The Effects of Spray Sunscreen on MG63 Cancer Cell Lines. Anthony Williams Pittsburgh Central Catholic High School Grade 11

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1 The Effects of Spray Sunscreen on MG63 Cancer Cell Lines Anthony Williams Pittsburgh Central Catholic High School Grade 11

2 Sunscreen Meant to protect against harmful UV rays that can be carcinogenic Comes in spray or lotion form and in different SPF Can either reflect sunlight or absorb it Use is recommended by numerous medical organizations to prevent cancer brought on by harmful UV rays

3 ! Banana Boat Ultra Defense Continuous Clear Spray Sunscreen SPF 100 Active Ingredients: Avobenzone 3%, Homosalate 10%, Octisalate 5%, Octocrylene 10%, Oxybenzone 6%! Broad-spectrum UVA and UVB protection!!! SPF 100 blocks against 99% of UVB rays!!! Recommended by the Skin Cancer Foundation

4 Oxybenzone Chemical formula: C14H12O3 Organic compound Really soluble and is in a class of ketones Regular component of sunscreen 97% of Americans tested have oxybenzone in their systems Possible hormonal and photo allergenic affect Also been claimed that it is a carcinogen

5 Past studies on Oxybenzone Past studies have been conducted on Oxybenzone Past studies on cells and lab animals indicated that this chemical disrupts the human hormonal system (Nakagawa 2002, Schlumpf 2001, 2004, Kunz 2006, van Liempd 2007, and Ma 2003) Also poses a greater threat when mixed with other sunscreen chemicals Have been linked to forming free radical chemicals which leads to cell damage(allen 1996, Serpone 2002, Hanson 2006)

6 An Overview of Cancer Cells Cancer cells are cells that grow and divide at an irregular, unregulated pace.! Apoptosis does not occur in cancerous cells; their mutations are passed on to the second generation, eventually clustering and forming tumors.! Tumors can be malignant (aggressive) or benign.!!

7 MG63 Cancer Cell Line Human cancer cell line! Osteosarcoma cells,an aggressive form of bone cancer! Useful model to test the effects of variables on cancer cell proliferation!

8 Purpose To determine the effects of Spray Sunscreen on MG63 cell proliferation and survivorship

9 Hypotheses Null: The different concentrations of spray sunscreen will not have a significant effect on the proliferation and survivorship of the MG63 cells. Alternative: The different concentrations of spray sunscreen will have a significant effect on the proliferation and survivorship of the MG63 cells.

10 Materials Cryotank Spray Sunscreen (Banana Boat Ultra Defense Continuous Clear Spray Sunscreen SPF 100) 75mm 2 tissue culture treated flasks 25 mm 2 tissue culture treated flasks Fetal bovine serum (FBS) MG63 cancer Cell Line Trypsin-EDTA Pen/strep Macropipette + sterile macropipette tips (1 ml, 5 ml, 10, ml, 20 ml) Micropipettes + sterile tips DMEM Serum - 1% and Complete Media (4 mm L-glutamine, 4500 mg/l glucose, 1 mm sodium pyruvate, and 1500 mg/l sodium bicarbonate + [ 10% fetal bovine serum for complete]) 75 ml culture flask Incubator Nikon Inverted Microscope with imaging technology Laminar Flow Hood Laminar Flow Hood UV Sterilizing Lamp Sharpie pen Hemacytometer Sterile PBS Ethanol (70%) Sterile Water Purple Nitrile gloves

11 Procedure (Cell Line Culture) A 1 ml aliquot of MG63 cells from a Cryotank was used to inoculate 30 ml of 10% serum DMEM media in a 75mm 2 culture flask yielding a cell density of approximately 10 6 to 2x10 6 cells The media was replaced with 15 ml of fresh media to remove cryo-freezing fluid and incubated (37 C, 5% CO 2 ) for 2 days until a cell density of approximately 4x10 6 to 5x10 6 cells/ml was reached The culture was passed into 12 flasks in preparation for experiment and incubated for 2 days at 37 C, 5% CO 2

12 Procedure (Addition of Variable on Day 0) Procedure (Addition of Variable on Day 0) Cells from a T75 flask were resuspended after trypsinization to a density of approximately K/mL. 4 ml of 10% DMEM media was added to each T25 flask 0.5 ml of cell suspension was transferred to 12 T25 flasks. Flasks were placed back into incubator and cells were allowed to attach for several hours 0.1 ml of sunscreen (not the active ingredient) was added to 9.9 ml of media to create a 1% stock (stock A). Serial dilution to create a 0.1% (stock B). All were sterile filtered using a 0.22 micron filter. T25 flasks were removed from incubator and variable was added to reach desired concentrations

13 Procedure (Cell Counts) Day 1 and Day 3 Cell densities were determined as follows: The cells were trypsinized and collected into cell suspension. 20 µl aliquots were transferred to a Hemocytometer for quantification (eight total counts).

14 Concentrations High Low Control Cells 1 ml 1 ml 1 ml Media 3.95 ml 3.95 ml 4.0 ml Stock % stock 0.05 stock 0 ml 0.01% 0.001% 0%

15 Results (day 1) Cell! Counts! Per! Flask P-value < 10^-6 Control! 186,625 Low! 339,750 High! 507,625 Concentrations

16 Dunnett s Test Results (day 1) CONCENTRATI ONS T VALUE T crit Results HIGH Significant LOW Significant

17 Results (day 3) P-value < 10^-6 Cell! Counts! Per! Flask Control! 235,375 Low! 347,375 Region 1 High! 612,250 Concentrations

18 Dunnett s Test Results (day 3) CONCENTRATI ON T value T crit Results HIGH Significant LOW Significant

19 Conclusion The null hypothesis is rejected. The data strongly suggests that the different concentrations did have a significant effect on the cell survivorship and proliferation

20 Limitations Cell counts can vary Low number of flasks Only one cell line used Limited exposure time Only one sunscreen tested Cell health not observed

21 Project Improvements More flasks Use multiple cell lines More concentrations Longer exposure time Different sunscreens and SPFs Trypan blue assay

22 !! Citations/ Acknowledgements Mark Krotec, PTEI deltamethrin/appendix-j.pdf

23 Day 1 Anova

24 Day 3 Anova