El Haliem, Nesreen G. A.; Mohamed, Doha S.
Introduction and aim of the work
Aspartame is a widely used artificial sweetener consumed by hundreds of millions of people worldwide . Commercial names of the newly introduced Aspartame include ‘Nutra Sweet,’ ‘diet sweet,’ ‘canderil,’ and others. It is about 200 times sweeter than sucrose. It is found in more than 6000 products, for example soft drinks, candies, tabletop sweeteners, and some pharmaceuticals such as vitamins and sugar-free cough drops [2,3].
Aspartame is metabolized in the gastrointestinal tract into aspartic acid, phenylalanine, and methanol and is then later oxidized into formaldehyde and Formic A in many tissues. Formic acid was considered the principal metabolite responsible for the deleterious effects of acute intoxication by methanol in humans and animals .
Several studies on laboratory animals have been carried out to verify its toxicity. It was confirmed that aspartame was a multipotential carcinogenic agent . The intake of aspartame might impair the renal function . The effect of aspartame on the liver is still a matter of controversy, and some authors have found no change in the function of the hepatic microsomal enzymes . However, some histological changes were observed in fetal liver after the intake of aspartame during pregnancy .
PimPinella anisum is a member of the Umbelliferae and grows naturally in Egypt. It is found in North-Eastern Anatolia . The fruits of the anise plant are locally known as yansoon. The seeds of P. anisum contain 1.5–6% essential oil, 10–20% fixed oil, and 18% protein. The main constituent of the essential oil is anethole . The essential oil, aqueous, and ethanol extracts of P. anisum exert bronchodilator effects through their inhibitory effects on muscarinic receptors . P. anisum oil has led to hepatoprotective activity on carbon tetrachloride-induced liver injury in rats . It was reported that the essential oil of anise was highly effective as both a larvicidal and an ovicidal agent .
The present study was designed to evaluate the toxic effect of the chronic use of aspartame on the histological structure of the liver and kidney and the protective effect of P. anisum against the changes induced by aspartame.
Materials and methods
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): were used as a control group.
Group II (10 animals) received 250 mg/kg/day aspartame once daily for 2 months  through an intragastric tube. Dose consumptions between rats and humans were corrected by factor 5 as aspartame metabolizes in rats faster than that in humans .
Group III (10 animals) received 0.5 mg/kg/day P. anisum oil through an intraperitoneal injection 2 h before aspartame at the same previous dose and duration .
Preparation of materials
Aspartame was obtained from Al-Ameriya Pharma Company (Alexandria) in Egypt. It is available in the form of tablets, each one 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).
Specimens were taken from the kidney and liver of the control and treated animals and were fixed in 10% formalin for H&E stain .
Transmission electron microscopy
Immediately after sacrificing the animals, 10–12 small pieces 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 post fixed 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 one day, then at 45°C for another day, and finally at 60°C for three days . Semithin sections (0.5–1 um) were prepared using an LKB ultra microtome. Ultrathin sections (500–800 Å) 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 5min and examined using an electron microscope ‘Jeol JEM 1010’ (Japan) at the electron microscopic unit of Faculty of Medicine, Sohag University.
Morphometric and statistical analysis
Morphometric analysis. The image analyzer Leica ICC50 D-35578 Wetzlar (Germany). at the Histology Department Faculty of Medicine Sohag University was used to obtain the following morphometric data :
1. The mean distance between the visceral and the parietal layer of the renal corpuscle (urinary space) using H&E-stained sections at × 400 magnification.
2. The mean diameter of the lumen of the proximal convoluted tubule (rounded sections and lined with five cells) using H&E-stained sections at × 400 magnification.
3. The mean diameter of the lumen of the distal convoluted tubule (rounded sections and lined with eight cells) using H&E-stained sections at × 400 magnification.
The previous measurements were obtained in non overlapping ten fields in slides of five different rats in each group.
Statistical analysis. The data obtained were expressed as mean value ± SD and analyzed using an independent t-test. The significance of the data was determined by the P value; P > 0.05 was considered NS, P < 0.05 significant (S), and P < 0.001 highly significant (HS).
Control animals (group I): liver
Light microscopic examination showed that the liver was composed of the classical hepatic lobules (Fig. 1). Hepatocytes were polyhedral in shape, with sharply defined boundaries. They had an acidophilic cytoplasm and central rounded nuclei with one or two prominent nucleoli. Blood sinusoids were seen separating the cords and lined by flattened endothelial cells and von Kupffer cells (Fig. 2).
Ultrastructurally, hepatocytes had euochromatic rounded nuclei and the cytoplasm contained mitochondria, rough endoplasmic reticulum, smooth endoplasmic reticulum, peroxisomes, free ribosomes, and glycogen granules (Fig. 3). Von kupffer cells showed euochromatic irregular nuclei with prominent nucleoli and the cytoplasm contained mitochondria, rough endoplasmic reticulum, and lysosomes (Fig. 4).
Aspartame-treated rats (group II)
Light microscopic examination revealed that treatment with aspartame for 2 months led to major changes in the histological structure of the liver. Some hepatocytes displayed a vacuolated cytoplasm. Dilated congested blood sinusoids were observed. The von kupffer cells were frequently seen and became more prominent (Fig. 5).
Some hepatic lobules exhibited a disturbed parenchymal architecture and some hepatocytes had irregular nuclei and a vacuolated cytoplasm with ill-defined cell boundaries (Fig. 6).
Ultrastructurally, most of the hepatocytes revealed large vacuoles and areas of compactness of cytoplasmic organelles in their cytoplasm (Fig. 7). Some cells had irregular nuclei with clumps of heterochromatin, numerous vacuoles, and few mitochondria compared with the control (Fig. 8). The von kupffer cells revealed irregular nuclei and the cytoplasm contained multiple lysosomes, electron-dense bodies, and occasional vacuoles (Fig. 9).
Aspartame and P. anisum oil-treated rats (group III)
Light microscopic examination showed an obvious improvement in the affected hepatocytes compared with those treated with aspartame only. Most of the hepatocytes were more or less similar to those of the control group, whereas some cells had a slightly vacuolated cytoplasm (Fig. 10).
Ultrastructurally, most of the hepatocytes appeared more or less similar to those of the control group. However, some cells had few vacuoles in their cytoplasm compared with the group treated only with aspartame (Fig. 11). The von kupffer cells were more or less similar to those of the control group.
Control animals (group I): kidney
Light microscopic examination revealed that the renal cortex consisted of renal corpuscles, tubules, and minimal interstitial tissue in between. The renal corpuscles were composed of glomeruli surrounded by Bowman’s spaces. The proximal convoluted tubules appeared to be lined by acidophilic cuboidal epithelium with an apical brush border and enclosing a narrow lumen. The distal convoluted tubules were lined with acidophilic cuboidal epithelium surrounding a wider lumen (Fig. 12).
Ultrastructurally, the renal corpuscles consisted of glomerular fenestrated capillaries surrounded by podocytes that performed processes sharing in the formation of the filtration barrier (Fig. 13).
The lining cells of the proximal tubules rested on a thin basement membrane. The apical membrane was protruding into numerous microvilli. The intercellular junction between adjacent cells was observed at the lateral membrane. Their cytoplasm contained euochromatic rounded nuclei, pinocytotic vesicles, and numerous longitudinally arranged mitochondria between the basal infoldings (Fig. 14).
The distal renal tubule lining cells rested on a thin basement membrane. Their cytoplasms had euochromatic rounded nuclei, numerous mitochondria, free ribosomes, and basal infoldings (Fig. 15).
Aspartame-treated rats (group II)
Light microscopic examination revealed partial or complete loss of the brush border of proximal convoluted tubular epithelium, with widening of their lumens. Acidophilic material and interstitial cells’ infiltration appeared between the tubules (Fig. 16). Some cells of the renal tubules had pyknotic nuclei and a vacuolated cytoplasm. Exfoliated cells or cell remnants were observed in the lumen of some tubules. Some renal corpuscles with atrophic glomeruli and increased urinary space were observed (Fig. 17).
Ultrastructurally, the filtration barrier revealed a thickened basement membrane and the glomerular capillary endothelium was beaded (Fig. 18).
Most of the proximal convoluted epithelial cells showed heterochromatic nuclei with a dilated nuclear envelope. The cytoplasm was electron dense and contained mitochondria with destroyed cristae and numerous lysosomes. The apical microvilli were partially destroyed, with loss of the basal infoldings. Some cells detached from the underlying basal lamina and lost their intercellular junction with their adjacent cells (Fig. 19). Numerous ballooned mitochondria with partially destroyed cristae and irregular heterochromatic nuclei were observed in some cells. The basement membranes that surrounded most of the proximal tubules were thickened (Fig. 20).
The lining cells of the distal renal tubule revealed small heterochromatic nuclei, mitochondria with destroyed cristae, vacuoles, and lysosomes (Fig. 21).
Aspartame and P. anisum oil-treated rats (group III)
Light microscopic examination revealed that most of the renal tubules were more or less similar to those of the control group (Fig. 22).
Ultrastructurally, the filtration barrier thickness was more or less similar to that of the control group (Fig. 23). However, some cells lining the proximal tubules showed some lysosomes and vacuoles in their cytoplasm. These cells rested on a thin basement membrane with a preserved intercellular junction compared with the previous group treated only with aspartame (Fig. 24). The distal convoluted tubules appeared more or less similar to those of the control group (Fig. 25).
As regards the statistical study, there was a statistically highly significant increase in the mean distance between the visceral and the parietal layer of the Bowman’s capsule (urinary space) and the mean diameter of both proximal and distal convoluted tubule lumen in group II in comparison with the control group. In group III, these showed a highly significant decrease as compared with group II. All these data are presented in Table 1 and graphically illustrated in Histogram 1–3.
In the present work, the long-term administration of aspartame induced major alterations in the light and electron microscopic structures of both the liver and the kidney. Several possible mechanisms were considered to be involved in the hepatotoxic and nephrotoxic effects of aspartame.
It has been reported that after ingestion of aspartame, it is metabolized in the gastrointestinal tract into three constituents: aspartic acid, phenylalanine, and methanol and further breakdown products including formaldehyde and formic acid. Methanol has a slow rate of oxidation and its major effect is found mainly in the liver and the kidneys .
In the present study, cytoplasmic vacuoles in hepatocytes might be responsible for the physical changes in the structure of plasma membranes of protein and lipids of different organelles. This affected the Na+/K+ pump function and caused the accumulation of sodium and migration of water to the cells. This might occur as a result of the release of the free radicals secondary to the production of methanol and aspartic acid after aspartame ingestion . In agreement with our explanation, it was suggested that methanol could increase the lipid peroxidation products as well as the surface charge density . This might result in membrane liver cell damage. Some investigators have reported that these vacuoles most probably represented a cellular defense mechanism against toxic substances, in which these substances were aggregated in the vacuoles, thus preventing their interference with cellular metabolism . However, aspartame did not affect DNA-damaging activity in the rat hepatocyte .
It was suggested that the degenerative changes observed in the liver treated with aspartame might be inflammatory similar to a hepatitis-like condition. This was confirmed by other researchers as they found that the disturbance in the secretion and formation of coagulation factor VII and fibrinogen induced by aspartame caused prolonged hepatitis . However, aspartame might disrupt the delicate balance between a positively and a negatively charged amino acid residue in humans . This might lead to the formation of a salt bridge between these amino acid residues and facilitate autoantigen presentation and CD4 T-helper cell activation as well as a decrease in the serum concentrations of the growth hormone. This causes a decrease in the activity of several hepatic cytochromes P-450 and other drug-metabolizing enzymes. Finally, the patients developed autoimmune hepatitis.
The prominent appearance of von kupffer cells in some hepatic lobules of aspartame-treated rats was attributed to accumulated formaldehyde, which led to damage of the protein molecules. These molecules were recognized by specific detection sites on kupffer cells [24,25]. Some authors added that macrophages could destroy these proteins at a rate 100 times faster than proteins not treated with formaldehyde .
In the present work, damage of liver cells might be secondary to the activation of Kupffer cells that secrete tumor necrosis factor alpha, interleukins, reactive oxygen, nitrogen species, proteases, and prostaglandins. These mediators could act directly on hepatocytes to cause cell death. This was in accordance with others, where they proved that Kupffer cells might cause hepatocellular damage in acetaminophen hepatotoxicity [26,27]. In contrast, some authors found that Kupffer cells played a protective role in hepatic injury .
In the present study, it was observed that aspartame led to structural changes in both glomeruli and tubules. The appearance of acidophoilic material inside the tubules might be simulating those described as hyaline material in male rats . The presence of this material could indicate the accumulation of protein secondary to renal dysfunction. Similar findings were obtained in rat kidney after feeding with waste fat released from grilled chicken .
The renal cortex was more affected than other parts in the kidney as the cortex received most of the blood nutrient flow to the organ. Thus, when a blood-borne toxicant is delivered to the kidney, a high percentage of the toxin will reach the cortex .
In the present study, there was a highly significant increase in the mean diameter of the proximal and distal tubules’ lumen in aspartame-treated rats. It was suggested that aspartame induced apoptotic changes in most of the renal tubule lining cells that had hetrochromatic irregular nuclei, ballooned mitochondria with destroyed cristae, loss of brush borders, and secondary lysosomes. These changes could be secondary to DNA damage that occurred as a result of breakage and then cross linking occurred within the genetic material by formaldehyde exposure. The chronic use of aspartame led to damage of nucleic acids, mainly DNA .
However, in-vivo exposure of aspartame did not lead to chromosome aberrations in bone marrow cells of male Swiss mice .
The mitochondrial changes observed might be considered as early manifestations of apoptosis and an adaptive process to unfavorable environments such as excess exposure of the cell to free radicals . Some authors agreed with this explanation as they proved that methanol significantly increased the malondialdehyde level and caspase-3 activity . This caused an increase in the level of lipid peroxidation and activation of the intrinsic pathway of apoptosis, where mitochondria permeability increased due to the formation of a high conductance channel in the inner mitochondrial membrane. This led to the release of proapoptotic molecules into the cytoplasm that activated caspases, followed by the release of death-inducing molecules such as cytochrome C, apoptosis-inducing factor, and endonuclease G .
It was suggested that the disruption of the intercellular junctions and detachment of the cells from the basement membrane in the proximal tubules might be a precancerous sign . Multiple studies have shown a correlation between the reduction of the integrity of these junctions and tumor initiation and progression [38,39]. In agreement with our finding, it was proved that the changes in the genetic material caused by aspartame may lead to cancer such as brain tumor in humans [40,41]. In contrast to this, some authors reported that aspartame was safe and it was not mutagenic or clastogenic in animals [3,42].
In the present work, there was a highly significant increase in the mean distance between the visceral and the parietal layer of the renal corpuscle. This could be secondary to shrinkage of the glomerulus. It was suggested that the drug concentration in the blood was affected by capillary constriction, leading to a decrease in glomerular filtration of that drug, which minimized its effect and protected the tubular cells . This might cause shrinkage and atrophy of the glomeruli. At the same time, the mesangial cell processes may be retracted due to the contraction of their filaments, which might be stimulated by angiotensin II.
The thickening of the glomerular and tubular basement membrane might have resulted from an increase in the amount of collagenous fibers secondary to overproduction of collagen fibers after repeated damage to the epithelial cells with regeneration and secretions of the new fibers. However, this thickening might be due to an increase in the deposition of glycoproteins .
In this study, it was observed that the proximal tubules were affected more than the distal ones. This was explained by some authors by the fact that PCTs are the first to come into contact with the toxic agent after its filtration by the glomeruli .
The beading of glomerular capillary endothelium might result in a vascular leakage with migration of neutrophils and monocular cells into the interstitial tissue, which led to inflammatory cellular infiltration. In addition, formaldehyde could conjugate with human serum albumin and yield a new antigenic determinant with stimulation of the body to produce anti formaldehyde human serum antibodies and to raise the antigen memory cells. This led to sustained stimulation of the immune system .
The present study demonstrated that coadministration of P. anisum oil was effective in decreasing the toxic effect of aspartame on both the liver and the kidney of adult albino rats. This was observed by both light and electron microscopes. This improvement might be secondary to the antioxidant ability of anise, which attacks reactive oxygen species (ROS) 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% . 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 .
P. anisum increases vitamin C and E levels, which may lead to decreased oxidative stress as both vitamins are scavengers of the toxic free radicals generated with aspartame . The increase in vitamin C could be due to the protection of the existing vitamin C from oxidation to dehydroascorbic acid by some of the antioxidant phytochemicals present in the anise seeds. It was suggested that vitamin C might spare vitamin E and this might have led to an increase in the vitamin E level . Consistent with these suggestions, it was proved that aspartame decreases the activities of superoxide dismutase and catalase in different tissues .
However, the persistence of some histological changes in animals treated with P. anisum oil and aspartame might be a result of the increased production of ROS by aspartame, which overwhelms the capacity of intrinsic defense mechanisms in the cells. In addition to this, the P. anisum oil enrichment in our study might not be sufficient to protect both the liver and the kidney from the toxic effect of aspartame. Mechanisms other than the production of ROS might be implicated in aspartame-induced cellular damage. In accordance with these suggestions, some authors reported that aspartame could be metabolized in cell mitochondria, inducing mitochondrial and nuclear DNA damage and affecting its ability to produce inadequate and incomplete energy metabolism, which gives rise to highly damaging free radicals .
In conclusion, the results of our study showed that aspartame causes hepatorenal changes and consequently affects their functions. P. anisum oil provided direct protection from these changes; thus, the intake of aspartame should be restricted unless necessary and, if required, should be used for a short time. The optimum dose of anisum to exert good prophylactic effect against aspartame will need further investigations. To determine which chemical component in the P. anisum oil is responsible for its effects, chromatographic analysis of the oil should be performed and its ingredients should be studied in an aspartame-treated model.
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Conflicts of interest
There is no conflict of interest to declare.
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