Antioxidant Attenuation of Antioxidant Attenuation of Yeast MutagenesisYeast Mutagenesis

Peter ChekanPeter Chekan

Central Catholic High School Central Catholic High School

PittsburghPittsburgh

Ultraviolet lightUltraviolet light

Electromagnetic radiation Wavelength shorter than

that of visible light Greater energy for

wavelength Sun produces UV-light Can cause sunburn skin

and cancer Damages DNA

Oxidative stressOxidative stress

UV light can result in oxidation stress Increases oxidant production in animal cells Caused by an imbalance between the

production of reactive oxygen Results in cellular degeneration Could cause cancer Antioxidants are thought to counter

oxidative stress

Antioxidants Antioxidants

Molecules capable of slowing or preventing the oxidation of other molecules

Maybe able to prevent cancer and coronary heart disease

Body produces antioxidants Obtained through Diet Vitamin C

Vitamin CVitamin C

Antioxidant Enzyme cofactor Also known as Ascorbic

acid In Oranges, Strawberries,

and Grapefruit The disease Scurvy occurs from lack

of Vitamin C

Saccharomyces cerevisiaeSaccharomyces cerevisiae (Yeast)(Yeast)

Eukaryotic microorganism Unicellular 3–4 µm diameter Used in baking and production

of alcoholic beverages The most studied cellular model in research Cell cycle is similar to human cells Comparable DNA replication, recombination,

cell division and metabolism

PurposePurpose

Determine if the antioxidant Vitamin C can increase survivorship of UV-stressed cells

HypothesisHypothesis

Null HypothesisNull Hypothesis The vitamin C concentrations will not

significantly affect UV-stressed yeast survivorship rate

AlternativeAlternative The vitamin C concentrations will

significantly affect the yeast survivorship rate

Materials Materials Test tubes Test tube rack Micropipettes Pipette tips Yeast (Saccharomyces cerevisiae) YEPD Agar Plates YEPD Media (0.5% yeast extract,

2% Glucose, 2% Peptone) Vitamin C UV Lamp/hood

Proper safety equipment Water Spreader bar Vortex SDF

UV Safety Glasses

Procedure Procedure

1. Saccharomyces cerevisiae was grown overnight in sterilized YEPD media.

2. A sample of the overnight cultures was added to fresh media in a sterile sidearm flask.

3. The culture of yeast was incubated at 30°C until a density of 50 Klett spectrophotometer units was reached. This represents a cell density of approximately 107 cells per mL.

4. The culture was diluted in sterile dilution fluid to a concentration of approximately 105 cells per mL.

Procedure ContinuedProcedure Continued1. Jhjhjhjhjh2. A3. DF4. d

5. The following tubes were created

6. 0.1 mL of each suspension was plated onto YEPD agar plates (50 total plates)

7. The plates were exposed to assigned durations of UV radiation (0, 15, 30, 45, 60 sec)

YeastYeast SDFSDF Vit CVit C

0 % Vit C 0.01 ml 9.99 ml 0 ml

1% Vit C 0.01ml 8.99 ml 1 ml

Procedure ContinuedProcedure Continued1. H

2. G

3. F

4. F

5. F

6. F

7. F

8. Plates were incubated for 48 hours at room temperature.

9. Colonies were counted. Each colony was assumed to have arisen from one cell.

102 cells103 cells/mL

105 cells/mL

107 cells/mL (yeast)

100 uL100 uL

100 uL

Dilution Dilution ProcedureProcedure

Data Data 0%0%

0 sec0 sec 15 sec15 sec 30 sec30 sec 45 sec45 sec 60 sec60 sec

Trial 1Trial 1 7777 8585 6262 2727 33

Trial 2Trial 2 102102 8282 8383 3939 99

Trial 3Trial 3 7171 9090 7373 3232 1010

Trial 4Trial 4 8181 9090 7474 4646 33

1%1%

0 sec0 sec 15 sec15 sec 30 sec30 sec 45 sec45 sec 60 sec60 sec

Trial 1Trial 1 8080 104104 6767 5151 1010

Trial 2Trial 2 122122 100100 5151 4646 2424

Trial 3Trial 3 124124 8888 5858 3232 3636

Trial 4Trial 4 118118 9494 4949 3333 3232

Average Survivorship of Yeast

0

20

40

60

80

100

120

0 sec 15 sec 30 sec 45 sec 60 sec

Time of Exposure to UV

Num

ber

of

Co

lon

ies

0%

1%

P>0.05

P<0.05

P>0.05P<0.05

P= 1.22E-15 P= 1.22E-15 P<0.05P<0.05

P=1.21E-08P=1.21E-08P<0.05P<0.05

P=2.04E-07P=2.04E-07 P<0.05P<0.05

P<0.05P<0.05

P<0.05P<0.05

P<0.05 P<0.05

P<0.05

P<0.05

Dunnetts Test ResultsDunnetts Test Results

Dunnetts Test ResultsDunnetts Test Results

If P is < than 0.05 then the results are significant

Interpretations

UV light significantly effected yeast with or without Vit C

Statistical Analyses supports a rejection for 30 sec

Vit C appeared to improve survivorship of UV stressed cells (expect for 30 sec)

No interaction between variables Definitely an effect of UV light on yeast

ConclusionConclusion

The statistical analyses allows a REJECTION REJECTION (except for 15 sec) of the null hypothesis, indicating that vitamin C can significantly effect the survivorship rate for yeast

The vitamin C alone appeared positively effect yeast survivorship

Limitations/ExtensionsLimitations/Extensions

Use different concentrations of Vitamin C Use different types of antioxidants

(Lycopene, Vitamin A, Vitamin E) Expose to varying amounts of UV light Increase sample size Synchronize cell plating times more

effectively

References

http://en.wikipedia.org/wiki/Ultraviolet http://en.wikipedia.org/wiki/File:DNA_UV_mutation.

gif http://en.wikipedia.org/wiki/Vitamin_C http://en.wikipedia.org/wiki/File:Ambersweet_orange

s.jpg http://lpi.oregonstate.edu/infocenter/vitamins/vitamin

C/ http://en.wikipedia.org/wiki/Antioxidant http://www.nlm.nih.gov/medlineplus/

antioxidants.html