research article metallothionein-i/ii knockout mice

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Research Article Metallothionein-I/II Knockout Mice Aggravate Mitochondrial Superoxide Production and Peroxiredoxin 3 Expression in Thyroid after Excessive Iodide Exposure Na Zhang, 1 Lingyan Wang, 1 Qi Duan, 1 Laixiang Lin, 2 Mohamed Ahmed, 1 Tingting Wang, 1 and Xiaomei Yao 1 1 Department of Pathophysiology, School of Basic Medical Sciences, Tianjin Medical University, Tianjin 300070, China 2 Key Lab of Hormones and Development of Ministry of Health, Institute of Endocrinology, Metabolic Disease Hospital, Tianjin Medical University, Tianjin 300070, China Correspondence should be addressed to Xiaomei Yao; [email protected] Received 24 October 2014; Revised 13 January 2015; Accepted 11 February 2015 Academic Editor: Kaushik Biswas Copyright © 2015 Na Zhang et al. is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Purpose. We aim to figure out the effect of metallothioneins on iodide excess induced oxidative stress in the thyroid. Methods. Eight-week-old MT-I/II knockout (MT-I/II KO) mice and background-matched wild-type (WT) mice were used. Mitochondrial superoxide production and peroxiredoxin (Prx) 3 expression were measured. Results. In in vitro study, more significant increases in mitochondrial superoxide production and Prx 3 expression were detected in the MT-I/II KO groups. In in vivo study, significantly higher concentrations of urinary iodine level were detected in MT-I/II KO mice in 100 HI group. Compared to the NI group, there was no significant difference existing in serum thyroid hormones level in either groups ( > 0.05), while the mitochondrial superoxide production was significantly increased in 100 HI groups with significantly increased LDH activity and decreased relative cell viability. Compared to WT mice, more significant changes were detected in MT-I/II KO mice in 100 HI groups. No significant differences were detected between the NI group and 10 HI group in both the MT-I/II KO and WT mice groups ( > 0.05). Conclusions. Iodide excess in a thyroid without MT I/II protection may result in strong mitochondrial oxidative stress, which further leads to the damage of thyrocytes. 1. Introduction Iodine is a fundamental constituent of thyroid hormones and has stimulatory effects on hydrogen peroxide (H 2 O 2 ) generation, which is involved in oxidative stress [1, 2]. 150 g iodine is the daily requirement for thyroid hormone synthesis [3]. e International Council for the Control of Iodine Deficiency Disorders proposed that 150–299 g/day is adequate to cover the thyroid requirement, and the maximal allowable dietary dose of iodine is 1.0 mg/day for children and 2.0 mg/day for adults [4]. However, concentrations of iodine can be up to several hundredfold to thousandfold higher than the above amounts [5]. Intakes of up to 600 mg per day in the European Union and 1100mg per day in the United States are declared as tolerable for adults [6]. Iodide excess has been demonstrated to be toxic to the thyroid [710] and may lead to hypothyroidism, hyperthyroidism, euthyroid goiter, or thyroid autoimmunity [3, 1113], with oxidative stress being one of the underlying mechanisms [14, 15]. Animal models with different iodine intake dietary were established in mice [1618] and in rats [19, 20] to study the effects of excess iodine on the thyroid. Humans may get exposed to excess iodine through medications or diet. Substances involved in iodine excess include iodine-containing drugs, amiodarone being the prime offender. Approximately 9mg of iodine is released following a daily dose of 300 mg amiodarone. Other substances include iodpovidone used for topical disinfection and seaweed. Seaweed preparations containing up to 2 mg iodine per gram in protein-bound form and may cause dietary iodine intake to exceed 5000 mg per day in popula- tions with increased consumption [21, 22]. Contrast agents for conventional radiography or computer tomography may Hindawi Publishing Corporation Oxidative Medicine and Cellular Longevity Volume 2015, Article ID 267027, 11 pages http://dx.doi.org/10.1155/2015/267027

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Page 1: Research Article Metallothionein-I/II Knockout Mice

Research ArticleMetallothionein-III Knockout Mice AggravateMitochondrial Superoxide Production and Peroxiredoxin 3Expression in Thyroid after Excessive Iodide Exposure

Na Zhang1 Lingyan Wang1 Qi Duan1 Laixiang Lin2 Mohamed Ahmed1

Tingting Wang1 and Xiaomei Yao1

1Department of Pathophysiology School of Basic Medical Sciences Tianjin Medical University Tianjin 300070 China2Key Lab of Hormones and Development of Ministry of Health Institute of Endocrinology Metabolic Disease HospitalTianjin Medical University Tianjin 300070 China

Correspondence should be addressed to Xiaomei Yao jupx163com

Received 24 October 2014 Revised 13 January 2015 Accepted 11 February 2015

Academic Editor Kaushik Biswas

Copyright copy 2015 Na Zhang et alThis is an open access article distributed under theCreativeCommonsAttribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Purpose We aim to figure out the effect of metallothioneins on iodide excess induced oxidative stress in the thyroid MethodsEight-week-old MT-III knockout (MT-III KO) mice and background-matched wild-type (WT) mice were used Mitochondrialsuperoxide production and peroxiredoxin (Prx) 3 expression were measured Results In in vitro study more significant increases inmitochondrial superoxide production and Prx 3 expression were detected in the MT-III KO groups In in vivo study significantlyhigher concentrations of urinary iodine level were detected in MT-III KO mice in 100 HI group Compared to the NI groupthere was no significant difference existing in serum thyroid hormones level in either groups (119875 gt 005) while the mitochondrialsuperoxide production was significantly increased in 100HI groups with significantly increased LDH activity and decreased relativecell viability Compared to WT mice more significant changes were detected in MT-III KO mice in 100 HI groups No significantdifferences were detected between the NI group and 10 HI group in both the MT-III KO and WT mice groups (119875 gt 005)Conclusions Iodide excess in a thyroid without MT III protection may result in strong mitochondrial oxidative stress whichfurther leads to the damage of thyrocytes

1 Introduction

Iodine is a fundamental constituent of thyroid hormonesand has stimulatory effects on hydrogen peroxide (H

2O2)

generation which is involved in oxidative stress [1 2]150 120583g iodine is the daily requirement for thyroid hormonesynthesis [3] The International Council for the Control ofIodine Deficiency Disorders proposed that 150ndash299 120583gday isadequate to cover the thyroid requirement and the maximalallowable dietary dose of iodine is 10mgday for children and20mgday for adults [4] However concentrations of iodinecan be up to several hundredfold to thousandfold higherthan the above amounts [5] Intakes of up to 600mg per dayin the European Union and 1100mg per day in the UnitedStates are declared as tolerable for adults [6] Iodide excess hasbeen demonstrated to be toxic to the thyroid [7ndash10] and may

lead to hypothyroidism hyperthyroidism euthyroid goiteror thyroid autoimmunity [3 11ndash13] with oxidative stressbeing one of the underlying mechanisms [14 15] Animalmodels with different iodine intake dietary were establishedinmice [16ndash18] and in rats [19 20] to study the effects of excessiodine on the thyroid Humans may get exposed to excessiodine through medications or diet Substances involved iniodine excess include iodine-containing drugs amiodaronebeing the prime offender Approximately 9mg of iodine isreleased following a daily dose of 300mg amiodarone Othersubstances include iodpovidone used for topical disinfectionand seaweed Seaweed preparations containing up to 2mgiodine per gram in protein-bound form and may causedietary iodine intake to exceed 5000mg per day in popula-tions with increased consumption [21 22] Contrast agentsfor conventional radiography or computer tomography may

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2015 Article ID 267027 11 pageshttpdxdoiorg1011552015267027

2 Oxidative Medicine and Cellular Longevity

also lead to iodine excess Radiologists use up to 100 g ormore of iodine in complex organic molecules Erythrosinea food additive used to improve the colour of canned cherriesor of candies contains 57 iodine [2 3] Although iodizedsalt programs have made great progress in the global effort tocontrol iodine deficiency they need to be carefullymonitoredto ensure adequate iodine intake while avoiding iodine excess[20]

The mitochondria contain specific receptors for thyroidhormones and are an important source of reactive oxygenspecies (ROS) [22] Thyrocytes possess various enzymaticsystems such as peroxidise (GPx) catalases superoxidedismutases (SOD) and peroxiredoxins (Prxs) that contributeto limiting cellular injuries [23] Peroxiredoxin 3 (Prx 3) ispredominantly located in the mitochondria and is respon-sible for scavenging ROS Prx 3 can be the target for upto 90 of hydrogen peroxide generated in the matrix [24]Metallothioneins (MTs) have been demonstrated to havestrong antioxidative activity in protecting cells from thedamaging effects of ROSThey too are regarded as scavengersof ROS including superoxide and hydroxyl radicals [25 26]

In light of previous works documented on iodide inducedoxidative stress and thyrotoxicity we have demonstrated thetime-course and concentration-response effects of early acutehigh concentrations of iodide exposure on mitochondrialsuperoxide generation When endogenous antioxidant sys-tems were overwhelmed by high concentrations of iodideinduced ROS production they began attacking proteinslipid and nucleic acids [15] However the nature of iodideexcess induced oxidative stress in thyroid with or withoutMTIII remains to be determined

By targeting mitochondrial superoxide production andPrx 3 protein expression we aimed to figure out the effect ofhigh concentrations of iodide on mitochondrial superoxideproduction and Prx 3 expression in the thyroids of metal-lothionein III knockout (MT-III KO) mice in vitro and invivo We propose that iodide excess in a thyroid without MTIII protection may result in strong mitochondrial oxidativestress which further leads to the damage of thyrocytes

2 Methods

21 Animals andThyroid Cell Suspension Preparation Eight-week-old 129S7SvEvBrd-Mt1tm1BriMt2tm1BriJ (MT-IIIknockout MT-III KO) mice (number 002211 Jackson LabMaineUSA) and background-matchedwild-type (WT)micewere used In the in vitro study a thyroid cell suspension wasprepared A cell suspension (5 times 104 cellswell) was incubatedin medium RPMI-1640 with 10 fetal bovine serum (FBS)at 37∘C in an atmosphere comprising 5 CO

2and 95 air

and exposed to high concentrations of potassium iodide (KI)or 10minus3M H

2O2for 2 hours In vivo MT-III KO and WT

mice were randomly divided into control groups (normaliodide intake NI) 10 times iodide intake groups (10 HI) and100 times iodide intake groups (100 HI) NI groups receiveddeionized water and a normal diet (daily iodide uptake is15 120583gd) In the 10HI and 100HI groups 15 120583gd and 150 120583gdKI were added respectively to the deionized water for 14days before sacrifice Animal procedures were approved by

the Institutional Animal Care and Use Committee of TianjinMedical University and in accordance with the NIH Guide

22 Reagents MitoSOX Red (38-phenanthridinediamine5-(61015840-triphenylphosphoniumhexyl)-56-dihydro-6-phenyl)was purchased from Invitrogen (Invitrogen Life Technolo-gies CA USA) Anti-Peroxiredoxin 3 antibody waspurchased from Abcam (Abcam Cambridge MA USA)120573-Actin Rabbit mAb was purchased from Cell Signaling(Cell Signaling Technology Inc MA USA) 3-(45-Dimeth-ylthiazol-2-yl)-25-diphenyl-tetrazolium bromide (MTT)and TSH were purchased from Sigma (Sigma-AldrichMO USA) RPMI-1640 and FBS were purchased from GEHealthcare Life Sciences (Hyclone UT USA) All the otherchemicals made in China were of analytical grade

23 Cell Viability Assay Cell viability was evaluated withthe MTT assay A cell suspension (5 times 104 cellswell) wasincubated in medium RPMI-1640 with 10 fetal bovineserum (FBS) at 37∘C in an atmosphere comprising 5 CO

2

and 95 air and exposed to high concentrations of potassiumiodide (KI) or 10minus3M H

2O2for 2 hours in order to get a

final concentration of 10minus4M 10minus3M and 10minus2MThe groupwithout KI is the control group the well with only mediumin is regarded as blank group Subsequently 10 120583L of 3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide(MTT 5mgmL)was added in dark and incubated for 4 h andcovered with aluminium foil After incubation for 2 hoursthe supernatant was removed and 100120583L of DMSO wasadded to each well to dissolve the formazan crystals formedand was then shaken for 10min Absorbance was measuredby a spectrophotometer at 490 nm (Wallac 1420 VICTOR3PerkinElmer) [33 34]

24 Lactate Dehydrogenase (LDH) Assay LDH release inthe supernatant following different treatments was mea-sured using a cytotoxicity detection kit (LDH) (NanjingJiancheng Bioengineering Institute Jiangsu China) LDHis an oxidoreductase that catalyzes the interconversion oflactate and pyruvate LDH is a stable cytosolic enzyme thatupon membrane damage is released into cell culture mediaThe assay is based on the reduction of the 2-p-iodophenyl-3-nitrophenyl tetrazolium chloride (tetrazolium INT) to ared formazan which is specifically detected by colorimetric(450 nm) assay

25 Flow Cytometry A mitochondrial superoxide indicator(MitoSOX Red Invitrogen) was used to measure mitochon-drial superoxide production by flow cytometry In vivo thethyroid cell suspension of MT-III KO and WT mice wasprepared for (normal iodide intake NI) 10 times iodideintake groups (10 HI) and 100 times iodide intake groups(100 HI) For the in vitro study a thyroid cell suspensionwas prepared first The thyroid cell suspension was exposedto high concentrations of potassium iodide (KI) (10minus4M10minus3M and 10minus2M) or 10minus3M H

2O2for 2 hours Subse-

quently 5 120583M of MitoSOX was added and incubated for10min at 37∘C in the dark Then the cells were washed

Oxidative Medicine and Cellular Longevity 3

with Hankrsquos solution and were suspended in Hankrsquos solutionwith 1 BSA The fluorescence intensity of MitoSOX wasdetected by a FACSCalibur (BD Bioscience San Jose CA)and the excitationemission wavelength is 488 nm575 nmCollecting FL2 channel forward scattering (forward scatterFSC) and lateral scattering (side scatter SSC) data 10000cells were collected for each sample The control groupwithout MitoSOX was regarded as the blank zero group forstandardization [35ndash38]

26 Western Blot Analysis Whole cell proteins were assayedfor protein concentration using the bicinchoninic acid (BCA)protein assay kit (Beyotime Institute of BiotechnologyJiangsu China) 50120583g of protein was transferred to nitro-cellulose membrane followed by SDS polyacrylamide gelelectrophoresis The nitrocellulose membrane was incubatedovernight with primary antibodies Anti-Peroxiredoxin 3antibody (Abcam Cambridge MA USA) and horseradishperoxidase (HRP) conjugated secondary antibody developedby Immobilon Western Chemiluminescent HRP Substrate(Merck Millipore MA USA) To verify equal loading 120573-actin (1 1000) (Cell Signaling Technology Inc MA USA)was used as a loading control Blots were scanned as grayscaleimages and quantitated using Image J software (NIH) All theblot intensities were normalized with that of loading control120573-actin [39]

27 Determination of Urinary Iodine Concentrations andSerum Thyroid Hormones Spot urine samples of mice werecollected and urinary iodine concentrations were measuredby As-Ce catalytic spectrophotometry in the Key Lab ofHormones and Development Ministry of Health Institute ofEndocrinology Tianjin Medical University [40] Blood wasobtained 14 days after NI 10 HI or 100 HI intake Samplesof blood were centrifuged for 10min serum samples wereobtained and stored at minus80∘C for use Serum concentrationsof thyroxine (T

4) free thyroxine (FT

4) triiodothyronine

(T3) and free triiodothyronine (FT

3) were determined by

using the Direct Chemiluminescence Technology Compet-itive Immunoassay Kits (Siemens Healthcare DiagnosticInc)

28 Statistics The data was represented as mean plusmn SD Basedon the Kolmogorov-Smirnov (K-S) for normality test andLevene statistic for the test of homogeneity of variancesUrinary iodine concentrations were expressed as the medianand determined with the nonparametric Kruskal-Wallis testone-way ANOVA with the least significant difference (LSD)test was performed using SPSS 170 to determine the differ-ences between the groups A 119875 value of less than 005 wasconsidered to be statistically significant

3 Results

31 Effect of MT-III on High Concentrated Iodide ExposureInduced Mitochondrial Superoxide Production and Prx 3 Pro-tein Expression In Vitro To investigate the antioxidative roleof MT-III in oxidative stress induced by high concentrationsof iodide in the thyroid thyroid cell suspensions were

Table 1 The median urinary iodine concentrations (120583gL) of WTmice and MT-III KO mice

Group WT MT-III KONI 32029 plusmn 11216 42433 plusmn 13195

10 HI 359292 plusmn 51998lowast

394924 plusmn 64793lowast

100 HI 3047917 plusmn 412894lowast

4066635 plusmn 352046lowast

lowastCompared to the NI group (119875 lt 005) WT mice group compared to MT-III KO mice group (119875 lt 005)119873 = 10 for each group

prepared from the thyroid of MT-III KO mice and WTmice The cells were then exposed to high concentrationsof KI (10minus4M 10minus3M and 10minus2M) or 10minus3M H

2O2for 2

hours Following 10minus4M 10minus3M and 10minus2M of KI or 10minus3MH2O2exposure we showed a significant increase in mito-

chondrial superoxide production with increased MitoSOXRed fluorescence intensity (119875 lt 005) (Figure 1(d)) and anincrease in LDH release (119875 lt 005) (Figure 1(c)) with adecrease in relative cell viability (119875 lt 005) (Figure 1(a)) in thethyroid cell suspensions of both MT-III KO and WT micegroups Compared to WT mice group a more significantincrease of mitochondrial superoxide production increasein LDH release and decrease in a relative cell viability canbe detected in MT-III KO mice group (119875 lt 005) (Figures1(d) 1(c) and 1(a)) Compared to control group the Prx 3protein expressions were significantly increased in 10minus2MKIor 10minus3MH

2O2exposure group in bothMT-III KO andWT

mice groups (119875 lt 005) Compared to WT mice group therewas a significant increase of Prx 3 protein expression in thethyroid cell suspensions ofMT-III KOmicewith the increaseof KI concentration (119875 lt 005) (Figure 1(b))

32 Urinary Iodine Concentrations In Vivo In order toverify the model of iodine excess in vivo urinary iodineconcentrations were detected after the MT-III KO mice andWT mice had NI 10 HI and 100 HI for 14 days The medianurinary iodine concentrations in NI 10 HI and 100 HI wereshown in Table 1 The high iodide intake of 10 HI group and100HI group in bothMT-III KOmice andWTmice resultedin significant increases in urinary iodine concentration whencompared to theNI group (119875 lt 005) The urinary iodineconcentrations were increased as the iodine supply increasedto 10HI 100HI in bothWT andMT-III KOmiceMoreovercompared to 10 HI group the extent of increase in urinaryiodine concentration in 100 HI was significantly higher (119875 lt005) Compared to WT mice MT -III KO mice had a moreprominent increase of urinary iodine concentration in 100HIgroup (119875 lt 005)

33 Changes of Serum Thyroid Hormones Level in WT andMT-III KO Mice following NI 10 HI or 100 HI Intake for14 Days We demonstrated that compared to the NI groupthere was no significant difference existing in serum thyroidhormones level (T

3 T4 FT3 and FT

4) in either 10 HI

group or 100 HI group in both WT mice and MT-III KOmice (119875 gt 005) However compared to WT mice therewas a significant difference in serum thyroid hormones level

4 Oxidative Medicine and Cellular Longevity

120

100

80

60

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20

0

lowastlowast

Rela

tive v

iabi

lity

()

lowastlowast lowast

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WT miceMT-III KO mice

ControlKI KI KI

10minus4 M 10minus3 M 10minus2 M 10minus3 MH2O2

(a)

lowast lowastlowastlowast3

25

2

15

1

05

0Expr

essio

n of

Prx3

()

Prx 3

120573-actin

WT MT-III KO

WT miceMT-III KO mice

ControlKI KI KI

10minus4 M 10minus3 M 10minus2 M 10minus3 MH2O2

Con

trol

10minus4

M K

I

10minus3

M K

I

10minus2

M K

I

10minus3

MH2O2

Con

trol

10minus4

M K

I

10minus3

M K

I

10minus2

M K

I

10minus3

MH2O2

(b)

6543210

LDH

rele

ase

(Um

g pr

otei

n)

lowast lowast lowast lowast lowast lowastlowast lowast

times103

WT miceMT-III KO mice

ControlKI KI KI

10minus4 M 10minus3 M 10minus2 M 10minus3 MH2O2

(c)

lowastlowast lowast

lowast lowastlowast

lowastlowast120

100806040200M

itoSO

X flu

ores

cenc

e in

tens

ityW

TM

T-II

I KO

Control 262

Control 3644

10minus4 M KI 3754

10minus4 M KI 4931

10minus3 M KI 4925

10minus3 M KI 6447

10minus2 M KI 578

10minus2 M KI 7172

10minus3 M H2 9827

10minus3 M H2O28932

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 101 102 103 104 101 102 103 104100 100

WT miceMT-III KO mice

ControlKI KI KI

10minus4 M 10minus3 M 10minus2 M 10minus3 MH2O2

O2

(d)

Figure 1 Oxidative and antioxidative effect of high concentrations of KI on the mitochondria of thyrocytes in MT-III KO mice and WTmice (a) Decreased relative viability induced by high concentrations of KI (10minus4M 10minus3M and 10minus2M) or 10minus3MH

2O2in the thyroid cells

of MT-III KOmice byMTT assay (119873 = 8) (b) Representative western blot of Prx 3 (27 kDa) 120573-actin (45 kDa) was used as a loading controlDensitometric analysis showed a significant increase (lowast119875 lt 005) in Prx 3 expression compared to untreated control in the thyroid cells ofeither WT or MT-III KO mice (c) Increased LDH release following 2 h of high concentration of KI or H

2O2exposure in the thyroid cells

of MT-III KO mice (119873 = 8) (d) High concentration of KI or H2O2induced increased mitochondrial superoxide production in the thyroid

cells of WT and MT-III KO mice Histogram analysis was performed on the mean fluorescence intensity of MitoSOX Red as measured byflow cytometry Experiments were repeated 3 times with similar results Data are represented as mean plusmn SD One-way ANOVA with the LSDtest was used lowast119875 lt 005 compared with the control group of WT or MT-III KO mice respectively 119875 lt 005WTmice versus MT-III KOmice under the same treatment

(FT4) in 100 HI group in MT-III KO mice (119875 lt 005)

(Table 2)

34 High Iodide Intake Induced Increased MitochondrialSuperoxide Production and Peroxiredoxin 3 Expression inThyroid In Vivo To clarify the effect of iodide excess onmitochondrial superoxide production and Prx 3 protein

expression in thyroid of MT-III KO mice in vivo micewere divided into normal iodide intake (NI) 10 times iodideintake (10 HI) and 100 times iodide intake (100 HI) groupsCompared to the NI group the mitochondrial superoxideproduction measured by flow cytometry Prx 3 proteinexpression detected bywestern blot analysis and LDH releasein 100 HI group were significantly increased in both MT-III

Oxidative Medicine and Cellular Longevity 5

Table 2 Changes of serum thyroid hormones level (nmolL) in WT and MT-III KO mice following NI 10 HI or 100 HI intake for 14 days

Group T3 T4 FT3 FT4

WTNI 072 plusmn 013 2608 plusmn 1421 480 plusmn 042 2856 plusmn 451

10 HI 053 plusmn 009 3350 plusmn 445 426 plusmn 041 3142 plusmn 095

100 HI 071 plusmn 013 3470 plusmn 1324 461 plusmn 025 3320 plusmn 455

MT-III KONI 058 plusmn 015 2005 plusmn 468 404 plusmn 077 2569 plusmn 097

10 HI 069 plusmn 013 2253 plusmn 779 478 plusmn 005 2722 plusmn 103

100 HI 080 plusmn 022 1773 plusmn 676 501 plusmn 073 2399 plusmn 192lowast

lowastIn 100 HI group compared to WT mice there was a significant difference in serum thyroid hormones level (FT4) in MT-III KO mice (119875 lt 005)119873 = 8 foreach group

KO and WT groups (119875 lt 005) Compared to WT groupmore significant increases in the mitochondrial superoxideproduction Prx 3 protein expression and LDH release weredetected in MT-III KO group (119875 lt 005) There were nosignificant differences between the NI group and 10 HI groupin bothMT-III KO andWTmice (119875 gt 005) Comparedwiththe NI group relative cell viability of 100 HI group decreasedin both the MT-III KO and WT mice groups (119875 lt 005)Compared to the WT group a more significant decrease inrelative cell viability was detected in MT-III KO group (119875 lt005) No significant difference of relative cell viability wasdetected between NI group and 10 HI group in both MT-IIIKO and WT mice groups (119875 gt 005) (Figure 2)

4 Discussion

Iodide excess has been recognized as a risk factor for thedevelopment of thyroid disease in humans and animals [7ndash13] One of the underlying mechanisms is that the increasedoxidative stress induced by iodide excess cannot be balancedby endogenous antioxidant systems [14 15] In the presentstudy we focused the research on excessive iodide inducedmitochondrial superoxide production and Prx 3 expressionin the thyroid with or without MT III in vitro and in vivo

In a previous study [15] we showed that the concentrationresponse of KI induced a decrease in cell viability in FRTLcells by MTT assay compared to the control group therelative viability of 10minus5M 10minus4M 10minus3M and 10minus2M KIexposure groupswas significantly decreased at 24 hHoweverno significant decrease in the 10minus6M or 10minus7M KI exposuregroups was observed A strong stimulatory effect of iodidewas detected in 10minus4MKI and in 10minus3MKI exposure groupsat the 2 h time point [15] Iodide had a stimulatory effect onH2O2generation and when increased H

2O2is considered

as a limiting step in the biosynthesis of thyroid hormones[1] The normal relative cell viability in the 10minus6M or 10minus7MKI exposure groups can be explained by the physiologicaleffect of iodide which is necessary for the biosynthesis ofthyroid hormones [15] We used KI concentrations of 10minus4Mand above in the in vitro study and NI 10 HI and 100 HIin the in vivo study In bovine thyroid slices the maximalstimulation effect on H

2O2generation was obtained after 2 h

of preincubation with 10minus4M KI [1] It was reported that in

vitro the acute toxic effects of high iodide doses in humanthyroid follicles significantly increased the percentage ofnecrotic cells in 10minus5Mand doubledwith 10minus3Mas comparedto values measured at 10minus7M [10] Iodide concentrations of10minus4M or greater may inhibit the iodothyronine synthesisand thyroid hormone secretion [41]The concentrations from10minus5 to 10minus3M were from 100 to 10000 times higher thanthe normal iodine plasma levels estimated to be 10minus7M ineuthyroid human beings [42]

In the present study we detected that in 100 HI intakegroup there is a significant change of FT

4in MT-III KO

mice compared to WT mice however the values remainwithin the normal range in thyroid function The NI and10 HI groups in both WT and MT-III KO mice wereunder normal conditions and the thyroid function was notaffected Iodine is essential for the synthesis of the thyroidhormones In most individuals the decreased productionof thyroid hormones is only transient and resumes afteradaptation to the acute Wolff-Chaikoff effect [43] A detailedanalysis reveals a persistent drop of serum T

4and T

3of

25 and 15 respectively and a rise of TSH of 2mULAlthough no clinical signs of thyroid dysfunction or goiterwere found sonographic thyroid volume is slightly increased[2 29ndash32 44 45] Exposure to high concentrations ofiodine may decrease the release of thyroid hormone shownby the increase in the serum level of TSH [27 28 46](Table 3)

Thyroid toxicity of iodide excess has been demonstratedin vitro and in animals fed with an iodide-rich diet [42] Weshowed that high concentrated iodide induced cytotoxicity inthyrocytes significantly increasingmitochondrial superoxideanion production in the thyroid in high concentrated iodideexposure in vitro and in 100 HI diet in vivo in bothMT-III KO and WT mice which leads to the decreasedrelative cell viability in the thyroid It is suggested thatoxidative stress is the underlying mechanism in iodide excessinduced apoptosis in thyroid cells [42] Consistent withprevious reports [42 47ndash49] we showed that there is astrong increase of superoxide anion production in FRTLcells following high concentrations of iodide exposure [15]Serrano-Nascimento et al also observed that iodide excessincreased ROS production in thyrocytes and mitochondriawere the source of superoxide anion production [50] Theoxidative attack can be explained by the oxidative effect

6 Oxidative Medicine and Cellular Longevity

0

20

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NI 10 HI 100 HI

Rela

tive v

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WT miceMT-III KO mice

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NI

NI

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10 HI

100 HI

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Expr

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)

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lowast

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Prx 3

120573-actin

(b)

NI 10 HI 100 HI

WT miceMT-III KO mice

0

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LDH

rele

ase (

Um

g pr

otei

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lowastlowast

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0

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NI 10 HI 100 HIMito

SOX

fluor

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nce i

nten

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WT miceMT-III KO mice

lowast

lowast

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MT-

III K

O

NI 1681

NI 2051 10 HI 2514 100 HI 6133

100 HI 478710 HI 1819

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

(d)

Figure 2 Mitochondrial superoxide production Prx 3 protein expression and LDH release and relative viability in the thyroid of WT andMT-III knockout mice following NI 10 HI or 100 HI diet for 14 days (a) 100 HI intake decreased the relative viability in thyroid of MT-IIIKO and WT mice (b) Representative western blot of Prx 3 (27 kDa) 120573-actin (45 kDa) was used as a loading control Densitometric analysisshowed a significant increase in Prx 3 expression in 100 HI group in the thyroid of either WT or MT-III KO mice especially in MT-IIIKO mice (c) 100 HI increased the LDH release in the thyroid of MT-III KO and WT mice (d) 100 HI intake increased the mitochondrialsuperoxide production in thyroid of WT and MT-III KO mice Histogram analysis was performed on the mean fluorescence intensity ofMitoSOX Red as measured by flow cytometry Experiments were repeated 3 times with similar results Data are represented as mean plusmn SD(119873 = 10group) One-way ANOVA with the LSD test was used lowast119875 lt 005 compared with the control group of WT or MT-III KO micerespectively 119875 lt 005WTmice compared with the MT-III KO mice under the same treatment

of HI diet on the thyroid gland ROS include free radi-cals such as the superoxide anion hydroxyl radicals andthe nonradical hydrogen peroxide Whether cells die fromoxidative stress induced apoptosis depends on the balancebetween the generation of oxidant species and the antioxidantsystem

In addition we demonstrated that no significant differ-ence was detected between NI group and 10 HI group in bothMT-III KO and WT mice groups This can be explained bythe fact that under basal conditions thyroid epithelial cellsproduce moderate amounts of ROS that are physiologicallyrequired for thyroid hormone synthesis [14] Physiologically

Oxidative Medicine and Cellular Longevity 7

Table 3 Documented literatures of iodide intake and changes of serum thyroxine (T4) and triiodothyronine (T3) concentrations

Dosage of daily iodine intake Time Subject Changes of serum T4 T3 concentrations Reference250 or 500 120583g 14 days Normal volunteers No change [27]1500 120583g 14 days Normal volunteers T4 T3 darr TSH uarr [27]

50 or 250mg 13 days Normal subjects T4 T3 darr TSH uarr(within normal range) [28]

27mg 4 weeks Normal volunteers T4 within the normal range except fortwo subjects TSH uarr [29]

Single doses of 10 30 50 and 100mg andthen daily doses of 10 15 30 50 or 100mg 12 days Euthyroid volunteers T3 T4 darr TSH uarr [30]

190mg 10 days Euthyroid volunteers T3 T4 darr TSH uarr [31]114mg 3ndash7 weeks Normal controls T3 T4 darr TSH uarr [32]

114mg 3ndash7 weeks Thyrotoxic patientsT4 T3 uarr in some casesother patients remained

euthyroid even after 6 weeks[32]

114mg 2 weeks Hypothyroid patients onthyroxine replacement

No consistent change(T4 uarr at 1 and 14 days TSH darr the first day) [32]

mitochondrial ROS generation is primarily due to electronsleaking from the electron transport chain (ETC) to generatesuperoxide radical Superoxide from the electron transportchain and other sources is converted by MnSOD to H

2O2

which is then metabolized by Prx 3 to water [47]Besides we demonstrated that high concentrated iodide

induces significant increased expression of Prx 3 whichsuggests that the increase of Prx 3 expression following highconcentrated iodide exposure orHI diet in the thyroidmay bethe protective response against high ROS generation Prx 3 isan indispensable ROS scavenger that protects against oxida-tive damage and subsequent apoptosis [51ndash53] It is reportedthat Prx 3 has an essential role in regulating oxidation-induced apoptosis and can be a potential therapeutic targetin castrate-independent prostate cancer [52] Iodide hada stimulatory effect on H

2O2generation in thyroid slices

[1] At high concentrations (above 01mM) H2O2induces

apoptosis in thyroid cells [1] When iodide is in excess ascompared to tyrosine residues it reacts with the iodoniumcation formed by iodide oxidation to give molecular iodineExcess molecular iodine then induced apoptosis throughgeneration of free radicals [54] H

2O2generation is a limiting

step in thyroid hormone biosynthesis it is essential foriodide oxidation by thyroperoxidase [14 55 56] H

2O2can

be eliminated by Prx 3 In mammals Prx 3 is targeted to themitochondrial matrix It is estimated that Prx 3 will be thetarget for up to 90 of hydrogen peroxide generated in themitochondrial matrix [57] mitochondrial peroxiredoxins (3andor 5) can reduce H

2O2to water through reducing equiv-

alents provided by thiol-containing proteins [58 59] butthe mechanisms involved are unclear [24 59] Drechsel andPatel demonstrated that thioredoxin (Trxs)peroxiredoxin(Prxs) (Prx 3 and Prx 5) is the major contributing systemto H2O2removal in brain mitochondria [60] Prxs and Trxs

are important liver antioxidant proteins and play importantroles in maintaining liver redox homeostasis [61] It isreported that chronic ethanol exposure increases NADPHoxidase activity and increases the production of superoxide

and hydrogen peroxide [62ndash64] Thioredoxin scavenges andreduces ROS via peroxiredoxins [65] Endoplasmic reticulumstress disrupts the electron transport chain leading to theincreased production of ROS [48 49] Bae et al reportedthat the antioxidant role of Prx 3 was evident from thefindings that pyrazole-induced protein carbonylation and theformation of 4-HNE adducts and MDA in the liver weremarkedly increased in Prx 3minusminus mice compared with wild-type mice [66]

A high urinary excretion of iodine in a spot urine sampleis the ultimate proof of iodine excess in vivo [2] We showedthe urinary iodine concentration increased as the iodidesupply increased significantly higher urinary iodine concen-tration can be detected in 100 HI than 10 HI and in MT-IIIKOmice than inWTmiceThis data indicates the adaptationof the thyroid gland to iodine excess and that the excessiveiodine is excreted from the urine in order to maintain thenormal function of thyroid This makes the studies in vivodifferent from studies in vitro In addition to the urinaryexcretion of iodine there are many control mechanisms ofthe thyroid to counteract iodide excess These include thesodium-iodide symporter which controls the limiting stepof thyroid hormone synthesis the Wolff-Chaikoff effect anarrest of thyroid hormone secretion from stores in the colloida preferential secretion of the less active T

4over T

3 and

dumping of excess iodine by secreting it in nonhormonalform [2]

Moreover we demonstrated that significant increasesof mitochondrial superoxide production and Prx 3 proteinexpression can be detected in MT-III KO mice group Thisdata indicated the protective role of MT-III against highconcentrations of iodide induced mitochondrial superoxideproduction in the thyrocytes MT has been demonstratedto play a key role in the detoxification of heavy metalsprotecting against various forms of oxidative injury by scav-enging oxygen free radicals MTs belong to the group ofintracellular cysteine-rich metal-binding proteins which iswidely expressed in cells (nuclei andor cytoplasm) of various

8 Oxidative Medicine and Cellular Longevity

organs and tumors [67ndash69] MTs play a protective role inpreventing against intoxication with heavy metals such asPb Hg Cu and Cd [67 70] All 20 cysteine sulfur atomsare involved in the radical quenching process and the rateconstant for the reaction of hydroxyl radical with MT isabout 340-fold higher than that with GSH [71 72] There arefour MT isoforms in mammals MT-I and MT-II are mainlyinvolved in the protection of tissue against metal toxicitiesoxidative stresses and apoptosis It has been demonstratedthat the rabbit liver metallothionein-1 which contains zincandor cadmium ions appeared to scavenge free hydroxyland superoxide radicals [25 26] Under low levels of iodideorganification in goitrous-modified tissue of thyroid glandmetallothionein may provide a partial compensatory effecton prooxidative processes [73 74] Although no cell lineor animal model overexpressing MT in the thyroid hasbeen found yet documented evidence of MT in protectingagainst oxidative stress in cardiac-specific metallothionein-overexpressed transgenic mice has been demonstrated Caiet al reported that metallothionein as a potent antioxi-dant prevented the development of diabetic cardiomyopathythrough the inhibition of the mitochondrial cytochromec-mediated caspase-3 activation pathway [75] Sun et alreported that through doxorubicin induced chronic toxi-city in a metallothionein-overexpressed transgenic mouseheart the antioxidant action of MT is highly responsiblefor this cardioprotection [76] Ceylan-Isik et al reportedthat the cardiac-specific overexpression of MT rescues LPS-induced cardiac contractile and intracellular Ca2+ dysfunc-tions through the alleviation of ROSoxidative stress stresssignaling activation and endoplasmic reticulum (ER) stress[77] They also reported that the cardiac-specific overexpres-sion of metallothionein protects against nicotine exposure-induced cardiac contractile dysfunction and fibrosis possiblythrough inhibition of ROS accumulation and apoptosis [78]Zhang et al suggest that cardiac overexpression of metalloth-ionein prevents cold exposure-induced cardiac anomaliespossibly through the attenuation of myocardial fibrosis [79]Hu et al demonstrated that cardiac-specific overexpression ofmetallothionein protects against cigarette smoking exposure-induced myocardial contractile and mitochondrial damagefavoring a role of lessened apoptosis in a metallothionein-induced beneficial effect against side-stream smoke exposure[80] Besides metallothionein has been shown to regulateapoptosis and proliferation Overexpression of metalloth-ionein frequently occurs in human tumors and is related totumor progression [81 82] High expression of MT has beenobserved in different types of cancer and has been consideredas a potential prognostic marker in cancers of the breastpancreas skin and cervix [82ndash86] Further studies will beattempted to study the oxidative and antioxidative stress onan engineered cell line overexpressing metallothionein

5 Conclusion

Thepresent study provides evidence that high concentrationsof iodide may lead to a significant increase in mitochondrialsuperoxide production and Prx 3 expression in both invitro and in vivo studies MT-III KO mice aggravated

mitochondrial superoxide production and peroxiredoxin 3expression in the thyroid after excessive iodide exposureMT-III may potentially protect the thyroid against highconcentrations of iodide induced oxidative stress

Abbreviations

MT-III KO Metallothionein III knockoutMTs MetallothioneinsWT Wild-typeROS Reactive oxygen speciesPrx PeroxiredoxinKI Potassium iodideNI Normal iodide intakeHI High iodide intakeLDH Lactate dehydrogenaseH2O2 Hydrogen peroxide

SOD Superoxide dismutaseMTT 3-(45-Dimethylthiazol-2-yl)-25-

diphenyltetrazoliumbromide

K-S Kolmogorov-SmirnovLSD Least significant differenceT4 Thyroxine

T3 Tri-iodothyronine

FT4 Free thyroxine

FT3 Free tri-iodothyronine

Conflict of Interests

The authors declare that they have no competing interests

Acknowledgments

Thiswork is supported by theNational Natural Science Foun-dation of China (no 81273009) the Tianjin Science amp Tech-nology Council Grant of China (no 09JCYBJC11700) andthe Tianjin Educational amp Scientific Grant (no 20050107)This paper has been edited by one of the authors MohamedAhmed who is from USA and now is studying in TianjinMedical University China

References

[1] B Corvilain L Collyn J van Sande and J E DumontldquoStimulation by iodide of H

2O2generation in thyroid slices

from several speciesrdquo The American Journal of PhysiologymdashEndocrinology and Metabolism vol 278 no 4 pp E692ndashE6992000

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

[3] E Roti and E Degli Uberti ldquoIodine excess and hyperthy-roidismrdquoThyroid vol 11 no 5 pp 493ndash500 2001

[4] World Health Organization and United Nations ChildrenrsquosFund ldquoInternational Council for Control of Iodine DeficiencyDisorders 1999 progress towards the elimination of iodinedeficiency disorders (IDD)rdquo WHO Booklet World HealthOrganization 1999

Oxidative Medicine and Cellular Longevity 9

[5] A M Leung and L E Braverman ldquoConsequences of excessiodinerdquo Nature Reviews Endocrinology vol 10 no 3 pp 136ndash142 2013

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

[7] IMahmoud I ColinM CMany and J-F Denef ldquoDirect toxiceffect of iodide in excess on iodine-deficient thyroid glandsepithelial necrosis and inflammation associated with lipofuscinaccumulationrdquo Experimental and Molecular Pathology vol 44no 3 pp 259ndash271 1986

[8] J E Fradkin and J Wolff ldquoIodide-induced thyrotoxicosisrdquoMedicine vol 62 no 1 pp 1ndash20 1983

[9] J Golstein and J E Dumont ldquoCytotoxic effects of iodide onthyroid cells difference between rat thyroid FRTL-5 cell andpri-mary dog thyrocyte responsivenessrdquo Journal of EndocrinologicalInvestigation vol 19 no 2 pp 119ndash126 1996

[10] 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

[11] W Teng Z Shan X Teng et al ldquoEffect of iodine intakeon thyroid diseases in Chinardquo The New England Journal ofMedicine vol 354 no 26 pp 2783ndash2793 2006

[12] P Laurberg K M Pedersen A Hreidarsson N Sigfusson EIversen and P R Knudsen ldquoIodine intake and the patternof thyroid disorders a comparative epidemiological study ofthyroid abnormalities in the elderly in iceland and in JutlandDenmarkrdquo Journal of Clinical Endocrinology and Metabolismvol 83 no 3 pp 765ndash769 1998

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

[14] 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

[15] 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

[16] H Han P Xin L Zhao et al ldquoExcess iodine and high-fatdiet combination modulates lipid profile thyroid hormoneand hepatic LDLr expression values in micerdquo Biological TraceElement Research vol 147 no 1ndash3 pp 233ndash239 2012

[17] S-J Zhao Y Ye F-J Sun E-J Tian and Z-P Chen ldquoTheimpact of dietary iodine intake on lipid metabolism in micerdquoBiological Trace Element Research vol 142 no 3 pp 581ndash5882011

[18] 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

[19] V Pitsiavas P Smerdely M Li and S C Boyages ldquoAmiodaroneinduces a different pattern of ultrastructural change in thethyroid to iodine excess alone in both the BBW rat and theWistar ratrdquo European Journal of Endocrinology vol 137 no 1pp 89ndash98 1997

[20] M B Zimmermann ldquoIodine deficiency and excess in childrenworldwide status in 2013rdquo Endocrine Practice vol 19 no 5 pp839ndash846 2013

[21] K Mussig ldquoIodine-induced toxic effects due to seaweed con-sumptionrdquo in Comprehensive Handbook of Iodine V R PreedyG N Burrow and R R Watson Eds pp 897ndash908 ElsevierAmsterdam The Netherlands 2009

[22] A-M G Psarra S Solakidi and C E Sekeris ldquoThe mito-chondrion as a primary site of action of steroid and thyroidhormones presence and action of steroid and thyroid hormonereceptors in mitochondria of animal cellsrdquo Molecular andCellular Endocrinology vol 246 no 1-2 pp 21ndash33 2006

[23] Y Song N Driessens M Costa et al ldquoRoles of hydrogenperoxide in thyroid physiology and diseaserdquo Journal of ClinicalEndocrinology and Metabolism vol 92 no 10 pp 3764ndash37732007

[24] A G Cox C C Winterbourn and M B Hampton ldquoMito-chondrial peroxiredoxin involvement in antioxidant defenceand redox signallingrdquo Biochemical Journal vol 425 no 2 pp313ndash325 2010

[25] B Ruttkay-Nedecky L Nejdl J Gumulec et al ldquoThe role ofmetallothionein in oxidative stressrdquo International Journal ofMolecular Sciences vol 14 no 3 pp 6044ndash6066 2013

[26] N Thirumoorthy A Shyam Sunder K T Manisenthil KumarM Senthil kumarGNKGanesh andMChatterjee ldquoA reviewof metallothionein isoforms and their role in pathophysiologyrdquoWorld Journal of Surgical Oncology vol 9 article 54 2011

[27] 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

[28] M Saberi and R D Utiger ldquoAugmentation of thyrotropinresponses to thyrotropin releasing hormone following smalldecreases in serum thyroid hormone concentrationsrdquo TheJournal of Clinical Endocrinology and Metabolism vol 40 no3 pp 435ndash441 1975

[29] H Namba S Yamashita H Kimura et al ldquoEvidence of thyroidvolume increase in normal subjects receiving excess iodiderdquoTheJournal of Clinical Endocrinology and Metabolism vol 76 no 3pp 605ndash608 1993

[30] E Sternthal L Lipworth B Stanley C Abreau S L Fang andL E Braverman ldquoSuppression of thyroid radioiodine uptakeby various doses of stable iodiderdquo The New England Journal ofMedicine vol 303 no 19 pp 1083ndash1088 1980

[31] A G Vagenakis B Rapoport F Azizi G I Portnay L EBraverman and S H Ingbar ldquoHyperresponse to thyrotropinreleasing hormone accompanying small decreases in serumthyroid hormone concentrationsrdquoThe Journal of Clinical Inves-tigation vol 54 no 4 pp 913ndash918 1974

[32] G Philippou D A Koutras G Piperingos A Souvatzoglouand S D Moulopoulos ldquoThe effect of iodide on serum thyroidhormone levels in normal persons in hyperthyroid patientsand in hypothyroid patients on thyroxine replacementrdquoClinicalEndocrinology vol 36 no 6 pp 573ndash578 1992

[33] L Z Ali Salim R Othman M A Abdulla et al ldquoThymo-quinone inhibits murine leukemia WEHI-3 cells in vivo and invitrordquo PLoS ONE vol 9 no 12 Article ID e115340 2014

[34] X Wang J Ge K Wang J Qian and Y Zou ldquoEvaluation ofMTT assay for measurement of emodin-induced cytotoxicityrdquoASSAY and Drug Development Technologies vol 4 no 2 pp203ndash207 2006

10 Oxidative Medicine and Cellular Longevity

[35] C Lv C Wu Y-h Zhou Y Shao G Wang and Q-y WangldquoAlpha lipoic acid Modulated high glucose-induced rat mesan-gial cell dysfunction viamTORp70S6K4E-BP1 pathwayrdquo Inter-national Journal of Endocrinology vol 2014 Article ID 65858914 pages 2014

[36] L Liu X Hu G-Y Cai et al ldquoHigh glucose-induced hyper-trophy of mesangial cells is reversed by connexin43 overex-pression via PTENAktmTOR signalingrdquo Nephrology DialysisTransplantation vol 27 no 1 pp 90ndash100 2012

[37] D D Harrison-Findik S Z Lu E M Zmijewski J Jonesand M C Zimmerman ldquoEffect of alcohol exposure on hepaticsuperoxide generation and hepcidin expressionrdquoWorld Journalof Biological Chemistry vol 4 no 4 pp 119ndash130 2013

[38] M Wang Y Wang J He et al ldquoAlbumin induces neuroprotec-tion against ischemic stroke by altering Toll-like receptor 4 andregulatory T cells in micerdquo CNS and Neurological DisordersmdashDrug Targets vol 12 no 2 pp 220ndash227 2013

[39] X Yao ldquoEffect of zinc exposure on HNE and GLT-1 in spinalcord culturerdquo NeuroToxicology vol 30 no 1 pp 121ndash126 2009

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

[41] G P Becks M C Eggo and G N Burrow ldquoRegulation ofdifferentiated thyroid function by iodide preferential inhibitoryeffect of excess iodide on thyroid hormone secretion in sheepthyroid cell culturesrdquo Endocrinology vol 120 no 6 pp 2569ndash2575 1987

[42] M Vitale T Di Matola F DrsquoAscoli et al ldquoIodide excessinduces apoptosis in thyroid cells through a p53-independentmechanism involving oxidative stressrdquo Endocrinology vol 141no 2 pp 598ndash605 2000

[43] P Pramyothin AM Leung E N Pearce A OMalabanan andL E Braverman ldquoClinical problem-solving A hidden solutionrdquoTheNew England Journal of Medicine vol 365 no 22 pp 2123ndash2127 2011

[44] W Jubiz S Carlile and L D Lagerquist ldquoSerum thyrotropinand thyroid hormone levels in humans receiving chronicpotassium iodiderdquo The Journal of Clinical Endocrinology andMetabolism vol 44 no 2 pp 379ndash382 1977

[45] A G Vagenakis P Downs L E Braverman A Burger andS H Ingbar ldquoControl of thyroid hormone secretion in normalsubjects receiving iodidesrdquoThe Journal of Clinical Investigationvol 52 no 2 pp 528ndash532 1973

[46] M Safran andL E Braverman ldquoEffect of chronic douchingwithpolyvinylpyrrolidone-iodine on iodine absorption and thyroidfunctionrdquo Obstetrics and Gynecology vol 60 no 1 pp 35ndash401982

[47] K Newick B Cunniff K Preston et al ldquoPeroxiredoxin 3 is aredox-dependent target of thiostrepton in malignant mesothe-lioma cellsrdquo PLoS ONE vol 7 no 6 Article ID e39404 2012

[48] A Deniaud O S El Dein E Maillier et al ldquoEndoplasmicreticulum stress induces calcium-dependent permeability tran-sition mitochondrial outer membrane permeabilization andapoptosisrdquo Oncogene vol 27 no 3 pp 285ndash299 2008

[49] A Gorlach P Klappa and T Kietzmann ldquoThe endoplasmicreticulum folding calcium homeostasis signaling and redoxcontrolrdquo Antioxidants and Redox Signaling vol 8 no 9-10 pp1391ndash1418 2006

[50] 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 symporter

expression and activity involves the PI3KAkt signaling path-wayrdquo Endocrinology vol 155 no 3 pp 1145ndash1156 2014

[51] Y-G Wang L Li C-H Liu S Hong and M-J ZhangldquoPeroxiredoxin 3 is resistant to oxidation-induced apoptosis ofHep-3b cellsrdquoClinical and Translational Oncology vol 16 no 6pp 561ndash566 2014

[52] H C Whitaker D Patel W J Howat et al ldquoPeroxiredoxin-3is overexpressed in prostate cancer and promotes cancer cellsurvival by protecting cells fromoxidative stressrdquoBritish Journalof Cancer vol 109 no 4 pp 983ndash993 2013

[53] L Li Y G Zhang and C L Chen ldquoAnti-apoptotic role ofperoxiredoxin III in cervical cancer cellsrdquo FEBS Open Bio vol3 pp 51ndash54 2013

[54] 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

[55] L I Nadolnik Z V Niatsetskaya and S V Lupachyk ldquoEffectof oxidative stress on rat thyrocyte iodide metabolismrdquo CellBiochemistry and Function vol 26 no 3 pp 366ndash373 2008

[56] A C Freitas Ferreira L de Carvalho Cardoso D Rosenthaland D Pires de Carvalho ldquoThyroid Ca2+NADPH-dependentH2O2generation is partially inhibited by propylthiouracil and

methimazolerdquo European Journal of Biochemistry vol 270 no 11pp 2363ndash2368 2003

[57] G R Sue Z C Ho and K Kim ldquoPeroxiredoxins a historicaloverview and speculative preview of novel mechanisms andemerging concepts in cell signalingrdquo Free Radical Biology andMedicine vol 38 no 12 pp 1543ndash1552 2005

[58] M S Seo S W Kang K Kim I C Baines T H Lee and SG Rhee ldquoIdentification of a new type of mammalian perox-iredoxin that forms an intramolecular disulfide as a reactionintermediaterdquo Journal of Biological Chemistry vol 275 no 27pp 20346ndash20354 2000

[59] Z A Wood L B Poole and P A Karplus ldquoPeroxiredoxinevolution and the regulation of hydrogen peroxide signalingrdquoScience vol 300 no 5619 pp 650ndash653 2003

[60] D A Drechsel and M Patel ldquoRespiration-dependent H2O2

removal in brain mitochondria via the thioredoxinperoxire-doxin systemrdquoThe Journal of Biological Chemistry vol 285 no36 pp 27850ndash27858 2010

[61] W H Watson X Yang Y E Choi D P Jones and J P KehrerldquoThioredoxin and its role in toxicologyrdquo Toxicological Sciencesvol 78 no 1 pp 3ndash14 2004

[62] G Ekstrom and M Ingelman-Sundberg ldquoRat liver microso-mal NADPH-supported oxidase activity and lipid peroxida-tion dependent on ethanol-inducible cytochrome P-450 (P-450IIE1)rdquo Biochemical Pharmacology vol 38 no 8 pp 1313ndash1319 1989

[63] J Rashba-Step N J Turro and A I Cederbaum ldquoIncreasedNADPH- and NADH-dependent production of superoxideand hydroxyl radical by microsomes after chronic ethanoltreatmentrdquoArchives of Biochemistry and Biophysics vol 300 no1 pp 401ndash408 1993

[64] V Thakur M T Pritchard M R McMullen Q Wang andL E Nagy ldquoChronic ethanol feeding increases activation ofNADPH oxidase by lipopolysaccharide in rat Kupffer cellsrole of increased reactive oxygen in LPS-stimulated ERK12activation andTNF-120572 productionrdquo Journal of Leukocyte Biologyvol 79 no 6 pp 1348ndash1356 2006

Oxidative Medicine and Cellular Longevity 11

[65] HNakamuraAMitsui and J Yodoi ldquoThioredoxin overexpres-sion in transgenic micerdquo Methods in Enzymology vol 347 pp436ndash440 2002

[66] 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

[67] A Bieniek B Pula A Piotrowska et al ldquoExpression of metal-lothionein III andKi-67 antigen in various histological types ofbasal cell carcinomardquo Folia Histochemica et Cytobiologica vol50 no 3 pp 352ndash357 2012

[68] P Dziegiel ldquoExpression of metallothioneins in tumor cellsrdquoPolish Journal of Pathology vol 55 no 1 pp 3ndash12 2004

[69] M Oslash Pedersen A Larsen M Stoltenberg and M PenkowaldquoThe role of metallothionein in oncogenesis and cancer prog-nosisrdquo Progress inHistochemistry and Cytochemistry vol 44 no1 pp 29ndash64 2009

[70] W Braun M Vasak A H Robbins et al ldquoComparison ofthe NMR solution structure and the X-ray crystal structure ofrat metallothionein-2rdquo Proceedings of the National Academy ofSciences of the United States of America vol 89 no 21 pp 10124ndash10128 1992

[71] P JThornalley andM Vasak ldquoPossible role formetallothioneinin protection against radiation-induced oxidative stress Kinet-ics andmechanism of its reaction with superoxide and hydroxylradicalsrdquo Biochimica et Biophysica Acta Protein Structure andMolecular Enzymology vol 827 no 1 pp 36ndash44 1985

[72] M A Schwarz J S Lazo J C Yalowich et al ldquoMetallothioneinprotects against the cytotoxic and DNA-damaging effects ofnitric oxiderdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 92 no 10 pp 4452ndash4456 1995

[73] Y Luo A Kawashima and Y Ishido ldquoIodine excess as anenvironmental risk factor for autoimmune thyroid diseaserdquoInternational Journal of Molecular Sciences vol 15 pp 12895ndash12912 2014

[74] H I Falrsquofushinsrsquoka L L Hnatyshyna andOI Osadchuk ldquoTraceelements storage peculiarities and metallothionein contentin human thyroid gland under iodine deficiency euthyroidnodular goiterrdquo Ukrainskii Biokhimicheskii Zhurnal vol 86 pp107ndash113 2014

[75] L Cai Y H Wang G H Zhou et al ldquoAttenuation bymetallothionein of early cardiac cell death via suppression ofmitochondrial oxidative stress results in a prevention of diabeticcardiomyopathyrdquo Journal of the American College of Cardiologyvol 48 no 8 pp 1688ndash1697 2006

[76] X Sun Z Zhou and Y J Kang ldquoAttenuation of doxorubicinchronic toxicity in metallothionein-overexpressing transgenicmouse heartrdquo Cancer Research vol 61 no 8 pp 3382ndash33872001

[77] A F Ceylan-Isik P Zhao B F Zhang X Xiao G Su and J RenldquoCardiac overexpression of metallothionein rescues cardiaccontractile dysfunction and endoplasmic reticulum stress butnot autophagy in sepsisrdquo Journal of Molecular and CellularCardiology vol 48 no 2 pp 367ndash378 2010

[78] N Hu R Guo X Han B Zhu and J Ren ldquoCardiac-specificoverexpression of metallothionein rescues nicotine-inducedcardiac contractile dysfunction and interstitial fibrosisrdquoToxicol-ogy Letters vol 202 no 1 pp 8ndash14 2011

[79] Y Zhang N Hu Y Hua K L Richmond F Dong and JRen ldquoCardiac overexpression of metallothionein rescues coldexposure-induced myocardial contractile dysfunction through

attenuation of cardiac fibrosis despite cardiomyocyte mechani-cal anomaliesrdquo Free Radical Biology and Medicine vol 53 no 2pp 194ndash207 2012

[80] N Hu X Han E K Lane F Gao Y Zhang and J RenldquoCardiac-specific overexpression of metallothionein rescuesagainst cigarette smoking exposure-induced myocardial con-tractile and mitochondrial damagerdquo PLoS ONE vol 8 no 2Article ID e57151 2013

[81] M T Brazao-Silva S V Cardoso P R de Faria et al ldquoAdenoidcystic carcinoma of the salivary gland a clinicopathologicalstudy of 49 cases and of metallothionein expression with regardto tumour behaviourrdquo Histopathology vol 63 no 6 pp 802ndash809 2013

[82] K J Schmitz H Lang G Kaiser et al ldquoMetallothioneinoverexpression and its prognostic relevance in intrahepaticcholangiocarcinoma and extrahepatic hilar cholangiocarci-noma (Klatskin tumors)rdquo Human Pathology vol 40 no 12 pp1706ndash1714 2009

[83] S E Theocharis A P Margeli J T Klijanienko and G PKouraklis ldquoMetallothionein expression in human neoplasiardquoHistopathology vol 45 no 2 pp 103ndash118 2004

[84] STheocharis J Klijanienko C Giaginis et al ldquoMetallothioneinexpression in mobile tongue squamous cell carcinoma associa-tions with clinicopathological parameters and patient survivalrdquoHistopathology vol 59 no 3 pp 514ndash525 2011

[85] S-K Tai O J-K Tan V T-K Chow et al ldquoDifferentialexpression of metallothionein 1 and 2 isoforms in breast cancerlines with different invasive potential identification of a novelnon-silent metallothionein-1H mutant variantrdquo The AmericanJournal of Pathology vol 163 no 5 pp 2009ndash2019 2003

[86] Y Hishikawa T Koji D K Dhar S Kinugasa M Yamaguchiand N Nagasue ldquoMetallothionein expression correlates withmetastatic and proliferative potential in squamous cell carci-noma of the oesophagusrdquo British Journal of Cancer vol 81 no4 pp 712ndash720 1999

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Page 2: Research Article Metallothionein-I/II Knockout Mice

2 Oxidative Medicine and Cellular Longevity

also lead to iodine excess Radiologists use up to 100 g ormore of iodine in complex organic molecules Erythrosinea food additive used to improve the colour of canned cherriesor of candies contains 57 iodine [2 3] Although iodizedsalt programs have made great progress in the global effort tocontrol iodine deficiency they need to be carefullymonitoredto ensure adequate iodine intake while avoiding iodine excess[20]

The mitochondria contain specific receptors for thyroidhormones and are an important source of reactive oxygenspecies (ROS) [22] Thyrocytes possess various enzymaticsystems such as peroxidise (GPx) catalases superoxidedismutases (SOD) and peroxiredoxins (Prxs) that contributeto limiting cellular injuries [23] Peroxiredoxin 3 (Prx 3) ispredominantly located in the mitochondria and is respon-sible for scavenging ROS Prx 3 can be the target for upto 90 of hydrogen peroxide generated in the matrix [24]Metallothioneins (MTs) have been demonstrated to havestrong antioxidative activity in protecting cells from thedamaging effects of ROSThey too are regarded as scavengersof ROS including superoxide and hydroxyl radicals [25 26]

In light of previous works documented on iodide inducedoxidative stress and thyrotoxicity we have demonstrated thetime-course and concentration-response effects of early acutehigh concentrations of iodide exposure on mitochondrialsuperoxide generation When endogenous antioxidant sys-tems were overwhelmed by high concentrations of iodideinduced ROS production they began attacking proteinslipid and nucleic acids [15] However the nature of iodideexcess induced oxidative stress in thyroid with or withoutMTIII remains to be determined

By targeting mitochondrial superoxide production andPrx 3 protein expression we aimed to figure out the effect ofhigh concentrations of iodide on mitochondrial superoxideproduction and Prx 3 expression in the thyroids of metal-lothionein III knockout (MT-III KO) mice in vitro and invivo We propose that iodide excess in a thyroid without MTIII protection may result in strong mitochondrial oxidativestress which further leads to the damage of thyrocytes

2 Methods

21 Animals andThyroid Cell Suspension Preparation Eight-week-old 129S7SvEvBrd-Mt1tm1BriMt2tm1BriJ (MT-IIIknockout MT-III KO) mice (number 002211 Jackson LabMaineUSA) and background-matchedwild-type (WT)micewere used In the in vitro study a thyroid cell suspension wasprepared A cell suspension (5 times 104 cellswell) was incubatedin medium RPMI-1640 with 10 fetal bovine serum (FBS)at 37∘C in an atmosphere comprising 5 CO

2and 95 air

and exposed to high concentrations of potassium iodide (KI)or 10minus3M H

2O2for 2 hours In vivo MT-III KO and WT

mice were randomly divided into control groups (normaliodide intake NI) 10 times iodide intake groups (10 HI) and100 times iodide intake groups (100 HI) NI groups receiveddeionized water and a normal diet (daily iodide uptake is15 120583gd) In the 10HI and 100HI groups 15 120583gd and 150 120583gdKI were added respectively to the deionized water for 14days before sacrifice Animal procedures were approved by

the Institutional Animal Care and Use Committee of TianjinMedical University and in accordance with the NIH Guide

22 Reagents MitoSOX Red (38-phenanthridinediamine5-(61015840-triphenylphosphoniumhexyl)-56-dihydro-6-phenyl)was purchased from Invitrogen (Invitrogen Life Technolo-gies CA USA) Anti-Peroxiredoxin 3 antibody waspurchased from Abcam (Abcam Cambridge MA USA)120573-Actin Rabbit mAb was purchased from Cell Signaling(Cell Signaling Technology Inc MA USA) 3-(45-Dimeth-ylthiazol-2-yl)-25-diphenyl-tetrazolium bromide (MTT)and TSH were purchased from Sigma (Sigma-AldrichMO USA) RPMI-1640 and FBS were purchased from GEHealthcare Life Sciences (Hyclone UT USA) All the otherchemicals made in China were of analytical grade

23 Cell Viability Assay Cell viability was evaluated withthe MTT assay A cell suspension (5 times 104 cellswell) wasincubated in medium RPMI-1640 with 10 fetal bovineserum (FBS) at 37∘C in an atmosphere comprising 5 CO

2

and 95 air and exposed to high concentrations of potassiumiodide (KI) or 10minus3M H

2O2for 2 hours in order to get a

final concentration of 10minus4M 10minus3M and 10minus2MThe groupwithout KI is the control group the well with only mediumin is regarded as blank group Subsequently 10 120583L of 3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide(MTT 5mgmL)was added in dark and incubated for 4 h andcovered with aluminium foil After incubation for 2 hoursthe supernatant was removed and 100120583L of DMSO wasadded to each well to dissolve the formazan crystals formedand was then shaken for 10min Absorbance was measuredby a spectrophotometer at 490 nm (Wallac 1420 VICTOR3PerkinElmer) [33 34]

24 Lactate Dehydrogenase (LDH) Assay LDH release inthe supernatant following different treatments was mea-sured using a cytotoxicity detection kit (LDH) (NanjingJiancheng Bioengineering Institute Jiangsu China) LDHis an oxidoreductase that catalyzes the interconversion oflactate and pyruvate LDH is a stable cytosolic enzyme thatupon membrane damage is released into cell culture mediaThe assay is based on the reduction of the 2-p-iodophenyl-3-nitrophenyl tetrazolium chloride (tetrazolium INT) to ared formazan which is specifically detected by colorimetric(450 nm) assay

25 Flow Cytometry A mitochondrial superoxide indicator(MitoSOX Red Invitrogen) was used to measure mitochon-drial superoxide production by flow cytometry In vivo thethyroid cell suspension of MT-III KO and WT mice wasprepared for (normal iodide intake NI) 10 times iodideintake groups (10 HI) and 100 times iodide intake groups(100 HI) For the in vitro study a thyroid cell suspensionwas prepared first The thyroid cell suspension was exposedto high concentrations of potassium iodide (KI) (10minus4M10minus3M and 10minus2M) or 10minus3M H

2O2for 2 hours Subse-

quently 5 120583M of MitoSOX was added and incubated for10min at 37∘C in the dark Then the cells were washed

Oxidative Medicine and Cellular Longevity 3

with Hankrsquos solution and were suspended in Hankrsquos solutionwith 1 BSA The fluorescence intensity of MitoSOX wasdetected by a FACSCalibur (BD Bioscience San Jose CA)and the excitationemission wavelength is 488 nm575 nmCollecting FL2 channel forward scattering (forward scatterFSC) and lateral scattering (side scatter SSC) data 10000cells were collected for each sample The control groupwithout MitoSOX was regarded as the blank zero group forstandardization [35ndash38]

26 Western Blot Analysis Whole cell proteins were assayedfor protein concentration using the bicinchoninic acid (BCA)protein assay kit (Beyotime Institute of BiotechnologyJiangsu China) 50120583g of protein was transferred to nitro-cellulose membrane followed by SDS polyacrylamide gelelectrophoresis The nitrocellulose membrane was incubatedovernight with primary antibodies Anti-Peroxiredoxin 3antibody (Abcam Cambridge MA USA) and horseradishperoxidase (HRP) conjugated secondary antibody developedby Immobilon Western Chemiluminescent HRP Substrate(Merck Millipore MA USA) To verify equal loading 120573-actin (1 1000) (Cell Signaling Technology Inc MA USA)was used as a loading control Blots were scanned as grayscaleimages and quantitated using Image J software (NIH) All theblot intensities were normalized with that of loading control120573-actin [39]

27 Determination of Urinary Iodine Concentrations andSerum Thyroid Hormones Spot urine samples of mice werecollected and urinary iodine concentrations were measuredby As-Ce catalytic spectrophotometry in the Key Lab ofHormones and Development Ministry of Health Institute ofEndocrinology Tianjin Medical University [40] Blood wasobtained 14 days after NI 10 HI or 100 HI intake Samplesof blood were centrifuged for 10min serum samples wereobtained and stored at minus80∘C for use Serum concentrationsof thyroxine (T

4) free thyroxine (FT

4) triiodothyronine

(T3) and free triiodothyronine (FT

3) were determined by

using the Direct Chemiluminescence Technology Compet-itive Immunoassay Kits (Siemens Healthcare DiagnosticInc)

28 Statistics The data was represented as mean plusmn SD Basedon the Kolmogorov-Smirnov (K-S) for normality test andLevene statistic for the test of homogeneity of variancesUrinary iodine concentrations were expressed as the medianand determined with the nonparametric Kruskal-Wallis testone-way ANOVA with the least significant difference (LSD)test was performed using SPSS 170 to determine the differ-ences between the groups A 119875 value of less than 005 wasconsidered to be statistically significant

3 Results

31 Effect of MT-III on High Concentrated Iodide ExposureInduced Mitochondrial Superoxide Production and Prx 3 Pro-tein Expression In Vitro To investigate the antioxidative roleof MT-III in oxidative stress induced by high concentrationsof iodide in the thyroid thyroid cell suspensions were

Table 1 The median urinary iodine concentrations (120583gL) of WTmice and MT-III KO mice

Group WT MT-III KONI 32029 plusmn 11216 42433 plusmn 13195

10 HI 359292 plusmn 51998lowast

394924 plusmn 64793lowast

100 HI 3047917 plusmn 412894lowast

4066635 plusmn 352046lowast

lowastCompared to the NI group (119875 lt 005) WT mice group compared to MT-III KO mice group (119875 lt 005)119873 = 10 for each group

prepared from the thyroid of MT-III KO mice and WTmice The cells were then exposed to high concentrationsof KI (10minus4M 10minus3M and 10minus2M) or 10minus3M H

2O2for 2

hours Following 10minus4M 10minus3M and 10minus2M of KI or 10minus3MH2O2exposure we showed a significant increase in mito-

chondrial superoxide production with increased MitoSOXRed fluorescence intensity (119875 lt 005) (Figure 1(d)) and anincrease in LDH release (119875 lt 005) (Figure 1(c)) with adecrease in relative cell viability (119875 lt 005) (Figure 1(a)) in thethyroid cell suspensions of both MT-III KO and WT micegroups Compared to WT mice group a more significantincrease of mitochondrial superoxide production increasein LDH release and decrease in a relative cell viability canbe detected in MT-III KO mice group (119875 lt 005) (Figures1(d) 1(c) and 1(a)) Compared to control group the Prx 3protein expressions were significantly increased in 10minus2MKIor 10minus3MH

2O2exposure group in bothMT-III KO andWT

mice groups (119875 lt 005) Compared to WT mice group therewas a significant increase of Prx 3 protein expression in thethyroid cell suspensions ofMT-III KOmicewith the increaseof KI concentration (119875 lt 005) (Figure 1(b))

32 Urinary Iodine Concentrations In Vivo In order toverify the model of iodine excess in vivo urinary iodineconcentrations were detected after the MT-III KO mice andWT mice had NI 10 HI and 100 HI for 14 days The medianurinary iodine concentrations in NI 10 HI and 100 HI wereshown in Table 1 The high iodide intake of 10 HI group and100HI group in bothMT-III KOmice andWTmice resultedin significant increases in urinary iodine concentration whencompared to theNI group (119875 lt 005) The urinary iodineconcentrations were increased as the iodine supply increasedto 10HI 100HI in bothWT andMT-III KOmiceMoreovercompared to 10 HI group the extent of increase in urinaryiodine concentration in 100 HI was significantly higher (119875 lt005) Compared to WT mice MT -III KO mice had a moreprominent increase of urinary iodine concentration in 100HIgroup (119875 lt 005)

33 Changes of Serum Thyroid Hormones Level in WT andMT-III KO Mice following NI 10 HI or 100 HI Intake for14 Days We demonstrated that compared to the NI groupthere was no significant difference existing in serum thyroidhormones level (T

3 T4 FT3 and FT

4) in either 10 HI

group or 100 HI group in both WT mice and MT-III KOmice (119875 gt 005) However compared to WT mice therewas a significant difference in serum thyroid hormones level

4 Oxidative Medicine and Cellular Longevity

120

100

80

60

40

20

0

lowastlowast

Rela

tive v

iabi

lity

()

lowastlowast lowast

lowast lowast

lowast

WT miceMT-III KO mice

ControlKI KI KI

10minus4 M 10minus3 M 10minus2 M 10minus3 MH2O2

(a)

lowast lowastlowastlowast3

25

2

15

1

05

0Expr

essio

n of

Prx3

()

Prx 3

120573-actin

WT MT-III KO

WT miceMT-III KO mice

ControlKI KI KI

10minus4 M 10minus3 M 10minus2 M 10minus3 MH2O2

Con

trol

10minus4

M K

I

10minus3

M K

I

10minus2

M K

I

10minus3

MH2O2

Con

trol

10minus4

M K

I

10minus3

M K

I

10minus2

M K

I

10minus3

MH2O2

(b)

6543210

LDH

rele

ase

(Um

g pr

otei

n)

lowast lowast lowast lowast lowast lowastlowast lowast

times103

WT miceMT-III KO mice

ControlKI KI KI

10minus4 M 10minus3 M 10minus2 M 10minus3 MH2O2

(c)

lowastlowast lowast

lowast lowastlowast

lowastlowast120

100806040200M

itoSO

X flu

ores

cenc

e in

tens

ityW

TM

T-II

I KO

Control 262

Control 3644

10minus4 M KI 3754

10minus4 M KI 4931

10minus3 M KI 4925

10minus3 M KI 6447

10minus2 M KI 578

10minus2 M KI 7172

10minus3 M H2 9827

10minus3 M H2O28932

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 101 102 103 104 101 102 103 104100 100

WT miceMT-III KO mice

ControlKI KI KI

10minus4 M 10minus3 M 10minus2 M 10minus3 MH2O2

O2

(d)

Figure 1 Oxidative and antioxidative effect of high concentrations of KI on the mitochondria of thyrocytes in MT-III KO mice and WTmice (a) Decreased relative viability induced by high concentrations of KI (10minus4M 10minus3M and 10minus2M) or 10minus3MH

2O2in the thyroid cells

of MT-III KOmice byMTT assay (119873 = 8) (b) Representative western blot of Prx 3 (27 kDa) 120573-actin (45 kDa) was used as a loading controlDensitometric analysis showed a significant increase (lowast119875 lt 005) in Prx 3 expression compared to untreated control in the thyroid cells ofeither WT or MT-III KO mice (c) Increased LDH release following 2 h of high concentration of KI or H

2O2exposure in the thyroid cells

of MT-III KO mice (119873 = 8) (d) High concentration of KI or H2O2induced increased mitochondrial superoxide production in the thyroid

cells of WT and MT-III KO mice Histogram analysis was performed on the mean fluorescence intensity of MitoSOX Red as measured byflow cytometry Experiments were repeated 3 times with similar results Data are represented as mean plusmn SD One-way ANOVA with the LSDtest was used lowast119875 lt 005 compared with the control group of WT or MT-III KO mice respectively 119875 lt 005WTmice versus MT-III KOmice under the same treatment

(FT4) in 100 HI group in MT-III KO mice (119875 lt 005)

(Table 2)

34 High Iodide Intake Induced Increased MitochondrialSuperoxide Production and Peroxiredoxin 3 Expression inThyroid In Vivo To clarify the effect of iodide excess onmitochondrial superoxide production and Prx 3 protein

expression in thyroid of MT-III KO mice in vivo micewere divided into normal iodide intake (NI) 10 times iodideintake (10 HI) and 100 times iodide intake (100 HI) groupsCompared to the NI group the mitochondrial superoxideproduction measured by flow cytometry Prx 3 proteinexpression detected bywestern blot analysis and LDH releasein 100 HI group were significantly increased in both MT-III

Oxidative Medicine and Cellular Longevity 5

Table 2 Changes of serum thyroid hormones level (nmolL) in WT and MT-III KO mice following NI 10 HI or 100 HI intake for 14 days

Group T3 T4 FT3 FT4

WTNI 072 plusmn 013 2608 plusmn 1421 480 plusmn 042 2856 plusmn 451

10 HI 053 plusmn 009 3350 plusmn 445 426 plusmn 041 3142 plusmn 095

100 HI 071 plusmn 013 3470 plusmn 1324 461 plusmn 025 3320 plusmn 455

MT-III KONI 058 plusmn 015 2005 plusmn 468 404 plusmn 077 2569 plusmn 097

10 HI 069 plusmn 013 2253 plusmn 779 478 plusmn 005 2722 plusmn 103

100 HI 080 plusmn 022 1773 plusmn 676 501 plusmn 073 2399 plusmn 192lowast

lowastIn 100 HI group compared to WT mice there was a significant difference in serum thyroid hormones level (FT4) in MT-III KO mice (119875 lt 005)119873 = 8 foreach group

KO and WT groups (119875 lt 005) Compared to WT groupmore significant increases in the mitochondrial superoxideproduction Prx 3 protein expression and LDH release weredetected in MT-III KO group (119875 lt 005) There were nosignificant differences between the NI group and 10 HI groupin bothMT-III KO andWTmice (119875 gt 005) Comparedwiththe NI group relative cell viability of 100 HI group decreasedin both the MT-III KO and WT mice groups (119875 lt 005)Compared to the WT group a more significant decrease inrelative cell viability was detected in MT-III KO group (119875 lt005) No significant difference of relative cell viability wasdetected between NI group and 10 HI group in both MT-IIIKO and WT mice groups (119875 gt 005) (Figure 2)

4 Discussion

Iodide excess has been recognized as a risk factor for thedevelopment of thyroid disease in humans and animals [7ndash13] One of the underlying mechanisms is that the increasedoxidative stress induced by iodide excess cannot be balancedby endogenous antioxidant systems [14 15] In the presentstudy we focused the research on excessive iodide inducedmitochondrial superoxide production and Prx 3 expressionin the thyroid with or without MT III in vitro and in vivo

In a previous study [15] we showed that the concentrationresponse of KI induced a decrease in cell viability in FRTLcells by MTT assay compared to the control group therelative viability of 10minus5M 10minus4M 10minus3M and 10minus2M KIexposure groupswas significantly decreased at 24 hHoweverno significant decrease in the 10minus6M or 10minus7M KI exposuregroups was observed A strong stimulatory effect of iodidewas detected in 10minus4MKI and in 10minus3MKI exposure groupsat the 2 h time point [15] Iodide had a stimulatory effect onH2O2generation and when increased H

2O2is considered

as a limiting step in the biosynthesis of thyroid hormones[1] The normal relative cell viability in the 10minus6M or 10minus7MKI exposure groups can be explained by the physiologicaleffect of iodide which is necessary for the biosynthesis ofthyroid hormones [15] We used KI concentrations of 10minus4Mand above in the in vitro study and NI 10 HI and 100 HIin the in vivo study In bovine thyroid slices the maximalstimulation effect on H

2O2generation was obtained after 2 h

of preincubation with 10minus4M KI [1] It was reported that in

vitro the acute toxic effects of high iodide doses in humanthyroid follicles significantly increased the percentage ofnecrotic cells in 10minus5Mand doubledwith 10minus3Mas comparedto values measured at 10minus7M [10] Iodide concentrations of10minus4M or greater may inhibit the iodothyronine synthesisand thyroid hormone secretion [41]The concentrations from10minus5 to 10minus3M were from 100 to 10000 times higher thanthe normal iodine plasma levels estimated to be 10minus7M ineuthyroid human beings [42]

In the present study we detected that in 100 HI intakegroup there is a significant change of FT

4in MT-III KO

mice compared to WT mice however the values remainwithin the normal range in thyroid function The NI and10 HI groups in both WT and MT-III KO mice wereunder normal conditions and the thyroid function was notaffected Iodine is essential for the synthesis of the thyroidhormones In most individuals the decreased productionof thyroid hormones is only transient and resumes afteradaptation to the acute Wolff-Chaikoff effect [43] A detailedanalysis reveals a persistent drop of serum T

4and T

3of

25 and 15 respectively and a rise of TSH of 2mULAlthough no clinical signs of thyroid dysfunction or goiterwere found sonographic thyroid volume is slightly increased[2 29ndash32 44 45] Exposure to high concentrations ofiodine may decrease the release of thyroid hormone shownby the increase in the serum level of TSH [27 28 46](Table 3)

Thyroid toxicity of iodide excess has been demonstratedin vitro and in animals fed with an iodide-rich diet [42] Weshowed that high concentrated iodide induced cytotoxicity inthyrocytes significantly increasingmitochondrial superoxideanion production in the thyroid in high concentrated iodideexposure in vitro and in 100 HI diet in vivo in bothMT-III KO and WT mice which leads to the decreasedrelative cell viability in the thyroid It is suggested thatoxidative stress is the underlying mechanism in iodide excessinduced apoptosis in thyroid cells [42] Consistent withprevious reports [42 47ndash49] we showed that there is astrong increase of superoxide anion production in FRTLcells following high concentrations of iodide exposure [15]Serrano-Nascimento et al also observed that iodide excessincreased ROS production in thyrocytes and mitochondriawere the source of superoxide anion production [50] Theoxidative attack can be explained by the oxidative effect

6 Oxidative Medicine and Cellular Longevity

0

20

40

60

80

100

120

NI 10 HI 100 HI

Rela

tive v

iabi

lity

()

WT miceMT-III KO mice

lowastlowast

(a)

0

02

04

06

08

1

12

NI

NI

10 HI

10 HI

100 HI

100 HI NI 10 HI 100 HI

Expr

essio

n of

Prx

3 (

)

WT miceMT-III KO mice

lowast

lowast

WT MT-III KO

Prx 3

120573-actin

(b)

NI 10 HI 100 HI

WT miceMT-III KO mice

0

1000

2000

3000

4000

LDH

rele

ase (

Um

g pr

otei

n)

lowastlowast

(c)

0

20

40

60

80

NI 10 HI 100 HIMito

SOX

fluor

esce

nce i

nten

sity

WT miceMT-III KO mice

lowast

lowast

WT

MT-

III K

O

NI 1681

NI 2051 10 HI 2514 100 HI 6133

100 HI 478710 HI 1819

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

(d)

Figure 2 Mitochondrial superoxide production Prx 3 protein expression and LDH release and relative viability in the thyroid of WT andMT-III knockout mice following NI 10 HI or 100 HI diet for 14 days (a) 100 HI intake decreased the relative viability in thyroid of MT-IIIKO and WT mice (b) Representative western blot of Prx 3 (27 kDa) 120573-actin (45 kDa) was used as a loading control Densitometric analysisshowed a significant increase in Prx 3 expression in 100 HI group in the thyroid of either WT or MT-III KO mice especially in MT-IIIKO mice (c) 100 HI increased the LDH release in the thyroid of MT-III KO and WT mice (d) 100 HI intake increased the mitochondrialsuperoxide production in thyroid of WT and MT-III KO mice Histogram analysis was performed on the mean fluorescence intensity ofMitoSOX Red as measured by flow cytometry Experiments were repeated 3 times with similar results Data are represented as mean plusmn SD(119873 = 10group) One-way ANOVA with the LSD test was used lowast119875 lt 005 compared with the control group of WT or MT-III KO micerespectively 119875 lt 005WTmice compared with the MT-III KO mice under the same treatment

of HI diet on the thyroid gland ROS include free radi-cals such as the superoxide anion hydroxyl radicals andthe nonradical hydrogen peroxide Whether cells die fromoxidative stress induced apoptosis depends on the balancebetween the generation of oxidant species and the antioxidantsystem

In addition we demonstrated that no significant differ-ence was detected between NI group and 10 HI group in bothMT-III KO and WT mice groups This can be explained bythe fact that under basal conditions thyroid epithelial cellsproduce moderate amounts of ROS that are physiologicallyrequired for thyroid hormone synthesis [14] Physiologically

Oxidative Medicine and Cellular Longevity 7

Table 3 Documented literatures of iodide intake and changes of serum thyroxine (T4) and triiodothyronine (T3) concentrations

Dosage of daily iodine intake Time Subject Changes of serum T4 T3 concentrations Reference250 or 500 120583g 14 days Normal volunteers No change [27]1500 120583g 14 days Normal volunteers T4 T3 darr TSH uarr [27]

50 or 250mg 13 days Normal subjects T4 T3 darr TSH uarr(within normal range) [28]

27mg 4 weeks Normal volunteers T4 within the normal range except fortwo subjects TSH uarr [29]

Single doses of 10 30 50 and 100mg andthen daily doses of 10 15 30 50 or 100mg 12 days Euthyroid volunteers T3 T4 darr TSH uarr [30]

190mg 10 days Euthyroid volunteers T3 T4 darr TSH uarr [31]114mg 3ndash7 weeks Normal controls T3 T4 darr TSH uarr [32]

114mg 3ndash7 weeks Thyrotoxic patientsT4 T3 uarr in some casesother patients remained

euthyroid even after 6 weeks[32]

114mg 2 weeks Hypothyroid patients onthyroxine replacement

No consistent change(T4 uarr at 1 and 14 days TSH darr the first day) [32]

mitochondrial ROS generation is primarily due to electronsleaking from the electron transport chain (ETC) to generatesuperoxide radical Superoxide from the electron transportchain and other sources is converted by MnSOD to H

2O2

which is then metabolized by Prx 3 to water [47]Besides we demonstrated that high concentrated iodide

induces significant increased expression of Prx 3 whichsuggests that the increase of Prx 3 expression following highconcentrated iodide exposure orHI diet in the thyroidmay bethe protective response against high ROS generation Prx 3 isan indispensable ROS scavenger that protects against oxida-tive damage and subsequent apoptosis [51ndash53] It is reportedthat Prx 3 has an essential role in regulating oxidation-induced apoptosis and can be a potential therapeutic targetin castrate-independent prostate cancer [52] Iodide hada stimulatory effect on H

2O2generation in thyroid slices

[1] At high concentrations (above 01mM) H2O2induces

apoptosis in thyroid cells [1] When iodide is in excess ascompared to tyrosine residues it reacts with the iodoniumcation formed by iodide oxidation to give molecular iodineExcess molecular iodine then induced apoptosis throughgeneration of free radicals [54] H

2O2generation is a limiting

step in thyroid hormone biosynthesis it is essential foriodide oxidation by thyroperoxidase [14 55 56] H

2O2can

be eliminated by Prx 3 In mammals Prx 3 is targeted to themitochondrial matrix It is estimated that Prx 3 will be thetarget for up to 90 of hydrogen peroxide generated in themitochondrial matrix [57] mitochondrial peroxiredoxins (3andor 5) can reduce H

2O2to water through reducing equiv-

alents provided by thiol-containing proteins [58 59] butthe mechanisms involved are unclear [24 59] Drechsel andPatel demonstrated that thioredoxin (Trxs)peroxiredoxin(Prxs) (Prx 3 and Prx 5) is the major contributing systemto H2O2removal in brain mitochondria [60] Prxs and Trxs

are important liver antioxidant proteins and play importantroles in maintaining liver redox homeostasis [61] It isreported that chronic ethanol exposure increases NADPHoxidase activity and increases the production of superoxide

and hydrogen peroxide [62ndash64] Thioredoxin scavenges andreduces ROS via peroxiredoxins [65] Endoplasmic reticulumstress disrupts the electron transport chain leading to theincreased production of ROS [48 49] Bae et al reportedthat the antioxidant role of Prx 3 was evident from thefindings that pyrazole-induced protein carbonylation and theformation of 4-HNE adducts and MDA in the liver weremarkedly increased in Prx 3minusminus mice compared with wild-type mice [66]

A high urinary excretion of iodine in a spot urine sampleis the ultimate proof of iodine excess in vivo [2] We showedthe urinary iodine concentration increased as the iodidesupply increased significantly higher urinary iodine concen-tration can be detected in 100 HI than 10 HI and in MT-IIIKOmice than inWTmiceThis data indicates the adaptationof the thyroid gland to iodine excess and that the excessiveiodine is excreted from the urine in order to maintain thenormal function of thyroid This makes the studies in vivodifferent from studies in vitro In addition to the urinaryexcretion of iodine there are many control mechanisms ofthe thyroid to counteract iodide excess These include thesodium-iodide symporter which controls the limiting stepof thyroid hormone synthesis the Wolff-Chaikoff effect anarrest of thyroid hormone secretion from stores in the colloida preferential secretion of the less active T

4over T

3 and

dumping of excess iodine by secreting it in nonhormonalform [2]

Moreover we demonstrated that significant increasesof mitochondrial superoxide production and Prx 3 proteinexpression can be detected in MT-III KO mice group Thisdata indicated the protective role of MT-III against highconcentrations of iodide induced mitochondrial superoxideproduction in the thyrocytes MT has been demonstratedto play a key role in the detoxification of heavy metalsprotecting against various forms of oxidative injury by scav-enging oxygen free radicals MTs belong to the group ofintracellular cysteine-rich metal-binding proteins which iswidely expressed in cells (nuclei andor cytoplasm) of various

8 Oxidative Medicine and Cellular Longevity

organs and tumors [67ndash69] MTs play a protective role inpreventing against intoxication with heavy metals such asPb Hg Cu and Cd [67 70] All 20 cysteine sulfur atomsare involved in the radical quenching process and the rateconstant for the reaction of hydroxyl radical with MT isabout 340-fold higher than that with GSH [71 72] There arefour MT isoforms in mammals MT-I and MT-II are mainlyinvolved in the protection of tissue against metal toxicitiesoxidative stresses and apoptosis It has been demonstratedthat the rabbit liver metallothionein-1 which contains zincandor cadmium ions appeared to scavenge free hydroxyland superoxide radicals [25 26] Under low levels of iodideorganification in goitrous-modified tissue of thyroid glandmetallothionein may provide a partial compensatory effecton prooxidative processes [73 74] Although no cell lineor animal model overexpressing MT in the thyroid hasbeen found yet documented evidence of MT in protectingagainst oxidative stress in cardiac-specific metallothionein-overexpressed transgenic mice has been demonstrated Caiet al reported that metallothionein as a potent antioxi-dant prevented the development of diabetic cardiomyopathythrough the inhibition of the mitochondrial cytochromec-mediated caspase-3 activation pathway [75] Sun et alreported that through doxorubicin induced chronic toxi-city in a metallothionein-overexpressed transgenic mouseheart the antioxidant action of MT is highly responsiblefor this cardioprotection [76] Ceylan-Isik et al reportedthat the cardiac-specific overexpression of MT rescues LPS-induced cardiac contractile and intracellular Ca2+ dysfunc-tions through the alleviation of ROSoxidative stress stresssignaling activation and endoplasmic reticulum (ER) stress[77] They also reported that the cardiac-specific overexpres-sion of metallothionein protects against nicotine exposure-induced cardiac contractile dysfunction and fibrosis possiblythrough inhibition of ROS accumulation and apoptosis [78]Zhang et al suggest that cardiac overexpression of metalloth-ionein prevents cold exposure-induced cardiac anomaliespossibly through the attenuation of myocardial fibrosis [79]Hu et al demonstrated that cardiac-specific overexpression ofmetallothionein protects against cigarette smoking exposure-induced myocardial contractile and mitochondrial damagefavoring a role of lessened apoptosis in a metallothionein-induced beneficial effect against side-stream smoke exposure[80] Besides metallothionein has been shown to regulateapoptosis and proliferation Overexpression of metalloth-ionein frequently occurs in human tumors and is related totumor progression [81 82] High expression of MT has beenobserved in different types of cancer and has been consideredas a potential prognostic marker in cancers of the breastpancreas skin and cervix [82ndash86] Further studies will beattempted to study the oxidative and antioxidative stress onan engineered cell line overexpressing metallothionein

5 Conclusion

Thepresent study provides evidence that high concentrationsof iodide may lead to a significant increase in mitochondrialsuperoxide production and Prx 3 expression in both invitro and in vivo studies MT-III KO mice aggravated

mitochondrial superoxide production and peroxiredoxin 3expression in the thyroid after excessive iodide exposureMT-III may potentially protect the thyroid against highconcentrations of iodide induced oxidative stress

Abbreviations

MT-III KO Metallothionein III knockoutMTs MetallothioneinsWT Wild-typeROS Reactive oxygen speciesPrx PeroxiredoxinKI Potassium iodideNI Normal iodide intakeHI High iodide intakeLDH Lactate dehydrogenaseH2O2 Hydrogen peroxide

SOD Superoxide dismutaseMTT 3-(45-Dimethylthiazol-2-yl)-25-

diphenyltetrazoliumbromide

K-S Kolmogorov-SmirnovLSD Least significant differenceT4 Thyroxine

T3 Tri-iodothyronine

FT4 Free thyroxine

FT3 Free tri-iodothyronine

Conflict of Interests

The authors declare that they have no competing interests

Acknowledgments

Thiswork is supported by theNational Natural Science Foun-dation of China (no 81273009) the Tianjin Science amp Tech-nology Council Grant of China (no 09JCYBJC11700) andthe Tianjin Educational amp Scientific Grant (no 20050107)This paper has been edited by one of the authors MohamedAhmed who is from USA and now is studying in TianjinMedical University China

References

[1] B Corvilain L Collyn J van Sande and J E DumontldquoStimulation by iodide of H

2O2generation in thyroid slices

from several speciesrdquo The American Journal of PhysiologymdashEndocrinology and Metabolism vol 278 no 4 pp E692ndashE6992000

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

[3] E Roti and E Degli Uberti ldquoIodine excess and hyperthy-roidismrdquoThyroid vol 11 no 5 pp 493ndash500 2001

[4] World Health Organization and United Nations ChildrenrsquosFund ldquoInternational Council for Control of Iodine DeficiencyDisorders 1999 progress towards the elimination of iodinedeficiency disorders (IDD)rdquo WHO Booklet World HealthOrganization 1999

Oxidative Medicine and Cellular Longevity 9

[5] A M Leung and L E Braverman ldquoConsequences of excessiodinerdquo Nature Reviews Endocrinology vol 10 no 3 pp 136ndash142 2013

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

[7] IMahmoud I ColinM CMany and J-F Denef ldquoDirect toxiceffect of iodide in excess on iodine-deficient thyroid glandsepithelial necrosis and inflammation associated with lipofuscinaccumulationrdquo Experimental and Molecular Pathology vol 44no 3 pp 259ndash271 1986

[8] J E Fradkin and J Wolff ldquoIodide-induced thyrotoxicosisrdquoMedicine vol 62 no 1 pp 1ndash20 1983

[9] J Golstein and J E Dumont ldquoCytotoxic effects of iodide onthyroid cells difference between rat thyroid FRTL-5 cell andpri-mary dog thyrocyte responsivenessrdquo Journal of EndocrinologicalInvestigation vol 19 no 2 pp 119ndash126 1996

[10] 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

[11] W Teng Z Shan X Teng et al ldquoEffect of iodine intakeon thyroid diseases in Chinardquo The New England Journal ofMedicine vol 354 no 26 pp 2783ndash2793 2006

[12] P Laurberg K M Pedersen A Hreidarsson N Sigfusson EIversen and P R Knudsen ldquoIodine intake and the patternof thyroid disorders a comparative epidemiological study ofthyroid abnormalities in the elderly in iceland and in JutlandDenmarkrdquo Journal of Clinical Endocrinology and Metabolismvol 83 no 3 pp 765ndash769 1998

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

[14] 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

[15] 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

[16] H Han P Xin L Zhao et al ldquoExcess iodine and high-fatdiet combination modulates lipid profile thyroid hormoneand hepatic LDLr expression values in micerdquo Biological TraceElement Research vol 147 no 1ndash3 pp 233ndash239 2012

[17] S-J Zhao Y Ye F-J Sun E-J Tian and Z-P Chen ldquoTheimpact of dietary iodine intake on lipid metabolism in micerdquoBiological Trace Element Research vol 142 no 3 pp 581ndash5882011

[18] 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

[19] V Pitsiavas P Smerdely M Li and S C Boyages ldquoAmiodaroneinduces a different pattern of ultrastructural change in thethyroid to iodine excess alone in both the BBW rat and theWistar ratrdquo European Journal of Endocrinology vol 137 no 1pp 89ndash98 1997

[20] M B Zimmermann ldquoIodine deficiency and excess in childrenworldwide status in 2013rdquo Endocrine Practice vol 19 no 5 pp839ndash846 2013

[21] K Mussig ldquoIodine-induced toxic effects due to seaweed con-sumptionrdquo in Comprehensive Handbook of Iodine V R PreedyG N Burrow and R R Watson Eds pp 897ndash908 ElsevierAmsterdam The Netherlands 2009

[22] A-M G Psarra S Solakidi and C E Sekeris ldquoThe mito-chondrion as a primary site of action of steroid and thyroidhormones presence and action of steroid and thyroid hormonereceptors in mitochondria of animal cellsrdquo Molecular andCellular Endocrinology vol 246 no 1-2 pp 21ndash33 2006

[23] Y Song N Driessens M Costa et al ldquoRoles of hydrogenperoxide in thyroid physiology and diseaserdquo Journal of ClinicalEndocrinology and Metabolism vol 92 no 10 pp 3764ndash37732007

[24] A G Cox C C Winterbourn and M B Hampton ldquoMito-chondrial peroxiredoxin involvement in antioxidant defenceand redox signallingrdquo Biochemical Journal vol 425 no 2 pp313ndash325 2010

[25] B Ruttkay-Nedecky L Nejdl J Gumulec et al ldquoThe role ofmetallothionein in oxidative stressrdquo International Journal ofMolecular Sciences vol 14 no 3 pp 6044ndash6066 2013

[26] N Thirumoorthy A Shyam Sunder K T Manisenthil KumarM Senthil kumarGNKGanesh andMChatterjee ldquoA reviewof metallothionein isoforms and their role in pathophysiologyrdquoWorld Journal of Surgical Oncology vol 9 article 54 2011

[27] 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

[28] M Saberi and R D Utiger ldquoAugmentation of thyrotropinresponses to thyrotropin releasing hormone following smalldecreases in serum thyroid hormone concentrationsrdquo TheJournal of Clinical Endocrinology and Metabolism vol 40 no3 pp 435ndash441 1975

[29] H Namba S Yamashita H Kimura et al ldquoEvidence of thyroidvolume increase in normal subjects receiving excess iodiderdquoTheJournal of Clinical Endocrinology and Metabolism vol 76 no 3pp 605ndash608 1993

[30] E Sternthal L Lipworth B Stanley C Abreau S L Fang andL E Braverman ldquoSuppression of thyroid radioiodine uptakeby various doses of stable iodiderdquo The New England Journal ofMedicine vol 303 no 19 pp 1083ndash1088 1980

[31] A G Vagenakis B Rapoport F Azizi G I Portnay L EBraverman and S H Ingbar ldquoHyperresponse to thyrotropinreleasing hormone accompanying small decreases in serumthyroid hormone concentrationsrdquoThe Journal of Clinical Inves-tigation vol 54 no 4 pp 913ndash918 1974

[32] G Philippou D A Koutras G Piperingos A Souvatzoglouand S D Moulopoulos ldquoThe effect of iodide on serum thyroidhormone levels in normal persons in hyperthyroid patientsand in hypothyroid patients on thyroxine replacementrdquoClinicalEndocrinology vol 36 no 6 pp 573ndash578 1992

[33] L Z Ali Salim R Othman M A Abdulla et al ldquoThymo-quinone inhibits murine leukemia WEHI-3 cells in vivo and invitrordquo PLoS ONE vol 9 no 12 Article ID e115340 2014

[34] X Wang J Ge K Wang J Qian and Y Zou ldquoEvaluation ofMTT assay for measurement of emodin-induced cytotoxicityrdquoASSAY and Drug Development Technologies vol 4 no 2 pp203ndash207 2006

10 Oxidative Medicine and Cellular Longevity

[35] C Lv C Wu Y-h Zhou Y Shao G Wang and Q-y WangldquoAlpha lipoic acid Modulated high glucose-induced rat mesan-gial cell dysfunction viamTORp70S6K4E-BP1 pathwayrdquo Inter-national Journal of Endocrinology vol 2014 Article ID 65858914 pages 2014

[36] L Liu X Hu G-Y Cai et al ldquoHigh glucose-induced hyper-trophy of mesangial cells is reversed by connexin43 overex-pression via PTENAktmTOR signalingrdquo Nephrology DialysisTransplantation vol 27 no 1 pp 90ndash100 2012

[37] D D Harrison-Findik S Z Lu E M Zmijewski J Jonesand M C Zimmerman ldquoEffect of alcohol exposure on hepaticsuperoxide generation and hepcidin expressionrdquoWorld Journalof Biological Chemistry vol 4 no 4 pp 119ndash130 2013

[38] M Wang Y Wang J He et al ldquoAlbumin induces neuroprotec-tion against ischemic stroke by altering Toll-like receptor 4 andregulatory T cells in micerdquo CNS and Neurological DisordersmdashDrug Targets vol 12 no 2 pp 220ndash227 2013

[39] X Yao ldquoEffect of zinc exposure on HNE and GLT-1 in spinalcord culturerdquo NeuroToxicology vol 30 no 1 pp 121ndash126 2009

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

[41] G P Becks M C Eggo and G N Burrow ldquoRegulation ofdifferentiated thyroid function by iodide preferential inhibitoryeffect of excess iodide on thyroid hormone secretion in sheepthyroid cell culturesrdquo Endocrinology vol 120 no 6 pp 2569ndash2575 1987

[42] M Vitale T Di Matola F DrsquoAscoli et al ldquoIodide excessinduces apoptosis in thyroid cells through a p53-independentmechanism involving oxidative stressrdquo Endocrinology vol 141no 2 pp 598ndash605 2000

[43] P Pramyothin AM Leung E N Pearce A OMalabanan andL E Braverman ldquoClinical problem-solving A hidden solutionrdquoTheNew England Journal of Medicine vol 365 no 22 pp 2123ndash2127 2011

[44] W Jubiz S Carlile and L D Lagerquist ldquoSerum thyrotropinand thyroid hormone levels in humans receiving chronicpotassium iodiderdquo The Journal of Clinical Endocrinology andMetabolism vol 44 no 2 pp 379ndash382 1977

[45] A G Vagenakis P Downs L E Braverman A Burger andS H Ingbar ldquoControl of thyroid hormone secretion in normalsubjects receiving iodidesrdquoThe Journal of Clinical Investigationvol 52 no 2 pp 528ndash532 1973

[46] M Safran andL E Braverman ldquoEffect of chronic douchingwithpolyvinylpyrrolidone-iodine on iodine absorption and thyroidfunctionrdquo Obstetrics and Gynecology vol 60 no 1 pp 35ndash401982

[47] K Newick B Cunniff K Preston et al ldquoPeroxiredoxin 3 is aredox-dependent target of thiostrepton in malignant mesothe-lioma cellsrdquo PLoS ONE vol 7 no 6 Article ID e39404 2012

[48] A Deniaud O S El Dein E Maillier et al ldquoEndoplasmicreticulum stress induces calcium-dependent permeability tran-sition mitochondrial outer membrane permeabilization andapoptosisrdquo Oncogene vol 27 no 3 pp 285ndash299 2008

[49] A Gorlach P Klappa and T Kietzmann ldquoThe endoplasmicreticulum folding calcium homeostasis signaling and redoxcontrolrdquo Antioxidants and Redox Signaling vol 8 no 9-10 pp1391ndash1418 2006

[50] 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 symporter

expression and activity involves the PI3KAkt signaling path-wayrdquo Endocrinology vol 155 no 3 pp 1145ndash1156 2014

[51] Y-G Wang L Li C-H Liu S Hong and M-J ZhangldquoPeroxiredoxin 3 is resistant to oxidation-induced apoptosis ofHep-3b cellsrdquoClinical and Translational Oncology vol 16 no 6pp 561ndash566 2014

[52] H C Whitaker D Patel W J Howat et al ldquoPeroxiredoxin-3is overexpressed in prostate cancer and promotes cancer cellsurvival by protecting cells fromoxidative stressrdquoBritish Journalof Cancer vol 109 no 4 pp 983ndash993 2013

[53] L Li Y G Zhang and C L Chen ldquoAnti-apoptotic role ofperoxiredoxin III in cervical cancer cellsrdquo FEBS Open Bio vol3 pp 51ndash54 2013

[54] 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

[55] L I Nadolnik Z V Niatsetskaya and S V Lupachyk ldquoEffectof oxidative stress on rat thyrocyte iodide metabolismrdquo CellBiochemistry and Function vol 26 no 3 pp 366ndash373 2008

[56] A C Freitas Ferreira L de Carvalho Cardoso D Rosenthaland D Pires de Carvalho ldquoThyroid Ca2+NADPH-dependentH2O2generation is partially inhibited by propylthiouracil and

methimazolerdquo European Journal of Biochemistry vol 270 no 11pp 2363ndash2368 2003

[57] G R Sue Z C Ho and K Kim ldquoPeroxiredoxins a historicaloverview and speculative preview of novel mechanisms andemerging concepts in cell signalingrdquo Free Radical Biology andMedicine vol 38 no 12 pp 1543ndash1552 2005

[58] M S Seo S W Kang K Kim I C Baines T H Lee and SG Rhee ldquoIdentification of a new type of mammalian perox-iredoxin that forms an intramolecular disulfide as a reactionintermediaterdquo Journal of Biological Chemistry vol 275 no 27pp 20346ndash20354 2000

[59] Z A Wood L B Poole and P A Karplus ldquoPeroxiredoxinevolution and the regulation of hydrogen peroxide signalingrdquoScience vol 300 no 5619 pp 650ndash653 2003

[60] D A Drechsel and M Patel ldquoRespiration-dependent H2O2

removal in brain mitochondria via the thioredoxinperoxire-doxin systemrdquoThe Journal of Biological Chemistry vol 285 no36 pp 27850ndash27858 2010

[61] W H Watson X Yang Y E Choi D P Jones and J P KehrerldquoThioredoxin and its role in toxicologyrdquo Toxicological Sciencesvol 78 no 1 pp 3ndash14 2004

[62] G Ekstrom and M Ingelman-Sundberg ldquoRat liver microso-mal NADPH-supported oxidase activity and lipid peroxida-tion dependent on ethanol-inducible cytochrome P-450 (P-450IIE1)rdquo Biochemical Pharmacology vol 38 no 8 pp 1313ndash1319 1989

[63] J Rashba-Step N J Turro and A I Cederbaum ldquoIncreasedNADPH- and NADH-dependent production of superoxideand hydroxyl radical by microsomes after chronic ethanoltreatmentrdquoArchives of Biochemistry and Biophysics vol 300 no1 pp 401ndash408 1993

[64] V Thakur M T Pritchard M R McMullen Q Wang andL E Nagy ldquoChronic ethanol feeding increases activation ofNADPH oxidase by lipopolysaccharide in rat Kupffer cellsrole of increased reactive oxygen in LPS-stimulated ERK12activation andTNF-120572 productionrdquo Journal of Leukocyte Biologyvol 79 no 6 pp 1348ndash1356 2006

Oxidative Medicine and Cellular Longevity 11

[65] HNakamuraAMitsui and J Yodoi ldquoThioredoxin overexpres-sion in transgenic micerdquo Methods in Enzymology vol 347 pp436ndash440 2002

[66] 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

[67] A Bieniek B Pula A Piotrowska et al ldquoExpression of metal-lothionein III andKi-67 antigen in various histological types ofbasal cell carcinomardquo Folia Histochemica et Cytobiologica vol50 no 3 pp 352ndash357 2012

[68] P Dziegiel ldquoExpression of metallothioneins in tumor cellsrdquoPolish Journal of Pathology vol 55 no 1 pp 3ndash12 2004

[69] M Oslash Pedersen A Larsen M Stoltenberg and M PenkowaldquoThe role of metallothionein in oncogenesis and cancer prog-nosisrdquo Progress inHistochemistry and Cytochemistry vol 44 no1 pp 29ndash64 2009

[70] W Braun M Vasak A H Robbins et al ldquoComparison ofthe NMR solution structure and the X-ray crystal structure ofrat metallothionein-2rdquo Proceedings of the National Academy ofSciences of the United States of America vol 89 no 21 pp 10124ndash10128 1992

[71] P JThornalley andM Vasak ldquoPossible role formetallothioneinin protection against radiation-induced oxidative stress Kinet-ics andmechanism of its reaction with superoxide and hydroxylradicalsrdquo Biochimica et Biophysica Acta Protein Structure andMolecular Enzymology vol 827 no 1 pp 36ndash44 1985

[72] M A Schwarz J S Lazo J C Yalowich et al ldquoMetallothioneinprotects against the cytotoxic and DNA-damaging effects ofnitric oxiderdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 92 no 10 pp 4452ndash4456 1995

[73] Y Luo A Kawashima and Y Ishido ldquoIodine excess as anenvironmental risk factor for autoimmune thyroid diseaserdquoInternational Journal of Molecular Sciences vol 15 pp 12895ndash12912 2014

[74] H I Falrsquofushinsrsquoka L L Hnatyshyna andOI Osadchuk ldquoTraceelements storage peculiarities and metallothionein contentin human thyroid gland under iodine deficiency euthyroidnodular goiterrdquo Ukrainskii Biokhimicheskii Zhurnal vol 86 pp107ndash113 2014

[75] L Cai Y H Wang G H Zhou et al ldquoAttenuation bymetallothionein of early cardiac cell death via suppression ofmitochondrial oxidative stress results in a prevention of diabeticcardiomyopathyrdquo Journal of the American College of Cardiologyvol 48 no 8 pp 1688ndash1697 2006

[76] X Sun Z Zhou and Y J Kang ldquoAttenuation of doxorubicinchronic toxicity in metallothionein-overexpressing transgenicmouse heartrdquo Cancer Research vol 61 no 8 pp 3382ndash33872001

[77] A F Ceylan-Isik P Zhao B F Zhang X Xiao G Su and J RenldquoCardiac overexpression of metallothionein rescues cardiaccontractile dysfunction and endoplasmic reticulum stress butnot autophagy in sepsisrdquo Journal of Molecular and CellularCardiology vol 48 no 2 pp 367ndash378 2010

[78] N Hu R Guo X Han B Zhu and J Ren ldquoCardiac-specificoverexpression of metallothionein rescues nicotine-inducedcardiac contractile dysfunction and interstitial fibrosisrdquoToxicol-ogy Letters vol 202 no 1 pp 8ndash14 2011

[79] Y Zhang N Hu Y Hua K L Richmond F Dong and JRen ldquoCardiac overexpression of metallothionein rescues coldexposure-induced myocardial contractile dysfunction through

attenuation of cardiac fibrosis despite cardiomyocyte mechani-cal anomaliesrdquo Free Radical Biology and Medicine vol 53 no 2pp 194ndash207 2012

[80] N Hu X Han E K Lane F Gao Y Zhang and J RenldquoCardiac-specific overexpression of metallothionein rescuesagainst cigarette smoking exposure-induced myocardial con-tractile and mitochondrial damagerdquo PLoS ONE vol 8 no 2Article ID e57151 2013

[81] M T Brazao-Silva S V Cardoso P R de Faria et al ldquoAdenoidcystic carcinoma of the salivary gland a clinicopathologicalstudy of 49 cases and of metallothionein expression with regardto tumour behaviourrdquo Histopathology vol 63 no 6 pp 802ndash809 2013

[82] K J Schmitz H Lang G Kaiser et al ldquoMetallothioneinoverexpression and its prognostic relevance in intrahepaticcholangiocarcinoma and extrahepatic hilar cholangiocarci-noma (Klatskin tumors)rdquo Human Pathology vol 40 no 12 pp1706ndash1714 2009

[83] S E Theocharis A P Margeli J T Klijanienko and G PKouraklis ldquoMetallothionein expression in human neoplasiardquoHistopathology vol 45 no 2 pp 103ndash118 2004

[84] STheocharis J Klijanienko C Giaginis et al ldquoMetallothioneinexpression in mobile tongue squamous cell carcinoma associa-tions with clinicopathological parameters and patient survivalrdquoHistopathology vol 59 no 3 pp 514ndash525 2011

[85] S-K Tai O J-K Tan V T-K Chow et al ldquoDifferentialexpression of metallothionein 1 and 2 isoforms in breast cancerlines with different invasive potential identification of a novelnon-silent metallothionein-1H mutant variantrdquo The AmericanJournal of Pathology vol 163 no 5 pp 2009ndash2019 2003

[86] Y Hishikawa T Koji D K Dhar S Kinugasa M Yamaguchiand N Nagasue ldquoMetallothionein expression correlates withmetastatic and proliferative potential in squamous cell carci-noma of the oesophagusrdquo British Journal of Cancer vol 81 no4 pp 712ndash720 1999

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Oxidative Medicine and Cellular Longevity

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Page 3: Research Article Metallothionein-I/II Knockout Mice

Oxidative Medicine and Cellular Longevity 3

with Hankrsquos solution and were suspended in Hankrsquos solutionwith 1 BSA The fluorescence intensity of MitoSOX wasdetected by a FACSCalibur (BD Bioscience San Jose CA)and the excitationemission wavelength is 488 nm575 nmCollecting FL2 channel forward scattering (forward scatterFSC) and lateral scattering (side scatter SSC) data 10000cells were collected for each sample The control groupwithout MitoSOX was regarded as the blank zero group forstandardization [35ndash38]

26 Western Blot Analysis Whole cell proteins were assayedfor protein concentration using the bicinchoninic acid (BCA)protein assay kit (Beyotime Institute of BiotechnologyJiangsu China) 50120583g of protein was transferred to nitro-cellulose membrane followed by SDS polyacrylamide gelelectrophoresis The nitrocellulose membrane was incubatedovernight with primary antibodies Anti-Peroxiredoxin 3antibody (Abcam Cambridge MA USA) and horseradishperoxidase (HRP) conjugated secondary antibody developedby Immobilon Western Chemiluminescent HRP Substrate(Merck Millipore MA USA) To verify equal loading 120573-actin (1 1000) (Cell Signaling Technology Inc MA USA)was used as a loading control Blots were scanned as grayscaleimages and quantitated using Image J software (NIH) All theblot intensities were normalized with that of loading control120573-actin [39]

27 Determination of Urinary Iodine Concentrations andSerum Thyroid Hormones Spot urine samples of mice werecollected and urinary iodine concentrations were measuredby As-Ce catalytic spectrophotometry in the Key Lab ofHormones and Development Ministry of Health Institute ofEndocrinology Tianjin Medical University [40] Blood wasobtained 14 days after NI 10 HI or 100 HI intake Samplesof blood were centrifuged for 10min serum samples wereobtained and stored at minus80∘C for use Serum concentrationsof thyroxine (T

4) free thyroxine (FT

4) triiodothyronine

(T3) and free triiodothyronine (FT

3) were determined by

using the Direct Chemiluminescence Technology Compet-itive Immunoassay Kits (Siemens Healthcare DiagnosticInc)

28 Statistics The data was represented as mean plusmn SD Basedon the Kolmogorov-Smirnov (K-S) for normality test andLevene statistic for the test of homogeneity of variancesUrinary iodine concentrations were expressed as the medianand determined with the nonparametric Kruskal-Wallis testone-way ANOVA with the least significant difference (LSD)test was performed using SPSS 170 to determine the differ-ences between the groups A 119875 value of less than 005 wasconsidered to be statistically significant

3 Results

31 Effect of MT-III on High Concentrated Iodide ExposureInduced Mitochondrial Superoxide Production and Prx 3 Pro-tein Expression In Vitro To investigate the antioxidative roleof MT-III in oxidative stress induced by high concentrationsof iodide in the thyroid thyroid cell suspensions were

Table 1 The median urinary iodine concentrations (120583gL) of WTmice and MT-III KO mice

Group WT MT-III KONI 32029 plusmn 11216 42433 plusmn 13195

10 HI 359292 plusmn 51998lowast

394924 plusmn 64793lowast

100 HI 3047917 plusmn 412894lowast

4066635 plusmn 352046lowast

lowastCompared to the NI group (119875 lt 005) WT mice group compared to MT-III KO mice group (119875 lt 005)119873 = 10 for each group

prepared from the thyroid of MT-III KO mice and WTmice The cells were then exposed to high concentrationsof KI (10minus4M 10minus3M and 10minus2M) or 10minus3M H

2O2for 2

hours Following 10minus4M 10minus3M and 10minus2M of KI or 10minus3MH2O2exposure we showed a significant increase in mito-

chondrial superoxide production with increased MitoSOXRed fluorescence intensity (119875 lt 005) (Figure 1(d)) and anincrease in LDH release (119875 lt 005) (Figure 1(c)) with adecrease in relative cell viability (119875 lt 005) (Figure 1(a)) in thethyroid cell suspensions of both MT-III KO and WT micegroups Compared to WT mice group a more significantincrease of mitochondrial superoxide production increasein LDH release and decrease in a relative cell viability canbe detected in MT-III KO mice group (119875 lt 005) (Figures1(d) 1(c) and 1(a)) Compared to control group the Prx 3protein expressions were significantly increased in 10minus2MKIor 10minus3MH

2O2exposure group in bothMT-III KO andWT

mice groups (119875 lt 005) Compared to WT mice group therewas a significant increase of Prx 3 protein expression in thethyroid cell suspensions ofMT-III KOmicewith the increaseof KI concentration (119875 lt 005) (Figure 1(b))

32 Urinary Iodine Concentrations In Vivo In order toverify the model of iodine excess in vivo urinary iodineconcentrations were detected after the MT-III KO mice andWT mice had NI 10 HI and 100 HI for 14 days The medianurinary iodine concentrations in NI 10 HI and 100 HI wereshown in Table 1 The high iodide intake of 10 HI group and100HI group in bothMT-III KOmice andWTmice resultedin significant increases in urinary iodine concentration whencompared to theNI group (119875 lt 005) The urinary iodineconcentrations were increased as the iodine supply increasedto 10HI 100HI in bothWT andMT-III KOmiceMoreovercompared to 10 HI group the extent of increase in urinaryiodine concentration in 100 HI was significantly higher (119875 lt005) Compared to WT mice MT -III KO mice had a moreprominent increase of urinary iodine concentration in 100HIgroup (119875 lt 005)

33 Changes of Serum Thyroid Hormones Level in WT andMT-III KO Mice following NI 10 HI or 100 HI Intake for14 Days We demonstrated that compared to the NI groupthere was no significant difference existing in serum thyroidhormones level (T

3 T4 FT3 and FT

4) in either 10 HI

group or 100 HI group in both WT mice and MT-III KOmice (119875 gt 005) However compared to WT mice therewas a significant difference in serum thyroid hormones level

4 Oxidative Medicine and Cellular Longevity

120

100

80

60

40

20

0

lowastlowast

Rela

tive v

iabi

lity

()

lowastlowast lowast

lowast lowast

lowast

WT miceMT-III KO mice

ControlKI KI KI

10minus4 M 10minus3 M 10minus2 M 10minus3 MH2O2

(a)

lowast lowastlowastlowast3

25

2

15

1

05

0Expr

essio

n of

Prx3

()

Prx 3

120573-actin

WT MT-III KO

WT miceMT-III KO mice

ControlKI KI KI

10minus4 M 10minus3 M 10minus2 M 10minus3 MH2O2

Con

trol

10minus4

M K

I

10minus3

M K

I

10minus2

M K

I

10minus3

MH2O2

Con

trol

10minus4

M K

I

10minus3

M K

I

10minus2

M K

I

10minus3

MH2O2

(b)

6543210

LDH

rele

ase

(Um

g pr

otei

n)

lowast lowast lowast lowast lowast lowastlowast lowast

times103

WT miceMT-III KO mice

ControlKI KI KI

10minus4 M 10minus3 M 10minus2 M 10minus3 MH2O2

(c)

lowastlowast lowast

lowast lowastlowast

lowastlowast120

100806040200M

itoSO

X flu

ores

cenc

e in

tens

ityW

TM

T-II

I KO

Control 262

Control 3644

10minus4 M KI 3754

10minus4 M KI 4931

10minus3 M KI 4925

10minus3 M KI 6447

10minus2 M KI 578

10minus2 M KI 7172

10minus3 M H2 9827

10minus3 M H2O28932

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 101 102 103 104 101 102 103 104100 100

WT miceMT-III KO mice

ControlKI KI KI

10minus4 M 10minus3 M 10minus2 M 10minus3 MH2O2

O2

(d)

Figure 1 Oxidative and antioxidative effect of high concentrations of KI on the mitochondria of thyrocytes in MT-III KO mice and WTmice (a) Decreased relative viability induced by high concentrations of KI (10minus4M 10minus3M and 10minus2M) or 10minus3MH

2O2in the thyroid cells

of MT-III KOmice byMTT assay (119873 = 8) (b) Representative western blot of Prx 3 (27 kDa) 120573-actin (45 kDa) was used as a loading controlDensitometric analysis showed a significant increase (lowast119875 lt 005) in Prx 3 expression compared to untreated control in the thyroid cells ofeither WT or MT-III KO mice (c) Increased LDH release following 2 h of high concentration of KI or H

2O2exposure in the thyroid cells

of MT-III KO mice (119873 = 8) (d) High concentration of KI or H2O2induced increased mitochondrial superoxide production in the thyroid

cells of WT and MT-III KO mice Histogram analysis was performed on the mean fluorescence intensity of MitoSOX Red as measured byflow cytometry Experiments were repeated 3 times with similar results Data are represented as mean plusmn SD One-way ANOVA with the LSDtest was used lowast119875 lt 005 compared with the control group of WT or MT-III KO mice respectively 119875 lt 005WTmice versus MT-III KOmice under the same treatment

(FT4) in 100 HI group in MT-III KO mice (119875 lt 005)

(Table 2)

34 High Iodide Intake Induced Increased MitochondrialSuperoxide Production and Peroxiredoxin 3 Expression inThyroid In Vivo To clarify the effect of iodide excess onmitochondrial superoxide production and Prx 3 protein

expression in thyroid of MT-III KO mice in vivo micewere divided into normal iodide intake (NI) 10 times iodideintake (10 HI) and 100 times iodide intake (100 HI) groupsCompared to the NI group the mitochondrial superoxideproduction measured by flow cytometry Prx 3 proteinexpression detected bywestern blot analysis and LDH releasein 100 HI group were significantly increased in both MT-III

Oxidative Medicine and Cellular Longevity 5

Table 2 Changes of serum thyroid hormones level (nmolL) in WT and MT-III KO mice following NI 10 HI or 100 HI intake for 14 days

Group T3 T4 FT3 FT4

WTNI 072 plusmn 013 2608 plusmn 1421 480 plusmn 042 2856 plusmn 451

10 HI 053 plusmn 009 3350 plusmn 445 426 plusmn 041 3142 plusmn 095

100 HI 071 plusmn 013 3470 plusmn 1324 461 plusmn 025 3320 plusmn 455

MT-III KONI 058 plusmn 015 2005 plusmn 468 404 plusmn 077 2569 plusmn 097

10 HI 069 plusmn 013 2253 plusmn 779 478 plusmn 005 2722 plusmn 103

100 HI 080 plusmn 022 1773 plusmn 676 501 plusmn 073 2399 plusmn 192lowast

lowastIn 100 HI group compared to WT mice there was a significant difference in serum thyroid hormones level (FT4) in MT-III KO mice (119875 lt 005)119873 = 8 foreach group

KO and WT groups (119875 lt 005) Compared to WT groupmore significant increases in the mitochondrial superoxideproduction Prx 3 protein expression and LDH release weredetected in MT-III KO group (119875 lt 005) There were nosignificant differences between the NI group and 10 HI groupin bothMT-III KO andWTmice (119875 gt 005) Comparedwiththe NI group relative cell viability of 100 HI group decreasedin both the MT-III KO and WT mice groups (119875 lt 005)Compared to the WT group a more significant decrease inrelative cell viability was detected in MT-III KO group (119875 lt005) No significant difference of relative cell viability wasdetected between NI group and 10 HI group in both MT-IIIKO and WT mice groups (119875 gt 005) (Figure 2)

4 Discussion

Iodide excess has been recognized as a risk factor for thedevelopment of thyroid disease in humans and animals [7ndash13] One of the underlying mechanisms is that the increasedoxidative stress induced by iodide excess cannot be balancedby endogenous antioxidant systems [14 15] In the presentstudy we focused the research on excessive iodide inducedmitochondrial superoxide production and Prx 3 expressionin the thyroid with or without MT III in vitro and in vivo

In a previous study [15] we showed that the concentrationresponse of KI induced a decrease in cell viability in FRTLcells by MTT assay compared to the control group therelative viability of 10minus5M 10minus4M 10minus3M and 10minus2M KIexposure groupswas significantly decreased at 24 hHoweverno significant decrease in the 10minus6M or 10minus7M KI exposuregroups was observed A strong stimulatory effect of iodidewas detected in 10minus4MKI and in 10minus3MKI exposure groupsat the 2 h time point [15] Iodide had a stimulatory effect onH2O2generation and when increased H

2O2is considered

as a limiting step in the biosynthesis of thyroid hormones[1] The normal relative cell viability in the 10minus6M or 10minus7MKI exposure groups can be explained by the physiologicaleffect of iodide which is necessary for the biosynthesis ofthyroid hormones [15] We used KI concentrations of 10minus4Mand above in the in vitro study and NI 10 HI and 100 HIin the in vivo study In bovine thyroid slices the maximalstimulation effect on H

2O2generation was obtained after 2 h

of preincubation with 10minus4M KI [1] It was reported that in

vitro the acute toxic effects of high iodide doses in humanthyroid follicles significantly increased the percentage ofnecrotic cells in 10minus5Mand doubledwith 10minus3Mas comparedto values measured at 10minus7M [10] Iodide concentrations of10minus4M or greater may inhibit the iodothyronine synthesisand thyroid hormone secretion [41]The concentrations from10minus5 to 10minus3M were from 100 to 10000 times higher thanthe normal iodine plasma levels estimated to be 10minus7M ineuthyroid human beings [42]

In the present study we detected that in 100 HI intakegroup there is a significant change of FT

4in MT-III KO

mice compared to WT mice however the values remainwithin the normal range in thyroid function The NI and10 HI groups in both WT and MT-III KO mice wereunder normal conditions and the thyroid function was notaffected Iodine is essential for the synthesis of the thyroidhormones In most individuals the decreased productionof thyroid hormones is only transient and resumes afteradaptation to the acute Wolff-Chaikoff effect [43] A detailedanalysis reveals a persistent drop of serum T

4and T

3of

25 and 15 respectively and a rise of TSH of 2mULAlthough no clinical signs of thyroid dysfunction or goiterwere found sonographic thyroid volume is slightly increased[2 29ndash32 44 45] Exposure to high concentrations ofiodine may decrease the release of thyroid hormone shownby the increase in the serum level of TSH [27 28 46](Table 3)

Thyroid toxicity of iodide excess has been demonstratedin vitro and in animals fed with an iodide-rich diet [42] Weshowed that high concentrated iodide induced cytotoxicity inthyrocytes significantly increasingmitochondrial superoxideanion production in the thyroid in high concentrated iodideexposure in vitro and in 100 HI diet in vivo in bothMT-III KO and WT mice which leads to the decreasedrelative cell viability in the thyroid It is suggested thatoxidative stress is the underlying mechanism in iodide excessinduced apoptosis in thyroid cells [42] Consistent withprevious reports [42 47ndash49] we showed that there is astrong increase of superoxide anion production in FRTLcells following high concentrations of iodide exposure [15]Serrano-Nascimento et al also observed that iodide excessincreased ROS production in thyrocytes and mitochondriawere the source of superoxide anion production [50] Theoxidative attack can be explained by the oxidative effect

6 Oxidative Medicine and Cellular Longevity

0

20

40

60

80

100

120

NI 10 HI 100 HI

Rela

tive v

iabi

lity

()

WT miceMT-III KO mice

lowastlowast

(a)

0

02

04

06

08

1

12

NI

NI

10 HI

10 HI

100 HI

100 HI NI 10 HI 100 HI

Expr

essio

n of

Prx

3 (

)

WT miceMT-III KO mice

lowast

lowast

WT MT-III KO

Prx 3

120573-actin

(b)

NI 10 HI 100 HI

WT miceMT-III KO mice

0

1000

2000

3000

4000

LDH

rele

ase (

Um

g pr

otei

n)

lowastlowast

(c)

0

20

40

60

80

NI 10 HI 100 HIMito

SOX

fluor

esce

nce i

nten

sity

WT miceMT-III KO mice

lowast

lowast

WT

MT-

III K

O

NI 1681

NI 2051 10 HI 2514 100 HI 6133

100 HI 478710 HI 1819

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

(d)

Figure 2 Mitochondrial superoxide production Prx 3 protein expression and LDH release and relative viability in the thyroid of WT andMT-III knockout mice following NI 10 HI or 100 HI diet for 14 days (a) 100 HI intake decreased the relative viability in thyroid of MT-IIIKO and WT mice (b) Representative western blot of Prx 3 (27 kDa) 120573-actin (45 kDa) was used as a loading control Densitometric analysisshowed a significant increase in Prx 3 expression in 100 HI group in the thyroid of either WT or MT-III KO mice especially in MT-IIIKO mice (c) 100 HI increased the LDH release in the thyroid of MT-III KO and WT mice (d) 100 HI intake increased the mitochondrialsuperoxide production in thyroid of WT and MT-III KO mice Histogram analysis was performed on the mean fluorescence intensity ofMitoSOX Red as measured by flow cytometry Experiments were repeated 3 times with similar results Data are represented as mean plusmn SD(119873 = 10group) One-way ANOVA with the LSD test was used lowast119875 lt 005 compared with the control group of WT or MT-III KO micerespectively 119875 lt 005WTmice compared with the MT-III KO mice under the same treatment

of HI diet on the thyroid gland ROS include free radi-cals such as the superoxide anion hydroxyl radicals andthe nonradical hydrogen peroxide Whether cells die fromoxidative stress induced apoptosis depends on the balancebetween the generation of oxidant species and the antioxidantsystem

In addition we demonstrated that no significant differ-ence was detected between NI group and 10 HI group in bothMT-III KO and WT mice groups This can be explained bythe fact that under basal conditions thyroid epithelial cellsproduce moderate amounts of ROS that are physiologicallyrequired for thyroid hormone synthesis [14] Physiologically

Oxidative Medicine and Cellular Longevity 7

Table 3 Documented literatures of iodide intake and changes of serum thyroxine (T4) and triiodothyronine (T3) concentrations

Dosage of daily iodine intake Time Subject Changes of serum T4 T3 concentrations Reference250 or 500 120583g 14 days Normal volunteers No change [27]1500 120583g 14 days Normal volunteers T4 T3 darr TSH uarr [27]

50 or 250mg 13 days Normal subjects T4 T3 darr TSH uarr(within normal range) [28]

27mg 4 weeks Normal volunteers T4 within the normal range except fortwo subjects TSH uarr [29]

Single doses of 10 30 50 and 100mg andthen daily doses of 10 15 30 50 or 100mg 12 days Euthyroid volunteers T3 T4 darr TSH uarr [30]

190mg 10 days Euthyroid volunteers T3 T4 darr TSH uarr [31]114mg 3ndash7 weeks Normal controls T3 T4 darr TSH uarr [32]

114mg 3ndash7 weeks Thyrotoxic patientsT4 T3 uarr in some casesother patients remained

euthyroid even after 6 weeks[32]

114mg 2 weeks Hypothyroid patients onthyroxine replacement

No consistent change(T4 uarr at 1 and 14 days TSH darr the first day) [32]

mitochondrial ROS generation is primarily due to electronsleaking from the electron transport chain (ETC) to generatesuperoxide radical Superoxide from the electron transportchain and other sources is converted by MnSOD to H

2O2

which is then metabolized by Prx 3 to water [47]Besides we demonstrated that high concentrated iodide

induces significant increased expression of Prx 3 whichsuggests that the increase of Prx 3 expression following highconcentrated iodide exposure orHI diet in the thyroidmay bethe protective response against high ROS generation Prx 3 isan indispensable ROS scavenger that protects against oxida-tive damage and subsequent apoptosis [51ndash53] It is reportedthat Prx 3 has an essential role in regulating oxidation-induced apoptosis and can be a potential therapeutic targetin castrate-independent prostate cancer [52] Iodide hada stimulatory effect on H

2O2generation in thyroid slices

[1] At high concentrations (above 01mM) H2O2induces

apoptosis in thyroid cells [1] When iodide is in excess ascompared to tyrosine residues it reacts with the iodoniumcation formed by iodide oxidation to give molecular iodineExcess molecular iodine then induced apoptosis throughgeneration of free radicals [54] H

2O2generation is a limiting

step in thyroid hormone biosynthesis it is essential foriodide oxidation by thyroperoxidase [14 55 56] H

2O2can

be eliminated by Prx 3 In mammals Prx 3 is targeted to themitochondrial matrix It is estimated that Prx 3 will be thetarget for up to 90 of hydrogen peroxide generated in themitochondrial matrix [57] mitochondrial peroxiredoxins (3andor 5) can reduce H

2O2to water through reducing equiv-

alents provided by thiol-containing proteins [58 59] butthe mechanisms involved are unclear [24 59] Drechsel andPatel demonstrated that thioredoxin (Trxs)peroxiredoxin(Prxs) (Prx 3 and Prx 5) is the major contributing systemto H2O2removal in brain mitochondria [60] Prxs and Trxs

are important liver antioxidant proteins and play importantroles in maintaining liver redox homeostasis [61] It isreported that chronic ethanol exposure increases NADPHoxidase activity and increases the production of superoxide

and hydrogen peroxide [62ndash64] Thioredoxin scavenges andreduces ROS via peroxiredoxins [65] Endoplasmic reticulumstress disrupts the electron transport chain leading to theincreased production of ROS [48 49] Bae et al reportedthat the antioxidant role of Prx 3 was evident from thefindings that pyrazole-induced protein carbonylation and theformation of 4-HNE adducts and MDA in the liver weremarkedly increased in Prx 3minusminus mice compared with wild-type mice [66]

A high urinary excretion of iodine in a spot urine sampleis the ultimate proof of iodine excess in vivo [2] We showedthe urinary iodine concentration increased as the iodidesupply increased significantly higher urinary iodine concen-tration can be detected in 100 HI than 10 HI and in MT-IIIKOmice than inWTmiceThis data indicates the adaptationof the thyroid gland to iodine excess and that the excessiveiodine is excreted from the urine in order to maintain thenormal function of thyroid This makes the studies in vivodifferent from studies in vitro In addition to the urinaryexcretion of iodine there are many control mechanisms ofthe thyroid to counteract iodide excess These include thesodium-iodide symporter which controls the limiting stepof thyroid hormone synthesis the Wolff-Chaikoff effect anarrest of thyroid hormone secretion from stores in the colloida preferential secretion of the less active T

4over T

3 and

dumping of excess iodine by secreting it in nonhormonalform [2]

Moreover we demonstrated that significant increasesof mitochondrial superoxide production and Prx 3 proteinexpression can be detected in MT-III KO mice group Thisdata indicated the protective role of MT-III against highconcentrations of iodide induced mitochondrial superoxideproduction in the thyrocytes MT has been demonstratedto play a key role in the detoxification of heavy metalsprotecting against various forms of oxidative injury by scav-enging oxygen free radicals MTs belong to the group ofintracellular cysteine-rich metal-binding proteins which iswidely expressed in cells (nuclei andor cytoplasm) of various

8 Oxidative Medicine and Cellular Longevity

organs and tumors [67ndash69] MTs play a protective role inpreventing against intoxication with heavy metals such asPb Hg Cu and Cd [67 70] All 20 cysteine sulfur atomsare involved in the radical quenching process and the rateconstant for the reaction of hydroxyl radical with MT isabout 340-fold higher than that with GSH [71 72] There arefour MT isoforms in mammals MT-I and MT-II are mainlyinvolved in the protection of tissue against metal toxicitiesoxidative stresses and apoptosis It has been demonstratedthat the rabbit liver metallothionein-1 which contains zincandor cadmium ions appeared to scavenge free hydroxyland superoxide radicals [25 26] Under low levels of iodideorganification in goitrous-modified tissue of thyroid glandmetallothionein may provide a partial compensatory effecton prooxidative processes [73 74] Although no cell lineor animal model overexpressing MT in the thyroid hasbeen found yet documented evidence of MT in protectingagainst oxidative stress in cardiac-specific metallothionein-overexpressed transgenic mice has been demonstrated Caiet al reported that metallothionein as a potent antioxi-dant prevented the development of diabetic cardiomyopathythrough the inhibition of the mitochondrial cytochromec-mediated caspase-3 activation pathway [75] Sun et alreported that through doxorubicin induced chronic toxi-city in a metallothionein-overexpressed transgenic mouseheart the antioxidant action of MT is highly responsiblefor this cardioprotection [76] Ceylan-Isik et al reportedthat the cardiac-specific overexpression of MT rescues LPS-induced cardiac contractile and intracellular Ca2+ dysfunc-tions through the alleviation of ROSoxidative stress stresssignaling activation and endoplasmic reticulum (ER) stress[77] They also reported that the cardiac-specific overexpres-sion of metallothionein protects against nicotine exposure-induced cardiac contractile dysfunction and fibrosis possiblythrough inhibition of ROS accumulation and apoptosis [78]Zhang et al suggest that cardiac overexpression of metalloth-ionein prevents cold exposure-induced cardiac anomaliespossibly through the attenuation of myocardial fibrosis [79]Hu et al demonstrated that cardiac-specific overexpression ofmetallothionein protects against cigarette smoking exposure-induced myocardial contractile and mitochondrial damagefavoring a role of lessened apoptosis in a metallothionein-induced beneficial effect against side-stream smoke exposure[80] Besides metallothionein has been shown to regulateapoptosis and proliferation Overexpression of metalloth-ionein frequently occurs in human tumors and is related totumor progression [81 82] High expression of MT has beenobserved in different types of cancer and has been consideredas a potential prognostic marker in cancers of the breastpancreas skin and cervix [82ndash86] Further studies will beattempted to study the oxidative and antioxidative stress onan engineered cell line overexpressing metallothionein

5 Conclusion

Thepresent study provides evidence that high concentrationsof iodide may lead to a significant increase in mitochondrialsuperoxide production and Prx 3 expression in both invitro and in vivo studies MT-III KO mice aggravated

mitochondrial superoxide production and peroxiredoxin 3expression in the thyroid after excessive iodide exposureMT-III may potentially protect the thyroid against highconcentrations of iodide induced oxidative stress

Abbreviations

MT-III KO Metallothionein III knockoutMTs MetallothioneinsWT Wild-typeROS Reactive oxygen speciesPrx PeroxiredoxinKI Potassium iodideNI Normal iodide intakeHI High iodide intakeLDH Lactate dehydrogenaseH2O2 Hydrogen peroxide

SOD Superoxide dismutaseMTT 3-(45-Dimethylthiazol-2-yl)-25-

diphenyltetrazoliumbromide

K-S Kolmogorov-SmirnovLSD Least significant differenceT4 Thyroxine

T3 Tri-iodothyronine

FT4 Free thyroxine

FT3 Free tri-iodothyronine

Conflict of Interests

The authors declare that they have no competing interests

Acknowledgments

Thiswork is supported by theNational Natural Science Foun-dation of China (no 81273009) the Tianjin Science amp Tech-nology Council Grant of China (no 09JCYBJC11700) andthe Tianjin Educational amp Scientific Grant (no 20050107)This paper has been edited by one of the authors MohamedAhmed who is from USA and now is studying in TianjinMedical University China

References

[1] B Corvilain L Collyn J van Sande and J E DumontldquoStimulation by iodide of H

2O2generation in thyroid slices

from several speciesrdquo The American Journal of PhysiologymdashEndocrinology and Metabolism vol 278 no 4 pp E692ndashE6992000

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

[3] E Roti and E Degli Uberti ldquoIodine excess and hyperthy-roidismrdquoThyroid vol 11 no 5 pp 493ndash500 2001

[4] World Health Organization and United Nations ChildrenrsquosFund ldquoInternational Council for Control of Iodine DeficiencyDisorders 1999 progress towards the elimination of iodinedeficiency disorders (IDD)rdquo WHO Booklet World HealthOrganization 1999

Oxidative Medicine and Cellular Longevity 9

[5] A M Leung and L E Braverman ldquoConsequences of excessiodinerdquo Nature Reviews Endocrinology vol 10 no 3 pp 136ndash142 2013

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

[7] IMahmoud I ColinM CMany and J-F Denef ldquoDirect toxiceffect of iodide in excess on iodine-deficient thyroid glandsepithelial necrosis and inflammation associated with lipofuscinaccumulationrdquo Experimental and Molecular Pathology vol 44no 3 pp 259ndash271 1986

[8] J E Fradkin and J Wolff ldquoIodide-induced thyrotoxicosisrdquoMedicine vol 62 no 1 pp 1ndash20 1983

[9] J Golstein and J E Dumont ldquoCytotoxic effects of iodide onthyroid cells difference between rat thyroid FRTL-5 cell andpri-mary dog thyrocyte responsivenessrdquo Journal of EndocrinologicalInvestigation vol 19 no 2 pp 119ndash126 1996

[10] 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

[11] W Teng Z Shan X Teng et al ldquoEffect of iodine intakeon thyroid diseases in Chinardquo The New England Journal ofMedicine vol 354 no 26 pp 2783ndash2793 2006

[12] P Laurberg K M Pedersen A Hreidarsson N Sigfusson EIversen and P R Knudsen ldquoIodine intake and the patternof thyroid disorders a comparative epidemiological study ofthyroid abnormalities in the elderly in iceland and in JutlandDenmarkrdquo Journal of Clinical Endocrinology and Metabolismvol 83 no 3 pp 765ndash769 1998

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

[14] 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

[15] 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

[16] H Han P Xin L Zhao et al ldquoExcess iodine and high-fatdiet combination modulates lipid profile thyroid hormoneand hepatic LDLr expression values in micerdquo Biological TraceElement Research vol 147 no 1ndash3 pp 233ndash239 2012

[17] S-J Zhao Y Ye F-J Sun E-J Tian and Z-P Chen ldquoTheimpact of dietary iodine intake on lipid metabolism in micerdquoBiological Trace Element Research vol 142 no 3 pp 581ndash5882011

[18] 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

[19] V Pitsiavas P Smerdely M Li and S C Boyages ldquoAmiodaroneinduces a different pattern of ultrastructural change in thethyroid to iodine excess alone in both the BBW rat and theWistar ratrdquo European Journal of Endocrinology vol 137 no 1pp 89ndash98 1997

[20] M B Zimmermann ldquoIodine deficiency and excess in childrenworldwide status in 2013rdquo Endocrine Practice vol 19 no 5 pp839ndash846 2013

[21] K Mussig ldquoIodine-induced toxic effects due to seaweed con-sumptionrdquo in Comprehensive Handbook of Iodine V R PreedyG N Burrow and R R Watson Eds pp 897ndash908 ElsevierAmsterdam The Netherlands 2009

[22] A-M G Psarra S Solakidi and C E Sekeris ldquoThe mito-chondrion as a primary site of action of steroid and thyroidhormones presence and action of steroid and thyroid hormonereceptors in mitochondria of animal cellsrdquo Molecular andCellular Endocrinology vol 246 no 1-2 pp 21ndash33 2006

[23] Y Song N Driessens M Costa et al ldquoRoles of hydrogenperoxide in thyroid physiology and diseaserdquo Journal of ClinicalEndocrinology and Metabolism vol 92 no 10 pp 3764ndash37732007

[24] A G Cox C C Winterbourn and M B Hampton ldquoMito-chondrial peroxiredoxin involvement in antioxidant defenceand redox signallingrdquo Biochemical Journal vol 425 no 2 pp313ndash325 2010

[25] B Ruttkay-Nedecky L Nejdl J Gumulec et al ldquoThe role ofmetallothionein in oxidative stressrdquo International Journal ofMolecular Sciences vol 14 no 3 pp 6044ndash6066 2013

[26] N Thirumoorthy A Shyam Sunder K T Manisenthil KumarM Senthil kumarGNKGanesh andMChatterjee ldquoA reviewof metallothionein isoforms and their role in pathophysiologyrdquoWorld Journal of Surgical Oncology vol 9 article 54 2011

[27] 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

[28] M Saberi and R D Utiger ldquoAugmentation of thyrotropinresponses to thyrotropin releasing hormone following smalldecreases in serum thyroid hormone concentrationsrdquo TheJournal of Clinical Endocrinology and Metabolism vol 40 no3 pp 435ndash441 1975

[29] H Namba S Yamashita H Kimura et al ldquoEvidence of thyroidvolume increase in normal subjects receiving excess iodiderdquoTheJournal of Clinical Endocrinology and Metabolism vol 76 no 3pp 605ndash608 1993

[30] E Sternthal L Lipworth B Stanley C Abreau S L Fang andL E Braverman ldquoSuppression of thyroid radioiodine uptakeby various doses of stable iodiderdquo The New England Journal ofMedicine vol 303 no 19 pp 1083ndash1088 1980

[31] A G Vagenakis B Rapoport F Azizi G I Portnay L EBraverman and S H Ingbar ldquoHyperresponse to thyrotropinreleasing hormone accompanying small decreases in serumthyroid hormone concentrationsrdquoThe Journal of Clinical Inves-tigation vol 54 no 4 pp 913ndash918 1974

[32] G Philippou D A Koutras G Piperingos A Souvatzoglouand S D Moulopoulos ldquoThe effect of iodide on serum thyroidhormone levels in normal persons in hyperthyroid patientsand in hypothyroid patients on thyroxine replacementrdquoClinicalEndocrinology vol 36 no 6 pp 573ndash578 1992

[33] L Z Ali Salim R Othman M A Abdulla et al ldquoThymo-quinone inhibits murine leukemia WEHI-3 cells in vivo and invitrordquo PLoS ONE vol 9 no 12 Article ID e115340 2014

[34] X Wang J Ge K Wang J Qian and Y Zou ldquoEvaluation ofMTT assay for measurement of emodin-induced cytotoxicityrdquoASSAY and Drug Development Technologies vol 4 no 2 pp203ndash207 2006

10 Oxidative Medicine and Cellular Longevity

[35] C Lv C Wu Y-h Zhou Y Shao G Wang and Q-y WangldquoAlpha lipoic acid Modulated high glucose-induced rat mesan-gial cell dysfunction viamTORp70S6K4E-BP1 pathwayrdquo Inter-national Journal of Endocrinology vol 2014 Article ID 65858914 pages 2014

[36] L Liu X Hu G-Y Cai et al ldquoHigh glucose-induced hyper-trophy of mesangial cells is reversed by connexin43 overex-pression via PTENAktmTOR signalingrdquo Nephrology DialysisTransplantation vol 27 no 1 pp 90ndash100 2012

[37] D D Harrison-Findik S Z Lu E M Zmijewski J Jonesand M C Zimmerman ldquoEffect of alcohol exposure on hepaticsuperoxide generation and hepcidin expressionrdquoWorld Journalof Biological Chemistry vol 4 no 4 pp 119ndash130 2013

[38] M Wang Y Wang J He et al ldquoAlbumin induces neuroprotec-tion against ischemic stroke by altering Toll-like receptor 4 andregulatory T cells in micerdquo CNS and Neurological DisordersmdashDrug Targets vol 12 no 2 pp 220ndash227 2013

[39] X Yao ldquoEffect of zinc exposure on HNE and GLT-1 in spinalcord culturerdquo NeuroToxicology vol 30 no 1 pp 121ndash126 2009

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

[41] G P Becks M C Eggo and G N Burrow ldquoRegulation ofdifferentiated thyroid function by iodide preferential inhibitoryeffect of excess iodide on thyroid hormone secretion in sheepthyroid cell culturesrdquo Endocrinology vol 120 no 6 pp 2569ndash2575 1987

[42] M Vitale T Di Matola F DrsquoAscoli et al ldquoIodide excessinduces apoptosis in thyroid cells through a p53-independentmechanism involving oxidative stressrdquo Endocrinology vol 141no 2 pp 598ndash605 2000

[43] P Pramyothin AM Leung E N Pearce A OMalabanan andL E Braverman ldquoClinical problem-solving A hidden solutionrdquoTheNew England Journal of Medicine vol 365 no 22 pp 2123ndash2127 2011

[44] W Jubiz S Carlile and L D Lagerquist ldquoSerum thyrotropinand thyroid hormone levels in humans receiving chronicpotassium iodiderdquo The Journal of Clinical Endocrinology andMetabolism vol 44 no 2 pp 379ndash382 1977

[45] A G Vagenakis P Downs L E Braverman A Burger andS H Ingbar ldquoControl of thyroid hormone secretion in normalsubjects receiving iodidesrdquoThe Journal of Clinical Investigationvol 52 no 2 pp 528ndash532 1973

[46] M Safran andL E Braverman ldquoEffect of chronic douchingwithpolyvinylpyrrolidone-iodine on iodine absorption and thyroidfunctionrdquo Obstetrics and Gynecology vol 60 no 1 pp 35ndash401982

[47] K Newick B Cunniff K Preston et al ldquoPeroxiredoxin 3 is aredox-dependent target of thiostrepton in malignant mesothe-lioma cellsrdquo PLoS ONE vol 7 no 6 Article ID e39404 2012

[48] A Deniaud O S El Dein E Maillier et al ldquoEndoplasmicreticulum stress induces calcium-dependent permeability tran-sition mitochondrial outer membrane permeabilization andapoptosisrdquo Oncogene vol 27 no 3 pp 285ndash299 2008

[49] A Gorlach P Klappa and T Kietzmann ldquoThe endoplasmicreticulum folding calcium homeostasis signaling and redoxcontrolrdquo Antioxidants and Redox Signaling vol 8 no 9-10 pp1391ndash1418 2006

[50] 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 symporter

expression and activity involves the PI3KAkt signaling path-wayrdquo Endocrinology vol 155 no 3 pp 1145ndash1156 2014

[51] Y-G Wang L Li C-H Liu S Hong and M-J ZhangldquoPeroxiredoxin 3 is resistant to oxidation-induced apoptosis ofHep-3b cellsrdquoClinical and Translational Oncology vol 16 no 6pp 561ndash566 2014

[52] H C Whitaker D Patel W J Howat et al ldquoPeroxiredoxin-3is overexpressed in prostate cancer and promotes cancer cellsurvival by protecting cells fromoxidative stressrdquoBritish Journalof Cancer vol 109 no 4 pp 983ndash993 2013

[53] L Li Y G Zhang and C L Chen ldquoAnti-apoptotic role ofperoxiredoxin III in cervical cancer cellsrdquo FEBS Open Bio vol3 pp 51ndash54 2013

[54] 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

[55] L I Nadolnik Z V Niatsetskaya and S V Lupachyk ldquoEffectof oxidative stress on rat thyrocyte iodide metabolismrdquo CellBiochemistry and Function vol 26 no 3 pp 366ndash373 2008

[56] A C Freitas Ferreira L de Carvalho Cardoso D Rosenthaland D Pires de Carvalho ldquoThyroid Ca2+NADPH-dependentH2O2generation is partially inhibited by propylthiouracil and

methimazolerdquo European Journal of Biochemistry vol 270 no 11pp 2363ndash2368 2003

[57] G R Sue Z C Ho and K Kim ldquoPeroxiredoxins a historicaloverview and speculative preview of novel mechanisms andemerging concepts in cell signalingrdquo Free Radical Biology andMedicine vol 38 no 12 pp 1543ndash1552 2005

[58] M S Seo S W Kang K Kim I C Baines T H Lee and SG Rhee ldquoIdentification of a new type of mammalian perox-iredoxin that forms an intramolecular disulfide as a reactionintermediaterdquo Journal of Biological Chemistry vol 275 no 27pp 20346ndash20354 2000

[59] Z A Wood L B Poole and P A Karplus ldquoPeroxiredoxinevolution and the regulation of hydrogen peroxide signalingrdquoScience vol 300 no 5619 pp 650ndash653 2003

[60] D A Drechsel and M Patel ldquoRespiration-dependent H2O2

removal in brain mitochondria via the thioredoxinperoxire-doxin systemrdquoThe Journal of Biological Chemistry vol 285 no36 pp 27850ndash27858 2010

[61] W H Watson X Yang Y E Choi D P Jones and J P KehrerldquoThioredoxin and its role in toxicologyrdquo Toxicological Sciencesvol 78 no 1 pp 3ndash14 2004

[62] G Ekstrom and M Ingelman-Sundberg ldquoRat liver microso-mal NADPH-supported oxidase activity and lipid peroxida-tion dependent on ethanol-inducible cytochrome P-450 (P-450IIE1)rdquo Biochemical Pharmacology vol 38 no 8 pp 1313ndash1319 1989

[63] J Rashba-Step N J Turro and A I Cederbaum ldquoIncreasedNADPH- and NADH-dependent production of superoxideand hydroxyl radical by microsomes after chronic ethanoltreatmentrdquoArchives of Biochemistry and Biophysics vol 300 no1 pp 401ndash408 1993

[64] V Thakur M T Pritchard M R McMullen Q Wang andL E Nagy ldquoChronic ethanol feeding increases activation ofNADPH oxidase by lipopolysaccharide in rat Kupffer cellsrole of increased reactive oxygen in LPS-stimulated ERK12activation andTNF-120572 productionrdquo Journal of Leukocyte Biologyvol 79 no 6 pp 1348ndash1356 2006

Oxidative Medicine and Cellular Longevity 11

[65] HNakamuraAMitsui and J Yodoi ldquoThioredoxin overexpres-sion in transgenic micerdquo Methods in Enzymology vol 347 pp436ndash440 2002

[66] 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

[67] A Bieniek B Pula A Piotrowska et al ldquoExpression of metal-lothionein III andKi-67 antigen in various histological types ofbasal cell carcinomardquo Folia Histochemica et Cytobiologica vol50 no 3 pp 352ndash357 2012

[68] P Dziegiel ldquoExpression of metallothioneins in tumor cellsrdquoPolish Journal of Pathology vol 55 no 1 pp 3ndash12 2004

[69] M Oslash Pedersen A Larsen M Stoltenberg and M PenkowaldquoThe role of metallothionein in oncogenesis and cancer prog-nosisrdquo Progress inHistochemistry and Cytochemistry vol 44 no1 pp 29ndash64 2009

[70] W Braun M Vasak A H Robbins et al ldquoComparison ofthe NMR solution structure and the X-ray crystal structure ofrat metallothionein-2rdquo Proceedings of the National Academy ofSciences of the United States of America vol 89 no 21 pp 10124ndash10128 1992

[71] P JThornalley andM Vasak ldquoPossible role formetallothioneinin protection against radiation-induced oxidative stress Kinet-ics andmechanism of its reaction with superoxide and hydroxylradicalsrdquo Biochimica et Biophysica Acta Protein Structure andMolecular Enzymology vol 827 no 1 pp 36ndash44 1985

[72] M A Schwarz J S Lazo J C Yalowich et al ldquoMetallothioneinprotects against the cytotoxic and DNA-damaging effects ofnitric oxiderdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 92 no 10 pp 4452ndash4456 1995

[73] Y Luo A Kawashima and Y Ishido ldquoIodine excess as anenvironmental risk factor for autoimmune thyroid diseaserdquoInternational Journal of Molecular Sciences vol 15 pp 12895ndash12912 2014

[74] H I Falrsquofushinsrsquoka L L Hnatyshyna andOI Osadchuk ldquoTraceelements storage peculiarities and metallothionein contentin human thyroid gland under iodine deficiency euthyroidnodular goiterrdquo Ukrainskii Biokhimicheskii Zhurnal vol 86 pp107ndash113 2014

[75] L Cai Y H Wang G H Zhou et al ldquoAttenuation bymetallothionein of early cardiac cell death via suppression ofmitochondrial oxidative stress results in a prevention of diabeticcardiomyopathyrdquo Journal of the American College of Cardiologyvol 48 no 8 pp 1688ndash1697 2006

[76] X Sun Z Zhou and Y J Kang ldquoAttenuation of doxorubicinchronic toxicity in metallothionein-overexpressing transgenicmouse heartrdquo Cancer Research vol 61 no 8 pp 3382ndash33872001

[77] A F Ceylan-Isik P Zhao B F Zhang X Xiao G Su and J RenldquoCardiac overexpression of metallothionein rescues cardiaccontractile dysfunction and endoplasmic reticulum stress butnot autophagy in sepsisrdquo Journal of Molecular and CellularCardiology vol 48 no 2 pp 367ndash378 2010

[78] N Hu R Guo X Han B Zhu and J Ren ldquoCardiac-specificoverexpression of metallothionein rescues nicotine-inducedcardiac contractile dysfunction and interstitial fibrosisrdquoToxicol-ogy Letters vol 202 no 1 pp 8ndash14 2011

[79] Y Zhang N Hu Y Hua K L Richmond F Dong and JRen ldquoCardiac overexpression of metallothionein rescues coldexposure-induced myocardial contractile dysfunction through

attenuation of cardiac fibrosis despite cardiomyocyte mechani-cal anomaliesrdquo Free Radical Biology and Medicine vol 53 no 2pp 194ndash207 2012

[80] N Hu X Han E K Lane F Gao Y Zhang and J RenldquoCardiac-specific overexpression of metallothionein rescuesagainst cigarette smoking exposure-induced myocardial con-tractile and mitochondrial damagerdquo PLoS ONE vol 8 no 2Article ID e57151 2013

[81] M T Brazao-Silva S V Cardoso P R de Faria et al ldquoAdenoidcystic carcinoma of the salivary gland a clinicopathologicalstudy of 49 cases and of metallothionein expression with regardto tumour behaviourrdquo Histopathology vol 63 no 6 pp 802ndash809 2013

[82] K J Schmitz H Lang G Kaiser et al ldquoMetallothioneinoverexpression and its prognostic relevance in intrahepaticcholangiocarcinoma and extrahepatic hilar cholangiocarci-noma (Klatskin tumors)rdquo Human Pathology vol 40 no 12 pp1706ndash1714 2009

[83] S E Theocharis A P Margeli J T Klijanienko and G PKouraklis ldquoMetallothionein expression in human neoplasiardquoHistopathology vol 45 no 2 pp 103ndash118 2004

[84] STheocharis J Klijanienko C Giaginis et al ldquoMetallothioneinexpression in mobile tongue squamous cell carcinoma associa-tions with clinicopathological parameters and patient survivalrdquoHistopathology vol 59 no 3 pp 514ndash525 2011

[85] S-K Tai O J-K Tan V T-K Chow et al ldquoDifferentialexpression of metallothionein 1 and 2 isoforms in breast cancerlines with different invasive potential identification of a novelnon-silent metallothionein-1H mutant variantrdquo The AmericanJournal of Pathology vol 163 no 5 pp 2009ndash2019 2003

[86] Y Hishikawa T Koji D K Dhar S Kinugasa M Yamaguchiand N Nagasue ldquoMetallothionein expression correlates withmetastatic and proliferative potential in squamous cell carci-noma of the oesophagusrdquo British Journal of Cancer vol 81 no4 pp 712ndash720 1999

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Behavioural Neurology

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Disease Markers

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OncologyJournal of

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Oxidative Medicine and Cellular Longevity

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 4: Research Article Metallothionein-I/II Knockout Mice

4 Oxidative Medicine and Cellular Longevity

120

100

80

60

40

20

0

lowastlowast

Rela

tive v

iabi

lity

()

lowastlowast lowast

lowast lowast

lowast

WT miceMT-III KO mice

ControlKI KI KI

10minus4 M 10minus3 M 10minus2 M 10minus3 MH2O2

(a)

lowast lowastlowastlowast3

25

2

15

1

05

0Expr

essio

n of

Prx3

()

Prx 3

120573-actin

WT MT-III KO

WT miceMT-III KO mice

ControlKI KI KI

10minus4 M 10minus3 M 10minus2 M 10minus3 MH2O2

Con

trol

10minus4

M K

I

10minus3

M K

I

10minus2

M K

I

10minus3

MH2O2

Con

trol

10minus4

M K

I

10minus3

M K

I

10minus2

M K

I

10minus3

MH2O2

(b)

6543210

LDH

rele

ase

(Um

g pr

otei

n)

lowast lowast lowast lowast lowast lowastlowast lowast

times103

WT miceMT-III KO mice

ControlKI KI KI

10minus4 M 10minus3 M 10minus2 M 10minus3 MH2O2

(c)

lowastlowast lowast

lowast lowastlowast

lowastlowast120

100806040200M

itoSO

X flu

ores

cenc

e in

tens

ityW

TM

T-II

I KO

Control 262

Control 3644

10minus4 M KI 3754

10minus4 M KI 4931

10minus3 M KI 4925

10minus3 M KI 6447

10minus2 M KI 578

10minus2 M KI 7172

10minus3 M H2 9827

10minus3 M H2O28932

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 101 102 103 104 101 102 103 104100 100

WT miceMT-III KO mice

ControlKI KI KI

10minus4 M 10minus3 M 10minus2 M 10minus3 MH2O2

O2

(d)

Figure 1 Oxidative and antioxidative effect of high concentrations of KI on the mitochondria of thyrocytes in MT-III KO mice and WTmice (a) Decreased relative viability induced by high concentrations of KI (10minus4M 10minus3M and 10minus2M) or 10minus3MH

2O2in the thyroid cells

of MT-III KOmice byMTT assay (119873 = 8) (b) Representative western blot of Prx 3 (27 kDa) 120573-actin (45 kDa) was used as a loading controlDensitometric analysis showed a significant increase (lowast119875 lt 005) in Prx 3 expression compared to untreated control in the thyroid cells ofeither WT or MT-III KO mice (c) Increased LDH release following 2 h of high concentration of KI or H

2O2exposure in the thyroid cells

of MT-III KO mice (119873 = 8) (d) High concentration of KI or H2O2induced increased mitochondrial superoxide production in the thyroid

cells of WT and MT-III KO mice Histogram analysis was performed on the mean fluorescence intensity of MitoSOX Red as measured byflow cytometry Experiments were repeated 3 times with similar results Data are represented as mean plusmn SD One-way ANOVA with the LSDtest was used lowast119875 lt 005 compared with the control group of WT or MT-III KO mice respectively 119875 lt 005WTmice versus MT-III KOmice under the same treatment

(FT4) in 100 HI group in MT-III KO mice (119875 lt 005)

(Table 2)

34 High Iodide Intake Induced Increased MitochondrialSuperoxide Production and Peroxiredoxin 3 Expression inThyroid In Vivo To clarify the effect of iodide excess onmitochondrial superoxide production and Prx 3 protein

expression in thyroid of MT-III KO mice in vivo micewere divided into normal iodide intake (NI) 10 times iodideintake (10 HI) and 100 times iodide intake (100 HI) groupsCompared to the NI group the mitochondrial superoxideproduction measured by flow cytometry Prx 3 proteinexpression detected bywestern blot analysis and LDH releasein 100 HI group were significantly increased in both MT-III

Oxidative Medicine and Cellular Longevity 5

Table 2 Changes of serum thyroid hormones level (nmolL) in WT and MT-III KO mice following NI 10 HI or 100 HI intake for 14 days

Group T3 T4 FT3 FT4

WTNI 072 plusmn 013 2608 plusmn 1421 480 plusmn 042 2856 plusmn 451

10 HI 053 plusmn 009 3350 plusmn 445 426 plusmn 041 3142 plusmn 095

100 HI 071 plusmn 013 3470 plusmn 1324 461 plusmn 025 3320 plusmn 455

MT-III KONI 058 plusmn 015 2005 plusmn 468 404 plusmn 077 2569 plusmn 097

10 HI 069 plusmn 013 2253 plusmn 779 478 plusmn 005 2722 plusmn 103

100 HI 080 plusmn 022 1773 plusmn 676 501 plusmn 073 2399 plusmn 192lowast

lowastIn 100 HI group compared to WT mice there was a significant difference in serum thyroid hormones level (FT4) in MT-III KO mice (119875 lt 005)119873 = 8 foreach group

KO and WT groups (119875 lt 005) Compared to WT groupmore significant increases in the mitochondrial superoxideproduction Prx 3 protein expression and LDH release weredetected in MT-III KO group (119875 lt 005) There were nosignificant differences between the NI group and 10 HI groupin bothMT-III KO andWTmice (119875 gt 005) Comparedwiththe NI group relative cell viability of 100 HI group decreasedin both the MT-III KO and WT mice groups (119875 lt 005)Compared to the WT group a more significant decrease inrelative cell viability was detected in MT-III KO group (119875 lt005) No significant difference of relative cell viability wasdetected between NI group and 10 HI group in both MT-IIIKO and WT mice groups (119875 gt 005) (Figure 2)

4 Discussion

Iodide excess has been recognized as a risk factor for thedevelopment of thyroid disease in humans and animals [7ndash13] One of the underlying mechanisms is that the increasedoxidative stress induced by iodide excess cannot be balancedby endogenous antioxidant systems [14 15] In the presentstudy we focused the research on excessive iodide inducedmitochondrial superoxide production and Prx 3 expressionin the thyroid with or without MT III in vitro and in vivo

In a previous study [15] we showed that the concentrationresponse of KI induced a decrease in cell viability in FRTLcells by MTT assay compared to the control group therelative viability of 10minus5M 10minus4M 10minus3M and 10minus2M KIexposure groupswas significantly decreased at 24 hHoweverno significant decrease in the 10minus6M or 10minus7M KI exposuregroups was observed A strong stimulatory effect of iodidewas detected in 10minus4MKI and in 10minus3MKI exposure groupsat the 2 h time point [15] Iodide had a stimulatory effect onH2O2generation and when increased H

2O2is considered

as a limiting step in the biosynthesis of thyroid hormones[1] The normal relative cell viability in the 10minus6M or 10minus7MKI exposure groups can be explained by the physiologicaleffect of iodide which is necessary for the biosynthesis ofthyroid hormones [15] We used KI concentrations of 10minus4Mand above in the in vitro study and NI 10 HI and 100 HIin the in vivo study In bovine thyroid slices the maximalstimulation effect on H

2O2generation was obtained after 2 h

of preincubation with 10minus4M KI [1] It was reported that in

vitro the acute toxic effects of high iodide doses in humanthyroid follicles significantly increased the percentage ofnecrotic cells in 10minus5Mand doubledwith 10minus3Mas comparedto values measured at 10minus7M [10] Iodide concentrations of10minus4M or greater may inhibit the iodothyronine synthesisand thyroid hormone secretion [41]The concentrations from10minus5 to 10minus3M were from 100 to 10000 times higher thanthe normal iodine plasma levels estimated to be 10minus7M ineuthyroid human beings [42]

In the present study we detected that in 100 HI intakegroup there is a significant change of FT

4in MT-III KO

mice compared to WT mice however the values remainwithin the normal range in thyroid function The NI and10 HI groups in both WT and MT-III KO mice wereunder normal conditions and the thyroid function was notaffected Iodine is essential for the synthesis of the thyroidhormones In most individuals the decreased productionof thyroid hormones is only transient and resumes afteradaptation to the acute Wolff-Chaikoff effect [43] A detailedanalysis reveals a persistent drop of serum T

4and T

3of

25 and 15 respectively and a rise of TSH of 2mULAlthough no clinical signs of thyroid dysfunction or goiterwere found sonographic thyroid volume is slightly increased[2 29ndash32 44 45] Exposure to high concentrations ofiodine may decrease the release of thyroid hormone shownby the increase in the serum level of TSH [27 28 46](Table 3)

Thyroid toxicity of iodide excess has been demonstratedin vitro and in animals fed with an iodide-rich diet [42] Weshowed that high concentrated iodide induced cytotoxicity inthyrocytes significantly increasingmitochondrial superoxideanion production in the thyroid in high concentrated iodideexposure in vitro and in 100 HI diet in vivo in bothMT-III KO and WT mice which leads to the decreasedrelative cell viability in the thyroid It is suggested thatoxidative stress is the underlying mechanism in iodide excessinduced apoptosis in thyroid cells [42] Consistent withprevious reports [42 47ndash49] we showed that there is astrong increase of superoxide anion production in FRTLcells following high concentrations of iodide exposure [15]Serrano-Nascimento et al also observed that iodide excessincreased ROS production in thyrocytes and mitochondriawere the source of superoxide anion production [50] Theoxidative attack can be explained by the oxidative effect

6 Oxidative Medicine and Cellular Longevity

0

20

40

60

80

100

120

NI 10 HI 100 HI

Rela

tive v

iabi

lity

()

WT miceMT-III KO mice

lowastlowast

(a)

0

02

04

06

08

1

12

NI

NI

10 HI

10 HI

100 HI

100 HI NI 10 HI 100 HI

Expr

essio

n of

Prx

3 (

)

WT miceMT-III KO mice

lowast

lowast

WT MT-III KO

Prx 3

120573-actin

(b)

NI 10 HI 100 HI

WT miceMT-III KO mice

0

1000

2000

3000

4000

LDH

rele

ase (

Um

g pr

otei

n)

lowastlowast

(c)

0

20

40

60

80

NI 10 HI 100 HIMito

SOX

fluor

esce

nce i

nten

sity

WT miceMT-III KO mice

lowast

lowast

WT

MT-

III K

O

NI 1681

NI 2051 10 HI 2514 100 HI 6133

100 HI 478710 HI 1819

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

(d)

Figure 2 Mitochondrial superoxide production Prx 3 protein expression and LDH release and relative viability in the thyroid of WT andMT-III knockout mice following NI 10 HI or 100 HI diet for 14 days (a) 100 HI intake decreased the relative viability in thyroid of MT-IIIKO and WT mice (b) Representative western blot of Prx 3 (27 kDa) 120573-actin (45 kDa) was used as a loading control Densitometric analysisshowed a significant increase in Prx 3 expression in 100 HI group in the thyroid of either WT or MT-III KO mice especially in MT-IIIKO mice (c) 100 HI increased the LDH release in the thyroid of MT-III KO and WT mice (d) 100 HI intake increased the mitochondrialsuperoxide production in thyroid of WT and MT-III KO mice Histogram analysis was performed on the mean fluorescence intensity ofMitoSOX Red as measured by flow cytometry Experiments were repeated 3 times with similar results Data are represented as mean plusmn SD(119873 = 10group) One-way ANOVA with the LSD test was used lowast119875 lt 005 compared with the control group of WT or MT-III KO micerespectively 119875 lt 005WTmice compared with the MT-III KO mice under the same treatment

of HI diet on the thyroid gland ROS include free radi-cals such as the superoxide anion hydroxyl radicals andthe nonradical hydrogen peroxide Whether cells die fromoxidative stress induced apoptosis depends on the balancebetween the generation of oxidant species and the antioxidantsystem

In addition we demonstrated that no significant differ-ence was detected between NI group and 10 HI group in bothMT-III KO and WT mice groups This can be explained bythe fact that under basal conditions thyroid epithelial cellsproduce moderate amounts of ROS that are physiologicallyrequired for thyroid hormone synthesis [14] Physiologically

Oxidative Medicine and Cellular Longevity 7

Table 3 Documented literatures of iodide intake and changes of serum thyroxine (T4) and triiodothyronine (T3) concentrations

Dosage of daily iodine intake Time Subject Changes of serum T4 T3 concentrations Reference250 or 500 120583g 14 days Normal volunteers No change [27]1500 120583g 14 days Normal volunteers T4 T3 darr TSH uarr [27]

50 or 250mg 13 days Normal subjects T4 T3 darr TSH uarr(within normal range) [28]

27mg 4 weeks Normal volunteers T4 within the normal range except fortwo subjects TSH uarr [29]

Single doses of 10 30 50 and 100mg andthen daily doses of 10 15 30 50 or 100mg 12 days Euthyroid volunteers T3 T4 darr TSH uarr [30]

190mg 10 days Euthyroid volunteers T3 T4 darr TSH uarr [31]114mg 3ndash7 weeks Normal controls T3 T4 darr TSH uarr [32]

114mg 3ndash7 weeks Thyrotoxic patientsT4 T3 uarr in some casesother patients remained

euthyroid even after 6 weeks[32]

114mg 2 weeks Hypothyroid patients onthyroxine replacement

No consistent change(T4 uarr at 1 and 14 days TSH darr the first day) [32]

mitochondrial ROS generation is primarily due to electronsleaking from the electron transport chain (ETC) to generatesuperoxide radical Superoxide from the electron transportchain and other sources is converted by MnSOD to H

2O2

which is then metabolized by Prx 3 to water [47]Besides we demonstrated that high concentrated iodide

induces significant increased expression of Prx 3 whichsuggests that the increase of Prx 3 expression following highconcentrated iodide exposure orHI diet in the thyroidmay bethe protective response against high ROS generation Prx 3 isan indispensable ROS scavenger that protects against oxida-tive damage and subsequent apoptosis [51ndash53] It is reportedthat Prx 3 has an essential role in regulating oxidation-induced apoptosis and can be a potential therapeutic targetin castrate-independent prostate cancer [52] Iodide hada stimulatory effect on H

2O2generation in thyroid slices

[1] At high concentrations (above 01mM) H2O2induces

apoptosis in thyroid cells [1] When iodide is in excess ascompared to tyrosine residues it reacts with the iodoniumcation formed by iodide oxidation to give molecular iodineExcess molecular iodine then induced apoptosis throughgeneration of free radicals [54] H

2O2generation is a limiting

step in thyroid hormone biosynthesis it is essential foriodide oxidation by thyroperoxidase [14 55 56] H

2O2can

be eliminated by Prx 3 In mammals Prx 3 is targeted to themitochondrial matrix It is estimated that Prx 3 will be thetarget for up to 90 of hydrogen peroxide generated in themitochondrial matrix [57] mitochondrial peroxiredoxins (3andor 5) can reduce H

2O2to water through reducing equiv-

alents provided by thiol-containing proteins [58 59] butthe mechanisms involved are unclear [24 59] Drechsel andPatel demonstrated that thioredoxin (Trxs)peroxiredoxin(Prxs) (Prx 3 and Prx 5) is the major contributing systemto H2O2removal in brain mitochondria [60] Prxs and Trxs

are important liver antioxidant proteins and play importantroles in maintaining liver redox homeostasis [61] It isreported that chronic ethanol exposure increases NADPHoxidase activity and increases the production of superoxide

and hydrogen peroxide [62ndash64] Thioredoxin scavenges andreduces ROS via peroxiredoxins [65] Endoplasmic reticulumstress disrupts the electron transport chain leading to theincreased production of ROS [48 49] Bae et al reportedthat the antioxidant role of Prx 3 was evident from thefindings that pyrazole-induced protein carbonylation and theformation of 4-HNE adducts and MDA in the liver weremarkedly increased in Prx 3minusminus mice compared with wild-type mice [66]

A high urinary excretion of iodine in a spot urine sampleis the ultimate proof of iodine excess in vivo [2] We showedthe urinary iodine concentration increased as the iodidesupply increased significantly higher urinary iodine concen-tration can be detected in 100 HI than 10 HI and in MT-IIIKOmice than inWTmiceThis data indicates the adaptationof the thyroid gland to iodine excess and that the excessiveiodine is excreted from the urine in order to maintain thenormal function of thyroid This makes the studies in vivodifferent from studies in vitro In addition to the urinaryexcretion of iodine there are many control mechanisms ofthe thyroid to counteract iodide excess These include thesodium-iodide symporter which controls the limiting stepof thyroid hormone synthesis the Wolff-Chaikoff effect anarrest of thyroid hormone secretion from stores in the colloida preferential secretion of the less active T

4over T

3 and

dumping of excess iodine by secreting it in nonhormonalform [2]

Moreover we demonstrated that significant increasesof mitochondrial superoxide production and Prx 3 proteinexpression can be detected in MT-III KO mice group Thisdata indicated the protective role of MT-III against highconcentrations of iodide induced mitochondrial superoxideproduction in the thyrocytes MT has been demonstratedto play a key role in the detoxification of heavy metalsprotecting against various forms of oxidative injury by scav-enging oxygen free radicals MTs belong to the group ofintracellular cysteine-rich metal-binding proteins which iswidely expressed in cells (nuclei andor cytoplasm) of various

8 Oxidative Medicine and Cellular Longevity

organs and tumors [67ndash69] MTs play a protective role inpreventing against intoxication with heavy metals such asPb Hg Cu and Cd [67 70] All 20 cysteine sulfur atomsare involved in the radical quenching process and the rateconstant for the reaction of hydroxyl radical with MT isabout 340-fold higher than that with GSH [71 72] There arefour MT isoforms in mammals MT-I and MT-II are mainlyinvolved in the protection of tissue against metal toxicitiesoxidative stresses and apoptosis It has been demonstratedthat the rabbit liver metallothionein-1 which contains zincandor cadmium ions appeared to scavenge free hydroxyland superoxide radicals [25 26] Under low levels of iodideorganification in goitrous-modified tissue of thyroid glandmetallothionein may provide a partial compensatory effecton prooxidative processes [73 74] Although no cell lineor animal model overexpressing MT in the thyroid hasbeen found yet documented evidence of MT in protectingagainst oxidative stress in cardiac-specific metallothionein-overexpressed transgenic mice has been demonstrated Caiet al reported that metallothionein as a potent antioxi-dant prevented the development of diabetic cardiomyopathythrough the inhibition of the mitochondrial cytochromec-mediated caspase-3 activation pathway [75] Sun et alreported that through doxorubicin induced chronic toxi-city in a metallothionein-overexpressed transgenic mouseheart the antioxidant action of MT is highly responsiblefor this cardioprotection [76] Ceylan-Isik et al reportedthat the cardiac-specific overexpression of MT rescues LPS-induced cardiac contractile and intracellular Ca2+ dysfunc-tions through the alleviation of ROSoxidative stress stresssignaling activation and endoplasmic reticulum (ER) stress[77] They also reported that the cardiac-specific overexpres-sion of metallothionein protects against nicotine exposure-induced cardiac contractile dysfunction and fibrosis possiblythrough inhibition of ROS accumulation and apoptosis [78]Zhang et al suggest that cardiac overexpression of metalloth-ionein prevents cold exposure-induced cardiac anomaliespossibly through the attenuation of myocardial fibrosis [79]Hu et al demonstrated that cardiac-specific overexpression ofmetallothionein protects against cigarette smoking exposure-induced myocardial contractile and mitochondrial damagefavoring a role of lessened apoptosis in a metallothionein-induced beneficial effect against side-stream smoke exposure[80] Besides metallothionein has been shown to regulateapoptosis and proliferation Overexpression of metalloth-ionein frequently occurs in human tumors and is related totumor progression [81 82] High expression of MT has beenobserved in different types of cancer and has been consideredas a potential prognostic marker in cancers of the breastpancreas skin and cervix [82ndash86] Further studies will beattempted to study the oxidative and antioxidative stress onan engineered cell line overexpressing metallothionein

5 Conclusion

Thepresent study provides evidence that high concentrationsof iodide may lead to a significant increase in mitochondrialsuperoxide production and Prx 3 expression in both invitro and in vivo studies MT-III KO mice aggravated

mitochondrial superoxide production and peroxiredoxin 3expression in the thyroid after excessive iodide exposureMT-III may potentially protect the thyroid against highconcentrations of iodide induced oxidative stress

Abbreviations

MT-III KO Metallothionein III knockoutMTs MetallothioneinsWT Wild-typeROS Reactive oxygen speciesPrx PeroxiredoxinKI Potassium iodideNI Normal iodide intakeHI High iodide intakeLDH Lactate dehydrogenaseH2O2 Hydrogen peroxide

SOD Superoxide dismutaseMTT 3-(45-Dimethylthiazol-2-yl)-25-

diphenyltetrazoliumbromide

K-S Kolmogorov-SmirnovLSD Least significant differenceT4 Thyroxine

T3 Tri-iodothyronine

FT4 Free thyroxine

FT3 Free tri-iodothyronine

Conflict of Interests

The authors declare that they have no competing interests

Acknowledgments

Thiswork is supported by theNational Natural Science Foun-dation of China (no 81273009) the Tianjin Science amp Tech-nology Council Grant of China (no 09JCYBJC11700) andthe Tianjin Educational amp Scientific Grant (no 20050107)This paper has been edited by one of the authors MohamedAhmed who is from USA and now is studying in TianjinMedical University China

References

[1] B Corvilain L Collyn J van Sande and J E DumontldquoStimulation by iodide of H

2O2generation in thyroid slices

from several speciesrdquo The American Journal of PhysiologymdashEndocrinology and Metabolism vol 278 no 4 pp E692ndashE6992000

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

[3] E Roti and E Degli Uberti ldquoIodine excess and hyperthy-roidismrdquoThyroid vol 11 no 5 pp 493ndash500 2001

[4] World Health Organization and United Nations ChildrenrsquosFund ldquoInternational Council for Control of Iodine DeficiencyDisorders 1999 progress towards the elimination of iodinedeficiency disorders (IDD)rdquo WHO Booklet World HealthOrganization 1999

Oxidative Medicine and Cellular Longevity 9

[5] A M Leung and L E Braverman ldquoConsequences of excessiodinerdquo Nature Reviews Endocrinology vol 10 no 3 pp 136ndash142 2013

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

[7] IMahmoud I ColinM CMany and J-F Denef ldquoDirect toxiceffect of iodide in excess on iodine-deficient thyroid glandsepithelial necrosis and inflammation associated with lipofuscinaccumulationrdquo Experimental and Molecular Pathology vol 44no 3 pp 259ndash271 1986

[8] J E Fradkin and J Wolff ldquoIodide-induced thyrotoxicosisrdquoMedicine vol 62 no 1 pp 1ndash20 1983

[9] J Golstein and J E Dumont ldquoCytotoxic effects of iodide onthyroid cells difference between rat thyroid FRTL-5 cell andpri-mary dog thyrocyte responsivenessrdquo Journal of EndocrinologicalInvestigation vol 19 no 2 pp 119ndash126 1996

[10] 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

[11] W Teng Z Shan X Teng et al ldquoEffect of iodine intakeon thyroid diseases in Chinardquo The New England Journal ofMedicine vol 354 no 26 pp 2783ndash2793 2006

[12] P Laurberg K M Pedersen A Hreidarsson N Sigfusson EIversen and P R Knudsen ldquoIodine intake and the patternof thyroid disorders a comparative epidemiological study ofthyroid abnormalities in the elderly in iceland and in JutlandDenmarkrdquo Journal of Clinical Endocrinology and Metabolismvol 83 no 3 pp 765ndash769 1998

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

[14] 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

[15] 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

[16] H Han P Xin L Zhao et al ldquoExcess iodine and high-fatdiet combination modulates lipid profile thyroid hormoneand hepatic LDLr expression values in micerdquo Biological TraceElement Research vol 147 no 1ndash3 pp 233ndash239 2012

[17] S-J Zhao Y Ye F-J Sun E-J Tian and Z-P Chen ldquoTheimpact of dietary iodine intake on lipid metabolism in micerdquoBiological Trace Element Research vol 142 no 3 pp 581ndash5882011

[18] 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

[19] V Pitsiavas P Smerdely M Li and S C Boyages ldquoAmiodaroneinduces a different pattern of ultrastructural change in thethyroid to iodine excess alone in both the BBW rat and theWistar ratrdquo European Journal of Endocrinology vol 137 no 1pp 89ndash98 1997

[20] M B Zimmermann ldquoIodine deficiency and excess in childrenworldwide status in 2013rdquo Endocrine Practice vol 19 no 5 pp839ndash846 2013

[21] K Mussig ldquoIodine-induced toxic effects due to seaweed con-sumptionrdquo in Comprehensive Handbook of Iodine V R PreedyG N Burrow and R R Watson Eds pp 897ndash908 ElsevierAmsterdam The Netherlands 2009

[22] A-M G Psarra S Solakidi and C E Sekeris ldquoThe mito-chondrion as a primary site of action of steroid and thyroidhormones presence and action of steroid and thyroid hormonereceptors in mitochondria of animal cellsrdquo Molecular andCellular Endocrinology vol 246 no 1-2 pp 21ndash33 2006

[23] Y Song N Driessens M Costa et al ldquoRoles of hydrogenperoxide in thyroid physiology and diseaserdquo Journal of ClinicalEndocrinology and Metabolism vol 92 no 10 pp 3764ndash37732007

[24] A G Cox C C Winterbourn and M B Hampton ldquoMito-chondrial peroxiredoxin involvement in antioxidant defenceand redox signallingrdquo Biochemical Journal vol 425 no 2 pp313ndash325 2010

[25] B Ruttkay-Nedecky L Nejdl J Gumulec et al ldquoThe role ofmetallothionein in oxidative stressrdquo International Journal ofMolecular Sciences vol 14 no 3 pp 6044ndash6066 2013

[26] N Thirumoorthy A Shyam Sunder K T Manisenthil KumarM Senthil kumarGNKGanesh andMChatterjee ldquoA reviewof metallothionein isoforms and their role in pathophysiologyrdquoWorld Journal of Surgical Oncology vol 9 article 54 2011

[27] 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

[28] M Saberi and R D Utiger ldquoAugmentation of thyrotropinresponses to thyrotropin releasing hormone following smalldecreases in serum thyroid hormone concentrationsrdquo TheJournal of Clinical Endocrinology and Metabolism vol 40 no3 pp 435ndash441 1975

[29] H Namba S Yamashita H Kimura et al ldquoEvidence of thyroidvolume increase in normal subjects receiving excess iodiderdquoTheJournal of Clinical Endocrinology and Metabolism vol 76 no 3pp 605ndash608 1993

[30] E Sternthal L Lipworth B Stanley C Abreau S L Fang andL E Braverman ldquoSuppression of thyroid radioiodine uptakeby various doses of stable iodiderdquo The New England Journal ofMedicine vol 303 no 19 pp 1083ndash1088 1980

[31] A G Vagenakis B Rapoport F Azizi G I Portnay L EBraverman and S H Ingbar ldquoHyperresponse to thyrotropinreleasing hormone accompanying small decreases in serumthyroid hormone concentrationsrdquoThe Journal of Clinical Inves-tigation vol 54 no 4 pp 913ndash918 1974

[32] G Philippou D A Koutras G Piperingos A Souvatzoglouand S D Moulopoulos ldquoThe effect of iodide on serum thyroidhormone levels in normal persons in hyperthyroid patientsand in hypothyroid patients on thyroxine replacementrdquoClinicalEndocrinology vol 36 no 6 pp 573ndash578 1992

[33] L Z Ali Salim R Othman M A Abdulla et al ldquoThymo-quinone inhibits murine leukemia WEHI-3 cells in vivo and invitrordquo PLoS ONE vol 9 no 12 Article ID e115340 2014

[34] X Wang J Ge K Wang J Qian and Y Zou ldquoEvaluation ofMTT assay for measurement of emodin-induced cytotoxicityrdquoASSAY and Drug Development Technologies vol 4 no 2 pp203ndash207 2006

10 Oxidative Medicine and Cellular Longevity

[35] C Lv C Wu Y-h Zhou Y Shao G Wang and Q-y WangldquoAlpha lipoic acid Modulated high glucose-induced rat mesan-gial cell dysfunction viamTORp70S6K4E-BP1 pathwayrdquo Inter-national Journal of Endocrinology vol 2014 Article ID 65858914 pages 2014

[36] L Liu X Hu G-Y Cai et al ldquoHigh glucose-induced hyper-trophy of mesangial cells is reversed by connexin43 overex-pression via PTENAktmTOR signalingrdquo Nephrology DialysisTransplantation vol 27 no 1 pp 90ndash100 2012

[37] D D Harrison-Findik S Z Lu E M Zmijewski J Jonesand M C Zimmerman ldquoEffect of alcohol exposure on hepaticsuperoxide generation and hepcidin expressionrdquoWorld Journalof Biological Chemistry vol 4 no 4 pp 119ndash130 2013

[38] M Wang Y Wang J He et al ldquoAlbumin induces neuroprotec-tion against ischemic stroke by altering Toll-like receptor 4 andregulatory T cells in micerdquo CNS and Neurological DisordersmdashDrug Targets vol 12 no 2 pp 220ndash227 2013

[39] X Yao ldquoEffect of zinc exposure on HNE and GLT-1 in spinalcord culturerdquo NeuroToxicology vol 30 no 1 pp 121ndash126 2009

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

[41] G P Becks M C Eggo and G N Burrow ldquoRegulation ofdifferentiated thyroid function by iodide preferential inhibitoryeffect of excess iodide on thyroid hormone secretion in sheepthyroid cell culturesrdquo Endocrinology vol 120 no 6 pp 2569ndash2575 1987

[42] M Vitale T Di Matola F DrsquoAscoli et al ldquoIodide excessinduces apoptosis in thyroid cells through a p53-independentmechanism involving oxidative stressrdquo Endocrinology vol 141no 2 pp 598ndash605 2000

[43] P Pramyothin AM Leung E N Pearce A OMalabanan andL E Braverman ldquoClinical problem-solving A hidden solutionrdquoTheNew England Journal of Medicine vol 365 no 22 pp 2123ndash2127 2011

[44] W Jubiz S Carlile and L D Lagerquist ldquoSerum thyrotropinand thyroid hormone levels in humans receiving chronicpotassium iodiderdquo The Journal of Clinical Endocrinology andMetabolism vol 44 no 2 pp 379ndash382 1977

[45] A G Vagenakis P Downs L E Braverman A Burger andS H Ingbar ldquoControl of thyroid hormone secretion in normalsubjects receiving iodidesrdquoThe Journal of Clinical Investigationvol 52 no 2 pp 528ndash532 1973

[46] M Safran andL E Braverman ldquoEffect of chronic douchingwithpolyvinylpyrrolidone-iodine on iodine absorption and thyroidfunctionrdquo Obstetrics and Gynecology vol 60 no 1 pp 35ndash401982

[47] K Newick B Cunniff K Preston et al ldquoPeroxiredoxin 3 is aredox-dependent target of thiostrepton in malignant mesothe-lioma cellsrdquo PLoS ONE vol 7 no 6 Article ID e39404 2012

[48] A Deniaud O S El Dein E Maillier et al ldquoEndoplasmicreticulum stress induces calcium-dependent permeability tran-sition mitochondrial outer membrane permeabilization andapoptosisrdquo Oncogene vol 27 no 3 pp 285ndash299 2008

[49] A Gorlach P Klappa and T Kietzmann ldquoThe endoplasmicreticulum folding calcium homeostasis signaling and redoxcontrolrdquo Antioxidants and Redox Signaling vol 8 no 9-10 pp1391ndash1418 2006

[50] 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 symporter

expression and activity involves the PI3KAkt signaling path-wayrdquo Endocrinology vol 155 no 3 pp 1145ndash1156 2014

[51] Y-G Wang L Li C-H Liu S Hong and M-J ZhangldquoPeroxiredoxin 3 is resistant to oxidation-induced apoptosis ofHep-3b cellsrdquoClinical and Translational Oncology vol 16 no 6pp 561ndash566 2014

[52] H C Whitaker D Patel W J Howat et al ldquoPeroxiredoxin-3is overexpressed in prostate cancer and promotes cancer cellsurvival by protecting cells fromoxidative stressrdquoBritish Journalof Cancer vol 109 no 4 pp 983ndash993 2013

[53] L Li Y G Zhang and C L Chen ldquoAnti-apoptotic role ofperoxiredoxin III in cervical cancer cellsrdquo FEBS Open Bio vol3 pp 51ndash54 2013

[54] 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

[55] L I Nadolnik Z V Niatsetskaya and S V Lupachyk ldquoEffectof oxidative stress on rat thyrocyte iodide metabolismrdquo CellBiochemistry and Function vol 26 no 3 pp 366ndash373 2008

[56] A C Freitas Ferreira L de Carvalho Cardoso D Rosenthaland D Pires de Carvalho ldquoThyroid Ca2+NADPH-dependentH2O2generation is partially inhibited by propylthiouracil and

methimazolerdquo European Journal of Biochemistry vol 270 no 11pp 2363ndash2368 2003

[57] G R Sue Z C Ho and K Kim ldquoPeroxiredoxins a historicaloverview and speculative preview of novel mechanisms andemerging concepts in cell signalingrdquo Free Radical Biology andMedicine vol 38 no 12 pp 1543ndash1552 2005

[58] M S Seo S W Kang K Kim I C Baines T H Lee and SG Rhee ldquoIdentification of a new type of mammalian perox-iredoxin that forms an intramolecular disulfide as a reactionintermediaterdquo Journal of Biological Chemistry vol 275 no 27pp 20346ndash20354 2000

[59] Z A Wood L B Poole and P A Karplus ldquoPeroxiredoxinevolution and the regulation of hydrogen peroxide signalingrdquoScience vol 300 no 5619 pp 650ndash653 2003

[60] D A Drechsel and M Patel ldquoRespiration-dependent H2O2

removal in brain mitochondria via the thioredoxinperoxire-doxin systemrdquoThe Journal of Biological Chemistry vol 285 no36 pp 27850ndash27858 2010

[61] W H Watson X Yang Y E Choi D P Jones and J P KehrerldquoThioredoxin and its role in toxicologyrdquo Toxicological Sciencesvol 78 no 1 pp 3ndash14 2004

[62] G Ekstrom and M Ingelman-Sundberg ldquoRat liver microso-mal NADPH-supported oxidase activity and lipid peroxida-tion dependent on ethanol-inducible cytochrome P-450 (P-450IIE1)rdquo Biochemical Pharmacology vol 38 no 8 pp 1313ndash1319 1989

[63] J Rashba-Step N J Turro and A I Cederbaum ldquoIncreasedNADPH- and NADH-dependent production of superoxideand hydroxyl radical by microsomes after chronic ethanoltreatmentrdquoArchives of Biochemistry and Biophysics vol 300 no1 pp 401ndash408 1993

[64] V Thakur M T Pritchard M R McMullen Q Wang andL E Nagy ldquoChronic ethanol feeding increases activation ofNADPH oxidase by lipopolysaccharide in rat Kupffer cellsrole of increased reactive oxygen in LPS-stimulated ERK12activation andTNF-120572 productionrdquo Journal of Leukocyte Biologyvol 79 no 6 pp 1348ndash1356 2006

Oxidative Medicine and Cellular Longevity 11

[65] HNakamuraAMitsui and J Yodoi ldquoThioredoxin overexpres-sion in transgenic micerdquo Methods in Enzymology vol 347 pp436ndash440 2002

[66] 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

[67] A Bieniek B Pula A Piotrowska et al ldquoExpression of metal-lothionein III andKi-67 antigen in various histological types ofbasal cell carcinomardquo Folia Histochemica et Cytobiologica vol50 no 3 pp 352ndash357 2012

[68] P Dziegiel ldquoExpression of metallothioneins in tumor cellsrdquoPolish Journal of Pathology vol 55 no 1 pp 3ndash12 2004

[69] M Oslash Pedersen A Larsen M Stoltenberg and M PenkowaldquoThe role of metallothionein in oncogenesis and cancer prog-nosisrdquo Progress inHistochemistry and Cytochemistry vol 44 no1 pp 29ndash64 2009

[70] W Braun M Vasak A H Robbins et al ldquoComparison ofthe NMR solution structure and the X-ray crystal structure ofrat metallothionein-2rdquo Proceedings of the National Academy ofSciences of the United States of America vol 89 no 21 pp 10124ndash10128 1992

[71] P JThornalley andM Vasak ldquoPossible role formetallothioneinin protection against radiation-induced oxidative stress Kinet-ics andmechanism of its reaction with superoxide and hydroxylradicalsrdquo Biochimica et Biophysica Acta Protein Structure andMolecular Enzymology vol 827 no 1 pp 36ndash44 1985

[72] M A Schwarz J S Lazo J C Yalowich et al ldquoMetallothioneinprotects against the cytotoxic and DNA-damaging effects ofnitric oxiderdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 92 no 10 pp 4452ndash4456 1995

[73] Y Luo A Kawashima and Y Ishido ldquoIodine excess as anenvironmental risk factor for autoimmune thyroid diseaserdquoInternational Journal of Molecular Sciences vol 15 pp 12895ndash12912 2014

[74] H I Falrsquofushinsrsquoka L L Hnatyshyna andOI Osadchuk ldquoTraceelements storage peculiarities and metallothionein contentin human thyroid gland under iodine deficiency euthyroidnodular goiterrdquo Ukrainskii Biokhimicheskii Zhurnal vol 86 pp107ndash113 2014

[75] L Cai Y H Wang G H Zhou et al ldquoAttenuation bymetallothionein of early cardiac cell death via suppression ofmitochondrial oxidative stress results in a prevention of diabeticcardiomyopathyrdquo Journal of the American College of Cardiologyvol 48 no 8 pp 1688ndash1697 2006

[76] X Sun Z Zhou and Y J Kang ldquoAttenuation of doxorubicinchronic toxicity in metallothionein-overexpressing transgenicmouse heartrdquo Cancer Research vol 61 no 8 pp 3382ndash33872001

[77] A F Ceylan-Isik P Zhao B F Zhang X Xiao G Su and J RenldquoCardiac overexpression of metallothionein rescues cardiaccontractile dysfunction and endoplasmic reticulum stress butnot autophagy in sepsisrdquo Journal of Molecular and CellularCardiology vol 48 no 2 pp 367ndash378 2010

[78] N Hu R Guo X Han B Zhu and J Ren ldquoCardiac-specificoverexpression of metallothionein rescues nicotine-inducedcardiac contractile dysfunction and interstitial fibrosisrdquoToxicol-ogy Letters vol 202 no 1 pp 8ndash14 2011

[79] Y Zhang N Hu Y Hua K L Richmond F Dong and JRen ldquoCardiac overexpression of metallothionein rescues coldexposure-induced myocardial contractile dysfunction through

attenuation of cardiac fibrosis despite cardiomyocyte mechani-cal anomaliesrdquo Free Radical Biology and Medicine vol 53 no 2pp 194ndash207 2012

[80] N Hu X Han E K Lane F Gao Y Zhang and J RenldquoCardiac-specific overexpression of metallothionein rescuesagainst cigarette smoking exposure-induced myocardial con-tractile and mitochondrial damagerdquo PLoS ONE vol 8 no 2Article ID e57151 2013

[81] M T Brazao-Silva S V Cardoso P R de Faria et al ldquoAdenoidcystic carcinoma of the salivary gland a clinicopathologicalstudy of 49 cases and of metallothionein expression with regardto tumour behaviourrdquo Histopathology vol 63 no 6 pp 802ndash809 2013

[82] K J Schmitz H Lang G Kaiser et al ldquoMetallothioneinoverexpression and its prognostic relevance in intrahepaticcholangiocarcinoma and extrahepatic hilar cholangiocarci-noma (Klatskin tumors)rdquo Human Pathology vol 40 no 12 pp1706ndash1714 2009

[83] S E Theocharis A P Margeli J T Klijanienko and G PKouraklis ldquoMetallothionein expression in human neoplasiardquoHistopathology vol 45 no 2 pp 103ndash118 2004

[84] STheocharis J Klijanienko C Giaginis et al ldquoMetallothioneinexpression in mobile tongue squamous cell carcinoma associa-tions with clinicopathological parameters and patient survivalrdquoHistopathology vol 59 no 3 pp 514ndash525 2011

[85] S-K Tai O J-K Tan V T-K Chow et al ldquoDifferentialexpression of metallothionein 1 and 2 isoforms in breast cancerlines with different invasive potential identification of a novelnon-silent metallothionein-1H mutant variantrdquo The AmericanJournal of Pathology vol 163 no 5 pp 2009ndash2019 2003

[86] Y Hishikawa T Koji D K Dhar S Kinugasa M Yamaguchiand N Nagasue ldquoMetallothionein expression correlates withmetastatic and proliferative potential in squamous cell carci-noma of the oesophagusrdquo British Journal of Cancer vol 81 no4 pp 712ndash720 1999

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OncologyJournal of

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Oxidative Medicine and Cellular Longevity

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Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 5: Research Article Metallothionein-I/II Knockout Mice

Oxidative Medicine and Cellular Longevity 5

Table 2 Changes of serum thyroid hormones level (nmolL) in WT and MT-III KO mice following NI 10 HI or 100 HI intake for 14 days

Group T3 T4 FT3 FT4

WTNI 072 plusmn 013 2608 plusmn 1421 480 plusmn 042 2856 plusmn 451

10 HI 053 plusmn 009 3350 plusmn 445 426 plusmn 041 3142 plusmn 095

100 HI 071 plusmn 013 3470 plusmn 1324 461 plusmn 025 3320 plusmn 455

MT-III KONI 058 plusmn 015 2005 plusmn 468 404 plusmn 077 2569 plusmn 097

10 HI 069 plusmn 013 2253 plusmn 779 478 plusmn 005 2722 plusmn 103

100 HI 080 plusmn 022 1773 plusmn 676 501 plusmn 073 2399 plusmn 192lowast

lowastIn 100 HI group compared to WT mice there was a significant difference in serum thyroid hormones level (FT4) in MT-III KO mice (119875 lt 005)119873 = 8 foreach group

KO and WT groups (119875 lt 005) Compared to WT groupmore significant increases in the mitochondrial superoxideproduction Prx 3 protein expression and LDH release weredetected in MT-III KO group (119875 lt 005) There were nosignificant differences between the NI group and 10 HI groupin bothMT-III KO andWTmice (119875 gt 005) Comparedwiththe NI group relative cell viability of 100 HI group decreasedin both the MT-III KO and WT mice groups (119875 lt 005)Compared to the WT group a more significant decrease inrelative cell viability was detected in MT-III KO group (119875 lt005) No significant difference of relative cell viability wasdetected between NI group and 10 HI group in both MT-IIIKO and WT mice groups (119875 gt 005) (Figure 2)

4 Discussion

Iodide excess has been recognized as a risk factor for thedevelopment of thyroid disease in humans and animals [7ndash13] One of the underlying mechanisms is that the increasedoxidative stress induced by iodide excess cannot be balancedby endogenous antioxidant systems [14 15] In the presentstudy we focused the research on excessive iodide inducedmitochondrial superoxide production and Prx 3 expressionin the thyroid with or without MT III in vitro and in vivo

In a previous study [15] we showed that the concentrationresponse of KI induced a decrease in cell viability in FRTLcells by MTT assay compared to the control group therelative viability of 10minus5M 10minus4M 10minus3M and 10minus2M KIexposure groupswas significantly decreased at 24 hHoweverno significant decrease in the 10minus6M or 10minus7M KI exposuregroups was observed A strong stimulatory effect of iodidewas detected in 10minus4MKI and in 10minus3MKI exposure groupsat the 2 h time point [15] Iodide had a stimulatory effect onH2O2generation and when increased H

2O2is considered

as a limiting step in the biosynthesis of thyroid hormones[1] The normal relative cell viability in the 10minus6M or 10minus7MKI exposure groups can be explained by the physiologicaleffect of iodide which is necessary for the biosynthesis ofthyroid hormones [15] We used KI concentrations of 10minus4Mand above in the in vitro study and NI 10 HI and 100 HIin the in vivo study In bovine thyroid slices the maximalstimulation effect on H

2O2generation was obtained after 2 h

of preincubation with 10minus4M KI [1] It was reported that in

vitro the acute toxic effects of high iodide doses in humanthyroid follicles significantly increased the percentage ofnecrotic cells in 10minus5Mand doubledwith 10minus3Mas comparedto values measured at 10minus7M [10] Iodide concentrations of10minus4M or greater may inhibit the iodothyronine synthesisand thyroid hormone secretion [41]The concentrations from10minus5 to 10minus3M were from 100 to 10000 times higher thanthe normal iodine plasma levels estimated to be 10minus7M ineuthyroid human beings [42]

In the present study we detected that in 100 HI intakegroup there is a significant change of FT

4in MT-III KO

mice compared to WT mice however the values remainwithin the normal range in thyroid function The NI and10 HI groups in both WT and MT-III KO mice wereunder normal conditions and the thyroid function was notaffected Iodine is essential for the synthesis of the thyroidhormones In most individuals the decreased productionof thyroid hormones is only transient and resumes afteradaptation to the acute Wolff-Chaikoff effect [43] A detailedanalysis reveals a persistent drop of serum T

4and T

3of

25 and 15 respectively and a rise of TSH of 2mULAlthough no clinical signs of thyroid dysfunction or goiterwere found sonographic thyroid volume is slightly increased[2 29ndash32 44 45] Exposure to high concentrations ofiodine may decrease the release of thyroid hormone shownby the increase in the serum level of TSH [27 28 46](Table 3)

Thyroid toxicity of iodide excess has been demonstratedin vitro and in animals fed with an iodide-rich diet [42] Weshowed that high concentrated iodide induced cytotoxicity inthyrocytes significantly increasingmitochondrial superoxideanion production in the thyroid in high concentrated iodideexposure in vitro and in 100 HI diet in vivo in bothMT-III KO and WT mice which leads to the decreasedrelative cell viability in the thyroid It is suggested thatoxidative stress is the underlying mechanism in iodide excessinduced apoptosis in thyroid cells [42] Consistent withprevious reports [42 47ndash49] we showed that there is astrong increase of superoxide anion production in FRTLcells following high concentrations of iodide exposure [15]Serrano-Nascimento et al also observed that iodide excessincreased ROS production in thyrocytes and mitochondriawere the source of superoxide anion production [50] Theoxidative attack can be explained by the oxidative effect

6 Oxidative Medicine and Cellular Longevity

0

20

40

60

80

100

120

NI 10 HI 100 HI

Rela

tive v

iabi

lity

()

WT miceMT-III KO mice

lowastlowast

(a)

0

02

04

06

08

1

12

NI

NI

10 HI

10 HI

100 HI

100 HI NI 10 HI 100 HI

Expr

essio

n of

Prx

3 (

)

WT miceMT-III KO mice

lowast

lowast

WT MT-III KO

Prx 3

120573-actin

(b)

NI 10 HI 100 HI

WT miceMT-III KO mice

0

1000

2000

3000

4000

LDH

rele

ase (

Um

g pr

otei

n)

lowastlowast

(c)

0

20

40

60

80

NI 10 HI 100 HIMito

SOX

fluor

esce

nce i

nten

sity

WT miceMT-III KO mice

lowast

lowast

WT

MT-

III K

O

NI 1681

NI 2051 10 HI 2514 100 HI 6133

100 HI 478710 HI 1819

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

(d)

Figure 2 Mitochondrial superoxide production Prx 3 protein expression and LDH release and relative viability in the thyroid of WT andMT-III knockout mice following NI 10 HI or 100 HI diet for 14 days (a) 100 HI intake decreased the relative viability in thyroid of MT-IIIKO and WT mice (b) Representative western blot of Prx 3 (27 kDa) 120573-actin (45 kDa) was used as a loading control Densitometric analysisshowed a significant increase in Prx 3 expression in 100 HI group in the thyroid of either WT or MT-III KO mice especially in MT-IIIKO mice (c) 100 HI increased the LDH release in the thyroid of MT-III KO and WT mice (d) 100 HI intake increased the mitochondrialsuperoxide production in thyroid of WT and MT-III KO mice Histogram analysis was performed on the mean fluorescence intensity ofMitoSOX Red as measured by flow cytometry Experiments were repeated 3 times with similar results Data are represented as mean plusmn SD(119873 = 10group) One-way ANOVA with the LSD test was used lowast119875 lt 005 compared with the control group of WT or MT-III KO micerespectively 119875 lt 005WTmice compared with the MT-III KO mice under the same treatment

of HI diet on the thyroid gland ROS include free radi-cals such as the superoxide anion hydroxyl radicals andthe nonradical hydrogen peroxide Whether cells die fromoxidative stress induced apoptosis depends on the balancebetween the generation of oxidant species and the antioxidantsystem

In addition we demonstrated that no significant differ-ence was detected between NI group and 10 HI group in bothMT-III KO and WT mice groups This can be explained bythe fact that under basal conditions thyroid epithelial cellsproduce moderate amounts of ROS that are physiologicallyrequired for thyroid hormone synthesis [14] Physiologically

Oxidative Medicine and Cellular Longevity 7

Table 3 Documented literatures of iodide intake and changes of serum thyroxine (T4) and triiodothyronine (T3) concentrations

Dosage of daily iodine intake Time Subject Changes of serum T4 T3 concentrations Reference250 or 500 120583g 14 days Normal volunteers No change [27]1500 120583g 14 days Normal volunteers T4 T3 darr TSH uarr [27]

50 or 250mg 13 days Normal subjects T4 T3 darr TSH uarr(within normal range) [28]

27mg 4 weeks Normal volunteers T4 within the normal range except fortwo subjects TSH uarr [29]

Single doses of 10 30 50 and 100mg andthen daily doses of 10 15 30 50 or 100mg 12 days Euthyroid volunteers T3 T4 darr TSH uarr [30]

190mg 10 days Euthyroid volunteers T3 T4 darr TSH uarr [31]114mg 3ndash7 weeks Normal controls T3 T4 darr TSH uarr [32]

114mg 3ndash7 weeks Thyrotoxic patientsT4 T3 uarr in some casesother patients remained

euthyroid even after 6 weeks[32]

114mg 2 weeks Hypothyroid patients onthyroxine replacement

No consistent change(T4 uarr at 1 and 14 days TSH darr the first day) [32]

mitochondrial ROS generation is primarily due to electronsleaking from the electron transport chain (ETC) to generatesuperoxide radical Superoxide from the electron transportchain and other sources is converted by MnSOD to H

2O2

which is then metabolized by Prx 3 to water [47]Besides we demonstrated that high concentrated iodide

induces significant increased expression of Prx 3 whichsuggests that the increase of Prx 3 expression following highconcentrated iodide exposure orHI diet in the thyroidmay bethe protective response against high ROS generation Prx 3 isan indispensable ROS scavenger that protects against oxida-tive damage and subsequent apoptosis [51ndash53] It is reportedthat Prx 3 has an essential role in regulating oxidation-induced apoptosis and can be a potential therapeutic targetin castrate-independent prostate cancer [52] Iodide hada stimulatory effect on H

2O2generation in thyroid slices

[1] At high concentrations (above 01mM) H2O2induces

apoptosis in thyroid cells [1] When iodide is in excess ascompared to tyrosine residues it reacts with the iodoniumcation formed by iodide oxidation to give molecular iodineExcess molecular iodine then induced apoptosis throughgeneration of free radicals [54] H

2O2generation is a limiting

step in thyroid hormone biosynthesis it is essential foriodide oxidation by thyroperoxidase [14 55 56] H

2O2can

be eliminated by Prx 3 In mammals Prx 3 is targeted to themitochondrial matrix It is estimated that Prx 3 will be thetarget for up to 90 of hydrogen peroxide generated in themitochondrial matrix [57] mitochondrial peroxiredoxins (3andor 5) can reduce H

2O2to water through reducing equiv-

alents provided by thiol-containing proteins [58 59] butthe mechanisms involved are unclear [24 59] Drechsel andPatel demonstrated that thioredoxin (Trxs)peroxiredoxin(Prxs) (Prx 3 and Prx 5) is the major contributing systemto H2O2removal in brain mitochondria [60] Prxs and Trxs

are important liver antioxidant proteins and play importantroles in maintaining liver redox homeostasis [61] It isreported that chronic ethanol exposure increases NADPHoxidase activity and increases the production of superoxide

and hydrogen peroxide [62ndash64] Thioredoxin scavenges andreduces ROS via peroxiredoxins [65] Endoplasmic reticulumstress disrupts the electron transport chain leading to theincreased production of ROS [48 49] Bae et al reportedthat the antioxidant role of Prx 3 was evident from thefindings that pyrazole-induced protein carbonylation and theformation of 4-HNE adducts and MDA in the liver weremarkedly increased in Prx 3minusminus mice compared with wild-type mice [66]

A high urinary excretion of iodine in a spot urine sampleis the ultimate proof of iodine excess in vivo [2] We showedthe urinary iodine concentration increased as the iodidesupply increased significantly higher urinary iodine concen-tration can be detected in 100 HI than 10 HI and in MT-IIIKOmice than inWTmiceThis data indicates the adaptationof the thyroid gland to iodine excess and that the excessiveiodine is excreted from the urine in order to maintain thenormal function of thyroid This makes the studies in vivodifferent from studies in vitro In addition to the urinaryexcretion of iodine there are many control mechanisms ofthe thyroid to counteract iodide excess These include thesodium-iodide symporter which controls the limiting stepof thyroid hormone synthesis the Wolff-Chaikoff effect anarrest of thyroid hormone secretion from stores in the colloida preferential secretion of the less active T

4over T

3 and

dumping of excess iodine by secreting it in nonhormonalform [2]

Moreover we demonstrated that significant increasesof mitochondrial superoxide production and Prx 3 proteinexpression can be detected in MT-III KO mice group Thisdata indicated the protective role of MT-III against highconcentrations of iodide induced mitochondrial superoxideproduction in the thyrocytes MT has been demonstratedto play a key role in the detoxification of heavy metalsprotecting against various forms of oxidative injury by scav-enging oxygen free radicals MTs belong to the group ofintracellular cysteine-rich metal-binding proteins which iswidely expressed in cells (nuclei andor cytoplasm) of various

8 Oxidative Medicine and Cellular Longevity

organs and tumors [67ndash69] MTs play a protective role inpreventing against intoxication with heavy metals such asPb Hg Cu and Cd [67 70] All 20 cysteine sulfur atomsare involved in the radical quenching process and the rateconstant for the reaction of hydroxyl radical with MT isabout 340-fold higher than that with GSH [71 72] There arefour MT isoforms in mammals MT-I and MT-II are mainlyinvolved in the protection of tissue against metal toxicitiesoxidative stresses and apoptosis It has been demonstratedthat the rabbit liver metallothionein-1 which contains zincandor cadmium ions appeared to scavenge free hydroxyland superoxide radicals [25 26] Under low levels of iodideorganification in goitrous-modified tissue of thyroid glandmetallothionein may provide a partial compensatory effecton prooxidative processes [73 74] Although no cell lineor animal model overexpressing MT in the thyroid hasbeen found yet documented evidence of MT in protectingagainst oxidative stress in cardiac-specific metallothionein-overexpressed transgenic mice has been demonstrated Caiet al reported that metallothionein as a potent antioxi-dant prevented the development of diabetic cardiomyopathythrough the inhibition of the mitochondrial cytochromec-mediated caspase-3 activation pathway [75] Sun et alreported that through doxorubicin induced chronic toxi-city in a metallothionein-overexpressed transgenic mouseheart the antioxidant action of MT is highly responsiblefor this cardioprotection [76] Ceylan-Isik et al reportedthat the cardiac-specific overexpression of MT rescues LPS-induced cardiac contractile and intracellular Ca2+ dysfunc-tions through the alleviation of ROSoxidative stress stresssignaling activation and endoplasmic reticulum (ER) stress[77] They also reported that the cardiac-specific overexpres-sion of metallothionein protects against nicotine exposure-induced cardiac contractile dysfunction and fibrosis possiblythrough inhibition of ROS accumulation and apoptosis [78]Zhang et al suggest that cardiac overexpression of metalloth-ionein prevents cold exposure-induced cardiac anomaliespossibly through the attenuation of myocardial fibrosis [79]Hu et al demonstrated that cardiac-specific overexpression ofmetallothionein protects against cigarette smoking exposure-induced myocardial contractile and mitochondrial damagefavoring a role of lessened apoptosis in a metallothionein-induced beneficial effect against side-stream smoke exposure[80] Besides metallothionein has been shown to regulateapoptosis and proliferation Overexpression of metalloth-ionein frequently occurs in human tumors and is related totumor progression [81 82] High expression of MT has beenobserved in different types of cancer and has been consideredas a potential prognostic marker in cancers of the breastpancreas skin and cervix [82ndash86] Further studies will beattempted to study the oxidative and antioxidative stress onan engineered cell line overexpressing metallothionein

5 Conclusion

Thepresent study provides evidence that high concentrationsof iodide may lead to a significant increase in mitochondrialsuperoxide production and Prx 3 expression in both invitro and in vivo studies MT-III KO mice aggravated

mitochondrial superoxide production and peroxiredoxin 3expression in the thyroid after excessive iodide exposureMT-III may potentially protect the thyroid against highconcentrations of iodide induced oxidative stress

Abbreviations

MT-III KO Metallothionein III knockoutMTs MetallothioneinsWT Wild-typeROS Reactive oxygen speciesPrx PeroxiredoxinKI Potassium iodideNI Normal iodide intakeHI High iodide intakeLDH Lactate dehydrogenaseH2O2 Hydrogen peroxide

SOD Superoxide dismutaseMTT 3-(45-Dimethylthiazol-2-yl)-25-

diphenyltetrazoliumbromide

K-S Kolmogorov-SmirnovLSD Least significant differenceT4 Thyroxine

T3 Tri-iodothyronine

FT4 Free thyroxine

FT3 Free tri-iodothyronine

Conflict of Interests

The authors declare that they have no competing interests

Acknowledgments

Thiswork is supported by theNational Natural Science Foun-dation of China (no 81273009) the Tianjin Science amp Tech-nology Council Grant of China (no 09JCYBJC11700) andthe Tianjin Educational amp Scientific Grant (no 20050107)This paper has been edited by one of the authors MohamedAhmed who is from USA and now is studying in TianjinMedical University China

References

[1] B Corvilain L Collyn J van Sande and J E DumontldquoStimulation by iodide of H

2O2generation in thyroid slices

from several speciesrdquo The American Journal of PhysiologymdashEndocrinology and Metabolism vol 278 no 4 pp E692ndashE6992000

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

[3] E Roti and E Degli Uberti ldquoIodine excess and hyperthy-roidismrdquoThyroid vol 11 no 5 pp 493ndash500 2001

[4] World Health Organization and United Nations ChildrenrsquosFund ldquoInternational Council for Control of Iodine DeficiencyDisorders 1999 progress towards the elimination of iodinedeficiency disorders (IDD)rdquo WHO Booklet World HealthOrganization 1999

Oxidative Medicine and Cellular Longevity 9

[5] A M Leung and L E Braverman ldquoConsequences of excessiodinerdquo Nature Reviews Endocrinology vol 10 no 3 pp 136ndash142 2013

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

[7] IMahmoud I ColinM CMany and J-F Denef ldquoDirect toxiceffect of iodide in excess on iodine-deficient thyroid glandsepithelial necrosis and inflammation associated with lipofuscinaccumulationrdquo Experimental and Molecular Pathology vol 44no 3 pp 259ndash271 1986

[8] J E Fradkin and J Wolff ldquoIodide-induced thyrotoxicosisrdquoMedicine vol 62 no 1 pp 1ndash20 1983

[9] J Golstein and J E Dumont ldquoCytotoxic effects of iodide onthyroid cells difference between rat thyroid FRTL-5 cell andpri-mary dog thyrocyte responsivenessrdquo Journal of EndocrinologicalInvestigation vol 19 no 2 pp 119ndash126 1996

[10] 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

[11] W Teng Z Shan X Teng et al ldquoEffect of iodine intakeon thyroid diseases in Chinardquo The New England Journal ofMedicine vol 354 no 26 pp 2783ndash2793 2006

[12] P Laurberg K M Pedersen A Hreidarsson N Sigfusson EIversen and P R Knudsen ldquoIodine intake and the patternof thyroid disorders a comparative epidemiological study ofthyroid abnormalities in the elderly in iceland and in JutlandDenmarkrdquo Journal of Clinical Endocrinology and Metabolismvol 83 no 3 pp 765ndash769 1998

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

[14] 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

[15] 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

[16] H Han P Xin L Zhao et al ldquoExcess iodine and high-fatdiet combination modulates lipid profile thyroid hormoneand hepatic LDLr expression values in micerdquo Biological TraceElement Research vol 147 no 1ndash3 pp 233ndash239 2012

[17] S-J Zhao Y Ye F-J Sun E-J Tian and Z-P Chen ldquoTheimpact of dietary iodine intake on lipid metabolism in micerdquoBiological Trace Element Research vol 142 no 3 pp 581ndash5882011

[18] 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

[19] V Pitsiavas P Smerdely M Li and S C Boyages ldquoAmiodaroneinduces a different pattern of ultrastructural change in thethyroid to iodine excess alone in both the BBW rat and theWistar ratrdquo European Journal of Endocrinology vol 137 no 1pp 89ndash98 1997

[20] M B Zimmermann ldquoIodine deficiency and excess in childrenworldwide status in 2013rdquo Endocrine Practice vol 19 no 5 pp839ndash846 2013

[21] K Mussig ldquoIodine-induced toxic effects due to seaweed con-sumptionrdquo in Comprehensive Handbook of Iodine V R PreedyG N Burrow and R R Watson Eds pp 897ndash908 ElsevierAmsterdam The Netherlands 2009

[22] A-M G Psarra S Solakidi and C E Sekeris ldquoThe mito-chondrion as a primary site of action of steroid and thyroidhormones presence and action of steroid and thyroid hormonereceptors in mitochondria of animal cellsrdquo Molecular andCellular Endocrinology vol 246 no 1-2 pp 21ndash33 2006

[23] Y Song N Driessens M Costa et al ldquoRoles of hydrogenperoxide in thyroid physiology and diseaserdquo Journal of ClinicalEndocrinology and Metabolism vol 92 no 10 pp 3764ndash37732007

[24] A G Cox C C Winterbourn and M B Hampton ldquoMito-chondrial peroxiredoxin involvement in antioxidant defenceand redox signallingrdquo Biochemical Journal vol 425 no 2 pp313ndash325 2010

[25] B Ruttkay-Nedecky L Nejdl J Gumulec et al ldquoThe role ofmetallothionein in oxidative stressrdquo International Journal ofMolecular Sciences vol 14 no 3 pp 6044ndash6066 2013

[26] N Thirumoorthy A Shyam Sunder K T Manisenthil KumarM Senthil kumarGNKGanesh andMChatterjee ldquoA reviewof metallothionein isoforms and their role in pathophysiologyrdquoWorld Journal of Surgical Oncology vol 9 article 54 2011

[27] 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

[28] M Saberi and R D Utiger ldquoAugmentation of thyrotropinresponses to thyrotropin releasing hormone following smalldecreases in serum thyroid hormone concentrationsrdquo TheJournal of Clinical Endocrinology and Metabolism vol 40 no3 pp 435ndash441 1975

[29] H Namba S Yamashita H Kimura et al ldquoEvidence of thyroidvolume increase in normal subjects receiving excess iodiderdquoTheJournal of Clinical Endocrinology and Metabolism vol 76 no 3pp 605ndash608 1993

[30] E Sternthal L Lipworth B Stanley C Abreau S L Fang andL E Braverman ldquoSuppression of thyroid radioiodine uptakeby various doses of stable iodiderdquo The New England Journal ofMedicine vol 303 no 19 pp 1083ndash1088 1980

[31] A G Vagenakis B Rapoport F Azizi G I Portnay L EBraverman and S H Ingbar ldquoHyperresponse to thyrotropinreleasing hormone accompanying small decreases in serumthyroid hormone concentrationsrdquoThe Journal of Clinical Inves-tigation vol 54 no 4 pp 913ndash918 1974

[32] G Philippou D A Koutras G Piperingos A Souvatzoglouand S D Moulopoulos ldquoThe effect of iodide on serum thyroidhormone levels in normal persons in hyperthyroid patientsand in hypothyroid patients on thyroxine replacementrdquoClinicalEndocrinology vol 36 no 6 pp 573ndash578 1992

[33] L Z Ali Salim R Othman M A Abdulla et al ldquoThymo-quinone inhibits murine leukemia WEHI-3 cells in vivo and invitrordquo PLoS ONE vol 9 no 12 Article ID e115340 2014

[34] X Wang J Ge K Wang J Qian and Y Zou ldquoEvaluation ofMTT assay for measurement of emodin-induced cytotoxicityrdquoASSAY and Drug Development Technologies vol 4 no 2 pp203ndash207 2006

10 Oxidative Medicine and Cellular Longevity

[35] C Lv C Wu Y-h Zhou Y Shao G Wang and Q-y WangldquoAlpha lipoic acid Modulated high glucose-induced rat mesan-gial cell dysfunction viamTORp70S6K4E-BP1 pathwayrdquo Inter-national Journal of Endocrinology vol 2014 Article ID 65858914 pages 2014

[36] L Liu X Hu G-Y Cai et al ldquoHigh glucose-induced hyper-trophy of mesangial cells is reversed by connexin43 overex-pression via PTENAktmTOR signalingrdquo Nephrology DialysisTransplantation vol 27 no 1 pp 90ndash100 2012

[37] D D Harrison-Findik S Z Lu E M Zmijewski J Jonesand M C Zimmerman ldquoEffect of alcohol exposure on hepaticsuperoxide generation and hepcidin expressionrdquoWorld Journalof Biological Chemistry vol 4 no 4 pp 119ndash130 2013

[38] M Wang Y Wang J He et al ldquoAlbumin induces neuroprotec-tion against ischemic stroke by altering Toll-like receptor 4 andregulatory T cells in micerdquo CNS and Neurological DisordersmdashDrug Targets vol 12 no 2 pp 220ndash227 2013

[39] X Yao ldquoEffect of zinc exposure on HNE and GLT-1 in spinalcord culturerdquo NeuroToxicology vol 30 no 1 pp 121ndash126 2009

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

[41] G P Becks M C Eggo and G N Burrow ldquoRegulation ofdifferentiated thyroid function by iodide preferential inhibitoryeffect of excess iodide on thyroid hormone secretion in sheepthyroid cell culturesrdquo Endocrinology vol 120 no 6 pp 2569ndash2575 1987

[42] M Vitale T Di Matola F DrsquoAscoli et al ldquoIodide excessinduces apoptosis in thyroid cells through a p53-independentmechanism involving oxidative stressrdquo Endocrinology vol 141no 2 pp 598ndash605 2000

[43] P Pramyothin AM Leung E N Pearce A OMalabanan andL E Braverman ldquoClinical problem-solving A hidden solutionrdquoTheNew England Journal of Medicine vol 365 no 22 pp 2123ndash2127 2011

[44] W Jubiz S Carlile and L D Lagerquist ldquoSerum thyrotropinand thyroid hormone levels in humans receiving chronicpotassium iodiderdquo The Journal of Clinical Endocrinology andMetabolism vol 44 no 2 pp 379ndash382 1977

[45] A G Vagenakis P Downs L E Braverman A Burger andS H Ingbar ldquoControl of thyroid hormone secretion in normalsubjects receiving iodidesrdquoThe Journal of Clinical Investigationvol 52 no 2 pp 528ndash532 1973

[46] M Safran andL E Braverman ldquoEffect of chronic douchingwithpolyvinylpyrrolidone-iodine on iodine absorption and thyroidfunctionrdquo Obstetrics and Gynecology vol 60 no 1 pp 35ndash401982

[47] K Newick B Cunniff K Preston et al ldquoPeroxiredoxin 3 is aredox-dependent target of thiostrepton in malignant mesothe-lioma cellsrdquo PLoS ONE vol 7 no 6 Article ID e39404 2012

[48] A Deniaud O S El Dein E Maillier et al ldquoEndoplasmicreticulum stress induces calcium-dependent permeability tran-sition mitochondrial outer membrane permeabilization andapoptosisrdquo Oncogene vol 27 no 3 pp 285ndash299 2008

[49] A Gorlach P Klappa and T Kietzmann ldquoThe endoplasmicreticulum folding calcium homeostasis signaling and redoxcontrolrdquo Antioxidants and Redox Signaling vol 8 no 9-10 pp1391ndash1418 2006

[50] 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 symporter

expression and activity involves the PI3KAkt signaling path-wayrdquo Endocrinology vol 155 no 3 pp 1145ndash1156 2014

[51] Y-G Wang L Li C-H Liu S Hong and M-J ZhangldquoPeroxiredoxin 3 is resistant to oxidation-induced apoptosis ofHep-3b cellsrdquoClinical and Translational Oncology vol 16 no 6pp 561ndash566 2014

[52] H C Whitaker D Patel W J Howat et al ldquoPeroxiredoxin-3is overexpressed in prostate cancer and promotes cancer cellsurvival by protecting cells fromoxidative stressrdquoBritish Journalof Cancer vol 109 no 4 pp 983ndash993 2013

[53] L Li Y G Zhang and C L Chen ldquoAnti-apoptotic role ofperoxiredoxin III in cervical cancer cellsrdquo FEBS Open Bio vol3 pp 51ndash54 2013

[54] 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

[55] L I Nadolnik Z V Niatsetskaya and S V Lupachyk ldquoEffectof oxidative stress on rat thyrocyte iodide metabolismrdquo CellBiochemistry and Function vol 26 no 3 pp 366ndash373 2008

[56] A C Freitas Ferreira L de Carvalho Cardoso D Rosenthaland D Pires de Carvalho ldquoThyroid Ca2+NADPH-dependentH2O2generation is partially inhibited by propylthiouracil and

methimazolerdquo European Journal of Biochemistry vol 270 no 11pp 2363ndash2368 2003

[57] G R Sue Z C Ho and K Kim ldquoPeroxiredoxins a historicaloverview and speculative preview of novel mechanisms andemerging concepts in cell signalingrdquo Free Radical Biology andMedicine vol 38 no 12 pp 1543ndash1552 2005

[58] M S Seo S W Kang K Kim I C Baines T H Lee and SG Rhee ldquoIdentification of a new type of mammalian perox-iredoxin that forms an intramolecular disulfide as a reactionintermediaterdquo Journal of Biological Chemistry vol 275 no 27pp 20346ndash20354 2000

[59] Z A Wood L B Poole and P A Karplus ldquoPeroxiredoxinevolution and the regulation of hydrogen peroxide signalingrdquoScience vol 300 no 5619 pp 650ndash653 2003

[60] D A Drechsel and M Patel ldquoRespiration-dependent H2O2

removal in brain mitochondria via the thioredoxinperoxire-doxin systemrdquoThe Journal of Biological Chemistry vol 285 no36 pp 27850ndash27858 2010

[61] W H Watson X Yang Y E Choi D P Jones and J P KehrerldquoThioredoxin and its role in toxicologyrdquo Toxicological Sciencesvol 78 no 1 pp 3ndash14 2004

[62] G Ekstrom and M Ingelman-Sundberg ldquoRat liver microso-mal NADPH-supported oxidase activity and lipid peroxida-tion dependent on ethanol-inducible cytochrome P-450 (P-450IIE1)rdquo Biochemical Pharmacology vol 38 no 8 pp 1313ndash1319 1989

[63] J Rashba-Step N J Turro and A I Cederbaum ldquoIncreasedNADPH- and NADH-dependent production of superoxideand hydroxyl radical by microsomes after chronic ethanoltreatmentrdquoArchives of Biochemistry and Biophysics vol 300 no1 pp 401ndash408 1993

[64] V Thakur M T Pritchard M R McMullen Q Wang andL E Nagy ldquoChronic ethanol feeding increases activation ofNADPH oxidase by lipopolysaccharide in rat Kupffer cellsrole of increased reactive oxygen in LPS-stimulated ERK12activation andTNF-120572 productionrdquo Journal of Leukocyte Biologyvol 79 no 6 pp 1348ndash1356 2006

Oxidative Medicine and Cellular Longevity 11

[65] HNakamuraAMitsui and J Yodoi ldquoThioredoxin overexpres-sion in transgenic micerdquo Methods in Enzymology vol 347 pp436ndash440 2002

[66] 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

[67] A Bieniek B Pula A Piotrowska et al ldquoExpression of metal-lothionein III andKi-67 antigen in various histological types ofbasal cell carcinomardquo Folia Histochemica et Cytobiologica vol50 no 3 pp 352ndash357 2012

[68] P Dziegiel ldquoExpression of metallothioneins in tumor cellsrdquoPolish Journal of Pathology vol 55 no 1 pp 3ndash12 2004

[69] M Oslash Pedersen A Larsen M Stoltenberg and M PenkowaldquoThe role of metallothionein in oncogenesis and cancer prog-nosisrdquo Progress inHistochemistry and Cytochemistry vol 44 no1 pp 29ndash64 2009

[70] W Braun M Vasak A H Robbins et al ldquoComparison ofthe NMR solution structure and the X-ray crystal structure ofrat metallothionein-2rdquo Proceedings of the National Academy ofSciences of the United States of America vol 89 no 21 pp 10124ndash10128 1992

[71] P JThornalley andM Vasak ldquoPossible role formetallothioneinin protection against radiation-induced oxidative stress Kinet-ics andmechanism of its reaction with superoxide and hydroxylradicalsrdquo Biochimica et Biophysica Acta Protein Structure andMolecular Enzymology vol 827 no 1 pp 36ndash44 1985

[72] M A Schwarz J S Lazo J C Yalowich et al ldquoMetallothioneinprotects against the cytotoxic and DNA-damaging effects ofnitric oxiderdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 92 no 10 pp 4452ndash4456 1995

[73] Y Luo A Kawashima and Y Ishido ldquoIodine excess as anenvironmental risk factor for autoimmune thyroid diseaserdquoInternational Journal of Molecular Sciences vol 15 pp 12895ndash12912 2014

[74] H I Falrsquofushinsrsquoka L L Hnatyshyna andOI Osadchuk ldquoTraceelements storage peculiarities and metallothionein contentin human thyroid gland under iodine deficiency euthyroidnodular goiterrdquo Ukrainskii Biokhimicheskii Zhurnal vol 86 pp107ndash113 2014

[75] L Cai Y H Wang G H Zhou et al ldquoAttenuation bymetallothionein of early cardiac cell death via suppression ofmitochondrial oxidative stress results in a prevention of diabeticcardiomyopathyrdquo Journal of the American College of Cardiologyvol 48 no 8 pp 1688ndash1697 2006

[76] X Sun Z Zhou and Y J Kang ldquoAttenuation of doxorubicinchronic toxicity in metallothionein-overexpressing transgenicmouse heartrdquo Cancer Research vol 61 no 8 pp 3382ndash33872001

[77] A F Ceylan-Isik P Zhao B F Zhang X Xiao G Su and J RenldquoCardiac overexpression of metallothionein rescues cardiaccontractile dysfunction and endoplasmic reticulum stress butnot autophagy in sepsisrdquo Journal of Molecular and CellularCardiology vol 48 no 2 pp 367ndash378 2010

[78] N Hu R Guo X Han B Zhu and J Ren ldquoCardiac-specificoverexpression of metallothionein rescues nicotine-inducedcardiac contractile dysfunction and interstitial fibrosisrdquoToxicol-ogy Letters vol 202 no 1 pp 8ndash14 2011

[79] Y Zhang N Hu Y Hua K L Richmond F Dong and JRen ldquoCardiac overexpression of metallothionein rescues coldexposure-induced myocardial contractile dysfunction through

attenuation of cardiac fibrosis despite cardiomyocyte mechani-cal anomaliesrdquo Free Radical Biology and Medicine vol 53 no 2pp 194ndash207 2012

[80] N Hu X Han E K Lane F Gao Y Zhang and J RenldquoCardiac-specific overexpression of metallothionein rescuesagainst cigarette smoking exposure-induced myocardial con-tractile and mitochondrial damagerdquo PLoS ONE vol 8 no 2Article ID e57151 2013

[81] M T Brazao-Silva S V Cardoso P R de Faria et al ldquoAdenoidcystic carcinoma of the salivary gland a clinicopathologicalstudy of 49 cases and of metallothionein expression with regardto tumour behaviourrdquo Histopathology vol 63 no 6 pp 802ndash809 2013

[82] K J Schmitz H Lang G Kaiser et al ldquoMetallothioneinoverexpression and its prognostic relevance in intrahepaticcholangiocarcinoma and extrahepatic hilar cholangiocarci-noma (Klatskin tumors)rdquo Human Pathology vol 40 no 12 pp1706ndash1714 2009

[83] S E Theocharis A P Margeli J T Klijanienko and G PKouraklis ldquoMetallothionein expression in human neoplasiardquoHistopathology vol 45 no 2 pp 103ndash118 2004

[84] STheocharis J Klijanienko C Giaginis et al ldquoMetallothioneinexpression in mobile tongue squamous cell carcinoma associa-tions with clinicopathological parameters and patient survivalrdquoHistopathology vol 59 no 3 pp 514ndash525 2011

[85] S-K Tai O J-K Tan V T-K Chow et al ldquoDifferentialexpression of metallothionein 1 and 2 isoforms in breast cancerlines with different invasive potential identification of a novelnon-silent metallothionein-1H mutant variantrdquo The AmericanJournal of Pathology vol 163 no 5 pp 2009ndash2019 2003

[86] Y Hishikawa T Koji D K Dhar S Kinugasa M Yamaguchiand N Nagasue ldquoMetallothionein expression correlates withmetastatic and proliferative potential in squamous cell carci-noma of the oesophagusrdquo British Journal of Cancer vol 81 no4 pp 712ndash720 1999

Submit your manuscripts athttpwwwhindawicom

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Oxidative Medicine and Cellular Longevity

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Page 6: Research Article Metallothionein-I/II Knockout Mice

6 Oxidative Medicine and Cellular Longevity

0

20

40

60

80

100

120

NI 10 HI 100 HI

Rela

tive v

iabi

lity

()

WT miceMT-III KO mice

lowastlowast

(a)

0

02

04

06

08

1

12

NI

NI

10 HI

10 HI

100 HI

100 HI NI 10 HI 100 HI

Expr

essio

n of

Prx

3 (

)

WT miceMT-III KO mice

lowast

lowast

WT MT-III KO

Prx 3

120573-actin

(b)

NI 10 HI 100 HI

WT miceMT-III KO mice

0

1000

2000

3000

4000

LDH

rele

ase (

Um

g pr

otei

n)

lowastlowast

(c)

0

20

40

60

80

NI 10 HI 100 HIMito

SOX

fluor

esce

nce i

nten

sity

WT miceMT-III KO mice

lowast

lowast

WT

MT-

III K

O

NI 1681

NI 2051 10 HI 2514 100 HI 6133

100 HI 478710 HI 1819

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

(d)

Figure 2 Mitochondrial superoxide production Prx 3 protein expression and LDH release and relative viability in the thyroid of WT andMT-III knockout mice following NI 10 HI or 100 HI diet for 14 days (a) 100 HI intake decreased the relative viability in thyroid of MT-IIIKO and WT mice (b) Representative western blot of Prx 3 (27 kDa) 120573-actin (45 kDa) was used as a loading control Densitometric analysisshowed a significant increase in Prx 3 expression in 100 HI group in the thyroid of either WT or MT-III KO mice especially in MT-IIIKO mice (c) 100 HI increased the LDH release in the thyroid of MT-III KO and WT mice (d) 100 HI intake increased the mitochondrialsuperoxide production in thyroid of WT and MT-III KO mice Histogram analysis was performed on the mean fluorescence intensity ofMitoSOX Red as measured by flow cytometry Experiments were repeated 3 times with similar results Data are represented as mean plusmn SD(119873 = 10group) One-way ANOVA with the LSD test was used lowast119875 lt 005 compared with the control group of WT or MT-III KO micerespectively 119875 lt 005WTmice compared with the MT-III KO mice under the same treatment

of HI diet on the thyroid gland ROS include free radi-cals such as the superoxide anion hydroxyl radicals andthe nonradical hydrogen peroxide Whether cells die fromoxidative stress induced apoptosis depends on the balancebetween the generation of oxidant species and the antioxidantsystem

In addition we demonstrated that no significant differ-ence was detected between NI group and 10 HI group in bothMT-III KO and WT mice groups This can be explained bythe fact that under basal conditions thyroid epithelial cellsproduce moderate amounts of ROS that are physiologicallyrequired for thyroid hormone synthesis [14] Physiologically

Oxidative Medicine and Cellular Longevity 7

Table 3 Documented literatures of iodide intake and changes of serum thyroxine (T4) and triiodothyronine (T3) concentrations

Dosage of daily iodine intake Time Subject Changes of serum T4 T3 concentrations Reference250 or 500 120583g 14 days Normal volunteers No change [27]1500 120583g 14 days Normal volunteers T4 T3 darr TSH uarr [27]

50 or 250mg 13 days Normal subjects T4 T3 darr TSH uarr(within normal range) [28]

27mg 4 weeks Normal volunteers T4 within the normal range except fortwo subjects TSH uarr [29]

Single doses of 10 30 50 and 100mg andthen daily doses of 10 15 30 50 or 100mg 12 days Euthyroid volunteers T3 T4 darr TSH uarr [30]

190mg 10 days Euthyroid volunteers T3 T4 darr TSH uarr [31]114mg 3ndash7 weeks Normal controls T3 T4 darr TSH uarr [32]

114mg 3ndash7 weeks Thyrotoxic patientsT4 T3 uarr in some casesother patients remained

euthyroid even after 6 weeks[32]

114mg 2 weeks Hypothyroid patients onthyroxine replacement

No consistent change(T4 uarr at 1 and 14 days TSH darr the first day) [32]

mitochondrial ROS generation is primarily due to electronsleaking from the electron transport chain (ETC) to generatesuperoxide radical Superoxide from the electron transportchain and other sources is converted by MnSOD to H

2O2

which is then metabolized by Prx 3 to water [47]Besides we demonstrated that high concentrated iodide

induces significant increased expression of Prx 3 whichsuggests that the increase of Prx 3 expression following highconcentrated iodide exposure orHI diet in the thyroidmay bethe protective response against high ROS generation Prx 3 isan indispensable ROS scavenger that protects against oxida-tive damage and subsequent apoptosis [51ndash53] It is reportedthat Prx 3 has an essential role in regulating oxidation-induced apoptosis and can be a potential therapeutic targetin castrate-independent prostate cancer [52] Iodide hada stimulatory effect on H

2O2generation in thyroid slices

[1] At high concentrations (above 01mM) H2O2induces

apoptosis in thyroid cells [1] When iodide is in excess ascompared to tyrosine residues it reacts with the iodoniumcation formed by iodide oxidation to give molecular iodineExcess molecular iodine then induced apoptosis throughgeneration of free radicals [54] H

2O2generation is a limiting

step in thyroid hormone biosynthesis it is essential foriodide oxidation by thyroperoxidase [14 55 56] H

2O2can

be eliminated by Prx 3 In mammals Prx 3 is targeted to themitochondrial matrix It is estimated that Prx 3 will be thetarget for up to 90 of hydrogen peroxide generated in themitochondrial matrix [57] mitochondrial peroxiredoxins (3andor 5) can reduce H

2O2to water through reducing equiv-

alents provided by thiol-containing proteins [58 59] butthe mechanisms involved are unclear [24 59] Drechsel andPatel demonstrated that thioredoxin (Trxs)peroxiredoxin(Prxs) (Prx 3 and Prx 5) is the major contributing systemto H2O2removal in brain mitochondria [60] Prxs and Trxs

are important liver antioxidant proteins and play importantroles in maintaining liver redox homeostasis [61] It isreported that chronic ethanol exposure increases NADPHoxidase activity and increases the production of superoxide

and hydrogen peroxide [62ndash64] Thioredoxin scavenges andreduces ROS via peroxiredoxins [65] Endoplasmic reticulumstress disrupts the electron transport chain leading to theincreased production of ROS [48 49] Bae et al reportedthat the antioxidant role of Prx 3 was evident from thefindings that pyrazole-induced protein carbonylation and theformation of 4-HNE adducts and MDA in the liver weremarkedly increased in Prx 3minusminus mice compared with wild-type mice [66]

A high urinary excretion of iodine in a spot urine sampleis the ultimate proof of iodine excess in vivo [2] We showedthe urinary iodine concentration increased as the iodidesupply increased significantly higher urinary iodine concen-tration can be detected in 100 HI than 10 HI and in MT-IIIKOmice than inWTmiceThis data indicates the adaptationof the thyroid gland to iodine excess and that the excessiveiodine is excreted from the urine in order to maintain thenormal function of thyroid This makes the studies in vivodifferent from studies in vitro In addition to the urinaryexcretion of iodine there are many control mechanisms ofthe thyroid to counteract iodide excess These include thesodium-iodide symporter which controls the limiting stepof thyroid hormone synthesis the Wolff-Chaikoff effect anarrest of thyroid hormone secretion from stores in the colloida preferential secretion of the less active T

4over T

3 and

dumping of excess iodine by secreting it in nonhormonalform [2]

Moreover we demonstrated that significant increasesof mitochondrial superoxide production and Prx 3 proteinexpression can be detected in MT-III KO mice group Thisdata indicated the protective role of MT-III against highconcentrations of iodide induced mitochondrial superoxideproduction in the thyrocytes MT has been demonstratedto play a key role in the detoxification of heavy metalsprotecting against various forms of oxidative injury by scav-enging oxygen free radicals MTs belong to the group ofintracellular cysteine-rich metal-binding proteins which iswidely expressed in cells (nuclei andor cytoplasm) of various

8 Oxidative Medicine and Cellular Longevity

organs and tumors [67ndash69] MTs play a protective role inpreventing against intoxication with heavy metals such asPb Hg Cu and Cd [67 70] All 20 cysteine sulfur atomsare involved in the radical quenching process and the rateconstant for the reaction of hydroxyl radical with MT isabout 340-fold higher than that with GSH [71 72] There arefour MT isoforms in mammals MT-I and MT-II are mainlyinvolved in the protection of tissue against metal toxicitiesoxidative stresses and apoptosis It has been demonstratedthat the rabbit liver metallothionein-1 which contains zincandor cadmium ions appeared to scavenge free hydroxyland superoxide radicals [25 26] Under low levels of iodideorganification in goitrous-modified tissue of thyroid glandmetallothionein may provide a partial compensatory effecton prooxidative processes [73 74] Although no cell lineor animal model overexpressing MT in the thyroid hasbeen found yet documented evidence of MT in protectingagainst oxidative stress in cardiac-specific metallothionein-overexpressed transgenic mice has been demonstrated Caiet al reported that metallothionein as a potent antioxi-dant prevented the development of diabetic cardiomyopathythrough the inhibition of the mitochondrial cytochromec-mediated caspase-3 activation pathway [75] Sun et alreported that through doxorubicin induced chronic toxi-city in a metallothionein-overexpressed transgenic mouseheart the antioxidant action of MT is highly responsiblefor this cardioprotection [76] Ceylan-Isik et al reportedthat the cardiac-specific overexpression of MT rescues LPS-induced cardiac contractile and intracellular Ca2+ dysfunc-tions through the alleviation of ROSoxidative stress stresssignaling activation and endoplasmic reticulum (ER) stress[77] They also reported that the cardiac-specific overexpres-sion of metallothionein protects against nicotine exposure-induced cardiac contractile dysfunction and fibrosis possiblythrough inhibition of ROS accumulation and apoptosis [78]Zhang et al suggest that cardiac overexpression of metalloth-ionein prevents cold exposure-induced cardiac anomaliespossibly through the attenuation of myocardial fibrosis [79]Hu et al demonstrated that cardiac-specific overexpression ofmetallothionein protects against cigarette smoking exposure-induced myocardial contractile and mitochondrial damagefavoring a role of lessened apoptosis in a metallothionein-induced beneficial effect against side-stream smoke exposure[80] Besides metallothionein has been shown to regulateapoptosis and proliferation Overexpression of metalloth-ionein frequently occurs in human tumors and is related totumor progression [81 82] High expression of MT has beenobserved in different types of cancer and has been consideredas a potential prognostic marker in cancers of the breastpancreas skin and cervix [82ndash86] Further studies will beattempted to study the oxidative and antioxidative stress onan engineered cell line overexpressing metallothionein

5 Conclusion

Thepresent study provides evidence that high concentrationsof iodide may lead to a significant increase in mitochondrialsuperoxide production and Prx 3 expression in both invitro and in vivo studies MT-III KO mice aggravated

mitochondrial superoxide production and peroxiredoxin 3expression in the thyroid after excessive iodide exposureMT-III may potentially protect the thyroid against highconcentrations of iodide induced oxidative stress

Abbreviations

MT-III KO Metallothionein III knockoutMTs MetallothioneinsWT Wild-typeROS Reactive oxygen speciesPrx PeroxiredoxinKI Potassium iodideNI Normal iodide intakeHI High iodide intakeLDH Lactate dehydrogenaseH2O2 Hydrogen peroxide

SOD Superoxide dismutaseMTT 3-(45-Dimethylthiazol-2-yl)-25-

diphenyltetrazoliumbromide

K-S Kolmogorov-SmirnovLSD Least significant differenceT4 Thyroxine

T3 Tri-iodothyronine

FT4 Free thyroxine

FT3 Free tri-iodothyronine

Conflict of Interests

The authors declare that they have no competing interests

Acknowledgments

Thiswork is supported by theNational Natural Science Foun-dation of China (no 81273009) the Tianjin Science amp Tech-nology Council Grant of China (no 09JCYBJC11700) andthe Tianjin Educational amp Scientific Grant (no 20050107)This paper has been edited by one of the authors MohamedAhmed who is from USA and now is studying in TianjinMedical University China

References

[1] B Corvilain L Collyn J van Sande and J E DumontldquoStimulation by iodide of H

2O2generation in thyroid slices

from several speciesrdquo The American Journal of PhysiologymdashEndocrinology and Metabolism vol 278 no 4 pp E692ndashE6992000

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

[3] E Roti and E Degli Uberti ldquoIodine excess and hyperthy-roidismrdquoThyroid vol 11 no 5 pp 493ndash500 2001

[4] World Health Organization and United Nations ChildrenrsquosFund ldquoInternational Council for Control of Iodine DeficiencyDisorders 1999 progress towards the elimination of iodinedeficiency disorders (IDD)rdquo WHO Booklet World HealthOrganization 1999

Oxidative Medicine and Cellular Longevity 9

[5] A M Leung and L E Braverman ldquoConsequences of excessiodinerdquo Nature Reviews Endocrinology vol 10 no 3 pp 136ndash142 2013

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

[7] IMahmoud I ColinM CMany and J-F Denef ldquoDirect toxiceffect of iodide in excess on iodine-deficient thyroid glandsepithelial necrosis and inflammation associated with lipofuscinaccumulationrdquo Experimental and Molecular Pathology vol 44no 3 pp 259ndash271 1986

[8] J E Fradkin and J Wolff ldquoIodide-induced thyrotoxicosisrdquoMedicine vol 62 no 1 pp 1ndash20 1983

[9] J Golstein and J E Dumont ldquoCytotoxic effects of iodide onthyroid cells difference between rat thyroid FRTL-5 cell andpri-mary dog thyrocyte responsivenessrdquo Journal of EndocrinologicalInvestigation vol 19 no 2 pp 119ndash126 1996

[10] 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

[11] W Teng Z Shan X Teng et al ldquoEffect of iodine intakeon thyroid diseases in Chinardquo The New England Journal ofMedicine vol 354 no 26 pp 2783ndash2793 2006

[12] P Laurberg K M Pedersen A Hreidarsson N Sigfusson EIversen and P R Knudsen ldquoIodine intake and the patternof thyroid disorders a comparative epidemiological study ofthyroid abnormalities in the elderly in iceland and in JutlandDenmarkrdquo Journal of Clinical Endocrinology and Metabolismvol 83 no 3 pp 765ndash769 1998

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

[14] 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

[15] 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

[16] H Han P Xin L Zhao et al ldquoExcess iodine and high-fatdiet combination modulates lipid profile thyroid hormoneand hepatic LDLr expression values in micerdquo Biological TraceElement Research vol 147 no 1ndash3 pp 233ndash239 2012

[17] S-J Zhao Y Ye F-J Sun E-J Tian and Z-P Chen ldquoTheimpact of dietary iodine intake on lipid metabolism in micerdquoBiological Trace Element Research vol 142 no 3 pp 581ndash5882011

[18] 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

[19] V Pitsiavas P Smerdely M Li and S C Boyages ldquoAmiodaroneinduces a different pattern of ultrastructural change in thethyroid to iodine excess alone in both the BBW rat and theWistar ratrdquo European Journal of Endocrinology vol 137 no 1pp 89ndash98 1997

[20] M B Zimmermann ldquoIodine deficiency and excess in childrenworldwide status in 2013rdquo Endocrine Practice vol 19 no 5 pp839ndash846 2013

[21] K Mussig ldquoIodine-induced toxic effects due to seaweed con-sumptionrdquo in Comprehensive Handbook of Iodine V R PreedyG N Burrow and R R Watson Eds pp 897ndash908 ElsevierAmsterdam The Netherlands 2009

[22] A-M G Psarra S Solakidi and C E Sekeris ldquoThe mito-chondrion as a primary site of action of steroid and thyroidhormones presence and action of steroid and thyroid hormonereceptors in mitochondria of animal cellsrdquo Molecular andCellular Endocrinology vol 246 no 1-2 pp 21ndash33 2006

[23] Y Song N Driessens M Costa et al ldquoRoles of hydrogenperoxide in thyroid physiology and diseaserdquo Journal of ClinicalEndocrinology and Metabolism vol 92 no 10 pp 3764ndash37732007

[24] A G Cox C C Winterbourn and M B Hampton ldquoMito-chondrial peroxiredoxin involvement in antioxidant defenceand redox signallingrdquo Biochemical Journal vol 425 no 2 pp313ndash325 2010

[25] B Ruttkay-Nedecky L Nejdl J Gumulec et al ldquoThe role ofmetallothionein in oxidative stressrdquo International Journal ofMolecular Sciences vol 14 no 3 pp 6044ndash6066 2013

[26] N Thirumoorthy A Shyam Sunder K T Manisenthil KumarM Senthil kumarGNKGanesh andMChatterjee ldquoA reviewof metallothionein isoforms and their role in pathophysiologyrdquoWorld Journal of Surgical Oncology vol 9 article 54 2011

[27] 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

[28] M Saberi and R D Utiger ldquoAugmentation of thyrotropinresponses to thyrotropin releasing hormone following smalldecreases in serum thyroid hormone concentrationsrdquo TheJournal of Clinical Endocrinology and Metabolism vol 40 no3 pp 435ndash441 1975

[29] H Namba S Yamashita H Kimura et al ldquoEvidence of thyroidvolume increase in normal subjects receiving excess iodiderdquoTheJournal of Clinical Endocrinology and Metabolism vol 76 no 3pp 605ndash608 1993

[30] E Sternthal L Lipworth B Stanley C Abreau S L Fang andL E Braverman ldquoSuppression of thyroid radioiodine uptakeby various doses of stable iodiderdquo The New England Journal ofMedicine vol 303 no 19 pp 1083ndash1088 1980

[31] A G Vagenakis B Rapoport F Azizi G I Portnay L EBraverman and S H Ingbar ldquoHyperresponse to thyrotropinreleasing hormone accompanying small decreases in serumthyroid hormone concentrationsrdquoThe Journal of Clinical Inves-tigation vol 54 no 4 pp 913ndash918 1974

[32] G Philippou D A Koutras G Piperingos A Souvatzoglouand S D Moulopoulos ldquoThe effect of iodide on serum thyroidhormone levels in normal persons in hyperthyroid patientsand in hypothyroid patients on thyroxine replacementrdquoClinicalEndocrinology vol 36 no 6 pp 573ndash578 1992

[33] L Z Ali Salim R Othman M A Abdulla et al ldquoThymo-quinone inhibits murine leukemia WEHI-3 cells in vivo and invitrordquo PLoS ONE vol 9 no 12 Article ID e115340 2014

[34] X Wang J Ge K Wang J Qian and Y Zou ldquoEvaluation ofMTT assay for measurement of emodin-induced cytotoxicityrdquoASSAY and Drug Development Technologies vol 4 no 2 pp203ndash207 2006

10 Oxidative Medicine and Cellular Longevity

[35] C Lv C Wu Y-h Zhou Y Shao G Wang and Q-y WangldquoAlpha lipoic acid Modulated high glucose-induced rat mesan-gial cell dysfunction viamTORp70S6K4E-BP1 pathwayrdquo Inter-national Journal of Endocrinology vol 2014 Article ID 65858914 pages 2014

[36] L Liu X Hu G-Y Cai et al ldquoHigh glucose-induced hyper-trophy of mesangial cells is reversed by connexin43 overex-pression via PTENAktmTOR signalingrdquo Nephrology DialysisTransplantation vol 27 no 1 pp 90ndash100 2012

[37] D D Harrison-Findik S Z Lu E M Zmijewski J Jonesand M C Zimmerman ldquoEffect of alcohol exposure on hepaticsuperoxide generation and hepcidin expressionrdquoWorld Journalof Biological Chemistry vol 4 no 4 pp 119ndash130 2013

[38] M Wang Y Wang J He et al ldquoAlbumin induces neuroprotec-tion against ischemic stroke by altering Toll-like receptor 4 andregulatory T cells in micerdquo CNS and Neurological DisordersmdashDrug Targets vol 12 no 2 pp 220ndash227 2013

[39] X Yao ldquoEffect of zinc exposure on HNE and GLT-1 in spinalcord culturerdquo NeuroToxicology vol 30 no 1 pp 121ndash126 2009

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

[41] G P Becks M C Eggo and G N Burrow ldquoRegulation ofdifferentiated thyroid function by iodide preferential inhibitoryeffect of excess iodide on thyroid hormone secretion in sheepthyroid cell culturesrdquo Endocrinology vol 120 no 6 pp 2569ndash2575 1987

[42] M Vitale T Di Matola F DrsquoAscoli et al ldquoIodide excessinduces apoptosis in thyroid cells through a p53-independentmechanism involving oxidative stressrdquo Endocrinology vol 141no 2 pp 598ndash605 2000

[43] P Pramyothin AM Leung E N Pearce A OMalabanan andL E Braverman ldquoClinical problem-solving A hidden solutionrdquoTheNew England Journal of Medicine vol 365 no 22 pp 2123ndash2127 2011

[44] W Jubiz S Carlile and L D Lagerquist ldquoSerum thyrotropinand thyroid hormone levels in humans receiving chronicpotassium iodiderdquo The Journal of Clinical Endocrinology andMetabolism vol 44 no 2 pp 379ndash382 1977

[45] A G Vagenakis P Downs L E Braverman A Burger andS H Ingbar ldquoControl of thyroid hormone secretion in normalsubjects receiving iodidesrdquoThe Journal of Clinical Investigationvol 52 no 2 pp 528ndash532 1973

[46] M Safran andL E Braverman ldquoEffect of chronic douchingwithpolyvinylpyrrolidone-iodine on iodine absorption and thyroidfunctionrdquo Obstetrics and Gynecology vol 60 no 1 pp 35ndash401982

[47] K Newick B Cunniff K Preston et al ldquoPeroxiredoxin 3 is aredox-dependent target of thiostrepton in malignant mesothe-lioma cellsrdquo PLoS ONE vol 7 no 6 Article ID e39404 2012

[48] A Deniaud O S El Dein E Maillier et al ldquoEndoplasmicreticulum stress induces calcium-dependent permeability tran-sition mitochondrial outer membrane permeabilization andapoptosisrdquo Oncogene vol 27 no 3 pp 285ndash299 2008

[49] A Gorlach P Klappa and T Kietzmann ldquoThe endoplasmicreticulum folding calcium homeostasis signaling and redoxcontrolrdquo Antioxidants and Redox Signaling vol 8 no 9-10 pp1391ndash1418 2006

[50] 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 symporter

expression and activity involves the PI3KAkt signaling path-wayrdquo Endocrinology vol 155 no 3 pp 1145ndash1156 2014

[51] Y-G Wang L Li C-H Liu S Hong and M-J ZhangldquoPeroxiredoxin 3 is resistant to oxidation-induced apoptosis ofHep-3b cellsrdquoClinical and Translational Oncology vol 16 no 6pp 561ndash566 2014

[52] H C Whitaker D Patel W J Howat et al ldquoPeroxiredoxin-3is overexpressed in prostate cancer and promotes cancer cellsurvival by protecting cells fromoxidative stressrdquoBritish Journalof Cancer vol 109 no 4 pp 983ndash993 2013

[53] L Li Y G Zhang and C L Chen ldquoAnti-apoptotic role ofperoxiredoxin III in cervical cancer cellsrdquo FEBS Open Bio vol3 pp 51ndash54 2013

[54] 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

[55] L I Nadolnik Z V Niatsetskaya and S V Lupachyk ldquoEffectof oxidative stress on rat thyrocyte iodide metabolismrdquo CellBiochemistry and Function vol 26 no 3 pp 366ndash373 2008

[56] A C Freitas Ferreira L de Carvalho Cardoso D Rosenthaland D Pires de Carvalho ldquoThyroid Ca2+NADPH-dependentH2O2generation is partially inhibited by propylthiouracil and

methimazolerdquo European Journal of Biochemistry vol 270 no 11pp 2363ndash2368 2003

[57] G R Sue Z C Ho and K Kim ldquoPeroxiredoxins a historicaloverview and speculative preview of novel mechanisms andemerging concepts in cell signalingrdquo Free Radical Biology andMedicine vol 38 no 12 pp 1543ndash1552 2005

[58] M S Seo S W Kang K Kim I C Baines T H Lee and SG Rhee ldquoIdentification of a new type of mammalian perox-iredoxin that forms an intramolecular disulfide as a reactionintermediaterdquo Journal of Biological Chemistry vol 275 no 27pp 20346ndash20354 2000

[59] Z A Wood L B Poole and P A Karplus ldquoPeroxiredoxinevolution and the regulation of hydrogen peroxide signalingrdquoScience vol 300 no 5619 pp 650ndash653 2003

[60] D A Drechsel and M Patel ldquoRespiration-dependent H2O2

removal in brain mitochondria via the thioredoxinperoxire-doxin systemrdquoThe Journal of Biological Chemistry vol 285 no36 pp 27850ndash27858 2010

[61] W H Watson X Yang Y E Choi D P Jones and J P KehrerldquoThioredoxin and its role in toxicologyrdquo Toxicological Sciencesvol 78 no 1 pp 3ndash14 2004

[62] G Ekstrom and M Ingelman-Sundberg ldquoRat liver microso-mal NADPH-supported oxidase activity and lipid peroxida-tion dependent on ethanol-inducible cytochrome P-450 (P-450IIE1)rdquo Biochemical Pharmacology vol 38 no 8 pp 1313ndash1319 1989

[63] J Rashba-Step N J Turro and A I Cederbaum ldquoIncreasedNADPH- and NADH-dependent production of superoxideand hydroxyl radical by microsomes after chronic ethanoltreatmentrdquoArchives of Biochemistry and Biophysics vol 300 no1 pp 401ndash408 1993

[64] V Thakur M T Pritchard M R McMullen Q Wang andL E Nagy ldquoChronic ethanol feeding increases activation ofNADPH oxidase by lipopolysaccharide in rat Kupffer cellsrole of increased reactive oxygen in LPS-stimulated ERK12activation andTNF-120572 productionrdquo Journal of Leukocyte Biologyvol 79 no 6 pp 1348ndash1356 2006

Oxidative Medicine and Cellular Longevity 11

[65] HNakamuraAMitsui and J Yodoi ldquoThioredoxin overexpres-sion in transgenic micerdquo Methods in Enzymology vol 347 pp436ndash440 2002

[66] 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

[67] A Bieniek B Pula A Piotrowska et al ldquoExpression of metal-lothionein III andKi-67 antigen in various histological types ofbasal cell carcinomardquo Folia Histochemica et Cytobiologica vol50 no 3 pp 352ndash357 2012

[68] P Dziegiel ldquoExpression of metallothioneins in tumor cellsrdquoPolish Journal of Pathology vol 55 no 1 pp 3ndash12 2004

[69] M Oslash Pedersen A Larsen M Stoltenberg and M PenkowaldquoThe role of metallothionein in oncogenesis and cancer prog-nosisrdquo Progress inHistochemistry and Cytochemistry vol 44 no1 pp 29ndash64 2009

[70] W Braun M Vasak A H Robbins et al ldquoComparison ofthe NMR solution structure and the X-ray crystal structure ofrat metallothionein-2rdquo Proceedings of the National Academy ofSciences of the United States of America vol 89 no 21 pp 10124ndash10128 1992

[71] P JThornalley andM Vasak ldquoPossible role formetallothioneinin protection against radiation-induced oxidative stress Kinet-ics andmechanism of its reaction with superoxide and hydroxylradicalsrdquo Biochimica et Biophysica Acta Protein Structure andMolecular Enzymology vol 827 no 1 pp 36ndash44 1985

[72] M A Schwarz J S Lazo J C Yalowich et al ldquoMetallothioneinprotects against the cytotoxic and DNA-damaging effects ofnitric oxiderdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 92 no 10 pp 4452ndash4456 1995

[73] Y Luo A Kawashima and Y Ishido ldquoIodine excess as anenvironmental risk factor for autoimmune thyroid diseaserdquoInternational Journal of Molecular Sciences vol 15 pp 12895ndash12912 2014

[74] H I Falrsquofushinsrsquoka L L Hnatyshyna andOI Osadchuk ldquoTraceelements storage peculiarities and metallothionein contentin human thyroid gland under iodine deficiency euthyroidnodular goiterrdquo Ukrainskii Biokhimicheskii Zhurnal vol 86 pp107ndash113 2014

[75] L Cai Y H Wang G H Zhou et al ldquoAttenuation bymetallothionein of early cardiac cell death via suppression ofmitochondrial oxidative stress results in a prevention of diabeticcardiomyopathyrdquo Journal of the American College of Cardiologyvol 48 no 8 pp 1688ndash1697 2006

[76] X Sun Z Zhou and Y J Kang ldquoAttenuation of doxorubicinchronic toxicity in metallothionein-overexpressing transgenicmouse heartrdquo Cancer Research vol 61 no 8 pp 3382ndash33872001

[77] A F Ceylan-Isik P Zhao B F Zhang X Xiao G Su and J RenldquoCardiac overexpression of metallothionein rescues cardiaccontractile dysfunction and endoplasmic reticulum stress butnot autophagy in sepsisrdquo Journal of Molecular and CellularCardiology vol 48 no 2 pp 367ndash378 2010

[78] N Hu R Guo X Han B Zhu and J Ren ldquoCardiac-specificoverexpression of metallothionein rescues nicotine-inducedcardiac contractile dysfunction and interstitial fibrosisrdquoToxicol-ogy Letters vol 202 no 1 pp 8ndash14 2011

[79] Y Zhang N Hu Y Hua K L Richmond F Dong and JRen ldquoCardiac overexpression of metallothionein rescues coldexposure-induced myocardial contractile dysfunction through

attenuation of cardiac fibrosis despite cardiomyocyte mechani-cal anomaliesrdquo Free Radical Biology and Medicine vol 53 no 2pp 194ndash207 2012

[80] N Hu X Han E K Lane F Gao Y Zhang and J RenldquoCardiac-specific overexpression of metallothionein rescuesagainst cigarette smoking exposure-induced myocardial con-tractile and mitochondrial damagerdquo PLoS ONE vol 8 no 2Article ID e57151 2013

[81] M T Brazao-Silva S V Cardoso P R de Faria et al ldquoAdenoidcystic carcinoma of the salivary gland a clinicopathologicalstudy of 49 cases and of metallothionein expression with regardto tumour behaviourrdquo Histopathology vol 63 no 6 pp 802ndash809 2013

[82] K J Schmitz H Lang G Kaiser et al ldquoMetallothioneinoverexpression and its prognostic relevance in intrahepaticcholangiocarcinoma and extrahepatic hilar cholangiocarci-noma (Klatskin tumors)rdquo Human Pathology vol 40 no 12 pp1706ndash1714 2009

[83] S E Theocharis A P Margeli J T Klijanienko and G PKouraklis ldquoMetallothionein expression in human neoplasiardquoHistopathology vol 45 no 2 pp 103ndash118 2004

[84] STheocharis J Klijanienko C Giaginis et al ldquoMetallothioneinexpression in mobile tongue squamous cell carcinoma associa-tions with clinicopathological parameters and patient survivalrdquoHistopathology vol 59 no 3 pp 514ndash525 2011

[85] S-K Tai O J-K Tan V T-K Chow et al ldquoDifferentialexpression of metallothionein 1 and 2 isoforms in breast cancerlines with different invasive potential identification of a novelnon-silent metallothionein-1H mutant variantrdquo The AmericanJournal of Pathology vol 163 no 5 pp 2009ndash2019 2003

[86] Y Hishikawa T Koji D K Dhar S Kinugasa M Yamaguchiand N Nagasue ldquoMetallothionein expression correlates withmetastatic and proliferative potential in squamous cell carci-noma of the oesophagusrdquo British Journal of Cancer vol 81 no4 pp 712ndash720 1999

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Oxidative Medicine and Cellular Longevity

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Evidence-Based Complementary and Alternative Medicine

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Page 7: Research Article Metallothionein-I/II Knockout Mice

Oxidative Medicine and Cellular Longevity 7

Table 3 Documented literatures of iodide intake and changes of serum thyroxine (T4) and triiodothyronine (T3) concentrations

Dosage of daily iodine intake Time Subject Changes of serum T4 T3 concentrations Reference250 or 500 120583g 14 days Normal volunteers No change [27]1500 120583g 14 days Normal volunteers T4 T3 darr TSH uarr [27]

50 or 250mg 13 days Normal subjects T4 T3 darr TSH uarr(within normal range) [28]

27mg 4 weeks Normal volunteers T4 within the normal range except fortwo subjects TSH uarr [29]

Single doses of 10 30 50 and 100mg andthen daily doses of 10 15 30 50 or 100mg 12 days Euthyroid volunteers T3 T4 darr TSH uarr [30]

190mg 10 days Euthyroid volunteers T3 T4 darr TSH uarr [31]114mg 3ndash7 weeks Normal controls T3 T4 darr TSH uarr [32]

114mg 3ndash7 weeks Thyrotoxic patientsT4 T3 uarr in some casesother patients remained

euthyroid even after 6 weeks[32]

114mg 2 weeks Hypothyroid patients onthyroxine replacement

No consistent change(T4 uarr at 1 and 14 days TSH darr the first day) [32]

mitochondrial ROS generation is primarily due to electronsleaking from the electron transport chain (ETC) to generatesuperoxide radical Superoxide from the electron transportchain and other sources is converted by MnSOD to H

2O2

which is then metabolized by Prx 3 to water [47]Besides we demonstrated that high concentrated iodide

induces significant increased expression of Prx 3 whichsuggests that the increase of Prx 3 expression following highconcentrated iodide exposure orHI diet in the thyroidmay bethe protective response against high ROS generation Prx 3 isan indispensable ROS scavenger that protects against oxida-tive damage and subsequent apoptosis [51ndash53] It is reportedthat Prx 3 has an essential role in regulating oxidation-induced apoptosis and can be a potential therapeutic targetin castrate-independent prostate cancer [52] Iodide hada stimulatory effect on H

2O2generation in thyroid slices

[1] At high concentrations (above 01mM) H2O2induces

apoptosis in thyroid cells [1] When iodide is in excess ascompared to tyrosine residues it reacts with the iodoniumcation formed by iodide oxidation to give molecular iodineExcess molecular iodine then induced apoptosis throughgeneration of free radicals [54] H

2O2generation is a limiting

step in thyroid hormone biosynthesis it is essential foriodide oxidation by thyroperoxidase [14 55 56] H

2O2can

be eliminated by Prx 3 In mammals Prx 3 is targeted to themitochondrial matrix It is estimated that Prx 3 will be thetarget for up to 90 of hydrogen peroxide generated in themitochondrial matrix [57] mitochondrial peroxiredoxins (3andor 5) can reduce H

2O2to water through reducing equiv-

alents provided by thiol-containing proteins [58 59] butthe mechanisms involved are unclear [24 59] Drechsel andPatel demonstrated that thioredoxin (Trxs)peroxiredoxin(Prxs) (Prx 3 and Prx 5) is the major contributing systemto H2O2removal in brain mitochondria [60] Prxs and Trxs

are important liver antioxidant proteins and play importantroles in maintaining liver redox homeostasis [61] It isreported that chronic ethanol exposure increases NADPHoxidase activity and increases the production of superoxide

and hydrogen peroxide [62ndash64] Thioredoxin scavenges andreduces ROS via peroxiredoxins [65] Endoplasmic reticulumstress disrupts the electron transport chain leading to theincreased production of ROS [48 49] Bae et al reportedthat the antioxidant role of Prx 3 was evident from thefindings that pyrazole-induced protein carbonylation and theformation of 4-HNE adducts and MDA in the liver weremarkedly increased in Prx 3minusminus mice compared with wild-type mice [66]

A high urinary excretion of iodine in a spot urine sampleis the ultimate proof of iodine excess in vivo [2] We showedthe urinary iodine concentration increased as the iodidesupply increased significantly higher urinary iodine concen-tration can be detected in 100 HI than 10 HI and in MT-IIIKOmice than inWTmiceThis data indicates the adaptationof the thyroid gland to iodine excess and that the excessiveiodine is excreted from the urine in order to maintain thenormal function of thyroid This makes the studies in vivodifferent from studies in vitro In addition to the urinaryexcretion of iodine there are many control mechanisms ofthe thyroid to counteract iodide excess These include thesodium-iodide symporter which controls the limiting stepof thyroid hormone synthesis the Wolff-Chaikoff effect anarrest of thyroid hormone secretion from stores in the colloida preferential secretion of the less active T

4over T

3 and

dumping of excess iodine by secreting it in nonhormonalform [2]

Moreover we demonstrated that significant increasesof mitochondrial superoxide production and Prx 3 proteinexpression can be detected in MT-III KO mice group Thisdata indicated the protective role of MT-III against highconcentrations of iodide induced mitochondrial superoxideproduction in the thyrocytes MT has been demonstratedto play a key role in the detoxification of heavy metalsprotecting against various forms of oxidative injury by scav-enging oxygen free radicals MTs belong to the group ofintracellular cysteine-rich metal-binding proteins which iswidely expressed in cells (nuclei andor cytoplasm) of various

8 Oxidative Medicine and Cellular Longevity

organs and tumors [67ndash69] MTs play a protective role inpreventing against intoxication with heavy metals such asPb Hg Cu and Cd [67 70] All 20 cysteine sulfur atomsare involved in the radical quenching process and the rateconstant for the reaction of hydroxyl radical with MT isabout 340-fold higher than that with GSH [71 72] There arefour MT isoforms in mammals MT-I and MT-II are mainlyinvolved in the protection of tissue against metal toxicitiesoxidative stresses and apoptosis It has been demonstratedthat the rabbit liver metallothionein-1 which contains zincandor cadmium ions appeared to scavenge free hydroxyland superoxide radicals [25 26] Under low levels of iodideorganification in goitrous-modified tissue of thyroid glandmetallothionein may provide a partial compensatory effecton prooxidative processes [73 74] Although no cell lineor animal model overexpressing MT in the thyroid hasbeen found yet documented evidence of MT in protectingagainst oxidative stress in cardiac-specific metallothionein-overexpressed transgenic mice has been demonstrated Caiet al reported that metallothionein as a potent antioxi-dant prevented the development of diabetic cardiomyopathythrough the inhibition of the mitochondrial cytochromec-mediated caspase-3 activation pathway [75] Sun et alreported that through doxorubicin induced chronic toxi-city in a metallothionein-overexpressed transgenic mouseheart the antioxidant action of MT is highly responsiblefor this cardioprotection [76] Ceylan-Isik et al reportedthat the cardiac-specific overexpression of MT rescues LPS-induced cardiac contractile and intracellular Ca2+ dysfunc-tions through the alleviation of ROSoxidative stress stresssignaling activation and endoplasmic reticulum (ER) stress[77] They also reported that the cardiac-specific overexpres-sion of metallothionein protects against nicotine exposure-induced cardiac contractile dysfunction and fibrosis possiblythrough inhibition of ROS accumulation and apoptosis [78]Zhang et al suggest that cardiac overexpression of metalloth-ionein prevents cold exposure-induced cardiac anomaliespossibly through the attenuation of myocardial fibrosis [79]Hu et al demonstrated that cardiac-specific overexpression ofmetallothionein protects against cigarette smoking exposure-induced myocardial contractile and mitochondrial damagefavoring a role of lessened apoptosis in a metallothionein-induced beneficial effect against side-stream smoke exposure[80] Besides metallothionein has been shown to regulateapoptosis and proliferation Overexpression of metalloth-ionein frequently occurs in human tumors and is related totumor progression [81 82] High expression of MT has beenobserved in different types of cancer and has been consideredas a potential prognostic marker in cancers of the breastpancreas skin and cervix [82ndash86] Further studies will beattempted to study the oxidative and antioxidative stress onan engineered cell line overexpressing metallothionein

5 Conclusion

Thepresent study provides evidence that high concentrationsof iodide may lead to a significant increase in mitochondrialsuperoxide production and Prx 3 expression in both invitro and in vivo studies MT-III KO mice aggravated

mitochondrial superoxide production and peroxiredoxin 3expression in the thyroid after excessive iodide exposureMT-III may potentially protect the thyroid against highconcentrations of iodide induced oxidative stress

Abbreviations

MT-III KO Metallothionein III knockoutMTs MetallothioneinsWT Wild-typeROS Reactive oxygen speciesPrx PeroxiredoxinKI Potassium iodideNI Normal iodide intakeHI High iodide intakeLDH Lactate dehydrogenaseH2O2 Hydrogen peroxide

SOD Superoxide dismutaseMTT 3-(45-Dimethylthiazol-2-yl)-25-

diphenyltetrazoliumbromide

K-S Kolmogorov-SmirnovLSD Least significant differenceT4 Thyroxine

T3 Tri-iodothyronine

FT4 Free thyroxine

FT3 Free tri-iodothyronine

Conflict of Interests

The authors declare that they have no competing interests

Acknowledgments

Thiswork is supported by theNational Natural Science Foun-dation of China (no 81273009) the Tianjin Science amp Tech-nology Council Grant of China (no 09JCYBJC11700) andthe Tianjin Educational amp Scientific Grant (no 20050107)This paper has been edited by one of the authors MohamedAhmed who is from USA and now is studying in TianjinMedical University China

References

[1] B Corvilain L Collyn J van Sande and J E DumontldquoStimulation by iodide of H

2O2generation in thyroid slices

from several speciesrdquo The American Journal of PhysiologymdashEndocrinology and Metabolism vol 278 no 4 pp E692ndashE6992000

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

[3] E Roti and E Degli Uberti ldquoIodine excess and hyperthy-roidismrdquoThyroid vol 11 no 5 pp 493ndash500 2001

[4] World Health Organization and United Nations ChildrenrsquosFund ldquoInternational Council for Control of Iodine DeficiencyDisorders 1999 progress towards the elimination of iodinedeficiency disorders (IDD)rdquo WHO Booklet World HealthOrganization 1999

Oxidative Medicine and Cellular Longevity 9

[5] A M Leung and L E Braverman ldquoConsequences of excessiodinerdquo Nature Reviews Endocrinology vol 10 no 3 pp 136ndash142 2013

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

[7] IMahmoud I ColinM CMany and J-F Denef ldquoDirect toxiceffect of iodide in excess on iodine-deficient thyroid glandsepithelial necrosis and inflammation associated with lipofuscinaccumulationrdquo Experimental and Molecular Pathology vol 44no 3 pp 259ndash271 1986

[8] J E Fradkin and J Wolff ldquoIodide-induced thyrotoxicosisrdquoMedicine vol 62 no 1 pp 1ndash20 1983

[9] J Golstein and J E Dumont ldquoCytotoxic effects of iodide onthyroid cells difference between rat thyroid FRTL-5 cell andpri-mary dog thyrocyte responsivenessrdquo Journal of EndocrinologicalInvestigation vol 19 no 2 pp 119ndash126 1996

[10] 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

[11] W Teng Z Shan X Teng et al ldquoEffect of iodine intakeon thyroid diseases in Chinardquo The New England Journal ofMedicine vol 354 no 26 pp 2783ndash2793 2006

[12] P Laurberg K M Pedersen A Hreidarsson N Sigfusson EIversen and P R Knudsen ldquoIodine intake and the patternof thyroid disorders a comparative epidemiological study ofthyroid abnormalities in the elderly in iceland and in JutlandDenmarkrdquo Journal of Clinical Endocrinology and Metabolismvol 83 no 3 pp 765ndash769 1998

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

[14] 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

[15] 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

[16] H Han P Xin L Zhao et al ldquoExcess iodine and high-fatdiet combination modulates lipid profile thyroid hormoneand hepatic LDLr expression values in micerdquo Biological TraceElement Research vol 147 no 1ndash3 pp 233ndash239 2012

[17] S-J Zhao Y Ye F-J Sun E-J Tian and Z-P Chen ldquoTheimpact of dietary iodine intake on lipid metabolism in micerdquoBiological Trace Element Research vol 142 no 3 pp 581ndash5882011

[18] 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

[19] V Pitsiavas P Smerdely M Li and S C Boyages ldquoAmiodaroneinduces a different pattern of ultrastructural change in thethyroid to iodine excess alone in both the BBW rat and theWistar ratrdquo European Journal of Endocrinology vol 137 no 1pp 89ndash98 1997

[20] M B Zimmermann ldquoIodine deficiency and excess in childrenworldwide status in 2013rdquo Endocrine Practice vol 19 no 5 pp839ndash846 2013

[21] K Mussig ldquoIodine-induced toxic effects due to seaweed con-sumptionrdquo in Comprehensive Handbook of Iodine V R PreedyG N Burrow and R R Watson Eds pp 897ndash908 ElsevierAmsterdam The Netherlands 2009

[22] A-M G Psarra S Solakidi and C E Sekeris ldquoThe mito-chondrion as a primary site of action of steroid and thyroidhormones presence and action of steroid and thyroid hormonereceptors in mitochondria of animal cellsrdquo Molecular andCellular Endocrinology vol 246 no 1-2 pp 21ndash33 2006

[23] Y Song N Driessens M Costa et al ldquoRoles of hydrogenperoxide in thyroid physiology and diseaserdquo Journal of ClinicalEndocrinology and Metabolism vol 92 no 10 pp 3764ndash37732007

[24] A G Cox C C Winterbourn and M B Hampton ldquoMito-chondrial peroxiredoxin involvement in antioxidant defenceand redox signallingrdquo Biochemical Journal vol 425 no 2 pp313ndash325 2010

[25] B Ruttkay-Nedecky L Nejdl J Gumulec et al ldquoThe role ofmetallothionein in oxidative stressrdquo International Journal ofMolecular Sciences vol 14 no 3 pp 6044ndash6066 2013

[26] N Thirumoorthy A Shyam Sunder K T Manisenthil KumarM Senthil kumarGNKGanesh andMChatterjee ldquoA reviewof metallothionein isoforms and their role in pathophysiologyrdquoWorld Journal of Surgical Oncology vol 9 article 54 2011

[27] 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

[28] M Saberi and R D Utiger ldquoAugmentation of thyrotropinresponses to thyrotropin releasing hormone following smalldecreases in serum thyroid hormone concentrationsrdquo TheJournal of Clinical Endocrinology and Metabolism vol 40 no3 pp 435ndash441 1975

[29] H Namba S Yamashita H Kimura et al ldquoEvidence of thyroidvolume increase in normal subjects receiving excess iodiderdquoTheJournal of Clinical Endocrinology and Metabolism vol 76 no 3pp 605ndash608 1993

[30] E Sternthal L Lipworth B Stanley C Abreau S L Fang andL E Braverman ldquoSuppression of thyroid radioiodine uptakeby various doses of stable iodiderdquo The New England Journal ofMedicine vol 303 no 19 pp 1083ndash1088 1980

[31] A G Vagenakis B Rapoport F Azizi G I Portnay L EBraverman and S H Ingbar ldquoHyperresponse to thyrotropinreleasing hormone accompanying small decreases in serumthyroid hormone concentrationsrdquoThe Journal of Clinical Inves-tigation vol 54 no 4 pp 913ndash918 1974

[32] G Philippou D A Koutras G Piperingos A Souvatzoglouand S D Moulopoulos ldquoThe effect of iodide on serum thyroidhormone levels in normal persons in hyperthyroid patientsand in hypothyroid patients on thyroxine replacementrdquoClinicalEndocrinology vol 36 no 6 pp 573ndash578 1992

[33] L Z Ali Salim R Othman M A Abdulla et al ldquoThymo-quinone inhibits murine leukemia WEHI-3 cells in vivo and invitrordquo PLoS ONE vol 9 no 12 Article ID e115340 2014

[34] X Wang J Ge K Wang J Qian and Y Zou ldquoEvaluation ofMTT assay for measurement of emodin-induced cytotoxicityrdquoASSAY and Drug Development Technologies vol 4 no 2 pp203ndash207 2006

10 Oxidative Medicine and Cellular Longevity

[35] C Lv C Wu Y-h Zhou Y Shao G Wang and Q-y WangldquoAlpha lipoic acid Modulated high glucose-induced rat mesan-gial cell dysfunction viamTORp70S6K4E-BP1 pathwayrdquo Inter-national Journal of Endocrinology vol 2014 Article ID 65858914 pages 2014

[36] L Liu X Hu G-Y Cai et al ldquoHigh glucose-induced hyper-trophy of mesangial cells is reversed by connexin43 overex-pression via PTENAktmTOR signalingrdquo Nephrology DialysisTransplantation vol 27 no 1 pp 90ndash100 2012

[37] D D Harrison-Findik S Z Lu E M Zmijewski J Jonesand M C Zimmerman ldquoEffect of alcohol exposure on hepaticsuperoxide generation and hepcidin expressionrdquoWorld Journalof Biological Chemistry vol 4 no 4 pp 119ndash130 2013

[38] M Wang Y Wang J He et al ldquoAlbumin induces neuroprotec-tion against ischemic stroke by altering Toll-like receptor 4 andregulatory T cells in micerdquo CNS and Neurological DisordersmdashDrug Targets vol 12 no 2 pp 220ndash227 2013

[39] X Yao ldquoEffect of zinc exposure on HNE and GLT-1 in spinalcord culturerdquo NeuroToxicology vol 30 no 1 pp 121ndash126 2009

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

[41] G P Becks M C Eggo and G N Burrow ldquoRegulation ofdifferentiated thyroid function by iodide preferential inhibitoryeffect of excess iodide on thyroid hormone secretion in sheepthyroid cell culturesrdquo Endocrinology vol 120 no 6 pp 2569ndash2575 1987

[42] M Vitale T Di Matola F DrsquoAscoli et al ldquoIodide excessinduces apoptosis in thyroid cells through a p53-independentmechanism involving oxidative stressrdquo Endocrinology vol 141no 2 pp 598ndash605 2000

[43] P Pramyothin AM Leung E N Pearce A OMalabanan andL E Braverman ldquoClinical problem-solving A hidden solutionrdquoTheNew England Journal of Medicine vol 365 no 22 pp 2123ndash2127 2011

[44] W Jubiz S Carlile and L D Lagerquist ldquoSerum thyrotropinand thyroid hormone levels in humans receiving chronicpotassium iodiderdquo The Journal of Clinical Endocrinology andMetabolism vol 44 no 2 pp 379ndash382 1977

[45] A G Vagenakis P Downs L E Braverman A Burger andS H Ingbar ldquoControl of thyroid hormone secretion in normalsubjects receiving iodidesrdquoThe Journal of Clinical Investigationvol 52 no 2 pp 528ndash532 1973

[46] M Safran andL E Braverman ldquoEffect of chronic douchingwithpolyvinylpyrrolidone-iodine on iodine absorption and thyroidfunctionrdquo Obstetrics and Gynecology vol 60 no 1 pp 35ndash401982

[47] K Newick B Cunniff K Preston et al ldquoPeroxiredoxin 3 is aredox-dependent target of thiostrepton in malignant mesothe-lioma cellsrdquo PLoS ONE vol 7 no 6 Article ID e39404 2012

[48] A Deniaud O S El Dein E Maillier et al ldquoEndoplasmicreticulum stress induces calcium-dependent permeability tran-sition mitochondrial outer membrane permeabilization andapoptosisrdquo Oncogene vol 27 no 3 pp 285ndash299 2008

[49] A Gorlach P Klappa and T Kietzmann ldquoThe endoplasmicreticulum folding calcium homeostasis signaling and redoxcontrolrdquo Antioxidants and Redox Signaling vol 8 no 9-10 pp1391ndash1418 2006

[50] 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 symporter

expression and activity involves the PI3KAkt signaling path-wayrdquo Endocrinology vol 155 no 3 pp 1145ndash1156 2014

[51] Y-G Wang L Li C-H Liu S Hong and M-J ZhangldquoPeroxiredoxin 3 is resistant to oxidation-induced apoptosis ofHep-3b cellsrdquoClinical and Translational Oncology vol 16 no 6pp 561ndash566 2014

[52] H C Whitaker D Patel W J Howat et al ldquoPeroxiredoxin-3is overexpressed in prostate cancer and promotes cancer cellsurvival by protecting cells fromoxidative stressrdquoBritish Journalof Cancer vol 109 no 4 pp 983ndash993 2013

[53] L Li Y G Zhang and C L Chen ldquoAnti-apoptotic role ofperoxiredoxin III in cervical cancer cellsrdquo FEBS Open Bio vol3 pp 51ndash54 2013

[54] 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

[55] L I Nadolnik Z V Niatsetskaya and S V Lupachyk ldquoEffectof oxidative stress on rat thyrocyte iodide metabolismrdquo CellBiochemistry and Function vol 26 no 3 pp 366ndash373 2008

[56] A C Freitas Ferreira L de Carvalho Cardoso D Rosenthaland D Pires de Carvalho ldquoThyroid Ca2+NADPH-dependentH2O2generation is partially inhibited by propylthiouracil and

methimazolerdquo European Journal of Biochemistry vol 270 no 11pp 2363ndash2368 2003

[57] G R Sue Z C Ho and K Kim ldquoPeroxiredoxins a historicaloverview and speculative preview of novel mechanisms andemerging concepts in cell signalingrdquo Free Radical Biology andMedicine vol 38 no 12 pp 1543ndash1552 2005

[58] M S Seo S W Kang K Kim I C Baines T H Lee and SG Rhee ldquoIdentification of a new type of mammalian perox-iredoxin that forms an intramolecular disulfide as a reactionintermediaterdquo Journal of Biological Chemistry vol 275 no 27pp 20346ndash20354 2000

[59] Z A Wood L B Poole and P A Karplus ldquoPeroxiredoxinevolution and the regulation of hydrogen peroxide signalingrdquoScience vol 300 no 5619 pp 650ndash653 2003

[60] D A Drechsel and M Patel ldquoRespiration-dependent H2O2

removal in brain mitochondria via the thioredoxinperoxire-doxin systemrdquoThe Journal of Biological Chemistry vol 285 no36 pp 27850ndash27858 2010

[61] W H Watson X Yang Y E Choi D P Jones and J P KehrerldquoThioredoxin and its role in toxicologyrdquo Toxicological Sciencesvol 78 no 1 pp 3ndash14 2004

[62] G Ekstrom and M Ingelman-Sundberg ldquoRat liver microso-mal NADPH-supported oxidase activity and lipid peroxida-tion dependent on ethanol-inducible cytochrome P-450 (P-450IIE1)rdquo Biochemical Pharmacology vol 38 no 8 pp 1313ndash1319 1989

[63] J Rashba-Step N J Turro and A I Cederbaum ldquoIncreasedNADPH- and NADH-dependent production of superoxideand hydroxyl radical by microsomes after chronic ethanoltreatmentrdquoArchives of Biochemistry and Biophysics vol 300 no1 pp 401ndash408 1993

[64] V Thakur M T Pritchard M R McMullen Q Wang andL E Nagy ldquoChronic ethanol feeding increases activation ofNADPH oxidase by lipopolysaccharide in rat Kupffer cellsrole of increased reactive oxygen in LPS-stimulated ERK12activation andTNF-120572 productionrdquo Journal of Leukocyte Biologyvol 79 no 6 pp 1348ndash1356 2006

Oxidative Medicine and Cellular Longevity 11

[65] HNakamuraAMitsui and J Yodoi ldquoThioredoxin overexpres-sion in transgenic micerdquo Methods in Enzymology vol 347 pp436ndash440 2002

[66] 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

[67] A Bieniek B Pula A Piotrowska et al ldquoExpression of metal-lothionein III andKi-67 antigen in various histological types ofbasal cell carcinomardquo Folia Histochemica et Cytobiologica vol50 no 3 pp 352ndash357 2012

[68] P Dziegiel ldquoExpression of metallothioneins in tumor cellsrdquoPolish Journal of Pathology vol 55 no 1 pp 3ndash12 2004

[69] M Oslash Pedersen A Larsen M Stoltenberg and M PenkowaldquoThe role of metallothionein in oncogenesis and cancer prog-nosisrdquo Progress inHistochemistry and Cytochemistry vol 44 no1 pp 29ndash64 2009

[70] W Braun M Vasak A H Robbins et al ldquoComparison ofthe NMR solution structure and the X-ray crystal structure ofrat metallothionein-2rdquo Proceedings of the National Academy ofSciences of the United States of America vol 89 no 21 pp 10124ndash10128 1992

[71] P JThornalley andM Vasak ldquoPossible role formetallothioneinin protection against radiation-induced oxidative stress Kinet-ics andmechanism of its reaction with superoxide and hydroxylradicalsrdquo Biochimica et Biophysica Acta Protein Structure andMolecular Enzymology vol 827 no 1 pp 36ndash44 1985

[72] M A Schwarz J S Lazo J C Yalowich et al ldquoMetallothioneinprotects against the cytotoxic and DNA-damaging effects ofnitric oxiderdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 92 no 10 pp 4452ndash4456 1995

[73] Y Luo A Kawashima and Y Ishido ldquoIodine excess as anenvironmental risk factor for autoimmune thyroid diseaserdquoInternational Journal of Molecular Sciences vol 15 pp 12895ndash12912 2014

[74] H I Falrsquofushinsrsquoka L L Hnatyshyna andOI Osadchuk ldquoTraceelements storage peculiarities and metallothionein contentin human thyroid gland under iodine deficiency euthyroidnodular goiterrdquo Ukrainskii Biokhimicheskii Zhurnal vol 86 pp107ndash113 2014

[75] L Cai Y H Wang G H Zhou et al ldquoAttenuation bymetallothionein of early cardiac cell death via suppression ofmitochondrial oxidative stress results in a prevention of diabeticcardiomyopathyrdquo Journal of the American College of Cardiologyvol 48 no 8 pp 1688ndash1697 2006

[76] X Sun Z Zhou and Y J Kang ldquoAttenuation of doxorubicinchronic toxicity in metallothionein-overexpressing transgenicmouse heartrdquo Cancer Research vol 61 no 8 pp 3382ndash33872001

[77] A F Ceylan-Isik P Zhao B F Zhang X Xiao G Su and J RenldquoCardiac overexpression of metallothionein rescues cardiaccontractile dysfunction and endoplasmic reticulum stress butnot autophagy in sepsisrdquo Journal of Molecular and CellularCardiology vol 48 no 2 pp 367ndash378 2010

[78] N Hu R Guo X Han B Zhu and J Ren ldquoCardiac-specificoverexpression of metallothionein rescues nicotine-inducedcardiac contractile dysfunction and interstitial fibrosisrdquoToxicol-ogy Letters vol 202 no 1 pp 8ndash14 2011

[79] Y Zhang N Hu Y Hua K L Richmond F Dong and JRen ldquoCardiac overexpression of metallothionein rescues coldexposure-induced myocardial contractile dysfunction through

attenuation of cardiac fibrosis despite cardiomyocyte mechani-cal anomaliesrdquo Free Radical Biology and Medicine vol 53 no 2pp 194ndash207 2012

[80] N Hu X Han E K Lane F Gao Y Zhang and J RenldquoCardiac-specific overexpression of metallothionein rescuesagainst cigarette smoking exposure-induced myocardial con-tractile and mitochondrial damagerdquo PLoS ONE vol 8 no 2Article ID e57151 2013

[81] M T Brazao-Silva S V Cardoso P R de Faria et al ldquoAdenoidcystic carcinoma of the salivary gland a clinicopathologicalstudy of 49 cases and of metallothionein expression with regardto tumour behaviourrdquo Histopathology vol 63 no 6 pp 802ndash809 2013

[82] K J Schmitz H Lang G Kaiser et al ldquoMetallothioneinoverexpression and its prognostic relevance in intrahepaticcholangiocarcinoma and extrahepatic hilar cholangiocarci-noma (Klatskin tumors)rdquo Human Pathology vol 40 no 12 pp1706ndash1714 2009

[83] S E Theocharis A P Margeli J T Klijanienko and G PKouraklis ldquoMetallothionein expression in human neoplasiardquoHistopathology vol 45 no 2 pp 103ndash118 2004

[84] STheocharis J Klijanienko C Giaginis et al ldquoMetallothioneinexpression in mobile tongue squamous cell carcinoma associa-tions with clinicopathological parameters and patient survivalrdquoHistopathology vol 59 no 3 pp 514ndash525 2011

[85] S-K Tai O J-K Tan V T-K Chow et al ldquoDifferentialexpression of metallothionein 1 and 2 isoforms in breast cancerlines with different invasive potential identification of a novelnon-silent metallothionein-1H mutant variantrdquo The AmericanJournal of Pathology vol 163 no 5 pp 2009ndash2019 2003

[86] Y Hishikawa T Koji D K Dhar S Kinugasa M Yamaguchiand N Nagasue ldquoMetallothionein expression correlates withmetastatic and proliferative potential in squamous cell carci-noma of the oesophagusrdquo British Journal of Cancer vol 81 no4 pp 712ndash720 1999

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

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Disease Markers

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BioMed Research International

OncologyJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

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PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

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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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Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 8: Research Article Metallothionein-I/II Knockout Mice

8 Oxidative Medicine and Cellular Longevity

organs and tumors [67ndash69] MTs play a protective role inpreventing against intoxication with heavy metals such asPb Hg Cu and Cd [67 70] All 20 cysteine sulfur atomsare involved in the radical quenching process and the rateconstant for the reaction of hydroxyl radical with MT isabout 340-fold higher than that with GSH [71 72] There arefour MT isoforms in mammals MT-I and MT-II are mainlyinvolved in the protection of tissue against metal toxicitiesoxidative stresses and apoptosis It has been demonstratedthat the rabbit liver metallothionein-1 which contains zincandor cadmium ions appeared to scavenge free hydroxyland superoxide radicals [25 26] Under low levels of iodideorganification in goitrous-modified tissue of thyroid glandmetallothionein may provide a partial compensatory effecton prooxidative processes [73 74] Although no cell lineor animal model overexpressing MT in the thyroid hasbeen found yet documented evidence of MT in protectingagainst oxidative stress in cardiac-specific metallothionein-overexpressed transgenic mice has been demonstrated Caiet al reported that metallothionein as a potent antioxi-dant prevented the development of diabetic cardiomyopathythrough the inhibition of the mitochondrial cytochromec-mediated caspase-3 activation pathway [75] Sun et alreported that through doxorubicin induced chronic toxi-city in a metallothionein-overexpressed transgenic mouseheart the antioxidant action of MT is highly responsiblefor this cardioprotection [76] Ceylan-Isik et al reportedthat the cardiac-specific overexpression of MT rescues LPS-induced cardiac contractile and intracellular Ca2+ dysfunc-tions through the alleviation of ROSoxidative stress stresssignaling activation and endoplasmic reticulum (ER) stress[77] They also reported that the cardiac-specific overexpres-sion of metallothionein protects against nicotine exposure-induced cardiac contractile dysfunction and fibrosis possiblythrough inhibition of ROS accumulation and apoptosis [78]Zhang et al suggest that cardiac overexpression of metalloth-ionein prevents cold exposure-induced cardiac anomaliespossibly through the attenuation of myocardial fibrosis [79]Hu et al demonstrated that cardiac-specific overexpression ofmetallothionein protects against cigarette smoking exposure-induced myocardial contractile and mitochondrial damagefavoring a role of lessened apoptosis in a metallothionein-induced beneficial effect against side-stream smoke exposure[80] Besides metallothionein has been shown to regulateapoptosis and proliferation Overexpression of metalloth-ionein frequently occurs in human tumors and is related totumor progression [81 82] High expression of MT has beenobserved in different types of cancer and has been consideredas a potential prognostic marker in cancers of the breastpancreas skin and cervix [82ndash86] Further studies will beattempted to study the oxidative and antioxidative stress onan engineered cell line overexpressing metallothionein

5 Conclusion

Thepresent study provides evidence that high concentrationsof iodide may lead to a significant increase in mitochondrialsuperoxide production and Prx 3 expression in both invitro and in vivo studies MT-III KO mice aggravated

mitochondrial superoxide production and peroxiredoxin 3expression in the thyroid after excessive iodide exposureMT-III may potentially protect the thyroid against highconcentrations of iodide induced oxidative stress

Abbreviations

MT-III KO Metallothionein III knockoutMTs MetallothioneinsWT Wild-typeROS Reactive oxygen speciesPrx PeroxiredoxinKI Potassium iodideNI Normal iodide intakeHI High iodide intakeLDH Lactate dehydrogenaseH2O2 Hydrogen peroxide

SOD Superoxide dismutaseMTT 3-(45-Dimethylthiazol-2-yl)-25-

diphenyltetrazoliumbromide

K-S Kolmogorov-SmirnovLSD Least significant differenceT4 Thyroxine

T3 Tri-iodothyronine

FT4 Free thyroxine

FT3 Free tri-iodothyronine

Conflict of Interests

The authors declare that they have no competing interests

Acknowledgments

Thiswork is supported by theNational Natural Science Foun-dation of China (no 81273009) the Tianjin Science amp Tech-nology Council Grant of China (no 09JCYBJC11700) andthe Tianjin Educational amp Scientific Grant (no 20050107)This paper has been edited by one of the authors MohamedAhmed who is from USA and now is studying in TianjinMedical University China

References

[1] B Corvilain L Collyn J van Sande and J E DumontldquoStimulation by iodide of H

2O2generation in thyroid slices

from several speciesrdquo The American Journal of PhysiologymdashEndocrinology and Metabolism vol 278 no 4 pp E692ndashE6992000

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

[3] E Roti and E Degli Uberti ldquoIodine excess and hyperthy-roidismrdquoThyroid vol 11 no 5 pp 493ndash500 2001

[4] World Health Organization and United Nations ChildrenrsquosFund ldquoInternational Council for Control of Iodine DeficiencyDisorders 1999 progress towards the elimination of iodinedeficiency disorders (IDD)rdquo WHO Booklet World HealthOrganization 1999

Oxidative Medicine and Cellular Longevity 9

[5] A M Leung and L E Braverman ldquoConsequences of excessiodinerdquo Nature Reviews Endocrinology vol 10 no 3 pp 136ndash142 2013

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

[7] IMahmoud I ColinM CMany and J-F Denef ldquoDirect toxiceffect of iodide in excess on iodine-deficient thyroid glandsepithelial necrosis and inflammation associated with lipofuscinaccumulationrdquo Experimental and Molecular Pathology vol 44no 3 pp 259ndash271 1986

[8] J E Fradkin and J Wolff ldquoIodide-induced thyrotoxicosisrdquoMedicine vol 62 no 1 pp 1ndash20 1983

[9] J Golstein and J E Dumont ldquoCytotoxic effects of iodide onthyroid cells difference between rat thyroid FRTL-5 cell andpri-mary dog thyrocyte responsivenessrdquo Journal of EndocrinologicalInvestigation vol 19 no 2 pp 119ndash126 1996

[10] 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

[11] W Teng Z Shan X Teng et al ldquoEffect of iodine intakeon thyroid diseases in Chinardquo The New England Journal ofMedicine vol 354 no 26 pp 2783ndash2793 2006

[12] P Laurberg K M Pedersen A Hreidarsson N Sigfusson EIversen and P R Knudsen ldquoIodine intake and the patternof thyroid disorders a comparative epidemiological study ofthyroid abnormalities in the elderly in iceland and in JutlandDenmarkrdquo Journal of Clinical Endocrinology and Metabolismvol 83 no 3 pp 765ndash769 1998

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

[14] 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

[15] 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

[16] H Han P Xin L Zhao et al ldquoExcess iodine and high-fatdiet combination modulates lipid profile thyroid hormoneand hepatic LDLr expression values in micerdquo Biological TraceElement Research vol 147 no 1ndash3 pp 233ndash239 2012

[17] S-J Zhao Y Ye F-J Sun E-J Tian and Z-P Chen ldquoTheimpact of dietary iodine intake on lipid metabolism in micerdquoBiological Trace Element Research vol 142 no 3 pp 581ndash5882011

[18] 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

[19] V Pitsiavas P Smerdely M Li and S C Boyages ldquoAmiodaroneinduces a different pattern of ultrastructural change in thethyroid to iodine excess alone in both the BBW rat and theWistar ratrdquo European Journal of Endocrinology vol 137 no 1pp 89ndash98 1997

[20] M B Zimmermann ldquoIodine deficiency and excess in childrenworldwide status in 2013rdquo Endocrine Practice vol 19 no 5 pp839ndash846 2013

[21] K Mussig ldquoIodine-induced toxic effects due to seaweed con-sumptionrdquo in Comprehensive Handbook of Iodine V R PreedyG N Burrow and R R Watson Eds pp 897ndash908 ElsevierAmsterdam The Netherlands 2009

[22] A-M G Psarra S Solakidi and C E Sekeris ldquoThe mito-chondrion as a primary site of action of steroid and thyroidhormones presence and action of steroid and thyroid hormonereceptors in mitochondria of animal cellsrdquo Molecular andCellular Endocrinology vol 246 no 1-2 pp 21ndash33 2006

[23] Y Song N Driessens M Costa et al ldquoRoles of hydrogenperoxide in thyroid physiology and diseaserdquo Journal of ClinicalEndocrinology and Metabolism vol 92 no 10 pp 3764ndash37732007

[24] A G Cox C C Winterbourn and M B Hampton ldquoMito-chondrial peroxiredoxin involvement in antioxidant defenceand redox signallingrdquo Biochemical Journal vol 425 no 2 pp313ndash325 2010

[25] B Ruttkay-Nedecky L Nejdl J Gumulec et al ldquoThe role ofmetallothionein in oxidative stressrdquo International Journal ofMolecular Sciences vol 14 no 3 pp 6044ndash6066 2013

[26] N Thirumoorthy A Shyam Sunder K T Manisenthil KumarM Senthil kumarGNKGanesh andMChatterjee ldquoA reviewof metallothionein isoforms and their role in pathophysiologyrdquoWorld Journal of Surgical Oncology vol 9 article 54 2011

[27] 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

[28] M Saberi and R D Utiger ldquoAugmentation of thyrotropinresponses to thyrotropin releasing hormone following smalldecreases in serum thyroid hormone concentrationsrdquo TheJournal of Clinical Endocrinology and Metabolism vol 40 no3 pp 435ndash441 1975

[29] H Namba S Yamashita H Kimura et al ldquoEvidence of thyroidvolume increase in normal subjects receiving excess iodiderdquoTheJournal of Clinical Endocrinology and Metabolism vol 76 no 3pp 605ndash608 1993

[30] E Sternthal L Lipworth B Stanley C Abreau S L Fang andL E Braverman ldquoSuppression of thyroid radioiodine uptakeby various doses of stable iodiderdquo The New England Journal ofMedicine vol 303 no 19 pp 1083ndash1088 1980

[31] A G Vagenakis B Rapoport F Azizi G I Portnay L EBraverman and S H Ingbar ldquoHyperresponse to thyrotropinreleasing hormone accompanying small decreases in serumthyroid hormone concentrationsrdquoThe Journal of Clinical Inves-tigation vol 54 no 4 pp 913ndash918 1974

[32] G Philippou D A Koutras G Piperingos A Souvatzoglouand S D Moulopoulos ldquoThe effect of iodide on serum thyroidhormone levels in normal persons in hyperthyroid patientsand in hypothyroid patients on thyroxine replacementrdquoClinicalEndocrinology vol 36 no 6 pp 573ndash578 1992

[33] L Z Ali Salim R Othman M A Abdulla et al ldquoThymo-quinone inhibits murine leukemia WEHI-3 cells in vivo and invitrordquo PLoS ONE vol 9 no 12 Article ID e115340 2014

[34] X Wang J Ge K Wang J Qian and Y Zou ldquoEvaluation ofMTT assay for measurement of emodin-induced cytotoxicityrdquoASSAY and Drug Development Technologies vol 4 no 2 pp203ndash207 2006

10 Oxidative Medicine and Cellular Longevity

[35] C Lv C Wu Y-h Zhou Y Shao G Wang and Q-y WangldquoAlpha lipoic acid Modulated high glucose-induced rat mesan-gial cell dysfunction viamTORp70S6K4E-BP1 pathwayrdquo Inter-national Journal of Endocrinology vol 2014 Article ID 65858914 pages 2014

[36] L Liu X Hu G-Y Cai et al ldquoHigh glucose-induced hyper-trophy of mesangial cells is reversed by connexin43 overex-pression via PTENAktmTOR signalingrdquo Nephrology DialysisTransplantation vol 27 no 1 pp 90ndash100 2012

[37] D D Harrison-Findik S Z Lu E M Zmijewski J Jonesand M C Zimmerman ldquoEffect of alcohol exposure on hepaticsuperoxide generation and hepcidin expressionrdquoWorld Journalof Biological Chemistry vol 4 no 4 pp 119ndash130 2013

[38] M Wang Y Wang J He et al ldquoAlbumin induces neuroprotec-tion against ischemic stroke by altering Toll-like receptor 4 andregulatory T cells in micerdquo CNS and Neurological DisordersmdashDrug Targets vol 12 no 2 pp 220ndash227 2013

[39] X Yao ldquoEffect of zinc exposure on HNE and GLT-1 in spinalcord culturerdquo NeuroToxicology vol 30 no 1 pp 121ndash126 2009

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

[41] G P Becks M C Eggo and G N Burrow ldquoRegulation ofdifferentiated thyroid function by iodide preferential inhibitoryeffect of excess iodide on thyroid hormone secretion in sheepthyroid cell culturesrdquo Endocrinology vol 120 no 6 pp 2569ndash2575 1987

[42] M Vitale T Di Matola F DrsquoAscoli et al ldquoIodide excessinduces apoptosis in thyroid cells through a p53-independentmechanism involving oxidative stressrdquo Endocrinology vol 141no 2 pp 598ndash605 2000

[43] P Pramyothin AM Leung E N Pearce A OMalabanan andL E Braverman ldquoClinical problem-solving A hidden solutionrdquoTheNew England Journal of Medicine vol 365 no 22 pp 2123ndash2127 2011

[44] W Jubiz S Carlile and L D Lagerquist ldquoSerum thyrotropinand thyroid hormone levels in humans receiving chronicpotassium iodiderdquo The Journal of Clinical Endocrinology andMetabolism vol 44 no 2 pp 379ndash382 1977

[45] A G Vagenakis P Downs L E Braverman A Burger andS H Ingbar ldquoControl of thyroid hormone secretion in normalsubjects receiving iodidesrdquoThe Journal of Clinical Investigationvol 52 no 2 pp 528ndash532 1973

[46] M Safran andL E Braverman ldquoEffect of chronic douchingwithpolyvinylpyrrolidone-iodine on iodine absorption and thyroidfunctionrdquo Obstetrics and Gynecology vol 60 no 1 pp 35ndash401982

[47] K Newick B Cunniff K Preston et al ldquoPeroxiredoxin 3 is aredox-dependent target of thiostrepton in malignant mesothe-lioma cellsrdquo PLoS ONE vol 7 no 6 Article ID e39404 2012

[48] A Deniaud O S El Dein E Maillier et al ldquoEndoplasmicreticulum stress induces calcium-dependent permeability tran-sition mitochondrial outer membrane permeabilization andapoptosisrdquo Oncogene vol 27 no 3 pp 285ndash299 2008

[49] A Gorlach P Klappa and T Kietzmann ldquoThe endoplasmicreticulum folding calcium homeostasis signaling and redoxcontrolrdquo Antioxidants and Redox Signaling vol 8 no 9-10 pp1391ndash1418 2006

[50] 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 symporter

expression and activity involves the PI3KAkt signaling path-wayrdquo Endocrinology vol 155 no 3 pp 1145ndash1156 2014

[51] Y-G Wang L Li C-H Liu S Hong and M-J ZhangldquoPeroxiredoxin 3 is resistant to oxidation-induced apoptosis ofHep-3b cellsrdquoClinical and Translational Oncology vol 16 no 6pp 561ndash566 2014

[52] H C Whitaker D Patel W J Howat et al ldquoPeroxiredoxin-3is overexpressed in prostate cancer and promotes cancer cellsurvival by protecting cells fromoxidative stressrdquoBritish Journalof Cancer vol 109 no 4 pp 983ndash993 2013

[53] L Li Y G Zhang and C L Chen ldquoAnti-apoptotic role ofperoxiredoxin III in cervical cancer cellsrdquo FEBS Open Bio vol3 pp 51ndash54 2013

[54] 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

[55] L I Nadolnik Z V Niatsetskaya and S V Lupachyk ldquoEffectof oxidative stress on rat thyrocyte iodide metabolismrdquo CellBiochemistry and Function vol 26 no 3 pp 366ndash373 2008

[56] A C Freitas Ferreira L de Carvalho Cardoso D Rosenthaland D Pires de Carvalho ldquoThyroid Ca2+NADPH-dependentH2O2generation is partially inhibited by propylthiouracil and

methimazolerdquo European Journal of Biochemistry vol 270 no 11pp 2363ndash2368 2003

[57] G R Sue Z C Ho and K Kim ldquoPeroxiredoxins a historicaloverview and speculative preview of novel mechanisms andemerging concepts in cell signalingrdquo Free Radical Biology andMedicine vol 38 no 12 pp 1543ndash1552 2005

[58] M S Seo S W Kang K Kim I C Baines T H Lee and SG Rhee ldquoIdentification of a new type of mammalian perox-iredoxin that forms an intramolecular disulfide as a reactionintermediaterdquo Journal of Biological Chemistry vol 275 no 27pp 20346ndash20354 2000

[59] Z A Wood L B Poole and P A Karplus ldquoPeroxiredoxinevolution and the regulation of hydrogen peroxide signalingrdquoScience vol 300 no 5619 pp 650ndash653 2003

[60] D A Drechsel and M Patel ldquoRespiration-dependent H2O2

removal in brain mitochondria via the thioredoxinperoxire-doxin systemrdquoThe Journal of Biological Chemistry vol 285 no36 pp 27850ndash27858 2010

[61] W H Watson X Yang Y E Choi D P Jones and J P KehrerldquoThioredoxin and its role in toxicologyrdquo Toxicological Sciencesvol 78 no 1 pp 3ndash14 2004

[62] G Ekstrom and M Ingelman-Sundberg ldquoRat liver microso-mal NADPH-supported oxidase activity and lipid peroxida-tion dependent on ethanol-inducible cytochrome P-450 (P-450IIE1)rdquo Biochemical Pharmacology vol 38 no 8 pp 1313ndash1319 1989

[63] J Rashba-Step N J Turro and A I Cederbaum ldquoIncreasedNADPH- and NADH-dependent production of superoxideand hydroxyl radical by microsomes after chronic ethanoltreatmentrdquoArchives of Biochemistry and Biophysics vol 300 no1 pp 401ndash408 1993

[64] V Thakur M T Pritchard M R McMullen Q Wang andL E Nagy ldquoChronic ethanol feeding increases activation ofNADPH oxidase by lipopolysaccharide in rat Kupffer cellsrole of increased reactive oxygen in LPS-stimulated ERK12activation andTNF-120572 productionrdquo Journal of Leukocyte Biologyvol 79 no 6 pp 1348ndash1356 2006

Oxidative Medicine and Cellular Longevity 11

[65] HNakamuraAMitsui and J Yodoi ldquoThioredoxin overexpres-sion in transgenic micerdquo Methods in Enzymology vol 347 pp436ndash440 2002

[66] 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

[67] A Bieniek B Pula A Piotrowska et al ldquoExpression of metal-lothionein III andKi-67 antigen in various histological types ofbasal cell carcinomardquo Folia Histochemica et Cytobiologica vol50 no 3 pp 352ndash357 2012

[68] P Dziegiel ldquoExpression of metallothioneins in tumor cellsrdquoPolish Journal of Pathology vol 55 no 1 pp 3ndash12 2004

[69] M Oslash Pedersen A Larsen M Stoltenberg and M PenkowaldquoThe role of metallothionein in oncogenesis and cancer prog-nosisrdquo Progress inHistochemistry and Cytochemistry vol 44 no1 pp 29ndash64 2009

[70] W Braun M Vasak A H Robbins et al ldquoComparison ofthe NMR solution structure and the X-ray crystal structure ofrat metallothionein-2rdquo Proceedings of the National Academy ofSciences of the United States of America vol 89 no 21 pp 10124ndash10128 1992

[71] P JThornalley andM Vasak ldquoPossible role formetallothioneinin protection against radiation-induced oxidative stress Kinet-ics andmechanism of its reaction with superoxide and hydroxylradicalsrdquo Biochimica et Biophysica Acta Protein Structure andMolecular Enzymology vol 827 no 1 pp 36ndash44 1985

[72] M A Schwarz J S Lazo J C Yalowich et al ldquoMetallothioneinprotects against the cytotoxic and DNA-damaging effects ofnitric oxiderdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 92 no 10 pp 4452ndash4456 1995

[73] Y Luo A Kawashima and Y Ishido ldquoIodine excess as anenvironmental risk factor for autoimmune thyroid diseaserdquoInternational Journal of Molecular Sciences vol 15 pp 12895ndash12912 2014

[74] H I Falrsquofushinsrsquoka L L Hnatyshyna andOI Osadchuk ldquoTraceelements storage peculiarities and metallothionein contentin human thyroid gland under iodine deficiency euthyroidnodular goiterrdquo Ukrainskii Biokhimicheskii Zhurnal vol 86 pp107ndash113 2014

[75] L Cai Y H Wang G H Zhou et al ldquoAttenuation bymetallothionein of early cardiac cell death via suppression ofmitochondrial oxidative stress results in a prevention of diabeticcardiomyopathyrdquo Journal of the American College of Cardiologyvol 48 no 8 pp 1688ndash1697 2006

[76] X Sun Z Zhou and Y J Kang ldquoAttenuation of doxorubicinchronic toxicity in metallothionein-overexpressing transgenicmouse heartrdquo Cancer Research vol 61 no 8 pp 3382ndash33872001

[77] A F Ceylan-Isik P Zhao B F Zhang X Xiao G Su and J RenldquoCardiac overexpression of metallothionein rescues cardiaccontractile dysfunction and endoplasmic reticulum stress butnot autophagy in sepsisrdquo Journal of Molecular and CellularCardiology vol 48 no 2 pp 367ndash378 2010

[78] N Hu R Guo X Han B Zhu and J Ren ldquoCardiac-specificoverexpression of metallothionein rescues nicotine-inducedcardiac contractile dysfunction and interstitial fibrosisrdquoToxicol-ogy Letters vol 202 no 1 pp 8ndash14 2011

[79] Y Zhang N Hu Y Hua K L Richmond F Dong and JRen ldquoCardiac overexpression of metallothionein rescues coldexposure-induced myocardial contractile dysfunction through

attenuation of cardiac fibrosis despite cardiomyocyte mechani-cal anomaliesrdquo Free Radical Biology and Medicine vol 53 no 2pp 194ndash207 2012

[80] N Hu X Han E K Lane F Gao Y Zhang and J RenldquoCardiac-specific overexpression of metallothionein rescuesagainst cigarette smoking exposure-induced myocardial con-tractile and mitochondrial damagerdquo PLoS ONE vol 8 no 2Article ID e57151 2013

[81] M T Brazao-Silva S V Cardoso P R de Faria et al ldquoAdenoidcystic carcinoma of the salivary gland a clinicopathologicalstudy of 49 cases and of metallothionein expression with regardto tumour behaviourrdquo Histopathology vol 63 no 6 pp 802ndash809 2013

[82] K J Schmitz H Lang G Kaiser et al ldquoMetallothioneinoverexpression and its prognostic relevance in intrahepaticcholangiocarcinoma and extrahepatic hilar cholangiocarci-noma (Klatskin tumors)rdquo Human Pathology vol 40 no 12 pp1706ndash1714 2009

[83] S E Theocharis A P Margeli J T Klijanienko and G PKouraklis ldquoMetallothionein expression in human neoplasiardquoHistopathology vol 45 no 2 pp 103ndash118 2004

[84] STheocharis J Klijanienko C Giaginis et al ldquoMetallothioneinexpression in mobile tongue squamous cell carcinoma associa-tions with clinicopathological parameters and patient survivalrdquoHistopathology vol 59 no 3 pp 514ndash525 2011

[85] S-K Tai O J-K Tan V T-K Chow et al ldquoDifferentialexpression of metallothionein 1 and 2 isoforms in breast cancerlines with different invasive potential identification of a novelnon-silent metallothionein-1H mutant variantrdquo The AmericanJournal of Pathology vol 163 no 5 pp 2009ndash2019 2003

[86] Y Hishikawa T Koji D K Dhar S Kinugasa M Yamaguchiand N Nagasue ldquoMetallothionein expression correlates withmetastatic and proliferative potential in squamous cell carci-noma of the oesophagusrdquo British Journal of Cancer vol 81 no4 pp 712ndash720 1999

Submit your manuscripts athttpwwwhindawicom

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

Page 9: Research Article Metallothionein-I/II Knockout Mice

Oxidative Medicine and Cellular Longevity 9

[5] A M Leung and L E Braverman ldquoConsequences of excessiodinerdquo Nature Reviews Endocrinology vol 10 no 3 pp 136ndash142 2013

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

[7] IMahmoud I ColinM CMany and J-F Denef ldquoDirect toxiceffect of iodide in excess on iodine-deficient thyroid glandsepithelial necrosis and inflammation associated with lipofuscinaccumulationrdquo Experimental and Molecular Pathology vol 44no 3 pp 259ndash271 1986

[8] J E Fradkin and J Wolff ldquoIodide-induced thyrotoxicosisrdquoMedicine vol 62 no 1 pp 1ndash20 1983

[9] J Golstein and J E Dumont ldquoCytotoxic effects of iodide onthyroid cells difference between rat thyroid FRTL-5 cell andpri-mary dog thyrocyte responsivenessrdquo Journal of EndocrinologicalInvestigation vol 19 no 2 pp 119ndash126 1996

[10] 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

[11] W Teng Z Shan X Teng et al ldquoEffect of iodine intakeon thyroid diseases in Chinardquo The New England Journal ofMedicine vol 354 no 26 pp 2783ndash2793 2006

[12] P Laurberg K M Pedersen A Hreidarsson N Sigfusson EIversen and P R Knudsen ldquoIodine intake and the patternof thyroid disorders a comparative epidemiological study ofthyroid abnormalities in the elderly in iceland and in JutlandDenmarkrdquo Journal of Clinical Endocrinology and Metabolismvol 83 no 3 pp 765ndash769 1998

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

[14] 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

[15] 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

[16] H Han P Xin L Zhao et al ldquoExcess iodine and high-fatdiet combination modulates lipid profile thyroid hormoneand hepatic LDLr expression values in micerdquo Biological TraceElement Research vol 147 no 1ndash3 pp 233ndash239 2012

[17] S-J Zhao Y Ye F-J Sun E-J Tian and Z-P Chen ldquoTheimpact of dietary iodine intake on lipid metabolism in micerdquoBiological Trace Element Research vol 142 no 3 pp 581ndash5882011

[18] 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

[19] V Pitsiavas P Smerdely M Li and S C Boyages ldquoAmiodaroneinduces a different pattern of ultrastructural change in thethyroid to iodine excess alone in both the BBW rat and theWistar ratrdquo European Journal of Endocrinology vol 137 no 1pp 89ndash98 1997

[20] M B Zimmermann ldquoIodine deficiency and excess in childrenworldwide status in 2013rdquo Endocrine Practice vol 19 no 5 pp839ndash846 2013

[21] K Mussig ldquoIodine-induced toxic effects due to seaweed con-sumptionrdquo in Comprehensive Handbook of Iodine V R PreedyG N Burrow and R R Watson Eds pp 897ndash908 ElsevierAmsterdam The Netherlands 2009

[22] A-M G Psarra S Solakidi and C E Sekeris ldquoThe mito-chondrion as a primary site of action of steroid and thyroidhormones presence and action of steroid and thyroid hormonereceptors in mitochondria of animal cellsrdquo Molecular andCellular Endocrinology vol 246 no 1-2 pp 21ndash33 2006

[23] Y Song N Driessens M Costa et al ldquoRoles of hydrogenperoxide in thyroid physiology and diseaserdquo Journal of ClinicalEndocrinology and Metabolism vol 92 no 10 pp 3764ndash37732007

[24] A G Cox C C Winterbourn and M B Hampton ldquoMito-chondrial peroxiredoxin involvement in antioxidant defenceand redox signallingrdquo Biochemical Journal vol 425 no 2 pp313ndash325 2010

[25] B Ruttkay-Nedecky L Nejdl J Gumulec et al ldquoThe role ofmetallothionein in oxidative stressrdquo International Journal ofMolecular Sciences vol 14 no 3 pp 6044ndash6066 2013

[26] N Thirumoorthy A Shyam Sunder K T Manisenthil KumarM Senthil kumarGNKGanesh andMChatterjee ldquoA reviewof metallothionein isoforms and their role in pathophysiologyrdquoWorld Journal of Surgical Oncology vol 9 article 54 2011

[27] 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

[28] M Saberi and R D Utiger ldquoAugmentation of thyrotropinresponses to thyrotropin releasing hormone following smalldecreases in serum thyroid hormone concentrationsrdquo TheJournal of Clinical Endocrinology and Metabolism vol 40 no3 pp 435ndash441 1975

[29] H Namba S Yamashita H Kimura et al ldquoEvidence of thyroidvolume increase in normal subjects receiving excess iodiderdquoTheJournal of Clinical Endocrinology and Metabolism vol 76 no 3pp 605ndash608 1993

[30] E Sternthal L Lipworth B Stanley C Abreau S L Fang andL E Braverman ldquoSuppression of thyroid radioiodine uptakeby various doses of stable iodiderdquo The New England Journal ofMedicine vol 303 no 19 pp 1083ndash1088 1980

[31] A G Vagenakis B Rapoport F Azizi G I Portnay L EBraverman and S H Ingbar ldquoHyperresponse to thyrotropinreleasing hormone accompanying small decreases in serumthyroid hormone concentrationsrdquoThe Journal of Clinical Inves-tigation vol 54 no 4 pp 913ndash918 1974

[32] G Philippou D A Koutras G Piperingos A Souvatzoglouand S D Moulopoulos ldquoThe effect of iodide on serum thyroidhormone levels in normal persons in hyperthyroid patientsand in hypothyroid patients on thyroxine replacementrdquoClinicalEndocrinology vol 36 no 6 pp 573ndash578 1992

[33] L Z Ali Salim R Othman M A Abdulla et al ldquoThymo-quinone inhibits murine leukemia WEHI-3 cells in vivo and invitrordquo PLoS ONE vol 9 no 12 Article ID e115340 2014

[34] X Wang J Ge K Wang J Qian and Y Zou ldquoEvaluation ofMTT assay for measurement of emodin-induced cytotoxicityrdquoASSAY and Drug Development Technologies vol 4 no 2 pp203ndash207 2006

10 Oxidative Medicine and Cellular Longevity

[35] C Lv C Wu Y-h Zhou Y Shao G Wang and Q-y WangldquoAlpha lipoic acid Modulated high glucose-induced rat mesan-gial cell dysfunction viamTORp70S6K4E-BP1 pathwayrdquo Inter-national Journal of Endocrinology vol 2014 Article ID 65858914 pages 2014

[36] L Liu X Hu G-Y Cai et al ldquoHigh glucose-induced hyper-trophy of mesangial cells is reversed by connexin43 overex-pression via PTENAktmTOR signalingrdquo Nephrology DialysisTransplantation vol 27 no 1 pp 90ndash100 2012

[37] D D Harrison-Findik S Z Lu E M Zmijewski J Jonesand M C Zimmerman ldquoEffect of alcohol exposure on hepaticsuperoxide generation and hepcidin expressionrdquoWorld Journalof Biological Chemistry vol 4 no 4 pp 119ndash130 2013

[38] M Wang Y Wang J He et al ldquoAlbumin induces neuroprotec-tion against ischemic stroke by altering Toll-like receptor 4 andregulatory T cells in micerdquo CNS and Neurological DisordersmdashDrug Targets vol 12 no 2 pp 220ndash227 2013

[39] X Yao ldquoEffect of zinc exposure on HNE and GLT-1 in spinalcord culturerdquo NeuroToxicology vol 30 no 1 pp 121ndash126 2009

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

[41] G P Becks M C Eggo and G N Burrow ldquoRegulation ofdifferentiated thyroid function by iodide preferential inhibitoryeffect of excess iodide on thyroid hormone secretion in sheepthyroid cell culturesrdquo Endocrinology vol 120 no 6 pp 2569ndash2575 1987

[42] M Vitale T Di Matola F DrsquoAscoli et al ldquoIodide excessinduces apoptosis in thyroid cells through a p53-independentmechanism involving oxidative stressrdquo Endocrinology vol 141no 2 pp 598ndash605 2000

[43] P Pramyothin AM Leung E N Pearce A OMalabanan andL E Braverman ldquoClinical problem-solving A hidden solutionrdquoTheNew England Journal of Medicine vol 365 no 22 pp 2123ndash2127 2011

[44] W Jubiz S Carlile and L D Lagerquist ldquoSerum thyrotropinand thyroid hormone levels in humans receiving chronicpotassium iodiderdquo The Journal of Clinical Endocrinology andMetabolism vol 44 no 2 pp 379ndash382 1977

[45] A G Vagenakis P Downs L E Braverman A Burger andS H Ingbar ldquoControl of thyroid hormone secretion in normalsubjects receiving iodidesrdquoThe Journal of Clinical Investigationvol 52 no 2 pp 528ndash532 1973

[46] M Safran andL E Braverman ldquoEffect of chronic douchingwithpolyvinylpyrrolidone-iodine on iodine absorption and thyroidfunctionrdquo Obstetrics and Gynecology vol 60 no 1 pp 35ndash401982

[47] K Newick B Cunniff K Preston et al ldquoPeroxiredoxin 3 is aredox-dependent target of thiostrepton in malignant mesothe-lioma cellsrdquo PLoS ONE vol 7 no 6 Article ID e39404 2012

[48] A Deniaud O S El Dein E Maillier et al ldquoEndoplasmicreticulum stress induces calcium-dependent permeability tran-sition mitochondrial outer membrane permeabilization andapoptosisrdquo Oncogene vol 27 no 3 pp 285ndash299 2008

[49] A Gorlach P Klappa and T Kietzmann ldquoThe endoplasmicreticulum folding calcium homeostasis signaling and redoxcontrolrdquo Antioxidants and Redox Signaling vol 8 no 9-10 pp1391ndash1418 2006

[50] 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 symporter

expression and activity involves the PI3KAkt signaling path-wayrdquo Endocrinology vol 155 no 3 pp 1145ndash1156 2014

[51] Y-G Wang L Li C-H Liu S Hong and M-J ZhangldquoPeroxiredoxin 3 is resistant to oxidation-induced apoptosis ofHep-3b cellsrdquoClinical and Translational Oncology vol 16 no 6pp 561ndash566 2014

[52] H C Whitaker D Patel W J Howat et al ldquoPeroxiredoxin-3is overexpressed in prostate cancer and promotes cancer cellsurvival by protecting cells fromoxidative stressrdquoBritish Journalof Cancer vol 109 no 4 pp 983ndash993 2013

[53] L Li Y G Zhang and C L Chen ldquoAnti-apoptotic role ofperoxiredoxin III in cervical cancer cellsrdquo FEBS Open Bio vol3 pp 51ndash54 2013

[54] 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

[55] L I Nadolnik Z V Niatsetskaya and S V Lupachyk ldquoEffectof oxidative stress on rat thyrocyte iodide metabolismrdquo CellBiochemistry and Function vol 26 no 3 pp 366ndash373 2008

[56] A C Freitas Ferreira L de Carvalho Cardoso D Rosenthaland D Pires de Carvalho ldquoThyroid Ca2+NADPH-dependentH2O2generation is partially inhibited by propylthiouracil and

methimazolerdquo European Journal of Biochemistry vol 270 no 11pp 2363ndash2368 2003

[57] G R Sue Z C Ho and K Kim ldquoPeroxiredoxins a historicaloverview and speculative preview of novel mechanisms andemerging concepts in cell signalingrdquo Free Radical Biology andMedicine vol 38 no 12 pp 1543ndash1552 2005

[58] M S Seo S W Kang K Kim I C Baines T H Lee and SG Rhee ldquoIdentification of a new type of mammalian perox-iredoxin that forms an intramolecular disulfide as a reactionintermediaterdquo Journal of Biological Chemistry vol 275 no 27pp 20346ndash20354 2000

[59] Z A Wood L B Poole and P A Karplus ldquoPeroxiredoxinevolution and the regulation of hydrogen peroxide signalingrdquoScience vol 300 no 5619 pp 650ndash653 2003

[60] D A Drechsel and M Patel ldquoRespiration-dependent H2O2

removal in brain mitochondria via the thioredoxinperoxire-doxin systemrdquoThe Journal of Biological Chemistry vol 285 no36 pp 27850ndash27858 2010

[61] W H Watson X Yang Y E Choi D P Jones and J P KehrerldquoThioredoxin and its role in toxicologyrdquo Toxicological Sciencesvol 78 no 1 pp 3ndash14 2004

[62] G Ekstrom and M Ingelman-Sundberg ldquoRat liver microso-mal NADPH-supported oxidase activity and lipid peroxida-tion dependent on ethanol-inducible cytochrome P-450 (P-450IIE1)rdquo Biochemical Pharmacology vol 38 no 8 pp 1313ndash1319 1989

[63] J Rashba-Step N J Turro and A I Cederbaum ldquoIncreasedNADPH- and NADH-dependent production of superoxideand hydroxyl radical by microsomes after chronic ethanoltreatmentrdquoArchives of Biochemistry and Biophysics vol 300 no1 pp 401ndash408 1993

[64] V Thakur M T Pritchard M R McMullen Q Wang andL E Nagy ldquoChronic ethanol feeding increases activation ofNADPH oxidase by lipopolysaccharide in rat Kupffer cellsrole of increased reactive oxygen in LPS-stimulated ERK12activation andTNF-120572 productionrdquo Journal of Leukocyte Biologyvol 79 no 6 pp 1348ndash1356 2006

Oxidative Medicine and Cellular Longevity 11

[65] HNakamuraAMitsui and J Yodoi ldquoThioredoxin overexpres-sion in transgenic micerdquo Methods in Enzymology vol 347 pp436ndash440 2002

[66] 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

[67] A Bieniek B Pula A Piotrowska et al ldquoExpression of metal-lothionein III andKi-67 antigen in various histological types ofbasal cell carcinomardquo Folia Histochemica et Cytobiologica vol50 no 3 pp 352ndash357 2012

[68] P Dziegiel ldquoExpression of metallothioneins in tumor cellsrdquoPolish Journal of Pathology vol 55 no 1 pp 3ndash12 2004

[69] M Oslash Pedersen A Larsen M Stoltenberg and M PenkowaldquoThe role of metallothionein in oncogenesis and cancer prog-nosisrdquo Progress inHistochemistry and Cytochemistry vol 44 no1 pp 29ndash64 2009

[70] W Braun M Vasak A H Robbins et al ldquoComparison ofthe NMR solution structure and the X-ray crystal structure ofrat metallothionein-2rdquo Proceedings of the National Academy ofSciences of the United States of America vol 89 no 21 pp 10124ndash10128 1992

[71] P JThornalley andM Vasak ldquoPossible role formetallothioneinin protection against radiation-induced oxidative stress Kinet-ics andmechanism of its reaction with superoxide and hydroxylradicalsrdquo Biochimica et Biophysica Acta Protein Structure andMolecular Enzymology vol 827 no 1 pp 36ndash44 1985

[72] M A Schwarz J S Lazo J C Yalowich et al ldquoMetallothioneinprotects against the cytotoxic and DNA-damaging effects ofnitric oxiderdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 92 no 10 pp 4452ndash4456 1995

[73] Y Luo A Kawashima and Y Ishido ldquoIodine excess as anenvironmental risk factor for autoimmune thyroid diseaserdquoInternational Journal of Molecular Sciences vol 15 pp 12895ndash12912 2014

[74] H I Falrsquofushinsrsquoka L L Hnatyshyna andOI Osadchuk ldquoTraceelements storage peculiarities and metallothionein contentin human thyroid gland under iodine deficiency euthyroidnodular goiterrdquo Ukrainskii Biokhimicheskii Zhurnal vol 86 pp107ndash113 2014

[75] L Cai Y H Wang G H Zhou et al ldquoAttenuation bymetallothionein of early cardiac cell death via suppression ofmitochondrial oxidative stress results in a prevention of diabeticcardiomyopathyrdquo Journal of the American College of Cardiologyvol 48 no 8 pp 1688ndash1697 2006

[76] X Sun Z Zhou and Y J Kang ldquoAttenuation of doxorubicinchronic toxicity in metallothionein-overexpressing transgenicmouse heartrdquo Cancer Research vol 61 no 8 pp 3382ndash33872001

[77] A F Ceylan-Isik P Zhao B F Zhang X Xiao G Su and J RenldquoCardiac overexpression of metallothionein rescues cardiaccontractile dysfunction and endoplasmic reticulum stress butnot autophagy in sepsisrdquo Journal of Molecular and CellularCardiology vol 48 no 2 pp 367ndash378 2010

[78] N Hu R Guo X Han B Zhu and J Ren ldquoCardiac-specificoverexpression of metallothionein rescues nicotine-inducedcardiac contractile dysfunction and interstitial fibrosisrdquoToxicol-ogy Letters vol 202 no 1 pp 8ndash14 2011

[79] Y Zhang N Hu Y Hua K L Richmond F Dong and JRen ldquoCardiac overexpression of metallothionein rescues coldexposure-induced myocardial contractile dysfunction through

attenuation of cardiac fibrosis despite cardiomyocyte mechani-cal anomaliesrdquo Free Radical Biology and Medicine vol 53 no 2pp 194ndash207 2012

[80] N Hu X Han E K Lane F Gao Y Zhang and J RenldquoCardiac-specific overexpression of metallothionein rescuesagainst cigarette smoking exposure-induced myocardial con-tractile and mitochondrial damagerdquo PLoS ONE vol 8 no 2Article ID e57151 2013

[81] M T Brazao-Silva S V Cardoso P R de Faria et al ldquoAdenoidcystic carcinoma of the salivary gland a clinicopathologicalstudy of 49 cases and of metallothionein expression with regardto tumour behaviourrdquo Histopathology vol 63 no 6 pp 802ndash809 2013

[82] K J Schmitz H Lang G Kaiser et al ldquoMetallothioneinoverexpression and its prognostic relevance in intrahepaticcholangiocarcinoma and extrahepatic hilar cholangiocarci-noma (Klatskin tumors)rdquo Human Pathology vol 40 no 12 pp1706ndash1714 2009

[83] S E Theocharis A P Margeli J T Klijanienko and G PKouraklis ldquoMetallothionein expression in human neoplasiardquoHistopathology vol 45 no 2 pp 103ndash118 2004

[84] STheocharis J Klijanienko C Giaginis et al ldquoMetallothioneinexpression in mobile tongue squamous cell carcinoma associa-tions with clinicopathological parameters and patient survivalrdquoHistopathology vol 59 no 3 pp 514ndash525 2011

[85] S-K Tai O J-K Tan V T-K Chow et al ldquoDifferentialexpression of metallothionein 1 and 2 isoforms in breast cancerlines with different invasive potential identification of a novelnon-silent metallothionein-1H mutant variantrdquo The AmericanJournal of Pathology vol 163 no 5 pp 2009ndash2019 2003

[86] Y Hishikawa T Koji D K Dhar S Kinugasa M Yamaguchiand N Nagasue ldquoMetallothionein expression correlates withmetastatic and proliferative potential in squamous cell carci-noma of the oesophagusrdquo British Journal of Cancer vol 81 no4 pp 712ndash720 1999

Submit your manuscripts athttpwwwhindawicom

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

Page 10: Research Article Metallothionein-I/II Knockout Mice

10 Oxidative Medicine and Cellular Longevity

[35] C Lv C Wu Y-h Zhou Y Shao G Wang and Q-y WangldquoAlpha lipoic acid Modulated high glucose-induced rat mesan-gial cell dysfunction viamTORp70S6K4E-BP1 pathwayrdquo Inter-national Journal of Endocrinology vol 2014 Article ID 65858914 pages 2014

[36] L Liu X Hu G-Y Cai et al ldquoHigh glucose-induced hyper-trophy of mesangial cells is reversed by connexin43 overex-pression via PTENAktmTOR signalingrdquo Nephrology DialysisTransplantation vol 27 no 1 pp 90ndash100 2012

[37] D D Harrison-Findik S Z Lu E M Zmijewski J Jonesand M C Zimmerman ldquoEffect of alcohol exposure on hepaticsuperoxide generation and hepcidin expressionrdquoWorld Journalof Biological Chemistry vol 4 no 4 pp 119ndash130 2013

[38] M Wang Y Wang J He et al ldquoAlbumin induces neuroprotec-tion against ischemic stroke by altering Toll-like receptor 4 andregulatory T cells in micerdquo CNS and Neurological DisordersmdashDrug Targets vol 12 no 2 pp 220ndash227 2013

[39] X Yao ldquoEffect of zinc exposure on HNE and GLT-1 in spinalcord culturerdquo NeuroToxicology vol 30 no 1 pp 121ndash126 2009

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

[41] G P Becks M C Eggo and G N Burrow ldquoRegulation ofdifferentiated thyroid function by iodide preferential inhibitoryeffect of excess iodide on thyroid hormone secretion in sheepthyroid cell culturesrdquo Endocrinology vol 120 no 6 pp 2569ndash2575 1987

[42] M Vitale T Di Matola F DrsquoAscoli et al ldquoIodide excessinduces apoptosis in thyroid cells through a p53-independentmechanism involving oxidative stressrdquo Endocrinology vol 141no 2 pp 598ndash605 2000

[43] P Pramyothin AM Leung E N Pearce A OMalabanan andL E Braverman ldquoClinical problem-solving A hidden solutionrdquoTheNew England Journal of Medicine vol 365 no 22 pp 2123ndash2127 2011

[44] W Jubiz S Carlile and L D Lagerquist ldquoSerum thyrotropinand thyroid hormone levels in humans receiving chronicpotassium iodiderdquo The Journal of Clinical Endocrinology andMetabolism vol 44 no 2 pp 379ndash382 1977

[45] A G Vagenakis P Downs L E Braverman A Burger andS H Ingbar ldquoControl of thyroid hormone secretion in normalsubjects receiving iodidesrdquoThe Journal of Clinical Investigationvol 52 no 2 pp 528ndash532 1973

[46] M Safran andL E Braverman ldquoEffect of chronic douchingwithpolyvinylpyrrolidone-iodine on iodine absorption and thyroidfunctionrdquo Obstetrics and Gynecology vol 60 no 1 pp 35ndash401982

[47] K Newick B Cunniff K Preston et al ldquoPeroxiredoxin 3 is aredox-dependent target of thiostrepton in malignant mesothe-lioma cellsrdquo PLoS ONE vol 7 no 6 Article ID e39404 2012

[48] A Deniaud O S El Dein E Maillier et al ldquoEndoplasmicreticulum stress induces calcium-dependent permeability tran-sition mitochondrial outer membrane permeabilization andapoptosisrdquo Oncogene vol 27 no 3 pp 285ndash299 2008

[49] A Gorlach P Klappa and T Kietzmann ldquoThe endoplasmicreticulum folding calcium homeostasis signaling and redoxcontrolrdquo Antioxidants and Redox Signaling vol 8 no 9-10 pp1391ndash1418 2006

[50] 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 symporter

expression and activity involves the PI3KAkt signaling path-wayrdquo Endocrinology vol 155 no 3 pp 1145ndash1156 2014

[51] Y-G Wang L Li C-H Liu S Hong and M-J ZhangldquoPeroxiredoxin 3 is resistant to oxidation-induced apoptosis ofHep-3b cellsrdquoClinical and Translational Oncology vol 16 no 6pp 561ndash566 2014

[52] H C Whitaker D Patel W J Howat et al ldquoPeroxiredoxin-3is overexpressed in prostate cancer and promotes cancer cellsurvival by protecting cells fromoxidative stressrdquoBritish Journalof Cancer vol 109 no 4 pp 983ndash993 2013

[53] L Li Y G Zhang and C L Chen ldquoAnti-apoptotic role ofperoxiredoxin III in cervical cancer cellsrdquo FEBS Open Bio vol3 pp 51ndash54 2013

[54] 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

[55] L I Nadolnik Z V Niatsetskaya and S V Lupachyk ldquoEffectof oxidative stress on rat thyrocyte iodide metabolismrdquo CellBiochemistry and Function vol 26 no 3 pp 366ndash373 2008

[56] A C Freitas Ferreira L de Carvalho Cardoso D Rosenthaland D Pires de Carvalho ldquoThyroid Ca2+NADPH-dependentH2O2generation is partially inhibited by propylthiouracil and

methimazolerdquo European Journal of Biochemistry vol 270 no 11pp 2363ndash2368 2003

[57] G R Sue Z C Ho and K Kim ldquoPeroxiredoxins a historicaloverview and speculative preview of novel mechanisms andemerging concepts in cell signalingrdquo Free Radical Biology andMedicine vol 38 no 12 pp 1543ndash1552 2005

[58] M S Seo S W Kang K Kim I C Baines T H Lee and SG Rhee ldquoIdentification of a new type of mammalian perox-iredoxin that forms an intramolecular disulfide as a reactionintermediaterdquo Journal of Biological Chemistry vol 275 no 27pp 20346ndash20354 2000

[59] Z A Wood L B Poole and P A Karplus ldquoPeroxiredoxinevolution and the regulation of hydrogen peroxide signalingrdquoScience vol 300 no 5619 pp 650ndash653 2003

[60] D A Drechsel and M Patel ldquoRespiration-dependent H2O2

removal in brain mitochondria via the thioredoxinperoxire-doxin systemrdquoThe Journal of Biological Chemistry vol 285 no36 pp 27850ndash27858 2010

[61] W H Watson X Yang Y E Choi D P Jones and J P KehrerldquoThioredoxin and its role in toxicologyrdquo Toxicological Sciencesvol 78 no 1 pp 3ndash14 2004

[62] G Ekstrom and M Ingelman-Sundberg ldquoRat liver microso-mal NADPH-supported oxidase activity and lipid peroxida-tion dependent on ethanol-inducible cytochrome P-450 (P-450IIE1)rdquo Biochemical Pharmacology vol 38 no 8 pp 1313ndash1319 1989

[63] J Rashba-Step N J Turro and A I Cederbaum ldquoIncreasedNADPH- and NADH-dependent production of superoxideand hydroxyl radical by microsomes after chronic ethanoltreatmentrdquoArchives of Biochemistry and Biophysics vol 300 no1 pp 401ndash408 1993

[64] V Thakur M T Pritchard M R McMullen Q Wang andL E Nagy ldquoChronic ethanol feeding increases activation ofNADPH oxidase by lipopolysaccharide in rat Kupffer cellsrole of increased reactive oxygen in LPS-stimulated ERK12activation andTNF-120572 productionrdquo Journal of Leukocyte Biologyvol 79 no 6 pp 1348ndash1356 2006

Oxidative Medicine and Cellular Longevity 11

[65] HNakamuraAMitsui and J Yodoi ldquoThioredoxin overexpres-sion in transgenic micerdquo Methods in Enzymology vol 347 pp436ndash440 2002

[66] 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

[67] A Bieniek B Pula A Piotrowska et al ldquoExpression of metal-lothionein III andKi-67 antigen in various histological types ofbasal cell carcinomardquo Folia Histochemica et Cytobiologica vol50 no 3 pp 352ndash357 2012

[68] P Dziegiel ldquoExpression of metallothioneins in tumor cellsrdquoPolish Journal of Pathology vol 55 no 1 pp 3ndash12 2004

[69] M Oslash Pedersen A Larsen M Stoltenberg and M PenkowaldquoThe role of metallothionein in oncogenesis and cancer prog-nosisrdquo Progress inHistochemistry and Cytochemistry vol 44 no1 pp 29ndash64 2009

[70] W Braun M Vasak A H Robbins et al ldquoComparison ofthe NMR solution structure and the X-ray crystal structure ofrat metallothionein-2rdquo Proceedings of the National Academy ofSciences of the United States of America vol 89 no 21 pp 10124ndash10128 1992

[71] P JThornalley andM Vasak ldquoPossible role formetallothioneinin protection against radiation-induced oxidative stress Kinet-ics andmechanism of its reaction with superoxide and hydroxylradicalsrdquo Biochimica et Biophysica Acta Protein Structure andMolecular Enzymology vol 827 no 1 pp 36ndash44 1985

[72] M A Schwarz J S Lazo J C Yalowich et al ldquoMetallothioneinprotects against the cytotoxic and DNA-damaging effects ofnitric oxiderdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 92 no 10 pp 4452ndash4456 1995

[73] Y Luo A Kawashima and Y Ishido ldquoIodine excess as anenvironmental risk factor for autoimmune thyroid diseaserdquoInternational Journal of Molecular Sciences vol 15 pp 12895ndash12912 2014

[74] H I Falrsquofushinsrsquoka L L Hnatyshyna andOI Osadchuk ldquoTraceelements storage peculiarities and metallothionein contentin human thyroid gland under iodine deficiency euthyroidnodular goiterrdquo Ukrainskii Biokhimicheskii Zhurnal vol 86 pp107ndash113 2014

[75] L Cai Y H Wang G H Zhou et al ldquoAttenuation bymetallothionein of early cardiac cell death via suppression ofmitochondrial oxidative stress results in a prevention of diabeticcardiomyopathyrdquo Journal of the American College of Cardiologyvol 48 no 8 pp 1688ndash1697 2006

[76] X Sun Z Zhou and Y J Kang ldquoAttenuation of doxorubicinchronic toxicity in metallothionein-overexpressing transgenicmouse heartrdquo Cancer Research vol 61 no 8 pp 3382ndash33872001

[77] A F Ceylan-Isik P Zhao B F Zhang X Xiao G Su and J RenldquoCardiac overexpression of metallothionein rescues cardiaccontractile dysfunction and endoplasmic reticulum stress butnot autophagy in sepsisrdquo Journal of Molecular and CellularCardiology vol 48 no 2 pp 367ndash378 2010

[78] N Hu R Guo X Han B Zhu and J Ren ldquoCardiac-specificoverexpression of metallothionein rescues nicotine-inducedcardiac contractile dysfunction and interstitial fibrosisrdquoToxicol-ogy Letters vol 202 no 1 pp 8ndash14 2011

[79] Y Zhang N Hu Y Hua K L Richmond F Dong and JRen ldquoCardiac overexpression of metallothionein rescues coldexposure-induced myocardial contractile dysfunction through

attenuation of cardiac fibrosis despite cardiomyocyte mechani-cal anomaliesrdquo Free Radical Biology and Medicine vol 53 no 2pp 194ndash207 2012

[80] N Hu X Han E K Lane F Gao Y Zhang and J RenldquoCardiac-specific overexpression of metallothionein rescuesagainst cigarette smoking exposure-induced myocardial con-tractile and mitochondrial damagerdquo PLoS ONE vol 8 no 2Article ID e57151 2013

[81] M T Brazao-Silva S V Cardoso P R de Faria et al ldquoAdenoidcystic carcinoma of the salivary gland a clinicopathologicalstudy of 49 cases and of metallothionein expression with regardto tumour behaviourrdquo Histopathology vol 63 no 6 pp 802ndash809 2013

[82] K J Schmitz H Lang G Kaiser et al ldquoMetallothioneinoverexpression and its prognostic relevance in intrahepaticcholangiocarcinoma and extrahepatic hilar cholangiocarci-noma (Klatskin tumors)rdquo Human Pathology vol 40 no 12 pp1706ndash1714 2009

[83] S E Theocharis A P Margeli J T Klijanienko and G PKouraklis ldquoMetallothionein expression in human neoplasiardquoHistopathology vol 45 no 2 pp 103ndash118 2004

[84] STheocharis J Klijanienko C Giaginis et al ldquoMetallothioneinexpression in mobile tongue squamous cell carcinoma associa-tions with clinicopathological parameters and patient survivalrdquoHistopathology vol 59 no 3 pp 514ndash525 2011

[85] S-K Tai O J-K Tan V T-K Chow et al ldquoDifferentialexpression of metallothionein 1 and 2 isoforms in breast cancerlines with different invasive potential identification of a novelnon-silent metallothionein-1H mutant variantrdquo The AmericanJournal of Pathology vol 163 no 5 pp 2009ndash2019 2003

[86] Y Hishikawa T Koji D K Dhar S Kinugasa M Yamaguchiand N Nagasue ldquoMetallothionein expression correlates withmetastatic and proliferative potential in squamous cell carci-noma of the oesophagusrdquo British Journal of Cancer vol 81 no4 pp 712ndash720 1999

Submit your manuscripts athttpwwwhindawicom

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

Page 11: Research Article Metallothionein-I/II Knockout Mice

Oxidative Medicine and Cellular Longevity 11

[65] HNakamuraAMitsui and J Yodoi ldquoThioredoxin overexpres-sion in transgenic micerdquo Methods in Enzymology vol 347 pp436ndash440 2002

[66] 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

[67] A Bieniek B Pula A Piotrowska et al ldquoExpression of metal-lothionein III andKi-67 antigen in various histological types ofbasal cell carcinomardquo Folia Histochemica et Cytobiologica vol50 no 3 pp 352ndash357 2012

[68] P Dziegiel ldquoExpression of metallothioneins in tumor cellsrdquoPolish Journal of Pathology vol 55 no 1 pp 3ndash12 2004

[69] M Oslash Pedersen A Larsen M Stoltenberg and M PenkowaldquoThe role of metallothionein in oncogenesis and cancer prog-nosisrdquo Progress inHistochemistry and Cytochemistry vol 44 no1 pp 29ndash64 2009

[70] W Braun M Vasak A H Robbins et al ldquoComparison ofthe NMR solution structure and the X-ray crystal structure ofrat metallothionein-2rdquo Proceedings of the National Academy ofSciences of the United States of America vol 89 no 21 pp 10124ndash10128 1992

[71] P JThornalley andM Vasak ldquoPossible role formetallothioneinin protection against radiation-induced oxidative stress Kinet-ics andmechanism of its reaction with superoxide and hydroxylradicalsrdquo Biochimica et Biophysica Acta Protein Structure andMolecular Enzymology vol 827 no 1 pp 36ndash44 1985

[72] M A Schwarz J S Lazo J C Yalowich et al ldquoMetallothioneinprotects against the cytotoxic and DNA-damaging effects ofnitric oxiderdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 92 no 10 pp 4452ndash4456 1995

[73] Y Luo A Kawashima and Y Ishido ldquoIodine excess as anenvironmental risk factor for autoimmune thyroid diseaserdquoInternational Journal of Molecular Sciences vol 15 pp 12895ndash12912 2014

[74] H I Falrsquofushinsrsquoka L L Hnatyshyna andOI Osadchuk ldquoTraceelements storage peculiarities and metallothionein contentin human thyroid gland under iodine deficiency euthyroidnodular goiterrdquo Ukrainskii Biokhimicheskii Zhurnal vol 86 pp107ndash113 2014

[75] L Cai Y H Wang G H Zhou et al ldquoAttenuation bymetallothionein of early cardiac cell death via suppression ofmitochondrial oxidative stress results in a prevention of diabeticcardiomyopathyrdquo Journal of the American College of Cardiologyvol 48 no 8 pp 1688ndash1697 2006

[76] X Sun Z Zhou and Y J Kang ldquoAttenuation of doxorubicinchronic toxicity in metallothionein-overexpressing transgenicmouse heartrdquo Cancer Research vol 61 no 8 pp 3382ndash33872001

[77] A F Ceylan-Isik P Zhao B F Zhang X Xiao G Su and J RenldquoCardiac overexpression of metallothionein rescues cardiaccontractile dysfunction and endoplasmic reticulum stress butnot autophagy in sepsisrdquo Journal of Molecular and CellularCardiology vol 48 no 2 pp 367ndash378 2010

[78] N Hu R Guo X Han B Zhu and J Ren ldquoCardiac-specificoverexpression of metallothionein rescues nicotine-inducedcardiac contractile dysfunction and interstitial fibrosisrdquoToxicol-ogy Letters vol 202 no 1 pp 8ndash14 2011

[79] Y Zhang N Hu Y Hua K L Richmond F Dong and JRen ldquoCardiac overexpression of metallothionein rescues coldexposure-induced myocardial contractile dysfunction through

attenuation of cardiac fibrosis despite cardiomyocyte mechani-cal anomaliesrdquo Free Radical Biology and Medicine vol 53 no 2pp 194ndash207 2012

[80] N Hu X Han E K Lane F Gao Y Zhang and J RenldquoCardiac-specific overexpression of metallothionein rescuesagainst cigarette smoking exposure-induced myocardial con-tractile and mitochondrial damagerdquo PLoS ONE vol 8 no 2Article ID e57151 2013

[81] M T Brazao-Silva S V Cardoso P R de Faria et al ldquoAdenoidcystic carcinoma of the salivary gland a clinicopathologicalstudy of 49 cases and of metallothionein expression with regardto tumour behaviourrdquo Histopathology vol 63 no 6 pp 802ndash809 2013

[82] K J Schmitz H Lang G Kaiser et al ldquoMetallothioneinoverexpression and its prognostic relevance in intrahepaticcholangiocarcinoma and extrahepatic hilar cholangiocarci-noma (Klatskin tumors)rdquo Human Pathology vol 40 no 12 pp1706ndash1714 2009

[83] S E Theocharis A P Margeli J T Klijanienko and G PKouraklis ldquoMetallothionein expression in human neoplasiardquoHistopathology vol 45 no 2 pp 103ndash118 2004

[84] STheocharis J Klijanienko C Giaginis et al ldquoMetallothioneinexpression in mobile tongue squamous cell carcinoma associa-tions with clinicopathological parameters and patient survivalrdquoHistopathology vol 59 no 3 pp 514ndash525 2011

[85] S-K Tai O J-K Tan V T-K Chow et al ldquoDifferentialexpression of metallothionein 1 and 2 isoforms in breast cancerlines with different invasive potential identification of a novelnon-silent metallothionein-1H mutant variantrdquo The AmericanJournal of Pathology vol 163 no 5 pp 2009ndash2019 2003

[86] Y Hishikawa T Koji D K Dhar S Kinugasa M Yamaguchiand N Nagasue ldquoMetallothionein expression correlates withmetastatic and proliferative potential in squamous cell carci-noma of the oesophagusrdquo British Journal of Cancer vol 81 no4 pp 712ndash720 1999

Submit your manuscripts athttpwwwhindawicom

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

Page 12: Research Article Metallothionein-I/II Knockout Mice

Submit your manuscripts athttpwwwhindawicom

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