MINERAL OIL EFFECTS ON YEAST MUTAGENESIS Luke Giannetta

MINERAL OIL EFFECTS ON YEAST MUTAGENESIS Luke Giannetta Second Year in PJAS Central Catholic High School 10 th Grade

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

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

LIQUID PARAFFIN u General formula u Paraffin isomers Cn. C 2 n+2 H

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

LYSINE u Lysine codons AAA, AAG u (-) lysine yeast mutants used in research u Lys 2 mutants are missing an enzyme function within the lysine biosynthesis pathway u Result: Cells require lysine supplementation

AMES TEST u Developed by Bruce Ames (c. 1970) u Tests for mutagenic/anti-mutagenic properties of various chemicals u Used a (-)-histidine mutant Salmonella (single-point substitution) Bacteria cannot synthesize histidine due to mutation u Exposure to suspected mutagen correlated with increased reversion (mutation) rate u Visible colonies appearing on complete (-)Histidine media evidence of mutation through reversion u Obviously, a lower limit on mutation u 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 u Short-wavelength electromagnetic waves u greater energy than visible light u wavelengths range from 400 nm to 10 nm u Sun’s UV rays absorbed by ozone layer u Causes direct DNA damage (mutagen)

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

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

MATERIALS u (-) Lysine agar plates 1% yeast nitrogen base w/o amino acids 2% glucose 1 m. M amino acid mix 1. 5% agar u YEPD plates u UV light hood (LD-50 on yeast is 30 s) u Sterile dilution fluid (SDF) 10 m. M KH 2 PO 4, 1 m. M Mg. SO 4 1 m. M Ca. Cl 2, 100 m. M Na. Cl u SDF Test Tubes u Sterile pipette tips, microplates u u u 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 1. Strain of yeast (-) Lys phenotype grown for 2 days in YEPD media. 2. Sterile yeast pellet washed with SDF to remove any residual nutrients (lysine) 3. Stock re-suspended and stored in com. (-) Lys media for 2 days 4. A 100% stock and a 10% sub-stock of the mineral oil were made by diluting the variable with sterile water. 5. The pellet in SDF was re-suspended.

PROCEDURE (CONT’D) 6. The following ingredients were pipetted into sterile microtubules Water Variable Yeast Total Volume 0. 8 m. L 0. 2 m. L 1 m. L 0. 1% 0. 7 m. L 0. 1 m. L (of 10% substock) 0. 2 m. L 1% 0. 79 m. L 0. 01 m. L 0. 2 m. L 10% 0. 7 m. L 0. 1 m. L 0. 2 m. L 1 m. L 0%

PROCEDURE (CONT’D) 7. Cells were allowed to sit for 25 mins. 8. 0. 1 m. L 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 m. L of each tube was split into two 0. 2 m. L aliquots and plated onto 2 complete (-) Lys plates 10. Plates were incubated for 5 days at 32 °C 11. Colonies counted and recorded.

UV EXPOSURE PROCEDURE (-) Lys Yeast Regular Yeast u 0. 1 m. L of suspended Saccharomyces ceravisae cells were plated on each of 12 YEPD plates u Plates were exposed to UV light in groups of three at 0, 15, 30, and 45 seconds Plates were incubated for three days at 32 °Celsius u Plates incubated for three days at 32 °Celsius Colonies were counted, each colony assumbed to have arisen from a single cell u Colonies were counted, each colony assumed to have arisen from a single cell u 0. 1 m. L of suspended (-) Lys cells were plated on each of 12 complete (-) Lys plates u Plates exposed to UV light in groups of three – at 0, 15, 30, and 45 seconds u u

(-) LYS UV EXPOSURE RESULTS UV Light Effects on (-) Lys Yeast 37. 5 # of Colonies P value = 0. 000132 30. 0 22. 5 15. 0 7. 5 0. 0 0 sec 15 sec 30 sec Exposure Time (s) 45 sec

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 Not Significant

REGULAR YEAST UV EXPOSURE RESULTS UV Light Effects on Yeast Survivorship 90 P-value = 0. 000454 # of Colonies P value = 0. 000454 80 70 60 50 40 30 0 15 30 Exposure Time (s) 45

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 Significant

MINERAL OIL EXPOSURE RESULTS Mineral Oil Effect on Mutagenesis # of Colonies 4 P Value = 0. 032446 3 2 1 0 0. 0% 0. 1% 1. 0% Mineral Oil Concentration 10. 0%

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 Not Significant 0% vs. 10% 2. 63208 Not Significant

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

LIMITATIONS AND EXTENSIONS Limitations u Slightly out-of-synced plating which leads to slightly different exposure times to mineral oil u Inability to control the exact amount of cells on each plate (minor difference overshadow by massive amount of cells) u Slight positioning differences in UV Oven Inability to account for cell deaths due UV Light

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

SOURCES u http: //www. britannica. com/EBchecked/topic/442584/paraffin-hydrocarbon u https: //shop. riteaid. com/rite-aid-pharmacy-mineral-oil-usp-16 -fl-oz-1 -pt-473 -ml 0033067 u http: //www. cancer. org/cancercauses/othercarcinogens/generalinformation aboutcarcinogens/known-and-probable-human-carcinogens u http: //www. aacr. org/home/public--media/aacr-in-the-news. aspx? d=2740 u http: //dontdip. tamu. edu/ingredients. htm u http: //www. sciencemag. org/content/274/5286/430. abstract? ijkey=7 dd 94 e 096 ea 54 9 bac 90 bce 0 ec 51 acb 6422 cbb 1 a 4&keytype 2=tf_ipsecsha u http: //www. iarc. fr/en/media-centre/pr/2004/pr 154. html

ANOVA Seconds Exposed 0 15 30 45 91 74 69 49 80 74 46 45 92 80 50 39 Wild Type Yeast Anova: Single Factor SUMMARY Groups Count Sum Average Variance Column 1 3 263 87. 66667 44. 33333 Column 2 3 228 76 12 Column 3 3 165 55 151 Column 4 3 133 44. 33333 25. 33333 ANOVA Source of Variation SS df MS Between Groups 3478. 917 3 1159. 639 Within Groups 465. 3333 8 58. 16667 3944. 25 11 Total F P-value 19. 93649 F crit 0. 000454 4. 066180551

MINERAL OIL EFFECTS ON YEAST MUTAGENESIS Luke Giannetta Second Year in PJAS Central Catholic High School 10 th Grade