Protective effects of selenium on methimazole nephrotoxicity in adult rats and their offspring

Experimental and Toxicologic Pathology 63 (2011) 553–561

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Protective effects of selenium on methimazole nephrotoxicity in adult rats and their offspring Ibtissem Ben Amara a , Afef Troudi a,1 , Elmouldi Garoui a,1 , Ahmed Hakim b , Tahia Boudawara c , Khaled Mounir Zeghal b , Najiba Zeghal a,∗ a

Animal Physiology Laboratory, Faculty of Science, BP1171, 3000 Sfax, Tunisia Laboratory of Pharmacology, Faculty of Medicine, 3029 Sfax, Tunisia c Anatomopathology Laboratory, CHU Habib Bourguiba, 3029 Sfax, Tunisia b

a r t i c l e

i n f o

Article history: Received 6 November 2009 Accepted 16 April 2010 Keywords: Methimazole Selenium Kidney Creatinine clearance Antioxidant activities Nephrotoxicity Rats

a b s t r a c t This study aims to investigate the improving effects of selenium on methimazole-induced kidney impairments in adult rats and their pups. The animals were randomly divided into four groups of six each: group I served as control which received standard diet; group II received only methimazole in drinking water as 250 mg/l; group III received both methimazole (250 mg/l, orally) and selenium (0.5 mg/kg of diet); group IV served as a positive control and received selenium (0.5 mg/kg of diet) as sodium selenite (Na2 SeO3 ). Treatments were started from the 14th day of pregnancy until day 14 after delivery. In the methimazoletreated group, body and absolute kidney weights decreased in pups and their mothers when compared to control. Daily urine volume, plasma creatinine levels were higher, while urinary levels were lower than in control. Besides, antioxidant enzyme activities, superoxide dismutase, catalase and glutathione peroxidase decreased. Lipid peroxidation recorded an increase revealed by high kidney malondialdehyde levels, while those of plasma and urinary uric acid showed a significant decline. Methimazole-treated rat kidneys exhibited leucocytic infiltrations, vascular congestion and narrowed Bowman’s space. Coadministration of selenium through diet improved all the parameters cited above in adult rats and their progeny. Nevertheless, the distorted histoarchitecture in rat kidney was alleviated by selenium treatment. It can then be concluded that selenium is an important protective element that may be used as a dietary supplement against kidney impairments. © 2010 Elsevier GmbH. All rights reserved.

1. Introduction It is widely acknowledged that hypothyroidism is characterized by mental, behavioural, and circulatory disturbances (Larsen et al., 2003). Hence, various methods have been used to induce congenital hypothyroidism in animals, including the complete removal or destruction of the thyroid gland by surgical thyroidectomy ˜ 1986), radio-thyroidectomy using isotopes (Ruiz-Marcos and Ipina, (Cocks et al., 1970), the restriction of iodine in the diet (Li et al., 2007), and more recently through the administration of antithyroid drugs (Cano-Europa et al., 2009). The administration of an antithyroid drug such as methimazole to pregnant and breast-feeding women with Graves’ hyperthyroidism is resorted to for the purpose of reducing the risk of this pathology in mothers (Mandel and Cooper, 2001). Yet methimazole,

∗ Corresponding author at: Animal Physiology Laboratory, Life Sciences Department, BP1171, 3000 Sfax, Tunisia. Tel.: +216 74 274 600; fax: +216 74 274 437. E-mail addresses: [email protected], naj [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.2010.04.007

transmitted through placenta or milk, has been shown to expose the fetuses/neonates to a risk of hypothyroidism (Marchant and Alexander, 1972; Marchant et al., 1977). Treatment with methimazole may also have side effects such as liver cirrhosis, skin irritation, allergies, pharyngitis with fever and nephritis (Edward, 1992). In fact, methimazole-induced alterations in kidney function are characterized by signs of injury such as changes in relative kidney weights, urinary volume and the fractional excretion of potassium (Schmitt et al., 2003). This antithyroid drug can also evoke a high potential for antioxidant imbalance (Alturfan et al., 2007). Very often, methimazole associated with oxidative stress increases free radicals production and reduces the capacity of the antioxidative defense (Cano-Europa et al., 2008; Ben Amara et al., 2009). However, the supplementation of antioxidants can be useful to inhibit oxidative damage. The interest in selenium pharmacology and biochemistry has thereby increased since it possesses biological and antioxidant properties (Meotti et al., 2004). In fact, selenium supplementation has proven to be effective not only in improving the selenium status and the immune function of renal patients (Smith and Temple, 1997) but also in decreasing oxidative stress products in several tissues (Atif et al., 2008; El-Sharaky et al.,

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Table 1 Daily food and water consumption, methimazole and selenium quantities ingested by lactating rats controls or treated with methimazole (MMI), methimazole + selenium (MMI + Se) or selenium (Se) from the 14th day of pregnancy until day 14 after delivery. Parameters and treatments Average feed intake (g/day/rat) Average water consumption (ml/day/rat) Ingested MMI quantities (mg/day/rat) Ingested Se quantities (␮g/day/rat)

Control 32.001 ± 2.704 41.034 ± 2.352 – 5.728 ± 0.420

MMI 15.630 32.150 8.050 2.370

MMI + Se ± ± ± ±

0.800a , e 1.702a , d 0.112 0.430

49.600 32.370 8.092 33.232

± ± ± ±

Se 2.432a , d ; b , e 2.897a , d 0.724 1.629a , e ; b , e

52.423 ± 2.830a , e 40.318 ± 3.242 – 35.123 ± 1.896a , e

Values are expressed as means ± S.D. for six animals in each group comparisons are made between two groups: a Control group and groups treated with methimazole (MMI), methimazole + selenium (MMI + Se) or selenium (Se). b Methimazole (MMI) group and group treated with methimazole + selenium (MMI + Se). d The symbols represent statistical significance (** p < 0.01). e The symbols represent statistical significance (*** p < 0.001).

2007; Steinbrenner and Sies, 2009). Humans usually take up selenium through their diet, predominantly from cereals, fish and meat (Steinbrenner and Sies, 2009). In fact, inorganic selenium species are generally used as a nutritional source (Rock et al., 2001) and selenite is effectively incorporated into placenta during pregnancy and transferred to pups during lactation (Anan et al., 2009). To our knowledge, there are no studies carried out on adult and suckling rats describing methimazole-induced oxidative stress in kidney during late pregnancy and early postnatal periods. Furthermore, the protective role that selenium plays in methimazoleinduced nephrotoxicity has not yet been investigated. Therefore, in this work, we assesses, first, the effects of methimazole on renal parameters, lipid peroxidation as well as on antioxidant enzymatic and non-enzymatic activities and, subsequently, the ability of selenium to improve and protect kidney function. 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 Adult Wistar rats, weighing about 140 g, were purchased from the Central Pharmacy (SIPHAT, Tunisia). They were housed at ambient temperature 22 ± 3 ◦ C in a 12-h light/dark cycle and a minimum relative humidity of 40%. Commercial diet (SICO, Sfax, Tunisia) and tap water were given ad libitum. The concentration of selenium in standard diet (0.17 mg/kg of diet) was determined, after mineralization, by the Electrothermic Atomic Absorption Spectrometry technique (ET-AAS). Measurements were performed on a Perkin–Elmer 5100/Zeeman Atomic Absorption Spectrometer with a 196-nm wavelength. The general guidelines for the use and care of living animals in scientific investigations were followed (Council of European Communities, 1986). The handling of the animals was approved by the Tunisian Ethical Committee for the Care and Use of laboratory animals.

Twenty-four pregnant female rats of Wistar strain were randomly divided into four groups of six each. Group I served as a negative control (0.17 Na2 SeO4 mg/kg of diet); Group II received orally 250 mg/l of methimazole C4 H6 N2 S; animals of group III were treated orally with methimazole (250 mg/l of drinking water) and 0.5 mg/kg of selenium added to their diet as Na2 SeO3 ; Group IV served as positive control (0.5 Na2 SeO3 mg/kg of diet). Treatments were started from the 14th day of pregnancy until day 14 after delivery. The dose of methimazole and the start of treatment were chosen according to Schwartz et al. (1997), since methimazole induced the classical picture of hypothyroidism without lethal effects. The selenium dose (0.5 mg/kg of diet) used in our experiments and in other findings would give high protection against hypothyroidism (Golstein et al., 1988) and stress conditions (Ognjanovic et al., 2008). Pregnant female rats were allowed to deliver spontaneously three weeks after coitus. No delay was observed in the delivery of the treated groups. Within 24 h after birth, the litters were reduced to 8 pups for each mother (four males and four females if possible) to ensure standardized nutrition and maternal care (Fishbeck and Rasmussen, 1987). So, 192 pups were sacrificed on postnatal day 14. During the lactating period, the dams’ food and water intake was recorded daily and the ingested methimazole and selenium quantities calculated (Table 1). Urinary samples, collected into bottles within 24-h cycles, were obtained from each animal housed in a specially designed metabolic cage where faecal contamination was avoided. The urinary volume of 14-day-old rats was calculated by taking away the 24-h urine volume of mothers kept with their pups and of those kept alone in metabolic cages. The volume of each sample was recorded and centrifuged at 3000 × g for 5 min. On day 14 after delivery, the animals were anesthetized with chloral hydrate by intra peritoneal way. Forty-eight (48) pups of each group were weighed. Blood was collected from the brachial artery of pups and via the aortic puncture of adult rats. Plasma samples were drawn from blood after centrifugation at 2200 × g. Both plasma and urine samples were kept at −20 ◦ C until analysis. Kidneys (right or left), removed from adipose tissue and surrenal glands, were taken from mothers and their offspring and their absolute and relative weights recorded. Some kidney samples, taken from each mother or pup, were homogenized (10%, w/v) in phosphate buffer (0.1 M, pH 7.4) and centrifuged at 10,000 × g for 10 min. The resulting and clear supernatants were used for biochemical assays.

2.3. Experimental design

2.4. Biochemical estimations

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.

2.4.1. Protein quantification Kidney protein contents were measured according to the method of Lowry et al. (1951) using bovine serum albumin as standard.

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2.4.2. Malondialdehyde measurement The kidney malondialdehyde concentrations, a lipid peroxidation index, were determined spectrophotometrically according to Draper and Hadley (1990). Briefly, an aliquot of kidney extract supernatant was mixed with 1 ml of 5% trichloroacetic acid and centrifuged at 2500 × g for 10 min. An amount of 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 malondialdehyde values were calculated using 1,1,3,3-tetraethoxypropane as standard and expressed as nmol of malondialdehyde/g of kidney.

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2.4.5.2. Total superoxide dismutase activity. Superoxide dismutase activity was measured according to the method of Beutler (1984). One hundred microliters of the supernatant were mixed with 1.5 ml of a Tris–HCl buffer (pH 8.5) and 1000 ␮l of 15 mM pyrogallol and then incubated at 25 ◦ C for 10 min. The reaction was determined by adding 50 ␮l of 1 N HCl and the absorbance was measured at 440 nm. One unit was determined as the amount of enzyme that inhibited the oxidation of pyrogallol by 50%. The activity was expressed as U/mg protein. 2.4.5.3. Glutathione peroxidase activity. The assessment of glutathione peroxidase activity was determined using a commercial kit (catalog No. RS 505; Randox, Ltd.). Glutathione peroxidase catalyzes the oxidation of reduced glutathione by cumene hydroperoxide. In the presence of reduced glutathione reductase and nicotinamide adenine dinucleotide phosphate reduced form (NADPH), the oxidized reduced glutathione is immediately converted to the reduced form with a concomitant oxidation of NADPH–NADP+ . The decrease in absorbance at 340 nm was measured (Randox, 1996). The enzyme activity was expressed as nmol of reduced glutathione oxidized/min/mg protein.

2.4.3. Reduced glutathione Kidney reduced glutathione 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 was added to compounds containing sulfhydryl groups. Briefly, 3 ml of sulfosalicylic acid (4%) were added to 500 ␮l of kidney homogenate in phosphate buffer for deproteinisation and the mixture was centrifuged at 2500 × g for 15 min; Ellman’s reagent was then added to 500 ␮l of supernatant. The absorbance was measured at 412 nm after 10 min. Total reduced glutathione content was expressed as mg/g of kidney.

2.4.6. Biochemical estimation in plasma and urine Urine and plasma levels of creatinine and uric acid were measured by colorimetric methods using commercial reagent kits (Ref: 20151 and 20091) respectively, purchased from Biomaghreb (Ariana, Tunis, Tunisia).

2.4.4. Measurement of kidney vitamin E Vitamine E (␣-tocopherol) level was assayed by the extraction method of Katsanidis and Addis (1999) using high-performance liquid chromatography (HPLC). Roughly, the kidney was deproteinised with alcohol containing ␣-tocopherol acetate as an internal standard and extraction was performed using hexane. HPLC analysis was carried out using a reverse-phase analytical column (5 ␮m C18; 4.6 mm × 150 mm, HypersilTM , Supelco, USA) with UV monitoring at 292 nm.

2.4.7. Creatinine clearance Creatinine clearance was calculated according to the formula (Charrel, 1991): Creatinine clearance =

2.4.5. Antioxidant enzyme activities The enzyme activities were determined within the same day of collecting in fresh kidney homogenates diluted in phosphate buffer:

U·V P

where U is the urinary creatinine level, V the volume of urine sample collected within 24 h and P the plasma creatinine concentration.

2.4.5.1. Catalase activity. Catalase activity was assayed by the method of Aebi (1984). The 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. This activity was calculated in terms of ␮mol H2 O2 consumed/min/mg of protein.

2.5. Histological studies Some kidneys, intended for histological examination, were immediately fixed in formalin (10%) solution for 48 h and processed in a series of graded ethanol, then embedded in paraffin, serially sectioned at 3 ␮m and stained with hematoxylin–eosin for light microscopy examination (Gabe, 1968).

Table 2 Body weight (g), absolute (g) and relative (mg/g) kidney weights of mothers and their offspring controls or treated with methimazole (MMI), methimazole + selenium (MMI + Se) or selenium (Se) from the 14th day of pregnancy until day 14 after delivery. Parameters and treatments

Controls

MMI

MMI + Se

Se

Body weights (g) Mothers (n = 6) Offspring (n = 48)

170.981 ± 6.217 22.100 ± 0.248

168.500 ± 5.885 12.710 ± 0.150a,e

177.166 ± 5.036 19.172 ± 1.140a,d

203.667 ± 9.500a,d 24.000 ± 0.490

Absolute kidney weights (g) Mothers (n = 6) Offspring (n = 48)

0.631 ± 0.053 0.117 ± 0.013

0.555 ± 0.063a,c 0.072 ± 0.010a,e

0.657 ± 0.089 0.128 ± 0.010

0.754 ± 0.050a,c 0.128 ± 0.015

Relative kidney weights (mg/g bw) Mothers (n = 6) Offspring (n = 48)

3.750 ± 0.747 5.712 ± 0.649

3.244 ± 0.449 5.721 ± 0.740

2.275 ± 0.121a,d 6.795 ± 0.870a,d

3.630 ± 0.298 5.260 ± 0.371

Values are expressed as means ± S.D. Number of determinations: mothers (n = 6); offspring (n = 48) in each group. Comparisons are made between two groups: a Control group and groups treated with methimazole (MMI), methimazole + selenium (MMI + Se) or selenium (Se). b Methimazole (MMI) group and group treated with methimazole + selenium (MMI + Se). c The symbol represents statistical significance (* p < 0.05). d The symbols represent statistical significance (** p < 0.01). e The symbols represent statistical significance (*** p < 0.001).

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Fig. 1. Urinary volume of mothers (A) and their offspring (B), controls (CT) or treated with methimazole (MMI), methimazole + selenium (MMI + Se) or selenium (Se) from the 14th day of pregnancy until day 14 after delivery. Comparisons are made between two groups: (a) control group and groups treated with methimazole (MMI), methimazole + selenium (MMI + Se) or selenium (Se); (b) methimazole (MMI) group and group treated with methimazole + selenium (MMI + Se); the symbols represent statistical significance: (c) (* p < 0.05), (e) (*** p < 0.001); number of determinations: (n = 6).

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 (methimazole, methimazole + selenium, selenium) vs (negative controls)] and [methimazole + selenium] vs [methimazole, selenium]. 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 51 and 22%, respectively, in methimazole-treated mothers (Table 1) contrarily to (methimazole + selenium)-treated group where food intake increased significantly (p < 0.001). In the selenium-treated group, food intake and ingested selenium quantities increased by 43 and 84%, respectively. 3.2. Body and kidney weights In the methimazole-treated group, a significant decrease in body (−42%) and absolute kidney weights (−38%) was obtained in 14-day-old rats (p < 0.001). However, when selenium was supple-

Fig. 2. Creatinine clearance of mothers (A) and their offspring (B), controls (CT) or treated with methimazole (MMI), methimazole + selenium (MMI + Se) or selenium (Se) from the 14th day of pregnancy until day 14 after delivery. Comparisons are made between two groups: (a) control group and groups treated with methimazole (MMI), methimazole + selenium (MMI + Se) or selenium (Se); (b) methimazole (MMI) group and group treated with methimazole + selenium (MMI + Se); the symbols represent statistical significance: (d) (** p < 0.01), (e) (*** p < 0.001); number of determinations: (n = 6).

mented to diet, a partial recovery occurred in body weight (+34%), while that of absolute kidney reached normal values (Table 2). On the other hand, body and kidney weights of rats which have received 0.5 selenium mg/kg of diet were not significantly changed when compared to negative control. 3.3. Creatinine levels in plasma and urine Our results showed a constellation of disorders in the renal function of the methimazole-treated group illustrated by an increased urine output (Fig. 1A and B) and changes in creatinine and acid uric levels (Table 3). In fact, the 24-h urine volume in treated mothers and their pups was higher than in control (p < 0.001), while an amelioration of the parameters cited above was observed in the (methimazole + selenium) group. Creatinine levels in methimazole-treated mothers and their pups were higher in plasma (+25%; +27%) and lower in urine (−25; −36%), respectively than those of control. So, after methimazole treatment, we have found a reduction in creatinine clearance, an indicator of glomerular dysfunction in adult rats (−25%) and their pups (−23%) (Fig. 2A and B). Co-treatment with selenium improved these parameters. 3.4. Uric acid levels in plasma and urine Results indicated that uric acid levels were lower in the plasma and urine of methimazole-treated mothers (−33; −30%) and their offspring (−24; −37%) when compared to control (Table 3). The 0.5 mg/kg supplementation of selenium to diet restored these parameters to near normal values (p < 0.05). In positive controls (selenium group), the urinary excretion of this nitrogenous waste

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Table 3 Plasma and urinary levels of creatinine and uric acid of mothers and their offspring controls or treated with methimazole (MMI), methimazole + selenium (MMI + Se) or selenium (Se) from the 14th day of pregnancy until day 14 after delivery. Parameters and treatments

Creatinine

Uric acid

Plasma (␮mol/l)

Urine (␮mol/l)

Plasma (␮mol/l)

Controls Mothers (8) Offspring (8)

127.830 ± 3.836 85.432 ± 3.544

4167.731 ± 333.788 6677.697 ± 260.670

292.293 ± 13.754 212.415 ± 9.445

MMI Mothers (8) Offspring (8)

169.804 ± 12.193a , e 117.230 ± 5.539a , e

3141.697 ± 190.196a , e 4271.606 ± 344.639a , e

196.35 ± 12.621a , e 162.732 ± 6.718a , c

MMI + Se Mothers (8) Offspring (8)

145.425 ± 8.462a , c ; b , c 93.487 ± 7.000a , c ; b , e

3620.796 ± 196.829a , d ; b , e 6041.726 ± 165.841a , d ; b , e

266.113 ± 4.803a , c 180.88 ± 2.161a , d ; b , d

Se Mothers (8) Offspring (8)

132.494 ± 4.580a , c 97.303 ± 5.007a , d

3815.827 ± 508.776 6062.925 ± 154.039a , c

317.73 ± 20.546a , d 232.05 ± 14.574

Urine (␮mol/l) 1458.8 ± 49.457 1948.8 ± 200.547 1022 ± 98.199a , e 1218 ± 330.708a , e 1293.6 ± 67.409a , d ; b , d 1790.88 ± 64.903b , c 1603 ± 100.858 2200.8 ± 70.279a , c

Values are expressed as means ± S.D. Comparisons are made between two groups: a Control group and groups treated with methimazole (MMI), methimazole + selenium (MMI + Se) or selenium (Se). b Methimazole (MMI) group and group treated with methimazole + selenium (MMI + Se). c The symbols represent statistical significance (* p < 0.05). d The symbols represent statistical significance (** p < 0.01). e The symbols represent statistical significance (*** p < 0.001).

tive controls (Table 4). The supplementation of selenium alleviated lipid peroxidation induced by methimazole treatment and significantly modulated the malondialdehyde levels in the kidney of both mothers and pups.

product (uric acid) increased in both mothers (+10%) and their pups (+21%).

3.5. Lipid peroxidation in kidney

3.6. Enzymatic antioxidants (glutathione peroxidase, catalase and superoxide dismutase)

Our results revealed an increase of lipid peroxidation in the kidney of the methimazole-treated group as evidenced by the enhanced malondialdehyde levels in the kidney homogenates of mothers (+8%) and their offspring (+9%) when compared to nega-

In the kidney homogenates of methimazole-treated rats, glutathione peroxidase, catalase and superoxide dismutase activities

Table 4 Malonedialdehyde levels, enzymatic antioxidants activities (glutathione peroxidase, catalase and superoxide dismutase), non-enzymatic antioxidants contents (reduced glutathione and vitamin E) of mothers and their offspring controls or treated with methimazole (MMI), methimazole + selenium (MMI + Se) or selenium (Se) from the 14th day of pregnancy until day 14 after delivery. Parameters and treatments

Controls

MMI

MMI + Se

Malonaldialdehyde˛ Mothers (8) Offspring (8)

90.471 ± 1.921 90.316 ± 0.648

98.793 ± 2.438a , d 98.764 ± 1.673a , d

96.925 ± 4.058a , c 92.040 ± 1.997a , c

89.454 ± 1.997 87.155 ± 1.612

Catalaseˇ Mothers (8) Offspring (8)

27.615 ± 3.147 23.700 ± 1.951

13.311 ± 0.667a , c 16.406 ± 0.5413a , d

23.327 ± 2.705b , d 20.611 ± 2.193a , c , b , d

28.092 ± 4.031 25.944 ± 4.425

Superoxide dismutase Mothers (8) Offspring (8)

14.235 ± 1.280 16.145 ± 1.510

9.949 ± 1.492a , e 9.278 ± 0.765a , e

13.816 ± 1.361b , e 12.023 ± 1.127a , c ; b , d

15.447 ± 1.756 14.780 ± 0.556

Glutathione peroxidaseı Mothers (8) Offspring (8)

76.384 ± 16.868 57.251 ± 5.515

33.646 ± 5.013a , e 39.871 ± 3.148a , e

61.227 ± 5.880a , c ; b , d 48.038 ± 4.078a , c ; b , d

Reduced glutathione Mothers (8) Offspring (8)

11.106 ± 0.417 9.395 ± 1.552

5.967 ± 0.367a , e 5.907 ± 0.121a , e

9.960 ± 0.549a , d ; b , e 8.773 ± 0.478b , e

12.470 ± 0.902a , c 11.870 ± 0.905a , c

0.335 ± 0.100 0.387 ± 0.029

0.167 ± 0.027a , d 0.103 ± 0.015a , e

0.236 ± 0.005a , c ; b , c 0.279 ± 0.101b , c

0.555 ± 0.145 0.418 ± 0.027

Vitamin Eε Mothers (8) Offspring (8)

Values are expressed as means ± S.D. Number of determinations: (n = 8). Comparisons are made between two groups: a Control group and groups treated with methimazole (MMI), methimazole + selenium (MMI + Se) or selenium (Se). b Methimazole (MMI) group and group treated with methimazole + selenium (MMI + Se). c The symbols represent statistical significance (* p < 0.05). d The symbols represent statistical significance (** p < 0.01). e The symbols represent statistical significance (*** p < 0.001). ␣ Malonaldialdehyde: nmol of MDA/g tissue. ␤ Catalase: ␮moles H2 O2 degraded/min/mg protein. ␥ Superoxide dismutase: units/mg protein. ␦ Glutathione peroxidase: nmol of GSH/min/mg protein. ␺ Reduced glutathione: mg/g tissue. ␧ Vitamin E: mg/g tissue.

Se

100.316 ± 11.412a , e 67.290 ± 4.263a , e

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Fig. 3. Histological findings in renal tissue of adult rats from the four experimental groups: (A) Control group—normal renal architecture; (B) methimazole group—methimazole-treated group showed a reduction of the glomerular Bowman’s space, vacuoles formation, leucocytic infiltrations and an extensive vascular congestion (indicated by arrows);(C) methimazole + selenium group—histological damage decreased; (D) selenium group—selenium-treated group showed normal renal picture as in control group. Hematoxylin–eosin, ×400. ) Bowman’s space; ( ) tubular lumen; (—) vascular congestion; ( ) leucocytic infiltrations; ( ) vacuoles formation. Arrows indicate: (

decreased significantly by 30, 52 and 30% in mothers and by 56, 31 and 42% in their pups, respectively, when compared to negative controls (Table 4). The administration of selenium ameliorated these enzymes activities in the (methimazole + selenium) group. Treatment with selenium alone increased kidney glutathione peroxidase activity (p < 0.001) level in mothers and their offspring.

3.7. Non-enzymatic antioxidants (reduced glutathione and vitamin E) The levels of non-enzymatic antioxidants such as reduced glutathione and vitamin E markedly decreased in mothers (−46; −50%) and pups (−37; −73%), respectively, in the methimazole-exposed group, when compared to controls. However, the supplemented selenium maintained the antioxidants levels to those of control (Table 4).

3.8. Kidney histological studies Kidney histological studies showed numerous abnormalities in methimazole-treated mothers (Fig. 3B) as well as in their pups (Fig. 4B) detected in glomeruli and in convoluted tubules, when compared to controls (Figs. 3A and 4A). The kidney of methimazole-treated mothers exhibited a narrowed Bowman’s space and vascular congestion inside glomeruli (Fig. 3B1 ) and between tubules (Fig. 3B2 ). Furthermore, convoluted tubules were closed (Fig. 3B2 ) and/or open showing vacuoles formation which indicate a beginning of necrosis step (Fig. 3B3 ). Moreover in their pups, we have observed tubular obstruction and cell necrosis (Fig. 4B). Infiltrations of lymphocyte and polynuclear cells were also occurred particularly between tubules in mothers as well as in their pups (Figs. 3B3 and 4B1 ). After the supplementation of selenium, the kidney histoarchitecture was improved (Figs. 3C and 4C). In the selenium-treated group, the kidney his-

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Fig. 4. Histological findings in renal tissue of 14-day-old-rats from controls (A) and whose mothers were treated with methimazole (B), methimazole + selenium (C) or selenium (D). (A) Controls group—normal renal architecture; (B) methimazole group—methimazole-treated group showed a reduction of the glomerular Bowman’s space, leucocytic infiltrations, cell necrosis and an extensive vascular congestion (indicated by arrows); (C) methimazole + selenium group—histological damage decreased; (D) selenium group—selenium-treated group showed normal renal picture as in control group. Hematoxylin–eosin, ×400. ) Bowman’s space; ( ) tubular lumen; (—) vascular congestion; ( ) leucocytic infiltrations; ( ) cell necrosis. Arrows indicate: (

tological aspect was similar to that of controls (Figs. 3D and 4D). 4. Discussion Our investigation allowed us to notice that the exposure of rats to methimazole during late pregnancy and early postnatal periods affected the body and organ weights of their offspring. The reduction of daily food consumption, observed in the methimazoletreated group, supports these findings. Furthermore, according to Bradley et al. (1972), renal growth reduction mainly results from a decrement in the length of the proximal and distal renal tubule, whereas glomerular growth is retarded due to the decrease of total body growth. In our case, co-administration of selenium to the methimazole-treated group improved body and kidney growth. This improvement could be attributed to an increase in daily food consumption and ingested selenium quantities by lactating rats, as

demonstrated by our results and reported by previous studies of Navarro-Alarcon and Cabrera-Vique (2008) who showed that the supranutritional intakes of selenium activated growth and development. In the current investigation, the oral administration of methimazole to lactating rats induced a reduction in the glomerular filtration rate objectified by a reduced creatinine clearance and a decline of a 24-h urinary excretion of creatinine. Our results are in agreement with previous studies which demonstrated that hypothyroidism was associated with decreases in the glomerular filtration rate and renal plasma flow (Skowsky and Kikuchi, 1978). The impairment in glomerular function, observed in the methimazole-treated group, was accompanied by an increase in a 24-h urine output both in mothers and their offspring as found by our study. According to Schmitt et al. (2003), methimazole-induced hypothyroidism was associated with an altered abundance of renal sodium entry pathways along with an increased fractional sodium excretion, reduced

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vasopressin secretion and polyuria. The presence of selenium with methimazole alleviated its harmful effect on all the above measured parameters. Indeed, El-Demerdash (2004) demonstrated that selenium was beneficial in decreasing creatinine levels in several rat tissues. Also, sodium selenite is able to maintain a functional renal state in the case of intoxication (Rudenko et al., 1998) via its important antioxidant properties. Another biochemical marker employed in this study to evaluate kidney function was uric acid levels in plasma and urine. Under our experimental conditions, we noted a decrease in plasma uric acid levels and an increase in urinary excretion of the methimazoletreated group. Impairment in kidney function could probably occur via kidney oxidative damage. In fact, uric acid in blood is the most important antioxidant (Ames et al., 1981). This compound is the end product of purine catabolism and can reduce oxidative stress by scavenging various reactive oxygen species (Regoli and Winste, 1999). When produced in excess, it causes tissue injury, including lipid peroxidation, DNA damage and enzyme inactivation (DalPizzol et al., 2001). Previous reports of Bagchi et al. (1995) and Baskol et al. (2007) demonstrated that hypothyroidism might generate reactive oxygen species. Moreover, the activities of antioxidant enzymes depend on the magnitude of the oxidative stress and hence on the dose of stressor. In fact, Iciek et al. (2000) reported that methimazole had the strongest antioxidative properties at the concentration level of 0.5 mM. The protective effects gradually decreased at higher and lower concentrations of this drug. In this regard, with the methimazole dose tested in our study, the organism became unable to counteract the high reactive oxygen species production. In fact, the increased malondialdehyde levels, the major product of lipid peroxidation, and the decline in the levels of ␣-tocopherol (vitamin E) suggested that methimazole treatment probably induced oxidative stress in the kidney tissue of mothers and their pups. Indeed, the reduction of ␣-tocopherol, the most effective chain breaking lipid soluble antioxidant present in cell membranes that plays a major role in maintaining their integrity, increased methimazole likelihood to cause oxidative damage. Our results are in line with previous findings of Banudevi et al. (2006) who found similar changes in the kidney of adult rats exposed to Polychlorinated biphenyls. In our experimental finding, the co-administration of selenium to the methimazole-treated group restored malondialdehyde and vitamin E 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). Indeed, Ithayarasi and Devi (1997) and El-Demerdash (2001) demonstrated that selenium maintained the activities of antioxidant enzymes to near normal levels, thus emphasizing its effects as an antioxidant. Our investigation demonstrated that the co-administation of selenium also improved enzymatic (glutathione peroxidase, superoxide dismutase and catalase) and non-enzymatic (glutathione) antioxidants activities, demonstrating the role of this compound against oxidative stress probably generated by methimazole exposure in rat kidney. So, the increases of selenium-dependent antioxidant enzymes activities such as glutathione peroxidase might decrease free radical-mediated lipid peroxidation and regenerate reduced glutathione (Gan et al., 2002). Therefore, this trace element could be useful as a free radical scavenger compound against stress conditions in several tissues, including the kidney. Our histopathological data substantiate kidney dysfunction. There were leucocytic infiltrations considered, according to AbdelRahman and Zaki (1992), as a prominent response of the body tissue facing any injurious impacts. Renal lesions were also characterized by vascular congestion as well as tubular obstruction. These processes are proposed to extend the initial injury to the renal tubules, as reported by Sutton et al. (2002). We have also described tubu-

lar cell necrosis in pups. These modifications could be due to the accumulation of free radicals resulting from an increased lipid peroxidation in the renal tissues of the methimazole-treated group. Thus, the improvement of the methimazole-induced histological alterations could be attributed to the antioxidant efficacy of selenium. It can be concluded from the present study that methimazoleinduced hypothyroidism in pregnant and lactating rats and their pups is associated with marked alterations in their renal function and in enzymatic and non-enzymatic components. Our results show that selenium, a nutritional antioxidant, improves total antioxidant status. They also suggest that it is an important nephroprotective element that may be used as a dietary supplement against oxidative damage induced by hypothyroidism.

Acknowledgments This work was supported by the DGRST grants (Appui à la Recherche Universitaire de Base ARUB 99/UR/08-73), Tunisia. The authors are indebted to Mrs Serra Ben Saleh for her skillful technical assistance in vitamin E measurement and to Mr Chedli Bouzid for his assistance in histolological techniques. We also wish to extend our thanks to Mr Bejaoui Hafedh, teacher of English at Sfax Faculty of Science, who has helped to proofread and edit this paper.

References Abdel-Rahman M, Zaki TZ. Cytotoxic action of malathion on renal and hepatic tissues of mice. J Egypt Germ Soc Zool 1992;8:105–14. Aebi H. Catalase in vitro. Method Enzymol 1984;105:121–6. Alturfan AA, Zengin E, Dariyrli N, Alturfan EE, Gumustas MK, Aytac E, et al. Investigation of zinc and copper levels in methimazole-induced hypothyroidism: relation with the oxidant-antioxidant status. Folia Biol 2007;53:183–8. Ames BN, Cathcart R, Schwiers E. Uric acid provides an antioxidant defense in humans against oxidant and radical caused aging and cancer: a hypothesis. Proc Natl Acad Sci USA 1981;78:6858–62. Anan Y, Ogra Y, Somekawa L, Suzuki KT. Effects of chemical species of selenium on maternal transfer during pregnancy and lactation. Life Sci 2009;84: 888–93. Atif F, Yousuf S, Agrawal SK. Restraint stress-induced oxidative damage and its amelioration with selenium. Eur J Pharmacol 2008;600:59–63. Banudevi S, Krishnamoorthy G, Venkataraman P, Vignesh C, Aruldhas MM, Arunakaran J. Role of ␣-tocopherol on antioxidant status in liver, lung and kidney of PCB exposed male albino rats. Food Chem Toxicol 2006;44:2040–6. Bagchi D, Bagchi M, Hassoun EA, Stohs SJ. In vitro and in vivo generation of reactive oxygen species, DNA damage and lactate dehydrogenase leakage by selected pesticides. Toxicology 1995;104:129–40. Baskol G, Atmaca H, Tanriverdi F, Baskol M, Kocer D, Bayram F. Oxidative stress and enzymatic antioxidant status in patients with hypothyroidism before and after treatment. Exp Clin Endocrinol Diabetes 2007;115:522–6. Ben Amara I, Fetoui H, Guermazi F, Zeghal N. Dietary selenium addition improves cerebrum and cerebellum impairments induced by methimazole in suckling rats. Int J Dev Neurosci 2009;27:719–26. Beutler E. Red blood cell metabolism: a manual of biochemical methods. Orlando, FL: Grune Stratton Inc; 1984. p. 83–5. Bradley SE, Bradley GP, Stephan F. Role of structural imbalance in the pathogenesis of renal dysfunction in the hypothyroid rat. Trans Assoc Am Phys 1972;85:344–52. Cano-Europa E, Pérez-Severiano F, Vergara P, Ortiz-Butrón R, Ríos C, Segovia J, et al. Hypothyroidism induces selective oxidative stress in amygdala and hippocampus of rat. Metab Brain Dis 2008;23:275–87. Cano-Europa E, Blas-Valdivia V, Franco-Colin M, Gallardo-Casas CA, OrtizButron R. Methimazole-induced hypothyroidism causes cellular damage in the spleen, heart, liver, lung and kidney. Acta Histochem 2009., doi:10.1016/j.acthis.009.07.004. Charrel M. Urée et créatinine. In: Sémiologie biochimique [urea and creatinin]. Paris: Ellipses Publisher; 1991. p. 124–8. Council of European Communities: Council instructions about the protection of living animals used in scientific investigations. Official Journal of the European Communities (JO 86/609/CEE) L358, 1986; 1–18. Cocks JA, Balázs R, Johnson AL, Eayrs JT. Effect of thyroid hormone on the biochemical maturation of the rat brain: conversion of glucose-carbon into amino acids. J Neurochem 1970;17:1275–85. Dal-Pizzol F, Klamt F, Bernard RJS, Moreira JCF. Mitogenic signalling mediated by oxidants in retinal treated Sertoli cells. Free Radic Res 2001;35:749–55. Draper HH, Hadley M. Malondialdehyde determination as index of lipid peroxidation. Methods Enzymol 1990;186:421–31.

I. Ben Amara et al. / Experimental and Toxicologic Pathology 63 (2011) 553–561 Edward AC. Thyroid hormones and drugs that affect the thyroid. In: Smith CM, Reynard AM, editors. Text-book of pharmacology. Philadelphia, PA: W.B. Saunders Company; 1992. p. 652–6. El-Demerdash FM. Antioxidant effect of vitamin E and selenium on lipid peroxidation, enzyme activities and biochemical parameters in rats exposed to aluminium. J Trace Elem Med Biol 2004;18:113–21. El-Demerdash FM. Effects of selenium and mercury on the enzymatic activities and lipid peroxidation in brain, liver, and blood of rats. J Environ Sci Health B 2001;36:489–99. Ellman GL. Tissue sulfhydryl groups. Arch Biochem Biophys 1959;82:70–7. El-Sharaky AS, Newairy AA, Badreldeen MM, Ewedaa SM, Sheweita SA. Protective role of selenium against renal toxicity induced by cadmium in rats. Toxicology 2007;235:185–93. Fishbeck KL, Rasmussen KM. Effect of repeated cycles on maternal nutritional status, lactational performance and litter growth in ad libitum-fed and chronically foodrestricted rats. J Nutr 1987;117:1967–75. Gabe M. Techniques histologiques. In: Masson, editor. Paris, 1968; p. 838–41. Gan L, Liu Q, Xu HB, Zhu YS, Yang XL. Effects of selenium overexposure on glutathione peroxidase and Thioredoxin reductase gene expressions and activities. Biol Trace Elem Res 2002;89:165–75. Golstein J, Corvilain B, Lamy F, Paquer D, Dumont JE. Effects of a selenium deficient diet on thyroid function of normal and perchlorate treated rats. Acta Endocrinol 1988;118:495–502. Iciek M, Polak M, Włodek L. Effect of thiol drugs on the oxidative hemolysis in human erythrocytes. Acta Pol Pharm 2000;57:449–54. Ithayarasi AP, Devi CS. Effect of alpha-tocopherol on lipid peroxidation in isoproterenol induced myocardial infarction in rats. Indian J Physiol Pharmacol 1997;41:369–76. Jollow DJ, Mitchell JR, Zampaglione N, Gillete JR. Bromobenzene induced liver necrosis: protective role of glutathione and evidence for 3,4-bromobenzeneoxide as the hepatotoxic intermediate. Pharmacology 1974;11:151–69. Katsanidis E, Addis PB. Novel HPLC analysis of tocopherols, tocotrienols, and cholesterol in tissue. Free Radic Biol Med 1999;27:1137–40. Larsen PR, Davies TF, Schlumberger MJ, Hay ID. Thyroid physiology and diagnostic evaluation of patients with thyroid disorders. In: Larsen PR, Kronenberg HM, Melmed S, Polonsky KS, editors. Williams textbook of endocrinology. Philadelphia: Saunders; 2003. p. 331–73. Li JQ, Wang X, Yan YQ, Wang KW, Qin DK, Xin ZF, et al. The effects on foetal brain development in the rat of a severely iodine deficient diet derived from an endemic area: observations on the first generation. Physiol Behav 2007;91:299–303. Lowry OH, Rosebrugh NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951;193:265–75.

561

Mandel SJ, Cooper DS. The use of antithyroid drugs in pregnancy and lactation. J Clin Endocrinol Metab 2001;86:2354–9. Marchant B, Brownlie BE, Hart DM, Horton PW, Alexander WD. The placental transfer of propylthiouracil, methimazole and carbimazole. J Clin Endocrinol Metab 1977;45:1187–93. Marchant B, Alexander WD. The thyroid accumulation, oxidation and metabolic fate of 35 S-methimazole in the rat. Endocrinology 1972;91:747–56. Meotti FC, Stangherlin E, Zeni G, Nogueira CW, Rocha JBT. Protective role of aryl and alkyl diselenide on lipid peroxidation. Environ Res 2004;94:276–82. Navarro-Alarcon M, Cabrera-Vique C. Selenium in food and the human body: a review. Sci Total Environ 2008;400:115–41. Ognjanovic BI, Markovic SD, Pavlovic SZ, Zikic RV, Stajn AS, Saicic ZS. Effect of chronic cadmium exposure on antioxidant defense system in some tissues of rats: protective effect of selenium. Physiol Res 2008;57:403–11. Randox Laboratories, Ltd. Radicales Libres, United Kingdom, Crumlin, 1996; 1–16. Regoli F, Winste GW. Quantification of total oxidant scavenging capacity of antioxidants for peroxynitrite peroxyl radicals and hydroxyl radicals. Toxicol Appl Pharmacol 1999;156:96–105. Rock MJ, Kincaid RL, Carstens GE. Effects of prenatal source and level of dietary selenium on passive immunity and thermometabolism of newborn lambs. Small Rum Res 2001;40:129–38. Rudenko SS, Bodnar BM, Kukharchuk OL, Mahalias VM, Rybshchka MM, Ozerova IO, et al. Effect of selenium on the functional state of white rat kidney in aluminium cadmium poisoning. Ukr Biokhim Zh 1998;70:98–105. ˜ SL. Hypothyroidism affects preferentially the dendritic denRuiz-Marcos A, Ipina sities on the more superficial region of pyramidal neurons of the rat cerebral cortex. Brain Res 1986;393:259–62. Schmitt R, Klussmann E, Kahl T, Ellison DH, Bachmann S. Renal expression of sodium transporters and aquaporin-2 in hypothyroid rat. Am J Physiol Renal Physiol 2003;284:1097–104. Schwartz HL, Ross ME, Oppenheimer JH. Lack of effect of thyroid hormone on late fetal rat brain development. Endocrinology 1997;138:3119–24. Skowsky WR, Kikuchi TA. The role of vasopressin in the impaired water excretion of myxedema. Am J Med 1978;64:613–21. Smith A, Temple K. Selenium metabolism and renal disease. J Renal Nutr 1997;7:72–89. Steinbrenner H, Sies H. Protection against reactive oxygen species by selenoproteins. Biochim Biophys Acta 2009., doi:10.1016/j.bbagen.2009.02.014. Sutton TA, Fisher CJ, Molitoris BA. Microvascular endothelial injury and dysfunction during ischemic acute renal failure. Kidney Int 2002;62:1539–49.