Aspartame

Brayden Callison, Rylee Wimpey, and Max Faulhaber

Introduction

Aspartame is an artificial sweetener used in many soft drinks, foods, and medicines initially believed to be an initiative for decreased obesity and diabetic rates (Czarnecka et al., 2021). Aspartame is consumed daily by many, including pregnant women. Studies show that aspartame can cause damage to the structure of the placenta, increasing the oxidative stress, ultimately damaging the cells surrounding the embryo that provide it with nutrients (Chen et al., 2024). A previous study done on aspartame’s effects on development of zebrafish embryos resulted in negative morphological effects such as implications on body length, pigmentation, and neutrophil production (Wu et al., 2024). Additionally, Kormsing et al. (2020) performed a study using chicken embryos injected with aspartame at concentrations 10, 20, and 30 mg/mL. In that study, Kormsing et al. (2020) found that higher doses caused increased mortality and developmental abnormalities like brain malformations, abnormal heart looping, and absent limb buds. Since mortality rates were high at higher concentrations, it is important to study the effects of aspartame lower concentrations. In doing so, we can assess its effects on external morphology in a living embryo.

This study investigates the effects of exposure to aspartame on the morphological development of a chicken embryo. Our experiment aims to analyze weight, body length, eyeball diameter, limb length, and morphological abnormalities in embryotic chickens exposed to aspartame via injection into the yolk. We predicted that the weight of the treated embryos would be lower, and the overall development would be slower.

Methods

Chicken Embryotic Selection

Treatment (12 mg/mL aspartame in PBS saline): 20 eggs
Vehicle Control (PBS saline): 15 eggs
Negative Control: 10 eggs

Administration of Aspartame

Day 3: Removal of eggs from the incubator.
- Cleansed eggs using ethanol and Kimwipes
- Injection of 0.1 mL aspartame solution into treatment eggs.
- Injection 0.1 mL PBS saline in vehicle control eggs
Day 10: Observation of Embryotic Development
- Taped egg
- Cut small window in taped section
- Removed embryo from egg and membrane
- Positioned embryo on black background
- Imaging taken on iPhone 15
- Data collected through ImageJ
- Statistical ANOVA testing, and post-hoc Tukey's testing ran

Results

Undeveloped Embryos (Prior to Incubation):

3 treated embryos
2 vehicle control embryos
1 negative control embryo

Statistical Testing Using ANOVA:

Difference shown among body weight of groups (F= 10, df=2, p<0.01).
Difference shown among the body length of groups (F=4.1, df=2, p=0.02).
Difference shown among the HH stages of groups (F=10, df= 2, p<0.01).

Statistical Testing Using Post-hoc Tukey's Test:

Body Weight:
- Significant increase in weight of the vehicle control group compared to the negative control group (p=0.02).
- No significant difference from the weights of the vehicle control group and the aspartame group (p=0.23).

Body Length:
- Moderate increase in body length of embryos of the vehicle control group compared to the negative control group (p=0.15).
- No significant increase in body length of aspartame group compared to the vehicle control group (p=0.60).
HH Stages:
- No indication of increase in developmental stages of vehicle control group compared to negative control group (p=0.23).
- Significant increase in developmental stages of the aspartame group compared to the vehicle control group (p=0.02).

Figure 1. Comparison of embryotic weight among vehicle control groups (PBS saline), negative control groups, and aspartame groups (12 mg/mL aspartame in PBS saline). The dots represent raw data, and the adject dot with bars to the left of each group represents the mean and error bar (standard deviation).

Figure 2. Comparison of chicken embryotic body length among a vehicle control group (PBS saline), a negative control group, and an aspartame group (12 mL/mg aspartame in PBS saline). The dots represent raw data, and the adject dot with bars to the left of each group represents the mean and error bar (standard deviation).

Figure 3. Comparison of HH developmental stages among a vehicle control group (PBS saline), a negative control group, and an aspartame group (12 mL/mg aspartame in PBS saline). The dots represent raw data, and the adject dot with bars to the left of each group represents the mean and error bar (standard deviation).

Discussion

Summary

The vehicle control group showed a significant increase in weight from the negative control group, while the aspartame group showed no significant difference in weight compared to the vehicle control group. This suggests that the alteration in weight is due to injection of the PBS saline rather than the aspartame
We did not find support for our hypothesis, as aspartame treatment resulted in minimal observable deformities or alteration in weight. Additionally, in opposition to our hypothesis, we found an increase in the speed of development of chicken embryos as determined by progressive HH stages.

Literature Analysis

Prior findings are in agreement with our results of stable morphological development; Kormsing et al. (2020) found only developmental defects at high doses between10 to 30 mg/mL. Lower dose groups in their study showed little to no morphological change.
Additionally, prior findings are in agreement to our results of higher HH stages among treatment groups; Al-Qudsi & Al-Rashdi (2022) reported that artificial sweeteners altered early vitelline vessel development in chick embryos by affecting the vascular branching which suggest quicker development.

Acknowledgement

The unique data collection of HH staging done in this study compared to other previously done studies was a strength in illustrating developmental differences in the embryotic chicken after the same incubation period.
Solution concentration differed from other previously done studies which allowed our study to see if an alteration in development would be noticeable, while remaining low enough in concentration to keep mortality rates low

Limitations to Study: Small Sample Size
Future Directions:
- Analysis of long-term effects of aspartame on developmental pacing
- Repetition of study using larger sample size

References

Chen, Y. C., Yeh, Y. C., Lin, Y. F., Hsu, S. Y., Nacis, J. S., Hsu, J. W., & Hsieh, R. H. (2024). Aspartame intake during pregnancy induces placental dysfunction through impaired mitochondrial function and biogenesis modulation. Placenta, 158, 285–292. https://doi.org/10.1016/j.placenta.2024.11.003

Czarnecka, K., Pilarz, A., Rogut, A., Maj, P., Szymańska, J., Olejnik, Ł., & Szymański, P. (2021). Aspartame-True or False? Narrative Review of Safety Analysis of General Use in Products. Nutrients, 13(6). https://doi.org/10.3390/nu13061957

Fatma M.S.N. Al-Qudsi, & Amna Al-Rashdi. (2022). Artificial Sweeteners Alter Early Vitelline Vessel Development in Chick Embryo: Life Sciences-Biology. International Journal of Life Science and Pharma Research, 11(3), 50–60. https://doi.org/10.22376/ijpbs/lpr.2021.11.3.L50-L60

Nattharin Kormsing, Yadaridee Viravud, Jantima Roongruangchai, Vasana Plakornkul, & Thanaporn Rungruang (2020). Teratogenic Effects of Aspartame Exposure of Chick Embryonic Development. Rangsit Graduate Research Conference : RGRC, 15((2563)), 2731–2736. https://rsujournals.rsu.ac.th/index.php/rgrc/article/view/1780

Wu, Y., et al. (2024). “Evaluation of aspartame effects at environmental concentration on early development of Zebrafish: Morphology and transcriptome1.” Enviromental Pollution, 361. DOI:10.1016/j.envpol.2024.124792

Author Contributions

Our entire team designed the experiment. M.F collected and organized data. B.C analyzed data and constructed figures. R.W produced written formatting of results. M.F summarized discussion of results. B.C compared results to previous literature. R.W discussed limitations and suggested future directions.

Page updated

Google Sites

Report abuse