Download Ca /Nicotinamide Adenine Dinucleotide Phosphate

0013-7227/01/$03.00/0
Printed in U.S.A.
The Journal of Clinical Endocrinology & Metabolism 86(9):4339 – 4343
Copyright © 2001 by The Endocrine Society
Ca2ⴙ/Nicotinamide Adenine Dinucleotide PhosphateDependent H2O2 Generation Is Inhibited by Iodide
in Human Thyroids
LUCIENE C. CARDOSO, DENISE C. L. MARTINS, MARCIA D. L. FIGUEIREDO, DORIS ROSENTHAL,
MARIO VAISMAN, ALICE H. D. VIOLANTE, AND DENISE P. CARVALHO
Instituto de Biofı́sica Carlos Chagas Filho (L.C.C., M.D.L.F., D.R., D.P.C.); and Serviço de Endocrinologia (D.C.L.M., M.V.,
A.H.D.V.), Hospital Universitário Clementino Fraga Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
21949-900
A calcium and NAD(P)H-dependent H2O2-generating activity
has been studied in paranodular thyroid tissues from four
patients with cold thyroid nodules and from nine diffuse toxic
goiters. H2O2 generation was detected both in the particulate
(P 3,000 g) and in the microsomal (P 100,000 g) fractions of
paranodular tissue surrounding cold thyroid nodules (PN),
with the same biochemical properties described for NADPH
oxidase found in porcine and human thyroids. In PN tissues,
the particulate NADPH oxidase activity (224 ⴞ 38 nmol
H2O2䡠hⴚ1䡠mgⴚ1 protein) was similar to that described for the
porcine thyroid enzyme. However, no NADPH oxidase activity
was detectable in the particulate fractions from eight diffuse
toxic goiter patients treated with iodine before surgery; all
but one also received propylthiouracil or methimazole in the
preoperative period. Thyroid cytochrome c reductase (diffuse
toxic goiters ⴝ 438 ⴞ 104 nmol NADPⴙ䡠hⴚ1䡠mgⴚ1 protein; PN ⴝ
78 ⴞ 10 nmol NADPⴙ䡠hⴚ1䡠mgⴚ1 protein) and thyroperoxidase
(diffuse toxic goiters ⴝ 621 ⴞ 179 U䡠gⴚ1 protein; PN ⴝ 232 ⴞ 121
U䡠gⴚ1 protein) activities were unaffected by iodide. Thus, the
human NADPH oxidase seems to be inhibited by iodinated
compounds in vivo and probably is an enzyme involved in the
Wolff-Chaikoff effect. Our findings reinforce the hypothesis
that thyroid NADPH oxidase is responsible for the production
of H2O2 necessary for thyroid hormone biosynthesis. (J Clin
Endocrinol Metab 86: 4339 – 4343, 2001)
H
In human thyroid tissues, the presence of a Ca2⫹/
NAD(P)H-dependent H2O2 generator similar to the porcine
thyroid NADPH oxidase was only recently characterized
(15). More recently the human and porcine enzyme cDNA
have been cloned (16, 17). However, further molecular identification of this thyroid enzyme has not yet been achieved.
Iodide, apart from inhibiting thyroid hormone biosynthesis, has also been known for a long time for its properties in
inhibiting thyroid hormone release and reducing thyroid
gland vascularity (18, 19). So patients with diffuse toxic goiters (DTG) are almost routinely prepared for thyroidectomy
with iodine in the preoperative period.
The aim of the present study was to further characterize
the human NADPH oxidase activity using paranodular tissues from cold thyroid nodules and to evaluate whether the
use of iodine before surgery in patients with DTG could
impair NADPH oxidase activity, leading to diminished thyroid hormone biosynthesis. We have found a human thyroid
NADPH oxidase activity, in paranodular tissues, that is similar to the enzyme already characterized in porcine glands.
In DTG patients treated with iodine for 10 –15 d, all thyroid
serum hormone levels were decreased at the day of surgery,
compared with serum hormone levels before treatment. Furthermore, there has been a marked inhibition of the Ca2⫹/
NADPH-dependent H2O2 generation in the particulate (P
3,000 g) fraction of thyroid samples obtained from iodinetreated patients. Thus, our results reinforce the concept that
thyroid NADPH oxidase inhibition might occur during the
Wolff-Chaikoff effect.
2O2
GENERATION IS a limiting step of thyroid hormones biosynthesis, as previously shown in dog thyroid slices (1, 2). In fact, the H2O2 generator system has been
localized at the apical cell surface in rat and pig open follicles
(3, 4) and in intact rat follicles (5). Biochemical studies have
confirmed that in porcine thyroid glands H2O2 is generated
by an NADPH:O2 oxidoreductase, the so-called thyroid
NADPH oxidase (6 – 8). This enzyme is a flavoprotein dependent on calcium for activity (7–9) and is activated by ATP
(7). We have previously shown that in pig thyrocytes
NADPH oxidase activity is induced by TSH, an effect that is
mimicked by forskolin, and cAMP analogs (10). In dog thyrocytes, the expression of the H2O2 generator was also shown
to be induced by TSH via the cAMP cascade (11). The H2O2
generator, NADPH oxidase, therefore seems to be a thyrocyte differentiation marker (10, 11), just as thyroglobulin and
thyroperoxidase are.
Inhibition of protein iodination and thus of thyroid hormone biosynthesis by iodide or iodinated compounds seems
to be owing to the inhibition of hydrogen peroxide generation, both in dog and in human thyrocytes (12, 13). Hence, the
fact that thyroid NADPH oxidase is irreversibly inhibited in
vitro by iodide and 2-iodohexadecanal, a thyroid iodocompound that probably mediates the Wolff-Chaikoff effect, is
another evidence that this is the enzyme responsible for H2O2
generation associated with thyroid hormonogenesis (14).
Abbreviations: DTG, Diffuse toxic goiters; FAD, Flavin adenine dinucleotide; HRP, horseradish peroxidase; SSKI, saturated solution of iodine; TPO, thyroperoxidase.
4339
4340
The Journal of Clinical Endocrinology & Metabolism, September 2001, 86(9):4339 – 4343
Materials and Methods
Materials
NADPH and lyophilized horseradish peroxidase (HRP, grade 1) were
purchased from Boehringer (Mannheim, Germany); Scopoletin, cytochrome c, and Flavin adenine dinucleotide (FAD) were obtained from
Sigma (St. Louis, MO).
Patients
We studied paranodular thyroid tissue samples obtained from four
female patients with cold nodules and normal serum T4, T3, and TSH
levels who did not receive any treatment before surgery. Eight patients
with DTG (females 27– 42 yr old) programmed for thyroidectomy received saturated solution of iodine (SSKI, 5 drops three times per day)
for 10 –15 d before surgery. The patients received either propylthiouracil
(PTU) (500 –900 mg/d, n ⫽ 6) or methimazole (MMI) (25–50 mg/d, n ⫽
2) until the day before surgery. Another patient with DTG (21 yr old) was
allergic to antithyroid drugs and received only SSKI (5 drops three times
per day) for 7 d and propanolol (80 mg/d) before surgery. The patients
gave their informed consent, and the study has been approved by the
Institutional Human Research Committee.
Thyroid tissue samples were obtained at thyroidectomy and either
freshly processed for NADPH oxidase and cytochrome c reductase
measurements or stored at ⫺20 C for further thyroperoxidase (TPO)
extraction and activity evaluation.
Serum hormone levels
Blood samples were collected before starting treatment (basal), 7 d
afterward, and on the day of surgery. Total T4, total T3, and TSH (third
generation) were measured using a solid-phase, chemiluminescent enzyme immunoassay (IMMULITE). Free T4 and reverse T3 were measured by RIA. All kits were purchased from Diagnostic Products Corp.
(Los Angeles, CA).
Thyroid samples processing
For NADPH oxidase and cytochrome c reductase preparations, fresh
human thyroid tissue samples (1 g) were cleaned from fibrous tissue or
hemorrhagic areas, minced, and homogenized in sodium phosphate
buffer, pH 7.2, containing 0.25 m sucrose, 0.5 mm dithiothreitol (DTT),
and 1 mm EGTA, using an Ultra-Turrax (Staufen, Germany). The homogenate was filtered through cheesecloth. The particulate fraction was
collected by centrifugation at 3,000 g for 15 min at 4 C and resuspended
in 3 ml 50 mm sodium phosphate buffer, pH 7.2, containing 0.25 m
sucrose and 2 mm MgCl2 (buffer A). The pellet was washed twice with
3 ml of buffer A and centrifuged at 3,000 g for 15 min at 4 C. The last
pellet (P 3,000 g) was gently resuspended in 1 ml buffer A. The supernatant of the first centrifugation was centrifuged at 100,000 g for 1 h at
4 C. The pellet (microsomal fraction, P 100,000 g) was washed twice in
2 ml buffer A, and gently resuspended in 0.5 ml buffer A. Protein
concentrations were measured by the method of Bradford (20), using
BSA as standard. The particulate fractions (P 3,000 g and P 100,000 g)
were incubated with 1 N NaOH (30 min, 20 C) to dissolve particulates
before protein determination.
For TPO preparation, thyroid tissue samples (1 g) were cleaned from
fibrous tissue or hemorrhagic areas, minced, and homogenized in 50 mm
Tris-HCl buffer pH 7.2, containing 1 mm potassium iodide, using an
Ultra-Turrax (Ika). The homogenate was centrifuged at 100,000 g for 1 h
at 4 C, and the pellet was resuspended in 2 ml digitonin (1% wt/vol).
The mixture was incubated at 4 C for 24 h and then centrifuged at 100,000
g for 1 h at 4 C. The supernatant containing solubilized TPO was used
for the iodide-oxidation assays, as previously described (21).
Ca2⫹ and NADPH-dependent H2O2 generating system:
NADPH oxidase activity
H2O2 formation was measured by incubating samples of the thyroid
particulate fractions (P 3,000 g and P 100,000 g) at 30 C in 1 ml 170 mm
sodium phosphate buffer, pH 7.4, containing 1 mm sodium azide, 1 mm
EGTA, 1 ␮m FAD, 1.5 mm CaCl2, as previously described (15). The
reaction was started by adding 0.2 mm NADPH; aliquots of 100 ␮l were
Cardoso et al. • Iodide Inhibits Thyroid NADPH Oxidase
collected at intervals up to 20 min and mixed with 10 ␮l 3 N HCl to stop
the reaction and destroy the remaining NADPH. Calcium-dependent
thyroid NADH oxidase activity was also evaluated in the particulates of
one DTG tissue sample, by adding 0.2 mm NADH to initiate the reaction.
Initial rates of H2O2 formation were determined from eight aliquots of
each assay by following the decrease in 0.4 ␮m scopoletin fluorescence
in the presence of HRP (0.5 ␮g/ml) in 200 mm phosphate buffer, pH 7.8,
in a Hitachi (Tokyo, Japan) spectrofluorimeter (F 4000), as previously
described (9). The excitation and emission wavelengths were 360 and 460
nm, respectively. All measurements were performed on at least three
samples from each particulate preparation and expressed as nmoles
H2O2䡠h. Specific activities were expressed per milligram protein (nmoles
H2O2䡠h⫺1䡠mg⫺1 protein) in the thyroid P 3,000 g and P 100,000 g
fractions.
To evaluate the activation of the enzyme by phosphate, the concentration of phosphate in the reaction mixture was increased from 50 to 200
mm. To determine the Ca2⫹-dependence of H2O2 generation, parallel
samples were assayed without Ca2⫹, in the presence of 1 mm EGTA.
Thyroid NADPH-cytochrome c reductase activity
Aliquots of human thyroid particulate fractions (P 3,000 g and P
100,000 g) were incubated, at 30 C, in 1 ml 50 mm sodium phosphate
buffer (pH 7.2) containing 1 mm sodium azide, 1.2 mm EGTA, and 0.1
mm cytochrome c. The reaction was started by adding 0.1 mm NADPH.
The initial NADPH concentration was measured at 340 nm in a U-3300
(Hitachi) double-beam spectrophotometer, using a molar absorption
coefficient of 6.2 ⫻ 103 M⫺1 cm⫺1. Aliquots of 100 ␮l were taken at
intervals and mixed with 10 ␮l 2 mm dithiothreitol and 100 ␮l 2% SDS
to stop the reaction. The initial rates of cytochrome c-dependent NADPH
oxidation were determined from eight aliquots of each assay by following the decrease in NADPH fluorescence at pH 8.0 in a Hitachi
spectrofluorimeter (F 4000), as previously described (10). The excitation
and emission wavelengths were 340 and 453 nm, respectively. NADPHcytochrome c reductase activity was expressed as nanomoles of NADPH
oxidized per hour and milligram of protein in the thyroid P 3,000 g and
P 100,000 g fractions (nmoles NADP⫹䡠h⫺1䡠mg⫺1 protein).
Thyroperoxidase iodide-oxidation activity
Thyroid peroxidase iodide-oxidation assays were performed using 12
mm iodine in 50 mm phosphate buffer (pH 7.4), and glucose-glucose
oxidase as the hydrogen peroxide (H2O2) generating system, as previously described (21, 22). The increase in absorbency at 353 nm (⌬A353)
was followed for 4 min on a U-3300 (Hitachi) double-beam spectrophotometer. The TPO activity was estimated from the ⌬A353/min determined from the linear portion of the reaction curve. One unit of iodide
oxidation activity is defined as ⌬A353/min (U) ⫽ 1.0, and activity was
related to the protein concentration in the enzyme preparation (U/g⫺1
protein).
Statistical analysis
Statistical analysis of intergroup enzyme activities [DTG and paranodular tissue surrounding cold nodules (PN)] was performed using the
Mann-Whitney test. Phosphate effect on enzyme activity was evaluated
by the Kruskal-Wallis ANOVA followed by the Dunn multiple comparison test. Serum hormone levels at different periods of iodine treatment were analyzed by ANOVA for repeated measures followed by
the Bonferroni multiple comparison test. Results are expressed as
mean ⫾ se.
Results
Serum thyroid hormone levels in patients with DTG
Serum total and free T4, total T3, and rT3 levels in DTG
patients receiving iodine for 7 d or 10 –15 d were compared
with those found before iodine treatment. All serum hormonal levels were already significantly decreased after 7 d
of iodine treatment, and persistently diminished until the
Cardoso et al. • Iodide Inhibits Thyroid NADPH Oxidase
The Journal of Clinical Endocrinology & Metabolism, September 2001, 86(9):4339 – 4343 4341
TABLE 1. Serum thyroid hormone levels in patients with DTG treated with SSKI in the preoperative period
Basal
7d
Surgery (10 –15 d)
Total T4 ␮g/dl
(4 –13)a
Free T4 ng/dl
(0.8 –2.0)a
T3 ng/dl
(70 –210)a
rT3 ng/dl
(0.09 – 0.35)a
14.5 ⫾ 1.7
11.0 ⫾ 1.6b
10.1 ⫾ 1.6d
2.7 ⫾ 0.5
2.0 ⫾ 0.4b
1.7 ⫾ 0.3d
226.1 ⫾ 25.6
164.2 ⫾ 19.8c
149.2 ⫾ 18.8d
0.63 ⫾ 0.15
0.40 ⫾ 0.10b
0.34 ⫾ 0.11c
Results are expressed as mean ⫾ SEM.
a
Normal range; b P ⬍ 0.05; c P ⬍ 0.01;
d
P ⬍ 0.001.
FIG. 1. Thyroid NADPH oxidase activity. A Ca2⫹- and NADPHdependent H2O2 generation was measured in human thyroid samples
obtained from four paranodular to cold nodule tissues (PN) and from
eight iodine-treated patients with diffuse toxic goiter (iodine). H2O2
generation was measured in 170 mM sodium phosphate buffer, pH 7.4,
containing 1 mM sodium azide, 1 mM EGTA, 1 ␮M FAD, and 1.5 mM
CaCl2. The reaction was started by adding 0.2 mM NADPH; aliquots
of 100 ␮l were collected at intervals up to 20 min and mixed with 10
␮l 3 N HCl to stop the reaction. Initial rates of H2O2 formation were
determined from eight aliquots of each assay by following the decrease
in 0.4 ␮M scopoletin fluorescence in the presence of HRP (0.5 ␮g/ml)
in 200 mM phosphate buffer, pH 7.8, in a Hitachi spectrofluorimeter
(F 4000, excitation ⫽ 360 and emission ⫽ 460 nm). A, Particulate
3,000 g fraction (P 3,000 g). B, Microsomal fraction (P 100,000 g).
Enzyme activity is expressed as mean of at least two measurements
in each particulate preparation.
day of surgery (10 –15 d after beginning of SSKI treatment;
Table 1).
Calcium and NADPH-dependent H2O2 generation activities
in human thyroid tissues
In the absence of Ca2⫹, no NADPH-dependent H2O2 generating activity was found in any of the particulate fractions
studied. In our assays, NADPH oxidase activity significantly
increased in the presence of high concentrations (200 mm) of
phosphate anions (456 ⫾ 47 nmol H2O2䡠h⫺1/ml⫺1), compared with the activity found in 50 mm phosphate (208 ⫾ 35
H2O2䡠h⫺1/ml⫺1, P ⬍ 0.05), confirming findings previously
reported for pig and human thyroids (15). Therefore, in all
following experiments, rates of H2O2 formation were measured using human thyroid particulate fractions in 170 mm
phosphate buffer pH 7.4.
NADPH/Ca2⫹-dependent H2O2 generation activities in
paranodular thyroid tissues were found in both the P 3,000
g and P 100,000 g fractions, in contrast to pig thyroids in
which no detectable activity is found in the P 100,000 g
fraction.
scribed for porcine thyroid particulate, also enriched in thyroid plasma membranes. On the other hand, H2O2 generation
was either undetectable or low in the P 3,000 g obtained from
DTG patients treated with iodine (Fig. 1A). However, there
were no significant differences between the microsomal fraction (P 100,000 g) NADPH oxidase activities in paranodular
(200 ⫾ 36 nmol H2O2䡠h⫺1䡠mg⫺1 protein) or DTG samples
(235 ⫾ 41 nmol H2O2䡠h⫺1䡠mg⫺1 protein), independent of
iodine treatment received before surgery (Fig. 1B). NADH
oxidase activity was also undetectable in the P 3,000 g obtained from one DTG sample and represented 50% of the
NADPH oxidase activity found in the P 100,000 g of the same
DTG (NADH ⫽ 77; NADPH ⫽ 139 nmol H2O2䡠h⫺1䡠mg⫺1
protein), as previously described (15). In the DTG patient
who received neither PTU nor MMI before surgery, NADPH
oxidase activity was also undetectable in the P 3,000 g fraction and was within the normal range in the P 100,000 g
fraction (100 nmol H2O2䡠h⫺1䡠mg⫺1 protein).
Cytochrome c reductase activity
Cytochrome c reductase is another enzymatic system,
which was proposed as capable of generating H2O2 in the
thyroid gland. So we evaluated the effect of iodine treatment
on this enzyme activity. We found no effect of iodine treatment on cytochrome c reductase activity, either in the P 3,000
g fraction (DTG ⫽ 438 ⫾ 104 nmol NADP⫹䡠h⫺1䡠mg⫺1 protein;
PN ⫽ 78 ⫾ 10 nmol NADP⫹䡠h⫺1䡠mg⫺1 protein, n ⫽ 2) or in
the P 100,000 g fraction (DTG ⫽ 515 ⫾ 49 nmol NADP⫹䡠
h⫺1䡠mg⫺1 protein; PN ⫽ 216 ⫾ 84 nmol NADP⫹䡠h⫺1䡠mg⫺1
protein, n ⫽ 2) (Fig. 2A and Fig. 2B). In fact, the cytochrome
c reductase activity seems to be higher in thyroid tissues
obtained from DTG patients than in PN tissues.
Thyroperoxidase activity
Total TPO iodide-oxidation activities in DTG tissues were
not significantly different from that in paranodular tissues
(DTG ⫽ 621 ⫾ 179 U/g⫺1 protein; PN ⫽ 232 ⫾ 121 U/g⫺1
protein, Fig. 2C). Furthermore, there was no significant difference between DTG-TPO activities in both the thyroid P
3,000 g (618 ⫾ 288 U/g⫺1 protein) and P 100,000 g fractions
(1328 ⫾ 673 U/g⫺1 protein), indicating that iodine treatment
does not block TPO activity, at least irreversibly. In the DTG
patient who received neither PTU nor MMI before surgery,
TPO was also within the normal range (695 U/g⫺1 protein).
Discussion
NADPH oxidase activity is inhibited by iodine treatment
In paranodular thyroid tissues, NAPDH oxidase H2O2generating levels found in the P 3,000 g fraction (224 ⫾ 38
nmol H2O2䡠h⫺1䡠mg⫺1 protein) were similar to those de-
Iodine organification and thyroid hormone biosynthesis
are dependent on thyroperoxidase and on H2O2 as cofactor.
The H2O2 supply has been demonstrated to be a limiting step
for thyroid hormone biosynthesis (2). The cDNA for the
4342
The Journal of Clinical Endocrinology & Metabolism, September 2001, 86(9):4339 – 4343
FIG. 2. Thyroid cytochrome c reductase and thyroperoxidase activities. NADPH oxidation by cytochrome c reductase and TPO iodide
oxidation activities were measured in human thyroid samples obtained from four paranodular to cold nodule tissues (PN) and from
eight iodine-treated patients with diffuse toxic goiter (iodine). Cytochrome c reductase activity was measured in the presence of 1 mM
sodium azide, 1.2 mM EGTA, and 0.1 mM cytochrome c. The reaction
was started by adding 0.1 mM NADPH; aliquots of 100 ␮l were taken
at intervals and mixed with 10 ␮l 2 mM dithiothreitol and 100 ␮l 2%
SDS to stop the reaction. NADPH oxidation was determined from
eight aliquots of each assay by following the decrease in NADPH
fluorescence at pH 8.0 in a Hitachi spectrofluorimeter (F 4000, excitation ⫽ 340 and emission ⫽ 453 nm). A, Particulate 3,000 g fraction
(P 3,000 g). B, Microsomal fraction (P 100,000 g). All measurements
were performed on at least two samples from each particulate preparation. C, TPO activity was measured using 12 mM KI in 50 mM
phosphate buffer (pH 7.4), and glucose-glucose oxidase as the hydrogen peroxide (H2O2) generating system. The increase in absorbance
at 353 nm (⌬A353) was followed for 4 min on a U-3300 (Hitachi)
double-beam spectrophotometer, and enzyme activity was estimated
from the ⌬A353/min determined from the linear portion of the reaction
curve. One unit of iodide oxidation activity is defined as ⌬A353/min
(U) ⫽ 1.0. Results are expressed as mean ⫾ SE.
enzyme responsible for H2O2 generation in the thyroid gland
has only recently been cloned in porcine and human thyroids
(16, 17). Nevertheless, some biochemical properties of the
thyroid H2O2-generating enzyme, NADPH oxidase, have already been defined in porcine and more recently in human
thyroids (15). Leseney et al. (15) reported a Ca2⫹ and
NAD(P)H-dependent H2O2-generating activity in human
thyroids, but a very low NADPH oxidase activity in the P
3,000 g fraction was found in their study. In the present
study, however, the human thyroid NADPH oxidase activity
found in the P 3,000 g fraction of PN tissues was similar to
that of the porcine thyroid enzyme, probably because patients with cold thyroid nodules did not receive any iodine
treatment before surgery (9, 15, 23). Furthermore, in pig
thyroids NADPH oxidase activity is predominantly found in
the P 3,000 g fraction, whereas in human thyroid tissues the
enzymatic activity was present both in the microsomal and
P 3,000 g fractions, as previously reported (15). It has previously been demonstrated that thyroid NADPH oxidase
Cardoso et al. • Iodide Inhibits Thyroid NADPH Oxidase
activity (10) and mRNA expression (16) are significantly
increased by TSH in porcine thyrocytes. Thus, in thyroid
samples obtained from DTG, in which the TSH intracellular
pathway is hyperstimulated, an increased NADPH oxidase
activity could be found.
Thyroid autoregulation by iodine involves the inhibition
of several steps of thyroid metabolism, such as iodide transport, hormone secretion, and adenylate cyclase activity (24,
25). However, other mechanisms have been proposed to
explain iodine inhibition of its own organification. Previous
studies have shown that high, unphysiological, iodide concentrations can inhibit thyroperoxidase in vitro (26). However, other studies have shown that both in canine and human thyrocytes, the Wolff-Chaikoff effect could have been
caused, at least in part, by impaired H2O2 generation, although the authors did not evaluate thyroid NADPH oxidase
activity, an ill-defined enzymatic system at the time (12, 13).
Then it was reported that the porcine NADPH oxidase could
be irreversibly inhibited in vitro by iodide and 2-iodohexadecanal, a thyroid iodocompound probably involved in the
thyroid autoregulation by iodine (14). These findings indicated the participation of NADPH oxidase inhibition in the
decreased iodine organification caused by iodide. Our
present findings of undetectable NADPH oxidase activity in
the P 3,000 g fraction from thyroid tissues exposed to high in
vivo levels of iodide show that a decreased H2O2 generation
might be important for the hormone biosynthesis inhibition
promoted by iodine treatment in human thyroids. NADPH
oxidase inhibition occurred despite the fact that PTU was
administered to the patients during the period of iodine
treatment, suggesting that iodide could act directly on the
enzyme in vivo, as previously described in vitro (14) and/or
that PTU was not sufficient to prevent lipid iodination
completely.
NADH oxidase activity was also inhibited in the P 3,000
g obtained from one patient treated with iodine, indicating
that thyroid NADPH and NADH oxidase activities could
correspond to a single enzyme responsible for Ca2⫹-dependent H2O2 generation in the thyroid, as previously suggested
(15).
NADPH oxidase activity is modulated by calcium; however, enzyme synthesis is dependent on the cAMP cascade
(10, 16, 27). Excess of iodide decreases cAMP in thyroid cells,
but the strong inhibitory effect of iodide on thyroid NADPH
oxidase, which was found only in the P 3,000 g fraction of
DTG, is probably not owing to only decreased enzyme synthesis because microsomal fraction NADPH oxidase activity
was similar in DTG and PN tissues. Furthermore, TPO whose
synthesis is also regulated by cAMP was not inhibited, at
least irreversibly, either in the P 3,000 g or in the microsomal
fractions. Besides, cytochrome c reductase, a putative thyroid
H2O2-generating system, was unaffected by iodine treatment, which shows, together with the previous findings, that
this enzyme is not inducible by TSH (10), that cytochrome c
reductase is not involved in H2O2 generation linked to thyroid hormonogenesis. Furthermore, we presume no direct
irreversible effect of PTU or MMI on thyroid NADPH oxidase in vivo because this enzyme activity was undetectable
in the P 3,000 g from a goiter that received only iodine and
propanolol before surgery. Moreover, NADPH oxidase ac-
Cardoso et al. • Iodide Inhibits Thyroid NADPH Oxidase
The Journal of Clinical Endocrinology & Metabolism, September 2001, 86(9):4339 – 4343 4343
tivity was normal in the microsomal fraction obtained from
the DTG patients treated with either PTU or MMI. Thus, we
believe there is a direct iodide/iodocompound effect on the
thyroid NADPH oxidase.
It has recently been demonstrated that in rats there is a
decrease in the sodium/iodide symporter 24 h after excess
iodide, which could explain the escape from iodine administration (28). In our patients, serum hormone levels were
significantly decreased 7 d after the start of iodine treatment
and remained diminished until the time of surgery, indicating that there was no escape from the iodine effect even after
15 d of treatment. Thus, it is possible that NADPH oxidase
inhibition at the site for thyroid hormonogenesis might contribute to the maintained lower thyroid hormone serum level
when iodine is administered to patients with DTG.
In conclusion, our present findings reinforce the hypothesis that thyroid NADPH oxidase is the enzyme responsible
for the production of H2O2 necessary for thyroid hormone
biosynthesis and shows unequivocally its role in thyroid
autoregulation.
Acknowledgments
Received July 7, 2000. Accepted May 7, 2001.
Address all correspondence and requests for reprints to: Denise Pires
de Carvalho, Instituto de Biofı́sica Carlos Chagas Filho, CCS-Bloco GCidade Universitária, Ilha do Fundão, Rio de Janeiro, Brazil 21949-900,
Brasil. E-mail: [email protected].
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
References
1. Ahn CS, Rosenberg IN 1970 Iodine metabolism in thyroid slices: effects of
TSH, dibutyryl cyclic 3⬘,5⬘-AMP, NaF and prostaglandin E1. Endocrinology
86:396 – 405
2. Corvilain B, Van Sande J, Laurent E, Dumont JE 1991 The H2O2-generating
system modulates protein iodination and the activity of the pentose phosphate
pathway in dog thyroid. Endocrinology 128:779 –785
3. Björkman U, Ekholm R, Denef JF 1981 Cytochemical localization of hydrogen
peroxide in isolated thyroid follicles. J Ultrastruct Res 74:105–115
4. Björkman U, Ekhlom R 1984 Generation of H2O2 in isolated porcine thyroid
follicles. Endocrinology 115:392–398
5. Labato MA, Briggs RT 1985 Cytochemical localization of hydrogen peroxide
generating sites in the rat thyroid gland. Tissue Cell 17:889 –900
6. Virion A, Michot JL, Dème D, Kaniewski J, Pommier J 1984 NADPHdependent H2O2 generation and peroxidase activity in thyroid particulate
fraction. Mol Cell Endocrinol 36:95–105
7. Nakamura Y, Ogihara S, Ohtaki S 1987 Activation by ATP of calciumdependent NADPH-oxidase generating hydrogen peroxide in thyroid plasma
membranes. J Biochem 102:1121–1132
8. Dupuy C, Kaniewski J, Dème D, Pommier J, Virion A 1989 NADPH-depen-
21.
22.
23.
24.
25.
26.
27.
28.
dent H2O2 generation catalyzed by thyroid plasma membranes. Studies with
electron scavengers. Eur J Biochem 185:597– 603
Dème D, Virion A, Aı̈t-Hammou N, Pommier J 1985 NADPH-dependent
generation of H2O2 in a thyroid particulate fraction requires Ca2⫹. FEBS Lett
186:107–110
Carvalho DP, Dupuy C, Gorin Y, et al. 1996 The Ca2⫹- and reduced nicotinamide adenine dinucleotide phosphate-dependent hydrogen peroxide generating system is induced by thyrotropin in porcine thyroid cells. Endocrinology 137:1007–1012
Raspé E, Dumont JE 1995 Tonic modulation of dog thyrocyte H2O2 generation
and I⫺ uptake by thyrotropin through the cyclic adenosine 3⬘,5⬘-monophosphate cascade. Endocrinology 136:965–973
Corvilain B, Laurent E, Lecomte M, Van Sande J, Dumont JE 1994 Role of
the cyclic adenosine 3⬘,5⬘-monophosphate and the phosphatidylinositolCa2⫹ cascades in mediating the effects of thyrotropin and iodide on hormone synthesis and secretion in human thyroid slices. J Clin Endocrinol
Metab 79:152–159
Corvilain B, Van Sande J, Dumont J 1988 Inhibition by iodide of iodide
binding to proteins: the “Wolff-Chaikoff” effect is caused by inhibition of H2O2
generation. Biochem Biophys Res Comm 154:1287–1292
Ohayon R, Boeynaems JM, Braekman JC, Van den Bergen H, Gorin Y, Virion
A 1994 Inhibition of thyroid NADPH-oxidase by 2-iodohexadecanal in a cellfree system. Mol Cell Endocrinol 99:133–141
Leseney AM, Dème D, Dupuy C, et al. 1999 Biochemical characterization of
a Ca2⫹/NAD(P)H-dependent H2O2 generator in human thyroid tissue. Biochimie (Paris) 81:373–380
Dupuy C, Ohayon R, Valent A, Noel-Hudson MS, Dème D, Virion A 1999
Purification of a novel flavoprotein involved in the thyroid NADPH oxidase.
Cloning of the porcine and human cDNAs. J Biol Chem 274:37265–37269
De Deken X, Wang D, Many MC, et al. 2000 Cloning of two human thyroid
cDNAs encoding new members of the NADPH oxidase family. J Biol Chem
275:23227–23233
Robuschi G, Manfredi A, Salvi M, et al. 1986 Effect of sodium ipodate and
iodide on freeT4 and free T3 concentration in patients with Graves’ disease. J
Endocrinol Invest 9:287–291
Roti E, Robuschi G, Manfredi A, et al. 1985 Comparative effects of sodium
ipodate and iodide on serum thyroid hormone concentrations in patients with
Graves’ disease. Clin Endocrinol (Oxf) 22:489 – 496
Bradford MM 1976 A rapid and sensitive method for the quantification of
microgram quantities of proteins utilizing the protein-dye binding. Anal Biochem 72:248 –252
Carvalho DP, Rego KGM, Rosenthal D 1994 Thyroid peroxidase in dyshormonogenetic goiters with organification and thyroglobulin defects. Thyroid
4:421– 426
Moura EG, Rosenthal D, Carvalho-Guimarães DP 1989 Thyroid peroxidase
activity in human nodular goiters. Braz J Med Biol Res 22:31–39
Dupuy C, Dème D, Kaniewski J, Pommier J, Virion A 1988 Ca2⫹ regulation
of thyroid NADPH-dependent H2O2 generation. FEBS Lett 233:74 –78
Wolff J 1989 Excess iodide inhibits the thyroid by multiple mechanisms. Adv
Exp Med Biol 261:211–244
Pisarev MA 1985 Thyroid autoregulation. J Endocrinol Invest 8:475– 484
Pommier J, Dème D, Nunez J 1973 Effect of iodide concentration on thyroxine
synthesis catalysed by thyroid peroxidase. Eur J Biochem 37:406 – 414
Gorin Y, Ohayon R, Carvalho DP, et al. 1996 Solubilization and characterization of a thyroid Ca2⫹-dependent and NADPH-dependent K3Fe(CN)6 reductase. Relationship with the NADPH-dependent H2O2 generating system.
Eur J Biochem 240:807– 814
Eng PHK, Cardona GR, Fang SL, et al. 1999 Escape from the acute WolffChaikoff effect is associated with a decrease in thyroid sodium/iodide
symporter messenger ribonucleic acid and protein. Endocrinology 140:
3404 –3410