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

2. 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).

3. 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

4. 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)

5. 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

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

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

8. 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

9. 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.

10. 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.

11. 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

12. 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).

15. 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

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

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

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

19. 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.

20. References Mark Krotec, PTEI Phil Campbell, Ph.D.
Donald B. DeFranco, Ph.D.
667
scientists-in-the-middle-of-the-bpa-debate/
bisphenol-a-plastics/

21. ANOVA Analysis: Day 1

22. ANOVA Analysis: Day 2