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

Leonilla Elbling^,1, Rosa-Maria Weiss, Olga Teufelhofer^*, Maria Uhl, Siegfried Knasmueller, Rolf Schulte-Hermann^*, Walter Berger and Michael Micksche

Institute for Cancer Research,
^* Division Oncological Toxicology,
Environmental Toxicology Group,
Division of Applied and Experimental Oncology, Medical University of Vienna, Vienna, Austria

¹ Correspondence: Institute of Cancer Research, Division of Applied and Experimental Oncology, Medical University of Vienna, Borschkegasse 8a, Vienna 1090, Austria. E-mail: elbling.leonilla{at}meduniwien.ac.at

SPECIFIC AIMS

There is considerable interest in the health benefits of green tea (GT) extracts and its major catechin, (–)-epigallocatechin-3-gallate (EGCG), which have mainly been attributed to the protection against oxidative stress. Studies on cytotoxic/oxidant activities of EGCG have received much less attention and have recently been ascribed to in vitro artifacts. Aims of the present study were to assess both oxidant/toxic and antioxidant activities of GT extracts and EGCG in vitro, with specific attention to the contribution of the H₂O₂ generated.

PRINCIPAL FINDINGS

1. Cytotoxicity and genotoxicity of GT extracts and EGCG
GT extracts, as well as EGCG, induced cell death (necrosis and apoptosis) after 24 h treatment. The analyzed cell lines were the human promyelocytic leukemic HL60 cells of the granulocyte/monocyte/macrophage lineage which exhibit phagocytic activity and responsiveness to chemotactic stimuli, and the murine RAW 264.7 cells which resemble (as a standard monocytic/macrophage-like cell line) primary cultured macrophages in many aspects. The LC₅₀ of the two differently EGCG-enriched GT extracts were of 14.7 vs. 107.8 and 20.8 vs. 234.5 µg/mL and of EGCG 37.2 vs. 180.6 µM for RAW 264.7 vs. HL60 cells, respectively. We addressed in detail the quality and pitfalls of commonly used cytotoxicity assays (Trypan blue exclusion, MTT-assay) to determine cytotoxic effects of GT extracts and EGCG concentrations above a critical level (EGCG>200 or >700 µM). The assays tested tend to detect false positive viability due to both the specific form of cell death induced by GT extracts and EGCG (Trypan blue impermeable cells) and the production of formazan without cells (MTT-assay).

The lowest concentration of EGCG which induced cell death in RAW 264.7 cells (10 µM) increased DNA damage in the alkaline comet assay after 1 h and enhanced micronuclei formation after 24 h. For HL60 cells, however, the genotoxic EGCG concentration of 20 µM was far below the lowest cytotoxic concentration of 100 µM. Lethal effects were induced within the first two hours of the higher enriched GT extract (GTE) and EGCG treatment and DNA damage was readily detectable within 15min. Comet tail induction reached plateau at 100 µM EGCG.

2. Oxidative stress induction by GTE and EGCG and the contribution of H₂O₂
Oxidative stress (DCFH-DA assay after 1 h treatment) was induced in both cell lines at the dose levels which were also genotoxic (10 µg/mL GTE and 20 µM EGCG). Concentration-dependent DCF-fluorescence reached a plateau at 200 µM EGCG. The magnitude of the oxidative and DNA-damaging effect of 10 µM EGCG corresponded to that of 3 µM and 11 µM of exogenously added H₂O₂, respectively. The comparison of the effect of EGCG with that of exogenous H₂O₂ added at the concentrations generated by EGCG solubilization (FOX-assay performed within 15 min) (Fig. 1 A, B) showed EGCG to be significantly more cyto- and genotoxic and pro-oxidant than the H₂O₂ formed (Fig. 1C-E ).

View larger version (54K): [in this window] [in a new window]   Figure 1. H2O2 content formed upon treatment with EGCG and oxidant potency of EGCG as compared with the respective H2O2 concentrations. A, B) Within 15 min after addition of EGCG at the indicated concentrations, H2O2 content was measured in HBSS and RPMI-1640 + 10% FCS in presence and absence of HL60 cells by the FOX-assay. C, D) Comparison of DCF-fluorescence and CT-formation after treatment with EGCG and the respective formed H2O2 concentrations in presence and absence of cells. E) Apotosis induction after treatment with EGCG and the respective formed H2O2 concentration. Photomicrographs of cytospin prepared and Hoechst 33258 stained cells. Bars indicate 20 µm.

3. Absence of protection against H₂O₂-induced oxidative stress by EGCG
The potency of EGCG to scavenge H₂O₂ and/or protect from H₂O₂-induced stress was assessed in absence and presence of HL60 cells. In this model we could not detect any antioxidant activity of µM EGCG concentrations. Rather, and depending on the applied H₂O₂ concentrations and the assay performed, EGCG concentrations of 1 µM and 10 µM increased the H₂O₂ content in solution (FOX-assay) (Fig. 2 A) and enhanced the oxidative and genotoxic effects induced by H₂O₂ (Fig. 2B, C ).

View larger version (17K): [in this window] [in a new window]   Figure 2. Lack of antioxidant potency of EGCG against H2O2-induced oxidative stress. A) EGCG at the indicated concentrations without H2O2 (black square) and with H2O2 at 10 µM (triangle down) and 20 µM (triangle up) were added to HBSS without cells and the H2O2 content was measured by FOX-assay within 15 min upon addition of EGCG at the indicated concentrations. B, C) EGCG at the indicated concentrations without H2O2 (filled square) and 10 µM H2O2 (triangle down) as well as 20 µM H2O2 (triangle up) were added to HL60 cells and DCF-fluorescence and CT-formation was determined. EGCG was added 30 min before H2O2 treatment.

CONCLUSIONS AND SIGNIFICANCE

EGCG, a flavonoid phytochemical, represents the most abundant catechin component in green tea leaves. Several health benefits of green tea have been proposed and attributed to its free radical scavenging and antioxidant activities. However, the beneficial effects of green tea on human diseases, including cancer, are still inconclusive, and toxic and pro-oxidant effects induced by GT extracts and EGCG in vitro as observed earlier were attributed to cell culture artifacts and claimed to be irrelevant for the in vivo situation.

In the present study we demonstrate that the cyto- and genotoxicity as well as pro-oxidant effects induced by EGCG occur at pharmacologically relevant concentrations. In contrast to previous reports, only the combination of catalase with superoxide dismutase inhibited these effects completely. This observation indicates, in accordance with a recent report, that superoxide (SO) represents an additional toxic specimen generated when EGCG is dissolved. Both cell models, although differing in their extent of response toward GT extracts and EGCG, did not differ with regard to the lowest effective concentrations. This indicates, in contrast to previous reports and presumably depending on the cell systems used, that cellular characteristics other than malignancy are decisive factors determining sensitivity to EGCG. It has to be pointed out that the lowest toxic concentrations were below the range where generation of H₂O₂ could be detected in EGCG-containing solutions and media. In contrast to earlier reports, the toxic effect of EGCG was significantly higher than that of the generated H₂O₂ amounts added exogenously. These results also suggest that the toxic effect of EGCG cannot be fully ascribed to H₂O₂. Rather, additional effects of EGCG such as SO formation should be considered.

Antioxidant actions of EGCG have been attributed both to direct scavenging and/or chelation of redox-active metal ions, to the inhibition of reactive oxygen species (ROS) generation and of redox-sensitive transcription factors, as well as to the induction of antioxidant enzymes. In our hands, neither nM nor µM concentrations of EGCG caused antioxidant effects, but rather showed an oxidative stress-enhancing activity in the H₂O₂ stress model, both under cell-free conditions and in presence of cells. Discrepancies of our findings with previous reports may be caused by pitfalls in reading of cytotoxicity assays (compare above) at critical concentrations but also by cell-specific differences in resistance mechanisms and in the metabolism affecting availability and the actual toxic effects of EGCG.

Experimental investigations and epidemiological studies on the protective activities of green tea and EGCG belong to a field of research which produces numerous controversial reports of either promising or disadvantageous results. In accordance with our in vitro results, no convincing indication of an antioxidant activity in vivo has been demonstrated yet. Our contribution confirms the presence of a pro-oxidant activity of EGCG at µM concentrations near the peak serum concentrations found after tea consumption but below those generating detectable amounts of H₂O₂. We hypothesize that EGCG concentrations below 1µM as achieved during common tea consumption may induce H₂O₂ at low levels similar to those generated in response to physiological stimuli. Such low levels would not cause cell damage but contribute to physiological ROS-mediated signaling and thus may account for the proposed beneficial effects of tea consumption.

Our data suggest that detailed mechanistic studies of the effects of GT extracts and EGCG should be performed in vivo before excessive intake and/or topical application of GT products can be recommended to healthy and/or diseased persons.

View larger version (28K): [in this window] [in a new window]   Figure 3. Schematic diagram illustrating the redox and the lack of antioxidant potency of EGCG. The oxidative effect of EGCG (2) exceeds that of the H2O2 concentrations (3) generated in solution in absence (1) and presence of cells (2). H2O2 added in presence of EGCG to solutions in absence (4) and presence (5) of cells is not scavenged but rather increased and oxidative cell injuries are increased (5) as compared with those induced by H2O2 alone (6). Instead of the µM ROS-inducing EGCG concentrations, a ROS-signaling is suggested as one beneficial mechanism of nM EGCG concentrations in vivo (7).

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2915fje;

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