Duox2 (and probablyDuox1Reactive oxygen species (ROS) 1 have emerged as important molecules involved in regulating essential cell functions, such as growth and differentiation (1). NAD(P)H oxidases are a major source of ROS. Phagocyte oxidase is the oxidase that has been investigated most thoroughly (2). It catalyzes the production of superoxide by the one-electron reduction of oxygen, using NADPH as the electron donor. The catalytic moiety of the phagocyte NADPH oxidase is gp91phox , a plasma membraneassociated flavohemoprotein. Recently, it was discovered that gp91 phox belongs to a family consisting of several very similar oxidases. Seven NOX (NADPH oxidase) and DUOX/ThOX (dual oxidase/thyroid oxidase) genes have been identified that encode different NADPH oxidases with differing mRNA tissue expression. The Nox family comprises gp91phox , now known as Nox2; Nox1, which is predominantly expressed in the colon (3); Nox3, cloned from fetal kidney (4); Nox4 found in the kidney cortex (5, 6); and Nox5, expressed in the testis, spleen, and lymph nodes (7). In addition to the basic structure of gp91 phox , Nox5 has a long intracellular N-terminal domain with four calcium binding sites implicated in its Ca 2ϩ -dependent activation (8).The biological functions of these Nox proteins are now under investigation. They are involved in signal transduction related to cell growth and cancer (9 -11) and to angiogenesis (12).Duox1 and Duox2 are large homologues of Nox2 with an N-terminal extension comprising two EF-hand motifs, an additional transmembrane helix, and a peroxidase homology ectodomain (see Fig. 4A) (13,14). DUOX genes have been identified in the thyroid gland, where they are strongly expressed (13,14). However, the DUOX are also expressed on the mucosal surfaces of the trachea and the bronchi (15) and in the airway epithelial cells (16,17), where it has been suggested that Duox1 is the isoform responsible for acid production and secretion in airways (16) and plays a critical role in mucin expression (18). DUOX2 was also expressed throughout the digestive tract, where it was found to be functional (19,20), in addition to the salivary gland and rectum (15).It has been suggested that Duox2, which was identified by purifying thyroid NADPH oxidase, may constitute the catalytic core of this enzyme and generate the H 2 O 2 used by Tpo to catalyze the biosynthesis of thyroid hormones at the apical surface of the thyrocytes (13). Although no functional Duoxbased H 2 O 2 -generating system has yet been reconstituted (21), this proposal is corroborated by a recent report of permanent and severe congenital hypothyroidism in a patient with a bial-* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.¶ Recipient of a fellowship from the Commissariat à l'Energie Atomique (Paris, France).ʈ Recipients of a fellowship from the National Education, Research, an...

NADPH oxidase 4 (NOX4) belongs to the NOX family that generates reactive oxygen species (ROS). Function and tissue distribution of NOX4 have not yet been entirely clarified. To date, in the thyroid gland, only DUOX1/2 NOX systems have been described. NOX4 mRNA expression, as shown by real-time PCR, was present in normal thyroid tissue, regulated by TSH and significantly increased in differentiated cancer tissues. TSH increased the protein level of NOX4 in human thyroid primary culture and NOX4-dependent ROS generation. NOX4 immunostaining was detected in normal and pathologic thyroid tissues. In normal thyroid tissue, staining was heterogeneous and mostly found in activated columnar thyrocytes but absent in quiescent flat cells. Papillary and follicular thyroid carcinomas displayed more homogeneous staining. The p22(phox) protein that forms a heterodimeric enzyme complex with NOX4 displayed an identical cellular expression pattern and was also positively regulated by TSH. ROS may have various biological effects, depending on the site of production. Intracellular NOX4-p22(phox) localization suggests a role in cytoplasmic redox signaling, in contrast to the DUOX localization at the apical membrane that corresponds to an extracellular H(2)O(2) production. Increased NOX4-p22(phox) in cancer might be related to a higher proliferation rate and tumor progression but a role in the development of tumors has to be further studied and established in the future.

Ionizing radiation (IR) causes not only acute tissue damage, but also late effects in several cell generations after the initial exposure. The thyroid gland is one of the most sensitive organs to the carcinogenic effects of IR, and we have recently highlighted that an oxidative stress is responsible for the chromosomal rearrangements found in radio-induced papillary thyroid carcinoma. Using both a human thyroid cell line and primary thyrocytes, we investigated the mechanism by which IR induces the generation of reactive oxygen species (ROS) several days after irradiation. We focused on NADPH oxidases, which are specialized ROS-generating enzymes known as NOX/DUOX. Our results show that IR induces delayed NADPH oxidase DUOX1-dependent H 2 O 2 production in a dose-dependent manner, which is sustained for several days. We report that p38 MAPK, activated after IR, increased DUOX1 via IL-13 expression, leading to persistent DNA damage and growth arrest. Pretreatment of cells with catalase, a scavenger of H 2 O 2 , or DUOX1 down-regulation by siRNA abrogated IR-induced DNA damage. Analysis of human thyroid tissues showed that DUOX1 is elevated not only in human radio-induced thyroid tumors, but also in sporadic thyroid tumors. Taken together, our data reveal a key role of DUOX1-dependent H 2 O 2 production in long-term persistent radio-induced DNA damage. Our data also show that DUOX1-dependent H 2 O 2 production, which induces DNA double-strand breaks, can cause genomic instability and promote the generation of neoplastic cells through its mutagenic effect.ionizing radiation | oxidative stress | NADPH oxidase | thyroid |

Here, using two human BRAF-mutated thyroid cell lines and a rat thyroid cell line expressing BRAF in a conditional manner, we show that NOX4 upregulation is controlled at the transcriptional level by the oncogene via the TGF-β/Smad3 signaling pathway. Importantly, treatment of cells with NOX4-targeted siRNA downregulates BRAF-induced NIS repression. Innovation and Conclusion: Our results establish a link between BRAF and NOX4, which is confirmed by a comparative analysis of NOX4 expression in human (TCGA) and mouse thyroid cancers. Remarkably, analysis of human and murine BRAF-mutated thyroid tumors highlights that the level of NOX4 expression is inversely correlated to thyroid differentiation suggesting that other genes involved in thyroid differentiation in addition to NIS might be silenced by a mechanism controlled by NOX4-derived ROS. This study opens a new opportunity to optimize thyroid cancer therapy. Antioxid. Redox Signal. 26, 864-877.

Hydrogen peroxide (H2O2) is a crucial substrate for thyroid peroxidase, a key enzyme involved in thyroid hormone synthesis. However, as a potent oxidant, H2O2 might also be responsible for the high level of oxidative DNA damage observed in thyroid tissues, such as DNA base lesions and strand breakages, which promote chromosomal instability and contribute to the development of tumours. Although the role of H2O2 in thyroid hormone synthesis is well established, its precise mechanisms of action in pathological processes are still under investigation. The NADPH oxidase/dual oxidase family are the only oxidoreductases whose primary function is to produce reactive oxygen species. As such, the function and expression of these enzymes are tightly regulated. Thyrocytes express dual oxidase 2, which produces most of the H2O2 for thyroid hormone synthesis. Thyrocytes also express dual oxidase 1 and NADPH oxidase 4, but the roles of these enzymes are still unknown. Here, we review the structure, expression, localization and function of these enzymes. We focus on their potential role in thyroid cancer, which is characterized by increased expression of these enzymes.

During childhood, the thyroid gland is one of the most sensitive organs to the carcinogenetic effects of ionizing radiation that may lead to papillary thyroid carcinoma (PTC) associated with RET/PTC oncogene rearrangement. Exposure to ionizing radiation induces a transient "oxidative burst" through radiolysis of water, which can cause DNA damage and mediates part of the radiation effects. H 2 O 2 is a potent DNAdamaging agent that induces DNA double-strand breaks, and consequently, chromosomal aberrations. Irradiation by 5 Gy X-ray increased extracellular H 2 O 2 . Therefore, we investigated the implication of H 2 O 2 in the generation of RET/PTC1 rearrangement after X-ray exposure. We developed a highly specific and sensitive nested reverse transcription-PCR method. By using the human thyroid cell line HTori-3, previously found to produce RET/PTC1 after γ-irradiation, we showed that H 2 O 2 , generated during a 5 Gy X-ray irradiation, causes DNA double-strand breaks and contributes to RET/PTC1 formation. Pretreatment of cells with catalase, a scavenger of H 2 O 2 , significantly decreased RET/PTC1 rearrangement formation. Finally, RET/PTC chromosomal rearrangement was detected in HTori-3.1 cells after exposure of cells to H 2 O 2 (25 μmol/L), at a dose that did not affect the cell viability. This study shows for the first time that H 2 O 2 is able to cause RET/PTC1 rearrangement in thyroid cells and consequently highlights that oxidative stress could be responsible for the occurrence of RET/PTC1 rearrangement found in thyroid lesions even in the absence of radiation exposure.Cancer Res; 70(10); 4123-32. ©2010 AACR.

Aims: The dual oxidase 2 (DUOX2) protein belongs to the NADPH oxidase (NOX) family. As H 2 O 2 generator, it plays a key role in both thyroid hormone biosynthesis and innate immunity. DUOX2 forms with its maturation factor, DUOX activator 2 (DUOXA2), a stable complex at the cell surface that is crucial for the H 2 O 2 -generating activity, but the nature of their interaction is unknown. The contribution of some cysteine residues located in the N-terminal ectodomain of DUOX2 in a surface protein-protein interaction is suggested. We have investigated the involvement of different cysteine residues in the formation of covalent bonds that could be of critical importance for the function of the complex. Results: We report the identification and the characterization of an intramolecular disulfide bond between cys-124 of the N-terminal ectodomain and cys-1162 of an extracellular loop of DUOX2, which has important functional implications in both export and activity of DUOX2. This intramolecular bridge provides structural support for the formation of interdisulfide bridges between the N-terminal domain of DUOX2 and the two extracellular loops of its partner, DUOXA2. Innovation: Both stability and function of the maturation factor, DUOXA2, are dependent on the oxidative folding of DUOX2, indicating that DUOX2 displays a chaperone-like function with respect to its partner. Conclusions: The oxidative folding of DUOX2 that takes place in the endoplasmic reticulum (ER) appears to be a key event in the trafficking of the DUOX2/DUOXA2 complex as it promotes an appropriate conformation of the N-terminal region, which is propitious to subsequent covalent interactions with the maturation factor, DUOXA2. Antioxid. Redox Signal. 23,[724][725][726][727][728][729][730][731][732][733]

Introduction: Human papillomavirus (HPV) is associated with more human cancers than any other virus. Many studies have investigated the association between bladder cancer and HPV but the results remain controversial. The aim of the present study is to evaluate whether HPV have an etiological role in bladder carcinogenesis among Moroccan patients. Methodology: Forty-eight fresh biopsies (43 bladder tumors and 5 non-tumor samples) were collected for this purpose. Nested PCR with the consensus MY09/MY11 and GP5