Effects of Bisphenol A on Cancer Cell Proliferation Anthony DiBello 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 WILL NOT significantly affect cancer cell proliferation Alternative: BPA exposure WILL significantly affect cancer cell proliferation

Materials Cryotank Three 75 cm2 tissue culture treated flasks Twelve 25 cm2 tissue culture treated flasks 
 Fetal bovine serum (FBS) 
 MG63 Osteosarcoma Cancer Cell Line 
 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]) 
 10 mg Bisphenol A 75 ml culture flask Incubator Nikon Inverted Microscope Laminar Flow Hood 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/mL, the cells were passed into two flasks and incubated for 2 days at 37° C, 5% CO2.

Procedure: Proliferation Experiment – Day 0 (Addition of Variable) Cells were trypsinized and suspended in 20 mL of media, creating a density of approximately 1x10^5 cells per flask 0.5 ml of the cell suspension was added to 25 cm2 tissue culture treated flasks containing 4.5 mL 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% CO2 for the remainder of the study.

Concentration Chart Low (10^-6 M) High (10^-5 M) Cell Suspension Low (10^-6 M) High (10^-5 M) Cell Suspension 0.5 mL Media 4.5 mL 4 mL Variable 0 mL 0.5 mL (10^-5 stock) 0.5 mL (10^-4 stock) Total 5 mL

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).

Dunnett’s Test Results Concentration T-Value T-Critical (0.05) Variation Day 1 - 10^-6 M Insignificant 10^-5 M Day 2 6.13 3.47 Significant 10.16

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 Extensions 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. Different exposures of BPA can be used (longer exposures and greater concentrations) Test BPA exposure on other cell lines (C2C12, 3T3) 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. DeFranco, Ph.D. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2967230/#!po=81.6 667 http://www.northcarolinahealthnews.org/2012/04/02/local- scientists-in-the-middle-of-the-bpa-debate/ https://www.scientificamerican.com/article/just-how-harmful-are- bisphenol-a-plastics/

ANOVA Analysis: Day 1

ANOVA Analysis: Day 2