©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Inhibition of Catechol O-Methyltransferase-catalyzed O-Methylation of 2- and 4-Hydroxyestradiol by Quercetin
POSSIBLE ROLE IN ESTRADIOL-INDUCED TUMORIGENESIS (*)

(Received for publication, August 23, 1995; and in revised form, October 17, 1995)

Bao Ting Zhu (§) Joachim G. Liehr (¶)

From the Department of Pharmacology and Toxicology, The University of Texas Medical Branch, Galveston, Texas 77555-1031

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Catecholestrogens have been postulated to mediate the induction of kidney tumors by estradiol in male Syrian hamsters. In this study, we examined the mechanism of inhibition by quercetin of the catechol O-methyltransferase-catalyzed O-methylation of catecholestrogens as a basis for the previously reported enhancement of estradiol-induced tumorigenesis by this flavonoid. In hamsters treated with 50 µg of [6,7-^3H]estradiol, quercetin increased concentrations of 2- and 4-hydroxyestradiol in kidney by 80 and 59%, respectively. In animals treated with two 10-mg estradiol implants, quercetin also decreased by 63-65% the urinary excretion of 2- and 4-hydroxyestradiol monomethyl ethers. Taken together, these results demonstrate the in vivo inhibition of the O-methylation of catecholestrogens by quercetin. S-Adenosyl-L-homocysteine, produced by the methylation of catecholestrogens, noncompetitively inhibited the O-methylation of 2- and 4-hydroxyestradiol by hamster kidney cytosolic catechol O-methyltransferase (IC approximately 10-14 µM). Due to the rapid O-methylation of quercetin itself, quercetin decreased renal concentrations of S-adenosyl-L-methionine by approximately 25% in control or estradiol-treated hamsters and increased concentrations of S-adenosyl-L-homocysteine by 5-15 nmol/g of wet tissue, which was estimated to cause a 30-70% inhibition of the enzymatic O-methylation of catecholestrogens. Quercetin or fisetin (a structural analog) inhibited the O-methylation of 2- and 4-hydroxyestradiol by a competitive plus noncompetitive mechanism (IC approximately 2-5 µM). These results suggest that the in vivo O-methylation of catecholestrogens is inhibited more by S-adenosyl-L-homocysteine than by quercetin. The accumulation of 2- and 4-hydroxyestradiol during co-administration of estradiol and quercetin may enhance metabolic redox cycling of catecholestrogens and thus estradiol-induced kidney tumorigenesis.


INTRODUCTION

The chronic administration of natural or synthetic estrogens such as estradiol (E(2)) (^1)or diethylstilbestrol to male Syrian hamsters induces kidney tumors with an incidence approaching 100%(1) . Estrogens are complete carcinogens, i.e. tumor initiators and promoters in this animal model, which is thus useful for studying the multiple roles of estrogens in the development of hormone-associated cancers. The metabolic redox cycling of CE metabolites or of diethylstilbestrol between their hydroquinone and quinone forms has been established as a mechanism of metabolic activation(2) , because this process generates potentially mutagenic free radicals in addition to chemically reactive estrogen semiquinone/quinone intermediates(3, 4, 5) . These reactive chemical species mediate damage to DNA and other cellular components in the target organ of carcinogenesis in analogy to some classical chemical carcinogens (4, 5, 6, 7, 8) and thus may participate in the initiation of tumors. This mechanistic hypothesis has been probed by the co-administration of quercetin to hamsters treated chronically with E(2)(9) , because this flavonoid is a good inhibitor of the in vitro O-methylation of CE by purified porcine liver COMT(9) . Quercetin itself is also an excellent substrate for COMT(10) . The inhibition of the enzymatic conversion of CE to methoxyestrogens in hamster kidney by quercetin was expected to result in an accumulation of CE, the substrates for metabolic redox cycling, and thus to enhance the induction of renal tumors by E(2). Consistent with this hypothesis, a diet supplemented with quercetin significantly enhanced the severity of E(2)-induced tumorigenesis(9) , but it did not enhance (rather, slightly decreased) the induction of tumors by diethylstilbestrol, (^2)which may directly undergo metabolic redox cycling without initial conversion to catechol metabolites(2) . The enhancement of E(2)-induced kidney tumorigenesis by quercetin also contrasts sharply with the inhibition by this flavonoid of tumors in several other animal models. For instance, this flavonoid decreases the incidence of 7,12-dimethylbenz(a)anthracene- and N-nitrosomethylurea-induced mammary tumors in rats (11) and azoxymethanol-induced colonic neoplasms in mice(12) . Taken together, these observations suggest that the selective enhancement by quercetin of E(2) (but not diethylstilbestrol)-induced kidney tumorigenesis in hamsters may be due to a specific potentiating effect rather than to a nonspecific co-carcinogenesis by this flavonoid. The inhibition of renal O-methylation of 2- and 4-OH-E(2), which may accumulate in kidneys of hamsters treated with E(2) and quercetin, may increase the concentrations of substrates for metabolic redox cycling of CE(2) , increase the production of potentially mutagenic free radicals(3, 4, 5) , and thereby potentiate E(2)-induced renal tumorigenesis.

To evaluate the in vivo inhibition of the O-methylation of CE metabolites by quercetin, we first determined the concentrations of unmetabolized CE in kidney and of methoxyestrogens (the O-methylated CE) in urine of hamsters treated with E(2) alone or in combination with quercetin. Because of the rapid metabolic O-methylation of quercetin itself(10) , we examined the effect of this flavonoid on the pools of SAM, the cofactor and methyl donor in the O-methylation of CE, and of SAH, the demethylated product of SAM(10) . Kinetic analyses of the effect of SAH, a potential noncompetitive inhibitor, on the COMT-catalyzed O-methylation of CE were carried out with a cytosolic COMT preparation from hamster kidney to evaluate the contribution of increased tissue levels of SAH to the in vivo inhibition of O-methylation of CE. These kinetic studies were also expected to assist in understanding the nature of the noncompetitive component of inhibition of the O-methylation of CE by quercetin as observed previously(9) . Finally, we have also studied the in vitro inhibitory effect of quercetin and fisetin on the O-methylation of CE catalyzed by cytosolic COMT of hamster kidney and also determined the circulating and tissue levels of unmetabolized quercetin available as an inhibitor.

Our study demonstrates that administration of quercetin to hamsters inhibited the O-methylation of CE metabolites in kidney and increased renal concentrations of these reactive estrogen metabolites. The correlation of increased concentrations of CE metabolites in kidney of quercetin-treated hamsters with increased severity of E(2)-induced renal tumorigenesis observed previously (9) supports the postulated role of these reactive estrogen metabolites in the induction of hormonal cancers.


MATERIALS AND METHODS

Chemicals

The chemicals used in this study were obtained from the following sources. Quercetin (3,3`,4`,5,7-pentahydroxyflavone), hesperetin (3`,5,7-trihydroxy-4`-methoxyflavonone), 2-OH-E(2), 4-OH-E(2), SAM, SAH, dithiothreitol, porcine liver COMT (1720 units/mg of protein, purified by affinity column procedure), beta-glucuronidase (Type IX-A), and sulfatase (Type H-1) from Sigma. Fisetin (3,3`,4`,7-tetrahydroxyflavone) was from Aldrich, and [methyl-^3H]SAM (specific activity, 11.2-13.5 Ci/mmol) and [6,7-^3H]E(2) (specific activity, 53.5 Ci/mmol) were from DuPont NEN. A special diet supplemented with 3% quercetin (by weight) was prepared by Dyets (Houston, TX). To determine the stability of quercetin in the chow, its content was determined by HPLC and was found to be 97 ± 2% of the initial concentrations after the chow was kept at 4 °C for up to 3 months.

Animals

2-4-month-old male Syrian hamsters, purchased from Harlan Sprague-Dawley (Houston, TX), were housed in a facility accredited by the American Association for Accreditation of Laboratory Animal Care and had free access to rodent chow and water throughout the experiment. The animals were allowed to acclimatize for at least one week prior to any experimentation.

Preparation of Hamster Kidney Cytosolic Fractions

All procedures were carried out at 0-4 °C. Kidneys from 2-month-old male Syrian hamsters were homogenized in 1.4% potassium chloride solution containing 10 mM EDTA, pH 7.4. Tissue homogenates were centrifuged at 9,000 times g for 10 min, and supernatants were pooled and filtered through two layers of cheesecloth to remove lipid clots. The filtrates were then recentrifuged at 105,000 times g (4 °C) for 60 min, and the supernatant cytosolic fractions were filtered (0.45-µm pore size). The proteins in the filtrate were precipitated by slowly adding ice-cold ethanol to a final concentration of 80%. The protein precipitates were collected by centrifugation at 9,000 times g for 10 min, and then resuspended in 10 mM Tris-HCl (pH 7.4) to a protein concentration of 2 mg/ml. Aliquots of these cytosolic preparations were stored at -80 °C.

Inhibition of COMT-catalyzed O-Methylation of CE by Quercetin and SAH

The COMT-catalyzed O-methylation of CE was carried out as described previously(10, 13, 14) . The reaction mixture consisted of 250-500 µg of cytosolic protein from hamster kidney, 1.2 mM MgCl(2), 200 µM SAM iodide (containing 0.5 µCi of [methyl-^3H]SAM), 1 mM dithiothreitol, and varying concentrations of CE in a final volume of 1.0 ml of Tris-HCl buffer (50 mM, pH 7.4). The reaction was started by addition of cytosolic protein of hamster kidney, and carried out at 37 °C for 30 min. The reaction was arrested by rapidly cooling to ice temperatures. The reaction mixture was then immediately extracted with 7 ml of ice-cold n-heptane. After centrifugation at 1000 times g for 10 min, 3-ml aliquots of the organic extracts were analyzed for radioactivity content by liquid scintillation counting (Beckman Instruments, model LS 5000TD).

Urinary Concentrations of Methoxyestrogen Metabolites

2-4-monthold male Syrian hamsters received for 1 week either a control diet or a diet supplemented with 3% quercetin (by weight). The animals were then treated with two subcutaneous implants of 10 mg of E(2). Portions (100 µl) of the first 48-h urine samples following E(2) implantation were collected in metabolic cages (purchased from Lab Products Inc., Maywood, NJ) and were extracted 3 times with 7 ml of diethyl ether for the determination of concentrations of unconjugated methoxyestrogens. For the determination of total methoxyestrogen pools (unconjugated and conjugated metabolites), 100-µl aliquots of urine were first hydrolyzed at 37 °C for 24 h by beta-glucuronidase (10,000 units) and sulfatase (2000 units) in a final volume of 0.5 ml of sodium acetate buffer (0.5 M, pH 6.0). The enzymatic hydrolyses were arrested by the addition of 1 ml of 1 M citric acid and 0.1 ml of 5 N HCl. The mixtures were then extracted 3 times with 7 ml of diethyl ether. The combined ether extracts were evaporated to dryness under a stream of nitrogen gas and then assayed by gas chromatography as described previously(15, 16) .

Tissue Concentrations of 2-OH-E(2) and 4-OH-E(2)

To avoid variations caused by ingestions of differing amounts of quercetin by individual hamsters, 30 mg of quercetin (in 2 ml of syrup) was administered to each hamster (8 weeks old) by intragastric intubation. Control animals received 2 ml of syrup (vehicle). 15 min after quercetin or vehicle administration, each hamster received an intraperitoneal injection of 50 µg of E(2) (in 50 µl of corn oil, containing 100 µCi of [6,7-^3H]E(2)). The animals were decapitated 1 h after E(2) injection, and the kidneys and livers were immediately removed and washed 2-3 times in ice-cold normal saline solution. Two kidneys or 0.4-0.6 g of liver tissues were weighed and homogenized for 1-2 min in 3 ml of 0.2 N HCl containing 5 mM ascorbic acid. The homogenates were extracted 3 times with 10 ml of ethyl acetate saturated with 0.2 N HCl. The pooled extracts were treated with 1 g of anhydrous sodium sulfate for 30 min to remove any water component, transferred to another container, and dried under a stream of nitrogen gas. After the dried extracts were redissolved in 200 µl of methanol, 800 µl of 100 mM Tris base buffer (pH 8.3) and 25 mg of neutral alumina were added to adsorb CE metabolites. The adsorbed CE were eluted with 0.3 N HCl, extracted with ethyl acetate (saturated with 0.2 N HCl), and separated on thin-layer chromatographic plates as described previously(17, 18, 19) . The overall extraction efficiency (approximately 40%) was determined based on the extraction efficiency of known amounts of ^14C-labeled 2-OH-E(2) and 4-OH-E(2) added to the tissues from untreated animals.

Tissue Concentrations of SAM and SAH

Tissue concentrations of SAM and SAH were determined according to procedures described by Chun et al.(20) . Briefly, kidneys were immediately removed from decapitated animals, rinsed in cold saline and blotted on filter paper. About 1 g of freshly excised kidney tissue was homogenized in 1.5 M HCl solution (1:4 (w:v)). After centrifugation at 3000 times g for 15 min, the deproteinized supernatant was chromatographed using a Dowex 50 (H) (resin bed, 4 times 0.5 cm) column pre-equilibrated with 0.1 M HCl to adsorb SAM and SAH. SAM and SAH were then recovered by elution of the column with 10 ml of 6 M HCl. Thiodiglycol (20 µl) was added to the 6 M HCl eluate prior to evaporation under reduced pressure. Samples were then redissolved in 1 ml of water and analyzed by HPLC as described previously(20) .

Tissue Concentrations of Quercetin

4-week-old male hamsters received a diet supplemented with 3% quercetin for 2 weeks or 6.5 months. Blood samples (1-3 ml), obtained by cardiac puncture of animals anesthetized with CO(2), were centrifuged at approximately 3000 times g for 10 min. Aliquots (300 µl) of supernatant plasma were transferred to tubes containing 1 ml of 0.2 M Tris base buffer (pH 8.2), 5 mM ascorbic acid, and 100 mg of neutral alumina. After the cardiac puncture, the animals were immediately decapitated and kidneys were removed, weighed, and homogenized in 4 volumes (v:w) of 30% aqueous methanol. Tris base buffer (0.5 ml, pH 8.2) containing 5 mM ascorbic acid and 100 mg of neutral alumina were added. The neutral alumina (which adsorbs quercetin) was precipitated by a brief centrifugation, and the supernatant was removed with a Pasteur pipette. The neutral alumina precipitates were washed 3 times with 5 ml of 20 mM Tris base solution containing 0.2% EDTA. Quercetin was eluted from the neutral alumina with 300 µl of 0.25 M HCl-50% methanol solution and analyzed by HPLC using a reversed phase C(18) column (150 times 4.6 mm, particle size 5 µM, Rainin Instrument Co., Torrance, CA) with UV detection at 340 nm. The HPLC system consisted of a Waters model 510 pump, a model 501 solvent delivery system, a model 490 multi-wavelength detector, and a model 740 data module. The mobile phase was 50% aqueous methanol containing 10 mM KH(2)PO(4), adjusted to pH 2.40 with H(3)PO(4).


RESULTS

Effect of Quercetin on Estrogen Metabolite Concentrations in Tissue and Urine

Tissue Concentrations of 2-OH-E(2) and 4-OH-E(2)

1 h after an intraperitoneal injection of 50 µg of [6,7-^3H]E(2) to male Syrian hamsters, concentrations of 2-OH-E(2) and 4-OH-E(2) in kidney were 2.7 ± 0.4 and 1.3 ± 0.3 ng/g of wet tissue, respectively, and corresponding concentrations in liver were 2.1 ± 0.3 and 0.6 ± 0.2 ng/g of wet tissue, respectively (Fig. 1). When hamsters were treated with 30 mg of quercetin by intragastric intubation 15 min prior to the intraperitoneal injection of [6,7-^3H]E(2), concentrations of 2-OH-E(2) and 4-OH-E(2) in kidney were increased by 80% (p < 0.01) and 59% (p < 0.01), respectively, and corresponding concentrations in liver were increased by 48% (p < 0.05) and 59%, respectively (Fig. 1).


Figure 1: Effect of quercetin on concentrations of 2-OH-E(2) and 4-OH-E(2) in kidney (left panel) and in liver (right panel) of male Syrian hamsters treated with an intraperitoneal injection of 50 µg E(2). 6-week-old animals received 30 mg of quercetin (in 2 ml syrup) or vehicle alone by intragastric intubation 15 min prior to an intraperitoneal injection of 50 µg of E(2) (in 50 µl of corn oil, containing 100 µCi of [6,7-^3H]E(2)). The concentrations of 2-OH-E(2) and 4-OH-E(2) were determined as described under ``Materials and Methods.'' Values are expressed as means ± S.D. (n = 4; *, p < 0.05;**, p < 0.01 by Student's t test).



Urinary Excretion of Methoxyestrogen Metabolites

The urinary excretion of 2- and 4-methoxyestradiol in the first 48 h after implantation of two 10-mg E(2) pellets to male hamsters was 9.2 ± 4.1 and 1.4 ± 0.9 µg/24 h, respectively (Fig. 2). Pretreatment of hamsters with a diet supplemented with 3% quercetin for 2 weeks decreased the urinary excretion of 2- and 4-methoxyestradiol in the first 48 h by 65% (p < 0.05) and 53%, respectively (Fig. 2).


Figure 2: Effect of a 3% quercetin dietary supplement on the urinary excretion of 2- and 4-methoxyestradiol (2- and 4-MeO-E(2), respectively) in the first 48 h following implantation of two pellets of 10 mg of E(2) to male Syrian hamsters. 8-week-old hamsters received a regular rodent chow or a diet supplemented with 3% quercetin (by weight) for 1 week prior to administration of E(2). Methoxyestrogen metabolites in the first 48-h urine were extracted with ethyl acetate and analyzed by gas chromatography with electron-capture detection as described under ``Materials and Methods.'' Values are expressed as means ± S.D. (n = 4; *, p < 0.05 by Student's t test).



In summary, concentrations of CE metabolites in kidney of male hamsters injected with 50 µg of [6,7-^3H]E(2) were comparable to those in liver. Treatment of hamsters with quercetin significantly increased CE concentrations in kidney and concomitantly decreased urinary concentrations of methoxyestrogens. Taken together, these results demonstrate an in vivo inhibition by quercetin of the O-methylation of CE metabolites during co-treatment of hamsters with E(2).

Inhibition of the O-Methylation of CE Metabolites by SAH

In Vitro Inhibition of the O-Methylation of CE by SAH

The in vitro O-methylation of 2-OH-E(2) or 4-OH-E(2) (at 10 and 40 µM concentrations) by hamster kidney cytosolic COMT was inhibited by the addition of SAH in a concentration-dependent manner. SAH inhibited the O-methylation of two different concentrations (10 and 40 µM) of 2-OH-E(2) or 4-OH-E(2) with very similar inhibition potencies (IC values of approximately 10-14 µM; Fig. 3). The rates of O-methylation of 2.5-50 µM 2-OH-E(2) or 4-OH-E(2) in the absence of inhibitors were of typical hyperbolic patterns and reached plateau rates at about 30-50 µM substrate concentrations (Fig. 4, inset). The K(m) values for 2-OH-E(2) and 4-OH-E(2) were 4.6-4.8 and 9.5-11.3 µM, respectively, and corresponding V(max) values were 82.3-84.6 and 66.7-68.8 pmol/mg of protein/min, respectively ( Fig. 4and Table 1and Table 2). In the presence of varying concentrations (5, 10, 20, and 40 µM) of SAH, the maximal velocities (V(max)) for the O-methylation of 2-OH-E(2) and 4-OH-E(2) were inhibited in a concentration-dependent manner, whereas the corresponding K(m) values for 2- and 4-OH-E(2) substrates were not altered ( Fig. 4and Table 2), indicating a pure noncompetitive mechanism of enzyme inhibition with respect to CE substrates.


Figure 3: Effect of addition of SAH on the O-methylation of 2-OH-E(2) (upper panel) and 4-OH-E(2) (lower panel) catalyzed by hamster kidney cytosolic COMT. The incubation conditions are described under ``Materials and Methods.'' The final concentration of SAM was 50 µM (containing approximately 0.5 µCi of [methyl-^3H]SAM). The rates of O-methylation of 10 or 40 µM 2-OH-E(2) in the absence of SAH were 56.4 or 75.0 pmol/mg of protein/min, respectively, and corresponding rates for 10 or 40 µM 4-OH-E(2) were 32.5 or 54.6 pmol/mg of protein/min, respectively. Rates of O-methylation in the presence of different concentrations of SAH were expressed as percent of control values. Each value is the mean of replicate determinations. The intra-assay variations were within 6.5%.




Figure 4: Double-reciprocal plots of the O-methylation of 2.5-50 µM 2-OH-E(2) (upper panel) or 4-OH-E(2) (lower panel) catalyzed by hamster kidney cytosolic COMT in the absence or presence of various concentrations of SAH. The left inset illustrates the COMT-catalyzed O-methylation of 2-OH-E(2) or 4-OH-E(2) as a function of substrate concentration. The incubation conditions are described under ``Materials and Methods.'' The final concentration of SAM was 50 µM (containing approximately 0.5 µCi of [^3H-methyl]SAM). Each value is the mean of replicate determinations. The intra-assay variations were within 8.5%.







To determine the mechanism of the noncompetitive inhibition of CE O-methylation by SAH, we examined the possibility of decreased interaction of the methyl donor SAM with COMT in the presence of SAH. The effect of varying concentrations of SAM on the rates of O-methylation of 50 µM 2-OH-E(2) by kidney cytosolic COMT exhibited a hyperbolic curve pattern, with the K(m) value for SAM approximately 7 µM (Fig. 5). In the presence of 5, 10, and 20 µM SAH, the apparent K(m) values for SAM in the O-methylation of 50 µM 2-OH-E(2) were increased proportionally to the SAH concentrations present, whereas the V(max) values were not altered (Fig. 5), thus indicating that SAH competitively inhibited the interaction of SAM with COMT.


Figure 5: Double-reciprocal plot of the effect of varying SAM concentrations on the O-methylation of 50 µM 2-OH-E(2) by hamster kidney cytosolic COMT in the absence or presence of SAH. The left inset illustrates the rates of COMT-catalyzed O-methylation of 50 µM 2-OH-E(2) as a function of SAH concentrations. The incubation conditions are described under ``Materials and Methods.'' Each value is the mean of replicate determinations. The intra-assay variations were within 8.5%.



Tissue Concentrations of SAM and SAH

In male Syrian hamsters, the renal concentrations of SAM and SAH were 42.2 ± 5.1 and 16.6 ± 4.2 nmol/g of wet tissue, respectively, with an average SAH/SAM ratio of 0.38 (Table 1). Treatment of hamsters with a diet supplemented with 3% quercetin for 2 weeks lowered renal SAM concentrations by approximately 25% (p < 0.05), and concomitantly increased SAH levels by 37% (p < 0.05) and 89% (p < 0.01) in control or estrogen-treated hamsters, respectively, resulting in 1.9-2.6-fold increases in the average SAH/SAM ratios (Table 1). Treatment of hamsters with E(2) alone did not significantly influence renal SAM and SAH concentrations (Table 1).

In summary, the addition of SAH strongly inhibited the in vitro O-methylation of 2-OH-E(2) and 4-OH-E(2) catalyzed by hamster kidney cytosolic COMT with IC values of approximately 10-14 µM. Enzyme kinetic analyses revealed the inhibition of CE O-methylation by SAH to be purely noncompetitive with respect to CE substrate, but purely competitive with respect to the methyl donor SAM. Treatment of hamsters with E(2) and a 3% quercetin supplement in the diet decreased the renal pool of SAM by 25% and almost doubled SAH concentrations in the kidney. The marked increase in renal SAH levels induced by the quercetin co-treatment is estimated to inhibit the COMT-catalyzed O-methylation of CE metabolites by approximately 30-70% based on the in vitro inhibiting activity of SAH.

Inhibition of the O-Methylation of CE Metabolites by Quercetin

Inhibition of the in Vitro O-Methylation of CE by Quercetin

The in vitro O-methylation of 10 µM 2-OH-E(2) or 4-OH-E(2) by hamster kidney cytosolic COMT was inhibited by the addition of quercetin or its structural analog, fisetin, in a concentration-dependent manner (Fig. 6). Quercetin or fisetin displayed similar inhibition potencies for the O-methylation of 10 µM 2-OH-E(2) (both IC values approximately 8 µM; Fig. 6, upper panel) or 4-OH-E(2) (both IC values approximately 2 µM; Fig. 6, lower panel). In contrast, hesperetin, a monomethylated flavonoid, showed little or no inhibitory effect. In the presence of varying concentrations of quercetin or fisetin, the V(max) values of this enzymatic O-methylation of 2-OH-E(2) and 4-OH-E(2) were markedly inhibited in a concentration-dependent manner ( Fig. 7and Table 2). The marked decreases in V(max) values in the presence of flavonoids indicated a substantial contribution by a noncompetitive mechanism of enzyme inhibition. In addition to the observed decreases in V(max) values, the K(m) values were simultaneously increased in the presence of quercetin or fisetin ( Fig. 7and Table 2), thus indicating a mixed (competitive plus noncompetitive) mechanism of enzyme inhibition as reported previously with a purified porcine liver COMT preparation(4) .


Figure 6: Effect of addition of quercetin, fisetin and hesperetin on the O-methylation of 10 µM 2-OH-E(2) or 4-OH-E(2) catalyzed by hamster kidney cytosolic COMT. The incubation conditions are described under ``Materials and Methods.'' The rates of the O-methylation of 10 µM 2-OH-E(2) and 4-OH-E(2) in the absence of flavonoids were 54.2 and 30.8 pmol/mg of protein/min, respectively, and were considered to be 100%. Rates of the O-methylation of CE in the presence of different concentrations of quercetin, fisetin, or hesperetin are expressed as percent of control. Each value is the mean of two to three determinations. Intra-assay variations were within 11%.




Figure 7: Double-reciprocal plots of the O-methylation of 2.5-40 µM 2-OH-E(2) and 4-OH-E(2) catalyzed by hamster kidney cytosolic COMT in the absence or presence of two different concentrations of quercetin or fisetin. The incubation conditions are described under ``Experimental Procedures.'' Each value is the mean of replicate determinations. Intra-assay variations were within 7%.



Tissue Levels of Quercetin

The concentrations of unmetabolized quercetin in plasma, kidney, and liver of hamsters treated with a dietary supplement of 3% quercetin for 2 weeks were 0.22 ± 0.19 µM, 0.43 ± 0.37, and 0.37 ± 0.22 nmol/g of wet tissue, respectively (Table 3). Similar concentrations of unmetabolized quercetin were obtained in plasma, liver, and kidney of hamsters treated with this 3% quercetin diet for 6.5 months (Table 3). In contrast, concentrations in tissues from animals on a control diet were below the detection limit (<0.05 µM quercetin in plasma or <0.05 nmol of quercetin/g of wet tissue; Table 3).



In summary, quercetin inhibited the COMT-catalyzed O-methylation of 2-OH-E(2) and 4-OH-E(2)in vitro by a competitive plus noncompetitive mechanism. The concentrations of quercetin in circulation and in tissues were low relative to the concentrations required for inhibiting the enzymatic O-methylation of CE in vitro. A comparison of the in vitro inhibition of CE O-methylation by SAH and quercetin with their available tissue concentrations suggests that the direct inhibition of COMT by quercetin may be less pronounced in vivo, whereas the inhibition by SAH may be a dominant mechanism.


DISCUSSION

Our data show that administration of quercetin to male Syrian hamsters treated with E(2) increased concentrations of CE metabolites in kidney (the target organ of tumorigenesis) and concomitantly decreased excretion of methoxyestrogens in the urine. These results demonstrate an in vivo inhibition of the O-methylation of CE metabolites during co-treatment of hamsters with quercetin and E(2). Our results also show that treatment of hamsters with quercetin decreased concentrations of SAM (the cofactor for COMT-catalyzed O-methylation reactions) and concomitantly increased concentrations of SAH (the demethylated product of SAM), and thereby markedly increased the SAH/SAM ratios in hamster kidney.

The CE concentrations in the kidney of male hamsters injected with 50 µg of [6,7-^3H]E(2) were comparable with those in liver. In contrast, the enzyme activities in kidney catalyzing the 2- and 4-hydroxylation of E(2) are at least 1 order of magnitude lower than those in liver(21) . These relatively high concentrations of CE metabolites in kidney may be explained in part by the lower detoxifying enzyme activities in this organ compared with those in liver(22) . The larger increase in renal CE concentrations compared with hepatic concentrations in male hamsters co-treated with quercetin and E(2) also suggests that detoxification of CE in liver may remain intact, whereas that in kidney may be compromised. Second, the concentrations of endogenous catecholamines (substrates and competitive inhibitors of COMT-catalyzed O-methylation) in hamster kidney are more than 40-fold higher than those in liver(14) , which inhibits the O-methylation of CE (14) and thereby may contribute to the high levels of CE in kidney. Finally, in addition to direct aromatic hydroxylation of parent estrogen, CE may be formed by metabolic deconjugation of estrogen conjugates such as estrogen glucuronides and methyl ethers(16) . This metabolic deconjugation has been shown to be an important source of CE production in hamster kidney but is less important in liver compared with hepatic CE production by direct hydroxylation of parent estrogens(16) .

Our study clearly demonstrates that the COMT-catalyzed O-methylation of CE is inhibited by quercetin via two different mechanisms, i.e. the direct inhibition of COMT by quercetin and the indirect inhibition by elevated tissue concentrations of SAH. Quercetin itself is a substrate for COMT (10) and thus competitively inhibits the O-methylation of CE substrates by competing for the methylating enzyme. Although SAH inhibited COMT in a noncompetitive fashion with respect to CE substrates, the kinetic analysis revealed that SAH competitively inhibited the association of the methyl donor SAM with the methylating enzyme. Thus, SAH may decrease concentrations of the COMTbulletSAM complex and increase those of the COMTbulletSAH complex. A decrease in the concentration of the COMTbulletSAM complex is consistent with a decrease in the V(max) value and unchanged K(m) value (a noncompetitive inhibition). This noncompetitive inhibition by SAH also explains the noncompetitive component of enzyme inhibition by quercetin or fisetin in vitro, because the O-methylation of either flavonoid will increase the concentrations of SAH. In addition to these two mechanisms, an approximately 25% decrease in renal pools of SAM (the methyl donor) during quercetin administration may also be a contributing factor for the decreased metabolism of CE by O-methylation in vivo.

In hamster kidney, the inhibition of CE O-methylation by SAH likely dominates over the direct inhibition by quercetin for the following reasons. (i) Despite the chronic administration of a high dose of quercetin to animals (3% in the diet), plasma or tissue concentrations of unmetabolized quercetin do not exceed 0.5 nmol/ml or g of wet tissue, respectively. Quercetin has previously been shown to undergo rapid O-methylation and/or other conjugation reactions(10) . The low concentrations of unmetabolized quercetin in blood and in tissues observed in this study are in close agreement with previous studies(23) . (ii) The marked increase in renal concentrations of SAH and in SAH/SAM ratios during treatment with quercetin makes it likely that inhibition by SAH is the dominant form of inhibition in quercetin-treated animals. The increase in tissue levels of SAH during 3% quercetin treatment results from rapid and extensive O-methylation of this flavonoid as demonstrated previously (10) . Based on our enzyme kinetic studies, the magnitude of increase in renal SAH concentrations (approximately 10 µM) is estimated to significantly inhibit the metabolic O-methylation of CE in vivo. Thus, it is suggested that a markedly increased demand on the circulating one-carbon pool due to the O-methylation of quercetin or other catechols may result in an increase in tissue pools of SAH and a concomitant inhibition of the O-methylation in vivo of CE metabolites. This metabolic change may be the basis of the previously observed increase in the severity of kidney tumorigenesis in hamsters treated with E(2) and quercetin (9) and supports the postulated role of CE, in particular 4-OH-E(2), in the induction of estrogen-associated tumors(4, 5) . CE have previously been shown to undergo metabolic redox cycling, a process to generate potentially mutagenic free radicals in addition to other chemically reactive species such as estrogen semiquinones and quinones(3, 6, 7, 8) . Details of this mechanism of DNA damage induced by redox cycling of CE are discussed in a recent review (5) .

In summary, the administration of quercetin to male Syrian hamsters treated with E(2) inhibits the O-methylation of CE metabolites and thereby increases their concentrations in tissues and decreases urinary excretion of methoxyestrogen conjugates. The increase in CE concentrations together with the previously reported increase in the severity of E(2)-induced kidney tumorigenesis in hamsters is taken as evidence in support of a critical role of redox cycling of CE metabolites and free radical generation in the induction of hormone-associated cancers.


FOOTNOTES

*
This work was supported by Grant CA 43233 from the National Institutes of Health, National Cancer Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Laboratory for Cancer Research, Dept. of Chemical Biology and Pharmacognosy, Rutgers, The State University of New Jersey, Piscataway, NJ 08855-0789.

To whom correspondence and reprints requests should be addressed. Tel.: 409-772-9624; Fax: 409-772-9642.

(^1)
The abbreviations used are: E(2), estradiol; 2- and 4-OH-E(2), 2- and 4-hydroxyestradiol, respectively; CE, catecholestrogen(s); COMT, catechol O-methyltransferase; SAM, S-adenosyl-L-methionine; SAH, S-adenosyl-L-homocysteine; HPLC, high-performance liquid chromatography.

(^2)
B. T. Zhu and J. G. Liehr, unpublished results.


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