1. Vitamin E's Effects on Yeast's Mutagenesis Rate Kristopher Sabatini 12 th Grade at CCHS 5 th Year at PJAS

2. Previous Studies ● Mixed and contradictory studies ● Positive anticancer effects on bladder and breast cancer ● No effect on kidney, lung nor cardiac cancer ● Linked to increase in prostate cancer in high doses ● Overall – beneficial to general health

3. Vitamin E ● Common Anti-oxidant ● Found in nut oils and green vegetables ● Lipid Soluble and hydrophobic ● Variety of Isomers ● Alpha and gamma tocopherols– most useful to humans

4. Ultraviolet Rays Light waves that have shorter wavelengths, thus greater energy, than visible light They range from 400nm to 10nm Given off from the sun but most are absorbed by the ozone layer Mutagen – Direct DNA Damage

5. Yeast Commonly used model Tolerant and safe to culture Has similar reproduction, metabolism, and chemistry as other more advanced eukaryotic cells Saccharomyces cerevisiae Special Strain, unable to produce Lysine

6. Lysine a - Ketoglutarate AcCoA CoA HC Synthase Homocirate LYS7 Water Homoaconitate Homoisocitrate a - Ketoadipate a- Aminoadipate a- Aminodipate Semialdehye SaccharopinaLysine LYS4 LYS12 aAA- Aminotranfease LYS2 LYS9 LYS1 NAD NADH CO2 Glutamate a-Ketoglutrate ATP PP NADPH NADP Glutamate NADPH NADP Water NADP; NADP a- Ketoglutarate Lysine’s codons are AAA and AAG There are defined minus lysine yeast mutants used in research. Lys 2 mutants are missing an enzyme function within the lysine biosynthesis pathway. Result – cells require lysine supplementation

7. Ames Test - Developed to test the mutagenic and anti-mutagenic properties of various chemicals by Bruce Ames in 1970s. - Ames used a minus histidine mutant Salmonella (single point substitution). Bacteria cannot synthesize histidine due to this mutation. - Exposure to suspected mutagen correlated with increased reversion (mutation) rate. - Visible colonies appearing on complete (-His) media evidence of mutation through reversion - Obviously, a lower limit on mutation rate, because only 1 DNA site in genome assayed.

8. Modified Ames Test (-) Lys Yeast – Eukaryote The number of reverted colonies of yeast can be correlated with the rate of mutation. A reversion at that point can result in a reversion back to wild type yeast (lys +). Mutagen substitution – UV light instead of heat

9. Objective ● To determine what effects Vitamin E has on Yeast's Mutagenesis Rate

10. Hypothesis ● Vitamin E will not have an effect on Yeast's Mutagenesis Rate

11. 45 (-) Lysine YEPD agar plates(1% yeast extract, 2% peptone, 2% dextrose, 1.5% agar) UV Light Oven (LD-50 on Yeast is 30 seconds) Sterile dilution fluid [SDF] (10mM KH2PO4, 10mM K2HPO4, 1mM MgSO4,.1mM CaCl2, 100mM NaCl) Klett spectrophotometer Sterile pipette tips and Micropipettes Vortex Sidearm flask Spreader bar Ethanol Micro burner (-) Lysine Saccharomyces cerevisiae (John Wolford lab, CMU) Rubber Gloves Test tubes Microtubes Test Tube Rack SDF Test Tubes Vitamin E Oil Solution Materials

12. Procedure 1. A strain of yeast (-) Lys phenotype was grown for 2 days in YEPD media. 2. 1 day prior to experimentation the media was removed from the cell pellet and replaced with 3 mL of SDF. 3. The Vitamin was sterile filtered 4. The stock solution was sterilized with a 0.22 syringe micron-filter. 5. The pellet in SDF was resuspended. 6. The following ingredients were pipetted into sterile mircotubes. (Percents are by volume compared to stock solution) Two Tubes per concentration were used as 1.5 mL were needed WaterVariableYeastVolume Tubes 1 and 2 0. 8 mL0 mL0.2 mL1 mL Tubes 3 and 4 0.79 mL0.01 mL0.2 mL1 mL Tubes 5 and 6 0. 7 mL0.1 mL0.2 mL1 mL

13. 7. The cells were allowed to sit for 15 min. 8. 0.1 mL aliquots were spread onto 45 complete (-) Lys (15 each) agar plates (necessary to show cells that have reverted through mutation to wild type + lys ). 9. The plates were exposed to UV light for either 0, 15, 30 seconds 10. The plates were allowed to incubate for 3 days at 32 o C. 11. The colonies were counted and recorded. Each colony assumed to have arisen from 1 cell.

14. Results

15. ANOVA Statistical Analysis Alpha Value = 0.05 TestP-ValueInterpretation Two Factor ANOVA0.0000239948038879 2 Significant Single Factor ANOVA on isolated on UV 1.49668808058975E -08 Insignificant Single Factor ANOVA on isolated Vitamin E 0.806861423820598Significant

16. Dunnett's Test TestT-ValueInterpretati on 0s 0% vs 0s 1%1.46Insignificant 0s 0% vs 0s 10%1.65Insignificant 15s 0% vs 15s 1%8.36Significant 15s 0% vs 15s 10%11.98Significant 30s 0% vs 30s 1%6.78Significant 30s 0% vs 30s 10%6.56Significant T- crit = 3.29

17. Conclusion ● The Null Hypothesis can be rejected in both the 1% and 10% groups ● The insignificant variance in the no exposure group fits expectations (no mutagen thus little mutation) ● In each of the two other exposure groups, the Vitamin yielded significant results when compared back to the control of that group ● The lower colony counts in the 30s groups is may be due to UV light outright killing the colonies ● This results can be contributed to Vitamin E's anti-oxidant abilities combating the free radicals formed by UV stress

18. Limitations and Extensions ● Limitations ● Unsynced plating which leads to slightly different exposure times to Vitamin E ● Inability to control the exact amount of cells on each plate (minor difference overshadow by massive amount of cells) ● Slight positioning differences in UV Oven ● Inability to account for cell deaths due UV Light ● Extensions ● Different model ● Reduce lag time with lab hands ● Almar Blue Assay to account for cell deaths

19. Sources http://www.uic.edu/classes/phar/phar332/Clinical_Cases/vitamin%20cases/vitamin%2 0E/Vitamin%20E%20Chemistry.htm http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/A/AmesTest.html http://theoncologist.alphamedpress.org/content/6/3/298.full http://student.ccbcmd.edu/~gkaiser/biotutorials/protsyn/mutate.html http://jama.ama-assn.org/content/306/14/1549 http://ods.od.nih.gov/factsheets/vitamine/ http://www.huffingtonpost.com/craig-cooper/vitamine-e-prostate-cancer- _b_1016922.html