Research ArticleActivation of the Nrf2-Keap 1 Pathway in Short-TermIodide Excess in Thyroid in Rats

Tingting Wang1 Xue Liang1 Iruni Roshanie Abeysekera1 Umar Iqbal1 Qi Duan1

Gargi Naha1 Laixiang Lin2 and Xiaomei Yao1

1Department of Physiology and Pathophysiology School of Basic Medical Sciences Tianjin Medical University Tianjin 300070 China2Key Laboratory of Hormones and Development (Ministry of Health) Tianjin Key Laboratory of Metabolic DiseasesTianjin Metabolic Diseases Hospital amp Tianjin Institute of Endocrinology Tianjin Medical University Tianjin 300070 China

Correspondence should be addressed to Xiaomei Yao jupx163com

Received 29 August 2016 Revised 29 October 2016 Accepted 23 November 2016 Published 4 January 2017

Academic Editor Ayman M Mahmoud

Copyright copy 2017 Tingting Wang et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Wistar rats were randomly divided into groups of varying iodide intake normal iodide 10 times high iodide and 100 times highiodide onDays 7 14 and 28 Insignificant changeswere observed in thyroid hormone levels (119901 gt 005) Urinary iodine concentrationand iodine content in the thyroid glands increased after high consumption of iodide from NI to 100HI (119901 lt 005) The urinaryiodine concentration of the 100HI group on Days 7 14 and 28 was 60ndash80 times that of the NI groupThemitochondrial superoxideproduction and expressions of Nrf2 Srx and Prx 3 all significantly increased while Keap 1 significantly decreased in the 100 HIgroup when compared to the NI or 10HI group on Days 7 14 and 28 (119901 lt 005) Immunofluorescence staining results showed thatNrf2 was localized in the cytoplasm in NI group Although Nrf2 was detected in both cytoplasm and nucleus in 10HI and 100HIgroups a stronger positive staining was found in the nucleus We conclude that the activation of the Nrf2-Keap 1 antioxidativedefense mechanism may play a crucial role in protecting thyroid function from short-term iodide excess in rats

1 Introduction

Iodine being a critical constituent of thyroid hormones isessential for normal growth and development in all verte-brates [1 2] During thyroid hormone synthesis there isa constant production of reactive oxygen species (ROS)especially hydrogen peroxide (H2O2) which is subsequentlyutilized for the oxidation of iodide [3] Although the basallevel of ROS production is important for maintaining thy-roid hormone biosynthesis iodide excess may increase theproduction of ROS in thyrocytes [4 5] Higher amounts ofROS can cause oxidative stress by damaging the cellular com-ponents and affecting organelle integrity [5] The increasedgeneration of ROS triggered by iodide excess is responsiblefor its cytotoxic effect on thyrocytes [6 7]

Thomopoulos reported that hyperthyroidism maydevelop in around 10 of patients with excess iodine andthat it may occur several years after the initiation of iodine

excess [8] Wolff reported that chronic ingestion of morethan ten times the daily requirement of iodide or iodide-generating organic compounds could lead to iodide goiter incertain subjects [9] In the thyroid slices of several speciesexcess iodide is known to stimulate the generation of H2O2[10] This occurs when the latter is in the presence of either300 120583M KI in dog thyroid slices or 100 120583M KI in bovinethyroid slices [10]

Possible molecular mechanisms responsible for excessiodide-induced ROS production are described as belowWhen iodide is in excess as compared with tyrosine residuesit reacts with the iodonium cation formed by iodide oxidationto give molecular iodine Excess molecular iodine inducesapoptosis through an increased generation of free radicals[11] Various types of iodolipids are produced when iodinebinds to membrane lipids which is considered to be themain mechanism of free radical-induced damage The mito-chondria contain specific receptors for the thyroid hormones

HindawiOxidative Medicine and Cellular LongevityVolume 2017 Article ID 4383652 13 pageshttpsdoiorg10115520174383652

2 Oxidative Medicine and Cellular Longevity

and much of the ROS production occurs here via oxidativephosphorylation [3 12] ROS include free radicals such assuperoxide anions hydroxyl radicals and H2O2

Nuclear factor erythroid 2-related factor 2 (Nrf2) is atranscription factor which is vital in regulating the expressionof some antioxidative enzymes such as hemeoxygenase-1thioredoxin peroxiredoxins (Prxs) and Sulfiredoxin (Srx)[13] Nrf2 is released from the Nrf2-Keap 1 (Kelch-likeECH-associated protein 1) complex and translocated to thenucleus after the initiation of oxidative stress [14] Srx arecently discovered member of the oxidoreductases familycontributes to cellular redox balance Previous studies haveshown Srx to be the only enzyme which catalyses ATP-dependent reduction of the hyperoxidized form of Prxs [1516] Prxs are important peroxidases that reduce peroxides[17 18] Peroxiredoxin 3 (Prx 3) is a critical scavenger formitochondrial H2O2 Also mitochondria contain 30 timesmore Prx 3 than glutathione peroxidase [19]

In the present study we aim to investigate the effect ofnormal iodide intake (NI) 10 times high iodide intake (10HI) and 100 times high iodide intake (100 HI) on Days 7 14and 28 on the antioxidative action of Srx and Prx 3 via Nrf2-Keap 1 pathway in the thyroid of rats

2 Methods

21 Animals and Diet A total of 216 Wistar rats (eight weeksold) at SPF level weighing 29636 plusmn 853 g were randomlyassigned to NI 10 HI and 100 HI groups Along with thenormal diet the NI group (with the addition of deionizedwater) 10 HI and 100 HI groups received different dosagesof potassium iodide in the deionized water resulting in thefollowing daily iodide intake 75 120583gd 75 120583gd and 750120583gdrespectively [20] The rats were sacrificed after iodide intakefor a week two weeks and four weeks at ages of 9 10 and12 weeks respectively (they are collectively referred to as Day7 Day 14 and Day 28) In this study a total of two rats diedin the NI group and none died in any other group Animalprocedures were approved by the Institutional Animal Careand Use Committee of Tianjin Medical University (thenumber is SYXK (Jin) 2014-0004) which is in accordancewith the NIH Guide

22 Reagent Anti-Peroxiredoxin-3 antibody (ab16751)and anti-Keap 1 antibody (ab66620) were purchased fromAbcam (Abcam Cambridge MA USA) Nrf2 (H-300)sc-13032 Sulfiredoxin (FL-137) sc-99076 goat anti-rabbitIgG-HRP sc-2004 goat anti-mouse IgG-HRP sc-2005and goat anti-rabbit IgG-PE sc-3739 were bought fromSanta Cruz (Santa Cruz Biotechnology Inc CA USA)MitoSOX Red (38-phenanthridinediamine5-(61015840-triphenyl-phosphoniumhexyl)-56-dihydro-6-phenyl) mitochondrialsuperoxide indicator (M36008) was purchased fromInvitrogen (Invitrogen Life Technologies CA USA) 120573-Actin(AA128) was purchased from Beyotime (Beyotime Instituteof Biotechnology Jiangsu China) RPMI-1640 and fetalbovine serum (FBS) were purchased from GE HealthcareLife Sciences (HyClone UT USA) Immobilon WesternChemiluminescent HRP Substrate (WBKLS0100) was

purchased from Millipore (Merck Millipore MA USA) Allthe other chemicals made in China were of analytic grade[21]

23 Thyroid Weight Measurement Following the intake ofNI 10 HI and 100HI the body weight and the thyroid weightwere measured when the rats were sacrificed on Days 7 14and 28 We calculated the ratio of the thyroid weightbodyweight (milligrams per 100 gram body) according to theratio of the viscera (viscerabody weight) [21 22] The ratswere anesthetized at appropriate concentrations (10 chloralhydrate 03mL100 g) Skin subcutaneous tissue fascia andmuscles of the anterior neck were removed and the thyroidgland was exposed In order to ensure the integrity of thethyroid gland it was removed carefully with a trachea ringThe fascia covering thyroid gland was stripped and thethyroid gland was collected under stereoscopic microscopecarefully

24 Measurement of Urinary Iodine Concentration and IodineContent in the Thyroid Glands Urine samples were collectedusingmetabolic cages for 24 hours the day before the ratsweresacrificed Thyroid tissue homogenates were prepared Uri-nary iodine concentration and iodine content in the thyroidglands were measured by As-Ce catalytic spectrophotometryin the Key Lab of Hormones and Development Ministry ofHealth Institute of Endocrinology Tianjin Medical Univer-sity [23] The iodine standard solution and samples wereadded to the test tubes (15mm times 150mm) respectively1mL ammonium persulfate was added and then mixed anddigested for 60 minutes at 100∘C After the test tubes werecooled down 25mL of arsenious acid solution was addedandmixed Consequently 03mL cerium sulfate solution wasadded and mixed every 30 s The absorbance at 400 nm wasmeasured with a spectrophotometer

25 Serum Thyroid Hormones Levels Measurement Bloodsamples were taken from the carotid artery and then cen-trifuged for 10min at 2000 rmin to obtain the samples ofthe serum After that the samples were stored at minus80∘Cfor further analysis Levels of serum total thyroxine (TT4)total triiodothyronine (TT3) free thyroxine (FT4) and freetriiodothyronine (FT3) were all determined using a chemi-luminescent immunoassay technique All kits for thyroidfunction were purchased from Siemens (Siemens HealthcareDiagnostics Products Limited Llanberis UK)

26 Flow Cytometry MitoSOX Red was used to measuremitochondrial superoxide production by flow cytometryThe thyroid cell suspension of Wistar rats was prepared5 120583M of MitoSOX Red was added and the suspension wasincubated for 10min at 37∘C in the dark Flow cytometry wascarried out using a FACSCalibur (BD Bioscience San JoseCA) Collecting FL2 channel forward scattering (forwardscatter FSC) and lateral scattering (side scatter SSC) data10000 cells were collected for each sampleThe control groupwithout MitoSOX was regarded as the blank zero group forstandardization [24]

Oxidative Medicine and Cellular Longevity 3

Table 1 Changes of the median urinary iodine concentration (120583gL) of Wistar rats following the intake of NI 10 HI and 100 HI on Days 714 and 28 (119873 = 6 for each group) All data is presented as the median (range)

Group The median urinary iodine concentration (120583gL)Day 7 Day 14 Day 28

NI 3212 (1500ndash3517) 3582 (3002ndash3738) 3330 (1886ndash4135)10 HI 30525lowast (15928ndash37980) 35325lowast (14870ndash40560) 26280lowast (16510ndash34045)100 HI 264892lowast (58566ndash421704) 244610lowast (176075ndash298748) 226632lowast (83425ndash298162)lowastCompared to the NI group (119901 lt 005)Compared to the 10 HI group (119901 lt 005)

27 Western Blot Analysis The bicinchoninic acid proteinassay kit (Beyotime Institute of Biotechnology JiangsuChina) was used to detect the concentration of proteins50120583g proteins were separated by SDS-PAGE and transferredto the PVDF membrane Subsequently the membrane wasblocked for 1 hour at room temperature using 5 nonfatmilk Then the membrane was incubated overnight at 4∘Cwith primary antibodies followed by horseradish peroxidaseconjugated secondary antibodiesThe proteins were detectedby Immobilon Western Chemiluminescent HRP Substrate120573-Actin was used as a loading control Blots were scannedas gray scale images and quantified using Image J software(NIH) All the blot intensities were normalized with that ofthe loading control 120573-actin

28 Immunofluorescence Staining The thyroid tissues werefirst embedded with an Optimal Cutting Temperature (OCT)compound at minus80∘C Following which the tissues were frozenin a cryostat machine and cut into frozen sections for 5 120583m atminus20∘C The slices were incubated with 5 FBS for 60min atroom temperature Subsequently they were incubated witha primary antibody [Nrf2 (1 100) or Srx (1 100)] at 4∘Covernight After three washes with PBS the second antibodywas linked to fluorophores (goat anti-rabbit IgG-PE) Thenucleus was stained for 5minwithHoechst 33258 (50120583L) andwashed 3 times with PBS MitoSOX Red was used to measuremitochondrial superoxide production Using a Zeiss LSM510 confocal microscope fluorescent images of the preparedslides were obtained

29 Statistics The data of urinary iodine concentration(Table 1) showed a skewed distribution and were expressedas the median Differences between groups were evaluated bynonparametric KruskalndashWallis test If the latter test showedsignificant differences between groups the individual groupswere compared with the control group by the Nemenyitests using SPSS 220 A 119901 value of lt005 was consideredstatistically significant [25]

The other data was expressed as mean plusmn SD Differencesbetween groups were evaluated by one-way analysis of vari-ance (ANOVA) if this test showed significant differencesbetween groups the individual groups were compared withthe control group by Least Significant Difference (LSD)test using SPSS 220 A 119901 value of lt005 was consideredstatistically significant

3 Results

31 Effects of the Iodide Intake (NI 10 HI and 100 HI) on theRatio of Thyroid WeightBody Weight on Days 7 14 and 28The parameters body weight thyroid weight and the ratio ofthe thyroid weightbody weight were not significantly alteredfollowing the intake of NI 10 HI and 100 HI on Days 7 14and 28 (119901 gt 005) (Figure 1)

32 Effects of the Iodide Intake (NI 10 HI and 100 HI)on Urinary Iodine Concentration and Iodine Content in theThyroid Glands on Days 7 14 and 28 The median urinaryiodine concentration and their ranges for NI 10 HI and 100HI on Days 7 14 and 28 are illustrated in Table 1 In allthe different time periods there was a significant increase inthe urinary iodine concentration among 10 HI and 100 HIwhen compared to the NI group (119901 lt 005) In additionthe urinary iodine concentration of the 100 HI group alsoincreased significantly when compared to 10 HI (119901 lt 005)Between any of the iodide intake groups (NI 10 HI and100 HI) there was no significant difference in the urinaryiodine concentration on Day 14 and Day 28 when comparedto Day 7 (119901 gt 005) Furthermore there was no significantdifference between Day 14 and Day 28 (119901 gt 005) Whenthe intake of iodide was increased from NI to 10 HI andfurther to 100 HI a simultaneous increase in the urinaryiodine concentration was also observed on Days 7 14 and28 The urinary iodine concentration in the 10 HI group wasapproximately 10 times that of the NI group whereas theurinary iodine concentration in the 100 HI group was about60ndash80 times that of the NI group on Days 7 14 and 28

In our study we demonstrated that the iodine content inthe thyroid glands was significantly increased in the 100 HIgroup when compared to the NI group on Days 7 14 and 28(119901 lt 005) Between any of the iodide intake groups (NI 10HI and 100 HI) there was no significant difference foundin the iodine content in the thyroid glands on Days 7 14and 28 (119901 gt 005) When the intake of iodide was increasedfrom NI to 10 HI and further to 100 HI the iodine content inthe thyroid glands increased gradually on Days 7 14 and 28(Table 2)

33 Effects of the Iodide Intake (NI 10 HI and 100 HI) on theChanges of SerumThyroid Hormones Levels on Days 7 14 and28 There were no significant alterations in TT3 TT4 FT3

4 Oxidative Medicine and Cellular Longevity

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Figure 1 Effects of the iodide intake (NI 10 HI and 100 HI) on (a) the body weight (b) the thyroid weight and (c) the ratio of the thyroidweightbody weight on Days 7 14 and 28 All data is presented as mean plusmn SD (119873 = 6 for each group) Statistical analyses were performed byone-way analysis of variance (ANOVA) with the Least Significant Difference (LSD) test

and FT4 levels following the intake ofNI 10HI and 100HI onDays 7 14 and 28 (119901 gt 005) Moreover for all three dosagesof iodide intake there were no significant differences in anyof the serum thyroid hormones levels on Day 14 and Day 28when compared to Day 7 (119901 gt 005) In addition there wereno significant changes between Day 14 and Day 28 in serumthyroid hormones levels (TT3 TT4 FT3 and FT4) (119901 gt 005)(Table 3)

34 Effects of the Iodide Intake (NI 10 HI and 100 HI) on theChanges of Mitochondrial Superoxide Production on Days 7

14 and 28 On Days 7 14 and 28 when compared to the NIgroup the mitochondrial superoxide production in the 10 HIgroup showed no significant increase (119901 gt 005) Howeverthere was a significant increase in the mitochondrial super-oxide production in the 100 HI group (119901 lt 005) Whencompared to the 10 HI there was a significant increase in themitochondrial superoxide production in the 100 HI group(119901 lt 005) In the NI and 10 HI groups the mitochondrialsuperoxide production on Days 7 14 and 28 showed nosignificant difference (119901 gt 005) However in the 100 HIgroup compared to Day 7 there was a significant increase in

Oxidative Medicine and Cellular Longevity 5

Table 2 Changes of iodine content in the thyroid glands(120583g100mg) of Wistar rats following the intake of NI 10 HI and 100HI onDays 7 14 and 28 (119873 = 6 for each group) All data is presentedas the mean plusmn SD

Group Iodine content in the thyroid glands (120583g100mg)Day 7 Day 14 Day 28

NI 6219 plusmn 594 6012 plusmn 813 6337 plusmn 90810 HI 12125 plusmn 1048 12645 plusmn 893 15233 plusmn 1077100 HI 13353 plusmn 861lowast 13636 plusmn 964lowast 17845 plusmn 874lowastlowastCompared with NI group (119901 lt 005)

Table 3 Changes of serum thyroid hormones levels of Wistar ratsfollowing the intake of NI 10 HI and 100 HI on Days 7 14 and 28(119873 = 6 for each group) All data is presented as the mean plusmn SD

Group The levels of serum thyroid hormonesTT3

(nmolL)TT4

(nmolL)FT3

(pmolL)FT4

(pmolL)Day 7

NI 118 plusmn 015 9077 plusmn 552 396 plusmn 010 2203 plusmn 17010 HI 119 plusmn 008 9154 plusmn 774 377 plusmn 026 2302 plusmn 206100 HI 107 plusmn 011 8917 plusmn 903 378 plusmn 011 2008 plusmn 543

Day 14NI 115 plusmn 007 9583 plusmn 520 392 plusmn 016 2234 plusmn 37110 HI 112 plusmn 008 8615 plusmn 252 363 plusmn 048 2019 plusmn 192100 HI 119 plusmn 004 9154 plusmn 331 384 plusmn 046 2098 plusmn 250

Day 28NI 113 plusmn 006 9417 plusmn 238 389 plusmn 013 2025 plusmn 57210 HI 118 plusmn 008 9077 plusmn 378 398 plusmn 057 2115 plusmn 534100 HI 114 plusmn 022 8769 plusmn 635 379 plusmn 046 2117 plusmn 543

mitochondrial superoxide production on Day 28 (119901 lt 005)Similarly compared to Day 14 there was also a significantincrease in mitochondrial superoxide production on Day 28(119901 lt 005) Accordingly the fluorescent intensity ofMitoSOXRed onDay 28 gradually increased after the increased dosagesof iodide intake fromNI to 100 HI which was consistent withour results of flow cytometry (Figure 2)

35 Effects of the Iodide Intake (NI 10 HI and 100 HI) on theChanges of Nrf2 Keap 1 Srx and Prx 3 Expressions on Days 714 and 28 On Days 7 14 and 28 when compared to the NIgroup the expressions of Nrf2 Keap 1 Srx and Prx 3 showedno significant differences in the 10 HI group (119901 gt 005)the expressions of Nrf2 Srx and Prx 3 were significantlyincreased while Keap 1 was notably decreased in the 100 HIgroup (119901 lt 005) On Days 7 14 and 28 when comparedto the 10 HI group the expressions of Nrf2 Srx and Prx 3were significantly increased while Keap 1 was significantlydecreased in the 100 HI group (119901 lt 005) (Figure 3)

36 Effects of the Iodide Intake (NI 10 HI and 100 HI) on theChanges of Immunofluorescence Staining onDays 7 14 and 28Following the increased iodide intake from NI to 10 HI and

further to 100 HI the expressions of Nrf2 and Srx intensifiedIn the NI group Nrf2 was localized in the cytoplasm on Days7 14 and 28 In the 10 HI group the positive staining ofNrf2 can be observed in both the nucleus and the cytoplasmMoreover in the 100 HI group a stronger positive stainingof Nrf2 can be detected in the nucleus on Days 7 14 and 28(Figure 4(a)) Srx positive staining was only located in thecytoplasm on Days 7 14 and 28 (Figure 4(b))

4 Discussion

In our study we found that there were no significant alter-ations in TT3 TT4 FT3 and FT4 levels following the intakeofNI 10HI and 100HI onDays 7 14 and 28 (119901 gt 005)Thereare very efficient homeostatic mechanisms that resist changesin circulating T3 and T4 levels in response to iodide excessDue to the compensatory mechanisms such as the Wolff-Chaikoff effect the changes in T3 and T4 levels following anincrease in iodide intake are minimal and usually transientin nature [26ndash28] The Wolff-Chaikoff effect relies on a high(ge10minus3 molar) intracellular concentration of iodide Duringinitial exposure excess iodide is transported by the sodium-iodide symporter (NIS) into the cellsWhen intracellular con-centration reaches at least 10minus3 molar iodide organificationis blocked [27 29] Moreover the regulatory mechanismsinclude modulation of blood flow enzyme activity geneexpression and transport proteins in signaling pathways [230 31] In a study conducted by Eng et al 16 Wistar ratswere given 2000120583g of iodide acutely it was observed thatthere was a significant decrease in serum T4 and T3 levelson Day 1 of the study Subsequently on Day 6 both serumT4 and T3 levels returned to normal ranges However inboth cases the serum TSH levels remained unchanged [32]Mooij et al observed no significant changes in serum thyroidhormones levels when female Wistar rats were given 100 120583giodide daily for 18 weeks [33] Paul et al demonstrated thatwhen normal volunteers received 1500120583g of supplementaliodine daily for 14 days a small decrease in serum T3 andT4 concentrations with compensatory increase of TSH wasdetected although all values remained within the normalranges [34] However the presence of handicaps such as anincreased autoimmune susceptibility fetal period extremesof age pregnancy lactation or an active pathological entitysignificantly impair these mechanisms [1 35ndash37]

Our study demonstrated that both the urinary iodineconcentration and the iodide intake increased simultaneouslyfrom NI to 10 HI and further to 100 HI Interestingly wefound that the urinary iodine concentration of the 10 HIgroup was approximately 10 times that of the NI groupwhereas the urinary iodine concentration of the 100HI groupwas about 60ndash80 times that of the NI group on Days 7 14and 28 The urinary iodine concentration is regarded as asensitive indicator of iodine status because approximately90 of ingested iodide is excreted in the urine [38 39]Thereis an increase for the maintenance of thyroid homeostasisas well as the steady state of the internal environment ofthe body The thyroid gland has adaptation mechanisms thatreduce iodide metabolism when the supply is abundant thus

6 Oxidative Medicine and Cellular Longevity

1793 2066 4897

1773 2264 5291

1806 2373 7360

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lowast998771998779

(b)

Figure 2 Continued

Oxidative Medicine and Cellular Longevity 7

NI 10 HI 100 HI

Day 28

20120583m20120583m20120583m

(c)

Figure 2 Effects of the iodide intake (NI 10 HI and 100 HI) on the changes of mitochondrial superoxide production on Days 7 14 and28 (a) There was a significant increase in the mitochondrial superoxide production in the 100 HI group on Days 7 14 and 28 (119901 lt 005)The difference was more significant on Day 28 when compared to Day 7 or Day 14 (b) Histogram analysis was performed on the meanfluorescence intensity of MitoSOX Red All data is presented as the mean plusmn SD (119873 = 6 for each group) Statistical analyses were performedby one-way analysis of variance (ANOVA) with the Least Significant Difference (LSD) test lowast119901 lt 005 versus the NI group on Days 7 14 and28 respectively 119901 lt 005 versus the 10 HI group on Days 7 14 and 28 respectively 998771119901 lt 005 versus the Day 7 group in the 100 HI group119901 lt 005 versus the Day 14 in the 100 HI group Experiments were repeated 3 times with similar results (c) There was a significant increasein the mitochondrial superoxide production in the 100 HI group on Day 28 and was observed by confocal microscopy Scale bar 20120583m

avoiding thyrotoxicosisThere are several mechanisms whichinclude a direct inhibitory effect of iodide in the thyroid itselfand inhibition by iodide of its own organification (Wolff-Chaikoff effect) its transport thyroid hormones secretioncAMP formation in response to TSH and several othermetabolic steps [40] We suggest that all the protectivemechanisms may ensure the excessive iodide intake of the 10HI group on Days 7 14 and 28 be eliminated from urineTheurinary iodine concentration of the 100 HI group on Days7 14 and 28 was about 60ndash80 times that of the NI groupalthough the thyroid function was normal This leads us topropose the idea that excessive iodide accumulated in thebody may trigger the oxidative and antioxidative signalingpathway to maintain the normal thyroid function

We demonstrated that the production of mitochondrialsuperoxide significantly increased on Days 7 14 and 28 inthe 100 HI group This is consistent with our previous studyon metallothionein-III knockout mice [24] Joanta et alreported that the initiation of free radical production wasobserved after giving a high dose of iodide [3] Serrano-Nascimento et al demonstrated that an increased mitochon-drial superoxide production was shown in response to NaI(10minus6M to 10minus3M) treatment in PCCl3 thyroid cells by usingMitoSOX Red [41] Mitochondria are potent producers ofsuperoxide from complexes I and III of the electron transportchain Mitochondrial superoxide production is a major causeof cellular oxidative damage [42] Physiologically ROS arenot necessarily harmful because they are continuously bal-anced by the process of hormone synthesis and the endoge-nous antioxidant system [1] Excess ROS are generated duringthe trapping oxidation and organification of excessive iodinein thyrocytes which could lead to increased oxidative stress[1]

The Nrf2-Keap 1 pathway is the chief cytoprotectivemechanism in response to oxidative stress caused by ROSDuring normal and balanced redox homeostasis the Nrf2function is inhibited because of constant proteasomal degra-dation after ubiquitination of the protein This is regulated

through the binding of the inhibitor protein Keap 1 [13 14]It is reported that Srx activation via Nrf2 dependent pathwayprotects fromoxidative liver injury throughPyrazole [43] andalcohol in mice [44] Similar findings in lung tissues haveshown that there is a marked increase in the expressions ofSrx and Prx 3 in human squamous cell carcinoma [45] Thissuggests that these proteins may play a protective role againstoxidative injury Also the pathway including Keap 1 Nrf2and ARE-mediated protein expression plays a very criticalrole in protecting cells fromoxidative stress [46 47] Focusingon the pathway we demonstrated that the expression of Nrf2was significantly increased while Keap 1 was significantlydecreased in the 100 HI group when compared to the NIor 10 HI group on Days 7 14 and 28 Similarly Ajiboyeet al showed that rats treated with Chalcone dimers notonly increased the expression of Nrf2 but also suppressedcytoplasmic Keap 1 expression [48] Yang et al also showedthat the downregulation of Keap 1 level may be responsiblefor the overactivation of Nrf2 [49] Pang et al showed thatcaffeic acid prevents acetaminophen-induced liver injury byactivating the Nrf2-Keap 1 antioxidative defense system [50]

In order to verify whether high levels of expressionof Nrf2 and its nuclear translocation can upregulate someantioxidant enzymes in the thyroid gland the expressions ofPrx 3 and Srx following the intake of NI 10 HI and 100 HIon Days 7 14 and 28 were measured We demonstrated thatthe expressions of Srx and Prx 3 in the 100 HI group weresignificantly increased when compared to the NI group or 10HI group on Days 7 14 and 28 The possible explanationsare described as below Firstly Srx is a cytosolic proteinthat is able to translocate to sites where hyperoxidized(inactivated) Prx 3 is located Therefore it engages itself inthe reactivation of Prx 3 under oxidative conditions [51]Secondly Prx 3 is a typical 2-Cys Peroxiredoxin locatedexclusively in the mitochondrial matrix it is the principalperoxidase responsible for protecting cells from oxidativedamage by reducing peroxides such as H2O2 [52] Finallymitochondria contain 30 times more Prx 3 than glutathione

8 Oxidative Medicine and Cellular Longevity

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lowast lowast

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NI 10 HI 100 HI NI 10 HI 100 HI NI 10 HI 100 HI

(d)

Figure 3 Effects of the iodide intake (NI 10 HI and 100 HI) on the changes of (a) Nrf2 (b) Keap 1 (c) Srx and (d) Prx 3 expressions onDays 7 14 and 28 Representative western blot and histograms of densitometric analyses normalized for the relative 120573-actin content All datais presented as the mean plusmn SD (119873 = 6 for each group) Statistical analyses were performed by one-way analysis of variance (ANOVA) withthe Least Significant Difference (LSD) test lowast119901 lt 005 versus the NI group on Days 7 14 and 28 respectively 119901 lt 005 versus the 10 HI groupon Days 7 14 and 28 respectively Experiments were repeated 3 times with similar results

Oxidative Medicine and Cellular Longevity 9

NI

10 HI

100 HI

Hoechst Nrf2 Merge HoechstHoechst

Day 7 Day 28 Day 14

Nrf2 Merge Nrf2 Merge

Day 7 Day 7 Day 14 Day 14 Day 28 Day 28 20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

(a)

Hoechst Srx Merge Hoechst Srx Merge Hoechst Srx Merge

NI

10 HI

100 HI

Day 7 Day 28 Day 14 Day 7 Day 7 Day 14 Day 14 Day 28 Day 28 20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

(b)

Figure 4 Effect of the iodide intake (NI 10 HI and 100 HI) on the changes of immunofluorescence staining on Days 7 14 and 28 (a) In theNI group Nrf2 (red) was localized in the cytoplasm on Days 7 14 and 28 In the 10 HI group the positive staining of Nrf2 can be observedin both the nucleus and the cytoplasm Moreover in the 100 HI group a stronger positive staining of Nrf2 can be detected in the nucleus onDays 7 14 and 28 the nucleus was dyed with Hoechst (blue) (b) Srx (red) positive staining was located in the cytoplasm the nucleus wasdyed with Hoechst (blue) Scale bar 20120583m

peroxidase Prx 3 can be classified as an important regulatorof mitochondrial H2O2 [22] The elevated expression of Prx3 is associated with the blockage of apoptosis increasingcell proliferation and is related to adaptive responses whichare all required to maintain mitochondrial function [53 54]Bae et al have suggested that Prx 3 and Srx jointly protectmice from Pyrazole-induced oxidative liver injury in a Nrf2-dependent manner [43]

The novelty we demonstrated in the present study is thatiodide excess induced both oxidative stress and antioxidativedefense increases throughNrf2-Keap 1 pathway in the thyroidgland from rats We extended our established mechanismsby applying the Nrf2-Keap 1 pathway to set up a bridgebetween oxidative stress and antioxidative defense inducedby iodide excess in the thyroid gland In our previous studywe have established that oxidative stress induced by acutehigh concentrations of iodide in FRTL cells significantlyincreases mitochondrial superoxide production [55] Theinhibitors of themitochondrial respiratory chain complexes I

and III are involved inmitochondrial superoxide productionWe demonstrated that exposure to 100120583M KI for 2 hourssignificantly increased mitochondrial superoxide produc-tion enhanced by either 05120583M Rotenone (an inhibitorof mitochondrial complex I) or 10 120583M Antimycin A (aninhibitor of complex III) [56] We illustrated that 300 120583MPTU (an inhibitor of TPO) attenuated the excessive iodide-induced mitochondrial superoxide production We showedthat 30 120583M KClO4 (a competitive inhibitor of iodide trans-port) relieved the production the mitochondrial superoxideinduced by iodide excess We displayed that 10mUmL TSHcan inhibit excessive iodide-induced strong mitochondrialsuperoxide production [55] MT-I and MT-II are mainlyinvolved in the protection of tissue against oxidative stresseswe indicated that metallothionein-III knockout mice aggra-vated mitochondrial superoxide production in thyroid afterexcessive iodide exposure [24] In addition we demonstratedthat both the oxidative stress and the antioxidative defenseincreased simultaneously after high dosages of iodide intake

10 Oxidative Medicine and Cellular Longevity

Oxidative stress

(a)

(b)

(c)

Antioxidative defense

Wist

ar ra

ts Day 7Day 14Day 28

Normal iodideintake

Normal urinaryiodine

Normal thyroidfunction

10 times iodideintake

10 times urinaryiodine

Normal thyroidfunction

100 times iodideintake

60ndash80 timesurinary iodine

Normal thyroidfunction

Keap 1 Nrf2

Keap 1

Nrf2

Srx

Nucleus

Prx 3

SrxARE

Mitochondria

AntioxidativeOxidativestress

Oxidative Antioxidative

Oxidative Antioxidative

Under physiological conditions(normal iodide intake)

Excessive iodide intake(10 times iodide intake)

Excessive iodide intake(100 times iodide intake)

defense defenseuarrstressuarr

defenseuarruarrstressuarruarr

Figure 5 Proposed mechanisms in the present study (a) The urinary iodine concentration and thyroid function of Wistar rats weredetected (b) The activation of the Nrf2-Keap 1 pathway induced by iodide excess in the thyroid (c) The balance between oxidative stressand antioxidative defense under physiological conditions and excessive iodide intake

Oxidative Medicine and Cellular Longevity 11

We suggested that the Nrf2-Keap 1 pathway is vital for thebalance between oxidative stress and antioxidative defenseinduced by iodide excess in the thyroid gland

Excessive iodide stimulated the Nrf2-Keap 1 pathwayand enhanced the antioxidative defense We found thatthe urinary iodine concentration of the 100 HI group wasabout 60ndash80 times that of the NI group on Days 7 14and 28 however the thyroid functions were normal Weproposed that the excessive iodide accumulated in the bodymay trigger the Nrf2-Keap 1 pathway to maintain a normalthyroid functionWe demonstrated that the Nrf2moves fromthe cytoplasm to the nucleus under the microscope withsignificantly increased expressions of Nrf2 Srx and Prx 3 andnotably decreased Keap 1 when exposed to high iodide Thissuggests that excessive iodide stimulates the disassociation ofNrf2 from Keap 1 and assists Nrf2 to penetrate the nucleusThen Nrf2 attaches to the antioxidant response element(ARE) to activate the expression of the antioxidative genesSrx and Prx 3 resulting in an enhanced antioxidative defenseinduced by high iodide By activation of the Nrf2-Keap 1pathway there is a proportional increase in oxidative stressand antioxidative defense in response to iodide excess It isto be noted that the thyroid function was normal in the 10HI group and 100 HI in present study Inspired by the reportby Poncin et al [6] we proposed that there should be abalance between oxidative stress and antioxidative defense inresponse to iodide excess in the 10 HI and the 100 HI groups

5 Conclusion

In conclusion our results highlight that the activation ofNrf2-Keap 1 Srx and Prx 3 antioxidative defense mecha-nisms may play a crucial role in protecting the thyroid fromiodide excess induced oxidative stress on Days 7 14 and 28(Figure 5)

Abbreviations

NI Normal iodide intakeHI High iodide intakeROS Reactive oxygen speciesH2O2 Hydrogen peroxideNrf2 Nuclear factor erythroid 2-related factor 2Keap 1 Kelch-like ECH-associated protein 1ARE Antioxidant response elementPrx PeroxiredoxinSrx SulfiredoxinPrx 3 Peroxiredoxin 3TT4 Total thyroxineTT3 Total triiodothyronineFT4 Free thyroxineFT3 Free triiodothyronineTSH ThyrotropinLSD Least significant difference

Disclosure

This manuscript has been edited by Umar Iqbal IruniRoshanie Abeysekera Gargi Naha and Tejas Bharadwaj all

of whom are currently studying at the International School ofMedicine Tianjin Medical University China

Competing Interests

The authors declare that there is no conflict of interestsregarding the publication of this article

Acknowledgments

This work is supported by National Natural Science Founda-tion of China (nos 81273009 and 81270182) Tianjin Scienceamp Technology Council Grant of China (nos 16JCYBJC26100and 09JCYBJC11700) and International Studentrsquos Science ampTechnology Innovation Project (Scientific Research Project)of Tianjin Medical University

References

[1] Y Luo A Kawashima Y Ishido et al ldquoIodine excess as anenvironmental risk factor for autoimmune thyroid diseaserdquoInternational Journal of Molecular Sciences vol 15 no 7 pp12895ndash12912 2014

[2] A E Hussein A M Abbas G A El Wakil A Z Elsamanoudyand A A El Aziz ldquoEffect of chronic excess iodine intakeon thyroid function and oxidative stress in hypothyroid ratsrdquoCanadian Journal of Physiology and Pharmacology vol 90 no5 pp 617ndash625 2012

[3] A E Joanta A Filip S Clichici S Andrei and D DaicoviciuldquoIodide excess exerts oxidative stress in some target tissues ofthe thyroid hormonesrdquoActa Physiologica Hungarica vol 93 no4 pp 347ndash359 2006

[4] SMorandMChaaraoui J Kaniewski et al ldquoEffect of iodide onnicotinamide adenine dinucleotide phosphate oxidase activityand Duox2 protein expression in isolated porcine thyroidfolliclesrdquo Endocrinology vol 144 no 4 pp 1241ndash1248 2003

[5] J J Lazaro A Jimenez D Camejo et al ldquoDissecting the inte-grative antioxidant and redox systems in plant mitochondriaEffect of stress and S-nitrosylationrdquo Frontiers in Plant Sciencevol 4 article 460 2013

[6] S Poncin A-C GerardM Boucquey et al ldquoOxidative stress inthe thyroid gland from harmlessness to hazard depending onthe iodine contentrdquo Endocrinology vol 149 no 1 pp 424ndash4332008

[7] M Karbownik and A Lewinski ldquoThe role of oxidative stressin physiological and pathological processes in the thyroidgland possible involvement in pineal-thyroid interactionsrdquoNeuroendocrinology Letters vol 24 no 5 pp 293ndash303 2003

[8] P Thomopoulos ldquoHyperthyroidism due to excess iodinerdquo LaPresse Medicale vol 31 no 35 pp 1664ndash1669 2002

[9] J Wolff ldquoIodide goiter and the pharmacologic effects of excessiodiderdquoTheAmerican Journal of Medicine vol 47 no 1 pp 101ndash124 1969

[10] B Corvilain L Collyn J Van Sande and J E Dumont ldquoStim-ulation by iodide of H2O2 generation in thyroid slices fromseveral speciesrdquoAmerican Journal of PhysiologymdashEndocrinologyand Metabolism vol 278 no 4 pp E692ndashE699 2000

[11] M-C Many C Mestdagh M-F Van den Hove and J-F DenefldquoIn vitro study of acute toxic effects of high iodide doses inhuman thyroid folliclesrdquo Endocrinology vol 131 no 2 pp 621ndash630 1992

12 Oxidative Medicine and Cellular Longevity

[12] J Van Sande G Grenier C Willems and J E DumontldquoInhibition by iodide of the activation of the thyroid cyclic 3101584051015840-AMP systemrdquo Endocrinology vol 96 no 3 pp 781ndash786 1975

[13] R Young J D Hayes K Brown C R Wolf and C B AWhitelaw ldquoPeroxiredoxin gene expression signatures in liverreflect toxic insultrdquo Assay and Drug Development Technologiesvol 8 no 4 pp 512ndash517 2010

[14] Q Ma ldquoRole of Nrf2 in oxidative stress and toxicityrdquo AnnualReview of Pharmacology and Toxicology vol 53 pp 401ndash4262013

[15] B Biteau J Labarre and M B Toledano ldquoATP-dependentreduction of cysteine-sulphinic acid by S cerevisiae sulphire-doxinrdquo Nature vol 425 no 6961 pp 980ndash984 2003

[16] L Wu H Jiang H A Chawsheen et al ldquoTumor promoter-induced sulfiredoxin is required for mouse skin tumorigenesisrdquoCarcinogenesis vol 35 no 5 pp 1177ndash1184 2014

[17] C C Winterbourn and M B Hampton ldquoThiol chemistryand specificity in redox signalingrdquo Free Radical Biology andMedicine vol 45 no 5 pp 549ndash561 2008

[18] J W Baty M B Hampton and C C Winterbourn ldquoProteomicdetection of hydrogen peroxide-sensitive thiol proteins inJurkat cellsrdquo Biochemical Journal vol 389 no 3 pp 785ndash7952005

[19] T-S Chang C-S Cho S Park S YuW K Sang and G R SueldquoPeroxiredoxin III a mitochondrion-specific peroxidase regu-lates apoptotic signaling by mitochondriardquo Journal of BiologicalChemistry vol 279 no 40 pp 41975ndash41984 2004

[20] P Thomopoulos ldquoHyperthyroidism due to excess iodinerdquo LaPresse Medicale vol 31 no 35 pp 1664ndash1669 2002

[21] S Chung X-H Liao C Di Cosmo et al ldquoDisruption of themelanin-concentrating hormone receptor 1 (MCH1R) affectsthyroid functionrdquo Endocrinology vol 153 no 12 pp 6145ndash61542012

[22] G Ozguner and O Sulak ldquoSize and location of thyroid gland inthe fetal periodrdquo Surgical and Radiologic Anatomy vol 36 no4 pp 359ndash367 2014

[23] P L Jooste and E Strydom ldquoMethods for determination ofiodine in urine and saltrdquo Best Practice amp Research ClinicalEndocrinology amp Metabolism vol 24 no 1 pp 77ndash88 2010

[24] N Zhang L Wang Q Duan et al ldquoMetallothionein-IIIknockoutmice aggravatemitochondrial superoxide productionand peroxiredoxin 3 expression in thyroid after excessive iodideexposurerdquoOxidative Medicine and Cellular Longevity vol 2015Article ID 267027 11 pages 2015

[25] P I Gus D Marinho S Zelanis et al ldquoA case-control studyon the oxidative balance of 50 autologous serum eye dropsrdquoOxidative Medicine and Cellular Longevity vol 2016 Article ID9780193 5 pages 2016

[26] P Laurberg C Cerqueira L Ovesen et al ldquoIodine intake as adeterminant of thyroid disorders in populationsrdquo Best Practiceamp Research Clinical Endocrinology amp Metabolism vol 24 no 1pp 13ndash27 2010

[27] H Burgi ldquoIodine excessrdquo Best Practice amp Research ClinicalEndocrinology amp Metabolism vol 24 no 1 pp 107ndash115 2010

[28] E Roti and E D Uberti ldquoIodine excess and hyperthyroidismrdquoThyroid vol 11 no 5 pp 493ndash500 2001

[29] A M Leung and L E Braverman ldquoConsequences of excessiodinerdquo Nature Reviews Endocrinology vol 10 no 3 pp 136ndash142 2014

[30] K Wang Y N Sun J Y Liu et al ldquoThe impact of iodineexcess on thyroid hormone biosynthesis and metabolism in

ratsrdquoBiological Trace Element Research vol 130 no 1 pp 72ndash852009

[31] M Michalkiewicz L J Huffman J M Connors and G AHedge ldquoAlterations in thyroid blood flow induced by varyinglevels of iodine intake in the ratrdquo Endocrinology vol 125 no 1pp 54ndash60 1989

[32] P H K Eng G R Cardona S-L Fang et al ldquoEscape fromthe acute Wolff-Chaikoff effect is associated with a decrease inthyroid sodiumiodide symporter messenger ribonucleic acidand proteinrdquo Endocrinology vol 140 no 8 pp 3404ndash3410 1999

[33] P Mooij H J De Wit and H A Drexhage ldquoA high iodineintake in Wistar rats results in the development of a thyroid-associated ectopic thymic tissue and is accompanied by a lowthyroid autoimmune reactivityrdquo Immunology vol 81 no 2 pp309ndash316 1994

[34] T Paul B Meyers R J Witorsch et al ldquoThe effect of smallincreases in dietary iodine on thyroid function in euthyroidsubjectsrdquoMetabolism vol 37 no 2 pp 121ndash124 1988

[35] X Teng Z Shan W Teng C Fan H Wang and R GuoldquoExperimental study on the effects of chronic iodine excess onthyroid function structure and autoimmunity in autoimmune-prone NODH-2h4 micerdquo Clinical and Experimental Medicinevol 9 no 1 pp 51ndash59 2009

[36] E Swist Q Chen C Qiao D Caldwell H Gruber and KA Scoggan ldquoExcess dietary iodine differentially affects thyroidgene expression in diabetes thyroiditis-prone versusmdashresistantBioBreeding (BB) ratsrdquoMolecular Nutrition and Food Researchvol 55 no 12 pp 1875ndash1886 2011

[37] L Liu D Wang P Liu et al ldquoThe relationship between iodinenutrition and thyroid disease in lactating women with differentiodine intakesrdquo British Journal of Nutrition vol 114 no 9 pp1487ndash1495 2015

[38] S A Skeaff C D Thomson and R S Gibson ldquoMild iodinedeficiency in a sample of New Zealand school childrenrdquoEuropean Journal of Clinical Nutrition vol 56 no 12 pp 1169ndash1175 2002

[39] M B Zimmermann P L Jooste and C S Pandav ldquoIodine-deficiency disordersrdquo The Lancet vol 372 no 9645 pp 1251ndash1262 2008

[40] J Wolff ldquoExcess iodide inhibits the thyroid by multiple mecha-nismsrdquoAdvances in ExperimentalMedicine andBiology vol 261pp 211ndash244 1989

[41] C Serrano-Nascimento S D S Teixeira J P Nicola R TNachbar A M Masini-Repiso and M T Nunes ldquoThe acuteinhibitory effect of iodide excess on sodiumiodide symporterexpression and activity involves the PI3KAkt signaling path-wayrdquo Endocrinology vol 155 no 3 pp 1145ndash1156 2014

[42] M D Brand C Affourtit T C Esteves et al ldquoMitochondrialsuperoxide production biological effects and activation ofuncoupling proteinsrdquo Free Radical Biology andMedicine vol 37no 6 pp 755ndash767 2004

[43] S H Bae S H Sung H E Lee et al ldquoPeroxiredoxin IIIand sulfiredoxin together protect mice from pyrazole-inducedoxidative liver injuryrdquo Antioxidants amp Redox Signaling vol 17no 10 pp 1351ndash1361 2012

[44] S H Bae S H Sung E J Cho et al ldquoConcerted actionof sulfiredoxin and peroxiredoxin I protects against alcohol-induced oxidative injury inmouse liverrdquoHepatology vol 53 no3 pp 945ndash953 2011

[45] Y S Kim H L Lee K B Lee et al ldquoNuclear factor E2-related factor 2 dependent overexpression of sulfiredoxin and

Oxidative Medicine and Cellular Longevity 13

peroxiredoxin III in human lung cancerrdquo Korean Journal ofInternal Medicine vol 26 no 3 pp 304ndash313 2011

[46] H-Y Cho A E Jedlicka S P M Reddy et al ldquoRole of NRF2in protection against hyperoxic lung injury in micerdquo AmericanJournal of Respiratory Cell and Molecular Biology vol 26 no 2pp 175ndash182 2002

[47] T W Kensler N Wakabayashi and S Biswal ldquoCell survivalresponses to environmental stresses via the Keap1-Nrf2-AREpathwayrdquo Annual Review of Pharmacology and Toxicology vol47 pp 89ndash116 2007

[48] TO AjiboyeM T Yakubu andA TOladiji ldquoElectrophilic andReactive Oxygen Species Detoxification Potentials of ChalconeDimers is Mediated by Redox Transcription Factor Nrf-2rdquoJournal of Biochemical and Molecular Toxicology vol 28 no 1pp 11ndash22 2014

[49] X Yang D Wang Y Ma et al ldquoContinuous activation of Nrf2and its target antioxidant enzymes leads to arsenite-inducedmalignant transformation of human bronchial epithelial cellsrdquoToxicology and Applied Pharmacology vol 289 no 2 pp 231ndash239 2015

[50] C Pang Z Zheng L Shi et al ldquoCaffeic acid preventsacetaminophen-induced liver injury by activating the Keap1-Nrf2 antioxidative defense systemrdquo Free Radical Biology andMedicine vol 91 pp 236ndash246 2016

[51] Y H Noh J Y Baek W Jeong S G Rhee and T-S ChangldquoSulfiredoxin translocation into mitochondria plays a crucialrole in reducing hyperoxidized peroxiredoxin IIIrdquo The Journalof Biological Chemistry vol 284 no 13 pp 8470ndash8477 2009

[52] B Cunniff A N Wozniak P Sweeney K DeCosta and N HHeintz ldquoPeroxiredoxin 3 levels regulate a mitochondrial redoxsetpoint in malignant mesothelioma cellsrdquo Redox Biology vol3 pp 79ndash87 2014

[53] P-J Chua E-H Lee Y Yu G W-C Yip P-H Tan and B-HBay ldquoSilencing the Peroxiredoxin III gene inhibits cell prolifera-tion in breast cancerrdquo International Journal of Oncology vol 36no 2 pp 359ndash364 2010

[54] B Cunniff K Newick K J Nelson et al ldquoDisabling mito-chondrial peroxide metabolism via combinatorial targetingof peroxiredoxin 3 as an effective therapeutic approach formalignant mesotheliomardquo PLOS ONE vol 10 no 5 Article IDe0127310 2015

[55] X Yao M Li J He et al ldquoEffect of early acute high con-centrations of iodide exposure on mitochondrial superoxideproduction in FRTL cellsrdquo Free Radical Biology and Medicinevol 52 no 8 pp 1343ndash1352 2012

[56] L Wang Q Duan T Wang et al ldquoMitochondrial respiratorychain inhibitors involved in ROS production induced by acutehigh concentrations of iodide and the effects of sod as aprotective factorrdquo Oxidative Medicine and Cellular Longevityvol 2015 Article ID 217670 14 pages 2015

Submit your manuscripts athttpswwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

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BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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ObesityJournal of

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Research and TreatmentAIDS

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Oxidative Medicine and Cellular Longevity 3

Table 1 Changes of the median urinary iodine concentration (120583gL) of Wistar rats following the intake of NI 10 HI and 100 HI on Days 714 and 28 (119873 = 6 for each group) All data is presented as the median (range)

Group The median urinary iodine concentration (120583gL)Day 7 Day 14 Day 28

NI 3212 (1500ndash3517) 3582 (3002ndash3738) 3330 (1886ndash4135)10 HI 30525lowast (15928ndash37980) 35325lowast (14870ndash40560) 26280lowast (16510ndash34045)100 HI 264892lowast (58566ndash421704) 244610lowast (176075ndash298748) 226632lowast (83425ndash298162)lowastCompared to the NI group (119901 lt 005)Compared to the 10 HI group (119901 lt 005)

27 Western Blot Analysis The bicinchoninic acid proteinassay kit (Beyotime Institute of Biotechnology JiangsuChina) was used to detect the concentration of proteins50120583g proteins were separated by SDS-PAGE and transferredto the PVDF membrane Subsequently the membrane wasblocked for 1 hour at room temperature using 5 nonfatmilk Then the membrane was incubated overnight at 4∘Cwith primary antibodies followed by horseradish peroxidaseconjugated secondary antibodiesThe proteins were detectedby Immobilon Western Chemiluminescent HRP Substrate120573-Actin was used as a loading control Blots were scannedas gray scale images and quantified using Image J software(NIH) All the blot intensities were normalized with that ofthe loading control 120573-actin

28 Immunofluorescence Staining The thyroid tissues werefirst embedded with an Optimal Cutting Temperature (OCT)compound at minus80∘C Following which the tissues were frozenin a cryostat machine and cut into frozen sections for 5 120583m atminus20∘C The slices were incubated with 5 FBS for 60min atroom temperature Subsequently they were incubated witha primary antibody [Nrf2 (1 100) or Srx (1 100)] at 4∘Covernight After three washes with PBS the second antibodywas linked to fluorophores (goat anti-rabbit IgG-PE) Thenucleus was stained for 5minwithHoechst 33258 (50120583L) andwashed 3 times with PBS MitoSOX Red was used to measuremitochondrial superoxide production Using a Zeiss LSM510 confocal microscope fluorescent images of the preparedslides were obtained

29 Statistics The data of urinary iodine concentration(Table 1) showed a skewed distribution and were expressedas the median Differences between groups were evaluated bynonparametric KruskalndashWallis test If the latter test showedsignificant differences between groups the individual groupswere compared with the control group by the Nemenyitests using SPSS 220 A 119901 value of lt005 was consideredstatistically significant [25]

The other data was expressed as mean plusmn SD Differencesbetween groups were evaluated by one-way analysis of vari-ance (ANOVA) if this test showed significant differencesbetween groups the individual groups were compared withthe control group by Least Significant Difference (LSD)test using SPSS 220 A 119901 value of lt005 was consideredstatistically significant

3 Results

31 Effects of the Iodide Intake (NI 10 HI and 100 HI) on theRatio of Thyroid WeightBody Weight on Days 7 14 and 28The parameters body weight thyroid weight and the ratio ofthe thyroid weightbody weight were not significantly alteredfollowing the intake of NI 10 HI and 100 HI on Days 7 14and 28 (119901 gt 005) (Figure 1)

32 Effects of the Iodide Intake (NI 10 HI and 100 HI)on Urinary Iodine Concentration and Iodine Content in theThyroid Glands on Days 7 14 and 28 The median urinaryiodine concentration and their ranges for NI 10 HI and 100HI on Days 7 14 and 28 are illustrated in Table 1 In allthe different time periods there was a significant increase inthe urinary iodine concentration among 10 HI and 100 HIwhen compared to the NI group (119901 lt 005) In additionthe urinary iodine concentration of the 100 HI group alsoincreased significantly when compared to 10 HI (119901 lt 005)Between any of the iodide intake groups (NI 10 HI and100 HI) there was no significant difference in the urinaryiodine concentration on Day 14 and Day 28 when comparedto Day 7 (119901 gt 005) Furthermore there was no significantdifference between Day 14 and Day 28 (119901 gt 005) Whenthe intake of iodide was increased from NI to 10 HI andfurther to 100 HI a simultaneous increase in the urinaryiodine concentration was also observed on Days 7 14 and28 The urinary iodine concentration in the 10 HI group wasapproximately 10 times that of the NI group whereas theurinary iodine concentration in the 100 HI group was about60ndash80 times that of the NI group on Days 7 14 and 28

In our study we demonstrated that the iodine content inthe thyroid glands was significantly increased in the 100 HIgroup when compared to the NI group on Days 7 14 and 28(119901 lt 005) Between any of the iodide intake groups (NI 10HI and 100 HI) there was no significant difference foundin the iodine content in the thyroid glands on Days 7 14and 28 (119901 gt 005) When the intake of iodide was increasedfrom NI to 10 HI and further to 100 HI the iodine content inthe thyroid glands increased gradually on Days 7 14 and 28(Table 2)

33 Effects of the Iodide Intake (NI 10 HI and 100 HI) on theChanges of SerumThyroid Hormones Levels on Days 7 14 and28 There were no significant alterations in TT3 TT4 FT3

4 Oxidative Medicine and Cellular Longevity

0

50

100

150

200

250

300

350

400

450

500

NI 10 HI 100 HI

Day 7Day 14

Day 28

Body

wei

ght (

g)

(a)

Day 7Day 14

Day 28

0

5

10

15

20

25

30

NI 10 HI 100 HI

Thyr

oid

wei

ght (

mg)

(b)

Day 7Day 14

Day 28

0

1

2

3

4

5

6

7

8

9

NI 10 HI 100 HI

Ratio

of t

hyro

id w

eigh

tbod

y w

eigh

t (m

g10

0 g)

(c)

Figure 1 Effects of the iodide intake (NI 10 HI and 100 HI) on (a) the body weight (b) the thyroid weight and (c) the ratio of the thyroidweightbody weight on Days 7 14 and 28 All data is presented as mean plusmn SD (119873 = 6 for each group) Statistical analyses were performed byone-way analysis of variance (ANOVA) with the Least Significant Difference (LSD) test

and FT4 levels following the intake ofNI 10HI and 100HI onDays 7 14 and 28 (119901 gt 005) Moreover for all three dosagesof iodide intake there were no significant differences in anyof the serum thyroid hormones levels on Day 14 and Day 28when compared to Day 7 (119901 gt 005) In addition there wereno significant changes between Day 14 and Day 28 in serumthyroid hormones levels (TT3 TT4 FT3 and FT4) (119901 gt 005)(Table 3)

34 Effects of the Iodide Intake (NI 10 HI and 100 HI) on theChanges of Mitochondrial Superoxide Production on Days 7

14 and 28 On Days 7 14 and 28 when compared to the NIgroup the mitochondrial superoxide production in the 10 HIgroup showed no significant increase (119901 gt 005) Howeverthere was a significant increase in the mitochondrial super-oxide production in the 100 HI group (119901 lt 005) Whencompared to the 10 HI there was a significant increase in themitochondrial superoxide production in the 100 HI group(119901 lt 005) In the NI and 10 HI groups the mitochondrialsuperoxide production on Days 7 14 and 28 showed nosignificant difference (119901 gt 005) However in the 100 HIgroup compared to Day 7 there was a significant increase in

Oxidative Medicine and Cellular Longevity 5

Table 2 Changes of iodine content in the thyroid glands(120583g100mg) of Wistar rats following the intake of NI 10 HI and 100HI onDays 7 14 and 28 (119873 = 6 for each group) All data is presentedas the mean plusmn SD

Group Iodine content in the thyroid glands (120583g100mg)Day 7 Day 14 Day 28

NI 6219 plusmn 594 6012 plusmn 813 6337 plusmn 90810 HI 12125 plusmn 1048 12645 plusmn 893 15233 plusmn 1077100 HI 13353 plusmn 861lowast 13636 plusmn 964lowast 17845 plusmn 874lowastlowastCompared with NI group (119901 lt 005)

Table 3 Changes of serum thyroid hormones levels of Wistar ratsfollowing the intake of NI 10 HI and 100 HI on Days 7 14 and 28(119873 = 6 for each group) All data is presented as the mean plusmn SD

Group The levels of serum thyroid hormonesTT3

(nmolL)TT4

(nmolL)FT3

(pmolL)FT4

(pmolL)Day 7

NI 118 plusmn 015 9077 plusmn 552 396 plusmn 010 2203 plusmn 17010 HI 119 plusmn 008 9154 plusmn 774 377 plusmn 026 2302 plusmn 206100 HI 107 plusmn 011 8917 plusmn 903 378 plusmn 011 2008 plusmn 543

Day 14NI 115 plusmn 007 9583 plusmn 520 392 plusmn 016 2234 plusmn 37110 HI 112 plusmn 008 8615 plusmn 252 363 plusmn 048 2019 plusmn 192100 HI 119 plusmn 004 9154 plusmn 331 384 plusmn 046 2098 plusmn 250

Day 28NI 113 plusmn 006 9417 plusmn 238 389 plusmn 013 2025 plusmn 57210 HI 118 plusmn 008 9077 plusmn 378 398 plusmn 057 2115 plusmn 534100 HI 114 plusmn 022 8769 plusmn 635 379 plusmn 046 2117 plusmn 543

mitochondrial superoxide production on Day 28 (119901 lt 005)Similarly compared to Day 14 there was also a significantincrease in mitochondrial superoxide production on Day 28(119901 lt 005) Accordingly the fluorescent intensity ofMitoSOXRed onDay 28 gradually increased after the increased dosagesof iodide intake fromNI to 100 HI which was consistent withour results of flow cytometry (Figure 2)

35 Effects of the Iodide Intake (NI 10 HI and 100 HI) on theChanges of Nrf2 Keap 1 Srx and Prx 3 Expressions on Days 714 and 28 On Days 7 14 and 28 when compared to the NIgroup the expressions of Nrf2 Keap 1 Srx and Prx 3 showedno significant differences in the 10 HI group (119901 gt 005)the expressions of Nrf2 Srx and Prx 3 were significantlyincreased while Keap 1 was notably decreased in the 100 HIgroup (119901 lt 005) On Days 7 14 and 28 when comparedto the 10 HI group the expressions of Nrf2 Srx and Prx 3were significantly increased while Keap 1 was significantlydecreased in the 100 HI group (119901 lt 005) (Figure 3)

36 Effects of the Iodide Intake (NI 10 HI and 100 HI) on theChanges of Immunofluorescence Staining onDays 7 14 and 28Following the increased iodide intake from NI to 10 HI and

further to 100 HI the expressions of Nrf2 and Srx intensifiedIn the NI group Nrf2 was localized in the cytoplasm on Days7 14 and 28 In the 10 HI group the positive staining ofNrf2 can be observed in both the nucleus and the cytoplasmMoreover in the 100 HI group a stronger positive stainingof Nrf2 can be detected in the nucleus on Days 7 14 and 28(Figure 4(a)) Srx positive staining was only located in thecytoplasm on Days 7 14 and 28 (Figure 4(b))

4 Discussion

In our study we found that there were no significant alter-ations in TT3 TT4 FT3 and FT4 levels following the intakeofNI 10HI and 100HI onDays 7 14 and 28 (119901 gt 005)Thereare very efficient homeostatic mechanisms that resist changesin circulating T3 and T4 levels in response to iodide excessDue to the compensatory mechanisms such as the Wolff-Chaikoff effect the changes in T3 and T4 levels following anincrease in iodide intake are minimal and usually transientin nature [26ndash28] The Wolff-Chaikoff effect relies on a high(ge10minus3 molar) intracellular concentration of iodide Duringinitial exposure excess iodide is transported by the sodium-iodide symporter (NIS) into the cellsWhen intracellular con-centration reaches at least 10minus3 molar iodide organificationis blocked [27 29] Moreover the regulatory mechanismsinclude modulation of blood flow enzyme activity geneexpression and transport proteins in signaling pathways [230 31] In a study conducted by Eng et al 16 Wistar ratswere given 2000120583g of iodide acutely it was observed thatthere was a significant decrease in serum T4 and T3 levelson Day 1 of the study Subsequently on Day 6 both serumT4 and T3 levels returned to normal ranges However inboth cases the serum TSH levels remained unchanged [32]Mooij et al observed no significant changes in serum thyroidhormones levels when female Wistar rats were given 100 120583giodide daily for 18 weeks [33] Paul et al demonstrated thatwhen normal volunteers received 1500120583g of supplementaliodine daily for 14 days a small decrease in serum T3 andT4 concentrations with compensatory increase of TSH wasdetected although all values remained within the normalranges [34] However the presence of handicaps such as anincreased autoimmune susceptibility fetal period extremesof age pregnancy lactation or an active pathological entitysignificantly impair these mechanisms [1 35ndash37]

Our study demonstrated that both the urinary iodineconcentration and the iodide intake increased simultaneouslyfrom NI to 10 HI and further to 100 HI Interestingly wefound that the urinary iodine concentration of the 10 HIgroup was approximately 10 times that of the NI groupwhereas the urinary iodine concentration of the 100HI groupwas about 60ndash80 times that of the NI group on Days 7 14and 28 The urinary iodine concentration is regarded as asensitive indicator of iodine status because approximately90 of ingested iodide is excreted in the urine [38 39]Thereis an increase for the maintenance of thyroid homeostasisas well as the steady state of the internal environment ofthe body The thyroid gland has adaptation mechanisms thatreduce iodide metabolism when the supply is abundant thus

6 Oxidative Medicine and Cellular Longevity

1793 2066 4897

1773 2264 5291

1806 2373 7360

NI 10 HI 100 HID

ay 7

Day

14

Day

28

250

200

150

100

50

0

Cou

nt

0 102 103 104 105

Comp-PE-A PE-A

250

200

150

100

50

0

Cou

nt

0 102 103 104 105

Comp-PE-A PE-A

250

200

150

100

50

0

Cou

nt

0 102 103 104 105

Comp-PE-A PE-A

250

200

150

100

50

0

Cou

nt

0 102 103 104 105

Comp-PE-A PE-A

250

200

150

100

50

0

Cou

nt

0 102 103 104 105

Comp-PE-A PE-A

250

200

150

100

50

0

Cou

nt

0 102 103 104 105

Comp-PE-A PE-A

250

200

150

100

50

0

Cou

nt

0 102 103 104 105

Comp-PE-A PE-A

250

200

150

100

50

0

Cou

nt

0 102 103 104 105

Comp-PE-A PE-A

250

200

150

100

50

0

Cou

nt

0 102 103 104 105

Comp-PE-A PE-A

(a)

0

10

20

30

40

50

60

70

80

NI 10 HI 100 HI

Day 7Day 14Day 28

Mito

SOX

fluor

esce

nce i

nten

sity

lowastlowast

lowast998771998779

(b)

Figure 2 Continued

Oxidative Medicine and Cellular Longevity 7

NI 10 HI 100 HI

Day 28

20120583m20120583m20120583m

(c)

Figure 2 Effects of the iodide intake (NI 10 HI and 100 HI) on the changes of mitochondrial superoxide production on Days 7 14 and28 (a) There was a significant increase in the mitochondrial superoxide production in the 100 HI group on Days 7 14 and 28 (119901 lt 005)The difference was more significant on Day 28 when compared to Day 7 or Day 14 (b) Histogram analysis was performed on the meanfluorescence intensity of MitoSOX Red All data is presented as the mean plusmn SD (119873 = 6 for each group) Statistical analyses were performedby one-way analysis of variance (ANOVA) with the Least Significant Difference (LSD) test lowast119901 lt 005 versus the NI group on Days 7 14 and28 respectively 119901 lt 005 versus the 10 HI group on Days 7 14 and 28 respectively 998771119901 lt 005 versus the Day 7 group in the 100 HI group119901 lt 005 versus the Day 14 in the 100 HI group Experiments were repeated 3 times with similar results (c) There was a significant increasein the mitochondrial superoxide production in the 100 HI group on Day 28 and was observed by confocal microscopy Scale bar 20120583m

avoiding thyrotoxicosisThere are several mechanisms whichinclude a direct inhibitory effect of iodide in the thyroid itselfand inhibition by iodide of its own organification (Wolff-Chaikoff effect) its transport thyroid hormones secretioncAMP formation in response to TSH and several othermetabolic steps [40] We suggest that all the protectivemechanisms may ensure the excessive iodide intake of the 10HI group on Days 7 14 and 28 be eliminated from urineTheurinary iodine concentration of the 100 HI group on Days7 14 and 28 was about 60ndash80 times that of the NI groupalthough the thyroid function was normal This leads us topropose the idea that excessive iodide accumulated in thebody may trigger the oxidative and antioxidative signalingpathway to maintain the normal thyroid function

We demonstrated that the production of mitochondrialsuperoxide significantly increased on Days 7 14 and 28 inthe 100 HI group This is consistent with our previous studyon metallothionein-III knockout mice [24] Joanta et alreported that the initiation of free radical production wasobserved after giving a high dose of iodide [3] Serrano-Nascimento et al demonstrated that an increased mitochon-drial superoxide production was shown in response to NaI(10minus6M to 10minus3M) treatment in PCCl3 thyroid cells by usingMitoSOX Red [41] Mitochondria are potent producers ofsuperoxide from complexes I and III of the electron transportchain Mitochondrial superoxide production is a major causeof cellular oxidative damage [42] Physiologically ROS arenot necessarily harmful because they are continuously bal-anced by the process of hormone synthesis and the endoge-nous antioxidant system [1] Excess ROS are generated duringthe trapping oxidation and organification of excessive iodinein thyrocytes which could lead to increased oxidative stress[1]

The Nrf2-Keap 1 pathway is the chief cytoprotectivemechanism in response to oxidative stress caused by ROSDuring normal and balanced redox homeostasis the Nrf2function is inhibited because of constant proteasomal degra-dation after ubiquitination of the protein This is regulated

through the binding of the inhibitor protein Keap 1 [13 14]It is reported that Srx activation via Nrf2 dependent pathwayprotects fromoxidative liver injury throughPyrazole [43] andalcohol in mice [44] Similar findings in lung tissues haveshown that there is a marked increase in the expressions ofSrx and Prx 3 in human squamous cell carcinoma [45] Thissuggests that these proteins may play a protective role againstoxidative injury Also the pathway including Keap 1 Nrf2and ARE-mediated protein expression plays a very criticalrole in protecting cells fromoxidative stress [46 47] Focusingon the pathway we demonstrated that the expression of Nrf2was significantly increased while Keap 1 was significantlydecreased in the 100 HI group when compared to the NIor 10 HI group on Days 7 14 and 28 Similarly Ajiboyeet al showed that rats treated with Chalcone dimers notonly increased the expression of Nrf2 but also suppressedcytoplasmic Keap 1 expression [48] Yang et al also showedthat the downregulation of Keap 1 level may be responsiblefor the overactivation of Nrf2 [49] Pang et al showed thatcaffeic acid prevents acetaminophen-induced liver injury byactivating the Nrf2-Keap 1 antioxidative defense system [50]

In order to verify whether high levels of expressionof Nrf2 and its nuclear translocation can upregulate someantioxidant enzymes in the thyroid gland the expressions ofPrx 3 and Srx following the intake of NI 10 HI and 100 HIon Days 7 14 and 28 were measured We demonstrated thatthe expressions of Srx and Prx 3 in the 100 HI group weresignificantly increased when compared to the NI group or 10HI group on Days 7 14 and 28 The possible explanationsare described as below Firstly Srx is a cytosolic proteinthat is able to translocate to sites where hyperoxidized(inactivated) Prx 3 is located Therefore it engages itself inthe reactivation of Prx 3 under oxidative conditions [51]Secondly Prx 3 is a typical 2-Cys Peroxiredoxin locatedexclusively in the mitochondrial matrix it is the principalperoxidase responsible for protecting cells from oxidativedamage by reducing peroxides such as H2O2 [52] Finallymitochondria contain 30 times more Prx 3 than glutathione

8 Oxidative Medicine and Cellular Longevity

000

020

040

060

080

100

120

140

NI 10 HI 100 HI

Expr

essio

n of

Nrf2

(Nrf2

120573-a

ctin

)

120573-Actin

Nrf2

Day 7 Day 14 Day 28

lowast lowast

lowast

Day 7Day 14

Day 28

NI 10 HI 100 HI NI 10 HI 100 HI NI 10 HI 100 HI

(a)

000

020

040

060

080

100

120

NI 10 HI 100 HI

lowast

lowast

Expr

essio

n of

Kea

p 1

(Kea

p 1120573

-act

in)

120573-Actin

Keap 1

Day 7 Day 14 Day 28

lowast

Day 7Day 14

Day 28

NI 10 HI 100 HI NI 10 HI 100 HI NI 10 HI 100 HI

(b)

120573-Actin

SrxNI 10 HI 100 HI NI 10 HI 100 HI NI 10 HI 100 HI

000

010

020

030

040

050

060

NI 10 HI 100 HI

Expr

essio

n of

Srx

(Srx

120573-a

ctin

)

Day 7 Day 14 Day 28

lowast

lowast

lowast

Day 7Day 14

Day 28

(c)

120573-Actin

Prx 3

lowast

000

020

040

060

080

100

120

140

NI 10 HI 100 HI

Expr

essio

n of

Prx

3 (P

rx 3

120573-a

ctin

)

Day 7 Day 14 Day 28

lowastlowast

Day 7Day 14

Day 28

NI 10 HI 100 HI NI 10 HI 100 HI NI 10 HI 100 HI

(d)

Figure 3 Effects of the iodide intake (NI 10 HI and 100 HI) on the changes of (a) Nrf2 (b) Keap 1 (c) Srx and (d) Prx 3 expressions onDays 7 14 and 28 Representative western blot and histograms of densitometric analyses normalized for the relative 120573-actin content All datais presented as the mean plusmn SD (119873 = 6 for each group) Statistical analyses were performed by one-way analysis of variance (ANOVA) withthe Least Significant Difference (LSD) test lowast119901 lt 005 versus the NI group on Days 7 14 and 28 respectively 119901 lt 005 versus the 10 HI groupon Days 7 14 and 28 respectively Experiments were repeated 3 times with similar results

Oxidative Medicine and Cellular Longevity 9

NI

10 HI

100 HI

Hoechst Nrf2 Merge HoechstHoechst

Day 7 Day 28 Day 14

Nrf2 Merge Nrf2 Merge

Day 7 Day 7 Day 14 Day 14 Day 28 Day 28 20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

(a)

Hoechst Srx Merge Hoechst Srx Merge Hoechst Srx Merge

NI

10 HI

100 HI

Day 7 Day 28 Day 14 Day 7 Day 7 Day 14 Day 14 Day 28 Day 28 20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

(b)

Figure 4 Effect of the iodide intake (NI 10 HI and 100 HI) on the changes of immunofluorescence staining on Days 7 14 and 28 (a) In theNI group Nrf2 (red) was localized in the cytoplasm on Days 7 14 and 28 In the 10 HI group the positive staining of Nrf2 can be observedin both the nucleus and the cytoplasm Moreover in the 100 HI group a stronger positive staining of Nrf2 can be detected in the nucleus onDays 7 14 and 28 the nucleus was dyed with Hoechst (blue) (b) Srx (red) positive staining was located in the cytoplasm the nucleus wasdyed with Hoechst (blue) Scale bar 20120583m

peroxidase Prx 3 can be classified as an important regulatorof mitochondrial H2O2 [22] The elevated expression of Prx3 is associated with the blockage of apoptosis increasingcell proliferation and is related to adaptive responses whichare all required to maintain mitochondrial function [53 54]Bae et al have suggested that Prx 3 and Srx jointly protectmice from Pyrazole-induced oxidative liver injury in a Nrf2-dependent manner [43]

The novelty we demonstrated in the present study is thatiodide excess induced both oxidative stress and antioxidativedefense increases throughNrf2-Keap 1 pathway in the thyroidgland from rats We extended our established mechanismsby applying the Nrf2-Keap 1 pathway to set up a bridgebetween oxidative stress and antioxidative defense inducedby iodide excess in the thyroid gland In our previous studywe have established that oxidative stress induced by acutehigh concentrations of iodide in FRTL cells significantlyincreases mitochondrial superoxide production [55] Theinhibitors of themitochondrial respiratory chain complexes I

and III are involved inmitochondrial superoxide productionWe demonstrated that exposure to 100120583M KI for 2 hourssignificantly increased mitochondrial superoxide produc-tion enhanced by either 05120583M Rotenone (an inhibitorof mitochondrial complex I) or 10 120583M Antimycin A (aninhibitor of complex III) [56] We illustrated that 300 120583MPTU (an inhibitor of TPO) attenuated the excessive iodide-induced mitochondrial superoxide production We showedthat 30 120583M KClO4 (a competitive inhibitor of iodide trans-port) relieved the production the mitochondrial superoxideinduced by iodide excess We displayed that 10mUmL TSHcan inhibit excessive iodide-induced strong mitochondrialsuperoxide production [55] MT-I and MT-II are mainlyinvolved in the protection of tissue against oxidative stresseswe indicated that metallothionein-III knockout mice aggra-vated mitochondrial superoxide production in thyroid afterexcessive iodide exposure [24] In addition we demonstratedthat both the oxidative stress and the antioxidative defenseincreased simultaneously after high dosages of iodide intake

10 Oxidative Medicine and Cellular Longevity

Oxidative stress

(a)

(b)

(c)

Antioxidative defense

Wist

ar ra

ts Day 7Day 14Day 28

Normal iodideintake

Normal urinaryiodine

Normal thyroidfunction

10 times iodideintake

10 times urinaryiodine

Normal thyroidfunction

100 times iodideintake

60ndash80 timesurinary iodine

Normal thyroidfunction

Keap 1 Nrf2

Keap 1

Nrf2

Srx

Nucleus

Prx 3

SrxARE

Mitochondria

AntioxidativeOxidativestress

Oxidative Antioxidative

Oxidative Antioxidative

Under physiological conditions(normal iodide intake)

Excessive iodide intake(10 times iodide intake)

Excessive iodide intake(100 times iodide intake)

defense defenseuarrstressuarr

defenseuarruarrstressuarruarr

Figure 5 Proposed mechanisms in the present study (a) The urinary iodine concentration and thyroid function of Wistar rats weredetected (b) The activation of the Nrf2-Keap 1 pathway induced by iodide excess in the thyroid (c) The balance between oxidative stressand antioxidative defense under physiological conditions and excessive iodide intake

Oxidative Medicine and Cellular Longevity 11

We suggested that the Nrf2-Keap 1 pathway is vital for thebalance between oxidative stress and antioxidative defenseinduced by iodide excess in the thyroid gland

Excessive iodide stimulated the Nrf2-Keap 1 pathwayand enhanced the antioxidative defense We found thatthe urinary iodine concentration of the 100 HI group wasabout 60ndash80 times that of the NI group on Days 7 14and 28 however the thyroid functions were normal Weproposed that the excessive iodide accumulated in the bodymay trigger the Nrf2-Keap 1 pathway to maintain a normalthyroid functionWe demonstrated that the Nrf2moves fromthe cytoplasm to the nucleus under the microscope withsignificantly increased expressions of Nrf2 Srx and Prx 3 andnotably decreased Keap 1 when exposed to high iodide Thissuggests that excessive iodide stimulates the disassociation ofNrf2 from Keap 1 and assists Nrf2 to penetrate the nucleusThen Nrf2 attaches to the antioxidant response element(ARE) to activate the expression of the antioxidative genesSrx and Prx 3 resulting in an enhanced antioxidative defenseinduced by high iodide By activation of the Nrf2-Keap 1pathway there is a proportional increase in oxidative stressand antioxidative defense in response to iodide excess It isto be noted that the thyroid function was normal in the 10HI group and 100 HI in present study Inspired by the reportby Poncin et al [6] we proposed that there should be abalance between oxidative stress and antioxidative defense inresponse to iodide excess in the 10 HI and the 100 HI groups

5 Conclusion

In conclusion our results highlight that the activation ofNrf2-Keap 1 Srx and Prx 3 antioxidative defense mecha-nisms may play a crucial role in protecting the thyroid fromiodide excess induced oxidative stress on Days 7 14 and 28(Figure 5)

Abbreviations

NI Normal iodide intakeHI High iodide intakeROS Reactive oxygen speciesH2O2 Hydrogen peroxideNrf2 Nuclear factor erythroid 2-related factor 2Keap 1 Kelch-like ECH-associated protein 1ARE Antioxidant response elementPrx PeroxiredoxinSrx SulfiredoxinPrx 3 Peroxiredoxin 3TT4 Total thyroxineTT3 Total triiodothyronineFT4 Free thyroxineFT3 Free triiodothyronineTSH ThyrotropinLSD Least significant difference

Disclosure

This manuscript has been edited by Umar Iqbal IruniRoshanie Abeysekera Gargi Naha and Tejas Bharadwaj all

of whom are currently studying at the International School ofMedicine Tianjin Medical University China

Competing Interests

The authors declare that there is no conflict of interestsregarding the publication of this article

Acknowledgments

This work is supported by National Natural Science Founda-tion of China (nos 81273009 and 81270182) Tianjin Scienceamp Technology Council Grant of China (nos 16JCYBJC26100and 09JCYBJC11700) and International Studentrsquos Science ampTechnology Innovation Project (Scientific Research Project)of Tianjin Medical University

References

[1] Y Luo A Kawashima Y Ishido et al ldquoIodine excess as anenvironmental risk factor for autoimmune thyroid diseaserdquoInternational Journal of Molecular Sciences vol 15 no 7 pp12895ndash12912 2014

[2] A E Hussein A M Abbas G A El Wakil A Z Elsamanoudyand A A El Aziz ldquoEffect of chronic excess iodine intakeon thyroid function and oxidative stress in hypothyroid ratsrdquoCanadian Journal of Physiology and Pharmacology vol 90 no5 pp 617ndash625 2012

[3] A E Joanta A Filip S Clichici S Andrei and D DaicoviciuldquoIodide excess exerts oxidative stress in some target tissues ofthe thyroid hormonesrdquoActa Physiologica Hungarica vol 93 no4 pp 347ndash359 2006

[4] SMorandMChaaraoui J Kaniewski et al ldquoEffect of iodide onnicotinamide adenine dinucleotide phosphate oxidase activityand Duox2 protein expression in isolated porcine thyroidfolliclesrdquo Endocrinology vol 144 no 4 pp 1241ndash1248 2003

[5] J J Lazaro A Jimenez D Camejo et al ldquoDissecting the inte-grative antioxidant and redox systems in plant mitochondriaEffect of stress and S-nitrosylationrdquo Frontiers in Plant Sciencevol 4 article 460 2013

[6] S Poncin A-C GerardM Boucquey et al ldquoOxidative stress inthe thyroid gland from harmlessness to hazard depending onthe iodine contentrdquo Endocrinology vol 149 no 1 pp 424ndash4332008

[7] M Karbownik and A Lewinski ldquoThe role of oxidative stressin physiological and pathological processes in the thyroidgland possible involvement in pineal-thyroid interactionsrdquoNeuroendocrinology Letters vol 24 no 5 pp 293ndash303 2003

[8] P Thomopoulos ldquoHyperthyroidism due to excess iodinerdquo LaPresse Medicale vol 31 no 35 pp 1664ndash1669 2002

[9] J Wolff ldquoIodide goiter and the pharmacologic effects of excessiodiderdquoTheAmerican Journal of Medicine vol 47 no 1 pp 101ndash124 1969

[10] B Corvilain L Collyn J Van Sande and J E Dumont ldquoStim-ulation by iodide of H2O2 generation in thyroid slices fromseveral speciesrdquoAmerican Journal of PhysiologymdashEndocrinologyand Metabolism vol 278 no 4 pp E692ndashE699 2000

[11] M-C Many C Mestdagh M-F Van den Hove and J-F DenefldquoIn vitro study of acute toxic effects of high iodide doses inhuman thyroid folliclesrdquo Endocrinology vol 131 no 2 pp 621ndash630 1992

12 Oxidative Medicine and Cellular Longevity

[12] J Van Sande G Grenier C Willems and J E DumontldquoInhibition by iodide of the activation of the thyroid cyclic 3101584051015840-AMP systemrdquo Endocrinology vol 96 no 3 pp 781ndash786 1975

[13] R Young J D Hayes K Brown C R Wolf and C B AWhitelaw ldquoPeroxiredoxin gene expression signatures in liverreflect toxic insultrdquo Assay and Drug Development Technologiesvol 8 no 4 pp 512ndash517 2010

[14] Q Ma ldquoRole of Nrf2 in oxidative stress and toxicityrdquo AnnualReview of Pharmacology and Toxicology vol 53 pp 401ndash4262013

[15] B Biteau J Labarre and M B Toledano ldquoATP-dependentreduction of cysteine-sulphinic acid by S cerevisiae sulphire-doxinrdquo Nature vol 425 no 6961 pp 980ndash984 2003

[16] L Wu H Jiang H A Chawsheen et al ldquoTumor promoter-induced sulfiredoxin is required for mouse skin tumorigenesisrdquoCarcinogenesis vol 35 no 5 pp 1177ndash1184 2014

[17] C C Winterbourn and M B Hampton ldquoThiol chemistryand specificity in redox signalingrdquo Free Radical Biology andMedicine vol 45 no 5 pp 549ndash561 2008

[18] J W Baty M B Hampton and C C Winterbourn ldquoProteomicdetection of hydrogen peroxide-sensitive thiol proteins inJurkat cellsrdquo Biochemical Journal vol 389 no 3 pp 785ndash7952005

[19] T-S Chang C-S Cho S Park S YuW K Sang and G R SueldquoPeroxiredoxin III a mitochondrion-specific peroxidase regu-lates apoptotic signaling by mitochondriardquo Journal of BiologicalChemistry vol 279 no 40 pp 41975ndash41984 2004

[20] P Thomopoulos ldquoHyperthyroidism due to excess iodinerdquo LaPresse Medicale vol 31 no 35 pp 1664ndash1669 2002

[21] S Chung X-H Liao C Di Cosmo et al ldquoDisruption of themelanin-concentrating hormone receptor 1 (MCH1R) affectsthyroid functionrdquo Endocrinology vol 153 no 12 pp 6145ndash61542012

[22] G Ozguner and O Sulak ldquoSize and location of thyroid gland inthe fetal periodrdquo Surgical and Radiologic Anatomy vol 36 no4 pp 359ndash367 2014

[23] P L Jooste and E Strydom ldquoMethods for determination ofiodine in urine and saltrdquo Best Practice amp Research ClinicalEndocrinology amp Metabolism vol 24 no 1 pp 77ndash88 2010

[24] N Zhang L Wang Q Duan et al ldquoMetallothionein-IIIknockoutmice aggravatemitochondrial superoxide productionand peroxiredoxin 3 expression in thyroid after excessive iodideexposurerdquoOxidative Medicine and Cellular Longevity vol 2015Article ID 267027 11 pages 2015

[25] P I Gus D Marinho S Zelanis et al ldquoA case-control studyon the oxidative balance of 50 autologous serum eye dropsrdquoOxidative Medicine and Cellular Longevity vol 2016 Article ID9780193 5 pages 2016

[26] P Laurberg C Cerqueira L Ovesen et al ldquoIodine intake as adeterminant of thyroid disorders in populationsrdquo Best Practiceamp Research Clinical Endocrinology amp Metabolism vol 24 no 1pp 13ndash27 2010

[27] H Burgi ldquoIodine excessrdquo Best Practice amp Research ClinicalEndocrinology amp Metabolism vol 24 no 1 pp 107ndash115 2010

[28] E Roti and E D Uberti ldquoIodine excess and hyperthyroidismrdquoThyroid vol 11 no 5 pp 493ndash500 2001

[29] A M Leung and L E Braverman ldquoConsequences of excessiodinerdquo Nature Reviews Endocrinology vol 10 no 3 pp 136ndash142 2014

[30] K Wang Y N Sun J Y Liu et al ldquoThe impact of iodineexcess on thyroid hormone biosynthesis and metabolism in

ratsrdquoBiological Trace Element Research vol 130 no 1 pp 72ndash852009

[31] M Michalkiewicz L J Huffman J M Connors and G AHedge ldquoAlterations in thyroid blood flow induced by varyinglevels of iodine intake in the ratrdquo Endocrinology vol 125 no 1pp 54ndash60 1989

[32] P H K Eng G R Cardona S-L Fang et al ldquoEscape fromthe acute Wolff-Chaikoff effect is associated with a decrease inthyroid sodiumiodide symporter messenger ribonucleic acidand proteinrdquo Endocrinology vol 140 no 8 pp 3404ndash3410 1999

[33] P Mooij H J De Wit and H A Drexhage ldquoA high iodineintake in Wistar rats results in the development of a thyroid-associated ectopic thymic tissue and is accompanied by a lowthyroid autoimmune reactivityrdquo Immunology vol 81 no 2 pp309ndash316 1994

[34] T Paul B Meyers R J Witorsch et al ldquoThe effect of smallincreases in dietary iodine on thyroid function in euthyroidsubjectsrdquoMetabolism vol 37 no 2 pp 121ndash124 1988

[35] X Teng Z Shan W Teng C Fan H Wang and R GuoldquoExperimental study on the effects of chronic iodine excess onthyroid function structure and autoimmunity in autoimmune-prone NODH-2h4 micerdquo Clinical and Experimental Medicinevol 9 no 1 pp 51ndash59 2009

[36] E Swist Q Chen C Qiao D Caldwell H Gruber and KA Scoggan ldquoExcess dietary iodine differentially affects thyroidgene expression in diabetes thyroiditis-prone versusmdashresistantBioBreeding (BB) ratsrdquoMolecular Nutrition and Food Researchvol 55 no 12 pp 1875ndash1886 2011

[37] L Liu D Wang P Liu et al ldquoThe relationship between iodinenutrition and thyroid disease in lactating women with differentiodine intakesrdquo British Journal of Nutrition vol 114 no 9 pp1487ndash1495 2015

[38] S A Skeaff C D Thomson and R S Gibson ldquoMild iodinedeficiency in a sample of New Zealand school childrenrdquoEuropean Journal of Clinical Nutrition vol 56 no 12 pp 1169ndash1175 2002

[39] M B Zimmermann P L Jooste and C S Pandav ldquoIodine-deficiency disordersrdquo The Lancet vol 372 no 9645 pp 1251ndash1262 2008

[40] J Wolff ldquoExcess iodide inhibits the thyroid by multiple mecha-nismsrdquoAdvances in ExperimentalMedicine andBiology vol 261pp 211ndash244 1989

[41] C Serrano-Nascimento S D S Teixeira J P Nicola R TNachbar A M Masini-Repiso and M T Nunes ldquoThe acuteinhibitory effect of iodide excess on sodiumiodide symporterexpression and activity involves the PI3KAkt signaling path-wayrdquo Endocrinology vol 155 no 3 pp 1145ndash1156 2014

[42] M D Brand C Affourtit T C Esteves et al ldquoMitochondrialsuperoxide production biological effects and activation ofuncoupling proteinsrdquo Free Radical Biology andMedicine vol 37no 6 pp 755ndash767 2004

[43] S H Bae S H Sung H E Lee et al ldquoPeroxiredoxin IIIand sulfiredoxin together protect mice from pyrazole-inducedoxidative liver injuryrdquo Antioxidants amp Redox Signaling vol 17no 10 pp 1351ndash1361 2012

[44] S H Bae S H Sung E J Cho et al ldquoConcerted actionof sulfiredoxin and peroxiredoxin I protects against alcohol-induced oxidative injury inmouse liverrdquoHepatology vol 53 no3 pp 945ndash953 2011

[45] Y S Kim H L Lee K B Lee et al ldquoNuclear factor E2-related factor 2 dependent overexpression of sulfiredoxin and

Oxidative Medicine and Cellular Longevity 13

peroxiredoxin III in human lung cancerrdquo Korean Journal ofInternal Medicine vol 26 no 3 pp 304ndash313 2011

[46] H-Y Cho A E Jedlicka S P M Reddy et al ldquoRole of NRF2in protection against hyperoxic lung injury in micerdquo AmericanJournal of Respiratory Cell and Molecular Biology vol 26 no 2pp 175ndash182 2002

[47] T W Kensler N Wakabayashi and S Biswal ldquoCell survivalresponses to environmental stresses via the Keap1-Nrf2-AREpathwayrdquo Annual Review of Pharmacology and Toxicology vol47 pp 89ndash116 2007

[48] TO AjiboyeM T Yakubu andA TOladiji ldquoElectrophilic andReactive Oxygen Species Detoxification Potentials of ChalconeDimers is Mediated by Redox Transcription Factor Nrf-2rdquoJournal of Biochemical and Molecular Toxicology vol 28 no 1pp 11ndash22 2014

[49] X Yang D Wang Y Ma et al ldquoContinuous activation of Nrf2and its target antioxidant enzymes leads to arsenite-inducedmalignant transformation of human bronchial epithelial cellsrdquoToxicology and Applied Pharmacology vol 289 no 2 pp 231ndash239 2015

[50] C Pang Z Zheng L Shi et al ldquoCaffeic acid preventsacetaminophen-induced liver injury by activating the Keap1-Nrf2 antioxidative defense systemrdquo Free Radical Biology andMedicine vol 91 pp 236ndash246 2016

[51] Y H Noh J Y Baek W Jeong S G Rhee and T-S ChangldquoSulfiredoxin translocation into mitochondria plays a crucialrole in reducing hyperoxidized peroxiredoxin IIIrdquo The Journalof Biological Chemistry vol 284 no 13 pp 8470ndash8477 2009

[52] B Cunniff A N Wozniak P Sweeney K DeCosta and N HHeintz ldquoPeroxiredoxin 3 levels regulate a mitochondrial redoxsetpoint in malignant mesothelioma cellsrdquo Redox Biology vol3 pp 79ndash87 2014

[53] P-J Chua E-H Lee Y Yu G W-C Yip P-H Tan and B-HBay ldquoSilencing the Peroxiredoxin III gene inhibits cell prolifera-tion in breast cancerrdquo International Journal of Oncology vol 36no 2 pp 359ndash364 2010

[54] B Cunniff K Newick K J Nelson et al ldquoDisabling mito-chondrial peroxide metabolism via combinatorial targetingof peroxiredoxin 3 as an effective therapeutic approach formalignant mesotheliomardquo PLOS ONE vol 10 no 5 Article IDe0127310 2015

[55] X Yao M Li J He et al ldquoEffect of early acute high con-centrations of iodide exposure on mitochondrial superoxideproduction in FRTL cellsrdquo Free Radical Biology and Medicinevol 52 no 8 pp 1343ndash1352 2012

[56] L Wang Q Duan T Wang et al ldquoMitochondrial respiratorychain inhibitors involved in ROS production induced by acutehigh concentrations of iodide and the effects of sod as aprotective factorrdquo Oxidative Medicine and Cellular Longevityvol 2015 Article ID 217670 14 pages 2015

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Oxidative Medicine and Cellular Longevity

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Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

4 Oxidative Medicine and Cellular Longevity

0

50

100

150

200

250

300

350

400

450

500

NI 10 HI 100 HI

Day 7Day 14

Day 28

Body

wei

ght (

g)

(a)

Day 7Day 14

Day 28

0

5

10

15

20

25

30

NI 10 HI 100 HI

Thyr

oid

wei

ght (

mg)

(b)

Day 7Day 14

Day 28

0

1

2

3

4

5

6

7

8

9

NI 10 HI 100 HI

Ratio

of t

hyro

id w

eigh

tbod

y w

eigh

t (m

g10

0 g)

(c)

Figure 1 Effects of the iodide intake (NI 10 HI and 100 HI) on (a) the body weight (b) the thyroid weight and (c) the ratio of the thyroidweightbody weight on Days 7 14 and 28 All data is presented as mean plusmn SD (119873 = 6 for each group) Statistical analyses were performed byone-way analysis of variance (ANOVA) with the Least Significant Difference (LSD) test

and FT4 levels following the intake ofNI 10HI and 100HI onDays 7 14 and 28 (119901 gt 005) Moreover for all three dosagesof iodide intake there were no significant differences in anyof the serum thyroid hormones levels on Day 14 and Day 28when compared to Day 7 (119901 gt 005) In addition there wereno significant changes between Day 14 and Day 28 in serumthyroid hormones levels (TT3 TT4 FT3 and FT4) (119901 gt 005)(Table 3)

34 Effects of the Iodide Intake (NI 10 HI and 100 HI) on theChanges of Mitochondrial Superoxide Production on Days 7

14 and 28 On Days 7 14 and 28 when compared to the NIgroup the mitochondrial superoxide production in the 10 HIgroup showed no significant increase (119901 gt 005) Howeverthere was a significant increase in the mitochondrial super-oxide production in the 100 HI group (119901 lt 005) Whencompared to the 10 HI there was a significant increase in themitochondrial superoxide production in the 100 HI group(119901 lt 005) In the NI and 10 HI groups the mitochondrialsuperoxide production on Days 7 14 and 28 showed nosignificant difference (119901 gt 005) However in the 100 HIgroup compared to Day 7 there was a significant increase in

Oxidative Medicine and Cellular Longevity 5

Table 2 Changes of iodine content in the thyroid glands(120583g100mg) of Wistar rats following the intake of NI 10 HI and 100HI onDays 7 14 and 28 (119873 = 6 for each group) All data is presentedas the mean plusmn SD

Group Iodine content in the thyroid glands (120583g100mg)Day 7 Day 14 Day 28

NI 6219 plusmn 594 6012 plusmn 813 6337 plusmn 90810 HI 12125 plusmn 1048 12645 plusmn 893 15233 plusmn 1077100 HI 13353 plusmn 861lowast 13636 plusmn 964lowast 17845 plusmn 874lowastlowastCompared with NI group (119901 lt 005)

Table 3 Changes of serum thyroid hormones levels of Wistar ratsfollowing the intake of NI 10 HI and 100 HI on Days 7 14 and 28(119873 = 6 for each group) All data is presented as the mean plusmn SD

Group The levels of serum thyroid hormonesTT3

(nmolL)TT4

(nmolL)FT3

(pmolL)FT4

(pmolL)Day 7

NI 118 plusmn 015 9077 plusmn 552 396 plusmn 010 2203 plusmn 17010 HI 119 plusmn 008 9154 plusmn 774 377 plusmn 026 2302 plusmn 206100 HI 107 plusmn 011 8917 plusmn 903 378 plusmn 011 2008 plusmn 543

Day 14NI 115 plusmn 007 9583 plusmn 520 392 plusmn 016 2234 plusmn 37110 HI 112 plusmn 008 8615 plusmn 252 363 plusmn 048 2019 plusmn 192100 HI 119 plusmn 004 9154 plusmn 331 384 plusmn 046 2098 plusmn 250

Day 28NI 113 plusmn 006 9417 plusmn 238 389 plusmn 013 2025 plusmn 57210 HI 118 plusmn 008 9077 plusmn 378 398 plusmn 057 2115 plusmn 534100 HI 114 plusmn 022 8769 plusmn 635 379 plusmn 046 2117 plusmn 543

mitochondrial superoxide production on Day 28 (119901 lt 005)Similarly compared to Day 14 there was also a significantincrease in mitochondrial superoxide production on Day 28(119901 lt 005) Accordingly the fluorescent intensity ofMitoSOXRed onDay 28 gradually increased after the increased dosagesof iodide intake fromNI to 100 HI which was consistent withour results of flow cytometry (Figure 2)

35 Effects of the Iodide Intake (NI 10 HI and 100 HI) on theChanges of Nrf2 Keap 1 Srx and Prx 3 Expressions on Days 714 and 28 On Days 7 14 and 28 when compared to the NIgroup the expressions of Nrf2 Keap 1 Srx and Prx 3 showedno significant differences in the 10 HI group (119901 gt 005)the expressions of Nrf2 Srx and Prx 3 were significantlyincreased while Keap 1 was notably decreased in the 100 HIgroup (119901 lt 005) On Days 7 14 and 28 when comparedto the 10 HI group the expressions of Nrf2 Srx and Prx 3were significantly increased while Keap 1 was significantlydecreased in the 100 HI group (119901 lt 005) (Figure 3)

36 Effects of the Iodide Intake (NI 10 HI and 100 HI) on theChanges of Immunofluorescence Staining onDays 7 14 and 28Following the increased iodide intake from NI to 10 HI and

further to 100 HI the expressions of Nrf2 and Srx intensifiedIn the NI group Nrf2 was localized in the cytoplasm on Days7 14 and 28 In the 10 HI group the positive staining ofNrf2 can be observed in both the nucleus and the cytoplasmMoreover in the 100 HI group a stronger positive stainingof Nrf2 can be detected in the nucleus on Days 7 14 and 28(Figure 4(a)) Srx positive staining was only located in thecytoplasm on Days 7 14 and 28 (Figure 4(b))

4 Discussion

In our study we found that there were no significant alter-ations in TT3 TT4 FT3 and FT4 levels following the intakeofNI 10HI and 100HI onDays 7 14 and 28 (119901 gt 005)Thereare very efficient homeostatic mechanisms that resist changesin circulating T3 and T4 levels in response to iodide excessDue to the compensatory mechanisms such as the Wolff-Chaikoff effect the changes in T3 and T4 levels following anincrease in iodide intake are minimal and usually transientin nature [26ndash28] The Wolff-Chaikoff effect relies on a high(ge10minus3 molar) intracellular concentration of iodide Duringinitial exposure excess iodide is transported by the sodium-iodide symporter (NIS) into the cellsWhen intracellular con-centration reaches at least 10minus3 molar iodide organificationis blocked [27 29] Moreover the regulatory mechanismsinclude modulation of blood flow enzyme activity geneexpression and transport proteins in signaling pathways [230 31] In a study conducted by Eng et al 16 Wistar ratswere given 2000120583g of iodide acutely it was observed thatthere was a significant decrease in serum T4 and T3 levelson Day 1 of the study Subsequently on Day 6 both serumT4 and T3 levels returned to normal ranges However inboth cases the serum TSH levels remained unchanged [32]Mooij et al observed no significant changes in serum thyroidhormones levels when female Wistar rats were given 100 120583giodide daily for 18 weeks [33] Paul et al demonstrated thatwhen normal volunteers received 1500120583g of supplementaliodine daily for 14 days a small decrease in serum T3 andT4 concentrations with compensatory increase of TSH wasdetected although all values remained within the normalranges [34] However the presence of handicaps such as anincreased autoimmune susceptibility fetal period extremesof age pregnancy lactation or an active pathological entitysignificantly impair these mechanisms [1 35ndash37]

Our study demonstrated that both the urinary iodineconcentration and the iodide intake increased simultaneouslyfrom NI to 10 HI and further to 100 HI Interestingly wefound that the urinary iodine concentration of the 10 HIgroup was approximately 10 times that of the NI groupwhereas the urinary iodine concentration of the 100HI groupwas about 60ndash80 times that of the NI group on Days 7 14and 28 The urinary iodine concentration is regarded as asensitive indicator of iodine status because approximately90 of ingested iodide is excreted in the urine [38 39]Thereis an increase for the maintenance of thyroid homeostasisas well as the steady state of the internal environment ofthe body The thyroid gland has adaptation mechanisms thatreduce iodide metabolism when the supply is abundant thus

6 Oxidative Medicine and Cellular Longevity

1793 2066 4897

1773 2264 5291

1806 2373 7360

NI 10 HI 100 HID

ay 7

Day

14

Day

28

250

200

150

100

50

0

Cou

nt

0 102 103 104 105

Comp-PE-A PE-A

250

200

150

100

50

0

Cou

nt

0 102 103 104 105

Comp-PE-A PE-A

250

200

150

100

50

0

Cou

nt

0 102 103 104 105

Comp-PE-A PE-A

250

200

150

100

50

0

Cou

nt

0 102 103 104 105

Comp-PE-A PE-A

250

200

150

100

50

0

Cou

nt

0 102 103 104 105

Comp-PE-A PE-A

250

200

150

100

50

0

Cou

nt

0 102 103 104 105

Comp-PE-A PE-A

250

200

150

100

50

0

Cou

nt

0 102 103 104 105

Comp-PE-A PE-A

250

200

150

100

50

0

Cou

nt

0 102 103 104 105

Comp-PE-A PE-A

250

200

150

100

50

0

Cou

nt

0 102 103 104 105

Comp-PE-A PE-A

(a)

0

10

20

30

40

50

60

70

80

NI 10 HI 100 HI

Day 7Day 14Day 28

Mito

SOX

fluor

esce

nce i

nten

sity

lowastlowast

lowast998771998779

(b)

Figure 2 Continued

Oxidative Medicine and Cellular Longevity 7

NI 10 HI 100 HI

Day 28

20120583m20120583m20120583m

(c)

Figure 2 Effects of the iodide intake (NI 10 HI and 100 HI) on the changes of mitochondrial superoxide production on Days 7 14 and28 (a) There was a significant increase in the mitochondrial superoxide production in the 100 HI group on Days 7 14 and 28 (119901 lt 005)The difference was more significant on Day 28 when compared to Day 7 or Day 14 (b) Histogram analysis was performed on the meanfluorescence intensity of MitoSOX Red All data is presented as the mean plusmn SD (119873 = 6 for each group) Statistical analyses were performedby one-way analysis of variance (ANOVA) with the Least Significant Difference (LSD) test lowast119901 lt 005 versus the NI group on Days 7 14 and28 respectively 119901 lt 005 versus the 10 HI group on Days 7 14 and 28 respectively 998771119901 lt 005 versus the Day 7 group in the 100 HI group119901 lt 005 versus the Day 14 in the 100 HI group Experiments were repeated 3 times with similar results (c) There was a significant increasein the mitochondrial superoxide production in the 100 HI group on Day 28 and was observed by confocal microscopy Scale bar 20120583m

avoiding thyrotoxicosisThere are several mechanisms whichinclude a direct inhibitory effect of iodide in the thyroid itselfand inhibition by iodide of its own organification (Wolff-Chaikoff effect) its transport thyroid hormones secretioncAMP formation in response to TSH and several othermetabolic steps [40] We suggest that all the protectivemechanisms may ensure the excessive iodide intake of the 10HI group on Days 7 14 and 28 be eliminated from urineTheurinary iodine concentration of the 100 HI group on Days7 14 and 28 was about 60ndash80 times that of the NI groupalthough the thyroid function was normal This leads us topropose the idea that excessive iodide accumulated in thebody may trigger the oxidative and antioxidative signalingpathway to maintain the normal thyroid function

We demonstrated that the production of mitochondrialsuperoxide significantly increased on Days 7 14 and 28 inthe 100 HI group This is consistent with our previous studyon metallothionein-III knockout mice [24] Joanta et alreported that the initiation of free radical production wasobserved after giving a high dose of iodide [3] Serrano-Nascimento et al demonstrated that an increased mitochon-drial superoxide production was shown in response to NaI(10minus6M to 10minus3M) treatment in PCCl3 thyroid cells by usingMitoSOX Red [41] Mitochondria are potent producers ofsuperoxide from complexes I and III of the electron transportchain Mitochondrial superoxide production is a major causeof cellular oxidative damage [42] Physiologically ROS arenot necessarily harmful because they are continuously bal-anced by the process of hormone synthesis and the endoge-nous antioxidant system [1] Excess ROS are generated duringthe trapping oxidation and organification of excessive iodinein thyrocytes which could lead to increased oxidative stress[1]

The Nrf2-Keap 1 pathway is the chief cytoprotectivemechanism in response to oxidative stress caused by ROSDuring normal and balanced redox homeostasis the Nrf2function is inhibited because of constant proteasomal degra-dation after ubiquitination of the protein This is regulated

through the binding of the inhibitor protein Keap 1 [13 14]It is reported that Srx activation via Nrf2 dependent pathwayprotects fromoxidative liver injury throughPyrazole [43] andalcohol in mice [44] Similar findings in lung tissues haveshown that there is a marked increase in the expressions ofSrx and Prx 3 in human squamous cell carcinoma [45] Thissuggests that these proteins may play a protective role againstoxidative injury Also the pathway including Keap 1 Nrf2and ARE-mediated protein expression plays a very criticalrole in protecting cells fromoxidative stress [46 47] Focusingon the pathway we demonstrated that the expression of Nrf2was significantly increased while Keap 1 was significantlydecreased in the 100 HI group when compared to the NIor 10 HI group on Days 7 14 and 28 Similarly Ajiboyeet al showed that rats treated with Chalcone dimers notonly increased the expression of Nrf2 but also suppressedcytoplasmic Keap 1 expression [48] Yang et al also showedthat the downregulation of Keap 1 level may be responsiblefor the overactivation of Nrf2 [49] Pang et al showed thatcaffeic acid prevents acetaminophen-induced liver injury byactivating the Nrf2-Keap 1 antioxidative defense system [50]

In order to verify whether high levels of expressionof Nrf2 and its nuclear translocation can upregulate someantioxidant enzymes in the thyroid gland the expressions ofPrx 3 and Srx following the intake of NI 10 HI and 100 HIon Days 7 14 and 28 were measured We demonstrated thatthe expressions of Srx and Prx 3 in the 100 HI group weresignificantly increased when compared to the NI group or 10HI group on Days 7 14 and 28 The possible explanationsare described as below Firstly Srx is a cytosolic proteinthat is able to translocate to sites where hyperoxidized(inactivated) Prx 3 is located Therefore it engages itself inthe reactivation of Prx 3 under oxidative conditions [51]Secondly Prx 3 is a typical 2-Cys Peroxiredoxin locatedexclusively in the mitochondrial matrix it is the principalperoxidase responsible for protecting cells from oxidativedamage by reducing peroxides such as H2O2 [52] Finallymitochondria contain 30 times more Prx 3 than glutathione

8 Oxidative Medicine and Cellular Longevity

000

020

040

060

080

100

120

140

NI 10 HI 100 HI

Expr

essio

n of

Nrf2

(Nrf2

120573-a

ctin

)

120573-Actin

Nrf2

Day 7 Day 14 Day 28

lowast lowast

lowast

Day 7Day 14

Day 28

NI 10 HI 100 HI NI 10 HI 100 HI NI 10 HI 100 HI

(a)

000

020

040

060

080

100

120

NI 10 HI 100 HI

lowast

lowast

Expr

essio

n of

Kea

p 1

(Kea

p 1120573

-act

in)

120573-Actin

Keap 1

Day 7 Day 14 Day 28

lowast

Day 7Day 14

Day 28

NI 10 HI 100 HI NI 10 HI 100 HI NI 10 HI 100 HI

(b)

120573-Actin

SrxNI 10 HI 100 HI NI 10 HI 100 HI NI 10 HI 100 HI

000

010

020

030

040

050

060

NI 10 HI 100 HI

Expr

essio

n of

Srx

(Srx

120573-a

ctin

)

Day 7 Day 14 Day 28

lowast

lowast

lowast

Day 7Day 14

Day 28

(c)

120573-Actin

Prx 3

lowast

000

020

040

060

080

100

120

140

NI 10 HI 100 HI

Expr

essio

n of

Prx

3 (P

rx 3

120573-a

ctin

)

Day 7 Day 14 Day 28

lowastlowast

Day 7Day 14

Day 28

NI 10 HI 100 HI NI 10 HI 100 HI NI 10 HI 100 HI

(d)

Figure 3 Effects of the iodide intake (NI 10 HI and 100 HI) on the changes of (a) Nrf2 (b) Keap 1 (c) Srx and (d) Prx 3 expressions onDays 7 14 and 28 Representative western blot and histograms of densitometric analyses normalized for the relative 120573-actin content All datais presented as the mean plusmn SD (119873 = 6 for each group) Statistical analyses were performed by one-way analysis of variance (ANOVA) withthe Least Significant Difference (LSD) test lowast119901 lt 005 versus the NI group on Days 7 14 and 28 respectively 119901 lt 005 versus the 10 HI groupon Days 7 14 and 28 respectively Experiments were repeated 3 times with similar results

Oxidative Medicine and Cellular Longevity 9

NI

10 HI

100 HI

Hoechst Nrf2 Merge HoechstHoechst

Day 7 Day 28 Day 14

Nrf2 Merge Nrf2 Merge

Day 7 Day 7 Day 14 Day 14 Day 28 Day 28 20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

(a)

Hoechst Srx Merge Hoechst Srx Merge Hoechst Srx Merge

NI

10 HI

100 HI

Day 7 Day 28 Day 14 Day 7 Day 7 Day 14 Day 14 Day 28 Day 28 20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

(b)

Figure 4 Effect of the iodide intake (NI 10 HI and 100 HI) on the changes of immunofluorescence staining on Days 7 14 and 28 (a) In theNI group Nrf2 (red) was localized in the cytoplasm on Days 7 14 and 28 In the 10 HI group the positive staining of Nrf2 can be observedin both the nucleus and the cytoplasm Moreover in the 100 HI group a stronger positive staining of Nrf2 can be detected in the nucleus onDays 7 14 and 28 the nucleus was dyed with Hoechst (blue) (b) Srx (red) positive staining was located in the cytoplasm the nucleus wasdyed with Hoechst (blue) Scale bar 20120583m

peroxidase Prx 3 can be classified as an important regulatorof mitochondrial H2O2 [22] The elevated expression of Prx3 is associated with the blockage of apoptosis increasingcell proliferation and is related to adaptive responses whichare all required to maintain mitochondrial function [53 54]Bae et al have suggested that Prx 3 and Srx jointly protectmice from Pyrazole-induced oxidative liver injury in a Nrf2-dependent manner [43]

The novelty we demonstrated in the present study is thatiodide excess induced both oxidative stress and antioxidativedefense increases throughNrf2-Keap 1 pathway in the thyroidgland from rats We extended our established mechanismsby applying the Nrf2-Keap 1 pathway to set up a bridgebetween oxidative stress and antioxidative defense inducedby iodide excess in the thyroid gland In our previous studywe have established that oxidative stress induced by acutehigh concentrations of iodide in FRTL cells significantlyincreases mitochondrial superoxide production [55] Theinhibitors of themitochondrial respiratory chain complexes I

and III are involved inmitochondrial superoxide productionWe demonstrated that exposure to 100120583M KI for 2 hourssignificantly increased mitochondrial superoxide produc-tion enhanced by either 05120583M Rotenone (an inhibitorof mitochondrial complex I) or 10 120583M Antimycin A (aninhibitor of complex III) [56] We illustrated that 300 120583MPTU (an inhibitor of TPO) attenuated the excessive iodide-induced mitochondrial superoxide production We showedthat 30 120583M KClO4 (a competitive inhibitor of iodide trans-port) relieved the production the mitochondrial superoxideinduced by iodide excess We displayed that 10mUmL TSHcan inhibit excessive iodide-induced strong mitochondrialsuperoxide production [55] MT-I and MT-II are mainlyinvolved in the protection of tissue against oxidative stresseswe indicated that metallothionein-III knockout mice aggra-vated mitochondrial superoxide production in thyroid afterexcessive iodide exposure [24] In addition we demonstratedthat both the oxidative stress and the antioxidative defenseincreased simultaneously after high dosages of iodide intake

10 Oxidative Medicine and Cellular Longevity

Oxidative stress

(a)

(b)

(c)

Antioxidative defense

Wist

ar ra

ts Day 7Day 14Day 28

Normal iodideintake

Normal urinaryiodine

Normal thyroidfunction

10 times iodideintake

10 times urinaryiodine

Normal thyroidfunction

100 times iodideintake

60ndash80 timesurinary iodine

Normal thyroidfunction

Keap 1 Nrf2

Keap 1

Nrf2

Srx

Nucleus

Prx 3

SrxARE

Mitochondria

AntioxidativeOxidativestress

Oxidative Antioxidative

Oxidative Antioxidative

Under physiological conditions(normal iodide intake)

Excessive iodide intake(10 times iodide intake)

Excessive iodide intake(100 times iodide intake)

defense defenseuarrstressuarr

defenseuarruarrstressuarruarr

Figure 5 Proposed mechanisms in the present study (a) The urinary iodine concentration and thyroid function of Wistar rats weredetected (b) The activation of the Nrf2-Keap 1 pathway induced by iodide excess in the thyroid (c) The balance between oxidative stressand antioxidative defense under physiological conditions and excessive iodide intake

Oxidative Medicine and Cellular Longevity 11

We suggested that the Nrf2-Keap 1 pathway is vital for thebalance between oxidative stress and antioxidative defenseinduced by iodide excess in the thyroid gland

Excessive iodide stimulated the Nrf2-Keap 1 pathwayand enhanced the antioxidative defense We found thatthe urinary iodine concentration of the 100 HI group wasabout 60ndash80 times that of the NI group on Days 7 14and 28 however the thyroid functions were normal Weproposed that the excessive iodide accumulated in the bodymay trigger the Nrf2-Keap 1 pathway to maintain a normalthyroid functionWe demonstrated that the Nrf2moves fromthe cytoplasm to the nucleus under the microscope withsignificantly increased expressions of Nrf2 Srx and Prx 3 andnotably decreased Keap 1 when exposed to high iodide Thissuggests that excessive iodide stimulates the disassociation ofNrf2 from Keap 1 and assists Nrf2 to penetrate the nucleusThen Nrf2 attaches to the antioxidant response element(ARE) to activate the expression of the antioxidative genesSrx and Prx 3 resulting in an enhanced antioxidative defenseinduced by high iodide By activation of the Nrf2-Keap 1pathway there is a proportional increase in oxidative stressand antioxidative defense in response to iodide excess It isto be noted that the thyroid function was normal in the 10HI group and 100 HI in present study Inspired by the reportby Poncin et al [6] we proposed that there should be abalance between oxidative stress and antioxidative defense inresponse to iodide excess in the 10 HI and the 100 HI groups

5 Conclusion

In conclusion our results highlight that the activation ofNrf2-Keap 1 Srx and Prx 3 antioxidative defense mecha-nisms may play a crucial role in protecting the thyroid fromiodide excess induced oxidative stress on Days 7 14 and 28(Figure 5)

Abbreviations

NI Normal iodide intakeHI High iodide intakeROS Reactive oxygen speciesH2O2 Hydrogen peroxideNrf2 Nuclear factor erythroid 2-related factor 2Keap 1 Kelch-like ECH-associated protein 1ARE Antioxidant response elementPrx PeroxiredoxinSrx SulfiredoxinPrx 3 Peroxiredoxin 3TT4 Total thyroxineTT3 Total triiodothyronineFT4 Free thyroxineFT3 Free triiodothyronineTSH ThyrotropinLSD Least significant difference

Disclosure

This manuscript has been edited by Umar Iqbal IruniRoshanie Abeysekera Gargi Naha and Tejas Bharadwaj all

of whom are currently studying at the International School ofMedicine Tianjin Medical University China

Competing Interests

The authors declare that there is no conflict of interestsregarding the publication of this article

Acknowledgments

This work is supported by National Natural Science Founda-tion of China (nos 81273009 and 81270182) Tianjin Scienceamp Technology Council Grant of China (nos 16JCYBJC26100and 09JCYBJC11700) and International Studentrsquos Science ampTechnology Innovation Project (Scientific Research Project)of Tianjin Medical University

References

[1] Y Luo A Kawashima Y Ishido et al ldquoIodine excess as anenvironmental risk factor for autoimmune thyroid diseaserdquoInternational Journal of Molecular Sciences vol 15 no 7 pp12895ndash12912 2014

[2] A E Hussein A M Abbas G A El Wakil A Z Elsamanoudyand A A El Aziz ldquoEffect of chronic excess iodine intakeon thyroid function and oxidative stress in hypothyroid ratsrdquoCanadian Journal of Physiology and Pharmacology vol 90 no5 pp 617ndash625 2012

[3] A E Joanta A Filip S Clichici S Andrei and D DaicoviciuldquoIodide excess exerts oxidative stress in some target tissues ofthe thyroid hormonesrdquoActa Physiologica Hungarica vol 93 no4 pp 347ndash359 2006

[4] SMorandMChaaraoui J Kaniewski et al ldquoEffect of iodide onnicotinamide adenine dinucleotide phosphate oxidase activityand Duox2 protein expression in isolated porcine thyroidfolliclesrdquo Endocrinology vol 144 no 4 pp 1241ndash1248 2003

[5] J J Lazaro A Jimenez D Camejo et al ldquoDissecting the inte-grative antioxidant and redox systems in plant mitochondriaEffect of stress and S-nitrosylationrdquo Frontiers in Plant Sciencevol 4 article 460 2013

[6] S Poncin A-C GerardM Boucquey et al ldquoOxidative stress inthe thyroid gland from harmlessness to hazard depending onthe iodine contentrdquo Endocrinology vol 149 no 1 pp 424ndash4332008

[7] M Karbownik and A Lewinski ldquoThe role of oxidative stressin physiological and pathological processes in the thyroidgland possible involvement in pineal-thyroid interactionsrdquoNeuroendocrinology Letters vol 24 no 5 pp 293ndash303 2003

[8] P Thomopoulos ldquoHyperthyroidism due to excess iodinerdquo LaPresse Medicale vol 31 no 35 pp 1664ndash1669 2002

[9] J Wolff ldquoIodide goiter and the pharmacologic effects of excessiodiderdquoTheAmerican Journal of Medicine vol 47 no 1 pp 101ndash124 1969

[10] B Corvilain L Collyn J Van Sande and J E Dumont ldquoStim-ulation by iodide of H2O2 generation in thyroid slices fromseveral speciesrdquoAmerican Journal of PhysiologymdashEndocrinologyand Metabolism vol 278 no 4 pp E692ndashE699 2000

[11] M-C Many C Mestdagh M-F Van den Hove and J-F DenefldquoIn vitro study of acute toxic effects of high iodide doses inhuman thyroid folliclesrdquo Endocrinology vol 131 no 2 pp 621ndash630 1992

12 Oxidative Medicine and Cellular Longevity

[12] J Van Sande G Grenier C Willems and J E DumontldquoInhibition by iodide of the activation of the thyroid cyclic 3101584051015840-AMP systemrdquo Endocrinology vol 96 no 3 pp 781ndash786 1975

[13] R Young J D Hayes K Brown C R Wolf and C B AWhitelaw ldquoPeroxiredoxin gene expression signatures in liverreflect toxic insultrdquo Assay and Drug Development Technologiesvol 8 no 4 pp 512ndash517 2010

[14] Q Ma ldquoRole of Nrf2 in oxidative stress and toxicityrdquo AnnualReview of Pharmacology and Toxicology vol 53 pp 401ndash4262013

[15] B Biteau J Labarre and M B Toledano ldquoATP-dependentreduction of cysteine-sulphinic acid by S cerevisiae sulphire-doxinrdquo Nature vol 425 no 6961 pp 980ndash984 2003

[16] L Wu H Jiang H A Chawsheen et al ldquoTumor promoter-induced sulfiredoxin is required for mouse skin tumorigenesisrdquoCarcinogenesis vol 35 no 5 pp 1177ndash1184 2014

[17] C C Winterbourn and M B Hampton ldquoThiol chemistryand specificity in redox signalingrdquo Free Radical Biology andMedicine vol 45 no 5 pp 549ndash561 2008

[18] J W Baty M B Hampton and C C Winterbourn ldquoProteomicdetection of hydrogen peroxide-sensitive thiol proteins inJurkat cellsrdquo Biochemical Journal vol 389 no 3 pp 785ndash7952005

[19] T-S Chang C-S Cho S Park S YuW K Sang and G R SueldquoPeroxiredoxin III a mitochondrion-specific peroxidase regu-lates apoptotic signaling by mitochondriardquo Journal of BiologicalChemistry vol 279 no 40 pp 41975ndash41984 2004

[20] P Thomopoulos ldquoHyperthyroidism due to excess iodinerdquo LaPresse Medicale vol 31 no 35 pp 1664ndash1669 2002

[21] S Chung X-H Liao C Di Cosmo et al ldquoDisruption of themelanin-concentrating hormone receptor 1 (MCH1R) affectsthyroid functionrdquo Endocrinology vol 153 no 12 pp 6145ndash61542012

[22] G Ozguner and O Sulak ldquoSize and location of thyroid gland inthe fetal periodrdquo Surgical and Radiologic Anatomy vol 36 no4 pp 359ndash367 2014

[23] P L Jooste and E Strydom ldquoMethods for determination ofiodine in urine and saltrdquo Best Practice amp Research ClinicalEndocrinology amp Metabolism vol 24 no 1 pp 77ndash88 2010

[24] N Zhang L Wang Q Duan et al ldquoMetallothionein-IIIknockoutmice aggravatemitochondrial superoxide productionand peroxiredoxin 3 expression in thyroid after excessive iodideexposurerdquoOxidative Medicine and Cellular Longevity vol 2015Article ID 267027 11 pages 2015

[25] P I Gus D Marinho S Zelanis et al ldquoA case-control studyon the oxidative balance of 50 autologous serum eye dropsrdquoOxidative Medicine and Cellular Longevity vol 2016 Article ID9780193 5 pages 2016

[26] P Laurberg C Cerqueira L Ovesen et al ldquoIodine intake as adeterminant of thyroid disorders in populationsrdquo Best Practiceamp Research Clinical Endocrinology amp Metabolism vol 24 no 1pp 13ndash27 2010

[27] H Burgi ldquoIodine excessrdquo Best Practice amp Research ClinicalEndocrinology amp Metabolism vol 24 no 1 pp 107ndash115 2010

[28] E Roti and E D Uberti ldquoIodine excess and hyperthyroidismrdquoThyroid vol 11 no 5 pp 493ndash500 2001

[29] A M Leung and L E Braverman ldquoConsequences of excessiodinerdquo Nature Reviews Endocrinology vol 10 no 3 pp 136ndash142 2014

[30] K Wang Y N Sun J Y Liu et al ldquoThe impact of iodineexcess on thyroid hormone biosynthesis and metabolism in

ratsrdquoBiological Trace Element Research vol 130 no 1 pp 72ndash852009

[31] M Michalkiewicz L J Huffman J M Connors and G AHedge ldquoAlterations in thyroid blood flow induced by varyinglevels of iodine intake in the ratrdquo Endocrinology vol 125 no 1pp 54ndash60 1989

[32] P H K Eng G R Cardona S-L Fang et al ldquoEscape fromthe acute Wolff-Chaikoff effect is associated with a decrease inthyroid sodiumiodide symporter messenger ribonucleic acidand proteinrdquo Endocrinology vol 140 no 8 pp 3404ndash3410 1999

[33] P Mooij H J De Wit and H A Drexhage ldquoA high iodineintake in Wistar rats results in the development of a thyroid-associated ectopic thymic tissue and is accompanied by a lowthyroid autoimmune reactivityrdquo Immunology vol 81 no 2 pp309ndash316 1994

[34] T Paul B Meyers R J Witorsch et al ldquoThe effect of smallincreases in dietary iodine on thyroid function in euthyroidsubjectsrdquoMetabolism vol 37 no 2 pp 121ndash124 1988

[35] X Teng Z Shan W Teng C Fan H Wang and R GuoldquoExperimental study on the effects of chronic iodine excess onthyroid function structure and autoimmunity in autoimmune-prone NODH-2h4 micerdquo Clinical and Experimental Medicinevol 9 no 1 pp 51ndash59 2009

[36] E Swist Q Chen C Qiao D Caldwell H Gruber and KA Scoggan ldquoExcess dietary iodine differentially affects thyroidgene expression in diabetes thyroiditis-prone versusmdashresistantBioBreeding (BB) ratsrdquoMolecular Nutrition and Food Researchvol 55 no 12 pp 1875ndash1886 2011

[37] L Liu D Wang P Liu et al ldquoThe relationship between iodinenutrition and thyroid disease in lactating women with differentiodine intakesrdquo British Journal of Nutrition vol 114 no 9 pp1487ndash1495 2015

[38] S A Skeaff C D Thomson and R S Gibson ldquoMild iodinedeficiency in a sample of New Zealand school childrenrdquoEuropean Journal of Clinical Nutrition vol 56 no 12 pp 1169ndash1175 2002

[39] M B Zimmermann P L Jooste and C S Pandav ldquoIodine-deficiency disordersrdquo The Lancet vol 372 no 9645 pp 1251ndash1262 2008

[40] J Wolff ldquoExcess iodide inhibits the thyroid by multiple mecha-nismsrdquoAdvances in ExperimentalMedicine andBiology vol 261pp 211ndash244 1989

[41] C Serrano-Nascimento S D S Teixeira J P Nicola R TNachbar A M Masini-Repiso and M T Nunes ldquoThe acuteinhibitory effect of iodide excess on sodiumiodide symporterexpression and activity involves the PI3KAkt signaling path-wayrdquo Endocrinology vol 155 no 3 pp 1145ndash1156 2014

[42] M D Brand C Affourtit T C Esteves et al ldquoMitochondrialsuperoxide production biological effects and activation ofuncoupling proteinsrdquo Free Radical Biology andMedicine vol 37no 6 pp 755ndash767 2004

[43] S H Bae S H Sung H E Lee et al ldquoPeroxiredoxin IIIand sulfiredoxin together protect mice from pyrazole-inducedoxidative liver injuryrdquo Antioxidants amp Redox Signaling vol 17no 10 pp 1351ndash1361 2012

[44] S H Bae S H Sung E J Cho et al ldquoConcerted actionof sulfiredoxin and peroxiredoxin I protects against alcohol-induced oxidative injury inmouse liverrdquoHepatology vol 53 no3 pp 945ndash953 2011

[45] Y S Kim H L Lee K B Lee et al ldquoNuclear factor E2-related factor 2 dependent overexpression of sulfiredoxin and

Oxidative Medicine and Cellular Longevity 13

peroxiredoxin III in human lung cancerrdquo Korean Journal ofInternal Medicine vol 26 no 3 pp 304ndash313 2011

[46] H-Y Cho A E Jedlicka S P M Reddy et al ldquoRole of NRF2in protection against hyperoxic lung injury in micerdquo AmericanJournal of Respiratory Cell and Molecular Biology vol 26 no 2pp 175ndash182 2002

[47] T W Kensler N Wakabayashi and S Biswal ldquoCell survivalresponses to environmental stresses via the Keap1-Nrf2-AREpathwayrdquo Annual Review of Pharmacology and Toxicology vol47 pp 89ndash116 2007

[48] TO AjiboyeM T Yakubu andA TOladiji ldquoElectrophilic andReactive Oxygen Species Detoxification Potentials of ChalconeDimers is Mediated by Redox Transcription Factor Nrf-2rdquoJournal of Biochemical and Molecular Toxicology vol 28 no 1pp 11ndash22 2014

[49] X Yang D Wang Y Ma et al ldquoContinuous activation of Nrf2and its target antioxidant enzymes leads to arsenite-inducedmalignant transformation of human bronchial epithelial cellsrdquoToxicology and Applied Pharmacology vol 289 no 2 pp 231ndash239 2015

[50] C Pang Z Zheng L Shi et al ldquoCaffeic acid preventsacetaminophen-induced liver injury by activating the Keap1-Nrf2 antioxidative defense systemrdquo Free Radical Biology andMedicine vol 91 pp 236ndash246 2016

[51] Y H Noh J Y Baek W Jeong S G Rhee and T-S ChangldquoSulfiredoxin translocation into mitochondria plays a crucialrole in reducing hyperoxidized peroxiredoxin IIIrdquo The Journalof Biological Chemistry vol 284 no 13 pp 8470ndash8477 2009

[52] B Cunniff A N Wozniak P Sweeney K DeCosta and N HHeintz ldquoPeroxiredoxin 3 levels regulate a mitochondrial redoxsetpoint in malignant mesothelioma cellsrdquo Redox Biology vol3 pp 79ndash87 2014

[53] P-J Chua E-H Lee Y Yu G W-C Yip P-H Tan and B-HBay ldquoSilencing the Peroxiredoxin III gene inhibits cell prolifera-tion in breast cancerrdquo International Journal of Oncology vol 36no 2 pp 359ndash364 2010

[54] B Cunniff K Newick K J Nelson et al ldquoDisabling mito-chondrial peroxide metabolism via combinatorial targetingof peroxiredoxin 3 as an effective therapeutic approach formalignant mesotheliomardquo PLOS ONE vol 10 no 5 Article IDe0127310 2015

[55] X Yao M Li J He et al ldquoEffect of early acute high con-centrations of iodide exposure on mitochondrial superoxideproduction in FRTL cellsrdquo Free Radical Biology and Medicinevol 52 no 8 pp 1343ndash1352 2012

[56] L Wang Q Duan T Wang et al ldquoMitochondrial respiratorychain inhibitors involved in ROS production induced by acutehigh concentrations of iodide and the effects of sod as aprotective factorrdquo Oxidative Medicine and Cellular Longevityvol 2015 Article ID 217670 14 pages 2015

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

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Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Oxidative Medicine and Cellular Longevity 5

Table 2 Changes of iodine content in the thyroid glands(120583g100mg) of Wistar rats following the intake of NI 10 HI and 100HI onDays 7 14 and 28 (119873 = 6 for each group) All data is presentedas the mean plusmn SD

Group Iodine content in the thyroid glands (120583g100mg)Day 7 Day 14 Day 28

NI 6219 plusmn 594 6012 plusmn 813 6337 plusmn 90810 HI 12125 plusmn 1048 12645 plusmn 893 15233 plusmn 1077100 HI 13353 plusmn 861lowast 13636 plusmn 964lowast 17845 plusmn 874lowastlowastCompared with NI group (119901 lt 005)

Table 3 Changes of serum thyroid hormones levels of Wistar ratsfollowing the intake of NI 10 HI and 100 HI on Days 7 14 and 28(119873 = 6 for each group) All data is presented as the mean plusmn SD

Group The levels of serum thyroid hormonesTT3

(nmolL)TT4

(nmolL)FT3

(pmolL)FT4

(pmolL)Day 7

NI 118 plusmn 015 9077 plusmn 552 396 plusmn 010 2203 plusmn 17010 HI 119 plusmn 008 9154 plusmn 774 377 plusmn 026 2302 plusmn 206100 HI 107 plusmn 011 8917 plusmn 903 378 plusmn 011 2008 plusmn 543

Day 14NI 115 plusmn 007 9583 plusmn 520 392 plusmn 016 2234 plusmn 37110 HI 112 plusmn 008 8615 plusmn 252 363 plusmn 048 2019 plusmn 192100 HI 119 plusmn 004 9154 plusmn 331 384 plusmn 046 2098 plusmn 250

Day 28NI 113 plusmn 006 9417 plusmn 238 389 plusmn 013 2025 plusmn 57210 HI 118 plusmn 008 9077 plusmn 378 398 plusmn 057 2115 plusmn 534100 HI 114 plusmn 022 8769 plusmn 635 379 plusmn 046 2117 plusmn 543

mitochondrial superoxide production on Day 28 (119901 lt 005)Similarly compared to Day 14 there was also a significantincrease in mitochondrial superoxide production on Day 28(119901 lt 005) Accordingly the fluorescent intensity ofMitoSOXRed onDay 28 gradually increased after the increased dosagesof iodide intake fromNI to 100 HI which was consistent withour results of flow cytometry (Figure 2)

35 Effects of the Iodide Intake (NI 10 HI and 100 HI) on theChanges of Nrf2 Keap 1 Srx and Prx 3 Expressions on Days 714 and 28 On Days 7 14 and 28 when compared to the NIgroup the expressions of Nrf2 Keap 1 Srx and Prx 3 showedno significant differences in the 10 HI group (119901 gt 005)the expressions of Nrf2 Srx and Prx 3 were significantlyincreased while Keap 1 was notably decreased in the 100 HIgroup (119901 lt 005) On Days 7 14 and 28 when comparedto the 10 HI group the expressions of Nrf2 Srx and Prx 3were significantly increased while Keap 1 was significantlydecreased in the 100 HI group (119901 lt 005) (Figure 3)

36 Effects of the Iodide Intake (NI 10 HI and 100 HI) on theChanges of Immunofluorescence Staining onDays 7 14 and 28Following the increased iodide intake from NI to 10 HI and

further to 100 HI the expressions of Nrf2 and Srx intensifiedIn the NI group Nrf2 was localized in the cytoplasm on Days7 14 and 28 In the 10 HI group the positive staining ofNrf2 can be observed in both the nucleus and the cytoplasmMoreover in the 100 HI group a stronger positive stainingof Nrf2 can be detected in the nucleus on Days 7 14 and 28(Figure 4(a)) Srx positive staining was only located in thecytoplasm on Days 7 14 and 28 (Figure 4(b))

4 Discussion

In our study we found that there were no significant alter-ations in TT3 TT4 FT3 and FT4 levels following the intakeofNI 10HI and 100HI onDays 7 14 and 28 (119901 gt 005)Thereare very efficient homeostatic mechanisms that resist changesin circulating T3 and T4 levels in response to iodide excessDue to the compensatory mechanisms such as the Wolff-Chaikoff effect the changes in T3 and T4 levels following anincrease in iodide intake are minimal and usually transientin nature [26ndash28] The Wolff-Chaikoff effect relies on a high(ge10minus3 molar) intracellular concentration of iodide Duringinitial exposure excess iodide is transported by the sodium-iodide symporter (NIS) into the cellsWhen intracellular con-centration reaches at least 10minus3 molar iodide organificationis blocked [27 29] Moreover the regulatory mechanismsinclude modulation of blood flow enzyme activity geneexpression and transport proteins in signaling pathways [230 31] In a study conducted by Eng et al 16 Wistar ratswere given 2000120583g of iodide acutely it was observed thatthere was a significant decrease in serum T4 and T3 levelson Day 1 of the study Subsequently on Day 6 both serumT4 and T3 levels returned to normal ranges However inboth cases the serum TSH levels remained unchanged [32]Mooij et al observed no significant changes in serum thyroidhormones levels when female Wistar rats were given 100 120583giodide daily for 18 weeks [33] Paul et al demonstrated thatwhen normal volunteers received 1500120583g of supplementaliodine daily for 14 days a small decrease in serum T3 andT4 concentrations with compensatory increase of TSH wasdetected although all values remained within the normalranges [34] However the presence of handicaps such as anincreased autoimmune susceptibility fetal period extremesof age pregnancy lactation or an active pathological entitysignificantly impair these mechanisms [1 35ndash37]

Our study demonstrated that both the urinary iodineconcentration and the iodide intake increased simultaneouslyfrom NI to 10 HI and further to 100 HI Interestingly wefound that the urinary iodine concentration of the 10 HIgroup was approximately 10 times that of the NI groupwhereas the urinary iodine concentration of the 100HI groupwas about 60ndash80 times that of the NI group on Days 7 14and 28 The urinary iodine concentration is regarded as asensitive indicator of iodine status because approximately90 of ingested iodide is excreted in the urine [38 39]Thereis an increase for the maintenance of thyroid homeostasisas well as the steady state of the internal environment ofthe body The thyroid gland has adaptation mechanisms thatreduce iodide metabolism when the supply is abundant thus

6 Oxidative Medicine and Cellular Longevity

1793 2066 4897

1773 2264 5291

1806 2373 7360

NI 10 HI 100 HID

ay 7

Day

14

Day

28

250

200

150

100

50

0

Cou

nt

0 102 103 104 105

Comp-PE-A PE-A

250

200

150

100

50

0

Cou

nt

0 102 103 104 105

Comp-PE-A PE-A

250

200

150

100

50

0

Cou

nt

0 102 103 104 105

Comp-PE-A PE-A

250

200

150

100

50

0

Cou

nt

0 102 103 104 105

Comp-PE-A PE-A

250

200

150

100

50

0

Cou

nt

0 102 103 104 105

Comp-PE-A PE-A

250

200

150

100

50

0

Cou

nt

0 102 103 104 105

Comp-PE-A PE-A

250

200

150

100

50

0

Cou

nt

0 102 103 104 105

Comp-PE-A PE-A

250

200

150

100

50

0

Cou

nt

0 102 103 104 105

Comp-PE-A PE-A

250

200

150

100

50

0

Cou

nt

0 102 103 104 105

Comp-PE-A PE-A

(a)

0

10

20

30

40

50

60

70

80

NI 10 HI 100 HI

Day 7Day 14Day 28

Mito

SOX

fluor

esce

nce i

nten

sity

lowastlowast

lowast998771998779

(b)

Figure 2 Continued

Oxidative Medicine and Cellular Longevity 7

NI 10 HI 100 HI

Day 28

20120583m20120583m20120583m

(c)

Figure 2 Effects of the iodide intake (NI 10 HI and 100 HI) on the changes of mitochondrial superoxide production on Days 7 14 and28 (a) There was a significant increase in the mitochondrial superoxide production in the 100 HI group on Days 7 14 and 28 (119901 lt 005)The difference was more significant on Day 28 when compared to Day 7 or Day 14 (b) Histogram analysis was performed on the meanfluorescence intensity of MitoSOX Red All data is presented as the mean plusmn SD (119873 = 6 for each group) Statistical analyses were performedby one-way analysis of variance (ANOVA) with the Least Significant Difference (LSD) test lowast119901 lt 005 versus the NI group on Days 7 14 and28 respectively 119901 lt 005 versus the 10 HI group on Days 7 14 and 28 respectively 998771119901 lt 005 versus the Day 7 group in the 100 HI group119901 lt 005 versus the Day 14 in the 100 HI group Experiments were repeated 3 times with similar results (c) There was a significant increasein the mitochondrial superoxide production in the 100 HI group on Day 28 and was observed by confocal microscopy Scale bar 20120583m

avoiding thyrotoxicosisThere are several mechanisms whichinclude a direct inhibitory effect of iodide in the thyroid itselfand inhibition by iodide of its own organification (Wolff-Chaikoff effect) its transport thyroid hormones secretioncAMP formation in response to TSH and several othermetabolic steps [40] We suggest that all the protectivemechanisms may ensure the excessive iodide intake of the 10HI group on Days 7 14 and 28 be eliminated from urineTheurinary iodine concentration of the 100 HI group on Days7 14 and 28 was about 60ndash80 times that of the NI groupalthough the thyroid function was normal This leads us topropose the idea that excessive iodide accumulated in thebody may trigger the oxidative and antioxidative signalingpathway to maintain the normal thyroid function

We demonstrated that the production of mitochondrialsuperoxide significantly increased on Days 7 14 and 28 inthe 100 HI group This is consistent with our previous studyon metallothionein-III knockout mice [24] Joanta et alreported that the initiation of free radical production wasobserved after giving a high dose of iodide [3] Serrano-Nascimento et al demonstrated that an increased mitochon-drial superoxide production was shown in response to NaI(10minus6M to 10minus3M) treatment in PCCl3 thyroid cells by usingMitoSOX Red [41] Mitochondria are potent producers ofsuperoxide from complexes I and III of the electron transportchain Mitochondrial superoxide production is a major causeof cellular oxidative damage [42] Physiologically ROS arenot necessarily harmful because they are continuously bal-anced by the process of hormone synthesis and the endoge-nous antioxidant system [1] Excess ROS are generated duringthe trapping oxidation and organification of excessive iodinein thyrocytes which could lead to increased oxidative stress[1]

The Nrf2-Keap 1 pathway is the chief cytoprotectivemechanism in response to oxidative stress caused by ROSDuring normal and balanced redox homeostasis the Nrf2function is inhibited because of constant proteasomal degra-dation after ubiquitination of the protein This is regulated

through the binding of the inhibitor protein Keap 1 [13 14]It is reported that Srx activation via Nrf2 dependent pathwayprotects fromoxidative liver injury throughPyrazole [43] andalcohol in mice [44] Similar findings in lung tissues haveshown that there is a marked increase in the expressions ofSrx and Prx 3 in human squamous cell carcinoma [45] Thissuggests that these proteins may play a protective role againstoxidative injury Also the pathway including Keap 1 Nrf2and ARE-mediated protein expression plays a very criticalrole in protecting cells fromoxidative stress [46 47] Focusingon the pathway we demonstrated that the expression of Nrf2was significantly increased while Keap 1 was significantlydecreased in the 100 HI group when compared to the NIor 10 HI group on Days 7 14 and 28 Similarly Ajiboyeet al showed that rats treated with Chalcone dimers notonly increased the expression of Nrf2 but also suppressedcytoplasmic Keap 1 expression [48] Yang et al also showedthat the downregulation of Keap 1 level may be responsiblefor the overactivation of Nrf2 [49] Pang et al showed thatcaffeic acid prevents acetaminophen-induced liver injury byactivating the Nrf2-Keap 1 antioxidative defense system [50]

In order to verify whether high levels of expressionof Nrf2 and its nuclear translocation can upregulate someantioxidant enzymes in the thyroid gland the expressions ofPrx 3 and Srx following the intake of NI 10 HI and 100 HIon Days 7 14 and 28 were measured We demonstrated thatthe expressions of Srx and Prx 3 in the 100 HI group weresignificantly increased when compared to the NI group or 10HI group on Days 7 14 and 28 The possible explanationsare described as below Firstly Srx is a cytosolic proteinthat is able to translocate to sites where hyperoxidized(inactivated) Prx 3 is located Therefore it engages itself inthe reactivation of Prx 3 under oxidative conditions [51]Secondly Prx 3 is a typical 2-Cys Peroxiredoxin locatedexclusively in the mitochondrial matrix it is the principalperoxidase responsible for protecting cells from oxidativedamage by reducing peroxides such as H2O2 [52] Finallymitochondria contain 30 times more Prx 3 than glutathione

8 Oxidative Medicine and Cellular Longevity

000

020

040

060

080

100

120

140

NI 10 HI 100 HI

Expr

essio

n of

Nrf2

(Nrf2

120573-a

ctin

)

120573-Actin

Nrf2

Day 7 Day 14 Day 28

lowast lowast

lowast

Day 7Day 14

Day 28

NI 10 HI 100 HI NI 10 HI 100 HI NI 10 HI 100 HI

(a)

000

020

040

060

080

100

120

NI 10 HI 100 HI

lowast

lowast

Expr

essio

n of

Kea

p 1

(Kea

p 1120573

-act

in)

120573-Actin

Keap 1

Day 7 Day 14 Day 28

lowast

Day 7Day 14

Day 28

NI 10 HI 100 HI NI 10 HI 100 HI NI 10 HI 100 HI

(b)

120573-Actin

SrxNI 10 HI 100 HI NI 10 HI 100 HI NI 10 HI 100 HI

000

010

020

030

040

050

060

NI 10 HI 100 HI

Expr

essio

n of

Srx

(Srx

120573-a

ctin

)

Day 7 Day 14 Day 28

lowast

lowast

lowast

Day 7Day 14

Day 28

(c)

120573-Actin

Prx 3

lowast

000

020

040

060

080

100

120

140

NI 10 HI 100 HI

Expr

essio

n of

Prx

3 (P

rx 3

120573-a

ctin

)

Day 7 Day 14 Day 28

lowastlowast

Day 7Day 14

Day 28

NI 10 HI 100 HI NI 10 HI 100 HI NI 10 HI 100 HI

(d)

Figure 3 Effects of the iodide intake (NI 10 HI and 100 HI) on the changes of (a) Nrf2 (b) Keap 1 (c) Srx and (d) Prx 3 expressions onDays 7 14 and 28 Representative western blot and histograms of densitometric analyses normalized for the relative 120573-actin content All datais presented as the mean plusmn SD (119873 = 6 for each group) Statistical analyses were performed by one-way analysis of variance (ANOVA) withthe Least Significant Difference (LSD) test lowast119901 lt 005 versus the NI group on Days 7 14 and 28 respectively 119901 lt 005 versus the 10 HI groupon Days 7 14 and 28 respectively Experiments were repeated 3 times with similar results

Oxidative Medicine and Cellular Longevity 9

NI

10 HI

100 HI

Hoechst Nrf2 Merge HoechstHoechst

Day 7 Day 28 Day 14

Nrf2 Merge Nrf2 Merge

Day 7 Day 7 Day 14 Day 14 Day 28 Day 28 20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

(a)

Hoechst Srx Merge Hoechst Srx Merge Hoechst Srx Merge

NI

10 HI

100 HI

Day 7 Day 28 Day 14 Day 7 Day 7 Day 14 Day 14 Day 28 Day 28 20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

(b)

Figure 4 Effect of the iodide intake (NI 10 HI and 100 HI) on the changes of immunofluorescence staining on Days 7 14 and 28 (a) In theNI group Nrf2 (red) was localized in the cytoplasm on Days 7 14 and 28 In the 10 HI group the positive staining of Nrf2 can be observedin both the nucleus and the cytoplasm Moreover in the 100 HI group a stronger positive staining of Nrf2 can be detected in the nucleus onDays 7 14 and 28 the nucleus was dyed with Hoechst (blue) (b) Srx (red) positive staining was located in the cytoplasm the nucleus wasdyed with Hoechst (blue) Scale bar 20120583m

peroxidase Prx 3 can be classified as an important regulatorof mitochondrial H2O2 [22] The elevated expression of Prx3 is associated with the blockage of apoptosis increasingcell proliferation and is related to adaptive responses whichare all required to maintain mitochondrial function [53 54]Bae et al have suggested that Prx 3 and Srx jointly protectmice from Pyrazole-induced oxidative liver injury in a Nrf2-dependent manner [43]

The novelty we demonstrated in the present study is thatiodide excess induced both oxidative stress and antioxidativedefense increases throughNrf2-Keap 1 pathway in the thyroidgland from rats We extended our established mechanismsby applying the Nrf2-Keap 1 pathway to set up a bridgebetween oxidative stress and antioxidative defense inducedby iodide excess in the thyroid gland In our previous studywe have established that oxidative stress induced by acutehigh concentrations of iodide in FRTL cells significantlyincreases mitochondrial superoxide production [55] Theinhibitors of themitochondrial respiratory chain complexes I

and III are involved inmitochondrial superoxide productionWe demonstrated that exposure to 100120583M KI for 2 hourssignificantly increased mitochondrial superoxide produc-tion enhanced by either 05120583M Rotenone (an inhibitorof mitochondrial complex I) or 10 120583M Antimycin A (aninhibitor of complex III) [56] We illustrated that 300 120583MPTU (an inhibitor of TPO) attenuated the excessive iodide-induced mitochondrial superoxide production We showedthat 30 120583M KClO4 (a competitive inhibitor of iodide trans-port) relieved the production the mitochondrial superoxideinduced by iodide excess We displayed that 10mUmL TSHcan inhibit excessive iodide-induced strong mitochondrialsuperoxide production [55] MT-I and MT-II are mainlyinvolved in the protection of tissue against oxidative stresseswe indicated that metallothionein-III knockout mice aggra-vated mitochondrial superoxide production in thyroid afterexcessive iodide exposure [24] In addition we demonstratedthat both the oxidative stress and the antioxidative defenseincreased simultaneously after high dosages of iodide intake

10 Oxidative Medicine and Cellular Longevity

Oxidative stress

(a)

(b)

(c)

Antioxidative defense

Wist

ar ra

ts Day 7Day 14Day 28

Normal iodideintake

Normal urinaryiodine

Normal thyroidfunction

10 times iodideintake

10 times urinaryiodine

Normal thyroidfunction

100 times iodideintake

60ndash80 timesurinary iodine

Normal thyroidfunction

Keap 1 Nrf2

Keap 1

Nrf2

Srx

Nucleus

Prx 3

SrxARE

Mitochondria

AntioxidativeOxidativestress

Oxidative Antioxidative

Oxidative Antioxidative

Under physiological conditions(normal iodide intake)

Excessive iodide intake(10 times iodide intake)

Excessive iodide intake(100 times iodide intake)

defense defenseuarrstressuarr

defenseuarruarrstressuarruarr

Figure 5 Proposed mechanisms in the present study (a) The urinary iodine concentration and thyroid function of Wistar rats weredetected (b) The activation of the Nrf2-Keap 1 pathway induced by iodide excess in the thyroid (c) The balance between oxidative stressand antioxidative defense under physiological conditions and excessive iodide intake

Oxidative Medicine and Cellular Longevity 11

We suggested that the Nrf2-Keap 1 pathway is vital for thebalance between oxidative stress and antioxidative defenseinduced by iodide excess in the thyroid gland

Excessive iodide stimulated the Nrf2-Keap 1 pathwayand enhanced the antioxidative defense We found thatthe urinary iodine concentration of the 100 HI group wasabout 60ndash80 times that of the NI group on Days 7 14and 28 however the thyroid functions were normal Weproposed that the excessive iodide accumulated in the bodymay trigger the Nrf2-Keap 1 pathway to maintain a normalthyroid functionWe demonstrated that the Nrf2moves fromthe cytoplasm to the nucleus under the microscope withsignificantly increased expressions of Nrf2 Srx and Prx 3 andnotably decreased Keap 1 when exposed to high iodide Thissuggests that excessive iodide stimulates the disassociation ofNrf2 from Keap 1 and assists Nrf2 to penetrate the nucleusThen Nrf2 attaches to the antioxidant response element(ARE) to activate the expression of the antioxidative genesSrx and Prx 3 resulting in an enhanced antioxidative defenseinduced by high iodide By activation of the Nrf2-Keap 1pathway there is a proportional increase in oxidative stressand antioxidative defense in response to iodide excess It isto be noted that the thyroid function was normal in the 10HI group and 100 HI in present study Inspired by the reportby Poncin et al [6] we proposed that there should be abalance between oxidative stress and antioxidative defense inresponse to iodide excess in the 10 HI and the 100 HI groups

5 Conclusion

In conclusion our results highlight that the activation ofNrf2-Keap 1 Srx and Prx 3 antioxidative defense mecha-nisms may play a crucial role in protecting the thyroid fromiodide excess induced oxidative stress on Days 7 14 and 28(Figure 5)

Abbreviations

NI Normal iodide intakeHI High iodide intakeROS Reactive oxygen speciesH2O2 Hydrogen peroxideNrf2 Nuclear factor erythroid 2-related factor 2Keap 1 Kelch-like ECH-associated protein 1ARE Antioxidant response elementPrx PeroxiredoxinSrx SulfiredoxinPrx 3 Peroxiredoxin 3TT4 Total thyroxineTT3 Total triiodothyronineFT4 Free thyroxineFT3 Free triiodothyronineTSH ThyrotropinLSD Least significant difference

Disclosure

This manuscript has been edited by Umar Iqbal IruniRoshanie Abeysekera Gargi Naha and Tejas Bharadwaj all

of whom are currently studying at the International School ofMedicine Tianjin Medical University China

Competing Interests

The authors declare that there is no conflict of interestsregarding the publication of this article

Acknowledgments

This work is supported by National Natural Science Founda-tion of China (nos 81273009 and 81270182) Tianjin Scienceamp Technology Council Grant of China (nos 16JCYBJC26100and 09JCYBJC11700) and International Studentrsquos Science ampTechnology Innovation Project (Scientific Research Project)of Tianjin Medical University

References

[1] Y Luo A Kawashima Y Ishido et al ldquoIodine excess as anenvironmental risk factor for autoimmune thyroid diseaserdquoInternational Journal of Molecular Sciences vol 15 no 7 pp12895ndash12912 2014

[2] A E Hussein A M Abbas G A El Wakil A Z Elsamanoudyand A A El Aziz ldquoEffect of chronic excess iodine intakeon thyroid function and oxidative stress in hypothyroid ratsrdquoCanadian Journal of Physiology and Pharmacology vol 90 no5 pp 617ndash625 2012

[3] A E Joanta A Filip S Clichici S Andrei and D DaicoviciuldquoIodide excess exerts oxidative stress in some target tissues ofthe thyroid hormonesrdquoActa Physiologica Hungarica vol 93 no4 pp 347ndash359 2006

[4] SMorandMChaaraoui J Kaniewski et al ldquoEffect of iodide onnicotinamide adenine dinucleotide phosphate oxidase activityand Duox2 protein expression in isolated porcine thyroidfolliclesrdquo Endocrinology vol 144 no 4 pp 1241ndash1248 2003

[5] J J Lazaro A Jimenez D Camejo et al ldquoDissecting the inte-grative antioxidant and redox systems in plant mitochondriaEffect of stress and S-nitrosylationrdquo Frontiers in Plant Sciencevol 4 article 460 2013

[6] S Poncin A-C GerardM Boucquey et al ldquoOxidative stress inthe thyroid gland from harmlessness to hazard depending onthe iodine contentrdquo Endocrinology vol 149 no 1 pp 424ndash4332008

[7] M Karbownik and A Lewinski ldquoThe role of oxidative stressin physiological and pathological processes in the thyroidgland possible involvement in pineal-thyroid interactionsrdquoNeuroendocrinology Letters vol 24 no 5 pp 293ndash303 2003

[8] P Thomopoulos ldquoHyperthyroidism due to excess iodinerdquo LaPresse Medicale vol 31 no 35 pp 1664ndash1669 2002

[9] J Wolff ldquoIodide goiter and the pharmacologic effects of excessiodiderdquoTheAmerican Journal of Medicine vol 47 no 1 pp 101ndash124 1969

[10] B Corvilain L Collyn J Van Sande and J E Dumont ldquoStim-ulation by iodide of H2O2 generation in thyroid slices fromseveral speciesrdquoAmerican Journal of PhysiologymdashEndocrinologyand Metabolism vol 278 no 4 pp E692ndashE699 2000

[11] M-C Many C Mestdagh M-F Van den Hove and J-F DenefldquoIn vitro study of acute toxic effects of high iodide doses inhuman thyroid folliclesrdquo Endocrinology vol 131 no 2 pp 621ndash630 1992

12 Oxidative Medicine and Cellular Longevity

[12] J Van Sande G Grenier C Willems and J E DumontldquoInhibition by iodide of the activation of the thyroid cyclic 3101584051015840-AMP systemrdquo Endocrinology vol 96 no 3 pp 781ndash786 1975

[13] R Young J D Hayes K Brown C R Wolf and C B AWhitelaw ldquoPeroxiredoxin gene expression signatures in liverreflect toxic insultrdquo Assay and Drug Development Technologiesvol 8 no 4 pp 512ndash517 2010

[14] Q Ma ldquoRole of Nrf2 in oxidative stress and toxicityrdquo AnnualReview of Pharmacology and Toxicology vol 53 pp 401ndash4262013

[15] B Biteau J Labarre and M B Toledano ldquoATP-dependentreduction of cysteine-sulphinic acid by S cerevisiae sulphire-doxinrdquo Nature vol 425 no 6961 pp 980ndash984 2003

[16] L Wu H Jiang H A Chawsheen et al ldquoTumor promoter-induced sulfiredoxin is required for mouse skin tumorigenesisrdquoCarcinogenesis vol 35 no 5 pp 1177ndash1184 2014

[17] C C Winterbourn and M B Hampton ldquoThiol chemistryand specificity in redox signalingrdquo Free Radical Biology andMedicine vol 45 no 5 pp 549ndash561 2008

[18] J W Baty M B Hampton and C C Winterbourn ldquoProteomicdetection of hydrogen peroxide-sensitive thiol proteins inJurkat cellsrdquo Biochemical Journal vol 389 no 3 pp 785ndash7952005

[19] T-S Chang C-S Cho S Park S YuW K Sang and G R SueldquoPeroxiredoxin III a mitochondrion-specific peroxidase regu-lates apoptotic signaling by mitochondriardquo Journal of BiologicalChemistry vol 279 no 40 pp 41975ndash41984 2004

[20] P Thomopoulos ldquoHyperthyroidism due to excess iodinerdquo LaPresse Medicale vol 31 no 35 pp 1664ndash1669 2002

[21] S Chung X-H Liao C Di Cosmo et al ldquoDisruption of themelanin-concentrating hormone receptor 1 (MCH1R) affectsthyroid functionrdquo Endocrinology vol 153 no 12 pp 6145ndash61542012

[22] G Ozguner and O Sulak ldquoSize and location of thyroid gland inthe fetal periodrdquo Surgical and Radiologic Anatomy vol 36 no4 pp 359ndash367 2014

[23] P L Jooste and E Strydom ldquoMethods for determination ofiodine in urine and saltrdquo Best Practice amp Research ClinicalEndocrinology amp Metabolism vol 24 no 1 pp 77ndash88 2010

[24] N Zhang L Wang Q Duan et al ldquoMetallothionein-IIIknockoutmice aggravatemitochondrial superoxide productionand peroxiredoxin 3 expression in thyroid after excessive iodideexposurerdquoOxidative Medicine and Cellular Longevity vol 2015Article ID 267027 11 pages 2015

[25] P I Gus D Marinho S Zelanis et al ldquoA case-control studyon the oxidative balance of 50 autologous serum eye dropsrdquoOxidative Medicine and Cellular Longevity vol 2016 Article ID9780193 5 pages 2016

[26] P Laurberg C Cerqueira L Ovesen et al ldquoIodine intake as adeterminant of thyroid disorders in populationsrdquo Best Practiceamp Research Clinical Endocrinology amp Metabolism vol 24 no 1pp 13ndash27 2010

[27] H Burgi ldquoIodine excessrdquo Best Practice amp Research ClinicalEndocrinology amp Metabolism vol 24 no 1 pp 107ndash115 2010

[28] E Roti and E D Uberti ldquoIodine excess and hyperthyroidismrdquoThyroid vol 11 no 5 pp 493ndash500 2001

[29] A M Leung and L E Braverman ldquoConsequences of excessiodinerdquo Nature Reviews Endocrinology vol 10 no 3 pp 136ndash142 2014

[30] K Wang Y N Sun J Y Liu et al ldquoThe impact of iodineexcess on thyroid hormone biosynthesis and metabolism in

ratsrdquoBiological Trace Element Research vol 130 no 1 pp 72ndash852009

[31] M Michalkiewicz L J Huffman J M Connors and G AHedge ldquoAlterations in thyroid blood flow induced by varyinglevels of iodine intake in the ratrdquo Endocrinology vol 125 no 1pp 54ndash60 1989

[32] P H K Eng G R Cardona S-L Fang et al ldquoEscape fromthe acute Wolff-Chaikoff effect is associated with a decrease inthyroid sodiumiodide symporter messenger ribonucleic acidand proteinrdquo Endocrinology vol 140 no 8 pp 3404ndash3410 1999

[33] P Mooij H J De Wit and H A Drexhage ldquoA high iodineintake in Wistar rats results in the development of a thyroid-associated ectopic thymic tissue and is accompanied by a lowthyroid autoimmune reactivityrdquo Immunology vol 81 no 2 pp309ndash316 1994

[34] T Paul B Meyers R J Witorsch et al ldquoThe effect of smallincreases in dietary iodine on thyroid function in euthyroidsubjectsrdquoMetabolism vol 37 no 2 pp 121ndash124 1988

[35] X Teng Z Shan W Teng C Fan H Wang and R GuoldquoExperimental study on the effects of chronic iodine excess onthyroid function structure and autoimmunity in autoimmune-prone NODH-2h4 micerdquo Clinical and Experimental Medicinevol 9 no 1 pp 51ndash59 2009

[36] E Swist Q Chen C Qiao D Caldwell H Gruber and KA Scoggan ldquoExcess dietary iodine differentially affects thyroidgene expression in diabetes thyroiditis-prone versusmdashresistantBioBreeding (BB) ratsrdquoMolecular Nutrition and Food Researchvol 55 no 12 pp 1875ndash1886 2011

[37] L Liu D Wang P Liu et al ldquoThe relationship between iodinenutrition and thyroid disease in lactating women with differentiodine intakesrdquo British Journal of Nutrition vol 114 no 9 pp1487ndash1495 2015

[38] S A Skeaff C D Thomson and R S Gibson ldquoMild iodinedeficiency in a sample of New Zealand school childrenrdquoEuropean Journal of Clinical Nutrition vol 56 no 12 pp 1169ndash1175 2002

[39] M B Zimmermann P L Jooste and C S Pandav ldquoIodine-deficiency disordersrdquo The Lancet vol 372 no 9645 pp 1251ndash1262 2008

[40] J Wolff ldquoExcess iodide inhibits the thyroid by multiple mecha-nismsrdquoAdvances in ExperimentalMedicine andBiology vol 261pp 211ndash244 1989

[41] C Serrano-Nascimento S D S Teixeira J P Nicola R TNachbar A M Masini-Repiso and M T Nunes ldquoThe acuteinhibitory effect of iodide excess on sodiumiodide symporterexpression and activity involves the PI3KAkt signaling path-wayrdquo Endocrinology vol 155 no 3 pp 1145ndash1156 2014

[42] M D Brand C Affourtit T C Esteves et al ldquoMitochondrialsuperoxide production biological effects and activation ofuncoupling proteinsrdquo Free Radical Biology andMedicine vol 37no 6 pp 755ndash767 2004

[43] S H Bae S H Sung H E Lee et al ldquoPeroxiredoxin IIIand sulfiredoxin together protect mice from pyrazole-inducedoxidative liver injuryrdquo Antioxidants amp Redox Signaling vol 17no 10 pp 1351ndash1361 2012

[44] S H Bae S H Sung E J Cho et al ldquoConcerted actionof sulfiredoxin and peroxiredoxin I protects against alcohol-induced oxidative injury inmouse liverrdquoHepatology vol 53 no3 pp 945ndash953 2011

[45] Y S Kim H L Lee K B Lee et al ldquoNuclear factor E2-related factor 2 dependent overexpression of sulfiredoxin and

Oxidative Medicine and Cellular Longevity 13

peroxiredoxin III in human lung cancerrdquo Korean Journal ofInternal Medicine vol 26 no 3 pp 304ndash313 2011

[46] H-Y Cho A E Jedlicka S P M Reddy et al ldquoRole of NRF2in protection against hyperoxic lung injury in micerdquo AmericanJournal of Respiratory Cell and Molecular Biology vol 26 no 2pp 175ndash182 2002

[47] T W Kensler N Wakabayashi and S Biswal ldquoCell survivalresponses to environmental stresses via the Keap1-Nrf2-AREpathwayrdquo Annual Review of Pharmacology and Toxicology vol47 pp 89ndash116 2007

[48] TO AjiboyeM T Yakubu andA TOladiji ldquoElectrophilic andReactive Oxygen Species Detoxification Potentials of ChalconeDimers is Mediated by Redox Transcription Factor Nrf-2rdquoJournal of Biochemical and Molecular Toxicology vol 28 no 1pp 11ndash22 2014

[49] X Yang D Wang Y Ma et al ldquoContinuous activation of Nrf2and its target antioxidant enzymes leads to arsenite-inducedmalignant transformation of human bronchial epithelial cellsrdquoToxicology and Applied Pharmacology vol 289 no 2 pp 231ndash239 2015

[50] C Pang Z Zheng L Shi et al ldquoCaffeic acid preventsacetaminophen-induced liver injury by activating the Keap1-Nrf2 antioxidative defense systemrdquo Free Radical Biology andMedicine vol 91 pp 236ndash246 2016

[51] Y H Noh J Y Baek W Jeong S G Rhee and T-S ChangldquoSulfiredoxin translocation into mitochondria plays a crucialrole in reducing hyperoxidized peroxiredoxin IIIrdquo The Journalof Biological Chemistry vol 284 no 13 pp 8470ndash8477 2009

[52] B Cunniff A N Wozniak P Sweeney K DeCosta and N HHeintz ldquoPeroxiredoxin 3 levels regulate a mitochondrial redoxsetpoint in malignant mesothelioma cellsrdquo Redox Biology vol3 pp 79ndash87 2014

[53] P-J Chua E-H Lee Y Yu G W-C Yip P-H Tan and B-HBay ldquoSilencing the Peroxiredoxin III gene inhibits cell prolifera-tion in breast cancerrdquo International Journal of Oncology vol 36no 2 pp 359ndash364 2010

[54] B Cunniff K Newick K J Nelson et al ldquoDisabling mito-chondrial peroxide metabolism via combinatorial targetingof peroxiredoxin 3 as an effective therapeutic approach formalignant mesotheliomardquo PLOS ONE vol 10 no 5 Article IDe0127310 2015

[55] X Yao M Li J He et al ldquoEffect of early acute high con-centrations of iodide exposure on mitochondrial superoxideproduction in FRTL cellsrdquo Free Radical Biology and Medicinevol 52 no 8 pp 1343ndash1352 2012

[56] L Wang Q Duan T Wang et al ldquoMitochondrial respiratorychain inhibitors involved in ROS production induced by acutehigh concentrations of iodide and the effects of sod as aprotective factorrdquo Oxidative Medicine and Cellular Longevityvol 2015 Article ID 217670 14 pages 2015

Submit your manuscripts athttpswwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

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Diabetes ResearchJournal of

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Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

6 Oxidative Medicine and Cellular Longevity

1793 2066 4897

1773 2264 5291

1806 2373 7360

NI 10 HI 100 HID

ay 7

Day

14

Day

28

250

200

150

100

50

0

Cou

nt

0 102 103 104 105

Comp-PE-A PE-A

250

200

150

100

50

0

Cou

nt

0 102 103 104 105

Comp-PE-A PE-A

250

200

150

100

50

0

Cou

nt

0 102 103 104 105

Comp-PE-A PE-A

250

200

150

100

50

0

Cou

nt

0 102 103 104 105

Comp-PE-A PE-A

250

200

150

100

50

0

Cou

nt

0 102 103 104 105

Comp-PE-A PE-A

250

200

150

100

50

0

Cou

nt

0 102 103 104 105

Comp-PE-A PE-A

250

200

150

100

50

0

Cou

nt

0 102 103 104 105

Comp-PE-A PE-A

250

200

150

100

50

0

Cou

nt

0 102 103 104 105

Comp-PE-A PE-A

250

200

150

100

50

0

Cou

nt

0 102 103 104 105

Comp-PE-A PE-A

(a)

0

10

20

30

40

50

60

70

80

NI 10 HI 100 HI

Day 7Day 14Day 28

Mito

SOX

fluor

esce

nce i

nten

sity

lowastlowast

lowast998771998779

(b)

Figure 2 Continued

Oxidative Medicine and Cellular Longevity 7

NI 10 HI 100 HI

Day 28

20120583m20120583m20120583m

(c)

Figure 2 Effects of the iodide intake (NI 10 HI and 100 HI) on the changes of mitochondrial superoxide production on Days 7 14 and28 (a) There was a significant increase in the mitochondrial superoxide production in the 100 HI group on Days 7 14 and 28 (119901 lt 005)The difference was more significant on Day 28 when compared to Day 7 or Day 14 (b) Histogram analysis was performed on the meanfluorescence intensity of MitoSOX Red All data is presented as the mean plusmn SD (119873 = 6 for each group) Statistical analyses were performedby one-way analysis of variance (ANOVA) with the Least Significant Difference (LSD) test lowast119901 lt 005 versus the NI group on Days 7 14 and28 respectively 119901 lt 005 versus the 10 HI group on Days 7 14 and 28 respectively 998771119901 lt 005 versus the Day 7 group in the 100 HI group119901 lt 005 versus the Day 14 in the 100 HI group Experiments were repeated 3 times with similar results (c) There was a significant increasein the mitochondrial superoxide production in the 100 HI group on Day 28 and was observed by confocal microscopy Scale bar 20120583m

avoiding thyrotoxicosisThere are several mechanisms whichinclude a direct inhibitory effect of iodide in the thyroid itselfand inhibition by iodide of its own organification (Wolff-Chaikoff effect) its transport thyroid hormones secretioncAMP formation in response to TSH and several othermetabolic steps [40] We suggest that all the protectivemechanisms may ensure the excessive iodide intake of the 10HI group on Days 7 14 and 28 be eliminated from urineTheurinary iodine concentration of the 100 HI group on Days7 14 and 28 was about 60ndash80 times that of the NI groupalthough the thyroid function was normal This leads us topropose the idea that excessive iodide accumulated in thebody may trigger the oxidative and antioxidative signalingpathway to maintain the normal thyroid function

We demonstrated that the production of mitochondrialsuperoxide significantly increased on Days 7 14 and 28 inthe 100 HI group This is consistent with our previous studyon metallothionein-III knockout mice [24] Joanta et alreported that the initiation of free radical production wasobserved after giving a high dose of iodide [3] Serrano-Nascimento et al demonstrated that an increased mitochon-drial superoxide production was shown in response to NaI(10minus6M to 10minus3M) treatment in PCCl3 thyroid cells by usingMitoSOX Red [41] Mitochondria are potent producers ofsuperoxide from complexes I and III of the electron transportchain Mitochondrial superoxide production is a major causeof cellular oxidative damage [42] Physiologically ROS arenot necessarily harmful because they are continuously bal-anced by the process of hormone synthesis and the endoge-nous antioxidant system [1] Excess ROS are generated duringthe trapping oxidation and organification of excessive iodinein thyrocytes which could lead to increased oxidative stress[1]

The Nrf2-Keap 1 pathway is the chief cytoprotectivemechanism in response to oxidative stress caused by ROSDuring normal and balanced redox homeostasis the Nrf2function is inhibited because of constant proteasomal degra-dation after ubiquitination of the protein This is regulated

through the binding of the inhibitor protein Keap 1 [13 14]It is reported that Srx activation via Nrf2 dependent pathwayprotects fromoxidative liver injury throughPyrazole [43] andalcohol in mice [44] Similar findings in lung tissues haveshown that there is a marked increase in the expressions ofSrx and Prx 3 in human squamous cell carcinoma [45] Thissuggests that these proteins may play a protective role againstoxidative injury Also the pathway including Keap 1 Nrf2and ARE-mediated protein expression plays a very criticalrole in protecting cells fromoxidative stress [46 47] Focusingon the pathway we demonstrated that the expression of Nrf2was significantly increased while Keap 1 was significantlydecreased in the 100 HI group when compared to the NIor 10 HI group on Days 7 14 and 28 Similarly Ajiboyeet al showed that rats treated with Chalcone dimers notonly increased the expression of Nrf2 but also suppressedcytoplasmic Keap 1 expression [48] Yang et al also showedthat the downregulation of Keap 1 level may be responsiblefor the overactivation of Nrf2 [49] Pang et al showed thatcaffeic acid prevents acetaminophen-induced liver injury byactivating the Nrf2-Keap 1 antioxidative defense system [50]

In order to verify whether high levels of expressionof Nrf2 and its nuclear translocation can upregulate someantioxidant enzymes in the thyroid gland the expressions ofPrx 3 and Srx following the intake of NI 10 HI and 100 HIon Days 7 14 and 28 were measured We demonstrated thatthe expressions of Srx and Prx 3 in the 100 HI group weresignificantly increased when compared to the NI group or 10HI group on Days 7 14 and 28 The possible explanationsare described as below Firstly Srx is a cytosolic proteinthat is able to translocate to sites where hyperoxidized(inactivated) Prx 3 is located Therefore it engages itself inthe reactivation of Prx 3 under oxidative conditions [51]Secondly Prx 3 is a typical 2-Cys Peroxiredoxin locatedexclusively in the mitochondrial matrix it is the principalperoxidase responsible for protecting cells from oxidativedamage by reducing peroxides such as H2O2 [52] Finallymitochondria contain 30 times more Prx 3 than glutathione

8 Oxidative Medicine and Cellular Longevity

000

020

040

060

080

100

120

140

NI 10 HI 100 HI

Expr

essio

n of

Nrf2

(Nrf2

120573-a

ctin

)

120573-Actin

Nrf2

Day 7 Day 14 Day 28

lowast lowast

lowast

Day 7Day 14

Day 28

NI 10 HI 100 HI NI 10 HI 100 HI NI 10 HI 100 HI

(a)

000

020

040

060

080

100

120

NI 10 HI 100 HI

lowast

lowast

Expr

essio

n of

Kea

p 1

(Kea

p 1120573

-act

in)

120573-Actin

Keap 1

Day 7 Day 14 Day 28

lowast

Day 7Day 14

Day 28

NI 10 HI 100 HI NI 10 HI 100 HI NI 10 HI 100 HI

(b)

120573-Actin

SrxNI 10 HI 100 HI NI 10 HI 100 HI NI 10 HI 100 HI

000

010

020

030

040

050

060

NI 10 HI 100 HI

Expr

essio

n of

Srx

(Srx

120573-a

ctin

)

Day 7 Day 14 Day 28

lowast

lowast

lowast

Day 7Day 14

Day 28

(c)

120573-Actin

Prx 3

lowast

000

020

040

060

080

100

120

140

NI 10 HI 100 HI

Expr

essio

n of

Prx

3 (P

rx 3

120573-a

ctin

)

Day 7 Day 14 Day 28

lowastlowast

Day 7Day 14

Day 28

NI 10 HI 100 HI NI 10 HI 100 HI NI 10 HI 100 HI

(d)

Figure 3 Effects of the iodide intake (NI 10 HI and 100 HI) on the changes of (a) Nrf2 (b) Keap 1 (c) Srx and (d) Prx 3 expressions onDays 7 14 and 28 Representative western blot and histograms of densitometric analyses normalized for the relative 120573-actin content All datais presented as the mean plusmn SD (119873 = 6 for each group) Statistical analyses were performed by one-way analysis of variance (ANOVA) withthe Least Significant Difference (LSD) test lowast119901 lt 005 versus the NI group on Days 7 14 and 28 respectively 119901 lt 005 versus the 10 HI groupon Days 7 14 and 28 respectively Experiments were repeated 3 times with similar results

Oxidative Medicine and Cellular Longevity 9

NI

10 HI

100 HI

Hoechst Nrf2 Merge HoechstHoechst

Day 7 Day 28 Day 14

Nrf2 Merge Nrf2 Merge

Day 7 Day 7 Day 14 Day 14 Day 28 Day 28 20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

(a)

Hoechst Srx Merge Hoechst Srx Merge Hoechst Srx Merge

NI

10 HI

100 HI

Day 7 Day 28 Day 14 Day 7 Day 7 Day 14 Day 14 Day 28 Day 28 20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

(b)

Figure 4 Effect of the iodide intake (NI 10 HI and 100 HI) on the changes of immunofluorescence staining on Days 7 14 and 28 (a) In theNI group Nrf2 (red) was localized in the cytoplasm on Days 7 14 and 28 In the 10 HI group the positive staining of Nrf2 can be observedin both the nucleus and the cytoplasm Moreover in the 100 HI group a stronger positive staining of Nrf2 can be detected in the nucleus onDays 7 14 and 28 the nucleus was dyed with Hoechst (blue) (b) Srx (red) positive staining was located in the cytoplasm the nucleus wasdyed with Hoechst (blue) Scale bar 20120583m

peroxidase Prx 3 can be classified as an important regulatorof mitochondrial H2O2 [22] The elevated expression of Prx3 is associated with the blockage of apoptosis increasingcell proliferation and is related to adaptive responses whichare all required to maintain mitochondrial function [53 54]Bae et al have suggested that Prx 3 and Srx jointly protectmice from Pyrazole-induced oxidative liver injury in a Nrf2-dependent manner [43]

The novelty we demonstrated in the present study is thatiodide excess induced both oxidative stress and antioxidativedefense increases throughNrf2-Keap 1 pathway in the thyroidgland from rats We extended our established mechanismsby applying the Nrf2-Keap 1 pathway to set up a bridgebetween oxidative stress and antioxidative defense inducedby iodide excess in the thyroid gland In our previous studywe have established that oxidative stress induced by acutehigh concentrations of iodide in FRTL cells significantlyincreases mitochondrial superoxide production [55] Theinhibitors of themitochondrial respiratory chain complexes I

and III are involved inmitochondrial superoxide productionWe demonstrated that exposure to 100120583M KI for 2 hourssignificantly increased mitochondrial superoxide produc-tion enhanced by either 05120583M Rotenone (an inhibitorof mitochondrial complex I) or 10 120583M Antimycin A (aninhibitor of complex III) [56] We illustrated that 300 120583MPTU (an inhibitor of TPO) attenuated the excessive iodide-induced mitochondrial superoxide production We showedthat 30 120583M KClO4 (a competitive inhibitor of iodide trans-port) relieved the production the mitochondrial superoxideinduced by iodide excess We displayed that 10mUmL TSHcan inhibit excessive iodide-induced strong mitochondrialsuperoxide production [55] MT-I and MT-II are mainlyinvolved in the protection of tissue against oxidative stresseswe indicated that metallothionein-III knockout mice aggra-vated mitochondrial superoxide production in thyroid afterexcessive iodide exposure [24] In addition we demonstratedthat both the oxidative stress and the antioxidative defenseincreased simultaneously after high dosages of iodide intake

10 Oxidative Medicine and Cellular Longevity

Oxidative stress

(a)

(b)

(c)

Antioxidative defense

Wist

ar ra

ts Day 7Day 14Day 28

Normal iodideintake

Normal urinaryiodine

Normal thyroidfunction

10 times iodideintake

10 times urinaryiodine

Normal thyroidfunction

100 times iodideintake

60ndash80 timesurinary iodine

Normal thyroidfunction

Keap 1 Nrf2

Keap 1

Nrf2

Srx

Nucleus

Prx 3

SrxARE

Mitochondria

AntioxidativeOxidativestress

Oxidative Antioxidative

Oxidative Antioxidative

Under physiological conditions(normal iodide intake)

Excessive iodide intake(10 times iodide intake)

Excessive iodide intake(100 times iodide intake)

defense defenseuarrstressuarr

defenseuarruarrstressuarruarr

Figure 5 Proposed mechanisms in the present study (a) The urinary iodine concentration and thyroid function of Wistar rats weredetected (b) The activation of the Nrf2-Keap 1 pathway induced by iodide excess in the thyroid (c) The balance between oxidative stressand antioxidative defense under physiological conditions and excessive iodide intake

Oxidative Medicine and Cellular Longevity 11

We suggested that the Nrf2-Keap 1 pathway is vital for thebalance between oxidative stress and antioxidative defenseinduced by iodide excess in the thyroid gland

Excessive iodide stimulated the Nrf2-Keap 1 pathwayand enhanced the antioxidative defense We found thatthe urinary iodine concentration of the 100 HI group wasabout 60ndash80 times that of the NI group on Days 7 14and 28 however the thyroid functions were normal Weproposed that the excessive iodide accumulated in the bodymay trigger the Nrf2-Keap 1 pathway to maintain a normalthyroid functionWe demonstrated that the Nrf2moves fromthe cytoplasm to the nucleus under the microscope withsignificantly increased expressions of Nrf2 Srx and Prx 3 andnotably decreased Keap 1 when exposed to high iodide Thissuggests that excessive iodide stimulates the disassociation ofNrf2 from Keap 1 and assists Nrf2 to penetrate the nucleusThen Nrf2 attaches to the antioxidant response element(ARE) to activate the expression of the antioxidative genesSrx and Prx 3 resulting in an enhanced antioxidative defenseinduced by high iodide By activation of the Nrf2-Keap 1pathway there is a proportional increase in oxidative stressand antioxidative defense in response to iodide excess It isto be noted that the thyroid function was normal in the 10HI group and 100 HI in present study Inspired by the reportby Poncin et al [6] we proposed that there should be abalance between oxidative stress and antioxidative defense inresponse to iodide excess in the 10 HI and the 100 HI groups

5 Conclusion

In conclusion our results highlight that the activation ofNrf2-Keap 1 Srx and Prx 3 antioxidative defense mecha-nisms may play a crucial role in protecting the thyroid fromiodide excess induced oxidative stress on Days 7 14 and 28(Figure 5)

Abbreviations

NI Normal iodide intakeHI High iodide intakeROS Reactive oxygen speciesH2O2 Hydrogen peroxideNrf2 Nuclear factor erythroid 2-related factor 2Keap 1 Kelch-like ECH-associated protein 1ARE Antioxidant response elementPrx PeroxiredoxinSrx SulfiredoxinPrx 3 Peroxiredoxin 3TT4 Total thyroxineTT3 Total triiodothyronineFT4 Free thyroxineFT3 Free triiodothyronineTSH ThyrotropinLSD Least significant difference

Disclosure

This manuscript has been edited by Umar Iqbal IruniRoshanie Abeysekera Gargi Naha and Tejas Bharadwaj all

of whom are currently studying at the International School ofMedicine Tianjin Medical University China

Competing Interests

The authors declare that there is no conflict of interestsregarding the publication of this article

Acknowledgments

This work is supported by National Natural Science Founda-tion of China (nos 81273009 and 81270182) Tianjin Scienceamp Technology Council Grant of China (nos 16JCYBJC26100and 09JCYBJC11700) and International Studentrsquos Science ampTechnology Innovation Project (Scientific Research Project)of Tianjin Medical University

References

[1] Y Luo A Kawashima Y Ishido et al ldquoIodine excess as anenvironmental risk factor for autoimmune thyroid diseaserdquoInternational Journal of Molecular Sciences vol 15 no 7 pp12895ndash12912 2014

[2] A E Hussein A M Abbas G A El Wakil A Z Elsamanoudyand A A El Aziz ldquoEffect of chronic excess iodine intakeon thyroid function and oxidative stress in hypothyroid ratsrdquoCanadian Journal of Physiology and Pharmacology vol 90 no5 pp 617ndash625 2012

[3] A E Joanta A Filip S Clichici S Andrei and D DaicoviciuldquoIodide excess exerts oxidative stress in some target tissues ofthe thyroid hormonesrdquoActa Physiologica Hungarica vol 93 no4 pp 347ndash359 2006

[4] SMorandMChaaraoui J Kaniewski et al ldquoEffect of iodide onnicotinamide adenine dinucleotide phosphate oxidase activityand Duox2 protein expression in isolated porcine thyroidfolliclesrdquo Endocrinology vol 144 no 4 pp 1241ndash1248 2003

[5] J J Lazaro A Jimenez D Camejo et al ldquoDissecting the inte-grative antioxidant and redox systems in plant mitochondriaEffect of stress and S-nitrosylationrdquo Frontiers in Plant Sciencevol 4 article 460 2013

[6] S Poncin A-C GerardM Boucquey et al ldquoOxidative stress inthe thyroid gland from harmlessness to hazard depending onthe iodine contentrdquo Endocrinology vol 149 no 1 pp 424ndash4332008

[7] M Karbownik and A Lewinski ldquoThe role of oxidative stressin physiological and pathological processes in the thyroidgland possible involvement in pineal-thyroid interactionsrdquoNeuroendocrinology Letters vol 24 no 5 pp 293ndash303 2003

[8] P Thomopoulos ldquoHyperthyroidism due to excess iodinerdquo LaPresse Medicale vol 31 no 35 pp 1664ndash1669 2002

[9] J Wolff ldquoIodide goiter and the pharmacologic effects of excessiodiderdquoTheAmerican Journal of Medicine vol 47 no 1 pp 101ndash124 1969

[10] B Corvilain L Collyn J Van Sande and J E Dumont ldquoStim-ulation by iodide of H2O2 generation in thyroid slices fromseveral speciesrdquoAmerican Journal of PhysiologymdashEndocrinologyand Metabolism vol 278 no 4 pp E692ndashE699 2000

[11] M-C Many C Mestdagh M-F Van den Hove and J-F DenefldquoIn vitro study of acute toxic effects of high iodide doses inhuman thyroid folliclesrdquo Endocrinology vol 131 no 2 pp 621ndash630 1992

12 Oxidative Medicine and Cellular Longevity

[12] J Van Sande G Grenier C Willems and J E DumontldquoInhibition by iodide of the activation of the thyroid cyclic 3101584051015840-AMP systemrdquo Endocrinology vol 96 no 3 pp 781ndash786 1975

[13] R Young J D Hayes K Brown C R Wolf and C B AWhitelaw ldquoPeroxiredoxin gene expression signatures in liverreflect toxic insultrdquo Assay and Drug Development Technologiesvol 8 no 4 pp 512ndash517 2010

[14] Q Ma ldquoRole of Nrf2 in oxidative stress and toxicityrdquo AnnualReview of Pharmacology and Toxicology vol 53 pp 401ndash4262013

[15] B Biteau J Labarre and M B Toledano ldquoATP-dependentreduction of cysteine-sulphinic acid by S cerevisiae sulphire-doxinrdquo Nature vol 425 no 6961 pp 980ndash984 2003

[16] L Wu H Jiang H A Chawsheen et al ldquoTumor promoter-induced sulfiredoxin is required for mouse skin tumorigenesisrdquoCarcinogenesis vol 35 no 5 pp 1177ndash1184 2014

[17] C C Winterbourn and M B Hampton ldquoThiol chemistryand specificity in redox signalingrdquo Free Radical Biology andMedicine vol 45 no 5 pp 549ndash561 2008

[18] J W Baty M B Hampton and C C Winterbourn ldquoProteomicdetection of hydrogen peroxide-sensitive thiol proteins inJurkat cellsrdquo Biochemical Journal vol 389 no 3 pp 785ndash7952005

[19] T-S Chang C-S Cho S Park S YuW K Sang and G R SueldquoPeroxiredoxin III a mitochondrion-specific peroxidase regu-lates apoptotic signaling by mitochondriardquo Journal of BiologicalChemistry vol 279 no 40 pp 41975ndash41984 2004

[20] P Thomopoulos ldquoHyperthyroidism due to excess iodinerdquo LaPresse Medicale vol 31 no 35 pp 1664ndash1669 2002

[21] S Chung X-H Liao C Di Cosmo et al ldquoDisruption of themelanin-concentrating hormone receptor 1 (MCH1R) affectsthyroid functionrdquo Endocrinology vol 153 no 12 pp 6145ndash61542012

[22] G Ozguner and O Sulak ldquoSize and location of thyroid gland inthe fetal periodrdquo Surgical and Radiologic Anatomy vol 36 no4 pp 359ndash367 2014

[23] P L Jooste and E Strydom ldquoMethods for determination ofiodine in urine and saltrdquo Best Practice amp Research ClinicalEndocrinology amp Metabolism vol 24 no 1 pp 77ndash88 2010

[24] N Zhang L Wang Q Duan et al ldquoMetallothionein-IIIknockoutmice aggravatemitochondrial superoxide productionand peroxiredoxin 3 expression in thyroid after excessive iodideexposurerdquoOxidative Medicine and Cellular Longevity vol 2015Article ID 267027 11 pages 2015

[25] P I Gus D Marinho S Zelanis et al ldquoA case-control studyon the oxidative balance of 50 autologous serum eye dropsrdquoOxidative Medicine and Cellular Longevity vol 2016 Article ID9780193 5 pages 2016

[26] P Laurberg C Cerqueira L Ovesen et al ldquoIodine intake as adeterminant of thyroid disorders in populationsrdquo Best Practiceamp Research Clinical Endocrinology amp Metabolism vol 24 no 1pp 13ndash27 2010

[27] H Burgi ldquoIodine excessrdquo Best Practice amp Research ClinicalEndocrinology amp Metabolism vol 24 no 1 pp 107ndash115 2010

[28] E Roti and E D Uberti ldquoIodine excess and hyperthyroidismrdquoThyroid vol 11 no 5 pp 493ndash500 2001

[29] A M Leung and L E Braverman ldquoConsequences of excessiodinerdquo Nature Reviews Endocrinology vol 10 no 3 pp 136ndash142 2014

[30] K Wang Y N Sun J Y Liu et al ldquoThe impact of iodineexcess on thyroid hormone biosynthesis and metabolism in

ratsrdquoBiological Trace Element Research vol 130 no 1 pp 72ndash852009

[31] M Michalkiewicz L J Huffman J M Connors and G AHedge ldquoAlterations in thyroid blood flow induced by varyinglevels of iodine intake in the ratrdquo Endocrinology vol 125 no 1pp 54ndash60 1989

[32] P H K Eng G R Cardona S-L Fang et al ldquoEscape fromthe acute Wolff-Chaikoff effect is associated with a decrease inthyroid sodiumiodide symporter messenger ribonucleic acidand proteinrdquo Endocrinology vol 140 no 8 pp 3404ndash3410 1999

[33] P Mooij H J De Wit and H A Drexhage ldquoA high iodineintake in Wistar rats results in the development of a thyroid-associated ectopic thymic tissue and is accompanied by a lowthyroid autoimmune reactivityrdquo Immunology vol 81 no 2 pp309ndash316 1994

[34] T Paul B Meyers R J Witorsch et al ldquoThe effect of smallincreases in dietary iodine on thyroid function in euthyroidsubjectsrdquoMetabolism vol 37 no 2 pp 121ndash124 1988

[35] X Teng Z Shan W Teng C Fan H Wang and R GuoldquoExperimental study on the effects of chronic iodine excess onthyroid function structure and autoimmunity in autoimmune-prone NODH-2h4 micerdquo Clinical and Experimental Medicinevol 9 no 1 pp 51ndash59 2009

[36] E Swist Q Chen C Qiao D Caldwell H Gruber and KA Scoggan ldquoExcess dietary iodine differentially affects thyroidgene expression in diabetes thyroiditis-prone versusmdashresistantBioBreeding (BB) ratsrdquoMolecular Nutrition and Food Researchvol 55 no 12 pp 1875ndash1886 2011

[37] L Liu D Wang P Liu et al ldquoThe relationship between iodinenutrition and thyroid disease in lactating women with differentiodine intakesrdquo British Journal of Nutrition vol 114 no 9 pp1487ndash1495 2015

[38] S A Skeaff C D Thomson and R S Gibson ldquoMild iodinedeficiency in a sample of New Zealand school childrenrdquoEuropean Journal of Clinical Nutrition vol 56 no 12 pp 1169ndash1175 2002

[39] M B Zimmermann P L Jooste and C S Pandav ldquoIodine-deficiency disordersrdquo The Lancet vol 372 no 9645 pp 1251ndash1262 2008

[40] J Wolff ldquoExcess iodide inhibits the thyroid by multiple mecha-nismsrdquoAdvances in ExperimentalMedicine andBiology vol 261pp 211ndash244 1989

[41] C Serrano-Nascimento S D S Teixeira J P Nicola R TNachbar A M Masini-Repiso and M T Nunes ldquoThe acuteinhibitory effect of iodide excess on sodiumiodide symporterexpression and activity involves the PI3KAkt signaling path-wayrdquo Endocrinology vol 155 no 3 pp 1145ndash1156 2014

[42] M D Brand C Affourtit T C Esteves et al ldquoMitochondrialsuperoxide production biological effects and activation ofuncoupling proteinsrdquo Free Radical Biology andMedicine vol 37no 6 pp 755ndash767 2004

[43] S H Bae S H Sung H E Lee et al ldquoPeroxiredoxin IIIand sulfiredoxin together protect mice from pyrazole-inducedoxidative liver injuryrdquo Antioxidants amp Redox Signaling vol 17no 10 pp 1351ndash1361 2012

[44] S H Bae S H Sung E J Cho et al ldquoConcerted actionof sulfiredoxin and peroxiredoxin I protects against alcohol-induced oxidative injury inmouse liverrdquoHepatology vol 53 no3 pp 945ndash953 2011

[45] Y S Kim H L Lee K B Lee et al ldquoNuclear factor E2-related factor 2 dependent overexpression of sulfiredoxin and

Oxidative Medicine and Cellular Longevity 13

peroxiredoxin III in human lung cancerrdquo Korean Journal ofInternal Medicine vol 26 no 3 pp 304ndash313 2011

[46] H-Y Cho A E Jedlicka S P M Reddy et al ldquoRole of NRF2in protection against hyperoxic lung injury in micerdquo AmericanJournal of Respiratory Cell and Molecular Biology vol 26 no 2pp 175ndash182 2002

[47] T W Kensler N Wakabayashi and S Biswal ldquoCell survivalresponses to environmental stresses via the Keap1-Nrf2-AREpathwayrdquo Annual Review of Pharmacology and Toxicology vol47 pp 89ndash116 2007

[48] TO AjiboyeM T Yakubu andA TOladiji ldquoElectrophilic andReactive Oxygen Species Detoxification Potentials of ChalconeDimers is Mediated by Redox Transcription Factor Nrf-2rdquoJournal of Biochemical and Molecular Toxicology vol 28 no 1pp 11ndash22 2014

[49] X Yang D Wang Y Ma et al ldquoContinuous activation of Nrf2and its target antioxidant enzymes leads to arsenite-inducedmalignant transformation of human bronchial epithelial cellsrdquoToxicology and Applied Pharmacology vol 289 no 2 pp 231ndash239 2015

[50] C Pang Z Zheng L Shi et al ldquoCaffeic acid preventsacetaminophen-induced liver injury by activating the Keap1-Nrf2 antioxidative defense systemrdquo Free Radical Biology andMedicine vol 91 pp 236ndash246 2016

[51] Y H Noh J Y Baek W Jeong S G Rhee and T-S ChangldquoSulfiredoxin translocation into mitochondria plays a crucialrole in reducing hyperoxidized peroxiredoxin IIIrdquo The Journalof Biological Chemistry vol 284 no 13 pp 8470ndash8477 2009

[52] B Cunniff A N Wozniak P Sweeney K DeCosta and N HHeintz ldquoPeroxiredoxin 3 levels regulate a mitochondrial redoxsetpoint in malignant mesothelioma cellsrdquo Redox Biology vol3 pp 79ndash87 2014

[53] P-J Chua E-H Lee Y Yu G W-C Yip P-H Tan and B-HBay ldquoSilencing the Peroxiredoxin III gene inhibits cell prolifera-tion in breast cancerrdquo International Journal of Oncology vol 36no 2 pp 359ndash364 2010

[54] B Cunniff K Newick K J Nelson et al ldquoDisabling mito-chondrial peroxide metabolism via combinatorial targetingof peroxiredoxin 3 as an effective therapeutic approach formalignant mesotheliomardquo PLOS ONE vol 10 no 5 Article IDe0127310 2015

[55] X Yao M Li J He et al ldquoEffect of early acute high con-centrations of iodide exposure on mitochondrial superoxideproduction in FRTL cellsrdquo Free Radical Biology and Medicinevol 52 no 8 pp 1343ndash1352 2012

[56] L Wang Q Duan T Wang et al ldquoMitochondrial respiratorychain inhibitors involved in ROS production induced by acutehigh concentrations of iodide and the effects of sod as aprotective factorrdquo Oxidative Medicine and Cellular Longevityvol 2015 Article ID 217670 14 pages 2015

Submit your manuscripts athttpswwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

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Diabetes ResearchJournal of

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Research and TreatmentAIDS

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Oxidative Medicine and Cellular Longevity 7

NI 10 HI 100 HI

Day 28

20120583m20120583m20120583m

(c)

Figure 2 Effects of the iodide intake (NI 10 HI and 100 HI) on the changes of mitochondrial superoxide production on Days 7 14 and28 (a) There was a significant increase in the mitochondrial superoxide production in the 100 HI group on Days 7 14 and 28 (119901 lt 005)The difference was more significant on Day 28 when compared to Day 7 or Day 14 (b) Histogram analysis was performed on the meanfluorescence intensity of MitoSOX Red All data is presented as the mean plusmn SD (119873 = 6 for each group) Statistical analyses were performedby one-way analysis of variance (ANOVA) with the Least Significant Difference (LSD) test lowast119901 lt 005 versus the NI group on Days 7 14 and28 respectively 119901 lt 005 versus the 10 HI group on Days 7 14 and 28 respectively 998771119901 lt 005 versus the Day 7 group in the 100 HI group119901 lt 005 versus the Day 14 in the 100 HI group Experiments were repeated 3 times with similar results (c) There was a significant increasein the mitochondrial superoxide production in the 100 HI group on Day 28 and was observed by confocal microscopy Scale bar 20120583m

avoiding thyrotoxicosisThere are several mechanisms whichinclude a direct inhibitory effect of iodide in the thyroid itselfand inhibition by iodide of its own organification (Wolff-Chaikoff effect) its transport thyroid hormones secretioncAMP formation in response to TSH and several othermetabolic steps [40] We suggest that all the protectivemechanisms may ensure the excessive iodide intake of the 10HI group on Days 7 14 and 28 be eliminated from urineTheurinary iodine concentration of the 100 HI group on Days7 14 and 28 was about 60ndash80 times that of the NI groupalthough the thyroid function was normal This leads us topropose the idea that excessive iodide accumulated in thebody may trigger the oxidative and antioxidative signalingpathway to maintain the normal thyroid function

We demonstrated that the production of mitochondrialsuperoxide significantly increased on Days 7 14 and 28 inthe 100 HI group This is consistent with our previous studyon metallothionein-III knockout mice [24] Joanta et alreported that the initiation of free radical production wasobserved after giving a high dose of iodide [3] Serrano-Nascimento et al demonstrated that an increased mitochon-drial superoxide production was shown in response to NaI(10minus6M to 10minus3M) treatment in PCCl3 thyroid cells by usingMitoSOX Red [41] Mitochondria are potent producers ofsuperoxide from complexes I and III of the electron transportchain Mitochondrial superoxide production is a major causeof cellular oxidative damage [42] Physiologically ROS arenot necessarily harmful because they are continuously bal-anced by the process of hormone synthesis and the endoge-nous antioxidant system [1] Excess ROS are generated duringthe trapping oxidation and organification of excessive iodinein thyrocytes which could lead to increased oxidative stress[1]

The Nrf2-Keap 1 pathway is the chief cytoprotectivemechanism in response to oxidative stress caused by ROSDuring normal and balanced redox homeostasis the Nrf2function is inhibited because of constant proteasomal degra-dation after ubiquitination of the protein This is regulated

through the binding of the inhibitor protein Keap 1 [13 14]It is reported that Srx activation via Nrf2 dependent pathwayprotects fromoxidative liver injury throughPyrazole [43] andalcohol in mice [44] Similar findings in lung tissues haveshown that there is a marked increase in the expressions ofSrx and Prx 3 in human squamous cell carcinoma [45] Thissuggests that these proteins may play a protective role againstoxidative injury Also the pathway including Keap 1 Nrf2and ARE-mediated protein expression plays a very criticalrole in protecting cells fromoxidative stress [46 47] Focusingon the pathway we demonstrated that the expression of Nrf2was significantly increased while Keap 1 was significantlydecreased in the 100 HI group when compared to the NIor 10 HI group on Days 7 14 and 28 Similarly Ajiboyeet al showed that rats treated with Chalcone dimers notonly increased the expression of Nrf2 but also suppressedcytoplasmic Keap 1 expression [48] Yang et al also showedthat the downregulation of Keap 1 level may be responsiblefor the overactivation of Nrf2 [49] Pang et al showed thatcaffeic acid prevents acetaminophen-induced liver injury byactivating the Nrf2-Keap 1 antioxidative defense system [50]

In order to verify whether high levels of expressionof Nrf2 and its nuclear translocation can upregulate someantioxidant enzymes in the thyroid gland the expressions ofPrx 3 and Srx following the intake of NI 10 HI and 100 HIon Days 7 14 and 28 were measured We demonstrated thatthe expressions of Srx and Prx 3 in the 100 HI group weresignificantly increased when compared to the NI group or 10HI group on Days 7 14 and 28 The possible explanationsare described as below Firstly Srx is a cytosolic proteinthat is able to translocate to sites where hyperoxidized(inactivated) Prx 3 is located Therefore it engages itself inthe reactivation of Prx 3 under oxidative conditions [51]Secondly Prx 3 is a typical 2-Cys Peroxiredoxin locatedexclusively in the mitochondrial matrix it is the principalperoxidase responsible for protecting cells from oxidativedamage by reducing peroxides such as H2O2 [52] Finallymitochondria contain 30 times more Prx 3 than glutathione

8 Oxidative Medicine and Cellular Longevity

000

020

040

060

080

100

120

140

NI 10 HI 100 HI

Expr

essio

n of

Nrf2

(Nrf2

120573-a

ctin

)

120573-Actin

Nrf2

Day 7 Day 14 Day 28

lowast lowast

lowast

Day 7Day 14

Day 28

NI 10 HI 100 HI NI 10 HI 100 HI NI 10 HI 100 HI

(a)

000

020

040

060

080

100

120

NI 10 HI 100 HI

lowast

lowast

Expr

essio

n of

Kea

p 1

(Kea

p 1120573

-act

in)

120573-Actin

Keap 1

Day 7 Day 14 Day 28

lowast

Day 7Day 14

Day 28

NI 10 HI 100 HI NI 10 HI 100 HI NI 10 HI 100 HI

(b)

120573-Actin

SrxNI 10 HI 100 HI NI 10 HI 100 HI NI 10 HI 100 HI

000

010

020

030

040

050

060

NI 10 HI 100 HI

Expr

essio

n of

Srx

(Srx

120573-a

ctin

)

Day 7 Day 14 Day 28

lowast

lowast

lowast

Day 7Day 14

Day 28

(c)

120573-Actin

Prx 3

lowast

000

020

040

060

080

100

120

140

NI 10 HI 100 HI

Expr

essio

n of

Prx

3 (P

rx 3

120573-a

ctin

)

Day 7 Day 14 Day 28

lowastlowast

Day 7Day 14

Day 28

NI 10 HI 100 HI NI 10 HI 100 HI NI 10 HI 100 HI

(d)

Figure 3 Effects of the iodide intake (NI 10 HI and 100 HI) on the changes of (a) Nrf2 (b) Keap 1 (c) Srx and (d) Prx 3 expressions onDays 7 14 and 28 Representative western blot and histograms of densitometric analyses normalized for the relative 120573-actin content All datais presented as the mean plusmn SD (119873 = 6 for each group) Statistical analyses were performed by one-way analysis of variance (ANOVA) withthe Least Significant Difference (LSD) test lowast119901 lt 005 versus the NI group on Days 7 14 and 28 respectively 119901 lt 005 versus the 10 HI groupon Days 7 14 and 28 respectively Experiments were repeated 3 times with similar results

Oxidative Medicine and Cellular Longevity 9

NI

10 HI

100 HI

Hoechst Nrf2 Merge HoechstHoechst

Day 7 Day 28 Day 14

Nrf2 Merge Nrf2 Merge

Day 7 Day 7 Day 14 Day 14 Day 28 Day 28 20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

(a)

Hoechst Srx Merge Hoechst Srx Merge Hoechst Srx Merge

NI

10 HI

100 HI

Day 7 Day 28 Day 14 Day 7 Day 7 Day 14 Day 14 Day 28 Day 28 20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

(b)

Figure 4 Effect of the iodide intake (NI 10 HI and 100 HI) on the changes of immunofluorescence staining on Days 7 14 and 28 (a) In theNI group Nrf2 (red) was localized in the cytoplasm on Days 7 14 and 28 In the 10 HI group the positive staining of Nrf2 can be observedin both the nucleus and the cytoplasm Moreover in the 100 HI group a stronger positive staining of Nrf2 can be detected in the nucleus onDays 7 14 and 28 the nucleus was dyed with Hoechst (blue) (b) Srx (red) positive staining was located in the cytoplasm the nucleus wasdyed with Hoechst (blue) Scale bar 20120583m

peroxidase Prx 3 can be classified as an important regulatorof mitochondrial H2O2 [22] The elevated expression of Prx3 is associated with the blockage of apoptosis increasingcell proliferation and is related to adaptive responses whichare all required to maintain mitochondrial function [53 54]Bae et al have suggested that Prx 3 and Srx jointly protectmice from Pyrazole-induced oxidative liver injury in a Nrf2-dependent manner [43]

The novelty we demonstrated in the present study is thatiodide excess induced both oxidative stress and antioxidativedefense increases throughNrf2-Keap 1 pathway in the thyroidgland from rats We extended our established mechanismsby applying the Nrf2-Keap 1 pathway to set up a bridgebetween oxidative stress and antioxidative defense inducedby iodide excess in the thyroid gland In our previous studywe have established that oxidative stress induced by acutehigh concentrations of iodide in FRTL cells significantlyincreases mitochondrial superoxide production [55] Theinhibitors of themitochondrial respiratory chain complexes I

and III are involved inmitochondrial superoxide productionWe demonstrated that exposure to 100120583M KI for 2 hourssignificantly increased mitochondrial superoxide produc-tion enhanced by either 05120583M Rotenone (an inhibitorof mitochondrial complex I) or 10 120583M Antimycin A (aninhibitor of complex III) [56] We illustrated that 300 120583MPTU (an inhibitor of TPO) attenuated the excessive iodide-induced mitochondrial superoxide production We showedthat 30 120583M KClO4 (a competitive inhibitor of iodide trans-port) relieved the production the mitochondrial superoxideinduced by iodide excess We displayed that 10mUmL TSHcan inhibit excessive iodide-induced strong mitochondrialsuperoxide production [55] MT-I and MT-II are mainlyinvolved in the protection of tissue against oxidative stresseswe indicated that metallothionein-III knockout mice aggra-vated mitochondrial superoxide production in thyroid afterexcessive iodide exposure [24] In addition we demonstratedthat both the oxidative stress and the antioxidative defenseincreased simultaneously after high dosages of iodide intake

10 Oxidative Medicine and Cellular Longevity

Oxidative stress

(a)

(b)

(c)

Antioxidative defense

Wist

ar ra

ts Day 7Day 14Day 28

Normal iodideintake

Normal urinaryiodine

Normal thyroidfunction

10 times iodideintake

10 times urinaryiodine

Normal thyroidfunction

100 times iodideintake

60ndash80 timesurinary iodine

Normal thyroidfunction

Keap 1 Nrf2

Keap 1

Nrf2

Srx

Nucleus

Prx 3

SrxARE

Mitochondria

AntioxidativeOxidativestress

Oxidative Antioxidative

Oxidative Antioxidative

Under physiological conditions(normal iodide intake)

Excessive iodide intake(10 times iodide intake)

Excessive iodide intake(100 times iodide intake)

defense defenseuarrstressuarr

defenseuarruarrstressuarruarr

Figure 5 Proposed mechanisms in the present study (a) The urinary iodine concentration and thyroid function of Wistar rats weredetected (b) The activation of the Nrf2-Keap 1 pathway induced by iodide excess in the thyroid (c) The balance between oxidative stressand antioxidative defense under physiological conditions and excessive iodide intake

Oxidative Medicine and Cellular Longevity 11

We suggested that the Nrf2-Keap 1 pathway is vital for thebalance between oxidative stress and antioxidative defenseinduced by iodide excess in the thyroid gland

Excessive iodide stimulated the Nrf2-Keap 1 pathwayand enhanced the antioxidative defense We found thatthe urinary iodine concentration of the 100 HI group wasabout 60ndash80 times that of the NI group on Days 7 14and 28 however the thyroid functions were normal Weproposed that the excessive iodide accumulated in the bodymay trigger the Nrf2-Keap 1 pathway to maintain a normalthyroid functionWe demonstrated that the Nrf2moves fromthe cytoplasm to the nucleus under the microscope withsignificantly increased expressions of Nrf2 Srx and Prx 3 andnotably decreased Keap 1 when exposed to high iodide Thissuggests that excessive iodide stimulates the disassociation ofNrf2 from Keap 1 and assists Nrf2 to penetrate the nucleusThen Nrf2 attaches to the antioxidant response element(ARE) to activate the expression of the antioxidative genesSrx and Prx 3 resulting in an enhanced antioxidative defenseinduced by high iodide By activation of the Nrf2-Keap 1pathway there is a proportional increase in oxidative stressand antioxidative defense in response to iodide excess It isto be noted that the thyroid function was normal in the 10HI group and 100 HI in present study Inspired by the reportby Poncin et al [6] we proposed that there should be abalance between oxidative stress and antioxidative defense inresponse to iodide excess in the 10 HI and the 100 HI groups

5 Conclusion

In conclusion our results highlight that the activation ofNrf2-Keap 1 Srx and Prx 3 antioxidative defense mecha-nisms may play a crucial role in protecting the thyroid fromiodide excess induced oxidative stress on Days 7 14 and 28(Figure 5)

Abbreviations

NI Normal iodide intakeHI High iodide intakeROS Reactive oxygen speciesH2O2 Hydrogen peroxideNrf2 Nuclear factor erythroid 2-related factor 2Keap 1 Kelch-like ECH-associated protein 1ARE Antioxidant response elementPrx PeroxiredoxinSrx SulfiredoxinPrx 3 Peroxiredoxin 3TT4 Total thyroxineTT3 Total triiodothyronineFT4 Free thyroxineFT3 Free triiodothyronineTSH ThyrotropinLSD Least significant difference

Disclosure

This manuscript has been edited by Umar Iqbal IruniRoshanie Abeysekera Gargi Naha and Tejas Bharadwaj all

of whom are currently studying at the International School ofMedicine Tianjin Medical University China

Competing Interests

The authors declare that there is no conflict of interestsregarding the publication of this article

Acknowledgments

This work is supported by National Natural Science Founda-tion of China (nos 81273009 and 81270182) Tianjin Scienceamp Technology Council Grant of China (nos 16JCYBJC26100and 09JCYBJC11700) and International Studentrsquos Science ampTechnology Innovation Project (Scientific Research Project)of Tianjin Medical University

References

[1] Y Luo A Kawashima Y Ishido et al ldquoIodine excess as anenvironmental risk factor for autoimmune thyroid diseaserdquoInternational Journal of Molecular Sciences vol 15 no 7 pp12895ndash12912 2014

[2] A E Hussein A M Abbas G A El Wakil A Z Elsamanoudyand A A El Aziz ldquoEffect of chronic excess iodine intakeon thyroid function and oxidative stress in hypothyroid ratsrdquoCanadian Journal of Physiology and Pharmacology vol 90 no5 pp 617ndash625 2012

[3] A E Joanta A Filip S Clichici S Andrei and D DaicoviciuldquoIodide excess exerts oxidative stress in some target tissues ofthe thyroid hormonesrdquoActa Physiologica Hungarica vol 93 no4 pp 347ndash359 2006

[4] SMorandMChaaraoui J Kaniewski et al ldquoEffect of iodide onnicotinamide adenine dinucleotide phosphate oxidase activityand Duox2 protein expression in isolated porcine thyroidfolliclesrdquo Endocrinology vol 144 no 4 pp 1241ndash1248 2003

[5] J J Lazaro A Jimenez D Camejo et al ldquoDissecting the inte-grative antioxidant and redox systems in plant mitochondriaEffect of stress and S-nitrosylationrdquo Frontiers in Plant Sciencevol 4 article 460 2013

[6] S Poncin A-C GerardM Boucquey et al ldquoOxidative stress inthe thyroid gland from harmlessness to hazard depending onthe iodine contentrdquo Endocrinology vol 149 no 1 pp 424ndash4332008

[7] M Karbownik and A Lewinski ldquoThe role of oxidative stressin physiological and pathological processes in the thyroidgland possible involvement in pineal-thyroid interactionsrdquoNeuroendocrinology Letters vol 24 no 5 pp 293ndash303 2003

[8] P Thomopoulos ldquoHyperthyroidism due to excess iodinerdquo LaPresse Medicale vol 31 no 35 pp 1664ndash1669 2002

[9] J Wolff ldquoIodide goiter and the pharmacologic effects of excessiodiderdquoTheAmerican Journal of Medicine vol 47 no 1 pp 101ndash124 1969

[10] B Corvilain L Collyn J Van Sande and J E Dumont ldquoStim-ulation by iodide of H2O2 generation in thyroid slices fromseveral speciesrdquoAmerican Journal of PhysiologymdashEndocrinologyand Metabolism vol 278 no 4 pp E692ndashE699 2000

[11] M-C Many C Mestdagh M-F Van den Hove and J-F DenefldquoIn vitro study of acute toxic effects of high iodide doses inhuman thyroid folliclesrdquo Endocrinology vol 131 no 2 pp 621ndash630 1992

12 Oxidative Medicine and Cellular Longevity

[12] J Van Sande G Grenier C Willems and J E DumontldquoInhibition by iodide of the activation of the thyroid cyclic 3101584051015840-AMP systemrdquo Endocrinology vol 96 no 3 pp 781ndash786 1975

[13] R Young J D Hayes K Brown C R Wolf and C B AWhitelaw ldquoPeroxiredoxin gene expression signatures in liverreflect toxic insultrdquo Assay and Drug Development Technologiesvol 8 no 4 pp 512ndash517 2010

[14] Q Ma ldquoRole of Nrf2 in oxidative stress and toxicityrdquo AnnualReview of Pharmacology and Toxicology vol 53 pp 401ndash4262013

[15] B Biteau J Labarre and M B Toledano ldquoATP-dependentreduction of cysteine-sulphinic acid by S cerevisiae sulphire-doxinrdquo Nature vol 425 no 6961 pp 980ndash984 2003

[16] L Wu H Jiang H A Chawsheen et al ldquoTumor promoter-induced sulfiredoxin is required for mouse skin tumorigenesisrdquoCarcinogenesis vol 35 no 5 pp 1177ndash1184 2014

[17] C C Winterbourn and M B Hampton ldquoThiol chemistryand specificity in redox signalingrdquo Free Radical Biology andMedicine vol 45 no 5 pp 549ndash561 2008

[18] J W Baty M B Hampton and C C Winterbourn ldquoProteomicdetection of hydrogen peroxide-sensitive thiol proteins inJurkat cellsrdquo Biochemical Journal vol 389 no 3 pp 785ndash7952005

[19] T-S Chang C-S Cho S Park S YuW K Sang and G R SueldquoPeroxiredoxin III a mitochondrion-specific peroxidase regu-lates apoptotic signaling by mitochondriardquo Journal of BiologicalChemistry vol 279 no 40 pp 41975ndash41984 2004

[20] P Thomopoulos ldquoHyperthyroidism due to excess iodinerdquo LaPresse Medicale vol 31 no 35 pp 1664ndash1669 2002

[21] S Chung X-H Liao C Di Cosmo et al ldquoDisruption of themelanin-concentrating hormone receptor 1 (MCH1R) affectsthyroid functionrdquo Endocrinology vol 153 no 12 pp 6145ndash61542012

[22] G Ozguner and O Sulak ldquoSize and location of thyroid gland inthe fetal periodrdquo Surgical and Radiologic Anatomy vol 36 no4 pp 359ndash367 2014

[23] P L Jooste and E Strydom ldquoMethods for determination ofiodine in urine and saltrdquo Best Practice amp Research ClinicalEndocrinology amp Metabolism vol 24 no 1 pp 77ndash88 2010

[24] N Zhang L Wang Q Duan et al ldquoMetallothionein-IIIknockoutmice aggravatemitochondrial superoxide productionand peroxiredoxin 3 expression in thyroid after excessive iodideexposurerdquoOxidative Medicine and Cellular Longevity vol 2015Article ID 267027 11 pages 2015

[25] P I Gus D Marinho S Zelanis et al ldquoA case-control studyon the oxidative balance of 50 autologous serum eye dropsrdquoOxidative Medicine and Cellular Longevity vol 2016 Article ID9780193 5 pages 2016

[26] P Laurberg C Cerqueira L Ovesen et al ldquoIodine intake as adeterminant of thyroid disorders in populationsrdquo Best Practiceamp Research Clinical Endocrinology amp Metabolism vol 24 no 1pp 13ndash27 2010

[27] H Burgi ldquoIodine excessrdquo Best Practice amp Research ClinicalEndocrinology amp Metabolism vol 24 no 1 pp 107ndash115 2010

[28] E Roti and E D Uberti ldquoIodine excess and hyperthyroidismrdquoThyroid vol 11 no 5 pp 493ndash500 2001

[29] A M Leung and L E Braverman ldquoConsequences of excessiodinerdquo Nature Reviews Endocrinology vol 10 no 3 pp 136ndash142 2014

[30] K Wang Y N Sun J Y Liu et al ldquoThe impact of iodineexcess on thyroid hormone biosynthesis and metabolism in

ratsrdquoBiological Trace Element Research vol 130 no 1 pp 72ndash852009

[31] M Michalkiewicz L J Huffman J M Connors and G AHedge ldquoAlterations in thyroid blood flow induced by varyinglevels of iodine intake in the ratrdquo Endocrinology vol 125 no 1pp 54ndash60 1989

[32] P H K Eng G R Cardona S-L Fang et al ldquoEscape fromthe acute Wolff-Chaikoff effect is associated with a decrease inthyroid sodiumiodide symporter messenger ribonucleic acidand proteinrdquo Endocrinology vol 140 no 8 pp 3404ndash3410 1999

[33] P Mooij H J De Wit and H A Drexhage ldquoA high iodineintake in Wistar rats results in the development of a thyroid-associated ectopic thymic tissue and is accompanied by a lowthyroid autoimmune reactivityrdquo Immunology vol 81 no 2 pp309ndash316 1994

[34] T Paul B Meyers R J Witorsch et al ldquoThe effect of smallincreases in dietary iodine on thyroid function in euthyroidsubjectsrdquoMetabolism vol 37 no 2 pp 121ndash124 1988

[35] X Teng Z Shan W Teng C Fan H Wang and R GuoldquoExperimental study on the effects of chronic iodine excess onthyroid function structure and autoimmunity in autoimmune-prone NODH-2h4 micerdquo Clinical and Experimental Medicinevol 9 no 1 pp 51ndash59 2009

[36] E Swist Q Chen C Qiao D Caldwell H Gruber and KA Scoggan ldquoExcess dietary iodine differentially affects thyroidgene expression in diabetes thyroiditis-prone versusmdashresistantBioBreeding (BB) ratsrdquoMolecular Nutrition and Food Researchvol 55 no 12 pp 1875ndash1886 2011

[37] L Liu D Wang P Liu et al ldquoThe relationship between iodinenutrition and thyroid disease in lactating women with differentiodine intakesrdquo British Journal of Nutrition vol 114 no 9 pp1487ndash1495 2015

[38] S A Skeaff C D Thomson and R S Gibson ldquoMild iodinedeficiency in a sample of New Zealand school childrenrdquoEuropean Journal of Clinical Nutrition vol 56 no 12 pp 1169ndash1175 2002

[39] M B Zimmermann P L Jooste and C S Pandav ldquoIodine-deficiency disordersrdquo The Lancet vol 372 no 9645 pp 1251ndash1262 2008

[40] J Wolff ldquoExcess iodide inhibits the thyroid by multiple mecha-nismsrdquoAdvances in ExperimentalMedicine andBiology vol 261pp 211ndash244 1989

[41] C Serrano-Nascimento S D S Teixeira J P Nicola R TNachbar A M Masini-Repiso and M T Nunes ldquoThe acuteinhibitory effect of iodide excess on sodiumiodide symporterexpression and activity involves the PI3KAkt signaling path-wayrdquo Endocrinology vol 155 no 3 pp 1145ndash1156 2014

[42] M D Brand C Affourtit T C Esteves et al ldquoMitochondrialsuperoxide production biological effects and activation ofuncoupling proteinsrdquo Free Radical Biology andMedicine vol 37no 6 pp 755ndash767 2004

[43] S H Bae S H Sung H E Lee et al ldquoPeroxiredoxin IIIand sulfiredoxin together protect mice from pyrazole-inducedoxidative liver injuryrdquo Antioxidants amp Redox Signaling vol 17no 10 pp 1351ndash1361 2012

[44] S H Bae S H Sung E J Cho et al ldquoConcerted actionof sulfiredoxin and peroxiredoxin I protects against alcohol-induced oxidative injury inmouse liverrdquoHepatology vol 53 no3 pp 945ndash953 2011

[45] Y S Kim H L Lee K B Lee et al ldquoNuclear factor E2-related factor 2 dependent overexpression of sulfiredoxin and

Oxidative Medicine and Cellular Longevity 13

peroxiredoxin III in human lung cancerrdquo Korean Journal ofInternal Medicine vol 26 no 3 pp 304ndash313 2011

[46] H-Y Cho A E Jedlicka S P M Reddy et al ldquoRole of NRF2in protection against hyperoxic lung injury in micerdquo AmericanJournal of Respiratory Cell and Molecular Biology vol 26 no 2pp 175ndash182 2002

[47] T W Kensler N Wakabayashi and S Biswal ldquoCell survivalresponses to environmental stresses via the Keap1-Nrf2-AREpathwayrdquo Annual Review of Pharmacology and Toxicology vol47 pp 89ndash116 2007

[48] TO AjiboyeM T Yakubu andA TOladiji ldquoElectrophilic andReactive Oxygen Species Detoxification Potentials of ChalconeDimers is Mediated by Redox Transcription Factor Nrf-2rdquoJournal of Biochemical and Molecular Toxicology vol 28 no 1pp 11ndash22 2014

[49] X Yang D Wang Y Ma et al ldquoContinuous activation of Nrf2and its target antioxidant enzymes leads to arsenite-inducedmalignant transformation of human bronchial epithelial cellsrdquoToxicology and Applied Pharmacology vol 289 no 2 pp 231ndash239 2015

[50] C Pang Z Zheng L Shi et al ldquoCaffeic acid preventsacetaminophen-induced liver injury by activating the Keap1-Nrf2 antioxidative defense systemrdquo Free Radical Biology andMedicine vol 91 pp 236ndash246 2016

[51] Y H Noh J Y Baek W Jeong S G Rhee and T-S ChangldquoSulfiredoxin translocation into mitochondria plays a crucialrole in reducing hyperoxidized peroxiredoxin IIIrdquo The Journalof Biological Chemistry vol 284 no 13 pp 8470ndash8477 2009

[52] B Cunniff A N Wozniak P Sweeney K DeCosta and N HHeintz ldquoPeroxiredoxin 3 levels regulate a mitochondrial redoxsetpoint in malignant mesothelioma cellsrdquo Redox Biology vol3 pp 79ndash87 2014

[53] P-J Chua E-H Lee Y Yu G W-C Yip P-H Tan and B-HBay ldquoSilencing the Peroxiredoxin III gene inhibits cell prolifera-tion in breast cancerrdquo International Journal of Oncology vol 36no 2 pp 359ndash364 2010

[54] B Cunniff K Newick K J Nelson et al ldquoDisabling mito-chondrial peroxide metabolism via combinatorial targetingof peroxiredoxin 3 as an effective therapeutic approach formalignant mesotheliomardquo PLOS ONE vol 10 no 5 Article IDe0127310 2015

[55] X Yao M Li J He et al ldquoEffect of early acute high con-centrations of iodide exposure on mitochondrial superoxideproduction in FRTL cellsrdquo Free Radical Biology and Medicinevol 52 no 8 pp 1343ndash1352 2012

[56] L Wang Q Duan T Wang et al ldquoMitochondrial respiratorychain inhibitors involved in ROS production induced by acutehigh concentrations of iodide and the effects of sod as aprotective factorrdquo Oxidative Medicine and Cellular Longevityvol 2015 Article ID 217670 14 pages 2015

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

8 Oxidative Medicine and Cellular Longevity

000

020

040

060

080

100

120

140

NI 10 HI 100 HI

Expr

essio

n of

Nrf2

(Nrf2

120573-a

ctin

)

120573-Actin

Nrf2

Day 7 Day 14 Day 28

lowast lowast

lowast

Day 7Day 14

Day 28

NI 10 HI 100 HI NI 10 HI 100 HI NI 10 HI 100 HI

(a)

000

020

040

060

080

100

120

NI 10 HI 100 HI

lowast

lowast

Expr

essio

n of

Kea

p 1

(Kea

p 1120573

-act

in)

120573-Actin

Keap 1

Day 7 Day 14 Day 28

lowast

Day 7Day 14

Day 28

NI 10 HI 100 HI NI 10 HI 100 HI NI 10 HI 100 HI

(b)

120573-Actin

SrxNI 10 HI 100 HI NI 10 HI 100 HI NI 10 HI 100 HI

000

010

020

030

040

050

060

NI 10 HI 100 HI

Expr

essio

n of

Srx

(Srx

120573-a

ctin

)

Day 7 Day 14 Day 28

lowast

lowast

lowast

Day 7Day 14

Day 28

(c)

120573-Actin

Prx 3

lowast

000

020

040

060

080

100

120

140

NI 10 HI 100 HI

Expr

essio

n of

Prx

3 (P

rx 3

120573-a

ctin

)

Day 7 Day 14 Day 28

lowastlowast

Day 7Day 14

Day 28

NI 10 HI 100 HI NI 10 HI 100 HI NI 10 HI 100 HI

(d)

Figure 3 Effects of the iodide intake (NI 10 HI and 100 HI) on the changes of (a) Nrf2 (b) Keap 1 (c) Srx and (d) Prx 3 expressions onDays 7 14 and 28 Representative western blot and histograms of densitometric analyses normalized for the relative 120573-actin content All datais presented as the mean plusmn SD (119873 = 6 for each group) Statistical analyses were performed by one-way analysis of variance (ANOVA) withthe Least Significant Difference (LSD) test lowast119901 lt 005 versus the NI group on Days 7 14 and 28 respectively 119901 lt 005 versus the 10 HI groupon Days 7 14 and 28 respectively Experiments were repeated 3 times with similar results

Oxidative Medicine and Cellular Longevity 9

NI

10 HI

100 HI

Hoechst Nrf2 Merge HoechstHoechst

Day 7 Day 28 Day 14

Nrf2 Merge Nrf2 Merge

Day 7 Day 7 Day 14 Day 14 Day 28 Day 28 20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

(a)

Hoechst Srx Merge Hoechst Srx Merge Hoechst Srx Merge

NI

10 HI

100 HI

Day 7 Day 28 Day 14 Day 7 Day 7 Day 14 Day 14 Day 28 Day 28 20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

(b)

Figure 4 Effect of the iodide intake (NI 10 HI and 100 HI) on the changes of immunofluorescence staining on Days 7 14 and 28 (a) In theNI group Nrf2 (red) was localized in the cytoplasm on Days 7 14 and 28 In the 10 HI group the positive staining of Nrf2 can be observedin both the nucleus and the cytoplasm Moreover in the 100 HI group a stronger positive staining of Nrf2 can be detected in the nucleus onDays 7 14 and 28 the nucleus was dyed with Hoechst (blue) (b) Srx (red) positive staining was located in the cytoplasm the nucleus wasdyed with Hoechst (blue) Scale bar 20120583m

peroxidase Prx 3 can be classified as an important regulatorof mitochondrial H2O2 [22] The elevated expression of Prx3 is associated with the blockage of apoptosis increasingcell proliferation and is related to adaptive responses whichare all required to maintain mitochondrial function [53 54]Bae et al have suggested that Prx 3 and Srx jointly protectmice from Pyrazole-induced oxidative liver injury in a Nrf2-dependent manner [43]

The novelty we demonstrated in the present study is thatiodide excess induced both oxidative stress and antioxidativedefense increases throughNrf2-Keap 1 pathway in the thyroidgland from rats We extended our established mechanismsby applying the Nrf2-Keap 1 pathway to set up a bridgebetween oxidative stress and antioxidative defense inducedby iodide excess in the thyroid gland In our previous studywe have established that oxidative stress induced by acutehigh concentrations of iodide in FRTL cells significantlyincreases mitochondrial superoxide production [55] Theinhibitors of themitochondrial respiratory chain complexes I

and III are involved inmitochondrial superoxide productionWe demonstrated that exposure to 100120583M KI for 2 hourssignificantly increased mitochondrial superoxide produc-tion enhanced by either 05120583M Rotenone (an inhibitorof mitochondrial complex I) or 10 120583M Antimycin A (aninhibitor of complex III) [56] We illustrated that 300 120583MPTU (an inhibitor of TPO) attenuated the excessive iodide-induced mitochondrial superoxide production We showedthat 30 120583M KClO4 (a competitive inhibitor of iodide trans-port) relieved the production the mitochondrial superoxideinduced by iodide excess We displayed that 10mUmL TSHcan inhibit excessive iodide-induced strong mitochondrialsuperoxide production [55] MT-I and MT-II are mainlyinvolved in the protection of tissue against oxidative stresseswe indicated that metallothionein-III knockout mice aggra-vated mitochondrial superoxide production in thyroid afterexcessive iodide exposure [24] In addition we demonstratedthat both the oxidative stress and the antioxidative defenseincreased simultaneously after high dosages of iodide intake

10 Oxidative Medicine and Cellular Longevity

Oxidative stress

(a)

(b)

(c)

Antioxidative defense

Wist

ar ra

ts Day 7Day 14Day 28

Normal iodideintake

Normal urinaryiodine

Normal thyroidfunction

10 times iodideintake

10 times urinaryiodine

Normal thyroidfunction

100 times iodideintake

60ndash80 timesurinary iodine

Normal thyroidfunction

Keap 1 Nrf2

Keap 1

Nrf2

Srx

Nucleus

Prx 3

SrxARE

Mitochondria

AntioxidativeOxidativestress

Oxidative Antioxidative

Oxidative Antioxidative

Under physiological conditions(normal iodide intake)

Excessive iodide intake(10 times iodide intake)

Excessive iodide intake(100 times iodide intake)

defense defenseuarrstressuarr

defenseuarruarrstressuarruarr

Figure 5 Proposed mechanisms in the present study (a) The urinary iodine concentration and thyroid function of Wistar rats weredetected (b) The activation of the Nrf2-Keap 1 pathway induced by iodide excess in the thyroid (c) The balance between oxidative stressand antioxidative defense under physiological conditions and excessive iodide intake

Oxidative Medicine and Cellular Longevity 11

We suggested that the Nrf2-Keap 1 pathway is vital for thebalance between oxidative stress and antioxidative defenseinduced by iodide excess in the thyroid gland

Excessive iodide stimulated the Nrf2-Keap 1 pathwayand enhanced the antioxidative defense We found thatthe urinary iodine concentration of the 100 HI group wasabout 60ndash80 times that of the NI group on Days 7 14and 28 however the thyroid functions were normal Weproposed that the excessive iodide accumulated in the bodymay trigger the Nrf2-Keap 1 pathway to maintain a normalthyroid functionWe demonstrated that the Nrf2moves fromthe cytoplasm to the nucleus under the microscope withsignificantly increased expressions of Nrf2 Srx and Prx 3 andnotably decreased Keap 1 when exposed to high iodide Thissuggests that excessive iodide stimulates the disassociation ofNrf2 from Keap 1 and assists Nrf2 to penetrate the nucleusThen Nrf2 attaches to the antioxidant response element(ARE) to activate the expression of the antioxidative genesSrx and Prx 3 resulting in an enhanced antioxidative defenseinduced by high iodide By activation of the Nrf2-Keap 1pathway there is a proportional increase in oxidative stressand antioxidative defense in response to iodide excess It isto be noted that the thyroid function was normal in the 10HI group and 100 HI in present study Inspired by the reportby Poncin et al [6] we proposed that there should be abalance between oxidative stress and antioxidative defense inresponse to iodide excess in the 10 HI and the 100 HI groups

5 Conclusion

In conclusion our results highlight that the activation ofNrf2-Keap 1 Srx and Prx 3 antioxidative defense mecha-nisms may play a crucial role in protecting the thyroid fromiodide excess induced oxidative stress on Days 7 14 and 28(Figure 5)

Abbreviations

NI Normal iodide intakeHI High iodide intakeROS Reactive oxygen speciesH2O2 Hydrogen peroxideNrf2 Nuclear factor erythroid 2-related factor 2Keap 1 Kelch-like ECH-associated protein 1ARE Antioxidant response elementPrx PeroxiredoxinSrx SulfiredoxinPrx 3 Peroxiredoxin 3TT4 Total thyroxineTT3 Total triiodothyronineFT4 Free thyroxineFT3 Free triiodothyronineTSH ThyrotropinLSD Least significant difference

Disclosure

This manuscript has been edited by Umar Iqbal IruniRoshanie Abeysekera Gargi Naha and Tejas Bharadwaj all

of whom are currently studying at the International School ofMedicine Tianjin Medical University China

Competing Interests

The authors declare that there is no conflict of interestsregarding the publication of this article

Acknowledgments

This work is supported by National Natural Science Founda-tion of China (nos 81273009 and 81270182) Tianjin Scienceamp Technology Council Grant of China (nos 16JCYBJC26100and 09JCYBJC11700) and International Studentrsquos Science ampTechnology Innovation Project (Scientific Research Project)of Tianjin Medical University

References

[1] Y Luo A Kawashima Y Ishido et al ldquoIodine excess as anenvironmental risk factor for autoimmune thyroid diseaserdquoInternational Journal of Molecular Sciences vol 15 no 7 pp12895ndash12912 2014

[2] A E Hussein A M Abbas G A El Wakil A Z Elsamanoudyand A A El Aziz ldquoEffect of chronic excess iodine intakeon thyroid function and oxidative stress in hypothyroid ratsrdquoCanadian Journal of Physiology and Pharmacology vol 90 no5 pp 617ndash625 2012

[3] A E Joanta A Filip S Clichici S Andrei and D DaicoviciuldquoIodide excess exerts oxidative stress in some target tissues ofthe thyroid hormonesrdquoActa Physiologica Hungarica vol 93 no4 pp 347ndash359 2006

[4] SMorandMChaaraoui J Kaniewski et al ldquoEffect of iodide onnicotinamide adenine dinucleotide phosphate oxidase activityand Duox2 protein expression in isolated porcine thyroidfolliclesrdquo Endocrinology vol 144 no 4 pp 1241ndash1248 2003

[5] J J Lazaro A Jimenez D Camejo et al ldquoDissecting the inte-grative antioxidant and redox systems in plant mitochondriaEffect of stress and S-nitrosylationrdquo Frontiers in Plant Sciencevol 4 article 460 2013

[6] S Poncin A-C GerardM Boucquey et al ldquoOxidative stress inthe thyroid gland from harmlessness to hazard depending onthe iodine contentrdquo Endocrinology vol 149 no 1 pp 424ndash4332008

[7] M Karbownik and A Lewinski ldquoThe role of oxidative stressin physiological and pathological processes in the thyroidgland possible involvement in pineal-thyroid interactionsrdquoNeuroendocrinology Letters vol 24 no 5 pp 293ndash303 2003

[8] P Thomopoulos ldquoHyperthyroidism due to excess iodinerdquo LaPresse Medicale vol 31 no 35 pp 1664ndash1669 2002

[9] J Wolff ldquoIodide goiter and the pharmacologic effects of excessiodiderdquoTheAmerican Journal of Medicine vol 47 no 1 pp 101ndash124 1969

[10] B Corvilain L Collyn J Van Sande and J E Dumont ldquoStim-ulation by iodide of H2O2 generation in thyroid slices fromseveral speciesrdquoAmerican Journal of PhysiologymdashEndocrinologyand Metabolism vol 278 no 4 pp E692ndashE699 2000

[11] M-C Many C Mestdagh M-F Van den Hove and J-F DenefldquoIn vitro study of acute toxic effects of high iodide doses inhuman thyroid folliclesrdquo Endocrinology vol 131 no 2 pp 621ndash630 1992

12 Oxidative Medicine and Cellular Longevity

[12] J Van Sande G Grenier C Willems and J E DumontldquoInhibition by iodide of the activation of the thyroid cyclic 3101584051015840-AMP systemrdquo Endocrinology vol 96 no 3 pp 781ndash786 1975

[13] R Young J D Hayes K Brown C R Wolf and C B AWhitelaw ldquoPeroxiredoxin gene expression signatures in liverreflect toxic insultrdquo Assay and Drug Development Technologiesvol 8 no 4 pp 512ndash517 2010

[14] Q Ma ldquoRole of Nrf2 in oxidative stress and toxicityrdquo AnnualReview of Pharmacology and Toxicology vol 53 pp 401ndash4262013

[15] B Biteau J Labarre and M B Toledano ldquoATP-dependentreduction of cysteine-sulphinic acid by S cerevisiae sulphire-doxinrdquo Nature vol 425 no 6961 pp 980ndash984 2003

[16] L Wu H Jiang H A Chawsheen et al ldquoTumor promoter-induced sulfiredoxin is required for mouse skin tumorigenesisrdquoCarcinogenesis vol 35 no 5 pp 1177ndash1184 2014

[17] C C Winterbourn and M B Hampton ldquoThiol chemistryand specificity in redox signalingrdquo Free Radical Biology andMedicine vol 45 no 5 pp 549ndash561 2008

[18] J W Baty M B Hampton and C C Winterbourn ldquoProteomicdetection of hydrogen peroxide-sensitive thiol proteins inJurkat cellsrdquo Biochemical Journal vol 389 no 3 pp 785ndash7952005

[19] T-S Chang C-S Cho S Park S YuW K Sang and G R SueldquoPeroxiredoxin III a mitochondrion-specific peroxidase regu-lates apoptotic signaling by mitochondriardquo Journal of BiologicalChemistry vol 279 no 40 pp 41975ndash41984 2004

[20] P Thomopoulos ldquoHyperthyroidism due to excess iodinerdquo LaPresse Medicale vol 31 no 35 pp 1664ndash1669 2002

[21] S Chung X-H Liao C Di Cosmo et al ldquoDisruption of themelanin-concentrating hormone receptor 1 (MCH1R) affectsthyroid functionrdquo Endocrinology vol 153 no 12 pp 6145ndash61542012

[22] G Ozguner and O Sulak ldquoSize and location of thyroid gland inthe fetal periodrdquo Surgical and Radiologic Anatomy vol 36 no4 pp 359ndash367 2014

[23] P L Jooste and E Strydom ldquoMethods for determination ofiodine in urine and saltrdquo Best Practice amp Research ClinicalEndocrinology amp Metabolism vol 24 no 1 pp 77ndash88 2010

[24] N Zhang L Wang Q Duan et al ldquoMetallothionein-IIIknockoutmice aggravatemitochondrial superoxide productionand peroxiredoxin 3 expression in thyroid after excessive iodideexposurerdquoOxidative Medicine and Cellular Longevity vol 2015Article ID 267027 11 pages 2015

[25] P I Gus D Marinho S Zelanis et al ldquoA case-control studyon the oxidative balance of 50 autologous serum eye dropsrdquoOxidative Medicine and Cellular Longevity vol 2016 Article ID9780193 5 pages 2016

[26] P Laurberg C Cerqueira L Ovesen et al ldquoIodine intake as adeterminant of thyroid disorders in populationsrdquo Best Practiceamp Research Clinical Endocrinology amp Metabolism vol 24 no 1pp 13ndash27 2010

[27] H Burgi ldquoIodine excessrdquo Best Practice amp Research ClinicalEndocrinology amp Metabolism vol 24 no 1 pp 107ndash115 2010

[28] E Roti and E D Uberti ldquoIodine excess and hyperthyroidismrdquoThyroid vol 11 no 5 pp 493ndash500 2001

[29] A M Leung and L E Braverman ldquoConsequences of excessiodinerdquo Nature Reviews Endocrinology vol 10 no 3 pp 136ndash142 2014

[30] K Wang Y N Sun J Y Liu et al ldquoThe impact of iodineexcess on thyroid hormone biosynthesis and metabolism in

ratsrdquoBiological Trace Element Research vol 130 no 1 pp 72ndash852009

[31] M Michalkiewicz L J Huffman J M Connors and G AHedge ldquoAlterations in thyroid blood flow induced by varyinglevels of iodine intake in the ratrdquo Endocrinology vol 125 no 1pp 54ndash60 1989

[32] P H K Eng G R Cardona S-L Fang et al ldquoEscape fromthe acute Wolff-Chaikoff effect is associated with a decrease inthyroid sodiumiodide symporter messenger ribonucleic acidand proteinrdquo Endocrinology vol 140 no 8 pp 3404ndash3410 1999

[33] P Mooij H J De Wit and H A Drexhage ldquoA high iodineintake in Wistar rats results in the development of a thyroid-associated ectopic thymic tissue and is accompanied by a lowthyroid autoimmune reactivityrdquo Immunology vol 81 no 2 pp309ndash316 1994

[34] T Paul B Meyers R J Witorsch et al ldquoThe effect of smallincreases in dietary iodine on thyroid function in euthyroidsubjectsrdquoMetabolism vol 37 no 2 pp 121ndash124 1988

[35] X Teng Z Shan W Teng C Fan H Wang and R GuoldquoExperimental study on the effects of chronic iodine excess onthyroid function structure and autoimmunity in autoimmune-prone NODH-2h4 micerdquo Clinical and Experimental Medicinevol 9 no 1 pp 51ndash59 2009

[36] E Swist Q Chen C Qiao D Caldwell H Gruber and KA Scoggan ldquoExcess dietary iodine differentially affects thyroidgene expression in diabetes thyroiditis-prone versusmdashresistantBioBreeding (BB) ratsrdquoMolecular Nutrition and Food Researchvol 55 no 12 pp 1875ndash1886 2011

[37] L Liu D Wang P Liu et al ldquoThe relationship between iodinenutrition and thyroid disease in lactating women with differentiodine intakesrdquo British Journal of Nutrition vol 114 no 9 pp1487ndash1495 2015

[38] S A Skeaff C D Thomson and R S Gibson ldquoMild iodinedeficiency in a sample of New Zealand school childrenrdquoEuropean Journal of Clinical Nutrition vol 56 no 12 pp 1169ndash1175 2002

[39] M B Zimmermann P L Jooste and C S Pandav ldquoIodine-deficiency disordersrdquo The Lancet vol 372 no 9645 pp 1251ndash1262 2008

[40] J Wolff ldquoExcess iodide inhibits the thyroid by multiple mecha-nismsrdquoAdvances in ExperimentalMedicine andBiology vol 261pp 211ndash244 1989

[41] C Serrano-Nascimento S D S Teixeira J P Nicola R TNachbar A M Masini-Repiso and M T Nunes ldquoThe acuteinhibitory effect of iodide excess on sodiumiodide symporterexpression and activity involves the PI3KAkt signaling path-wayrdquo Endocrinology vol 155 no 3 pp 1145ndash1156 2014

[42] M D Brand C Affourtit T C Esteves et al ldquoMitochondrialsuperoxide production biological effects and activation ofuncoupling proteinsrdquo Free Radical Biology andMedicine vol 37no 6 pp 755ndash767 2004

[43] S H Bae S H Sung H E Lee et al ldquoPeroxiredoxin IIIand sulfiredoxin together protect mice from pyrazole-inducedoxidative liver injuryrdquo Antioxidants amp Redox Signaling vol 17no 10 pp 1351ndash1361 2012

[44] S H Bae S H Sung E J Cho et al ldquoConcerted actionof sulfiredoxin and peroxiredoxin I protects against alcohol-induced oxidative injury inmouse liverrdquoHepatology vol 53 no3 pp 945ndash953 2011

[45] Y S Kim H L Lee K B Lee et al ldquoNuclear factor E2-related factor 2 dependent overexpression of sulfiredoxin and

Oxidative Medicine and Cellular Longevity 13

peroxiredoxin III in human lung cancerrdquo Korean Journal ofInternal Medicine vol 26 no 3 pp 304ndash313 2011

[46] H-Y Cho A E Jedlicka S P M Reddy et al ldquoRole of NRF2in protection against hyperoxic lung injury in micerdquo AmericanJournal of Respiratory Cell and Molecular Biology vol 26 no 2pp 175ndash182 2002

[47] T W Kensler N Wakabayashi and S Biswal ldquoCell survivalresponses to environmental stresses via the Keap1-Nrf2-AREpathwayrdquo Annual Review of Pharmacology and Toxicology vol47 pp 89ndash116 2007

[48] TO AjiboyeM T Yakubu andA TOladiji ldquoElectrophilic andReactive Oxygen Species Detoxification Potentials of ChalconeDimers is Mediated by Redox Transcription Factor Nrf-2rdquoJournal of Biochemical and Molecular Toxicology vol 28 no 1pp 11ndash22 2014

[49] X Yang D Wang Y Ma et al ldquoContinuous activation of Nrf2and its target antioxidant enzymes leads to arsenite-inducedmalignant transformation of human bronchial epithelial cellsrdquoToxicology and Applied Pharmacology vol 289 no 2 pp 231ndash239 2015

[50] C Pang Z Zheng L Shi et al ldquoCaffeic acid preventsacetaminophen-induced liver injury by activating the Keap1-Nrf2 antioxidative defense systemrdquo Free Radical Biology andMedicine vol 91 pp 236ndash246 2016

[51] Y H Noh J Y Baek W Jeong S G Rhee and T-S ChangldquoSulfiredoxin translocation into mitochondria plays a crucialrole in reducing hyperoxidized peroxiredoxin IIIrdquo The Journalof Biological Chemistry vol 284 no 13 pp 8470ndash8477 2009

[52] B Cunniff A N Wozniak P Sweeney K DeCosta and N HHeintz ldquoPeroxiredoxin 3 levels regulate a mitochondrial redoxsetpoint in malignant mesothelioma cellsrdquo Redox Biology vol3 pp 79ndash87 2014

[53] P-J Chua E-H Lee Y Yu G W-C Yip P-H Tan and B-HBay ldquoSilencing the Peroxiredoxin III gene inhibits cell prolifera-tion in breast cancerrdquo International Journal of Oncology vol 36no 2 pp 359ndash364 2010

[54] B Cunniff K Newick K J Nelson et al ldquoDisabling mito-chondrial peroxide metabolism via combinatorial targetingof peroxiredoxin 3 as an effective therapeutic approach formalignant mesotheliomardquo PLOS ONE vol 10 no 5 Article IDe0127310 2015

[55] X Yao M Li J He et al ldquoEffect of early acute high con-centrations of iodide exposure on mitochondrial superoxideproduction in FRTL cellsrdquo Free Radical Biology and Medicinevol 52 no 8 pp 1343ndash1352 2012

[56] L Wang Q Duan T Wang et al ldquoMitochondrial respiratorychain inhibitors involved in ROS production induced by acutehigh concentrations of iodide and the effects of sod as aprotective factorrdquo Oxidative Medicine and Cellular Longevityvol 2015 Article ID 217670 14 pages 2015

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

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Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Oxidative Medicine and Cellular Longevity 9

NI

10 HI

100 HI

Hoechst Nrf2 Merge HoechstHoechst

Day 7 Day 28 Day 14

Nrf2 Merge Nrf2 Merge

Day 7 Day 7 Day 14 Day 14 Day 28 Day 28 20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

(a)

Hoechst Srx Merge Hoechst Srx Merge Hoechst Srx Merge

NI

10 HI

100 HI

Day 7 Day 28 Day 14 Day 7 Day 7 Day 14 Day 14 Day 28 Day 28 20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

20120583m

(b)

Figure 4 Effect of the iodide intake (NI 10 HI and 100 HI) on the changes of immunofluorescence staining on Days 7 14 and 28 (a) In theNI group Nrf2 (red) was localized in the cytoplasm on Days 7 14 and 28 In the 10 HI group the positive staining of Nrf2 can be observedin both the nucleus and the cytoplasm Moreover in the 100 HI group a stronger positive staining of Nrf2 can be detected in the nucleus onDays 7 14 and 28 the nucleus was dyed with Hoechst (blue) (b) Srx (red) positive staining was located in the cytoplasm the nucleus wasdyed with Hoechst (blue) Scale bar 20120583m

peroxidase Prx 3 can be classified as an important regulatorof mitochondrial H2O2 [22] The elevated expression of Prx3 is associated with the blockage of apoptosis increasingcell proliferation and is related to adaptive responses whichare all required to maintain mitochondrial function [53 54]Bae et al have suggested that Prx 3 and Srx jointly protectmice from Pyrazole-induced oxidative liver injury in a Nrf2-dependent manner [43]

The novelty we demonstrated in the present study is thatiodide excess induced both oxidative stress and antioxidativedefense increases throughNrf2-Keap 1 pathway in the thyroidgland from rats We extended our established mechanismsby applying the Nrf2-Keap 1 pathway to set up a bridgebetween oxidative stress and antioxidative defense inducedby iodide excess in the thyroid gland In our previous studywe have established that oxidative stress induced by acutehigh concentrations of iodide in FRTL cells significantlyincreases mitochondrial superoxide production [55] Theinhibitors of themitochondrial respiratory chain complexes I

and III are involved inmitochondrial superoxide productionWe demonstrated that exposure to 100120583M KI for 2 hourssignificantly increased mitochondrial superoxide produc-tion enhanced by either 05120583M Rotenone (an inhibitorof mitochondrial complex I) or 10 120583M Antimycin A (aninhibitor of complex III) [56] We illustrated that 300 120583MPTU (an inhibitor of TPO) attenuated the excessive iodide-induced mitochondrial superoxide production We showedthat 30 120583M KClO4 (a competitive inhibitor of iodide trans-port) relieved the production the mitochondrial superoxideinduced by iodide excess We displayed that 10mUmL TSHcan inhibit excessive iodide-induced strong mitochondrialsuperoxide production [55] MT-I and MT-II are mainlyinvolved in the protection of tissue against oxidative stresseswe indicated that metallothionein-III knockout mice aggra-vated mitochondrial superoxide production in thyroid afterexcessive iodide exposure [24] In addition we demonstratedthat both the oxidative stress and the antioxidative defenseincreased simultaneously after high dosages of iodide intake

10 Oxidative Medicine and Cellular Longevity

Oxidative stress

(a)

(b)

(c)

Antioxidative defense

Wist

ar ra

ts Day 7Day 14Day 28

Normal iodideintake

Normal urinaryiodine

Normal thyroidfunction

10 times iodideintake

10 times urinaryiodine

Normal thyroidfunction

100 times iodideintake

60ndash80 timesurinary iodine

Normal thyroidfunction

Keap 1 Nrf2

Keap 1

Nrf2

Srx

Nucleus

Prx 3

SrxARE

Mitochondria

AntioxidativeOxidativestress

Oxidative Antioxidative

Oxidative Antioxidative

Under physiological conditions(normal iodide intake)

Excessive iodide intake(10 times iodide intake)

Excessive iodide intake(100 times iodide intake)

defense defenseuarrstressuarr

defenseuarruarrstressuarruarr

Figure 5 Proposed mechanisms in the present study (a) The urinary iodine concentration and thyroid function of Wistar rats weredetected (b) The activation of the Nrf2-Keap 1 pathway induced by iodide excess in the thyroid (c) The balance between oxidative stressand antioxidative defense under physiological conditions and excessive iodide intake

Oxidative Medicine and Cellular Longevity 11

We suggested that the Nrf2-Keap 1 pathway is vital for thebalance between oxidative stress and antioxidative defenseinduced by iodide excess in the thyroid gland

Excessive iodide stimulated the Nrf2-Keap 1 pathwayand enhanced the antioxidative defense We found thatthe urinary iodine concentration of the 100 HI group wasabout 60ndash80 times that of the NI group on Days 7 14and 28 however the thyroid functions were normal Weproposed that the excessive iodide accumulated in the bodymay trigger the Nrf2-Keap 1 pathway to maintain a normalthyroid functionWe demonstrated that the Nrf2moves fromthe cytoplasm to the nucleus under the microscope withsignificantly increased expressions of Nrf2 Srx and Prx 3 andnotably decreased Keap 1 when exposed to high iodide Thissuggests that excessive iodide stimulates the disassociation ofNrf2 from Keap 1 and assists Nrf2 to penetrate the nucleusThen Nrf2 attaches to the antioxidant response element(ARE) to activate the expression of the antioxidative genesSrx and Prx 3 resulting in an enhanced antioxidative defenseinduced by high iodide By activation of the Nrf2-Keap 1pathway there is a proportional increase in oxidative stressand antioxidative defense in response to iodide excess It isto be noted that the thyroid function was normal in the 10HI group and 100 HI in present study Inspired by the reportby Poncin et al [6] we proposed that there should be abalance between oxidative stress and antioxidative defense inresponse to iodide excess in the 10 HI and the 100 HI groups

5 Conclusion

In conclusion our results highlight that the activation ofNrf2-Keap 1 Srx and Prx 3 antioxidative defense mecha-nisms may play a crucial role in protecting the thyroid fromiodide excess induced oxidative stress on Days 7 14 and 28(Figure 5)

Abbreviations

NI Normal iodide intakeHI High iodide intakeROS Reactive oxygen speciesH2O2 Hydrogen peroxideNrf2 Nuclear factor erythroid 2-related factor 2Keap 1 Kelch-like ECH-associated protein 1ARE Antioxidant response elementPrx PeroxiredoxinSrx SulfiredoxinPrx 3 Peroxiredoxin 3TT4 Total thyroxineTT3 Total triiodothyronineFT4 Free thyroxineFT3 Free triiodothyronineTSH ThyrotropinLSD Least significant difference

Disclosure

This manuscript has been edited by Umar Iqbal IruniRoshanie Abeysekera Gargi Naha and Tejas Bharadwaj all

of whom are currently studying at the International School ofMedicine Tianjin Medical University China

Competing Interests

The authors declare that there is no conflict of interestsregarding the publication of this article

Acknowledgments

This work is supported by National Natural Science Founda-tion of China (nos 81273009 and 81270182) Tianjin Scienceamp Technology Council Grant of China (nos 16JCYBJC26100and 09JCYBJC11700) and International Studentrsquos Science ampTechnology Innovation Project (Scientific Research Project)of Tianjin Medical University

References

[1] Y Luo A Kawashima Y Ishido et al ldquoIodine excess as anenvironmental risk factor for autoimmune thyroid diseaserdquoInternational Journal of Molecular Sciences vol 15 no 7 pp12895ndash12912 2014

[2] A E Hussein A M Abbas G A El Wakil A Z Elsamanoudyand A A El Aziz ldquoEffect of chronic excess iodine intakeon thyroid function and oxidative stress in hypothyroid ratsrdquoCanadian Journal of Physiology and Pharmacology vol 90 no5 pp 617ndash625 2012

[3] A E Joanta A Filip S Clichici S Andrei and D DaicoviciuldquoIodide excess exerts oxidative stress in some target tissues ofthe thyroid hormonesrdquoActa Physiologica Hungarica vol 93 no4 pp 347ndash359 2006

[4] SMorandMChaaraoui J Kaniewski et al ldquoEffect of iodide onnicotinamide adenine dinucleotide phosphate oxidase activityand Duox2 protein expression in isolated porcine thyroidfolliclesrdquo Endocrinology vol 144 no 4 pp 1241ndash1248 2003

[5] J J Lazaro A Jimenez D Camejo et al ldquoDissecting the inte-grative antioxidant and redox systems in plant mitochondriaEffect of stress and S-nitrosylationrdquo Frontiers in Plant Sciencevol 4 article 460 2013

[6] S Poncin A-C GerardM Boucquey et al ldquoOxidative stress inthe thyroid gland from harmlessness to hazard depending onthe iodine contentrdquo Endocrinology vol 149 no 1 pp 424ndash4332008

[7] M Karbownik and A Lewinski ldquoThe role of oxidative stressin physiological and pathological processes in the thyroidgland possible involvement in pineal-thyroid interactionsrdquoNeuroendocrinology Letters vol 24 no 5 pp 293ndash303 2003

[8] P Thomopoulos ldquoHyperthyroidism due to excess iodinerdquo LaPresse Medicale vol 31 no 35 pp 1664ndash1669 2002

[9] J Wolff ldquoIodide goiter and the pharmacologic effects of excessiodiderdquoTheAmerican Journal of Medicine vol 47 no 1 pp 101ndash124 1969

[10] B Corvilain L Collyn J Van Sande and J E Dumont ldquoStim-ulation by iodide of H2O2 generation in thyroid slices fromseveral speciesrdquoAmerican Journal of PhysiologymdashEndocrinologyand Metabolism vol 278 no 4 pp E692ndashE699 2000

[11] M-C Many C Mestdagh M-F Van den Hove and J-F DenefldquoIn vitro study of acute toxic effects of high iodide doses inhuman thyroid folliclesrdquo Endocrinology vol 131 no 2 pp 621ndash630 1992

12 Oxidative Medicine and Cellular Longevity

[12] J Van Sande G Grenier C Willems and J E DumontldquoInhibition by iodide of the activation of the thyroid cyclic 3101584051015840-AMP systemrdquo Endocrinology vol 96 no 3 pp 781ndash786 1975

[13] R Young J D Hayes K Brown C R Wolf and C B AWhitelaw ldquoPeroxiredoxin gene expression signatures in liverreflect toxic insultrdquo Assay and Drug Development Technologiesvol 8 no 4 pp 512ndash517 2010

[14] Q Ma ldquoRole of Nrf2 in oxidative stress and toxicityrdquo AnnualReview of Pharmacology and Toxicology vol 53 pp 401ndash4262013

[15] B Biteau J Labarre and M B Toledano ldquoATP-dependentreduction of cysteine-sulphinic acid by S cerevisiae sulphire-doxinrdquo Nature vol 425 no 6961 pp 980ndash984 2003

[16] L Wu H Jiang H A Chawsheen et al ldquoTumor promoter-induced sulfiredoxin is required for mouse skin tumorigenesisrdquoCarcinogenesis vol 35 no 5 pp 1177ndash1184 2014

[17] C C Winterbourn and M B Hampton ldquoThiol chemistryand specificity in redox signalingrdquo Free Radical Biology andMedicine vol 45 no 5 pp 549ndash561 2008

[18] J W Baty M B Hampton and C C Winterbourn ldquoProteomicdetection of hydrogen peroxide-sensitive thiol proteins inJurkat cellsrdquo Biochemical Journal vol 389 no 3 pp 785ndash7952005

[19] T-S Chang C-S Cho S Park S YuW K Sang and G R SueldquoPeroxiredoxin III a mitochondrion-specific peroxidase regu-lates apoptotic signaling by mitochondriardquo Journal of BiologicalChemistry vol 279 no 40 pp 41975ndash41984 2004

[20] P Thomopoulos ldquoHyperthyroidism due to excess iodinerdquo LaPresse Medicale vol 31 no 35 pp 1664ndash1669 2002

[21] S Chung X-H Liao C Di Cosmo et al ldquoDisruption of themelanin-concentrating hormone receptor 1 (MCH1R) affectsthyroid functionrdquo Endocrinology vol 153 no 12 pp 6145ndash61542012

[22] G Ozguner and O Sulak ldquoSize and location of thyroid gland inthe fetal periodrdquo Surgical and Radiologic Anatomy vol 36 no4 pp 359ndash367 2014

[23] P L Jooste and E Strydom ldquoMethods for determination ofiodine in urine and saltrdquo Best Practice amp Research ClinicalEndocrinology amp Metabolism vol 24 no 1 pp 77ndash88 2010

[24] N Zhang L Wang Q Duan et al ldquoMetallothionein-IIIknockoutmice aggravatemitochondrial superoxide productionand peroxiredoxin 3 expression in thyroid after excessive iodideexposurerdquoOxidative Medicine and Cellular Longevity vol 2015Article ID 267027 11 pages 2015

[25] P I Gus D Marinho S Zelanis et al ldquoA case-control studyon the oxidative balance of 50 autologous serum eye dropsrdquoOxidative Medicine and Cellular Longevity vol 2016 Article ID9780193 5 pages 2016

[26] P Laurberg C Cerqueira L Ovesen et al ldquoIodine intake as adeterminant of thyroid disorders in populationsrdquo Best Practiceamp Research Clinical Endocrinology amp Metabolism vol 24 no 1pp 13ndash27 2010

[27] H Burgi ldquoIodine excessrdquo Best Practice amp Research ClinicalEndocrinology amp Metabolism vol 24 no 1 pp 107ndash115 2010

[28] E Roti and E D Uberti ldquoIodine excess and hyperthyroidismrdquoThyroid vol 11 no 5 pp 493ndash500 2001

[29] A M Leung and L E Braverman ldquoConsequences of excessiodinerdquo Nature Reviews Endocrinology vol 10 no 3 pp 136ndash142 2014

[30] K Wang Y N Sun J Y Liu et al ldquoThe impact of iodineexcess on thyroid hormone biosynthesis and metabolism in

ratsrdquoBiological Trace Element Research vol 130 no 1 pp 72ndash852009

[31] M Michalkiewicz L J Huffman J M Connors and G AHedge ldquoAlterations in thyroid blood flow induced by varyinglevels of iodine intake in the ratrdquo Endocrinology vol 125 no 1pp 54ndash60 1989

[32] P H K Eng G R Cardona S-L Fang et al ldquoEscape fromthe acute Wolff-Chaikoff effect is associated with a decrease inthyroid sodiumiodide symporter messenger ribonucleic acidand proteinrdquo Endocrinology vol 140 no 8 pp 3404ndash3410 1999

[33] P Mooij H J De Wit and H A Drexhage ldquoA high iodineintake in Wistar rats results in the development of a thyroid-associated ectopic thymic tissue and is accompanied by a lowthyroid autoimmune reactivityrdquo Immunology vol 81 no 2 pp309ndash316 1994

[34] T Paul B Meyers R J Witorsch et al ldquoThe effect of smallincreases in dietary iodine on thyroid function in euthyroidsubjectsrdquoMetabolism vol 37 no 2 pp 121ndash124 1988

[35] X Teng Z Shan W Teng C Fan H Wang and R GuoldquoExperimental study on the effects of chronic iodine excess onthyroid function structure and autoimmunity in autoimmune-prone NODH-2h4 micerdquo Clinical and Experimental Medicinevol 9 no 1 pp 51ndash59 2009

[36] E Swist Q Chen C Qiao D Caldwell H Gruber and KA Scoggan ldquoExcess dietary iodine differentially affects thyroidgene expression in diabetes thyroiditis-prone versusmdashresistantBioBreeding (BB) ratsrdquoMolecular Nutrition and Food Researchvol 55 no 12 pp 1875ndash1886 2011

[37] L Liu D Wang P Liu et al ldquoThe relationship between iodinenutrition and thyroid disease in lactating women with differentiodine intakesrdquo British Journal of Nutrition vol 114 no 9 pp1487ndash1495 2015

[38] S A Skeaff C D Thomson and R S Gibson ldquoMild iodinedeficiency in a sample of New Zealand school childrenrdquoEuropean Journal of Clinical Nutrition vol 56 no 12 pp 1169ndash1175 2002

[39] M B Zimmermann P L Jooste and C S Pandav ldquoIodine-deficiency disordersrdquo The Lancet vol 372 no 9645 pp 1251ndash1262 2008

[40] J Wolff ldquoExcess iodide inhibits the thyroid by multiple mecha-nismsrdquoAdvances in ExperimentalMedicine andBiology vol 261pp 211ndash244 1989

[41] C Serrano-Nascimento S D S Teixeira J P Nicola R TNachbar A M Masini-Repiso and M T Nunes ldquoThe acuteinhibitory effect of iodide excess on sodiumiodide symporterexpression and activity involves the PI3KAkt signaling path-wayrdquo Endocrinology vol 155 no 3 pp 1145ndash1156 2014

[42] M D Brand C Affourtit T C Esteves et al ldquoMitochondrialsuperoxide production biological effects and activation ofuncoupling proteinsrdquo Free Radical Biology andMedicine vol 37no 6 pp 755ndash767 2004

[43] S H Bae S H Sung H E Lee et al ldquoPeroxiredoxin IIIand sulfiredoxin together protect mice from pyrazole-inducedoxidative liver injuryrdquo Antioxidants amp Redox Signaling vol 17no 10 pp 1351ndash1361 2012

[44] S H Bae S H Sung E J Cho et al ldquoConcerted actionof sulfiredoxin and peroxiredoxin I protects against alcohol-induced oxidative injury inmouse liverrdquoHepatology vol 53 no3 pp 945ndash953 2011

[45] Y S Kim H L Lee K B Lee et al ldquoNuclear factor E2-related factor 2 dependent overexpression of sulfiredoxin and

Oxidative Medicine and Cellular Longevity 13

peroxiredoxin III in human lung cancerrdquo Korean Journal ofInternal Medicine vol 26 no 3 pp 304ndash313 2011

[46] H-Y Cho A E Jedlicka S P M Reddy et al ldquoRole of NRF2in protection against hyperoxic lung injury in micerdquo AmericanJournal of Respiratory Cell and Molecular Biology vol 26 no 2pp 175ndash182 2002

[47] T W Kensler N Wakabayashi and S Biswal ldquoCell survivalresponses to environmental stresses via the Keap1-Nrf2-AREpathwayrdquo Annual Review of Pharmacology and Toxicology vol47 pp 89ndash116 2007

[48] TO AjiboyeM T Yakubu andA TOladiji ldquoElectrophilic andReactive Oxygen Species Detoxification Potentials of ChalconeDimers is Mediated by Redox Transcription Factor Nrf-2rdquoJournal of Biochemical and Molecular Toxicology vol 28 no 1pp 11ndash22 2014

[49] X Yang D Wang Y Ma et al ldquoContinuous activation of Nrf2and its target antioxidant enzymes leads to arsenite-inducedmalignant transformation of human bronchial epithelial cellsrdquoToxicology and Applied Pharmacology vol 289 no 2 pp 231ndash239 2015

[50] C Pang Z Zheng L Shi et al ldquoCaffeic acid preventsacetaminophen-induced liver injury by activating the Keap1-Nrf2 antioxidative defense systemrdquo Free Radical Biology andMedicine vol 91 pp 236ndash246 2016

[51] Y H Noh J Y Baek W Jeong S G Rhee and T-S ChangldquoSulfiredoxin translocation into mitochondria plays a crucialrole in reducing hyperoxidized peroxiredoxin IIIrdquo The Journalof Biological Chemistry vol 284 no 13 pp 8470ndash8477 2009

[52] B Cunniff A N Wozniak P Sweeney K DeCosta and N HHeintz ldquoPeroxiredoxin 3 levels regulate a mitochondrial redoxsetpoint in malignant mesothelioma cellsrdquo Redox Biology vol3 pp 79ndash87 2014

[53] P-J Chua E-H Lee Y Yu G W-C Yip P-H Tan and B-HBay ldquoSilencing the Peroxiredoxin III gene inhibits cell prolifera-tion in breast cancerrdquo International Journal of Oncology vol 36no 2 pp 359ndash364 2010

[54] B Cunniff K Newick K J Nelson et al ldquoDisabling mito-chondrial peroxide metabolism via combinatorial targetingof peroxiredoxin 3 as an effective therapeutic approach formalignant mesotheliomardquo PLOS ONE vol 10 no 5 Article IDe0127310 2015

[55] X Yao M Li J He et al ldquoEffect of early acute high con-centrations of iodide exposure on mitochondrial superoxideproduction in FRTL cellsrdquo Free Radical Biology and Medicinevol 52 no 8 pp 1343ndash1352 2012

[56] L Wang Q Duan T Wang et al ldquoMitochondrial respiratorychain inhibitors involved in ROS production induced by acutehigh concentrations of iodide and the effects of sod as aprotective factorrdquo Oxidative Medicine and Cellular Longevityvol 2015 Article ID 217670 14 pages 2015

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

10 Oxidative Medicine and Cellular Longevity

Oxidative stress

(a)

(b)

(c)

Antioxidative defense

Wist

ar ra

ts Day 7Day 14Day 28

Normal iodideintake

Normal urinaryiodine

Normal thyroidfunction

10 times iodideintake

10 times urinaryiodine

Normal thyroidfunction

100 times iodideintake

60ndash80 timesurinary iodine

Normal thyroidfunction

Keap 1 Nrf2

Keap 1

Nrf2

Srx

Nucleus

Prx 3

SrxARE

Mitochondria

AntioxidativeOxidativestress

Oxidative Antioxidative

Oxidative Antioxidative

Under physiological conditions(normal iodide intake)

Excessive iodide intake(10 times iodide intake)

Excessive iodide intake(100 times iodide intake)

defense defenseuarrstressuarr

defenseuarruarrstressuarruarr

Figure 5 Proposed mechanisms in the present study (a) The urinary iodine concentration and thyroid function of Wistar rats weredetected (b) The activation of the Nrf2-Keap 1 pathway induced by iodide excess in the thyroid (c) The balance between oxidative stressand antioxidative defense under physiological conditions and excessive iodide intake

Oxidative Medicine and Cellular Longevity 11

We suggested that the Nrf2-Keap 1 pathway is vital for thebalance between oxidative stress and antioxidative defenseinduced by iodide excess in the thyroid gland

Excessive iodide stimulated the Nrf2-Keap 1 pathwayand enhanced the antioxidative defense We found thatthe urinary iodine concentration of the 100 HI group wasabout 60ndash80 times that of the NI group on Days 7 14and 28 however the thyroid functions were normal Weproposed that the excessive iodide accumulated in the bodymay trigger the Nrf2-Keap 1 pathway to maintain a normalthyroid functionWe demonstrated that the Nrf2moves fromthe cytoplasm to the nucleus under the microscope withsignificantly increased expressions of Nrf2 Srx and Prx 3 andnotably decreased Keap 1 when exposed to high iodide Thissuggests that excessive iodide stimulates the disassociation ofNrf2 from Keap 1 and assists Nrf2 to penetrate the nucleusThen Nrf2 attaches to the antioxidant response element(ARE) to activate the expression of the antioxidative genesSrx and Prx 3 resulting in an enhanced antioxidative defenseinduced by high iodide By activation of the Nrf2-Keap 1pathway there is a proportional increase in oxidative stressand antioxidative defense in response to iodide excess It isto be noted that the thyroid function was normal in the 10HI group and 100 HI in present study Inspired by the reportby Poncin et al [6] we proposed that there should be abalance between oxidative stress and antioxidative defense inresponse to iodide excess in the 10 HI and the 100 HI groups

5 Conclusion

In conclusion our results highlight that the activation ofNrf2-Keap 1 Srx and Prx 3 antioxidative defense mecha-nisms may play a crucial role in protecting the thyroid fromiodide excess induced oxidative stress on Days 7 14 and 28(Figure 5)

Abbreviations

NI Normal iodide intakeHI High iodide intakeROS Reactive oxygen speciesH2O2 Hydrogen peroxideNrf2 Nuclear factor erythroid 2-related factor 2Keap 1 Kelch-like ECH-associated protein 1ARE Antioxidant response elementPrx PeroxiredoxinSrx SulfiredoxinPrx 3 Peroxiredoxin 3TT4 Total thyroxineTT3 Total triiodothyronineFT4 Free thyroxineFT3 Free triiodothyronineTSH ThyrotropinLSD Least significant difference

Disclosure

This manuscript has been edited by Umar Iqbal IruniRoshanie Abeysekera Gargi Naha and Tejas Bharadwaj all

of whom are currently studying at the International School ofMedicine Tianjin Medical University China

Competing Interests

The authors declare that there is no conflict of interestsregarding the publication of this article

Acknowledgments

This work is supported by National Natural Science Founda-tion of China (nos 81273009 and 81270182) Tianjin Scienceamp Technology Council Grant of China (nos 16JCYBJC26100and 09JCYBJC11700) and International Studentrsquos Science ampTechnology Innovation Project (Scientific Research Project)of Tianjin Medical University

References

[1] Y Luo A Kawashima Y Ishido et al ldquoIodine excess as anenvironmental risk factor for autoimmune thyroid diseaserdquoInternational Journal of Molecular Sciences vol 15 no 7 pp12895ndash12912 2014

[2] A E Hussein A M Abbas G A El Wakil A Z Elsamanoudyand A A El Aziz ldquoEffect of chronic excess iodine intakeon thyroid function and oxidative stress in hypothyroid ratsrdquoCanadian Journal of Physiology and Pharmacology vol 90 no5 pp 617ndash625 2012

[3] A E Joanta A Filip S Clichici S Andrei and D DaicoviciuldquoIodide excess exerts oxidative stress in some target tissues ofthe thyroid hormonesrdquoActa Physiologica Hungarica vol 93 no4 pp 347ndash359 2006

[4] SMorandMChaaraoui J Kaniewski et al ldquoEffect of iodide onnicotinamide adenine dinucleotide phosphate oxidase activityand Duox2 protein expression in isolated porcine thyroidfolliclesrdquo Endocrinology vol 144 no 4 pp 1241ndash1248 2003

[5] J J Lazaro A Jimenez D Camejo et al ldquoDissecting the inte-grative antioxidant and redox systems in plant mitochondriaEffect of stress and S-nitrosylationrdquo Frontiers in Plant Sciencevol 4 article 460 2013

[6] S Poncin A-C GerardM Boucquey et al ldquoOxidative stress inthe thyroid gland from harmlessness to hazard depending onthe iodine contentrdquo Endocrinology vol 149 no 1 pp 424ndash4332008

[7] M Karbownik and A Lewinski ldquoThe role of oxidative stressin physiological and pathological processes in the thyroidgland possible involvement in pineal-thyroid interactionsrdquoNeuroendocrinology Letters vol 24 no 5 pp 293ndash303 2003

[8] P Thomopoulos ldquoHyperthyroidism due to excess iodinerdquo LaPresse Medicale vol 31 no 35 pp 1664ndash1669 2002

[9] J Wolff ldquoIodide goiter and the pharmacologic effects of excessiodiderdquoTheAmerican Journal of Medicine vol 47 no 1 pp 101ndash124 1969

[10] B Corvilain L Collyn J Van Sande and J E Dumont ldquoStim-ulation by iodide of H2O2 generation in thyroid slices fromseveral speciesrdquoAmerican Journal of PhysiologymdashEndocrinologyand Metabolism vol 278 no 4 pp E692ndashE699 2000

[11] M-C Many C Mestdagh M-F Van den Hove and J-F DenefldquoIn vitro study of acute toxic effects of high iodide doses inhuman thyroid folliclesrdquo Endocrinology vol 131 no 2 pp 621ndash630 1992

12 Oxidative Medicine and Cellular Longevity

[12] J Van Sande G Grenier C Willems and J E DumontldquoInhibition by iodide of the activation of the thyroid cyclic 3101584051015840-AMP systemrdquo Endocrinology vol 96 no 3 pp 781ndash786 1975

[13] R Young J D Hayes K Brown C R Wolf and C B AWhitelaw ldquoPeroxiredoxin gene expression signatures in liverreflect toxic insultrdquo Assay and Drug Development Technologiesvol 8 no 4 pp 512ndash517 2010

[14] Q Ma ldquoRole of Nrf2 in oxidative stress and toxicityrdquo AnnualReview of Pharmacology and Toxicology vol 53 pp 401ndash4262013

[15] B Biteau J Labarre and M B Toledano ldquoATP-dependentreduction of cysteine-sulphinic acid by S cerevisiae sulphire-doxinrdquo Nature vol 425 no 6961 pp 980ndash984 2003

[16] L Wu H Jiang H A Chawsheen et al ldquoTumor promoter-induced sulfiredoxin is required for mouse skin tumorigenesisrdquoCarcinogenesis vol 35 no 5 pp 1177ndash1184 2014

[17] C C Winterbourn and M B Hampton ldquoThiol chemistryand specificity in redox signalingrdquo Free Radical Biology andMedicine vol 45 no 5 pp 549ndash561 2008

[18] J W Baty M B Hampton and C C Winterbourn ldquoProteomicdetection of hydrogen peroxide-sensitive thiol proteins inJurkat cellsrdquo Biochemical Journal vol 389 no 3 pp 785ndash7952005

[19] T-S Chang C-S Cho S Park S YuW K Sang and G R SueldquoPeroxiredoxin III a mitochondrion-specific peroxidase regu-lates apoptotic signaling by mitochondriardquo Journal of BiologicalChemistry vol 279 no 40 pp 41975ndash41984 2004

[20] P Thomopoulos ldquoHyperthyroidism due to excess iodinerdquo LaPresse Medicale vol 31 no 35 pp 1664ndash1669 2002

[21] S Chung X-H Liao C Di Cosmo et al ldquoDisruption of themelanin-concentrating hormone receptor 1 (MCH1R) affectsthyroid functionrdquo Endocrinology vol 153 no 12 pp 6145ndash61542012

[22] G Ozguner and O Sulak ldquoSize and location of thyroid gland inthe fetal periodrdquo Surgical and Radiologic Anatomy vol 36 no4 pp 359ndash367 2014

[23] P L Jooste and E Strydom ldquoMethods for determination ofiodine in urine and saltrdquo Best Practice amp Research ClinicalEndocrinology amp Metabolism vol 24 no 1 pp 77ndash88 2010

[24] N Zhang L Wang Q Duan et al ldquoMetallothionein-IIIknockoutmice aggravatemitochondrial superoxide productionand peroxiredoxin 3 expression in thyroid after excessive iodideexposurerdquoOxidative Medicine and Cellular Longevity vol 2015Article ID 267027 11 pages 2015

[25] P I Gus D Marinho S Zelanis et al ldquoA case-control studyon the oxidative balance of 50 autologous serum eye dropsrdquoOxidative Medicine and Cellular Longevity vol 2016 Article ID9780193 5 pages 2016

[26] P Laurberg C Cerqueira L Ovesen et al ldquoIodine intake as adeterminant of thyroid disorders in populationsrdquo Best Practiceamp Research Clinical Endocrinology amp Metabolism vol 24 no 1pp 13ndash27 2010

[27] H Burgi ldquoIodine excessrdquo Best Practice amp Research ClinicalEndocrinology amp Metabolism vol 24 no 1 pp 107ndash115 2010

[28] E Roti and E D Uberti ldquoIodine excess and hyperthyroidismrdquoThyroid vol 11 no 5 pp 493ndash500 2001

[29] A M Leung and L E Braverman ldquoConsequences of excessiodinerdquo Nature Reviews Endocrinology vol 10 no 3 pp 136ndash142 2014

[30] K Wang Y N Sun J Y Liu et al ldquoThe impact of iodineexcess on thyroid hormone biosynthesis and metabolism in

ratsrdquoBiological Trace Element Research vol 130 no 1 pp 72ndash852009

[31] M Michalkiewicz L J Huffman J M Connors and G AHedge ldquoAlterations in thyroid blood flow induced by varyinglevels of iodine intake in the ratrdquo Endocrinology vol 125 no 1pp 54ndash60 1989

[32] P H K Eng G R Cardona S-L Fang et al ldquoEscape fromthe acute Wolff-Chaikoff effect is associated with a decrease inthyroid sodiumiodide symporter messenger ribonucleic acidand proteinrdquo Endocrinology vol 140 no 8 pp 3404ndash3410 1999

[33] P Mooij H J De Wit and H A Drexhage ldquoA high iodineintake in Wistar rats results in the development of a thyroid-associated ectopic thymic tissue and is accompanied by a lowthyroid autoimmune reactivityrdquo Immunology vol 81 no 2 pp309ndash316 1994

[34] T Paul B Meyers R J Witorsch et al ldquoThe effect of smallincreases in dietary iodine on thyroid function in euthyroidsubjectsrdquoMetabolism vol 37 no 2 pp 121ndash124 1988

[35] X Teng Z Shan W Teng C Fan H Wang and R GuoldquoExperimental study on the effects of chronic iodine excess onthyroid function structure and autoimmunity in autoimmune-prone NODH-2h4 micerdquo Clinical and Experimental Medicinevol 9 no 1 pp 51ndash59 2009

[36] E Swist Q Chen C Qiao D Caldwell H Gruber and KA Scoggan ldquoExcess dietary iodine differentially affects thyroidgene expression in diabetes thyroiditis-prone versusmdashresistantBioBreeding (BB) ratsrdquoMolecular Nutrition and Food Researchvol 55 no 12 pp 1875ndash1886 2011

[37] L Liu D Wang P Liu et al ldquoThe relationship between iodinenutrition and thyroid disease in lactating women with differentiodine intakesrdquo British Journal of Nutrition vol 114 no 9 pp1487ndash1495 2015

[38] S A Skeaff C D Thomson and R S Gibson ldquoMild iodinedeficiency in a sample of New Zealand school childrenrdquoEuropean Journal of Clinical Nutrition vol 56 no 12 pp 1169ndash1175 2002

[39] M B Zimmermann P L Jooste and C S Pandav ldquoIodine-deficiency disordersrdquo The Lancet vol 372 no 9645 pp 1251ndash1262 2008

[40] J Wolff ldquoExcess iodide inhibits the thyroid by multiple mecha-nismsrdquoAdvances in ExperimentalMedicine andBiology vol 261pp 211ndash244 1989

[41] C Serrano-Nascimento S D S Teixeira J P Nicola R TNachbar A M Masini-Repiso and M T Nunes ldquoThe acuteinhibitory effect of iodide excess on sodiumiodide symporterexpression and activity involves the PI3KAkt signaling path-wayrdquo Endocrinology vol 155 no 3 pp 1145ndash1156 2014

[42] M D Brand C Affourtit T C Esteves et al ldquoMitochondrialsuperoxide production biological effects and activation ofuncoupling proteinsrdquo Free Radical Biology andMedicine vol 37no 6 pp 755ndash767 2004

[43] S H Bae S H Sung H E Lee et al ldquoPeroxiredoxin IIIand sulfiredoxin together protect mice from pyrazole-inducedoxidative liver injuryrdquo Antioxidants amp Redox Signaling vol 17no 10 pp 1351ndash1361 2012

[44] S H Bae S H Sung E J Cho et al ldquoConcerted actionof sulfiredoxin and peroxiredoxin I protects against alcohol-induced oxidative injury inmouse liverrdquoHepatology vol 53 no3 pp 945ndash953 2011

[45] Y S Kim H L Lee K B Lee et al ldquoNuclear factor E2-related factor 2 dependent overexpression of sulfiredoxin and

Oxidative Medicine and Cellular Longevity 13

peroxiredoxin III in human lung cancerrdquo Korean Journal ofInternal Medicine vol 26 no 3 pp 304ndash313 2011

[46] H-Y Cho A E Jedlicka S P M Reddy et al ldquoRole of NRF2in protection against hyperoxic lung injury in micerdquo AmericanJournal of Respiratory Cell and Molecular Biology vol 26 no 2pp 175ndash182 2002

[47] T W Kensler N Wakabayashi and S Biswal ldquoCell survivalresponses to environmental stresses via the Keap1-Nrf2-AREpathwayrdquo Annual Review of Pharmacology and Toxicology vol47 pp 89ndash116 2007

[48] TO AjiboyeM T Yakubu andA TOladiji ldquoElectrophilic andReactive Oxygen Species Detoxification Potentials of ChalconeDimers is Mediated by Redox Transcription Factor Nrf-2rdquoJournal of Biochemical and Molecular Toxicology vol 28 no 1pp 11ndash22 2014

[49] X Yang D Wang Y Ma et al ldquoContinuous activation of Nrf2and its target antioxidant enzymes leads to arsenite-inducedmalignant transformation of human bronchial epithelial cellsrdquoToxicology and Applied Pharmacology vol 289 no 2 pp 231ndash239 2015

[50] C Pang Z Zheng L Shi et al ldquoCaffeic acid preventsacetaminophen-induced liver injury by activating the Keap1-Nrf2 antioxidative defense systemrdquo Free Radical Biology andMedicine vol 91 pp 236ndash246 2016

[51] Y H Noh J Y Baek W Jeong S G Rhee and T-S ChangldquoSulfiredoxin translocation into mitochondria plays a crucialrole in reducing hyperoxidized peroxiredoxin IIIrdquo The Journalof Biological Chemistry vol 284 no 13 pp 8470ndash8477 2009

[52] B Cunniff A N Wozniak P Sweeney K DeCosta and N HHeintz ldquoPeroxiredoxin 3 levels regulate a mitochondrial redoxsetpoint in malignant mesothelioma cellsrdquo Redox Biology vol3 pp 79ndash87 2014

[53] P-J Chua E-H Lee Y Yu G W-C Yip P-H Tan and B-HBay ldquoSilencing the Peroxiredoxin III gene inhibits cell prolifera-tion in breast cancerrdquo International Journal of Oncology vol 36no 2 pp 359ndash364 2010

[54] B Cunniff K Newick K J Nelson et al ldquoDisabling mito-chondrial peroxide metabolism via combinatorial targetingof peroxiredoxin 3 as an effective therapeutic approach formalignant mesotheliomardquo PLOS ONE vol 10 no 5 Article IDe0127310 2015

[55] X Yao M Li J He et al ldquoEffect of early acute high con-centrations of iodide exposure on mitochondrial superoxideproduction in FRTL cellsrdquo Free Radical Biology and Medicinevol 52 no 8 pp 1343ndash1352 2012

[56] L Wang Q Duan T Wang et al ldquoMitochondrial respiratorychain inhibitors involved in ROS production induced by acutehigh concentrations of iodide and the effects of sod as aprotective factorrdquo Oxidative Medicine and Cellular Longevityvol 2015 Article ID 217670 14 pages 2015

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Oxidative Medicine and Cellular Longevity 11

We suggested that the Nrf2-Keap 1 pathway is vital for thebalance between oxidative stress and antioxidative defenseinduced by iodide excess in the thyroid gland

Excessive iodide stimulated the Nrf2-Keap 1 pathwayand enhanced the antioxidative defense We found thatthe urinary iodine concentration of the 100 HI group wasabout 60ndash80 times that of the NI group on Days 7 14and 28 however the thyroid functions were normal Weproposed that the excessive iodide accumulated in the bodymay trigger the Nrf2-Keap 1 pathway to maintain a normalthyroid functionWe demonstrated that the Nrf2moves fromthe cytoplasm to the nucleus under the microscope withsignificantly increased expressions of Nrf2 Srx and Prx 3 andnotably decreased Keap 1 when exposed to high iodide Thissuggests that excessive iodide stimulates the disassociation ofNrf2 from Keap 1 and assists Nrf2 to penetrate the nucleusThen Nrf2 attaches to the antioxidant response element(ARE) to activate the expression of the antioxidative genesSrx and Prx 3 resulting in an enhanced antioxidative defenseinduced by high iodide By activation of the Nrf2-Keap 1pathway there is a proportional increase in oxidative stressand antioxidative defense in response to iodide excess It isto be noted that the thyroid function was normal in the 10HI group and 100 HI in present study Inspired by the reportby Poncin et al [6] we proposed that there should be abalance between oxidative stress and antioxidative defense inresponse to iodide excess in the 10 HI and the 100 HI groups

5 Conclusion

In conclusion our results highlight that the activation ofNrf2-Keap 1 Srx and Prx 3 antioxidative defense mecha-nisms may play a crucial role in protecting the thyroid fromiodide excess induced oxidative stress on Days 7 14 and 28(Figure 5)

Abbreviations

NI Normal iodide intakeHI High iodide intakeROS Reactive oxygen speciesH2O2 Hydrogen peroxideNrf2 Nuclear factor erythroid 2-related factor 2Keap 1 Kelch-like ECH-associated protein 1ARE Antioxidant response elementPrx PeroxiredoxinSrx SulfiredoxinPrx 3 Peroxiredoxin 3TT4 Total thyroxineTT3 Total triiodothyronineFT4 Free thyroxineFT3 Free triiodothyronineTSH ThyrotropinLSD Least significant difference

Disclosure

This manuscript has been edited by Umar Iqbal IruniRoshanie Abeysekera Gargi Naha and Tejas Bharadwaj all

of whom are currently studying at the International School ofMedicine Tianjin Medical University China

Competing Interests

The authors declare that there is no conflict of interestsregarding the publication of this article

Acknowledgments

This work is supported by National Natural Science Founda-tion of China (nos 81273009 and 81270182) Tianjin Scienceamp Technology Council Grant of China (nos 16JCYBJC26100and 09JCYBJC11700) and International Studentrsquos Science ampTechnology Innovation Project (Scientific Research Project)of Tianjin Medical University

References

[1] Y Luo A Kawashima Y Ishido et al ldquoIodine excess as anenvironmental risk factor for autoimmune thyroid diseaserdquoInternational Journal of Molecular Sciences vol 15 no 7 pp12895ndash12912 2014

[2] A E Hussein A M Abbas G A El Wakil A Z Elsamanoudyand A A El Aziz ldquoEffect of chronic excess iodine intakeon thyroid function and oxidative stress in hypothyroid ratsrdquoCanadian Journal of Physiology and Pharmacology vol 90 no5 pp 617ndash625 2012

[3] A E Joanta A Filip S Clichici S Andrei and D DaicoviciuldquoIodide excess exerts oxidative stress in some target tissues ofthe thyroid hormonesrdquoActa Physiologica Hungarica vol 93 no4 pp 347ndash359 2006

[4] SMorandMChaaraoui J Kaniewski et al ldquoEffect of iodide onnicotinamide adenine dinucleotide phosphate oxidase activityand Duox2 protein expression in isolated porcine thyroidfolliclesrdquo Endocrinology vol 144 no 4 pp 1241ndash1248 2003

[5] J J Lazaro A Jimenez D Camejo et al ldquoDissecting the inte-grative antioxidant and redox systems in plant mitochondriaEffect of stress and S-nitrosylationrdquo Frontiers in Plant Sciencevol 4 article 460 2013

[6] S Poncin A-C GerardM Boucquey et al ldquoOxidative stress inthe thyroid gland from harmlessness to hazard depending onthe iodine contentrdquo Endocrinology vol 149 no 1 pp 424ndash4332008

[7] M Karbownik and A Lewinski ldquoThe role of oxidative stressin physiological and pathological processes in the thyroidgland possible involvement in pineal-thyroid interactionsrdquoNeuroendocrinology Letters vol 24 no 5 pp 293ndash303 2003

[8] P Thomopoulos ldquoHyperthyroidism due to excess iodinerdquo LaPresse Medicale vol 31 no 35 pp 1664ndash1669 2002

[9] J Wolff ldquoIodide goiter and the pharmacologic effects of excessiodiderdquoTheAmerican Journal of Medicine vol 47 no 1 pp 101ndash124 1969

[10] B Corvilain L Collyn J Van Sande and J E Dumont ldquoStim-ulation by iodide of H2O2 generation in thyroid slices fromseveral speciesrdquoAmerican Journal of PhysiologymdashEndocrinologyand Metabolism vol 278 no 4 pp E692ndashE699 2000

[11] M-C Many C Mestdagh M-F Van den Hove and J-F DenefldquoIn vitro study of acute toxic effects of high iodide doses inhuman thyroid folliclesrdquo Endocrinology vol 131 no 2 pp 621ndash630 1992

12 Oxidative Medicine and Cellular Longevity

[12] J Van Sande G Grenier C Willems and J E DumontldquoInhibition by iodide of the activation of the thyroid cyclic 3101584051015840-AMP systemrdquo Endocrinology vol 96 no 3 pp 781ndash786 1975

[13] R Young J D Hayes K Brown C R Wolf and C B AWhitelaw ldquoPeroxiredoxin gene expression signatures in liverreflect toxic insultrdquo Assay and Drug Development Technologiesvol 8 no 4 pp 512ndash517 2010

[14] Q Ma ldquoRole of Nrf2 in oxidative stress and toxicityrdquo AnnualReview of Pharmacology and Toxicology vol 53 pp 401ndash4262013

[15] B Biteau J Labarre and M B Toledano ldquoATP-dependentreduction of cysteine-sulphinic acid by S cerevisiae sulphire-doxinrdquo Nature vol 425 no 6961 pp 980ndash984 2003

[16] L Wu H Jiang H A Chawsheen et al ldquoTumor promoter-induced sulfiredoxin is required for mouse skin tumorigenesisrdquoCarcinogenesis vol 35 no 5 pp 1177ndash1184 2014

[17] C C Winterbourn and M B Hampton ldquoThiol chemistryand specificity in redox signalingrdquo Free Radical Biology andMedicine vol 45 no 5 pp 549ndash561 2008

[18] J W Baty M B Hampton and C C Winterbourn ldquoProteomicdetection of hydrogen peroxide-sensitive thiol proteins inJurkat cellsrdquo Biochemical Journal vol 389 no 3 pp 785ndash7952005

[19] T-S Chang C-S Cho S Park S YuW K Sang and G R SueldquoPeroxiredoxin III a mitochondrion-specific peroxidase regu-lates apoptotic signaling by mitochondriardquo Journal of BiologicalChemistry vol 279 no 40 pp 41975ndash41984 2004

[20] P Thomopoulos ldquoHyperthyroidism due to excess iodinerdquo LaPresse Medicale vol 31 no 35 pp 1664ndash1669 2002

[21] S Chung X-H Liao C Di Cosmo et al ldquoDisruption of themelanin-concentrating hormone receptor 1 (MCH1R) affectsthyroid functionrdquo Endocrinology vol 153 no 12 pp 6145ndash61542012

[22] G Ozguner and O Sulak ldquoSize and location of thyroid gland inthe fetal periodrdquo Surgical and Radiologic Anatomy vol 36 no4 pp 359ndash367 2014

[23] P L Jooste and E Strydom ldquoMethods for determination ofiodine in urine and saltrdquo Best Practice amp Research ClinicalEndocrinology amp Metabolism vol 24 no 1 pp 77ndash88 2010

[24] N Zhang L Wang Q Duan et al ldquoMetallothionein-IIIknockoutmice aggravatemitochondrial superoxide productionand peroxiredoxin 3 expression in thyroid after excessive iodideexposurerdquoOxidative Medicine and Cellular Longevity vol 2015Article ID 267027 11 pages 2015

[25] P I Gus D Marinho S Zelanis et al ldquoA case-control studyon the oxidative balance of 50 autologous serum eye dropsrdquoOxidative Medicine and Cellular Longevity vol 2016 Article ID9780193 5 pages 2016

[26] P Laurberg C Cerqueira L Ovesen et al ldquoIodine intake as adeterminant of thyroid disorders in populationsrdquo Best Practiceamp Research Clinical Endocrinology amp Metabolism vol 24 no 1pp 13ndash27 2010

[27] H Burgi ldquoIodine excessrdquo Best Practice amp Research ClinicalEndocrinology amp Metabolism vol 24 no 1 pp 107ndash115 2010

[28] E Roti and E D Uberti ldquoIodine excess and hyperthyroidismrdquoThyroid vol 11 no 5 pp 493ndash500 2001

[29] A M Leung and L E Braverman ldquoConsequences of excessiodinerdquo Nature Reviews Endocrinology vol 10 no 3 pp 136ndash142 2014

[30] K Wang Y N Sun J Y Liu et al ldquoThe impact of iodineexcess on thyroid hormone biosynthesis and metabolism in

ratsrdquoBiological Trace Element Research vol 130 no 1 pp 72ndash852009

[31] M Michalkiewicz L J Huffman J M Connors and G AHedge ldquoAlterations in thyroid blood flow induced by varyinglevels of iodine intake in the ratrdquo Endocrinology vol 125 no 1pp 54ndash60 1989

[32] P H K Eng G R Cardona S-L Fang et al ldquoEscape fromthe acute Wolff-Chaikoff effect is associated with a decrease inthyroid sodiumiodide symporter messenger ribonucleic acidand proteinrdquo Endocrinology vol 140 no 8 pp 3404ndash3410 1999

[33] P Mooij H J De Wit and H A Drexhage ldquoA high iodineintake in Wistar rats results in the development of a thyroid-associated ectopic thymic tissue and is accompanied by a lowthyroid autoimmune reactivityrdquo Immunology vol 81 no 2 pp309ndash316 1994

[34] T Paul B Meyers R J Witorsch et al ldquoThe effect of smallincreases in dietary iodine on thyroid function in euthyroidsubjectsrdquoMetabolism vol 37 no 2 pp 121ndash124 1988

[35] X Teng Z Shan W Teng C Fan H Wang and R GuoldquoExperimental study on the effects of chronic iodine excess onthyroid function structure and autoimmunity in autoimmune-prone NODH-2h4 micerdquo Clinical and Experimental Medicinevol 9 no 1 pp 51ndash59 2009

[36] E Swist Q Chen C Qiao D Caldwell H Gruber and KA Scoggan ldquoExcess dietary iodine differentially affects thyroidgene expression in diabetes thyroiditis-prone versusmdashresistantBioBreeding (BB) ratsrdquoMolecular Nutrition and Food Researchvol 55 no 12 pp 1875ndash1886 2011

[37] L Liu D Wang P Liu et al ldquoThe relationship between iodinenutrition and thyroid disease in lactating women with differentiodine intakesrdquo British Journal of Nutrition vol 114 no 9 pp1487ndash1495 2015

[38] S A Skeaff C D Thomson and R S Gibson ldquoMild iodinedeficiency in a sample of New Zealand school childrenrdquoEuropean Journal of Clinical Nutrition vol 56 no 12 pp 1169ndash1175 2002

[39] M B Zimmermann P L Jooste and C S Pandav ldquoIodine-deficiency disordersrdquo The Lancet vol 372 no 9645 pp 1251ndash1262 2008

[40] J Wolff ldquoExcess iodide inhibits the thyroid by multiple mecha-nismsrdquoAdvances in ExperimentalMedicine andBiology vol 261pp 211ndash244 1989

[41] C Serrano-Nascimento S D S Teixeira J P Nicola R TNachbar A M Masini-Repiso and M T Nunes ldquoThe acuteinhibitory effect of iodide excess on sodiumiodide symporterexpression and activity involves the PI3KAkt signaling path-wayrdquo Endocrinology vol 155 no 3 pp 1145ndash1156 2014

[42] M D Brand C Affourtit T C Esteves et al ldquoMitochondrialsuperoxide production biological effects and activation ofuncoupling proteinsrdquo Free Radical Biology andMedicine vol 37no 6 pp 755ndash767 2004

[43] S H Bae S H Sung H E Lee et al ldquoPeroxiredoxin IIIand sulfiredoxin together protect mice from pyrazole-inducedoxidative liver injuryrdquo Antioxidants amp Redox Signaling vol 17no 10 pp 1351ndash1361 2012

[44] S H Bae S H Sung E J Cho et al ldquoConcerted actionof sulfiredoxin and peroxiredoxin I protects against alcohol-induced oxidative injury inmouse liverrdquoHepatology vol 53 no3 pp 945ndash953 2011

[45] Y S Kim H L Lee K B Lee et al ldquoNuclear factor E2-related factor 2 dependent overexpression of sulfiredoxin and

Oxidative Medicine and Cellular Longevity 13

peroxiredoxin III in human lung cancerrdquo Korean Journal ofInternal Medicine vol 26 no 3 pp 304ndash313 2011

[46] H-Y Cho A E Jedlicka S P M Reddy et al ldquoRole of NRF2in protection against hyperoxic lung injury in micerdquo AmericanJournal of Respiratory Cell and Molecular Biology vol 26 no 2pp 175ndash182 2002

[47] T W Kensler N Wakabayashi and S Biswal ldquoCell survivalresponses to environmental stresses via the Keap1-Nrf2-AREpathwayrdquo Annual Review of Pharmacology and Toxicology vol47 pp 89ndash116 2007

[48] TO AjiboyeM T Yakubu andA TOladiji ldquoElectrophilic andReactive Oxygen Species Detoxification Potentials of ChalconeDimers is Mediated by Redox Transcription Factor Nrf-2rdquoJournal of Biochemical and Molecular Toxicology vol 28 no 1pp 11ndash22 2014

[49] X Yang D Wang Y Ma et al ldquoContinuous activation of Nrf2and its target antioxidant enzymes leads to arsenite-inducedmalignant transformation of human bronchial epithelial cellsrdquoToxicology and Applied Pharmacology vol 289 no 2 pp 231ndash239 2015

[50] C Pang Z Zheng L Shi et al ldquoCaffeic acid preventsacetaminophen-induced liver injury by activating the Keap1-Nrf2 antioxidative defense systemrdquo Free Radical Biology andMedicine vol 91 pp 236ndash246 2016

[51] Y H Noh J Y Baek W Jeong S G Rhee and T-S ChangldquoSulfiredoxin translocation into mitochondria plays a crucialrole in reducing hyperoxidized peroxiredoxin IIIrdquo The Journalof Biological Chemistry vol 284 no 13 pp 8470ndash8477 2009

[52] B Cunniff A N Wozniak P Sweeney K DeCosta and N HHeintz ldquoPeroxiredoxin 3 levels regulate a mitochondrial redoxsetpoint in malignant mesothelioma cellsrdquo Redox Biology vol3 pp 79ndash87 2014

[53] P-J Chua E-H Lee Y Yu G W-C Yip P-H Tan and B-HBay ldquoSilencing the Peroxiredoxin III gene inhibits cell prolifera-tion in breast cancerrdquo International Journal of Oncology vol 36no 2 pp 359ndash364 2010

[54] B Cunniff K Newick K J Nelson et al ldquoDisabling mito-chondrial peroxide metabolism via combinatorial targetingof peroxiredoxin 3 as an effective therapeutic approach formalignant mesotheliomardquo PLOS ONE vol 10 no 5 Article IDe0127310 2015

[55] X Yao M Li J He et al ldquoEffect of early acute high con-centrations of iodide exposure on mitochondrial superoxideproduction in FRTL cellsrdquo Free Radical Biology and Medicinevol 52 no 8 pp 1343ndash1352 2012

[56] L Wang Q Duan T Wang et al ldquoMitochondrial respiratorychain inhibitors involved in ROS production induced by acutehigh concentrations of iodide and the effects of sod as aprotective factorrdquo Oxidative Medicine and Cellular Longevityvol 2015 Article ID 217670 14 pages 2015

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

12 Oxidative Medicine and Cellular Longevity

[12] J Van Sande G Grenier C Willems and J E DumontldquoInhibition by iodide of the activation of the thyroid cyclic 3101584051015840-AMP systemrdquo Endocrinology vol 96 no 3 pp 781ndash786 1975

[13] R Young J D Hayes K Brown C R Wolf and C B AWhitelaw ldquoPeroxiredoxin gene expression signatures in liverreflect toxic insultrdquo Assay and Drug Development Technologiesvol 8 no 4 pp 512ndash517 2010

[14] Q Ma ldquoRole of Nrf2 in oxidative stress and toxicityrdquo AnnualReview of Pharmacology and Toxicology vol 53 pp 401ndash4262013

[15] B Biteau J Labarre and M B Toledano ldquoATP-dependentreduction of cysteine-sulphinic acid by S cerevisiae sulphire-doxinrdquo Nature vol 425 no 6961 pp 980ndash984 2003

[16] L Wu H Jiang H A Chawsheen et al ldquoTumor promoter-induced sulfiredoxin is required for mouse skin tumorigenesisrdquoCarcinogenesis vol 35 no 5 pp 1177ndash1184 2014

[17] C C Winterbourn and M B Hampton ldquoThiol chemistryand specificity in redox signalingrdquo Free Radical Biology andMedicine vol 45 no 5 pp 549ndash561 2008

[18] J W Baty M B Hampton and C C Winterbourn ldquoProteomicdetection of hydrogen peroxide-sensitive thiol proteins inJurkat cellsrdquo Biochemical Journal vol 389 no 3 pp 785ndash7952005

[19] T-S Chang C-S Cho S Park S YuW K Sang and G R SueldquoPeroxiredoxin III a mitochondrion-specific peroxidase regu-lates apoptotic signaling by mitochondriardquo Journal of BiologicalChemistry vol 279 no 40 pp 41975ndash41984 2004

[20] P Thomopoulos ldquoHyperthyroidism due to excess iodinerdquo LaPresse Medicale vol 31 no 35 pp 1664ndash1669 2002

[21] S Chung X-H Liao C Di Cosmo et al ldquoDisruption of themelanin-concentrating hormone receptor 1 (MCH1R) affectsthyroid functionrdquo Endocrinology vol 153 no 12 pp 6145ndash61542012

[22] G Ozguner and O Sulak ldquoSize and location of thyroid gland inthe fetal periodrdquo Surgical and Radiologic Anatomy vol 36 no4 pp 359ndash367 2014

[23] P L Jooste and E Strydom ldquoMethods for determination ofiodine in urine and saltrdquo Best Practice amp Research ClinicalEndocrinology amp Metabolism vol 24 no 1 pp 77ndash88 2010

[24] N Zhang L Wang Q Duan et al ldquoMetallothionein-IIIknockoutmice aggravatemitochondrial superoxide productionand peroxiredoxin 3 expression in thyroid after excessive iodideexposurerdquoOxidative Medicine and Cellular Longevity vol 2015Article ID 267027 11 pages 2015

[25] P I Gus D Marinho S Zelanis et al ldquoA case-control studyon the oxidative balance of 50 autologous serum eye dropsrdquoOxidative Medicine and Cellular Longevity vol 2016 Article ID9780193 5 pages 2016

[26] P Laurberg C Cerqueira L Ovesen et al ldquoIodine intake as adeterminant of thyroid disorders in populationsrdquo Best Practiceamp Research Clinical Endocrinology amp Metabolism vol 24 no 1pp 13ndash27 2010

[27] H Burgi ldquoIodine excessrdquo Best Practice amp Research ClinicalEndocrinology amp Metabolism vol 24 no 1 pp 107ndash115 2010

[28] E Roti and E D Uberti ldquoIodine excess and hyperthyroidismrdquoThyroid vol 11 no 5 pp 493ndash500 2001

[29] A M Leung and L E Braverman ldquoConsequences of excessiodinerdquo Nature Reviews Endocrinology vol 10 no 3 pp 136ndash142 2014

[30] K Wang Y N Sun J Y Liu et al ldquoThe impact of iodineexcess on thyroid hormone biosynthesis and metabolism in

ratsrdquoBiological Trace Element Research vol 130 no 1 pp 72ndash852009

[31] M Michalkiewicz L J Huffman J M Connors and G AHedge ldquoAlterations in thyroid blood flow induced by varyinglevels of iodine intake in the ratrdquo Endocrinology vol 125 no 1pp 54ndash60 1989

[32] P H K Eng G R Cardona S-L Fang et al ldquoEscape fromthe acute Wolff-Chaikoff effect is associated with a decrease inthyroid sodiumiodide symporter messenger ribonucleic acidand proteinrdquo Endocrinology vol 140 no 8 pp 3404ndash3410 1999

[33] P Mooij H J De Wit and H A Drexhage ldquoA high iodineintake in Wistar rats results in the development of a thyroid-associated ectopic thymic tissue and is accompanied by a lowthyroid autoimmune reactivityrdquo Immunology vol 81 no 2 pp309ndash316 1994

[34] T Paul B Meyers R J Witorsch et al ldquoThe effect of smallincreases in dietary iodine on thyroid function in euthyroidsubjectsrdquoMetabolism vol 37 no 2 pp 121ndash124 1988

[35] X Teng Z Shan W Teng C Fan H Wang and R GuoldquoExperimental study on the effects of chronic iodine excess onthyroid function structure and autoimmunity in autoimmune-prone NODH-2h4 micerdquo Clinical and Experimental Medicinevol 9 no 1 pp 51ndash59 2009

[36] E Swist Q Chen C Qiao D Caldwell H Gruber and KA Scoggan ldquoExcess dietary iodine differentially affects thyroidgene expression in diabetes thyroiditis-prone versusmdashresistantBioBreeding (BB) ratsrdquoMolecular Nutrition and Food Researchvol 55 no 12 pp 1875ndash1886 2011

[37] L Liu D Wang P Liu et al ldquoThe relationship between iodinenutrition and thyroid disease in lactating women with differentiodine intakesrdquo British Journal of Nutrition vol 114 no 9 pp1487ndash1495 2015

[38] S A Skeaff C D Thomson and R S Gibson ldquoMild iodinedeficiency in a sample of New Zealand school childrenrdquoEuropean Journal of Clinical Nutrition vol 56 no 12 pp 1169ndash1175 2002

[39] M B Zimmermann P L Jooste and C S Pandav ldquoIodine-deficiency disordersrdquo The Lancet vol 372 no 9645 pp 1251ndash1262 2008

[40] J Wolff ldquoExcess iodide inhibits the thyroid by multiple mecha-nismsrdquoAdvances in ExperimentalMedicine andBiology vol 261pp 211ndash244 1989

[41] C Serrano-Nascimento S D S Teixeira J P Nicola R TNachbar A M Masini-Repiso and M T Nunes ldquoThe acuteinhibitory effect of iodide excess on sodiumiodide symporterexpression and activity involves the PI3KAkt signaling path-wayrdquo Endocrinology vol 155 no 3 pp 1145ndash1156 2014

[42] M D Brand C Affourtit T C Esteves et al ldquoMitochondrialsuperoxide production biological effects and activation ofuncoupling proteinsrdquo Free Radical Biology andMedicine vol 37no 6 pp 755ndash767 2004

[43] S H Bae S H Sung H E Lee et al ldquoPeroxiredoxin IIIand sulfiredoxin together protect mice from pyrazole-inducedoxidative liver injuryrdquo Antioxidants amp Redox Signaling vol 17no 10 pp 1351ndash1361 2012

[44] S H Bae S H Sung E J Cho et al ldquoConcerted actionof sulfiredoxin and peroxiredoxin I protects against alcohol-induced oxidative injury inmouse liverrdquoHepatology vol 53 no3 pp 945ndash953 2011

[45] Y S Kim H L Lee K B Lee et al ldquoNuclear factor E2-related factor 2 dependent overexpression of sulfiredoxin and

Oxidative Medicine and Cellular Longevity 13

peroxiredoxin III in human lung cancerrdquo Korean Journal ofInternal Medicine vol 26 no 3 pp 304ndash313 2011

[46] H-Y Cho A E Jedlicka S P M Reddy et al ldquoRole of NRF2in protection against hyperoxic lung injury in micerdquo AmericanJournal of Respiratory Cell and Molecular Biology vol 26 no 2pp 175ndash182 2002

[47] T W Kensler N Wakabayashi and S Biswal ldquoCell survivalresponses to environmental stresses via the Keap1-Nrf2-AREpathwayrdquo Annual Review of Pharmacology and Toxicology vol47 pp 89ndash116 2007

[48] TO AjiboyeM T Yakubu andA TOladiji ldquoElectrophilic andReactive Oxygen Species Detoxification Potentials of ChalconeDimers is Mediated by Redox Transcription Factor Nrf-2rdquoJournal of Biochemical and Molecular Toxicology vol 28 no 1pp 11ndash22 2014

[49] X Yang D Wang Y Ma et al ldquoContinuous activation of Nrf2and its target antioxidant enzymes leads to arsenite-inducedmalignant transformation of human bronchial epithelial cellsrdquoToxicology and Applied Pharmacology vol 289 no 2 pp 231ndash239 2015

[50] C Pang Z Zheng L Shi et al ldquoCaffeic acid preventsacetaminophen-induced liver injury by activating the Keap1-Nrf2 antioxidative defense systemrdquo Free Radical Biology andMedicine vol 91 pp 236ndash246 2016

[51] Y H Noh J Y Baek W Jeong S G Rhee and T-S ChangldquoSulfiredoxin translocation into mitochondria plays a crucialrole in reducing hyperoxidized peroxiredoxin IIIrdquo The Journalof Biological Chemistry vol 284 no 13 pp 8470ndash8477 2009

[52] B Cunniff A N Wozniak P Sweeney K DeCosta and N HHeintz ldquoPeroxiredoxin 3 levels regulate a mitochondrial redoxsetpoint in malignant mesothelioma cellsrdquo Redox Biology vol3 pp 79ndash87 2014

[53] P-J Chua E-H Lee Y Yu G W-C Yip P-H Tan and B-HBay ldquoSilencing the Peroxiredoxin III gene inhibits cell prolifera-tion in breast cancerrdquo International Journal of Oncology vol 36no 2 pp 359ndash364 2010

[54] B Cunniff K Newick K J Nelson et al ldquoDisabling mito-chondrial peroxide metabolism via combinatorial targetingof peroxiredoxin 3 as an effective therapeutic approach formalignant mesotheliomardquo PLOS ONE vol 10 no 5 Article IDe0127310 2015

[55] X Yao M Li J He et al ldquoEffect of early acute high con-centrations of iodide exposure on mitochondrial superoxideproduction in FRTL cellsrdquo Free Radical Biology and Medicinevol 52 no 8 pp 1343ndash1352 2012

[56] L Wang Q Duan T Wang et al ldquoMitochondrial respiratorychain inhibitors involved in ROS production induced by acutehigh concentrations of iodide and the effects of sod as aprotective factorrdquo Oxidative Medicine and Cellular Longevityvol 2015 Article ID 217670 14 pages 2015

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Oxidative Medicine and Cellular Longevity 13

peroxiredoxin III in human lung cancerrdquo Korean Journal ofInternal Medicine vol 26 no 3 pp 304ndash313 2011

[46] H-Y Cho A E Jedlicka S P M Reddy et al ldquoRole of NRF2in protection against hyperoxic lung injury in micerdquo AmericanJournal of Respiratory Cell and Molecular Biology vol 26 no 2pp 175ndash182 2002

[47] T W Kensler N Wakabayashi and S Biswal ldquoCell survivalresponses to environmental stresses via the Keap1-Nrf2-AREpathwayrdquo Annual Review of Pharmacology and Toxicology vol47 pp 89ndash116 2007

[48] TO AjiboyeM T Yakubu andA TOladiji ldquoElectrophilic andReactive Oxygen Species Detoxification Potentials of ChalconeDimers is Mediated by Redox Transcription Factor Nrf-2rdquoJournal of Biochemical and Molecular Toxicology vol 28 no 1pp 11ndash22 2014

[49] X Yang D Wang Y Ma et al ldquoContinuous activation of Nrf2and its target antioxidant enzymes leads to arsenite-inducedmalignant transformation of human bronchial epithelial cellsrdquoToxicology and Applied Pharmacology vol 289 no 2 pp 231ndash239 2015

[50] C Pang Z Zheng L Shi et al ldquoCaffeic acid preventsacetaminophen-induced liver injury by activating the Keap1-Nrf2 antioxidative defense systemrdquo Free Radical Biology andMedicine vol 91 pp 236ndash246 2016

[51] Y H Noh J Y Baek W Jeong S G Rhee and T-S ChangldquoSulfiredoxin translocation into mitochondria plays a crucialrole in reducing hyperoxidized peroxiredoxin IIIrdquo The Journalof Biological Chemistry vol 284 no 13 pp 8470ndash8477 2009

[52] B Cunniff A N Wozniak P Sweeney K DeCosta and N HHeintz ldquoPeroxiredoxin 3 levels regulate a mitochondrial redoxsetpoint in malignant mesothelioma cellsrdquo Redox Biology vol3 pp 79ndash87 2014

[53] P-J Chua E-H Lee Y Yu G W-C Yip P-H Tan and B-HBay ldquoSilencing the Peroxiredoxin III gene inhibits cell prolifera-tion in breast cancerrdquo International Journal of Oncology vol 36no 2 pp 359ndash364 2010

[54] B Cunniff K Newick K J Nelson et al ldquoDisabling mito-chondrial peroxide metabolism via combinatorial targetingof peroxiredoxin 3 as an effective therapeutic approach formalignant mesotheliomardquo PLOS ONE vol 10 no 5 Article IDe0127310 2015

[55] X Yao M Li J He et al ldquoEffect of early acute high con-centrations of iodide exposure on mitochondrial superoxideproduction in FRTL cellsrdquo Free Radical Biology and Medicinevol 52 no 8 pp 1343ndash1352 2012

[56] L Wang Q Duan T Wang et al ldquoMitochondrial respiratorychain inhibitors involved in ROS production induced by acutehigh concentrations of iodide and the effects of sod as aprotective factorrdquo Oxidative Medicine and Cellular Longevityvol 2015 Article ID 217670 14 pages 2015

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom