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Estrogen effects on thyroid iodide uptake and thyroperoxidase activity in normal and ovariectomized rats

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Estrogen effects on thyroid iodide uptake and thyroperoxidase activity in normal and ovariectomized rats
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  steroids 71 (2006) 653–659 available at www.sciencedirect.com journal homepage: www.elsevier.com/locate/steroids Estrogen effects on thyroid iodide uptake andthyroperoxidase activity in normal andovariectomized rats L´ ıvia P. Lima, In ˆ es A. Barros, Patr´ ıcia C. Lisb ˆ oa, Renata L. Ara´ ujo, Alba C.M. Silva,Doris Rosenthal, Andrea C.F. Ferreira, Denise P. Carvalho ∗ Laborat´ orio de Fisiologia End´ ocrina, Instituto de Biof ´ ısica Carlos Chagas Filho,Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil a r t i c l e i n f o  Article history: Received 24 August 2005Received in revised form 9 March2006Accepted 13 March 2006Published on line 9 June 2006 Keywords: 17-  -Estradiol benzoateOvariectomyThyroid iodide uptakeThyroperoxidase a b s t r a c t Sex steroids interfere with the pituitary–thyroid axis function, although the reports havebeen controversial and no conclusive data is available. Some previous reports indicate thatestradiol might also regulate thyroid function through a direct action on the thyrocytes.In this report, we examined the effects of low and high doses of estradiol administeredto control and ovariectomized adult female rats and to pre-pubertal females. We demon-strate that estradiol administration to both intact adult and pre-pubertal females causesa significant increase in the relative thyroid weight. Serum T3 is significantly decreased inovariectomized rats, and is normalized by estrogen replacement. Neither doses of estrogenproduced a significant change in serum TSH and total T4 in ovariectomized, adult intactand pre-pubertal rats. The highest, supraphysiological, estradiol dose produced a signifi-cant increase in thyroid iodide uptake in ovariectomized and in pre-pubertal rats, but notin control adult females. Thyroperoxidase activity was significantly higher in intact adultrats treated with both estradiol doses and in ovariectomized rats treated with the highestestradiol dose. Since serum TSH levels were not significantly changed, we suggest a directaction of estradiol on the thyroid gland, which depends on the age and on the previousgonad  status  of the animal.© 2006 Elsevier Inc. All rights reserved. 1. Introduction The prevalence of thyroid diseases is higher in women thanin men, and in the postmenopausal period a further increasein the incidence of thyroid dysfunction is detected [1,2]. Estro- genpossiblystimulatesthyrotrophin(TSH)secretion;howeversome previous reports indicate that this hormone might alsoregulatethyroidfunctionthroughadirectactiononthyrocytes[3–8]. The findings that the administration of estradiol stim- ∗ Corresponding author at : Instituto de Biof ´ısica Carlos Chagas Filho, UFRJ, CCS-Bloco G-Cidade Universit´aria, Ilha do Fund ˜ ao, Rio de  Janeiro, 21949-900, Brazil. Tel.: +55 21 25626552; fax: +55 21 22808193.E-mail address: dencarv@biof.ufrj.br (D.P. Carvalho). ulates thyroid radioiodine uptake in rats that were ovariec-tomized and hypophysectomized [9] support the hypothesis of a direct effect of estrogen on the thyroid. More recently,the presence of estrogen receptors has been demonstrated inbothhumanandratthyroidglands[10–12].Moreover,estradiol increasestheFRTL-5ratthyroidcelllineproliferationeitherinthepresenceorabsenceofTSH[3].Estradiolalsoregulatesthy- roid cell proliferation in vivo, since ovariectomy limits thyroidgrowth induced by low iodide diet, and sex steroids increase 0039-128X/$ – see front matter © 2006 Elsevier Inc. All rights reserved.doi:10.1016/j.steroids.2006.03.007  654  steroids 71 (2006) 653–659 thyroid cell proliferation induced by TSH during sexual matu-ration in rats [13,14]. Although estradiol directly stimulates thyroid iodideuptake in vivo [9], a reduction of the sodium-iodide sym- porter (NIS) gene expression, as well as of iodide uptakeweredetectedinestradiol-treatedFRTL-5thyroidcells[3].Pre- vious in vivo measurements of thyroid radioiodide uptakehave usually been done 2, 24 or 72h after radioiodineadministration. However, immediately after iodide entersinto thyrocytes it can be incorporated into proteins and theradioactivity content in the gland should thus correspondto both iodide transport and organification activities. Wehave recently [15] demonstrated that the measurement of radioiodide uptake 15min after iodide administration reflectsiodide transport through the sodium-iodide symporter with-out the influence of in vivo thyroid iodine organificationactivity [15]. Using this novel approach we aimed in this report to evaluate the effect of estradiol on NIS function invivo.Since estradiol might regulate iodide uptake due to itsaction on NIS, which is a protein that plays an importantrole on thyroid hormone biosynthesis, we were interested onthe effect of this steroid on another thyroid hormone biosyn-theticpathway.Atpresent,thereisnodataintheliteratureonthe possible regulatory effect of estrogen on thyroperoxidase.Thyroperoxidase (TPO) is a key enzyme in thyroid hormonebiosynthesis that catalyzes iodide oxidation, organificationand the coupling of iodotyrosines to produce T3 and T4 [16].Furthermore, autoimmune thyroid disease is more frequentin women and TPO is the main thyroid auto antigen [17,18]. Hence,wehypothesizedwhetherestradiolcouldregulateTPOactivity in vivo.In this report, we examined the effects of low and highdoses of estradiol on thyroid function in intact and ovariec-tomized adult female rats, and in pre-pubertal females. Wedemonstrate, for the first time, that both thyroperoxidase andshort-termiodideuptakearestimulatedbyestrogenadminis-tration; however the effects depend not only on the estradioldose administered but also on the age and the previous gonadstatus of the animal. 2. Experimental 2.1. Animal treatment Adult (80–90 days of age, 200–230g) and pre-pubertal femaleWistar rats (40 days of age, 70–90g) were housed under con-trolled conditions of temperature (24 ± 1 ◦ C) and light (12hlight starting at 7:00 a.m.). The estrous cycle was evaluated bydaily vaginal cytology, and all the adult female rats includedin the study had regular cycle for at least 2 weeks beforestarting the experiments. All experiments were conducted inaccordancewithstandardsofanimalcaredefinedbytheInsti-tutional Committee.A group of adult animals was ovariectomized at the begin-ning of the experimental period and the control group wassham-operated. Ovariectomized (OVX) rats received vehicle(sesame oil), 0.7 or 14  g estradiol benzoate/100g b.w., s.c.(1,3,5[10]-estratriene-3, 17-  -diol 3-benzoate, Sigma ChemicalCo., MO, USA) daily for 10 days before sacrifice. The wholeexperimental period corresponded to 3 weeks.Intact adult female rats were divided into control (C) andestradiol-treated rats that received vehicle (sesame oil), 0.7 or14  gestradiolbenzoate/100gb.w.,s.c.,dailyfor10daysbeforesacrifice.Thepre-pubertalfemalerats,receivedvehicle(sesameoil),0.7 or 14  g estradiol benzoate/100g b.w., s.c., daily for 10 daysbefore sacrifice starting on the 30th day of age.Ratsfromallexperimentalgroupsweresacrificed24hafterthe last injection. 2.2. TPO preparation TPO extraction from rat thyroids was performed as previouslydescribed [15,19]. Pools of two rat thyroids were minced and homogenized in 0.5ml 50mM Tris–HCl buffer, pH 7.2, con-taining 1mM KI, using an Ultra-Turrax homogenizer (Staufen,Germany). The homogenate was centrifuged at 100,000 × g ,4 ◦ C for 1h. The pellet was suspended in 0.5ml digitonin (1%,w/v)andincubatedat4 ◦ Cfor24htosolubilizetheperoxidase.Thedigitonin-treatedsuspensionwascentrifugedat100,000g,4 ◦ C for 1h, and the supernatant containing the solubilizedTPO was used for the assays. Protein content was determinedby the method of Bradford [20]. 2.3. Thyroid peroxidase activity The TPO iodide-oxidation activity was measured as previ-ously described [15,19,21–24]. The assay mixture contained: 1.0ml of freshly prepared 50mM sodium phosphate buffer,pH 7.4, containing 24mM KI and 11mM glucose, and increas-ing amounts (20–80  l) of solubilized TPO. The final volumewas adjusted to 2.0ml with 50mM sodium phosphate buffer,pH 7.4, and the reaction was started by the addition of 10  lof 0.1% glucose oxidase (Boehringer Grade I). The increasein absorbance at 353nm (tri-iodide production) was regis-tered for 4min on a Hitachi spectrophotometer (U-3300).The    A 353nm  /min was determined from the linear portionof the reaction curve and one unit (U) of TPO correspondsto    A 353nm  /min=1.0. The enzymatic activity is expressed asU/g protein. 2.4. Short term radioiodide uptake—NIS activity We have previously demonstrated that the measurementof radioiodide uptake 15min after  125 I–NaI administration(short-term iodide uptake) reflects iodide transport throughthe sodium-iodide symporter without the influence of in vivothyroid iodine organification activity [15], since methylmer- captoimidazole (MMI) administration prior to radioiodineinjection does not modify the measurement of iodide uptake.Thus, in order to evaluate the in vivo NIS function using thy-roid radioiodine uptake measurements without the influenceofTPOiodineorganificationreaction,theanimalsreceivedNa- 125 I(250,000dpm,i.p.,Amersham,Buckinghamshire,England)15min before decapitation. The thyroids were removed andweighed.Theradioactivityofthethyroidglandswasmeasuredusing a gamma counter (LKB) and expressed as percentage of total  125 I injected per mg of thyroid.  steroids 71 (2006) 653–659  655 Table 1 – Effect of ovariectomy and estradiol replacement on serum total T4, T3, TSH and estradiol concentrations Treatment Serum T4 (  g/dl) Serum T3 (ng/dl) Serum TSH (ng/ml) Serum estradiol (pg/ml) Control 4.06  ±  0.90 51.7  ±  8.41 1.77  ±  0.58 51.8  ±  10.73OVX 3.54  ±  0.99 38.3  ±  6.84 * 1.61  ±  0.71 36.9  ±  4.28Eb 0.7  g/100g 3.71  ±  1.25 53.6  ±  6.53 2.25  ±  1.03 44.2  ±  5.50Eb 14  g/100g 2.94  ±  0.60 50.5  ±  7.21 2.21  ±  0.97 406.9  ±  90.31 * Hormone results are expressed as mean ± standard error. ∗  p <0.05 vs. other groups. 2.5. Hormone measurements SerumTSHdeterminationwasdonebyaspecificRIAusingpri-maryantibodiesforratTSH.RatTSHforiodinationandprepa-ration of the standard curve was provided by the NationalHormone and Peptide Program/NIDDK (Bethesda, MD, USA).TSH was iodinated by the chloramine T method and  125 I TSHspecific activity was 32 × 105dpm. Intra- and interassay coef-ficients of variation were 7.7 and 6.5%, respectively, and thesensitivity was 0.63ng/ml. Total serum T3 and T4 concentra-tions were assayed using commercial radioimmunoassay kitsusing   125 I as tracer (Immunotech, Prague, Czech Republic). T 3 sensitivity was of 0.1nmol/l, and inter- and intra assay coef-ficients of variation varied from 8.3 to 8.6% and from 2.9 to3.3%, respectively. T 4  sensitivity was of 13nmol/l and inter-andintraassaycoefficientsofvariationvariedfrom5.6to8.6%and 4 to 5.1%, respectively. Rat hormone free serum was usedin the standard curves for total T 3 , T 4  and TSH. Serum totalestradiol was measured using a commercial radioimmunoas-say kit using   125 I as tracer, and standard curves with humanserum were used (Immunotech, Marseille, France). Estradiolsensitivity was of 4pg/ml (14.7pmol/l) and inter- and intraassay coefficients of variation varied from 4.1 to 14.4% and 3.1to 15.1%, respectively. Serum T4, T3 and estradiol measure-mentswereperformedusingthepresenceofspecificantibod-ies adhered to the internal surface of propylene tubes. 2.6. Statistical analysis All the experiments were repeated at least three times, using at least five animals per group in each experiment. Data areexpressed as mean ± standard error. Thyroperoxidase activ-ity, radioiodide uptake, serum TSH and estradiol concentra-tions were analyzed by nonparametric analysis of variance,Kruskal–Wallis, followed by the multiple comparison test of Dunn. The results of thyroid weight and serum total T3 andT4wereanalyzedbyone-wayanalysisofvariance,followedbytheNewman–Keulsmultiplecomparisontest.Thedifferenceswere considered significant when  p <0.05. 3. Results In the OVX and intact adult groups, the administration of aphysiological dose of estradiol (0.7  g/100g b.w.) maintainedtheestradiolserumconcentrationinthemeanvaluesfoundinthe control groups (Tables 1 and 2). In all the rats that received thesupraphysiologicaldoseof17-  -estradiol(14  g/100gb.w.)serum concentrations of estradiol were significantly higherthan in the respective control groups (Tables 1 and 2).Serum TSH levels were not significantly changed byovariectomy or by the administration of both doses of estra-diol to OVX rats, although serum TSH concentrations weresomewhat heterogeneous among the groups (Table 1). 17-  -Estradiol administration in both the physiological and supra-physiological doses did not alter serum TSH levels in intactadult and pre-pubertal female rats (Table 2). Moreover, serum TSH was significantly lower in all the pre-pubertal rats thanin the adult female groups (Table 2).Serum T4 did not change after ovariectomy or when bothdoses of estradiol were administered to OVX, intact adult andpre-pubertal rats (Tables 1 and 2).Ovariectomy significantly decreased serum T3, and bothdosesofestradiolwereabletorestoreserumT3levels(Table1). Table 2 – Effect of estradiol administration to pre-pubertal and adult female rats on serum total T4, T3, TSH and estradiolconcentrations Treatment Serum T4 (  g/dl) Serum T3 (ng/dl) Serum TSH (ng/ml) Serum estradiol (pg/ml) Pre-pubertalControl 5.42  ±  1.96 57.2  ±  7.25 1.36  ±  0.41 ** 29.4  ±  2.41Eb 0.7  g/100g 3.96  ±  2.11 67.8  ±  4.82 1.33  ±  0.45 ** 33.4  ±  8.39Eb 14  g/100g 2.39  ±  0.44 67.2  ±  12.9 1.23  ±  0.43 ** 355  ±  163.4 * AdultControl 4.11  ±  1.20 55.2  ±  8.07 2.67  ±  0.36 38.6  ±  13.42Eb 0.7  g/100g 3.68  ±  0.67 54.0  ±  9.00 2.73  ±  0.18 46.5  ±  10.42Eb 14  g/100g 3.51  ±  0.72 48.2  ±  4.60 2.79  ±  0.68 604.3  ±  223.50 * Serum hormone levels results are expressed as mean ± standard error. ∗  p <0.05 vs. other groups. ∗∗  p <0.05 vs. the respective adult groups.  656  steroids 71 (2006) 653–659 Table 3 – Effect of ovariectomy and estradiol replacement on body weight (g), thyroid weight (mg), and relative thyroidweight (mg/100g b.w.) Treatment Body weight (g) Thyroidweight (mg)Relative thyroid weight(mg/100g b.w.) Control 218  ±  5.6 10.9  ±  0.46 5.9  ±  0.15OVX 249  ±  6.1 * 12.8  ±  0.84 5.2  ±  0.24 * Eb 0.7  g/100g 240  ±  6.0 13.6  ±  1.21 6.9  ±  0.37Eb 14  g/100g 243  ±  4.3 13.6  ±  1.97 6.6  ±  0.88Results are expressed as mean ± standard error. ∗  p <0.05 vs. control group. Estradiol administration did not change serum T3 in bothintact and pre-pubertal rats (Table 2).Thyroid weight relative to body weight was lower in theOVXgroup(Table3),mainlybecauseovariectomycausedasig- nificant increase in body weight. In both groups that receivedestradiol, thyroid weight was increased, although not signifi-cantly, while their body weight were similar to the OVX group(Table 3). Estradiol significantly increased the relative thy- roid weight in the pre-pubertal (lowest estradiol dose) andintact adult (both estradiol doses) rats (Fig. 1), whereas the sex steroid caused no significant change in their body weight(pre-pubertals:  C =84 ± 1.6; Eb 0.7=75 ± 3.9; Eb 14=81 ± 6.2g;adults: C =218 ± 4.5;Eb0.7=214 ± 9.3;Eb14=229 ± 5.4g).More-over, the relative thyroid weight in the pre-pubertal controlrats was higher than in the intact adults (  p <0.05). Fig. 1 – Thyroid weight (mg) relative to body weight (100g)in pre-pubertal and adult female rats. The thyroid weight was measured in the following groups: pre-pubertal controlgroup (treated with vehicle,  n =16), pre-pubertal Eb 0.7group (treated with 0.7  g Eb/100g b.w., s.c., for 10 days, n =15), pre-pubertal Eb 14 group (treated with 14  g Eb/100g b.w., s.c., for 10 days,  n =15), adult control group (treatedwith vehicle,  n =18), adult Eb 0.7 group (treated with 0.7  gEb/100g b.w., s.c., for 10 days,  n =15) and adult Eb 14 group(treated with 14  g Eb/100g b.w., s.c., for 10 days,  n =15).Results are expressed as the mean ± S.E.M.  †  p <0.01 vs.pre-pubertal control and  *  p <0.001 vs. adult control. The short-term radioiodide uptake was not significantlydifferent between control and OVX groups, although a slightdecrease was observed (Fig. 2). The lowest estradiol dose sig- nificantly increased thyroid radioiodide uptake when com-pared to the OVX group and the administration of the highestdose of estradiol to OVX rats significantly increased thyroidradioiodide uptake when compared to the control and OVXgroups (Fig. 2). Radioiodide uptake also increased in the pre- pubertal group that received the highest dose of estradiol,when compared to the pre-pubertal control group, whereasestradiol administration to adult intact rats caused no changein the thyroid radioiodide uptake (Fig. 3).TPO activity did not change after ovariectomy or after theadministration of the lowest dose of estradiol (Fig. 4). In con- trast, the administration of a high estradiol dose to OVX ani- Fig. 2 – Thyroid iodide uptake 15min after radioiodideadministration. The rats received 100  l of saline solutioncontaining  125 I–NaI (250,000dpm, i.p.) 15min beforesacrifice. After 15min the thyroids were removed andweighed, and the radioactivity was measured andexpressed as percentage of the  125 I injected/mg thyroid.The thyroid iodide uptake was measured in the followinggroups: Control (sham-operated that received vehicle, n =17), OVX (rats sacrificed after 3 weeks of ovariectomy,treated with vehicle,  n =18), Eb 0.7 (OVX rats treated with0.7  g Eb/100g b.w., s.c. for the last 10 days beforesacrificed,  n =8), Eb 14 (OVX rats treated with 14  g Eb/100g b.w., s.c. for the last 10 days before sacrificed,  n =7). Resultsare expressed as mean ± S.E.M.  ◦  p <0.05 vs. OVX group, *  p <0.05 vs. OVX and control groups.  steroids 71 (2006) 653–659  657 Fig. 3 – Thyroid iodide uptake 15min after radioiodideadministration. The rats received 100  l of saline solutioncontaining  125 I–NaI (125,000dpm, i.p. in pre-pubertal ratsand 250,000dpm, i.p. in adult rats) 15min before sacrifice.After 15min the thyroids were removed and weighed, andthe radioactivity was measured and expressed aspercentage of the  125 I injected/mg thyroid. The thyroidiodide uptake 15min after radioiodide administration wasmeasured in the following groups: pre-pubertal controlgroup (treated with vehicle,  n =10), pre-pubertal Eb 0.7group (treated with 0.7  g Eb/100g b.w, s.c., for 10 days, n =10), pre-pubertal Eb 14 group (treated with 14  gEb/100g b.w., s.c., for 10 days,  n =10), adult control group(treated with vehicle,  n =9), adult Eb 0.7 group (treated with0.7  g Eb/100g b.w., s.c., for 10 days,  n =9) and adult Eb 14group (treated with 14  g Eb/100g b.w., s.c., for 10 days, n =7). Results are expressed as the mean ± S.E.M. of eachgroup.  *  p <0.05 vs. pre-pubertal control group. mals significantly increased TPO activity (Fig. 4). TPO activi- ties were similar between pre-pubertal and adult female rats(Fig. 5). Estradiol administration did not change TPO activity in pre-pubertal rats, while in intact adults TPO was increasedby both doses of estradiol; however the difference was statis-ticallysignificantonlyintheratsthatreceivedthelowestdose(Fig. 5). 4. Discussion Although estradiol might influence TSH secretion in vitro [25]and in vivo [26], in the present study ovariectomy and estra- dioladministrationdidnotsignificantlyalterserumTSHlevelsin adult female rats treated with estradiol for 10 days. Prob-ably, these different findings are due to the protocols used,since older rats (160 days-old) were treated with estrogen fora longer period of time (30 days) in this previous report [26].Pre-pubertal rats had significantly lower serum TSH whencompared to intact adult female rats, and serum estradiol lev-els did not differ statistically from the adult control animals.Furthermore, the administration of physiological and supra-physiological doses of estradiol did not increase TSH in theseimmatureanimalsprobablyeitherduetotheshort-termestra- Fig. 4 – Thyroperoxidase iodide-oxidation activity. The TPOiodide-oxidation activity was measured as previouslydescribed [6,11,20] in control (sham-operated that received  vehicle,  n =32), OVX (3 weeks ovariectomy, treated with vehicle for the last 10 days before sacrifice,  n =12), Eb 0.7(OVX rats treated with 0.7  g Eb/100g b.w., s.c., for 10 days before sacrifice,  n =12), Eb 14 (ovariectomized rats treatedwith 14  g Eb/100g b.w., s.c., for 10 days before sacrifice, n =11). Results are expressed as the mean ± S.E.M.  *  p <0.001 vs. other groups.Fig. 5 – The TPO iodide-oxidation activity was measured aspreviously described [6,11,20] in the following groups: pre-pubertal control group (treated with vehicle,  n =9),pre-pubertal Eb 0.7 group (treated with 0.7  g Eb/100g b.w.,s.c., for 10 days,  n =8), pre-pubertal Eb 14 group (treatedwith 14  g Eb/100g b.w. during 10 days,  n =10), adult control group (treated with vehicle,  n =19), adult Eb 0.7group (treated with 0.7  g Eb/100g b.w., s.c., for 10 days, n =14) and adult Eb 14 group (treated with 14  g Eb/100g b.w., s.c., for 10 days,  n =15). Results are expressed as themean ± S.E.M. of each group.  *  p <0.05 vs. control group.
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