(Received for publication, August 23, 1995; and in revised form, October 17, 1995)
From the
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-H]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.
The chronic administration of natural or synthetic estrogens
such as estradiol (E) (
)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
(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
. Consistent with this
hypothesis, a diet supplemented with quercetin significantly enhanced
the severity of E
-induced tumorigenesis(9) , but it
did not enhance (rather, slightly decreased) the induction of tumors by
diethylstilbestrol, (
)which may directly undergo metabolic
redox cycling without initial conversion to catechol
metabolites(2) . The enhancement of E
-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
(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
, which may
accumulate in kidneys of hamsters treated with E
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
-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 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-induced renal tumorigenesis observed previously (9) supports the postulated role of these reactive estrogen
metabolites in the induction of hormonal cancers.
Figure 1:
Effect
of quercetin on concentrations of 2-OH-E and 4-OH-E
in kidney (left panel) and in liver (right
panel) of male Syrian hamsters treated with an intraperitoneal
injection of 50 µg E
. 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
(in 50 µl of corn oil, containing 100 µCi of
[6,7-
H]E
). The concentrations of
2-OH-E
and 4-OH-E
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).
Figure 2:
Effect of a 3% quercetin dietary
supplement on the urinary excretion of 2- and 4-methoxyestradiol (2-
and 4-MeO-E, respectively) in the first 48 h following
implantation of two pellets of 10 mg of E
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
. 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-H]E
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
.
Figure 3:
Effect of addition of SAH on the O-methylation of 2-OH-E (upper panel) and
4-OH-E
(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-
H]SAM). The rates of O-methylation of 10 or 40 µM 2-OH-E
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
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 (upper panel) or 4-OH-E
(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
or 4-OH-E
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
[
H-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 by kidney cytosolic COMT exhibited a hyperbolic curve pattern,
with the K
value for SAM approximately 7
µM (Fig. 5). In the presence of 5, 10, and 20
µM SAH, the apparent K
values for SAM
in the O-methylation of 50 µM 2-OH-E
were increased proportionally to the SAH concentrations present,
whereas the V
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 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
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%.
In summary, the addition of SAH
strongly inhibited the in vitro O-methylation of 2-OH-E and 4-OH-E
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
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.
Figure 6:
Effect of addition of quercetin, fisetin
and hesperetin on the O-methylation of 10 µM 2-OH-E or 4-OH-E
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
and
4-OH-E
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 and 4-OH-E
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%.
In summary, quercetin inhibited the
COMT-catalyzed O-methylation of 2-OH-E and
4-OH-E
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.
Our data show that administration of quercetin to male Syrian
hamsters treated with E 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
. 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-H]E
were comparable with those in liver. In contrast, the enzyme
activities in kidney catalyzing the 2- and 4-hydroxylation of E
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
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 COMTSAM complex and increase those of the COMT
SAH
complex. A decrease in the concentration of the COMT
SAM complex
is consistent with a decrease in the V
value and
unchanged K
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 and quercetin (9) and supports the
postulated role of CE, in particular 4-OH-E
, 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 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
-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.