The Effects of UVA on Vitamin D Treated

The Effects of UVA on Vitamin D Treated Cells Daniel Love Central Catholic High School Grade 11

Oxidative Stress • Caused by X-Rays and UV Rays • Stress causes an increase in free radical production • Cell degeneration possible • Other effects include an increased risk of cancer or death

Ultra Violet Radiation � Radiates � Most � Have from the sun. radiation is stopped by the ozone layer shorter wavelengths than visible light, thus are more powerful � Waves range from 100 nm to 400 nm

Effects of UV Radiation � � In humans, causes sunburn, nausea, sun stroke and possibly skin cancer. FDA protection methods include sunscreen, hats, sunglasses, and antiradiation clothing. Can possibly cause dimers in a cell’s DNA, which leads to replication errors and mutations. �

Staphylococcus epidermidis � Gram positive bacteria. � Common surface symbiont in many mammals (including humans). � Most forms considered non-pathogenic. � Potentially pathogenic � Forms biofilms

Vitamin D � A group of fat-soluble secosteroids. � The body can synthesize it with adequate sun exposure. � Effects of supplementation are uncertain. � Needed for bone growth. � Liquid vitamin D is measured in IUs, which is the measurement of concentration. 4, 000 IUs per m. L.

Vitamin D Toxicity � Also called hypervitaminosis D. � Results � Can from excess vitamin D supplements. cause liver or kidney conditions. � Main consequence is a build-up of calcium in the bloodstream, known as Hypercalcemia

Purpose The purpose of this experiment is to determine whether vitamin D will significantly remediate the effects of UV radiation on S. epidermidis

Hypotheses Null Hypothesis- Vitamin D will have no significant effect on the survivorship of UV stressed Staph. Alternate Hypothesis- Vitamin D will have a significant effect on the survivorship of UV stressed Staph.

Materials � LB agar plates (0. 5% yeast extract, 1% tryptone, 1% sodium chloride) � Staphylococcous epidermidis � Sterile Dilution Fluid [SDF] (100 m. M KH 2 PO 4, 100 m. M K 2 HPO 4, 10 m. M Mg. SO 4, 1 m. M Na. Cl) � Sterile test tubes � Sterile spreader bars � Incubator � Ethanol � Bunsen � Vortex � Vitamin burner D (liquid supplement) � Micropipettes � Sterile Tips � Klett Spectrophotometer � Labeling tape � Labconco UVC Hood (254 nm UVC 0. 7 -0. 9 cm 2 at working surface) � UVA 50 watt lamp

Procedure UVA 1. Bacteria (Staph) was grown overnight in sterile LB Media. 2. A sample of the overnight culture was added to fresh media in a sterile sidearm flask. 3. The culture was placed in an incubator (37°C) until a density of 50 Klett spectrophotometer units was reached. This represents a cell density of approximately 10⁸ cells/m. L. 4. The cell concentration was then diluted to 10³ cells/m. L. 5. 0. 1 m. L of the cell concentration was added to the agar plate and exposed to UVA light at varying times. 6. The plates were incubated at 37°C overnight. 7. The resulting cell colonies were counted the next day. Each colony was assumed to have risen from one cell.

UVA Effects on S. epidermidis 237 245 248 251 P-Value=0. 309 Number of Cell Colonies 261 UVA Stressed Cells 0 5 10 Time of Exposure (min) 20 30

Procedure UVC 1. Bacteria (Staph) was grown overnight in sterile LB Media. 2. A sample of the overnight culture was added to fresh media in a sterile sidearm flask. 3. The culture was placed in an incubator (37°C) until a density of 50 Klett spectrophotometer units was reached. This represents a cell density of approximately 10⁸ cells/m. L. 4. Concentrations of Vitamin D were made in separate tubes with concentrations of 0% (control), 1%, and 10%. 5. The cell concentration was then diluted and added to each tube. The cells were exposed to the vitamin D for ten minutes 6. 0. 1 m. L was then plated from each tube. 7. The cells were then exposed to timed amounts of UVC radiation (0 s, 2 s, 5 s, 10 s, and 20 s) 8. The cells were incubated at 37°C overnight. 9. The resulting cell colonies were counted the next day. All colonies were assumed to have risen from one cell

Concentration chart Concentration 0% (Control) 1% 10% S. epidermidis 0. 1 m. Ls SDF 9. 9 m. Ls 9. 8 m. Ls 8. 9 m. Ls Vitamin D 0 m. Ls 0. 1 m. Ls 1 m. L Final Volume 10 m. Ls

Vitamin D UVC Remediation Effects P-Value (Whole Graph=9. 05239 E-56) P-Value=0. 00037 282 285 249 P-Value=0. 848 257 Number of Cell Colonies 249 Interaction=0. 0029 256 P-Value=0. 651 153 171 175 P-Value=9. 952 E-05 96 54 45 P-Value=0. 554 9 0 s 2 s 5 s UVC Exposure Time (sec) 0% D 10% D 10 s 12 20 UVC 10

Dunnett’s Test T-Crit = 1. 94 Concentration T-Value Significance 0 UVC, 1% Vitamin D 0. 54 Insignificant 0 UVC, 10% Vitamin D 4. 33 Significant 10 UVC, 1% Vitamin D 4. 64 10 UVC, 10% Vitamin D 1 Significant Insignificant

S. epidermidis Survivorship P-Value=9. 05239 E-56 350 LD 50= 5 UVC LD 50= 6 UVC LD 50=5. 5 UVC Number of Colonies 300 250 200 0% 150 1% 100 50 0 0 UVC 2 UVC 5 UVC 10 UVC Exposure time (sec) 20 UVC

Conclusions � The null hypothesis was rejected for concentrations of 1% Vitamin D with a 10 second Exposure. � Null Hypothesis can be accepted for all other concentrations � 1% Vitamin D was able to significantly remediate the UVC radiation. � UVA is much weaker than UVC and has a higher kill time.

Limitations � UVA radiation was not strong enough � UVA exposures weren’t long enough � Only 6 replicates � Only 4 exposure times � Only 1 wavelength used (UVC 250 nm) � Plating may not have been synchronized � Cannot analyze the health or growth rate of cells that recovered from radiation

Extensions � More replicates and concentrations � More wavelengths � Longer exposure times for UVA in order to generate a kill curve � Use UVB instead of UVA � Conduct an agar infusion test to simulate longer exposure

References http: //www. epa. gov/sunwise/doc/uvradiation. html http: //hps. org/hpspublications/articles/uv. html http: //earthobservatory. nasa. gov/Features/UVB/ http: //www. skincancer. org/prevention/uva-and-uvb http: //www. who. int/uv/faq/whatisuv/en/index 2. html http: //www. who. int/uv/uv_and_health/en/