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DETERMINING THE EFFECTS OF ARTIFICIAL SWEETENERS AS TERATOGENS ON CHICK
EMBRYO CELL MORPHOLOGY
Alexandria Emery Katharine Meola Katrina Nikitsina Lorraine Salterelli
Introduction
Teratogen: any factor that causes malformations of an embryo
There are many types of teratogens from the environment, drugs, or infectious agents.
Examples: Alcohol, nicotine, caffeine, mercury, cat feces, pesticides, and chlamydia.
Increasing populations means increasing pregnancies along with high costs of healthcare illustrates the importance of proper prenatal care
Artificial Sweeteners as Possible Teratogens
Why is this important? 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 People use this freely without fear of possible consequences Currently approved by the FDA, though controversy remains as
amount consumed is increased in diet Previous studies have shown risks to using substitutes
including increasing risks for cancer, or increasing cravings for sweets
Effects: altering or halting development, ending with malformations or death of the developing embryo, or tumor growth within the consumer.
Artificial Sweeteners
Sucrose Sugar Disaccharide made from
glucose and fructose Used as a source of
energy and to increase sweetness of foods
Form of added sugar which should be restricted to less than 25% of energy intake2
Sucralose Splenda 0 calories Not readily broken down
by body: passes through unmetabolized1
No energy gain Widely believed to be the
safest alternative
Artificial Sweeteners Aspartame
Equal Low calories Negligent energy gain Not used in cooking Made of amino acid Phenylalanine Denatures in high temperatures3
Saccharin Sweet N' Low 0 calories No energy gain Used for over 100 years Studied intensively Concerns arise from study
that found correlation of use with bladder tumors in male mice4
Why study the chick embryo as a model for the human embryo?
First studied by Aristotle Tissue differentiation lead to disproving
preformation, supporting epigenesis5
Eggshell parts can be replaced by clear material to allow a visual of the development
Short developmental period Eggs may be frozen to halt development until
ready for use Cheap to maintain Similar development
Chick Embryo
Figure 1: Comparison of embryos Chick and human embryo similarities made by Haeckel in 1874
Hypotheses1. 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 solutionsOf the four experimental groups, we predict saccharin will have the greatest negative effects on the embryo development.
Objectives
1. To determine if the sweeteners will have an effect at the cellular level on the cell morphology.
2. To determine if there is a visible, larger morphological effect on the organ systems.
3. To determine how different the effects of the different sweeteners are.
Methods
Solutions were made by adding sugar to the Ringer’s Solution
Methods
Eggs were injected with sugar solutions and weighed before incubated for 7 days
Figure 2: Aspartame High 1 Egg After Injection
Figure 3: Eggs weighed before incubation
Methods Chick Eggs were: 1. Placed in freezer after 7 days of
incubation to ensure death 2. Weighed again3. Cracked open to observe embryos
Embryos were:1. Weighted and measured for length
2. Preserved in ethanol until
observations were completed
Figure 4: Eggs after being placed in freezer
Methods
Homogenization occurred and cells were stained with methylene blue and eosin stains
Figure 5: Eosin staining
Results Deformations and mutations
Table 2: Physical deformations and mutations observed
Results
Figure 7: Untreated vs. Aspartame High Concentration
Sucrose LowSucrose High
Figure 6: Embryos injected with Sucrose High and Low Concentrations
Results
Control
Aspartame High Sucralose Low
Saccharin Low
Cell Staining: Eosin
Figure 8: Eosin staining of cells from different groups
Results
Sucralose Low
Saccharin Low
Sucrose Low
Control
Cell Staining: Methylene Blue
Figure 9: Methylene blue staining of cells from different groups
ResultsInitial
weight
after 7 days
incubation
chang
e
Untreated 65.094 61.089 -4.005
Ringer’s
solution
66.658 63.160 -3.498
Aspartame H 66.619 62.805 -3.814
Aspartame L 66.316 62.040 -4.276
Saccharin H 66.865 63.030 -3.835
Saccharin L 69.813 65.430 -4.382
Sucralose H 62.628 58.800 -3.828
Sucralose L 65.901 61.960 -3.941
Sucrose H 66.201 62.810 -3.391
Sucrose L 69.558 66.080 -3.478Table 2: Average Weight of Embryos in Ovo and their Change
Untreat
ed
Aspart
ame 0
.6M
Aspart
ame 2
.2M
Saccha
rin 0.6
M
Saccha
rin 2.2
M
Sucros
e 0.6M
Sucros
e 2.2M
Sucrol
ose 0.6
M
Sucrol
ose 2.2
M
Ringer
's 0.9%
Salin
e0
0.51
1.52
2.53
3.54
4.5
Solutions
∆ Eg
g M
ass
Change in Egg Weight
Figure 10: Average Weight of Change in Egg Weight after the Incubation Period. Error is shown in SD bars. [F(9) = 1.36, P = 0.22]
Results
Figure 11: Average Body Weight of Chick Embryos. Error is shown in SD bars. [F(7) = 2.73, P = 0.032]
Untreated Aspartame 0.6M
Aspartame 2.2M
Saccharin 0.6M
Saccharin 2.2M
Sucrose 0.6M Sucrose 2.2M Sucrolose 0.6M
Sucrolose 2.2M
Ringer's 0.9% Saline
0.00
0.50
1.00
1.50
2.00
2.50
3.00
Solutions
Embr
yo W
eigh
t
Embryo Weight
Results
Figure 12: The Significant Mean Difference of the Embryo Length
Tukey Test: Embryo Weight
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)
Results
Untreated Aspartame 0.6M
Aspartame 2.2M
Saccharin 0.6M
Saccharin 2.2M
Sucrose 0.6M Sucrose 2.2M Sucrolose 0.6M
Sucrolose 2.2M
Ringer's 0.9% Saline
0
0.5
1
1.5
2
2.5
3
3.5
4
Solutions
Embr
yo L
engt
h
Figure 13: Average Body Length of Chick Embryos. Error is shown in SD bars. [F(7) = 3.04, P = 0.021]
Embryo Length
Results
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 (
cm)
Figure 14: The Significant Mean Difference of the Embryo Length
Tukey Test: Embryo Length
Figure 15: Average Cell Size. Error is shown in SD bars. [F(5) = 10.54, P = 1.82 X 10-7 ]
Untreated Aspartame 0.6M
Aspartame 2.2M
Saccharin 0.6M
Saccharin 2.2M
Sucrose 0.6M
Sucrose 2.2M
Sucrolose 0.6M
Sucrolose 2.2M
Ringer's 0.9% Saline
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
10.00
Solutions
Cell
Size
Results
Cell Size
Results
Sucrose High Sucrose Low Saccharin High Saccharin Low Sucralose Low Control Aspartame Low0.00
2.00
4.00
6.00
8.00
10.00
12.00
Cell
Dia
met
er (
µ)
Figure 16: The Significant Mean Difference of the Cell Size
Tukey Test: Cell Size
Conclusion
Lowest Body Weight: Sucralose High
Smallest Body Length: Sucralose High
Irregular shaped cells: Aspartame Low Sucralose
Highest Body Weight: Saccharin High
Largest Body Length:Saccharin High
Enlarged Cell Size:Sucrose High Sucrose Low
ConclusionA 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 experiment7 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 is time consuming and was not able to be performed, but might provide information on how irregular shapes were produced
References4. Calorie Control Council [Internet]. Saccharin. cited 2014 Mar 9. Available from:
http://www.caloriecontrol.org/sweeteners-and-lite/sugar-substitutes/saccharin2. Dietary Reference Intakes for Energy, Carbohydrate, Fiber, Fat, Fatty Acids, Cholesterol,
Protein, and Amino Acids (Macronutrients)[Internet]. 2005. National Academy of Sciences. Institute of Medicine. Food and Nutrition Board. Available from: http://www.nal.usda.gov/fnic/DRI//DRI_Energy/R1-26.pdf
6. Gilbert, S F 1997. Developmental Biology.http://9e.devbio.com/about.php.5. Gilbert SF. Developmental Biology. 6th edition. Sunderland (MA): Sinauer Associates;
2000. Comparative Embryology. Available from: http://www.ncbi.nlm.nih.gov/books/NBK9974/
1. 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.pdf
3. 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/FINAL_Aspartame%20Brochure_Web%20Version_11-2011.pdf
7. 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.
Acknowledgements Dr. Rosch Dr. Stone Kutztown University