Inhibitory activity of a green tea extract and some of its constituents on multidrug resistance-associated protein 2 functionality.

Green tea extracts (GTE) might modulate ABC transporter gene expression or function. This may be relevant in the treatment of cancer or in influencing intestinal drug permeability. To gain more insight on the influence of a GTE on secretory transport proteins we investigated the influence of GTE and several green tea components on the mRNA expression level of P-glycoprotein (P-gp) and multidrug resistance-associated protein 2 (MRP2) in human gastrointestinal epithelial LS-180 cells. Furthermore, the functional activity of MRP2, using glutathione methylfluorescein (GS-MF) or [3H]methotrexate (MTX) as substrate, was investigated in canine kidney cells stably overexpressing human MRP2 (MDCK-MRP2). GTE, at a concentration of 0.01 mg/mL, did not increase mRNA expression of P-gp or MRP2 in LS-180 cells. Functional assays in MDCK-MRP2 cells using GS-MF did not show any effect of 0.01 mg/mL GTE on MRP2 activity. In the same cell line the cellular accumulation of MTX (a specific substrate of MRP2) was significantly increased with the MRP-specific inhibitor MK-571 or with 1 mg/mL GTE, but not with 0.1 mg/mL. The green tea components (-)-epigallocatechin gallate, (-)-epigallocatechin, theanine, or caffeine, each in corresponding concentrations to the respective concentration of GTE, did not show any effect on MRP2 function. These data demonstrate that the mRNA expression patterns of P-gp and MRP2 in LS-180 cells are not altered by 0.01 mg/mL of GTE. However, MRP2 function was inhibited by 1 mg/mL GTE, whereas none of the green tea components tested were responsible for this effect.

Toxicity of green tea extracts and their constituents in rat hepatocytes in primary culture.

Recent reports on sporadic cases of liver disorders (acute hepatitis, icterus, hepatocellular necrosis) after ingestion of dietary supplements based on hydro-alcoholic extracts from green tea leaves led to restrictions of the marketing of such products in certain countries of the EU. Since green tea is considered to exert a number of beneficial health effects, and, therefore, green tea products are widely used as dietary supplements, we were interested in the possible mechanism of hepatotoxicity of green tea extracts and in the components involved in such effects. Seven hours after seeding on collagen, rat hepatocytes in primary culture were treated with various hydro-alcoholic green tea extracts (two different native 80% ethanolic dry extracts and an 80% ethanolic dry extract cleared from lipophilic compounds). Cells were washed, and reduction of resazurin, used as a viability parameter monitoring intact mitochondrial function, was determined. It was found that all seven green tea extracts examined enhanced resazurin reduction significantly at a concentration range of 100-500 microg/ml medium, while a significant decrease was observed at 1-3mg/ml medium. Decreased levels were concomitant with abundant necrosis as observed by microscopic inspection of the cultures and with increased leakage of lactate dehydrogenase activity from the cells. In a separate series of experiments, the green tea constituents (-)-epicatechin, (-)-epigallocatechin-3-gallate, caffeine and theanine were tested at concentrations reflecting their levels in a typical green tea extract. Synthetic (+)-epigallocatechin (200 microM) was used for comparison. Cytotoxicity was found with (-)-epigallocatechin-3-gallate only. The concomitant addition of 0.25 mM ascorbate/0.05 mM alpha-tocopherol had no influence on cytotoxicity. In conclusion, our results suggest that high concentrations of green tea extract can exert acute toxicity in rat liver cells. (-)-Epigallocatechin-3-gallate seems to be a key constituent responsible for this effect. The relatively low bioavailability of catechins reported after oral exposure to green tea argues, however, against a causal role of these constituents in the reported liver disorders.

Bioactivity of well-defined green tea extracts in multicellular tumor spheroids.

The effect of green tea extracts (GTE) of a reproducible, well-defined composition on cellular viability, proliferation, and antioxidant defense was investigated in multicellular spheroids derived from WiDr human colon adenocarcinoma cells. The maximum GTE concentration investigated, i.e. 100 micro g GTE/ml, was equivalent to the plasma concentration commonly measured in humans drinking 6-10 cups of green tea per day. This GTE concentration lead to a substantial retardation of spheroid volume growth with diameters reaching only half the size of untreated aggregates. Flow cytometric analysis and immunocytochemistry showed an enhanced accumulation of cells in G2/M and in the non-proliferating compartment, respectively. The emergence of central necrosis occurred at larger spheroid diameters compared to control conditions leading to a significant increase (p<0.05) in the thickness of the viable cell rim (mean +/- SD) from 240+/-49.9 micro m to 294+/-69.5 micro m. This was associated with an elevation of the intracellular GSH concentration and, thus, of cellular antioxidant defense, as shown by HPLC analysis. A considerable toxicity, however, was found at these GTE levels in single cells. Cells did not adhere to culture dishes nor did they aggregate to form spheroids when plated as a suspension with GTE already in the culture medium. The findings show that green tea constituents interfere with early phases of tumorigenesis at a cellular level, e.g., by reducing cell-substratum and cell-cell interaction, enhancing G2/M arrest, and retarding spheroid volume growth. The differences in GTE effects between single cells and cell spheroids underline the importance of inclusion of spheroids in pharmaco-/toxicological testing.