MINERAL OIL EFFECTS ON

YEAST MUTAGENESIS

Luke GiannettaSecond Year in PJAS

Central Catholic High School10th Grade

Unrefined mineral oil is a known carcinogen(American Cancer Society) Studies by:

International Agency for Research on CancerNational Toxicology Program

Principal Ingredients D-alpha tocopherol (Vitamin E) Liquid Paraffin

MINERAL OIL

?QUESTION

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

General formula CnC2n+2H

Paraffin isomers

LIQUID PARAFFIN

Saccharomyces cerevisiae Commonly used model Tolerant and safe to culture Has similar reproduction,

metabolism, and chemistryas other more advanced eukaryotic cells

(-) Lys Special strain unable to produce lysine

CELL MODEL

Lysine codons AAA, AAG

(-) lysine yeast mutantsused in research

Lys 2 mutants are missingan enzyme functionwithin the lysine biosynthesis pathway

Result: Cells require lysine supplementation

LYSINE

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

AMES TEST-Lys yeast

Lys+

MODIFIEDAMES TEST

The number of reverted colonies of yeastcan be correlated with the rate of

mutation.

A reversion at that pointcan result in a reversion

back to wild type yeast (lys +)

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)

ULTRAVIOLET (UV) RAYS

PURPOSE To determine the effects of mineral oil

(suspected mutagen) on the mutagenesis rateof (-) 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 HypothesisMineral oil will have a significant effect onthe mutagenesis rate of yeast.

(-) Lysine agar plates1% yeast nitrogen basew/o amino acids2% glucose1 mM amino acid mix1.5% 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

MATERIALS

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 mediafor 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

6. The following ingredients were pipetted into sterile microtubules

PROCEDURE (CONT’D)

Water Variable YeastTotal

Volume

0% 0.8 mL 0 mL 0.2 mL 1 mL

0.1% 0.7 mL0.1 mL(of 10%

substock)0.2 mL 1 mL

1% 0.79 mL 0.01 mL 0.2 mL 1 mL

10% 0.7 mL 0.1 mL 0.2 mL 1 mL

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.

PROCEDURE (CONT’D)

(-) Lys 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 at32 °Celsius

Colonies were counted, each colony assumed to have arisen from a single cell

UV EXPOSURE PROCEDURE

0 sec 15 sec 30 sec 45 sec0.0

7.5

15.0

22.5

30.0

37.5

(-) LYS UV EXPOSURE RESULTS#

of

Colo

nie

s

UV Light Effects on (-) Lys Yeast

P value = 0.000132

Exposure Time (s)

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

DUNNETT’S TEST RESULTS

T-Critical = 3.75

P-value = 0.000454

REGULAR YEAST UV EXPOSURE RESULTS

# o

f C

olo

nie

s

UV Light Effects on Yeast Survivorship

0 15 30 4530

40

50

60

70

80

90

P value = 0.000454

Exposure Time (s)

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

DUNNETT’S TEST RESULTS

T-Critical = 3.75

0.0% 0.1% 1.0% 10.0%0

1

2

3

4

MINERAL OIL EXPOSURE RESULTS

Mineral Oil Effect on Mutagenesis

# o

f C

olo

nie

s

Mineral Oil Concentration

P Value = 0.032446

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

DUNNETT’S TEST RESULTS

T-Critical = 2.88

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

CONCLUSIONS

Limitations

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

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.

LIMITATIONS AND EXTENSIONS

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/generalinformationaboutcarcinogens/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=7dd94e096ea549bac90bce0ec51acb6422cbb1a4&keytype2=tf_ipsecsha

http://www.iarc.fr/en/media-centre/pr/2004/pr154.html

SOURCES

ANOVA

Seconds Exposed 0 15 30 45 Wild Type Yeast

91 74 69 49

80 74 46 45

92 80 50 39

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 F P-value F crit

Between Groups 3478.917 3 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 GiannettaSecond Year in PJAS

Central Catholic High School10th Grade