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The effect of aspartame on the pituitary thyroid axis of adult male albino rat and the possible protective effect of Pimpinella anisum oil: histological and immunohistochemical study

El Haliem, Nesreen G.A.

The Egyptian Journal of Histology: March 2013 - Volume 36 - Issue 1 - p 195–205
doi: 10.1097/01.EHX.0000425919.49371.ec
Original articles

Background: Aspartame is a synthetic sweetener. Its metabolites can be toxic to many organs such as liver and kidney. Pimpinella anisum (P. anisum) has been used for different purposes as an antioxidant, hepatoprotective, and anti-inflammatory agent.

Aim: The present work was carried out to study the histological changes in the pituitary thyroid axis of adult male albino rats after aspartame treatment and the possible role of P. anisum in minimizing these changes.

Materials and methods: Twenty-five adult male Albino rats were used. They were divided into three groups: group I was the control group, group II received 250 mg/kg/day aspartame once daily for 2 months, and group III received prophylactic P. anisum oil 0.5 ml/kg/day once daily, followed by aspartame after 2 h for 2 months. At the end of the experiment, the rats were sacrificed. The thyroid and pituitary gland tissue samples were processed for light microscopic and electron examination. Also, an immunohistochemical study was carried out for the detection of thyrotrophs.

Results: Light microscopic examination of aspartame-treated animal showed loss of architecture of the thyroid gland. Many follicles were small in size and others had disrupted wall and detached cells in their lumens. Some thyrocyte had pyknotic nuclei and deeply stained vacuolated cytoplasm. There was a highly significant increase in the number of positive immunostained thyroid-stimulating hormone cells. Most cells in pars distalis were hypertrophied with eccentric nuclei and a large negative Golgi image. The thyrotrophs and somatotrophs had dilated cisternae of rough endoplasmic reticulum, destroyed mitochondria, and few secretory granules. Some cells with secretory granules of both somatotrophs and thyrotrophs were frequently observed. The administration of P. anisum induced improvements in the degenerative changes of this axis.

Conclusion: From this study, it could be concluded that prolonged consumption of aspartame induced disturbance in the pituitary thyroid axis. The use of P. anisum decreased the toxic effect of aspartame.

Department of Histology, Faculty of Medicine, Sohag University, Sohag, Egypt

Correspondence to Nesreen G.A. El Haliem,Department of Histology, Faculty of Medicine, Sohag University, Sohag, Egypt Tel: +20 100 656 6743; fax: +20 934 602 963; e-mail: nesreengamal2000@yahoo.com

Received July 25, 2012

Accepted October 3, 2012

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Introduction and aim

Aspartame is used as a nonsugar sweetener in food and beverages and as a noncaloric additive in diabetes and obesity diets. It was discovered accidentally in 1965 by James Schlatter, and approved for use by the Food and Drug Administration in the 1970s 1. Aspartame is metabolized in the gastrointestinal tract of rodents, nonhuman primates, and humans into its three constituents: aspartic acid (40%), phenylalanine (50%), and methanol (10%) 2. Aspartic acid and phenylalanine are amino acids, and they are responsible for disturbance in the level of several neurotransmitters in the brain 3,4. Some researches have reported that aspartame does not produce any endocrine-like activities 5,6, whereas others have reported that its metabolites decrease the secretion of some pituitary hormones 7.

Nowadays, there is an increase in the use of herbal medicine instead of chemical compounds such as Pimpinella anisum (P. anisum) seeds 8,9.

P. anisum has been used as a traditional aromatic herb in many drinks and baked foods because of the presence of volatile oils in its fruits. In traditional medicine, aniseed has been used for the treatment of nausea, abdominal colic, insomnia, and epilepsy 10,11. Moreover, it plays a major role in controlling excess oxidative radical formation at cell membranes 12. The principal constituents of P. anisum are anise oil (1–4%). The major component of anise oil, transanethole (75–90%), is responsible for its characteristic taste and smell and is considered as an active estrogenic agent. Other constituents include coumarins (umbelliferone, umbelliprenine, bergapten, and scopoletin), lipids (fatty acids, β-amyrin, and stigmasterol), and flavonoids 13.

Considerable previous work was focused on the effects of aspartame on the pituitary–gonadal axis 14. In contrast, data on aspartame action on the pituitary–thyroid axis are lacking.

Therefore, the aim of the present study was to determine the effect of long-term aspartame administration on the histological structure of this axis and the possible protective role of P. anisum on these effects.

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Materials and methods

Materials

A total of 25 adult male Albino rats were used in the present study and they weighed between 150 and 200 g. The animals were divided into three groups.

  • Group 1 (five animals) was used as a control group.
  • Group II (10 animals) received 250 mg/kg/day aspartame once daily for 2 months 15 through an orogastric tube. Dose consumptions between rats and humans were corrected by factor 5 as aspartame metabolizes in rats faster than that in humans 16.
  • Group III (10 animals) received 0.5 mg/kg/dayP. anisum oil through an intraperitoneal injection 2 h before aspartame at the same previous dose and duration 17.
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Preparation of materials

Aspartame was obtained from Al-Ameriya Pharma Company (Alexandria, Egypt). It is available in the form of tablets, each containing 20 mg of aspartame. The tablets were dissolved in distilled water and administered to the rats. P. anisum oil was obtained from a spices and aromatics shop in Sohag (Egypt).

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Light microscopy

Specimens were taken from the thyroids and pituitaries of the control and treated animals. They were fixed in 10% formalin for H&E stain and immunohistochemical staining for the β-chain of thyroid stimulating hormone (TSH).

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Immunohistochemical technique

It was carried out following the avidin–biotin–peroxidase complex method. Sections of pituitaries were deparaffinized in xylene, rehydrated in ethyl alcohol, and washed twice in distilled water. Endogenous peroxidases were blocked by treatment with 5% hydrogen peroxidase for 10 min. For antigen retrieval, the sections were microwaved twice (5 min each) in a microwave oven in a citrate buffer (10 mmol/l sodium citrate buffer, pH 6.0) and heated in a microwave oven (at a power of 750 W). Immunoperoxidase staining for TSH was carried out using a monoclonal antibody at a dilution of 1:200 (Thermo Fisher, Fremont, California, USA). This antibody recognizes thyrotrophes by cytoplasmic reactions. The sections were incubated with the primary antibody for 60 min at 25°C and then they were washed twice with PBS. They were incubated for 30 min with the biotinylated secondary antibody, followed by washing in PBS and a 30-min incubation in streptavidin peroxidase. A mixture of a DAB chromogen+DAB substrate at a ratio 20 μl : 1 ml was added to the sections and then incubated for 10 min. The slides were counterstained with Mayer’s hematoxylin, dehydrated, and mounted with DPX (BDH Ltd, Poole, UK) 18. The cytoplasmic site of the reaction stained brown, whereas the nuclei appeared blue.

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Transmission electron microscopy

Immediately after the animals were sacrificed, pituitaries were fixed in 5% gluteraldehyde for 24 h. The specimens were then washed in three to four changes of cacodylate buffer (pH 7.2) for 20 min at every change and postfixed in 1% osmium tetroxide for 2 h. Then, they were washed in four changes of cacodylate buffer for 20 min each. Dehydration was carried out using ascending grades of alcohol (30, 50, 70, 90, and absolute alcohol) each for 2 h. They were cleared in propylene oxide and then embedded in Epon 812 using a gelatin capsule. These samples were kept in an incubator at 35°C for 1 day, then at 45°C for another day, and finally at 60°C for 3 days 18. Semithin sections (0.5–1 μm) were prepared using an ultra microtome Reichert Supernova (Leica, Wetzlar, Germany). Ultrathin sections (500–800 Aº) from selected areas of trimmed blocks were prepared and collected on copper grids. The ultrathin sections were then contrasted in uranyle acetate for 10 min and lead citrate for 5 min and examined using an electron microscope Jeol TEM 1010 (Tokyo, Japan) at the electron microscopic unit of Faculty of Medicine, Sohag University.

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Morphometric and statistical analysis

Morphometric analysis

The image analyzer Leica ICC50 (Leica) at the Histology Department, Faculty of Medicine, Sohag University, was used to obtain the following morphometric data:

  • Diameter of thyroid follicles using H&E-stained sections at ×400 magnification. The follicular diameters of 50 follicles, in 10 random fields, of the thyroid gland of each rat of the different groups were measured. The measurements were obtained using Digimizer PC Image Analysis Software (version 4.0, 2011; MedCalc Software, Mariakerke, Belgium).
  • Number of positive-stained thyrotrophs in TSH-immunostained sections at ×400 magnification. The number was obtained in nonoverlapping 10 fields in each slide of five different rats in each group.

The mean value and SD of these data obtained for each group were calculated.

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Statistical analysis

Statistical analysis was carried out using the SPSS version 9/PC program (USA). Comparison of significance between the different groups was carried out using a independent t-test. The significance of the data was determined by the P value; P value greater than 0.05 was considered nonsignificant, P<0.05 as significant, and P<0.001 as highly significant.

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Results

Control animals (group I)

Light microscopic examination

The thyroid gland consisted of numerous follicles and interfollicular stroma. The follicle consisted of follicular epithelium and intrafollicular substances. They were uniformly distributed and variable sized as large ones were near the capsule, whereas small ones were toward the center of the gland. They were lined by a single layer of cuboidal epithelium. The follicles were filled with uniformly distributed colloid. In some follicles, colloid was the absorbing type with peripheral vacuoles. Parafollicular cells appeared polygonal with a granular, weakly acidophilic cytoplasm, which was paler and larger than that of the follicular cells. They were located individually or in small groups, both within the follicular wall and in the interfollicular spaces (Fig. 1).

Figure 1

Figure 1

The pars distalis was formed of anastomosing cords or groups of cells separated by blood sinusoids. The cells were of two types: chromophobes and chromophils. The chromophobes had rounded vesicular, relatively large nuclei, and a pale cytoplasm. Two types of chromophils were observed: acidophils and basophils. The somatotrophs were small cells with large nuclei and their cytoplasm was engorged with dense granules. The thyrotrophs were branched cells present in close relation with adjacent blood capillaries and had very fine secretory granules uniformly distributed in their cytoplasm (Fig. 2).

Figure 2

Figure 2

TSH-immunopositive cells were often present in the central part of the pars distalis as small groups or single cells in close proximity to the blood capillaries. TSH immunopositivity was observed as a diffuse granular brown cytoplasmic stain. They varied in size and appearance (polygonal, elongated, or ovoid) (Fig. 3).

Figure 3

Figure 3

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Electron microscopic examination

Somatotrophs were polygonal or oval and sometimes irregular in shape. Their cytoplasm contained rough endoplasmic reticulum (RER), mitochondria, a well-developed Golgi apparatus and secretory granules of variable sizes, but with the same density (Fig. 4). Thyrotrophs were distinguished from other anterior pituitary cells by their small size, angular shape, and small cytoplasmic granules. They contained euchromatic oval nuclei, RER, mitochondria, long slender processes, and small secretory granules (Fig. 5).

Figure 4

Figure 4

Figure 5

Figure 5

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Treated animals

Aspartame-treated animals (group II)

Light microscopic examination: After aspartame treatment, there were various degrees of degenerative changes. There was loss of normal architecture of the thyroid gland with excess connective tissue stroma. Most of the follicles were small sized and almost devoid of colloid (Fig. 6). Others were filled with colloid of no pinocytotic activity. The wall of some follicles was interrupted. The lining cells of the many follicles had pyknotic nuclei and a deeply stained acidophilic cytoplasm, whereas others had a vacuolated cytoplasm and destroyed membrane. Desquamated epithelial cells and mitotic figures were frequently observed in some follicles (Fig. 7).

Figure 6

Figure 6

Figure 7

Figure 7

The pars distalis contained many cells with eccentric nuclei and numerous pale-staining variable-sized vacuoles in their cytoplasm. Some cells had a large negative Golgi image with eccentric nuclei and others had a rarified cytoplasm (Fig. 8).

Figure 8

Figure 8

Immunohistochemical study showed that TSH-positive staining cells were larger and more numerous compared with the control group, either in clusters or as singles. Some of these cells had negatively staining vacuoles in their cytoplasm (Fig. 9).

Figure 9

Figure 9

Electron microscopic examination: Somatotrophs with euchromatic nuclei and dilated RER were frequently observed (Fig. 10).

Figure 10

Figure 10

The majority of thyrotrophs had irregular euchromatic nuclei with clumps of heterochromatin and dilated perinuclear space and the mitochondria were swollen, with partially destroyed cristae. The secretory granules were in the excretory phase as most of them were in contact with the cell membrane (Fig. 11).

Figure 11

Figure 11

The degenerated thyrotrophs had small eccentric irregular hyperchromatic nuclei, dilated sacuoles of the Golgi body, few secretory granules, and dilated RER cisternae filled with moderate electron density material (Fig. 12). Some cells contained two types of granules; one as large as those of somatotrophs and others as small as those of thyrotrophs were also observed (Fig. 13).

Figure 12

Figure 12

Figure 13

Figure 13

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Combined treated animals (group III)

Light microscopic examination: The histological changes in the thyroid gland decreased in comparison with the group treated with aspartame. Most of the thyroid follicles were more or less similar to those of the controls apart from some thyroctes with a vacuolated cytoplasm. Small-sized follicles were still observed. They were lined with cuboidal epithelium and filled with colloid of the absorbing type (Fig. 14). Most cells of the pars distalis were similar to those of the control group and decreased in size in comparison with the aspartame-treated group. Some cells with a vacuolated cytoplasm were still observed (Fig. 15).

Figure 14

Figure 14

Figure 15

Figure 15

TSH-immunopositive cells decreased in number in comparison with those in group II and were more or less similar as group I (Fig. 16).

Figure 16

Figure 16

Electron microscopic examination: Somatotrophs and thyrotrophs appeared similar to those of the control group, containing euchromatic nuclei, mitochondria, RER, Golgi bodies, and secretory granules (Figs. 17 and 18).

Figure 17

Figure 17

Figure 18

Figure 18

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Statistical results

In the statistical study, there was a statistically highly significant decrease in the mean diameter of the thyroid follicles and a highly significant increase in the mean number of positive immunoreactive thyrotrophs in group II in comparison with the control group. In group III, there was a highly significant increase and decrease in the mean diameter of the follicles and number of thyrotrophs, respectively, compared with group II, whereas there was a non significant decrease in the diameter of the thyroid follicle in comparison with group I. All these data were presented in Table 1 and graphically illustrated in Chart 19 and 20 and 2.

Table 1

Table 1

Chart 19

Chart 19

Chart 20

Chart 20

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Discussion

The results obtained from the present study clearly showed that the chronic administration of aspartame induced histological changes in the pituitary thyroid axis. Light microscopic examination of thyroid glands showed marked loss of architecture. Many follicles showed apoptotic changes where most of the lining epithelial cells had pyknotic nuclei and a deeply stained acidophilic cytoplasm. Necrotic cells with vacuolated cytoplasm and destroyed cell membrane were less observed. Numerous desquamated cells appeared in the lumen of some follicles. There was a statistically highly significant decrease in the mean follicular diameter as most of the follicles appeared smaller in size, with no colloid. These follicles might be newly formed, stimulated, or atrophic follicles. These degenerative changes could be attributed to the direct toxic effect of aspartame on thyrocytes. This might occur as a result of the release of the free radicals secondary to the production of methanol and aspartic acid after the ingestion of aspartame. The accumulation of these radicals exerts a potent damaging effect on the cell 19. In agreement with our explanation, many studies have proved that methanol could increase the lipid peroxidation products and activate the intrinsic pathway of apoptosis 20. Furthermore, some studies have speculated that the methanol accumulated in the mitochondria and could influence the oxidative phosphorylation 21. This is associated with depletion of the ATP stores in the cells and low concentrations of glucose that could inhibit the synthesis and release of thyroid hormones from thyroid follicular epithelial cells 22. In agreement with our findings, it has been suggested that aspartame interferes with the thyroid function at the glandular level as well as at the peripheral level by inhibiting the conversion of T4 into T3 in the liver and kidney secondary to their damage by its toxicity 23,24. Moreover, it has been reported that aspartame decreases serotonin level through an increased supply of phenylalanine 25. Serotonin is necessary for the synthesis or the release of T3, and activates thyroid follicular cells by enhancing them to extend pseudopodia and engulf thyroglobulin from the follicular lumen 26,27.

The widening of the interfollicular space might be because of the presence of a large number of atrophic follicles. Excess connective tissue stroma could be a result of the increase in the amount of collagenous fibers secondary to the overproduction of collagen fibers after repeated damage to the epithelial cells with regeneration and secretions of the new fibers. However, this thickening might be because of an increase in the deposition of glycoproteins 28.

In the present work, most of the cells in the pituitary glands of rats treated with aspartame had a vacuolated cytoplasm and some of them were apparently larger in size. Immunohistochemically, there was a statistically highly significant increase in the number of TSH-positive staining cells compared with the control group. This was in agreement with other studies that have found thyrotroph hyperplasia in long-standing hypothyroid rats 29. These findings were confirmed ultrastructurally as most thyrotrophs showed signs of hyperactivity such as irregular nuclei and few secretory granules that mostly exocytosed. Other cells developed degenerative changes such as irregular hyperchromatic nuclei, dilated sacuoles of Golgi bodies, and numerous variable-sized dilated RER cisternae filled with fine material of low electron density. These changes might occur in chronically stimulated thyrotrophs secondary to the decrease in thyroid hormone production by the affected thyroid, which led to cellular exhaustion. In agreement with this suggestion, some studies have reported that aspartame might form strong complexes with zinc and could lead to a disruption in the balance of the complex ion and also in its homeostasis 30. Zinc levels were significantly positively correlated with free T3 levels; thus, its deficiency could decrease the production of thyroid hormone 31. Similar cells were observed in the pituitaries of experimentally induced hypothyroid rats 32,33 and in patients with untreated primary hypothyroidism 34. However, stimulating thyrotrophs might be a result of the direct effect of aspartame on hypothalamus, which increased the secretion of thyrotropin-releasing hormone. In agreement with this theory, it was reported that the decreased level of serotonin caused by aspartame might have led to dysregulation of the hypothalamus–pituitary–thyroid axis 35. In contrast to this, some authors found that aspartame caused a dose-dependent increase in the brain tumor necrotizing factor-α 36 that inhibits this axis to respond to decreased thyroxin 37.

The mitochondrial changes observed in this study might be considered as early manifestations of apoptosis and adaptive process to unfavorable environments such as excess exposure of the cell to free radicals 21. Some authors agreed with this explanation as they proved that methanol significantly increased the malondialdehyde level and caspase-3 activity 36.

The appearance of some cells that contained two types of secretory granules with a predominance of those similar to thyrotrophs might be a way to increase the number of the latter through the process called transdifferentiation. These bihormonal cells could play an adaptive role in situations of physiologic demand for hypersecretion of a specific hormone. Similar cells were found in the pituitary of thyroidectomized and experimental hypothyroid rats, which were called somatothyrotroph cells 38,39. Some authors have reported the same changes in other cells in pars distalis as somato/mamotrophs 40, somato/gonadotrophs 41,42, cortico/mamotroph 43, cortico/gonadotrophs 44, or cortico/thyrotrophs 45. In agreement with these findings, some studies have found a relation between both somatotrophs and thyrotrophs. They have reported that Pit-1, a transcription factor necessary for the expression of both the growth hormone (GH) and the βTSH-subunit genes 46,47, is produced in both somatotrophs and thyrotrophs 48, and thyroid hormones exert their feedback effect on both cells 49. This was confirmed by the presence of dilated RER cisternae and few secretory granules in the cytoplasm of somatotrophs that represent their hyperactivity. The same relation was proved by some authors who found that thyroxin replacement led not only to a decrease in plasma TSH levels but also an enhanced GH response to growth releasing hormone stimulation 40. Indeed, numerous studies have concluded that thyroid hormones are important regulators of both GH and TSH gene expression 50,51.

The present study found that the administration of P. anisum oil was effective in decreasing the toxic effect of aspartame on the pituitary thyroid axis. Most of the thyroid follicles were more or less similar to the control group apart from a few thyrocytes with a vacuolated cytoplasm. Statistically, there was no significant decrease in the mean follicular diameter compared with the control group.

This improvement might be secondary to the antioxidant ability of anise, which attacks reactive oxygen species and thus neutralizes their harmful effects on the tissues. It was found that anise could have a radical scavenging effect, inhibiting H2O2-chelating and Fe2+-chelating activity by more than 70% 52. The polyphenols present in the anise seeds could donate electrons and react with free radicals to convert them into more stable products and terminate the free radical chain reaction, whereas other compounds in the oil act as chain-breaking agents in lipid peroxidation 53. It was proved that the antioxidant activities of P. anisum could be activated by its positive effect on vitamin C and E levels 54. The increase in vitamin C could be because of the protection of the existing vitamin C from oxidation to dehydroascorbic acid by some of the antioxidant phytochemicals present in the anise seeds. However, vitamin C has a positive effect on cell activity and increases O2 consumption, and as a result, stimulates the thyroid gland, which plays a major role in the metabolism. Some studies have reported that there is a positive correlation between thyroxin secretion and body weight in the presence of vitamin C. This increases the metabolism of phenylalanine and tyrosine, which are the main amino acids in thyroid hormone synthesis, maintains GH secretion, and increases the basal metabolism 55.

In the present study, there was a highly significant decrease in the number of thyrotrophs compared with those of the aspartame group that might be secondary to improvement in the histological structure of the thyroid gland and restoring the low serotonin level induced by aspartame. In agreement with this suggestion, some studies have reported that P. anisum could inhibit serotonin reuptake and thereby increase the levels of serotonin in synaptic clefts 56. Furthermore, some studies have reported that flavonoids, one of the components of P. anisum, could improve the toxic effect of aspartame on the thyroid gland, where it has been known to act as a naturally occurring selective estrogen receptor modulator. It might show affinity for estrogen receptor-α 57 in livers that respond to estrogen by regulating liver function 58,59, as increased the production and secretion of more heavily sialyated thyroid binding globulins and reduced theirclearance 60,61.

In the light of these findings, it was concluded that aspartame disrupted the pituitary–thyroid axis function in male rats. It affected the thyroid follicular structure and consequently led to a feedback stimulation of pituitary cells. However, P. anisum was found to reduce its toxic effects on the thyroid gland and prevent its secondary effect on the pituitary. Further investigations should be performed in order to determine the bioactive principles of the essential oil (transanethole, anisaldehyde, estragole, anisketone) and the molecular mechanism responsible for its protective action. In addition, further studies should be carried out on the synergistic action of P. anisum and other antioxidants to overcome the remaining histological changes with P. anisum alone.

Table

Table

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Acknowledgements

Conflicts of interest

There is no conflict of interest to declare.

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Keywords:

aspartame; Pimpinella anisum; thyroid follicle; thyroid stimulating hormone; thyrotrophs

© 2013 The Egyptian Journal of Histology