European Journal of Nutrition

, Volume 46, Issue 2, pp 70–78 | Cite as

Effect of the olive oil phenol hydroxytyrosol on human hepatoma HepG2 cells

Protection against oxidative stress induced by tert-butylhydroperoxide
  • Luis GoyaEmail author
  • Raquel Mateos
  • Laura Bravo



Scientific evidence suggests that olive oil’s beneficial effects are related to the high level of antioxidants, including phenolic compounds such as hydroxytyrosol. In vivo studies have shown that olive oil HTy is bioavailable and its biological activities, similar to those reported for other natural antioxidants such as quercetin, include prevention of LDL oxidation. Previous studies from our laboratory have shown that HTy and other phenolics in olive oil are absorbed and metabolized by cultured human hepatoma HepG2 cells where glucuronidated and methylated conjugates were the main derivatives formed, resembling the metabolic profile of olive oil phenols observed in human plasma and urine.

Aim of the study

The effect of olive oil phenol (HTy) on cell viability and redox status of cultured HepG2 cells, and the protective effect of HTy against an oxidative stress induced by tert-butylhydroperoxide (t-BOOH) were investigated.


Lactate dehydrogenase activity as marker for cell integrity, concentration of reduced glutathione (GSH), generation of reactive oxygen species (ROS) and activity of the antioxidant enzyme glutathione peroxidase (GPx) as markers of redox status and determination of malondialdehyde (MDA) as marker of lipid peroxidation were measured.


No changes in cell integrity or intrinsic antioxidant status resulted from a direct treatment with 10–40 μM HTy. Pre-treatment of HepG2 with 10–40 μM HTy for 2 or 20 h completely prevented cell damage as well as the decrease of reduced glutathione and increase of malondialdehyde evoked by t-BOOH in HepG2 cells. Reactive oxygen species generation and the significant increase of glutathione peroxidase activity induced by t-BOOH were greatly reduced when cells were pretreated with HTy.


The results clearly show that treatment of HepG2 cells with the olive oil phenolic HTy may positively affect their antioxidant defense system, favoring cell integrity and resistance to cope with a stressful situation.


bioactive compounds dietary antioxidants olive oil phenolics liver cell culture antioxidant defenses biomarkers for oxidative stress 



R. Mateos was a postdoctoral fellow from the Ministerio de Educación y Ciencia. We thank Dr. J.L. Espartero (University of Seville, Spain) for kindly providing hydroxytyrosol.


  1. 1.
    Bravo L (1998) Polyphenols: chemistry, dietary sources, metabolism and nutritional significance. Nutr Rev 56:317–333CrossRefGoogle Scholar
  2. 2.
    Visioli F, Galli C (1998) The effect of minor constituents of olive oil on cardiovascular disease: new findings. Nutr Rev 56:142–147CrossRefGoogle Scholar
  3. 3.
    Mateos R, Espartero JL, Trujillo M, Ríos JJ, León-Camacho M, Alcudia F, Cert A (2001) Determination of phenols, flavones, and lignans in virgin olive oils by solid-phase extraction and high-performance liquid chromatography with diode array ultraviolet detection. J Agric Food Chem 49:2185–2192CrossRefGoogle Scholar
  4. 4.
    Owen R, Mier W, Giacosa A, Hull WE, Spiegelhalder B, Bartsch H (2000) Identification of lignans as major components in the phenolic fraction of olive oil. Clin Chem 46:976–988Google Scholar
  5. 5.
    Visioli F, Galli C, Bornet F, Mattei A, Patelli R, Galli G, Caruso D (2000) Olive oil phenolics are dose-dependently absorbed in humans. FEBS Lett 468:159–160CrossRefGoogle Scholar
  6. 6.
    Caruso D, Visioli F, Patelli R, Galli C, Galli G (2001) Urinary excretion of olive oil phenols and their metabolites in humans. Metab Clin Exp 50:1426–1428Google Scholar
  7. 7.
    Vissers MH, Zock PL, Roodenburg AJ, Leenen R, Katan MB (2002) Olive oil phenols are absorbed in humans. J Nutr 132:409–417Google Scholar
  8. 8.
    Miró-Casas E, Covas M-I, Fito M, Farré-Albaladejo M, Marrugat J, de la Torre R (2003) Tyrosol and hydroxytyrosol are absorbed from moderate and sustained doses of virgin olive oil in humans. Eur J Clin Nutr 57:186–190CrossRefGoogle Scholar
  9. 9.
    D’Angelo S, Manna C, Migliardi V, Mazzoni O, Morrica P, Capasso G, Pontoni G, Galletti P, Zappia V (2001) Pharmacokinetics and metabolism of hydroxytyrosol, a natural antioxidant from olive oil. Drug Metab Dispos 29:1492–1498Google Scholar
  10. 10.
    Tuck KL, Freeman MP, Hayball PJ, Stretch GL, Stupans I (2001) The in vivo fate of hydroxytyrosol and tyrosol, antioxidant phenolic constituents of olive oil, following intravenous and oral dosing of labelled compounds to rats. J Nutr 131:1993–1996Google Scholar
  11. 11.
    Tuck KL, Hayball PJ, Stupans I (2002) Structural characterization of the metabolites of hydroxytyrosol, the principal phenolic component in olive oil, in rats. J Agric Food Chem 50:2404–2409CrossRefGoogle Scholar
  12. 12.
    Saija A, Trombetta D, Tomaino A, Lo Cascio R, Princi P, Uccella N, Bonina F, Castelli F (1998) In vitro evaluation of the antioxidant activity and biomembrane interaction of the plant phenols oleuropein and hydroxytyrosol. Int J Pharm 166:123–133CrossRefGoogle Scholar
  13. 13.
    Gordon MH, Paiva-Martins F, Almeida M (2001) Antioxidant activity of hydroxytyrosol acetate compared with that of other olive oil polyphenols. J Agric Food Chem 49:2480–2485CrossRefGoogle Scholar
  14. 14.
    De la Puerta R, Martínez-Domínguez ME, Ruiz-Gutierrez V, Flavill JA, Hoult JR (2001) Effects of virgin olive oil phenolics on scavenging of reactive nitrogen species and upon nitrergic neurotransmission. Life Sci 69:1213–1222CrossRefGoogle Scholar
  15. 15.
    Manna C, Galletti P, Cucciolla V, Moltedo O, Leone A, Zappia V (1997) The protective effect of the olive oil polyphenols (3,4-dihydroxyphenyl)-ethanol counteracts reactive oxygen metabolite-induced cytotoxicity in Caco-2 cells. J Nutr 127:286–292Google Scholar
  16. 16.
    Mateos R, Domínguez MM, Espartero JL, Cert A (2003) Antioxidant effect of phenolic compounds, α-tocopherol, and other minor components in virgin olive oil. J Agric Food Chem 51:7170–7175CrossRefGoogle Scholar
  17. 17.
    Salami M, Galli C, De Angelis L, Visioli F (1995) Formation of F2-isoprostanes in oxidized low-density lipoprotein: inhibitory effect of hydroxytyrosol. Pharmacol Res 31:275–279CrossRefGoogle Scholar
  18. 18.
    Caruso D, Berra B, Giavarini F, Cortesi N, Fedeli E, Galli G (1999) Effect of virgin olive oil phenolic compounds on in vitro oxidation of human low density lipoproteins. Nutr Metab Cardiovasc Dis 9:102–107Google Scholar
  19. 19.
    Scaccini C, Nardini M, D’Aquino M, Gentili V, Di Felice M, Tomassi G (1992) Effect of dietary oils on lipid peroxidation and on antioxidant parameters of rat plasma and lipoprotein fractions. J Lipid Res 33:627–633Google Scholar
  20. 20.
    Coni E, Di Benedetto R, Di Pasquale M, Masella R, Modesti D, Mattei R, Carlini EA (2000) Protective effect of oleuropein, an olive oil biophenol, on low density lipoprotein oxidizability in rabbits. Lipids 35:45–54CrossRefGoogle Scholar
  21. 21.
    Mateos R, Goya L, Bravo L (2005) Metabolism of the olive oil phenols hydroxytyrosol, tyrosol and hydroxytyrosyl acetate by human hepatoma HepG2 cells. J Agric Food Chem 53:9897–9905CrossRefGoogle Scholar
  22. 22.
    Alía M, Mateos R, Ramos S, Lecumberri E, Bravo L, Goya L (2006) Influence of quercetin and rutin on growth and the antioxidant defense system in a human hepatoma cell line (HepG2). Eur J Nutr 45:19–28CrossRefGoogle Scholar
  23. 23.
    Baraldi PG, Simoni D, Manfredini S, Menziani E (1983) Preparation of 3,4-dihydroxy-1-benzeneethanol: a reinvestigation. Liebigs Ann Chem 684–686Google Scholar
  24. 24.
    Alía M, Ramos S, Mateos R, Bravo L, Goya L (2005) Response of the antioxidant defense system to t-butyl hydroperoxide and hydrogen peroxide in a human hepatoma cell line (HepG2). J Biochem Mol Toxicol 19:119–128CrossRefGoogle Scholar
  25. 25.
    Alía M, Ramos S, Mateos R, Bravo L, Goya L (2006) Quercetin protects human hepatoma cell line (HepG2) against oxidative stress induced by tertbutyl hydroperoxide. Toxicol Appl Pharm 212:110–118CrossRefGoogle Scholar
  26. 26.
    Vasault A (1987) Lactate dehydrogenase. UV-method with pyruvate and NADH. In: Bergmeyer HV (ed) Methods of enzymatic analysis. Weinheim, Verlag-Chemie, pp. 118–133Google Scholar
  27. 27.
    Welder AA, Acosta D (1994) Enzyme leakage as an indicator of cytotoxicity in culture cells. In: Tyson CA, Franzier JM (eds) In vitro toxicity indicators: methods in toxicology. Academic press, New York, pp. 46–49Google Scholar
  28. 28.
    Hissin PJ, Hilf R (1976) A fluorometric method for determination of oxidised and reduced glutathione in tissues. Anal Biochem 74:214–226CrossRefGoogle Scholar
  29. 29.
    Mateos R, Goya L, Bravo L (2004) Determination of malondialdehyde (MDA) by high-performance liquid chromatography as the 2,4-dinitrophenylhydrazine derivative. A marker for oxidative stress in cell cultures of human hepatoma HepG2. J Chromatogr B 805:33–39CrossRefGoogle Scholar
  30. 30.
    Bradford MM (1976) A rapid and sensitive method for the quantification of microgram quantities of protein, utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefGoogle Scholar
  31. 31.
    Wang H, Joseph JA (1999) Quantifying cellular oxidative stress by dichlorofluorescein assay using microplate reader. Free Rad Biol Med 27:612–616CrossRefGoogle Scholar
  32. 32.
    Gunzler WA, Kremers H, Flohe L (1974) An improved coupled test procedure for glutathione peroxidase. Klin Chem Klin Biochem 12:444–448Google Scholar
  33. 33.
    Petroni A, Blasevich M, Salami M, Papini N, Montedoro GF, Galli C (1995) Inhibition of platelet aggregation and eicosanoid production by phenolic components of olive oil. Throm Res 78:151–160CrossRefGoogle Scholar
  34. 34.
    De la Puerta R, Ruiz-Gutierrez V, Hoult RS (1999) Inhibition of leukocyte 5-lipoxygenase by phenolics from virgin olive oil. Biochem Pharmacol 57:445–449CrossRefGoogle Scholar
  35. 35.
    Manna C, Galletti P, Cucciolla V, Montedoro G, Zappia V (1999) Olive oil hydroxytyrosol protects human erythrocytes against oxidative damages. J Nutr Biochem 10:159–165CrossRefGoogle Scholar
  36. 36.
    Chen L, Yang X, Jiao H, Zhao B (2002) Tea catechins protect against lead-induced cytotoxicity, lipid peroxidation, and membrane fluidity in HepG2 cells. Toxicol Sci 69:149–156CrossRefGoogle Scholar
  37. 37.
    Murakami C, Hirakawa Y, Inui H, Nakano Y, Yoshida H (2002) Effects of epigallocatechin 3-O-gallate on cellular antioxidative system in HepG2 cells. J Nutr Sci Vitaminol 48:89–94Google Scholar
  38. 38.
    Murakami C, Hirakawa Y, Inui H, Nakano Y, Yoshida H (2002) Effect of tea catechins on cellular lipid peroxidation and cytotoxicity in HepG2 cells. Biosci Biotechnol Biochem 66:1559–1562CrossRefGoogle Scholar
  39. 39.
    Scharf G, Prustomersky S, Knasmuller S, Schulte-Hermann R, Huber WW (2003) Enhancement of glutathione and g-glutamylcysteine synthetase, the rate limiting enzyme of glutathione synthesis, by chemoprotective plant-derived food and beverage components in the human hepatoma cell line HepG2. Nutr Cancer 45:74–83CrossRefGoogle Scholar
  40. 40.
    Rodgers EH, Grant MH (1998) The effect of the flavonoids, quercetin, myricetin and epicatechin on the growth and enzyme activities of MCF7 human breast cancer cells. Chem Biol Interact 116:213–228CrossRefGoogle Scholar
  41. 41.
    Viña J (1990) Glutathione: metabolism and physiological functions. CRC Press, BostonGoogle Scholar
  42. 42.
    Myhrstad MC, Carlsen H, Nordstrom O, Blomhoff R, Moskaug JO (2002) Flavonoids increase the intracellular glutathione level by transactivation of the gamma-glutamylcysteine synthetase catalytical subunit promoter. Free Rad Biol Med 32:386–393CrossRefGoogle Scholar
  43. 43.
    Pilz J, Meineke I, Gleiter CH (2000) Measurement of free and bound malondialdehyde in plasma by high-performance liquid chromatography as the 2,4-dinitrophenylhydrazine derivative. J Chromatogr B 742:315–325Google Scholar
  44. 44.
    Suttnar J, Cermak J, Dyr E (1997) Solid-phase extraction in malondialdehyde analysis. Anal Biochem 249:20–23CrossRefGoogle Scholar
  45. 45.
    Suttnar J, Masova L, Dyr E (2001) Influence of citrate and EDTA anticoagulants on plasma malondialdehyde concentrations estimated by high-performance liquid chromatography. J Chromatogr B 751:193–119Google Scholar
  46. 46.
    Courtois F, Delvin E, Ledoux M, Seidman E, Lavoie JC, Levy E (2002) The antioxidant BHT normalizes some oxidative effects of iron + ascorbate on lipid metabolism in Caco-2 cells. J Nutr 132:1289–1292Google Scholar
  47. 47.
    LeBel CP, Ishiropoulos H, Bondy SC (1992) Evaluation of the probe 2′,7′-dichlorofluorescin as an indicator of reactive oxygen species formation and oxidative stress. Chem Res Toxicol 5:227–231CrossRefGoogle Scholar
  48. 48.
    Ursini F, Maiorino M, Brigelius-Flohé R, Aumann KD, Roveri A, Schomburg D, Flohé L (1995) Diversity of glutathione peroxidases. Methods Enzymol 252:38–114CrossRefGoogle Scholar
  49. 49.
    Duthie GG, Duthie SJ, Kyle JAM (2000) Plant polyphenols in cancer and heart disease: implications as nutritional antioxidants. Nutr Res Rev 13:79–106CrossRefGoogle Scholar
  50. 50.
    Röhrdanz E, Ohler S, Tran-Thi Q-H, Kahl R (2002) The phytoestrogen daidzein affects the antioxidant enzyme system of rat hepatoma H4IIE cells. J Nutr 132:370–375Google Scholar

Copyright information

© Steinkopff Verlag Darmstadt 2007

Authors and Affiliations

  1. 1.Depto. de Metabolismo y Nutrición, Instituto del Frío (CSIC)C/José Antonio Novais, 10 Ciudad UniversitariaMadridSpain
  2. 2.CIFA Venta del Llano, IFAPAMenjíbarSpain

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