Effect of aspartame in bone formation of young female rats

Abstract


Introduction
Aspartame is a synthetic non saccharide sweetener and it has a sweeter taste than sucrose by 200 times (1).Aspartame was discovered in 1965 and then the Food and Drug Administration (FDA) approved it using in the 1981 (2).Aspartame is an ingredient of nearly 6000 commercial products widespread in all over the world, whether they are food or pharmaceutical products (3).Aspartame has brand names such as Equal and NutraSweet, and it used widely in many products, especially those classified as diet foods, which include breakfast cereal products, chewing gum, yogurt, instant coffee, and pharmaceuticals (4).It is a methyl ester from aspartic acid with phenylalanine dipeptide (5).By actions of the esterase and the peptidases, aspartame can hydrolyze it absorbed through the gut, and released its components aspartic acid 40%, phenylalanine 50% and methanol 10%.At high doses and prolonged aspartame consumption, these releasing components have harmful effect and it may be more toxic than aspartame (6).At first, methanol is oxidized to formaldehyde, which is then converted in the liver to formic acid.In fact, both formaldehyde and formic acid are known as harmful substances for the liver and damage its cells (7,8).Also, the superoxide anion and the hydrogen peroxide are produced through the aspartame hydrolysis, which caused the denaturation of protein that affect the structures of enzymes (9).Carcinomas in renal pelvis, was observed in the females' ureter and breast, who consumed excessive amounts of aspartame (10), and in males malignant of peripheral nerves was noted (11,12).Some studies have showed, that consumption of soft drink which contain caloric sweeteners causes a negative effect on health, and these effects may extend to bone, since adolescents who consumed soft drink showed a reduction in the mineral density of bone also growing in bone fractures risks (13,14).
We aimed in current research to study the effect of aspartame in bone formation of young ages was the goal of the present study, since the bone growth is greater.

Ethical approve
The approval of ethics for the current study was obtained from the laboratory animals housed in the College of Veterinary Medicine of Mosul University with a number of approvals UM.VET.2022.029.

Laboratory animals and experimental design
Twenty-four females rat 1.5 months old, weighting 90-100 gm were maintained under controlled conditions involved, 12h -12h light-dark in the animal's house in College of Veterinary Medicine /University of Mosul.The rats were given the standard diet and water, and were grouped into four groups, each group included six rats.Group 1 (G1) as control group were given distilled water.Group 2 (G2) rats were given aspartame 0.4 g/kg/day orally.Group 3 (G3) rats were given aspartame 0.8 g/kg/day orally.Group 4 (G4) rats were given aspartame 1.2 g/kg/day orally.

Collection of the blood
After the experimental period 30 days blood samples were drawn and collected from the optical vein, and left to clot, then centrifuged for a duration 15 minutes, the serum was isolated and saved in -18ºC in order to estimate the biochemical parameters (15).

Biochemical examination
Vitamin D specific for rats and Insulin Like Growth Factor -1 specific for rats were estimated in serum by using ELISA kits (Elabscience-company) (16).Biomerieux kit method of Belfeld and Goldbery was used to determine alkaline phosphatase activity in serum (17).Serum calcium, inorganic phosphate and albumin were estimated by spectrophotometer kits using Biolabo, France (18)(19)(20).

Histopathological of bone
Rats were killed and left femurs were excised and cleaned, and fixed in 10 % formaldehyde in the phosphatebuffered solution, followed by decalcification by formic acid-sodium citrate solution, then dehydrated by the alcohol and cleared by the xylol after that embedded in the paraffin and sectioned into 5 µm by using a rotatory microtome.The slides were stained using hematoxylin and eosin (21).

Statistical analysis
The one-way analysis of variance (ANOVA) test was used to analyze all data, which were expressed as means standard deviation (SD), using SPSS selector test used for detecting the statistical significance between all groups at P≤0.05 probability (22).

Results
The results indicated that there was a significant elevation P≤0.05 in vitamin D and IGF-1 levels in the all groups that gave 0.4 g/kg, 0.8 g/kg and 1.2 g/kg in comparison to control group.However, serum vitamin D and IGF-1 levels between the group that treated 0.8 g/kg and the group that treated 1.2 g/kg did not differ significantly as shown in table 1.Also, a significant elevation P≤0.05 in ALP activity was noticed in treated groups when comparison to control group, so this elevation was proportional to the rise in the doses.In addition, serum calcium showed a significantly P≤0.05 decrease in the groups that treated with 0.4 g/kg and 0.8 g/kg respectively, and a significant P≤0.05 increase in the group that was treated with 1.2 g/kg when compared to the control groups.However, serum inorganic phosphate was not changed significantly in all groups in comparison to control group.Serum albumin significantly elevated P≤0.05 in treated groups in comparison to control group (Table 2).
In the histopathological examination, the bone sections of control group revealed, normal thickness of trabecular bone, normal osteogenic tissue, and regular vacuole between bony tissue and osteogenic tissue as shown in figure 1. Treated rats with 0.4 g/kg of aspartame showed little thickness of trabecular bone and osteogenic tissue, regular vacuole between bony and osteogenic tissue as shown figure 2. The group 3 that treated with 0.8 g/kg aspartame showed a slight thin trabecular bone and poor developed osteogenic tissue, as well as a wide vacuole between bony and osteogenic tissue as shown in figure 3. The left femur of rats that treated by 1.2 g/kg of aspartame showed the negative effects that seen through the very little thickness of trabecular bone (TB), low quantity of osteogenic tissue, wide vacuole between bony tissue and osteogenic tissue, and reducing osteoblasts as shown in figure 4.

Discussion
Administration of aspartame to the young rats caused an elevation in vitamin D levels, and this elevation was proportional to the dose of aspartame that was used in this work.That excess doses from vitamin D in serum may affect bone tissues, and this was confirmed in the histological changes that were seen when the rats were treated with 1.2 g/kg, as shown in fig.4, which showed the negative effects by reducing bone density and very little thickness of trabecular bone.Our results about the negative effect of high levels of vitamin D in bone tissues agree with results from Laure et al., that high doses of vitamin D are related to bone loss since high doses of vitamin D may inhibit PTH hormone through its action on the parathyroid gland or through its enhancement of calcium absorption from the intestine, affecting bone formation and the activity of osteoclast cells (23).Also, the administration of aspartame caused a significant elevation in serum IGF-1.This increase in IGF-1 may result from the increase of the metabolites of aspartame after its ingestion.It is converted to aspartic acid, phenylalanine, and methanol which agreement with study by Sun et al., that documented an increase in IGF-1 levels in aspartame intake group (24).The increase in aspartic acid and phenylalanine levels, which are essential amino acids for protein and peptide synthesis, is also important for binding proteins that correlate with IGF levels (25).The activity of ALP was elevated when the dose of aspartame increased, which agrees with (26).This elevation in alkaline phosphatase may be related to bone turnover and increased bone resorption (27,28).Alkaline phosphatase used as an indicator of the osteoblast activity; it is a useful to monitor formation of bone (29,30).Alkaline phosphatase is considered a good biomarker for bone formation, and it elevated in several metabolic disorders and in osteoporosis (31).Alkaline phosphatase plays a great role in the hard tissue, also it reduces the pyrophosphate level in the extracellular.It also has an effect on the calcification of bone metabolism (32,33).The elevation in alkaline phosphatase activity in this study also agreement with findings of Sary et al., since they reported that aspartame led to increases in ALT, AST and ALP activities (34).These findings were supported by the histological findings, which seen reduced bone density and very little thickness of trabecular bone.Serum calcium was reduced after being given 0.4 g/kg of aspartame; this may refer to the negative effect of aspartame.However, serum calcium was elevated when the dose of aspartame was increased by 0.8 g/kg and also by 1.2 g/kg.This elevation may be attributed to the high levels of vitamin D, because vitamin D enhances calcium absorption in the intestine (35).High level of vitamin D in serum causes a high concentration of calcium in the blood, and this case leads to soft tissue calcification (36,37).Calcium is a major element and has biological functions in the body that are regulated in serum through the resorption and excretion through the kidney and the absorption via the intestine along with the exchanges with the bony tissue, which is regarded as a reservoir for calcium and phosphate (38,39).On the other hand, the high level of calcium after 0.8 g/kg also with 1.2 g/kg aspartame administration may be due to the stimulation of the PTH by low calcium level, so this causes calcium release from the bone in order to normalize calcium concentration (40).This study showed that oral administration of aspartame caused an increase of albumin concentration this elevation was proportional to the increasing aspartame dose.These results are consistent with the findings of Sary et al. since they noticed a very significant elevation in serum albumin and total protein in both female and male rats after treatment with aspartame.This may refer to the negative effect of aspartame (34).The elevation in serum albumin may also be attributed to the level of aspartic acid and phenylalanine that were produced from aspartame hydrolysis.Both amino acids are occurred in the proteins (41).

Conclusions
The current study concluded, that aspartame taken at high doses causes harmful effects on bone formation, while low doses have moderate effects.

Figure 1 :
Figure 1: The photomicrograph for the left femur of the control group reveals the trabecular bone TB thickness, and the osteogenic tissue OT, regular vacuole between bony tissue and osteogenic tissue.H&E stain, 100X.

Figure 2 :
Figure 2: The photomicrograph in the left femur of the group 1 (treated with aspartame 0.4 g/kg) reveals the little thickness in trabecular bone TB and osteogenic tissue OT, regular vacuole between bony tissue and osteogenic tissue.H&E stain, 100X.

Figure 3 :
Figure 3: The photomicrograph in the left femur of the group 2 (treated with aspartame 0.8 g/kg) reveals the little thickness of trabecular bone TB and low quantity of osteogenic tissue OT, wide vacuole between bony tissue and osteogenic tissue.H&E stain, 100X.

Figure 4 :
Figure 4: The photomicrograph in the left femur of the group 3 (treated with aspartame 1.2 g/kg) reveals the very little thickness of trabecular bone TB and low quantity of osteogenic tissue OT, wide vacuole between bony tissue and osteogenic tissue.H&E stain, 100X.

Table 1 :
Aspartame effects in serum vitamin D and IGF-1

Table 2 :
Aspartame Effects in serum ALP activity, calcium, inorganic phosphate and albumin The values expressed as mean± SD for 6 animals/group.Letters that different in the vertically mean a significant variance P≤0.05.