Neonatal hypothyroidism caused by maternal nicotine exposure is reversed by higher T3 transfer by milk after nicotine withdraw

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Highlights

FCT 5926 No. of Pages 1, Model 5G

26 May 2011

" Thyroid dysfunction is induced by maternal nicotine exposure during lactation. " The postnatal hypothyroidism induced by nicotine isnormalized by higher T3 transfer through the milk. " The iodine uptake is strongly influenced by nicotine in both mothers and pups duringlactation period.

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Food and Chemical Toxicology xxx (2011) xxx–xxx

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FCT 5926 No. of Pages 7, Model 5G

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Neonatal hypothyroidism caused by maby higher T3 transfer by milk after nico

Elaine de Oliveira a, Egberto Gaspar de Moura a, AnSilvio Claudio-Neto b, Alex Christian Manhães b, Ma Laboratory of Endocrine Physiology, Biology Institute, State University of Rio de Janb Laboratory of Neurophysiology, Biology Institute, State University of Rio de Janeiro,

a r t i c l e i n f o

Article history:

a b s t r a c t

Maternal nicotine expos

Contents lists av

Food and Che

journal homepage: www.e

Received 25 January 2011Accepted 27 April 2011

25nicotine withdrawal. Here s26T3, r27imp le28illed d29mar r30At t d31TSH d32ina s3315 a c-34otin c-35rec f36ease

Available online xxxx

Keywords:LactationNicotineIodideThyroid hormonesDeiodinase

(D1 and D2), and serumbirth, a minipump wassaline. Animals were kdecreased T4 and mamD1 and mammary D2.2 h-RAIU and increasedTSH, but increased deiodwas higher at both daytion was affected by nicotine withdrawal, pupsT3 in relation with incr

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57s.58f59e60-61n62g63n64also related to the presence of neonatal goiter (Chanoine et al.,651991). Normal thyroid hormone status is essential for a child’s neu-66rological development and even small disruptions in the mother’s67thyroid status during gestation may cause intellectual and behav-68ioral abnormalities in her children (Axelstad et al., 2008).69There are multiple mechanisms by which smoking can affect70TH levels. Although still unclear, a nicotine-induced sympathetic71activation could have a role. Tobacco smoke contains thiocyanate,72a substance with a potential goitrogenic effect (Meberg and73Marstein, 1986) through inhibition of the function of the so-74dium-iodide symporter (NIS) in the basal membrane of the thy-75roid gland, through iodide organification and through the76increase of the efflux of iodide (Laurberg et al., 2004). In areas77with borderline low iodide intake, smoking during pregnancy

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1. Introduction

Cigarette smoking has multiple effects on the thyroid gland.has both stimulatory and inhibitory effects and can be considerea risk factor for the development of thyroid disease (Utiger, 1998Epidemiological studies show controversy regarding serum TSH ismokers; some studies report lower levels (Christensen et a1984; Ericsson and Lindgrade, 1991; Fisher et al., 1997; Knudseet al., 2002), whereas others have found no effect (Seyler et a1986; Winternitz and Quillen, 1977; Müller et al., 1995). The prevalence of non-toxic goiter is higher in smokers than in nonsmoker(Hegedus et al., 1985; Lio et al., 1989). Studies have demonstratethat passive smoking is accompanied by higher thyroid hormon(TH) levels (Carrillo et al., 2009; Metsios et al., 2007). On the othe

0278-6915/$ - see front matter � 2011 Elsevier Ltd. All rights reserved.doi:10.1016/j.fct.2011.04.040

Abbreviations: TH, thyroid hormones; NIS, sodium-iodide symporter; TSthyrotropin; T3, triiodothyronine; T4, thyroxine; NIC, nicotine; C, control; RAIradioiodine uptake; type 1 D1, deiodinase; D2, type 1 deiodinase; OATP, Organanion transporter polypeptides; NTCP, Na(+)/taurocholate co-transporting polpeptide; MCT, monocarboxylate transporter; PRL, prolactin.⇑ Corresponding author. Address: Departamento de Ciências Fisiológicas,

andar, Instituto de Biologia, Universidade do Estado do Rio de Janeiro, Av. 28 dsetembro, 87, Rio de Janeiro, RJ 20551-030, Brazil. Tel.: +55 21 28688334; fax: +521 28688029.

E-mail addresses: [email protected], [email protected] (P.C. Lisboa).

Please cite this article in press as: de Oliveira, E., et al. Neonatal hypothyroidmilk after nicotine withdraw. Food Chem. Toxicol. (2011), doi:10.1016/j.fct

rnal nicotine exposure is reversedne withdraw

Paula Santos-Silva a, Cíntia Rodrigues Pinheiro a,na Cottini Fonseca Passos a, Patricia Cristina Lisboa a,

, Brazilzil

leads to neonatal hypothyroidism that can be returned to euthyroidism afte, we examined the transfer of iodine through milk, deiodinase activitieT4 and TSH in rat offspring after maternal exposure to nicotine. One day aftelanted to dams releasing nicotine (NIC), 6 mg/kg/day for 13 days or vehicat the day 15 and 21 of lactation. At day 15, NIC-treated dams showe

y 2 h-radioiodine uptake (RAIU) and increase of TSH, thyroid 2 h-RAIU, livehe cessation of NIC-exposure, pups displayed decreased T3, T4 and thyroi

. At weaning (21-postnatal day), NIC-treated dams recovered their T4 anse level in the liver and mammary gland. Milk T3 content in NIC-treated damnd 21, and thyroid function was recovered at the day 21. Thus, thyroid fune in both mothers and pups, suggesting a primary hypothyroidism. After niovered thyroid function probably due to the increased lactational transfer od mammary gland deiodinase activities.

� 2011 Elsevier Ltd. All rights reserve

hand, there is evidence that smoking causes hypothyroidismthrough the increase in thiocyanate levels in Hashimoto patient(Fukayama et al., 1992).

Maternal smoking also changes the thyroid function of infantChildren of parents who smoke have higher cord concentrations oserum thyroglobulin and thiocyanate at birth and at 1 year of ag(Karakaya et al., 1987), with no difference in TH levels. Other studies have found higher serum thyroxine (T4) and lower thyrotropi(TSH) in children delivered at term by smoking mothers (Meberand Marstein, 1986). Cigarette smoking during pregnancy has bee

able at ScienceDirect

ical Toxicology

evier .com/locate / foodchemtox

ism caused by maternal nicotine exposure is reversed by higher T3 transfer by.2011.04.040

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2 E. de Oliveira et al. / Food and Chemical Toxicology xxx (2011) xxx–xxx

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uses thyroid enlargement and increases serum thyroglobulin ine newborn (Chanoine et al., 1991). Smoking mothers have low-iodide content in their breast milk and urine, and the offspringve lower urinary iodide content. In smoking mothers, iodinensfer through breast milk is negatively correlated to the con-

ntration of urinary cotinine, the main nicotine metaboliteurberg et al., 2004). Therefore, it is possible that nicotine re-ces iodine transfer through maternal milk.Even though most studies have stated that thiocyanate is the

ti-thyroid component of tobacco (Laurberg et al., 2004), we havemonstrated, in rats, a thyroid dysfunction in neonates whoseothers were treated with nicotine during lactation. Early nicotineposure caused neonatal primary hypothyroidism (at the end ofcotine exposure) and programmed for secondary hypothyroid-

at adulthood (Oliveira et al., 2009). It is remarkable that ataning, after nicotine withdrawal, these animals presented nor-

al serum TH and TSH levels, a very quick recovery.To understand the mechanism involved in the development of

yroid dysfunction in the progeny and in the recovery of thyroidnction after nicotine withdrawal, the present study was designedevaluate whether maternal nicotine exposure during lactationects maternal thyroid function, triiodothyronine (T3) transferthe pups through the milk and iodothyronine deiodinase

tivities.

Materials and methods

Animal use and experimental procedures were approved by the Animal Cared Use Committee of the Biology Institute of the State University of Rio de JaneiroUA/189/2007 and CEUA/015/2009), which based its analysis on the principlesmulgated by Brazilian Law n.� 11.794/2008 (Marques et al., 2009). Wistar ratsre kept in a temperature-controlled room (25 ± 1 �C) with artificial light–dark cy-s (lights on 07:00, lights off 19:00). Three-month old, virgin female rats were

d maintained in our laboratory.

. Experimental model of maternal nicotine exposure during lactation

Two days after birth, 24 lactating rats were randomly assigned to one of the fol-ing groups:Nicotine (NIC, n = 12) – dams were anesthetized with thiopental; a 3 � 6 cm

a on the back was shaved, and an incision was made to permit s.c. insertion ofotic minipumps (Alzet, 2ML2, Los Angeles, CA, USA). Pump implantation oc-

red during the postnatal day 2 (PN2) because, according to the manufacturer’sommendation, a pump must be filled with the solution of interest and immersedsaline for 24 h to release substances continuously and homogeneously. Pumpsre prepared with nicotine free-base (Sigma, St Louis, MO, USA) diluted in NaCl% solution, to release an initial dose of 6 mg/kg of nicotine per day (duringdays of lactation), as previously described (Oliveira et al., 2009). We chose toform nicotine exposure through subcutaneous osmotic minipump infusion toid the adverse effects of nicotine peaks. In our model, plasma nicotine levelsre similar to those observed in moderate to heavy smokers, at approximatelyng/ml (Lichtensteiger et al., 1988). Cotinine milk and serum levels were previ-sly measured using this experimental model (Oliveira et al., 2010a; Oliveiraal., 2010b).

Control (C, n = 12) – dams were implanted with osmotic minipumps containingly saline solution.

At birth (PN1), litters were adjusted to 6 male pups to maximize lactation per-mance. Throughout lactation, body weight (dams and pups) and maternal foodake were monitored daily. Dams and offspring were killed at 15 and 21 days oftation by rapid decapitation, with no prior anesthesia because anesthesia affectshormone profile. We used a total of 24 dams, i.e., 6 C and 6 NIC dams for 15 days

d 6 C and 6 NIC dams for 21 days as well as 36 C-pups and 36 NIC-pups for eachdied period. Milk samples were collected by manual extraction. For this purpose,

s were separated from litters for a period of 2 h before milking (Bonomo et al.,05). After 90 min they were injected with oxytocin (5UI/ml, sc, Eurofarma, São

lo, SP, Brazil). After 30 min, they were lightly anesthetized (pentobarbitalmg/kg BW), and milk was extracted by gently stripping the thoracic and abdom-l teats. We obtained 0.5–1.0 mL of milk samples from each lactating rat. Samplesre sonicated and frozen at �20 �C for further analysis. Blood samples from dams

d offspring were collected, centrifuged to obtain serum (2,000g, 20 min, 4 �C) andividually kept at �20 �C until assay.

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ease cite this article in press as: de Oliveira, E., et al. Neonatal hypothyroidism cailk after nicotine withdraw. Food Chem. Toxicol. (2011), doi:10.1016/j.fct.2011.

. Study of mammary and thyroid glands 125I uptake (RAIU)

At the 15th and 21st days of lactation, dams were separated from their pups for. After this period, the dams received a single intraperitoneal (i.p.) injection con-ning 2.22 � 104 Bq of 125I (CNEN, RJ, Brazil), and the pups were allowed to nurse2 h (Polanska et al., 2006). After sacrifice, the thyroid (dams and pups) and mam-ry glands were excised and weighed to evaluate the RAIU (radioiodine uptake).

e 125I was individually evaluated in a gamma-counter (Gamma Wizard, model70–001, PerkinElmer/Las Inc., Boston, MA, USA). Uptake was expressed as%I/mg of tissue.

. Deiodinase activity determination

The measurement of type 1 and 2 iodothyronine deiodinase (D1 and D2) activ-s was based on the release of radioiodide from [50-125I]-reverse T3 (27.8 MBq/– PerkinElmer/NEN, Boston, MA, USA) as previously reported (Lisboa et al.,

03b). The 50-deiodinase or outer-ring deiodinase assays occurred in phosphateffer containing 1 mM EDTA, pH 6.9 in the presence of 1.5 lM reverse T3 andmM DTT for D1 or 2 nM reverse T3 and 40 mM DTT for D2. To distinguish D1

d D2 activity, assays were performed in the presence of 1 mM PTU (to inhibit) or 100 nM of T4 (to suppress D2). Free 125I of enzymatic deiodination wasted from Dowex 50 W-X2 columns (100–200 mesh hydrogen form BioRad, Rich-nd, CA, EUA) with 10% acetic acid. Deiodination percentage in the presence of the

zyme was around 10–20%. The amount of free 125I in the blank was less than2% of the total radioactivity in the reaction mixture. Specific enzyme activitys expressed in nanomoles, picomoles or femtomoles of rT3 deiodinated/h.mgprotein. Protein in the mammary gland and liver was measured by the methodscribed by Bradford (1976).

. Serum and milk hormone levels

Free T3 (serum and milk) and serum free T4 were determined by radioimmuno-ay (RIA), using a commercial kit (ICN Pharmaceuticals, Inc, NY) with an assaysitivity of 0.045 ng/dL (T4) and 0.06 pg/mL (T3). Intra-assay variations were% (T4) and 3.5% (T3). TSH was measured by specific RIA, using a rat TSH kit sup-ed by the National Institutes of Health (NIH, MD, USA) and was expressed inms of the reference preparation provided (RP-3). The intra-assay variation was%, with 0.18 ng/ml as the lower limit of detection.

180. Statistical analysis

181Data were reported as mean ± SEM. GraphPad Prism 5 was used for statistical182alyses and graphics (GraphPad softwares, Inc., La Jolla, CA, USA). RAIU and TSH183re analyzed by the unpaired test of Mann–Whitney. Other experimental data184re analyzed using the unpaired Student’s t-test with the significance level set185p < 0.05. Regarding offspring analyses, litter was used as the experimental unit186tead of using individual animals: for RAIU, we considered the average of values187m 6 pups of the same litter; for serum hormones, we used the average of values188m 2 pups of the same litter.

189Results

190During nicotine exposure (i.e. from day 2 until day 15), NIC191ms exhibited no change in body weight or food intake. However,192C dams showed lower food intake at weaning (C: 46.6 ± 4.9 vs.193C: 33.6 ± 4.5 g; n = 12/group, p < 0.05). As shown in Table 1,194cotine treatment affects serum TH in mothers only at the end

le 1yroid function of nicotine-treated dams and pups during lactation.

15 days 21 days

C NIC C NIC

MothersThyroid weight (mg) 16.7 ± 1.9 13.5 ± 0.6 18.3 ± 2.4 18.8 ± 2.3FT3 (pg/mL) 2.8 ± 0.6 2.5 ± 0.4 1.4 ± 0.2 1.2 ± 0.1FT4 (ug/dL) 3.4 ± 0.6 2.9 ± 0.3* 3.1 ± 0.5 3.3 ± 0.3TSH (ng/mL) 2.0 ± 0.2 3.7 ± 0.7* 3.7 ± 0.4 4.4 ± 0.4

OffspringThyroid weight (mg) 2.6 ± 0.2 2.8 ± 0.0.2 5.7 ± 0.8 5.3 ± 0.57FT3 (pg/mL) 2.2 ± 0.2 1.5 ± 0.1* 2.9 ± 0.3 2.1 ± 0.4FT4 (ug/dL) 7.3 ± 0.2 5.1 ± 0.2* 8.8 ± 1.1 8.6 ± 0.8TSH (ng/mL) 1.6 ± 0.1 2.2 ± 0.2* 1.73 ± 0.1 1.18 ± 0.3

lues represent mean ± SEM of 6 rats per group.< 0.05.

used by maternal nicotine exposure is reversed by higher T3 transfer by04.040

195 of nicotine exposure: they presented lower T4 (�16%, p < 0.05) and196 higher TSH (+89%, p < 0.05).197 In agreement with our previous results (Oliveira et al., 2009),198 NIC offspring presented lower serum T3 (�32%, p < 0.05), lower199 T4 (�30%, p < 0.05) and higher TSH (+38%, p < 0.05) in PN15. At200 weaning (PN21), these pups showed no significant changes in TH201 concentration or serum TSH.202 Fig. 1 depicts thyroid and mammary gland RAIU from nicotine-203 exposed dams. At the end of nicotine exposure only, NIC dams204 showed higher thyroid 125I uptake (7-fold increase, p < 0.05; fig.205 1A) and lower mammary RAIU (�41%, p < 0.05; fig. 1C). No thyroid206 weight difference (Table 1) was observed at 15 or 21 days of207 lactation.208 Fifteen days-old NIC-pups displayed lower thyroid RAIU (NIC:209 0.00399 ± 0.001 vs. C: 0.00668 ± 0.001% 125I uptake/mg, n = 6 per210 group, p = 0.04). However, they did not present this alteration at211 weaning. The thyroid weight of suckling pups was not changed212 in the two periods analyzed (Table 1).213 NIC dams showed higher milk T3 at both time points (15 days –214 NIC: 5.87 ± 0.5 vs. C: 3.02 ± 0.6 pg/mL, p = 0.008; 21 days – NIC:215 7.69 ± 0.9 vs. C: 3.28 ± 0.4 pg/mL, p = 0.003), as depicted in fig. 2.216 At the end of the nicotine exposure (15 days of lactation), we217 detected higher mammary D2 activity (3.6-fold increase, p < 0.05;218 fig. 3C) and liver D1 activity (+36%, p < 0.05; fig. 3E). At weaning,219 we observed higher mammary D1 (+48%, p < 0.05; fig. 3B), mam-220 mary D2 (+86%, p < 0.05; fig. 3D) and liver D1 (+56%, p < 0.05; fig.221 3F) activities in NIC dams.

222 4. Discussion

223 To the best of our knowledge, there have been few experimental224 studies focusing on the effects of nicotine exposure exclusively

225during the early postnatal period. This time period is of special rel-226evance because, in general, women quit smoking during pregnancy227(Giglia et al., 2006; Polanska et al., 2006), but some studies show228that women who stop smoking during gestation often relapse dur-229ing lactation (McBride and Pirie, 1990; O’Campo et al., 1992;230Hannöver et al., 2008). Lactation is a critical period of life in which231important cognitive and neurological developments occur. During232this period, the mother’s milk represents the primary source of233nutrition and iodine supply that is essential for neonatal thyroid234function. Thus, in the present model we choose to study only lac-235tation, with an amount of nicotine corresponding to that used by236heavy smokers (Lichtensteiger et al., 1988).237Previously, we reported that maternal postnatal nicotine treat-238ment leads to primary hypothyroidism, higher adiposity and high-239er serum leptin in the offspring, but that after nicotine withdrawal,240those alterations were reversed to baseline (Oliveira et al., 2009). In241fact, in this experimental model, some aspects have previously242been evaluated such as milk production and energy, body compo-243sition, serum lipid and protein profiles, prolactin (PRL) and adrenal244hormones of dams and pups and these parameters were clearly245distinct when comparing the nicotine-exposed group to the with-246drawal group (Oliveira et al., 2010b).247NIC dams had no change in body weight gain or food consump-248tion during nicotine exposure, although after nicotine withdrawal,249lactating rats displayed a lower food intake. This result was sur-250prising because people who quit smoking often gain weight. How-251ever, as we previously showed that NIC dams had hyperleptinemia252at weaning (Oliveira et al., 2010b), this leptin change is compatible253with the maternal hypophagia. As those mothers did not lost254weight, it is possible that the hypophagia was not sufficient in255intensity or duration to cause measurable weight variation. Also,256as those animals were hypothyroid at the end of nicotine adminis-

21st lactation day

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E. de Oliveira et al. / Food and Chemical Toxicology xxx (2011) xxx–xxx 3

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C NIC

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Fig. 1. Iodide content in thyroid (A and B) and mammary gland (C and D) in lactatof 6 mothers per group. ⁄p < 0.05 vs (C).

Please cite this article in press as: de Oliveira, E., et al. Neonatal hypothyroidmilk after nicotine withdraw. Food Chem. Toxicol. (2011), doi:10.1016/j.fct

C NIC

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ats NIC-treated or saline-treated during lactation. Results are expressed as mean ± SE

ism caused by maternal nicotine exposure is reversed by higher T3 transfer by.2011.04.040

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266withdrawal induced compensatory changes in thyroid function.267Th268pu269ble270the milk. At 15 days of lactation, despite no change in thyroid271we272er273is274in

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4 E. de Oliveira et al. / Food and Chemical Toxicology xxx (2011) xxx–xxx

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tion, it is probable that they still had a lower metabolic rate andat the small reduction in food intake was counterbalanced by thepometabolism.We have identified lactation as a sensitive period and nicotinethe factor responsible for the main alterations observed in thy-

id function caused by smoking in pups and dams. Nicotine expo-re exclusively during lactation caused important changes inaternal thyroid function observable on the last day of treatmentat were normalized at weaning, suggesting that the nicotine

15th lactation day

C NIC

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. 2. Milk T3 content in lactating rats NIC-treated or saline-treated at 15 (A) and 21< 0.05 vs (C).

C NIC

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. 3. Mammary D1 and D2 activities at 15 (A and C) and 21 days (B and D) in lactatinivity at 15 and 21 days respectively. Results are expressed as mean ± SEM of 6 moth

ease cite this article in press as: de Oliveira, E., et al. Neonatal hypothyroidism cailk after nicotine withdraw. Food Chem. Toxicol. (2011), doi:10.1016/j.fct.2011.

21st lactation day

C NIC

0

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6

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(pg/

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days of lactation. Results are expressed as mean ± SEM of 6 mothers per group.

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ts NIC-treated or saline-treated during lactation. Pannels E and F show liver D1per group. ⁄p < 0.05 vs (C).

ose changes in maternal thyroid function may influence thep’s thyroid function in both periods. We cannot discard a possi-direct effect of nicotine in the pups, as it is transferred through

ight, NIC dams presented lower serum T4, higher TSH and high-thyroid RAIU. In contrast, no alteration was found at weaning. Itwell known that TSH stimulates NIS gene expression and

creases iodine uptake by thyroid cells (Riedel et al., 2001).

used by maternal nicotine exposure is reversed by higher T3 transfer by04.040

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E. de Oliveira et al. / Food and Chemical Toxicology xxx (2011) xxx–xxx 5

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Therefore, the higher TSH of NIC lactating rats could be responsiblfor the higher thyroid RAIU shown in the present study. Concerning maternal T4, lower serum concentration could be caused blower production. This reduction could be the result of a defecin thyroid hormone biosynthesis or secretion or of a higher peripheral deiodination. Regarding mammary iodine uptake, NIC damshowed lower 125I uptake only at the end of nicotine exposursuggesting another inhibitory effect of nicotine directly on thmammary gland or perhaps simply that the mammary gland competes with the higher thyroid iodine uptake. This lower supply oiodine to pups through maternal milk could contribute to the thyroid hypofunction previously observed in suckling pups (Oliveiret al., 2009) and confirmed in the present study, where the pupthyroid had a lower RAIU.

In the presence of nicotine (at 15 days) or after its removal (a21 days), maternal milk had higher T3 content. In both periods olactation, NIC mothers showed higher liver D1 activity. In maladult rats, nicotine treatment did not change serum T3, T4, TSor liver D1 activity (Colzani et al., 1998). We have suggested thathese differences result from physiologic responses typical of thlactation period. However, although Colzani et al. (1998) usethe same minipumps infusion as in our study, they designedmodel of moderate smoking (2 mg/kg/day) and also performethe study over a shorter period of exposure (7 days).

Recently, we showed that lactating rats presented higher serumleptin only after nicotine withdrawal, at weaning (Oliveira et a2010b). As leptin stimulates liver D1 activity (Cusin et al., 2000Lisboa et al., 2003a), it is conceivable that the increase in D1 activity of NIC mothers at weaning is due to hyperleptinemia. Higher lver D1 and mammary D2 activities can contribute to thmaintenance of tissue T3 generation, which maintains the higheT3 transfer through the milk of NIC dams. In addition, at weaninthe higher mammary D1 activity also contributes to the highemilk T3. On the last day of nicotine exposure, higher D2 activitin the mammary gland can be explained by the hyperprolactinemia observed in the NIC group (Oliveira et al., 2010b) as PRL specifically up-regulates mammary deiodinase during this particulaperiod (Capuco et al., 2008). Because leptin may also stimulatD1 and D2 activities in the mammary gland (Lisboa et al., 2003bLisboa et al., 2006), it is possible that the higher serum leptin iNIC dams at weaning is responsible for the higher D1 and Dactivities.

Corroborating previous findings (Oliveira et al., 2009), maternanicotine exposure leads to lower serum T3 and T4 in NIC pups a15 days-old. These animals showed lower thyroid RAIU withouchanging the thyroid weight. It is important to consider that thyroid RAIU after 2 h reflects iodide uptake and organification (NIfunction + thyroid peroxidase activity) (Ferreira et al., 2005), sois possible that, in addition to the lower supply of radioactive iodine, because RAIU was lower in the mammary gland, the lower iodide content could also be due to lower iodide organification. Thchange was not found after nicotine removal in weaned pupAmong several tobacco compounds, it is well known that thiocyanate inhibits NIS function in the thyroid and in the lactating mammary gland. Here, we showed that nicotine can also be an inhibitoof NIS.

It is known that leptin stimulates ‘‘in vivo’’ thyroidal RAIU ieuthyroid rats (de Oliveira et al., 2007). Paradoxically, the puppresented lower thyroid RAIU and hyperleptinemia at 15 daysold. We have previously shown that chronic leptin administratio

The higher T3 transfer through the milk at PN15 and PN21,could be related to the higher T4 deiodination on the mammary

Please cite this article in press as: de Oliveira, E., et al. Neonatal hypothyroidmilk after nicotine withdraw. Food Chem. Toxicol. (2011), doi:10.1016/j.fct

gland, which could also simultaneously provide more iodine ithe milk. Those adaptive changes may help to reverse the neonatathyroid dysfunction caused by nicotine exposure, and they mahave been preserved by evolution through natural selection.

The secretion of TH through milk requires the transport of thhormone across the mammary gland. This process is mediated bnon-specific TH transporters that include OATP (Organic aniotransporter polypeptides), NTCP (Na(+)/taurocholate co-transporing polypeptide) and MCT (monocarboxylate transporter) (Visseet al., 2011). Some studies have shown that PRL increases NTCexpression in purified basolateral liver plasma membran(Ganguly et al., 1994; Wood et al., 2005), and rats treated witbromocriptine (hypoprolactinemic ones) showed lower NTCexpression (Ganguly et al., 1994). As NIC dams present hyperprolactinemia at the end of nicotine exposure (Oliveira et al. 2010bit is possible that the higher T3 content in the milk is a consequence of an increase in NTCP expression in mammary tissucausing higher T3 transport.

The data from this study show that maternal nicotine exposurinduces some anti-thyroid effects, which are followed by physiological changes that seem to act to protect pups against the deveopment of hypothyroidism. To our knowledge, no experimental oclinical study addresses whether such mechanisms (for examplincrease of milk T3 content) occur in lactating mothers who havhypothyroidism or in lactating mothers who are smokers.

We have to be cautious in direct comparison between rodenstudies and the possible effects in humans, as there are differencein the transport and metabolism of thyroid hormones between ratand humans, especially concerning T3 production. In humans,greater proportion of T3 is produced peripherally, whereas in rodents a greater proportion is produced by the thyroid. In additionrats are much more likely to develop hypothyroidism than human(Hard 1998). Nevertheless, it is important to consider thaalthough there are differences, there are many similarities betweethese two species. It is important to develop studies to evaluatparameters that are not possible to evaluate in humans, such amaternal nicotine exposure during lactation and the end-pointsuch as tissue deiodinases.

In conclusion, we have demonstrated that maternal nicotinexposure exclusively during lactation induces important changein dams and pups, with different patterns during exposure anduring the withdrawal period. Such changes could be caused by drect nicotine action after its transfer through the breast milk and/oby secondary mechanisms related to maternal alterations induceby the nicotine treatment. The exposure to nicotine during the lactation period leads to anti-thyroid effects in dams and neonatewhich is reversed after nicotine withdrawal. Thus, we propose thaduring nicotine withdrawal, the development of maternal adaptivmechanisms maintains the euthyroid status of the progeny. Thphysiological importance of the current findings is to proposemechanistic explanation for these adaptive responses, involvinmammary gland deiodinases. Although generalizations to the human population should be made with care due to the differencebetween species, the present data serve as an alert that maternasmoking can alter the iodide supply during lactation, leading tdeleterious alterations in the neonates with an impact on the future development of the thyroid function.

Conflict of Interest

399Authors declare that there no conflict of interest.

4006. Uncited references

401( Passos et al. (2001). Q1

ism caused by maternal nicotine exposure is reversed by higher T3 transfer by.2011.04.040

402 Acknowledgment

403 This research was supported by the ‘‘Thyroid Department of the404 Brazilian Society of Endocrinology and Metabolism’’ (Departamen-405 to de Tireóide da Sociedade Brasileira de Endocrinologia e Metab-406 ologia), the ‘‘National Council for Scientific and Technological407 Development’’ (Conselho Nacional de Desenvolvimento Científico408 e Tecnológico-CNPq), the ‘‘Carlos Chagas Filho Research Founda-409 tion of the State of Rio de Janeiro’’ (Fundação Carlos Chagas Filho410 de Amparo à Pesquisa do Estado do Rio de Janeiro-FAPERJ) and411 Coordination for the Enhancement of Higher Education Personnel412 (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior –413 CAPES). All authors are grateful to Miss Monica Moura, Ulisses Ris-414 so Siqueira and Carlos Alberto Sampaio Guimarães for technical415 assistance.

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Please cite this article in press as: de Oliveira, E., et al. Neonatal hypothyroidism caused by maternal nicotine exposure is reversed by higher T3 transfer bymilk after nicotine withdraw. Food Chem. Toxicol. (2011), doi:10.1016/j.fct.2011.04.040