The Effects of EGCG on Microbiological SurvivorshipSean BaileyGrade 11, CCHS2nd Year in PJAS

This Experiment• to determine whether or not epigallocatechin-3-gallate has a anti-carcinogenic effect on Saccharomyces cerevisiae.

Previous Studies• EGCG has been studied extensively by medical

researchers, due to its relatively low cost, abundance, and high potential for antioxidant properties.

• A search of the PubMed database for the phrase “egcg cancer” results in 1273 hits, indicating its widespread research.

• Of note is a study testing EGCG’s effect on Candida albicans, another strain of yeast, published in the Canadian Journal of Microbiology in 2009, which was found to have inhibitory effects on mutagenesis.

EGCG• Epigallocatechin-3-gallate is the

most abundant catechin in green tea

• is a strong catechin and antioxidant, and thus has the potential to inhibit mutagenesis, preventing cancer

• other common antioxidants include ascorbic acid, melatonin, and anthocyanin, which is found in red wine

Saccharomyces cerevisiae• commonly known as yeast

• simple to use and easy to culture

• used often as a first line of research into anticancer drugs because it mimics the human body’s cellular processes with a great deal of homology

—Lysine• The strain of yeast used in this experiment

were unable to produce lysine, a key nutrient involved in cell growth.

• These mutants do not have enzymes that function in the production of lysine.

• Thus, the cells require lysine in order to grow.

• They were also grown in agar that did not contain lysine, rendering the cells unable to grow under normal conditions.

Mutagenesis• DNA is contained in the nucleus of eukaryotic cells.

• UV radiation is a mutagen, meaning it changes the DNA of the cells.

• The —lysine yeast would not survive if it weren’t for mutagenesis.

• The UV radiation mutates the yeast’s DNA, causing it in some cases to produce lysine and grow.

• Thus, survivorship of the cells can be used as a crude measure of mutagenesis, i.e. if cells grow, it means they mutated to produce lysine

Ames Test• developed by Bruce Ames in the 1970s

• cultured salmonella that did not produce histidine in media that did not contain histidine

• exposed the salmonella to a mutagen

• growth of salmonella colonies indicated that the mutagen caused reversion of the histidine-producing codons in the DNA of the cell

• minus lysine test is a modified Ames test

Hypotheses• The null hypothesis is that epigallocatechin-3-

gallate will not have a significant effect on the reversion rate of Saccharomyces cerevisiae which have been stressed by ultraviolet radiation.

• The alternate hypothesis is that epigallocatechin-3-gallate will have a significant effect on the reversion rate of Saccharomyces cerevisiae which have been stressed by ultraviolet radiation.

Materials• —lysine agar plates• sterile dilution fluid• Klett

spectrophotometer• micropipettes (p1000,

p20)• sterile pipette tips• vortex• sidearm flask• spreader bar• ethanol• burner

• —lysine S. cerevisiae• UV light hood• sterile rubber gloves• test microtubes• microtube rack• Epigallocatechin-3-

gallate, 50mg (procured from Santa Cruz Biotechnology)

Procedure1. A strain of S. cerevisiae —lys phenotype was

grown for 2 days in YEPD media to a density of approximately 10^8 cells per ml

2. 4 days prior to experimentation the media was removed from the cell pellet and replaced with 3 mL of SDF.

3. Prior to removal, a series of washes were conducted with SDF to remove any residual lysine nutrients

4. The pellet in SDF was resuspended, and stored in —lys media for 2 days.

5. A stock solution of 1% EGCG was made.

Procedure (cont’d)6. A set of dilutions were prepared from the stock

solution and sterile water in the following concentrations:1. 0% EGCG: 0.2 mL S. cerevisiae, 0.8 mL

sterile water2. 0.01% EGCG: 0.2 mL S. cerevisiae, 0.79 mL

sterile water, 0.01 mL 1% EGCG stock3. 0.1% EGCG: 0.2 mL S. cerevisiae, 0.7 mL

sterile water, 0.1 mL 1% EGCG stock7. The cells infused with the solutions for 20

minutes.8. Eight 0.1 mL aliquots per experimental group

were spreadplated onto waiting —lys agar plates.

Procedure (cont’d)• The plates were transported to the Mellon

Institute, Carnegie Mellon’s biology laboratory, taking great pains to keep constant their temperature.

• Each concentration of EGCG had 8 plates each exposed to 0, 15, and 30 seconds of UV radiation.

• The plates were then transported back to Central Catholic, incubated at 37° C for four days.

• The plates were removed from the incubator, colonies were counted, and data was recorded. Each colony was assumed to have arisen from one single cell.

• The plates were then sterilized with bleach and safely discarded.

ANOVA and Dunnett’s Test• An ANOVA (ANalysis Of VAriance) is a statistical test

to determine if one group differs significantly against the other.

• Each group of data is averaged and compared to each other group, and a probability is generated (P-value).

• To determine which group had a significant effect if such an effect is gathered, a Dunnett’s pairwise test is performed, which compares each individual data set.

• A single factor ANOVA was performed on each timeset (0 seconds, 15 seconds, and 30 seconds) and Dunnett’s tests were performed where significance was found.

Results - Graph and ANOVAs

0 seconds 15 seconds 30 seconds0

7.5

15

22.5

30

37.50% EGCG 0.01% EGCG 0.1% EGCG

P values0s: 0.91415s: 0.02930s: 0.012

Effect of EGCG on Yeast Reversion Rate

Results - Dunnett’s Test

T value Significant?

15s, 0% vs 0.01% 1.81 NO

15s, 0% vs 0.1% 12.38 YES

30s, 0% vs 0.01% 0.00 NO

30s, 0% vs 0.1% 9.15 YES

T-crit: 2.31

Conclusions• The null hypothesis was rejected for the 0.1%

concentration of EGCG, as the colony count varied significantly across both times.

• The null hypothesis was accepted for the 0.01% concentration of EGCG.

• At the higher concentration, EGCG had a significant effect on mutagenesis, reducing the amount of reversions.

Limitations and Extensions

Limitations• slight variations in the amount of cells per plate• synchronicity of plates• even exposure to UV light sources• crudeness of Ames test• temperature variations due to transport of plates

in cold weatherExtensions

• test a wider variety of concentrations• use a stain assay like Trypan Blue to count cells

killed by UV radiation• test different mutagens

Sources• Evenson, Nikki and Phyllis Braun. The effects of tea polyphenols

on Candida albicans: inhibition of biofilm formation and proteasome inactivation. 2009, Canadian Journal of Microbiology.

• Santa Cruz Biotechnology. Material Safety Data Sheet for EGCG.

• Matuo, R., et. al. Saccharomyces cerevisiae as a model system to study the response to anticancer agents. 2012, Cancer Chemotherapy Pharmacology Journal.

• Stevens, S. K., et. al. The anticancer ruthenium complex KP1019 induces DNA damage, leading to cell cycle delay and cell death in Saccharomyces cerevisiae. 2013, Mol Pharmacology.

• Wikimedia Commons Image Library. EGCG molecule and green tea.

Thanks and CreditsI would like to take this opportunity to formally thank Carnegie Mellon University, my sponsor Mark Krotec, and Central Catholic High School.

Graphs were generated using matplotlib.Statistics were done in R.