The Effects of Atrazine on MG-63 Cancer Cells

1. Jonah Duch Grade 11 Central Catholic High School The Effects of Atrazine on MG-63 Cancer Cells

2. An Overview of CancerCells • 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 secondgeneration, eventually clustering and forming tumors. • Tumors can be malignant (aggressive) orbenign.

3. MG63 Cancer CellLine • Human cancer cell line • Osteosarcoma cells, an aggressive form of bone cancer • Useful model to test the effects of variables on cancer cellproliferation

4. Atrazine • An organic compound widely used as aherbicide • Used to prevent broadleaf weeds in crops such as maize and sugarcane and on turf, such as lawns and golf courses • Prepared from cyanuric chloride, which is treated sequentially with ethylamine and isopropylamine

5. Banned in the European Union in 2004 because of its persistent groundwater contamination • In the United States it is one of the most widely used herbicides, with 76 million pounds of it applied each year, in spite of the restriction that used to be imposed • It would be very difficult or impossible to ingest a toxic dose from natural sources. • Is a Xenohormone, it mimics the actions of a particular hormone Atrazine

6. Type of xenohormone that imitates estrogen • Can be either natural or synthetic chemical compounds • Widely used in industrial compounds which have estrogenic effects on a living organism • Can mimic the effects of endogenous estrogen and thus have been implicated in the precocious puberty and other disorders of the reproductive system Overview of Xenoestrogens

7. Purpose To determine the effect of atrazine exposure on cancer cell proliferation.

8. Hypothesis NullHypothesis Atrazine WILL NOT have an effect on cancer proliferation. AlternativeHypothesis Atrazine WILL significantly effect the proliferationand survivorship ofcancer.

9. Materials • Cryotank • 75mm2 tissue culture treatedflasks • 25 mm2 tissue culture treatedflasks • Fetal bovine serum(FBS) • MG63 Osteosarcoma Cancer CellLine • Trypsin-EDTA • Pen/strep • Macropipette + sterile macropipette tips(1 mL, 5 mL, 10, mL, 20mL) • Micropipettes + steriletips • DMEMSerum-1% andCompleteMedia (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 cultureflask • Atrazine (4%) • Incubator • Evos Imaging System • Laminar FlowHood • Laminar Flow Hood UV SterilizingLamp • Sharpiepen • Hemacytometer • SterilePBS • Ethanol • SterileWater • Gloves

10. Procedure: CellCulturing • A 1 mL aliquot of MG63 cells from a Cryotank was used to inoculate 30 mL of 10% serum DMEM media in two75mm2 culture flask yielding a cell density of approximately 106 to 2x106cells. • The media was replaced with 15 mL of fresh media to remove cryo-freezing fluid and incubated (37° C, 5% CO2) for 2 days until a cell density of approximately 4x106 to 5x106 cells/mL wasreached. • The culture was passed into two sets of 3 flasksin preparationfor experiment and incubated for 2 days at 37° C, 5%CO2.

11. Procedure: Proliferation Experiment- Day 0 (Addition ofVariable) • After trypsinization, cells from all of the flasks were pooled into 1common 75mm2 flask (cell density of approximately 1 millioncells/mL). • 0.1 mL of the cell suspension was added to twelve 25 mm2 tissue culture treated flasks containing 5 mL of DMEM (com) media, creating a cell density of approximately 105 cells perflask. • Two stock solutions of atrazine were created using sterile water: 10^-4x and 10^-6x. X = the concentration of the undiluted Atrazineproduct. The following concentrations of atrazine (next page) were added to the flasks. The cells were incubated at 37°C, 5% CO2 for the remainder of the study. •

12. Concentrations ofVariable

13. Procedure- Days 1 and2 • Day 1 and Day2 • Cell densities were determined asfollows: • The cells were trypsinized and collected into cell suspension. • 20 µl aliquots were transferred to a Hemacytometerfor • quantification (eightcounts perflask). • Day 1 and Day2 • Evos imaging system was used to take imagesof • representative areas of eachflask.

14. Atrazine Concentration vs. Cell Proliferation

15. Dunnett’s TestResults

16. Proliferation Results: Day1 Control High Low

17. Proliferation Results: Day2 High Low Control

18. Conclusions • The null hypothesis can be rejected • All concentrations did appear to have significant effects and reduce proliferation • The higher concentration of variable appeared to have a greater negative impact on the cells

19. FutureChanges Limitations Extensions • Use a wider rangeofconcentrations • Test the effects of atrazine on other celllines • Test synergistic effects of atrazine • Hemacytometer countsin proliferation experiment can vary • Cell culture health canvary • Low number ofreplicates • Lagtime

20. WorksCited • Southern AGInc • http://www.ncbi.nlm.nih.gov/pubmed/22914 097 • http://www.ncbi.nlm.nih.gov/pubmed/21425 949 • http://www.ncbi.nlm.nih.gov/pubmed/19778091 • https://www.ncbi.nlm.nih.gov/pubmed/18942551

22. Statistical Analyses of the Proliferation Results • ANOVA • Statistical analysis that allows a comparison of multiple means in a data set • Dunnett’stest • Determines significant variance between the control group and experimentalgroup.