Determining the Effects of Artificial Sweeteners as Teratogens on Chick Embryo Cell Morphology

Alexandria Emery, Katharine Meola, Katrina Nikitsina, Lorraine SalterelliDepartment of Biology, Kutztown University, Kutztown, PA, 19530

Introduction• Teratogens are defined as any chemical or substance that can cause

malformations within a developing embryo. • Many exist that pose a threat, although they are currently approved by

government agencies.• Sweeteners and sugar substitutes are currently approved by the FDA,

though controversy about their safety remains.• Epidemic levels of obesity cause an increase in use of artificial

sweeteners • Women are especially prone to dieting and use, possibly more so when

weight gain is expected due to pregnancy • Previous studies have shown risks to using substitutes including

increasing risks for cancer, or increasing cravings for sweets • Possible effects include altering or halting development, ending with

malformations or death of the developing embryo, or tumor growth within the consumer.

• Increasing populations means increasing pregnancies along with high costs of healthcare illustrates the importance of proper prenatal car

• Sugar substitutes that were tested:1. Sucrose

• Common table sugar2. Sucralose (Splenda):

• widely believed to be the safest artificial sweetener• Not readily broken down by body: passes through unmetabolized

(IFIC, 2009)• No energy gain

3. Aspartame (Equal): • Negligent energy gain• Denatures at high temperatures (IFIC, 20011)

4. Saccharin (Sweet N’ Low):• used for over a century, and the most studied• Concerns arise from study that found correlation of use with bladder

tumors in male mice3

• Chick embryo as a Model organism

Hypotheses 1. Changes in the cell morphology will appear at the macroscopic level and at the cellular level.

Null: There will be no changes in cell morphology at either macroscopic or cellular levels

2. The damage to embryos will be increased in those that receive the higher concentrated sugar solutions

Null: The damage observed will be equal in those that received low and high concentrated solutions

Results

Table 1: Commonly observed malformations when observing embryos

Conclusion

Lowest Body Weight: Highest Body Weight: Sucralose High Saccharin High

Smallest Body Length: Largest Body Length:Sucralose High Saccharin High

Irregular shaped cells: Enlarged Cell Size:Aspartame Low Sucrose HighSucralose Sucrose Low

• Body weight and length was observed to have decreased in higher concentrations of sweetener solutions, with saccharin as an exception.

• All treatment groups caused an enlarged cell size when compared with the untreated control group.• Solutions may have caused an increase water intake to dilute sweeteners.

• A significant difference was found among the groups for body weight, body length, and cell size. • Aspartame: produced differences among all observations.• Sucralose Low may be harmful because of its effects on body weight and

length• Saccharin High might affect body weight. Low concentrations may effect

body height.• Sucrose may cause low body weight and stunt growth. However, this was not

observed in an experiment (Shafet et al. 2012) that found an increase in body weight of chicks injected with carbohydrates

• A follow-up of immunofluorescence stain would reveal if there were damages to the cytoskeleton. This technique was not able to be performed due to time constraints, but might provide information on how irregular cell shapes were produced.

• Our first hypothesis was supported• The groups varied from controls in different aspects, but the large variance and

significant differences found among all measurements seems to indicate that all were somewhat affected by the sweeteners

• These implications may lead to sweeteners being re-evaluated for safety of consumption by government agencies,• Further studies supporting risks when using sweeteners may help eliminate

them from products and on market shelves.• A major improvement in this study in future replications and trials would be to

greatly increase sample size.

Literature Cited

Calorie Control Council [Internet]. Saccharin. cited 2014 Mar 9. Available from: http://www.caloriecontrol.org/sweeteners-and-lite/sugar-substitutes/saccharinGilbert SF. 1997. Developmental Biology.http://9e.devbio.com/about.php.International Food Information Council [Internet]. 2009. Everything You Need to Know About Sucralose. cited 2014 Mar 9. Available from: http://www.foodinsight.org/Content/5519/Sucralose%20cons%20piece_web.pdfInternational Food Information Council [Internet]. 2011. Everything You Need to Know About Aspartame. cited 2014 Mar 9. Available from: http://www.foodinsight.org/Content/3848/FINAL_Aspartame%20Brochure_Web%20Version_11-2011.pdfShafey, TM, Alodan, MA, Al-Ruqaie, IM, Abouheif, MA. 2012. In ovo feeding of carbohydrates and incubated at a high incubation temperature on hatchability and glycogen status of chicks. South African Journal of Animal Science. 42(3), 210-220.

Acknowledgement

We would like to thank the Biology Department at Kutztown University, and Dr. Rosch and Dr. Stone for providing help and support as well as advice.

Research Objectives

• The primary objectives of this research experiment are to:• To determine if the sweeteners will have an effect at the cellular level on

the cell morphology. • To determine if there is a visible, larger morphological effect on the organ

systems.• To determine how different the effects of the different sweeteners are.

Solution External Abnormalities

Control Bulging eyes, Hemorrhaging

0.9% Saline Smaller embryo

0.6 M Saccharin

No development, Bacterial Infection, Hard Yolk

2.2 M Saccharin

No development or tiny embryo, Bacterial Infection, Hard Yolk, Hemorrhaging

0.6 M Aspartame

Tiny embryo, Bacterial Infection, Hemorrhaging

2.2 M Aspartame

Tiny embryo

0.6 M Sucrose

Bacterial Infection, Hemorrhaging

2.2 M Sucrose

Tiny embryo

0.6 M Sucralose

Tiny embryo, Hard yolk

2.2 M Sucralose

Bacterial Infection, No development

Significant differences were found among groups in embryo weight and length, as well as cell size.

Figure 1: An ANOVA found a significant difference between the above groups in embryo weight [F(7) = 2.73, p = 0.032]

Figure 2: An ANOVA found a significant difference between the above groups in embryo length [F(7) = 3.04, p = 0.021]

Figure 3: Muscle cells from tissue sample stained with eosin (left) and methylene blue (right) from different groups

Figure 4: An ANOVA found a significant difference between the above groups in cell size [F(5) = 10.54, p = 1.82 X 10-7 ]

Control Aspartame Low

Aspartame High

Sucrose High

Sucralose High

Sucralose Low

Sucrose Low

Ringers0.00

0.50

1.00

1.50

2.00

2.50

3.00

Wei

ght

(g)

Control Aspartame Low

Aspartame High

Sucrose High

Sucralose High

Sucralose Low

Sucrose Low

Ringers0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

Leng

th (c

m)

Sucrose High Sucrose Low Saccharin High

Saccharin Low

Sucralose Low

Control Aspartame Low

0.00

2.00

4.00

6.00

8.00

10.00

12.00

Cell

Dia

met

er (µ

)

Control

Aspartame High Sucralose Low

Saccharin Low

Sucralose Low

Saccharin Low

Sucrose Low

Control

Figure 1: Comparison of embryos Chick and human embryo similarities made by Haeckel in 1874. Chicks are inexpensive, easy to maintain and have a short developmental period

Methods• A 0.9% Ringer’s saline solution was made, and used as an injection control

and base in creating sweetener solutions in low (0.6M) and high (2.2M) concentrations.

• Four eggs were assigned to each of the ten groups, aseptically injected, weighed, and then incubated for seven days.

• On the seventh day, eggs were reweighed. Embryos were anesthetized and killed in a freezer.

• Eggs were opened to observe embryo weight, length, and malformations. Embryos were preserved in ethanol.

• Tissue samples were homogenized and stained with eosin and methylene blue to observe cell morphology.

Figure 1: performing injections Figure 2: Weighing eggs

Figure 3: Staining cells