Part I：Spatiotemporal Expression and Enzymatic Activity of Zebrafish D-Amino Acid Oxidase Part II：Sodium Benzoate Induced Toxicity during Zebrafish Development

• Main Authors: Mei-Yun Huang, 黃美韻
• Other Authors: Ming-Kai Chern
• Format: Others
• Language: zh-TW
• Published: 2004
• Online Access: http://ndltd.ncl.edu.tw/handle/22296841573083290151

碩士 === 淡江大學 === 生命科學研究所碩士班 === 94 === D-amino acid oxidase (DAO) is a FAD-containing enzyme which has been characterized in microorganisms and animals. On early periods, the studies of DAO were focused on analysis of enzymatic activity, large-scale production of DAO enzyme, to produce antibiotic and in treatment of patients who had amino acid metabolism disorders. Recently, the distribution and physiological functions of DAOs have become an important issue to be addressed. Sodium benzoate (SB) is a competitive inhibitor of DAO in vitro. SB is a widespreadly used preservative on cosmetic industry and food. It was also used in the treatment of hyperammonaemia in patients with inborn errors of the urea cycle. However, little is unknown about whether SB possessed potential toxicities during early development. Here, we used zebrafish as a model to characterize the enzymatic activity analysis of zebrafish DAO (ZDAO) and to test SB-induced toxicity during early embryonic development of zebrafish. We chose zebrafish as our animal model for the following reasons: large amount and transparent eggs, easy to observe during organogenesis. In the first part of this thesis, we cloned and sequenced a ZDAO gene. In terms of amino acid sequence comparison, ZDAO shared 93% identity with the carp’s DAO. A 39 kDa 6xHis-ZDAO fusion protein was induced and purified in Escherichia coli expression system. The specific enzymatic activity of 6xHis-ZDAO fusion protein was 0.40±0.27 U/mg. The enzymatic specific activities of endogenous ZDAO at different developmental stages were also measured. The ZDAO enzymatic specific activities at 3dpf was 0.148±0.025 U/mg, reached to the optimal at 6dpf (0.323±0.040 U/mg) and gradually decreased to 0.019±0.005 U/mg at adult fish. These results indicated that ZDAO expression profile was dynamics. In the second part of this thesis, we treated zebrafish with different concentrations of SB. After low dosage SB (1~1000 ppm) treatment, the zebrafish embryos exhibited 100% survival rates. As the exposure dosages increased, the survival rates decreased. No embryos were survival after treatment with 2000 ppm of SB. Morphological defects were observed, including hatching gland abnormalities, gut abnormalities and edema in pericardial sac. In addition to the predominant morphological defects in gut and heart, we also found that SB-treated embryos had the following defects that revealed by using antibodies against mature neurons (anti-acetylated-tubulin), sensory neurons (Zn12), motor neurons (Znp1 and Zn5), neuromuscular junctions (Znp1 and α-BTX) and muscle fibers (F59), including: disturbed muscle fibers, missing of neuromast, prolonged RB (Rohon-Beard) neuron lifespan and dispersed trunk motoneuron projections and neuromuscular junctions. Based on these observations, we conclude that SB is able to induce toxicities on sensory neurons, muscle fibers, motor neurons and neuromuscular junctions during early embryonic development of zebrafish.