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)

4. Nonsteroidal ER Activators Xenoestrogens or Environmental Estrogens  Type of xenohormone that imitates estrogen  Commonly used in industrial compounds which have estrogenic effects on a living organisms  Industrial chemicals or their byproducts with estrogen activity  Effects on human health are controversial:  Recent studies suggest that long term exposure (BPA) may cause cancer  Examples: Bisphenol (BPA) and Polychlorobiphenyls (PCBs)

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 not have an effect on the stem cell behavior. (Proliferation/ Differentiation)  Alternate Hypothesis: Atrazine will have a significant effect on the stem cell behavior. (Proliferation/ Differentiation) What effect does Xenoestrogen (Atrazine) have on Stem Cell proliferation and differentiation?

7. Materials  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  Cryotank  75mm2 tissue culture treated flasks  25 mm2 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, 4500 mg/L glucose, 1 mM sodium pyruvate, and 1500 mg/L sodium bicarbonate + [ 10% fetal bovine serum for complete])

8. Procedure A. Cell Culturing 1. A 1mL aliquot of C2C12 cells to inject, create 30 mL of 10% media in two culture flasks yielding a cell density of approximately 2x10 6 to 2x10 cells in each 75 mm^2 2. The media was removed and 15 mL of fresh media was added in order to remove any contamination 3. The flasks were incubated at 37 degrees Celsius for 2 days or until cell density of 4x10 6 to 5x10 6 cells/mL was reached 4. The culture was passed into new flasks and incubated for 2 days at 37 degrees Celsius and 5% CO2

9. Procedure Cont. B. Cell Passing 1. Media was removed from each 75mm 2 flask 2. 2 mL of trypsin was added to each flask to wash the surface 3. After trypsin was removed, 1 mL of FRESH trypsin was added to each, a process called trypsinization 4. The flasks were incubated for 4 minutes 5. In order to quench the reaction, 13 mL of fresh DMEM 10% was added, creating a cell density of approximately 1 million cells/mL 6. 2 mL of the cell suspension was added to six 25 mm 2 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 10 5 cells per flask

10. Procedure Cont. C. Variable Stock Solution 1. Two stock solutions of atrazine were created using sterile water: 1/100x and 1/10,000x. X = the concentration of the undiluted Atrazine product: i. High (1/100): 0.1 mL of 4% Atrazine added to 9.9 mL sterile water yielding a total of 5 mL ii. Low (1/10,000): 0.1 mL of High solution added to 9.9 mL sterile water yielding a total of 5 mL 2. The following concentrations of the variable (next page) were added to each 25 mm 2 flask (2 flasks created per variable) 3. The flasks were incubated at 37 degrees Celsius with 5% CO2

11. Chart of Concentrations 0 (Control)10-6X10-4X Stock Solution00.05 µL of 1/10,000 stock 0.05 µL of 1/100 stock Fresh Media5 mL4.950 mL Total5 mL

12. Procedure Cont. D. Cell Wells 1. 1 mL of 1% DMEM media was added to each well (12 wells in total) 2. The well was split into three sections: 4-control, 4-low, 4-high 3. 10 µL of distilled water was added to the control section 4. 10 µL of the low stock solution was added to the low section 5. 10 µL of the high stock solution was added to the high section 6. The well was incubated at 37 degrees Celsius with 5% CO2

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

14. C2C12 Proliferation Results P-value= 2.85E-12 P-value= 1.83E-14

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

16. Day 3 (Flask) Differentiation Analysis ControlLowHigh

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

18. Day 3 (Well) Differentiation Analysis Control LowHigh

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 Variable Concentration T ValueInterpretation Low3.01659076Significant High1.263358523Not Significant Day 1: T Crit= 2.67 Variable Concentration T ValueInterpretation Low2.670741014Not Significant High3.160143817Significant Day 3: T Crit= 2.67

22. Conclusion The results of the Anova test and the Dunnett’s statistical analysis and the qualitative anaylis support the conclusion that: In all 3 concentrations of the variable, the null hypothesis can be rejected in both cases of day 1 and 3: 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  Health status of cells can vary  Transport issues with cells between incubators  Lag time in cell counting can cause inaccurate data  Only used qualitative assay of differentiation  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  Utilize quantitative assay (MyoD expression)  CyQUANT™ Cell Proliferation Assay  More quantitative than counting cells on a Hemocytometer  Fluorescent dye binds to nucleic acid in the cell

24. Sources  http://www.atcc.org/ATCCAdvancedCatalogSearch/ProductDetails/tabid/45 2/Default.aspx?ATCCNum=crl-1772&Template=cellBiology http://www.atcc.org/ATCCAdvancedCatalogSearch/ProductDetails/tabid/45 2/Default.aspx?ATCCNum=crl-1772&Template=cellBiology  http://www.environmentalhealthnews.org/ehs/news/2011/2011- 1123atrazine-tied-to-menstrual-irregularities http://www.environmentalhealthnews.org/ehs/news/2011/2011- 1123atrazine-tied-to-menstrual-irregularities  http://www.ncbi.nlm.nih.gov/pubmed/20184662 http://www.ncbi.nlm.nih.gov/pubmed/20184662  http://eartotheground.typepad.com/weblog/2010/06/the-herbicide-atrazine- has-estrogenic-effects-and-interferes-with-metamorphosis-of-frogs.html http://eartotheground.typepad.com/weblog/2010/06/the-herbicide-atrazine- has-estrogenic-effects-and-interferes-with-metamorphosis-of-frogs.html  http://www.epa.gov/opp00001/reregistration/atrazine/atrazine_update.htm http://www.epa.gov/opp00001/reregistration/atrazine/atrazine_update.htm