Effects of Phenanthrene, A Polycyclic Aromatic Hydrocarbon on Zebrafish (Danio Rerio) Early Development

Polycyclic Aromatic Hydrocarbons (P AH) are ubiquitous environmental contaminants released from different sources such as forest fires and ocean oil seeps, as well as land based runoff and creosote. These sources of P AHs are both natural and anthropogenic. Some P AHs have been known to be carcinogenic and mutagenic. In this project, the effects of phenanthrene, a P AH compound. on early embryogenesis involving somitogenesis (development of somites) were studied using zebrafish (Danio rerio) early embryos as an experimental system. The developmental stage relevant to this project was the segmentation stage, during which time somites develop. In early embryos, somite patterning is under the influence of Wnt signaling. Although the process of somitogenesis remains to be fully understood, the "clock and wavefront" model is widely accepted and the process involves a variety of cellular and molecular interactions.\ud This research shows the developmental stage specific effects of phenanthrene on somitogenesis. Other developmental abnormalities due to phenanthrene exposure were also noticed and documented; these abnormalities included pericardiallyolk sac edema and scoliosis. Embryos treated at 2 hours post fertilization (hpt) were most sensitive to phenanthrene exposure and displayed abnormal development of somites and showed a decreased total somite number. Embryos treated at 8 and 12 hpf did not show such abnormalities, suggesting there is a critical time in development when somitogenesis is most affected by phenanthrene. Involvement of the Wnt signaling pathway in somitogenesis with an apparent increase in nuclear ~-catenin was tested through pharmacological and immunological studies. l-Azakenpaullone, an inhibitor of Glycogen Synthase Kinase-3~ (GSK-3~), was used to target a specific step in the pathway. The results from embryos exposed to this inhibitor were similar to embryos exposed to phenanthrene. Immunostaining of embryos exposed to both compounds reveals nuclear accumulation of ~-catenin, suggesting that phenanthrene may be causing abnormalities by disrupting a specific step in the Wnt signaling pathway. Preliminary Western blot analysis using antibodies to phosphorylated forms of GSK-3~ (anti-Ser 9GSK-3~) also supports this notion