Green tea extract and (−)‐epigallocatechin‐3‐gallate, the major tea catechin, exert oxidant but lack antioxidant activities

Green tea is the most widely consumed beverage. It has attained high reputation as a health‐promoting dietary component ascribed to the antioxidant activity of (−)‐epigallocatechin‐3‐gallate (EGCG), its main polyphenolic constituent. Evidence is increasing that tea constituents can be cell damaging and pro‐oxidant themselves. These effects were suggested to be due to spontaneous H2O2 generation by polyphenols in solution. In the present study, we investigated the oxidant and antioxidant properties of green tea extracts (GTE) and of EGCG by means of the rodent macrophage‐like RAW 264.7 and human promyelocytic leukemic HL60 cell lines. The results obtained show that both under cell‐free conditions and in the presence of cells the oxidant activities of GTE and EGCG exceeded those of spontaneously generated H2O2 (FOX assay). Increase of intracellular oxidative stress was indicated by 2′,7′‐dichlorofluorescin probing, and the enhanced genotoxicity was demonstrated by the alkaline comet assay and by the micronucleus assay (cytokinesis block). Time‐ and dose‐dependent induction of cell death was monitored by trypan blue exclusion, MTT assay, and Hoechst staining. Furthermore, in our systems in vitro, EGCG neither directly scavenges H2O2 nor mediates other antioxidant activities but rather increased H2O2‐induced oxidative stress and DNA damage. In conclusion, our data suggest that detailed mechanistic studies on the effects of GTE and EGCG should be performed in vivo before excessive intake and/or topical application of green tea products can be recommended to healthy and/or diseased persons.

[1]  A. Kong,et al.  Epigallocatechin-3-gallate-induced stress signals in HT-29 human colon adenocarcinoma cells. , 2003, Carcinogenesis.

[2]  Chung S. Yang Inhibition of carcinogenesis by tea , 1997, Nature.

[3]  S. Rhee Redox signaling: hydrogen peroxide as intracellular messenger , 1999, Experimental & Molecular Medicine.

[4]  M. Fenech,et al.  Anti-genotoxic effects of tea catechins against reactive oxygen species in human lymphoblastoid cells. , 2004, Mutation research.

[5]  G. Rothe,et al.  Flow cytometric assays of oxidative burst activity in phagocytes. , 1994, Methods in enzymology.

[6]  J. Avruch,et al.  Mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation. , 2001, Physiological reviews.

[7]  B. Frei,et al.  Tea Catechins and Polyphenols: Health Effects, Metabolism, and Antioxidant Functions , 2003, Critical reviews in food science and nutrition.

[8]  R. Eckert,et al.  Green Tea Polyphenol Stimulates a Ras, MEKK1, MEK3, and p38 Cascade to Increase Activator Protein 1 Factor-dependent Involucrin Gene Expression in Normal Human Keratinocytes* , 2002, The Journal of Biological Chemistry.

[9]  Sandra N. Mohr,et al.  Pharmacokinetics of tea catechins after ingestion of green tea and (-)-epigallocatechin-3-gallate by humans: formation of different metabolites and individual variability. , 2002, Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology.

[10]  G. Yang,et al.  Effect of black and green tea polyphenols on c-jun phosphorylation and H(2)O(2) production in transformed and non-transformed human bronchial cell lines: possible mechanisms of cell growth inhibition and apoptosis induction. , 2000, Carcinogenesis.

[11]  S. Azam,et al.  Prooxidant property of green tea polyphenols epicatechin and epigallocatechin-3-gallate: implications for anticancer properties. , 2004, Toxicology in vitro : an international journal published in association with BIBRA.

[12]  Nagi B. Kumar,et al.  Green tea polyphenols and cancer chemoprevention: multiple mechanisms and endpoints for phase II trials. , 2004, Nutrition reviews.

[13]  E. LaVoie,et al.  Identification of oxidation products of (-)-epigallocatechin gallate and (-)-epigallocatechin with H(2)O(2). , 2000, Journal of agricultural and food chemistry.

[14]  D. Dickinson,et al.  Green Tea Polyphenol Causes Differential Oxidative Environments in Tumor versus Normal Epithelial Cells , 2003, Journal of Pharmacology and Experimental Therapeutics.

[15]  Xiaofeng Meng,et al.  Stability, cellular uptake, biotransformation, and efflux of tea polyphenol (-)-epigallocatechin-3-gallate in HT-29 human colon adenocarcinoma cells. , 2002, Cancer research.

[16]  H. Fujiki,et al.  Sealing effects of (-)-epigallocatechin gallate on protein kinase C and protein phosphatase 2A. , 1997, Biophysical chemistry.

[17]  G. Loo,et al.  Effects of epigallocatechin gallate and quercetin on oxidative damage to cellular DNA. , 2000, Mutation research.

[18]  Hong Zhang,et al.  Oxidation-triggered c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein (MAP) kinase pathways for apoptosis in human leukaemic cells stimulated by epigallocatechin-3-gallate (EGCG): a distinct pathway from those of chemically induced and receptor-mediated apoptosis. , 2002, The Biochemical journal.

[19]  Chung S. Yang,et al.  Involvement of multidrug resistance-associated proteins in regulating cellular levels of (-)-epigallocatechin-3-gallate and its methyl metabolites. , 2003, Biochemical and biophysical research communications.

[20]  S. Kaakkola,et al.  Characteristics of catechol O-methyl-transferase (COMT) and properties of selective COMT inhibitors. , 1992, Progress in drug research. Fortschritte der Arzneimittelforschung. Progres des recherches pharmaceutiques.

[21]  Chung S. Yang,et al.  Mechanisms of cancer prevention by tea constituents. , 2003, The Journal of nutrition.

[22]  K. Shirouzu,et al.  Analysis of cell growth inhibitory effects of catechin through MAPK in human breast cancer cell line T47D. , 2002, International journal of oncology.

[23]  Z. Y. Wang,et al.  Tea and cancer. , 1993, Journal of the National Cancer Institute.

[24]  W. Berger,et al.  Promyelocytic HL60 cells express NADPH oxidase and are excellent targets in a rapid spectrophotometric microplate assay for extracellular superoxide. , 2003, Toxicological sciences : an official journal of the Society of Toxicology.

[25]  G. Yang,et al.  Inhibition of growth and induction of apoptosis in human cancer cell lines by tea polyphenols. , 1998, Carcinogenesis.

[26]  H. Forman,et al.  Redox signaling: thiol chemistry defines which reactive oxygen and nitrogen species can act as second messengers. , 2004, American journal of physiology. Cell physiology.

[27]  Chi-Tang Ho,et al.  Identification and characterization of methylated and ring-fission metabolites of tea catechins formed in humans, mice, and rats. , 2002, Chemical research in toxicology.

[28]  D. Ross,et al.  Cell-specific metabolism in mouse bone marrow stroma: studies of activation and detoxification of benzene metabolites. , 1992, Molecular pharmacology.

[29]  T. McMillan,et al.  The green tea polyphenol, epigallocatechin‐3‐gallate, protects against the oxidative cellular and genotoxic damage of UVA radiation , 2002, International journal of cancer.

[30]  R. K. Bhattacharya,et al.  Anticlastogenic, antigenotoxic and apoptotic activity of epigallocatechin gallate: a green tea polyphenol. , 2003, Mutation research.

[31]  B. Halliwell,et al.  Artifacts in cell culture: rapid generation of hydrogen peroxide on addition of (-)-epigallocatechin, (-)-epigallocatechin gallate, (+)-catechin, and quercetin to commonly used cell culture media. , 2000, Biochemical and biophysical research communications.

[32]  Laishun Chen,et al.  Human salivary tea catechin levels and catechin esterase activities: implication in human cancer prevention studies. , 1999, Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology.

[33]  A. Bürkle,et al.  Induction of poly(ADP-ribosyl)ation and DNA damage in human peripheral lymphocytes after treatment with (-)-epigallocatechin-gallate. , 2003, Mutation research.

[34]  Hideyuki Ito,et al.  Antibacterial Activity of Hydrolyzable Tannins Derived from Medicinal Plants against Helicobacter pylori , 2004, Microbiology and immunology.

[35]  C. Guillemette,et al.  The UDP-glucuronosyltransferase 1A9 Enzyme Is a Peroxisome Proliferator-activated Receptor α and γ Target Gene* , 2003, The Journal of Biological Chemistry.

[36]  M. Fenech The in vitro micronucleus technique. , 2000, Mutation research.

[37]  Xiaofeng Meng,et al.  Enzymology of methylation of tea catechins and inhibition of catechol-O-methyltransferase by (-)-epigallocatechin gallate. , 2003, Drug metabolism and disposition: the biological fate of chemicals.

[38]  B. Frei,et al.  Antioxidant activity of tea polyphenols in vivo: evidence from animal studies. , 2003, The Journal of nutrition.

[39]  D. August,et al.  Ingestion of green tea rapidly decreases prostaglandin E2 levels in rectal mucosa in humans. , 1999, Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology.

[40]  G. Yen,et al.  Modulation of Tea and Tea Polyphenols on Benzo(a)pyrene-induced DNA Damage in Chang Liver Cells , 2004, Free radical research.

[41]  B. Halliwell,et al.  Contribution of hydrogen peroxide to the cytotoxicity of green tea and red wines. , 2003, Biochemical and biophysical research communications.

[42]  C Helma,et al.  A public domain image-analysis program for the single-cell gel-electrophoresis (comet) assay. , 2000, Mutation research.

[43]  D. Heimburger,et al.  Micronutrients and cancer therapy. , 2004, Nutrition reviews.

[44]  H. Nakagawa,et al.  Generation of hydrogen peroxide primarily contributes to the induction of Fe(II)-dependent apoptosis in Jurkat cells by (-)-epigallocatechin gallate. , 2004, Carcinogenesis.

[45]  R. Tice,et al.  Single cell gel/comet assay: Guidelines for in vitro and in vivo genetic toxicology testing , 2000, Environmental and molecular mutagenesis.

[46]  J. Buttriss,et al.  A review of the epidemiological evidence for the ‘antioxidant hypothesis’ , 2004, Public Health Nutrition.

[47]  J. Hou,et al.  ESR study on the structure-antioxidant activity relationship of tea catechins and their epimers. , 1999, Biochimica et biophysica acta.

[48]  M. Segal,et al.  Hydrogen peroxide as a diffusible signal molecule in synaptic plasticity , 2004, Molecular Neurobiology.

[49]  M. Koo,et al.  Growth inhibition and cell cycle arrest effects of epigallocatechin gallate in the NBT‐II bladder tumour cell line , 2004, BJU international.

[50]  J. Stone An assessment of proposed mechanisms for sensing hydrogen peroxide in mammalian systems. , 2004, Archives of biochemistry and biophysics.

[51]  R. Dashwood,et al.  Inhibition of beta-catenin/Tcf activity by white tea, green tea, and epigallocatechin-3-gallate (EGCG): minor contribution of H(2)O(2) at physiologically relevant EGCG concentrations. , 2002, Biochemical and biophysical research communications.

[52]  J. Lin,et al.  Cancer chemoprevention by tea polyphenols through mitotic signal transduction blockade. , 1999, Biochemical pharmacology.

[53]  May-Chen Kuo,et al.  Blood and urine levels of tea catechins after ingestion of different amounts of green tea by human volunteers. , 1998, Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology.

[54]  A. Murakami,et al.  Synergistic suppression of superoxide and nitric oxide generation from inflammatory cells by combined food factors. , 2003, Mutation research.

[55]  T. Nakayama Suppression of hydroperoxide-induced cytotoxicity by polyphenols. , 1994, Cancer research.

[56]  A. Furukawa,et al.  (-)-Epigallocatechin gallate causes oxidative damage to isolated and cellular DNA. , 2003, Biochemical pharmacology.

[57]  Epigallocatechin-3-gallate is absorbed but extensively glucuronidated following oral administration to mice. , 2003, The Journal of nutrition.

[58]  R. Tice,et al.  A simple technique for quantitation of low levels of DNA damage in individual cells. , 1988, Experimental cell research.

[59]  N. Miyata,et al.  Scavenging mechanisms of (-)-epigallocatechin gallate and (-)-epicatechin gallate on peroxyl radicals and formation of superoxide during the inhibitory action. , 1999, Free radical biology & medicine.

[60]  S. Mandel,et al.  Cell signaling pathways in the neuroprotective actions of the green tea polyphenol (‐)‐epigallocatechin‐3‐gallate: implications for neurodegenerative diseases , 2004, Journal of neurochemistry.

[61]  C. Guillemette,et al.  The UDP-glucuronosyltransferase 1A9 enzyme is a peroxisome proliferator-activated receptor alpha and gamma target gene. , 2003, The Journal of biological chemistry.

[62]  Sushil K. Jain,et al.  Effect of vitamin B6 on oxygen radicals, mitochondrial membrane potential, and lipid peroxidation in H2O2-treated U937 monocytes. , 2004, Free radical biology & medicine.

[63]  W. Dröge Free radicals in the physiological control of cell function. , 2002, Physiological reviews.

[64]  J. Packer,et al.  Green tea catechins partially protect DNA from (.)OH radical-induced strand breaks and base damage through fast chemical repair of DNA radicals. , 2001, Carcinogenesis.

[65]  K. Fukuchi,et al.  Production of hydrogen peroxide and methionine sulfoxide by epigallocatechin gallate and antioxidants. , 2001, Anticancer research.

[66]  C. Ioannides,et al.  Evaluation of the antigenotoxic potential of monomeric and dimeric flavanols, and black tea polyphenols against heterocyclic amine-induced DNA damage in human lymphocytes using the Comet assay. , 2002, Mutation research.

[67]  Chi-Tang Ho,et al.  Delivery of Tea Polyphenols to the Oral Cavity by Green Tea Leaves and Black Tea Extract , 2004, Cancer Epidemiology Biomarkers & Prevention.

[68]  M. Lebwohl,et al.  Scavenging of hydrogen peroxide and inhibition of ultraviolet light-induced oxidative DNA damage by aqueous extracts from green and black teas. , 1999, Free radical biology & medicine.

[69]  D. Dickinson,et al.  Roles of Catalase and Hydrogen Peroxide in Green Tea Polyphenol-Induced Chemopreventive Effects , 2004, Journal of Pharmacology and Experimental Therapeutics.