Xenoestrogen Effects on Stem Cell Behavior

Xenoestrogen Effects on Stem Cell BehaviorMaria DeRenzoOakland Catholic High SchoolGrade 10

Tissue EngineeringDevelopment 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

C2C12Subclone of the mus musculus (mouse) myoblast cell lineDifferentiates rapidly, forming contractile myotubes and produces characteristic muscle proteinsMouse stem cell line is used as a model in many tissue engineering experimentsUsed to study the differentiation of non-muscle cells (stem cells) to skeletal muscle cellsExpresses muscle proteins and the androgen receptor (AR)

Nonsteroidal ER ActivatorsXenoestrogens or Environmental EstrogensType of xenohormone that imitates estrogenCommonly used in industrial compounds which have estrogenic effects on a living organismsIndustrial 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)

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

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? Materials75 mL culture flaskIncubatorNikon Inverted Microscope with imaging technologyLaminar Flow HoodLaminar Flow Hood UV Sterilizing LampSharpie penHemacytometerSterile PBSC2C12 Stem Cell LineEthanol (70%)Sterile WaterNitrile gloves

Cryotank75mm2 tissue culture treated flasks25 mm2 tissue culture treated flasksFetal bovine serum (FBS)Trypsin-EDTAAtrazinePen/strepMacropipette + sterile macropipette tips (1 mL, 5 mL, 10, mL, 20 mL)Micropipettes + sterile tipsDMEM 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])

ProcedureCell CulturingA 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 reachedThe culture was passed into new flasks and incubated for 2 days at 37 degrees Celsius and 5% CO2

Procedure Cont.Cell PassingMedia was removed from each 75mm2 flask2 mL of trypsin was added to each flask to wash the surfaceAfter 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 quench 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 SolutionTwo 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 mLThe 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 with 5% CO2

Chart of Concentrations0 (Control)10-6X10-4XStock Solution0

0.05 L of 1/10,000 stock0.05 L of 1/100 stock

Fresh Media5 mL4.950 mL4.950 mLTotal 5 mL5 mL 5 mL Procedure Cont.Cell Wells1 mL of 1% DMEM media was added to each well (12 wells in total)The well was split into three sections: 4-control, 4-low, 4-high10 L of distilled water was added to the control section10 L of the low stock solution was added to the low section10 L of the high stock solution was added to the high sectionThe well was incubated at 37 degrees Celsius with 5% CO2

Counting Procedure (Days 1 and 3)Day 1: 1 flask representing each variable was used to determine cell density of for the first counting dayThe cells in each flask were trypsinized and 2 mL of fresh media were added to the individual flasks in order to stop the reactionFor each flask: eight 25 l 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-12P-value=1.83E-14

Day 1(Flask)Differentiation Analysis

ControlLow

HighDay 3 (Flask)Differentiation Analysis

Control

Low

HighDay 1 (Well)Differentiation Analysis

Control

Low

HighDay 3 (Well)Differentiation Analysis

Control

Low

HighAnova: Single FactorAnalysis 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

Dunnetts TestQuantitative 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 Dunnets Test ResultsVariable ConcentrationT ValueInterpretationLow3.01659076SignificantHigh1.263358523Not Significant Day 1: T Crit= 2.67Variable ConcentrationT ValueInterpretationLow2.670741014Not SignificantHigh3.160143817Significant Day 3: T Crit= 2.67Conclusion The results of the Anova test and the Dunnetts 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 proliferationAtrazine in high concentrations majorly decreases the cell proliferationHowever, time of exposure seems to be a major factor in the growth rate

LimitationsFuture ExtensionsHealth status of cells can varyTransport issues with cells between incubatorsLag time in cell counting can cause inaccurate dataOnly used qualitative assay of differentiation Test greater concentrations of AtrazineUse other cell lines such as MG63 Cancer cellsPerform experiment over longer period of timeTest to see if exposure time is a factorUtilize 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

Sourceshttp://www.atcc.org/ATCCAdvancedCatalogSearch/ProductDetails/tabid/452/Default.aspx?ATCCNum=crl-1772&Template=cellBiologyhttp://www.environmentalhealthnews.org/ehs/news/2011/2011-1123atrazine-tied-to-menstrual-irregularitieshttp://www.ncbi.nlm.nih.gov/pubmed/20184662http://eartotheground.typepad.com/weblog/2010/06/the-herbicide-atrazine-has-estrogenic-effects-and-interferes-with-metamorphosis-of-frogs.htmlhttp://www.epa.gov/opp00001/reregistration/atrazine/atrazine_update.htm