Department of Pharmacology and Toxicology, University of Otago, Dunedin, New Zealand
Received August 4, 2003; accepted September 22, 2003
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ABSTRACT |
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Key Words: epigallocatechin gallate; CYP19; CYP3A; CYP1A; catechol O-methyltransferase.
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INTRODUCTION |
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Numerous studies with either green-tea extract or specific members of the catechin family have postulated a wide variety of potential mechanisms of action, such as estrogen receptor (ER) antagonism (Komori et al., 1993), proapoptotic (Morre et al., 2000
), antiangiogenic (Kondo et al., 2002
) and anti-oxidative (Guo et al., 1999
). None of these mechanisms have been 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 estradiol-induced responses in both uterotropic and ER
reporter gene assays (Goodin et al., 2002
). Alternatively, alterations in the synthesis and/or metabolism of estradiol may be involved in the antitumor actions of the catechins. Therefore, aromatase (CYP19) would be a key target to examine, as it is responsible for the synthesis of estrogens from androgens and aromatase expression in breast tumor epithelial cells positively correlates with cellular proliferation (Lu et al., 1996
). CYP3A and CYP1A are also important targets, as these CYP450 isoforms are responsible for the formation of the antiestrogenic metabolite, 2-hydroxyestradiol (Kerlan et al., 1992
; Shou et al., 1997
). This metabolite is then rapidly converted by catechol O-methyltransferase (COMT) to 2-methoxyestradiol, which displays antiangiogenic properties (Fotsis et al., 1994
; Klauber et al., 1997
). Therefore, the overall balance of the activity of these enzymes is important in the regulation of estradiol-mediated breast tumor growth.
The only previous reports of catechin-mediated alterations of CYP450 isoforms are restricted to either in vitro investigations or to in vivo studies in which animals were treated with EC, (+)-catechin, or herbal supplements containing a mixture of various plant extracts (Lhoste et al., 2003; Muto et al., 2001
; Ryu and Chung, 2003
; Siegers et al., 1982
; Wang et al., 1988
). Therefore, the aim of the present study was to determine if either EGCG or ECG would modulate the effect of enzymes involved in the synthesis and metabolism of estradiol, namely, aromatase, CYP1A, CYP3A, or COMT, at doses that were previously reported to cause tumor regression in athymic mice (Liao et al., 1995
). Since COMT inhibition has led to an increase in the renal carcinogenic potential of estradiol (Zhu and Liehr, 1994
), the effect of these two catechins on renal enzymes was also determined. Additionally, catechin-mediated changes in hepatic CYP2E1 were characterized, as this enzyme is expressed in breast tumors (Iscan et al., 2001
) and also activates numerous carcinogens (Constan et al., 1999
; Sohn et al., 2001
). EGCG and ECG were used in this investigation, as catechins that contain a gallate group in the 3' position demonstrate potent inhibition of CYP450 in vitro (Muto et al., 2001
; Wang et al., 1988
).
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MATERIALS AND METHODS |
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Animals.
Female Swiss-Webster mice (56 weeks old) were purchased from Hercus Taieri Resource Unit (Dunedin). All procedures were approved by the University of Otago Animal Ethics Committee (#96/01). The animals were housed in micro-isolator cages on shredded paper bedding and had continuous access to Reliance rodent diet (Dunedin, NZ) and water. They were maintained at 2124°C with a 12-h light/dark cycle and allowed to acclimatize for one week before experimentation. Mice were randomly assigned to the various treatment groups, which consisted of 9 mice in each group. Mice were dosed with EGCG (12.5, 25, or 50 mg/kg/day, ip), ECG (12.5 or 25 mg/kg, ip), or saline control (8 ml/kg) for 7 days. To control for potential catechin-mediated weight loss, saline control mice were pair-fed. Mice were sacrificed by CO2 inhalation 24 h following the final catechin dose. The doses and route of administration used were based on previous work, which demonstrated that EGCG (50 mg/kg/day, 14 days, ip) caused tumor regression in a murine MCF-7 cell implant model (Liao et al., 1995). Mice used as positive controls were treated with ß-naphthoflavone (80 mg/kg, ip, 3 consecutive days), dexamethasone (75 mg/kg, ip, 4 consecutive days), or acetone (4.8 g/kg, po, 16 h prior to necropsy).
Plasma markers of hepatic and renal injury.
Plasma ALT activity and BUN were used as indicators of hepatic and renal damage, respectively. Immediately following euthanasia, blood was collected from the inferior vena cava and stored on ice. Plasma ALT activity was determined kinetically, using a Sigma diagnostic kit, and the results are expressed as IU/l. BUN was determined using a Sigma diagnostic kit, and the results are expressed as mg/dl.
Liver histology.
Liver slices were obtained from the distal portion of the 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 µm sections, and stained with hematoxylin and eosin for examination by light microscopy. The examiner of the slides was blind to the various treatment groups.
Tissue preparation.
Hepatic and renal cytosolic extracts and microsomes were prepared from individual mice by differential centrifugation (Guengerich, 1989). Ovarian tissue was pooled from 3 mice and microsomes were prepared as described (Kellis and Vickery, 1984
). Protein concentration of the resulting microsomes or cytosolic extract was immediately determined by the bicinchoninic acid method (Smith et al., 1985
). The microsomes and cytosolic extracts were stored at -80°C until experimentation.
Aromatase catalytic activity.
The release of tritiated water from [3H]-androstenedione was used as an indicator of aromatase activity, as this assay has been validated as an appropriate indicator of murine ovarian aromatase activity (Toda et al., 2001). The assay was conducted as described (Vinggaard et al., 2000
) with the following modifications. The incubation mixture contained 100 nM [3H]-androstenedione, 25 µg protein, 250 µM NADPH, and 50 mM KH2PO4 buffer in a total volume of 500 µl. Samples were preincubated for 2 min at 37°C, and the reaction was initiated by the addition of NADPH. 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 x g for 15 min. One hundred µl of the aqueous phase was transferred to scintillation vials and the radioactivity was counted on a Beckman LS3801 scintillation counter. Results are expressed as pmol/mg/h.
CYP1A catalytic activity.
Ethoxyresorufin O-deethylation was used to determine changes in the catalytic activity of CYP1A (Ryan and Levin, 1990) and was performed as described previously (Bray et al., 2002
). Results are expressed as nmol/mg/min.
CYP3A catalytic activity.
Erythomycin N-demethylation was used as a selective probe for changes in the catalytic activity of CYP3A (Wrighton et al., 1985) and was performed as described previously (Bray et al., 2002
). Results are expressed as nmol/mg/min.
CYP2E1 catalytic activity.
Aniline hydroxylation was used as a selective probe for changes in the catalytic activity of CYP2E1 and was performed as described previously (Inder et al., 1999). Results are expressed as nmol/mg/min.
COMT catalytic activity.
Methylation of 4-nitrocatechol was used as a selective probe for changes in COMT activity (Herblin, 1973) with the following modifications. Hepatic or renal cytosolic extract (2 mg) was incubated with 25 µM 4-nitrocatechol, 0.2 mM SAM, 0.01 M MgCl2, and 1 mM Tris-HCl buffer (pH 7.0) in a total volume of 2 ml. Samples were preincubated for 2 min at 37°C and a 60-min incubation period was initiated by the addition of SAM. The reaction was terminated by addition of 12 N NaOH and the samples were centrifuged at 1000 x g for 10 min. Absorbance was determined at 520 nm and the results are expressed as nmol/mg/min.
Electrophoresis and Western blotting.
SDSPAGE was performed as previously described (Bray et al., 2002). Briefly, 10 µg of microsomal protein was loaded onto a 10% gel with a 4% stacking gel. Polypeptide levels of CYP1A, CYP3A, and CYP2E1 were quantified by Western immunoblotting as previously described (Bray et al., 2002
). Upon transfer to a nitrocellulose membrane, proteins were incubated with CYP1A, CYP3A, or CYP2E1 antirat primary antibody. Each antibody was proven by the manufacturer to cross-react with the specific mouse CYP450 isoform examined. After washing, membranes were incubated with antirabbit IgG (an alkaline phosphate conjugate) secondary antibody. The bands were visualized using the NBT/BCIP system and quantified by scanning densitometry.
Statistical analysis.
Individual groups were analyzed using a two-way ANOVA coupled with the Student-Newman-Keuls post hoc test with p < 0.05 as the minimum requirement for a statistically significant difference.
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RESULTS |
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DISCUSSION |
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Due to the hepatotoxicity and mortality that occurred, enzymatic activities were only performed following the administration of lower doses of the catechins. Aromatase was initially examined, as it is responsible for the synthesis of estradiol from testosterone, and a catechin-specific inhibition of aromatase in vitro has previously been reported. Specifically, Satoh et al. (2002), reported IC50 values of 60 µM and 100 µM for EGCG and EGC, respectively, while 100 µM of ECG only produced 20% inhibition. However, other groups have reported that EGC (Kao et al., 1998
) and (-)-catechin fail to inhibit aromatase (Campbell and Kurze, 1993
). Our in vivo results support a catechin-specific inhibition of aromatase, since EGCG (12.5 or 25 mg/kg) inhibited ovarian aromatase activity by 56%, while ECG did not alter aromatase activity. A decrease in aromatase in vivo would reduce the production of estradiol and thus decrease estradiol-mediated events. Therefore, aromatase inhibition may be partly responsible for the antitumor actions of EGCG in rodent models of mammary cancer. However, other mechanisms are likely to be involved in the antitumor effect produced by EGCG, as other aromatase inhibitors have been reported to cause a decrease in tumor growth but not tumor regression (Yue and Brodie, 1993
). Further studies examining the effect of EGCG on breast tumor aromatase activity in a murine model will be conducted to confirm this hypothesis.
The effect of EGCG on breast tumor growth could also be due to changes in estradiol metabolism via the modulation of either CYP1A, CYP3A, or COMT. Our results demonstrated that EGCG increased the catalytic activity of hepatic, but not renal CYP3A, and ECG decreased the catalytic activity of hepatic CYP1A, while renal CYP1A remained below detectable levels. The renal enzymatic results are not unexpected: we have previously reported that renal CYP1A is undetectable (Bray et al., 2001), while rodent renal CYP3A remains at low but detectable levels following administration of the classic inducer, dexamethasone (Zerilli et al., 1998
). While there are no other in vivo studies with EGCG or ECG, two studies have measured CYP450 activity in male rats following treatment with either (+)-catechin or EC. Specifically, Siegers et al. (1982)
demonstrated that (+)-catechin (200 mg/kg/day, 7 or 28 days, po) did not alter total hepatic CYP450 or CYP2E1 activity in Sprague-Dawley rats. However, Lhoste et al. (2003)
reported that both (+)-catechin and EC (3035 mg/rat/day, 14 days, po) decreased CYP2E1 (37 and 25%, respectively) in male F344 rats. The discrepancy in the response following (+)-catechin in rats may be due to the fact that Lhoste et al. (2003)
administered the catechins in 10% ethanol in order to simulate catechin exposure from drinking wine. However, it appears as though the effect of catechins on CYP2E1 activity is both catechin- and species-specific, as we demonstrated a modest increase in hepatic CYP2E1 following EGCG. It is worth noting that the majority of the in vivo data is not supported by in vitro inhibition studies. Specifically, studies, using bacterial cells transfected with plasmids for specific human CYP450 isoforms, demonstrated that both ECG and EGCG acted as either mixed or noncompetitive inhibitors of CYP1A1, CYP1A2, CYP2A6, CYP2C9, CYP2E1, and CYP3A4 (Muto et al., 2001
). ECG exhibited the greatest potency against CYP1A2 as 10 µM caused 50% inhibition. However, the effect of ECG on CYP1A in vivo is exhibited via a decrease in protein and not through competitive enzyme inhibition, as CYP1A polypeptide levels and catalytic activity were both decreased to the same extent. Since EGCG, but not ECG induces weight loss and hepatotoxicity in mice, it is not surprising that their effects on CYP450 isoforms in vivo are distinct both from each other and in in vitro experiments.
This catechin-specific modulation of CYP450 isoforms may in part explain why EGCG, but not ECG, was able to decrease tumor growth in mice (Liao et al., 1995). Specifically, EGCG (25 mg/kg) was a weak inducer of hepatic CYP3A activity and polypeptide levels. This is important, as CYP3A and CYP1A are responsible for the formation of approximately 85% of 2-hydroxyestradiol, which is then rapidly converted to 2-methoxyestradiol by COMT (Kerlan et al., 1992
; Shou et al., 1997
). Importantly, EGCG did not inhibit COMT activity as has been demonstrated with the in vivo administration of other gallate-containing compounds (Mannisto and Kaakkola, 1999
). Therefore, the rapid conversion to 2-methoxyestradiol should not be altered by EGCG, thus allowing the induction of CYP3A to shift the metabolism toward the production of 2-methoxyestradiol. Since nM and µM concentrations of 2-methoxyestradiol disrupt microtubule formation (DAmato et al., 1994
), inhibit angiogenesis and induce apoptosis (Lakhani et al., 2003
), small increases in 2-methoxyestradiol may have significant inhibitory effects on breast tumor growth.
Overall, the modest increase in CYP3A activity, paired with the 56% inhibition of aromatase, may work together to both decrease estradiol production and shift the metabolism of estradiol toward the production of antiestrogenic and antiangiogenic metabolites. However, CYP3A is also responsible for the formation of 1520% of 4-hydroxyestradiol, a metabolite with estrogenic properties (Weisz et al., 1992). Therefore, to determine the exact effect on estradiol metabolism, future studies will measure the production of estradiol and its metabolites following EGCG treatment in tumor-bearing mice. Additionally, future studies will also determine the specific isoform of CYP3A (i.e., 3A11, 3A41, and/or 3A44) that is increased by EGCG.
In addition to examining the enzymes responsible for the synthesis and metabolism of estradiol, the modulatory effect on CYP2E1 by EGCG and ECG was also determined, as this enzyme plays an important role in activation of numerous carcinogens (Constan et al., 1999; Sohn et al., 2001
) and is also expressed in breast tumors (Iscan et al., 2001
). Therefore, this examination would demonstrate if catechin administration had the potential to produce drug/chemical interactions. While ECG failed to alter CYP2E1, EGCG (25 mg/kg/day, 7days) increased CYP2E1 catalytic activity by 37% and polypeptide levels by 22%. Unfortunately this increase in CYP2E1 occurred following the same dose of EGCG that modulated aromatase and CYP3A. This small increase in CYP2E1 is unlikely to significantly increase the activation of carcinogens. However, the combined increase in CYP2E1 and CYP3A may cause drug-interactions with substances, such as acetaminophen, which utilize both of these enzymes in their metabolism (Kostrubsky et al., 1997
; Raucy et al., 1989
). This effect may be limited to the mouse, as, in the male rat, CYP2E1 is either unchanged (Siegers et al., 1982
) or decreased by (+)-catechin and EC (Lhoste et al., 2003
). However, further work needs to be performed to determine the extent of this potential species-specific response.
In summary, this is the first study to examine both hepatic and renal enzymes responsible for the synthesis and metabolism of estradiol following in vivo administration of specific catechins. The resulting catechin-specific alteration in ovarian aromatase and hepatic CYP3A by EGCG may prove to be an important component of the mechanism of EGCG-mediated breast tumor suppression. However, this result is tempered by the finding that high doses of EGCG produce significant hepatotoxicity and mortality in female mice.
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ACKNOWLEDGMENTS |
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NOTES |
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