1. How Does HCT Prevent Cellular Aflatoxin Accumulation? Performed by: Tristan Hayes Dr. Jennifer Stewart

2. What is Aflatoxin? Aflatoxin-B1 –Produced by the fungus Aspergillus –Contaminates grains, wheat, soybean products, and nuts (especially under hot humid conditions) Accumulates in Liver and Lungs –Aflatoxicosis (liver damage) –Carcinogenic (liver cancer) –Caused 23 pet deaths in 2005-2006 in USA Source: http://www.fda.gove/oc/po/firmrecalls/diamond12_05.html

3. How does it work? Aflatoxin-B1 attacks in the cell in two ways

4. What is HCT? Hydroxy cis-terpenone (HCT) –Synthesized by Dr. Zhou in VCU Chem Dept.

5. HCT decreases cellular accumulation of aflatoxin

6. Our Mission To determine how HCT prevents cellular accumulation of aflatoxin-B1

7. Clue Cell Accumulation of AF-B1 blocked by transport inhibitors such as Probenecid:

8. Hypothesis HCT could block the binding of aflatoxin to transporter proteins. –Proteins in the plasma or microsomal membranes, which one?

9. Plans Measure Aflatoxin binding to plasma membranes and microsomal membranes Determine if HCT prevents binding

10. Isolation of Cell Membranes Harvest the cells by pouring out the media, washing with buffer, and scraping the cells. Homogenize Cells (Sonication) Centrifuge at 1000x g to pellet nucleus Centrifuge at 23,000x g to pellet membrane Centrifuge at 100,000 x g to pellet microsomes

11. Measure Total Cellular Protein (BioRad) Plasma membranes and Microsomes. Measure H-AFB1 Binding HAFB1 HCT or Vehicle Membranes Mix and then Incubate Collect onto Filters, Wash Count Membrane-bound H-AFB1 in Scintillation Counter Isolation of Cell Membranes

12. HCT and Unlabeled AFB1 decreased binding of 3 HAFB1 to Plasma Membranes Probenicid had no effect suggesting binding to proteins other than organic anion transporter proteins (OATs).

13. HCT and Unlabeled AFB1 decreased binding of 3 HAFB1 to Microsomes ATP had no effect, suggesting that binding is not energy dependant.

14. HCT and Unlabeled AFB1 decreased binding of 3 HAFB1 to Hep G2 Human Liver Cells. ATP and AMP had no effect, suggesting binding is not energy dependant.

15. Conclusions HCT decreased binding of 3HAFB1 to membrane and microsomal proteins. –This finding provides insight into the protective effects of Hydroxy Cis-Terpenone against Aflatoxin-B1.