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