Thyroid Ca 2+ /NADPH-dependent H 2 O 2 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 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 partially inhibited thyroid 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. References 1. Taurog, A. (1996) Hormone synthesis: thyroid iodine metabolism. In The Thyroid: a Fundamental and Clinical Text (Braverman, L.E. & Utiger, R.D., eds), 7th edn, pp. 47–80. Lippincott-Raven, New York, USA. 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. 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(2001) Tyrosine cross-linking of extracellular matrix is catalyzed by DUOX, a multidomain oxi- dase/peroxidase with homology to the phagocyte oxidase subunit gp91phox. J. Cell Biol. 154, 879–891. 27. Deken, X.D., Wang, D., Dumont, J.E. & Miot, F. (2002) Char- acterization of ThOX proteins as components of the thyroid H 2 O 2 -generating system. Exp. Cell Res. 273, 187–196. 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