• RASHA N. ABU-AJAMIEH Department of Pharmacology and Biomedical Sciences, Faculty of Pharmacy and Medical Sciences, University of Petra, Amman, Jordan
  • BAYAN Y. GHANIM University of Petra Pharmaceutical Center (UPPC), University of Petra, Amman, Jordan
  • OMAR S. GAMMOH Department of Pharmacy, Faculty of Health Sciences, American University of Madaba, Madaba, Jordan
  • NIDAL A. QINNA Department of Pharmacology and Biomedical Sciences, Faculty of Pharmacy and Medical Sciences, University of Petra, Amman, Jordan, University of Petra Pharmaceutical Center (UPPC), University of Petra, Amman, Jordan


Objective: Acetaminophen (APAP) overdose contributes to liver damage through modulation of pro-apoptotic processing. This study evaluated the involvement of caspase/Bax factors in APAP hepatotoxicity in vivo and in vitro.

Methods: The involvement of caspase/Bax factors in APAP hepatotoxicity was evaluated in BALB/c mice and on isolated primary mouse hepatocytes. In vitro MTT assay was carried out on primary cultured mouse hepatocytes treated with APAP (2.5, 5, 10 mmol) and Annexin V/PI staining was employed to cell suspension for imaging under fluorescence microscopy. In addition, caspase-3 concentrations were determined in cell lysates. In vivo, mice were treated with a toxic dose of APAP (700 mg/kg) and immunodetection of Bax was made by Western Blot. Vitamin C (Vit C) was used as a hepato-protectant due to its known antioxidant activities.

Results: In vitro dose-dependent increase in mitochondrial electron transport capacity was evident in isolated mouse primary hepatocytes incubated with the high dose of APAP (10 mmol) compared to both nontreated cells and cells pre-treated with Vitamin C (Vit C) (0.5 mmol) (p<0.05). Apoptosis was confirmed in hepatocytes through Annexin V staining after APAP treatment and the signal was reduced when hepatocytes were pre-treated with Vit C. In addition, caspase-3 concentration was decreased in cells pretreated with Vit C prior to APAP exposure. In vivo, Bax immunodetection utilizing western blotting was increased in mice treated with the toxic dose of APAP (700 mg/kg) and attenuated through pre-treatment with Vit C.

Conclusion: Modulation of apoptotic caspase/Bax pathway is present in hepatocytes undergoing APAP-induced toxicity.

Keywords: Liver injury, Hepatoprotection, Hepatotoxicity, Cell proliferation, Programmed cell death, Mitochondrial stress


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1. Michaut A, Moreau C, Robin MA, Fromenty B. Acetaminophen-induced liver injury in obesity and nonalcoholic fatty liver disease. Liver Int 2014;34:e171–9.
2. Ghanem CI, Perez MJ, Manautou JE, Mottino AD. Acetaminophen from liver to brain: new insights into drug pharmacological action and toxicity. Pharmacol Res 2016;109:119-31.
3. Nagai H, Matsumaru K, Feng G, Kaplowitz N. Reduced glutathione depletion causes necrosis and sensitization to tumor necrosis factor-?–induced apoptosis in cultured mouse hepatocytes. Hepatology 2002;36:55–64.
4. Jaeschke H, Xie Y, McGill MR. Acetaminophen-induced liver injury: from animal models to humans. J Clin Transl Hepatol 2014;2:153–61.
5. Reed JC. Bcl-2 on the brink of breakthroughs in cancer treatment. Nat Publ Gr 2018;25:3–6.
6. Goldar S, Khaniani MS, Derakhshan SM, Baradaran B. Molecular mechanisms of apoptosis and roles in cancer development and treatment. Asian Pacific J Cancer Prev 2015;16:2129–44.
7. Mariño G, Kroemer G. Mechanisms of apoptotic phosphatidylserine exposure. Cell Res 2013;23:1247–8.
8. Kale MA, Bindu SM, Khadkikar P. Role of antioxidants and nutrition in oxidative stress: a review. Int J Appl Pharm 2015;7:1–4.
9. Al Shaker HA, Qinna NA, Badr M, Al Omari MMH, Idkaidek N, Matalka KZ, et al. Glucosamine modulates propranolol pharmacokinetics via intestinal permeability in rats. Eur J Pharm Sci 2017;105:137–43.
10. Al Shaker HA, Qinna NA, Al Hroub H, Al Omari MMH, Badwan AA. RP-HPLC–UV method for the quantification of propranolol in rat’s serum and Krebs buffer using one-step protein precipitation. Acta Chromatogr 2017;30:1–6.
11. Beebe SJ, Buescher ES, Stark RH, Schoenbach KH. Nanosecond pulsed electric field (nsPEF) application effects on human cells: intracellular membrane disruption and apoptosis induction. IEEE 2002;30:251.
12. Berridge MV, Tan AS. Characterization of the cellular reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT): subcellular localization, substrate dependence, and involvement of mitochondrial electron transport in MTT reduction. Arch Biochem Biophys 1993;303:474–82.
13. Riss TL, Moravec RA, Niles AL, Duellman S, Benink HA, Worzella TJ, et al. Cell viability assays. eli lilly and company and the national center for advancing translational sciences; 2004.
14. Paiva GS, Taft CA, Carvalho MC, De Souza IA, Barbosa da Silva EC, Cavalcanti KP, et al. A comparative study of the effects of vitamins C and E in the development of sarcoma 180 in mice. J Cancer 2014;4:724–6.
15. McGill MR, Williams CD, Xie Y, Ramachandran A, Jaeschke H. 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 2012;264:387–94.
16. Qinna NA, Ghanim BY. Chemical induction of hepatic apoptosis in rodents. J Appl Toxicol 2019;7:178–90.
17. Kavanagh TJ, Rademacher PM, Nelson SD, Stamper BD, Mohar I, White CC. Acetaminophen-induced liver damage in mice is associated with gender-specific adduction of peroxiredoxin-6. Redox Biol 2014;2:377–87.
18. Qinna NA, Shubbar MH, Matalka KZ, Al-Jbour N, Ghattas MA, Badwan AA. Glucosamine enhances paracetamol bioavailability by reducing its metabolism. J Pharm Sci 2015;104:257–65.
19. Tripathy D, Grammas P. Acetaminophen inhibits neuronal inflammation and protects neurons from oxidative stress. J Neuroinflammation 2009;6:10.
20. Ott M, Gogvadze V, Orrenius S, Zhivotovsky B. Mitochondria, oxidative stress and cell death. Apoptosis 2007;12:913–22.
21. Jaeschke H, Ramachandran A. Oxidant stress and lipid peroxidation in acetaminophen hepatotoxicity. React Oxyg Species 2018;5:145–58.
22. Kon K, Kim JS, Jaeschke H, Lemasters JJ. Mitochondrial permeability transition in acetaminophen-induced necrosis and apoptosis of cultured mouse hepatocytes. Hepatology 2004;40:1170–9.
23. Rai Y, Pathak R, Kumari N, Sah DK, Pandey S, Kalra N, et al. Mitochondrial biogenesis and metabolic hyperactivation limits the application of MTT assay in the estimation of radiation induced growth inhibition. Sci Rep 2018;8:1531.
24. Adeneye A, Olagunju J. Protective effect of oral ascorbic acid (vitamin c) against acetaminophen-induced hepatic injury in rats. Afr J Biomed Res 2010;11:55–61.
25. Abdulkhaleq FM, Alhussainy TM, Badr MM, Abu Khalil AA, Gammoh O, Ghanim BY, et al. Anti-oxidative stress effects of vitamin C, E and B 12, and their combination can protect the liver against acetaminophen-induced hepatotoxicity in rats. Drug Des Dev Ther 2018;12:3525–33.
26. Abdulrazzaq AM, Badr M, Gammoh O, Abu Khalil AA, Ghanim BY, Alhussainy TM, et al. Hepatoprotective actions of ascorbic acid, alpha-lipoic acid and silymarin or their combination against acetaminophen-induced hepatotoxicity in rats. Medicina (B Aires) 2019;55:181.
27. Mellidou I, Kanellis AK. Genetic control of ascorbic acid biosynthesis and recycling in horticultural crops. Front Chem 2017;5:1–8.
28. Birben E, Sahiner UM, Sackesen C, Erzurum S, Kalayci O. Oxidative stress and antioxidant defense. World Allergy Organ J 2012;5:9–19.
29. Adikwu E, Deo O. Hepatoprotective effect of vitamin C (Ascorbic Acid). Pharmacol Pharm 2013;4:84–92.
30. Jayadi T, Widiasmoko B. Curcumin benefits as antioxidant, anti inflammation and antiapoptosis ameliorate paracetamol toxicity. Asian J Pharm Clin Res 2018;11:1–3.
31. Rajeshkumar S, Kayalvizhi D. Antioxidant and hepatoprotective effect of aqueous and ethanolic extracts of important medicinal plant pongamia pinnata (Family: Leguminoseae). Asian J Pharm Clin Res 2015;8:67–70.
32. Jaeschke H, Bajt ML. Intracellular signaling mechanisms of acetaminophen-induced liver cell death. Toxicol Sci 2006;89:31–41.
33. Smith T, Engelbrecht L, Smith C. Anti-inflammatory cellular targets on neutrophils elucidated using a novel cell migration model and confocal microscopy: a clinical supplementation study. J Inflamm 2018;15:1–10.
34. Yiang GTT, Yu YLL, Lin KTT, Chen JNN, Chang WJJ, WEI CWW. Acetaminophen induces JNK/p38 signaling and activates the caspase-9-3-dependent cell death pathway in human mesenchymal stem cells. Int J Mol Med 2015;36:485–92.
35. Saile B, DiRocco P, Dudas J, El-Armouche H, Sebb H, Eisenbach C, et al. IGF-I induces DNA synthesis and apoptosis in rat liver hepatic stellate cells (HSC) but DNA synthesis and proliferation in rat liver myofibroblasts (rMF). Lab Invest 2004;84:1037–49.
36. Bajt ML, Farhood A, Lemasters JJ, Jaeschke H. Mitochondrial bax translocation accelerates DNA fragmentation and cell necrosis in a murine model of acetaminophen hepatotoxicity. J Pharmacol Exp Ther 2008;324:8–14.
37. Sharma S, Singh RL, Kakkar P. Modulation of Bax/Bcl-2 and caspases by probiotics during acetaminophen-induced apoptosis in primary hepatocytes. Food Chem Toxicol 2011;49:770–9.
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How to Cite
ABU-AJAMIEH, R. N., B. Y. GHANIM, O. S. GAMMOH, and N. A. QINNA. “HEPATOCYTE APOPTOSIS INDUCTION BY ACETAMINOPHEN THROUGH MODULATION OF CASPASE/BAX PATHWAY IN MICE”. International Journal of Pharmacy and Pharmaceutical Sciences, Vol. 12, no. 11, Nov. 2020, pp. 47-52, doi:10.22159/ijpps.2020v12i11.39141.
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