Thyroid hormones, tri-iodothyronine (T3) and tetraiodothyronine (T4) are crucial in regulating the growth and the differentiation of many tissues and organs such as the maturation and function of the nervous system [1]. Several reports have been published on the relation between hyperthyroidism and oxidative stress. In fact, high concentrations of thyroid hormones stimulate free radical formation in mitochondria by affecting oxygen metabolism [2]. Although reactive oxygen species play an important role in physiological mechanisms extremely reactive oxygen radicals can cause severe oxidative damage to molecules [3].

Hyperthyroidism is known to stimulate ROS generation and induce oxidative stress in many tissues by disturbing the endogenous prooxidant/ anti-oxidant equilibrium [4]. Others in vivo and in vitro studies indicate that thyroid hormones have a considerable impact on oxidative stress. Similarly, Xing et al. show that hyperthyroidism increased MDA (malondialdehyde) and glutathione (GSH) levels in cerebral, hepatic and cardiac tissues of rat [5]. Indeed, hyperthyroidism accelerates several physiologic processes, a fact which is reflected in the increased renal blood flow, hypertrophic and hyperplastic tubuli, and increased glomerular filtration rate [6]. Other findings reported that hyper metabolism due to hyperthyroidism is associated with an increase in formation of free radicals and lipid peroxide levels in many organs [7]. It is reported that not only hyperthyroidism but also hypothyroidism lead to changes in oxidant and antioxidant systems [8,9]. The action of thyroid hormones (thyroxine, T4; triiodothyronine, T3) on brain development and function is gaining renewed interest. In fact, mammalian brain maturation was stimulated by thyroid hormones [9]. For several years, a special attention was paid to the dietary regimen and more particularly to natural products (fruits, vegetables) for their capacities to reduce oxidative damages induced by various medicals terms as hypo and hyperthyroidism, as medicinal plant we find Eruca sativa . This plant is an annual and biannual herb and is one of the varieties of mustard. The plant originated in the Mediterranean region but is presently found round the world. This plant has a wide spread medicinal use. Traditionally, its use as astringent, diuretic, digestive, emollient, tonic, depurative, laxative, rubefacient and stimulant is well- documented [10,11].

Eruca sativa has several antioxidant constituents including glucosinolates, flavonoides, carotenoids [12]. Several reports are found in the literature about the antifungal activity of these compounds on postharvest pathogens [13,14].

In this study, we aimed to investigate whether a dietary regimen enriched for 7 weeks of Eruca sativa oil, could prevent hyperthyroidism induced by levothyroxin injection as a synthetic thyroid hormone that is chemically identical to thyroxine (T4) in male rat. Serum levels of TSH, FT4, calcium, creatinine, urea, proteins and uric acid and activities of antioxidant enzymes (Superoxide-dismutase, catalase, glutathione peroxidase) in thyroid, kidney and brain were measured.

Essential oil extraction of Eruca sativa

The essential oils were extracted by hydrodistillation of dried plant material (100 g of Eruca sativa ) in 500 ml of distilled water) using a Clevenger-type apparatus for 4.5 h as described in the European Pharmacopeia. The essential oil was collected, dried under anhydrous sodium sulphate and stored at 4°C until used [15].

Experimental design

3-month-old Wistar male rats, about 160 g body weight, fed on 15% proteins food pellets (SNA, Sfax, Tunisia), were kept in a breeding farm, at 22°C, with a stable hygrometry, under constant photoperiod.

The rats were divided into 4 batches: (C) was control group, (L) levothyroxin treated animals injected (i.p) (20 mg/kg), (ES) was a group treated with Eruca sativa oil during 7 weeks by forcible feeding and (ES+L) was a group previously treated with Eruca sativa oil by forcible feeding and injected (i.p) by levothyroxin for 3 days.

At the end of this experimental period, all animals were rapidly sacrificed by decapitation in ordrer to minimize the handling stress. Blood serum was obtained by centrifugation (1500 xg, 15 min, 4°C). Thyroid, kidney and brain were stored at -80°C until use.

The experimental protocol was performed according to the European convention for the protection of vertebrate animals used for experimental and other scientific purposes (Council of Europe No 123, Strasbourg) and approved by the ethics committee for research on laboratory animal use of our institution.

Biochemical assay

Levels of FT4, TSH, calcium, creatinine, urea, proteins, urea and acid uric in serum were determined by kit methods Spinreact.

The level of lipid peroxidation was measured as thiobarbituric acid reactive substances (TBARS), according to Yagi’s method [16]. For the assay, 125 μl of supernatant (S1) were mixed with 175 μl of 20% trichloroacetic acid containing 1% butyl-hydroxytoluene and centrifuged (1000 xg, 10 min, 4°C). Then, 200 μl of supernatant (S2) was mixed with 40 μl of HCl (0.6 M) and 160 μl of thiobarbituric acid (0.72 mM) and the mixture was heated at 80°C for 10 min. The absorbance was measured at 530 nm. The amount of TBARS was calculated using an extinction coefficient of 156 mM^-1 cm^-1 and expressed as n moles/mg protein.

The total superoxide-dismutase (SOD) activity was determined by measuring its ability to inhibit the photoreduction of nitrobluetetrazolium (NBT) [17]. One unit of SOD represents the amount inhibiting the photoreduction of NBT by 50%. The activity is expressed as units/mg protein, at 25°C.

Glutathione-peroxidase (GPX) activity was assayed according to the method of Flohe and Gunzler. [18]. The activity at 25°C was expressed as μ moles of GSH oxidized/min/g protein.

Catalase (CAT) activity was measured according to Aebi [19]. The reaction mixture (1 ml) contained 100 mM phosphate buffer (pH=7), 100 mM H₂O₂ and 20 μl (about 1-1.5 mg of protein) of liver. H₂O₂ decomposition was followed at 25°C by measuring the decrease in absorbance at 240 nm for 1 min. Besides, the enzyme activity was calculated using an extinction coefficient of 0.043 mM^-1 cm^-1 and expressed in international units (I.U.) i.e., in μ moles H₂O₂ destroyed /min/mg protein.

Protein content in tissue extracts was determined according to Lowry’s method [20] using bovine serum albumine as standard.

Gas chromatography-mass chromatography (GC-MC)

The essential oils were analysed using an Agilent-Technologies 6890 N Network GC system equipped with a flame ionization detector and HP-5MS capillary column (30 m × 0.25 mm, film thickness 0.25 μm; Agilent-Technologies, Little Falls, CA, USA). The injector and detector temperatures were set at 220 and 290°C, respectively. The column temperature was programmed from 80 to 220°C at a rate of 4°C/min, with the lower and upper temperatures being held for 3 and 10 min, respectively. The flow rate of the carrier gaz (Helium) was 1.0 ml/min. A sample of 1.0 μl was injected, using split mode (split ratio, 1:100). All quantifications were carried out using a built-in data-handling program provided by the manufacturer of the gas chromatograph. The identity of the components was assigned by comparison of their retention indices, relative to n- alkanes indices and GC-MS spectra from a home-made library, constructed based on the analyses of reference oils, laboratory-synthesized components and commercial available standards [21,22].

As shown in Figure 1, hyperthyroidism induced a significant decrease of TSH and a significant increase of FT4 in serum respectively by 66% and +148% (Figure 1) as compared to controls. In the same group our data show a significant increase by +31% in calcitonine level as other biomarkers of thyroid dysfunction.

Figure 1: Plasma free thyroxin (FT4) (pmol/l) (A) and TSH levels (uIU/ml) (B) after 7 weeks and 3 days of treatment in: controls (C), Levothyroxin-treated rats (L), Eruca sativa given rats (ES) and rats previously given Eruca sativa then treated by Levothyroxin (L+ ES). Values are the mean of 6 measurements ± SD. ** Significant difference as compared to control rats (C) (p ≤ 0.01). ++ Significant difference when comparing (L+ ES) groups to L group.

As compared to controls, levothyroxin affected kidney function which is evidenced by an increase levels of creatinine and urea levels by +50% and +72%, respectively and a decrease levels of proteins and acid uric by -29% and -23% respectively in serum (Figures 2 and 3).

Figure 2: Serum creatinine (µmol/l) (A), Urea mmol/l (B) levels after 7 weeks and 3 days of treatment in: controls (C), Levothyroxin-treated rats (L), Eruca sativa given rats (ES) and rats previously given Eruca sativa then treated by Levothyroxin (L+ ES). Values are the mean of 6 measurements ± SD. ** Significant difference as compared to control rats (C) (p ≤ 0.01). ++ Significant difference when comparing (L+ ES) groups to L group.

Figure 3: Serum proteins (g/l) (A), uric acid (µmol/l) (B) and calcium(mmol/l) (C) levels after 7 weeks and 3 days of treatment in: controls (C), Levothyroxin-treated rats ( L), Eruca sativa given rats (ES) and rats previously given Eruca sativa then treated by Levothyroxin  (L+ ES). Values are the mean of 6 measurements ± SD. ** Significant difference as compared to control rats (C) (p ≤ 0.01). ++ Significant difference when comparing (L+ ES) groups to L group.

Interestingly, these adverse effects of levothyroxine were much alleviated in animals previously treated with Eruca sativa oil.

The TBARS level in thyroid, kidney and brain increased in levothyroxin-treated rats as compared to controls by +87%, +133% and +69% respectively (Tables 1 and 2). SOD activities in kidney, brain and thyroid were significantly decreased by 91%, 80% and by 48% respectively. Again, when rats were previously treated with the Eruca sativa oil this effect of levothyroxin poisoning was much reduced. Activities of GPX and CAT were found to be respectively reduced (by 50% and 54% respectively) in thyroid and (by 77% and 44%, respectively) in kidney and by 66% and 48% in brain of levothyroxintreated rats, as compared to controls (Tables 3 and 4). These changes were alleviated when the rats were previously treated with Eruca sativa oil.

Table 1: TBARS (nmol/mg protein) levels in thyroid, brain and kidney after 7 weeks and 3 days of treatmentin: controls (C), Levothyroxin-treated rats (L), Eruca sativa given rats (ES) and rats previously given Eruca sativa then treated by Levothyroxin  (L+ES). Values are the mean of 6 measurements ± SD. ** Significant difference as compared to control rats (C) (p ≤ 0.01). ++ Significant difference when comparing (L+ES) groups to L group. (p ≤ 0.01).

Table 2: SOD (U/mg protein) levels in thyroid, brain and kidney after 7 weeks and 3 days of treatment in: controls (C), Levothyroxin-treated rats ( L), Eruca sativa given rats (ES) and rats previously given Eruca sativa then treated by Levothyroxin (L+ES). Values are the mean of 6 measurements ± SD. ** Significant difference as compared to control rats (C) (p ≤ 0.01). ++ Significant difference when comparing L+ES groups to L group. (p ≤ 0.01).

Table 3: GPX (U/mg protein) levels in thyroid, brain and kidney after 7 weeks and 3 days of treatment in: controls (C), Levothyroxin-treated rats (L), Eruca sativa given rats (ES) and rats previously given Eruca sativa then treated by Levothyroxin (L+ES). Values are the mean of 6 measurements ± SD. **Significant difference as compared to control rats (C) (p ≤ 0.01). ++ Significant difference when comparing L+ES groups to L group. (p ≤ 0.01).

Table 4: CAT (U/mg protein) levels in thyroid, brain and kidney after 7 weeks and 3 days of treatment in: controls (C), Levothyroxin-treated rats (L), Eruca sativa given rats (ES) and rats previously given Eruca sativa then treated by Levothyroxin (L+ES).

GC/MS analysis: The results obtained with the GC/MS analysis of the oil are depicted in Table 5 that shows the identified constituents, the percentage composition, retention time and retention index. The essential oil was found to contain 32 constituents representing 98.87% of the total essential oil while minor constituents (1.13%) of the oil remained unidentified. The main constituents were found, 2- Phenylethyl, isothiocyanate (30.59%), Methyl stearate (20.31%), Eucalyptol (15.61%), 5-methylthiopentanonitrile (6.87) and Camphor (6.34%).

Table 5: GC/MS chromatogram of Eruca sativa essential oil. TR^b: Retension Time (min) KI^a: Retention Index (kovats).

The thyroid gland is an organ that contribute in regulating the body’s metabolism and other body functions and maintain body homeostatic. This means, high concentrations of thyroid hormones play an important role in inducing oxidative stress in many organs by stimulating free radical formation in mitochondria, affecting oxygen metabolism and the antioxidant defence system [23]. In our study, the injection of levothyroxine, as a synthetic thyroid hormone that is chemically identical to thyroxine (T4) and is naturally secreted by the follicular cells of the thyroid gland, was used to induce hyperthyroidism. The effectiveness of levothyroxine administration in inducing hyperthyroidism was confirmed by significant increase in FT4 together with obvious decrease in TSH circulating levels. Also, an increase in lipid peroxidation in thyroid was also signaled after this treatment. This is can be explained in the light of previous findings by the considerable impact of thyroid hormones on oxidative stress and that thyroid hormones act on mitochondria by regulating energy metabolism, and mitochondria are a major source of intracellular free radicals [24]. Moreover, this thyroid dysfunction can be also associated with hyper-metabolic state which caused mainly by oxidative stress which induced damage to molecules, then affected many organs. It seems that when thyroid hormones increase metabolic systems of the body in general, the resulting increase in reactive oxygen types inevitably leads to lipid peroxidation [25]. On the other hand, hyperthyroidism leads to changes in both oxidant and antioxidant systems. In this finding, a significant elevation of lipid peroxidation, together with the decrease of SOD, GPX and CAT activities in levothyroxin treated animals compared to controls was reported, showing that the increase in free radicals signaled by could not be overcame by antioxidant system which affected thyroid and others organs.

Because hyperthyroidism affect also nutrient absorption and use by the body, an increase level of calcium was detected in this finding.

Levothyroxin damage also kidney by reducing glomerular filtration rate, an increase level of creatinine and urea and a decrease level of proteins in blood were noted. As other renal markers we find in blood of treated rats, a decrease of uric acid level, as the end product of purine catabolism, exerts antioxidative properties and may participate to the defense against an oxidative stress by scavenging various ROS [26]. Many findings reported that when the thyroid is either hyper-or hypo-functioning, changes in different clinical renal parameters such as glomerular filtration rate, urinary protein, creatinine ratio and markers of tubular function can occur. Vice versa, kidney disease influences circulating thyroid hormones [27]. Besides, an increase of lipid peroxidation level and superoxide dismutase activity together with reduction of CAT and GPX activities in kidney were reported in this data which contribute to the worsening of the oxidative stress. Our results are supported by others findings which recorded that hyperthyroidism increases glomerular filtration rate up to 18% in hyperthyroid rats [28]. According to Xing [29], renal failure progresses in hyperthyroidism due to the damage caused by glomerulosclerosis, proteinuria and oxidative stress. Levothyroxine induced also oxidative stress in brain, because thyroid hormones are essential for brain maturation, and for brain function. This data show also that oxidative stress in brain is evidenced by the increase level in lipid peroxidation and the inhibition of the enzymatic defense system mainly that brain is a large consumer of oxygen and being poor in anti-oxidant defense systems. Thyroid hormone deficiency, even of short duration may lead to irreversible brain damage, the consequences of which depend on the specific timing of onset and duration of thyroid hormone deficiency [30]. Many studies have linked oxidative stress to thyroid cancer by showing its association with abnormally regulated oxidative or antioxidative molecules [31]. The data obtained in the present study showed Eruca sativa oil to have a potent antioxidant activity and protect against levothyroxine induced damages in thyroidal state, kidney and brain. We found that Eruca sativa oil treatment was able to restore TSH, FT4, creatinine, urea, proteins, uric acid and calcitonin levels in plasma. Similar results were reported in mercuric chloride treated rats by Abassi et al. [32] who emphasized the alleviating effect of Eruca sativa on renal damage by reducing creatinine, urea and lipid peroxidation in blood. Eruca sativa effects could be able to reduce lipid peroxidation and restored the levels of SOD, CAT and GPX activities in thyroid, kidney and brain. The possible factors behind the protective effects of Eruca sativa is related to its contents of bioactive substances such as glucosinolates, flavonoids, carotenoids, vitamin C and phenolic compounds [33,34].

Tabert et al. [35] indicated the main antioxidant in E. sativa seed extract to be glucoerucin. The antioxidant activity of glucoerucin plausibly implicates free radical scavenging activity and an ability to induce phase-II metabolizing enzymes [36]. Eruca sativa oil, with its potent free radical scavenging ability was expected to protect the biomolecules from free radical induced damage. This latter might also be expected to exclude Fe³⁺ as a strategy to control ROS. Consistent to report of Khoobchandani [37], our study also indicated that the beneficial effects of Eruca sativa oil can be attributed to scavenging free radical’s properties. It is important to note that the curative and preventive properties of medicinal plants are perhaps due to the presence of various secondary metabolites such as alkaloids, flavonoids, glycosides, phenols, saponins, sterols and phospholipids all known to be efficient radical scavengers [38]. As for the second reason, our research, demonstrate the presences of 32 compounds identified in the essential oil of Eruca sativa , accounting 98.87% of the total essential oil and contributed beneficial effects against hypertroidism [39,40].

Furthermore, Bansal et al. [41] have proven that the antioxidant activity of the essential oil could be attributed in part to the presence of compounds such as 3-Cyclohexene-1-methanol and camphor and its ability to decompose free radicals by quenching reactive oxygen species and trapping radicals before reaching their cellular targets [42].

Eruca sativa oil prevent thyroid, kidney and brain from damage induced by levothyroxin by scavenging ROS by its antioxidants major components. Obtained by GC-MS. Further studies on the isolation of more of antioxidants are in progress.

The authors declare that there is no conflict of interest.