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Am J Physiol Endocrinol Metab
278: E692–E699, 2000.
Stimulation by iodide of H2O2 generation
in thyroid slices from several species
B. CORVILAIN,1,2 L. COLLYN,1 J. VAN SANDE,1 AND J. E. DUMONT1
de Recherche Interdisciplinaire en Biologie Humaine et Nucléaire,
School of Medicine, and 2Department of Endocrinology, Erasme University Hospital,
Free University of Brussels, 1070 Brussels, Belgium
1Institut
Wolff-Chaikoff effect; thyroid hormone synthesis; iodide toxicity; pentose phosphate pathway
THYROID HORMONE SYNTHESIS in the thyroid requires
iodide, thyroglobulin, and an oxidation system to oxidize iodide and to iodinate tyrosyl groups in thyroglobulin and couple them into iodothyronines (13, 23). This
oxidation system is constituted by a thyroperoxidase that
oxidizes iodide in the presence of H2O2 and an ill defined
H2O2 generating system using NADPH as coenzyme.
The metabolism of iodide in the thyroid gland makes
the most efficient use of an iodine supply that is often
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E692
scarce and intermittent. But the thyroid also has
adaptation mechanisms that reduce iodine metabolism
when the supply is abundant, thus avoiding thyrotoxicosis. These include direct inhibitory effects of iodide in
the thyroid itself, and inhibition by iodide of its own
organification (Wolff-Chaikoff effect), its transport, thyroid hormone secretion, cAMP formation in response to
thyroid-stimulating hormone (TSH), and several other
metabolic steps (29). We also previously observed an
inhibitory effect of iodide on H2O2 generation in response to various agonists. Because, when iodide supply is sufficient, H2O2 generation is the limiting step for
iodide organification, it was concluded that the WolffChaikoff effect was caused by the inhibitory effect of
iodide on H2O2 generation (11). However, in unstimulated dog thyroid slices, a small inconstant stimulatory
effect of iodide on H2O2 generation was observed. Until
then, the only stimulatory effect reported with iodide
was on [1-14C]glucose oxidation in some species [sheep
(14, 18), cattle (17), and to a lesser extent dogs (18, 25)].
This effect was attributed to an increase in the NADP1/
NADPH ratio, but its physiological significance was
unknown (16).
The present study was initiated to determine whether
the small iodide stimulatory effect on H2O2 generation
previously observed in dog thyroid was also observed in
other species. Because we have demonstrated recently
in dog and human thyroid that the activity of the
pentose phosphate pathway is controlled by the rate of
NADPH oxidation, which itself depends on the rate of
H2O2 generation (10, 12), we investigated mainly species in which a stimulatory effect of iodide on [1-14C]glucose oxidation had been demonstrated previously. We
analyzed this effect for various iodide concentrations
and various kinetics to compare those conditions with
those in which the classical inhibitory effect on H2O2
generation is observed. Because it is well known that
iodide depletion may influence thyroid response to
iodide (6), we also studied in dog thyroids whether the
size of the iodine pool, i.e., previous iodine supply,
might influence the H2O2 response to iodide. The
results suggest that iodide stimulates the generation of
its cosubstrate H2O2, which limits its oxidation and
thus its own metabolism.
MATERIALS AND METHODS
Products. Horseradish peroxidase type II, homovanillic
acid, 12-O-tetradecanoylphorbol 13-acetate (TPA), and bovine
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Corvilain, B., L. Collyn, J. Van Sande, and J. E.
Dumont. Stimulation by iodide of H2O2 generation in thyroid
slices from several species. Am J Physiol Endocrinol Metab
278: E692–E699, 2000.—The regulation of thyroid metabolism by iodide involves numerous inhibitory effects. However,
in unstimulated dog thyroid slices, a small inconstant stimulatory effect of iodide on H2O2 generation is observed. The
only other stimulatory effect reported with iodide is on
[1-14C]glucose oxidation, i.e., on the pentose phosphate pathway. Because we have recently demonstrated that the pentose phosphate pathway is controlled by H2O2 generation, we
study here the effect of iodide on basal H2O2 generation in
thyroid slices from several species. Our data show that in
sheep, pig, bovine, and to a lesser extent dog thyroid, iodide
had a stimulatory effect on H2O2 generation. In horse and
human thyroid, an inconstant effect was observed. We demonstrate in dogs that the stimulatory effect of iodide is greater in
thyroids deprived of iodide, raising the possibility that differences in thyroid iodide pool may account, at least in part, for
the differences between the different species studied. This
represents the first demonstration of an activation by iodide
of a specialized thyroid function. In comparison with conditions in which an inhibitory effect of iodide on H2O2 generation is observed, the stimulating effect was observed for lower
concentrations and for a shorter incubation time with iodide.
Such a dual control of H2O2 generation by iodide has the
physiological interest of promoting an efficient oxidation of
iodide when the substrate is provided to a deficient gland and
of avoiding excessive oxidation of iodide and thus synthesis of
thyroid hormones when it is in excess. The activation of H2O2
generation may also explain the well described toxic effect of
acute administration of iodide on iodine-depleted thyroids.
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STIMULATION BY IODIDE OF H2O2 GENERATION IN THYROID
IP3) over the sum of radioactivity in inositol phosphates and
phosphatidylinositol.
Results are expressed as the means 6 SE of the results of
$3 pooled experiments, and in each experiment, measurements were made at least on duplicate incubation flasks. The
biochemical measurements, but not the relative effects of the
agents tested, may vary greatly from one tissue to another.
Therefore, to pool the results of different experiments, the
effects of the agonists were expressed as a percentage of the
control value. For statistical analysis, the results of individual experiments were pooled and analyzed by ANOVA
followed by Dunnett’s or Bonferroni multiple comparison
tests. When results of separate experiments were pooled to
estimate stimulation or inhibition factors, statistical significance was calculated on the logarithms of the ratios of
experimental to control in each experiment. This procedure
has been shown to normalize the distribution of metabolic
variables in thyroid (15).
RESULTS
Effect of iodide on H2O2 generation in human, horse,
sheep, and dog thyroid slices. The initial experiments
were performed on human thyroid slices and thyroid
from three animal species. Various concentrations of
iodide were added in the preincubation and incubation
media for experiments performed on human, horse, and
dog thyroid and only in the incubation medium for
those performed on sheep thyroid. As shown in Table 1,
the iodide effect on basal H2O2 generation depends on
the species considered. In human and horse thyroid
slices, iodide had only a small and inconstant stimulatory effect on H2O2 generation. In human thyroid slices,
the basal production of H2O2 was very low and frequently below the detection limit. Only experiments in
which the basal production of H2O2 was estimated to be
reliable were taken into account (.20 ng H2O2 · 100 mg
wet wt21 · 120 min21). Stimulation by iodide was significant in two experiments out of five with a maximal
stimulation of 210% for an iodide concentration of 1025
M. However, when all the experiments were pooled, the
effect of iodide on H2O2 generation was not statistically
significant whatever the concentration used. The same
level of stimulation without statistically significant
results was obtained in horse thyroid slices. In dog
thyroid slices, iodide had a small but constant stimulatory effect on H2O2 generation. This effect was maximal
for an iodide concentration of 1025 M but remained
Table 1. Effect of iodide on H2O2 production in horse, dog, and sheep thyroid slices
H2O2 Production, %Control
Addition
Human
Horse
Dog
Sheep
None
KI 1026 M
KI 1025 M
KI 1024 M
KI 1023 M
KI 1025 M 1 MMI
TSH 10 mU/ml
100 (5)
132.7 6 10.1 (5)
135.4 6 19.2 (5)
110.8 6 22.4 (5)
72.0 6 18.4 (4)
ND
718 6 139.0 (5)
100 (5)
107.0 6 5.1 (4)
130.1 6 8.2 (5)
130.4 6 28.2 (4)
119.7 6 26.4 (5)
89.5 6 5.4 (4)
ND
100 (5)
114.2 6 11.2 (5)
160.7 6 19.1 (5)*
158.1 6 11.8 (5)*
ND
90.8 6 4.1 (5)
714.8 6 118.2 (5)
100 (8)
112.5 6 10.2 (7)
185.7 6 26.7 (8)*
192.0 6 30.5 (6)*
ND
90.0 6 11.2 (7)
356.4 6 89.9 (8)
Results are means 6 SE; number of experiments for each condition is given in parentheses. Length of incubation was 2 h for human, horse,
and sheep and 1 h for dog thyroid slices. Basal values for H2O2 production for 100 mg wet wt (wwt) ranges were 21–61, 1040–2741, 261–731,
and 43–1426 ng for human, horse, dog, and sheep thyroid slices, respectively. MMI, methimazole 1024 M; ND, not done; 100, 100% of control
value. * P , 0.05 vs. basal. Thyroid-stimulating hormone (TSH)-stimulated values are given only for comparison.
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TSH were purchased from Sigma Chemical (St. Louis, MO),
carbamylcholine from K and K (Plainview, NY), ionomycin
from Calbiochem-Berhing (La Jolla, CA), and forskolin (FSK)
from Hoechst Pharmaceuticals (Bombay, India). All other
reagents were of the purest grade commercially available.
Tissues. Human thyroid tissue was obtained from euthyroid patients undergoing lobectomies for resection of solitary
cold nodules. Only the healthy normal-looking nonnodular
tissue was used within 10–30 min of surgical resection.
Sheep, pig, bovine, and horse thyroids were obtained from
freshly killed animals at a local slaughterhouse. Dog thyroids
were obtained from dogs 610 kg otherwise used for cardiological experiments. Dogs with decreased iodide pool were obtained and treated for 6 wk with propylthiouracil (2 3 150
mg/day), strumazol (2 3 80 mg/day), and NaClO4 (2 3 1
g/day). Not to interfere with iodide metabolism during the
experiment, treatment with propylthiouracil and strumazol
was stopped 48 h before and the treatment with NaClO4 24 h
before the experiment. On the day of the experiment, dogs
were anesthetized with pentobarbital, and the thyroid lobes
were resected. Thyroids were cut into thin slices of ,50 mg
wet weight (wet wt) with a Stadie-Riggs microtome.
H2O2 determinations. Slices were preincubated in KrebsRinger HEPES (KRH) medium supplemented with 8 mM
glucose and 0.5 g/l of BSA and then transferred to fresh
medium containing 0.1 mg/ml horseradish peroxidase type II,
440 µM homovanillic acid, and the tested agonists. The
fluorescence of the medium was measured 90 min later except
when stipulated otherwise (315 and 425 nm excitation and
emission wavelengths respectively) (2).
cAMP measurements. Slices were preincubated at 37°C for
1 h under an atmosphere of 95% O2-5% CO2 (vol/vol) in 2 ml of
Krebs-Ringer bicarbonate (KRB) supplemented with 8 mM
glucose and 0.5 g/l of BSA. For the test incubation of 1 h,
medium was supplemented with 100 µM Ro 20–1724, a
cAMP-specific phosphodiesterase inhibitor (22). cAMP was
measured according to Brooker et al. (7).
Inositol phosphate measurements. Slices were preincubated for 4 h at 37°C in a medium similar to cAMP measurements but with 20 µCi/ml 3H-labeled inositol (specific activity
10–20 Ci/mmol, Amersham). The slices were then transferred
to fresh unlabeled medium to which 10 mM lithium was
added after 15 min. After an additional 5 min, the tested
agents were added. The [3H]inositol phosphates were isolated
by stepwise chromatography on a AG1-X8 resin (formate
form, 100–200 µm mesh; Biorad, Watford, UK) Incorporation
of [3H]inositol into the total phosphatidylinositide pool was
quantitated after chloroform/methanol extraction of lipids
from the pellet (3). Results are expressed as the percentage of
radioactivity incorporated in inositol phosphates (IP1 1 IP2 1
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STIMULATION BY IODIDE OF H2O2 GENERATION IN THYROID
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weak compared with that obtained with 10 mU/ml
TSH. In sheep thyroid slices, an iodide concentration of
1024 M nearly doubled H2O2 generation, which reached
.50% of the value obtained with 10 mU/ml TSH. In all
species, this stimulation was prevented by adding
methimazole (1024 M) to the preincubation and incubation media.
Effect of increasing time of preincubation with iodide
on H2O2 generation. The data presented in Table 1
clearly show that the effect of iodide on H2O2 generation
is dependent on its concentration and on the species
studied. Because in human thyroid the basal level of
H2O2 generation was too frequently below the detection
limit, further experiments were performed on other
species. The following set of experiments was performed to evaluate the kinetics of this effect in the three
previous species and in two others (pig and bovine). The
concentration of iodide for which the maximal stimulatory effect had been observed was used for those
experiments (1025 M for dog thyroid experiments and
1024 M for other species). In all species but horse,
preincubation with iodide increased its stimulatory
effect on H2O2 generation. In horse thyroid slices, even
a preincubation of 4 h did not increase its stimulatory
effect (data not shown). In bovine thyroid slices, a
preincubation of 4 h results in a quite undetectable
basal level of H2O2 generation. The maximal measurable stimulation was therefore obtained after 2 h of
preincubation and reached 444 6 220% for an iodide
concentration of 1024 M (P , 0.05). In three species
(dog, pig, and sheep) the kinetics of the iodide effect on
H2O2 generation were analyzed in more detail (Fig. 1).
In dog and pig thyroid slices, the maximal effect was
nearly reached after 1 h of preincubation with iodide.
Further increase of the length of preincubation resulted in only a minor additional increase of H2O2
generation. After 4 h of preincubation, the maximal
effect reached 186.9 6 18.3% of the control value in dog
thyroid slices and 408 6 58% of the control in pig
thyroid slices. In sheep thyroid slices, the kinetic of
preincubation with iodide showed a progressive increase of H2O2 generation with the time of preincubation. H2O2 generation after 4 h of preincubation reached
606 6 76% of the control value. Compared with the
stimulation obtained by TSH, the iodide stimulatory
effect is weak in dog thyroids and of the same order of
magnitude or even more potent in pig and sheep
thyroid, respectively. As previously demonstrated, iodide inhibited H2O2 generation in dog TSH-stimulated
slices (11), whereas in sheep and pig TSH-stimulated
slices, iodide induced a further increase of H2O2 generation. Both effects were reversed in the presence of
methimazole.
Effect of increasing iodide concentrations on H2O2
generation in dog and pig thyroid slices after preincubation of 4 h in presence of iodide. Because we had shown
that increasing the time of preincubation may increase
the stimulatory effect of iodide, we studied the effect of
various concentrations of iodide after a preincubation
of 4 h. Contrary to observations made with 1 h of
preincubation, in dog thyroid, after 4 h of preincuba-
Fig. 1. Effect of increasing length of preincubation with iodide on
H2O2 generation in pig (A), sheep (B), and dog (C) thyroid slices. All
slices were preincubated for 4 h. Iodide was added in preincubation
media after various time intervals. Ranges of basal values for H2O2
generation for 100 mg wet wt were (in ng) 48–170 for pig, 57–67 for
sheep, and 182–601 for dog thyroid slices, respectively. * P , 0.05 vs.
control; ** P , 0.01 vs. control. Conditions detailed on bottom of
figure are related to incubation. Methimazole (MMI, 1024 M) was
added during preincubation and incubation. Open bars, no KI in
preincubation or incubation; other bars, various times of KI presence
in preincubation: cross-hatched, 0 h; hatched, 1 h; back-hatched, 2 h;
vertical, 4 h.
STIMULATION BY IODIDE OF H2O2 GENERATION IN THYROID
Fig. 2. Effect of increasing iodide concentration on H2O2 generation
in pig (A) and dog (B) thyroid slices. After preincubation of 4 h in
presence of iodide, slices were incubated for 90 min with same
concentration of iodide. Ranges of basal values for H2O2 production
for 100 mg wet wt were (in ng) 33–60 for pig and 122–345 for dog
thyroid slices, respectively. * P , 0.05 vs. control; ** P , 0.01 vs.
control. MMI, 1024 M
Fig. 3. Effect of increasing length of preincubation with iodide (3 3
1024 M) on H2O2 generation in dog thyroid slices. All slices were
preincubated for 6 h. Control slices were preincubated and incubated
without iodide. Iodide was added in preincubation and incubation
media. Length of preincubation with iodide ranged from 0 h (iodide
present only during incubation) to 6 h. Basal values for H2O2
production for 100 mg wet wt ranged from 45 to 146 ng. * P , 0.05 vs.
control; ** P , 0.01 vs. control.
biphosphate (PIP2) cascade. Ionomycin, a divalent cation ionophore that allows extracellular Ca21 to enter
the cells, and TPA, a pharmacological probe for diacylglycerol-regulated protein kinase C, were used to assess
the separate effects of the activation of each branch of
the PIP2 cascade. In sheep thyroid slices, activation of
the PIP2 cascade by carbamylcholine, enhancement
of Ca21 intracellular levels by ionophore, and activation
of protein kinase C by TPA all stimulated H2O2 generation. Conversely, the agents stimulating the cAMP
cascade exerted an insignificant inhibition on H2O2
generation. TSH had a biphasic effect, inhibiting H2O2
generation at 1 mU/ml and increasing it at 10 mU/ml.
Such biphasic effects previously observed in human
thyroid slices corresponded to the stimulation of the
cAMP cascade at the lower concentration of TSH and of
the PIP2-phospholipase C (PLC) cascade at the higher
concentration, respectively (10). In pig thyroid, H2O2
generation was stimulated by low concentration of TSH
(1 mU/ml), an insignificant stimulating effect was
observed with FSK, and no effect was observed with
carbamylcholine. In dog thyroid slices, as previously
described (12), H2O2 generation was stimulated by both
the cAMP cascade and the PIP2-PLC cascade (Table 2).
Effect of iodide on PIP2 cascade in sheep, pig, and dog
thyroid slices. Because H2O2 generation in sheep and
pig thyroid is controlled by the PIP2 cascade, we studied
here the effect of iodide on the [3H]inositol phosphate
generation in those two species. Iodide (1024 M) in the
preincubation and incubation media did not modify
[3H]inositol phosphate generation, whereas the effect of
iodide on H2O2 generation in sheep and pig thyroid was
clearly demonstrated. In the same set of experiments,
10 mU/ml TSH in pig and 1025 M carbachol in sheep
were moderate and potent stimulators, respectively, of
the PIP2 cascade. Even though both cascades positively
control H2O2 generation in dog thyroid slices, we decided to test the effect of iodide only on the PIP2
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tion, H2O2 generation exhibited a biphasic curve in the
presence of an increasing concentration of iodide (Fig.
2). As already shown in Fig. 1, a significant stimulatory
effect of iodide could be obtained for an iodide concentration of 1025 M. However, for a higher concentration of
iodide, an inhibitory effect on H2O2 generation was
observed that became statistically significant for an
iodide concentration of 1023 M. This effect was also
reversed in the presence of methimazole. The situation
was quite different in pig thyroid slices, where no
inhibitory action of iodide after a 4-h preincubation was
observed whatever the concentration of iodide used.
Effect of increasing time of preincubation with high
concentration of iodide on H2O2 generation in dog
thyroid slices. In the presence of high concentrations of
iodide (3 3 1024 M), H2O2 generation exhibited a
biphasic curve when plotted against the time of preincubation (Fig. 3). The production of H2O2 was maximal
after 1 h of preincubation with iodide (136 6 13% of the
basal level) and then decreased to reach a maximal
inhibition after 6 h (55 6 10% of the basal level).
Effects of agents controlling either phosphatidylinositol P2 or cAMP cascade on generation of H2O2 in sheep,
pig, and dog thyroid slices. FSK was chosen to test the
role of the cAMP cascade. Carbamylcholine was chosen
to test the role of the whole phospatidylinositol 4,5
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STIMULATION BY IODIDE OF H2O2 GENERATION IN THYROID
Table 2. Effects of agents controlling either PIP2 or
cAMP cascade on generation of H2O2 in sheep,
pig, and dog thyroid slices
H2O2 Production, %Control
Sheep
Pig
Dog
None
TSH
(1 mU/ml)
TSH
(10 mU/ml)
Cchol
(1025 M)
FSK (1025 M)
Ionomycin
(1026 M)
TPA
(531026 M)
100 (14)
100 (5)
100 (10)
286.4671.7 (5)†
367.9684.5 (3)†
85.867.7 (9)
189.7617.5 (9)†
711.36108.9 (5)† 607.96113.8 (5)†
341.8658.9 (12)† 116.4616.9 (5)
72.969.6 (9)
131.0627.4 (5)
979.26354.7 (6)†
533.5690 (5)†
608.06123.3 (9)† 922.76182.0 (5)† 772.96254.9 (5)†
238.4626.9 (9)†
615.3654.7 (5)†
5706157.2 (5)†
Results are means 6 SE; number of experiments for each condition
is given in parentheses. Ranges of basal values for H2O2 production
for 100 mg wwt/90 min (in ng) were 53–1105 in sheep, 48–209 in pig,
and 178–2699 in dog thyroid slices, respectively. † P , 0.01 vs. basal.
PIP2, phosphatidylinositol 4,5 biphosphate; Cchol, carbamylcholine;
FSK, forskolin; TPA, 12-O-tetradecanoylphorbol 13-acetate; 100,
100% of control value.
cascade because it had already been demonstrated in
this species that iodide does not stimulate basal cAMP
generation (26); iodide does not stimulate the PIP2
cascade either (Table 3).
Effect of iodide on cAMP cascade in sheep thyroid
slices. Iodide (1025 M) added during the 1-h preincubation and during the incubation decreased basal cAMP
generation from 85.7 6 10.1 pmol · 100 mg wet wt21 · 60
min21 in control slices to 61.3 6 6.1 pmol · 100 mg wet
wt21 · 60 min21 in iodide-treated slices (P , 0.05).
Effect of thyroid iodine pool on H2O2 generation
induced by iodide. Dogs with depleted thyroid iodine
Table 3. Effects of iodide, TSH, and Cchol on inositol
phosphate generation in sheep, pig, and dog
thyroid slices
DISCUSSION
Thyroid hormone synthesis is characterized by the
requirement of a scarce substrate (iodide) and a toxic
cofactor (H2O2). The thyroid cell generates large
amounts of H2O2, which is toxic for any cell because of
its transformation into various O2
2 -derived free radicals
(9, 19, 28). A process of apoptosis has also recently been
described in vitro when the thyrocyte is exposed to
H2O2 (24). The thyroid cell protects itself by regulating
Addition
KI
(1024 M)
Cchol
(1025 M)
H2O2 , %Control
IP, %Control
313 6 102
119 6 7
707 6 508
1,158 6 59†
H2O2 , %Control
IP, %Control
425 6 108
104 6 11
H2O2 , %Control
IP, %Control
154 6 25.5
99 6 4
TSH
(10 mU/ml)
Sheep
Pig
505 6 185
358 6 43*
Dog
800 6 182
948 6 225†
Results are means 6 SE; n 5 3 for sheep experiments and n 5 2 for
pig and dog experiments. * P , 0.05 vs. basal; † P , 0.01 vs. basal.
Ranges of basal values for H2O2 generation (in ng/100 mg wet wt)
were 52–251 in sheep, 130–141 in pig, and 133–1148 in dog thyroid
slices, respectively. Ranges of basal values for inositol phosphate (IP)
generation were (in cpm/100 mg wet wt) 384–1054 in sheep, 2700–
2960 in pig, and 13349–21561 in dog thyroid slices, respectively.
H2O2 stimulated values are given only for comparison.
Fig. 4. Effect of thyroid iodine pool on H2O2 generation induced by
iodide. Ranges of basal values for H2O2 production for 100 mg wet wt
were 261–731, 268–838, and 166–244 ng for control thyroids and
thyroids with decreased and increased iodine pool, respectively. * P ,
0.05 vs. control; ** P , 0.01 vs. control.
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Addition
pool were obtained as described in MATERIALS AND
Other dogs were obtained and treated with 1
ml lipiodol intramuscularly (480 mg iodine/ml) 10 days
before the experiment to increase the thyroid iodine
pool. As shown in Fig. 4, the stimulatory effect of iodide
on H2O2 generation was dependent on the iodine status
of the gland. In comparison with control thyroids, in
iodine-depleted thyroids the H2O2 response to iodide
was stronger and more sensitive, starting at 1026 M,
peaking at 3 3 1026 M, and then decreasing. The
maximal stimulation was 322 6 55% of the control
value and nearly reached the value obtained with 10
mU/ml TSH (478 6 168% of the control value). In dogs
treated with lipiodol, no effect whatever of iodide was
observed with the iodide concentration used.
Effect of washing with or without methimazole on
stimulatory effect of iodide on H2O2 generation in pig
thyroid slices. All the slices were preincubated for 2 h in
the presence of iodide (1024 M) and then washed for 15
min in KRH to remove excess iodide. H2O2 generation
was determined either immediately or after additional
washing with or without methimazole for a period of
time ranging from 1 to 20 h. When the washing was
done without methimazole, the stimulatory effect of
iodide decreased with time but was still present after 5
h. In the presence of methimazole, the effect of iodide
decreased more rapidly and disappeared after 3 h. After
a washing period of 20 h, a strong inhibitory effect of
iodide on H2O2 generation could be observed. This effect
is partially relieved if the washing has been done in the
presence of methimazole (P , 0.01) (Fig. 5).
METHODS.
STIMULATION BY IODIDE OF H2O2 GENERATION IN THYROID
its production of H2O2 according to what is required for
thyroid hormone synthesis. It is able to increase its
H2O2 production up to 10-fold in the presence of a high
concentration of TSH or other agents that stimulate
thyroid metabolism. H2O2 not used for thyroid hormone
synthesis is destroyed in the thyroid, as in other cells,
by several enzymes of the glutathione peroxidase family and by catalase (5). We had already demonstrated
that large concentrations of iodide inhibited the H2O2
generation stimulated by various agonists, but until
now, a possible action of iodide on basal H2O2 production had never been thoroughly investigated. All the
effects of iodide on thyroid metabolism described so far
are inhibitory, apart from the effect on glucose metabolism. Actually, various data had already demonstrated
a stimulatory effect of iodide on basal [1-14C]glucose
oxidation in several species [strong in sheep and bovine
thyroid (18, 14, 17), weaker in dog thyroid (18, 25)]. The
present data establish that iodide can also stimulate
H2O2 generation in several species. This observation is
consistent with our previous data showing that
[1-14C]glucose oxidation in dog and human thyroids is
controlled by the rate of H2O2 generation (12, 10). The
magnitude of the iodide effect was greatly dependent on
the species under study and the experimental conditions. As expected, sensitivity to the effect of iodide on
H2O2 generation was maximal in sheep, the species
known to be the most sensitive to its effect on [1-14C]glucose oxidation. The stimulatory effect was also strong
in pig and bovine thyroid but weaker in human, dog,
and horse thyroid. The stimulatory effect of iodide on
H2O2 generation was prevented by methimazole, suggesting that it is mediated by an oxidative derivative of
iodide. Apart from the fact that both require a functional iodide oxidation system, the stimulatory and
inhibitory effects of iodide are clearly distinct; they are
species dependent, observable in different experimental conditions, and have an opposite physiological meaning.
In some species (pig and sheep), the stimulatory
effect of iodide predominates, and demonstration of the
inhibitory effect requires special experimental procedures such as an increased incubation time. In those
species, the strong iodide stimulation on H2O2 generation further increases the effect of TSH. Conversely, in
other species (dog and human), the inhibitory effect
predominates and the stimulatory effect of iodide is
weaker. In these species, iodide inhibits the effect of
TSH on H2O2 generation, as demonstrated here and
previously (10, 12). This inhibition was attributed to
the strong inhibitory effect of iodide on the activation of
the cAMP cascade by TSH upstream and downstream
of cAMP (10, 12). In species where both effects coexist,
the inhibitory effect is observed earlier and for lower
iodide concentrations. In dog thyroid, for a given preincubation time with iodide, iodide can stimulate or
inhibit basal production of H2O2 according to its concentration. In the same experimental system, we had
previously shown that high concentrations of iodide
(.1024 M) are needed to demonstrate the WolffChaikoff effect (11), the same as the concentration
needed here to inhibit the basal production of H2O2 and
a concentration 100 times higher than the concentration needed to observe a stimulatory effect. Finally, in
dog and pig thyroid, the first stimulatory effect is
followed much later by an inhibition. In dog thyroid,
iodide can stimulate or inhibit basal production according to the length of preincubation with iodide. In pig
thyroid slices, when the stimulatory effect of iodide is
initiated, it persists after 5 h of washing in the absence
of iodide. However, after 20 h of washing, an inhibitory
effect can be observed, suggesting a late synthesis of an
inhibitory compound. Because the inhibitory effect is
partially relieved when the washing is done in the
presence of methimazole, this suggests that the synthesis of this inhibitory compound requires a functional
peroxidase.
The physiological role of the stimulatory and inhibitory effects is also different. Inhibition by excess iodide
of H2O2 generation and, therefore, of iodide oxidation
and thyroid hormone synthesis is a rapid mechanism to
prevent excessive thyroid hormone secretion. It precedes the later classical negative feedback mechanism
of excess thyroid hormones on TSH secretion and
thyroid stimulation. On the other hand, activation of
H2O2 generation at low concentrations of iodide, especially in iodide-deficient animals, will tie the generation of the toxic but necessary H2O2 with the availability of substrate and thus could represent a remarkable
adaptation mechanism. Activation or induction of the
enzymes necessary for the metabolism of a substrate by
the substrate itself is a widespread strategy in bacterial and eukaryote metabolism.
To test this hypothesis we attempted in the current
study to evaluate the extent to which iodide depletion
or repletion may influence the H2O2 response of the
gland to a small iodide load. Compared with control
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Fig. 5. Effect of various times of washing with or without MMI on
stimulatory effect of iodide on H2O2 generation in pig thyroid slices.
All slices were preincubated for 2 h with or without iodide (control
slices). Time of washing ranged from 0 h (no washing) to 20 h. When
washing was done with MMI, it was also added in incubation
medium. Basal values for H2O2 production for 100 mg wet wt were
determined for each time of washing and ranges were 30–194 ng/100
mg wet wt when no washing was done and 49–151, 45–166, 81–216,
28–167, and 40–119 ng after 1, 3, 5, 8, and 20 h of washing,
respectively. Statistical comparisons were made for each time interval with corresponding control (same time of washing and incubation
without iodide). * P , 0.05 vs. control; ** P , 0.01 vs. control. BME,
Eagle’s basal medium.
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STIMULATION BY IODIDE OF H2O2 GENERATION IN THYROID
iodide stimulatory effect on [1-14C]glucose oxidation
actually results from an increased H2O2 generation
through NADPH oxidation by the still unknown H2O2
generating system. As for [1-14C]glucose oxidation, great
interspecies variation exists. The stimulatory effect
was greater in sheep, pig, and bovine than in dog
thyroids and inconstant in human and horse thyroids.
This may be due to either genetic or environmental
factors; however, because we demonstrated in dog
thyroid that iodide pool greatly influences the H2O2
response to iodide, it seems quite possible that different
diets may account, at least in part, for differences
between species. Whatever the mechanism involved,
the stimulatory action of iodide on H2O2 generation
makes physiological sense. In the absence of iodide,
there is no need to generate toxic H2O2. Conversely, in
its presence, increased H2O2 generation stimulates a
more efficient oxidation of iodide. When compared with
the inhibitory actions of iodide, this effect is obtained
earlier and for lower concentrations, suggesting a physiological role at least as important as that attributed to
the classical inhibitory effects. All our results can be
viewed teleologically as a means of gearing H2O2 generation to iodide supply. However, when the iodine thyroid
pool is or becomes sufficient, as in dogs treated with
lipiodol or after long preincubation time with iodide or
for high concentration of iodide, the thyroid protects
itself against excess iodine organification by decreasing
H2O2 production, in keeping with the classical WolffChaikoff effect.
We thank C. Maenhaut for organization of the in vivo treatments
and C. Massart for excellent technical help.
This work was supported by the Fonds de la Recherche Scientifique Médicale, the Ministère de la Politique Scientifique (PAI) and
the Fondation Tournai-Solvay.
B. Corvilain is a fellow of the Erasmus Foundation (ULB, Brussels).
Address for reprint requests and other correspondence: B. Corvilain, IRIBHN, School of Medicine, Campus Erasme, Bat C, Route
de Lennik 808, 1070 Brussels, Belgium (E-mail: [email protected]).
Received 28 May 1999; accepted in final form 12 November 1999.
REFERENCES
1. Belshaw BE and Becker DV. Necrosis of follicular cells and
discharge of thyroidal iodine induced by administering iodide to
iodine-deficient dogs. J Clin Endocrinol Metab 36: 466–474,
1973.
2. Benard B and Brault J. Production de peroxide dans la
thyroı̈de. Union Med Can 100: 701–705, 1971.
3. Berridge MJ. Rapid accumulation of inositol trisphosphate
reveals that agonists hydrolyse polyphosphoinositides instead of
phosphatidylinositol. Biochem J 212: 849–858, 1983.
4. Bjorkman U and Ekholm R. Generation of H2O2 in isolated
porcine thyroid follicles. Endocrinology 115: 392–398, 1984.
5. Björkman U and Ekholm R. Hydrogen peroxide degradation
and glutathione peroxidase activity in cultures of thyroid cells.
Mol Cell Endocrinol 111: 99–107, 1995.
6. Brabant G, Bergmann P, Kirsh CM, Kohrle J, Hesch RD,
and von zur Muhlen A. Early adaptation of thyrotropin and
thyroglobulin secretion to experimentally decreased iodine supply in man. Metabolism 41: 1093–1096, 1992.
7. Brooker G, Harper JF, Terasaki WL, and Moylan RD. RIA of
cyclic AMP and cyclic GMP. Adv Cyclic Nucleotide Res 10: 1–33,
1979.
8. Contempre B, Denef JF, Dumont JE, and Many MC. Selenium deficiency aggravates the necrotizing effects of a high
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.6 on May 7, 2017
dogs, the effect of iodide on H2O2 generation was
strongly increased in thyroid with a decreased iodine
pool but was absent in thyroid with an increased iodine
pool. This suggests that thyroid iodide content is a
major determinant of the H2O2 response of the gland to
a small iodide load. However, we cannot exclude the
possibility that other mechanisms may play a role,
because in our model, iodine deprivation coexists with
hypothyroidism and chronic stimulation by TSH.
We therefore addressed the question of the mechanisms involved in this action of iodide. As a first step,
we defined, by using various agonists, which cascade is
implicated in the control of H2O2 generation in sheep
and pig thyroid. The present data establish that in
sheep thyroid, as already observed in human (10), calf
(20), and pig thyroid (4), H2O2 generation is activated
only by the PIP2 cascade, whereas in these species the
cAMP cascade has an unimportant (4) or even an
inhibitory effect on H2O2 generation (10, 20). As already
described, both cascades activate H2O2 generation in
dog thyroid slices (12). We could not observe any effect
of iodide on the PIP2 cascade in pig, sheep, and dog
thyroid. It is unlikely, therefore, that the action of
iodide on basal H2O2 generation is mediated by an
activation of the PIP2 cascade. Iodide inhibited basal
cAMP generation in sheep thyroid slices. Because
cAMP exerts a small negative control on H2O2 generation in sheep thyroid, iodide may partly stimulate H2O2
generation by relieving this negative control. Even if
this mechanism possibly plays a role in sheep thyroid,
it cannot be involved in dog thyroid, in which the cAMP
cascade positively controls H2O2 generation, or in pig
thyroid, where cAMP cascade does not exert a negative
control. It is possible that other untested metabolic
pathways could be involved; therefore, the classical
metabolic cascades are not involved in the stimulatory
effect of iodide, contrary to what was observed previously for the inhibitory effect of iodide on H2O2 generation that obtains both at the level of intracellular signal
generation and at the level of the H2O2 generating
system (10, 12). This effect of iodide, like many others,
is inhibited by methimazole, which suggests that the
effect is not direct but is secondary to the generation of
an oxidized form of iodine, previously called X-I (27).
Conversely, it is also possible that in vivo, the increase in H2O2 synthesis induced by iodide in iodinedepleted thyroid may have a toxic role in the cell. A
necrosis of follicular cells was already described after
administration of iodide to iodine-deficient dogs but not
to control dogs (1). A necrotizing effect of iodide was also
described in iodine-deficient rats and mice (8, 21). The
toxicity of iodide was aggravated in cases of selenium
deficiency, a circumstance in which defenses against
H2O2 are reduced due to a decreased activity of glutathione peroxidase (8). Our data are in keeping with the
hypothesis that some of these toxic effects induced by
iodide in iodide-deficient thyroids may be partly related
to the toxicity of H2O2.
In conclusion, iodide stimulates H2O2 generation in
several species. Our experiments suggest that in dog,
bovine, and sheep thyroid, the previously observed
STIMULATION BY IODIDE OF H2O2 GENERATION IN THYROID
9.
10.
11.
12.
14.
15.
16.
17.
18.
19. Jonas SK, Riley PA, and Willson RL. Hydrogen peroxide
cytotoxicity. Biochem J 264: 651–655, 1989.
20. Lippes HA and Spaulding SW. Peroxide formation and glucose
oxidation in calf thyroid slices: regulation by protein kinase-C
and cytosolic free calcium. Endocrinology 118: 1306–1311, 1986.
21. Mahmoud I, Colin I, Many MC, and Denef JF. Direct toxic
effect of iodide in excess on iodine-deficient thyroid glands:
epithelial necrosis and inflammation associated with lipofuscin
accumulation. Exp Mol Pathol 44: 259–271, 1986.
22. Miot F, Erneux C, Wells JN, and Dumont JE. The effects of
alkylated xanthines on cyclic AMP accumulation in dog thyroid
slices exposed to carbamylcholine. Mol Pharmacol 25: 261–266,
1984.
23. Nunez J and Pommier J. Formation of thyroid hormones.
Vitam Horm 39: 175–229, 1982.
24. Riou C, Tonoli H, Bernier-Valentin F, Rabilloud R, Fonlupt
P, and Rousset B. Susceptibility of differentiated thyrocytes in
primary cultures to undergo apoptosis after exposure to hydrogen peroxide: relation with the level of expression of apoptosis
regulatory proteins, Bcl-2 and Bax. Endocrinology 140: 1990–
1997, 1999.
25. Tseng FY, Rani CSS, and Field JB. Effect of iodide on glucose
oxidation and 32P incorporation into phospholipids stimulated by
different agents in dog thyroid. Endocrinology 124: 1450–1455,
1989.
26. Van Sande J, Cochaux P, and Dumont JE. Further characterization of the iodide inhibitory effect on the cyclic AMP system in
dog thyroid slices. Mol Cell Endocrinol 40: 181–192, 1985.
27. Van Sande J and Dumont JE. Effects of thyrotropin, PG E1
and iodide on cyclic 38, 58-AMP concentration in dog thyroid slices.
Biochim Biophys Acta 313: 320–328, 1973.
28. Wiseman H and Halliwell B. Damage to DNA by reactive
oxygen and nitrogen species: role in inflammatory disease and
progression to cancer. Biochem J 313: 17–29, 1996.
29. Wolff J. Excess iodide inhibits the thyroid by multiple mechanisms. Adv Exp Med Biol 261: 211–244, 1989.
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.6 on May 7, 2017
13.
iodide dose in iodine deficient rats. Endocrinology 132: 1866–
1868, 1993.
Corvilain B, Contempre B, Longombe AO, Goyens P, Gervy
Decoster C, Lamy F, Vanderpas JB, and Dumont JE.
Selenium and the thyroid: how the relationship was established.
Am J Clin Nutr 57: 244S–248S, 1993.
Corvilain B, Laurent E, Lecomte M, Van Sande J, and
Dumont JE. Role of the cyclic adenosine 38, 58-monophosphate
and the phosphatidylinositol-Ca21 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, 1994.
Corvilain B, Van Sande J, and Dumont JE. Inhibition by
iodide of iodide binding to proteins: the ‘‘Wolff-Chaikoff’’ effect is
caused by inhibition of H2O2 generation. Biochem Biophys Res
Commun 154: 1287–1292, 1988.
Corvilain B, Van Sande J, Laurent E, and Dumont JE. The
H2O2-generating system modulates protein iodination and the
activity of the pentose phosphate pathway in dog thyroid.
Endocrinology 128: 779–785, 1991.
DeGroot LJ. Current views on formation of thyroid hormones.
N Engl J Med 272: 243–250, 1965.
Dumont JE. Effet in vitro de l’iodure et de la diiodotyrosine sur
le shunt hexosemonophosphate thyroı̈dien. Compt Rend Soc
Biol(Paris) 155: 2225–2228, 1961.
Dumont JE. Action de l’hormone thyréotrope sur le métabolisme énergétique du tissu tyroı̈dien: effets in vitro de l’hormone.
Bull Soc Chim Biol 46: 1131–1134, 1964.
Dumont JE. The action of thyrotropin on thyroid metabolism.
Vitam Horm 29: 287–412, 1971.
Green WJ. Further studies of the effects of inorganic iodide on
thyroidal intermediary metabolism in vitro. Endocrinology 79:
1–9, 1966.
Jarrett R and Field JB. A comparison of the effects of
thyrotropin and sodium iodide on the oxidation of glucose-1-14C
and the levels of triphosphopyridine nucleotides in thyroid slices
from several species. Endocrinology 75: 711–715, 1964.
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