Wide distribution of [3H](-)-epigallocatechin gallate, a cancer preventive tea polyphenol, in mouse tissue.

Masami Suganuma 0 2 Sachiko Okabe 0 2 Masumi Oniyama 0 2 Yukiko Tada 0 2 Hideyuki Ito 0 1 2 Hirota Fujiki 0 2 0 Abbreviations: EC , (-)-epicatechin; ECG, (-)-epicatechin gallate; EGC, (-)- epigallocatechin; EGCG, (-)-epigallocatechin gallate 1 Faculty of Pharmaceutical Sciences, Okayama University , Tsushima, Okayama 700-0082 , Japan 2 Saitama Cancer Center Research Institute , Ina, Kitaadachi-gun, Saitama 362-0806 2To whom correspondence should be addressed Email: The increasing recognition of green tea and tea polyphenols as cancer preventives has created a need for a study of their bioavailability. For this purpose, we synthesized [3H] (-)-epigallocatechin gallate ([3H]EGCG) with a specific activity of 48.1 GBq/mmol and directly administered the solution into the stomachs of CD-1 female or male mice. Radioactivity in the digestive tract, various organs, blood, urine and feces was measured with an oxidizer at various times after administration and significant radioactivity was found in the previously reported target organs of EGCG and green tea extract (digestive tract, liver, lung, pancreas, mammary gland and skin), as well as other organs (brain, kidney, uterus and ovary and testes) in both sexes. Incorporation of radioactivity in the cells was confirmed by microautoradiography. Within 24 h, 6.6 (females) and 6.4% (males) of total administered radioactivity was excreted in the urine and 37.7 and 33.1% in feces. HPLC analysis of urine from both sexes revealed that 0.03-0.59% of administered [3H]EGCG, along with at least five metabolites, was excreted. In addition, we found that a second, equal administration to female mice after a 6 h interval enhanced tissue levels of radioactivity in blood, brain, liver, pancreas, bladder and bone 4-6 times above those after a single administration. These results suggest that frequent consumption of green tea enables the body to maintain a high level of tea polyphenols and this paper is the first pharmacological evidence of a wide distribution of [3H]EGCG in mouse organs, indicating a similar wide range of target organs for cancer prevention in humans. - In 1987, our results showing inhibition of tumor promotion by ()-epigallocatechin gallate (EGCG), a tea polyphenol with a molecular weight of 458, opened up the study of the cancer preventive properties of EGCG and other tea polyphenols (1,2). The inhibitory effects of EGCG and green tea extract on carcinogenesis in various organs in rodents have now been demonstrated over the past decade. Specifically, EGCG and green tea extract in drinking water effectively inhibit carcinogenesis in the esophagus, glandular stomach, duodenum, colon, liver, pancreas, lung, breast and skin (313) and EGCG in drinking water also inhibits metastasis of B16 melanoma cells into the lungs (14). At present, both EGCG and green tea extract are acknowledged practical cancer preventive agents (15), but to fully appreciate their significance, it was necessary to study how EGCG and green tea extract in drinking water are incorporated into various organs, along with their metabolism. Tea (Camellia sinensis) is widely consumed throughout the world as black tea, oolong tea and green tea. Approximately 26% of the solid weight of green tea extract is tea polyphenols and EGCG constitutes 11% of these (16). The composition and content of tea polyphenols undergo changes during the fermentation process: The content of EGCG in black tea, the most fermented tea, is less than in green tea, whereas the content of theaflavins is higher in black tea. All this was known and, moreover, a prospective cohort study in Saitama Prefecture revealed the cancer preventive effects of drinking green tea in humans (17). However, the bioavailability and metabolic fate of tea polyphenols have not been fully understood. A few papers had reported on HPLC analysis of tea polyphenols in plasma and urinary samples of humans or rats after ingestion of green tea (1821). These papers demonstrated that tea polyphenols, including EGCG, ()-epigallocatechin (EGC) and ()-epicatechin (EC), were incorporated into the blood only a few hours after oral administration and excreted into urine as conjugated forms, e.g. glucuronide and sulfate. However, a more detailed study of their bioavailability, using radiolabeled tea polyphenols, was needed. Accordingly, we recently obtained [3H]EGCG, labeled with 3H with a specific activity of 48.1 GBq/mmol, as a tool for a bioavailability study (Figure 1). The 3H label in this EGCG was quite stable: it did not, for example, exchange with 3H2O after incubation for 24 h (Amersham, UK, personal communication). First, we studied the distribution of [3H]EGCG in mouse organs by oral administration: Radioactivity was found in various organs, including many where inhibition of carcinogenesis by EGCG or green tea extract has already been shown. HPLC analyses of urine samples showed that small amounts of [3H]EGCG were excreted in urine. A second, equal administration of [3H]EGCG after a 6 h interval increased the radioactivity in blood and various organs. This paper provides the first evidence that the radioactivity of [3H]EGCG is distributed in various organs, a finding supported by microautoradiography. Materials and methods Chemicals 4(n)-[3H]()-EGCG was labeled with tritium gas (Amersham, Aylesbury, UK). The radiochemical purity of [3H]EGCG was 92.9% by analysis by HPLC and 3H NMR. The positions labeled with 3H were in one of the aromatic rings estimated from the 3H NMR; it was not possible to specifically identify the sites. The 3H label in EGCG was quite stable, confirming that it did not exchange with water over a period of 24 h. The specific activity of [3H]EGCG was 48.1 GBq/mmol and the EGCG used for all the experiments was 99.7% pure. Animals Female and male CD-1 mice, 7 weeks old, were obtained from Charles River Japan (Kanagawa, Japan). Mice were given no nourishment for ~15 h before gastric intubation. Distribution of radioactivity in mice after a single administration of [3H]EGCG Each mouse was given 200 m l 0.05% EGCG solution containing 3.7 MBq [3H]EGCG by gastric tube and then kept in a metabolic cage with controlled temperature, humidity and light. At various intervals after administration, samples of digestive tract, blood and various organs were taken for measurements of radioactivity. Digestive tract. The digestive tract was divided into three parts, stomach, small intestine and colon. Total radioactivity of each part, including dietary content, was measured with an oxidizer (Packard Japan, Tokyo, Japan) (22). The oxidizer converts 3H radioactivity in tissue samples to 3H2O in a process of perfect combustion. The 3H2O was measured with a scintillation counter (LS 6500; Beckman Instrument, Fullerton, CA). Blood. Radioactivity in blood was measured with an oxidizer using filter paper blotted with 50 m l blood. Various organs. Radioactivity of various organs was measured with an oxidizer using (...truncated)