Thyroid Ca
2+
/NADPH-dependent H
2
O
2
generation ispartially inhibited
by propylthiouraciland methimazole
Andrea C. Freitas Ferreira, Luciene de Carvalho Cardoso, Doris Rosenthal and Denise Pires de Carvalho
Laborato
´
rio de Fisiologia Endo
´
crina, Instituto de Biofı
´
sica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Brazil
H
2
O
2
generation is a limiting step in thyroid hormone bio-
synthesis. Biochemical studies have confirmed that H
2
O
2
is
generated by a thyroid Ca
2+
/NADPH-dependent oxidase.
Decreased H
2
O
2
availability may be another mechanism
of inhibition of thyroperoxidase activity produced by thio-
ureylene compounds, as propylthiouracil (PTU) and
methimazole (MMI) are antioxidant agents. Therefore, we
analyzed whether PTU or MMI could scavenge H
2
O
2
or
inhibit thyroid NADPH oxidase activity in vitro. Our results
show that PTU and thiourea did not significantly scavenge
H
2
O
2
. However, MMI significantly scavenged H
2
O
2
at high
concentrations. Only MMI was able to decrease the amount
of H
2
O
2
generated by the glucose–glucose oxidase system.
On the other hand, both PTU and MMI were able to
partially inhibit thyroid NADPH oxidase activity in vitro.As
PTU did not scavenge H
2
O
2
under the conditions used here,
we presume that this drug may directly inhibit thyroid
NADPH oxidase. Also, at the concentration necessary to
inhibit NADPH oxidase activity, MMI did not scavenge
H
2
O
2
, also suggesting a direct effect of MMI on thyroid
NADPH oxidase. In conclusion, this study shows that
MMI, but not PTU, is able to scavenge H
2
O
2
in the
micromolar range and that both PTU and MMI can impair
thyroid H
2
O
2
generation in addition to their potent thyro-
peroxidase inhibitory effects.
Keywords: antithyroid drugs; H
2
O
2
;NADPHoxidase;
thyroid.
The mechanism by which antithyroid drugs, such as
propylthiouracil (PTU) and 1-methyl-2-mercaptoimidazole
or methimazole (MMI), block thyroid hormone biosyn-
thesis has been well studied [1]. Both are known to inhibit
thyroperoxidase (TPO), a key enzyme of thyroid hormone
biosynthesis. Magnusson et al. [2] suggest that inhibition of
TPO by thioureylene drugs occurs through competition
with H
2
O
2
for oxidized iodine, and Davidson et al.[3]
propose that these drugs are able to block iodination by
trapping oxidized iodine. However, the results obtained by
Engler et al. [4] indicate that inactivation of TPO by MMI
and PTU involves a reaction between these drugs and the
oxidized TPO heme group, which is produced by the
interaction between TPO and H
2
O
2
. In addition, Taurog
and Dorris [5] suggest that the inhibition of iodination
produced by PTU involves competition between this drug
and tyrosine residues of thyroglobulin for oxidized iodine.
Decreased H
2
O
2
availability may be an additional
mechanism of inhibition of TPO-catalyzed reactions pro-
duced by thioureylene compounds, as PTU and MMI seem
to be antioxidant agents in vitro [6–8]. Ross et al.[9]have
shown that PTU and MMI do not alter superoxide
synthesis and that PTU does not affect the synthesis of
hydroxyethyl radicals and the generation of hydroxyl
radicals. However, Hicks et al. [8] have demonstrated that
PTU scavenges hydroxyl radicals at the serum free drug
levels commonly attained during PTU therapeutic use. In
addition, Cohen et al. [6] suggest that MMI and thiourea
can cause loss of H
2
O
2
.
H
2
O
2
generation is a limiting step in thyroid hormone
biosynthesis [10,11], and biochemical studies have con-
firmed that H
2
O
2
is generated by a thyroid NADPH
oxidase [12–14]. Two genes probably involved in thyroid
H
2
O
2
generation have recently been cloned [15,16]; they
encode two novel flavoproteins, thyroid oxidases 1 and 2
(ThOX1 and ThOX2), which have a peroxidase domain of
undefined physiological significance. As impaired H
2
O
2
availability decreases thyroid hormone biosynthesis [17],
and the proteins involved in thyroid H
2
O
2
generation have
peroxidase domains, another possible mechanism of action
of PTU and MMI is inhibition of thyroid NADPH oxidase
activity.
The aim of this study was to evaluate a possible H
2
O
2
scavenging effect of PTU and MMI, which may be involved
in their inhibition of TPO, and to analyze whether PTU or
MMI inhibits thyroid NADPH oxidase activity in vitro.
Materials and Methods
Chemicals
NADPH, glucose oxidase (grade I), lyophilized horseradish
peroxidase (HRP, grade I) and glucose oxidase (grade I)
Correspondence to D. Pires de Carvalho, Laborato
´
rio de Fisiologia
Endo
´
crina, Instituto de Biofı
´
sica Carlos Chagas Filho,
Universidade Federal do Rio de Janeiro, CCS, Bloco G,
Ilha do Funda
˜
o, Rio de Janeiro, RJ, Brazil.
Fax: + 55 21 2280 8193, Tel.: + 55 21 590 7147,
E-mail: dencarv@biof.ufrj.br
Abbreviations: PTU, propylthiouracil; MMI, 1-methyl-2-mercapto-
imidazole or methimazole; TPO, thyroperoxidase; HRP, horseradish
peroxidase.
(Received 9 January 2003, revised 10 March 2003,
accepted 14 March 2003)
Eur. J. Biochem. 270, 2363–2368 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03576.x
were purchased from Boehringer (Mannheim, Germany).
Scopoletin, digitonin, cytochrome c, MMI, 6-n-propylthio-
uracyl (PTU), thiocarbamide (thiourea) and FAD were
obtained from Sigma Chemical Co. (St Louis, MO, USA).
CaCl
2
was purchased from Mallinckrodt, and Tris
(hydroxymethyl)aminomethane and H
2
O
2
were from
Merck(RiodeJaneiro,RJ,Brazil).
TPO preparation
TPO was extracted from human thyroid tissue samples
obtained from diffuse toxic goiters during thyroidectomy, as
described by Moura et al. [18] and Carvalho et al.[19].After
cleaning on an ice-cooled glass plate, thyroid tissue samples
(1 g) were minced and homogenized in 3 mL 50 m
M
Tris/
HCl buffer, pH 7.2, containing 1 m
M
KI, using an Ultra-
Turrax homogenizer (Staufen). The homogenate was cen-
trifuged at 100 000 g,4°C for 1 h. The pellet was suspended
in 2 mL digitonin (1%, w/v) and incubated at 4 °Cfor24h
to solubilize TPO. The digitonin-treated suspension was
centrifuged at 100 000 g,4°C for 1 h, and the supernatant
containing solubilized TPO was used for the assays.
Inhibition of TPO iodide-oxidizing activity
TPO iodide-oxidizing activity was measured as previously
described [18,19]. The control assay mixture contained
1.0 mL freshly prepared 50 m
M
sodium phosphate buffer,
pH 7.4, containing 24 m
M
KI, 11 m
M
glucose, and the
amount of solubilized TPO that produced iodide-oxidizing
activity of 0.1 DA
353
Æmin
)1
. The final volume was adjusted
to 2.0 mL with 50 m
M
sodium phosphate buffer, pH 7.4,
and the reaction was started by the addition of 10 lL0.1%
glucose oxidase. The increase in A
353
(tri-iodide production)
was registered for 4 min on a Hitachi spectrophotometer
(U-3300; Tokyo, Japan). To test the inhibitory effects, the
desired concentration of PTU, MMI or thiourea was added
to the assay mixture before the final volume was adjusted to
2 mL. The DA
353
Æmin
)1
in the presence or absence of
inhibitors was determined from the linear portion of the
reaction curve.
The inhibitory potency was expressed as the concentra-
tion necessary to produce 50% inhibition of the original
peroxidase activity (IC
50
). Each compound was tested in at
least three series of experiments, in which 8–12 different
concentrations were assayed.
H
2
O
2
-trapping effect
To study if PTU, MMI and thiourea are able to scavenge
H
2
O
2
,4.0m
M
H
2
O
2
was incubated in the absence or
presence of 10 l
M
PTU, 4 l
M
MMI and 2 l
M
thiourea
(respective IC
50
values for TPO iodide-oxidizing activity)
and 100 l
M
PTU, 40 l
M
MMI and 20 l
M
thiourea
(respective IC
100
values for TPO iodide oxidizing activity).
Aliquots of 100 lL were then added to 1 mL 0.2
M
sodium
phosphate buffer, pH 7.8, containing scopoletin (5.0 m
M
)
and HRP (5 lgÆmL
)1
). Fluorescence was measured in a
Hitachi spectrofluorimeter (F4000; excitation wave-
length ¼ 360 nm, emission wavelength ¼ 460 nm), as pre-
viously described [20]. The fluorescence measurements were
plotted against H
2
O
2
concentrations.
In vivo, the thyroid gland generates H
2
O
2
gradually, so an
enzymatic system (glucose–glucose oxidase) was used as a
model to test the ability of PTU or MMI to interfere with
progressive H
2
O
2
production in vitro.PTU(10l
M
or
100 l
M
) or MMI (4 l
M
or 40 l
M
) was incubated in the
presence of 11 m
M
glucose, and the final volume was
adjusted to 2.0 mL with 50 m
M
sodium phosphate buffer,
pH 7.4. The reaction was started by the addition of 10 lL
1mgÆL
)1
glucose oxidase. This concentration of glucose
oxidase in the presence of 11 m
M
glucose produces H
2
O
2
-
generating activity similar to that produced in vitro by
porcine and human thyroid NADPH oxidase, the enzyme
responsible for thyroid H
2
O
2
production in vivo [21,22].
Aliquots of 100 lL of the reaction mixture were transferred
to test tubes 0, 5, 10 and 15 min after the addition of glucose
oxidase. Then, 1 mL 0.2
M
sodium phosphate buffer,
pH 7.8, containing scopoletin (5.0 m
M
)andHRP
(5 lgÆmL
)1
), was added, and the fluorescence was measured
as described above. H
2
O
2
production proportional to
scopoletin fluorescence decrement was plotted against time.
Thyroid NADPH oxidase preparation
For thyroid NADPH oxidase preparations, fresh human
thyroid tissue paranodular to cold nodules (1 g) was cleaned
from fibrous tissue or hemorrhagic areas, minced and
homogenized in sodium phosphate buffer, pH 7.2, contain-
ing 0.25
M
sucrose, 0.5 m
M
dithiothreitol and 1 m
M
EGTA,
using an Ultra-Turrax. The homogenate was filtered
through cheesecloth. The particulate fraction was collected
by centrifugation at 3000 g for 15 min at 4 °Cand
resuspended in 3 mL 50 m
M
sodium phosphate buffer,
pH 7.2, containing 0.25
M
sucrose and 2 m
M
MgCl
2
(buffer
A). The pellet was washed twice with 3 mL buffer A and
centrifuged at 3000 g for 15 min at 4 °C. The last pellet
(P3000 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,
P100 000 g) was washed twice in 2 mL buffer A, and gently
resuspended in 0.5 mL buffer A.
Inhibition of NADPH oxidase activity
H
2
O
2
formation was measured by incubating aliquots of
human thyroid particulate fractions (either P3000 or
P100 000 g)at30°C in 1 mL 170 m
M
sodium phosphate,
pH 7.4, containing 1 m
M
sodium azide, 1 m
M
EGTA, 1 l
M
FAD and 1.5 m
M
CaCl
2
. To test the inhibitory effects, the
desired amounts of PTU or MMI were added to the assay
mixture before adjustment of the final volume to 1 mL. The
reaction was started by adding 0.2 m
M
NADPH; aliquots of
100 lL were collected at intervals up to 20 min and mixed
with 10 lL3
M
HCltostopthereactionanddestroythe
remaining NADPH. The amount of H
2
O
2
in each sample
was measured in 200 m
M
phosphate buffer (pH 7.8) by
following the decrease in 0.4 l
M
scopoletin fluorescence in
the presence of HRP (0.5 lgÆmL
)1
) in a Hitachi spectro-
fluorimeter as previously described [23,24]. H
2
O
2
production
(nmol H
2
O
2
Æh
)1
ÆmL
)1
) in the presence or absence of these
drugs was determined from the linear portion of the
reaction curve, and the results were expressed as percentage
of control.
2364 A. C. Freitas Ferreira et al.(Eur. J. Biochem. 270) Ó FEBS 2003
Results
Inhibition of TPO iodide-oxidizing activity
The already described concentrations of PTU and MMI
necessary to produce 50% inhibition of TPO-mediated
thyroglobulin iodination were 19.5 l
M
and 10 l
M
, respect-
ively [1]. Under our experimental conditions, we have found
similar differences in the IC
50
values for the PTU
(9.8 ± 1.1 l
M
) and MMI (3.8 ± 0.2 l
M
) inhibitory effects
on the TPO iodide-oxidizing reaction. Thiourea produced
50% inhibition of the initial TPO iodide-oxidizing activity
at a concentration of 2.3 ± 0.2 l
M
. Thus, in our experi-
mental conditions, thiourea and MMI are more potent TPO
inhibitors than PTU.
H
2
O
2
-trapping effect
To further evaluate the possible mechanism of TPO
inhibition by PTU, MMI and thiourea, we tested whether
they were able to scavenge H
2
O
2
in vitro. Our results show
that PTU and thiourea at either IC
50
(PTU ¼ 10 l
M
,
thiourea ¼ 2 l
M
)orIC
100
(PTU ¼ 100 l
M
, thio-
urea ¼ 20 l
M
) did not significantly scavenge H
2
O
2
.On
the other hand, MMI significantly scavenged H
2
O
2
when
the concentration of IC
100
(40 l
M
) was added (Fig. 1).
Furthermore, PTU did not scavenge H
2
O
2
generated by the
glucose–glucose oxidase system, and MMI was able to
scavenge H
2
O
2
generated by glucose–glucose oxidase only
at IC
100
(Fig. 2A,B).
Inhibition of NADPH oxidase activity
Both PTU and MMI partiallyinhibitedthyroid NADPH
oxidase activity in vitro (Fig. 3). As PTU did not scavenge
H
2
O
2
in the conditions used here, we presume that it inhibits
thyroid NADPH oxidase directly (Fig. 3A). At the concen-
tration necessary to inhibit NADPH oxidase activity in vitro
(Fig. 3B), MMI did not significantly scavenge H
2
O
2
,also
suggesting a direct effect of MMI on thyroid NADPH
oxidase.
Although the kinetics of NADPH oxidase inhibition by
antithyroid drugs seem to differ (Fig. 3), the curve analysis
by the statistical curve-fitting package
ENZFITTER
(Elsevier-
Biosoft, Cambridge, UK) showed that PTU is as potent as
MMI in inhibiting this enzyme. PTU produced 50%
inhibition of the initial NADPH oxidase activity at a
concentration of 26.3 l
M
, with residual activity equal to
Fig. 1. Study of the H
2
O
2
-trapping effect of PTU, MMI and thiourea.
H
2
O
2
concentration was measured after incubation with or without
PTU, MMI and thiourea, as follows: 4.0 l
M
H
2
O
2
was incubated in
the presence or absence of 100 l
M
PTU, 40 l
M
MMI and 20 l
M
thiourea (IC
100
for TPO iodide-oxidizing activity). Then, aliquots of
100 lL were transferred to a tube, and 1 mL 0.2
M
sodium phosphate
buffer, pH 7.8, containing scopoletin (5.0 l
M
)andHRP(5lgÆmL
)1
)
was added. Fluorescence was measured in a Hitachi (F4000) spectro-
fluorimeter (excitation at 360 nm, emission at 460 nm). Results are
expressed as mean ± SEM obtained in at least three different
experiments. Data were analyzed by parametric one-way analysis of
variance followed by Newman-Keuls multiple comparison test.
*P < 0.05 when compared with control, PTU and thiourea.
Fig. 2. Effect of PTU and MMI on H
2
O
2
produced by glucose–glucose
oxidase system. Glucose (11 m
M
) was incubated in the presence or
absence of 100 l
M
PTU or 40 l
M
MMI (IC
100
for TPO iodide-oxi-
dizing activity), and the final volume was adjusted to 2.0 mL with
50 m
M
sodium phosphate buffer, pH 7.4. The reaction was started by
the addition of 10 lL1mgÆL
)1
glucose oxidase. (A) Aliquots of
100 lL were transferred to the test tube 15 min after glucose oxidase
addition. (B) Aliquots of 100 lL were transferred to the test tube 0, 5,
10 and 15 min after glucose oxidase addition. Then, in both (A) and
(B), scopoletin solution (1 mL 0.2
M
sodium phosphate buffer, pH 7.8,
containing 5.0 l
M
scopoletin and 5 lgÆmL
)1
HRP) was added. The
fluorescence was measured in a Hitachi (F4000) spectrofluorimeter
(excitation 360 nm, emission 460 nm). The graph shows H
2
O
2
concentrations plotted against time. Results are expressed as
mean ± SEM obtained in three different experiments.
Ó FEBS 2003 Thyroid NADPH oxidase inhibition by PTU and MMI (Eur. J. Biochem. 270) 2365
17.1% of control, whereas we have found an IC
50
for
MMI of 31.7 l
M
, with a residual activity equal to 45.2% of
control (Fig. 3).
As shown in Fig. 2, PTU did not interfere with the
generation of H
2
O
2
by glucose–glucose oxidase; however, a
slight decrease in the amount of H
2
O
2
generated by
NADPHoxidaseisshownwithbothPTUandMMI
(Fig. 4).
Discussion
Hicks et al. [8] showed that PTU acts as a highly efficient
scavenger of hydroxyl radicals and an efficient inhibitor of
lipid peroxidation at the free drug levels attained in serum at
a dose of 300 mgÆday
)1
. On the other hand, we show that
PTU did not interact with H
2
O
2
. Thus, as both PTU and
thiourea neither scavenge H
2
O
2
added to the incubation
mixture nor impair H
2
O
2
generated by the glucose–glucose
oxidase system, inhibition of the TPO iodide-oxidizing
reaction produced by these drugs may be due to a direct
effect on TPO activity only. On the other hand, it is possible
that the inhibition of thyroid hormone biosynthesis by
MMI in vivo is due to both a direct effect on TPO activity
and its ability to scavenge H
2
O
2
. In fact, the amount of
H
2
O
2
generated by the thyroid NADPH oxidase enzymatic
system in vitro is similar to that produced by the glucose–
glucose oxidase system used here, so it is possible that MMI
also decreases the availability of H
2
O
2
produced by
NADPH oxidase in vivo [21,22]. However, the fact that
MMI is a more potent TPO inhibitor than PTU cannot be
explained by its ability to destroy H
2
O
2
,becausethe
concentrations of H
2
O
2
present under the assay conditions
of the iodide oxidizing reaction are in the millimolar range
and MMI does not seem to interfere with H
2
O
2
at the
concentration necessary to inhibit 50% of TPO iodide
oxidizing activity.
Ross et al. [9] suggested that inhibition of neutrophil-
mediated hypochlorous acid formation and A1PI inativa-
tion are the mechanisms by which PTU and MMI protect
against neutrophil-mediated tissue injury in a variety of
pathological conditions. Weetman et al. [7] showed that
MMI, at the concentrations found in the thyroid gland of
patients with toxic diffuse goiters treated with carbimazole,
inhibits the production of oxygen radicals by monocytes
and reduces the production of H
2
O
2
by the same cells, which
may be related to the immunosuppressive action of the drug
in vivo and in vitro. In this study, we showed that
methimazole scavenges H
2
O
2
. It is possible that the ability
of MMI to destroy H
2
O
2
contributes to its immunosup-
pressive effects. However, Imseis et al.[25]showedthatthe
therapeutic efficacy of
131
I in hyperthyroid patients was
reduced by pretreatment with propylthiouracyl but not with
methimazole, which contradicts the antioxidative effect of
MMI demonstrated in our study. Therefore, the mechanism
of protection against
131
I radiation promoted by PTU
remains undefined.
Surprisingly, both PTU and MMI inhibited thyroid
NADPH oxidase H
2
O
2
generation activity in vitro.
Although they did not completely inhibit NADPH oxidase
activity, it is possible that this effect contributes to inhibition
of thyroid hormone biosynthesis in vivo. However, the
concentrations necessary to inhibit thyroid NADPH oxidase
were higher than those used to inhibit TPO activity in vitro.
A peroxidase domain has been found in the sequence
encoding two recently cloned flavoproteins that correspond
to thyroid oxidases (ThOX1 and ThOX2) [15,16,26,27].
Thus, PTU and MMI may interact with the peroxidase
domain of ThOX proteins, leading to alterations in their
Fig. 3. Inhibition of NADPH oxidase activity by PTU and MMI.
NADPH oxidase activity was measured in the presence of different
PTU (A) or MMI (B) concentrations, as follows: the amount of solu-
bilized NADPH oxidase producing a fixed H
2
O
2
-forming activity was
assayed at 30 °C in the presence of 1 mL 170 m
M
sodium phosphate,
pH 7.4, containing 1 m
M
sodium azide, 1 m
M
EGTA, 1 l
M
FAD and
1.5 m
M
CaCl
2
. The reaction was started by adding 0.2 m
M
NADPH;
aliquots of 100 lL were collected at intervals up to 20 min and mixed
with 10 lL3
M
HCl to stop the reaction and destroy the remaining
NADPH. The amount of H
2
O
2
in each sample was measured in
200 m
M
phosphate buffer (pH 7.8) by following the decrease in 0.4 l
M
scopoletin fluorescence in the presence of HRP (0.5 lgÆmL
)1
)ina
Hitachi spectrofluorimeter (F4000). The excitation and emission
wavelengths were 360 and 460 nm, respectively. Activity (nmol
H
2
O
2
ÆmL
)1
Æh
)1
) in the presence or absence of inhibitors was deter-
mined from the linear portion of each reaction curve and plotted
against different PTU and MMI concentrations. The results were
expressed as percentage of control (mean of two separate experiments).
Inhibitory curves were analyzed by the statistical curve-fitting package
ENZFITTER
(Elsevier-Biosoft, Cambridge, UK).
2366 A. C. Freitas Ferreira et al.(Eur. J. Biochem. 270) Ó FEBS 2003
structures, so that the oxidation of NADPH and thus H
2
O
2
generation would be impaired.
In conclusion, this study shows that MMI, but not PTU,
is able to scavenge H
2
O
2
in the micromolar range and that
both PTU and MMI may impair thyroid H
2
O
2
generation.
However, the inhibitory effect on H
2
O
2
generation was
partial and could only complement their known potent TPO
inhibitory effects.
Acknowledgements
This work was supported by grants from Conselho Nacional de
Desenvolvimento Cientı
´
fico e Tecnolo
´
gico (CNPq) and Fundac¸ a
˜
o
Carlos Chagas Filho de Amparo a
`
Pesquisa do Estado do Rio de Janeiro
(FAPERJ). We are grateful for the technical assistance of Norma Lima
de Arau´ jo Faria, Advaldo Nunes Bezerra and Wagner Nunes Bezerra.
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Fig. 4. Effect of PTU and MMI on the H
2
O
2
produced bythyroid NADPH oxidase. NADPH oxidase activity was measured in the presence or
absence of (A) 10 or (B) 100 l
M
PTU and (A) 4 or (B) 40 l
M
MMI (IC
50
or IC
100
for TPO iodide-oxidizing activity, respectively), as follows: the
amount of solubilized NADPH oxidase producing a fixed H
2
O
2
-forming activity was assayed at 30 °C in the presence of 1 mL 170 m
M
sodium
phosphate, pH 7.4, containing 1 m
M
sodium azide, 1 m
M
EGTA, 1 l
M
FAD and 1.5 m
M
CaCl
2
. The reaction was started by adding 0.2 m
M
NADPH; aliquots of 100 lL were collected at intervals up to 20 min and mixed with 10 lL3
M
HCl to stop the reaction and destroy the remaining
NADPH. The amount of H
2
O
2
in each sample was measured in 200 m
M
phosphate buffer (pH 7.8) by following the decrease in 0.4 l
M
scopoletin
fluorescence in the presence of HRP (0.5 lgÆmL
)1
) in a Hitachi spectrofluorimeter (F4000). The excitation and emission wavelengths were 360 and
460 nm, respectively. The graphs show H
2
O
2
produced bythyroid NADPH oxidase 15 min after the addition of NADPH, and the insert shows
H
2
O
2
concentrations plotted against time. Results are expressed as mean ± SEM obtained in three different experiments.
Ó FEBS 2003 Thyroid NADPH oxidase inhibition by PTU and MMI (Eur. J. Biochem. 270) 2367
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2368 A. C. Freitas Ferreira et al.(Eur. J. Biochem. 270) Ó FEBS 2003
. Thyroid Ca
2+
/NADPH-dependent H
2
O
2
generation is partially inhibited
by propylthiouracil and methimazole
Andrea C. Freitas Ferreira,. Brazil
H
2
O
2
generation is a limiting step in thyroid hormone bio-
synthesis. Biochemical studies have confirmed that H
2
O
2
is
generated by a thyroid Ca
2+
/NADPH-dependent