Protective effect of betulin and betulinic acid on acetaminophen and ethanol-induced cytotoxicity and reactive oxygen species production in HepG2 cells
More details
Hide details
Department of Virology and Immunology, Maria Curie-Skłodowska University, Lublin, Poland
Agnieszka Szuster-Ciesielska   

Department of Virology and Immunology, Maria Curie-Skłodowska University, Akademicka 19, 20-033 Lublin, Poland
J Pre Clin Clin Res. 2010;4(2):96–100
Natural triterpenoids such as ursolic and oleanolic acids have been detected to suppress enzymes which play a role in liver damage, e.g. cytochrome P450, cytochrome b5, CYP1A and CYP2A, and to increase the antioxidant substances glutathione, metallothioneins, and glutathione-S-transferase, with simultaneous protective effects on liver mitochondria. To test the hypothesis that plant triterpenes may also induce mechanisms leading to reduced production of reactive oxygen species after induction with ethanol and acetaminophen, the cytoprotective effect of two plant triterpenes, betulin and its oxidized form, betulinic acid, was compared. Acetaminophen alone at concentrations between 25-250 µg/ml exhibited dose-dependent cytotoxicity for HepG2 cells. When a mixture of 25 µg/ml of acetaminophen was mixed with different concentrations of 5-50 mM of ethanol, an additional increase in the toxicity was observed, depending on the ethanol concentration used. Acetaminophen alone was detected as a strong inducer of superoxide anion in HepG2 cells. Moreover, the mixture of acetaminophen with ethanol induced significantly more superoxide anion than acetaminophen and ethanol alone. When betulin or betulinic acid were preincubated with HepG2 cells the level of superoxide anion and hydrogen peroxide was significantly lower than in respective controls. Betulin, in comparison to betulinic acid, was a stronger inhibitor of oxidative burst.
Patocka J: Biologically active pentacyclic triterpenes and their current medicine significance. J Appl Biomed 2003, 1, 7-12.
Zdzisińska B, Rzeski W, Paduch R, Szuster-Ciesielska A, Kaczor J, Wejksza K, Kandefer-Szerszeń M: Differential effect of betulin and betulinic acid on cytokine production in human whole blood cell cultures. Pol J Pharmacol 2003, 55, 235-238.
Liu J, Liu Y, Klaassen CD: The effect of Chinese hepatoprotective medicines on experimental liver injury in mice. J Ethnopharmacol 1994, 42, 183-191.
Liu J, Liu Y, Klaassen CD: Protective effect of oleanolic acid against chemical-induced acute necrotic liver injury in mice. Zhongguo Yao Li Xue Bao 1995, 16, 97-102.
Liu Y, Krepplel H, Liu J, Choudhuri S, Klaassen CD: Oleanolic acid protects against cadmium hepatotoxicity by inducing metallothionein. J Pharmacol Exp Ther 1993, 266, 400-406.
Liu J, Liu Y, Klaassen CD: The effect of 10 triterpenoid compounds on experimental liver injury in mice. Fundam Appl Toxicol 1994, 2, 34-40.
Miura N, Matsumoto Y, Miyairi S, Nishiyama S, Nagamura A: Protective effects of triterpene compounds against the cytotoxicity of cadmium in HepG2 cells. Mol Pharmacol 1999, 56, 1324-1328.
Liu J, Liu Y, Madhu C, Klaassen CD: Protective effect of oleanolic acid on acetaminophen-induced hepatotoxicity in mice. J Pharmacol Exp Ther 1993, 266, 1607-1613.
Saleem M, Alam A, Arifin S, Shah M.S, Ahmed B, Sultana S: Lupeol, a triterpene, inhibits early responses of tumor promotion induced by benzoyl peroxide in murine skin. Pharmacol Res 2001, 43, 127-134.
Saraswat B, Visen PK, Aggarwal DP: Ursolic acid isolated from Eucalyptus tereticornis protects against ethanol toxicity in isolated rat hepatocytes. Phytother Res 2000, 14, 163-166.
Yamashita K, Lu H, Lu J, Cen G, Yokoyama T, Sagara Y, Manabe M, Kodama H: Effect of three triterpenoids, lupeol, betulin, and betulinic acid on the stimulus-induced superoxide generation and tyrosyl phosphorylation of proteins in human neutrophils. Clin Chim Acta 2002, 325, 91-96.
Larson AM, Polson J, Fontana RJ, Dawern TJ, Lalani E, Hynan LS, Reisch JS, Schiodt EV, Ostapowicz G, Shakil AO, Lee MW: Acute Liver Failure Study Group. Acetaminophen-induced acute liver failure: results of a United States multicenter, prospective study. Hepatology 2005, 42, 1364-1372.
Jaeschke H: Role of inflammation in the mechanism of acetaminopheninduced hepatotoxicity. Expert Opin Drug Metab Toxicol 2005, 1, 389- 397.
Gujral JS, Hinson JA, Farhood A, Jaeschke H: NADPH oxidase derived oxidant stress is critical for neutrophil cytotoxicity during endotoxemia. Am J Physiol Gastrointest Liver Physiol 2004, 287, G243-G252.
Zimmerman HJ, Maddrey WC: Acetaminophen (paracetamol) hepatotoxicity with regular intake of alcohol: analysis of instances of therapeutic misadventure. Hepatology 1995, 22, 767-773.
Lauterburg B, Corcoran G, Mitchell J: Mechanism of action of Nacetylcysteine in the protection against hepatotoxicity of acetaminophen in rats in vivo. J Clin Invest 1983, 71, 980-991.
Liang Q, Sheng Y, Ji L, Min Y, Xia Y, Wang Z: Acetaminophen-induced cytotoxicity in human normal liver L-02 cells and the protection of antioxidants. Toxicol Mech Methods 2010, 20, 273-278.
Szuster-Ciesielska A, Kandefer-Szerszeń M: Protective effects of betuli and betulinic acid against ethanol-induced cytotoxicity in HepG2 cells. Pharmacol Rep 2005, 57, 588-595.
Pick E: Microassays for superoxide and hydrogen peroxide production and nitroblue tetrazolium reduction using and enzyme immunoassay microplate reader. Methods Enzymol 1986, 132, 414-419.
Jaeschke H, Gores GJ, Cederbaum AI, Hinson JA, Pessayre D, Lemasters JJ: Mechanisms of hepatotoxicity. Toxicol Sci 2002, 65, 166-176.
Mitchell JR, Jollow DJ, Potter WZ, Davis DC, Gillette JR, Brodie BB: Acetaminophen-induced hepatic necrosis.I.Role of drug metabolism. J Pharmacol Exp Ther 1973, 187, 185-194.
Reid AB, Kurten RC, McCullough SS, Brock RW, Hinson JA: Mechanisms of acetaminophen-induced hepatotoxicity: role of oxidative stress and mitochondrial permeability transition in freshly isolated mouse hepatocytes. J Pharmacol Exp Ther 2005, 312, 509-516.
Neuman MG: Synergistic signaling for apoptosis in vitro by ethanol and acetaminophen. Alcohol 2002, 27, 89-98.
Cheung C, Yu AM, Ward JM, Krausz KW, Akiyama TE, Feigenbaum L, Gonzalez FJ: The CYP2E1-humanized transgenic mouse: role of CYP2E1 in acetaminophen hepatotoxicity. Drug Metab Disposition 2005, 33, 449-457.
Stopiński M, Mrozińska M: Paracetamol (acetaminofen) – dawki skuteczne i bezpieczne (in Polish). Paracetamol (Acetaminophen) – effective and safe doses. Przew Lek 2003, 6, 88-95.