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First published on October 4, 2005; DOI: 10.1124/dmd.104.003434

0090-9556/06/3401-8-11$20.00
DMD 34:8-11, 2006

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SHORT COMMUNICATION

DOSE-DEPENDENT LEVELS OF EPIGALLOCATECHIN-3-GALLATE IN HUMAN COLON CANCER CELLS AND MOUSE PLASMA AND TISSUES

Joshua D. Lambert, Mao-Jung Lee, Lauren Diamond, Jihyeung Ju, Jungil Hong, Mousumi Bose, Harold L. Newmark, and Chung S. Yang

Department of Chemical Biology, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, New Jersey

(Received January 7, 2005; accepted September 23, 2005)


   Abstract
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 Abstract
Materials and Methods
 Results and Discussion
 References
 
Epigallocatechin-3-gallate (EGCG; molecular formula: C22H18011)is the most abundant catechin in green tea (Camellia sinensis Theaceae). Both EGCG and green tea have been shown to have cancer-preventive activity in a number of animal models, and numerous mechanisms have been proposed based on studies with human cell lines. EGCG has been shown to undergo extensive biotransformation to yield methylated and glucuronidated metabolites in mice, rats, and humans. In the present study, we determined the concentration-dependent uptake of EGCG by HT-29 human colon cancer cells (20–600 µM) and the dose dependence of EGCG plasma and tissue levels after a single dose of EGCG (50–2000 mg/kg i.g.) to male CF-1 mice. The cytosolic levels of EGCG were linear with respect to extracellular concentration of EGCG after treatment of HT-29 cells for 2 h (915.3–6851.6 µg/g). In vivo, EGCG exhibited a linear dose relationship in the plasma (0.03–4.17 µg/ml), prostate (0.01–0.91 µg/g), and liver (0.09-18.3 µg/g). In the small intestine and colon, however, the levels of EGCG plateaued between 500 and 2000 mg/kg i.g. These results suggest that absorption of EGCG from the small intestine is largely via passive diffusion; however, at high concentrations, the small intestinal and colonic tissues become saturated. The levels of 4''-O-methyl-EGCG and 4',4''-di-O-methyl-EGCG parallel those of EGCG with respect to dose. The present study provides information with respect to what concentrations of EGCG are achievable in mice and may guide dose selection for future cancer chemoprevention studies with EGCG.

Epigallocatechin-3-gallate (EGCG; Fig. 1) is the most abundant and widely studied catechin in green tea (Camellia sinensis Theaceae). Both green tea and EGCG have been reported to have cancer-preventive activities in a number of animal models (Yang et al., 2002Go; Lambert and Yang, 2003Go). Numerous potential mechanisms have been proposed to account for these activities, including inhibition of growth factor receptor signaling, inhibition of activator protein-1 and nuclear factor {kappa}B-mediated transcription, inhibition of the proteasome, and others (Liang et al., 1997Go; Masuda et al., 2001Go; Nam et al., 2001Go; Yang et al., 2001Go). In most studies, the concentrations needed to observe these activities typically range from 1 to 100 µM: concentrations that exceed those found in rodent and human plasma by 10- to 100-fold (Lambert and Yang, 2003Go).



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  		FIG. 1. Dose-dependent cytosolic concentration of EGCG in HT-29 human colon adenocarcinoma cells. Cells were exposed to 20 to 600 µM EGCG in HBSS, pH 7.4 for 2 h. The cytosolic fraction was prepared and EGCG levels were determined by HPLC. Each point represents the mean of n = 4. Error bars represent the standard deviation.

 
Chow et al. (2001Go) have previously reported, in one of the only careful dose-finding studies, that after oral administration of EGCG to humans, a linear increase in plasma concentrations and area under the curve was observed over a dose range of 200 to 600 mg. However, administration of 800 mg of EGCG resulted in a greater than predicted plasma Cmax and area under the curve. The authors hypothesized that this increase might be due to saturation of factors (e.g., phase II enzymes, efflux pumps, etc.) that limit bioavailability of EGCG.

EGCG has been shown to undergo extensive phase II metabolism including glucuronidation and methylation in vitro and in vivo (Lambert et al., 2003Go; Lu et al., 2003aGo,bGo). We have previously reported on the active efflux of EGCG by multidrug resistance-related protein (Mrp) 1 and 2 from human cancer cell lines in vitro (Hong et al., 2003Go). Additionally, epicatechin-3-gallate has been reported to be a substrate of monocarboxylate transporters (Hong et al., 2003Go; Vaidyanathan and Walle, 2003Go). Monocarboxylate transporters represent potential saturable factors that may limit absorption of EGCG from the gut and its availability to the plasma, and may explain the phenomena observed by Chow et al. (2001Go).

We have previously determined the pharmacokinetic parameters of EGCG in the mouse and ascertained that this species is more similar to humans in terms of EGCG bioavailability than is the rat (Lambert et al., 2003Go). In the present study, we examined the dose dependence of plasma and tissues levels of EGCG in mice. Based on energy consumption calculations, the doses selected (50–2000 mg/kg i.g.) include the range used by Chow et al. (2001Go) in their study and extend the range by a 10-fold greater dose. Herein, we report the results of our study.


   Materials and Methods
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 Abstract
 Materials and Methods
Results and Discussion
 References
 
Chemicals. EGCG (100% pure) was provided by Tokyo Food Techno Co. (Fujieda City, Japan). ß-D-Glucuronidase (G-7896; EC 3.2.1.31 [EC] , from Escherichia coli with 9 x 106 U/g solid) and sulfatase (S-9754; EC 3.1.6.1 [EC] , from Abalone entrails with 2.3 x 10 5 U/g solid) were purchased from Sigma-Aldrich (St. Louis, MO). All other reagents were of the highest grade commercially available. Dosing solutions of EGCG were prepared in 0.9% NaCl containing 15% ethanol. For analytical purposes, a standard stock solution of EGCG, epigallocatechin, epicatechin, and epicatechin-3-gallate (10 µg/ml each) was prepared in 0.2% ascorbic acid-0.005% EDTA (pH 3.8) and stored at –80°C.

Cell Culture and Treatment. HT-29 human colon adenocarcinoma cells (The American Type Culture Collection, Manassas, VA) were maintained in log-phase growth in McCoy's 5A medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 0.1 mg/ml streptomycin at 37°C in 95% relative humidity and 5% CO2. Dose-dependent cellular uptake of EGCG over a dose range of 20 to 600 µM for 2 h was determined using previously described methods (Hong et al., 2003Go). In brief, once cells reached 70% confluence in six-well plates, medium was replaced with Hanks' balanced salt solution (HBSS; pH 7.4). Cells were allowed to equilibrate for 2 h; then, fresh HBSS containing 100 mM ascorbic acid and the desired concentration of EGCG was added. After incubation at 37°C for 2 h, cells were washed three times with phosphate-buffered saline and scraped into 2% ascorbic acid. The cells were sonicated with a probe sonicator and centrifuged at 14,000 rpm to pellet cell debris. The supernatant (i.e., cytosol) was transferred to a new tube. The protein content was determined by the method of Bradford (1976Go), and the supernatant was combined with an equal volume of ice-cold methanol. After centrifugation, 50 µl of the supernatant was analyzed by HPLC with electrochemical detection.

Animals. Male CF-1 mice (30–35 g) were purchased from Charles River Laboratories, Inc. (Wilmington, MA) and allowed to acclimate for at least 1 week before the start of the experiment. The mice were housed 10 per cage and maintained in air-conditioned quarters with a room temperature of 20 ± 2°C, relative humidity of 50 ± 10%, and an alternating 12-h light/dark cycle. Animals were given Purina rodent chow (Research Diets, New Brunswick, NJ) and water ad libitum and fasted overnight before treatment.

Treatment and Sample Collection. After an overnight fast, mice (six per group) were given a single dose of EGCG (50–2000 mg/kg i.g.) and sacrificed at 50 or 180 min. Blood was collected from anesthetized animals by cardiac puncture and plasma was isolated by centrifugation at 500g for 15 min. Plasma was combined with a 1/10th volume of ascorbate preservative (20% ascorbic acid-0.1% EDTA) and stored at –80°C for later analysis. The lungs, liver, colon, small intestine, and prostate were collected, washed in 0.9% NaCl, and frozen at –80°C for later analysis.

Quantification of EGCG and Metabolites. Plasma levels of EGCG and its metabolites were analyzed as previously reported (Chen et al., 1997Go). In brief, 100 µl of plasma was hydrolyzed with 1 U of sulfatase and 250 U of ß-glucuronidase at 37°C for 45 min, and extracted with methylene chloride and ethyl acetate. The ethyl acetate fractions were pooled and dried under vacuum. Samples were reconstituted in 10% aqueous acetonitrile and analyzed by HPLC.

Tissue samples were prepared and analyzed by our standard methods (Lambert et al., 2003Go). In brief, samples were homogenized in 2 volumes of ice-cold 2% ascorbic acid using a mechanical Dounce homogenizer, and 200-µl aliquots were hydrolyzed, extracted, and analyzed in a manner identical to plasma. EGCG and its methylated metabolites were identified by comparing samples with the retention times of authentic standards. Quantification was based on comparison of peak heights with standard plasma containing a known amount of compound.

Sample Analysis. EGCG levels were analyzed using an HPLC apparatus consisting of two ESA model 580 dual-piston pumps (ESA, Inc., Chelmsford, MA), a Waters model 717plus refrigerated autosampler (Waters, Milford, MA), and an ESA 5500 Coulochem electrode array system (CEAS). The potentials of the CEAS were set at –100, 100, 300, and 500 mV. Separation was achieved using a binary mobile phase of solvent A (0.03 M NaH2PO4, pH 3.35, containing 1.75% acetonitrile and 0.125% tetrahydrofuran) and solvent B [0.015 M NaH2PO4, pH 3.45, containing 58.5% acetonitrile and 6.25% tetrahydrofuran as previously reported (Chen et al., 1997Go)].


   Results and Discussion
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 Abstract
 Materials and Methods
 Results and Discussion
References
 
Incubation of HT-29 cells, a commonly used cell line for drug uptake studies, with concentrations of EGCG ranging from 20 to 600 µM resulted in a linear increase in the cytosolic concentration of EGCG (Fig. 1). Although we have previously shown that these cells express Mrp pumps capable of effluxing EGCG from the cytosol, and UDP-glucuronosyltransferase and catechol-O-methyltransferase activity capable of biotransforming EGCG (Hong et al., 2002Go), our present results suggest that these are not saturated under the present conditions.

To extend these in vitro studies, we examined the dose-concentration relation of EGCG following intragastric administration of EGCG to CF-1 mice. The doses used are approximately equivalent to the consumption of 2 to 53 cups of green tea (185 mg of EGCG per cup) by a 70-kg person. The dose conversion from humans to mice was based on scaling dose to daily caloric intake (i.e., 2000 kcal/day for humans and 12 kcal/day for mice) using the following equation (Schneider et al., 2004Go). From mg/kg in human to mg/kcal: (Dosemg/kg · body weightkg)/2000 kcal = Dosemg/kcal and from mg/kcal to mg/kg in mice: (Dosemg/kcal · 12 kcal)/body weightkcal = Dosemg/kg.

No acute toxicity was observed after single-dose administration of up to 2000 mg/kg i.g. A linear increase in the plasma concentration of EGCG was observed 50 and 180 min after administration of 50 to 2000 mg/kg i.g. EGCG to male CF-1 mice (Fig. 2). The concentration of total and unconjugated EGCG in the plasma ranged from 0.03 to 4.17 µg/ml and 0.01 to 0.37 µg/ml, respectively. Plasma levels were higher at 50 min than at 180 min, and EGCG was largely conjugated as the glucuronide (45–90%); these observations are consistent with our previous pharmacokinetics study in the mouse (Lambert et al., 2003Go). There was no apparent relationship between dose and the percentage of conjugated versus unconjugated EGCG. Interestingly, we did not observe the greater than linear increase in the plasma concentrations of EGCG as was previously reported in humans at high doses (Chow et al., 2001Go). It is unclear whether this is a species difference or is the result of the statistical variability in human studies.



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  		FIG. 2. Concentration of total EGCG in the plasma, liver, prostate, small intestine, and colon of male CF-1 mice 50 or 180 min after treatment with a single dose of EGCG (50–2000 mg/kg i.g.). For doses 50, 200, 500, and 2000 mg/kg, the points represent the average of two independent experiments with an overall n = 12. For doses 100 and 1000 mg/kg, the points represent the average of one independent experiment with n = 6. Error bars represent the standard error of the mean.

 
The concentration of EGCG in the prostate and liver also showed a linear relationship to dose (Fig. 2). EGCG at doses of 50 to 2000 mg/kg i.g. resulted in liver and prostate concentrations of 0.09 to 18.3 µg/g and 0.01 to 0.91 µg/g, respectively. Prostate concentrations for most doses were below the limit of detection at 180 min (data not shown). As we have previously reported, EGCG was in the unconjugated form in the tissues (Lambert et al., 2003Go). By contrast, levels of EGCG in the small intestine and colon were curvilinear with respect to EGCG dose (Fig. 2). These levels plateaued between 500 and 2000 mg/kg i.g. and resulted in maximal concentrations of 16.1 to 162.9 µg/g and 0.5 to 23.4 µg/g in the small intestine and colon, respectively. As we have previously observed, the levels in the small intestine were higher at 50 min, whereas those in the colon were higher at 180 min. The colonic concentration at 180 min after the 1000 mg/kg dose was greater than expected. We believe that this point is anomalous and is the result of incomplete washing of the colon samples in this treatment group. This resulted in residual fecal matter in the sample, which led to higher than expected levels. The dose-dependent levels of 4''-O-methyl-EGCG and 4',4''-di-O-methyl-EGCG mirrored those of EGCG in the liver (0.05–0.49 µg/g and 0.09–0.14 µg/g, respectively), the prostate (0.01–0.12 µg/g and 0.002–0.03 µg/g), the small intestine (0.32–1.74 µg/g for 4''-O-methyl-EGCG), and the colon (0.04–0.27 µg/g for 4''-O-methyl-EGCG).

It is possible that at the higher doses (i.e., 500 and 2000 mg/kg i.g.), EGCG has saturated the small intestinal and colonic tissues, resulting in a plateau of the levels in these tissues. EGCG continues to cross the intestinal barrier, however, due to the lower concentrations in the plasma and other tissues and the higher saturation point of EGCG in these tissues: this would manifest as a linear increase in plasma or tissue concentration with respect to dose. In addition, the linear increase in plasma and other tissues may represent a saturation of the active elimination of EGCG by the small intestine (via Mrp2) or the liver (via biliary secretion). Overwhelming hepatic clearance would allow a greater amount of EGCG to exit the portal circulation and enter the systemic circulation. Further studies are needed to determine the dose-dependent absorptive capacity of the small intestine and its saturation point for EGCG. The fact that in vitro uptake studies using HT-29 cells did not predict the saturation of small intestinal and colon levels of EGCG suggests the limitation of this cell line as a model for the intestinal barrier. Likely this is because the model fails to take into account the impact of hepatic clearance, the presence of intestinal mucosal layer, and other factors.

To conclude, we have determined the dose dependence of EGCG levels in HT-29 human colon adenocarcinoma cells and in CF-1 mice following single-dose oral administration of EGCG. Cytosolic EGCG levels were linear with respect to extracellular concentration in vitro. In vivo, the plasma, liver, and prostate levels of EGCG exhibited a linear dose-concentration relationship: the dose-concentration relationship in the small intestine and colon was curvilinear and plateaued between 500 and 2000 mg/kg i.g. These doses were not acutely toxic, and these data should be useful in designing future cancer chemoprevention studies.


   Footnotes
 
Supported by National Institutes of Health Grant CA88961 (to C.S.Y.).

Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.

doi:10.1124/dmd.104.003434.

ABBREVIATIONS: EGCG, epigallocatechin-3-gallate; Mrp, multidrug resistance-related protein; i.g., intragastric; HBSS, Hanks' balanced salt solution; HPLC, high-performance liquid chromatography.

Address correspondence to: Dr. Chung S. Yang, Department of Chemical Biology, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, 164 Frelinghuysen Rd., Piscataway, NJ 08854-8020. E-mail: csyang{at}rci.rutgers.edu


   References
Top
 Abstract
 Materials and Methods
 Results and Discussion
 References
 


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Hong J, Lambert JD, Lee SH, Sinko PJ, and Yang CS (2003) Involvement of multidrug resistance-associated proteins in regulating cellular levels of (–)-epigallocatechin-3-gallate and its methyl metabolites. Biochem Biophys Res Commun 310: 222–227.[CrossRef][Medline]

Hong J, Lu H, Meng X, Ryu JH, Hara Y, and Yang CS (2002) Stability, cellular uptake, biotransformation and efflux of tea polyphenol (–)-epigallocatechin-3-gallate in HT-29 human colon adenocarcinoma cells. Cancer Res 62: 7241–7246.[Abstract/Free Full Text]

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