Minimal oxidative load: a prerequisite for thyroid cell function

in Journal of Endocrinology
View More View Less
  • Unité de Morphologie Expérimentale, Université Catholique de Louvain, UCL-5251, 52 Avenue E. Mounier, B-1200 Bruxelles, Belgium

In addition to reactive oxygen species (ROS) produced by mitochondria during aerobic respiration, thyrocytes are continuously producing H2O2, a key element for hormonogenesis. Because nothing is known about ROS implication in normal non-stimulated cells, we studied their possible involvement in thyrocytes incubated with a potent antioxidant, N-acetylcysteine (NAC). NAC, which blocked the production of intracellular ROS, also decreased dual oxidases, thyroperoxidase, pendrin, and thyroglobulin protein and/or gene expression. By contrast, Na+/I symporter mRNA expression was unaffected. Among antioxidant systems, peroxiredoxin (PRDX) five expression was reduced by NAC, whereas peroxiredoxin three increased and catalase remained unchanged. In vivo, the expression of both dual oxidases and peroxiredoxin five proteins was also decreased by NAC. In conclusion, when intracellular ROS levels drop below a basal threshold, the expression of proteins involved in thyroid cell function is hampered. This suggests that keeping ROS at a minimal level is required for safeguarding thyrocyte function.

Abstract

In addition to reactive oxygen species (ROS) produced by mitochondria during aerobic respiration, thyrocytes are continuously producing H2O2, a key element for hormonogenesis. Because nothing is known about ROS implication in normal non-stimulated cells, we studied their possible involvement in thyrocytes incubated with a potent antioxidant, N-acetylcysteine (NAC). NAC, which blocked the production of intracellular ROS, also decreased dual oxidases, thyroperoxidase, pendrin, and thyroglobulin protein and/or gene expression. By contrast, Na+/I symporter mRNA expression was unaffected. Among antioxidant systems, peroxiredoxin (PRDX) five expression was reduced by NAC, whereas peroxiredoxin three increased and catalase remained unchanged. In vivo, the expression of both dual oxidases and peroxiredoxin five proteins was also decreased by NAC. In conclusion, when intracellular ROS levels drop below a basal threshold, the expression of proteins involved in thyroid cell function is hampered. This suggests that keeping ROS at a minimal level is required for safeguarding thyrocyte function.

Introduction

Reactive species include both reactive oxygen species (ROS) and reactive nitrogen species (RNS). In all cell types, ATP generation by mitochondrial aerobic respiration generates ROS as by-products of oxidative phosphorylations. In physiological conditions, ROS in excess must be constantly detoxified by antioxidant systems to avoid cell damage. In thyrocytes, H2O2 is a ROS that is required for hormonogenesis. It is produced from dual oxidases (DUOX1/2) (Dupuy et al. 1999, De Deken et al. 2002) at the apical pole of the cell, where it oxidizes iodide (actively transported across the basal membrane by Na+/I symporter (NIS, product of SLC5A5) (Dai et al. 1996)) into iodine in a reaction catalyzed by thyroperoxidase (TPO). Iodine is then incorporated into tyrosine residues of thyroglobulin (TG), again by TPO. Hence, H2O2 and free radicals are continuously produced in thyroid cells, even in physiological conditions (Denef et al. 1996, De Deken et al. 2002, Schweizer et al. 2008). Obviously, when ROS are overproduced in pathological situations, they may exert a wide range of actions, usually deleterious (Adler et al. 1999, Blokhina et al. 2003). To preserve cell integrity, several protective systems against ROS, such as peroxiredoxins (PRDX), catalase, and glutathione peroxidases are heavily expressed and active in thyrocytes. Thus, PRDX5 and glutathione peroxidase are increased in goitrous thyroids, both in human and rodents (Mano et al. 1997, Mutaku et al. 2002, Gerard et al. 2005, Poncin et al. 2008a). Cells can rely on various antioxidant tools to maintain the oxidative stress (OS) in strict limits to avoid OS becoming eventually harmful. Among them, selenium, which is required for glutathione peroxidase activity, has been shown to exert protective effects in various situations where OS is elevated, while its defect is deleterious (Contempre et al. 1993, 2004, Schomburg & Kohrle 2008, Schweizer et al. 2008).

Up to now, nothing has been reported about the physiological implication of ROS in normal resting thyrocytes. To answer this question, we analyzed the in vitro and in vivo expression of proteins involved in thyroid cell function in the presence of N-acetylcysteine (NAC), an agent supposed to deprive cells from intracellular ROS, because of potent antioxidant properties. We also investigated the regulation of antioxidant systems when the cell oxidative load is reduced.

Materials and Methods

Cell cultures

PCCL3 cells (a continuous line of non-transformed rat thyroid follicular cells, Fusco et al. 1987) were a gift from Dr F. Miot (Université Libre de Bruxelles, IRIBHN, Brussels, Belgium), and human thyroids from multinodular goiters were obtained at surgery after patients gave their informed consent. The study was performed after approval from the ethical committee had been received. Cells were cultured as previously reported (Poncin et al. 2008b). NAC (5, 2.5, 1, or 0.5 mM; Sigma–Aldrich) was added for 3 days in the cell medium containing 0.5% newborn calf serum and 1 mU/ml TSH. All experiments were repeated at least twice.

ROS production

Thyrocytes were incubated in multichamber glass slides in appropriate medium. ROS production was measured using a fluorescent dye, 2′, 7′ dichlorofluorescein diacetate (DCFH-DA, Molecular Probes, Paisley, UK). Phosphate buffer saline (PBS, pH 7.4)-washed thyroid cells were incubated in Krebs–Ringer–HEPES (KRH) medium (pH 7.4) containing DCFH-DA (25 μM) at 37 °C for 1 h. The excess of dye was removed by two washes with PBS. Cells were stained with Hoechst for 20 min and rinsed in PBS. Cover slides were mounted in fluorescent mounting medium (DakoCytomation, Carpinteria, CA, USA) for microscopic observation. ROS production was visualized on a fluorescent microscope equipped with a digital camera (Zeiss, Zaventem, Belgium).

Viability assay

Cell viability was assessed using the Alamar blue assay (Biosource International, Camarillo, CA, USA), as previously described (Gerard et al. 2006).

Apoptosis detection

Caspase activity was measured by using a CaspACE FITC-VAD-fmk in situ marker (Promega) that binds activated caspases, according to the manufacturer's instructions. Briefly, cells were incubated with 20 μM FITC-VAD-fmk at 37 °C for 20 min. Cells were then washed twice with PBS, dyed with Hoechst for 20 min, and rinsed in PBS. Cells were fixed in 10% buffered formalin for 30 min and rinsed with PBS. Cover slides were mounted in fluorescent mounting medium for microscopic observation. Cells treated with staurosporin (5 μM, Sigma) were used as positive controls.

Nitrite assay

Nitrite accumulation in the medium of human thyrocytes was measured by the Griess reaction using a commercially available kit (Promega).

Quantitative PCR

For each condition, cells from six individual wells were suspended in TriPure isolation reagent (Roche Diagnostics GmbH). Total RNA purification and reverse transcription were performed as previously described (Gerard et al. 2008). Quantitative PCR was performed in an iCycler apparatus (IQ5, Bio-Rad). cDNAs (2 μl) were mixed with 500 nM of each selected primers (Table 1) and SYBR green reaction mix (Bio-Rad) in a final volume of 25 μl. Reactions were performed as follows: 95 °C/1.5 min, followed by 40 cycles of 95°C/15 s, annealing temperature (Table 1)/45 s, and 81 °C/15 s. Amplification levels were normalized to those of β-actin. All melting curves were analyzed for each PCR to avoid genomic DNA amplification.

Table 1

Forward and reverse primers and annealing temperatures

Primer forwardPrimer reverseAnnealing T (°C)
Actin5′-CATCCTGCGTCTGGACCT-3′5′-AGGAGGAGCAATGATCTTGAT-3′62
r DUOX1/25′-GTGGCTGGAGGGAGCCAT-3′5′-CCGTGAACAGACTCCTGT-3′60
r TPO5′-CAGGTTTTGGTGGGAGAA-3′5′-CTGCACACTCATTAACATCTT-3′58
r NIS5′-GCGCTGCGACTCTCCCACTGAC-3′5′-GGCGGTAGAAGATCGGCAAGAAGA-3′60
r Pendrin5′-CATCAAGACACATCTCCGTTGGCCCT-3′5′-GGTACTTCCGTTACCACTGGGC-3′60
r TG5′-GCAGAACAACCACCATCACTGGAGC-3′5′-TGGCACTGGGGACTCTGGACTTGAC-3′59
h DUOX1/25′-GTGGCTGGCTGACATCAT-3′5′-TGCAGGGAGTTGAAGAA-3′58
h TPO5′-CACGATGCAGAGAAACCTCAA-3′5′-ATAGACTGGAGGGAGCCAT-3′60
h NIS 5′-ACCGCGCCCCACCTCTTTCTTATT-3′5′-CCCCCTCCTGATTCTGGTTGTTG-3′62
h Pendrin5′-TGGAACATCAAGACATATCTCAGTTG-3′5′-TGCTGCTGGATACGAGAAAGTG-3′60
h TG5′-CGCCTGGCGGCTCAGTCTACCTT-3′5′-AGCAGTTTCTGCGTGGGAG-3′60

Western blotting

Thyrocytes were suspended in Laemmli buffer (50 mM Tris–HCl, pH 6.8, 2% SDS, 10% glycerol), containing a protease inhibitor cocktail (Sigma), and were sonicated during 30 s. Protein concentration was determined using a BCA protein assay kit (Pierce, Rockfort, IL, USA). DUOXs (antibody provided by F. Miot, IRIBHN), TPO (antibody provided by J. Ruf, Université de la Méditerranée, Marseille, France), PRDX5 (antibody provided by B. Knoops, UCL, Louvain La Neuve), and β-actin (Sigma) western blottings were performed as previously described (Gerard et al. 2006). Membranes were blocked for 1 h at room temperature (RT) in PBS (pH 7.4), 5% non-fat dry milk, 0.1% Tween, and incubated overnight at 4 °C with the primary antibody at a dilution of 1/4000 (DUOXs, TPO), 1/10 000 (PRDX5), or 1/2000 (β-actin). Membranes were incubated for 1 h at RT with EnVision (1/200, DakoCytomation) peroxidase-labeled secondary antibody, and visualized with enhanced chemiluminescence (SuperSignal West Pico, Pierce) on CLXposure TM films (Pierce). Western blots were scanned and quantified by densitometry using the NIH Scion Image Analysis Software (National Institutes of Health, Bethesda, MA, USA). Densitometric values were normalized to β-actin expression.

PRDX5 immunofluorescence

Thyrocytes were cultured in multichamber glass slides in appropriate medium. They were fixed for 30 min in 4% paraformaldehyde, rinsed once with PBS, permeabilized for 15 min in a PBS–Triton 1% solution at RT, and washed with PBS supplemented with 1% BSA (PBS–BSA). Cells were then incubated for 1 h with PRDX5 primary antibody (1/75) at RT. After being washed in PBS, a FITC-conjugated secondary antibody was added for 1 h at RT at a dilution of 1/30 (anti-rabbit; DakoCytomation). Cover slides were mounted in fluorescent-mounting medium for microscopic observation.

Animals and treatments

NMRI mice of 2-months-old received a standard diet. Animals were given i.p. injected of saline solution of NAC (100 mg/kg per day) for 4 days. Mice were housed and handled according to Belgian regulation of Laboratory Animal Welfare.

Preparation of tissue samples for microscopy

Five animals of each group (control and NAC) were anesthetized with pentothal and thyroid lobes were dissected. One thyroid lobe was fixed in paraformaldehyde (4% in PBS) for 24 h and embedded in paraffin. Thick sections (5 μm) were used for PRDX5 immunohistochemistry. The second lobe was frozen and cryostat sections (5 μm) were used for DUOXs immunohistochemistry.

DUOXs and PRDX5 immunohistochemistry

Tissue sections were washed with PBS–BSA and incubated at RT for 30 min with normal goat serum (1/50, Vector Labs, Burlingham, CA, USA) in PBS–BSA. Slides were then incubated at RT for 1 h with DUOXs (1/75) or PRDX5 (1/250) primary antibodies. The antibody was detected using Envision (DakoCytomation) or ABC kit (Vector labs) for DUOXs and PRDX5 detection respectively. The peroxidase activity was revealed using AEC (DakoCytomation) as substrate. Sections were counterstained with Mayer's hematoxylin.

Data analysis and statistics

Data were expressed as mean ±s.e.m., n=6 for all assays. All experiments were repeated at least twice. Statistical analyses were performed using ANOVA followed by Tukey–Kramer multiple comparison test (GraphPad InStat, San Diego, CA, USA), or by unpaired t-test. P<0.05 was considered as statistically significant.

Results

NAC reduces intracellular ROS production without inducing apoptosis or affecting cell viability

In human thyrocytes, ROS, as detected by DCFH-DA fluorescence (Fig. 1A), were mainly observed in cytoplasm granules. NAC (1 mM)-treated cells showed a marked decrease in fluorescence. Identical results were obtained in PCCL3 cells (data not shown).

Figure 1
Figure 1

NAC reduces intracellular ROS production without inducing apoptosis or affecting cell viability. ROS production was assessed in control- and NAC-treated cells using DCFH-DA fluorescence (A, upper panel). Nuclei were stained with Hoechst (A, lower panel). Scale bars, 20 μm. Nitrite accumulation in the culture medium of human thyrocytes (B, left panel) was assessed using Griess reaction. Results are expressed as mean±s.e.m. of six (n=6) individual wells of one representative experiment. *P<0.05 versus control cells. Nos2 mRNA expression in human thyrocytes was analyzed by qPCR (B, right panel). Values, adjusted to the β-actin signal, are expressed as mean ±s.e.m. of one representative experiment (n=6). Apoptosis was detected using a CaspACE FITC-VAD-fmk in situ marker (C) in staurosporin (5 μM)-treated cells used as positive control, in control cells, and in NAC-treated cells. Nuclei were stained with Hoechst (lower panel). Scale bars, 25 μm. Cell viability was assessed using Alamar Blue assay (D). Full colour version of this figure available via .

Citation: Journal of Endocrinology 201, 1; 10.1677/JOE-08-0470

In human thyrocytes, nitrite levels (Fig. 1B, left panel), the stable end product of NO generation, were low in media from control cells and slightly increased in NAC (1 mM)-treated cells (1.6-fold, P<0.05). Nos2 mRNA expression was not influenced by NAC (Fig. 1B, right panel).

In staurosporin-treated cells, used as positive control for apoptosis, all nuclei were labeled with CaspACE FITC-VAD-fmk marker (Fig. 1C). By contrast, apoptosis was detected neither in control, nor in NAC (1 mM)-treated cells. Cell viability was therefore unaffected by NAC (Fig. 1D).

NAC reduces the expression of proteins involved in thyroid cell function

In human thyrocytes, DUOXs and TPO proteins were detected by Western blot (Table 2). NAC at concentrations ranging from 0.5 to 5 mM strongly decreased the expression of both proteins. As no difference was observed from one concentration to another, 1 mM was selected as reference concentration in all experiments. NAC also decreased Duoxs and Tpo mRNA expressions, both in human and PCCL3 cells (Table 3). By contrast, NAC had no effect on Nis mRNA expression. Tg and pendrin mRNAs were also downregulated in PCCL3 cells, but not in human cells. Thus, except NIS, all thyroid differentiation genes studied in this work were downregulated in ROS-deprived cells.

Table 2

DUOXs and TPO protein expressions in human thyrocytes. DUOXs and TPO protein expressions in human thyrocytes were analyzed by western blot

Human
ControlNAC 1 mM
DUOXs2.39±0.381.17±0.38*
TPO3.69±0.720.98±0.82*

Values, normalized to β-actin, are expressed as mean ±s.e.m. of one representative experiment (n=6). *P<0.05 versus control cells.

Table 3

Relative mRNA expression of thyroid genes. Duoxs, Tpo, Nis, Tg, and pendrin mRNA levels were measured by qRT-PCR in human thyrocytes and PCCL3 cells

HumanPCCL3
ControlNAC 1 mMControlNAC 1 mM
Duoxs1.61±0.440.93±0.27*1.09±0.130.62±0.14*
Tpo0.72±0.110.15±0.05*0.81±0.190.34±0.08*
Nis1.61±0.531.59±0.380.89±0.170.79±0.14
Tg0.88±0.260.85±0.110.63±0.160.33±0.1*
Pendrin0.83±0.180.46±0.080.74±0.090.35±0.05*

Values, normalized to β-actin, are expressed as mean ±s.e.m. of one representative experiment (n=6). *P<0.05 versus control cells.

NAC differentially affects the expression of PRDX5 and PRDX3

Both in human and PCCL3 cells, NAC decreased PRDX5 protein expression by 2.6-folds (P<0.05, Fig. 2A). The analysis of PRDX5 protein expression by immunofluorescence (Fig. 2B) showed a cytoplasmic and granular pattern, suggesting a mitochondrial localization of the protein (Banmeyer et al. 2005). By contrast, NAC induced a twofold increase in PRDX3 protein expression (P<0.05, Fig. 2C). Catalase protein expression remained unchanged (Fig. 2D).

Figure 2
Figure 2

NAC differentially regulates the expression of antioxidant enzymes in human thyrocytes. PRDX5 (A), PRDX3 (C), and catalase (D) protein expressions were analyzed by western blot. Densitometric values normalized to β-actin are expressed as mean ±s.e.m. of one representative experiment (n=6). *P<0.05 versus control cells. PRDX5 protein (B) localization was analyzed by immunofluorescence in control- and NAC-treated cells. Scale bars: 20 μm. Full colour version of this figure available via .

Citation: Journal of Endocrinology 201, 1; 10.1677/JOE-08-0470

NAC decreases DUOXs and PRDX5 expressions in mouse thyroids

In mouse thyroids, DUOXs was detected at the apical pole of thyrocytes (Fig. 3A). In NAC- treated mice, DUOXs immunostaining was more faint and detected in few cells (Fig. 3B). A PRDX5 signal (Fig. 3C) was present in the cytosol and in nuclei, as previously reported (Poncin et al. 2008a). In NAC-treated mice (Fig. 3D), the signal was strongly reduced, which is in accordance with the above reported in vitro observations.

Figure 3
Figure 3

NAC decreases DUOX and PRDX5 expressions in mouse thyroids. DUOXs protein expression was detected by immunohistochemistry on frozen sections. In control thyroids (A), DUOXs was detected at the apical pole of the cells (arrow). In NAC-treated mice (B), DUOXs was barely detected only in few cells. PRDX5 protein expression was detected by immunohistochemistry on paraffin sections. In control thyroids (C), PRDX5 was detected in the cytoplasm and in nuclei. In NAC-treated cells (D), the staining was greatly decreased. Scale bars, 10 μm. Full colour version of this figure available via .

Citation: Journal of Endocrinology 201, 1; 10.1677/JOE-08-0470

Discussion

This study shows that PCCL3 and human thyroid cells produce a given amount of intracellular ROS in basal conditions, likely as a result of physiological aerobic metabolism. But thyrocytes are somewhat unique in that they produce H2O2 that, along with iodine, is a key element for TH synthesis. Thus, ROS in the thyroid are definitively more than just by-products of aerobic respiration. They are in fact intrinsically involved in endocrine function. H2O2 is produced outside the cell by NADPH oxidases, DUOXs localized at the apical pole of the thyrocyte (De Deken et al. 2002). The hormone synthesis then occurs in the follicular lumen, close to the apical pole. Until now, H2O2 has always been detected in culture media (Bjorkman & Ekholm 1992, Fortemaison et al. 2005), never inside the cell. Here, we show for the first time that ROS are physiologically produced into cells without provoking significant cell damage, despite the fact that basal DNA damage in thyrocytes is higher than in other tissues (Krohn et al. 2007, Maier et al. 2007). H2O2 is likely one of these ROS as theDCFH-DA probe mainly reacts with H2O2. Besides DUOXs produced H2O2, other possible sources of intracellular ROS include mitochondria and peroxisomes (Bedard & Krause 2007). It is likely that ROS other than H2O2, such as hydroxyl radicals and anion superoxide, are produced in physiological conditions, but likely in very low amounts, as suggested by previous models (Denef et al. 1996). Further studies are required to find out the source and the type of intracellular ROS, and to sort out those resulting from aerobic metabolism as in every other organs, and those specifically involved in hormone synthesis.

NAC may decrease cell OS, either directly, as a source of sulfhydryl groups that neutralize ROS, or indirectly as glutathione precursor by restoring glutathione levels (GSH) (Gillissen & Nowak 1998). The present study shows that NAC decreases intracellular ROS to very low levels without inducing apoptosis or affecting cell viability. This marked decrease in intracellular ROS levels is associated with decreased DUOXs, TPO, TG, and pendrin expressions, but not Nis mRNA expression. Such effects on DUOXs expression were also observed in vivo. As NAC may also influence GSH intracellular levels, it is possible that some specific biochemical processes might be altered by glutathionylation, i.e., the reversible formation of disulfide bonds between protein cysteines and glutathione, as recently suggested (Cao et al. 2005, le-Donne et al. 2007). Although, ROS are sometimes known to act as transduction signals, for instance in cells stimulated by ligands (cytokines, growth factors), or in pathological situations (Lander 1997, Finkel 1998, Adler et al. 1999), it is the first time that the maintenance of a minimal intracellular oxidative load is shown to be required for safeguarding endocrine functions. This has already been suggested in pancreatic cells or in the lung (Lenzen 2008), but without any convincing demonstration. Thus, when the oxidative load in thyrocytes drops below a minimal threshold, various actors involved in the TH synthesis are also downregulated. This might eventually be relevant in terms of thyroid function in patients treated with NAC, but as far as we know, there is no robust data yet reporting such interactions in the literature.

In addition to ROS, thyrocytes also produce RNS in low amounts (basal nitrite release) associated with NOS2 and NOS3 expressions (Colin et al. 1995), suggesting a role for NO in normal thyroids. In contrast with ROS, NAC increased NO production without affecting Nos2 mRNA expression. The interpretation of this result is somewhat difficult as NAC-induced effects on NOS2 expression remains a matter of debate. It may either increase, or decrease, or have no effect on NOS2 expression and NO release (Ramasamy et al. 1999, Vos et al. 1999, Chen et al. 2000, Zafarullah et al. 2003). Nevertheless, NO levels are particularly low when compared with those produced for instance in cells incubated with Th1 cytokines (Kasai et al. 1995, van den Hove et al. 2002, Gerard et al. 2006). Although this slight increase in NO production may appear odd in cells treated with NAC, one should keep in mind that superoxide anions, which are constantly produced by mitochondria during aerobic respiration (Bedard & Krause 2007), react with NO in normal cells to form low amounts of nitrotyrosines. Although this remains to be proven, it could be speculated that in case of NAC-induced ROS suppression, superoxide anions are no more available in sufficient amounts to react with NO. Consequently, NO might then be released in culture media in slightly greater amounts.

As the oxidative load was deeply affected by NAC, it made sense to analyze how antioxidant systems react in such conditions. Our results indicate that they are actually influenced by intracellular ROS, especially PRDXs whose expression is known to be regulated by OS (Bast et al. 2002). In our study, PRDX5 expression decreased, while PRDX3 increased in NAC-treated cells, indicating that both proteins are differently regulated in conditions of ROS depletion. By contrast, catalase protein expression remained unchanged, which is in line with the low enzymatic activity previously reported in the thyroid (Nadolnik & Valentyukevich 2007). Thus, in accordance with previous results (Gerard et al. 2005, Poncin et al. 2008a), our present data confirm that PRDXs 5 and 3 are in fact highly regulated in the thyroid, suggesting that they may play important roles in detoxification processes along side the glutathione peroxidase system (Nadolnik & Valentyukevich 2007).

In conclusion, our results showed for the first time that in physiological conditions, thyroid cells produce intracytoplasmic ROS. When their intracellular levels drop very low, as in NAC-treated cells, the expression of important proteins involved in TH synthesis is hampered. This strongly suggests that the maintenance of the oxidative load above a minimum threshold is required to safeguard the function of thyroid cells.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

This work was supported by the grant no. 3.4552.08 (Fonds National de la Recherche Scientifique (FNRS-FRSM)). A-C Gérard is a postdoctoral researcher (Fonds National de la Recherche Scientifique).

References

  • Adler V, Yin Z, Tew KD & Ronai Z 1999 Role of redox potential and reactive oxygen species in stress signaling. Oncogene 18 61046111.

  • Banmeyer I, Marchand C, Clippe A & Knoops B 2005 Human mitochondrial peroxiredoxin 5 protects from mitochondrial DNA damages induced by hydrogen peroxide. FEBS Letter 579 23272333.

    • Search Google Scholar
    • Export Citation
  • Bast A, Wolf G, Oberbaumer I & Walther R 2002 Oxidative and nitrosative stress induces peroxiredoxins in pancreatic beta cells. Diabetologia 45 867876.

    • Search Google Scholar
    • Export Citation
  • Bedard K & Krause KH 2007 The NOX, family of ROS-generating NADPH oxidases: physiology and pathophysiology. Physiological Reviews 87 245313.

  • Bjorkman U & Ekholm R 1992 Hydrogen peroxide generation and its regulation in FRTL-5 and porcine thyroid cells. Endocrinology 130 393399.

  • Blokhina O, Virolainen E & Fagerstedt KV 2003 Antioxidants, oxidative damage and oxygen deprivation stress: a review. Annals of Botany 91 179194.

    • Search Google Scholar
    • Export Citation
  • Cao X, Kambe F, Lu X, Kobayashi N, Ohmori S & Seo H 2005 Glutathionylation of two cysteine residues in paired domain regulates DNA binding activity of Pax-8. Journal of Biolgical Chemistry 280 2590125906.

    • Search Google Scholar
    • Export Citation
  • Chen G, Wang SH & Warner TD 2000 Regulation of iNOS mRNA levels in endothelial cells by glutathione, a double-edged sword. Free Radical Research 32 223234.

    • Search Google Scholar
    • Export Citation
  • Colin IM, Nava E, Toussaint D, Maiter DM, vanDenhove MF, Luscher TF, Ketelslegers JM, Denef JF & Jameson JL 1995 Expression of nitric oxide synthase isoforms in the thyroid gland: evidence for a role of nitric oxide in vascular control during goiter formation. Endocrinology 136 52835290.

    • Search Google Scholar
    • Export Citation
  • Contempre B, Denef JF, Dumont JE & Many MC 1993 Selenium deficiency aggravates the necrotizing effects of a high iodide dose in iodine deficient rats. Endocrinology 132 18661868.

    • Search Google Scholar
    • Export Citation
  • Contempre B, de Escobar GM, Denef JF, Dumont JE & Many MC 2004 Thiocyanate induces cell necrosis and fibrosis in selenium- and iodine-deficient rat thyroids: a potential experimental model for myxedematous endemic cretinism in central Africa. Endocrinology 145 9941002.

    • Search Google Scholar
    • Export Citation
  • Dai G, Levy O & Carrasco N 1996 Cloning and characterization of the thyroid iodide transporter. Nature 379 458460.

  • De Deken X, Wang D, Dumont JE & Miot F 2002 Characterization of ThOX proteins as components of the thyroid H(2)O(2)-generating system. Experimental Cell Research 273 187196.

    • Search Google Scholar
    • Export Citation
  • Denef JF, Many MC & van den Hove MF 1996 Iodine-induced thyroid inhibition and cell necrosis: two consequences of the same free-radical mediated mechanism? Molecular Cellular Endocrinology 121 101103.

    • Search Google Scholar
    • Export Citation
  • Dupuy C, Ohayon R, Valent A, Noel-Hudson MS, Deme D & Virion A 1999 Purification of a novel flavoprotein involved in the thyroid NADPH oxidase. Cloning of the porcine and human cdnas. Journal of Biolgical Chemistry 274 3726537269.

    • Search Google Scholar
    • Export Citation
  • le-Donne I, Rossi R, Giustarini D, Colombo R & Milzani A 2007 S-glutathionylation in protein redox regulation. Free Radical Biology and Medicine 43 883898.

    • Search Google Scholar
    • Export Citation
  • Finkel T 1998 Oxygen radicals and signaling. Current Opinion in Cell Biology 10 248253.

  • Fortemaison N, Miot F, Dumont JE & Dremier S 2005 Regulation of H2O2 generation in thyroid cells does not involve Rac1 activation. European Journal Endocrinology 152 127133.

    • Search Google Scholar
    • Export Citation
  • Fusco A, Berlingieri MT, Di Fiore PP, Portella G, Grieco M & Vecchio G 1987 One- and two-step transformations of rat thyroid epithelial cells by retroviral oncogenes. Molecular and Cellular Biology 7 33653370.

    • Search Google Scholar
    • Export Citation
  • Gerard AC, Many MC, Daumerie C, Knoops B & Colin IM 2005 Peroxiredoxin 5 expression in the human thyroid gland. Thyroid 15 205209.

  • Gerard AC, Boucquey M, van den Hove MF & Colin IM 2006 Expression of TPO and ThOXs in human thyrocytes is downregulated by IL-1α/IFN-γ, an effect partially mediated by nitric oxide. American Journal of Physiology-Endocrinology and Metabolism 291 E242E253.

    • Search Google Scholar
    • Export Citation
  • Gerard AC, Poncin S, Caetano B, Sonveaux P, Audinot JN, Feron O, Colin IM & Soncin F 2008 Iodine deficiency induces a thyroid stimulating hormone-independent early phase of microvascular reshaping in the thyroid. American Journal of Pathology 172 748760.

    • Search Google Scholar
    • Export Citation
  • Gillissen A & Nowak D 1998 Characterization of N-acetylcysteine and ambroxol in anti-oxidant therapy. Respiratory Medicine 92 609623.

  • van den Hove MF, Stoenoiu MS, Croizet K, Couvreur M, Courtoy PJ, Devuyst O & Colin IM 2002 Nitric oxide is involved in interleukin-1alpha-induced cytotoxicity in polarised human thyrocytes. Journal of Endocrinology 173 177185.

    • Search Google Scholar
    • Export Citation
  • Kasai K, Hattori Y, Nakanishi N, Manaka K, Banba N, Motohashi S & Shimoda S 1995 Regulation of inducible nitric oxide production by cytokines in human thyrocytes in culture. Endocrinology 136 42614270.

    • Search Google Scholar
    • Export Citation
  • Krohn K, Maier J & Paschke R 2007 Mechanisms of disease: hydrogen peroxide, DNA damage and mutagenesis in the development of thyroid tumors. Nature Clinical Practice. Endocrinology & Metabolism 3 713720.

    • Search Google Scholar
    • Export Citation
  • Lander HM 1997 An essential role for free radicals and derived species in signal transduction. FASEB Journal 11 118124.

  • Lenzen S 2008 Oxidative stress: the vulnerable beta-cell. Biochemical Society Transactions 36 343347.

  • Maier J, van SH, van OC, Paschke R, Weiss RE & Krohn K 2007 Iodine deficiency activates antioxidant genes and causes DNA damage in the thyroid gland of rats and mice. Biochemica and Biophysica Acta 1773 990999.

    • Search Google Scholar
    • Export Citation
  • Mano T, Shinohara R, Iwase K, Kotake M, Hamada M, Uchimuro K, Hayakawa N, Hayashi R, Nakai A & Ishizuki Y 1997 Changes in free radical scavengers and lipid peroxide in thyroid glands of various thyroid disorders. Hormone and Metabolic Research 29 351354.

    • Search Google Scholar
    • Export Citation
  • Mutaku JF, Poma JF, Many MC, Denef JF & van den Hove MF 2002 Cell necrosis and apoptosis are differentially regulated during goitre development and iodine-induced involution. Journal of Endocrinology 172 375386.

    • Search Google Scholar
    • Export Citation
  • Nadolnik LI & Valentyukevich OI 2007 Peculiarities of the antioxidant status of the thyroid gland. Bulletin of Experimental Biology and Medicine 144 529531.

    • Search Google Scholar
    • Export Citation
  • Poncin S, Gerard AC, Boucquey M, Senou M, Calderon PB, Knoops B, Lengele B, Many MC & Colin IM 2008a Oxidative stress in the thyroid gland: from harmlessness to hazard depending on the iodine content. Endocrinology 149 424433.

    • Search Google Scholar
    • Export Citation
  • Poncin S, Lengele B, Colin IM & Gerard AC 2008b Differential interactions between Th1/Th2, Th1/Th3, and Th2/Th3 cytokines in the regulation of TPO and DUOX expression, and of thyroglobulin secretion in thyrocytes in vitro. Endocrinology 149 15341542.

    • Search Google Scholar
    • Export Citation
  • Ramasamy S, Drummond GR, Ahn J, Storek M, Pohl J, Parthasarathy S & Harrison DG 1999 Modulation of expression of endothelial nitric oxide synthase by nordihydroguaiaretic acid, a phenolic antioxidant in cultured endothelial cells. Molecular Pharmacology 56 116123.

    • Search Google Scholar
    • Export Citation
  • Schomburg L & Kohrle J 2008 On the importance of selenium and iodine metabolism for thyroid hormone biosynthesis and human health. Molecular Nutrition & Food Research 52 12351246.

    • Search Google Scholar
    • Export Citation
  • Schweizer U, Chiu J & Kohrle J 2008 Peroxides and peroxide-degrading enzymes in the thyroid. Antioxidants & Redox Signaling 10 15771592.

  • Vos TA, Van GH, Tuyt L, De Jager-Krikken A, Leuvenink R, Kuipers F, Jansen PL & Moshage H 1999 Expression of inducible nitric oxide synthase in endotoxemic rat hepatocytes is dependent on the cellular glutathione status. Hepatology 29 421426.

    • Search Google Scholar
    • Export Citation
  • Zafarullah M, Li WQ, Sylvester J & Ahmad M 2003 Molecular mechanisms of N-acetylcysteine actions. Cellular and Molecular Life Sciences 60 620.

If the inline PDF is not rendering correctly, you can download the PDF file here.

 

      Society for Endocrinology

Sept 2018 onwards Past Year Past 30 Days
Abstract Views 0 0 0
Full Text Views 628 312 24
PDF Downloads 191 85 5
  • View in gallery

    NAC reduces intracellular ROS production without inducing apoptosis or affecting cell viability. ROS production was assessed in control- and NAC-treated cells using DCFH-DA fluorescence (A, upper panel). Nuclei were stained with Hoechst (A, lower panel). Scale bars, 20 μm. Nitrite accumulation in the culture medium of human thyrocytes (B, left panel) was assessed using Griess reaction. Results are expressed as mean±s.e.m. of six (n=6) individual wells of one representative experiment. *P<0.05 versus control cells. Nos2 mRNA expression in human thyrocytes was analyzed by qPCR (B, right panel). Values, adjusted to the β-actin signal, are expressed as mean ±s.e.m. of one representative experiment (n=6). Apoptosis was detected using a CaspACE FITC-VAD-fmk in situ marker (C) in staurosporin (5 μM)-treated cells used as positive control, in control cells, and in NAC-treated cells. Nuclei were stained with Hoechst (lower panel). Scale bars, 25 μm. Cell viability was assessed using Alamar Blue assay (D). Full colour version of this figure available via .

  • View in gallery

    NAC differentially regulates the expression of antioxidant enzymes in human thyrocytes. PRDX5 (A), PRDX3 (C), and catalase (D) protein expressions were analyzed by western blot. Densitometric values normalized to β-actin are expressed as mean ±s.e.m. of one representative experiment (n=6). *P<0.05 versus control cells. PRDX5 protein (B) localization was analyzed by immunofluorescence in control- and NAC-treated cells. Scale bars: 20 μm. Full colour version of this figure available via .

  • View in gallery

    NAC decreases DUOX and PRDX5 expressions in mouse thyroids. DUOXs protein expression was detected by immunohistochemistry on frozen sections. In control thyroids (A), DUOXs was detected at the apical pole of the cells (arrow). In NAC-treated mice (B), DUOXs was barely detected only in few cells. PRDX5 protein expression was detected by immunohistochemistry on paraffin sections. In control thyroids (C), PRDX5 was detected in the cytoplasm and in nuclei. In NAC-treated cells (D), the staining was greatly decreased. Scale bars, 10 μm. Full colour version of this figure available via .

  • Adler V, Yin Z, Tew KD & Ronai Z 1999 Role of redox potential and reactive oxygen species in stress signaling. Oncogene 18 61046111.

  • Banmeyer I, Marchand C, Clippe A & Knoops B 2005 Human mitochondrial peroxiredoxin 5 protects from mitochondrial DNA damages induced by hydrogen peroxide. FEBS Letter 579 23272333.

    • Search Google Scholar
    • Export Citation
  • Bast A, Wolf G, Oberbaumer I & Walther R 2002 Oxidative and nitrosative stress induces peroxiredoxins in pancreatic beta cells. Diabetologia 45 867876.

    • Search Google Scholar
    • Export Citation
  • Bedard K & Krause KH 2007 The NOX, family of ROS-generating NADPH oxidases: physiology and pathophysiology. Physiological Reviews 87 245313.

  • Bjorkman U & Ekholm R 1992 Hydrogen peroxide generation and its regulation in FRTL-5 and porcine thyroid cells. Endocrinology 130 393399.

  • Blokhina O, Virolainen E & Fagerstedt KV 2003 Antioxidants, oxidative damage and oxygen deprivation stress: a review. Annals of Botany 91 179194.

    • Search Google Scholar
    • Export Citation
  • Cao X, Kambe F, Lu X, Kobayashi N, Ohmori S & Seo H 2005 Glutathionylation of two cysteine residues in paired domain regulates DNA binding activity of Pax-8. Journal of Biolgical Chemistry 280 2590125906.

    • Search Google Scholar
    • Export Citation
  • Chen G, Wang SH & Warner TD 2000 Regulation of iNOS mRNA levels in endothelial cells by glutathione, a double-edged sword. Free Radical Research 32 223234.

    • Search Google Scholar
    • Export Citation
  • Colin IM, Nava E, Toussaint D, Maiter DM, vanDenhove MF, Luscher TF, Ketelslegers JM, Denef JF & Jameson JL 1995 Expression of nitric oxide synthase isoforms in the thyroid gland: evidence for a role of nitric oxide in vascular control during goiter formation. Endocrinology 136 52835290.

    • Search Google Scholar
    • Export Citation
  • Contempre B, Denef JF, Dumont JE & Many MC 1993 Selenium deficiency aggravates the necrotizing effects of a high iodide dose in iodine deficient rats. Endocrinology 132 18661868.

    • Search Google Scholar
    • Export Citation
  • Contempre B, de Escobar GM, Denef JF, Dumont JE & Many MC 2004 Thiocyanate induces cell necrosis and fibrosis in selenium- and iodine-deficient rat thyroids: a potential experimental model for myxedematous endemic cretinism in central Africa. Endocrinology 145 9941002.

    • Search Google Scholar
    • Export Citation
  • Dai G, Levy O & Carrasco N 1996 Cloning and characterization of the thyroid iodide transporter. Nature 379 458460.

  • De Deken X, Wang D, Dumont JE & Miot F 2002 Characterization of ThOX proteins as components of the thyroid H(2)O(2)-generating system. Experimental Cell Research 273 187196.

    • Search Google Scholar
    • Export Citation
  • Denef JF, Many MC & van den Hove MF 1996 Iodine-induced thyroid inhibition and cell necrosis: two consequences of the same free-radical mediated mechanism? Molecular Cellular Endocrinology 121 101103.

    • Search Google Scholar
    • Export Citation
  • Dupuy C, Ohayon R, Valent A, Noel-Hudson MS, Deme D & Virion A 1999 Purification of a novel flavoprotein involved in the thyroid NADPH oxidase. Cloning of the porcine and human cdnas. Journal of Biolgical Chemistry 274 3726537269.

    • Search Google Scholar
    • Export Citation
  • le-Donne I, Rossi R, Giustarini D, Colombo R & Milzani A 2007 S-glutathionylation in protein redox regulation. Free Radical Biology and Medicine 43 883898.

    • Search Google Scholar
    • Export Citation
  • Finkel T 1998 Oxygen radicals and signaling. Current Opinion in Cell Biology 10 248253.

  • Fortemaison N, Miot F, Dumont JE & Dremier S 2005 Regulation of H2O2 generation in thyroid cells does not involve Rac1 activation. European Journal Endocrinology 152 127133.

    • Search Google Scholar
    • Export Citation
  • Fusco A, Berlingieri MT, Di Fiore PP, Portella G, Grieco M & Vecchio G 1987 One- and two-step transformations of rat thyroid epithelial cells by retroviral oncogenes. Molecular and Cellular Biology 7 33653370.

    • Search Google Scholar
    • Export Citation
  • Gerard AC, Many MC, Daumerie C, Knoops B & Colin IM 2005 Peroxiredoxin 5 expression in the human thyroid gland. Thyroid 15 205209.

  • Gerard AC, Boucquey M, van den Hove MF & Colin IM 2006 Expression of TPO and ThOXs in human thyrocytes is downregulated by IL-1α/IFN-γ, an effect partially mediated by nitric oxide. American Journal of Physiology-Endocrinology and Metabolism 291 E242E253.

    • Search Google Scholar
    • Export Citation
  • Gerard AC, Poncin S, Caetano B, Sonveaux P, Audinot JN, Feron O, Colin IM & Soncin F 2008 Iodine deficiency induces a thyroid stimulating hormone-independent early phase of microvascular reshaping in the thyroid. American Journal of Pathology 172 748760.

    • Search Google Scholar
    • Export Citation
  • Gillissen A & Nowak D 1998 Characterization of N-acetylcysteine and ambroxol in anti-oxidant therapy. Respiratory Medicine 92 609623.

  • van den Hove MF, Stoenoiu MS, Croizet K, Couvreur M, Courtoy PJ, Devuyst O & Colin IM 2002 Nitric oxide is involved in interleukin-1alpha-induced cytotoxicity in polarised human thyrocytes. Journal of Endocrinology 173 177185.

    • Search Google Scholar
    • Export Citation
  • Kasai K, Hattori Y, Nakanishi N, Manaka K, Banba N, Motohashi S & Shimoda S 1995 Regulation of inducible nitric oxide production by cytokines in human thyrocytes in culture. Endocrinology 136 42614270.

    • Search Google Scholar
    • Export Citation
  • Krohn K, Maier J & Paschke R 2007 Mechanisms of disease: hydrogen peroxide, DNA damage and mutagenesis in the development of thyroid tumors. Nature Clinical Practice. Endocrinology & Metabolism 3 713720.

    • Search Google Scholar
    • Export Citation
  • Lander HM 1997 An essential role for free radicals and derived species in signal transduction. FASEB Journal 11 118124.

  • Lenzen S 2008 Oxidative stress: the vulnerable beta-cell. Biochemical Society Transactions 36 343347.

  • Maier J, van SH, van OC, Paschke R, Weiss RE & Krohn K 2007 Iodine deficiency activates antioxidant genes and causes DNA damage in the thyroid gland of rats and mice. Biochemica and Biophysica Acta 1773 990999.

    • Search Google Scholar
    • Export Citation
  • Mano T, Shinohara R, Iwase K, Kotake M, Hamada M, Uchimuro K, Hayakawa N, Hayashi R, Nakai A & Ishizuki Y 1997 Changes in free radical scavengers and lipid peroxide in thyroid glands of various thyroid disorders. Hormone and Metabolic Research 29 351354.

    • Search Google Scholar
    • Export Citation
  • Mutaku JF, Poma JF, Many MC, Denef JF & van den Hove MF 2002 Cell necrosis and apoptosis are differentially regulated during goitre development and iodine-induced involution. Journal of Endocrinology 172 375386.

    • Search Google Scholar
    • Export Citation
  • Nadolnik LI & Valentyukevich OI 2007 Peculiarities of the antioxidant status of the thyroid gland. Bulletin of Experimental Biology and Medicine 144 529531.

    • Search Google Scholar
    • Export Citation
  • Poncin S, Gerard AC, Boucquey M, Senou M, Calderon PB, Knoops B, Lengele B, Many MC & Colin IM 2008a Oxidative stress in the thyroid gland: from harmlessness to hazard depending on the iodine content. Endocrinology 149 424433.

    • Search Google Scholar
    • Export Citation
  • Poncin S, Lengele B, Colin IM & Gerard AC 2008b Differential interactions between Th1/Th2, Th1/Th3, and Th2/Th3 cytokines in the regulation of TPO and DUOX expression, and of thyroglobulin secretion in thyrocytes in vitro. Endocrinology 149 15341542.

    • Search Google Scholar
    • Export Citation
  • Ramasamy S, Drummond GR, Ahn J, Storek M, Pohl J, Parthasarathy S & Harrison DG 1999 Modulation of expression of endothelial nitric oxide synthase by nordihydroguaiaretic acid, a phenolic antioxidant in cultured endothelial cells. Molecular Pharmacology 56 116123.

    • Search Google Scholar
    • Export Citation
  • Schomburg L & Kohrle J 2008 On the importance of selenium and iodine metabolism for thyroid hormone biosynthesis and human health. Molecular Nutrition & Food Research 52 12351246.

    • Search Google Scholar
    • Export Citation
  • Schweizer U, Chiu J & Kohrle J 2008 Peroxides and peroxide-degrading enzymes in the thyroid. Antioxidants & Redox Signaling 10 15771592.

  • Vos TA, Van GH, Tuyt L, De Jager-Krikken A, Leuvenink R, Kuipers F, Jansen PL & Moshage H 1999 Expression of inducible nitric oxide synthase in endotoxemic rat hepatocytes is dependent on the cellular glutathione status. Hepatology 29 421426.

    • Search Google Scholar
    • Export Citation
  • Zafarullah M, Li WQ, Sylvester J & Ahmad M 2003 Molecular mechanisms of N-acetylcysteine actions. Cellular and Molecular Life Sciences 60 620.