TITLE:
Zebrafish as an in Vivo Screen for Early Black Cranberry Proanthocyanidin Biomolecular Activity
AUTHORS:
Nicholas J. Macedo, Catherine C. Neto, Anne M. Liberty, Tracie L. Ferreira
KEYWORDS:
Zebrafish; In Vivo; Proanthocyanidins
JOURNAL NAME:
American Journal of Molecular Biology,
Vol.4 No.2,
March
28,
2014
ABSTRACT:
Antioxidants have
been widely studied in various naturally occurring substances as a bioavailable
cancer prevention treatment. Proanthocyanidins (PACs), which are abundant
polyphenols in Early Black (EB) Cranberry (Vaccinium macrocarpon), are readily available
and we have shown their anticancer activity in several cancer cell
lines. This work focused on the activity of these compounds when incorporated
into the zebrafish (Danio rerio)
system. We began investigating the in
vivo effect of these phytochemicals, the protective role of several other
cranberry compounds, and the metabolic activity of the vertebrate model
organism. Proanthocyanidin fractions were separated from fresh EB Cranberry
fruit by chromatography on Sephadex LH-20 in order to acquire a workable
stock solution in DMSO. Various concentrations of proanthocyanidins in solution
were tested against fish ranging in age from 1-cell stage to adult level of
growth. Acridine orange apoptosis indicator dye was incorporated into the
treatment protocol, and it was observed that irregular epithelial cell death
was occurring in treated embryos but not in the control group. Further
apoptosis assays were carried out utilizing Dihydroethidium (DHE) superoxide
sensitive dye in the treatment protocol. Fluorescing red nuclei were visible
along the outer surface of the epithelium cell layer; an indication of
superoxide release within cells leading to the nicking of DNA within the
nucleus. It was also possible to screen for superoxide release in PACs treated
CCD-CO18 and HT-29 cells using confocal microscopy and cell apoptosis was
investigated by trypan blue cytotoxicity assay; cell apoptosis results were
statistically significant as confirmed by ANOVA analysis. Results indicate that
the phytochemicals may induce apoptosis in rapidly dividing cells.