Toxicity of methimazole on femoral bone in suckling rats: Alleviation by selenium

Experimental and Toxicologic Pathology 64 (2012) 187–195

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Toxicity of methimazole on femoral bone in suckling rats: Alleviation by selenium Ibtissem Ben Amara a , Afef Troudi a,1 , Nejla Soudani a,1 , Fadhel Guermazi b , Najiba Zeghal a,∗ a b

Animal Physiology Laboratory, Sfax Faculty of Science, BP 1171, 3000 Sfax, Tunisia Nuclear Medicine Service, CHU Habib Bourguiba of Sfax, 3029 Sfax, Tunisia

a r t i c l e

i n f o

Article history: Received 13 April 2010 Accepted 10 August 2010 Keywords: Selenium Methimazole Suckling rats Bone Antioxidants enzymes Thyroid hormone Hypothyroidism Histoarchitecture

a b s t r a c t Aims: Selenium has a pharmacological properties and it is well considered as an antioxidant. The present study investigated the potential ability of selenium, used as a nutritional supplement, to alleviate bone impairments in suckling rats whose mothers were treated with methimazole, an antithyroid drug. Main methods: Female Wistar rats were randomly divided into four groups of six each: group I served as control which received standard diet; group II were rendered hypothyroid by administration of methimazole (250 mg L−1 in their drinking water); group III received both methimazole (250 mg L−1 in their drinking water) and selenium (0.5 mg kg−1 of diet); group IV received 0.5 Na2 SeO3 mg kg−1 of diet. Treatments were started from the 14th day of pregnancy until day 14 after delivery. Key findings: Methimazole treatment decreased femur length and weight in 14-day-old rats, when compared to controls. Femur antioxidant enzyme activities, superoxide dismutase, catalase and glutathione peroxidase decreased. Lipid peroxidation recorded an increase revealed by high femur malondialdehyde levels. Methimazole also caused a significant decrease in calcium and phosphorus levels in bone. Yet, in plasma and urine, they increased and decreased inversely. Besides, plasma total tartrate-resistant acid phosphatase was enhanced, while total alkaline phosphatase was reduced. Co-administration of selenium through diet improved the biochemical parameters cited above. Nevertheless, distorted histoarchitecture revealed in hypothyroid rat femur was alleviated by Se treatment. Significance: The present study suggests that selenium is an important protective element that may be used as a dietary supplement protecting against bone impairments. © 2010 Elsevier GmbH. All rights reserved.

1. Introduction Bone is a specialized connective tissue, which forms the framework of the body. Various physiological conditions can adversely affect femoral bone metabolism. For instance, food deprivation (Fetoui et al., 2006), iodine and/or selenium (Se) deficiency (Nekrasova et al., 2006; Moreno-Reyes et al., 2001, 2006; Ren et al., 2007), pollutants such as fluoride (Bouaziz et al., 2004), pesticides (Mahjoubi Samet et al., 2005), goitrogenic agents like thiocyanate (Ghorbel et al., 2008) and antithyroid drugs (Pahuja and De Luca, 1982) affects bone maturation. These compounds are able to interfere, directly or indirectly, with the synthesis of thyroid hormones which fundamentally determine the development and growth of many organs, including the bone. In fact, thyroid hormones are necessary for skeleton maturation (Lioté and Orcel, 2000), the linear growth, maintenance of bone mass and calcium-phosphorus home-

∗ Corresponding author at: Animal Physiology Laboratory, Life Sciences Department, Sfax Faculty of Science, BP1171, 3000 Sfax, Tunisia. Tel.: +216 74 274 600; fax: +216 74 274 437. E-mail addresses: naj [email protected], [email protected] (N. Zeghal). 1 These authors contributed equally to this work. 0940-2993/$ – see front matter © 2010 Elsevier GmbH. All rights reserved. doi:10.1016/j.etp.2010.08.005

ostasis (Bassett and Williams, 2003). However, a decreased thyroid function in youth retards growth and delays skeletal maturation and increases fracture risk (Harvey et al., 2002). Moreover, thyroid impairment in fetuses/neonates can occur after treatment of their pregnant and breast-feeding dams with antithyroid drugs like methimazole (MMI), widely used to manage hyperthyroidism associated with Grave’s disease (Zakrzewski, 2008). Yet, MMI reduces the risk of this pathology in dams (Mandel and Cooper, 2001) but is transmitted through placenta or milk (Marchant et al., 1977; Marchant and Alexander, 1972), thus exposing progeny to a risk of hypothyroidism (Dussault and Ruel, 1987). Consequently, MMI represents a pathogenesis factor by inducing hypothyroidism and bone disorders in fetuses and neonates. Several studies have demonstrated that MMI, associated with oxidative stress and cellular damage, is the consequence of both increased production of free radical and reduced capacity of the antioxidative defence system (Das and Chainy, 2004; Sarandol et al., 2005). Free radicals production is believed to induce bone related diseases by suppressing bone formation and stimulating bone resorption. Indeed, antioxidants deficiency has a negative impact on bone mass (Ramajayam et al., 2007). So, there is growing evidence that oxidative stress contributes to bone damage, whereas antioxidants may prevent the undesired oxidative dam-

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age induced by reactive oxygen species in the bone tissue (Brzoska et al., 2009). In fact, organisms are equipped with several lines of antioxidant defence against oxidative damage. The defence lines act either as non-enzymatic antioxidant defences, such as vitamins C and E, or as enzymatic antioxidant defences, such as scavenger enzymes (SOD, GPx) (Halliwell and Gutteridge, 2007). Accordingly, Ramajayam et al. (2007) have reported a protective role of antioxidants (vitamins C and E) against oxidative damage in bone tissue. However, selenium-containing molecules may be better nucleophiles (and therefore antioxidants) than classical antioxidants (Arteel and Sies, 2001). In fact, selenium, an essential element in almost all biological systems, has pharmacological properties and it is well considered as an antioxidant (Meotti et al., 2004). Several selenoproteins are expressed in bone tissue and are important in bone metabolism (Ebert and Jakob, 2007). In general, oral supplementation is a better option for administration of antioxidants as therapeutic molecules (Subudhi et al., 2009). Selenium, an essential trace element of fundamental importance for animals and humans, is obtained from dietary sources including meat, fish and eggs which contribute as the major part of dietary Se in several countries such as Greece, Portugal and Japan (Pappa et al., 2006; Ventura et al., 2007; Haratake et al., 2007). In this respect, Combs (2001) indicates that an adequate adult diet should have at least 40 ␮g/day of Se to support the maximum expression of seleno-enzymes. Inorganic Se compounds, used throughout the experiment, are generally provided as a nutritional source (Rock et al., 2001), being easily absorbed by duodenum (Ognjanovic et al., 2008). Selenite compounds, according to Anan et al. (2009), are effectively incorporated into placenta during pregnancy and transferred to pups during lactation. In recent reports, Se supplementation has been proved to have protective effects against some pathological states such as MMIinduced cerebrum and cerebellum impairment (Ben Amara et al., 2009), cancer (Steinbrenner and Sies, 2009), diabetes (Kiersztan et al., 2007), Kashin-Beck osteoarthropathy (Zou et al., 2009), atherosclerosis and possibly osteoporosis (Ebert and Jakob, 2007). Based on these facts, we hypothesize that Se supplementation could be a useful method to protect against bone impairment, thus allowing further exploration of its protective potential. This could be therefore a step forward in the protection of human fetuses/neonates against hypothyroidism and its adverse effects, including bone impairment. To our knowledge, there are no studies carried out on suckling rats describing methimazole-induced oxidative stress and bone disorders during late pregnancy and early postnatal periods. Besides, the protective role of selenium on methimazole-induced femur toxicity has not yet been investigated. So, we first assessed the effects of MMI, an antithyroid drug, on bone toxicity and maturity of suckling rats and, subsequently, the ability of Se supplementation to improve and protect bone maturation and development in those rats whose dams were treated with MMI.

2. Materials and methods 2.1. Chemicals Sodium selenite (Na2 SeO3 ), methimazole (C4 H6 N2 S), glutathione (oxidized and reduced), nicotinamide adenine dinucleotide phosphate reduced form (NADPH), 5,5 -dithio-bis-2nitrobenzoic acid (DTNB) and thiobarbituric acid (TBA) were purchased from Sigma (St. Louis; MO, USA). All other chemicals were of analytical grade and were purchased from standard commercial suppliers.

2.2. Animals and diet Male and female Wistar rats weighing 180 ± 10 g were purchased from the Central Pharmacy (SIPHAT, Tunisia). They were kept in polypropylene cages in normal housing conditions at ambient temperature 22 ± 3 ◦ C with a 12-h light/dark cycle and a minimum relative humidity of 40%. Rats were fed a commercial rodent diet purchased from Industrial Society of Nutrients (SICO, Sfax, Tunisia). Diet iodine content (0.720 ␮g iodine g−1 of diet) was determined in basal diet, after acid mineralization, using the catalytic method of Sandell and Kolthoff (1937). The concentration of Se in standard diet (0.17 mg kg−1 of diet) was also determined by us, after acid mineralization, by the electrothermic atomic absorption spectrometry (ET-AAS) technique previously followed for food samples by Kumpulainen et al. (1983) and described by Ekholm et al. (2007). Briefly, 0.5 g of diet was digested in a mixed acid of HNO3 , HClO4 and H2 SO4 . Se was reduced to Se IV with 3 M HCl, chelated with ammonium pyrrolidine dithiocarbamate and extracted into methylisobutylketone for the determination. Measurements were performed on a Perkin-Elmer 5100/Zeeman Atomic Absorption Spectrometer with a 196-nm wavelength. After a one-week acclimatization in the laboratory conditions, pairs of male and virgin female rats were kept overnight in each cage. Pregnant female rats were inspected daily by the presence of the vaginal plug which indicated day zero of pregnancy. 2.3. Experimental procedure Twenty-four pregnant females were randomly divided into four groups of six each. The first group served as a control (0.17 mg Na2 SeO3 kg−1 of diet); the second group (MMI) received in their drinking water 250 mg L−1 of methimazole C4 H6 N2 S (dissolved in distilled water); animals of the third group MMI + Se were treated orally with methimazole (250 mg L−1 in their drinking water) and 0.5 mg kg−1 of Se added to their diet as Na2 SeO3 (mixed with pellet diet); the fourth group (Se) received 0.5 mg Na2 SeO3 kg−1 of diet. Treatments were started from the 14th day of pregnancy until the 14th day postnatal. Hence, our treatment groups were as follows: Group 1: sodium selenite (0.17 mg kg−1 of diet; negative control). Group 2: methimazole (250 mg L−1 in their drinking water). Group 3: methimazole (250 mg L−1 in their drinking water) + sodium selenite (0.5 mg kg−1 of diet). Group 4: sodium selenite (0.5 mg kg−1 of diet; positive control). The dose of methimazole (250 mg L−1 of drinking water) and the beginning of the treatment (the 14th day of pregnancy) were chosen according to Schwartz et al. (1997), since MMI-induced hypothyroidism without lethal effects. The Se dose (0.5 mg kg−1 of diet) used in our experiments and in other findings gave high protection against hypothyroidism (Golstein et al., 1988; Ben Amara et al., 2009) and stress conditions (Ognjanovic et al., 2008; Ben Amara et al., 2009; Soudani et al., 2010). The rats were allowed to deliver spontaneously 3 weeks after coitus. In the first day postnatal, the number of pups born and their sex were recorded. Each litter was culled to eight pups for each mother (four males and four females if possible) as it has been shown that this procedure maximizes the lactation performance (Fishbeck and Rasmussen, 1987). MMI, Se and iodine quantities ingested by lactating rats were determined daily after measuring drinking water and food consumption, respectively (Table 1). The experimental procedures were carried out according to the general guidelines on the use of living animals in scientific investigations (Council of European Communities, 1986) and approved by the Ethics Committee of Sciences Faculty of Sfax and

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the Tunisian Ministry of Higher Education Scientific Research and Technology. Forty-eight pups of each group were euthanized in the 14th day postnatal after anesthesia with chloral hydrate by intraperitoneal route and weighed on an electronic balance (Denver Instrument, APX-203, USA) sensitive to the nearest 0.1 mg. Urine was taken from bladder. Blood was collected from brachial artery and centrifuged at 2200 × g for 10 min. Plasma samples were drawn and kept at −80 ◦ C until analysis. Femurs were dissected out and the surrounding muscles and tissues were removed. All femur samples were weighed. Some of them were intended for histological examination and the others were stored at −80 ◦ C until biochemical analyses. 100 mg of bone samples were taken from femoral region and homogenized with 2 ml of 0.1 M Tris–HCl buffer (pH 7.2) using mortar and pestle according to Ramajayam et al. (2007). The homogenates were centrifuged at 10,000 × g for 30 min at 4 ◦ C and the supernatant was used for biochemical estimations. 2.4. Biochemical assays 2.4.1. Plasma thyroid hormones Plasma-free T3 (Ref: 1579) and T4 (Ref: 1363) levels were determined by radio-immunoassay using kits from Immunotech (Marseille, France). 2.4.2. Calcium and phosphorus levels in plasma, urine and femurs Calcium and phosphorus levels were determined in femurs, after nitric acid mineralization, in plasma and urine using commercial reagents kits (Biocon, Ref 2004, 1904, respectively). 2.4.3. Plasma total alkaline phosphatase (ALP) and acid phosphatase (ACP) levels The plasma levels of total alkaline phosphatase (ALP) and total tartrate-resistant acid phosphatase (ACP) were determined, respectively, by a colorimetric method (Elitech diagnostics SEES FRANCE, Ref PASL-0500; Biomerieux FRANCE, Ref 746419901). 2.4.4. Protein quantification The femur protein contents were measured according to the method of Lowry et al. (1951) using bovine serum albumin as standard. 2.4.5. Malondialdehyde (MDA) measurement in bone Malondialdehyde (MDA) levels are a quantitative measure of lipid peroxidation, which in turn results from oxidative stress. The femur MDA levels were determined spectrophotometrically according to Draper and Hadley (1990). Briefly, an aliquot of bone extract supernatant was mixed with 1 ml of 5% trichloroacetic acid (TCA) and centrifuged at 2500 × g for 10 min. 1 ml of thiobarbituric acid reagent (0.67%) was added to 500 ␮l of supernatant and heated at 90 ◦ C for 15 min. The mixture was then cooled and measured for absorbance at 532 nm using a spectrophotometer (Jenway UV-6305, Essex, England). The MDA values were calculated using 1,1,3,3-tetraethoxypropane as standard and expressed as nmoles of MDA/g of tissue. 2.4.6. Reduced glutathione level (GSH) in bone The femur GSH contents were determined by Ellman’s method (1959), modified by Jollow et al. (1974) based on the development of a yellow colour when 5,5-dithiobis-2 nitro benzoic acid (DTNB) was added to compounds containing sulfhydryl groups. Briefly, 3 ml of sulfosalicylic acid (4%) was added to 500 ␮l of femur homogenate in phosphate buffer for deproteinisation. The mixture was centrifuged at 2500 × g for 15 min. Then Ellman’s reagent was added

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to 500 ␮l of supernatant. The absorbance was measured at 412 nm after 10 min. Total GSH content was expressed as ␮g/g of tissue. 2.4.7. Antioxidant enzyme activities 2.4.7.1. Catalase (CAT) activity. Catalase (CAT) activity was assayed by the method of Aebi (1984). Enzymatic reaction was initiated by adding an aliquot of 20 ␮l of the homogenized tissue and the substrate (H2 O2 ) to a concentration of 0.5 M in a medium containing 100 mM phosphate buffer, pH 7.4. Changes in absorbance were recorded at 240 nm. CAT activity was calculated in terms of ␮mol H2 O2 consumed/min/mg of protein. 2.4.7.2. Total superoxide dismutase activity (SOD). Superoxide dismutase (SOD) activity was estimated according to Beauchamp and Fridovich (1971). There action mixture contained 50 mM of tissue homogenates in potassium phosphate buffer (pH 7.4), 0.1 mM lmethionine, 2 mM riboflavine and 75 mM NitroBlue Tetrazolium (NBT). The developed blue colour reaction was measured at 560 nm. Units of SOD activity were expressed as the amount of enzyme required to inhibit the reduction of NBT by 50% and the activity was expressed as U/mg of protein. 2.4.7.3. Glutathione peroxidase activity (GPx). Glutathione peroxidase (GPx) activity was measured according to Flohe and Gunzler (1984). GPx catalyzes the oxidation of GSH by cumene hydroperoxide. In the presence of GSH reductase and NADPH, the oxidized GSH is immediately converted to the reduced form with a concomitant oxidation of NADPH–NADP+ . The decrease in absorbance at 340 nm was measured (Randox Laboratories, 1996). The enzyme activity was expressed as nmoles of GSH oxidized/min/mg protein. 2.5. Histological studies Femurs, intended for histological examination, were taken and immediately demineralized for 72 h in acetic acid (1.7 mol L−1 ) according to Talbot et al. (2001), then fixed for 48 h in formalin (10%) solution, embedded in paraffin, serially sectioned at 5 ␮m and stained with hematoxylin–eosin for light microscopy examination (Gabe, 1968). 2.6. Statistical analysis The data were analyzed using the statistical package program Stat view 5 Soft Ware for Windows (SAS Institute, Berkley, CA). Statistical analysis was performed using one-way Analysis of Variance (ANOVA) followed by Fisher’s Protected Least Significant Difference (PLSD) test as a post hoc test for comparison between groups [treated groups (MMI, MMI + Se, Se) vs (controls)] and [MMI + Se] vs [MMI, Se]. Correlation coefficients, using the correlation matrix and regression analysis, were performed as described by Kleinbaum et al. (1998). All values were expressed as mean ± S.D. Differences were considered significant if p < 0.05. 3. Results 3.1. Food intake Food and water consumption was reduced by 50% and 20% respectively in MMI-treated dams, while in MMI + Se treated group, a partial recovery occurred only in food intake (+63%) when compared to MMI-treated group. In Se-treated group, food intake, ingested Se and iodine quantities increased by 37%, 79% and 40%, respectively (Table 1).

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Table 1 Daily food and water consumption, MMI, Se and iodine quantities ingested by lactating rats controls or treated with MMI, MMI + Se or Se from the 14th day of pregnancy until the 14th day postnatal. Measurements

Groups Control 30.70 ± 40.20 ± – 5.23 ± 21.16 ±

Food consumption (g/day/rat) Water consumption (mL/day/rat) Ingested MMI quantitiesa (mg/day/rat) Ingested Se quantitiesa (␮g/day/rat) Ingested iodinea (␮g/day/rat)

MMI 1.67 1.59

15.23 32.27 8.50 2.59 10.80

0.53 1.27

MMI + Se ± ± ± ± ±

0.61*** 0.89*** 0.18 0.50 0.41***

41.54 30.31 7.58 20.77 29.91

± ± ± ± ±

Se ,+++,†

2.80** 1.70*** 0.42++ 1.40*** 2.02**,+++

48.89 ± 40 ± – 24.44 ± 35.20 ±

2.61*** 2.22 1.30*** 1.88***

Values are mean ± SD for six rats in each group. MMI or Se group vs control group: **P < 0.01, ***P < 0.001; MMI group vs (MMI + Se) group: ++ P < 0.01, +++ P < 0.001; Se group vs (MMI + Se) group: † P < 0.05. a Quantities of MMI, Se and I ingested by each lactating dam were calculated from water and diet intake (which were measured every day) respectively. Table 2 Body weight, femur weight and length, calcium and phosphorus levels in bone, plasma and urine of 14-day-old rats controls or whose dams were treated with MMI, MMI + Se or Se from the 14th day of pregnancy until the 14th day postnatal. Measurements

Groups Control

Body weights (g) Femur weights (g) Femur lengths (mm) Bone levels (mg/g) Calcium Phosphorus

22.10 ± 0.25 0.09 ± 0.002 12.25 ± 0.11 144.82 ± 2.94 51.60 ± 1.58

MMI

MMI + Se

Se

12.71 ± 0.15*** 0.04 ± 0.001*** 8.84 ± 0.16*

19.17 ± 1.15*++†† 0.05 ± 0.005**+††† 9.34 ± 1.42*†

48.79 ± 7.49*** 27.91 ± 4.28***

68.72 ± 12.92***++††† 35.86 ± 6.17*+

24 ± 0.49 0.10 ± 0.02 12.65 ± 1.13 153.53 ± 11.91 33.68 ± 4.82*

Plasma levels (mg/L) Calcium Phosphorus

58.52 ± 1.36 89.97 ± 5.80

75.03 ± 1.837*** 35.39 ± 2.08***

66.52 ± 8.06+† 43.48 ± 3.46**++

72.22 ± 9.10 46.06 ± 2.48*

Urinary levels (mg/L) Calcium Phosphorus

86.13 ± 1.88 211.91 ± 6.12

66.21 ± 1.25** 577.02 ± 17.43***

70.31 ± 3.38*+ 333.73 ± 88.01*++†

70.70 ± 1.31* 252.21 ± 64.71

Values are mean ± SD. MMI or Se group vs control group: *P < 0.05, **P < 0.01, ***P < 0.001; (MMI + Se) group vs MMI group: + P < 0.05, ++ P < 0.01; (MMI + Se) group vs Se group: † P < 0.05, †† P < 0.01, ††† P < 0.001. Number of determinations: Body weight (n = 48); femur lengths and weights (n = 36); calcium and phosphorus levels in bone, plasma and urine (n = 6).

3.2. Body weight, femur weight and length In MMI group, a decrease of 42% in the weight of 14-day-old rats was noted on euthanasia day (Table 2). In the same group, a significant decrease in femur weight (−56%) and length (−28%) was also obtained (Table 2). When Se was supplemented to the diet of MMI-treated rats, a partial recovery occurred in body weight (+34%), femur weight (+20%) and length (+5%). Body weight, femur weight and length of rats which received only 0.5 mg of Se kg−1 of diet were not significantly changed, when compared to control. A positive correlation was observed between plasma FT3 levels and femur weight or length (r = 0.75, p < 0.003; r = 0.91, p < 0.0003, respectively) in the MMI + Se group (Table 3). 3.3. Thyroid hormone levels After MMI treatment, FT3 and FT4 levels decreased in pups by 37% and 64%, respectively. Se supplemented to the diet of MMItreated dams provoked a 16% increase of FT4 levels in their pups, while plasma FT3 levels exceeded normal values when compared to

MMI-treated group (Fig. 1). In Se-treated group, plasma FT4 levels in pups increased by 23% while plasma FT3 was not significantly changed, when compared to controls. A positive correlation was found between Se quantities ingested by dams of MMI + Se group and plasma FT3 levels (r = 0.93, p < 0.0001) while a negative correlation was observed between FT3 and the ratio FT4 /FT3 (r = −0.83, p < 0.0003) (Table 3). 3.4. Calcium and phosphorus levels in bone, plasma and urine The exposure of dams to MMI (250 mg L−1 ) altered the bone mineral composition of their offspring. Indeed compared to controls, a decline in calcium (−66%) and phosphorus (−46%) contents in bone was noted. Besides, calcium levels increased by 22% plasma but decreased by 23% in urine. While, phosphorus decreased by 61% in plasma and increased by 63% in urine (Table 2). In MMI + Se group we observed a 29% increase in calcium and a 22% phosphorus contents in bone while calcium levels showed a significant fall in plasma (−11%) and a significant rise in urine (+6%) contrarily to phosphorus (+19%) and (−42%), respectively,

Table 3 Correlation between plasma triiodothyronine levels (FT3 ) or ingested Se quantities with selected variables in 14-day-old rats of (MMI + Se) group.

Plasma FT3 levels vs bone weight (g) Plasma FT3 levels vs bone length (mm) Plasma FT3 levels vs alkaline phosphatase (U/L) Plasma FT3 levels vs the ratio (FT4 / FT3 ) Ingested Se quantities vs plasma FT3 levels (pg/ml) Ingested Se quantities vs alkaline phosphatase (U/L) *

Correlation coefficient*

95% Confidence interval

0.75 0.91 0.91 −0.83 0.94 0.84

0.30–0.92 0.82–0.98 0.84–0.97 −0.95 to −0.50 0.78–0.98 0.55–0.91

Correlation coefficients, using the correlation matrix and regression analysis, were performed as described by Kleinbaum et al. (1998).

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Fig. 1. Plasma levels of free thyroxine (FT4 ), free triiodothyronine (FT3 ), total tartrate-resistant acid phosphatase (ACP) and total alkaline phosphatase (ALP) in 14-day-old rats controls or whose mothers were treated with MMI, MMI + Se or Se from the 14th day of pregnancy until the 14th day postnatal. MMI treated groups vs control group: *P < 0.05, **P < 0.01, ***P < 0.001; (MMI + Se) group vs MMI group: ++ P < 0.01, +++ P < 0.001; (MMI + Se) group vs Se group: †† P < 0.01, ††† P < 0.001. Number of determinations: FT4 and FT3 (n = 6); ALP and ACP (n = 8).

when compared to MMI group. In Se-treated group, bone mineral contents were not significantly changed when compared to controls.

improved these biomarkers in MMI + Se group when compared to MMI group (Fig. 1). A positive correlation was observed between Se quantities ingested by dams of MMI + Se group and ALP levels (r = 0.84, p < 0.002) and also between plasma FT3 and ALP levels (r = 0.91, p < 0.0001) (Table 3).

3.5. Plasma levels of acid phosphatase (ACP) and alkaline phosphatase (ALP)

3.6. Lipid peroxidation in bone Biochemical markers such as total tartrate-resistant acid phosphatase (ACP), which reflected bone resorption, increased by 58%, while total alkaline phosphatase (ALP), which reflected bone formation, was significantly reduced (p < 0.05). Supplementation of Se

Our results revealed an increase of lipid peroxidation in the femur of MMI-treated group as evidenced by the enhanced MDA levels in the femur homogenates of suckling rats (+14%)

Table 4 Malondialdehyde levels, enzymic antioxidants activities (glutathione peroxidase, catalase and superoxide dismutase), non-enzymic antioxidants contents (reduced glutathione) in femur of 14-day-old rats controls or whose dams were treated with MMI, MMI + Se or Se from the 14th day of pregnancy until the 14th day postnatal. Parameters and treatments a

Malondialdehyde Glutathioneb Glutathione peroxidasec Catalased Superoxide dismutasee

Control 49.08 30.35 30.54 17.48 16.87

± ± ± ± ±

MMI 1.25 4.66 1.12 1.48 1.38

57.02 20.08 22.36 13.74 7.89

MMI + Se ± ± ± ± ±

1.71** 1.82*** 1.53*** 1.50** 0.58***

49.08 28.04 28.97 15.69 12.09

± ± ± ± ±

Se ++

2.64 2.77++ 3.19++ 0.55+ 1.18+++†††

47.76 32.36 30.40 16.87 17.16

± ± ± ± ±

2.74 4.57 2.18 0.75 0.51

Values are mean ± SD. MMI or Se group vs control group: **P < 0.01; ***P < 0.001; (MMI + Se) group vs MMI group: + P < 0.05, ++ P < 0.01, +++ P < 0.001; (MMI + Se) group vs Se group: ††† P < 0.001. a Malondialdehyde: nmoles of MDA/g tissue. b Glutathione: ␮g/g tissue. c Glutathione peroxidase: nmoles of GSH/min/mg protein. d Catalase: ␮mol H2 O2 degraded/min/mg protein. e Superoxide dismutase: units/mg protein.

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when compared to negative controls (Table 4). Supplementation of Se alleviated lipid peroxidation induced by MMI treatment and modulated significantly the MDA levels in femur of pups. 3.7. Non-enzymic antioxidants (GSH) The levels of non-enzymic GSH antioxidant were significantly decreased in pups (−34%) in MMI-exposed group, when compared to controls. However, selenium supplemented to the MMI-treated group maintains the level of this antioxidant to that of controls (Table 4).

Treated rats displayed fewer, thinner, and fragmented bone trabeculae (Fig. 3B) compared to the control group where the proliferative zone (PZ) contained flattened chondrocytes in columns or clusters parallel to the growth axis; the largest proliferative cells differentiated to form hypertrophic chondrocytes (Fig. 2A). In addition, bone trabeculae in the primary spongium of euthyroid rats were markedly developed and were organized parallel to the columns of proliferating chondrocytes (Fig. 3A). When Se was supplemented to the diet of MMI-treated rats (Figs. 2C and 3C), the histological aspects of the femur sections were partially reversed when compared to the control group. In Se-treated group, the histological aspects of the femur were similar to those of controls (Figs. 2D and 3D).

3.8. Femur antioxidant enzyme (GPx, CAT and SOD) activities In the femur homogenate of MMI-treated rats, glutathione peroxidase (GPx), catalase (CAT) and superoxide dismutase (SOD) activities decreased significantly by 27%, 21% and 53% in pups when compared to negative controls (Table 4). The administration of Se ameliorated enzyme activities (CAT, GPx and SOD) in (MMI + Se) group. 3.9. Histological studies The biochemical modifications cited above were correlated with our histological studies (Figs. 2 and 3). In fact, in the femur sections of MMI-treated rats, proliferating chondrocytes failed to form discreet columns. Also, hypertrophic chondrocyte differentiation and neovascularization in this region were greatly diminished (Fig. 2B).

4. Discussion In this study, the exposure of rats to MMI during late pregnancy and early postnatal periods decreased in the pups’ body weight, femur weight and length. It seemed that MMI-treatment affected the appetite of female rats. Growth impairment could also be attributed to the reduction of plasma thyroid hormone levels of suckling rats, thus verifying that these hormones are crucial for optimal bone growth in humans and rats (Ohlsson et al., 1993). In fact, they play an important role in skeletal development by regulating growth and endochondral bone formation (Underwood and Van Wyk, 1992). Se supplementation in the diet of MMI-treated group improved body growth and bone formation. This improvement could be attributed to the increase of plasma FT3 levels. This

Fig. 2. Femur histological sections of 14-day-old rats controls (A) and whose dams were treated with MMI (B), MMI + Se (C) or Se (D) from the 14th day of pregnancy until the 14th day postnatal. Optic microscopy; hematoxylin–eosin stain; magnification ×200. RZ: reserve zone; PZ: proliferative zone; HZ: hypertrophic zone; PS: primary spongium region.

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Fig. 3. Bone trabeculae in the primary spongium (indicated by the arrows) of 14-day-old rats controls (A) and whose dams were treated with MMI (B), MMI + Se (C) or Se (D) from the 14th day of pregnancy until the 14th day postnatal. Optic microscopy; hematoxylin–eosin stain; magnification ×400.

is in agreement with the positive correlation obtained between Se quantities ingested by dams and plasma FT3 levels. On the other hand, growth amelioration could probably be due to an increase in daily food consumption as well as Se and iodine quantities ingested by lactating rats when compared to MMI group, as demonstrated by our results and reported by previous studies of Navarro-Alarcon and Cabrera-Vique, 2008 who showed that the supranutritional intakes of Se were recommended for normal growth and development. In fact, Se contained in the diet of pregnant and lactating rats is transferred either through placenta or breast milk from dams to their fetuses and newborn as reported in rats by Archimbaud et al. (1992) and Anan et al. (2009). Se is incorporated into the tissues as newly synthesized seleno-proteins (Ognjanovic et al., 2008), especially in bone (Dreher et al., 1998). In fact, several selenoproteins, such as glutathione peroxidases, thioredoxin reductases, selenoprotein P, are expressed in mesenchymal stem and bone cells (Ebert and Jakob, 2007). These selenoproteins were involved in the antioxidant defence system. Pathological consequences of hypothyroidism could point to a high potential for antioxidant imbalance (Alturfan et al., 2007). This pathology might generate reactive oxygen species (Baskol et al., 2007), reduce specifically cellular thiol reserve in most tissues and alter glutathione/GSH-Px content (Carmeli et al., 2008). In the present study, exposure rats to MMI alone during late pregnancy and early postnatal period’s increases lipid peroxidation in femur and decreases its GSH content. This is consistent with previous reports which demonstrated that MMI induces critical oxidative damages in several tissues such as heart and muscle (Venditti et al.,

1997), hippocampus and amygdala (Cano-Europa et al., 2008), cerebrum and cerebellum (Ben Amara et al., 2009), spleen, liver, lung and kidney (Cano-Europa et al., 2009). The co-administration of Se to MMI-treated group restores MDA and GSH levels to near normal values. This could be explained by the important role of selenium in preventing lipid peroxidation and in protecting the integrity and functioning of tissues and cells (Ognjanovic et al., 2008). Recent studies have shown that free radicals production such as H2 O2 or xanthine oxidase generated superoxide anions which are able to inhibit osteoblastic differentiation in mouse (Mody et al., 2001) and rabbit marrow cells (Bai et al., 2004). Nevertheless, these super oxide anions are found to stimulate osteoclast differentiation and bone resorption (Ha et al., 2004; Lean et al., 2004). In the current investigation, biochemical markers such as total tartrateresistant acid phosphatase (ACP), which reflects bone resorption, increases, while total alkaline phosphatase (ALP), which reflects bone formation, is reduced in pups of MMI-treated group, compared to controls. Indeed, the net activity of bone-resorbing cells is more accentuated than that of bone-forming cells, as a result of oxidative stress (Wauquier et al., 2009). Our results are in line with previous findings of Orlando et al. (1993) who found similar changes in plasma ACP levels of hyperthyroid women treated with MMI. The co-treatment with Se ameliorates bone formation by increasing the levels of plasma ALP. The positive correlation observed in our study and in others (Davis et al., 2008) between ingested Se quantities and alkaline phosphatase activity supports these findings. This oligoelement exhibits a protective role against bone impairment via their antioxidant properties.

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Selenium is principally essential part of the glutathione peroxidases (GPx) which provide a defence against oxidative stress by catalyzing the reduction of organic hydroperoxides. According to McPherson (1994), the last antioxidant enzyme and others such as superoxide dismutase (SOD) and catalase (CAT) constitute mutually a supportive team of defense against reactive oxygen species. Our results showed a decrease of GPx total SOD and catalase activities in suckling rats whose mothers were treated with MMI compared to control, suggesting the induction of oxidative stress by MMI. In the (MMI + Se)-treated group, the increase of SOD, catalase and selenium-dependent antioxidant enzyme activities such as GPx might decrease free radical-mediated lipid peroxidation generated by MMI. Likewise, Ahmad et al. (2005) showed that the mechanistic approach of selenium supplementation against oxidative stress involved an increase in the antioxidant enzyme defences, such as catalase, GPx and SOD. Furthermore, recent reports of Ebert and Jakob (2007) suggest that antioxidative selenoenzymes neutralized reactive oxygen species to avoid damage of the genome and proteome which otherwise would cause loss of cellular functions and induction of apoptosis and senescence. We also know that the bone is a composite material consisting of collagen fibers and hydroxyapatite crystals containing inorganic components, mainly calcium and phosphorus. In our study, hypothyroidism, induced by MMI, delayed the bone growth of pups, which was reflected by an altered bone mineral composition, especially the decreasing calcium and phosphorus contents. These alterations could be attributed to the removal of calcium and phosphorus from bone tissue, due to reactive oxygen species probably generated by MMI. These results are in agreement with previous studies of Arai et al. (2007) who reported that oxidative stress affected the mineralization of bone. In fact, reactive oxygen species production is particularly involved in mineral tissue homeostasis and contributes mostly to bone remodelling by promoting bone resorption (Garrett et al., 1990). Se supplementation through diet may probably regulate bone mineral composition as reported by Ebert and Jakob (2007). Indeed, Wauquier et al. (2009) suggest that antioxidants, including Se, should be investigated as a potential approach for the treatment of bone loss. Disorders in bone formation, reflected by changes in its mineralization and turnover rate, were confirmed by histological studies. In fact, hypothyroidism induced by MMI caused growth retardation due to growth plate dysgenesis in which hypertrophic chondrocyte differentiation failed to progress. There were growth plate structure abnormalities with disorganized proliferating chondrocyte columns, cartilage matrix and a reduced hypertrophic chondrocyte differentiation. Impaired vascular invasion at the primary spongium was also observed by us and by others (Lewinson et al., 1994). These modifications could probably be due to the accumulation of free radicals resulting from an increased lipid peroxidation in the femur tissues of the MMI-treated group. In fact, Garrett et al. (1990) demonstrated the relationships between oxygen-derived free radicals and bone histological disorders. Se supplementation improved the histological features of the femur evidenced by its return to normal aspects in which the phenomena of proliferation must have been involved. 5. Conclusion This is the first report which demonstrates that methimazole causes oxidative damage in bone tissue of suckling rats. Coadministration of selenium through diet improved MMI-induced changes in osteomineral metabolism, bone histoarchitecture, enzymatic (superoxide dismutase, catalase, and glutathione peroxidase) and non-enzymatic (glutathione) activities. As a result, selenium could be a useful method to protect against bone impairment. The protective effect exhibited by Se supplementation can involve the

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