How Does HCT Prevent Cellular Aflatoxin Accumulation?

Performed by:

Tristan Hayes

Dr. Jennifer Stewart

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

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

What is HCT?

• Hydroxy cis-terpenone (HCT)– Synthesized by Dr. Zhou in VCU Chem Dept.

HCT decreases cellular accumulation of aflatoxin

0 10 20 30 40 50 600

10

20

30

V

HCT

Time (min)

[3 H]

AF

B1

Up

tak

e (

fmo

l/1

06c

ell

s)

Our Mission• To determine how HCT prevents cellular

accumulation of aflatoxin-B1

Clue• Cell Accumulation of AF-B1 blocked by

transport inhibitors such as Probenecid:

0 5000

20

40

60

80

1000 2000

Probenecid [M]

% In

hib

itio

n

Hypothesis• HCT could block the binding of aflatoxin to

transporter proteins.– Proteins in the plasma or microsomal membranes, which one?

Plans

• Measure Aflatoxin binding to plasma membranes and microsomal membranes

• Determine if HCT prevents binding

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 nucleusCentrifuge at 23,000x g to pellet membraneCentrifuge at 100,000 x g to pellet microsomes

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

*0

50

100

*

*

** P < 0.001

* P < 0.01

TREATMENT

[3H

] A

FB

1 (%

Co

ntr

ol)

HCT and UnlabeledAFB1 decreased binding of 3HAFB1 to Plasma Membranes

Probenicid had no effect suggesting binding to proteins other than organic anion transporter proteins (OATs).

*0

50

100

*

*

** P < 0.001

* P < 0.05

TREATMENT

[3H

] A

FB1

(% C

ontr

ol)

HCT and UnlabeledAFB1 decreased binding of 3HAFB1 to Microsomes

ATP had no effect, suggesting that binding is not energy dependant.

*

Control M

AFB1-40

M

HCT-40

Amp 5

0mM

ATP 50

mM

0

50

100

*

*

** P < 0.001

* P < 0.01

TREATMENT

[3H

] A

FB

1 (%

Co

ntr

ol) HCT and UnlabeledAFB1 decreased binding of 3HAFB1 to Hep G2 Human Liver Cells.

ATP and AMP had no effect, suggesting binding is not energy dependant.

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.