Effects of Bisphenol A on Cancer Cell Proliferation

Effects of Bisphenol A on Cancer Cell Proliferation Anthony Di. Bello Central Catholic High School Grade 11

Cancer Overview • Cancer cells grow and divide at an unregulated pace (also known as proliferation). • Apoptosis (programmed cell death) does not occur in cancer cells. Extended passing on of this mutation eventually causes tumors to form. • Tumors can be metastatic (invasive of other body tissues) or benign (relatively harmless).

MG 63 Cell Line • Human cancer cell line • Osteosarcoma cells (aggressive form of bone cancer) • Useful model for testing the effects of variables on the proliferation of cancer cells

Bisphenol A • Originally produced as a synthetic estrogen • Endocrine-Disrupting Chemical used in the production of: • Polycarbonate plastic (beverage bottles, infant feeding bottles, food containers) • Epoxy resins (protective lining for canned food)

Bisphenol A and Cancer • As a xenoestrogen, BPA may contain chemical factors that either cause cancer, promote cancerous growth, inhibit cancer growth, or alter the progression/phenotype of cancer. • Some cancers are promoted by the work of xenohormones (including xenoestrogens). • Mimic the actions of a certain hormone (estrogen) • Might promote cancer growth

Purpose • To determine the effect of BPA exposure on cancer cell proliferation

Hypotheses • Null: BPA exposure • Alternative: BPA WILL NOT exposure WILL significantly affect cancer cell proliferation

Materials Cryotank 
 Three 75 cm 2 tissue culture treated flasks
 Twelve 25 cm 2 tissue culture treated flasks 
 Fetal bovine serum (FBS) 
 MG 63 Osteosarcoma Cancer Cell Line 
 Macropipette + sterile macropipette tips (1 m. L, 5 m. L, 10, m. L, 20 m. L) 
 • Micropipettes + sterile tips 
 • DMEM Serum - 1% and Complete Media (4 m. M Lglutamine, 4500 mg/L glucose, 1 m. M sodium pyruvate, and 1500 mg/L sodium bicarbonate + [ 10% fetal bovine serum for complete]) 
 • 10 mg Bisphenol A • • 75 ml culture flask • Incubator • Nikon Inverted Microscope • Laminar Flow Hood UV Sterilizing Lamp • Labeling Tape • Hemocytometer
 • Sterile PBS
 • Ethanol (70% and 100%) • Purple Nitrile gloves • Trypsin-EDTA • Pen/strep

Procedure: Cell Culture • MG 63 cells were thawed and grown for four days. • When flasks reached a density of approximately 1 million cells/m. L, the cells were passed into two flasks and incubated for 2 days at 37° C, 5% CO 2.

Procedure: Proliferation Experiment – Day 0 (Addition of Variable) • Cells were trypsinized and suspended in 20 m. L of media, creating a density of approximately 1 x 10^5 cells per flask • 0. 5 ml of the cell suspension was added to 25 cm 2 tissue culture treated flasks containing 4. 5 m. L of DMEM (com) media. • Two stock solutions of BPA were created using pure ethanol and DMEM media: 10^-4 M and 10^-5 M. • Experimental flasks were exposed to Bisphenol A and all flasks were incubated. (2 flasks per concentration x 3 concentrations x 2 days = 12 flasks total) • The cells were incubated at 37°C, 5% CO 2 for the remainder of the study.

Concentration Chart 0 Low (10^-6 M) High (10^-5 M) Cell Suspension 0. 5 m. L Media 4. 5 m. L 4 m. L Variable 0 m. L 0. 5 m. L (10^-5 stock) 0. 5 m. L (10^-4 stock) Total 5 m. L

Procedure: Cell Density • Day 1 and Day 2 ▫ For each flask, cell densities were determined as follows: • The cells were trypsinized and collected into cell suspension. • 10 μl aliquots were transferred to a Hemocytometer for quantification (eight counts per flask).

700000 BPA Exposure on MG 63: Day 1 P value = 0. 219 Cells /Flask 600000 533125 500000 [VALUE] 481250 10^-6 M 10^-5 M 400000 300000 0 M BPA Concentration (Molarity)

800000 BPA Exposure on MG 63: Day 2 632500 700000 Cells/Flask 600000 451250 P value = 6. 88 E-09 500000 331875 400000 300000 200000 100000 0 0 M 10^-6 M BPA Concentration (Molarity) 10^-5 M

Dunnett’s Test Results Concentration T-Value T-Critical (0. 05) Variation Day 1 - - - 10^-6 M - - Insignificant 10^-5 M - - Insignificant Day 2 - - 10^-6 M 6. 13 3. 47 Significant 10^-5 M 10. 16 3. 47 Significant -

Images: Day 1 0 M Low (10^-6 M) High (10^-5 M)

Images: Day 2 0 M 10^-6 M 10^-5 M

Conclusions • The effect of BPA exposure on cancer cell proliferation was significant for both concentrations on day two only. • Null hypothesis rejected.

Future Changes Limitations • It is unlikely that all cell suspensions were perfectly homogenous. • Pipetting at various stages of experimentation was not perfectly synchronized. • BPA needed to be dissolved in pure ethanol, potentially harming cell growth. Extensions • Different exposures of BPA can be used (longer exposures and greater concentrations) • Test BPA exposure on other cell lines (C 2 C 12, 3 T 3) • Use an Ames test to determine mutagenic capabilities of BPA on a non-cancer cell model.

References • Mark Krotec, PTEI • Phil Campbell, Ph. D. • Donald B. De. Franco, Ph. D. • https: //www. ncbi. nlm. nih. gov/pmc/articles/PMC 2967230/#!po=81. 6 667 • http: //www. northcarolinahealthnews. org/2012/04/02/localscientists-in-the-middle-of-the-bpa-debate/ • https: //www. scientificamerican. com/article/just-how-harmful-arebisphenol-a-plastics/

ANOVA Analysis: Day 1

ANOVA Analysis: Day 2