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

Vitamin E's Effects on Yeast's Mutagenesis Rate

Kristopher Sabatini

12th Grade at CCHS

5th Year at PJAS

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

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

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

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

Lysine a - Ketoglutarate

AcCoACoA

HCSynthase

Homocirate

LYS7Water

Homoaconitate

Homoisocitrate

a - Ketoadipate

a- Aminoadipate

a- AminodipateSemialdehye

SaccharopinaLysine

LYS4

LYS12

aAA- Aminotranfease

LYS2

LYS9

LYS1

NADNADHCO2

Glutamatea-Ketoglutrate

ATPPPNADPHNADP

GlutamateNADPHNADPWater

NADP; NADPa- 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

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.

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

Objective

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

Hypothesis

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

• 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

Procedure1. 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 filtered4. 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

Water Variable Yeast Volume

Tubes 1 and 2

0. 8 mL 0 mL 0.2 mL 1 mL

Tubes 3 and 4

0.79 mL 0.01 mL 0.2 mL 1 mL

Tubes 5 and 6

0. 7 mL 0.1 mL 0.2 mL 1 mL

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 32o C.

11. The colonies were counted and recorded. Each colony assumed to have arisen from 1 cell.

Results

0s 15s 30s0

5

10

15

20

25

30

35

40

45

Vitamin E's Effects on Yeast's Mutagenesis Rate

0.00%1.00%10.00%

Exposure Time

Colo

nies

ANOVA Statistical AnalysisAlpha Value = 0.05

Test P-Value Interpretation

Two Factor ANOVA 0.00002399480388792

Significant

Single Factor ANOVA on isolated on UV

1.49668808058975E-08

Insignificant

Single Factor ANOVA on isolated Vitamin E

0.806861423820598

Significant

Dunnett's Test

Test T-Value Interpretation

0s 0% vs 0s 1% 1.46 Insignificant

0s 0% vs 0s 10% 1.65 Insignificant

15s 0% vs 15s 1%

8.36 Significant

15s 0% vs 15s 10%

11.98 Significant

30s 0% vs 30s 1%

6.78 Significant

30s 0% vs 30s 10%

6.56 Significant

T- crit = 3.29

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

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

Sources

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