, Jaeschke H (2016) Oxidative stress during acetaminophen hepatotoxicity: Sources, pathophysiological role and therapeutic potential. Redox Biol 10:148–156
Gujral JS, Farhood A, Bajt ML, Jaeschke H (2003) Neutrophils aggravate acute liver injury during obstructive cholestasis in bile duct-ligated mice. Hepatology 38:355–363
Gujral JS, Liu J, Farhood A, Hinson JA, Jaeschke H (2004) Functional importance of ICAM-1 in the mechanism of neutrophil-induced liver injury in bile duct-ligated mice. Am J Physiol Gastrointest Liver Physiol 286:G499-507
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
Hasegawa T, Malle E, Farhood A, Jaeschke H (2005) Generation of hypochlorite-modified proteins by neutrophils during ischemia-reperfusion injury in rat liver: attenuation by ischemic preconditioning. Am J Physiol Gastrointest Liver Physiol 289:G760-767
Holt MP, Cheng L, Ju C (2008) Identification and characterization of infiltrating macrophages in acetaminophen-induced liver injury. J Leukoc Biol 84:1410–1421
Imaeda AB, Watanabe A, Sohail MA, Mahmood S, Mohamadnejad M, Sutterwala FS, Flavell RA, Mehal WZ (2009) Acetaminophen-induced hepatotoxicity in mice is dependent on Tlr9 and the Nalp3 inflammasome. J Clin Invest 119:305–314
Jaeschke H (2003) Molecular mechanisms of hepatic ischemia-reperfusion injury and preconditioning. Am J Physiol Gastrointest Liver Physiol 284:G15-26
Jaeschke H (2006) Mechanisms of Liver Injury. II. Mechanisms of neutrophil-induced liver cell injury during hepatic ischemia-reperfusion and other acute inflammatory conditions. Am J Physiol Gastrointest Liver Physiol 290:G1083–G1088
Jaeschke H (2008) Innate immunity and acetaminophen-induced liver injury: why so many controversies? Hepatology 48:699–701
Jaeschke H (2015) Acetaminophen: dose-dependent drug hepatotoxicity and acute liver failure in patients. Dig Dis 33:464–471
Jaeschke H, Liu J (2007) Neutrophil depletion protects against murine acetaminophen hepatotoxicity: another perspective. Hepatology 45:1588–1589
Jaeschke H, Ramachandran A (2020) Mechanisms and pathophysiological significance of sterile inflammation during acetaminophen hepatotoxicity. Food Chem Toxicol 138:111240
Jaeschke H, Smith CW (1997) Mechanisms of neutrophil-induced parenchymal cell injury. J Leukoc Biol 61:647–653
Jaeschke H, Farhood A, Smith CW (1990) Neutrophils contribute to ischemia/reperfusion injury in rat liver in vivo. FASEB J 4:3355–3359
Jaeschke H, Williams CD, Ramachandran A, Bajt ML (2012) Acetaminophen hepatotoxicity and repair: the role of sterile inflammation and innate immunity. Liver Int 32:8–20
Jaeschke H, Ramachandran A, Chao X, Ding WX (2019) Emerging and established modes of cell death during acetaminophen-induced liver injury. Arch Toxicol 93:3491–3502
James LP, McCullough SS, Knight TR, Jaeschke H, Hinson JA (2003) Acetaminophen toxicity in mice lacking NADPH oxidase activity: role of peroxynitrite formation and mitochondrial oxidant stress. Free Radic Res 37:1289–1297
Jollow DJ, Mitchell JR, Potter WZ, Davis DC, Gillette JR, Brodie BB (1973) Acetaminophen-induced hepatic necrosis. II. Role of covalent binding in vivo. J Pharmacol Exp Ther 187:195–202
Ju C, Reilly TP, Bourdi M, Radonovich MF, Brady JN, George JW, Pohl LR (2002) Protective role of Kupffer cells in acetaminophen-induced hepatic injury in mice. Chem Res Toxicol 15:1504–1513
Kaiser SK, Dart RC (2022) The Roles of Antidotes in Emergency Situations. Emerg Med Clin North Am 40:381–394
Kubes P, Mehal WZ (2012) Sterile inflammation in the liver. Gastroenterology 143:1158–1172
Laskin DL, Gardner CR, Price VF, Jollow DJ (1995) Modulation of macrophage functioning abrogates the acute hepatotoxicity of acetaminophen. Hepatology 21:1045–1050
Lawson JA, Farhood A, Hopper RD, Bajt ML, Jaeschke H (2000) The hepatic inflammatory response after acetaminophen overdose: role of neutrophils. Toxicol Sci 54:509–516
Liu ZX, Han D, Gunawan B, Kaplowitz N (2006) Neutrophil depletion protects against murine acetaminophen hepatotoxicity. Hepatology 43:1220–1230
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)). Methods 25:402–408
Marques PE, Amaral SS, Pires DA, Nogueira LL, Soriani FM, Lima BH, Lopes GA, Russo RC, Avila TV, Melgaço JG, Oliveira AG, Pinto MA, Lima CX, De Paula AM, Cara DC, Leite MF, Teixeira MM, Menezes GB (2012) Chemokines and mitochondrial products activate neutrophils to amplify organ injury during mouse acute liver failure. Hepatology 56:1971–1982
Marques PE, Oliveira AG, Pereira RV, David BA, Gomides LF, Saraiva AM, Pires DA, Novaes JT, Patricio DO, Cisalpino D, Menezes-Garcia Z, Leevy WM, Chapman SE, Mahecha G, Marques RE, Guabiraba R, Martins VP, Souza DG, Mansur DS, Teixeira MM, Leite MF, Menezes GB (2015) Hepatic DNA deposition drives drug-induced liver injury and inflammation in mice. Hepatology 61:348–360
Masubuchi Y, Nakayama J, Watanabe Y (2011) Sex difference in susceptibility to acetaminophen hepatotoxicity is reversed by buthionine sulfoximine. Toxicology 287:54–60
McConnachie LA, Mohar I, Hudson FN, Ware CB, Ladiges WC, Fernandez C, Chatterton-Kirchmeier S, White CC, Pierce RH, Kavanagh TJ (2007) Glutamate cysteine ligase modifier subunit deficiency and gender as determinants of acetaminophen-induced hepatotoxicity in mice. Toxicol Sci 99:628–636
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, 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
Mitchell JR, Jollow DJ, Potter WZ, Gillette JR, Brodie BB (1973) Acetaminophen-induced hepatic necrosis. IV. Protective role of glutathione. J Pharmacol Exp Ther 187:211–217
Nguyen NT, Umbaugh DS, Sanchez-Guerrero G, Ramachandran A, Jaeschke H (2022) Kupffer cells regulate liver recovery through induction of chemokine receptor CXCR2 on hepatocytes after acetaminophen overdose in mice. Arch Toxicol 96:305–320
Piperno E, Berssenbruegge DA (1976) Reversal of experimental paracetamol toxicosis with N-acetylcysteine. Lancet 2(7988):738–739
Ramachandran A, Jaeschke H (2018) Acetaminophen toxicity: novel insights into mechanisms and future perspectives. Gene Expr 18:19–30
Ramachandran A, Jaeschke H (2019) Acetaminophen hepatotoxicity. Semin Liver Dis 39:221–234
Rohrer PR, Rudraiah S, Goedken MJ, Manautou JE (2014) Is nuclear factor erythroid 2-related factor 2 responsible for sex differences in susceptibility to acetaminophen-induced hepatotoxicity in mice? Drug Metab Dispos 42:1663–1674
Roth K, Strickland J, Copple BL (2020) Regulation of macrophage activation in the liver after acute injury: Role of the fibrinolytic system. World J Gastroenterol 26:1879–1887
Rumack BH, Bateman DN (2012) Acetaminophen and acetylcysteine dose and duration: past, present and future. Clin Toxicol (phila) 50:91–98
Saito C, Yan HM, Artigues A, Villar MT, Farhood A, Jaeschke H (2010a) Mechanism of protection by metallothionein against acetaminophen hepatotoxicity. Toxicol Appl Pharmacol 242:182–190
Saito C, Zwingmann C, Jaeschke H (2010b) Novel mechanisms of protection against acetaminophen hepatotoxicity in mice by glutathione and N-acetylcysteine. Hepatology 51:246–254
Stravitz RT, Lee WM (2019) Acute liver failure. Lancet 394(10201):869–881
Subramanya SB, Venkataraman B, Meeran MFN, Goyal SN, Patil CR, Ojha S (2018) Therapeutic potential of plants and plant derived phytochemicals against acetaminophen-induced liver injury. Int J Mol Sci 19:3776
Umbaugh DS, Nguyen NT, Jaeschke H, Ramachandran A (2021) Mitochondrial membrane potential drives early change in mitochondrial morphology after acetaminophen exposure. Toxicol Sci 180:186–195
Williams CD, Bajt ML, Farhood A, Jaeschke H (2010a) Acetaminophen-induced hepatic neutrophil accumulation and inflammatory liver injury in CD18-deficient mice. Liver Int 30:1280–1292
Williams CD, Farhood A, Jaeschke H (2010b) Role of caspase-1 and interleukin-1beta in acetaminophen-induced hepatic inflammation and liver injury. Toxicol Appl Pharmacol 247:169–178
Williams CD, Antoine DJ, Shaw PJ, Benson C, Farhood A, Williams DP, Kanneganti TD, Park BK, Jaeschke H (2011) Role of the Nalp3 inflammasome in acetaminophen-induced sterile inflammation and liver injury. Toxicol Appl Pharmacol 252:289–297
Williams CD, Bajt ML, Sharpe MR, McGill MR, Farhood A, Jaeschke H (2014) Neutrophil activation during acetaminophen hepatotoxicity and repair in mice and humans. Toxicol Appl Pharmacol 275:122–133
Yang W, Tao Y, Wu Y, Zhao X, Ye W, Zhao D, Fu L, Tian C, Yang J, He F, Tang L (2019) Neutrophils promote the development of reparative macrophages mediated by ROS to orchestrate liver repair. Nat Commun 10:1076
You Q, Holt M, Yin H, Li G, Hu CJ, Ju C (2013) Role of hepatic resident and infiltrating macrophages in liver repair after acute injury. Biochem Pharmacol 86:836–843
Zhang C, Feng J, Du J, Zhuo Z, Yang S, Zhang W, Wang W, Zhang S, Iwakura Y, Meng G, Fu YX, Hou B, Tang H (2018) Macrophage-derived IL-1α promotes sterile inflammation in a mouse model of acetaminophen hepatotoxicity. Cell Mol Immunol 15:973–982
Acknowledgements
This work was funded in part by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) grants R01 DK102142 (HJ) and DK125465 (AR), and National Institute of General Medicine (NIGMS) funded Liver Disease COBRE grants P20 GM103549 and P30 GM118247 (HJ). DSU was supported by a National Institutes of Health predoctoral fellowship (F31 DK134197).
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Nguyen, N.T., Umbaugh, D.S., Smith, S. et al. Dose-dependent pleiotropic role of neutrophils during acetaminophen-induced liver injury in male and female mice. Arch Toxicol 97, 1397–1412 (2023). https://doi.org/10.1007/s00204-023-03478-4
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DOI: https://doi.org/10.1007/s00204-023-03478-4