Vitamin C Attenuation of Smoke Stress

Will MaherCentral Catholic High School

Terms To Know•       Secondhand Smoke- smoke that is inhaled involuntarily, or passively, by someone who is not smoking  

•       Passive Smoking- the involuntary inhalation of smoke from tobacco products 

Cigarette Smoke• Cigarette smoke has harmful effects- releases toxins, irritants, etc. 

• Main pollutants are acetone, carbon monoxide, arsenic, benzene, butane, cyanide, nicotine, lead, formaldehyde, and many others    

How Smoke Kills the Cells in Your Lungs

FANCD2 (Gene coding for protein)

Secondhand Smoke is exposed into the air

The smoke is inhaled and gets into the

victims lungs

Once in the lungs, the smoke attacks

FANCD2 

FANCD2• FANCD2 - a human gene

that is used in the lung • Low levels of FANCD2 can

cause damage to the body’s DNA, triggering the overgrowth of cells (Cancer)

• When DNA is damaged, FANCD2 is the protein that repairs the DNA

Antioxidants•Antioxidant- a molecule capable of preventing the oxidation of other molecules

•Oxidation- a chemical reaction that transfers electrons from a substance to an oxidizing agent

•Oxidation reactions can produce free radicals, which can damage cells

Antioxidants

• Vitamin C• Vitamin E• Glutathione 

• Citrus Fruits• Strawberries•Wheat• Carrots• Spinach

Foods Containing Antioxidants

Vitamin C

The Recommended Dietary Allowance, or RDA, of Vitamin C for an adult male is 90 mg/day and for an adult female 75 mg/day

Experimental Cell Model

• Saccharomyces cerevisiae- a common yeast

• Great ease of manipulation in the laboratory (single-celled microbe)

• Similar biochemistry, genetics, cell cycle to other eukaryotes, including human cells

• MOST STUDIED CELL IN THE WORLD

ColoniesPetri Dish

Agar

Purpose

• To find agents that could help in the repair and growth of cells, especially human cells, after secondhand smoke exposure

Null Hypothesis

• The Vitamin C will not have a significant effect on the survivorship of yeast cells after secondhand smoke exposure 

Materials• YEPD agar plates (1% yeast

extract, 2% glucose, 1.5% agar)

• YEPD media (1% yeast extract, 2% peptone, 2% glucose)

• Sterile capped test tube sterile dilution fluid (SDF) (10 mM KH2PO4, 10 mM K2HPO4, 1 mM MgSO4, 0.1 mM CaCl2, 100 mM NaCl)

• Micropipettes• Permanent marker

• Plate spreader• Ethanol• Bunsen Burner• Klett Spectrophotometer• Incubator• Sidearm flask• Saccharomyces cerevisiae• RX Choice Liquid Vitamin C

(500mg Vit. C, 83mg Sodium)

• Wave Cigarettes

Procedure

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

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

3. The culture was placed in a shaking water bath (30oC) until a density of 50 Klett spectrophotometer units was reached. This represents a cell density of 107 cells/ml.

Procedure cont’d4.   The tubes contained:

Tube 1 Tube 2 Tube 3

SDF 9.9 mL 9.8 mL 8.9 mL

Vitamin C10% Stock

0 mL 0.1 mL 1.0 mL

Yeast Culture 0.1 mL 0.1 mL 0.1 mL

[Vit. C] 0% 0.1% 1%

Procedure cont’d5. The tubes were vortexed and then 0.1 mL aliquots from

each tube were spread onto YEPD plates.6. Plates from each tube were then exposed for 0, 60, and

180 seconds of smoke. An aluminum foil chimney was created and the lids of the plates were removed. The plates were inverted, placed onto the chimney, and a lit cigarette was placed within the foil chimney.

Procedure cont’d

7. The plates were incubated at 30C for 48 hours.8. The resulting colonies were counted. Each colony was assumed to have arisen from one cell.

p= 0.81

p=0.34

p=0.02

Dunnett’s Test

• Dunnett’s Test purpose- to determine if there is a significant variation between the variable groups and the control group

Dunnett’s Testa = .05t-critical = 3.03

0.1% Exposed for 180 Seconds of Smoke

1.34 Insignificant

1% Exposed for 180 Seconds of Smoke

3.37 Significant

Interpretation1. Smoke exposure appeared to significantly reduce the survivorship in the control and Vitamin C groups.

2. Vitamin C did not appear to significantly affect survivorship for no smoke and 60 seconds of exposure.

3. At 180 seconds of exposure, 1% Vitamin C appeared to significantly increase survivorship of yeast, while 0.1% did not yield a significant change in survivorship.

4. The two factor ANOVA strongly indicated that there was no interaction between the variable groups.

(Two Factor P-value= 0.65)

Conclusion

• The null hypothesis that Vitamin C would not increase survivorship of smoke-stressed cells was accepted for 60 seconds of exposure and for the concentration of 0.1% with 180 seconds of exposure.  

• The null hypothesis was rejected for the 1% Vitamin C group exposed to 180 seconds of smoke.

Limitations

• Due to physically holding the plates above the smoke chamber, some of the plates could have had more direct smoke exposure than others. 

• The yeast culture may not have been spread evenly in every plate.

• The cigarette may burn unevenly through time, varying smoke density and composition.

• Plating was not exactly synchronized, might result in slight variation of counts.

Extensions• Have a team of people synchronize the spread plating

• More controlled smoke chamber for exposure• More variations of exposure times• More variations of Vitamin C concentrations• Other agents besides Vitamin C could be tested

• A new cigarette could be burned for each new plate better controlling the variations of smoke density

Works Cited

• Scientific American Magazine• American Public Health Assoc. APHA.org• American Heart Assoc. americanheart.org• atlasgeneticsoncology.org • lungsusa.org• Sciencedaily.com