1. EFFECTS OF METHYLHEXANAMINE (DMAA) ON C2C12 AND 3T3 STEM CELLS Cameron Franz Pittsburgh Central Catholic High School Grade 11

2. Tissue Engineering TE is the development and manipulation of artificial implants, laboratory grown tissues, and genetically engineered cells and/or molecules to replace or support the function of defective or injured body parts It has the potential to replace or supplement the function of tissues destroyed or compromised in any variety of ways, including: Inherent design flaws Hereditary/congenital defects or conditions Disease Trauma Damage from an individual’s environment Aging TE has great potential for supplementing muscle tissue. 2

3. Stem Cells Unspecialized cells capable of renewing themselves through cell division. Under certain physiologic or experimental conditions, they can be induced to become tissue or organ specific cells with special functions. Stem cells offer a number of new potentials for treating disease 3

4. C2C12 Stem Cells Subclone of the mus musculus (mouse) myoblast cell line. A model type of stem cell line that was discovered in 1977 through experimentation of murine thigh muscle growth after a crush injury. Differentiates rapidly, forming contractile myotubes and produces characteristic muscle proteins. 4

5. 3T3 Stem Cells Cell line established from Swiss mouse embryo tissue standard fibroblast cell line Do not differentiate, produce ECM components in connective tissue Often used in the cultivation of keratinocytes, with the 3T3 cells secreting growth factors favorable to these kinds of cells. 5

6. Methylhexanamine (DMAA) Commonly marketed as a nutritional supplement or workout aid It has recently been subject to multiple bans by both governments and Sports Authorities The US Food and Drug Administration declared that methylhexanamine was potentially dangerous and did not qualify as a legal dietary supplement It warned supplement makers that it was illegal to market the drug However, it is still found in many supplements According to the FDA Numerous adverse events and at least 5 deaths have been reported in association with methylhexanamine-containing dietary supplements 6

7. DMAA (Continued) It is an indirect sympathomimetic drug Acts as a stimulant It constricts blood vessels and thus has effects on the heart, lungs, and reproductive organs It also causes bronchodilation, inhibits peristalsis in the intestines, and has diuretic effects 7

8. Purpose To determine the effect of DMAA exposure on C2C12 and 3T3 cell proliferation 8

9. Hypothesis Null: DMAA WILL NOT have an effect on the proliferation of C2C12 cells and 3T3 cells Alternative: DMAA WILL significantly effect the proliferation of C2C12 cells and 3T3 cells 9

10. Materials Cryotank Two 75mm2 tissue culture treated flasks 24 25mm2 tissue culture treated flasks Fetal bovine serum (FBS) C2C12 Myoblastic Stem Cell Line 3T3 Stem Cell Line Trypsin-EDTA Pen/strep Macropipette + sterile macropipette tips (1 mL, 5 mL, 10, mL, 20 mL) Micropipettes + sterile tips DMEM Media - 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]) 10 Methylhexanamine (DMAA) Powder 99.89% 75 mL culture flask Incubator Nikon Inverted Microscope 24 well plate Laminar Flow Hood Laminar Flow Hood UV Sterilizing Lamp Labeling Tape Hemocytometer Sterile PBS Ethanol (70% and 100%) Purple Nitrile gloves

11. Procedure A 1 mL aliquot of C2C12 cells and 3T3 cells from a Cryotank was used to inoculate 30 mL of 10% serum DMEM media in two 75mm2 culture flask yielding a cell density of approximately 106 to 2x106 cells. The media was replaced with 15 mL of fresh media to remove cryo-freezing fluid and incubated (37° C, 5% CO2) for 2 days until a cell density of approximately 4x106 to 5x106 cells/mL was reached. The culture was passed into two sets of 2 flasks in preparation for experiment and incubated for 2 days at 37° C, 5% CO2. 11

12. Procedure (Continued) After trypsinization, cells from all of the flasks were pooled into 1 common 75mm2 flask (cell density of approximately 1 million cells/mL). 0.2 mL of the cell suspension was added to eight 25 mm2 tissue culture treated flasks containing 5 mL of DMEM (com) media, creating a cell density of approximately 105 cells per flask. Concentrations of 0.1x, 1x, and 10x DMAA were created based on a 20mg dose by diluting DMAA Powder in media. The resulting solutions where sterile filtered. 2 T25 flasks of each concentration where created for both cell lines. 12

13. Cell Counts where obtained through the following: The cells were trypsinized and collected into cell suspension. 25 μl aliquots were transferred to a Hemocytometer for quantification (eight counts per flask). Counts were then taken on days 1 and 3

14. Stat Analysis ANOVA Short for Analysis of Variance Statistical test to find variance between and within groups If the P-Value is smaller than the Alpha Value (0.05), the analysis is significant Dunnett’s Test Follow up to an ANOVA Statistical test to find the source of variance If the T-value is larger then the T-Crit, the effect is significant

15. Effects of DMAA on 3T3 Stem Cells P-value=0.272 P-value=0.0097 (SIG.)

16. Dunnett’s Test: Day 3 3T3 Variable Concentrations T-ValueInterpretation T-Crit=2.306 DMAA 0.1X 0.167 not significant X 0.712 not significant 10X 3.12 SIGNIFICANT

17. Effects of DMAA on C2C12 Stem Cells P-value=0.0179 (SIG.) P-value=0.0001 (SIG.)

18. Dunnett’s Test: Day 1 C2C12 Variable Concentrations T-ValueInterpretation T-Crit=2.306 DMAA 0.1X 0.502 not significant X 3.16 SIGNIFICANT 10X 0.743 not significant

19. Dunnett’s Test: Day 3 C2C12 Variable Concentrations T-ValueInterpretation T-Crit=2.306 DMAA 0.1X 0.275 not significant X 1.15 not significant 10X 2.654 SIGNIFICANT

20. Conclusions The null hypothesis may be rejected for both 3T3 and C2C12 stem cells based on the results of the ANOVAs The Dunnett’s Tests show significant variation in the 10X for 3T3 on Day 3, in the 1X for C2C12 Day 1, and the 10X for C2C12 Day 3

21. Limitations Variation in counts using Hemocytometers Cell clumping Limited Number of DMAA exposures Limited number of replicates

22. Extensions More trials Studying effects on cell differentiation Different models Adding more variable concentrations and exposure times Synergistic effects of different variables (i.e. Caffeine and DMAA)

23. Resources & Acknowledgments Mr. Mark Krotec, PTEI Dr. Phil Campbell http://www.fda.gov/Food/DietarySupplements/QA DietarySupplements/ucm346576.htm http://www.fda.gov/Food/DietarySupplements/QA DietarySupplements/ucm346576.htm http://www.nytimes.com/2013/04/13/business/fd a-issues-warning-on-workout-booster.html?_r=0 http://www.nytimes.com/2013/04/13/business/fd a-issues-warning-on-workout-booster.html?_r=0 http://www.nutrivitashop.com/dmaa.html http://www.forbes.com/sites/robertglatter/2013/0 4/12/is-dmaa-dangerous-to-your-health/ http://www.forbes.com/sites/robertglatter/2013/0 4/12/is-dmaa-dangerous-to-your-health/ 23

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