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

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

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.

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

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

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?

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, 4500 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

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

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

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

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

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

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

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

Day 1(Flask) Differentiation Analysis Control Low High

Day 3 (Flask) Differentiation Analysis Control Low High

Day 1 (Well) Differentiation Analysis Control Low High

Day 3 (Well) Differentiation Analysis Control Low High

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

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

Dunnet’s Test Results Day 1: T Crit= 2.67 Variable Concentration T Value Interpretation Low 2.670741014 Not Significant High 3.160143817 Significant Day 3: T Crit= 2.67 Variable Concentration T Value Interpretation Low 3.01659076 Significant High 1.263358523 Not Significant

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

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

Sources 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.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://www.epa.gov/opp00001/reregistration/atrazine/atrazine_update.htm