1 RETINOIC ACID MODULATION OF THYROID DUAL OXIDASE ...

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RETINOIC ACID MODULATION OF THYROID DUAL OXIDASE ACTIVITY 1

IN RATS AND ITS IMPACT ON THYROID IODINE ORGANIFICATION 2

3

1Mônica Mühlbauer,

1Alba Cenélia Matos da Silva,

1Michelle Porto Marassi, 4

2Alexandre Lopes Lourenço,

1Andrea Claudia Freitas Ferreira

and

1Denise Pires de 5

Carvalho. 6

7

8

1Laboratório de Fisiologia Endócrina, Instituto de Biofísica Carlos Chagas Filho; 9

2Laboratório de Produtos Naturais, Instituto de Farmacologia Básica e Clínica; 10

Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil. 11

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Correspondence: 13

Andrea Claudia Freitas Ferreira 14

Instituto de Biofísica Carlos Chagas Filho 15

CCS - Bloco G - Cidade Universitária, Ilha do Fundão 16

Rio de Janeiro, 21949-900, Brasil 17

tel : 55 21 25626552 18

fax : 55 21 22808193 19

e-mail : [email protected] 20

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Short title: Effect of retinoic acid on thyroid DuOx activity 22

Key words: retinoic acid (RA), dual oxidase (DuOx), thyroperoxidase (TPO), sodium-23

iodide symporter (NIS), thyroid. 24

of 27 Accepted Preprint first posted on 1 April 2010 as Manuscript JOE-09-0421

Copyright © 2010 by the Society for Endocrinology.

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ABSTRACT 25

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The sodium iodide symporter (NIS) mediates iodide uptake into the thyrocytes, 27

which is important for the diagnosis and therapy of thyroid disorders. Decreased ability 28

to uptake iodide in thyroid carcinomas reduces the efficacy of radioiodine therapy, and 29

retinoic acid (RA) treatment re-induces iodide uptake. The effectiveness of treatment 30

not only depends on iodide uptake, but also on the ability of thyrocytes to organify 31

iodine, which is catalyzed by thyroperoxidase (TPO) in the presence of H2O2. Our goal 32

was to determine the influence of RA on thyroid iodide uptake, iodine organification, 33

and TPO and DuOx activities. Normal rats were treated with all-trans-RA or 13-cis-RA 34

(100 or 1500µg/100g b.w., s.c.) for 14 and 28 days. The 2h thyroid radioiodine content 35

significantly decreased in rats treated with all-trans-RA 100µg/100g b.w. for 14 days. In 36

this group, NIS function and TPO activity were unchanged, whereas DuOx activity was 37

significantly decreased, which might have contributed to the decrease in iodine 38

organification. Both doses of 13-cis-RA for 28 days increased the 15 min thyroid 39

radioiodine uptake, while the 2h radioiodide uptake increased only in rats treated with 40

the highest dose of 13-cis-RA. While TPO activity did not change, H2O2 generation was 41

increased in this group and serum T4 levels were normal. Since radioiodine half-life in 42

the thyroid gland is important for treatment efficacy, our results highlight the 43

importance of correctly choosing the RA isomer, the time and the dose of treatment, in 44

order to improve the efficacy of radioiodine therapy. 45

46

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INTRODUCTION 47

The content of iodine in the thyroid gland depends on the presence of proteins 48

that are necessary for iodide uptake through the basolateral membrane of thyrocytes and 49

its incorporation into the acceptor protein thyroglobulin (Tg) at the apical surface of 50

these cells (DeGroot LJ & Niepomniszcze 1977; Kopp 2005). The sodium-iodide 51

symporter (NIS) is responsible for iodide uptake through the thyroid cell basolateral 52

membrane against its electrochemical gradient. At the apical pole of thyrocytes, 53

intracellular iodide is transported through pendrin (PDS) (Royaux et al. 2000) into the 54

follicular lumen and then incorporated into Tg. Iodide oxidation and organification 55

occur at the apical surface of the follicular cell and these reactions are catalyzed by 56

thyroperoxidase (TPO) in the presence of hydrogen peroxide. Thus, thyroid iodide 57

organification depends on TPO activity, which is modulated by the concentration of 58

iodide, and hydrogen peroxide (DeGroot LJ & Niepomniszcze 1977; Kopp 2005). In the 59

presence of sufficient amounts of iodine, the limiting step for thyroid hormone 60

biosynthesis is the availability of H2O2, which is generated by thyroid dual oxidase 61

(DuOx) (Corvilain et al. 1991; Dupuy et al. 1999). 62

Treatment with RA was shown to increase NIS, TPO and Thyroglobulin (Tg) 63

mRNA levels in cancer cell lines and to decrease NIS mRNA levels and iodide uptake 64

in normal rat thyroid cell line (Schmutzler et al. 1997). In normal thyroid cell line, RA 65

reduces TSH receptor mRNA levels and increases TPO and Tg mRNA levels 66

(Schmutzler et al. 1997; Kurebayashi et al. 2000). On the other hand, some authors 67

demonstrated that RA suppress the accumulation of TPO and Tg mRNA stimulated by 68

TSH in a time- and dose-dependent manner in cultures of human thyrocytes (Arai et al. 69

1991; del Senno et al. 1993; Namba et al. 1993; del Senno et al. 1994). However, the 70

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possible effect of retinoic acid on thyroid dual oxidase (DuOx) has not been assessed so 71

far. 72

RA is widely used in dermatological treatment and thyroid cancer management 73

(Vershoore et al. 1993; Coelho et al. 2004), although the effects of this drug on normal 74

thyroid function are poorly defined. We have recently demonstrated that 13-cis-retinoic 75

acid is able to increase thyroid radioiodide uptake in normal female rats (Silva et al. 76

2009), however the effect on the thyroid of male rats was not evaluated. 77

Therefore, the aim of the present study was to analyze the influence of RA 78

treatment on thyroid iodide uptake, serum T4, TPO and DuOx activities in male rats. 79

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MATERIALS AND METHODS 81

Materials 82

All-trans- and 13-cis-retinoic acid were purchased from Sigma Chemical Co. 83

(St. Louis, MO). Tris(hydroxymethyl)aminomethan, glucose and potassium iodide were 84

from Merck (Rio de Janeiro, RJ, Brazil). Fetal bovine serum was purchased from 85

Cultilab (Rio de Janeiro, RJ, Brazil). Glucose oxidase (Grade I) was from Boehringer 86

(Mannheim, Germany) and Na- 125

I was purchased from Amersham (Buckinghamshire, 87

England). 88

89

Animals 90

Adult male Wistar rats weighing 250-300g were housed under controlled 91

conditions of temperature (24 ± 1ºC) and light (12h light starting at 7:00 a.m.) with 92

water and food ad libitum. The study conforms to the Guide for the Care and Use of 93

Laboratory Animals published by the US National Institutes of Health (NIH Publication 94

No.85-23, revised 1996) and was approved by the Institutional Animal Welfare 95

Committee (CAUAP/UFRJ). 96

Rats were randomly divided into the following groups: control, all-trans-retinoic 97

acid (all-trans-RA) and 13-cis-retinoic acid (13-cis-RA). Retinoic acid was 98

administered in the dose of 100µg/kg body weight (B.W.), subcutaneously, daily, for 14 99

days according to Coya et al. (1997) and in the doses 100µg/kg and 1500µg/kg body 100

weight for 28 days. The dose of 1500µg/kg body weight is similar to that used in the 101

treatment of thyroid cancer in humans (Coelho et al. 2004). Both all-trans- and 13-cis-102

RA were dissolved in DMSO and suspended in propylene glycol (1:1, v/v). Rats from 103

all experimental groups were sacrificed 24 h after the last injection. 104

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TPO preparation 106

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TPO extraction from rat thyroids was performed as previously described (Moura 108

et al. 1989; Ferreira et al. 2005). Pools of two rat thyroids were minced and 109

homogenized in 0.5 ml 50 mM Tris-HCl buffer, pH 7.2, containing 1 mM KI, using an 110

Ultra-Turrax homogenizer (Staufen, Germany). The homogenate was centrifuged at 111

100,000 g, 4oC for 1 h. The pellet was suspended in 0.5 ml digitonin (1%, w/v) and 112

incubated at 4oC for 24 h to solubilize the peroxidase. The digitonin-treated suspension 113

was centrifuged at 100,000 g, 4oC for 1 h, and the supernatant containing the solubilized 114

TPO was used for the assays. Protein content was determined by the method of 115

Bradford (1976). 116

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Thyroid peroxidase activity 118

119

The TPO iodide-oxidation activity was measured as previously described, 120

(Nakashima and Taurog 1978; Pommier 1978; Moura et al. 1989; Carvalho et al. 1994; 121

Ferreira et al. 2005). The assay mixture contained: 1.0 ml of freshly prepared 50 mM 122

sodium phosphate buffer, pH 7.4, containing 24 mM KI and 11 mM glucose, and 123

increasing amounts (20 to 80 µl) of solubilized TPO. The final volume was adjusted to 124

2.0 ml with 50 mM sodium phosphate buffer, pH 7.4, and the reaction was started by 125

the addition of 10 µl of 0.1% glucose oxidase (Boehringer Grade I). The increase in 126

absorbance at 353 nm (tri-iodide production) was registered for 4 min on a Hitachi 127

spectrophotometer (U-3300). The ∆A353nm/min was determined from the linear portion 128

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of the reaction curve and one unit (U) of TPO corresponds to ∆A353nm/min = 1.0. The 129

enzymatic activity is expressed as U/g protein. 130

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DuOx preparation 132

For dual oxidase (DuOx) preparation, the excised thyroid glands remained at 4 C 133

for 24h in 50 mM sodium phosphate buffer, pH 7.2, containing 0.25 M sucrose, 0.5 mM 134

DTT, 1 mM EGTA, 5µg/ml aprotinin and 34,8µg/ml PMSF before homogenization. 135

Then, the homogenate was centrifuged at 100,000 g for 35 min at 4°C and resuspended 136

in 0.5 ml 50 mM sodium phosphate buffer, pH 7.2, containing 0.25 M sucrose, 2 mM 137

MgCl2, 5µg/ml aprotinin and 34,8µg/ml PMSF and stored at –20°C until H2O2 138

generation measurements. 139

140

Ca2+ and NADPH-dependent H2O2 generating activity – Dual oxidase activity 141

H2O2 production was measured as previously described (Leseney et al. 1999; 142

Cardoso et al. 2001). We incubated samples of thyroid particulate fractions at 30°C, in 1 143

ml 170 mM sodium phosphate buffer, pH 7.4, containing 1 mM sodium azide, 1 mM 144

EGTA, 1 µM FAD and 1.5 mM CaCl2. The addition of 0.2 mM NADPH started the 145

reaction; aliquots of 100 µl were collected at intervals up to 20 minutes, and mixed with 146

10 µl 3 N HCl to stop the reaction and destroy the remaining NADPH. Initial rates of 147

H2O2 formation were determined from eight aliquots of each assay by following the 148

decrease in 0.4 µM scopoletin fluorescence in the presence of HRP (0.5 µg/ml) in 200 149

mM phosphate buffer, pH 7.8, in a Hitachi spectrofluorimeter (F 4000). The excitation 150

and emission wavelengths were 360 and 460 nm, respectively. Specific activities were 151

expressed per mg protein (nmoles H2O2.h-1

.mg-1

protein). 152

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Thyroid radioiodide content 154

We have previously shown that the measurement of radioiodide uptake 15 min 155

after 125

I-NaI administration (short-term iodide uptake) reflects iodide transport through 156

the sodium-iodide symporter, without influence of in vivo thyroid iodine organification 157

activity (Ferreira et al. 2005; Lima et al. 2006). Thus, in order to evaluate the in vivo 158

NIS function, the animals received Na-125

I (3700 Bq, i.p., Amersham, 159

Buckinghamshire, England) 15 min before decapitation. We also administered Na-125

I 160

(3700 Bq, i.p., Amersham, Buckinghamshire, England) 2 hours before decapitation, in 161

order to evaluate both iodide transport and in vivo organification activities (thyroid 162

iodine accumulation). In the thyroid, iodine organification is catalyzed by TPO and 163

consists of binding of iodine to tyrosil residues of thyroglobulin, an essential step of 164

thyroid hormone biosynthesis. The radioactivity of the thyroid glands was measured 165

using a gamma counter (LKB) and expressed as percentage of total 125

I injected per mg 166

of thyroid. 167

168

T4 measurement 169

Serum total T4 concentrations were measured using commercial kits 170

(radioimmunoassay, Diagnostic Systems Laboratories Inc., Texas, USA or 171

eletrochemoluminescence, Roche Diagnostics, Indianapolis, USA). T4 172

radioimmunoassay sensitivity was of 0.4 µg/dl and inter- and intra assay coefficients of 173

variation varied from 5.6 to 8.6% and 4 to 5.1%, respectively. T4 174

eletrochemoluminescence assay sensitivity was of 0.42 µg/dL and intra assay 175

coefficients of variation was of 4.2 %. 176

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Statistical analysis 179

The experiments were repeated at least two times, using at least 3 animals per 180

group in each experiment, so a total number of at least 6 animals per group was 181

achieved. Results are expressed as mean ± SEM and were analyzed by unpaired t-test. 182

Differences were considered significant when p<0.05. 183

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RESULTS 184

14 days retinoic acid treatment 185

Short-term (15 minutes) thyroid radioiodide uptake was unchanged by the 186

treatment with both isomers of RA in the low dose administered (100µg/100g b.w.) for 187

two weeks (figure 1A), suggesting that retinoic acid did not affect NIS function in the 188

period of time and dose used. On the other hand, treatment with all-trans-retinoic acid 189

for two weeks produced a significant decrease in thyroid iodine accumulation (2h) 190

(Figure 1B). TPO activity was not affected by the treatment with either isomers of RA 191

for two weeks when compared to the control group, as shown in figure 1C. However, 192

administration of the low dose of all-trans-retinoic acid (100µg/100g b.w.) for two 193

weeks led to a significant decrease in H2O2 formation while treatment with 13-cis-194

retinoic acid had no effect (Figure 1D), suggesting an inhibitory effect of all-trans-195

retinoic acid on DuOx activity. Despite decreased DuOx activity after 14 days of RA 196

treatment, total serum T4 concentration remained unchanged (Table 1). 197

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28 days retinoic acid treatment 199

All-trans-retinoic acid treatment did not affect 15 minutes thyroid radioiodide 200

uptake (Figure 2A). However, treatment with 13-cis-retinoic acid for 28 days 201

significantly increased NIS function in both doses (Figure 2B), as previously described 202

for thyroid cancers (Coelho et al. 2004) and female rats (Silva et al. 2009). 203

Treatment with 13-cis-RA at the higher dose (1500µg/100g b.w.) for four weeks 204

produced a significant increase in the 2h thyroid radioiodine content (Figure 2D), which 205

was unchanged by treatment with all-trans-RA (Figure 2C). 206

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While TPO activity was not affected by retinoic acid treatment in both doses 207

(Figures 3A and 3B), DuOx activity was significantly increased in the thyroid of rats 208

treated with 13-cis-RA in the dose of 1500µg/100g b.w. for four weeks (Figure 3D), but 209

not with all-trans-RA (Figure 3C). Total serum T4 did not differ among groups (Table 210

2). 211

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DISCUSSION 213

214

We have shown herein that retinoic acid may affect thyroid function not only by 215

interfering with NIS function, but also by modulating dual oxidase activity. It is well 216

documented that retinoic acid stimulates thyroid iodide uptake in human thyroid 217

follicular carcinoma cells (Schmutzler 2001), thyroid cancer (Coelho et al. 2004) and 218

thyroid gland of female rats (Silva et al. 2009). We now demonstrate that, although 28 219

days of treatment with 13-cis-retinoic acid stimulates radioiodide uptake, in fact, 14 220

days of all-trans-retinoic acid decreased thyroid iodide content, what has been shown to 221

be related to decreased thyroid iodide organification. DuOx activity was reduced in this 222

group while NIS function and TPO activity were not affected. It is known that the 223

availability of H2O2 is a limiting step for iodine organification (Corvilain et al. 1991), 224

so the decreased radioiodine content 2h after its administration might be related to the 225

decreased DuOx activity. This result highlights the importance of the duration of 226

treatment with retinoic acid and the isomer used in order to achieve the desirable effect. 227

It is known that radioiodide uptake per se is important although not sufficient for its 228

therapeutic effectiveness. 229

On the other hand, 13-cis-retinoic acid significantly increased radioiodide uptake 230

in both doses used when the rats were treated for 28 days. Increased NIS function after 231

RA treatment is in agreement with data shown by Schmutzler et al. (2002). However, 232

the increased radioiodine content was only detected in rats treated with the highest dose 233

of 13-cis-retinoic acid. This result reinforces the concept that an increase in NIS 234

function does not necessarily lead to increased thyroid radioiodine content. In this 235

sense, H2O2 generation was increased in rats treated with the highest dose of 13-cis-236

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retinoic acid for 28 days, showing again the importance of dual oxidase activity as a 237

limiting step for iodine organification. 238

The effects of retinoic acid treatment on the thyroid of male rats were similar to 239

those found in female rats, as previously described by our group (Silva et al. 2009), 240

suggesting no gender specific difference regarding thyroid response to RA treatment. 241

To our knowledge, we have reported herein for the first time the regulatory 242

effect of retinoic acid on dual oxidase activity. As reviewed by Lambeth et al. (2007), 243

Duox1 and Duox2 expression are regulated in both thyroid and non-thyroid cells by a 244

promoter region within −150 bp of the first exon of Duox1 and within −250 bp of 245

Duox2. In dog and pig thyrocytes, cAMP, which is a pathway downstream of the TSH 246

receptor, increase Duox protein and mRNA (Dupuy et al. 1999; De Deken et al. 2000; 247

Morand et al. 2003). However, no data about a retinoic acid responsive element in Duox 248

promoter are available. Pailler-Rodde et al. (1991) have shown that retinoic acid 249

induces an increase in PKC activity. Since Duox is a calcium-dependent NADPH 250

oxidase (Dupuy et al. 1999; Carvalho et al. 1996; Ferreira et al. 2003; Ginabreda et al. 251

2008) 13-cis-retinoic acid could also increase Duox activity by a stimulatory effect on 252

the calcium pathway. Nevertheless, a direct effect of retinoic acid on dual oxidase 253

promoter cannot be excluded. Further experiments are needed to elucidate the 254

mechanism underlying the effect of retinoic acid on dual oxidase activity. 255

Even though thyroid iodine organification was significantly decreased in rats 256

treated with all-trans retinoic acid for 14 days and increased in rats treated with 13-cis 257

retinoic acid for 28 days, serum T4 levels remained unchanged. Our data suggest that 258

compensatory mechanisms might take place in order to avoid significant changes in 259

serum T4 levels. 260

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In conclusion, our data suggest that treatment with RA produces direct effects on 261

thyroid function that are dose, isomer and time-dependent, suggesting that the efficacy 262

of retinoic acid to improve radioiodine therapy might be influenced by these factors. 263

Treatment with 100µg/100g b.w. of all-trans-retinoic acid for 2 weeks reduced thyroid 264

iodine content while treatment with 1500µg/100g b.w. of 13-cis-retinoic acid increase 265

thyroid iodine content. So, it might be important to evaluate serum thyroid hormones 266

and TSH levels in patients under treatment with retinoic acid for dermatological and 267

oncological purposes, especially in areas with a low iodine intake and in patients with 268

possible sub clinical thyroid dysfunction. 269

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DECLARATION OF INTEREST 270

We declare that there is no conflict of interest that could be perceived as 271

prejudicing the impartiality of the research reported. 272

273

FUNDING 274

This work was supported by grants from CNPq, Departamento de 275

Tireóide/SBEM, FAPERJ/SUS and FAPERJ/Pensa Rio. 276

277

ACKNOWLEDGEMENTS 278

We are grateful for the technical assistance of Norma Lima de Araújo Faria, 279

Advaldo Nunes Bezerra and Wagner Nunes Bezerra. The authors declare that there is no 280

conflict of interest that would prejudice the impartiality of this scientific work. Mônica 281

Mühlbauer, Alba Cenélia Matos da Silva, Michelle Porto Marassi were reciepients of 282

fellowships from CAPES and CNPq. 283

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400

FIGURE LEGENDS 401

402

Figure 1: Effect of all-trans-retinoic acid and 13-cis-retinoic acid for 14 days, in the 403

dose of 100µg/kg b.w., daily injections, sc, on some parameters of thyroid function. A: 404

Short-term iodide uptake 15 minutes after radioiodide administration [Control (n=16), 405

All-trans-RA (n=19), 13-cis-RA (n=19)]. B: Thyroid iodine accumulation 2 hour after 406

radioiodide administration [Control (n=11), All-trans-RA (n=13), 13-cis-RA (n=13)]. * 407

p<0.05 vs. Control and vs. 13-cis-RA100µg/kg b.w.. C: Thyroperoxidase iodide-408

oxidation activity [Control (n=27), All-trans-RA (n=32), 13-cis-RA (n=31)]. D: DuOx 409

Ca++

-dependent H2O2-generating activity [Control (n=12), All-trans-RA (n=10), 13-cis-410

RA (n=12)]. * p<0.0001 vs. control and 13-cis-RA. Measurements were performed as 411

described in Material and Methods. Data are expressed as mean ± S.E.M. 412

413


doses of 100 and 1500µg/kg b.w., daily injections, sc, on thyroid iodide uptake (15 min) 415

and accumulation (2h). A: Short-term iodide uptake 15 minutes after radioiodide 416

administration in rats treated with all-trans-retinoic acid [Control (n=17), All-trans-417

RA100µg/kg BW (n=11), All-trans-RA1500µg/kg b.w. (n=7)]. B: Short-term iodide 418

uptake 15 minutes after radioiodide administration in rats treated with 13-cis-retinoic 419

acid [Control (n=17), 13-cis-RA100µg/kg b.w. (n=11), 13-cis-RA1500µg/kg b.w. 420

(n=7)]. * p<0.05 vs. control. C: Thyroid iodine accumulation 2 hour after radioiodide 421

administration in rats treated with all-trans-retinoic acid [Control (n=24), All-trans-422

RA100µg/kg b.w. (n=12), All-trans-RA1500µg/kg b.w. (n=14)]. D: Thyroid iodine 423

accumulation 2 hour after radioiodide administration in rats treated with 13-cis-retinoic 424

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22

acid [Control (n=24), 13-cis-RA100µg/kg b.w. (n=12), 13-cis-RA1500µg/kg b.w. 425

(n=14)]. * p<0.05 vs. Control. Measurements were performed as described in Material 426

and Methods. Data are expressed as mean ± S.E.M. 427

428


doses of 100 and 1500µg/kg b.w., daily injections, sc, on thyroperoxidase and DuOx 430

activities. A: Thyroperoxidase iodide-oxidation activity in the thyroid of rats treated 431

with all-trans-retinoic acid [Control (n=26), All-trans-RA100µg/kg b.w. (n=23), All-432

trans-RA1500µg/kg b.w. (n=7)]. B: Thyroperoxidase iodide-oxidation activity in the 433

thyroid of rats treated with 13-cis-retinoic acid [Control (n=26), 13-cis-RA100µg/kg 434

b.w. (n=22), 13-cis-RA1500µg/kg b.w. (n=7)]. C: DuOx Ca++

-dependent H2O2-435

generating activity in the thyroid of rats treated with all-trans-retinoic acid [Control 436

(n=17), All-trans-RA100µg/kg b.w. (n=9), All-trans-RA1500µg/kg b.w. (n=10)]. D: 437

DuOx Ca++

-dependent H2O2-generating activity in the thyroid of rats treated with 13-438

cis-retinoic acid [Control (n=17), 13-cis-RA100µg/kg b.w. (n=7), 13-cis-RA1500µg/kg 439

b.w. (n=10)]. * p<0.05 vs. Control and vs. 13-cis-RA100µg/kg b.w. Measurements were 440

performed as described in Material and Methods. Data are expressed as mean ± S.E.M. 441

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FIGURE 1

A B

14 days

Control All-trans-RA 13-cis-RA 0

25

50

75

100

125

15m

in 12

5 I upt

ake

(% C

ontr

ol)

Control 0

2h 12

5 I upt

ake

(% C

ontr

ol)

All-trans-RA 13-cis-RA

C

Control 0

1000

2000

3000

4000

TPO

act

ivity

(U/g

pro

tein

)

All-trans-RA 13-cis-RA

D

All-trans-RA 13-cis-RA Control 0

100

200

DuO

x ac

tivity

(n

mol

H2O

2.h-1

.mg-

1 pro

tein

)

*

25

50

75

100

125

*

of 27

B

28 days

Control 100 1500

15 m

in 12

5 I upt

ake

(% C

ontr

ol)

All-trans-RA (µg/kg BW)

Control 0

100

200

* *

13-cis-RA (µg/kg BW)

A

FIGURE 2

100 1500

0

50

100

150

2h 12

5 I upt

ake

(% C

ontr

ol)

Control

C

100 1500


D

2h 12

5 I upt

ake

(% C

ontr

ol)

Control


100 1500

15 m

in 12

5 I upt

ake

(% C

ontr

ol)

0

100

200

0

50

100

150 *

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FIGURE 3

Control 0

1000

2000

3000

TPO

act

ivity

(U/g

pro

tein

)

Control 0

1000

2000

3000 TP

O a

ctiv

ity (U

/g p

rote

in)

100 1500

All-trans-RA (µg/kg BW) 13-cis-RA (µg/kg BW)

100 1500

Control

DuO

x ac

tivity

(n

mol

H2O

2.h-1

.mg-

1 pro

tein

)

100 1500


Control 0

100

200

300 *

DuO

x ac

tivity

(n

mol

H2O

2.h-1

.mg-

1 pro

tein

)


100 1500

B

28 days

A

C D

0

100

200

300

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1 RETINOIC ACID MODULATION OF THYROID DUAL OXIDASE ...

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