1. Sodium Fluoride Effects on Yeast Mutagenesis
Joshua Egan
Third Year in PJAS
Pittsburgh Central Catholic High School
11th Grade

2. Sodium Fluoride
Inorganic ionic compound, dissolving to give separated Na+ and F− ions
Often added to municipal drinking water and certain food products for the purposes of maintaining dental health
Tap water, tooth paste, etc.
Classed as toxic by both inhalation (of dusts or aerosols) and ingestion
Shown to affect the heart and circulatory system

3. Problem
Sodium fluoride is included in consumable products across the globe with unknown effects on internal microflora.

4. Is sodium fluoride a potential mutagen?
Question
Is sodium fluoride a potential mutagen?

5. Cell Model Saccharomyces cerevisiae Commonly used model
Tolerant and safe to culture
Has similar reproduction, metabolism, and chemistry as other more advanced eukaryotic cells
(-) Lys Special strain unable to produce lysine

6. DNA and Mutations Deoxyribonucleic Acid
Transcribed by RNA which is used by the Ribosome to assemble proteins from amino acids
Eukaryotes have DNA inside their nuclei
Mutations are changes made to the cell's genome, and are mostly harmless
Can be caused by radiation, viruses, chemical mutagens, and random errors in DNA replication  

7. Lysine Lysine codons AAA, AAG
(-) lysine yeast mutants used in research
(-) Lys mutants are missing an enzyme function within the lysine biosynthesis pathway
Result: Cells require lysine supplementation

8. Ames Test
Used a (-) histidine mutant Salmonella (single-point substitution) Bacteria cannot synthesize histidine due to mutation
Exposure to suspected mutagen correlated with increased reversion (mutation) rate
A lower limit on mutation, assayed only 1 DNA site in genome

9. Ames Test

10. Modified Ames Test
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+)

11. Purpose
To determine the effects of sodium fluoride (suspected mutagen) on the mutagenesis rate of (-) Lys yeast.

12. Hypotheses Null Hypothesis: Alternate Hypothesis:
Sodium Fluoride will not have a significant effect on the mutagenesis rate of yeast.
Alternate Hypothesis:
Sodium Fluoride will have a significant effect on the mutagenesis rate of yeast.

13. Materials (-) Lysine agar plates Spreader bar Ethanol Micro burner
1% yeast nitrogen base w/o amino acids
Ethanol
Micro burner
2% glucose
(-) Lysine Saccharomyces cerevisiae
1 mM amino acid mix
1.5% agar
Rubber gloves
Sterile dilution fluid (SDF) 10 mM KH2PO4, 1 mM MgSO4, 1 mM CaCl2, 100 mM NaCl
Test tubes
Microtubes
SDF Test Tubes
Test tube rack
Macro/micropipettes
Sodium fluoride powder
Sterile pipette tips, microplates
Incubator
Vortex
Side-arm flask

14. Procedure
Strain of yeast (-) Lys phenotype grown for 2days in YEPD media.
Sterile yeast pellet washed with SDF to remove any residual nutrients (lysine) and allowed to sit and acclimate
Stock re-suspended and stored in com. (-) Lys media for 2 days
A 1% sub-stock of the sodium fluoride was made by diluting the variable with sterile water.
Sterilized through a 0.22 micron syringe filter
The pellet was re-suspended in SDF

15. Procedure (cont.)
The following ingredients were pipetted into sterile 1.5 mL microtubules:
Water
Variable
Yeast
Total Volume 0%
0.9 mL
0 mL
0.4 mL
1.3 mL
0.01%
0.887 mL
0.013 mL (of 1% sub-stock)
0.1%
0.77 mL
0.13 mL

16. Procedure (cont.) The cells were allowed to sit for 15 minutes
0.2 mL aliquots were spread onto complete (-) Lys agar plates (necessary to show cells that have reverted through mutation to wild type (+) Lys)
12 plates for each concentration, totaling 36 plates
All plates were allowed to incubate for 5 days at 32 ̊C
The colonies were counted and recorded. Each colony assumed to have arisen from 1 cell

17. P-value for all data: 4.73 × 10-3 (significant)

18. ANOVA/Dunnett's Test Anova: Dunnett's Test:
Statistical test that allows for the comparison of means of different groups, to determine significant variation
Utilize p-values as measures of significance
Confidence interval of the likelihood of seeing the same effect produced by the same variable with replication
P>0.05: not significant
P<0.05: significant
Dunnett's Test:
A test used to find out which variable groups produced significant variation compared to a control
If T-value is greater than the T-critical, variations are considered significant

19. Dunnett’s Test Results
T Value
Result
(T-Crit: 2.920)
0% vs. 0.01%
0.914
Not significant
0% vs. 0.1%
3.433
Significant

20. Interpretation of Results
The null hypothesis can be rejected
The alternate hypothesis can be accepted
After running the Dunnett’s Test, it was found that at 0.1% concentration the sodium fluoride had a significant positive effect on the rate of mutagenesis

21. Limitations and Extensions
Slightly out-of-synced plating which leads to slightly different exposure times to sodium fluoride
Surprisingly small # of revertants (major problem)
Inability to control the exact amount of cells on each plate (minor difference overshadow by massive amount of cells)
Slight positioning differences in the incubation process
Limited concentration exposures

22. Limitations and Extensions
Testing different models
Repeat experiment with larger concentration of cells (account for small number of revertants)
Different concentrations of the variable
Reduce lag time with lab assistants
Trypan Blue Assay to account for cell deaths
A future experiment testing sodium fluoride’s effects on mammalian and cancerous cell lines to see if it promotes uncontrollable growth

23. Sources https://pubchem.ncbi.nlm.nih.gov/compound/sodium_flu oride
Lec22.pdf
management/datopic.html
prevention/risk/myths/fluoridated-water-fact-sheet

24. ANOVA