Inhibition of thyroid peroxidase by dietary flavonoids

Article abstract of DOI:10.1021/tx950076m

Flavonoids are widely distributed in plant-derived foods and possess a variety of biological activities including antithyroid effects in experimental animals and humans. A structure-activity study of 13 commonly consumed flavonoids was conducted to evaluate inhibition of thyroid peroxidase (TPO), the enzyme that catalyzes thyroid hormone biosynthesis. Most flavonoids tested were potent inhibitors of TPO, with IC₅₀ values ranging from 0.6 to 41 μM. Inhibition by the more potent compounds, fisetin, kaempferol, naringenin, and quercetin, which contain a resorcinol moiety, was consistent with mechanism-based inactivation of TPO as previously observed for resorcinol and derivatives. Other flavonoids inhibited TPO by different mechanisms, such as myricetin and naringin, showed noncompetitive inhibition of tyrosine iodination with respect to iodine ion and linear mixed-type inhibition with respect to hydrogen peroxide. In contrast, biochanin A was found to be an alternate substrate for iodination. The major product, 6,8- diiodo-biochanin A, was characterized by electrospray mass spectrometry and ¹H-NMR. These inhibitory mechanisms for flavonoids are consistent with the antithyroid effects observed in experimental animals and, further, predict differences in hazards for antithyroid effects in humans consuming dietary flavonoids. In vivo, suicide substrate inhibition, which could be reversed only by de novo protein synthesis, would be long-lasting. However, the effects of reversible binding inhibitors and alternate substrates would be temporary due to attenuation by metabolism and excretion. The central role of hormonal regulation in growth and proliferation of thyroid tissue suggests that chronic consumption of flavonoids, especially suicide substrates, could play a role in the etiology of thyroid cancer.

Full text of DOI:10.1021/tx950076m

16
Chem. Res. Toxicol. 1996, 9, 16-23
Articles
In h ibition of Th yr oid P er oxid a se by Dieta r y F la von oid s
Rao L. Divi† and Daniel R. Doerge*
National Center for Toxicological Research, J efferson, Arkansas 72079
Received May 1, 1995X
Flavonoids are widely distributed in plant-derived foods and possess a variety of biological
activities including antithyroid effects in experimental animals and humans. A structure-
activity study of 13 commonly consumed flavonoids was conducted to evaluate inhibition of
thyroid peroxidase (TPO), the enzyme that catalyzes thyroid hormone biosynthesis. Most
flavonoids tested were potent inhibitors of TPO, with IC50 values ranging from 0.6 to 41 µM.
Inhibition by the more potent compounds, fisetin, kaempferol, naringenin, and quercetin, which
contain a resorcinol moiety, was consistent with mechanism-based inactivation of TPO as
previously observed for resorcinol and derivatives. Other flavonoids inhibited TPO by different
mechanisms, such as myricetin and naringin, showed noncompetitive inhibition of tyrosine
iodination with respect to iodine ion and linear mixed-type inhibition with respect to hydrogen
peroxide. In contrast, biochanin A was found to be an alternate substrate for iodination. The
major product, 6,8-diiodo-biochanin A, was characterized by electrospray mass spectrometry
1
and H-NMR. These inhibitory mechanisms for flavonoids are consistent with the antithyroid
effects observed in experimental animals and, further, predict differences in hazards for
antithyroid effects in humans consuming dietary flavonoids. In vivo, suicide substrate
inhibition, which could be reversed only by de novo protein synthesis, would be long-lasting.
However, the effects of reversible binding inhibitors and alternate substrates would be
temporary due to attenuation by metabolism and excretion. The central role of hormonal
regulation in growth and proliferation of thyroid tissue suggests that chronic consumption of
flavonoids, especially suicide substrates, could play a role in the etiology of thyroid cancer.
with the antithyroid effects of flavonoids observed in
humans (goiter) and experimental animals (12, 13);
however, the mechanism by which flavonoids block
thyroid hormone synthesis is not known. The present
study evaluates the effects of 13 commonly consumed
flavonoids on TPO to elucidate these mechanisms.
In tr od u ction
Flavonoids (see Figure 1) are a class of naturally
occurring, low molecular weight benzo-γ-pyrone deriva-
tives ubiquitously distributed in the plant kingdom.
Common human and animal foods contain from traces
to several grams of flavonoids per kilogram fresh weight
(1). Flavonoids display diverse biological and pharma-
cological properties, e.g., anti-inflammatory, antiallergic,
antiviral, pro- or antimutagenic, pro- or anticarcinogenic,
antibacterial, and antioxidant effects (2). Flavonoids
inhibit many enzymes including thyroid peroxidase
(TPO)1 (3) and 5′-deiodinase (4), the key enzymes of
thyroid hormone synthesis, aldose reductase (5), ATPases
(6), neutrophil NADPH oxidase (7), and phosphodi-
esterases (8). Consumption of flavonoids by experimental
animals reduces both iodide ion uptake and iodide ion
incorporation into thyroid hormones (9, 10). In vitro,
several flavonoids reduce iodide ion uptake as well as
incorporation in porcine thyroid slices and inhibited TPO-
dependent iodination (3, 11). These data are consistent
Ma ter ia ls a n d Meth od s
Baicalein, biochanin A, catechin, fisetin, flavanone, flavone,
and guaiacol were purchased from Aldrich Chemical Co. (Mil-
waukee, WI); kaempferol, morin, myricetin, naringenin, narin-
gin, quercetin, rutin, horseradish peroxidase, chloroperoxidase,
and lactoperoxidase were obtained from Sigma Chemical Co.
(St. Louis, MO). Bovine myeloperoxidase (MPO) was obtained
from ExOxEmis Co. (San Antonio, TX), and yeast cytochrome c
peroxidase (CcP) was a generous gift from Dr. J ames Erman,
N. Illinois State University. The concentration of H2O2, ob-
tained from Sigma, was determined by iodometric titration (14),
and dilutions were made daily. All chemicals were used without
further purification. Stock solutions of the flavonoids were
prepared by dissolution in ethanol just before use.
Porcine TPO was purified according to the method of Van
Zyl and Van Der Walt (17) with certain modifications. Porcine
thyroid glands obtained from a local slaughterhouse were freed
from extraneous matter and sliced into small pieces. The slices
were homogenized in 50 mM Tris-HCl and 0.5 mM KI buffer
(pH 8.0) containing catalase (500 units/L), and microsomes were
prepared by the method of Hosoya and Morrison (16). The
microsomes were incubated with 0.25% sodium deoxycholate
and 5 mM CHAPS in Tris-HCl-KI buffer at 4 °C for overnight.
The detergent-solubilized suspension was centrifuged at 100000g
for 1 h, and the supernatant was fractionated between 25% and
55% ammonium sulfate saturation. The precipitate was dis-
* Corresponding author, at Tel (501) 543-7943, FAX (501) 543-7720,
E-mail DDOERGE@FDANT.NCTR.FDA.GOV.
† Supported by a fellowship from the Oak Ridge Institute for Science
and Education administered through an interagency agreement be-
tween the U.S. Department of Energy and the U.S. Food and Drug
Administration.
X Abstract published in Advance ACS Abstracts, November 1, 1995.
1 Abbreviations: CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-
1-propanesulfonate; CPO, chloroperoxidase; CcP, cytochrome c per-
oxidase; ES/MS, electrospray ionization mass spectrometry; HRP,
horseradish peroxidase; IC50, concentration producing 50% inhibition
of activity; LPO, bovine lactoperoxidase; MIT, 3-iodotyrosine; MPO,
bovine myeloperoxidase; TPO, porcine thyroid peroxidase.
0893-228x/96/2709-0016$12.00/0 © 1996 American Chemical Society
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Thyroid Peroxidase Inhibition by Flavonoids
Chem. Res. Toxicol., Vol. 9, No. 1, 1996 17
F igu r e 1. Flavonoid structures.
solved in Tris-HCl-KI buffer containing catalase and dialyzed
against the same buffer. The dialyzed enzyme preparation was
subjected to trypsin (0.02%) digestion for 1 h at room temper-
ature. The digestion was stopped with soybean trypsin inhibitor
(0.02%), the mixture was centrifuged at 100000g for 1 h, and
the supernatant was fractionated again with ammonium sulfate
as described above. The precipitate from ammonium sulfate
fractionation was dissolved in and dialyzed against 0.02 M
phosphate buffer (pH 6.8) containing 0.1 mM KI and catalase
(500 units/L). The enzyme solution was concentrated by ultra-
filtration (MWCO ) 30 kDa) and subjected to gel filtration on
a Bio-Gel A 1.5 m column (2.0 × 40 cm). The column was
equilibrated and eluted with 0.02 M phosphate buffer (pH 6.8).
The fractions containing guaiacol oxidation activity were pooled
and rechromatographed on a hydroxylapatite column (1.8 × 20
cm) equilibrated with 0.02 M phosphate buffer (pH 6.8) contain-
ing 0.1 mM KI. The enzyme was eluted with a linear gradient
of 0.02-0.25 M phosphate buffer (pH 6.8) containing 0.1 mM
KI. The inclusion of catalase in the buffer during homogeniza-
tion and initial purification, and 3-[(3-cholamidopropyl)dim-
ethylammonio]-1-propanesulfonate (CHAPS), a zwitterionic
surfactant, for solubilization greatly increased the yield (ca. 6
mg of purified enzyme/ kg thyroid slices) of the purified enzyme.
The Rz value of the enzyme preparations used in the current
studies ranged from 0.32 to 0.42. The concentration of the
enzyme was measured spectrophotometrically by using the
extinction coefficient 1.12 × 105 M-1cm-1 at 412 nm (17).
Guaiacol oxidation activity was determined at pH 7.0 as
described previously (18).
In h ibition Assa ys. TPO (2.0 nM) was incubated with
tyrosine (150 µM), iodide ion (150 µM), and hydrogen peroxide
(50 µM) with different fixed concentrations of flavonoid, and
iodination to 3-iodotyrosine (MIT) was monitored spectropho-
tometrically at 289 nm. MIT was quantified using HPLC for
those flavonoids with absorbance maxima that interfered with
spectrophotometric monitoring. It was determined that MIT
formation rates were identical within experimental error whether
determined by the UV or HPLC methods, although the UV
method was more precise. HPLC analysis was performed
immediately using a Radial Compression Module equipped with
a NovaPak C18 cartridge (5 × 100 mm, 4-µm particle size,
Waters Associates, Milford, MA) using UV detection at 289 nm
essentially as described earlier (18).
Tim e Cou r se of In a ctiva tion of TP O. Those flavonoids
that inactivated TPO by more than 50% in the above experiment
were studied further. TPO (1.0-5.0 µM) was incubated with
kaempferol, 8-40 µM; morin, 10-50 µM; naringenin, 6-30 µM;
or quercetin, 10-50 µM at 25 ( 0.1 °C in 0.1M phosphate buffer
(pH 7.4), and hydrogen peroxide (200 µM) was added to initiate
the reaction. Aliquots were withdrawn at various time points
and diluted 1000- to 2000-fold, and the remaining enzyme
activity was determined. The half-times for inactivation were
estimated graphically from plots of activity remaining vs time
(cf. Figure 3 inset and ref 20). The inactivation of TPO by the
solvent ethanol (final concentration <2%, v/v) and H2O2 was
minimal during the incubation period.
Hyd r ogen P er oxid e Con cen tr a tion Dep en d en ce of In -
a ctiva tion . TPO (1.0 µM) was incubated with excess narin-
genin (150 µM), and enzyme activity was titrated by addition
of varying but limiting concentrations of hydrogen peroxide.
After 4 min of incubation, aliquots were withdrawn and diluted,
and residual activity was measured. Soret spectral changes
were monitored by second derivative UV-vis spectrophotometry
to maximize sensitivity using a Hewlett-Packard 8452A diode
array spectrophotometer as previously described (19).
Stea d y-Sta te Kin etics of In h ibition of TP O-Ca ta lyzed
Tyr osin e Iod in a tion by Myr icetin a n d Na r in gin . The
inhibition of TPO (1.25-2.5 nM) by myricetin (0-2.0 µM) or
naringin (0-6.0 µM) was studied in the presence of varying
concentrations of either iodide ion (0-100 µM) or H2O2 (0-160
µM) at fixed concentrations of H2O2 (25 µM) and iodide ion (150
µM), respectively. MIT formation was monitored spectropho-
tometrically at 289 nm.
Iod in a tion of Bioch a n in A. TPO (2.5 nM) was incubated
with varying concentrations of biochanin A (0-50 µM) at a fixed
concentration of iodide ion (250 µM) in 0.1 M phosphate buffer
(pH 7.4) at 25 ( 0.1 °C, and the reaction was started by addition
of hydrogen peroxide (50 µM). The increase in absorbance at
290 nm was monitored for 1-2 min spectrophotometrically.
Competitive iodination kinetics were determined in the
presence of an enzymatic hydrogen peroxide-generating system.
Biochanin A, TPO (2.5 nM), iodide ion (150 µM), tyrosine (150
µM), glucose (1.25 mM), glucose oxidase (10 nM), and biochanin
A (0-20 µM) were incubated in 0.1 M phosphate buffer (pH 7.4)
at 25 ( 0.1 °C. The reaction was followed by monitoring MIT
formation.
For determination of reaction products, biochanin A (50 µM)
was incubated with TPO (2.5 nM), iodide ion (0.4 mM), and H2O2
(50 µM) at 25 ( 0.1 °C in 0.1 M phosphate buffer (pH 7.4). After
3 min of incubation, biochanin A and its products were extracted
with 2 volumes of ethyl acetate. This was repeated twice before
concentrating the combined aliquots of the ethyl acetate extract
by Speed Vac (Savant Instruments Inc., Farmingdale, NY). The
concentrate was reconstituted with 400 µL of HPLC mobile
phase. HPLC analysis of 20-40 µL aliquots was performed
In a ctiva tion Exp er im en ts. TPO or lactoperoxidase (LPO,
1.0 µM) was preincubated with the flavonoid (100-150 µM) for
1 min prior to initiation of the reaction by the addition of
hydrogen peroxide (200 µM). After 4 min of incubation, aliquots
were removed and diluted 1000- to 2000-fold, and the residual
enzyme activity was measured using the tyrosine iodination
assay as described previously (19). It was determined that,
following dilution, the concentration of flavonoid remaining had
a minimal effect on the enzyme activity assay. A minimum of
triplicate analyses was performed.
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18 Chem. Res. Toxicol., Vol. 9, No. 1, 1996
Divi and Doerge
Ta ble 1. Effect of F la von oid s on TP O Activitya
compound
IC50 (µM)b
% inactivationc
flavanone
flavone
rutin
>1500
2.1 ( 0.13
6.8 ( 0.34
36.5 ( 4.26
42.9 ( 6.32
2.3 ( 1.52
8.3 ( 1.76
31.1 ( 3.97
14.6 ( 2.01
72.4 ( 5.89
63.9 (12.59
52.2 ( 6.43
76.9 ( 10.67
8.9 ( 1.02
>2000
40.6 ( 3.88
36.4 ( 3.86
12.6 ( 1.56
8.3 ( 0.93
6.3 ( 0.63
6.2 ( 0.84
2.7 ( 0.99
2.4 ( 0.64
2.1 ( 0.82
1.2 ( 0.48
0.6 ( 0.18
catechin
naringin
baicalein
fisetin
biochanin A
naringenin
quercetin
morin
kaempferol
myricetin
a
The assay system containing TPO (2.0 nM), tyrosine (0.15
mM), iodide ion (0.15 mM), and the appropriate concentration of
test compound was preincubated in phosphate buffer (pH 7.4) for
2 min. The conditions for HPLC analysis are described in Materials
F igu r e 2. Second-derivative Soret spectra of native TPO and
flavonoid-inactivated TPO. TPO (1.0 µM) was incubated with
or without kaempferol (150 µM)/naringenin (150 µM) and H2O2
(200 µM). After 4 min of incubation, spectra were obtained
between 380 and 460 nm. Spectra 1, 2, and 3 are native TPO,
kaempferol-inactivated TPO, and naringenin-inactivated TPO,
respectively.
b
and Methods. IC50 values (mean ( SD) are based on 2-5
experiments in which the compounds were tested at 8-12 con-
centrations in duplicate. c Inactivation experiments were carried
out in the presence of TPO (1.0 µM), test compound (150 µM), and
H2O2 (200 µM) at 25 °C in 0.1 M phosphate buffer (pH 7.4). After
2-3 min of incubation, aliquots of the reaction mixture were
withdrawn and diluted 1000 to 2000-fold, and tyrosine iodination
activity was measured spectrophotometrically.
mum at 428 nm (a red shift of 15 nm) and about 60-
70% decrease in the Soret absorbance. Similar changes
were observed during inactivation of the enzyme in the
presence of catechin, fisetin, morin, or quercetin. In
contrast, analogous incubations with flavanone, flavone,
myricetin, or naringin resulted in no spectral changes.
Control experiments in which the flavonoid was omitted
from the incubation mixture resulted in no change in
absorbance. It is likely that the 390/393 nm peaks
observed were due to the presence of oxidized flavonoids.
The levels formed were low in these cases because of the
enzyme inactivation that occurred; however, with phenol
and pyrogallol, which are good substrates, prominent
absorptions in this spectral region are observed (data not
shown). As predicted for mechanism-based inhibitiors
(20), a linear relationship was observed between the
concentration of kaempferol, morin, naringenin, or quer-
cetin versus the product of inactivation half-times and
the flavonoid concentration. Values for ki and Ki ranged
from 1.4 to 6.7 and 6.2 to 12.9, respectively. A typical
plot obtained with naringenin is shown in Figure 3. The
saturation kinetics observed are consistent with forma-
tion of a reversible complex between enzyme and the
flavonoid prior to inactivation. The apparent enzyme-
inhibitor dissociation constants (Ki, Table 2) for the
flavonoids are approximately one-half to one-third the
apparent Km value for iodide ion (22.4 µM, tyrosine
iodination) determined under similar conditions for TPO.
The apparent maximal inactivation rate constants (ki)
for these flavonoids are ca. 700- to 3000-fold lower than
the turnover number for the normal enzyme reaction
with iodide ion (tyrosine iodination, ∼4800 min-1).
Figure 4 shows the titration of TPO activity by varying
limiting concentrations of H2O2 in the presence of nar-
ingenin under aerobic or minimal oxygen conditions.
Inactivation was increased by hypoxia at all H2O2 con-
centrations tested. Extrapolation of the linear portion
of the curves yielded approximate partition ratio values
of 8 and 42, respectively, in hypoxic and aerobic condi-
tions. Addition of other TPO substrates (e.g., pyrogallol,
iodide ion) reduced the inactivation of TPO by kaempferol
(see Table 3). The presence of pyrogallol, a potent
substrate for TPO, in the reaction mixture at g150 µM
completely protected the enzyme from inactivation by
kaempferol. The presence of iodide ion (5.0 mM) in the
using a reversed phase column (3 cm × 2 mm, 3 µm particle
size, Perkin Elmer, Norwalk, CT) using 60% acetonitrile in H2O
with a flow rate of 1.0 mL/min. The detection was carried out
at 230 nm, and UV spectra (220-350 nm) were obtained using
a Spectra Focus rapid scanning detector (Spectra Physics, San
J ose, CA). Individual product peaks were collected for further
analysis (see below).
The products of the biochanin A iodination were investigated
by using ES/MS with a VG Platform mass spectrometer (Fisons
Instruments, Altrincham, U.K.). The mass spectrometer was
operated at the following conditions: capillary voltage 2.63 kV,
HV lens voltage 0.57 kV, cone voltage 45-75 V, and source
temperature 60 °C; and scanning was carried out at an m/ z
range of 100-600 at a rate of 1.49 s/scan for both positive and
negative ion acquisitions. A 10 µL sample containing ca. 150
ng of the products (separated off-line by LC as described above)
was introduced by flow injection analysis using a syringe pump
delivering 50% acetonitrile in water at 10 µL/min.
The major product of biochanin A described above was further
1
characterized by H-NMR (Bruker AM1 spectrometer operated
at 500.13 MHz) in CDCl3.
Resu lts
All of the flavonoids tested except flavanone and
flavone inhibited tyrosine iodination by TPO, but with
markedly different potencies (see Table 1). IC50 values
ranged from 0.6 to 40.6 µM with the following order of
potency: myricetin > kaempferol > morin > quercetin
> naringenin > biochanin A > fisetin > baicalein >
naringin > catechin > rutin. Subsequent studies were
undertaken to determine the mechanisms for the ob-
served inhibitory effects.
Tim e-Dep en d en t In a ctiva tion of TP O by F la -
von oid s. Incubation of TPO with kaempferol, morin,
naringenin, or quercetin in the presence of H2O2 showed
time-dependent loss of enzyme activity in an apparent
first-order process. No loss of activity was observed in
the absence of H2O2, and the rate of inactivation was
dependent on the concentration of the flavonoid. Inac-
tivation was associated with spectral changes in the Soret
absorbance band (Figure 2). Native TPO had an absorp-
tion maximum at 413 nm, whereas naringenin- or
kaempferol-inactivated TPO had an absorption maxi-
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Thyroid Peroxidase Inhibition by Flavonoids
Chem. Res. Toxicol., Vol. 9, No. 1, 1996 19
F igu r e 3. Kinetics of inactivation of TPO by naringenin.
Inset: TPO was incubated with (b) naringenin (6 µM) or without
(O) naringenin and hydrogen peroxide (200 µM) at 25 ( 0.1 °C
in phosphate buffer (pH 7.4), and the activity remaining at
various time points was determined (n ) 6). Half-times for
inactivation of TPO (1.0-5.0 µM) by naringenin (6.0-30.0 µM)
were determined graphically from the time course of inactiva-
tion.
F igu r e 4. 4. H2O2 dependence of inactivation of TPO by
naringenin under aerobic and hypoxic conditions. TPO (1.0 µM)
was incubated with naringenin (150 µM) in 0.1 M phosphate
buffer (pH 7.4) at 25 ( 0.1 °C, and the enzyme activity was
titrated by addition of limiting amounts of hydrogen peroxide
(9 ). Minimal oxygen conditions (b) were maintained by flushing
with nitrogen and keeping the solutions under nitrogen atmo-
sphere during the reaction. Partition ratios were estimated by
extrapolating the linear portion of the curves to the abscissa
(42 and 8 µM). Inset: Correlation between H2O2 added and the
inactivation of TPO by naringenin (b) and loss of Soret absor-
bance (O).
Ta ble 2. Kin etic Con sta n ts for th e In a ctiva tion of TP O
by F la von oid sa
compound
Ki (µM)
ki (min-1
)
kaempferol
morin
naringenin
quercetin
6.23
12.87
7.06
2.64
1.36
6.68
1.69
Ta ble 3. In a ctiva tion of TP O by Ka em p fer ol in th e
P r esen ce a n d Absen ce of Alter n a te Su bstr a tesa
activity remaining
7.09
reaction conditions
TPO
(% of control)
a
Reaction mixtures consisting of TPO (1.0-5.0 µM), kaempferol
99.2 ( 3.17
97.4 ( 5.86
96.3 ( 4.89
18.2 ( 8.96
96.8 ( 4.59
99.6 ( 3.92
63.8 ( 8.46
94.3 ( 7.29
(8.0-40.0 µM)/morin (10.0-50.0 µM)/naringenin (6.0-30.0 µM)/
quercetin (12.0-60.0 µM), and H2O2 (0.2 mM) were incubated at
25 ( 0.1 °C in 0.1 M phosphate buffer (pH 7.4), and aliquots were
withdrawn at various time points and residual activity of TPO
was measured. The ratio of TPO/flavonoid was maintained at
0.125, 0.1, 0.167, and 0.083, respectively, for kaempferol, morin,
naringenin, and quercetin. Half-time for inactivation was deter-
mined from the time course plots as previously described (19, 20).
TPO + H2O2
TPO + KAE
TPO + KAE + H2O2
TPO + I-
TPO + PYG
TPO + KAE + I- + H2O2
TPO + KAE + PYG + H2O2
a
TPO (1.0 µM) was incubated with 5.0 mM iodide ion, 150 µM
kaempferol (KAE), 200 µM pyrogallol (PYG), and 200 µM hydrogen
peroxide (H2O2) in the combinations indicated at 25 ( 0.1 °C in
0.1 M phosphate buffer (pH 7.4) for 4 min, aliquots were with-
drawn and diluted 1000- to 2000-fold, and the tyrosine iodination
activity was measured. The values are mean ( SD (n ) 6).
reaction mixture partially protected the enzyme against
inactivation.
Dialysis of inactivated TPO was performed in order to
distinguish between covalent or reversible binding of
naringenin or kaempferol. Dialysis of enzyme alone
resulted in 2-8% loss of enzyme activity with little or
no change in the spectral properties. Incubation of TPO
with the flavonoids in the absence of H2O2 caused no
significant changes in either activity or spectra compared
to control incubation. However, in the presence of H2O2,
kaempferol or naringenin produced ∼70% inactivation
of TPO and ∼65% decrease in the Soret absorbance that
remained after dialysis.
Ta ble 4. In a ctiva tion of Va r iou s P er oxid a ses by
Na r in gen in /Qu er cetin a
% inactivation
(naringenin)
% inactivation
(quercetin)
peroxidase
LPO
TPO
MPO
CPO
CcP
73.2 ( 2.36
82.7 ( 3.18
48.5 ( 1.45
24.8 ( 9.83
24.6 ( 15.43
2.0 ( 8.25
61.4 ( 6.32
73.3 ( 4.96
51.8 ( 6.32
17.1 ( 10.53
61.0 ( 8.67
43.7 ( 7.93
HRP
a
In a ctiva tion of Differ en t P er oxid a ses by Na r in -
gen in /Qu er cetin . Table 4 lists the activities of different
enzymes remaining after incubation of these enzymes
with naringenin or quercetin in the presence of H2O2.
TPO, LPO, and CcP were found to be more susceptible
to inactivation by quercetin compared to other enzymes,
although significant inactivation of MPO and HRP was
observed.
Peroxidases (1.0 µM) except CcP were incubated with narin-
genin or quercetin (150 µM) and H2O2 (200 µM) for 3 min at 25 °C
in 0.1 M phosphate buffer (pH 7.4). In the case of CcP, 5.0 µM of
enzyme was incubated with 250 µM test compound. Aliquots were
withdrawn and diluted 1000-fold, and residual guaiacol oxidation
activity was determined. The values given are mean ( SD (n )
4). The activity in the absence of test compound, but containing
5% ethanol, was taken as 100%.
Effect of Myr icetin a n d Na r in gin on th e Kin etics
of TP O-Ca ta lyzed Tyr osin e Iod in a tion . Myricetin
and naringin, the glycoside of naringenin, did not inac-
tivate TPO, but low concentrations did inhibit tyrosine
iodination. The iodide ion concentration dependence of
TPO-catalyzed tyrosine iodination was measured in
incubations with myricetin and naringin. For both
compounds, the inhibition was noncompetitive with
respect to iodide ion concentration since Vmax, but not Km,
was affected. The Dixon plots shown in Figure 5A,C
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20 Chem. Res. Toxicol., Vol. 9, No. 1, 1996
Divi and Doerge
F igu r e 6. Time course of tyrosine iodination in the presence
of biochanin A. The reaction was carried out in the presence of
2.5 nM TPO, 150 µM iodide ion, 150 µM tyrosine, 1.25 mM
D-glucose, 10 nM glucose oxidase, and 0 µM (b), 4.0 µM (9), 8.0
µM (2), 12.0 µM (1), or 20.0 µM (() biochanin A at 25 ( 0.1 °C.
The reaction was started by addition of glucose oxidase to the
reaction mixture. MIT formed was monitored by LC as described
in Materials and Methods.
F igu r e 5. 5. Dixon plots showing the inhibition of TPO-
catalyzed tyrosine iodination by myricetin and naringin. (A) The
incubation system contained 2.5 nM TPO, 150 µM tyrosine,
12.5-100 µM ((-b) iodide ion, 25 µM H2O2, and 0-1.6 µM
myricetin. In (B) 2.5 nM TPO, 150 µM tyrosine, 150 µM iodide
ion, 10-160 µM H2O2 ((-b) and 0-2.5 µM myricetin were
present. (C) 1.25 nM TPO, 150 µM tyrosine, 25 µM H2O2, 12.5-
166.7 µM ((-b), iodide ion, and 0-6.0 µM naringin were present
in the incubation mixture. In (D), 2.5 nM TPO, 150 µM iodide
ion, 150 µM tyrosine, 5-80 µM H2O2 ((-b), and 0-6.0 µM
naringin were present.
Sch em e 1. Iod in a tion of Bioch a n in A by TP O
yielded an apparent value for the enzyme-inhibitor
dissociation constant, Ki, of 1.4 and 4.8 µM, respectively,
for myricetin and naringin when measured in incubations
containing 25 µM H2O2. These Ki values are ca. 5- to
16-fold lower than the respective Km for iodide ion.
mined using the Michaelis-Menten relationship to be
17.30 µM and 0.0283 AU/min at a fixed concentration of
iodide ion and H2O2.
Ch a r a cter iza tion of Bioch a n in A P r od u cts Usin g
ES/MS a n d NMR. TPO catalyzed the conversion of
biochanin A into at least three products as determined
by HPLC-UV. The peaks were collected off-line and
analyzed by ES/MS in positive and negative ion modes.
The major product (retention time 11.9 min) gave a
deprotonated molecule at nominal mass 535 Da with
prominent fragment ions at m/ z 521 (M - CH2) and 127
(I-). In positive ion mode, the protonated molecule had
a nominal mass of 537 Da, consistent with a diiodinated
derivative. Both minor products were similarly analyzed
and were consistent with isomeric monoiodinated [(M -
H)-, m/ z 409; (M + H)+, m/ z 411]. Insufficient amounts
of purified monoiodinated products precluded additional
means of structure proof.
The H2O2 concentration dependence of TPO-catalyzed
tyrosine iodination was also measured in incubations
with myricetin and naringin. The Dixon plots (Figure
5B,D) of the reciprocal rate of tyrosine iodination as a
function of flavonoid concentration yielded families of
curves intersecting above the x-axis. This pattern is
consistent with either competivive or linear mixed-type
inhibition (21), and an apparent Ki value of 0.54 and 1.7
µM, respectively, for myricetin and naringin was obtained
when measured at 150 µM iodide ion. Replots of the
Dixon plot slopes revealed that myricetin was a mixed-
type inhibitor and naringin was a competitive inhibitor
with respect to H2O2 concentration. The Ki values are
20- to 63-fold lower than the Km determined for H2O2 (34
µM).
The structure of diiododinated product was further
identified using 1H-NMR. All proton resonances for
biochanin A (see Scheme 1) were tentatively assigned as
follows: 3.83 (s, 3H, He); 5.45 (bs, 1H, Hf); 6.28 (d, 1H,
Ha, J Ha,Hg ) 2.2 Hz); 6.35 (d, 1H, Hg); 6.96 (d, 2H, Hd,
J Hc,Hd ) 6.7 Hz); 7.43 (d, 2H, Hc); 7.84 (s, 1H, Hb); 12.90
(s, 1H, Hh). The assignments for Hc and Hd were
confirmed by selective NOE irradiation experiments:
NOE was observed for Hc upon irradiation of Hb and for
Hd upon irradiation of He; the resonances for Ha and Hg
were assigned based on known effects of o-hydroxyl
groups and the observed 2.2 Hz coupling. The assign-
ment of Hf was based on the observed line broadening
expected for a hydrogen bonded proton. Hh was assigned
based on the expected acidity and its resulting extreme
downfield resonance. The diiodinated product had simi-
lar proton resonances except that the two protons in the
resorcinol moiety (Ha and Hg), and no others, were
eliminated (data not shown).
In h ibition of TP O-Ca ta lyzed Tyr osin e Iod in a tion
by Bioch a n in A. Figure 6 shows the time course of
iodination of tyrosine in the presence of different con-
centrations of biochanin A when glucose/glucose oxidase
was used to produce a steady state concentration of H2O2.
The presence of biochanin A in the reaction mixture
resulted in initial blockade of iodination followed by a
linear increase in MIT formation after the lag phase. The
length of the lag phase was dependent on the concentra-
tion of biochanin A. However, the rate of MIT formation
following lag phase was unchanged from the control rate.
TPO-catalyzed iodination of bioichanin A (0-50 µM) was
measured at fixed concentrations of iodide ion (250 µM)
and H2O2 (50 µM). Reactions were started by the
addition of H2O2 to the reaction mixture and monitored
spectrophotometrically at 290 nm. Reciprocal velocities
(1/∆A per minute) were plotted against the reciprocal of
biochanin A concentration, and Km and Vmax were deter-
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Thyroid Peroxidase Inhibition by Flavonoids
Chem. Res. Toxicol., Vol. 9, No. 1, 1996 21
Sch em e 2. P r op osed F or m a tion a n d Rea ction s of
F la von oid Ra d ica ls
Discu ssion
The flavonoids investigated in this study fit into several
broad groups based on IC50 values for inhibition of TPO-
catalyzed tyrosine iodination (see Table 1). While it is
not valid to compare IC50 values for compounds with
unknown inhibition mechanisms, the ability to differenti-
ate grossly flavonoid potency was a useful screening
procedure in the initial phase of this study. In previous
work, we have demonstrated several mechanisms can be
responsible for inhibition of TPO- and LPO-catalyzed
reactions: suicide inactivation (19), rapid equilibrium
(reversible) binding (22), and alternate substrate com-
petition for the enzymatic iodinating intermediate (18,
23). These experimental approaches were used to de-
termine inhibition mechanisms and structure-activity
relationships for these typical dietary flavonoids.
below); the change in going from a hydroxypyranone ring
in kaempferol to a hydroxydihydropyran in catechin
produced a marked reduction in IC50 and TPO inactiva-
tion; however, the same change from the hydroxypyra-
none in kaempferol to a dihydropyranone in naringenin
produced a minimal change in inactivation potency.
Reversible (rapid equilibrium) inhibition of TPO-
catalyzed tyrosine iodination was observed in the pres-
ence of myricetin and naringin. Figure 5 shows the
Dixon plots for inhibition of tyrosine iodination in the
presence of varying amounts of the two substrates, iodide
ion (A, C) or H2O2 (B, D). The inhibition by myricetin
and naringin was consistent with a noncompetitive
mechanism with respect to iodide ion and linear mixed-
type with respect to H2O2 (21). In both noncompetitive
and linear mixed-type mechanisms, inhibition results
from the binding of inhibitor and substrate to different
sites or enzyme forms. A minimal mechanism consistent
with these kinetics and the known properties of peroxi-
dases is shown in Scheme 2. In this proposed mecha-
nism, myricetin and naringin interact with TPO com-
pounds I and II but not EOI (the enzymatic iodinating
species) or native TPO. Further interpretation of the
inhibition kinetics in this multisubstrate ping-pong
system is beyond the scope of this discussion.
When the more potent inhibitors were investigated
further, it was seen that naringenin, quercetin, morin,
and kaempferol caused irreversible inactivation of TPO
(see Table 1, column 3). The kinetics of irreversible
enzyme inactivation (see Table 2 and Figure 3), the
altered visible absorbance spectrum of inactivated TPO
(Figure 2), and the correspondence of spectral changes
with loss of enzymatic activity (Figure 4) were consistent
with mechanism-based inhibition as previously described
for the action of resorcinol and derivatives on TPO and
LPO (19). In the previously proposed mechanism, reac-
tive resorcinol radicals are formed in the active site by
compound I-mediated oxidation of the phenolic suicide
substrate, and inactivation occurs by covalent binding of
these radicals to catalytic amino acid radical(s) on
compound II (19). This conclusion was supported by the
observed binding of 10 mol of resorcinol/mol of LPO
inactivated. The spectral changes observed for flavonoid-
inactivated TPO (see Figure 2) are similar to those seen
in resorcinol-inactivated TPO and LPO, suggesting a
common mechanism of inactivation. At the present time,
the cause for the observed spectral changes is not clear
but may result from heme modification in addition to
active site amino acid residues. The lack of commercial
sources for radiolabeled flavonoids precluded further
investigation of the covalent binding of flavonoids to TPO.
In agreement with our previous study, a free resorcinol
moiety is present in all of the flavonoids (naringenin,
quercetin, morin, kaempferol) that cause substantial
inactivation (>40%) under the conditions (see Table 1).
The smaller amounts of inactivation, observed for fla-
vonoids that do not contain a resorcinol moiety (flavone,
flavanone, baicalein, fisetin), were similar to the low level
inactivation caused by the action of monophenolic radi-
cals (e.g., guaiacol), as was observed in the previous study
(19).
Figure 6 shows the effects of increasing amounts of
biochanin A on TPO-catalyzed tyrosine iodination in the
presence of a continuous source of H2O2 produced by
glucose/glucose oxidase. As the concentration of biocha-
nin A was increased, the length of the lag phase in-
creased. Following the lag, tyrosine iodination resumed
at the control rate. During the lag phase, or in the
absence of tyrosine, biochanin A (λmax 262, 322 nm) was
converted to products with red-shifted UV absorption
maxima (λmax 280, 340 nm), and HPLC analysis demon-
strated the formation of at least three products with
altered UV spectra (data not shown). The identity of the
1
However, the presence of a resorcinol moiety in fla-
vonoids was not sufficient to cause substantial TPO
inactivation, as rutin, biochanin A, and myricetin were
weak inactivators. Possible modifying factors for these
compounds are as follows: the presence of a sugar moiety
on the resorcinol hydroxyl group in naringin completely
blocked the inactivation observed for the aglycon, nar-
ingenin; the presence of a sugar moiety in the pyranone
ring of rutin; the presence of a pyrogallol moiety in the
2-aryl substituent of myricetin appears to make this
flavonoid a potent substrate and competitive inhibitor of
TPO-catalyzed reactions; biochanin A, in which the aryl
substituent is attached to the 3-position of the pyranone
ring, is an alternate substrate inhibitor of iodination (see
major product was established by ES/MS and H-NMR
as the diiodinated resorcinol derivative shown in Scheme
1. The two minor products observed by HPLC were found
to be monoiodinated, presumably resulting from iodina-
tion of either position on the resorcinol ring. These data
are consistent with alternate substrate inhibition of
iodination as previously reported for inhibition of TPO-
catalyzed iodination reactions by ethylenethiourea and
N,N′-disubstituted benzimidazole-2-thiones (18, 23). In
this mechanism, competition between tyrosine and the
alternate substrate for the enzymatic iodinating species
(EOI, see Scheme 2) results initially in complete blockade
of tyrosine iodination because of the higher affinity for
biochanin A. However, after the alternate substrate is
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22 Chem. Res. Toxicol., Vol. 9, No. 1, 1996
Divi and Doerge
consumed, tyrosine iodination resumes at an unchanged
rate.
flavonoid inhibitors, especially at high doses, could elicit
prolonged blockade of thyroid hormone synthesis. Any
compound that causes chronic inhibition of thyroid
hormone synthesis is a potential thyroid carcinogen that
acts by long-term stimulation of thyroid growth induced
by elevated levels of thyroid stimulating hormone (TSH)
(27). Although there is considerably less evidence of a
role for TSH stimulation in the etiology of thyroid cancer
in humans as opposed to experimental animals, three
case-control studies in the U.S. consistently showed
thyroid cancer was strongly associated with preexisting
goiter and thyroid nodules (for a review, see ref 27).
However, studies relating iodide intake levels and human
thyroid cancer in endemic goiter regions of the world are
equivocal (27). Nevertheless, our results do suggest that
the consumption of foods rich in flavonoids, especially
those that inactivate TPO, has the potential to induce
goiter and in this manner may be involved in the etiology
of human thyroid cancer.
Resorcinol-mediated inactivation was previously in-
vestigated for a series of peroxidases, and it was deter-
mined that TPO, LPO, and CcP were highly susceptible,
but HRP and other peroxidases were resistant (19).
These observations were consistent with the presence of
catalytic amino acid radicals in the susceptible peroxi-
dases but not in HRP. A similar trend was seen in the
present study for naringenin, except that MPO was also
significantly inactivated (see Table 4). This latter ob-
servation could be consistent with the suggestion that
MPO also forms protein-centered radicals following reac-
tion with H2O2 (24). Inactivation by quercetin was
highest for TPO, LPO, and CcP, but significant inactiva-
tion of all peroxidases tested occurred, including HRP.
Most compounds tested inhibited the TPO-catalyzed
oxidation of guaiacol with IC50s of a magnitude similar
to that seen for tyrosine iodination. However, low
concentrations of myricetin and naringin (<10 µM)
caused a stimulation of guaiacol oxidation, and the
kinetics showed an increase in Vmax (data not shown).
These observations are consistent with preferential TPO-
mediated oxidation, by either compound I or compound
II, of the flavonoid to a phenoxyl radical that abstracts a
hydrogen atom from guaiacol to form the guaiacol radical
which produces the colored oxidation product, presum-
ably a dimeric species (see Scheme 2, ref 25). Regenera-
tion of the flavonoid completes a catalytic process that
drives guaiacol oxidation at an increased rate due to
higher enzymatic turnover of the flavonoid relative to
guaiacol.
Ack n ow led gm en t. We gratefully acknowledge the
assistance of M. I. Churchwell and J . Deck in obtaining
mass and NMR spectra, respectively.
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TX950076M
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