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Experimental Biology and Medicine 227:141-146 (2002)
© 2002 Society for Experimental Biology and Medicine

ORIGINAL ARTICLE

Generation of Oxygen Free Radicals in Thyroid Cells and Inhibition of Thyroid Peroxidase

Masahiro Sugawara1,*, Yoshinobu Sugawara*, Katherine Wen* and Cecilia Giulivi{dagger}

* The Division of Endocrinology and Metabolism, West Los Angeles Veterans Affairs Medical Center and the Department of Medicine, University of California, Los Angeles, School of Medicine, Los Angeles, California 90073; and
{dagger} The Department of Molecular Pharmacology and Toxicology, School of Pharmacy, University of Southern California, Los Angeles, California 90033


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We examined whether superoxide (O2-) is produced as a precursor of hydrogen peroxide (H2O2) in cultured thyroid cells using the cytochrome c method and the electron paramagnetic resonance (EPR) method. No O2- or its related radicals was detected in thyroid cells under the physiological condition. The presence of quinone, 2,3-dimethoxy-l-naphthoquinone (DMNQ), or 2-methyl-1, 4-naphthoquinone (menadione), in the medium produced O2- and hydroxyl radicals (OH•); the amount of H2O2 generation was also increased. Incubation of follicles with DMNQ or menadione inhibited iodine organification (a step of thyroid hormone formation) and its catalytic enzyme, thyroid peroxidase (TPO). This inhibition should be caused by reactive oxygen species because the two quinones, particularly DMNQ, exert their effect through the generation of reactive oxygen species. It is speculated that the site-specific inactivation of TPO might have occurred at the heme-linked histidine residue of the TPO molecule, a critical amino acid for enzyme activity because OH• (vicious free radicals) can be formed at the iron-linked amino acid. TPO mRNA level and electrophoretic mobility of TPO were not inhibited by quinones. Our study suggests that thyroid H2O2 is produced by divalent reduction of oxygen without O2- generation. If thyroid cells happen to be exposed to significant amount of reactive oxygen species, TPO and subsequent thyroid hormone formation are inhibited.

Key Words: thyroid peroxidase • superoxide • hydrogen peroxide • quinones • thyroid cells


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The thyroid gland produces H2O2 by the NADPH oxidase system of the apical membrane (1–3) and it utilizes H2O2 as a substrate of thyroid peroxidase (TPO) for thyroid hormone formation. The thyroid gland contains superoxide dismutase (SOD) (4–6), which converts O2- to H2O2. However, whether the thyroid cell actually produces O2- has been controversial; conflicting results have been published (7–9). In a simplified cell-free experiment, incubation of lactperoxidase (an analog of TPO) with excess H2O2 in the presence of iodide has been shown to produce O2- and hydroxyl radicals (OH•) (10). Whether this phenomenon actually happens in the thyroid cell has not been tested. It is conceivable that the thyroid gland is exposed to oxygen free radicals during radiation therapy to the neck (ionized radiation) (11,12), acute bacterial infection of the thyroid gland (respiratory burst from activated leukocytes) (13), and ras-related thyroid tumors (as an O2- signal from mutated ras oncogen) (14). However, the effect of oxygen free radicals on thyroid cell function, particularly on thyroid hormone formation, is still unknown. In this study, we first examined whether thyroid cells produce O2- under the physiological condition. Then, intracellular oxygen free radicals were generated by menadione or 2,3-dimethoxy-l-naphthoquinone (DMNQ) (15,16), and the effects of oxygen free radicals on iodine organification and cellular TPO were examined.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Primary Culture of Porcine Thyroid Follicles.
Fresh porcine thyroid tissue were procured from a local abattoir (Farmer John Co., Los Angeles, CA) and follicles were isolated by collagenase digestion as described (17). Cultured porcine follicles were used to examine generation of reactive oxygen species, iodide uptake, iodine organification, TPO activity, TPO immunoblot, and TPO mRNA levels. The culture conditions were described previously (17). For the demonstration of reactive oxygen species, cultured FRTL-5 rat thyroid cells were also used.

Biochemical Analysis
Reagents.
Reagents and culture medium were obtained from Sigma Chemical Co. (St. Louis, MO) unless specified. DMNQ was obtained from Oxis International (Portland, OR).

Measurement of H2O2 production.
The content of H2O2 in the medium was measured by the homovanillic acid method (18). Porcine follicles or FRTL-5 rat thyroid cells cultured in 12-well plates up to 80% confluency were washed twice with Tyrode salt solution. The incubation medium for the H2O2 assay contained testing agents, 440 µM homovanillic acid, and 0.5 units/ml horseradish peroxidase in a total volume of 1.0 ml of Tyrode salt solution containing 1.8 mM CaCl2, pH 7.4. Incubation was done for 1 hr at 37°C by floating plates in a water bath without a cover lid. The amount of H2O2 generated during incubation was measured in an Amicon fluorometer at the excitation and emission wavelengths of 315 nm and 425 nm, respectively (18). All reagents used for experiments were tested for the presence of nonspecific interference in the homovanillic acid method.

Measurement of O2- by the reduction of acetylated cytochrome c.
The amount of O2- generated during incubation was measured by the degree of the reduction of acetylated cytochrome c (19). The advantage of using acetylated cytochrome c over nonacetylated cytochrome c is to eliminate interference by cytochrome c reductase as previously described (8,19). Porcine thyroid follicles or FRTL-5 cells cultured in 12-well plates were washed twice with Tyrode salt solution. Then, 1 ml of Tyrode salt solution containing agents to be tested was added to each well with or without 30 µg of SOD. The reaction for the O2- assay was initiated by adding acetylated cytochrome c with a final concentration of 80 µM, and incubation was carried out for 1 hr at 37°C in a water bath without a cover lid. At the end of incubation, the medium was removed and centrifuged at 10,000g for 2 min. The absorbance of the supernatant was measured at the wavelength of 550 nm (20) in a 160 U Shimadzu double beam spectrophotometer (Shimadzu, Kyoto, Japan). Nonspecific reduction of cytochrome c by testing agents and SOD without cells was examined and subtracted in this assay system. The amount of O2- produced was calculated based on the extinction coefficient of 21 x 103 M-1 cm-1 (20).

Electron paramagnetic resonance (EPR) spectroscopy.
To confirm superoxide production in the thyroid cell, cultured FRTL-5 rat thyroid cells were used for EPR experiment because these cells were convenient for transporting to the experimental site. Oxygen free radicals were detected by EPR in conjunction with the spin trapping technique. The spin trap used was 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) because it diffuses through the plasma membrane (21). DMPO was purified by repeated charcoal filtration until the EPR spectrum of the spin trap alone was signal-free. The reaction mixture contained FRTL-5 cells (1 x 106 cells), 20 µmol/L menadione, and 200 mM DMPO in 1 ml of phosphate-buffered saline (PBS). In some experiments, 1 µM ionomycin was used without menadione. The reaction mixtures, after 6–60 min of incubation, were transferred into sealed capillary ends of Pasteur pipettes. EPR spectra were recorded at 9.81 GHz on an ECS spectrometer (Burker, Billerica, MA). Measurement was carried out with a 100 kHz field modulation at room temperature. Instrument settings were as follows: 20 mW microwave power, 0.5 G modulation amplitude, 1.3 s time constant, 18 G/min scan rate, and 100 G sweep width.

Effects of menadione and DMNQ on iodide uptake and iodine organification.
Porcine follicles (8000 follicles/well) were plated in 12-well culture plates and cultured in the presence of 1 mU/ml bovine thyroid stimulating hormone (TSH) and 0.1 µM KI for 1 week. After washing, follicles were incubated with 0–25 µM menadione or DMNQ, 10 pmol NaI, and 0.1 µCi of Na125I for 1 hr in the presence of 1 ml of Tyrode salt solution. To determine iodide uptake, follicles were washed with Tyrode salt solution, and the radioactivity of follicles was counted. Iodine organification, which represents intrafollicular protein iodination, was measured by precipitating thyroid protein with 10% trichloloacetic acid as previously described (22). The results of iodide uptake and iodine organification were expressed as percentages of 125I-iodine taken by the follicles and thyroid protein, respectively, from the medium.

TPO activity in thyroid follicles.
Follicles were cultured in 6-well plates (16,000 follicles/well) in the presence of 1 µU/ml TSH and 0.1 µM KI for 8 days. The presence of KI greater than 5 µM tends to disturb the shape of follicles in our culture system. We chose eighth day of culture because TPO activity became relatively stable at this time (23). Follicles in two wells of 6-well culture plates were pooled, washed twice with PBS, pH 7.0, and sonicated. The pellet was then centrifuged at 100,000g for 60 min at 4°C;, reconstituted to 1 ml with PBS, sonicated, and used for measurement of TPO activity by the modified method of guaiacol oxidation (24). The reaction mixture for TPO assay contained PBS buffer, 100–200 µl of sonicated 100,000g pellet, 12 µmol guaiacol, and 880 nmol H2O2 in a total volume of 1 ml. The reaction was started by adding H2O2, and the increase in absorbance was measured at OD of 470 nm in a Shimadzu spectrophotometer. This crude TPO sample gave a linear increase in TPO activity up to 300 µl of the volume. The DNA content in the 100,000g pellet of the sonicate was measured by the mithramycin fluorescent method (25). TPO activity was expressed as guaiacol units per milligram of DNA; one guaiacol unit was arbitrarily defined as an increase in OD of 1.0 at 1 min.

TPO mRNA measurement.
TPO mRNA levels were measured by competitive RT-PCR as we described previously (23).

Immunoblot analysis of TPO.
Porcine polyclonal TPO antibody was kindly provided by Dr. Alvin Taurog (University of Texas). TPO samples were prepared from thyroid follicles in the presence of protease inhibitors. Electrophoresis of the sonicated 100,000g pellet of follicle (2 µg of protein) was performed under reducing condition using Novex apparatus (Novex, San Diego, CA). Immunoblot was done using Novex Western blot reagents.

Statistical Analysis.
The significant differences of the mean values were analyzed by the Dunnett multiple comparison test when the experimental groups were compared with the corresponding control group. Also, the unpaired Student's t test was used when the mean values of two groups were compared.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
O2- Generation in Thyroid Cells.
As shown in Table IGo, O2- was not detected in the control group of thyroid follicles (no quinone) by the cytochrome c method (Table IGo). To confirm the above results, we employed EPR with DMPO as a spin trapping agent. The medium in control cells (2 x 106), even after 60 min of incubation, did not show EPR signals (Fig. 1AGo). Intracellular EPR signal after washing cells was also absent (results not shown). Ionomycin, an agent known to increase H2O2 generation in dog thyroid cells (26), did not show any specific EPR signal (Fig. 1BGo). When quinone was added to the medium, O2- was detected in thyroid follicles (Table IGo) and in FRTL-5 cells (Fig. 1CGo). The medium from the cells incubated with 20 µM menadione exhibited EPR signals (Fig. 1CGo). The EPR signals consisted of a quartet with line intensities of 1:2:2:1. Based on these characteristics and hyperfine coupling constants (aH = aN = 14.9G), this spectrum was assigned to 5-hydroxy-2, 2-dimethyl-3-pyrrolidinyloxyl (DMPO-OH), the spin adduct from DMPO and hydroxyl radicals. The DMPO-OH adduct can be formed either by direct trapping of the hydroxyl radical or by rapid breakdown of DMPO-OOH, the adduct formed by O2- (27). The addition of 5 µM SOD to the medium completely quenched the signals, suggesting that the signals were mainly derived from O2-.


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  		Table I. Generation of O2- and H2O2 in Porcine Thyroid Follicles by DMNQ and Menadione
 


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  		Figure 1. EPR spectra in FRTL-5 cells. (A) Control cells. (B) Cells treated with 0.1 µM ionomycin for 60 min. (C) Cells treated with 25 µM menadione for 60 min. An aliquot was taken from the medium for this experiment.

 
H2O2 Generation in Thyroid Cells.
Both quinones (DMNQ and menadione) increased H2O2 production in a dose-response manner in cultured follicles (Table IGo). FRTL-5 cells also showed a similar response to DMNQ or menadione for H2O2 production (results not shown).

Effects of Quinones on Iodide Uptake and Iodine Organification.
Figure 2Go shows 125I iodide uptake and 125I iodine organification by thyroid follicles in the presence of 20 µM DMNQ or menadione. The presence of DMNQ caused a significant inhibition of iodine organification without affecting iodide uptake. Menadione showed inhibition of iodide uptake and iodine organification. To examine whether this inhibition was caused by H2O2 alone, we added H2O2 (10 and 20 nmol) directly to the medium every 10 min. The concentration of H2O2 was chosen based on the amount of H2O2 generated by DMNQ. The addition of 10 or 20 nmol H2O2 six times did not enhance or inhibit intrafollicular iodination.



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  		Figure 2. Effects of DMNQ and menadione on iodide uptake and iodine organification in porcine thyroid follicles. Follicular cells were exposed to 20 µM DMNQ or menadione for 60 min in the presence of Na125I-KI mixture. The results were expressed as percentages of 125I taken by follicles (iodide uptake) and bound thyroid protein (iodine organification). The results are the means ± SD of triplicate samples of one of representative experiments.

 
Effects of Quinones on Cellular TPO.
To further investigate the mechanism of decreased iodine organification, we measured cellular TPO activity after thyroid follicles were exposed to DMNQ or menadione for 2 hr. Figure 3Go shows a profound inhibition of TPO activity in cultured thyroid follicles when concentrations of quinones were greater than 10 µM. We also measured TPO mRNA levels by competitive RT-PCR in thyroid follicles that were treated with DMNQ for 2 hr followed by washing and culturing for another 48 hr. TPO mRNA levels in the control and DMNQ-treated groups were 12.1 ng/µg poly(A)+ RNA and 18.5 ng/µg poly(A)+ RNA (mean of the two experiments), respectively. Electrophoretic mobility of TPO protein showed the same pattern and the same staining intensity between control cells and DMNQ-treated cells by immunoblotting (results not shown).



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  		Figure 3. Cellular TPO activities in porcine thyroid follicles after exposure to DMNQ or menadione. Follicular cells were exposed to 0–20 µM DMNQ or menadione for 2 hr in the presence of 0.1 µM KI. Cellular TPO activity was measured in the sonicate of 100,000g pellet by guaiacol assay. The results are the means ± SD of triplicate samples of one of the representative experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The thyroid gland contains tissue specific NADPH oxidase (2,3) and produces H2O2. It is theoretically possible that H2O2 can be derived from O2- because SOD, an enzyme to convert O2- to H2O2, is also present in the thyroid cell (4–6). Nakamura et al. (7,8) used thyroid cell membrane fraction and demonstrated O2- production; however, there was no O2- detected in the medium of the intact thyroid cell (8). They proposed that O2- is produced inside of the plasma membrane where O2- is converted to H2O2 by SOD followed by diffusion of H2O2 through the plasma membrane (8). We have tried to demonstrate intracellular O2- by analyzing the EPR spectra from control cells (FRTL-5 cells) incubated with 200 µM DMPO for 15 min followed by washing. The EPR spectra from whole-cell suspension did not show O2- or O2--derived signals, suggesting no intracellular O2- formation in the physiological condition. The concept of O2- as the initial product for thyroid H2O2 has been questioned by Dupuy et al. (9). Jenzer et al. (10) showed production of O2- and OH• by incubating lactoperoxidase with excessive H2O2, which generates free radicals by the Fenton reaction (iron-mediated H2O2 breakdown). However, their finding cannot be extrapolated to the cellular level because the high concentrations of H2O2 used in their experiment should not prevail in the thyroid cell. Our present study could not demonstrate O2- formation under the physiological condition in the medium and cells. Even addition of ionomycin, a stimulator of thyroid H2O2 in dog thyroid cells (26), failed to show O2- in the thyroid cell (Fig. 1BGo). The question can be raised that the methodology is not sensitive enough to pick up O2- generation. However, if thyroid H2O2 production depends on solely from O2- or has stoichiometry relationship with O2-, EPR should have picked up O2- as the detection limit of free radicals by the EPR method is micromolar ranges (10). The thyroid gland has been shown to posses calcium and TSH dependent NADPH oxidase (2,3). In contrast to leukocyte NADPH oxidase that can produce O2-, thyroid NADPH oxidase appears to produce H2O2 by divalent reduction of oxygen without O2- generation.

When DMNQ or menadione was used, O2- formation was demonstrated in the medium (Table IGo); this was confirmed by the EPR experiment (Fig. 1CGo). The mechanism of quinone-mediated O2- generation is different from the classic NADPH oxidase system. Quinones are reduced by intracellular redox enzymes and transfer one electron to oxygen for formation of O2- (16). In addition, DMNQ and menadione were the potent agents to augment H2O2 production in the thyroid cell (Table IGo). The most important finding in this study was that the two quinones displayed antithyroid activity by inhibiting iodine organification, a step of thyroid hormone formation, and its catalytic enzyme, TPO. How did the two quinones inactivate cellular TPO? The action of menadione can be derived from the generation of reactive oxygen species and/or arylation of critical nucleophil, whereas the action of DMNQ is specifically mediated solely through the generation of reactive oxygen species (15,16). Thus, inactivation of TPO by DMNQ indicates that reactive oxygen species are responsible for TPO inactivation. How do reactive oxygen species inhibit TPO? Two mechanisms are possible: formation of compound III due to excessive H2O2 or O2-, and free radical-mediated TPO inactivation (28). The former causes reversible TPO inactivation and the latter causes irreversible TPO inactivation (28). Compound III formation is impossible to demonstrate in the cellular system. Furthermore, removal of quinone by washing follicles did not restore TPO activity, suggesting that this is irreversible TPO inhibition. We speculate that oxygen free radicals attacked the active site of TPO, causing inactivation of the catalytic site of the enzyme. Theoretically, this is quite possible. In the TPO molecule, proximal and distal histidine residues play a critical role for catalytic activity of TPO because the former is linked to the iron center of the heme and the latter is close to the peroxide-binding pocket (29). Vicious free radical (hydroxyl radical) formation is facilitated by the presence of iron known as Fenton reaction; thus, histidine residues linked to the heme can be the specific target by oxygen free radicals. Jenzer et al. (10,28) also proposed the same mechanism for inactivation of lactoperoxidase by free radicals in their cell-free experiments. Nevertheless, the generation of excessive amounts of reactive oxygen species can have a negative effect on thyroid hormone formation. Thus, the presence of SOD and other antioxidants in the thyroid cell becomes important for maintenance of thyroid hormone formation if oxygen free radials are produced accidentally. We have not examined the effects of SOD and catalase on TPO activity in our culture system because the two enzymes are not permeable to the membrane and quinones produce oxygen free radicals primarily intracellularly. Reactive oxygen species did not have inhibitory effect on TPO mRNA levels or electrophoretic mobility of TPO molecule as long as the exposure time was short. This suggests that transcription process and antibody binding sites of TPO molecules are not inhibited by reactive oxygen species. Although the two quinones are not the products of human body, they are useful to examine the effect of oxygen free radicals in vitro.

Are there any clinical conditions involving O2- and its related radicals in the thyroid gland? Ionized radiation releases reactive oxygen species from the water molecule (11). Thus, neck cancer patients who undergo radiation therapy may be exposed to significant amounts of reactive oxygen species in the thyroid gland. Administration of 131I-iodine to rats showed an increase in lipid peroxidation in the thyroid gland, suggesting the generation of oxygen free radicals by ß radiation (12). Activated leukocytes due to infection generate reactive oxygen species from the leukocyte NADPH oxidase system, and this phenomenon is known as respiratory burst (13). Thus, the thyroid gland of patients with acute bacterial thyroiditis is exposed to reactive oxygen species. Mutation of the ras oncogen is one of the most common events in initiating tumors, including thyroid tumors (30). It has been shown that activated ras oncogen forms a large amount of O2- as its message signal (14). Thus, it is conceivable that thyroid tumors linked to the mutated ras oncogen produce oxygen free radicals. There are many naturally occurring quinones isolated from biological tissues (16). Also, chemotherapeutic drugs (adriamycine, daunorubicin, and mitomycine), acetaminophen (Tylenol), and air pollutants (cigarette smoke and automobile exhaust) are common source of quinones. Some of the quinones have a great potential to induce the generation of oxygen free radicals (16). Whether these quinones or quinone products exhibit organ-specific toxicity to the thyroid gland is unknown.


   Acknowledgments
 
We thank Dr. Alvin Taurog, University of Texas Southeastern Medical Center, for providing us with porcine thyroid peroxidase antibody.


   Footnotes
 
This study was supported by Veterans Affairs Medical Research Funds.

1 To whom requests for reprints should be addressed at West Los Angeles Veterans Affairs, Medical Center (111M), 11301 Wilshire Boulevard, Los Angeles, CA 90073. E-mail: msugawar{at}ucla.edu Back


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received for publication April 20, 2001. Accepted for publication October 22, 2001.




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