Epigallocatechin Gallate Modulates CYP450 Isoforms in the Female Swiss-Webster Mouse

TOXICOLOGICAL SCIENCES 76, 262–270 (2003) DOI: 10.1093/toxsci/kfh001 Epigallocatechin Gallate Modulates CYP450 Isoforms in the Female Swiss-Webster Mouse Mette G. Goodin and Rhonda J. Rosengren1 Department of Pharmacology and Toxicology, University of Otago, Dunedin, New Zealand Downloaded from https://academic.oup.com/toxsci/article/76/2/262/1686012 by guest on 28 September 2021 Received August 4, 2003; accepted September 22, 2003 and epicatechin (EC). There has been scientific interest in this This study was designed to determine the effect of the in vivo chemical family due to their various pharmacological actions. administration of epigallocatechin gallate (EGCG) and epicat- For example, the catechins inhibit the proliferation of human echin gallate (ECG) on enzymes involved in the synthesis and breast cancer cells (Kavanagh et al., 2001; Morre et al., 2000; metabolism of estradiol. EGCG (12.5, 25, or 50 mg/kg/day, ip) or ECG (12.5 or 25 mg/kg/day, ip) was administered to female Swiss- Valcic et al., 1996) and epidemiological studies have suggested Webster mice for 7 days. The chemicals were well tolerated by the that green tea consumption is linked to a decrease in both the mice with the exception of EGCG given at 50 mg/kg, which rate of development and recurrence of breast cancer (Nakachi resulted in severe hepatic necrosis and a 67% mortality rate. et al., 1998). Additionally, in the rat DMBA mammary carci- Following the administration of nontoxic doses of EGCG and nogenesis model, the administration of green-tea extract has ECG, aromatase (CYP19), CYP3A, CYP1A, and catechol decreased both tumor number (Tanaka et al., 1997) and weight O-methyltransferase (COMT) were measured. Additionally, the (Kavanagh et al., 2001). However, the effect of purified cat- activity of CYP2E1 was determined, since this CYP450 isoform is echins on tumor growth has only been examined in mice. The important in the bioactivation of numerous carcinogens. The re- results demonstrated that EGCG caused regression of MCF-7 sults demonstrated that ovarian aromatase activity was inhibited 56% by EGCG (25 and 12.5 mg/kg), but not ECG, while hepatic cell implanted tumors and reduced the growth of implanted CYP3A catalytic activity and polypeptide levels were increased prostate tumors (Liao et al., 1995). The effect on prostate and 47 ؎ 2%, respectively, by 25 mg/kg of EGCG. tumors was catechin-specific as EC, EGC, and ECG all failed 4 ؎ 31 However, ECG (but not EGCG) inhibited CYP1A catalytic activ- to decrease tumor growth. However, EGCG was the only ity and polypeptide levels (31 ؎ 5 and 47 ؎ 5%, respectively). catechin examined in the mammary tumor model. Overall, the Hepatic and renal COMT, as well as renal CYP3A remained results demonstrate that EGCG may have beneficial character- unchanged following catechin dosing. Hepatic CYP2E1 catalytic istics that could be exploited in the treatment of breast cancer. ؎ activity and polypeptide levels were significantly increased (37 Numerous studies with either green-tea extract or specific ؎ 3 and 22 3%) following administration of EGCG (25 mg/kg). members of the catechin family have postulated a wide variety These results indicate that EGCG modulates enzymes responsible of potential mechanisms of action, such as estrogen receptor for both the synthesis and metabolism of estradiol, which may provide a potential mechanism for the reported action of EGCG, (ER) antagonism (Komori et al., 1993), proapoptotic (Morre et reported action as an inhibitor of breast tumor growth. al., 2000), antiangiogenic (Kondo et al., 2002) and anti-oxida- Key Words: epigallocatechin gallate, CYP19, CYP3A, CYP1A, tive (Guo et al., 1999). None of these mechanisms have been catechol O-methyltransferase. conclusively proven in either breast cancer cells or breast tumor models. However, ER antagonism is not likely to be involved, since EGCG, EGC, and ECG all failed to antagonize ␣ The catechins are a group of polyphenolic chemicals that estradiol-induced responses in both uterotropic and ER re- belong to the flavanol family, which is a subclass of flavonoids. porter gene assays (Goodin et al., 2002). Alternatively, alter- Flavonoids are plant-derived chemicals that are contained in a ations in the synthesis and/or metabolism of estradiol may be variety of foods and beverages (Arts et al., 2000). In these involved in the antitumor actions of the catechins. Therefore, sources, the predominant catechins are epigallocatechin gallate aromatase (CYP19) would be a key target to examine, as it is (EGCG), epicatechin gallate (ECG), epigallocatechin (EGC), responsible for the synthesis of estrogens from androgens and aromatase expression in breast tumor epithelial cells positively 1 correlates with cellular proliferation (Lu et al., 1996). CYP3A To whom correspondence should be addressed at the Department of Phar- macology and Toxicology, 18 Frederick Street, Adams Building, University and CYP1A are also important targets, as these CYP450 iso- of Otago, Dunedin, New Zealand. Fax: ϩ64 3 479 9140. E-mail: forms are responsible for the formation of the antiestrogenic [email protected]. metabolite, 2-hydroxyestradiol (Kerlan et al., 1992; Shou et Toxicological Sciences 76(2), © Society of Toxicology 2003; all rights reserved. al., 1997). This metabolite is then rapidly converted by cate- 262 EGCG MODULATES CYP450 ISOFORMS 263 chol O-methyltransferase (COMT) to 2-methoxyestradiol, methasone (75 mg/kg, ip, 4 consecutive days), or acetone (4.8 g/kg, po, 16 h which displays antiangiogenic properties (Fotsis et al., 1994; prior to necropsy). Klauber et al., 1997). Therefore, the overall balance of the Plasma markers of hepatic and renal injury. Plasma ALT activity and activity of these enzymes is important in the regulation of BUN were used as indicators of hepatic and renal damage, respectively. Immediately following euthanasia, blood was collected from the inferior vena estradiol-mediated breast tumor growth. cava and stored on ice. Plasma ALT activity was determined kinetically, using The only previous reports of catechin-mediated alterations a Sigma diagnostic kit, and the results are expressed as IU/l. BUN was of CYP450 isoforms are restricted to either in vitro investiga- determined using a Sigma diagnostic kit, and the results are expressed as tions or to in vivo studies in which animals were treated with mg/dl. EC, (ϩ)-catechin, or herbal supplements containing a mixture Liver histology. Liver slices were obtained from the distal portion of the of various plant extracts (Lhoste et al., 2003; Muto et al., 2001; left lateral lobe and the tissue was fixed for at least 48 h in 10% neutral buffered formalin. The samples were then embedded in paraffin, cut into 5 ␮m Ryu and Chung, 2003; Siegers et al., 1982; Wang et al., 1988). Downloaded from https://academic.oup.com/toxsci/article/76/2/262/1686012 by guest on 28 September 2021 sections, and stained with hematoxylin and eosin for examination by light Therefore, the aim of the present study was to determine if microscopy. The examiner of the slides was blind to the various treatment either EGCG or ECG would modulate the effect of enzymes groups. involved in the synthesis and metabolism of estradiol, namely, Tissue preparation. Hepatic and renal cytosolic extracts and microsomes aromatase, CYP1A, CYP3A, or COMT, at doses that were were prepared from individual mice by differential centrifugation (Guengerich, previously reported to cause tumor regression in athymic mice 1989). Ovarian tissue was pooled from 3 mice and microsomes were prepared (Liao et al., 1995). Since COMT inhibition has led to an as described (Kellis and Vickery, 1984). Protein concentration of the resulting microsomes or cytosolic extract was immediately determined by the bicincho- increase in the renal carcinogenic potential of estradiol (Zhu ninic acid method (Smith et al., 1985). The microsomes and cytosolic extracts and Liehr, 1994), the effect of these two catechins on renal were stored at –80°C until experimentation. enzymes was also determined. Additionally, catechin-mediated Aromatase catalytic activity. The release of tritiated water from [3H]- changes in hepatic CYP2E1 were characterized, as this enzyme androstenedione was used as an indicator of aromatase activity, as this assay is expressed in breast tumors (Iscan et al., 2001) and also has been validated as an appropriate indicator of murine ovarian aromatase activates numerous carcinogens (Constan et al., 1999; Sohn et activity (Toda et al., 2001). The assay was conducted as described (Vinggaard et al., 2000) with the following modifications. The incubation mixture con- al., 2001). EGCG and ECG were used in this investigation, as 3 tained 100 nM [ H]-androstenedione, 25 ␮g protein, 250 ␮M NADPH, and 50 catechins that contain a gallate group in the 3Ј position dem- mM KH2PO4 buffer in a total volume of 500 ␮l. Samples were preincubated onstrate potent inhibition of CYP450 in vitro (Muto et al., for 2 min at 37°C, and the reaction was initiated by the addition of NADPH. 2001; Wang et al., 1988). After 10 min, the reaction was terminated by the addition of chloroform and 0.9% NaCl. The samples were vortexed for 30 s and then centrifuged at 1700 ϫ g for 15 min. One hundred ␮l of the aqueous phase was transferred to MATERIALS AND METHODS scintillation vials and the radioactivity was counted on a Beckman LS3801 scintillation counter. Results are expressed as pmol/mg/h. Chemicals. EGCG, ECG, ␤-naphthoflavone, dexamethasone, acetone, CYP1A catalytic activity. Ethoxyresorufin O-deethylation was used to NADPH, alanine aminotransferase (ALT) kit, blood urea nitrogen (BUN) kit, determine changes in the catalytic activity of CYP1A (Ryan and Levin, 1990) S-adenosyl-L-methionine (SAM), antirabbit IgG, erythromycin, aniline, and and was performed as described previously (Bray et al., 2002). Results are ethoxyresorufin were purchased from Sigma Chemical Co. (St. Louis, MO). expressed as nmol/mg/min. 3 [ H]-androstenedione (25.9 Ci/mmol) was purchased from New England Nu- CYP3A catalytic activity.

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