| Reactive oxygen species (ROS) are generated by many exogenous and endogenous sources. They are highly reactive and can attack biomolecules such as proteins, lipids and DNA, causing damage to living cells. DNA is subjected to constant attack by many damage inducing agents including ROS, but constant repair and maintenance by multiple DNA repair pathways helps to maintain genomic stability. The cell is also protected from the effects of ROS by antioxidants. However, ROS are implicated in cell signaling as important primary and secondary messengers. Many critical transcription factors involved in embryonic development are sensitive to the redox condition in the cell and so reductive stress can be just as dangerous as oxidative stress. Indeed, this laboratory has shown that the position and type of oxidative damage can affect binding of at least one specific transcription factor to its consensus sequence. Based on this evidence, it is possible that ROS might have a role in embryonic development of zebrafish embryos. In the following thesis, we have attempted to answer some basic questions pertaining to the role of ROS in zebrafish embryogenesis. We first asked whether some parts of the zebrafish embryo were more sensitive to ROS than others. We found that the head region of the 24 hours post fertilization (hpf) embryo showed a significant increase in 8-oxoG levels when treated with hydrogen peroxide (H2O2), but we were not able to detect any difference in 8-oxoG levels or localization in earlier embryonic stages by standard fluorescence microscopy. Since AP endonuclease 1(Apex1) is the main enzyme in the base excision repair (BER) pathway that repairs oxidative damage to DNA, we observed the effects of knocking down Apex1 and found that the knockdown embryos exhibited higher levels of 8-oxoG. Finally, we showed that antioxidants such as N-acetylcysteine (NAC) rescued neither peroxide-treated nor Apex1 knockdown embryos. In fact, NAC treatment sensitized embryos subjected to peroxide. NAC did not change 8-oxoG levels in peroxide-treated embryos. In conclusion, this thesis serves as a starting point for the investigation into the role of ROS in zebrafish embryonic development. |
