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
 Paraffin
isomers
CnC2n+2H
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
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 mL
0 mL
0.2 mL
1 mL
0.1%
0.7 mL
0.1 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
0%
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

0.1 mL of suspended (-) Lys cells were
plated on each of 12 complete (-) Lys
plates
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 exposed to UV light in groups
of three – at 0, 15, 30, and 45 seconds

Plates were incubated for three days at
32 °Celsius

Plates incubated for three days at
32 °Celsius

Colonies were counted, each colony
assumbed to have arisen from a single
cell

Colonies were counted, each colony
assumed to have arisen from a single
cell
(-) 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
P value = 0.000454
# of Colonies
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

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
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.
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
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
0.000454
F crit
4.066180551
MINERAL OIL EFFECTS
ON
YEAST MUTAGENESIS
Luke Giannetta
Second Year in PJAS
Central Catholic High School
10th Grade