The Effects of EGCG on Microbiological Survivorship. Sean Bailey Grade 11, CCHS 2nd Year in PJAS

Transcription

1 The Effects of EGCG on Microbiological Survivorship Sean Bailey Grade 11, CCHS 2nd Year in PJAS

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

3 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.

4 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

5 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

6 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.

7 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

8 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

9 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.

10 Materials lysine agar plates sterile dilution fluid Klett spectrophotometer micropipettes (p1000, p20) sterile pipette tips vortex sidearm flask spreader bar ethanol lysine S. cerevisiae UV light hood sterile rubber gloves test microtubes microtube rack Epigallocatechin-3- gallate, 50mg (procured from Santa Cruz Biotechnology) burner

11 Procedure 1. 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.

12 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 water % EGCG: 0.2 ml S. cerevisiae, 0.79 ml sterile water, 0.01 ml 1% EGCG stock % EGCG: 0.2 ml S. cerevisiae, 0.7 ml sterile water, 0.1 ml 1% EGCG stock 7. 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.

13 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.

14 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.

15 Results - Graph and ANOVAs Effect of EGCG on Yeast Reversion Rate 30 0% EGCG 0.01% EGCG 0.1% EGCG P values 0s: s: s: seconds 15 seconds 30 seconds

16 Results - Dunnett s Test T-crit: 2.31 T value Significant? 15s, 0% vs 0.01% 1.81 NO 15s, 0% vs 0.1% YES 30s, 0% vs 0.01% 0.00 NO 30s, 0% vs 0.1% 9.15 YES

17 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.

18 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 weather Extensions test a wider variety of concentrations use a stain assay like Trypan Blue to count cells killed by UV radiation test different mutagens

19 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.

20 Thanks and Credits I 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.