1. Determining the Effects of Artificial Sweeteners as Teratogens on Chick Embryo Cell
Morphology
Alexandria Emery, Katharine Meola, Katrina Nikitsina, Lorraine Salterelli
Department 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 sugar
2. 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 High
Sucralose 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/saccharin
Gilbert 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/Sucr
alose%20cons%20piece_web.pdf
International Food Information Council [Internet]. 2011. Everything You Need to Know About
Aspartame. cited 2014 Mar 9. Available from: http://www.foodinsight.org/Content/3848/FINA
L_Aspartame%20Brochure_Web%20Version_11-2011.pdf
Shafey, 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 ]
-0.50
0.00
0.50
1.00
1.50
2.00
2.50
3.00
Control Aspartame
Low
Aspartame
High
Sucrose
High
Sucralose
High
Sucralose
Low
Sucrose
Low
Ringers
Weight(g)
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
Control Aspartame
Low
Aspartame
High
Sucrose
High
Sucralose
High
Sucralose
Low
Sucrose
Low
Ringers
Length(cm)
0.00
2.00
4.00
6.00
8.00
10.00
12.00
14.00
Sucrose High Sucrose LowSaccharin HighSaccharin LowSucralose Low Control Aspartame Low
CellDiameter(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