Zebrafish as an in Vivo Screen for Early Black Cranberry Proanthocyanidin Biomolecular Activity

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 anti cancer 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.