Tylenol's Influence on Yeast Mutagenesis - Chloe Stiles 9th Grade at Oakland Catholic High School
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Tylenol’s Influence on Yeast
Mutagenesis
Chloe Stiles
9th Grade at Oakland Catholic High School
Tylenol
• Form used during this experiment
– Ingredients
• Acetaminophen USP (160 mg)
• Butylparaben
• FD&C Red 40 (CI 16035)
• Cherry Flavoring
• Glycerin
• High Fructose Corn Syrup
• Microcrystalline Cellulose
• Carboxymethlylcellulose Sodium
• Propylene Glycol
• Purified Water
• Sodium Benzoate
• Sorbitol Solution
• Sucralose
• Xanthan GumAcetaminophen – Prescription drug – Most common drug ingredient in America – Used in many pain reliever drugs • Tylenol • Panadol • Feverall
Previous Studies • Doses over 325 mg can be toxic to liver • Acetaminophen doses greater than 2275 mg per day may increase the chances of the blood thinning effect
Yeast • Common cell model • Tolerant and safe to culture • Has similar reproduction, metabolism, and chemistry as other more advanced eukaryotic cells • Saccharomyces cerevisiae – Special strain that is unable to produce Lysine was used – Lys 2 (-)
Lysine
Ames Test • Developed to test the mutagenic and anti-mutagenic properties of various chemicals by Bruce Ames in 1970s • Ames minus histidine mutant Salmonella (single point substitution) • 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
Sample Mutagenesis
AssayModified 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 +)
Ultraviolet Rays • Light waves that have shorter wavelengths, thus greater energy, than visible light • They range from 400nm to 100nm • Given off from the sun but most are absorbed by the ozone layer • Can cause skin cancer, sunburn, and sun stroke • Mutagen – Direct DNA Damage
Question
Does Tylenol (Acetaminophen) have
significant mutagenic properties?
Objective
To assess the mutagenicity of Tylenol
(Acetaminophen)Hypothesis • Null Hypothesis: Tylenol will not have a significant effect on Yeast's Mutagenesis Rate • Alternate Hypothesis: Tylenol will significantly increase Yeast’s Mutagenesis Rate
Materials
• Com – Lys plates (Yeast • Vortex
Nitrogen base 1%, Dextrose • Sidearm flask
2%, 1.5% agar, complete • Spreader bar
amino acid mix(minus lysine)
• Ethanol
100mg/L)
• Micro burner
• UV Light Hood
– 254 nm light 0.7-0.9 microwatts • (-) Lysine yeast(John Wolford
per cm2 lab, CMU)
• Sterile dilution fluid [SDF] • Rubber Gloves
(10mM KH2PO4, 10mM • Test tubes
K2HPO4, 1mM MgSO4, .1mM • Microtubes
CaCl2, 100mM NaCl)
• Test Tube Rack
• Klett spectrophotometer
• SDF Test Tubes
• Sterile pipette tips and
• Tylenol
MicropipettesProcedure 1. A strain of yeast (-) Lys phenotype was grown for 2 days in YEPD media 2. A series of washes with SDF were performed on the sterile yeast pellet to remove any residual nutrients (lysine) 3. A 3.5% Tylenol extract was sterilized through a 0.22 micron syringe filters 4. The pellet in SDF was re-suspended 5. The following ingredients were pipetted into sterile microtubes. (Percents are by volume compared to stock solution)
Test Tube Ingredients
0% .01% .05% .1%
Microbe 0.2mL 0.2mL 0.2mL 0.2mL
SDF 0.8mL 0.75mL 0.55mL 0.3mL
Tylenol 0mL 0.05mL 0.25mL 0.5mL
Total 1mL 1mL 1mL 1mLProcedure (Continued)
6. The cells were allowed to sit for 15 minutes
7. 0.1 mL and 0.2 mL aliquots were spread onto 48
complete (-) Lys (24 each) agar plates (necessary to
show cells that have reverted through mutation to
wild type (+) Lys)
8. 25 plates were made using the control
concentration and were exposed to UV light for
either 0, 5, 10, 20, or 30 seconds
9. All plates were allowed to incubate for 5 days at
32˚C
10. The colonies were counted and recorded. Each
colony assumed to have arisen from 1 cellTylenol’s Effects on Yeast
Mutagenesis Rate
1.4
P-value =0.359
Avg. Number of Colonies
1.2 P-value =0.621
1
0.8
0.6 0.1 mL
0.2 mL
0.4
0.2
0
0% 0.05% 0.01% 0.10%
Concentrations of TylenolUV’s Effects on Yeast’s Mutagenesis
Rate P-value =0.0049
120
Avg. Number of Colonies
P-value =8.56E-06
100
80
60
0.1 mL
40 0.2 mL
20
0
0 sec 5 sec 10 sec 20 sec 30 sec
UV Exposure TimeDunnett’s Test
UV Light
• Alpha= .05 T-crit= 3.48
Test T-Value Interpretation
5 sec 24.61632 Significant
10 sec 16.77975 Significant
20 sec 5.171686 Significant
30 sec 0.293589 Not significantConclusions • The null hypothesis can be rejected for the 5 sec, 10 sec, and 20 sec UV light groups • All three concentrations (0.05%, 0.01%, and 0.1%) in the 0.1mL and 0.2mL groups did not have a significant effect, so the null can be accepted • The lower colony counts in the 30 sec group may be due to UV light toxicity • UV light appears to be a stronger mutagen than Tylenol
Limitations • Plating synchronization • UVC was used • Low mutagenesis background rate • Slight positioning differences in UV Oven • Inability to account for cell deaths caused by UV Light
Extensions • Different model • Reduce lag time with lab assistants • Trypan Blue Assay to account for cell deaths • A future experiment testing Tylenol’s effects on mammalian and cancerous cell lines to see if it promotes uncontrollable growth
References • http://www.medicinenet.com/acetaminophen/article.htm • http://www.medicinenet.com/tylenol_liver_damage/article.htm • http://www.fda.gov/forconsumers/consumerupdates/ucm168830.htm • http://emedicine.medscape.com/article/820200-overview • http://www.nlm.nih.gov/medlineplus/ency/article/002598.htm • http://www.mayoclinic.org/drugs-supplements/acetaminophen-oral- route-rectal-route/description/drg-20068480
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