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

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

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

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

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

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  

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

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

Ames Test

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+)

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

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.

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

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

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

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

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

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

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

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

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

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

Sources https://pubchem.ncbi.nlm.nih.gov/compound/sodium_flu oride http://www.biology-pages.info/A/AmesTest.html https://www.britannica.com/science/yeast-fungus https://explorable.com/anova http://www.stat.purdue.edu/~xuanyaoh/stat350/xyMar23 Lec22.pdf https://ori.hhs.gov/education/products/n_illinois_u/data management/datopic.html https://www.cancer.gov/about-cancer/causes- prevention/risk/myths/fluoridated-water-fact-sheet

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