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

Determining the Effects of Artificial Sweeteners as Teratogens on Chick Embryo Cell MorphologyAlexandria Emery Katharine Meola Katrina Nikitsina Lorraine Salterelli

Introduction

Teratogen: any factor that causes malformations of an embryoThere 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 TeratogensWhy is this important?Epidemic levels of obesity cause an increase in use of artificial sweetenersWomen are especially prone to dieting and use, possibly more so when weight gain is expected due to pregnancyPeople use this freely without fear of possible consequencesCurrently approved by the FDA, though controversy remains as amount consumed is increased in dietPrevious studies have shown risks to using substitutes including increasing risks for cancer, or increasing cravings for sweetsEffects: altering or halting development, ending with malformations or death of the developing embryo, or tumor growth within the consumer.

Artificial SweetenersSucroseSugarDisaccharide made from glucose and fructoseUsed as a source of energy and to increase sweetness of foodsForm of added sugar which should be restricted to less than 25% of energy intake2SucraloseSplenda0 caloriesNot readily broken down by body: passes through unmetabolized1No energy gainWidely believed to be the safest alternative

Artificial SweetenersAspartameEqualLow caloriesNegligent energy gainNot used in cookingMade of amino acid PhenylalanineDenatures in high temperatures3

SaccharinSweet N' Low0 caloriesNo energy gainUsed for over 100 yearsStudied intensivelyConcerns 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 AristotleTissue differentiation lead to disproving preformation, supporting epigenesis5Eggshell parts can be replaced by clear material to allow a visual of the developmentShort developmental periodEggs may be frozen to halt development until ready for useCheap to maintainSimilar developmentChick Embryo

Figure 1: Comparison of embryos Chick and human embryo similarities made byHaeckel 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 solutionsNull: The damage observed will be equal in those that received low and high concentrated solutions

Of 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 Ringers 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 Low

Sucrose HighFigure 6: Embryos injected with Sucrose High and Low Concentrations

Results

ControlAspartame HighSucralose Low Saccharin LowCell Staining: Eosin Figure 8: Eosin staining of cells from different groups

Results

Sucralose Low

Saccharin Low

Sucrose Low

ControlCell Staining: Methylene Blue Figure 9: Methylene blue staining of cells from different groups

ResultsInitial weightafter 7 days incubationchangeUntreated65.09461.089-4.005Ringers solution66.65863.160-3.498Aspartame H66.61962.805-3.814Aspartame L66.31662.040-4.276Saccharin H66.86563.030-3.835Saccharin L69.81365.430-4.382Sucralose H62.62858.800-3.828Sucralose L65.90161.960-3.941Sucrose H66.20162.810-3.391Sucrose L69.55866.080-3.478

Table 2: Average Weight of Embryos in Ovo and their Change 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]

ResultsFigure 11: Average Body Weight of Chick Embryos. Error is shown in SD bars. [F(7) = 2.73, P = 0.032]Embryo Weight

ResultsFigure 12: The Significant Mean Difference of the Embryo LengthTukey Test: Embryo Weight

Results Figure 13: Average Body Length of Chick Embryos. Error is shown in SD bars. [F(7) = 3.04, P = 0.021]Embryo Length

ResultsFigure 14: The Significant Mean Difference of the Embryo LengthTukey Test: Embryo Length

Figure 15: Average Cell Size. Error is shown in SD bars. [F(5) = 10.54, P = 1.82 X 10-7 ]ResultsCell Size

ResultsFigure 16: The Significant Mean Difference of the Cell Size Tukey Test: Cell Size

ConclusionLowest 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 lengthSaccharin 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 carbohydratesA 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.pdf6. 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.pdf3. 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.pdf7. 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. RoschDr. Stone Kutztown University