Acetaminophen or paracetamol (APAP) overdose is a common cause of liver injury. Silymarin (SLM) is a hepatoprotective agent widely used for treating liver injury of different origin. In order to evaluate the possible beneficial effects of SLM, Balb/c mice were pretreated with SLM (100 mg/kg b.wt. per os) once daily for three days. Two hours after the last SLM dose, the mice were administered APAP (300 mg/kg b.wt. i.p.) and killed 6 (T6), 12 (T12) and 24 (T24) hours later. SLM-treated mice exhibited a significant reduction in APAP-induced liver injury, assessed according to AST and ALT release and histological examination. SLM treatment significantly reduced superoxide production, as indicated by lower GSSG content, lower HO-1 induction, alleviated nitrosative stress, decreased p-JNK activation and direct measurement of mitochondrial superoxide production in vitro. SLM did not affect the APAP-induced decrease in CYP2E1 activity and expression during the first 12 hrs. Neutrophil infiltration and enhanced expression of inflammatory markers were first detected at T12 in both groups. Inflammation progressed in the APAP group at T24 but became attenuated in SLM-treated animals. Histological examination suggests that necrosis the dominant cell death pathway in APAP intoxication, which is partially preventable by SLM pretreatment. We demonstrate that SLM significantly protects against APAP-induced liver damage through the scavenger activity of SLM and the reduction of superoxide and peroxynitrite content. Neutrophil-induced damage is probably secondary to necrosis development.

Clinical and Transplant Pathology Department Institute for Clinical and Experimental Medicine Prague Czech Republic

Department of Metabolism and Diabetes Institute for Clinical and Experimental Medicine Prague Czech Republic

Department of Pharmacology Faculty of Medicine and Dentistry Palacky University Olomouc Olomouc Czech Republic

Department of Veterinary Science Faculty of Agrobiology Food and Natural Resources Czech University of Life Sciences Prague Prague Czech Republic

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Rumack BH. Acetaminophen misconceptions. Hepatology, 2004; 40(1): 10–5. doi: 10.1002/hep.20300 PubMed DOI

Prescott LF. Hepatotoxicity of mild analgesics. Br J Clin Pharmacol, 1980; 10 Suppl 2: 373S–379S. PubMed PMC

Kuriakose GC and Kurup MG. Antioxidant and hepatoprotective activity of Aphanizomenon flos-aquae Linn against paracetamol intoxication in rats. Indian J Exp Biol, 2010; 48(11): 1123–30. PubMed

Jaeschke H, McGill MR, and Ramachandran A. Oxidant stress, mitochondria, and cell death mechanisms in drug-induced liver injury: lessons learned from acetaminophen hepatotoxicity. Drug Metab Rev, 2012; 44(1): 88–106. doi: 10.3109/03602532.2011.602688 PubMed DOI PMC

Knight TR, Ho YS, Farhood A, and Jaeschke H. Peroxynitrite is a critical mediator of acetaminophen hepatotoxicity in murine livers: protection by glutathione. J Pharmacol Exp Ther, 2002; 303(2): 468–75. doi: 10.1124/jpet.102.038968 PubMed DOI

Knight TR and Jaeschke H. Peroxynitrite formation and sinusoidal endothelial cell injury during acetaminophen-induced hepatotoxicity in mice. Comp Hepatol, 2004; 3 Suppl 1: S46. PubMed PMC

Masubuchi Y, Suda C, and Horie T. Involvement of mitochondrial permeability transition in acetaminophen-induced liver injury in mice. J Hepatol, 2005; 42(1): 110–6. doi: 10.1016/j.jhep.2004.09.015 PubMed DOI

Bajt ML, Ramachandran A, Yan HM, Lebofsky M, Farhood A, Lemasters JJ, et al. Apoptosis-inducing factor modulates mitochondrial oxidant stress in acetaminophen hepatotoxicity. Toxicol Sci, 2011; 122(2): 598–605. doi: 10.1093/toxsci/kfr116 PubMed DOI PMC

Gujral JS, Knight TR, Farhood A, Bajt ML, and Jaeschke H. Mode of cell death after acetaminophen overdose in mice: apoptosis or oncotic necrosis? Toxicol Sci, 2002; 67(2): 322–8. PubMed

Jaeschke H, McGill MR, Williams CD, and Ramachandran A. Current issues with acetaminophen hepatotoxicity—a clinically relevant model to test the efficacy of natural products. Life Sci, 2011; 88(17–18): 737–45. doi: 10.1016/j.lfs.2011.01.025 PubMed DOI PMC

Wagner H, Diesel P, and Seitz M. [The chemistry and analysis of silymarin from Silybum marianum Gaertn]. Arzneimittelforschung, 1974; 24(4): 466–71. PubMed

Comelli MC, Mengs U, Schneider C, and Prosdocimi M. Toward the definition of the mechanism of action of silymarin: activities related to cellular protection from toxic damage induced by chemotherapy. Integr Cancer Ther, 2007; 6(2): 120–9. doi: 10.1177/1534735407302349 PubMed DOI

Nayak SS, Jain R, and Sahoo AK. Hepatoprotective activity of Glycosmis pentaphylla against paracetamol-induced hepatotoxicity in Swiss albino mice. Pharm Biol, 2011; 49(2): 111–7. doi: 10.3109/13880209.2010.501084 PubMed DOI

Jain NK and Singhai AK. Protective effects of Phyllanthus acidus (L.) Skeels leaf extracts on acetaminophen and thioacetamide induced hepatic injuries in Wistar rats. Asian Pac J Trop Med, 2011; 4(6): 470–4. doi: 10.1016/S1995-7645(11)60128-4 PubMed DOI

Sabina EP, Pragasam SJ, Kumar S, and Rasool M. 6-gingerol, an active ingredient of ginger, protects acetaminophen-induced hepatotoxicity in mice. Zhong Xi Yi Jie He Xue Bao, 2011; 9(11): 1264–9. PubMed

Wu M, Katta A, Gadde MK, Liu H, Kakarla SK, Fannin J, et al. Aging-associated dysfunction of Akt/protein kinase B: S-nitrosylation and acetaminophen intervention. PLoS One, 2009; 4(7): e6430 doi: 10.1371/journal.pone.0006430 PubMed DOI PMC

Jaeschke H, Xie Y, and McGill MR. Acetaminophen-induced Liver Injury: from Animal Models to Humans. J Clin Transl Hepatol, 2014; 2(3): 153–61. doi: 10.14218/JCTH.2014.00014 PubMed DOI PMC

Guengerich FP, Martin MV, Sohl CD, and Cheng Q. Measurement of cytochrome P450 and NADPH-cytochrome P450 reductase. Nat Protoc, 2009; 4(9): 1245–51. doi: 10.1038/nprot.2009.121 PubMed DOI PMC

Bustamante E, Soper JW, and Pedersen PL. A high-yield preparative method for isolation of rat liver mitochondria. Anal Biochem, 1977; 80(2): 401–8. PubMed

Ide T, Tsutsui H, Kinugawa S, Utsumi H, Kang D, Hattori N, et al. Mitochondrial electron transport complex I is a potential source of oxygen free radicals in the failing myocardium. Circ Res, 1999; 85(4): 357–63. PubMed

Wardman P. Fluorescent and luminescent probes for measurement of oxidative and nitrosative species in cells and tissues: progress, pitfalls, and prospects. Free Radic Biol Med, 2007; 43(7): 995–1022. doi: 10.1016/j.freeradbiomed.2007.06.026 PubMed DOI

Zhang DW, Shao J, Lin J, Zhang N, Lu BJ, Lin SC, et al. RIP3, an energy metabolism regulator that switches TNF-induced cell death from apoptosis to necrosis. Science, 2009; 325(5938): 332–6. doi: 10.1126/science.1172308 PubMed DOI

Chen X, Chen H, Deng R, and Shen J. Pros and cons of current approaches for detecting peroxynitrite and their applications. Biomed J, 2014; 37(3): 120–6. doi: 10.4103/2319-4170.134084 PubMed DOI

McGill MR and Jaeschke H. Metabolism and disposition of acetaminophen: recent advances in relation to hepatotoxicity and diagnosis. Pharm Res, 2013; 30(9): 2174–87. doi: 10.1007/s11095-013-1007-6 PubMed DOI PMC

Bunchorntavakul C and Reddy KR. Acetaminophen-related hepatotoxicity. Clin Liver Dis, 2013; 17(4): 587–607, viii. doi: 10.1016/j.cld.2013.07.005 PubMed DOI

Cheung C, Yu AM, Ward JM, Krausz KW, Akiyama TE, Feigenbaum L, et al. The cyp2e1-humanized transgenic mouse: role of cyp2e1 in acetaminophen hepatotoxicity. Drug Metab Dispos, 2005; 33(3): 449–57. doi: 10.1124/dmd.104.002402 PubMed DOI

Lee SS, Buters JT, Pineau T, Fernandez-Salguero P, and Gonzalez FJ. Role of CYP2E1 in the hepatotoxicity of acetaminophen. J Biol Chem, 1996; 271(20): 12063–7. PubMed

Xie W, Wang M, Chen C, Zhang X, and Melzig MF. Hepatoprotective effect of isoquercitrin against acetaminophen-induced liver injury. Life Sci, 2016; 152: 180–9. doi: 10.1016/j.lfs.2016.04.002 PubMed DOI

Al-Rasheed N, Faddah L, Al-Rasheed N, Bassiouni YA, Hasan IH, Mahmoud AM, et al. Protective Effects of Silymarin, Alone or in Combination with Chlorogenic Acid and/or Melatonin, Against Carbon Tetrachloride-induced Hepatotoxicity. Pharmacogn Mag, 2016; 12(Suppl 3): S337–45. doi: 10.4103/0973-1296.185765 PubMed DOI PMC

Miguez MP, Anundi I, Sainz-Pardo LA, and Lindros KO. Hepatoprotective mechanism of silymarin: no evidence for involvement of cytochrome P450 2E1. Chem Biol Interact, 1994; 91(1): 51–63. PubMed

Manov I, Motanis H, Frumin I, and Iancu TC. Hepatotoxicity of anti-inflammatory and analgesic drugs: ultrastructural aspects. Acta Pharmacol Sin, 2006; 27(3): 259–72. doi: 10.1111/j.1745-7254.2006.00278.x PubMed DOI

Du K, Ramachandran A, and Jaeschke H. Oxidative stress during acetaminophen hepatotoxicity: Sources, pathophysiological role and therapeutic potential. Redox Biol, 2016; 10: 148–156. doi: 10.1016/j.redox.2016.10.001 PubMed DOI PMC

Cederbaum AI. Cytochrome P450 2E1-dependent oxidant stress and upregulation of anti-oxidant defense in liver cells. J Gastroenterol Hepatol, 2006; 21 Suppl 3: S22–5. PubMed

Ishii T, Itoh K, Takahashi S, Sato H, Yanagawa T, Katoh Y, et al. Transcription factor Nrf2 coordinately regulates a group of oxidative stress-inducible genes in macrophages. J Biol Chem, 2000; 275(21): 16023–9. PubMed

Gardner CR, Heck DE, Yang CS, Thomas PE, Zhang XJ, DeGeorge GL, et al. Role of nitric oxide in acetaminophen-induced hepatotoxicity in the rat. Hepatology, 1998; 27(3): 748–54. doi: 10.1002/hep.510270316 PubMed DOI

Michael SL, Mayeux PR, Bucci TJ, Warbritton AR, Irwin LK, Pumford NR, et al. Acetaminophen-induced hepatotoxicity in mice lacking inducible nitric oxide synthase activity. Nitric Oxide, 2001; 5(5): 432–41. doi: 10.1006/niox.2001.0385 PubMed DOI

Hinson JA, Bucci TJ, Irwin LK, Michael SL, and Mayeux PR. Effect of inhibitors of nitric oxide synthase on acetaminophen-induced hepatotoxicity in mice. Nitric Oxide, 2002; 6(2): 160–7. doi: 10.1006/niox.2001.0404 PubMed DOI

Serviddio G, Bellanti F, Stanca E, Lunetti P, Blonda M, Tamborra R, et al. Silybin exerts antioxidant effects and induces mitochondrial biogenesis in liver of rat with secondary biliary cirrhosis. Free Radic Biol Med, 2014; 73: 117–26. doi: 10.1016/j.freeradbiomed.2014.05.002 PubMed DOI

Patel N, Joseph C, Corcoran GB, and Ray SD. Silymarin modulates doxorubicin-induced oxidative stress, Bcl-xL and p53 expression while preventing apoptotic and necrotic cell death in the liver. Toxicol Appl Pharmacol, 2010; 245(2): 143–52. doi: 10.1016/j.taap.2010.02.002 PubMed DOI

Serviddio G, Bellanti F, Giudetti AM, Gnoni GV, Petrella A, Tamborra R, et al. A silybin-phospholipid complex prevents mitochondrial dysfunction in a rodent model of nonalcoholic steatohepatitis. J Pharmacol Exp Ther, 2010; 332(3): 922–32. doi: 10.1124/jpet.109.161612 PubMed DOI

Koksal E, Gulcin I, Beyza S, Sarikaya O, and Bursal E. In vitro antioxidant activity of silymarin. J Enzyme Inhib Med Chem, 2009; 24(2): 395–405. doi: 10.1080/14756360802188081 PubMed DOI

Dambach DM, Watson LM, Gray KR, Durham SK, and Laskin DL. Role of CCR2 in macrophage migration into the liver during acetaminophen-induced hepatotoxicity in the mouse. Hepatology, 2002; 35(5): 1093–103. doi: 10.1053/jhep.2002.33162 PubMed DOI

Holt MP, Cheng L, and Ju C. Identification and characterization of infiltrating macrophages in acetaminophen-induced liver injury. J Leukoc Biol, 2008; 84(6): 1410–21. doi: 10.1189/jlb.0308173 PubMed DOI PMC

Jaeschke H. Innate immunity and acetaminophen-induced liver injury: why so many controversies? Hepatology, 2008; 48(3): 699–701. doi: 10.1002/hep.22556 PubMed DOI

Bajt ML, Farhood A, and Jaeschke H. Effects of CXC chemokines on neutrophil activation and sequestration in hepatic vasculature. Am J Physiol Gastrointest Liver Physiol, 2001; 281(5): G1188–95. doi: 10.1152/ajpgi.2001.281.5.G1188 PubMed DOI

Gujral JS, Farhood A, Bajt ML, and Jaeschke H. Neutrophils aggravate acute liver injury during obstructive cholestasis in bile duct-ligated mice. Hepatology, 2003; 38(2): 355–63. doi: 10.1053/jhep.2003.50341 PubMed DOI

Jaeschke H, Farhood A, Bautista AP, Spolarics Z, Spitzer JJ, and Smith CW. Functional inactivation of neutrophils with a Mac-1 (CD11b/CD18) monoclonal antibody protects against ischemia-reperfusion injury in rat liver. Hepatology, 1993; 17(5): 915–23. PubMed

Xie Y, Williams CD, McGill MR, Lebofsky M, Ramachandran A, and Jaeschke H. Purinergic receptor antagonist A438079 protects against acetaminophen-induced liver injury by inhibiting p450 isoenzymes, not by inflammasome activation. Toxicol Sci, 2013; 131(1): 325–35. doi: 10.1093/toxsci/kfs283 PubMed DOI PMC

Jaeschke H and Lemasters JJ. Apoptosis versus oncotic necrosis in hepatic ischemia/reperfusion injury. Gastroenterology, 2003; 125(4): 1246–57. PubMed

Moriwaki K and Chan FK. RIP3: a molecular switch for necrosis and inflammation. Genes Dev, 2013; 27(15): 1640–9. doi: 10.1101/gad.223321.113 PubMed DOI PMC

Cho YS, Challa S, Moquin D, Genga R, Ray TD, Guildford M, et al. Phosphorylation-driven assembly of the RIP1-RIP3 complex regulates programmed necrosis and virus-induced inflammation. Cell, 2009; 137(6): 1112–23. doi: 10.1016/j.cell.2009.05.037 PubMed DOI PMC

He S, Wang L, Miao L, Wang T, Du F, Zhao L, et al. Receptor interacting protein kinase-3 determines cellular necrotic response to TNF-alpha. Cell, 2009; 137(6): 1100–11. doi: 10.1016/j.cell.2009.05.021 PubMed DOI

Vanlangenakker N, Vanden Berghe T, and Vandenabeele P. Many stimuli pull the necrotic trigger, an overview. Cell Death Differ, 2012; 19(1): 75–86. doi: 10.1038/cdd.2011.164 PubMed DOI PMC

Ramachandran A, McGill MR, Xie Y, Ni HM, Ding WX, and Jaeschke H. Receptor interacting protein kinase 3 is a critical early mediator of acetaminophen-induced hepatocyte necrosis in mice. Hepatology, 2013; 58(6): 2099–108. doi: 10.1002/hep.26547 PubMed DOI PMC

Dara L, Johnson H, Suda J, Win S, Gaarde W, Han D, et al. Receptor interacting protein kinase 1 mediates murine acetaminophen toxicity independent of the necrosome and not through necroptosis. Hepatology, 2015; 62(6): 1847–57. doi: 10.1002/hep.27939 PubMed DOI PMC

Feng S, Yang Y, Mei Y, Ma L, Zhu DE, Hoti N, et al. Cleavage of RIP3 inactivates its caspase-independent apoptosis pathway by removal of kinase domain. Cell Signal, 2007; 19(10): 2056–67. doi: 10.1016/j.cellsig.2007.05.016 PubMed DOI

Yang X, Chao X, Wang ZT, and Ding WX. The end of RIPK1-RIPK3-MLKL-mediated necroptosis in acetaminophen-induced hepatotoxicity? Hepatology, 2016; 64(1): 311–2. doi: 10.1002/hep.28263 PubMed DOI