Iodine incorporation into thyroglobulin is dependent on the activities of both thyroperoxidase (TPO) and thyroid dual oxidase 2 (DuOx2). Although TPO expression is decreased in some thyroid nodular lesions, DuOx1 and 2 mRNA expressions are maintained, but DuOx H 2 O 2 -generating activity has never been evaluated in such tumors. Our goal was to determine DuOx activity in hypofunctioning lesions of the thyroid. We evaluated H 2 O 2 generation by DuOx in 12 paranodular to cold nodule samples, 17 non-toxic multinodular goiters (MNG; 33 samples), 3 papillary carcinomas (PC; 4 samples), 3 follicular carcinomas (FC; 4 samples), and 10 follicular adenomas. DuOx activity was detected in all paranodular tissues (121G23 nmol H 2 O 2 /h per mg protein), but was undetectable (!1 nmol H 2 O 2 generated) in all PC, two out of four FC samples and seven out of ten adenomas. In 11 MNG at least two different areas of the goiter have been evaluated, and in 5 of these goiters one of the samples had DuOx activity below the limit of detection. The coefficient of variation in MNG samples ranged from 11.3 to 57.2%. Interestingly, in all the adenomas studied, TPO activity (486G142 U/g protein, nZ8) was well within the range found in paranodular tissues (414G116 U/g protein, nZ3). We found a significant negative correlation between DuOx and TPO activities, suggesting that these enzymes are regulated in opposite directions, at least in thyroid tumors.

Objective: The aim of the present study was to compare the effects of iopanoic acid (IOP) or a saturated solution of potassium iodide (SSKI) administration to patients with toxic diffuse goiters (TDG). Design: Patients with TDG are treated with thionamides and high doses of iodine preoperatively. In this study, two types of preoperative drug regimens were used: propylthiouracil or methimazole plus SSKI for 10 -15 days ðn ¼ 8Þ or IOP for 7 days ðn ¼ 6Þ: Methods: Serum thyroid hormones (total and free thyroxine (T 4 ), total tri-iodothyronine (T 3 ) and reverse T 3 (rT 3 ), were evaluated after 7 days of either SSKI or IOP treatment, and after 10 -15 days of SSKI administration. During thyroidectomy, samples of thyroid gland were obtained to evaluate thyroperoxidase and thyroid H 2 O 2 -generating activities. Results: Serum total T 3 was significantly decreased after 7 days of either treatment, and serum rT 3 was significantly increased in IOP-treated patients. Serum total and free T 4 were unaffected by 7 days of IOP treatment, but decreased after 7 days of SSKI treatment, although significantly diminished levels were only reached after a further 3-8 days of SSKI administration. During both drug regimens, serum TSH remained low (SSKI: 0:159^0:122; IOP: 0:400^0:109 mU=ml). Thyroperoxidase activity was significantly lower in thyroid samples from patients treated with SSKI for 10-15 days than in the thyroid glands from IOP-treated patients. However, thyroid H 2 O 2 generation was inhibited in samples from patients treated with either IOP or SSKI. Conclusions: We show herein that IOP treatment can be effective in the management of hyperthyroidism and that this drug inhibits thyroid NADPH oxidase activity, just as previously described for SSKI, probably due to its iodine content.

We report herein the study of two siblings (DESM and DSM) with hypothyroidism, goiter, and positive perchlorate discharge tests (50% and 70%) in a family (M) with no history of consanguinity. Thyroid gland histology showed a predominance of hyperactive follicles, with high epithelial cells and variable colloid content. Thyroid peroxidase iodide oxidation (DESM, 1034; DSM, 1064 U/g protein) and albumin iodination (DESM, 16; DSM, 8 nmol I/mg protein) activities were within the normal range. Tg content was normal in both glands compared with that in diffuse toxic goiter (DESM, 28; DSM, 17; diffuse toxic goiter, 19 mg/g tissue), and Tg could be normally iodinated by thyroid peroxidase in vitro (DESM, 3.4; DSM, 4.3; diffuse toxic goiter, 6.3 nmol I/mg Tg). Thyroid cytochrome c reductase activities in these goiters were higher than that in paranodular tissues (DESM, 473; DSM, 567; paranodular tissues, 78 nmol NADP(+)/h/mg protein). However, thyroid NADPH oxidase activities were very low both in the particulate 3,000 x g (DESM, 4.8; DSM, 44; paranodular tissues, 224 nmol H(2)O(2)/h/mg protein) and in the particulate 100,000 x g fractions (DESM, 40; DSM, 47; paranodular tissues, 200 nmol H(2)O(2)/h/mg protein). Thus, a decreased Ca(2+)/NAD(P)H-dependent H(2)O(2) generation is the probable cause of the organification defect in these goiters.

A calcium and NAD(P)H-dependent H(2)O(2)-generating activity has been studied in paranodular thyroid tissues from four patients with cold thyroid nodules and from nine diffuse toxic goiters. H(2)O(2) generation was detected both in the particulate (P 3,000 g) and in the microsomal (P 100,000 g) fractions of paranodular tissue surrounding cold thyroid nodules (PN), with the same biochemical properties described for NADPH oxidase found in porcine and human thyroids. In PN tissues, the particulate NADPH oxidase activity (224 +/- 38 nmol H(2)O(2) x h(-1) x mg(-1) protein) was similar to that described for the porcine thyroid enzyme. However, no NADPH oxidase activity was detectable in the particulate fractions from eight diffuse toxic goiter patients treated with iodine before surgery; all but one also received propylthiouracil or methimazole in the preoperative period. Thyroid cytochrome c reductase (diffuse toxic goiters = 438 +/- 104 nmol NADP(+) x h(-1) x mg(-1) protein; PN = 78 +/- 10 nmol NADP(+) x h(-1) x mg(-1) protein) and thyroperoxidase (diffuse toxic goiters = 621 +/- 179 U x g(-1) protein; PN = 232 +/- 121 U x g(-1) protein) activities were unaffected by iodide. Thus, the human NADPH oxidase seems to be inhibited by iodinated compounds in vivo and probably is an enzyme involved in the Wolff-Chaikoff effect. Our findings reinforce the hypothesis that thyroid NADPH oxidase is responsible for the production of H(2)O(2) necessary for thyroid hormone biosynthesis.

RESUMONo presente estudo avaliamos a atividade geradora de peróxido de hidrogênio (H 2 O 2 ) em frações particuladas de tireóides suínas e humanas. Inicialmente, analisamos as propriedades bioquímicas da NADPH-oxidase -enzima geradora de H 2 O 2 -localizada na membrana apical da célula tireóidea suína. Nossos resultados demonstram que a atividade geradora de H 2 O 2 na tireóide suína ocorre, principalmente, na fração de membrana apical tireóidea (P 3.000g). Entretanto, no P 3.000g a enzima NADPH-oxidase é apenas parcialmente cálcio-dependente, ao contrário do que acontece em frações purificadas de membrana tireóidea suína, nas quais a enzima é completamente dependente de cálcio, conforme estudos anteriores. Nossos resultados confirmam os previartiente descritos para a NADPH-oxidase tireóidea suína. Em tecidos tireóideos humanos, a geração de H 2 O 2 ocorreu, tanto na fração microsomal (P 100.000g) quanto na fração de membrana apical (P 3.000g). Nossos dados revelam ainda que a NADPH-oxidase humana é completamente cálcio-dependente, ativada por altas concentrações de fosfato e parece ser tão ativa na glândula humana quanto na suína. Além disto, a enzima humana é dependente de adenina-flavina-dinucleotídeo (FAD) no meio de reação, ou seja, parece ser uma flavoproteína, assim como a proteína suína. ABSTRACTIn the present study we evaluated the enzyme responsible for hydrogen peroxide (H 2 O 2 ) generation in porcine and human thyroid glands. First, we analyzed the biochemical properties of the hydrogen peroxide generating enzyme (NADPH-oxidase), localized in the apical membrane of porcine thyroid cells. Our results showed that the H 2 O 2 generating activity in porcine thyroids occurs mainly in the apical membrane fraction (P 3.000g). In the porcine P 3.000g, thyroid NADPH-oxidase was partially calcium-dependent; however, in a purified porcine thyroid membrane fraction the enzyme is completely calcium-dependent, as previously determined. These data agree with those already reported for the porcine enzyme. In humans, H 2 O 2 generation occurred both in the microsomal (P 100.000g) and in the apical membrane fractions (P 3.000g). Our data reveal that NADPH-oxidase seems to be as active in human thyroid glands as in porcine thyroids; both are activated by phosphate and calcium in high concentrations. Furthermore, the human NADPH-oxidase is completely calcium-dependent and requires flavine adenine dinucleotide (FAD) in the reaction mixture, suggesting the human NADPH-oxidase to be a flavoenzyme, as the porcine protein.