1. Xenoestrogen Effects on Stem Cell Behavior
Maria DeRenzo
Oakland Catholic High School
Grade 10

2. Tissue Engineering
Development and manipulation of artificial implants, laboratory-grown tissues, genetically engineered cells and/or molecules
Purpose: to replace or support the function of defective parts of the body

3. C2C12 Subclone of the mus musculus (mouse) myoblast cell line
Differentiates rapidly, forming contractile myotubes and produces characteristic muscle proteins
Mouse stem cell line is used as a model in many tissue engineering experiments
Used to study the differentiation of non- muscle cells (stem cells) to skeletal muscle cells
Expresses muscle proteins and the androgen receptor (AR)
AR- DNA binding transcription factor which regulates gene expression.

4. Xenoestrogens (Synthetic Estrogen)
Industrially made compounds
Type of xenohormone that imitates estrogen
Commonly used in industrial compounds which have estrogenic effects on a living organisms

5. Atrazine Organic compound
Commonly used as an herbicide, or weed-killer
Harmful if consumed directly by human
Environmental Hazards:
Can travel through soil and ground water which later is used as drinking water
Harmful to aquatic organisms because the pesticide travels by run off water and climatic factors

6. Key Question Hypotheses
Null Hypothesis: The xenoestrogen, Atrazine, will increase the stem cell behavior. (Proliferation/ Differentiation)
Hypothesis: Atrazine will decrease the stem cell behavior. (Proliferation/ Differentiation)
What effect does Xenoestrogen (Atrazine) have on Stem Cell differentiation?

7. Materials Cryotank 75mm2 tissue culture treated flasks
Fetal bovine serum (FBS)
Trypsin-EDTA
Atrazine
Pen/strep
Macropipette + sterile macropipette tips (1 mL, 5 mL, 10, mL, 20 mL)
Micropipettes + sterile tips
DMEM Serum - 1% and Complete Media (4 mM L-glutamine, mg/L glucose, 1 mM sodium pyruvate, and 1500 mg/L sodium bicarbonate + [ 10% fetal bovine serum for complete])
75 mL culture flask
Incubator
Nikon Inverted Microscope with imaging technology
Laminar Flow Hood
Laminar Flow Hood UV Sterilizing Lamp
Sharpie pen
Hemacytometer
Sterile PBS
C2C12 Stem Cell Line
Ethanol (70%)
Sterile Water
Nitrile gloves

8. Procedure Cell Culturing
A 1mL aliquot of C2C12 cells to inject, create 30 mL of 10% media in two culture flasks yielding a cell density of approximately 2x106 to 2x10 cells in each 75 mm^2
The media was removed and 15 mL of fresh media was added in order to remove any contamination
The flasks were incubated at 37 degrees Celsius for 2 days or until cell density of 4x106 to 5x106 cells/mL was reached
The culture was passed into new flasks and incubated for 2 days at 37 degrees Celsius

9. Procedure Cont. Cell Passing Media was removed from each 75mm2 flask
2 mL of trypsin was added to each flask to wash the surface
After trypsin was removed, 1 mL of FRESH trypsin was added to each, a process called trypsinization
The flasks were incubated for 4 minutes
In order to stop the reaction, 13 mL of fresh DMEM 10% was added, creating a cell density of approximately 1 million cells/mL
2 mL of the cell suspension was added to six 25 mm2 flasks that already contained 3 mL of fresh DMEM 10% media, yielding a total of 5 mL in each flask and creating a cell density of approximately 105 cells per flask

10. Procedure Cont.
Variable Stock Solution
Two stock solutions of atrazine were created using sterile water: 1/100x and 1/10,000x.
X = the concentration of the undiluted Atrazine product.
High (1/100): 0.1 mL of 4% Atrazine added to 9.9 mL sterile water yielding a total of 5 mL
Low (1/10,000): 0.1 mL of High solution added to 9.9 mL sterile water yielding a total of 5 mL
The following concentrations of the variable (next page) were added to each 25 mm2 flask (2 flasks created per variable)
The flasks were incubated at 37 degrees Celsius for the next couple days in order to gather accurate results

11. Chart of Concentrations
0 (Control)
10-6x
0-4x
Stock Solution
0.05 µL of 1/10,000 stock
0.05 µL of 1/100 stock
Fresh Media
5 mL
4.950 mL
Total

12. Procedure Cont. Cell Wells
After one day of growth in 10% serum DMEM, media was removed and replacd with 1 mL of 1% DMEM media was added to each well (12 wells in total)
The well plate was split into three sections: 4-control, 4-low, 4-high
10 µL of distilled water was added to the control section
10 µL of the low stock solution was added to the low section
10 µL of the high stock solution was added to the high section
The well plate was incubated at 37 degrees Celsius, 5% Co2

13. Counting Procedure (Days 1 and 3)
1 flask representing each variable was used to determine cell density of for the first counting day
The cells in each flask were trypsinized and 2 mL of fresh media were added to the individual flasks in order to quench the reaction
For each flask: eight 25 µl aliquots samples were transferred to
hemacytometers to count under the microscope
Day 3:
The same procedure was done using the day 3 flasks

14. C2C12 Proliferation Results
P-value=
2.85E-12
P-value=
1.83E-14
Cell Count (Cells/flask)
Variable Concentration

15. Day 1(Flask) Differentiation Analysis
Control
Low
High

16. Day 3 (Flask) Differentiation Analysis
Control
Low
High

17. Day 1 (Well) Differentiation Analysis
Control
Low
High

18. Day 3 (Well) Differentiation Analysis
Control
Low
High

19. Anova: Single Factor Analysis of Variation
Statistical test that asses the differences between means (averages)
Used when the experiment involves three or more levels of a single independent variable
Tests hypotheses about the average of a dependent variable across different groups
Determines whether or not null hypothesis can be rejected or accepted

20. Dunnett’s Test
Quantitative statistical analysis that compares means (averages)
All variation groups are compared to reference group (control)
Identifies variation groups with means that significantly differ from the reference group

21. Dunnet’s Test Results Day 1: T Crit= 2.67 Variable Concentration
T Value
Interpretation
Low
Not Significant
High
Significant
Day 3: T Crit= 2.67
Variable Concentration
T Value
Interpretation
Low
Significant
High
Not Significant

22. Conclusion
The results of the Anova test and the Dunnett’s statistical analysis support the conclusion that:
Atrazine in low concentrations significantly increases the cell proliferation
Atrazine in high concentrations majorly decreases the cell proliferation
However, time of exposure seems to be a major factor in the growth rate

23. Limitations Future Extensions Test greater concentrations of Atrazine
Use other cell lines such as MG63 Cancer cells
Perform experiment over longer period of time
Test to see if exposure time is a factor

24. Sources
2/Default.aspx?ATCCNum=crl-1772&Template=cellBiology
1123atrazine-tied-to-menstrual-irregularities
has-estrogenic-effects-and-interferes-with-metamorphosis-of-frogs.html