Presenter: Marilyn Pruitt Advisor: Dr. M. Whalen Department of Chemistry

1. EFFECTS OF COMBINATIONS OF ORGANOCHLORINE COMPOUNDS ON THE CYTOTOXIC FUNCTION OF HUMAN NATURAL KILLER CELLS Presenter: Marilyn Pruitt Advisor: Dr. M. Whalen Department of Chemistry Tennessee State University

2. Introduction Cancer has become one of the main causes of death among males and females of all races and ages. Natural Killer (NK)cells have a central role in stopping the spread of tumor cells in the body. NK cells are white blood cells that are capable of killing tumor cells. When NK cells are inactive or absent in the body the tumor cells spread and overtake that area eventually spreading to the whole organ, thus causing cancer.

3. Studies have shown that some pesticides increase the incidence of tumors in humans and animals. Some of these pesticides are still widely used in the U.S. and others while banned in the U.S. are still available in other countries and on the black market. This study investigates what effects pesticides have on blocking the function of NK cells in the body.

4. Natural killer cells are innate immune cells that control certain microbial infections and tumors. The function of natural killer cells is regulated by a balance between signals transmitted by activating receptors, which recognize ligands on tumors and virus-infected cells, and inhibitory receptors specific for major histocompatibility complex class I molecules.

5. Chromium release assays of organochlorine compounds have shown that at certain concentrations these compounds are inhibitors of human NK cells’ ability to kill tumor cells. • Dichlorodiphenyltrichloroethane (DDT) • Dichlorodiphenyldichloroethylene (DDE) • Alpha Chlordane • Gamma Chlordane

6. DDT Dichlorodiphenyltrichloroethane (DDT) is an insecticide and was widely used after WWII. DDT was banned from the US in 1973 although it is still used in other parts of the world. DDT was found to be useful against mosquitoes after the malaria outbreak in the late 1940s. DDT is not metabolized very rapidly by animals; instead, it is deposited and stored in the fatty tissues.

7. The biological half-life of DDT is about eight years; that is, it takes about eight years for an animal to metabolize half of the amount it assimilates. If ingestion continues at a steady rate, DDT builds up within the animal over time.

8. DDE Dichlorodiphenyldichloroethylene (DDE) is a degradation product of DDT and is found as an impurity in DDT. DDE has no commercial use. A study in humans showed that women who had high amounts of a form of DDE in their breast milk were unable to breast feed their babies for as long as women who had little DDE in the breast milk.

9. Another study in humans showed that women who had high amounts of DDE in breast milk had an increased chance of having premature babies.

10. Chlordane Chlordane was used as a pesticide in the United States from 1948 to 1988 on crops, lawns, and gardens. Most uses of chlordane were discontinued in 1987; however, manufacture for export still continues. a-Chlordane is the cis isomer and g-Chlordane is the trans isomer. Chlordane entered the environment when it was used as a pesticide on crops, on lawns and gardens, and to control termites.

11. Chlordane sticks strongly to soil particles at the surface and is not likely to enter groundwater. It can stay in the soil for over 20 years. • Most chlordane leaves soil by evaporation to the air. It breaks down very slowly. Chlordane doesn't dissolve easily in water. • It builds up in the tissues of fish, birds, and mammals.

12. a-Chlordane g-Chlordane cis trans

13. Methods Cell Preparation: NK cells are prepared by incubating buffy coats obtained from the American Red Cross with an antibody cocktail, that removes all white blood cells except NK cells for 40 min to 1 h. NK cells were collected by applying a density gradient.

14. Cell Treatment and Viability: NK cells were exposed to compounds, (DDT, DDE, a-chlordane, and g-chlordane) for 24 h or 6 d. Cell viability was determined by trypan blue exclusion.

15. Chromium Release Assay: • K562 tumor cells were loaded with 51Cr. 100 mL of 51Cr loaded tumor cells were then mixed with 100 mL control or treated purified NK cells in the wells of a 96 well test plate. The plate was centrifuged. After a 2 h incubationa 100 mL aliquot of the supernatant was removed and counted for radioactivity. Tumor cell lysis is determined as followed: • (Sample cpm – spontaneous cpm) • (maximum cpm – spontaneous cpm) • x 100 = % lysis

16. Figure 1 • 24 hour chromium release assay of 2.5 mM a-Chlordane and 2.5 mM g-Chlordane shows that the control has 22.1% lysis, a-Chlordane has 17.8% lysis, g-Chlordane has 21.9% lysis and in combination (a- & g-Chlordane) has 20.7% lysis. • The 24 hour assay of a-Chlordane & g-Chlordane does not show any significant combination effect of the compounds on the NK cells at the given concentrations.

17. Figure 1

18. Figure 2 • 6 day chromium release assay of 2.5 mM a-Chlordane and 2.5 mM g-Chlordane shows that the control has 14.3% lysis, a-Chlordane has 13.4% lysis, g-Chlordane has 11.9% lysis and in combination (a- & g-Chlordane) has 12.0% lysis. • The 6 day assay of a-Chlordane & g-Chlordane also shows no significant combination effect of the compounds on the NK cells at the given concentrations.

19. Figure 2

20. Figure 3 • 48 hour chromium release assay of 2.5 mM DDT and 5.0 mM DDE shows that the control has 62.1% lysis, DDT has 72.6% lysis, DDE has 64.6% lysis and in combination (DDT & DDE) has 61.5% lysis. • The 48 hour assay of DDT & DDE shows no combination effect of the compounds on the NK cells at the given concentrations.

21. Figure 3

22. Figure 4 • 6 day chromium release assay of 2.5 mM DDT and 5.0 mM DDE shows that the control has 48.9% lysis, DDT has 50.2% lysis, DDE has 38.9% lysis and in combination (DDT & DDE) has 29.3% lysis. • The 6 day assay of DDT & DDE shows a significant combination effect of the compounds on the NK cells at the given concentrations than the compounds alone.

23. Figure 4

24. Summary Chromium release assays of 2.5 mM a-Chlordane & 2.5 mM g-Chlordane were shown not to have a significant combination effect on NK cells after 24 hours or 6 day testing. Chromium release assays of 2.5 mM DDT & 5.0 mM DDE were shown to have a significant combination effect on NK cells 6 day testing. 6 day assay of DDT and DDE showed the greatest combination effect on the NK cells.

25. Acknowledgements Center of Excellence S.T.A.R.S. Program Prof. M. Wade Prof. T. Gary Chemistry Department Prof. M. Whalen Adrian Reed