Mineral Oil Effects on Yeast Mutagenesis Luke Giannetta Second Year in PJAS Central Catholic High School 10th Grade

Mineral Oil Unrefined mineral oil is a known carcinogen (American Cancer Society) Studies by: International Agency for Research on Cancer National Toxicology Program Principal Ingredients D-alpha tocopherol (Vitamin E) Liquid Paraffin

? Question Does commercially sold mineral oil affect the mutagenic rate of yeast significantly?

Liquid Paraffin General formula CnC2n+2H Paraffin isomers

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

Lysine Lysine codons AAA, AAG (-) 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 by Bruce Ames (c. 1970) Tests for mutagenic/anti-mutagenic properties of various chemicals 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 Visible colonies appearing on complete (-)Histidine media  evidence of mutation through reversion Obviously, a lower limit on mutation Assayed only 1 DNA site in genome

Ames Test -Lys yeast Lys+

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 +)

Ultraviolet (UV) Rays Short-wavelength electromagnetic waves greater energy than visible light wavelengths range from 400nm to 10nm Sun’s UV rays absorbed by ozone layer Causes direct DNA damage (mutagen)

Purpose To determine the effects of mineral oil (suspected mutagen) on the mutagenesis rate of (-) Lys yeast To determine the effects of UV light (known mutagen) on the mutagenesis of (-) Lys and the survivorship of wild-type yeast

Hypotheses Null Hypothesis Mineral oil will not have a significant effect on the mutagenesis rate of yeast. Alternate Hypothesis Mineral oil will have a significant effect on the mutagenesis rate of yeast.

Materials (-) Lysine agar plates 1% yeast nitrogen base w/o amino acids 2% glucose 1 mM amino acid mix 1.5% agar YEPD plates UV light hood (LD-50 on yeast is 30 s) Sterile dilution fluid (SDF) 10 mM KH2PO4, 1 mM MgSO4 1 mM CaCl2, 100 mM NaCl SDF Test Tubes Sterile pipette tips, microplates Vortex Side-arm flask Spreader bar Ethanol Micro burner (-) Lysine Saccharomyces cerevisiae Wild type (+) Lys Saccharomyces cerevisae Rubber gloves Test tubes Microtubes Test tube rack Rite Aid ® Mineral Oil

Procedure Strain of yeast (-) Lys phenotype grown for 2 days in YEPD media. Sterile yeast pellet washed with SDF to remove any residual nutrients (lysine) Stock re-suspended and stored in com. (-) Lys media for 2 days A 100% stock and a 10% sub-stock of the mineral oil were made by diluting the variable with sterile water. The pellet in SDF was re-suspended.

Procedure (cont’d) The following ingredients were pipetted into sterile microtubules Water Variable Yeast Total Volume 0% 0.8 mL 0 mL 0.2 mL 1 mL 0.1% 0.7 mL 0.1 mL (of 10% substock) 1% 0.79 mL 0.01 mL 10% 0.1 mL

Procedure (cont’d) 7. Cells were allowed to sit for 25 mins. 8. 0.1 mL of each tube plated onto 6 complete (-) Lys plates (necessary to show cells that have reverted via mutation to wild-type (+) Lys) 9. Remaining 0.4 mL of each tube was split into two 0.2 mL aliquots and plated onto 2 complete (-) Lys plates 10. Plates were incubated for 5 days at 32 °C 11. Colonies counted and recorded. Each colony assumed to have arisen from a single cell.

UV exposure Procedure (-) Lys Yeast Regular Yeast 0.1 mL of suspended (-) Lys cells were plated on each of 12 complete (-) Lys plates Plates exposed to UV light in groups of three – at 0, 15, 30, and 45 seconds Plates were incubated for three days at 32 °Celsius Colonies were counted, each colony assumbed to have arisen from a single cell Regular Yeast 0.1 mL of suspended Saccharomyces ceravisae cells were plated on each of 12 YEPD plates Plates were exposed to UV light in groups of three at 0, 15, 30, and 45 seconds Plates incubated for three days at 32 °Celsius Colonies were counted, each colony assumed to have arisen from a single cell

(-) LYS UV Exposure Results UV Light Effects on (-) Lys Yeast P value = 0.000132 # of Colonies Exposure Time (s)

Dunnett’s Test Results T-Critical = 3.75 Test T Value Interpretation 0 sec vs. 15 sec 4.8805 Significant 0 sec vs. 30 sec 2.6103 Not Significant 0 sec vs. 45 sec 0.7945

REGULAR YEAST UV EXPOSURE RESULTS UV Light Effects on Yeast Survivorship P-value = 0.000454 P value = 0.000454 # of Colonies Exposure Time (s)

Dunnett’s Test Results T-Critical = 3.75 Test T Value Interpretation 0 sec vs. 15 sec 1.87356 Not Significant 0 sec vs. 30 sec 5.245857 Significant 0 sec vs. 45 sec 6.958826

Mineral Oil Exposure REsults Mineral Oil Effect on Mutagenesis P Value = 0.032446 # of Colonies Mineral Oil Concentration

Dunnett’s Test Results T-Critical = 2.88 Test T Value Interpretation 0% vs. 0.1% 0.21934 Not Significant 0% vs. 1%sec 1.97406 0% vs. 10% 2.63208

Conclusions None of the concentrations of mineral oil showed ability to significantly affect the rate of yeast mutagenesis Null Hypothesis was accepted Alternate Hypothesis was rejected

Limitations and Extensions Slightly out-of-synced plating which leads to slightly different exposure times to mineral oil 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

Limitations and Extensions Different model Reduce lag time with lab assistants Trypan Blue Assay to account for cell deaths A future experiment testing mineral oil's effects on mammalian and cancerous cell lines to see if it promotes uncontrollable growth.

Sources http://www.britannica.com/EBchecked/topic/442584/paraffin-hydrocarbon https://shop.riteaid.com/rite-aid-pharmacy-mineral-oil-usp-16-fl-oz-1-pt-473-ml-0033067 http://www.cancer.org/cancer/cancercauses/othercarcinogens/generalinformationaboutcarc inogens/known-and-probable-human-carcinogens http://www.aacr.org/home/public--media/aacr-in-the-news.aspx?d=2740 http://dontdip.tamu.edu/ingredients.htm http://www.sciencemag.org/content/274/5286/430.abstract?ijkey=7dd94e096ea549bac90bc e0ec51acb6422cbb1a4&keytype2=tf_ipsecsha http://www.iarc.fr/en/media-centre/pr/2004/pr154.html

ANOVA Seconds Exposed 15 30 45 Wild Type Yeast 91 74 69 49 80 46 92 50 15 30 45 Wild Type Yeast 91 74 69 49 80 46 92 50 39 Anova: Single Factor SUMMARY Groups Count Sum Average Variance Column 1 3 263 87.66667 44.33333 Column 2 228 76 12 Column 3 165 55 151 Column 4 133 25.33333 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 3478.917 1159.639 19.93649 0.000454 4.066180551 Within Groups 465.3333 8 58.16667 Total 3944.25 11  

Mineral Oil Effects on Yeast Mutagenesis Luke Giannetta Second Year in PJAS Central Catholic High School 10th Grade