Oxidative stress produced by the dietary or chemical substrates is one of the major causes of liver cell injury. In this study, we compared the effects of two dietary antioxidants, α-tocopherol (α-T) and β-carotene (β-C) against tert-butyl hydroperxide (tBHP)-induced oxidative stress in human hepatoma HepG2 cells. Cell proliferation, lipid peroxidation (LPO), cellular lactate dehydrogenase (LDH), [3H]-aflatoxin B1(AFB1)-DNA adduct formation, and cytochrome P450 2E1 (CYP2E1) expression were determined after antioxidants were added to the tBHP-stressed cells. When compared to an ethanol-based control, all biomarkers for the cell damage were significantly increased by treatments. Treatments of β-C or the combination of two antioxidants at 50 ppm for 48 h enhanced cell proliferation (P < 0.05) compared to tBHP control. The antioxidative and cytoprotective actions of α-T and β-C, alone or in combination, were associated with modulation of microsomal CYP2E1 expression, corresponding to the regulation of LPO production (P < 0.0001). Our results indicate that α-T and β-C may contribute differently to protection of cellular membrane disruption in CYP2E1-expressing HepG2 cells. Moreover, the combination of α-T and β-C appears to impel the greater protection of pathogenic processes of oxidative stress in liver.
Alia M Sonia 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 Toxicol19: 119–128.
2.
Blomhoff R (2005) Dietary antioxidants and cardiovascular disease. Curr Opin Lipidol16: 47–54.
3.
Chen HW Chiang T Wang CY Lii CK (2000) Inhibition of tert-butyl hydroperoxide-induced cell membrane bleb formation by alpha-tocopherol and glutathione. Food Chem Toxicol38: 1089–1096.
4.
Frei B (1994) Reactive oxygen species and antioxidant vitamins: mechanisms of action. Am J Med97: 5S–13S.
5.
Glascott PA Gilfor E Farber JL (1992) Effects of vitamin E on the killing of cultured hepatocytes by tert-butyl hydroperoxide. Mol Pharmacol41: 1155–1162.
6.
Glascott PA Gilfor E Farber JL (1995) Relationship of the metabolism of vitamins C and E in cultured hepatocytes treated with tert-butyl hydroperoxide. Mol Pharmacol48: 80–88.
7.
Guengerich FP Kim DH Iwasaki M (1991) Role of human cytochrome P-450 IIE1 in the oxidation of many low molecular weight cancer suspects. Chem Res Toxicol4: 168–179.
8.
Guiardelli A Cattabeni F Cantoni A (1997) Alternative mechanisms for hydroperoxide-induced DNA single strand breakage. Free Radic Res26: 537–547.
9.
Gumpricht E Dahl R Devereaux MW Sokol RJ (2004) Beta-carotene prevents bile acid-induced cytotoxicity in the rat hepatocyte: Evidence for an antioxidant and anti-apoptotic role of beta-carotene in vitro. Pediatr Res55: 814–821.
10.
Hosotani K Kawahata A Koyama K Murakami C Yoshida H Yamaji R (2004) Effect of carotenoids and ascorbic acid of Japanese persimmons on cellular lipid peroxidation in HepG2 cells. Biofactors21: 241–245.
11.
Imlay JM Linn S (1988) DNA damage and oxygen radical toxicity. Science240: 1302–1309.
12.
Jimenez-Lopez JM Cederbaum AI (2004) Green tea polyphenol epigallocatechin-3-gallate protects HepG2 cells against CYP2E1-dependent toxicity. Free Radic Biol Med36: 359–370.
13.
Kawanishi S Oikawa S Murata M (2005) Evaluation for safety of antioxidant chemopreventive agents. Antioxid Redox Signal7: 1728–1739.
14.
Koop DR (1992) Oxidative and reductive metabolism by cytochrome P450 2E1. FASEB J6: 724–730.
15.
Lee M Bae MA (2007) Docosahexaenoic acid induces apoptosis in CYP2E1-containing HepG2 cells by activating the c-Jun N-terminal protein kinase related mitochondrial damage. J Nutr Biochem18: 348–354.
16.
Liu RH (2004) Potential synergy of phytochemicals in cancer prevention: mechanism of action. J Nutr134: 3479S–3485S.
17.
Mace K Aguilar F Wang JS Vautravers P Gómez-Lechón M Gonzalez FJ (1997) Aflatoxin B1-induced DNA adduct formation and p53 mutation in CYP450-expressing human liver cell lines. Carcinogenesis18: 1291–1297.
18.
Mari M Cederbaum AI (2001) Induction of catalase, alpha and microsomal glutathione S-transferase in CYP2E1-overexpressing HepG2 cells and protection against short-term oxidative stress. Hepatology33: 652–661.
19.
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 Pharmacol62: 705–712.
20.
Martin KR Failla ML Smith JC (1996) Beta-carotene and lutein protect HepG2 human liver cells against oxidant-induced damage. J Nutr126: 2098–2106.
21.
Meydani M (2004) Vitamin E modulation of cardiovascular disease. Ann N Y Acad Sci1031: 271–279.
22.
Mihara M Uchiyama M (1983) Effects of antioxidants on the TBA reaction of various rat liver homogenates. Biochem Med30: 131–134.
23.
Naito Y Shimozawa M Kuroda M Nakabe N Manabe H Katada K (2005) Tocotrienols reduce 25-hydroxycholesterol-induced monocyte-endothelial cell interaction by inhibiting the surface expression of adhesion molecules. Atherosclerosis180: 19–25.
24.
Nordmann R Ribiere C Rouach H (1992) Implication of free radical mechanisms in ethanol-induced cellular injury. Free Radic Biol Med12: 219–240.
25.
Paterson E Gordon MH Niwat C George TW Parr L Waroonphan S Lovegrove JA (2006) Supplementation with fruit and vegetable soups and beverages increases plasma carotenoid concentrations but does not alter markers of oxidative stress or cardiovascular risk factors. J Nutr136: 2849–2855.
26.
Robertson G Leclercq I Farrell GC (2001) Nonalcoholic steatosis and steatohepatitis II. Cytochrome P-450 enzymes and oxidative stress. Am J Physiol Gastrointest Liver Physiol281: 1135–1139.
27.
Sharma S Stutzman JD Kelloff GJ Steele VE (1994) Screening of potential chemopreventive agents using biochemical markers of carcinogenesis. Cancer Res54: 5848–5855.
28.
Sies H (1997) Antioxidant in Disease Mechanisms and Therapy Advances in Pharmacology.Academic press: SanDiego, USA. p. 320.
29.
Silvers KJ Couch LH Rorke EA Howanrd PC (1997) Role of nitroreductases but not cytochrome P450 in the metabolic activation of 1-Nitropyrene in the HepG2 human hepatoblastoma cell line. Biochem Pharmacol54: 927–936.
30.
Thurman RG (1973) Induction of hepatic microsomal reduced nicotinamide adenine dinucleotide phosphate-dependent production of hydrogen peroxide by chronic prior treatment with ethanol. Mol Pharmacol9: 670–675.
31.
Tirmenstein MA Gie X Elkins CA Fariss MW (1999) Administration of the tris salt of tocopheryl hemisuccinate inactivates CYP2E1, enhances microsomal tocopherol levels and protects against carbon tetrachiloride-induced hepatoxicity. Free Radic Biol Med26: 825–835.
32.
Trombino S Serini S Di Nicuolo F Celleno L Andò S Picci N (2004) Antioxidant effect of ferulic acid in isolated membranes and intact cells: synergistic interactions with alpha-tocopherol, beta-carotene, and ascorbic acid. J Agric Food Chem52: 2411–2420.
33.
Woods JA Bilton RF Young AJ (1999) β-Carotene enhances hydrogen peroxide-induced DNA damage in human hepatocellular HepG2 cells. FEBS Lett449: 255–258.
34.
Wu D Cederbaum AI (2005) Oxidative stress mediated toxicity exerted by ethanol-inducible CYP2E1. Toxicol Appl Pharmacol207: 70–76.
35.
Xu Y Leo MA Lieber CS (2003) Lycopene attenuates arachidonic acid toxicity in HepG2 cells overexpressing CYP2E1. Biochem Biophys Res Commun303: 745–750.
36.
Yagi K Ohtsuka K Ohishi N (1985) Lipid peroxidation caused by chinoform-ferric chelate in cultured neural retinal cells. Experientia41: 1561–1563.
37.
Zima T Kalousová M (2005) Oxidative stress and signal transduction pathways in alcoholic liver disease. Alcohol Clin Exp Res29: 110S–115S.
