Liv.52 protects HepG2 cells from oxidative damage induced by tert-butyl hydroperoxide

  • S. Vidyashankar
  • S. K Mitra
  • Krishna S. Nandakumar
Article

Abstract

Oxidative stress induced by toxicants is known to cause various complications in the liver. Herbal drug such as Liv.52 is found to have hepatoprotective effect. However, the biochemical mechanism involved in the Liv.52 mediated protection against toxicity is not well elucidated using suitable in vitro models. Hence, in the present study, the hepatoprotective effect of Liv.52 against oxidative damage induced by tert-butyl hydroperoxide (t-BHP) in HepG2 cells was evaluated in order to relate in vitro antioxidant activity with cytoprotective effects. Cytotoxicity was measured by MTT assay. Antioxidant effect of Liv.52 was determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, ferric-reducing antioxidant power (FRAP) assay, and lipid peroxidation and measurement of non-enzymic and antioxidant enzymes in HepG2 cells exposed to t-BHP over a period of 24 h. The results obtained indicate that t-BHP induced cell damage in HepG2 cells as shown by significant increase in lipid peroxidation as well as decreased levels of reduced glutathione (GSH). Liv.52 significantly decreased toxicity induced by t-BHP in HepG2 cells. Liv.52 was also significantly decreased lipid peroxidation and prevented GSH depletion in HepG2 cells induced by t-BHP. Therefore, Liv.52 appeared to be important for cell survival when exposed to t-BHP. The protective effect of Liv.52 against cell death evoked by t-BHP was probably achieved by preventing intracellular GSH depletion and lipid peroxidation. The results showed protective effect of Liv.52 against oxidative damage induced in HepG2 cells. Hence, taken together, these findings derived from the present study suggest the beneficial effect of Liv.52 in regulating oxidative stress induced in liver by toxicants.

Keywords

Liv.52 Liver Oxidative stress HepG2 cells tert-butyl hydroperoxide Hepatoprotection 

Notes

Acknowledgment

Authors thank Executive Director for supporting this study.

References

  1. 1.
    Tiwari AK (2004) Antioxidants: new-generation therapeutic base for treatment of polygenic disorders. Curr Sci 86:1092–1102Google Scholar
  2. 2.
    Willcox JK, Ash SL, Catignani GL (2004) Antioxidants and prevention of chronic disease. Crit Rev Fd Sci Nutr 44:275–295CrossRefGoogle Scholar
  3. 3.
    Ballinger SW (2005) Mitochondrial dysfunction in cardiovascular disease. Fr Rad Biol Med 28:1278–1295CrossRefGoogle Scholar
  4. 4.
    Adachi M, Ishii H (2002) Role of mitochondria in alcoholic liver injury. Fr Rad Biol Med 32:487–491CrossRefGoogle Scholar
  5. 5.
    Vitaglione P, Morisco F, Caporaso N, Fogliano V (2004) Dietary antioxidant compounds and liver health. Crit Rev Fd Sci Nutr 44:575–586CrossRefGoogle Scholar
  6. 6.
    Jaeschke H, Gores GJ, Cederbaum AI, Hinson JA, Pessayre D, Lemasters JJ (2002) Forum-mechanisms of hepatotoxicity. Toxicol Sci 65:166–176CrossRefPubMedGoogle Scholar
  7. 7.
    Mitra SK (2000) Herbal products for liver diseases. Hepatology 31:546–547CrossRefPubMedGoogle Scholar
  8. 8.
    Husseini HF, Alavian SM, Heshmat R, Heydari MR, Abolmaali K (2005) The efficacy of Liv.52 on liver cirrhotic patients: a randomized, double blind placebo-controlled first approach. Phytomedicine 12:619–624CrossRefGoogle Scholar
  9. 9.
    Mandal SC, Saraswathi B, Kumar CK, Mohana Lakshmi S, Maiti BC (2000) Protective effect of leaf extract of Ficus hispidea L against paracetamol-induced hepatotoxicity in rats. Phytother Res 14:457–459CrossRefPubMedGoogle Scholar
  10. 10.
    Sandhir R, Gill KD (1999) Hepatoprotective effects of Liv. 52 on ethanol induced liver damage in rats. Indian J Expt Biol 37:762–766Google Scholar
  11. 11.
    Gopumadhavan S, Jagadeesh S, Chauhan L, Kulkarni RD (1993) Protective effect of Liv.52 on alcohol induced fetotoxicity. Alcohol Clin Exp Res 17:1089–1092CrossRefPubMedGoogle Scholar
  12. 12.
    Chauhan BL, Kulkarni RD (1991) Effect of Liv.52 a herbal preparation on absorption and metabolism of ethanol in humans. Eur J Clin Pharmacol 40:189–191PubMedGoogle Scholar
  13. 13.
    Mitra SK, Varma SR, Godavarthi A, Nandakumar KS (2008) Liv. 52 regulates ethanol induced PPAR γ and TNF α expression in HepG2 cells. Mol Cell Biochem 315:9–15CrossRefPubMedGoogle Scholar
  14. 14.
    Stanner SA, Hughes J, Kelly CNM, Buttriss J (2004) A review of the epidemiological evidence for the ‘antioxidant hypothesis’. Public Health Nutr 7:407–422CrossRefPubMedGoogle Scholar
  15. 15.
    Scalbert A, Manach C, Morand C, Remesy C, Jimenez L (2005) Dietary polyphenols and the prevention of diseases. Crit Rev Fd Sci Nutr 45:287–306CrossRefGoogle Scholar
  16. 16.
    Knasmuller S, Mersch-Sundermann V, Kevekordes S, Darroudi F, Huber WW, Hoelzl C, Bichler J, Majer BJ (2004) Use of human-derived liver cell lines for the detection of environmental and dietary genotoxicants; current state of knowledge. Toxicology 198:315–328CrossRefPubMedGoogle Scholar
  17. 17.
    Mersch-Sundermann V, Knasmuller S, Wu XJ, Darroudi F, Kassie F (2004) Use of a human-derived liver cell line for the detection of cytoprotective, antigenotoxic and cogenotoxic agents. Toxicology 198:329–340CrossRefPubMedGoogle Scholar
  18. 18.
    Kahkonen MP, Hopia AI, Vuorela HJ, Rauha JP, Pihlaja K, Kujala TS (1999) Antioxidant activity of plant extracts containing phenolic compounds. J Agric Food Chem 47:3954–3962CrossRefPubMedGoogle Scholar
  19. 19.
    Sanchez-Moreno C, Larrauri JA, Saura-Calixto F (1998) A procedure to measure the antiradical efficiency of polyphenols. J Sci Food Agric 76:270–276CrossRefGoogle Scholar
  20. 20.
    Chu YH, Chang CL, Hsu HF (2000) Flavonoid content of several vegetables and their antioxidant activity. J Food Agric 80:561–566CrossRefGoogle Scholar
  21. 21.
    Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65:55–63CrossRefPubMedGoogle Scholar
  22. 22.
    Ohakawa H, Ohishi U, Yagi K (1979) Assay for lipid peroxides in animal tissues by thiobarbituric reaction. Anal Biochem 95:145–149Google Scholar
  23. 23.
    Anderson ME (1985) Determination of glutathione and glutathione disulfide in biological samples. Methods Enzymol 113:548–555CrossRefPubMedGoogle Scholar
  24. 24.
    Flohe L, Otting O (1984) Superoxide dismutase assays. Methods Enzymol 105:93–104CrossRefPubMedGoogle Scholar
  25. 25.
    Aebi H (1984) Catalase in vitro. Methods Enzymol 105:121–126CrossRefPubMedGoogle Scholar
  26. 26.
    Flohe L, Gunzler WA (1984) Assays of glutathione peroxidase. Methods Enzymol 105:114–121CrossRefPubMedGoogle Scholar
  27. 27.
    Rice-Evans CA, Miller NJ, Paganga G (1997) Antioxidant properties of phenolic compounds. Trends Plant Sci 2:152–159CrossRefGoogle Scholar
  28. 28.
    Ferguson LR (2001) Role of plant polyphenols in genomic stability. Mutat Res 475(1–2):89–111PubMedGoogle Scholar
  29. 29.
    Sies H, Summer KH (1975) Hydroperoxide-metabolizing systems in rat liver. Eur J Biochem 57(2):503–512CrossRefPubMedGoogle Scholar
  30. 30.
    Bellomo G, Jewel SA, Thor H, Orrenius S (1982) Regulation of intracellular calcium compartmentation: studies with isolated hepatocytes and t-butylhydroperoxide. Proc Natl Acad Sci 79:6842–6846CrossRefPubMedGoogle Scholar
  31. 31.
    Rush GF, Gorski JR, Ripple MG, Sowinski J, Bugelski P, Hewitt WR (1985) Organic hydroperoxide-induced lipid-peroxidation and cell-death in isolated hepatocytes. Toxicol Appl Pharmacol 78:473–483CrossRefPubMedGoogle Scholar
  32. 32.
    Jewell SA, Di Monte D, Richelmi P, Bellomo G, Orrenius S (1986) tert- butylhydroperoxide-induced toxicity in isolated hepatocytes: contribution of thiol oxidation and lipid peroxidation. J Biochem Toxicol 1:13–22CrossRefPubMedGoogle Scholar
  33. 33.
    Nicotera P, McConkey D, Svensson SA, Bellomo G, Orrenius S (1988) Correlation between cytosolic Ca2+ concentration and cytotoxicity in hepatocytes exposed to oxidative stress. Toxicology 52:55–63CrossRefPubMedGoogle Scholar
  34. 34.
    Masaki N, Kyle ME, Farber JL (1989) tert-butyl hydroperoxide kills cultured hepatocytes by peroxidizing membrane lipids. Arc Biochem Biophys 269:390–399CrossRefGoogle Scholar
  35. 35.
    Buc-Calderon P, Latour I, Roberfroid M (1991) Biochemical changes in isolated hepatocytes exposed to tert-butyl hydroperoxide. Implications for its cytotoxicity. Cell Biol Toxicol 7:129–143CrossRefPubMedGoogle Scholar
  36. 36.
    Thabrew MI, Hughes RD, McFarlane IG (1997) Screening of hepatoprotective plant components using a HepG2 cell cytotoxicity assay. J Pharm Pharmacol 49:1132–1135PubMedGoogle Scholar
  37. 37.
    Kinjo J, Hirakawa T, Tsuchihashi R, Nagao T, Okawa M, Nohara T, Okabe H (2003) Hepatoprotective constituents in plants. 14 Effects of soyasapogenol B, sophoradiol, and their glucuronides on the cytotoxicity of tert-butyl hydroperoxide to HepG2 cells. Biol Pharm Bull 26:1357–1360CrossRefPubMedGoogle Scholar
  38. 38.
    Alia M, Ramos S, Mateos R, Bravo L, Goya L (2005) Response of the antioxidant defense system to tert-butyl hydroperoxide and hydrogen peroxide in a human hepatoma cell line (HepG2). J Biochem Mol Toxicol 19:119–128CrossRefPubMedGoogle Scholar
  39. 39.
    Alia M, Ramos S, Mateos R, Granado-Serrano AB, Bravo L, Goya L (2006) Quercetin protects human hepatoma HepG2 against oxidative stress induced by tert-butyl hydroperoxide. Toxicol Appl Pharmacol 212:110–118CrossRefPubMedGoogle Scholar
  40. 40.
    Lee HU, Bae EA, Han MJ, Kim DH (2005) Hepatoprotective effect of 20(S)- ginsenosides Rg3 and its metabolite 20(S)-ginsenoside Rh2 on tert-butyl hydroperoxide-induced liver injury. Biol Pharm Bull 28:1992–1994CrossRefPubMedGoogle Scholar
  41. 41.
    Lee HU, Bae EA, Han MJ, Kim NJ, Kim DH (2005) Hepatoprotective effect of ginsenoside Rb1 and compound K on tert-butyl hydroperoxide-injury. Liver Int 25:1069–1073CrossRefPubMedGoogle Scholar
  42. 42.
    Martin C, Martinez R, Navarro R, Ruiz-Sanz JI, Lacort M, Ruiz-Larrea MB (2001) tert-Butyl hydroperoxide-induced lipid signaling in hepatocytes: involvement of glutathione and free radicals. Biochem Pharmacol 62:705–712CrossRefPubMedGoogle Scholar
  43. 43.
    Kedderis GL (1996) Biochemical basis of hepatocellular injury. Toxicol Pathol 24:77–83CrossRefPubMedGoogle Scholar
  44. 44.
    Castell JV, Gomez-Lechon MJ, Ponsoda X, Bort R (1997) In vitro investigation of the molecular mechanisms of hepatotoxicity. In: Castell JV, Gomez-Lechon MJ (eds) In vitro methods in pharmaceutical research. Academic Press, London, pp 375–410Google Scholar

Copyright information

© Springer Science+Business Media, LLC. 2009

Authors and Affiliations

  • S. Vidyashankar
    • 1
  • S. K Mitra
    • 1
  • Krishna S. Nandakumar
    • 1
  1. 1.Research and Development The Himalaya Drug Company BangaloreIndia

Personalised recommendations