Therefore, we analyzed whether PTU or MMI could scavenge H2O2or inhibit thyroid NADPH oxidase activity in vitro.. On the other hand, both PTU and MMI were able to partially inhibit thyro
Trang 1Thyroid Ca2+/NADPH-dependent H2O2 generation is partially inhibited
by propylthiouracil and 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
H2O2generation is a limiting step in thyroid hormone
bio-synthesis Biochemical studies have confirmed that H2O2is
generated by a thyroid Ca2+/NADPH-dependent oxidase
Decreased H2O2 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 H2O2or
inhibit thyroid NADPH oxidase activity in vitro Our results
show that PTU and thiourea did not significantly scavenge
H2O2 However, MMI significantly scavenged H2O2at high
concentrations Only MMI was able to decrease the amount
of H2O2generated 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 H2O2under 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
H2O2, 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 H2O2 in the micromolar range and that both PTU and MMI can impair thyroid H2O2generation in addition to their potent thyro-peroxidase inhibitory effects
Keywords: antithyroid drugs; H2O2; NAD PH oxidase; 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 H2O2 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 H2O2 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 H2O2 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 H2O2
H2O2 generation is a limiting step in thyroid hormone biosynthesis [10,11], and biochemical studies have con-firmed that H2O2 is generated by a thyroid NADPH oxidase [12–14] Two genes probably involved in thyroid
H2O2 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 H2O2 availability decreases thyroid hormone biosynthesis [17], and the proteins involved in thyroid H2O2generation 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 H2O2 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)
Trang 2were purchased from Boehringer (Mannheim, Germany).
Scopoletin, digitonin, cytochrome c, MMI,
6-n-propylthio-uracyl (PTU), thiocarbamide (thiourea) and FADwere
obtained from Sigma Chemical Co (St Louis, MO, USA)
CaCl2 was purchased from Mallinckrodt, and Tris
(hydroxymethyl)aminomethane and H2O2 were from
Merck (Rio de Janeiro, 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 mMTris/
HCl buffer, pH 7.2, containing 1 mMKI, using an
Ultra-Turrax homogenizer (Staufen) The homogenate was
cen-trifuged at 100 000 g, 4C for 1 h The pellet was suspended
in 2 mL digitonin (1%, w/v) and incubated at 4C for 24 h
to solubilize TPO The digitonin-treated suspension was
centrifuged at 100 000 g, 4C 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 mMsodium phosphate buffer,
pH 7.4, containing 24 mM KI, 11 mM glucose, and the
amount of solubilized TPO that produced iodide-oxidizing
activity of 0.1 DA353Æmin)1 The final volume was adjusted
to 2.0 mL with 50 mMsodium phosphate buffer, pH 7.4,
and the reaction was started by the addition of 10 lL 0.1%
glucose oxidase The increase in A353(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 DA353Æmin)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% inhibiconcentra-tion of the original
peroxidase activity (IC50) Each compound was tested in at
least three series of experiments, in which 8–12 different
concentrations were assayed
H2O2-trapping effect
To study if PTU, MMI and thiourea are able to scavenge
H2O2, 4.0 mM H2O2 was incubated in the absence or
presence of 10 lM PTU, 4 lM MMI and 2 lM thiourea
(respective IC50 values for TPO iodide-oxidizing activity)
and 100 lM PTU, 40 lM MMI and 20 lM thiourea
(respective IC100values for TPO iodide oxidizing activity)
Aliquots of 100 lL were then added to 1 mL 0.2Msodium
phosphate buffer, pH 7.8, containing scopoletin (5.0 mM)
and HRP (5 lgÆmL)1) Fluorescence was measured in a
Hitachi spectrofluorimeter (F4000; excitation
wave-length¼ 360 nm, emission wavelength ¼ 460 nm), as
pre-viously described [20] The fluorescence measurements were
plotted against HO concentrations
In vivo, the thyroid gland generates H2O2gradually, so an enzymatic system (glucose–glucose oxidase) was used as a model to test the ability of PTU or MMI to interfere with progressive H2O2 production in vitro PTU (10 lM or
100 lM) or MMI (4 lM or 40 lM) was incubated in the presence of 11 mM glucose, and the final volume was adjusted to 2.0 mL with 50 mMsodium phosphate buffer,
pH 7.4 The reaction was started by the addition of 10 lL
1 mgÆL)1 glucose oxidase This concentration of glucose oxidase in the presence of 11 mMglucose produces H2O2 -generating activity similar to that produced in vitro by porcine and human thyroid NADPH oxidase, the enzyme responsible for thyroid H2O2 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 0.2M sodium phosphate buffer,
pH 7.8, containing scopoletin (5.0 mM) and HRP (5 lgÆmL)1), was added, and the fluorescence was measured
as described above H2O2 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 0.25Msucrose, 0.5 mMdithiothreitol and 1 mMEGTA, using an Ultra-Turrax The homogenate was filtered through cheesecloth The particulate fraction was collected
by centrifugation at 3000 g for 15 min at 4C and resuspended in 3 mL 50 mM sodium phosphate buffer,
pH 7.2, containing 0.25Msucrose and 2 mMMgCl2(buffer A) The pellet was washed twice with 3 mL buffer A and centrifuged at 3000 g for 15 min at 4C 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 4C 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
H2O2 formation was measured by incubating aliquots of human thyroid particulate fractions (either P3000 or P100 000 g) at 30C in 1 mL 170 mMsodium phosphate,
pH 7.4, containing 1 mMsodium azide, 1 mMEGTA, 1 lM FADand 1.5 mMCaCl2 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 mMNADPH; aliquots of
100 lL were collected at intervals up to 20 min and mixed with 10 lL 3M HCl to stop the reaction and destroy the remaining NADPH The amount of H2O2in each sample was measured in 200 mM phosphate buffer (pH 7.8) by following the decrease in 0.4 lMscopoletin fluorescence in the presence of HRP (0.5 lgÆmL)1) in a Hitachi spectro-fluorimeter as previously described [23,24] H2O2production (nmol H2O2Æh)1ÆmL)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
Trang 3Inhibition 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 lMand 10 lM,
respect-ively [1] Under our experimental conditions, we have found
similar differences in the IC50 values for the PTU
(9.8 ± 1.1 lM) and MMI (3.8 ± 0.2 lM) 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 lM Thus, in our
experi-mental conditions, thiourea and MMI are more potent TPO
inhibitors than PTU
H2O2-trapping effect
To further evaluate the possible mechanism of TPO
inhibition by PTU, MMI and thiourea, we tested whether
they were able to scavenge H2O2in vitro Our results show
that PTU and thiourea at either IC50 (PTU¼ 10 lM,
thiourea¼ 2 lM) or IC100 (PTU¼ 100 lM,
thio-urea¼ 20 lM) did not significantly scavenge H2O2 On
the other hand, MMI significantly scavenged H2O2when
the concentration of IC100 (40 lM) was added (Fig 1)
Furthermore, PTU did not scavenge H2O2generated by the
glucose–glucose oxidase system, and MMI was able to
scavenge H2O2generated by glucose–glucose oxidase only
at IC100(Fig 2A,B)
Inhibition of NADPH oxidase activity Both PTU and MMI partially inhibited thyroid NADPH oxidase activity in vitro (Fig 3) As PTU did not scavenge
H2O2in 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 H2O2, 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 packageENZFITTER (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 , with residual activity equal to
Fig 1 Study of the H 2 O 2 -trapping effect of PTU, MMIand 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 ) and HRP (5 lgÆmL)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 MMIon 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 lL 1 mgÆL)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)1HRP) 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.
Trang 417.1% of control, whereas we have found an IC50 for
MMI of 31.7 lM, 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 H2O2by glucose–glucose oxidase; however, a
slight decrease in the amount of HO generated by
NAD PH oxidase is shown with both PTU and MMI (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 H2O2 Thus, as both PTU and thiourea neither scavenge H2O2 added to the incubation mixture nor impair H2O2generated 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 H2O2 In fact, the amount of
H2O2generated 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 H2O2 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 H2O2, because the concentrations of H2O2present under the assay conditions
of the iodide oxidizing reaction are in the millimolar range and MMI does not seem to interfere with H2O2 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 H2O2by 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 H2O2 It is possible that the ability
of MMI to destroy H2O2 contributes to its immunosup-pressive effects However, Imseis et al [25] showed that the therapeutic efficacy of 131I 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 131I radiation promoted by PTU remains undefined
Surprisingly, both PTU and MMI inhibited thyroid NADPH oxidase H2O2 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 FADand
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 lL 3 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 Activity (nmol
H 2 O 2 ÆmL)1Æh)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).
Trang 5structures, so that the oxidation of NADPH and thus H2O2
generation would be impaired
In conclusion, this study shows that MMI, but not PTU,
is able to scavenge H2O2in the micromolar range and that
both PTU and MMI may impair thyroid H2O2generation
However, the inhibitory effect on H2O2 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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