Artificial Sweeteners Mutagenic Effects. Warren Austin Pittsburgh Central Catholic High School Second Year at PJAS

Transcription

1 Artificial Sweeteners Mutagenic Effects Warren Austin Pittsburgh Central Catholic High School Second Year at PJAS

2 Artificial Sweeteners Maybe derived from naturally occurring substances Excessive sweetness that leaves an after taste in mouth Powder or liquid form Commonly found in bake goods, diet beverages, puddings and jello Consumed by diabetics and those seeking weight loss

3 Previous Studies Male rats developed cancerous tumors in the bladder after intense exposure to cyclamate and saccharin The FDA performed further tests No link has been discovered between human cancer and saccharin

4 Previous Studies Continued The University of Rochester tested yeast cultured in the presence of an abundance of saccharin The yeast had dose dependent increases within one gene or multiple genes Cell viability reduced from no exposure to high exposure of saccharin However, cell concentration was not reduced with increased exposure

5 Sweet N Low Commonly used zero calorie liquid artificial sweetener on food market Ingredients include water, soluble saccharin, with Benzonic Acid (0.054%) and Methyl Paraben (0.046%) as Preservatives Saccharin Significant factor of artificial sweeteners Questions have risen if saccharin causes cancer if consumed in abundance

6 Question Does the saccharin in the Sweet N Low artificial sweetener product cause it to have significant mutagenic properties?

7 Yeast, Saccharomyces cervisiae Common cell model used in laboratory studies Safe organism that is prolific in growth and culturing Reproduction, metabolism, and chemistry is similar to those of other more advanced eukaryotic cells (Lys 2-) A special strain that is unable to produce Lysine

8 Lysine L-lysine- an essential amino acid Codons for amino acid sequence are AAA and AAG In particular, Lys 2 mutants are missing an essential enzyme function that is present within the lysine biosynthesis pathway

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10 The Ames Test Developed by Bruce Ames, who tested the mutagenic and anti-mutagenic properties of various chemicals He used a minus histidine Salmonella, a single point mutation Higher exposure to suspected mutagen correlated with increased reversion (mutation) rate With visible colonies appearing on complete (-His) media evidence of mutation through revision Interpreted as a lower limit on mutation rate because only 1 DNA site in the genome was tested

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12 A Modified Form of the Ames (-Lys) yeast eukaryote Test The number of reverted colonies of yeast on the agar plates can be correlated with the rate of mutation A reversion at that point can result in a reversion back to wild type yeast (Lys+) Artificial sweetener Sweet N Low used a mutagen substitution

13 Ultraviolet Rays Has a shorter wavelength but higher frequency than visible light Ranges from 40 and 400 nm Naturally radiates from the sun, and most of the light is absorbed by the ozone layer, with small amounts reaching the surface of the earth Various laboratory equipment can produce UV light for experimental test A mutagen because it can alter the DNA of organisms and mammals

14 Purpose Assess the mutagenicity of an artificial sweetener

15 Hypothesis Null Hypothesis: Sweet N Low will not have a significant effect on Yeast s Mutagenesis Rate. Alternative Hypothesis: Sweet N Low will significantly increase Yeast s Mutagenesis Rate.

16 Materials Com (-Lys) agar plates (Yeast Nitrogen base 1%, dextrose 2%, 1.5% agar, complete amino acid mix (minus lysine) 100mg/L UV Light Hood (LD-50 on Yeast in 30 Seconds) (-) Lysine Saccharomyces cerevisiae (John Wolford lab, CMU) Liquid Sweet N Low artificial sweetener SDF (Sterile dilution fluid) (10mM KH2PO4, 10mM K2HPO4, 1mM MgSO4, 1mM CaCl2, 100mM NaCl) Klett spectrophotometer Sterile pipette tips and Micropipettes Vortex Centrifuge

17 Procedure A strain of yeast (-) Lys phenotype was grown for several days in YEPD media A series of washes with SDF were performed on the sterile yeast pellet to remove any residual nutrients (lysine) A 0.2% liquid Sweet N Low artificial sweetener was sterilized through a 0.22 micron syringe filter The pellet in SDF was re-suspended The following ingredients were pipetted into sterile micro tubes. (Percents are by volume compared to stock solution)

18 Table of Liquid Concentrations Contents 0% 0.01% 0.05% 0.1% Sterile Water 0.8 ml 0.75 ml 0.55 ml 0.30 ml Variable 0 ml 0.05 ml 0.25 ml 0.50 ml Yeast 0.2 ml 0.2 ml 0.2 ml 0.2 ml Total Volume 1 ml 1 ml 1 ml 1 ml

19 Procedure (Continued) 6. The cells were allowed to sit for 5 minutes ml aliquots were spread onto 12 complete (-Lys) (3 plates for each of the 4 concentrations) agar plates (necessary to show cells that have reverted through mutation to wild type (+) lys) ml aliquots were spread onto 12 complete (-Lys) (3 plates for each of the 4 concentrations) agar plates (necessary to show cells that have reverted through mutation to wild type (+) lys) 9. The cells were allowed to sit for 10 minutes ml aliquots were spread onto 12 complete (-Lys) (3 plates for each of the 4 concentrations) agar plates (necessary to show cells that have reverted through mutation to wild type (+) lys)

20 Procedure (Continued) ml aliquots were spread onto 12 complete (-Lys) (3 plates for each of the 4 concentrations) agar plates (necessary to show cells that have reverted through mutation to wild type (+) lys) plates of 0.1mL of Yeast and 2 plates of 0.2mL were exposed to UV light for each of the following time increment: 0, 5, 10, 20, 30 seconds, 13. All plates were allowed to incubate for 4 days at 32C 14. The colonies were counted and recorded. Each colony assumed to have arisen from 1 cell.

21 Sweet N Low Mutagenic Effects 7 Yeast's Mutagenesis Rate of 5 Minute Exposure to Sweetener % 0.01% 0.05% 0.10%

22 Statistical Test for 5 min Exposure

23 Sweet N Low Mutagenic Effects 7 Yeast's Mutagenesis Rate of 10 Minute Exposure to Sweetener % 0.01% 0.05% 0.10%

24 Statistical Test for 10 min Exposure

25 Graphical Results of UV 400 UV Light s Effects of Yeast's Mutagenesis Rate sec 5 sec 10 sec 20 sec 30 sec

26 Statistical Test for UV Light Exposure

27 Dunnett s Test for UV Test Exposure Time of UV Exposure T-Value Interpretation 5 sec Significant 10 sec Significant 20 sec Significant 30 sec Insignificant T Critical Value: 3.48 Alpha:.05

28 Conclusions The Null Hypothesis can be accepted for the 0.01%, 0.05%, and 0.1% Sweet N Low artificial sweetener, and 30 sec UV light groups. The Null Hypothesis can be rejected for 5 sec, 10 sec, and 20 sec UV light groups. Through the experiment, it is evident that the concentrations of artificial sweetener did not have significant mutagenic effects. The lower colony counts in 30 sec group UV light exposure may be due to UV light toxicity UV light appears to be a stronger mutagen than Sweet N Low

29 Limitations Extensions Cell death? UV hood and positioning Exposure time Low reversion rate Increase cell density (increase cell reversion rate) Increase reversion time LD50 of drug Select different gene

30 Sources