Báo cáo khoa học: Thyroid Ca2+/NADPH-dependent H2O2 generation is partially inhibited by propylthiouracil and methimazole

doi:10.1046/j.1432-1033.2003.03576.x

Eur. J. Biochem. 270, 2363–2368 (2003) (cid:1) FEBS 2003

Thyroid 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

partially inhibit thyroid NADPH oxidase activity in vitro. As PTU did not scavenge H2O2 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 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 H2O2 generation in addition to their potent thyro- peroxidase inhibitory effects.

Keywords: antithyroid drugs; H2O2; NADPH oxidase; thyroid. H2O2 generation is a limiting step in thyroid hormone bio- synthesis. Biochemical studies have confirmed that H2O2 is 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 H2O2 or inhibit thyroid NADPH oxidase activity in vitro. Our results show that PTU and thiourea did not significantly scavenge H2O2. However, MMI significantly scavenged H2O2 at high concentrations. Only MMI was able to decrease the amount of H2O2 generated by the glucose–glucose oxidase system. On the other hand, both PTU and MMI were able to

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.

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

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 H2O2 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 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

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)

Chemicals

NADPH, glucose oxidase (grade I), lyophilized horseradish peroxidase (HRP, grade I) and glucose oxidase (grade I)

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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). CaCl2 was purchased from Mallinckrodt, and Tris (hydroxymethyl)aminomethane and H2O2 were from Merck (Rio de Janeiro, RJ, Brazil).

TPO preparation

containing scopoletin (5.0 mM)

In vivo, the thyroid gland generates H2O2 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 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 mM sodium 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 mM glucose 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 0.2 M sodium phosphate buffer, pH 7.8, 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.

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 mM Tris/ HCl buffer, pH 7.2, containing 1 mM KI, using an Ultra- Turrax homogenizer (Staufen). The homogenate was cen- trifuged at 100 000 g, 4 (cid:2)C for 1 h. The pellet was suspended in 2 mL digitonin (1%, w/v) and incubated at 4 (cid:2)C for 24 h to solubilize TPO. The digitonin-treated suspension was centrifuged at 100 000 g, 4 (cid:2)C for 1 h, and the supernatant containing solubilized TPO was used for the assays. Thyroid NADPH oxidase preparation

Inhibition of TPO iodide-oxidizing activity

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 mM dithiothreitol and 1 mM 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 (cid:2)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 buffer A and centrifuged at 3000 g for 15 min at 4 (cid:2)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 (cid:2)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. TPO iodide-oxidizing activity was measured as previously described [18,19]. The control assay mixture contained 1.0 mL freshly prepared 50 mM sodium 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 mM sodium 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.

Inhibition of NADPH oxidase activity

The inhibitory potency was expressed as the concentra- tion necessary to produce 50% inhibition 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

spectrofluorimeter (F4000;

H2O2 formation was measured by incubating aliquots of human thyroid particulate fractions (either P3000 or P100 000 g) at 30 (cid:2)C in 1 mL 170 mM sodium phosphate, pH 7.4, containing 1 mM sodium azide, 1 mM EGTA, 1 lM FAD and 1.5 mM CaCl2. 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 mM 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 H2O2 in each sample was measured in 200 mM phosphate buffer (pH 7.8) by following the decrease in 0.4 lM scopoletin fluorescence in the presence of HRP (0.5 lgÆmL)1) in a Hitachi spectro- fluorimeter as previously described [23,24]. H2O2 production (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. 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 IC100 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 mM) and HRP (5 lgÆmL)1). Fluorescence was measured in a Hitachi excitation wave- length ¼ 360 nm, emission wavelength ¼ 460 nm), as pre- viously described [20]. The fluorescence measurements were plotted against H2O2 concentrations.

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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 lM and 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

or (PTU ¼ 100 lM,

Fig. 2. Effect of PTU and MMI on H2O2 produced by glucose–glucose oxidase system. Glucose (11 mM) was incubated in the presence or absence of 100 lM PTU or 40 lM MMI (IC100 for TPO iodide-oxi- dizing activity), and the final volume was adjusted to 2.0 mL with 50 mM 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 lM 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 H2O2 time. Results are expressed as concentrations plotted against mean ± SEM obtained in three different experiments.

To further evaluate the possible mechanism of TPO inhibition by PTU, MMI and thiourea, we tested whether they were able to scavenge H2O2 in vitro. Our results show that PTU and thiourea at either IC50 (PTU ¼ 10 lM, thiourea ¼ 2 lM) thio- IC100 urea ¼ 20 lM) did not significantly scavenge H2O2. On the other hand, MMI significantly scavenged H2O2 when the concentration of IC100 (40 lM) was added (Fig. 1). Furthermore, PTU did not scavenge H2O2 generated by the glucose–glucose oxidase system, and MMI was able to scavenge H2O2 generated by glucose–glucose oxidase only at IC100 (Fig. 2A,B).

Inhibition of NADPH oxidase activity

Fig. 1. Study of the H2O2-trapping effect of PTU, MMI and thiourea. H2O2 concentration was measured after incubation with or without PTU, MMI and thiourea, as follows: 4.0 lM H2O2 was incubated in the presence or absence of 100 lM PTU, 40 lM MMI and 20 lM thiourea (IC100 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 lM) 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.

Both PTU and MMI partially inhibited thyroid NADPH oxidase activity in vitro (Fig. 3). As PTU did not scavenge H2O2 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 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 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 lM, with residual activity equal to

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NADPH 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 H2O2 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 H2O2. In fact, the amount of H2O2 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 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 H2O2 present 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.

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 H2O2-forming activity was assayed at 30 (cid:2)C in the presence of 1 mL 170 mM sodium phosphate, pH 7.4, containing 1 mM sodium azide, 1 mM EGTA, 1 lM FAD and 1.5 mM CaCl2. The reaction was started by adding 0.2 mM 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 H2O2 in each sample was measured in 200 mM phosphate buffer (pH 7.8) by following the decrease in 0.4 lM 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 H2O2ÆmL)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).

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 H2O2 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 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.

17.1% 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).

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 As shown in Fig. 2, PTU did not interfere with the generation of H2O2 by glucose–glucose oxidase; however, a slight decrease in the amount of H2O2 generated by

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Fig. 4. Effect of PTU and MMI on the H2O2 produced by thyroid NADPH oxidase. NADPH oxidase activity was measured in the presence or absence of (A) 10 or (B) 100 lM PTU and (A) 4 or (B) 40 lM MMI (IC50 or IC100 for TPO iodide-oxidizing activity, respectively), as follows: the amount of solubilized NADPH oxidase producing a fixed H2O2-forming activity was assayed at 30 (cid:2)C in the presence of 1 mL 170 mM sodium phosphate, pH 7.4, containing 1 mM sodium azide, 1 mM EGTA, 1 lM FAD and 1.5 mM CaCl2. The reaction was started by adding 0.2 mM 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 H2O2 in each sample was measured in 200 mM phosphate buffer (pH 7.8) by following the decrease in 0.4 lM 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 H2O2 produced by thyroid NADPH oxidase 15 min after the addition of NADPH, and the insert shows H2O2 concentrations plotted against time. Results are expressed as mean ± SEM obtained in three different experiments.

2. Magnusson, R.P., Taurog, A. & Dorris, M.L. (1984) Mechanism of iodide-dependent catalytic activity of thyroid peroxidase and lactoperoxidase. J. Biol. Chem. 259, 197–205.

3. Davidson, B., Soodak, M., Neary, J.T., Strout, H.V., Kieffer, J.D., Mover, H. & Maloof, F. (1978) The irreversible inactivation of thyroid peroxidase by methylmercaptoimidazole, thiouracil, and propylthiouracil in vitro and its relationship to in vivo findings. Endocrinology 103, 871–882.

structures, so that the oxidation of NADPH and thus H2O2 generation would be impaired.

4. Engler, H., Taurog, A. & Nakashima, T. (1982) Mechanism of inactivation of thyroid peroxidase by thioureylene drugs. Biochem. Pharmacol. 31, 3801–3806.

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 may impair thyroid H2O2 generation. However, the inhibitory effect on H2O2 generation was partial and could only complement their known potent TPO inhibitory effects.

Acknowledgements

5. Taurog, A. & Dorris, M.L. (1989) A reexamination of the pro- posed inactivation of thyroid peroxidase in the rat thyroid by propylthiouracil. Endocrinology 124, 3038–3042.

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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